Reverse smoking is a common practice in some parts of India,
rrhereby the light,ed end of a homemade cigar is held inside t,he mouth.
Pindborg, et al. (64) conducted an epidemiological survey of 10,169
villagers in the Srikakulam distric.t of south India and found that
43.8 percent of those interviewed practiced reverse smoking. Leuko.
plakia wan found in 8.8 percent of reverse smokers compared to 0.1
percent in nonsmokers. The 10 pat,ients found to have oral cancer
were all reverse smokers. Reddy, et al. (68) found t,hat reverse smoking
was practiced by 73 of 100 patients with oral cancer. Reddy, et al.
(66, 67) reported chara&eristic histologic findings of the oral cav$
in biopsies obtained from reverse smokers. In t,wo other studies fronl
India, changes in the ult,rast,ructure of the oral mucosa of chewers
(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<O.OOl). 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 relat,ionships were demonstrated for the amount smoked
and alcohol consumpt.ion 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 !I.0 for female cigarette smokers compared to
nonsmokers ( P <O.OOl ) .
Stell (83) conducted :I 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 ins 3.48 comparerl
to nonsmokers. The relative risk n-as 1.34 for smokers of pipes a~1
cigars.

Moore (.5,9) reported the occurwnre of second primary cancers in
203 smokers who had heen surgically treated for cancer of the oral
cavity. pharynx. or larynx, without recurrence for a period of 3 years.

76


Within an average followup period of 7 years, 40 percent of the 122
patients who continued to smoke develolwd second primary GLIKCI`S
of the upper respiratory or digwtivr tract, but only 6 percent of the
patients who stopped smoking developed second cancers. A total of
i0 patients wit11 cancer of the I;~rynx underwent laryngectomy. Of
the 16 who continued to snloke. tllree devcloprd a second cancer,
whereas none of the 3-& ex-smokers without a larynx derelolwd a
second primary malignamy.

Cancer of the Pancreas

Hirayama (37) reported a signiticant association between cigarette
smoking and the derelopnlent of cancer of the pancreas. The mortality
ratios were 2.05 (P<O.OOl) for 111en and 1.9 (l'<O.OS) 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 I-nited States. The strongest
associations appeared to be with cigarette smoking and cert.ain
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<O.OOl) .
The mortality ratio of 1.07 for men who smoked compared to non-
smoke,rs 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 (39) 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 Vnitrd States,
Denmark, England, and Wales. Increasing rates of bladder cancer
tvere observed in populations characterized bv an increase in cigarette
smoking among successive birth cohorts. The association was con-
sistent in both men and Tomen, and was also consistent for different
uationalitirs and urban and rural groups. These findings suggest a
rausal role for cigarette smoking in the development. of bladder cancer.

In a retrospective study from Germany. Fischer (27) examined the
smoking habits of 18% men with bladder cancer and a control group of
1:M 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 rnen
with bladder cancer were nonsmokers.

Xipell (103) studied renal nodules in 250 patients in Australia w]le
came to autopsy. Benign adenonias were ,thc most common lesions
and were found in 22 percent of the patients. The remaining nodules
were cysts, thrombosed veins, abscesses, granulomas, and metastatie
lesions. A statistically significant difference between the smoking
habits of those with adenomas and those with the miscellaneous lesions
was reported (P<O.OU). All the adenomas were found in smokers.

Cole, et al. (14) conducted a retrospective study of 461 persons
with transitional or squamous cell carcinoma of the lower urinary
tract. After the dam \rere controlled for cigarette smoking, occupy.
tional exposure appe,ared to comribute t,o 18 percent of the lower
urinary tract cancer among men aged 20 to 80 compared to the 39
percent, attributed to cigarette smoking in men in a previous
report (1.5).

Werf-Messing and Kaalen (200) examined the association of
occupational exposure and smoking in the development of bladder
cancer in 346 males in the Netherlands who had this disease. The
smoking habits of cancer and control patients in each group were
nearly identical; however. patients \vith bladder cancer had a longer
exposure to hazardous \vorking conditions than did controls.

Experimental Carcinogenesis

Experimental studies, mainly in aninlals. have added to an under-
standing of many of the processes involved in tobacco carc.inogenesis.
Possible mechanisms of chemical carcillogenesis were reviewed by
Miller and JIiller (158), Ryser (7'6'). and Leone (51). Electron spin
resonance studies of carcinogenesis were reviewed by Smartz (87).
Franke (28) discusset the posible role of Ilydrophobic interactions
of polycyclic aromatic hydrocarbons with 7)rotein in chemical CRT-
cinogenesis. Chemiral carcinogencsis in Syrian hamsters was reviewed
by Shubik (89) and Hornburger (.?a).

