experimental production of carcinoma with...

Experimental Production of Carcinoma with Cigarette Tar"

ERNESTL. WYNDER,| EVARTSA. GRAHAM,ANDADELEB. CRONINGER

(Department of Surgery, Washington Unirersity School of Medicine, St. Louis, ilo., and the Dirision of'Clinical

Inrestigation of the Sloan-Kettering Institute of the Memorial Centerfor Cancer and AlliedDiseases, New York, N.Y.)

The increasing frequency of primary cancer ofthe lung in many parts of the world has arousedgreat interest in this condition and has stimulateda search for an explanation. In 1950, Wynder andGraham (40), on the basis of a clinical and statistical investigation, presented evidence of a realassociation between lung cancer and smoking, especially of cigarettes. These data have been wellsubstantiated by a large-scale British study byDoll and Hill (9,10). Both studies showed that therisk of developing cancer of the lung increases indirect proportion to the amount of smoking. Tenother recent studies reached similar conclusions(3, 11, 15, 20, 21, 24, 26, 33, 34, 42). In 1952, TheCouncil of International Organizations of MedicalSciences convened a symposium on the endemiolo-gy of lung cancer and agreed that the present evidence points to a relationship between lung cancerand cigarette smoking (12).

Tobacco is also thought to play some role in theproduction of cancer of the larynx, oral cavity, andesophagus. Although the studies of those relationships are not so complete as the studies on lungcancer, the collected data are suggestive (33, 41).

The increasing incidence of bronchiogenic carcinoma and the available evidence relating smoking to it and possibly to cancer of other sites led usto undertake the experimental work reported here.This investigation is directed toward determiningin laboratory animals whether there are carcinogenic factors in cigarette smoke.

PREVIOUSINVESTIGATIONS

Many attempts have been made with tobaccoproducts to induce cancers in a variety of experimental animals. The first was reported by Broschin 1900 (4). He painted guinea pigs with tobacco"juice" for an unknown period of time and de-

* This investigation was supported by a research grant fromthe National Cancer Institute of the National Institutes ofHealth, Public Health Service.

t Present Address: Memorial Center for Cancer and AlliedDiseases, 444 East Sixty-eighth Street, New York 21, N.Y.

Received for publication June 8, 1953.

scribed epithelial proliferation. Subsequently,many different approaches to the problem wereundertaken with various types of tobacco, different methods of tar preparation, and different species of animals. Many of those studies were carriedon for too brief a period of time or with too fewanimals to be regarded as significant. Hoffmannand his associates (17),forinstance,painted animalsfor only 14 days, at which time they noted hairloss. Wacker and Schmincke (37) observed proliferation of epithelium in rabbits' ears 21 days

after a subcutaneous injection of pipe tar.The first recorded experiment with mice and

with tobacco tars as the suspected carcinogen wasthe one just cited by Hoffmann and co-workers. Themore detailed of the subsequent studies are listedin Table 1. This table attempts to summarize themethods used in the various studies and the results obtained. In many instances the method ofstudy was not described in sufficient detail to giveall the information considered essential.

From this survey of the literature it is foundthat, before our study, all the previous attempts toproduce experimental cancer in mice with tobaccoproducts were successful in the production of onlyseven epidermoid cancers of the skin.

Several investigators attempted to induce pulmonary tumors in mice with tobacco smoke.Lorenz and co-workers (22) obtained negative results in this manner. Campbell (5), and especiallyEssenberg (13), however, claim to have found asignificantly higher percentage of pulmonaryadenomas in the experimental than in the controlgroup. It is doubtful that such a finding is important. At any rate, so far these methods have notinduced true bronchiogenic carcinomas.

The majority of the investigators working withtobacco tars used rabbits as the experimental animals (14, 23, 27-32, 36). In view of the fact thatthe present work deals with mice we shall onlybriefly list some of the studies with rabbits. Rofforeported the production of carcinomas in rabbitears after painting the ears with a distillate oftobacco (28, 30, 32). Sugiura (36), in attempting

855

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TABLE1. SUMMARYOFMETHODOLOGYOFSOMESTUDIESONTOBACCOTARASAPOSSIBLECARCINOGEN

Author Type of Tobacco Method of Tar Collection Temperature Portion of Tar UsedNumberof Mice

FORMICE

Solvent

Helwig (16), 1928 Not given Tarry extract from bowls ofburned briar pipes

Unknown Denicotinized tar Ether with olÃ¬veoil

Not given Distillate produced at temperatures of 400Â°-500Â°C.and tarry product extracted with ether orchloroform

400Â°-500Â°C. Extracts 50 Chloroform orether

Bogen and Loomis(2), 19SÂ«

Kentucky Bur-ley tobacco

Destructive distillation andextraction with HiSOÂ«,alcohol, and ether

Not given 50 per cent whole tar solution inglycerine

1ÃŽ Glycerine

Cooper (7), 193Â« Not given Intermittently puffed andexhaled pipe tobacco; theresulting tar condensatewas then distilled

