Keynote Address

iii

Smokeless tobacco use is increasing throughout the United States,

especially among the most vulnerable of our citizens—the children.

With more than 30,000 new cases of oral cancer reported in 1991 for

the United States alone, it is time that use of smokeless tobacco take its

rightful place next to cigarette smoking, in National and world consciousness, as a serious health risk that must be stopped.

As many of you recall, smokeless tobacco use as a health issue first

became a subject of focus in the United States about 6 years ago. The media

reported extensively at that time on the tragic consequences of smokeless

tobacco use—particularly on cases of oral cancer among young men.

In 1986, the Surgeon General released a report, The Health Consequences

of Using Smokeless Tobacco, which concluded that oral use of smokeless

tobacco represents a significant health risk, is not a safe substitute for

cigarette smoking, can cause cancer and a number of noncancerous oral

conditions, and can lead to nicotine addiction and dependence. Also,

Congress passed the Comprehensive Smokeless Tobacco Act of 1986, which

banned smokeless tobacco advertising on television and radio and required

that three health warnings be rotated on smokeless tobacco packages and in

advertisements.

Regrettably, the subsequent downturn in sales of smokeless tobacco

products was not sustained. Statistics from the 1991 Federal Trade Commission Report to Congress on smokeless tobacco sales and advertising expenditures paint a grim picture: The weight of smokeless tobacco products sold

overall rose by more than 2 million pounds in 1989, to 117.5 million

pounds, following a 3-year decline. In 1990, sales increased by 5 million

pounds, to 122.5, and in 1991, sales again rose by almost 3 million pounds,

to 125.1 million. Three companies—U.S. Tobacco, Conwood, and

Pinkerton—together control over 80 percent of the smokeless tobacco

market share in the United States.

The most disturbing trend is in sales of moist snuff, now the most

popular—and the most dangerous—form of smokeless tobacco. While oral

cancer has been shown to occur several times more frequently among snuff

users than among non-users, for some cancer sites in the mouth, the disease

may occur as much as 50 times more frequently among long-term snuff

users.

Sales of moist snuff have risen steadily in recent years, while sales of

other forms of smokeless tobacco have fluctuated or have declined. Sales of

moist snuff climbed to 45.0 million pounds in 1989, a 25 percent increase

Keynote Address

Based on HHS Secretary Sullivan’s keynote address to the First International Conference on

Smokeless Tobacco, Columbus, Ohio, April 10-13, 1991.
Smoking and Tobacco Control Monograph No. 2

iv

over the 35.9 million pounds sold in 1986. In 1990 and 1991, sales reached

52.9 million pounds, a 47 percent increase over 1986 (Figure 1). The U.S.

Distribution Journal reported that the volume of moist snuff sales has risen 70

percent in the last 10 years.

Our most recent surveys of smokeless tobacco use nationwide revealed

that in 1988, 25.8 percent of males between the ages of 12 and 17 had tried

some form of smokeless tobacco, and 6.6 percent had used smokeless

tobacco in the preceding month.

Newer evidence suggests the problem is becoming much more serious.

For example, according to a 1991 survey by the Illinois Department of

Public Health, nearly half of all high school-aged males across the state have

tried smokeless tobacco in some form. About 16 percent of Illinois high

school juniors are regular users, and in rural communities, the figure rises to

28 percent. Some children begin ST use as early as the second, third, or

fourth grade!

The culture of smokeless tobacco use in the United States has to a large

extent centered on sports, particularly baseball. When impressionable

youngsters see their heroes openly using smokeless tobacco on the playing

field, there is a powerful incentive to try it. These young people also begin

to correlate smokeless tobacco use with athletic excellence.

When I addressed the Major League Baseball team physicians and

trainers in December of 1990, I suggested that no effort aimed at ending the

use of smokeless tobacco in America would succeed without the support of

the Major League Baseball community. I called upon baseball officials to

develop a program with the goal of eventually eliminating smokeless tobacco use in professional baseball and in America as a whole.

I am pleased to report that the baseball community has responded

positively and boldly. On March 7, 1991, as a first step in the effort to

disassociate professional baseball from smokeless tobacco use, Baseball

Commissioner Fay Vincent announced that the use of smokeless tobacco

will be forbidden in four rookie and class A minor leagues—the levels at

which most young men enter professional baseball.

The Commissioner also announced stepped-up efforts to educate

players about the health risks of smokeless tobacco use and to help users

quit. Major League Baseball and the National Cancer Institute have teamed

up to produce “Beat the Smokeless Habit,” a guide for baseball players of all

ages to learn about the dangers of smokeless tobacco use and how to quit.

Copies of the guide have been distributed to every major and minor league

player as well as to 40,000 college baseball players. I commend Commissioner Vincent for his actions and will be following the progress of his

efforts with great interest.

Now we must urge officials and coaches at the college, high school, and

youth league levels to ban the use of smokeless tobacco in baseball and in

other sports activities under their jurisdictions.
Keynote Address

v

Figure 1

Annual consumption of spitting tobacco, United States, 1961-1991

1961 1966 1971 1976 1981 1986 1991

Year

0

20

40

60

80

100

120

140

Combined

Chewing 

Tobacco

Snuff

Pounds (millions)
Smoking and Tobacco Control Monograph No. 2

vi

This brings us to another significant element that serves to encourage

young people to begin using smokeless tobacco—promotion and advertising

by the tobacco companies.

I would like to note that expenditures by the tobacco companies to

promote and advertise smokeless tobacco use rose sharply—by more than

19 percent—in 1989, according to the Federal Trade Commission. The

largest single category of expenditure, nearly a quarter of total promotion

and advertising expenditures for smokeless tobacco, was devoted to sports

and sporting events. Another 18.5 percent was devoted to expenses involved in the insidious practice of distributing free samples.

The tobacco companies indeed play a major role in the promotion of

sporting events. Philip Morris, known for its Virginia Slims Tennis Tournaments, is now the number one event sponsor in sports, according to The

Sporting News, spending about $100 million annually. RJR Nabisco spends

about $40 million. The tobacco product these two companies promote, of

course, is cigarettes.

It is not surprising, therefore, that when The Sporting News last year

compiled a list of who they believe to be the 100 most powerful people in

sports in the United States, the marketing vice presidents of Philip Morris

and RJR Nabisco were ranked 29th and 49th, respectively.

This represents a sad commentary on the state of sports in America.

Without question, by sponsoring sporting events, the tobacco companies

are trading on the prestige and image of the athletes to barter their deadly

products. They are using the vigor and energy of these athletes as a subtle—

but incorrect and dishonest—message that tobacco use is compatible with

good health. And, all too often, that message is aimed at young people,

women, minorities, and blue-collar workers.

We must question seriously the values that allow activities ostensibly

representing the essence of fun, fitness, and health to be exploited to such

a large degree by the merchants of suffering, disease, and death.

The time has come to end the association of tobacco and sports in this

country and around the world. The time has come for promoters of and

participants in sporting events to be held accountable for the fact that when

they accept money from the tobacco companies, they are promoting not

only fun and games—they are promoting disease and death.

It is immoral for civilized societies to condone the promotion and

advertising of products that, when used as intended, cause disability and

death.

I am all too aware that such activities are allowed under current law in

the United States and elsewhere. And I am, quite frankly, disgusted that

this remains the case. We react with horror and outrage when we hear of

suffering and death overseas. Yet, at the same time, we not only allow our

citizens to suffer and die from the poisonous chemicals contained in tobacco, we condone expenditures of vast amounts of money by the tobacco

companies to encourage it!
Keynote Address

vii

In 1990, I urged the tobacco industry to voluntarily withdraw from

direct sponsorship of sporting events. Obviously, my plea fell on indifferent

ears.

If the tobacco companies will not adhere to this country’s strong

philosophy of voluntary corporate responsibility, then it is up to our citizens to provide the incentive. As individuals, Americans can send a message

to the tobacco companies in the only language they appear to understand—

the language of money. The message is that we will no longer financially

support promoters of sporting events and others who would encourage our

children to use addictive substances that will ruin their health and send

them to an early grave. And I urge public and private institutions to refrain

from allowing their facilities to be used for tobacco company-sponsored

sporting events.

The disgraceful tradeoff in America between profits and good health

must stop! But it will stop only when our citizens rise up and say,

“Enough—no more!”

I urge individuals and organizations throughout the Nation and the

world to join me in the expression of anger and resolve. Let this be the

beginning of an all-out effort.

Make no mistake: the continuing battle against tobacco use will be

long and difficult. But it is a battle that can be won, must be won, and will

be won. Together, we will win it.

Louis W. Sullivan, M.D.

Secretary

Department of Health

and Human Services
Smoking and Tobacco Control Monograph No. 2

viii
Foreword

ix

Tobacco use in the United States has gone through many stages. Initially tobacco was used by Native Americans in religious ceremonies, with

apparently little or no routine usage. The colonists, however, adopted

tobacco for secular uses and quickly established it as a trading commodity.

Toward the end of the 19th century, smoking tobacco was found primarily

in cigars, pipes, or roll-your-own cigarettes; less than 3 percent of all tobacco

consumed was in the form of manufactured cigarettes. By contrast, more

than 50 percent was consumed as chewing tobacco.

The changing social acceptance of the manufactured cigarette, together

with the use of milder, blended tobaccos, beginning around 1913, allowed

tobacco users to absorb nicotine more quickly and efficiently through

inhalation than via absorption through the oral mucosa. Thus, by the end

of World War I, cigarette smoking had become not only socially acceptable

but even fashionable—at least for men. By the early 1920’s, tobacco consumed in the form of cigarettes had increased considerably, accounting for

fully 25 percent of all tobacco being used in the United States.

The 1920’s and 1930’s saw a major increase in the number of male

smokers, with the 1940’s and 1950’s characterized as the beginning of this

country’s lung cancer epidemic. These time frames illustrate the 20- to 30-

year lag time between the initiation of regular smoking and a clinical

diagnosis of lung cancer. This lag time was observed again with the increased use of cigarettes by women in the 1940’s and 1950’s and the subsequent increase in lung cancer among women in the 1970’s.

Social norms have played a major role in the history of tobacco use. In

the early part of this century, many antitobacco laws were based on morals

and religion.1 During the latter half of the 20th century, however, medical

science has provided the foundation for a rational public health policy on

tobacco use. When the landmark 1964 Surgeon General’s Report identified

smoking as the major cause of lung cancer, smoking rates dropped. Subsequent reports from the Surgeon General have reinforced the public’s consciousness of the multitude of hazards in exposure to tobacco smoke—for

both the smoker and the nonsmoker.

Paradoxically, it was the heightened awareness of smoking hazards in

the 1970’s and early 1980’s that prompted some people, looking for a safe

alternative to cigarettes, to begin using a product that the industry labeled

“smokeless” tobacco. Early advertising campaigns pitched these products as

a safe alternative because they did not contain the major health-threatening

Foreword

1 The use of spitting tobacco fell into social disfavor immediately after the turn of the century

because of concern that exposure to saliva increased the spread of tuberculosis, a major

uncontrolled cause of death at that time. Many towns and cities enacted local ordinances

against spitting in public.
Smoking and Tobacco Control Monograph No. 2

x

ingredient of cigarettes—smoke. Millions of consumers succumbed to this

faulty logic, and the use of spitting tobacco (chewing tobacco and snuff, or

ST) spread rapidly, particularly among young adult and adolescent males, as

Figure 1 illustrates.

Unfortunately, the tobacco industry’s ability to increase demand

through new and innovative marketing strategies has always outpaced the

ability of the scientific community to document the adverse health effects

resulting from product use. The ST industry began to aggressively advertise

and promote new product lines, often using well-known figures in sports

and entertainment to pitch these products on television during prime time.

The use of TV and radio for cigarette advertising had been banned by the

Congress since 1971, but the legislation did not address other tobacco

products.

Particularly alarming is that ST advertising has been most effective with

boys and young men. Although the marketing of ST products is legal,

current marketing strategies are persuading many individuals to become

regular users before reaching 18, the legal age to buy the products in most

states. One advertising campaign (see Figure 2) used National Football

League stars to teach “beginners” how to work up to stronger brands of

spitting tobacco. In the wake of these promotions, the consumption of

spitting tobacco, especially moist snuff, has continued to rise. From

1972 to 1991, total U.S. consumption of spitting tobacco has risen from

99 million pounds per year to 125 million pounds. There has been an

alarming 40 percent increase in consumption of moist snuff, the most

dangerous form of spitting tobacco.

The increase in ST consumption stands in sharp contrast to the significant decline in consumption observed in all other tobacco categories. Over

the past decade alone, total cigarette sales have decreased by more than

130 billion cigarettes, and our national per capita consumption is at its

lowest level since the early 1940’s (Figure 3). Because of the 20- to 30-year

lag time between large-scale exposure to a carcinogenic agent and subsequent increases in cancer mortality, the trends in ST consumption predict a

public health problem in the making, unless significant behavior changes

can be achieved soon.

In 1981 National Cancer Institute (NCI) investigator Deborah Winn

and colleagues published a seminal study that pointed to a link between oral

snuff dipping and oral cancer. Later that year, after reviewing the evidence,

the Institute announced that it believed that snuff use was a cause of oral

cancer. In 1984 the Smoking and Tobacco Control Program of NCI issued a

Request for Applications, soliciting research on the use of spitting tobacco,

and the following year NCI funded the first clinical trials aimed at intervening in ST use.

The year 1985 saw a flurry of activity related to spitting tobacco. In

February, the National Cancer Advisory Board issued a resolution on smokeless tobacco, in which it stated that the NCAB “considers the use of smokeless tobacco to pose a serious and increasing health risk.” The board also
xi

Figure 1Prevalence of the use of chewing tobacco (left) and snuff (right) among males, 1970 and 1985

Source: 1970 National Health Interview Survey and 1985 Current Population Survey

< 20 20-29 30-39 40-49 50+ All Ages

0

1234567

1970

1985

Percentage

< 20 20-29 30-39 

Percentage

0

123

Percentage

Percentage
Smoking and Tobacco Control Monograph No. 2

xii

Figure 2

ST ad aimed at “beginners”

recommended that the Surgeon General undertake a complete review of the

scientific evidence on ST’s health effects, and that the Office on Smoking

and Health include ST use as part of its ongoing surveillance activities.

Subsequently, the Office on Smoking and Health sponsored a supplement to

the Current Population Survey to collect data on current usage of smokeless

tobacco. The Surgeon General at that time, Dr. C. Everett Koop, appointed a

PHS Advisory Committee to report on ST’s health consequences.

In September 1985, the International Agency for Research on Cancer

(IARC) in Lyon, France, issued a report that concluded, “In aggregate, there

is sufficient evidence that oral use of smokeless tobacco is carcinogenic to

humans.” Then, in January 1986, an NIH-NCI Consensus Development

Conference on Smokeless Tobacco was held; the participants concluded

that there was strong evidence from human studies to link snuff use with

cancer.
xiii

81 82 83 84 85 86 87 88 89 90 91

500525

550575

600625

650

Billions of Cigarettes

Millions of Pounds

81 82 83 84 85 

253035404550

Figure 3U.S. tobacco consumption of cigarettes (left) and moist snuff (right), 1981 to 1991

Source: U.S. Department of Agriculture

Millions of Pounds

Billions of Cigarettes
Smoking and Tobacco Control Monograph No. 2

xiv

The Surgeon General’s Advisory Committee Report, compiled and

published under NCI auspices, was issued in April and concluded that

“oral use of smokeless tobacco represents a significant health risk. It is not

a safe substitute for smoking cigarettes. It can cause cancer and a number

of noncancerous oral conditions and can lead to nicotine addiction and

dependence.”

The committee also concluded that evidence related to the carcinogenic

potential of chewing tobacco was limited, but the evidence on oral use of

snuff was more than sufficient to conclude that it is carcinogenic to humans.

The PHS Advisory Committee also noted that snuff contained N'-

nitrosamines at levels 100 times higher than the levels permitted under

Federal regulations for all other ingested consumer products in the United

States. The levels of nicotine absorbed during ST use were found to be high

and to remain elevated for most of the day. Later in the same year, Congress passed the Comprehensive Smokeless Tobacco Health Education Act of

1986, which banned advertising on broadcast media and required rotating

warning labels on ST products and in print advertising.

In 1989, NCI issued its first monograph on this subject, Smokeless

Tobacco Use in the United States, which reinforced the overall conclusion of

the 1986 Report of the Surgeon General, that “there is no safe form of

tobacco use.” The monograph also contained results from the Current

Population Survey sponsored by the Office on Smoking and Health, which

found that ST use varied considerably by region of the country, with rates

among adult males in many southern states exceeding 12 percent, reaching

23 percent in West Virginia.

In April 1991, NCI cosponsored the First International Conference on

Smokeless Tobacco. From this conference, the world learned that ST use is a

problem in many countries, and the types of ST used are diverse, but the

potential for addiction and oral cancer and other untoward health effects

are omnipresent. This monograph presents a selection of peer-reviewed

papers from that valuable conference.

We have made significant advances in our understanding of the health

consequences associated with ST use, but much more remains to be done.

NCI is committed to the continued support of research on spitting tobacco,

with the eventual goal of making the use of this deadly and addictive

product an extinct behavior. Research alone, however, will not solve this

growing health threat. Thus, the reduction of ST use is a high priority

within ASSIST—the American Stop Smoking Intervention Study for Cancer

Prevention, a 17-state, NCI-funded, demonstration project that began last

October. Over the next 7 years, ASSIST is expected to reach more than

90 million individuals, including 20 million tobacco users. While ASSIST is

aimed primarily at cigarette smokers, an estimated 1 million or more regular

ST users will be targeted for the project interventions.
Foreword

xv

We have made great progress over the past 40 years in making the

United States a smoke-free society. Our task will not be complete however,

until we make the nation free of tobacco use in all forms. To accomplish

this will require the full cooperation of public and private agencies, research

and service organizations, professionals and laypeople, working together

toward this common goal. To do otherwise would invite unnecessary

suffering, disease, and death.

Antonia C. Novello, M.D., M.P.H.

Surgeon General
Smoking and Tobacco Control Monograph No. 2

xvi
Contents

xvii

Keynote Address ................................................................................ iii

Foreword ............................................................................................ ix

Acknowledgments ............................................................................. xxiii

Overview and Summary .................................................................... xxxix

Chapter 1. Epidemiologyi ................................................................................ 1

The Smokeless Tobacco Problem:

Risk Groups in North America .................................................... 3

Elbert D. Glover

Penny N. Glover

Surveillance of and Knowledge About

Cancer Associated With Smokeless Tobacco Use ....................... 11

Deborah M. Winn

Smokeless Tobacco Use in India ................................................. 19

P.C. Gupta

Smokeless Tobacco in Professional Baseball:

Patterns of Players’ Use ............................................................... 26

V.L. Ernster

D.G. Grady

L. Stillman

M. Walsh

J.C. Greene

Chapter 2. Clinical and Pathological Effects i............................................ 39

Oral Mucosal Lesions: Clinical Findings in Relation to

Smokeless Tobacco Use Among U.S. Baseball Players ................ 41

John C. Greene

Virginia L. Ernster

Deborah G. Grady

Paul B. Robertson

Margaret M. Walsh

Linda A. Stillman

Contents

Page
Smoking and Tobacco Control Monograph No. 2

xviii

Chapter 2 (continued)

Smokeless Tobacco Use in India:

Effects on the Oral Mucosa ........................................................... 51

P.R. Murti

P.C. Gupta

R.B. Bhonsle

D.K. Daftary

F.S. Mehta

J.J. Pindborg

Smokeless Tobacco-Associated

Epithelial and Langerhans Cell Changes ...................................... 66

Troy E. Daniels

John S. Greenspan

Laisheng Chou

Deborah G. Grady

Walter W. Hauck

John C. Greene

Virginia L. Ernster

The Use of Smokeless Tobacco in Sweden .................................... 74

Jan Bergström

Periodontal Effects Associated With the

Use of Smokeless Tobacco: Results After 1 Year............................ 78

P.B. Robertson

V. Ernster

M. Walsh

J. Greene

D. Grady

W. Hauck

Chapter 3. Carcinogenesisi ................................................................................ 87

Oral Carcinoma and Smokeless Tobacco Use:

A Clinical Profile ........................................................................... 91

W. Frederick McGuirt

Anna Wray

Chemical Composition of Smokeless Tobacco Products .............. 96

Klaus D. Brunnemann

Dietrich Hoffmann

Carcinogenesis of Smokeless Tobacco .......................................... 109

Dietrich Hoffmann

Abraham Rivenson

Stephen S. Hecht
Contents

xix

Chapter 3 (continued)

Oncogenes in Head and Neck Cancer........................................... 119

Kenneth D. Somers

Suzanne E. Glickman

Suzanne L. Cartwright

Bryan G. Stoll

Stella L. Maurizio

Gary L. Schechter

Oncogenes in Tobacco-Induced Oral Cancer ............................... 128

Madhav G. Deo

Dhananjaya Saranath

Metabolism and Macromolecular Binding of NNK and NNN,

Important Carcinogens in Smokeless Tobacco ............................. 141

Stephen S. Hecht

Role of Nicotine as a Cofactor in

Smokeless Tobacco Carcinogenesis ............................................... 153

Christopher A. Squier

Georgia K. Johnson

Perinatal Carcinogenesis by Constituents of Smokeless Tobacco:

Animal Models and Potential Human Risk................................... 161

Lucy M. Anderson

Jerry M. Rice

Interaction Between Smokeless Tobacco-Related Carcinogens

And Human Papillomaviruses in the Pathogenesis of

Oral Dysplasia and Cancer ............................................................ 175

Joel M. Palefsky

John S. Greenspan

Troy E. Daniels

Jennifer Berline

Deborah G. Grady

Virginia L. Ernster

Identification of Human Papillomavirus DNA in

Smokeless Tobacco Keratoses and Premalignant and

Malignant Oral Lesions, by PCR Amplification With

Consensus Sequence Primers ........................................................ 183

Robert O. Greer, Jr.

Kenneth R. Shroyer

Louise Crosby

Lipids as Factors in the Cell Response to Tobacco Components . 191

George S. Schuster

Scott Lubas

Thomas R. Dirksen

John F. Erbland
Smoking and Tobacco Control Monograph No. 2

xx

Chapter 3 (continued)

Role of Viruses in Oral Carcinogenesis ......................................... 202

No-Hee Park

Byung-Moo Min

Sheng-Lin Li

Henry M. Cherrick

Jay Doniger

Chapter 4 Nicotine Effects and Addictioni .................................................. 217

Pharmacology of Smokeless Tobacco Use: Nicotine Addiction

And Nicotine-Related Health Consequences ................................ 219

Neal L. Benowitz

Recent Advances in Understanding the Actions

Of Nicotine in the Central Nervous System ................................. 229

Paul B.S. Clarke

Dependence on Smokeless Tobacco.............................................. 239

Martin J. Jarvis

Chapter 5. Preventioni ....................................................................................... 245

Marketing Smokeless Tobacco: Implications for

Preventive Education .................................................................... 247

Carol N. D’Onofrio

Applying the Social Inoculation Model to a Smokeless Tobacco

Use Prevention Program With Little Leaguers .............................. 260

Richard I. Evans

Bettye E. Raines

J. Greg Getz

Chapter 6. Cessationi .......................................................................................... 277

Enough Snuff: Smokeless Tobacco Cessation from Behavioral,

Clinical, and Public Health Perspectives....................................... 279

Herbert H. Severson

Smokeless Tobacco Cessation and Nicotine

Reduction Therapy ........................................................................ 291

Elbert D. Glover

Penny N. Glover

Role of Dentists in Cessation Counseling: Survey Findings ........ 297

Kathleen L. Schroeder

David E. Heisel

An Intervention Study of Tobacco Habits Among

Rural Indian Villagers .................................................................... 301

Mira B. Aghi

P.C. Gupta

F.S. Mehta

J.J. Pindborg
Contents

xxi

Chapter 7. Policyi ................................................................................................ 313

WHO Strategies To Curb Smokeless Tobacco:

A Global Perspective...................................................................... 315

Roberto Masironi

Legal and Administrative Strategies for Control

And Prevention of the Use of Smokeless Tobacco ........................ 323

Seamus O’Hickey

National Cancer Institute’s Role in Reducing Tobacco Use ......... 330

Robert Mecklenburg

Chapter 8. Recommendations i......................................................................... 335

Recommendations for the Control of Smokeless Tobacco ........... 337

Robert Mecklenburg

R. Craig Stotts

Indexi ........................................................................................................................ 351
Smoking and Tobacco Control Monograph No. 2

xxii
xxiii

Smokeless Tobacco or Health: An International Perspective was developed

under the general editorship of the Smoking and Tobacco Control Program

(STCP), Donald R. Shopland, Coordinator, National Cancer Institute,

Bethesda, Maryland. The senior scientific editor was R. Craig Stotts,

Dr.P.H., R.N., Research Fellow, NCI Division of Cancer Prevention and

Control. The consulting scientific editors were Kathleen L. Schroeder,

D.D.S., M.Sc., West Virginia University Schools of Medicine and Dentistry,

and David M. Burns, M.D., Professor of Medicine, University of California, San Diego.

Special recognition is due Dr. Schroeder for her role in planning and

organizing the First International Conference on Smokeless Tobacco, from

which these monograph papers were developed. Members of the committee

that assisted Dr. Schroeder in planning the conference are listed near the

end of this section.

The staff of the NCI Smoking and Tobacco Control Program

gratefully acknowledges the authors who made this monograph

possible. Attributions for each chapter, in alphabetical order,

are as follows:

Acknowledgments

Chapter 1 Epidemiology Virginia L. Ernster, Ph.D.

Department of Epidemiology and Biostatistics

School of Medicine

Department of Dental Public Health and Hygiene

School of Dentistry

University of California, San Francisco

San Francisco, California

Elbert D. Glover, Ph.D.

Tobacco Research Center

Mary Babb Randolph Cancer Center

West Virginia University Health Sciences Center

Morgantown, West Virginia

Penny N. Glover, M.Ed.

Tobacco Research Center

Mary Babb Randolph Cancer Center

West Virginia University Health Sciences Center

Morgantown, West Virginia
Smoking and Tobacco Control Monograph No. 2

xxiv

Chapter 1 (continued) Deborah G. Grady, M.D.

Department of Epidemiology and Biostatistics

Department of Medicine, School of Medicine

Department of Dental Public Health and Hygiene

School of Dentistry

University of California, San Francisco

San Francisco, California

John C. Greene, D.M.D., M.P.H., I.A.C.D.

Department of Dental Public Health and Hygiene

Dean, School of Dentistry

University of California, San Francisco

San Francisco, California

Prakesh C. Gupta, D.Sc., F.A.C.E.

Basic Dental Research Unit and

WHO Collaborating Centre for

Oral Cancer Prevention

Tata Institute of Fundamental Research

Colaba, Bombay, India

Linda A. Stillman, M.P.H.

Department of Dental Public Health and Hygiene

University of California, San Francisco

San Francisco, California

Deborah M. Winn, Ph.D.

National Center for Health Statistics

Centers for Disease Control

Hyattsville, Maryland

Chapter 2 Clinical and Jan M. Bergström, Odont. Dr.

Pathological Department of Periodontology

Effects Karolinska Institutet

School of Dentistry

Huddinge, Sweden

R.B. Bhonsle, B.D.S.

Basic Dental Research Unit and

WHO Collaborating Centre for

Oral Cancer Prevention

Tata Institute of Fundamental Research

Colaba, Bombay, India

Laisheng Chou

Division of OIral Pathology

Department of Stomatology

School of Dentistry

University of California, San Francisco

San Francisco, California
xxv

Chapter 2 (continued) D.K. Daftary, M.D.S.

Basic Dental Research Unit and

WHO Collaborating Centre for

Oral Cancer Prevention

Tata Institute of Fundamental Research

Colaba, Bombay, India

Troy E. Daniels, D.D.S., M.S.

Division of Oral Pathology

Department of Stomatology

School of Dentistry

University of California, San Francisco

San Francisco, California

Virginia L. Ernster, Ph.D.

Department of Epidemiology and Biostatistics

School of Medicine

Department of Dental Public Health and Hygiene

School of Dentistry

University of California, San Francisco

San Francisco, California

Deborah G. Grady, M.D.

Department of Epidemiology and Biostatistics

Department of Medicine, School of Medicine

Department of Dental Public Health and Hygiene

School of Dentistry

University of California, San Francisco

San Francisco, California

John C. Greene, D.M.D., M.P.H., I.A.C.D.

Department of Dental Public Health and Hygiene

Dean, School of Dentistry

University of California, San Francisco

San Francisco, California

John S. Greenspan, B.D.S., Ph.D.

Division of Oral Pathology

Department of Stomatology

School of Dentistry

University of California, San Francisco

San Francisco, California

Prakesh C. Gupta, D.Sc., F.A.C.E.

Basic Dental Research Unit and

WHO Collaborating Centre for

Oral Cancer Prevention

Tata Institute of Fundamental Research

Colaba, Bombay, India

Acknowledgments
Smoking and Tobacco Control Monograph No. 2

xxvi

Chapter 2 (continued) Louis S. Hansen, D.D.S.

Division of Oral Pathology

Department of Stomatology

School of Dentistry

University of California, San Francisco

San Francisco, California

Walter W. Hauck, Ph.D.

Department of Epidemiology and Biostatistics

School of Medicine

University of California, San Francisco

San Francisco, California

F.S. Mehta, D.M.D.

Basic Dental Research Unit and

WHO Collaborating Centre for

Oral Cancer Prevention

Tata Institute of Fundamental Research

Colaba, Bombay, India

P.R. Murti, M.D.S.

Basic Dental Research Unit and

WHO Collaborating Centre for

Oral Cancer Prevention

Tata Institute of Fundamental Research

Colaba, Bombay, India

J.J. Pindborg, D.D.S., Dr. Odont.

Department of Oral Pathology

Royal Dental College

Rigshospitalet

Copenhagen, Denmark

Paul B. Robertson, D.D.S.

Dean, Faculty of Dentistry

University of British Columbia

Vancouver, Canada

Linda A. Stillman, M.P.H.

Department of Dental Public Health and Hygiene

School of Denistry

University of California, San Francisco

San Francisco, California

Margaret M. Walsh, Ed.D.

Department of Dental Public Health and Hygiene

School of Denistry

University of California, San Francisco

San Francisco, California
xxvii

Chapter 3 Carcinogenesis Lucy M. Anderson, Ph.D.

Division of Cancer Etiology

National Cancer Institute

Frederick, Maryland

Jennifer Berline

Division of Oral Pathology,

Department of Stomatology

School of Dentistry

University of California, San Francisco

San Francisco, California

Klaus D. Brunnemann, Ph.D.

Naylor Dana Institute for Disease Prevention

American Health Foundation

Valhalla, New York

Steven G. Carmella

Naylor Dana Institute for Disease Prevention

American Health Foundation

Valhalla, New York

Suzanne L. Cartwright

Head and Neck Tumor Biology Program

Departments of Microbiology/Immunology

and Otalaryngology-Head and Neck Surgery

Norfolk, Virginia

Henry M. Cherrick, D.D.S., M.S.D.

School of Dentistry

University of California, Los Angeles

Los Angeles, California

Louise K. Crosby, M.A.

Division of Oral Pathology and Oncology

University of Colorado

Denver, Colorado

Troy E. Daniels, D.D.S., M.S.

Division of Oral Pathology

Department of Stomatology

School of Dentistry

University of California, San Francisco

San Francisco, California

Madhev G. Deo, M.D., Ph.D., F.A.M.S., F.N.A.

Cancer Research Institute

Parel, Bombay, India

Thomas R. Dirksen, D.D.S., M.S., Ph.D.

Department of Oral Biology

School of Dentistry

Medical College of Georgia

Augusta, Georgia

Acknowledgments
Smoking and Tobacco Control Monograph No. 2

xxviii

Chapter 3 (continued) Jay Doniger, Ph.D.

Georgetown University School of Medicine

Washington, D.C.

John F. Erbland, Ph.D., D.M.D.

Department of Oral Biology

Medical College of Georgia

School of Dentistry

Augusta, Georgia

Virginia L. Ernster, Ph.D.

Department of Epidemiology and Biostatistics

School of Medicine

Department of Dental Public Health and Hygiene

School of Dentistry

University of California, San Francisco

San Francisco, California

Suzanne E. Glickman

Head and Neck Tumor Biology Program

Departments of Microbiology/Immunology

and Otolaryngology-Head and Neck Surgery

Eastern Virginia Medical School

Norfolk, Virginia

Deborah G. Grady, M.D.

Department of Epidemiology and Biostatistics

Department of Medicine, School of Medicine

Department of Dental Public Health and Hygiene

School of Dentistry

University of California, San Francisco

San Francisco, California

John S. Greenspan, B.D.S., Ph.D.

Division of Oral Pathology

Department of Stomatology

School of Dentistry

University of California, San Francisco

San Francisco, California

Robert O. Greer, Jr., D.D.S., Sc.D.

Department of Pathology, School of Medicine

Division of Oral Pathology and Oncology

School of Dentistry

University of Colorado

Denver, Colorado

Stephen S. Hecht, Ph.D.

Naylor Dana Institute for Disease Prevention

American Health Foundation

Valhalla, New York
xxix

Chapter 3 (continued) Dietrich K. Hoffmann, Ph.D.

Naylor Dana Institute for Disease Prevention

American Health Foundation

Valhalla, New York

Georgia K. Johnson

Dows Institute for Dental Research

College of Dentistry

University of Iowa

Iowa City, Iowa

Sheng-Lin Li, D.D.S.

School of Dentistry

University of California, Los Angeles

Los Angeles, California

Scott Lubas

Department of Oral Biology

Medical College of Georgia

School of Dentistry

Augusta, Georgia

Stella L. Maurizio

Head and Neck Tumor Biology Program

Departments of Microbiology/Immunology

and Otolaryngology-Head and Neck Surgery

Eastern Virginia Medical School

Norfolk, Virginia

W. Frederick McGuirt, M.D.

Department of Otolaryngology

Bowman Gray School of Medicine

Wake Forest University

Winston-Salem, North Carolina

Byung-Moo Min, D.D.S., Ph.D.

School of Dentistry

University of California, Los Angeles

Los Angeles, California

Sharon E. Murphy, Ph.D.

Naylor Dana Institute for Disease Prevention

American Health Foundation

Valhalla, New York

Joel M. Palefsky, M.D.

Division of Oral Pathology,

Department of Stomatology

School of Dentistry

Department of Laboratory Medicine

School of Medicine

University of California, San Francisco

San Francisco, California

Acknowledgments
Smoking and Tobacco Control Monograph No. 2

xxx

Chapter 3 (continued) No-Hee Park, D.M.D., Ph.D.

School of Dentistry and

Jonsson Comprehensive Cancer Center

University of California, Los Angeles

Los Angeles, California

Jerry M. Rice, Ph.D.

Division of Cancer Etiology

National Cancer Institute

Frederick, Maryland

Abraham Rivenson, M.D.

Naylor Dana Institute for Disease Prevention

American Health Foundation

Valhalla, New York

Dhananjaya Saranath, Ph.D.

Cancer Research Institute

Parel, Bombay, India

Gary L. Schechter, M.D.

Head and Neck Tumor Biology Program

Departments of Microbiology/Immunology

and Otolaryngology-Head and Neck Surgery

Eastern Virginia Medical School

Norfolk, Virginia

George S. Schuster, D.D.S., M.S., Ph.D.

Department of Oral Biology

Medical College of Georgia

School of Dentistry

Augusta, Georgia

Kenneth R. Shroyer, M.D., Ph.D.

Department of Pathology

University of Colorado School of Medicine

Denver, Colorado

Kenneth D. Somers, Ph.D.

Head and Neck Tumor Biology Program

Departments of Microbiology/Immunology

and Otolaryngology-Head and Neck Surgery

Eastern Virginia Medical School

Christopher A. Squier, Ph.D.

Dows Institute for Dental Research

College of Dentistry

University of Iowa

Iowa City, Iowa
xxxi

Chapter 3 (continued) Bryan G. Stoll

Head and Neck Tumor Biology Program

Departments of Microbiology/Immunology

and Otolaryngology-Head and Neck Surgery

Eastern Virginia Medical School

Norfolk, Virginia

Anna M. Wray, M.D.

Department of Otolaryngology

Bowman Gray School of Medicine

Wake Forest University

Winston-Salem, North Carolina

Chapter 4 Nicotine Effects Neal L. Benowitz, M.D.

and Addiction University of California, San Francisco

San Francisco, California

Paul B.S. Clarke, M.A., Ph.D.

Department of Pharmacology and Therapeutics

McGill University

Montreal, Canada

Martin J. Jarvis, M.Phil.

Imperial Cancer Research Fund

Health Behaviour Unit

Institute of Psychiatry

London, U.K.

Chapter 5 Prevention Carol N. D’Onofrio, Dr.P.H.

School of Public Health

University of California, Berkeley

Berkeley, California

Richard I. Evans, Ph.D.

Department of Psychology

University of Houston

Houston, Texas

J. Greg Getz, Ph.D.

Department of Psychology

University of Houston

Houston, Texas

Bettye E. Raines

Department of Psychology

University of Houston

Houston, Texas

Chapter 6 Cessation Mira B. Aghi, Ph.D.

Basic Dental Research Unit

Tata Institute of Fundamental Research

Bombay, India

Acknowledgments
Smoking and Tobacco Control Monograph No. 2

xxxii

Chapter 6 (continued) Elbert D. Glover, Ph.D.

Tobacco Research Center

Mary Babb Randolph Cancer Center

West Virginia University Health Sciences Center

Morgantown, West Virginia

Penny N. Glover, M.Ed.

Tobacco Research Center

Mary Babb Randolph Cancer Center

West Virginia University Health Sciences Center

Morgantown, West Virginia

Prakesh C. Gupta, D.Sc., F.A.C.E.

Basic Dental Research Unit and

WHO Collaborating Centre for

Oral Cancer Prevention

Tata Institute of Fundamental Research

Colaba, Bombay, India

David E. Heisel, D.D.S., M.B.A.

Columbus Health Department

Community Dental Programs

Columbus, Ohio

F.S. Mehta, D.M.D.

Basic Dental Research Unit and

WHO Collaborating Centre for

Oral Cancer Prevention

Tata Institute of Fundamental Research

Colaba, Bombay, India

J.J. Pindborg, D.D.S., Dr. Odont.

Department of Oral Pathology

Royal Dental College

Rigshospitalet, Copenhagen, Denmark

Kathleen L. Schroeder, D.D.S., M.Sc.

Tobacco Research Center

Mary Babb Randolph Cancer Center

Department of Oral Pathology

West Virginia University Schools of

Medicine and Dentistry

Morgantown, West Virginia

Herbert H. Severson, Ph.D.

Oregon Research Institute

Eugene, Oregon

Chapter 7 Policy Roberto Masironi, Ph.D.

Tobacco or Health Programme

World Health Organization

Geneva, Switzerland
xxxiii

Chapter 7 (continued) Robert E. Mecklenburg, D.D.S., M.P.H.

Consultant, National Cancer Institute

National Institutes of Health

Bethesda, Maryland

Seamus O’Hickey, M.Sc., B.D.S., F.I.C.D., D.P.A.

Department of Health

Dublin, Ireland

Chapter 8 Recommendations Robert E. Mecklenburg, D.D.S., M.P.H.

Consultant, National Cancer Institute

National Institutes of Health

Bethesda, Maryland

R. Craig Stotts, Dr.P.H., R.N.

Division of Cancer Prevention and Control

National Cancer Institute

National Institutes of Health

Bethesda, Maryland

The editors gratefully acknowledge also the following distinguished scientists, researchers, and others, both in and outside

government, who contributed critical reviews:

Jerry E. Bouquot, D.D.S., M.S.D.

Department of Oral Pathology

Head and Neck Diagnosis of West Virginia

West Virginia University Schools of Medicine and Dentistry

Morgantown, West Virginia

Moon S. Chen, Jr., Ph.D., M.P.H.

Department of Preventive Medicine

Ohio State University

Columbus, Ohio

Arden G. Christen, D.D.S., M.S.D, M.A., F.A.C.D.

Preventive/Community Dentistry

Indiana University School of Dentistry

Indianapolis, Indiana

Richard I. Evans, Ph.D.

Department of Psychology

University of Houston

Houston, Texas

Jacquelyn L. Fried, R.D.H., M.S.

Department of Dental Hygiene

College of Dental Surgery

University of Maryland at Baltimore, Dental School

Baltimore, Maryland

Acknowledgments
Smoking and Tobacco Control Monograph No. 2

xxxiv

Elbert D. Glover, Ph.D.

Tobacco Research Center

Mary Babb Randolph Cancer Center

West Virginia University Health Sciences Center

Morgantown, West Virginia

John C. Greene, D.M.D., M.P.H., F.A.C.D.

Department of Dental Public Health and Hygiene

Dean, School of Dentistry

University of California, San Francisco

San Francisco, California

Robert O. Greer, Jr., D.D.S., Sc.D.

Division of Oral Pathology and Oncology

University of Colorado Health Sciences Center

Denver, Colorado

Stephen S. Hecht, Ph.D.

Naylor Dana Institute for Disease Prevention

American Health Foundation

Valhalla, New York

David E. Heisel, D.D.S., M.B.A.

Community Dental Programs

Columbus Health Department

Columbus, Ohio

Jack E. Henningfield, Ph.D.

Clinical Pharmacology Branch

National Institute on Drug Abuse

Addiction Research Center

Baltimore, Maryland

Dietrich K. Hoffmann, Ph.D.

Naylor Dana Institute for Disease Prevention

American Health Foundation

Valhalla, New York

Elizabeth C. Lenz, D.D.S.

Department of Oral Pathology

West Virginia University School of Dentistry

Morgantown, West Virginia

James L. McDonald, Jr., Ph.D.

Preventive/Community Dentistry

Indiana University School of Dentistry

Indianapolis, Indiana

Robert E. Mecklenburg, D.D.S., M.P.H.

Consultant, National Cancer Institute

National Institutes of Health

Bethesda, Maryland
xxxv

Rawhi Omar, M.D., Ph.D.

Department of Pathology

West Virginia Universoty School of Medicine

Morgantown, West Virginia

No-Hee Park, D.M.D., Ph.D.

School of Dentistry and

Jonsson Comprehensive Cancer Center

University of California, Los Angeles

Los Angeles, California

Kathleen L. Schroeder, D.D.S., M.Sc.

Tobacco Research Center

Mary Babb Randolph Cancer Center

Department of Oral Pathology

West Virginia University Schools of

Medicine and Dentistry

Morgantown, West Virginia

George S. Schuster, D.D.S., Ph.D., M.S.

Department of Oral Biology/Microbiology

School of Dentistry, Medical College of Georgia

Augusta, Georgia

Herbert H. Severson, Ph.D.

Oregon Research Institute

Eugene, Oregon

Christopher A. Squier, Ph.D.

Dows Institute for Dental Research

College of Dentistry

University of Iowa

Iowa City, Iowa

R. Craig Stotts, Dr.P.H., R.N.

Division of Cancer Prevention and Control

National Cancer Institute

National Institutes of Health

Bethesda, Maryland

Deborah M. Winn, Ph.D.

National Center for Health Statistics

Centers for Disease Control

Hyattsville, Maryland

Acknowledgments
Smoking and Tobacco Control Monograph No. 2

xxxvi

Following are members of the Conference Planning Committee, who participated in organizing the First International Conference on Smokeless Tobacco:

Donald F. Bowers, D.D.S., M.S.D., F.A.C.D.

Ohio State University

College of Dentistry

Columbus, Ohio

Moon S. Chen, Jr., Ph.D., M.P.H.

Department of Preventive Medicine

Ohio State University

Columbus, Ohio

Arden G. Christen, D.D.S., M.S.D., M.A., F.A.C.D.

Preventive/Community Dentistry

Indiana University

School of Dentistry

Indianapolis, Indiana

Michael W. Easley, D.D.S., M.P.H., F.A.C.D.

Health Care Division—Oral Products

Clinical Operations and Biometrics Section

Procter & Gamble Company

Sharon Woods Technical Center

Cincinnati, Ohio

Richard I. Evans, Ph.D.

Department of Psychology

University of Houston

Houston, Texas

Elbert D. Glover, Ph.D.

Tobacco Research Center

Mary Babb Randolph Cancer Center

West Virginia University Health Sciences Center

Morgantown, West Virginia

John C. Greene, D.M.D., M.P.H., F.A.C.D.

University of California

School of Dentistry

San Francisco, California

Prakash C. Gupta, D.Sc., F.A.C.E.

Basic Dental Research Unit and

WHO Collaborating Centre for

Oral Cancer Prevention

Tata Institute of Fundamental Research

Bombay, India

Dietrich K. Hoffmann, Ph.D.

Naylor Dana Institute for Disease Prevention

American Health Foundation

Valhalla, New York
xxxvii

James L. McDonald, Ph.D.

Preventive/Community Dentistry

Indiana University

School of Dentistry

Indianapolis, Indiana

Robert E. Mecklenburg, D.D.S., M.P.H.

Consultant, National Cancer Institute

National Institutes of Health

Bethesda, Maryland

Herbert H. Severson, Ph.D.

Oregon Research Institute

Eugene, Oregon

Margaret M. Titterington, M.S.

Public Education Department

American Cancer Society

Ohio Division, Inc.

Dublin, Ohio

David S. Yohn, Ph.D., M.P.H.

Ohio State University

Comprehensive Cancer Center

Columbus, Ohio

Sponsors of the smokeless tobacco conference were the

following:

American Fund for Dental Health

Marion Merrell Dow, Inc.

National Institute of Dental Research (grant no. R13 DE/CA-09884-01)

National Cancer Institute (grant no. R13 DE/CA-09884-01)

U.S. Public Health Service, Office of International Health

Ohio State University Comprehensive Cancer Center

American Cancer Society, Ohio Division

West Virginia University, Mary Babb Randolph Cancer Center

Nationwide Insurance

Ohio Dental Association

Acknowledgments
Smoking and Tobacco Control Monograph No. 2

xxxviii

The editor acknowledges the contributions of the following

staff members of R.O.W. Sciences, Inc., Rockville, Maryland, who

provided technical and editorial assistance in the preparation of

this monograph:

Richard H. Amacher, Project Manager

Jill Aughenbaugh, Indexer

Douglas Bishop, Art Director

Margaret Bowman, Copy Editor

Jean Brady, Research Assistant

Karen Bray, Technical Assistant

Caroline Caldwell, Graphics Specialist

Rebecca Charton, Librarian

Traci Cherrier, Research Assistant

Catherine Godfrey, Word Processing Specialist

Faye Grant, Administrative Secretary

Chris Louthan, Research Assistant

Steve Luckabaugh, Word Processing Specialist

Kay Marshall, Copy Editor

Chérie Melat, Graphics Specialist

Frances Nebesky, Copy Editor

Adrienne Plater, Word Processing Specialist

Theodora Radcliffe, Copy Editor, Senior Indexer

Barbara Shine, Managing Editor

Neil Swan, Science Writer

Donna Tharpe, Copy Editor

Sonia Van Putten, Word Processing Specialist

Debra Waugh, Copy Editor

Larry Wentzel, Graphics Specialist

Ronald W. Wolf, Coordinator, Senior Copy Editor
xxxix

BRIEF HISTORICAL

PERSPECTIVE

Overview and Summary

Large-scale consumption of tobacco has been a significant lifestyle factor in America for centuries (Robert, 1967; US DHHS,

1992). Prior to the beginning of the 20th century, tobacco was consumed in

the form of spitting tobacco (chewing tobacco and snuff), smoked as cigars,

or loose tobacco smoked in pipes or in hand-rolled cigarettes (Shopland and

Haenlein, in press; US DHHS, 1990). Consumption of machine-made

cigarettes was almost nonexistent, and spitting tobacco was the dominate

form of use, accounting for nearly 60 percent of all tobacco consumed on a

per capita basis.

Figure 1 depicts tobacco consumption in the United States, by major

product category, for the past 100 years (Milmore and Conover, 1956; US

Department of Agriculture). This data is in pounds of tobacco consumed

per adult rather than in individual units such as cigars or cigarettes, to allow

direct comparisons between categories. The data are also presented in a

cumulative fashion to show better the change in individual product use over

time and in relation to other categories.

What is clearly evident is that, for much of the latter part of the 19th

century and the early decades of the 20th, chewing tobacco was the single

predominate product (Milmore and Conover, 1956). When one recognizes

that, during the early part of this century, tobacco use was primarily a male

phenomena, if these data were expressed in terms of male consumption

only, total pounds per person would be nearly double. In 1890, on a per

adult basis, chewing tobacco accounted for 4 lbs of the 7.3 lbs of total

tobacco used annually. What is also evident is that cigarette smoking did

not begin to penetrate significantly into American society until after 1910,

when R.J. Reynolds introduced the first blended cigarette—Camel. Camel

cigarettes, first marketed in 1913, differed substantially from other brands at

that time in that they contained different types and blends of tobacco and

had a pH that prevented absorption of nicotine through the oral mucosa

(National Cancer Institute, 1991). In contrast to smokeless tobacco users,

smokers of these new cigarettes had to inhale the smoke to achieve sufficient quantities of nicotine in the blood stream.

Camel was also the first nonregional cigarette, and Camel’s introduction was accompanied by a then massive $1.5 million national advertising

and marketing campaign. Consumption of Camel cigarettes immediately

increased, providing one of the first documented cases to demonstrate that

advertising could create demand for a product where no previous demand

existed. Other manufacturers followed by introducing such brands as Lucky

Strike and Chesterfield; for decades these brands dominated the domestic

U.S. cigarette market.

Consumption of machine-manufactured cigarettes increased dramatically following World War I; by 1935, more tobacco was being consumed in

the form of cigarettes than all other products combined (US DHHS, 1990).
xl

Figure 1Trends in per capita tobacco consumption by major product category, United States, 1890 to 1990
xli

As the popularity of cigarettes continued to increase, consumption of

noncigarette tobacco products, especially smokeless tobacco, declined.

Per capita cigarette tobacco consumption peaked in the early 1950’s (as

did total per capita tobacco consumption), probably in response both to the

first scientific studies linking cigarette smoking to lung cancer and to the

introduction and aggressive promotion of filter cigarettes by the cigarette

industry in response to these early scientific findings (US DHHS, 1990).

Filter cigarettes, which had previously accounted for less than 1 percent of

all cigarettes consumed in the United States in 1950, had captured 50

percent of the market by the end of the decade, and today account for over

90 percent of all cigarette sales (US DHHS, 1989a). In comparison to their

nonfilter counterparts, filter cigarettes generally contain less tobacco.

For decades following the introduction of the mass-produced and massmarketed cigarettes, consumption and prevalence of smokeless tobacco had

been on the decline (Milmore and Conover, 1956). However, the latter part

of the 1970’s and the early part of the 1980’s, saw major increases in ST use

(Connolly, 1986; US DHHS, 1986). This increase was the result of renewed

and more aggressive advertising by the ST industry that accompanied the

introduction of innovative products such as Skoal Bandits—and the use of

well-known sport and entertainment personalities in ST promotions

(Ernster, 1989). Personalities such as football stars Walt Garrison and Terry

Bradshaw; baseball greats George Brett, Sparky Lyle, Carlton Fisk, and Bobby

Murcer; and country-and-western singer Charlie Daniels, obviously appealed

to a very young and impressionable audience (Ernster, 1989). Furthermore,

use of broadcast media to promote ST was not prohibited under the existing

Congressional legislation that had governed cigarettes since 1971; thus, the

ST industry was free to use television to recruit a large and relatively untapped market of new users. Because the themes and images used appealed

primarily to children and adolescents, increases in ST consumption that

occurred during the 1980’s was primarily confined to these age groups

(Marcus et al., 1989; Rouse, 1989). Where previously little or no use of ST

was seen among adolescents, prevalence of ST use among older teens increased between 250 and 300 percent between 1970 and 1985 (Marcus et al.,

1989) (See Figures 2 and 3 in the Foreword).

Compared with cigarettes, we know much less about the factors influencing ST use. Only in the past few years have the research results elucidated the many facets of ST use and means to intervene in this process

(US DHHS, 1986, 1989a, and 1990).

This monograph represents the most recent major attempt to bring

together the important research findings of the last few years. Previous

compilations of ST research have been the U.S. Surgeon General’s Report in

1986 (US DHHS, 1986) and a monograph published by the National Cancer

Institute in 1989, titled Smokeless Tobacco Use in the United States (US DHHS,

1989b). The present monograph will use the model established by the two

previous publications in presenting as broad a picture of the ST problem as

possible. Sections in this monograph describe the epidemiology, clinical

and pathological effects, carcinogenesis, nicotine effects and addiction,

Overview and Summary
Smoking and Tobacco Control Monograph No. 2

xlii

EPIDEMIOLOGY

CLINICAL AND

PATHOLOGICAL

EFFECTS

CARCINOGENESIS

prevention, cessation, and policy research findings in the area of ST use.

Finally, recommendations based on research and compiled by experts in the

field is presented.

From an international perspective, the use of ST is not as prevalent

as cigarette smoking, although some countries experience relatively high ST

use rates. Although ST use in Europe is practically nonexistent, Sweden has

one of the highest rates of use in the world. Prevalence is also high in India,

the United States, and Canada. Most countries, however, unless they have

banned ST, are experiencing growth in ST consumption.

ST use is typically a male habit, but is prevalent in women in certain

geographic areas as well as within some cultures and populations. For

example, some Native American tribes have 45 percent ST prevalence rates

among adolescent females (Schinke et al., 1989; US DHHS, 1986). Among

some elderly women in rural southeastern United States, high prevalence

rates of dry snuff contributed to a 50-fold increase in risk for oral cancer

among long-term users, according to one study (Winn et al., 1981). Nationally, use among women in the United States is less than 0.5 percent,

whereas among adult males regular use exceeds 6 percent. Among younger

white males, 30 to 33 percent are current users in many regions of the

country (Rouse, 1989).

Baseball players in the United States are often ST users. Ernster and

colleagues (chapter 1) found that 37 percent had used ST in the past month.

This high-profile group of ST users is highly influential, because they act as

role models for youth.

Historically, the highest rates of use in the United States have been

among residents in rural areas, but recent studies have documented an

increasing prevalence rate among urban and suburban residents. White

males are the most likely group to use ST, whereas Hispanics and Asians

traditionally have had very low rates of use. However, given ST’s recent

reach into urban areas and into all education and income strata, this problem is becoming more difficult to relegate to only a few segments of society.

ST use is rapidly becoming a significant public health problem not only for

the United States but also for other countries of the world.

The presence of oral lesions is common place among many ST

users. Among snuff users, oral lesions have been found in 67

percent of baseball players who were year-round users and in 32

percent of baseball players who used snuff only during the baseball season

(Greene et al., chapter 2). Other common findings among ST users are

gingival recession, loss of tooth structure, and leukoplakia. In Swedish data

on snuff use, Bergström (chapter 2) found five- to sixfold increases in risk of

developing cancer at the site of snuff placement and a frequent occurrence

of irreversible gingival recession.

Earlier studies have conclusively established the use of ST as a

cause of oral cancer. The studies in this monograph on carcinogenesis build

earlier studies and expand our knowledge of possible mechanisms of tumor

development.
xliii

Carcinogenesis is a complex, multistage process, and data are just now

beginning to emerge that permit a better understanding of this complex

process for ST use. This monograph describes the effects of tobacco-specific

N-nitrosamines on live tissue, specifically the development of tumors. ST

contains much higher levels of N-nitrosamines than are permitted in any

other consumer product (US DHHS, 1986) and are believed, from a number

of studies, to play a major role in ST carcinogenesis.

Anderson and Rice (chapter 3) have reported results from their study

suggesting that 4-(methylnitrosamino)-1-(3-pyridyl)-1-bitanone in ST may

be a potential source of cancer in the children of ST users. Their findings

clearly warrant additional investigation including longitudinal epidemiological studies to determine whether the father’s use of ST carries a risk of

cancer for descendants.

Nicotine’s main activity in the system of a smoker or ST user has been

thought to be in its addictive properties. However, Hoffmann and colleagues and Squier (chapter 3) found nicotine to also have a role as a potentiator in the carcinogenic process in ST users.

Other studies in this monograph have found oral cancers in ST users

among whom some had human papillomavirus, some had herpes simplex

virus, and some had neither virus present. These results may indicate that,

in some individuals, the viruses may be co-factors in carcinogenicity, but for

others the viruses are not necessary for tumors to develop. The role of two

other ST constituents, N-nitrosonornicotine , a tobacco-associated nitrosamine, and 12-O-tetradecanoylphorbol-13-acetate , a tumor promoter, also

have been found to affect cell growth and behavior.

The multiple adverse health effects of smoking, including nico

tine addiction (US DHHS, 1988), have been documented in a

series of authoritative reports by the U.S. Surgeon General and others. This

monograph presents new research results on nicotine consumption in ST

users. Although many studies have found cigarettes and ST to be similarly

addictive, some differences remain in these two major forms of nicotine

administration.

Among ST users, the blood level of nicotine is very steady following

initial administration and remains quite constant during the day—probably

the result of the prolonged contact of tobacco with the oral cavity. For this

reason, the transdermal nicotine patch may be an improved cessation

method for ST users compared with nicotine gum, as the patch more closely

mimics the stable nicotine levels commonly seen in ST users.

Typical health education methods may not work when applied to ST

prevention. D’Onofrio (chapter 5) describes the success of marketing by the

ST industry and recommends that ST prevention efforts incorporate some of

these strategies, including the narrow targeting of messages for high-risk

groups, attractive packaging of educational programs, product diversification (focusing on “micro-markets”), using educators who are culturally

acceptable to the target group, and providing education programs that react

NICOTINE EFFECTS

AND ADDICTION

PREVENTION

Overview and Summary
Smoking and Tobacco Control Monograph No. 2

xliv

quickly to changes in promotions and marketing strategies by the tobacco

industry.

One example of the strategies that D’Onofrio recommends is found in

the work of Evans and colleagues who used the Little League as a channel

for delivering messages about the dangers of ST use (chapter 5). Based on

the social inoculation theory originally used by these investigators in their

work on smoking, the current study makes extensive use of formative

evaluations to guide midcourse corrections in materials, educational strategies, and methods for maintaining a high level of participation in the study.

Cessation interventions for ST use have demonstrated mixed results.

Although the withdrawal symptoms appear to be less severe for ST users

than for smokers, other factors hamper higher success rates. One factor is

the practice of combining ST use with smoking; when one form of tobacco

use is prohibited or discouraged, the user often temporarily switches to the

other. Thus, ST cessation programs must also include smoking as a targeted

behavior.

One advantage that ST cessation efforts have over smoking cessation is

the presence of an easily observable clinical marker. A lesion in the mouth

where the ST users places tobacco is very common; showing the user this

lesion serves as a strong impetus to quit and remain abstinent.

Pharmacological adjuncts hold some promise for improving cessation

rates. Although nicotine gum has provided some degree of success, especially when used as part of a comprehensive effort, one problem with gum

appears to be using it correctly. The transdermal nicotine patch has several

advantages over the gum, including its ease of use and its ability to maintain a steady level of nicotine with only minimal effort by the user. Like

nicotine gum, however, the patch’s potential as a cessation aid is greatest

only when used as part of a complete program that addresses behavioral and

physiological aspects of ST addiction.

The role of health care professionals in ST cessation interventions

deserves much more attention than it has previously received. Dentists and

oral hygienists see many people who are at high risk for becoming ST users

or who are already users. Yet, very few dentists have someone in their office

teach prevention of ST use, refer patients to an outside cessation program, or

prescribe nicotine replacement therapy.

In a survey by Schroeder and Heisel (chapter 6), dentists reported a

willingness to provide pamphlets to their patients using ST, they also

reported three major barriers to ST cessation counseling: (1) lack of training

in counseling, (2) lack of insurance coverage for this activity, and (3) frustration from attempting to counsel about ST cessation.

Some researchers have found that “low-intensity” counseling by health

care professionals is very effective and efficient (US DHHS, 1990). The lowintensity approach requires asking whether the patient uses tobacco and, if

so, recommending that the patient quit, giving reasons related to that

specific patient’s health, suggesting the patient set a quit date, providing

CESSATION
xlv

POLICY

Overview and Summary

self-help materials, and scheduling a follow-up visit or phone call. In about

3 minutes, this intervention can provide a cue to action, strategies for

quitting, and plans for follow-up—all considered essential elements of any

full-fledged cessation program.

As mentioned previously, ST use is relatively common in some countries but in many, its use is unknown. The European Community is considering a total ban on ST products, which may be feasible since the industry

has not been able to develop a strong consumer base. The World Health

Organization (WHO) recommends that ST products be banned in countries

where ST use is nonexistent and that, in all countries, a social climate

unfavorable to ST use should be established and maintained. Other strategies recommended by WHO include (1) the use of taxation as a means of

reducing ST accessibility to young people and providing a financial base for

health education programs, (2) restrictions on use in public places and

worksites, and (3) prominent health warnings on packages.

In January 1991, NCI convened an expert panel of ST researchers,

health care, and public health professionals. This panel developed a set of

recommendations that was later approved by the First International Conference on Smokeless Tobacco and that presents the most important actions

that are needed to prevent and control the use of ST. This document is

presented in this monograph and is offered as a resource for persons who

want to become involved in this very important area of tobacco control

(chapter 8).

In the United States, the National Cancer Institute has led

the federal government’s efforts in preventing ST use among

youth and in helping adult users to quit (US DHHS, 1990). NCI

has funded eight major research studies, six on prevention and two on

cessation, that have resulted in improvements in our knowledge and understanding of ST problem in general, as well as the efficacy of specific intervention approaches.

As the scientific understanding of ST use has grown, one fact has

become increasingly clear: Those factors that encourage, establish and

maintain cigarette smoking are essentially identical for ST. Thus, the same

strategies now being recommended to control cigarette smoking should

also be applied to reduce ST use. These strategies employ a dual approach

that targets both the user and the social environment. This approach was

detailed in the National Cancer Institutes first Smoking and Tobacco Control Monograph titled Strategies to Control Tobacco Use in the United States: A

Blueprint for Public Health Action in the 1990’s.

The state of the art in controlling tobacco use combines multiple

environmental changes with multiple programs directed to individuals in

different stages of the initiation and cessation process. Just as is the case in

cigarette smoking, this approach recognizes that no single approach is best

for all ST users and that different users are most attracted to and most

affected by different programs. Perhaps most important, it recognizes that

no single intervention channel can reach all users and that no single time is

COMPREHENSIVE

STRATEGIES TO

CONTROL ST USE
Smoking and Tobacco Control Monograph No. 2

xlvi

best for individuals to make an attempt to quit. Comprehensive strategies

are characterized by the delivery of persistent and inescapable messages to

quit, or not start, coupled with continuously available support for individual

cessation efforts provided through multiple channels, and reinforced by

environmental incentives to not use ST.

As with cigarette smoking, ST use primarily starts during adolescence.

Unlike cigarette smoking, however, ST use is almost exclusively a male

phenomena. The processes involved in ST initiation and cessation can be

viewed as a cycle (Figure 2) with transition from regular use to dependence

occurring during late adolescence and early adulthood. Experimentation

and initial use are heavily influenced by issues, such as peer pressure and

role modeling, during adolescent development. Dependent use develops

when the individual incorporates the personal psychological and sociological utility of ST use into methods by which they function in and cope with

the adult world. Although a significant number of adolescents may experiment with ST—including a certain fraction of adolescent females—most

never become regular ST users and some who do adopt regular use quit

before they become dependent.

Just as the process of quitting smoking is cyclical with smokers making

many attempts to stop before finally succeeding, users of ST follow a similar

cycle with many attempts to quit. Among cigarette smokers in the United

States, about one-third attempt to quit each year but only about 10 percent

of these succeed (Pierce and Hatziandreu, 1989). Although comparable

national data on ST behavior is unavailable, data from NCI’s eight intervention studies on ST use indicate ST users report experiencing the same withdrawal symptoms and problems of relapse as those seen in cigarette smokers

(National Cancer Institute, 1990). Clearly, then, ST users who have unsuccessfully tried to quit need to be motivated to try again. A useful

conceptualization of the cessation process is one whereby users cycle

through various stages of cessation, and with each attempt, gain additional

experience that can ultimately contribute to long-term success.

One goal in the overall strategy to control ST use, therefore, is to move

users from one stage of the cycle to another, rather than using long-term

cessation as the only goal and outcome measure. ST dependency is not a

sudden process but one that begins gradually with experimentation starting

in early adolescence or even preadolescence. Although the process is nearly

identical to that seen in cigarette smoking, a number of regional studies

have reported first use of ST occurring much earlier, especially among

certain populations of Native Americans where even pre-school-aged children use ST (Schinke et al., 1989)

As shown in Figure 3, the first step in the process is thinking about

using ST, and as individuals move through their teenage years, a certain

fraction change from believing they will never use ST to contemplation of

use. The images presented by advertising as well as the examples of older

siblings and adults—especially adult role models such as athletes and wellknown entertainers—are powerful inducements for children to perceive ST
xlvii

use as a means of entry into the adult world. Counter-advertising that

creates a negative image of the user can often help offset these influences.

The change from thinking about using ST to experimentation does not

necessarily lead to regular use, but as the individual gains experience with

ST, this transition clearly becomes more likely. Factors that help move the

individual from thinking to experimenting include the wide-spread availability of ST products, the ease with which children can purchase ST, promotional distribution of free samples—many of which are given to teens

despite local laws, and advertising. The change from occasional experimentation to regular use is critical, because with regular use the adolescent

develops experience that imparts psychological and sociological utility to

the user. The ability to purchase ST products easily, the personal and social

rewards, and peer acceptance, are all critical to establishing regular use.

Figure 2

Processes of ST initiation and cessation

Overview and Summary
Smoking and Tobacco Control Monograph No. 2

xlviii

Progressing from regular to dependent use requires that the utility of ST

to persist after the pervasive anxieties of adolescents dissipate. For utility to

continue, however, ST use has to be allowed in those situations when the

user wants to use the product, such as during work as a means to relieve

stress. If ST use is not permitted in the workplace or the user is in a whitecollar occupation, the behavior may be frowned on by coworkers. In these

instances the user must develop alternative methods for handling stress.

For the confirmed smoker, the cyclical pattern of not thinking about

quitting (precontemplation), thinking about quitting (contemplation), and

attempting to quit, generates a new set of nonsmokers each time they pass

through the cycle (Prochaska and DiClemente, 1986). The same process is

Figure 3

Factors that influence adolescent progression into adult ST use
xlix

Figure 4

ST cessation process

Overview and Summary

true for ST users. This process is illustrated in Figure 4 and identifies specific

intervention processes that can influence these stages. Interventions are

provided as examples to show possible interactions between intervention

approaches and not meant to be all inclusive.

Many environmental influences and programs for controlling tobacco

use (both cigarettes and ST) are intended to influence the user at different

stages in this cycle. Public information campaigns that present the risks

associated with ST use move individuals from the precontemplation to

contemplation, as is personalizing the risks through physicians’ or dentists’

warnings. Other reasons important to the user include concern about

addiction and setting a good example to others, especially children if the

user is a parent.
Smoking and Tobacco Control Monograph No. 2

l

The move from thinking about quitting to making a serious attempt is

often triggered by a variety of environmental stimuli. Two factors that

contributed to the decline in ST use in the mid-1980’s, even though this

decline was temporary, was concern about the health risks and Congressionally mandated warning labels on ST products and in advertising. As with

cigarettes, cost is an important consideration that contributes to cessation

attempts. Another powerful inducement to quit is a dentist’s warning to a

patient when the dentist finds leukoplakia at the site of ST placement.

Social factors may also play different roles in motivating quitting

behavior among ST users compared with cigarette smokers. Increasingly,

smokers are feeling more socially ostracized as fewer public settings permit

smoking indoors (Pertschuk and Shopland, 1989; US DHHS, 1991). ST users

can be surreptitious users, thereby avoiding social sanctions, resulting in less

motivation to quit.

The major barriers to long-term cessation remain difficult to alter and,

with the exception of addiction, are part of the user’s social and economic

environment. The barriers include social norms and workplace rules that

encourage ST use; the positive behavior of peers, family members, and

friends toward ST use; and periods of stress that facilitate relapse.

R. Craig Stotts, Dr. P.H., R.N.

Donald R. Shopland

Division of Cancer Prevention and Control

National Cancer Institute

REFERENCES

Connolly, G.N., Winn, D.M., Hecht, S.S.,

Henningfield, J.E., Walker, B., Hoffmann, D. The

reemergence of smokeless tobacco. New England

Journal of Medicine 314: 1020-1027, 1986.

Ernster, V.L. Advertising and promotion of smokeless tobacco products. National Cancer Institute

Monographs No. 8: 87-94, 1989.

Marcus, A.C., Crane, L.A., Shopland, D.R., Lynn,

W.R. Use of smokeless tobacco in the United

States: Recent estimates from the Current Population Survey. National Cancer Institute Monographs

8: 17-23, 1989.

Milmore, B.K., Conover, A.G. Tobacco consumption

in the United States, 1880-1955. Tobacco Smoking

patterns in the United States, W. Haenszel, M.B.

Shimkin, and H.P. Miller (editors). Washington,

DC: U.S. Government Printing Office, 1956.

(Public Health Monograph No. 45).

Novotny, T.E., Pierce, J.P., Fiore, M.C., Davis, R.M.

Smokeless tobacco use in the United States: The

Adult Use of Tobacco Surveys. National Cancer

Institute Monographs 8: 25-28, 1989.

Pertschuk, M., Shopland, D.R. (editors). Major Local

Smoking Ordinances in the United States. A Detailed

Matrix of the Provisions of Workplace, Restaurant, and

Public Places Smoking Ordinances. U.S. Department

of Health and Human Services, National Institutes

of Health, National Cancer Institute. NIH Publication No. 90-479, 1989.

Pierce, J., Hatziandreu, E. Report of the 1986 Adult Use

of Tobacco Survey. U.S. Department of

Health and Human Services, Public Health Service,

Centers for Disease Control. Publication No. 10491,

1989.

Prochaska, J.O., DiClemente, C.C. Towards a

comprehensive model of change. In: Treating

Behaviors, W.R. Miller and H. Heather (editors).

New York: Plenum Press, 1986, pp. 3-27.

Robert, J.C. The Story of Tobacco in America. Chapel

Hill, NC: The University of North Carolina Press,

1967.

Rouse, B.A. Epidemiology of smokeless tobacco use: A

national study. National Cancer Institute Monographs

8: 29-33; 1989.
li

Schinke, S.P., Schilling, R.F., Gilchrist, L.D., Ashby,

M.R., Kitajima, E. Native youth and smokeless

tobacco: Prevalence rates, gender differences, and

descriptive characteristics. National Cancer Institute

Monographs 8: 39-42, 1989.

Shopland, D.R., Haenlein, M. Use of epidemiology

in lung cancer control. Prevention and

Treatment of Lung Cancer. In press.

U.S. Department of Agriculture. Tobacco Situation

and Outlook Report. U.S. Department of Agriculture, Economic Research Service, Series TS 218.

Various years, 1955 to 1992.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986.

U.S. Department of Health and Human Services. The

Health Consequences of Smoking: Nicotine Addiction.

A Report of the Surgeon General. U.S. Department of

Health and Human Services, Public Health Service,

Centers for Disease Control, Center for Health

Promotion and Education, Office on Smoking and

Health. DHHS Publication No. (CDC) 88-846,

1988.

U.S. Department of Health and Human Services.

Reducing the Health Consequences of Smoking: 25

Years of Progress. A Report of the Surgeon General,

1989. U.S. Department of Health and Human

Services, Public Health Service, Center for Disease

Control, Center for Chronic Disease Prevention

and Health Promotion, Office on Smoking and

Health. DHHS Publication No. (CDC) 89-8411,

1989a.

U.S. Department of Health and Human Services.

Smokeless Tobacco Use in the United States. U.S.

Department of Health and Human Services, Public

Health Service, National Institutes of Health,

National Cancer Institute. NIH Publication No. 89-

3055, 1989b.

U.S. Department of Health and Human Services.

Smoking, Tobacco and Cancer Program Status Report,

1985-1989. U.S. Department of Health and Human

Services, Public Health Service, National Institutes of

Health, National Cancer Institute. NIH Publication

No. 90-3107, 1990.

U.S. Department of Health and Human Services.

Strategies to Control Tobacco Use in the United States: A

Blueprint for Public Health Action in the 1990’s. U.S.

Department of Health and Human Services, Public

Health Service, National Institutes of Health,

National Cancer Institute. NIH Publication No. 92-

3316, 1991.

U.S. Department of Health and Human Services.

Smoking and Health in the Americas. A 1992 Report of

the Surgeon General in Collaboration with the Pan

American Health Organization. U.S. Department of

Health and Human Services, Public Health Service,

Center for Disease Control, DHHS Publication No.

(CDC) 92-8419, 1992.

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W., Toledo,

A., Fraumeni, J.F., Jr. Snuff dipping and oral cancer

among women in the southern United States. New

England Journal of Medicine 304: 745-749, 1981.

Overview and Summary
Smoking and Tobacco Control Monograph No. 2

lii
Chapter 1

1

Chapter 1

Epidemiology

CONTENTS The Smokeless Tobacco Problem:

Risk Groups in North America

Elbert D. Glover and Penny N. Glover ................................................ 3

Introduction ................................................................................. 3

Historical Perspective ................................................................... 3

Prevalence..................................................................................... 4

Groups at Risk .............................................................................. 7

Further Research ........................................................................... 8

References ..................................................................................... 9

Surveillance of and Knowledge About

Cancer Associated With Smokeless Tobacco Use

Deborah M. Winn ............................................................................... 11

Introduction ................................................................................. 11

Trends in Oral Cancer Incidence and Mortality .......................... 12

Public Knowledge of ST and Cancer Links .................................. 15

Research Needs ............................................................................. 16

References ..................................................................................... 17

Smokeless Tobacco Use in India

P.C. Gupta ........................................................................................... 19

Introduction ................................................................................. 19

Types of ST Used .......................................................................... 20

Effects of ST Use ........................................................................... 23

Effects of Education...................................................................... 24

Conclusion ................................................................................... 24

References ..................................................................................... 25

Smokeless Tobacco in Professional Baseball:

Patterns of Player’s Use

V.L. Ernster, D.G. Grady, L. Stillman, M. Walsh, and J.C. Greene ..... 26

Introduction ................................................................................. 26

Methods........................................................................................ 26

Results........................................................................................... 27

Discussion..................................................................................... 34

References ..................................................................................... 36
Smoking and Tobacco Control Monograph No. 2

2
Chapter 1

3

The Smokeless Tobacco Problem:

Risk Groups in North America

Elbert D. Glover and Penny N. Glover

ABSTRACT Tobacco consumption—most notably smokeless tobacco use—has become one of the

fastest growing detrimental health habits in North America. Adolescents still perceive ST as a

safe alternative to cigarettes. This paper provides state-specific information about the prevalence of ST use in the United States. A review of the literature reveals that the groups at highest

risk are white youth and young adults, aged 10 to 30, with the most vulnerable being those

living in the southern United States. It is suggested that further research in several areas is

needed for the effectiveness of smokeless tobacco control to be enhanced.

Columbus discovered Native Americans using tobacco in various forms (e.g.,

dipping, chewing, rubbing, smoking), and on his return, he introduced

tobacco to the Old World. Actually many of his crew were viewed as being

possessed by the devil when they expelled smoke from their nostrils; consequently, they were imprisoned (Christen and Glover, 1987; Christen et al.,

1982; Penn, 1902). Once Britain established its colonies in North America,

the use of ST became deeply entrenched. Before 1900, the dominant form

of tobacco used in North America was smokeless tobacco. Three events

occurred that began to move North American tobacco users from smokeless

tobacco to cigarette smoking: (1) the invention of the cigarette rolling

machine, which allowed for mass production of cigarettes; (2) the

INTRODUCTION Perhaps one of the fastest growing detrimental health habits in

North America over the past few years has been the use of smokeless tobacco. There has been an upswing in the popularity of ST among young

adults and children. As a result, the topic has captured the attention of the

North American press and public as health professionals and legislators seek

to alert the populace about health problems associated with the use of

smokeless tobacco (Glover et al., 1988).

There are two types of ST—snuff and chewing tobacco. Snuff is a finely

ground tobacco of which the user places a pinch (called a dip or rub) in the

gingival groove. Snuff can be dry, moist, or in sachets (tea bag-like

pouches). The most common position to place snuff is in the mandibular

labial mucosa (cuspid to cuspid); however, this is more common in many

European countries, especially Sweden. In European countries, sniffing

(inhaling) dry snuff through the nostrils is more common than in North

America. Chewing tobacco comes in the form of loose leaf, plug, or twist,

and the user places a bolus of tobacco (a golf ball-sized piece) inside the

cheek. Typically, whenever a user chews tobacco, one will see an extended

cheek. This manuscript provides state-specific information regarding the

prevalence of ST and reviews the groups at risk for ST use (Christen and

Glover, 1987; Christen et al., 1982; Penn, 1902; Smokeless Tobacco Research

Council, 1984; USDA, 1969; Vogues, 1984).

The use of ST in North America appears to have originated with

Native Americans. On his trip to the New World, Christopher

HISTORICAL

PERSPECTIVE
Smoking and Tobacco Control Monograph No. 2

4

postulation of the germ theory, which at the time created a fear that tuberculosis could be spread by spitting tobacco juices; and (3) World War I. Cigarettes were offered and given freely to American combat soldiers, allowing a

nation to become hooked on tobacco. Since the turn of the century, smokeless tobacco use declined until the 1970’s when, through clever advertising

by the tobacco industry, it began to experience a resurgence (Glover et al.,

1984; Harper, 1980; Smight, 1981). The annual increase of 10 to 11 percent

continued until 1986, when two significant events occurred: (1) a consensus

development conference on smokeless tobacco was held in Bethesda, Maryland (Consensus Conference, 1986); and (2) the Surgeon General’s first

report on smokeless tobacco was published (US DHHS, 1986). These reports

helped create a national awareness of ST’s effect on health for the first time,

along with the much publicized case of Sean Marsee, product labeling, and

an advertisement ban. As a result, tobacco sales declined over the next

18 mo (1986 to 1987). Sales and usage bounced back by 1988.

Today, it appears that tobacco use continues to increase, especially

among young people. Specifically, moist snuff (e.g., Copenhagen, Skoal),

sachets (e.g., Skoal Bandits, Renegades), and loose leaf tobacco (e.g., Red Man,

Chattanooga Chew, Levi Garrett) are the smokeless tobacco products that

young adults and youth are using (Glover et al., 1988). On the other hand,

dry snuff (e.g., Bruton, Dental Snuff), plug (e.g., Bull of the Woods, Red Man

Plug), and twist (e.g., Mammoth Cave, Samson) are declining in use. These

last products are used primarily by older adults (Glover et al., 1988).

There are an estimated 10 to 12 million ST users in the United States

alone (Consensus Conference, 1986; US DHHS, 1986). Youth are using

smokeless tobacco products at alarming rates. This resurgence of popularity

is attributed to innovative advertising campaigns by tobacco companies.

Sports figures promote the product in an attempt to erase the old, unsanitary

image of the habit and replace it with a “macho” image (Christen and

Glover, 1981; Glover et al., 1981, 1982, and 1988). Because of public pressure, the tobacco companies stopped using current and former sports personalities to promote their products in 1985 (Consensus Conference, 1986; US

DHHS, 1986). Today, the tobacco companies focus their advertisements on

young white males, using masculine role models in activities such as fishing,

hunting, rock climbing, and white-water rafting (Glover et. al., 1988).

Children tend to model the behavior and accept the values of significant

others (e.g., parents, teachers, coaches); consequently, these sport figures are

contemporary role models (Glover, 1978).

substantial emphasis by advertisers on the 18- to 20-yr-old group (Maxwell,

1980). However, younger people (as young as 10 to 12) also appear to be

influenced by the trend of ST use (Christen, 1980; Hunter et al., 1986; Marty

et al., 1986; Schroeder et al., 1987). Some reports indicate that smokeless

tobacco is sometimes consumed by individuals younger than age 10 (US

DHHS, 1986). A statewide study of 5,392 Texas children in grades 7 through

12 reported that approximately 9 percent were regular ST users (Schaefer et

al., 1985). In an Oklahoma statewide study, approximately 13 percent of

PREVALENCE The average consumer of ST is in the 18- to 30-yr age bracket, with
Chapter 1

5

third grade males and 22 percent of fifth grade males reported regular use of

smokeless tobacco (Glover, 1990). These percentages increased to approximately 22, 33, and 39 percent among the 7th, 9th, and 11th grade males,

respectively. In two studies in Oregon, 9 percent of 7th grade males,

19 percent of 9th grade males, and 23 percent of 11th grade males reported

using smokeless tobacco daily (Lichtenstein et al., 1985; Severson et al.,

1985).

In all the aforementioned studies, females reported a low level of ST

use— approximately 1 to 3 percent—which is typical of female use in

prevalence studies. However, North American Indians report a much higher

use among both males and females (Schinke et al., 1989). Actually, gender

is not a predictor of smokeless tobacco use among Native Americans. African Americans and Mexican Americans also report a low rate of smokeless

tobacco use. The typical user can be described as a white, rural or suburban,

young adult male (Hunter et al., 1980; Marty et al., 1986). These data are

not limited to the South and West; northern and eastern states on occasion

have reported similar rates of use. However, the use of smokeless tobacco

tends to be regional in that some areas report higher rates of use. In Ohio,

for example, a survey of adults indicated that 10 percent of the males

regularly use some form of snuff or chewing tobacco, with another 5 percent

being previous smokeless tobacco users (Schroeder and Chen, 1985). In a

Massachusetts study of 5,078 students, 16 percent of the males and 2 percent of the females reported using smokeless tobacco “once or twice”

(McCarty and Krakow, 1985). In the only national survey among college

students, it was reported that 22 percent of collegiate males are users of

smokeless tobacco, whereas 2 percent of females reported using smokeless

tobacco (Glover et al., 1986). Although there are no national youth surveys

on ST use, a summary of self-reported data on 43,000 students in grades 4

through 11 in 16 locations in the United States and 1 in Canada reported

that 40 to 60 percent of males had tried smokeless tobacco and 10 to

20 percent of older males reported recent use (Boyd et al., 1987).

Eight surveys conducted in Canada, Colorado, Georgia, Nebraska, and

Oregon indicated that about 8 to 10 percent of the young males (aged 5 to

19) were regular users of smokeless tobacco (Glover et al., 1984). A survey in

Pitt County, North Carolina (the county that produces more flue-cured

tobacco than any other county in the United States), noted a prevalence of

15 percent (Glover et al., 1987).

As shown in Table 1, recent population surveys estimate the rate of

smokeless tobacco use for U.S. males age 16 and older (1985 data) at 5.5 percent and for U.S. males age 18 and older (1987 data) at 6.2 percent (Marcus

et al., 1989).

The lowest reported use of smokeless tobacco in the United States was

in the Northeast (2.3 percent), and the highest reported use was in the

South (8.3 percent) (Marcus et al., 1989). As shown in Tables 2 and 3, West

Virginia had the highest prevalence (23.1 percent), and Washington, D.C.,

showed the lowest (Marcus et al., 1989).
Smoking and Tobacco Control Monograph No. 2

6

Table 1

Prevalence rates from population surveys

1985 1987

Snuff 1.9% 3.2%

ChewingTobacco 3.9 4.1

All ST 5.5 6.2

Source: Marcus et al., 1989.

Table 2

States with the highest prevalence of ST use

State Prevalence

West Virginia 23.1%

Mississippi 16.5

Wyoming 15.7

Arkansas 14.7

Montana 13.7

Kentucky 13.6

Oklahoma 11.0

Tennessee 10.3

New Mexico 10.2

Table 3

States with the lowest prevalence of ST use

State Prevalence

Washington, D.C. 0.0%

New Jersey 0.1

Hawaii 0.2

Massachusetts 0.2

Connecticut 0.3

Maryland 0.4

Rhode Island 0.5

New York 0.5

Delaware 0.6
Chapter 1

7

The previous data by Marcus, although reported in an NCI monograph

in 1989, were collected in 1985. More recently, patterns of tobacco use were

surveyed by the Youth Risk Behavior Surveillance System (Morbidity and

Mortality Weekly Report, 1991). This survey used a three-stage sample

design to obtain a probability sample of 11,631 students in grades 9 through

12 in 50 states, the District of Columbia, Puerto Rico, and the Virgin Islands.

Twenty-two states are noted and reported current use (30 d preceding the

survey) at 10 percent. Tables 4 and 5 illustrate the highest and the lowest ST

use among states.

GROUPS AT RISK From gleaning the literature, it becomes obvious that the groups

at highest risk are white youth and young adults, aged 10 to 30. The tobacco companies aim their advertisements and marketing at these groups

(Maxwell, 1980). Moreover, the profile indicates that the southern region is

the most vulnerable (Marcus et al., 1989). In addition, smokeless tobacco

Table 4

Highest reported ST use among states in Youth Risk Behavior Surveillance System

(1990)

State Prevalence

West Virginia 20%

South Dakota 19

Oklahoma 16

Kentucky 15

Alabama 14

Nebraska 14

Colorado 13

New Mexico 13

Pennsylvania 13

Table 5

Lowest reported ST use among states in Youth Risk Behavior Surveillance System

(1990)

State Prevalence

Washington, D.C. 1%

Massachusetts 7

New York 7

New Hampshire 8

North Carolina 8

Utah 8

South Carolina 9

Wisconsin 10
Smoking and Tobacco Control Monograph No. 2

8

use is two to three times more prevalent among blue-collar workers than

among white-collar workers.

In our concern for young, white, blue-collar males, it is important that

we not ignore females. Virtually all the studies reported ST use by females

with only one line and moved on to males’ use. At one time we had few

female smokers because smoking was neither fashionable nor acceptable.

Today, women are smoking at the same rates as men, and it is estimated

that there will be more female smokers than male by the year 1996. Today,

certain brands of cigarettes are marketed specifically to women; in the

future, women may have their own snuff and chewing brands.

Finally, Native Americans (in Canada and the United States, especially

Alaska) report disturbing rates of smokeless tobacco use without regard to

gender. It appears that Native Americans are in need of education and

funds to combat this major health problem.

FURTHER

RESEARCH

Several areas must be addressed if we are to understand better the health

menace of smokeless tobacco.

• There is a need for well-designed studies of the prevalence of ST use

based on continuing national and international probability samples.

This would allow us to monitor trends.

• These studies should include the following: (1) Identifying the type

of tobacco used as moist, dry, or sachet snuff tobacco or loose leaf,

plug, or twist chewing tobacco allows us to determine where the

problem exists. Because of a lack of knowledge of ST, many researchers lump all products under one category. Yet, in research on smoking tobacco, we know exactly where we stand relative to cigarettes,

pipes, and cigars. If data are collected for ST generically, we do not

know where the problem exists relative to the type. (2) Age of users.

(3) Gender of users. It is critical to recognize women as potential

users and collect data on women as well as men. Currently, women

do not use ST products in significant numbers. However, if we delay

until we have a problem, we then become crisis-oriented. As we

collect data, it is critical to collect data on female usage to monitor

trends. (4) Region of users. (5) Concomitant cigarette smoking.

• Longitudinal studies are important.

• Operational definitions of users, regular users, heavy users, light users,

and ex-users should be set to permit useful comparisons. Only one

study has attempted to quantify these terms on the basis of nicotine

consumption (Marcus et al., 1989; Schroeder et al., 1988).

• Studies should validate self-reports.

• A national survey for persons aged 12 and under would be beneficial;

however, this type of national study is complicated and filled with

compliance problems. Moreover, it would be a monumental task to

convince an agency to fund such a study.
Chapter 1

9

REFERENCES

Boyd, G., Ary, D.V., Wirt, R., et al. Use of smokeless

tobacco among children and adolescents in the

United States. Preventive Medicine 16: 402-421,

1987.

Christen, A.G. The case against smokeless tobacco:

Five facts for the health professional to consider.

Journal of the American Dental Association 101: 464-

469, 1980.

Christen, A.G., Glover, E.D. Smokeless tobacco:

Seduction of youth. World Smoking and Health 6:

20-24, 1981.

Christen, A.G., Glover, E.D. History of smokeless

tobacco use in the United States. Health Education

18(3): 6-11, 13, 1987.

Christen, A.G., Swanson, B.Z., Glover, E.D.,

Henderson, A.H. Smokeless tobacco: The folklore

and social history of snuffing, sneezing, dipping,

and chewing. Journal of the American Dental

Association 105: 821-829, 1982.

Consensus Conference. Health implications of

smokeless tobacco use. Journal of the American

Medical Association 255(8): 1045-1048, 1986.

Glover, E.D. Modeling: A powerful change agent.

Journal of School Health 48: 175-176, 1978.

Glover, E.D. Current research in smokeless tobacco.

Presented at the 14th Commonwealth and

International Conference on Physical Education,

Sport, Health, and Dance, Auckland, New Zealand,

1990.

Glover, E.D., Christen, A.G., Henderson, A.H. Just a

pinch between the cheek and gum. Journal of

School Health 51: 415-418, 1981.

Glover, E.D., Christen, A.G., Henderson, A.H.

Smokeless tobacco and the adolescent male.

Journal of Early Adolescence 2: 1-13, 1982.

Glover, E.D., Edwards, S.W., Christen, A.G.,

Finnicum, P. Smokeless tobacco research: An

interdisciplinary approach. Health Values 8: 21-25,

1984.

Glover, E.D., Johnson, R., Laflin, M., Edwards, S.W.,

Christen, A.G. Smokeless tobacco use trends

among college students in the United States.

World Smoking and Health 11(1): 4-9, 1986.

Glover, E.D., O’Brien, K., Holbert, D. Prevalence of

smokeless tobacco use in Pitt County, North

Carolina. International Journal of Addictions 22(6):

557-565, 1987.

Glover, E.D., Schroeder, K.L., Henningfield, J.E.,

Severson, H.H., Christen, A.G. An interpretative

review of smokeless tobacco research in the United

States: Part I. Journal of Drug Education 18(4): 285-

310, 1988.

Harper, S. In tobacco, where there’s smokeless fire.

Advertising Age 85, 1980.

Hunter, S.M., Croft, J.B., Burke, G.L., Parker, F.C.,

Webber, L.S., Berenson, G.S. Longitudinal

patterns of cigarette smoking and smokeless

tobacco use in adolescents: The Bogalusa Heart

Study. American Journal of Public Health 76: 193-

195, 1986.

Hunter, S.M., Webber, L.S., Berenson, G.S. Cigarette

smoking: Bogalusa Heart Study. Preventive

Medicine 9: 710-712, 1980.

Lichtenstein, E., Severson, H.H., Friedman, L.S.,

Ary, D.V. Chewing tobacco use by adolescents:

Prevalence and relation to cigarette smoking.

Addictive Behaviors 9: 351-355, 1985.

Marcus, A.C., Crane, L.A., Shopland, D.R.,

Lynn, W.R. Use of smokeless tobacco in the

United States: Recent estimates from the Current

Population Survey. National Cancer Institute

Monographs 8: 17-24, 1989.

Marty, P.J., McDermott, R.J., Williams, T. Patterns of

smokeless tobacco use in a population of high

school students. American Journal of Public Health

76(17): 190-192, 1986.

Maxwell, J.C. Maxwell Manufactured Products:

Chewing snuff is growth segment. Tobacco

Reporter 107: 32-33, 1980.

McCarty, D., Krakow, M. More than “just a pinch”:

The use of smokeless tobacco among Massachusetts students. Report by the Massachusetts

Department of Public Health, Division of Drug

Rehabilitation. January 28, 1985.

Morbidity and Mortality Weekly Report. Current

tobacco, alcohol, marijuana, and cocaine use

among high school students: United States.

Morbidity and Mortality Weekly Report 40(38): 659-

663, 1991.

Penn, W.A. The Soverane Herbe: A History of Tobacco.

New York: Grant Richards, 1902, pp. 1-326.

Schaefer, S.P., Henderson, A.H., Glover, E.D.,

Christen, A.G. Patterns of use and incidence of

smokeless tobacco consumption in school-aged

children. Archives of Otolaryngology 111: 639-642,

1985.

Schinke, S.P., Schilling, R.F., II, Gilchrist, L.D.,

Ash, M.R., Kitajima, E. Native youth and

smokeless tobacco: Prevalence rates, gender

differences, and descriptive characteristics.

National Cancer Institute Monographs 8: 39-42,

1989.

Schroeder, K.L., Chen, M.S. Smokeless tobacco and

blood pressure. New England Journal of Medicine

312: 919, 1985.

Schroeder, K.L., Chen, M.S., Iaderosa, G.R.,

Glover, E.D., Edmundson, E.W. Proposed

definition of a smokeless tobacco user based on

potential nicotine consumption. Addictive

Behaviors 13: 395-400, 1988.
Schroeder, K.L., Iaderosa, G.B., Chen, M.S.,

Glover, E.D., Edmundson, E.W. Bimodal initiation

of smokeless tobacco usage: Implications for

cancer education. Journal of Cancer Education 2(1):

1-7, 1987.

Severson, H.H., Lichtenstein, E., Gallison, C. A pinch

instead of a puff: Implications of chewing tobacco

for addictive processes. Bulletin of Social Psychology

and Addictive Behaviors 4: 85-92, 1985.

Smight, T.A. A man’s chew. Nutshell 43, 1981.

Smokeless Tobacco Research Council. Smokeless

Tobacco. Peekskill, NY: Smokeless Tobacco

Research Council, 1984.

U.S. Department of Agriculture. Tobacco in the

United States. Misc. Publ. 867. Washington, DC:

U.S. Department of Agriculture, 1969.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986.

Vogues, E. Tobacco Encyclopedia. Mainz: Germany

Tobacco International, 1984, p. 293.
Epidemiological studies of smokeless tobacco and cancer continue to show that smokeless

Surveillance of and Knowledge About

Cancer Associated With

Smokeless Tobacco Use

Deborah M. Winn

ABSTRACT

tobacco increases oral cancer risk and possibly the risk of other head and neck cancers,

suggesting a continuing need to monitor oral cancer trends and to communicate health risks to

the public. Cancers of the gum and buccal mucosa, the sites most strongly associated with

smokeless tobacco, show no change in incidence since ST use increased in the 1970’s; however,

it may be premature to expect a rise in oral cancer as a result of smokeless tobacco use. Soft

tissue lesions, including oral precancers, are common in ST users. The majority of adult U.S.

male users and non-users of smokeless tobacco know that smokeless tobacco use increases the

risk of mouth and throat cancer, although older men (age 65 and older) are less informed.

A few studies suggest that many adolescents are aware of cancer risks from ST. However,

smokeless tobacco use remains high among youth and young men despite their knowledge of

health consequences.

INTRODUCTION Epidemiological evidence, as well as studies of carcinogenesis

involving the tobacco-specific nitrosamines contained in smokeless tobacco

(Hecht and Hoffmann, 1988), have implicated ST as a cause of cancers of the

oral cavity and pharynx. The epidemiological evidence is well documented

in reviews and evaluations, including the report of an advisory committee

to the U.S. Surgeon General (US DHHS, 1986a), the International Agency for

Research on Cancer (IARC, 1985), and other documents written up to about

1986 (US DHHS, 1986b), when Federal legislation was passed requiring

warning labels on smokeless tobacco products and banning advertising on

radio and TV. Since the mid-1980’s, work on carcinogenesis has continued,

including the development of potential biological markers of exposure to

smokeless tobacco (Carmella, 1990).

In addition to the case control and other studies used as the basis for

conclusions in the mid-1980’s about the carcinogenicity of ST products,

several recent epidemiological case control studies of oral cancer (Blot et al.,

1988; Spitz et al., 1988; Stockwell and Lyman, 1986), summarized elsewhere

(Winn, in press), have examined cancer risks from smokeless tobacco use.

All three studies showed that oral cancer risks were elevated among users of

smokeless tobacco, with relative risks, the estimate of the ratio of oral and

pharyngeal cancer risks in smokeless tobacco users vs. non-users, ranging

from 2.3 to 11.2, suggesting moderate to strong associations between ST use

and oral cancer sites. However, in one study (Spitz et al., 1988) use of

smoking tobacco could not be ruled out as responsible for the association,

and the findings had only marginal statistical significance. The other two,

both using population-based cancer registries for case ascertainment (ensuring complete enumeration of cases in specific geographic areas), could rule

out cigarette smoking and chance as explanations for the findings. In the

study that examined numerous head and neck anatomic sites (Stockwell and
Smoking and Tobacco Control Monograph No. 2

12

Lyman, 1986), ST use was linked to elevated risks in all cancer sites studied,

controlling for smoking and other factors; statistically significant excesses

were observed for the gum and buccal mucosa, salivary glands, and larynx.

In that report, tobacco use histories were obtained through medical record

review. Most patients had tobacco use recorded. However, no information

was given to determine whether tobacco use data were obtained with equal

accuracy in the control group, consisting of patients with other cancers, as

for the cases with a head and neck cancer. The third report used interview

data for information on tobacco use.

Concern over health effects from smokeless tobacco use has prompted

interest in whether oral cancer is becoming epidemic in areas where ST use

is common. An additional area of importance to the public health community is the prevention of new smokeless tobacco use and cessation by

current users. One aspect of the effort is public education, and public

education relies on information about gaps in public knowledge. Both of

these issues are examined in greater depth in this report.

TRENDS IN

ORAL CANCER

INCIDENCE AND

MORTALITY

Examination of geographic and time trends in cancer incidence

and mortality has been useful in identifying or highlighting causes

of cancer. For example, the higher mortality rates for malignant

melanoma and other skin cancers as latitude decreases (and sun

exposure increases) are clearly evident from U.S. maps of cancer mortality

based on deaths from 1950 to 1980 (Pickle et al., 1987).

Maps also were important in identifying an epidemic area of oral cancer

mortality among women in the southeastern United States (Mason et al.,

1975). Interest in this epidemic of high oral cancer mortality among

women led to an ecological study (Blot and Fraumeni, 1977) correlating oral

cancer mortality with industrial and demographic characteristics and

subsequently to a case control study. In that case control study (Winn et al.,

1981), it was found that ST use was associated with a fourfold increased risk

of oral and pharyngeal cancer. Risks for the gum and buccal mucosa were

especially elevated and estimated to increase the risk by close to fiftyfold.

Smokeless tobacco was thought to account for 87 percent of the gum and

buccal mucosal cancer among women in the epidemic area.

This epidemic is on the downswing as shown by maps of cancer mortality that cover a longer period, for example, from 1950 to 1980, and highlight trends across decades (Mason et al., 1975). These maps show that oral

cancer death among women is becoming less common in the southeastern

United States and more common in some urban areas elsewhere in the

United States. One may infer that the use of snuff, the probable cause of the

epidemic in the southeastern United States, is no longer common among

women there. U.S. production figures show a decline in demand for dry

snuff (US DHHS, 1986a), the type of smokeless tobacco most commonly

used by the women in the study.

Incidence as well as mortality data are available for examination of time

trends. According to data from the Surveillance, Epidemiology, and End

Results (SEER) program from 1947 to 1984 (Devesa et al., 1987), the incidence of oral cancer among women increased by more than 50 percent
Chapter 1

13

during the four decades to 5.3/100,000 women. Although the incidence

rate during this interval remained fairly constant for men across all ages, at

about 12.3 to 13.5/100,000 men, this stability obscures an increase in

incidence among younger cohorts and a decrease among older cohorts.

Devesa and coworkers (1987) suggest that adoption of cigarette smoking

among women might account for the rise in incidence of oral cancer. They

also suggest that the decrease in older men was consistent with gradual

declines in ST use and pipe smoking during the century, and that cigarettes

and, possibly, alcohol might account for increases among the younger age

groups.

It is difficult to correlate tobacco use patterns with oral cancer incidence

and mortality because of the relatively sparse data on tobacco use patterns

from small geographic units such as counties. Also, the long latency for

cancers makes it difficult to know when to expect a change in incidence

after a population changes its risk-factor habits. Nevertheless, available

cancer surveillance data have been examined for changes in cancer occurrence.

Partly inspired by concern over potential rises in oral cancer associated

with ST use by youth, several letters to the editors of medical journals were

published presenting data on tongue cancer from cancer hospital records

and from U.S. incidence and mortality data bases (Davis and Severson, 1987;

Depue, 1986; Schantz et al., 1984; Shemen, 1984). In all of these reports,

tongue cancer rose when measured by increases in incidence or mortality or

as a proportion of cancers seen at cancer centers. However, it has not been

possible to elucidate the causes of this increase.

If smokeless tobacco use among young men is leading to an epidemic of

oral cancer, then it might be expected that a rise in incidence might be

detected or be clearly evident for cancers of the gum and buccal mucosa

because of the strong relative risks associated with ST and this cancer site.

Figure 1 shows the incidence of oral cancer and the incidence of gum and

buccal mucosal cancer (a subset of all oral cancers) from 1973 to 1987

among men under age 50 from the SEER program, which includes data from

population-based cancer registries covering about 10 percent of the U.S.

population. Gum and buccal mucosal cancer incidence essentially was

unchanged during this 15-yr period, in spite of a corresponding rise in ST

use, although oral cancer in general has been increasing among younger

men. Although there was no increase in the incidence of gum and buccal

mucosal cancer during this period, it should be noted that baseline rates of

these cancers are very low in younger adults (< 0.5/100,000 new cancers per

year in men under age 50); oral cancer typically occurs at older ages (62.4 is

the median age of diagnosis [Young et al., 1981]); and case control studies

suggest that most individuals with oral cancer associated with ST use have

used the products for a long time. This would suggest that it may be too

early to detect any cancer consequences of increased ST use.

Oral mucosal lesions are common in ST users. In surveys of adolescents, lesions ranging from small local mucosal changes involving slight

color and texture modifications to more significant color changes and deep
Smoking and Tobacco Control Monograph No. 2

14

0

0.5

1

1.5

2

2.5

3

Oral cancer

Gum/buccal mucosal 

cancer

Incidence Rate per 100,000 Persons

1973-75 1976-78 1979-81 1982-84 1985-87

Years

Source: SEER Program, 1973 to 1987

Figure 1

Incidence of oral cancer: males under age 50, 1973 to 1987

Source: SEER Program, 1973 to 1987.

furrowing have been observed in between 23 and 63 percent of smokeless

tobacco users (Centers for Disease Control, 1988; Greer and Poulson, 1983;

Offenbacher and Weathers, 1985; Poulson et al., 1984), far exceeding those

among non-users (Offenbacher and Weathers, 1985). The more serious

lesion, leukoplakia, has been noted in small proportions of young users. In

one study, leukoplakia was observed in 5.0 percent of high school football

players in Alabama, in contrast to only 0.1 percent among non-users (Creath

et al., 1988). Leukoplakia had a prevalence of 46.0 percent among ST-using

professional baseball players, whereas only 1.4 percent of non-users had

leukoplakia (Ernster et al., 1990).

The transformation of leukoplakia to frank carcinoma is a concern. In

one study in India (Gupta et al., 1980), malignant transformation of leukoplakia occurred at a rate of 0.9/1,000 among persons ages 35 to 54 and 10.2/

1,000/yr among older persons. The authors summarized the literature on

transformation rates, which suggests that between 0.13 and 10.0 percent of

leukoplakias will transform, but the studies involved differing followup

periods and lesion definitions.
Chapter 1

15

Few other recent studies on this subject have been conducted. There

are variations in the risk of transformation to cancer by type of leukoplakia

(Gupta et al., 1989), and precise estimates of transformation rates for nonAsian populations are lacking. Some information on the prevalence of oral

soft tissue lesions from 1957 to 1973 is available, based on data from large

oral cancer screening clinics, some of which covered more than half of the

target communities (Bouquot and Gorlin, 1986). However, currently in the

United States, leukoplakia (or other precancerous lesions) is not reportable

to population-based cancer or other registries, so statistical data on prevalence, incidence, and trends over time for leukoplakia are not obtainable.

National prevalence estimates for leukoplakia and ST-associated lesions will

be available on the completion of the Third National Health and Nutrition

Examination Survey conducted by the Centers for Disease Control’s National Center for Health Statistics.

PUBLIC

KNOWLEDGE

OF ST AND

CANCER LINKS

About 6 percent of U.S. adult men use smokeless tobacco, according

to data from the National Health Interview Survey (NHIS) of Cancer

Epidemiology and Control conducted in 1987 by the National

Center for Health Statistics; the survey indicated that smokeless

tobacco use by women overall was negligible (0.5 percent) (Schoenborn and

Boyd, 1989). The NHIS is a continuous, multipurpose, cross-sectional survey

used to obtain national estimates of health characteristics through household interviews. The survey has a multistage, probability, cluster sample

design. In the 1987 NHIS, members of the U.S. public were asked questions

about whether they thought that snuff and chewing tobacco increase the

risk of mouth and throat cancer. The percentages of U.S. men reporting

that these products increase cancer risks were similar for snuff (79.9 percent)

and for chewing tobacco (83.8 percent). Among tobacco chewers, 71.5 percent thought that their habit increases risk, compared to 85.1 percent

among non-users of chewing tobacco. The corresponding figures concerning risks due to snuff use were 85.1 percent and 80.6 percent for snuff users

and non-users, respectively. Figure 2 shows that knowledge was inversely

related to age, with more younger men being informed; 80 percent or more

of those under age 65 knew of mouth and throat cancer risks, compared to

about 70 percent of older men.

The NHIS survey covered adults only, but several regional studies,

generally conducted in areas where ST use among youth is common, suggest

that two-thirds or more of adolescents understand that ST use can cause

cancer. Missouri users in grades 5, 8, and 12 were similar to non-users in

knowing that smokeless tobacco causes mouth cancer (75 and 80 percent,

respectively) (Brownson et al., 1990); furthermore, knowledge of health risks

improved with age. A Texas survey (Schaefer et al., 1985) showed that

67 percent of high school students surveyed thought that snuff and chewing

tobacco cause cancer. In another study, in Alabama (Creath et al., 1988),

93.7 percent of male high school football players and 92.5 percent of those

who used ST were aware that using smokeless tobacco could be harmful to

health; “cancer” was the most common response to a question about how

health was affected by these products. These data suggest a fairly high level
Smoking and Tobacco Control Monograph No. 2

16

Figure 2

Males who report that smokeless tobacco increases cancer risk, United States, 1987

of knowledge among all ages that smokeless tobacco causes cancer. However, the relation of health knowledge to behavior is uncertain; it seems

clear that knowledge alone is insufficient to reduce significant ST use in

these populations.

RESEARCH NEEDS This report suggests some important research needs:

• Longitudinal studies of persons with ST-associated oral lesions are

needed to examine the natural history of these lesions, including

their continuance, regression, and progression. Factors influencing

the natural history of the lesions must be determined.

• Definitions and classifications of oral lesions vary in studies involving

mucosal examinations and should be standardized, possibly through

an international consensus conference. The same definitions should

be used by all investigators to allow comparison of results across

studies.

Non-chewer

0

20

40

60

80

100

Percentage Reporting Use Increases Cancer Risk

Uses chewing 

tobacco

Non-snuff user Uses snuff

Current Use of Smokeless Tobacco

Source: NCHS, NHIS, 1987.

ages 17-34 ages 35-64 ages 65 and older
Chapter 1

17

• Cancer incidence and mortality surveillance systems should continue

to monitor oral cancer trends in view of the increasing numbers of

smokeless tobacco users and the potential for rises in oral cancer

incidence rates.

• Better data are needed on the transformation rates for leukoplakia in

U.S. populations.

• Finally, more data are needed on how knowledge of health risks and

health education can be used to prevent ST use and encourage effective cessation efforts.

REFERENCES

Blot, W.J., Fraumeni, J.F., Jr. Geographic patterns of

oral cancer in the United States: Etiologic

implications. Journal of Chronic Disease 30: 745-

757, 1977.

Blot, W.J., McLaughlin, J.K., Winn, D.M., et al.

Smoking and drinking in relation to oral and

pharyngeal cancer. Cancer Research 48: 3282-3287,

1988.

Bouquot, J.E., Gorlin, R.J. Leukoplakia, lichen

planus, and other oral keratoses in 23,616 white

Americans over the age of 35 years. Oral Surgery,

Oral Medicine, Oral Pathology 61: 373-381, 1986.

Brownson, R.C., Dilorenzo, T.M., Tuinen, M.V.,

Finder, W.W. Patterns of cigarette and smokeless

tobacco use among children and adolescents.

Preventive Medicine 19: 170-180, 1990.

Carmella, S.G., Kagan, S.S., Kagan, M., et al. Mass

spectrometric analysis of tobacco-specific nitrosamine hemoglobin adducts in snuff dippers,

smokers and non-smokers. Cancer Research 50:

5438-5445, 1990.

Centers for Disease Control. Prevalence of oral

lesions and smokeless tobacco use in Northern

Plains Indians. Morbidity and Mortality Weekly

Report 37: 608-611, 1988.

Creath, C.J., Shelton, W.O., Wright, J.T., Bradley,

D.H., Feinstein, R.A., Wisniewski, J.F. The

prevalence of smokeless tobacco use among

adolescent male athletes. Journal of the American

Dental Association 116: 43-48, 1988.

Davis, S., Severson, R.K. Increasing incidence of

cancer of the tongue in the United States among

young adults. Lancet 2: 910-911, 1987.

Depue, R.H. Rising mortality from cancer of the

tongue in young white males. New England Journal

of Medicine 315: 647, 1986.

Devesa, S.S., Silverman, D.T., Young, J.L., Pollack,

E.D., Brown, C.C., Horm, J.W., Percy, C.L., Myers,

M.H., McKay, F.W., Fraumeni, J.F., Jr. Cancer

incidence and mortality trends among whites in

the United States, 1947-1984. Journal of the

National Cancer Institute 79: 701-770, 1987.

Ernster, V.L., Grady, D.G., Greene, J.C., Walsh, M.,

Robertson, P.B., Daniels, T., Benowitz, N., Seigel,

D., Gerbert, B., Hauck, W. Smokeless tobacco use

and health effects among baseball players. Journal

of the American Medical Association 264: 218-224,

1990.

Greer, R.O., Poulson, T.C. Oral tissue alterations

associated with the use of smokeless tobacco by

teen-agers: Part 1. Clinical findings. Oral Surgery

56: 275-284, 1983.

Gupta, P.C., Bhonsle, R.B., Murti, P.R., et al. An

epidemiologic assessment of cancer risk in oral

precancerous lesions in India with special

reference to nodular leukoplakia. Cancer 63: 2247-

2252, 1989.

Gupta, P.C., Mehta, F.S., Daftary, D.K., et al.

Incidence rates of oral cancer and natural history

of oral precancerous lesions in a 10-year follow-up

study of Indian villagers. Community Dental and

Oral Epidemiology 8: 287-333, 1980.

Hecht, S.S., Hoffmann, D. Tobacco-specific nitrosamines: An important group of carcinogens in

tobacco and tobacco smoke. Carcinogenesis 9: 875-

884, 1988.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosamines (volume 37). Lyon:

IARC, 1985.

Mason, T.J., McKay, F.W., Hoover, R., et al. Atlas of

Cancer Mortality for U.S. Counties: 1950-1969. U.S.

Department of Health, Education, and Welfare;

Public Health Service; National Institutes of

Health. NIH Publication No. 75-780, 1975.

Offenbacher, S., Weathers, D.R. Effects of smokeless

tobacco on the periodontal, mucosal, and caries

status of adolescent males. Journal of Oral

Pathology 14: 169-181, 1985.

Pickle, L.W., Mason, T.J., Howard, N., et al. Atlas of

U.S. Cancer Mortality Among Whites: 1950-1980.

U.S. Department of Health and Human Services,

Public Health Service, National Institutes of

Health. NIH Publication No. 87-2900, 1987.
Smoking and Tobacco Control Monograph No. 2

18

Poulson, T.C., Lindenmuth, J.E., Greer, R.O., Jr. A

comparison of the use of smokeless tobacco in

rural and urban teenagers. CA—A Cancer Journal

for Clinicians 34: 248-261, 1984.

Schaefer, S.D., Henderson, A.H., Glover, E.D.,

Christen, A.G. Patterns of use and incidence of

smokeless tobacco consumption in school-age

children. Archives of Otolaryngology 111: 639-642,

1985.

Schantz, S.P., Byers, R.M., Goepfert, J. Tobacco and

cancer of the tongue in young adults. Journal of

the American Medical Association 259: 1943-1944,

1988.

Schoenborn, C.A., Boyd, G. Smoking and other

tobacco use: United States, 1987. National Center

for Health Statistics. Vital Health Statistics 10(169):

9, 1989.

Shemen, L.J., Klotz, J., Schottenfeld, D., Strong, E.W.

Increase of tongue cancer in young men. Journal of

the American Medical Association 252: 1857, 1984.

Spitz, M.R., Fueger, J.J., Goepfert, J., et al. Squamous

cell carcinoma of the upper aerodigestive tract: A

case comparison analysis. Cancer 61: 203-208,

1988.

Stockwell, H.G., Lyman, G.H. Impact of smoking

and smokeless tobacco on the risk of cancer of the

head and neck. Head and Neck Surgery 9: 104-110,

1986.

U.S. Department of Health and Human Services.

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health. NIH Publication No. 86-2874, 1986a.

U.S. Department of Health and Human Services.

Health Implications of Smokeless Tobacco Use:

National Institutes of Health Consensus Development

Conference (volume 6, no. 1). U.S. Department of

Health and Human Services, Public Health Service,

National Institutes of Health, Office of Medical

Applications of Research, 1986b.

Winn, D.M. Recent research directions: Smokeless

tobacco and aerodigestive cancers. In: The Biology

and Prevention of Aerodigestive Tract Cancer,

G. Newell and W.K. Hong (Editors). New York:

Plenum Publishing, 1992 (in press).

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W.,

Toledo, A., Fraumeni, J.F., Jr. Snuff dipping and

oral cancer among women in the southern United

States. New England Journal of Medicine 304: 745-

749, 1981.

Young, J.L., Jr., Percy, C.L., Asire, A.J. (Editors).

Surveillance, Epidemiology, and End Results:

Incidence and Mortality Data, 1973-1977.

National Cancer Institute Monographs 57: 66, 1981.

DHHS Publication No. (NIH) 81-2330.
Chapter 1

19

Smokeless tobacco use is very common in India and neighboring countries. The betel quid

Smokeless Tobacco Use in India

P.C. Gupta

ABSTRACT

chewing habit and its variants predominate, but several other forms of ST also are popular. The

major use of ST is in the form of custom-made preparations from individual ingredients for

immediate use. In recent years several commercial ST products have been marketed, with

backing by intense advertising and promotion campaigns. It has been possible to educate rural

Indian populations and thereby persuade them to reduce their tobacco use; such education has

significantly decreased the risk of oral cancer.

INTRODUCTION Smokeless tobacco use may be of two kinds: oral use and nasal

use. In India and Southeast Asia, nasal use is uncommon; therefore, only

oral use is described in this paper.

In India, the neighboring countries, and some other countries of the

southeast region, smokeless tobacco use is very common, although it has

declined over time. Trend data on the prevalence of ST use are not available, but inferences may be drawn from the data on total tobacco consumption. Analysis of the per capita amount of tobacco consumed in both

smoked and smokeless tobacco forms in India over the past 40 years has

shown that smoking has increased considerably, but smokeless tobacco has

declined from its position as the dominant form (Sanghvi, 1989).

Reliable prevalence data from some selected parts of India became

available during the late 1960’s and early 1970’s, when large cross-sectional,

house-to-house surveys of tobacco habits were conducted in rural populations (Mehta et al., 1969 and 1972). Table 1 shows the sample size and the

prevalence of overall tobacco use (smoking and chewing), by sex, among

individuals aged 15 and over. It is clear that tobacco use is very common in

India. Among men it is uniformly high, but among women there is great

variability. The areas surveyed were selected with specific objectives and

thus may not represent the whole of India. They are, however, widely

dispersed areas and do represent a large part of the country.

Table 2 shows the prevalence of smokeless tobacco use among nonsmokers and smokers, and the overall prevalence of use. The prevalence of

ST use varied markedly in different regions, although in general it was

comparable between smokers and nonsmokers. In most places, ST use was

found to be more common among women.

A high prevalence of smokeless tobacco use in India may be somewhat

surprising because tobacco was introduced in India, like everywhere else, by

the Europeans about 400 years ago, primarily as a substance for smoking.

To learn the reasons for the popularity of ST use, one has to look at some

ancient cultural practices of this region.

1 Supported solely by the National Institutes of Health, under Indo-U.S. Fund Research

Agreement no. 01-022-N.

1
Smoking and Tobacco Control Monograph No. 2

20

Table 1

Prevalence of tobacco use (smoking and smokeless) in India,

from population-based, house-to-house studies

Tobacco Users, Percentage

Area Sample Size Men Women All Over Age 15

Bhavnagar 10,071 71% 15% 44%

Ernakulam 10,287 81 39 59

Strikakulam 10,169 81 33 56

Singhbhum 10,048 81 33 56

Darabhanga 10,340 78 51 64

Pune 101,761 62 49 64

TYPES OF

ST USED

The most common methods of ST use in India are betel quid chewing

and its variants. Betel quid chewing is an ancient practice—several

millennia old. Betel quid is mentioned in ancient religious texts, medicinal

treatises, literary works, and old stone inscriptions. It has always been a part

of religious, social, and cultural rituals, and the practice enjoys complete

social acceptance, even today.

Traditionally betel quid consisted of betel leaf, pieces of areca nut, a few

drops of lime (calcium hydroxide), several condiments, sweetening, and

flavoring agents, depending on regional practices and individual preferences. After tobacco was introduced in India in the 17th century, it became

an ingredient of the betel quid. Through its association with a socially

accepted practice, ST use became widespread. Currently almost all habitual

users of betel quid use it with tobacco. The habit is widespread in all parts

of India and is practiced by both men and women.

Until recently, the habit of betel quid chewing was not recognized as an

ST habit. In the literature, betel quid chewing was generally referred to as

“betel nut” chewing. This terminology was probably responsible for the

erroneous impression that the nut used in the betel quid was the main

active substance for carcinogenesis. The term “betel nut” is a misnomer,

because the nut used in the betel quid is from the palm Areca catachu and

therefore should be called “areca nut” (Burton-Bradly, 1979).

The betel quid chewing habit evolved into several variants such as

chewing of mawa, khaini, mainpuri tobacco, and, more recently, various

brands of commercially manufactured and marketed pan masala. Probably a

major reason for the popularity of these variants is the perishability of the

betel leaf, a fresh green leaf from the betel vine, in which various ingredients of the quid are smeared and wrapped. The tenderness and freshness of

the leaf are highly prized, and therefore betel leaf does not last for more

than a few days. Betel vine is said to be a delicate plant, requiring much

care and attention, and cannot be grown everywhere. The leaf is thus

difficult to obtain in places distant from betel-growing areas.
Chapter 1

21

Table 2

ST use among men and women, smokers and nonsmokers

Men, Percentage Women, Percentage

Area Smokers Nonsmokers Overall Smokers Nonsmokers Overall

Gujurat 10% 24% 15% 0% 15% 15%

Kerala 33 42 36 43 38 38

Srikakulam 16 16 16 4 8 6

Darabhanga 52 56 54 8 12 11

Singhbhum 22 47 31 25 28 27

Maharashtra 30 58 56 0 49 49

Although the betel quid itself, as well as various combinations of its

ingredients, can be chewed with or without the inclusion of tobacco, most

habitual chewers include tobacco in their quid. This is understandable

because tobacco is the only addictive substance among the betel quid

ingredients. This point was not recognized, however, until evidence

emerged from house-to-house, cross-sectional studies of large samples of

rural populations (Mehta et al., 1969 and 1972).

Table 3 shows the prevalence of chewing habits in some areas where

chewing was reported to be popular. The table shows the prevalence of

chewing among nonsmokers, the proportion of chewers who exclude

tobacco from their quid, and those who include tobacco. Smokers are

excluded from the table. The data readily confirm that, although chewing

habits were popular, the proportion of those who excluded tobacco from

their quid was minuscule. In different areas, different kinds of chewing

habits prevailed; for example, the predominant chewing substance in

Ernakulam district was betel quid, whereas in Pune district it was tobacco

and lime.

The tobacco-plus-lime mixture is probably the most common variant of

the betel quid. The mixture is known as khaini in the northern part of

India, and it is popular in other parts as well. Tobacco-plus-lime is the most

common form of tobacco used in Pune district.

A user typically carries a double-mouth box, the larger part containing

tobacco flakes and the smaller one lime (calcium hydroxide) paste. To

prepare the quid, the user places a small amount of tobacco in the palm; a

dash of lime is flicked by a thumb or forefinger, and it is mixed and rubbed

vigorously with the tobacco in the hand. The mixture is then ready for use

and is placed in the mouth. Some individuals may add pieces of areca nut

as well.

The exact placement of the tobacco-plus-lime mixture in the mouth

varies by geographic area. In Pune district, the mixture is often placed in the

canine region; in Darabhanga, in the labial groove; and in Singhbhum

district, many users prefer to keep it on the tongue. The most common sites

of oral cancers and precancers also vary correspondingly in those regions.
Smoking and Tobacco Control Monograph No. 2

22

Table 3

Comparison of individuals who use tobacco in their chewing quid

with those who do not

In Ernakulam In Singhbhum In Maharashtra

(n=10,287) (n=10,048) (n=101,761)

Number of Chewersa 2,699 1,334 51,835

With tobacco 99% 97% 99%

Without tobacco 1% 3% 1%

a Without any smoking habit.

Mawa is another variant of betel quid that contains areca nut, tobacco,

and lime. Mawa is popular in Bhavnagar district and nearby areas, but in

other areas it may be known by different names. By weight, more than

90 percent of mawa is areca nut. It is prepared immediately prior to use and

is generally purchased from kiosks that sell betel quid and other tobacco

products. The vendor places small pieces of sun-cured areca nut (5.5 g) on a

piece of cellophane (10 to 13 cm), adds tobacco flakes (0.4 g), and sprinkles

a few drops of a solution of calcium hydroxide. The mixture is then tied as

a round ball in the cellophane wrapper and given to the customer. A user

typically rubs the cellophane ball vigorously on the palm for a couple of

minutes, ostensibly to homogenize the mawa mixture. (More likely, this

action produces greater availability of free nicotine through the action of

calcium hydroxide on tobacco.) The user then opens the cellophane,

removes any veins of tobacco leaf, and puts the mixture into the mouth.

One quid may be chewed for 10 to 20 min. Some users may chew only half

of the quid at one time (Sinor et al., 1990).

The most recent variant on betel quid is pan masala, a manufactured

item containing areca nut and other ingredients common in betel quid;

some brands contain dehydrated and powdered betel leaves. Pan masala is

generally available in two types—with tobacco and without tobacco—sold

under the same brand name. The single name for both choices affords a

significant marketing advantage to the manufacturer. Since there are no

restrictions on advertising a consumer product that contains no tobacco,

pan masala without tobacco is vigorously advertised and promoted, without

restriction, even on the government-controlled electronic media. Pan

masala with tobacco, however, carries the same brand name and therefore

gets considerable benefit from the unrestricted advertisement and promotion of the nontobacco counterpart.

The vigorous, high-profile advertising of pan masala has prompted

manufacturers who rarely advertised their chewing products before to start

advertising heavily as well. As a result, advertisements of commercially

manufactured and marketed ST products are common on the roads, in

magazines, and in videotapes. It is easy to discern the target group: urban

individuals with education, traditional values, and disposable income.

Recently, working women and middle-class housewives also seem to have

become a specific target of such advertisements.
Chapter 1

23

New and more effective ways of advertising and promoting are constantly explored and employed. For example, in the videocassette versions

of Indian films, animation is used to superimpose dancing tobacco products

on the movie so that viewers cannot avoid the commercial. The March

1991 issue of several magazines contained not only an advertisement of a

particular brand of chewing tobacco, but also, for the first time, a free

product sample pasted on the advertisement.

As a result of such high-profile advertising, ST use is increasing rapidly

in the stratum of society from which it had almost disappeared—among

individuals with college education who are in business and in middle and

high-level management positions. No hard data are yet available, but cans

and sachets of smokeless tobacco are becoming more common in public

places where well-educated people are seen, for example, in airport lounges.

Medical practitioners are reporting a rapid increase in the incidence of oral

submucous fibrosis, a chronic debilitating disease with no known cure,

believed to be caused by areca-nut chewing (Bhonsle et al., 1987; Sinor et

al., 1990).

