o  More data are also needed on cigarette flavor additives and their
combustion products. Flavoring agents and additives should be
studied by cigarette companies for carcinogenicity and toxicity
before their commercial use is permitted, and the results of such
  studies should be made available.

o  Research should be done on the distribution, partitioning, and
penetration of lower "tar" and nicotine cigarette smoke in the
lung, with consideration of potential changes in smoking
patterns by those who smoke lower "tar" and nicotine ciga-
  rettes. Cigarette smoking-machines currently in use and the
techniques by which animals inhale cigarette smoke in research
models may not be representative of the human situation
because human smokers are able to take larger, more frequent,
and higher velocity puffs. To conduct meaningful assays of
cigarette yields and the biological activity of cigarette smoke, it
must be determined how smokers actually smoke various types
of commercial cigarettes. When this information is available, it
  will be possible to design smoking-machines that yield more
  accurate estimates of human risk.

o  Controlled studies are needed to determine the role of nicotine
  as a primary reinforcer in cigarette smoking and to determine
  whether there are other chemicals in addition to nicotine that
may contribute to or reinforce the smoking habit. By analyzing
  the mechanisms whereby nicotine reinforces smoking behavior,
  it may be possible to design more efficacious methods of
  smoking cessation.
o  Research should be conducted to define what effects modifica-
  tions of the physical and chemical properties of leaf tobaccos
  have on the pharmacology of cigarette smoke. Since tobacco
  culturing and curing practices are continually changing, it is
  important to determine whether such changes as the use of new
  pesticides also alter the composition and biological activity of
  cigarette smoke.

o Standardized experimental cigarettes have frequently proved
unpalatable and unacceptable for behavioral research. Proto-
type cigarettes should be especially designed to deliver a wide
range of constituent concentrations, particularly those that
approximate commercial cigarettes. This would allow research-
ers to predict the behavior of smokers of new types of cigarettes
more accurately.

26


Section 2. PHARMACOLOGY AND
       TOXICOLOGY


CONTENTS

Introduction

Experimental Systems for Assay of Relative Risks of
Cigarette Smoking
Lung Cancer
     Animal Models

Lung Carcinogens in Cigarette Smoke
Polycyclic Aromatic Hydrocarbons
Tobacco-Specific N-Nitrosamines
Other Mutagenic or Co-mutagenic Agents
Weak Acids
Nicotine
Polonium 210

    Volatile N-Nitrosamines
Bladder Cancer
Laryngeal Cancer
Other Cancers

Early End Points Suggestive of Carcinogenic Potential
Chronic Obstructive Lung Disease
Sudden Death Due to Cardiovascular Disease
   Animal Models
   Nicotine
  Carbon Monoxide
  Other Agents

Complications of Pregnancy and Early Childhood
Nonspecific End Points of Toxicologic Significance
  Reduction of Lung Defense Mechanisms
  Induction of Microsomal Oxidase
  Changes in Genetic Status
  Changes in Immune Status

composition of Smokes From Various Types of Cigarettes
Smoking-Machine Design
Dependence of Smoke Composition on Cigarette Design
     Filters
     Ventilation
    Tobacco Variety
    Agricultural Practice
    Reconstituted Sheet and Modified Tobaccos

29


Additives

Variations in Human Smoking Behavior

Research Needs

Surveillance of New Cigarettes
Determination of Parameters of Human Cigarette
Smoking

Evaluation of Health Effects of Nicotine
The Effects of Smoking-Machine Parameters on
Relative and Absolute Yields of Smoke Components
From Various Types of Cigarettes
Influence of Raw Product Modification on
Pharmacology of Cigarette Smoke
Physical and Chemical Properties of Smoke From
Cigarettes Delivering Less Than 10 mg of "Tar"
Development and Validation of Analytical Methods
Other Research Needs

Summary

References

LIST OF FIGURES

Figure l.-Effect of cigarette smoke differing in selected
chemical components on pancreatic elastase levels in
beagle dogs after a f5OO-day exposure protocol of 12
cigarettes per day, 7 days per week

LIST OF TABLES

Table I.-Major toxic agents in the gas phase of cigarette
smoke (unaged)

Table 2.-Major toxic agents in the particulate matter of
cigarette smoke (unaged)


Table 3.Ahefficient.a and standard deviations of
coefficients for Prediction Model 10

Table 4.-Comparison of mutagenic and tumor-promoting
activity of fractions of cigarette smoke condensate

31


lntroductlon

Tobacco and tobacco smoke are very complex mixtures. In 1968,
Stedman (155) reported that they contained more than 1,200 clearly
identified substances in addition to a number of polymer classes, such
as pigments, resins, and proteins, that were not resolved into specific
compounds. Since that time, many additional compounds have been
isolated; at least a thousand additional constituents were found in
tobacco and tobacco smoke in the following 10 years (67). Cigarette
smoke components arise through distillation of volatile and semivola-
tile materials from the leaf and from the pyrolytic decomposition of
leaf constituents. In addition, nonvolatile components of tobacco leaf
can be transferred to the smoke without degradation. Thus, the
components of smoke are very diverse. Many suspected or proved toxic
agents have been identified in the gas phase (Table 1) or in the
particulate matter (Table 2) of smoke (290). It is not surprising that
chronic exposure to such a complex mixture will lead to a variety of
pharmacologic and toxicologic responses.

