Tumorigenesis with 1,2-Benzanthracene, DeoxycholicAcid, and 3@.Methylcholanthrene*

MICHAEL KLEINt

(Deparimerd of Anatomy, University of Tennessee Medical Units, Memphis, Tennessee)

SUMMARY

Male mice belonging to strain B6AFi/J were given fifteen injections by stomachtube of 1,@-benzanthnacene, deoxycholic acid, on 3-methylcholanthrene, each suspended in methocel-Aerosol O.T. Injections were started on the 7th—8th day post partum.Control mice received no treatment, on methocel-Aerosol O.T. alone. Some of the micewere killed at median ages of from 340 to 444 days, others at median ages of 547—600days.

Many of the mice exposed to benzanthracene had pulmonary adenomas and hepatomas at autopsy. On the other hand, the mice treated with benzanthnacene and killedat a median age of 547 days all bore hepatomas. In addition, almost all had pulmonaryadenomas. This demonstrates that benzanthnacene was highly carcinogenic when treatment with this agent was instituted during infancy. This conclusion is reinforced by thethe observation of numerous tumors in the lungs and livers of mice that received onlytwo doses of benzanthracene during infancy. Although occasional pulmonary adenomasand hepatomas were found among the mice exposed repeatedly to deoxycholic acid, acomparison of tumor yields between these mice and the untreated controls, or the micetreated with methocel-Aerosol O.T. only, indicated a lack of carcinogenic activity fordeoxycholic acid. Many pulmonary adenomas, hepatomas, and forestomach papillomaswere noted in the mice exposed to methylcholanthrene. Several of the mice bone lymphocytic neoplasms, and others developed adenocarcinoma of the large intestine. Amore potent carcinogenic effect was produced by repeated treatment with methylcholanthrene than with benzanthnacene.

Although the hydrocarbon 1,@-benzanthraceneis considered generally to be noncarcinogenic (6,9, 10, 17—@0),or weakly so (@,6, 8), several investigatons have observed this compound to be activein initiating skin tumonigenesis in the mouse (4, 5,18). White and Eschenbrenner (@4) added 1,@-benzanthracene to the diet and observed that twoout of six rats developed multiple hepatomaswhich were considered to have been induced. Ina series of experiments, Steiner and co-workers

a Supported by research grant CA-04097-05 from the National Cancer Institute, National Institutes of Health, U.S.Public Health Service.

t Presentaddress:Bio-AssaySection,CarcinogenesisStudies Branch of the National Cancer Institute, Bethesda 14,Maryland.

Received for publication July 13, 1968.

(@1—@3)observed that subcutaneous injection ofthis compound alone was followed by the development of sarcomas in as many as 55 per cent of themice. Klein (1@) injected 1,@-benzanthracene andcroton oil into mice intramuscularly and obtainedseveral hemangioendotheliomas and one fibnosancoma at the injection sites. He concluded thatthese tumors were probably induced. In a series ofexperiments with different chemical agents including 3-methylcholanthrene, N-@ fluorenylacetamide, and urethan, each administered orally to infant mice, Klein (13—15)demonstrated that theyoung mouse was especially susceptible to carcinogenesis. Advantage was taken of the increasedsensitivity offered by this technic by feeding thequestionable carcinogen benzanthrácene to young'mice in the present investigation. Under these cm

1701

Susceptibility of Strain B6AF1/J Hybrid Infant Mice to

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Cancer Research Vol. @3,November 1963170@

cumstances, a potent tumorigenic influence wasdemonstrated in both the lungs and liver.

A number of investigators have exposed miceor rats to deoxycholic acid and found this agent tobe noncancinogenic or weakly carcinogenic (6, 1@).In two experiments, however, a significant increasein incidence of spindle-celled sarcomas (1) andlung tumors (16) was obtained following injectionof deoxycholic acid into mice. It is known that thepotent carcinogen 3-methylcholanthrene may beproduced by an in vitro pyrolysis of deoxycholicacid followed by hydrogenation (@,5),although itwould appear from the general lack of tumors reported for the latter compound that such a conversion either does not occur in vivo or does soinfrequently. The occasional report of a tumorigenic effect produced by deoxycholic acid suggested that a similar effect might be obtained morereadily by using an especially sensitive testingprocedure such as that provided by institutingtreatment during infancy. Thus, this agent wasadministered repeatedly to young mice in thepresent investigation. However, no enhancementin tumorigenesis was found even after a prolongedperiod of observation.