There are also several methods of ST use that cannot be termed variants

of betel quid chewing. One of them is use of manufactured snuff, which is

common in the Western Region. Dry snuff is meant for nasal use, but oral

use is more common in India. There is a difference in variety as well; finer

snuff is for nasal use and coarser snuff for oral use. Manufacturers market

snuff as tapkeer, but local names may differ. One method of using dry snuff

is oral application with a dry twig, identical to the snuff dipping described

among women in North Carolina (Winn et al., 1981).

Mishri is a powdered form of roasted tobacco. It is common in Maharashtra and central regions of India, especially among women. People begin

using mishri as a dentifrice, but it soon turns into an addiction. A typical

user applies mishri to the teeth and gums several times a day.

Tobacco is also used in the form of gudakhu, a paste made of tobacco

and molasses. This is common in the eastern region. Creamy snuff, common in Goa, is a manufactured item marketed in toothpaste-like tubes. Its

marketing technique exploits the prevailing misconception that tobacco is

good for the teeth and gums. There are several herbal and medicinal tooth

powders that contain tobacco.

EFFECTS OF

ST USE

The most extensively studied and best documented health consequence

of ST use in India is oral cancer. Numerous case-control and some

cohort studies have clearly demonstrated the causal role of smokeless

tobacco in oral cancer. This subject has already been reviewed and evaluated in depth (IARC, 1984). Interestingly, the experimental as well as

epidemiological evidence with respect to areca nut chewing has been

assessed as inadequate to demonstrate carcinogenicity (Gupta et al., 1982;

IARC, 1984).

A less studied health consequence is the effect of smokeless tobacco use

on reproduction. Two studies have indicated that ST use during pregnancy

leads to significantly lower birth weight, higher placenta weight, and higher

infant mortality (Krishnamurty, 1989).
Smoking and Tobacco Control Monograph No. 2

24

Overall health consequences of smokeless tobacco use in India have

been assessed through examination of the relative risk of all-cause mortality

among tobacco users. This was done through cohort studies in two areas.

In one area where bidi smoking and betel quid chewing were prevalent, the

age-adjusted excess risk among smokers was 40 percent (p < 0.05). Surprisingly, even among chewers who did not smoke, the excess risk was 30 percent (p < 0.05). There was no clue, however, as to specific causes of this

excess mortality among chewers (except oral cancer) because of a lack of

information on the causes of death (Gupta et al., 1984a).

In another area, where reverse smoking was practiced, the age-adjusted

excess mortality was close to 100 percent (Gupta et al., 1984b). An attempt

has been made to estimate excess mortality attributable to tobacco use. The

estimate is that every year, 630,000 adult deaths occur prematurely because

of tobacco use in India (Gupta, 1988). Because of insufficient information,

it is possible to categorize only 56 percent of these excess deaths according

to cause (Notani et al., 1989).

EFFECTS OF

EDUCATION

Can the tobacco habits in rural populations be changed through

educational efforts, and would such efforts result in any health benefit?

An answer to this question has been provided by an intervention study

among rural Indian populations in three areas of India. More than 36,000

tobacco users were interviewed about tobacco use and examined for the

presence of oral cancer and precancerous lesions. All these individuals were

educated about the health hazards of tobacco use. Personal communication

was provided by the examining dentist and a social scientist. Subjects also

were educated through the use of documentary films, posters, newspaper

articles, radio messages, and folk-art theater.

Considerable social science research was carried out to assess why

people begin using tobacco, why they continue using it, what they perceive

as the health effects of using tobacco, and what influences could help them

discontinue its use. This research was continuous, and feedback was incorporated into the education program. The educational campaign was also

continuous; interviews and examinations were carried out yearly. An

assessment after 5 yr of followup showed that a significantly higher percentage of people in the intervention cohort stopped or reduced their tobacco

use (smoking as well as ST use) than in the control cohort. Consequently,

the incidence of precancerous lesions decreased substantially in the intervention cohort compared to the control cohort. Higher quit rates were

achieved among smokeless tobacco users and thus the reduction in risk was

also greater (Gupta et al., 1986a and 1986b). Similar results were reported

after 8 yr of followup (Gupta et al., 1989).

CONCLUSION On the whole, this discussion shows that the problem of smokeless

tobacco use in India is quite different from that in industrialized countries.

The health consequences of ST use are very serious but, except for oral

cancer, are not as well understood as the consequences of cigarette smoking.

It is possible to educate the population about the health risks of smokeless

tobacco. Such an effort would result in a significant health benefit to the

population.
Chapter 1

25

REFERENCES

Bhonsle, R.B., Murti, P.R., Gupta, P.C., Mehta, F.S.,

Sinor, P.N., Irani, R.R., Pindborg, J.J. Regional

variations in oral submucous fibrosis in India.

Community Dentistry and Oral Epidemiology 15: 225-

229, 1987.

Burton-Bradly, B.G. Is betel chewing carcinogenic?

Lancet 2: 903, 1979.

Gupta, P.C. Health consequences of tobacco use in

India. World Smoking and Health 13: 5-10, 1988.

Gupta, P.C., Aghi, M.B., Bhonsle, R.B., et al. Intervention study of chewing and smoking habits

for primary prevention of oral cancer among

12,212 Indian villagers. In: Tobacco: A Major

International Health Hazard, D.G. Zaridze and R.

Peto (Editors). IARC Scientific Publications,

No. 74. Lyon: International Agency for Research

on Cancer, 1986b.

Gupta, P.C., Bhonsle, R.B., Mehta, F.S., Pindborg, J.J.

Mortality experience in relation to tobacco

chewing and smoking habits from a 10-year

follow-up study in Ernakulam district, Kerala.

International Journal of Epidemiology 13: 184-187,

1984a.

Gupta, P.C., Mehta, F.S., Pindborg, J.J. Mortality

among reverse chutta smokers in South India.

British Medical Journal 289: 865-866, 1984b.

Gupta, P.C., Mehta, F.S., Pindborg, J.J., et al.

Intervention study for primary prevention of oral

cancer among 36,000 Indian tobacco users. Lancet

1: 1235-1238, 1986a.

Gupta, P.C., Mehta, F.S., Pindborg, J.J., et al. A

primary prevention study of oral cancer among

Indian villagers: Eight-year follow-up results. In:

Evaluating Effectiveness of Primary Prevention of

Cancer, M. Hakama, V. Beral, J.W. Cullen, and

D.M. Parkin (Editors). IARC Scientific Publications, No. 103. Lyon: International Agency for

Research on Cancer, 1989, pp. 149-156.

Gupta, P.C., Pindborg, J.J., Mehta, F.S. Comparison

of carcinogenicity of betel quid with and without

tobacco: An epidemiological review. Ecology of

Disease 1: 213-219, 1982.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosamines (volume 37). Lyon:

IARC, 1984.

Krishnamurty, S. Tobacco use in pregnancy and

reproductive outcome. In: Tobacco and Health:

The Indian Scene, L.D. Sanghvi and P.N. Notani

(Editors). Bombay: UICC-Tata Memorial Centre,

1989.

Mehta, F.S., Gupta, P.C., Daftary, D.K., Pindborg, J.J.,

Choksi, S.K. An epidemiologic study of oral

cancer and precancerous conditions among

101,761 villagers in Maharashtra, India. International Journal of Cancer 10: 134-141, 1972.

Mehta, F.S., Pindborg, J.J., Gupta, P.C., Daftary, D.K.

Epidemiologic and histologic study of oral cancer

and leukoplakia among 50,915 villagers in India.

Cancer 24: 832-849, 1969.

Notani, P.N., Jayant K., Sanghvi, L.D. Assessment of

mortality and morbidity due to tobacco use in

India. In: Tobacco and Health: The Indian Scene,

L.D. Sanghvi and P.N. Notani (Editors). Bombay:

UICC-Tata Memorial Centre, 1989.

Sanghvi, L.D. Tobacco related cancer. In: Tobacco

and Health: The Indian Scene, L.D. Sanghvi and

P.N. Notani (Editors). Bombay: UICC-Tata

Memorial Centre, 1989.

Sinor, P.N., Gupta, P.C., Bhonsle, R.B., Murti, P.R.,

Daftary, D.K., Mehta, F.S., Pindborg, J.J. A casecontrol study of oral submucous fibrosis with

special reference to the etiologic role of areca nut.

Journal of Oral Pathology and Medicine 19: 94-98,

1990.

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W.,

Toledo, A., Fraumeni, J.F. Snuff dipping and oral

cancer among women in the southern United

States. New England Journal of Medicine 304: 745-

749, 1981.
Smoking and Tobacco Control Monograph No. 2

26

Smokeless tobacco use was examined in 2,009 professional baseball players as part of a

Smokeless Tobacco in Professional

Baseball: Patterns of Players’ Use

V.L. Ernster, D.G. Grady, L. Stillman, M. Walsh, and J.C. Greene

ABSTRACT

study of oral and other health effects of ST use conducted at spring training in 1988, 1989, and

1990. According to questionnaire data, 37.5 percent of participants had used ST within the

previous week. Among current-week users, the median age at first ST use was 17 yr, median

duration of use was 5 yr, median amount of use per day was 1 h, and median cotinine level was

92.8 ng/mL. The reported ST product usually used was more often a snuff than a chewing

tobacco brand (77.1 vs. 19.7 percent, respectively). Of current-week users, 49 percent were

year-round users. The latter used ST more hours per day, had used ST more recently, were more

likely to use snuff, and had higher serum cotinine levels than did seasonal users. Similar

differences were seen in comparisons of snuff to chewing tobacco users. Most ST users

considered ST harmful to health; however, snuff users were more likely to report ST as “very”

harmful, and year-round snuff users most likely to have noticed oral effects of use. Snuff users

appeared more motivated to quit than did chewing tobacco users.

INTRODUCTION Over the 3-yr period from 1988 through 1990, we collected data on

smokeless tobacco use among professional baseball players as part of a study

of oral and other health effects of ST use. A description of the overall study

design and highlights of the findings from the first year of the study have

been published elsewhere (Ernster et al., 1990). Here we provide a detailed

description of ST use patterns in professional baseball players, based on the

combined data from all 3 yr of the study.

METHODS During spring training of 1988, 1989, and 1990, all players and coaching

staff of the seven Major League Baseball teams and their associated minor

league teams that conduct spring training in Phoenix and Tucson, Arizona,

were eligible to participate in the study. Participants completed a questionnaire that provided detailed information about their patterns of ST use, as

well as demographic and other data. In all 3 yr of the study, information

was collected on age at initiation of ST use, duration of use, amount used,

and type and brand of ST used most often. Analyses of type (snuff or

chewing tobacco), brand, and amount (cans of snuff or pouches of chewing

tobacco used per week) were based on the reported type usually used.

Because some participants who usually used one type of ST (snuff or chew)

also used the other type, we also calculated the number of hours of ST use

per day as a combined measure of amount of use. Information about

differences in ST use during the baseball season compared with the offseason were collected only in years 2 and 3 of the study, and data on perceptions of health effects and attempts to quit using ST were collected only in

year 2. A question about perceived readiness to quit using ST was added in

year 3.

1 Supported by National Institute of Dental Research grant no. DE-08547-02.

1
Chapter 1

27

On the basis of their self-reported ST use, we classified participants as

non-users (those who had never used ST or who had never used it more

than once a month in the past), former users (those who had used ST more

than once a month in the past but not within the previous month), and

current users (those who had used ST more than once per month and had

used it within the previous month). Current users were further divided into

current-month users (those who had used ST within the past month but not

within the past week) and current-week users (those who had used ST

within the past week). Unless otherwise indicated, the results reported here

for current ST users pertain only to current-week users.

During the first 2 yr of the study, blood was drawn for biochemical

validation of self-reported ST use. Low serum cotinine levels (< 12 ng/mL)

together with normal serum thiocyanate levels (< 85 mmol/L) were considered biochemical evidence of no use of tobacco. According to this standard,

95 percent of participants in the first year of the study from whom blood

samples were available and who reported that they neither used ST nor

smoked cigarettes (357 of 376) were classified biochemically as non-users.

Given the accuracy of self-reported use status and because blood was collected from only about 80 percent of players in the first year of the study,

50 percent of players in the second year, and none of the players in the

third year, ST use status in this paper is based on self-report. Data on

cotinine levels are for those players from whom blood was obtained in the

first 2 yr of the study. Finally, information on most recent ST use was

collected only in the first 2 yr of the study.

For univariate statistical analyses, standard t tests and χ2 tests were

performed, as appropriate. We have based the p values for the multivariate

analyses on the standard errors of log odds ratios, adjusted for covariates by

means of multiple logistic analysis (Hosner and Lemeshow, 1989).

RESULTS The distribution of the participants by age, race, education, cigarette

smoking, and alcohol use at the time of entry into the study is shown in

Table 1. These are the combined baseline data from players who were new

to the study in each of the 3 yr, a total of 2,039 players (1,109 first seen in

1988, 532 first seen in 1989, and 398 first seen in 1990). A majority of the

players were aged 20 to 29 yr (76.3 percent), were white (68.1 percent), and

had had at least some college education (75.2 percent). Current cigarette

smoking was rare (3.1 percent), and alcohol consumption was generally

moderate (21.3 percent were nondrinkers and 72.5 percent consumed fewer

than 14 drinks per week).

At baseline, 37.5 percent of participants reported having used ST within

the past week and 3.4 percent within the past month but not the past week;

12.6 percent reported themselves to be former ST users and 46.5 percent

non-users (Figure 1). We deleted from the analysis 30 players for whom ST

use status was not determined. Characteristics of ST use among currentweek users, again based on the combined baseline data, are shown in

Table 2. The median age at first use of ST was 17 yr, median duration of use

was 5 yr, and median time ST was used per day was 1 h. With respect to

brand usually used, 77.1 percent of players named a snuff brand and only
Smoking and Tobacco Control Monograph No. 2

28

Table 1

Baseline demographic and other characteristics from all 3 yr combined

Number a Percentageb

Age

< 20 yr 241 11.9%

20 to 24 1,125 55.6

25 to 29 419 20.7

30 to 34 129 6.4

35 to 39 48 2.4

≥ 40 63 3.1

Total 2,025 100.1

Race

White 1,387 68.1

Hispanic 350 17.2

Black 269 13.2

Asian 25 1.2

Other 5 0.2

Total 2,036 99.9

Education

Elementary 43 2.2

Some high school 76 3.8

High school graduate 375 18.8

Some college 1,041 52.3

College graduate 455 22.9

Total 1,990 100.0

Cigarette Smoking

Never-smoker 1,779 88.1

Former smoker 177 8.8

Current smoker 63 3.1

Total 2,019 100.0

Alcohol Consumption

Nondrinker 429 21.3

< 14 drinks per week 1,459 72.5

14 to 20 drinks per week 85 4.2

> 20 drinks per week 40 2.0

Total 2,013 100.0

a Totals vary because of missing data.

b Totals vary from 100 percent because of rounding.

19.7 percent a brand of chewing tobacco; 3.1 percent listed both. Among

those who usually used snuff, Copenhagen and Skoal (69.5 percent and

20.8 percent, respectively) were the most popular brands named, and among

those who usually used chew, Levi Garrett and Red Man (46.9 percent and

40.7 percent, respectively) were the most popular. Among snuff users,

39.7 percent used less than one can per week, 35.8 percent used one to three
Chapter 1

29

Figure 1

Distribution of subjects (n=2,009) at baseline examination, by ST use

cans per week, and 24.4 percent used more than three cans per week (median = two cans per week). Among chewing tobacco users 48.9 percent

used less than one pouch per week, 31.1 percent used one to three pouches

per week, and 20.0 percent used more than three pouches per week (median

= 1.5 pouches per week). Data on most recent ST use and cotinine levels

were available for participants who entered the study in 1988 or 1989.

Among current-week users, nearly one-third (30.7 percent) reported using ST

within the preceding hour (median = 3.8 h since last use), and 76.1 percent

had serum cotinine levels 25 ng/mL (median = 92.8 ng/mL).

Because data on seasonal use were collected only in years 2 and 3 of the

study, analyses of characteristics associated with seasonal vs. year-round use

are confined to all current ST users seen in year 2 of the study, whether or

not they had been in the study in year 1 (n=397), plus all current users new

to the study in year 3 (n=160). These data showed that about one-half of

current-week users (51 percent) used ST almost exclusively during the

baseball season (“I rarely use it during off-season”).

Given the accepted addictiveness of tobacco use, it was curious to us

that fully half of the current-week users were able to refrain from ST during

the off-season. We therefore decided to compare characteristics of seasonal

and year-round ST users in our study (Table 3). There were no significant

differences in age or race, but year-round users were significantly more likely

to have started using ST at an early age (mean age at initiation of use was

16.2 vs. 17.4 yr for year-round and seasonal users, respectively), to have used

ST for a longer period of time (7.0 vs. 5.1 yr), to currently use ST more hours

per day (2.1 vs. 0.9 h), to have used ST more recently (10.5 vs. 25.8 h since

last use), to use snuff (90.0 percent vs. 66.4 percent), and to be white

(87.4 vs. 77.0 percent).
Smoking and Tobacco Control Monograph No. 2

30

Table 2

ST use characteristics in current-week usersa

Numberb Percentagec

Age at First Use

< 10 yr 18 2.4%

10 to 14 141 18.7

15 to 19 444 59.0

≥ 20 150 19.9

Total 753 100.0

Duration of Use

≤ 3 yr 206 27.9

4 to 6 275 37.2

7 to 9 119 16.1

≥ 10 139 18.8

Total 739 100.0

Hours in Mouth

0.0 to 0.5 h/d 186 27.2

> 0.5 to 1.0 157 23.0

> 1.0 to 1.5 93 13.6

> 1.5 to 2.0 67 9.8

> 2.0 to 4.0 123 18.0

≥ 4.0 57 8.3

Total 683 99.9

Type of ST Usually Used

Snuff 567 77.1

Chew 145 19.7

Both equally 23 3.1

Total 735 99.9

Brand of Snuff Usually Used

Copenhagen 394 69.5

 Skoal 118 20.8

 Hawken 37 6.5

 Other 18 3.2

 Total 567 100.0

Brand of Chew Usually Used

 Levi Garrett 68 46.9

 Red Man 59 40.7

 Other 18 12.4

 Total 145 100.0

Amount of Use

Snuff (cans/wk)d

 ≤ 1 216 39.7

 > 1 to 3 195 35.8

 > 3 133 24.4

 Total 544 99.9

Footnotes at end of table.
Chapter 1

31

Table 2 (continued)

Numberb Percentagec

Amount of Use (continued)

Chew (pouches/wk)e

 ≤ 1 66 48.9%

 > 1 to 3 42 31.1

 > 3 27 20.0

 Total 135 100.0

Time Since Last Usef

> 24 h 86 15.9

> 12 to 24 134 24.8

> 1 to 12 155 28.6

≤ 1 166 30.7

Total 541 100.0

Cotinine (ng/mL)f

0 to 25 99 23.9

> 25 to 75 87 21.0

> 75 to 200 133 32.0

> 200 96 23.1

Total 415 100.0

a Includes baseline data for current-week users from all 3 yr combined for all variables

except cotinine, most recent use, and seasonal use.

b Totals vary because of missing data.

c Totals vary from 100 percent because of rounding.

d Includes only subjects who usually use snuff.

e Includes only subjects who usually use chew.

f Includes all current-week users seen in year 1 plus all new current-week players from

year 2.

We then attempted to determine which of these ST use variables were

independently associated with seasonal use by constructing a multiple

logistic regression model with seasonal use as the dependent variable and

age, race, age at first use, amount, type, duration, and most recent use as the

predictor variables. This analysis was confined to year 2 users, the only

group with data on both seasonal use and most recent use. Only hours of

use per day (p < 0.0001) and type of ST usually used (p=0.0008) were independently associated with seasonal use in the multivariate model. Duration

of use may also be independently associated with seasonal use (p=0.066).

Analyses of brand and amount used in relation to seasonality of use were

performed separately for snuff and for chewing tobacco users (Table 4).

Among year-round snuff users, Copenhagen was more commonly used and

Hawken less commonly used than among seasonal snuff users; and yearround users were much more likely than seasonal users to report use of more

than three cans per week. There were no significant brand differences

between year-round and seasonal chewing tobacco users, but the former

were much more likely to report use of more than three pouches per week.
Smoking and Tobacco Control Monograph No. 2

32

Table 3

Characteristics of seasonal and year-round ST users seen in years 2 and 3 and

association of each characteristic with year-round use

Univariate Multivariate

Year-Round Seasonal Analysisa Analysisb

(n) Mean (n) Mean p

Age, yr (270) 24.1 (282) 24.1 (0.92) (0.13)

Age at First Use, yr (271) 16.2 (281) 17.4 (0.0001) (0.29)

Duration of Use, yr (266) 7.0 (275) 5.1 (< 0.0001) (0.066)

Hours of Use/Day (256) 2.1 (251) 0.9 (< 0.0001) (0.0001)

Time Since Last Use, hc (162) 10.5 (178) 25.8 (0.0001) (0.096)

(n) Percentage (n) Percentage

Raced (0.009) (0.785)

White (235) 87.4% (213) 77.0%

Black (19) 7.0 (27) 9.9

Latino (14) 5.2 (32) 11.4

Other (1) 0.4 (5) 1.8

Type of ST Usually Usede (< 0.0001) (0.0008)

Snuff (238) 90.0 (180) 66.4

Chew (22) 8.2 (82) 30.4

Both (5) 1.8 (9) 3.2

a Univariate p values are from two-tailed t tests for continuous variables (age, age at first

use, duration of use, hours of use per day, and time since last use) and from χ2 tests for

categorical variables (race and type usually used).

b Multivariate p values are from the multivariate logistic regression model with seasonal

use as the dependent variable and all variables in the table as predictors. The multivariate analysis was confined to current-week ST users seen in year 2 (n=298), because

information on recency of use was not available for players seen in year 3.

c Includes only current-week ST users seen in year 2.

d Players in the “other” category were excluded from both univariate and multivariate

analyses.

e Players who reported using both snuff and chewing tobacco were excluded from both

univariate and multivariate analyses.

Finally, serum cotinine levels were significantly higher in year-round users

than in seasonal users (199.2 vs. 71.4 ng/mL, respectively).

The fact that year-round users were more likely than seasonal users to

use snuff suggested that it might be a more addictive product than chewing

tobacco. We therefore compared snuff and chew users in terms of several

surrogate measures of addiction, using the combined baseline data from all

3 yr of the study. Compared with chewing tobacco users, snuff users used

ST more hours per day, had used ST more recently, had higher serum

cotinine levels, and were more likely to be year-round users (Table 5).
Chapter 1

33

Table 4

Additional characteristics of seasonal and year-round ST users

Seasonal Year-Round pa

Brand ST Usually Used

Snuff (n=186) (n=243) 0.0001

Copenhagen 62.9% 76.5%

Skoal 22.0 18.9

Hawken 12.4 2.0

Other 2.7 2.5

Chew (n=85) (n=22) 0.68

Levi Garrett 51.8 45.4

Red Man 29.4 27.3

Other 18.8 27.3

Amount Used

Snuff (cans/wk) (n=182) (n=241) < 0.0001

≤ 1 63.2 24.1

1 to 3 29.7 39.0

> 3 7.1 36.9

Chew (pouches/wk) (n=84) (n=22) 0.023

≤ 1 56.0 36.4

1 to 3 32.1 27.3

> 3 11.9 36.4

Serum Cotinine (ng/mL) (n=121) (n=95)

mean=71.4 mean=199.2 < 0.0001

a Based on χ2 test.

We found no significant differences in age, race, age at initiation, or duration of ST use between snuff and chewing tobacco users among these

professional baseball players.

Finally, we examined perceptions of health effects of ST and attitudes

toward quitting among current-week users, distributed by seasonality of use

and type of ST used (Table 6). Year-round snuff users were more likely to

report having noticed sores, white patches, or gum problems where ST is

placed in the mouth (39.5 percent) than were chew users or seasonal snuff

users. When asked how harmful to their health they thought ST use to be,

only a small proportion indicated that they didn’t know or that it was “not

at all” harmful. However, both year-round and seasonal snuff users were

more likely than chewing tobacco users to think that ST is “very harmful”

and nearly twice as likely to indicate that they were thinking about quitting

ST use in the next 12 mo. Snuff users reported more quit attempts to date

than chewing tobacco users and scored somewhat higher on a quit ladder, a

scale of 1 to 10 that measures readiness to quit.
Smoking and Tobacco Control Monograph No. 2

34

Table 5

Characteristics of current snuff and chewing tobacco users

Type of ST Usually Used

Snuff Chewing Tobacco

Mean pa

Use/Day, h 1.9 1.1 0.0004

Hours Since Last Use 14.1 29.3 < 0.0001

Serum Cotinine, ng/mLb 149.6 46.7 < 0.0001

Age, yr 24.1 24.6 0.29

Age at Initiation 17.1 17.0 0.80

Duration of Use, yr 6.1 6.1 0.93

Percentage

Year-Round Usersc 56.6% 20.6%

Race

White 83.2 80.6 0.87

Black 8.3 9.0

Latino 7.8 9.7

Other 0.7 0.7

a Based on t test for continuous variables; χ2 test for race. b Serum cotinine analysis is based on all current-week ST users seen in year 1 plus all

current-week users seen for the first time in year 2.

c Seasonal use analysis is based on all current-week ST users seen in year 2 plus all

current-week users seen for the first time in year 3.

DISCUSSION This report, based on combined baseline data collected from 2,009

Major League and minor league baseball players from 1988 through 1990,

confirms our earlier findings, based on the 1988 data alone (Ernster et al.,

1990), of a high rate of ST use in this population (37.5 percent). Two other

studies of Major League Baseball players surveyed in 1987 reported comparable results: Connolly and coworkers (1988) found that 34 percent of 265

players who completed questionnaires at spring training in Florida were selfreported current ST users, and Wisniewski and Bartolucci (1989) found that

45.6 percent of the 528 players on 25 teams who responded to a mail survey

were current users. In all three studies, snuff was found to be the preferred

form of ST; a snuff brand was reported as the product usually used by 77

percent of ST users in our study and 71 percent of users surveyed by

Connolly and coworkers.

The prevalence of ST use found among professional baseball players is

much higher than that reported for young men in the general population

(5.9 percent among men aged 20 to 29 in 1986, with much regional variation) (Bauman et al., 1989; Marcus et al., 1989; Novotny et al., 1989; Rouse,

1989), and generally much higher than reported in school-based studies of

adolescent males (Ary, 1989; Ary et al., 1987; Boyd et al., 1987; Brownson
Chapter 1

35

Table 6

Perceptions of health effects and attitudes toward quitting among current ST users,

by seasonality of use and type of ST useda

 Seasonal Use Year-Round Use

Chew Snuff Chew Snuff

“How harmful do you think that chewing/dipping (n=65) (n=131) (n=18) (n=172)

tobacco is for your health?”

Not at all harmful/don’t know 4.6% 12.2% 5.6% 5.8%

Slightly/somewhat harmful 72.3 56.5 72.2 61.1

Very harmful 23.1 31.3 22.2 33.1

“Have you ever noticed sores, white patches, or gum (n=65) (n=131) (n=18) (n=172)

problems where you hold the tobacco in your mouth?”

Affirmative responses 16.9 22.1 22.2 39.5

“Are you seriously thinking about quitting chewing/ (n=64) (n=129) (n=18) (n=172)

dipping tobacco in the next 12 months?”

Affirmative responses 34.4 58.1 33.3 60.2

“How many times have you seriously tried to quit?” (n=60) (n=111) (n=18) (n=157)

0.4 1.7 1.1 1.7

Score on quitting ladderb (n=47) (n=93) (n=10) (n=142)

4.7 5.8 4.0 5.7

a Results are based on all current-week users seen in year 2, with the exception of “score on quitting

ladder,” which is based on all current-week smokeless tobacco users seen in year 3.

b Measures readiness to quit using smokeless tobacco; values range from 0 (“no thought of quitting”) to

10 (“taking action to quit”).

et al., 1990; Colburn et al., 1989; Creath et al., 1988; Glover et al., 1986;

Jones and Moberg, 1988; Kegeles et al., 1989; Leopardi et al., 1989; Murray

et al., 1988). Only Native Americans have comparably high rates in the

United States (Bruerd, 1990; Hall and Dexter, 1988; Schinke et al., 1989).

However, it appears that about one-half of users in our study who reported

use within the past week were seasonal users, which means that the prevalence of year-round use may be closer to 18 or 19 percent in this group. On

the one hand, the ability to “take or leave” ST during the off-season might

be seen as an indication that many individuals can use ST without becoming addicted. On the other hand, year-round users differ from seasonal

users in ways that suggest ST is an addictive product for many individuals.

Year-round users use ST more hours per day, use more cans of snuff or

pouches of chew, have used ST more recently, and have higher serum

cotinine levels than seasonal users.

Year-round users in our study were also more likely to usually use snuff

than were seasonal users, which suggests that snuff may be more addictive

than chewing tobacco. When we compared current ST users who usually
Smoking and Tobacco Control Monograph No. 2

36

used snuff to those who usually used chewing tobacco, our findings were

similar to those reported in the comparison of year-round to seasonal users.

Snuff users used ST more hours per day than chewing tobacco users, had

used ST more recently, and had higher serum cotinine levels. Not surprisingly, they were also more likely to be year-round users. Among snuff users,

those who used ST year-round were more likely to use the Copenhagen

brand and less likely to use the Hawken brand than seasonal users. Thus,

snuff users, particularly users of the most popular brands, seem much less

able to restrict their ST use to the baseball season than chewing tobacco

users. These findings are interesting in light of our earlier report of a much

higher risk of leukoplakia in snuff users than in chewing tobacco users, and

the lower risk of leukoplakia in users of Hawken than in users of other snuff

brands (Grady et al., 1990).

Finally, snuff users seem to have a greater awareness of the health

hazards of ST use and to be more motivated to quit than chewing tobacco

users. Snuff users were more likely to think their ST use might be “very”

harmful to their health, were much more likely to indicate that they were

seriously thinking about quitting in the next 12 mo, and had higher scores

on readiness to quit. Compared with chewing tobacco users and seasonal

snuff users, year-round snuff users were also more likely to report having

noticed sores, white patches, or gum problems where they held tobacco in

the mouth. Given their own subjective experience, and our earlier findings

of a much greater risk of oral lesions for snuff users, the perception of

greater adverse health risks on the part of snuff users than of chewing

tobacco users may be well founded.

Our findings underscore the importance, for future studies, of distinguishing between snuff and chewing tobacco users and, where applicable,

between year-round and seasonal ST users. If only because intensity of use

appears to differ markedly by ST type and seasonal use, separate analyses

should be performed to distinguish health risks associated with these different types of use.

ACKNOWLEDGMENTS We gratefully acknowledge the support of the management, trainers,

players, coaches, and medical staff of the following professional baseball teams: California

Angels, Chicago Cubs, Cleveland Indians, Milwaukee Brewers, Oakland Athletics, San Francisco

Giants, and Seattle Mariners. We also thank Jana Murray, Patricia Lee, and Maureen Morris for

their invaluable efforts.

REFERENCES

Ary, D.V. Use of smokeless tobacco among male

adolescents: Concurrent and prospective relationships. National Cancer Institute Monographs 8: 49-

55, 1989.

Ary, D.V., Lichtenstein, E., Severson, H.H. Smokeless

tobacco use among male adolescents: Patterns,

correlates, predictors, and the use of other drugs.

Preventive Medicine 16: 385-401, 1987.

Bauman, K.E., Koch, G.G., Fisher, L.A., Bryan, E.S.

Use of smokeless tobacco by age, race, and gender

in ten standard metropolitan statistical areas of

the southeast United States. National Cancer

Institute Monographs 8: 35-37, 1989.

Boyd, G.D., et al. Use of smokeless tobacco among

children and adolescents in the United States.

Preventive Medicine 16: 402-421, 1987.
Chapter 1

37

Brownson, R.C., Dilorenzo, T.M., Van Tuinen, M., et

al. Patterns of cigarette and smokeless tobacco use

among children and adolescents. Preventive

Medicine 19: 170-180, 1990.

Bruerd, B. Smokeless tobacco use among Native

American school children. Public Health Reports

105: 196-201, 1990.

Colborn, J.W., Cummings, K.M., Michalek, A.M.

Correlates of adolescents’ use of smokeless

tobacco. Health Education Quarterly 16: 91-100,

1989.

Connolly, G.N., Orleans, C.T., Kogan, M. Use of

smokeless tobacco in major-league baseball. New

England Journal of Medicine 318: 1281-1284, 1988.

Creath, C.J., Bradley, D.H., Shelton, W.O., et al. The

prevalence of smokeless tobacco use among

adolescent male athletes. Journal of the American

Dental Association 116: 43-48, 1988.

Ernster, V.L., Grady, D.G., Greene, J.C., Walsh, M.,

Robertson, P.B., Daniels, T., Benowitz, N., Siegel,

D., Gerbert, B., Hauck, W. Smokeless tobacco use

and health effects among baseball players. Journal

of the American Medical Association 264: 218-224,

1990.

Glover, E.D., Johnson, R., Laflin, M., et al. Smokeless

tobacco use trends among college students in the

United States. World Smoking and Health 11: 4-9,

1986.

Grady, D., Greene, J., Daniels, T.E., Ernster, V.L.,

Robertson, P.B., Hande, W., Greenspan, D.,

Greenspan, J.S., Silverman, S. Oral mucosal lesions

found in smokeless tobacco users. Journal of the

American Dental Association 121: 117-123, 1990.

Hall, R.L., Dexter, D. Smokeless tobacco use and

attitudes toward smokeless tobacco among Native

Americans and other adolescents in the northwest.

American Journal of Public Health 78: 1586-1588,

1988.

Hosner, D.W., Lemeshow, S. Applied Logistic

Regression. New York: John Wiley and Sons, 1989.

Jones, R.B., Moberg, D.P. Correlates of smokeless

tobacco use in a male adolescent population.

American Journal of Public Health 78: 61-63, 1988.

Kegeles, S.S., Burleson, J.A., Miozza, J. Cigarette and

smokeless tobacco use among Connecticut

adolescents. American Journal of Public Health 79:

1413-1414, 1989.

Leopardi, E.A., Poulson, T.C., Neiger, B.L., et al. A

report of two smokeless tobacco surveys and

associated intervention strategies among Utah

adolescents. Journal of Cancer Education 4: 125-

134, 1989.

Marcus, A.C., Crane, L.A., Shopland, D.R., Lynn,

W.R. Use of smokeless tobacco in the United

States: Recent estimates from the Current

Population Survey. National Cancer Institute

Monographs 8: 17-23, 1989.

Murray, D.M., Roche, L.M., Goldman, A.I.,

Whitbeck, J. Smokeless tobacco use among ninth

graders in a north-central metropolitan population: Cross-sectional and prospective associations

with age, gender, race, family structure, and other

drug use. Preventive Medicine 17: 449-460, 1988.

Novotny, T.E., Pierce, J.P., Fiore, M.C., Davis, R.M.

Smokeless tobacco use in the United States: The

Adult Use of Tobacco Surveys. National Cancer

Institute Monographs 8: 25-28, 1989.

Rouse, B.A. Epidemiology of smokeless tobacco use:

A national study. National Cancer Institute

Monographs 8: 29-33, 1989.

Schinke, S.P., Schilling, R.F. II, Gilchrist, L.D.,

Ashby, M.R., Kitajima, E. Native youth and ST:

Prevalence rates, gender differences, and descriptive characteristics. National Cancer Institute

Monographs 8: 39-42, 1989.

Wisniewski, J.F., Bartolucci, A.A. Comparative

patterns of smokeless tobacco usage among major

league baseball personnel. Journal of Oral

Pathology and Medicine 18: 322-326, 1989.
Smoking and Tobacco Control Monograph No. 2

38
Chapter 2

39

Chapter 2

Clinical and Pathological Effects

CONTENTS Oral Mucosal Lesions: Clinical Findings in Relation to

Smokeless Tobacco Use Among U.S. Baseball Players

John C. Greene, Virginia L. Ernster, Deborah G. Grady,

Paul B. Robertson, Margaret M. Walsh, and Linda A. Stillman .......... 41

Introduction ................................................................................. 41

Methods........................................................................................ 42

Results........................................................................................... 43

Discussion..................................................................................... 48

References ..................................................................................... 49

Smokeless Tobacco Use in India:

Effects on Oral Mucosa

P.R. Murti, P.C. Gupta, R.B. Bhonsle, D.K. Daftary, F.S. Mehta,

and J.J. Pindborg .................................................................................. 51

Introduction ................................................................................. 51

Prevalence of ST Use..................................................................... 52

Effects of Betel Quid Chewing ..................................................... 52

Other ST Use and Effects .............................................................. 60

Discussion..................................................................................... 62

References ..................................................................................... 63

Smokeless Tobacco-Associated Epithelial

And Langerhans Cell Changes

Troy E. Daniels, Louis S. Hansen, John S. Greenspan, Laisheng Chou,

Deborah G. Grady, Walter W. Hauck, John C. Greene, and

Virginia L. Ernster ................................................................................ 66

Introduction ................................................................................. 66

Subjects and Methods .................................................................. 67

Epithelial Changes ....................................................................... 68

LC Changes .................................................................................. 68

Discussion..................................................................................... 69

References ..................................................................................... 72

The Use of Smokeless Tobacco in Sweden

Jan Bergström ...................................................................................... 74

Introduction ................................................................................. 74

Epidemiological Aspects ............................................................... 74

Effects on Oral Health .................................................................. 75

References ..................................................................................... 77
Smoking and Tobacco Control Monograph No. 2

40

Periodontal Effects Associated With the Use of

Smokeless Tobacco: Results After 1 Year

P.B. Robertson, V. Ernster, M. Walsh, J. Greene, D. Grady,

and W. Hauck ......................................................................................... 78

Introduction .................................................................................... 78

Materials and Methods.................................................................... 79

Results.............................................................................................. 80

Discussion........................................................................................ 84

References ........................................................................................ 86
Chapter 2

41

We have been conducting a 3-yr study of the health effects of smokeless tobacco use by

Oral Mucosal Lesions: Clinical Findings

In Relation to Smokeless Tobacco Use

Among U.S. Baseball Players1

John C. Greene, Virginia L. Ernster, Deborah G. Grady, Paul B. Robertson,

Margaret M. Walsh, and Linda A. Stillman

ABSTRACT

members of Major League and minor league baseball teams. Findings from the first year (1988),

involving 1,109 players, were previously reported. This report presents data about ST usage and

associated oral mucosal lesions among 894 additional players who first entered the study in

1989 (n=523) and 1990 (n=371). The 1989 and 1990 data were obtained by the same study

team, using the same study methods, as in 1988. Findings in the two sets of data are highly

consistent. There was a significant association between the prevalence of oral mucosal lesions

and the frequency, amount, and recency of ST use and the type and brand used. Players who

used ST year round had a much higher prevalence of lesions (67 percent) than those who used

it primarily during the baseball season (32 percent). Among those who used snuff year round,

72 percent had lesions, compared to 44 percent of seasonal users. Year-round snuff users who

used four or more cans per week had a lesion prevalence of 88 percent.

INTRODUCTION We previously reported findings from the first year (1988) of a 3-yr

study of the health effects of smokeless tobacco use among members of

Major League and minor league baseball teams (Ernster et al., 1990; Grady et

al., 1990 and 1991). Our primary purpose has been to study the association

of ST use with various oral health and other health measures: oral mucosal

lesions; gingival inflammation and recession; dental caries, erosion, attrition, and staining; pulse and blood pressure; and total and high-density

lipoproteins. The study was designed to detect the effects of the type and

brand of ST used and the amounts and length of use, controlling for potentially confounding variables such as age, alcohol use, cigarette smoking, and

oral hygiene practices. For efficiency, we chose an adult population known

to have a high prevalence of ST use—professional baseball players.

Ernster and coworkers (1990) described the overall study design and

provided an overview of findings involving 1,109 players examined during

the first year. Grady and colleagues (1990) presented a more extensive

analysis of the mucosal lesion findings from the same study as they relate to

a number of variables. Because the long-term use of ST has been strongly

associated with oral cancer in previous studies (IARC, 1985; US DHHS, 1986;

Winn et al., 1981), the oral mucosal lesions that are associated with ST use

are of special interest and concern.

In this paper we present new data on oral mucosal lesions from 894

players examined for the first time in the second and third years of the

study. We also examine the effects of seasonality of ST use on the prevalence and severity of oral mucosal lesions.

1 Supported by National Institute of Dental Research grant no. DE-08547-02.
Smoking and Tobacco Control Monograph No. 2

42

METHODS A detailed description of the study methods is presented by Ernster and

coworkers elsewhere in this volume. The study population consisted of

professional baseball players and other male team personnel from seven

Major League teams and their associated minor league teams who participated in spring training in and around Phoenix, Arizona. People who

agreed to take part in the study were asked to complete a questionnaire; a

study staff member was present to assist in filling out the questionnaire, if

needed. Players then were asked to rinse their mouths with water to remove

any traces of tobacco, and to undergo an oral examination. The participants

were instructed not to inform the examiners of their ST use status. The

examination consisted of an inspection of the oral mucosa and a standardized clinical examination of the periodontium and the dentition by trained

examiners calibrated for diagnostic uniformity. Unless otherwise indicated,

the present analysis is confined to players who first entered the study in

1989 (n=523) and 1990 (n=371).

Because self-reports of ST use were more than 90 percent accurate,

according to serum cotinine and thiocyanate verification in year 1, the

smokeless tobacco use status reported here is derived from questionnaire

data. On the basis of self-reports, the players were classified as never or

minor users, former users, current-month users, or current-week users.

Abnormalities of the oral mucosa were categorized and recorded as

(1) leukoplakia/erythroplakia—any white, opaque, or leathery-appearing

plaque not clinically characteristic of another type of white lesion, or any

red patch not clinically identifiable as another type of red lesion, or

(2) other mucosal abnormalities—i.e., interdental hyperkeratosis, retromolar

hyperkeratosis, other white changes, aphthous ulcer, other mucosal ulcers,

pigmented lesions, fibroma, or tongue changes. The leukoplakia/erythroplakia lesions were also classified into four degrees of severity: (1) no or

slight color change, with a change in texture; (2) color and texture change,

but no thickening; (3) color and texture change, with mild to moderate

thickening; (4) no normal color, severe texture change, and heavy thickening. These categories are similar to the ones developed by Greer and

Poulson (1983) from a system published by Axéll and coworkers (1976).

Because erythroplakia was not noted in the current study group, we refer to

the oral mucosal lesions either as leukoplakia or, simply, as lesions.

Study participants were asked to indicate the type and brand of ST they

usually used. They were also asked when they first started using ST and how

long they had been using it. Participants’ exposure to ST was recorded in

terms of cans per week for snuff, and pouches per week for chew. When a

player reported using both snuff and chew, but used one type usually and

the other only occasionally, the exposure was counted as the dominant type

for purposes of determining dose-response. We calculated hours per day

that either snuff or chew was kept in the mouth as a combined measure of

ST use. Recency of use was reported in hours since ST was last used. Because recency of use was not recorded in the third year of the study, multivariate logistic regression analysis of recency data was performed on secondyear data only.
Chapter 2

43

During the first year of the study, it became obvious that some players

used ST only during the regular baseball season, whereas others used it year

round. Therefore, seasonality of use was included in the questionnaire for

the second and third years, and the effects of this variable are reported here.

Seasonality analyses include all current-week ST users examined in year 2

(n=389), some of whom had been examined for the other variables in year 1

and are not otherwise included in this report, and all current-week users

examined in year 3 but not in year 2 (n=156).

RESULTS Baseline demographic and other characteristics (age, race, educational

level, cigarette smoking, alcohol consumption) of the 894 study participants

were very similar in percentages to those of the entire study population

from all 3 yr combined (see Ernster et al., this volume). Most of the study

subjects (76 percent) were between the ages of 20 and 29, nearly 70 percent

were white, and 77 percent had some college education. Only 2.0 percent

of subjects reported that they were current smokers, and only 4.7 percent

reported heavy alcohol consumption. Approximately two-thirds indicated

that they visited their dentist to have their teeth cleaned at least once every

12 mo.

As we reported earlier (Ernster et al., 1990; Grady et al., 1990), the

prevalence of ST use among professional baseball players is very high. Data

on ST use status were available for 879 players in this study. Nearly 37 percent of these players (323) reported using ST within the 7 d prior to the

examination and thus were classified as current-week users. Most of those

had used ST in the previous 24 h. Oral mucosal lesions were found in

51.7 percent of the current-week users, 3.5 percent of former users, and

2.9 percent of non-users (odds ratio = 36.0 for current-week users vs. nonusers). The association between severity of the most severe lesion and ST

use status is shown in Table 1. Most of the lesions (76.7 percent) were

degree 1 or 2 in severity. All of the degree 3 and degree 4 lesions were found

in current-week users.

The prevalence of lesions according to characteristics of ST use in

current-week users is shown in Table 2. Prevalence of lesions increased with

earlier first use, with longer duration of use, and with more recent use.

Lesion prevalence increased as the exposure to ST increased, whether measured by the amount used per week or by hours ST was held in the mouth

each day. Among snuff users, 40 percent of those who used one can or less

than two cans per week had lesions, whereas 84.7 percent of those who used

more than three cans per week had lesions. A similar trend was seen among

tobacco chewers: 8 percent of those who used up to one pouch per week

had lesions, while 27 percent of those who used more than three pouches

per week had lesions. When prevalence of lesions was analyzed according

to the number of hours of ST use per day, 30 percent of those using ST for

0.5 h/d or less had lesions, whereas 85 percent of those using ST for more

than 4.0 h/d had lesions.

The prevalence of lesions in current-week users, according to the type

and brand of ST used, is shown in Table 3. Most of the ST users usually used

snuff, and the snuff users had a much higher risk of developing oral lesions
Smoking and Tobacco Control Monograph No. 2

44

Table 1

Severity of most severe lesion, by ST use status

Degree of Severity of Lesionsa

1 2 34

n (%) n (%) n (%) n (%) Total

Use Status

Never/minor 10 (83.3) 2 (16.7) 0 (0.0) 0 (0.0) 12

Former 3 (75.0) 1 (25.0) 0 (0.0) 0 (0.0) 4

Current month 2 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 2

Current week 63 (37.7) 61 (36.5) 40 (24.0) 3 (1.8) 167

Total 78 64 40 3 185

a Lesions were graded from 1 to 4 by increasing degree of severity.

than chew users (61.2 percent vs. 14.8 percent). Of the players who used

Copenhagen brand snuff, 72 percent had a lesion at the time of the examination, compared with only 11 percent of the Hawken brand users.

Multivariate logistic regression analyses were based on year 2 data only,

because recency of use was not recorded in year 3. Variables included in the

analysis were duration, recency, and amount of use; age when ST use began;

type and brand of ST; and seasonality of use. Amount of use (hours per

day), recency of use, and use of Copenhagen and Skoal brands were independently and significantly associated with the presence of oral lesions

(Table 4). Lesion prevalence increased with increasing amounts of use (odds

ratio [OR]=1.46 per hour of use per day; p=0.009). Lesion prevalence decreased with time since last use of ST (OR=0.96 for each additional hour

since last use; p=0.0012). The odds of having a lesion were eightfold higher

for users of Copenhagen snuff than for users of chew (OR=8.05; p < 0.0001),

and more than sixfold higher for users of Skoal than for users of chew

(OR=6.78; p=0.0003). Duration of use and age when ST use was begun

showed no independent effect on lesion prevalence. The odds ratio for

lesion prevalence among year-round users compared to seasonal users was

elevated but was not statistically significant (OR=1.46; p=0.23).

Of the current-week users, 277 (51.2 percent) used ST only during the

baseball season, and 264 (48.8 percent) used it year round. Of the seasonal

users, about one-third (32.1 percent) had lesions, in contrast to two-thirds

(66.7 percent) of the year-round users. The distribution of seasonal and

year-round ST users with lesions according to various ST use characteristics

is shown in Table 5. The percentage of participants with lesions was greater

for year-round users than for seasonal users at virtually all levels of each use

characteristic.

Table 6 shows the prevalence of lesions in seasonal and year-round

current-week users, according to type and brand of ST usually used. Except

for Hawken brand snuff, for which there were only 5 year-round users,
Chapter 2

45

Table 2

Prevalence of leukoplakia, by ST use characteristics in current-week users

(n=323)

 na Percentage With Leukoplakia

Age at First Use

< 10 yr 13 69.2%

10-14 75 57.3

15-19 185 51.9

≥ 20 49 36.7

Duration of Use

≤ 3 yr 98 37.8

4-6 122 52.5

7-9 45 68.9

≥ 10 52 59.6

Time Since Last Usedb

> 24 h 26 11.5

> 12-24 36 22.2

> 1-12 37 51.4

≤ 1 57 79.0

Amount of Use

Snuffc

 (cans/wk)

≤ 1 95 40.0

> 1-3 89 65.2

> 3 72 84.7

Chewd (pouches/wk)

≤ 1 26 7.7

> 1-3 17 17.6

> 3 11 27.3

Hours ST in Mouth/Day

0.0-0.5 76 30.3

> 0.5-1.0 65 36.9

> 1.0-1.5 37 54.0

> 1.5-2.0 26 61.5

> 2.0-4.0 66 74.2

> 4.0 26 84.6

a Category totals differ because of missing values.

b Information not collected in year 3, so these values are for year 2 subjects only.

c Includes only subjects who usually used snuff.

d Includes only subjects who usually used chew.

prevalence was greater in the year-round user group for each type and brand

usually used. Nearly three-fourths (72 percent) of those who used snuff year

round had lesions, compared to 44 percent of seasonal snuff users. Players

who used chew were more likely to be seasonal users, while those who used

snuff were more likely to be year-round users.
Smoking and Tobacco Control Monograph No. 2

46

Table 3

Prevalence of leukoplakia, by type and brand of ST used, current-week users

(n=323)

na Percentage With Leukoplakia

Type of ST Usually Used

Snuff 258 61.2%

Chew 54 14.8

Brand of ST Usually Used

Snuff

Copenhagen 177 72.3

Skoal 54 42.6

Hawken 18 11.1

Chew

Red Man 21 14.3

Levi Garrett 24 16.7

a Category totals differ because of missing values.

Table 4

Association of various risk factors with the presence of oral lesions in current ST

usersa

Risk per: Odds Ratio p

Duration Year of 1.00 0.91

ST use

Amount Hours/day 1.46 0.0093

of ST use

Initiation Year of age 1.03 0.49

ST use begun

Recency Hours since 0.96 0.0012

ST last used

Type/Brand

Chew (referent) 1.00 —

Copenhagen 8.05 < 0.0001

Skoal 6.78 0.0003

Hawken 3.24 0.16

Seasonality

Seasonal (referent) 1.00 —

Year round 1.46 0.23

a This multivariate regression analysis includes all current-week users seen in year 2 for

whom data on all variables were available; n=290.
Chapter 2

47

Table 5

Prevalence of leukoplakia in current-week ST users (n=545),a

 by seasonality of

use and use characteristics

Seasonal Year-Round

Current-Week ST Users Current-Week ST Users

Percentage With Percentage With

nb Leukoplakia nb Leukoplakia

All Current-Week

ST Usersc 277 32.1% 264 66.7%

Age at First Use

< 10 yr 3 66.7 12 75.0

10-14 34 20.6 72 65.3

15-19 174 32.8 137 67.2

≥ 20 65 33.8 43 65.1

Duration of Use

≤ 3 yr 104 36.5 46 54.4

4-6 105 31.4 102 64.7

7-9 28 28.6 47 76.6

≥ 10 34 26.5 64 70.3

Time Since Last Usedd

> 24 h 46 10.9 14 21.4

> 12-24 47 27.8 33 30.3

> 1-12 36 34.0 46 71.7

≤ 1 47 51.1 64 87.5

Hours ST in Mouth/Day

0.0-0.5 111 26.1 42 40.5

> 0.5-1.0 70 35.7 48 56.2

> 1.0-1.5 25 40.0 41 65.8

> 1.5-2.0 15 33.3 26 76.9

> 2.0-4.0 21 52.4 64 79.7

> 4.0 5 20.0 28 82.1

Amount of Use

Snuffe

 (cans/wk)

≤ 1 112 33.9 55 52.7

> 1-3 55 61.8 91 70.3

> 3 13 61.5 88 87.5

Chewf

 (pouches/wk)

≤ 1 47 4.3 8 0.0

> 1-3 26 11.5 6 50.0

> 3 9 22.2 8 25.0

a Includes some players examined in year 1 who are not otherwise included in this report.

b Category totals differ because of missing values.

c Seasonality data were not available for four current-week users.

d Information not collected in year 3, so these values are for year 2 subjects only.

e Includes only persons who usually used snuff.

f

 Includes only persons who usually used chew.
Smoking and Tobacco Control Monograph No. 2

48

Table 6

Prevalence of leukoplakia in current-week users (n=545), by seasonality of use

and by type and brand of ST used

Seasonal Year-Round

Current-Week ST Users Current-Week ST Users

Percentage With Percentage With

na Leukoplakia na Leukoplakia

Type of ST Usually Used

Snuff 184 44.0% 236 72.0%

Chew 82 8.5 22 22.7

Brand of ST Usually Used

Snuff

Copenhagen 117 52.1 179 76.5

Skoal 39 33.3 46 63.0

Hawken 23 17.4 5 0.0

Chew

Red Man 24 12.5 6 16.7

Levi Garrett 42 4.8 10 30.0

Other 16 12.5 6 16.7

a Category totals differ because of missing values.

Univariate analysis showed that lesion prevalence was significantly

increased among year-round ST users (OR=4.17; p < 0.0001), compared to

seasonal users. However, as shown in Table 4, seasonality showed no

statistically significant independent effect on lesion prevalence.

Finally, lesions found in year-round ST users were more likely to be

severe (29.6 percent were degree 3 or 4 leukoplakia) than those in seasonal

users (6.7 percent degree 3 or 4 leukoplakia).

DISCUSSION Questionnaire and clinical examination data from 894 new participants

in the second and third years of this 3-yr study of professional baseball

players yielded findings that were highly consistent with the previously

published results from the first year (Ernster et al., 1990; Grady et al., 1990).

The prevalence of current-week ST use in this new group of players was

36.7 percent, and oral lesions were found in 51.7 percent of current-week

users, yielding an odds ratio of 36.0 for users compared with non-users. The

prevalence of oral lesions increased with the amount used (whether expressed in hours per day or in cans or pouches per week), with recency of

last use, with use of snuff instead of chew, and with use of certain brands of

snuff. All of these trends are very consistent with and confirmatory of our

earlier reports on first-year findings from our study of 1,109 different

players.

That ST users are at increased risk of oral lesions has been shown by

other investigators (US DHHS, 1986). However, no other group has had as

large a sample of adults or examined the effects of so many variables related

to patterns of use.
Chapter 2

49

Lesion prevalence associated with the use of Copenhagen and Skoal

brands of snuff continued to be several times greater than prevalence with

the use of chew, as we have noted previously. Our previously published

first-year results were the first to show an increased risk of oral lesions

associated with recency of use and with type and brand used. Other investigators have previously reported an increased risk of oral lesions with increasing amounts of ST used (Greer and Poulson, 1983; Hirsch et al., 1982;

Poulson et al., 1984; Wolfe and Carlos, 1987).

Information on the prevalence and severity of oral mucosal lesions

related to seasonality of ST use has not previously been reported. The data

gathered on seasonality of use in years 2 and 3 of this study show that about

half of the current ST users in this population are year-round users, while

the other half use ST only during the baseball season. Year-round users were

much more likely to have lesions (66.7 percent) than were seasonal users

(32.1 percent). It might be expected that, because the oral exams were made

at the beginning of the season, seasonal users would naturally be less likely

to have developed a white keratotic mucosal response. However, when the

effects of other variables are taken into account, seasonality was not a

statistically significant predictor of lesion prevalence in the multivariate

model shown in Table 4; in that model the ST use variables of amount,

recency, and type of ST were shown to have statistically significant independent effects. These findings are not unexpected, because there is no real

reason to believe that seasonality should have an effect on lesion prevalence

apart from the amount and type of ST used. It is interesting to note that of

those who reported using ST year round, 91.5 percent used snuff, while of

those who used ST only during the baseball season, 69.2 percent used snuff.

Furthermore, of the year-round snuff users, 77.9 percent used Copenhagen,

whereas 65.4 percent of the seasonal snuff users did so.

Thus, the greater prevalence of lesions among year-round users compared to seasonal users appears to result largely from increased and recent

exposure to certain types and brands of ST. In keeping with this, the lesions

found in year-round users tended to be more severe. From other evidence

gathered in this study (Ernster et al., this volume), it appears that year-round

users are more likely to have difficulty quitting and to be addicted. For

these reasons, year-round ST users should be preferentially targeted for

documenting long-term health effects of such use and for cessation

intervention.

REFERENCES

Axéll, T., Mörnstad, H., Sundström, B. The relation

of the clinical picture to the histopathology of

snuff dipper’s lesions in a Swedish population.

Journal of Oral Pathology 5: 229-236, 1976.

Ernster, V.L., Grady, D.G., Greene, J.C., Walsh, M.,

Robertson, P.B., Daniels, T., Benowitz, N., Siegel,

D., Gerbert, B., Hauck, W. Smokeless tobacco use

and health effects among baseball players. Journal

of the American Medical Association 264: 218-224,

1990.

Grady, D.G., Ernster, V.L., Stillman, L., Greene, J.C.,

Daniels, T.E., Silverman, S. A surprise with

smokeless tobacco oral lesions. Journal of the

American Dental Association 122: 62-64, 1991.

Grady, D.G., Greene, J.C., Daniels, T.E., Ernster, V.L.,

Robertson, P.B., Hauck, W., Greenspan, D.,

Greenspan, J.S., Silverman, S. Oral mucosal

lesions found in smokeless tobacco users. Journal

of the American Dental Association 121: 117-123,

1990.
Smoking and Tobacco Control Monograph No. 2

50

Greer, R.O., Jr., Poulson, T.C. Oral tissue alterations

associated with the use of smokeless tobacco by

teenagers. Oral Surgery, Oral Medicine, Oral

Pathology 56: 275-284, 1983.

Hirsch, J.M., Neyden, G., Thilander, H. A clinical,

histomorphological and histochemical study on

snuff-induced lesions of varying severity. Journal

of Oral Pathology 11: 387-398, 1982.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosamines (volume 37). Lyon:

IARC, 1985, pp. 37-135.

Poulson, T.C., Lindenmuth, J.E., Greer, R.O., Jr. A

comparison of the use of smokeless tobacco in

rural and urban teenagers. CA—A Cancer Journal

for Clinicians 34: 248-261, 1984.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication (NIH) No. 86-2874, 1986.

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W.,

Toledo, A., Fraumeni, J.F., Jr. Snuff dipping and

oral cancer among women in the southern United

States. New England Journal of Medicine 304: 745-

749, 1981.

Wolfe, M.D., Carlos, J.P. Oral health effects of

smokeless tobacco use in Navajo Indian adolescents. Community Dentistry and Oral Epidemiology

15: 230-235, 1987.
Chapter 2

51

Smokeless tobacco is used in diverse forms in India for chewing, sucking in the mouth until

Smokeless Tobacco Use in India:

Effects on Oral Mucosa1

P.R. Murti, P.C. Gupta, R.B. Bhonsle, D.K. Daftary,

F.S. Mehta, and J.J. Pindborg

ABSTRACT

it becomes bland, or applying over the teeth and gums. Tobacco is chewed most commonly in

betel quid. ST use results in oral cancer, precancer, and other oral lesions. Tobacco smoking

and other factors such as nutrition and viruses like HPV and HSV may modify the effect of ST

use. There is a need to study the exact role of other factors in the causation of oral cancer.

Long-term studies have demonstrated that in most instances oral cancer arises from precancerous lesions or conditions. The methods currently available to identify cancer potential in

precancer, however, have limited usefulness. Therefore, research should be extended in that

direction.

INTRODUCTION Tobacco was introduced to India by Portuguese traders about 1600.

Although tobacco was initially smoked, it later was used in smokeless form

as well. Of the 400 million individuals aged 15 and over in India, 47 percent use tobacco and 16 percent use tobacco in smokeless forms; of the

250 million kg of tobacco consumed each year, 86 percent is used for

smoking, 13 percent in smokeless forms other than snuff, and 1 percent as

snuff (Sanghvi, 1989).

Smokeless tobacco is used in diverse forms in different regions of India

for chewing, holding in the mouth, or applying over teeth and gums. ST is

chewed, more often in betel quid (pan), consisting of betel leaves (Piper

betle), areca nut (Areca catechu), slaked lime, and catechu (Acacia catechu);

with areca nut and slaked lime (e.g., mainpuri tobacco, mawa, pan masala);

and less commonly by itself. A mixture of tobacco and slaked lime (khaini)

is kept in the mouth and sucked. Other products like roasted and powdered

tobacco (mishri), dry snuff (bajjar), or tobacco paste with molasses (gudhaku)

are applied over teeth and gums. Creamy snuff is used initially as a dentifrice but soon turns into an addiction.

Betel quid chewing is widespread all over India, whereas most other

uses are popular in specific geographic regions. A link between betel quid

chewing and oral cancer was suspected as early as 1908 (Bentall, 1908; Fells,

1908), and by the late 1960’s, several studies had demonstrated the association between betel quid chewing and other forms of tobacco use with oral

cancer in India (Orr, 1933; Sanghvi et al., 1955; Shanta and Krishnamurthy,

1959; Wahi, 1968).

Several oral lesions are associated with ST use, of which oral cancer and

precancer are the most serious. Since 1966 we have conducted several crosssectional and prospective epidemiological studies on oral cancer and

1 Supported solely by funds from the National Institutes of Health, through Indo-U.S. Fund

Research Agreement no. 01-022-N.
Smoking and Tobacco Control Monograph No. 2

52

precancer (Bhonsle et al., 1976; Daftary et al., 1980; Gupta et al., 1980;

Mehta et al., 1971 and 1972) in different geographic areas chosen for specific tobacco habits. In this paper, we review oral mucosal lesions associated

with some common forms of ST use in India.

This paper discusses oral cancer and precancerous lesions and conditions associated with chewing betel quid with tobacco in Ernakulam district

in Kerala State. The effects of chewing mainpuri tobacco, mawa, the use of

tobacco-plus-lime mixture, and other habits practiced elsewhere in India are

also described briefly.

PREVALENCE

OF ST USE

There are no nationwide surveys on the prevalence of tobacco use in

India. Table 1 gives the prevalence of ST use reported among 255,194

individuals in 9 areas in some major studies: The reported prevalence

ranges from 11 to 49 percent. Except in Singhbhum, Pune, and Mainpuri

districts, chewing betel quid with tobacco was widespread. Chewing tobacco without any other ingredient was practiced in two areas by 3 and

7 percent of the individuals. However, there are differences in the selection

of samples, methodology, and the age groups of the individuals in these

studies.

EFFECTS OF

BETEL QUID

CHEWING

Chewing of betel quid (betel leaf, areca nut, slaked lime, sweeteners,

and flavoring) is an Indian habit dating back more than 2,000 years.

Sometime after its introduction, tobacco became an important constituent of betel quid, and currently most habitual users chew betel quid

with tobacco (Figure 1). Various tobacco preparations (e.g., zarda or kiwam)

that can be used in the betel quid are commercially available. Regional

differences in the choice of material and the way in which betel quid is

chewed abound.

Betel quid chewing inevitably stains the mucosa bright red as a result of

the formation of o-quinone from water-soluble polyphenols, notably

leucocynidins, at an alkaline pH of 8 to 9 via secondary reactions

(Jayalakshmi and Mathew, 1980). These stains can be washed clean or will

disappear if the habit is discontinued. It is not uncommon, however, to

find habitual chewers with perpetually stained mucosa.

Pan Chewer’s

Lesion

Among heavily addicted betel quid chewers, a thick, brownish-black

encrusted lesion was observed on the buccal mucosa and the mandibular groove corresponding to the site of the quid placement. This could

be scraped off with a piece of gauze. The annual age-adjusted incidence rate

of this lesion was 28.0 per 1,000 male betel quid chewers and 17.4 per 1,000

among females (Gupta et al., 1980).

These lesions show a pale-staining, parakeratin-like surface layer of

epithelium containing round nuclear remnants, ballooning vacuolated cells,

and consistent epithelial hyperplasia. Pan chewer’s lesion is a thick encrustation, but it disappears when the habit is discontinued. The lesion does

not seem to exhibit any cancer potential. Of the 532 lesions observed over a

3-yr period, 26 percent remained persistent, 45 percent regressed spontaneously, and 29 percent recurred (Gupta et al., 1980).
Chapter 2

53

Table 1

Prevalence of some ST habits in India, by area

Predominant

n Prevalence Habita

Ernakulam 10,287 37% Betel quid

Srikakulam 10,169 11 Betel quid

Singhbhum 10,048 28 Tobacco-plus-lime

Darbhanga 10,340 16 Betel quid

Bhavnagar 10,071 13 Betel quid, dry snuff

Pune 101,761 49 Tobacco-plus-lime, mishri

Mainpuri 35,000 30b Mainpuri tobacco, pattiwala

Lucknow 10,000 13c Betel quid

Ahmedabad 57,718 47d Betel quid

a Many smoked as well.

b 7 percent mainpuri tobacco. c 3 percent tobacco alone.

d 7 percent tobacco alone.

Source: Mehta et al., 1971 and 1972; Wahi, 1968; Pindborg et al., 1972; Smith et al.,

1975.

Epithelial

Dysplasia

Epithelial dysplasia is generally assumed to indicate the malignant

potential of a lesion. Dysplastic lesions exhibited malignant transformation rates that were 15 times higher than nondysplastic lesions (Gupta

et al., 1980). In a 10-yr followup study of 16 dysplastic lesions in betel

quid chewers, 8 dysplastic lesions in those who chewed and smoked, and

18 dysplastic lesions in smokers only, 19 percent, 25 percent, and 6 percent,

respectively, progressed to cancer, and 25 percent, 50 percent, and 22 percent, respectively, regressed. The remaining dysplastic lesions either remained stationary or changed their clinical characteristics.

Leukoplakia Leukoplakia is the most common precancerous lesion, initially hypothesized as precancerous mainly because of its coexistence with cancer. In a

study of 650 oral cancers in India, 32 percent had coexistent leukoplakia

(Paymaster, 1956). Furthermore, leukoplakias and oral cancers share the

same etiologic agent, tobacco, and they occur at strikingly similar locations.

For example, oral cancer and leukoplakias among betel quid chewers generally occur on the buccal mucosa. The most important evidence of

leukoplakia’s precancerous nature, however, is its excess risk for cancer as

demonstrated in prospective studies (see below).

Epidemiology The prevalence of leukoplakia in Ernakulam district was 17 per 1,000.

Prevalence was 18 per 1,000 among betel quid chewers and 61 per 1,000

among those who chewed and smoked (Mehta et al., 1969). The annual

age-adjusted incidence rate was 2.1 per 1,000 person-years among men and

1.3 per 1,000 among women; among betel quid chewers, the incidence was

2.5 per 1,000 among men and 3.0 per 1,000 among women. The incidence

rate was highest (6.0) in those who smoked and chewed (Gupta et al., 1980).
Smoking and Tobacco Control Monograph No. 2

54

Figure 1

Constituents of betel quid in Kerala: (a) betel leaf; (b) shell lime; (c) raw areca

nut; (d) tobacco. Note that catechu is not used in Kerala.

Clinical Aspects Leukoplakia is a raised white patch on the oral mucosa that measures

5 mm or more and that cannot be scraped off and cannot be attributed to

any other diagnosable disease (Mehta et al., 1969). Leukoplakias are classified as homogeneous, ulcerated, or nodular types. Homogeneous leukoplakia is characterized by raised plaque formation consisting of plaque

(Figure 2, top) or groups of plaques varying in size and with irregular edges.

Ulcerated leukoplakia consists of an area of ulceration that is sometimes

surrounded by keratinized areas, pigmentation, or both (Figure 2, middle)

and generally occurs among smokers of bidi (a kind of cheap smoking stick).

Nodular leukoplakia consists of many small white nodules on an erythematous base (Figure 2, bottom). About 94 percent of the leukoplakias among

betel quid chewers are the homogeneous type. The risk for cancer varies

with different clinical types of leukoplakia (see below).

In Ernakulam district, leukoplakias occurred more often among men,

with the male-to-female ratio being 4:1 (Mehta et al., 1971). The average

age of individuals with this lesion was 47.8 yr. Generally the location of

leukoplakias varies according to where tobacco is held in the mouth. For

example, those associated with betel quid chewing occurred more often on

the posterior part of the buccal mucosa and the mandibular groove, whereas

leukoplakias associated with tobacco-plus-lime and mishri occur in the

premolar region of the buccal mucosa and the labial mucosa.

Histology Leukoplakias associated with betel quid chewing showed hyperorthokeratosis in 82 percent and hyperparakeratosis in 12 percent. Hyperparakeratosis was seen somewhat more frequently in those who chewed betel

quid and smoked (23 percent) or who smoked only (24 percent) (Mehta et

al., 1969). Epithelial dysplasia was observed in 15 percent of the betel quidassociated leukoplakias compared with 5 and 8 percent in those associated

with the combined habit of chewing and smoking or smoking only. In

regard to epithelial thickness, atrophy was observed in 75 percent of the
Chapter 2

55

Figure 2

Homogeneous leukoplakia in a betel quid chewer (top); ulcerated leukoplakia in

a bidi smoker (middle); nodular leukoplakia in a betel quid chewer (bottom).

betel quid-associated leukoplakias compared with 40 and 31 percent, respectively, in those associated with the combined habit or with smoking only.

Orthokeratinization occurred generally in combination with epithelial

atrophy, whereas parakeratinization was more commonly associated with

hyperplastic epithelium.
Smoking and Tobacco Control Monograph No. 2

56

Natural History In a 10-yr followup study of 94 betel quid-associated leukoplakias,

7 percent progressed to oral cancer, in contrast to only 4 percent of the

85 leukoplakias associated with combined betel quid chewing and smoking

and none of the 45 leukoplakias associated with smoking only (Gupta et al.,

1980). The higher rate of malignant transformation in betel quid-associated

leukoplakias may be caused by epithelial atrophy. The malignant transformation rate was highest in nodular leukoplakias (21.0 percent) and in

homogeneous leukoplakias (1.7 percent) (Gupta et al., 1980). Leukoplakias

associated with betel quid chewing were less persistent (25 percent), regressed more often (57 percent), and showed higher recurrence rates than

did smoking-associated leukoplakias. In another 8-yr followup study, the

relative risk (RR) for the development of oral cancer among all oral precancerous lesions and conditions was highest (RR=3,243.2) for nodular leukoplakia (Gupta et al., 1989); the RR for homogeneous (RR=25.6) and ulcerated

(RR=43.6) leukoplakia were also high and significant.

Oral Cancer Oral cancer is common in India. According to weighted averages from

six cancer registries in India, oral cancer is the second and third most

common cancer among men and women, respectively (Indian Council of

Medical Research, 1989). Contributing to the problem of high morbidity,

patients with this disease often seek medical attention in a late clinical

stage, consequently having a poor prognosis.

Epidemiology In a cross-sectional study among 10,287 villagers in Ernakulam district,

all 12 oral cancers occurred among those who chewed betel quid; 6 of these

patients smoked as well (Mehta et al., 1969). In a 10-yr followup study of

the above sample, the overall age-adjusted incidence was 16 per 100,000

person-years, 23 per 100,000 among betel quid chewers, and 32 per 100,000

in those who chewed and smoked (Gupta et al., 1980). Several extensive

independent assessments have confirmed the causal role of betel quid

chewing and oral cancer (IARC, 1985; National Institutes of Health, 1986;

US DHHS, 1986; WHO, 1984). About 30 percent of the oral cancers in this

part of the world are attributable to betel quid chewing and an additional

50 percent to the combined habit of chewing and smoking (WHO, 1984).

In India, most of the habitual betel quid chewers include tobacco. Betel

quid, however, may be chewed without tobacco. As several investigators in

the past were not explicit about this, there was some confusion about the

carcinogenicity of betel quid with and without tobacco. The question has

been extensively reviewed, and the conclusions are that the relative risk for

oral cancer in those who chewed betel quid without tobacco was insignificant or significantly lower than for those who chewed betel quid with

tobacco (Gupta et al., 1982). Overall, there is inadequate evidence that the

habit of chewing betel quid without tobacco is carcinogenic for humans

(IARC, 1985).

Clinical Aspects Oral cancer occurs more frequently among men than among women.

In a sample of 2,007 oral cancers seen at the main cancer treatment center

in Kerala, the male-to-female ratio was 1.8:1 (Nair et al., 1988); the mean age

for men was 57.1 yr and for women, 58.6. The bucccal mucosa is the most

commonly involved location (Figure 3), and in a sample of 2,007 oral
Chapter 2

57

Figure 3

Buccal carcinoma in a betel quid chewer

cancers, 50 percent were located in the buccal mucosa and 24 percent on

the tongue. The frequency of buccal mucosal involvement among men was

slightly higher (53 percent) than that among women (45 percent). This may

be because men smoke and chew, whereas women generally chew betel quid

only. The mean ages of patients with cancer of the buccal mucosa and the

tongue were quite similar, 56.9 and 55.8 yr, respectively, among men, and

58.5 and 56.5 yr among women.

Squamous cell carcinoma is the most common cancer, accounting for

95 to 98 percent of all oral malignancies in India. In Kerala, 7 percent of the

2,007 oral cancers reported were verrucous carcinomas (Nair et al., 1988).

This is a variant of squamous cell carcinoma that is broad based and locally

invasive; generally it does not metastasize.

Natural History In most instances oral cancer arises from precancerous lesions or

conditions. In a 10-yr followup study in Ernakulam district, all 12 oral

cancers arose from a precancerous condition (Gupta et al., 1980), whereas in

another 8-yr followup study, 15 of the 19 oral cancers developed from

precancer (RR=69.2) (Gupta et al., 1989).

Unfortunately, patients in India seek medical attention in the later

stages of disease. Of the 2,007 oral cancers described above, only 12 percent

were localized lesions and most of the remaining ones were extensive (Nair

et al., 1988).

Intervention Oral cancer is amenable to primary and secondary prevention. In

Ernakulam district, after 5 yr of educational intervention among 12,000

tobacco users, 10 percent of the men and 15 percent of the women discontinued their betel quid chewing, and 26 percent of men and 31 percent of

women reduced betel quid use (Gupta et al., 1986). This resulted in a

significant drop in the incidence of leukoplakia among betel quid chewers.

The rate ratios that indicate the protective effect of educational intervention

were 0.51 for men and 0.19 for women. After 8 yr the cessation rate
Smoking and Tobacco Control Monograph No. 2

58

increased to 13 percent among men and 18 percent among women, and the

rate of reduction increased to 35 percent among men and 39 percent among

women (Gupta et al., 1990). Correspondingly, the number of observed

leukoplakias among betel quid chewers was 27.0 among men vs. 79.7 predicted, and 49.0 among women vs. 163.9 predicted. A further increase in

the rates of cessation and reduction in tobacco use and a decrease in the

incidence of leukoplakia also were observed after 10 yr of intervention.

Submucous

Fibrosis

Submucous fibrosis is a chronic condition marked by mucosal rigidity of

various intensity caused by a fibroelastic transformation of the

juxtaepithelial layer that results in progressive inability to open the mouth

(Figure 4). When the tongue is involved, it is shrunken, and its mobility

may be restricted. Occasional pharyngeal and esophageal involvement also

has been observed. Diagnosis of the condition is based on the presence of

palpable fibrous bands. Submucous fibrosis occurs predominantly among

Indians at home and abroad and, to a lesser extent, other Asiatics. Areca nut

chewing in any form is currently believed to be involved in the pathogenesis of submucous fibrosis (Bhonsle et al., 1987; Mehta et al., 1972; Sinor et

al., 1990). Because areca nut, like tobacco, is an ingredient in betel quid,

and tobacco is known to contain carcinogens, this condition is discussed

here. Furthermore, submucous fibrosis is a high-risk precancerous condition

(Gupta et al., 1989) in which the malignant potential is the result of epithelial atrophy and the action of carcinogens (Pindborg et al., 1984).

Epidemiology The prevalence and the incidence rates of submucous fibrosis are high

in Ernakulam district relative to rates in other areas of India. The overall

prevalence was 351 per 100,000, and prevalence was highest among betel

quid chewers (1,090 per 100,000) (Pindborg et al., 1968). This condition

was not seen among people who do not chew betel quid. The overall ageadjusted incidence rate was 7 per 100,000 person-years among men and

17 per 100,000 among women; all new cases of submucous fibrosis developed among betel quid chewers (Gupta et al., 1980). In comparison to the

incidence rates, the prevalence rates seem too large (prevalence = incidence

x duration).

Submucous fibrosis affects both sexes, but a definite female predominance was observed in Ernakulam district (Gupta et al., 1980; Pindborg et

al., 1968). Such predominance, however, seemed to depend on the type and

extent of areca nut chewing habits among men and women in different

areas (Bhonsle et al., 1987). Although there is some regional variation, this

condition generally occurs between the ages of 20 and 40 yr. The mean age

of patients in Ernakulam district (51 yr) was higher than the mean age of

patients in Pune district (37 yr) (Bhonsle et al., 1987).

Clinical Aspects Submucous fibrosis commonly affects the buccal mucosa, retromolar

areas, and the soft palate. The frequency of their involvement, however,

varies from one geographic area to another, probably depending on the

variations in the method of areca nut chewing (Bhonsle et al., 1987). The

earliest and most common sign of this condition is blanching of the oral

mucosa that imparts a marblelike appearance (Pindborg et al., 1980). When

the disease is fully developed, palpable fibrous bands develop in the buccal
Chapter 2

59

Figure 4

Limited oral opening in a patient with submucous fibrosis. Note the shrunken

appearance of the tongue, the absence of lingual papillae, and associated leukoplakia (L).

mucosa, soft palate, and the rima oris. The bands run vertically in the

buccal mucosa and are circular around the rima oris. As the disease

progresses, the mucosa becomes stiff and the oral opening may be restricted.

Petechial spots resulting from the breakdown of connective tissue support

to the vasculature were observed in 11 percent of patients, according to

Bhonsle and coworkers (1981). Furthermore, submucous fibrosis is often

associated with leukoplakia (Figure 4), oral cancer, and pigmentation

changes (Pindborg et al., 1968 and 1984). Most of the patients complained

of a burning sensation, often aggravated by spicy food; another common

complaint is excessive or decreased salivation.

Histology Epithelial atrophy, juxtaepithelial hyalinization, and varying density of

collagen are the common features in submucous fibrosis. A notable feature

is the presence of epithelial dysplasia in 26 percent of the cases (Pindborg et

al., 1984).

Natural History

regress, either spontaneously or with cessation of betel quid chewing. The

most serious aspect of this condition is its malignant potential. In a 17-yr

followup study of 66 patients with submucous fibrosis, malignant transformation was observed in 0.4 percent at the end of 10 yr (Gupta et al., 1980),

which increased to 4.5 percent at the end of 15 yr (Pindborg et al., 1984)

and to 7.6 percent at the end of 17 yr (Murti et al., 1985). All five individuals in whom cancer developed were women who chewed betel quid with

tobacco. In another 8-yr followup study of 12,000 tobacco users, the RR of

malignant transformation for submucous fibrosis, compared with that for

tobacco users without any oral mucosal lesion or condition, was 397.3

(Gupta et al., 1989). There is no effective cure for submucous fibrosis;

however, the condition appears to be amenable to primary prevention

(Murti et al., 1990).

Unlike other precancerous lesions, submucous fibrosis does not
Smoking and Tobacco Control Monograph No. 2

60

Oral Lichen Planus Lichen planus is primarily a dermatologic disorder. Mucosal

surfaces may be affected along with cutaneous lesions; the mucosal lesions

may occur alone; or one may precede the other. The oral mucosa is more

commonly affected, and oral lesions are suspected of having some cancer

potential. In Ernakulam district, oral lichen planus was found to be strongly

associated with betel quid chewing (Bhonsle et al., 1979; Gupta et al., 1980;

Pindborg et al., 1972). This paper describes oral lichen planus as a result of

betel quid use and its malignant potential.

Epidemiology Oral lichen planus was diagnosed on the basis of presence of

Wickham’s striae. The overall prevalence of oral lichen planus was 1.5 percent; it was highest (3.2 percent) among betel quid chewers, and lowest

(0.3 percent) in non-users of tobacco (Pindborg et al., 1972). The annual

age-adjusted incidence rate per 1,000 person-years was 2.1 among men and

2.5 in women who chewed betel quid vs. 0.6 among men and 0.9 in women

who were not users of tobacco (Gupta et al., 1980). The RR for oral lichen

planus among betel quid chewers was 6.2 for men and 4.9 for women

(Bhonsle et al., 1979). Oral lesions thus showed a strong association with

betel quid chewing, although tobacco use is not regarded as an etiologic

factor for oral lichen planus.

Clinical Aspects Oral lichen planus occurred predominantly among women. The

buccal mucosa was the most favored location; the lesions occurred in

diverse morphological forms such as reticular, annular, linear, erosive or

ulcerated, and pigmented forms. Of these, 20 percent were erosive or

ulcerated lesions (Pindborg et al., 1972).

Natural History The malignant potential of oral lichen planus over a 10-yr period

(mean, 5.1 yr) was assessed in 722 individuals with the condition. Oral

cancer developed in three patients (0.4 percent) who had erosive (atrophic)

lesions; two of them were betel quid chewers and the third was a smoker

(Murti et al., 1986). In another 8-yr followup study of 344 individuals with

oral lichen planus, the RR for malignant transformation was 15.8, which

was not significant (p > 0.05) (Gupta et al., 1989). Oral lichen planus often

regressed and sometimes recurred. The regression rates were highest in nonusers of tobacco and lowest in those with the combined habits of chewing

and smoking (Gupta et al., 1980). These observations support the hypothesis that tobacco plays some role in this disease.

OTHER ST USE

AND EFFECTS

Tobacco Chewing

Chewing tobacco without any other ingredient does not seem to

be common in India. In two cross-sectional studies, the prevalence of leukoplakia among tobacco chewers was 7.3 percent in

Lucknow (Pindborg et al., 1972) and 12.7 percent in Ahmedabad

(Smith et al., 1975).

Mainpuri Tobacco Mainpuri tobacco contains tobacco, slaked lime, finely cut areca

nut, cloves, camphor, and other flavoring agents. This product is widely

used in Mainpuri district in Uttar Pradesh and nearby areas, where it is

known by different names, and is strongly associated with leukoplakia and

oral cancer.
Chapter 2

61

Leukoplakia The overall prevalence of leukoplakia in Mainpuri district was 5,160 per

100,000; it was 26,740 per 100,000 in mainpuri tobacco users and 15,160

among those who used pattiwalla tobacco (sun-cured tobacco chewed with

or without slaked lime and without areca nut), which is another popular

brand of ST used in that region. Among those who used both types of

tobacco, the prevalence was 80,000 per 100,000 (Wahi et al., 1970).

Oral Cancer The prevalence of oral cancer in Mainpuri district was 99 per 100,000.

Prevalence was 781 per 100,000 among mainpuri tobacco users, 117 per

100,000 in pattiwalla tobacco users, and 413 per 100,000 in those who used

both forms. Prevalence was 36 per 100,000 in non-users of tobacco (Wahi,

1968).

Mawa Chewing Mawa consists of areca nut shavings sprinkled with watery slaked

lime. A little sun-cured tobacco is added, and the product is packed in a

cellophane paper and sold. In recent years mawa chewing has gained

considerable popularity in Bhavnagar district and adjoining areas in Gujarat.

Mawa chewing is strongly associated with oral submucous fibrosis

(Sinor et al., 1990). For example, the RR for submucous fibrosis for all forms

of areca nut chewing was 109.6, 106.4 for mawa chewing, and 780.0 for

chewing mawa and betel quid. Clinically, submucous fibrosis in mawa

chewers differs in regard to age, sex, and location from those observed

among betel quid chewers.

Tobacco-Plus-Lime Sun-cured tobacco and slaked lime is used in Maharashtra and

other states in northern and eastern India where it is known as khaini. A

small quantity of tobacco and slaked lime is held in the palm and rolled

with the thumb. In Maharashtra, the mixture is placed in the premolar

region of the mandibular groove (Bhonsle et al., 1979); in Bihar and Uttar

Pradesh, the mixture is held in the lower labial groove. It is sucked from

time to time until it becomes bland.

Tobacco-Plus-Lime

User’s Lesion

A thick, yellowish-white lesion, occasionally with loose tags of

tissue, occurs where tobacco-plus-lime is held in the mouth by

patients in Maharashtra. Prevalence among 101,761 villagers in

Maharashtra was 2.9 percent (Bhonsle et al., 1979), and use occurred more

often among men. As this lesion is similar in appearance to leukoplakia, it

may be misdiagnosed. However, unlike leukoplakia this lesion can be

scraped off; it will disappear if tobacco-plus-lime use is discontinued.

The histological characteristics include pale, parakeratin-like surface

layers of epithelium containing round nuclear remnants, ballooning vacuolated cells, and epithelial hyperplasia (Bhonsle et al., 1979).

Tobacco-plus-lime user’s lesion as a specific entity appears to be a

counterpart of the pan chewer’s lesion (described above). This lesion does

not show any malignant potential.

Leukoplakia Tobacco-plus-lime use is associated with both leukoplakia and oral cancer

among patients in Maharashtra (Mehta et al., 1972). The prevalence of

leukoplakia was 1,442 per 100,000 among tobacco-plus-lime users and
Smoking and Tobacco Control Monograph No. 2

62

6,337 per 100,000 among those who also smoked. Generally, leukoplakias

associated with this habit occurred in the premolar region of the mandibular

groove where the tobacco-plus-lime mixture is held.

Oral Cancer Oral cancer was also observed among tobacco-plus-lime users. Prevalence

was 28 per 100,000 among tobacco-plus-lime users and 186 per 100,000 in

those who also smoked. As with leukoplakia, cancer generally occurred at

the site of placement of the tobacco-plus-lime, and sometimes leukoplakia

coexisted with it.

Mishri Compared with use of betel quid or tobacco-plus-lime, mishri use produced

fewer leukoplakias and no oral cancers. The prevalence of leukoplakia

among mishri users in Maharashtra was 190 per 100,000. Although no oral

cancers were detected among mishri users, short-term experimental tests

have indicated mutagenic potential of mishri (Kulkarni et al., 1987).

Bajjar In Gujarat, dry snuff known as bajjar (described above as mishri) is applied

over teeth and gums by 8 percent of 10,071 individuals (Mehta et al., 1971).

Very few leukoplakias were observed among bajjar users; the prevalence was

280 per 100,000, and these leukoplakias did not show any cancer potential.

Gudhaku

teeth, but the product becomes addictive. Gudhaku was used by 8.3 percent

of the 10,048 individuals in Singhbhum district in Bihar, but no oral lesions

were observed among them.

Miscellaneous

Products

Gudhaku is a tobacco paste with molasses. Initially, it is used to clean

Among other ST products, pan masala and creamy snuff are increasingly becoming popular all over the country. Pan masala contains

pieces of areca nut, slaked lime, catechu, and powdered tobacco. Pan masala

comes in attractive tins and foil packs, making it appealing and convenient

to use. Undoubtedly the aggressive advertising in the print and the electronic media contribute to its popularity. Animal experimental studies

indicated the genotoxic nature of pan masala. Creamy snuff is initially used

as a dentifrice but becomes an addictive substance. There are, however, no

studies to date on the effects of these products on human oral mucosa.

DISCUSSION Betel quid chewing is the most common form of ST use in India.

Extensive investigations in several cross-sectional and prospective epidemiological studies showed a strong association between many forms of ST use,

oral cancer, precancer, and other oral mucosal lesions. Of the several ST

forms, mawa and pan masala are of relatively recent origin, but there is an

upswing in their use. Mawa chewing was demonstrated to cause oral submucous fibrosis (Sinor et al., 1990). There is practically no information,

however, on the effects of pan masala on human oral mucosa.

Smokeless tobacco is an independent risk for oral cancer and precancer.

There is also an additive effect of using more than one type of ST product

and a synergistic effect with smoking (Gupta et al., 1980; Mehta et al., 1969;

Pindborg et al., 1984; Sinor et al., 1990; Wahi et al., 1968 and 1970; WHO,

1984).
Chapter 2

63

Although the pathogenic effects of ST in oral cancer and precancerous

lesions are clear, the role of other factors that may modify the effect of ST

use in India is not fully understood. Such factors may include nutrition,

viruses, especially HPV and HSV, and the role of oncogenes. Furthermore,

several animal studies and epidemiological studies have shown that vitamins A, C, and E, retinoic acid, carotenoids, riboflavin, selenium, and zinc

may influence the risk of oral cancer independently or by modifying the

effect of tobacco. There are several hospital-based studies with short-term

followup on chemoprevention with vitamin A and beta-carotene. Population-based studies with long-term followup, however, are highly desirable

and will be helpful in the development of practical chemopreventive

measures.

The finding that oral cancer is generally preceded by precancer facilitates the early detection of cancer. Although nodular leukoplakia and

submucous fibrosis are demonstrated to be a high-risk lesion and condition,

respectively, the difficulty lies in identifying which particular lesion would

progress to cancer. The conventional approach of using epithelial dysplasia

as a marker has been very helpful. There is, however, a need to develop

other markers based on advanced methodologies. In recent times

flowcytometric methods have been tried and reported to be promising, but

additional long-term studies are essential to demonstrate the potential of

such methods.

REFERENCES

Bentall, W.C. Cancer in Travancore, South India. A

summary of 1,700 cases. British Journal of Medicine

2: 1428-1431, 1908.

Bhonsle, R.B., Murti, P.R., Daftary, D.K., Mehta, F.S.

An oral lesion in tobacco-lime users in Maharashtra,

India. Journal of Oral Pathology 8: 47-52, 1979.

Bhonsle, R.B., Murti, P.R., Gupta, P.C., Mehta, F.S.

Reverse dhumti smoking in Goa: An epidemiologic

study of 5,449 villagers for oral precancerous

lesions. Indian Journal of Cancer 13: 301-305, 1976.

Bhonsle, R.B., Murti, P.R., Gupta, P.C., Mehta, F.S.,

Sinor, P.N., Irani, R.R., Pindborg, J.J. Regional

variations in oral submucous fibrosis in India.

Community Dentistry and Oral Epidemiology 15: 225-

229, 1987.

Bhonsle, R.B., Murti, P.R., Pindborg, J.J., Daftary, D.K.,

Mehta, F.S. Focal vascular dilatation and petechiae

in oral submucous fibrosis. Scandinavian Journal of

Dental Research 89: 270-274, 1981.

Bhonsle, R.B., Pindborg, J.J., Gupta, P.C., Murti, P.R.,

Mehta, F.S. Incidence rate of oral lichen planus

among Indian villagers. Acta Dermato-Venereologica

(Stockholm) 59: 255-257, 1979.

Daftary, D.K., Bhonsle, R.B., Murti, P.R., Pindborg, J.J.,

Mehta, F.S. An oral lichen planus-like lesion in

Indian betel-tobacco chewers. Scandinavian Journal

of Dental Research 88: 244-249, 1980.

Fells, A. Cancer of the mouth in southern India, with

an analysis of 209 operations. British Journal of

Medicine 2: 1357-1358, 1908.

Gupta, P.C., Bhonsle, R.B., Murti, P.R., Daftary, D.K.,

Mehta, F.S., Pindborg, J.J. An epidemiologic

assessment of cancer risk in oral precancerous

lesions in India with special reference to nodular

leukoplakia. Cancer 63: 2247-2252, 1989.

Gupta, P.C., Mehta, F.S., Daftary, D.K., Pindborg, J.J.,

Bhonsle, R.B., Jalnawalla, P.N., Sinor, P.N., Pitkar,

V.K., Murti, P.R., Irani, R.R., Shah, H.T., Kadam,

P.M., Iyer, K.S.S., Iyer, H.M., Hegde, A.K.,

Chandrashekar, V.K., Shroff, B.C., Sahiar, B.E.,

Mehta, M.N. Incidence rates of oral cancer and

natural history of oral precancerous lesions in a 10-

year follow-up study of Indian villagers. Community Dentistry and Oral Epidemiology 8: 287-333,

1980.

Gupta, P.C., Mehta, F.S., Pindborg, J.J., Aghi, M.B.,

Bhonsle, R.B., Daftary, D.K., Murti, P.R., Shah,

H.T., Sinor, P.N. Intervention study for primary

prevention of oral cancer among 36,000 Indian

tobacco users. Lancet 1(8492): 1235-1239, 1986.

Gupta, P.C., Mehta, F.S., Pindborg, J.J., Aghi, M.B.,

Bhonsle, R.B., Murti, P.R., Daftary, D.K. A primary

prevention study of oral cancer among Indian

villagers: 8-year follow-up results. In: Evaluating

Effectiveness of Primary Prevention of Cancer,

M. Hakama, V. Beral, J.W. Cullen, and D.M. Parkin

(Editors). Lyon: IARC, 1990, pp. 149-156.
Smoking and Tobacco Control Monograph No. 2

64

Gupta, P.C., Pindborg, J.J., Mehta, F.S. Comparison

of carcinogenicity of betel quid with and without

tobacco: An epidemiological review. Ecology of

Disease 1: 213-219, 1982.

Indian Council of Medical Research. National Cancer

Registry: Annual Report. New Delhi: Indian

Council of Medical Research, 1989.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosamines (volume 37). Lyon:

IARC, 1985.

Jayalakshmi, A., Mathew, A.G. Chemical composition and processing. In: The Areca Nut Palm,

K.V.A. Bavappa, M.K. Nair, and T. Premkumar

(Editors). Kasargod, India: Central Plantation

Crops Research Institute, 1980, p. 241.

Kulkarni, J.R., Sarkar, S., Bhide, S.V. Mutagenecity of

abstracts of brown and black mishri, pyrolysed

products of tobacco using short-term tests.

Mutagenesis 2: 263-266, 1987.

Mehta, F.S., Gupta, P.C., Daftary, D.K., Pindborg, J.J.

Choksi, S.K. An epidemiologic study of oral cancer

and precancerous conditions among 101,761

villagers in Maharashtra, India. International

Journal of Cancer 10: 134-141, 1972.

Mehta, F.S., Pindborg, J.J., Gupta, P.C., Daftary, D.K.

Epidemiologic and histologic study of oral cancer

and leukoplakia among 50,915 villagers in India.

Cancer 24: 832-849, 1969.

Mehta, F.S., Pindborg, J.J., Hamner, J.E. III., et al.

Report on Investigations of Oral Cancer and Precancerous Conditions in Indian Rural Populations, 1966-69.

Copenhagen: Munksgaard, 1971.

Murti, P.R., Bhonsle, R.B., Pindborg, J.J., Daftary,

D.K., Gupta, P.C., Mehta, F.S. Malignant transformation rate in oral submucous fibrosis over a 17-yr

period. Community Dentistry and Oral Epidemiology

13: 340-341, 1985.

Murti, P.R., Daftary, D.K., Bhonsle, R.B., Gupta, P.C.,

Mehta, F.S., Pindborg, J.J. Malignant potential of

oral lichen planus: Observations in 722 patients

from India. Journal of Oral Pathology 15: 71-77,

1986.

Murti, P.R., Gupta, P.C., Bhonsle, R.B., Daftary, D.K.,

Mehta, F.S., Pindborg, J.J. Effect on the incidence

of oral submucous fibrosis of intervention in the

areca nut chewing habit. Journal of Oral Pathology

and Medicine 19: 99-100, 1990.

Nair, M.K., Sankaranarayanan, R., Padmanabhan,

T.K., Padmakumari, G. Clinical profile of 2,007

oral cancers in Kerala, India. Annals of Dentistry

47: 23-26, 1988.

National Institutes of Health. Health Implications of

Smokeless Tobacco Use. National Institutes of

Health Consensus Development Conference

Statement. Vol. 6. No. 1., 1986.

Orr, I.M. Oral cancer in betel nut-chewers in

Travancore: Its aetiology, pathology and treatment. Lancet 2: 575-580, 1933.

Paymaster, J.C. Cancer of the buccal mucosa:

I. Clinical study of 650 cases in Indian patients.

Cancer 9: 431-435, 1956.

Pindborg, J.J., Bhonsle, R.B., Murti, P.R., Gupta, P.C.,

Daftary, D.K., Mehta, F.S. Incidence rate and early

forms of oral submucous fibrosis. Oral Surgery,

Oral Medicine, Oral Pathology 50: 40-44, 1980.

Pindborg, J.J., Kiaer, J., Gupta, P.C., Chawla, T.N.

Studies in oral leukoplakia among 10,000 persons

in Lucknow, India, with special reference to

tobacco and betel nut. Bulletin of the World Health

Organization 47: 13-19,1972.

Pindborg, J.J., Mehta, F.S., Daftary, D.K., Gupta, P.C.,

Bhonsle, R.B. Prevalence of oral lichen planus

among 7,639 Indian villagers in Kerala, South

India. Acta Dermato-Venereologica (Stockholm) 52:

216-220, 1972.

Pindborg, J.J., Mehta, F.S., Gupta, P.C., Daftary, D.K.

Prevalence of oral submucous fibrosis among

50,915 Indian villagers. British Journal of Cancer

22: 646-654, 1968.

Pindborg, J.J., Murti, P.R., Bhonsle, R.B., Gupta, P.C.,

Daftary, D.K., Mehta, F.S. Oral submucous fibrosis

as a precancerous condition. Scandinavian Journal

of Dental Research 92: 224-229, 1984.

Sanghvi, L.D. Tobacco-related cancers. In: Tobacco

and Health: The Indian Scene, L.D. Sanghvi and

P. Notani (Editors). Bombay: Tata Memorial

Center, 1989, pp. 9-15.

Sanghvi, L.D., Rao, K.C.M., Khanolkar, V.R.

Smoking and chewing of tobacco in relation to

the cancer of the upper alimentary tract. British

Journal of Cancer 1: 111-114, 1955.

Shanta, V., Krishnamurthy, S.A. A study of

aetiological factors in oral squamous cell carcinoma. British Journal of Cancer 13: 382-388, 1959.

Sinor, P.N., Gupta, P.C., Murti, P.R., Bhonsle, R.B.,

Daftary, D.K., Mehta, F.S., Pindborg, J.J. A casecontrol study of oral submucous fibrosis with

special reference to the etiologic role of areca nut.

Journal of Oral Pathology and Medicine 19: 94-98,

1990.

Smith, L.W., Bhargava, K., Mani, N.J., Malaowalla,

A.M., Silverman, S., Jr. Oral cancer and precancerous lesions in 57,518 industrial workers of

Gujarat, India. Indian Journal of Cancer 12: 118-

123, 1975.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986.
Chapter 2

65

Wahi, P.N. The epidemiology of oral and oropharyngeal cancer: A report of the study in

Mainpuri district, Uttar Pradesh, India. Bulletin of

the World Health Organization 38: 495-521, 1968.

Wahi, P.N., Mittal, V.P., Lahiri, B., Luthra, U.K., Seth,

R.K., Arora, G.D. Epidemiological study of

precancerous lesions of the oral cavity: A preliminary report. Indian Journal of Medical Research 58:

1361-1391, 1970.

World Health Organization. Control of oral cancer

in developing countries: Report of WHO meeting.

Bulletin of the World Health Organization 62: 817-

830, 1984.
Smoking and Tobacco Control Monograph No. 2

66

Smokeless tobacco-associated oral mucosal lesions from professional baseball players were

1 This study was supported by National Institute of Dental Research grant no. DE-08547.

ABSTRACT

studied for histopathologic changes and changes in Langerhans cell (LC) density and antigen

expression. Four types of epithelial change were found: hyperparakeratosis, hyperorthokeratosis, pale surface staining, and basal cell hyperplasia. These changes were associated with

the type of ST used (snuff or chewing tobacco) but not with the duration (years) or amount

(hours per day) of ST use. The thickness of the hyperkeratotic layer in a specimen correlated

directly with the amount of ST use. Immunohistochemical analysis with four monoclonal

antibodies detected fewer LC in lesion specimens than in autologous control specimens. The

average reduction in LC was 58 percent (range, 3 to 95 percent). There were no significant

differences in the LC reduction identified by the four different marker antigens. Specimens

with all types of epithelial change showed similar reductions in LC. Our data indicate that

snuff causes a greater variety and severity of epithelial change than does chewing tobacco, and

that smokeless tobacco reduces the number of LC at its site of contact with the oral mucosa.

INTRODUCTION Smokeless tobacco contains carcinogens (Hoffmann et al., 1987),

and an association between snuff use and oral carcinoma has been observed

(Peacock et al., 1960; Sundström et al., 1982; US DHHS, 1986; Winn et al.,

1981). ST use results in oral mucosal lesions, usually described as leukoplakia (Grady et al., 1990; Pindborg and Renstrup, 1963), that can become

dysplastic (Andersson et al., 1989; Greer et al., 1986; Hirsh et al., 1982;

Jungell and Malmström, 1985; Kaugars et al., 1989; Roed-Petersen and

Pindborg, 1973; Smith et al., 1970) and can undergo malignant transformation (Roed-Petersen and Pindborg, 1973; Sundström et al., 1982). Histopathologic studies of ST-associated oral mucosal lesions have found hyperkeratosis in most specimens and various frequencies of dysplasia and carcinoma (Andersson et al., 1989; Axéll et al., 1976; Greer et al., 1986; Hirsh et

al., 1982; Jungell and Malmström, 1985; Kaugars et al., 1989; Pindborg and

Renstrup, 1963; Pindborg et al., 1980; Roed-Petersen and Pindborg, 1973;

Smith et al., 1970).

Langerhans cells are dendritic cells that migrate from the bone marrow

to reside in epithelium throughout the body. They perform important

immunologic functions by participating in cutaneous and mucosal immune

reactions that can have both local and systemic effects. Changes in the

number of LC can affect systemic immune responses (Toews et al., 1980),

and the localized absence of LC in the mucosa may be associated with

colonization by viruses and fungi (Daniels et al., 1987). Localized reductions in the number of LC might impair skin or mucosal immunologic

Smokeless Tobacco-Associated Epithelial

And Langerhans Cell Changes

Troy E. Daniels, Louis S. Hansen, John S. Greenspan, Laisheng Chou,

Deborah G. Grady, Walter W. Hauck, John C. Greene, and

Virginia L. Ernster

1
Chapter 2

67

SUBJECTS AND

METHODS

protection against mutagens or opportunistic viruses and have a role in

epithelial dysplasia and carcinoma (Viac et al., 1990).

A 3-yr study of the oral and systemic effects of ST use on professional

baseball players provided us an opportunity to examine patterns of histopathologic change, and possible effects on LC numbers and antigen expression, in oral epithelial lesions associated with the use of ST (Ernster et al.,

1990). This paper summarizes the results of studies presented in detail

elsewhere (Daniels et al., 1992 and in press).

We conducted oral examinations among 1,632 members of seven

Major League and minor league professional baseball teams during

spring training in 1988 (Ernster et al., 1990) and 1989. Participants were

asked to identify all ST products they used and to indicate the types and

brands they used most often. Of the players examined, 40 percent had used

ST within the past week and were classified as current ST users. Previous

detailed analysis of the 1988 data showed that 75 percent of current users

reported a brand of snuff as their usual ST product, 21 percent reported

chewing tobacco as their usual product, and the remainder did not indicate

a preference (Ernster et al., 1990).

At oral examination, 350 of the players (21 percent) had mucosal

lesions, usually consisting of an ill-defined white area on the upper or lower

labial, alveolar, or buccal mucosa; 94 percent of the lesions were in mucosal

sites adjacent to the lower jaw. There were 138 biopsy specimens from 129

current ST users that were satisfactory for morphological evaluation.

The average age of the players who had biopsies was 25 yr. By selfreport, 126 of the players who had biopsies stated their usual brand of ST as

follows: 6 (5 percent) used chewing tobacco; 90 (71 percent) used

Copenhagen brand snuff; 27 (21 percent) used other brands of snuff; and 3

(2 percent) used a combination of ST products. The average duration of ST

use by the players whose lesions were biopsied was 7.3 yr. Other details

about these players and methods of histopathologic examination and

grading are described elsewhere (Daniels et al., in press).

We selected 17 pairs of lesion and autologous control specimens for LC

studies. LC were identified by the use of monoclonal antibodies against the

T6 antigen, usually considered to be intrinsically expressed by LC, and

against three class II HLA-D gene products: HLA-DR, HLA-DP, and HLA-DQ.

We expressed mean LC counts as number of LC per millimeter of epithelial

surface (LC/mm) and as number of LC per square millimeter of epithelial

cross-section (LC/mm2

). The calculation in terms of sectional area of epithelium compensates for the difference in thickness between some of the lesion

specimens and their autologous controls. We expressed the numbers of LC

in the paired lesion and control specimens from each individual as a percentage reduction of LC in the lesion specimen compared with its control.

The selection of cases and immunohistochemical methods are described

elsewhere (Daniels et al., 1989 and 1992).
Smoking and Tobacco Control Monograph No. 2

68

EPITHELIAL Each specimen had a predominant pattern of epithelial change:

CHANGES • Hyperparakeratosis (HPK), characterized by increased epithelial

thickness and keratinization, with nuclei remaining in most of the

surface epithelial cells;

• Hyperorthokeratosis (HOK), characterized by increased keratinization,

the presence of a granular cell layer, and absence of nuclei in the

surface epithelial cells;

• Pale surface staining (PSS) of the epithelium, characterized by a pale

staining zone of hyperkeratosis having ill-defined epithelial cell

boundaries with few nuclei, no granular cell layer, and frequent

vacuoles; or

• Basal cell hyperplasia (BCH), characterized by cells with a basilar

pattern extending through a larger-than-usual portion of the epithelium, widened rete processes, thinning of the epithelium over the

connective tissue papillae, and little or no hyperkeratosis.

Of the 132 specimens from snuff users, 56 percent had HPK, 35 percent

had PSS, 5 percent had HOK, and 4 percent had BCH. The thickness of

hyperkeratosis was grade 1 (slight) in 32 percent of the lesions, grade 2

(moderate) in 50 percent, and grade 3 (severe) in 18 percent. Of the six

specimens from chewing tobacco users, all had HPK and the hyperkeratosis

was grade 1 in five and grade 2 in the sixth. The grade of hyperkeratosis and

the amount of ST use, in estimated hours per day, were highly correlated

(p=0.005, one-way analysis of variance). No significant correlations were

observed between the four patterns of epithelial change in the specimens

and the age of the players, the duration of ST use, or the number of hours of

ST use per day.

None of the HPK, PSS, or HOK specimens showed any signs of dysplasia. Among the five BCH specimens, we observed slight epithelial dysplasia

(BCH and cellular atypia) in only one, taken from a subject who had used

snuff for 3 yr. Of the remaining BCH specimens, two were from players who

reported using snuff for 3 yr or less, and two were from players who used

snuff for 5 yr. All five used Copenhagen brand snuff.

In addition to the changes in epithelial maturation, we observed

koilocytic cells (Koss and Durfee, 1956) in 12 percent of the specimens,

which resemble those seen in papillomavirus-infected epithelium of the

uterine cervix (zur Hausen and Schneider, 1987). We also observed a chevron pattern of keratinization (Pindborg et al., 1980) in 6 percent of the

specimens, all of the PSS type.

LC CHANGES Langerhans cells were readily identified within the epithelium by their

intense staining, dendrites, and generally suprabasal location. Those identified by their HLA-DP and -DQ antigens tended to have shorter, blunter

dendritic processes than those identified by their T6 and HLA-DR antigens.

Epithelial cell expression of HLA-DR antigen was not observed in either

lesion or control specimens.
Chapter 2

69

The number of LC expressing each of the four antigens was fairly uniform in each of the four types of pathological lesions. Comparison of the

mean number of LC in all types of lesions identified by all four antigens

showed that there were fewer LC in the lesion specimens (means of 6 LC/mm

and 10 LC/mm2) than in the control specimens (means of 14 LC/mm and

30 LC/mm2).

The 17 pairs of lesion and autologous control specimens showed an

average reduction in LC per millimeter of 58 percent (range, 3 to 95 percent).

That each pair showed a difference in the same direction is highly significant

(p < 0.0001 by the sign test) (Conover, 1980). In all but one pair of specimens, there were fewer LC per square millimeter in each lesion specimen

than in its corresponding control, by an average of 60 percent. Control

specimens averaged 0.4 mm in thickness. The lesion specimens with PSS

were the thickest at 0.7 mm, and those with HPK were the next thickest at

0.6 mm. Both of those types showed slightly greater reductions in LC per

square millimeter than in LC per millimeter, as a result of differences in

epithelial thickness between lesion and control specimens. However, similar

reductions in LC were seen in HOK specimens, which were not thicker than

their autologous controls, and in BCH specimens, which do not have hyperkeratosis.

DISCUSSION

Epithelial

Changes

Our findings suggest that the epithelial response in snuff users is

different from that in chewing tobacco users. Snuff was associated

with a greater variety and severity of epithelial change than chewing

tobacco. Snuff has been reported to be more likely than chewing

tobacco to cause oral lesions and to cause more severe lesions (Grady et al.,

1990), possibly because of differences in pH, chemical composition, or

moisture content (Hoffmann et al., 1977). However, caution should be used

in interpreting our findings about possible differences between snuff and

chewing tobacco, because there were only six chewing tobacco users among

the subjects who had biopsies.

Chewing tobacco users have a lower rate of mucosal lesions than snuff

users, and a smaller percentage of players use chewing tobacco than use

snuff (Ernster et al., 1990). Previous histopathologic studies of ST lesions in

Europe and North America have mainly examined snuff-associated lesions.

Two studies (Greer et al., 1986; Kaugars et al., 1989) included snuffassociated and chewing tobacco-associated lesions but did not analyze the

findings separately (Table 1).

Some of these previous histopathologic studies showed the prevalence

of epithelial dysplasia to range from 2 to 18 percent (Andersson et al., 1989;

Greer et al., 1986; Hirsch et al., 1982; Jungell and Malmström, 1985; RoedPetersen and Pindborg, 1973; Smith et al., 1970), whereas other studies did

not note dysplasia (Axéll et al., 1976; Pindborg et al., 1980; Pindborg and

Renstrup, 1963), and one included only specimens having dysplasia

(Kaugars et al., 1989). Several authors have described ST-associated oral

carcinomas (Peacock et al., 1960; Roed-Petersen and Pindborg, 1973; Smith

et al., 1970; Sundström et al., 1982; Winn et al., 1981) (Table 1). The

prevalence of dysplasia in our study was < 1 percent. It is not known,
70

Table 1Data summary: histopathologic studies of snuff-associated oral lesions

Mean Epithelial 

 Authors Year Country n Age Chevrons Pindborg and Renstrup 1963 Denmark 12 60 Smith et al. 1970 United States 657 62 Roed-Petersen and Pindborg 1973 Denmark 31 58 yes Axéll et al. 1976 Sweden 114 50 yes Pindborg et al. 1980 Denmark 12 59 12 (100%) Sundström et al. 1982 Sweden 23 76 Hirsch et al. 1982 Sweden 50 41 Jungell and Malmström 1985 Finland 21 19 1 (5%) Greer et al.a 1986 United States 45 41 42 (93%) 

Kaugars et al.a 1989 United States 108 56 1Andersson et al. 1989 Sweden 252 36 yes Daniels et al.a 1991 United States 129 25 8 (6%) 

a Includes chewing tobacco use.
Chapter 2

71

however, whether similar criteria were used for the diagnosis of dysplasia in

the other studies. Differences in rates of dysplasia might also be attributable

to differences in the populations studied, types of ST used, duration and

amount of ST use, placement site of ST, and variations in concurrent smoking and alcohol use.

Although the establishment of consistent and reproducible morphological criteria for epithelial dysplasia is not yet possible, evidence of disturbed

epithelial maturation and the presence of atypical cells are considered risks

for malignant transformation (WHO Collaborating Centre for Oral Precancerous Lesions, 1978). BCH, as observed alone in four of the five BCH

specimens in this study, does not by itself define dysplasia but is a commonly observed feature in dysplastic epithelium. All of the BCH specimens

in this study were associated with a single brand of snuff, but the significance of this observation is not yet established.

We have demonstrated a variety of morphological changes in the

epithelium that contacts ST. Although the lesions from this group of

generally young men were all benign and only one exhibited signs of

dysplasia, that specimen was from a player who had been using snuff for

only 3 yr. Our data indicate that of the various forms of ST, snuff is more

frequently associated with development of oral mucosal lesions and with a

greater variety and severity of epithelial changes than chewing tobacco.

Langerhans Cells Our present data are consistent with previous observations that

mucosal or cutaneous lesions that are or may develop into carcinoma are

associated with a significant reduction in T6+ (CD1+) Langerhans cells

(Barton et al., 1989; Meissner et al., 1986; Pitigala-Arachchi et al., 1989;

Smolle et al., 1986; Viac et al., 1990). In vivo chemical carcinogenesis

experiments have suggested that loss of LC during tumor promotion may

impair immunologic protection against skin tumors (Halliday et al., 1988).

We have shown that the number of LC per millimeter in ST-associated

oral lesions is on average 58 percent less than the number in autologous

control tissue. This reduction in the number of LC in lesional tissue is not

accounted for by differences in epithelial thickness, because the number of

LC is similarly reduced when measured per square millimeter. Nonkeratinized areas of the oral mucosa generally have more LC than keratinized areas

have (Cruchley et al., 1989; Daniels, 1984), but no study has investigated

changes in the number of LC at sites undergoing increasing keratinization.

Epidemiological studies have shown that cigarette smoking is associated

with an increased risk for cancer of the uterine cervix (Winkelstein, 1990).

A recent study (Barton et al., 1988) observed a dose-response relationship

between the number of cigarettes smoked daily and reduction in LC counts

in the cervical epithelium. The counts were reduced in both clinically

normal and lesional epithelium in the cervix. The amounts of reduction

noted in the above studies were similar to those noted in our study.

The pathogenesis of oral mucosal carcinoma is unknown, but viruses

and immunologic changes may be cofactors. There is clear evidence of

participation by a few types of human papillomavirus (HPV) in the
Smoking and Tobacco Control Monograph No. 2

72

development of most cervical carcinomas (zur Hausen and Schneider, 1987),

but a consistent relationship has not been established for oral carcinoma

(Scully, 1988). Koilocytic epithelial cells (Koss and Durfee, 1956) are associated with HPV infection (zur Hausen and Schneider, 1987), and those cells

have been observed in some of the specimens from previous studies of STassociated lesions (Andersson et al., 1989; Daniels et al., in press; Greer et al.,

1986; Hirsch et al., 1982; Pindborg and Renstrup, 1963) but without examination for HPV. Viral DNA from diverse types of HPV has been identified by

in situ hybridization methods in only small proportions of ST lesions, oral

mucosal dysplasias and carcinomas (Greer et al., 1990), and by polymerase

chain reaction methods in less than half of oral carcinomas (Palefsky et al.,

1991). LC are intraepithelial antigen-processing cells that are crucial in

epithelial immunity, and their number is greatly reduced at the site of

contact with ST (Daniels et al., 1989 and 1992). This localized reduction or

loss of immunologic function may contribute to colonization of the mucosal sites by opportunistic viruses as noted in oral hairy leukoplakia (Daniels

et al., 1987).

ST reduces the number of LC at the site of its contact with the mucosa.

This illustrates one form of host response to the constituents of that complex material. More knowledge is needed about the constituents of various

types of ST that are responsible for morphological epithelial changes, the

immunologic sequelae of ST-induced LC reduction, and the presence of

cofactors that may lead to the development of malignant neoplasia.

REFERENCES

Daniels, T.E. Human mucosal Langerhans cells:

Postmortem identification of regional variations

in oral mucosa. Journal of Investigative Dermatology

82: 21-24, 1984.

Daniels, T., Chou, L., Greenspan, J., et al. Langerhans cells in smokeless tobacco-associated oral

mucosal lesions (abstract). Journal of Dental

Research 68: 947, 1989.

Daniels, T.E., Chou, L.S., Greenspan, J.S., et al.

Reduction of Langerhans cells in smokeless

tobacco-associated oral mucosal lesions. Journal of

Oral Pathology and Medicine 21: 100, 1992.

Daniels, T.E., Greenspan, D., Greenspan, J.S., et al.

Absence of Langerhans cells in oral hairy leukoplakia, an AIDS-associated lesion. Journal of

Investigative Dermatology 89: 178-182, 1987.

Daniels, T.E., Hansen, L.S., Greenspan, J.S., et al.

Histopathology of smokeless tobacco lesions in

professional baseball players: Associations with

different types of tobacco. Oral Surgery, Oral

Medicine, Oral Pathology, in press.

Ernster, V.L., Grady, D., Greene, J.C., Walsh, M.,

Robertson, P.B., Daniels, T., Benowitz, N.,

Siegel, D., Gerbert, B., Hanck, W. Smokeless

tobacco use and health effects among professional

baseball players. Journal of the American Medical

Association 264: 218-224, 1990.

Andersson, G., Axéll, T., Larsson, Å. Histologic

changes associated with the use of loose and

portion-bag packed Swedish moist snuff: A

comparative study. Journal of Oral Pathology and

Medicine 18: 491-497, 1989.

Axéll, T., Mörnstad, H., Sundström, B. The relation

of the clinical picture to the histopathology of

snuff dipper’s lesions in a Swedish population.

Journal of Oral Pathology 5: 229-236, 1976.

Barton, S.E., Hollingworth, A., Maddox, P.H., et al.

Possible cofactors in the etiology of cervical

intraepithelial neoplasia: An immunopathologic

study. Journal of Reproductive Medicine 34: 613-616,

1989.

Barton, S.E., Maddon, P.H., Jenkins, D., et al. Effect

of cigarette smoking on cervical epithelial

immunity: A mechanism for neoplastic change?

Lancet 2: 652-654, 1988.

Conover, W.J. Practical Nonparametric Statistics (2nd

edition). New York: John Wiley and Sons, 1980,

pp. 122-128.

Cruchley, A.T., Williams, D.M., Farthing, P.M., et al.

Regional variation in Langerhans cell distribution

and density in normal human oral mucosa

determined using monoclonal antibodies against

CD1, HLADR, HLADQ and HLADP. Journal of Oral

Pathology and Medicine 18: 510-516, 1989.
Chapter 2

73

Grady, D., Greene, J.C., Daniels, T.E., Ernster, V.L.,

Robertson, P.B., Hauck, W., Greenspan, D.,

Greenspan, J.S., Silverman, S. Oral mucosal lesions

found in smokeless tobacco users. Journal of the

American Dental Association 121: 117-123, 1990.

Greer, R.O., Eversole, L.R., Crosby, L.K. Detection of

human papillomavirus-genomic DNA in oral

epithelial dysplasias, oral smokeless tobaccoassociated leukoplakias, and epithelial malignancies. Journal of Oral and Maxillofacial Surgery 48:

1201-1205, 1990.

Greer, R.O., Poulson, T.C., Boone, M.E., et al.

Smokeless tobacco-associated oral changes in juvenile, adult and geriatric patients: Clinical and

histomorphologic features. Gerodontics 2: 87-98,

1986.

Halliday, G.M., Odling, K.A., Ruby, J.C., et al.

Suppressor cell activation and enhanced skin allograft survival after tumor promoter but not

initiator induced depletion of cutaneous Langerhans cells. Journal of Investigative Dermatology 90:

293-297, 1988.

Hirsch, J.M., Heyden, G., Thilander, H. A clinical,

histomorphological and histochemical study on

snuff-induced lesions of varying severity. Journal of

Oral Pathology 11: 387-398, 1982.

Hoffmann, D., Adams, J.D., Lisk, D., et al. Toxic and

carcinogenic agents in dry and moist snuff. Journal

of the National Cancer Institute 79: 1281-1286, 1987.

Jungell, P., Malmström, M. Snuff-induced lesions in

Finnish recruits. Scandinavian Journal of Dental

Research 93: 442-447, 1985.

Kaugars, G.E., Mehailescu, W.L., Gunsolley, J.C.

Smokeless tobacco use and oral epithelial dysplasia. Cancer 64: 1527-1530, 1989.

Koss, L.G., Durfee, G.R. Unusual patterns of

squamous epithelium of the uterine cervix:

Cytologic and pathologic study of koilocytic

atypia. Annals of the New York Academy of Science

63: 1245-1261, 1956.

Meissner, K., Haftek, M., Arlot, M., et al. Quantitative analysis of T6-positive Langerhans cells in

human skin cancers. Virchows Archives A [Pathology and Anatomy] 410: 57-63, 1986.

Palefsky, J., Berline, J., Seid, K., et al. Heterogeneity

of HPV types detected in invasive oral cancer

(abstract). Journal of Dental Research 70: 271, 1991.

Peacock, E.E., Greenberg, B.G., Brawley, B.W. The

effect of snuff and tobacco on the production of

oral carcinoma: An experimental and epidemiological study. Annals of Surgery 151: 542-550, 1960.

Pindborg, J.J., Renstrup, G. Studies in oral leukoplakias: II. Effect of snuff on oral epithelium. Acta

Dermato-Venereologica 43: 271-276, 1963.

Pindborg, J.J., Reibel, J., Roed-Petersen, B., et al.

Tobacco-induced changes in oral leukoplakic epithelium. Cancer 45: 2330-2336, 1980.

Pitigala-Arachchi, A., Crane, I.J., Scully, C., et al.

Epithelial dendritic cells in pathological human

oral tissues. Journal of Oral Pathology and Medicine

18: 11-16, 1989.

Roed-Petersen, B., Pindborg, J.J. A study of Danish

snuff-induced oral leukoplakias. Journal of Oral

Pathology 2: 301-313, 1973.

Scully, C., Cox, M.F., Prime, S.S., et al. Papillomaviruses: The current status in relation to oral

disease. Oral Surgery 65: 526-532, 1988.

Smith, J.F., Mincer, H.A., Hopkins, K.P., et al. Snuffdipper’s lesion, a cytological and pathological

study in a large population. Archives of Otolaryngology 92: 450-456, 1970.

Smolle, J., Soyer, H.P., Ehall, R., et al. Langerhans

cell density in epithelial skin tumors correlates

with epithelial differentiation but not with the

peritumoral infiltrate. Journal of Investigative

Dermatology 87: 477-479, 1986.

Sundström, B., Mörnstad, H., Axéll, T. Oral

carcinomas associated with snuff dipping. Journal

of Oral Pathology 11: 245-251, 1982.

Toews, G.B., Bergstresser, P.R., Streilein, J.W., et al.

Epidermal Langerhans cell density determines

whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. Journal of

Immunology 124: 445-453, 1980.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986.

Viac, J., Guérin-Reverchon, I., Chardonnet, Y., et al.

Langerhans cells and epithelial cell modifications

in cervical intraepithelial neoplasia: Correlation

with human papillomavirus infection. Immunobiology 180: 328-338, 1990.

WHO Collaborating Centre for Oral Precancerous

Lesions. Definition of leukoplakia and related

lesions: An aid to studies on oral precancer. Oral

Surgery 46: 518-539, 1978.

Winkelstein, W. Smoking and cervical cancer—

current status: A review. American Journal of

Epidemiology 131: 945-957, 1990.

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W.,

Toledo, A., Fraumeni, J.F., Jr. Snuff dipping and

oral cancer among women in the southern United

States. New England Journal of Medicine 304: 745-

749, 1981.

zur Hausen, H., Schneider, A. The role of papillomaviruses in human anogenital cancer. In: Papovaviridae: The Papillomaviruses (volume 2), N.P.

Salzman and P.M. Howley (Editors). New York:

Plenum Press, 1987, pp. 245-263.
Smoking and Tobacco Control Monograph No. 2

74

Twenty-five years of observation of increasing consumption of moist snuff in Sweden have

The Use of Smokeless Tobacco

In Sweden

Jan Bergström

ABSTRACT

undoubtedly offered some experience. Use of moist snuff is inevitably associated with a more

or less severe mucosal lesion, with a typically wrinkled appearance at the site of placement of

the tobacco quid. The rate of aggravation and the risk for malignant change are conclusions

not readily deduced from the information presently available. For a better understanding of

such complex problems, prospective studies are warranted.