TABLE l.-Major toxic agents in the gas pham of cigarette
      smoke (unaged)*

Di~thylnitmaamine
EthYlmethytnitroMmine
Diethylnitmsandne
Nitnxopyrrdidine
other nitmoaminen
(4 compotmde)
Hydrrdw
Vinyl chloride
urethane
Folldd&@
Hydmseneyanide
Aaolein
Aeetaldehyde
Nitrogen oxides (NO&
Ammonia
Pyridii
Carton monoxide

C
C
TI
CT Qc
`X T
Ei
T
T?d
TM
T


TABLE 2.-Major toxic agents in the particulate matter of
     cigarette smoke (unaged)*

B-+h~
bbiethmne
Ber&jjuonulthene
Bellr+~thInceDe
other polymlclear erometic hydm
carbons (>26 compound@
JXben&jJecridhe
Diben&h~dine
Dibe.nz&gJcuhemie
m=
Fl~OthlX
B-ofaimw
other polymldsv -tic by&o-
carbons (>lO compounds)
NapMtmlenea
l-Methylindokd
MbhthylarbsFales
Other neutrel compoundn
catechol
b & CMethyka~
0th c&e&oh (>4 mmpounde)
U&OtWlphC3ldSIWldhd6
N'-Nitmmnombtine
other nonvoletik nitmeeminea
&N~htbyluniM
OthW-tiCMUilE8
Unknown nitro compound8
Polonium-210
Niid mmpour&
Cedmium compounds
Aneaic
Nkotine
Mioor t4baan 8lkabida
I%mol
Crcaols (3 eompouoda)

TI
TI
TI
TI


TI
TI
TI
TI
ac
cot
ccc


cd:
cot
COG
cd2
cot
cd2
ccc
cot
cd2
C
C
Bc
Bc
Bc
C
C
C
C
T
T
CT
CT

  ?
l-10 M
0.349 H
o.m.2 pg
  ?

u)-r16ocB
80-4oM
?
?
loo-250 !q
?
o-26 w
?
?
0.62-1.2 pci
1@600 w
9-70 ng
l-26 M
0.1-20 mg
0.01-02 mg
l&206 #tg
10-166 R

?
6 M
0s P2
0.1 pg

Experimental Systems for Assay of Relative Risks of Cigarette
Smoklng
Lung Cancer

Animul Models

The mouse skin carcinogenesis assay is thus far the most fruitful
method of evaluating smoke condensates from different types of
cigarettes for carcinogenic potency for the human lung (46,51,89,106).

34


This model for the development of cancer dates back to 1915 (191). A
large body of laboratory experience has provided consistent evidence
for the quantitative validity of this relationship. Procedures providing
good dose-response relationships are in use in many laboratories.
Assays can be standardized to give relatively consistent results within
a laboratory, and probably among laboratories (62, 63,64, 65).
The assay depends on a number of similarities between the
laboratory model and human experience. The epithelium of both the
skin and lung is directly exposed to the presumptive carcinogenic
agent-in this case, cigarette smoke or cigarette smoke condensate.
Babbit and mouse skin develop tumors after exposure to coal tar, a
known occupational carcinogen. Mouse skin assays have predicted
occupational induction of human lung cancer by bis-chloromethyl ether
w, 170.

It is conceivable that the mouse skin carcinogenesis assay may give a
misleading measure of the relative risk of various types of cigarettes.
Skin is covered with a lipid film, and the pilo-sebaceous apparatus is
particularly suited for penetration of lipid materials into the skin. In
contrast, the airway surface is covered by an aqueous film and might
be less readily penetrated by fat-soluble materials. There is no
evidence, however, that such a difference is important. Indeed, the
response of mouse skin to different types of experimental cigarettes is
roughly parallel to the response of hamster larynx to the same
materials (49,50,189).
The hamster larynx has been used for comparative studies of
different types of cigarettes (17, 50, 52). Invasive carcinomas of the
larynx were induced in 37 percent of inbred hamsters exposed to
cigarette smoke for 59 to 30 weeks. Both the cancer incidence and the
incidence of other epithelial changes were dose related. Exposure of
rats and mice to cigarette smoke for up to 21/2 years resulted in a small
incidence of respiratory tract tumors, primarily pulmonary adenomas
(44, 68, 72). Cigarette smoke `produced changes in cultured human
gastric epithelial cells suggestive of malignancy (158).

Experience in man and with the mouse skin system indicates that
two or more distinct classes of carcinogenic stimuli lead to the
occurrence of tumors (16, 26, 48). Tumor initiators appear to alter the
genetic constitution of the cell; tumor promoters accelerate and
enhance the neoplastic expression of previously initiated cells. Both
may play a role in the induction of tumors. Other types of coca&n+
gens may also play a role in the induction of mouse skin tumors by
cigarette smoke condensate (16, 74,89,176). If similar mechanisms act
in man, it may not be possible to differentiate between a human
carcinogen in the conventional sense and a cocarcinogen or tumor

35


promoter acting on a diverse population already exposed to low levels
of a variety of tumor initiators.

Two prominent classes of tumor initiators are found in smoke
condensates of commercial cigarettes-polycyclic aromatic hydrocar-
bons (PAH) and tobacco-specific nitrosamines (TSNA), Other car&+
gens or tumor initiators are present in cigarette smoke as well;
however, they appear to be less significant because they either are less
potent or are present at lower concentrations than are PAH or TSNA.

Polycyclic Aromatic Hydrocarbons

A large variety of PAH molecules are formed by the pyrolytic
process during combustion of the cigarette (87, 105). Of the PAHs,
be&a&rene (BaP) is the most prominent and has been studied most
intensively. Chemical assays for BaP in smoke condensates are well
established, and it has been suggested that such assays can serve as
indicators of production of all of the PAHs. This appears to be
generally true. Among smoke condensates from 98 experimental
cigarettes,  the correlation  coefficient between BaP and
bem$a]anthracene content was 0.78 (15). Although highly signiicant,
the value is sufficiently low to indicate that real differences do exist in
the ratios of these cyclic molecules in the various cigarette smokes.
Nevertheless, BaP appears to be the most important single member of
this class of compounds, taking into consideration both its concentra-
tion and its relative carcinogenic potency.