370Â°-590Â°C.; I Tobacco tar of 400Â°-500Â°C.â€”480Â°-700Â°C. wooden pipes

A. 10 per cent in alcohol solution

B. 30 per cent solution in glycerol

C. Neutral fraction freed frombases or phenols using either1. 50 per cent solution in

benzene2. 10 per cent solution in

glycerolD. Original tar dissolved in

glycerol and applied hot at55Â°C

II Tobacco tar of 700Â°-800Â°C.-

Alcohol, benzene, andglycerol

Schuerch andWin-terstein(35),1935Taki

(19),1937Campbell

(6),1939Sugiura

(38),1940Flory

(14),1941ShubiM

1950Wynder,

Graham,andCroninger,1953CigareUnknownNot

givenMixture

of American and Turkish leaf tobac

cosEntire

leaf andstem ofcuredKentucky

tobaccoCoarsely

groundtobacco,

Â«.CigarettesCigarettesSmoke

of cigars collectedincottonwhich was ex

tracted withchloroform.Thesolution was denico

tinized with 2 percentHCIExtract

from tobacco pipesdissolved in ether and filtered, thendenicotinizedwith

HCITar

condensate (partial)ofartificiallysmoked ciga

rettesDistillationÂ«t10OÂ°-SOOÂ°C.

and redistilled at500Â°-900Â°C.Destructive

distillation at700Â°C. for 6-8hoursyielding

3fractionsContinuous

suctionappliedtoclay pipes at520Â°-730Â°

C.Tar

extracted with acetonefrom smokedcigarettestubsWhole

tarry condensate ofintermittently puffed cigarettesNot

givenNot

givenNot

giveniooÂ°-oooÂ°

c.120Â°-700Â°

C.520Â°-7SOÂ°

C.Not

givenBurning

tip â€”S16Â°-599Â°C.:smoke

â€”26.7Â°-60.0Â°C.;combustionâ€”

968Â°C.(max.).68Â«Â°

C. (av.)wooden

pipes; 25 per cent solution in alcohol

III Tobacco tar of 400Â°-500Â°C.â€”clay pipes; 50 per centsolutioninalcoholI

25 per cent tar in alcohol solutionII

Tar with nicotine appliedfor3months then used denicotin

izedtarIIIDenicotinized tarextractedwith

chloroformIVDenicotinizedtarextractheat-ed

to 130Â°C. prior to paintingV DenicotinizedtarVI

Tar extract distilled at 100Â°C.VIITar extract distilled at160Â°-170Â°

C.VIIITar extract distilled at 180Â°C.IXTar extract distilled at 300Â°C.Denicotinized

tarTar

condensateI

A. DistillateÂ» applied for90daysB.

After 90daysI.Watery 8uid of first dis

tillateÃŒ.Oily liquid of first distil

late3.Second distillate.500Â°-900Â°

C.C.Cholesterol dietduringpaintingII

Oily liquid of first andseconddistillatesIII

Control animals using coaltarI

350Â°-700Â°C.destructive distillateIIDenicotinized 350Â°-700Â°C. de

structivedistillateIIIDenicotinized 120Â°-350Â°C.destructivedistillateIVMixture of watery parts of distillatesjfVControl group; coal tar and benzene in equalpartsDenicotinized

whole pipetarI

Tar extractII Tar extract applied for alimitedperiod

followed byapplicationsofcroton oil for duration of ex

perimentI

50 per cent whole tar in acetone1.Tar2.

Tar/crotonII

Acetonecontrols1.Acetone4.

Acetone/crotonIII0.3 per cent methylcholanthreneI)

It100100100100100100100100100104231864457074030C5730

DBA40304Â«7Â«SOU6030SO8131SO14UAlcoholChloroformEtherNoneNoneNoneNoneAcetoneU.S.P.

acetone;white mineral oil(usedwithcrotonoil)

* Applied by painting the skin t P. Shubik, personal communication.



TABLE Iâ€”Continued

Strain and InitialAge ofApplicationNot

givenNot

givenNot

givenNot

givenFrequency

and DosageofApplication*3

times a week; dosage notgivenNot

given2

times a week; dosage notgiven8

day intervals; dosage notDuration

ofApplicationslyr.8

mos.Over

1yr.Over

16 mos.ResultsUlcÃ©rationUlcÃ©rationNegativeOneepithelioma from 10 per cent tar (pro-AuthorHelwig

(16),1988Bogen

and Loomia(2),19SÃ•Cooper

(7), 1932

in 16 months

NotgivenNot

givenNot

given2

times weekly, dosage notgiven

1 times aweckÃ¯

times aweek2

or S times aweek2

times a weekNot given3 times aweekNot

givenNotgiven2



mos.

348daysÂ«65

days635

days270

daysÂ«mos.190days6

mos.480daysNot

givenÂ«0

mos.NegativeNegativeAtrophy

of skin with slight hyperkeratosis Schuereh and \Vin-and perivascular infiltration terstein (35)

Same as I1935Same

asIAtrophy

of skin and chronicinflammationHair

lossNegativeLoss ofhairLoss

of haÃ¯rand atrophy of skinAtrophy of skin nnd hairloss3

papillomu (1st observed at 85 days); Taki (19), 19372 squamous cellepitheliomasOne

typical epithelioma at 16-20 mos. Campbell (6),1939

Bagg albino mice, 2-S 4 times a week; O OÃgm/ 90-300 daysmonths old dose

A. NegativeB. After 90 days

1. No results2. No results3. One squamous carcinoma

C. No accelerating influence

Sugiura (36), 1940

C57 blacks and DBA mice,age of initial applicationnot given

Bagg albino

cholesterol dietâ€”90-180 daysC57 black 80-475 days; No tumors

DBA died too early forresults

75-S09 days 75 per cent developed progressively growingtumors with 31 squamous cell carcinomas

White mice (not an inbredstrain); age at initial application notgivenWhite

mice (not inbred); ageat initial application not

givenC

strain; 3monthsCAFi,

8-12 weeks5

times a week 18 mos.015 gm/ dose

.018.015"

.805

times a week; 0.013 gm/ 18 mos.paintingTwice

weekly 9mos.Tar:

3 times weekly; 0.040 24 mos. maximumgin/painting

Croton oil: 3 times weeklytar /acetone plus 1 painting/week of 5 per centcroton oil/mineral oilPapillomas

CÂ»4 in <ÃŒ115 mos.8 Â¡n7J-11mos.1

in 14 mos.