The carcinogenicity of methylcholanthnene inmice and in other species is well known (6, 19).In addition, methyicholanthrene has been shownto be active in hepatocarcinogenesis when administered to mice beginning in infancy (13) or asnewborns (11). In the present study, sucklingmice were again given oral injections of methylcholanthrene. This was done to provide a referencefor evaluating the carcinogenic potency of orallyadministered benzanthracene or deoxycholic acidwith that of a known potent carcinogen such asmethylcholanthrene, and to confirm the hepatocarcinogenic action of the latter compound in adifferent strain of mouse. Many tumors were notedamong the mice treated with methylcholanthrene. The details of this experiment and thosereferred to earlier are reported in the present communication.

MATERIALS AND METHODSThe investigation consisted of two related ex

periments, run concurrently, both using strainB6AF1/J infant male hybrid mice obtained from across between C57BL/6J females' and A/J males.'The litters derived from these matings were assigned at random to different groups. ExperimentI consisted of five groups of 40 mice each andExperiment II of five groups of twenty mice eachat the start of treatment. A major difference between the two experiments was the median age of

the mice at death. Those in Experiment I werekilled at median ages of from 340 to 444 days.Since some of the agents employed in this investigation—in particular, 1,@-benzanthracene and deoxycholic acid—are considered by many investigators to be weak carcinogens, at best, the micein Experiment II were kept under observationlonger (median age at death, 547—600days), sinceit was considered probable that more time wouldbe required to demonstrate carcinogenic activityfor these agents.

Beginning with the 7th—8th day post partum,the mice were treated repeatedly by stomach tubewith one of the following chemical agents: 1,@-benzanthracene,2 deoxycholic acid,3 or 3-methylcholanthrene.' These were suspended in 0.1 percent methocel-Aenosol O.T. (dioctyl ester of sodium sulfo-succinic acid) and administered 3 timesweekly, 0.05 mi/dose. In one group the mice received a total of only two doses of benzanthracenegiven on alternate days. For controls, some of themice were treated with 0.1 per cent methocelAerosol O.T. alone, whereas others were given notreatment. Treated mice were returned to theirrespective mothers immediately following each exposure to agent. The mice were weaned at about5 weeks of age.

The specific treatments scheduled for the different groups in each experiment follow. The micereceived a total of fifteen doses of agent in allgroups under treatment except group 10, in whichthey received only two doses.

Experiment IGroup 1, no treatment.Group @,0.1 per cent methocel-Aerosol O.T.

only.Group 3, 3 per cent 1,@-benzanthracene in

methocel-Aerosol O.T.Group 4, 3 per cent deoxycholic acid in metho

cel-Aerosol O.T.Group 5, 3 per cent 3-methylcholanthrene in

methocel-Aerosol O.T.Experiment II

Group 6, no treatment.Group 7, 0.1 per cent methocel-Aerosol O.T.Group 8, 3 per cent benzanthracene in metho

cel-Aerosol O.T.Group 9, 3 per cent deoxycholic acid in metho

eel-Aerosol O.TGroup 10, 3 per cent benzanthracene in metho

eel-Aerosol O.T.

C Obtained from Eastman Organic Chemicals, Rochester 8,

N.Y. The sampleusedin this investigation,on analysis,showeda melting point of 161°—162°C.

3 Obtained from Matheson-Coleman & Bell Divn., The

Matheson Co., East Rutherford, N.J1 Obtained from Roscoe B. Jackson Memorial Laboratory,

Bar Harbor, Maine.

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Exp.NO.GaourNO.