Epidemiological, clinical, and autopsy data from studies of humans
have est,ablished c*ip:lrette smoking as the major cause of lung cancer
in the 7-nitcd States. One of the reasons it has not been possible to

78


,nlwacterize fully the mechanisms responsible for this causal relation-
&l) is the lack of an ideal animal nlodel in which to study respiratory
tract carcinogenesis in the laboratorg. Exposing animals to cigarette
woke in a closed chamber does not rrl)licate the kinds of exposure
waking humans receive, although some recently developed smoking
Awbers provide conditions similar to tlw c~sposure experienced by
human smokers. Many anhal .-j arc obligatory 11osr breathers and,
in them, a large portion of the particulate phase of cigarette smoke
~n;ly be removed bv turbulent ljrecipitation in the nasal ljassages or
hryis before rcncliiiig tlic sites in tlw lung ilrost coninionly exposed
in humans . ,1ucrbacb. et al. (.i) first dcliloilstl,:lt,etl tliat malignant
hng tunlors could be l)rodwetl in snlokill g clogs \VllO \vere taught to
snlokc through a tracheostoma. Scvcral inrc~stipators hart recently
rsr\uGned resl)irntorv tYilCt c~ilIYiIl(~g~lleSis in ;tilinuls using intra-
tr:~c~lle:~l instillations of cllrltlical c~rc~inoge~~s fo11nt1 in c~igarrttc smoke.
iwlnding benzo( a) pywie and ITH-dibenz (dg) carbazolc. Tumors
rtwilting from this type of trwltnwnt art frqurntly sinlilar to lung
t~111ors found in humans (24 .iJ. .1-J. .16. ;I';". 80).

Harris, et al. (03) examined the acute nlt.rastrlwtural efiects of
1wnzo (a ) pyrene carried on ferric oxide part,icles on the trachco-
t)rowhial rpithclium of the Svrian Golden Ilamster. Trsh substances
wre administered br intrntrache,ll instillation. Ferric oxide. alone
resulted in some f&al replacement of columnar epithelium wit.h
~)olygonal basal cells. This effert. was reversed by termination of the
treatment . After treatment with benzo (a) pyrene and ferric~ oxide.
fwal replacement of the columnar cells with pleomorphic ~11s oc-
wrred. These pleomorphic cells had t.he ultrastructural fe.atures of
:ttypical squamous ~11s and v-erc similar to thr hyperplastic tpithelial
~11s described in the bronchi of smoking do,? and the neoplastic
~~l~amous cells found in hlmlan lwonchogenic carcinoma.

111 an extension of this Ptudy, Harris? et al. ($2) reporte,d t.hat' vita-
Cl1 -1 deficiency or the application of benzo(a) pyrene-ferric. oxide
througl~ intratr~chcal instillation resulted in squamous met.apl:& of
the tl*acheil. I{otIl lesions appeared to he morphologically similar by
light n~icmxwq~~, but at the ultrastructural level significant differ-
ices were obser\-ed. Squnn~~~ nwtnplasia induced by lWllzo(a) py~-
rent-ferric oxide was c~haractrrizecl bv defects in thr basement,
~11embranc. enlarged nuclei witIr cytoplasnlic invaginat.ions, and
l~leonlorphic nucleoli not well following vitamin h deficiency.

Scllakumar and Shubik (80) treated Goldt~n Syrian hamsters with
\`mklv intratrachrul instillations of XI-dihenz(c.p) carbazolc (7H-
l)lN`j snsp~~~drd with q11a1 anloullts of ferric oxide in a saline solu-
h. 011~ gt`0tl1) of 3s llamsters 1va.s t.rented with 45 111g. of the
(`;lfGnopen :uld a sct~oiid group was trrated with 15 mp. JIore than
G pcrceilt. of the aiiinra15 in each group developed respiratory tract

79


tumors. Most of the tlumorr: occurred in the, major airways and were
squ:1*110us cell carcinomas -. Adenocarcinomas and annplastic. carcino,nas
were found less frequently.