02

papillomas in 14 mos.1 cancer in 17mos.Negativencers

Flory (14), 1941

1 in 8.5 mos.

0

0

13 in 126-256 daysShubik.t

1950Pmpil-

lomas Cancers(no.) (no.)

I \\holr tar in acetone Wynder, Graham,(1:1 ratio) and Croninger.

1. Tar 48 36 19532. Tar/croton 13 3

II Acetone controls1. Acetone 0 02. Acetone /croton 0 0

III 0 3 per cent methyl- 25 Ucholanthrene



858 Cancer Research

*, ,

to repeat Roffo's work, obtained negative results,

whereas Flory (14) in a similar large-scale experiment obtained papillomas and several lesionswhich he designated as "carcinomatoids" in rabbit

ears after application of a tobacco distillate (24,39).Roffo also used rats as experimental animals.

He implanted pills of tobacco tar into rat bladdersand noted cancer-like growths after 8 months (29).McNally (25) applied a water-soluble product ofcigarette smoke to the mucous membrane of theoral cavity and to the tongue and skin of rats. Allanimals were lost within 4 months due to the toxic-ity of the material, and no significant changes werenoted.

MATERIALS AND METHODSThe method of study employed in this investigation is pre

sented in some detail because the varying results of differentworkers may be a reflection of different methods used. In this

SMOKING APPARATUS

A - SMOKING ARMB - CONDENSING FLASKC - TARD -DRY ICE-95%ETHANOLE -COTTON FILTERF - TIMERG - VACUUM

'

:

CHART1.â€”Schematic drawing of smoking apparatus

study the guiding principle in collecting the tobacco tars wasto simulate human smoking habits as closely and as practicallyas possible. It was felt that any change in temperature or theremoval of any fraction from the smoke might alter the effectof the resulting tars. Therefore, we used a smoke condensateobtained from smoking cigarettes intermittently, and, unlikemost other investigators, we did not remove the nicotine fromthe tars in the initial phases of the experiment.

Tobaccoused.â€”Apopular brand of domestic cigarettes wasused.

Smoking apparatus.â€”Thetobacco tars were collected in thefollowing manner. Sixty cigarettes were placed in a battery ofglass smoking arms (A) containing separate holders for tencigarettes each and lighted with a multiple jet gas burner(Chart 1). The smoking arms were rotated and changed as thecigarettes burned down, keeping the apparatus filled with 6Qcigarettes at all times after the original loading. The arms wereplaced in position on glass candelabra-like receivers on each ofthe two smoking units. These receivers were jointed and connected to a series of three 2,000 ml.-glass flasks (B) and connecting tubes on each unit. A vacuum was created by a smallelectrically driven air pump (G) (1.8 cu. ft. displacement offree air per minute at a vacuum of 27 in. of mercury). At 18-second intervals an open-shut valve, geared to a three r.p.m. ihorsepower motor (F), was automatically opened for a 2-second period, allowing the vacuum from the air pump to beapplied to the condensing flasks and ultimately to the burning

JUÃ‚

cigarettes. The condensing flasks were enclosed in insulatedstainless steel tanks filled with 95 per cent ethanol and dryice (D) to lower the temperature to approximately â€”60Â°C.The smoke that failed to condense inside the cooled tanksthen passed on to a third flask outside each unit and insulated with an asbestos jacket. The two units met at acommon source of vacuum, at which point a cotton filter(E) was placed in the line to protect the motors from tarcorrosion. The smoking time per cigarette averaged from

<L_7j_to 8 minutes or from 28 to 32j>uffs per cigarette at arate of three 2-second puffs per minute. The cigarettes wereburned down to approximately a 2-2.5-cm. length.

As the smoke condensed, a dark brown viscous liquidformed and adhered to the glass apparatus with diminishingdensity to the third set of flasks. After 50 cartons weresmoked, all of the tar contained in the glassware above thecotton filter was washed clean with approximately 300 cc. ofacetone by dismantling and corking the separate glasswarepieces. This tar-acetone mixture was then stored at â€”7Â°C. inglass-stoppered bottles until made up into solutions.

Preparation of tarsfor application.â€”Solutionswere made upfresh monthly from 50 cartons of cigarettes. The choice of acetone as a solvent for these tars was based primarily on the factthat acetone was known to be noncarcinogenic and also because it was found to be the best dissolving medium for thissubstance.

The tar-acetone mixture was poured into 150-ml. porcelainevaporating dishes, which had been previously weighed; thesewere placed in an exhaust hood containing a rotating fan for 6hours and were then left overnight. The average weight of theresidual tar obtained in this manner was 9.7 gm/200 cigarettes.An equivalent volume of acetone was added to each gm. of residual tar and this mixture (1 cc. acetone to 1 gm. tar) wasplaced in 125-ml. Pyrex glass-stoppered bottles and stored atâ€”7Â°C. until used. When used, small amounts of these solu

tions were repoured into other bottles to reduce the evaporation of the acetone as much as possible.

