CONTENTS

Introduction

Epidemiologic Studies
Background
Epidemiologic Studies
  Discussion

Pathologic Studies
  Discussion

Experimental Chemical Carcinogenesis

Tumor Initiation and &carcinogens

Organ-Specific Carcinogens

Carbon Monoxide in Cigarette Smoke

Smokers' Compensation

Transplacental Carcinogenesis

Flavor Additives

Conclusions and Recommendations

Summary

References

LIST OF FIGURES

Figure l.-Lung cancer mortality ratios, by amount of
"tar" and nicotine in cigarette smoke


Figure 2.-Lung cancer mortality ratios, by number of
cigarettes smoked per day and amount of "tar" and
nicotine in cigarette smoke

Figure 3.-Relative risks of lung cancer in long-term filter
smokers (LTF) compared with nonfilter smokers (NF) by
number of cigarettes smoked per day, males

Figure I.-Decrease of be@a]pyrene in the smoke of
U.S. nonfilter cigarettes (36 mm)

Figure &-Percent of sections with advanced lesions by
smoking habit in two periods

LIST OF TABLES

Table l.-American Cancer Society Matched Groups Study

Table 2.-Known carcinogenic agents in the gas phase of
cigarette smoke

Table 3.-Tumor-initiating agents in the particulate phase
of tobacco smoke

Table I.-&carcinogenic agents in the particulate matter
of tobacco smoke

Table 5.-Organ-specific carcinogens in the particulate
matter of cigarette smoke

Table 6.-Carbon monoxide in smoke of cigarettes

78


lntroductlm

Research indicates that cigarette smoking causes cancer of the lung,
larynx, oral cavity, and esophagus, and is significantly associated with
pancreas, urinary bladder, and kidney cancer in both men and women
(102, 108, 104. This conclusion is based on epidemiologic, pathologic,
and experimental evidence collected over the past half-century.
A quarter-century ago lung cancer was found to be related
quantitatively to cigarette "tar" cumulatively inhaled. This finding,
along with much other evidence, led to the production and widespread
use of today's lower "tar" and nicotine cigarettes.2
The evidence summarized in this section demonstrates that lower
"tar" and nicotine cigarettes produce lower rates of lung cancer than
do their higher "tar," higher nicotine predecessors, but smokers of
lower "tar" and nicotine cigarettes still have much higher cancer
morbidity and mortality rates than do nonsmokers, as well as a higher
incidence of other diseases associated with smoking.

One important research concern is to identify the human carcinogen-
ic chemical or chemicals in the particulate and gas phases of cigarette
smoke. Multiple metabolic transformations are available in the human
body for the several thousand chemicals in cigarette smoke, a number
of which could lead to carcinogenic activity in model animal systems.
Another important research concern is that changes in cigarette
composition to reduce "tar," nicotine, and possibly even total smoke
exposure may inadvertently increase, or fail to decrease, those
chemical constituents still largely unidentified that contribute to
cardiovascular and pulmonary diseases, pregnancy complications, and
fetal and perinatal deaths.

A third area of concern is that the animal model systems used to
predict human disease from cigarette smoking require additional study
and correlation with the human situation, if these models are to serve
as a basis for modifying cigarette composition. When disease-produc-
ing chemicals are identified, their reduction or elimination should be
associated in the animal models with a decrease in the disease(s)
predicted and without untoward effects.

This section summarizes data on the human cancers associated with
lower "tar" and nicotine cigarettes, as compared with the "standard"
cigarette of the 1939s or 1949s. In addition, it compares pathologic
(autopsy) studies on bronchi of cigarette smokers of a quartercentury
ago with bronchi of lower "tar" and nicotine cigarette smokers.
Further, the section describes the identification, metabolism, and
possible mechanisms of action of certain carcinogenic chemicals in both
the particulate and the gas phases of cigarette smoke. Finally, the

79


section presents a series of conclusions and recommendations for
research.

Epldemlologic Studies
Background

It has been established that cigarette smoking causes cancer of
various organs including the lung, oral cavity, esophagus, and larynx,
as well as exhibiting a significant association with cancer of the pan-
creas, bladder and kidney (102). Epidemiological studies, both retrospec-
tive and prospective, have shown a dose-response effect; that is, risk
increases with the length of time the individual has smoked and with
the number of cigarettes consumed. Such studies have demonstrated
that, upon cessation of the smoking habit, risk for developing these
cancers declines; the slope of the decline depends on the duration and
extent of the former habit. For an individual who has smoked more
than 20 cigarettes per day for more than 20 years, no reduction in risk
of cancer development is noted for at least 3 years; however, the risk
decreases thereafter and, after 10 years of cessation, begins to
approach that of one who has never smoked.

From these epidemiological observations, it has been predicted that a
smoker's caner risk would be reduced if the "tar" yield of a cigarette
were reduced, provided that the individual does not compensate by
more frequent and deeper inhalation of lower "tar" cigarettes.

The trend toward cigarettes with lower "tar" and nicotine started
more than 25 years ago with the introduction of a number of filter
brands. This trend continued over the years with a greater number of
filter brands on the market. Since the early 1970s there has been a
rapid increase in production of cigarettes with 15 mg or less "tar" and
1.0 mg or less nicotine. By 1930, brands with these characteristics are
expected to account for more than 40 percent of total sales (70). In
1950, the average cigarette had 40 mg "tar" and 2.2 mg nicotine.
Today's filter cigarettes average about 14 mg "tar" and 1.0 mg
nicotine. The downward trend, particularly in terms of "tar" in filter
cigarettes, is continuing. There are increasing numbers of cigarettes
yielding 10 mg "tar" or less, and these have only one-fourth the "tar"
yields common 30 years ago. Although total consumption has increased
from 365 billion cigarettes in 1950 to 620 billion cigarettes in 1979,
consumption per capita by persons 18 years of age and over has
decreased by 5 percent in recent years-from 4,143 cigarettes in 1973
to 3,924 cigarettes in 19'79 (101), reflecting the 30 million smokers who
have quit (75). On the other hand, the proportion of smokers who
reported that they smoke 26 or more cigarettes per day increased from
23 percent in 1970 to 28 percent in 1978.


