Health Hazards

of Nitrite

Inhalants

U.S. DEPARMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Health Hazards of Nitrite Inhalants

Editors:

Harry W. Haverkos, M.D.

Divlsion of Clinical Research

National Institute on Drug Abuse

John A. Dougherty, Ph.D.

Veterans Administration Medical Center

Lexington, KY

NIDA Research Monograph 83

1988

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

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Temple Universlty School of Medicine

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Rensselaer Polytechnic Institute

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RICHARD E. BELLEVILLE, Ph.D.

NB Associates, Health Sciences

RockviIle, Maryland

KARST J. BESTEMAN

Alcohol and Drug Problems Association

of North America

Washington, D.C

GILBERT J. BOTVIN, Ph.D.

Cornell University Medical College

New York, New York

JOSEPH V. BRADY, Ph.D.

The Johns Hopkins Uiverrsity School of

Medicine

Baltimore, Maryland

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Washington University School of

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MARY L. JACOBSON

National Federation of Parents for

Drug-Free Youth

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REESE T. JONES, M.D.

Langley Porter Neuropsychiatric lnstitute

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DENISE KANDEL, Ph.D.

College of Physicians and Surgeons of

Columbia University

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HERBERT KLEBER, M.D.

Yale University School of Medicine

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RICHARD RUSSO

New Jersey State Department of Health

Trenton, New Jersey

NIDA Research Monograph Series

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Director, NIDA

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Acting Associate Director for Science, NIDA

Parklawn Building, 5600 Fishers Lane, Rockville, Maryland 20857
Health Hazards of Nitrite Inhalants
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from a technical

review on the extent of use and health hazards of nitrite inhalants

that took place on March 31, 1987, in Rockville, MD. The meeting

was sponsored by the Office of Science and the Division of Clinical

Research, National Institute on Drug Abuse.

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text as either previously published or adapted from previously

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may be used or reproduced without permission from the Institute or

the authors. Citation of the source is appreciated.

Opinions expressed in this volume are those of the authors and do

not necessarily reflect the opinions or official policy of the National

Institute on Drug Abuse or any other part of the U.S. Department of

Health and Human Services.

The U.S. Government does not endorse or favor any specific commercial product or company. Trade or company names appearing in this

publication are used only because they are considered essential in the

context of the studies reported herein. Other companies, not named,

may also manufacture the products mentioned.

DHHS publication number (ADM)89-1573

Printed 1988, Reprinted 1989

NIDA Research Monographs are indexed in the Index Medicus. They

are selectively included in the coverage of American Statistics Index,

BioSciences Information Service, Chemical Abstracts, Current

Contents, Psvchological Abatracts, and Psvchopharmacology Abstracts.

iv
Contents

Page

Preface vii

Nitrite Inhalants: Historical Perspective 1

Guy R. Newell, Margaret R. Spitz, and

Michael B. Wilson

The Fate and Toxicity of Butyl Nitrites 15

Roger P. Maickel

The Acute Toxicity of Nitrite Inhalants 28

Ronald W. Wood

Indications From Animal and Chemical Experiments of a

Carcinogenic Role for lsobutyl Nitrite 3 9

Sidney S. Mirvish, Martin D. Ramm, and

David M. Babcook

Toxicity of Inhaled lsobutyl Nitrite in BALB/c Mice: Systemic

and lmmunotoxic Studies 50

Daniel M. Lewis and Dennis W. Lynch

Altered T-Cell Helper/Suppressor Ratio in Mice Chronically

Exposed to Amyl Nitrite 5 9

Jesse S. Ortiz and Vilma L. Rivera

Effects of Nitrites on the Immune System of Humans 75

Elizabeth M. Dax, James E. Nagel, W. Robert Lange,

William H. Adler, and Jerome H. Jaffe

v
Page

Deliberate Inhalation of lsobutyl Nitrite During Adolescence: A

Descriptive Study 81

Richard H. Schwartz

Nitrite Inhalants: Contemporary Patterns of Abuse 86

W. Robert Lange, Elizabeth M. Dax, Charles A. Haertzen,

Frederick R. Snyder, and Jerome H. Jaffe

Epidemiologic Studies-Kaposi’s Sarcoma Vs. Opportunistic

Infections Among Homosexual Men With AIDS 96

Harry W. Haverkos

List of NIDA Research Monographs 106

vi
Preface

The National Institute on Drug Abuse (NIDA) is concerned about the

acquired immunodeficiency syndrome (AIDS) for two reasons. First,

intravenous drug abusers constitute approximately 25 percent of

reported AIDS patients in the United States. Human immunodeficiency virus (HIV) infection is transmitted among intravenous drug

abusers primarily when contaminated needles and other paraphernalia

used to inject drugs are shared. Infected drug abusers are capable of

transmitting HIV to their sexual partners during sexual contact and

to their unborn children during pregnancy. Second, the use of drugs

may expedite disease progression by further decreasing immune

function or by acting as the vehicle of transmission of other infectious agents, such as hepatitis B virus, which may also be immunosuppressive. Drugs of abuse may promote the development of

malignancies. Nitrite inhalants, commonly used by homosexual men,

have been associated with Kaposi’s sarcoma (KS) in AIDS. The

mechanism of action of nitrites as a cofactor in KS, if any, has yet

to be elucidated.

On March 31, 1987, NIDA sponsored a technical review entitled “The

Extent of Use and Health Hazards of Nitrite Inhalants.“ Approximately 25 scientists attended the meeting in Rockville, MD. The

purpose of the workshop was to review the status of research

regarding nitrite inhalants and their potential public health implications and to set directions for future studies. In part, this meeting

was called to provide up-to-date information about nitrite inhalants

for NIDA to respond to a congressional request for information as

part of the Anti-Drug Abuse Act of 1986.

This monograph is a collection of presentations delivered at the

meeting. The chapters are organized to present the history of nitrite

use; the basic biochemical, pharmacologic, and toxicologic effects of

various forms of alkyl nitrites; the effects of nitrite use on the

vii
immune system; and epidemiological findings associating nitrite use

with KS in AIDS. The purpose of this introduction is to provide an

overview of issues and controversies raised at the meeting that are

covered in more detail in individual chapters.

There are many controversial issues surrounding nitrites, not the

least of which are their current regulatory status. As Guy Newell

discusses in his historical presentation, amyl nitrite is a prescription

drug, but butyl nitrite, with effects very similar if not identical to

the amyl congener, is not considered to be a drug because it is

marketed as a “room odorizer.” Proposals to regulate the sale of

butyl nitrites have been periodically considered by various agencies of

the Federal Government.

A series of metabolic and toxicity studies of four nitrite butyl esters

in mice was presented by Roger Maickel. The lethality of these

compounds was related to rapid hydrolysis to nitrite ions with the

subsequent oxidation of hemoglobin to methemoglobin. However,

methemoglobin formation cannot account for all toxic effects. While

there were wide differences in toxicity, the relative toxic potencies

of the four butyl nitrite isomers found in “room odorizers" were

maintained under a variety of experimental conditions and routes of

administration.

The acute toxicity of nitrites in animals and man was reviewed by

Ronald Wood. Skin and tracheobronchial irritations (especially about

the nose and lips), burns from accidental ignition, headaches,

hypotension, cyanosis, methemoglobinemia, intoxication, and the

development of habitual use patterns are possible adverse effects of

nitrite inhalation.

Much of the discussion at the meeting focused on two possible

pharmacologic mechanisms by which nitrites may be involved in the

genesis of KS in AIDS: carcinogenicity and immunosuppression.

Although dependent on an unproven and controversial mechanism,

nitrites have been hypothesized to interact with organic amines and

amides in vivo to form significant amounts of highly carcinogenic

N-nitrosamines. Direct data on the carcinogenicity of nitrites in

animals are sparse. However, the in vitro studies of lipid peroxidation and the finding of route-dependent in vivo formation of

nitrosamines from amyl nitrite and methylaniline in mice, reported by

viii
Sidney Mirvish, suggest that the possibility warrants serious

investigation. Interestingly, the areas where absorbed concentrations

of volatile nitrites would be expected to be highest--the skin

surrounding the nose and in the nasal/pulmonary mucosa-are also

reported to be the areas in which KS occurs in persons with AIDS.

This association logically leads to the hypothesis that there is a

causal relationship between nitrites and KS, perhaps mediated by the

formation of N-nitroso compounds. How HIV infection initiates or

promotes this process is not clear.

Besides participating in the formation of carcinogenic metabolites,

alkyl nitrites may increase the likelihood of KS by altering immune

function. Dan Lewis, Jesse Ortiz, and Elizabeth Dax studied the

effects of nitrites on immunologic function using variations on two

basic strategies: first, examination of effects on immunologic

components (lymphocyte numbers, thymus weight, etc.) and second,

measurement of effects of nitrite pretreatment on the responsiveness

of immune system components to challenge with various adjuvants,

mitogens, and antigens. Given the complexity of the immune systems

in mice and humans, the possible variations in nitrite exposure

parameters, the large number of dependent variables and sampling

times to choose from, and the diversity of analytic methods available,

one might have predicted in advance the divergent results obtained.

Lewis reported no significant detrimental effects on the immune

systems of mice from exposures to 300 parts per million isobutyl

nitrite vapor for 13 weeks. However, the other two researchers

showed different patterns of decreases in T-lymphocyte numbers and

changes in immune functions after 21 weeks of intranasal amyl nitrite

in mice (Ortiz, this volume) or after 13 sessions of amyl nitrite

inhalation in human volunteers (Dax, this volume). lt is apparent

that there is much to learn before the relevance of nitrites to

disease processes, such as KS, is understood.

Much has been written about the extent of nitrite use among

homosexual men. In addition, household and high school surveys have

quantitated use of nitrites among adolescents and young adults, but

these surveys have not distinguished use by sexual orientation. In

this monograph two surveys of nitrite inhalant use by drug abusers

are presented. Richard Schwartz presents data collected from

adolescents at a residential drug treatment community in suburban

Virginia. Schwartz also assesses the rates of acute toxicity attributed to nitrites among adolescent abusers. Robert Lange presents

ix
data concerning nitrite use among intravenous drug abusers in

treatment from six regions of the United States and among homosexual men in Baltimore. Lange suggests that nitrite use is decreasing among homosexual men because of the fear of an association with

AIDS. Nitrite use among intravenous drug abusers is not as extensive

as among homosexual men and is not apparently changing.

The unique epidemiology of KS in AIDS suggests that a cofactor is

necessary to explain its pathogenesis. Harry Haverkos reviewed the

existing epidemiologic studies of nitrite use and KS in homosexual

men and found inconclusive results; nitrite use is associated with KS

in some studies but not in others. In discussing the variables that

may be responsible for the varied results, he ruled out bloodborne

infectious agents and focused attention on drug use and sexually

transmitted microbial agents as the most likely places to look for the

KS cofactor. His discussion points out the difficulties of interpreting

questionnaire data when sample sizes are small and methods,

populations, and questions vary. Tighter control over survey

conditions and standardized methods would make these studies more

efficient, but may be impractical to achieve.

An open discussion moderated by Dr. Newell followed the presentations at the workshop. More epidemiologic and laboratory studies

are needed to assess the role, if any, of nitrite inhalants as a

cofactor in AIDS-related KS. There is a need to develop questionnaires for studies that access nitrite exposure over one’s lifetime,

analogous to pack-years in cigarette usage. Despite the epidemiologic

associations with KS, butyl nitrite has never been tested as a carcinogen. Such studies should be conducted. Studies of animal

models infected with retroviruses and challenged with large quantities

of nitrites before, during, and/or after retrovirus infection would be

useful.

More research is needed to determine the dose-response curve of

nitrite inhalation in humans. Many individuals use other drugs, such

as alcohol, marijuana, and/or cocaine, with nitrites. What are the

effects of these drugs in combination?

Nitrite inhalants are important drugs of abuse in the United States.

Their association with KS and AIDS raises an important scientific

question about possible synergistic reactions between viruses and

chemicals in the development of cancer. It is our hope that this

x
monograph will stimulate interest in nitrite inhalant research and

attract investigators who can conduct the multidisciplinary research

necessary to address the scientific questions raised at this NIDA

technical review.

Harry W. Haverkos, M.D.

Clinical Medicine Branch

Division of Clinical Research

National Institute on Drug Abuse

Rockville, MD

John A. Dougherty, Ph.D.

Veterans Administration Medical Center

Lexington, KY

xi
Nitrite Inhalants: Historical

Perspective

Guy R. Newell, Margaret R. Spitz and

Michael B. Wilson

INTRODUCTION

Early studies of Kaposi’s sarcoma (KS), manifesting as part of the

acquired immune deficiency syndrome (AIDS), showed that many

people with KS had used volatile nitrites. In the body, nitrites may

be converted to N-nitroso compounds, which are known to be

mutagens, teratogens, and strong carcinogens. In addition, studies

found nitrites to have potent immunosuppressive properties. These

facts led us to consider volatile nitrites as possible etiologic

cofactors in AIDS-related KS.

THERAPEUTIC USE OF NITRITES

In 1859, the vasodilatory effect that follows inhalation of amyl nitrite

was described. This led to its first therapeutic use in 1867 as relief

from angina pectoris (Brunton 1867). The original form of the drug

was glass ampules enclosed in mesh: they were called pearls. When

crushed between the fingers, they made a popping sound; hence, the

colloquialism “poppers” evolved. Butyl nitrite was also investigated in

the late 1880s. Although it has the same properties as amyl nitrite,

it was never used clinically (Brunton 1897). lsobutyl nitrite is a

third member of this family of compounds, and the vasodilatory

effects of these three aliphatic nitrites-amyl, butyl, and isobutylwere found to be similar (Haley 1980). Nitrites exert their vasodilatory effects by relaxing the muscles in blood vessel walls (Nickerson

1975). Vasodilation of the cerebral blood vessels causes an increase

in intracranial pressure, producing a euphoric effect or “high.” These

aliphatic nitrites were also reported to enhance sexual performance

(Everett 1975; Hollister 1975).

1
TOXICITY OF VOLATILE NlTRITES

In spite of having a wide range of human toxicities, amyl nitrite was

considered safe for therapeutic use (table 1). The Food and Drug

Administration (FDA) eliminated the prescription requirement in

September 1960. The pharmaceutical industry first presented

testimony regarding significant abuse of amyl nitrite among young

adults (Lubell 1964). Following that testimony in 1969, the FDA

reinstated the prescription requirement. However, abuse of this drug

continued in spite of the FDA’s regulation. The National Institute on

Drug Abuse noted 13 emergency room admissions resulting from

nitrite side effects in 1976, and it investigated 84 related complaints

that year (Reed 1979).

CARClNOGENIClTY OF N-NITROSO COMPOUNDS

Nitrite, present as a natural impurity in salt, was a key ingredient in

curing meats for thousands of years. Many foods are naturally rich

in nitrates, which are converted to nitrites by bacteria. Nitrite can

combine with other chemicals called amides or amines to form a

family of compounds called nitrosamines. These substances as a

group are among the most potent carcinogens known. Nitrosamines

are not permitted to be knowingly added to food at any level,

although it is likely that some foods contain small amounts of this

carcinogenic substance. In 1978, the FDA set the amount of

allowable nitrite to be added to bacon at 120 ppm and required that

bacon be free of preformed nitrosamines at 10 ppb, the lowest

amount detectable by available technology.

Magee and Barnes first reported the carcinogenicity of

N-nistrosodimethylamine (NDMA) in 1956 (Magee and Barnes 1956).

In all, 290 N-nitroso compounds have been tested for carcinogenicity,

and 252 (87 percent) of these have shown such activity (Bogovski and

Bogovski 1981). These compounds are active carcinogens in 39

different animal species; no species tested is resistant to their

carcinogenic effects (Preussmann 1983). Experiments on the

comparative metabolism of N-nitrosamines using animal and human

tissue provide convincing evidence of similar activating metabolic

pathways (Bogovski and Bogovski 1981). Thus, humans are most

likely to be susceptible to the carcinogenic effects of these

compounds. Organic nitrites, including amyl, butyl, and isobutyl

nitrite, are mutagenic according to the Ames test with or without

metabolic activation: N-nitroso compounds have the ability to induce

2
TABLE 1. Toxicity of inhaled volatile nitrites

System or Illness Effects

Central Nervous System

Cardiovascular System

Hematologic System

Ocular Organs

Pulmonary Organs

Cancer

Light-headedness, weakness, nausea,

ataxia, delirium, headache (shortlived “nitrite headaches” and prolonged pulsatile headaches), syncope

(after profound vasodilation), tolerance, sedation, and anesthesia.

(Last two effects reported for animals

exposed to high concentrations.)

Profound hypotension and cutaneous

flushing followed by reflex vasoconstriction and tachycardia, and transient

electrocardiographic changes (inverted

T waves and depressed ST segments).

Methemoglobinemia (documented after

oral and parenteral administration and

repeatedly mentioned as a possibility

after inhalation; not reported in association with hedonistic use of these

substances), and normocytic normochromic anemia after exposure in an industrial

setting.

Increased intraocular pressure and supraorbital pain.

Irritation (reported for animals exposed to

high concentrations).

Inhaled nitrites could interact freely with

endogenous trivalent nitrogen compounds

to produce nitrosamines that are known to

be carcinogens.

SOURCE: Adapted from Sigell et al. 1978, Copyright 1978, the American Psychiatric

Association.

3
cancer after only one dose (Jorgensen 1982). These are among the

most potent known chemical carcinogens in animals.

lMMUNOSUPPRESSIVE EFFECTS OF VOLATILE NITRITES

In 1982, Goedert et al. found that the helper:suppressor ratio of

peripheral blood lymphocytes was lower among users of volatile

nitrite than among nonusers. This was the first indication that these

compounds had the potential to suppress the immune system in

humans. However, the possibility that poppers were only a surrogate

marker for other factors that were highly correlated with popper use

was not ruled out.

We demonstrated the immunosuppressive effects of nitrites on human

peripheral blood leukocytes in vitro (Hersh et al. 1983). Lymphocyte

blastogenesis, cell-mediated cytotoxicity, and monocyte adherence

were all Suppressed by concentrations of isobutyl nitrite that were

noncytotoxic in terms of cell viability. lsobutyl nitrite inhibited the

induction of a- and b-interferon, which also might contribute to

susceptibility to infectious diseases and manifestation of cancers in

users of these substances. These in vitro effects of isobutyl nitrite

were evident after only 2 hours of exposure of human lymphocytes in

culture, suggesting that prolonged exposure may not be necessary for

immunosuppressive and carcinogenic effects to occur. Cellular

cytotoxiclty was demonstrated (Jacobs et al. 1983) as was depression

of murine natural killer cells (Lotzova et al. 1984). We suggested

that continued use of these substances might interfere with attempts

to correct the immune system by other agents and, therefore, that

their use should be condemned.

NITRITES AS RECREATlONAL DRUGS

Butyl and isobutyl nitrites do not fit the definition of a food, drug,

or cosmetic as specified by the Federal Food, Drug, and Cosmetic Act

from which the FDA derives its regulatory authority. Therefore, they

are not subject to regulation by the FDA Because amyl nitrite was

no longer available after 1969 without a prescription, there was a

proliferation of different brands of butyl and isobutyl nitrite during

the early 1970s. Most commercial preparations contained some of

each type, along with unspecified impurities (Ostrow 1982; Hersh et

al. 1983).

In the 1970s. reports of nitrite use for augmentation of sexual

experiences along with descriptions of their aphrodisiac properties

4
began (Louria 1970; Pearlman and Adams 1970; Everett 1972; Gay and

Sheppard 1972; Dimijian 1978; Hollister 1975). lsraelstam et al. (1978)

reported that amyl nitrite was known to have been used recreationally as early as the 1960s. They report on one man in San

Francisco who tried amyl nitrite in 1963. However, the popper craze

really only began during the years 1974 to 1977. By 1979, over 5

million people in the United States used these drugs more than once

a week (Mayer 1983).

Sigell et al, (1978) reported that the sale of nonprescription volatile

nitrites, commonly referred to as poppers, was a large business. An

estimated total of $50 million a year was made from the sales of

more than 100,000 bottles a week in just one city. Further, they

claimed that, prior to 1978, male homosexuals indulged in the use of

volatile nitrites more than any other group. A guide to homosexual

lovemaking asserts that the use of amyl nitrite has “passed into every

comer of gay life.” Nitrites were used primarily during sexual

activity because they reduce social and sexual inhibitions, heighten

sexual arousal, relax the anal sphincter, and are thought to prolong

orgasm (Lubell 1964, Sigell 1978; lsraelstam et al. 1978). The

chronology of volatile nitrite availability and use as it could relate to

AIDS-related KS was described (Newell et al. 1984; Newell et al.

1985b) and is given in table 2.

NITRITE USE REPORTED IN THREE CASE-CONTROL STUDlES

In December 1981, we began accepting referrals of individuals with

possible AIDS either with or without KS and opportunistic infections

(Ol). One hundred thirty-five lifestyle questionnaires were completed

by the first 145 referrals. At one extreme, 31 patients had either KS

or Ol as severe manifestations of AIDS; at the other extreme, 29

referrals were symptom-free and served as controls (Newell et al.

1985b). In spite of the small numbers in our study, we found a

statistically significant increased risk of 5.5 (95 percent confidence

interval 1.1-28.7) for KS/Ol patients who had used volatile nitrites

compared with symptom-free (SF) controls (Newell et al. 1985b). In

addition, there was a smooth dose-response gradient of 1.0 for those

who never used nitrites, 4.0 for those who occasionally used, and 6.3

for those who used them frequently (table 3). Predictably, there was

considerable confounding among several risk factors. In particular,

use of nitrites was confounded by such activities as marijuana use,

attending bathhouses, and active fisting (insertion of fist into the

rectum). The estimated risk of developing KS for each factor was

5
TABLE 2. Chronology of volatile nitrite availability and use

Year Availability and Use

1859

1867

1880s

1960

1963

1960s

1969

1970

1974

1976

1977

1978

1979

1980

1981

Flushing of skin with amyl nitrite described

Therapeutic use of amyl nitrite for angina pectoris

Butyl nitrite investigated but not used clinically

Amyl nitrite prescription requirement eliminated by FDA

First report of recreational use

Recreational use common among young adults

Amyl nitrite prescription requirement reinstated by FDA

Street brands of butyl and isobutyl nitrite used;

JAMA reports use for sexual augmentation

Popper craze said to have begun

$50 million sales per year in one city

Nitrites permeate gay life

Three cases of KS/PCP found in retrospect

Over 5 million people used>once per week

19 cases of KS/PCP found in retrospect

56 cases of KS/PCP reported

Nitrite use suspected associated with KS/PCP

SOURCE: Adapted from Newell et al. 1985a, Copyright 1985, the American Journal of

Medicine.

high after taking the others into account. For example, stratification

of nitrite use by prior history of syphilis (table 4) resulted in odds

ratios of 1.0, 3.0, and 5.0 (never, occasional, and frequent use,

6
respectively) among the 28 subjects with no prior history of syphilis

as well as among the 32 subjects with a prior history of syphilis.

This suggested that use of volatile nitrites was a significant risk

factor for developing KS, independent of a prior history of syphilis.

We also found that increasing use of nitrites paralleled sexual activity

(table 5).

Marmor et al. (1982) compared 20 KS patients with 40 homosexual

controls drawn from a physician’s practice and matched them for race

and age. The major predisposing risk factors for development of KS

among the patients were amyl nitrite use and number of sex partners

per month. Marmor found that development of KS was associated

with the use of amyl nitrite but not with the use of butyl nitrite.

We believe that butyl nitrite was the predominant form of nitrite

used, but amyl nitrite was endorsed because of the way the question

regarding nitrite use was phrased, as Marmor and associates

suggested. As noted above, the effects of amyl, butyl, and isobutyl

nitrites are the same (Haley 1980). One of Marmor’s patients

reported using nitrites only once, 3 years prior to diagnosis of his

KS.

The Centers for Disease Control (CDC) study (Jaffe et al. 1983)

compared 50 KS or Ol patients with 120 homosexual controls selected

from either a venereal disease clinic (78 controls), or a physician’s

practice (42 controls). In the CDC study, nitrite use was extensive

(95 to 96 percent) among patients and both control groups. However,

lifetime usage was significantly greater among patients than among

either of the control groups. In all three studies, controls were

drawn from physician practices or clinics, and so did not necessarily

represent the male homosexual population at large. However, since

the controls were probably in poorer health than individuals in the

ideal referent population would be, risk estimates would be biased in

favor of unity. Thus, any differences found would be minimum risk

estimates. This source of bias was discussed in each paper.

Comparable findings from the three studies are shown in table 6. In

all studies, use of nitrites by cases was more prevalent than use

among the controls. If one discards the one individual reported by

Marmor et al. (1982), who only used nitrites once, the percent of use

among the cases in the three studies is 95, 96, and 94 percent. In

any epidemic, it is not unusual for some patients not to give a clear

history of exposure. The comparable prevalence of exposure among

the control groups used by Jaffe et al. (1983) does not militate

against nitrites as a potential contributing factor for development of

7
TABLE 3. Nitrite use as a risk factor for Kaposi’s sarcoma/

opportunistic infections (KS/Ol)

95 Percent

KS/Ol Controls Odds Confid.

Nitrite Use Number Percent Number Percent Ratio Interval

Never 2 6.5 8 27.6 1.0 -

Occasional 7 22.5 7 24.1 4.0 0.6 - 26.0

Frequent 22 71.0 14 48.3 6.3 1.2 - 34.0

Overall Odds Ratio 5.5 1.1 - 28.7

NOTE: Never=once only or once per year to allow for the experimenter; occasional=once per 6 months to once per month; frequent=once per month or

more.

SOURCE: Newell et al. 1985b, Copyright 1985. Oxford University Press.