‘TOTAL

NO.Incat

‘TREATM,ENTtMzcz

wzmMzrn@ts

AGS ATDLATE

ALLMIca

(DATA)Hepatomas

PerNo.centPulmonary

adenomas

PerNo.centL.ympbo

cyticneoplasms

PerNo.centReticulum

cellneoplasms

type AFore

stomachpapillomas

———

PerNo.centLarge

intestine

adeno.carcinomas

— —

PerNo.centNo.PercentII

IIIIIIII1@

2@S45

6789

1039

38894035

2020201920None

O.1%MAonlyS%BAinMA8%DOCAinMA3%MCAinMA

None0.1%MAonly3%BAinMA3%DOCAinMA3%BAinMA0

0180

17

22203

160

046

049

101010016804

1087

435

67197

1710

269510

100

30359537850

0004

220000

000

11

10100000

1100

000000

8800

000000

020

22

000000

050

63

000000

0002

000000

0006

00000441

444487437840

600547547547

568

KLEIN—Susceptibtlily of Infant Mice to Tumorigenesis 1703

In view of the similarity between these two expeniments, the data from each were combined unden the same headings in Table 1.

The animals were kept in stainless-steel, hanging cages in an air-conditioned room and fedPurina Laboratory Chow and tap water adlibitum.Mice were checked daily, and cachexic animalswere killed by cervical dislocation, as were thesurvivors subsequently. All mice were autopsied,and grossly visible tumors and other macroscopicpathology were recorded. An occasional animalwas lost because of cannibalism on advanced post

10 per cent incidence of pulmonary adenomas wasobserved among untreated male mice of this strainwhen these were killed at a median age of 441 days(15). In the same study, another group of infantmales was treated with the solvent methocelAerosol O.T. alone, and a pulmonary tumor mcidence of @6per cent was recorded during a comparable observation period. These lung tumors andthose in the untreated controls, in most instances,occurred as small, solitary nodules. Aside from oneadditional tumor (a reticulum-cell neoplasm, typeA, observed in the small intestine of a mouse

TABLE 1

Suscm'TIBILrrY OF INFANT B6AF1/J HYBRID M@s.LEMIcE TO TUMOR INDUCTION FoLLowING REPEATED ORAL ADMINISTRATION OF 1,2-BENZANTILRACENE (BA), DEoxycHouc ACID (DOCA), AND 8-METHYLCHOLANTHRENE

(MCA) EACH SUSPENDED IN METHOCEL-AEROSOL O.T. (MA)

a Survivors at time first tumor was observed in the group.t Thetreatedmicereceiveda totaloffifteendoseseach,exceptin group10,whereonlytwodoseswereadministered(0.05

ml/dose).@ Data for these two groups were obtained from a previous report (15).

mortem cha1@iges.These mice are not included inthe totals in Table 1. Organs containing tumors ormasses suspected of being tumors were excised,fixed in Tellyesniczky's fluid, and the tissues prepared for subsequent staining with hematoxylinand eosin. All tumor diagnoses were confirmedhistologically.

RESULTS

Experiment I.—Carcinogenicity of benzanthnacene, deoxycholic acid, and methylcholanthnene inB6A.F,/J male mice autopsied at a median age of340 or 437 days (Table 1).

The present experiment is part of a continuingstudy on chemical carcinogenesis with the use ofinfant male mice of the B6AF,/J hybrid strain. A

treated with methocel-Aerosol), no other neoplasms were noted among the mice describedabove. These data are listed in Table 1 undergroups 1 and@

The mice in group 3 were exposed repeatedlyto benzanthracene beginning with infancy andwere autopsied at a median age of 437 days. Theincidence of pulmonary adenomas for these micenose to 95 per cent. Thirty-six of the 37 tumorbearers bore multiple tumor nodules, an average of3 per lung. In addition to the increased yield oflung tumors, eighteen of the 39 mice in this groupdeveloped hepatomas, an average of @.1per tumorbearer. Two other mice in the group were observedwith solitary forestomach papillomas, and onewith a reticulum-cell neoplasm, type A, found in

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an inguinal lymph node. Similar tumors have beendescribed in detail by Dunn (3).

Few tumors were observed among the micetreated with deoxycholic acid and killed at a median age of 437 days (group 4). These tumors wereall pulmonary adenomas, a total of five beingnoted in the four tumor-bearing mice in the group.