Epidemiologic Studies

Three epidemiologic studies-by the American Cancer Society, the
American Health Foundation, and the National Cancer Institute-
have evaluated the effect of lower "tar" and nicotine cigarettes on
lung cancer mortality.

The American Cancer Society conducted a prospective study in
which more than a million men and women in 25 States were enrolled
in 1959 and traced for 13 years. Subjects completed a questionnaire on
smoking habits upon enrollment, and the survivors completed another
questionnaire in 1965. An analysis of mortality from lung cancer was
made for two 6-year periods: July 1960 to June 1966 and July 1966 to
June 1972. The analysis included males and females who, in 1959-69
and in 1965, reported either that they had never smoked regularly or
that they smoked cigarettes regularly but never smoked cigars or pipes
regularly (36).

On each questionnaire, subjects reported the brand that they usually
smoked. From this information and from various reports of "tar" and
nicotine published in the years in which the questionnaires were
completed, subjects were classified as high "tar" and nicotine (T/N)
smokers, medium T/N smokers, and low T/N smokers. In the first
period, high T/N brands were defined as cigarettes with 26.8 or more
mg of "tar" and 2.0 or more mg of nicotine. Low T/N was defined as
brands with less than 17.6 mg "tar" and less than 1.2 mg nicotine. The
medium T/N category was between these two groups. By the time the
second questionnaire was distributed, there had been an increase in the
number of filter brands on the market and a general lowering of T/N
levels. Low T/N was defined in the same way as in the first period, but
the high T/N category had to be reset at a somewhat lower level.
Smokers in the three groups were compared by a matched groups
analysis. In this procedure, the groups were matched by age and other
factors, including number of cigarettes smoked per day, age at which
smoking began, race, urban or rural residence, occupational exposures,
education, income, and prior history of lung cancer or heart disease.
To be counted in the study, at least one person in each of the three
T/N groups had to be matched on all the variables mentioned above.
The adjusted number of lung cancer deaths was obtained by dividing
the number of deaths in each triad by the lowest number in each of the
three groups. The adjusted numbers of deaths were then summarized
for each of the three T/N groups.
Table 1 shows the number of subjects and the unadjusted and
adjusted number of lung cancer deaths in the high, medium, and low
T/N groups by sex and time period. In both sexes, deaths were fewest
in the low T/N group.
Figure 1 shows the lung cancer mortality ratios based upon the
adjusted number of lung cancer deaths. The number of adjusted deaths
for high T/N smokers was set at 1.00, and the adjusted number of lung

81


TABLE I.-American Cancer Society Matched Groupa Study

                 fw        Medium        LQW
sex           Period         T/N          T/N         T/N

Male
Male
Female
Female

Male         l960-l666         567          466          166
Mele        1666-1672         437          666           7s
Female        1960-1966          66          82           al
Female        1%&1972          89          149           44

  Male         196&11)86        l22.4         117.4         lOL0
  Awe         196&1m         S.6          a.6         76.6
  Female        1960-1966         4s.3          41.4          27.4
  Female       l9M-1972         68.1          42.2          lx2

SOURCE: Eemmoed et al. (ss).

cancer deaths for medium and low T/N smokers was compared with it.
The mortality ratio for male low T/N smokers was 0.83 and 0.79 in the
two time periods; for females, it was 0.57 and 0.62. The mortality from
lung cancer in low T/N cigarette smokers for both sexes over the
combined time periods was 26 percent lower than for high T/N
smokers. The mortality ratio for smokers of medium T/N cigarettes
was lower than for high T/N, but greater than for the low T/N
smokers.

Low T/N smokers had mortality ratios considerably higher than men
and women who had never smoked. In men, the mortality ratio of
nonsmokers for lung cancer was only 9 percent of that of the low T/N
smokers; in women, the nonsmoker rate was 49 percent as high in the
fit 6-year period and 22 percent as high in the second 6year period.

It is important to note that the T/N level of the brand of cigarettes
smoked was not as significant as the number of cigarettes smoked. The
adjusted number of deaths in men and women who smoked fewer than
20 high T/N cigarettes per day was compared with those who smoked
20 or more low T/N cigarettes per day. Figure 2 shows the mortality
ratios. The less-than-2O-cigarettes-perday high T/N smokers had
mortality ratios from 67 percent to 27 percent lower than the men and
women who smoked 20 or more low T/N cigarettes per day.

A retrospective study of lower "tar" and nicotine cigarettes was
conducted by the American Health Foundation (111). Data on lung
cancer cases in white males and females were collected, and interviews
were conducted in hospitals in six U.S. cities between 1969 and 1976.
Control cases were selected from patients in the same hospitals on the
basis of an absence of a history of tobacco-related diseases.

82


           Men
  Period1           Peliod2
1.00             1.00

         women
  Period1           Period2
1.00             1.00

"TAR" AND NICOTINE IN CIGARETTE WOKE

FIGURE l.-Lung cancer mortality ratios, by amount of "tar"
and nicotine in cigarette smoke
NOTE:H-hi&Y-msdnm;L-br.
SOUBCE: lid at al. (se).

Cigarette smokers were classified as long-term filter smokers (those
who smoked filter cigarettes currently and for at least 10 years) and
nonfilter smokers (current smokers of nonfilter brands).