TABLE 4. Nitrite use and history of syphilis

History of

Syphilis

Frequency of

Nitrite Use KS/Ol SF

Odds

Ratio

Never 1 6 1.0

No Occasional 2 4 3.0

Frequent 7 8 5.3

Never 1 2 1.0

Yes Occasional 5 3 3.3

Frequent 15 6 5.0

NOTE: Never=once only or once per year to allow for the experimenter: occasional=once per 6 months to once per month: frequent=once per month or more

Often

8
TABLE 5. Nitrite use by sexual activity

Nitrite Use

Average Number Average Number

Partners Different Partners

per Week per Week

Never 2.3 1.7

Occasional 3.1 2.0

Frequent 4.0 2.6

NOTE: Never=once only or once per year to allow for the experimenter; occasional=once per 6 months to once per month: frequent once per month or more

Often.

the disease. As noted above, the lifetime use was significantly

greater among cases than among both control groups. Also, Marmor

et al. (1982) noted that passive exposure at homosexual discotheques

was reported by many subjects. Laboratory studies showed that one

brief exposure to human lymphocytes in culture was sufficient to

cause immunologic damage (Hersh et al. 1983).

TABLE 6. Prevalence of nitrite use among three case-control

studies

Study

Cases

Number

Used Percent

Controls

Number

Used Percent

Marmor et al. 1982 20 100 40 68

Jaffe et al. 1983 50 96 120 96.95

Newell et al. 1985 31 94 29 72

COHORT EFFECT OF NlTRlTE EXPOSURE

Fifty percent of all patients in these studies with AIDS-related KS/Ol

were between 30 and 39 years of age, and 90 percent of all patients

were men between the ages of 20 and 49. The cohort of men 30 to

39 years old (mid-age 35) in 1980 would have been 20 to 29 years old

9
(mid-age 25) in 1970 when volatile nitrites became available on the

open market. Further analyses of our data showed a dose-response

gradient among patients in the 30 to 39 age group, but not in the

other two age groups (table 7). The numbers of subjects in the

younger and older age groups were small.

TABLE 7. Odds ratios for nitrite use by age at referral

Nitrite Use

Age at Referral

20-29 30-39 40-49

Never 1.0 1.0 1.0

Occasional 6.0 4.0 1.0

Frequent 2.8 9.3 3.0

NOTE: Never=once only or once per year to allow for the experimenter; occasional=once per 6 months to once per month: frequent=once per month or more

often.

Nevertheless, the increased risk clustered in the predicted age group.

This cohort would have reached mid-age of 30 years during 1975

when nitrite use was reported to be very common among male

homosexuals. The age of the cohort of men who were developing

KS/Ol is related to the history of nitrite availability and use shown

in table 8.

Four logical times for initial exposure to nitrites were: (1) 1965, the

central year from 1960 to 1969 during which amyl nitrite was

available without a prescription; (2) 1970, when street-variety nitrites

became available; (3) 1974, when the literature began reporting its

use among homosexual males; and (4) 1977, by which time its use was

repotted to be very common. Onset of the KS epidemic was reported

in mid-1981. We believe that 1965 is too soon for significant

exposure to have occurred, thus the lag time of 16 years is probably

too long. At the other extreme, the 4 years between 1977 and 1981

may be too short for the development of cancer, although it is

possible if the carcinogen were potent enough or the exposure were

highly concentrated. Metabolic products of inhaled nitrites may

satisfy both of these criteria. The bulk of the literature suggests

10
that nitrite use had saturated the gay male population by 1974.

Thus, a better estimate for onset of exposure would be a peak in

1974 followed by the epidemic reported in 1981, a span of 7 years.

TABLE 8. Time intervals from exposure to nitrites to first report

of AIDS epidemic

Year

Availability and Use Onset of Epidemic

of Nitrites Reported 1981

1965 Amyl nitrite available

1960- 1969

16 years

1970 Street-variety

“poppers” available

11 years

1974 “Popper” craze

widely recognized

7 years

1977 Nitrites reported “in

every corner of gay life”

4 years

SUMMARY

There are important reasons for considering nitrite inhalation as a

factor in the development of AIDS-related KS in young male

homosexuals. These are (1) the pharmacologic properties of amyl,

butyl, and isobutyl nitrites, which are toxic; (2) the mutagenic,

teratogenic, and carcinogenic products resulting from metabolism of

N-nitroso compounds; (3) the potent carcinogenicity of N-nitroso

compounds in 39 different animal species: and (4) the deleterious

effects of volatile nitrites on human lymphocytes both in vitro and in

vivo .

Specifically related to this epidemic, there are additional reasons for

pursuing the connection between nitrite inhalation and development of

KS. These include: (1) the timing of the production and sales of

volatile nitrites for use as recreational drugs and the subsequent

outbreak of the AIDS epidemic (7 to 10 years); (2) the extensive use

of nitrites among male homosexuals; (3) the virtual universal history

of nitrite use by young male homosexuals in whom KS has developed

11
during the past 3 years; and (4) the age group in which KS is

developing is consistent with a cohort initially exposed 7 to 10 years

ago.

CONCLUSION

We conclude that nitrite use may contribute to the development of

AIDS-related KS among male homosexuals. lmmunosuppression may

allow expression of human immunodeficiency virus that was previously suppressed. The interaction of nitrites with other identified

risk factors is yet to be elucidated.

REFERENCES

Bogovski, P., and Bogovski, S. Animal species in which n-nitroso

compounds induce cancer. Int J Cancer 27:471-474, 1981.

Brunton, T.L. On the use of nitrite of amyl in angina pectoris.

Lancet 2:97-98, 1867.

Brunton, T.L Lectures on the actions of medicines. New York

Macmillan, 1897. 673 pp.

Dimijian, G.G. Contemporary drug abuse. In: Goth, A., ed. Medical

Pharmacology. St. Louis: C.V. Mosby, 1978. pp. 297-330.

Everett, G.M. Effects of amyl nitrite (“poppers”) on sexual

experience. Med Aspects Hum Sex 6:146-151, 1972.

Everett, G.M. Amyl nitrite (“poppers”) as an aphrodisiac. In:

Sandler, M., and Gessa, G.L., eds. Sexual Behavior: Pharmacology

and Biochemistry. New York: Raven Press, 1975. pp. 97-98.

Gay, G.R., and Sheppard, C.W. Sex in the “drug culture.” Med

Aspects Hum Sex 6:28-47, 1972.

Goedert, J.J.; Wallen, WC.; Mann, D.L; Strong, D.M.; Neuland, C.Y.;

Greene, M.H.; Murray, C.; Fraumeni, J.F., Jr.: and Blattner, W.A.

Amyl nitrite may alter T lymphocytes in homosexual men. Lancet

1:412-416, 1982.

Haley, T.J. Review of the physiological effects of amyl, butyl, and

isobutyl nitrites. Clin Toxicol 16(3):317-329, 1980.

Hersh, E.M.; Reuben, J.M.; Bogerd, H.; Rosenblum, M.; Bielski, M.;

Mansell, P.W.A.; Rios, A.; Newell, G.R.; and Sonnenfeld, G. Effect

of the recreational agent isobutyl nitrite on human peripheral blood

leukocytes and on in vitro interferon production. Cancer Res

43:1365-1371, 1983.

Hollister, LE. The mystique of social drugs and sex. In: Sandler,

M., and Gessa, G.L, eds. Sexual Behavior Pharmacology and

Biochemistry. New York: Raven Press, 1975. pp. 85-92.

12
Israelstam, S.; Lambert, S.; and Oki, G. Poppers, a new recreational

drug craze. Can Psychiatr Assoc J 23:493-495, 1978.

Jacobs, R.F.; Marmer, D.J.; Steele, R.W.; and Hogue, T.R. Cellular

immunotoxicity of amyl nitrite. J Toxicol Clin Toxicol

20(5):421-449, 1983.

Jaffe, H.W.; Choi, K; Thomas, P.A.; Haverkos, H.W.; Auerbach, D.M.;

Guinan, M.E.; Rogers, M.F.; Spira, T.J.; Darrow, W.W.; Kramer, M.A.;

Friedman, S.M.; Monroe, J.M.; Friedman-Kien, A.E.; Laubenstein,

LJ.; Marmor, M.; Safai, B.; Dritz, S.K.; Crispi, S.J.; Fannin, S.L;

Orkwis, J.P.; Kelter, A.; Rushing, W.R.; Thacker, S.B.; and Curran,

J.W. National case-control study of Kaposi’s sarcoma and

pneumocystis carinii pneumonia in homosexual men: Part 1.

Epidemiologic results. Ann Intern Wed 99:145-151, 1983.

Jorgensen, K.A. Amyl nitrite and Kaposi’s sarcoma in homosexual

men. N Engl J Med 307:893-894, 1982.

Lotzova, E.; Savary, C.A.; Hersh, E.M.; Khan, A.A.; and Rosenblum,

M. Depression of murine natural killer cell cytotoxicity by isobutyl

nitrite. Cancer lmmunol lmmunother 17(2):130-134, 1984.

Louria, D.B. Sexual use of amyl nitrite. Med Aspects Hum Sex 4:89,

1970.

Lubell, I. Correspondence with Burroughs Wellcome Co., 1964, U.S.

Food and Drug Administration, 1967. (May be obtained from

Medical Information Department, Burroughs Wellcome Co., telephone

919-248-3000.)

Magee, P.N., and Barnes, J.M. The production of malignant primary

hepatic turnouts in the rat by feeding dimethylnitrosamine. Br J

Cancer 10:114-122, 1956.

Marmor, M.; Friedman-Kien, A.E.; Laubenstein, L; Byrum, R.D.;

William, D.C.; Donofrio, S.; and Dubin, N. Risk factors for Kaposi’s

sarcoma in homosexual men. Lancet 1:1083-1087, 1982.

Mayer, K.H. Medical consequences of the inhalation of volatile

nitrites. In: Ostrow, D.G.; Sandholzer, T.A.; Felman, Y.M., eds.

Sexually Transmitted Diseases in Homosexual Men. New York:

Plenum Medical Book Co., 1983. pp. 237-242.

Newell, G.R.; Adams, S.C.; Mansell, P.W.A.; and Hersh, E.M.

Toxicity, immunosuppressive effects and carcinogenic potential of

volatile nitrites: Possible relationship to Kaposi’s sarcoma.

Pharmacotherapy 4:284-291, 1984.

Newell, G.R.; Mansell, P.W.A.; Spitz, M.R.; Reuben, J.M.; Hersh, E.M.

Volatile nitrites: Use and adverse effects related to the current

epidemic of the acquired immune deficiency syndrome. Am J Med

78:811-816, 1985a.

13
Newell, G.R.; Mansell, P.W.A.; Wilson, M.B.; Lynch, H.K.; Spitz, M.R.;

and Hersh, E.M. Risk factor analysis among men referred for

possible acquired immune deficiency syndrome. Prev Med 14:81-91,

1985b.

Nickerson, M. Vasodilator drugs. In: Gilman, A.G., and Goodman,

LS., eds. The Pharmacologic Basis of Therapeutics. New York:

Macmillan, 1985. pp. 727-743.

Ostrow, D. Kaposi’s sarcoma. An interview with Dr. James Curran,

CDC Kaposi’s task force director. Homosexual Health Reports

1:3-8, 1982.

Pearlman, J.T., and Adams, G.L Amyl nitrite inhalation fad. JAMA

212:160, 1970.

Preussmann, R. Public health significance of environmental

N-nitroso compounds. In: Egan, H., ed. Environmental

Carcinogens: Selected Methods of Analysis. Vol. 6, N-Nitroso

Compounds. IARC scientific publication no. 45. Lyon, France:

International Agency for Research on Cancer, 1983. pp. 3-17.

Reed, D. The multi-million-dollar mystery high. Christopher Street

2:21-27, 1979.

Sigell, L.T.; Kapp, F.T.; Fusaro, G.A.; Nelson, E.D.; Falck, R.S.

Popping and snorting volatile nitrites: A current fad for getting

high. Am J Psychiatry 135:1216-1218, 1978.

AUTHORS

Guy R. Newell, M.D.

Margaret R. Spitz, M.D., M.P.H.

Michael B. Wilson, M.Ed. (Deceased)

Department of Cancer Prevention

and Control

The University of Texas

M.D. Anderson Hospital

1515 Holcombe Boulevard

Houston, TX 77030

14
The Fate and Toxicity of Butyl

Nitrites

Roger P. Maickel

INTRODUCTION

Volatile organic nitrites have evolved over the past quarter century

as popular street drugs, inhaled to after consciousness and intensify

sexual experiences (Sigell et al. 1978). Originally, the specific

compound used for this purpose was amyl nitrite, used in the

treatments of angina and poisoning by hydrogen sulfide or hydrogen

cyanide (Stine et al. 1978). This agent, which is primarily iso-pentyl

nitrite, was removed from the category of prescription drugs by the

Federal Drug Administration (FDA) in 1960; however, by 1968, abusive

use had become so common that it was returned to prescription

status (Israelstam et al. 1978).

Subsequently, the ready availability of butyl nitrites, used in a

variety of commercial applications (intermediates for perfumes,

antifreeze preparations, etc.), made these compounds an attractive

substitute. The pharmacology of the butyl nitrites is similar to that

of amyl nitrite; indeed, they were once considered as alternative

agents for the therapy of angina (Brunton 1897). They are presently

available from a variety of sources (pornography shops, “head” shops,

mail order catalogues) and can be obtained under a wide variety of

suggestive trade names (table 1). Nickerson et al. (1979) discussed

the pharmacology and uses of these compounds; a related report

discusses their abusive use and some biomedical effects (Pryor et al.

1980).

NATURE OF BUTYL NITRATES

The term “butyl nitrites” refers to four compounds, the nitrous acid

esters of n-butyl, isobutyl, sec-butyl, and tert-butyl alcohols. One of

15
TABLE 1. Current trade names for butyl nitrites

Aroma of Men Heart On

Ban Apple Gas Highball

Bang Jac Aroma

Bolt Liquid Increase

Bullet Looker Room

Climax Mama Poppers

Crypt Tonight Oz

Cum RUSH

Discorama Satan’s Scent

Hardware Toilet Water

the problems in dealing with these substances is the fact that

commercially available samples are generally not pure. In fact,

analysis of such materials in this laboratory indicated that the labeled

component was actually from 44 to 96 percent of the sample (table

2). A sample of “RUSH,” sold as a “room odorizer” by Gemini Books,

Lafayette, IN, was found to be primarily isobutyl nitrite (IBN). The

compounds are rather unstable unless stored at low temperatures and

TABLE 2. GLC analysis of commercials available butyl nitrites

Compound Authentic Compound Maior Containment

Percent R.T.* R.T.*

(minutes)

Percent

(minutes)

lsobutyl nitrate (lBN)a63 4.7 26 6.6

n-butyl nitrite (NBN)a

79 5.5 13 6.1

sec-butyi nitrite (SBN)a44 4.0 56 5.6

tert-butyl nitrite (TBN)a96 4.6 4 3.0

RUSHb

63 4.7 17 6.6

*Retention time.

aPfaltz and Bauer, Inc., Stamford, CT.

bPacific Western Distributing Corp.. San Frandsco, CA.

SOURCE: GLC analysis as reported by Maickel and McFadden (1979).

16
in the absence of light, air, and water (Noyes 1943); the primary

breakdown products are the corresponding alcohols. When prepared

in the laboratory by the method of Noyes (1943), IBN, n-butyl nitrite

(NBN), and set-butyl nitrite (SBN) can be produced with >99 percent

purity: tert-butyl nitrite (TBN) requires a simple distillation process

to achieve a similar level of purity (Maickel and McFadden 1979).

STABILITY STUDIES

All butyl nitrites are believed to be rapidly hydrolyzed in vivo,

yielding the corresponding alcohols and nitrite ions (Sutton 1963).

Some idea of the rapidity of in vivo degradation of these compounds

may be observed in mice exposed to the butyl nitrites through

inhalation of concentrations sufficient to cause death within 10 to 20

minutes (i.e., 3,000, 2,000, 4,000, and 50,000 ppm of IBN, NBN, SBN,

and TBN, respectively). No detectable levels (less than 1 .0 ug/ml)

could be found of IBN, NBN, or SBN. Under these conditions, the

blood level of TBN was 16.7 ± 3.2 (SEM) u g/ml.

Since plasma levels of the butyl nitrites were so low even when mice

were exposed to lethal doses, it was felt that the stabilii of these

compounds should be assessed under various test conditions.

Accordingly, a study was performed of the stability of the various

butyl nitrites in 0.1 M phosphate buffer (pH=7.4) at 37 °C. The

results (table 3) showed that rapid decomposition of these compounds

occurred under such conditions. Three of the compounds (IBN, NBN,

and SBN) had monophasic decay curves with half-lives of 14.0, 15.2,

and 21.8 minutes, respectively, while TBN showed a blphasic decay

curve with a half-life of 5.6 minutes for the first phase, and 16.2

minutes for the second phase. All four compounds decayed according

to first order kinetics.

When similar studies were performed using mouse plasma (rather than

phosphate buffer) under similar conditions, even more rapid degradation was observed (table 4). All four compounds showed a rapid

initial decay phase (with a half-life of 2.8 to 3.7 seconds) for the

first 10 to 20 seconds, followed by a second, slower phase (with a

half-life of 75 to 219 minutes) that extended out to >3.0 hours. The

transition from first to second phases occurred at a concentration of

approximately 0.1 mg/ml for all four butyl nitrites and the decay

curves were first order in both phases.

Finally, similar studies were performed to examine the stability of

the butyl nitrites when incubated with mouse whole blood at 37 °C

17
TABLE 3. Stability of butyl nitrites in phosphate buffer (pH = 7.4)

at 37 °C

Compound

Half-Life Minutes)

Phase I Phase II

IBN 14.0±0.7

NBN 15.2±1.8

SBN 21 .8±1.9

TBNa

5.6±0.4 16.2±2.7

aPhase l/Phase II transition at approximately 12 minutes.

NOTE: Butyl nitrites (1 mg/ml) were added to 0.1 M phosphate buffer (pH-7.4) in

septum-stoppered viaIs in a shaking incubatur at 37 °C. Samples were

withdrawn at 2 to 5 minute intervals for GLC analysis as described by

McFadden et al. 1981. Data were analyzed by feast-squares regress&n analysis;

values are reported as mean ± SEM (n=5).

TABLE 1. Stability of butyl nitrites in mouse plasma at 37 °C

Compound

Half-Life (Minutes)

Phase I Phase II

SBNa

2.79±0.16 219±65

TBNa2.82±0.23 127±28

NBN” 3.41±0.70 162±50

lBNa3.66±0.26 75±21

aPhase l/Phase II transition at approximately 15 seconds.

NOTE: Butyl nitrites (1 mg/ml) were added to mouse plasma in septum-stoppered vials

in a shaking incubator at 37 °C. Samples were withdrawn at 5-second to 10-

minute intervals for GLC analysis as described by McFadden et at. (1981). Data

ware analyzed by least-squares regression analysis: values are reported as mean

± SEM (n=6-7).

(table 5). Under these conditions, the rates of decay for all four

compounds were extremely rapid. NBN and IBN decayed in a monophasic fashion with half-life values <2 seconds, while SBN and TBN

showed biphasic decay curves with initial phase half-life values of <3

seconds and second phase decay values of 6.1 and 20 minutes,

respectively. Thus, the breakdown of butyl nitrites was relatively

16
TABLE 5. Stability of butyl nitrites in mouse whole blood

at 37 °C

Compound

Half-Life Minutes)

Phase I Phase II

NBN <1.0

IBN 1.60±0.05

SBNa 1.91±0.06 6.1±1.6

TBNa 2.72±0.26 20.2±2.3

aPhase l/Phase II transition at approximately 15 seconds.

NOTE: Butyl nitrites (1 mg/ml) were added to mouse whole blood in septum-stoppered

vials in a shaking incubator at 37 °C. Samples were withdrawn at 5 to 10

second intervals for GLC analysis as described by McFadden at al. (1981). Data

were analyzed by least-squares regression analysis; values are reported as

time ± SEM (n=4).

rapid in aqueous conditions and markedly enhanced in the presence

of mouse plasma and even more so in the presence of whole blood.

Osterloh and Goldfield (1984) have shown that NBN is rapidly

converted to n-butanol, nitrite and nitrate in water; the rapidity of

breakdown increased as the pH was lowered from 9 to 4. In whole

blood, NBN was rapidly converted to butanol and nitrate with

formation of methemoglobin; in plasma, these authors found NBN to

be relatively stable.

TOXICITY

The resultant effects of the presence of erythrocytes, coupled with

earlier observations that IP dosage of butyl nitrites (but not the

corresponding butyl alcohols) to mice resulted in discoloration of the

blood (Maickel and McFadden 1979), suggested that methemoglobinemia was a significant factor in the toxicity of the butyl nitrites.

Accordingly, a series of studies were performed to examine the ability

of butyl nitrites to produce methemoglobin in hemolyzed mouse

erythrocytes. The results of these studies are presented in table 6,

showing NBN as the most potent compound in this regard, closely

followed by IBN, the most commonly available butyl nitrite in

19
TABLE 6. Butyl nitrite-induced methemoglobin formation in mouse

hemolyzed erythrocytes in vitro

MH 5 0 Relative

Compound (95% Confidence Linmits) Poetency

NBN 131 (180-99) 1.00

IBN 174 (211-148) 0.75

SBN 202 (295-149) 0.65

TBN 5072 (6508-4089) 0.03

NOTE: MH values are the concentration ( g butyl nitrites whole blood) necessary

to oxidize 50 percent of the Hb to metHb. Values and 95 percent confidence

limits ware datermined by the dose-response analysis of Goldstein (1964).

SOURCE: McFadden et al. 1981, Copyright 1981, Purdue University, Department of

Pharmacology and Toxicity.

commercially (street drug) available preparations. TBN was less

potent by more than an order of magnitude.

Similar data were obtained from studies in which methemoglobin

levels were determined in mice exposed to lethal concentrations of

the four butyl nitrites in dynamic inhalation chambers. In such a

test situation, death occurred in 10 to 20 minutes at the exposure

concentrations used. As seen in table 7, TBN is much less potent

than the other three butyl nitrites, with required lethal

concentrations at an order of magnitude greater. Even at this

concentration, a significantly lower level of methemoglobin was

produced by TBN as compared to the levels produced by NBN, IBN,

and SBN.

One interesting aspect of these compounds is the relatively consistent

lethal potency when given by various routes. Table 8 displays a

summary of this data as derived from LD, determinations. NBN was

the most potent compound, regardless of route of administration,

with IBN next in potency in every case. With IP dosage, SBN and

TBN were virtually identical, while with PO dosage, TBN was a bit

more potent than SBN. On inhalation, TBN was clearly the least toxic

substance by almost an order of magnitude. Similar results with NBN

have been repotted by Osterloh and Goldfield (1985).

20
TABLE 7. Levels of methemoglobin in mice exposed to lethal

concentrations of butyl nitrites

Compound

Exposure

Concentration

(ppm)

Percent

Methemoglobin

NBN 2,000 97.4 ± 2.4

IBN 3,000 03.5 ± 1.7

SBN 4.000 83.1 ± 3.0

TBN 50,000 83.2 ± 2.2

NOTE: Concentrations of butyl nitrites were determined by GLC as reported by

McFadden and Maickel (1981). Methemoglobin levels were determined as

described by Frankel et al. (1970); values are mean ± SEM (n=4-5).

SOURCE: McFadden et al. 1981, Copyright 1981, Purdue University, Department of

Pharmacology and Toxicology.

Additional studies have also been performed to see if various

treatments could reduce or prevent the toxicity of these compounds.

Since most commercially available preparations contain IBN, and

several of these commercial preparations have been implicated in

human poisonings via the oral route (Dixon et al. 1981), IBN was used

as the test agent. The lethality of a PO dose of 400 mg/kg of IBN

could be significantly reduced by pretreating mice with methylene

blue (50 mg/kg, IP) or toluidine blue (50 mg/kg, IP), but not with

ascorbic acid (200 mg/kg, IP) or sodium selenite (3 mg/kg, IP). In

contrast, mice given 300 mg/kg, PO of IBN, showed no significant

antidotal effects in response to 100 percent oxygen, ascorbic acid,

methylene blue, or sodium selenite (McFadden and Maickel 1982).

These data would suggest that methemoglobinemia produced by butyl

nitrites plays a significant role in the lethality caused by these

compounds.

Further, in mice pretreated with methylene blue (50 mg/kg, IP) 15

minutes prior to a 45-minute period of exposure to 500 ppm of butyl

nitrites, methylene blue completely prevented methemoglobinemia

produced by NBN, IBN, and SBN. Under these conditions, TBN did

not produce any significant methemoglobinemia (McFadden et al.

1961). Similarly, methylene blue pretreatment significantly increased

the median lethal time values for survival when mice were exposed to

higher concentrations of all four butyl nitrites (table 9). In this

regard, Klonne et al. (1987) have reported single exposure LC50

21
TABLE 8. Comparative potency (lethality) of butyl nitrites administered by different routes

IP Dosage (mg/kg) PO Dosage (mg/kg)

Compound LD50 R.P. LD 5 0 R.P.

Inhalation (ppm)

L C 5 0 R.P.

NBN 158 1.00 180 1.00 567 1.00

IBN 184 0.86 279 0.65 1,033 0.55

SBN 592 0.27 428 0.42 1,753 0.32

TBN 613 0.26 336 0.54 10,852 0.05

NOTE: IP deaths @ 0.5 hr.; PO deaths @ 2.0 hr.; Inhalation deaths @ 1.0 hr.; R.P.=relative potancy where most potent compound=1.00.

Data on IP dosage from Maickel and McFadden (1979); on PO dosage from McFadden and Maickel (1982); and on inhalation

from McFadden et al. (1981).
TABLE 9. Effects of methylene blue pretreatment on survival of mice exposed to butyl nitrites

LD50 (Minutes) Ratio Methylene

Compound Control Methylene Blue (Methylene Blue to Control) (ppm)

IBN 1500 20.5

NBN 1000 24.0

SBN 2000 43.0

TBN 50000 32.7

232.1 11.3

187.0 7.8

261.5 6.1

68.6 2.1

NOTE: Mice were dosed with 50 mg/kg, IP, methylene blue 15 minutes prior to butyl nitrite exposure. LT50 values were determined

by probit analysis.

SOURCE: McFadden et al. 1981, Copyright 1981, Purdue University, Department of Pharmacology and Toxicology.
values in rats for: ethyl, methyl, n-propyl, n-butyl, isopentyl, and

isobutyl nitrites to be 160, 176, 300, 420, 716, and 777 ppm,

respectively.