The mice in group 5 were exposed repeatedly tomethylcholanthrene. Many of these animals diedrelatively early in the experiment, the median ageat death for all the mice being 340 days. Some of theanimals appeared cachexic prior to death, and atautopsy the lungs of these mice were found tocontain numerous tumor nodules. A potent tumonigenie effect was observed for methyleholanthrene,neoplasms appearing in the lungs, liver, fonestomach, thymus, and large intestine. All 35 of themice developed multiple lung tumors that werediagnosed histologically as pulmonary adenomas.Frequently, the lungs were crowded with tumornodules 1—@mm. in diameter and larger. Seventeenof the mice (49 per cent) bore hepatomas, an average of @.6per tumor-bearer Papillomas of theforestomach were noted in 63 per cent of the mice.These tumors were multiple in fifteen of the@animals, the average being @.4tumors per tumorbearer. Four of the mice in this group (11 per cent)had large mediastinal tumors diagnosed as lymphocytic neoplasms in the region of the thymus.Upon histologic examination, it was noted thatthe thymus and surrounding tissues were completely replaced by a large, compact mass of smallto medium-sized lymphocytic cells. Tumors of thelarge intestine in the region of the caecum wereobserved in two of the mice. Both neoplasms werediagnosed histologically as adenocarcinomas. Thetumors replaced many of the intestinal glands,invaded the submucosa extensively, and also penetrated into the musculanis.

Experiment II.—Carcinogenicity of benzanthnacene and deoxycholic acid in B6AF1/J male miceautopsied at a median age of 547 or 568 days(Table 1).

The untreated mice in group 6 and those treatedwith methocel-Aenosol only in group 7 served ascontrols for this experiment. Hepatomas and pulmonary adenomas were observed in both groups,the tumors appearing as solitary nodules in oven50 pen cent of the susceptible mice. Lymphocyticneoplasms, characterized by large tumors in thevicinity of the thymus, were also seen among several of the mice The incidence of hepatomas andlymphocytic neoplasms in each case was 10 percent for both groups, whereas that for pulmonaryadenomas was 30 or 35 per cent (Table 1, groupsOand 7, respectively) The median age at,death for

the mice in group 6 was 600 days, as comparedwith 547 days for group 7.

A considerable increase in incidence of hepatomas and pulmonary adenomas was noted amongthe mice treated with benzanthracene, in group 8.Thus, hepatomas were found in 100 pen cent of themice. At the same time, nineteen of the twentymice in the group bone pulmonary adenomas. Themedian age at death was the same for this groupas for the methocel-Aerosol-treated controls ingroup 7. Ninety per cent of the mice with hepatomas had more than one such neoplasm pen liver,the average being 3.@tumors per mouse. All nineteen of the mice with pulmonary adenomas hadmultiple tumors, an average of 3.5 per mouse.Aside from these tumors and the hepatomas, noother neoplasms were noted.

Few tumors were observed in the mice treatedwith deoxycholic acid and autopsied at a medianage of 547 days (group 9). Only three mice out

of nineteen bone hepatomas, all solitary tumors,whereas seven bone pulmonary adenomas, an average of 1.3 per tumor-bearer.

The mice in group 10 received two doses ofbenzanthnacene as compared with fifteen doses ingroup 8. The median age at death was slightlylonger for the former mice (568 days, as comparedwith 547 days). Eighty per cent bore hepatomas,an average of 1.8 per tumor-bearer, whereas allbut three of the mice (85 per cent )bore pulmonaryadenomas, an average of 3.1 per mouse. No otherneoplasms were noted in this group.

Purity of benz[a]anthracene.—T@ie marked carcinogenic activity demonstrated for 1,@-benz[a]anthracene in this investigation contrasted sharplywith the striking lack of carcinogenic influencenoted by others. The possibility was consideredthat perhaps some impunity in the sample administered rather than the 1,@-benz[a]anthracenewas responsible for the tumonigenic effect. Anextensive chemical analysis of the original samplewas therefore conducted with the following results:

Thin-layer chromatography, with a solvent system of Skelly F :diethyl ether :glacial acetic acid inthe ratio of 50 :50 : 1 and with a supporting mediumof silica gel G, showed one spot when the samplewas dissolved in hexane. The Re' value was 0.79.One peak, slightly unsymmetrical, was observedwith gas chromatography. The sample was dissolved in chloroform and the column used wasSilicone DOW II on Chromasonb W. The ultraviolet spectrum was identical with the publishedspectrum of 1,@-benz[a]anthracene.4 In the latter

4 R. A. Friedel and M. Orchin, Ultraviolet Spectra of

Aromatic Compounds. New York: John Wiley and Sons,. Inc.,1951 (spectrum number 499).