Experimental conditions.â€”Testsfor the amount of vacuumapplied on the smoking cigarette were made at three stages ofburning, i.e., at the start of burning, at midpoint, and at buttstage. In the first stage the magnitude of the vacuum at thestart of the puff was zero; it increased to a maximum value ofâ€”9 mm. of water and then fell to an average of â€”8mm. Thisvalue was found to reach a maximum of â€”7mm. and an average of â€”6mm. in the second stage, and a maximum of â€”5mm.with an average of â€”4mm. in the butt stage.

Temperatures of the burning cigarette:1 All the temperaturestudies were made with iron constantan thermocouples withwelded hot junctions. No. 26 gauge duplex glass-insulated wirewith a combined resistance of 1.4 ohms/ft at 21.1Â°C. and aNo. 28 gauge duplex enamel insulated wire with a combined resistance of 2.2 ohms/ft at 21.1Â°C. were used. The tempera

tures were registered on a suspended galvanometer potentiometer-type null Imliintv, calibrated from 0 to 2,000Â°F. Theburning tip temperatures depended on the penetration of thethermocouple into the combustion zone. The results of 60 temperature readings ranged from 316 to 599Â°C. The average ofthe 60 temperature readings was 438Â°C. with a room temperature of 26.7Â°C.

Combustion temperatures of the cigarette: Combustiontemperatures were obtained by inserting 28 gauge thermocouples up through the mouth end approximately three-quarters of the length of the cigarette and by then smoking thecigarette to the point where the hot junction of the thermocouple would pass through the combustion zone. Tempera-

1Temperature studies were carried out by the Filio Sales& Engineering Co., St. Louis, Mo.

W



>WYNDER et al.â€”Experimental Carcinoma with Cigarette Tar 859

Â»J

e***

tures up to 966Â°C. were obtained by this method. The averagereading, however, was 682Â°C.

Temperatures of the smoke as distinguished from that of theburning cigarette: Twenty-eight gauge iron constantan thermocouples were inserted in the receiving end of the cigarettearm, and the cigarettes were smoked down to the short buttstage. No appreciable rise in the temperature of the smoke wasnoticed until the cigarettes were consumed to butt length. Thetemperatures of the smoke ranged from 26.7 to 60Â°C.

Animals used.â€”Onehundred fifty-six CAFi mice (genetically homogeneous FI hybrids from the cross A/Cloudman inbredstrain males by BALB/C inbred strain females obtained fromthe Roscoe B. Jackson Memorial Laboratory at Bar Harbor,Maine), with an equal distribution of males and females, werestarted at 8-12 weeks of age. VK4AUt_Â»T'

All the mice were housed in wjre-mesh cages containing 12animals each, and were fed a diet of Rockland mouse pellets,a rolled oat supplement, and water ad libitum. Toward the endof the experiment, animals developing lesions were segregatedand placed in individual glass jars.

Subgroups: The CAFi mice were originally divided intotwo groups, i.e., one group of 112 mice painted with tobaccotars in acetone and a second group of 44 mice receiving acetonealone. Preliminary studies on 60 Swiss mice showed that theanimals were sensitive to tars containing nicotine, particularlyin concentrated solutions. Being cognizant of the sensitivity ofmice to tobacco tars, we began the CAFi group cautiously usingdilute solutions, the concentrations of which were gradually in-

greased. The tarred group was started with tri-weekly gaint-of a residual tar/acetone solution containing 1 part of tar

3 of acetone. The dosages were gradually increased to'.1 and finally to 1:1 ratio in 2 months; the latter dosageaveraged 40_ing of tar/acetone solution per painting. -^ "Â¡

Prior to each painting the entire dorsal area of each mousewas shaved of excess hair with an electric clipper, and oneSaturated brushful of the tar/acetone mixture was applied tothe shaved area with a No. 5 camel's hair brush ; several strokes

were used, beginning at the nape of the neck and working backward to the tail region medially and laterally. The control animals were shaved and painted with acetone in the samemanner.* ~") Seven months after onset of painting, 81 of the 102 surviv-

yi ing tarred mice and 14 of the 42 surviving controls on acetone.^^7^'pwere segregated and given an additional painting per week of

1 per cent croton resin in acetone (Berenblum [1]) in order todetermine the cocarcinogenic properties of croton resin in relation to tobacco tars. Each subgroup contained an equal numberof males and females. The croton resin solution was found tobe extremely irritating to the skin of mice and caused widespread ulcÃ©ration.Dosages were reduced without relief of thiscondition. Two months later these animals were changed to a5 per cent solution of croton oil (Croton Oil N. F. VII, Magnus,Mabee, and Reynard, Inc., New York City) in mineral oil(pure Superla No. 34, Standard Oil Co. of Ind.), which, although much less irritating to the skin, seemed to induce severediarrhea.

V' j In the 15th moni Â¡iof painting a few of the tar/acetone andtar/croton/acetone animals were placed on a denicotinized 1:1tar/acetone solution, prepared by extracting the nicotine with1 per cent hydrochloric acid. This treatment was used onlywhen the animal had an open ulcerating lesion. When the lesionscabbed over, the whole tar/acetone solution was reapplied.Whereas most of the animals with lesions were kept on thewhole tar/acetone solution throughout the experiment, it wasoccasionally necessary to resort to this less toxic treatmentwith denicotinized solutions in order to preserve the animal.Abo, as the animals became weakened by old age or disease, thetar paintings were decreased by painting merely the lesion it-

self. When a lesion had become a grossly positive carcinoma,the tar paintings were stopped completely. The animal wasthen allowed to live free of paintings and was sacrificed onlywhen death appeared imminent.