The contribution of BaP or PAH in general to mouse skin
carcinogenesis by cigarette smoke condensate cannot be fully mea-
sured at this time. Wynder and Hoffmann (188) found a correlation
between BaP levels and carcinogenic activity of smoke condensates
from several types of cigarettes. A much larger series of experimental
cigarettes was studied in the smoking and health program of the
National Cancer Institute. No significant dependence of carcinogenic
potency on BaP content was observed (6&63,64,65). The relationship
between chemical composition of the experimental smoke condensates
and the biological activity of this series was examined extensively by
Bayne (15). He employed the linear terms, squared terms, and all
interaction terms between any 2 of 10 independent variables. Starting
with a 66-&m regression equation, he searched for simpler prediction
models that would provide useful estimates of carcinogenic activity.
The simplest model (Table 3) that retained good predictability
contained nine terms. The interaction of BaP with the nicotine term
was one that appeared important.

BaP and other tumor initiators are particularly important because
humans are already exposed to a number of initiators in the
environment. The effect of initiators is cumulative and irreversible.
Hence, any additional exposure to initiators such as the PAH might be
expected to increase tumor incidence in smokers.

36


TABLE 3.-Coeffkienta and standard deviations of coefficients
      for Prediction Model 10

                                        t3taDdIud deviatioo
       Terms.             Coefficienta            of aleffiientd


   1 Intercept                2.667                 0292
   2C                   4.792 E-2              0.234 Is2
    2cI                   4.66s E-4               0.406 El
   1 PH                   4.464 E-l              OMO El-1
    5vwA                  1.242 E-l               0.555 Fe-1
    6NxN                245oEl-5              0.668E-5
   7 pH x pH               2.662 J3-2              0.875 E-2
   8NxpH                -7.078 E-t               1.664 E-4
    9NxBAP               -1.T70 EL9              0.2TIE4
bs:; Cbmmhdon (m&&y); VWA-very wed mcih (u&g); N-nicoh (n@g); and BAP-bearo[+ymm

Born Bayme (26).

Tobacco-Specific N-Nitrosamines

During tobacco curing, fermentation, and burning, nornicotine gives
rise to N'-nitrosonomicotine (NNN), nicotine to NNN and to 4-(N-
methyl-N-nitrosamino)-l-(3-pyridil)-1-butanone (NNK), and anatabine
to N'-nitrosoanatabine (NAT). NNN is a moderately active carcinogen,
inducing tumors in the respiratory tract of mice, rats, and hamsters.
NNK is a strong carcinogen, inducing lung carcinoma in each of the
three animal species (75, 84, 86). The concentration of these carcino-
gens in cigarette smoke is very high in comparison with usual
environmental exposures, being 1 to 35 ppm in tobacco and 1 to 9 erg in
the smoke of a cigarette (57). These tobacco-specific N-nitrosamines
may play a role in the development of several types of human cancer.
NNN is metabolically activated by human liver microsomes (76) and,
together with NNK and NAT, may be formed in wivo from the tobacco
alkaloids.

Other Mutagenic or Co-mutagenic Agents

It is generally believed that tumor initiators are mutagens that can
be detected by one or more short-term biological assays (2, 103). A
number of fractions of cigarette smoke condensate are positive in the
Ames assay system (93, 101). The agents responsible for this activity
have not been fully identified, but probably include products of protein
pyrolysis (119). Ames test activity, however, does not predict the
activity of fractions in the mouse skin carcinogenesis assay. Fractions
of smoke condensate that show activity as complete carcinogens (89) or
in a promotion assay that would detect skin carcinogens as well as
tumor promoters (24) are not correspondingly active in the Ames
system (Table 4). It cannot be determined whether the unidentified
mutagens in cigarette smoke are an important cause of lung cancer in

37


TABLE 4.-Comparison of mutagenic and tumor-promoting
     activity of fractions of cigarette smoke condensate

whole eondeneate
Beeollatiblted
Bmea before, insoluble
Baaee after, iduble
Bmen, ether soluble
Bass, water soluble
week acid.9$ iMobible
Weak ad4 e&r soluble
Strong acidq iaaohble
Strong aci4 ether soluble
strong aei& water Eoluble
Neutrala, 80% methuwl dubie
Neutde, cyclobexane mluble
Neutrala, nitrome~ duble

100
89
21
26
11
1
30
5
2
<1
<2
2
51
2

humans; however, added exposure to any tumor initiators probably
carries an incremental risk of cancer.

Weak Acids

Cigarette smoke contains weak organic acids that exhibit tumor-
promoting or cocarcinogenic activity (24,74,176). The concentration of
very weak acids in cigarette smoke condensates was one of the terms
predictive of the skin carcinogenic activity of smoke condensates
(Table 3). Of the weak acids, catechol appears to be the most important
on the basis of concentration and activity (74,176).

It is probable that the weakly acidic constituents of smoke act as
tumor promoters or cocarcinogens rather than as tumor initiators, This
is true for phenols and for catechol (27, 176). There is no reason to
believe that tumor promoters or other types of cocarcinogens exhibit
either a cumulative or an irreversible effect. Indeed, for tumor
promotion in mouse skin by croton oil, clear thresholds for frequency of
application and for the amount of promoter in each applied dose are
apparent (26). If this is also true for man, the risk of very small doses
of weak acids might be negligible. Phenol (1.26, 188), but not catechol
(29), can be selectively removed by filters. The extent to which the
cocarcinogenic weak acids are reduced by selective filtration cannot be
determined at this time.