OTHER STUDIES

Finally, several studies have been performed to examine the

subchronic toxicology of butyl nitrites in mice by inhalation

(McFadden and Maickel 1985). Mice were exposed to IBN (400 ppm),

NBN (300 ppm), SBN (500 ppm), or TBN (1000 ppm) for 7 hours daily

for 60 days. Under these conditions, survival rates were 80 to 95

percent. During the first 30 days of exposure, treated animals had

significantly lower body weights than untreated controls; during the

second 30 days of exposure, the body weights of mice exposed to

IBN, NBN, or SBN returned to control values, while those of mice

exposed to TBN continued to be significantly lower than those of

controls. A summary of organ weights of these mice is shown in

table 10. Liver weight was significantly increased only in the IBNtreated mice, kidney weight in those treated with IBN and SBN, and

lung weight in all except the mice exposed to TBN. In contrast,

spleen weights were significantly elevated in mice exposed to all four

butyl nitrites, presumably reflecting the adverse effects of butyl

nitrites on erythrocytes.

Selected enzymes were also determined in these mice. Plasma

isocitric dehydrogenase was significantly depressed in mice exposed to

TBN, liver microsomal glucose-6-phosphatase was significantly

depressed in mice exposed to IBN, and liver microsomal cytochrome

P-450 was significantly depressed in mice exposed to NBN (McFadden

and Maickel 1985).

SUMMARY

In summary, all four butyl nitrites were found to be moderately toxic

compounds in mice, especially when given PO, TBN was the least

toxic and also the least potent in producing methemoglobinemia.

24
TABLE 10. Effects of 60 days exposure to butyl nitrites on organ weights of mice

Proportion of Body Weight (Butyl Nitrite/Control)

Compound Kidney Liver Lung Spleen

IBN 1.15a 1.14a

1.74a2.57a

NBN 1.04 0.98 1.33a 1.82a

SBN 1.12a

0.96 1.11 1.27a

TBN 0.97 0.96 1.60a

1.31a

a

significantly greater than control.

NOTE: Mice were treated as described in McFadden and Maickel (1985).
REFERENCES

Brunton, T. Lectures on the Action of Medicines. New York:

MacMillan Co., 1897. pp. 332-343.

Dixon, D.S.; Reisch, RF.; and Santinga, P.H. Fatal

methemoglobinemia resulting from ingestion of isobutyl nitrite, a

“room odorizer” widely used for recreational purposes. J Forensic

Sci 26:593-597, 1981.

Frankel, S.; Reitman, S.; and Sonnenwirth, A., eds. Gradwhol’s

Clinical Laboratory Methods and Diagnosis. St. Louis, MO: C.V.

Mosby Co., 1970. pp. 406-401.

Goldstein, A. Biostatistics. An introductory Text. New York: The

Macmillan Co., 1964. pp. 272.

Israelstam, S.; Lambert, S.; and Oki, G. Poppers, a new recreational

drug craze. Can J Psychiatry 23:493-495, 1978.

Klonne, DR.; Ulrich, C.E.; Weismann, J.; and Morgan, A.K Acute

inhalation toxicity of aliphatic (Cl-C,) nitrites in rats. Fundam

Appl Toxicol 8:101-196, 1987.

Maickel, R.P.; and McFadden, D.P. Acute toxicology of butyl nitrites

and butyl alcohols. Res Commun Chem Pathol Pharmacol 26:75-83,

1979.

McFadden, D-P., and Maickel, R.P. Butyl nitrites - an example of

hazardous, noncontrolled recreational drugs. Res Commun Subst

Abuse 3:233-236, 1982.

McFadden, D.P., and Maickel, R.P. Subchronic toxicology of butyl

nitrites in mice by inhalation. J Appl Toxicol 5:134-139, 1985.

McFadden, D.P.; Carlson, G.P.; and Maickel, R.P. The role of

methemoglobin in acute butyl nitrite toxicity in mice. Fundam Appl

Taxicol 1:448-451, 1981.

Nickerson, M.; Parker, J.O.; Lowry, T.P.; and Swenson, E.W. Isobutyl

Nitrite and Related Compounds. San Francisco: Pharmex Ltd.,

1979. 96 pp.

Noyes, W. n-Butyl nitrite. In: Adams, R.; Conant, J.B.; Clarke, H.T.;

and Kamm, O., eds. Organic Synthesis Collections. II. New York:

J. Wiley and Sons, 1943. pp. 108-109.

Osterloh, J., and Goldfield, D. Butyl nitrite transformation in vitro,

chemical nitrosation reactions, and mutagenesis. J Anal Toxicol

8:164-169, 1984.

Ostertoh, J., and Goldfield, D. Uptake of inhaled n-butyl nitrite and

in vivo transformation in rats. J Pharm Sci 74:780-782, 1985.

Pryor, G.T.; Howd, R.A.; Bingham, L.R.; Rebert, C.S.; and Jensen, R.A.

Biomedical Studies on the Effects of Abused Inhalant Mixtures.

Final Report. National Institute on Drug Abuse Contract 271-277-

3402, 1980.

26
Sigell, L.; Kapp, F.; Fusaro, G.; Nelson, E; and Falck, R. Popping and

snorting volatile nitrites: A current fad for getting high. Am J

Psychiatry 135:1216-1218, 1978.

Stine, R.; Slosberg, B.; and Beacham, B. Hydrogen sulfide

intoxication. A case report and discussion of treatment. Ann

Intern Med 85:756-758, 1976.

Sutton, W.L Aliphatic nitro compounds, nitrates, nitrites, alkyl

nitrites. In: Fassett, D.W., and Irish, D., eds. Industrial Hygiene

and Toxicology. Vol. II. New York: Interscience, 1963. pp. 414-

438.

AUTHOR

Roger P. Maickel, Ph.D.

Department of Pharmacology

and Toxicology

School of Pharmacy and

Pharmacal Sciences

Purdue University

West Lafayette, IN 47907

27
The Acute Toxicity of Nitrite

Inhalants

Ronald W. Wood

INTRODUCTION

Alkyl nitrites produce a variety of effects when inhaled. Except in

the context of anginal pain or as an antidote to cyanide poisoning,

these effects cannot be construed as having therapeutic utility. A

number of demonstrable risks accompany the inhalation of these

materials, and this chapter will review the acute hazards associated

with the nonmedical use of these chemicals. Aside from the spectrum

of effects desired by the user, there are less desirable “side effects”

as well as frank injuries associated with the use of these products,

including skin and tracheobronchial irritation; bum injuries; acute

toxicity mediated by hypokinetic anoxia, methemoglobinemia, and

associated disorders of blood and blood-forming organs; and the

induction of a substance abuse disorder.

DESIRED ACUTE EFFECTS AND UNDESIRED SIDE EFFECTS

Inhalation of high concentrations of nitrites relaxes smooth muscle:

the consequent intense peripheral vasodilation produces flushing, a

fall in blood pressure, and a reflex increase in heart rate to

maintain perfusion of vital organs (Haley 1980). These effects are

accompanied by feelings of warmth, rapid pulse, and throbbing

sensations. Volatile nitrites are frequently used as an adjunct to

sexual behavior, because of their smooth muscle effects, nitrites can

reduce sphincter tone and alter tumescence. The vasodilation is

accompanied by heat loss and a subsequent chill. Headache, nausea,

and fainting are common sequelae of nitrite inhalation.

28
Skin and Tracheobronchial Irritation

Skin contact with commercial products containing butyl nitrite can

produce a crusty lesion at the site. Repetitive use of the material

can lead to a proliferation of these lesions around the nose and lip

(Fisher et al. 1981; Fisher 1984; Romaguera and Grimalt 1982) and

has been reported around the penis, the scrotum, and elsewhere (Bos

et al. 1985). The latter report suggests that a true allergic response

to these materials may occur.

The irritating properties of these materials are not confined to the

skin and have manifested themselves in tracheobronchitis with

erythema of sufficient severity to require hospitalization, and with

complaints of cough, fever, mild hemoptysis, and exertional dyspnea

(Covalla et al. 1981). Subchronic toxicity evaluations support

concern about lung injury (McFadden and Maickel1985; Lynch et al.

1985).

Burn Injuries

Aikyl nitrites are flammable and explosive. At least one bum injury

has been reported through the National injury Information Clearing

House (NEISS data base) of the Consumer Product Safety Commission

(CPSC), following the use of a room odorizer product near a candle.

“This incident involved a 26- yearold male who opened a small bottle

on a stand when it ignited, flamed up, and burned him. During this

accident, the victim spilled some of the gymnasium room odorizer

onto parts of the living room furnishings. As a result, part of an

ottoman and small sections of the carpet were damaged. lt is

believed that the liquid gymnasium room odorizer ignited, as its

vapors came into contact with a lighted cigarette in a nearby ash

tray. The victim received bums on the left side of his nose and an

area of his left cheek. The victim was also singed on his right arm,

forehead and some parts of his hair” (CPSC memo from Schmettzer to

Perez, July 11, 1977).

The materials are labeled with a warning of this hazard, which Is

not to be underestimated; small quantities have been responsible for

refrigerator explosions in laboratories, and larger quantities have

been implicated in the largest fire in San Francisco since the

earthquake of 1998 (Turner 1981). Despite these hazards, many of

the products packaged in small vials with shrink-fit plastic comply

with U.S. Postal Service regulations for the shipment of small

quantities of flammable and explosive materials.

29
Acute Toxicity

Whether inhaled or swallowed, nitrites can produce anoxic states.

The administration of nitrites by any route can produce profound

methemoglobinemia. Tissue anoxemia can result from

methemoglobinemia (Darling and Roughton 1942; Lester and

Greenburg 1944). Ascorbic acid and methylene blue may be effective

prophylactic agents for nitrite poisoning if administered promptly, but

laboratory studies offer little support for this in the case of isobutyl

nitrite ingestion (McFadden and Maickel 1982). Prolonged

administration of the materials may lead to several disorders of the

Mood and blood-forming organs, including Heinz-body hemolytic

anemia and splenomegaly (Romeril and Concannon 1981); increased

spleen weight has been observed in mice chronically exposed to butyl

nitrites (McFadden and Maickel 1985). The administration of nitrites

can also produce hypokinetic anoxia, an oxygen starvation of vital

organs secondary to sustained profound peripheral vasodilation,

pooling of Mood in the extremities, and impaired vascular return

(Wilkins et al. 1937).

A number of human anecdotes report on the acute toxicity of

volatile nitrite “room odorizer” products and provide correlative

observations that substantiate concern aroused by experimental work

with laboratory animals. Severe methemoglobinemia can result from

deliberate inhalation of room odorizer products (Home et al. 1979;

Shesser et al. 1981; Guss et al. 1985). Ingestion can produce a more

rapid and malignant methemoglobinemia than can inhalation and can

be lethal (Dixon et al. 1981; Shesser et al. 1980; Shesser et al. 1981;

Smith et al. 1980; Wason et al. 1980).

The acute toxicity of the alkyl nitrites has received attention in the

laboratory and is relevant to the regulation of these materials in

interstate commerce, including packaging and labeling requirements.

A summary of these experimental findings is presented in table 1.

Room odorizer products are currently considered to be “toxic” by

inhalation or ingestion, although the oral toxicity of the material has

not been evaluated adequately according to the CPSC. The materials

display an unusually steep lethality function so that, in the effective

range, small increments in dose produce large changes in the number

of resultant deaths (Wood and Cox 1981; Klonne et al. 1987). In

addition, there is a very narrow margin of safety between

behaviorally effective and lethal concentrations (Rees et al. 1986).

30
Users estimate they can derive 40 “doses” from a typical room

odorizer, yielding approximately 0.2 ml per self-administration

(lsraelstam et al. 1978). This estimate is neither a dose nor an

exposure concentration, but a rate of loss of a volatile material from

an open container. There have been no studies that describe either

the absorbed dose or the exposure concentrations that are effective

in producing smooth muscle relaxation, alterations in cardiovascular

function, behavioral impairment, or self-administration under the brief

exposure conditions typical of self-administration. However, Pryor et

al. (1989) did expose rats to isobutyl nitrite for 15 to 60 seconds, at

concentrations that increased gradually in the exposure chamber. The

LC50s expressed as a peak concentration ranged from 4.5 to 4.8

percent. The slope of the lethality function was influenced by the

rate of change of concentration. A constant concentration-time

product relationship was not obtained in this study, or in that of

Klonne et al. (1987), suggesting that the dramatic acute effects of

the agent may alter the pharmacokinetics and exaggerate the toxicity

of these agents.

For some of the aliphatic nitrites, the alcohol from which they are

synthesized may contribute significantly to their toxicity when

injected or ingested. In addition, the alkyl nitrites are metabolized

to nitrite ions and the corresponding alcohol, which may lead to

delayed deaths or hepatotoxicity. n-Butyl alcohol has an oral

median lethal dose (LD50) for the male rat of 790 mg/kg (Purchase

1969), in contrast to 13,600 mg/kg for ethanol (Smyth et al. 1941).

In an investigation of several highly pure butyi nitrites and their

alcohols given to mice intraperitoneally, set- and tert-butyl nitrites

were found to have significant delayed toxicity; the toxicity of the

alcohols by the same route was also delayed, and the LD50 ranged

from 254 (n-butyl) to 544 mg/kg (isobutyl) 7 days after

administration (Maickel and McFadden 1979).

SUBSTANCE ABUSE DISORDERS

There is clear evidence that volatile nitrites are used as drugs of

abuse. National surveys indicate that high school seniors and adults

not only have used alkyl nitrites as drugs, but also that 7.9 to 11.1

percent of high school seniors from 1979 to 1985 reported having

tried these drugs in their lifetime (Johnston et al. 1986). The

incidence of deliberate use by homosexual men has been greater and

is discussed in more detail shortly. Lowry (1980) conservatively

estimated that 250 million recreational doses a year were consumed

in the United States (Lowry 1982).

31
Volatile nitrites are persistently self-administered by people.

lsraelstam et al. (1978) interviewed 150 users of isobutyl nitrite and

reported that users who administered nitrites did so from three to

six times per “occasion”; the number of occasions ranged from only

once to four times a week. The duration that this frequency of selfadministration was maintained was unspecified. Goedert et al. (1982),

in an attempt to determine if volatile nitrites alter immune function,

studied 17 men; 10 were described as regular users, inhaling nitrites

from 1 to 20 times per month; 4 had used nitrites for longer than 6

years. Romeril and Concannon (1981) presented two cases, each of

which reported 20 sniffs per occasion, two to three times per week,

for either 3 or 24 months. Fisher et al. (1981) reported on two men

who stated that they were “in the habit” of inhaling butyl nitrite and

continued to do so during the 6-week period that they were seen by

a physician.

According to the nosological scheme of the Diagnostic and Statistical

Manual (DSM Ill) of the American Psychiatric Association, one

definitional criterion of “substance abuse” is a duration of

pathological use for at least 1 month; several of the cases described

above meet this criterion. Other than the duration of use,

definitional criteria for “pathological use” vary with the substance

abused but may include episodes of complication due to substance

intoxication, e.g., alcoholic blackouts, opioid overdose; need for daily

use of the substance for adequate functioning; and continuation of

substance use despite a serious physical disorder that the individual

knows is exacerbated by use of the substance. Examples from the

literature fulfilling these criteria follow.

Compications Due to Substance Intoxication

Shesser et al. (1981) report a hospital admission following several

hours of continuous inhalation of an isobutyl nitrite preparation.

The patient was alert and combative, and the parents sought to have

the patient admitted to the hospital because of mental status change

and cyanosis. The patient admitted to having had several drinks of

alcohol. Treatment of the patient with oxygen and intravenous

methylene blue and ascorbic acid resulted in alleviation of the

cyanosis; with the patient becoming calmer and reoriented. Covalla

et al. (1981) report a hospital admission for severe tracheobronchitis

following the.patient’s inhalation, with a friend, of two bottles of

LockerRoom in a week’s time. Hi friend developed a similar but less

severe illness. The cases in the following paragraph also constitute

complications of substance intoxication.

32
Continuation of Substance Use Despite a Serious Physical Disorder

Exacerbated by Use of the Substance

Home et al. (1979) report emergency room admission on two

separate occasions of a 25year-old man after he inhaled butyl

nitrite. The occurrence of clinically significant methemoglobinemia

was not sufficient to deter self-administration in this individual. In

a survey of 255 experienced users (Lowry 1979), 10 percent had

experienced nasal irritation at least once, and 5 percent had

experienced nausea or temporary loss of erection. These negatlve

effects were associated with “overuse” (emphasis Lowry’s). Fisher et

al. (1981) report several cases of facial dermatitis due to butyl

nitrite inhalation. Two of the three cases reported that they were

“in the habit of inhaling butyl nitrite,” and continued to do so

during the 6-week period in which the dermatitis was evident. The

skin lesions cleared when nitrite use was terminated.

Need for Daily Use of the Substance for Adequate Functioning

Sigell et al. (1978) interviewed an unspecified number of users, some

of whom claimed that they were no longer able to perform sexually

without the use of these drugs. Everett (1975) has made a similar

assertion.

CONCLUSION

Research funded by the National Institute on Drug Abuse (NIDA) has

demonstrated that drugs abused repetitively by humans are selfadministered by animals; drugs that are abused sporadically by

humans (most hallucinogens), or not at all (major tranquilizers), are

not taken by laboratory animals. Thus, there is a pharmacologic sine

qua non for the ability of drugs to maintain self-administration: in

the absence of intrinsic abuse potential, humans will not persistently

abuse a drug. Although there have been no laboratory investigations

using animal self administration preparations, volatile nitrites have

abuse potential because they would not generate persistent human

self-administration in its absence.

The extrapharmacologic determinants of substance abuse are of great

importance; in this case, the pattern of distribution, availability, and

promotion of materials in commerce play predominant roles in

elevating the abuse liability of the volatile nitrites. The pattern of

distribution of volatile nitrites is typical of a free market with access

to the mails and the right to advertise. The interruption of this

33
pattern of distribution would reduce the abuse liability of these

agents.

TABLE 1. Acute toxicity of akyl nitrites

Compound Species LD5 0 /LC5 0 Reference

Oral

n-Butyl Rat 83 mg/kg Wood and Cox 1981

(79.5-86.5) Federal Hazardous

Substances Act protocol

Ethanol vehicle

(50 mg/kg="highly toxic")

n-Butyl Mouse 180 mg/kg McFadden and Maickel 1982

(0-288) (2-hour LD50)

sec-Butyl 428 mg/kg

(358-469)

tert-Butyl 336 mg/kg

(292-447)

Isobutyl 279 mg/kg

(0-610)

n-Butyl Mouse 171 mg/kg McFadden and Maickel 1982

(27-249) (7-day LD50)

sec-Butyl 423 mg/kg

(393-456)

tert-Butyl 308 mg/kg

(220-426)

Isobutyl 205 mg/kg

(5-311)

Intraperitoneal

Amyl Mouse 130 mg/kg Dewey et al. 1973

(111-152)

n-Butyl Mouse 158 mg/kg Mcfadden and Maickel 1979

(127-197) (30-minute LD50)

sec-Butyl 592 mg/kg

(476-734)

tert-Butyl 625 mg/kg

(520-750)

Isobutyl 169 mg/kg

(139-199)

Intravenous

Amyl Mouse 51 mg/kg Dewey et al. 1973

(38-68) (24-hours LD50)

34
TABLE 1. (Continued)

Compound Species LD5 0 /LC5 0 Reference

Inhalation

Isbutyl Mouse 1346 ppm Rees et al. 1986

(1219-1473) (30-minute exposures)

n-butyl 949 ppm

(897-1001)

Isoamyl 1430 ppm

(1302-1559)

Isobutyl Mouse 1033 ppm McFadden et al. 1981

(843-1234) (60-minute exposures)

n-butyl 567 ppm

(531-625)

sec-Butyl 1753 ppm

(1552-1964)

tert-Butyl 10852 ppm

(626-15408)

Methyl Rat 176 ppm Klonne et al. 1987

(169-183) (4-hour exposures)

Ethyl 160 ppm

(151-169)

n-Propyl 300 ppm

(293-308)

n-Butyl 4210 ppm

(410-431)

Isobutyl 777 ppm

(747-809)

Isopentyl 716 ppm

(Isoamyl) (702-731)

n-Butyl Rat 1470 ppm CPSC memo fro Perez to

(1226-1823) Preuss, September 24, 1979

Isobutyl 1000 ppm Federal Hazardous

(815-1255) Substances Act protocol

Isoamyl Rat 1118 ppm (1-hour exposures)

(797-1493)

Note: LD50=Median lethal dose (lethal to 50 percent of test subjects).

LC50=Lethal concentration, 50 percent.

35
REFERENCES

Bos, J.D.; Jansen, F.C.; and Timmer, J.G. Allergic contact

dermatitis to amyl nitrite (“poppers”). Contact Dermetitis

12(2):109, 1985.

Covalla, J.R.; Strimlan, C.V.; and Lech, J.G. Severe

tracheobronchitis from inhalation of an isobutyl nitrite

preparation. Drug lntell Clin Pham, 15(1):51-52, 1981.

Darling, R.C., and Roughton, F.J.W. The effect of methemoglobin

on the equilibrium between oxygen and hemoglobin. Am J Physiol

137:56-68, 1942.

Dewey, W.L; Tucker, LS.; Prange, A; Spaulding, T.; and Chau, T.T.

Some behavioral and toxicological effects of amyl nitrite. Res

Commun Chem Pathol Pharmacol 5(3):889-892, 1973.

Dixon, D.S.; Reisch, R.F.; and Santinga, P.H. Fatal

methemoglobinemia resulting from ingestion of isobutyl nitrite, a

“room odorizer” widely used for recreational purposes. J Forensic

Sci 26(3):587-593, 1981.

Everett, G.M. Amyl nitrite (“poppers”) as an aphrodisiac. In:

Sandler, M., and Gessa, G.L., eds. Sexual Behavior

Pharmacology and Biochemistry. New York: Raven Press, 1975.

pp. 97-98.

Fisher, A.A. “Poppers” or “snappers” dermatitis in homosexual men.

Cutis 34(2):118-122, 1984.

Fisher, A.A.; Brancaccio, R.R.; and Jelinek, J.E. Facial dermatitis in

men due to inhalation of butyl nitrite. Cutis 27(2):146, 152-153,

1981.

Goedert, J.J.; Neuland, C.Y.; Wallen, W.C.; Greene, M.H.; Mann, D.L;

Murray, C.; Strong, D.M.; Fraumeni, J.F., Jr.: and Blattner, W.A.

Amyl nitrite may alter T lymphocytes in homosexual men. Lancet

1982-1:412-416, 1982.

Guss, D.A; Normann, S.A.; and Manoguerra, A.S. Clinically

significant methemoglobinemia from inhalation of isobutyl nitrite.

Am J Emerg Med 3(1):46-47, 1985.

Haley, T.J. Review of the physiological effects of amyl, butyl, and

isobutyl nitrites. Clin Toxicol 16(3):317-329, 1980.

Horne, M.K 3d.; Waterman, M.R.; Simon, L.M.; Garriott, J.C.; and

Foerster, E.H. Methemoglobinemia from sniffing butyl nitrite.

Ann lntern Med 91(3):417-418, 1979.

Israelstarn, S.; Lambert, S.; and Oki, G. Use of isobutyl nitrite as

a recreational drug. Br J Addict 73(3):319-320, 1978.

36
Johnston, L.D.; O’Malley, P.M.; and Bachman, J.G. Drug Use Among

American High School Students, College Students, and Other

Young Adults. National Trends Through 1985. National lnstitute

on Drug Abuse. DHHS Pub. No. (ADM) 86-1450. Washington, DC:

Supt. of Docs., U.S. Govt. Print. Off., 1986. 237 pp.

Klonne, D.R.; Ulrich, C.E.; Weissman, J.; and Morgan, AK Acute

inhalation toxicity of aliphatic (Cl-C5 nitrites in rats. Fundam

Appl Toxicol 8:101-106, 1987.

Lester, D., and Greenburg, L.A. The comparative anoxemic effects

from carbon monoxide and methemoglobin. J Pharmacol Exp Ther

81:182-188, 1944.

Lowry, T.P. The volatile nitrites as sexual drugs: A user survey.

J Sex Educ Ther 5:8-10, 1979.

Lowry, T.P. Neurophysiological aspects of amyl nitrite. J

Psychedelic Drugs 12:73-74, 1980

Lowry, T.P. Psychosexual aspects of the volatile nitrites. J

Psychoactive Drugs 14:77-79, 1982.

Lynch, D.W.; Moorman, W.J.; Burg, J.R.; Phipps, F.C.; Lewis, T.R.;

Khan, A; Lewis, D.M.; Chandler, F.W.; Kimbrough, R.D.; and

Spira, T.J. Subchronic inhalation toxicity of lsobutyl nitrite in

BALB/c mice. I. Systemic toxicity. J Taxicol Environ Health

15:823-833, 1985.

Maickel, R.P., and McFadden, D.P. Acute toxicology of butyl

nitrites and butyl alcohols. Res Commun Chem Pathol Pharmacol

28(1):75-83, 1979.

McFadden, D.P.; Carlson, G.P.; and Maickel, R.P. The role of

methemoglobin in acute butyl nitrite toxicity in mice. Fundam

Appl Toxicol 1(6):448-451, 1981.

McFadden, D.P., and Maickel, R.P. Comparative toxicology of butyl

nitrites in mice. Toxicol Appl Pharmocol 48:A43, 1979.

McFadden, D.P., and Maickel, R.P. Butyl nitrttes-an example of

hazardous, noncontrolled recreational drugs. Res Commun Subst

Abuse 3(2):223-236, 1982.

McFadden, D.P., and Maickel, R.P. Subchronic toxicology of butyl

nitrites in mice by inhalation. J Appl Toxicol 5(3):134-139, 1985.

Pryor, G.T.; Howd, R.A; Bingham, L.R.; Rebert, C.S.; and Jensen,

R.A Biomedical studies on the effects of abused inhalant

mixtures. SRI International Project LSU-5956. Final Report.

National Institute on Drug Abuse Contract No. 271-77-3402.

February 14, 1980. pp. 3659.

Purchase, I.F.H. Studies in kaffircom malting and brewing. XXII.

The acute toxicity of some fusel oils found in Bantu beer. SuidAfr Tydskr Geneesk 53:795-798, 1969.