INTRODUCTION Smokeless tobacco in the form of moist snuff seems to be a variety

of tobacco that by tradition is related to Sweden. From a historical point of

view, the use of smokeless tobacco or snuff has been known in Sweden since

the early 1600’s. Snuff seems to have been appreciated since, in a report

dated 1638, it is stated that ”there is nobody neither man nor woman who

does not snuff or drink tobacco” (P. Brake, report to Axel Oxenstierna, Royal

Library, Stockholm). At that time, however, snuffing tobacco probably

meant to inhale it into one’s nose, as was the custom elsewhere in Europe.

Later on, from the early 1800’s and onward, moist snuff for intraoral use

became a specifically Swedish fashion.

At first, the use of moist oral snuff had social implications: whereas

nasal snuff was used among the upper class and the bourgeoisie, oral moist

snuff was the habit of the simple people. Later, however, use of the former

variety declined, while the latter increased and became a widespread habit,

remaining so into the 1920’s when it was gradually replaced by cigarette

smoking. In some parts of the country, snuff dipping remained a frequent

habit. In the mid-1960’s there was a revival of the habit, starting among

young people, especially high school students and team athletes. It is not

well known why this old habit reemerged, but it may be speculated that it

replaced cigarette smoking among those who were either theoretically aware

of the risks of smoking (students) or practically aware that smoking actually

interfered with their physical performance (sportsmen).

EPIDEMIOLOGICAL

ASPECTS

Since 1965 there has been a steady increase in the use of moist

snuff in Sweden. It is further evident that the increase has

been concentrated among young men. According to a study among

Stockholm schoolchildren in 1970, 4 percent of 12- and 13-yr-olds,

10 percent of 15- and 16-yr-olds, and 35 percent of students aged 16 to

18 dipped snuff (Bergström et al., 1975); all of the snuff dippers were male.

Furthermore, 80 percent of the students exhibited typical lesions of the

mucosa—sometimes including gingival recession at the site of placement.

According to the Swedish National Smoking and Health Association,

20 to 25 percent of Swedish men used snuff daily or occasionally during the

period 1985 to 1988 (Ramström, 1985; Ramström and Tibblin, 1986, 1987,

and 1988). The most recent report, based on 1988 data, shows that 35 percent of males in the 16 to 24 age group are regular or occasional users
Chapter 2

75

(Table 1). The frequency of snuff use decreases with increasing age. Still,

the habit is nearly totally limited to males, with a predominance among the

lower educational stratum of the population (Table 2). As observed by

Nordgren and Ramström (1990), the trend in Sweden over the past two-anda-half decades clearly indicates an emerging pattern of oral snuff use among

young Swedish males.

According to reports over the past 5 yr, snuff dipping among young

men is usually not associated with regular smoking. Only 13 percent are

regular consumers of both smoked and smokeless tobacco. Interestingly, it

appears that snuff dipping has, to some extent, replaced smoking: 37 percent of the 18- to 34-yr-old and 61 percent of the 35- to 70-yr-old dippers

claim to be former smokers. Ramström (1990) found that, among smokers

who turn to snuff dipping, nearly twice as many quit smoking as compared

to ”pure smokers.” Thus there seems to be some evidence that snuff dipping in Sweden may be a result of shifting tobacco habits, serving as a

substitute for smoking and thus as a means of smoking cessation. The

number of young dippers who later turn to smoking seems to be relatively

small. Some 15 percent of current smokers report that they started their

tobacco use as snuff dippers. Among older smokers, the percentage of those

starting as snuff dippers is even smaller (4 percent).

The most common adverse oral effect associated with the use of

moist snuff is a lesion at the site of the quid placement, which has

EFFECTS ON

ORAL HEALTH

been described by several investigators (Andersson and Axéll, 1989; Axéll et

al., 1976; Frithiof et al., 1983; Mörnstad and Axéll, 1989). The clinical

severity of the lesion is usually scored on a four-point scale based mainly on

the wrinkling pattern of the mucosa. There is some evidence that the

severity of lesions associated with moist snuff increases with age, with

duration of use, with exposure, and to some extent with brand (some brands

seem to be more hazardous than others) (Axéll et al., 1976; Hirsch et al.,

1982). A variety of snuff available on the Swedish market is the portionpacked snuff in paper bags. The bag allows diffusion of the active components, without being dissolved itself. Although presently only 10 percent of

dippers, at most, regularly use portion-packed snuff, and they may not be

fully comparable to dippers who use ordinary loose snuff, it appears that use

of the portion packs may be less harmful (Andersson and Axéll, 1989;

Andersson et al., 1989).

Another clinical feature associated with Swedish use of oral snuff is

localized gingival recession at the buccal aspect of teeth adjacent to the

place of the quid—usually the lateral incisor. Although repeatedly observed,

gingival recession has been the object of little study. This condition should

be further studied because it is an irreversible sign and probably reflects the

accumulated impact on the mucosa. According to Andersson and Axéll

(1989) there seems to be a positive correlation between the severity of the

lesion and the magnitude of the gingival recession. It might be speculated

that gingival recession is an indirect manifestation secondary to contractile

actions within the mucosal lesion, rather than a direct reaction of the

marginal gingiva. This needs to be substantiated, however.
Smoking and Tobacco Control Monograph No. 2

76

Table 1

Snuff habits among Swedish men (1988), by age group

Percentage at Each Level of Use

Age 16 to 24 Age 25 to 34 Age 35 to 54 Age 55 to 74

(n=213) (n=196) (n=433) (n=312)

Daily User 29% 20% 13% 14%

Occasional User 6851

Non-User 65 72 82 83

Source: Ramström and Tibblin, 1988.

Table 2

Snuff habits among Swedish men (1988), by age and education

Percentage at Each Level of Use

Age 18 to 29 Age 30 to 49 Age 50 to 70

Low High Low High Low High

Educationa Educationb Educationa Educationb Educationa Educationb

(n=69) (n=101) (n=133) (n=151) (n=260) (n=63)

Daily User 37% 18% 18% 10% 21% 0%

Occasional User 10 1 6 6 2 0

Non-User 54 81 77 84 77 100

a Low = 9 yr schooling.

b High = ≥12 yr schooling.

Source: Ramström and Tibblin, 1988.

The histopathological pattern associated with snuff-induced lesions has

been investigated by Axéll et al. (1976) and Hirsch et al. (1982). They found

a high incidence of slight dysplasia, but the histomorphologic pattern was

not predictable from the clinical state. The occurrence of oral cancer associated with snuff dipping in Swedish populations has been addressed by Axéll

et al. (1978) and Sundström et al. (1982), who report a five- or sixfold

increase in the risk of developing cancer at the site of snuff placement.

Prospective, systematic studies are needed to yield more information about

the development and aggravation of snuff-induced lesions and their potential transformation into dysplastic precancerous or cancerous manifestations.
Chapter 2

77

REFERENCES

Andersson, G., Axéll, T. Clinical appearance of

lesions associated with the use of loose and

portion-bag packed Swedish moist snuff: A

comparative study. Journal of Oral Pathology and

Medicine 18: 2-7, 1989.

Andersson, G., Axéll, T., Larsson, Å. Histologic

changes associated with the use of loose and

portion-bag packed Swedish moist snuff: A

comparative study. Journal of Oral Pathology and

Medicine 18: 491-497, 1989.

Axéll, T., Mörnstad, H., Sundström, B. The relation

of the clinical picture to the histopathology of

snuff dipper’s lesions in a Swedish population.

Journal of Oral Pathology 5: 229-236, 1976.

Axéll, T., Mörnstad, H., Sundström, B. Snuff and

cancer of the oral cavity. L Kartidningen 75: 2224-

2226, 1978.

Bergström, J., Larsson, K., Pschorn, E., Randahl, P.,

Öman, P. Occurrence of Snuff Dipping Among

Children in Stockholm and Its Periodontal Effects

(Swedish). Stockholm: School of Dentistry,

Karolinska Institutet, 1975.

Frithiof, L., Anneroth, G., Larson, U., Sederholm, C.

The snuff-induced lesion: A clinical and morphological study of Swedish material. Acta

Odontologica Scandinavica 41: 53-64, 1983.

Hirsch, I.M., Heyden, G., Thilander, H. A clinical

histomorphological and histochemical study on

snuff-induced lesions of varying severity. Journal

of Oral Pathology 11: 387-398, 1982.

Mörnstad, H., Axéll, T. Clinical picture of snuff

dipper’s lesion in Swedes. Community Dentistry

and Oral Epidemiology 17: 97-101, 1989.

Nordgren, P., Ramström, L. Moist snuff in Sweden:

Tradition and evolution. British Journal of

Addiction 85: 1107-1112, 1990.

Ramström, L. Tobacco Habits in Sweden 1985

(Swedish). Stockholm: Swedish National

Smoking and Health Association, 1985.

Ramström, L. Smokeless tobacco: A potential

gateway to smoking? In: Tobacco and Health

1990: The Global War. Proceedings of the 7th World

Conference on Tobacco and Health, B. Durston and

K. Jamrozik (Editors). Perth: Health Department

of Western Australia, 1990.

Ramström, L., Tibblin, H. Tobacco Habits in Sweden

1986 (Swedish). Stockholm: Swedish National

Smoking and Health Association, 1986.

Ramström, L., Tibblin, H. Tobacco Habits in Sweden

1987 (Swedish). Stockholm: Swedish National

Smoking and Health Association, 1987.

Ramström, L., Tibblin, H. Tobacco Habits in Sweden

1988 (Swedish). Stockholm: Swedish National

Smoking and Health Association, 1988.

Sundström, B., Mörnstad, H., Axéll, T. Oral

carcinomas associated with snuff dipping. Journal

of Oral Pathology 11: 245-251, 1982.
Smoking and Tobacco Control Monograph No. 2

78

This report of the oral consequences of smokeless tobacco use among 280 professional

Periodontal Effects Associated With

The Use of Smokeless Tobacco:

Results After 1 Year1

P.B. Robertson, V. Ernster, M. Walsh, J. Greene,

D. Grady, and W. Hauck

ABSTRACT

baseball players examined in both 1988 and 1989 compares findings in 133 non-users with no

mucosal changes, 63 users without ST-induced mucosal lesions, and 84 users with mucosal

lesions. Subjects completed questionnaires on smokeless tobacco use, rinsed their mouths

under supervision, and were cautioned not to discuss their use of tobacco with the dental

examiners. They then received an oral examination that included recording of all mucosal

abnormalities, missing teeth, decayed and filled surfaces, extrinsic stain, plaque index, gingival

index, pocket depth, attachment loss, and gingival recession. In addition, loss of cervical tooth

structure was assessed at the 1989 examinations. In both years, about 92 percent of mucosal

lesions in users affected the mandibular teeth at sites where the ST quid was placed. These sites

exhibited a significantly greater frequency of recession than did the sites not adjacent to lesions

in users or similar sites in non-users. By 1989, 36 percent of sites adjacent to lesions in users

showed at least 1 mm of recession and 25 percent showed at least 2 mm of recession, compared

with 12 and 3 percent, respectively, in non-users. Moreover, an apical shift in the position of

the gingival margin of users in sites adjacent to lesions averaged -0.36 mm during the 1-yr

period, whereas sites of non-users and users without lesions were unchanged. Loss of tooth

structure in areas of recession appeared to result from mechanical abrasion rather than

chemical erosion from products of smokeless tobacco, because exposed root surfaces of nonusers were affected by cervical depressions as often as sites adjacent to lesions. Compared with

sites of non-users, extrinsic stain was more frequent in sites adjacent to lesions in users, but

more than 80 percent of these sites did not show stain. Missing teeth, previous caries experience, levels of plaque and gingivitis, pocket depths, and occurrence of severe forms of periodontitis were not related to ST use.

INTRODUCTION We previously described the oral consequences of smokeless

tobacco use among baseball players examined during the 1988 spring

training season in Arizona (Ernster et al., 1990; Grady et al., 1990; Robertson

et al., 1990). More than 50 percent of these team members reported using

ST, and 39 percent were using ST during the week prior to the examinations.

Among these current-week users, 46 percent had oral mucosal lesions,

primarily in mandibular sites where the smokeless tobacco quid was placed.

Poor oral hygiene and gingivitis were not related to the development of oral

lesions. All periodontal measurements on mesial surfaces were similar, and

severe forms of periodontal disease were equally rare in non-users, users

without lesions, and users with lesions. However, buccal sites adjacent to

mucosal lesions in ST users showed significantly greater recession and

related attachment loss than in sites not adjacent to lesions in users or

comparable sites in non-users.

1 Supported by National Institute of Dental Research grant no. DE-08547.
Chapter 2

79

The study reported here compared changes in oral and periodontal

measurements among smokeless tobacco non-users, ST users without mucosal lesions, and ST users with mucosal lesions, who were examined in both

1988 and 1989.

MATERIALS AND

METHODS

The subjects were 280 Major League and minor league baseball

team members who were examined during both the 1988 and

1989 spring training seasons in Arizona. The study protocol has been described in detail (Ernster et al., 1990; Robertson et al., 1990). Briefly, each of

the subjects completed a comprehensive questionnaire that included a basic

demographic profile and dental history and elicited information on the use

of smokeless tobacco or other tobacco products. The 1988 examinations

included cotinine and thiocyanate determinations to confirm self-reported

tobacco use and showed that the self-report was highly accurate in defining

use status. Before entering the oral examination area, the subjects were asked

not to discuss their use of tobacco with the oral examiners, and all subjects

rinsed their mouths repeatedly with water to ensure that no traces of ST

remained in the mouth. Mucosal and gingival pathology were recorded,

including a complete description of oral lesions. This analysis considers

measurements obtained on the buccal surfaces of a subset of 12 teeth: maxillary and mandibular first molars (tooth numbers 3, 14, 19, 30), maxillary and

mandibular first premolars (tooth numbers 5, 12, 21, 28), and maxillary and

mandibular central incisors (tooth numbers 8, 9, 24, 25). Measurements in

both years included the presence or absence of caries, restorations, and

extrinsic stain. The plaque index (PI) (Silness and Löe, 1964), gingival index

(GI) (Löe and Silness, 1963), pocket depth measured from the gingival margin, and position of the gingival margin measured from the cementoenamel

junction were recorded. All probing measurements were recorded to the

nearest millimeter. Attachment levels were derived from the pocket depth

and gingival margin measurements. Recession was defined as displacement

of the gingival margin at least 1 mm and at least 2 mm apical to the

cementoenamel junction. In addition, the presence of cervical erosion or

abrasion was recorded in 1989 because loss of tooth structure was frequently

observed in association with areas of recession during the 1988 examinations.

We recorded all buccal sites that showed a loss of tooth structure resulting in

smooth, hard depressions adjacent to the cementoenamel junction. No

distinction was made between loss of tooth substance by a chemical process

(erosion) or by excessive mechanical wear (abrasion).

Prior to both spring seasons, the examiners participated in an extensive

training and calibration exercise that included patients with stain, cervical

caries, and moderate adult periodontitis with some areas of recession. Differences between examiners were discussed and resolved and the examination

process was repeated until at least 90 percent agreement between any two

examiners was achieved for all measurements. A final calibration session was

conducted with 12 patients in 1988 and 10 patients in 1989. Average percentage of agreement between all examiners for buccal surfaces was essentially equal in both years and ranged from 98 percent (cervical caries) to

93 percent (PI). Average kappa statistics (Fleiss and Chilton, 1983) for any

measurement were never lower than 0.78.
Smoking and Tobacco Control Monograph No. 2

80

Measurements were expressed as the percentage of total sites that

showed caries, stain, cervical depressions, visible plaque (PI > 1), and gingival bleeding (GI > 1), and the mean and standard deviation for age, missing

teeth, percentage of decayed and filled teeth, pocket depth, attachment loss,

and position of the gingival margin. Where players were used as the unit of

analysis (non-users, users without lesions, and users with lesions), differences between categories were evaluated by χ2

, and differences between

mean scores were evaluated by Wilcoxon tests.

To assess the effects of ST on the tooth and adjacent tissue where the

tobacco quid was held, the data were also analyzed by tooth site in four

categories: sites in the mouths of subjects who were not using ST and did

not have any oral mucosal lesions in either 1988 or 1989 (non-user, no

lesion); sites in the mouths of subjects who used ST in 1988 and 1989 but

did not have any oral mucosal lesions in 1989 (user, no lesion); sites in the

mouths of subjects who used ST in 1988 and 1989 and had a mucosal lesion

in 1989 not adjacent to the site (user, nonadjacent lesion); and sites in the

mouths of subjects who used ST in 1988 and 1989 and had a lesion on the

mucosa in 1989 that was immediately adjacent to the site (user, adjacent

lesion). The analyses of differences between sites were evaluated by χ2,

which was corrected for the interclass correlation that resulted from use of

multiple tooth sites in the same player (Donner and Donald, 1988).

RESULTS Of 280 team members who were seen in 1988 and 1989, 133 subjects

were non-users of ST and showed no mucosal lesions, 63 subjects were users

but did not show a mucosal lesion in 1989, and 84 subjects were users and

did show a mucosal lesion in 1989. Of the 63 users without lesions in 1989,

46 had also been without a lesion in 1988, and 54 of the 84 users with

lesions in 1989 also had shown lesions in 1988. For 1989, the age, race,

average number of missing teeth, and percentage of all buccal surfaces that

were decayed or filled are shown in Table 1. There were no differences in

age among the three subject groups. A higher proportion of users than nonusers—with and without lesions—were white. Values for missing teeth and

previous caries experience were very low and reflected the excellent general

dental health and regular dental care exhibited by these team members.

About 65 percent of all subjects showed no missing teeth, and most of the

tooth loss in remaining subjects was associated with premolar extraction

prior to orthodontic therapy.

A total of 104 oral lesions were identified in the 84 ST users with lesions

in 1989, 67 of whom had 1 lesion, and 17 of whom had 2 or more separate

lesions. These lesions were characterized by white, slightly raised, and

irregularly corrugated changes in the mucobuccal fold, typical of ST use

(Grady et al., 1990; Greer and Poulson, 1983; Greer et al., 1986; Pindborg

and Renstrup, 1963). Although most lesions terminated coronally at the

mucogingival junction, the color of the adjacent gingiva was usually more

pale than surrounding areas. On average, each lesion involved 4.6 ±1.9

teeth. Lesion patterns among teeth of ST users with lesions in 1988 and

1989 are shown in Table 2. The majority of lesions in both years showed a

bilateral pattern involving the mandibular central incisors. The second

most common site for lesions was in the mandibular left quadrant, followed
Chapter 2

81

Table 1

Age, race, missing teeth, and decayed and filled teeth in 1989 among team

members examined in 1988 and 1989

 Race Decayed and

n Agea White Black Latino Missing Teetha Filled Teetha

Non-Users 133 25.7 ± 5.8 57% 26% 16% 3.0 ± 3.5 2.4 ± 3.8%

Users Without

 Lesions 63 25.5 ± 3.9 83 8 10 2.8 ± 3.6 2.9 ± 3.9

Users With

Lesions 84 25.3 ± 5.5 82 14 4 3.0 ± 3.8 3.6 ± 4.6

a Mean ± SD.

Table 2

Lesion patterns among teeth of smokeless tobacco users with lesions in 1988 and

1989

1988 1989

Bilateral Mandibular Anterior 46% 45%

Bilateral Mandibular Posterior 2 9

Unilateral Mandibular Right 16 18

Unilateral Mandibular Left 32 20

Maxillary 4 8

by the mandibular right quadrant. The maxilla was not a preferred site to

hold the tobacco quid, and maxillary lesions were infrequent. Despite

comments from users that they tried to move the quid around the mouth in

anticipation of the 1989 examinations, the general patterns of lesions in

both years remained fairly constant, although in 1989 there was a slight

shift of lesions to the maxilla.

The remaining dental and periodontal results are given only for the

mandibular teeth where more than 90 percent of mucosal lesions were

located. The percentage of mandibular surfaces that showed visible plaque,

gingival bleeding, and extrinsic stain for each site group in 1988 and 1989 is

shown in Figure 1. About 20 to 28 percent of all surfaces showed visible

plaque, and there were no large or statistically significant differences between the groups in either year, nor were there significant changes from

1988 to 1989 in any group. A similar lack of significant differences was

observed in gingival bleeding, which occurred in < 10 percent of
Smoking and Tobacco Control Monograph No. 2

82

Figure 1

The percentage of mandibular buccal surfaces in 1988 and 1989 that showed visible plaque

(PI > 1), gingival bleeding (GI > 1), and extrinsic stain for sites in non-users with no STinduced mucosal lesions in 1989 (A), sites in users with no lesion (B), sites in users with a

lesion elsewhere but not adjacent to the site (C), and sites in users with a lesion immediately

adjacent to the site (D).

 * Significant difference from non-user with no lesion sites.

surfaces in all groups. Severe forms of gingivitis were observed infrequently

and were equally distributed among subject groups. However, sites in users

with lesions had a significantly greater frequency of extrinsic stain than sites

in non-users at both the 1988 (p=0.04) and 1989 (p=0.03) examinations.

Extrinsic stain was seen most frequently in sites with an adjacent lesion, and

the number of these sites that exhibited stain increased significantly

(p=0.01) from the 1988 to 1989 examination.

Because probing measurements were made to the nearest millimeter, we

were concerned that, in cases where the gingival margin was positioned near

the cementoenamel junction, very slight apical or coronal movement might

distort the change in number of sites defined as showing recession. Thus, in

Figure 2 we have expressed recession as the percentage of all mandibular

surfaces with at least 1 mm, and at least 2 mm of displacement of the

gingival margin apical to the cementoenamel junction for each site group at

both examinations. Using the 1-mm threshold, we observed a significant

increase (p=0.04) relative to non-users in the percentage of sites with recession in sites of users with nonadjacent lesions in 1989 and sites of users with
Chapter 2

83

an adjacent lesion in 1988 and 1989 (p=0.02). In addition, nonadjacent and

adjacent sites showed significant increases (p=0.01) in the frequency of

recession from 1988 to 1989. Using the 2-mm threshold, we found sites

adjacent to lesions at both examinations showed a significant increase

(p=0.03) in the frequency of recession compared with non-users. The

frequency of recession in these sites also increased significantly (p=0.01)

from 1988 to 1989. In general, incisor and premolar teeth showed more

recession than molar teeth in all site categories. However, patterns of

recession for incisor, premolar, and molar teeth were similar to those shown

in Figure 2, at both the 1-mm and 2-mm thresholds. All tooth types adjacent to ST-induced lesions showed a significantly greater frequency of

recession than did the same tooth types in non-users, users without lesions,

and users with a lesion not adjacent to the tooth.

The average position of the gingival margin relative to the cementoenamel junction in 1988 was 0.5±0.8 mm for sites of non-users, 0.4±0.8 mm

for sites of users without lesions, 0.4±0.9 mm for sites not adjacent to

lesions in users, and 0.0±0.8 mm for sites adjacent to lesions in users. In

1989, no changes were observed in the average position of the gingival

margin in the same sites of non-users or users without lesions. Sites in users

with lesions that were not adjacent to the lesion showed a slight apical

displacement (-0.11±0.5 mm) during the 1-yr period. However, a significant

(p=0.02) apical shift in the gingival margin, which averaged -0.36±0.6 mm

during the 1-yr period, was observed in sites adjacent to lesions in users. By

1989, 36 percent of sites adjacent to lesions in users showed at least 1 mm of

recession and 25 percent showed 2 mm of recession compared with 12 and

3 percent, respectively, of sites with recession in non-users. About 98 percent of sites in non-users and all sites in users with 2 mm of recession in

1988 also showed recession in 1989.

Loss of cervical tooth structure recorded at the 1989 examination

occurred primarily on the root surface in areas of recession. The majority

showed hard, smooth, and often highly polished depressions in the root

surface that were clinically consistent with mechanical abrasion. The

percentage of all mandibular buccal surfaces with loss of cervical tooth

substance and the percentage of mandibular buccal surfaces with recession

that also showed loss of tooth substance is given in Figure 3. Cervical

depressions were more frequent in sites of users with an adjacent lesion than

all other sites, although differences were not statistically significant. The

finding was consistent with the higher frequency of recession in sites

adjacent to lesions. However, sites with recession that also showed loss of

cervical tooth substance occurred at least as often in non-users as in users

with and without lesions, and there appeared to be no relationship between

ST use and cervical depressions in sites with existing recession. Because

these cervical depressions were common on exposed root surfaces of all

subjects and were seen as often in sites that were not adjacent to ST-induced

mucosal lesions as in lesion-adjacent sites, we have used the term abrasion in

Figure 3 to suggest that this loss of tooth substance is not related to chemical decalcification from products of smokeless tobacco.
Smoking and Tobacco Control Monograph No. 2

84

Figure 2

The percentage of mandibular buccal surfaces in 1988 and 1989 that showed recession of

1 mm or greater and recession of 2 mm or greater for the categories of tooth sites described

in Figure 1.

No significant differences in pocket depth were found among the four

groups of sites, and the average pocket depth for all subjects was

1.7±0.6 mm. Fewer than 1 percent of all buccal sites showed pocket depths

> 6 mm, which were distributed proportionally among sites in non-users,

and in users with and without lesions. About 95 percent of attachment loss

observed in the subjects was a function of recession, and no severe forms of

early onset or rapidly progressive periodontal disease were observed in any

of the team members.

* Significant difference from non-user with no lesion sites.

** Significant difference from non-user with no lesion sites as well as from 1988 to 1989.

DISCUSSION The primary oral consequences of using ST for at least 1 yr are increased risk of mucosal lesions among mandibular teeth where the quid is

most often placed and increased frequency of gingival recession affecting

buccal tooth surfaces adjacent to ST-induced lesions. Compared with sites

of non-users, extrinsic stain was also more frequent in ST users, adjacent to

lesions, but more than 80 percent of these sites did not show stain. Missing

teeth, previous caries experience, levels of plaque and gingivitis, pocket

depths, and occurrence of severe forms of periodontitis were not related to

the use of smokeless tobacco.
Chapter 2

85

Figure 3

Percentage of mandibular buccal sites that showed abrasion in 1989 and the

percentage of mandibular buccal sites with recession ≥ 1 mm that also showed

abrasion in 1989 for the tooth sites in Figure 1.

Recession associated with ST use is permanent and progressive, and it

affects about one-quarter to one-third of sites with adjacent mucosal lesions.

In the 1-yr period, few new areas of recession were observed in non-users,

whereas about 20 percent of sites adjacent to lesions in ST users showed

recession in 1989 but not 1988. The recession presumably results from

localized damage to gingival tissue by products of the ST quid, particularly

in areas with a thin or absent alveolar housing (Löst, 1984; O’Leary et al.,

1971; Robertson et al., 1990). In addition to esthetic problems, these areas

of recession may develop thermal sensitivity and root caries (Gorman,

1967).

Exposed root surfaces are also at risk for abrasion or erosion. Abrasion

is the loss of tooth substance by abnormal mechanical wear, primarily as a

result of toothbrushing or use of highly abrasive dentifrices. Erosion is the

loss of tooth substance by chemical processes that do not involve bacterial

action. We had hypothesized that products of the ST quid, held against the

surfaces of the teeth for considerable periods of time, might result in loss of

some tooth structure by erosion. This seems not to be the case, however, as

loss of enamel was rare in all groups, and exposed root surfaces of non-users

and users without lesions were affected by cervical depressions at least as

often as sites adjacent to lesions. Thus, this loss of root structure appears to

be caused by abrasion.
Smoking and Tobacco Control Monograph No. 2

86

We conclude that, although ST use in young subjects with good oral

hygiene and regular professional care does not result in severe forms of

periodontal disease, such use is associated with a significantly increased risk

for permanent and progressive recession in areas adjacent to ST-induced

mucosal lesions.

ACKNOWLEDGMENTS We gratefully acknowledge the support of the Office of the Commissioner

of Baseball and the management, trainers, coaches, and players of the California Angels,

Chicago Cubs, Cleveland Indians, Milwaukee Brewers, Oakland Athletics, San Francisco Giants,

and Seattle Mariners. We also acknowledge the invaluable efforts of Jana Murray for coordinating the study, Joan Drues for data entry and management, and students in the periodontology

graduate and dental hygiene programs at UCSF who played a major role in all phases of the

studies in Arizona.

REFERENCES

Greer, R.O., Poulson, T.C., Boone, M.E., et al.

Smokeless tobacco-associated oral changes in

juvenile adult and geriatric patients: Clinical and

histomorphic features. Gerodontics 2: 87-98, 1986.

Löe, H., Silness, J. Periodontal disease in pregnancy:

I. Prevalence and severity. Acta Odontologica

Scandinavica 21: 533-551, 1963.

Löst, C. Depth of alveolar bone dehiscences in

relation to gingival recessions. Journal of Clinical

Periodontology (Copenhagen) 11: 583-589, 1984.

O’Leary, T.J., Drake, R.B., Crump, P.P., et al. The

incidence of recession in young males: A further

study. Journal of Periodontology 42: 264-275, 1971.

Pindborg, J.J., Renstrup, G. Studies in oral leukoplakias: II. Effect of snuff on oral epithelium.

Acta Dermato-Venereologica (Stockholm) 43: 271-

276, 1963.

Robertson, P.B., Walsh, M., Greene, J., et al.

Periodontal effects associated with the use of

smokeless tobacco. Journal of Periodontology 61:

438-443, 1990.

Silness, J., Löe, H. Periodontal disease in pregnancy:

II. Correlation between oral hygiene and

periodontal condition. Acta Odontologica

Scandinavica 22: 121-135, 1964.

Donner, A., Donald, A. The statistical analysis of

multiple binary measurements. Journal of Clinical

Epidemiology 9: 899-904, 1988.

Ernster, V.L., Grady, D., Greene, J.C., Walsh, M.,

Robertson, P.B., Daniels, T., Benowitz, N., Siegel,

D., Gerbut, B., Hauck, W. Smokeless tobacco:

Prevalence of use and health effects among

professional baseball players. Journal of the

American Medical Association 264: 218-224, 1990.

Fleiss, J.L., Chilton, N.W. The measurement of

interexaminer agreement on periodontal disease.

Journal of Periodontal Research 18: 601-608, 1983.

Gorman, W.J. Prevalence and etiology of gingival

recession. Journal of Periodontology 38: 316-340,

1967.

Grady, D., Greene, J., Daniels, T.E., Ernster, V.L.,

Robertson, P.B., Hauck, W., Greenspan, D.,

Greenspan, J.S., Silverman, S. Oral mucosal lesions

in smokeless tobacco users. Journal of the American

Dental Association 121: 117-123, 1990.

Greer, R.O., Poulson, T.C. Oral tissue alterations

associated with the use of smokeless tobacco by

teenagers. Oral Surgery, Oral Medicine, Oral

Pathology 56: 275-284, 1983.
Chapter 3

87

Chapter 3

Carcinogenesis

CONTENTS Oral Carcinoma and Smokeless Tobacco Use:

A Clinical Profile

W. Frederick McGuirt and Anna Wray .................................................. 91

Introduction .................................................................................... 91

Patients ............................................................................................ 91

Field Cancerization ......................................................................... 92

Discussion........................................................................................ 93

References ........................................................................................ 95

Chemical Composition of Smokeless Tobacco Products

Klaus D. Brunnemann and Dietrich Hoffmann ..................................... 96

Introduction .................................................................................... 96

Chemical Composition ................................................................... 97

Carcinogenic Agents in ST .............................................................. 97

Carcinogenic N-Nitrosamines ....................................................... 100

TSNA .............................................................................................. 101

Control of Carcinogens in ST ....................................................... 104

References ...................................................................................... 106

Carcinogenesis of Smokeless Tobacco

Dietrich Hoffmann, Abraham Rivenson, and Stephen S. Hecht ......... 109

Introduction .................................................................................. 109

Carcinogens in Smokeless Tobacco .............................................. 111

N-Nitrosamines ............................................................................. 113

TSNA .............................................................................................. 113

Discussion...................................................................................... 115

References ...................................................................................... 116

Oncogenes in Head and Neck Cancer

Kenneth D. Somers, Suzanne E. Glickman, Suzanne L. Cartwright,

Bryan G. Stoll, Stella L. Maurizio, and Gary L. Schechter .................... 119

Introduction .................................................................................. 119

Materials and Methods.................................................................. 120

Results............................................................................................ 120

Discussion...................................................................................... 123

References ...................................................................................... 126
Smoking and Tobacco Control Monograph No. 2

88

Oncogenes in Tobacco-Induced Oral Cancer

Madhav G. Deo and Dhananjava Saranath ......................................... 128

Introduction .................................................................................. 128

Oncogene Amplification ............................................................... 129

Expression of Cellular Oncogenes ................................................ 132

RFLP Analysis of L-myc and H-ras Oncogenes .............................. 133

H-ras Point Mutations ................................................................... 134

Identification of a Functional Oncogenes .................................... 136

Conclusions and Future Prospects ................................................ 137

References ...................................................................................... 139

Metabolism and Macromolecular Binding of NNK and

NNN, Important Carcinogens in Smokeless Tobacco

Stephen S. Hecht, Steven G. Carmella, and Sharon E. Murphy .......... 141

Introduction .................................................................................. 141

Metabolism of NNK and NNN ...................................................... 142

Detection of NNK/NNN-Hemoglobin Adducts............................. 146

Gaps in Assessing the Role of NNK and NNN .............................. 150

Summary ....................................................................................... 151

References ...................................................................................... 151

Role of Nicotine as a Cofactor in

Smokeless Tobacco Carcinogenesis

Christopher A. Squier and Georgia K. Johnson ................................... 153

Introduction .................................................................................. 153

In Vivo Studies with Nicotine ....................................................... 153

In Vitro Studies with Nicotine ...................................................... 154

Clinical Implications of Studies with Nicotine............................. 155

Future Directions........................................................................... 159

References ...................................................................................... 159

Perinatal Carcinogenesis by Constituents of Smokeless

Tobacco: Animal Models and Potential Human Risk

Lucy M. Anderson and Jerry M. Rice.................................................... 161

Introduction .................................................................................. 161

Sensitivity and Susceptibility Factors ............................................ 161

Tobacco-Specific Nitrosamines in Animal Models ....................... 163

Other ST Constituents................................................................... 165

Potential Risk and Future Research ............................................... 166

References ...................................................................................... 169

Interaction Between Smokeless Tobacco-Related

Carcinogens and Human Papillomaviruses in the

Pathogenesis of Oral Dysplasia and Cancer

Joel M. Palefsky, John S. Greeenspan, Tray E. Daniels,

Jennifer Berline, Deborah G. Grady, and Virginia Ernster................... 175

Introduction .................................................................................. 175

Methods......................................................................................... 177

Results............................................................................................ 178

Discussion...................................................................................... 178

References ...................................................................................... 181
Chapter 3

89

Identification of Human Papillomavirus DNA in

Smokeless Tobacco Keratosis and Premalignant and

Malignant Oral Lesions, by PCR Amplification

with Consensus Sequence Primus

Robert O. Greer, Jr., Kenneth R. Shroyer, and Louise Crosby.............. 183

Introduction .................................................................................. 183

Materials and Methods.................................................................. 184

PCR ................................................................................................ 184

Results............................................................................................ 185

Discussion...................................................................................... 185

References ...................................................................................... 189

Lipids as Factors in the Cell Response to

Tobacco Components

George S. Schuster, Scott Lubas, Thomas R. Dirksen, and

John F. Erbland ..................................................................................... 191

Introduction .................................................................................. 191

Methods......................................................................................... 193

PKC Results .................................................................................... 194

Discussion...................................................................................... 197

References ...................................................................................... 200

Role of Viruses in Oral Carcinogenesis

No-Hee Park, Byung-Moo Min, Sheng-Lin Li, Henry M. Cherrick,

and Jay Doniger .................................................................................... 202

Introduction .................................................................................. 202

Methods......................................................................................... 203

Results............................................................................................ 206

Discussion...................................................................................... 211

References ...................................................................................... 214
Smoking and Tobacco Control Monograph No. 2

90
Chapter 3

91

The clinical profile of 116 patients with oral cavity cancer who used smokeless tobacco

Oral Carcinoma and Smokeless Tobacco

Use: A Clinical Profile

W. Frederick McGuirt and Anna Wray

ABSTRACT

INTRODUCTION

PATIENTS

exclusive of other known carcinogens, such as smoking tobacco and alcohol, is discussed. The

patients, whose average age was 78.4 yr and average use 55.5 yr, had a 1:23 male-to-female ratio

and a 14.5:1 white-to-black ratio, both different from national rates for patients with oral cavity

cancers. The 116 patients’ course illustrates the field cancerization concept: In addition to the

referral lesion, 55 percent (64/116) had leukoplakia, erythroplasia, dysplasia, or carcinoma in

situ previously, at the time of presentation, or after initial therapy. A second malignant oral

lesion developed in 18 percent (21/116); 35 percent (41/116) had local recurrence despite

predominantly clear margins earlier. Of the 91 patients with documented followup, 45 died of

or with cancer. These distressing figures may, unfortunately, be duplicated in another 20 to 40

years in a different population group—current young male smokeless tobacco users.

Not only is tobacco an integral part of the local economy in North

Carolina, but also its use in all forms is pervasive in the population served

by the Wake Forest University Medical Center. Smokeless tobacco use,

especially, is endemic to the three or four generations living in our geographic region. Review of our tumor registry files yielded 150 cases of oral

cavity cancer in smokeless tobacco users—4.9 percent of our total head and

neck cancer caseload. Smokeless tobacco was used exclusive of other known

oral carcinogens, such as smoking tobacco and alcohol, by 116 of the 150

patients. These 116 cases form the basis of this report, in which we profile

the patients, the nature of the disease process, its treatment, and outcome.

The patient population reported here differed in several ways from the

national profile of oral cancer patients. Oral cavity carcinoma in the United

States has a 2:1 male-to-female ratio (Boring et al., 1991). This ratio is consistent with our new head and neck cancer patient population (1,393/699) but

higher than our oral cancer male-to-female ratio of 483/361 (1.3:1). This oral

cavity cancer ratio was strongly influenced (that is, reduced) by our patients

who used smokeless tobacco only. This group of patients had a 1:23 male-tofemale ratio, representing disease occurrence almost exclusively in females.

A second epidemiological aberration seen in the pool of smokeless

tobacco-associated oral cancers relates to the most common tumor sites.

The mobile tongue and floor of the mouth are the most common sites of

oral cavity carcinoma. In our series of smokeless-tobacco-only users, the

cancers occurred most frequently in the buccal and gingival areas, the

regions where the quid was held. Ninety-four percent of patients (109/116)

had their cancers at these sites.

The average age of these patients was 78.4 yr, with 83 percent (96/116)

older than 65 yr and 27 percent (31/116) older than 80 yr. These patients

were older than the overall head and neck cancer patients seen in our

institution, who averaged 63 yr during the same study period.
Smoking and Tobacco Control Monograph No. 2

92

The duration of smokeless tobacco use before presentation for treatment averaged 55.5 yr, with 81 percent of patients (86/106) having a history

of use > 40 yr.

A further epidemiological aberration occurred in the racial distribution

of patients. A 14.5:1 (116:8) ratio of white to black was seen, compared with

a 7.5:1 (1,845:247) ratio of all head and neck cancer cases and a population

distribution frequency of 3.5:1 in the geographic region served (U.S. Census

figures, 1990 to 1991). These figures support the epidemiological study of

Winn et al. (1981) from the same geographic region but a different referral

base. The use of snuff—and cancer cases attributed to it—was reported by

Winn and coworkers to be a phenomenon of white women.

These factors of sex, race, and age are results of sociological trends well

recognized and reported previously. Snuff use in the region until the mid1970’s was predominantly a practice of white females, who began to use

snuff at an early age in the 1930’s and 1940’s. This was a period of declining smokeless tobacco use and increased smoking. The social attitudes of

that time discouraged women’s smoking, but in the rural South, women

continued privately to use smokeless tobacco. Dry, powdered snuff (i.e.,

Scotch snuff) has been the predominant form used by these women, in

contrast to the flavored moist strips of tobacco preferred by today’s young

male users.

Whereas the now elderly women with long-term smokeless tobacco use

form the patient population reported here, the obvious concern is that the

young males currently using smokeless tobacco may well be the future

patients who will be profiled if they continue their chronic use. The addictive qualities of the product would imply this to be the expected outcome.

Oral carcinoma in a high school athlete from Oklahoma who was a

regular user of smokeless tobacco has become the focal point for an educational effort spearheaded by the American Academy of Otolaryngology-Head

and Neck Surgery (AAO-HNS). Oral carcinoma in the male population

under age 30 has not yet been seen by personal experience or become a

national problem, according to a questionnaire mailed to members of the

American Society of Head and Neck Surgery and selected AAO-HNS members, who are the physicians most likely to be treating such patients. The

youngest patient to date in our experience was aged 40; the next youngest,

aged 43.

FIELD CANCERIZATIONPatients with cancers attributed to smokeless tobacco illustrate the

field cancerization concept. In addition to the referral lesion,

adjacent and distant mucosal lesions exist synchronously or develop

metachronously. In the field cancerization model, a diffuse surface area of

mucous membrane is bathed by the carcinogens and placed at risk. Logically, the highest risk area for smokeless tobacco use is where the quid is

held, but adjacent areas and distal sites are continuously bathed by saliva

containing the carcinogenic agents that leach from the quid. Our patients

with smokeless tobacco-related oral cancers often exhibited adjacent and

distant mucous membrane changes. Varying degrees of abnormality,

compatible with the evolutionary transformation to malignancy, were seen.
Chapter 3

93

These abnormal changes included clinical leukoplakia and erythroplasia as

well as histologically varying degrees of dysplasia and carcinoma in situ.

Fifty-five percent of patients in our series (64/116) had had these types of

lesions excised previously, had them at the time of their presentation, or

developed them subsequent to therapy. Further evidence for an evolutionary transformation of a condemned mucosa was the high incidence (18

percent) of second or metachronous malignant oral lesions that developed

in these patients. A third indicator of this evolutionary transformation was

the 35 percent incidence (41/116) of local cancer recurrence, despite predominantly clear margins on histological examination (28 of 33 surgical

specimens, 85 percent). These lesions were most likely not recurrences of

the original cancer but, rather, malignant transformations of previously

exposed mucous membranes at risk for new cancers in the same region.

DISCUSSION The disease course is most often silently and indolently progressive.

Patients may note a change or abnormality within the mouth, but the symptoms are usually minimal and a delay in seeking evaluation occurs. Fortythree percent of patients (48/111) had symptoms for 3 mo or longer before

presentation. The silent but progressive nature of the disease is further

illustrated by the 53 percent (62/116) incidence of advanced stage III and

stage IV lesions at the time of diagnosis, of which one-third (34 percent,

21/62) had been symptomatic for less than 2 mo.

Although the premalignant changes of leukoplakia and erythroplasia

have a slow transformation and progression, with only minimal symptoms,

once the smokeless tobacco-related cancers are established, they act in aggressive fashion, similar to other oral cavity cancers. This is confirmed by the

high incidence of stage III and stage IV disease. The advanced stages were

attributable to regional metastases in 27 percent of the cases, to locally

advanced disease (T3-T4) in 43 percent of the cases, and in many cases to

both characteristics.

Although many of these smokeless tobacco-related tumors have a

verruciform appearance, they should never be considered, on clinical

grounds alone, to be the less aggressive verrucous carcinoma described

histologically by Ackerman (1948). Although 24 percent (28/116) of our

patients had verruciform lesions, less than one-fifth of those, or 4.3 percent

overall (5/116), had verrucous carcinomas by the histological criteria of

Ackerman.

Localized stage I and stage II carcinomas may be treated equally well

with either surgery or irradiation. Therapy of advanced stage III and stage IV

cases usually required both surgery and irradiation. The surgical therapy in

advanced stage lesions may result in a significant alteration in form and

function of the oral cavity. The major problems of postsurgical resection

relate to the loss of the mandibular-dental function of mastication and the

oral-buccal-lingual function of deglutition. The addition of irradiation to

surgery in these advanced cases or as the primary modality of therapy for

early lesions causes morbidity related to the resulting xerostomia and the

lifelong need for frequent dental care and rehabilitation. The use of irradiation and the advanced age of the patients reduce the success rate of mandibular reconstruction.
Smoking and Tobacco Control Monograph No. 2

94

Mandibular resection results in cosmetic disfigurement, the degree

usually being directly proportional to the amount of bone resected and how

far anteriorly the resection proceeds. In general, lateral mandibular defects

are usually left unreconstructed. Anterior defects, though, require reconstruction, not only to correct severe cosmetic problems, but also to correct

basic functional problems. Various reconstructive methods and synthetic

replacement prostheses have been used. Our current preference is to use

microvascular anastomotic techniques for restoration by a composite free

flap. This provides soft-tissue support and bone with its own direct blood

supply.

There is a general lack of success in fitting dentures that allow adequate

mastication for solid food, even when the mandibular defect is restored by

bone grafting or a metal bar. The future use of implanted prosthetic devices

holds promise for nonirradiated cases, but such devices are still contraindicated in irradiated bone.

Intraoral soft tissue loss has been handled in various ways. In the

majority of our cases, primary closure of the defect is performed, because

patients have better function if sensate, normally lubricated, moist membranes are present. Tethering of the tongue is to be avoided if possible. The

tongue dysfunction is more often seen secondary to reconstruction of the

floor of the mouth. Lateral tongue flaps and skin grafts used to close the

anterior and lateral floor-of-mouth defects frequently contract and form scar

tissue that restricts movement of the oral structures. When oral tissue must

be replaced, use of the platysma myocutaneous flap is our most effective

method because of the pliability and thinness of the flap tissue. Unless the

resection has been quite extensive, other myocutaneous flaps often result in

an adynamic and obstructive tissue mass in the oral cavity.

Long-term followup was available for 91 patients; 49.5 percent of these

(45/91) have died of or with cancer. There was a linear survival rate correlating to the stage of lesion, and survival was better for patients with buccal

lesions than for those with alveolar lesions.

In summary, oral cavity carcinoma associated with smokeless tobacco

use has been a disease of elderly, white women with histories of long-term

(> 40 yr) snuff use. The disease is indolent and progressive, and it manifests

a high rate of associated leukoplakia, metachronous second primary cancers

of the oral cavity, and a high local recurrence rate, all manifestations of the

field cancerization phenomenon. The tumor is aggressive, with a high

incidence of advanced local disease, bony mandibular involvement, and

regional metastases. A midrange (43 percent) 3-yr cure rate results. Therapy

should be aggressive, but it is associated with significant morbidity related

to cosmetic effects, mastication, deglutition, xerostomia, and dental complications. Prevention through education of young potential users of smokeless tobacco promises to be the most effective therapeutic measure.
Chapter 3

95

REFERENCES

Ackerman, L.V. Verrucous carcinoma of the oral

cavity. Surgery 23: 670-678, 1948.

Boring C.C., Squires T.S., Tung T. Cancer statistics,

1991. CA—A Journal for Clinicians 41: 19-36, 1991.

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W.,

Toledo, A., Fraumani, J.F., Jr. Snuff dipping and

oral cancer among women in the southern United

States. New England Journal of Medicine 304: 745-

749, 1981.
Smoking and Tobacco Control Monograph No. 2

96

To date, 28 carcinogens have been identified in smokeless tobacco. In addition to certain

Chemical Composition of

Smokeless Tobacco Products1

Klaus D. Brunnemann and Dietrich Hoffmann

ABSTRACT

volatile aldehydes, traces of benzo[a]pyrene, certain lactones, urethan, hydrazine, metals,

polonium-210, and uranium-235 and -238 can be found in ST. However, the major contributors to the carcinogenicity of chewing tobacco and of snuff are the N-nitrosamines, especially

the tobacco-specific N-nitrosamines. The latter are formed from the Nicotiana alkaloids during

tobacco processing. In the United States, daily exposure to carcinogenic nitrosamines for snuff

users is at least 250 times higher than for those who do not use tobacco. Although there has

been a decline in the concentrations of nitrosamines in U.S. and Swedish ST products during

the past decade, this trend is not evident for all snuff brands. One new snuff brand contains

extremely high concentrations of carcinogenic nitrosamines. This observation adds to the

urgency of the recommendation of the World Health Organization to regulate harmful

substances in chewing tobacco and snuff. Similarly, flavorants and additives to tobacco should

be controlled.

INTRODUCTION In the United States, we differentiate between four primary types

of smokeless tobacco: Three are chewing tobaccos, namely loose leaf (scrap

leaf), plug, and twist or roll; the fourth is oral snuff. Loose leaf chewing

tobacco accounted for 52.7 percent of the U.S. output of total ST products in

1988 (124.5 million lb) (USDA, 1990). Loose leaf chewing tobacco consists

primarily of air-cured tobacco and, in most cases, is heavily treated with

licorice and sugars. Plug tobacco (7.2 percent of 1988 ST production) is the

oldest form of chewing tobacco. Plug tobacco is produced from the heavier

grades of leaves harvested from the top of the plant, freed from stems,

immersed in a mixture of licorice and sugar, pressed into a plug, covered by

a wrapper leaf, and reshaped. Plug tobacco is kept between cheek and gum

and is chewed in bites. Twist or roll tobacco is less important (1.1 percent of

1988 U.S. production). Twist tobacco is made from cured burley, and airand fire-cured leaves, which are flavored and twisted to resemble a decorative rope or pigtail.

The only U.S. tobacco product with increasing consumption is oral

snuff (39.0 percent of the U.S. smokeless tobacco production in 1988). Dry

snuff is made primarily from Kentucky and Tennessee fire-cured tobaccos.

The initial curing requires several weeks and goes through multiple phases.

In contrast to most other tobacco products, snuff undergoes an additional

fermentation process. Dry snuff is processed into a powdered substance that

may contain flavor and aroma additives, including spices. U.S. dry snuff,

which is taken orally, is similar to European nasal snuff.

Moist snuff consists primarily of air- and fire-cured tobaccos and contains tobacco stems as well as leaves that are powdered into fine particles

(containing between 20 and 55 percent moisture). Many brands of moist

1

 Supported by National Cancer Institute grant no. CA-29580.
Chapter 3

97

snuff are flavored with wintergreen, but mint and raspberry also are popular.

Since about 1975, the consumption of moist snuff has been steadily growing

in all parts of the United States, except for a temporary decline immediately

after the Surgeon General’s report on smokeless tobacco in 1986 (US DHHS,

1986). During the past 3 yr, the manufacture of moist snuff has again

steadily risen by more than 13 percent (Smyth, 1989; USDA, 1990). Oral use

of snuff, also termed “snuff dipping,” means placing a pinch of the tobacco

between the cheek or lip and the gums or beneath the tongue.

CHEMICAL

COMPOSITION

Extensive literature on the chemistry of tobacco, beginning with

Brückner (1936), has led to our current knowledge that natural

tobacco contains at least 3,050 different components (Robert, 1988). The

quantitative composition of tobacco undergoes substantial changes during

processing for smokeless tobaccos. In curing, the starch content of the

leaves declines drastically, and the reducing sugars increase by 100 percent.

Protein and nicotine decrease slightly. Fermentation of cured tobacco

causes the contents of carbohydrates and polyphenols in the leaves to

diminish. The bulk of the processed tobacco leaf before fermentation

consists of carbohydrates (about 50 percent) and proteins. Other major

components are alkaloids (0.5 to 5.0 percent) with nicotine as the predominant compound (85 to 95 percent of total alkaloids), terpenes (0.1 to 3.0

percent), polyphenols (0.5 to 4.5 percent), phytosterols (0.1 to 2.5 percent),

carboxylic acids (0.1 to 0.7 percent), alkanes (0.1 to 0.4 percent), aromatic

hydrocarbons, aldehydes, ketones, amines, nitriles, N- and O-heterocyclic

hydrocarbons, pesticides, alkali nitrates (0.01 to 5.00 percent), and at least

30 metallic compounds (International Agency for Research on Cancer, 1985;

Wynder and Hoffman, 1967). The given percentages apply to the Nicotiana

tabacum species, which is grown in North America and throughout the

world, but not to N. rustica, which is cultivated in parts of Eastern Europe

and Asia Minor. The leaves of N. rustica may contain up to 12 percent

nicotine (McMurtrey et al., 1942). Many ST formulations use plant extracts

or chemicals as flavoring agents (LaVoie et al., 1989; Mookherjee, 1988;

Robert, 1988; Sharma et al., 1991). Such additives may include methyl or

ethyl salicylate, β-citronellol, 1,8-cineole, menthol, benzyl benzoate, and

possibly coumarin (Figure 1) (LaVoie et al., 1989; Sharma et al., 1991).

However, most of the flavor additives are present in only small amounts;

their formulations remain trade secrets.

CARCINOGENIC

AGENTS IN ST

Until now, 28 tumorigenic agents have been isolated and identified in smokeless tobacco products (Table 1). These include some

carcinogenic polynuclear aromatic hydrocarbons (PAH), especially

benzo[a]pyrene (B[a]P). PAH originate primarily from polluted air

(Campbell and Lindsay, 1956 and 1957; Wynder and Hoffmann, 1967) and,

in the case of plug tobacco and snuff, probably also from fire-curing. In

fact, the highest reported values for B[a]P were found in snuff at levels of

up to 90 ppb (Ough, 1976).

The α- and β-angelica lactones have been reported in natural tobacco

(Robert, 1988). These tumorigenic agents may also be added to ST as part of

the flavoring mixtures made from plant extracts. A minor group of polyphenols in tobacco are the coumarins, of which scopoletin is the major
Smoking and Tobacco Control Monograph No. 2

98

Figure 1

Flavor compounds identified in snuff tobacco

representative (Figure 2) (Wynder and Hoffmann, 1967). Thus, it is not

surprising that tobacco also contains the parent compound, coumarin

(≤ 600 ppm). It is known that the fermentation of food and beverages leads

to the formation of urethan (Ough, 1976). Therefore, it is not unexpected

that burley tobacco, which is fermented, contains up to 400 ppm of urethan

(Schmoltz et al., 1978).

The most abundant carcinogens in smokeless tobacco are some volatile

aldehydes (Table 1). Although formaldehyde, acetaldehyde, and croton

aldehyde are weakly carcinogenic, they contribute most likely to the carcinogenic potential of smokeless tobacco (Weybrew and Stephens, 1962). It is
Chapter 3

99

Table 1

Carcinogenic agents in tobacco

Type of Tobaccoa Concentrationb (ng/g)

Benzo[a]pyrene NT, S > 0.1 - 90.0

α-Angelica Lactone NT present

β-Angelica Lactone NT present

Coumarin NT 600

Ethylcarbamate CT 310 - 375

Volatile Aldehydes

Formaldehyde NT, S 1,600 - 7,400

Acetaldehyde NT, S 1,400 - 27,400

Croton aldehyde S 200 - 2,400

Nitrosamines

Nitrosodimethylamine CT, S ND - 270

Nitrosopyrrolidine CT, S ND - 760

Nitrosopiperidine CT, S ND - 110

Nitrosomorpholine CT, S ND - 690

Nitrosodiethanolamine CT, S 40 - 6,800

Nitrosamino Acids

Nitrososarcosine S ND - 2,500

3-(Methylnitrosamino)- CT, S 200 - 65,700

propionic acid

4-(Methylnitrosamino)- CT, S ND - 9,100

butyric acid

Nitrosoazetadine-2- CT 4 - 140

carboxylic acid

Tobacco-Specific Nitrosamines

N'-Nitrosonornicotine CT, S 400 - 147,000

4-(Methylnitrosamino)-1- CT, S ND - 18,000

(3-pyridyl)-1-butanone

4-(Methylnitrosamino)-1- S present

(3-pyridyl)-1-butanol

N'-Nitrosoanabasine SM, S present - 560

Inorganic Compounds

Hydrazine SM 14 - 51

Arsenic NT 500 - 900

Nickel SM, S 180 - 2,700

Cadmium SM 700 - 790

(pCi/g)

Polonium-210 NT, S 0.16 - 1.22

Uranium-235 S 2.4

Uranium-238 S 1.91

a NT, natural tobacco; SM, smoking tobacco; S, snuff; CT, chewing tobacco.

b ND, not detected.
Smoking and Tobacco Control Monograph No. 2

100

Figure 2

Coumarins in tobacco

known that tobacco contains a sizeable spectrum of alkyl aldehydes, which

contribute to its scent. In commercial U.S. snuff brands, formaldehyde and

acetaldehyde were each found up to 7,400 ppb, and croton aldehyde up to

2,400 ppb (Sharma et al., 1991).

Both air- and fire-cured tobaccos contain hydrazine. In burley leaves

treated with the sucker growth inhibitor maleic hydrazide, the hydrazine

content was significantly higher (Liu et al., 1974). Like other plant products, tobacco contains trace amounts of nickel, cadmium, and arsenic.

These animal carcinogens were found in concentrations up to 2,700 ppb.

Uranium-235 and -238 were reported only in Indian snuff, each at about

2 pCi/g tobacco (Sharma et al., 1985). The radioactive polonium-210, which

decays to yield the human carcinogen radon, originates in U.S. tobacco

from soil that is fertilized with phosphates rich in radium-226 (Tso et al.,

1986), or from airborne particles that were taken up by the glandular hair

(trichomes) of the tobacco leaf (Martell, 1974). In U.S. commercial snuff, we

found between 0.16 and 1.22 pCi/g of polonium-210 (Hoffmann et al.,

1987).

CARCINOGENIC

N-NITROSAMINES

The most detailed studies on carcinogens in smokeless tobacco

have been reported for N-nitrosamines. These agents are present

in fresh green leaf in only minute amounts and are primarily formed during

curing, fermentation, and aging from secondary or tertiary amines and

nitrite or nitrogen oxides. Basically, in smokeless tobacco there are three

types of nitroso compounds: volatile nitrosamines, nitrosamino acids, and

tobacco-specific N-nitrosamines (TSNA). In addition, smokeless tobacco

contains N-nitrosodiethanolamine (NDELA), which is formed from

diethanolamine, a contamination product in tobacco. Table 2 presents data

on carcinogenic volatile N-nitrosamines (VNA) in various smokeless tobacco

types from the United States, Sweden, and other European countries

(Andersen et al., 1989; Brunnemann et al., 1985; Chamberlain et al., 1988;

Hoffman et al., 1987; International Agency for Research on Cancer, 1985;
Chapter 3

101

Table 2

Major volatile N-nitrosamines in smokeless tobacco, 1981 to 1990

Country Tobacco Samples NDMAa NPYRa NMORa

Type (n) (μg/kg) (μg/kg) (μg/kg)

United Moist snuff 32 3.8 -215.0 7.4 - 360.0 ND - 690.0

States Dry snuff 3 ND - 19.0 72.0 - 148.0 ND - 39.0

Chewing tobacco 6 64.0 0.8 0.6

Sweden Moist snuff 98 0.1 - 50.0 ND - 95.0 ND - 44.0

Chewing tobacco 4 0.2 0.8 0.4

Norway Moist snuff 2 130.0 8.9 32.0

Denmark Chewing tobacco 8 5.5 16.0 ND

United Nasal snuff 5 4.5 - 82.0 1.5 - 130.0 ND

Kingdom Moist snuff 7 6.0 - 82.0 64.0 - 860.0 ND - 1.5

Germany Nasal snuff 7 2.0 - 42.0 5.0 - 75.0 ND

a NDMA, nitrosodimethylamine; NPYR, nitrosopyrrolidine; NMOR, nitrosomorpholine;

ND, not detected. Single numbers represent mean of all samples.

Tricker and Preussmann, 1989). In general, the highest amounts of VNA are

found in moist and dry snuff, N-nitrosodimethylamine up to 265 ppb and

N-nitrosopyrrolidine up to 760 ppb. N-Nitrosomorpholine (NMOR), a

strong animal carcinogen, has been detected only in those U.S. snuff brands

that were packed in containers lined with a morpholine-containing wax

coating (Brunnemann et al., 1982).

Like volatile amines, the amino acids in tobacco, and probably also the

proteins with secondary amino groups, are amenable to N-nitrosation.

Since 1983, numerous studies have reported the presence of nitrosamino

acids in smokeless tobacco (Brunnemann et al., 1983; Djordjevic, 1989;

Ohshima, 1985; Tricker and Preussmann, 1989 and 1990). Until now, 10

nitrosamino acids have been identified in smokeless tobacco. Of these,

nitrosoproline, nitrosothioproline, and iso-NNAC are not carcinogenic;

nitrososarcosine, 3-(methylnitrosamino)propionic acid, 4-(methylnitrosamino)butyric acid, and N-nitrosoazetadine-2-carboxylic acid are known

carcinogens; and the remainder of the identified nitrosamino acids have so

far not been bioassayed. (See Table 3.) The concentration of the

nitrosamino acids depends on the nitrate or nitrite content of the tobacco as

well as on the processing and aging of the tobacco.

The most powerful carcinogens in smokeless tobacco derive from the

N-nitrosation of the Nicotiana alkaloids, especially from nicotine and

nornico-tine. They are formed during the curing, fermentation, and aging

of tobacco. These carcinogens are present in tobacco, tobacco smoke, and

TSNA
Smoking and Tobacco Control Monograph No. 2

102

Table 3

Major N-nitrosamino acids in smokeless tobacco, 1989 to 1991a

Country and Samples NSARb MNPAb MNBAb NPROb iso-NNACb

Tobacco Type (n) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g)

United States

Moist snuff 10 ND - 2.5 2.2 - 66.0 0.09 - 9.10 1.3 - 60.0 0.05 - 21.00

Chewing tobacco 1 nd 0.6 0.03 0.2 0.01

Dry snuff 3 nd 1.2 - 4.5 0.14 - 0.46 3.0 - 8.1 0.05 - 0.21

Sweden

Moist snuff 8 0.01 - 0.68 1.0 - 3.3 0.05 - 0.23 0.63 - 8.30 0.04 - 0.11

United Kingdom

Moist snuff 7 0.03 - 1.10 1.4 - 19.0 0.06 - 8.00 0.33 - 5.00 nd

Nasal snuff 5 ND - 0.04 1.0 - 2.8 0.10 - 0.28 2.7 - 8.7 nd

Germany

Nasal snuff 7 ND - 0.09 0.49 - 4.30 0.08 - 0.41 0.77 - 7.50 nd

a Adapted from Djordjevic et al., 1989; Hoffmann et al., 1991; Tricker and Preussmann, 1989.

b NSAR, N-nitrososarcosine; MNPA, 3-(methylnitrosamino)propionic acid; MNBA, 4-(methylnitrosamino)butyric acid; NPRO, N-nitrosoproline; iso-NNAC, 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid;

ND, not detected; nd, not determined.

in environmental tobacco smoke. Of the seven TSNA identified in ST

(Figure 3), N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-

(3-pyridyl)-1-butanone (NNK) are the only known carcinogens in tobacco

that induce oral tumors in laboratory animals. N'-nitrosoanabasine,

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 4-(methylnitrosamino)-

4-(3-pyridyl)butanol are carcinogenic in mice or rats (Hoffmann et al., this

volume). The high carcinogenic potency and high levels of TSNA have

prompted in-depth investigations on the formation and concentration of

the alkaloid-derived nitrosamines in the various tobacco products

(Table 4). As for the other nitrosamines, the nitrate or nitrite content and

the various steps of processing are the determining factors for the yields of

carcinogenic TSNA in ST products. According to analytical studies, NNN,

nitrosoanabasine, and nitrosoanatabine are formed primarily from the

corresponding secondary amines at the early stages of the tobacco processing, whereas TSNA such as NNK are formed from the tertiary amine nicotine

(Figure 3) and occur at the later stage of tobacco curing and fermentation

(Spiegelhalder and Fisher, 1991). This observation provides a partial explanation of the abundance of TSNA in snuff.

The carcinogenic risk associated with oral ST use and the major contributions of TSNA to this risk are underscored by a number of analytical data.

In 1981, the National Research Council estimated the daily exposure of U.S.

residents to carcinogenic nitrosamines and found the average nonsmoker is

exposed to about 1 μg and the smoker of 20 cigarettes per day to about 11 to

12 μg of carcinogenic nitrosamines (U.S. National Research Council, 1981).
Chapter 3

103

Table 4

TSNA in smokeless tobacco, 1981 to 1989

Country and Samples NNNa NATa NABa NNKa

Tobacco Type (n) (μg/g) (μg/g) (μg/g) (μg/g)

United States

Moist snuff 16 0.83 - 64.00 0.24 - 215.00 0.01 - 6.70 0.08 - 8.30

Chewing tobacco 2 0.67 - 1.50 0.7 - 2.4b 0.11 - 0.38

Dry snuff 6 9.4 - 55.0 11 - 40 0.5 - 1.2 0.88 - 14.00

Sweden

Moist snuff 8 2.0 - 6.1 0.9 - 2.4 0.04 - 0.14 0.61 - 1.70

Canada

Moist snuff 2 50 - 79 152 - 170 4.0 - 4.8 3.2 - 5.8

Plug 1 2.1 1.7b 0.24

Germany

Plug 2 1.4 - 2.1 0.36 - 0.55b 0.03 - 0.04

Nasal snuff 7 2.8 - 19 1.0 - 5.8a 0.58 - 6.40

India

Chewing tobacco 4 0.47 - 0.85 0.40 - 0.50b 0.13 - 0.23

Zarda 11 0.40 - 79.00 0.78 - 99b 0.22 - 24.00

USSR

Nass 4 0.12 - 0.52 0.04 - 0.33b 0.02 - 0.11

United Kingdom

Moist snuff 7 1.1 - 52.0 2.0 - 65.0b 0.4 - 13.0

Nasal snuff 5 3.0 - 16.0 1.8 - 2.5b 0.97 - 4.30

a NNN, N-nitrosonornicotine; NAT, N-nitrosoanatabine; NAB, N-nitrosoanabasine; NNK,

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

b Contains NAB.

On the basis of 1986 data for the two leading U.S. snuff brands, which had

about 90 percent of the market share, the average snuff dipper, who consumes snuff at 10 g/d, is exposed to an additional 270 to 280 μg of carcinogenic nitrosamines (Hoffmann et al., 1987). Most of the nitrosamines are

extracted from the tobacco during snuff dipping, as is reflected in data from

saliva analysis (Hoffmann and Adams, 1981; Nair et al., 1985; Oesterdahl

and Slorach, 1988; Paladino et al., 1986; Sipahimalani et al., 1984). In

addition, it is strongly indicated that additional amounts of TSNA are

endogenously formed during chewing (Nair et al., 1985). Recently we

estimated that the average snuff dipper has a lifetime exposure to about

0.70 mmol/kg body weight of NNN and 0.03 mmol/kg body weight of NNK.

These levels compare with 1.6 mmol/kg body weight of a mixture of NNN

and NNK that induced tumors in the mouths of rats after oral swabbing

(Hecht et al., 1986; Hoffmann et al., 1990).
Smoking and Tobacco Control Monograph No. 2

104

Figure 3

Formation of tobacco-specific N-nitrosamines

The chemical-analytic data and the results from bioassays and

epidemiological studies (Hoffmann et al., 1992; Preston-Martin,

1991; Winn et al., 1981) strongly support the World Health

CONTROL OF

CARCINOGENS

IN ST

Organization’s recommendation that, short of getting people to cease using

tobacco, the harmful agents in chewing tobacco and snuff must be reduced

(WHO, 1988). The history of the snuff analyses in the United States and

Sweden has shown that a drastic reduction of the major carcinogens in ST

products is feasible.

In 1981, the U.S. Environmental Protection Agency mandated a ban of

maleic hydrazide diethanolamine (MH-30) for use on tobacco (US EPA,

1981). The diethanolamine part of this sucker growth inhibitor gives rise to

the carcinogen NDELA (Brunnemann and Hoffmann, 1981). Following the

ban of MH-30, the NDELA concentrations in smokeless tobacco declined, as

shown by our monitoring of leading brands of snuff and chewing tobaccos.

The reduction of NDELA values occurred gradually between 1981 and 1990,

from 6,840 ppb to 94 ppb in snuff and from 224 ppb to 74 ppbin chewing

tobacco (Brunnemann and Hoffmann, 1991). The concentration of the

strongly carcinogenic NMOR in a snuff brand fell from 690 ppb in 1981 to a

nondetectable level (< 2 ppb) in 1990 with the elimination of traces of

morpholine in the packaging (Brunnemann et al., 1982; Brunnemann and

Hoffmann, 1991).
Chapter 3

105

While the reduction or disappearance of NDELA and NMOR was possible through the elimination of their precursors, this approach is not

feasible for the reduction of nitrosamino acid and TSNA levels, because

proteins and alkaloids, the precursors for these carcinogens, are integral

parts of the tobacco. Nevertheless, elimination of nitrate-rich ribs and stems

of certain tobacco varieties and changes in ST processing, especially of snuff,

can lead to a major reduction of nitrosamines. Using NNN as an indicator

for levels of TSNA, we have confirmed its gradual decrease in the two U.S.

moist snuff brands that account for more than 85 percent of the current

market share. In 1980, we reported 26.5 ppm and 39 ppm of NNN for

brands A and B, respectively; in 1990, these levels had decreased to 10.4 and

9.6 ppm, respectively. In Sweden, the average NNN value for the leading

five snuff brands in 1980 amounted to 11.4 ppm and in 1990 for three

leading brands to 5.4 ppm. Two new snuff brands introduced in 1989 and

1990 on the U.S. market had NNN values of 4.1 and 3.2 ppm, respectively.

Because the volatile nitrosamines and the nitrosamino acids are formed

during the preparation of snuff by mechanisms similar to those leading to

TSNA, their concentrations also have been reduced.

These observations strongly support the concept that product modifications can lead to a significant reduction of nitrosamines in smokeless

tobacco (Table 5). Therefore, it was rather surprising that another snuff

brand introduced in the United States in 1989 and 1990 contained extremely high concentrations of TSNA and other carcinogenic nitrosamines,

in fact the highest ever reported (Table 5; see brand D). The increased pH of

this snuff (7.7 to 8.2), compared with other U.S. brands (5.6 to 7.3), suggests

that changes in manufacturing were possibly intended to facilitate the

absorption of nicotine through the oral mucosa. Unprotonated nicotine,

which increases steadily with increased pH above 6.2, is absorbed more

rapidly than protonated nicotine (Brunnemann and Hoffmann, 1974;

US DHHS, 1988).

The latter finding underscores the WHO recommendation to have the

harmful substances in ST subject to governmental control (WHO, 1988), at

least as it concerns the United States. Regulating agencies should be encouraged also to evaluate the flavor components and other chemical additives

that are used in the manufacture of smokeless tobacco products. Any agents

that are teratogenic or genotoxic should be banned.
Smoking and Tobacco Control Monograph No. 2

106

Table 5

Alkaloids and N-nitroso compounds in moist snuff brands, 1990 to 1991a

United States Sweden

Brand A Brand B Brand C Brand Db Brand Ec Three Brands

Moisture, % 56.0 57.8 51.8 50.00 - 57.8 51.9 46.6 - 54.2

pH 7.11 7.30 5.61 7.72 - 8.17 7.36 7.67 - 7.90

Nicotine, % 2.04 2.17 2.15 1.22 - 2.21 1.47 1.13 - 1.25

Total Alkaloids, % 2.18 2.32 2.32 1.32 - 2.38 1.59 1.24 - 1.41

Volatile Nitrosamines,d

 ng/g

NDMA NDg NDg NDg 147 - 265 NDg 51 - 63

NPYR 44 59 120 245 - 757 NDg NDg - 155

Nitrosamino Acids,e μg/g

NSAR 0.06 0.06 NDh 0.4 - 2.5 0.10 0.03 - 0.68

MNPA 5.13 3.62 2.72 8.9 - 65.7 2.20 3.10 - 3.28

MNBA 0.47 0.26 0.09 1.9 - 9.1 0.20 0.19 - 0.23

Total 5.70 3.90 2.80 11.2 - 77.3 2.50 3.30 - 4.20

TSNA,f μg/g

NNN 10.40 9.57 4.14 21 - 147 3.20 5.24 - 5.67

NNK 2.19 3.14 1.24 6 - 18 0.70 1.37 - 2.08

NAT + NAB 9.76 7.90 2.97 22 - 115 2.00 2.58 - 3.47

Total 22.30 20.60 8.30 48 - 280 5.90 9.20 - 11.20

a All values are based on dry weight. Total alkaloids include nicotine, nornicotine, mysomine, anatabine,

anabasine, 2,3'-dipyridyl, and cotinine. b Range of five samples bought in different stores in Texas.

c Snuff in sachets imported from Sweden.

d NDMA, N-nitrosodimethylamine; NPYR, N-nitrosopyrrolidine. e ND, not detected < 0.005 μg/g. f NSAR, N-nitrososarcosine; MNPA, 3-(methylnitrosamino)propionic acid; MNBA, 4-(methylnitrosamino)butyric acid.

g ND, not detected < 0.01 μg/g. h TSNA, tobacco-specific N-nitrosamines; NNN, N'-nitrosonornicotine; NNK, 4-(methylnitrosamino)-1-(3-

pyridyl)-1-butanone; NAT, N'-nitrosoanatabine; NAB, N'-nitrosoanabasine.

ACKNOWLEDGMENT We thank Ilse Hoffmann and Jennifer Johnting for their editorial assistance.

Mookherjee, B.D., Wilson, R.A. Tobacco constituents: Their importance in

REFERENCES

Andersen, R.A., Burton, H.R., Fleming, P.D., et al.

Effect of storage conditions on nitrosated,

acetylated and oxidized pyridine alkaloid derivatives in smokeless tobacco products. Cancer

Research 49: 5895-5900, 1989.

Brückner, H. Die Biochemie des Tabaks und der

Tabakverarbeitung. Berlin: Verlagsbuchhandlung

Paul Parey, 1936.

Brunnemann, K.D., Genoble, L., Hoffmann, D.

N-nitrosamines in chewing tobacco: An international comparison. Journal of Agricultural and Food

Chemistry 33: 1178-1181, 1985.

Brunnemann, K.D., Hoffmann, D. The pH of

tobacco smoke. Food and Cosmetics Toxicology 12:

115-124, 1974.

Brunnemann, K.D., Hoffmann, D. Assessment of the

carcinogenic N-nitrosodiethanolamine in tobacco

products and tobacco smoke. Carcinogenesis 2:

1123-1127, 1981.
Chapter 3

107

Brunnemann, K.D., Hoffmann, D. Decreased

concentrations of N-nitrosodiethanolamine and

N-nitrosomorpholine in commercial tobacco

products. Journal of Agricultural and Food Chemistry

39: 207-208, 1991.

Brunnemann, K.D., Scott, J.C., Hoffmann, D.

N-Nitrosomorpholine and other volatile

N-nitrosamines in snuff tobacco. Carcinogenesis 3:

693-696, 1982.

Brunnemann, K.D., Scott, J.C., Hoffmann, D.

N-Nitrosoproline, an indicator for N-nitrosation of

amines in tobacco. Journal of Agricultural and Food

Chemistry 31: 905-909, 1983.

Campbell, J.M., Lindsey, A.J. Polycyclic hydrocarbons extracted from tobacco: The effect upon

total quantities found in smoking. British Journal

of Cancer 10: 649-652, 1956.

Campbell, J.M., Lindsey, A.J. Polycyclic aromatic

hydrocarbons in snuff. Chemical Industry July 6,

1957, p. 951.

Chamberlain, W.J., Schlotzhauer, W.S., Chortyk, O.T.

Chemical composition of commercial tobacco

products. Journal of Agricultural and Food Chemistry

36: 48-50, 1988.

Djordjevic, M.V., Brunnemann, K.D., Hoffmann, D.

Identification and analysis of a nicotine-derived

N-nitrosamino acid and other nitrosamino acids in

tobacco. Carcinogenesis 10: 1725-1731, 1989.

Hecht, S.S., Rivenson, A., Braley, J., et al. Induction

of oral cavity tumors in F-344 rats by tobaccospecific nitrosamines in snuff. Cancer Research 49:

3063-3069, 1986.

Hoffmann, D., Adams, J.D. Carcinogenic tobaccospecific N-nitrosamines in snuff and in the saliva

of snuff dippers. Cancer Research 41: 4305-4308,

1981.

Hoffmann, D., Adams, J.D., Lisk, D., et al. Toxic and

carcinogenic agents in dry and moist snuff. Journal

of the National Cancer Institute 79: 1281-1286, 1987.

Hoffmann, D., Brunnemann, K.D., Venitt, S.

Carcinogenic nitrosamines in oral snuff. Lancet

1988, p. 1232.

Hoffmann, D., Djordjevic, M.V., Brunnemann, K.D.

On the control of toxic substances in smokeless

tobacco. Journal of Smoking-Related Disease 2: 165-

172, 1991.

Hoffmann, D., Rivenson, A., Chung, F.L., et al.

Relevance of nicotine-derived N-nitrosamines in

tobacco carcinogenesis. In: Effects of Nicotine on

Biological Systems, F. Adlkofer and K. Thurau

(Editors). Boston: Birkhäuser, 1990, pp. 89-101.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel Quid and Areca-Nut Chewing;

and Some Related Nitrosamines (volume 37). Lyon:

IARC, 1985.

LaVoie, E.J., Tucciarone, P., Kagan, M., et al.

Quantitative analysis of steam distillates and

aqueous extracts of smokeless tobacco. Journal of

Agricultural and Food Chemistry 37: 154-157, 1989.

Liu, Y.Y., Schmeltz, I., Hoffmann, D. Quantitative

analysis of hydrazine in tobacco and cigarette

smoke. Analytical Chemistry 46: 885-889, 1974.

Martell, E.A. Radioactivity of tobacco trichromes

and insoluble cigarette smoke particles. Nature

249: 215-217, 1974.

McMurtrey, J.E., Jr., Bacon, C.W., Ready, D.

Growing tobacco as a source of nicotine. USDA

Technical Bulletin 820: 1-38, 1942.

Mookherjee, B.D., Wilson, R.A. Tobacco constituents: Their importance in flavor and fragrance

chemistry. Recent Advances in Tobacco Science 14:

114-168, 1988.

Nair, J., Ohshima, H., Friesen, M., et al. Tobaccospecific and betel nut-specific N-nitroso compounds: Occurrence in saliva and urine of betel

quid chewers and formation in vitro by

nitrosation of betel quid. Carcinogenesis 6: 295-

303, 1985

Oesterdahl, B.G., Slorach S. Tobacco-specific

N-nitrosamines in the saliva of habitual snuff

dippers. Food Additives and Contamination 57: 581-

586, 1988.

Ohshima, H., Nair, J., Bourgarde, M.-C., et al.

Identification and occurrence of two new

N-nitrosamino acids in tobacco products: 3-(Nnitroso-N-methylamino)propionic acid and 4-(Nnitroso-N-methylamino)butyric acid. Cancer

Letters 26: 153-162, 1985.

Ough, C.S. Ethyl carbamate in fermented beverages

and foods: I. Naturally occurring ethyl carbamate. Journal of Agricultural and Food Chemistry 24:

323-328, 1976.

Palladino, G., Adams, J.D., Brunnemann, K.D., et al.

Snuff dipping in college students: A clinical

profile. Military Medicine 151: 342-346, 1986.

Preston-Martin, S. Evaluation of the evidence that

tobacco-specific nitrosamines (TSNA) cause cancer

in humans. Critical Review of Toxicology 21: 295-

298, 1991.

Robert, N.L. Natural tobacco flavor. Recent Advances

in Tobacco Science 14: 49-81, 1988.

Schmeltz, I., Chiang, K.G., Hoffmann, D. Formation

and determination of ethyl carbamate in tobacco

and tobacco smoke. Journal of Analytical Toxicology 2: 265-268, 1978.

Sharma, A.K., Prokopczyk, B., Hoffmann, D.

Supercritical fluid extraction of moist snuff.

Journal of Agricultural and Food Chemistry 39: 508-

510, 1991.

Sharma, P.K., Lal, N., Nagpaul, K.K. Study of trace

amounts of U in snuff. Health Physics 48: 811-812,

1985.
Smoking and Tobacco Control Monograph No. 2

108

Sipahimalani, A.T., Chadka, M.S., Bhide, S.V., et al.

Detection of N-nitrosamines in the saliva of

habitual chewers of tobacco. Food Chemistry and

Toxicology 22: 261-264, 1984.

Smyth, J.H. Moist snuff sales gain. Tobacco Reporter

116 (8): 30-31, 1989.

Spiegelhalder, B., Fischer, S. Formation of tobaccospecific nitrosamines. Critical Reviews of Toxicology

21: 241, 1991.

Tricker, A.R., Preussmann, R. Preformed nitrosamines in smokeless tobacco. In: Tobacco and

Cancer: Perspectives in Preventive Research, A.P.

Maskens, R. Molimard, R. Preussmann, and J.W.

Wilmer (Editors). Amsterdam: Excerpta Medica,

1989, pp. 35-47.

Tricker, A.R., Preussmann, R. Exposure to nicotinederived N-nitrosamines from smokeless tobacco

and evidence against endogenous formation. In:

Effects of Nicotine on Biological Systems, F. Adlkofer

and K. Thurau (Editors). Boston: Birkhäuser,

1990, pp. 109-113.

Tso, T.C., Harley, N., Alexander, L.T. Source of lead210 and polonium-210 in tobacco. Science 153:

880-882, 1966.

U.S. Department of Agriculture. Tobacco Situation and

Outlook 213: 18, 1990.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. 86-2874, 1986.

U.S. Department of Health and Human Services. The

Health Consequences of Smoking: Nicotine Addiction.

A Report of the Surgeon General. U.S. Department of

Health and Human Services, Public Health Service,

Centers for Disease Control, Center for Health

Promotion and Education, Office on Smoking and

Health. DHHS Publication No. (CDC) 88-8406, 1988.

U.S. Environmental Protection Agency. Maleic

hydrazine: Notification of issuance of intent to

suspend pesticide registrations. Federal Register

46(179): 45999-46000, 1981.

U.S. National Research Council. The Health Effects

of Nitrate, Nitrite and N-nitroso Compounds.

Washington, DC: U.S. National Research Council,

1981, p. 51.

Weybrew, J.A., Stephens, R.L. Survey of the carbonyl

contents of tobacco. Tobacco Science 6: 53-57, 1962.

Winn, D.M., Blot, W.J., Shy, C.M.,Picke, L.W.,

Toledo, A., Fraumeni, J.F. Snuff dipping and oral

cancer among women in the southern United States.

New England Journal of Medicine 304: 745-749, 1981.

World Health Organization. Smokeless Tobacco

Control. Technical Report Series 773. Geneva:

WHO, 1988.

Wynder, E.L., Hoffmann, D. Tobacco and Tobacco Smoke:

Studies in Experimental Carcinogenesis. New York:

Academic Press, 1967.
Chapter 3

109

Smokeless tobacco induces tumors in the oral cavity of rats. Of the 28 known carcinogens

Carcinogenesis of Smokeless Tobacco1

Dietrich Hoffmann, Abraham Rivenson, and Stephen S. Hecht

1

 Supported by National Cancer Institute grant no. CA-29580.

ABSTRACT

in tobacco, the major contributors to the carcinogenic activity of ST are the nitrosamines,

especially the tobacco-specific nitrosamines (TSNA). Among seven TSNA that have been

identified in ST, N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone (NNK) are the most potent carcinogens. A total dose of 450 μg NNK is sufficient to

induce tumors in rats, and 420 μg NNK suffice to elicit tumors in mice. A mixture of NNN and

NNK causes oral tumors in rats at a dose comparable to that ingested by a lifelong snuff dipper.

In accordance with the recommendations of the World Health Organization, harmful substances

in ST should be reduced and should be subject to governmental control and regulations.

INTRODUCTION Epidemiological investigations have revealed that tobacco chewers

and snuff dippers face an increased risk for cancer of the oral cavity and

pharynx. Chewing of smokeless tobacco has also been linked with cancer of

the nasal cavity, esophagus, pancreas, and urinary bladder (International

Agency for Research on Cancer, 1985; Kabat et al., 1986; Winn, 1992).

Topical application of extracts from tobacco and from snuff has induced

tumors in mouse skin. Such extracts have also exhibited cocar-cinogenic

activity (IARC, 1985; US DHHS, 1986). In some bioassays, ST products were

tested for tumorigenicity by painting the oral cavity with extracts, by

implantation, or by repeated insertion of chewing tobacco or snuff into the

cheek pouch (Table 1) (Gothoskar et al., 1975; Hecht et al., 1986;

Homburger, 1971; Homburger et al., 1976; Kandarkar et al., 1981; Peacock et

al., 1960). Although most of these bioassays have led to epithelial hyperplasia in the mouth or cheek pouches, none of them actually induced oral

tumors.

Hirsch and Thilander (1981) developed a new method for the bioassay

of ST in the oral cavity of laboratory animals. A canal is created in the lower

lip of rats by surgery, and snuff is inserted and exchanged twice daily

(Hirsch and Thilander, 1981). The first assays with this technique led to

hyperplasia, dysplasia, and other lesions in the lip canal and oral cavity but

not to oral tumors (Hirsch and Johansson, 1983; Hirsch and Thilander,

1981). Subsequently, other investigators modified the lip canal methodology and assayed commercial snuff. In one case, 2 of 32 rats treated with

snuff developed epithelial tumors in the lip canal and 1 rat had a papilloma

of the palate (Hecht et al., 1986). Johansson and colleagues (1989) induced

five oral tumors and two nasal tumors in a group of 29 rats in a long-term

assay with snuff. Together with experimentally induced herpes simplex virus

type 1 (HSV-1) infection, snuff treatment caused squamous cell carcinoma

of the oral cavity in two of seven rats (Hirsch et al., 1984). Park and associates (1986) repeatedly infected the buccal pouches of hamsters with either
Smoking and Tobacco Control Monograph No. 2

110

Table 1

Bioassays for the induction of oral tumors with ST

Materiala Animalb Method Result Reference

CT Hamster Implantation into cheek No oral tumors Peacock et al., 1960

pouch

CT Hamster Thrice weekly insertion No neoplastic Kandarkar et al., 1981

of CT with lime into changes

cheek pouch

CT Rat Painting of oral cavity No oral tumors Gothoskar et al., 1975

with extract twice weekly

S Hamster, M Feeding of S with diet (20%) No oral tumors Homburger et al., 1976

S Hamster Insertion in cheek pouch No tumors Peacock et al., 1960

S Hamster Daily attachment of No oral tumors Homburger, 1971

cartridge containing 0.5 g

S to lower incisors for

30 min; 1 yr

S Rat, M Twice daily painting of oral No oral tumors Hecht et al., 1986

cavity and lips with

concentrated S extract

S Rat Lip canal; twice daily Epithelial Hirsch and Thilander,

insertion of 0.2 g S; hyperplasia and 1981

up to 18 to 22 mo dysplasia, no

oral tumors

S Rat, M Lip canal; one daily 3/32 oral tumors Hecht et al., 1986

insertion of 0.05 g S;

up to 25 mo

S Rat Lip canal; twice daily 0/10 oral tumors Hirsch et al., 1984

insertion of 0.2 g S;

up to 18 mo

As above plus HSV-1 2/7 oral tumors

infection

S Rat Lip canal; twice daily 5/29 oral and lip Johansson et al., 1989

insertion of 0.1 g; tumors

up to 20 mo 2/29 nasal tumors

S Hamster Insertion twice daily 0/15 oral tumors Park et al., 1986

of 0.15 g S into buccal

pouch; up to 6 mo

As above plus HSV-1 10/20 oral carcinoma

infection

As above plus HSV-2 11/20 oral carcinoma

infection

a CT, chewing tobacco; S, snuff.

b M, males.
Chapter 3

111

HSV-1 or HSV-2 and inserted 150 mg of commercial snuff into the infected

pouches twice daily. After 6 mo, 10 of 20 hamsters inoculated with HSV-1

and exposed to snuff developed invasive squamous cell carcinoma in the

buccal pouch; in the group treated with HSV-2 and snuff, 11 of 20 hamsters

developed carcinoma in the buccal pouch. None of the animals treated

with either virus type or with snuff alone developed oral tumors within

6 mo (Park et al., 1986).

Thus, bioassays support the epidemiological observation that the longterm use of ST leads to cancer in the oral cavity. The next goal in explaining

the causes of oral cancer by tobacco is the identification of those agents

among the more than 3,050 known tobacco constituents (Roberts, 1988)

that make smokeless tobacco carcinogenic. Until now, 28 known carcinogens have been reported in processed tobacco (Table 2) (Brunnemann and

Hoffmann, 1992; Hoffmann et al., 1991; IARC, 1987 and 1988).

CARCINOGENS

IN SMOKELESS

TOBACCO

The contamination of processed tobacco with benzo[a]pyrene and

other carcinogenic polynuclear aromatic hydrocarbons (PAH) stems

mostly from polluted air (Campbell and Lindsey, 1956 and 1957;

Wynder and Hoffmann, 1967). However, the levels of PAH appear to be too

low to make a significant contribution to the carcinogenicity of snuff in the

oral cavity (IARC, 1973). The possible carcinogenic effect of α- and βangelica lactones cannot be evaluated at this time, because these tobacco

constituents have not yet been assayed by oral application. Coumarin

applied to rats as a dietary component induced tumors in the bile duct but

not in the upper digestive tract (IARC, 1976). Urethan (ethyl carbamate),

when given in the drinking water to mice, induces primarily lung tumors; in

rats, urethan causes tumors at multiple sites, but predominantly hepatomas.

In hamsters, urethan causes tumors of the forestomach and melanotic

tumors of the skin (IARC, 1975).

Three carcinogenic volatile aldehydes have been detected in smokeless

tobacco; their concentrations in oral snuff are lower than in other processed

tobacco types and products (Brunnemann and Hoffmann, 1992; Wynder

and Hoffmann, 1967). Acetaldehyde, for example, is found in the major

tobacco types in amounts up to 270,000 ng/g (270 ppm) (Weybrew and

Stephens, 1962), yet the data reported for U.S. commercial snuff showed

about 1/10 of those concentrations (Table 2). The International Agency for

Research on Cancer (1987) regards formaldehyde as an animal carcinogen

and as probably carcinogenic to humans. After inhalation of formaldehyde

(14.3 ppm), rats developed squamous cell carcinoma of the nasal cavity

(Albert et al., 1982; IARC, 1982 and 1987). Inhalation of acetaldehyde

produced adenocarcinoma and squamous cell carcinoma of the nasal

mucosa in rats and laryngeal carcinoma in hamsters (IARC, 1987). Acetaldehyde is also known to inhibit the repair of DNA lesions (Grafström et al.,

1986). Croton aldehyde, fed to rats in the drinking water (0.6 mM), induced

benign and malignant tumors of the liver (Chung et al., 1986). Data are

lacking for the carcinogenicity of volatile aldehydes in the upper digestive

tract, but one would assume that these components contribute to the

carcinogenicity of ST.
Smoking and Tobacco Control Monograph No. 2

112

Table 2

Carcinogenic agents in tobacco

IARC Evaluation of Evidence

of Carcinogenicitya

Tobacco Concentration, In Laboratory In

Typeb ng/gc Animals Humans

Benzo[a]pyrene NT, S > 0.1 - 90.0 Sufficient Probable

α-Angelica Lactone NT Present

β-Angelica Lactone NT Present

Coumarin NT 600 Limited

Ethyl Carbamate CT 310 - 375 Sufficient

Volatile Aldehydes

Formaldehyde NT, S 1,600 - 7,400 Sufficient Probable

Acetaldehyde NT, S 1,400 - 27,400 Sufficient

Crotonaldehyde S 200 - 2,400

Nitrosamines

Nitrosodimethylamine CT, S ND - 270 Sufficient Probable

Nitrosopyrrolidine CT, S ND - 760 Sufficient

Nitrosopiperidine CT, S ND - 110 Sufficient

Nitrosomorpholine CT, S ND - 690 Sufficient

Nitrosodiethanolamine CT, S 40 - 6,800 Sufficient

Nitrosamino Acids

Nitrososarcosine S ND - 2,500 Sufficient

3-(Methylnitrosamino)- CT, S 200 - 65,700

propionic acid

4-(Methylnitrosamino)- CT, S ND - 9,100

butyric acid

Nitrosoazetadine-2- CT 4 - 140

carboxylic acid

Tobacco-Specific Nitrosamines

N'-Nitrosonornicotine CT, S 400 - 147,000 Sufficient

4-(methylnitrosamino)- CT, S ND - 18,000 Sufficient

 1-(3-pyridyl)-1-butanone

4-(methylnitrosamino)- S Present

1-(3-pyridyl)-1-butanol

N'-Nitrosoanabasine SM, S Present - 560 Limited

Inorganic compounds

Hydrazine SM 14 - 51 Sufficient Inadequate

Arsenic NT 500 - 900 Inadequate Sufficient

Nickel SM, S 180 - 2,700 Sufficient Sufficient

Cadmium SM 700 - 790 Sufficient Probable

p Ci/g

Polonium-210 NT, S 0.16 - 1.22 Sufficient Sufficient

Uranium-235 and -238 S 2.4, 1.91

a Absence of a designation indicates that IARC has not evaluated.

b NT, natural tobacco; SM, smoking tobacco; S, snuff; CT, chewing tobacco.

c ND, not determined.
Chapter 3

113

There is no information on the possible contribution of inorganic

carcinogenic ST constituents to the increased oral cancer risk of chewers and

snuff dippers. However, tobacco chewers have a higher level of trace

amounts of some metals in the oral mucosa than do nontobacco users

(Robertson and Bray, 1988). Of special concern is the human carcinogen

polonium-210, which is a decay product of radon (IARC, 1988). Data on the

polonium-210 content of oral tissues are needed before one can consider the

carcinogenic effect of polonium-210 on the oral cavity of chewers.

N-NITROSAMINES Processing of tobacco to chewing tobacco or snuff yields several

types of nitrosamines. Precursors to such carcinogens are nitrate or nitrite,

the amino acids and proteins of the tobacco, and the Nicotiana alkaloids.

These constituents form volatile nitrosamines (VNA), nitrosamino acids

(NNA), and tobacco-specific nitrosamines (TSNA), respectively. In addition,

residues of morpholine and diethanolamine from tobacco contaminants can

serve as precursors for the corresponding nitrosamines.

Nitrosamines are generally organ-specific carcinogens: they induce

benign and malignant tumors in specific organs, independent of site and

mode of application (Table 3). The VNA are powerful animal carcinogens.

For example, a daily dose of 40 μg of nitrosodimethylamine in the drinking

water during the lifetime of rats induces liver tumors in 50 percent of the

animals (Peto et al., 1984). None of the nitrosamines listed in Table 3,

except nitrosodiethanolamine, are known to induce oral cavity tumors.

Nitrosodiethanolamine causes tumors of the upper aerodigestive tract in

hamsters in addition to tracheal tumors after swabbing of the oral cavity

with an aqueous solution (Hoffmann et al., 1983).

So far, 10 nitrosamino acids have been identified in ST (Brunnemann

and Hoffmann, 1991). Only six of these have been assayed for carcinogenicity. Nitrosoproline and nitrosothioproline are inactive in mice and rats.

Nitrososarcosine and 3-(methylnitrosamino)propionic acid induce liver

tumors in mice or rats, whereas 4-(methylnitrosamino)butyric acid induces

bladder cancer in rats (Preussmann and Stewart, 1984; Rivenson et al.,

1989). None of the nitrosamino acids have been assayed by topical application to the oral cavity, although their relatively high concentrations in snuff

(up to 65 ppm) make such tests highly desirable.

TSNA The most important carcinogens in ST are TSNA. They are formed by

nitrosation of the Nicotiana alkaloids during curing, fermenting, and aging

of the leaves. Seven TSNA have been identified in ST (Brunnemann and

Hoffmann, this volume). Two of these, N'-nitrosonornicotine (NNN) and 4-

(methylnitrosamino)-1-(3-pyridine)-1-butanol (NNAL), are powerful carcinogens in mice, rats, and hamsters, inducing tumors in the lung, upper

aerodigestive tract, or pancreas (Table 4). N'-Nitrosoanabasine is a weak

esophageal carcinogen in rats. N'-Nitrosoanatabine (NAT) and 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid (iso-NNAC) are not carcinogenic and

4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) has not been

bioassayed.
Smoking and Tobacco Control Monograph No. 2

114

Table 3

Organ-specific carcinogenicity of nitrosamines in mice, rats, and hamsters

Major Target Organs for Carcinogenicity

Syrian

Mice Rats Golden Hamsters

Nitrosodimethylamine Liver, lung Liver, kidney Liver

Nitrosopyrrolidine Lung Liver Lung, trachea

Nitrosopiperidine Lung, forestomach Esophagus, liver Trachea, nasal cavity

Nitrosomorpholine Liver, lung Liver, nasal cavity Trachea, nasal cavity

Nitrosodiethanolamine Liver Trachea, nasal cavity

Table 4

Carcinogenicity of TSNA

Animal Route of Principal Dose,

(Strain) Application Target Organ mmol/Animal

TSNAa

NNN Mouse Topical (TI)b None 0.028

Mouse (A/J) Intraperitoneal Lung 0.1

Rat (F-344) Subcutaneous Nasal cavity, 0.2 - 3.4

esophagus

Rat (F-344) Oral Esophagus, 1.0 - 3.6

nasal cavity

Rat Oral Nasal cavity 8.8

(Sprague-Dawley)

Syrian golden Subcutaneous Trachea, 0.9 - 2.1

 hamster nasal cavity

NNAL Mouse (A/J) Intraperitoneal Lung 0.12

Rat (F-344) Subcutaneous Lung, pancreas 0.32

NAB Rat (F-344) Oral Esophagus 3 - 12

Syrian golden Subcutaneous None 2

hamster

NAT Rat (F-344) Subcutaneous None 2.8

 iso-NNAC Mouse (A/J) Intraperitoneal None 0.2

Source: Hecht and Hoffmann (1989), except iso-NNAC, Rivenson et al. (1989).

a For data on NNK, see Table 20-5.

b TI, tumor-initiating assay with TPA as promoter; NNN, N'-nitrosonornicotine; NNAL,

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NAB, N'-nitrosoanabasine; NAT,

N'-nitrosoanatabine; iso-NNAC, 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid.
Chapter 3

115

The most powerful carcinogen in ST is 4-(methylnitrosamino)-1-(3-

pyridyl)-1-butanone (NNK) (Table 5). NNK induces lung cancer and tumors

of the nasal cavity and liver in rats. When NNK is administered in drinking

water to rats, it also induces malignant tumors of the exocrine pancreas. In

fact, NNK and its enzymatic reduction product NNAL are the only pancreas

carcinogens known to occur in ST and in tobacco smoke. This finding is

significant because both tobacco smokers and ST users have an increased

risk for cancer of the exocrine pancreas (IARC, 1985; US DHHS, 1986 and

1989). NNK is also a weak transplacental carcinogen in mice and hamsters,

resulting in lung tumors in offspring (Anderson et al., 1989; Anderson and

Rice, this volume; Correa et al., 1990). Perhaps the most important aspect

of NNK is its high carcinogenic potency; 450 μg induce tumors in rats

(Belinsky et al., 1990).

NNN and NNK as an admixture have induced oral tumors in laboratory

animals, when an aqueous solution of these TSNA was used to swab the oral

cavity of rats twice daily for up to 131 wk (Hecht et al., 1986). The total

dose applied amounted to about 97 mg of NNN (1,400 μmol/kg) and 19 mg

of NNK (240 μmol/kg). Based on the analytical data for the best-selling U.S.

snuff brand in 1980 (Hoffmann and Adams, 1981), consumption of 10 g of

snuff per day exposes the oral cavity of a snuff dipper during a lifetime to

about 5,700 mg of NNN (460 μmol/kg) and to about 360 mg of NNK

(25 μmol/kg). This comparison supports the concept that the TSNA greatly

contribute to the increased risk of snuff dippers for cancer of the oral cavity.

The estimate of exposure of snuff dippers to the carcinogenic NNN and NNK

did not consider the likely event that additional amounts of TSNA are

formed endogenously during chewing (Tsuda and Kurasima, 1991).

DISCUSSION The bioassay data strongly support the epidemiological observation

that ST is carcinogenic to humans. Twenty-eight carcinogens have been

identified in chewing tobacco and snuff. The high concentrations of Nnitrosamines in ST, and especially the high levels of TSNA, are of great

concern. The TSNA derive exclusively from the tobacco alkaloids, predominantly from the pharmacoactive nicotine, and are formed during tobacco

processing. A bioassay has shown that a mixture of NNN and NNK induces

oral tumors in rats. The orally applied amounts of NNN and NNK are

comparable to the cumulative doses to which a snuff dipper is exposed

during a lifetime.

Emphasis should be placed on educating the public to the hazards of

tobacco chewing and snuff dipping. Because of the millions of ST users

throughout the world, urgent support for the recommendations of the

World Health Organization to regulate the harmful substances in ST is

needed (WHO, 1988). As discussed in Brunnemann and Hoffmann (1992),

significant reductions in the unacceptably high concentrations of carcinogens in tobacco, especially those of the nitrosamines, are feasible.
Smoking and Tobacco Control Monograph No. 2

116

Table 5

Carcinogenicity of NNK in laboratory animals

Route of Principal Dose,

Application Target Organ mmol/Animal Reference

Mouse Topical (TI)a Skin 0.028 Hecht and Hoffmann, 1989

(Sencar)

Oral Lung, nasal 0.6 - 1.2 Prokopczyk et al., 1992

cavity, liver

Mouse Intraperitoneal Lung 0.01 - 0.12 Hecht and Hoffmann, 1989

(A/J) Morse et al., 1991

Rat Subcutaneous Lung, nasal 0.2 - 2.8 Hecht and Hoffmann, 1989

(F-344) cavity, liver

Lung, nasal 0.0025 - 3.6 Belinsky et al., 1990

cavity

Oral (in drinking Lung, liver, 0.075 - 0.31 Hecht and Hoffmann, 1989

water) pancreas

Oral (by gavage) Liver, lung 1.3 Lijinsky et al., 1990

Intravesical Lung, liver 1.5 Lijinsky et al., 1990

Syrian Subcutaneous Trachea, lung, 0.005 - 0.9 Hoffmann et al., 1991

Golden nasal cavity

Hamsters

a TI = tumor-initiating assay with TPA as a promoter.

REFERENCES

Albert, R.E., Sellakumar, A.R., Laskin, S., et al.

Gaseous formaldehyde and hydrogen chloride

induction of nasal cancer in the rat. Journal of the

National Cancer Institute 68: 597-603, 1982.

Anderson, L.M., Hecht, S.S., Dixon, D.E., et al.

Evaluation of the transplacental tumorigenicity of

the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mice. Cancer

Research 49: 3770-3775, 1989.

Belinsky, S.A., Foley, J.F., White, C.M., et al. Doseresponse relationship between O6

-methylguanine

formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Research

50: 3772-3780, 1990.

Brunnemann, K.D., Hoffmann, D. Analytical studies

on tobacco-specific N-nitrosamines in tobacco and

tobacco smoke. Critical Reviews of Toxicology 2:

235-240, 1991.

Campbell, J.M., Lindsey, A.J. Polycyclic hydrocarbons extracted from tobacco: The effect upon

total quantities found in smoking. British Journal

of Cancer 10: 649-652, 1956.

Campbell, J.M., Lindsey, A.J. Polycyclic hydrocarbons in cigar smoke. British Journal of Cancer 11:

192-195, 1957.

Chung, F.-L., Tanaka, T., Hecht, S.S. Induction of

liver tumors in F344 rats by crotonaldehyde.

Cancer Research 46: 1285-1289, 1986.

Correa, A., Joshi, P.A., Castonguay, A., et al. The

tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is an active

transplacental carcinogen in Syrian golden

hamsters. Cancer Research 50: 3435-3438, 1990.

Gothoskar, S.V., Sant, S.M., Ranadive, K.J. Effect of

tobacco and lime on oral mucosa of rats fed on

vitamin B deficient diet. Indian Journal of Cancer

12: 424-429, 1975.
Chapter 3

117

Grafström, R.C., Curren, R.D., Yang, L.L. Acetaldehyde-induced inhibition of DNA repair and

potentiation of N-nitroso compounds-induced

mutagenesis in cultured human cells. Progress in

Clinical and Biological Research 209A: 255-264,

1986.

Hecht, S.S., Hoffmann, D. The relevance of tobaccospecific nitrosamines to human cancer. Cancer

Surveys 8: 273-294, 1989.

Hecht, S.S., Rivenson, A., Braley, J., et al. Induction

of oral cavity tumors in F344 rats by tobaccospecific nitrosamines and snuff. Cancer Research

46: 4162-4166, 1986.

Hirsch, J.-M., Johansson, S.L. Effect of long-term

application of snuff on the oral mucosa: An

experimental study in the rat. Journal of Oral

Pathology 12: 187-198, 1983.

Hirsch, J.-M., Johansson, S.L., Vahlne, A. Effect of

snuff and herpes simplex virus-1 on rat oral mucosa:

Possible associations with the development of

squamous cell carcinoma. Journal of Oral Pathology

13: 52-62, 1984.

Hirsch, J.-M., Thilander, H. Snuff-induced lesions of

the oral mucosa: An experimental model in the

rat. Journal of Oral Pathology 10: 342-353, 1981.

Hoffmann, D., Adams, J.D. Carcinogenic tobaccospecific N-nitrosamines in snuff and in the saliva

of snuff dippers. Cancer Research 41: 4305-4308,

1981.

Hoffmann, D., Castonguay, A., Rivenson, A., et al.

Comparative carcinogenicity and metabolismof 4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone and

N'-nitrosonornicotine in Syrian golden hamsters.

Cancer Research 41: 2386-2393, 1981.

Hoffmann, D., Djordjevic, M.V., Brunnemann, K.D.

On the control of toxic substances in smokeless

tobacco. Journal of Smoking-Related Disease 3: 165-

172, 1991.

Hoffmann, D., Rivenson, A., Adams, J.D. Effects of

route of administration and dose on the carcinogenicity of N-nitrosodiethanolamine in the Syrian

golden hamster. Cancer Research 43: 2521-2524,

1983.

Homburger, F. Mechanical irritation, polycyclic

hydrocarbons, and snuff. Effects on facial skin,

cheek pouch, and oral mucosa in Syrian hamsters.

Archives of Pathology 91: 411-417, 1971.

Homburger, F., Hsueh, S.-S., Russfield, A.B., et al.

Absence of carcinogenic effects of chronic feeding

of snuff in inbred Syrian hamsters. Toxicology and

Applied Pharmacology 35: 515-521, 1976.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk to Humans: Benzo[a]pyrene (volume 3). Lyon:

IARC, 1973, pp. 91-136.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk to Humans: Urethane (volume 7). Lyon:

IARC, 1975, pp. 111-140.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk to Humans: Coumarin (volume 10). Lyon:

IARC, 1976, pp. 113-119.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk to Humans: Formaldehyde (volume 29). Lyon:

IARC, 1982, pp. 345-389.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosamines (volume 37). Lyon:

IARC, 1985.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risks to Humans: Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs (volume 1

to 42, supplement 7). Lyon: IARC, 1987.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk to Humans: Man-Made Mineral Fibres and

Radon (volume 43). Lyon: IARC, 1988.

Johansson, S.L., Hirsch, J.-M., Larsson, P.-A., et al.

Snuff-induced carcinogenesis: Effect of snuff in

rats initiated with 4-nitroquinoline-N-oxide.

Cancer Research 49: 3063-3069, 1989.

Kabat, G.C., Dieck, G.S., Wynder, E.L. Bladder

cancer in nonsmokers. Cancer 57: 362-367, 1986.

Kandarkar, S.V., Hasgekar, N.N., Sirsat, S.M. Optical

and ultrastructural pathology of vitamin A

pretreated hamster cheek pouch exposed to lime

[Ca(OH)2] and tobacco over total lifespan.

Neoplasma 28: 729-737, 1981.

Lijinsky, W., Farnsworth, D., Farrelly, J., et al.

Tumors of the bladder and other organs induced

by intravesical administration of N-nitroso

compounds to rats. Annual Report, Basic Research

in Progress NCI, Frederick Cancer Research and

Development Center, September 1990, pp. 76-78.

Morse, M.A., Eklind, K.I., Hecht, S.S., et al. Structure-activity relationship for inhibition of 4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone

lung tumorigenesis by arylalkyl isothiocyanates in

A/J mice. Cancer Research 51: 1846-1850, 1991.

Park, N.H., Sapp, J.P., Herbosa, E.G. Oral cancer

induced in hamsters with herpes simplex infection

and simulated snuff dipping. Oral Surgery, Oral

Medicine, Oral Pathology 62: 164-168, 1986.

Peacock, E.D., Jr., Greenberg, B.G., Brawley, B.W.

The effect of snuff and tobacco on the production

of oral carcinoma: An experimental and epidemiological study. Annals of Surgery 151: 542-550,

1960.
Smoking and Tobacco Control Monograph No. 2

118

Peto, R., Gray, R., Brantom, P., et al. Nitrosamine

carcinogenesis in 5120 rodents: Chronic administration of sixteen different concentrations of

NDEA, NDMA, NPYR and NPIP in the water of

4,440 inbred rats, with parallel studies on NDEA

alone of the effect of age of starting (3, 6 or 20

weeks) and of species (rats, mice or hamsters).

IARC Scientific Publications 57: 627-665, 1984.

Preussmann, R., Stewart, B.W. N-Nitroso carcinogens. In: Chemical Carcinogens (volume 132), E.C.

Searle (Editor). Washington, DC: American

Chemical Society, 1984, pp. 643-828. (American

Chemical Society Monographs)

Prokopczyk, B., Rivenson, A., Hoffmann, D. Comparative carcinogneicity of 3-(methylnitrosamino)propionitrile, and 4-(methylnitrosamino)-1-

(3-pyridyl)-1-butanone upon local application to

mouse skin and rat oral mucosa. Cancer Letters 60:

153-157, 1991.

Rivenson, A., Djordjevic, M.V., Amin, S., et al.

Bioassay in A/J mice of some N-nitrosamines.

Cancer Letters 47: 111-114, 1989.

Roberts, D.L. Natural tobacco flavor. Recent Advances

in Tobacco Science 14: 49-81, 1988.

Robertson, J.B., Bray, J.T. Development of validation

test for self-reported abstinence from smokeless

tobacco products: Preliminary results. Preventive

Medicine 17: 496-502, 1988.

Tsuda, M., Kurashima, Y. Tobacco smoking,

chewing and snuff dipping: Factors contributing

to the endogenous formation of N-nitroso

compounds. Critical Reviews of Toxicology 21: 243-

253, 1991.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986.

U.S. Department of Health and Human Services.

Reducing the Health Consequences of Smoking: 25

Years of Progress. A Report of the Surgeon General.

U.S. Department of Health and Human Services,

Public Health Service, Centers for Disease Control,

Center for Chronic Disease Prevention and Health

Promotion, Office on Smoking and Health. DHHS

Publication No. (CDC) 89-8411, 1989.

Weybrew, J.A., Stephens, R.L. Survey of carbonyl

contents of tobacco. Tobacco Science 6: 53-57,

1962.

World Health Organization. Smokeless Tobacco

Control. Report of a WHO Study Group. Geneva:

WHO, 1988. World Health Organization

Technical Report 773.

Wynder, E.L., Hoffmann, D. Tobacco and Tobacco

Smoke. Studies in Experimental Carcinogenesis. New

York: Academic Press, 1967.
Chapter 3

119

Oncogenes in Head and Neck Cancer1

Kenneth D. Somers, Suzanne E. Glickman, Suzanne L. Cartwright,

Bryan G. Stoll, Stella L. Maurizio, and Gary L. Schechter

1 Supported by Smokeless Tobacco Research Council grant no. 0112 and the Medical College of

Hampton Roads Foundation.

ABSTRACT Squamous cell carcinomas of the head and neck (SCCHN) were analyzed for activated

oncogenes by DNA transfection assay and Southern blot hybridization. Transforming activity

was not detected in SCCHN DNAs (n=31) by two DNA transfection assays. A cluster of protooncogenes (int-2, hst-1, bcl-1) localized to chromosome 11 band q13 was amplified two- to

eightfold in approximately 30 percent of SCCHN (n=45); c-erbB-1 was amplified in 10 percent

of the same tumors. Adjacent clinically and histologically normal tissue from the same patients

had single proto-oncogene copy number. No amplification or rearrangement of c-erbB-2/HER2,

c-myc, N-myc, N-ras, H-ras-1, or K-ras-2 was detected in any of the SCCHN. The 11q13

amplicon in SCCHN consisted of int-2, hst-1, and bcl-1, but did not include c-sea, also on

11q13, or extend to the collagenase gene locus (11q21-22), c-ets-1 (11q23), or H-ras-1 (11p15.5).

The data implicate amplification of int-2/hst-1/bcl-1 as one of the genetic alterations underlying

head and neck tumor formation.

INTRODUCTION Proto-oncogenes are normal cellular genes known to function in

the control of cell growth and differentiation. When mutated or abnormally expressed, proto-oncogenes can be activated to oncogenic potential

resulting in aberrant cell signaling and unrestrained cell proliferation

(Bishop, 1991). Increasingly, activated oncogenes are being identified as

one of the genetic alterations underlying human tumor pathogenesis. The

most informative studies derive from the genetic analysis of colorectal

cancer in which the stepwise accumulation of genetic damage in the form of

activation of proto-oncogenes and inactivation of tumor suppressor genes

leads to tumor development and progression (Fearon and Vogelstein, 1990).

There are few reports of activated oncogenes in head and neck tumors.

Activated ras genes have been detected in < 10 percent of head and neck

tumors (Howell et al., 1990; Sheng et al., 1990). We reported amplification

of the int-2 proto-oncogene in squamous cell carcinomas of the head and

neck (SCCHN) (Somers et al., 1990) and recently demonstrated that hst-1 is

frequently co-amplified with int-2 in SCCHN; int-2 and hst-1 are in the

fibroblast growth factor family (Dickson et al., 1990), whose members

function in angiogenesis and as mitogenic growth factors (reviewed in

Burgess and Maciag, 1989). Although the expression and function of int-2

and hst-1 in SCCHN is uncertain, amplification of these genes in head and

neck tumors implicates their role in tumor formation. In this study, we

analyzed SCCHN for biologically active oncogenes by DNA transfection and

extended our analysis of the int-2 gene amplification unit in SCCHN.
Smoking and Tobacco Control Monograph No. 2

120

Surgical specimens were obtained from patients with histologically

identified SCCHN. Adjacent normal tissue was obtained whenever

possible. Tissue specimens were stored at -70 °C for subsequent

DNA extraction. Human laryngeal SCC cell lines UMSCC10A,

UMSCC10B, and UMSCC16 were provided by T.E. Carey, and Hep 2

was obtained from the American Type Culture Collection (ATCC).

MATERIALS

AND METHODS

Tissue

Specimens and

DNA Extraction

Cell lines were grown in Eagle’s minimum essential medium supplemented

with 10 percent fetal bovine serum. High molecular weight DNA was

prepared as described by Somers and coworkers (1990).

Transfection

Assays

DNA transfection was performed by the calcium phosphate precipitation technique (Graham and van der Eb, 1973) using NIH3T3 mouse

fibroblasts as recipients. Foci of morphologically transformed cells were

counted after 14 to 21 days. The nude mouse tumorigenicity assay was

performed as described (Fasano et al., 1984) using pSV2neo as a dominant

selectable marker, thereby permitting G418 antibiotic selection of stable

transfectants. Positive control DNA for transfection assays was extracted

from H-ras-transformed NIH3T3 cells, 44-9. DNA extracted from human

diploid fibroblasts or placenta served as the negative control. DNA extracted

from primary transfectants and tumors was used in a second cycle transfection and examined for human DNA sequences by Southern blot analysis

with the human Alu repetitive DNA probe BLUR 8.

Southern Blot

Analysis

DNA (10 μg) was digested with EcoRI or BamHI, separated by electrophoresis in 0.8 percent agarose gels, and blotted to nylon filters

(Biotrans, ICN). Filters were baked for 2 h at 80 °C. Filters were

prehybridized for 1 h at 42 °C in 50 percent formamide, 5X SSC (1X SSC is

0.15 M NaCl and 0.015 M sodium citrate), 5X Denhardt’s solution, 0.05 M

sodium phosphate (pH 6.5), 0.1 percent sodium dodecyl sulfate (SDS), and

250 μg/mL denatured salmon sperm DNA. Hybridization was performed

with 1x106 cpm of heat-denatured 32P-labeled oncogene DNA probes per

milliliter of prehybridization solution overnight at 42 °C. Filters were

washed for 20 min at room temperature in 2X SSC and 0.1 percent SDS and

at 50 °C for 30 min in 0.1X SSC and 0.1 percent SDS and then exposed at

-70 °C to Kodak XAR-5 film with an intensifying screen. DNA probes used

for hybridization included the following: int-2 probe SS6 (Casey et al., 1986)

provided by C. Dickson; hst-1 probe pORF1 (Taira et al., 1987) provided by

M. Terada; bcl-1 probe b (Tsujimoto et al., 1985) provided by Y. Tsujimoto;

c-sea probe p6.2 (Williams et al., 1988) provided by M. Hayman; and human

collagenase (CLG) probe pCllase 1 (Whitman et al., 1986) obtained from

ATCC. Probes for c-ets-1, H-ras-1, K-ras-2, N-ras, N-myc, c-myc, c-erbB-1, and

c-erbB-2 were described previously (Somers et al., 1990). Cloned insert DNA

(50 ng) was labeled with [32P]dCTP by the random primer method to a

specific activity of 5x107

 cpm/μg of DNA.

RESULTS

Transforming

Activity of

SCCHN DNA

We used two general oncogene detection techniques, the NIH3T3

focus assay and the nude mouse tumorigenicity assay, to analyze

proto-oncogene activation in 31 primary or metastatic SCCHN and

four human cell lines derived from SCC of the larynx (Hep 2,

UMSCC10A, UMSCC10B, and UMSCC16). Provisional evidence for
Chapter 3

121

the transfer of oncogenes by DNA transfection could not be confirmed by

analysis of transformant or nude mouse tumor DNAs for the presence of

human Alu repetitive DNA sequences (data not shown). Control transfections of NIH3T3 cells with 44-9 DNA containing activated H-ras produced

foci of morphologically transformed cells and induced tumors in nude mice

with a latency of 1 to 2 wk.

Southern Blot

Analysis of

Activated

Oncogenes

In an alternative approach, DNA extracted from SCCHN and tumorderived cell lines was analyzed for amplified or rearranged protooncogenes by Southern blot hybridization. An example of such an

analysis using probes corresponding to int-2, c-ets-1, N-ras, and

N-myc is shown in Figure 1. int-2 was amplified two- to fivefold in two

hypopharyngeal tumors (lanes 2 and 7), two tongue tumors (lanes 5 and

12), and one laryngeal tumor (lane 8), relative to human placenta DNA used

as a single copy int-2 gene control (Figure 1, top). int-2 was amplified

threefold in A431 epidermoid carcinoma cells used as a positive control.

The blot was stripped and reprobed with either c-ets-1 (Figure 1, middle) or a

mixture of N-ras and N-myc probes (Figure 1, bottom). No alterations of cets-1, N-myc, or N-ras were detected in any of the tumors. Using this type of

analysis, we subjected a panel of SCCHN to Southern blot analysis using

oncogene probes. The results are summarized in Table 1. int-2, hst-1, and

bcl-1 were amplified in approximately 30 percent of SCCHN, whereas c-erbB1 was amplified in 10 percent of the tumors. No amplification or rearrangement of c-erbB-2/HER2, c-myc, N-myc, N-ras, H-ras-1, or K-ras-2 was detected

in any of the SCCHN (Table 1).

Gene

Amplification

on 11q13

We demonstrated previously amplification of the int-2 protooncogene in SCCHN (Somers et al., 1990). As shown in Table 1,

int-2, hst-1, and bcl-1 were the most frequently amplified protooncogenes in SCCHN. To define accurately the region of DNA amplification

on 11q13, we analyzed two other gene loci, bcl-1 and c-sea, which have also

been mapped to 11q13. Figure 2 shows a representative BamHI digest of

tumor and matched normal DNA from six patients serially probed with

int-2, hst-1, bcl-1, and c-sea. The results demonstrate that the int-2, hst-1, and

bcl-1 genes are amplified together in a laryngeal and pharyngeal tumor but

not in adjacent normal tissue from the same patients. The degree of amplification generally ranged between two- and eightfold. The c-sea proto-oncogene, which also maps to 11q13, was not amplified in any of the tumors.

In an effort to characterize the region of DNA amplification, filters were

reprobed with the collagenase gene (CLG) located at segment 11q21-22

(Figure 2). No amplification of the CLG gene was detected in tumors where

int-2, hst-1, and bcl-1 were clearly amplified.

A map of chromosome 11 and the region of amplification is shown in

Figure 3. The 11q13 amplicon contains four previously recognized

oncogenes (int-2, hst-1, bcl-1, c-sea). Their location and map distances are

shown. hst-1 is located 35 kb from int-2, and bcl-1 is approximately 1,000 kb

proximal to the centromere from int-2 (Nguyen et al., 1988). The precise

location of c-sea relative to the other loci in 11q13 is unknown. We examined a group of int-2 amplified head and neck tumors for amplification of

hst-1, bcl-1, and c-sea. The results of this analysis are depicted in Figure 3.
Smoking and Tobacco Control Monograph No. 2

122

Figure 1

Amplification of int-2 in SCCHN. DNA (10 μg) was digested with EcoRI and

analyzed by Southern blot hybridization sequentially with the int-2 probe (top),

c-ets-1 probe (middle), and mixed N-ras and N-myc probes (bottom). Lanes 1 to

12: DNA from 12 separate tumors derived from the tongue (T), hypopharynx

(H), tonsil (TL), and larynx (L); A431: DNA from A431 cells; HP, human placenta

DNA. Numbers on the right indicate DNA fragment sizes in kb detected by the

probes.

Eleven of 12 tumors contained DNA coamplified for the int-2 and hst-1

gene; one tumor of the larynx exhibited int-2, but not hst-1, amplification.

Eight of the int-2 amplified tumors were evaluated for bcl-1 and c-sea DNA

amplification. All eight contained amplified bcl-1 DNA, whereas none of

eight had amplified c-sea DNA.
Chapter 3

123

Table 1

Frequency of oncogene amplification in head and neck squamous cell carcinoma

Chromosome

Location Total Amplification

Oncogene

int-2 11 q13 45 14 (31%)

hst-1 11 q13 39 11 (28%)

bcl-1 11 q13 28 8 (29%)

c-sea 11 q13 29 0

c-ets-1 11 q23 24 0

H-ras-1 11 p15.5 33 0

K-ras-2 12 p12.1 21 0

N-ras 1 p22 24 0

N-myc 2 p24 24 0

c-myc 8 q24 21 0

c-erbB-1 7 p12-13 21 2 (10%)

c-erbB-2 17 q11-12 21 0

DISCUSSION In this study we examined a series of SCCHN for activated oncogenes.

We were unable to detect transforming activity in 31 SCCHN DNAs by

transfection assays. Detection of activated oncogenes by the biological

transformation assay may underestimate the frequency of activated

oncogenes (Barbacid, 1987). Few studies have examined head and neck

cancer for activated ras genes. Tadokoro and associates (1989) detected

activated H-ras-1 by DNA transfection in two cell lines established from

metastatic lymph nodes of patients with palatal or floor-of-mouth SCC.

Activated K-ras-2 was detected in 1 gingival SCC of 11 tumor DNAs tested by

DNA transfection (Howell et al., 1990). Recently, the use of allele-specific

oligonucleotide hybridization assays or RNase A mismatch cleavage analysis

has significantly increased the detection of mutated ras genes in human

malignancies (Bos, 1989). Using polymerase chain reaction and oligonucleotide hybridization to detect ras mutations at codons 12, 13, and 61, Sheng

and associates (1990) detected a mutation at codon 12 of the H-ras-1 gene in

2 of 54 head and neck tumors. No mutations were detected at positions 12,

13, and 61 of the K-ras-2 gene or at positions 12 and 61 of the N-ras gene

(Sheng et al., 1990). Although further studies are needed to confirm these

findings, the combined data indicate that ras mutations are an uncommon

genetic alteration in SCCHN.