33


Nicotine

Nicotine exhibits neither complete carcinogenic activity nor tumor-
promoting activity. The nicotine content of cigarette smoke condensate
did not affect its carcinogenic activity when suspended in beeswax-
tricaprylin pellets implanted in rat lungs (48); however, in mouse skin
bioassays, this alkaloid is an important cocarcinogen (20). Not only is
nicotine active in models with other compounds such as BaP and 12-O-
tetradecanoylphorbol-X%-acetate (TPA), but also the measured carcino-
genic potency of cigarette smoke condensates appears to depend on the
nicotine content of the "tar." Of all of the individual compounds of
smoke condensates assayed in the smoking and health program of the
National Cancer Institute, nicotine was most closely related to
carcinogenic activity (62, 63, 64, 65). In the simplest predictive model
developed by Bayne, every term but one involved nicotine concentra-
tion, pH, or the concentration of crude condensate (Table 3). The
availability of nicotine to the tissues depends on the pH and concentra-
tion of condensate. Hence, available nicotine was a factor of all but one
term of the prediction model.

Nicotine may also play a role in the development of oral cancer in
tobacco chewers. Aqueous extracts or unburned tobacco exhibit tumor-
promoting activity when tested on mouse skin. This activity depends
on the presence of nicotine acting together with a fraction having a
molecular weight greater than 13,000 daltons (21). In addition, nicotine
gives rise to carcinogenic N-nitrosamines during tobacco chewing (84).

Data of Morosco and Coeringer (122) suggest that nicotine reduced
serum alpharantitrypsin activity and elevated pancreatic elastase
levels in dogs exposed to cigarette smoke. These workers believe that
interference with the protease-protease inhibitor balance may be a
factor in carcinogenesis (123).

It must be pointed out that the relationship between carcinogenic
activity of smoke condensates and their nicotine contents may be
caused in part by the conversion of nicotine to tobacco-specific
nitrosamines or to the co-occurrence of nicotine and some other
unidentified carcinogen. For example, the nicotine level of tobacco is
dependent on the amount of nitrate fertilizer used in tobacco culture
(166). High levels of tobacco-specific nitrosamines were found in the
unburned tobaccos usually raised with high levels of nitrogen fertilizer
(77). The level of volatile nitrosamines in cigarette smoke also depends
on nitrate fertilizer (170). One may postulate that the nicotine level of
cigarette smoke condensates is an indicator of such nitrogenous
carcinogens that were not measured directly. At present, however,
there is no direct evidence that this is the case. In any event, the
carcinogenic activity of mixtures of pure BaP and TPA are enhanced
by the concomitant application of nicotine under conditions such that
nitrosamine formation would not be expected (20).

39


Whether the cocarcinogenic effects of nicotine are important for
man is a matter of speculation. Tumor-promoting activity of croton oil
exhibits a threshold both for frequency of application and for the
quantity of agent present with any given treatment (26). The animal
studies in which nicotine acts as a cocarcinogen employ nearly lethal
levels of nicotine administered once or twice a day. In contrast,
smokers are exposed to a large number of low doses of nicotine daily. If
a threshold amount of nicotine per dose is required for cocarcinogenic
activity, human smokers may not be affected in a manner similar to
that of the mouse skin system.

Polonium 210

There have been repeated suggestions that "PO might contribute to
the carcinogenic activity of cigarette smoke in man (137). Polonium
levels in tobacco result primarily from the use of phosphate fertilizers
that are contaminated with radium decay products, particularly mPb,
a precursor of ~OPO (162,168). Very little uOPo is found in tobacco leaf,
but some is transferred to the smoke. Yields of 10 to 15 fCi of alpha
emitters were recently reported for experimental cigarettes and 490
fCi/gm for commercial cigarette smoke condensate (36). Most of the
radioactivity was due to insoluble forms of ~Po. Cancer may arise
from a single affected cell. It has been suggested that small amounts
of insoluble nOPo concentrated in small areas might deliver an effective
carcinogenic dose to a target cell (112). Harley et al. (71), however,
found very few "hot spots" in the lungs of deceased smokers. Based on
human experience with radon daughters, they assumed a lifetime risk
of lung cancer of 1 x 10-Z for a dose of one rad/year. At most, the
radioactivity they detected was estimated to explain only 10 percent of
the lung cancers suffered by cigarette smokers. They consider
polonium 210 a questionable risk factor in human carcinogenesis.

Polonium 210 contamination of tobacco can be effectively reduced by
selection of plant types and sources of phosphate fertilizer, and by
removal using chelating agents (71,171).

Volatile N-Nitrosamines

Tobacco smoke contains a number of secondary and tertiary amines.
These amines, together with nitrogen oxides, may give rise to the in
&uo formation of nitrosamines. Although the formation of most
nitrosamines is favored at low pH (llO), a small amount of volatile
nitrosamines is found in cigarette smoke and may be formed in the
lungs under normal conditions (30, 84, 170). The volatile N-nitrosa-
mines are organ-specific carcinogens, which in mice give rise to tumors
of the liver and kidney. At present, there is no reason to assume that
volatile nitrosamines cause lung cancer in smokers. Nevertheless, it is
prudent to limit the presence of any carcinogen in cigarette smoke.


Volatile nitrosamines in smoke can be reduced by selective filtration
and by limiting the nitrate content of tobaccos (SO, 121).