Relative risks for filter smokers and nonfilter smokers were
computed by number of cigarettes smoked per day. Figure 3 shows the
relative risk of the male filter smokers as a percent of the risk for
nonfilter smokers. The percentages ranged from 61 to 39. Females
showed the same pattern, with the relative risk for long-term filter
smokers ranging from 33 to 79 percent of the nonfilter group. Only in
the heaviest smoking category (a small number of cases) ware the
relative risks the same.
This risk ratio of filter smokers to nonfilter smokers remained low
when the data were adjusted for factors such as duration of smoking,
amount of cigarette smoking, age, and alcohol consumption.
The American Health Foundation study also analyzed the risk of
larynx cancer for long-term filter smokers versus that for nonsmokers.
There were many fewer cases of larynx cancer than of lung cancer, but
the same general pattern was observed. In men, the relative risk for
long-term filter smokers was between 50 percent and 75 percent of the

83


Perk41
1.00

Park42

20+ <20
A DAY   A DAY

LOW    HIQH
TIN    TIN

20+ <20
A DAY   A DAY

LOW    HIQH
TIH    TIN

20+ <20      20+ <2tl
A DAY   A DAY     A DAY   A DAY

LOW    HIQH      LOW    HIQH
TIN    TIN       TIN    TIN

FIGURE t.-Lung cancer mortality ratios, by number of
cigarettes smoked per day and amount of "Yar" and &tine in
cigarette smoke
mJEcE: Elelemd et al. (.3@.

risk for nonfilter smokers in various number-of-cigarettmoked-per-
day categories. Women showed the same pattern.

A third epidemiologic study was conducted in Austria (63). This
project, part of an international study of smoking by the National
Cancer Institute, analyzed data on a sample of 414 lung cancer patients
and 823 controls. Cigarettes were categorized into three groups by T/N
level: Group I, cigarettes with "tar" yields below 15 mg; Group II, 15 to
24 mg "tar"; and Group III, 25 mg or more "tar." These groups were
assigned values of 1, 2, and 3, respectively, to indicate average
exposure.

The average "tar" exposure in cancer patients (2.596) was signifi-
cantly higher than for controls (2.026). Scores for total "tar" exposure
were computed as the product of the number of cigarettes smoked per
day, the number of years smoked, and the "tar" level (1, 2, or 3).

84


LTF NF      LTF Ns      LTF NF      LTF NF      LTF NF

l-10        11-20        21-30        314         41 +

AMY        AMY        A DAY        AMY        A DAY

FIGURE 3

I.-Relative risks of hmg cancer in long-term filter

smokers (LTF) compared with nonfilter smokers (NF) by number
of cigarettes smoked per day, ndea
SOUIOCIE: wyedw aed &elhM (my

Relative risks were then computed by these scores. These risks
increased directly with "tar" exposure scores, from 1.6 for scares lower
than 566 to a relative risk of 6.1 for scores higher than 5,600.

Cigarette smoke condensate of present cigarettes pro&as fewer
tumors on mouse skin than did that of cigarettes tested some 30 yeam
ago (109). This difference is probably because today's cigarettes
contain more tobacco stems and more reconstituted tobaccos and have
cigarette paper with higher porosity, all contributing to smoke
condensate that is less tumorigenic to the experimental animal.
Changes in chemical composition of the smoke may be a factor. Using
just one chemical component as a carcinogenic indicator, researchers
have shown that benzo[ajpyrene (BaP) content is significantly lower in
today's cigarettes than in cigarettes of 36 years ago (Figure 4) (49).

85


r

12-

E 1.0-
E
8
&
? 0.6-
%
P



0.6 -

I      I      I       I      I      I
1955      1960      1965      1970      1975      1960
              YEARS OF PURCHASE

FIGURE 4.-Decream of benzo[a]pyrene in the smoke of U.S.
nonfilter cigadtm (&I!5 mm)
SOUECE: Hoffmule std. (4s).

Many brands of cigarettes classified as lower "tar" and nicotine were
introduced in the 1970s and had a remarkable growth in sales. The
average "tar" in lower "tar" and nicotine brands in 1978 was about 10
mg. Many brands of cigarettes classified as lower "tar" and nicotine in
studies reported in the 1960s and early 1970s would be classified as
medium "tar" and nicotine cigarettes in the 1980s. Therefore, it might
be assumed that cigarettes with lower "tar" and nicotine yields afford
even lower cancer risks. But this is not necessarily true. Studies of
smoking patterns suggest that smokers of the lower "tar" and nicotine
cigarettes tend to inhale more deeply (44,98), have higher amounts of
carboxyhemoglobin than predicted (106), and have higher than expe&
ed carbon monoxide in their exhaled breath (54). On the other hand,
the lower "tar" and nicotine cigarettes of 1980 have as little as one-
fourth the "tar" and nicotine of the cigarettes of 1950, and even if
some compensation takes place, actual net smoker exposure is probably
much lower.

There is evidence that machines that measure "tar" and nicotine
content are not suitable for measurements of smoke from lower "tar"
and nicotine cigarettes with perforated filter tips (62) and that the

86


"tar" and nicotine in the inhaled smoke may be more than indicated by
the test proc43dures.
Epidemiological studies thus far have only studied cohorts who
began their smoking careers with the old nonfilter, high "tar" and
nicotine cigarette. Only in the years to come can we determine the risk
of those individuals who began smoking with lower "tar" and nicotine
cigarettes, and it is important to study this risk.
As the "tar" yields of cigarettes decrease further, it is probable that
flavor additives will be increasingly used. Their potential biologic
activities need to be investigated and monitored on an ongoing basis.
Epidemiological data in addition to chemical and biological findings
show the reduced risk among lower "tar" and nicotine cigarette
smokers, which was predicted because of chemical and biological data
previously known. No such clear demonstration of effect exists,
however, for cardiovascular disease, chronic obstructive pulmonary
dii, or pregnancy. The character and mechanisms of smoke
components causing these diseases probably differ significantly from
those acting in carcinogenesis.