RESULTSGeneral observations.â€”The whole tar/acetone

solution promoted a general thickening of the skin,but rarely produced lasting areas of complete hairloss. The tar/croton group showed more hair loss,thickening, reddening, and proliferation of epithelium than the regular tar group. The acetone/croton group showed hair loss and thickening andscabbing of the skin, but not to so great a degreeas those animals on tar/croton. The skin of theacetone controls remained soft, pliable, and normal throughout the experiment.

A difference in the rate of hair regrowth wasnoted between the experimental and controlgroups. Tarred animals regrew hair rapidly between paintings and required regular shaving. Thetar/croton and acetone/croton animals remaineddenuded for longer periods, showing spotty andsometimes complete hair loss. The acetone controls always kept a short stubble of hair; yet theyregrew hair at a much slower rate than the animalson tar or on croton oil. Histologie sections on aseparate group of CAFi mice at 10 days showedhyperplasia, and hyperkeratosis in 75 per cent anddisappearance of sebaceous glands in 100 per cent ofthe animals painted with the tar/acetone solution.2 None of these changes were observed in animals painted with acetone alone.

Little difficulty was encountered because ofdrug intoxication, since, as described above, theanimals' tolerance to nicotine was increased. In

one instance, however, unintentional evaporationof a solution apparently led to an increased concentration of nicotine and brought about a numberof deaths in the tar/croton oil group. At the onsetof the painting procedure, the mice appeared to bein a partially drugged state and exhibited occasional fine tremors following paintings. These tremorsand stuporous states were rarely observed afterabout 2 months of application, but frequently reappeared in old age until the dosages weredecreased.

Papilloma formation.â€”Of 81 tarred CAFi mice,59 per centâ€”26 females and 22 malesâ€”developedpapillomas. The earliest appearance was observedduring the 33d week, with a mean time of 56 weeks.Of the papillomas, 8.6 per cent regressed. Thelesions were uniformly distributed over the paintedarea. Twenty-three animals had more than onepapilloma (Table 2; Chart 2).

A typical papilloma appeared as a firm, low,2Dr. Victoria Suntzeff, personal observation.



860 Cancer Research

TABLE 2 week; the mean time of appearance was 71 weeks.APPEARANCEOFLESIONSIN CAFi MICE AFTERPAINT- Of 62 mice still alive in the tar group at 12

INGWITHCONDENSEDCIGARETTETAB, months, the time at which the first gross cancerCROTONOn, ANDACETONE* was observed, 58 per cent developed cancer.

pApiiLOMAstCARciuoHAst Twenty-five of the carcinomas occurred in fe-NO. MOB.or (GROSS) (GROSS) males and 11 in male mice. The females,althoughGaorp*

ALIVE PAINTING No. of mice No. of mice .1 â€¢ â€¢i . i i,Tsi i their average weight was about 4 grams less than

T/CÂ§ 31 1 that of the males, had a longer survival time in theA 801A/CÂ§TT/CAA/CTT/CAA/CTT/CAA/CTT/CAA/CTT/CAA/CTT/CAA/CT/CAA/CTT/CA/CT

T/CAA/C14(Â¡9812814(19312814Â«218Â«812508ÃŒS11478221141719926418783Ifiiv

30

09S1888810101010li121212141414141616161618181818JA202020202222aaXX222424242431821532273910706050iâ€¢

40â€¢1I

302

*20102

Â°-^^^Popi

Homo/^^^^~1

.^^1^rJ

S^S

1^rVCorcinomo^S

If

Iâ€”*lililÃ04

8 12 16 2O24Number

of Months ofApplicationâ€”

â€¢â€”CAF. mtce (81) Cigarette tot/Acetone20-.-. CAFJ mice (50)Acetoni1CHAHT

2.â€”Month of first gross appearance ofpapillomasandcarcinomas in CAFi mice painted with a cigarettetar/4310j,

acetone solution, and a group of CAFi mice paintedwithjacetone alone. All of these lesions were subsequentlyprovedhistologically.

In two instances, where a cancer was foundinatumor which appeared grossly to be a papilloma, themonth481148ISofdeath was taken as the time of first appearance of thecancer.312tar

group than the males. Of 41 male and 40femalemice36

malestartedon experiment, at 12 months 34fe-and

only 28 male mice were alive. At183-'months the respective figures were 26 and 19.Inthe

control group twice as many males lived for1848

ISmonths

asfemales.1Ofthe cancers, the majority â€”27 (75 percent)â€”had previous papillomatous growths; however,anumi>er

arose from localized ulceratine sites. TwoÂ«T = tar/acetone, T/C = lar/croton oil/acetone, A = acetone, A/C =

acetone/croton oil.t This column represents cumulative totals on the first gross appearance

of papillomas.t This column represents cumulative totals on the first gross appearance

of carcinomas, all of which later were verified histologically. In two cases cancer was shown microscopically to be present in tumors that appeared grosslyto be papillomas. In these two cases the month of histologie verificationwas taken as the date of first appearance.

5 Croton oil was started in the seventh month.

broad-based, clearly outlined wart, varying in sizefrom 0.1 to 0.4 cm. in diameter. In most cases thesewarts were well-defined nodules, occasionally budding before sloughing and showing definitecraters.

