Reverse smoking is a common practice in some parts of India,
whereby the lighted end of a homemade cigar is held inside the mouth,
Pindborg, et al. (64) conducted an epidemiological survey of 10,169
villagers in the Srikakulam district of south India and found that
43.8 percent of those interviewed practiced reverse smoking. Leuko.
plakia was found in 8.8 percent of reverse smokers compared to 01
percent in nonsmokers. The 10 patients found to have oral cancer
were all reverse smokers. Reddy, et al. (68) found that reverse smokin
was practiced by 73 of 100 patients with oral cancer. Reddy, et al,
(66, 67) reported characteristic histologic findings of the oral cavity
in biopsies obtained from reverse smokers. In two other studies fron,
India, changes in the ultrastructure of the oral mucosa of chewerg
(54) and smokers (65) are described.

Cancer of the Esophagus

In the Japanese prospective study, Hirayama (37) reported that
male smokers had a mortality ratio for cancer of the esophagus of
2.24 compared to nonsmokers (P< 0.001). Martinez (57) studied the
relationship between smoking in various forms and the development
of cancer of the esophagus in a retrospective study of 179 patients,
Dose-response relationships were demonstrated for the amount smoked
and alcohol consumption and the development of cancer of the
esophagus.

Cancer of the Larynx

The mortality ratios for cancer of the larynx in the large Japanese
prospective study were reported by Hirayama (37) to be 11.0 for male
cigarette smokers and 9.0 for female cigarette smokers compared to
nonsmokers (P<0.001).

Stell (85) conducted a retrospective study of 190 patients with
carcinoma of the larynx. Only 13 percent of the patients were non-
smokers or ex-smokers compared with 41 percent of the controls. The
relative risk ratio for heavy cigarette smokers was 3.48 compared
to nonsmokers. The relative risk was 1.34 for smokers of pipes and
cigars,

Moore (59) reported the occurrence of second primary cancers in
203 smokers who had been surgically treated for cancer of the oral
cavity, pharynx. or larynx. without recurrence for a period of 8 years.

76
Within an average followup period of 7 years, 40 percent of the 122
patients who continued to smoke developed second primary cancers
of the upper respiratory or digestive tract, but only 6 percent of the
patients who stopped smoking developed second cancers. A total of
50 patients with cancer of the larynx underwent laryngectomy. Of
the 16 who continued to smoke. three developed a second cancer,
whereas none of the 34 ex-smokers without a larynx developed a
second primary malignancy.

Cancer of the Pancreas

Hirayama (37) reported a significant association between cigarette
smoking and the development of cancer of the pancreas. The mortality
ratios were 2.05 (P<0.001) for men and 1.9 (P<0.05) for women.

Krain (47) reviewed a number of environmental factors that may
be associated with the 15 percent annual increase in the death rate
from cancer of the pancreas found in the United States. The strongest
associations appeared to be with cigarette smoking and certain
occupational exposures.

Cancer of the Kidney and Urinary Bladder

Hirayama (37) reported a mortality ratio of 2.71 for cancer of
the kidney and bladder in women who smoke cigarettes (P<0.001).
The mortality ratio of 1.07 for men who smoked. compared to non-
smokers was not significant ; however, the few deaths from this cancer
among men in the Japanese study did not allow conclusions to be
drawn.

Hoover and Cole (89) examined the strength of the association
between cigarette smoking and the development of bladder cancer in
successive birth cohorts of men and women in the United States,
Denmark, England, and Wales. Increasing rates of bladder cancer
were observed in populations characterized by an increase in cigarette
smoking among successive birth cohorts. The association was con-
sistent in both men and women, and was also consistent for different
nationalities and urban and rural groups. These findings suggest a
causal role for cigarette smoking in the development of bladder cancer.

In a retrospective study from Germany. Fischer (27) examined the
smoking habits of 162 men with bladder cancer and a control group of
198 men who had benign prostatic hypertrophy. The relative risk

77
ratio was 6.4 for smokers of fewer than 15 cigarettes a day, and 27.5
for smokers using more than this amount. Only 3 percent of the mep
with bladder cancer were nonsmokers.