Pathologic Studier

Histological changes in the tracheobronchial tree in noncancer
patients can be observed at autopsy in direct proportion to the number
of cigarettes smoked per day during life. Lung cancer patients have
the most advanced histological changes in their remaining epithelium
(4, 6). Ex-smok ers who quit for at least 5 years show greatly reduced
histologic changes. This finding, together with the observation of cells
with disintegrating nuclei in the epithelial lining, suggests that a
healing process has taken place in these cases (5).
To evaluate the effect of smoking lower "tar" and nicotine
cigarettes on histologic changes in bronchial epithelium, male patients
who died of causes other than lung cancer in 1970-77 were compared
with those who died in 195569 (3). None of the men who died in the
later period could have, in the last 5 to 10 years of their lives, smoked
cigarettes that were as high in "tar" and nicotine content as the
cigarettes smoked by men who died in the earlier period. Sections from
the tracheobronchial tree of 211 men who died in the earlier period and
of 234 men who died in the later period were put in random order for
microscopic study. A total of 20,424 sections were read, an average of
46 sections per patient. Histologic changes studied included basal cell
hyperplasia, loss of cilia, and occurrence of cells with atypical nuclei.
Smokers had these changes far more frequently than did nonsmokers,
and within each group the percent with these changes increased with
the reported number of cigarettes smoked per day. Nonsmokers in both
time periods had about the same proportion of these changes. But in
each smoking category (adjusted for age), the men who died in 1970-77

87


NEVER <l    l-2    2+

NEVER <1     l-2    2+

SMOKED PACK PACKS PACKS        SMOKED PACK  PACKS PACKS
REG.   ADAY ADAY ADAY          REG.   A DAY   ADAY ADAY

FIGURE a--Percent of sections with advanced lesiona by
smoking habit in two periods

had far fewer histological changes than those who died in 1955-69.
Figure 5 shows the percentages with the most advanced histologic
change recorded (carcinoma-in-situ) in the 1955-69 and 1979-7'7 groups.
These changes were not found in nonsmokers in either group, and they
were found far more frequently in smokers in the 195569 cases than in
the 19'70-77 cases. In two-pack-aday smokers, 225 percent of the
195540 group had this advanced change, compared with only 2.2
percent of the twopack-aday smokers in the 19'7&77 group.

Discussion

Epidemiologic and experimental pathologic studies yield some
evidence that filter, lower "tar" and nicotine cigarettes produce fewer
neoplasms than the nonfilter cigarettes of 25 to 39 years ago. While it
is not always possible to directly extrapolate data on animal experi-
mental carcinogenesis studies to man, the data summarized in this
section show the predicted lower mouse skin tumorigenesis of filtered,
lower "tar" and nicotine cigarette "tar" on an equal weight basis. Po&


mortem studies of the human lung further support the finding that the
filter, lower "tar" and nicotine cigarettes are less oncogenic than the
nonfilter cigarettes of 25 to 50 years ago.

Experlmental Chemical Carcinogenesls

While epidemiologic, pathologic, and experimental studies all point
to polycyclic hydrocarbons within the "tar" moiety of inhaled cigarette
smoke as potential carcinogens for man, additional work is needed to
determine whether nicotine plays a major role as a cocarcinogen.
Further, nicotine and nor-nicotine give rise to two carcinogenic
nitrosamines that are found only in tobacco products. Tables 2,3,4, and
5 list known carcinogenic agents in both the particulate and the gas
phases of cigarette smoke.

Russell (90) recently suggested that a lower "tar," medium nicotine
cigarette would be more attractive to smokers and tend to promote
their use while minimizing health risk. This action cannot be supported
without further research on nicotine's effects in carcinogenesis.
Studies should address not only nicotine carcinogenesis, but also the
chemical's effects on the cardiovascular, gastrointestinal, endocrine,
and central nervous systems. Nicotine has been found to have potent
physiologic effects on these systems.

The following discussion briefly considers the probable routes of
metabolism and binding to critical cellular components of the chemi-
cals in the particulate and gas phases of cigarette smoke thought most
likely to be carcinogenic for man.

Most procarcinogens are metabolized through a mixed function
oxidase system, which is composed of the hemoprotein cytochrome P-
450, NADPHdependent cytochrome P-450 reductase, and phospholip-
id. Various forms of P-450 have been characterized immunologically
(99), and some have been separated electrophoretically (78). The amino
acid composition and partial sequences of some forms of P-450 have
been elucidated recently (15). A treatise on the physicochemical
characteristics and physiological function of P-450 has also appeared
(78). The different forms of P-450 may have differential effects in the
production of metabolites (99, 81, 91). Metabolic activation of most
carcinogens by the P-450 mediated oxygenases is considered to afford
structures that are strong electrophilea and thus prone to attach to
cellular nucleophiles, including proteins, nucleic acids, and other
macromolecules (21, 72, 73).

Polycyclic aromatic hydrocarbons present in tobacco smoke are
typified by ben@a]pyrene (BaP). BaP is found in the soil and
atmospheric particulates of cities, with relatively high concentrations
around highways, airports, factories, and similar installations (51).
Since it occurs in pyrolysis products, such materials as soot, tar, and
charcoal-broiled or thoroughly roasted foods all have measurable