37
Rees, D.C.; Coggeshall, E.M.; Dragan, Y.; Breen, T.J.; and Balster,

R.L Acute effects of some volatile nitrites on motor

performance and lethality in mice. Neurobehav Toxicol Teratol

8:139-142, 1986.

Romaguera, C., and Grimalt, F. Contact dermatitis from nasal

sprays and amyl nitrite. Contact Dermatitis 8(4):286-267, 1982.

Romeril, K.R., and Concannon, A.J. Heinz body haemolytic anaemia

after sniffing volatile nitrites. Med J Aust 1(6):302-303, 1981.

Shesser, R.; Dixon, D.; Allen, Y.; Mitchell, J.; and Edelstein, S.

Fatal methemoglobinemia from butyl nitrite ingestion. Ann

Intern Med 92(1):131-132, 1980.

Shesser, R.; Mitchell, J.; and Edelstein, S. Methemoglobinemia from

isobutyl nitrite preparations. Ann Emerg Med 10(5):262-264, 1981.

Sigell, LT.: Kapp, F.T.; Fusaro, GA.; Nelson, E.D.; and Falck, R.S.

Popping and snorting volatile nitrites: A current fad for getting

high. Am J Psychiatry 135(10):1216-1218, 1978.

Smith, M.; Stair, T.; and Rolnick, M.A. Butyl nitrite and a suicide

attempt. Ann Intern Med 92(5):714-720, 1980.

Smyth, H.F.; Seaton, J.; and Fischer, L. The single dose toxicity of

some glycols and derivatives. J Ind Hyg Toxicol 23:259-268, 1941.

Turner, W. San Francisco fire worst since ‘06: Razes 25 buidings.

New York Times, July 10, 198l.

Wason, S.; Detsky, A.S.; Platt, O.S.; and Lovejoy, F.H., Jr.

lsobutyl nitrite toxicicol by ingestion. Ann lntern Med 92(5):637-

638, 1980.

Wilkins, R.W.; Haynes, F.W.; and Weiss, S. The role of the venous

system in circulatory collapse induced by sodium nitrite. J Clin

Invest 16:85-91, 1937.

Wood, R.W., and Cox, C. Acute oral toxicity of butyl nitrite. J

Appl Taxicol 1(1):30-31, 1981.

ACKNOWLEDGEMENTS

This work was supported by grants DA04438 and KOZ-DA0017 from

the National Institute on Drug Abuse.

AUTHOR

Ronald W. Wood, Ph.D.

Research Associate Professor

Department of Environmental Medicine

New York University Medical Center

550 First Avenue

New York, NY 10016

38
Indications From Animal and

Chemical Experiments of a

Carcinogenic Role for IsoButyl Nitrite

Sidney S. Mirvish, Martin O. Ramm, and

David M. Babcook

INTRODUCTlON

We have been studying the in vivo formation of carcinogenic

nitrosamines and nitrosamides (which together constitute the highly

carcinogenic N-nitroso compounds) after exposure of laboratory

animals to the atmospheric pollutant nitrogen dioxide (NO2). In the

course of those experiments, we found that the nitrite ester of

cholesterol participated in the formation of nitrosamines. This

finding is consistent with suggestions that simple nitrite esters may

form similar carcinogenic compounds in man. In this chapter we

describe experiments evaluating the carcinogenic potential of amyl

nitrite and isobutyl nitrite (IBN), a major component of abused room

odorizers, and speculate on the possible relationship between nitrite

ester abuse and the development of Kaposi’s sarcoma (KS) in patients

with Acquired lmmunodeficiency Syndrome (AIDS). We first

summarize our data on NO2 and describe the nitrite ester studies, and

then discuss the possible connections between KS and nitrite ester

abuse.

NITROSAMINE FORMATION DUE TO EXPOSURE TO NO2

In chemical systems, NO2 reacts with amines to form nitrosamines.

On this basis we studied whether the same reactions could occur in

vivo. We exposed rats to NO2 in air (50 ppm for 4 hours), gavaged

them with the secondary amine morpholine, and later analyzed the

carcasses for the corresponding nitrosamine, N-nitrosomorpholine

(NMOR). The final stage of the analysis involved gas chromatography

of the NMOR, with detection by Thermal Energy Analysis (GC-TEA),

a system that can detect as little as 0.1 ng nitrosamine. While no

NMOR was detected, the carcasses did contain a “nitrosating agent”

39
that reacted with morpholine in dichloromethane or ether solution to

produce NMOR in vitro (Mirvish et al. 1981). All subsequent studies

were done on Swiss mice. The nitrosating agent was soluble in lipids

and occurred only in the skin; most of it persisted in the body for 24

hours after the NO2 exposure (Mirvish et al. 1983). The nitrosating

agent was shown to be produced by direct contact of NO2 with the

skin. Later, these findings provided some ideas about the possible

route of exposure to ISN vapor.

To identify the nitrosating agent derived from NO2 we examined the

nature of the precursor in skin lipids that reacted with NO2 to

produce this agent. About 40 percent of the precursor was associated

with the triglyceride fraction and 60 percent was identified as

cholesterol (Mirvish et al. 1986). These findings were discovered by

separation of the lipids on thin-layer and high-performance liquid

chromatography, with analysts of each fraction for a nitrosating agent

precursor. Cholesterol reacted with NO2 to produce the known

nitrite ester, cholesteryl-3-B-nitrite. Synthetic cholesteryl nitrite

reacted with morpholine by the scheme shown below to give 13

percent yields of NMOR. We showed that the cholesterol-derived

nitrosating agent in mouse skin was almost certainly cholesteryl

nitrite. These findings agreed with previous studies showing that

nitrite esters (including IBN) react fairly readily with secondary

amines to form nitrosamines (Doyle et al. 1983; Dabora et al. 1984;

Loeppky et al. 1984).

RONO + R'2NH -> ROH + R'2NNO

Thus far, we have described in vitro reactions of the N02derived

nitrosating agent to form nitrosamines. To be significant, these

reactions must also be shown to occur in vivo. To test for this, we

applied one of the secondary amines, morpholine or N-methylaniline,

to the skin of mice 20 hours after they were exposed to 50 ppm NO2

in air for 4 hours. After another 0.5 to 2.0 hours, the mice were

killed and the skins were analyzed for NMOR or N-nitroso-Nmethylaniline (NMA) by GC-TEA. We did not observe significant

NMOR, but did detect NMA, with maximum values of 18±7 nmol/mouse

(mean ± SD) for 6 mice killed 40 minutes after the amine was applied

(Mirvish and Ramm 1987). In a similar experiment, the mice were

shaved 30 minutes after methylaniline was painted, and the hair and

shaved skin were analyzed separately for NMA. The hair contained

87 percent of the NMA. This demonstrates the importance of direct

access of NO2 to the skin lipids, which should be more facile for

lipids in hair than for those in the skin itself. We attribute the

40
formation of NMA, but not NMOR, mainly to the lipid solubility of

N-methylaniline, which enables this amine to react with the skin

nitrosating agent (Morpholine is not extractable by lipids at neutral

pH.) The analyses included checks to ensure that the nitrosamines

were not produced during the workup.

Lipid Peroxidation By Cholesteryl Nitrite

Nitrite esters (but not nitrate esters) absorb ultraviolet light

between 320 and 380 nm, which helps explain why nitrite esters are

decomposed by light or heat to yield alkoxy radicals and nitric oxide

(Smith 1988):

light

RON0 -> RO + NO

This reaction proceeds readily, with a free energy of 37 kcal/mol

(Gingras and Waters 1954). For this reason, methyl nitrite is used to

initiate radical reactions in studies on atmospheric pollution

(Magalhaes and Chalk 1986). lt struck us that such a reaction of

cholesteryl nitrite could be biologically significant, because radical

production is closely linked with the promotion stage of carcinogenesis (Kensler and Taffe 1986). Lipid peroxidation is a measure of

radical production In the lipid phase of tissues and also shows

associations with tumor promotion. For these reasons, we tested

whether cholesteryl nitrite could catalyze the peroxidation of methyl

linoleate, used as a model for unsaturated lipids (Mirvish et al.

1987).

One gram of methyl linoleate, with and without the addiiion of 50

mg cholesteryl nitrite, was allowed to autoxidize in a sealed

dessicator (20 cm in diameter) for up to 7 days. To measure

autoxidation, we determined conjugated dienes end compounds (malondialdehyde plus endoperoxides) that react with thiobarbituric acid

(TBA) (Buege and Aust 1978).

Table 1 shows that cholesteryl nitrite significantly increased the

production of both conjugated dienes and TBA-reacting substances.

In a preliminary experiment, painting a cholesteryl nitrite solution

(12.5 mg/250 I acetone) once on the skins of mice produced a

significant increase after 1 to 2 days in TBA-reacting material in the

skin lipids (4.5 ± 0.2 (four mice, mean SE) compared to 0.1 ± 0

(two mice) in mice painted with acetone alone). As explained earlier,

41
TABLE 1. Effect of the nitrite esters cholesteryl nitrite and IBN on autoxidation of methyl linoleatea

Yield of peroxidized material (nmol/mg methyl linoleate,

mean ± SD)

Exposure Number Conjugated diene test TBA test

Nitrite time o f Ester Ester Ester Ester

ester (days) exposures absent present absent present

Cholesteryl

nitrite

IBN

a

lBN results are based on weight of methyl linoleate at the beginning of the experiment. because some IBN distilled into the

linoleate. Cholesteryl nitrite results are based on weights at the time of measurement.

b

*Results were sign’ificantly greater than those for corresponding samples without addition of nittrite ester, with p<0.05b or <0.01c.
these results suggest that cholesteryl nitrite could promote carcinogenesis in mouse skin.

In Vivo Formation of the Nitrosamine NMA from Amyl Nitrite

This and the following section describe our studies on amyl nitrite

and IBN, which were based on our findings about cholesteryl nitrite.

The studies in this section demonstrated that amyl nitrite can react

to produce a nitrosamine in vivo (Mirvish et al., in press; Mirvish and

Ramm 1987).

As in the studies on skin NSA, N-methylaniline was chosen as the

test amine, mainly because it is lipid-extractable from water at

neutral pH and, hence, could react with the lipid-soluble nitrite ester,

amyl nitrite. Adult male Swiss mice were treated with methylaniline

and amyl nitrite, with each agent delivered to a different site in the

body. In the main experiment, methylaniline (250 mg/ml water/kg

body weight) was injected IP and, 30 minutes later, amyl nitrite (40

mg/ml corn oil/kg body weight) was gavaged. After another 30 or 60

minutes, the mice were killed and the carcasses were analyzed for

NMA, using the methods described earlier. For six mice killed after

30 minutes, we obtained 480 ± 310 nmol NMA/mouse (mean ± SD).

Six mice killed after 60 minutes yielded 380 ± 100 nmol NMA/mouse.

The results at 30 minutes were 18 times higher than those for NMA

produced from the N02derived skin nitrosating agent.

In a different procedure, methylaniline in water was gavaged, amyl

nitrite in corn oil was injected IP 30 minutes later, and the mice

were killed after another 30 minutes. In four mice, the NMA yield

was only 6 ± 6 nmol/mouse. When amyl nitrite, but not methylaniline, was administered to four mice as in our main experiment, and

an amount of methylaniline similar to that injected IP was added to

each homogenate before the workup, NMA yield was only 5 ± 3

nmol/mouse, indicating that most NMA in the main experiment was

produced in vivo and not during the workup.

Four mice were treated as in the first experiment, with 30 minutes

elapsing between dosage with amyl nitrite and sacrifice, and certain

tissues were analyzed for NMA. These yielded 630 ± 140 (stomach

contents), 90 ± 2 (stomach wall), 9 ± 3 (liver), 5 ± 3 (intestines plus

contents), and 0.5 ± 0.3 (Mood) nmol NMAI/mouse. Hence, most NMA

was produced at the site of delivery of the amyl nitrite. These

results demonstrate that simple nitrite esters can produce large

yields of nitrosamine in vivo, if a lipid-soluble amine is supplied. In

43
humans, exposure to volatile nitrite esters could produce

carcinogenic N-nitroso compounds from endogenous or exogenous

amines, or, perhaps, from N-substituted amides (which can be more

lipid-soluble than most amines).

In Vitro Enhancement of Lipid Peroxidation by IBN

We have described our finding that cholesteryl nitrite enhanced the

autoxidation of methyl linoleate. We have now repeated this

experiment using IBN. Since IBN is volatile, methyl linoleate (0.5 g

in an evaporating dish), was placed in a sealed glass tank (8 x 21 x

28 cm) also containing a beaker with 2 ml IBN. After 4 to 5 days,

some IBN had distilled into the linoleate and IBN in the beaker was

replenished. As a control, methyl linoleate was stored in a similar

tank without IBN. Table 1 shows that IBN significantly enhanced the

formation of lBA-reading material but (unlike cholesteryl nitrite) did

not affect (or even reduce) the level of conjugated dienes. This

indicates that IBN increased the prcduction of endperoxides, but not

that of hydrcperoxides.

In conclusion, the nitrosamine formation and lipid peroxidation

experiments suggest that IBN could be carcinogenic in man, either

because it produced carcinogenic N-nitroso compounds in vivo, or

because it enhanced lipid peroxidation in vitro and hence could be a

tumor promoter.

POSSIBLE ASSOClATlON BETWEEN EXPOSURE TO IBN AND KS IN

AIDS

At about the time we were studying cholesteryl nitrite, Newell et al.

(1985) proposed that IBN could produce N-nitroso compounds in vivo,

which in turn could potentiate the induction of AIDS or KS

associated with AIDS. After the role of human immunodeficiency

virus (HIV) in AIDS was discovered, emphasis shifted to the second

of these hypotheses.

About 15 to 25 percent of U.S. homosexual males with AIDS have

developed KS; this cancer is often the cause of death (Leu and

Odermatt 1985; Safai et al. 1985; Biggar et al. 1985). Heterosexual

AIDS patients have a much lower incidence of KS (Haverkos et al.

1985). KS also occurs in immunosuppressed patients who do not have

AIDS, but at far lower incidences than in homosexual AIDS patients

(Friedman-Kien et al. 1982). Hence, KS in AIDS appears to be

associated with the lifestyle of homosexual men in the United States.

44
Haverkos et al. (1985) reported a significant association of KS in

homosexual male AIDS patients with their exposure to IBN measured

as total days of use.

There is evidence both for and against nitrites being involved in the

genesis of AlDS associated KS. In addition to Involvement in the

formation of nitrosamines, IBN is mutagenic in the Ames test

(Osterloh and Goldfield 1984), and Dax (this volume) reports an

immunosuppressive action of IBN that could contribute to cancer

induction. However, three points argue against the involvement of

IBN in the etiology of AIDS associated KS. First, the increased

incidence of KS in heterosexual AIDS patients in Africa is unlikely to

be associated with nitrite abuse (Kestens et al. 1985; Marquart 1986).

Second, there is some suggestion that concurrent cytomegalovirus

infection is associated with KS in AIDS (Safai et al. 1985). Third,

amyl nitrite was used for many years to treat angina pectoris without

any evidence of a carcinogenic effect.

Possible Routes of Access of IBN to the Tissue of Origin for KS

KS is a cancer of the endothelium of blood vessels (Leu and

Odermatt 1985) or lymph vessels (Beckstead et al. 1985; Jones et al.

1986). The main sites of origin for KS in AIDS are the dermis of

the skin or (less often) the submucosa of the oral cavity (Safai et

al. 1985; Volberding 1986). Common sites in the skin for AIDSassociated KS are the upper body, arms, and head, including the tip

of the nose. The tumor is often multicentric in origin, illustrating

the potency of the etiologic agents (Safai et al. 1985).

We favor the hypothesis that, if IBN is involved, its route of access

is by direct contact to the skin or oral mucosa with airborne IBN

vapor. This could occur directly when IBN is sniffed, which would

explain the site at the tip of the nose, or by general exposure to IBN

vapor inhaled by the subject or by others in bars, bathhouses or

homes (Lauritzen and Wilson 1986). (These places often smell

strongly of IBN, as reported at this meeting.) Hence the location of

the sites for AIDS-associated KS support our hypothesis about direct

contact with IBN vapor. This idea was suggested by our finding that

exposure to NO2 (which, like IBN vapor, is a lipid-soluble gas) results

in nitrosating agent formation only in the skin.

Another possibilit y is that IBN vapors are absorbed by the lungs and

travel to the skin via the bloodstream. In support of this view,

sniffed IBN does reach the peripheral blood vessels, where it

45
produces vasodilation (Needleman et al. 1985), and the brain, where

it produces a “high” sensation. This view could be valid, even

though IBN may only persist in the body for about 1 minute after it

is sniffed (the time for which the flushing and high feeling persists).

Needs for Research on IBN in Laboratory Animals

Research on the action of IBN in experimental animals would serve

to establish more firmly whether IBN is a likely factor in the

etiology of KS, and perhaps B-cell lymphoma (Levine et al. 1984;

Biggar et at. 1985; Ziegler et al. 1 984), associated with AIDS.

Specifically, we propose that research should be undertaken: (1) to

determine whether IBN can be an initiator and/or a promoter of

carcinogenesis; (2) to determine the route of access of IBN to the

dermis; and (3) to establish the mechanisms of any carcinogenic

effects of IBN.

With respect to point (1), initiation of carcinogenesis is a single,

rapid event, probably involving a mutation of the DNA. Promotion of

carcinogenesis is a second stage and usually involves chronic

exposure to the active agent. In man, lBN could initiate carcinogenesis via the formation of N-nitroso compounds or by a direct

reaction with DNA, and HIV could promote KS development, probably

via its immunosuppressive action. Alternatively, HIV could initiate

and IBN could promote KS development, perhaps via its effect on

lipid peroxidation. A determination of whether IBN initiates or

promotes carcinogenesis would help epidemiologists decide whether to

estimate IBN exposure before or after infection with HIV.

Hence, we recommend carcinogenesis tests to determine whether IBN

is: (1) a complete carcinogen, (2) a promoter of carcinogenesis,

especially in the skin, and (3) a cocarcinogen with viruses, including,

if possible, HIV in chimpanzees and feline leukemia virus in cats.

These tests should be done with IBN administered systemically and by

exposure to its vapors.

We further recommend metabolic studies, which should also be

performed with IBN given systemically and by exposure to its vapors.

These studies will establish the conditions under which various

N-nitroso compounds and nonvolatile nitrite esters (which could be

long-lasting indirect sources of N-nitroso compounds) are produced

from IBN; determine the distribution and metabolism of IBN in the

body, including its conversion to inorganic nitrite (which is also a

vasodilator); determine whether tissue lipids can be peroxidized by

46
IBN; and test for DNA damage caused by IBN, including the induction

of single-strand breaks and base alkylation (since these types of

damage could result in tumor initiation).

REFERENCES

Beckstead, J.H.; Wood, C.; and Fletcher, V. Evidence for the origin

of Kaposi’s sarcoma in lymphatic endothelium. Am J Pathol

119:294-300, 1985.

Biggar, R.J.; Horm, J.; Lubin, J.H.; Goedert, J.J.; Greene, M.H.; and

Fraumeni, J.F. Cancer trends in a population at risk of acquired

immunodeficiency syndrome. J Natl Cancer lnst 74:793-797, 1985.

Buege, J.A., and Aust, S.D. Microsomal lipid peroxidation. Methods

Enzymol 52:302-310, 1978.

Dabora, R.; Molina, M.; NG, V.; Wishnok, J.S.; and Tannenbaum, S.R.

Nitrosation by alkyl nitrites: Catalysis by inorganic salts. In:

O’Neili; I.K.; von Borstel, R.C.; Miller, C.T.; Long, J.; and Bartsch,

H., eds. N-Nitros Compounds: Occurrence, Biological Effects and

Relevance to Human Cancer. IARC Sci. Pub. No. 57. Lyon,

France: International Agency for Research on Cancer, 1984. pp.

311-316.

Doyle, M.P.; Terpstra, J.W.; Pickering, R.A.; and LePoire, D.M.

Hydrolysis, nitrosyl exchange, and synthesis of alkyl nitrites. J

Org Chem 48:3379-3382, 1983.

Friedman-Kien, A.E.; Laubenstein, L.J.; Rubinstein, P.; BuimoviciKlein, E.; Marmor, M.; Stahl, R.; Spigland, I.; Kwang, S.K.; and

Zolla-Pazner, S. Disseminated Kaposi’s sarcoma in homosexual men.

Ann Intern Med 96:693-700, 1982.

Gingras, B.A., and Waters, WA. The thermal decomposition of octyl

nitrite in hydrocarbon solvents. J Chem Soc:3508-3511, 1954.

Haverkos, H.W.; Pinsky, P.F.; Drotman, D.P.; and Bregman, D.J.

Disease manifestation among homosexual men with acquired

immunodeficiency syndrome: A possible role of nitrites in Kaposi’s

sarcoma. Sex Transm Dis 12:203-208, 1985.

Jones, R.R.; Spaull, J.; Spry, C.; and Jones, E.W. Histogenesis of

Kaposi’s sarcoma in patients with and without acquired immune

deficiency syndrome (AIDS). J Clin Pathol 39:742-749, 1986.

Kensler, T.W., and Taffe, B.G. Free radicals in tumor promotion.

Adv Free Radical Biol Med 2:347-387, 1986.

Kestens, L; Melbye, M.; Biggar, R.J.; Stevens, W.J.; Piot, P.; De

Muynck, A.; Taelman, H.; De Feyter, M.; Paluku, L; and Gigase,

P.L Endemic African Kaposi’s sarcoma is not associated with

immunodeficiency. Int J Cancer 36:49-54, 1985.

47
Lauritzen, J., and Wilson, H. Death Rush: Poppers and AIDS. New

York: Pagan Press, 1988. 84 pp.

Leu, H.F., and Odermatt, B. Multicentric angiosarcoma (Kaposi’s

sarcoma). Virchows Arch 408:29-41, 1985.

Levine, AM.; Meyer, P.R.; Begandy, M.K; Parker, J.W.; Taylor, C.R.;

Irwin, L; and Lukes, R.J. Development of B-cell lymphoma in

homosexual men. Ann Intern Med 100:7-13, 1984.

Loeppky, R.N.; Tomasik , W.; and Millard, T.G. Ester-mediated

nitrosamine formation from nitrite and secondary or tertiary

amines. In: O'Neill, I.K; Von Borstel, R.C.; Miller, C.T.; Long, J.;

and Bartsch, H., eds. N-Nitroso Compound: Occurence, Biological

Effects and Relevance to Human Cancer. IARC Sci. Pub. No. 57.

Lyon, France: International Agency for Research on Cancer, 1984.

pp. 353-364.

Magalhaes, A.M.T., and Chalk, P.M. Determination of the purity of

methyl nitrite. Anal Chim Acta 182:251-255, 1986.

Marquart, KH. AIDS associated Kaposi’s sarcoma in Africa. Br Med

J 292:484, 1986.

Mirvish, S.S.; Babcook, D.M.; Deshpande, AD.; and Nagel, D.L

Identification of cholesterol as a mouse skin lipid that reacts with

nitrogen dioxide to yield a nitrosating agent (NSA), and of

cholesteryl nitrite as the NSA produced in a chemical system from

cholesterol. Cancer Lett 31:97-104, 1988.

Mirvish, S.S.; lssenberg, P.; and Sams, J.P. A study of N-nitrosomorpholine synthesis in rodents exposed to nitrogen dioxide and

morpholine. In: Scanlan, R.A., and Tannenbaum, S., eds. NNitroso Compounds, ACS Symposium Series 174. Washington, DC

American Chemical Society, 1981. pp. 181-191.

Mirvish, S.S., and Ramm, M.D. Demonstration of in vivo formation of

the nitrosamine N-nitroso-N-methylanlline from amyl nitrite.

Cancer Lett 38:125-129, 1987.

Mirvish, S.S.; Ramm, M.D.; Babcook, D.M.; Sams, J.P.; and Panigot,

M. Lipidic nitrosating agents produced from atmospheric nitrogen

dioxide and in vivo nitrosation by amyl nitrite. In: Bartch, H.;

O’Neill, I.E.; and Schulte-Hennann, R., eds. The Relevence of NNitroso Compounds to Human Cancer. lARC Sci. Pub. No. 841.

Lyon, France: International Agency for Research on Cancer, 1987.

pp. 315-318.

Mirvish, S.S.; Sams, J.P.; and lssenberg, P. The nitrosating agent in

mice exposed to nitrogen dioxide: Improved extraction method and

localization to the skin. Cancer Res 43:2550-2554, 1983.
Needleman, P.; Corr; P.B.; and Johnson, E.M. Drugs used for the

treatment of angina: Organic nitrates, calcium channel blockers,

and B-adrenergic antagonists. In: Gilman, AG.; Goodman, LS.;

Rail, T.W.; and Murad, F., eds. The Pharmcological Besis of

Theraputics. 7th ed. New York: MacMillan, 1985. pp. 806-826.

Newell, G.R.; Mansell, P.W.; Spitz, M.R.; Reuben, J.M.; and Hersh,

E.M. Volatile nitrites: Use and adverse effects related to the

current epidemic of the acquired immune deficiency syndrome. Am

J Med 78:811-816, 1985.

Osterloh, J., and Goldfield, D. Butyl nitrite transformation in vitro:

Chemical nitrosation reaction and mutagenesis. J Anal Taxicol

8:164-189, 1984.

Safai, B.; Johnson, KG.; Myskowski, P.L; Koziner, B.; Yang, S.Y.;

Cunningham-Rundles, S.; Godbold, J.H.; and Dupont, B. The natural

history of Kaposi’s sarcoma in the acquired immunodeficiency

syndrome. Ann Intern Med 103:744-750, 1985.

Smith, PAS. The Chemistry of Open-Chain Organic Nitrogen

Compounds. Vol. 2. New York: WA Benjamin, 1988. p. 488.

Volberding, PA Kaposi’s sarcoma and the acquired immunodeficiency syndrome. Med Clin N Am 70:665-675, 1988.

Ziegler, J.L; Beckstead, J.A; Volberding, PA.; Abrams, D.I.; Levine,

A.M.; Lukes, R.J.; Gill, P.S.; Burkes, R.L; Meyer, P.R.; Metroka,

C.E.; Mouradian, J.; Moore, A; Riggs, S.A; Butler, J.J.; Cabanillas,

F.C.; Hemh, E.; Newell, G.R.; Laubenstein, LJ.; Knowles, D.;

Odajnyk, C.; Raphael, B.; Koziner, B.; Urmacher, C.; and Clarkson,

B.D. Non-Hodgkin’s lymphoma in 90 homosexual men. N Engl J

Med 311:565-570, 1984.