Proto-oncogenes can be activated to oncogenic potential by mutational

alterations or overexpression (Bishop, 1991). Overexpression of oncogenes

at the level of gene amplification is a frequent observation in human tumors

(Schwab and Amler, 1990). In certain tumors, oncogene amplification is

associated with a more aggressive tumor phenotype and a poor clinical

prognosis. Notable examples of oncogene amplification correlated with

decreased survival include N-myc amplification in neuroblastomas (Brodeur

et al., 1984; Seeger et al., 1985) and c-erbB-2/HER2 amplification in breast

and ovarian cancers (Slamon et al., 1989).
Smoking and Tobacco Control Monograph No. 2

124

Figure 2

Coamplification of int-2, hst-1, and bcl-1 in SCCHN. DNA (10 μg) was digested

with BamHI and the filter probed sequentially with int-2, hst-1, bcl-1, c-sea, and

CLG probes. Tumor (T) and adjacent normal tissue (N) from individual patients

with tumors of the larynx (L), floor of mouth (FOM), and pharynx (PX). A431

and HP are the same as in Figure 1. Numbers on the right indicate DNA fragment sizes in kb detected by the probes.
Chapter 3

125

Figure 3

Map of chromosome 11 with the region of gene amplification in band q13 expanded. The

table on the right indicates the presence (+) or absence (-) and frequency of amplification of

specific gene loci on chromosome 11 in a panel of SCCHN.

There are few studies that report oncogene amplification and overexpression in head and neck cancer. Amplification of N-myc and N-ras was

observed in approximately 40 percent of 23 cases of oral cavity SCC

(Saranath et al., 1989). We were unable to confirm these findings. Increased transcription of c-myc was detected in 14 SCCHN (Field and

Spandidos, 1987) and in 8 of 9 oral and laryngeal SCC (Riviere et al., 1990),

but the number of cases is too small to make any meaningful clinical correlations. Results of the present study reveal the coamplification of int-2, hst1, and bcl-1 in about 30 percent of SCCHN. The three genes comprise a

rather large amplicon on 11q13. We have broadly defined the limits of the

11q13 amplicon in SCCHN to include int-2, hst-1, and bcl-1, but not c-sea,

which also maps to 11q13. The amplicon does not extend to the collagenase locus 11q21-22, the c-ets-1 locus 11q23, or the pepsinogen cluster

mapped to 11p11-q13 (Somers et al., 1990). Further work is needed to map

more accurately the size of the 11q13 amplicon and to identify additional

genes that reside in this region. It is also important to define the selective

mechanisms that drive and maintain the 11q13 amplicon in SCCHN.

Because int-2 and hst-1 are members of the FGF gene family, one model

would predict that overexpression of int-2 or hst-1 might confer a growth

advantage on the tumor cell. Studies of int-2/hst-1 gene expression in

SCCHN are needed to address this possibility. However, data from other

studies have shown low or undetectable levels of int-2 or hst-1 transcripts in
Smoking and Tobacco Control Monograph No. 2

126

human breast cancer, regardless of any amplification of the gene (Fantl et

al., 1990). Alternatively, maintenance of the 11q13 amplicon would be

driven by the expression of an unidentified gene on 11q13. Ultimately, it

will be necessary to express int-2 and hst-1cDNAs in squamous epithelial

cells of the upper aerodigestive tract and examine these cells for properties

that distinguish the malignant tumor phenotype.

Finally, it is important that amplification of int-2, hst-1, and bcl-1 on

11q13 may be involved in different human tumors. One or more of the

three proto-oncogenes within the amplicon have been reported to be

amplified in breast carcinoma (Ali et al., 1989; Theillet et al., 1990), melanoma (Adelaide et al., 1988), esophageal carcinoma (Tsuda et al., 1989),

stomach cancer (Yoshida et al., 1988), and SCCHN (Berenson et al., 1989;

Merritt et al., 1990; Somers et al., 1990; Zhou et al., 1988). Thus, the elucidation of the role of the 11q13 amplicon in SCCHN might provide clues in

understanding the development and progression of other human malignancies. Clearly, this marker in SCCHN should encourage future studies to

determine if 11q13 gene amplification might be useful to predict tumor

behavior and disease progression.

REFERENCES

Adelaide, J., Mattei, M-G., Marics, I., Raybaud, F.,

Planche, J., de Lapeyriere, O., Birnbaum, D.

Chromosomal localization of the hst oncogene and

its co-amplification with the int-2 oncogene in

human melanoma. Oncogene 2: 413-416, 1988.

Ali, I.U., Merlo, G., Callahan, R., Lidereau, R. The

amplification unit on chromosome 11q13 in

aggressive primary human breast tumors entails

the bcl-1, int-2 and hst loci. Oncogene 4: 89-92,

1989.

Barbacid, M. ras genes. Annual Review of Biochemistry

56: 79-82, 1987.

Berenson, J.R., Yang, J., Mickel, R.A. Frequent

amplification of the bcl-1 locus in head and neck

squamous cell carcinomas. Oncogene 4: 1111-1116,

1989.

Bishop, J.M. Molecular themes in oncogenesis. Cell

64: 235-248, 1991.

Bos, J.L. ras oncogenes in human cancer: A review.

Cancer Research 49: 4682-4689, 1989.

Brodeur, G.M., Seeger, R.C., Schwab, M., Varmus,

H.E., Bishop, J.M. Amplification of N-myc in

untreated human neuroblastomas correlates with

advanced disease stage. Science 224: 1121-1124,

1984.

Burgess, W.H., Maciag, T. The heparin binding

(fibroblast) growth factor family proteins. Annual

Review of Biochemistry 58: 575-606, 1989.

Casey, G., Smith, R., McGillivray, D., Peters, G.,

Dickson, C. Characterization and chromosome

assignment of the human homolog of int-2: A

potential proto-oncogene. Molecular and Cellular

Biology 6: 502-510, 1986.

Dickson, C., Acland, P., Smith, R., Dixon, M., Deed,

R., MacAllan, D., Walther, W., Fuller-Pace, F.,

Kiefer, P., Peters, G. Characterization of int-2: A

member of the fibroblast growth factor family.

Journal of Cell Science (Suppl.) 13: 87-96, 1990.

Fantl, V., Richards, M.A., Smith, R., Lammie, G.A.,

Johnstone, G., Allen, D., Gregory, W., Peters, G.,

Dickson, C., Barnes, D.M. Gene amplification on

chromosome band 11q13 and oestrogen receptor

status in breast cancer. European Journal of Cancer

26: 423-429, 1990.

Fasano, O., Birnbaum, D., Edlund, L., Fogh, J.,

Wigler, M. New human transforming genes

detected by a tumorigenicity assay. Molecular and

Cellular Biology 4: 1695-1705, 1984.

Fearon, E.R., Vogelstein, B. A genetic model for

colorectal tumorigenesis. Cell 61: 759-767, 1990.

Field, J.K., Spandidos, D.A. Expression of oncogenes

in human tumors with special reference to the

head and neck region. Journal of Oral Pathology

16: 97-197, 1987.

Graham, F.L., van der Eb, A.J. A new technique for

the assay of infectivity of human adenovirus 5

DNA. Virology 52: 456-467, 1973.

Howell, R.E., Wong, F.S.H., Fenwick, R.G. A

transforming Kirsten ras oncogene in an oral

squamous carcinoma. Journal of Oral Pathology

and Medicine 19: 301-305, 1990.

Merritt, W.D., Weissler, M.C., Turk, B.F., Gilmer,

T.M. Oncogene amplification in squamous cell

carcinoma of the head and neck. Archives of

Otolaryngology—Head and Neck Surgery 116: 1394-

1398, 1990.
Chapter 3

127

Nguyen, C., Roux, D., Mattei, M-G., de Lapeyriere,

O., Goldfarb, M., Birnbaum, D., Jordan, B.R. The

FGF-related oncogenes hst and int-2, and the bcl-1

locus are contained within one megabase in band

q13 of chromosome 11, while the fgf-5 oncogene

maps to 4q21. Oncogene 3: 703-708, 1988.

Riviere, A., Wilckens, C., Loning, T. Expression of

c-erbB2 and c-myc in squamous epithelial and

squamous cell carcinomas of the head and neck

and the lower female genital tract. Journal of Oral

Pathology and Medicine 19: 408-413, 1990.

Saranath, D., Panchal, R.G., Nair, R., Mehta, A.R.,

Sanghavi, V., Sumegi, J., Klein, G., Deo, M.G.

Oncogene amplification in squamous cell

carcinoma of the oral cavity. Japanese Journal of

Cancer Research 80: 430-437, 1989.

Schwab, M., Amler, L.C. Amplification of cellular

oncogenes: A predictor of clinical outcome in

human cancer. Genes Chromosomes Cancer 1: 181-

193, 1990.

Seeger, R.C., Brodeur, G.M., Sather, H., Dalton, A.,

Siegel, S.E., Wong, K.Y., Hammond, D. Association of multiple copies of N-myc oncogene with

rapid progression of neuroblastomas. New England

Journal of Medicine 313: 1111-1116, 1985.

Sheng, Z.M., Barrois, M., Klijanienko, J., Micheau, C.,

Richard, J.M., Riou, G. Analysis of the c-Ha-ras-1

gene for deletion, mutation, amplification and

expression in lymph node metastases of human

head and neck carcinomas. British Journal of

Cancer 62: 398-404, 1990.

Slamon, D.J., Godolphin, W., Jones, L.A., Holt, J.A.,

Wong, S.G., Keith, D.E., Levin, W.J., Stuart, S.G.,

Udove, J., Ullrich, A., Press, M.F. Studies of the

HER2/neu proto-oncogene in human breast and

ovarian cancer. Science 244: 707-712, 1989.

Somers, K.D., Cartwright, S.L., Schechter, G.L.

Amplification of the int-2 gene in human head and

neck squamous cell carcinomas. Oncogene 5: 915-

920, 1990.

Tadokoro, K., Ueda, M., Ohshima, T., Fujita, K.,

Rikimaru, K., Takahashi, N., Enomoto, S.,

Tsuchida, N. Activation of oncogenes in human

oral cancer cells: A novel codon 13 mutation of

c-H-ras-1 and concurrent amplification of c-erbB-1

and c-myc. Oncogene 4: 499-505, 1989.

Taira, M., Yoshida, T., Miyagawa, K., Sakamoto, H.,

Terada, M., Sugimura, T. DNA sequence of

human transforming gene hst and identification

of the coding sequence required for transforming

activity. Proceedings of the National Academy of

Sciences 84: 2980-2984, 1987.

Theillet, C., Adnane, J., Szepetowski, P., Simon, M-P.,

Jeanteur, P., Birnbaum, D., Gaudray, P. BCL-1

participates in the 11q13 amplification found in

breast cancer. Oncogene 5: 147-149, 1990.

Tsuda, T., Tahara, E., Kajiyama, G., Sakamoto, H.,

Terada, M., Sugimura, T. High incidence of

coamplification of hst-1 and int-2 genes in human

esophageal carcinomas. Cancer Research 49: 5505-

5508, 1989.

Tsujimoto, Y., Jaffe, E., Cossman, J., Gorham, J.,

Nowell, P.C., Croce, C.M. Clustering of

breakpoints on chromosome 11 in human B cell

neoplasms with the t(11;14) chromosome

translocation. Nature 315: 340-343, 1985.

Whitham, S.E., Murphy, G., Angel, P., Rahmsdorf,

H-J., Smith, B.J., Lyons, A., Harris, T.J.R.,

Reynolds, J.J., Herrlich, P., Docherty, J.P. Comparison of human stromelysin and collagenase by

cloning and sequence analysis. Biochemistry

Journal 240: 913-916, 1986.

Williams, B.P., Shipley, J.M., Spurr, N.K., Smith,

D.R., Hayman, M.J., Goodfellow, P.N. A human

sequence homologous to v-sea maps to chromosome 11, band q13. Oncogene 3: 345-348, 1988.

Yoshida, M.C., Wada, M., Satoh, H., Yoshida, T.,

Sakamoto, H., Miyagawa, K., Yokota, J., Koda, T.,

Kakinuma, M., Sugimura, T., Terada, M. Human

HST1 (HSTF1) gene maps to chromosome band

11q13 and coamplifies with the INT2 gene in

human cancer. Proceedings of the National Academy

of Sciences 85: 4861-4864, 1988.

Zhou, D.J., Casey, G., Cline, M.J. Amplification of

human int-2 in breast cancers and squamous cell

carcinomas. Oncogene 2: 279-282, 1988.
Smoking and Tobacco Control Monograph No. 2

128

This paper reviews the status and implications of oncogenes in oral cancer caused by

Oncogenes in Tobacco-Induced

Oral Cancer

Madhav G. Deo and Dhananjaya Saranath

ABSTRACT

smokeless tobacco (chewing), which accounts for 40 percent of malignancies in India. A

variety of abnormalities consisting of amplification, overexpression, point mutation, deletion,

and rearrangement of oncogenes, particularly of myc and ras families, are seen in more than

90 percent of the cancers in India. A longitudinal study of oncogenes at various stages of oral

carcinogenesis, particularly in leukoplakias and early cancers, will not only throw light on the

molecular biology of malignant transformation but also provide clues to the early diagnosis of

this cancer through molecular biology techniques.

INTRODUCTION Oral cancer, which is among the 10 most prevalent cancers in the

world, is a common cancer in South and Southeast Asia, where the habit of

tobacco chewing is widely prevalent. In India, tobacco chewing is a major

cause of cancer mortality, accounting for about 40 percent of the total

malignancies. Clinical, epidemiological, and laboratory studies indicate a

causal relationship between prolonged tobacco chewing and oral cancer,

smokeless tobacco being the sine qua non (Gupta et al., 1987; Jussawalla

and Deshpande, 1971). A latent period of 5 to 15 yr is common, and almost

every tobacco-related oral cancer is preceded by a phase of leukoplakia.

Several constituents of tobacco, such as tobacco-specific N-nitrosamines, polycyclic aromatic hydrocarbons, and polonium-210 (a radioactive alpha emitter), are capable of inducing preneoplastic and neoplastic

changes in the oral cavity (IARC, 1985; Mattson and Winn, 1989). Although much information is available about their chemistry, the critical link

between the tobacco-specific carcinogens and the cellular macromolecules

leading to the malignant phenotype is an enigma. Tobacco-specific nitrosamines are known to form DNA adducts, resulting in mutations that could

profoundly affect cellular genes (Hecht et al., 1988).

The importance of proto-oncogenes in cellular growth and differentiation is well documented (Bishop, 1987; Klein and Klein, 1985). One of the

mechanisms involved in the process of carcinogenesis is the activation of

proto-oncogenes (oncogenes), which has been observed in a number of

human and experimental cancers (Klein, 1988). Illegitimate activation of

proto-oncogenes may occur by various mechanisms, including gene amplification, chromosomal translocation, point mutation, and retroviral insertion, that in turn contribute to tumor development and progression in

several systems (Klein, 1988).

This review elucidates the various molecular lesions that involve

oncogenes in oral cancers. Such studies can be grouped into four categories:

(1) examination of genomic DNA of the primary tumor for oncogene
Chapter 3

129

amplification; (2) aberrant oncogene expression as seen by an increase in

mRNA transcript level or alteration in the transcript size or an increase in

the protein product; (3) allelic loss, rearrangement, and point mutation; and

(4) detection, isolation, and cloning of a functional oncogene in oral cancers

in a transfection assay. The methodology used in our studies is outlined in

Figure 1.

ONCOGENE

AMPLIFICATION

We investigated the status of the commonly amplified oncogenes

of the myc and ras families in 102 primary oral tumor tissues

(Saranath et al., 1989). All tumors were squamous cell carcinomas (SCCs)

involving various regions: buccal mucosa (46 patients), lower alveolus

 (29 patients), tongue (23 patients), and floor of the mouth (4 patients). All

patients had chewed tobacco regularly for 10 to 15 yr. Although histological grading varied from well to poorly differentiated, the majority of the

tumors were large and in an advanced stage (TNM III/IV). Lymph node

metastases were frequent, particularly in the T3 and T4 lesions. However,

none of the patients showed distant metastasis. Peripheral blood cells

(PBCs) from the patients were analyzed for oncogene involvement. PBCs

from healthy volunteers and human placental DNA were included in the

study as controls. Vimentin, a single copy gene, was used as an internal

control gene.

Southern hybridization analysis of the EcoRI-digested DNA samples

revealed a 3-fold to 10-fold amplification of c-myc, N-myc, K-ras, or N-ras in

49 of the 102 (48 percent) primary oral tumor tissues screened (Table 1).

H-ras and L-myc oncogenes were not amplified in any of the samples. The

controls—PBC DNA from the patients and healthy volunteers and normal

human placental DNA—showed the presence of a single copy of the

oncogenes.

Amplification of myc oncogenes was observed in 37 of the 102 patients.

N-myc and c-myc were amplified in 26 percent (27 of 102) and 21 percent

(21 of 102) of the patients, respectively (Table 1). An unusual finding was

the coamplification of c-myc and N-myc in 11 of 37 (30 percent) of the

patients. It has been hypothesized that the products of myc family

oncogenes, c-myc, N-myc, and L-myc, down-regulate each other (Alt et al.,

1986). As a consequence, it is predicted that tumor cells would have amplification or increased expression of only one of the myc oncogenes. Indeed,

this appears to be the rule. However, oral cancer seems to be unique in this

respect, with the two myc oncogenes coamplified in a number of patients.

However, it is not possible from these studies to know if the coamplification

represents the existence of two different clones that each contain a single

amplified myc. Alternatively, the two myc oncogenes could be amplified in

the same cell. Coexpression of c-myc and N-myc has been reported in the

early undifferentiated pre-β cells in mice (Zimmerman et al., 1986), implying coexpression during a particular developmental stage or in a particular

differentiation window of the cell. In our studies, amplification bore no

correlation with clinicopathological features such as degree of differentiation, tumor size, TNM staging, nodal metastasis, or recurrence.
Smoking and Tobacco Control Monograph No. 2

130

Figure 1

Flow chart of methods used to study oncogene aberrations in oral cancers

Opinion is divided on the status of c-myc amplification or its increased

expression in oral cancer. Although Berenson and colleagues (1989) found

no amplification, Yokota and coworkers (1986) reported a fivefold to eightfold amplification of the oncogene in SCCs of the head and neck. They also

found that, compared with the primary tumor, the metastatic tumor tissue

showed higher myc amplification. Field et al. (1986) observed an elevated

c-myc mRNA expression correlating well with the stage of the disease. In

another study, tumors with poor prognosis exhibited increased c-myc

oncoprotein (Field et al., 1989). Oncogenes from the myc family have been

involved in regulating cellular proliferation, and their amplification is

associated with certain aggressive tumors (Alitalo et al., 1985).

Our data also demonstrate amplification of K-ras or N-ras in 33 percent

(34 of 102) of the total samples analyzed. K-ras showed a threefold to

eightfold amplification in 18 percent of the samples, whereas N-ras was

amplified in 28 percent (Table 1). Amplification of ras oncogene did not

correlate with any clinical parameters, including nodal metastasis. H-ras

was not amplified. Other investigators also have observed no amplification

of H-ras in oral cancer (Sheng et al., 1990; Sommers et al., 1990).

Oral tumor tissue

Homogenization

RNA extraction DNA extraction

Northern hybridization analysis/

dot blot analysis

Southern hybridization analysis

Amplification

of oncogenes

Gene 

rearrangement 

in restriction 

length 

polymorphism 

studies

Allelic 

loss of 

fragment

Point mutation

by polymerase

chain reaction 

analysis

Identification

of functional

oncogene

NIH3T3

transfection assay

Cotransfection with

oral tumor tissue DNA

and SV2 gene 

followed by analysis 

of transfected clones 

for

Nude mouse

tumorigenicity

Soft agar

cloning

Identification 

of known 

oncogenes

Presence 

of human 

sequence

neo
Chapter 3

131

Table 1

Oncogene amplification in oral cancersa

Cellular DNA Samples

Range of Fragment Amplified

Oncogenes Amplification (kb) n (%)

c-myc 3-6 12.7 21 (21)

N-myc 3-10 3.8 27 (26)

L-myc 1 10.0, 6.6 0

K-ras 3-8 2.7 18 (18)

N-ras 3-9 7.2 28 (28)

H-ras 1 23-30 0

EGF-R 3-8 7.8, 7.0, 6.1, 19 (29)

5.6, 3.7, 2.5,

2.1, 1.9, 1.6,

and 1.3

a Total number of samples analyzed was 102 for myc and ras and 66 for EGF-R gene.

In our study, coamplification of multiple oncogenes was an important

feature, being observed in 24 of the 49 samples showing amplification

(Table 2). Two oncogenes were coamplified in 10 tumor tissues and 3 in an

additional 10 samples; 5 patients showed concurrent amplification of the

four oncogenes—c-myc, N-myc, K-ras, and N-ras. Multiple oncogene amplification indicates the complex, multistage process of oral carcinogenesis, and

it implies alternate or simultaneous activation of the different oncogenes in

oral carcinogenesis.

There are very few studies on the status of other oncogenes in oral

cancer. In the studies of Sommers and colleagues (1990), amplification of

int-2 oncogene correlated well with frequency of recurrence. However, no

correlation was observed with clinical staging or histological grading of the

malignancy. Berenson and coworkers (1989) have reported a 2-fold to

10-fold amplification of the bcl-1 oncogene in poorly differentiated tumors,

but no correlation was seen with the TNM staging.

The epidermal growth factor-receptor (EGF-R) gene is considered to be

the proto-oncogene of the erb-B gene of avian erythroblastosis virus, which

is involved in transformation of chicken cells (Downward et al., 1984). Its

ligand EGF is a potent mitogen for a variety of cells. EGF-R is commonly

elevated in squamous cell carcinomas. In our study, in which all tumors

were SCCs, the EGF-R gene was amplified in 29 percent (19 of 66) of the

samples screened (Table 1). Preliminary results, from mRNA dot blot hybridization, indicate that amplification is associated with increased expression of the gene. Amplification and overexpression of the EGF-R gene,

through interaction with the ligand, could be an additional step in oral

carcinogenesis.
Smoking and Tobacco Control Monograph No. 2

132

Table 22-2

Amplification of oncogenes c-myc, N-myc, N-ras, and K-ras

Number of Patientsa

Total c-myc N-myc N-ras K-ras

Number of Patients 102

Patients Showing 49b 21 27 28 18

Oncogene Amplification

One Oncogene 24 9 5 7 3

Two Oncogenes 10

myc/myc 1

ras/ras 7 (c-myc/K-ras 1; N-myc/K-ras 1; N-myc/K-ras 5)

ras/ras 2

Three Oncogenes 10

myc/myc/ras 5 (N-myc/c-myc/K-ras 1; N-myc/c-myc/N-ras 4)

myc/ras/ras 5 (N-myc/N-ras/K-ras 5)

Four Oncogenes 5

a In many patients, more than one oncogene was amplified. The number derived by

addition of the individual figures is therefore higher than the total number of positive

samples.

Expression of cellular oncogenes in oral cancers has been investigated through a variety of techniques, such as Northern hybridization/dot blot analysis for mRNA transcript in tumor tissue, in situEXPRESSION

OF CELLULAR

ONCOGENES

hybridization for detecting mRNA specific to oncogenes, and immunohistochemical demonstration of oncoproteins using monoclonal antibodies.

Using dot blot hybridization, Spandidos and coworkers (1985) reported

multiple transcriptional activation of H-ras, K-ras, and c-myc in oral cancers

compared with normal tissue and premalignant pleomorphic salivary

adenomas. The authors also observed a significant increase in c-myc

oncogene expression in advanced stages III and IV compared with stages I

and II. Recently, the authors also demonstrated correlation of increased

c-myc expression with poor prognosis. No correlation was seen with the

patient age, sex, TNM staging, site of tumor, histopathological grading,

lymph node metastasis, or encapsular rupture of the tumor (Field et al.,

1989).

Using immunohistochemical techniques and oncoprotein-specific

monoclonals, Azuma and colleagues (1987) studied ras p21 protein in oral

cancers. Although 59 of 121 specimens reacted positively to Y13-259-ras

p21-specific Mab, 44 oral leukoplakias and 58 normal mucosa showed a

negative staining. Only 43 patients with oral cancer were followed up.
Chapter 3

133

In these persons, ras p21 expression correlated well with poor prognosis and

only weakly with regional lymph node metastasis. Furthermore, ras p21

expression was higher in the patients with a tobacco habit.

RFLP ANALYSIS

OF L-myc AND

H-ras ONCOGENES

L-myc

Researchers have examined restriction fragment length polymorphism (RFLP) in a number of human cancers to establish a

specific association of a particular allele with an increased

incidence of cancer, suggesting genetic predisposition to the

cancer, or as an indicator of clinical behavior, in particular,

H-ras

metastatic potential or prognosis. In our studies, we used Southern hybridization analysis of 76 oral cancer primary tumor tissues and PBCs from the

corresponding patients, and PBCs from 101 healthy volunteers, to classify

the Indian population into three genotypes—L-L, S-L, S-S—according to the

polymorphic patterns defined by the two L-myc alleles (Saranath et al.,

1990). The pattern of the alleles in the PBC DNA of the patients was identical to the corresponding tumor DNA. The relative ratios of the three genotypes in the oral cancer patients were not significantly different from those

seen in the healthy Indian population (χ2

=3.06, df=2, p > 0.25), implying no

predisposition to oral cancer by the presence of either allele. The L and S

alleles were equally distributed in the population, with the frequency of

each allele being 0.50, consistent with the Hardy-Weinberg law.

A striking correlation was observed between the RFLP pattern and the

stage of differentiation, as well as the size of the tumors (Saranath et al.,

1990). Thus, a preponderance of the S-fragment was observed in the moderate to poorly differentiated tumors (χ2=4.97, df=1, p < 0.05) and the larger

sized (> 4 cm) tumors (χ2=5.65, df=1, p < 0.025). These observations suggest

that the S allele product may be arresting the cells in a particular differentiation window, providing a further proliferative advantage to the cells, resulting in the larger sized tumors. There is also the possibility for involvement

of another gene in proximity to the S-allele, or for a partially different

S-fragment-coded L-myc protein, or a crucial role for the regulatory region of

the S-fragment protein in the tumors. Lung and kidney tumors associated

with S-fragment show aggressive behavior and higher tendency for metastasis (Kakehi and Yoshida, 1989; Kawashima et al., 1987). However, this is not

the case with malignancies of other organs (Ikeda et al., 1988).

The H-ras locus includes a hypervariable region, designated as variable

tandem repetition region (VTR), consisting of a series of 28 base pair repeats

3' to the gene (Capon et al., 1983). The VTR has been implicated in the

regulation of the H-ras oncogene (Spandidos and Holmes, 1987). RFLP of

the human H-ras oncogene has been ascribed to changes in the size of the

VTR and can be defined by several restriction enzymes, including BamHI,

PvuII, and TaqI (Capon et al., 1983; Pierotti et al., 1986).

In our studies, the status of H-ras locus was investigated in 62 patients

with oral cancers (Saranath et al., 1991a). Southern blot analysis on BamHI

digestion of the tumor tissue DNA revealed 23 patients with H-ras heterozygosity. BamHI digestion identified restriction fragments ranging from 6.6 kb

to 8.6 kb. The allelic heterozygosity was better resolved by PvuII and TaqI

digestion. The former yielded an invariable fragment of 2.6 kb and variable
Smoking and Tobacco Control Monograph No. 2

134

fragments ranging from 2.7 kb to 5.3 kb. TaqI digestion of the samples and

hybridization with the VTR region probe (1 kb MspI-digested, VTR-specific

H-ras probe) resolved four variable fragments of 2.4 kb, 3.0 kb, 3.6 kb, and

5.1 kb. In three samples, TaqI restriction analysis also demonstrated presence of a unique VTR rearrangement as indicated by 2.1 kb, 0.9 kb, and

0.6 kb fragments, substituting a 3.6 kb fragment, implying additional TaqI

sites. Such a variant VTR fragment could be generated by either mutational

events creating the TaqI sites or reiteration of the existing TaqI site present

18 bp from the 3' end of the VTR region during its amplification/duplication. This rearrangement, which suggests altered function of the VTR, could

be one of the molecular lesions in tobacco-induced oral cancer.

Analysis of matched tumor tissue and PBC DNA from the same patient

demonstrated tumor-associated loss of one of the allelic fragments in 7 of

23 patients (30 percent) with H-ras heterozygosity. Similar observations

have been made by Howell and colleagues (1989) in oral SCCs. H-ras allelic

loss has been reported in a number of human cancers (Ali et al., 1987);

however, its implication in carcinogenesis is not yet clear. Tumor suppressor genes or anti-oncogenes have been implicated in the pathogenesis of

some human tumors (Klein, 1988). Ali and coworkers (1987) identified a

putative suppressor gene on chromosome 11, in the vicinity of the H-ras

locus, localized between the β-globin and parathyroid hormone loci. The

loss of H-ras allele observed in our patients also may encompass functional

loss of the potent tumor suppressor gene on chromosome 11, further influencing the process of oral carcinogenesis in the patients. Alternatively, the

loss could involve the normal H-ras gene, giving a selective functional

advantage to the mutated H-ras allele involved in cell transformation and a

consequent influence in oral carcinogenesis. In this respect, it is significant

that 6 of 7 patients showing H-ras mutation at codon 12.2 also exhibited

associated loss of the wild type gene. Such a loss may not be evident in

RFLP studies or if the alleles are in a homozygous state.

H-ras POINT

MUTATIONS

Point mutations of ras oncogenes are observed in a variety of human

cancers (Bos, 1989). We employed the polymerase chain reaction

(PCR) technique for in vitro amplification of specific sequences followed by

allele-specific oligonucleotide hybridization to examine ras activation by

point mutations in 57 primary oral tumors (Saranath et al., 1991b). The

mutational activation was studied in all three ras family members (K-ras,

N-ras, and H-ras) at codons 12, 13, and 61, the codons affected in human

cancers (Bos, 1989). Suitable primers were used to amplify sequences of

111 bp and 178 bp for the regions flanking codons 12, 13, and 61, respectively. The amplified sequences were initially screened with sets of mixed

probes, each set covering one nucleotide position of a particular codon.

Probes used to identify mutations in H-ras have been described elsewhere

(Saranath et al., 1991b). On indication of a mutation in the sample DNA, a

duplicate PCR of the genomic DNA was performed, and the two independently amplified DNAs were screened simultaneously with a set of single

oligonucleotide probes specific for the nucleotide position. The presence of

wild type codons was screened for in every set.
Chapter 3

135

Mutations were detected in 20 to 57 (35 percent) of the samples and

were restricted exclusively to the H-ras oncogene at codons 12, 13, and

61 (Table 3). None of the samples showed mutations in the K-ras or

N-ras oncogenes. The mutations demonstrated an equal number (11 each)

of nucleotide transitions and transversions. The mutations were seen

primarily in codons 61.2 and 12.2. Nine samples showed A —> G transition

in codon 61.2 resulting in glutamine to arginine substitution. G —> T

transversion was observed in seven samples at codon 12.2 with glycine to

valine substitution. Two patients concurrently carried mutations at codons

61.2 and 12.2. Six samples with a point mutation at codon 12.2 and two

samples at codon 61.2 also demonstrated loss of the corresponding wild

type codons, as judged by the absence of signals on allele-specific oligonucleotide hybridization. In addition, three novel mutations, not yet

reported in human malignancies, were seen. These include the G —> A

(glycine to serine) substitution at codon 12.2, G —> A (glycine to aspartate)

at codon 13.2, and G —> T (glutamine to histidine) (three cases) at codon

61.3.

In contrast to the very high frequency (35 percent) of H-ras mutations

we observed, Sheng and coworkers (1990) detected mutations in only

3.7 percent (2 of 54) of their samples. Although mutations in H-ras occurred

in the studies by Sheng and colleagues, the mutations were restricted to

codon 12. Similarly, Johnson and coworkers (personal communication,

1991) and Rumsby and colleagues (1990) very rarely encountered mutations

in oral SCCs from patients in the United Kingdom. Thus, it is clear that

mutational frequencies in oral cancers in the West are infrequent, and

perhaps only codon 12 is affected. The differences in oral cancers in India

and the West may be attributed to differing tobacco habits. Whereas

tobacco chewing appears to be the prime factor in the development of oral

cancer in India, alcohol consumption contributes substantially in the West,

where smoking also is a confounding factor in the pathogenesis of oral

cancer. The mode of tobacco usage, strain or species of tobacco used, and

curing process (which differs between East and West) also may contribute to

these differences.

There appears to be, to a certain extent, organ specificity in activation

of ras family oncogenes (Bos, 1989). Thus, N-ras point mutations are seen

primarily in hematological malignancy, whereas in cancers of the lung,

colon, and pancreas, K-ras frequently is affected. H-ras mutations are

observed in bladder cancers (Bos, 1989) and experimentally induced skin

tumors (Quintanilla et al., 1986). No explanation is readily available for this

phenomenon, but it could be attributable to relative expression of different

ras oncogenes in various organs (Leon et al., 1987). In lung cancer, another

important tobacco-related cancer, mutation is observed primarily in codon

12 of K-ras (Slebos et al., 1990). However, in tobacco-related oral cancer,

both codons 12 and 61 of H-ras are affected. In experimentally induced

animal tumors, carcinogens show positional preference for ras mutations.

Nitrosamines and DMBA preferentially affect codons 12 and 61, respectively

(Quintanilla et al., 1986; Zarbl et al., 1985).
Smoking and Tobacco Control Monograph No. 2

136

Table 3

H-ras mutations

Number of

Patients Number of Patients With

 Codon With Wild Amino Amplification of Oncogenesa

Position of Number of Type Signal Nucleotide Acid

Nucleotide Patients Missing Change Change L-myc N-myc K-ras N-ras

12.1 1 0 GGC—>AGC Gly—>Ser 1 N N N

12.2b 7 6 GGC—>GTC Gly—>Val N N N 1

13.2 1 0 GGC—>GAC Gly—>Asp N N N N

61.2b 9 2 CAG—>CGG Gln—>Arg 3 2 N 2

61.2 1 0 CAG—>CTG Gln—>Leu N N N N

61.3 3 0 CAG—>CAT Gln—>His N 2 2 2

a N = nil.

b Two patients showed mutations at both codon 12.2 and 61.2.

As mentioned earlier, tobacco contains several potent mutagens and

carcinogens such as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Nitrosamines are known to produce

metabolites capable of binding DNA, resulting in products such as

O6-methylguanine, which can lead to miscoding during DNA replication

(Topal, 1988). In India, tobacco is chewed generally as betel quid, which

contains areca nut, slaked lime, catechu, and flavoring agents in addition to

tobacco. Whereas mutation of codon 12 could be attributable to tobaccospecific nitrosamines, other chemical constituents may be responsible for

mutation at codon 61. This aspect needs further elucidation.

IDENTIFICATION

OF A FUNCTIONAL

ONCOGENE

NIH3T3 mouse fibroblast transfection combined with the nude

mice tumorigenicity assay is widely used to identify activated

oncogenes (Shih and Weinberg, 1982). Using this system,

Friedman and colleagues (1983) reported the presence of transforming genes

in DNA from head and neck SCCs. However, the oncogene has yet to be

characterized. Recently, Howell and coworkers (1990) identified the K-ras

oncogene in head and neck SCCs from primary and secondary NIH-transformed cells. The oncogene was isolated from only 1 of the 11 tumors

studied by the investigators.

The NIH3T3 transfection assay shows a bias toward detection of ras

oncogenes. However, with the use of selectable markers such as SV2neo gene,

in cotransfection followed by tumorigenicity assays, several novel genes,

besides the ras family oncogenes, also have been detected in human cancers

(Fasano et al., 1984).

We recently carried out the NIH3T3 cotransfection assay, using oral

tumor tissue DNA and SV2neo selectable marker gene, followed by nude

mouse tumorigenicity assay. Calf thymus DNA and H-ras pEJ6.6 clone were

used as negative and positive controls, respectively. The H-ras oncogenetransfected cells induced tumors in the nude mice within 3 to 5 weeks.
Chapter 3

137

These tumors and the cell lines established from them showed the presence

of H-ras DNA in Southern hybridization analysis as well as a 1.2 kb H-ras

RNA transcript in Northern hybridization analysis, validating the absence of

technology errors in our studies.

Transfection assay was carried out with oral tumor DNA from three

patients. The samples from the three patients induced transformation of

NIH-3T3 cells, forming colonies in soft agar. The transfected cells induced

tumors in nude mice within 5 to 10 wk. Further, DNA isolated from the

nude mice tumors hybridized with human Alu Blur-2 probe in Southern

analysis and cell lines established from the tumors retained the human Alu

sequences. However, DNA isolated from oral tumor DNA transfected nude

mice tumors, as well as their established tumor cell lines, revealed no hybridization with any of the ras or myc oncogenes. This was surprising

because N-ras was amplified in two of the original primary tumors and one

of them also showed H-ras point mutation at codon 12.2; the third tumor

did not show any aberration of ras or myc oncogenes. As mentioned earlier,

positive results were obtained in three of three oral tumor samples. Generally, a maximum of 30 percent transformation rate has been observed in

NIH3T3 transfection/nude mouse tumorigenicity assays, using DNA from

solid tumors (Krontiris and Cooper, 1981). Very high transfection and nude

mice tumorigenicity frequency, in our studies, indicate the possibility of a

highly potent, tobacco-induced activated oncogene, different from the myc

or ras oncogenes. The situation may be similar to the presence of N-myc

oncogene in neuroblastomas or L-myc in small cell lung carcinomas. We are

currently in the process of cloning this gene from the nude mouse tumors to

isolate, sequence, and identify the gene.

CONCLUSIONS

AND FUTURE

PROSPECTS

The observation that more than 90 percent of the oral cancers caused

by smokeless tobacco (chewing) show some sort of oncogene aberration (Table 4) indicates that tobacco-induced oncogene changes are

closely linked to the pathogenesis of these cancers. The occurrence of

multiple oncogene aberrations suggests that the process of malignant

transformation results, probably, as a consequence of diverse molecule

alterations. In our studies, the functional lesions in oncogenes could be

grouped into (1) overproduction of oncogene effector molecules through

amplification or overexpression of the genes, (2) production of abnormal

oncogene products through point mutations, and (3) deletion of normal

oncogene allele or putative tumor suppressor, as evidenced by allelic loss.

These perturbations may act independently or in concert, resulting in

deregulation of cellular growth.

A distinct difference emerges in the mode of activation of oncogenes in

oral cancers in the Indian population and Western countries. Low levels of

amplification of myc and ras oncogenes and low incidence of point mutations are observed in the West. Comparative studies of molecular biology of

oral cancers vis-a-vis the varied tobacco habits and the associated confounding factors, such as alcohol in the West and different constituents of betel

quid in India, may throw light on the pathogenic mechanisms responsible

for these differences. Studies in suitable laboratory models using individual

agents may be equally fruitful.
Smoking and Tobacco Control Monograph No. 2

138

Table 4

Oncogenes in oral cancers

Summary Analysis Percentage Comments

Amplification 48.0%

c-myc, N-myc, K-ras, N-ras

(n=102)

Point Mutations 23.0 Excluding 12% samples

H-ras showing, in addition,

(n=57) amplification/allelic loss/

gene rearrangement

H-ras Allelic Loss 14.5 Excluding 21.5% samples

or VTR Rearrangement showing other oncogene

 (n=62; heterozygous=23) aberrations

EGF-R Amplification 9.0 Excluding 20% samples

(n=66) showing other oncogene

aberrations

Samples Showing at Least One 94.5

Oncogene Aberration

Transfection assays, in our studies, indicate involvement of a highly

potent activated oncogene, outside the ras and myc families, in tobaccoinduced oral carcinogenesis. The oncogene must be fully characterized. A

high incidence of point mutations is observed in Indian patients at codons

12 and 61 of the H-ras oncogene. Some of the tobacco-specific carcinogens,

such as nitrosamines, which form DNA adducts, are known to have positional preference for inducing point mutation in ras oncogenes. The precise

role of point mutation, which is an early event in certain human malignancies, in the pathogenesis of oral cancer needs further elucidation.

Some tobacco chewers develop oral cancer at a relatively young age, a

situation reminiscent of smoking and lung cancer. There is evidence that an

autosomal gene, showing Mendelian codominant inheritance, is responsible

for early-onset lung cancers (Bonney, 1990; Sellers et al., 1990). Interaction

of the gene, which has yet to be characterized, with smoking accounts for

69 percent and 47 percent of lung cancers occurring at ages 50 and 60,

respectively. Although this in no way negates the crucial role of smoking

and should not be a deterrent to antitobacco efforts, similar studies should

be conducted with tobacco chewers.

Our study comprised primarily large and advanced tumors. To get an

idea of the absolute and relative importance of these perturbations, it is

essential to investigate the status of oncogenes in a longitudinal study

encompassing different stages of tobacco carcinogenesis, including leukoplakia. Such a study is also required to define the molecular counterpart of
Chapter 3

139

precancerous lesions to identify leukoplakias that run a high risk of malignant transformation. In this respect, a retrospective study, using PCR

techniques to examine paraffin blocks of leukoplakias and oral cancers in

different stages of evolution, would be fruitful. Such studies are currently in

progress in our laboratory.

REFERENCES

Ali, I.U., Liderau, R., Theillet, C., Callahan, R.

Reduction to homozygosity of genes on chromosome 11 in human breast neoplasia. Science 238:

185-188, 1987.

Alitalo, K., Koskinen, P., Makela, T.P., Saksela, K.,

Sistonen, L., Winqvist, R. myc oncogene: Activation and amplification. Biochimica et Biophysica

Acta 907: 1-32, 1985.

Alt, F.W., DePinho, R., Zimmerman, K., Legouy, E.,

Hatton, K., Ferrier, P., Tesfaye, A., Yancopoulos, G.,

Nisen, P. The human myc gene family. Cold Spring

Harbor Symposia on Quantitative Biology LI: 931-941,

1986.

Azuma, M., Furumoto, N., Kawamata, H., Yoshida,

H., Yanagawa, T., Yura, Y., Hayashi, Y., Takegawa,

Y., Sato, M. The relation of ras oncogene product

p21 expression to clinicopathological status

criteria and clinical outcome in squamous cell

head and neck cancer. Cancer Journal 1: 375-380,

1987.

Berenson, J.R., Yang, J., Mickel, R.A. Frequent

amplification of the bcl-1 locus in head and neck

squamous cell carcinomas. Oncogene 4: 1111-1116,

1989.

Bishop, J.M. The molecular genetics of cancer.

Science 235: 311, 1987.

Bonney, G.E. Interactions of genes, environment

and life-style in lung cancer development. Journal

of the National Cancer Institute 82: 1236-1237, 1990.

Bos, J.L. ras oncogenes in human cancer: A review.

Cancer Research 49: 4682-4689, 1989.

Capon, D., Chen, E., Levinson, A., Seeburg, P.,

Goeddel, D. Complete nucleotide sequences of the

t24 human bladder carcinoma oncogene and its

normal homologue. Nature 302: 33-37, 1983.

Downward, J., Yarden, Y., Mayeot, S. Close similarity

of epidermal growth factor receptor and v-erbB

oncogene protein sequences. Nature 37: 521-528,

1984.

Fasano, O., Birnbaum, D., Edlund, L., Fogh, J.,

Wigler, M. New human transforming genes

detected by a tumorigenicity assay. Molecular and

Cellular Biology 4: 1695-1705, 1984.

Field, J.K., Lamothe, A., Spandidos, D.A. Clinical

relevance of oncogene expression in head and

neck tumors. Anticancer Research 6: 595-600, 1986.

Field, J.K., Spandidos, D.A., Stell, P.M., Vaughan,

E.D., Evan, J.I., Moore, J.P. Elevated expression of

the c-myc oncoprotein correlates with poor

prognosis in head and neck squamous cell

carcinoma. Oncogene 4: 1463-1468, 1989.

Friedman, W.H., Rosenblum, B., Lowenstein, P.,

Thornton, H., Katsantonis, G., Green, M.

Oncogenes: Preliminary studies in head and neck

cancer. Laryngoscope 93: 1441-1444, 1983.

Gupta, P.C., Mehta, F.S., Pindborg, J.J., Aghi, M.B.,

Bhonsle, R.B., Murti, P.R. An educational

intervention study for tobacco chewing and

smoking habits among Indian villagers. In:

Smoking and Health, A.M. Hisamichi and S.

Tominaga (Editors). Amsterdam: Excerpta

Medica, 1987, pp. 623-627.

Hecht, S., Spratt, T.E., Trushin, N. Evidence for

4-(3-pyridyl)-4-oxobutylation of DNA in F344 rats

treated with the tobacco-specific nitrosamines

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

and N'-nitrosonornicotine. Carcinogenesis 9: 161-

165, 1988.

Howell, R.E., Wong, F.S.H., Fenwick, R.G. Loss of

Harvey ras heterozygosity in oral squamous

carcinoma. Journal of Oral Pathology and Medicine

18: 79-83, 1989.

Howell, R.E., Wong, F.S.H., Fenwick, R.G. A

transforming Kirsten ras oncogene in an oral

squamous carcinoma. Journal of Oral Pathology

and Medicine 19: 301-305, 1990.

Ikeda, I., Ishizaka, Y., Ochiai, M., Sakai, R., Itabashi,

M., Onda, U., Sugimura, T., Nagao, M. No

correlation between l-myc restriction fragment

length polymorphism and malignancy of human

colorectal cancers. Japanese Journal of Cancer

Research 79: 674-676, 1988.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosamines (volume 37). Lyon:

IARC, 1985.

Jussawalla, D.J., Deshpande, V.A. Evaluation of

cancer risk in tobacco chewers and smokers: An

epidemiologic assessment. Cancer 28: 469-476,

1971.
Smoking and Tobacco Control Monograph No. 2

140

Kakehi, Y., Yoshida, O. Restriction fragment length

polymorphism of the L-myc gene and susceptibility

to metastasis in renal cancer patients. International

Journal of Cancer 43: 391-394, 1989.

Kawashima, K., Shikama, H., Imoto, K., Izawa, M.,

Naruke, T., Okahayashi, K., Nishimura, S. Close

correlation between restriction fragment length

polymorphism of the L-myc gene and metastasis of

human lung cancer to the lymph nodes and other

organs. Proceedings of the National Academy of

Sciences 85: 2353-2356, 1987.

Klein, G. Oncogenes and tumor suppressor genes.

Reviews in Oncology 1. Acta Oncologica 27: 427-

437, 1988.

Klein, G., Klein, E. Evolution of tumors and the

impact of molecular oncology. Nature 315: 190-

195, 1985.

Krontiris, T.G., Cooper, G.M. Transforming activity

of human tumor DNA. Proceedings of the National

Academy of Sciences 78: 1181-1184, 1981.

Mattson, M.E., Winn, D.M. Smokeless tobacco:

Association with increased cancer risk. National

Cancer Institute Monographs 8: 13-16, 1989.

Pierotti, M., Radice, P., Biunno, I., Borrello, M.,

Cattadori, M., Porta, G. Detection of two TaqI

polymorphisms in the VTR region of the human

H-ras-1 oncogene. Cytogenetics and Cell Genetics

43: 174-180, 1986.

Quintanilla, M., Brown, K., Ramsden, D., Balmain, A.

Carcinogen-specific mutation and amplification of

Ha-ras during mouse skin carcinogenesis. Nature

322: 78-82, 1986.

Rumsby, G., Carter, R.L., Gusterson, B.A. Low

incidence of ras oncogene activation in human

squamous cell carcinomas. British Journal of Cancer

61: 365-368, 1990.

Saranath, D., Panchal, R.G., Nair, R., Mehta, A.R.,

Sanghavi, V., Sumegi, J., Klein, G., Deo, M.G.

Oncogene amplification in squamous cell

carcinoma of the oral cavity. Japanese Journal of

Cancer Research 80: 430-437, 1989.

Saranath, D., Panchal, R.G., Nair, R., Mehta, A.R.,

Sanghavi, V., Deo, M.G. Restriction fragment

length polymorphism of the L-myc gene in oral

cancer patients. British Journal of Cancer 61: 530-

533, 1990.

Saranath, D., Bhoite, L.T., Mehta, A.R., Sanghavi, V.,

Deo, M.G. Loss of allelic heterozygosity at the

Harvey ras locus in human oral carcinomas.

Journal of Cancer Research and Clinical Oncology 117:

484-488, 1991a.

Saranath, D., Chang, S.E., Bhoite, L.T., Panchal, R.G.,

Kerr, I.B., Mehta, A.R., Johnson, N.W., Deo, M.G.

High frequency mutation in codons 12 and 61 of

H-ras oncogene in chewing tobacco-related human

oral carcinoma in India. British Journal of Cancer

63: 573-578, 1991b.

Sellers, T.A., Bailey-Wilson, J.E., Elston, R.C., Wilson,

A.F., Elston, G.Z., Ooi, W.L., Rothschild, H.

Evidence for Mendelian inheritance in the

pathogenesis of lung cancer. Journal of the

National Cancer Institute 82: 1272-1279, 1990.

Sheng, Z.M., Barrois, M., Klijanienko, J., Micheau,

C., Richard, J.M., Riou, G. Analysis of the c-Haras-1 gene for deletion, mutation, amplification

and expression in lymph node metastasis of

human head and neck carcinomas. British Journal

of Cancer 62: 398-404, 1990.

Shih, C., Weinberg, R.A. Isolation of a transforming

sequence from a human bladder carcinoma cell

line. Cell 29: 161-169, 1982.

Slebos, R.J.C., Kibbelaar, R.E., Dalesio, O., Kooistra,

A., Stam, J., Meijer, C.J.L.M., Wagenaar, S.S.,

Vanderschueren, R.G., Zandwijk, N., Mooi, W.J.,

Bos, J.L., Rodenhuis, S. H-ras oncogene activation

as a prognostic marker in adenocarcinoma of the

lung. New England Journal of Medicine 323: 561-

565, 1990.

Sommers, K.D., Cartwright, S.L., Schechter, G.L.

Amplification of the int-2 gene in human head

and neck squamous cell carcinoma. Oncogene 5:

915-920, 1990.

Spandidos, D.A., Holmes, L. Transcriptional

enhancer activity in the variable tandem repeat

DNA sequence downstream of the human Ha-ras1 gene. FEBS Letters 218: 41-46, 1987.

Spandidos, D.A., Lamothe, A., Field, J. Multiple

transcriptional activation of cellular oncogenes in

human head and neck solid tumours. Anticancer

Research 5: 221-224, 1985.

Topal, M.D. DNA repair, oncogenes and carcinogenesis. Carcinogenesis 9: 691-696, 1988.

Yokota, J., Tsunetsugu-Yokota, Y., Baltitora, H.,

Leferre, C., Cline, M.J. Alterations of myc, myb

and ras-ha protooncogene in cancers are frequent

and show clinical correlation. Science 231: 261-

265, 1986.

Zarbl, H., Sukumar, S., Arthur, A.V., Martin-Zanca,

D., Barbacid, M. Direct mutagenesis of Ha-ras-1

oncogenes by n-nitroso-n-methylurea during

initiation of mammary carcinogenesis in rats.

Nature 315: 382-385, 1985.

Zimmerman, K.A., Yancopoulos, G.D., Collum, R.G.,

Russell, K.S., Kohl, N.E., Denis, K.A., Nau, M.M.,

Witte, O.N., Toran-Allerand, D., Gee, C.E., Minna,

J.D., Alt, F.W. Differential expression of myc

family genes during murine development. Nature

319: 780-783, 1986.
Chapter 3

141

This paper describes the pathways of metabolic activation and macromolecular binding of

Metabolism and Macromolecular

Binding of NNK and NNN,

Important Carcinogens in

Smokeless Tobacco1

Stephen S. Hecht, Steven G. Carmella, and Sharon E. Murphy

ABSTRACT

two tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and

N'-nitrosonornicotine (NNN). NNK and NNN are important because of their tumorigenicity in

the oral cavity and their presence in significant quantities in snuff and the saliva of snuff

dippers. Studies in cultured rat oral tissue have shown that both compounds are metabolically

activated by α-hydroxylation. Methylation of DNA has been detected in rat oral tissue

incubated with NNK. Human oral tissue also can metabolize these nitrosamines. Analysis of

hemoglobin isolated from snuff dippers has demonstrated the presence of adducts resulting

from the metabolic activation of NNK and NNN. Together, these results support the hypothesis

that NNK and NNN are involved in the induction of oral cancer by snuff dipping.

1 These studies were supported by National Cancer Institute grants no. CA-29580 and no.

CA-44377.

INTRODUCTION Epidemiological studies have established that snuff dipping is a

cause of oral cancer (IARC, 1985; Winn et al., 1981). Two tobacco-specific

nitrosamines—4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and

N'-nitrosonornicotine (NNN)—are the most prevalent strong carcinogens in

oral snuff and the saliva of snuff dippers (Hoffman and Adams, 1981;

Hoffman et al., 1987). A mixture of NNK and NNN swabbed daily for life in

the oral cavities of rats induced a significant incidence of oral tumors (Hecht

et al., 1986). The total dose was similar to that encountered by snuff dippers

(Hecht and Hoffman, 1989). NNK and NNN are the only tobacco constituents known to induce oral tumors in animals. These data support the

hypothesis that NNK and NNN are causative factors in human oral cancer

induced by snuff dipping (Hecht and Hoffman, 1989).

In this paper, we summarize our current understanding of the mechanisms by which NNK and NNN are metabolized to intermediates that bind

to cellular macromolecules such as DNA and hemoglobin. We focus on data

that relate to oral cavity carcinogenesis. The metabolism of carcinogens to

DNA-reactive compounds is called metabolic activation. Its hallmark is the

generation of electrophilic intermediates that can bind to nucleophilic

centers in DNA, RNA, or protein. Binding to DNA with formation of covalently attached residues called “DNA adducts” is important in carcinogenesis. DNA adducts have miscoding properties and can interfere with the

normal processes of replication. The presence of DNA adducts in protooncogenes can lead to their activation; the presence of DNA adducts in

tumor suppressor genes can lead to their inactivation. Both processes are
Smoking and Tobacco Control Monograph No. 2

142

involved in the transformation of normal cells to neoplastic cells and the

eventual development of tumors. Although hemoglobin adducts, in contrast to DNA adducts, are not believed to play any part in tumor development, they can serve as surrogates for estimating levels of DNA adducts in

human tissues.

METABOLISM OF

NNK AND NNN

Figure 1 summarizes metabolic pathways of NNK and NNN.

Metabolites have been identified in the urine of rats, mice, and

hamsters and in in vitro experiments with cultured tissues or subcellular

fractions (Hecht and Hoffman, 1988; Hecht et al., 1983). There are three

major metabolism pathways for NNK—pyridine N-oxidation to give NNK-1-

N-oxide (marked 1 in Figure 1), carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and α-hydroxylation to the unstable

α-hydroxy-NNK derivatives 6 and 7. α-Hydroxylation is the major metabolic activation pathway of NNK because compounds 6 and 7 spontaneously decompose to the electrophiles methyl diazohydroxide (12) and 4-(3-

pyridyl)-4-oxobutyl diazohydroxide (14) with simultaneous formation of the

keto aldehyde (11) and formaldehyde (13). Methyl diazohydroxide reacts

with DNA, producing a mixture of methylated DNA bases among which

7-methylguanine, O6-methylguanine, and O4-methylthymidine have been

identified in the tissues of animals treated with NNK. The diazohy-droxide

(14) reacts with DNA, producing adducts of unknown structure that release

4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 20) upon acid hydrolysis. This

process is referred to as pyridyloxobutylation. Both diazohydroxides also

react with hemoglobin, producing globin adducts (Carmella and Hecht,

1987). The adduct formed from reaction of 14 with globin releases HPB

upon mild base hydrolysis. The aldehyde 11 and HPB, which are initial

products of α-hydroxylation of NNK, undergo further oxidation and reduction in vivo and in certain in vitro systems producing keto acid 17, diol 21,

and hydroxy acid 22.

NNAL is formed from NNK in most tissues and also methylates and

pyridyloxobutylates DNA (Hecht and Trushin, 1988). These results occur

either by reconversion to NNK or by α-hydroxylation. The latter pathways

are not illustrated in Figure 1, but give rise to diol 21 and hydroxy acid 22.

NNAL also undergoes pyridine N-oxidation producing NNAL-N-oxide 8.

Another pathway of NNAL metabolism not shown in Figure 1 is formation

of its O-glucuronide, which is a major metabolite in rodent urine (Morse et

al., 1990).

Metabolism of NNN occurs by pyridine-N-oxidation to NNN-1-N-oxide

2, by denitrosation and oxidation yielding norcotinine 3, and by hydroxylation of the four positions of its pyrrolidine ring. The hydroxylation products 4 and 5 are minor metabolites and appear to be stable. The α-hydroxylation products 9 and 10 are major metabolites and are unstable. Compound 9 spontaneously decomposes to the same diazohydroxide 14 that is

formed from methyl hydroxylation of NNK. Therefore, NNN also pyridyloxobutylates DNA and globin to give adducts that release HPB upon hydrolysis (Carmella and Hecht, 1987). Compound 10 generates the hydroxy

aldehyde 18, which exists in the cyclic form as lactol 19. The initial
143

Figure 1Metabolic pathways of NNK and NNN in rodentsSource: Hecht et al., 1991. Copyright 1991, CRC Press; used with permission.
Smoking and Tobacco Control Monograph No. 2

144

products of NNN α-hydroxylation, HPB and lactol 19, undergo further

oxidation, producing keto acid 17 and hydroxy acid 22. These two acids are

good indicators of the extent of α-hydroxylation of NNN by the two pathways illustrated in Figure 1.

Methylation and pyridyloxobutylation of DNA, via 12 and 14, are the

two most likely pathways by which NNK would initiate tumorigenesis. The

importance of the DNA methylation pathway in NNK tumorigenesis has

been demonstrated in certain systems, whereas the role of the pyridyloxobutylation pathway is less clear (Bebinsky et al., 1989); Hecht et al., 1990.

For NNN, which cannot methylate, DNA pyridyloxobutylation is likely to be

important in tumorigenesis, but the mechanism is unclear at present.

In Rat Oral Tissue The metabolism of NNK and NNN can be conveniently studied

by incubation of the tritium-labeled compounds with rat oral tissue

(Murphy et al., 1990). Oral tissue is obtained from the insides of both

cheeks, the roof of the mouth, and the inside of the lips. It is trimmed of

muscle and connective tissue. Pieces of tissue are placed epithelial side up

on a dry filter in a 60-mm culture dish and are then covered with 5 mL of

Williams’ medium with additives. They are incubated with [5-3H]NNK or

[5-3H]NNN, which have tritium at the 5 position of the pyridine ring.

Incubations are carried out for various periods up to 24 h; tissue viability is

excellent under these conditions. At the end of the incubation, the medium

is analyzed for metabolites by high-performance liquid chromatography

(HPLC) with radioflow detection. Figure 2 illustrates the time course of

metabolite formation from 1 μM NNK or NNN in rat oral tissue. The major

products of NNK metabolism were NNAL and NNK-1-N-oxide (1 in Figure 1). α-Hydroxylation produced keto acid (17) and HPB. Metabolism

experiments with HPB demonstrated that the keto acid was formed from

both keto aldehyde (11) and HPB, indicating that both α-hydroxylation

pathways of NNK were operative. α-Hydroxylation of NNN was six times

greater than α-hydroxylation of NNK. Keto acid (17) and HPB were the

major α-hydroxylation products. In contrast to pyridine-N-oxidation of

NNK, pyridine-N-oxidation of NNN was barely detectable. These results

indicate that metabolic activation of NNN to intermediates capable of

binding to DNA exceeded metabolic activation of NNK in cultured rat oral

tissue.

DNA binding was examined in these cultured tissues, and 7-methylguanine was detected in oral tissue incubated with NNK labeled with tritium

in the methyl group. This is consistent with the metabolic activation

pathway that produces keto aldehyde (11) and keto acid (17). Although

O6-methylguanine is known to be formed by the same pathway, it was not

detectable under these conditions. It was also not possible to detect HPB in

acid hydrolysates of DNA isolated from rat oral tissue incubated with

[5-3H]NNK or [5-3H]NNN. Surprisingly, in rat esophagus, which metabolizes

NNN to a similar extent by α-hydroxylation as oral tissue, HPB was detected

in acid hydrolysates of isolated DNA (Murphy et al., 1990b). These results

suggest that cellular constituents of oral tissue other than DNA may have a

particular affinity for reaction with diazohydroxide (14).
Chapter 3

145

Figure 2

Formation of NNK and NNN metabolites by cultured rat oral tissue as a function

of time

Source: Murphy et al., 1990b. Copyright 1990, Cancer Research; used with permission.

The enzymology of NNK and NNN activation in oral tissue also requires

further study. Cytochrome P450 isozymes are believed to be responsible for

the α-hydroxylation of these nitrosamines, but the forms present in oral

tissue have not been characterized. Comparative studies of NNK and NNN
Smoking and Tobacco Control Monograph No. 2

146

metabolism in rat oral tissue indicated that at least two different enzymes

were involved in α-hydroxylation (Murphy et al., 1990b).

In Human

Oral Tissue

Limited information is available on the metabolism of NNK and NNN

by human oral tissue. In one study, buccal mucosa obtained at immediate autopsy was cultured with labeled NNK or NNN (Cantonguay et al.,

1983). NNK was extensively converted to NNAL, as in all human tissues

examined to date. Hydroxy acid (22 in Figure 21-1) produced by α-hydroxylation of NNAL, was also detected. Similar results have been obtained in

ongoing experiments with oral tissue obtained surgically. NNN was metabolized to NNN-1-N-oxide and to hydroxy acid (22). α-Hydroxylation of both

NNK and NNN was far less extensive in human oral tissue than in rat oral

tissue. However, it is not clear whether this is attributable to differences in

the conditions under which the rat and human tissues were obtained and

cultured.

No information is available on oncogene activation by NNK or NNN in

oral cavity tumors. This information could be useful in assessing their role

in human oral cancer. Amplification of several oncogenes including c-myc,

N-myc, N-ras, K-ras, and int-2 have been observed in carcinomas of the oral

cavity (Saranath et al., 1989; Somers et al., 1990).

DETECTION

OF NNK/NNNHEMOGLOBIN

ADDUCTS

As illustrated in Figure 1, the diazohydroxide 14, which is formed

from both NNK and NNN, forms adducts in globin that release HPB

upon mild-base hydrolysis. This was established by experiments in

which labeled NNK or NNN was administered to rats (Carmella and

Hecht, 1987). For NNK, approximately 0.1 percent of the dose was bound to

hemoglobin, and about 20.0 percent of this could be released as HPB. The

half-life of the adduct in rats is approximately 9 d. As shown in Figure 3,

the formation of the HPB-releasing adduct was linear over doses ranging

from 15 to 10,000 μg/kg/d, administered i.p. for 4 d (Murphy et al., 1990a).

The release of HPB from lung and liver DNA by acid hydrolysis also increased over this dose range, but the increase was not linear. No data are

available for comparison of oral tissue DNA adduct formation to hemoglobin adducts.

Measurement of hemoglobin adducts in humans could serve as a

biomarker of the internal dose of a given electrophile (Ehrenberg and

Osterman-Golkar, 1976). In this case, the electrophile of interest is the

diazohydroxide 14. Its formation would be specifically related to uptake

and metabolic activation of NNN and NNK, in contrast to methyl diazohydroxide 12, which could have many environmental sources. Advantages

of hemoglobin adducts as surrogates for DNA adducts include the ready

availability of hemoglobin in quantities sufficient for accurate analysis and

the long lifetime of the erythrocyte in humans (120 d), which permits

integration of dose (Ehrenberg and Osterman-Golkar, 1976). Because HPB is

easily released from hemoglobin upon base hydrolysis and can be separated

from the protein by extraction, it appeared to be a suitable compound for

analysis.
Chapter 3

147

Figure 3

Log log plot of HPB released from globin, after base hydrolysis, and from lung

and liver DNA, after hydrolysis. Globin and DNA were obtained from rats

treated with [5-3

H]NNK (15 to 10,000 μg/kg/d, for 4 d).

Source: Murphy et al., 1990b. Copyright 1990, Cancer Research; used with permission.

Figure 4 summarizes the scheme used for analysis of HPB released from

human hemoglobin (Carmella et al., 1990). The key step is derivati-zation

to its pentafluorobenzoate, which can be analyzed with high sensitivity

(detection limit, approximately 1 fmol) by gas chromatography-negative ion

chemical ionization mass spectrometry (GC-NICI-MS) with selective ion

monitoring. With this method, HPB can readily be detected in samples of

human hemoglobin that have been dialyzed and treated with mild base.
Smoking and Tobacco Control Monograph No. 2

148

Figure 4

Scheme for analysis of HPB in hydrolysates of human hemoglobin

Source: Carmella et al., 1990. Copyright 1990, Cancer Research; used with permission.

Figure 5 illustrates results from analysis of hemoglobin of snuff dippers,

smokers, and nonsmokers (Carmella et al., 1990). In this group, snuff

dippers had the highest adduct levels; it is not known whether this will be a

general phenomenon. The mean adduct level observed in snuff dippers was

higher than expectations based on studies with rats. The effective daily dose

of NNK and NNN producing HPB in these snuff dippers could be estimated

as approximately 0.07 μg/kg/d. Injection of rats i.p. with 1 μg/kg NNK for

5 wk resulted in 517±3.2 fmol/g HPB. The higher levels of HPB-releasing

adducts in snuff dippers than in rats could result from the endogenous

formation of NNN and NNK. NNN may be formed by nitrosa-tion of nornicotine, which is present in tobacco or produced in metabolism of nicotine. NNK could be formed by nitrosation of nicotine. The wide variation
Chapter 3

149

Figure 5

Levels of HPB released from hemoglobin of snuff dippers, smokers, and

nonsmokers

Source: Carmella et al., 1990. Copyright 1990, Cancer Research; used with permission.

in levels of hemoglobin adducts among the snuff dippers suggests that there

are differences in individual capacities for metabolic activation of NNK and

NNN and, possibly, related differences in risk.

The presence of HPB-releasing adducts in snuff dippers’ hemoglobin

supports our hypothesis that NNK and NNN are causative agents for oral

cancer in snuff dippers. The data demonstrate that snuff dippers can metabolically activate NNK and NNN to diazohydroxide (14 in Figure 1).
Smoking and Tobacco Control Monograph No. 2

150

It will be important to investigate the presence of HPB-releasing adducts in

oral DNA from snuff dippers and to determine whether their levels correlate

with levels of hemoglobin adducts. A method for the analysis of HPB

released by acid hydrolysis of human DNA has recently been developed

(Foiles et al., 1991).

GAPS IN

ASSESSING

THE ROLE OF

NNK AND NNN

Although the metabolic activation pathways of NNK and NNN have

been characterized in a number of tissues, there are relatively limited

data for the oral cavity. It would be important to determine which

P450 isozymes are involved in NNK and NNN metabolism in the rat

oral cavity as well as in human oral tissues. This would allow one to more

accurately assess human risk by determining an individual’s level of expression of these particular isozymes in oral tissues. The DNA adducts that

result from the metabolic activation of NNK and NNN in oral tissue have

not been thoroughly examined. More sensitive methods are needed to

detect these adducts. It would be useful to understand the spectrum of

adducts formed from these nitrosamines in oral tissue; the presence of

particular adducts could perhaps be related to oncogene activation, tumor

suppressor gene inactivation, and risk for oral cancer.

The characteristics of the model in which a mixture of NNK and NNN

induces oral tumors upon lifetime swabbing in the rat oral cavity should be

more clearly defined. Is NNK or NNN responsible for the tumorigenic effect,

or are both compounds necessary? Does irritation caused by continuous

swabbing contribute to the tumorigenic effect? Could the protocol be

varied in some way to decrease the latent period and improve tumor yield in

order to provide a more practical model? It is not clear how the route of

administration affects tumor incidence in this model. Lung tumors were

observed, consistent with the organospecificity of NNK, but no tumors of

the esophagus and nasal cavity were observed. They might have been

expected, given the known target tissues of NNK and NNN.

The potent pulmonary tumorigenicity of NNK suggests that snuff

dippers may be at risk for lung cancer. This should be investigated.

The role of cofactors in tumorigenesis by NNK and NNN, as components of a complex mixture like snuff, is not clear. Snuff extract inhibited

the tumorigenicity of NNK and NNN (Hecht et al., 1986). This could be

attributable to the inhibitory effect of nicotine on their metabolic activation

or to other compounds in the extract. Snuff is tumorigenic when instilled

in the rat lip canal; the precise role of NNK and NNN in this model is not

clear (Hecht et al., 1986). It seems likely that the irritant properties of snuff

would enhance NNK and NNN tumorigenesis by increasing cell replication.

This should be tested. Viruses may also play a role as cofactors in NNK and

NNN tumorigenesis (Park et al., 1986).

Hemoglobin adducts appear to provide a method for assessing the

metabolic activation of NNK and NNN in humans. What contributes to a

high adduct level and what are its consequences? Does endogenous

nitrosation increase the dose of NNK and NNN beyond what is found in

snuff? Or are high adduct levels strictly a function of an individual’s ability
Chapter 3

151

to metabolically activate the compounds? What are the separate roles of

NNK and NNN in contributing to adduct levels? These questions must be

answered before the hemoglobin adduct data can be fully interpreted.

SUMMARY The evidence that NNK and NNN play a role in human oral cancer

induced by snuff is strong. Both compounds are present in significant

amounts in snuff and in the saliva of snuff dippers. They are metabolically

activated in snuff dippers to intermediates that bind to hemoglobin. They

cause oral tumors in rats and are metabolically activated by rat and human

oral tissue. Although there are many questions about the mechanisms by

which snuff causes oral tumors in rats and humans, there is no doubt that

the presence of NNK and NNN in snuff is an unacceptable risk to people

who choose to use these products.

REFERENCES

Belinsky, S.A., Devereux, T.R., Maronpot, R.R., et al.

Relationship between the formation of promutagenic adducts and the activation of the K-ras

proto-oncogene in lung tumors from A/J mice

treated with nitrosamines. Cancer Research 49:

5305-5311, 1989.

Carmella, S.G., Hecht, S.S. Formation of hemoglobin

adducts upon treatment of F344 rats with the

tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine. Cancer Research 47: 2626-2630,

1987.

Carmella, S.G., Kagan, S.S., Kagan, M., et al. Mass

spectrometric analysis of tobacco-specific nitrosamine hemoglobin adducts in snuff dippers,

smokers, and nonsmokers. Cancer Research 50:

5438-5445, 1990.

Castonguay, A., Stoner, G.D., Schut, H.A.J., et al.

Metabolism of tobacco-specific N'-nitrosamines by

cultured human tissues. Proceedings of the National

Academy of Sciences 80: 6694-6697, 1983.

Ehrenberg, L., Osterman-Golkar, S. Alkylation of

macromolecules for detecting mutagenic agents.

Teratogenesis, Carcinogenesis, and Mutagenesis 1:

105-127, 1976.

Foiles, P.G., Akerkar, S.A., Carmella, S.G., et al. Mass

spectrometric analysis of tobacco-specific nitrosamine DNA adducts in smokers and nonsmokers.

Chemical Research in Toxicology, 4: 364, 1991.

Hecht, S.S., Castonguay, A., Rivenson, A., et al.

Tobacco specific nitrosamines: Carcinogenicity,

metabolism, and possible role in human cancer.

Journal of Environmental Science and Health. Part C.

(CI) 1: 1-54, 1983a.

Hecht, S.S., Haley, N.J., and Hoffmann, D. Monitoring exposure to tobacco products by measurement

of nicotine metabolites and derived carcinogens.

In: Molecular Dosimetry and Human Cancer:

Analytical, Epidemiological, and Social Considerations,

J.D. Groopman and P.L. Skipper (Editors). Boca

Raton, FL: CRC Press, 1991, pp. 325-361.

Hecht, S.S., Hoffmann, D. Tobacco-specific nitrosamines: An important group of carcinogens in

tobacco and tobacco smoke. Carcinogenesis 9: 875-

884, 1988.

Hecht, S.S., Hoffmann, D. The relevance of tobaccospecific nitrosamines to human cancer. Cancer

Surveys 8: 273-294, 1989.

Hecht, S.S., Jordan, K.G., Choi, C.-I., et al. Effects of

deuterium substitution on the tumorigenicity of

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

and 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanol in A/J mice. Carcinogenesis 11: 1017-1020,

1990.

Hecht, S.S., Rivenson, A., Braley, J., et al. Induction

of oral cavity tumors in F344 rats by tobaccospecific nitrosamines and snuff. Cancer Research

46: 4162-4166, 1986.

Hecht, S.S., Trushin, N. DNA and hemoglobin

alkylation by 4-(methylnitrosamino)-1-(3-pyridyl)-

1-butanone (NNK) and 4-(methylnitrosamino)-1-

(3-pyridyl)-1-butanol (NNAL) in F344 rats.

Carcinogenesis 9: 1665-1668, 1988.

Hoffmann, D., Adams, J.D. Carcinogenic tobaccospecific N-nitrosamines in snuff and saliva of

snuff dippers. Cancer Research 41: 4305-4308,

1981.

Hoffmann, D., Adams, J.D., Lisk, D., Fiseune, I.,

Brunnemann, K. Toxic and carcinogenic agents

in dry and moist snuff. Journal of the National

Cancer Institute 79: 1281-1286, 1987.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosamines (volume 37). Lyon:

IARC, 1985, pp. 113-116.

Morse, M.A., Eklind, K.I., Toussaint, M., et al.

Characterization of a glucuronide metabolite of 4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone

and its dose dependent excretion in the urine of

mice and rats. Carcinogenesis 11: 1819-1823, 1990.
Smoking and Tobacco Control Monograph No. 2

152

Murphy, S.E., Heiblum, R., Trushin, N. Comparative

metabolism of N'-nitrosonornicotine and 4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone by

cultured rat oral tissue and esophagus. Cancer

Research 50: 4685-4691, 1990b.

Murphy, S.E., Palomino, A., Hecht, S.S., et al. Doseresponse study of DNA and hemoglobin adduct

formation by 4-(methylnitrosamino)-1-(3-pyridyl)-

1-butanone in F344 rats. Cancer Research 50: 5446-

5452, 1990a.

Park, N.H., Sapp, J.P., Herbosa, E.G. Oral cancer

induced in hamsters with herpes simplex infection

and simulated snuff-dipping. Oral Surgery, Oral

Medicine, Oral Pathology 62: 164-168, 1986.

Saranath, D., Panchal, R.G., Nair, R., et al.

Oncogene amplification in squamous cell

carcinoma of the oral cavity. Japanese Journal of

Cancer Research 80: 430-437, 1989.

Somers, K.D., Cartwright, S.L., Schechter, G.L.

Amplification of the int-2 gene in human head

and neck squamous cell carcinomas. Oncogene 5:

915-920, 1990.

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W.,

Toledo, A., Frameni, J.F., Jr. Snuff dipping and

oral cancer among women in southern United

States. New England Journal of Medicine 304: 745-

749, 1981.
Chapter 3

153

Nicotine is a constituent of all tobacco but is present at higher concentrations in smokeless

Role of Nicotine as a Cofactor

In Smokeless Tobacco Carcinogenesis1

Christopher A. Squier and Georgia K. Johnson

ABSTRACT

tobacco than in other forms. The ST user is likely to have nicotine come into contact more

frequently with the oral lining than is the smoker. Studies with animals suggest that the

presence of nicotine enhances the effect of known carcinogens in the development of oral

carcinoma. In vitro studies show that 2 percent and 6 percent nicotine applied topically to oral

mucosa causes epithelial damage and an increase in permeability. We suggest that the method

by which nicotine contributes to tumor development involves initial tissue damage, followed

by a reparative response, accompanied by increased permeability to carcinogens. Continued

exposure will lead to malignant transformation of the tissue.

INTRODUCTION Nicotine is the component of tobacco that causes the psychoactive

effects that lead to addiction as well as to a variety of physiological changes

(Benowitz, 1986 and 1988). The concentration of nicotine in tobacco

ranges from 1 to 2 percent of the dry weight of the processed leaf in cigarette tobacco (Gritz et al., 1981), between 1.45 and 8.00 percent in smokeless tobacco (snuff and plug tobacco) in the United States (Gritz et al., 1981),

and between 0.5 and 3.0 percent in snuff worldwide (Hoffmann and Adams,

1981). However, unlike smoking tobacco where only 15 to 20 percent of

nicotine is transferred into mainstream smoke (Schmeltz et al., 1979), which

is present intermittently in the oral cavity, all of the nicotine in ST is

present in the mouth for extended periods of time. The possibility that the

nicotine in smokeless tobacco may be a precursor of the tobacco-associated

nitrosamines, some of which are known carcinogens (Hoffmann and Adams,

1981), has been thoroughly explored (Hoffmann et al., 1985 and 1986).

However, little attention has been given to the effect that high levels of

topical nicotine might have on the oral mucosa.

IN VIVO STUDIES

WITH NICOTINE

In a series of studies using skin, Bock (1980) was able to show

that carcinogenesis by benzo[a]pyrene was enhanced by nicotine,

although nicotine did not initiate or promote tumors; nicotine acted as a

cofactor but was not a carcinogen by itself. We were able to demonstrate a

similar effect in oral mucosa (Chen and Squier, 1990) by painting hamster

cheek pouches with either 7,12-dimethylbenz[a]anthracene (DMBA) alone

or in combination with 6 percent nicotine for 12 wk. Animals treated with

DMBA and nicotine showed significantly (p < 0.001) more tumors and a

significantly (p < 0.05) greater than expected proportion of large tumors

(> 3 mm diameter) in the cheek pouch than hamsters treated with DMBA

alone. Animals treated with nicotine alone did not develop tumors. In a

similar experiment, using 6 percent nicotine and either N'-nitrosonornicotine (NNN) or 4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK),

1 Supported in part by National Institute of Dental Research grants no. R01-DE07930 and

R29-DE/AI10153-01.
Smoking and Tobacco Control Monograph No. 2

154

we have observed greater epithelial hyperkeratosis and dysplasia in the oral

mucosa and the presence of papillomas in gastric mucosa as compared with

treatment with nicotine alone (Chen and Squier, unpublished results).

Studies in which snuff was applied topically to the rat periodontium

have indicated decreased gingival blood flow (Schroeder and Milo, 1989)

and bone loss (Hill et al., 1990). However, other workers have reported

increased gingival blood flow in dogs after topical application of nicotine

(Johnson et al., 1989). These differences may represent a paradoxical

vasoactive effect of nicotine at various dose levels, but the studies do show

that nicotine can exert a local tissue effect.

IN VITRO

STUDIES WITH

NICOTINE

To examine the local acute effects of nicotine applied topically to

oral mucosa, we have developed an in vitro model system

(Figure 1). Nicotine is prepared in phosphate-buffered saliva (PBS)

at concentrations of 2 or 6 percent and placed in a circular rubber gasket

attached to the surface of specimens of oral mucosa with cyanoacrylate.

The tissue is maintained in culture medium and incubated at 37 °C for 1 or

2 h; controls are incubated with PBS alone at the same pH (7.5) and osmolarity (450 mosm) as the nicotine solution. The tissue consists of pig oral

mucosa taken at death from the gingiva, floor of the mouth, and the buccal

mucosa, which have a structure very similar to that of the corresponding

human tissue (Lesch et al., 1989). Specimens either are processed for morphological examination by light and electron microscopy, or the functional

capacity of the tissue is assessed by measure of the permeability. We accomplish this by clamping specimens between the halves of a glass perfusion

chamber and determining the amount of tritiated water that passes across

per unit time. From these data, a permeability constant (Kp) can be calculated (Siegal et al., 1971).

Morphologically, in all mucosal regions there was more damage after

treatment with 6 percent than with 2 percent nicotine and greater damage

after 1 h incubation with 6 percent nicotine than after 2 h with 2 percent

nicotine. Changes in controls were minimal. The epithelium of gingiva

(Figure 2) and floor-of-mouth mucosa (Figure 3) showed greater destruction

than buccal mucosa (Figure 4). Changes were usually more evident in the

deeper cellular layers than at the surface, and this type of damage was

particularly striking for gingiva. The changes represented acantholysis and

separation of epithelial strata, cell shrinkage, and nuclear pyknosis. It is

notable that the in vitro appearances frequently resemble those illustrated

for biopsies from oral mucosa of chronic snuff users (Andersson, 1991).

Those tissues treated with 2 percent nicotine that showed only slight

alterations at the light microscope level (Figures 3 and 5) revealed considerable ultrastructural changes when examined with the transmission electron

microscope. Changes included peripheral condensation of chromatin in cell

nuclei and the presence of frequent intracellular vacuoles (Figure 5, left) that

appear to represent damaged organelles such as mitochondria (Figure 5,

right) as well as discontinuities in the basal lamina. Such changes seem to

represent the initial cellular responses to nicotine and are of particular

importance because 2 percent nicotine is found in many types of snuff

worldwide.
Chapter 3

155

Figure 1

The model used to expose oral mucosa to topical nicotine

The pattern of damage may reflect the chemical action of the nicotine

molecule, as controls did not suggest any effect caused by pH or osmolarity.

Nicotine may disrupt phospholipid-containing membranes, such as those of

the deeper cell layers, whereas the superficial layers contain greater amounts

of glycolipid or pure lipid rather than phospholipid (Squier et al., 1991).

Measurement of permeability to tritiated water of tissues treated with

6 percent nicotine for 2 h (Table 1) revealed significant (p > 0.05) increases

in Kp values in all regions compared with controls incubated in PBS or

normal, untreated mucosa (values from Lesch and coworkers, 1989). Because the putative carcinogens in ST (the tobacco-associated nitrosamines)

are present in an aqueous environment and can be extracted by saliva

(Hecht et al., 1974), our results indicate that nicotine-induced damage could

increase the access of such carcinogens to the deeper, proliferative compartment of the oral epithelium.

likelihood of malignant transformation such that 4 percent of lesions may

become carcinoma (Schafer et al., 1983). The pathogenesis of these lesions

may reflect the acute epithelial damage brought about by the presence of

nicotine in tobacco that is applied to an area of oral mucosa. The initial

damage would evoke a hyperplastic reparative response and would be

associated with an increased permeability to any carcinogens. Chronic

irritation, as a result of continued placement of tobacco at the same site,

would lead to hyperkeratosis that appears as a white patch. Hyperplasia and

hyperkeratosis are protective responses of skin and oral mucosa to mechanical or chemical irritation. However, hyperkeratotic regions of skin (Grice,

1980) and oral mucosa (Squier et al., 1985) tend to show increased permeability so that tobacco-associated carcinogens will continue to enter the

lesional tissue. Thus, the tissue represents a site of elevated cell proliferation

that is exposed to higher levels of carcinogens than the adjacent normal

mucosa. This will increase the likelihood of development of carcinoma.

CLINICAL

IMPLICATIONS

OF STUDIES

WITH NICOTINE

One of the characteristic features of chronic ST use is the presence

of a hyperkeratotic oral mucosal lesion at the site of regular tobacco placement. Characterized clinically as a white lesion, leukoplakia, or snuff-dipper’s lesion, these lesions show an increased

Source: Squier and Lesch, 1988; used with permission.
Smoking and Tobacco Control Monograph No. 2

156

Figure 2

Sections of porcine gingiva after exposure for 2 h with PBS (left), 2 percent nicotine (center),

and 6 percent nicotine (right). Note the gradation of damage from left to right, all of which

occurs in the deeper cell layers (magnification x200).

Figure 3

Sections of porcine floor-of-mouth mucosa after exposure for 2 h with PBS (left), 2 percent

nicotine (center), and 6 percent nicotine (right). There is greater intercellular disruption after

treatment with 6 percent (right) than with 2 percent nicotine (center) (magnification x200).

Although this sequence of events is conceptual, it is consistent with

clinical evidence. When smokeless tobacco is first placed on the oral mucosa, there is erythema and, histologically, evidence of acute inflammation

(G.K. Johnson, personal communication). If a habitual tobacco user places

tobacco at a new site in the mouth, the original lesion usually resolves

clinically and histologically (Andersson, 1991), but another develops at the
Chapter 3

157

Figure 4

Sections of porcine buccal mucosa after exposure for 2 h with PBS (left), 2 percent nicotine

(center), and 6 percent nicotine (right). There is marked intercellular disruption after treatment with 6 percent nicotine, but the deepest cells show nuclear shrinkage and separation

with 2 percent nicotine (magnification x125).

new site of placement. Such events clearly implicate tobacco as the causative agent in pathological changes in the oral mucosa and lead to the

hypothesis that nicotine may be the comp onent that initiates the morphological changes described. The events proposed above would also be consistent with suggestions that ST carcinogenesis involves factors other than a

carcinogen (Park et al., 1988) and that the development of oral carcinoma

requires extended exposure to tobacco (Winn et al., 1981).

If nicotine is involved in the production of the snuff-induced lesion,

then the local effect of nicotine gum, often prescribed during smoking

cessation, might be of importance. In a study that evaluated gingival

response to nicotine chewing gum (2 mg or 4 mg per portion), Silver and

coworkers (1989) reported transient gingival blanching in all subjects.

When the gum was repeatedly held in the same location, vesicle formation

occurred that resolved when the gum was placed in different positions. The

absence of severe, prolonged changes in nicotine gum chewers (Christen et

al., 1985; Silver et al., 1989), in contrast to the tissue changes described in

our experiments, can probably be explained by the levels of nicotine exposure. Although bioavailability of nicotine may vary, nicotine concentration

in chewing gum (0.2 percent by weight) is almost an order less than that

found in typical ST products (0.5 to 3.0 percent nicotine) (Hoffmann and

Adams, 1981). Furthermore, the gum is typically used for only 5 to 6 h

daily. This represents a much lower nicotine exposure than that of individuals with an established ST habit who may continually use the product,

with its higher concentrations of nicotine, for more than 12 h/d (Andersson,

1991).
Smoking and Tobacco Control Monograph No. 2

158

Figure 5

Transmission electron micrographs of floor-of-mouth mucosa exposed for 2 h to

2 percent nicotine. Epithelial cells show condensation of nuclear chromatin (top,

magnification x17,000) and numerous vacuoles that appear to represent damaged cell organelles, including mitochrondria (bottom, x28,500 ).
Chapter 3

159

Table 1

Effect of nicotine on mucosal permeability to tritiated watera

Untreated PBS Nicotine

Control Control Treatedb

Tissue Region

Gingiva 364 ± 18 351 ± 54 667 ±77

Buccal mucosa 634 ± 19 692 ± 49 781 ± 46

Floor of mouth 808 ± 23 814 ± 46 971 ± 94

a Kp

 SEM x 10-7 cm/min. b Values significantly greater than controls (p > 0.05).

ACKNOWLEDGMENTS

FUTURE

DIRECTIONS

A logical consequence of the implication of nicotine in ST carcinogenesis would be to reduce its concentration to levels that do not

cause tissue damage. As this threshold is unknown, it is not possible to

predict whether such a strategy would still provide adequate nicotine levels

to offer the psychoactive effect that is the major reason for ST use while

limiting the pathological effects on the oral mucosa.

We thank Mary Kremer and Chuck Lesch for technical assistance.

Hecht, S.S., Ornaf, R.M., Hoffmann, D. Chemical

studies of tobacco smoke XXXIII. N'-nitrosonornicotine in tobacco: Analysis of possible

contributing factors and biologic implications.

Journal of the National Cancer Institute 54: 1237-

1244, 1974.

Hill, L.N., Milo, A.G., Schroeder, K.L. Bone loss

relative to gingival blood flow in snuff-treated

versus control rats (abstract 707). Journal of Dental

Research 69: 197, 1990.

Hoffmann, D., Adams, J.D. Carcinogenic tobaccospecific N-nitrosamines in snuff and in the saliva

of snuff dippers. Cancer Research 41: 4305-4308,

1981.

Hoffmann, D., Hecht, S.S., Melikian, A.A., Haley,

N.J., Brunnemann, K.D., Adams, J.D., Wynder,

E.L. Tumorigenic agents in tobacco products and

their uptake by chewers, smokers and nonsmokers. In: Biochemical and Molecular Epidemiology of

Cancer. New York: Alan R. Liss, 1986, pp. 191-

204.

Hoffmann, D., Lavoic, E.J., Hecht, S.S. Nicotine: A

precursor for carcinogens. Cancer Letters 26: 67-

75, 1985.

Johnson, G.K., Todd, G.L., Fung, E.Y.K. Effects of

nicotine on gingival blood flow (abstract 834).

Journal of Dental Research 68: 285, 1989.

Andersson, G. Snuff-induced changes associated

with the use of loose and portion-bag-packed

Swedish moist snuff: A clinical, histological, and

followup study. Swedish Dental Journal Suppl. 75:

1-88, 1991.

Benowitz, N.L. Clinical pharmacology of nicotine.

American Review of Medicine 37: 21-32, 1986.

Benowitz, N.W. Nicotine and smokeless tobacco.

CA—A Cancer Journal for Clinicians 38: 224-256,

1988.

Bock, F.G. Cocarcinogenic properties of nicotine.

Banbury Report 3: 129-139, 1980.

Chen, Y.-P., Squier, C.A. Effect of nicotine on 7,12-

dimethylbenz(a)anthracene carcinogenesis in

hamster cheek pouch. Journal of the National

Cancer Institute 82: 861-864, 1990.

Christen, A.G., Beiswanger, B.B., Mallatt, M.E., et al.

Effects of nicotine-containing chewing gum on

oral soft and hard tissues: A clinical study. Oral

Surgery, Oral Medicine, Oral Pathology 59: 37-42,

1985.

Grice, K.A. Transepidermal water loss in pathological

skin. In: The Pathophysiology of Skin, A. Jarrett

(Editor). London: Academic Press, 1980, p. 2147.

Gritz, E.R., Baer-Weiss, V., Benowitz, N.L., Yunakis,

H.V., Jarvik, M.E. Plasma nicotine and cotinine

concentrations in habitual smokeless tobacco

users. Clinical Pharmacology Therapy 30: 201-209,

1981.

REFERENCES
Smoking and Tobacco Control Monograph No. 2

160

Lesch, C.A., Squier, C.A., Cruckley, A., Williams,

D.M., Speight, P. The permeability of human oral

mucosa and skin to water. Journal of Dental

Research 68: 1345-1349, 1989.

Park, N.H., Akoto-Amanfu, E., Paik, D.I. Smokeless

tobacco carcinogenesis: The role of viral and other

factors. CA—A Cancer Journal for Clinicians 38:

224-233, 1988.

Schafer, W.G., Hine, M.K., Levy, B.M. A Textbook of

Oral Pathology (4th edition). Philadelphia: W.B.

Saunders, 1983, p. 104.

Schmeltz, I., Wenger, A., Hoffmann, D., Tso, T.C. On

the fate of nicotine during pyrolysis and in a

burning cigarette. Agricultural and Food Chemistry

27: 602-608, 1979.

Schroeder, K.L., Milo, A.G. Topically applied

smokeless tobacco effects on rat gingival blood

flow (abstract 879). Journal of Dental Research 68:

291, 1989.

Siegal, I.A., Hall, S.H., Stambaugh, R. Permeability of

the oral mucosa. In: Current Concepts of the

Histology of Oral Mucosa, C.A. Squier and J. Meyer

(Editors). Springfield, IL: Charles C. Thomas,

1971, pp. 274-186.

Silver, K.J., Sachs, D.P.L, Hottel, T.L. Gingival

response to nicotine polacrilex. Journal of the

American Dental Association 118: 53-56, 1989.

Squier, C.A., Hall, B.K. The permeability of hyperplastic oral epithelium. Journal of Oral Pathology

15: 357-362, 1985.

Squier, C.A., Wertz, P.W., Cox, P. Thin-layer

chromatographic analysis of lipids in different

layers of porcine epidermis and oral epithelium.

Archives of Oral Biology 36: 647-653, 1991.

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W.,

Toledo, A., Fraumeni, J.F., Jr. Snuff dipping and

oral cancer among women in the southern United

States. New England Journal of Medicine 304: 745-

749, 1981.
Chapter 3

161

Carcinogenic risk for human perinates from parental use of smokeless tobacco would be

Perinatal Carcinogenesis by Constituents

Of Smokeless Tobacco: Animal Models

And Potential Human Risk

Lucy M. Anderson and Jerry M. Rice

ABSTRACT

implied if (1) increased risk in organs distant from the oral site of exposure were demonstrated

in adults; (2) increased cancer risk from parental cigarette smoking were found; and (3) carcinogens in ST were active perinatal carcinogens in animal models. Epidemiological evidence

for the first two points is limited but increasing. In regard to the third point, in animal models,

the most potent carcinogen present in significant quantity in ST, 4-(methylnitrosamino)-1-

(3-pyridyl)-1-butanone (NNK), has been demonstrated to be a relatively potent carcinogen in

fetal hamsters, especially in the respiratory tract, and in newborn mice (lung and liver). NNK

was significantly but weakly positive in lungs and livers of fetal mice. Interpreted with other

studies on perinatal effects of nitrosamines and other carcinogens, the results with NNK are

consistent with metabolic activation in perinatal tissues being a limiting factor in risk for the

tobacco-specific nitrosamines and other smokeless tobacco carcinogens, such as benzo[a]pyrene

and N-nitrosodimethylamine. Investigation of this biochemistry with rodent tissue and human

or primate perinatal tissues or both is therefore of high priority in evaluation of human risk,

along with analysis of amniotic fluid and breast milk of ST users. The possibility of preconception effects, especially in males, predisposing to cancer in the offspring may be studied with

animal models. In addition, epidemiological studies of childhood and adult cancers as related

to the amount and kind of ST used by both parents are indicated.

INTRODUCTION Increasing use of smokeless tobacco by women of child-bearing age

(Orlandi and Boyd, 1989) presents the possibility of adverse effects, including cancer, for their children. Such a possibility exists if effective doses of

the toxic constituents of these topically applied products reach the fetus

transplacentally via blood circulation or the suckling infant through milk.

This possibility is confirmed by epidemiology linking ST with increased

cancer at sites other than the head and neck. Available evidence suggests an

association with cancers of the bladder, pancreas, and kidney (Bjalke and

Schuman, 1982; Goodman et al., 1986; Heuch et al., 1983; Kabat et al.,

1986). Both transplacental (Alaoui-Jamali et al., 1989; Anderson et al.,

1989a; Castonguay et al., 1984; Correa et al., 1990; Rossignol et al., 1989)

and transmammary (Diaz Gomez et al., 1986; LaVoie et al., 1987) passage of

tobacco product carcinogens occurs in rodents. In humans, an increase in

4-aminobiphenyl hemoglobin adducts in fetuses of smoking mothers was

recently demonstrated (Coghlin et al., 1991). It is therefore necessary to

consider risk of tumorigenesis in the fetuses and neonates of ST users.

SENSITIVITY AND

SUSCEPTIBILITY

FACTORS

A wide variety of chemicals cause tumors when delivered transplacentally to rodent fetuses or administered directly to newborns (Rice, 1981; Toth, 1968). The tumors that result are

mostly adult rather than embryonal type and present in tissues characteristic of the chemical and the species. Fetal sensitivity varies from minimal at

toxic adult doses to much greater than that observed in the adult (see

Anderson et al., 1985a). The most effective transplacental carcinogens are
Smoking and Tobacco Control Monograph No. 2

162

the rapidly penetrating, direct-acting alkylating agents, especially

N-ethylnitrosourea (ENU), to which the nervous systems of rat fetuses are

approximately fiftyfold more sensitive than those of the adult animal. In

mice, transplacental ENU not only initiated more lung and liver tumors in

fetuses than in adults at the same dose, but the prenatally initiated neoplasms became larger and more malignant (Branstetter et al., 1988 and

1989). ENU was also found to be a transplacental carcinogen in two nonhuman primates, patas and rhesus monkeys, causing, in addition to neoplasms of the vasculature, tumors that were not seen in the mothers or after

treatment of other adult monkeys (Rice et al., 1989), including hepatocellular carcinoma, leukemia, lung adenoma, nephroblastoma, and an assortment of tumors of the brain.

 These results indicate that animal fetuses, both rodent and primate,

have high innate sensitivity to tumor initiation by genotoxic carcinogens

that are in an activated state and an effective dose. The reasons for this

special sensitivity probably include high rates of cell division, which permits

fixation of lesions before repair can occur, and multiple divisions after

initiation to give rise to a large clone of initiated cells. Other factors, such as

limited ability to repair DNA damage, high proportion of undifferentiated

cells, and immaturity of immune surveillance, hormonal, and growth

control systems, have also been suggested to play a role, although convincing evidence is lacking.

More variable results are obtained for transplacental carcinogens that

are activated or detoxified or both by enzymes in the mother, the placenta,

or the fetus. Nitrosamines, in contrast to nitrosoureas, require metabolic

activation and are generally ineffective transplacental carcinogens; they

seem to elicit tumors in direct proportion to the capacity of fetal tissues to

activate them metabolically. Thus, N-nitrosodiethylamine (NDEA) causes

respiratory tumors in mice and hamsters with steadily increasing efficiency

as the end of gestation nears, correlated with the acquisition of an unusually

high ability to metabolize NDEA by fetal hamster lungs at term (Lofberg and

Tjalve, 1984).

Larger metabolism-dependent carcinogens such as polycyclic aromatic

hydrocarbons (PAHs) present an even more complex picture, because their

effectiveness may be limited by poor placental penetration (Neubert and

Tapken, 1988) or detoxification in the placenta (Pelkonen, 1985; Remmer,

1987). Nevertheless, certain PAHs, including 3-methylcholanthrene (MC)

and 7,12-dimethylbenz[a]anthracene (DMBA), are more active transplacentally than in adult rodents (Anderson et al., 1985b; Rice et al., 1978). In

mice, fetal risk of lung and liver carcinogenesis by MC is influenced strongly

by both maternal and fetal ability to respond to induction of a cytochrome

P450 (1A1) that metabolizes MC; maternal induction protected against and

fetal induction potentiated MC’s effect (Anderson et al., 1985b and 1989c).

Biochemical analysis of this model suggests that extensive induction in

maternal liver reduces the effective dose of unchanged carcinogen reaching

the fetuses, but in the fetus, the more limited induction in liver does not

outweigh the increased level of activation to ultimate carcinogen in the lung

(Chauhan et al., 1991; Miller et al., 1989, 1990a, and 1990b).
Chapter 3

163

 The newborn infant is a particularly vulnerable target for carcinogens,

often being more sensitive to carcinogens than adults (Toth, 1968) or than

late fetuses after an equivalent dose to the mother. The reasons for this

include high rates of cell division, as for fetuses, and long half-life of chemicals, because the newborn’s detoxifying enzymes are generally inadequate.

For the carcinogens in ST, newborn sensitivity would be of greatest concern

if these were secreted in milk in significant quantities. This would seem to

be a possibility, because tumorigenesis after transmammary delivery has

been demonstrated for a chemically diverse assortment of carcinogens

(Maekawa and Odashima, 1975; Mohr and Althoff, 1971; Nomura, 1973;

Nomura et al., 1974; Vesselinovitch et al., 1979).

Activated oncogenes have been found in transplacentally induced

tumors and show tissue specificity, but the type and incidence of activated

oncogenes were similar to those in tumors induced in the same tissues in

adults by these agents (Loktionov et al., 1990; Perantoni et al., 1987;

Sukumar and Barbacid, 1990; Yamasaki et al., 1987). Special fetal susceptibility does not appear to relate to unique features of oncogene activation.

 In some cases the appearance of prenatally initiated tumors may be

dependent on or hastened by postnatal exposure to tumor promoters. This

has been demonstrated for a variety of tumors in epithelial tissue and

several promoters (Anderson et al., 1985a; Diwan et al., 1989; Napalkov et

al., 1987; Suganuma et al., 1987). Hormones and tissue regeneration also

provided a promotional stimulus (Ogawa et al., 1982; Rice, 1981). On the

other hand, postnatal exposures to such diverse agents as hormones, barbiturates, growth factors, or nicotine sometimes suppressed tumor development (Alexandrov et al., 1989; Anderson et al., 1985a; Beniashvilli and

Zedginidze, 1989; Berger et al., 1987; Naito et al., 1985).

In sum, the perinate can present a high intrinsic sensitivity to tumorigenesis, which may be counterbalanced by maternal or placental detoxification, poor transplacental or transmammary delivery, limitations in metabolic activation, other poorly understood species- and organ-specific constraints, lack of necessary postnatal stimulation, or postnatal inhibitory

influences. No doubt other factors influencing the process in both directions remain to be discovered. All of these must be considered in evaluation

of potential human perinatal risk from ST.

TOBACCO-SPECIFIC

NITROSAMINES IN

ANIMAL MODELS

Cigarette smoke condensate administered to pregnant Syrian

hamsters caused a significant increase in tumors, including

neoplasms of the adrenal gland, pancreas, liver, respiratory

system, ovary, and connective tissue (Nicolov and Chernozemsky, 1979),

primarily in the female offspring. In short-term studies with mice, exposure

of mothers during pregnancy to mainstream or sidestream cigarette smoke

resulted in increased sister chromatid exchanges in fetal livers (Karube et al.,

1989), and extract of ST administered before and during gestation resulted

in reduced fetal weight, decreased ossification, and increased resorptions

(Paulson et al., 1991).

Other efforts have focused on the chemicals present in tobacco products. The carcinogens of greatest concern in ST are the tobacco-specific
Smoking and Tobacco Control Monograph No. 2

164

nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK),

N'-nitrosonornicotine (NNN), N'-nitrosoanabasine (NAB), and N'-nitrosoanatabine (NAT); all occur in ST in the parts-per-million range, being

especially high in snuff (Hecht and Hoffmann, 1988). Thus far, perinatal

carcinogenicity studies have been carried out only with NNK, the most

potent in adult rodents. NNK administered to pregnant Syrian golden

hamsters was detected in placenta, along with a twentyfold greater quantity

of its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL).

Both were present, at about one-half the placental level in the fetal lung,

and were found in amniotic fluid (Rossignol et al., 1989). Explants of

exposed fetal lung and trachea showed a high incidence of chromosomal

aberrations.

These findings predicted the positive outcome of the bioassay in hamsters (Correa et al., 1990); transplacental NNK caused a significant incidence

of respiratory tract tumors at doses of 50 to 300 mg/kg, with the greatest

effect shown in 61 percent of females after a single dose of 200 mg/kg on

the last day of gestation. There was also a significant number of pheochromocytomas of the adrenal glands and a few hepatocellular carcinomas and

pancreatic ductular adenomas. Thus NNK joins other nitrosamines as an

effective transplacental carcinogen of the respiratory system in hamsters,

again correlated with development of capacity for metabolic activation. The

fetal hamster lung and tracheal tissues were capable of metabolizing NNK in

vitro to a markedly increased extent over the last 4 d of gestation; the

amount of most metabolites doubled between days 14 and 15 (Rossignol et

al., 1989). In a comparison of methylated bases in DNA of lung explants

from fetuses and adults, fetal DNA contained slightly more O6

- and

N7-methylguanines. These findings are consistent with another report, that

fetal hamster lung metabolized NDEA at a threefold greater rate than adult

lung (Lofberg and Tjalve, 1984).

Metabolism of [14C]NNK was similarly examined in fetal C57BL/6 mice

(Castonguay et al., 1984). The compound and its metabolites were detected

in all fetal tissues and amniotic fluid. Macromolecule-bound label was

found in fetal nose and liver and to a lesser extent in lung and kidney. Fetal

liver, lung, and nasal tissue, incubated in vitro with NNK, formed the keto

acid product of αC-hydroxylation (carcinogen activation), but only at levels

3 to 4 percent those of the corresponding maternal tissue. Nasal tissue was

most active.

These biochemical findings suggested that NNK would be a positive but

weak transplacental carcinogen in the mouse, and bioassay confirmed this

prediction (Anderson et al., 1989b). Pregnant females of three mouse strains

of differing characteristics were given three NNK doses of 100 mg/kg. Transplacentally exposed offspring of strain A mice, highly susceptible to lung

tumor initiation, showed a tenfold increase in incidence of lung tumor

bearers among females and a twofold change in males, with a small effect

on multiplicity. Although of statistical significance, the degree of carcinogenicity was much less than that in the mothers, who exhibited a tumor

multiplicity of > 20. It is of interest that a greater effect was seen in the
Chapter 3

165

female offspring, as was noted above for both cigarette smoke condensate

and NNK in hamsters.

Progeny of C3H/He x C57BL/6 mice (C3B6F1

) and of outbred Swiss

[Cr:NIH(s)] mice, exposed transplacentally to NNK, were held for 18 mo and

included groups treated postnatally with barbital or polychlorinated biphenyls (PCBs) as tumor promoters. Prenatal NNK treatment of male C3B6F1

mice caused a significant increase in incidence of hepatocellular tumors

(40 vs. 17 percent in controls), with no further effect of barbital and PCBs.

Male offspring of Swiss mice had a low incidence of liver tumors (5 percent)

after transplacental NNK; this increased to 19 percent in those receiving

PCBs postnatally, a significant tumor-promotive effect. Although the

mothers again developed lung tumors in high multiplicity, there was no

increase in these in the C3B6F1

 or Swiss mice offspring.

The results from the three strains together show convincingly that NNK

is a relatively weak transplacental carcinogen in the mouse, affecting primarily fetal organs of high sensitivity. The postnatal effects of the PCBs in

Swiss mice are of special interest, indicating that, in certain genetic situations, tumors initiated prenatally by NNK could be promoted to expression

postnatally by exposure to other environmental agents.

NNK was also tested in neonatal Swiss mice, with 50 mg/kg doses given

five times between days 1 and 14. NNK was much more effective with

neonatal treatment than after transplacental exposure, causing liver tumors

in 57 percent of males and 14 percent of females (none in controls) and

lung tumors in 57 percent of males and 37 percent of females (21 to

22 percent in controls) (Anderson et al., 1991a). Here, the males were more

susceptible to lung tumorigenesis than were females. Although neonates

appear to be more sensitive than fetuses, the actual dose received may be

greater, because maternal and placental metabolism and the physical placental barrier probably reduce the amount of NNK reaching the fetus.

Overall the studies of perinatal carcinogenesis by NNK in hamsters and

mice show that this chemical is a weak (transplacentally in mice) to moderate (in neonatal mice and transplacentally in hamsters) perinatal tumorigen,

that its efficacy correlates with ability of target tissue to activate it metabolically, and that the tumors caused were those characteristic of the species

and strain.

OTHER ST

CONSTITUENTS

Other carcinogens are present in ST at lower levels than tobaccospecific nitrosamines (Hecht and Hoffmann, 1988) but could

contribute nonetheless to a perinatal carcinogenic effect. Of those found at

levels > 1 ppb, N-nitrosodimethylamine (NDMA) and benzo[a]pyrene (B[a]P)

have been tested for perinatal carcinogenicity. In rats, hamsters, and mice,

NDMA was a weak transplacental carcinogen, giving a significant but low

yield of tumors (Anderson et al., 1985a and 1989a). NDMA is quite fetotoxic, so only relatively low doses (5 to 30 mg/kg) could be tolerated for

testing. The limited prenatal effectiveness of NDMA as either a teratogen or

carcinogen may relate to lack of capacity for metabolic activation, because a

spontaneously reacting derivative, acetoxymethyl-methylnitrosamine, was

active as a teratogen (Platzek et al., 1983) and as a transplacental carcinogen

in rats (J.M. Rice, unpublished observations).
Smoking and Tobacco Control Monograph No. 2

166

In contrast, NDMA is highly carcinogenic in neonatal mice (Frei, 1970;

Toth et al., 1964), resulting in many lung and liver tumors. Coccia and

coworkers (1988) found that NDMA demethylase activity in neonatal Swiss

mouse liver, compared to adult liver, was two-thirds less, the rate of cell

division twenty-fivefold greater, the amount of O6

-methylguanine adduct in

DNA 2 and 4 h after a carcinogenic NDMA dose fourfold greater, and the

level of O6-methylguanine transferase repair enzyme fivefold less. Thus the

factors suggested as predisposing to tumor initiation in the neonate were all

confirmed.

B[a]P is a moderate transplacental carcinogen in mice, even though

transplacental passage of B[a]P may be limited (Neubert and Tapken, 1988).

Experiments in which B[a]P or its metabolites were injected directly into

mouse fetuses demonstrated that B[a]P itself caused lung tumors, but the

diol-epoxide metabolite, the ultimate carcinogenic form, was more effective,

suggesting that ability to activate B[a]P is limiting in the fetus (Rossi et al.,

1983). B[a]P, like the nitrosamines, is a potent carcinogen in neonatal mice

(Truhaut et al., 1966). As a highly soluble lipid compound, B[a]P would be

expected to partition readily into breast milk and has been quantified there

(LaVoie et al., 1987).

In contrast to B[a]P, DMBA is a potent transplacental carcinogen in the

rat (Rice et al., 1978). The possibility thus exists that other naturally occurring PAHs, including some that occur in tobacco products, may be significantly potent transplacentally. Most such substances are not readily available and have not been tested.

POTENTIAL RISK

AND FUTURE

RESEARCH

A conclusion from the assays of perinatal carcinogenesis by NNK

in animal models is that this chemical and similar ones in ST may

contribute to the risk of human perinatal carcinogenesis, depending on factors about which we have little or no information. Some of these

information gaps are as follows:

• Presence, ontogeny, and variability of cytochromes P450 activating

NNK, NDMA, and PAHs in human fetuses and placentas, especially

cytochrome P450 1A1/2, 2A3, 2D6, and 3A7

– Activation of PAH diols by cytochrome P450 3A7

– DNA and hemoglobin adducts as markers of activation

– Capacity of primate placenta to detoxify NNK, and influence of

other factors such as smoking and coexposure to other xenobiotics;

induction and competitive inhibition;

• Occurrence of aromatic amine carcinogens in ST;

• Toxicokinetics of NNK during pregnancy and lactation and modulating effects of other chemicals in nonhuman primate studies;

• Concentration of NNK and metabolites, other ST carcinogens in

amniotic fluid and breast milk of users;

• Preconception effects of NNK on cancer in animal models; and
Chapter 3

167

• Relations of childhood or adult cancers to use of ST by parents as

revealed by epidemiological studies.

These gaps might be bridged by application of current technologies, as

discussed below.

Biochemistry:

Carcinogen

Metabolism and

DNA Adducts

By extrapolation of conclusions reached with rodent models,

human perinatal risk as a result of carcinogens present in ST is

probably determined in part by the levels of activating cytochrome

P450 enzymes in the target tissues. Of cytochromes P450 in human

fetal liver, 40 to 85 percent is the fetus-specific P450 3A7, related but not

identical to the predominant isoform of this family in adult liver, 3A4

(Cresteil et al., 1985; Kitada et al., 1985a; Komori et al., 1989a and 1989b;

Wrighton and Vandenbranden, 1989). Which P450 ’s constitute the remaining 15 to 60 percent is an important unresolved question. P450 2D6 is

detectable in human fetal liver by both immunoblot and Northern blot,

although at low levels compared with adults (Treluyer et al., 1991). Low

levels of P450 1A1 gene expression were detected by use of the polymerase

chain reaction in the majority of a small sample of fetal adrenals, livers, and

lung, but not kidneys (Omiecinski et al., 1990). P450 1A2 mRNA was reported to be absent (Cresteil, reported in Anderson et al., 1991b); however, a

protein cross-reacting with an antibody to 1A2 was partially purified (Kitada

et al., 1990), and a 1A isoform, suggested to be 1A2, was recognized by a

monoclonal antibody to rat P450 1A1 in human fetal hepatocytes (Murray et

al., 1992). P450 2E1 was not expressed in fetal (Komori et al., 1989b) or

newborn (Morel et al., 1990) livers.

To relate these observations to carcinogens in ST, activation of NNK to

a mutagen in cell expression systems is supported by human cytochromes

P450 1A2, 2A3, 2D6, and 2E1 (Crespi et al., 1991). As noted above, in human

perinatal liver 2E1 is probably absent, 2D6 is present at low levels, and a P450

related to 1A2 may occur. Perinatal expression of 2A3 should be investigated, because it acts not only on NNK but also on NDMA (Crespi et al.,

1990). P450 3A7, the predominant human fetal form, also exhibits considerable NDMA demethylase activity (Kitada et al., 1985a).

With regard to PAHs such as B[a]P, B[a]P hydroxylase activity, generally

considered a detoxification step, correlated with levels of 3A7 and was

inhibitable by anti-3A7 antibody in human fetal livers (Kitada et al., 1985b

and 1987); phenols but not diols were formed (Blanck et al., 1983). It is not

certain which human P450 provides the initial activation of B[a]P to a diol;

both 2C8,9 (Cresteil et al., 1985) and 1A1 (Shimada et al., 1989b) have been

suggested. P450 1A1 may be effectively induced in human placentas by

smoking (Pasanen et al., 1990; Pasanen and Pelkonen, 1990), and placental

microsomes induced by smoking do produce B[a]P-7,8-diol (Blanck et al.,

1983). Whether ST brings about this induction is not known and should be

determined.

P450 3A4 carries out the further activation of B[a]P-7,8-diol to the ultimate diol-epoxide carcinogen (McManus et al., 1990; Shimada et al., 1989a).

Whether the fetal form, 3A7, also has this capacity should be determined. If
Smoking and Tobacco Control Monograph No. 2

168

so, PAH-diols formed by induced placenta might cross to the fetus where

they could be further activated by the P450 3A7 present in some abundance

in the fetal tissues.

In sum, for carcinogens in ST, there seems reason for concern about

tumor initiation in human fetuses by NDMA, as a result of its metabolism

by P450 3A7, and by PAHs, because P450 1A1 in fetal tissues and especially in

induced placenta could provide the initial activation, followed by possible

ultimate activation by the substantial levels of 3A7 in the fetus. Potential

effects of the more abundant NNK catalyzed by the low level of P450 2D6

present is more problematic; it is particularly important to discover whether

there is a 1A2-like protein present with activity toward NNK.

 DNA and hemoglobin adducts have been a popular exposure biomarker. Thus far, of the tobacco-related carcinogens, only 4-aminobiphenyl

adducts on hemoglobin have been detected in fetuses in association with

maternal cigarette smoking (Coghlin et al., 1991). The occurrence of aromatic amines in ST has not been reported and should be investigated.

O6

-methylguanine adducts of DNA from NNK or NDMA correlate with

tumorigenesis in fetal hamster and newborn mouse and are consistent with

the known oncogene mutations in the resultant tumors. Sensitive methods

for detection of this adduct in human tissues are becoming available

(Shields et al., 1990) and might profitably be applied to human fetuses and

newborns, as related to tobacco use and other parameters. O6-methyldeoxyguanosine has been detected in human placental DNA (Foiles et al., 1988).

The pyridyl-oxobutyl adduct could be an indicator of exposure specifically

to NNK. Repair and persistence of adducts after perinatal exposure will be

difficult to evaluate with human material, but this question might be

addressed with nonhuman primates.

Distribution and

Toxicokinetics

As noted above, NNK and a metabolite were present in the amniotic fluid of hamsters and mice. Fetuses both breathe and ingest

amniotic fluid. Human amniotic fluid has the capacity to activate carcinogens (Daubeze et al., 1986) and contains Ames-test mutagens, with a significantly higher level for heavy smokers (Rivrud et al., 1986). It might be

worthwhile to analyze human amniotic fluid for tobacco-specific nitrosamines and their metabolites.

The dose of NNK incurred by the fetus or nursing infant will be influenced significantly by metabolic clearance in maternal liver and possibly in

placenta, and both enzyme induction and competitive inhibition are possible. Are there NNK-metabolizing enzymes in placenta and, if so, are they

induced by cigarette smoking, snuff dipping, or both? This important

question is addressable now, by use of placentas from normal pregnancies to

search for relevant P450 isoforms (discussed above) and for measurement of

total NNK substrate conversion. This in vitro system could also be used to

study competitive inhibition of metabolism by chemicals such as hormones,

ethanol, pharmaceutical drugs, and industrial and household solvents.
Chapter 3

169

 The question of induction and inhibition in maternal liver, with an

influence on clearance, will be more difficult for researchers to address using

human tissue. A reasonable start may be made with nonhuman primates.

Epidemiology No epidemiological studies of cancer associated with parental use of

ST have been reported. Suggestions may be sought from the epidemiology

of cigarette smoking, which entails the same set of chemical carcinogens,

and from the animal model work described above. Reviews of the epidemiological evidence have been given by Preston-Martin (1989) and John et al.

(1991). Relative risks of 1.1 to 3.0 were frequently associated with maternal

or paternal cigarette smoking or both, with positive effects of statistical

significance in three studies: childhood leukemia associated with numbers

of cigarettes smoked daily by the mothers (Stjernfeldt et al., 1986); in a

prospective study, a relationship between all cancers and maternal smoking

(Golding et al., 1990); and increased adult lung cancers among children of

smoking mothers (Correa et al., 1983). Increased risk of hematopoietic

cancers in adulthood from maternal or paternal smoking (Sandler et al.,

1985) had a particularly strong association if both parents smoked. In

studies implicating fathers, there was a positive association between mothers

living with a smoker and brain tumors (Preston-Martin et al., 1982) and

between paternal smoking and rhabdomyosarcomas (Grufferman et al.,

1982). An effect of paternal smoking could imply (1) enhanced placental

penetration of the smoke constituents in nonsmoking mothers with

uninduced detoxification enzymes; (2) transmammary exposure of the

infant; (3) direct respiratory exposure of the infant; or (4) a germ cell effect

on the father.

There are corresponding possibilities for ST, especially snuff, that

epidemiologists should pursue, and both childhood and adult cancers

should be included. Populations in India and southeast Asia, where use of

ST by women is common, could be especially fruitful to study. In addition

to use of ST during pregnancy, use during lactation should receive attention,

in light of the high sensitivity of rodent neonates. Paternal use should also

be examined because preconception paternal exposures are becoming

increasingly implicated in various childhood cancers (Bunin et al., 1992),

with germ cell effects being the most likely mechanism. This possibility is

supported by the finding of increased tumors in the offspring of male

rodents exposed to carcinogens before conception (Mohr et al., 1989;

Turusov et al., 1989). Considering the high concentration of mutagens

present in some smokeless tobacco, epidemiological investigation of the

children of male as well as female users seems warranted.

REFERENCES

Alaoui-Jamali, M.A., Rossignol, G., Schuller, H.M.

Transplacental genotoxicity of a tobacco-specific Nnitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone in Syrian golden hamster. Mutation

Research 223: 65-72, 1989.

Alexandrov, V.A., Popovich, I.G., Anisimov, V.N.,

Napalkov, N.P. Influence of hormonal disturbances on transplacental and multigeneration

carcino-genesis in rats. IARC Scientific Publication

96: 35-49, 1989.
Smoking and Tobacco Control Monograph No. 2

170

Anderson, L.M., Donovan, P.J., Rice, J.M. Risk

assessment for transplacental carcinogenesis. In:

New Approaches in Toxicity Testing and Their

Applications in Human Risk Assessment, A.P. Li

(Editor). New York: Raven Press, 1985a, pp. 179-

202.

Anderson, L.M., Hagiwara, A., Kovatch, R.M., Rehm,

S., Rice, J.M. Transplacental initiation of liver,

lung, neurogenic, and connective tissue tumors by

N-nitroso compounds in mice. Fundamental and

Applied Toxicology 12: 604-620, 1989a.

Anderson, L.M., Hecht, S.S., Dixon, D.E., Dove, L.F.,

Kovatch, R.M., Amin, S., Hoffmann, D., Rice, J.M.

Evaluation of the transplacental tumorigenicity of

the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mice. Cancer

Research 49: 3770-3775, 1989b.

Anderson, L.M., Hecht, S.S., Kovatch, R.M., Amin, S.,

Hoffmann, D., Rice, J.M. Tumorigenicity of the

tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in infant mice.

Cancer Letters 58: 177-181, 1991a.

Anderson, L.M., Jones, A.B., Rice, J.M. Perinatal

carcinogenesis: Current directions (meeting

report). British Journal of Cancer 63: 1025-1028,

1991b.

Anderson, L.M., Jones, A.B., Riggs, C.W., Kovatch,

R.M. Modification of transplacental tumorigenesis

by 3-methylcholanthrene in mice by genotype at

the Ah locus and pretreatment with β-naphthoflavone. Cancer Research 49: 1676-1681, 1989c.

Anderson, L.M., Jones, A.B., Riggs, C.W., Ohshima,

M. Fetal mouse susceptibility to transplacental

lung and liver carcinogenesis by 3-methylcholanthrene: Positive correlation with responsiveness to

inducers of aromatic hydrocarbon metabolism.

Carcinogenesis 6: 1389-1393, 1985b.

Berger, M.R., Petru, E., Habs, M., Schmahl, D.

Influence of perinatal nicotine administration on

transplacental carcinogenesis in Sprague Dawley

rats by N-methylnitrosourea. British Journal of

Cancer 55: 37-40, 1987.

Beniashvili, D.S., Zedginidze, T.G. Significance of

modifying factors in the development of transplacental renal tumors in rabbits. IARC Scientific

Publication 96: 51-55, 1989.

Bjalke, E., Schuman, L.M. Chewing of tobacco and

use of snuff. Relationship to cancer of the pancreas and other sites in two prospective studies.

In: Proceedings of the 13th International Cancer

Congress. Seattle: 13th International Congress,

1982, p. 207.

Blanck, A., Rane, A., Toftgard, R., Gustafsson, J.

Biotransformation of benzo[a]pyrene and

7-ethoxyresorufin and heme-staining proteins in

microsomes from human fetal liver and placenta.

Biochemical Pharmacology 32: 1547-1552, 1983.

Branstetter, D.G., Stoner, G.D., Budd, C., Conran,

P.B., Goldblatt, P.J. Effect of gestational development on lung tumor size and morphology in the

mouse. Cancer Research 48: 379-386, 1988.

Branstetter, D.G., Stoner, G.D., Budd, C., Conran,

P.B., Goldblatt, P.J. Relationship between in utero

development of the mouse liver and tumor

development following transplacental exposure to

ethylnitrosourea. Cancer Research 49: 3620-3626,

1989.

Bunin, G., Noller, K., Rose, P., Smith, E. Carcinogenesis. In: Occupational and Environmental

Reproductive Hazards: A Guide for Clinicians, M.

Paul (Editor). Baltimore: Williams and Wilkins,

1992.

Castonguay, A., Tjalve, H., Trushin, N., Hecht, S.S.

Perinatal metabolism of the tobacco-specific

carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone in C57BL mice. Journal of the National

Cancer Institute 72: 1117-1126, 1984.

Chauhan, D.P., Miller, M.S., Owens, I.S., Anderson,

L.M. Gene expression, ontogeny and transplacental induction of hepatic UDP-glucuronosyl transferase activity in mice. Developmental Pharmacology and Therapeutics 16: 139-149, 1991.

Coccia, P., Salmona, M., Diomede, L., Citti, L.,

Marioni, L., Romano, M. Liver DNA alkylation

after a single carcinogenic dose of dimethylnitrosamine to newborn and adult CFW Swiss mice.

Chemico-Biological Interactions 68: 259-271, 1988.

Coghlin, J., Gann, P.H., Hammond, S.K., Skipper,

P.L., Taghizadeh, K., Paul, M., Tannenbaum, S.R.

4-Aminobiphenyl hemoglobin adducts in fetuses

exposed to the tobacco smoke carcinogen in

utero. Journal of the National Cancer Institute 83:

274-280, 1991.

Correa, E., Joshi, P.A., Castonguay, A., Schuller, H.M.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is an active

transplacental carcinogen in Syrian golden

hamsters. Cancer Research 50: 3435-3438, 1990.

Correa, P., Pickle, L.W., Fontham, E., Lin, Y.,

Haenszel, W. Passive smoking and lung cancer.

Lancet 2: 595-597, 1983.

Crespi, C.L., Penman, B.W., Gelboin, H.V.,

Gonzalez, F.J. A tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone, is activated by multiple human cytochrome P450s including the polymorphic human

cytochrome P450 2D6. Carcinogenesis 12: 1197-

1201, 1991.

Crespi, C.L., Penman, B.W., Leakey, J.A.E., Arlotto,

M.P., Stark, A., Parkinson, A., Turner, T., Steimel,

D., Rudo, K., Davies, R.L., Langenbach, R. Human

cytochrome P450 IIA3: cDNA sequence, role of the

enzyme in the metabolic activation of promutagens, comparison to nitrosamine activation by

human cytochrome P450 IIE1. Carcinogenesis 11:

1293-1300, 1990.
Chapter 3

171

Cresteil, T., Beaune, P., Kremers, P., Celier, C.,

Guengerich, F.P., Leroux, J. Immunoquantification

of epoxide hydrolase and cytochrome P-450

isozymes in fetal and adult human liver microsomes. European Journal of Biochemistry 151: 345-

350, 1985.