Cancer formation.â€”Of 81 tarred mice, 44.4 percent developed epidermoid cancer of the skin. Thefirst was noted grossly during the forty-second

cancers, which had been classified as papillomas atautopsy, were discovered upon histological examination. The cancers were always found only on thepainted area, and in advanced cases often spreadlaterally beyond the area of painting. Six mice hadtwo separate skin cancers. Prior to the development of the typical cancer, a localized thickeningand ridging of the epidermis surrounding thepapilloma in the shape of a rolled, puckering rimwas noted. During this process of initial invasion,the papilloma proper sloughed off and left a smallulcerating crater. These craters widened and continued to ulcerate. They frequently becamenecrotic and grossly infected. The rims and basesalso became more fully cratered and hardened. In



WYNDERet al.â€”Experimental Carcinoma with Cigarette Tar 861

the advanced stages the cancers appeared as grossly massive ulcers with definite rims (Figs. 1, 3, 5,7, 8,10,11,13, 14, 16, 18, 20). Histological examination of these lesions showed the usual characteristics of highly malignant epidennoid skin cancerâ€”downward invasion of epithelial cells with extension through the basement membrane, lack ofdifferentiation, and many typical mitotic figures(Figs. 2, 4, 9, 12, 15, 17).

Tar/croton oil.â€”The tar/croton oil group was

begun in the 7th month after the onset of tarapplication. Of 31 mice segregated into this group,42 per cent developed papillomas and 9.7 per centcarcinomas. The lesions did not appear earlierthan in the tarred group. The first papilloma wasnoted in the 8th month, and the first cancer inthe 16th month.

It is not possible to evaluate these data because77 per cent of the animals died between the 12thand 14th months, subsequent to marked weightloss. On one occasion, one group of twelve animalswas thought to have received an overdose of tar,resulting from excessive acetone evaporation.This group of animals died after having convulsions.

Acetone group.â€”Thirty animals comprised thisgroup. Nineâ€”30 per centâ€”were still living at the

end of 24 months, in contrast to 0 per cent in thetarred group. No lesions were noted in this groupof mice. The epidermis remained soft and pliablethroughout the experiment. The average weight ofanimals in this group through most of the studywas about 1 gm. more than in the tarred mice,although, at the onset of the experiment, the average weight of the mice in the tarred group was 2gm. greater than in the acetone control group.

Acetone /croton oil group.â€”A group of fourteen

mice originally treated only with acetone receivedan additional painting of croton oil per week starting at the 7th month. In this group three miceâ€”21per centâ€”were still living at 24 months. The epi

dermis became roughened and thickened followingrepeated applications of croton oil. No papillomasor carcinomas appeared in this group.

Methylcholanthrene group.â€”Twenty-five CAFj

mice were painted with a solution of 0.3 per centmethylcholanthrene in acetone 8 times a week.They all developed carcinomas within 4$ months.The first papilloma was noted during the 6thweek, and their average time of appearance was 7weeks. The respective figures for carcinoma formation were 12 and 16 weeks.

Transplantation.â€”Two of the cancers in the

tarred group have been successfully transplanted.One cancer was transplanted for 4 generations byDr. V. Suntzeff before it became grossly infected.

The method of transplantation was as follows: Thetumor was excised from the living host, and smallhealthy pieces were cut and placed in either apenicillin-streptomycin solution or a saline solution before being transplanted subcutaneouslywith a sterile trocar to the lateral chest region. Theother cancer was transplanted in our laboratorydirectly from the living animal to another CAFimouse with a sterile trocar without first placingthe tumor into a solution. The host and the mouseto receive the tumor were previously injected subcutaneously with 100 units of dihydrocillin;3 thisdose was repeated for 3 days after transplantation.For every third generation since the sixth generation, the tumor has been excised, minced, andsoaked in either dihydrocillin or syncrobin4 for 15-45 minutes prior to transplantation as a protectivemeasure against infection; subcutaneous injectionsof antibiotic have been omitted. Preliminary dataon the rate of growth of this tumor show thattransplants without the intermediary soaking inantibiotic grow to approximately 0.5-1 cm. in 15-21 days before ulcerating and being retransplant-ed, whereas transplants previously soaked in antibiotic grow more slowly (f-1 cm. hi size in 29-43

days). This tumor is still an active epidermoid carcinoma in the 13th generation with 100 per centtakes (Figs. 6 and 19).

DISCUSSIONEvaluation of method of present study.â€”The

basic principle in tar collection was to simulatehuman smoking habits as closely and as practicallyas possible. A popular brand of cigarettes wasused. The temperature factors were found to besimilar to those encountered in human smoking.The frequency of smoking (3 times a minute)is greater than that encountered in the averagehuman smoking. In view of the fact that this increase involved no significant temperaturechanges, the more frequent puffing rate waschosen for the practical purpose of obtaining the required amount of tar more rapidly and economically.

Since we assumed that the carcinogenic effect oftobacco tars might be owing to a summation effectof subthreshold carcinogens or that such a factormight be in the alkaloid fraction of the tars, thewhole condensate was used. Acetone, an established noncarcinogen, was found to be the bestsolvent for the tobacco tar and, therefore, the tarwas administered as a tar/acetone solution.

3Combination of streptomycin and penicillin by Upjohn& Co.

*Combination of streptomycin and penicillin by Schenley,Inc.



862 Cancer Research

We postulated that tobacco tars have only aweak carcinogenic activity and thus would exhibita long latent period. Therefore, we started with8-12-week old mice. Our results clearly indicatethat if all the mice had died during the first year ofpainting or if we had stopped the experiment atthat time, the data would have been much lesssignificant. For the same reason, we used a higherdosage schedule than had been used by previousinvestigators. Subsequent studies may show thatan additional increase in dosage may further accelerate tumor formation.

Preliminary studies indicated some difficultieswith the tar solution that contained nicotine. Thisproblem could be overcome, however, with the de-sensitization procedure employed.