Bladder Cancer

The induction of bladder cancer in animals has been studied
intensively over the past several decades. The bladder appears to be a
particularly sensitive target for agents that are metabolized in the
liver and excreted in the urine. Among the compounds known to
produce bladder cancer in both man and animals is P-naphthylamine.
The presence of Snaphthylamine in cigarette smoke has been demon-
strated (85), along with other carcinogenic aromatic amines (129). The
yield was so low, however, that they did not believe these agents
contributed significantly to the risk of bladder cancer in smokers.
The urine of 10 smokers and 21 nonsmokers was examined by
Yamasaki and Ames (192) for mutagens or for substances that were
converted to mutagens by rat liver microsomes. Increased levels of
mutagens were found in the urine of seven smokers, but in none of the
nonsmokers. If promutagens in urine are responsible for the bladder
cancers occurring in cigarette smokers, it is possible that certain
individuals are particularly sensitive to bladder carcinogenesis by
cigarette smoke. If true, this sensitivity may be exploited for disease
prevention. Large quantities of mutagen-containing urine can be
collected from sensitive individuals. Isolation and identification of the
promutagens might permit removal of the precursors from cigarette
smoke.

Laryngeal Cancer

Hamsters develop laryngeal cancer after long-term inhalation of
diluted cigarette smoke (17, 50, 52). The effect is dose related and has
been used to compare different cigarettes. Tobacco-specific nitrosa-
mines induce cancer in the trachea and lungs of hamsters and may be
of particular importance in the induction of human cancer of the
larynx (84). Other carcinogens and cocarcinogens of cigarette smoke
that are active in the mouse skin bioassay system may also contribute
to induction of laryngeal cancer. Both organ systems involve epithelial
tissue directly exposed to the carcinogenic mixture.

Other Cancers

Cigarette smoking is also associated with cancer of the kidney,
pancreas, oral cavity, and esophagus (173). No animal model of these
cancers has been developed to the point where it could be used for
quantitative comparisons of different types of cigarettes. Oral cavity
and esophageal tumors may be induced by direct exposure to smoke
carcinogens. NNN, when given in the drinking water of rats, induces
cancer of the esophagus (84). This finding suggests that tobacc+

41


specific nitrosamines may be active as "contact" carcinogens. Alterna-
tively, the carcinogens might be produced through metabolism at
distant sites, such as the liver, and then transported to the target site,
where they can be further activated. Pancreatic cancer was induced in
hamsters with diisopropylnitrosamine (134). This observation suggests
the possibility of a similar action of smoke nitrosamines. Any
carcinogen in cigarette smoke might contribute to induction of cancer
distant from the exposure site. To this extent, elimination of the
carcinogens causing lung cancer or bladder cancer would reduce the
induction of cancer in other organs as well.

Alcohol usage and cigarette smoking show synergistic effects in the
induction of cancer in the upper digestive tract (113,172). The effect of
alcohol in this circumstance may result from the induction of
microsomal enzymes, which are believed to metabolize carcinogens to
their active forms (213).

Early End Points Suggestive of Carcinogenic Potential

It is generally considered that the induction of cancer requires a
specific genotoxic event that may be preceded or followed by ill-
defined and less specific epigenetic changes that enhance the manifea-
tation of the genetic event (182). In the two-stage carcinogenesis
system of mouse skin, the first step-initiation-appears to be
genotoxic, and the second step-promotion-appears to be epigenetic.
Several other forms of cocarcinogenesis have been described (16).
Tobacco smoke owes its carcinogenic activity to several carcinogens
and cocarcinogens (24,87,176,188).

Agents capable of producing genetic change can often be detected
by mutagenesis assay systems (2). Most carcinogens are mutagens.
Conversely, agents capable of inducing mutations are suspect as
possible carcinogens. Cigarette smoke condensates and some of their
fractions are mutagenic in the Ames salmonella assay systems (93,
119). These fractions are clearly of interest because they possess the
capability of inducing genetic changes that might lead to tumor
formation. Mutagenesis assays may provide a basis for the quantita-
tive comparisons of new cigarettes when the relative importance of the
genetic and epigenetic factors in smoke-induced cancer is understood.
The Ames test gives poor results for fractions of smoke condensate
that appear to be most active in systems designed to detect tumor-
promoting activity (Table 4). Furthermore, mutagenesis assays of a
series of experimental cigarettes have not provided consistent results
(167). The complexity of carcinogenesis by tobacco smoke condensates
renders mutagenesis assays of uncertain value for quantitative
comparisons of relative carcinogenicity.

Several in vitro systems measure the transformation of normal cells
into malignant cells after exposure to carcinogens. These systems are
sensitive to both genetic and epigenetic processe s (90,186). Such assays

42


may prove to be useful short-term indicators of the relative potency of
different types of cigarette smoke. The toxicity of most experimental
smoke condensates may interfere with the conduct of such studi=,
however. Experimental cigarettes that yield smoke condensates with a
wide range of carcinogenic activity are now available. It should be
possible to determine the usefulness of in vitro systems with this
material. For organ-specific carcinogens, the DNA repair test is a good
predictor of relative carcinogenic activity (186).

Most chemicals that are carcinogenic to mouse skin selectively
destroy the sebaceous glands of the treated skin (23). The sebaceous
gland suppression assay is a good predictor of the activity of
experimental smoke condensates as carcinogens in mouse skin (22).

Chronic Obstructive Lung Disease

No animal models for chronic obstructive lung disease are
available to measure the potency of smoke from various types of
cigarettes. Long-term inhalation studies with hamsters, dogs, and
primates have not given rise to disease states comparable to emphyse-
ma observed in humans (17,50,52,114). In two experiments, Sprague-
Dawley and CD rats exposed to cigarette smoke for 6 to 26 months
developed emphysematous changes (104,12d. Similar results were not
reported in other long-term studies with rats (44,68).