ACNKOWLEDGMENTS

Research supported in part by grant ROl-CA32192 and core grant

CA-36727 from the National Institutes of Health.

AUTHORS

Sidney S. Mirvish, Ph.D.

Martin D. Ramm, B.S.

David M. Babcook, B.S.

The Eppley Institute for Research

in Cancer and Allied Diseases

University of Nebraska Medical Center

42nd and Dewey Avenue

Omaha, NE 88105

49
Toxicity of Inhaled lsobutyl

Nitrite in BALB/c Mice: Systemic

and lmmunotoxic Studies

Daniel M. Lewis and Dennis W. Lynch

INTRODUCTION

Initial epidemiologic studies of the outbreak of acquired immunodeficiency syndrome (AIDS) among homosexual men indicated that the

use of organic inhalant nitrites (poppers) such as amyl nitrite and

isobutyl nitrite (IBN) was a possible risk factor in the development of

this disease (Centers for Disease Control (CDC), 1982). Although

subsequent studies have shown that the human immunodeficiency virus

(HIV) is the etiological agent of AIDS (Popovich et al. 1984), the

possible role of inhalant nitrites as a cofactor in some of the

illnesses associated with this syndrome has not been excluded.

The use of inhalant nitrites has been reported to be associated with

altered ratios of T-helper and T-suppressor cells (Goedert et al.

1982), but other studies failed to confirm this observation (Kornfeld

et al. 1982). In vitro studies have been reported that show that

volatile nitrites can inhibit the responses of mononuclear cells (Hersh

et al. 1983; Jacobs et al. 1983), but the in vivo effects of these

compounds on the immune system have not yet been reported. The

pharmacological and toxicological properties of these compounds have

been reviewed (Haley 1980; Jackson 1979), and the reported effects

include dizziness, headache, tachycardia, syncope, hypotension,

increased intraocular pressure, and production of methemoglobinemia

(Bruckner and Peterson 1977). In addition, volatile nitrites have been

shown to be direct mutagens (Quinto 1980), and are highly flammable.

Thus, it is apparent that these chemicals are hazardous, but their

immunotoxic properties have not been fully evaluated.

The National Institute for Occupational Safety and Health (NIOSH),

as one of the Centers for Disease Control, was requested by the

CDC Task Force on AIDS Activity to perform an immunotoxicologic

50
evaluation of aliphatic nitrites utilizing the NIOSH High Risk

Exposure Chambers. IBN was selected as the test chemical primarily

because IBN could be obtained and maintained in a more chemically

pure state than amyl nitrite. IBN is used as an inhalant, and all

volatile nitrites are thought to have similar physiologic effects (Haley

1980). Mice were exposed to various concentrations of IBN and at

selected time intervals removed from the chamber and tested for

immunocompetency by a variety of techniques. Since there was a

general lack of toxicologic data on organic nitrites and on IBN in

particular, the subchronic inhalation toxicity of IBN was evaluated

concurrently with the immunotoxicity assessment. Exposures were

selected to mimic an occupational exposure setting in order to fill

this gap in the existing literature, rather than to conduct brief,

acute, high-level exposure to imitate exposure by nitrite abusers.

The results of that study are the subject of this report.

EXPERIMENTAL PROTOCOL AND METHODS

The exposure conditions and experimental protocol have been

described in detail previously (Lynch et al. 1985; Lewis et al. 1985).

Briefly, BALB/c mice were exposed to 300 ppm of IBN vapors in

inhalation chambers for 6.5 hours/day, 5 days/week, for up to 18

weeks. The IBN chamber concentrations were monitored hourly using

a Miran IA infrared analyzer (Foxboro-Wilkes, Norwalk, CT) and flow

rates adjusted to maintain exposure concentrations at the target

level. Individual body weights for exposed and control animals were

recorded weekly, and all mice were observed twice daily for clinical

signs, morbidity, and mortality. At selected time intervals, mice were

randomly selected and removed from the exposure chambers and killed

with an overdose of pentobarbital sodium. A blood sample was

obtained for hematological testing, and the spleen, liver, and thymus

removed and weighed. After weighing, the spleens were teased apart,

filtered through an 80-mesh wire screen, and the cell suspension

collected in Hanks balanced salts solution (HBSS). The cells were

washed in HBSS, counted, and divided into three portions. One

portion was used to enumerate antibody-producing cells (Dresser

1978) (a measure of B-cell function), one portion was used for the

lymphocyte blast transformation assay (LBT) (Luster et al. 1981) (a

measure of T-cell function), and the third portion was used to

enumerate the T-cell subpopulations by immunofluorescence staining

using monoclonal antibodies specific for mouse Thy-1.2, Lyt-1 , and

Lyt-2 antigens.

51
A separate group of animals was used to evaluate cellular immune

reactivity in vivo by means of a radiometric skin test procedure

(Luster et al. 1981). Briefly, 10 IBN-exposed and 10 control animals

were immunized wlth Freund’s complete adjuvant, which contains

mywbacteria, 21 days prior to sacrifice. The animals remained in the

exposure chambers during this time. Two days prior to sacrifice they

were injected with tritiated thymidine (1.0 uCi/g body weight).

One day later the animals were skin tested by injection of 10μl of

PPD antigen into the right ear and 10μl of saline into the left ear.

Twenty-four hours after skin testing, the mice were killed, the ears

excised, and the radioactivity of each ear determined. The ratio of

iritated thymldine counts in the right and left ear was calculated to

provide an index of antigen-induced inflammation.

RESULT AND DISCUSSION

lmmunotoxic effects may be associated with a decline in the general

health of an animal. For thii reason, the body weights of the mice

were monitored weekly during the exposure period, and at

termination of the experiment. No significant differences in weight

gain were noted during the exposure period and, as shown in table

1, the terminal body weights were not significantly different. Also

shown in table 1 are selected organ weights for mice exposed to 300

ppm of IBN for 13 weeks. The liver weights of exposed males and

the thymus weights of exposed females were statistically

significantly less than the controls, but these changes could not be

related to any gross or histopathological damage in the organs.

Histopathologic examinations were made of a number of tissues of

these animals, and the only agent-dose-related change noted was

focal hyperplasia of the bronchiolar epithelium.

Hematological evaluations of the mice included red cell counts

(RBC), white cell counts (WBC), hemoglobin, and methemoglobin

determinations. In table 2, the results of these analyses are shown.

As expected, the exposure to IBN did induce methemoglobin

formation. The exposed males also showed lower WBC values than

the controls, but these changes were not related to any observed

changes in the bone marrow cytology. Depressed WBC counts were

not seen in mice exposed to lower concentrations of IBN, nor in the

females exposed to 300 ppm; the significance of this observation is

unclear.

Several assays were performed to evaluate the immune system of the

exposed animals. To evaluate the antibody-producing capabilities of

52
TABLE1. Organ and body weights in mice exposed to 300 ppm of

IBN for 13 weeks

Exposure Methemoglbin

Group RBC WBC Hemoglbin (numbe pos/

(millions/ml3) (millions/ml3

) (thousands/ml3

) (g/dl) number tested)

Males

Exposed 9.5±0.8 2.4±0.8* 15.5±1.3 5/5

Control 9.0±1.1 6.4±1.9 16.0±0.5 0/5

Females

Exposed 9.0±0.9 5.4±1.7 15.9±0.4 3/5

Control 9.6±1.1 5.4±3.0 16.3±1.1 0/5

Exposure

Group Body Wt. (g) Liver (g) Spleen (mg) Thymus (mg)

Males

Exposed 31.4 1.94* 197.6 38.9

Control 31.7 2.14 199.5 40.1

Females

Exposed 25.4 1.53 231.3 33.0*

Control 24.9 1.56 186.3 45.2

*Statistically significant differnece versus controls, p<.05

NOTE: There was 15 mice/sex/groups.

TABLE 2. Heamtologic data from mice exposed to 300 ppm IBN

for 13 weeks

*Statisticaly significant from control, P<.05.

the mice, selected animals were immunized with sheep red blood cells,

and plaque assays for antibody-producing spleen cells were performed.

lt should be noted that the mice continued to be exposed to IBN

between the time they were immunized and the time the assay was

performed (4 days). As shown in table 3, the female mice exposed to

300 ppm IBN for 18 weeks had significantly higher numbers of

antibody-producing cells than did the controls. At no other time

period or dose level were there any statistically significant

53
differences Between exposed and control animals. These results do

demonstrate that mice can develop an antibody response (humoral

response) while being exposed to IBN.

TABLE 3. Enumeration of antibody-poducing spleen cells in mice

exposed to 300 ppm IBN for 18 weeks

*Statisticaly significant from control, P<.01.

To evaluate the cellular immune response, mice were immunized and

tested for a delayed hypersensitivity response using a radiometric

skin test assay. Thii assay was only performed on animals that

had been exposed to 50 ppm IBN for 7 or 13 weeks.

Positive skin test responses were obtained with both the IBNexposed and control animals (table 4). The exposed animals had

slightly higher mean values, but the differences were not

statistically significant. The results demonstrate that the animals

can develop a cellular immune response while being exposed to IBN.

The cellular immune responses were further evaluated using an in

vitro assay. The LBT assay, which measures the proliferative

response of splenic lymphocytes to known mitogens, was performed;

results are shown in table 5. Each mitogen was tested at several

concentrations; the results shown are for the optimal concentration

of each mitogen. Only animals exposed to 300 ppm IBN for 18

weeks showed any significant differences in the LBT assay, and, at

that time point and for each mitogen, the exposed animals were

consistently more responsive than the controls. lt is of interest to

note that in the control cultures, i.e., those cultures that received

no mitogen and measured the background level of proliferation by

the splenic lymphocytes, the IBN-exposed animals had significantly

higher levels of thymidine incorporation. lt has been shown that

54

Plaque Counts

(Mean number of Plaque-Forming Cells per 10° Spleen Cells)

Animals Number Exposed Control Exposed/Control

x 100

Males 5 206±63 199±57 104

Females 5 334±73 129±35 259*

Total 1 0 270±93 164±58 165
TABLE 4. Radiometric determination of the skin test sensitivity

of mice exposed to 50 ppm IBN for 7 or 13 weeks

Ratio of Tritiated Thymidine per mg Tissue of Antigen-Injected Ear/Saline-Injected

Ear. Mean ± SE

Weeks of Exposure

Treatment Number 7 13

Exposed 10 2.98±0.51 2.49±0.69

Controls 10 1.77±0.15 2.19±0.39

TABLE 5. Results of the LBT assay performed on mice exposed to

300 ppm IBN for 18 weeks

Mitogen Number Exposed Controls

Phytohemagglutinin 10 67815 ± 9477* 40288 ± 6177

Concanavalin A 1 0 64096 ± 12948 43628 ± 6811

Pokeweed Mitogen 10 9570 ± 1885* 2887 ± 536

Lipopolysaccharide 10 15724 ± 2312* 5718 ± 884

Control (no mitogen) 10 4596 ± 998* 1422 ± 166

*Statistically significantly difference from control p<.01

NOTE: Results expressed as the mean + SE of cpm of trithted thymidine

incorporated for the group. Each animal was tested in replicates of four and

the mean value for each animal averaged to give the group moan.

organic nitrites can stimulate guanylate cyclase and increase cyclic

guanosine monophosphate (cGMP) levels (Ignarro et al. 1981). An

increase in cGMP levels occurs during the initiation of a

proliferative response by lymphocytes (Hadden and Coffey 1982).

Thus, it is possible that the chronic exposure to IBN may have

altered the responsiveness of the lymphocytes to the mitogens or

caused the increased background proliferative response seen by

affecting the cGMP metabolism. The biological significance of an

enhanced LBT response is unclear. Additional studies are needed to

determine if this response correlates with resistance to infection,

incidence of autoimmune phenomena, or possibly an increased

incidence of tumors.

55
One of the first immunologic abnormalities recognized in AIDS

patients was an alteration in ratio of Tcell subpopulations,

i.e., an inversion of the ratio of T-helper to T-suppressor cells. For

this reason, we determined the percentage of spleen cells expressing

either Thy 1.2, Lyt-1, or Lyt-2 surface antigens. The Thy-l antigen

is common to all mature T-cells in the mouse. The Lyt-2 antigen is

analogous to the CD-8 antigen in man (the suppressor/cytotoxic Tcell subset), and the Lyt-1 antigen is found predominantly on the

helper/inducer subset of T-cells in mice but is expressed at low levels

on all peripheral T-cells (Ledbetter et al. 1961). Using a fluorescent

microscope, we could not accurately enumerate the helper

subpopulation. Thus, the ratio of helper to suppressor cells could

not be calculated. As shown in table 6, there was no difference in

percentages of cells expressing these markers in the IBN-exposed

animals. The ratio of Lyt-2 to Thy-l cells was not different, which

indirectly indicates that there was no depletion of the helper cell

subpopulation.

TABLE 6, Relative number of spleen cells expressing Thy-l.2

Lyt-1 or Lyt-2 antigens in mice exposed to 300 ppm IBN

for 18 weeks

(Mean ± SD)

Percentae of Cells Expressing Ratio of

Treatment Group Thy 1.2 Lyt-1 Lyt-2 Lyt-2/Thy-1.2

Exposed (10) 31.4±3.62

34.2±5.1 19.8±2.8 0.634±0.085

Control (10) 35.5±3.5 38.6±5.1 21.8±3.1 0.616±0.092

CONCLUSION

The results obtained in this study indicate that, at the levels tested,

IBN had no significant detrimental effect on the immune system of

mice. The toxic effects noted included methemoglobinemia and focal

hyperplasia of the bronchiolar epithelium. Thus, while IBN may not

be an immunotoxicant, it was not totally innocuous in this study.

Numerous other toxic effects of this chemical have been reported by

others (Haley 1960; Jackson 1979; Brudner and Peterson 1977), and it

should be considered a hazardous substance. Finally, inhalant nitrites

may not be responsible for the basic immune defects characteristic of

AIDS, but their role as a cofactor in some of the illnesses associated

with this syndrome cannot be ruled out.

56
REFERENCES

Bruckner, J.V., and Peterson, R.G. Review of the aliphatic and

aromatic hydrocarbons. In: Sharp, C.W., and Brehm, M.L, eds.

Review of Inhalants: Euphora to Dysfunction. National lnstitute

on Drug Abuse Research Monograph 15. DHEW Pub. No. (ADM)

77-553. Washington, D.C.: U.S. Govt. Print. Off., 1977. pp. 126

163.

Centers for Disease Control. Epidemiological aspects of the current

outbreak of Kaposi’s sarcoma and opportunistic infectfons. N Engl

J Med 306:248-252, 1982.

Dresser, D.W. Assays of immunoglobulin-secreting cells. In: Weir,

D.M., ed. Handbook of Experimentel lmmunology. 3rd ed. Oxford:

Blackwell, 1978. pp. 28.5-28.6.

Goedert, J.J.; Wallen, W.C.; Mann, D.L; Strong, D.M.; Neuland, C.Y.;

Greene, M.H.; Murray, C.; Fraumeni, J.F.; and Blattner, WA. Amyl

nitrite may alter T-lymphocytes in homosexual men. Lancet 1:412-

416, 1982.

Hadden, J.W., and Coffey, R.G. Cyclic nucleotides in mitogeninduced lymphocyte proliferation. lmmund Today 3:299-364, 1982.

Haley, T.J. Review of the physiological effects of amyl, butyl, and

isobutyl nitrites. Clin Toxicol 16:317-329, 1960.

Hersh, E.M.; Reuben, E.M.; Bogerd, H.; Rosenblum, M.; Bieoski, M.;

Mansell, P.W.A.; Rios, A.; Newell, G.R.; and Sonnenfeld, G. Effect

of the recreational agent isobutyl nitrite on human peripheral blood

leukocytes and on in vitro interferon production. Cancer Res

43:1365-1371, 1983.

Ignarro, LJ.; Lippton, H.; Edwards, J.C.; Baricos, W.H.; Hyman, A.L;

Kadowitz, P.J.; and Gruetter, C.A. Mechanism of vascular smooth

muscle relaxation by organic nitrates, nitrites, nitroprusside, and

nitric oxide: Evidence for the involvement of Snitrosothiols as

active intermediates. J Pharmacol Exp Ther 218:739-749, 1981.

Jackson, C.D. Volatile nitrite. NTP working paper. Jefferson, AR:

Office of Scientific Intelligence, National Center for Toxicological

Research, 1979. 22 pp.

Jacobs, R.F.; Manner, D.J.; Steele, R.W.; and Hogue, T.R. Cellular

immunotoxicity of amyl nitrite. J Toxicol Clin Toxicol 20:421-449,

1983.

Komfeld, H.; Vande Stouwe, R.A.; Lange, M.; Reddy, M.M.; and

Grieco, M.H. T-lymphocyte subpopulations in homosexual men. N

Engl J Med 307:729-731, 1982.

57
Ledbetter, J.A Evans, R.L; Lipinski, M.; Cunningham-Rundles, C.;

Good, RA; and Herzenberg, L.A. Evolutionary conservation of

surface molecules that distinguish T-lymphocyte helper/inducer and

cytotoxic suppressor subpopulations in mouse anf man. J Exp Med

153:310-323, 1981.

Lewis, D.M.; Keller, W.A, Lynch, D.W.; and Spira, TJ. Subchronic

inhalation toxicity of isobutyl nitrite in BALB/c mice. II. Immuno

toxicity studies. J TaxicoI Envrion Health 15:835-846, 1985.

Luster, M.I.; Dean, J.H.; and Moore, JA. Evaluation of immune

functions in toxicology. In: Hayes, W., ed. Methods in

Toxicology. New York Raven, 1981. pp. 516-586.

Lynch, D.W.; Moorman, WJ.; Burg, J.R.; Phlpps, F.C.; Lewis, T.R.;

Kahn, A; Lewis, D.M.; Chandler, F.W.; Kimbrough, R.D.; and Spira,

T. Subchronic inhalation toxicity of isobutyl nitrite in BALB/c

mice. I. Systemic toxicity studies. J Toxicol Environ Health

15:823-833, 1985.

Popovic, M.; Sarngadharan, M.G.; Read, E.; and Gallo, R.C.

Detection, isolation and continuous production of cytopathic

retroviruses (HTLV Ill) from patients with AIDS and pre-AIDS.

Science 224:497-500, 1984.

Quinto, I. Mutagenicity of alkinitrites in the salmonella test. Boll

Soc ital Biol Sper 56:816-820, 1980.

AUTHORS

Daniel M. Lewis, Ph.D.

Division of Respiratory Disease

studies

National Institute for Occupational

Safety and Health

944 Chestnut Ridge Road

Morgantown, WV 26505

Dennis W. Lynch, M.S.

Division of Biomedical and

Behavioral Studies

National Institute of Occupational

Safety and Health

Robert A. Taft Laboratories

4676 Columbia Parkway

Cincinnati, OH 45226

58
Altered T-Cell Helper/Suppressor

Ratio in Mice Chronically Exposed

to Amyl Nitrite

Jesse. Oritz and Vilma L. Rivera

INTRODUCTION

Amyl nitrite (AN) and other volatile nitrites have become popular

over-the-counter drugs that are inhaled to alter consciousness,

enhance meditation, stimulate dancing, and intensify sexual

experience.

The drug was first used in 1867 as a treatment for patients suffering

from anginal pain (Sigell et al. 1978). When the Food and Drug

Administration designated AN a prescription drug in 1968, butyl and

isobutyl nitrite (IBN) were used as substitutes on the street (Cohen

1981). Presently, these aliphatic nitrous acid esters are marketed in

small vials (10 to 30 ml) or in small, thin glass capsules (0.18 to 0.30

ml) covered by cloth webbing that may be crushed by the fingers

prior to inhalation. These are usually referred to as "poppers." In

the United States, approximately 5x107 to 15x107 ml per year of AN

are used (Haley 1980; Nickerson et al. 1979).

Dewey et al. (1973) examined the behavioral and toxicological effects

of AN in mice and dogs. When intravenous (IV) and intraperitoneal

(IP) routes of administration were employed, the medial lethal dose

(LDs50) and its 95 percent confidence index for AN in mice was found

to be 130 mg/kg by the IP route and 51 mg/kg by the IV route.

When 22 vials 300μl/vial) of AN were administered to dogs by

inhalation every 30 seconds, the animals exhibited Irritability,

tremors, ataxia, and defecation.

59
The Centers for Disease Control (CDC) (1983) reported that IBN had

no immunotoxic effect on mice exposed to vapors at concentrations of

20, 50, and 300 ppm for up to 18 weeks.

Other investigators have shown that inhalation of AN also relaxes

most smooth muscle structures, e.g., bronchial, gastrointestinal,

rectum proper, and internal anal sphincter muscles (Sigell et al. 1978;

Haley 1980; Nickerson et al. 1979). In humans, AN may be dangerous

to those who have had cerebral hemorrhage, hypotension, glaucoma,

and myocardial disease (except patients with artery spasm) due to its

hypotensive and tachycardiac effects (Jorgensen and Lawesson 1982).

Recent reports suggest that AN may be a contributing cofactor to

AIDS in the homosexual population (Jorgensen and Lawesson 1982;

Goedert et al. 1982; Digiovana and Safai 1981). Further, Goedert et

al. (1982) have suggested that use of nitrites may play a role in the

imbalance of T-cell ratios among the healthy homosexual population.

While Goedert’s hypothesis could not be substantiated by other

workers (Durack 1981; Jaffe et al. 1983), in vitro studies using IBN

have demonstrated impaired lymphocyte and monocyte function at 1

percent concentration but not at 0.5 percent (Hersh and Reuben 1983;

Mayer 1964; Newel et al. 1984). The results demonstrated that these

effects were irreversible after 24 hours of exposure. At lower

concentrations the drug inhibited lymphocyte blastogenesis, natural

killer cell activity, antibody-dependent lymphocyte-mediated

cytotoxicity, in vitro cell adherence, and transformation of monocytes

to macrophages. These agents also inhibited leucine, uridine, and

thymine incorporation and inhibition of interferon production by

fibroblasts. These inhibitory effects were detectable even at 0.01

percent concentrations. Further, Osterloh and Goldfield (1984)

showed that wmmon “street” preparations were effective nitrosating

agents at neutral pH. In a recent report presented in Atlanta at the

CDC International Conference on AIDS, Haverkos et al. (1985) studied

a group of AIDS patients and by multivariance analysis showed that

inhalation of nitrites was the most common factor in the genesis of

Kaposi’s sarcoma, a skin cancer commonly associated with AIDS.

In view of these reports, there is an ongoing reevaluation on the

effects of alkyl nitrites and their relationship to opportunistic

infections and Kaposi’s sarcoma in AIDS patients. As part of this

reevaluation, this project studied the in vivo effects of AN on T-cell

function.

60
METHOSDS AND MATERIALS

After a 5-day acclimation period, 4-week-old male Charles River Labs

CD-1 mice, weighing from 16 to 22 g, were placed in stainless steel

cages in a light and dark temperature controlled room. Paired mice

were then fed Purina Rodent Chow and distilled water ad libitum.

The mice were then exposed to distilled AN via inhalation 5 days a

week for 21 weeks. Preliminary exposure showed that one 10μl dose

of AN was lethal to mice. For this reason, animals were slowly

acclimated to the substance by increasing doses as shown in the

protocol. The control group was given the same amount of saline

solution (0.85 percent NaC). To insure inhalation, the AN or saline

was introduced intranasally to a single group of 50 mice using a

10 I micropipette. Mice were exposed as follows:

Exposure Time

Time Since

First Exposure Amount AN Cumulative

(Weeks) (Weeks) Given per Day Amount Given

(μl) (μl)

Following exposure, mice were weighed and randomly assigned to

group I or group II.

Group I: Listeria monocytogenes (LM) Challenge

A group of 20 mice (10 exposed and 10 control) was randomly

selected after a 13-week exposure time and challenged intranasally

with 50 μl of an ID50 dose of the facultative intracellular pathogenic

bacterium LM, containing 2.8x106 organisms per ml. These mice were

monitored for mortality death up to 14 days.

Group II: T-Cell Depletion

T-cell depletion was assessed with the remaining group of mice (14

control and 11 exposed) after a 21 -week exposure time. The

monoclonal antibodies assay was used to measure different T-cell

61
subpopulations. Total T cells (Thy-l), helper T cells (Lyt-2

negative), and killer T cells (Lyt-2 positive) were measured using

peripheral blood. The bound target antigens were revealed with a

fluorescein-labeled goat antirat immunoglobulin G (IgG) (no crossreactivity with mouse IgG) (Cappel Labs).

Blood was obtained by cardiac puncture on Iightly anesthetized mice

using a 23-gauge needle attached to a l-ml syringe containing 100

units of heparin. A semi-microtechnique was used to isolate the

peripheral blood lymphocytes (Ledbetter and Hersenbrg 1079).

Heparnized blood was fractionated by the Ficol-Hypaque gradient

centrifugation technique. The washed lymphocytes were Incubated for

45 minutes with the corresponding antibodies. Excess antibodies were

washed off and the pelleted cells resuspended with the corresponding

flourescien antibodies for 45 minutes. The reaction was

stopped by the addition of 2 percent paraformaldehyde and centrifuged, and the sediment was resuspended in 1 to 2 drops of

phosphate-buffered saline (PBS) containing 30 percent v/v glycerol.

These cells were observed under the fluorescent microscope.

The mice were then killed and the spleen, kidneys, lungs, and livers

collected. The organs were then washed with sterilized PBS, dried,

and weighed. Anatomical observations were made for gross pathology.

The organs were preserved in Formalin (10 percent) for further

studies.

STATISTICAL ANALYSIS

Student’s t test was used to test the null hypothesis that exposure of

mice to sublethal doses of AN does not affect the following

parameters: pathotoxicology (repeated measurement of weight, weight

gain, and organ/body weight ratio and cellular Immunity); and T-cell

differential count.

In addition, regression analysis was used to measure the relationship

between T-cell ratio and the independent variables of body weight

and weight gain in group 2.