The basic factors of the method, therefore, wereto collect the tar in a manner simulating humansmoking habits, to use the whole tar obtained, andto apply it in relatively large quantitites and for along time. This approach may be in part responsible for the greater production of cancer in thisinvestigation compared to that of previous studies.The possibility remains that the CAFi strain ofmice may be particularly susceptible to the carcinogenic effect of cigarette tars.

Effect of croton oil.â€”Subgroups were paintedwith croton oil to determine whether tumor formation could be speeded up, as had been indicatedby Berenblum (1). The CAFi mice received crotonoil in mineral oil once a week starting with the7th month after the painting with tobacco tarswas begun. Because of a high mortality duringthe 12th and 14th months in this group, thecroton-oil effect on tumor formation cannot beproperly evaluated from this experiment, althoughwithin the period of observation no acceleration ofcancer formation was noted.

Tobacco versus methylcholanthrene.â€”Althoughthis study establishes condensed cigarette smokeas a carcinogen for mouse epidermis, its activity isless potent than that of methylcholanthrene.Methylcholanthrene, of course, is a very potentcarcinogen, rather than a crude substance in whichsuspected carcinogens may be diluted.

Although crude tobacco tar is less potent as acarcinogen than methylcholanthrene for mouseepidermis on the basis of the number of cancersinduced and the length of the latent period, thefact remains that over 40 per cent of the animalspainted with tar from cigarette smoke developedcarcinomas, whereas no lesions were observed inthe control animals.

Animal versus human data.â€”Animal data donot necessarily confirm or deny human data, although historically much of our present under

standing of carcinogenesis is based on corollarystudies between clinical and laboratory research.The studies on coal tars from Pott to Yamagiwaand Ichikawa and to Cook, Hieger, and Kennawayserve as a classical example. In coal tar investigations experimental data confirmed the clinicaldata, and thus added import was given to both.

A similar relationship now exists in the tobaccotar field. Here, too, a clinical association betweensmoking and cancer seems established. It has beenshown that a condensate of this smoke may induceepidermoid cancer of the skin in the experimentalanimal. The suspected human carcinogen has thusbeen proved to be a carcinogen for a laboratoryanimal.

The most far-reaching effect of this observation,perhaps, does not lie in its immediate relationshipto human carcinogenesis, but, rather, in that theproven susceptibility of animals furnishes us witha working tool which may enable us to identify andisolate the carcinogenic agent(s) within the tars.

It is an interesting and perhaps significant factthat with the CAFi mice it was necessary to havean exposure to the tar for approximately one-halfof the life span of the animals (average 71 weeks).This agrees roughly with the previous finding ofWynder and Graham (40) that the maximum incidence of bronchiogenic carcinoma in the humanoccurs after 30-35 years of smokingâ€”about one-half of the human life span.

Tobacco as a carcinogen.â€”Itappears surprisingthat, in view of the detailed work carried on withcoal tars and its derivatives, relatively little workhas been done with tobacco tars. Because recentclinical data have placed increasing emphasis ontobacco as a human carcinogen, it is our hope thatmore investigators may approach the tobacco-cancer problem.

Although several hundred chemical and physical agents have been found to induce cancer in animals, each one of these agents seems to have aspecific carcinogenic action. Chronic traumatic irritation as such does not induce cancer to anysignificant degree. Therefore, in view of the positive animal and human data, we suspect thattobacco contains specific carcinogen (s).

Which fraction of the tar is carcinogenic is notyet known. Roffo (31) claimed to have identifiedbenzpyrene in tobacco tar, but this could not beconfirmed by Hirst and his co-workers (7), andmore recently that substance could not be detectedby Waller (38). An examination by Eby6 of thetobacco tar used in this study did not reveal anyspectroscopic evidence of the known carcinogenicaromatic hydrocarbons. Arsenic, an accepted hu-

â€¢J. Eby, personal communication.



WYNDERet al.â€”Experimental Carcinoma with Cigarette Tar 863

man carcinogen, is present in tobacco, but recentstudies by Daff and co-workers (8) based on thearsenic content of various types of Europeantobacco tends to place less emphasis on this inorganic element. Heat, cigarette paper, flavoring, andwetting agents have been suggested as etiologicfactors in the production of cancer, but it must benoted that clinical evidence has also pointed tocigar-smoking, pipe-smoking, and tobacco-chewing as possible factors in the production of cancerof the respiratory and alimentary tract.

The actual carcinogenic agent or agents in tobacco remain to be identified. Studies combiningchemical and biologic efforts leading to their identification are urgently needed. Should one be ableto identify definite carcinogens and succeed in removing them, or at least in reducing their quantityin tobacco, proper preventive methods would be athand. Such studies may further our understandingof human and animal carcinogenesis and may leadto the development of practical preventive measures against cancer.

SUMMARY AND CONCLUSIONS1. A cigarette tar condensate was obtained with

a smoking machine which simulated human smoking habits. The resulting tar was dissolved in acetone and applied to the backs of CAFi mice in adosage of 40 mg. of tar/acetone solution 3 times aweek. Control mice were painted with acetone.

2. Of 81 tarred mice, 59 per cent developedpapillomas. The first lesion was noted in the 33dweek, and the mean time of appearance was 56weeks.