Xipell (03) studied renal nodules in 250 patients in Australia who
came to autopsy. Benign adenomas were the most common lesions
and were found in 22 percent of the patients. The remaining nodules
were cysts, thrombosed veins, abscesses, granulomas, and metastatic
lesions. A statistically significant difference between the smoking
habits of those with adenomas and those with the miscellaneous lesions
was reported (P<0.012), All the adenomas were found in smokers,

Cole, et. al. (74) conducted a retrospective study of 461 persons
with transitional or squamous cell carcinoma of the lower urinary
tract. After the data were controlled for cigarette smoking, occupa-
tional exposure appeared to contribute to 18 percent of the lower
urinary tract cancer among men aged 20 to 89 compared to the 39
percent attributed to cigarette smoking in men in a previous
report (14).

Werf-Messing and Kaalen (100) examined the association of
occupational exposure and smoking in the development of bladder
cancer in 846 males in the Netherlands who had this disease. The
smoking habits of cancer and control patients in each group were
nearly identical; however, patients with bladder cancer had a longer
exposure to hazardous working conditions than did controls.

Experimental Carcinogenesis

Experimental studies, mainly in animals, have added to an under.
standing of many of the processes involved in tobacco carcinogenesis,
Possible mechanisms of chemical carcinogenesis were reviewed by
Miller and Miller (58), Ryser (76). and Leone (41). Electron spin
resonance studies of carcinogenesis were reviewed by Swartz (87),
Franke (28) discussed the posible role of hydrophobic interactions
of polycyclie aromatic hydrocarbons with protein in chemical car-
cinogenesis. Chemical carcinogenesis in Syrian hamsters was reviewed
by Shubik (82) and Homburger (38).

Respiratory Tract Carcinogenesis

Epidemiological, clinical, and autopsy data from studies of humans
have established cigarette smoking as the major cause of lung eancer
in the United States. One of the reasons it has not been possible to

78
characterize fully the mechanisms responsible for this causal relation-
ship is the lack of an ideal animal model in which to study respiratory
tract carcinogenesis in the laboratory. Exposing animals to cigarette
smoke in a closed chamber does not replicate the kinds of exposure
smoking humans receive, although some recently developed smoking
chambers provide conditions similar to the exposure experienced by
human smokers. Many animals are obligatory nose breathers and,
in them, a large portion of the particulate phase of cigarette smoke
may be removed by turbulent precipitation in the nasal passages or
larynx before reaching the sites in the lung most commonly exposed
m humans. Auerbach, et al. (3) first demonstrated that malignant
ling tumors could be produced in smoking dogs who were taught to
smoke through a tracheostoma. Several investigators have recently
examined respiratory tract carcinogenesis in animals using intra-
tracheal instilations of chemical carcinogens found in cigarette smoke,
including benzo(a)pyrene and (H-dibenz(d.g) carbazole. Tumors
resulting from this type of treatment are frequently similar to lung
tumors found in humans (24 32. 33. 36.77. SO),

Harris, et al. (33) examined the acute ultrastructural effects of
benzo(a)pyrene carried on ferric oxide particles on the tracheo-
bronchial epithelium of the Syrian Golden hamster. Test substances
were administered by intratracheal instillation. Ferric oxide alone
resulted in some focal replacement of columnar epithelium with
polygonal basal cells. This effect was reversed by termination of the
treatment. After treatment with benzo(a)pyrene and ferric oxide.
focal replacement of the columnar cells with pleomorphic cells oc-
curred. These pleomorphic cells had the ultrastructural features of
atypical squamous cells and were similar to the hyperplastic epithelial
cells described in the bronchi of smoking dogs and the neoplastic
“quamous cells found in human bronchogenic carcinoma,

Tn an extension of this study, Harris, et al. (32) reported that. vita-
min A deficiency or the application of benzo(a)pyrene-ferric oxide
through intratracheal instillation resulted in squamous metaplasia of
the trachea, Both lesions appeared to be morphologically similar by
light microscopy, but at the ultrastructural level significant differ-
neces were observed. Squamous metaplasia induced by benzo(a) py-
tene-ferric oxide was characterized by defects in the basement
membrane, enlarged nuclei with cytoplasmic invaginations, and
Pleomorphie nucleoli not seen following vitamin A deficiency.