Safiotti, et al. (77) examined t,he carcinogenic effects of henzo ( a~
pyrenc prepared as a slispension of fine crystalline part,icles at.
tached to ferric: oxide in a physiologic. saline solution and adminis.
tered by intratrnche.al applications to Syrian Golden hamsters, Vari.
ous concentrations of benzo( a) pyrenc and fe.rric oxide mere used iI,
single and multiple applications . A single administrat,ion of 3$.5 mgS
of benzo(a) 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 administrnt,io&
bronchogenic carcinomas including anaplastic and squamous cell tylRs
were induced in all dosage groups and a positive dose-response rela.
tionship was demonstrated.

Feron (24) studied reqkntory tract tumors in Syrian Golden
hamsters following tracheal instillations of furfural and/or benzo(a!
1)yrene. Of the 62 hamsters. 41 developed respiratory tract tumors of
which squamous cell carcinoma of the t,rachex was the most frequent
type observed. Furfural ir, combination with benzo (a) pyrene resulted
in :I liipher yield of tumors than was seen with benzo (a) pyrene alone.
Furfural alone possessed no carcinogenic activity.

Shabad (81) and one of his collaborators. Yanyshevn, produced
benigll and nlalignant epidrrmoid lung tunlors in rats following
single and nlnltiple administrations of bcnzo( a) pyrene by intra-
trachr>al instillation. Dose-response relationships were demonstrated.

Errperiments in Mice

Cigarette snloke condensate (CSC), various fractions of CSC? and
manly clwnlic*al coInpounds identified in CSC have been tested for
tunlorigvnic* ;lctiyity in mice. by a variety of nwthods, including skin
i):iinting and S11~JCllt~lleOll:` injections. complete mrcinogens and in-
cwiil)lcte 1.8 rcainogens. wl:icli include tumor initiators? tumor pro-
nwtew, and tmtlor ac~,*clerators llave been described. Several recent
studies lI:lve been conducted usin g mice as the experimental animal
which examine further the mechanisms involved in tobacco wrcino-
genesis.

IRC and O'SeilI (50) llwasnrrd the effect of duration and closnp(~
of lwnzo(n ) l)yrkbne applicntiolls on tllc rate of de\-elol)nlent of benip)
xnd nialig1Lant kin tiunors in niiw. The incidence rate for tuniol
fornlation \v:~s tlirectly proportional to both tinle and dose. TIRV
tl:lta caollforllled q"ite. c~lo:t~ly to l)o~tulatcd n~ntllen1:~tical models of
t11e rate of tllllwr dcwlop1llellt.

80


Davies and Whitehead (17) studied the effect of alt,ering the "tar?'
:uld nicotine ratio of cigarettes on experimental carcinogenesis. No
significant diEerence in tumor yield was found between condensates
obtained from the smoke of cigarettes containing 16.6 mg. "tar:: a,nd
1.75) mg. nicotine and other cigarettes containing 10.0 mg. "tar:' and
131 mg. nicotine.
Several st.udies by Bock, et al. (7? 8, 9) have examined t.he tumor
jwinotinp activity of a number of fractions of cigarett.e smoke con-
drusate (C'S(J). ,I number of subfractions of the neutral fraction
of (`SC were tested for tumor promoting activity in mice pretreated
\\ it11 i.l"-dinIcth?-lbenz (a) anthracene. as a tumor initiator (8). The
tnost polar subfractions and the fraction containing bcnzo(a)pyrene
were the lrlost actiw tumor promoting fractions. In anot1~e.r st,udy
(!,I, the weak acid fraction of CSC was found to be a very weak
complete carcinogen whiclL probably acts primarily as a tumor pro-
moting agent. The promoting activity depended primarily on the
non\-olatilv constituents of this frac.tion. More recently. Hock. et. al.
(7') reviewed the tumor promoting effects of CSC and extracts of
tobacco leaves. .I colubinntion of two subfractions of the tobacco ex-
tracts. as well as five major fractions of CSC, were found to have.
tul~lor pronioting activity. The fraction containing the polynuclear
aromatic hydrocarbons was found to be a complete carcinogen. Two
subfractions wew found to be strongly synergistic in their tumor pro-
lwting activity v-hen applied simultaneously to mouse skin.

Lszar, 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 %ar" or its
fractions.

Van Duuren, et al. (97) have suggested that. "cocarcinogenesis'? be
different,iated 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 t,reatment. with one agent followed
by single or repeat.ed treatment with il second agent. I-sing these
definitions, the authors found several tumor promoting agents to
pOSSess cocaminogenic activity.