a9


levels. BaP also has been identified in forest soils, in volcano effluents
(50), in marine sediments, and even in the deeper layers of soil from the
permafrost regions of the earth (52).
BaP was among the first polycyclic aromatic hydrocarbons isolated
from coal tar and has been used for various experimental purposes for
50 years.
On the basis of metabolic studies with phenanthrene, Boyland (16)
hypothesized that hydrocarbons were metabolized through arene oxide
or epoxide intermediates. Such intermediates could account for the
identification of phenols, dihydrodiols, premercapturic acids, and
mercapturic acids as metabolites of phenanthrene or naphthalene, all
depending on whether the epoxide reacted with water or glutathione
or rearranged nonenzymatically.
The information gathered from various experiment-a in vitro with
metabolites of BaP, DNA adducts, and presumed intermediates led to
the conclusion that both the dihydrodiol and epoxide moieties were
required for carcinogenic activation of BsP and other polycyclic
hydrocarbons. In the case of BaP, the potent carcinogenicity of the `I,%
dihydrodiol indicated that it was probably an intermediate toward the
final activated carcinogen (57).
A number of studies have substantiated the concept that a "hay"
region is involved in transformation of most polycyclic hydrocarbons to
the activated intermediate (7496,108).
The diol epoxide of BaP thus appears to be the metabolically derived
strong electmphile that is capable of reacting with critical constituents
in the cell. The reaction of this activated intermediate with nucleic
acids has been followed both in tivo and in vitro (59,61,76).
P1-450 is also a component of the enzyme system called aryl
hydrocarbon hydroxylase (AHH) (2). The major phenolic detoxification
product, 3-hydroxybe&a]pyrene, results from nonenzymatic rear-
rangement of the initial 2,3-epoxide formed by the P&i0 (112). The
phenols are amenable to conjugation by glucuronyl transferase or
sulfotransferase, leading to solubilization and more rapid excretion.
The available evidence suggests that in different strains of mice high
AHH inducibility leads to increased susceptibility to hydrocarbon-
induced tumors. The genetics of AHH inducibility in mice have been
thoroughly discussed (77, 78, 79). Attempts have been made to extend
some aspects of the AHH work to humans, despite the variability in
results noted in human populations (2).
Although there is currently more emphasis on the reactions of the
electrophilic species from carcinogens with nucleic acids, the binding of
carcinogens to proteins had been noted many years earlier (72). More
recent efforts have shown that ligandin, a hepatic protein that binds
anionic metabolites of glucocorticoids (67), also binds some carcinogens
such as polycyclic aromatic hydrocarbons and aminoazo dyes but not
aromatic amides (68).


Aromatic amines are found in tobacco smoke. These compounds are
formed during the burning of tobacco, including toludines, Znaphthyl-
amine, and unknown aminofluorenes. These compounds are also
activated through the P-450 system similar to that for the aromatic
hydrocarbons. Ring-hydroxylated products of aromatic amines appar-
ently are detoxification products. For most of the carcinogenic
aromatic amines or amides investigated, N-hydroxylation apparently
was the activation route.

Further reaction of the N-hydroxy compounds was found necessary
to afford forms capable of reacting with nucleic acids or proteins.
Acetate, glucuronide, sulfate, or even phosphate esters of the N-
hydroxy amide had the required characteristics; the products from in
vitro reactions with nucleic acid were the same as those isolated from
reactions in viva. In some but not all cases, the carcinogenicity of the
parent amide or amine roughly correlated with the enzyme levels in a
target organ.

One of the most readily obtained of the activated esters, N-acetoxy-
N-24luorenylacetamide (N-AcO-FAA) has been employed in many
model experiments to study effects on the structure and function of
nucleic acids. N-A&-FM forms a major adduct with DNA where
approximately 84 percent of the fluorene residue was linked to the C-g
of guanine by arylamidation, affording N-(deoxyguanosin43-yl)-2
fluorenylacetamide, which retained the N-a&y1 group (28).
An additional means for activation and adduct formation of
aromatic amine derivatives has been investigated by CM. King et al.
(58). An enzyme termed N-O-acyltransferase forms derivatives that
are quite reactive and readily form adducts with RNA.
More recent work points toward attachment by the activated
aromatic amines and amides to still other positions on the bases of
nucleic acids (10,55,56).

Numerous model studies with N-A&-FAA modified nucleic acids
have shown a change in function of the altered nucleic acid. However,
none has shown the exact role in the process of carcinogenesis; this
remains an area for further investigation (94).
Although the aminoazo dyes and aromatic amines or amides are
activated in a similar fashion and both bind to proteins, the proteins
involved differed somewhat (8,9, 68,97).

Relatively less emphasis has been placed recently on carcinogen-
protein interactions than on carcinogen-nucleic acid interactions. In
view of the essential function of the proteins, it seems their interac-
tions with carcinogens require more investigation.

N-Nitroso compounds found in tobacco smoke include those derived
from nicotine, nitrosonornicotine and related compounds, N-nitroso-
diethanolamine, and nitrosodimethylamine. Metabolic activation of
dialkylnitrosamines is necessary for expression of their toxic and
hepatocarcinogenic effects. Oxidative metabolism of dimethylnitrosa-

91


mine, for example, is accomplished by the liver microsomal P-450
system yielding an unstable (a-hydroxymethyl)methylnitrosamine,
which forms formaldehyde and an unstable methylnitrosamine. In
turn, this molecular species collapses with release of nitrogen and
formation of the methyl carbonium ion, CHa+, which alkylates
proteins, nucleic acids, and probably other cellular constituents. The
intermediacy of the (a-hydroxymethyl)methylnitrosamine is substanti-
ated by the potent mutagenicity and outstanding carcinogenicity of
the more stable (a-acetoxymethyl)methylnitrosamine (11). More recent
studies suggest that other oxidation pathways may also be involved
(66).

Tobacco and its resultant smoke contain two carcinogenic N-nitrosa-
mines that are formed from nicotine and nornicotine (Table 2) (46,47).
N-Nitrosonornicotine (NNN) gives rise to cw-hydroxy N-nitrosamines,
which are unstable and decompose finally to oxocarbonium ions, the
suspected ultimate carcinogenic forms of NNN. Most of the oxocarb+
nium ions react with water, yielding a keto alcohol and a hydroxyal-
dehyde (19). The other carcinogenic and tobacco specific N-nitrosamine
is 4-(N-methyl-N-nitrosamino)-l-(3-pyridyl)-1-butanone (NNK), which
is also a-hydroxylated. The methyl hydroxylation product gives rise via
an oxodiazohydroxide to the same carbonium ion as the 2'-hydroxyl-
ation of NNN (42).