Sellakumar and Shubik (80) treated Golden Syrian hamsters with
weekly intratracheal instillations of TH-dibenz(c.g)carbazole (7H-
DBC) suspended with equal amounts of ferric oxide in a saline solu-
tion. One group of 35 hamsters was treated with 45 mg. of the
‘arcinogen and a second group was treated with 15 mg. More than
So percent. of the animals in each group developed respiratory tract

79
tumors. Most of the tumors occurred in the major airways and were
squamous cell carcinomas. Adenocarcinomas and anaplastic carcino nas
were found less frequently.

Saffiotti, et al. (77) examined the carcinogenic effects of benzo (a)
pyrene prepared as a suspension of fine crystalline particles at.
tached to ferric oxide in a physiologic saline solution and adminis.
tered by intratracheal applications to Syrian Golden hamsters, Vari-
ous concentrations of benzo(a)pyrene and ferric oxide were used in
single and multiple applications. A single administration of 37.5 mg,
of benzo(2)pyrene with 12.5 mg. of ferric oxide resulted in five
bronchogenic carcinomas and five histologically benign respiratory
tumors in a total of 61 hamsters. Following multiple administrations,
bronchogenic carcinomas including anaplastic and squamous cell types
were induced in all dosage groups and a positive dose-response rela.
tionship was demonstrated.

Feron (24) studied respiratory tract tumors in Syrian Golden
hamsters following tracheal instillations of furfural and/or benzo(a)
pyrene. Of the 62 hamsters. 41 developed respiratory tract tumors of
which squamous cell carcinoma of the trachea was the most frequent
type observed. Furfural ir. combination with benzo(a) pyrene resulted
in a higher yield of tumors than was seen with benzo(a)pyrene alone.
Furfural alone possessed no carcinogenic activit y.

Shabad (87) and one of his collaborators, Yanysheva, produced
benign and malignant epidermoid lung tumors in rats following
single and multiple administrations of benzo(a)pyrene by intra-
tracheal instillation. Dose-response relationships were demonstrated.

Experiments in Mice

Cigarette smoke condensate (CSC), various fractions of CSC, and
many chenucal compounds identified in CSC have been tested for
tumorigenic activity in mice by a variety of methods, including skin
painting and suveutaneons injections. Complete carcinogens and in-
complete carcinogens, which include tumor initiators, tumor _pro-
moters, and tumor accelerators have been described. Several recent
studies have been conducted using mice as the experimental animal
which examine further the mechanisms involved in tobacco carcino-
genesis.

Lee and O'Neill (50) measured the effect of duration and dosage
of benzo(a)pyrene applications on the rate of development of benign
and malignant skin tumors in mice. The incidence rate for tumor
formation was directly proportional to both time and dose. These
data conformed quite closely to postulated mathematical models of
the rate of tumor development.

80
Davies and Whitehead (17) studied the-effect of altering the “tar”
and nicotine ratio of cigarettes on experimental carcinogenesis. No
significant difference in tumor yield was found between condensates
obtained from the smoke of cigarettes containing 16.6 mg. “tar” and
1.79 mg. nicotine and other cigarettes containing 10.0 mg. “tar” and
1,94 mg. nicotine.