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KLEIN—Susceptibility of Infant Mice to Tumorigenesis 1705

instance, the sample was dissolved in hexane.With reverse-phase ascending paper chromatography on mineral oil-embedded filter paper and witha solvent system of ethanol and methanol, 1 :1, atrace contaminant that fluoresced slightly underultraviolet was located below the highly fluonescent major component benz[a]anthnacene. Thesample itself was dissolved in hexane. The R@valuefor the major component, benz[a]anthnacene, was0.44, and for the minor component the value was0.18. It appeared that the impurity had about 1per cent of the fluorescence of the major spot.

Recrystallization, 3 X, of the commercial sampleof benz[a]anthracene from hot ethanol followed byreverse-phase chromatographic analysis of the purifled compound showed the same trace spot withabout the same relative degree of fluorescence ascompared with the major spot. This trace compound is not 1,@-benz[a]anthracene-7,1@-dione, thestarting material used in the synthesis of 1,@-beriz[a]anthracene by Eastman Kodak@ or naphthacene, a by-product that occasionally forms during the course of the reaction. Both these compounds migrated differently from the impuritywith the reverse-phase chromatographic system inuse.

DISCUSSION

Many tumors, especially pulmonary adenomasand hepatomas, were found among the mice afterrepeated treatment with 1,@-benzanthracene. Theincidence of lung tumors remained at 95 per centfor the mice killed at a median age of 437 or 547days (groups 3, 8). However, incidence of hepatomas increased with age from 46 to 100 per centin these two groups, respectively. It is apparentfrom these data that administration of 1,@-benzanthracene, when instituted during infancy, produced a potent carcinogenic effect on both thelungs and liver of the host mice. Reduction of thetotal administered dose of hydrocarbon from @.5mg. (groups 3, 8) to 3 mg. (group 10) still resultedin a high yield of pulmonary adenomas and hepatomas, providing additional evidence of the .cancinogenic potency of 1,@-benzanthracene under theconditions of testing employed. These findingssupport the data of Steiner and co-workers (@1—

@3),who showed that this compound was tumonigenie when injected subcutaneously into youngadult mice. Contrary to these results, a number ofinvestigators (2, 6, 8—10,17—20)have concludedthat benzanthracene possesses little or no carcinogenic activity. Steiner and Falk (@3)suggestedtwo reasons for the failure of some of these @nvestigators to obtain tuthors with benzanthnacene:(a) the use of too few animals and (b) too short a

period of observation. To this may now be addeda third—namely, the treatment of older ratherthan young animals such as were used in the present study. The fact that a compound consideredto be noncarcinogenic under one set of conditionsmay demonstrate some on appreciable carcinogenicactivity under other conditions is not surprising.However, this emphasizes the need for a comprehensive testing program when agents suspected ofbeing carcinogens or related chemically to knowncarcinogens are to be evaluated. Such a programcertainly ought to include long as well as shorterperiods of observation, sufficiently large groupsof animals, different routes of administration ofagents, different species including, perhaps, somenot now commonly employed in the laboratory,and infants (or newborns), as well as older animals.It is possible that some compounds now considened to be noncancinogenic, or questionably so,will show a moderate or high degree of cancinogenie activity when these are re-examined alongthe line suggested above.

The relationship of purity of 1,@-benz[a]anthracene to the findings in the present investigationwas discussed with Dr. Falk of the CarcinogenesisStudies Branch, National Cancer Institute. Thefollowing comments are derived from this discussiOn.5

1. Steiner and co-workers considered the possibility that highly potent impurities might penImps account for the occasional tumonigenic activity reported for benz[a]anthnacene. Thus thesame commercial product but from a differentbatch was tested for carcinogenicity, as was anotherobtained from England, and a synthetic sampleprepared in Dr. Falk's laboratory. All the samplespossessed carcinogenic activity. The present findings with commercial benz[a]anthracene thus confirm this carcinogenic activity.