3. Of 81 tarred mice, 44 per cent developed his-tologically proved carcinomas. The first carcinomawas observed in the 42d week, and the averagetime of appearance was 71 weeks. Of 62 mice aliveat 12 months, 58 per cent developed cancer. Seventy-one weeks constitutes approximately one-half ofthe life span of CAFi mice. This correspondsroughly with the fact already noted that in thehuman about 30-35 years of smoking, or approximately one-half the life span, are required for theproduction of bronchiogenic carcinoma.

4. One carcinoma was transplanted for 4 generations and another one is currently growing in the13th generation.

5. Control mice painted with acetone aloneshowed no skin lesions. At the end of 20 months ofpainting, 53 per cent were still living, compared to9.8 per cent in the group painted with tobaccotars.

6. The group of mice painted with croton oil inaddition to the tar, starting in the 7th month,cannot be properly evaluated because of a greater

number of deaths occurring during the 12th and14th months, although within the period of observation no acceleration of cancer formation wasnoted.

7. The group of mice started with acetone andreceiving croton oil beginning in the 7th monthshowed roughening and thickening of the epidermis, but no tumor formation was noted.

8. All CAFi mice painted with 0.3 per centsolution of methylcholanthrene in acetone developed cancer within 4| months. The first papillomaappeared during the 6th week, with average appearance during the 7th seek. The first carcinoma was observed during the 12th week, with amean time of appearance of 16 weeks.

9. The results obtained with CAFi mice establish condensed cigarette tar as a carcinogen formouse epidermis. These studies provide a tool todetermine and isolate the possible carcinogenicagent (s) within tobacco tar. At present it is notknown which fraction or fractions in tobacco tarsare carcinogenic. Combined chemical and biologicstudies are now in progress to search for suchagents. Such studies, in view of the corollary clinical data relating smoking to various types of cancer, appear urgent. They may result not only infurthering our knowledge of carcinogenesis, but inpromoting some practical aspects of cancer prevention.

ACKNOAVLEDGMENTSThe authors wish to express their gratitude to Galen B.

Cook, Mary Gay Crow, and Gertrude Cassel of WashingtonUniversity School of Medicine, Department of Surgery, St.Louis, Mo., for their technical assistance.

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2. BOGEN,E., and Looms, R. N. Tobacco Tar: An Experimental Investigation of Its Alleged Carcinogenic Action.Am. J. Cancer, 16:1515-21, 1932.

3. BRESLOW,L. Does Cigarette Smoking Cause Lung Cancer?California's Health, 9:1-3, 1951.

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8. DAFF, M. E.; DOLL,R.; and KENNAWAY,E. L. Cancer ofthe Lung in Relation to Tobacco. Brit. J. Cancer, 6:1-20,1951.



864 Cancer Research

9. DOLL, R., and HILL, A. B. Smoking and Carcinoma ofLung; Preliminary Report. Brit. Med. J., 2:739-48, 1950.

10. . A Study of the Aetiology of Carcinoma of theLung. Ibid., 2:1271-86, 1952.

11. DUNGAL,N. Lung Carcinoma in Iceland. Lancet, 2:245-47,1950.

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14. FLOHT, C. M. The Production of Tumors by TobaccoTars. Cancer Research, 1:262-76, 1941.

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Un-x

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38. WALLER,R. E. The Benzpyrene Content of Town Air.Brit. J. Cancer, 5:8-21, 1952.

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40. WTNDER,E. L., and GRAHAM,E. A. Tobacco Smoking asa Possible Etiologic Factor in Bronchiogenic Carcinoma:A Study of Six Hundred and Eighty-Four Proved Cases.J.A.M.A., 143:329-36, 1950.

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FIG. 1.â€”No.87 CAFi. 322 days. Beginning carcinoma.FIG. 2.â€”Photomicrograph of carcinoma. No. 87 CAFi

at 371 days. X52.FIG. 3.â€”No.87 CAFi. 371 days. Advanced carcinoma.FIG. 4.â€”Photomicrograph of carcinoma. No. 87 CAFi

at 371 days. X215.FIG. 5.â€”No. 87 CAFt. 371 days. Advanced carcinoma.FIG. 6.â€”Photomicrograph of fourth generation transplant

of tumor from No. 87 CAFi. X215.

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Fio. 7.â€”No.37 CAFi. Early carcinoma (2 separate lesions),540 days.

Fio. 8.â€”No.37 CAF]. Advanced carcinoma, 590 days.FIG. 9.â€”No. 87 CAFi. Photomicrograph of lower lesion

at 590 days. X285.FIG. 10.â€”No.16 CAF!. Early carcinoma, 494 days.FIG. 11.â€”No.16 CAFi. Advanced carcinoma, 564 days.FIG. 12.â€”No. 16 CAFi. Photomicrograph of carcinoma

at 564 days. X200.FIG. 13.â€”No.15 CAF!. Early carcinoma, 494 days.FIG. 14.â€”No.15 CAFi. Advanced carcinoma, 605 days.FIG. 15.â€”No. 15 CAFi. Photomicrograph of carcinoma

at 605 days. X200.



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15

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FIG. 16.â€”No.3 CAFi. Beginning carcinoma, 451 days.FIG. 17.â€”No. 3 CAFi. Photomicrograph of carcinoma

at 564 days. X210.FIG. 18.â€”No. 3 CAFi. Advanced carcinoma, 494 days.FIG. 19.â€”Photomicrograph of third generation transplant

of tumor from No. 3 CAFi. X210.FIG. 20.â€”No.3 CAFi. Advanced carcinoma, 564 days.



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1953;13:855-864. Cancer Res Ernest L. Wynder, Evarts A. Graham and Adele B. Croninger Experimental Production of Carcinoma with Cigarette Tar

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