Several studies by Bock, et al. (7, 8, 9) have examined the tumor
promoting activity of a number of fractions of cigarette smoke con-
densate (CSC). A number of subfractions of the neutral fraction
of CSC were tested for tumor promoting activity in mice pretreated
with 7,12-dimethylbenz(a)anthracene as a tumor initiator (8). The
most polar subfractions and the fraction containing venzo(a) pyrene
were the most active tumor promoting fractions. In another study
(9), the weak acid fraction of CSC was found to be a very weak
complete carcinogen which probably acts primarily as a tumor pro-
moting agent. The promoting activity depended primarily on the
nonvolatile constituents of this fraction. More recently, Bock, et al.
(7) reviewed the tumor promoting effects of CSC and extracts of
tobacco leaves. A combination of two subfractions of the tobacco ex-
tracts, as well as five major fractions of CSC, were found to have
tumor promoting activity. The fraction containing the polynuclear
aromatic hydrocarbons was found to be a complete carcinogen. Two
subfractions were found to be strongly synergistic in their tumor pro-
moting activity when applied simultaneously to mouse skin.

Lazar, et al. (49) found that hydroquinone applied to mouse skin
in conjunction with the active fractions of CSC accelerated the early
histologic changes that result from the application of “tar” or its
fractions.

Van Duuren, et al. (97) have suggested that “cocarcinogenesis” be
differentiated from “tumor promotion” defining “cocarcinogenesis”
as the production of malignant tumors by two or more agents applied
simultaneously or alternately in single or repeated doses to mouse skin
and “tumor promotion” as a single treatment with one agent followed
by single or repeated treatment with a second agent. Using these
definitions, the authors found several tumor promoting agents to
possess cocarcinogenic activity.

Roe, et al. (74) studied mechanisms of mouse skin carcinogenesis
using benzo(a)pyrene and a neutral fraction of OSC applied singly
or in various combinations with each other. Skin tumor incidence rates
Increased with the dose of applied material for both the neutral frae-
tion and benzo(a)pyrene. Mixtures of the neutral fraction with benzo-
(a)pyrene did not act independently in the production of malignant
skin tumors but synergistically, suggesting that some of the eompo-
nents of the neutral fraction act as cocarcinogens rather than as com-
plete carcinogens.

81
Schmahl (78) found a direct relationship between the dosage and
duration of subcutaneous injections of tobacco smoke condensates and
the development of sarcomas in rats.

Maenza, et al. (56) studied the effects of a combination of nickel
subsulfide (Ni,S.) and benzo(a)pyrene on sarcoma induction in rats,
The interval between administration of the carcinogen and the de.
velopment of sarcomas was significantly shorter (P<0.001) in male
Fischer rats given injections of a combination of 10 mg. of Ni,S. and
5 mg. of benzo(a)pyrene than in rats given either ingredient alone.
There appeared to be a synergistic interaction between nickel com-
pounds and the polycyclic aromatic hydrocarbons.

Healey, et al. (34) added further refinements to a technique for
measuring the nonspecific esterase activity of mouse skin following
applications of various chemical compounds. With few exceptions,
changes in esterase activity reflected the known tumor producing
activity of a number of polycyclic hydrocarbons and_ tobacec
condensates.

Sydnor, et al. (89) examined the effect of an aqueous extract of
cigarette smoke condensate on benzo(a)pyrene-induced sarcoma in
female Sprague-Dawley rats. Benzo(a)pyrene was injected subcu-
taneously in various concentrations of 12.5 pg. to 400 zg. per dose dis-
solved in sesame oil. Injections were given on alternate days fo
30 doses. The mean tumor induction time was accelerated in five of
seven groups given the aqueous extract of CSC in their drinking
water. Animals given any benzo(a)pyrene eventually developed sar-
comas at the site of injection. Dose-response relationships were demon-
strated for the concentration of benzo(a)pyrene administered. It
appeared that aqueous extracts of CSC contained one or more com-
ponents which functioned as cocarcinogens.