RESULTS

Host Resistance to LM

Lewis et al. (1983) demonstrated that there were no effects after

exposing mice to IBN vapors for 13 weeks. Since our animals were

62
exposed to IBN by the intranasal route (a slightly different method),

we decided to check our experimental animals for signs of immune

imbalance after 13 weeks by challenging them to an LD50 of LM.

Results shown in figure 1 indicate 20 percent of the exposed mice

and 40 percent of the controls survived this challenge; however, no

significant statistical differences (p>0.05) were observed in the

survival time of the two groups.

FlGURE 1. Days following LM challenge

NOTE: Effect of AN on mice survival following intranasal challenge with LM

(pathogenic). Significance was determined by Fisher’s exact test: p>0.05.

T-Cell Depletion

Results of T-cell differential counts are summarized in table 1.

Further, relationships between T-cell helper/suppressor ratio (T-cell

H/S ratio), body weight, and weight gain were analyzed (see table 2).

As seen in table 1, the total T cell and suppressor (Lyt-2 positive)

cells did not differ significantly between the two groups (p<0.1578

and p>0.4387, respectively). However, a significant decrease was

observed in helper (Lyt-2 negative) cells (p<0.0037). As a result,

there was a significant shift in the helper to suppressor ratio

between the two groups.

Furthermore, a significant degree of correlation (p<0.01) was found

between T-cell H/S ratio and the independent variables of body

weight and weight gain in the exposed group (table 3).

63
TABLE 1. Evaluation of T-cell different count folbwing AN exposure (group 2)

Total T-Cell % from Lyt-2 Negative

T-Lymphocyte Lyt-2 Negative Lyt-2 positive Lyt-2 Positive

Number (Mean ± SE) *(Mean ± SE) (Mean ± SE) *(Mean ± SE)

Control 41.22 37.74 24.60 1.53

(14) (±2.89) (±3.81) (±1.96) (±0.24)

Exposed 35.55 20.88 27.99 0.75

(11) (±2.37) (±3.44) (±4.18) (±0.10)

p (value) =>0.1578 =<0.0037 =>0.4387 =<0.005

*Student’s t test was used to mean values. Level of significance p<0.01.
Pathoxicologic Parameter

Mouse body weight and rate of weight gain after termination of each

exposure time are represented as means of each group (tables 2 and

4). A decrease In mean body weight was found after an accumulative

exposure time of 8 weeks (data not shown) and continued until the

end of the experiment (see table 2). After 21 weeks of exposure

time, both body weight and weight gain rate were significantly

decreased p<0.02; p<0.05, respectively). When the organ to body

weight ratio was evaluated (table 5), no significant differences were

found between the two groups. However, examination of lungs,

TABLE 2. Mouse body weight of group 2 following AN exposure

(T-cell depletion)

Control Exposed Level of

(±SE) (n=14) (±SE) (n=11) Significance*

Body Weight Mean 37.62 37.62 34.48 p<0.02

(g) (1.06) (1.12)

Weight Gain Mean 18.37 Text p<0.05

(g) (0.86) (1.11)

*Student’s t test was used to compare mean values. A value of p<0.05 was

considered stastically significantly. Total AN=655μl.

spleen, liver, and kidneys of mice exposed to AN showed extensive

damage to the lungs (table 6), which was statistically significant

(p<0.01). Lung damage consisted of (1) hemorrhagic spots; (2)

petechiae throughout the lobes; (3) collapsed consolidated sections,

which were red and showed the appearance of emphysema; and (4) air

pockets with large air bubbles. These gross pathological observations

were made in 10 of 11 mice. Spleens in 3 of 11 mice in the exposed

group were edematous and showed dark hemorrhagic spots. In the

control group, the lungs were normal in 11 mice and edematous in 3.

The spleens were normal in 11 mice and edematous without

hemorrhagic spots in 3 of the controls; and kidneys and livers

appeared normal in both groups.

65
TABLE 3. Relationships between T-cell H/S ratio, body weight, and

weight gain in mice following AN exposure

Group

Studied Correlation Coefficient (r)*

(n) T-cell H/S Ratio:Body Weight T-Cell H/S Ratio:Weight Gain

Control -0.471 (p>0.05) -0.308 (p>0.05)

(14)

Exposed 0.867 (p<0.01) 0.792 (p<0.01)

(11)

*Regression analysis was used to compare the relationships between the variables

mentioned above. A value of p<0.05 was considered to be statistically correlated.

DlSCUSSION

In summary, the results presented here demonstrate that exposure to

sublethal doses of AN can cause a significant decrease in helper cells,

thus resulting in an inverted T-cell H/S ratio. Mean body weight

decreased after 8 weeks and became statistically significant by the

end of 21 weeks of exposure. The cellular immunity parameter (Tcell H/S ratio) showed a statistically significant correlation coeffi

cient between T-cell H/S ratio and the independent variables of body

weight and weight-gain rate. Thus, as T-cell H/S ratio decreased,

weight gain also decreased. The gross pathological observation of

selected organs also showed statistically significant damage to the

lungs in the exposed group.

The fact that no correlations between T-cell H/S ratio and the

independent variables were detected in the control group indicates

that the positive correlation detected in the experimental group may

be a reflection of AN’s toxic effect.

Another parameter tested was the resistance of exposed mica to

pathogenic intracellular LM, used to assess the competence of Tlymphocytes and macrophages. Peak resistance was reached on day 6

for the control group (40 percent survival) but was delayed until day

14 (20 percent survival) in the exposed group.

66
TABLE 4. Mouse body weight and weight gain following AN exposure and challenge with pathogenic LM

*Student’s t test was used to compare mean values. A value of p<0.05 was considered statistically significant. Total AN=265 μl.

Control Exposed Level of

(±SE) (n=10) (±SE) (n=10) Significance*

Body Weight Mean 36.1 33.4 p<0.05

(g) (0.84) (0.82)

Weight Gain Mean 12.1 13.06 p<0.25

(g) (1.07) (0.78)
TABLE 5. Relative* and absolute** organ weights of group 2 following AN exposure

Group Spleen Lung Liver

Studied Organ Mean*** Mean*** Mean***

(n) Weight (±SE) (±SE) (±SE)

Control Relative 0.227±0.083 0.584±0.024 6.47±0.247

(14) Absolute 0.089±0.037 0.229±0.089 2.55±0.105

Exposed Relative 0.235±0.010 0.623±0.023 6.14±0.118

(11) Absolute 0.086±0.005 0.227±0.081 2.21±0.061

*Relative=orgsn weight/body weight.

**Absolute=organ weight.

***Studant’s t test was used to compare the mean value. A value of p>0.05 was not considered statistically significant.
TABLE 6. Pathological (gross) obsetvations of the lungs following

AN exposure (group 2)

Lungs

Normal Abnormal

Control (n=14) 11 3

Exposed (n=11) 1 10

Total 12 13

NOTE: Level of significance: p<0.01.

These results could be misleading since challenge to LM was done

after only 13 weeks of exposure time. Other workers have not shown

any effects after exposing mice for 13 weeks. The mice received a

total of only 385 I of AN, which is apparently not sufficient to alter

survival in vivo, although it might be extremely toxic in vitro. Had

this parameter been tested at 21 weeks, there could have been a

statistically significant difference.

Inhalation of AN produces effects similar to nitrous acid and Nnitroso compounds. Several studies (Maigetter et al. 1978;

Gardner et al. 1977; Fenter et al. 1973; Holt et al. 1979) have

reported depression of cellular immunity of various animal species

following NO2 exposure. Holt et al. (1979) exposed BALB/c mice to

10-ppm NO2 for 30 weeks, and examination of lung pathology showed

peripheral air spaces, emphysematous zones, large air spaces in the

peripheral lung, and diffuse interstitial pneumonia. Similar gross

appearances of the lungs, e.g., hemorrhagic areas, have been reported

in humans following exposure to large concentrations of NO2 (Parks

1974). Other studies have shown that NO2 exposure increases protein

content and glucose metabolism in lung tissue. Cytosolic enzyme

activities such as glucose-6-phosphate dehydrogenase, glutathione

peroxidase and reductase, and disulfite reductase are also increased in

the lung. Experiments with rats exposed to 5, 7, or 10 ppm NO2

resulted in an increase in glucose consumption via the Hexose

69
Monophosphate Shunt, increasing production of lactate and pyruvate

(Mustafa et al. 1980).

Haley’s (1980) review of the physiological effects of AN inhalation

mentions that subcutaneous administration of AN in rabbits produced

diuresis, albuminuria, and glycosuria. He also reports that after AN

inhalation, glucose and lactic acid were detected in the urine. Thus,

exposure of mice to AN could have produced the same physiological

efects as NO2 exposure and, as a consequence, the exposed group

may have had an increased lipid peroxidation and glucose consumption

followed by increased excretion of glucose compared to the control

group, leading to lower body weight and lower weight gain. lt has

also been shown that these aikyl compounds can be nitrosating

(Osterloh and Goldfield 1984), thus posing a further hazard in their

potential carcinogenic effect. Although this effect has been shown in

vitro, there are currently no data on the amounts of nitrosamines

formed in vivo in humans. However, this danger should not be

ignored.

The work presented here disagrees with the findings reported by the

Centers for Disease Control (1983), which attributes differences to

the methodologies and substances used. CDC researchers exposed

their animals to vapors of IBN for 18 weeks; whereas the present

study administered AN intranasally for 21 weeks. lt is possible that

immunotoxic effects do not reveal themselves until certain

concentrations and exposure times have elapsed. Prior to the start

of our experiments, we exposed a few animals to 5, 10, and 15 μI of

AN. Those animals receiving 10 and 15 μl of AN for the first time

died immediately after exposure. Animals receiving 5 μl became very

ill and one died within 24 hours; the rest survived. For this reason,

we administered AN in small doses and slowly increased the amount

given. Eventually the animals were able to tolerate 10 μl of AN

without any noticeable ill effects. Apparently animals do develop

tolerance to AN; however, felt that a maximum of 10 μl /day was a

reasonable sublethal dose.

Several reports in the medical literature suggest that AN might be an

inducer of Kaposi’s sarwma (Jorgensen and Lawesson 1982, Goedert et

al. 1982; Digiovana and Safai 1981; Haverkos et al. 1985) other

researchers refute the argument (Durack 1981; Jaffe et al. 1988).

More recent in vitro studies using IBN show this agent to be

immunotoxic (Hersh and Reuben 1983; Mayer 1984; Newell et al. 1984)

at very low concentrations. In addition, CDC’s latest epidemiological

report demonstrates a possible link between nitrites (poppers) and

70
Kaposi’s sarcoma. A survey of 87 AIDS victims found that

significantly more sufferers of Kaposi’s sarwma reported heavy use of

poppers than did sufferers of Pneumocystic carinii pneumonia.

CONCLUSION

The studies presented here show that chronic inhalation of AN can

lead to a decrease in helper cells, thus altering the T-cell H/S ratio,

which is the same phenomenon that occurs in AIDS victims. This

suggests a link between AN inhalation and cellular immunity

depression.

In view of the results reported here, there is an urgent need to

confirm these findings and to perform microscopic anatomy studies of

lung tissue and possibly other organs to elucidate the pathologic

effect of AN. In addition, challenge studies with pathogenic and

nonpathogenic organisms should be conducted after long-term

exposure, along with humoral immunity studies.

Since the termination of this study, various reports in the literature

have shown that a more appropriate reagent for measuring helper Tcell populations would be the L3T4 antigen (Swain et al. 1984). For

this reason we recommend that these results be confirmed using the

more recently discovered antigens. We further recommend that

persons prone to using alkyl nitrites should consider the possible

adverse health effects that could emanate from inhaling these

substances and thus should refrain from using them.

REFERENCES

Centers for Disease Control. An evaluation of the immunotoxic

potential of isobutyl nitrite. MMWR 32:457-464, 1983.

Cohen, S. When friends or patients ask about. . . The volatile

nitrites. JAMA 241:2077-2078, 1981.

Dewey, W.L; Tucker, LS.; Prange, A; Spaulding, T.; and Chann, T.T.

Some behavioral and toxicological effects of amyl nitrite. Res

Commun Chem Pathol Pharmacol 5:889-892, 1973.

Digiovana, J.J., and Safai, B. Kaposi’s sarcoma: Retrospective study

of 90 cases with particular emphasis on the familiar occurrence,

ethnic background and prevalence of other diseases. Am J Med

71:779-783, 1981.

Durack, D.T. Opportunisitic infections and Kaposi’s sarcoma

homosexual men. N Engl J Med 306:1465-1467, 198l.

71
Fenter, J.D.; Findlay, J.D.; Port, C.D.; Ehrlich, R.; and Coffin, D.L

Chronic exposure to nitrogen dioxide. Arch Environ Health 27:85-

89, 1973.

Gardner, D.E.; Coffin, D.L; Pinigin, MA; and Sidorenko, C.I. Role

of time as a factor in the toxicity of chemical compounds In

lntermittent and continuous exposure. J Taxied Environ Health

3:811-820, 1977.

Goedert, JJ.; Wallen, W.C.: and Mann, D.L Amyl nitrite may alter

lymphocytes in homosexual men. Lancet 1982-1:412-415, 1982.

Haley,T.J. Review of the physiological effects of amyl, butyl and

isobutyl nitrites. Clin Toxicol 16:317-329, 1986.

Haverkos, H.W.; Pinsky, P.F.; Drotman, P.D.; and Bregman, D.J.

Disease manifestation among homosexual men with acquired

immunodeficiency syndrome (AIDS): A possible role of nitrites in

Kaposi’s sacroma. Sex Transmited Dis 12:203-208, 1985.

Hersh, EM, and Reuben, J.M. Effect of the recreational agent

isobutyl nitrite on human peripheral blood leukocytes and an in

votro interferon production. Cawer Res 43:1365-1371, 1983.

Halt, P.G.; Finlay-Jones, L.M.; Keast, D.; and Papadimitrou, J.M.

lmmuniogical function in mice chronically exposed to nitrogen

dioxide (NO2) Environ Res 19:154-162, 1979.

Jaffe, H.W. et al. National case control study of Kaposi’s sarcoma

and Pneumocystis carinii pneumonia in homosexual men:

Epidemiologic results. Ann lntern Med 99:145-151, 1983.

Jorgensen, K.A., and Lawesson, S.O. Amyl nitrite and Kaposi’s

sarwma in homosexual men. N Engl J Med 307:893-894, 1982.

Ledbetter, J.A, and Hersenberg, L.A Xenogeneic monoclonal

antibodies to mouse lymphoid differentiation antigens. lmmunol Rev

47:63-90, 1979.

Lewis, D.M.; Koler, W.A; and Lynch, D.W. An immunotoxicological

study of isobutyl nitrite. Fed Proc 42(4):949, 1983.

Maigetter, R.Z.; Fenters, J.D.; Findlay, J.C.; Ehrlich, R.; and Gardner,

D.E. Effect of exposure to nitrogen dioxide on T and B cells in

mouse spleens. Toxicol Lett 2:157-161, 1978.

Mayer, K.H. Inhalation induced immunosuppression: Sniffing out the

volatile nitrite-AIDS connection. Pharmaco-therapy 4:235-236, 1984.

Mustafa, M.G.; Elsayed, N.; Urn, J.S.T.; and Postlelhwait, E. Effects

of nitrogen dioxide on lung metabolism. In: Grosjean D., ed.

Nitrogenous Air Polutants. Ann Arbor, MI: Ann Arbor Science

Pub, Inc., 1980. pp. 165-178.

Newel, C.R.; Adams, S.C.; Pharm, P.; Mansell, P.W.A; and Hersh, E.M.

Toxicity immunosuppressive effects and carcinogenic potential of

volatile nitrites: Possible relationship to Kaposi’s sarwma.

Pharmacotherapy 4:284-291, 1984.

72
Nickerson, M.; Parker, J.O.; Lowry, T.P; and Swenson E.W. Isobutyl

Nitrite and Related Compounds. San Frandsw: Pharmex Ltd.,

1979. 95 pp.

Osterioh, H., and Goldfield, D. Butyl nitrite transformation in vitro,

chemical nitrosation reactions and mutagenesis. J Anal Toxicol

8:164-169, 1984.

Parkes, W.R. Occupational Lung Disorders Noxious Gasses. London,

England: Butterworths Publishers, 1974. pp. 469-473.

Rosemblant, M. Personal communication. Boston Biomedical Researcl

Institute, Boston, MA, 1963.

Sigell, LT.; Kapp, F.T.; Fusaro, GA; Nelson, E.D.; and Falcok, R.S.

Popping and snorting volatile nitrites: A current fad for getting

high. Am J Psvchietry 135:1216-1218, 1978.

Swain, S.L; Dialynas, D.P.; Fitch, F.W.; and English, M. Monoclonal

antibody to L3T4, blocks the function of T-cells specific for class 2

major histocompatibility complex antigens. J Imunol 132:1118-

1123, 1964.

ACKNOWLEDGMENTS

Dr. Marlo Rosemblant of the Boston Biomedical Research Institute

gave technical advice end Dr. Warren Litsky of the University of

Massachusetts, Department of Environmental Sciences, reviewed the

manuscript of this chapter. Dr. Mark Jason of the Department of

Chemistry at Amherst College distilled and donated the pure AN

Dr. Eric Mark of the Microbiology Department at the University of

Massachusetts donated the antibodies for the monoclonal antibody

assays.

This work WaS supported in part by biomedical support grant RR07048

from the University of Massachusetts in Amherst.

AUTHORS

Jesse S. Ortiz, Dr.P.H.

Division of Public Health

University of Massachusetts

Amherst, MA 01003

73
Vilma L. Rivera, M.S.

Associate Hazardous Material

Specialist

State of California

Department of Health Services

Toxic Substances Control Division

107 South Broadway, Room 7011

Los Angeles, CA 90012

74
Effects of Nitrites on the Immune

System of Humans

Elizabeth M. Dax, James E. Nagel,

W. Robert Lange, William H. Adler, and

Jerome H. Jaffe

INTRODUCTlON

Kaposi’s sarcoma (KS) is a disease manifestation of unexpectedly high

occurrence in homosexual men with acquired immunodeficiency

syndrome (AIDS) and has been recognized as such since 1981 (Centers

for Disease Control 1981). In other groups of people with AIDS, the

occurrence of KS is less frequent (3 to 4 percent vs. approximately

14 percent in the homosexual group) (Centers for Disease Control

1986). Epidemiological evidence indicates that factors associated with

the presence of KS in patients with AIDS include sexual activity

(Jaffe et al. 1983). drug usage, particularly the use of volatile nitrites

(Haverkos et al. 1985), and a history of sexually transmitted diseases

and possibly their therapies (Marmor et al. 1982). The association

between nitrite abuse and the development of KS has been

demonstrated in some studies (Marmor et al. 1982; Haverkos et al.

1985) but has not been supported in the studies of Polk et al. (1987).

In some samples the use of volatile nitrites by homosexual men

approaches 100 percent (Israelstam et al. 1978; Marmor et al. 1982).

Conversely, it has been reported that few heterosexual patients with

AIDS use nitrites (Guinan et al. 1984). Thus, the question arises

whether the nitrites are surrogate markers of other factors involved

in the development of KS, or whether they, themselves, are cofactors

in the development of this disease in AIDS patients. It has been

suggested that nitrites may be involved in the development of KS by

intrinsic immunosuppressive properties.

There are several possible explanations of the nitrites’ ability to act

as cofactors in the development of KS in AIDS patients. Most

important, there is evidence that nitrites may compromise the immune

75
system. Goedert et al. (1982) found low helper:suppressor Tlymphocyte ratios in eight volunteer, homosexual men who regularly

used nitrite compared with seven volunteers who were not regular

users. The altered T-lymphocyte ratios were due to decreases in

helper cells as well as increases in suppressor cells. Cytomegalovirus

(CMV) antibody titers were high in 14 of the 15 volunteers but were

not related to either nitrite abuse or T-cell abnormalities. The

authors concluded that “nitrites may be immunosuppressive in the

setting of repeated viral antigenic stimulation, and may contribute to

the high frequency of KS and opportunistic infections in homosexual

men” (Goedert et al. 1982, p. 412). In another study, Hersh et al.

(1963) examined the effects of nitrites administered in vitro on

parameters of lymphocyte function. A dose response inhibition of in

vitro blastogenic response to mitogens, natural killer cell activity,

antibody-mediated cellular toxicity, and adherence and transformation

of monocytes to macrophages was observed. Again, the authors

suggested that nitrites have an immunosuppressive effect on mononuclear leucocyte function. Animal studies support the proposal that

nitrites are immunosuppressive (Lewis and Ortiz, this volume). To

date, immunological function following inhalation of nitrites has not

Been reported. This paper summarizes preliminary results of such a

study.

MATERIALS AND METHODS

Eight HIV-negative male volunteers gave informed consent to

participate in this study. They were in residence on the Addiction

Research Center ward. During the first week of the study two

Baseline immunologicol test batteries were run. Over 4 days of the

second week, each volunteer participated in 13 inhalation sessions

(0.18,0.3, and 0.46 ml amyl nitrite each three times, and four placebo

doses). The placebo, banana oil, was included in each inhalation

session with or wlthout amyl nitrite. The drugs were inhaled from a

4-liter flask connected to a collapsible breathing bag which contained

a known volume of air. Another inflow valve was opened when the

bag was fully collapsed in order to ensure full inhalation of the

nitrite dose. Blood was drawn for immunological tests immediately

following the last inhalation session, then at 24 hours, 96 hours, and

7 days.

Tests of immune status included white cell counts, absolute

lymphocyte numbers, lymphocyte subset representation, in vitro

lymphocyte responses to the mitogens Phytohemaglutanin (PHA),

Concanavalin A (ConA) and pokeweed mitogen (PWM), natural killer

76
cell activity, and in vitro polyclonal induction of immunoglobulin G

(IgG) and immunoglobulin M (IgM) synthesis.

The determination of lymphocyte subsets utilized monoclonal

antibodies against membrane markers. The OKT series of antibodies

were obtained from Ortho Diagnostics, and the Leu series were

obtained from Becton Dickinson. The stained cell preparations were

analyzed using an Ortho 50H Cytofluorograph (Nagel et al. 1981 b).

The in vitro responses to mitogens were determined by culturing 5 x

104 lymphocytes in 0.2 ml volumes in indivldual well microculture

plates. The cells were grown in RPMI 1640 (Grand Island, NY)

supplemented with 10 percent fetal calf serum with 100 ug penicillin

and 100 ug streptomycin per ml. Cultures were maintained for

72 hours with 1 Ci 32' H-Trd present for the last 18 hours. Results

are presented as the mean cpm for three replicative samples. Natural

killer cell activity was determined using Cr51 labeled K562 tumor

cells as targets. Peripheral blood lymphocytes were diluted to

provide effector to target (E:T) ratios of 100:1 to 0.78:1. The

resulting cytotoxicity curves were examined for linearity. Results are

expressed as specific cytotoxicity for the 12.51 E:T ratio previously

described (Nagel et al. 1981 c). In vitro immunoglobulin production

was analyzed using cultures of peripheral blood lymphocytes in the

complete media described above. PWM was used as a polyclonal

activator of immunoglobulin production (Nagel et al. 1981 a). The

quantitation of IgM and IgG content in culture supermates was

accomplished using an ELISA technique with specific enzyme-linked

anti-immunoglobulin reagents. Results are expressed as the peak

amount of IgM and IgG in different cultures stimulated with varying

doses of PWM.

RESULTS

The T-lymphocyte line associated with cell-mediated immunity carry

surface antigens recognized by the CD3, CD4, and CD8 antibodies for

the immature, T4 helper, and T8 suppressor cells, respectively. The

B-cell line lymphocytes, which produce immunoglobulins, are identified

with the Leu-12 antibody.

The representation of T-lymphocyte subsets as the percentage of total

lymphocytes was unchanged after amyl nitrite inhalation. Therefore,

there were only minor variations in the lymphocyte helper/suppressor

ratio (T4/T8) following inhalation of amyl nitrite. However, the ab

solute number of peripheral blood lymphocytes decreased significantly

following the inhalation protocol, then returned to baseline levels

77
after 24 hours, and increased over baseline levels at day 7. The

absolute numbers of CD3+ and CD8+ lymphocytes were significantly

decreased immediately following inhalation. Natural killer ceils (leu7+11) doubled in percentage representation between baseline levels

and the second day post-inhalation while the level of natural killer

cell activity showed an initial significant decrease, then returned to

baseline levels by day 4 post-inhalation. The percentage

representation of leu-12+B -lymphocyte showed an increase from 12.6

to 17.4 percent between day 4 and day 7, which corresponded to an

increase in the in vitro PWM-induced immunoglobulin synthesis. The

response to the T cell mitogens Con A and PHA was significantly

elevated on day 7 following exposure to nitrite. The results showed

a nitrite-induced lymphopenia resulting from relatively short exposure.

Further, a nonspecific, perhaps compensatory, immunostimulation

following exposure to amyl nitrite was compatible with the suggestion

that the nitrites may aggravate symptoms by enhancing HIV

replication. The nonspecific stimulation could mask or interfere with

specific immune responses to pathogens.

CONCLUSIONS

The results showed that exposure to amyl nitrite can induce changes

in immune function even after short exposure to moderate doses.

Several tests of immune function showed an “overshoot” over basal

activity at 7 days following nitrite inhalation after an initial

immunosuppression. A possible interpretation of the results would be

that the nitrites cause a cycling of immune activity between

suppressed and nonspecific stimulated levels. This situation might

result in a period of ‘immunosuppression followed by a proliferative

period in which virus-containing cells propagate in the presence of a

nondirected immunoresponse. In the community, nitrites are often

used in an episodic manner, which may facilitate such cyclic changes.

REFERENCES

Centers for Disease Control. Kaposi’s sarwma and Pneumocystitis

pneumonia among homosexual men-New York City and California.

MMWR 30:305-308, 1981.

Centers for Disease Control. Update: Acquired immunodeficiency

syndrome-United States. MMWR 35:757-766, 1986.

Goedert, J.J.; Neuland, C.Y.; Wallen, W.C.; Greene, M.H.; Mann, D.L.;

Murray, C.; Strong, D.M.; Fraumeni, J.F.; and Blattner, W.A. Amyl

nitrite may alter T lymphocytes in homosexual males. Lancet

20:412-416, 1982.