Alkylnitrosoureas afford alkylating moieties without the need for
metabolic activation; spontaneous decomposition occurs at alkaline pH
values. However, the organs affected by alkylnitrosoureas may vary,
depending on the route of administration and the animal model.

Nitrosomethylurea, most widely used in model experiments, can
cause tumors in brain, breast, stomach, liver, heart, skin, kidney,
intestinal tract, bladder, trachea, and peripheral nervous system (107);
administration to pregnant animals often leads to tumors of the
nervous system in the offspring many months later (SO).
The alkylating moiety (carbonium ion) formed from a nitroso
compound may attach to a variety of positions in the nucleic acids
bases, on the phosphate backbone, or on the ribose portion of the RNA.

Environmentally, nitrosamines and related structures represent a
problem, since they may be formed endogenously from secondary or
tertiary amines, amides, or ureas and nitrite, available from reduction
of nitrate by bacteria of the salivary plaque. Nitrate has a widespread
distribution in dietary vegetables and grains. Although each individual
has therefore the capacity to form nitroso compounds, endogenous
nitrosation can sometimes be inhibited by ascorbic acid, propyl gallate,
or other compounds that compete with the amine or amide for nitrous
acid. This is not a panacea, for ascorbic acid may enhance nitrosation of
certain amines (18). Furthermore, innocuous nitroso compounds, such
as nitrosoproline, or even some aliphatic nitro alcohols, can provide a
nitroso group to form carcinogenic nitrosamines or amides by transm-

92


trosation (26, 95). Although certain bacteria are instrumental in
formation of nitrosamines within the organism (.@), bacteria also
degrade nitrosamines (89), leading to a balance between endogenous
formation and decomposition of nitroso compounds. During the
chewing of tobacco, N-nitrosonornicotine is formed in the oral cavity
(41). Although it has not been demonstrated, it may be assumed that
under certain conditions the carcinogens NNN and NNK can also be
formed from nicotine in other organs or sites in man.

Another carcinogen, vinyl chloride, has also been identified in
tobacco smoke. Metabolically, vinyl chloride is activated through the P-
450 system by formation of a halogenated epoxide (7,.@, 113). Such an
epoxide may yield halogenated aldehydes or alcohols through rear-
rangement (45, 118) or through derivatives of glutathione through S-
transferase (4.5).

In summary, most of the identified carcinogens found in tobacco
smoke are activated through the P-450 system to electrophilic corn
pounds, which react with proteins, nucleic acids, perhaps lipids, and
other cellular constituents. Since there are many constituents of
tobacco smoke, only the activation pathways of BaP, typical aromatic
amines, nitrosamines, and vinyl chloride have been presented here. The
activation pathways of the other carcinogens found in tobacco smoke
may be similar.
Although the pathogenesis of several types of cancer, chronic
obstructive pulmonary diseases, and cardiovascular diseases is linked to
different tobacco smoke constituents, the epidemiologic associations
with cigarette smoking are dose related for each of these diseases (84,
86, S7, 58, 102). Thus, the first goal in production of a "less hazardous
cigarette" was to reduce total smoke delivery. Because the causal
relation between smoking and lung cancer was the first established,
primary emphasis was placed on reducing the carcinogenic "tar" of
cigarette smoke (110).

Tumor lnltlation and Cocarcinoge~~s

Inhalation studies with Syrian golden hamstem and bioassays on
mouse skin, rabbit ears, and the connective tissue of mice and rats have
clearly indicated that the major carcinogenicity of cigarette smoke
resides in its particulate phase (28, 18,109). Although the presence of
volatile carcinogens in the gas phase has been well established (Table
2), the models available at present do not allow detection of a
carcinogenic effect of the gas phase because of the low sensitivity of
the systems (98).

Extensive fractionation studies combined with bioassays have
supported the concept that the concentration in cigarette "tar" of
certain polynuclear aromatic hydrocarbons (PAH), which are known
human carcinogens (85, 69, 86), is too low to account for their activity


TABLE 2.-Known carcinogenic agents in the gas phsae of
     cigarette smoke*

DitMthytIli-
EthylmethylnitmsMine
Dbth~llti~ltt!
Nitmwpydiine
mher nirnd'
Hydrazine
Vinyl chloride
Aerylonitde
ZNhpropme
UMhlM

1
0.1
0
2
0
24
1
82
0.72
`al

as complete carcinogens. These PAH, however, are active as tumor
initiators and thus contribute to the induction of tumors by tobacco
"tar," which contains an abundance of cocarcinogens (2U, 49). Tables 3
and 4 list the tumor initiators and cocarcinogens in cigarette smoke
known at this time. Large-scale model studies on mouse skin and
inhalation studies with Syrian golden hamsters have shown that a
significant reduction of "tar" and a selective reduction of tumor
initiators and cocarcinogens will lead to a significant reduction of the
carcinogenic potential of cigarette smoke (IS, ZS, 24,29, SO, 91, 92, 48).
Recently, a study has indicated that nicotine (and possibly other
tobacco alkaloids) may be active as a cocarcinogen (14), while another
study did not show acrolein to have cocarcinogenic properties (27').
Further detailed investigations are required.

Organ-Specific Carcinogens

This approach toward the less hazardous cigarette has been criticized
by several groups as one-sided because it has been concerned only with
"tar," nicotine, and tumor initiators such as PAH and with coca&n+
gens, rather than with organ-specific carcinogens (85,88,102).
Table 5 lists the known organ-specific carcinogens. In the case of
polonium-210, a recent indepth study raises doubts on the significance
of =OPo as a factor contributing to lung cancer in smokers. Neverthe-
less, it may be prudent to reduce the =OPo content of tobacco products
w

Among the aromatic amines, certain individual compounds are
known human bladder carcinogens (e.g., Z-naphthylamine, 4-biphenyla-

94


TABLE 3.-Tumor-initiating agenta in the particulate phase of
       tobacco smoke*

CompoutKl

Relative activity aa
amplete eareiaogelP

NgAiguette

+++
+++
++
++
++
++
++
++

1040
  0.6
r10
a,
60
2
s-10
G
40-70
404
MO
  7
10
34
s
  0.1
  0.7

mine, and henzidine) (83). Doll (22) has discussed the aromatic amines
as likely contributors to the increased risk of cigarette smokers for
bladder cancer. These carcinogenic compounds are primarily pyrosyn-
thesized from the tobacco proteins (8492). Except,for the development
of a process to reduce the protein content of tobacco (IOO), no efforts
toward the reduction of aromatic amines in cigarette smoke have been
reported.