Aryl Hydrocarbon Hydronylase (AHH)

Certain of the chemical compounds found in the gas and particulate
phase of cigarette smoke are absorbed through the lung or oral cavity
into the general circulation. Possibly through such absorption some
chemical carcinogens are carried to target organs not directly exposed
to cigarette smoke. Some of these chemical compounds are probably
excreted unchanged while others are metabolized to various degrees
by enzyme systems present in the liver and many other tissnes. The
microsomal mixed-function oxidases are kev enzyme systems for the
metabolism of a wide variety of chemical compounds including the

82
chemical carcinogens found in cigarette smoke. Aryl hydrocarbon
hydroxylase (AHH) is a part of the cytochrome P-450 containing
microsomal enzyme system that is present in several tissues of humans
and many animal species. The activity of this enzyme system is in-
duced following exposure to the appropriate chemical stimulus. The
hydroxylation of polycyclic hydrocarbons results in the detoxification
of some and the activation of others to reactive carcinogenic forms.
An understanding of the role of AHH in the metabolism of chemical
carcinogens in man may help clarify some of the mechanisms involved
in tobacco carcinogenesis. Recently, several studies examined AHH
activity in animals and man.

Studies in Animals

Sydnor, et al. (88) found that an aqueous extract of CSC adminis-
tered in the drinking water of rats potentiated benzo(a)pyrene-
induced AHH activity in the liver. The liver AHH activity was
slightly increased by the aqueous extract of OSC alone.

Rondia and Gielen (75) reported that rats exposed to various levels
of carbon monoxide developed a decrease in AHH activity in liver
homogenates. The reduction in AHH activity developed after 120
hours exposure to levels of carbon monoxide which produced carboxy-
hemoglobin levels below 15 percent.

Welch, et al. (99) reported that the administration of benzo(a)-
pyrene to pregnant rats resulted in an increase of the in vitro AHH
activity of maternal liver, placenta, and fetal liver. A twentyfold
higher dose of benzo(a) pyrene was necessary for stimulation of AHH
activity in fetal liver than in the placenta or maternal liver.

Studies in Man

Levin, et al. (52) studied the induction of AHH activity in human
skin. Human foreskin obtained from circumcised children was main-
tained in tissue culture medium. Exposure to 10 2/M. of benzo(a) py-
rene for 16 hours led to a twofold to fivefold increase in the activity of
AHH in the exposed skin over control values.

Whitlock, et al. (701) reported the presence of AHH in human
lymphocytes. The AHH activity of lymphocytes compared to rat liver
or hamster embryo cells is relatively low. Treatment with pokeweed
mitogen alone increased AHH activity about twofold. However, a
threefold to eightfold greater AHH activity was found in cells treated
with the mitogen and benz(a)anthracene than in resting cells.

83
495-028 O-—72——7
Cell and Tissue Culture Studies

In studies of tobacco carcinogenesis, cigarette smoke condensate
(CSC), subfractions of CSC, and individual chemical compounds
found in CSC have been administered to a variety of animals using
several routes of administration. Tests on living animals are frequently
complicated and time consuming. Cell and tissue culture systems offer
an alternate tool for the study of carcinogenesis which, in some in-
stances, is relatively more rapid than animal testing. Specific enzyme
systems and other cellular functions can often be studied in greater
detail using these systems, Cells obtained from a variety of tissues and
animals can be grown or maintained in culture bottles when nourished
with an appropriate nutritive medium in a supportive atmosphere,
When these cultures are exposed to various chemical compounds,
changes can occur which may range from minor morphologic varia-
tions to malignant transformation or cell death. Toxic effects on cell
cultures must be differentiated from malignant transformation. Sey-
eral studies have recently examined the effect of cigarette smoke
condensate or individual polycyclic hydrocarbons found in CSC on
various cell and tissue culture systems.

Benedict, et al. (4) studied polycyclic hydrocarbon produced cyto-
toxicity, malignant transformation, and chromosome deformity in a
variety of cell lines derived from rats, hamsters, and human tumor
cells. The cytotoxic effect of benzo(a)pyrene was found to be related
to the aryl hydrocarbon hydroxylase activity (AHH) of the given
cell culture. Benzo(a)pyrene was cytotoxic to fetal rat hepatocytes,
but this effect was probably related to the action of the hydroxvlated
metabolite, 3-hydroxybenzo(a) pyrene, since the cytotoxicity was
blocked when the AHH system was overloaded with phenobarbital.
Cell strains not possessing AHH activity showed no cytotoxic effects
from benzo(a)pyrene alone: however, in the presence of fetal rat
hepatocytes possessing AHH activity. enough benzo(a)pyrene metabo-
lites were secreted into the medium to induce cytotoxic effects in the
normally resistent cell lines. In hamster secondary cultures, at the
chromosome level cytotoxicity was associated with chromatid breaks,
whereas malignant transformation was more closely related to
aneuploidy.