Daubeze, M., Cassand, P., Migaud, M.L., Garrigue, P.,

Leng, J.J., Narbonne, J.F. Metabolic activation of

benzo[a]pyrene by human amniotic fluid. Comptes

Rendus de l’Acadamie des Sciences 302: 625-628,

1986.

Diaz Gomez, M.I., Tamayo, D., Castro, J.A. Administration of N-nitrosodimethylamine, N-nitrosopyrrolidine, or N-nitrosonornicotine to nursing

rats: Their interactions with liver and kidney

nucleic acids from sucklings. Journal of the

National Cancer Institute 76: 1133-1136, 1986.

Diwan, B.A., Ohshima, M., Rice, J.M. Effects of

postnatal administration of tumour-promoting

barbiturates on the development of tumours

initiated by prenatal exposure of fetal rats and

mice to N-alkylnitrosoureas. IARC Scientific

Publication 96: 75-80, 1989.

Foiles, P.G., Miglietta, L.M., Akerkar, S.A., Everson,

R.B., Hecht, S.S. Detection of O6

-methyldeoxyguanosine in human placental DNA. Cancer

Research 48: 4184-4188, 1988.

Frei, J.V. Toxicity, tissue changes, and tumor

induction in inbred Swiss mice by methylnitrosoamine and -amide compounds. Cancer

Research 30: 11-17, 1970.

Golding, J., Paterson, M., Kinlen, L.J. Factors

associated with childhood cancer in a national

cohort study. British Journal of Cancer 62: 304-308,

1990.

Goodman, M.T., Morgenstern, H., Wynder, E.L. A

case-control study of factors affecting the development of renal cell cancer. American Journal of

Epidemiology 124: 926-941, 1986.

Grufferman, S., Wang, H.H., DeLong, E.R., Kimm,

S.Y.S., Delzell, E.S., Falletta, J.M. Environmental

factors in the etiology of rhabdomyosarcoma in

childhood. Journal of the National Cancer Institute

68: 107-113, 1982.

Hecht, S.S., Hoffmann, D. Tobacco-specific Nnitrosamines, an important group of carcinogens

in tobacco and tobacco smoke. Carcinogenesis 9:

875-884, 1988.

Heuch, I., Kvale, G., Jacobsen, B.K., Bjelke, E. Use of

alcohol, tobacco and coffee, and risk of pancreatic

cancer. British Journal of Cancer 48: 637-643, 1983.

John, E.M., Savitz, D.A., Sandler, D.P. Prenatal

exposure to parents’ smoking and childhood

cancer. American Journal of Epidemiology 133: 123-

132, 1991.

Kabat, G.C., Dieck, G.S., Wynder, E.L. Bladder cancer

in nonsmokers. Cancer 57: 362-367, 1986.

Karube, T., Odagirai, Y., Takemoto, K., Watanabe, S.

Analyses of transplacentally induced sister

chromatid exchanges and micronuclei in mouse

fetal liver cells following maternal exposure to

cigarette smoke. Cancer Research 49: 3550-3552,

1989.

Kitada, M., Kamataki, T., Itahashi, K., Rikihisa, T.,

Kanukobo, Y. Significance of cytochrome P-450

(P-450HFLa) of human fetal livers in the steroid

and drug oxidations. Biochemical Pharmacology 36:

453-456, 1987.

Kitada, M., Kamataki, T., Itahashi, K., Rikihisa, T.,

Kato, R., Kanakubo, Y. Purification and properties

of cytochrome P-450 from homogenates of

human fetal livers. Archives of Biochemistry and

Biophysics 241: 275-280, 1985a.

Kitada, M., Kamataki, T., Itahashi, K., Rikihisa, T.,

Kato, R., Kanakubo, Y. Immunochemical

examinations of cytochrome P-450 in various

tissues of human fetuses using antibodies to

human fetal cytochrome P-450, P-450 HFLa.

Biochemical and Biophysical Research Communications 131: 1154-1159, 1985b.

Kitada, M., Taneda, M., Ohta, K., Nagashima, K.,

Itahashi, K., Kamataki, T. Metabolic activation of

aflatoxin B1 and 2-amino-3-methylimidazo[4,5-f]-

quinoline by human adult and fetal livers. Cancer

Research 50: 2641-2645, 1990.

Komori, M., Nisho, K., Ohi, H., Kitada, M.,

Kamataki, T. Molecular cloning and sequence

analysis of cDNA containing the entire coding

region for human fetal liver cytochrome P-450.

Journal of Biochemistry 105: 161-163, 1989a.

Komori, M., Nishio, K., Fujitani, T., Ohi, H., Kitada,

M., Mima, S., Itahashi, K., Kamataki, T. Isolation

of a new human fetal liver cytochrome P450

cDNA clone: Evidence for expression of a limited

number of forms of cytochrome P450 in human

fetal livers. Archives of Biochemistry and Biophysics

272: 219-225, 1989b.

LaVoie, E.J., Stern, S.L., Choi, C., Reinhardt, J.,

Adams, J.D. Transfer of the tobacco-specific

carcinogens N'-nitrosonornicotine and 4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone

and benzo[a]pyrene in the milk of lactating rats.

Carcinogenesis 8: 433-437, 1987.

Lofberg, B., Tjalve, H. The disposition and metabolism of N-nitrosodiethylamine in adult, infant,

and foetal tissues of the Syrian golden hamster.

Acta Pharmacological et Toxicologica 54: 104-114,

1984.

Loktionov, A., Hollstein, M., Martel, M., Galendo,

D., Cabral, R.P., Tomatis, L., Yamasaki, Y. Tissuespecific activating mutations of Ha- and Ki-ras

oncogenes in skin, lung, and liver tumors induced

in mice following transplacental exposure to

DMBA. Molecular Carcinogenesis 3: 134-140, 1990.
Smoking and Tobacco Control Monograph No. 2

172

Maekawa, A., Odashima, S. Induction of tumors of

the nervous system in ACI/N rat with 1-butyl-1-

nitrosourea administered transplacentally,

neonatally, or via maternal milk. Gann 66: 175-

183, 1975.

McManus, M.E., Burgess, W.M., Veronese, M.E.,

Huggett, A., Quattrochi, L.C., Tukey, R.H. Metabolism of 2-acetylaminofluorene and benzo[a]pyrene

and activation of food-derived heterocyclic amine

mutagens by human cytochromes P-450. Cancer

Research 50: 3367-3376, 1990.

Miller, M.S., Jones, A.B., Anderson, L.M. The

formation of 3-methylcholanthrene-initiated lung

tumors correlates with induction of cytochrome

P450 IA1 by the carcinogen in fetal but not adult

mice. Toxicology and Applied Pharmacology 104:

235-245, 1990a.

Miller, M.S., Jones, A.B., Chauhan, D.P., Anderson,

L.M. Role of the maternal environment in

determining susceptibility to transplacentally

induced chemical carcinogenesis in mouse fetuses.

Carcinogenesis 11: 1979-1984, 1990b.

Miller, M.S., Jones, A.B., Chauhan, D.P., Park, S.S.,

Anderson, L.M. Differential induction of fetal

mouse liver and lung cytochromes P-450 by βnaphthoflavone and 3-methylcholanthrene.

Carcinogenesis 10: 875-883, 1989.

Mohr, U., Althoff, J. Carcinogenic activity of

aliphatic nitrosamines via the mother’s milk in the

offspring of Syrian golden hamsters. Proceedings of

the Society for Experimental Biology and Medicine 136:

1007-1009, 1971.

Mohr, U., Emura, M., Aufderheide, M., Riebe, M.,

Ernst, H. Possible role of genetic predisposition in

multigeneration carcinogenesis. IARC Scientific

Publication 89: 93-103, 1989.

Morel, F., Beaune, P.H., Ratanasavanh, D., Flinois, J.,

Yang, C.S., Guengerich, F.P., Guillouzo, A.

Expression of cytochrome P-450 enzymes in

cultured human hepatocytes. European Journal of

Biochemistry 191: 437-444, 1990.

Murray, G.I., Foster, C.O., Barnes, T.S., Weaver, R.J.,

Snyder, C.P., Ewen, S.W.B., Melvin, W.T., Burke,

M.D. Cytochrome P450 IA expression in adult and

human fetal liver. Carcinogenesis 13: 165-169,

1992.

Naito, M., Aoyama, H., Ito, A. Inhibitory effect of

phenobarbital on the development of gliomas in

WF rats treated neonatally with N-ethyl-Nnitrosourea. Journal of the National Cancer Institute

74: 725-728, 1985.

Napalkov, N.P., Likhachev, A., Anisimov, V.,

Loktionov, A., Zabezhinski, M., Ovsyannikov, A.,

Wahrendorf, J., Becher, H., Tomatis, L. Promotion

of skin tumours by TPA in the progeny of mice

exposed pre-natally to DMBA. Carcinogenesis 8:

381-385, 1987.

Neubert, D., Tapken, S. Transfer of benzo[a]pyrene

into mouse embryos and fetuses. Archives of

Toxicology 62: 236-239, 1988.

Nicolov, I.G., Chernozemsky, I.N. Tumors and

hyperplastic lesions in Syrian hamsters following

transplacental and neonatal treatment with

cigarette smoke condensate. Journal of Cancer

Research and Clinical Oncology 94: 249-256, 1979.

Nomura, T. Carcinogenesis by urethan via mother’s

milk and its enhancement of transplacental

carcinogenesis in mice. Cancer Research 33: 1677-

1683, 1973.

Nomura, T., Okamoto, E., Tateishi, N., Kimura, S.,

Isa, Y., Manabe, H., Sakamoto, Y. Tumor induction in the progeny of mice receiving 4-nitroquinoline 1-oxide and N-methyl-N-nitrosourethan

during pregnancy or lactation. Cancer Research 34:

3373-3378, 1974.

Ogawa, K., Yokokawa K., Tomoyori, T., Onoe, T.

Induction of α-glutamyltranspeptidase-positive

altered hepatocyte lesions by combination of

transplacental-initiation and postnatal-selection.

International Journal of Cancer 29: 333-336, 1982.

Omiecinski, C.H., Redlich, C.A., Costa, P. Induction

and developmental expression of cytochrome P450

IA1 messenger RNA in rat and human tissues:

Detection by the polymerase chain reaction.

Cancer Research 50: 4315-4321, 1990.

Orlandi, M.A., Boyd, G. Smokeless tobacco use

among adolescents: A theoretical overview.

National Cancer Institute Monographs 8: 5-12, 1989.

Pasanen, M., Haaparanta, T., Sundin, M., Sivonen,

P., Vakakangas, K., Raunio, H., Hines, R.,

Gustafsson, J., Pelkonen, L. Immunochemical and

molecular biological studies on human placental

cigarette smoke-inducible cytochrome P450-

dependent monooxygenase activities. Toxicology

62: 175-187, 1990.

Pasanen, M., Pelkonen, O. Human placental xenobiotic and steroid biotransformations catalyzed by

cytochrome P450, epoxide hydrolase, and glutathione S-transferase activities and their relationships to maternal cigarette smoking. Drug Metabolism Reviews 21: 427-461, 1990.

Paulson, R.M., Shanfel, J., Prause, L., Iranpour, S.,

Paulson, J.O. Pre-conceptional and post-conceptional tobacco effects on the CD-1 mouse fetus.

Journal of Craniofacial Genetics and Developmental

Biology 11: 48-58, 1991.

Pelkonen, O. Fetoplacental biochemistry—

xenobiotic metabolism and pharmacokinetics. In:

Occupational Hazards and Reproduction, K.

Hemminki, M. Sorsa, and V. Vainio (Editors).

Washington: Hemisphere Publishing, 1985, pp.

113-126.
Chapter 3

173

Perantoni, A.O., Rice, J.M., Reed, C.D., Watatani, M.,

Wenk, M.F. Activated neu oncogene sequences in

primary tumors of the peripheral nervous system

induced in rats by transplacental exposure to

ethylnitrosourea. Proceedings of the National

Academy of Sciences 84: 6317-6321, 1987.

Platzek, T., Bochert, G., Rahm, U. Embryotoxicity

induced by alkylating agents. Teratogenicity of

acetoxymethyl-methylnitrosamine: Dose-response

relationship, application route dependency and

phase specificity. Archives of Toxicology 52: 45-69,

1983.

Preston-Martin, S. Epidemiological studies of

perinatal carcinogenesis. IARC Scientific Publication

89: 289-313, 1989.

Preston-Martin, S., Yu, M.C., Benton, B., Henderson,

B.E. N-Nitroso compounds and childhood brain

tumors: A case-control study. Cancer Research 42:

5240-5245, 1982.

Remmer, H. Passively inhaled tobacco smoke: A

challenge to toxicology and preventive medicine.

Archives of Toxicology 61: 89-104, 1987.

Rice, J.M. Effects of prenatal exposure to chemical

carcinogens and methods for their detection. In:

Developmental Toxicology, C.A. Kimmel and J.

Buelke-Sam (Editors). New York: Raven Press,

1981, pp. 191-212.

Rice, J.M., Joshi, S.R., Shenefelt, R.E., Wenk, M.L.

Transplacental carcinogenic activity of 7,12-

dimethylbenz[a]anthracene. In: Carcinogenesis,

Polynuclear Aromatic Hydrocarbons (volume 3), P.W.

Jones and R.I. Freudenthal (Editors). New York:

Raven Press, 1978, pp. 413-422.

Rice, J.M., Rehm, S., Donovan, P.J., Perantoni, A.O.

Comparative transplacental carcinogenesis by

directly acting and metabolism dependent

alkylating agents in rodents and nonhuman

primates. IARC Scientific Publication 96: 17-34,

1989.

Rivrud, G.N., Berg, K., Anderson, D., Blowers, S., Bjro,

K. Mutagenic effect of amniotic fluid from smoking women at term. Mutation Research 171: 71-77,

1986.

Rossi, L., Barbieri, O., Sanguineti, M., Staccione, A.,

Santi, L.F., Santi, L. Carcinogenic activity of

benzo[a]pyrene and some of its synthetic derivatives by direct injection into the mouse fetus.

Carcinogenesis 4: 153-156, 1983.

Rossignol, G., Alaoui-Jamali, M.A., Castonguay, A.,

Schuller, H.M. Metabolism and DNA damage

induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone in fetal tissues of the Syrian golden

hamster. Cancer Research 49: 5671-5676, 1989.

Sandler, D.P., Everson, R.B., Wilcox, A.J., Browder,

J.P. Cancer risk in adulthood from early life

exposure to parents’ smoking. American Journal of

Public Health 75: 487-492, 1985.

Shields, P.G., Povey, A.C., Wilson, V.L., Weston, A.,

Harris, C.C. Combined high-performance liquid

chromatography/32P-postlabeling assay of N7

-

methyldeoxyguanosine. Cancer Research 50: 6580-

6584, 1990.

Shimada, T., Iwasaki, M., Martin, M.V., Guengerich,

F.G. Human liver microsomal cytochrome P-450

enzymes involved in the bioactivation of procarcinogens detected by umu gene response in

Salmonella typhimurium TA 1535/pSK1002. Cancer

Research 49: 3218-3228, 1989a.

Shimada, T., Martin, M.V., Pruess-Schwartz, D.,

Marnett, L.J., Guengerich, F.P. Role of individual

human cytochrome P-450 enzymes in the

bioactivation of benzo[a]pyrene, 7,8-dihydroxy7,8-dihydrobenzo[a]pyrene, and other dihydrodiol

derivatives of polycyclic aromatic hydrocarbons.

Cancer Research 49: 6304-6312, 1989b.

Stjernfeldt, M., Berglund, K., Lindsten, J.,

Ludvigsson, J. Maternal smoking during pregnancy and risk of childhood cancer. Lancet 1:

1350-1352, 1986.

Suganuma, M., Fujiki, H., Morino, K., Takayama, S.,

Sugimura, T. Tumor promoting activity of

teleocidin in skin and forestomach of mice

initiated transplacentally with 7,12-dimethylbenz[a]anthracene. Journal of Cancer Research and

Clinical Oncology 113: 123-125, 1987.

Sukumar, S., Barbacid, M. Specific patterns of

oncogene activation in transplacentally induced

tumors. Proceedings of the National Academy of

Sciences 87: 718-722, 1990.

Toth, B. Critical review of experiments in chemical

carcinogenesis using newborn animals. Cancer

Research 28: 727-738, 1968.

Toth, B., Magee, P.N., Shubik, P. Carcinogenesis

study with dimethylnitrosamine administered

orally to adult and subcutaneously to newborn

BALB/c mice. Cancer Research 24: 1712-1721,

1964.

Treluyer, J.M., Jacqx-Aigrain, E., Alvarez, F., Cresteil,

T. Expression of CYP2D6 in developing human

liver. European Journal of Biochemistry 202: 583-

588, 1991.

Truhaut, R., Lesca, P., Dechambre, R.P., GerardMarchant, P. Sur les modalites de manifestation

du pouvoir cancerogene du benzo-3-4-pyrene chez

la souris nouveau-nee ou traitee immediatement

apres le sevrage. Pathologie et Biologie (Paris) 14:

955-959, 1966.

Turusov, V.S., Cardis, E. Review of experiments of

multigeneration carcinogenicity: Discussion of

design, experimental models and analyses. IARC

Scientific Publication 89: 105-120, 1989.

Vesselinovitch, S.D., Rao, K.V.N., Mihailovich, N.

Transplacental and lactational carcinogenesis by

safrole. Cancer Research 39: 4378-4380, 1979.
Smoking and Tobacco Control Monograph No. 2

174

Wrighton, S.A., Vandenbranden, M. Isolation and

characterization of human fetal liver cytochrome

P450 HLp2: A third member of the P450 III gene

family. Archives of Biochemistry and Biophysics 268:

144-151, 1989.

Yamasaki, H., Hollstein, M., Martel, N., Galenda, D.,

Cabral, R.P., Tomatis, L. Transplacental induction

of a specific mutation in fetal Ha-ras and its

critical role in postnatal carcinogenesis. International Journal of Cancer 40: 818-822, 1987.
Chapter 3

175

Several lines of evidence point to a role for smokeless tobacco-related carcinogens in the

Interaction Between Smokeless TobaccoRelated Carcinogens and Human

Papillomaviruses in the Pathogenesis of

Oral Dysplasia and Cancer1

Joel M. Palefsky, John S. Greenspan, Troy E. Daniels, Jennifer Berline,

Deborah G. Grady, and Virginia L. Ernster

ABSTRACT

pathogenesis of oral cancer. Previous studies of oral cancer suggest that other factors may play

a role as well, including use of alcohol and infection with human papillomavirus (HPV). To

examine the role of HPV in the pathogenesis of ST-related oral cancer, we sought HPV DNA in

three different sets of formalin-fixed biopsy tissues, using the polymerase chain reaction (PCR)

technique: (1) nondysplastic oral lesions associated with short-term ST use, (2) oral cancers and

dysplasias associated with long-term use of ST, and (3) oral cancers and dysplasias not related to

the use of ST. In these studies HPV was not detected in early, nondysplastic lesions associated

with short-term use. However, HPV was detectable in approximately 30 percent of the oral

cancers—whether or not they were associated with ST use. These results suggested that HPV

infection may be one of several factors contributing to the pathogenesis of ST-related oral

cancer, and that HPV infection does not occur at the earliest stages of ST lesion development.

INTRODUCTION It has been estimated that 30 percent of all cancers in the United

States are associated with the use of tobacco in its various forms (Doll and

Peto, 1981; Wynder and Gori, 1977). Approximately 53 million Americans

are believed to smoke, and approximately 12 million to use smokeless

tobacco (Hecht and Hoffmann, 1988). Smoking is strongly associated with

malignancy of the respiratory tract, upper digestive tract, bladder, kidney,

and pancreas (Doll and Peto, 1981; Wynder and Gori, 1977). There is also a

large body of evidence linking smoking to oral cancer (Doll and Peto, 1981;

Wynder and Gori, 1977), and death rates from oral cancer are four times

higher among smokers than nonsmokers (Doll and Peto, 1976; Hammond,

1966; Rogot and Murray, 1980). Oral cancer is also strongly associated with

the use of smokeless tobacco, especially snuff (Consensus Conference, 1986;

IARC, 1985; US DHHS, 1986; Vogler et al., 1962; Winn, 1988); the incidence

of cancer of the cheek and gums has been shown to be as much as 50 times

higher among long-term ST users than among age-matched controls (Winn,

1986).

The mechanisms by which smoking and ST contribute to the development of oral cancer or its precursors are not yet known. One potential

mechanism is the direct effect of carcinogens in cigarette smoke or ST on

the oral epithelium. Tobacco products are known to contain a number of

substances that may give rise to carcinogens (Hecht and Hoffmann, 1988).

1 This study was supported by National Institute of Dental Research grant no. PO1 DE08547.
Smoking and Tobacco Control Monograph No. 2

176

The most important of these are nicotine, a tertiary amine, and nornicotine,

anabasine, and anatabine, which are secondary amines. These compounds

may react with nitrosating agents to form nitrosamines (NNN, NNK, NNA,

NAB, and NAT), which have been shown to be carcinogenic in mice, rats,

and hamsters (Hecht and Hoffmann, 1988; Hoffmann and Hecht, 1985).

Other potential carcinogens found in smoked tobacco products include

formaldehyde, acetaldehyde, croton aldehyde, and benzopyrene. The

mechanism by which nitrosamines and other potential carcinogens act to

promote malignancy is not yet known. One possibility is modification of

cellular DNA by nitrosamines. There are several mechanisms by which this

could occur; for example, nitrosamines may be α-hydroxylated by specific

cytochrome P450 isozymes to yield methyldiazohydroxide, which is capable

of methylating DNA to produce O6-methylguanine and other point mutations.

A second mechanism may involve induction of chromosomal DNA

breakage. It has been shown that tobacco-related products may induce

single-stranded DNA breaks because of the reduction of oxygen to superoxide radicals (Borish et al., 1987), and the resulting repair process may

increase the likelihood of a mutation (Richter et al., 1988). These mutations

may then contribute to carcinogenesis by altering the levels or functions of

genes involved in the control of cell growth.

In addition to the use of tobacco products, viral infection is increasingly

implicated in the pathogenesis of oral cancer. Oral cancer bears significant

resemblance to other mucosal epithelial cancers, such as cancer of the cervix

and anus, both of which have been associated with infection with human

papillomavirus (HPV) (Beaudenon et al., 1986; Boshart et al., 1984; Crum et

al., 1985; Lorincz et al., 1986 and 1987; Reid et al., 1987). The association

between HPV infection and mucosal cancer has been characterized best for

anogenital cancer. Of the more than 60 known HPV types, infection with

HPV types 16 and 18 is most closely associated with anogenital cancer and

high-grade intraepithelial neoplasia (grades 2-3); types 31, 33, and 35 are

associated with an intermediate risk of cancer; and types 6, 11, 42, 43, and

44 are associated with low-grade intraepithelial neoplasia (grade 1) and

condyloma, both of which carry a low risk of progression to invasive

disease.

The relationship between HPV infection and oral cancer was first

described in 1983, when cytopathic changes of HPV infection (koilocytosis)

similar to those seen in cervical HPV infection were noted in oral lesions

(Syrjanen et al., 1983). Subsequent to that report, several investigators have

looked for HPV DNA in oral lesions (Adler-Storthz et al., 1986; Dekmezian et

al., 1987; Kashima et al., 1990; Loning et al., 1985 and 1987; Lookingbill et

al., 1987; Maitland et al., 1987; Milde and Loning, 1986; Ostrow et al., 1987;

Scully et al., 1985; Syrjanen et al., 1988; de Villiers et al., 1985 and 1986),

using a wide variety of HPV DNA hybridization techniques with differing

sensitivities and specificities. In one study, involving dot blot hybridization,

HPV DNA was detected in 8 of 22 oral cancers (36 percent): type 16/18 in 3

cancers, type 6/11 in 1, and other types in 4 other cancers (Loning et al.,

1987). Using in situ hybridization, one group reported detection of HPV
Chapter 3

177

DNA in 6 of 51 (12 percent) oral cancer specimens, and 6 of 21 (29 percent)

oral dysplasia specimens (Syrjanen et al., 1988). Using Southern blot hybridization, another group detected HPV sequences in almost 50 percent of

invasive cancers, but also detected HPV in a similar proportion of normal

tissues (Maitland et al., 1987). Using a similar technique, Kashima et al.

reported that 7 of 74 (9 percent) were positive for HPV DNA. In this series

of tests, a wide range of types were identified, including HPV-16, -6, -57, -3,

and -13 (Kashima et al., 1990). Similar to the report by Maitland et al.

(1987), HPV DNA was detectable in 3 of 33 (9 percent) clinically normal

tissues obtained from the mucosal surface contralateral to the lesion. Taken

together, these studies suggest that HPV DNA is detectable in fewer than

half of the specimens of oral cancer, and that a wide range of HPV types

may be present. Moreover, detection of HPV DNA in clinically normal oral

tissues in proportions similar to those of the cancerous lesions in some

studies raises the question of the nature of the contribution of HPV to the

pathogenesis of oral lesions.

The techniques used in the studies described above, that is, Southern

blot hybridization, dot blot hybridization, and in situ hybridization, all have

limited sensitivity. Thus, the inability to detect HPV DNA in a higher

proportion of oral cancers and precancerous lesions may reflect a level of

HPV infection below the limit of the sensitivity of these tests or infection

with HPV types other than would be normally detected with currently

available probes. To address the question of the prevalence of HPV in oral

dysplasias and cancers, using DNA detection techniques that are both highly

sensitive and capable of detecting a large number of HPV types, we used the

PCR technique with HPV L1 consensus primers.

Studies of the role of HPV in ST-associated lesions provide a unique

opportunity to determine the stage at which HPV infection may begin to

play a role in the pathogenesis of oral cancer for two reasons: (1) the use of

ST products is clearly associated with low-grade lesions such as hyperkeratosis and hyperorthokeratosis, which may represent precursors to

dysplasia, given sufficient exposure; and (2) as described previously, use of

ST products is also clearly a risk factor for development of oral cancer.

Using in situ hybridization, Greer et al. (1990) studied 50 leukoplakias

associated with the use of ST, and found 2 (4 percent) containing HPV-6 and

3 (6 percent) containing HPV-2. Therefore, to address the question of the

stage of precancerous disease at which HPV may first be detected through

more sensitive techniques, we used PCR also to study ST-associated lesions

ranging from histologically normal to invasive cancer.

METHODS One hundred eight oral biopsies ranging from histologically normal to

invasive cancer were obtained, fixed in formalin, and embedded in paraffin.

To perform PCR, tissue sections 7 μm in thickness were cut from each paraffin block, and the paraffin was removed by suspending the section in 500 μL

of xylene in its original Eppendorf tube. The tissue was dried, resuspended in

100 μL of water containing proteinase K at a concentration of 100 μg/mL,

and digested overnight at 37 °C. Consensus primers for the detection of the

L1 region were employed as described elsewhere (Ting et al., 1990). Positive
Smoking and Tobacco Control Monograph No. 2

178

controls consisted of the amplification of human beta-hemoglobin DNA

from each tissue. Negative controls were used to minimize the possibility of

false-positives attributable to contamination from one specimen to another

in the laboratory, and consisted of amplification of each mixture with all

components of the reaction except target DNA. Fifty cycles of amplification

were performed with 800.0 μM dNTPs (United States Biochemical Corp.,

Cleveland, Ohio), 1.0 μM of each primer, 2.5 mM MgCl2, 0.5% Tween-20,

0.5% NP-40, and 50 units/mL AmpliTaq DNA polymerase (Perkin-Elmer

Cetus, Norwalk, Connecticut). Five microliters of 100 μL amplification

product were applied to a nylon membrane and the presence of HPV DNA

was sought with 32P-labeled consensus probes. Positive samples identified

positive with the consensus probes were then studied with probes specific

for HPV-6, -11, -16, -18, -31, and -33.

RESULTS To begin to understand the stages of oral disease at which HPV infection

DISCUSSION

may occur, we examined 52 biopsies obtained from professional baseball

players who were short-term users of ST. The tissues represented 26 biopsies

with histologically diagnosed lesions ranging from hyperparakeratosis to

basal cell hyperplasia (Daniels et al., this volume; Grady et al., 1991) and

26 histologically normal biopsies adjacent to the lesion, which served as

controls. None of the tissues demonstrated dysplastic changes. In these

studies HPV-33 could be detected in only one hyperparakeratotic lesion, and

HPV type 6/11 in the normal tissue adjacent to another hyperparakeratotic

lesion, which was itself HPV-negative.

To study the prevalence of HPV in high-grade lesions associated with

long-term use of ST, 10 high-grade dysplasia/oral cancer lesions and 4

histologically normal control tissues were obtained from the laboratory of

Dr. George Kaugars of the University of Virginia. In contrast to the low-grade

lesions obtained from the baseball players, 3 of 10 high-grade dysplasia/oral

cancer lesions (30 percent) associated with long-term use of ST were found to

be HPV-16 DNA-positive; none of the normal control tissues were positive

for HPV DNA.

Thirty-two oral cancer and dysplasia tissues not associated with use of

ST were studied, along with 10 negative controls. The results indicated that

8 of 25 of the oral cancer tissues (32 percent) were HPV DNA-positive and 3

of 7 (43 percent) of high-grade oral dysplasia were HPV DNA-positive

(Palefsky et al., unpublished data). The HPV types detected were found to be

heterogeneous; fewer than half of the HPV types detected were represented

by the common anogenital HPV types. In contrast to the oral dysplasia and

cancer tissues, none of seven fibromas and none of three normal tissues were

positive for HPV.

Studies of the prevalence of HPV in ST-associated lesions in short-term

and long-term users are not strictly comparable, since the latter represented

an older population from a specific geographic area of the United States.

Nevertheless, results of these studies suggest that the low-grade nondysplastic lesions associated with short-term ST use (< 5 yr) are not associated

with HPV, whereas high-grade ST-associated lesions contain HPV DNA in a

proportion similar to that seen in oral dysplasia and cancers that are not
Chapter 3

179

associated with ST use. These results suggest that HPV does not play a

significant role in the pathogenesis of ST-associated disease at its earliest

stages, but instead begins to be detectable in association with dysplasia.

While dysplastic and cancerous tissues not associated with use of ST were

also found to be associated with HPV, a large number of cases of oral cancer

and dysplasia were HPV-negative on PCR examination, consistent with

studies using less sensitive techniques. Oral cancer therefore appears to be a

more heterogeneous disease than cervical cancer with respect to etiology;

while HPV may play a significant role in the pathogenesis of oral cancer, it

is clear that it need not necessarily be present, and that other factors are

likely to be important as well.

Among other factors that may play a role in the pathogenesis of oral

cancer are genetic mutations. Reports have been published that document

the sequence of cellular changes at the molecular level that occur as a

benign colonic adenoma progresses to invasive colorectal cancer (Baker et

al., 1990; Fearon et al., 1990). In these studies, a series of genetic mutations

accumulated as the lesion progressed, and the accumulation of further

abnormalities was necessary for progression to the next stage of disease.

These included mutations in the ras oncogene, translocations of the short

arm of chromosome 5, and breakage of the long arm of chromosome 11. A

model such as this may also be applicable to the progressive changes that

occur in the oral mucosa as a lesion develops from benign to increasing

grades of dysplasia and, ultimately, to invasive cancer.

On the basis of the data described above, we propose a model for the

development of ST-associated cancer (Figure 1). In this model, ST-related

products are responsible for the development of low-grade nondysplastic

lesions, including hyperparakeratosis, hyperorthokeratosis, and basal cell

hyperplasia, that may progress to dysplasia with continued exposure. It is

not yet known if these low-grade lesions are associated with chromosomal

abnormalities or oncogenic mutations. With continued exposure to these

products, as well as other cofactors such as alcohol, further genetic damage

may accumulate, resulting in dysplasia. ST products may induce these

chromosomal changes via DNA adduct formation or chromosomal breakage.

The result of these changes may include activation of oncogenes such as ras

or inactivation of a member of the anti-oncogene family, such as the RB or

p53 proteins. We also propose that infection with HPV may represent one

of the cofactors that play a role, in conjunction with ST products, in the

pathogenesis of ST-related dysplasia and cancer. At this time, it is not

known if dysplasia occurs as a result of HPV infection or if HPV infects a

preestablished dysplastic lesion and potentiates its progression. However,

recent studies of HPV-16-transfected cells with the floating raft model of

epithelial differentiation have been conducted, in which transfection of

HPV-16 DNA has been shown to result in epithelial changes consistent with

dysplasia (McCance et al., 1988). This supports the hypothesis that dysplasia results from HPV infection.

Once established, HPV infection may contribute to the development

and/or progression of dysplasia in a number of ways. Among these are the

binding and inactivation of p53 gene product by HPV E6 protein, as well as
Smoking and Tobacco Control Monograph No. 2

180

Figure 1

Proposed schema for the development of ST-related oral cancer. In this schema,

several different etiologic factors may play a significant role. ST appears to play

an important role and may continue to do so at any subsequent stage. HPV does

not appear to play an important role at the earliest stages but may facilitate

development and progression of dysplasia in some ST-related lesions. Like ST,

HPV may also play a role in development of invasive cancer. A variety of other

factors such as ras mutations or loss of chromosomal arms may also play a role at

different stages of disease. The alterations depicted in this figure were adapted

from studies of the development of colon cancer (Fearon and Vogelstein, 1990);

similar studies of ST-related cancer have not yet been performed.

the inactivation of the retinoblastoma gene product by the HPV E7 protein

(Munger et al., 1989; Werness et al., 1990). In addition, other mechanisms

by which HPV contributes to the development of dysplasia almost certainly

exist and remain to be characterized.

In summary, ST-related oral cancer may result from cumulative genetic

damage induced by ST, with or without other cofactors. Among possible

significant cofactors is HPV infection, which does not appear to play a role

in pathogenesis of the earliest ST-related lesions, but may instead play a role

at later stages, beginning with dysplasia.

This model suggests that the development of ST-related cancers may

represent the end product of cooperation between a number of different

factors, including HPV infection. Prevention of cancer development must

therefore focus on interference with this process at a number of levels.

Cessation of exposure to ST-related products remains the most important

intervention, but interference with potential cofactors, including abstinence

from alcohol, may play an important role as well. Currently, no therapies

are available to interfere with HPV gene products, and the only method of

preventing the consequences of HPV infection is the removal of HPVinfected lesional tissue. Nevertheless, if and when therapies directed against

HPV do become available, they may constitute useful adjunctive measures

for those cases in which HPV infection can be demonstrated.

Effect 

of ST

HPV Infection HPV Infection

DNA 

hypomethylation

ras

mutation 18q loss

17p loss 

p53

Normal 

epithelium

Epithelial

hyperplasia

Early

dysplasia

Intermediate

dysplasia

Late

dysplasia Cancer
Chapter 3

181

Much remains to be learned about the pathogenesis of ST-related cancer

at the molecular level. The evidence for an important role for HPV in the

pathogenesis of oral cancer in general, and ST-related oral cancer in particular, remains incomplete; studies of larger numbers of dysplastic and cancerous oral tissues obtained from populations with well-matched controls are

needed. Such studies should include analysis of histologically normal

control tissues from matched control subjects, as well as of normal tissue

from the individuals with oral lesions. Standardized methods of HPV

detection are needed, as well as standardization of histopathologic criteria

for grading the lesions. Adequate in vitro models for studying the interaction between ST products, HPV, and alcohol are needed; the recently developed floating raft system may be very useful for this purpose. Further

research is needed to define which of the ST-related products are most toxic

to epithelial cells; whether and how HPV gene products interact with ST

products to induce dysplastic changes; characterization of chromosomal

changes induced by ST; characterization of the role of oncogenic activators,

or inactivation of anti-oncogenes in these cells; characterization of the HPV

types present in ST-related cancer; and characterization of the risk factors for

acquisition of HPV infection. Knowledge of cofactors that play a role along

with ST products will permit a more effective approach to the prevention of

ST-related dysplasia and cancer. Knowledge of the molecular mechanisms

of the pathogenesis of these diseases will also be of great value in preventing

progression or inducing regression of ST-related dysplasia, and in preventing

progression to cancer among those in whom dysplasia has already occurred.

REFERENCES

Adler-Storthz, K., Newland, J.R., Tessin, B.A., et al.

Identification of human papillomavirus types in oral

verruca vulgaris. Journal of Oral Pathology and

Medicine 15: 230-233, 1986.

Baker, S.J., Preisinger, A.C., Jessup, J.M., et al. p53

gene mutations occur in combination with 17p

allelic deletions as late events in colorectal

tumorigenesis. Cancer Research 50: 7717-7722, 1990.

Beaudenon, S., Kremsdorf, D., Croissant, O., et al. A

novel type of human papillomavirus associated with

genital neoplasias. Nature (London) 321: 246-249,

1986.

Borish, E.T., Pryor, W.A., Venugopal, S., et al. DNA

synthesis is blocked by cigarette tar-induced DNA

single-strand breaks. Carcinogenesis 8: 1517-1520,

1987.

Boshart, M., Gissmann, L., Ikenberg, H., et al. A new

type of papillomavirus: Its presence in genital

cancer biopsies and in cell lines derived from

cervical cancer. EMBO Journal 3: 1151-1157, 1984.

Consensus Conference. Health implications of

smokeless tobacco use. Journal of the American

Medical Association 255: 1045-1048, 1986.

Crum, C., Mitao, M.R., Levine, R., et al. Cervical

papillomaviruses segregate within morphologically

distinct precancerous lesions. Journal of Virology 54:

675-681, 1985.

Dekmezian, R.H., Batsakis, J.G., Goepfert, H. In situ

hybridization of papillomavirus DNA in head and

neck squamous cell carcinomas. Archives of

Otolaryngology and Head and Neck Surgery 113: 819-

821, 1987.

Doll, R., Peto, R. Mortality in relation to smoking:

20 years’ observations on male British doctors.

British Medical Journal 2: 1525-1536, 1976.

Doll, R., Peto, R. The causes of cancer: Quantitative

estimate of avoidable risks of cancer in the United

States today. Journal of the National Cancer Institute

66: 1191-1308, 1981.

Fearon, E.R., Vogelstein, B. A genetic model for

colorectal tumorigenesis. Cell 61: 759-767, 1990.

Grady, D., Greene, J., Ernster, V.L., et al. Short-term

changes a surprise with smokeless tobacco oral

lesions. Journal of the American Dental Association

122: 62-64, 1991.

Greer, R.O., Eversole, L.R., Crosby, L.K. Detection of

human papillomavirus-genomic DNA in oral

epithelial dysplasias, oral smokeless tobaccoassociated leukoplakias and epithelial malignancies. Journal of Oral and Maxillofacial Surgery 48:

1201-1205, 1990.

Hammond, E.C. Smoking in relation to the death

rates of one million men and women. National

Cancer Institute Monograph 19: 127-204, 1966.
Smoking and Tobacco Control Monograph No. 2

182

Hecht, S.S., Hoffmann, D. Tobacco-specific nitrosamines, an important group of carcinogens in

tobacco and tobacco smoke. Carcinogenesis 9: 875-

884, 1988.

Hoffmann, D., Hecht, S.S. Nicotine-derived N-nitrosamines and tobacco-related cancer: Current status

and future questions. Cancer Research 45: 935-944,

1985.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic Risk

of Chemicals to Humans: Tobacco Habits Other than

Smoking: Betel-Quid and Areca-Nut Chewing; and

Some Related Nitrosamines (volume 37). Lyon:

IARC, 1985, pp. 113-116.

Kashima, H.K., Kutcher, M., Kessis, T., et al. Human

papillomavirus in squamous cell carcinoma,

leukoplakia, lichen planus, and clinically normal

epithelium of the oral cavity. Annals of Otology,

Rhinology and Laryngology 99: 55-61, 1990.

Loning, T., Ikenberg, H., Becker, J., et al. Analysis of

oral papillomas, leukoplakia and invasive carcinomas for human papillomavirus type related DNA.

Journal of Investigative Dermatology 84: 417-420,

1985.

Lookingbill, D.P., Kreider, J.W., Howitt, M.K., et al.

Human papillomavirus type 16 in bowenoid

papulosis, intraoral papillomas, and squamous cell

carcinoma of the tongue. Archives of Dermatology

123: 363-368, 1987.

Lorincz, A.T., Lancaster, W., Temple, G. Cloning and

characterization of a new human papillomavirus

from a woman with dysplasia of the uterine cervix.

Journal of Virology 58: 225-229, 1986.

Lorincz, A.T., Quinn, A., Lancaster, W., et al. A new

type of papillomavirus associated with cancer of

the uterine cervix. Virology 159: 187-190, 1987.

Maitland, N.J., Cox, M.F., Lynas, C., et al. Detection

of human papillomavirus DNA in biopsies of

human oral tissue. British Journal of Cancer 56:

245-250, 1987.

McCance, D.J., Kopan, R., Fuchs, E., et al. Human

papillomavirus type 16 alters human epithelial cell

differentiation in vitro. Proceedings of the National

Academy of Sciences 85: 7169-7173, 1988.

Milde, K., Loning, T. The detection of papillomavirus DNA in oral papillomas and carcinomas:

Application of in situ hybridization with

biotinylated HPV 16 probes. Journal of Oral

Pathology 15: 292-296, 1986.

Munger, K., Werness, B.A., Dyson, N., et al. Complex

formation of human papillomavirus E7 proteins

with the retinoblastoma tumor suppressor gene

product. EMBO Journal 8: 4099-4105, 1989.

Ostrow, R.S., Manias, D.A., Fong, W.J., et al. A survey

of human cancers for human papillomavirus DNA

by filter hybridization. Cancer 59: 429-434, 1987.

Reid, R., Greenberg, M., Jenson, A.B., et al. Sexually

transmitted papillomaviral infections. I. The

anatomic distribution and pathologic grade of

neoplastic lesions associated with different viral

types. American Journal of Obstetrics and Gynecology

156: 212-222, 1987.

Richter, C., Park, J-W., Ames, B.N. Normal oxidative

damage to mitochondrial and nuclear DNA is

extensive. Proceedings of the National Academy of

Sciences 85: 6465-6467, 1988.

Rogot, E., Murray, J.L. Smoking and the causes of

death among U.S. veterans: 16 years of observation.

Public Health Reports 95: 213-222, 1980.

Scully, C., Prime, S., Maitland, N. Papillomaviruses:

Their role in oral disease. Oral Surgery, Oral Medicine,

Oral Pathology 60: 166-174, 1985.

Syrjanen, K., Syrjanen, S.M., Lamberg, M., et al.

Morphological and immunohistochemical evidence

suggesting human papillomavirus (HPV) involvement in oral squamous cell carcinogenesis. International Journal of Oral Surgery 12: 418-424, 1983.

Syrjanen, S.M., Syrjanen, K.J., Happonen, R-P. Human

papillomavirus (HPV) DNA sequences in oral

precancerous lesions and squamous cell carcinoma

demonstrated by in situ hybridization. Journal of

Oral Pathology 17: 273-278, 1988.

Ting, Y., Manos, M.M. In: PCR Protocols and Applications. New York: Academic Press, 1990, pp. 356-

367.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHSS Publication No. (NIH) 86-2874, 1986, pp. 33-58.

de Villiers, E-M., Neumann, C., Le, J-Y., et al. Infection

of the oral mucosa with defined types of human

papillomavirus. Medical Microbiology and Immunology 174: 287, 1986.

de Villiers, E-M., Wiedauer, H., Otto, H., et al.

Papillomavirus in human tongue carcinomas.

International Journal of Cancer 36: 575, 1985.

Vogler, W.R., Lloyd, J.W., Milmore, B.K. A retrospective study of etiologic factors in cancer of the

mouth, pharynx and larynx. Cancer 15: 246-258,

1962.

Werness, B.A., Levine, A.J., Howley, P.M. Association

of human papillomavirus types 16 and 18 E6

proteins with p53. Science 248: 76-79, 1990.

Winn, D.M. Smokeless tobacco and oral/pharynx

cancer: The role of cofactors. Banbury Report 23:

361-371, 1986.

Winn, D.M. Smokeless tobacco and cancer: The

epidemiologic evidence. CA—A Cancer Journal for

Clinicians 38: 236-243, 1988.

Wynder, E.L., Gori, G.B. Contribution of the

environment to cancer incidence: An epidemiologic exercise. Journal of the National Cancer Institute

58: 825-832, 1977.
Chapter 3

183

Identification of Human Papillomavirus

DNA in Smokeless Tobacco Keratoses

And Premalignant and Malignant

Oral Lesions, by PCR Amplification

With Consensus Sequence Primers1

Robert O. Greer, Jr., Kenneth R. Shroyer, and Louise Crosby

ABSTRACT Human papillomaviruses (HPVs), trophic for cutaneous and mucosal epithelium, have been

reported in association with benign, dysplastic, and malignant proliferations of the oral

mucous membrane. More than 60 HPV types have been recognized on the basis of significant

sequence divergence following molecular cloning as recombinant DNAs. To evaluate the role

of HPV in oral neoplasia, completion of large-scale studies is needed to determine the diversity

of papillomaviruses involved and studies implemented to identify patterns of transcription of

transforming sequences in high-risk HPV types with a trophism for the oral cavity. The

purpose of the current investigation was to test for the presence of HPVs in biopsies of oral

epithelial dysplasia, verrucous hyperplasia, smokeless tobacco keratoses, and squamous cell

carcinoma by examining lesional tissue by polymerase chain reaction (PCR) amplification

techniques using L1 consensus sequence primers. HPV sequences were detected in all four

categories of lesions and in no control samples. PCR amplification allows rapid and specific

detection of HPV in oral biopsy specimens and may enhance our ability to evaluate large

numbers of clinical samples and demonstrate a broad variety of HPV sequences and novel HPV

types in oral cancer and precancer.

INTRODUCTION Papillomaviruses are tenacious, persistent viruses capable of residing latent within host cells for extended periods. HPV is an epitheliotrophic

infectious agent with a genome consisting of approximately 7,900 base pairs

(bp) of double-stranded circular DNA encapsulated within an icosahedral

protein shell. HPV has been increasingly associated with mucosal cancers,

particularly carcinoma of the cervix (Howley, 1987; Vousden, 1989; zur

Hansen and Schneider, 1987). The cloned DNAs of a significant number of

human and animal papillomavirus DNAs have been sequenced completely,

and they are notable for their overall similarity in genetic organization, in

spite of substantial sequence variation and diversity in host and tissue

specificity, histological presentation, and malignant potential (Koutsky et

al., 1989; zur Hansen and Schneider, 1987). Although human

papillomaviruses are characterized by a low degree of pathogenicity, their

synergistic effect with other carcinogens, such as tobacco and alcohol,

enhances their carcinogenic potential.

1 This investigation was supported in part by funding from Smokeless Tobacco Research

Council grant no. 0278, National Cancer Institute grant no. CA-21098-14, and a grant from

the Sands House Foundation.
Smoking and Tobacco Control Monograph No. 2

184

In previous studies using in situ hybridization methods and dot blot

hybridization, we have demonstrated human papillomavirus in ST keratoses,

oral epithelial dysplasia, verrucous hyperplasia, verrucous carcinoma, and

squamous cell carcinoma (Greer et al., 1987, 1990a, and 1990b).

Recently we used a PCR DNA amplification system for the identification

of HPV type 16 E6 DNA sequence in formalin-fixed, paraffin-embedded

biopsy material from 61 oral precancers and cancers, including squamous

cell carcinoma, epithelial dysplasia, ST keratoses, verrucous hyperplasia, and

verrucous carcinoma (Shroyer and Greer, 1991). This PCR amplification

procedure allowed rapid and specific detection of low-abundance viruses

and single-copy genes in biopsy specimens. The PCR amplification technique was judged simple and highly sensitive for analyzing specific HPV

DNA sequences from archival material. The test can be correlated easily

with the histological appearance seen in adjacent sections.

Several investigators (Bauer et al., 1991; Manos et al., 1990; Resnick et

al., 1990) have developed a PCR DNA amplification system using two

distinct consensus oligonucleotide primer sets for the improved detection

and typing of a broad spectrum of human genital papillomavirus sequences

including those of novel viruses. Their systems incorporate one consensus

primer set designed to amplify the L1 domain from a wide variety of HPV

types and a second consensus primer set designed to amplify a domain

within the E6 open reading frame sequence of HPV. Resnick and associates

(1990) have demonstrated that when consensus and type-specific oligonucleotide probes are used in hybridization analysis of amplified products,

as few as 10 copies of HPV can be detected. The investigators have been

able also to amplify many other known genital HPV types including 6, 11,

16, 18, 31, 33, 35, 39, 40, 42, 45, and 51 to 59, as well as a large number of

other HPVs that have not yet been assigned a type.

In the current investigation, we analyzed paraffin-embedded tissue

sections from squamous cell cancers, oral epithelial dysplasias, verrucous

hyperplasias, and oral ST keratoses for the presence of HPV DNA by a PCRbased system, using consensus sequence primers that amplify an approximately 450-bp region of the L1 open reading frame. We also evaluated 18

PCR product bands in the four categories of lesions, using dot blot hybridization to confirm the presence of HPV-16 DNA.

PCR

MATERIALS

AND METHODS

Sections from 44 paraffin-embedded tissue blocks representing

8 squamous cell carcinomas, 11 oral epithelial dysplasias,

9 verrucous hyperplasias, and 16 ST keratoses were examined from the

pathology archives of Western States Regional Oral Pathology Laboratory

and the University of Colorado Oral Pathology Laboratory. All tissue

samples were obtained during 1990. Sections 5 to 10 μm in thickness were

cut from blocks and placed in 0.6-mL capped centrifuge tubes and prepared

for PCR amplification as described by Wright and Manos (1990).

Aliquots (10 μL) of the prepared samples were subjected to 40 cycles of

amplification, with HPV L1 consensus sequence primers MY11 and MY09

with β-globin primers PC04 and GH20 (Perkin-Elmer Cetus, Norwalk,

Connecticut), as previously described (Resnick et al., 1990). Recombinant
Chapter 3

185

plasmid-containing HPV-16 DNA (Oncor, Gaithersburg, Maryland) and

sections from cervical squamous cell carcinoma were used as positive controls. Great care was taken to avoid the possibility of sample contamination

during preparation. Amplification was evaluated by agarose gel electrophoresis (10 μL from each reaction), ethidium bromide staining, and visualization under ultraviolet light. Samples that failed to yield the 268-bp

β-globin amplification product were excluded from the study.

HPV negative controls included tissue samples that had been previously

characterized as negative for HPV DNA. In addition, multiple aqueous

controls were included with each experiment to monitor for the possibility

of reagent contamination.

Dot blot hybridization for HPV-16 was carried out on 18 cases with

BioRad’s Bio-Dot minifold apparatus for six replicate membranes (Sambrook

et al., 1989). DNA oligomer probes were end-labeled with γ-[32P]ATP with

hybridization carried out for 3 h at 56 °C in hybridization solution with

each of the radio-labeled probes. Subsequent autoradiographic exposure was

at -70 °C for 12 to 48 h.

RESULTS Biopsy specimens and positive controls were amplified with HPV L1 consensus primers (Figure 1). HPV amplification products were observed in

smokeless tobacco keratoses, squamous cell carcinomas, oral epithelial

dysplasia, and verrucous hyperplasias. From the total of 30 amplifiable

specimens, 8 showed evidence of infection with HPV. The data reported in

Table 1 represent the specific lesions analyzed and the determination of

whether or not HPV was present. Subsequently, 18 cases representing each

of the four categories of lesions were probed for HPV-16 using residual PCR

product via dot blot hybridization. Of 18 cases, 8 were positive for HPV-16:

3 ST keratoses, 2 squamous cell carcinomas, 2 epithelial dysplasias, and

1 verrucous hyperplasia. Two cases positive by generic probe analysis were

negative for HPV-16 and are currently being analyzed by dot blot for HPV-2,

-4, -6, -11, -18, -31, -33, and -35. The eight HPV-16-positive cases are shown

in Figure 2 in lanes A1, 2, and 3, B5, and C1 through 4.

DISCUSSION A distinct advantage of PCR lies in its ability to make large numbers of

copies of DNA sequences from targets that are present in minute quantities

in the original sample. As a result, the improved L1 consensus sequence

amplification method is significantly more sensitive in detecting amplified

types of HPV in oral tissue samples than in situ hybridization. Although the

PCR method applied here allows a demonstration of only generic HPV, it is

anticipated that we will subsequently be able to determine specific HPV

types through the use of type-specific DNA probes in hybridization analysis

of the PCR products. The increased sensitivity of the PCR method seems to

be critical in detecting HPV in small tissue fragments from the oral cavity

and in suboptimal tissue samples where low-level infections may be present.

One disadvantage of the PCR technique is the potential for obtaining false

positive results because of sample-to-sample contamination, or more importantly, because of the carryover of DNA from previous DNA amplifications

(Bauer et al., 1991). To minimize this risk, we used only single-designateduse pipettes and separated pre- and post-PCR samples and reagents throughout all stages of the investigation.
Smoking and Tobacco Control Monograph No. 2

186

Figure 1

PCR amplification with HPV L1 consensus sequence primers, representative cases. HPV L1

DNA was amplified over 40 cycles. β-Globin primers were included as an amplification

control. Amplified products were analyzed by 4 percent (wt/vol) agarose gel electrophoresis,

stained with ethidium bromide, and photographed under UV light. Molecular weight markers

(Hae III cut pBR322) are shown in the right margin. HPV L1 positive cases (A2-4, B3-6) show

a band at about 450 bp (arrow). β-Globin amplification products show a band at about 268

bp (arrowhead). HPV-16 plasmid DNA positive control (B9) and reagent controls (A11, B10,

11) are also shown.

Although the procedure may improve the likelihood of detecting a wide

variety of HPV sequences, including novel types, it still does not answer the

basic question of whether HPV assumes the function of a tumor promoter,

as suggested by Amtmann (1987), or a causal agent, as suggested by Chow

and associates (1987) and McCance (1988). To accomplish that, investigators must correlate expression of transforming sequences and lesional
Chapter 3

187

Table 1

Identification of HPV in oral biopsy specimens through PCR analysis

Pathological Diagnosis HPVa

Squamous Cell Carcinoma 2/8

Oral Epithelial Dysplasia 1/11

Verrucous Hyperplasia 2/9

ST Keratosis 3/16

a Positive cases over total number of amplifiable cases tested.

presentation with patient historical data, including specific etiologic influences such as alcohol consumption, smoking habits, wart history, racial

origin, age, and sex. While PCR is a highly sensitive technique for the

demonstration of HPV DNA, it provides no information on the functional

state of the viral genome. Investigation of the possible role of HPV in the

neoplastic transformation of the oral mucosa will require an understanding

of the pattern of gene expression in HPV DNA-positive cases. Such an

analysis in the past has been encumbered by considerable problems with

inadequate degrees of sensitivity in the detection of mRNA.

Another question to be answered is whether there is clinical significance in detecting HPV in the oral precancerous and cancerous lesion.

Nuovo and associates (1990), in a study of HPV DNA in penile lesions

histologically negative for condyloma, suggest that lesions with low copy

numbers of the virus may in fact be either exceedingly early or regressing.

They question whether these lesions, which are associated with small copy

numbers of the virus, are truly infectious. The same questions apply to the

oral cavity.

There is little doubt that HPV plays a significant role in the development of mucosal cancer. Accumulated evidence over the last several years

links specific HPV types to the various manifestations of anogenital infection (Bartholoma et al., 1991). The seven most prevalent anogenital HPV

types are HPV-6, -11, -16, -18, -31, -33, and -35. It is reasonable to suggest

that some of these same viruses play a significant role in the development

of oral cancer.

Because it is unlikely that all latent affected sites are eradicated with

treatment, such as incisional biopsy, complete surgical excision, or stripping, it is possible that once someone is infected with HPV, the virus is

harbored for life. It is also likely that HPV infections of the oral mucous

membranes are not completely curable by surgical excision.

Bauer and associates (1991) have suggested that there may be an agespecific prevalence for cervical HPV infection. The same postulate may be

made for HPV-infected oral mucous membranes. Although other genetic,

environmental, and exogenous variables may be implicated in the progression to oral dysplasia or neoplasia, infection with specific HPVs may very

well still be predictive of disease in certain subsets of patients. To determine
Smoking and Tobacco Control Monograph No. 2

188

Figure 2

Dot blot hybridization for HPV-16 with residual PCR product. Eight positive

cases are seen in lanes A1, 2, and 3, B5, and C1 through 4. A1, B5, and C4 are

weakly positive. A2, 3, and C1, 2, and 3 are strongly positive. HPV-16 plasmid

DNA positive control is D1.

whether HPV is responsible for preneoplastic or neoplastic mucosal changes,

long-term prospective studies are needed to aid understanding of the cause

of HPV oral infections in asymptomatic patients, patients who have immune disorders, and patients who are extensive users of exogenous agents

such as tobacco and alcohol.

Clearly, more work is needed to obtain a complete catalog of oral HPV

types. From an epidemiological standpoint, investigators need to determine

the prevalence of infections with multiple HPV types in the oral cavity.

Also, Bauer and associates (1991), in their evaluation of genital HPV infections, have shown that infections with multiple HPV types were underestimated both when a commercially available dot blot kit system was used and

when they used the PCR method. These investigators are currently developing a system that utilizes restriction endonuclease digestion of amplification

products to detect multiple infections.

There is little question that PCR will play a significant role in long-term

epidemiological studies of HPV. PCR offers the advantage of type detection

and high sensitivity in assessing the incidence and prevalence of HPV

infection. Further analysis via PCR is needed to determine the demographic

and behavioral risk factors for HPV infection in subsets of high-risk patients,
Chapter 3

189

such as tobacco and alcohol abusers, patients with immune deficits, and

perhaps even patients with long-term chronic oral infections, including

periodontal disease. Such prospective studies should allow us to more

adequately understand the natural history of HPV infection, its role in

mucous membrane disease, and potential clinical applications of HPV

detection by PCR methods.

Experimental data strongly suggest that, besides the presence of HPV

DNA in cervical epithelium, other events are required for full malignant

conversion of cells infected by so-called oncogenic HPVs. Bosch and coworkers (1990) have suggested that a change in the state of the viral DNA

may be one factor in progression toward malignancy. Lehn and associates

(1988) have suggested that in premalignant lesions of the cervix, viral DNA

is present as an episome, with a tendency toward integration in high-grade

cervical interepithelial neoplasias. Several studies have provided evidence

that HPV-16 and HPV-18 E6 and E7 genes can profoundly influence proliferation and differentiation capacities of rodent and human cells and tissue

culture. It is completely unknown, however, which cellular regulatory

mechanisms are responsible for either downregulation of HPV-18 E6 and E7

genes or growth inhibition of the nontumorigenic hybrid cells in vivo, and

whether these mechanisms are partially or completely different or identical

(Bosch et al., 1990). Future studies are needed to analyze whether there is a

causal relationship between the downregulation of the HPV-18 E6 and E7

gene expression and in vivo growth inhibition of nontumorigenic cells.

Identification of cellular genes that interfere with the expression of HPV

transforming genes will be required. Bosch and others suggest that this will

contribute to a better understanding of the cellular growth-regulatory

networks affected in HPV-associated carcinogenesis (Bosch et al., 1990).

REFERENCES

Amtmann, E., Randeria, J., Wayss, K. Interactions of

papillomaviruses with carcinogens and tumor

promoters. In: Cancer Cells 5, Papillomaviruses,

B.M. Steinberg, J.L. Brandsma, and L.B. Taichman

(Editors). Cold Spring Harbor, NY: Cold Spring

Harbor Laboratory Press, 1987, pp. 259-265.

Bartholoma, N.Y., Adelson, M.D., Forbes, B.A.

Evaluation of two commercially available nucleic

acid hybridization assays for detection and typing

of human papillomavirus in clinical specimens.

American Journal of Clinical Pathology 95: 21-29,

1991.

Bauer, H.M., Ting, Y., Greer, C., et al. Genital human

papillomavirus infection in female university

students as determined by a PCR-based method.

Journal of the American Medical Association 265: 472-

477, 1991.

Bosch, F.X., Schwarz, E., Boukamp, P., et al. Suppression in vivo of human papillomavirus type 18 E6-

E7 gene expression in nontumorigenic hela

fibroblast hybrid cells. Journal of Virology 64: 4743-

4754, 1990.

Bouck, N.P., Good, D.L., Polverini, P.J., et al.

Suppressor control of an inhibitor of angiogenesis.

In: Recessive Oncogenes and Tumor Suppression, W.

Cavenee, N. Hastie, and E. Stanbridge (Editors).

Cold Spring Harbor, NY: Cold Spring Harbor

Laboratory Press, 1989, pp. 179-185.

Chow, L.T., Hirochika, H., Nasseri, M., et al. Human

papillomavirus gene expression. In: Cancer Cells

5, Papillomaviruses, B.M. Steinberg, J.L. Brandsma,

and L.B. Taichman (Editors). Cold Spring Harbor,

NY: Cold Spring Harbor Laboratory Press, 1987,

pp. 55-72.

Elovic, A., Galli, S.J., Weller, P.F., et al. Production

of transforming growth factor-alpha by hamster

eosinophils. American Journal of Pathology 137:

1425-1434, 1990.

Greer, R.O., Douglas, J.M., Breese, L., et al. Evaluation of oral and laryngeal specimens for human

papillomavirus (HPV) DNA by dot blot hybridization. Journal of Oral Pathology and Medicine 19: 35-

38, 1990.
Smoking and Tobacco Control Monograph No. 2

190

Greer, R.O., Eversole, L.R., Crosby, L.K. Detection of

papillomavirus-genomic DNA in oral epithelial

dysplasias, oral smokeless tobacco-associated

leukoplakias, and epithelial malignancies. Journal

of Oral Maxillofacial Surgery 48: 1201-1205, 1990.

Greer, R.O., Eversole, L.R., Poulson, T.C., et al.

Identification of human papillomavirus DNA in

smokeless tobacco-associated keratoses from

juveniles, adults and older adults using immunocytochemical and in situ DNA hybridization

techniques. Gerodontics 3: 201-208, 1987.

Howley, P.M. The role of papillomaviruses in human

cancer. In: Important Advances in Oncology, V.T.

DeVita, Jr., S. Hellman, and J.A. Rosenberg

(Editors). Philadelphia: Lippincott, 1987, pp. 55-

73.

Koutsky, L.A., Galloway, D.A., Holmes, K.K. Epidemiology of genital human papillomavirus

infection. Epidemiology Review 10: 122-163, 1989.

Lehn, H., Villa, F., Marziona, M., et al. Physical state

and biologic activity of human papillomavirus

genomes in precancerous lesions of the female

genital tract. Journal of General Virology 69: 187-

196, 1988.

Manos, M.M., Ting, Y., Wright, D.K., et al. Use of

polymerase chain reaction amplification for the

detection of genital human papillomaviruses. In:

Cancer Cells 7, Molecular Diagnostics of Human

Cancer, M. Furth and M. Greaves (Editors). Cold

Spring Harbor, NY: Cold Spring Harbor Laboratory

Press, 1990, pp. 209-214.

McCance, D.J. Human papilloma virus (HPV)

infections in the aetiology of cervical cancer.

Cancer Survey 7: 499-506, 1988.

Nuovo, G., Hochman, H., Yehuda, D., et al.

Detection of human papillomavirus DNA in

penile lesions histologically negative for condyloma. American Journal of Surgical Pathology 14:

829-836, 1990.

Resnick, R.M., Cornelissen, M.T., Wright, D.K., et al.

Detection and typing of human papillomavirus in

archival cervical cancer specimens by DNA

amplification with consensus primers. Journal of

the National Cancer Institute 82: 1477-1484, 1990.

Sambrook, J., Fritsch, E.F., Maniafis, T. (Editors)

Molecular Cloning: A Laboratory Manual (2nd

edition). Cold Spring Harbor, NY: Cold Spring

Harbor Laboratory Press, 1989, pp. 1051-1957.

Shroyer, K.R., Greer, R.O. Detection of human

papillomavirus DNA by in situ DNA hybridization

and polymerase chain reaction in premalignant

and malignant oral lesions. Oral Surgery, Oral

Medicine, Oral Pathology 71: 708-713, 1991.

Ting, Y., Manos, M.M. Detection and typing of

genital human papillomaviruses. In: PCR

Protocols: A Guide to Methods and Applications, M.

Innis, D. Gelfand, J. Sninsky, and T. White

(Editors). San Diego: Academic Press, 1990, pp.

356-367.

Vousden, K.H. Human papillomaviruses and

cervical carcinoma. Cancer Cells. 1: 43-50, 1989.

Wright, D.K., Manos, M.M. Sample preparation for

paraffin-embedded tissues. In: PCR Protocols: A

Guide to Methods and Applications. M. Innis, D.

Gelfand, J. Sninsky, and T. White (Editors). San

Diego: Academic Press, 1990, pp. 153-158.

zur Hansen, H., Schneider, A. The role of

papillomaviruses in human anogenital cancer. In:

Papoviridae: The Papillomaviruses, volume 2, N.P.

Salzman and P.M. Howley (Editors). New York:

Plenum Press, 1987, pp. 245-264.
Chapter 3

191

Lipids are recognized as major components of or act in concert with components of the

Lipids as Factors in the

Cell Response to Tobacco Components1

George S. Schuster, Scott Lubas, Thomas R. Dirksen, and John F. Erbland

ABSTRACT

intracellular signaling systems that regulate cell growth and behavior. Extracellular modulators, by altering signaling-associated lipids, can affect cell responses. The current study

examines the effects of N'-nitrosonornicotine (NNN), a tobacco-associated nitrosamine, and a

tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA), on cell lipids and on protein

kinase C (PKC) activity in oral epithelial cells. Cells were exposed to NNN or TPA for 30 min

and homogenized, and then their PKC and non-PKC histone phosphotransferase activity was

assessed. NNN significantly increased PKC activity in the cells, especially in the particulate

fraction, whereas TPA produced a slight decrease in activity compared with dimethyl sulfoxide

(DMSO) solvent-treated cells. Both modulators produced increased non-PKC phosphotransferase activity compared with control cells. Lipid synthesis by cells during and after

exposure to the modulators was determined through use of [14C]acetate. Cells treated concurrently with modulator and [14C]acetate showed varied responses, depending on the lipid class

and dose of modulator. Generally both modulators stimulated labeling of phospholipids,

whereas NNN increased and TPA decreased diglyceride labeling. Fatty acid labeling was

decreased by both modulators, whereas triglyceride labeling was enhanced by TPA. Posttreatment labeling showed that modulator-induced changes were transient. The results indicate

that NNN can alter some components of the signaling pathway, notably PKC, and may affect

de novo synthesis of diglycerides, which are putative endogenous promoters.

INTRODUCTION Lipids are recognized as important participants in the responses of

cells to a variety of modulators, that is, substances that alter cell behavior.

Lipids function as components of cell membranes to which the modulators

bind, specifically or nonspecifically; they are components of specific receptors for tumor promoters, and they act as part of or in concert with components of the intracellular signaling systems that are activated after binding

of agents to specific receptors. These signaling pathways regulate cell

growth and behavior. For example, studies have demonstrated that tumor

promoters such as 12-0-tetradecanoylphorbol-13-acetate specifically bind to

receptors. In the case of TPA, these receptors are composed of Ca2+, phospholipid, and protein kinase C (PKC) (Konig et al., 1985). Receptor binding

then results in activation of the intracellular signaling pathways.

Intracellular signaling involves the closely interconnecting activities of

the inositol phosphate-Ca2+ and diacylglycerol (DAG)-PKC pathways, which

have been implicated in the control of cell proliferation. In these pathways

there is activation of certain proteins through phosphorylation of tyrosine

and hydrolysis of phosphatidylinositol-4,5-bisphosphate (PlP2) with formation of 1,2-diacylglycerol and inositol-1,4,5-triphosphate (IP3

). The diglyceride activates PKC, and IP3

 promotes increased cytosolic free calcium.

These latter two activities initiate a cascade of reactions that bring about

1 Funded by Smokeless Tobacco Research Council grant no. 0056 and National Institutes of

Health grant nos. DE-07143 and RR-05795.
Smoking and Tobacco Control Monograph No. 2

192

various cellular responses (Berridge, 1987). Thus any stimuli or cell reactions that affect components of these pathways can in turn alter cell behavior. In addition there are a variety of associated reactions, such as prostaglandin production, that may contribute to the signaling-induced responses (Parker, 1987).

Nitrosamines from smokeless tobacco are carcinogens that may act

independently or in synergy with other agents to induce esophageal, nasal,

and oral tumors (Hecht and Hoffmann, 1988). Indeed, synergy or promotion should be considered critical factors in the activities of some of these

nitrosamines, especially some of the weaker ones (Hecht and Hoffmann,

1988).

Previous studies in our laboratories have shown that modulators of cell

behavior such as retinoic acid (RA), TPA, and the nitrosamine N'-nitrosonornicotine may affect cell lipids and other components of the cell signaling

system, either directly or indirectly. Ringler and associates (1984) demonstrated that RA decreased formation of cholesterol, sphingomyelin, phosphatidylinositol, and phosphatidylserine and increased formation of triglycerides of normal and transformed hamster fibroblast cell types. In hamster

oral epithelial cells, RA also affected lipid synthesis from acetate, decreasing

synthesis of cholesterol and fatty acids but increasing that of triglycerides

and phosphatidylcholine. In these same cells, NNN enhanced acetate

incorporation into phosphatidylcholine and decreased its incorporation

into cholesterol (Schuster et al., 1988). TPA also has been shown to alter cell

lipids in a variety of cell types. For example, TPA altered incorporation and

release of arachidonic acid, especially that associated with the phospholipids

of U937 cells. The pattern of change was to a great extent dependent on the

state of cell differentiation (Wiederhold, 1988).

Additional studies have demonstrated that pretreatment of cells with

one modulator can alter the subsequent cell surface binding of another

modulator. For example, RA decreased binding of phorbol esters to oral

epithelial cells, whereas NNN enhanced binding significantly (Table 1).

These changes occurred at times that corresponded to the altered lipid

synthesis induced by RA (Schuster et al., 1986 and 1988). The opposite has

been shown to occur as well, that is, pretreatment of oral epithelial cells

with TPA enhanced binding of NNN to the cells by 89 percent. These

studies also demonstrated that, although TPA bound to receptors on the

cell, NNN binding did not appear to involve receptors.

The results of these various studies suggested a relation between cell

lipid metabolism and the extent and nature of cell behavior in the presence

of these modulators. They also indicated that nitrosamines and promoters

can interact to alter the cell responses to each other and that these may be

related to their effects on cell lipids. Thus, various cell responses and

perhaps tobacco-related carcinogenesis may be dependent on one agent

altering the response to others. The current studies describe the effects of

NNN and TPA on some components of the signaling system that may be

related to transformation-associated responses.
Chapter 3

193

Table 1

Specific binding of PDB to oral epithelial cells after pretreatment with RA or

NNN (percent control)a

RA NNN

Time of Pretreatment,

4 68b -

24 94 100

48 100 -

72 - 218b

168 - 170b

Source: Schuster et al., 1988.

a Specific binding of [3H]phorbol dibutyrate (PDB) to hamster buccal pouch cells (HCP)

was assayed after pretreatment of the cells for various periods with 10 μM NNN, 10 μM

retinoic acid (RA), or 0.1 percent dimethyl sulfoxide solvent (control). Percentage of

control was calculated based on pmol [3H]PDB specifically bound/106 viable cells.

Results were based on three samples per time point in three to five experiments.

b Different from control (based on pmols/106 cells), p < 0.05.

METHODS

Cells

The cells used were a line of cloned normal hamster buccal pouch (HCP)

cells, the characteristics of which are described elsewhere (Schuster et al.,

1990). The cells were maintained in Dulbecco’s modification of minimal

essential medium (DMEM) supplemented with 5 percent fetal bovine serum

(FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The same lot of

FBS (GIBCO) was used for all studies.

Protein Kinase

C Assay

HCP cell suspensions (2x106 cells) were exposed to 10 μM NNN or

0.1 μM TPA in DMSO such that the final concentration of the

solvent was 0.1 percent. Control cultures contained the same amount of

DMSO. Cells were incubated 30 min at 37 °C in an atmosphere of 95

percent air-5 percent CO2 and then harvested by centrifugation. PKC was

assayed by the methods described by Chida and associates (1988) and Ways

and associates (1987). Cells were suspended in buffer consisting of 20 mM

Tris-HCL (pH 7.4), 2 mM EDTA, 0.5 mM EGTA, 0.25 M sucrose, and

50 mg/mL phenylsulfonyl-methyl chloride (PMSF). They were disrupted by

50 strokes in a glass homogenizer, then centrifuged at 100,000 g for 1 h at

4 °C. The supernatant was separated, and the pellet resuspended in the

same buffer as described above except that it lacked sucrose but contained

0.1 percent Triton X-100.

The final assay mixture consisted of 20 mM Tris-HCL (pH 7.4), 5 mM

MgS04, 200 μg/mL histone (type V-S, Sigma), 10 μM ATP (including

1 μCi γ-[32P]ATP), and 200 μL of enzyme preparation. Activators consisted

of 64 μM phosphatidylserine, diglyceride (1.3 μM 1,2-sn-diolein), and 1 mM

CaCl2. The samples were incubated for 3 min at 30 °C. The reaction was

stopped by the addition of 1 mL of ice-cold 25 percent trichloroacetic acid

(TCA). Samples were filtered through membrane filters (Millipore type HA)

and then washed three times with the TCA. They were then counted in a
Smoking and Tobacco Control Monograph No. 2

194

liquid scintillation counter. Protein content of aliquots was measured by the

Bio-Rad assay. We determined specific activity for PKC by subtracting 32P

incorporation into histone in the absence of activators from that in the

presence of activators. The basal histone phosphotransferase activity (nonPKC) is the 32P incorporated into histone in the absence of activators. Activity was calculated on the amount of protein in duplicate aliquots of cell

supernatant or pellet.

Lipid Assays HCP cells were plated in 100-mm dishes at 5x106 cells/dish. After overnight attachment, the medium was removed by aspiration, and the plates

refed with medium containing TPA (0.1 μM, 0.2 μM, or 0.5 μM), NNN (10

μM, 20 μM, or 50 μM), or DMSO (0.1 percent). The medium also contained

10 μCi/mL [14C]acetate. At the end of 30 min, the media were removed, and

the cells were harvested. An identical set of plates was pretreated for 30 min

with 0.1 μM TPA or 10 μM NNN or 0.1 percent DMSO alone, but no isotope.

After 30 min, the plates were refed with medium containing 1 μCi/mL of

isotope but no modulator. These were incubated for an additional 4 h and

then harvested.

At the end of the respective labeling periods, the cells were washed three

times with saline, dislodged with Teflon scrapers, and harvested by centrifugation. Cell pellets were washed twice with saline, and the lipids extracted

by the method of Bligh and Dyer (1959). Lipids were separated on SG-81

paper by use of the solvent system described by Marinetti (1965) for neutral

lipids and the solvents of Rouser and coworkers (1970) for polar lipids.

Radioactive lipids were located by autoradiography, and the spots cut from

the paper and counted in a liquid scintillation spectrometer. Aliquots of the

cells were assayed for protein as described above, and the number of cells

counted in a hemocytometer. Results were compared by a t test based on

three to five experiments.

PKC RESULTS PKC activity was assayed after 30 min of exposure to the modulator,

when maximum NNN uptake was previously shown (Schuster et al.,

1990). The total PKC activity in cells exposed to NNN was about 1.5 times

that of control cells, 826±67 (SD) cpm/μg protein for NNN-exposed cells vs.

555±84 (SD) cpm/μg protein for control (DMSO-treated) cells (n=12). This

difference was significant (p < 0.05). PKC activity was present in both the

cytosol and particulate fractions, and NNN produced an apparent change in

the distribution of this activity (Table 2). The majority of activity was

present in the particulate fraction, and PKC activity in this fraction was

enhanced in response to NNN; TPA had a minimal effect on distribution at

this point.

Figure 1 shows the specific activity of PKC in the cell fractions after

30 min of modulator exposure. PKC activity was significantly reduced in the

cytosol after treatment with NNN compared with DMSO exposure, whereas it

was significantly increased in the particulate fraction. The increase in

overall as well as percent of activity in the particulate fraction suggests that

the major response to NNN occurs here, not only as de novo synthesis but

also as a shift from one site to the other. After 30 min of incubation, TPA

produced a slight overall decrease in total activity that was not statistically

significant.
Chapter 3

195

Table 2

Enzyme activity in cell fractions after modulator exposurea

Cell Fraction

 Cytosol Solubilized particulate

PKC activityb

Modulator

DMSO 24.8% 75.2%

TPA 16.8 83.2

NNN 2.3 97.7

Non-PKC histone phosphotransferase activityb

DMSO 51.6 48.4

TPA 41.9 58.1

NNN 48.4 51.6

a Percentage of protein kinase C (PKC) activity and non-PKC histone phosphotransferase

activity was assayed in cytosolic and solubilized particulate fractions of hamster buccal

pouch cells (HCP) following exposure of the cells to 10 μM N'-nitrosonornicotine (NNN),

0.l μM 12-0-tetradecanoylphorbol-13-acetate (TPA), or 0.1 percent dimethyl sulfoxide

solvent for 30 min.

b Mean of six experiments.

Non-PKC

Phosphotransferase

Both NNN and TPA did increase the levels of total non-PKC phosphotransferase activity somewhat. This was most notable in cells treated

with NNN, where total activity was increased to 1.5 times that of

DMSO-exposed cells (1,941±98 [SD] cpm/μg protein vs. 1,282±95 [SD] cpm/

μg protein) (n=12). The increase was significant (p < 0.05). This increase in

activity occurred in both cytosol and particulate fractions (Figure 2). The

TPA-produced increase in enzymic activity was less; 1,425±139 (SD) cpm/μg

protein for TPA-treated cells vs. 1,282±95 (SD) cpm/μg protein for DMSOtreated cells (n=12), but this change was evident in only the particulate

fraction (Figure 2). Non-PKC phosphotransferase activity generally was

about evenly distributed between the cytosol and particulate fractions.

NNN did not appear to affect this distribution, whereas TPA caused a modest shift of activity from cytosol to particulate fraction (Table 2).

Diglyceride

Formation

In addition to PKC, diglycerides are major components of cell signaling

pathways and may serve as endogenous promoters. Diglycerides may be

formed from phospholipids via the activities of phospholipases or synthesized de novo by cells. The current studies were directed at the latter

source. Formation of diglycerides and other lipid classes in direct response

to the modulators NNN and TPA was examined through labeling of the cells

in the presence of the modulator or through pretreating of the cells, removing the modulator, and then labeling the cells. The latter study was done to

permit assessment of the longevity of any responses. NNN increased cell

lipid labeling by nearly 15 percent compared with solvent-treated cells,

whereas TPA decreased labeling about 20 percent. This was not the result

of differences in cell number, because direct cell counts and protein
Smoking and Tobacco Control Monograph No. 2

196

Figure 1

The effects of 10 μM NNN, 0.1 μM TPA, or 0.1 percent DMSO on the specific

activity of protein kinase C were measured in cytosolic and solubilized particulate fractions of hamster buccal pouch cells after 30 min exposure to the modulators. Values are the mean of six experiments (±SD).

* p < 0.05 compared with DMSO-treated cells.

determination of aliquots from the samples revealed no significant differences in these parameters among the treatments.

Concurrent treatment and labeling of the cells with modulators and

isotope for 30 min produced various responses at modulator concentrations

equal to or above those previously shown (Schuster et al., 1986) to produce

changes in cell lipids, the response depending on the dose of modulator and

lipid class. Total cell lipid labeling and labeling of various classes by acetate

differed somewhat between experiments; thus, when responses in various

classes are taken as an aggregate of all experiments, some differences are not

statistically significant. However, the labeling of lipid classes at a given

concentration of modulator, compared with solvent-treated control cultures,

was generally very consistent between experiments (Figures 3 and 4). Thus,

phospholipid labeling was uniformly increased by both modulators.

Diglyceride labeling was decreased by TPA (Figure 4) and stimulated at all

except the highest dose of NNN (Figure 3). Cholesterol labeling was
Chapter 3

197

Figure 2

The effects of 10 μM NNN, 0.l μM TPA, or 0.1 percent DMSO on the specific

activity of non-PKC histone phosphotransferase were determined in cytosolic and

solubilized particulate fractions of HCP cells after 30 min exposure to the modulators. Values are the mean of six experiments (±SD).

* p < 0.05 compared with DMSO-treated cells.

moderately but consistently decreased by both modulators, whereas triglyceride labeling was greatly increased and fatty acid labeling decreased by TPA.

NNN decreased labeling of cholesterol esters.

Posttreatment labeling of cell lipids indicated that most of the alterations disappeared within 4 h after modulator removal, although labeling of

phospholipids in TPA-treated cells remained slightly but significantly

elevated as did that of sphingomyelin and glycolipids. At 4 h after NNN

exposure, there was significantly elevated labeling only in steroid precursors.

DISCUSSION Some tobacco-derived substances may affect cell behavior only marginally in normal time or concentration exposures, and cofactors may therefore

be critical for responses such as altered division or transformation to occur.

Many of these cell responses are lipid associated, such as signaling pathways,

or involve lipid-containing components, such as the cell membranes

(Schuster et al., 1986, 1988, and 1990). Therefore, if lipid metabolism is

altered it can affect the cell behavior.
Smoking and Tobacco Control Monograph No. 2

198

Figure 3

Relative effects of various concentrations of NNN on [14C]acetate incorporation

into lipids of HCP cells during 30 min exposure to the modulator plus 10 μCi/mL

[

14C]acetate. Treated cells are compared with cells exposed to 0.1 percent

DMSO, which were given a value of 1.00.

Although signaling pathway components such as PKC are usually

activated by binding of agents to specific receptors, NNN binds nonspecifically (Schuster et al., 1990). However, it does significantly affect specific

activity of both PKC and non-PKC histone phosphotransferase activity. PKC

activity is increased in the particulate fraction, whereas activity in the

cytosol decreases (Table 2 and Figure 1), suggesting that there may be a shift

in activity to the particulate fraction as well as an increase in activity in this

fraction. These results are similar to the shifts seen in MCF-7 cells in response to TPA and the diglyceride 1,2-dioctanoyl-sn-glycerol demonstrated

by Issandou and associates (1988). The TPA-associated decrease in PKC

activity in HCP cells was not significant, but a similar response in U937

cells, as shown by Ways and coworkers (1991), was more extensive, suggesting differences between cell types. The optimal time and dose of exposure

to the NNN and TPA were based on our previous studies with HCP cells as

well as studies by others (Issandou et al., 1988; Schuster et al., 1986; Ways et

al., 1991). The present results demonstrate that changes in PKC activity can

occur in oral epithelial cells in response to NNN, meaning that this nitrosamine and perhaps other modulators that do not bind to receptors are able

to activate this component of the intracellular signaling system.

The significance of the NNN-induced increase in non-PKC phosphotransferase activity is not clear at this time, but Ways and coworkers (1987)

suggest that such activity may mediate other effects produced by cell modulators.
Chapter 3

199

Figure 4

Relative effects of various concentrations of TPA on [14C]acetate incorporation

into cellular lipids during 30 min exposure to the modulator plus 10 μCi/mL

[

14C]acetate. Treated cells are compared with cells exposed to 0.1 percent

DMSO, which were given a value of 1.00.

Cell signaling pathways also involve lipids as direct mediators in the

responses to stimuli (Berridge, 1987). Diglycerides may serve as endogenous

promoters (Rozengurt, 1984). Although phospholipase activity likely is the

major source for diglycerides involved in cell signaling, neosynthesized

diglycerides are important in activation and downregulation of PKC and

altered mitogenic signaling (Chiarugi, 1989a and 1989b; Peter-Riesch et al.,

1988). Those studies showed increased diglyceride synthesis from glucose.

In contrast, significantly increased synthesis from acetate was not evident

in the HCP cells used in our studies, and indeed, TPA caused a significant

decrease in diglycerides from this source. This may be a result of specific

metabolic patterns of the HCP cell type; that is, the pathways used for

neosynthesis of diglycerides from acetate are not affected by NNN and are

downregulated by TPA. HCP cells apparently do not synthesize significant

quantities of lipids from radiolabeled glucose, even in media totally lacking

nonlabeled glucose (unpublished observations). Therefore, other pathways

for lipid production in these cells will have to be examined for their response to the various modulators, because others also have shown cellspecific differences (Chida et al., 1988; Harris et al., 1982; Hecht and

Hoffmann, 1988.) The changes in diglyceride formation appear to be

transient because prior studies with longer periods of TPA exposure and

labeling (Schuster et al., 1990) as well as the current posttreatment labeling

showed no differences from control cells.

Altered labeling of lipid classes other than the diglycerides during the

short term may relate to cells adjusting to the binding or embedment of the

modulators in the cell membrane. Frezard and coworkers (1989) found that,
Smoking and Tobacco Control Monograph No. 2

200

when TPA binds to cells, it becomes embedded in the cytoplasmic membranes. NNN does not bind to receptors but likely will at least transiently

affect membrane organization. Therefore, the changes seen in phospholipids and fatty acids may reflect the cells’ adjustment to the presence of

the modulators, a result consistent with previous studies showing TPAassociated increases in 18:2, 18:3, 20:3, and 20:4 fatty acids in these cells

(Schuster et al., 1990).

Although incorporation of acetate into cell lipids varied between

experiments, the patterns of response to modulators are consistent. The

variation may be the result of the nature of the HCP cells in culture. The

cells were plated so as to be loose monoloyers when used. However, even

when thoroughly dispersed for plating, these epithelial cells would tend to

stick together and settle to the surface of the dish in patches, resulting in

areas where the cells may be locally dense. In such a situation the cells in

the center of the clusters may be less active in their growth and synthetic

activities, including their responses to the modulators. In general, metabolic or growth rate-associated responses are likely to be affected by culture

density, resulting in interexperiment variability. Cell regulatory characteristics that have been shown to be density dependent include cyclic nucleotide

levels, availability of various receptors, and lipid metabolic pathways

(D’Armiento et al., 1973; Holley et al., 1978; Jetten et al., 1989; Ponec et al.,

1987).

The previous and current studies taken together suggest that NNN and

TPA can directly or indirectly affect the cell signaling pathways, including

some of the lipid components. They also suggest that the combined effects

of modulators may be a significant factor in the response of oral epithelial

cells to initiators and promoters. Finally, the results suggest that these cells

may differ from other cell types in some ways, necessitating the use of oral

epithelial cells to fully define responses to tobacco exposure.

REFERENCES

Berridge, M.J. Inositol lipids and cell proliferation.

Biochimica et Biophysica Acta 907: 33-45, 1987.

Bligh, E.G., Dyer, W.J. A rapid method of total lipid

extraction and purification. Canadian Journal of

Biochemistry and Physiology 37: 911-917, 1959.

Chiarugi, V., Bruni, P., Pasquali, F., et al. Synthesis of

diacylglycerol de novo is responsible for permanent

activation and down-regulation of protein kinase C

in transformed cells. Biochemical and Biophysical

Research Communications 164: 816-823, 1989b.

Chiarugi, V.P., Magnelli, L., Pasquali, F., et al. Signal

transduction in EF-J-ras-transformed cells: de novo

synthesis of diacylglycerol and subversion of

agonist-stimulated inositol lipid metabolism. FEBS

Letters 252: 129-134, 1989.

Chida, K., Kato, N., Yamada, S., et al. Protein kinase

C activities and bindings of a phorbol ester in 41

cell lines. Biochemical and Biophysical Research

Communications 157: 1-8, 1988.

D’Armiento, M., Johnson, G.S., Paston, I. Cyclic

AMP and growth of fibroblasts: Effect of environmental pH. Nature New Biology 242: 78-80, 1973.

Frezard, F., Garnier-Suillerot, A., Ballard, J., et al.

Membrane-phorbol ester interaction monitored by

circular dichroism. Biochimica et Biophysica Acta

979: 316-320, 1989.

Harris, C.C., Trump, B.F., Grafstrom, R., et al.

Differences in metabolism of chemical carcinogens

in cultured human epithelial cells. Journal of

Cellular Biochemistry 18: 285-294, 1982.

Hecht, S.S., Hoffmann, D. Tobacco-specific nitrosamines: An important group of carcinogens in

tobacco and tobacco smoke. Carcinogenesis 9: 875-

884, 1988.

Holley, R.W., Armour, R., Baldwin, J.H. Densitydependent regulation of growth of BSC-1 cells in

cell culture: Growth inhibitors formed by the cells.

Proceedings of the National Academy of Sciences 75:

1864-1866, 1978.
Chapter 3

201

Issandou, M., Bayard, F., Darbon, J.M. Inhibition of

MCF-7 cell growth by 12-0-tetradecanoylphorbol13-acetate and 1,2-dioctanoyl-sn-glycerol: Distinct

effects on protein kinase C activity. Cancer Research

48: 6943-6950, 1988.

Jetten, A.M., George, M.A., Nervi, C., et al. Increased

cholesterol sulfate and cholesterol sulfotransferase

activity in relation to the multi-step process of

differentiation in human epidermal keratinocytes.

Journal of Investigative Dermatology 92: 203-209,

1989.

Konig, B., DiNitto, P.A., Blumberg, P.M. Phospholipid and Ca2+ dependency of phorbol ester

receptors. Journal of Cellular Biochemistry 27: 255-

265, 1985.

Marinetti, G.V. Chromatography of lipids on

commercial silica gel loaded filter paper. Journal of

Lipid Research 6: 315-317, 1965.

Parker, J., Daniel, L.W., Waite, M. Evidence of

protein kinase C involvement in phorbol diesterstimulated arachidonic acid release and prostaglandin synthesis. Journal of Biological Chemistry

286: 5385-5393, 1987.

Peter-Riesch, B., Fathi, M., Schlegel, W., et al.

Glucose and carbachol generate 1,2-diacyl-glycerol

by different mechanisms in pancreatic islets.

Journal of Clinical Investigations 81: 1154-1161,

1988.

Ponec, M., Kempenaar, J., Boonstra, J. Regulation of

lipid synthesis in relation to keratinocyte differentiation capacity. Biochimica et Biophysica Acta 921:

512-521, 1987.

Ringler, M.B., Erbland, J.F., Singh, B.B., et al. Effects

of retinoic acid on 14C-acetate incorporation into

lipids of normal and transformed hamster fibroblasts. Experimental Cell Research 154: 171-180,

1984.

Rouser, G., Fleischer, S., Yamamoto, A. Twodimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5:

494-496, 1970.

Rozengurt, E., Rodriguez-Pena, A., Coombs, M., et al.

Diacylglycerol stimulates DNA synthesis and cell

division in mouse 3T3 cells: Role of Ca2+-sensitive

phospholipid-dependent protein kinase. Proceedings of the National Academy of Sciences 81: 5748-

5752, 1984.

Schuster, G.S., Erbland, J.F., Wyrick, S.D., et al. Oral

epithelial cell lipid synthesis in the presence of

retinoic acid or nitrosonornicotine. Journal of Oral

Pathology 15: 430-433, 1986.

Schuster, G.S., Erbland, J.F., Wyrick, S.D., et al.

Phorbol ester binding to oral epithelial cells in the

presence of retinoic acid on N'-nitrosonornicotine.

Cytobios 54: 53-60, 1988.

Schuster, G.S., Lubas, S., Erbland, J.F., et al. Binding

and uptake of N'-nitrosonornicotine by oral

epithelial cells. Journal of Oral Pathology and

Medicine 19: 114-118, 1990.

Ways, K.D., Dodd, R.C., Earp, H.S. Dissimilar effects

of phorbol ester and diacylglycerol derivatives on

protein kinase activity in the monoblastoid U937

cell. Cancer Research 47: 3344-3350, 1987.

Ways, K., Riddle, R., Ways, M., et al. Effect of

phorbol esters on cytosolic protein kinase C:

Content and activity in the human monoblastoid

U937 cell. Journal of Biological Chemistry 266: 1258-

1264, 1991.

Wiederhold, M.D., Anderson, K.M., Harris, J.E.

Labeling of lipids and phospholipids with 3Harachidonic acid and the biosynthesis of

eicosanoids in U937 cells differentiated by phorbol

ester. Biochimica et Biophysica Acta 959: 296-304,

1988.
Smoking and Tobacco Control Monograph No. 2

202

The role of herpes simplex virus and human papillomavirus in oral carcinogenesis was

Role of Viruses in Oral Carcinogenesis1

No-Hee Park, Byung-Moo Min, Sheng-Lin Li,

Henry M. Cherrick, and Jay Doniger

ABSTRACT

studied. Herpes simplex virus showed no carcinogenicity in vivo; repeated viral inoculation of

hamster buccal pouch mucosa failed to produce tumors or histopathologic evidence of

malignancy in pouches. However, herpes simplex virus demonstrated in vivo cocarcinogenicity; viral inoculation significantly enhanced the oncogenic capacity of benzo[a]pyrene, a

tobacco-chemical carcinogen, in the oral cavity of hamsters. Human papillomavirus types 16

and 18 demonstrated oncogenicity by transforming normal human oral keratinocytes. While

normal cells exhibited a limited lifespan, cells transformed by these viruses showed immortality

and altered morphology in comparison with their normal counterparts. The transformed HOK16A and B and HGK-18 cells contained intact type 16 or 18 human papillomaviral DNA

integrated into cellular chromosomes, respectively. Further, these cells expressed several viralspecific poly(A+)RNAs including viral E6/E7 polyadenylated RNAs. Notably, these cells

overexpressed cellular myc proto-oncogene compared to their normal counterparts; however,

the immortalized cell lines were not able to produce tumors in nude mice, indicating that the

cells are only partially transformed.

INTRODUCTION Clearly cigarette, cigar, and pipe smoking are causally associated

with oral cancer (US DHHS, 1982) as is snuff dipping (US DHHS, 1986).

The constituents of smoked tobacco tar and smokeless tobacco responsible

for oral cancer are the tobacco-specific N'-nitrosamines (TSNA) and

benzo[a]pyrene (B[a]P). TSNA are formed from nicotine and minor tobacco

alkaloids during aging, curing, and fermentation of tobacco (Hecht et al.,

1977). Among TSNA, high levels of N'-nitrosonornicotine (NNN) and

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are found in tobacco

(Hoffmann et al., 1984). Some studies, however, indicate a lack of linkage

between malignant changes and ST use by humans (Offenbacher and

Weathers, 1985). Moreover, several laboratory studies report failure to

develop oral malignancy with repeated intraoral placement of smokeless

tobacco in animals (Park et al., 1985; Shklar et al., 1985). Possible involvement of other factors such as alcohol, caffeine, and viruses has therefore

been postulated to be associated with development of tobacco-related oral

malignancies in humans. Among these factors, the role of viruses, especially

herpes simplex virus (HSV) and human papillomavirus (HPV), in oral

carcinogenesis has been studied in our laboratory.

Oral cancers appear to be associated with an increased immune response to HSV-1 (Shillitoe et al., 1982). The expression of HSV-1 genes has

been detected in oral cancer tissues (Eglin et al., 1983). HSV infection is

extremely prevalent, with up to 90 percent of individuals having antibodies

to HSV by age 10 (Overall, 1979). More than one-third of the world’s

population suffers from recurrent intraoral or orofacial herpetic infections

(National Institutes of Health, 1973). Individuals with latent HSV infection

in the sensory or autonomic ganglia actively shed infectious virions onto

1

 Supported in part by Smokeless Tobacco Research Council grants no. 0052 and no. 0231.
Chapter 3

203

oral mucosae, yet are without clinical symptoms, providing an opportunity

for HSV to interact with water-soluble components of smokeless tobacco in

the oral cavity. Tobacco and HSV are synergistic in developing precancerous

lesions in mice (Park et al., 1985). Furthermore, repeated HSV infection in

combination with simulated snuff dipping leads to oral cancer in animals

(Hirsch et al., 1983; Park et al., 1985). Also, HSV-1 infection significantly

increases the carcinogenic activity of 7,12-dimethylbenz[a]anthracene

(DMBA) in hamster buccal pouch mucosa by, in part, accelerating DMBAinduced activation of c-erb-B-1 proto-oncogene in the pouch epithelium (Oh

et al., 1989).

Human papillomavirus (HPV) is also linked to certain human malignancies. This association is based on the finding that up to 90 percent of cancer

tissues from genital lesions contain viral DNA (Durst et al., 1983). Of the

more than 60 genotypes of HPV, types 16 (HPV-16) and 18 (HPV-18), as well

as recently isolated types 31 (HPV-31) and 33 (HPV-33), are most frequently

associated with cervical cancer (Schwarz et al., 1985). In a high percentage

of cervical carcinomas and in cell lines derived from these cancers, HPV-16

and -18 DNAs are integrated into cellular chromosomes, whereas the viral

DNAs are generally retained as extrachromosomal episomes in premalignant

dysplastic lesions (McCance, 1986). As in cervical cancers, HPV is also

positively correlated with human oral malignancies, with up to 60 percent

of cancer tissues from oral biopsies containing viral DNA (Dekemezian et al.,

1987). Since the epithelia of oral and female genital mucosae are histologically similar, and both are continuously challenged by many environmental

factors, close association of HPV with the development of oral malignancies

is not surprising. Although human oral keratinocytes are undeniably major

target cells for HPV infection and HPV-induced tumorigenesis, the in vitro

transforming activity of HPV in human oral keratinocytes has never been

studied because of the unavailability of a suitable culture system.

In the present study, we demonstrate the in vivo cocarcinogenic effect

of HSV in the oral cavity of hamsters, and the carcinogenicity of HPV for

one of its target cells, the oral keratinocytes.

METHODS

Viruses, Plasmids,

And Primary

Culture of Oral

Keratinocytes

HSV-1 (F-strain; American Type Culture Collection, Rockville,

Maryland) was propagated in Vero cell monolayers with viral

titers being adjusted to 108

 plaque-forming units (PFU) per

milliliter. pMHPV-16d (a head-to-tail dimer of HPV-16 DNA

inserted into the BamHI cloning site of the plasmid pdMMTneo)

and pSHPV-18m (recombinant DNA containing single copy of

HPV-18 DNA inserted into EcoRI cloning site of plasmid PSV2neo) were

constructed as described elsewhere (Woodworth et al., 1989; Park et al.,

1991). The primary normal human oral keratinocytes (NHOK) and normal

human gingival keratinocytes (NHGK) were established from the excised

tissues of hard palate and retromolar areas of a healthy male volunteer as

described elsewhere (Park et al., 1991).
Smoking and Tobacco Control Monograph No. 2

204

To investigate the in vivo carcinogenicity of NNN, NNK, and

B[a]P, alone or in combination with HSV-1 inoculation, we

inoculated the right buccal pouch mucosa with HSV-1 or culture

medium (mock inoculation), as described elsewhere (Park et al.,

Animals, HSV-1

Inoculation, and

Application of

Carcinogens

1988), and applied either mineral oil or chemical carcinogens topically as

follows: group 1 (control), mock inoculation plus topical application of

mineral oil; group 2, HSV-1 inoculation plus topical application of mineral

oil; group 3, mock inoculation plus topical application of 1 percent NNK;

group 4, HSV-1 inoculation plus topical application of 1 percent NNK;

group 5, mock inoculation plus topical application of 1 percent NNN;

group 6, HSV-1 inoculation plus topical application of 1 percent NNN;

group 7, mock inoculation plus topical application of 1 percent B[a]P;

group 8, HSV-1 inoculation plus topical application of 1 percent B[a]P.

Approximately 100 μL of mineral oil, NNK, NNN, or B[a]P solution were

applied as described in Table 1. One hundred sixty hamsters were divided

into eight equal groups, and right pouch mucosae were inoculated with

HSV-1 (1x108

 PFU per pouch) or culture medium (mock inoculation).

Twenty-four hours after the inoculation, mineral oil, NNK, NNN, or B[a]P

was topically applied to the inoculated pouch mucosa, three times per week

for 15 (for B[a]P) or 20 (for mineral oil, NNK, and NNN) consecutive weeks.

Left pouches were used as internal controls. Since our preliminary study

showed that mild premalignant microscopic changes were developed by 15

wk and 20 wk of treatment of topical B[a]P and TSNA (NNN and NNK),

respectively, in hamster buccal pouches, the chemical carcinogens were

applied for 15 or 20 wk to demonstrate the cocarcinogenicity of HSV-1. The

pouches were examined once a week for the appearance of tumors, and the

animals were sacrificed at 30 wk after the initiation of topical application of

mineral oil or chemical carcinogens. The above data were obtained just

before the animals were sacrificed. Of animals receiving both HSV-1 inoculation and topical B[a]P, two hamsters died of HSV-1 encephalitis at 2 wk

after inoculation. No changes were observed from the left pouch mucosae

used as internal controls. At the end of the experimental period, the animals were sacrificed and the buccal pouches were fixed for light microscopic

examination. Although NNN and NNK are hydrophilic compounds, they

were dissolved in mineral oil to enhance the penetration of the compounds

into tissue.

Transformation

Of Primary NHOK

And NHGK With

HPV-16 and HPV-18

Primary NHOK and NHGK were transfected with pMHPV-16d

(or pdMMTneo) and pSHPV-18m (or pSV2neo ) using Lipofectin

reagent (BRL Life Technologies, Gaithersburg, Maryland). Two

G418-resistant cell colonies transfected with pMHPV-16d were

isolated, subcultured, and named HOK-16A and HOK-16B lines. One cell

colony transfected with pSHPV-18m was isolated and named HGK-18 (Park

et al., 1991).

Analysis of Cellular

DNA and RNA From

HPV-Transformed

Oral Keratinocytes

High molecular weight cellular DNA from NHOK, NHGK, HOK16A, HOK-16B, and HGK-18 lines was extracted. To determine

the presence of viral DNA and, if present, its copy number

per cell, cellular DNA were restricted and electrophoresed in

1 percent agarose, and Southern blot hybridization carried out under stringent condition using 32P-labeled 7.9-kbp total HPV-16 DNA or total HPV-18
Chapter 3

205

Table 1

Effect of tobacco-related chemical carcinogens and HSV-1, alone or in combination, on the

development of oral cancer in hamster buccal pouches

Effect on Right Buccal Pouches

Average Average Average

Number Number Number Number of Tumor Size Cumulative

of Pouches of Pouches of Discrete Tumors per (mm dia- Tumor Diameter

per Group With Tumors Tumors Pouch meter)a per Pouch(mm)a

Experimental

Groupb

1. Control

(mock

inoculation

+ TA of

mineral oil) 20 0 0 0 NA NA

2. HSV-1

inoculation

+ TA of

mineral oil 20 0 0 0 NA NA

3. Mock

inoculation

+ TA of 1%

NNK 20 0 0 0 NA NA

4. HSV-1

inoculation

+ TA of 1%

NNK 20 0 0 0 NA NA

5. Mock

inoculation

+ TA of 1%

NNN 20 0 0 0 NA NA

6. HSV-1

inoculation

+ TA of 1%

NNN 20 0 0 0 NA NA

7. Mock

inoculation

+ TA of 1%

B[a]P 20 4 6 1.5 0.5 0.75 ± 0.40

8. HSV-1

inoculation

+ TA of 1%

B[a]P 18 10c 30c 3.0c 0.8d 2.4 ± 0.5d

a NA: not applicable.

b TA: topical application.

c Significantly different (p < 0.05) from group 7 (Fisher’s exact test, double tailed).

d Significantly different (p < 0.05) from group 7 (Student’s t test, double pair).
Smoking and Tobacco Control Monograph No. 2

206

DNA probes. After hybridization, the filter was washed and exposed to

X-ray film. The physical state of viral DNA in the HOK-16A, HOK-16B, and

HGK-18 lines was determined by Southern blot hybridization analysis as

indicated in the figure legends. Northern blot hybridization was performed

to determine expression of HPV DNA, c-myc proto-oncogene, and β-actin

gene in the transformed cells (Park et al., 1991).

In Vivo Tumorigenicity of HPVTransformed Oral

Keratinocytes

NHOK, NHGK, HOK-16A, HOK-16B, and HGK-18 monolayer

cultures were trypsinized, resuspended in PBS, and subcutaneously injected into 25 athymic nude mice (nu/nu; 1x107

 cells/

0.1 mL per animal; five animals per cell type) 1 d after mice had

been X-irradiated (300R). All mice were injected on the right flank and

monitored twice weekly for the appearance of tumors over a period of more

than 3 mo.

RESULTS

Cocarcinogenicity

Of HSV-1 in B[a]PInduced Oral

Cancer

As we reported previously (Park et al., 1988), repeated HSV-1

inoculation induces neither tumors nor histopathologic changes

in hamster buccal pouch mucosa. The present data also show

that tumors or malignant histopathologic changes do not develop in the buccal pouch mucosa of animals receiving mineral

oil, NNN, or NNK, alone or in combination with repeated HSV-1

inoculation. The body weights of animals were not altered by HSV-1

inoculation and/or topical application of chemical carcinogens. However,

20 percent of hamsters receiving topical B[a]P along with mock inoculation

developed tumors in the right pouches, while 56 percent of animals treated

with topical B[a]P and HSV-1 inoculation developed tumors in the right

pouch mucosa (Table 1). HSV-1 inoculation also significantly hastened the

appearance of B[a]P-induced tumor formation. The tumors appeared 16 wk

after the initiation of topical B[a]P in animals receiving B[a]P plus mock

inoculation, but the tumors occurred 11 to 12 wk after initiation of B[a]P

treatment in animals treated with both topical B[a]P and HSV-1 inoculation.

The average size of tumors in animals receiving both HSV-1 and B[a]P was

significantly greater than in those receiving B[a]P treatment with mock

inoculation (Table 1).

Microscopic findings also show that HSV-1 inoculation induced more

malignant histopathologic changes in pouch mucosa receiving B[a]P. A

significantly higher number of pouches showed epithelial atypia and cancer

invasion in the group receiving both HSV-1 inoculation and topical B[a]P,

than in the group receiving mock inoculation and topical B[a]P (Figure 27-1

and Table 2). These data indicate that NNN and NNK are not carcinogenic

in hamster buccal pouches and that HSV-1 does not alter the noncarcinogenicity of these compounds in the pouch epithelium. B[a]P, alone, demonstrated weak carcinogenicity in buccal pouches, an effect that was significantly increased by HSV-1. These results also confirm that HSV-1 alone is

not carcinogenic, but can selectively enhance the oncogenicity of certain

chemical carcinogens in the oral cavity of hamsters.
Chapter 3

207

Table 2

Effect of tobacco-related chemical carcinogens and HSV-1, alone or in combination, on the

histopathologic changes of hamster buccal pouch mucosa

Histopathologic Changes of Right Pouch Mucosaa

Inflammatory Invasive

Granular Infiltrate Squamous

Hyper- Cell Layer in Lamina Epithelial Cell

keratosis Hyperplasia Acanthosis Propria Atypia Carcinoma

Experimental

Groupb

1. Control (mock

inoculation

+ TA of

mineral oil) 0/20 0/20 0/20 0/20 0/20 0/20

2. HSV-1

inoculation

+ TA of

mineral oil 0/20 0/20 0/20 0/20 0/20 0/20

3. Mock

inoculation +

TA of 1% NNK 10/20 5/20 5/20 0/20 0/20 0/20

4. HSV-1

inoculation +

TA of 1% NNK 9/20 6/20 6/20 0/20 0/20 0/20

5. Mock

inoculation +

TA of 1% NNN 11/20 5/20 7/20 0/20 0/20 0/20

6. HSV-1

inoculation +

TA of 1% NNN 10/20 7/20 6/20 0/20 0/20 0/20

7. Mock

inoculation +

TA of 1% B[a]P 11/20 9/20 9/20 7/20 4/20 1/20

8. HSV-1

inoculation +

TA of 1% B[a]P 12/18 10/18 12/18 15/18c 10/18c 4/18c

a The excised pouch tissues were fixed in 10% neutral formalin, sectioned in paraffin, and stained with

hematoxylin and eosin for light microscopic findings. Numerator is the number of pouches with the

described histopathologic changes; denominator is the number of pouches examined.

b TA: topical application.

c Significantly different (p < 0.05) from group 7 (Fisher’s exact test, double tailed).

Proliferation

Pattern and Morphology of Oral

Keratinocytes

NHOK, NHGK, and G418-resistant cell colonies transfected with

the vector plasmids (pdMMTneo or pSV2neo) were similar in their

morphology and could not be subcultured beyond the fifth to

sixth passage. The G418-resistant cell colonies transfected with

recombinant pMHPV-16d or pSHPV-18m plasmids, however, appear to be

immortal, these cells have now been maintained through 40 passages over
Smoking and Tobacco Control Monograph No. 2

208

Figure 1

Microphotographs representing the induction of hyperkeratosis, hyperplasia,

acanthosis, carcinoma in situ, and invasive cancer from the hamster buccal

pouches. A., control; B., hyperplasia and hyperkeratosis; C. and D., acanthosis

and hyperkeratosis; E., carcinoma in situ; F., invasive cancer (original magnification x100).

8 mo. The cells display keratinocyte morphology and are characterized by a

lack of stratification. These cells continue to proliferate and to retain an

undifferentiated morphology. The transformed cell lines proliferate faster

than NHOK or NHGK, have a cobblestone-like morphology, and establish a

higher density at confluence in comparison with the normal counterpart.

Viral DNA in HPVTransformed Oral

Keratinocytes

DNA from NHOK and NHGK did not hybridize to HPV-16 DNA

and HPV-18 DNA, respectively, indicating that NHOK and

NHGK did not contain HPV-16 and HPV-18 DNA, respectively.

DNA from HOK-16A, HOK-16B, and HGK-18 cell lines hybridized to the

viral probes, suggesting the presence of viral DNA in the immortalized cell

lines. Densitometric analysis showed that HOK-16A and HOK-16B cell lines
Chapter 3

209

contain approximately 40 and 25 copies of HPV-16 DNA per cell, respectively, while the HGK-18 cell line harbors about 10 copies of HPV-18 DNA

per cell (Figures 2 and 3).

After digestion of HOK-16A and HOK-16B cellular DNA with EcoRV, an

enzyme that does not cut the pMHPV-16d plasmid, Southern blot analysis

showed a single HPV-16-specific band, larger than 30 kbp, suggesting that

HPV DNA exists as an integrated form, not an episomal form, in the HOK16A and -16B cell lines. After BamHI digestion, which releases the HPV-16

DNA sequences from vector DNA, Southern analysis showed that HOK-16A

and -16B cell lines contained 7.9-kbp HPV-16 DNA genome, indicating an

integration of intact HPV-16 DNA into host chromosomes in these cell lines.

In addition to the expected 7.9-kbp complete HPV-16 DNA genome, the cell

lines contained rearranged HPV-16 DNA sequences; the hybridization of

HOK-16A and -16B DNA digested with BamHI showed multiple HPV-16-

specific bands that were bigger or smaller than 7.9-kbp (Figure 27-2). Similar to HOK-16A and -16B lines, HGK-18 DNA also contained intact and

integrated HPV-18 DNA; after EcoRI digestion, which releases HPV-18 DNA

from vector, Southern analysis showed 7.9-kbp band hybridized to 32P-HPV18 DNA. After digestion with SalI, an enzyme that does not cut the pSHPV18m plasmid, a single HPV-18-specific band larger than 30 kbp was seen,

suggesting that HPV-18 DNA exists as an integrated form in the HGK-18 cell

line (Figure 3).

To further assess the integration of rearranged viral sequences into

cellular genomic DNA, high molecular weight cellular DNA from the transformed, immortalized cell lines was double digested with two enzymes:

BamHI and EcoRV (for HOK-16A and HOK-16B DNA) or EcoRI and SalI (for

HGK-18 DNA). This treatment can generate smaller, viral-specific, rearranged DNA fragment(s) if EcoRV or SalI digestion sites exist in cellular

DNA covalently linked to viral DNA. The double restriction profiles of

HOK-16A and HGK-18 DNA were similar to BamHI and EcoRI restriction

profiles of the DNA, respectively, indicating no rearranged fragments with

junctions between cellular and viral DNA (Figures 2 and 3). This does not

rule out integration, because EcoRV or SalI restriction sites do not necessarily

exist in the rearranged HPV-16-specific BamHI fragments or HPV-18-specific

EcoRI fragments in HOK-16A DNA and HGK-18 DNA, respectively. However, the double digestion of HOK-16B DNA generated smaller rearranged

HPV-16-specific DNA fragments than were observed after the single BamHI

digestion (Figure 2 and 3), providing conclusive evidence for integration

into this line.

Northern blot hybridization using 32P-HPV-16 DNA probe revealed that

multiple HPV-16 poly(A+)RNAs were highly expressed from the HOK-16A

and HOK-16B cell lines, whereas HPV-16 poly(A+

)RNAs were not expressed

from NHOK (Figure 4). The intense 1.6-1.8-kbp band is characteristic of the

major HPV-16 E6/E7 message observed in HPV-16 immortalized human

keratinocyte and cervical epithelial cell lines (Figure 3). The HGK-18 cell line

also abundantly expressed HPV-18 E6/E7 polyadenylated RNAs (Figure 5).
Smoking and Tobacco Control Monograph No. 2

210

Figure 2

Southern blot hybridization analysis of cellular DNA of NHOK, HOK-16A, and

HOK-16B.

A: Determination of the presence and copy numbers of HPV-16 DNA per cell in HOK16A and HOK-16B cell lines. HPV-16 DNA (corresponding to 5, 25, and 125 copies of

viral DNA per cell which were mixed with carrier DNA [BamHI-digested 10 μg of NHOK

DNA]) and BamHI-digested cellular DNA (10 μg) extracted from NHOK, HOK-16A, and

HOK-16B were electrophoresed in 1% agarose gel. The fragmented DNA was then

transferred to a nitrocellulose filter and hybridized to 32P-labeled 7.9-kbp total HPV-16

DNA. The filter was washed and exposed to Kodak SB-5 X-ray film.

B: Determination of physical state of HPV-16 DNA in HOK-16A and HOK-16B cell lines.

10 μg of high molecular weight cellular DNA were digested with BamHI (B) and/or

EcoRV (E) restriction enzymes. BamHI enzyme separates vector from HPV-16

sequences, while EcoRV does not digest pMHPV-16d. The fragmented DNA was then

transferred to nitrocellulose filter and hybridized to 32P-labeled 7.9-kbp HPV-16 DNA.

The filter was washed and exposed to X-ray film.

Source: Park et al., 1991; used with permission.

Figures 4 and 5 show cellular myc proto-oncogene and β-actin gene

poly(A+)RNAs transcribed from NHOK, NHGK, and the immortalized

cell lines. There are three polyadenylated, hybridized c-myc RNAs

whose sizes are 5.2-kb, 2.4-kb, and 1.1-kb. Expression of the myc gene

Expression of

c-myc Protooncogene and

β-Actin Gene

from the HOK-16A, HOK-16B, and HGK-18 cell lines was notably higher

than that of the normal counterpart. The expression patterns of β-actin

gene from NHOK, NHGK, HOK-16A, HOK-16B, and HGK-18 cell lines were

somewhat similar and active. All cells expressed 2.0-kb mRNA in a similar

manner, indicating that the cells were metabolically active (Figures 4 and 5).
Chapter 3

211

Figure 3

Determination of the presence and physical state of HPV-18 DNA in HGK-18 and

HeLa cell lines. HPV-18 DNA (corresponding to 30 copies of viral DNA per cell

which were mixed with carrier DNA [EcoRI-digested 10 μg of NHGK DNA]) and

SalI (S) and/or EcoRI (E)-digested NHGK, HGK-18, HeLa cell DNAs were electrophoresed in 1% agarose gel. EcoRI enzyme separates vector from HPV-18

sequences, while SalI does not digest pSHPV-18m. The fragmented DNA was

transferred to a nitrocellulose filter and hybridized to 32P-labeled 7.9-kbp total

HPV-18 DNA. The filter was washed and exposed to X-ray film.

The immortalized HOK-16A, HOK-16B, and HGK-18 cell lines

were tested for tumorigenicity in nude mice. Mice injected with

either the immortalized cells or normal cells did not develop

Tumorigenicity of

HPV-Transformed

Cells

tumors after being monitored for more than 3 mo.

DISCUSSION Present data show that HSV-1 enhances the carcinogenicity of B[a]P, a

tobacco-chemical carcinogen. The exact mechanism of HSV-1 cocarcinogenicity remains speculative, but HSV-1 has been proposed to stimulate the

oncogenicity of chemical carcinogens by impairing the immunologic

response of the host, by interfering with cellular chemical detoxification, by

altering target cell permeability, or by causing proliferation of latent tumor

cells (Casto and DiPaolo, 1973). Since HSV infection has been associated

with chromosomal aberrations, mutations, and selective DNA amplification,

HSV might alter cellular DNA, making interaction with B[a]P more favorable. However, rigorous laboratory investigations must be carried out to

elucidate further the mechanism of HSV cocarcinogenicity.

Our results provide evidence that HPV-16 and HPV-18 participate in the

carcinogenesis in one of their in vivo target cells, oral keratinocytes. NHOK

and NHGK exhibited a limited in vitro lifespan, terminally differentiating

after five to six passages. Liposomal transfection of these primary oral

keratinocytes with pdMMTneo or pSV2neo plasmid did not extend their
Smoking and Tobacco Control Monograph No. 2

212

Figure 4

Northern blot hybridization analysis of poly(A+)RNAs of NHOK, HOK-16A, and

HOK-16B cell lines

A: Determination of the expression of HPV-16 from HOK-16A and HOK-16B cell lines.

Poly(A+)RNAs extracted from NHOK, HOK-16A, and HOK-16B cells were electrophoresed in 1.2% agarose gel containing 2.2 M formaldehyde, transferred to a nylon

filter, and hybridized to 32P-labeled 7.9-kbp total HPV-16 DNA. The filter was washed

and exposed to X-ray film.

B: The hybridized 32P-HPV-16 DNA was stripped from the nylon filter and rehybridized

with 32P-labeled v-myc oncogene probe. The filter was washed and exposed to X-ray

film.

C: Hybridized 32P-v-myc DNA was stripped from the filter and rehybridized to 32P-labeled

human β-actin gene. The filter was washed and exposed to X-ray film.

Source: Park et al., 1991; used with permission.

in vitro lifespan, but transfection with pMHPV-16d and pSHPV-18m, cloned

HPV-16 and HPV-18 DNA, respectively, recombined into vector plasmidconferred immortality. These data implicate the presence of HPV-16 or -18

DNA in the cells as a requirement for their immortality. This result supports

findings of other reports showing generation of immortalized human

keratinocytes by cloned HPV-16 or HPV-18 DNA (Kaur and McDougall,

1988; Woodworth et al., 1989). The transformation mechanism of keratinocytes by HPV-16 DNA transfection is not clear, but expression of HPV16 E6/E7 gene products has been proposed as being responsible for the

transformation (Kaur and McDougall, 1988). Our Northern blot analysis

demonstrates that the viral E6 and E7 open reading frames are expressed in
Chapter 3

213

Figure 5

Northern blot hybridization analysis of poly(A+)RNAs of NHGK, HGK-18, and

HeLa cell lines

A: Determination of the expression of HPV-18 from HGK-18 and HeLa cell lines.

Poly(A+)RNAs extracted from NHGK, HGK-18, and HeLa cells were electrophoresed in

1.2% agarose gel containing 2.2 M formaldehyde, transferred to a nylon filter, and

hybridized to 32P-labeled EcoRI-BamHI 2.4-kbp fragment of HPV-18 DNA. This 2.4-

kbp HPV-18 DNA fragment contains intact HPV-18 E6 and E7 genes which are

responsible for cell transformation. The filter was washed and exposed to X-ray film.

B: The hybridized 32P-HPV-18 DNA was stripped from the nylon filter and rehybridized

with 32P-labeled v-myc oncogene and 32P-labeled human β-actin gene probes. The

filter was washed and exposed to X-ray film.

the HOK-16A, HOK-16B, and HGK-18 cell lines, indicating that the transformation mechanism of oral keratinocytes by HPV-16 and HPV-18 could be

similar to that of other human cells.

The physical state of the viral DNA in the transformed cell lines in this

study is also similar to that reported elsewhere (Kaur and McDougall, 1988;

Woodworth et al., 1989). Southern blot hybridization analysis showed that

both HPV-16 and HPV-18 DNA are retained in integrated form in the HOK16B cell line and suggests the same for line HOK-16A and HGK-18 cell lines.

Integrated DNA might be absolutely necessary for the morphological transformation and immortalization of the cells, because HPV-16 DNA found in

benign lesions is always in the episomal form, whereas it is usually integrated into host chromosome in carcinomas, even though episomal viral

DNA can persist concurrently.
Smoking and Tobacco Control Monograph No. 2

214

Northern blot analysis of the immortalized cell lines shows that the

viral DNA, including the early region encoding the E6/E7 genes, is actively

transcribed. The E6 and E7 genes of HPV-16 and HPV-18 have been reported to be retained in many cancer cell lines (Schwarz et al., 1985) and

may be responsible, in part, for the immortality of the HOK-16A, HOK-16B,

and HGK-18 cell lines.

Since HPV-16 and -18 DNAs are integrated in cervical carcinomas and

these tumors appear to retain the HPV enhancer region, it is possible that

HPV insertion also results in an activation of certain cellular protooncogenes (Durst et al., 1987). In HeLa cell chromosomes, HPV-18 DNA has

been found to integrate into four specific chromosomes: chromosomes 8, 9,

5, and 22. Of these, chromosomes 8, 9, and 22 correspond to the location

of cellular myc, abl, and sis proto-oncogenes, respectively. Lazo and colleagues (1989) reported that the location of the HPV DNA integration site is

near the myc proto-oncogene, but the myc gene is not rearranged or amplified. It may, however, be expressed through a viral regulatory sequence in

its proximity. Both viral gene amplification and constitutive c-myc gene

expression might be important contributing factors to the immortalization

and proliferative properties of HeLa cells. Like HeLa cells, the c-myc gene is

overexpressed in HOK-16A, HOK-16B, and HGK-18 cell lines compared to

their normal counterpart. The role of the overexpressed c-myc gene products in the immortalized cell lines is unknown, but it, along with the expression of HPV-16 or HPV-18 sequences, could play a crucial role for the

immortalization of the cells. These results also suggest that the overexpression of c-myc gene is not sufficient for the cells to be tumorigenic. The

human oral keratinocyte cell lines immortalized by HPV-16 and HPV-18

provide, therefore, a useful model system for elucidating critical molecular

changes associated with oral carcinogenesis.

REFERENCES

Casto, B.C., DiPaolo, J.A. Virus, chemicals, and

cancer. Progress in Medical Virology 16: 1-47, 1973.

Dekmezian, R.H., Batsakis, J.G., Goepfert, H. In situ

hybridization of papillomavirus DNA in head and

neck squamous cell carcinomas. Archives of

Otolaryngology—Head and Neck Surgery 113: 819-

821, 1987.

Durst, M., Croce, C.M., Gissmann, L., Schwarz, E.,

Huebner, K. Papillomavirus sequences integrate

near cellular oncogenes in some cervical carcinomas. Proceedings of the National Academy of Sciences

84: 1070-1074, 1987.

Durst, M., Gissmann, L., Ikenburg, H., zur Hausen, H.

A papillomavirus DNA from a cervical carcinoma

and its prevalence in cancer biopsy samples from

different geographic regions. Proceedings of the

National Academy of Sciences 80: 3812-3815, 1983.

Eglin, R.P., Scully, C., Lehner, T., Ward-Booth, P.,

McGregor, I.A. Detection of RNA complementary

to herpes simplex virus in human oral squamous

cell carcinoma. Lancet 2: 766-768, 1983.

Hecht, S.S., Chen, C.B., Dong, M., Ornaf, R.M.,

Hoffmann, D., Ts’o, T.C. Chemical studies on

tobacco smoke: VI. Studies on nonvolatile

nitrosamines in tobacco. Beitrage Tabakforsch 9: 1-

6, 1977.

Hirsch, J.M., Johansson, S.L., Vahlne, A. Effect of

snuff and herpes simplex virus 1 on rat oral

mucosa: Possible associations with the development of squamous cell carcinoma. Journal of Oral

Pathology 12: 187-198, 1983.

Hoffmann, D., Brunnenmann, K.D., Adams, J.D.,

Hecht, S.S. Formation and analysis of N-nitrosamines in tobacco products and their endogenous formation in consumers. In: N-Nitroso

Compounds: Occurrence, Biological Effects and

Relevance to Human Cancer, I.K. O’Neill,

R.C. von Borsteel, C.T. Miller, J. Long, and H.