A number of pulmonary function tests have been evaluated as
measures of early lung disease in man (31, 61, 73, 100, 135, 154. Thus
far, similar tests have not proved useful as animal assays. They might,
however, be useful in comparing the effects of different types of
cigarettes on human smokers. Exposure of CD rats to whole tobacco
smoke for 6 months led to a loss of lung parenchymal tissue distal to
the terminal airways (124). This was indicated by a 21 percent decrease
in parenchymal tissue and 12 percent decrease in alveolar surface area.
Recent evidence suggests that emphysema results from a shift in the
balance of elastase production and elastase inhibition in the lung (97).
A few individuals with genetically determined very low levels of
alphal-antitrypsin, an elastase inhibitor, are particularly prone to
develop the disease (58). When purified elastase is instilled into the
lungs of dogs, emphysematous changes appear in as little as 96 minutes
(96,98).

Cigarette smoke can act on this system in two ways. In vitro tests
with cigarette smoke condensate show that this material suppressed
the antiprotease activity of human serum, pulmonary lavage fluid, and
purified human alphal-antitrypsin (94). The suppression of protease
inhibitors by cigarette smoke is blocked by the presence of phenolic
antioxidants, suggesting that oxidants or free radicals of the smoke
were responsible for the effect (107). In one study, the serum levels of
alphal-antitrypsin in smokers were higher than in nonsmokers (76).
Another study found, however, that immediately after smoking, serum


alphal-antitrypsin activity was reduced in smokers (95). Likewise, the
activity of alphai-antitrypsin in lung lavage fluid from Sprague
Dawley rats was reduced by 30 to 40 percent after 3 to 6 puffs of
cigarette smoke. Similar reductions were observed in lavage fluid from
the lower respiratory tract of asymptomatic smokers (58). Even
greater differences were seen between smokers and nonsmokers with
idiopathic pulmonary fibrosis. Cigarette smoke also stimulates the
release of elastase from macrophages in vitro and in &VU and from
polymorphonuclear leukocytes in vitro (19,1&9,185). Thus, smoke may
increase the elaboration of elastase in the lung and at the same time
suppress its inactivation. The techniques used in these studies could he
applied to smoke from various types of cigarettes; they might then
serve as short-term end points to evaluate relative cigarette risk.

Dogs exposed to cigarette smoke through tracheostomies for 600
days had significantly higher levels of pancreatic elastase than sham-
smoked controls (122). The greatest effects were seen in animals
exposed to higher nicotine cigarettes, although the blood carboxyhem-
oglobin levels were the same for both higher and lower nicotine
smokers (Figure 1). The lower nicotine cigarettes in this study were
produced by removal of the alkaloid by a commercial process (65). It
cannot he stated with confidence that other constituents were not
removed as well.

Sudden Death Due to Cardiovascular Disease

Animal Models

No animal model permitting the quantitative comparison of death
rates due to cardiovascular disease induced by different types of
cigarettes is presently available. Long-term inhalation studies using
smoke-exposed rats, hamsters, dogs, and primates have been conducted
(17,& 50,52,68,104,114). None has provided an end point comparable
to sudden death observed in human smokers. There are, however,
several avenues of investigation whose intermediate experimental
observations might indicate a mechanism for mortality caused by
cardiovascular effects. Much attention has been given to changes
induced by nicotine-induced catecholamine release (138, 156, 160).
Methods to follow these effects in animals are well established. Other
short-term end points being studied include lipoprotein levels (79),
alteration of arterial morphology (9, 10, 32, 111), and changes in
arachidonic acid metabolism (12, 82). These procedures might be
adapted for estimation of the relative potency of various types of
cigarettes, but there is no direct evidence that any of these changes are
either necessary or sufficient indicators of the risk of sudden death due
to heart disease.

44


  30-


-z
5  25-
g          Y
2
5  20-
Y
g  15-
?!
f `O-



   5-



   0

oooE32

    -
I   1,  I      t     I     1
   CODE13           colm?OL

FIGURE l.-Effect of cigarette smoke differing in selected
chemical components on pancreatic elastase levels in beagle dogs
after a 6OOday exposure protocol of 12 cigarettes per day, 7 days
per week. Bars indicate mean *SD. Animals exposed to code 32
(hiih-nicotine) and code 13 (low-nicotine) cigarettes diff'ered
significantly (p<O.O!5) in pancreatic elastase levels from
corresponding sham-posed controls. Sign&ant differences were
also observed (p<O.O5) between code 32 and code 13 cigarette
smokers (Student t-teat).

Nicotine

It has long been known that nicotine elevates blood pressure and
heart rate and may increase the onset of angina pectoris attacks. These
effects were summarized in the 1976 report, Tke Health Gmaquences
of Smoking (175). Nicotine readily passes through, biological mem-
branes. The level in the breast fluid of smoking women is similar to
that found in the plasma (81). The heart rate of fetuses of smoking
women is elevated, apparently caused by transplacental passage of
nicotine (127, 186). Thus, nicotine causes widespread effects in the
smoker.

An estimate of the relative potency of various cigarettes with
respect to the acute cardiovascular effects of nicotine can be deter-
mined by direct chemical assay of relative levels of nicotine in the
smoke. By measurement of urinary excretion of nicotine and its major

45


metabolite, cotinine, it is possible to estimate the individual smoker's
actual exposure to nicotine.