Diamond (79) studied the metabolism of benzo( a) pyrene and 7.12-
dimethylbenz(a)anthracene (DMB.A) in mouse, hamster, rat, monkey,
and human cell cultures. Metabolism of hydrocarbons to “alkali solu-
ble” and “water soluble” derivatives was measured. The results sug-
gested that the parent compounds were first metabolized to “alkali
extractable” derivatives and then to “water soluble” derivatives, All the
cell cultures tested which were sensitive to the growth-inhibitory
effects of benzo(a)pyrene or DMBA were able to metabolize these

84
carcinogenic hydrocarbons to “water soluble” derivatives. The data
are consistent with the hypothesis that metabolism of the carcinogen
isrequired for growth-inhibitory or cytotoxic effects.

Several authors have examined malignant transformation in cell
cultures. Inui and Takayama (4/) cultured hamster lung fibroblasts
and then exposed them to crude cigarette “tar” for a period of 3 hours.
Between 2 to 48 hours following this exposure, toxic effects of the
“tar”, including cell necrosis, swelling, vacuolization, and disintegra-
tion of cytoplasm were observed. The death of 40 to 70 percent of
the cells within 72 hours was followed hy the appearance of trans-
formed cells which grew at rapid rates. These transformed cells pro-
duced malignant tumors when inoculated in the cheek pouch of ham-
sters. Control] cell lines produced no changes when inoculated in a
similar fashion.

In a similar study by Di Paolo, et al. (2/), transformation of pri-
mary hamster cell cultures was induced by benzo(a) pyrene. 3-methyl-
cholanthrene, or 7,12-dimethylbenz(a)anthracene. Transformed cell
limes were established and subsequently inoculated in hamsters pro-
ducing malignant tumors at various sites. Characteristic chromosomal
changes in the transformed cells were also described.

An increase in proliferation and tumor production rate of L-Strain
cells produced by treatment with cigarette “tar” was studied by
Inui and Takayama (40). L-Strain cell cultures not exposed to “tar”
did not produce tumors when inoculated in C3H mice. After an
exposure to low concentrations of cigarette “tar” significant changes
occurred in the cultures characterized by enlarged cells with vacuolated
cytoplasm, giant cell formation, and accelerated growth rates. These
transformed cells produced tumors in 70 percent of injected C3H
mice.

Nagata (60) treated cell cultures obtained from kidneys of new-
born mice with 20-methylcholanthrene in various concentrations. Con-
trol cultures could not be maintained for long; however, the treated
cells formed two permanent cell lines which had a transformed mor-
phology and altered karyotypes. Epithelial carcinomas were produced
after the subcutaneous injection of these transformed cells into un-
conditioned newborn mice.

Freeman, et al. (29) isolated hamster-specific C-type RNA viruses
from tumors induced by cell cultures transformed by chemical car-
“mogens. Cell cultures were prepared from early passage hamster
embryo cells and treated for 7 days with 3-methylcholanthrene or cer-
tain fractions of cigarette smoke condensate. Following treatment,
morphologically transformed cell lines were isolated and maintained.
Subsequent inoculation in newborn hamsters produced malignant
tumors at the site of inoculation. New cell lines were established from
Soine of the resulting tumors. No infectious viruses were isolated from
vell lines prior to inoculation ; however, C-type RNA viruses were iso-

85
lated from tumors and from cell lines derived from tumors. The ay.
thors concluded that the chemical treatment and activation of viruses
appeared to be related events.