Roe. et al. (74) studied mechanisms of mouse skin cwc%logcwsis
using benzo(a)pyrene and a neutral fraction of (1% applied sillgl?:
or in \-arious combinations with each other. Skin tumor incidcncc rates
increased \\-it11 the dose of applietl material for both the nelltr:tl frar-
tion ilild lpllzo(a) pvrclne. JIisturrb of the ll~~utl?ll frX(*tiOll IVith ~)~~lZO-
(a)pyrelle did not &St intl(~pendently in the production of ill:lligllant
skill tulllors but synergistically, supyesting tll:lt SOlllCb Of th(' (`01111)0-
upntfj of tile llp~ltral fr:lctioii act :lS c~ocarviiiogeils IXtll?~ tll;tll as (`0111-
i'lctecRrcinogcns.

Bl


Schmiihl (7'8) found a direct relationship between the dosage and
durat.ion of subcutaneous injections of tobacco smoke condensates ano
the development of sarcomas in rats.

Maenza, et al. (56) studied t.he effects of a combination of nic.ke]
subsulfide ( Ni3S2) and benzo(a)pyrene on sarcoma induction in rats.
The interval bet,wcen administrat,ion of the carcinogen and the de-
velopment. of sarcomas was significantly short,er (P<O.OOl) 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 eit.her ingredient alone.
There appeared to be a synergistic interaction between nickel com-
pounds and t.he polycyclic aromatic hydrocarbons.

Healey, et al. (34) added further refinements to a technique for
measuring the nonspecific e&erase activity of mouse skin following
applications of various chemical compounds. With few exceptions.
changes in e&erase activity reflected the known tumor producing
activity of a number of polycyc.lic hydrocarbons and tobaccc
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 pg. per dose dis-
solved in sesame oil. Injections were given on alternate days foi
30 doses. The mea.n tumor induction time was accelerated in five 01
seven groups given the aqueous extract of CSC in their drinking
water. Animals given any benzo (a) pyre,ne eventually developed sar-
comas at the site of injection. Dose-response relationships were demon-
strated for the concent,ration of benzo( a) pyrene administered. It
appeared that aqueous extracts of CSC contained one or more corn-
ponents which functioned as cocarcinogens.

A~yl! Wydrocahm Hydrox~y74ue (,4aH)

Certain of the chemical conlpounds 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 probnbl!
excreted unchanged while others are nletabolized to various degree
by enzyme systems present in the 1ive.r and many other tissues. The
microsomal mixed-function oxidases are key enzyme systems for the
metabolisnl of a wide variety of chemical compounds including the


chemical carcinogens found in cigarette smoke. Aryl hydrocarbon
hydroxylase (AHH) is a part of the cytochrome P-%50 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 t.he appropriate chemical stimulus. The
hydroxylation of polycyclic hydrocarbons resu1t.s in the detoxific,ation
of some and the activation of others to reactive carcinogenic forms.
An understanding of the role of -1HH in the metabolism of chemical
carcinogens in man may help clarify some of the mechanisms involved
in tobacco carcinogenesis. Recently, several studies examined -1HH
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 t.o levels of carbon monoxide lvhich produced c.arboxy-
hemoglobin levels below 15 percent.
Welch, et al. (99) reported t.hat the administ.ration 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 st,imulation of AHH
activity in fetal liver than in the, placenta or mate,rnal liver.

Studies in Man

Levin, et al. (59) studied t.he induction of AHH actirit,y in human
skin. Human foreskin obtained from circumcised children was main-
tained in t,issue culture medium. Exposure to 10 ,/RI. 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. (101) reported the presence of AHH in human
lymphocytes. The AH11 activit.y of lymphocytes compared to rat liver
or hamster e,mbryo cells is relatively low. Treat,ment with pokeweed
mitogen alone inweased AHH activity about, twofold. Howeve,r: a
threefold to eightfold greater AHH activity was found in cells treat,e,d
with the mitogen and benz (a)anthracene than in resting cells.