A major group of organ-specific carcinogens in cigarette smoke are
the N-nitrosamines. The volatile nitrosamines, for which protein and
nitrate are precursors, can be selectively reduced by filtration (I 7). The
tobacco-specific N-nitrosamines in tobacco and in smoke are formed
during tobacco curing as well as during smoking. So far, N'-nitrosonor-
nicotine (NNN), 4-(N-methyl-N-nitrosamine)-l-(3-pyridyl)-l-butanone
(NNK), and N'mitrosoanatabine (NAT) have been identified. These
compounds are formed from the major tobacco alkaloids: nicotine
(NNN and NNK), nomicotine (NNN), and anatabine (NAT). The total
concentration of these three nitrosamines varies between 0.7 and 10.0
Irg/cigarette (47). NNN is a moderately active carcinogen in mice, rata,
and Syrian golden hamsters, whereas NNK is a strong carcinogen in
the respiratory tract of all three species; NAT has so far not been

95


TABLE 4.-Cocarcinogenic agents in the particulate matter of
       ~.~MCCO smoke*

L Neutral fraction
  pyreae 6)
  MethYlPyreaea (I)
  Fluomotheoe (-)
  Methylfluoraotheoed (+ ;I)
  ~tiAi,~~~~ (4
  B-mPP~ (+I
  other PAEra (+)
  Napbthalenen (-)
  1-Metbylinddea (-)
  BMethylcarhamlea (-)
  4#-Dichloraetilb (-)
  Other oeutral eompotmd8 (7)
IL Acidic fmetioo
  ~teelml (4
  ~Methyldechol (-)
  4aethylLxt4uM (-)
  cEkhykda?hol (-)
  4-o-Ropylateehd (7)

+
?
+
?
+
+
?
+
+
+
+
?

bioassayed. Although conclusive epidemiologic data are not available,
"NNN should be regarded for practical purposes as if it were
carcinogenic to humans" (53). Ftesearch programs on the reduction of
these tobacco-specific carcinogens in cigarette smoke and their possible
in tivo formation in the smoker from nicotine, nornicotine, anatabine,
and other tobacco alkaloids need to be undertaken.

A neglected area may be the reduction of other organ-specific
carcinogens in cigarette smoke, such as nitro-arenes and pesticides that
may give rise to carcinogens such aa maleic hydrazide diethanolamine
(MH-30).

Carbon Yonoxlde in Cigarette Smoke

Until a few years ago the reduction of carbon monoxide in cigarette
smoke had not been seriously studied. In fact, in 1976 a report from the
United Kingdom demonstrated that unperforated filter cigarettes can
deliver higher carbon monoxide values (13-18 mg/cig) than nonfilter


TABLE 5.-Orgamfic aucinogens in the particulate matter
      of cigarette smoke

L -
   N'-Ni~WlVliCOtiO8
  44.N.Me-thyl-N-oh-m-
   mino~l-(Myridyl~l.
   butaoooe
   N'-Nitmmnatabine
  Nitmsopiperidiw
  uokoowo u08yometrical

   Polollium-210
  Nickel mmpouoda
  tkdmim eompouod8
   UUkWWM
III. PImcrm
   Nitmmmbm
   UUkBOWM
IV. Kidney and bladder
  BNapbthyhine
  .&nioofluoreae
  x--h
   o-Toluidine
   uokoowo aroom& Mliuea
   o-Nitrotduene
  uokoowo oitm eootpouod8
  Di-n.butylniW
   otter oi-e8

0.14.0 *
1-m w

0.1-0.4 pg
0.24.6 erg
mne

?

0.03-1.3 pci
0-6oow
S-IO og
?

?
?

+
+
+

+

+
+
?
?

cigarettes (9-16 mg/cig) (105). This finding has been confirmed in both
Germany and the United States (49). An increasing number of the
cigarette brands sold in the United States have perforated filter tips,
at present amounting to approximately 50 percent. The filter perfora-
tion leads to air dilution of the smoke and to changes in the burning
profile of the cigarette, and thus, to a significant reduction of the
carbon monoxide content of the smoke (Table 6). Filter tip perforation
similarly reduces the nitrogen oxides in cigarette smoke (82).

Smokers' Compensation

Studies by Russell and his group (90, 98) and recently by Hill and
Marquardt (4.4) have demonstrated that many smokers who switch to
lower "tar" and nicotine cigarettes will compensate for the loss in
smoke nicotine (and possibly other agents) by intensifying their smoke
intake, puffing more frequently, and drawing larger volumes per puff.
In the case of cigarettes with perforated filter tips, the occlusion of the
filter vents by the fingertips may be an additional compensation

97


TABLE B.-Carbon monoxide in smoke of dgareth
                    carbon onmmide (olgk!igade)

                                             Pafaated
                   Nonfilter           nlter           filter

19Tl/1976       11.6-17.0
         (N * 8)
             9-16
         (N - 9)
            16-n
         (N - 7)
            14iw9.9
         W - le)

14.4-20.0
W - 28)
lal8
(N - 10)
las-23            -
0 - 17)
8.&las
W - W    (EJ)

technique that smokers may develop either intentionally or subcons-
ciously (62). These factors of "smoker compensation" must be consid-
ered in the evaluation of lower "tar" and nicotine cigarettes. Filtered,
lower "tar" and nicotine cigarettes that are less vulnerable to
increasing the smoke and nicotine deliveries are needed. Such producta
are envisioned by scientists in the tobacco health field. Attempting to
minimize smoker compensation by selectively reducing `%ar" and other
smoke compounds while maintaining nicotine yield may carry serious
disadvantages. First, maintaining nicotine delivery may reinforce
physiologic habituation, and interfere with smoking cessation attempts
(98). Second, nicotine gives rise to the tobacco-specific carcinogenic N-
nitrosamines, NNN and NNK, and nicotine itself may be carcinogenic
(see Experimental Chemical Carcinogenesis within this section). F'inal-
ly, nicotine is suspected to bc a major smoke constituent correlated
with the increased risk of cardiovascular disease among cigarette
smokers.