Bartsch (Editors). New York: Oxford University

Press, 1984, pp. 743-762.
Chapter 3

215

Kaur, P., McDougall, J.K. Characterization of primary

human keratinocytes transformed by human

papillomavirus type 18. Journal of Virology 62:

1917-1924, 1988.

Lazo, P.A., DiPaolo, J.A., Popescu, N.C. Amplification of the integrated viral transforming genes of

human papillomavirus 18 and its 5'-flanking

cellular sequence located near the myc protooncogene in HeLa cells. Cancer Research 49: 4305-

4310, 1989.

McCance, D. Human papillomaviruses and cancer.

Biochimica et Biophysica Acta 823: 195-205, 1986.

National Institutes of Health. Workshop on the

treatment and prevention of herpes simplex virus

infection. Journal of Infectious Diseases 127: 117-

119, 1973.

Offenbacher, S., Weathers, D.R. Effects of smokeless

tobacco on the periodontal, mucosal and caries

status of adolescent males. Journal of Oral

Pathology 14: 162-181, 1985.

Oh, J.S., Paik, D.-I., Christensen, R., Akoto-Amanfu,

E., Kim, K., Park, N.-H. Herpes simplex virus

enhances the 7,12-dimethylbenz[a]anthracene

(DMBA)-induced carcinogenesis and amplification

and overexpression of c-erb-B-1 proto-oncogene in

hamster buccal pouch epithelium. Oral Surgery,

Oral Medicine, Oral Pathology 68: 428-435, 1989.

Overall, J.C., Jr. Dermatologic Diseases. In: Antiviral

Agents and Viral Diseases of Man, G.J. Galasso,

T.C. Merigan, and R.A. Buchanan (Editors). New

York: Raven Press, 1979, pp. 305-384.

Park, N.-H., Akoto-Amanfu, E., Paik, D.I. Smokeless

tobacco carcinogenesis: The role of viral and other

factors. CA-A Cancer Journal for Clinicians 38: 248-

256, 1988.

Park, N.-H., Min, B.-M., Li, S.-L., Cherrick, H.M.,

Doniger, J. Immortalization of human oral

keratinocytes with type 16 human papillomavirus.

Carcinogenesis 12: 1627-1631, 1991.

Park N.-H., Niukian, K., Shklar, G. Combined effect

of herpes simplex virus and tobacco on the

histopathologic changes in lips of mice. Oral

Surgery, Oral Medicine, Oral Pathology 59: 154-158,

1985.

Schwarz, E., Freese, U.K., Gissmann, L., Mayer, W.,

Roggenburk, B., Stremlau, A., zur Hausen, H.

Structure and transcription of human

papillomavirus sequences in cervical carcinoma

cells. Nature (London) 314: 111-114, 1985.

Shillitoe, E.J., Greenspan, D., Greenspan, J.S.,

Hanson, L.S., Silverman, S., Jr. Neutralizing

antibody to herpes simplex virus type 1 in

patients with oral cancer. Cancer 49: 2315-2320,

1982.

Shklar, G., Niukian, K., Hassan, M., Herbosa, E.G.

Effects of smokeless tobacco and snuff on oral

mucosa of experimental animals. Journal of Oral

and Maxillofacial Surgery 43: 80-86, 1985.

U.S. Department of Health and Human Services.

Health Consequences of Smoking: Cancer. A Report

of the Surgeon General. U.S. Department of Health

and Human Services, Public Health Service, Office

on Smoking and Health. DHHS Publication No.

(PHS) 82-50179, 1982.

U.S. Department of Health and Human Services.

Health Implications of Smokeless Tobacco Use:

National Institutes of Health Consensus Development

Conference Statement. 6, No. 1. U.S. Department of

Health and Human Services, Public Health Service,

National Institutes of Health, 1986.

Woodworth, C.D., Doniger, J., DiPaolo, J.A.

Immortalization of human foreskin keratinocytes

by various human papillomavirus DNAs corresponds to their association with cervical carcinoma. Journal of Virology 63: 159-164, 1989.
Smoking and Tobacco Control Monograph No. 2

216
Chapter 4

217

Chapter 4

Nicotine Effects and Addiction

CONTENTS Pharmacology of Smokeless Tobacco Use:

Nicotine Addiction and Nicotine-Related

Health Consequences

Neal L. Benowitz .................................................................................. 219

Introduction ................................................................................. 219

Pharmacology of Nicotine ........................................................... 219

Nicotine and Cotinine Levels in Blood........................................ 220

Nicotine and ST Addiction ........................................................... 222

Health Consequences of Nicotine Exposure................................ 225

Conclusions .................................................................................. 227

References ..................................................................................... 227

Recent Advances in Understanding the Actions of

Nicotine in the Central Nervous System

Paul B.S. Clarke .................................................................................... 229

Introduction ................................................................................. 229

Nicotine’s Action in the CNS ....................................................... 229

Pharmacological Help for Quitting Tobacco Use ........................ 235

References ..................................................................................... 237

Dependence on Smokeless Tobacco

Martin J. Jarvis ..................................................................................... 239

Introduction ................................................................................. 239

Nicotine Uptake From Unusual Use of Tobacco.......................... 240

Nicotine Absorption From Dry Nasal Snuff ................................. 240

Nicotine Intake and Dependence in Sweden............................... 241

Cessation of Smokeless Tobacco Use ........................................... 243

Future Research Needs ................................................................. 243

References ..................................................................................... 244
Smoking and Tobacco Control Monograph No. 2

218
Chapter 4

219

Nicotine is easily absorbed from smokeless tobacco and could contribute to adverse health

Pharmacology of Smokeless Tobacco Use:

Nicotine Addiction and

Nicotine–Related Health Consequences

Neal L. Benowitz

ABSTRACT

consequences of ST use. Because nicotine is the cause of addiction to cigarettes, ST use may

have a similar addiction liability. Cessation of ST use is difficult for many people. The nicotine

withdrawal syndrome that follows the cessation of regular ST use supports the idea that some

users are addicted to ST. Experimental data in support of this proposition indicate that ST users

adjust their tobacco-consuming behavior to regulate levels of nicotine in the body. ST use

results in cardiovascular effects that are similar to those of cigarette smoking. Chronic systemic

exposure to nicotine from cigarette smoking may contribute to accelerated coronary and

peripheral vascular disease, acute cardiac ischemic events, delayed wound healing, reproductive

disturbances, peptic ulcer disease, and esophageal reflux. Insofar as nicotine contributes to the

adverse health effects of cigarette smoking, the nicotine in ST would be expected to present

similar hazards. Illness caused by systemic absorption of nicotine and other toxins from ST

should be considered a potential sequel to long-term tobacco use.

INTRODUCTION In addition to causing oral pathology, there is concern that habitual, long-term smokeless tobacco use produces systemic effects that might

adversely affect health. Of particular concern is exposure to nicotine, which

is present in large amounts in smokeless tobacco (Table 1). This paper

reviews the pharmacology of nicotine as related to the use and potential

health hazards of smokeless tobacco.

PHARMACOLOGY

OF NICOTINE

Nicotine is a tertiary amine composed of a pyridine and a

pyrrolidine ring. Nicotine binds to acetylcholine receptors at

ganglia and neuromuscular junctions and in the brain (Benowitz, 1988). In

its non-ionized form, nicotine freely permeates membranes, including the

buccal mucosa and the blood-brain barrier. As a weak base, nicotine is less

ionized and penetrates membranes more easily in alkaline solutions. Chewing tobacco and snuff, as well as nicotine gum, are buffered to an alkaline

pH to facilitate absorption of nicotine.

Nicotine is absorbed more slowly from ST than from tobacco smoke,

but peak venous levels are similar (Figure 1). Whereas blood levels of

nicotine fall rapidly after smoking, the concentrations plateau during and

after ST use, consistent with continued absorption even after the tobacco is

removed from the mouth (Benowitz et al., 1988). Possibly, continued

absorption of nicotine is attributable to release of nicotine from mucous

membranes and/or absorption of nicotine that has been swallowed. The

systemic absorption of nicotine per dose is greater with the use of chewing

tobacco (average 4.5 mg nicotine from an average dose of 7.9 g chewed for

1

 Supported in part by grants no. CA-32389, DA-02277, DA-01696, and RR-00083 from the

National Institutes of Health.

1
Smoking and Tobacco Control Monograph No. 2

220

Table 1

Nicotine content of cigarettes and smokeless tobacco

Nicotine in

Concentration Typical Nicotine in Dose Typically

of Nicotine Single Dose Single Dose Consumed in

(mg/g) (g Tobacco) (mg) 1 Day

Cigarettes 15.7 0.54 8.4 168 mg per

(15)a (13.3-26.9)b 20 cigarettes

Moist Snuff 10.5 1.4 14.5 157 mg per

(8)a (6.1-16.6)b 15 g

Chewing Tobacco 16.8 7.9 133.0 1,176 mg per

(2)a (8.1-24.5)b 70 g

a Number of brands tested.

b Range.

Source: Adapted from Benowitz et al., 1990.

30 min) or snuff (average 3.6 mg nicotine from 2.5 g moist snuff kept in the

mouth for 30 min) compared with that from smoking cigarettes (average

1.0 mg nicotine per cigarette). Individuals vary considerably in the amount

of nicotine reaching the systemic circulation, even when they use a fixed

dose of smokeless tobacco. For example, we found an eightfold range in

peak plasma nicotine concentration (range 4 to 33 ng/mL) among 10 subjects who placed 2.5 g snuff in the mouth for 30 min. The average systemic

bioavailability of nicotine from cigarettes and smokeless tobacco can be

estimated from the average systemic doses of nicotine absorbed, as described

above, and the data on the amount of nicotine in tobacco, as shown in

Table 1. Systemic bioavailability is estimated to be 12.0 percent,

14.0 percent, and 3.4 percent for cigarette smoke, oral snuff, and chewing

tobacco, respectively.

Nicotine is extensively metabolized by the liver (Benowitz, 1988). The

major metabolite is cotinine, which has been used as a marker for nicotine

intake. The half-life of nicotine averages 2 to 3 h. Consistent with this halflife, nicotine accumulates for 6 to 8 h throughout the day with regular ST

use, and the levels of nicotine persist overnight, even while the user sleeps

(Figure 2) (Benowitz et al., 1989).

NICOTINE AND

COTININE LEVELS

IN BLOOD

As Figure 1 shows, peak levels of nicotine after cigarette smoking

or single doses of oral snuff or chewing tobacco are similar.

Likewise, nicotine levels after the use of ST in sachets (Skoal

Bandits) and after nasal insufflation of fine, ground nasal snuff are similar,

although in the latter case absorption is very rapid, resembling absorption

from cigarette smoke (Russell et al., 1981 and 1985).
Chapter 4

221

Figure 1

Nicotine absorption rates from tobacco smoke and smokeless tobacco

Average blood nicotine levels in 10 men who smoked for 9 min (1.3 cigarettes), placed

2.5 g moist oral snuff in the mouth for 30 min, chewed an average of 7.9 g (range of 0.9 g

to 17.8 g) chewing tobacco for 30 min, and chewed 4 mg nicotine gum (two 2-mg pieces

of Nicorette) for 30 min. Studies were performed in the morning after overnight abstinence from tobacco.

Source: Adapted from Benowitz et al., 1988.

With regular daily cigarette smoking, blood or plasma levels of nicotine

sampled in the afternoon when those levels are at or near steady state

generally range from 10 to 50 ng/mL. In a research ward study of eight
Smoking and Tobacco Control Monograph No. 2

222

Figure 2

Circadian blood nicotine concentration with cigarette smoking, chewing tobacco,

and oral snuff

Blood nicotine concentrations in subjects who smoked cigarettes (closed circles), used

chewing tobacco (open circles), or used oral snuff (closed squares). Data are shown as

mean ± S.E. for eight subjects.

Source: Adapted from Benowitz et al., 1989; used with permission.

men, the levels of nicotine during ad libitum use of oral snuff (averaging

15.6±5.9 g/d) or chewing tobacco (averaging 72.9±21.6 g/d) were similar to

those observed with cigarette smokers (average 15.6±5.9 cigarettes/d)

(Figure 2) (Benowitz et al., 1989).

Using plasma cotinine as an indicator of daily nicotine consumption

from ST, one can compare consumption in different populations (Table 2).

Cotinine levels in our research volunteers were somewhat higher than those

measured in other groups. The college students and young men studied by

Gritz et al. (1981) and Biglan et al. (in press) had cotinine levels averaging

55 to 60 percent of those seen in our research subjects, the latter of whom

were, on average, much older. Professional baseball players had, in general,

much lower cotinine values, particularly among the chewing tobacco users

(Siegel et al., 1992). The lower level of cotinine in the baseball players

reflects intermittent use, often in conjunction with playing baseball.

NICOTINE AND

ST ADDICTION

People smoke cigarettes for the psychoactive effects of nicotine, and

it is presumed that smokeless tobacco is consumed for the same

reason. Nicotine may enhance the sense of well-being, produce arousal or

relaxation, help maintain vigilance, and reduce anxiety (Benowitz, 1988).
Chapter 4

223

Table 2

Serum cotinine in different populations of ST users

Plasma Cotinine (ng/mL)

Source Type (n) Mean Range

Gritz, 1981 Snuff 11 197a 14–556

Benowitz, 1989 Snuff 8 356b 182–868

Benowitz, 1989 Chew 8 354b 100–836

Siegel, 1992 Snuff 182 144c –

Siegel, 1992 Chew 48 82c –

Biglan, 1992 Snuff 20 217d –

a Afternoon measurement.

b Mean values throughout 24 h.

c Different times of day.

d Morning measurement.

Whether enhancement of performance and mood is the result of an intrinsic enhancing effect of nicotine, or of relief of symptoms of abstinence in

habitual users, is unclear. ST use is especially common among male athletes, some of whom believe that it enhances their athletic performance.

Studies of the effects of ST on reaction time in athletes, however, have not

confirmed any improvement in performance (Edwards and Glover, 1986).

Addiction to or dependence on smokeless tobacco can be defined as

compulsive use despite awareness of substantial reasons not to use it. Formal criteria for establishing drug dependence have been developed in the

Surgeon General’s Report, The Health Consequences of Smoking: Nicotine

Addiction (US DHHS, 1988). The primary criteria include highly controlled

or compulsive use, psychoactive effects, and drug-reinforced behavior.

Additional criteria include physical dependency, among several others. The

compulsive use of smokeless tobacco has been reported and is described

elsewhere in this volume. Nicotine from ST clearly has psychoactivity, and

nicotine withdrawal symptoms develop after sudden cessation of ST use

(Hatsukami et al., 1987).

That nicotine reinforces ST use has been shown recently in studies by

Biglan and coworkers (in press). In a laboratory setting, depriving young

men who regularly use smokeless tobacco from their tobacco for a period of

time resulted in a greater consumption of ST than in a condition under

which smokeless tobacco was given as a loading condition (Figure 3). The

level of ST consumption was such that, whether or not subjects were

preloaded with smokeless tobacco, after a period of ad libitum consumption,

serum nicotine levels were similar in all conditions. In a second study of

young men who regularly used snuff and smoked cigarettes, Biglan and

coworkers found that when ST use was restricted, subjects smoked more

cigarettes, and when cigarette use was restricted, subjects consumed more

smokeless tobacco, compared to when both forms of tobacco were available

ST Users
Smoking and Tobacco Control Monograph No. 2

224

Figure 3

Serum nicotine concentration after deprivation then ad libitum oral snuff use in

regular users

Condition I was on arrival at the laboratory after overnight tobacco abstinence. Condition II was at the end of a 2-h pre-experimental session when subjects were either

deprived of (Dep) or preloaded with smokeless tobacco (ST). Condition III was at the end

of the experimental session during which subjects were either deprived or had ad libitum

access to smokeless tobacco.

Source: Adapted from Biglan et al., in press.

(Biglan et al., in press). These studies indicate that habitual ST users are

titrating a level of nicotine in the body, as has been well described for

cigarette smokers, supporting the dependence criterion of drug-reinforced

behavior. That habitual ST use increases the likelihood of cigarette smoking

when smokeless tobacco is unavailable or undesirable because of social

constraints is supported by population survey data (Glover et al., 1989).

Thus, the criteria for addiction to nicotine are met for at least some ST

users. The 1986 Surgeon General’s Report on smokeless tobacco concluded

that ST is an addicting substance (US DHHS, 1986). It is unclear what

proportion of all ST users are addicted. Professional baseball players who

use smokeless tobacco intermittently, often only in association with playing

their game, for the most part do not seem to be addicted to ST.
Chapter 4

225

Nicotine has many actions on the human body (Benowitz, 1988)

(Table 3). The role of nicotine in producing these effects has been

established by studies of direct administration. In general, the

responses are consistent with activation of the sympathetic nerHEALTH

CONSEQUENCES

OF NICOTINE

EXPOSURE

vous system. Cardiovascular effects include heart rate acceleration (10 to

20 beats/min) and increased blood pressure (5 to 10 mmHg), similar to the

effects of cigarette smoking. Nicotine also increases the circulating levels of

catecholamines and free fatty acids, which may contribute to the increased

level of total cholesterol and decreased levels of high-density lipoprotein

cholesterol that are found in habitual cigarette smokers. Inhibition of

prostacyclin synthesis and other effects on platelets may enhance coagulation.

The potential adverse health consequences of nicotine may be summarized as follows:

• Nicotine intoxication;

• Accelerated coronary and peripheral vascular disease;

• Stroke;

• Hypertension (complications);

• Delayed wound healing;

• Reproductive or perinatal disorders (low birth weight, prematurity,

spontaneous abortion);

• Peptic ulcer disease; and

• Esophageal reflux.

For more details, see Benowitz (1988, 1991a, and 1991b) and US DHHS

(1986 and 1988).

The greatest concern for nicotine–related effects is acceleration or

aggravation of cardiovascular disease (Benowitz, 1991a). In a study of the

cardiovascular effects of daily ST use, we found that the most prominent

effects of nicotine—heart rate acceleration and increased urinary catecholamine excretion—were similar throughout the day in people smoking

cigarettes and those using smokeless tobacco (Benowitz et al., 1989). In

addition, urine sodium excretion was greater during use of smokeless

tobacco than during smoking, probably because of the absorption of sodium, which, in smokeless tobacco, acts as an alkaline buffer to facilitate

nicotine absorption. Insofar as nicotine contributes to adverse health effects

of cigarette smoking, nicotine in smokeless tobacco would be expected to

present similar hazards.

Nicotine could promote atherosclerotic vascular disease by contributing

to hyperlipidemia, endothelial injury, or both (Benowitz, 1991a). Although

cigarette smoking is not associated with an increased risk of hypertension,

complications of hypertension are more severe in people who also smoke

cigarettes (Isles et al., 1979). Nicotine may aggravate hypertension by

causing vasoconstriction. Case histories of patients with hypertension

aggravated by the use of smokeless tobacco have been reported (McPhaul et

al., 1984; Wells and Rustick, 1986), and one survey of college students

indicated that ST users had elevated blood pressure (Schroeder and Chen,
Smoking and Tobacco Control Monograph No. 2

226

Table 3

Actions of nicotine in humans

Affected Systems Effects

CNS Arousal or relaxation

Enhanced concentration, vigilance

Appetite suppression

Electroencephalographic changes

Cardiovascular Increased heart rate, cardiac contractility, blood pressure

Cutaneous vasoconstriction

Systemic venoconstriction

Increased muscle blood flow

Catecholamine release

Metabolic Lipolysis with fatty acid release

Increased energy expenditure

Endocrine Increased growth hormone

Adrenocorticotrophic hormone/cortisol

Vasopressin

Beta endorphins

Inhibition of prostacyclin synthesis

1985). However, a more recent study of ST use among baseball players

revealed no relationship between smokeless tobacco and blood pressure

(Ernster et al., 1990).

There is considerable evidence that nicotine may contribute to acute

cardiac ischemia, such as aggravating angina pectoris, precipitating unstable

angina or acute myocardial infarction, and even sudden death (Benowitz,

1991a). The possible mechanisms of nicotine effect include its systemic

hemodynamic effects (which increase myocardial work), enhancement of

thrombosis, induction of coronary vasoconstriction, and/or

arrhythmogenesis.

Whether habitual use of smokeless tobacco is associated with

hyperlipidemia, increased incidence of complications from hypertension,

accelerated atherosclerotic vascular disease, and/or increased risk of acute

cardiac ischemic events remains to be established in studies of large populations of users. Of interest in this regard is a recent study of users of smokeless tobacco that indicated they had a higher prevalence of hypercholesterolemia (when normalized for age and education) than did nonusers of tobacco (Tucker, 1989). Considering that the levels of nicotine in

the blood are similar in smokers and users of smokeless tobacco, it seems

likely that people with coronary artery disease are at increased risk from ST

use.

Other suspected adverse health effects of nicotine, particularly reproductive disorders and peptic ulcer disease, must also be considered as potential complications of habitual ST use.
Chapter 4

227

CONCLUSIONS The following may be concluded about nicotine and ST use: Systemic absorption and blood levels of nicotine are substantial and are often

comparable in ST users and cigarette smokers. Data from a few studies

performed to date indicate that ST use has the potential to produce dependency similar to that seen in cigarette smokers. However, it appears that in

some populations, such as baseball players who use smokeless tobacco only

intermittently, many ST users are not dependent. Smokeless tobacco use by

young people does pose a concern for later development of dependence on

cigarettes.

The health hazards known to be caused by cigarette smoking, and

suspected to be related to acute or chronic nicotine exposure, are expected

to be a hazard of habitual ST use as well. The major concerns are acceleration or aggravation of coronary artery disease and reproductive disorders.

REFERENCES

Benowitz, N.L. Pharmacologic aspects of cigarette

smoking and nicotine addiction. New England

Journal of Medicine 319: 1318-1330, 1988.

Benowitz, N.L. Nicotine and coronary heart disease.

Trends in Cardiovascular Medicine 1: 315-321,

1991a.

Benowitz, N.L. Nicotine replacement therapy during

pregnancy. Journal of the American Medical

Association 266: 3174-3177, 1991b.

Benowitz, N.L., Jacob, P. III, Yu, L. Daily use of

smokeless tobacco: Systemic effects. Annals of

Internal Medicine 111: 112-116, 1989.

Benowitz, N.L., Porchet, H., Jacob, P. III. Pharmacokinetics, metabolism, and pharmacodynamics of

nicotine. In: Nicotine Psychopharmacology:

Molecular, Cellular, and Behavioral Aspects,

S. Wonnacott, M.A.H. Russell, and I.P. Stolerman

(Editors). Oxford: Oxford University Press, 1990,

pp. 112-157.

Benowitz, N.L., Porchet, H., Sheiner, L., Jacob, P. III.

Nicotine absorption and cardiovascular effects

with smokeless tobacco use: Comparison with

cigarettes and nicotine gum. Clinical Pharmacology

and Therapeutics 44: 23-28, 1988.

Biglan, A., LaChance, P.A., Benowitz, N.L. Experimental analysis of the effects of smokeless tobacco

deprivation. Journal of Abnormal Psychology, in

press, 1992.

Edwards, S.W., Glover, E.D. Snuff and neuromuscular performance (letter). American Journal of Public

Health 76: 206, 1986.

Ernster, V.L., Grady, D.G., Greene, J.C., Walsh, M.,

Robertson, P., Daniels, T.E., Benowitz, N., Siegel,

D., Gerbert, B., Hauck, W.W. Smokeless tobacco

use and health effects among baseball players.

Journal of the American Medical Association 264: 218-

224, 1990.

Glover, E.D., Laflin, M., Edwards, S.W. Age of

initiation and switching patterns between

smokeless tobacco and cigarettes among college

students in the United States. American Journal of

Public Health 79: 207-208, 1989.

Gritz, E.R., Baer-Weiss, V., Benowitz, N.L., Van

Vunakis, H., Jarvik, M.E. Plasma nicotine and

cotinine concentration in habitual smokeless

tobacco users. Clinical Pharmacology and Therapeutics 30: 201-209, 1981.

Hatsukami, D.K., Gust, S.W., Keenan, R.M. Physiologic and subjective changes from smokeless

tobacco withdrawal. Clinical Pharmacology and

Therapeutics 41: 103-107, 1987.

Isles, C., Brown, J.J., Cummings, A.M., et al. Excess

smoking in malignant-phase hypertension. British

Medical Journal 1: 579-581, 1979.

McPhaul, M., Punzi, H.A., Sandy, A., Borganelli, M.,

Rude, R., Kaplan, N.M. Snuff-induced hypertension in pheochromocytoma. Journal of the

American Medical Association 252: 2860-2862,

1984.

Russell, M.A.H., Jarvis, M.J., Devitt, G., Feyerabend,

C. Nicotine intake by snuff users. British Medical

Journal 283: 814-817, 1981.

Russell, M.A.H., Jarvis, M.J., West, R.J., Feyerabend,

C. Buccal absorption of nicotine from smokeless

tobacco. Lancet 2: 1370, 1985.

Schroeder, K.L., Chen, M.S.J. Smokeless tobacco and

blood pressure. New England Journal of Medicine

312: 919, 1985.

Siegel, D., Benowitz, N., Ernster, V.L., Grady, D.G.,

Hauck, W.W. Smokeless tobacco, cardiovascular

risk factors, and nicotine and cotinine levels in

professional baseball players. American Journal of

Public Health, in press, 1992.
Smoking and Tobacco Control Monograph No. 2

228

Tucker, L.A. Use of smokeless tobacco, cigarette

smoking, and hypercholesterolemia. American

Journal of Public Health 79: 1048-1050, 1989.

U.S. Department of Health and Human Services.

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, National Institutes of Health, National

Cancer Institute. DHHS Publication No. (NIH)

86-2874, 1986.

U.S. Department of Health and Human Services.

Health Consequences of Smoking: Nicotine Addiction.

A Report of the Surgeon General, 1988. U.S.

Department of Health and Human Services, Public

Health Service, Centers for Disease Control,

Center for Health Promotion and Education,

Office on Smoking and Health. DHHS Publication

No. (CDC) 88-8406, 1988.

Wells, D.G., Rustick, J.M. Hypertension from

smokeless tobacco (letter). Anesthesiology 65: 339,

1986.
Chapter 4

229

There is now a consensus that nicotine is the key factor in habitual tobacco use and that it

Recent Advances in Understanding

The Actions of Nicotine in the

Central Nervous System

Paul B.S. Clarke1

ABSTRACT

is the drug’s actions on the central nervous system that are particularly important. Knowledge

of sites and mechanisms of nicotine’s CNS actions is advancing rapidly. Precisely which

actions of the drug contribute to its reinforcing properties remain to be identified, but the

participation of the mesolimbic dopamine pathway of the brain seems likely. Current pharmacological approaches to cessation of tobacco use have focused on nicotine replacement and

have not met with great success. The high relapse rates encountered with this treatment

approach (and with other current tobacco cessation techniques) may reflect the persistence of

learned associations acquired during tobacco use. These associations could be extinguished

(unlearned) through use of a drug that blocks the central actions of nicotine. Nicotine

blockade therapy thus represents an attractive but largely untried treatment approach.

INTRODUCTION Tobacco use is a major cause of preventable cancer. This review is

intended primarily for health workers involved in the prevention and

treatment of cancer. Its purpose is to provide an overview of recent advances in the understanding of nicotine’s actions in the central nervous

system and to highlight areas of uncertainty. This survey is selective and

does not cover certain fields that may prove extremely important, such as

the pharmacogenetic aspects of nicotine (Collins et al., 1990), the effects of

nicotine on development (Navarro et al., 1989), and the interactions of

nicotine with other drugs.

Two key questions serve as a framework for this review: (1) What does

nicotine do in the CNS to account for its central role in tobacco use? and

(2) Can pharmacologists help the tobacco user who wishes to quit?

NICOTINE’S

ACTION IN

THE CNS

As the Primary

Reinforcer

There is widespread agreement among scientists and clinicians that

most habitual smokers continue to smoke because they are dependent on nicotine. Nicotine dependence is thought to underlie the

use of smokeless tobacco as well. The evidence, which has been

reviewed at length elsewhere (Jaffe, 1990; US DHHS, 1988), can be

summarized as follows:

• Nicotine is consumed not only via tobacco smoke but also via smokeless tobacco, thus avoiding the many pyrolysis products contained in

smoke;

• Nicotine-free cigarettes generally are not smoked;

1 The author receives research funding from Fonds de la Recherche en Santé de Québec and the

Medical Research Council of Canada.
Smoking and Tobacco Control Monograph No. 2

230

• Cigarette smokers regulate their levels of nicotine in response to

pharmacological manipulations such as preloading with nicotine or

receptor antagonism;

• Nicotine is self-administered intravenously by humans and animals in

the laboratory;

• Self-administered doses of nicotine are psychoactive; and

• Habitual smokers experience withdrawal symptoms that can be

alleviated by administration of nicotine.

Central vs.

Peripheral Effects

Virtually all the known actions of nicotine appear to be mediated by nicotinic receptors (Clarke, 1987; US DHHS, 1988).

There are receptors for nicotine in the CNS and in the periphery. Precisely

which pharmacological actions of nicotine are important to tobacco smokers is unclear; however, it appears that the primary reinforcing actions of

nicotine occur within the CNS.

Most of the evidence for this claim comes from work in animals.

Almost all the behavioral effects of nicotine that have been investigated in

animals appear to be attributable to direct CNS actions (Clarke, 1987), and

recent evidence suggests that nicotine’s reinforcing properties are no exception. Several species of laboratory animals have been shown to self-administer intravenous infusions of nicotine voluntarily. Rates of nicotine selfadministration in rats were found to be markedly reduced after central

administration of the nicotinic receptor blocker chlorisondamine in a low

dose that was unlikely to act peripherally (Corrigall et al., 1992). Moreover,

as described in a later section of this paper, lesion studies in rats have

focused attention on a particular nerve pathway in the brain that seems to

be intimately connected with nicotine’s rewarding effects.

Central actions of nicotine also appear to be important in regulating

cigarette smoking by humans. In one study, acute administration of

mecamylamine, a centrally active nicotinic antagonist, altered smoking

behavior, whereas the nicotinic antagonist pentolinium, which does not

readily pass into the CNS, did not (Stolerman et al., 1973). In this shortterm experiment, mecamylamine actually increased indices of smoke intake,

and it seems likely that subjects smoked more in an attempt to overcome a

blockade of nicotine’s effects. In contrast, a preliminary report suggests that

chronic treatment with mecamylamine can dramatically reduce smoking

behavior and even permit highly dependent smokers to quit (Tennant et al.,

1984). There appear to be no reports of the effects of nicotinic antagonists

on consumption of smokeless tobacco.

Actions of nicotine in the peripheral nervous system may also contribute to the maintenance of tobacco use. If the evidence just reviewed suggests that the primary reinforcing effects of nicotine are likely attributable to

central actions of the drug, its peripheral actions may act as secondary

reinforcers. At doses obtained from cigarette smoke, nicotine appears to

exert significant actions at the autonomic ganglia and at certain sensory

nerve endings (but not on skeletal muscle). Jarvik and Assil (1988) have
Chapter 4

231

reported that the nicotinic receptor antagonist mecamylamine attenuates

the sensation produced by direct application of a nicotine solution to the

tongue. This ability of nicotine to stimulate sensory nerve endings is of

special interest, since sensory cues that occur in temporal proximity to

smoking behavior would most readily tend to acquire the properties of

secondary reinforcers. Consistent with this notion, Rose and colleagues

(1985) have demonstrated that sensory stimuli produced in the airways by

smoke inhalation can affect the immediate satisfaction derived from cigarettes. Although these findings are suggestive, nicotine is but one constituent of tobacco smoke, and the extent to which it contributes to secondary

reinforcement remains to be determined.

Subtypes of

CNS Nicotinic

Receptors

On pharmacological grounds, nicotinic receptors in the periphery

have long been subdivided into those occurring at the neuromuscular

junction and those found in the autonomic ganglia and adrenal

glands. With the advent of powerful immunological and molecular genetic

techniques, an extended family of nicotinic receptors has recently been

revealed. (See Deneris et al., 1991, for a review.) All known nicotinic

receptors comprise a number of subunits. Generally speaking, each subunit

is encoded by a different gene. An ever-growing number of such genes have

been identified. For example, in the rat, more than a dozen putative subunits have been found; each receptor subtype is made up of a unique permutation of subunits, and the potential for diversity is staggering.

One factor that may limit this diversity is incompatibility between

subunits. By injecting species of nicotinic receptor messenger RNA into frog

oocytes, it is possible to produce and test the electrophysiological properties

of “artificial” nicotinic receptors. The results of such studies show that

certain combinations of subunits do not work as nicotinic receptors (Deneris

et al., 1991).

In addition, receptor subunits (and, by implication, receptor subtypes)

are differentially regulated. Thus, receptor subunits tend to show characteristic anatomical patterns of expression. For example, receptor subunits

expressed in muscle are different from those expressed in the brain. The

richest diversity of nicotinic receptors probably occurs in the CNS, where

overlapping but unique distributions have been elegantly revealed by the

technique of in situ hybridization histochemistry (Wada et al., 1989). Not

only are the nicotinic receptor subtypes found in different places, but also at

different times; receptor subunits manifest characteristic patterns of expression during development, some disappearing before adulthood.

At present, we simply do not know how many receptor subtypes exist.

Considerable diversity is suggested not only by the several subunit-encoding

mRNA species detected in the brain (Deneris et al., 1991; Wada et al., 1989),

but also by findings at the protein level (Clarke et al., 1985; Schulz et al.,

1991).
Smoking and Tobacco Control Monograph No. 2

232

On a molecular level, the best studied nicotinic receptors are those

found in the electric organ of the electric ray and in mammalian

muscle. Each of these receptors comprises five subunits, which

together form the walls of an ion channel that transverses the cell

Nicotine’s

Complex Agonist

Actions at the

Receptor Level

membrane. Agonists such as nicotine and the neurotransmitter acetylcholine bind to certain of the subunits. Binding of an agonist induces the

receptor macromolecule to undergo a conformational change (i.e., a change

in shape), which in turn allows the channel to open and results in a net

flow of positively charged ions (cations) into the nerve cell. The entry of

these cations (primarily Na+) tends to depolarize the neuron, making firing

of the cell more likely. Calcium ions also may enter the cell in significant

numbers. Calcium plays an important role in intracellular signaling pathways, and the consequences of calcium entry can be complex. Most nicotinic receptors are believed to control the passage of positively charged ions

into the cell, thereby producing excitation. However, two reports in the last

5 yr suggest that some nicotinic receptors in the brain may be inhibitory (de

la Garza et al., 1987; Wong and Gallagher, 1989).

Within the receptor macromolecule, certain receptor subunits bind

nicotine (and other agonists). Other subunits, sometimes referred to as

“structural subunits,” do not, but their presence can indirectly influence

agonist binding (Deneris et al., 1991). Antagonists can act in several different ways: some tend to compete for the agonist recognition site, others

block the ion channel once it is opened, and yet others target additional

sites on the molecule (Changeux et al., 1984). The precise mode of action

depends on the drug and even on the type of nicotinic receptor in question.

The receptor macromolecule also presents a target for endogenous substances, notably certain peptides, exerting a modulatory role (Boksa and

Livett, 1984).

It is clear that the diversity of nicotinic receptors and our present state

of ignorance about them make generalization difficult. In some respects

each nicotinic receptor subtype is unique. In certain other respects there are

considerable characteristics in common. Furthermore, the extended family

of nicotinic receptors form part of a yet wider superfamily of ligand-gated ion

channels, and certain common organizing principles are beginning to

emerge. Studies of non-nicotinic members of this receptor superfamily

suggest that nicotinic receptors may well be modulated by a variety of

endogenous and pharmacological factors acting at a number of distinct sites

on the macromolecule.

Desensitization

Of Receptors

Activation of receptors by nicotine may lead to receptor desensitization. This is seen macroscopically as tachyphylaxis, in which the

tissue becomes refractory to further applications of the drug. First described

in the periphery, tachyphylaxis also occurs in central actions of nicotine.

Central-occurring tachyphylaxis is well established in animals (Clarke, 1987)

and also occurs in humans, although there are fewer human data available

(US DHHS, 1988).

Neuronal nicotinic receptors (e.g., those located in autonomic ganglia

or in the brain) are more sensitive than muscle nicotinic receptors to the
Chapter 4

233

activating and desensitizing actions of nicotine (Paton and Savini, 1968).

(Were this not so, tobacco use would be acutely harmful to one’s health.)

There appear to be considerable differences between putative subtypes of

CNS nicotinic receptors in their sensitivity to nicotine-induced activation

and desensitization (Couturier et al., 1990; Luetje and Patrick, 1991).

The implication is that doses of nicotine relevant to smoking and ST

consumption may selectively activate certain subtypes of CNS receptors,

leaving others unactivated and perhaps still other receptor subtypes in a

more or less permanent state of desensitization. Evidence for this is discussed below, in the context of central sites of action.

Nicotine’s ability to induce desensitization may underlie certain aspects

of tobacco use. Habitual cigarette smokers inhale intermittently and space

their cigarettes over time. This pattern of intermittent dosing may optimize

the tradeoff between receptor activation and desensitization. Habitual

smokers report that the first cigarette of the day is generally the most

satisfying (US DHHS, 1988); the period of abstinence imposed by sleep may

allow a large proportion of receptors to return from a desensitized to an

activatable state. In addition, the greater difficulty people have in quitting

cigarette smoking compared to giving up other forms of tobacco consumption may be due in part to the pharmacodynamic peculiarities of this form

of nicotine administration (Benowitz et al., 1990).

Locating Central

Sites of Action

Nicotinic receptors have been visualized most directly by autoradiography. Sections of brain tissue are mounted on microscope

slides and incubated with radioactive probes that selectively label nicotinic

receptors. The sections are then exposed to film, and the resulting images

reveal the neuroanatomical location of the receptors. In this way, nicotinic

receptors have been mapped in rat brain (Clarke et al., 1985; Schulz et al.,

1991; Swanson et al., 1987), and to some extent in human brain (Adem et

al., 1988). Nicotinic receptors are concentrated in a number of brain regions

and nuclei, the precise distribution depending on the receptor subtype in

question.

In animals, a variety of other approaches have been used to locate

central sites of nicotine action. Several of the brain areas that feature

prominently in nicotinic receptor autoradiographs have been studied

electrophysiologically, and many neurons in these areas respond to the local

application of nicotine. (For a review, see Clarke, 1990a.) Through recording the responses of neurons in brain tissue slices maintained in vitro, it has

been possible to show that many cells are directly acted upon by nicotine,

via receptors located on the cell body, on dendrites, or on both. By contrast, studies of transmitter release, again in vitro, have shown that nicotine

can also exert direct effects on nerve terminals (Chesselet, 1984).

It must be stressed that nicotine does not occur naturally in the body.

The endogenous agonist for nicotinic receptors is acetylcholine. Thus,

nicotine is often employed because it is a convenient cholinergic agonist. In

many cases, high doses of the drug are used which are of doubtful relevance

to tobacco use. Furthermore, a recent electrophysiological report suggests

that there is a prevalent subtype of nicotinic receptor in the brain that has
Smoking and Tobacco Control Monograph No. 2

234

so far eluded detection because it desensitizes extremely rapidly in the

presence of even low concentrations of nicotine (Couturier et al., 1990).

This receptor subtype, although physiologically important, may play no role

in nicotine dependence.

One way to study the pharmacological effects of “smoking doses” of

nicotine would be to expose animals to tobacco smoke. This has rarely been

attempted, partly because of practical difficulties, but also because such an

approach introduces unwanted chemicals and may be highly stressful.

Nevertheless, similar effects of smoke exposure and nicotine have been

reported (Fuxe et al., 1986).

Typically, nicotine is administered to conscious animals in single doses

which provide steady-state plasma levels similar to those measured in

habitual smokers. This sort of approach has been used to measure the

effects of nicotine on neuronal activity in the rat brain, as indicated by

uptake of radiolabeled 2-deoxyglucose (Grunwald et al., 1988; London et al.,

1988). By this index, nicotine activates a number of brain structures, and

the most affected structures are, with few exceptions, those that possess the

highest density of nicotinic receptors, as measured by autoradiography

using radiolabeled nicotine. These functional and receptor mapping studies

only roughly approximate actual smoking with the transient peaks that may

be attained through individual puffs. Taken together, however, they

strongly suggest that only certain nicotinic subtypes are significantly activated by doses of nicotine relevant to tobacco consumption.

Reinforcement

Through

Mesolimbic

Dopaminergic

Neurons

The reinforcing effects of nicotine in animals are of central origin

(see above). Nicotine activates a large number of brain regions,

many of which could conceivably participate in the drug’s reinforcing effects. However, one neuronal system calls for particular attention—the mesolimbic dopaminergic pathway. This nerve pathway is

one of several in the brain that secrete dopamine. Its cell bodies are located

in the ventral tegmental area (VTA) of the midbrain, and it projects to the

forebrain, where its major area of termination is the nucleus accumbens.

Animals will work hard to increase the release of dopamine from this latter

structure. This can be achieved either through electrical stimulation via

indwelling electrodes, or through the self-administration of certain drugs, of

which amphetamine is a good example (Wise and Rompre, 1989). Crudely,

then, this system can be looked upon as a “reward pathway,” and the

meager evidence available suggests this system serves a similar function in

humans.

The neurons that constitute the mesolimbic dopaminergic pathway

represent targets for nicotine (reviewed in Clarke 1990b and 1990c). Thus,

autoradiography has revealed nicotinic receptors both at the cell body/

dendrite level and on terminals in the nucleus accumbens. Electrophysiological experiments have shown that nicotine, applied directly, can increase the firing rate of these neurons, and biochemical studies have shown

that nicotine can act directly on terminals to promote the release of dopamine. In conscious rats, nicotine-induced dopamine release has been

documented in the nucleus accumbens. Furthermore, near-total destruction
Chapter 4

235

of the mesolimbic dopaminergic system markedly suppresses the rate of

nicotine self-administration. The latter observation suggests that nicotine,

like amphetamine, exerts its reinforcing effects to a large extent by activating the mesolimbic dopaminergic system.

All these findings are subject to an important caveat. The above studies

have firmly established that nicotine, given acutely, can stimulate the

release of mesolimbic dopamine. It remains an open question whether

nicotine would have the same effect, were it to be administered chronically

in such a way as to mimic the pharmacodynamic patterns of cigarette

smoking or tobacco chewing.

PHARMACOLOGICAL HELP

FOR QUITTING

TOBACCO USE

Limits of Current

Pharmacotherapies

Quitting the tobacco habit is notoriously difficult, and even

the best strategies are rather ineffective (US DHHS, 1988). The

most effective current methods for quitting are behavior-based,

and they place considerable demands on the time of health

personnel. For these reasons, it is worthwhile to seek a pharmacological aid for tobacco use cessation.

The only drug treatment readily available for those who wish

to quit using tobacco is nicotine replacement therapy. This approach

follows, logically enough, from the widely held view that habitual tobacco

use is a form of nicotine dependence. Although considerable success was

reported in early studies from cessation clinics using nicotine polacrilex

(nicotine gum), the cumulative results are less than dramatic. Lam and

coworkers (1987), in reviewing more than a dozen randomized, controlled

trials, concluded that nicotine gum was marginally more effective than

placebo gum, but in both cases, only a small minority of subjects remained

abstinent in the long run (23 percent and 13 percent, respectively, at

12 mo). Beside its modest efficacy, nicotine chewing gum is associated with

several undesirable side effects (Hughes, 1988).

As a form of drug delivery, the transdermal patch offers some distinct

advantages over nicotine gum. Nevertheless, the problem of efficacy remains. In a double-blind study, Abelin and colleagues (1989) reported a

statistically significant but small advantage to using nicotine patches; at the

end of 3 mo of treatment, 36 percent of subjects were abstinent or nearly so,

compared to 23 percent of controls receiving placebo. Rose and coworkers

(1990) reported higher rates of recidivism, with only 18 percent of subjects

remaining abstinent after 3 wk (compared to 6 percent of placebo controls).

In another recent study (Hurt et al., 1990), high rates of abstinence at 6 wk

were followed by substantial relapse, leading the authors to recommend

adjunctive behavioral intervention and training to address this problem.

Clearly, results to date with nicotine chewing gum and transdermal

patches have been disappointing. In the absence of other cessation strategies, they help only a minority of tobacco smokers achieve lasting abstinence. Can one hope that these treatments will be of more help for those

wishing to quit smokeless tobacco products? Nicotine polacrilex and the

transdermal patch release nicotine in a gradual manner that fails to mimic

the puff-by-puff exposure to nicotine that cigarette smoking affords
Smoking and Tobacco Control Monograph No. 2

236

(Benowitz et al., 1990). It has been hypothesized that, among many cigarette smokers, those transient boli of nicotine may play an important role in

the dependence process (Russell and Feyerabend, 1978). Blood nicotine

levels resulting from the use of ST (oral snuff, chewing tobacco), though, are

less transient in nature, and thus can be more closely modeled by gum or by

transcutaneous delivery (Benowitz et al., 1990). Nicotine gum and skin

patches offer complementary features. The former does not fully replace

blood nicotine levels, but offers the possibility of obtaining a boost of

nicotine when it is wanted; transdermal patches can deliver adequate total

amounts of nicotine, but administration is continuous.

Conditioning

And Effective

Pharmacotherapy

Many people give up the use of tobacco products for a few days

or weeks with little difficulty. Some even manage to remain

abstinent for a considerable time. Eventually, however, most

attempts to quit fail. The addiction can reassert itself by even a minor

relapse after years of abstinence. This “priming” effect is not unique to

nicotine, but has been reported also, for example, in cocaine abusers (Jaffe

et al., 1989).

Why does the abstinent individual remain susceptible to relapse? One

reason may be that ex-smokers remain partially tolerant to the nauseating

effects of nicotine and hence have less of a barrier to overcome than novice

smokers. The persistence of tolerance cannot, however, explain why after

the discomfort of quitting, and after experiencing the healthful, gustatory,

and economic advantages of cessation, so many tobacco users relapse.

Something is drawing them back.

This powerful force appears to be secondary reinforcement. Over the

course of their dependence, tobacco users are likely to associate many

stimuli with nicotine delivery. Many of these cues will initially be neutral,

but through repeated pairing with the primary reinforcer, some are likely to

assume reinforcing properties even in the absence of nicotine. The potency

of these secondary reinforcers is borne out by verbal reports of smokers. In

addition, laboratory studies have formally demonstrated the establishment

of secondary reinforcers in monkeys self-administering nicotine intravenously (Goldberg et al., 1981).

Nicotine Blockade

Therapy—

A Neglected

Approach

In my view, most tobacco users relapse because they retain the

well-learned connection between tobacco administration and

nicotine delivery. For example, an abstinent smoker knows that

he or she has only to start smoking a cigarette to experience

rapid reinforcement. This association, encoded in some form in the brain,

may underlie the phenomenon of craving. It follows that, to abjure the

tobacco habit permanently, one should extinguish these long-established

associations. Current smoking cessation methods do not provide for this

deprogramming (or, in psychological parlance, “extinction”).

The most effective extinction procedure would be one in which individuals intending to quit would be administered a drug that would block

the reinforcing effects of nicotine. They would then be encouraged to

continue to use tobacco products in their customary way for a limited time,

under the influence of the medication. It should be possible to achieve such
Chapter 4

237

a blockade by administering a nicotine receptor blocker that is centrally

active (Clarke, 1991; Stolerman, 1986). This approach has been largely

untried. Results to date, although preliminary, are encouraging (Tennant

et al., 1984). However, before nicotinic blockade therapy can be properly

tested in human subjects, there is a clear need for a nicotinic receptor

antagonist with a selective CNS action (Clarke, 1991).

REFERENCES

Abelin, T., Buehler, A., Muller, P., Vesanen, K.,

Imhof, P.R. Controlled trial of transdermal

nicotine patch in tobacco withdrawal. Lancet 1: 7-

10, 1989.

Adem, A., Jossan, S.S., d’Argy, R., Brandt, I., Winblad,

B., Nordberg, A. Distribution of nicotinic receptors

in human thalamus as visualized by 3

H-nicotine

and 3H-acetylcholine receptor autoradiography.

Journal of Neural Transmission 73: 77-83, 1988.

Benowitz, N.L., Porchet, H.C., Jacob, P. Pharmacokinetics, metabolism, and pharmacodynamics of

nicotine. In: Nicotine Psychopharmacology:

Molecular, Cellular, and Behavioural Aspects,

S. Wonnacott, M.A.H. Russell, and I.P. Stolerman

(Editors). Oxford: Oxford University Press, 1990,

pp. 112-157.

Boksa, P., Livett, B.G. Substance P protects against

desensitization of the nicotinic response in

isolated adrenal chromaffin cells. Journal of

Neurochemistry 42: 618-627, 1984.

Changeux, J.P., Devillers-Thiery, A., Chemouilli, P.

Acetylcholine receptor: An allosteric protein.

Science 225: 1335-1345, 1984.

Chesselet, M.-F. Presynaptic regulation of neurotransmitter release in the brain: Facts and

hypothesis. Neuroscience 12: 347-375, 1984.

Clarke, P.B.S. The central pharmacology of nicotine:

Electrophysiological approaches. In: Nicotine

Psychopharmacology: Molecular, Cellular, and

Behavioural Aspects, S.Wonnacott, M.A.H. Russell,

and I.P. Stolerman (Editors). Oxford: Oxford

University Press, 1990a, pp. 158-193.

Clarke, P.B.S. Dopaminergic mechanisms in the

locomotor stimulant effects of nicotine. Biochemical Pharmacology 40: 1427-1432, 1990b.

Clarke, P.B.S. Mesolimbic dopamine activation: The

key to nicotine reinforcement? CIBA Foundation

Symposia 152: 153-168, 1990c.

Clarke, P.B.S. Nicotine and smoking: A perspective

from animal studies. Psychopharmacology 92: 135-

143, 1987.

Clarke, P.B.S. Nicotinic receptor blockade therapy

and smoking cessation. British Journal of Addiction

86: 501-505, 1991.

Clarke, P.B.S., Schwartz, R.D., Paul, S.M., Pert, C.B.,

Pert, A. Nicotinic binding in rat brain: Autoradiographic comparison of 3

H-acetylcholine, 3Hnicotine, and 125I-alpha-bungarotoxin. Journal of

Neuroscience 5: 1307-1315, 1985.

Collins, A.C., Bhat, R.V., Pauly, J.R., Marks, M.J.

Modulation of nicotine receptors by chronic

exposure to nicotinic agonists and antagonists.

CIBA Foundation Symposia 152: 68-82, 1990.

Corrigall, W.A., Franklin, K.B.J., Coen, K.M., Clarke,

P.B.S. The mesolimbic dopaminergic system is

implicated in the reinforcing effects of nicotine.

Psychopharmacology, in press.

Couturier, S., Bertrand, D., Matter, J.M., Hernandez,

M.C., Bertrand, S., Millar, N., Valera, S., Barkas, T.,

Ballivet, M. A neuronal nicotinic acetylcholine

receptor subunit (alpha 7) is developmentally

regulated and forms a homo-oligomeric channel

blocked by alpha-BTX. Neuron 5: 847-856, 1990.

Deneris, E.S., Connolly, J., Rogers, S.W., Duvoisin, R.

Pharmacological and functional diversity of

neuronal nicotinic acetylcholine receptors. Trends

in Pharmacological Sciences 12: 34-40, 1991.

Fuxe, K., Andersson, K., Harfstrand, A., Agnati, L.F.

Increases in dopamine utilization in certain limbic

dopamine terminal populations after a short

period of intermittent exposure of male rats to

cigarette smoke. Journal of Neural Transmission 67:

15-29, 1986.

de la Garza, R., Bickford-Wimer, P.C., Hoffer, B.J.,

Freedman, R. Heterogeneity of nicotine actions in

the rat cerebellum: An in vivo electrophysiologic

study. Journal of Pharmacology and Experimental

Therapeutics 240: 689-695, 1987.

Goldberg, S.R., Spealman, R.D., Goldberg, D.M.

Persistent behavior at high rates maintained by

intravenous self-administration of nicotine.

Science 214: 573-575, 1981.

Grunwald, F., Schrock, H., Theilen, H., Biber, A.,

Kuschinsky, W. Local cerebral glucose utilization

of the awake rat during chronic administration of

nicotine. Brain Research 456: 350-356, 1988.

Hughes, J.R. Dependence potential and abuse

liability of nicotine replacement therapies.

Progress in Clinical and Biological Research 261: 261-

277, 1988.

Hurt, R.D., Lauger, G.G., Offord, K.P., Kottke, T.E.,

Dale, L.C. Nicotine-replacement therapy with use

of a transdermal nicotine patch: A randomized

double-blind placebo-controlled trial. Mayo Clinic

Proceedings 65: 1529-1537, 1990.

Jaffe, J., Cascella, N.G., Kumor, K.M., Sherer, M.A.

Cocaine-induced cocaine craving. Psychopharmacology 97: 59-64, 1989.
Smoking and Tobacco Control Monograph No. 2

238

Jaffe, J.H. Tobacco smoking and nicotine dependence. In: Nicotine Psychopharmacology: Molecular,

Cellular, and Behavioural Aspects, S. Wonnacott,

M.A.H. Russell, and I.P. Stolerman (Editors).

Oxford: Oxford University Press, 1990, pp. 1-37.

Jarvik, M.E., Assil, K.M. Mecamylamine blocks the

burning sensation of nicotine on the tongue.

Chemical Senses 13: 213-217, 1988.

Lam, W., Sze, P.C., Sacks, H.S., Chalmers, T.C. Metaanalysis of randomised controlled trials of nicotine

chewing gum. Lancet 2: 27-30, 1987.

London, E.D., Connolly, R.J., Szikszay, M., Wamsley,

J.K., Dam, M. Effects of nicotine on local cerebral

glucose utilization in the rat. Journal of Neuroscience 8: 3920-3928, 1988.

Luetje, C.W., Patrick, J. Both alpha- and betasubunits contribute to the agonist sensitivity of

neuronal nicotinic acetylcholine receptors. Journal

of Neuroscience 11: 837-845, 1991.

Navarro, H.A., Seidler, F.J., Schwartz, R.D., et al.

Prenatal exposure to nicotine impairs nervous

system development at a dose which does not

affect viability or growth. Brain Research Bulletin

23: 187-192, 1989.

Paton, W.D.M., Savini, E.C. The action of nicotine

on the motor endplate in the cat. British Journal of

Pharmacology 32: 360-380, 1968.

Rose, J.E., Tashkin, D.P., Ertle, A., Zinser, M.C., Lafer,

R. Sensory blockade of smoking satisfaction.

Pharmacology, Biochemistry and Behavior 23: 289-

293, 1985.

Rose, J.E., Levin, E.D., Behm, F.M., Adivi, C., Schur,

C. Transdermal nicotine facilitates smoking

cessation. Clinical Pharmacology and Therapeutics

47: 323-330, 1990.

Russell, M.A.H., Feyerabend, C. Cigarette smoking:

A dependence on high-nicotine boli. Drug

Metabolism Reviews 8: 29-57, 1978.

Schulz, D.W., Loring, R.H., Aizenman, E., Zigmond,

R.E. Autoradiographic localization of putative

nicotinic receptors in the rat brain using 125Ineuronal bungarotoxin. Journal of Neuroscience 11:

287-297, 1991.

Stolerman, I.P., Goldfarb, T., Fink, R., Jarvik, M.E.

Influencing cigarette smoking with nicotine

antagonists. Psychopharmacologia 28: 247-259,

1973.

Stolerman, I.P. Could nicotine antagonists be used

in smoking cessation? British Journal of Addiction

81: 47-53, 1986.

Swanson, L.W., Simmons, D.M., Whiting, P.J.,

Lindstrom, J. Immunohistochemical localization

of neuronal nicotinic receptors in the rodent

central nervous system. Journal of Neuroscience 7:

3334-3342, 1987.

Tennant, F.S., Jr., Tarver, A.L., Rawson, R.A. Clinical

evaluation of mecamylamine for withdrawal from

nicotine dependence. In: Problems of Drug

Dependence, 1983: Proceedings of the 45th Annual

Scientific Meeting, the Committee on Problems of Drug

Dependence, Inc., L.S. Harris (Editor). U.S. Department of Health and Human Services; Public

Health Service; Alcohol, Drug Abuse, and Mental

Health Administration. NIDA Research Monograph No. 49. DHHS Publication No. 84-1316,

1984, pp. 239-246.

U.S. Department of Health and Human Services.

Health Consequences of Smoking: Nicotine Addiction.

A Report of the Surgeon General, 1988. U.S. Department of Health and Human Services, Public

Health Service, Center for Chronic Disease

Prevention and Health Promotion, Office on

Smoking and Health. DHHS Publication No.

(CDC) 88-8406, 1988.

Wada, E., Wada, K., Boulter, J., Deneris, E.,

Heinemann, S., Patrick, J., Swanson, L.W.

Distribution of alpha 2, alpha 3, alpha 4, and

beta 2 neuronal nicotinic receptor subunit mRNAs

in the central nervous system: A hybridization

histochemical study in the rat. Journal of Comparative Neurology 284: 314-335, 1989.

Wise, R.A., Rompre, P.-P. Brain dopamine and

reward. Annual Review of Psychology 40: 191-225,

1989.

Wong, L.A., Gallagher, J.P. A direct nicotinic

receptor-mediated inhibition recorded intracellularly in vitro. Nature 341: 439-442, 1989.
Chapter 4

239

One of the key issues determining the threat to public health from smokeless tobacco is the

Dependence on Smokeless Tobacco

Martin J. Jarvis1

ABSTRACT

degree of dependence experienced by users. Data documenting nicotine absorption and

dependence on smokeless tobacco products are reviewed. Single case histories of individuals

who either chewed cigarette butts or brushed snuff into their gums provide evidence of

compulsive behavioral rituals accompanied by substantial uptake of the drug nicotine. Studies

of dependent users of moist oral or dry nasal snuff show rapid absorption of nicotine from a

single pinch and blood levels from normal use that parallel those seen in cigarette smokers.

Among users of Swedish oral snuff, blood nicotine levels and subjective dependence were

similar to those of cigarette smokers. These observations, which are derived from relatively few

subjects, indicate that nicotine intake exerts a controlling influence over self-administration of

smokeless tobacco products and that subjective dependence may be no less than among

cigarette smokers. Available data from short-term cessation have been interpreted as showing

that smokeless tobacco users experience less severe withdrawal effects than do cigarette

smokers. Further studies on cessation of smokeless tobacco are needed.

INTRODUCTION The potential burden to society from the use of smokeless tobacco

depends both on the threat it poses to health and on the users’ degree of

addiction. With cigarette smoking, we see hundreds of thousands of deaths

each year in the United States because tobacco smoke is exceptionally

damaging to health and because smokers, despite awareness and acceptance

of the risks, find it very difficult to overcome their addiction. If addiction to

oral and nasal snuff is less tenacious than addiction to cigarettes, smokeless

tobacco users will find it easier to quit. But if dependence on smokeless

tobacco turns out to be as strong as it is with tobacco smoke, the trends

toward increasing prevalence of use, particularly by the young, may create

problems in the future.

In considering the addictiveness of smokeless tobacco, the U.S. Surgeon

General drew on three lines of evidence (US DHHS, 1986). The first two

related to the presence of nicotine in smokeless tobacco and evidence of its

absorption. This led to the following conclusion: “Given the nicotine

content of smokeless tobacco, its ability to produce high and sustained

blood levels of nicotine, and the well-established data implicating nicotine

as an addictive substance, one may deduce that smokeless tobacco is capable

of producing addiction in users.” Direct evidence of addiction to smokeless

tobacco, which was the third line of evidence, was at that time rather

scanty, and comprised mainly reports of withdrawal symptoms upon

cessation of smokeless tobacco (Hatsukami et al., 1987) or nicotine gum use

(Hughes et al., 1986; West and Russell, 1985).

This paper follows a similar course, (1) reviewing our laboratory evidence of patterns of nicotine absorption from smokeless tobacco products

and (2) presenting some new data that attempt to link nicotine absorption

with the crucial issue of feelings of subjective dependence.

1 The support of the Medical Research Council and the Imperial Cancer Research Fund is

gratefully acknowledged.
Smoking and Tobacco Control Monograph No. 2

240

Frequently we are approached for advice about chronic psychiatric

patients who do not smoke but who use tobacco in unusual ways

and whose behavior puzzles those who care for them. Two such

cases are presented briefly here.

NICOTINE

UPTAKE FROM

UNUSUAL USE

OF TOBACCO

A woman of Indian origin, diagnosed as schizophrenic, had the habit

of brushing her gums with a toothbrush that had been dipped in dry nasal

snuff (Edwards, 1987). She spent up to 9 h/d in this activity, could not

explain why she did it, and became tense and irritable if deprived of snuff.

On a day when she was permitted to apply 1.4 g of snuff in this way for

half an hour in the morning, her blood nicotine rose from a baseline of

7.3 ng/mL to peak some 4 h later at 35.5 ng/mL, a value similar to that seen

in heavy cigarette smokers.

More recently, we were contacted about a mentally handicapped

resident at a long-stay hospital who regularly collected and consumed the

butt-ends from used cigarettes. Apparently the patient swallowed the

cigarette butts, and the expressed concern was more about the aesthetics of

the behavior and disruption of life on the ward than about potential addiction. A blood specimen was taken at 5 p.m. on a day when he had consumed six filter tips. The blood nicotine concentration was found to be

29.2 ng/mL, and the corresponding cotinine was 622.2 ng/mL. Again, the

nicotine level is comparable with that seen in dependent smokers, whereas

the cotinine concentration, at about double the average in smokers, reflects

extensive first-pass metabolism of swallowed nicotine by the liver, preventing nicotine from reaching the systemic circulation.

Evidence from single cases such as these has obvious limitations, and

explanations couched solely in behavioral terms rather than invoking

nicotine dependence cannot be completely ruled out. Nevertheless, that

such compulsive rituals should give rise to blood nicotine levels characteristic of cigarette smoking is suggestive.

NICOTINE

ABSORPTION

FROM DRY

NASAL SNUFF

Smokeless tobacco is used in Britain mainly as finely ground tobacco

sniffed into the nose. In a habitual user, there was an increase in

blood nicotine concentration of 21.1 ng/mL over 5 min from a single

pinch (Russell et al., 1980). This evidence of rapid absorption

prompted the study of a group of snuff users in the West of England (Russell

et al., 1981). Among seven daily users, the average trough blood nicotine

was 21.9 ng/mL, rising to 34.5 ng/mL after their taking a pinch of their

usual snuff in their usual way. Both the trough and the postdosing peak

were close to levels observed in a comparison group of smokers before and

after a cigarette (see Figure 1). In interpretation of the results, it was suggested that the similarity was unlikely to be coincidental (Russell et al.,

1981):

To find that one group of people who sniff powdered tobacco into their

noses have similar blood nicotine concentrations to those of another

group who burn it to inhale its smoke suggests that the concentration

of nicotine has some controlling influence. It would be a remarkable

coincidence if factors such as flavor, strength of tobacco, social influences, and so on just happened to produce similar blood nicotine
Chapter 4

241

Figure 1.

Average plasma nicotine levels before and after a pinch of snuff (n=7) or a

cigarette (n=13). Tobacco use had been normal for both groups up to the time of

the test.

Source: Russell et al., 1981; used with permission.

concentrations resulting from two such different behaviors. The most

plausible explanation is that the rituals of snuffing and smoking are

determined by the nicotine concentrations that they produce.

Several of these snuff users took unusually large multiple doses, as

practiced in snuff-taking competitions. The mean increment in plasma

nicotine observed was 54 ng/mL. In one subject, who was the current

British champion, a boost of 97 ng/mL was obtained over 12.5 min (Russell

et al., 1981). This man reported a very high degree of dependence. He said

that he “was always on it [snuff],” and even woke several times each night

to take a pinch.

NICOTINE

INTAKE AND

DEPENDENCE

IN SWEDEN

The above results paint a convincing picture of the use of smokeless

tobacco as a drug-taking behavior. However, subjective aspects of

dependence were not systematically studied. We have recently

examined nicotine absorption and measures of dependence in users

of Swedish moist oral snuff (Holm et al., in press). Snuff has a long tradition

in Sweden, and its use is currently on the increase, particularly among the

young (Nordgren and Ramström, 1990). About 20 percent of adult Swedish

males take snuff.

Two studies were conducted. The first examined absorption from a

single dose of 2 g kept in the mouth for 30 min. Among 10 subjects,

the peak increment in blood nicotine concentration, which averaged

14.6 ng/mL, was observed at 35 min, shortly after the snuff was discarded.

The pattern of absorption showed an average increase of 9.9 ng/mL in the

Before After

0

20

25

30

35

40

Plasma Nicotine (ng/mL)

Cigarette Smokers

Snuff Users
Smoking and Tobacco Control Monograph No. 2

242

first 10 min, with a slower rise thereafter, and a relatively flat plateau maintained up to 1 h. The two individuals with the most rapid absorption had

increments of 12.8 ng/mL and 16.2 ng/mL in the first 5 min of dosing.

These results, which are similar to findings from the United States

(Benowitz et al., 1988; Gritz et al., 1981), showed substantial absorption of

nicotine and indicated that a blood specimen taken between 5 and 15 min

after the snuff is discarded should adequately capture the peak blood nicotine level. Thus, these results were used to inform the design of the second

study, which combined measures of nicotine uptake and subjective dependence in regular snuff takers (n=27) and cigarette smokers (n=35).

On a day of normal snuff use or smoking, blood specimens were taken

from snuff users 5 to 15 min after they discarded a pinch of the subjects’

usual snuff taken in the usual way, and from cigarette smokers 1 min after

their smoking a cigarette of their regular brand. A questionnaire assessed

various aspects of dependence. The postdosing nicotine concentrations

were similar in the snuff users and smokers (36.6 ng/mL and 36.7 ng/mL,

respectively) and were also similar to the earlier findings with British dry

snuff users (Russell et al., 1981). On questionnaire measures of dependence,

there were no significant differences between the snuff users and smokers in

self-assessed addiction, craving for tobacco, or difficulty in giving up. The

majority in each case rated themselves as fairly or extremely addicted, they

frequently or always craved when without their snuff or cigarettes, and they

anticipated that giving up would be very difficult. However, the snuff users

found their habit much more enjoyable (16 of 27 rated it as “extremely

enjoyable,” compared with 3 of 35 smokers, p < 0.0001) and rated enforced

abstinence for an hour or two as more unpleasant. For their part, the

smokers were significantly more likely to have their first cigarette of the day

before tea or coffee than were the snuff users (p < 0.01).

The picture that emerges from these results is of regular snuff users and

cigarette smokers as being remarkably similar in both nicotine intake and

dependence. Latency to first tobacco use of the day, which is perhaps the

best indicator of dependence among cigarette smokers (Heatherton et al.,

1989), was shorter in the smokers, but it seems likely that this disparity

reflects the incompatibility between taking snuff and ingesting food or

drink rather than any difference in dependence. Of more interest is the

apparently greater enjoyment of snuff use than of smoking. Rather than

resulting from intrinsic differences in the rewarding qualities of the two

habits, it may be that cigarette smokers now carry with them a permanent

awareness of the health risk and the increasingly unfavorable climate of

social opinion. If snuff users, as seems likely, view their habit as safer than

smoking, they are that much freer to indulge without worry or guilt.

Limitations on the generalizability of these findings stem from the

small sample size and the possibility that both the snuff users and cigarette

smokers might not have been representative of the wider population of

users. Nevertheless, there is a strong indication that among groups of snuff

users and smokers matched for nicotine levels, subjective dependence is also

likely to be similar.
Chapter 4

243

The data reviewed so far lead to the conclusion that nicotine exerts a

controlling influence on the maintenance of smokeless tobacco use

and that regular users experience levels of dependence similar to

CESSATION OF

SMOKELESS

TOBACCO USE

those of tobacco smokers. But it does not necessarily follow that cessation

of smokeless tobacco use will be equally difficult. It could be the same,

more difficult, or less difficult. One factor that might contribute to making

cessation more difficult is the perception that risks to health from smokeless

tobacco are lower. Motivation to quit and level of dependence are not

entirely independent in their effects on probability of cessation. At a given

level of dependence, a lower level of concern about potential damage to

health will reduce the discomfort the person is prepared to tolerate to

achieve cessation. On the other hand, it has been suggested that the lack of

bolus effects and the lower frequency of reinforcement from smokeless

tobacco may make it easier to quit than cigarettes (Hatsukami, 1991; West

and Krafona, 1990).

Only limited data are available on acute withdrawal effects from smokeless tobacco. Hatsukami and associates (1987) reported a range of withdrawal symptoms, including craving for tobacco, and raised total scores on

self-rated and observer-rated checklists. However, the symptoms were less

intense and fewer in number than among a comparison group of cigarette

smokers. Decrements in performance measures have also been reported

after short-term withdrawal from smokeless tobacco (Keenan et al., 1989).

There is an urgent need for further studies in this area.

The scope for successful long-term cessation is even less clear. Among

adolescents, 30 percent of smokeless tobacco users believed that quitting

their habit would be very difficult, compared with 23 percent of cigarette

smokers (Brownson et al., 1990). In the 1986 Adult Use of Tobacco Survey

in the United States, 39 percent of current users of smokeless tobacco had

attempted to quit, and of these, 47 percent reported experiencing difficulty

in doing so (Novotny et al., 1989). The implications of these findings for

actual cessation are unclear. Few cessation studies have been undertaken,

and those on a small scale (Eakin et al., 1989; Glover, 1986).

FUTURE

RESEARCH NEEDS

A number of studies have examined the prevalence of smokeless

tobacco use, both in the general population and in subgroups

such as male adolescents (US DHHS, 1989). However, dependence, which in

conjunction with prevalence of use and health risks will determine the

extent of future morbidity and mortality, has received less attention. Available evidence indicates that dependence on smokeless tobacco may be no

less tenacious than dependence on cigarettes. This conclusion is based on a

limited range of studies on relatively few subjects. A clearer picture will

emerge if future surveys include a number of items to assess the degree of

subjective dependence. It would also be informative to look not only at

whether users of smokeless tobacco see their habit as a threat to health, but

also at how hazardous they perceive it to be in comparison with cigarette

smoking and the extent to which they see it as a personal risk. These factors

have important implications for motivation to quit.
Smoking and Tobacco Control Monograph No. 2

244

The extent and severity of withdrawal symptoms and ways of facilitating

long-term cessation of smokeless tobacco use are important areas for future

research. If the analogy with cigarette smoking is valid, the majority of those

who wish to quit will do so by themselves, without assistance. This means

that motivation derived from public health campaigns will be crucial, and

much will depend on epidemiologists’ clarifying issues such as the magnitude

of the excess mortality attributable to long-term use of smokeless tobacco.

For those who do seek help, there would seem to be a natural affinity

between smokeless tobacco use and nicotine replacement methods. Nicotine

chewing gum is itself an oral tobacco product, though purified of carcinogens. Nicotine skin patches, which give substantial plateau levels of blood

nicotine without rapid absorption and without providing a behavioral ritual,

may also have a part to play.

REFERENCES

Benowitz, N.L., Porchet, H., Sheiner, L., Jacob, P. III.

Nicotine absorption and cardiovascular effects

with smokeless tobacco use: Comparison with

cigarettes and nicotine chewing gum. Clinical

Pharmacology and Therapeutics 44: 23-28, 1988.

Brownson, R.C., DiLorenzo, T.M., Van Tuinen., M.,

Finger, W.W. Patterns of cigarette and smokeless

tobacco use among children and adolescents.

Preventive Medicine 19: 170-180, 1990.

Eakin, E., Severson, H., Glasgow, R.E. Development

and evaluation of a smokeless tobacco cessation

program: A pilot study. National Cancer Institute

Monographs 8: 95-101, 1989.

Edwards, J.G. An unusual case of nicotine dependence. Psychological Medicine 7: 779-781, 1987.

Glover, E.D. Conducting smokeless tobacco

cessation clinics. American Journal of Public Health

76: 207, 1986.

Gritz, E.R., Baer-Weiss, V., Benowitz, N.L., Van

Vanakis, H., Jarvik, M.E. Plasma nicotine and

cotinine concentrations in habitual smokeless

tobacco users. Clinical Pharmacology and Therapeutics 30: 201-209, 1981.

Hatsukami, D.K., Gust, S.W., Keenan, R.M. Physiologic and subjective changes from smokeless

tobacco withdrawal. Clinical Pharmacology and

Therapeutics 41: 103-107, 1987.

Hatsukami, D.K., Nelson, R., Jensen, J. Smokeless

tobacco: Current status and future directions.

British Journal of Addiction 86: 559-563, 1991.

Heatherton, T.F., Kozlowski, L.T., Frecker, R.C.,

Rickert, W.S., Robinson, J. Measuring the

heaviness of smoking using self-reported time to

the first cigarette of the day and number of

cigarettes smoked per day. British Journal of

Addiction 84: 791-800, 1989.

Holm, H., Jarvis, M.J., Russell, M.A.H., Feyerabend, C.

Nicotine intake and dependence in Swedish snuff

takers. Psychopharmacology, in press.

Hughes, J.R., Hatsukami, D.K., Skoog, K. Physical

dependence on nicotine gum: A placebo-substitution trial. Journal of the American Medical Association

255: 3277-3279, 1986.

Keenan, R.M., Hatsukami, D.K., Anton, D.J. The

effects of short-term smokeless tobacco deprivation

on performance. Psychopharmacology 98: 126-130,

1989.

Nordgren, P., Ramström, L. Moist snuff in Sweden:

Tradition and evolution. British Journal of Addiction

85: 1107-1112, 1990.

Novotny, T.E., Pierce, J.P., Fiore, M.C., Davis, R.M.

Smokeless tobacco use in the United States: The

Adult Use of Tobacco Surveys. National Cancer

Institute Monographs 8: 25-28, 1989.

Russell, M.A.H., Jarvis, M.J., Devitt, G., Feyerabend, C.

Nicotine intake by snuff users. British Medical

Journal 283: 814-817, 1981.

Russell, M.A.H., Jarvis, M.J., Feyerabend, C. A new age

for snuff? Lancet 1: 474-475, 1980.

U.S. Department of Health and Human Services.

Smokeless Tobacco Use in the United States: National

Cancer Institute Monograph No. 8. U.S. Department of

Health and Human Services, Public Health Service,

National Institutes of Health, National Cancer

Institute. NIH Publication No. 89-3055, 1989.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes of

Health, National Cancer Institute. DHHS Publication No. (NIH) 86-2874, 1986.

West, R., Krafona, K. Oral tobacco: Prevalence, health

risks, dependence potential and public policy.

British Journal of Addiction 85: 1097-1098, 1990.

West, R.J., Russell, M.A.H. Effects of withdrawal from

long-term nicotine gum use. Psychological Medicine

15: 891-893, 1985.
Chapter 5

245

Chapter 5

Prevention

CONTENTS Marketing Smokeless Tobacco:

Implications for Preventive Education

Carol N. D’Onofrio .............................................................................. 247

Introduction ................................................................................. 247

Current Preventive Education...................................................... 248

Health Education vs. Marketing .................................................. 249

Implications of ST Marketing....................................................... 251

References ..................................................................................... 256

Applying the Social Inoculation Model to a

Smokeless Tobacco Use Prevention Program

With Little Leaguers

Richard I. Evans, Bettye E. Raines, and J. Greg Getz ........................... 260

Introduction ................................................................................. 260

Evolution of the Strategy ............................................................. 261

Data-Based Interventions ............................................................. 264

The Houston Little League Project ............................................... 268

Potential Barriers .......................................................................... 270

Conclusion ................................................................................... 273

References ..................................................................................... 273
Smoking and Tobacco Control Monograph No. 2

246
Chapter 5

247

Efforts to prevent smokeless tobacco use are essentially reactions to a problem created by

Marketing Smokeless Tobacco:

Implications for Preventive Education

Carol N. D’Onofrio

ABSTRACT

the tobacco industry. Although the public health community has applied lessons learned

from 25 years of progress in preventing cigarette smoking, current preventive efforts do not

adequately counteract aggressive product marketing. In spite of fundamental differences

between health education and commercial marketing, examination of the industry’s success

can stimulate new ideas for ST education. Such analysis also reveals some idiosyncrasies of the

public health culture that may now limit the effectiveness of interventions to prevent ST use.

INTRODUCTION The marketing of moist snuff in the United States has been profitable for the U.S. Tobacco Company. After snuff was packaged in round tin

cans, sales more than doubled between 1974 and 1984 (Bantle, 1980; Negin,

1985). In 1983, the introduction of snuff in small premeasured pouches

boosted sales still higher (Dougherty, 1984; O’Connor, 1983; Tobacco

Reporter, 1983). The company gained entry to the Fortune 500 in 1985,

showing the greatest profit margin of any company its size or larger (Business Week, 1986; Mintz, 1986; U.S. Tobacco, 1985). During the past 6 yr,

snuff sales and profits have continued to climb (FTC, 1991; Smith, 1989),

moving U.S. Tobacco up 81 positions in rank among the Nation’s 500

largest corporations (U.S. Tobacco, 1988 and 1991). Citing its financial

strength and proven business strategy, the company confidently forecasts

continuing growth (U.S. Tobacco, 1990). Other sources concur, observing

that increased bans on cigarette smoking should benefit the ST industry

(Deveny, 1990; Ellis, 1989; Smyth, 1989).

Although this record is celebrated in the business world as a spectacular

success, health professionals view it with alarm. Tobacco contains known

carcinogens, and the link between snuff use and oral cancer has been firmly

established (IARC, 1985; Mattson and Winn, 1989; Office of Medical Applications of Research, 1986; US DHHS, 1986; Winn et al., 1981; WHO, 1988).

Sharp increases in ST use portend the emergence of a major public health

problem.

Fortunately, lessons learned from 25 yr of progress in the prevention

and control of cigarette smoking (US DHHS, 1989) have been applied to

smokeless tobacco with little delay. Scientific evidence about the health

consequences of snuff dipping and tobacco chewing has been consolidated,

reviewed, and publicized (IARC, 1985; Office of Medical Applications of

Research, 1986; US DHHS, 1986; WHO, 1988). Legislation has banned ST

advertising in the broadcast media, required warning labels on product

packages and advertisements, restricted the sale of ST to minors in many

1 Supported in part by National Cancer Institute grant no. CA-41733.

1
Smoking and Tobacco Control Monograph No. 2

248

states, and increased taxes on ST products (Connolly et al., 1986; Deveny,

1990; US DHHS, 1989 and 1990a). Research has been funded to develop

and test interventions to prevent ST use and encourage cessation (US DHHS,

1990b). In addition, many health agencies and professional groups have

produced educational materials for use in schools and community-based

tobacco control programs. Work in these areas continues.

Experience from antismoking campaigns indicates that, if these efforts

can be sustained, advances in science, policy, and public education will slow

the increase in ST prevalence (US DHHS, 1989; Warner, 1982, 1986, and

1989) and eventually turn the tide toward a decrease. However, there will

be a time lag, in part because of activities of the ST industry. Research on

the carcinogenic properties of ST and the health effects of its use will be

complicated by the continual introduction of new products and product

variations. Resulting gaps in scientific knowledge will be cited to confound

debates about additional policy proposals. Public education will struggle

not only against competing priorities in schools, communities, and the

media, but also against relentless ST marketing. Moreover, public health

workers in each of these areas will compete with each other for scarce

resources, while the ST industry will easily finance its expansion through fat

profits.

These realities imply that current public health strategies for preventing

and controlling ST use must be bolstered by creative new approaches.

Because promising directions for basic scientific research, public policy, and

cessation programs are discussed elsewhere in this monograph, this paper

concentrates on preventive education. First, the need for innovation is

emphasized through a brief review of current educational efforts and what

they can accomplish. This analysis is then contrasted with ST marketing to

identify some specific deficiencies in education, as well as some unconventional ways they might be remedied.

CURRENT

PREVENTIVE

EDUCATION

At present, education about ST includes public information, educational programs for youth, and some related education of teachers,

parents, and health professionals. Most youth education is delivered

through schools, often in conjunction with education aimed at smoking

prevention. No national data are available on the extent or quality of

school-based instruction about ST, but it certainly is less adequate than

antismoking education, which itself is highly variable (D’Onofrio, 1989; US

DHHS, 1989 and 1990a).

Many schools provide no health education course or regularly scheduled time for health education in the curriculum. In some schools, some

teachers discuss various health topics in science or other courses, but instruction is not systematic and tobacco use may not be covered. Schools

with organized health education programs generally teach about tobacco

and other substances in a special unit. Alcohol and illicit drugs usually

receive the greatest attention, although cigarette smoking causes more

deaths annually than all other substances combined (Warner, 1987).

Smokeless tobacco receives still shorter shrift; for example, when a single

50-min class period covers tobacco in all forms, it is likely that smoking, not
Chapter 5

249

smokeless tobacco, will be emphasized. In the war on drugs, most of the

nation’s schools do not identify tobacco as a major enemy.

On the positive side, several state-of-the-art ST prevention programs

have been developed and field-tested through research grants from the

National Cancer Institute (Boyd and Glover, 1989; US DHHS, 1989 and

1990b). These programs tend to parallel the more effective smoking prevention programs in that they are guided by a social influences model of smoking initiation and are typically directed to children between the ages of 10

and 14. With the exception of a program for 4-H Club members in California and one for Little League baseball teams in Texas, NCI-sponsored prevention programs are conducted in school classrooms by specially trained

health educators, college students, or regular teachers, sometimes with the

assistance of peer leaders. Their objectives are to provide information about

the short-term health and social consequences of ST use; correct misconceptions about the pervasiveness and social acceptability of the practice; reveal

how parental modeling, peer pressure, and the media promote ST use; and

teach resistance skills. Although evaluation of these programs is still under

way, preliminary analyses suggest that, like most theory-based smoking

prevention programs (Botvin et al., 1990; Ellickson and Bell, 1990; Evans et

al., 1981; Flay, 1985; Flay et al., 1985 and 1989; Gersick et al., 1988; Glynn,

1989; Graham et al., 1990; Hansen et al., 1988a and 1988b; Johnson et al.,

1986; Luepker et al., 1983; Murray et al., 1988 and 1989; Pentz et al., 1989a

and 1989b; Schinke and Gilchrist, 1984; Telch et al., 1982), they will have

positive albeit modest effects on youth least likely to use tobacco.

Although early smoking prevention programs failed to show behavioral

effects (Flay, 1985; Thompson, 1978), results from the first generation of ST

prevention studies are encouraging. Knowledge gained from efforts to

prevent cigarette smoking over the past quarter-century (US DHHS, 1989)

has been well applied to the ST problem. A challenge for the future is to

promote adoption of NCI-sponsored programs as they become available for

widespread use by schools and other organizations serving youth. The first

of these projects is now being disseminated (D’Onofrio et al., 1991), and

others will soon follow.

As these materials are used and others are developed, a second challenge will be to assure that the principles that led to progress continue to be

applied. Thus (1) program design should be guided by behavioral theory,

(2) content should extend well beyond the health dangers of ST use,

(3) participatory instructional methods should be used, (4) young people

should have an opportunity to develop and practice skills for resisting

temptations to use ST, (5) teachers should be trained in program delivery,

and (6) as appropriate, same-age or older peers should be recruited and

trained to assist (Glynn, 1989).

HEALTH

EDUCATION vs.

MARKETING

Despite early indications that preventive education is on a promising track, current educational efforts pale in comparison to ST

marketing. To a certain extent this will always be the case, for

health education and marketing differ in several fundamental respects.
Smoking and Tobacco Control Monograph No. 2

250

The goal of the smokeless tobacco industry is to enlarge the market for

its products, while each company aims to improve its market share. Increasing sales by a few percentage points is regarded as a triumph. Public health,

on the other hand, tries to protect entire populations. Because preventive

education is evaluated against this absolute standard, programs that reduce

tobacco use by a few percentage points are viewed as only marginally

effective.

Marketing goals are expressed in positive terms like “increase, capture,

build, and acquire.” These words convey and invite initiative. However, in

aiming to “reduce, curtail, decrease, and delay” ST use, the goals of public

health carry negative connotations. These differences are reflected in

themes and messages used in marketing and health education campaigns.

Whereas tobacco advertising urges positive and expansive action (“buy this,

try that”), health education warns, “don’t do it.”

A marketing campaign is initiated by a single corporation after a long

period of careful market analysis and product development. The campaign

typically is planned to run for several years, with intermediate objectives

delineating shorter phases within a well-orchestrated long-term strategy.

Resources are assured to execute the plan, which is sufficiently flexible to

respond to unanticipated threats or opportunities. Marketing budgets are

generous. In 1989, ST manufacturers spent $81 million advertising and

promoting their products (Federal Trade Commission, 1991).

In contrast, health education is conducted by multiple agencies, groups,

and individuals with a vast array of objectives and priorities. These priorities, the resources available to support them, and organizational leadership

shift with economic and political currents. Health care professionals who

recognize the need for initiatives to prevent ST use must struggle in this

milieu to convince others that a problem exists, but agencies differ in the

amount and quality of scientific evidence they require to include ST on their

agenda. Once an agency has acknowledged the problem, competition for

resources begins.

Agency plans for preventive education frequently are prepared for

review by fiscal decisionmakers. Program objectives are shaped not only by

the degree to which agency executives and staff understand the ST problem

and state-of-the-science approaches to prevention, but also by the agency’s

mission, commitments, assets, constituent expectations, and time pressures.

Optimal proposals for prevention therefore tend to be compromised in the

budgetmaking process. Once plans are approved, implementation is expected to begin immediately, often with little regard to what others are

doing.

Because agencies tend to plan independently, local, state, and national

resources for the prevention of ST use are accumulated sporadically. Educational programs and materials enter the field in an unsystematic and unpredictable flow. These interventions vary in the effectiveness with which they

target key issues in prevention, and many of them have not been fieldtested or evaluated prior to broad distribution. Although companies that
Chapter 5

251

market ST carefully select and limit their product lines, educational approaches to prevention proliferate with little quality control.

These factors complicate the knitting of fragmented educational elements into a coordinated master plan for prevention. When planning can

be accomplished, the result is not streamlined or efficient, but at best a

patchwork. In this pluralistic society, institutional autonomy is protected,

and enthusiasm for close interorganizational collaboration is frequently

lukewarm.

Commercial marketing and health education also differ in the products

that they offer. ST products are tangible and cheap; good health education

is neither. Moreover, while ST fosters habituation and dependence, health

education promotes independence, active decisionmaking, and informed

choices. If marketing persuades youth to try ST enough times, nicotine

helps to recruit permanent customers (Boyd et al., 1987; US DHHS, 1990b;

WHO, 1988). Health education is directed to a more fundamental and

continuing change called learning.

Nevertheless, those who market ST are clever in applying learning

principles and appealing to the basic needs of children and adolescents.

Advertising promotes the fantasy that, with just a can of snuff or a pouch of

chew, young people can satisfy their curiosity, demonstrate their maturity

and independence, enjoy adventures, belong to an admired group, and

enhance their social image. Youth must be highly sophisticated to view the

development of resistance skills as an achievement. And of course, learning

how to resist temptation, defer gratification, communicate effectively, win

friends, and make good decisions is much harder than taking a dip or a

chew, especially when the tobacco industry provides free samples and stepby-step instructions for use (Ernster, 1989).

Finally, ST marketing and health education are separated by deep

differences in the values and ethics that underlie attempts to influence

behavior. Human concern and conscience preclude health educators’

promoting products that harm the consumer and from using some strategies

that are effective in building business profits.

IMPLICATIONS OF

ST MARKETING

Education to prevent ST use is essentially reactive to a problem

created by the tobacco industry. For most of this century, the ST

market was stagnant or shrinking (Tye et al., 1987; US DHHS, 1986). National surveys conducted between 1964 and 1975 found the prevalence of

ST use to be fairly stable at less than 5 percent and use rates to be highest

among persons over age 50 (US DHHS, 1986). However, in the early 1970’s,

the industry extended its product lines and began aggressive marketing to

males between ages 18 and 30, with a “substantial emphasis on the 18 to

24 group” (Maxwell, 1980).

The results have been well documented. Dramatic increases in advertising budgets for moist snuff were soon paralleled by sharp increases in

production and sales (Rosenthal, 1985; US DHHS, 1986). National surveys

of adults conducted between 1985 and 1987 found startling shifts in patterns of ST use, with older adolescents and young adult males using the
Smoking and Tobacco Control Monograph No. 2

252

products at a higher rate than any other age group (Marcus et al., 1989;

Novotny, et al., 1989; Orlandi and Boyd, 1989). Although the ST industry

has steadfastly insisted that its products are meant for adults, national and

regional surveys also have reported high rates of use among young boys and

adolescents (Bauman et al., 1989; Boyd et al., 1987; Glover, 1986; Orlandi

and Boyd, 1989; Rouse, 1989). And whereas earlier ST use was largely

restricted to rural areas in the South and the West, it is now reported in all

regions of the country (Orlandi and Boyd, 1989).

The ST industry, health professionals, and other analysts all recognize

clever and aggressive marketing as a major force in these changes (Christen

et al., 1982; Connolly et al., 1986; Deveny, 1990; Glover et al., 1981 and

1982; Hunter et al., 1986; Shelton, 1984; Smyth, 1989; US DHHS, 1986; U.S.

Tobacco, 1985; WHO, 1988). Sophisticated marketing is also identified as a

major obstacle in achieving national health objectives for the prevention

and control of tobacco use (US DHHS, 1990a). Detailed descriptions of the

creative strategies employed by ST companies can be found in industry

publications, trade journals, news magazines, and the health literature

(Anderson et al., 1979; Braverman et al., 1989; Christen et al., 1982;

Connolly et al., 1986; Deveny, 1990; Dougherty, 1984; Ernster, 1986 and

1989; Feigelson, 1983; Glover et al., 1981 and 1982; Harper, 1980; Maxwell,

1983; Mintz, 1986; Negin, 1985; O’Conner, 1983; Rosenthal, 1985; Shelton,

1982; Tobacco Reporter, 1983; US Tobacco and Candy Journal, 1987).

Those responsible for preventive interventions have used this information to understand the parameters of the problem, to estimate its magnitude, to identify fruitful policy directions, and to acquaint youth with

persuasive advertising strategies. To date, however, knowledge about ST

marketing has not been systematically applied either in the design of

counteractive educational programs or in evaluation of current educational

efforts.

Such omissions leave health education continually defending against

aggressive ST marketing teams that are committed to keeping the competition under fire. To increase the chances for success, health educators need

to study the opposition, strengthen their defense, and develop new offensive moves. As the following examples illustrate, examining secrets of the

industry’s success can stimulate new ideas for ST education and reveal some

idiosyncrasies of the public health culture that may be limiting the effectiveness of current approaches to prevention.

Market Research Commercial marketing invests heavily in research and product

testing before a campaign is launched. Once a campaign is under way, its

effectiveness is evaluated very simply by changes in sales. In contrast,

public health provides few resources for market research to guide the design

of educational programs. Health education is initiated as soon as possible

and then subjected to rigorous evaluation. To do it right in public health,

educational interventions must be subjected to longitudinal randomized

trials in defined populations with biochemical validation of self-reports and
Chapter 5

253

appropriate units of statistical analysis. The effectiveness of ST use prevention might be increased through a shift of resources from extensive evaluation of educational programs after they are delivered, toward more frontend research to guide initial development and refinement.

Market Definition Although the ST industry has identified males aged 18 to 24 as

its primary marketing target (Maxwell, 1980), public health researchers have

assumed that prevention will be most effective during initial experimentation with dipping and chewing. Because the onset of cigarette smoking

peaks during the transition from elementary to middle or junior high school

(Flay et al., 1983), resources for ST education have been concentrated on

those aged 10 to 14. The need for preventive programs for younger children

is now recognized, but older adolescents and young adults are discussed

only as targets for ST cessation.

Nonetheless, among adults who use ST regularly, the median age of

initiation for both snuff and chewing tobacco is 19 (Novotny, 1989). Although this figure may drop with the maturing of youth who grew up and

began to use ST during intense marketing, it suggests that the initiation of

ST use may occur throughout adolescence and into early adulthood. Preliminary data from a California survey (California Department of Health

Services, 1990), presented in Table 1, further indicate that many young

adults who dip and chew are not yet addicted to daily use. Preventive

education, therefore, should be directed not only to children and young

adolescents, but also to high school students and to young men making the

transition into college or the workplace. Like ST marketing, health education should especially target young men entering blue collar occupations in

factories, on farms, or in industries such as lumber, steel, fishing, and

firefighting.

Market

Segmentation

Smokeless tobacco marketing is precisely targeted. According to an

executive of U.S. Tobacco:

We’ve built our business by identifying the “pockets” of Americans

whose lifestyles include smokeless tobacco . . . . Other consumer

product companies are beginning to realize what we’ve understood all

along. America is not necessarily a “mass-market”; it is more a collection of “micro-markets” or regional markets defined by a variety of

factors (including age, sex, occupation, and hobbies), which may be

broadly defined as consumer “lifestyle” (Africk, 1985).

To date, education to prevent ST use has been only modestly adapted

to reach different segments of the youth population. Current projects with

4-H, Little League, and Native Americans provide an important start in

tailoring prevention programs to the groups at high risk for ST use. However, within these market segments, youth are still treated very much alike.

Health education thus needs to seek additional sites and alternative models

for preventing ST use among youth at greatest risk. Like U.S. Tobacco, we

should learn to “fish where the fishing is good” (Deveny, 1990).
Smoking and Tobacco Control Monograph No. 2

254

Table 1

Current use of smokeless tobacco as a weighted percentage of California males,

aged 18 to 44, who have ever used chewing tobacco or snuff

Age 18 to 24 Age 25 to 44

Chewing Tobacco

n 172 393

Daily use 4.5% 6.6%

< daily use 18.9 9.6

Snuff

n 141 318

Daily use 7.9% 8.5%

< daily use 11.8 9.1

School-based programs treat all students as potential users, but most

girls and many boys do not like ST and never intend to use it. Teaching

such youngsters skills to resist ST offers probably is not the best use of

educational time and resources. The limited effectiveness of school-based

programs in preventing high-risk youth from using tobacco raises questions

about optimal use of classroom time. With increasing pressures on their

curricula, some schools already are reluctant to commit multiple sessions to

tobacco education. Youth groups that hold only weekly or monthly meetings also hesitate to devote a high proportion of their program time to

prevention of tobacco use. Ironically, this problem will be exacerbated if

rates of cigarette smoking and ST use among young people begin to fall.

Maintaining and increasing the support of schools for preventive

education thus requires the development of programs that are relevant to all

youth served and that advance basic organizational objectives. One possible

approach is to emphasize the rights of individuals to a tobacco-free environment and to present tobacco use as a social as well as a personal health

issue. Some programs already are teaching children how to ask others not

to use tobacco in their presence, to discourage friends from trying tobacco,

and to support users who are trying to quit (D’Onofrio, 1991; D’Onofrio et

al., 1991). Development of these skills in communication, social relations,

and social problem-solving nurtures children’s development while simultaneously establishing their social identity as non-users of tobacco.

An extension of this approach is to involve youth as active partners in

the planning, delivery, and evaluation of school- and community-based

prevention programs. Their creativity should be tapped in the design and

production of educational materials and presentations. Young people

should testify at city council meetings on proposed tobacco control ordinances. They should appear on radio and television to debate the issues,

discuss their efforts, and showcase the songs, poems, skits, and artwork they

have produced. They should be recruited to help with surveillance of ST

promotions. Youth also can participate effectively in sting operations to

identify merchants who illegally sell ST or distribute free samples to minors.
Chapter 5

255

Assuming responsibility for adult tasks satisfies young people’s curiosity, fosters their maturity and independence, provides them with new

experiences, and enables them to affiliate with admired adults, including

doctors, dentists, attorneys, community activists, media and sports personalities, law enforcement officers, and others. Recognition of their capabilities and talents also enhances the self-image of youth, while participation

with others in the fight against tobacco deepens their sense of community

and creates future leaders. By responding to the developmental needs of

children and youth, these activities lead to outcomes that are widely recognized as protective against all forms of substance use (Hawkins et al., 1985).

Product

Development

U.S. Tobacco tailors its products to promote their acceptance. Packaging snuff in round tin cans was instrumental in increasing consumer

awareness: having a round can of snuff in the back pocket of blue jeans

became a status symbol among young males (Negin, 1985). Skoal Bandits

were designed to provide new users with a gradual introduction to moist

snuff, as well as to overcome some of the messiness associated with ST use

and to increase its acceptability in the urban environment (Bantle, 1980;

Dougherty, 1984; Maxwell, 1983; Negin, 1985; O’Conner, 1983). Prevention programs might similarly benefit from more creative packaging to

attract interest, and from product diversification to encourage easy trial,

which leads to more regular use of stronger products. However, to avoid

dilution of educational efforts, such initiatives should be part of a larger

strategy designed to promote eventual adoption of more potent programs.

Another industry principle that merits consideration is product variation to assure consumer choice. Both ST products and the strategies used to

market them are tailored to diverse tastes and customs. The public health

community, though, attributes great importance to the generalizability of

preventive programs and their potential for widespread acceptance. Possibly, tailoring health education programs to a variety of micro-markets

would prove more effective than developing generic programs for mass

distribution. An additional principle borrowed from the ST industry indicates that such innovations may win quicker acceptance if they are introduced not as new educational products, but as extensions of programs

already known and popular (Smyth, 1989).

Product

Distributors

U.S. Tobacco considers its sales force a key component of success.

Nationwide in 1986, the company had 436 field representatives who

provide retailers and customers with one-on-one attention (U.S. Tobacco,

1986). Specific functions include assuring fresh products, promoting optimal display, demonstrating use, obtaining customer feedback, and identifying potential areas for development (Deveny, 1990; U.S. Tobacco, 1986 and

1990). These salesman also court retailers with recognition, prizes, tickets to

sporting events, and invitations to social gatherings.

In addition, representatives of U.S. Tobacco serve as “traveling billboards” for the company (Hawkins et al., 1985). Like chameleons, they

adapt to the local culture and provide exciting in-the-flesh advertisements

for chewing and dipping. We have encountered a salesman in southern

California driving a red Ferrari. In Bakersfield, a sales representative in a
Smoking and Tobacco Control Monograph No. 2

256

hard hat was observed distributing free samples in the oil fields. And in a

rural mountain county, we found an ST representative wearing a cowboy

hat and driving a pickup truck with country-and-western music on the radio

and a can of Copenhagen on the dash.

Those who market prevention programs rarely display such cultural

adaptability or such verve. Too often, these programs are simply delivered

to schools and other organizations with the expectation that they will be

used. Given numerous barriers in program dissemination (D’Onofrio, 1989;

Glynn, 1989), employing field representatives to nurture the adoption

process appears worth a try. As far as possible, these distributors of prevention should establish warm relations with program intermediaries and

consumers. In addition, their manner and their personage should demonstrate that tobacco-free lifestyles are both accessible and exciting to youth

and young adults within the context of unique community cultures.

Development

Of the Delivery

System

The promotion of smokeless tobacco is pervasive and continuous.

However, prevention education typically is presented only at

schools and for a limited time. To counter subtle, sophisticated,

and omnipresent promotion of tobacco products, adults who spend time

with children should be enlisted in the preventive effort. Ideally, they

should provide education spontaneously as issues about the sale, promotion, and use of smokeless tobacco arise in the course of daily life

(D’Onofrio, 1991). That is, when parents, youth leaders, and others observe

ST being used, they need to discuss the practice as dangerous and socially

unacceptable. During visits to the corner store, attendance at baseball

games and other events, or while watching the Indianapolis 500 on television, adults should point out how ST is marketed. In these conversations,

adults also need to help children understand why a bad product is so readily

available, why some good people use it, and why the Government does not

ban substances that are harmful. Answering children’s questions about ST is

likely to require explaining history, politics, government, law, economics,

and other complex aspects of society.

The educational challenge is formidable, but if young people are to

make wise decisions about ST, both as individuals and as community

members, adults in all walks of life must rise to the task. Nurturing the

positive development of children and youth through education is public

health’s most powerful approach to prevention.

REFERENCES

Africk, J. The right to choose. US Tobacco Review

1(3), Third Quarter, 1985.

Anderson, H., Boerger, G., Leslie, C. A tobacco boom

with no smoke. Newsweek, August 20, 1979, p. 67.

Bantle, L.F. Smokeless tobacco: A trend to watch.

Tobacco Journal International 4: 344-346, 1980.

Bauman, K.E., Koch, G.G., Fisher, L.A., Bryan, E.S.

Use of smokeless tobacco by age, race, and gender

in ten standard metropolitan statistical areas of the

southeast United States. National Cancer Institute

Monographs 8: 35-37, 1989.

Botvin, G.J., Baker, E., Filazzola, A.D., et al. A

cognitive-behavioral approach to substance abuse

prevention: One-year followup. Addictive

Behaviors 15: 47-63, 1990.

Boyd, G.M., Ary, D.V., Wirt, R., et al. Use of

smokeless tobacco among children and adolescents in the United States. Preventive Medicine 16:

402-421, 1987.

Boyd, G.M., Glover, E.D. Smokeless tobacco use by

youth in the U.S. Journal of School Health 59(5):

189-194, 1989.
Chapter 5

257

Braverman, M.T., D’Onofrio, C.N., Moskowitz, J.M.

Marketing smokeless tobacco in California

communities: Implications for health education.

National Cancer Institute Monographs 8: 79-85, 1989.

Business Week. What could burn the king of

smokeless tobacco. Business Week, March 17,

1986, p. 86.

California Department of Health Services. Tobacco

Use in California 1990: A Preliminary Report

Documenting the Decline of Tobacco Use. San Diego:

University of California, 1990.

Christen, A.G., Swanson, B.Z., Glover, E.D., et al.

Smokeless tobacco: The folklore and social history

of snuffing, sneezing, dipping, and chewing.

Journal of the American Dental Association 105: 821-

829, 1982.

Connolly, G.N., Winn, D.M., Hecht, S.S., et al. The

reemergence of smokeless tobacco. New England

Journal of Medicine 31(16): 1020-1027, 1986.

D’Onofrio, C.N. Making the case for cancer prevention in the schools. Journal of School Health 59(5):

225-230, 1989.

D’Onofrio, C.N. Tobacco Talk: Educating Young

Children About Tobacco. Santa Cruz, CA: Network

Publications, 1991.

D’Onofrio, C.N., Braverman, M.T., Moskowitz, J.M.,

et al. The Project 4-Health Tobacco Education

Program. School of Public Health, University of

California, Berkeley, 1991.

Deveny, K. With help of teens, snuff sales revive.

Wall Street Journal, May 3, 1990, p. B1.

Dougherty, P.H. Moving smokers to snuff. New York

Times, January 13, 1984.

Ellickson, P.L., Bell, R.M. Drug prevention in junior

high: A multi-site longitudinal test. Science

247(16): 1299-1305, 1990.

Ellis, J. Stocks picked by 1988’s A-Team smartly

outpaced the market’s record run. Money, October

1989, p. 122.

Ernster, V.L. Advertising and promotion of smokeless tobacco products. National Cancer Institute

Monographs 8: 87-94, 1989.

Ernster, V.L. Advertising of smokeless tobacco

products. Presented at the NIH Consensus

Development Conference on Health Implications

of Smokeless Tobacco Use, Bethesda, Maryland,

January 1986.

Evans, R.I., Rozelle, R.M., Maxwell, S.E., Raines, B.E.,

Dill C.A., Guthrie, T.J. Social modeling films to

deter smoking in adolescents: Results of a threeyear field investigation. Journal of Applied Psychology 66(4): 399-414, 1981.

Federal Trade Commission. Report to Congress,

Pursuant to the Comprehensive Smokeless

Tobacco Health Education Act of 1986. FTC,

Washington, D.C., 1991.

Feigelson, J. Skoal Bandit blitz kicks off N.Y. entry.

Advertising Age, August 8, 1983, p. 46.

Flay, B.R. Psychosocial approaches to smoking

prevention: A review of findings. Health Psychology 4(5): 449-488, 1985.

Flay, B.R., d’Avernas, J.R., Best, J.A., Kersell, M.W.,

Ryan, K.B. Cigarette smoking: Why young people

do it and ways of preventing it. In: Pediatric and

Adolescent Behavioral Medicine, P. Firestone and

P. McGrath (Editors). New York: Springer-Verlag,

1983, pp. 132-183.

Flay, B.R., Koepke, D., Thompson, S.J., Santi, S., Best,

J.A., Brown, K.S. Six-year followup of the first

Waterloo school smoking prevention trial.

American Journal of Public Health 79(10): 1371-

1376, 1989.

Flay, B.R., Ryan, K.B., Best, J.A., Brown, K.S., Kersell,

M.W., d’Arvenas, J.R., Zanna, M.P. Are socialpsychological smoking prevention programs

effective? The Waterloo Study. Journal of

Behavioral Medicine 8(1): 37-59, 1985.

Gersick, K.E., Grady, K., Snow, D.L. Social-cognitive

skill development with sixth graders and its initial

impact on substance use. Journal of Drug Education

18(1): 55-67, 1988.

Glover, E.D. Regional prevalence and patterns of

smokeless tobacco use. Presented at the NIH

Consensus Development Conference on Health

Implications of Smokeless Tobacco Use, Bethesda,

Maryland, January 1986.

Glover, E.D., Christen, A.G., Henderson, A.H. Just a

pinch between the cheek and gum. Journal of

School Health 51: 415-418, 1981.

Glover, E.D., Christen, A.C., Henderson, A.H.

Smokeless tobacco and the adolescent male.

Journal of Early Adolescence 2(1): 1-13, 1982.

Glynn, T.J. Essential elements of school-based

smoking prevention programs: Research results.

Journal of School Health 59: 181-188, 1989.

Graham, J.W., Johnson, C.A., Hansen, W.B., et al.

Drug use prevention programs, gender, and

ethnicity: Evaluation of three seventh-grade

project SMART cohorts. Preventive Medicine 19:

305-313, 1990.

Hansen, W.B., Johnson, C.A., Flay, B.R., Graham,

J.W., Sobel, J. Affective and social influences

approaches to the prevention of multiple

substance abuse among seventh grade students:

Results from Project SMART. Preventive Medicine

17: 135-154, 1988a.

Hansen, W.B., Malotte, C.K., Fielding, J.E. Evaluation of a tobacco and alcohol abuse prevention

curriculum for adolescents. Health Education

Quarterly 15(1): 93-114, 1988b.

Harper, S. In tobacco, where there’s smokeless

there’s fire. Advertising Age, June 23, 1980.
Smoking and Tobacco Control Monograph No. 2

258

Hawkins, D.J., Lishner, D.M., Catalano, R.F. Childhood predictors of adolescent substance abuse. In:

Etiology of Drug Abuse: Implications for Prevention,

C.L. Jones and R.J. Battjes (Editors). NIDA

Research Monograph 56, DHHS Publication (ADM)

85-1335. Washington, DC: Government Printing

Office, 1985.

Hunter, S.M., Croft, J.B., Burke, G.L., et al. Longitudinal patterns of cigarette smoking and smokeless

tobacco use in youth: The Bogalusa Heart Study.

American Journal of Public Health 76: 193-195, 1986.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosamines. (volume 37). Lyon:

IARC, 1985.

Johnson, C.A., Hansen, W.B., Collins, L.M., Graham,

J.W. High school smoking prevention: Results of

a three-year longitudinal study. Journal of

Behavioral Medicine 9(1): 439-452, 1986.

Luepker, R.V., Johnson, C.A., Murray, D.M.,

Pechacek, T.F. Prevention of cigarette smoking:

Three-year followup of an education program for

youth. Journal of Behavioral Medicine 6: 53-62,

1983.

Marcus, A.C., Crane, L.A., Shopland, D.R., Lynn,

W.R. Use of smokeless tobacco in the United

States: Recent estimates from the Current

Population Survey. National Cancer Institute

Monographs 8: 17-23, 1989.

Mattson, M.E., Winn, D.M. Smokeless tobacco:

Association with increased cancer risk. National

Cancer Institute Monographs 8: 13-16, 1989.

Maxwell, J.C. Chewing, snuff is growth segment.

Advertising Age, June 23, 1980.

Maxwell, J.C. Smokeless tobacco volume sales drop.

Advertising Age, June 16, 1986, p. 62.

Maxwell, J.C., Jr. Smokeless keeps growing: Cigars

keep declining. Tobacco International, July: 90-91,

1983.

Mintz, M. The artful dodgers. Washington Monthly,

October 1986, pp. 9-16.

Murray, D.M., Davis-Hearn, M., Goldman, A.I., Pirie,

P., Luepker, R.V. Four- and five-year followup

results from four seventh-grade smoking prevention strategies. Journal of Behavioral Medicine 11(4):

395-405, 1988.

Murray, D.M., Pirie, P., Luepker, R.V., Pallonen, V.

Five- and six-year followup results from four

seventh-grade smoking prevention strategies.

Journal of Behavioral Medicine 12: 207-218, 1989.

Negin, E. Just a pinch between your cheek and gum.

Public Citizen, Spring: 28-32, 1985.

Novotny, T.E., Pierce, J.P., Fiore, M.C., Davis, R.M.

Smokeless tobacco use in the United States: The

Adult Use of Tobacco Surveys. National Cancer

Institute Monographs 8: 25-28, 1989.

O’Conner, J.J. Bandits out to steal bite from

cigarettes. Advertising Age, June 1983, pp. 2, 27.

Office of Medical Applications of Research. Health

implications of smokeless tobacco use. Journal of

the American Medical Association 255: 1045-1048,

1986.

Orlandi, M.A., Boyd, G. Smokeless tobacco use

among adolescents: A theoretical overview.

National Cancer Institute Monographs 8: 5-12, 1989.

Pentz, M.A., Dwyer, J.H., MacKinnon, D.P., Flay,

B.R., Hansen, W.B., Wang, E.Y.I., Johnson, C.A. A

multicommunity trial for primary prevention of

adolescent drug abuse: Effects on drug use

prevalence. Journal of the American Medical

Association 261(22): 3259-3266, 1989a.

Pentz, M.A., MacKinnon, D.P., Dwyer, J.H., et al.

Longitudinal effects of the midwestern prevention

project on regular and experimental smoking in

adolescents. Preventive Medicine 18: 304-321,

1989b.

Rosenthal, J. Son of the Marlboro Man. Washington

Monthly, March 1985, p. 51.

Rouse, B.A. Epidemiology of smokeless tobacco use:

A national study. National Cancer Institute

Monographs 8: 29-33, 1989.

Schinke, S.P., Gilchrist, L.D. Preventing cigarette

smoking with youth. Journal of Primary Prevention

5: 48-56, 1984.

Shelton, A. Moist snuff leads American market.

Tobacco Reporter 111(7): 30-31, 1984.

Shelton, A. Smokeless sales continue to climb.

Tobacco Reporter 109(8): 42-44, 1982.

Smyth, J. Moist snuff sales gain. Tobacco Reporter

116(8): 30-31, 1989.

Telch, M.J., Killen, J.D., McAlister, A.L., et al. Longterm followup of a pilot project on smoking

prevention with adolescents. Journal of Behavioral

Medicine 5(1): 1-8, 1982.

Thompson, E.L. Smoking education programs,

1960-76. American Journal of Public Health 68: 250-

257, 1978.

Tobacco Reporter. Smokeless tobacco: The bright

star of the U.S. market. Tobacco Reporter 110(11):

68-69, 1983.

Tye, J.B., Warner, K.E., Glantz, S.A. Tobacco

advertising and consumption: Evidence of a

causal relationship. Journal of Public Health Policy,

Winter: 492-508, 1987.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986.
Chapter 5

259

U.S. Department of Health and Human Services.

Promoting Health/Preventing Disease: Year 2000

Objectives for the Nation (conference edition). U.S.

Department of Health and Human Services, Public

Health Service, 1990a.

U.S. Department of Health and Human Services.

Reducing the Health Consequences of Smoking: 25

Years of Progress. A Report of the Surgeon General.

U.S. Department of Health and Human Services,

Public Health Service, Centers for Disease Control,

Center for Chronic Disease Prevention and Health

Promotion, Office on Smoking and Health. DHHS

Publication No. (CDC) 89-8411, 1989.

U.S. Department of Health and Human Services.

Smoking, Tobacco, and Cancer Program: 1985-1989

Status Report, D.R. Shopland and M.M. Massey

(Editors). U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. NIH

Publication No. 90-3107, 1990b.

U.S. Tobacco. 75 years of marketing and advertising.

US Tobacco Review 2(1): 10-12, 1986.

U.S. Tobacco. U.S. Tobacco rises to Fortune 500

Status. US Tobacco Review 1(1): 1, 1985

U.S. Tobacco. UST, 1988 First Quarter Report.

Greenwich, CT: U.S. Tobacco, 1988.

U.S. Tobacco. UST, 1990 Annual Report. Greenwich,

CT: U.S. Tobacco, 1990.

US Tobacco and Candy Journal. Corporate sponsors

drive the right messages home. US Tobacco and

Candy Journal, July 27-August 24, 1987, pp. 46, 84.

Warner, K.E. Effects of the antismoking campaign:

An update. American Journal of Public Health 79(2):

144-151, 1989.

Warner, K.E. Health and economic implications of a

tobacco-free society. Journal of the American

Medical Association 258(15): 2080-2086, 1987.

Warner, K.E. Selling Smoke: Cigarette Advertising and

Public Health. Washington, DC: American Public

Health Association, 1986.

Warner, K.E., Murt, H.A. Impact of the antismoking

campaign on smoking prevalence: A cohort

analysis. Journal of Public Health Policy 3(4): 374-

390, 1982.

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W.,

Toledo, A., Fraumeni, J.F., Jr. Snuff dipping and

oral cancer among women in the Southern United

States. New England Journal of Medicine 304: 745-

749, 1981.

World Health Organization. Smokeless Tobacco

Control: Report of a WHO Study Group. Geneva,

Switzerland: WHO, 1988. WHO Technical Report

Series 773.
Smoking and Tobacco Control Monograph No. 2

260

This paper first considers the question of how research on smoking prevention among

Applying the Social Inoculation Model

To a Smokeless Tobacco Use Prevention

Program With Little Leaguers

Richard I. Evans, Bettye E. Raines, and J. Greg Getz

ABSTRACT

adolescents can become the basis for developing programs to prevent the use of smokeless

tobacco. Within this context, the evolution of the social inoculation strategy is discussed. The

paper encompasses procedures we used in developing, implementing, and evaluating such

programs, drawing from our current National Cancer Institute-supported project dealing with

prevention of ST use among Little League baseball players. Recommendations for public policy

related to prevention of ST use are offered.

INTRODUCTION The invitation to this conference provides an opportunity to

describe how health promotion investigators, employing a social influence

orientation, can consider the cross-application of well-studied models from

cigarette smoking to the emerging problem of smokeless tobacco use. To

introduce the discussion, we describe the evolution of the “social inoculation strategy” originated by our research group to prevent cigarette smoking

among adolescents (Evans, 1976; Evans et al., 1978 and 1981). Variations of

this approach have been extended to the prevention of use of harmful

substances such as alcohol and illicit drugs (Botvin and Wills, 1985; Flay,

1985), suggesting that its application to preventing ST use may help meet

stated research goals (Chassin et al., 1989; Evans and Raines, 1990). Our

current NCI-supported ST use prevention program, which involves a large

population of Little League baseball players, is designed to test the efficacy

of such a cross-application (Evans and Raines, 1990).

We emphasize both psychosocial and methodological barriers to the

successful development, implementation, and evaluation of tobacco use

prevention programs, drawing on our broader program of health-related

research and on the Little League baseball project specifically for examples.

We define methodological problems to include the structural-organizational

barriers inherent in research conducted in natural settings.

While we are pleased that our approach has proved valuable in prevention efforts, we are concerned that one technique of this overall prevention

strategy—”Just Say No” to resist peer pressure—has been taken out of

context and redirected in simplistic form as a formula for preventing all

substance abuse. In our original program, “Just Say No” was presented as a

resistance response to low-level peer pressure and was only one of a series of

1 Support provided in part by National Cancer Institute grants no. CA-41722 and no.

CA-41471.

1
Chapter 5

261

responses and strategies designed to address increasing levels of peer pressure as well as other social influences in the young adolescent’s environment. Because of the current pervasiveness of the catchphrase, we emphasize that “Just Say No” is not enough! Such generalized use of a single

component of prevention strategy is something about which prevention

program planners should be cautioned (Evans, 1988).

EVOLUTION OF

THE STRATEGY

Our smoking prevention research program evolved from a series of

studies during the 1960’s, in which we attempted to apply socialpsychological theories and strategies to problems in preventive dentistry

(i.e., effective toothbrushing and flossing among a population of junior high

school students). The results of these studies (Evans et al., 1970 and 1975)

were encouraging, including the successful use of a chemical measure, a

tooth-staining disclosant that allowed us to compare the effects on oral

hygiene of actual vs. reported toothbrushing behavior (Evans et al., 1968).

Several findings from these studies proved useful as we entered the next

phase of our research program:

• the probability that reported health behavior may not correlate with

actual behavior;

• the observation that fear arousal alone in persuasive messages may

not be the most effective means of influencing health enhancement

behavior;

• the fact that community institutions such as schools may lend themselves as settings for implementing persuasive health messages in a

systematic and controlled manner; and

• the importance of tailoring both content and style of health promotion messages to the target audiences in such settings.

In 1973 we began addressing why young adolescents begin smoking

cigarettes even when they are fully aware of the related health dangers. An

extensive survey of junior high students in the Houston Independent School

District (Evans and Raines, 1982) identified several problems related to

preventive education about smoking in the school setting. These problems

created barriers to effective communication of important health messages to

adolescents. A student survey indicated that smoking prevention programs

in the curriculum

• focused too heavily and inappropriately on fear arousal;

• emphasized the long-term consequences of cigarette smoking (e.g.,

heart disease and cancer), failing to recognize that young adolescents

are more present- than future-oriented;

• used audiovisuals (e.g., films, videotapes) and other materials in

general use that had not been evaluated for their relevance to adolescents; and

• were predicated on the notion that mere awareness of a health threat

leads to the desired health enhancement behaviors.
Smoking and Tobacco Control Monograph No. 2

262

According to this survey, not only were students unresponsive to such

programs, but in some cases, it appeared that the health education messages

delivered were counterproductive.

In addition to the student survey findings, the evaluation of concurrent

in-place school smoking prevention programs identified two underlying

problems that might be addressed in a well-planned intervention study:

(1) Other than their reliance on imparting information about the dangers of

a potentially harmful behavior, the programs rarely had any guiding theoretical conceptualization; and (2) in the development of interventions,

program planners did not seek feedback from their target audiences.

To address such barriers to the understanding and acceptance of important health messages and, more critically, to elicit appropriate behavioral

action as a result of exposure to the messages, we undertook a longitudinal

investigation using a large population of junior high school students as they

entered seventh grade. Because the development of our intervention

strategy is described elsewhere (Evans, 1982, 1984, and 1990; Evans et al.,

1984a and 1984b), in this paper we only outline the development of this

program.

Results from an initial survey that included fifth- through ninth-grade

students indicated a significant increase in experimental cigarette smoking

at about the time students entered junior high school. At the time, junior

high schools in Houston included grades seven through nine. Thirteen

junior high schools were selected for assignment to various experimental

conditions, and two were chosen as resource schools to be used in the

development and pretesting of procedures and materials.

We established priorities in developing our research program, dictating

that (1) it was guided by appropriate theoretical considerations and (2) it

reflected input from the target population. In addition to drawing on

various psychosocial-behavioral theories and models, which are described in

more detail below, we instituted a systematic process assessment of the

knowledge, experiences, perceptions, beliefs, behaviors, and terminology of

the target audience. From the beginning, we attempted to establish and

maintain a feedback loop, which we defined as the linkage between the

content of persuasive messages created for disease prevention research

programs and data from the target audiences (Evans et al., 1984a). We thus

relied heavily on formative evaluation as we developed and implemented

our intervention strategies (Evans et al., 1989).

We worked from the premise that tobacco use, although an age-related

behavior, occurs most often within the context of social interactions; and

although it involves the use of cognitive or knowledge structures, social

adaptation appears to override intellectual adaptation or knowledge in

decisionmaking. Social learning theory (e.g., Bandura, 1977; Evans, 1989)

appeared to be particularly relevant. As applied to smoking initiation, the

theory suggests that through observation children acquire expectations and

learned behaviors vis-à-vis smoking. We considered that vicariously learned

positive or negative consequences of cigarette smoking might be important

factors in the decision to start smoking. Smoking behavior and expressed
Chapter 5

263

smoking-related attitudes of peers, family, and media figures could be

expected to affect the adolescent’s smoking-related attitudes, beliefs, values,

expectations, and learned behaviors. Young adolescents often perceive

smoking as glamorous or as a behavior distinctive to adults. Because people

tend to imitate the actions of their models (Bandura and Huston, 1961), we

expected smokers in the young person’s social setting to have an influence

out of proportion to their numbers.

Adolescents tend to overestimate the proportion of individuals who

smoke. Subjects in our investigations believed that “almost everyone” in

their age group smoked, although the data indicated that only a relatively

small percentage actually did (Evans et al., 1984b). Perceptions of group

norms, of course, can constitute a vicarious peer pressure in influencing the

behavior of adolescents (e.g., Ajzen and Fishbein, 1980; Evans et al., 1988;

Fishbein and Ajzen, 1975). Recent studies (Graham et al., 1991; MacKinnon

et al., in press) demonstrate that modifying subjects’ perceptions of group

norms, as a component of prevention programs, may sometimes be stronger

than a peer pressure resistance training component. We therefore incorporated modifying perceptions to conform to reality into our social inoculation strategy.

Another concept for addressing the problem of smoking prevention is

the theory of reasoned action (Ajzen and Fishbein, 1980; Fishbein and

Ajzen, 1975). It proposes a framework for predicting behavioral intentions,

which are assumed to mediate and thus predict subsequent overt behavior.

This approach suggested empirically testable hypotheses that could reveal

important components of the development of smoking behavior.

Personality characteristics of the subject may also interact with social

influences to encourage smoking. Bandura (1977) lists three characteristics

that appear to facilitate imitative learning: (1) low self-esteem, (2) dependence or powerlessness, and (3) a history of receiving frequent rewards

contingent on engaging in an imitative behavior.

The model shown in Figure 1 reflects the possibility that both social

environmental and personality determinants contribute to the complex of

psychological predispositions producing an intention to smoke or to not

smoke. The actual decision on a particular occasion may, of course, depend

on the effect of situational social influences. Teaching adolescents to cope

with such influences might decrease the probability that they will smoke.

Ideally, a prevention program would incorporate all the components of

the model reflected in Figure 1. In the development of our research program, however, we encountered time constraints and barriers to access to

potential study populations in the school district in which we were working

that precluded programs designed to modify the social environment within

the schools. Therefore, inoculation against social influences on the individual to smoke became our primary focus. We described this initial socialinfluences approach in our 3-yr field investigation (e.g., Evans, 1976; Evans

et al., 1978; Evans et al., 1981; Evans et al., 1984a) as the “social inoculation

strategy.”
Smoking and Tobacco Control Monograph No. 2

264

Figure 1

A model of smoking-related social psychological processes that have impact on

behavior

In developing our interventions, we tried to create messages to

which adolescents would listen and on which they might act.

DATA-BASED

INTERVENTIONS

We were guided, to some extent, by Laswell’s classic social-communication

model (Laswell and Casey, 1946), McGuire’s (1969 and 1974) informationprocessing communication model, and, as previously mentioned, the model

of an ongoing feedback loop (Evans et al., 1984a) that allowed specific

tailoring of the program to the target audience. (See Figure 2.)