In studies of tobacco carcinogenesis, cigarette smoke condensate
(CSC), subfractions of (`SC, and individual chemical compounds
found in CSC have been administered to a variet'y of animals using
several routes of administration. Tests on living animals are frequently
complicated and time consuming. (`cl1 and tissue. culture systems offer
an alternate tool for the study of wrcinogcnesis which, in some in-
stances, is relatively more rapid than animal testing. Specific enzyme
systems and other wllular functions can often be studied in greater
detail using these systems. (`ells obtained from a variety of tissues and
animals c*an 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 transformatjon or ccl1 death. Toxic effects on cell
cultures must be ditfercntinted from Inalignant transformation. Sev-
eral studies have recently examined the effect of cigarette sInoke
condensate or individual polycyclic hydrocarbons found in CSC on
various cell and tissue culture systems.

Benedict, et al. (4) studied polycyclic hpdrocarbon produced cyto-
toxicity, Inalignant transformation. and chromosome deformity in a
variet,y of cell lines derived from rats. hamsters, and human tumor
cells. The cytotoxic effect of benzo(a)p~~ene was found to be related
to the aryl hydrocarbon IIxdroxylase activity (AHH) of the given
cell culture. Benzo( a)pyrene was qvtotosic to fetal rat hepatocytes,
but this effect was probably related to the action of the hydroxylated
Inetabolite, 3-1Iydroxybenzo (a) pyrem', since the cytotoxicituv was
blocked when the ,1HH system was o\-crloaded with phenobarbital.
Cell strains not possessing -iHE-I actilitp showed no cytotosic effects
from brnzo ( a) pyrene alone : however. in the presence of fetal rat
hepatocaytes 1)ossessinp A!HH activity. enough benzo( a) pyrene metabo-
lites were secreted into the medium to indncc caytotosic etTects in the
normally resistrnt cell linrs. In hamster secondary cultures, at the
(~IIroIIIosoIne level cytotosicity was associated rrith chromatic1 breaks?
whereas malignant tI.aIIsfl)I,nlatioIl was n1ore closely related to
aneuploidy.

T)ianIond ( 1.9) studied the IlIetabolisIII of bcnzo (a) pyrcne and i'.ld-
dimeth;vlbcnz ( a) anthrawnc ( 1)JIlZ.i ) in Ino~Ise, hnnIst cr, rat. monkey,
;In(l hI~m:In (~11 c~ultnrrs. JletaholisIii of 1Iyd1~0c:I1~1~01is to `*alkali soln-
blr" am1 "water solut~lt~" tlcri\nti\-es WIS measured. The results SUM-
gested tliat tlw l~arcnt ~wIiil~oIInds were first nwtabolizetl to "alkali
extractable" d(~rivittiVcs ant1 t hrIi to ,`\\-ntcI.soliil)le" tlerivati\-es. A111 the
cvll cultures tcstetl \vl~ic~li were sensitive to tlw el.o\~-tli-iiiliihit(~r~
etfcrts of lmzo( 3) ]`yI'eIlc 01' I)JI1{.1 wcrc able to metabolize tlwsc

84


carcinogenic hydrocarbons to "water soluble" derivatives. The data
are consistent with the hypothesis that metabolism of the carcinogen
is required for growth-inhibitory or cytotoxic effects.
Several authors have examined malignant transformation in cell
cultures. Inui and Takayama (41) 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 et'Yfects of the
bStar7', including cell necrosis, swelling. vacuolization, and disintegra-
tion of cytoplasm \wre obserj-ed. The death of 40 to 70 percent of
the cells within 72 hours was followetl l)y tlie appearance of trans-
formed cells which ~IIW- at rapid rates. These transformed cells pro-
duced malignant tumors n-hen inoculated in the cheek pouch of ham-
sters. Control cell lines produced 110 changes when inoculated in a
similar fashion.
In a similar study by 1% Paolo, et al. ($21). transformation of pri-
mary hamster cell cultures was induwd by benzo (a) pyrrne. X-methyl-
cholanthrenc, or `i,l~-dimethvllH~llz (a) anthrawne. Transformed cell
lines were established a~~1 subsequently inoculated in hamsters pro-
ducing malignant tumors at various sites. Characteristic chron~osonial
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 t,he 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 5%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 lZNA viruses
from tumors induced by cell cultures transformed by chemical car-
einogens. Cell cultures \vere prepared from early passage hamster
embryo cells a~~1 treated for 7 days wit,11 S-methylcl~olanthrene or cer-
tain fractions of cigarette smoke condensate. Following treatment,
tt~~~rpl~ologically transformed cell lines were isolated and maintained,
Sttbsequent inoculation in newborn hamsters produced malignant
tuJJJors at t,he site of inoculation. New cell lines WPIT established from
Seme of the resulting tumors. So infectious viruses were isolated from
~11 lines prior to inoculation; however. C-type RN,\ I-irusrs were iso-

                                                        85


lated from tumors and from cell lines derived from tumors. The au-
t,hors 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, suggest.ing a mechanisII1
whereby various tobacco fractions might be rapidly screened foI
tumor-promoting activity.