78
Guinan, M.E.; Thomas, P.A.; Pinsky, P.F.; Goodrich, J.T.; Selik, R.M.;

Jaffe, H.W.; Haverkos, H.W.; Noble, G.; and Curran, J.W.

Heterosexual and homosexual patients with acquired

immunodeficiency syndrome: A comparison of surveillance,

interview, and laboratory data. Ann lntern Med 100:213-218, 1984.

Haverkos, H.W.; Pinsky, P.F.; Drotman, D.P.; and Bregman, D.J.

Disease manifestation among homosexual men with acquired

immunodeficiency syndrome: A possible role of nitrites in Kaposi’s

sarcoma. Sex Transm Dis 12:203-208, 1985.

Hersh, E.M.; Reubin, J.M.; Bogerd, H.; Rosenblum, M.; Mansell, P.W.A.;

Rios, A.; Newell, G.R.; and Sonnenfeld, G. Effect of the

recreational agent isobutyl nitrite in human peripheral blood

lymphocytes and on in vitro interferon production. Cancer Res

43:1365-1371, 1983.

Israeltam, S.; Lambert, S.; and Oki, G. Poppers a new recreational

drug craze. Can J Psychietry 23:493-495, 1978.

Jaffe, H.W.; Bregman, D.J.; and Selik, R.M. Acquired immune

deficiency syndrome in the United States: The first 1,000 cases.

J Infect Dis 148:339-345, 1983.

Marmor, M.; Freidman-Kein, A.E.; Laubenstein, L.; Byrum, R.D.;

William, D.C.; D’Onofrio, S.; and Dubin, N. Risk factors for

Kaposi’s sarcoma in homosexual men. Lancet 1:1083-1067, 1982.

Nagel, J.E.; Chrest, F.J.; and Adler, W.H. Human B cell function in

normal individuals of various ages. I. In vitro enumeration of

immunoglobulin synthesizing cells and the comparison of the results

with numbers of peripheral B and T cells, mitogen responses, and

levels of serum immunoglobulins. Clin Exp lmmunol 44:646-653,

1981 a.

Nagel, J.E.; Chrest, F.J.; and Adler, W.H. Enumeration of T

lymphocyte subsets by monoclonal antibodies in young and aged

humans. J lmmunol 127:2086-2088, 1981b.

Nagel, J.E.; Collins, G.D.; and Adler, W.H. Spontaneous natural killer

cytotoxicity of K562 erythroleukemic cells in normal patients.

Cancer Res 41:2284-2288, 1981c.

Polk, B.F.; Fox, R.; Brookmeyer, R.; Kanchanaraksa, S.; Kaslow, R.;

Visscher, B.; Rinaldo, C.; and Phair, J. Predictors of the acquired

immunodeficiency syndrome developing in a cohort of seropositive

homosexual men. N Engl J Med 316:61-66, 1987.

79
AUTHORS

Elizabeth M. Dax, M.D., Ph.D.

W. Robert Lange, M.D., M.P.H.

Jerome H. Jaffe, M.D.

National institute on Drug Abuse

Addiction Research Center

P.O. Box 5180

Baltimore, MD 21224

James E. Nagel, M.D.

William H. Adler, M.D.

National Institute on Aging

Gerontology Research Center

Francis Scott Key Medical Center

4940 Eastern Avenue

Baltimore, MD 21224

80
Deliberate Inhalation of Isobutyl

Nitrite During Adolescence:

A Descriptive Study

Richard H. Schwartz

INTRODUCTION

In the past 10 years, isobutyl nitrite has become popular as a means

of getting “high” among heterosexual adolescents, particulariy those

who abuse other “gateway” drugs. Several surveys of adolescents and

young adults reflect butyl nitrite use in the United States. In 1966

approximately 9 percent of 3,000 American high school seniors

surveyed by a standardized indepth questionnaire on drug use patterns

stated that they had deliberately inhaled volatile nitrites at least one

time in order to self-intoxicate (“get high”) (Johnston et al. 1967).

In the same study, 1.6 percent of the survey respondents had used

nitrite inhalants within the previous 30-day period, and 0.5 percent of

the respondents used the drug daily. Annual use of isobutyl nitrite

increased from the previous 3-year period. In 1983, 3.6 percent of

high school seniors inhaled isobutyl nitrite, while 4.7 percent did so

in 1966. The National Institute on Drug Abuse (NIDA) Household

Survey of children 12 to 17 years old is in dose accord with the

high school senior survey: 9 percent of the 12- to 17-year-old

respondents admitted to lifetime use of isobutyl nitrite, and 4 percent

were current users (National Institute on Drug Abuse 1986). In New

York State, 6 percent of 1.3 million 7th to 12th grade students

surveyed inhaled isobutyl nitrite during the 1983 school year, 3

percent were current users, and 1 percent, or 9,000 students, did so

at least 10 times in the 30-day period prior to the large statewide

survey on drug use frequencies and patterns (New York State Division

of Substance Abuse Services 1984).

lsobutyl nitrite, a licit chemical sold allegedly as a room odorizer in

some pornography, record, and tape shops, and by mail order from

magazine advertisements, is a popular drug of abuse among American

teenagers. The drug is chemically similar to amyl nitrite, an inhaled

volatile liquid that is used in the emergency treatment of cyanide

81
poisoning. lt has a rather unpleasant chemical odor, which is said to

resemble sweaty socks or the smell of a men’s locker room.

lsobutyl nitrite is sold legally under various trade names. Among the

more common are Rush, Locker Room, Bullet, Quick Silver, Thrust,

Lightning Bolt, and Hardware, many of which have sexual

connotations. lt is estimated that 12 million bottles of the products

were sold between 1978 and 1983 (Freezer 1978). The most common

sources of these products are underground outlets such as “head

shops” and pornography shops.

Labels on some vials of isobutyl nitrite bear clear printed warnings to

“avoid inhalation” of the vapors One label even reads “Danger:

Excessive Use May Cause Euphoria.”

The immediate effect of inhaling isobutyl nitrite is a sensation of

warmth and a lightheaded feeling that adolescents call a “head rush.”

This phenomenon, which is due to profound vasodilation, lasts only a

few minutes. Common physiological effects include pounding

headaches, dizziness, blurred vision, and pressure in the eyes.

Postural hypotension leading to loss of consciousness can easily occur

if the drug is inhaled while standing. Prolonged exposure to the

vapors can cause irritation to the eyes and the tracheobronchial tree

(Covalla et al. 1981). lf isobutyl nitrite is accidentally spilled around

the nose during inhalation, it can cause dermatitis and pronounced

yellow crusting on the upper lip or in the nasal passages (Fisher et

al. 1981). In some individuals, the consequences of using this product

may be much more severe: some methemoglobin-reductase enzymedeficient persons develop severe methemoglobinemia or Heinz body

hemolytic anemia after sniffing isobutyl nitrite (Horne et al. 1979;

Bogart et al. 1988; Dixon et al. 1981).

PATIENT POPULATION AND STUDY DESIGN

We recently had an opportunity to evaluate the extent and

consequences of the use of isobutyl nitrite among a select group of

youth. The 173 persons in our study were patients in a drug

treatment facility which is a long-term therapeutic community for

chemically dependent adolescents. These young people were

predominantly white and middle class. The vast majority had a

diagnosis of primary dependency or abuse of cannabis or alcohol.

Most had also abused a wide array of mood-altering drugs including

inhalant drugs, LSD, PCP, and cocaine.

82
The data in our study were gathered from a 21-item questionnaire

about isobutyl nitrite use, answered by patients present at the

treatment facility on a single day.

After a general announcement about the purposes of the study and

the guidelines for voluntary participation, the questionnaire was given

to individuals attending a large group therapy session consisting of

approximately 200 patients. They were informed that the purpose was

for medical research only. Participation was entirely voluntary; there

were no rewards or consequences based on a client’s decision of

whether or not to participate. The confidential questionnaire had

been approved by the program director and the professional treatment

staff.

RESULTS

Seventy-four (43 percent) of the 173 respondents (85 percent were

male, 35 percent were female) to the survey reported that they had

abused isobutyl nitrite for the purpose of self-intoxication; however,

52 (30 percent) had used it less than 10 times each. An additional 16

(9 percent) had used it between 10 and 99 times. Six (3 percent)

estimated that they had inhaled isobutyl nitrite on more than 100

occasions. Of the 22 individuals who had inhaled the vapors more

than 10 times, 19 were male.

The 22 clients (mean age 17 years), who inhaled isobutyl nitrite at

least 10 times had almost all been introduced to the drug by a close

friend. The substance was almost always used by inhalation directly

from the vial.

lsobutyl nitrite had been inhaled in solitude on at least one occasion

by 71 percent of the 22 frequent users, a particularly dangerous

practice if the user was standing or driving a car at the time because

of the cardiovascular effects. Relatively few (17 percent) used the

drug before heterosexual intercourse and none admitted to use before

anal intercourse. The average cost of a 12 ml bottle of isobutyl

nitrite (Rush) was reported at $5 to $6. Most of the young people

obtained the drug from a friend or purchased it from “head shops”

(33 percent), record stores (17 percent), pornography shops (11

percent), or mail-order catalogues (6 percent).

Nearly half the users described their experiences with isobutyl nitrite

as “unpleasant” because of universal symptoms of lightheadedness and

dizziness, as well as rapid heartbeat, blurred vision, facial flushing,

83
and headache. Thirty-four percent reported severe pulsatile

headache, 17 percent reported burning in the nose, and 11 percent

reported nausea immediately after inhalation of the drug. Probably

because of the unpleasant odor and physiological effects of the

volatile vasodilator drug, the euphoric effect (“high”) was described

as only “fair to good” by 56 percent of the respondents.

CONCLUSION

We believe that the results reported may reflect the extent and side

effects of the use of isobutyl nitrite among adolescents in drug

treatment programs. Being honest and open about their drug

problems was an integral part of the treatment program that these

individuals were undergoing. Conclusions from three published studies

support the validity of self-reporting of drug use by adolescents

(Smart and Jarvis 1981; Needle et al. 1983; Benson and Holmberg

1985).

The widespread and apparently increasing use of isobutyl nitrite as a

euphoric agent by teenagers warrants consideration of banning the

sale of isobutyl nitrite.

REFERENCES

Benson, G., and Holmberg, M.B. Validity of questionnaires in

population studies on drug use. Acta Psychiatr Scand 71:9-18, 1985.

Bogart, L.; Bonsignore, J.; and Carvalho, A. Massive hemolysis

following inhalation of volatile nitrites. Am J Hernerol 22:327-329,

1986.

Covalla, J.R.; Strimlan, C.V.; and Lech, J.G. Severe tracheobronchitis

from inhalation of an isobutyl nitrite preparation. Drug Intell Clin

Pharm 15:51-52, 1981.

Dixon, D.S.; Reich, R.E.; and Santinga, B.A. Fatal methemoglobinemia resulting from ingestion of isobutyl nitrite, a “room

odorizer” widely used for recreational purposes. J Forensic Sci

26:587-593, 1981.

Fisher, A.A.; Brancaccio, R.R.; and Jelenek, J.E. Facial dermatitis in

men due to inhalation of butyl nitrite. Cutis 27:146, 152-153, 1981.

Freezer, W.J. lsobutyl Nitrite and Related Compounds. San

Francisco: Pharmex, Ltd., 1978.

Home, M.K.; Waterman, M.R.; Simon, L.M.; Garriott, J.C.; and

Foerster, E.H. Methemoglobinemia from sniffing butyl nitrite. Ann

Intern Med 91:417-418, 1979.

84
Johnston, L; Bachman, J.; O’Malley, P. Use of Licit and Illicit Drugs

for America’s High School Students 1975-1986. National Institute

on Drug Abuse. DHHS Pub. No. (ADM) 85-1394. Washington, DC:

Supt. of Docs., U.S. Govt. Print. Off., 1987. 159 pp.

National Institute on Drug Abuse. Highlights from the National

Survey on Drug Abuse: 1985. in: National Institute on Drug

Abuse. National Household Survey on Drug Abuse 1985. DHHS

Pub. No. (ADM) 87-1539. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1987. 73 pp.

Needle, R.; McCubbin, H.; Lorence, J.; and Hochhauser, M. Reliability

and validity of adolescent self-reported drug use in a family-based

study: A methodological report. Int J Addict 18:901-912, 1983.

New York State Division of Substance Abuse Services. Substance

Abuse Among New York State Public and Private School Students in

Grades 7 through 72, 1983. Albany, NY: New York State Division

of Substance Abuse Services, 1984. 74 pp.

Smart, R.G., and Jarvis, G.K. Do self report studies of drug use

really give dependable results. J Drug issues 23:83-92, 1981.

AUTHOR

Richard H. Schwartz, M.D.

Clinical Professor

Child Health and Development

George Washington University

School of Medicine

Washington, DC 20037

85
Nitrite Inhalants:

Contemporary Patterns of Abuse

W. Robert Lange, Elizabeth M. Dax

Charles A. Heartzen, Frederick R. Snyder,

and Jerome H.Jaffe

INTRODUCTION

Amyl nitrite has been used medically since 1867 in the management of

coronary insufficiency (Brunton 1867), and even though the agent is

used infrequently in clinical therapeutics at this time, it does have a

contemporary role in diagnostic cardiology and diagnostic radiology

and in the management of cyanide poisoning. The history of butyl

and isobutyl nitrites (collectively referred to hereafter as butyl

nitrite) is more recent. Whereas amyl nitrite is a prescription drug,

butyl nitrite is marketed as a room odorizer. Even though it has no

clinical or practical utility, it is widely available and has been

endowed with a certain mystique.

Beginning in the late 1960s and into the early 1970s, reports began to

surface that nitrite inhalants were being abused by both sexes in an

attempt to augment the physical pleasure of sexual intercourse

(Everett 1972). Soon nitrite inhalants became known as “poppers” (an

onomatopoetic street name derived from the sound of breaking an

amyl nitrite pearl), and abuse patterns took two distinct courses. As

a street drug, poppers were frequently used on social occasions, often

in discotheques, to promote a sense of abandon while dancing, to

expand creativity, to stimulate music appreciation, and to enhance

meditation (Sigell et al. 1978). Others used them on a more general

basis for a euphorigenic rush, an altered state of consciousness, and

even as a stimulant (Nickerson et al. 1979). There is no scientific

evidence, however, that any of these effects are associated with the

use of nitrite inhalants.

86
A more widespread pattern of use was associated with overt sexual

activities. Purported attributes included prolongation of penile erection and relaxation of rectal smooth muscle and anal sphincter tone,

thus facilitating intromission (Labataille 1975). Other alleged drug

effects included heightened libido, i.e., an aphrodisiac, prevention of

premature ejaculation, increased volume of ejaculate, and promotion

of a generally intensified or prolonged sexual experience (Israelstam

et al. 1978).

Although awareness of adverse health effects of nitrite abuse was

raised in the 1970s (Louria 1976), real concerns surfaced with the

advent of the acquired immunodeficiency syndrome (AIDS) epidemic,

and the growing body of data suggesting that the volatile nitrites

may influence immunomoduiation or otherwise be a cofactor in the

pathogenesis of human immunodeficiency virus (HIV) infection and

certain AIDS-related syndromes. For example, of the first five homosexual men with AIDS reported to the Centers for Disease Control,

all had used nitrite inhalants, but only one had abused intravenous

drugs and only two were reported to be promiscuous (Centers for

Disease Control 1981).

Contemporary patterns of abuse of nitrite inhalants are not clearly

delineated. On one hand, a readily available drug with the reputation

of being an aphrodisiac would appear to have appreciable abuse

liability. On the other, the close association to and growing concern

over AIDS might be expected to alter patterns of abuse. This study

was a descriptive survey of two populations with a high probability of

using nitrite inhalants.

METHODS

Two distinct high-risk populations were surveyed: predominantly

heterosexual substance abusers in six regions of the United States

and homosexual men in Baltimore.

Substance Abusers

Substance abusers were surveyed in Baltimore, MD, during two separate time frames: 1981 through 1984 (n=365) and February through

October 1986 (n=246). in addition, substance abusers in five other

regions of the United States were queried about nitrite abuse patterns as part of a national HIV seroprevalence survey conducted

between September 1986 and February 1987. The following regions

were surveyed: New York City (n=267); Tampa, FL (n=59);

87
San Antonio, TX (n=102); Denver, CO (n=99); and Los Angeles, CA

(n-409). The Baltimore participants during both time periods were

comprised of both volunteers for National Institute on Drug AbuseAddiction Research Center (NIDA-ARC) research studies as well as

clients in treatment for substance abuse disorders. The former

tended to be recreational drug users not physically dependent on any

substance at the time of their participation; the latter had heavier

drug use histories. The subjects in the five locations other than

Baltimore were all individuals recently enrolled in a drug treatment

program.

Male Homosexuals

Between April and August 1966, a sample of 80 male homosexuals was

studied. Of these, 20 were interviewed while participating in the

activities of a homosexual support group, and 60 were interviewed in

gay bars in Baltimore, MD, and Washington, DC. They were queried

specifically in regard to their current and past use of both amyl and

butyl nitrites, including methods and reasons for using these drugs as

well as associated drug effects and side effects.

For the purpose of this investigation, current use was defined as use

at any time during the 6 months prior to the survey. To assess

possible differences between nitrite inhalant users and nonusers, the

data were analyzed by either of two methods. Variables measured on

an interval scale were subjected to independent samples r-tests,

whereas categoricai variables were analyzed using the chi-square

statistic.

RESULTS

Substance Abusers

Table 1 describes the Baltimore data for the two time frames. Among

recreational users (NIDA-ARC clients), the prevalence rate of nitrite

use during the preceding 6 months was 11 percent for both periods.

The participants with the heavier drug use patterns (clients in

treatment) also had a higher frequency of nitrite use, with the

current prevalence rate being 22 percent. Nitrite inhalant use was

more common in whites than in members of minority groups (x2=6.80,

p<0.05). The sexual orientation of the earlier samples is not known,

but 7 percent of both 1986 samples admitted to a history of

88
TABLE 1. Prevalence of nitrite use in Baltimore substance abusers

Prevalence

Dates Mean Age White Male of Use

Sample Studied No. (Years) (Percent) (Percent) (Percent)

Early 1980s

ARC Volunteers 1981-84 203 32±6 5 6 90 11

Clients in

Treatment 1982-84 162 35±9 5 4 66 17

1986

ARC Volunteers 1986 6 3 30±7 40 90 11

Clients in

Treatment 1986 183 32±6 4 4 7 8 2 2

homosexuality, and there was no statistically significant association

between homosexual behavior and nitrite use in these groups.

ARC subjects were asked when they first began using nitrites in relation to other substances of potential abuse, and these results are

depicted in table 2. The inhalable nitrites tend to be a drug class

whose use is initiated much later in life, with the mean age of first

use at 25.6 years. This compares to an age of 14.6 years for glue,

the other principal inhalant of abuse.

TABLE 2. Age of first use for substances of abuse for Baltimore

ARC volunteers

Substance Mean Age ± SD

Alcohol 13.9 ± 3.6

Glue 14.6 ± 3.2

Benzedrine/Speed 17.2 ± 3.1

Marijuana 17.6 ± 5.5

Heroin 18.5 ± 3.5

Cocaine 20.4 ± 4.4

Nitrites 25.6 ± 7.8

89
Table 3 shows the geographic differences in nitrite abuse patterns.

Among abusers in treatment, use of nitrite inhalants ranged from a

high prevalence of 40 percent in Tampa to a low of 7 percent in the

New York City area (Brooklyn and Harlem). In all locations,

experience with amyl nitrite was reported more frequently than

experience with butyl nitrite.

TABLE 3. Proportion of exclusively heterosexual substance abusers

in treatment reporting nitrite inhalants, by residence

Number Queried

Exclusively EH Reporting

EH Reporting Nitrite Use

City or Region Total

Heterosexual

(EH) Nitrite Use (Percent)

Tampa 5 9 53 21 40

Denver 99 90 34 38

San Antonio 102 93 28 30

Baltimore 182 172 39 23

Los Angeles 409 397 55 14

New York city 267 241 16 7

Total 1,118 1,046 193 18

Among substance abusers, nitrite use was reported more frequently

among whites than blacks or Hispanics, and more frequently among

men than women. Thirty percent (142/475) of ,whites reported nitrite

use compared with 13 percent (31/246) of blacks and 10 percent

(35/350) of Hispanics. The highest rates of nitrite use for all six

regions were reported by whites. Twenty-one percent (159/743) of

men reported nitrite use compared to 16 percent (57/366) of women.

Within each region, men reported nitrite use more frequently than

women except in San Antonio. There, 14 of 30 women (47 percent)

and 12 of 72 men (29 percent) reported nitrite use.

Since these data were collected as part of an HIV antibody survey,

the data were analyzed to ascertain whether there were differences

in nitrite use on the basis of HIV seropositivity. Among substance

abusers in Baltimore, there was a significant association between

nitrite inhalant use and HIV seronegativity (X

 2=121.6, p<0.001), and

this was felt to be secondary to the ethnicity of the sample: HIV

90
seropositivity was independently associated with blacks, whereas

nitrite use was independently associated with whites. In New York,

there was a tendency for an association between nitrite use and seropositivity, and in Denver, there was a distinct association. However,

among the five HIV positives in Denver, three admitted to being

homosexual, and this likely confounds the data in an area with few

HIV-seropositives.

Male Homosexuals

The mean age of the homosexual sample was 30.0 years (range 18 to

56 years). The component enrolled in the support group tended to be

older (mean age 39.5 years, range 23 to 56 years) than those

recruited in gay bars. Fity-five (89 percent) admitted to ever

having used nitrite inhalants, and there were no demographic differences between users and nonusers. The mean age of first use among

the homosexuals interviewed was 22.2 years (range 18 to 42 years),

and the mean age of peak use was 25.4 years (range 17 to 45 years).

Of those who had used poppers, 43 (78 percent) reported use of only

amyl nitrite, and the remaining 12 (22 percent) had used both amyl

and butyl. Among those using both, there was a preference for amyl

nitrite, which was regarded as being more potent and less associated

with side effects. No homosexual subject in our sample admitted to

having used only butyl nitrite.

Patterns of nitrite use were assessed for the homosexual men surveyed in Baltimore. Only 13 users (23 percent) reported having used

nitrite inhalants at times other than during sexual activity, and this

was primarily while dancing. Among the various sexual behaviors, use

was most often associated with masturbation (64 percent). Twentysix (47 percent) had used nitrite inhalants concomitantly with one or

more other drugs, with concomitant marijuana being the most

frequent (96 percent).

Despite the propensity for using nitrites during overt sexual activity,

the predominant reason given by the homosexual group for using the

substance was to get high, rather than for any anticipated physiologic

effect. Twenty-five (45 percent) reported some side effect associated

with nitrite use, the most common being headache. Three participants related that nitrite use Caused them to lose their erections, and

none credited the drug with facilitating erection, heightening libido,

or enhancing sexual enjoyment.

91
Homosexuai subjects reported that the duration of the subjective

effects of nitrite inhalation was brief. The modal duration was 1

minute, with a range of 4 seconds to 15 minutes. The reported modal

frequency of we was once weekly, and the modal intensity of peak

use was four times per week. The style or technique of use was

correlated with the intensity of use, with heavy or current users

tending to inhale more deeply. They also reported a longer retention

of inhalation than did light users. The time interval since most

recent use ranged from 1 to 156 months, with a mean of 25.2 months,

and the mean period since peak use was 4.1 years (range 1 to 11

years). From the data obtained during this survey, it appears that

the use of nitrite inhalants within the gay community has decreased

appreciably in recent years. Health-related concerns were the most

frequently cited reasons for the discontinuance of the substance.

DISCUSSION

Ongoing surveillance in Baltimore afforded the opportunity to monitor

changing patterns of nitrite inhalant use among substance abusers.

Over the period since 1981 (a time which predates the AIDS epidemic)

through 1996, there appears to have been no change in the patterns

of nitrite use by substance abusers. Consistently, 11 percent of

recreational abusers participating in NIDA-ARC research protocols

have reported recent use of nitrite inhalants, as have approximately

20 percent of heavier drug users enrolling in treatment. Although it

is difficult to rule out homosexual behavior among male substance

abusers in the study, the use of nitrites by female substance abusers

suggests that male homosexuals are not the only users of nitrites.

Even though some evidence is emerging suggesting that parenteral

drug abusers are attempting to procure new needles when they are

available (Des Jarlais et al. 1985), there is still a basic recalcitrance

to modify their drug use in the face of the risk of overdose death,

or their needle-sharing behavior in the face of the AIDS epidemic.

Consequently, there is probably little reason to anticipate that this

high-risk group will modify its use of nitrites out of concern for the

effects of these substances on the immune system.

There appear to be ethnic as well as geographic variations in nitrite

abuse, and in some of the locations studied, these factors may

confound each other. For example, Denver and San Antonio had the

highest frequencies of use of poppers, and the proportions of these

92
samples which were white were 60 percent and 62 percent, respectively. New York City had the lowest frequency Of use, and that

sample was only 18 percent white.

Within populations that have, a propensity to misuse a wide spectrum

of drugs, nitrites appear to be substances whose abuse begins

relatively late in life. This may reflect either a delayed availability

or a delayed popularity of nitrites. Butyl nitrites were not generally

available before 1970; therefore, they were not available during

teenage years for men and wormen currently in their thirties and forties. Whereas the prevalence of nitrite inhalant use among substance

abusers has remained constant in addition to being initiated late,

other inhalant use such as glue sniffing has classically been a

phenomenon Of young abusers; and, baaed on ongoing surveillance by

the ARC, its abuse in the Baltimore area appears to be waning

appreciably.

The natural history of addiction included what has been referred to

as the “maturing-out phase” (Winick 1972). For a variety Of reasons,

many long-term, hardcore addicts either become abstinent or manifest

marked changes in their drug use patterns beginning in their late

twenties and continuing through their thirties. Many will abuse

alcohol more heavily during this period. The late use Of nitrites

suggests that these substances may, in sime drug users, be associated

with this process.