Transplacental Carclnogenesfs

The possible transplacental effect of cigarette smoking on carcino-
genesis should be investigated. Recently, it has been shown that
cigarette "tar" is an active transplacental carcinogen in Syrian golden
hamsters (80). Furthermore, a number of smoke constituents are active
as transplacental carcinogens in the experimental animal (25). These
include volatile N-nitrosamines, bewaJpyrene, o-toluidine, ethyl
carbamate, and vinyl chloride (87). Other major tobacco carcinogens
including the benzofluoranthenes, NNN, and NNK need to be bioas-

98


sayed for their transplacental activity and to be considered with
respect to lower "tar" cigarettes.

Flavor Additives

The development of lower "tar" and nicotine cigarettes has tended
to yield products that lacked the taste components to which the smoker
had become accustomed. In order to keep such products acceptable to
the consumer, the manufacturers reconstitute aroma or flavor. There
are several ways in which this can be achieved. Flavor extracts of
tobacco can be added to the lower-yield blends. Other plant extracts
can be used to supplement the flavor spectrum, synthetic flavors can
be added, or a combination of techniques can bc applied (64, 65).
Powdered cocoa, one flavoring additive that is probably used in U.S.
cigarettes, has been found to increase mouse skin tumorigenicity of the
"tar" from a standard experimental cigarette at each of two dose
levels (31).

The burning of cigarettes with flavor additives produced increased
and perhaps novel types of semivolatile agents, including traces of
mutagenic compounds. The mutagenic agents were found in the basic
fraction of the semivolatile portion obtained from heating the tobacco
mixtures. Chemically, the agents thus far identified were substituted
pyrazines and other aza-arenes with and without amino groups (64).

The exact delineation of the chemical structure of additives, their
pyrolytic products, the possible carcinogenic properties, and the
quantities found in smoke of lower "tar" cigarettes is urgently needed
in order to assure the consumer that the filter, lower "tar" and nicotine
cigarette does not carry additional or new health risks.

Conclusions and Recommendations

1. Both retrospective and prospective epidemiologic studies in man
have shown a dose-response relationship between cigarette smok-
ing and the occurrence of cancer of the lung, larynx, esophagus,
oral cavity, and bladder with a less clear quantitative relationship
to cancers of the pancreas and kidney. Smoke dose was measured
by various parameters, including numbers of cigarettes (daily or
lifetime), duration of habit, depth of inhalation, and number of
puffs per cigarette.

  The highest priority in the field of public health is that
individuals who have not started smoking should not begin and
that those who currently smoke should quit.
2. Those individuals who start smoking with a filter-tipped, lower
?ar" and nicotine cigarette, or who switch after a period of time
from high "tar" and nicotine cigarettes to the lower "tar" and
nicotine cigarettes, will have a lower incidence of lung cancer, but
an incidence far in excess of the nonsmoker.

99


  Specifically, high priority should be given to continued and
long-term retrospective and prospective epidemiologic studies on
all tobaccorelated diseases, with specific reference to brand of
cigarettes smoked, number of cigarettes, manner of smoking,
inhalation, etc., along with generation of data on "tar," nicotine,
carbon monoxide, and other chemical content, as determined by
the most up-to-date scientific methods. This same epidemiologic
survey should include studies of individuals in high-risk occupa-
tions, of groups such as teenagers, minorities, and people of
varying socioeconomic status, of men compared with women, and
of different ages at which smoking began. Concern expressed by
the group was, because cigarette composition in the United States
is changing rapidly, without continued, well-planned, long-term
studies, it will be difficult to know what effect the changing
composition is having on the health of the American people.
3. An administrative mechanism to focus major interest on tobacco
and the diseases caused by smoking tobacco should bc established.
Such a mechanism should include involvement of basic scientists,
epidemiologists, physicians, statisticians, social scientists, and
related experts concerned with smoking. There should be a stable
source of funding for both new and established investigators to
work together on tobacco and health problems over a period of
time, since the answers to the questions raised over the past
quarter-century will not come quickly, considering the magnitude
and duration of the problem in the United States.
  Moreover, institutions and programs should be encouraged to
train scientists for smoking research and to maintain a core group
of physicians, scientists, and educators to consider various aspects
of smoking research issues.
4. Additional work in carcinogenesis should be performed:
a. It should be determined whether nitrosamines are formed from
  cigarette smoke in the human body and, if so, whether they are
  formed in significant concentrations. A key concern is whether
  nicotine itself forms nitrosamines in biologically significant
  quantities following reaction with nitrous oxides. The role of
  nicotine in human carcinogenesis should be identified.
b. Tobacco additives and flavoring agents should be studied by
  appropriate methods for carcinogenicity and other toxicities,
  before their commercial use is permitted, and study data should
  bc made available to the appropriate agencies.
c. A continuing study of lower Yar" and nicotine cigarettes for
  carcinogenicity might detect changes resulting from new or
  different manufacturing practices or from new additives or
  flavoring agents that might act synergistically.
d. The gas phase of cigarette smoke should bc examined more fully
for carcinogenicity.