Dietz and Flaxman (20) studied the toxicit? of aromatic hydro-
carbons on normal human epidermal cells in vitro. Picccs of adult
human abdominal skin were maintained in tissue culture nwtlium aIId
exl)osed to ~-rIIetlIylcl~olantlII~eI~e and benzo (:I) pyrencs at a concentr:I.
tion of 1 ~p./ml, for a periotl of I days. The cultures IveIne then kept foI
an additional 3 months followinp exposure. So n~:Ilign:IIit transfornla.
tioii occu~wd: liowever, giant cells and a more disordei~ly pattern of
growth \\rre observed in the treated cultures weeks earlier than simil:IIa
changes in control cultures.

There is evidence that some chemical carcino,rrcns incliItlin~ certaill
of the polycyclic hydrocarbons found in cigarette smoke con(lensatc
are active t,ecnItse of the, reaction of the carcinogen 01' a reactive iw
taholite with cellulaI~ IIIac~ronIolccrIlrs. 1)IIncan7 et al. (2,`) stridictl a
series of l~iltlio~lCtirr polyc~yclic hydrocarbons jvith respect to their
IrwtabolisIIi and tendency to bintl with cellular 11X-1 and RSA\ in
II~onol:i\-rI2 cultuws of l)rill1ilry IiIouse embryo cells. -111 tlw tcstrtl
II~tlf~oc~;II~~~oiIs were nIc~talwlizrcl to "w:ItCI' soluble" nir~t:Il~olitcs :It
:?I~I~~.oxirn:itcl~ equal rates. -1 **bintling imlex" IT-as c:Ilculntetl to de-
ttlrniine the bindinp of various hydrocarbons to crllIIlar I)S-1 ai111
RS.1. Tlw gi'oiip of hyd~warbons with a lrigh "bin~ling index" con-
sisted of lwtent caI~cino~eiIs, wlIilr aIiotlIer grorI1) v-itlI IiiiIch Ion-w
\~:ilues for the "binding index" were wit11 t)IIt one c~swption now
(3 i~ciiioviis .
(`nrl:Z+nw, et al. (1.2) stiitlicvl the in vi\-0 t)intliIig of benzo( :I)pyr~w~


Alexandrov and Vendrely (I) found that cigarette smoke conden-
sate, the hexane-extracted fraction of CSC, and benzo(a)pyrene all
inhibited RNA 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-
terns of a lvide variety of animals. These compounds were recently
ident,ified in cigarette smoke. Only recently has an association been
found between exposure to N-nitrosamines and malignant tumors in
humans (55). N-nitrosamines are formed chemically by a reaction of
NO and NOz or nitrites with secondary amines. The chemical pre-
cursors of the N-nitrossmines have been identified in cigarette smoke
condensate (CSC) by a number of investigators. These studies were
reviewed by Wynder and Hoff mann (10s). More recently, Rhoades and
Johnson (69) developed a method for the determination of N-nitrosa-
mines in tobacco smoke condensate using gas chromatography. TWO
N-nitrosamines were found in CSC : one was identified as N-dimet,hyl-
nitrosamine (DMNA) and t,he other \vas believed to be N-methyl-
ethylnitrosamine (MENA) (43: 7'0). 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 lo~v-
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 "lozo" nitro-
gen soils

Tobacco type


Summary of Recent Cancer Findings

In atldition to the sununnr~~ presented in the introduction of this
eIlat)ter, based on previous reports of the healtll consequences of smok.
ing, the following statements arc made to ernplrasize the recent devel-
0pnient.s in the field :

1. Krcrnt rll)idrllliologicnl and autopsy studies from several eoun-
tries c*olifilnl that c+arette smoking is the major cause of lung
  cancer.

2. (`ontinued cigarette smoking by lmticnts following successful
surgical removal of iL cancer of the oral cavity, pharynx, or
larynx without tumor recurrence for a l'rriod of 3 years is asso-
ciated with a significant increase (I?< 0.001) in the risk of devd-
opinp a SWOII~ primary cancer of the ul)per respiratory or diges-
tive tracts compared to similar patients who discontinue smoking
at the time of their surgery.