Nicotine appears to have measurable effects on performance by
smokers (149, 183). This may account for the apparent role of nicotine
in the reported tendency of some individuals to compensate when
switched from higher to lower "tar" and nicotine cigarettes (60, 136,
14.4 146,147').

The effects of carbon monoxide in reducing the oxygen-carrying
capacity of the blood are well known. More recently a body of evidence
has linked carbon monoxide directly to disease states and to early end
points that might be predictive of disease (11,109). Aronow has shown
that carbon monoxide, along with nicotine, decreased the duration of
exercise achieved before angina (6, 7, 8). In his studies, a non-nicotine
cigarette made of Indian herbal leaves was employed. Smoke from
these cigarettes was more active than expected on the basis of its
carbon monoxide content. Aronow (6) attributed this effect to a
"tobacco component" other than nicotine or carbon monoxide. The
effect, however, could well have been caused by a specific herb
constituent. Models using pigeons, rabbits, pigs, and primates have
been employed to study early end points for carbon monoxide effects
(4, 11, 114). To the extent that carbon monoxide is responsible for
cardiovascular disease, determination of the relative potency of various
cigarettes in affecting cardiovascular disease can be made by chemical
assay of cigarette carbon monoxide yield.

Other Agents

It has been suggested that agents of tobacco smoke other than
nicotine and carbon monoxide contribute to its cardiovascular effects
(4,116). Until these agents are identified or an alternative explanation
for tobacco effects is established, animal models predictive of cardi+
vascular death in smokers will be important.

Complications of Pregnancy and Early Childhood

A full understanding of the potential effects of smoking on
pregnancy and early infancy is still being developed. Most of the
current information available was reviewed in the 1980 report, The
Health Cbnsequences of Smoti~ for Women (174). Maternal smoking
causes changes in the vascular structure of the placenta and increased
fetal heart rate (9,10,127,136). Maternal carboxyhemoglobin (HbCO)
is elevated in smokers, leading to an elevated fetal HbCO and thus to a
reduced oxygen content of the fetal blood (108).

Some, if not all, of the smoking-related complications of pregnancy
are attributed to nicotine and carbon monoxide (108). The relative

46


hazards of lower "tar" and nicotine cigarettes with respect to these
agents can be determined by chemical assays of carbon monoxide and
nicotine. Actual disease risk, however, will be affected by the delivered
dose of these constituents, which in turn depends upon the individual's
style of smoking. Other constituents of smoke might also contribute to
complications of pregnancy. Comparisons of various types of cigarettes
should be possible through epidemiological study, coupled perhaps with
evaluation of the vasculature of human placenta (9, IO).

Recent reports indicate that cigarette smoke might contain active
transplacental carcinogens (54, 125, 140). The importance of this in
human cancer will probably not be determined soon. No animal assays
have yet been applied to assess the relative health hazard of varying
cigarettes in transplacental carcinogenesis.

Nonspecific End Points of Toxicologic Siignificsnce

Cigarette smoke and its components cause several conditions that
may relate to human disease in nonspecific ways. Using assays with
these end points may provide useful measures of potential risks due to
smoking.

Reduction of Lung Lkfeme Mechanisms

Vapor-phase constituents of cigarette smoke inhibit ciliary motility
and mucous flow in experimental animals (IS, 14). With ciliary
paralysis, removal of other toxic materials from the lung will be
inhibited. Animal models suffer some limitations in attempts to
duplicate the human situation. For example, many of the ciliastatic
agents in the gas phase of smoke are absorbed in the upper airways of
man and may not reach areas in the lung where they could affect
bronchial cilia (45). Furthermore, the concentration of ciliatoxic agents
in cigarette smoke will depend on the amount of dilution of smoke by
air that occurs during inhalation. Accordingly, the interpretation of
animal studies requires care. Similar effects occur in humans, however.
Clearance of FesOc dust from the lungs of smokers is dramatically
slower than from the lungs of nonsmokers (97).

Induction of Mi.cmwmd o;cidase

Cigarette smokers metabolize several compounds more rapidly than
nonsmokers (58, 39, 99, 187). This effect is believed caused by the
induction of microsomal oxidases, which include aryl hydrocarbon
hydroxylase (AHH). The level of AHH itself is much higher in
placentas from smoking women than from nonsmokers (190, MI, 178).
Activation of these enzymes has also been observed in the lungs of
rata, hamsters, and mice exposed to cigarette smoke (1, 59). Guinea
pigs, in contrast, showed a reduction in pulmonary AHH after smoke
exposure (18). Induction of AHH activity appears to result from

47


systemic exposure to the smoke compounds themselves or to the
metabolites of those compounds. Some carcinogens, including PAI&
induce AHH (38). More important, the AHH system is involved in the
metabolic formation of ultimate carcinogens from procarcinogen
precursors (118). Cigarette smoke may play an indirect role in
carcinogenesis among smokers through this mechanism. Assay of the
inducibility of AHH as a measure of individual sensitivity to cigarette
smoke has not proved useful (115,128); however, screening of enzyme
activity in tissues of human or animal smokers of different types of
cigarettes might prove useful for indicating the relative potency of the
different cigarettes.

Changes in Genetic Status

To the extent that an early step of carcinogenesis involves genetic
change, one would expect that exposure to cigarette smoke might
cause detectable changes in genetic material. It is reported that heavy
smokers have higher incidences of chromosomal aberrations and higher
rates of sister chromatid exchange than do nonsmokers (91). Animal
models with such end points are feasible, but have not heen applied to
assays of the toxicity of various cigarettes.