Within the homosexual population surveyed in the BaltimoreWashington, DC, area, the current level of nitrite use (21 percent) is

the same as that observed in heavy drug users in the same locate (22

percent). The observation that nitrite use in the gay community is

waning is encouraging; however, it is of concern that many

homosexual men who continue to use nitrites do so for the

euphorigenic properties, and will often use poppers with other sub

stances of abuse, notably marijuana. A has been observed that

homosexual men who use recreational drugs are significantly more

likely to engage in high-risk sexual practices capable Of transmitting

HIV infection than are those who do not (Polk, unpublished data).

That both substance abusers and homosexual men use nitrite inhalants

primarily to get high, and that both appear to have much more

experience with amyl nitrite rather than the butyl derivative, were

unexpected findings.

We conclude that heterosexual drug abusers in the Baltimore area do

not appear to have modified their nitrite use appreciably in recent

93
years. however, data from this sample, which admittedly may not be

representative of the overall homosexual community, would suggest

that homosexual males probably have decreased their use of nitrite

inhalants. This appears to be a result of a greater awareness of

AIDS risk in this population, and a consequent trend to modify their

high-risk behavior.

REFERENCES

Brunton, T. On the use of nitrite of amyl in angina pectotis.

Lancet 2:97-98, 1867.

Centers for Disease Control. Pneumomcystis pneumonia-Los Angeles.

MMWR 36:250-252, 1981.

Des Jarlais, D.C.; Friedman, S.R.: and Hopkins, W. Risk reduction for

the acquired immunodeficiency syndrome among intravenous drug

users. Ann Intern Med 163:755-759, 1985.

Everett, G.M. Effects of amyl nitrite (“poppers”) on sexual experience. Med Aspects Hum Sexuality 6:146, 1972.

Israelstam, S.; Lambert, S.; and Oki, G. Poppers, a new recreational

drug craze. Can Psychiatr Assoc J 23:493-495, 1978.

Labatailte, L. Amyl nitrite employed in homosexual relations. Med

Aspects Hum Sexuality 9:122, 1975.

Louria, D.B. Hazards of sniffing amyl nitrite during sexual intercourse. JAMA 236:1622, 1976.

Nickerson, M.; Parker, J.O.; Lowry, T.P.; and Swenson, E.W. Isobuyl

Nitrite and Related Compounds. San Francisco: Pharmex, Ltd.,

1979. 95 pp.

Sigell, LT.; Kapp, F.T.; Fusaro, G.A.; Nelson, E.D.; and Falck, R.S.

Popping and snorting volatile nitrites: A current fad for getting

high. Am J Psychiaty 135:1216-1218, 1978.

Winck, C. Maturing out of narcotic addiction. Bull Narc 14:1-7,

1972.

AUTHORS

W. Robert Lange, M.D., M.P.H.

Elizabeth M. Dax, M.D., Ph.D.

Charles A. Haertzen, Ph.D.

Frederick R. Snyder, Ph.D.

Jerome H. Jaffe, M.D.

94
Addiction Research Center

National Institute on Drug Abuse

Francis Scott Key Medical Center

Baltimore, MD 21224

95
Epidemiologic Studies-Kaposi’s

Sarcoma Vs. Opportunistic

Infections Among Homosexual Men

With AIDS

Harry W. Haverkos

INTRODUCTION

Kaposi’s sarcoma (KS) is an important manifestation of AIDS. It is

the most common cancer reported among AIDS patients and its

occurrence among gay men in New York and California was one of

the earliest harbingers of the AIDS pandemic (Centers for Disease

Control 1991).

KS associated with AIDS is unique both clinically and epidemiologically. Unlike “classical” (or non-AIDS-related) KS among elderly

men, or “endemic” KS in some parts of Africa, KS in patients at risk

for AIDS is associated with human immunodeficiency virus (HlV)

infection and cellular immune impairment (Biggar et al. 1984; Stahl et

al. 1982). Furthermore, KS occurs much more commonly among gay

men than among non-gay AIDS patients and is the only AIDS-related

illness in which the proportion of cases has declined (Haverkos et al.

1985; Haverkos 1987b).

Nitrite inhalants were investigated as a possible cause of AIDS early

in the epidemic, partly because of the preponderance of homosexual

men who used nitrites among the early patients with AIDS. During

pilot testing of questionnaires in 1981, Centers for Disease Control

(CDC) investigators found that nitrites were used by nearly all

homosexual men with AIDS. In addition, a 1981 survey of 420 men

attending sexually transmitted diseases clinics showed that homosexual men reported use of nitrites far more frequently than did

heterosexual men, and that the amount of use directly correlated with

the number of different sexual partners (Centers for Disease Control

1982). In 1982, Marmor and colleagues repotted significant associations between use of amyl nitrite and sexual activity, and the

96
development of AIDS (all 20 patients in the study had KS) among

homosexual men (Marmor et al. 1982).

On the other hand, when Marmor et al. reanalyzed their data

incorporating other factors and using multivariate analysis, they found

that still other variables, including frequency of receptive anal

intercourse and antibody titers to cytomegalovirus appeared to

differentiate patients with AIDS from controls (Marmor 1984; Marmor

et al. 1994). Although AIDS patients used greater quantities of

nitrites than did controls in the initial CDC study, Jaffe showed that

nitrite inhalant use did not appear to be important in distinguishing

patients with AIDS from controls. Measures of sexual activity were

the best markers for AIDS in that study (Jaffe et al. 1993). In

addition, studies conducted by that National Institute for Occupational

Safety and Health (NIOSH) for the CDC showed no immunotoxic

reactions in mice exposed to isobutyl nitrite vapors (Lewis et al.

1985).

This paper reviews six epidemiologic studies conducted among gay

men with AIDS to examine the role of nitrite inhalants, not as a

cause of AIDS, but as a cofactor in the development of KS as a

major manifestation of AIDS. Aspects of each study reviewed

include: Basic study design, wording of the questionnaire regarding

nitrite inhalant use (kindly provided by each of the investigators),

and published results comparing nitrite inhalant use of KS patients

compared wlth those who developed opportunistic infections and not

KS. All six studies included questions about sociodermographic

variables, medical history, use of drugs, and sexual history. Patients

were invited to provide Mood and/or other specimens for laboratory

analysis. More details about the six studies are available in the

references.

REANALAYSIS OF CDC STUDIES

Basic Design: Eighty-seven gay men with AIDS (47 with KS, 20 with

Pneumocysts carinii pneumonia (PCP), and 20 with both diseases) had

participated in 3 different studies in 1981 and 1982 conducted by CDC

in large metropolitan areas. Patients were interviewed by CDC

personnel. HIV testing was not performed.

Questionnaire: There were 10 questions about nitrite inhalant use on

the questionnaires. Patients were asked if they had ever used

inhalant sexual stimulants or “poppers” prior to the onset of illness.

If patients responded “yes” to the first question, they were asked to

97
give the calendar year they first used them, the last year they used

them, how many months during that time period they used them, how

many days per average month of use, and the number of “sniffs” per

average day (or night) of use. Questions about the location of use

(bathhouses, discos, Other) and types of nitrites (ampules, labeled

bottles, unlabeled bottles) were asked. Respondents were asked to

identify specific brand names of labeled bottles used and to identify

Where they purchased unlabeled bottles of nitrites.

Results: Eighty-four of the 87 (97 percent) AIDS cases in the studies

reported nitrite inhalant use. The median frequency of use was 394

days. Sixty percent of KS patients and those with both diseases used

nitrites more than 394 days compared with 10 percent of PCP

patients. Compared with PCP patients, KS patients and those with

both diseases reported more sexual partners, more “recreational” drug

use, higher incomes, and higher rates of non-B hepatitis.

Multivariate analysis showed that the variable most strongly

associated with KS was the use of large quantities of nitrite inhalants

(Haverkos et al. 1985b).

MOUNT SINAI SCHOOL OF MEDECINE, NEW YORK

Basic Design: A longitudinal study of 42 homosexual men with

persistent generalized lymphadenopathy was started at Mt. Sinai

School of Medicine in 1981. Over the 4 1/2 years of the study, 12

patients (29 percent) have developed AIDS, 8 with KS. Questionnaires were self-administered.

Questionnaire: The questionnaire contained eight questions about

nitrite inhalant use and was based on the one developed by CDC.

Mt. Sinai investigators deleted questions on the brand names of

labeled bottles and place of purchase of unlabeled bottles and

changed the question about location of use in bathhouses to ask

about use during sex or at discos.

Results: All 42 participants had used nitrites. The amount of nitrite

use was statistically associated with the development of KS (MathurWagh et al. 1984; Mathur-Wagh et al. 1985).

UNIVERSITY OF CALIFORNIA AT SAN FRANCISCO

Basic Design: An AIDS case-control study among gay men in San

Francisco started in 1993 and evolved into a longitudinal study. By

April 1986, 158 men in the initial cohort had developed AIDS, 108 had

98
KS. Trained interviewers administered the questionnaires. This study

is unpublished, but was presented at the First International AIDS

Conference in Atlanta (Osmond et al. 1985) and at a National

Institute of Allergy and Infectious Diseases-sponsored meeting in

April 1986 (Haverkos 1987a).

Questionnaire: The questionnaire contained seven questions about

nitrite inhalant use: first and most recent nitrite use, number of

days or nights of use during an average month, and number of “hits”

on a typical day or night of use. lt also asked about the amount of

use in the last 30 days and last year.

Results: Patients with KS were 2.2 times more likely to report

greater than four “hits” per night than patients with opportunistic

infections reported. Patients with KS, compared to patients with

opportunistic infections, were more likely to report a large number of

sexual partners, practice analingus, use large quantities of

recreational drugs by routes other than intravenous, and receive

metronidazole therapy for intestinal parasites. Patients with

opportunistic infections were more likely to report syphilis and to use

drugs intravenously than were patients with KS. Multivariate analysis

showed that the variable most strongly associated with KS was the

use of more than four “hits” of nitrite inhalants per night of use.

NATIONAL CANCER INSTITUTE

Basic Design: Two hundred forty-five men (85 from Manhattan and

160 from Washington, DC) were prospectively followed starting in

1982. Self-administered questionnaires were given to participants at

entry and at followup visits. Nineteen have developed AIDS, 8 of

whom had KS (Goedert et al. 1986).

Questionnaire: The questionnaire contained 15 questions about nitrite

inhalants. Patients were asked if they ever used nitrite inhalants

(“poppers”), what year they first used them, and to estimate the

number of days of use during the past 30 days. The participants

were asked to complete a grid containing four questions each for

their use in 1982, 1981, and 1980. They were asked to estimate the

number of days of use in each year, the number of sniffs on a

typical day of use in that year, the types of nitrites (unlabeled brown

bottle, “snapper,” brand name) used at least twice a month during

that year, and whether nitrites were used during sex acts, at other

times, or both for each year. The questionnaire contained a listing

of examples of brand names of labeled bottles.

99
Results: “Nitrite inhalant use, measured as frequency during the

previous year or intensity (number of sniffs) during the previous 6

months, was not significantly associated with AIDS, Kaposi’s sarcoma,

or Pneomocystis pneumonia (Goedert et al. 1986, p. 332).” Several

variabies were associated with the development of KS, including

receptive fellatio, frequent enemas/rectal douches, high levels of

antibody to hepatitis B antigen, and use of methaquaalone (Goedert et

al. 1986).

MULTICENTER AIDS COHORT

Basic Design: The Multicenter AIDS Cohort Study (MACS) is a

prospective study of homosexual men in Baltimore/Washington,

Chicago, Los Angeles, and Pittsburgh supported by the National

Institutes of Health (NIH). Four thousand nine hundred fiftymen were enrolled in 1984 and are evaluated every 6 months.

Questionnaires are administered by trained interviewers. One

thousand eight hundred thirty-five were HIV seropositive on entry, 59

have developed AIDS (24 with KS) during a median followup of 15

months (Polk et al. 1987).

Questionnaire: The questionnaire contains eight items concerning

nitrites and concentrates on use in the previous week, 8 months, and

2 years. Emphasis is also placed on the use of nitrite inhalants

during sex in the previous 6 months and 2 years.

Results: No association between nitrite inhalant use and KS was

reported. Significant associations for KS were found for decreased

hemoglobin level, decreased T-helper lymphocyte count, and increased

immunoglobulin A levels (Polk et al. 1987).

SAN FRANCISCO HEPATITIS B COHORT STUDY—CDC

Basic design: In 1984, followup of 6,700 homosexual men enrolled In

a 1978 hepatitis B study was initiated. Ninety-six of the men were

found to have AIDS (54 with KS) and were available for interview.

Questionnaires were administered by trained personnel (Darrow et al.

1986, Darrow et al. 1987).

Questionnaire: The questionnaire contained three questions about

nitrite inhalant use as follows: "Before your onset date for AIDS,

about how many days in an average month would you use nitrite

inhalants: 1. unlabeled bottles, 2. labeled bottles, and 3. ampules?”

100
Results: No differences in nitrite use were found between disease

groups. The development of AIDS in men infected with HIV may be

related to duration of infection, older age, and repeated exposures to

HIV.

DISCUSSION

The results of epidemiologic studies concerning nitrite inhalant use

and the development of KS among homosexual men with AIDS are

inconclusive. Of the six studies reviewed in this paper, three find a

strong association between larger quantities of nitrite inhalant use

and KS, and three do not confirm the association.

The questionnaires used in the six studies vary significantly in the

amount of information sought concerning nitrite inhalant use. Studies

begun in 1981 and 1982 asked more detailed questions about nitrites

than later studies asked, possibly because nitrites were considered a

possible cause of AIDS early in the epidemic. After HIV was

discovered as the cause of AIDS, epidemiologic studies tended to

focus more on sexual activities than on nonintravenous drug use. In

addition, nitrite inhalant use among gay men may be declining since

the AIDS epidemic (Lange et al., in press). lf so, questions asked in

1985 and 1986 about nitrite use in the previous 6 months or 2 years

cannot reflect accurately the amount of nitrite use at earlier times,

and, possibly more important, nitrite use at the time of HIV

infection.

The differences in outcomes of these studies may be due to

differences in study methods. The sample s&es of the studies are

small. The largest study evaluated 150 AIDS patients, 100 with KS

(Osmond et al. 1985). The smallest study evaluated 12 patients, 8

with KS (Mathur-Wagh et al. 1984). The various patient recruitment

processes may have introduced unsuspected selection biases. Several

of the studies used self-interviews, others used trained personnel to

administer the questionnaires. Although there is no evidence to

suggest that self-interviews are less reliable than face to face

interviews, information bias must be considered in any study involving

personal items, such as sexual and drug history.

There have been several hypotheses concerning the possible

mechanism by which nitrite inhalants may promote KS in users

infected with HIV. First, nitrite inhalants may act directly on the

immune system (Goedert et al. 1982). Second, nitrosamines, among

the possible metabolites of nitrites, are carcinogenic (Jorgensen and

101
Lawesson 1982). Interactions of nitrites and mouse skin lipids have

been shown to produce potentially carcinogenic substances (Mirvish et

al. 1986). Third, the vasodilatory action of nitrite inhalants may, in

some way, promote KS, a malignancy of the endothelial cells lining

blood vessels. Finally, use of nitrites may only be a marker for

another cofactor, e.g., an as-yet “unidentified” microorganism.

National Cancer Institute investigators have proposed a hepatitii Alike organism as the KS cofactor (Weiss and Biggar 1986).

Several epidemiologic observations suggest that an infectious cofactor

is less likely than nitrites. The “unidentified” virus must be highly

associated with gay sexual activity. This would imply a sexually

transmitted and/or bloodborne agent. Clearly the KS cofactor is not

boodborne. Blood donors who develop KS have transmitted HIV

infection to recipients, but those recipients do not develop KS

(Curran et al. 1984). There is no documented evidence that the

cofactor is sexually transmitted; there is no consistent pattern of KS

transmission among clusters of homosexual men linked by sexual

contact and developing AIDS (Auerbach et al. 1984). KS has not

been reported in a female partner of a bisexual man with KS. The

strong association of KS with increased income in one study is

consistent with the argument that a drug is more likely to be the

cofactor than a sexually transmitted agent (Haverkos et al. 1985b).

RECOMMENDATIONS

More epidemiologic and laboratory studies are needed to ascertain the

role, if any, of nitrite inhalants as a cofactor in AIDS-related KS.

Investigators need to develop questions that quantitate lifetime nitrite

exposure and measure dose more accurately. (The concept of “packyears” to quantitate cigarette use may be a useful model.)

Questioning heterosexual AIDS patients who have KS about nitrite use

may be helpful. Study of animal models infected with retroviruses

and challenged with large quantities of nitrites before, during, and/or

after retrovirus infection would be useful. Finally, the U.S. Public

Health Service could organize a multiagency task force to resolve this

issue as was done for aspirin use and Reye’s syndrome.

REFERENCES

Auerbach, D.M.; Darrow, W.W.; Jaffe, H.W.; and Curran, J.W. Cluster

of cases of the acquired immune deficiency syndrome: Patients

linked by sexual contact. Am J Med 76:487-492, 1984.

102
Biggar, R.J.; Melbye, M.; Kestens, L; Samgadharan, M.G.; de Feyter,

M.; Blattner, W.A.; Gallo, R.C.; and Gigase, P.L Kaposi’s sarcoma

in Zaire is not associated with HTLV-III infection. N Engl J Med

311:1051-1052, 1984.

Centers for Disease Control. Kaposi’s sarcoma and Pneumocystis

pneumonia among homosexual men - New York City and California.

MMWR 30:305-308, 1981.

Centers for Disease Control. Epidemiologic aspects of the current

outbreak of Kaposi’s sarwma and opportunistic infections. N Engl

J Med 308:248-252, 1982.

Curran, J.W.; Lawrence, D.N.; Jaffe, H.W.; Kaplan, J.E.; Zyla, LD.;

Chamberland, M.; Weinstein, R.; Kung-Jong, L.; Schonberger, LB.;

Spira, T.J.; Alexander, W.J.; Swinger, G.; Ammann, A.; Solomon, S.;

Auerbach, D.; Mildvan, D.; Stoneburner, R.; Jason, J.M.; Haverkos,

H.W.; and Evatt, B.L Acquired immunodeficiency syndrome (AIDS)

associated with transfusions. N Engl J Med 310:69-75, 1984.

Darrow, W.W.; Byers, R.H.; Jaffe, H.W.; O’Malley, P.M.; Rutherford,

G.W.; and Echenberg, D.F. Cofactors in the development of AIDS

and AIDS-related conditions. Abstract presented at the Second

International Conference on AIDS, Paris, France, June 1986. p. 99.

Darrow, W.W.; Echenberg, D.F.; Jaffe, H.W.; O’Malley, P.M.; Byers,

R.H.; Getchell, J.P.; and Curran, J.W. Risk factors for human

immunodeficiency virus infections in homosexual men. Am J Public

Health 77:479-483, 1987.

Goedert, J.J.; Bigger, R.J.; Melbye, M.; Mann, D.L; Wilson, S.; Gail,

M.H.; Grossman, R.J.; DiGioia, R.A.; Sanchez, W.C.; Weiss, S.H.; and

Blattner, W.A. Effect of T4 count and cofactors on incidence of

AIDS in homosexual men infected with human immunodeficiency

virus. JAMA 257:331-334, 1986.

Goedert, J.J.; Neuland, C.Y.; Wallen, W.C.; Greene, M.H.; Mann, D.L;

Murray, C.; Strong, D.M.; Fraumeni, J.F.; and Blattner, W.A. Amyl

nitrite may alter T lymphocytes in homosexual men. Lancet 1:412-

416, 1982.

Haverkos, H.W. Factors associated with the pathogenesis of AIDS. J

Infect Dis 156:251-257, 1987a.

Haverkos, H.W. immunosuppression, nitrite inhalants, and Kaposi’s

sarcoma. Clin lmmunol News 8:46-47, 1987b.

Haverkos, H.W.; Drotman, D.P.; and Morgan, M. Prevalence of

Kaposi’s sarwma among patients with AIDS. N Engl J Med

312:1518, 1985a.

Haverkos, H.W.; Pinsky, P.F.; Drotman, D.P.; and Bregman, D.J.

Disease manifestation among homosexual men with acquired

immunodeficiency syndrome: A possible role of nitrites in Kaposi’s

sarcoma. Sex Transm Dis 12:203-208, 1985b.

103
Jaffe, H.W.; Choi, K; Thomas, P.A; Haverkos, H.W.; Auerbach, D.M.;

Gutnan, M.E.; Rogers, M.F.; Spira, T.J.; Darrow, W.W.; Kramer, M.A;

Friedman, S.M.; Monroe, J.M.; Friedman-Kien, A.E.; Laubenstein,

LJ.; Marmor, M.; Safai, B.; Dritz, S.K, Crispi, S.J.; Fannin, S.L;

Orkwis, J.P.; Kelter, A; Rushing, W.R.; Thacker, S.B.; and Curran,

J.W. National case-control study of Kaposi’s sarcoma and

Pneumocystis carinii pneumonia in homosexual men: 1.

Epidemiologic results. Ann Intern Med 99:145-151, 1983.

Jorgensen, K.A., and Lawesson, S-O. Amyl nitrite and Kapcsi’s

sarwma in homosexual men. N Engl J Med 307:893-894, 1982.

Lange, W.R.; Dax, E.M.; Haertzen, C.A Snyder, F.R.; and Jaffe, J.H.

Nitrite inhalants: Contemporary patterns of abuse. Am J Drug

Alcohol Abuse, in press (1988).

Lewis, D.M.; Keller, W.A.; Lynch, D.M.; and Spira,T.J. Subchronic

inhalation toxicity of isobutyi nitrite in Balb/c mice: II. Immum

toxicity studies. J Toxicol Environ Health 15:835-848, 1985.

Marmor, M. Epidemic Kaposi’s sarwma and sexual practices among

male homosexuals. In: The epidemic Kaposi's sarcoma and

opportunistic infections. New York: Masson, 1984. pp. 291-296.

Marmor, M.; Friedman-Kien, A.E.; Laubenstein, L.; Byrum, R.D.;

William, D.C.; D’onofrio, S.; and Dubin, N. Risk factors for

Kaposi’s sarwma in homosexual men. Lancet 1:1083-1087, 1982.

Marmor, M.; Friedman-Ken, A.E.; Zolla-Pazner, S.; Stahl, R.E.;

Rubinstein, P.; Laubenstein, L.; William, DC.; Klein, R.J.; and

Spigland, I. Kaposi’s sarcoma in homosexual men: A

seroepidemiologic case-control study. Ann Intern Med 100:809-815,

1984.

Mathur-Wagh, U.; Enlow, R.W.; Spigland, I.; Winchester, R.J.; Sacks,

H.S.; Rorat, E.; Yancovitz, S.R.; Klein, M.J.; William, D.C.; and

Mildvan, D. Longitudinal study of persistent generalized

lymphadenopathy in homosexual men: Relation to acquired

immunodificiency syndrome. Lancet 1:1033-1038, 1984.

Mathur-Wagh, U.; Mildvan, D.; and Senie, R.T. Follow-up at 4 1/2

years on homosexual men with generalized lymphadenopathy. N

Engl J Med 313:1542-1543, 1985.

Mirvish, S.S.; Babcock, D.M.; Deshpande, A.D.; and Nagel, D.L

Identification of cholesterol as a mouse skin lipid that reacts with

nitrogen dioxide to yield a nitrosating agent (NSA), and of

cholesteryl nitrite as the NSA produced in a chemical system from

cholesterol. Cancer Lett 31:97-104, 1980.

Osmond, D.; Moss, AR.; Bachetti, P.; Volberding, P.; Barre-Sinoussi,

F.; and Chermann, J-C. A case-control study of risk factors for

AIDS in San Francisco. In: International Conference on Acquired

lmmunodeficiency Syndrome (AIDS), Atlanta, GA, April 14-17, 1985.

104
Polk, F.; Fox, R.; Brookmeyer, R.; Kanchanaraksa, S; Kaslow, R.;

Visscher, B.; Rinaldo, C.; and Phair, J. Predictors of the acquired

immunodeficiency syndrome developing in a cohort of seropositive

men. N Engl J Med 316:62-66, 1987.

Stahl, R.E.; Friedman, A; Dublin, R.; Marmor, M.; and ZollaPazner, S. Immunologic abnormalities in homosexual men. Am J

Med 73:171-178, 1982.

Weiss, S.H., and Biggar, R.J. The epidemiology of human retrovirusassociated illnesses. Mt Sinai J Med 53:579-591, 1986.

ACKNOWLEGMENTS

Dr. D. Peter Drotman, Centers for Disease Control, reviewed the

manuscript.

AUTHOR

Harry W. Haverkos M.D.

Chief, Clinical Medicine Branch

National Institute on Drug Abuse

Parklawn Building, Room 10A-08

5600 Fishers Lane

Rockville, MD 20857

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40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES.

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42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS.

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48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF SMOKING BEHAVIOR. John Grabowski, Ph.D., and Catherine S. Bell, M.S.,

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57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING

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63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG

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71 OPlATE RECEPTOR SUBTYPES AND BRAIN FUNCTION. Roger M.

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72 RELAPSE AND RECOVERY IN DRUG ABUSE. Frank M. Tims,

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74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG ABUSE.

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75 PROGRESS IN OPIOID RESEARCH. PROCEEDINGS OF THE 1986

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76 PROBLEMS OF DRUG DEPENDENCE, 1986. PROCEEDINGS OF

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78 THE ROLE OF NEUROPLASTlClTY IN THE RESPONSE TO DRUGS.

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IN PRESS

77 ADOLESCENT DRUG ABUSE: ANALYSES OF TREATMENT

RESEARCH. Elizabeth R. Rahdert, Ph.D., and John Grabowski, Ph.D.,

eds.

80 NEEDLE-SHARING AMONG INTRAVENOUS DRUG ABUSERS:

NATIONAL AND INTERNATIONAL PERSPECTIVES. Robert J. Battjes,

D.S.W., and Roy W. Pickens, Ph.D., eds.

81 PROBLEMS OF DRUG DEPENDENCE, 1987. PROCEEDINGS OF

THE 49TH ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

111
82 OPIOIOS IN THE HIPPOCAMPUS. Jacqueline F. McGinty, Ph.D.,

and David P. Friedman, Ph.D., eds.

112

U.S. GOVERNMENT PRINTING OFFICE : 1988 - 215-721 : QL 3
DHHS Publication No. (ADM)89-1573

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1988, Reprinted 1989