Sivak and Van Duuren (83) developed a cell culture system that
responded with characteristic changes in cell morphology to the appli-
cation of various fractions of tobacco leaf extracts. Certain dose.
response characteristics were demonstrated, suggesting a mechanism
whereby various tobacco fractions might. be rapidly screened for
tumor-promoting activity.

Dietz and Flaxman (20) studied the toxicity of aromatic hydro.
carbons on normal human epidermal cells in vitro. Pieces of adult
human abdominal skin were maintained in tissue culture medium and
exposed to 3-methylcholanthrene and benzo(a)pyrene at a concentra-
tion of 1 »g./ml. for a period of + days. The cultures were then kept for
an additional 3 months following exposure. No malignant transforma.
tion occurred; however, giant cells and a more disorderly pattern of
growth were observed in the treated cultures weeks earlier than similar
changes in control cultures,

Binding of Polycyclie Hydrocarbons to DNA and RNA

There is evidence that some chemical carcinogens including certain
of the polyeyclic hydrocarbons found in cigarette smoke condensate
are active because of the reaction of the carcinogen or a reactive me.
tabolite with cellular macromolecules, Duncan, et al. (23) studied a
series of radioactive polycyclic hydrocarbons with respect to their
metabolism and tendency to bind with cellular DNA and RNA in
monolayer cultures of primary mouse embryo cells. All the tested
hydrocarbons were metabolized to “water soluble” metabolites at
approximately equal rates. A “binding index” was calculated to de-
termine the binding of various hydrocarbons to cellular DNA and
RNA. The group of hydrocarbons with a high “binding index” con-
sisted of potent carcinogens, while another group with much lower
values for the “binding index” were with but one exception non-
carcinogens.

Carlassare, et al. (7/2) studied the in vivo binding of benzo( a) pyrene
to DNA. Benzo(a) pyrene-“H was fed to male and female NCL mice
which were sacrificed after 15 hours. The DNA was extracted and
purified from the skin, spleen, and liver. The binding of benzo(a)py-
rene was greatest. in the liver and somewhat less in the spleen and
ekin, It was caleulated that the average molecular weight of DNA
was 6 million and that 1 molecule of benzo(a)pyrene was bound to
every 46.8 molecules of DN A in the liver, suggesting covalent binding
of henzo(a) pyrene to DNA.

86
Alexandrov and Vendrely (1) found that cigarette smoke conden-
sate, the hexane-extracted fraction of CSC, and benzo(a)pyrene all
inhibited RN A synthesis in mouse skin.

N-Nitrosamines in Tobacco Smoke

The largest number of chemical carcinogens which have been iden-
tified in cigarette smoke condensate are polycyclic hydrocarbons.
N-nitrosamine compounds known for many years to be potent. car-
cinogens have produced malignant tumors in a number of organ sys-
tems of a wide variety of animals. These compounds were recently
identified in cigarette smoke. Only recently has an association been
found between exposure to N-nitrosamines and malignant tumors in
humans (65). N-nitrosamines are formed chemically by a reaction of
NO and NO: or nitrites with secondary amines. The chemical pre-
cursors of the N-nitrosamines have been identified in cigarette smoke
condensate (CSC) by a number of investigators. These studies were
reviewed by Wynder and Hoffmann (102). More recently, Rhoades and
Johnson (69) developed a method for the determination of N-nitrosa-
mines in tobacco smoke condensate using gas chromotography. Two
N-nitrosamines were found in CSC: one was identified as N-dimethyl-
nitrosamine (DMNA) and the other was believed to be N-methy}-
ethylnitrosamine (MENA) (43, 70). The concentration of DMNA
per cigarette in nanograms was determined in condensates from ex-
perimental cigarettes made -from single tobacco varieties rather
than a tobacco blend. Each tobacco tested was grown in both a low-
and high-nitrogen soil. High-nitrogen soil conditions resulted in a
considerable increase in nitrosamines. A popular brand of nonfilter
cigarettes was also tested. These results are presented in table 2.