Ch.anges in Immune Status

Recent reports suggest that smoking causes changes in immune
function (56, 69, l&), but the contribution of these effects to major
disease states is unclear. Men with malignant melanoma who smoke
are more likely to develop metastases than are nonsmokers, perhaps as
a consequence of impaired immune systems (159).

ComposMon of Smokes From Various Types ot Cigarettes
Smoking-Machine Deign

Laboratory smoking-machine parameters historically have been
standardized to permit interlaboratory comparisons and to provide
reproducible baselines with which modified cigarettes can be com-
pared. Somewhat different parameters are used in different countries
(28). In the United States, the most widely used standards are those
employed by the Federal Trade Commission (163). The machines
deliver a 35 ml puff from the cigarette over a 2-second period with a
hell-shaped puff profile. The cigarettes are puffed once each minute to
the defined butt length of 23 mm (nonfiltered cigarettes), or to a butt
length 3 mm longer than the filter overwrap (filter-tipped cigarettes).
The butt length is different from cigarette to cigarette, according to
the length of the overwrap.

These parameters were established in 1967 when the great majority
of cigarettes consumed in the United States were nonfiltered and 70 or


85 mm in length. They were based, in part, on observed smoking
patterns in a limited number of human smokers. The types of
cigarettes smoked today are substantially different with respect to
length, paper porosity, pressure drop, "tar" and nicotine yield, and the
concentration of gas phase constituents.

Cigarette smoking-machines can be designed, however, to control
puff volume, frequency of puffing, duration of puff, the profile of puff
pressure over time, butt length, position of cigarette during and
between puffs (e.g., horizontal or vertical), and "restricted" or "free"
smoking between puffs (i.e., whether the butt end is closed or open).
The puff volume can be measured in terms of the air entering the
cigarette or the air plus combustion gases leaving the cigarette.
Smoking-machines could be designed to change the puff frequency and
the nature of the puffs during the course of smoking a single cigarette
(41,4a.

Human smoking patterns are diverse and span a wide range from
one individual to another (40,78,139). Some individuals compensate for
lower yield cigarettes by changing their style of smoking (80,139,1&
146,180). These changes can include increasing puff volume, duration,
or frequency, or changing the puff pressure profile. In summary,
human smoking behavior may be quite different from standard
smoking-machine behavior. Furthermore, the average smoker may
have a different smoking pattern for each different type of cigarette.
The chemical composition of smoke is affected by smoking-machine
parameters. "Tar" yield per puff depends on puff volume, puff
frequency, butt length, and the frequency of puffing at different
stages of cigarette consumption (188, 193,194). The concentrations of
several specific chemical constituents of "tar" are controlled by the
puff frequency, volume, and duration (Chortyk, O.T., and Schlot-
zhauer, W.S.S., personal communication). If the human smoking
pattern varies systematically with the type of cigarette, the relative
yield of various chemical constituents delivered to the smoker may
vary substantially from that measured by machine. Accordingly,
evaluation of the toxicological and pharmacologic potential of the
smokes from new types of cigarettes will require knowledge of the
manner in which those cigarettes are smoked by the consumer and of
the effect of smoking patterns on the composition of smoke.

Dependence of Smoke Composition on Cigarette Design

The composition of smokes from different types of cigarettes can be
described by absolute yields per cigarette or per puff, or by the
concentration of constituents per unit weight of "tar" or per unit
volume of smoke. Modifications of cigarette design can affect yield
(quantitative change) or composition of the smoke (qualitative
change). Information with respect to individual constituents is avail-
able for many modifications. However, modifications affecting the


concentration of one substance will also affect the levels of other
substances as well.

Because of the complexity of cigarette smoke, the full impact of any
cigarette modification on the composition of the smoke in either
absolute or relative terms can never be ascertained. For this reason,
bioassays with appropriate end points are essential to determine the
relative toxicities of new types of cigarettes. Several modifications of
cigarettes reduce the mouse skin carcinogenic activity of the smoke
condensate. These include choice of leaf variety, use of reconstituted
sheet, and use of tobacco substitutes.

The design characteristic of commercial cigarettes that most affects
the cigarette yield is the filter. In 1930, the "tar" yield of cigarettes, as
reported by the Federal Trade Commission or by advertisements,
ranged from 30 mg for unfiltered, king-sine cigarettes to as low as 0.1
mg for some filter-tipped brands (55). Filters selectively remove
nitrosamines and semivolatile phenols from the smoke (88, 120, 126,
188). Thus, not only the absolute delivery of these constituents but also
their relative concentration in cigarette "tar" depend on the filter.

A second major influence on the composition of cigarette smoke is
ventilation of the cigarette by the use of paper with a high degree of
porosity or by the presence of holes in the mouthpiece. When more air
is drawn through the paper or through the mouthpiece, the amount of
air drawn through the burning coal of the cigarette is reduced. This
effect will reduce the quantity of "tar." By altering the burn
temperature, it will also change the combustion process and thus the
composition of the smoke. Ventilation also dilutes the gas phase of the
smoke with air, causing a marked reduction in the concentration of gas
phase constituents in the smoke (66,83,126).

Tobacco Vark ty

A substantial collection of tobacco lines is available to plant
geneticists. These include 63 species related to tobacco and about 1,000
different tobacco varieties (164). The wealth of this material permita
genetic manipulation of the leaf, which could be used selectively to
enhance or to reduce the content of specific constituents. Among flue-
cured tobacco lines available at present, the nicotine concentration
varies from 0.2 to 4.75 percent (34). Among various burley lines the
concentration varies from 0.3 to 4.58 percent. The ranges could be
extended by agronomists, should that be desired. Changes in yield of
many other smoke constituents might be achieved by genetic modifica-
tion.

60