In discussing the development of our interventions, we might first

consider the source of the communication in terms of Laswell’s model. (See

Figure 3.) Instead of adults who could be perceived to represent power,

which adolescents might reject, our messages were delivered by adolescent

narrators, selected for their perceived acceptability by or attractiveness to

junior high school students. Although a certain amount of peer credibility

General Social Milieu

Smoking-related behavior and 

expressed attitudes of peers, 

siblings, parents, respected 

adults, and media figures

Personality Factors

• Low self-esteem

• Dependency or powerlessness

• Frequent reward for imitative 

 behavior

Negative Intention

General decision 

AGAINST smoking

Specific social 

influence to smoke on 

a specific occasion

Positive Intention

General decision in 

FAVOR of smoking

Behavior

Refrain from smoking

Behavior

Engage in smoking

The process of making 

socially mediated, 

specific decisions

Psychological Predispositions

Smoking-related attitudes,

beliefs, values, expectations, 

learned behaviors

Negative Positive
Chapter 5

265

Figure 2

A model of ST-related social psychological processes that have impact on behavior

Intrapersonal or

Personality Factors

• Dependency or powerlessness

• Frequency of reward for 

 imitative behavior

• Coping and resistance 

 strategies

• Low self-esteem

• Self-image

• Knowledge of social pressure

• Locus of control

• Self-efficacy

Prior Experience

Personal previous 

experience with

smokeless tobacco

Negative Intention

General decision AGAINST 

using smokeless tobacco

Positive Intention

General decision

in FAVOR of using 

smokeless tobacco

Behavior

Refrain from using 

smokeless tobacco

Behavior

Engage in the use of 

smokeless tobacco

The process of 

making socially 

mediated, specific 

decisions

Psychological Predispositions

Knowledge, beliefs, perceptions of social expectations, 

personal expectations, values, attitudes, and learned behaviors 

concerned with the use of smokeless tobacco

General Social 

Environment

• Smokeless 

 tobacco-related 

 behavior and 

 expressed attitudes

of peers, siblings, 

and parents

• Media influences

Specific Social Influence

To use smokeless tobacco 

on a specific occasion

= Intrapersonal 

 factors

= Cognitive

 process

= Environmental

 factors

was inherent in their appearance as actors, in their presentation, the narrators acted as information brokers rather than authority figures. Increased

credibility accrued through the narrators’ prefacing biopsychosocial information with such phrases as “The researchers have found . . .” or “The

researchers have asked me to tell you . . . .”
Smoking and Tobacco Control Monograph No. 2

266

Figure 3

Synthesis of three social-communication models: external properties

The next component of Laswell’s model addresses the message. Based

on the literature (Janis and Feshbach, 1953; Leventhal et al., 1965; Sutton,

1982) and findings of our previous research (Evans et al., 1970 and 1975;

Evans, 1979), that fear arousal does not provide adequate long-term motivation to accept or comply with health-related messages, we relegated fear

arousal as only one aspect of our messages. We thus moved away from

merely emphasizing the more global approach of the period (e.g., “Smoking

is bad for you.”) to include messages specific to the adolescent’s social and

developmental level (Evans, 1979; Evans and Raines, 1982; Leventhal et al.,

1965).

McGuire’s information-processing communication model (McGuire,

1969 and 1974) was particularly useful in developing our intervention. As

indicated by this model, to be effective, messages must be (1) attended to

and (2) comprehended. The content of the message must be (3) accepted

by the target audience as useful and personally applicable, and it must be

(4) retained if the individual is to (5) act on it, now or later. Obviously, if a

persuasive message is to be effective, the audience must attend to it, and it

must be couched in language or visual design that can be understood.

Failing to gain attention of the target audience appeared to be a major

problem of earlier school-based health education programs. Furthermore,

educational materials such as videotapes with a high fear arousal content, as

described earlier, emphasized biomedical information (e.g., the physiological effects of smoking on the body or the etiology of cancer) at a level

beyond the comprehension of the young adolescent audience. Although

the problem is fairly easy to identify, the solution (e.g., effective messages

that catch and hold the attention of a seventh-grade student or accurate

scientific information at an elementary school reading level) is substantially

more difficult. Employing standard educational methodology for reading

level assessment, we created persuasive messages and pretested them with

students at the resource schools. Their feedback ensured that the language

Objectives

SOURCE

Power

Attractiveness

Credibility

MESSAGE

Specificity

CHANNEL

Face-to-Face

Print

Electronic

Characteristics of Receiver

TARGET
Chapter 5

267

used was fully understood. We asked students representative of the study

population to assist us in writing scripts and acting out role-played situations, using their own words and actions. Our evaluations indicate that

target audiences perceive such scenes as realistic.

Although a wide range of media may be effectively employed as channels for persuasive communications with groups (e.g., school classes) (Weiss,

1969), in the early studies described here, pragmatic considerations mitigated for videotape as the primary delivery mode, with printed materials

(e.g., posters with scenes from the videotapes) serving as supplements.

Videotaped messages could be standardized for presentation across groups

and schools, thus averting the problem of differential treatment.

One effect of producing the intervention audiovisuals locally was the

target audiences’ perceived personalization of the communication. Local

scenes on the videotapes (e.g., school campuses, shopping malls) held the

attention of the students. Personalization often requires little extra effort by

the researcher. In a later multischool, multidistrict study, the students’

attention to a standardized sound/slide presentation of health resources in

their county was greatly increased when one picture of the specific school

was presented as the first slide (Evans et al., 1990).

Following McGuire’s model, after attention and comprehension,

acceptance and retention of the material are critical. To encourage acceptance, students were urged to become involved in discussions and feedback

sessions. Training, rehearsal, and role-playing reinforced personal involvement. A series of “booster” videotapes with supplemental materials such as

classroom posters was used over a 2-yr period after the primary treatment

series, allowing for the repetition, reinforcement, and reintegration of

material required for retention of persuasive messages that might not be

acted on immediately.

The messages in the videotapes and discussions were presented so as to

reinforce self-attributions and self-determinations of decisions to smoke or

not. Throughout the messages, the audiences are told, “You can decide for

yourself,” or “Here is some information that might help you decide,” but

they are not given the “correct” decision in a prescribed manner. Process

data indicate that students like this low-key, nondirective approach.

In addition to tailoring our intervention as described above, we also

employ a concept of behavioral inoculation instead of the concept of cognitive inoculation that McGuire (1961) directed at restoring or maintaining

beliefs and attitudes. In several papers (e.g., Evans, 1982, 1984, 1990), we

described this concept as the “social inoculation strategy.” The strategy

involves increasing resistance to social influences to smoke that children

and adolescents encounter by inoculating them with both knowledge and a

repertoire of social skills to help them resist such pressures. Included also

are such coping responses as “Just say no” to low peer pressure to smoke.

From the social inoculation strategy orientation, a response to a higher level

of peer pressure might be, “I thought you were my friend. Why do you

want to give me cancer?” In addition to training adolescents to recognize
Smoking and Tobacco Control Monograph No. 2

268

and cope with such overt social influences to smoke as peer pressure, this

approach addresses possible covert social influences, such as models who

smoke in ads or the individual’s perceptions of peer group smoking norms.

THE HOUSTON

LITTLE LEAGUE

PROJECT

Within the past decade, there has been a significant increase in ST

use, particularly among young males (Boyd and Glover, 1989; US

DHHS, 1986). As we considered extending our research program

into prevention of ST use, a number of factors contributed to the design of

our current project, which involves Little League baseball players as the

study population. (In fact, current NCI-supported projects are also directed

to other special population groups: 4-H Club members and Native

Americans.)

• Some evidence indicates that the tendency to use ST relates to the

tendency to smoke cigarettes and may involve some of the same

psychosocial mechanisms, suggesting that models and methodologies

developed for smoking prevention research might be applicable to ST

use prevention (Chassin et al., 1989; Evans and Raines, 1990).

• ST use appears to begin at an earlier age than that usually noted for

initial experimentation with cigarette smoking (Ary et al., 1987;

Bonaguro et al., 1986; Schaefer et al., 1985), suggesting the efficacy of

using a target group of preadolescent subjects.

• With the exception of Native American populations, in which females

and males report about equal use (Schinke et al., 1986), ST is used

primarily by males (US DHHS, 1986), suggesting the selection of a

potential study group that is primarily male.

• Heavy ST use appears to be closely associated with the public lifestyle

of a significant number of professional baseball players (Connolly et

al., 1988), suggesting possible imitative behavior, particularly by

sports-minded youth. About 28 percent of our current Little League

sample (aged 12 or younger) believes that more than half of professional players use ST. However, this perception is not a strong discriminator between never having used ST and having initiated ST use.

It remains to be determined whether perceived use of ST by professional baseball players influences use by older adolescents.

• The widely accepted belief that ST use is a relatively “safe alternative”

to smoking cigarettes (Schaefer et al., 1985) has implications at both

individual and program levels. Some evidence suggests that such

beliefs may increase the likelihood that adolescents who are non-users

will take up the practice in the future (Chassin et al., 1985).

Data from the first wave of our longitudinal investigation (n=1,141)

reveal that 23 percent of the Hispanics and 18 percent of the Anglos

believe that ST is less habit-forming than smoking. Eighty percent of

both Hispanics and Anglos believe that ST use is safer than cigarette

use.

If school administrators and curriculum developers also perceive ST to

be safer than cigarette smoking, and program planners perceive ST use
Chapter 5

269

as limited only to males and a lower health risk, there could be less

curriculum time and fewer ST prevention research programs (Evans et

al., 1990).

• Overt influences to smoke cigarettes appear to be primarily peer

influences, actual or perceived (Evans, 1984; Evans et al., 1988).

Apparently less important in influencing adolescents to smoke

cigarettes, covert influences to use ST may come from adult authority

figures (Ary et al., 1987), including coaches (Marty et al., 1986),

indicating that different resistance strategies may be needed in the

setting of athletics.

As a function, in part, of integrating these factors into a longitudinal

study design, we proposed a research program that applies our social inoculation strategy to an ST use prevention program for preadolescents (aged 9

to 12) and young adolescents (aged 13 to 15), within the context of

baseball-related activities, using as subjects Little League baseball and Senior

League baseball players who are, in our area, 95 percent male. Shifting from

a school-based setting to the baseball setting allows a concentrated focus on

ST use, including correction of misperceptions such as the relative safety of

its use, and provides for the involvement of both players and coaches.

The study has the support of Little League baseball at various administrative levels, including endorsement by the national executive director and

the Texas state director. At the local level, district administrators and league

presidents act as liaisons between our project staff and Little League teams.

We also have the cooperation of the local Major League team, the Houston

Astros.

The 5-yr project involves three stages:

• An assessment of the psychosocial-behavioral processes involved in

the initiation and use of ST among young people, aged 9 to 15, who

are members of active Little League and Senior League baseball teams;

• The development of a theory-guided, data-based preventiondeterrence program to be executed within Little League baseball

activities; and

• The implementation and evaluation of the prevention program in a

3-yr longitudinal study involving a cohort of Little League players

assigned, by team, to program (treatment) or measurement-only

(control) condition.

During the initial phase, the cross-sectional survey instrument was

developed, pretested, and administered to players in several Little League

administrative districts in and around Harris County. Data from the initial

cross-sectional survey indicated no significant differences among the participating districts. Therefore, the longitudinal phase involves approximately

180 Little League teams from one large Little League administrative district

in Harris County and Galveston County. The geopolitical division of the

district allows us to sample teams from both urban and rural areas with a
Smoking and Tobacco Control Monograph No. 2

270

wide range of socioeconomic markers while maintaining a simplified administrative approach that facilitates cooperation and averts the attrition

problems that often plague research studies.

The cross-sectional survey instrument operationalized approximately

20 hypothetical predictors of ST use. With a representative sample of Little

League players (n=57) drawn from several administrative districts, the

instrument was pretested for comprehensibility by young subjects (< 12 yr)

and administration time, including subject attention span and optimal

approaches for conducting surveys in field houses or Little League field

bleachers. Based on feedback from the pretest, items were modified, combined into a second instrument, and administered, under field conditions,

to a second representative sample of Little League players (n=273). In this

sample, about 85 percent were male, white, and 12 yr old or younger;

12.9 percent had tried ST at least once; 12.5 percent had tried smoking

cigarettes at least once; 2.2 percent had used ST during the previous week;

and 1.8 percent had smoked cigarettes during that same period.

The final form of instrument was administered as a cross-sectional

survey to a third representative sample of Little League players (n=293; age

range, 7 to 15 yr) to help shape the longitudinal measurement questionnaire

and to provide content for the intervention program. The overall responses

of this sample to four key questions are summarized as follows:

• Players who had tried smokeless tobacco at least once—14 percent.

• Players who answered “maybe” or “yes” to try in future—16 percent.

• Players reporting at least one of their friends uses ST—27 percent.

• Players reporting using ST during past week—5 percent.

It is interesting to note that reported use “. . . during the past week . . .,”

shown in Table 1, is similar for all three groups, although other substantial

differences appear to be age related. It should be noted, of course, that the

youngest and oldest groups are fairly small subsamples.

At present, we are carrying out the second year of the Little League

longitudinal study with a longitudinal sample (n=1,141) that was identified

at its initial measurement as 94.7 percent male with 99.0 percent of the

sample falling between the ages of 8 and 12. As for racial distribution,

74.7 percent are white, 11.1 percent Hispanic, 8.5 percent black, 2.5 percent

Native American, and 2.5 percent other.

POTENTIAL

BARRIERS

The investigator must be sensitive to the issue of relevance in the design

and implementation of any risk-behavior prevention program. For

example, our Little League study involves a very diverse population, and

interventions must be tailored to a wide range of demographic factors. In a

discussion of tailoring risk-behavior prevention programs to special populations, Orlandi (1986) listed several specific problems that we have found

relevant to our current study. Below is a brief discussion of some barriers

and examples of steps we have taken to address or eliminate the particular

problem:
Chapter 5

271

Table 1

Summary of findings from cross-sectional study, by age

Percentage, by Age Group

7 to 8 yr 9 to 11 yr 12 to 15 yr

(n=71) (n=149) (n=73)

Reported Use of ST

Tried it 3% 9% 33%

Will try it 10 13 26

Friends use it 10 18 64

Used it past week 6 5 7

• Use of language that is unfamiliar or uncomfortable for the target population—For example, it is sometimes difficult for an academic-oriented

researcher to communicate at the level required for interaction with

9-yr-old Little League players from varied socioeconomic backgrounds. As described previously, we use a feedback loop involving

children similar to the target group to assess the appropriateness of

language in terms of attention and comprehension.

• Use of printed materials that are too sophisticated—Printed intervention

materials are assessed by a member of our staff who is trained in

preparing written material for elementary students. Printed material

to be distributed to coaches and parents is reviewed by the Little

League district administrator, whose responsibilities include distributing effective written materials for Little League.

• Using individuals who are not well known in the community—In our Little

League program, we have involved professional baseball players who

are well known to Little League players and their adult sponsors. We

also have used the resources of the University of Houston baseball

coach and his staff, who develop training materials for Little League

and are highly respected. At all times, we keep outside communicators or interveners within the context of the game of baseball.

• Using unfamiliar motivational devices—In the Little League study, we

use motivational devices to encourage full participation and continued participation in the study. Motivational devices range from cold

drinks, if the program is being implemented during hot weather, to

free tickets to an Astros or University of Houston baseball game

(tickets were provided by the Astros or Houston Cougars). A major

motivator during the critical first year (when at least 75-percent team

participation was required for all testing and intervention occasions)

was a set of Skills and Drills Training Videotapes produced by Dr.

Bragg Stockton, coach of the University of Houston baseball team.

The videotapes were a highly significant motivator, and each League

that met the 75-percent criterion for participation received a set.
Smoking and Tobacco Control Monograph No. 2

272

• Conveying the impression that the program is not intended for long-term

adoption or that community leaders are not expected to participate—From

the beginning we have made it clear that the final, evaluated program

will be available to Little League baseball for general distribution.

Little League district personnel provide continuous feedback to the

research staff. Our field coordinator is a Little League district administrator with more than 25 yr of experience. The state director acts as

a consultant to the program.

A major barrier to developing ST use prevention programs is the perception that ST is relatively harmless in comparison with other drugs. Although all of the communities where we are working have active drug abuse

prevention programs (e.g., Chicken Club, Just Say No Club) to which

community members contribute time and financial resources, many individuals do not perceive ST, cigarettes, or alcohol as drugs in the same

category as the illicit drugs emphasized by many of the community programs. Most of these issues suggest the importance of developing and

maintaining rapport with the organizations participating in the project.

To address this issue, we are presently developing a videotape with the

theme, “Tobacco is a drug, too!”

A frequent barrier found by health promotion investigators is the

sample biasing effects of stringent informed consent measures that often

have been reported for both measurement and intervention components

(e.g., Evans et al., 1977). In our present school-based projects, we have

gained the consent of the school districts involved to include the prevention

program as part of the regular Texas Education Agency-approved curriculum. Under these guidelines, measurement and intervention activities can

proceed at the discretion of the school superintendent and the advisory

school board, given that all procedures and materials meet the guidelines

of the Texas Education Agency and the school district. The Little League

program involves obtaining parental consent. However, parents of the

players attend games and practice sessions and are generally far more

available for providing consent than is usual in school- or other

community-based programs. To date, we have not encountered serious

problems gaining informed consent for Little League players.

A taboo on any behavior (e.g., illicit drug use or, in the case of children

and adolescents, the use of alcohol or tobacco) remains a barrier to reliable

measurement of that behavior. To address this problem in our studies, we

emphasize the strict regulations regarding anonymity and confidentiality

and use strategies that have been shown to increase the validity of selfreports. Such strategies include the collection of saliva specimens under

“pipeline” conditions, a procedure we had developed earlier (US DHHS,

1986).

Many researchers place an emphasis on summative evaluation and,

under constraints of time or funding, ignore the formative evaluation

procedures that frequently are valuable in the development of interventions

and measurement instruments. Providing for continuous feedback so that

appropriate modifications can be made should be a high priority for the
Chapter 5

273

researcher, particularly in the implementation of programs in natural

community settings. Our use of formative evaluation has been described in

a paper that draws on its use in field settings with smoking prevention and

ST prevention research (Evans et al., 1989). Formative evaluation was used,

for example, in the development of our measurement instrument for the

longitudinal phase of the Little League program, to stringently revise the

original instrument and its administrative procedures. The first field draft of

the instrument was 45 pages long, 206 questions, and constructed at a thirdto fourth-grade reading level. The initial reaction of subjects was so resistant

we had to decrease its bulk, both through deleting some scales and reformatting so that the form looked less threatening. The revised instrument now

has 145 questions, is 18 pages long, and is scaled down to a reading level of

second to third grade to ensure comprehensibility for even less able readers.

Measurement time originally averaged 30 to 45 min; the revised questionnaire now takes 20 min or less, well within the attention span of the average

Little League player.

CONCLUSION We have addressed the potential for cross-application of wellevaluated smoking prevention programs to the newer issue of ST prevention

programs, drawing on our own extended program of research in smoking

prevention. We have discussed some of the barriers to successful development, implementation, and evaluation of such programs, including the

problems related to tailoring interventions for special populations. We also

reviewed some problems inherent in developing valid and reliable measurements in field-based studies, steps that might be taken to maintain a longitudinal study sample over time, and the value of formative evaluation

throughout the course of a research program. A final issue of special concern to ST prevention researchers is that, to many young adolescents and

their community leaders, tobacco—especially ST—does not fit within their

concept of “drug” in the development of drug abuse prevention programs.

We recommend that special emphasis be given to promoting the notion

that tobacco is a drug, too. With respect to the relative emphasis on smoking in contrast to smokeless tobacco as a health threat, it seems more

persuasive to no longer distinguish between smoking cigarettes and using

ST. We recommend that a more generic phrase, “tobacco and health,” be

used.

REFERENCES

Ajzen, I., Fishbein, M. Understanding Attitudes and

Predicting Social Behavior. Englewood Cliffs, NJ:

Prentice-Hall, 1980.

Ary, D.V., Lichtenstein, E., Severson, H.N. Smokeless

tobacco use among male adolescents: Patterns,

correlates, predictors and the use of the drug.

Preventive Medicine 16: 385-401, 1987.

Bandura, A. Social Learning Theory. Englewood Cliffs,

NJ: Prentice-Hall, 1977.

Bandura, A., Huston, A.C. Identification as a process

of incidental learning. Journal of Abnormal and

Social Psychology 63: 311-318, 1961.

Bonaguro, J.A., Pugh, M., Bonaguro, E.W. Multivariate analysis of smokeless tobacco use by adolescents in grades four through twelve. Health

Education 17: 4-7, 1986.

Botvin, G.J., Wills, J.A. Personal and social skills

training: Cognitive behavioral approaches to

substance abuse prevention. In: Prevention

Research: Deterring Drug Abuse Among Children and

Adolescents, C.S. Bell and R. Battjes (Editors). U.S.

Department of Health and Human Services; Public

Health Service; Alcohol, Drug Abuse, and Mental

Health Administration; National Institute on Drug

Abuse DHHS Pub. No. (ADM) 87-1334. 1985, pp.

8-49. (NIDA Research Mongraph 63)
Smoking and Tobacco Control Monograph No. 2

274

Boyd, G.M., Glover, E.D. Smokeless tobacco use by

youth. U.S. Journal of School Health 59: 189-194,

1989.

Chassin, L., Presson, C.C., Sherman, S.J., et al.

Adolescent smokeless tobacco use: Future research

needs. National Cancer Institute Monographs 8: 101-

105, 1989.

Chassin, L., Presson, C.C., Sherman, S.J., et al.

Psychosocial correlates of adolescent smokeless

tobacco use. Addictive Behaviors 10: 431-435, 1985.

Connolly, G.N., Orleans, C.T., Kogan, M. Use of

smokeless tobacco in major league baseball. New

England Journal of Medicine 381: 1281-1284, 1988.

Evans, R.I. Albert Bandura: The Man and His Ideas.

New York: Praeger Press, 1989.

Evans, R.I. Development of the social inoculation

strategy to deter smoking and related health

threatening behaviors in adolescents: Review and

an update. Paper presented at the International

Workshop on Prevention of Drug Addictions,

Alicante, Spain, December 3-5, 1990.

Evans, R.I. Fear is not enough: Modification of

behavior to prevent disease. Postgraduate Medicine

65: 195-197, 1979.

Evans, R.I. Health promotion: Science or ideology?

Health Psychology 7(3): 203-219, 1988.

Evans, R.I. Modifying health lifestyles in children

and adolescents: Development and evaluation of a

social psychological intervention. In: Handbook of

Psychology and Health: Issues in Child Health and

Adolescent Health (volume 2), A. Baum and J.E.

Singer (Editors). Hillsdale, NJ: Lawrence Erlbaum

Associates, 1982.

Evans, R.I. A social inoculation strategy to deter

smoking in adolescents. In: Behavioral Health: A

Handbook of Health Enhancement and Disease

Prevention, J.D. Matarazzo, S.M. Weiss, J.A. Herd,

N.E. Miller, and S.M. Weiss (Editors). New York:

John Wiley and Sons, 1984, pp. 765-774.

Evans, R.I. Smoking in children: Developing a social

psychological strategy of deterrence. Journal of

Preventive Medicine 5: 122-127, 1976.

Evans, R.I., Dratt, L.M., Raines, B.E., et al. Social

influences on smoking initiation: Importance of

distinguishing descriptive versus mediating process

variables. Journal of Applied Social Psychology 18:

925-943, 1988.

Evans, R.I., Hansen, W.B., Mittelmark, M.B. Increasing the validity of self-reports of smoking behavior

in children. Journal of Applied Psychology 62: 521-

523, 1977.

Evans, R.I., Raines, B.E. Applying a social psychological model across health promotion interventions:

Cigarettes to smokeless tobacco. In: Social

Influence: Processes and Prevention, J. Edwards,

R.S. Tindale, L. Heath, et al. (Editors). New York:

Plenum Press, 1990, pp. 143-157.

Evans, R.I., Raines, B.E. Control and prevention of

smoking in adolescents: A psychosocial perspective. In: Promoting Adolescent Health: A Dialog on

Research and Practice, T.J. Coates, A.C. Petersen,

and C. Perry (Editors). New York: Academic Press,

1982.

Evans, R.I., Raines, B.E., Hanselka, L.L. Developing

data-based communications in social psychological research: Adolescent smoking prevention.

Journal of Applied Social Psychology 14(3): 289-295,

1984a.

Evans, R.I., Raines, B.E., Owen, A.E. Adolescent

Health Promotion: The Galveston Project. Final

Report to the Bureau of Health Care Delivery and

Assistance, Division of Maternal and Child Health.

1990.

Evans, R.I., Raines, B.E., Owen, A.E. Formative

evaluation in school-based health promotion

investigations. Preventive Medicine 18: 229-234,

1989.

Evans, R.I., Rozelle, R.M., Lasater, T.M., et al. Fear

arousal, persuasion, and actual versus implied

behavior change: New perspective utilizing a reallife dental hygiene program. Journal of Personality

and Social Psychology 16(2): 220-227, 1970.

Evans, R.I., Rozelle, R.M., Lasater, T.M., et al. New

measure of effects of persuasive communications:

A chemical indicator of toothbrushing behavior.

Psychological Reports 23(2): 731-736, 1968.

Evans, R.I., Rozelle, R.M., Maxwell, S.E., et al. Social

modeling films to deter smoking in adolescents:

Results of a three-year field investigation. Journal

of Applied Psychology 66: 399-414, 1981.

Evans, R.I., Rozelle, R.M., Mittelmark, M.B., et al.

Deterring the onset of smoking in children:

Knowledge of immediate physiological effects and

coping with peer pressures, media pressure, and

parent modeling. Journal of Applied Social

Psychology 8: 126-135, 1978.

Evans, R.I., Rozelle, R.M., Noblitt, R., et al. Explicit

and implicit persuasive communication over time

to initiate and maintain behavior change: A new

perspective utilizing a real-life dental hygiene

program. Journal of Applied Social Psychology 5(2):

150-156, 1975.

Evans, R.I., Smith, C.K., Raines, B.E. Deterring

cigarette smoking in adolescents: A psychosocialbehavioral analysis of an intervention strategy.

In: Social Psychological Aspects of Health, A. Baum,

J. Singer, and S. Taylor (Editors). Hillsdale, NJ:

Lawrence Erlbaum Associates, 1984b.

Fishbein, M., Ajzen, I. Belief, Attitude, Intention, and

Behavior: An Introduction to Theory and Research.

Reading, MA: Addison-Wesley, 1975.

Flay, B.R. Psychosocial approaches to smoking

prevention: A review of findings. Health Psychology 4: 449-488, 1985.
Chapter 5

275

Graham, J.W., Marks, G., Hansen, W.B. Social

influence processes affecting adolescent substance

use. Journal of Applied Psychology 76(2): 291-298,

1991.

Janis, I.L., Feshbach, S. Effects of fear-arousing

communications. Journal of Abnormal and Social

Psychology 48: 78-92, 1953.

Laswell, H.D., Casey, R.D. Propaganda, Communication and Public Opinion. Princeton, NJ: Princeton

University, 1946.

Leventhal, H., Singer, R., Jones, S. Effects of fear and

specificity of recommendations upon attitudes and

behavior. Journal of Personality and Social Psychology 2: 20-29, 1965.

MacKinnon, D.P., Johnson, D.A., Pentz, M.A., et al.

Mediating mechanisms in a school-based drug

prevention program: First year effects of the

midwestern prevention project. Health Psychology,

in press.

Marty, P.J., McDermott, R.J., Williams, T. Patterns of

smokeless tobacco use in a population of high

school students. American Journal of Public Health

76: 190-192, 1986.

McGuire, W.J. Communication-persuasion models

for drug education: Experimental findings. In:

Research on Methods and Programs of Drug Education,

M. Goodstadt (Editor). Toronto, Canada: Addiction Research Foundation, 1974, pp. 1-26.

McGuire, W.J. The effectiveness or supportive

refutational defenses in immunizing and restoring

beliefs against persuasion. Sociometry 24: 184-197,

1961.

McGuire, W.J. The nature of attitudes and attitude

change. In: Handbook of Social Psychology: The

Individual in a Social Context (volume 3),

G. Lindzey and E. Aronson (Editors). Reading,

MA: Addison-Wesley, 1969, pp. 136-314.

Orlandi, M.A. Community-based health promotion:

A multicultural perspective. Journal of School

Health 56: 394-401, 1986.

Schaefer, S.D., Henderson, A.H., Glover, E.D., et al.

Pattern of use and incidence of smokeless tobacco

consumption in school-age children. Archives of

Otolaryngology–Head and Neck Surgery 111: 639-

642, 1985.

Schinke, S.P., Gilchrist, L.D., Schilling, R.F., et al.

Smokeless tobacco use among Native American

adolescents. New England Journal of Medicine 314:

1051, 1986.

Severson, H.N., Ary, D.V. Sampling bias due to

consent procedures with adolescents. Journal of

Addictive Behaviors 8(4): 433-437, 1983.

Sutton, S.R. Fear-arousing communications: A

critical examination of theory and research. In:

Social Psychology and Behavioral Medicine, J.R. Eiser

(Editor). New York: John Wiley and Sons, 1982,

pp. 303-337.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986.

Weiss, W. Effects of the mass media of communication. In: The Handbook of Social Psychology:

Applied Social Psychology (volume 5), G. Lindzey

and E. Aronson (Editors). Reading, MA: AddisonWesley, 1969, pp. 77-195.
Smoking and Tobacco Control Monograph No. 2

276
Chapter 6

277

Chapter 6

Cessation

CONTENTS Enough Snuff: ST Cessation From the Behavioral,

Clinical, and Public Health Perspectives

Herbert H. Severson ............................................................................ 279

Introduction ................................................................................ 279

Clinical ST Cessation Studies ...................................................... 280

Implications From Other ST Studies ........................................... 281

Unique Aspects of ST Cessation .................................................. 283

Summary of Clinical Work ......................................................... 284

Components of ST Cessation ...................................................... 284

Public Health Interventions ........................................................ 286

Summary ..................................................................................... 287

References .................................................................................... 288

Smokeless Tobacco Cessation and

Nicotine Reduction Therapy

Elbert D. Glover and Penny N. Glover ............................................... 291

Introduction ................................................................................ 291

Nicotine in Smokeless Tobacco................................................... 292

Cessation Programs ..................................................................... 292

References .................................................................................... 295

Role of Dentists in Cessation Counseling:

Survey Findings

Kathleen L. Schroeder and David E. Heisel ........................................ 297

Introduction ................................................................................ 297

Materials and Methods................................................................ 299

Results.......................................................................................... 299

Conclusions ................................................................................. 301

References .................................................................................... 304

An Intervention Study of Tobacco Habits

Among Rural Indian Villagers

Mira B. Aghi, P.C. Gupta, F.S. Mehta, and J.J. Pindborg .................... 307

Introduction ................................................................................ 307

Materials and Methods................................................................ 308

Discussion.................................................................................... 310

References .................................................................................... 311
Smoking and Tobacco Control Monograph No. 2

278
Chapter 6

279

Despite concerns over the high prevalence and detrimental health effects of regular

Enough Snuff: ST Cessation

From the Behavioral, Clinical, and

Public Health Perspectives

Herbert H. Severson

ABSTRACT

smokeless tobacco use by American males, there has been little research on assisting users to

quit. A review of research shows promising results by adapting smoking cessation procedures.

A review of clinical cessation studies and psychosocial studies provides support for development and assessment of ST cessation clinical and self-help programs. Distinctive aspects of ST

cessation include the high prevalence of oral lesions, need for an oral substitute during

withdrawal, the potential use of nicotine polacrilex as an adjunct, perception of ST as a safe

alternative to smoking, frequent use of both cigarettes and ST, and difficulty in using nicotinefading approaches. Specific components and measures involved in ST cessation are reviewed.

Support for public health interventions is provided by a recent study of ST cessation in HMO

dental clinics that demonstrated the efficacy of providing ST users advice to quit in the context

of health care delivery. Cessation materials have also been developed for special populations,

such as Native Americans and baseball players.

INTRODUCTION Over the past 6 yr, there has been a great deal of public interest in

and scientific attention to the detrimental health effects of smokeless

tobacco use. The publication of the Surgeon General’s report on ST use (US

DHHS, 1986a) and the publicity surrounding the NIH Consensus Development Conference on Smokeless Tobacco (US DHHS, 1986b) focused attention on these health concerns. These publications identified three major

health risks associated with the oral use of tobacco: oral cancer, development of leukoplakia and other oral health problems, and nicotine addiction.

These two documents increased public awareness and spurred Congress to

pass House Bill 99-252, the Smokeless Tobacco Act of 1986. This act instituted rotating health warnings on ST products and eliminated radio and

television ads for ST. Although the focus on ST increased public awareness

of health effects, the sales of ST products have increased steadily with only a

minor flat spot in the growth curve following the enactment of Public Law

99-252.

Because the majority of new users of ST products are young teen males,

recent research programs have focused on preventing young people from

taking up the use of snuff or chew through school-based prevention programs (Severson and Zoref, 1991). Despite these educational efforts, the use

of ST, especially moist snuff, is increasing, especially among male adolescents and young male adults (US DHHS, 1986a). As of 1986, there were

already an estimated 6 million regular users of ST in the United States (US

DHHS, 1986a). Unfortunately, there has been little effort toward assisting

current users in quitting their habitual use of snuff or chewing tobacco.

Despite the concern for potential negative health consequences, scientific study of ST cessation has lagged behind epidemiological, health, and

preventive efforts. To date, there are few published studies on ST cessation,

although several are currently in progress.
Smoking and Tobacco Control Monograph No. 2

280

This paper broadly reviews the field of smokeless tobacco cessation.

Published and unpublished studies are reviewed to provide a current assessment of ST cessation and guide future studies. Psychosocial studies that

provide information on ST use and can guide development and implementation of effective cessation programs are also considered. The paper concludes with a review of ongoing public health interventions that provide

direction on development of broad-based efforts to promote ST cessation.

CLINICAL ST

CESSATION

STUDIES

Although they are not well-controlled randomized clinical trials, they are

instructive.

The first published study of ST cessation was done by Glover (1986)

who adapted the American Cancer Society’s FreshStart Adult Smoking

Cessation Program for use with 41 adult ST users. He reported a 6-mo

abstinence rate of only 2.3 percent. However, these subjects were mandated

to attend the program, as they had been found in violation of school rules

at a college that prohibited the use of tobacco products. Given the nonvoluntary nature of the subject sample, it is not unexpected to find a low

success rate in cessation. Subsequent studies by Eakin and coworkers (1989)

and DiLorenzo and coworkers (1991) provide a more optimistic view of

the potential cessation rates that can be achieved by formal ST cessation

treatment.

Eakin and coworkers (1989) reported an intervention with adolescent

daily users, aged 14 to 18, who were recruited by referrals from counselors,

coaches, and teachers in Eugene, Oregon, high schools. The study had

25 chronic ST users with a quasi-experimental design in which 11 of the

25 subjects provided a comparison group by receiving delayed treatment

(3-wk delay). This behavioral treatment consisted of three 1-h small-group

meetings led by counselors. The multiple-component treatment was cognitive-behavioral in nature and focused on encouraging subjects to use coping

skills for cessation. Of the 21 subjects completing treatment, 9 were successful in quitting their ST use at the end of treatment. Self-reported quitting

was confirmed with saliva cotinine assessment, and subjects were followed

up at 6 mo after treatment. Long-term cessation rates were reduced to

12 percent at 6-mo followup; however, subjects not achieving abstinence

had a self-reported reduction of 45 percent in their daily use of ST from

baseline levels. The participants in this study who quit reported that, in

addition to the group sessions, the ongoing telephone calls and support by

the counselor were key elements in their success.

DiLorenzo and associates (1991) reported a multiple baseline design

intervention on nine adult males recruited for a behavioral ST cessation

program. Mean age of the subjects was 32 yr and the average length of use

of ST was 9.3 yr. Seven subjects completed the eight 1-h behavioral treatment sessions provided over a period of 7 wk in small groups of three

subjects each. Cue extinction, setting a target date for quitting, the use of a

buddy system, and relapse prevention were the primary components of the

Currently, there are only three published cessation studies involving

smokeless tobacco. Unfortunately, these studies had relatively small

samples and could be best characterized as pilot clinical research.
Chapter 6

281

intervention. Cue extinction involved identifying two or three situations

most strongly associated with ST use and breaking these associations by

refraining from taking a dip for 30 min. Cessation of ST use was associated

with the introduction of the program. Six subjects remained abstinent

through the treatment phase and remained abstinent at the 9-mo followup.

These data were confirmed by collateral sources. The program appears to be

quite successful, although the modest number of subjects dictates caution in

interpretation of this study.

The studies to date show that, for subjects who wish to quit, clinical

cessation techniques based on smoking cessation are modestly effective.

The 6-mo followup by Eakin and coworkers (1989) is discouraging, as there

was high level of relapse, although 43 percent of the subjects who completed the treatment were able to quit. DiLorenzo and associates (1991)

demonstrated the effectiveness of cue extinction procedures and the importance of getting the user to eliminate ST use first in situations in which the

person is usually cued to use ST. Unfortunately, the studies have modest

numbers of subjects and lack a comparison, no-treatment group.

There are several unpublished studies in cessation of ST use. Dorothy

Hatsukami and colleagues at the University of Minnesota report promising

results using nicotine polacrilex gum (Nicorette) as part of a behavioral

group treatment program for adult ST users. They found no difference

between the use of the gum ad libitum or on a fixed interval schedule, or

between the 2 mg and 4 mg doses, in initial or 3-mo followup quit rates.

Dr. Hatsukami reports that approximately 75 percent of the adult men had

quit ST at the end of treatment (Hatsukami, personal communication,

1991).

Other studies are in progress. These studies involve comparing active

and placebo nicotine gum as an adjunctive aid for a multicomponent

cessation program for adult ST users, as well as studies of adolescent and

adult cessation using ground mint leaf products as a snuff substitute.

From the discussion above of clinical studies to date, ST cessation

involves adapting smoking cessation procedures. By and large, ST treatment

programs have adapted standard cognitive-behavioral techniques used in

smoking cessation programs. Personal communication with counselors

indicates that self-help smoking cessation materials can be readily adapted

and used by ST users. In our study of adolescent users, we depended heavily

on the adaption of smoking cessation materials for each of the group sessions (Eakin et al., 1989). Further research and clinical experience are

needed to determine how ST cessation differs from smoking cessation.

IMPLICATIONS

FROM OTHER

ST STUDIES

Studies assessing ST quit efforts, health problems, and use patterns

provide additional information that can have direct implications on

the design and implementation of a cessation program.

Studies support the need for ST cessation. Men who use ST appear

interested in quitting. Researchers report that recruitment in current ongoing studies has not been a problem. In our current study assessing nicotine gum, 675 men responded to twice-weekly newspaper ads (for 7 wk)

Quit Attempts
Smoking and Tobacco Control Monograph No. 2

282

soliciting daily ST users for a cessation program. In interviews, 64 percent of

ST users have reported that they would make a serious quit attempt in the

next 6 mo (Severson et al., 1990a). Of those trying to quit, 68 percent

reported an average of four attempts each, and 77 percent of those quit

attempts were “cold turkey” efforts. Additional support comes from a

survey of dental patients in which 54 percent of ST users reported that they

would make a quit attempt in the next year (Severson et al., 1990b). Ary

and coworkers (1989) found that more than one-third of current male

adolescent ST users reported unsuccessful quit attempts. Novotny and

coworkers (1989) in a national probability sample found a large percentage

(39.1 percent) of adult ST users had made unsuccessful attempts to quit.

Most studies to date have been convenience samples or subsets of other

studies. There is a need for a general population survey of ST users with

regard to quit attempts, success rates of self-quitting, interest in cessation

assistance, and relapse of self-quitters.

Oral Health

Problems

We also know that regular users often experience oral health problems

that they can easily identify, and these can be used to motivate them

to institute a quit attempt. Two-thirds of regular daily ST users reported that

they had experienced health problems that they directly attributed to their

use of snuff (Severson et al., 1990a). These symptoms included bleeding

and sore gums, lesions, receding gums, and upset stomach from swallowing

the juice. Although the sample in the study of Severson and associates

(1990a) was a self-selected sample of adult daily ST users, there is ample

evidence of frequent oral health problems among daily users (Glover et al.,

1989; Schroeder, 1989; US DHHS, 1986a). Estimates are that more than

50 percent of regular users have at least a degree 1 (early-stage) leukoplakia

lesion (Glover et al., 1989). Our own study of dental patients revealed that

78 percent of ST users had detectable oral lesions, and these were distributed

evenly across three levels of severity (Little et al., in press). These lesions,

along with bleeding and receding gums (gingivitis), can motivate a user to

seek cessation assistance and provide an objective outcome from cessation,

because the lesions usually heal quickly after ST cessation.

Nicotine

Addiction

Studies of nicotine absorption by snuff and chewing tobacco users conclude that venous plasma nicotine levels are similar in users of ST and

cigarettes (Benowitz et al., 1988). Gritz and coworkers (1981) and Russell

and coworkers (1981) concluded that plasma nicotine and cotinine levels

for daily habitual snuff users were comparable to those of a group of heavy

smokers. Finally, it appears that physiological dependence develops regardless of whether nicotine is taken in through a cigarette, smokeless tobacco,

or nicotine polacrilex (Hughes and Hatsukami, 1986).

Withdrawal

Symptoms and

Substitutes

We also know that symptoms experienced during ST withdrawal

appear to be the same as those for smoking cessation (Hatsukami et

al., 1987; Severson et al., 1990a). Hatsukami and colleagues reported

that ST users experienced cravings, irritability, distractibility, and hunger,

but the symptoms were less intense and fewer in number than those of the

cigarette smokers. Self-report measures of withdrawal symptoms can be

readily adapted from smoking cessation programs.
Chapter 6

283

In response to their withdrawal symptoms, persons going through

treatment for ST addiction often request an oral substitute. Chewers report

using cinnamon sticks, gum, sunflower seeds, finely ground mint leaves, or

other chewed foodstuff substitutes for ST during withdrawal. Unpublished

case reports and ongoing cessation studies report success with ad libitum use

of a mint snuff-like product as an ST substitute. This product, which contains finely ground mint in tins, is used in the same way as snuff but provides no nicotine.

Cigarette

Smoking

We also know that there is a co-morbidity with cigarette smoking. In

studies we have done in ST cessation, we have found that 25 to 30 percent

of all regular ST users also use cigarettes (Eakin et al., 1989; Little et al., in

press; Severson, in press). This is important because we may get people to

quit ST but then increase their use of cigarettes. This would result in no net

gain in the health risk status of subjects, and they would remain addicted to

nicotine. The use of cigarettes by ST users has serious implications for

cessation efforts. A dental-based intervention described later in this paper

reported significantly lower cessation rates among men who use both

tobacco products than those who use ST exclusively (Hollis et al., in press).

Studies of psychosocial factors in ST use and physical dependence on

nicotine support the adoption of cognitive-behavioral multicomponent

smoking cessation programs for use in ST cessation. There is ample evidence that regular ST users make quit attempts and experience serious

withdrawal symptoms when quitting (Hatsukami et al., 1991). The plasma

nicotine levels for ST users approximate those of a regular smoker, and the

levels of nicotine dependence are similar for both groups. The use of cigarettes is high for ST users, and this factor can be problematic in getting a

person to quit ST use. Unfortunately, we also have much to learn. Although the preliminary evidence is that ST cessation rates are similar to

smoking cessation rates, the small sample sizes, self-selective nature of

subjects, the lack of control groups, and lack of long-term followup make

one cautious about any interpretation. Particular areas to focus on include

determining how cessation processes differ by level of ST use. Also, we do

not know the relapse rates because the followup on studies has been minimal. We know little about the use of nicotine replacement in cessation selfhelp quitting, effects of a health professional’s advice, or the effects of work

site restriction in use. In other words, although the field of smoking cessation has a well-established research base, ST cessation lags far behind in

answering the same questions.

UNIQUE ASPECTS

OF ST CESSATION

Smokeless tobacco use presents some unique issues that need to

be considered. First, we have to remember that snuff can be used

without other people being aware. This surreptitious behavior provides

some difficulty in monitoring the use of snuff by others and allows use in

situations where its use is not permitted (e.g., in school classrooms or on the

job).

Second, the frequent oral lesions experienced by ST users provide the

cessation counselor with a direct proximal measure of a physical problem

caused by ST use. For a cigarette smoker, it is often difficult to point out

direct evidence of detrimental health effects from smoking.
Smoking and Tobacco Control Monograph No. 2

284

Third, cigarettes can be used during ST withdrawal. As described above,

up to one-third of men also use cigarettes and may smoke during the ST

withdrawal process to minimize cravings.

Fourth, we know that ST is perceived as a safe alternative and that

perception may result in less motivation to quit. Chewers perceive ST use to

be less harmful than smoking, although most nonchewers disagree (Bauman

et al., 1989; Lichtenstein et al., 1984). Among youth, 86 percent regard ST

as a safe alternative to cigarette smoking (US DHHS, 1986c). There is a

higher acceptance of teenage ST use by parents than of smoking (Chassin et

al., 1985). Parents’ acceptance of their sons’ chewing or dipping contributes

to the general perception that this behavior is acceptable and encourages

continued use.

And fifth, some of the cessation approaches that are used for cigarette

smoking are not easily translated to ST because snuff and chewing tobacco

products are generally not packaged in individual doses. For example, it is

difficult to do nicotine-fading procedures (i.e., gradually reducing the

amount of bioavailable nicotine). The lack of a standard dose, as in a

cigarette, makes gradual reduction procedures difficult to self-monitor. The

known nicotine content of cigarettes, via government reports, makes nicotine exposure relatively easy to compute. However, no comparable data on

nicotine content of ST products are available, and individuals vary greatly in

what constitutes a single pinch or dip (Severson et al., 1990a). Nicotine

reduction procedures call for users to control the amount of nicotine exposure by either changing products or reducing the amount of snuff or chew

they put in their mouths; however, in practice these procedures are difficult

to implement because the nicotine content of the product is difficult to

ascertain and the dip size is subjective.

SUMMARY OF

CLINICAL WORK

From clinical and psychosocial studies, we can conclude smoking

cessation procedures can be adapted for use with ST users with

minor modification. Preliminary information supports ST cessation success

rates being similar to smoking cessation, but there are few published studies

to date. Oral substitutes appear to be important adjuncts in ST cessation.

Users report use of mint snuff or other oral substitutes in quitting programs.

Nicotine polacrilex gum has a topography of use that is very similar to the

topography of snuff or chew use, and its use may be better received by ST

users than by smokers as an aid in cessation. This is supported by

Hatsukami and our own experience where we found that ST users report

compliant use of nicotine polacrilex gum as a part of cessation. The new

transdermal nicotine patches could also be a valuable aid, but to date their

use has not been evaluated for ST users. Finally, there is a unique opportunity for self-exam of the mouth to increase motivation and provide feedback

on oral health recovery that accompanies cessation.

COMPONENTS OF

ST CESSATION

Adaptation of smoking cessation materials has been used in all

aspects of ST cessation. A brief review of some of these measures

and components may be useful. These include assessing motivation and

readiness to quit, oral health measures, and assessing addiction level.
Chapter 6

285

The most accurate way to measure addiction level would be to

measure nicotine exposure via plasma nicotine levels or urinary

Assessing

Addiction Level

cotinine levels. However, collecting blood or urine samples is invasive, and

laboratory procedures for measuring nicotine or cotinine in body fluids are

expensive. Unfortunately, the product packaging and lack of government

testing of ST products for nicotine content preclude easy measurement of

nicotine exposure. A parsimonious, but less accurate, procedure for estimating a subject’s nicotine exposure level is to take the available nicotine level of

their usual product and multiply that times number of tins per week. For

example, if Copenhagen is used, which is a very high nicotine product with

30.76 mg/g, and a user reported using one tin of snuff per day or seven tins

per week, his total nicotine exposure would be 30.76 mg/g x 34.02 g per tin x

seven tins per week = approximately 7,325 mg nicotine. Products such as

Skoal and Kodiak are considered medium in nicotine content with 10.7 mg/g

and 14.6 mg/g, respectively (Hoffman et al., 1986). The low-nicotine snuff

products include Hawken, Bandits, and Happy Days. Hawken, for example,

has 5.7 mg/g of nicotine. To date, no data have been collected that correlate

this computation of nicotine bioavailability and plasma nicotine levels;

however, it appears that identifying the ST product used by the individual

can assist in assessing addiction levels. Even if one does not compute the

total bioavailable nicotine, the number of tins per week can give a general

view of addiction. Schroeder and colleagues (1988) have suggested categorizing ST users as light, moderate, or heavy on the basis of the number of tins

used per week. A light user would consume one tin or pouch or less per

week, a moderate user would use one and one-half tins or pouches weekly,

and a heavy user would be a person who uses more than two tins or pouches

per week. This method is compromised by the wide disparity in the level of

bioavailable nicotine in the various products as noted above.

An alternative measure of addiction is suggested by Eakin and coworkers

(1989), who adapted the Fagerstrom Addiction Scale (Fagerstrom, 1978),

which has been used in smoking cessation. Cigarette-based questions were

converted to ST items (e.g., “I chew or dip the first thing in the morning

within 30 minutes of waking”; “I swallow the juice when I can’t spit”; “I

chew or dip where it is prohibited”; and “I crave ST even when I’m sick in

bed”). These items are scored plus or minus and the total of seven or more

positive responses indicates the person is heavily addicted to ST. Although

this measure is being used in a number of ongoing ST cessation studies, there

are currently no validity data with ST users.

Assessing

Readiness

To Quit

Another useful measure that can be adapted from smoking research is an

assessment of readiness of the individual to quit. This involves using the

Prochaska-DiClemente model (DiClemente et al., 1991; Prochaska and

DiClemente, 1983) in which they postulate that the process of cessation can

be conceptualized as along a continuum from precontemplation to contemplation to action in making an actual quit effort. The person’s readiness to

quit is assessed by questions such as, “Are you seriously considering quitting

your use of chew or snuff in the next 6 months?” An affirmative response

indicates the subject is at the contemplation stage. Agreement with statements such as, “I’m seriously going to quit my use of chew or snuff in the
Smoking and Tobacco Control Monograph No. 2

286

next month,” is an indication of readiness to make the commitment to quit.

An alternative procedure to a series of questions is a 10-step “contemplation

ladder” on which subjects indicate where they are in their readiness to quit

by placing themselves on the ladder (Biener and Abrams, 1991).

PUBLIC HEALTH

INTERVENTIONS

Parallel to individual cessation programs of a clinical nature are

public health cessation efforts. These efforts involve more population-based interventions that may be less effective in terms of quit rates

but are more likely to affect large numbers of individuals who use ST. In the

end, interventions of this type may have a much larger public health impact

by getting more people to quit (Severson, 1980). These interventions are

diverse and range from getting hygienists and dentists or physicians and

nurses to provide ST users direct advice to quit, to policy changes in restrictions of sale and use of ST products. Health care professionals can advise

patients to quit ST and provide materials regarding health risks in the

context of regular health care. These interventions are minimal in terms of

cost and time, but similar programs for smokers have been shown to have a

significant impact on tobacco use (Vogt et al., 1989). Public health interventions also include media messages, such as radio or television public

health messages, written materials, and information from voluntary groups

(e.g., American Cancer Society and American Lung Association), policy

changes such as work site restrictions on ST use, or restrictions on ST product sales to minors. Legislative actions such as increased taxation on ST

products or putting health warnings on products are also considered public

health interventions. Professional groups such as the American Dental

Association and Academy of Otolaryngology have been active in publishing

materials on the detrimental health effects of ST, and the distribution of

these materials could also be considered a public health intervention.

An example of a public health cessation program was a 4-yr randomized

trial that used dentists and hygienists as the providers of cessation advice

(Little et al., in press). Men (aged 15 to 65) who came into HMO dental

clinics for regular hygiene visits and identified themselves as ST users were

randomized either to intervention or usual care based on an identification

number. The dental office intervention involved first having a dentist or

hygienist ask the patient about his tobacco use. Second, the clinician

conducted an oral health exam and provided specific feedback on lesions in

the mouth. Third, the practitioner provided direct advice to the patient to

quit. The key element is for the dentist or hygienist or both to give a clear

personal message that they believe ST use is harmful and they want the

patient to quit. Fourth, patients were asked to watch a brief 10-min video

that was motivational in nature, asked to set a quit date, and given self-help

materials. There was also a 1-wk followup phone call by the hygienist to see

how they were doing.

The results of this intervention were encouraging. Table 1 shows that

the self-reported quit rate for the intervention group was 22 percent at the

3-mo followup. The usual care group had an overall cessation of only

14 percent. However, the cessation rates were very different for people who

used only ST and those who used both ST and cigarettes. For men who used
Chapter 6

287

Table 1

Three-month followup of dental intervention for ST users in an HMO

Baseline Status Self-Reported Quit Rates

of Users at 3-Mo Followup

Percentage n Percentage n

Intervention

 ST only 69% 170 26% 45

 ST and cigarettes 31 75 12 9

 Overall cessation 22 54

Usual Care

 ST only 70 191 17 33

 ST and cigarettes 30 81 3 5

 Overall cessation 14 38

ST and cigarettes, the cessation rates were only 12 percent in the intervention group and 3 percent in the usual care group. It appears that men who

used cigarettes were much less likely to be successful in giving up ST than

were subjects who do not smoke cigarettes. The good news is that very few

of the ST-only users reported subsequently smoking cigarettes. Only 3 percent of men who reported ST-only use at baseline and reported no ST use at

followup reported smoking cigarettes.

There is increased attention being paid to special ST populations such as

baseball players and Native Americans, among whom the use of ST by

females is similar to males (Schinke et al., 1987 and 1989). Baseball players

also represent a special group as ST use has long been associated with the

sport, and these professionals are highly visible role models for American

boys. Surveys report 34 to 39 percent of professional players report using ST

in the past week (Connolly et al., 1988; Ernster, 1989). A recent publication

by the National Cancer Institute is specifically targeted to baseball players

and provides a self-help cessation manual (Orleans et al., 1991). Materials

are needed that target other groups in which ST use is prevalent. Specific

materials for cowboys, minority groups, and specific vocational groups (i.e.,

wood products or other factory workers) are more likely to be effective.

Adolescent ST prevention programs have been developed under NCI funding and are being evaluated, but cessation programs for adolescent ST users

have been lacking. The recent publication of a self-help quit pamphlet by

the American Cancer Society (1991), and the first self-help manual for

quitting snuff and chew on your own (Severson, 1992), offer valuable aids to

counselors providing cessation advice to young chewers and dippers.

SUMMARY There is evidence emerging that men are interested in quitting their use

of ST and that cessation programs can be developed that are modestly

successful. The program content and success rates appear similar to multicomponent smoking cessation programs. The need for oral substitutes such

as mint snuff or nicotine polacrilex also appears important and suggests

adjunctive aids.
Smoking and Tobacco Control Monograph No. 2

288

There is a need for development and evaluation of cessation programs

for ST users. There is a large group of men who are addicted to the nicotine

in this tobacco product, estimated at 6 million American men (US DHHS,

1986a), but little has been done to assist them in quitting. There is a need

for self-help quitting materials, cessation groups, and public health interventions. Preliminary evidence of successful adaptation of cognitive-behavioral

smoking cessation programs is encouraging. That users report trying to quit

on their own provides further support for developing cessation materials

and providing access to cessation programs. Studies using nicotine replacement (nicotine polacrilex or transdermal nicotine patches) or oral substitutes such as mint snuff are needed to determine whether those adjuncts

significantly increase long-term cessation rates. There are unique aspects of

ST use that have not received adequate attention in cessation programs, and

components (such as an oral self-exam) may provide significant motivation

for the ST user to quit.

Public health interventions for ST users also show promise. Early results

of a dental office-based intervention provide impetus for developing and

implementing public health interventions that have proved successful in

getting cigarette smokers to quit (Little et al., 1992). These interventions

may involve health professionals providing direct advice to quit in the

context of regular health care delivery. Special interventions may be most

relevant for high ST user groups such as baseball players or wood product

workers or ethnic groups such as Native Americans where prevalence of use

is endemic. In sum, there is much to be done in ST cessation, but the

groundwork has been laid and the direction and preliminary results are

promising.

ACKNOWLEDGMENTS

REFERENCES

American Cancer Society. Smokeless tobacco: Check it

out, think it over, take it out, throw it out, snuff it out,

keep it out. Atlanta: American Cancer Society

1991, No. 2090.

Ary, D.V., Lichtenstein, E., Severson, H., et al. An indepth analysis of male adolescent smokeless

tobacco users: Interviews with users and their

fathers. Journal of Behavioral Medicine 12: 449-467,

1989.

Bauman, K.E., Koch, G.G., Lentz, G.M. Parent

characteristics, perceived health risk, and smokeless tobacco use among white adolescent males.

National Cancer Institute Monographs 8: 43-48, 1989.

Benowitz, N.L., Porchet, H., Sheiner, L., et al.

Nicotine absorption and cardiovascular effects

with smokeless tobacco use: Comparison with

cigarettes and nicotine gum. Clinical Pharmacology

and Therapeutics 44: 23-28, 1988.

Biener, L., Abrams, D.B. The contemplation ladder:

Validation of a measure of readiness to consider

smoking cessation. Health Psychology 10: 360-365,

1991.

Chassin, L., Presson, C.C., Sherman, C.J., et al.

Psychological correlates of adolescent smokeless

tobacco use. Addictive Behavior 10: 431-435, 1985.

Connolly, G.N., Orleans, C.T., Kogan, M. Use of

smokeless tobacco in major-league baseball. New

England Journal of Medicine 318: 1281-1284, 1988.

DiClemente, C.C., Prochaska, J.O., Fairhurst, S.K., et

al. The process of smoking cessation: An analysis

of pre-contemplation, contemplation, and

preparation stages of change. Journal of Consulting

and Clinical Psychology 59(2): 295-304, 1991.

DiLorenzo, T.M., Kern, T.G., Pieper, R.M. Treatment

of smokeless tobacco use through a formalized

cessation program. Behavior Therapy 22: 41-46,

1991.

 The author thanks Ed Lichtenstein for his helpful editing of this manuscript

and personal support for this professional endeavor.
Chapter 6

289

Eakin, E., Severson, H.H., Glasgow, R.E. Development and evaluation of a smokeless tobacco

cessation program: A pilot study. National Cancer

Institute Monographs 8: 95-100, 1989.

Ernster, V.L. Advertising and promotion of smokeless tobacco products. National Cancer Institute

Monographs 8: 87-94, 1989.

Fagerstrom, K.O. Measuring the degree of dependence in tobacco smoking with reference to

individualization of treatment. Addictive Behaviors

3: 235-241, 1978.

Glover, E.D. Conducting smokeless tobacco

cessation clinics. American Journal of Public Health

76: 207, 1986.

Glover, E.D., Schroeder, K.L., Hinningfield, J.E., et al.

An interpretive review of smokeless tobacco

research in the United States: Part I. Drug

Education 18(4): 305-330, 1989.

Gritz, E.R., Baer-Weiss, V., Benowitz, N.L., et al.

Plasma nicotine and cotinine concentrations in

habitual smokeless tobacco users. Clinical

Pharmacology and Therapeutics 30: 201-209, 1981.

Hatsukami, D.K., Gust, S.W., Keenan, R.M. Physiological and subjective changes from smokeless

tobacco withdrawal. Clinical Pharmacology and

Therapeutics 41: 103-107, 1987.

Hatsukami, D., Nelson, R., Jensen, J. Smokeless

tobacco: Current status and future directions.

British Journal of Addiction 86: 559-563, 1991.

Hoffman, N.D., Harley, N.H., Fisenne, I., et al.

Carcinogenic agents in snuff. Journal of the

National Cancer Institute 76(3): 9, 1986.

Hollis, J.F., Vogt, T.M., Stevens, V., et al. The tobacco

reduction and cancer control (TRACC) program:

Team approaches to counseling in medical and

dental settings. In: Tobacco and the Clinician:

Interventions for Medical and Dental Practice,

D.M. Burns, S. Cohen, E. Gritz, and T. Kottke

(Editors). U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute, 1992, in

press.

Hughes, J.R., Hatsukami, D.K. Signs and symptoms

of tobacco withdrawal. Archives of General

Psychiatry 43: 289-294, 1986.

Lichtenstein, E., Severson, H.H., Friedman, L.S., et al.

Chewing tobacco use by adolescents: Prevalence

and relation to cigarette smoking. Addictive

Behavior, 9: 351-355, 1984.

Little, S.J., Stevens, V., LaChance, P., et al. Smokeless

tobacco habits and oral mucosal lesions on dental

patients. Journal of Public Health Dentistry, in press.

Little, S.J., Stevens, V., Severson, H.H., Lichtenstein,

E. An effective smokeless tobacco intervention for

dental hygiene patients. Journal of Dental Hygiene

66(4): 185-190, 1992.

Novotny, T.E., Pierce, J.P., Fiore, M.C., Davis, R.M.

Smokeless tobacco use in the United States: The

Adult Use of Tobacco Surveys. National Cancer

Institute Monographs 8: 25-28, 1989.

Orleans, C.T., Connolly, G.N., Workman, S. Beat the

Smokeless Habit: A Game Plan for Success. U.S.

Department of Health and Human Services, Public

Health Service, National Institutes of Health,

National Cancer Institute. NIH Publication No.

92-3270, 1991.

Prochaska, J.O., DiClemente, C.C. Stages and

processes of self-change of smoking: Toward an

integrative model of change. Journal of Consulting

and Clinical Psychology 5: 390-395, 1983.

Russell, M.A.H., Jarvis, M.J., Devitt, G., et al.

Nicotine intake by snuff users. British Medical

Journal 283: 814-817, 1981.

Schinke, S.P., Gilchrist, L.D., Ashby, M.R. Pacific

Northwest Native American youth and smokeless

tobacco use. International Journal of the Addictions

22(9): 881-884, 1987.

Schinke, S.P., Shilling, R.F. II, Gilchrist, L.D., Ashby,

M.R., Kitajimar, E. Native youth and smokeless

tobacco: Prevalence rates, gender differences, and

descriptive characteristics. National Cancer

Institute Monographs 8: 39-42, 1989.

Schroeder, K.L. Oral and systemic concerns with

smokeless tobacco. Clinical Dentistry 2(9): 1-27,

1989.

Schroeder, K.L., Chen, M.S., Jr., Iaderosa, G.R., et al.

Proposed definition of a smokeless tobacco user

based on “potential” nicotine consumption.

Addictive Behaviors 13: 395-400, 1988.

Severson, H.H. Enough Snuff: A Manual for Quitting

Smokeless Tobacco on Your Own. Eugene, OR:

Rainbow Publications, 1992.

Severson, H.H. Psychosocial factors in the use of

smokeless tobacco and their implications for

P.L. 99-252. Journal of Public Health Dentistry

50(1): 90-97, 1990.

Severson, H.H. Smokeless tobacco: Risks, epidemiology and cessation. In: Nicotine Addiction:

Principles and Management, C.T. Orleans and

J. Slade (Editors). New York: Oxford University

Press, in press.

Severson, H.H., Eakin, E.G., Lichtenstein, E., et al.

The inside scoop on the stuff called snuff: An

interview study of 94 adult male smokeless

tobacco users. Journal of Substance Abuse 2: 77-85,

1990a.

Severson, H.H., Eakin, E.G., Stevens, V.J., et al.

Dental office practices for tobacco users: Independent practice and HMO clinics. American Journal

of Public Health 80(12): 1503-1505, 1990b.
Smoking and Tobacco Control Monograph No. 2

290

Severson, H.H., Zoref, L. Prevention and early

interventions for addictive behaviors: Health

promotion in the schools. In: Interventions for

Achievement and Behavior Problems, G. Stoner,

M. Shinn, and H. Walker (Editors). Silver Spring,

MD: National Association of School Psychologists,

1991, pp. 539-557.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report to the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986a.

U.S. Department of Health and Human Services.

Health Implications of Smokeless Tobacco Use:

National Institutes of Health Consensus Development

Conference. U.S. Department of Health and

Human Services, Public Health Service, National

Institutes of Health, Office of Medical Applications Research, 1986b.

U.S. Department of Health and Human Services.

Youth Use of Smokeless Tobacco: More Than a Pinch

of Trouble. Washington, DC: Office of the

Inspector General, 1986c.

Vogt, T.M., Lichtenstein, E., Ary, D., et al. Integrating tobacco intervention into a health maintenance organization: The TRACC Program. Health

Education Research 4: 125-135, 1989.
Chapter 6

291

Given the health and social consequences of smokeless tobacco, strategies to reduce and

Smokeless Tobacco Cessation and

Nicotine Reduction Therapy

Elbert D. Glover and Penny N. Glover

ABSTRACT

stop ST consumption and the occurrence of tobacco-related diseases warrant close examination

and analysis. This paper examines ST use as an addiction and presents various pharmacological

adjuncts for helping smokeless tobacco users with their addiction. Specifically, 2-mg nicotine

polacrilex, 4-mg nicotine polacrilex, and transdermal nicotine patches are examined as

potential pharmacological adjuncts for ST cessation.

INTRODUCTION For more than 150 years, it has been known that nicotine is

absorbed by various organs (Orfila, 1851; Glover et al., 1989). Since 1950 we

have known that the brain is the highest among all the organs in its ability

to absorb nicotine (Werle and Meyer, 1950). Larson and his colleagues have

extensively reviewed the various physiological and behavioral actions of

nicotine (Larson and Silvette, 1968 and 1971; Larson et al., 1961). Here we

provide the basic knowledge necessary for attempting smokeless tobacco

cessation with nicotine therapy.

Since the turn of the century, it has been suspected that the central

nervous system is the site of action for the tobacco effects sought by users

(Armstrong-Jones, 1929; Russell and Feyerabend, 1981). Nicotine is a

euphoriant (Henningfield and Nemeth-Coslett, 1988). In a series of abuse

liability studies, Henningfield and Nemeth-Coslett found that nicotine

produced dose-related increases in euphoria, as measured by the drug-liking

scale. Their data demonstrated that increased euphoriant scale scores

exceeded placebo values and were directly related to increased doses of

nicotine, which identifies nicotine as a drug that produces dependence. As

a psychoactive substance, nicotine exerts a number of effects on the brain

and CNS. Once nicotine is absorbed into the blood, it is rapidly distributed

and absorbed by the brain and other highly blood-perfused tissues

(Henningfield and Nemeth-Coslett, 1988).

Animal experiments have shown that nicotine induces some of the

same changes in brain energy utilization that have been observed with drugs

like cocaine (Henningfield and Nemeth-Coslett, 1988). From the first use of

tobacco, the self-administered dosage gradually escalates. Tolerance, the

need to take larger doses to obtain the same physiological effect, begins

during the earlier stages of drug dependence and is a major factor in dose

escalation (Henningfield and Nemeth-Coslett, 1988). Henningfield and

Nemeth-Coslett further report that the self-reported number of cigarettes

smoked from day 1 through year 8 reveals that several years of smoking are

required before a stable level of one to two packs of cigarettes per day is

reached.
Smoking and Tobacco Control Monograph No. 2

292

Johnston (1942), studying intravenous nicotine delivery in humans,

came to the following conclusions:

• Smoking tobacco is essentially a means of administering nicotine, just

as smoking opium was a means of administering morphine.

• Nicotine has a psychic action.

• The actions of intravenous nicotine are similar in temporal pattern

and quality to those of inhaled tobacco smoke.

• Smokers show the same attitude toward tobacco as do people addicted to other drugs, and their judgment was therefore biased in

giving an opinion of its effect on them.

Johnston’s conclusions have been largely confirmed by a variety of techniques, including strategies available only in recent years.

Similar conclusions have been published by the National Institute on

Drug Abuse, the Public Health Service, and an Advisory Committee to the

U.S. Surgeon General (NIDA, 1984; US DHHS, 1983, 1986, and 1987). Drug

addiction is most often defined as the behavior of repeatedly ingesting a

substance that results in the delivery of a behavior-modifying chemical to

the central nervous system (Schuster, 1980). The symptoms associated with

nicotine withdrawal—craving, irritability, increasingly frequent waking

from sleep, slowed heart rate, anxiety, impaired concentration, restlessness,

drowsiness, impatience, confusion, increased hunger, and impaired reaction

time—can undermine the tobacco user’s motivation to stop (American

Psychiatric Association, 1987). The stronger the dependence on nicotine,

the lower the likelihood of successful tobacco cessation.

NICOTINE IN

SMOKELESS

TOBACCO

Plasma nicotine and cotinine levels were measured in 12 ST users by

Gritz and colleagues (1981). The subjects were male college students

who used approximately one-third of a can of smokeless tobacco per

day and did not smoke cigarettes (Gritz et al., 1981). The mean plasma

nicotine concentration of the subjects prior to tobacco use was 2.9 ng/mL;

this increased to 21.6 ng/mL after using smokeless tobacco throughout the

day. The plasma nicotine and cotinine levels found in the ST users were

comparable to concentrations found among regular cigarette smokers (Gritz,

1981). One-third of a can of ST per day is considered light use (Schroeder et

al., 1988). Similarly, in England, Russell and colleagues (1980 and 1981b)

found plasma nicotine levels in nasal snuff users reaching levels similar to

those of heavy smokers, as well as exhibiting a more rapid rate of absorption

than noninhaling cigar smokers. Furthermore, Russell and coworkers found

plasma cotinine levels to average 23 percent higher among daily snuff users

than among smokers.

CESSATION

PROGRAMS

Currently, there is little reported knowledge about smokeless tobacco

cessation. Glover (1986) reported research on two smokeless tobacco

cessation programs adapting the American Cancer Society’s FreshStart

Program for cigarette smokers. A total of 41 subjects initially enrolled in

both programs (20 in the first and 21 in the second). Glover reported a
Chapter 6

293

2.3 percent success rate for the quit-ST clinics at 6 mo, compared with his

38.0 percent success rate at 6 mo for cigarette smokers. Of the 41 ST users

who attempted abstinence, only 1 of the participants was able to go for

more than 4 h during the waking hours without using ST. Interestingly, the

success of this one individual who did achieve long-term success (1-yr

abstinence) was accomplished through a successive reduction of ST intake

with nicotine polacrilex used as an adjunct. Nicotine gum after 6 mo was

faded out.

After this experience, Glover concluded that smokeless tobacco appears

to be more addictive than cigarette smoking. Given the results of the

cessation program in which Glover had adapted the FreshStart Program, in

1986 the American Cancer Society convened an advisory group to provide

guidance for a coordinated educational effort to develop an ST cessation

manual for high school seniors. After 18 mo of development and revision,

the cessation manual was completed and released in 1991. The manual is

available through local chapters of the American Cancer Society (American

Cancer Society, 1991). The self-help program relies heavily on the theoretical bases of social contracting, peer education, and relapse prevention

(Glover et al., in press). Similar programs have been developed by others

(Hatsukami, 1991a; Severson, 1987). All three are similar in presentation

with one noted exception: Hatsukami’s program uses nicotine polacrilex,

which may be the most appropriate way to attempt ST cessation (Glover,

1992b). Given that the ST user and the smoker evidence similar nicotine

and cotinine concentration levels (Gritz et al., 1981; Russell et al., 1980 and

1981), a similar therapy could be beneficial—specifically, nicotine reduction

therapy.

Nicotine

Reduction

Therapy

Both smokeless and smoking tobacco contain nicotine, which is the basis

for addiction (Glover et al., 1989). If we are to assist ST users, nicotine

reduction therapy (NRT) should be the basis for cessation programs, just

as it is the basis for effective programs for smoking cessation. A decade ago,

we used groups, education, counseling, social contracting, and social support to assist with smoking cessation (Glover et al., 1992b). Today, we are

using NRT to assist smokers with successful cessation (Hatsukami, 1991b;

Russell et al., 1983).

Both nicotine polacrilex and nicotine transdermal systems were developed in part to improve quit rates by helping to diminish withdrawal

symptoms (particularly nicotine craving). Both have proven to be effective

stop-smoking therapies. Because nicotine plasma and cotinine levels are

similar after cigarette or smokeless tobacco use and because the addictive

potential of ST can be as great as or greater than that of cigarettes, we need

to begin to investigate NRT for ST cessation—specifically, 2-mg and 4-mg

nicotine polacrilex and transdermal patches.

Nicotine

Polacrilex

(2-mg)

Nicotine gum—nicotine polacrilex—has consistently been shown in

clinical trials to be more effective than placebo or no intervention in terms

of 6-mo and 1-yr abstinence rates (Hatsukami, 1991b). Nicotine polacrilex

is successful with smokers when used in conjunction with a behavioral

modification program (Russell et al., 1983; US DHHS, 1989 and 1990;
Smoking and Tobacco Control Monograph No. 2

294

Wilson et al., 1988). Compliance problems are related to the patient’s

chewing correctly and chewing a sufficient number of pieces per day.

Apparently many physicians do not provide proper chewing instructions to

patients.

If used correctly, nicotine polacrilex can be an excellent pharmacological adjunct for smokeless tobacco cessation. Moreover, nicotine polacrilex is

more like ST than cigarette smoking, so it provides a mimic cue. The

method of absorption from the nicotine gum is similar to that of smokeless

tobacco. Nicotine blood plasma levels, cotinine levels, placement of

polacrilex and ST, and outward appearance (bulge in the gingival area while

the material is in use) are identical. If researchers are to investigate nicotine

polacrilex as a pharmacological aid for ST cessation, it is critical to avoid

errors similar to those made with smoking cessation. Potential methodological errors include the following:

• Not using the proper chewing technique;

• Underdosing—not using sufficient quantities;

• Using nicotine with liquids; and

• Not using nicotine polacrilex in conjunction with some other type of

behavior modification program (Cummings et al., 1988).

Nicotine

Polacrilex

(4-mg)

Nicotine polacrilex in the 4-mg dose is identical to 2-mg nicotine polacrilex except that it has twice the nicotine concentration. Currently, 4-mg

nicotine polacrilex is not generally available in the United States; however,

clinical trials have been conducted. Glover and colleagues (1992b) enrolled

more than 500 highly dependent smokers (Fagerstrom score of 7 or greater)

in a double-blind, placebo-controlled study. They found no significant

difference in cesssation rates between placebo and 2-mg nicotine polacrilex;

however, the 4-mg version was found to be more effective than either

placebo or the 2-mg dose with highly dependent smokers. Given the

similarity of nicotine and cotinine levels in ST users and highly dependent

smokers, it may well be that 4-mg gum is more effective with ST users than

is the 2-mg version.

Transdermal

Patch

The nicotine transdermal patch has promise for nicotine reduction

therapy. A rate-controlling nicotine transdermal system has demonstrated efficacy in smoking cessation, including significant relief from

nicotine craving (Transdermal Nicotine Study Group, 1991). The

transdermal nicotine patch should afford fewer compliance problems than

the nicotine gum because the patch is placed on the upper part of the body

only once a day. Three pharmaceutical companies have 24-h patches

approved by the Food and Drug Administration for smoking cessation, and

a 16-h patch is currently awaiting FDA approval.

The patch delivers a specific amount of nicotine to the central nervous

system every hour, allowing for a steady rate of nicotine absorption; consequently, withdrawal symptoms are minimized. This technique has been

found to be very promising for cigarette smokers (Transdermal Nicotine
Chapter 6

295

Study Group, 1991). Several transdermal nicotine systems are available, and

they vary in nicotine dose, drug delivery design and technology, absorption

and efficacy rates, and effects on skin. The patch eliminates the nicotine

absorption peaks and troughs associated with nicotine polacrilex use

(Hatsukami, 1991b). It is hoped that the new transdermal technology can

be applied for ST users with similar success.

Other

Pharmacological

Adjuncts

Several nicotine products that are in various phases of testing for

smoking cessation could one day be used for ST cessation:

• Nicotine nasal spray—a device that delivers nicotine via the nose

(Schneider, 1992);

• Oral inhaler—a device that delivers nicotine via the lungs (Glover et

al., 1992a); and

• Oral lozenge—a device that delivers nicotine via the oral mucosa.

Validating

Self-Reports

One obstacle to the use of nicotine reduction therapy with ST cessation

was the inability to validate self-reports. Previously, it was impossible

to distinguish chemically the nicotine in smokeless tobacco from the

nicotine in polacrilex gum. However, Peyton and Benowitz (1991) have

developed a method for validating self-reports using excretion of the alkaloids anabasine and anatabine in the urine of ST users. Their test allows the

researcher to validate self-reports for ST users attempting to quit with NRT.

In light of the available literature, NRT has potential benefits for smokeless tobacco cessation and deserves further study. NRT may well be our best

hope for ST users.

REFERENCES

American Cancer Society. Smokeless Tobacco: Check

It Out, Think It Out, Take It Out, Throw It Out, Snuff

It Out, Keep It Out. Atlanta: American Cancer

Society, 1991 (Publication 91-10M-No. 2090).

American Psychiatric Association. Diagnostic and

Statistical Manual of Mental Disorders, 3rd edition,

revised. Washington, DC: American Psychiatric

Association, 1987.

Armstrong-Jones, S. Tobacco, its use and abuse:

From the nervous and mental aspect. Practitioner:

6-19, 1929.

Cummings, S.R., Hansen, B., Richard, R.J., Stein, M.J.,

Coates, T.J. Internist and nicotine gum. Journal of

the American Medical Association 260: 1565-1569,

1988.

Glover, E.D. Conducting smokeless tobacco

cessation clinics. American Journal of Public Health

76: 207, 1986.

Glover, E.D., Glover, P.N., Nilsson, F., Sawe, U. To

determine the safety and efficacy of the Nicohaler

as an aid in smoking cessation. Presented at the

Eighth World Conference on Tobacco and Health,

Buenos Aires, Argentina, 1992a.

Glover, E.D., Schroeder, K.L., Henningfield, J.E.,

Severson, H.H., Christen, A.G. Smokeless tobacco

research in the United States: Part II. Journal of

Drug Education 19(1): 1-19, 1989.

Glover, E.D., Wadland, W., Sachs, D.P.L., Rennard,

S.I., Daughton, D., Pomerleau, O.F., Huster, W.J.,

Nowak, R.T., Rolf, C.N. The safety and efficacy of

4 mg Nicorette as compared to 2 mg Nicorette

versus placebo in highly dependent smokers.

Presented at the Eighth World Conference on

Tobacco and Health. Buenos Aires, Argentina,

1992b.

Glover, E.D., Wang, M., Glover, P.H. Development

of a high school self-help smokeless tobacco

cessation manual. Health Values, in press.

Gritz, E.R., Baer-Weiss, V., Benowitz, N.L., Van

Vunakis, J., Jarvik, M.E. Plasma nicotine and

cotinine concentrations in habitual smokeless

tobacco users. Clinical Pharmacology Therapy 30:

201-209, 1981.

Hatsukami, D. Tough Enough to Quit Using Snuff.

Minneapolis: Tobacco Research Laboratory,

University of Minnesota, 1991a.
Smoking and Tobacco Control Monograph No. 2

296

Hatsukami, D. Clinical trials with nicotine replacement therapies. Presented at the Centennial

Meeting of the American Psychological Association. San Francisco, 1991b.

Henningfield, J.E., Nemeth-Coslett, R. Nicotine

dependence: Interface between tobacco and

tobacco-related disease. Chest 93(Suppl): 37S-55S,

1988.

Johnston, L.M. Tobacco smoking and nicotine.

Lancet 2 (742): 743, 1942.

Larson, P.S., Haag, H.B., Silvette, H. Tobacco

Experimental and Clinical Studies: A Comprehensive

Account of the World Literature. Baltimore:

Williams and Wilkins, 1961.

Larson, P.S., Silvette, H. Tobacco Experimental and

Clinical Studies: A Comprehensive Account of the

World Literature. Supplement I. Baltimore:

Williams and Wilkins, 1968.

Larson, P.S., Silvette, H. Tobacco Experimental and

Clinical Studies: A Comprehensive Account of the

World Literature. Supplement II. Baltimore:

Williams and Wilkins, 1971.

National Institute on Drug Abuse. Drug Abuse and

Drug Abuse Research, the first in a series of triennial

reports to Congress from the Secretary, Department of Health and Human Services. Department

of Health and Human Services, National Institutes

of Health, 1984.

Orfila, B. Memoire sur la nicotine et sur la conicine.

Ann Hygiene Pub 45: 147-230, 1851.

Peyton, J., Benowitz, N.L. Excretion of the alkaloids

anabasine and anatabine in urine of smokeless

tobacco users and cigarette smokers: Biochemical

markers of smokeless tobacco use. Presented at the

First International Conference on Smokeless

Tobacco. Columbus, Ohio, April 10-13, 1991a.

Russell, M.A.H., Feyerabend, C. Cigarette smoking:

A dependence disorder. British Journal of Medical

Psychiatry 8(1): 29-37, 1981.

Russell, M.A.H., Jarvis, M.J., Feyerabend, C. A new

age for snuff. Lancet 1: 474-475, 1980.

Russell, M.A.H., Jarvis, M.J., Dewitt, C., Feyerabend,

C. Nicotine intake by snuff users. British Medical

Journal 283: 814-817, 1981.

Russell, M.A.H., Merriman, R., Stapleton, J., Taylor,

W. Effects of nicotine chewing gum as an adjunct

to general practitioners’ advice against smoking.

British Medical Journal 287: 1782-1785, 1983.

Schneider, N. The safety and efficacy of the nicotine

nasal solution as an aid for smoking cessation.

Presented at the Smoking Cessation Scientific

Meeting. Thundridge, England, 1992.

Schroeder, K.L., Chen, M.S., Iaderosa, G.R., Glover,

E.D., Edmundson, E.W. Proposed definition of a

smokeless tobacco user based on “potential”

nicotine consumption. Addictive Behaviors 13:

395-400, 1988.

Schuster, C.R. Addiction: Report of the Task Force

on Smoking. World Smoking and Health 5: 22,

1980.

Severson, H.H. Up to Snuff: A Handbook on Smokeless

Tobacco. Eugene, OR: Independent Video

Services, 1987.

Transdermal Nicotine Study Group. Transdermal

nicotine for smoking cessation. Journal of the

American Medical Association 22: 3133-3138, 1991.

U.S. Department of Health and Human Services.

Why People Smoke Cigarettes. U.S. Department of

Health and Human Services, Public Health Service.

PHS Publication No. (PHS) 83-50195, 1983.

U.S. Department of Health and Human Services.

Nicotine: Drug Abuse Research. Second in a series

of triennial reports to Congress from the Secretary

of Health and Human Services. DHHS Publication

No. (ADM) 87-1486, 1987.

U.S. Department of Health and Human Services.

Reducing the Health Consequences of Smoking: 25

Years of Progress. A Report of the Surgeon General.

U.S. Department of Health and Human Services,

Public Health Service, Centers for Disease Control,

Center for Chronic Disease Prevention and Health

Promotion, Office on Smoking and Health. DHHS

Publication No. (CDC) 89-8411, 1989.

U.S. Department of Health and Human Services.

Health Benefits of Smoking Cessation: A Report of the

Surgeon General. U.S. Department of Health and

Human Services, Public Health Service, Centers for

Disease Control, Center for Chronic Disease

Prevention and Health Promotion, Office on

Smoking and Health. DHHS Publication No.

(CDC) 90-8416, 1990.

U.S. Department of Health and Human Services.

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. NIH

Publication No. 86-2874, 1986.

Werle, E., Meyer, A. Uber den Abbau von

Tabakalkaloiden surch Tierische Gewebe.

Biochemische Zeitschrift 321: 221-235, 1950.

Wilson, D.M., Taylor, D.W., Gilbert, J.R., Best, J.A.,

Lindsay, E.A., Willms, D.G., Singer, J. A randomized trial of a family physician intervention for

smoking cessation. Journal of the American Medical

Association 260: 1570-1574, 1988.
Chapter 6

297

Dental professionals in public health and research have been instrumental in raising the

Role of Dentists in Cessation

Counseling: Survey Findings

Kathleen L. Schroeder and David E. Heisel

ABSTRACT

public’s awareness of smokeless tobacco, yet recent data indicate that the general dental

practitioners’ and specialists’ role in reducing ST use has been underestimated and underused.

A 73-item survey was mailed to 1,064 dentists in Central Ohio to ascertain their knowledge of

and receptivity to providing an ST or tobacco use intervention in a clinical setting. Of the 529

dentists responding to the survey, only 9 percent stated that they were effective at getting their

patients to stop using tobacco. Relative to ST cessation, 71 percent of the dentists would be

willing to provide educational pamphlets on ST to their patients; 6 percent consider prescribing

nicotine gum; 3 percent consider delegating prevention responsibility; and 15 percent consider

referring to an outside cessation program. Of the dentists who responded, 73 percent felt

cessation counseling was frustrating. Dentists considered that barriers to ST cessation counseling included their lack of training (66 percent) and lack of insurance coverage (73 percent).

The results indicate the need for further education in tobacco and cessation counseling for

dentists.

INTRODUCTION The dental professional’s role in reducing smokeless tobacco use

has been double-edged. Public health dentists and dental researchers

studying the effects of ST (Archard and Tarpley, 1979; Axéll et al., 1976;

Christen, 1980; Connolly, 1986; Greer, 1983; Hirsch et al., 1982; Hoge and

Kirkham, 1983; Offenbacher and Weathers, 1985; Park et al., 1985; Pindborg

and Renstrup, 1963; Poulson, 1983; Roed-Petersen and Pindborg, 1973;

Schroeder, 1989; Schroeder et al., 1985, 1988a, 1990; Schroeder and Chen,

1985; Squires, 1984; Van Wyk, 1976) report that dental professionals have

been among the most instrumental in scientific and public health awareness

of ST. Lobbying efforts by dental professionals have played a major role in

having the Federal Trade Commission change its final rules for warnings on

snuff and chewing tobacco (ADA News, 1986). Yet recent data indicate that

the dental practitioners’ role in reducing ST use by their patients and in the

community has been underestimated and underused (Fried and RubinsteinDeVore, 1990; Geboy, 1990; Schroeder, 1989 and 1990; Schroeder et al.,

1988b and 1990).

In the past 10 yr, we have found the increased use of snuff in countries

such as Sweden and increases in dipping and chewing in the United States,

particularly in high schools and with young adult males (Christen et al.,

1979; Greer and Poulson, 1983; Hoffman et al., 1986; Poulson et al., 1984).

Clinicians have noted associated oral problems such as dental caries, abrasion, gingivitis, gingival recession, periodontitis, leukoplakia, and oral

cancer (Christen, 1980; Greer and Poulson, 1983; Greer et al., 1986 and

1988; Hoffman et al., 1986; Hoge and Kirkham, 1983; Offenbacher and

Weathers, 1985; Pindborg and Renstrup, 1963; Roed-Petersen and Pindborg,

1 Supported in part by a grant to Dr. Schroeder from the American Heart Association,

Central Ohio Chapter.

1
Smoking and Tobacco Control Monograph No. 2

298

1973; Schroeder et al., 1985) with the increased use of ST. Often, ST-induced lesions are found to have a hyperkeratotic white patch (leukoplakia)

(Greer and Poulson, 1983; Greer et al., 1986 and 1988; Pindborg and

Renstrup, 1963; Roed-Petersen and Pindborg, 1973). Leukoplakia is a white

patch or plaque that cannot be characterized clinically or pathologically as

any other disease (WHO, 1978) and is considered a premalignant lesion that

has a transformation rate of more than 6 percent for either dysplasia or

carcinoma (US DHHS, 1986).

Furthermore, nicotine concentrations in the blood of ST users have

been shown to be as high as those in cigarette smokers, and exposure to

nicotine from ST is 8 to 10 times higher than from an average package of

cigarettes (Schroeder et al., 1988c and 1990). ST has further been related to

increases in heart rate and blood pressure (Edwards et al., 1987; Gritz et al.,

1981; Neal et al., 1988; Schroeder and Chen, 1985; Schroeder et al., 1988c

and 1990; Squires et al., 1984). Thus, ST is a potential risk factor for coronary and peripheral vascular disease, peptic ulcer, reproductive disorders,

and neuromuscular disease (US DHHS, 1986).

Health professionals in the past few years have become aware of the

health hazards of ST, following the enactment of P.L. 99-252 aimed at

decreasing ST use (Chen and Schroeder, 1990; Chen et al., 1991; Schroeder,

1990). The American Dental Association, American Medical Association,

American Cancer Society, American Heart Association, Federal Government,

and State dental health departments include ST information in their efforts

to control tobacco use (CDC, 1986; Chen and Schroeder, 1987). Recently,

the American Academy of Otolaryngology-Head and Neck Surgery began a

1-yr campaign with a comprehensive slide and lecture series (McGuirt, 1990)

and videotape (American Academy of Otolaryngology-Head and Neck

Surgery, 1990) offered to their members. The American Dental Association

now offers several educational brochures and videotapes (ADA, 1990a and

1990b), and even the Office of the Commissioner of Major League Baseball

worked with the National Cancer Institute to produce a booklet on ST

education (US DHHS, 1991). Despite the availability of information on the

health hazards of ST, in one screening only 10 percent of ST users reported

that their dentist informed them that they had lesions associated with ST

use (Schroeder et al., 1988c). Dental practitioners should actively provide

information on ST and initiate early detection and prevention protocols for

patients at high risk for ST use. This could include health questions regarding tobacco use, measurement of blood pressure, more thorough identification and detection of lesions, and involvement in tobacco cessation counseling in the office or by referral. To promote these efforts to the dental

community, the dental components of two major NCI tobacco use interventions, the Community Intervention Trial for Smoking Cessation (COMMIT,

1988 to 1993) and the American Stop Smoking Intervention Study for

Cancer Prevention (ASSIST, 1994 to 1998), provide a framework for the

dissemination of oral health and cessation counseling information and the

adoption of tobacco-related policies for community organizations

(Mecklenburg, 1989).
Chapter 6

299

Because of dentists’ prevention-oriented role, exemplified in their

effectiveness with using fluorides and reducing dental decay rates, dentists

could be an effective force in the prevention and reduction of tobacco use as

well. There are more than 140,000 dentists in the United States, who serve

most populations and treat more than 70 percent of the total population

within any 2-yr interval (e.g., 79 percent of working adults ages 18 to 64,

1985 to 1986) (Mecklenburg, 1989). Dentists have a significant amount of

public contact and reinforce other health professional and community

activities that are directed toward tobacco prevention and cessation. Dentists also can prescribe nicotine gum or other alternative nicotine delivery

therapies for assisting their patients in office-based cessation programs.

For the most part, there has been insufficient education for dental

professionals to make them aware of how they can assist in tobacco use

prevention and cessation. We do know that, to assist or implement any

cessation programs through dental practitioners, the knowledge and receptivity of dental professionals for providing an office intervention for tobacco

use must be understood and assessed. Therefore, the current study provides

information on the involvement and views of a large population of dentists,

representative of Central Ohio, toward ST prevention and cessation activities, as well as the attitudes and barriers among the dental community

relative to tobacco knowledge and cessation counseling.

MATERIALS

AND METHODS

A 73-item survey and a computer-readable response sheet were

mailed to 1,064 dentists who were Ohio Dental Association members within the Central Ohio area between April and June of 1989. Central

Ohio provides a representative sampling from urban, suburban, and rural

populations (Table 1). The mailing list for area dentists was obtained from

the Ohio Dental Association. Dentists who did not respond within 3 wk

were sent a second mailing.

The survey contained items on demographics, knowledge of tobacco,

cessation counseling practices, and attitudes about counseling, particularly

relative to ST. Prior to the mailing, the survey was pilot-tested with

30 dentists and dental students, with a repeat reliability of r=0.73-1.00.

The completed response sheets were scanned by computer, and data

were tabulated. Data analysis was performed with the Statistical Analysis

System (SAS) computer program.

RESULTS Responses to the survey were received from 529 dentists (50 percent of the

sample). Table 1 provides the demographics of the responding dentists.

Many dentists reported conducting thorough soft tissue exams on all

patients (88 percent) and followup visits for those with suspected lesions

(82 percent); 82 percent indicated that it was their responsibility to educate

the public about ST. However, other questions revealed limited ST counseling practices (Table 2). Although 82 percent believed that it was their

responsibility to influence others about ST use, only 44 percent of the

dentists discussed the subject of ST cessation during every visit or almost

every visit. The most commonly reported method of counseling that

dentists would be willing to provide was distribution of pamphlets; very few
Smoking and Tobacco Control Monograph No. 2

300

Table 1

Characteristics of dentists completing the survey

Percentage

Characteristic (n=529)

Age

25 to 35 yr 22%

36 to 45 29

46 to 55 19

> 55 29

Gender

Male 95

Female 5

Race

White 96

Asian 2

Declined to state 2

Professional Status

General dentist 85

Specialist 15

Practice Location

Urban 23

Suburban 53

Rural 24

Smoking Status

Never smoked 53

Former smoker 39

Current smoker 7

Chewing/Dipping Status

Never chewed/dipped 91

Former chewer/dipper 7

Current chewer/dipper 2

thought to prescribe nicotine gum, to delegate teaching prevention responsibility, or to refer the patient to an outside cessation program.

Dentists’ attitudes (Table 3) may contribute to the current state of

reduced counseling practices. Only 9 percent thought themselves effective

at getting patients to stop using tobacco, 26 percent forgot to discuss tobacco use, and 23 percent believed they had no time to counsel patients.

Many of the responding dentists felt that ST counseling was frustrating and

ineffective. Often they believed the tobacco users were not interested, and a

few dentists felt that some patients might leave the practice as a result of

their counseling efforts. However, the primary inhibitors of the interest in
Chapter 6

301

Table 2

Counseling practices

Percentage

Statement of Practice (n=529)

I bring up quitting or cutting down with patients who chew/dip.

Every visit/almost every visit 44%

Occasionally 48

Initial visit only 2

Never 3

I have other staff counsel patients about chewing.

Often or sometimes 36

Never 61

With regard to cessation programs for ST, I would be most willing to:

Prescribe nicotine-containing gum 6

Provide educational pamphlets on ST 71

Delegate teaching prevention responsibility 3

Refer to outside program 15

None of the above 3

I give pamphlets or educational materials on chewing/dipping.

Often or sometimes 36

Never 63

ST counseling (Table 3 and Figure 1) were inadequate training in counseling

(66 percent) and lack of insurance coverage for cessation counseling (73 percent). However, 68 percent of the dentists stated that they would participate in a continuing education program regarding ST prevention and

cessation.

The survey did reveal certain exam-related counseling practices that the

dentists offered to their patients (Table 4). A high percentage (82 to 90

percent) of dentists provided thorough soft tissue exams on all patients,

conducted followup visits on suspected lesions, and informed their patients

about caries, stains, gingivitis, and soft tissue changes caused by ST use.

However, only a small percentage of the dentists reported routinely asking

questions about tobacco use on health histories (35 percent) or screening for

blood pressure (17 percent).

CONCLUSIONS Results from this study of ST cessation counseling among Central

Ohio dentists were similar to Gerbert and colleagues’ study on dentists’

attitudes about smoking cessation (Gerbert et al., 1989). Both studies

revealed very limited counseling efforts by dentists with regard to tobacco

use. Both studies indicate similar reasons for these disappointing results.

In our study, many of the dentists indicated uncertainty toward their

role as counselors. Although 82 percent indicated that it was a dentist’s

responsibility to educate the public about ST, 26 percent reported they

forget to discuss it with their patients. This may be attributable, in part, to
Smoking and Tobacco Control Monograph No. 2

302

Table 3

Dentists’ attitudes about ST cessation counseling

Percentage Who Agree

With Statement

Statement (n=529)

Counseling against tobacco is frustrating. 66%

Tobacco users will leave practice. 14

I am not interested. 16

It is not covered by insurance. 73

If health insurance paid for counseling, I would spend more 52

time counseling tobacco users to quit.

I forget to bring it up. 26

I feel advice is not effective. 36

I feel tobacco users are not interested. 50

I have no time to counsel. 23

I have no training in counseling. 66

I am quite effective at getting patients to stop using tobacco. 9

the perception that ST does not represent a major health risk, for whatever

reason, to their patients. Although 93 percent believed cigarette smoking to

be very harmful to a smoker’s health, only 74 percent believed ST to be very

harmful to a user’s health. Additionally, many found counseling on tobacco

use to be frustrating. A lack of training in counseling was a prime reason for

feeling ineffective as cessation counselors. A smaller percentage believed

that tobacco users were not interested.

Another major barrier appeared to be a lack of insurance coverage for

preventive counseling. Economic incentives through insurance coverage

may promote increased counseling and prevention.

Dentists can have a more effective role in counseling patients about ST

and smoking. Dentists are “ideally positioned to counsel against the use of

cigarettes and smokeless tobacco products.” The dental encounter probably

constitutes a teachable moment, when the patient is receptive to counseling

about lifestyle issues. Past efforts by dentists to prevent oral disease ally

them with the preventive health care movement; active counseling in other

areas seems an appropriate adjunct. Because many adolescents and adults

visit the dentist each year, the dental visit is an excellent opportunity to

reach a captive audience (Gerbert et al., 1989; Schroeder, 1989).

The results of this study seem to warrant the following steps:

• Promotion of counseling courses in dental schools and continuing education—In our study, 66 percent of the respondents reported inadequate

counseling education. Access to counseling courses would enhance

the dental practitioners’ confidence in their ability to counsel effectively.
Chapter 6

303

Figure 1

Dentists’ barriers to ST cessation counseling

• Promotion of the use of dental staff for cessation counseling—Dental

hygienists and assistants have equal or greater access to the dental

patient (Fried and Rubinstein, 1990). Delegation of counseling duties

to staff would be cost-effective and result in additional time for other

dental procedures by the dentist.

• Reimbursement for preventive counseling by insurance companies—Costs

for private practitioners continue to escalate because of factors such as

infection control and Occupational Safety and Health Administration

guidelines. Most practitioners will hesitate to spend time counseling

without appropriate remuneration.

• Marketing all tobacco use as offensive and harmful—Although education

about ST has been increasing, some dentists still view ST as a safe

alternative to smoking. This is evident among the participating

dentists in the present survey. While 93 percent believed cigarette

smoking to be very harmful, fewer believed ST to be very harmful.

Among the indicators of the impact of P.L. 99-252 on decreasing ST use

is the presence of curricula on ST. This potential means of evaluation of the

impact of the law indicates that the curriculum directors, not only in

No insurance

No counseling training

Users not interested

Advice not effective

Forget to mention

No time to counsel

Dentist not interested

Users will leave

Percentage per Response

0 20 40 60 80 100

14%

16%

23%

26%

36%

50%

66%

73%
Smoking and Tobacco Control Monograph No. 2

304

Table 4

Exam-related counseling practices

Percentage Who

Perform Practice

Practice (n=529)

I ask specific questions regarding my patients’ tobacco habits

on health history. 35%

I conduct a thorough soft tissue exam on all patients. 88

I conduct followup visits on suspected initiated lesions. 82

I inform about caries, stains, gingivitis, and soft tissue changes

due to ST use. 90

I screen for blood pressure on all patients. 17

primary and secondary education (Chen et al., 1991) but also in dental and

medical schools (Fried and Rubinstein-DeVore, 1990; Geboy, 1990;

Schroeder, 1990), consider smokeless tobacco, tobacco in general, and

programs on cessation counseling of sufficient importance that faculty

should include these topics in courses.

This study reveals the importance of addressing tobacco use cessation

education in dental schools and dental hygiene programs in the future, and

the important role the oral health practitioner can play in controlling

tobacco use. The potential for dentists as tobacco cessation counselors and

for prescribing of alternative nicotine delivery systems, such as nicotine

gum, as an adjunct to their counseling should not be overlooked.

The authors thank Dr. Steven Margulies and Lawrence Hill for their assistance with the survey and preparation of the manuscript.

REFERENCES

ADA News. FTC issues rules on smokeless. ADA News

17(22): 1, 1986.

American Academy of Otolaryngology-Head and

Neck Surgery. Smokeless Tobacco: Is It Worth the

Risk? (Videotape) 1990.

American Dental Association. Smokeless Tobacco:

The Risks. (Videotape) 1990a.

American Dental Association. Smokeless Tobacco:

The Problem. (Videotape) 1990b.

Archard, H.O., Tarpley, T.M. Clinicopathologic and

histochemical characterization of submucosal

deposits in snuff dipper’s keratosis. Journal of Oral

Pathology 1: 3-11, 1979.

Axéll, T., Mörnstad, H., Sundström, B. The relation

of the clinical picture to the histopathology of

snuff dipper’s lesions in a Swedish population.

Journal of Oral Pathology 5: 229-236, 1976.

Centers for Disease Control. Smokeless Tobacco

Education Resources. Atlanta, GA: CDC, 1986.

Chen, M.S., Schroeder, K.L. Analysis of print and

audiovisual materials to prevent smokeless

tobacco use. Journal of Cancer Education 2(4): 239-

245, 1987.

Chen, M.S., Schroeder, K.L. An epilogue to evaluating the impact of P.L. 99-252 on decreasing

smokeless tobacco use. Journal of Public Health

Dentistry 50(1): 1-4, 1990.

Chen, M.S., Schroeder, K.L., Glover, E.D., Bonaguro,

J., Capwell, E.M. Tobacco use prevention in the

national school curricula: Implications of a

stratified random sample. Health Values 15(2):

3-9, 1991.

Christen, A.G. The case against smokeless tobacco:

Five facts for the health professional to consider.

Journal of the American Dental Association 101(9):

464-469, 1980.

ACKNOWLEDGMENTS
Chapter 6

305

Christen, A.G., McDaniel, R.K., Doran, J.E. Snuff

dipping and tobacco chewing in a group of Texas

college athletes. Texas Dental Journal 97: 6-10,

1979.

Connolly, G.N., Winn, D.M., Hecht, S.S., et al. The

reemergence of smokeless tobacco. New England

Journal of Medicine 314(16): 1020-1027, 1986.

Edwards, S.W., Glover, E.D., Schroeder, K.L. The

effects of smokeless tobacco on heart rate and

neuromuscular reactivity in athletes and nonathletes. The Physician and Sportsmedicine 15(7):

141-147, 1987.

Fried, J.L., Rubinstein, L. Attitudes and behaviors of

dental hygienists concerning tobacco use. Journal

of Public Health Dentistry 50(3): 172-177, 1990.

Fried, J.L., Rubinstein-DeVore, L. Tobacco use

cessation curricula in U.S. dental schools and

dental hygiene programs. Journal of Dental

Education 54(12): 730-735, 1990.

Geboy, M.J. Dental school-based continuing

education in tobacco use cessation counseling for

oral health care providers. Journal of Dental

Education 54(12): 736-738, 1990.

Gerbert, B., Coates, T., Zahn, E. Dentists as smoking

cessation counselors. Journal of the American Dental

Association 118(1): 29-32, 1989.

Greer, R.O., et al. Smokeless tobacco-associated oral

changes in juvenile, adult and geriatric patients:

Clinical and histomorphometrical features.

Gerodontics 2(3): 87-98, 1986.

Greer, R.O., Poulson, T.C. Oral tissue alterations

associated with the use of smokeless tobacco by

teenagers. Oral Surgery, Oral Medicine, Oral

Pathology 56(3): 275-284, 1983.

Greer, R.O., Schroeder, K.L., Crosby, L. Morphologic

and immunohistochemical evidence of human

papillomavirus capsid antigen in smokeless

tobacco keratoses from juveniles and adults.

Journal of Oral and Maxillofacial Surgery 46: 919-

929, 1988.

Gritz, E.R., et al. Plasma nicotine and cotinine

concentrations in habitual smokeless tobacco

users. Clinical Pharmacology Therapy 30(2): 201-

209, 1981.

Hirsch, J.M., et al. A clinical histomorphological and

histochemical study on snuff-induced lesions of

varying severity. Journal of Oral Pathology 11: 387-

398, 1982.

Hoffman, D., et al. Carcinogenic agents in snuff.

Journal of the National Cancer Institute 76: 435-436,

1986.

Hoge, H.W., Kirkham, D.B. Clinical management

and soft tissue reconstruction of periodontal

damage resulting from habitual use of snuff.

Journal of the American Dental Association 107: 744-

745, 1983.

McGuirt, W.F. Smokeless tobacco usage: A historical review and discussion of its health implications. Slide Lecture Series, American Academy of

Otolaryngology-Head and Neck Surgery Foundation, Alexandria, VA, 1990.

Mecklenburg, R.E. Tobacco in America in the

1990’s: The role of dentists, the dental team and

organized dentistry. In: Smoking Cessation:

Helping Dental Patients To Quit Smoking.

Proceedings of the First National Dental Symposium

on Smoking Cessation, A.G. Christen and J.L.

McDonald (Editors). Chicago: American Dental

Association, 1989.

Neal, C.J., Schroeder, K.L., Wenrick, C.J. Effects of

smokeless tobacco on peripheral cardiovascular

flow in young male adults. Journal of Dental

Research 67: 274, 1988.

Offenbacher, S., Weathers, D.R. Effects of smokeless

tobacco on the periodontal mucosal and caries

status of adolescent males. Journal of Oral

Pathology 14: 169-181, 1985.

Park, N.-H., Niukian, D., Shklar, G. Combined

effects of herpes simplex virus and tobacco on the

histopathologic changes in lips of mice. Oral

Surgery, Oral Medicine, Oral Pathology 59(2): 154-

158, 1985.

Pindborg, J.J., Renstrup, G. Studies in oral

leukoplakias: Effect of snuff on oral epithelium.

Acta Dermato-Venereologica (Stockholm) 43: 271-

276, 1963.

Poulson, T.C., Lindenmuth, J.E., Greer, R.O. A

comparison of the use of smokeless tobacco in

rural and urban teenagers. CA—A Cancer Journal

for Clinicians 34: 248-261, 1984.

Roed-Petersen, B., Pindborg, J.J. A study of Danish

snuff-induced oral leukoplakias. Journal of Oral

Pathology 2: 301-313, 1973.

Schroeder, K.L. Oral and systemic concerns with

smokeless tobacco. In: Clark’s Clinical Dentistry

(volume 2), J. Hardin (Editor). New York: J.B.

Lippincott, 1989.

Schroeder, K.L. P.L. 99-252: Implications for

dentists and their clinical practice. Journal of

Public Health Dentistry 50(1): 1-6, 1990.

Schroeder, K.L., Chen, M.S. Smokeless tobacco and

blood pressure. New England Journal of Medicine

312(14): 919, 1985.

Schroeder, K.L., Chen, M.S., Jr., Iaderosa, G.B.,

Glover, E.D., Edmundson, E.W. Proposed

definition of a smokeless tobacco user based on

potential nicotine consumption. Addictive

Behaviors 13: 395-400, 1988a.

Schroeder, K.L., Chen, M.S., Kuthy, R.A. Smokeless

tobacco: The new thing to chew on. Ohio Dental

Journal 59(178): 11-14, 1985.
Smoking and Tobacco Control Monograph No. 2

306

Schroeder, K.L., Glover, E.D., Zebracki, M. Comparison of snuff and cigarettes on cardiovascular

hemodynamics in young adults. In: Tobacco and

Health 1990: The Global War. Proceedings of the

Seventh World Conference on Tobacco and Health,

B. Durston and K. Jamrozik (Editors). Perth:

Cancer Foundation of Western Australia, 1990,

pp. 412-415.

Schroeder, K.L., Heisel, D.E., Margulies, S., Williams,

D. Dentists and dental students knowledge and

practices regarding smoking and smokeless

tobacco. Journal of Dental Research 69: 1005, 1990.

Schroeder, K.L., Neal, C.J., Chen, M.S. Alteration in

cardiovascular hemodynamics from smokeless

tobacco use in young adult males. Journal of

Dental Research 67: 274, 1988b.

Schroeder, K.L., Soller, H.A., Chen, M.S., Neal, C.J.,

Glover, E.D. Screening for smokeless tobaccoassociated lesions: Recommendations for the

dental practitioner. Journal of the American Dental

Association 116: 37-42, 1988c.

Squier, C.A. Smokeless tobacco and oral cancer: A

cause for concern? CA—A Cancer Journal for

Clinicians 34(5): 242-247, 1984.

Squires, W.G., et al. Hemodynamic effects of oral

smokeless tobacco in dogs and young adults.

Preventive Medicine 13: 195-206, 1984.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986.

U.S. Department of Health and Human Services.

Beat the Smokeless Habit: Game Plan for Success.

U.S. Department of Health and Human Services,

Public Health Service, National Institutes of

Health, National Cancer Institute. DHHS

Publication No. (NIH) 92-3270, 1991.

Van Wyk, C.W. Oral lesions caused by habits.

Forensic Science 7: 41-49, 1976.

World Health Organization Collaborating Center for

Oral Precancerous Lesions. Definition of leukoplakia and related lesions: An aid to studies on

oral precancer. Oral Surgery 46(4): 518-539, 1978.
Chapter 6

307

In a house-to-house screening survey, 36,000 tobacco chewers and smokers were selected

An Intervention Study of Tobacco

Habits Among Rural Indian Villagers

Mira B. Aghi, P.C. Gupta, F.S. Mehta, and J.J. Pindborg

ABSTRACT

from the rural population in three districts of three States in India—Kerala, Andhra, and

Gujarat. These individuals were interviewed about their tobacco habits and examined for the

presence of precancerous lesions in a baseline survey and then annually over 9 yr. They were

educated through personal communication as well as mass media to give up their tobacco

habits. The results have indicated consistently that it is possible to induce changes in the

tobacco habits of rural populations through educational efforts. This is further substantiated by

a significant decrease in the incidence of precancerous lesions. As most cancers in India are

reported to be preceded by precancer, this study demonstrates that the primary prevention of

oral cancer is feasible, effective, and practicable.

INTRODUCTION India is afflicted with the problem of oral cancer. The incidence of

oral cancer in India is higher than the incidence of most other cancers

(National Cancer Registry, 1988). The relationship between oral cancer and

tobacco habits is established as much beyond doubt as the relationship

between cigarette smoking and lung cancer, although it may not be as much

publicized as the latter. Thus, it appears logical that one way oral cancer can

be controlled is through intervention in the tobacco habits of people. The

disease can be arrested and cured by treatment, but an epidemic would be

impossible to control if tobacco habits were not checked.

As in the case of lung cancer, it is yet not known how much tobacco

use would actually result in oral cancer. It seems improbable that such a

guideline will be available in the near future. Therefore, it appears that our

best bet would be to help people give up the tobacco habit. The primary

health care delivery system ought to include such education.

The question then arises: How do we design such a system? Not all

forms of health care delivery meet with success. Unless a health care delivery system is designed in consideration of the target population, their

habits, beliefs, lifestyles, and knowledge, the system may be ineffective.

The Tata Institute of Fundamental Research has specifically designed

delivery of health care services to help people stop their tobacco habits. The

communication strategies were developed with the full involvement of the

target population. The project study went through two phases before it

undertook to intervene in the people’s tobacco habits. Phase I was a series

of cross-sectional field surveys undertaken to determine the prevalence rates

of oral precancerous lesions and their association with tobacco habits. The

total population surveyed was more than 155,000 (Bhonsle et al., 1976;

Mehta et al., 1969 and 1972). Phase II of the project was a 10-yr followup

1

 This research was supported solely by funds from the National Institutes of Health under PL480 research agreement no. 01-022-N.

1
Smoking and Tobacco Control Monograph No. 2

308

study of 30,000 of the original study population. The results obtained in

Phases I and II established that (1) oral cancer and precancerous lesions

occurred almost solely among those who smoked or chewed tobacco and (2)

oral cancer was almost always preceded by some kind of precancerous

lesions (Gupta et al., 1980).

On the basis of these results, an intervention study was launched. The

objectives of the study were to make people give up the tobacco habit and

to investigate any effect this might have on the incidence and regression

rates of precancerous lesions and thus on controlling oral cancer.

In the present study, the intervention efforts have been implemented

through a carefully designed comprehensive program in a specifically

delineated target population. Every individual in the target population was

examined annually to measure changes in the incidence and regression

patterns of the disease. The intervention program was carried out by a team

of professional dental surgeons and social scientists who were given special

training in conducting in-depth interviews, approaching and interacting

with the tobacco users in a considerate, sympathetic, warm, and positive

manner without condescending in any way.

MATERIALS

AND METHODS

Study Population

The study population was selected from Ernakulam district in the

State of Kerala, Bhavnagar district in the State of Gujarat, and

Srikakulam district in the State of Andhra Pradesh. A large

number of individuals were screened, and about 12,000 tobacco

users aged 15 and over were selected in each district. The tobacco habits

practiced in the study included smoking cigarettes, smoking bidis, smoking

chutta in conventional and reverse manner, and chewing tobacco with or

without betel quid (Aghi, 1989).

Intervention

Methods

A program of intervention was developed after appropriate pilot and

pretesting surveys. In-depth interviews of the participants were conducted to investigate (1) the reasons for their starting and continuing the

tobacco habit (such as when and how the habit started, who encouraged it,

was it a peer or a parent?); (2) the perceived implications of the habit in its

social, economic, and health aspects; and (3) possible reasons they would

give up their habits (Aghi, 1987).

In addition, pilot surveys were conducted to assess the communication

media facilities available to the target population that could be used to

convey the intervention message. The intervention program outlined a

timetable for employing different media of communication and regulating

the flow of information so as not to overwhelm the target population. Pilot

studies also helped to modify the messages whenever necessary, with a view

to making them easily understandable. All this was an ongoing process for

every followup.

This paper comprises the results of 10 yr. Each followup used strategies

of communicating messages to the target population that assisted and

enabled them to give up the tobacco habit. The unique feature of this

project has been its user-based philosophy, which is characterized by appropriate input, provided according to the stage of the subject, as follows:
Chapter 6

309

• Knowledge regarding association of tobacco habits and oral cancer

was imparted.

• This knowledge was further strengthened by visual reinforcement

through flipcharts, posters, films, etc., and a small amount of fear was

deliberately induced.

• Knowledge of the health benefits and other advantages of quitting

the habit were conveyed; for example, regression of lesions and

saving of money.

• Various possible methods of discontinuing the tobacco habit, such as

cold turkey, gradual reduction, postponing the first smoke or quid,

etc., were described and the most appropriate one for the subject was

suggested, depending on the psychological profile of the subject and

characteristics of the habit.

• Withdrawal symptoms were explained and emphasis placed on their

temporary nature.

• Appropriate praise and reinforcement were given and leadership roles

suggested for successful quitters.

Modes of

Communication

Personal

Communication

Personal communication involved one-to-one contact with the

target population with a view to helping them sort out doubts and

learn behaviors that would result in abstaining from tobacco. The

steps in personal communication were worked out in a logical

order, recognizing the dynamics of human psychology and what it

Films

Posters

Newspaper

Articles

Articles were published in newspapers to inform and educate people

about oral cancer. It was known that many members of the target

population do not read; nevertheless, the articles could be read by some,

including schoolchildren, who have been found to be important agents of

change in rural India.

takes for people to make decisions and change their attitudes and behavior.

Certain visual aids were used in the personal communication.

Two films were produced specifically for this phase of the study. The

objective of the first film was to give information and create awareness of

the relationship between tobacco habits and oral cancer. The objective of

the second film was to point out how habits are taken up and developed. In

addition, this film presents ways the habits can be given up. Film is a very

powerful medium for creating awareness and, when effectively utilized,

works to motivate behavior changes.

Posters were displayed to remind the target population that they ought

to be reconsidering their tobacco habits. Two kinds of posters were used,

one with a written message only and the other with a visual display along

with a written message. Slides were prepared from the posters and were

projected in movie theaters in the vicinity to serve the same objective. In

addition, handwritten posters summarizing the findings about the particular

village were left behind to remind subjects that they had been examined for

oral cancer.
Smoking and Tobacco Control Monograph No. 2

310

Other Media There were folk drama enactments with the objective of talking about

the prevalent superstitions and misconceptions about tobacco. In addition,

radio programs were designed to motivate people to give up their tobacco

habits in light of the fact that they might lead to oral cancer. The programs

were used to create an environment to help the target population think

about their tobacco habits.

Cessation Camps In the last two followups, we held cessation camps to address the

demand of our target population, who told us that they believed what we

said about tobacco being harmful and that they would like to give it up;

they wanted to know how to do it. In the cessation camps, detailed discussions were held to suggest the solutions to the problems encountered in

discontinuing the tobacco habit. Regular daily followup was maintained for

a few days after the camps were held.

Other Modes In addition to the above methods, clinics were run to address dental

complaints and thereby try to eliminate at least some of the reasons given

by users for practicing the habit. Also, exhibits and group meetings were

conducted to show common lesions and hold discussions that might help

the target population to ask questions and express their feelings and doubts.

Assessment

of Methods

The various methods of communication were assessed individually

through carefully designed questionnaire-based sample studies for their

impact relative to their objectives. Since all of the communication methods

could impinge on an individual’s decision to quit or reduce tobacco use, it is

hard to ascertain which actually led to the decision. A rank ordering of the

various inputs by the target population revealed that subjects have been

helped the most by one-to-one interaction; cessation camps had also aided

them immensely.

DISCUSSION The results of the study have been consistently positive (Table 1).

Considering that the target population was not innately motivated to give

up tobacco use, it is quite remarkable that such successful results were

obtained.

A detailed assessment of the project was made after 1 yr and after 5 yr.

After 1 yr, we found that the regression of lesions was significantly higher

among those who had stopped or reduced their tobacco use than among

individuals who did not do so (Mehta et al., 1982). After 5 yr, it was found

that a significantly higher percentage of individuals had stopped and

reduced their tobacco use than in the control cohort. As a consequence of

this increased stoppage and reduction in tobacco use, the incidence of oral

precancerous lesions decreased significantly and substantially in two of the

three study areas (Gupta et al., 1986b). Through a multiple logistic regression analysis, it was shown that the intervention efforts definitely helped

people stop their tobacco habits (Gupta et al., 1986a).

Some new questions now arise: Where do we go from here? How can

this project be used as a takeoff point for intervention at the national level

in India?
Chapter 6

311

Table 1

The cessation of tobacco use in successive followups

Percentage Stopping Tobacco Use

Bhavnagar Ernakulam Srikakulam

First Followup 4.1% 2.0% 4.9%

Second 7.6 4.4 10.9

Third 10.5 6.4 14.2

Fourth 11.6 7.6 15.4

Fifth 12.5 9.4 16.6

Sixth 14.1 11.2 17.0

Seventh 11.5 11.6 17.3

Eighth 12.2 12.3 17.4

Ninth 12.7 13.1 18.1

Tenth 13.6 14.0 18.8

We have examined the feasibility of training basic health care workers

to examine mouths for early detection of both precancerous and cancerous

lesions, and we have found this to be possible (Mehta et al., 1986). It would

be indeed worthwhile to assess the possibility of training these health

personnel to intervene in tobacco habits. If it is possible, the potential is

great. Most of the strategies of communication that were utilized effectively

in this project are those that involve the mass media or an approach that

can be used on a large scale. Undoubtedly, this needs to be validated.

REFERENCES

Aghi, M.B. Psychosocial aspects of acquisition and

cessation of tobacco habits in India. World

Smoking and Health Journal 12(2): 4-7, 1987.

Aghi, M.B. The tobacco tradition in India. World

Health, January-February, 1989.

Bhonsle, R.B., Murti, P.R., Gupta, P.C., et al. Reverse

dhumti smoking in Goa: An epidemiologic study

of 5,449 villagers for oral precancerous lesions.

Indian Journal of Cancer 13: 301-305, 1976.

Gupta, P.C., Mehta, F.S., Daftary, D.K., et al.

Incidence of oral cancer and natural history of oral

precancerous lesions in a 10-year followup study of

Indian villagers. Community and Oral Epidemiology

8: 287-333, 1980.

Gupta, P.C., Mehta, F.S., Pindborg, J.J., et al.

Intervention of tobacco chewing and smoking

habits (letter). American Journal of Public Health 76:

709, 1986.

Gupta, P.C., Mehta, F.S., Pindborg, J.J., et al.

Intervention study for primary prevention of oral

cancer among 36,000 Indian tobacco users. Lancet

1(8492): 1235-1239, 1986.

Mehta, F.S., Aghi, M.B., Gupta, P.C., et al. An

intervention study of oral cancer and precancer in

rural Indian populations: A preliminary report.

Bulletin of the World Health Organization 60: 441-

446, 1982.

Mehta, F.S., Gupta, P.C., Bhonsle, R.B., et al.

Detection of oral cancer using basic health

workers in an area of high oral cancer incidence in

India. Cancer Detection and Prevention 9: 219-225,

1986.

Mehta, F.S., Gupta, P.C., Daftary, D.K., et al. An

epidemiologic study of oral cancer and precancerous conditions among 101,761 villagers in

Maharashtra, India. International Journal of Cancer

10: 134-141, 1972.

Mehta, F.S., Pindborg, J.J., Gupta, P.C., et al.

Epidemiologic and histologic study of oral cancer

and leukoplakia among 50,915 villagers in India.

Cancer 24: 832-849, 1969.

National Cancer Registry. Annual Report 1985. New

Delhi: Indian Council of Medical Research, 1988.
Smoking and Tobacco Control Monograph No. 2

312
Chapter 7

313

Chapter 7

Policy

CONTENTS WHO Strategies To Curb Smokeless Tobacco:

A Global Perspective

Roberto Masironi ................................................................................. 315

Introduction ................................................................................. 315

WHO Action ................................................................................. 316

Areas of Legislation ...................................................................... 317

Present Action .............................................................................. 320

References ..................................................................................... 321

Legal and Administrative Strategies for

Control and Prevention of the

Use of Smokeless Tobacco

Seamus O’Hickey ................................................................................. 323

Introduction ................................................................................. 323

History .......................................................................................... 323

Strategy ......................................................................................... 324

Legislation .................................................................................... 324

Litigation ...................................................................................... 325

International Measures ................................................................ 327

Future Strategy ............................................................................. 328

Addendum .................................................................................... 329

References ..................................................................................... 329

National Cancer Institute’s Role in Reducing Tobacco Use

Robert Mecklenburg ............................................................................ 330

NCI Mission and Mandate ........................................................... 330

Tobacco Use Intervention Program ............................................. 331

Professional Roles ......................................................................... 331

Public and Private Partnerships ................................................... 332

Summary ...................................................................................... 333

References ..................................................................................... 334
Smoking and Tobacco Control Monograph No. 2

314
Chapter 7

315

Smokeless tobacco is a leading cause of disease in many developing countries where its use

WHO Strategies To Curb

Smokeless Tobacco: A Global Perspective

Roberto Masironi

ABSTRACT

is common, e.g., in the Indian subcontinent. Among the industrialized nations, Sweden and

the United States show increasing consumption trends, particularly among the young. Unless

its market expansion is stopped, smokeless tobacco may become a problem also in countries

where it is still practically unknown. Tobacco manufacturers have launched marketing

programs to promote the use of smokeless tobacco. The World Health Organization recommends that a preemptive ban on manufacture, import, promotion, advertising, and sale of

smokeless tobacco be introduced in countries where the product is not yet known. In countries

where smokeless tobacco is already widely used, various types of prohibition and control are

urged, in keeping with local circumstances. Australia, Hong Kong, Ireland, Israel, New Zealand,

Norway, Saudi Arabia, Singapore, and the United Kingdom have banned smokeless tobacco

products. The European Economic Community countries are about to adopt similar policies.

INTRODUCTION The use of smokeless tobacco has become a leading cause of disease

in some countries and may become so in others unless urgent preventive

action is taken. Smokeless tobacco is widely used in the Indian-Pakistani

subcontinent, where it is estimated that 100 million people are using it

(International Union Against Cancer, 1989; WHO, 1988). Under various

names and consisting of various combinations of tobacco and other ingredients, smokeless tobacco is also widely used in Afghanistan, Bangladesh,

Malaysia, the Islamic Republic of Iran, the Central Asian republics, Sri Lanka,

and Thailand (WHO, 1986 and 1988), as well as in Bhutan (Dr. Yok Heng

Tamang, personal communication, 1991). It is used, although to a lesser

extent than a few decades ago, by the native circumpolar populations of

Alaska and the Canadian arctic, but not Greenland (Hart Hansen et al., 1990).

Among the industrialized countries, Sweden has the highest per capita

consumption of smokeless tobacco, with 17 percent of its population using

it at least occasionally and almost 30 percent of young adult males using it

daily (Nordgren and Ramström, 1990). In the United States, more than

12 million people use smokeless tobacco, and 12 percent of young adult

males are daily users (Connolly, 1991; WHO, 1988).

In the rest of the world, however, smokeless tobacco is almost unknown.

In central and eastern Europe, nasal snuff was used in past centuries but

now very little of it is marketed. In Australia, smokeless tobacco represents

a minuscule 0.01 percent of all tobacco products consumed, and its use is

estimated to be limited to between 3,000 and 5,000 people, mainly miners,

oil riggers, farmers, and aborigines (Trade Practices Commission of Australia,

1989). In Japan, no smokeless tobacco is produced, and only a small amount

is imported (Dr. M. Chiba and S. Watanabe, letters, 1991). In China, it is also

practically unknown; very few people chew tobacco, and nasal snuff is used

only by some local inhabitants of Tibet (Prof. Weng Xin-zhi, letter, 1991).