3. The intratracheal administration of certain polycyvlic hydrocar-
hens found in cigarette smoke condensate results in the forma-
tion of annplastic and squamous cell wnwrs of the lung and re-
spiratory tract in hamsters and rats. Many of these tumors are
histologically sinlilar to the lung cancers found most frequently
in cigarette smokers.

4. The .application of ciprette, smoke condensate or polycyclic hy-
drocarbons to various cell cultures often results in transformation
to ~11s wit.h a more rapid and disorderly pnwth pattern. Trans-
formed veil lines frequently produce henign or malignant tumors
whet1 transplanted to exlwrimental allinluls.

.i. S-llit~~os~~nlit~rs 1u1vc hern identified in cigarette smoke. These
~~npounds are known to he lwtent vanwr ca~~sinp chemicals for
a variety of animals. Tlwy al~pear to he formed in higher concen-
t rations from tobaccos raised under hiphnitrogcn soil conditions.

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95


CHAPTER 4

Pregnancy


Contents

                                          Page
Introduction____--______________________----------------   103

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--- _____ --___- _______ --------__----
  Carbon ?Ilonoside-__---_-__---. -~_----- _----
Polycyclic IIydrocarbons__-___---------------
Studies in Humans

105

106

110

114
115
116
117

118
119
119
119
119

120
121
121
122

Cigarette Smoking and Fetal and Infant ?tf ortali ty
  Introduction--- ______-____ -_____- _____________  ___--   123
SpontaneousAbortion ___-_ --__---__-___-- ____ --__--__   123
   Spontaneous Abortion Summary- - - - - - _ _ - - - - - - - - - _   124
Stillbirth-_-------_--___--_--- ____ -_----__---__~----   124
   Stillbirth S~lmmary-----_-----_______ ____ ---___--   125

495-02s %73-8

99


Page

Cigarette Smoking and Infant Mortality-Con.
  Late Fetal and Neonatal Deaths-__--- ____ --_-_- _______.
   EpidemiologicalStudies --_______ - ____ -_-_----_-__
     Comparisons of the Mortality Risks of Low-
       Birt,h-Weight Infants Born to Smokers and
       Nonsmokers ___---_-____ -_-- ____ ----_-_--_
      Recent Studies _-----_-____ ___- _____ __-_-__-_
     Analysis of Previously Reported Studies- _ _ _ _ _ _ _
     Factors Which Influence Perinatal Mortality
      Other Than Smoking--------__-_---__- ____
    Experimental Studies

126
126

126
128
130

131

      Studiesin Animals ____ -__-----__-_-_--_- ____
      Studiesin Humans ____ ----_--__-_-__- _______
   Significance of the Association_ _ - _ _ _ _ _ - _ - - - - _ - _ _ _ _
   La.te Fetal and Neonatal Death Summary _ - - _ _ _ _ _ _
Sex Ratio_----______-_______________________-----------
  Summary__-_--_--___-___--_-_-__-_-_----___-_______
Congenital Malformations__---_-____________________- ____
Congenital Ma(formation Summary- - _ - - _ _ _ _ _ _ _ - _ _ - _ - - - _
Lactation

132
133
133
134
135
135
136
137

138
138
138

  Nicotine_-_---- _________ --_-- _____ -_--_----
  Influence on the Lactation Process __-__ _ - - _ - - _ _
  Presence of Nicotine in the Milk- - - _ - - _ - _ - - _ - _
Evidence for an Effect Upon the Nursing Off-
  spring__-____-_-_-______________________--
  Nitrosamines--- -_________ -_-- ___-_______ -__
Studies in Humans

138
138
139

139
139

      Nicotine and/or Tobacco Smoke---------_-----
      Influence on t,he Lactation Process __--__ _- _ - -_ _
      Presence of Nicotine in the Milk----_--_-----_
     Evidence for a Clinical Effect Upon the Off-
      spring--_-----__~_--_---------------------
      Vitamin C ______-_______ -_-__- ________ ------
Lactation Summary _____ -__-_--_----__~__-_--________
Preeclampsia ____- - _ _ _ _ - _ _ _ _ _ _ - _ - _ _ _ _ -_ _ _ _ _ _ - __ _ _ __ _ _ _ _ _ _
Sz~mmary___-_-__-___-______--_--_-_____--___--_--_-

139
139
140

140
141
141
142
142

References---_-~_---_------~-------------~---------~~~--   142

100