TaBLE 2.—N-dimethylnitrosamine (DMNA) content of condensates
obtained from several tobaccos grown in both “high” and ‘low’ nitro-
gen sorls

 

 

DMNA (nano-

Tobacco type Soil nitrogen grams per

cigarette)
Robinson_____.____________... Low nitrogen__.__._..-___.___. 0
Catterton_.........__...____..-__ do.__- 2 eee 5
Burley... 2222.88. do.._- 2 eee 3
Robinson_.____._____________- High nitrogen_._..______..___- 27
Catterton....._____..__....__.._._- do... eee 60
Burley eee eee eee do___. 2 ee 140
U.S. nonfilter.. 222-2222 8

 

Source: Johnson, D. E., Rhoades, J. W. (48).

87
Summary of Recent Cancer Findings

In addition to the summary presented in the introduction of this
chapter, based on previous reports of the health consequences of smok.
ing, the following statements are made to emphasize the recent devel.
opments in the field:

1.

Recent epidemiological and autopsy studies from several coun-
tries confirm that cigarette smoking is the major cause of lung
cancer,

-Continued cigarette smoking by patients following successful

surgical removal of a cancer of the oral cavity, pharynx, or
larynx without tumor recurrence for a period of 3 years is asso-
clated with a significant increase (P< 0.001) in the risk of devel-
oping a second primary cancer of the upper respiratory or diges-
tive tract compared to similar patients who discontinue smoking
at the time of their surgery.

. The intratracheal administration of certain polycyclic hydrocar-

bons found in cigarette smoke condensate results in the forma-
tion of anaplastic and squamous cell cancers of the lung and re-
spiratory tract in hamsters and rats, Many of these tumors are
histologically similar to the Iung cancers fonnd most frequently
in cigarette smokers.

» The application of cigarette smoke condensate or polycyclic hy-

drocarbons to various cell cultures often results in transformation
to cells with a more rapid and disorderly growth pattern. Trans-
formed cell lines frequently produce benign or malignant tumors
when transplanted to experimental animals.

. N-nitrosamines have been identified in cigarette smoke. These

compounds are known to be potent cancer causing chemicals for
a variety of animals. They appear to be formed in higher concen-
trations from tobaccos raised under high-nitrogen soil conditions.

Cancer References

(1) ALEXANpDROV, K., VENDERLY, C. Action de condensats de fumée du tabac

sur la synthese de RNA dans la peau des souris. (Action of tobacco
smoke condensate on the synthesis of RNA in mouse skin.) Chemico-
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95
CHAPTER 4

 

Pregnancy
Contents

Introduction

Smoking and Birth Weight
Epidemiological Studies
Cigarette Smoking and the Low-Birth-Weight Infant.
Evidence for a Causal Association Between Cigarette
Smoking and Small-for-Dates Infants__._________
Evidence for an Indirect Association Between Ciga-
rette Smoking and Small-for-Dates Infants
Experimental Studies
Studies in Animals
Tobacco Smoke
Nicotine

Polycyclic Hydrocarbons
Studies in Humans

Carbon Monoxide._.________.______________-

Polycyclic Hydrocarbons___.____.-_______ 8.

Vitamin B,. and Cyanide Detoxification

Vitamin C

Liming of the Influence of Cigarette Smoking on Birth

Weight._....----2--- 2 woe ee eee eee
Site of Action at the Tissue and Cellular Level
Significance of the Association
Birth Weight Summary

Cigarette Smoking and Fetal and Infant Mortality
Introduction

Spontaneous Abortion Summary
Stillbirth

495-028 O—73—_8

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Cigarette Smoking and Infant Mortality—Con.
Late Fetal and Neonatal Deaths
Epidemiological Studies_.....-----.-0..
Comparisons of the Mortality Risks of Low-
Birth-Weight Infants Born to Smokers and

Nonsmokers

Analysis of Previously Reported Studies________
Factors Which Influence Perinatal Mortality
Other Than Smoking
Experimental Studies
Studies in Animals

Evidence for a Clinical Effect Upon the Off-
spring

Lactation Summary
Preeclampsia

100

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