Unlike tobacco smoking, which is widespread in all countries of the world,

the use of smokeless tobacco is extremely uneven, being used extensively in

some countries but virtually unknown in many others.
Smoking and Tobacco Control Monograph No. 2

316

Tobacco manufacturers have launched well-orchestrated marketing

programs to promote the use of smokeless tobacco in countries where the

product is unknown. For instance, a tobacco company opened a factory in

Scotland in 1985 to produce moist snuff for sale to other countries of

Europe, Africa, and the Middle East. The factory was later closed as a result

of pressure from health groups (WHO, 1988). The same company had

established a regional office in Hong Kong to promote sales in the Pacific

area and in Asia; this office also closed in 1987 as a result of a ban on smokeless tobacco by the Hong Kong government that year (Dr. J. Mackay, letter,

1991).

The recent manufacture and promotion of new forms of smokeless

tobacco by transnational tobacco companies has increased the need for

urgent action to prevent the spread of the ST habit to areas where it is

currently unknown. Of particular concern is the fact that some new forms

of smokeless tobacco are being marketed in ways that appeal to children and

young people. In Denmark, for instance, 60 percent of children know what

smokeless tobacco is, and 11 percent of 15-yr-olds, particularly boys, have

tried it (Commission of the European Communities, 1991). While decreasing among adults, ST use is rising among young males in some developed

countries (see Tables 1 and 2).

In Finland, snuff use had almost disappeared by the mid-1970’s. After

enhanced marketing efforts by national tobacco companies, smokeless

tobacco consumption increased from 43,000 kg in 1981 to 105,000 kg in

1989. Users are mainly young people, particularly 16- to 18-yr-old boys. In

a national survey done in 1987, about 5 percent of boys reported occasional

or daily use (WHO [EURO], 1991).

WHO ACTION In the face of an impending new epidemic of tobacco-related diseases, and following reports on the health hazards of smokeless tobacco

(IARC, 1986; US DHHS, 1986), the World Health Organization called together an international group of experts to review the evidence and propose

strategies for national and international control of smokeless tobacco. The

recommendations of that group are summarized here. According to the

WHO expert group, the major objectives of any national smokeless tobacco

control program should be the following (WHO, 1988):

• In countries where smokeless tobacco is not known yet, prevent its

introduction, with special emphasis on preventing its use by children.

• In countries where smokeless tobacco is already in use, prevent any

increase, and reduce the prevalence of its use in the population.

• In all countries, establish and maintain a social climate unfavorable to

smokeless tobacco use.

The most effective means of preventing the emergence of new tobaccorelated problems in any country is to prevent the introduction of new

tobacco products rather than to allow them to be introduced and take

action only after the resultant health problems have become apparent.
Chapter 7

317

Table 1

U.S. smokeless tobacco use

 Percentage of Users

Teenaged 50-Yr-Old

 Males Males and Females

1970 0.3% 2.7%

1980 2.7 1.3

1985 6.0 —

Table 2

ST use in Sweden

Percentage of Users

Teenaged 55-Yr-Old

Males Females Males Females

1955 0% 0% 21% 0%

1986 38 5 13 1

Health education and public information are important components of

any national tobacco control strategy, but the most important preventive

measure is legislation. Voluntary agreements are ineffective, because tobacco

companies are likely to circumvent them. Because of the relatively large

variety of smokeless tobacco products and different historical patterns of

use, laws are enacted differently in different countries. Certain types of ST

products are banned in some countries, while other types are only restricted,

and still others remain unaffected. Table 3 contains a summary of the

situation in various countries, to the extent we could ascertain it from a

review of available legislative texts (WHO, 1991).

AREAS OF

LEGISLATION

The same arguments that apply to legislation for the control of

smoking also apply to smokeless tobacco. The possibility of adopting

legislation to control smokeless tobacco is actually more favorable than that

for the control of smoking. Indeed, while it is not possible to ban cigarette

smoking completely, since the use of cigarettes is too deeply ingrained in

the socioeconomic structure of most countries, there is an opportunity to

legislate against the introduction of smokeless tobacco products in the many

nations where they are not yet on the market.

Smokeless tobacco is not only a health problem of individual countries,

but an international problem as well. Fortunately, the experience gained in

more than 30 years of smoking control activities worldwide can be drawn

upon for planning and implementing smokeless tobacco control programs.

Some nations have already benefited from the experience of other nations

in dealing with smokeless tobacco. For instance, the governments of Hong

Kong, Ireland, Israel, New Zealand, and some Australian states enacted
Smoking and Tobacco Control Monograph No. 2

318

Table 3

Legislative action to control smokeless tobacco

Country Year Banned Import, Manufacture, Sale, and/or Promotion

Ireland 1985 Moist snuff only

United Kingdom 1986 All smokeless tobacco sale to minors

Israel 1986 All smokeless tobacco

Hong Kong 1987 All smokeless tobacco (except nasal snuff)

New Zealand 1987 All smokeless tobacco

Singapore 1987 All smokeless tobacco (except nasal snuff)

Tasmania 1986 All smokeless tobacco (except nasal snuff)

S. Australia 1986 “Sucking tobacco” (no other types)

Victoria 1987 All smokeless tobacco (except nasal snuff)

W. Australia 1987 All smokeless tobacco (except nasal snuff)

Australia (nationwide) 1989 Oral snuff

United States 1987 Sale only of all smokeless tobacco to minors

Norway 1989 “New” types of tobacco products (no ban on

“traditional” chewing and snuff)

United Kingdom 1990 Moist snuff (no ban on nasal snuff and chewing

tobacco) (overturned in December 1990, awaiting

new ruling)

Saudi Arabia 1990 Chewing and moist snuff

China (Taiwan) 1990 All smokeless tobacco

E.E.C. 1990 Moist snuff (to be enforced beginning in 1992)

legislation from 1985 through 1987 to prohibit the importation, manufacture, advertising, promotion, and sale of smokeless tobacco products (WHO,

1988). Other countries have learned from this experience: Norway, Saudi

Arabia, Singapore, the United Kingdom, other Australian states, and Taiwan

have recently banned smokeless tobacco products (BASP, 1990; Connolly,

1991; Trade Practices Commission of Australia, 1989). E.E.C. countries

other than the United Kingdom are about to adopt the same policy (BASP,

1990).

Mongolia and China are in the process of adopting legislation to ban or

restrict ST use (Dr. J. Mackay, letter, 1991). A new law on the control of

tobacco hazards in the People’s Republic of China has been drafted to read,

“All importation and production of chewing tobacco is banned” (Prof.

Weng Xin-zhi, letter, 1991).

According to the WHO expert group, legislation to control smokeless

tobacco is most effective in the following areas: ban or control of manufacture, import, promotion, and sale; taxation and other economic disincentives; restriction on use in public places and places of work; and health

warnings. These are described in more detail below.

Ban or Control

Of ST Commerce

and sale of ST products. In these countries, there is an opportunity, which

may never be repeated, to prevent such products from coming into use.

This approach, called a preemptive ban, has been widely publicized by the

World Health Organization through a worldwide press release (WHO, 1987).

In countries where smokeless tobacco is not yet used, steps should

be taken to prohibit by law the manufacture, import, promotion,
Chapter 7

319

In countries where smokeless tobacco use is already too well established

for a comprehensive ban of this nature to be feasible, legislation should at

least prohibit promotion of smokeless tobacco products through

• Direct and indirect advertising in all its forms;

• Sponsorship of sporting, artistic, and other events and media programs;

• Distribution of free samples of ST products;

• Use of sports and other popular personalities;

• Promotion of other products of the same name, packaging, and

design, including items like T-shirts and toys; and

• Any other form of promotion, including those introduced from

outside the country.

If advertising of smokeless tobacco products cannot be completely

prohibited, it should at least be restricted by law to ensure the following:

• The impression is not given that ST is a safe alternative to cigarette

smoking;

• Misleading links do not appear established between ST and positive

values like success, youth, sports, open air, and the like;

• Advertising is restricted as to the type of media used, the size and

layout of pictorial material, and so forth; and

• Health warnings appear prominently on ST packaging and on any

advertising and promotional material.

In countries where smokeless tobacco products are sold, sales to minors

should be prohibited by law. Vending machines should be banned or

allowed only in areas where unaccompanied minors have no access.

In many developing countries, smokeless tobacco is a cottage industry

and products are generally prepared by small local stores. In these countries, it is particularly important that major manufacturing industries are not

permitted to develop. Priority should, at the same time, be given to identifying and promoting replacement crops that will provide profitable employment and economic substitutes for tobacco.

Taxation and

Other Economic

Disincentives

The World Health Organization has always advocated the use of

increased taxation as a part of comprehensive programs for the

control of both smoking and ST products. There is sound evidence

that taxation can be used to discourage young people from starting to use

tobacco, and to encourage users to discontinue the habit, without decreasing government revenue. Ideally, a proportion of the tax increase should be

used to finance health education programs. If it cannot be prohibited

altogether, the importing of ST products can be discouraged through high

import duties.
Smoking and Tobacco Control Monograph No. 2

320

No government subsidies should be provided for any form of tobacco

growing, manufacturing, sale, or export. If this is not possible, the World

Health Organization recommends that governments ensure that no new

form of tobacco becomes eligible for subsidies.

Restrictions

On ST Use

Although not because of the same health criteria as those that relate to

passive smoking, the use of smokeless tobacco may nevertheless be

unpleasant to nearby non-users. Restrictions on ST use may be justified in

light of health and cleanliness problems associated with spitting and the

disposal of chewed tobacco.

Health Warnings In countries where the sale and promotion of smokeless tobacco

products are still allowed, health warnings should be mandatory on packages of such products as well as on any related advertising and promotional

material. Similar to the rotating health warnings that appear on cigarette

packages, ST products should carry health warnings about the harmful

health effects associated with use of the products. Examples are shown in

Table 4.

Table 4

Examples of health warnings on ST packages and advertisements

Country Warning

France Dangerous if abused.

Iceland Snuff and chewing tobacco may damage the mucous membranes.

Ireland This product may cause oral cancer.

Portugal Tobacco damages health and, in particular, causes cancer.

Greece Tobacco damages your health.

Sweden Warning: Snuff and chewing tobacco contain nicotine. Therefore,

snuff produces just as strong a dependence as tobacco

smoking. The buccal cavity, mucous membranes and

gums can be damaged and may require treatment.

United States Warning: This product may cause mouth cancer.

Warning: This product may cause gum disease and tooth loss.

Warning: This product is not a safe alternative to cigarettes.

PRESENT ACTION In addition to the measures recommended by the WHO group

of experts (WHO, 1988), further action is now under way, particularly in

Europe. At the First European Conference on Tobacco Policy, held in

Madrid under the auspices of both the World Health Organization and the

E.E.C., 10 strategies for a tobacco-free Europe have been recommended

(WHO [EURO], 1989). One of these reads, “To prohibit new methods of

nicotine delivery, and to block future tobacco industry marketing strategies.” This is a clear mandate to stop the development of a new hazardous

addiction in Europe.

The countries in eastern Europe represent a special case, as they are now

facing a new tobacco-related danger. These nations are emerging under new

sociopolitical systems involving free-market economies. The transnational
Chapter 7

321

tobacco companies are already exerting marketing and promotional pressure

on these countries, as was revealed at the conference, “A Tobacco-Free New

Europe,” held in Kazimierz, Poland, November 21 through 23, 1990, under

the auspices of WHO and the International Union Against Cancer. One

recommendation of the conference was that governments of eastern European countries be requested to ban the introduction of smokeless tobacco,

which is practically unknown in these nations (International Union Against

Cancer, 1991). The E.E.C. is also taking action. The Commission of the

European Communities has issued a directive that, if approved, would

require member states to ban the import, manufacture, and sale of moist

snuff starting in March 1992. Moist snuff is already prohibited from import

into Switzerland, which is not an E.E.C. country (Swiss Federal Office of

Public Health, 1987; Dr. B. Meili, letter, 1991).

In conclusion, it can be said that action is now being taken, both

nationally and internationally, to stem the spread of ST use. Until several

years ago, there was no legislation specifically to restrict smokeless tobacco;

now an increasing number of countries have adopted legislation for that

purpose.

REFERENCES

BASP, European Bureau for Action on Smoking

Prevention. A New Form of Smokeless Tobacco:

Moist Snuff. Commission of the European

Communities, Europe Against Cancer Programme,

Brussels, 1990.

Commission of the European Communities, Europe

Against Cancer Programme. “Skoal Bandits” et

autres “Catch” au Danemark. Information Circular,

12 March 1991, Brussels.

Connolly, G.N. Health effects and regulations of

smokeless tobacco products. In: A Tobacco-Free

New Europe: Proceedings of an International Union

Against Cancer Conference, Kazimierz, Poland, 21-23

November 1990, M.Wood and W. Zatonski

(Editors). Belfast: Ulster Cancer Foundation,

1991.

International Agency for Research on Cancer.

Evaluation of the Carcinogenic Risk of Chemicals to

Humans: Tobacco Habits Other Than Smoking; BetelQuid and Areca-Nut Chewing and Some Related

Nitrosamines (volume 37). Lyon: IARC, 1985.

International Agency for Research on Cancer.

Tobacco, A Major International Health Hazard,

D. Zaridze and R. Peto (Editors). IARC Scientific

Publication No. 74. Lyon, France: IARC, 1986.

International Union Against Cancer. Tobacco and

Health: Proceedings of a Workshop, Bombay, 15-16

April 1987. L.D. Sanghvi and P. Notani (Editors).

Geneva: International Union Against Cancer,

1989.

International Union Against Cancer. A Tobacco-Free

New Europe. Proceedings of an International Union

Against Cancer Conference, Kazimierz, Poland, 21-23

November 1990, M.Wood and W. Zatonski

(Editors). Belfast: Ulster Cancer Foundation,

1991.

Nordgren, P., Ranström, M. Moist snuff in Sweden:

Tradition and evolution. British Journal of

Addiction 85: 1107-1112, 1990.

Peterson, J.S., et al. Smokeless tobacco. In: Tobacco

and Health Proceedings of the International Union of

Health Education/World Health Organization

Circumpolar Conference on Tobacco and Health,

Yellowknife, NWT, Canada, April 4-6 1989, J.P. Hart

Hansen, B. Harvald, and E.M. Nuutinen (Editors).

Arctic Medical Research 49 (Suppl. 2): 32-38, 1990.

Swiss Federal Office of Public Health. Ordonnance sur

les Denrées Alimentaires. Bern: Chancellerie

Fédérale, 1987.

Trade Practices Commission of Australia. Smokeless

Tobacco Products. Trade Practices Act 1974, Trade

Practices Commission, Canberra, September 1989.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Insitutes

of Health, National Cancer Institute. NIH

Publication No. 86-2874, 1986.
Smoking and Tobacco Control Monograph No. 2

322

World Health Organization. WHO Study Group Calls

for “Pre-Emptive Ban” on Smokeless Tobacco. Press

Release WHO/17. Geneva: WHO, June 9, 1987.

World Health Organization. Smokeless Tobacco

Control. Technical Report Series No. 773. Geneva:

WHO, 1988.

World Health Organization (EURO). It Can Be Done:

A Smoke-Free Europe. Report of the First European

Conference on Tobacco Policy, Madrid, 7-11 November

1988. Copenhagen: WHO Regional Office for

Europe, 1989.

World Health Organization. Legislative Responses to

Tobacco Use. Geneva: WHO, 1991.

World Health Organization (EURO). Smokeless

Tobacco in Finland. Country Files, Tobacco or

Health Programme. Copenhagen: WHO Regional

Office for Europe, 1991.
Chapter 7

323

The Irish government considers that the public health is more important than the ecoLegal and Administrative Strategies for

Control and Prevention of the

Use of Smokeless Tobacco

Seamus O’Hickey

ABSTRACT

nomic effects of a fall in tobacco consumption over a period of years. For the past 25 years, the

Department of Health has operated a strategy, based on education and legislation, to reduce

demand for and supply of tobacco. Public support for the strategy has facilitated implementation of strong legal controls and prohibitions on tobacco sales and use. The importation,

manufacture, and distribution of oral smokeless tobacco are totally banned by law in Ireland.

The laws banning oral smokeless tobacco have twice been challenged in the courts by U.S.

Tobacco International, Inc., with success in one case. Currently, the ban is in place but awaits

final judgment. National laws of European Community Member States are subject to E.C.

legislation, which is based on the Treaty of Rome. Irish laws and decisions of Irish courts

therefore have a wider, European influence. The E.C. and some of its Member States have

adopted Irish antitobacco legislation as a model.

INTRODUCTION The legal prohibition of commerce in oral smokeless tobacco in

Ireland arose initially from official action by the U.S. Public Health Service

in the early 1980’s, which was a response to rising public concern in the

United States about the increasing use of the product there. Through the

agency of organizations such as the International Chief Dental Officers’

Conference of the International Dental Federation, supported by the World

Health Organization’s Oral Health Unit, public health dental officers around

the world were alerted by their American colleagues to the potential and

serious consequences to the health of the public if the American experience

were to be exported to and repeated in other countries.

Accordingly, in 1985 the Irish Minister for Health made a decision to

ban oral smokeless tobacco in the moist snuff form. This decision received

support shortly thereafter in the U.S. publication of The Health Consequences

of Using Smokeless Tobacco: A Report of the Advisory Committee to the Surgeon

General (US DHHS, 1986).

HISTORY For a quarter of a century, the Department of Health in Ireland has been

engaged in developing and implementing programs designed to reduce the

supply of tobacco products and the demand for them.

Tobacco use is the single most significant cause of death in middle age

in Ireland. Illnesses resulting from tobacco impose an enormous strain on

the health services. Among a total population of 3.5 million people, about

0.5 million days per year are spent in hospital as a result of tobacco-related

illnesses, at a cost of more than £15 ($25) per year for every person in the

country (Lyons, 1988). Other health care costs, disability costs, and costs of

time lost from work place an equally high burden on the economy.
Smoking and Tobacco Control Monograph No. 2

324

The Irish government, having examined and considered all aspects and

implications of the tobacco issue, decided that public health considerations

outweigh by far the economic impact that a reduction in tobacco consumption would cause over a number of years.

STRATEGY The Department of Health has implemented its antitobacco policy by

means of a double-pronged strategy. From the start, an education and

information program has been conducted in various ways through the mass

media, the educational system, and official and voluntary agencies. The

second part of the strategy is the legislative program by which legal controls

and/or prohibitions are in place with respect to tobacco advertising, sponsorship, promotion, labeling, smoking in public buildings and offices, and

selling tobacco products to children and young people.

Advertising of tobacco on television was prohibited in 1971 and on

radio in 1975. The education and information program increased the

public’s knowledge about the hazards associated with tobacco, and this in

turn led to widespread public support for measures to curtail tobacco use

even further in the interests of users and non-users alike.

Public opinion polls had shown such a high level of support for the

Department’s strategies that a new Tobacco Act (1988) was brought forward

to further curtail and restrict tobacco consumption. The 1988 Act is crucial

insofar as it contains the ban on importing, making, and distributing oral

smokeless tobacco.

LEGISLATION Ireland’s ban on oral smokeless tobacco is contained in the Tobacco

(Health Promotion and Protection) Act, 1988. The preamble to the Act

summarizes its provisions: (a) prohibition and restriction on the consumption of tobacco and restriction on the consumption of tobacco products in

designated areas and facilities; (b) restriction on the sale of tobacco products

to persons under 16 years of age; (c) prohibition on the importation, manufacture, and sale of certain tobacco products; (d) amendment of previous

legislation; and (e) provision for other connected matters.

Specifically, the ban on oral smokeless tobacco is contained in Section 6

of the 1988 Tobacco Act, as follows:

(1) Any person who imports, manufactures, sells or otherwise disposes

of, or offers for sale or other disposal, or advertises, an oral smokeless tobacco product shall be guilty of an offense and shall be

liable—

(a) on summary conviction, to a fine not exceeding £1,000, or

(b) on conviction on indictment to a fine not exceeding £10,000.

(2) In this section “oral smokeless tobacco product” means any product

or substance, made wholly or partly from tobacco, which is intended for use, unlit, by being placed in the mouth and kept there

for a period, or by being placed in the mouth and sucked or

chewed.

This is a comprehensive ban, which is feasible to implement in Ireland

where there has never been an oral snuff habit.
Chapter 7

325

As already mentioned, a somewhat similar type of ban was previously

introduced, in 1985. The first ban did not involve new legislation at that

time, as it was introduced under previous legislation (Health Act, 1947).

The 1947 Health Act, by virtue of its Section 66, allowed the Minister for

Health to prohibit the import, manufacture, sale, or other disposal of a

restricted article or substance when it was likely, when accessible to the

general public, to be used for purposes involving risks of serious injury to

health or body. The 1985 ban (Health [Restricted Article] Order, 1985) was

confined to the Skoal Bandit form of oral smokeless tobacco—that is, sachettype products.

In addition to the legislation mentioned above (i.e., the 1985 Regulations and the 1988 Act), there are a couple of other important pieces of

relevant legislation. The first of these is the Tobacco Products Act (1978),

which gives the Minister for Health extensive powers to control all aspects

of advertising, sponsorship, and promotion of tobacco products. The

second is the Tobacco (Number 2) Regulations (1986), which greatly extend

the Minister’s powers in implementing the 1978 Act.

LITIGATION The 1985 ban on oral smokeless tobacco, under the 1947 Health Act,

was challenged in the Irish High Court by U.S. Tobacco (Ireland) Limited

and U.S. Tobacco International, Inc. U.S. Tobacco won the case, on the

grounds that, when framing the Act, the legislature did not intend that it be

used for this particular type of purpose. The Court held that the Minister

for Health had exceeded his powers, and so the first ban was nullified.

Unfortunately, the lawyers had decided to fight the case without

reference to the health risks of smokeless tobacco use and to defend the ban

on purely legal technical grounds. With hindsight, this was seen as a

mistake. After the defeat in the High Court, a decision was made in the

Department of Health not to appeal to the Supreme Court, as the second

ban, in the new proposed Tobacco Act (1988), was well on its way toward

implementation as Section 6.

The second ban also was challenged in the Irish High Court by U.S.

Tobacco. The case was heard late in 1990, and the decision, which upheld

the ban, was given on February 22, 1991 (High Court, 1990, 1991).

It is instructive to study this second court case: U.S. Tobacco claimed

that Section 6 was invalid, as it breached the Treaty of Rome (1957), by

which the European Community was formed, at Article 30, and that it was

unenforceable because of noncompliance with E.C. Council Directives

83/189 as amended by 88/182 (E.C., 1983 and 1988), and 89/622 (E.C.,

1989)—the “Notification” and “Labelling” Directives, respectively.

Article 30 states: “Quantitative restrictions on imports and all measures

having equivalent effect shall, without prejudice to the following provisions, be prohibited between Member States.”

U.S. Tobacco contended that Section 6 offends the principle of proportionality in that the same objective could be achieved by a less repressive

measure than the ban. The corporation contended also that the current

legal restrictions in Ireland on smoking tobacco could be applied to
Smoking and Tobacco Control Monograph No. 2

326

smokeless tobacco and that they would be adequate to protect the health

and lives of humans in Ireland.

The law involved is European Community law. It covers three topics:

(a) Articles 30 and 36 of the Treaty of Rome, (b) the principle of proportionality, and (c) the rights of Member States when there are uncertainties in

current research. Article 30 of the Treaty is stated above. Article 36 is as

follows:

The provisions of Articles 30 to 34 shall not preclude prohibitions or

restrictions on imports, exports or goods in transit justified on grounds

of public morality, public policy or public security; the protection of

health and life of humans, animals or plants; the protection of national

treasures possessing artistic, historic or archeological value; or the

protection of industrial and commercial property. Such prohibitions or

restrictions shall not, however, constitute a means of arbitrary discrimination or a disguised restriction on trade between Member States.

The State agreed that Article 30 was breached but contended that

Article 36 gives an exception from it. Under Article 36, the case law is clear

that exceptions are justified on grounds of necessity for “protection of

health and life of humans.” Exceptions are not allowed, under the principle

of proportionality, if the health and life of humans can be as effectively

protected by less strict measures. Insofar as there are uncertainties in the

present state of research, Member States can decide the degree of protection

they wish to assure for the health and life of humans.

The evidence fell into two categories: first, the conclusions of authoritative bodies, and second, opinions of expert witnesses.

Conclusions of

Authoritative

Bodies

According to volume 37, page 116, of the International Agency for

Cancer Research Monographs (IARC, 1985), “There is sufficient

evidence that oral use of snuffs . . . is carcinogenic to humans.”

From the preface (page vii) to the report of the Advisory Committee to

the U.S. Surgeon General (US DHHS, 1986), “The scientific evidence is

strong that the use of snuff can cause cancer in humans.”

The World Health Organization Study Group (WHO, 1988b) stated,

“There is conclusive scientific evidence that the use of smokeless tobacco

causes cancer in humans” (page 18).

According to the First European Conference on Tobacco Policy (WHO,

1988a), “These products are associated with increased risk of contracting

oral cancer and other diseases of the mouth.”

Testimony of

Expert Witnesses

The expert testimony was extremely detailed and went on for

6 days. All of the State’s witnesses were of the opinion that there

is an association between oral smokeless tobacco and oral cancer. The judge

had no hesitation in accepting their evidence.

The evidence of U.S. Tobacco’s expert witnesses did not convince the

judge, as can be seen from his assessment, which took the form of answers

to three questions:
Chapter 7

327

(a) Was the ban justified when introduced in July 1988?

(b) If so, is it still justified?

(c) If the answers to (a) and (b) are “Yes,” could the objectives of the

ban be achieved by less restrictive means?

The judge’s answers were as follows:

(a) Yes, there was enough evidence that smokeless tobacco could cause

oral cancer. People had to be protected by ensuring it was not

available by means of a total ban.

(b) Yes, it was not proven there is a scientific controversy. The

plaintiff’s witnesses disagreed with authoritative bodies, but no

responsible body agrees with them—the evidence is the other way.

To impose the ban it was sufficient that there was evidence that

cancer might be caused or that the risk was increased. As the State

is obliged to protect its citizens, it had enough evidence to take

action. It would fail in its duty if it were to act otherwise.

(c) It is submitted that existing restrictions (on smoking tobacco)

would be adequate, but it is not submitted that they would be as

effective as the ban. They are not adequate, not sufficient, not as

effective, and are “a second best.”

E.C. Council

Directives

The Notification Directives (E.C., 1983 and 1988) require that draft

technical regulations be communicated to the E.C. Commission. Section 6, however, is not a technical regulation, specification, or standard.

Agricultural produce is excluded from the 1983 Directive, and the 1989

Directive did not come into force until January 1, 1989, and was not operable when the 1988 Act was passed in July 1988.

The Labelling Directive (E.C., 1989) is concerned only with harmonizing laws, etc., regarding health warnings on packages of tobacco products.

It does not affect the right of a Member State to lay down its own rules

concerning the import, sale, and consumption of tobacco products, for

example, a ban, which is not imposed for reasons of labelling but for the

public health.

Judgment In conclusion, and in the judge’s own words, “the plaintiff’s claim fails in

toto and must be dismissed” (High Court, 1990, 1991).

INTERNATIONAL

MEASURES

Irish legislation in relation to restrictions on sales, promotion, and

advertising of tobacco has served in the past as a model for

subsequent European Community laws, regulations, and directives. The

introduction to the E.C. of a similar legal ban on oral smokeless tobacco is

under consideration at present. It is, therefore, possible that before long

there will be an E.C. ban similar to the Irish one.

A number of other countries have already adopted measures aimed at

banning or controlling the importation, manufacture, and sale of smokeless

tobacco products.
Smoking and Tobacco Control Monograph No. 2

328

Within the E.C. and following the Irish initiative, in February 1988 the

United Kingdom announced its intention to ban moist snuff. Since March

1990 in the United Kingdom, “no person shall supply, offer to supply, agree

to supply, expose for supply or possess for supply any oral snuff” (excluding

nasal dry snuff) (Consumer Protection, 1989).

Belgium adopted a Royal Decree, effective December 31, 1990, which

lays down that “it is forbidden to market moist snuff tobacco in pouches

aimed at being placed as such in the mouth.”

Luxembourg and France impose restrictions on the advertising of

smokeless tobacco products. In France (under the Law Concerning Measure

Against Tobacco, 1976) and Belgium (under the 1990 Royal Decree), health

warnings are required on smokeless tobacco packages. France is currently

examining a draft law (Law Concerning Measure Against Alcohol and

Tobacco, 1990) aimed at banning all forms of direct or indirect advertising

of tobacco products.

Outside the European Community, Hong Kong, New Zealand, and

Israel have banned all forms of smokeless tobacco (Public Health and Municipal Services Ordinance, 1987; Toxic Substances Act [amendment], 1985;

and Ministry of Trade Industry Codes, 1986, respectively), as have Japan and

Taiwan (European Bureau, 1990). Singapore adopted measures banning the

importation and sale of chewing tobacco and moist snuff, and Saudi Arabia

has banned moist snuff and chewing tobacco (European Bureau, 1990). The

Isle of Man prohibits the importation of moist snuff under the Custom and

Excise Acts Orders (Amendment No. 2) of 1986. In the United States,

“Masterpiece Tobacs” was banned because it did not comply with the Food

and Drug Administration rules (European Bureau, 1990).

Iceland’s Act on Prevention of Use of Tobacco (1984) bans all tobacco

advertising and promotion. India, Canada, Sweden, and the United States

have enacted legislation that makes the inclusion of health warnings on

smokeless tobacco packages compulsory. Massachusetts was the first U.S.

state to declare moist snuff a hazardous substance, and a ban was considered

but not enacted because of the large consumer demand for the product

(European Bureau, 1990).

FUTURE

STRATEGY

In 1988, the First European Conference on Tobacco Policy (WHO,

1988b), organized jointly by WHO and the European Community,

produced a 10-point strategy for a “Europe without tobacco.” Point 8 of the

strategy concerns the banning of all new tobacco products containing

nicotine. The conference adopted the following recommendation for

strategy on this issue:

The participants endorsed the recommendations of the WHO Study

Group on Smokeless Tobacco Control and urged Member States to use

these recommendations as a basis for action. Particularly important are

the Study Group’s recommendations for a smokeless tobacco program:

• Where smokeless tobacco is not used, prevent its introduction, with

special emphasis on preventing its use by children;
Chapter 7

329

• Where smokeless tobacco is already used, act to reduce the prevalence of use in the population; and

• Establish or maintain a social climate unfavorable to smokeless

tobacco use.

ADDENDUM In March 1991, U.S. Tobacco lodged an appeal to the Supreme Court in

Ireland against the High Court judgment (1990). The basis of the appeal is

the contention that the judge in the High Court misdirected himself in law

and in fact. In the Supreme Court the appeal will be heard by the full court

of five judges sitting together. The outcome of this most important case is

awaited with great interest.

REFERENCES

Consumer Protection, the Oral Snuff (Safety)

Regulations, 1989. London: Her Majesty’s

Stationery Office, 1989.

European Bureau for Action on Smoking Prevention.

Report on a New Form of Smokeless Tobacco: Moist

Snuff. Brussels: European Bureau, December 1990.

European Community. Council Directive 83/189,

March 28, 1983 (laying down a procedure for the

provision of information in the field of technical

standards and regulations). E.C. Official Journal

No. L109, 26/04/83: 0008, 1983.

European Community. Council Directive 88/182,

March 22, 1988 (amending Directive 83/189). E.C.

Official Journal No. L081, 26/03/88: 0075, 1988.

European Community. Council Directive 89/622,

November 13, 1989 (on the approximation of the

laws, regulations, and administrative provisions of

the Member States concerning the labeling of

tobacco products). E.C. Official Journal No. L359,

08/12/89: 0001, 1989.

Health Act. Dublin: The Stationery Office, 1947.

Health (Restricted Article) Order. (S.I. No. 429 of

1985). Dublin: The Stationery Office, 1985.

High Court, 1990. No. 871P: Judgment of

Mr. Justice Blayney delivered the 22nd day of

February, 1991. Dublin: Irish Law Reports, 1991.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosomines (volume 37). Lyon:

IARC, 1985.

Lyons, M. Paper presented at joint meeting of

World Health Organization and USSR Institute of

Preventive Medicine, Moscow, September 1988.

Tobacco (Control of Advertising, Sponsorship and

Sales Promotion, No. 2) Regulations. (S.I. No. 107

of 1986). Dublin: The Stationery Office, 1986.

Tobacco (Health Promotion and Protection) Act.

Dublin: The Stationery Office, 1988.

Tobacco Products (Control of Advertising, Sponsorship and Sales Promotion) Act. Dublin: The

Stationery Office, 1978.

Treaty of Rome. Treaty establishing the European

Economic Community, signed in Rome, Italy, on

March 25, 1957. Luxembourg: Office for Official

Publications of the European Communities, 1957.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. Publication

No. DHHS (NIH) 86-2874, 1986.

World Health Organization. First European Conference on Tobacco Policy: It Can Be Done—A SmokeFree Europe. Regional Publications. European

Series; No. 30. Copenhagen: WHO, 1988a.

World Health Organization. Smokeless Tobacco

Control: Report of a WHO Study Group. Technical

Report Series 773. Geneva: WHO, 1988b.
Smoking and Tobacco Control Monograph No. 2

330

The tobacco industry exploits the public good—trading human lives, economic well-being,

National Cancer Institute’s Role

In Reducing Tobacco Use

Robert Mecklenburg

ABSTRACT

and the environment for profit. The industry knows that its earnings are built on consumer

addiction, loss of health, and death. Waking the public to the needless and despicable

consequences of a morally bankrupt industry requires determined action by a wide variety of

private and public sector organizations. Each organization, doing what it can do best, can help

turn public opinion from tolerance to outrage, and public behavior from use to abstinence.

The National Cancer Institute, through its research and dissemination of knowledge, is a vital

partner in this quest for a healthy society. This paper provides an overview of NCI’s mission

and current activities related to the control of smoking and smokeless tobacco use. Major

research and demonstration projects, health professional roles, and public and private-sector

NCI partnerships are described.

NCI MISSION

AND MANDATE

NCI aims to provide knowledge and guidance for reducing cancer

incidence and mortality in the United States and around the world.

Evidence strongly links tobacco use, primarily smoking, with many types of

cancers (US DHHS, 1982). About 30 percent of all cancers are attributed to

tobacco use. Both smoking and ST use increase the risk for cancer of the

mouth and pharynx about fourfold, with the relative risk for both tobacco

forms related to the duration and intensity of exposure (US DHHS, 1990a;

Winn, 1981).

Goals set by the U.S. Department of Health and Human Services include

an objective of reducing cigarette smoking to no more than 15 percent

among people aged 20 and older (US DHHS, 1991). The smokeless tobacco

objective is to reduce prevalence of use among males aged 12 through 24 to

no more than 4 percent (US DHHS, 1991). These two goals are also objectives for NCI’s Smoking and Tobacco Control Program.

One study suggests that 25 to 30 percent of all regular ST users also

smoke (Eakin et al., 1989). Children and youth may become addicted to ST,

then later become smokers. Smokers may perceive ST as a safe alternative to

smoking and change to ST for maintaining their levels of nicotine. Both

forms of tobacco are addictive, produce health problems, and create intervention challenges (US DHHS, 1986a). To be successful, strategies to meet

national health objectives must address both smoking and smokeless means

of nicotine administration.

These objectives call for action, but just any action will not do. It is

essential that major tobacco use interventions be supported by scientifically

sound, established methods. Thus, NCI developed an 18-yr plan to develop

hypotheses, define evaluation criteria, examine scientific evidence to date,

support well-designed studies in areas where evidence seemed insufficient,

and build large-scale intervention trials (US DHHS, 1990b).
Chapter 7

331

Since 1983, specific NCI research efforts in the Division of Cancer

Prevention and Control have been directed toward the development of scientifically sound and practical tobacco use intervenTOBACCO USEINTERVENTION

PROGRAM

tions. Through fiscal year 1990, nearly $250 million had been invested in

tobacco use research and control. Sixty studies on smoking intervention

have been completed or are in progress, and an additional eight studies

focus on ST (US DHHS, 1990b).

Of the eight ST studies, four are directed to students, one is limited to

dental clinic patients, one targets Little League youth, one targets Native

American youth, and one targets 4-H members. Preliminary results suggest

that methods found to be effective in helping smokers quit can achieve

similar results among ST users.

The Community Intervention Trial for Smoking Cessation (COMMIT) is

a large, controlled study of randomly assigned subjects. COMMIT involves,

directly or indirectly, more than 6 million people in 22 paired study and

control communities to test intervention methods that were found effective

during smaller studies (US DHHS, 1990b). COMMIT is a university-based

research project that includes schools; worksites; professional, religious,

civic, and government organizations; and the media. The COMMIT trials

began in 1989 and are scheduled to run until 1993. Although intended to

focus on heavy smokers, the study suggests approaches to the psychological

and social patterns and physical dependencies of ST users. Already invaluable baseline and operational information has been obtained, but future

results hold high promise.

Findings from COMMIT should significantly contribute to a larger

initiative, the America Stop Smoking Intervention Study for Cancer Prevention (ASSIST) (US DHHS, 1990b). ASSIST will have an impact on patterns of

tobacco use nationwide. States will administer contracts for organizing and

operating coalitions that promote a multifaceted approach to influencing

people to avoid and discontinue tobacco use. From 50 to 100 million

people are expected to be included in this project. An organizational phase

for ASSIST will run from 1991 to 1993, and an operational phase from 1993

to 1998.

ST was specifically identified in the original Request for Proposals that

was sent to the states for ASSIST. These guidelines defined smokers as

tobacco users rather than just individuals who smoke tobacco (cigarettes,

cigars, pipes). For purposes of ASSIST, ST users are included in the definition

of “smoker” (ASSIST Program Guidelines, October 1991, Glossary of Terms,

unpublished).

PROFESSIONAL

ROLES

The medical profession has great incentive to become involved in

smoking control. Physicians often see patients sicken and die from

tobacco-related diseases. Indeed, surveys of patients and physicians suggest

that from one-third to one-half of medical practitioners provide at least

some smoking intervention services during encounters with patients (Frank

et al., 1991; Gerbert et al., 1989; US DHHS, 1976). Surveys also indicate that

dentists are involved to a lesser degree (Geboy, 1989; Gerbert et al., 1989;
Smoking and Tobacco Control Monograph No. 2

332

Secker-Walker et al., 1989) but may not consistently use the best intervention methods (Secker-Walker et al., 1987).

NCI recognized that both the medical and dental professions are

strategically situated to provide effective intervention. About 70 percent of

Americans see a physician at least once a year (US DHHS, 1986b). About 63

percent of the population see a dentist each year, including 75 percent of

children and youth (Hayward et al., 1989). Studies suggest that selected

tobacco use interventions are effective when provided in the dental clinic

environment (Cohen et al., 1989) and are as effective as physician intervention services (Cohen et al., 1987).

The time that physicians and dentists spend with patients offers an

excellent opportunity to influence behavior. Medical and dental visits

provide many opportunities for one-to-one discussion about tobacco use

and health consequences and methods for quitting. Medical visits for

prenatal care, child health, and upper respiratory or cardiovascular conditions provide special opportunities to discuss reasons for quitting. Dental

checkups are usually performed at regular intervals and often are prevention

oriented. Dental visits also provide opportunities for effective interventions

because tobacco effects are commonly found in the mouth. Oral lesions

that are visible to patients lead to teachable moments that help motivate

patients to stop. Both physicians and dentists can prescribe nicotine replacement therapy, when indicated. Followup visits for many routine

dental services can also be used for followup of tobacco use interventions.

NCI has developed and conducted training programs for medical and

dental clinicians, focusing on train-the-trainer programs for medical and

oral health teams who are willing to teach colleagues. Dental education

institutions are encouraged to integrate key issues into their curricula and to

establish behavioral outcome objectives. The NCI manual, How To Help Your

Patients Stop Smoking, designed for physicians, does not specifically address

smokeless tobacco, but the clinical intervention methods can apply to ST

intervention (Glynn and Manley, 1990). The NCI manual, How To Help

Your Patients Stop Using Tobacco, is used with the oral health team and

educator training programs (Mecklenburg et al., 1990). The title and contents of this manual specifically refer to “tobacco use” rather than “smoking,” reflecting the importance of influencing patients to avoid and discontinue the use of ST as well as smoking tobacco.

PUBLIC AND

PRIVATE

PARTNERSHIPS

NCI recognizes that intervention successes are the product of a

partnership between the research community and educators,

community leaders, policymakers, and program administrators.

NCI promotes the reduction of tobacco use by collaborating with many

private and public organizations. Examples of NCI partnerships include the

following:

• NCI partnerships with the academic community are important to its

extramural research program. Research and education institutions

continually strengthen the scientific basis for rational patient care
Chapter 7

333

and public health action. COMMIT is an example of a universitybased tobacco use intervention research project.

• NCI works with public and private-sector organizations to form

effective community contacts. Private volunteer, business, and

community service government organizations have access to large

segments of the public. These organizations want assurances that

their efforts will be effective. ASSIST is an NCI collaboration with the

American Cancer Society, state health departments, and many other

groups with special skills in working with the public (for example,

business, religion, education and public service organizations, and the

media). NCI’s National Dental Tobacco-Free Steering Committee is

an alliance between NCI and 11 national dental organizations that

are committed to influencing the public to avoid and discontinue

tobacco use.

• Within the National Institutes of Health, NCI and the National

Institute of Dental Research are jointly planning and identifying

several cooperative ventures against ST and in support of the prevention and early detection of oral cancer, with each institute building

on its special strengths.

• NCI collaborates with other U.S. Public Health Service agencies, such

as the Office of Disease Prevention and Health Promotion for tobacco,

cancer, and oral health objectives for the year 2000; with the Centers

for Disease Control, especially its Office on Smoking and Health; the

National Center for Health Statistics; and more recently, the Health

Resources and Services Administration and Indian Health Service for

training of Public Health Service clinical personnel in tobacco use

interventions.

• NCI collaborates in tobacco control activities by other government

agencies, such as the Department of Veterans Affairs and the Department of Defense. These departments are working with many of the

same private and public-sector organizations noted above.

• Finally, NCI provides tobacco use intervention support to the World

Health Organization and to other countries. The Basic Dental Research Unit of the Tata Institute of Fundamental Research in India

deserves special recognition for its 25-yr effort to develop oral cancer

control methods, including pioneering work in developing tobacco

use interventions.

SUMMARY The NCI program, in collaboration with numerous organizations, is

influencing the public to reduce its use of tobacco. ST control is a significant element of NCI’s commitment. The mission of NCI with regard to

tobacco control is twofold: First, to strengthen the science base for intervention services through its epidemiological and biomedical research programs,

and second, to ensure that sound scientific methods reach the right people,

at the right time, and in ways that are most likely to create a united effort

for reducing cancer incidence and mortality and other tobacco-induced

health problems.
Smoking and Tobacco Control Monograph No. 2

334

REFERENCES

Cohen, S.J., Christen, A.G., Katz, B.P., Drook, C.A.,

Davis, B.J., Smith, D.M., Stookey, G.K. Counseling

medical and dental patients about cigarette

smoking: The impact of nicotine gum and chart

reminders. American Journal of Public Health 77:

313-316, 1987.

Cohen, S.J., Stookey, G.K., Katz, B.P., Drook, C.A.,

Christen, A.G. Helping smokers quit: A randomized controlled trial with private practice dentists.

Journal of the American Dental Association 118: 41-

45, 1989.

Eakin, E., Severson, H., Glasgow, R.E. Development

and evaluation of a smokeless tobacco cessation

program: A pilot study. National Cancer Institute

Monographs 8: 95-100, 1989.

Frank, E., Winkleby, M.A., Altman, D.G., Rockhill, B.,

Fortman, S.P. Predictors of physicians’ smoking

cessation advice. Journal of the American Medical

Association 266(22): 3139-3144, 1991.

Geboy, M.J. Dentists’ involvement in smoking

cessation counseling: A review and analysis.

Journal of the American Dental Association 118: 79-

83, 1989.

Gerbert, B., Coates, T., Zahnd, E., Richard, R.J.,

Cummings, S.R. Dentists as smoking cessation

counselors. Journal of the American Dental Association 118: 29-32, 1989.

Glynn, T.G., Manley, M.W. How To Help Your

Patients Stop Smoking: A National Cancer Institute

Manual for Physicians. U.S. Department of Health

and Human Services, Public Health Service,

National Institutes of Health. NIH Publication No.

90-3064, 1990.

Hayward, R.A., Meetz, H.K., Shapiro, M.F., Freeman,

H.E. Utilization of dental services: 1986 patterns

and trends. Journal of Public Health Dentistry 49(3):

147-152, 1989.

Mecklenburg, R.E., Christen, A.G., Gerbert, B., Gift,

H.C., Glynn, T.J., Jones, R.B., Lindsay, E., Manley,

M.W., Severson H. How To Help Your Patients Stop

Using Tobacco: A National Cancer Institute Manual

for the Oral Health Team. U.S. Department of

Health and Human Services, Public Health Service,

National Institutes of Health. NIH Publication No.

91-3191, 1990.

Secker-Walker, R.H., Hill, H.C., Solomon, L.J., Flynn,

B.S. Smoking cessation practices in dental offices.

Journal of Public Health Dentistry 47: 10-15, 1987.

Secker-Walker, R.H., Solomon, L.J., Hill, H.C. A

statewide survey of dentists’ smoking cessation

advice. Journal of the American Dental Association

118: 37-40, 1989.

U.S. Department of Health, Education, and Welfare.

Adult Use of Tobacco, 1975. Washington, DC: U.S.

Government Printing Office, 1976.

U.S. Department of Health and Human Services. The

Health Consequences of Smoking: Cancer. A Report of

the Surgeon General. U.S. Department of Health

and Human Services, Public Health Service, Office

on Smoking and Health. DHHS Publication No.

(PHS) 82-50179, 1982.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health, National Cancer Institute. DHHS

Publication No. (NIH) 86-2874, 1986a.

U.S. Department of Health and Human Services.

Clinical Opportunities for Smoking Intervention. U.S.

Department of Health and Human Services, Public

Health Service, National Institutes of Health. NIH

Publication No. 86-2178, 1986b.

U.S. Department of Health and Human Services.

Healthy People 2000: National Health Promotion and

Disease Prevention Objectives. U.S. Department of

Health and Human Services, Public Health Service.

DHHS Publication No. (PHS) 91-50212, 1991.

U.S. Department of Health and Human Services. The

Health Benefits of Smoking Cessation: A Report of the

Surgeon General. U.S. Department of Health and

Human Services, Public Health Service, Centers for

Disease Control, Center for Chronic Disease

Prevention and Health Promotion, Office on

Smoking and Health. DHHS Publication No.

(CDC) 90-8416, 1990a.

U.S. Department of Health and Human Services.

Smoking, Tobacco, and Cancer Program: 1985-1989

Status Report. U.S. Department of Health and

Human Services, Public Health Service, National

Insitututes of Health. NCI Publication No. 90-

3107, 1990b.

Winn, D.M., Blot, W.J., Shy, C.M., Pickle, L.W.,

Toledo, A., Fraumeni, J.F. Snuff dipping and oral

cancer among women in the southern United

States. New England Journal of Medicine 304: 745-

749, 1981.
Chapter 8

335

Chapter 8

Recommendations

CONTENTS Recommendations for the Control of Smokeless Tobacco

Robert Mecklenburg and R. Craig Stotts ............................................. 337

Background................................................................................... 337

Introduction ................................................................................. 338

Conclusions .................................................................................. 339

References ..................................................................................... 348

Appendix ...................................................................................... 350
Smoking and Tobacco Control Monograph No. 2

336
Chapter 8

337

In 1990, Dr. Joseph Cullen, former Deputy Director, Division of

Recommendations for the

Control of Smokeless Tobacco

Robert Mecklenburg and R. Craig Stotts

BACKGROUND

Cancer Prevention and Control, National Cancer Institute, and many others

who spoke at the Seventh World Congress on Tobacco and Health, appealed

for immediate and widespread action against tobacco use. These speakers

said that, although the research base needs strengthening, it is sufficient to

promote change now. In 1991, Dr. Louis Sullivan, Secretary of Health and

Human Services, when addressing the First International Conference on

Smokeless Tobacco, also called for public education, new policies, and

responsible action in the private and public sectors.

The recent rise in the use of ST in the United States, particularly the use

of moist snuff, is cause for alarm. We know that dreadful health consequences followed the increase in smoking, and we know the toll of ST use in

southeast Asia. It would be irresponsible to wait, for perhaps a quarter

century, until oral cancer and other health consequences associated with ST

use increase significantly. Nor can a wait-and-see attitude be justified when

oral cancer incidence already exceeds the incidence of leukemia, cervical

cancer, and other well-recognized cancers (American Cancer Society, 1992).

In January 1991, the National Cancer Institute and the National Institute of Dental Research convened a workshop of smokeless tobacco experts

(see Appendix) to plan a response to the rise in ST use and the concomitant

increase in risks for deaths from oral and other cancers in the United States

and many other countries. The group assessed research conducted since the

1986 NIH Consensus Development Conference and the 1986 report of the

Surgeon General’s Advisory Committee, reexamined earlier conclusions, and

proposed action based on current knowledge.

The NCI/NIDR workshop participants concluded that smokeless tobacco is addictive and that it causes, or is strongly associated with, adverse

effects on oral and systemic health. Participants found that research findings have reinforced conclusions of other expert committees about the

health effects of ST use. As a result of workshop deliberations, a document

describing objectives and strategies for smokeless tobacco control and

specific recommendations for action during the 1990’s was prepared for use

in the United States. Copies of the first draft were distributed to participants

of the First International Conference on Smokeless Tobacco to facilitate

consideration of issues raised during the conference and development of

globally oriented conclusions, objectives, and strategies. Scientific presentations and discussions during the conference broadened, clarified, and

strengthened the conclusions and recommendations provided by the U.S.

workshop. As with the U.S. document, the document from the international conference is not applicable to all countries or to all situations.
Smoking and Tobacco Control Monograph No. 2

338

Rather, it should serve as a menu from which individuals, groups, governments, or international health organizations may choose to address their

tobacco-related problems.

Many ST interventions should be conducted as part of smoking control

initiatives. Although smoking and smokeless tobacco differ in many ways,

they also share many characteristics: (1) both are methods for nicotine

administration; (2) they are often used sequentially or concurrently by the

same individual; (3) both adversely affect health and are potentially lethal;

and (4) they often are susceptible to similar prevention and cessation

interventions.

The STCP monograph on smokeless tobacco and, hence, these recommendations are a synthesis of the work of the U.S. workshop committee and

the participants of the First International Conference on Smokeless Tobacco.

This volume is based on the latest scientific research in the field. Decisionmakers and health care professionals are invited to read it carefully and

urged to implement as many of the recommendations as possible.

INTRODUCTION A workshop on the control of smokeless tobacco was sponsored by

the National Cancer Institute and the National Institute of Dental Research

on January 24, 1991, in Rockville, Maryland, USA. Participants at the NCI/

NIDR workshop included biomedical, epidemiological, and behavioral

researchers; public health professionals; health educators; and representatives from government offices and research institutes within the U.S. Public

Health Service. The workshop was convened to plan a response to the

increase in ST use in the United States and the association of ST use with

oral cancer and other health effects in the United States and other countries.

In the United States, during most of this century, the use of chewing

tobacco and oral snuff, collectively referred to as smokeless tobacco, was

seen primarily in rural areas and among particular occupational groups such

as miners and agricultural workers, and the prevalence of use was highest

among people over the age of 50 (US DHHS, 1986). In the 1970’s, production and promotion of ST products increased, and new product types were

introduced. Anecdotal reports and local and regional surveys show high

rates of use among children and adolescents in some areas, and national

surveys of adults indicate the highest prevalence now is among young adult

males (Orlandi and Boyd, 1989; Schoenborn and Boyd, 1989; US DHHS,

1986). Government data (U.S. Department of the Treasury, 1991) indicate

that sales of moist snuff are again increasing, after a brief period of decline

in 1986 and 1987, reaching 45 million pounds in 1989 and 52 million

pounds in 1990.

The workshop agreed that the conclusions of the 1986 NIH Consensus

Development Conference on Smokeless Tobacco Use—that is, that smokeless tobacco use is addictive and that it causes or is strongly associated with

many adverse effects on oral and systemic health—remain valid.

The findings of the 1986 Report of the Advisory Committee to the

Surgeon General, The Health Consequences of Using Smokeless Tobacco, were

summarized as follows (US DHHS, 1986):
Chapter 8

339

After a careful examination of the relevant epidemiologic, experimental, and clinical data, the committee concludes that the oral

use of smokeless tobacco represents a significant health risk. It is

not a safe substitute for smoking cigarettes. It can cause cancer and

a number of noncancerous oral conditions and can lead to nicotine

addiction and dependence.

The above conclusion was supported by the following specific determinations:

• The use of smokeless tobacco can cause cancer in humans;

• Use of smokeless tobacco can result in noncancerous and precancerous oral pathologies including leukoplakias, gingival recession, and

possibly gingivitis, dental caries, abrasion, and staining; and

• Smokeless tobacco contains nicotine and its use can lead to nicotine

addiction or dependence.

Similar conclusions were reached by the NIH Consensus Development

Conference, “The Health Implications of Smokeless Tobacco Use,” and, with

respect to carcinogenicity, by the International Agency for Research on

Cancer (Consensus Conference, 1986; IARC, 1985). The conclusions

reached by the Advisory Committee to the Surgeon General have been

supported by subsequent research and serve as the bases for the recommendations that follow.

Research is in progress to delineate the mechanisms of carcinogenesis

and other biological effects, to describe the time course of tobacco-related

oral cancer and other pathologies, to quantify risk, to estimate attributable

morbidity and mortality, to understand patterns of use, and to develop

appropriate means of intervening with current or potential ST users. Because of the nature of biological, epidemiological, and behavioral research,

some questions addressed by this research will not be fully answered for

many years.

The workshop participants also recognize and commend the work that

has already been carried out by health organizations and educators; advocacy groups; government agencies; biomedical, epidemiological, and behavioral researchers; individual activists; and members of the U.S. Congress.

These groups and individuals are responsible for slowing the acceleration in

ST use observed in the 1970’s and early 1980’s and have provided current

information for understanding and combating smokeless tobacco use.

Future efforts at smokeless tobacco control will build on this foundation.

 The following conclusions and recommendations summarize the

consensus of the participants in the NCI/NIDR workshop [with subsequent

organization and refinement of relevant information by the above-named

authors]:

CONCLUSIONS
Smoking and Tobacco Control Monograph No. 2

340

Whereas,

• Smokeless tobacco products (in the form of oral snuff) currently are

the only types of tobacco with an increase in use in the United States,

particularly among young persons (U.S. Department of the Treasury,

1991);

• ST use begins at a young age;

• The addictive substance in both smokeless tobacco and cigarette

smoke is nicotine, and there is a clear potential for ST to serve as a

gateway to cigarette smoking (or to reinforce previously established

nicotine use) as well as leading to the use of other addictive substances (e.g., alcohol and illicit drugs);

• The prevalence of ST use is high among vulnerable populations (e.g.,

blue collar workers, rural residents, youth, Native Americans);

• Smokeless tobacco products are marketed aggressively with special

targeting of vulnerable populations in the United States and abroad;

• The increase in ST use in the United States over the past decade,

especially among young people, has not abated;

• Users of smokeless tobacco are at increased risk for oral cancer,

noncancerous oral pathologies, and nicotine addiction;

• The biological effects and epidemiology of ST use are a recent focus of

research, and it is possible and probable that continued investigation

will identify further health risks and harmful effects;

• The epidemic of cigarette smoking and resulting disease and death

that has taken place during this century demonstrates the severe,

long-term impact on world health that can occur when use of an

addictive product gains wide public acceptance before the full extent

of its harmful effects is discovered;

Therefore,

This working group endorses the objectives and strategies described

below to reduce the use of smokeless tobacco throughout the

world. The following recommendations include endorsements,

extensions, and modifications of objectives and recommendations

formulated by the Public Health Service (US DHHS, 1991), the Tata

International Symposium on the Control of Tobacco-Related

Cancers and Other Diseases (1990), and the World Health Organization (1988).

Public Health

Objectives

Introduction

The Nation’s objectives for health for the year 2000 include

specific objectives for control of all tobacco, including smokeless tobacco (US DHHS, 1991). The risk reduction objective for

smokeless tobacco is to “reduce smokeless tobacco use by males

aged 12 through 24 to a prevalence of no more than 4 percent.”
Chapter 8

341

This working group strongly recommends that ST control activities

make use of existing channels and structures that currently focus on control

of cigarette smoking for the following reasons:

• Resources for public health activities are limited.

• Cigarette smoking remains the number one preventable cause of

death and disability in most countries, and there is no intent or

reason for ST control to compete for limited resources with smoking

control activities.

• Cigarette smoking control has a long and successful history, and it is

most efficient for ST control activities to function within these

existing channels, structures, and techniques whenever possible.

• Cigarette smoking and smokeless tobacco use are two methods of selfadministering the same addictive substance obtained from different

preparations of the same plant. The tobacco industry’s marketing

strategies for these products are complementary. Hence, the public

health response should also show a coordinated approach. The clear

and unambiguous message that should be disseminated to the public

is there is no safe form of tobacco.

A number of general considerations will influence the course of activities recommended to achieve the objectives for controlling ST use. Prominent among them are the following:

• Prevention of initiation of smokeless tobacco use by the young and

cessation by current users are both necessary to achieve a significant

reduction in prevalence.

• Campaigns or programs discouraging ST use should convey the

message that all forms of tobacco are unhealthy. Care must always be

taken that users of one tobacco product are not inadvertently persuaded to switch to another tobacco product that is perceived as a

safe alternative.

• All tobacco control efforts should include smokeless tobacco. Because

prevalence of ST use varies according to region, urbanicity, age,

ethnicity, and type of employment, the level and nature of the effort

devoted to ST control will depend on the target population.

• Although the recent increase in use of smokeless tobacco in the

United States and some other countries has occurred primarily among

adolescents and young adults, intervention efforts should not overlook older users and regions where there is a long tradition of tobacco

use.

• Organizations and agencies that conduct ST intervention programs

should collaborate with other private and public organizations

worldwide, to coordinate priorities and strategies and to maximize

impact for the resources available.
Smoking and Tobacco Control Monograph No. 2

342

Recommendations for specific target audiences and locations are listed

below.

• Increase public awareness of the hazards associated with smokeless

tobacco, especially among those segments of the population at

greatest risk for ST use.

• Increase awareness by parents of the hazards and signs of ST use.

• Promote a shift in public opinion and social norms among predisposed groups so that smokeless tobacco is viewed as socially unacceptable, undesirable, and an unhealthy alternative to smoking.

• Develop public support for policies and legislation that discourage the

use of smokeless tobacco.

• Involve professional athletes, celebrities, and other influential role

models in public education campaigns.

• Develop effective counteradvertising materials and make them widely

available to individuals, agencies, and organizations interested in

promoting an antitobacco message.

• Provide education and training to individuals willing to become

spokespersons for ST control.

• Provide materials and programs for influential organizations, such as

parent and teacher associations and other organizations that reach

youth, especially high-risk youth (e.g., Little League and Babe Ruth

Baseball, other sports leagues, the Scouts, Future Farmers of America).

• Provide broad tobacco-related curricula in multiple grades to reduce

the initiation of tobacco use, including smokeless tobacco.

• Include cessation support in school ST curricula for students and

provide cessation support for school employees.

• Adopt policies that prohibit the use of any form of tobacco by students, faculty, staff, and visitors on school property at any time and at

school-sponsored events in other locations.

• Provide prevention and cessation interventions in colleges and trade

schools.

• Ascertain tobacco use status of all patients and routinely provide

cessation advice, support, and followup.

• Be alert to the physical signs of ST use and use clinical observations,

particularly by providers of oral health services, as an opportunity to

encourage cessation.

• Adopt tobacco-free policies in health care facilities to prohibit the use

of any form of tobacco.

• Acquire continuing education training regarding ST use and

strengthen tobacco intervention skills.

Public

Awareness

Schools

Health

Services

Professionals
Chapter 8

343

• Provide students with training and require them to demonstrate

competency in tobacco use counseling.

• Disseminate established training programs and materials for health

professionals to strengthen tobacco use prevention skills (for example,

materials presented in Glynn and Manley, 1989; Glynn et al., 1990;

Mecklenburg et al., 1990).

• Implement formal tobacco policies to restrict or ban the sale and use

of all tobacco at the workplace.

• Provide information, encouragement, support, and incentives for

employees and their families to quit using smokeless tobacco.

• Involve labor unions in the planning and implementation of worksite

intervention programs.

• Use labor unions to educate their constituencies about the hazards of

tobacco use, including ST, and encourage cessation.

• Give special attention to schools and health care facilities as worksites

with prominence and influence that extend beyond their own employees.

• Emphasize efforts at worksites with populations at high risk for ST use.

• Assess extent of smokeless tobacco use and identify populations at risk.

• Identify currently available resources and intervention needs.

• Develop a formal state tobacco control plan that includes smokeless

tobacco (as recommended by the Association of State and Territorial

Health Officials, 1989).

• Encourage schools to adopt tobacco-free policies and curricula that

address all forms of tobacco use.

• Provide appropriate resources for agencies that have responsibilities for

smokeless tobacco surveillance and control.

• Support research and disseminate findings.

• Provide leadership in identifying research needs.

• Provide a clearinghouse for ST research, policy, and control activities.

• Promote transfer of scientific findings to public health educators,

communications experts, and activists for dissemination to the public.

• Coordinate smoking and smokeless tobacco control activities carried

out by PHS agencies and offices and promote communication among

all Government agencies involved in tobacco surveillance and control.

• Provide clear and prominent communications to the public about the

hazards of ST use.

Employers and

Worksites

States,

Counties, and

Municipalities

Federal

Government
Smoking and Tobacco Control Monograph No. 2

344

• Provide technical support to States, including surveillance data,

resource materials and listings, and current information on national

activities.

• Encourage antismoking activists to extend their activities to include

smokeless tobacco.

• Assist in recruitment of prominent spokespersons by endorsing

activities, providing thank you’s, or awarding special recognition.

• Provide resources for public education campaigns in the form of

media materials (public service announcements or news kits), resource listings, and background information.

• Provide resources for intervention delivery through resource listings,

guidelines for materials development, and copies of materials produced by PHS agencies.

• Assemble information and arguments needed to support legislative

initiatives and make such information available to activists.

• Include smokeless tobacco as part of tobacco education programs,

emphasizing that ST is not a safe alternative to cigarettes.

• When possible, make use of available tobacco education materials.

Develop new materials or adapt older ones, as needed.

• Work with departments of education, school districts, teachers,

administrators, and parent associations to elicit support for tobacco

education. Encourage national organizations of these groups to pass

formal position statements.

• Promote tobacco-free policies in schools as an essential component of

health education programs and interventions.

• Direct educational efforts to high-risk youth both in school and in

outside settings, such as trade schools, shop classes, 4-H Clubs, Future

Farmers of America, YMCA, Little League, and other youth organizations and clubs.

• Extend tobacco education to colleges, universities, and other

postsecondary educational institutions.

• Work for inclusion of interventions in smokeless tobacco as part of

professional education.

• Support continuing education programs to train professionals to

intervene with their patients.

• Promote inclusion of tobacco counseling skills in professional licensing requirements.

• Adopt formal tobacco control positions that specifically include

smokeless tobacco. Strengthen current position statements in light of

the increase in ST use that has occurred since 1986.

• Sponsor, support, or promote national- and community-level smokeless tobacco control programs and public education campaigns.

Health

Educators

Health

Professional

Organizations
Chapter 8

345

• Solicit advice from experts in the field to ensure scientific accuracy of

all statements regarding ST.

• Provide tobacco cessation services to employees and students.

• Enlist support of prominent individuals to serve as spokespersons.

• Provide training in advocacy skills to volunteers from the community.

• Work with local media to secure news coverage and adequate placement of public service announcements.

• Sponsor ST control events.

• Provide multiple and sustained messages to the public about the

dangers of smokeless tobacco and cigarettes.

• Direct public education materials and campaigns to groups at highest

risk; for example, televised broadcasts of sporting events provide

access to potential users.

• Use schools as a valuable channel of access to youth, but efforts to

reach youth should not be confined to schools.

• Enlist support of major sports associations to refuse involuntary

promotion (accepting free samples, using tobacco products in public

and while on camera, display of logos and tobacco product names at

sporting events, and acceptance of tobacco company sponsorships).

• Provide education and cessation counseling to professional athletes.

• Encourage organizations and groups with an interest in reducing ST

use to extend their resources by coordinating their activities whenever

possible.

• Participate in and support networks, newsletters, and other mechanisms to increase communication among individuals and organizations involved in tobacco control.

• Additional case control studies should be carried out in geographic

regions with high prevalence of use to quantify the risk for oral cancer

and to identify other cancers, such as lung cancer, for which smokeless tobacco could be a risk factor.

• On the basis of additional epidemiological research, investigators

should develop estimates of the attributable cancer risk from ST use.

• Researchers should develop biological markers to identify and quantify

ST use for application in epidemiological, clinical, and behavioral

research.

• Data are needed to assess patterns of smokeless tobacco use and

monitor trends. All national tobacco surveys, especially surveys of

youth, should include questions on ST use. Smokeless tobacco should

not be omitted from surveys of smaller populations without clear

evidence that it is not used.

Activists

All

Research

Priorities

Research,

Intervention,

and

Surveillance

Needs
Smoking and Tobacco Control Monograph No. 2

346

• Standardized questions about tobacco use, knowledge, and attitudes

should be developed and circulated to all agencies and organizations

involved in collecting survey data. Brand names of ST products

should be included to prevent confusion between snuff and chewing

tobacco.

• Because youth are at special risk, national surveys of youth should

include questions on perceived risks of ST use, amount of ST used, age

of initiation, quit attempts, and use of cigarettes.

• More information is needed on the relationship of smokeless tobacco

use to cigarette smoking, especially among young users.

• Researchers should identify motivations for ST use and barriers to

cessation within various populations to develop relevant intervention

strategies.

• Investigators should determine the ST knowledge and attitudes of key

persons (parents, teachers, coaches, youth group leaders, professional

athletes) who can function as intermediaries to bring health messages

to youth.

• Researchers should develop prevention and cessation materials

appropriate for target audiences, including youth, young adults,

athletes, people in high-risk occupations such as logging, people with

low literacy, Native Americans, and rural populations.

• Assessing the efficacy of prevention and cessation interventions for ST

control should continue.

• A centralized clearinghouse is needed to monitor the literature on

smokeless tobacco research, policy, media coverage, and intervention

activities and to aid in research dissemination.

• New surveillance sources for clinical data, such as professional organizations and associations, should be established.

• Research on warning labels is needed to determine how they can be

made more effective.

The following strategies are not endorsed by the U.S. Government or its

agencies. These recommendations were developed by smokeless tobacco control

experts from the United States and other countries, convened during the U.S.

workshop and the First International Conference.

The recommendation of the World Health Organization Study Group

on Smokeless Tobacco Control—that countries with no established practice

of smokeless tobacco use should ban manufacture, importation, sale, and

promotion of ST products—is supported. In countries where smokeless

tobacco use is already established, such as in the United States, it is recommended that the following legislative, regulatory, and policy objectives be

instituted as interim measures to help achieve the long-term goal of a

tobacco-free society.

Strategies
Chapter 8

347

• A significant proportion of public revenues derived from the sale of

tobacco products should be used for tobacco intervention-related

public education, research, and services.

• Taxes on smokeless tobacco should be made commensurate with

those on cigarettes, and future tax increases should be applied equally

to all tobacco products. The taxes should be indexed to the rate of

inflation.

• States should enact legislation to prevent the sale or distribution of

smokeless tobacco to minors, including sales through vending machines. Such legislation should include provisions for vigorous

enforcement.

• Relevant coalitions should develop and disseminate model State and

local legislation for tobacco control.

• Governments should eliminate or severely restrict all forms of tobacco

product advertising and promotion to which minors are likely to be

exposed, including the following:

– image-based ads with special appeal to young people (allow only

tombstone ads, i.e., black lettering against white background, until

a total advertising ban is achieved);

– placement of advertising in periodicals with large youth readerships

and in those with large minority readership;

– the use of ST product names, logos, or likenesses on other products

or packaging, especially on products such as toys, bubble gum, and

other child-oriented items;

– placement of outdoor advertising near schools, recreation areas, or

other places where young people congregate;

– display of smokeless tobacco at point of sale or adjacent to items

that typically appeal to children, such as candy, toys, and comic

books;

– distribution of ST samples through the mail;

– distribution of free ST samples in public places;

– smokeless tobacco advertising and promotion through sponsorship

of athletic, sporting, cultural, or entertainment events;

– display of promotional ads, company logos, product names, or

product representation at sporting and entertainment events,

especially when these events are televised.

• Recognizing that there is no safe level of N-nitrosamines in tobacco

products and that it may not be technically feasible to produce an ST

product free of N-nitrosamines, the Government should ban smokeless tobacco in the United States.

All Levels of

Government

Federal

Government
Smoking and Tobacco Control Monograph No. 2

348

• Until a total ban is achieved, ST manufacturers should be required to

seek approval of all additives used in their products from an appropriate Government agency. Furthermore, manufacturers should reduce

the amount of nicotine, nitrosamines, polonium, and other known

hazardous substances in their products to the lowest levels technically

possible. Smokeless tobacco manufacturers should be required to list

the additives in large, easily legible type on each ST container, on

billboard advertisements, and in print media.

• The set of mandatory rotating health warnings on smokeless tobacco

products and billboards should be expanded to include addiction as a

health risk and to indicate that health benefits accrue to users when

they quit. Such warnings should be prominently printed on each

individually packaged product, on billboards, and in print media.

• The Federal Government should not support, or give the appearance

of supporting, the production, manufacture, or sale of tobacco

products.

• Activists should monitor the level and nature of ST promotion,

especially noting violations of the industry’s voluntary code prohibiting promotion to minors.

• All health-related organizations and associations should issue policy

statements condemning the use of tobacco products, including ST,

and ensure that these statements are well publicized to their members

and the public. Such organizations should encourage their members

to become active in tobacco control activities and provide, through

their contacts with patients or clients, training, technical support, or

referral as needed.

• Health and life insurance companies should determine policy applicants’ use of smokeless tobacco, as well as cigarettes, to estimate risk

and determine insurability.

• Tobacco-control coalitions should assemble information and arguments needed to support legislative initiatives.

Other

Channels

REFERENCES

American Cancer Society. Cancer Facts & Figures—

1992. Atlanta, GA: American Cancer Society,

1992, p. 5.

Association of State and Territorial Health Officials.

Guide to Public Health Practice: State Health Agency

Tobacco Prevention and Control Plans. U.S. Department of Health and Human Services, Public Health

Service, National Institutes of Health, National

Cancer Institute. NIH Publication No. 90-1577,

October 1989.

Consensus Conference. Health applications

[implications] of smokeless tobacco use. Journal of

the American Medical Association 255(8): 1045-1048,

1986.

International Agency for Research on Cancer. IARC

Monographs on the Evaluation of the Carcinogenic

Risk of Chemicals to Humans: Tobacco Habits Other

Than Smoking; Betel-Quid and Areca-Nut Chewing;

and Some Related Nitrosamines (volume 37). Lyon:

IARC, 1985.

International Symposium on the Control of

Tobacco-Related Cancers and Other Diseases. Tata

Institute of Fundamental Research, Bombay,

India, January 15-19, 1990.

Orlandi, M.A., Boyd, G.M. Smokeless tobacco use

among adolescents: A theoretical overview.

National Cancer Institute Monographs 8: 87-94,

1989.
Chapter 8

349

Schoenborn, C.A., Boyd, G.M. Smoking and Other

Tobacco Use: United States, 1987. Vital and Health

Statistics, Series 10, No. 169. Hyattsville, MD: U.S.

Department of Health and Human Services, 1989.

U.S. Department of Health and Human Services. The

Health Consequences of Using Smokeless Tobacco: A

Report of the Advisory Committee to the Surgeon

General. U.S. Department of Health and Human

Services, Public Health Service, National Institutes

of Health. NIH Publication No. 86-2874, 1986.

U.S. Department of Health and Human Services.

Healthy People 2000: National Health Promotion and

Disease Prevention Objectives. U.S. Department of

Health and Human Services, Public Health Service.

DHHS Publication No. (PHS) 91-50212, 1991.

U.S. Department of the Treasury. Monthly Statistical

Release: Tobacco Products. Bureau of Alcohol,

Tobacco, and Firearms, Department of the

Treasury, 1986-1991, compiled by authors.

World Health Organization. Smokeless Tobacco

Control. WHO Technical Report Series 773.

Geneva: WHO, 1988.
Smoking and Tobacco Control Monograph No. 2

350

Myron Allukian, Jr., D.D.S., M.P.H.

Gayle M. Boyd, Ph.D., M.P.H.

Greg Connolly, Ph.D.

Stephen Corbin, D.D.S., M.P.H.

Mary Daum

Carol D’Onofrio, Ph.D., Dr.P.H. (Chair)

Virginia Ernster, Ph.D.

Thomas F. Glynn, Ph.D.

Jerome Goldstein, Ph.D., M.D.

Dietrich Hoffmann, Ph.D.

John Horm, M.Sc.

Susan Johnson

Matthew Kinnard, Ph.D.

Dushanka Kleinman, D.D.S., M.Sc.

Steven Marcus, Ph.D.

Jeffrey McKenna

Robert Mecklenburg, D.D.S., M.P.H.

C. Tracy Orleans, Ph.D.

Kathleen L. Schroeder, D.D.S., M.Sc.

Herbert Severson, Ph.D.

R. Craig Stotts, Dr.P.H., R.N.

Phillip Swango, Ph.D.

APPENDIX

U.S. Workshop

Participants
Index

351

Index

acanthosis 207-208

acetaldehyde 98-100, 111-112, 176

Act on Prevention of Use of Tobacco (Iceland)

328

β-actin gene 210, 212-213

addiction

to cigarettes 219, 223-224, 227, 229-230, 233,

239-243

to nicotine 153, 219-224, 227, 229, 239-242, 279,

282-283, 285, 291-293, 330

to ST 219-224, 227, 239-244, 282-283, 285,

291-293

to ST, baseball players 32, 35

addiction level assessment 285

additives to ST 97, 106, 136, 348

adolescents

attitudes about smoking and ST 261-263, 268,

272-273, 284

cessation programs 287

knowledge of ST health risks 11, 15-16

males, ST use 279

marketing of ST to 251, 316

oral lesions 13-14

preventive ST education 248-249, 253-254

smoking 260-263, 268-269

smoking prevention 249, 260-263, 266-267

ST intervention 280

ST prevention programs 260-261, 268-273, 287

ST use iv, x, 3-5, 251-253, 268-273, 317, 330, 338,

340-341

surveys 346

Adult Use of Tobacco Survey (1986) 243

advertising

ban 4, 11, 247, 348

of ST iii, vi, ix-x, xii, xiv, 247, 250-251, 318-319,

347

of ST, in India 19, 22-23

tobacco 324, 328

tobacco companies 4

age of ST users 4-5, 8, 253-254, 271, 338, 340-341

associated oral cancer patients 91-92, 138

baseball players 27-29, 32-34, 67-68, 81

baseball players, first use 26, 29-30, 32-34

initiation of ST use 45, 47, 253, 268

Swedish male snuff users 74-76

alcohol and ST use 13, 27-28, 71, 135, 175,

179-181, 183, 187-189, 202

aldehydes 96, 100, 142, 144

alkaloids in moist snuff 106

alveolar lesions 94

American Academy of Otolaryngology-Head

and Neck Surgery 92, 286, 298

American Cancer Society 280, 287, 292-293,

333

American Dental Association 286, 298

American Stop Smoking Intervention Study

for Cancer Prevention (ASSIST) xiv, 298,

331, 333

amino acids in tobacco 101, 136

amplification

DNA, head and neck tumors 121-122

gene 121, 125, 128, 131, 137

HPV DNA 178

oncogene 121, 123, 125, 129-132, 134, 136-137,

146

PCR 134, 183-189

α-angelica lactone 97, 99, 111-112

β-angelica lactone 97, 99, 111-112

anogenital cancer 176, 178

anogenital infection 187-188

antigen expression, Langerhans cells 67

antitobacco laws, U.S. ix

antitobacco legislation, Ireland 323-325

areca nut 20-23, 51-52, 54, 58, 136

Asians, ST use 28

assays

DNA transfection 119-120, 137-138

lipid 194-195

nude mice tumorigenicity 120, 136-137

of ST in oral cavity 109

perinatal carcinogenesis 166

protein kinase C 193-195

transfection 123, 129, 136-138

ASSIST

see American Stop Smoking Intervention

Study for Cancer Prevention

Astros 269, 271

athletic performance, effect of ST on 223

attitudes about smoking, adolescents 261-263

attitudes about ST use 268, 272-273

baijar 51, 62

ban on ST advertising iii, xiv, 347

ban on ST products 347

by countries 318, 327

Ireland 323-325

WHO recommendations 315, 318

basal cell hyperplasia 66, 68-69, 71, 178-179

baseball players iv, 26-36, 41-49, 260, 268-273

age 27-29, 32-34

age of first use 26, 29-30, 32-34

associated oral lesions 41-49, 66-72, 78-86

attempts to quit 26, 33, 35-36

brand of used 27-31, 33
352

Smoking and Tobacco Control Monograph No. 2

comparison of seasonal and year-round use 26,

29, 31-33, 35

cotinine levels 222

duration of use 30

epithelial changes in lesions 66-72

health effects of ST use 223

oral biopsies 178

patterns of ST use 26-36

perception of health effects 26, 33, 35

periodontal effects 78-86

prevalence of ST use 34-35, 249, 287-288

race 27-29, 32, 34

ST addiction 224, 227

ST cessation 287-288

ST prevention 249, 253, 260, 268-273

(see also Little League; Major League

Baseball)

“Beat the Smokeless Habit” iv

benzo[a]pyrene (B[a]P), in ST 96-97, 99, 153,

161, 166-167, 176

cocarcinogenicity 112, 202, 204, 206-207, 211

betel quid chewing 19-24, 51-62, 136-137, 308

bidi smoking 24, 54-55, 308

bioassays

of ST in oral cavity 109-111, 115

perinatal carcinogenesis 164

transplacental NNK 164

blacks 270

baseball players, ST use 28, 32, 34, 81

oral cancer, ST use 91

bladder, cancer of 175

blood nicotine levels 219-224, 226-227, 236,

239-242

brain, actions of nicotine in 219, 229-235

brands of ST 4, 8, 41-42, 44-49

used by baseball players 26-31, 33, 36

Bruton 4

buccal carcinoma 57

buccal effects of ST use 67, 78-85

buccal lesions 94

buccal mucosa 129

effect of nicotine on 157, 159

buccal mucosal cancer 11-14

buccal mucosal lesions 52-54, 56, 58

buccal pouch carcinoma 111

buccal pouch mucosa, hamsters 202-208

Bull of the Woods 4

c-myc gene, oncogene, and proto-oncogene

131, 210

(see also myc)

cancer

see specific sites, e.g., oral cancer

cancer risks associated with ST use,

knowledge about 11, 15-16

carcinogenesis of oral cavity

role of viruses 202-214

carcinogenesis of ST 109

role of nicotine 153-159

carcinogenesis, perinatal 161-169

carcinogens

in chewing tobacco 96, 99, 112

in smoking tobacco 175-176

in snuff 100

in ST 66, 96, 98, 100, 105, 111, 153, 155-156, 161,

163-169, 175-176, 192

in tobacco 99, 102, 128, 136, 156, 202, 211

newborn sensitivity to 163

transplacental 161-162, 164-166

carcinoma in situ 91, 93, 208

carcinoma, oral

see oral cancer

carcinomas of head and neck 119, 122, 125

carcinomas of oral cavity 94

cardiovascular effects of nicotine 219, 225-226

cardiovascular effects of ST use 219, 225-226

case control studies 345

cell lipids 191-192, 195-197, 199-200

cell modulators 191-192, 195-200

cell response to tobacco components 191-200

cell signaling 192, 195, 199-200

cellular DNA 204, 209-211

central nervous system

action of nicotine in 226, 229-235

effects of nicotine on 291-292, 294

nicotinic receptors 230-234

cervix

cancer of 71-72, 176, 179, 183, 185, 202, 214

HPV infection 68, 179-180, 187, 189, 203

lesions of 189

cessation camps, India 310

cessation counseling, dentists 297-304

cessation methods 331

cessation of cigarette smoking 75, 219, 233,

235-237, 239, 243-244, 279, 281-284, 292-293

cessation of ST use 219, 223, 235-237, 239,

243-244, 253, 279-288, 291-295, 297-304, 341-348

adoption of smoking cessation procedures 279,

281-285, 295

clinical studies 280-281

compared to smoking 239, 243-244, 282-284

cue extinction procedures 280-281

dental counseling 297-304

India 24

motivation 282, 284, 286, 288

public health interventions 280, 286-288

quit attempts 282-283

readiness to quit 285

relapse 229, 236, 281, 283

special populations 279, 287

use of cigarettes 283-284

withdrawal symptoms 282-283

cessation of tobacco use 219, 229, 235-237, 239,

243-244

India 310-311

relapse 229, 236, 281, 283

Chattanooga Chew 4
Index

353

chewing tobacco

addiction to 32

and oral cancer, India 128, 135-136, 138

and oral epithelial changes 66, 69, 71

associated lesions 66-67, 69, 71

baseball players’ use 42-49

brands used by baseball players 28-31, 33

cancer risk 109, 113

carcinogenic potential xiv

carcinogens in 96, 99, 112

compared to snuff 26, 32-36, 66-67, 69, 71

consumption v, x-xi

current use 254

description of 3

historical overview ix-xi

India 19-23, 51-52, 60, 308

nicotine absorption 219-222

nicotine content 219

nitrosamines in 101-103

plasma cotinine levels 220, 222-223

prevalence in U.S. 6, 338

professional baseball players 28-36

substitutes 283-284

types, India 20-23

types of, U.S. 3, 96

children, ST use iii-iv, 4-5, 316, 338

(see also adolescents)

cholesterol 192, 196, 198-199, 225

chromosomal abnormalities 179

chromosomal translocation 128, 179

chromosome 11 121, 125, 134, 179

chromosomes 123, 176, 179-180

chutta 308

cigarette smoking

addiction to 219-224, 227, 229-230, 233, 239-243

adolescents 260-263, 268-269

adverse health effects 219, 225-227

advertising ix-x

and cancer of cervix 71

blood nicotine levels 220-222, 227, 242

cancers from 13, 161, 175

carcinogens 175-176

cessation of 75, 219, 233, 235-237, 243-244, 279,

281-284, 292-293

compared to ST use 175, 219-227, 239-244

concomitant with ST use 8, 19-21, 27-28, 71, 135,

224, 283-284, 286-287

consumption of x, xiii

control of 317, 319, 338, 341

dependence on 223-224, 227, 229-230, 233,

239, 243

during ST cessation 283-284

historical overview ix-x, xxxix, 3-4

India 19-21, 51, 53, 56, 307-308

intervention 330-331

legislation 317

link with cancer 330

model of social psychological processes 264

NCI xiv, 330-333

nicotine absorption 219-222, 240-242, 282-283,

292, 298

nicotine content of cigarettes 219

perinatal effects 163

prevention programs 247-249, 260-263, 266-267,

307

relapse 229, 236, 281, 283

ST as substitute for 75

Sweden 74-75

withdrawal effects 239, 243-244

cocarcinogenicity, viruses 202-214

college students, ST use 5

colon cancer 135, 179-180

communication models 264, 266-267

communication modes, tobacco use

intervention, India 308-310

Community Intervention Trial for Smoking

Cessation (COMMIT) 298, 331, 333

Comprehensive Smokeless Tobacco Act (1986)

iii

Comprehensive Smokeless Tobacco Health

Education Act of 1986 xiv

concurrent cigarette smoking and ST use 8,

27-28, 135, 224, 283-284, 286-287

Conference on a Tobacco-Free New Europe

320

conferences on ST xii, 4, 320, 337-340

consensus sequence primers 183-186

Consensus Development Conference on

Smokeless Tobacco (1986) xii, 4, 337-340

control of smoking 338, 341

control of ST iii, 247, 337-348

Ireland 318, 323-329

legislation 316-319

WHO recommendations 315-316, 318

(see also preventive education programs)

control of tobacco 340-348

Copenhagen (snuff) 4, 28, 30-31, 33, 36, 44, 46,

48-49, 68, 285

coronary artery disease 219, 225-226

cotinine levels in ST users 220, 222-223, 282,

285, 292-294

cotinine levels of baseball players 26-27, 29,

31-36

coumarins 97-100, 111-112

creamy snuff 23, 51, 62

crotonaldehyde 98-99, 111-112, 176

Current Population Survey xii, xiv

cytochrome P450 145, 150, 162, 166-168, 176

dental effects of ST use 41, 74-76, 78-86, 282,

297

dental intervention 286-288, 297-304

Dental Snuff 4

dentists

education of 297, 299, 301-304

ST cessation counseling 297-304

tobacco use intervention 331-332
354

Smoking and Tobacco Control Monograph No. 2

dependence on nicotine 224, 229

(see also addiction to nicotine)

dependence on ST 223-224, 227, 229, 239-244

comparison of snuff users and cigarette smokers

219-222, 227, 239-244

subjective 241-243

(see also addiction to ST)

desensitization of nicotine 232-234

diazohydroxides 142, 144, 146, 149

diglycerides 191, 193, 195-196, 198-199

7,12-dimethylbenz[a]anthracene (DMBA)

135, 153, 202, 204

transplacental carcinogen 162, 166

dimethyl sulfoxide (DMSO) 191, 193-199

Division of Cancer Prevention and Control,

NCI

tobacco use intervention 331

DNA

adducts 128, 138, 141-143, 146, 148-151, 166,

168, 179

amplification 121-122, 178, 184-186

cellular 204, 209-211

HPV 175-180, 183-189, 202-203, 206, 208-211,

213-214

oncogene amplification 129

transfection assays 119-120, 137-138

tumor tissue 133-134

viral 203-204, 206, 208, 211, 213-214

dopamine 229, 234-235

dot blot hybridization 130-132, 176-177,

184-185, 188

drug addiction, compared with nicotine

291-292

drug dependence criteria 223

dry snuff 3-4, 8, 23, 62, 96, 101-103, 239

use by women 92

use in Great Britain 240

(see also nasal snuff)

dysplasia, ST-associated 66-71, 76, 91, 93,

175-181, 183-185, 187, 298

dysplastic lesions, oral 53-54, 59, 63

education, ST use 247-256, 297, 304, 342,

344-345

by dental professionals 297-298, 303-304

in schools 342, 344-345

medical and dental professions, tobacco use

intervention 331-332

of dental professionals 297, 299, 301-304

of health professionals 342

prevention/cessation 297, 301, 304

prevention in India 19, 24, 57, 307-311

public 341-342, 345

educational levels of ST users

baseball players 27-28

India 22-23

Sweden 76

endocrine effects of nicotine 226

epidemiological studies of oral cancer

risks 11

epithelial atrophy 55-56, 58-59

epithelial atypia 207

epithelial cell expression 68-69

epithelial changes in oral lesions 66, 68-70

snuff and chewing tobacco compared 69

epithelial dysplasia 53-54, 59, 63, 67-71

epithelial tissue, effects of nicotine

epithelial tumors 109

(see also oral epithelial cells)

erythroplakia 42

erythroplasia 91, 93

esophagus, cancer of 109, 150, 192

European Community (EC)

Council Directives 325, 327

ST policy and legislation 315, 321, 323, 326-328

Fagerstrom Addiction Scale 285

fatty acids 191, 197-200

fear arousal in smoking and ST prevention

261, 266

Federal government, tobacco control 343-344,

347-348

Federal Trade Commission Report to

Congress 1991, ST sales and advertising iii

females

cigarette smoking ix

health effects of ST use, India 52-53, 56-58, 60

oral cancer incidence and mortality 12-13

oral cancer, ST use 91-92

prevalence of ST use 5, 8, 12, 15, 268, 317

ST use in India 19-21, 23

ST use in North Carolina 23

fetus

sensitivity to carcinogens 161-163, 165

field cancerization 91-92, 94

First European Conference on Tobacco Policy

320, 326, 328

First International Conference on Smokeless

Tobacco (1991) iii, xiv, 337-338, 346

floor-of-mouth mucosa, effect of nicotine on

156, 158-159

floor-of-mouth tumors 123-124, 129

Food and Drug Administration 294, 328

formaldehyde 98-100, 111-112, 142, 176

4-H Club, ST prevention programs 249, 253,

268, 331, 344

FreshStart Adult Smoking Cessation Program

280, 292-293

gender of ST users

see females; males

gene amplification 121, 125, 128, 131, 137

genes

β-actin 210, 212-213

herpes simplex virus 202-207, 211

myc 210

genetic mutations, role in oral cancer

135-136, 179

gingiva

effect of nicotine on 156-157, 159

gingival index 79-80, 82
Index

355

gingival recession from ST use 74-76, 78-79,

82-86, 282, 297, 339

gingivitis 41, 78, 82, 84, 282, 297, 339

governments, role in tobacco control 320,

343-344, 347-348

Great Britain, ST use 240, 242

gudakhu 23, 51, 62

gum cancer 11-14

hamster buccal pouch cells 193, 195-196,

202-208

Happy Days 285

Hawken 30-31, 33, 36, 44-46, 48, 285

head and neck cancers 11

head and neck tumors, oncogenes 119, 123,

125

health consequences of nicotine exposure

219, 225-227

health consequences of ST use, knowledge of

11, 15-17

health education 247-256, 266

health educators, ST control 344

health effects of smoking 219, 225-227

health effects of ST use 41-49, 219, 225-227,

247, 279, 282, 298, 337-339

baseball players 67

baseball players’ perception of 26, 33, 35-36

India 21, 23-24

Sweden 74-76

health services professionals 342, 344

health warning labels iii, xiv, 4, 11, 247, 279,

318-320, 327-328, 346, 348

HeLa cells 210-211, 214

hemoglobin adducts 142, 146-151, 166, 168

herpes simplex virus (HSV) 51, 63, 109, 111

and B[a]P 202, 204-211

cocarcinogenicity 202, 204, 206-207, 211

Hispanics, baseball players, ST use 28, 32, 34,

81

Hispanics, ST use 268, 270

histopathologic studies of ST oral lesions

66-67, 69-70

historical overview of tobacco use xxxix

history of ST use, North America xxxix, 3-4

HLA-D gene products 67-68

Houston Little League project 268-273

How To Help Your Patients Stop Smoking

332

How To Help Your Patients Stop Using

Tobacco 332

human oral tissue

metabolism of NNN and NNK in 141, 146-151

human papillomavirus (HPV) 51, 63, 68, 71-72

DNA 175-180, 183-189, 202-203, 206, 208-211,

213-214

L1 consensus sequence primers 183-186

relationship to oral cancer and dysplasia 175-181,

183-185, 187, 203

role in oral carcinogenesis 202-214

hybridization 123, 176-177

dot blot 130-132, 176-177, 184-185, 188

in situ 132, 231

Northern blot 130, 132, 137, 206, 209, 212-214

Southern blot 120-122, 129-130, 137, 176-177,

204, 206, 209-210, 213

hydrazine 96, 99-100, 112

4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)

142-144, 146-150

α-hydroxylation of NNN and NNK 142, 144-146

hypercholesterolemia 225-226

hyperkeratosis 42, 66, 69, 154-155, 177, 207-208

hyperlipidemia 225-226

hyperorthokeratosis 54, 66, 68-69, 177, 179

hyperparakeratosis 54, 66, 68-69, 178-179

hyperplasia 66, 68-69, 71, 109, 155, 183-185, 187,

207-208

hypertension 225-226

in situ hybridization 132, 176-177, 184-185, 231

India

oral cancer 128, 135, 137-138

smoking 51, 53-54

tobacco chewing 128, 135-136, 138

tobacco consumption 51, 60

tobacco habits 307-311

India, ST use 19-25, 51-63

advertising and promotion of ST products 19,

22-23

betel quid chewing 51-62

chewing tobacco 51-52, 60

education/intervention 24, 57-58

female/male use 19-21, 23

health effects 23-24

marketing of ST 19, 22-23

oral cancer 51-53, 56-63

oral lesions 51-62

prevalence of 19-21, 51-53, 60

snuff 51, 53, 62

tobacco consumption 19-23

types of ST used 20-23, 51

International Agency for Research on Cancer

(IARC) xii, 111-112, 326, 339

International Union Against Cancer 321

intervention programs xiv, 280-281

ASSIST 331, 333

COMMIT 331, 333

design of 261-273

India 24, 57-58, 307-311

National Cancer Institute 330-333

physicians and dentists 331-332

recommendations 338, 341-348

role of dentists 286-287, 297-304

(see also cessation; prevention)

intracellular signaling 191, 198

intraoral soft tissue loss 94

Ireland, control and prevention of ST

323-328

anti-ST legislation 323-325, 327-328
356

Smoking and Tobacco Control Monograph No. 2

education and information program 324

litigation 325-327

“Just Say No” 260, 267, 272

keratinization 68, 71

keratinocytes, oral 203, 207-214

keratoses, ST 183-185, 187

keto acid 142, 144-145, 164

khaini 20-21, 51, 61

kidney, cancer of 175

knowledge of ST health risks 11, 15-17,

341-342

Kodiak 285

koilocytic cells 68

koilocytosis 176

L1 consensus sequence primers 183-186

lactones 96

Langerhans cell changes in ST users 66, 68-69,

71

larynx, cancer of 111, 121-123

Laswell’s social-communication model 264,

266

Latinos

see Hispanics

legislation 11, 247, 317-318

Ireland 323-328

lesions, oral, ST-associated 11, 13, 36, 91, 139,

155, 157, 175, 177-178, 181, 183, 187, 189, 282,

292

baseball players and ST use 41-49, 66-72, 78-85

incidence of in India 307-308, 310-311

mucosal 66-67, 69, 71, 74-76, 78-86

patterns 80-81

precancerous, India 307-308, 310-311

regression of 310

site of 74-76, 78, 80-81

Sweden 74-76

leukoplakia

in baseball players 42, 45-49

prevalence 14-15

transformation to carcinoma 14-15, 17

ST use 36, 66, 72, 91, 93-94, 128, 132, 138-139,

155, 177, 279, 282, 297-298, 339

ST use, India 53-62

Levi Garrett 4, 28, 30, 33, 46, 48

lip canal methodology 109

lipid assays 194-195

lipids 191-200

Little League 331, 342, 344

ST preventive education 249, 253

ST use prevention program 260, 268-273

liver tumors 111, 113, 115, 162

loose-leaf chewing tobacco 3-4, 8, 96

lung cancer ix, 115, 135, 138, 150, 162, 164

lymph node metastasis 129, 132-133

mainpuri tobacco 20, 51-53, 60

Major League Baseball, Office of

Commissioner

ST education 298

Major League Baseball players, ST use iv,

26-36, 41-49, 67, 79, 222, 224, 268-269, 271

maleic hydrazide diethanolamine

(MH-30) 105

males

cigarette smoking ix

health effects of ST use, India 52-54, 56-58, 60

knowledge of ST risks 15-16

oral cancer iii, 13-14, 91

ST use iii-iv, x-xi, 5, 8, 15, 252, 268, 279, 281-282,

288, 315-317, 330

ST use, India 19-21

ST use, Sweden 74-76

tobacco use, India 19-21

malignant transformation of lesions 14-15,

17, 53, 56-57, 59-60, 66, 71, 74, 76, 92-93, 128,

137, 139, 153, 165, 298

Mammoth Cave 4

mandibular buccal surfaces 80-85

mandibular resection 94

market research 252

marketing of ST x, 4, 7, 8, 247, 249-253, 255-256,

316, 318-320, 328

India 19, 22-23

snuff 247, 251, 255

marketing of ST prevention programs

249-256

Masterpiece Tobacs 328

mawa 20, 22, 51-52, 61-62

McGuire’s information-processing

communication model 264, 266-267

mecamylamine 230-231

medical profession, tobacco use intervention

332

mesolimbic dopaminergic neurons 229,

234-235

metabolic activation pathways of NNN and

NNK 141-146, 149-151

metabolic effects of nicotine 226

metachronous malignant oral lesions 93

methylation in NNK tumorigenesis 144

3-methylcholanthrene (MC) 162

O6-methylguanine 136, 142-144, 164, 166, 168,

176

4-(methylnitrosamino)butyric acid (MNBA)

99, 101-102, 106, 112-113

3-(methylnitrosamino)propionic acid (MNPA)

99, 101-102, 106, 112-113

4-(methylnitrosamino)-4-(3-pyridyl)butanol

102

4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanol (iso-NNAL) 102, 104, 112-114,

142-146, 164

4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone (NNK) 99, 102-104, 106, 109, 112,

115-116, 136, 141-151, 153, 161, 164-168, 175,

202

metabolic activation of 141-146, 149-151
Index

357

perinatal carcinogenesis 161, 164-168

transplacental bioassays 164-168

tumorigenesis 141-146, 149-151

4-(methylnitrosamino)-4-(3-pyridyl)butyric

acid (iso-NNAC) 101-102, 104, 113-114

minor league baseball players, ST use iv, 67,

79

mint snuff substitute 283-284, 287

mishri 23, 51, 53-54, 62

model of ST-related oral cancer 179-180

moist snuff 3-4, 8, 92, 96-97, 101-103, 105-106,

239, 241-242, 279, 337-338

ban in Ireland 323

ban in U.K. 328

consumption of x, xiii

E.C. restrictions 321

marketing of 247, 251, 255

nicotine absorption from 219-221

nicotine content in 219

sales iii-iv

use in Sweden 74-75

mortality from oral cancer 12-13

mortality from tobacco use, India 24

mucosal cancers 176, 183, 187

mucosal lesions 41-49, 51-62, 66-67, 69, 71,

74-75, 78-86

mutations, oral cancers 135-136, 176, 179-180

myc gene 210

myc oncogenes 125, 128-129, 132-133, 136-137

myc proto-oncogene 202, 206, 210, 214

nasal cavity, cancer of 109, 115, 150, 192

nasal snuff 19, 23, 74, 101-102

addiction to 239

consumption 315

nicotine absorption 220, 240

(see also snuff)

National Cancer Advisory Board resolution

on ST x

National Cancer Institute

ASSIST xiv, 331, 333

collaborative intervention and prevention

efforts iv, 330-333

COMMIT 298, 331, 333

Division of Cancer Prevention and Control 331

First International Conference on ST xiv

mission and mandate 320, 333

monograph on ST (1989) xiv

NCI/NIDR workshop on ST (1991) 337-340

reduction of tobacco use 330-333

smoking intervention programs xiv

Smoking and Tobacco Control Program (STCP) x,

330

ST use prevention programs 249, 260, 268, 287,

298

National Center for Health Statistics 15

National Health and Nutrition Examination

Survey 15

National Health Interview Survey (NHIS) 15

National Institute of Dental Research

NCI/NIDR workshop on ST (1991) 337-340

Native Americans

ST cessation 287-288

ST use 3, 5, 8, 35, 253, 268, 270, 331, 340, 346

tobacco use ix

NCI

see National Cancer Institute

NCI/NIDR workshop on control of ST (1991)

337-340

neonates, effects of ST use on

sensitivity to carcinogens 165-166

neoplasia

role of HPV 176, 183, 189

neoplastic changes 128, 187

N-ethylnitrosourea (ENU) 162

Nicorette 221, 281

Nicotiana 96-97, 101, 113

nicotine

absorption from nicotine gum 294

absorption from ST and cigarettes xiv, 219-222,

239-242, 282-283, 285, 291-292, 294, 298

actions in CNS 226, 229-235

addiction, measurement 285

addiction to and dependence on iii, 219-224, 227,

229-230, 233, 236, 239-242, 279, 282-283,

291-293, 330, 339-340

blood levels of 219-224, 226-227, 236, 239-242

brain 291

cardiovascular effects 219, 225-226

cigarette smoking 219-223, 229-230, 233, 239-242

content of ST products 284, 292-293

desensitization of 232-234

effects on oral mucosa 153-159

fading procedures 279, 284

health consequences 219, 225-227

in CNS 291-292, 294

in ST 104, 106, 115, 176, 219-223, 239-242

metabolic and endocrine effects 226

pharmacology of 219-224, 229-235

psychoactive effects 223, 230

reinforcement effects 223, 229-230, 234-236

role in ST carcinogenesis 115, 153-159, 176

withdrawal 219, 223, 239, 243-244, 292

nicotine blockade therapy 229, 236-237

nicotine polacrilex gum 219, 235, 239, 244,

279, 281, 284, 287, 291, 293-295, 297, 299, 304

absorption of nicotine 221

gingival response 157, 159

nicotine reduction therapy 284, 291, 293, 295,

299, 304

nicotine replacement therapy 229, 235-237,

332

nicotine transdermal patch

see transdermal nicotine patch

nicotine withdrawal 292

nicotinic receptors 230-234
358

Smoking and Tobacco Control Monograph No. 2

NIH Consensus Development Conference on

Smokeless Tobacco (1986) xii, 4, 279, 337-340

NIH3T3 assays 120, 130, 136

1947 Health Act (Ireland) 325

nitrosamines xiv, 99, 102, 109, 112, 135-136, 138,

141, 145, 150, 162-164, 175, 191-192, 198

organ-specific carcinogenicity 113-114

nitrosamino acids, in ST 99-102, 105-106,

112-113

nitrosoazetadine-2-carboxylic acid 99, 112

nitrosoproline 113

nitrosothioproline 113

N-nitrosamines xiv, 96, 100-102, 112-115, 347

N-nitrosoanabasine (NAB) 99, 102-104, 106,

112-114, 164, 176

N-nitrosoanatabine (NAT) 102-104, 106,

113-114, 164, 176

N-nitrosodiethanolamine (NDELA) 99-100,

105, 112, 114

N-nitrosodiethylamine (NDEA) 162, 164

N-nitrosodimethylamine (NDMA) 99, 101, 106,

112-114, 165

fetal and neonatal carcinogen 161-162, 166-168

N-nitrosomorpholine (NMOR) 99, 101, 105,

112, 114

N'-nitrosonornicotine (NNN) 99, 102, 104-106,

109, 112-115, 136, 141-146, 148-151, 153, 164,

175, 191-200, 202

metabolic activation of 141-146, 149-151

N-nitrosopiperidine 99, 112, 114

N-nitrosoproline (NPRO) 102

N-nitrosopyrrolidine (NPYR) 99, 101, 106, 112,

114

N-nitrososarcosine (NSAR) 99, 101-102, 106,

112-113

non-PKC phosphotransferase activity 191,

195, 197-198

North America, history of ST use 3-4

Northern blot hybridization 130, 132, 137,

206, 209, 212-214

nude mouse tumorigenicity assays 120, 136,

137

Office on Smoking and Health xii, xiv

oncogene amplification, head and neck

tumors 123, 125

oncogene amplification, oral cavity

carcinomas 146

oncogenes

head and neck tumors 119, 123

myc 125, 128-129, 132-133, 136-137

ras 119, 123, 125, 128-129, 131-132, 134-137,

179-180

transplacental 163

oncogenes in oral cancer 128-139

aberrations 130, 137

amplification 121, 123, 125, 129-132, 134, 136,

138, 146

expression 128-129, 132, 135

myc and ras families 134, 136, 138

opportunistic viruses 67, 72

oral cancer

alcohol use 135, 137, 175, 179-181

and snuff use, Sweden 76

and snuff use x, xii, 66, 70

causative factors in ST 149

epidemiological studies of risks 11

genetic mutations 128-130, 134-135, 137-138,

179

genetic predisposition 133

histopathologic studies 66-67, 69

incidence and mortality 12-14

incidence, India 23-24, 307-309

knowledge of risks 11, 15-17

link with snuff use 247

model for development of 179-180

oncogenes 128-139, 146

pathogenesis of 135, 138, 175-181

role of HPV and HSV (viruses) 202-214

site of 21

ST-associated iii, x, 11-15, 66-67, 69-72, 75-76,

337-338, 345

ST-associated, India 51-53, 56-63

oral cancer, non-ST-related 175, 178-179

oral carcinogenesis 128, 131, 134, 138, 141

role of viruses 202-214

oral cavity carcinoma

bioassays 109-110

squamous cell 109-110, 135-136

(see also oral cancer)

oral cavity tumors

role of NNK and NNN 109, 115, 146, 150-151

oral dysplasia

see dysplasia, ST-associated

oral effects of ST use 67, 78-86

oral epithelial cells

response to tobacco exposure 175-176, 191-193,

198, 200

oral keratinocytes 203, 207-214

oral lesions 66-72, 74-75, 78-86, 91, 155, 157, 175,

177-178, 181, 183, 187, 189

mucosal 13

prevalence 15

ST-associated 11-16, 36, 279, 281-283, 297, 332

ST-associated, India 51-63

oral lichen planus 59-60

oral moist snuff

use in Sweden 74-75

oral mucosa 187

effect of nicotine 153-159

oral mucosal lesions 66-67, 69, 71, 78-86

of baseball players and ST use 41-49

ST use, India 51-62

oral snuff 19, 23, 338

addiction to 219-224, 239, 241-242

dry 96, 101

moist 96-97, 101

nicotine absorption 220-222, 241

types of, U.S. 96
Index

359

(see also snuff)

oral submucous fibrosis 23

oral tissue

metabolism of NNN and NNK in, human and rat

141-151

P.L. 99-252 298, 303

packaging of ST products 255

pan chewer’s lesion 52, 61

pan masala 20, 22, 51, 62

pancreas, cancer of 109, 135, 161, 175

papillomas 109

papillomavirus

see human papillomavirus

pathogenesis of oral cancer 135, 138, 175-181

role of HPV 175-181

pattiwala 61

peptic ulcers 219, 225-226

perception of ST health effects 3, 26

baseball players 26, 33, 35-36

perinatal ST carcinogenesis 161-169

periodontal effects of ST use 74-76, 78-86

periodontitis 78-79, 84, 297

peripheral nervous system, nicotine action in

230-231

pharmacological adjuncts for ST cessation

291, 295

pharmacology of nicotine addiction 219-224,

229-235, 240-241

pharmacology of ST use 219-224, 240-241

pharmacotherapies for cessation of tobacco

use 235-237, 244

(see also cessation)

pharynx, cancer of 11-12, 121, 123, 330

phospholipids 191, 195-200

physicians, tobacco use intervention 330-332

placement of ST 3, 11, 21, 52-54

plasma cotinine levels 220, 222-223, 282,

292-294

plasma nicotine levels 219-224, 226-227, 236,

239-242, 282-283, 285, 292-294, 298

plug tobacco 4, 8, 96, 103, 153

point mutation 128-130, 134-135, 137-138

polonium-210 96, 99-100, 112, 128

polyadenylated RNAs 202, 209-210, 212-213

polychlorinated biphenyls (PCBs) 165

polycyclic aromatic hydrocarbons 128

polymerase chain reaction (PCR) 123, 130,

134, 175, 177, 179, 183-189

polynuclear aromatic hydrocarbons (PAH)

97, 111, 162, 166-168

porcine oral mucosa

effects of nicotine on 156-157

preadolescent ST use prevention program

269

precancerous lesions 11, 15, 139, 177, 187, 298

ST-associated, India 24, 51-62, 307-308, 310-311

herpes simplex virus 203

(see also by type, e.g., leukoplakia)

predisposition to smoking and ST use 263-

265

prevalence of oral lesions

see oral lesions

prevalence of ST use

see smokeless tobacco use

preventive education and programs

adaptation of smoking prevention to ST 247, 249,

260, 268, 273

baseball players iv

cigarette smoking 247, 249, 260-263, 266-267

for adolescents/school-based 248-249, 254, 342,

344

India 19, 24, 57-59, 307-311

marketing of 249-256

NCI programs 330-333

research 252-253, 260-273

role of dentists 297-304

ST use 247-256, 260-261, 268-273, 279, 287,

341-348

(see also intervention programs)

Prochaska-DiClemente model of readiness to

quit 285

professional baseball

see baseball players; Major League Baseball

promotion of ST products 4, 7-8, 247, 249-253,

255-256

control of 346-348

India 19, 22-23

tobacco companies vi

promotion of ST prevention programs

249-256

protein kinase C

activity in oral epithelial cells 191, 193-199

proto-oncogenes, head and neck tumors 119,

123

psychoactive effects of nicotine 153, 159, 223,

230, 291-292

psychosocial reasons for smoking and ST use

263-265, 268-269

public awareness of ST health effects 279,

341-342

public health cessation interventions 280,

286-288

pyridyloxobutylation 142, 144

quid placement

see site of ST placement

quit ladder 33, 35

race of ST users 80-81, 270

baseball players 27-29, 32

(see also blacks; whites)

ras mutations, head and neck cancers

ras oncogenes 125, 128-129, 131-132, 134,

136-137, 179-180

rat oral tissue

activation of NNN and NNK in 144-146, 150-151

receptors, nicotinic 230-234

recession, gingival 74-76, 78-79, 82-86
360

Smoking and Tobacco Control Monograph No. 2

recommendations for control of ST 337-348

First International Conference on ST 337-338,

346

NCI/NIDR workshop 337-340

WHO 340, 346

Red Man 4, 28, 30, 33, 46, 48

reduction of tobacco use

see intervention; prevention

regional use of ST 5-8, 12, 252

reinforcing effects of nicotine 223, 229-230,

234-236

relapse of tobacco use 229, 236, 281, 283

Renegades 4

reproduction, effects of ST use on 23, 161-169,

225-226

research

smoking prevention 252-253, 261-268

ST prevention programs 252-253, 268-273

research needs 8, 16-17, 243-244, 339-340,

345-346

restriction fragment length polymorphism

(RFLP) 133-134

retinoic acid (RA) 192-193

risk groups for ST use 3, 7, 340

risk of cancer from ST use 340, 345

risk, perinatal, from ST use 161-169

risks for oral cancer

case control studies 11

knowledge of 11, 15

RNA 130, 137

mRNA 129, 132

polyadenylated 202, 209-210, 212-213

rodent fetal tumors 161-163, 165

roll tobacco 96

sachet ST 3-4, 8, 220, 325

sales of ST iii-iv, 247, 251, 279

Samson 4

schools

health education 266

preventive ST education 248-249, 254, 279, 342,

344

smoking prevention programs 248, 261-262,

266-267

seasonal use of ST compared to year-round

use 26, 29, 31-36

seasonality of ST use 41-49

self-help cessation manuals and programs

287-288, 293

serum cotinine levels 220, 222-223

serum cotinine levels of baseball players

26-27, 29, 31-36

serum nicotine levels 219-224, 226-227, 236,

239-242

Seventh World Congress on Tobacco and

Health 337

site of oral tumors 91

site of ST placement 3, 11, 21, 52-54, 71, 74-76,

78, 80, 84, 92

Skoal 4, 28, 30, 33, 44, 46, 48-49, 285

Skoal Bandits 4, 220, 255, 325

smokeless tobacco (ST)

advertising/promotion iii, vi, ix-x, xii, 318-320

ban on ST advertising iii

brands 4, 41-42, 44-46, 49

brands used by baseball players 26-31, 33, 36

carcinogenesis 109, 153-159

carcinogens in 66, 96-97, 105, 111, 115, 161,

163-169, 175-176, 192

chemical composition 96-97

commerce, ban and control of 318-319, 323

control of 247, 315-319, 337-348

historical overview ix-xi, xxxix, 3-4

legislation 317-318, 323-329

manufacture 315-316, 318, 321

marketing and promotion of x, 247, 249-256,

316, 318-320, 328

marketing of, India 19, 22-23

nicotine content 153, 159, 284-285, 292-293, 298

nitrosamines in 101-103, 105, 161-164, 192

NNN and NNK 102-103, 141-151, 161, 164-168

reduction of harmful agents 105

sale of iii-iv, 247, 251, 279, 315, 318-319

sports promotion of ST 4

substitutes 281-284, 287

taxation 248, 319, 347

tumorigenicity 97

types, India 20-23

types of 3, 8, 41-42, 44-46, 49

(see also chewing tobacco; snuff)

Smokeless Tobacco Act of 1986 279

smokeless tobacco (ST) use

addiction to 32, 35, 219-224, 227, 239-244,

282-283, 285, 291-293

addiction to, pharmacology of 219-224, 240-241

adolescent use 3-4, 248-249, 251-253, 268-273,

279-280, 284, 287, 340-341

adverse health effects 219, 225-227

age of first use 45, 47

age of users 253-254, 268, 271, 338, 340-341

and alcohol use 135, 175, 179-181, 183, 188-189

and concomitant cigarette smoking 19-21, 27-28,

75, 224, 279, 283-284, 286-287, 292, 302-303,

330

and herpes simplex virus 109, 111

and oral cancer iii, x, 23, 94, 111, 113, 175, 179-

180, 326, 330, 337-338, 345

as substitute for smoking 75

associated epithelial and Langerhans cell changes

66, 68-71

associated oral carcinomas, clinical profile 91

associated oral lesions 66-72, 74-76, 78-86

association with sports iv

athletes’ use 222-223

Australia 315

baseball players iv, 26-36, 66-72, 78-86, 222, 268,

baseball players’ use and associated oral lesions

41-49

blood nicotine levels 219-223, 240-242
Index

361

cancer sites other than head and neck 161

cardiovascular effects 219, 225-226

cessation of use 219, 223, 235-237, 243-244,

279-288, 291-295, 297-304, 341-348

children iii

China 315

compared to cigarette smoking 135, 219-227,

239-244

conferences and workshops on 337-340

Denmark 316

dental intervention 297-298, 301-304

dependence on 223-224, 227, 229, 239-244

duration of use 45-47, 66-68

effect on athletic performance 223

females 5, 8, 12, 15, 92, 94, 161, 268

Finland 316

Great Britain 240, 242

health effects of use 23-24, 279, 282, 298, 337-339

health hazards in developing countries 319

HPV and oral cancer 175-180, 183-189

India 19-25, 51-63, 307-311, 315

intervention programs 24, 268-273, 297-304, 332,

337, 341-348

intervention study, India 307-311

Japan 315

knowledge of health risks 11

Langerhans cell changes 66, 68-69, 71

leukoplakia 282, 297-298

males iii-iv, x-xi, 5, 8, 13, 252, 254, 268, 279,

281-282, 288

model of social psychological processes 265

NCI role in reducing use 330-333

nicotine absorption 219-223, 239-242, 282-283,

285, 292, 295, 298

nicotine addiction 282-283, 285, 291-293

nicotine effects 219, 223

not a safe alternative to smoking iii, xiv, 339, 344

oral cancer 11-13, 66-67, 69, 72, 91, 175-181, 247,

297

oral lesions 91, 175, 177-178, 181, 332

patterns of use 251, 338, 345

perception as safe alternative to smoking ix, 268,

272, 279, 284, 302-303, 319, 330

perception of habit 242-243

perception of health effects 3, 15, 26, 33, 35

perinatal risk 161-169

periodontal effects 74-76, 78-86

placement site 71, 74-76, 78, 80, 84

preadolescent 269

prevalence of lesions 41-49

prevalence of use, U.S. iii-iv, v, x-xii, 3-7, 251,

254, 279, 315, 317, 330, 338

prevention programs 260, 268-273, 279, 341-348

prevention research 252-253, 268-273

preventive education 24, 247-256, 260-273, 297,

301, 342, 344-345

professional baseball players 26-36

psychoactive effects 223

quit attempts 33, 35, 282

regional 5-7, 252, 338

reproductive effects 23, 161-169

restrictions on use 320, 324

risk groups, North America 3

risk reduction 340

surveys 251-252, 254

Sweden 74-76, 239, 241-242, 317

withdrawal symptoms 223, 239, 243-244,

282-283

worldwide consumption 315

(see also chewing tobacco; snuff)

smoking

see cigarette smoking

Smoking and Tobacco Control Program

(STCP) x, xiv

smoking cessation applied to ST cessation

279, 281, 283

smoking prevention programs 247, 249,

260-263, 266-267

adaptation to ST prevention 247, 249, 260, 268,

273

adolescents 249, 260-263, 266-267

development of intervention 261-267

research 252-253, 261-268

smoking tobacco

carcinogenic agents in 112, 176

nicotine content 153

perinatal effects 163

snuff

addiction to 219-224, 239-243

and oral cancer iii, 12-14, 66, 76, 151, 247

and oral epithelial changes 66, 69

associated lesions 67, 70, 76

associated oral carcinoma, clinical profile

baseball players 27-36, 41-49

brands used by baseball players 27-31, 33, 36

carcinogens in xiv, 96, 99-100, 112

compared to chewing tobacco 26, 32-36, 66-67,

69, 71

concomitant smoking 75

description of 3

dipping 74-75, 97, 103, 141, 148-151

dry 23, 96, 101-103, 240, 242

flavoring agents 97-98

Great Britain 240, 242

health effects of using 282

increased cancer risk 115, 150, 175

increased use 297

India 19, 23, 51, 53, 62

induction of oral tumors 109, 141, 149-151

marketing of moist 247, 251, 255

moist 96-97, 101-103, 105-106, 219-220, 239,

241-242, 279, 316, 337-338

moist oral 74-75

nasal 19, 23, 74, 101-103, 240, 315

nicotine absorption 219-222, 240-242, 292, 294

nicotine content 106, 153, 155, 219
362

Smoking and Tobacco Control Monograph No. 2

nitrosamines in 101, 103, 105-106

oral 19, 23, 220-222, 323, 325-326

placement 3

plasma cotinine levels 220, 222-223

prevalence of use v, x-xi, xiii, 6, 254, 297, 316

sales iii-iv

substitutes 281, 283-284, 287

Sweden 74-75, 239, 241-242

TSNA in 105-106

tumorigenicity of 97, 150

types of, U.S. 3-4, 96, 103

use by women 12, 94

users compared to cigarette smokers 219-222, 227,

240, 242

users’ perception of habit 242

(see also smokeless tobacco)

snuff-dipper’s lesion 155, 157

social communication models 264, 266-267

social inoculation strategy 260, 263, 267, 269

social psychological processes

smoking-related model 264

ST-related model 264

sodium excretion in ST users 225

soft tissue lesions 11, 15

(see also oral lesions)

Southern blot hybridization 119-122, 129-130,

137, 176-177, 204, 206, 209-210, 213

special populations, interventions 270, 273

spitting tobacco v, ix-x, xiv

(see also smokeless tobacco)

sporting events sponsored by tobacco

companies vi-vii

sports, association with ST iv, vi-vii, x

(see also baseball)

sports associations, tobacco control 345

sports, effect of ST on 223

squamous cell carcinoma 57, 109-110, 119,

122-123, 125, 129, 131, 134-136, 183-185, 187,

207

ST

see smokeless tobacco

stain, dental, from ST use 78-82, 84

staining, epithelial 66, 68-69

states, prevalence of ST use 3, 5-7

states, tobacco control 343, 347

STCP

see Smoking and Tobacco Control Program

submucous fibrosis 58-59, 61-62

Sullivan, Dr. Louis 337

Surgeon General

see U.S. Surgeon General

Surveillance, Epidemiology, and End Results

(SEER) program 12-13

surveys

adolescents 346

India, tobacco use 307-308

knowledge of health risks of ST use 15-16

Little League 269-273

of dentists 299-301

recommendations 345-346

ST use xii, xiv, 5-8, 15, 251-252, 254, 345

Sweden

carcinogens in ST 105-106

ST use 3, 74-76, 239, 241-242, 315

synergistic causal agents of oral lesions

alcohol and ST 135, 202

viruses and ST 202

tachyphylaxis 232

tapkeer 23

Tata Institute of Fundamental Research

(India) 307, 333

Tata International Symposium on the

Control of Tobacco-Related Cancers and

Other Diseases (1990) 340

taxation of ST products 248, 319, 347

12-O-tetradecanoylphorbol-13-acetate (TPA)

191-200

Texas Education Agency 272

tobacco

cancer from use of 128, 134, 175

carcinogens in 99, 109, 112, 115, 136, 202, 211

cell response to 191-200

chemical composition 97

control of 340-348

industry 316, 330

new products 316

unusual use of 240

Tobacco Act (Ireland) 324

tobacco companies

advertising/promotion iii, vi, ix-x, 4, 7

expenditures vi

sales iii

sporting events, sponsorship vi-vii

tobacco control 340-348

legislation 317

national, international 316-317, 321

program objectives 316, 333

tobacco-plus-lime mixture 21, 52-54, 61

tobacco-plus-lime-user’s lesion 61

Tobacco Products Act (1978) (Ireland) 325

Tobacco Regulations (1986) (Ireland) 325

tobacco-related illness 321, 323, 330-331

tobacco-specific N-nitrosamines (TSNA)

99-100, 102-106, 109, 113-115, 128, 136, 202

perinatal effects 161, 163-165

tobacco use

cessation 229, 235-237, 239

cessation and preventive education 297, 299

historical overview ix, xxxix, 3-4

India 10-23, 51, 58, 60, 307-311

intervention by dentists 297-304

interventions 297-304, 307, 330-332

mortality from 323

prevention programs 260-263, 273
Index

363

reduction/intervention, NCI 330-332

tongue abnormalities and tumors 13, 42,

56-59, 121, 129

training, cessation counseling 297, 299,

301-304

training of health professionals, intervention

332, 342

transdermal nicotine patch 235, 244, 284, 291,

293-295

transfection assays 123, 129, 136-138

transformation rates, malignancy

see malignant transformation of lesions

transnational tobacco companies 316, 321

transplacental carcinogens 161-162, 164-166

transplacental tumors 163-165

triglycerides 191-192, 197-199

tumor promoters 191-192

tumor site, ST-associated oral cancer 91, 93

tumorigenesis

metabolic activation of NNN and NNK 141-146,

149-151

oral cavity 141, 150

perinate 163

tumorigenic agents in ST 97

tumorigenicity assays 109, 136-137

tumors, head and neck 119, 123, 125

tumors, oral 110-111, 130, 192

development of 94, 128, 153

tumors, transplacental 163-165

twist chewing tobacco 4, 8

twist tobacco 96

U.S. Department of Health and Human

Services

tobacco reduction goals 330, 337

United Kingdom 318, 328

U.S. Environmental Protection Agency

ban on MH-30 105

U.S. Public Health Service 338, 340, 343-344

U.S. ST consumption 3, 315

U.S. Surgeon General

report on smokeless tobacco (1986) iii, xii, xiv, 4,

11, 97, 239, 279, 323, 326, 337

report on smoking (1988) 223

report on smoking and health (1964) ix

U.S. Tobacco Company iii, 247, 253, 255

U.S. tobacco consumption, figures of v, xi, xiii

U.S. Tobacco International, Inc. 323, 325

uranium-235, -238 96, 99-100, 112

urethan 96-97, 111

urinary bladder, cancer of 109

uterine cervix

cancer of 71-72

papillomavirus 68

verruciform lesions 93

verrucous carcinoma 57, 184

verrucous hyperplasia 183-185, 187

videotapes, use in interventions 266-267, 271

Vincent, Fay, Major League Baseball

Commissioner iv

viral DNA 203-204, 206, 208, 211, 213-214

viruses

cofactors in tumorigenesis 150

HPV 175-180, 183-189, 202-214

herpes simplex 51, 63, 109, 111, 202-214

role in oral carcinogenesis 202-214

volatile aldehydes 98-99, 111, 112

volatile N-nitrosamines (VNA) 100-101,

105-106, 113

warning labels iii, xiv, 4, 11, 247, 279, 318-320,

327-328, 346

white patch

see leukoplakia

whites

baseball players, ST use 27-29, 32, 34, 80-81

oral cancer, ST use 91-92, 94

ST use 3, 5, 7, 43, 270

withdrawal from ST use 223, 239, 243-244,

282-283, 292-293

compared to cigarette smoking 243-244

withdrawal symptoms 282-283, 292-293

workplace, ST control 343

workshops on ST 337-340, 346

World Health Organization

expert group 316, 318, 320

ST control recommendations 96, 104-105, 109,

115, 315-316, 318, 320, 340, 346

Study Group on Smokeless Tobacco Control 326,

328, 346

year 2000 public health objectives 340

year-round ST use compared to seasonal use

26, 29, 31-36

year-round use of ST 41, 44-49

youth, ST use

(see adolescents)

Youth Risk Behavior Surveillance System 7