@. The compounds suspected as impurities in

elude 1,@-benz[a]anthracene-7,1@-dione, 1,@-benz[a]anthracene-7,1@-diol, and naphthacene. Of thethree, naphthacene already has been tested andreported to be noncareinogenic (6) Although published data on the carcinogenicity of the other twocompounds appear to be lacking, it is probable thatthese two derivatives of 1,@-benz[a1anthnacenepossess little, if any, carcinogenic activity. It maybe pertinent here to cite the recent findings ofHeidelbenger et al. (7) on the carcinogenic potencyof different derivatives of 1,@,5,6-dlbenzanthracone. The latter investigators found that the 3—4and 9—10anthraquinones were noncarcinogenicwhen applied repeatedly to the skin of mice orwhen injected subcutaneously.

A H. L. FaIk personal communication.

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Under these circumstances, it appears justifiable to attribute the carcinogenic influence notedin the present study to benz[a]anthracene ratherthan to the trace impurity in the sample administered.

Deoxycholic acid, unlike benzanthnacene, didnot influence carcinogenesis when fed to youngmice (Table 1, groups 4, 9). Aside from an occasional report ascribing tumonigenic activity to deoxycholic acid (1, 16), most investigators havefound the latter compound to be noncarcinogenicon weakly so (6, 1@). Kennaway@ tried unsuccessfully to confirm the marked carcinogenicity ofdeoxycholic acid reported earlier from his labonatony (1). Although experimental conditions wereessentially the same in the repeat experiment, thedeoxycholic acid had to be dissolved in a differentsample of sesame oil, since the original solvent wascompletely used up. This suggested to Kennaway@that the marked carcinogenic activity observedinitially in his laboratory for deoxycholic acid wasprobably due to the presence of a co-carcinogenin the particular batch of sesame oil. Since the useof infant mice provides a sensitive test for carcinogenesis (13—15), failure to enhance tumorigenesisin the present investigation adds strong support tothe view that deoxycholic acid is noncarcinogenicin this species.

Many of the mice treated with methylcholanthrene developed hepatomas, pulmonary adenomas, and forestomach papillomas (Table 1, group5). In addition, some bore lymphocytic neoplasmsand adenocarcinomas of the large intestine. Theeffectiveness of this hydrocarbon as a hepatocarcinogen in strain B6AF,/J confirms a similar obsenvation made with mice related to strain A/He(13) and thus demonstrates that the action is notstrain-specific. Occasional hepatomas have alsobeen induced in mice belonging to strains C57BLand C3H when these were given one subcutaneousinjection of methyleholanthrene as newborns (11).

Although essentially the same incidence of lungtumors was obtained for benzanthracene as formethylcholanthrene, other data demonstrate thelatter compound to be a more potent carcinogenin the infant mouse. Thus, an average of threetumors per lung was noted in the case of benzanthracene, whereas the lungs of mice treated withmethylcholanthrene were crowded with adenomas.No attempt was made to obtain a precise count ofthe latter tumors. The larger number of mice withforestomach papillomas following treatment withmethylcholanthrene provided another indicationof the greaten carcinogenic potency of this agent

when compared with benzanthnacene (Table 1,groups 3, 5). This conclusion is reinforced furtherby the fact that median age at death was 340 dayafor the mice treated with methylcholanthrene ascompared with 437 days for those given benzanthracene. Extension of median age at death to 547days for mice treated with the latter compounddid not eventuate in a greater tumorigenic response in the forestomach as shown by the absenceof tumors in this organ (group 8). In addition toforestomach, skin with its stratified squamousepithelium has also been shown to be refractory tothe tumonigenic action of benzanthracene alone( 6, 18, 19) . However, skin tumonigenesis has been

initiated with this agent in the mouse (4, 5, 18).A similar finding has not yet been reported for theforestomach, but this is anticipated.

ACKNOWLEDGMENTS

The author is deeply indebted to Dr. Joseph A. Ontko andMr. Wells It Moorehead of the Department of Biochemistry,University of Tennessee Medical Units, for their splendidcooperation and able efforts in completing the chemical anal..yses reported in this study. Also, the author is most gratefulto Dr. Hans Falk, Chief, Carcinogenesis Studies Branch, National Cancer Institute, for his discussion relative to the chemi..cal purity of benz[a]anthracene. Finally, many thanks are dueMrs. Sue Little and Miss Bettye Jennings, and to Mr. WilliamE. Wright, dental student in the University of Tennessee College of Dentistry, for their able technical assistance.

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KLEIN—Suscepttbthty of Infant Mice to Tumorigene.i@

on March 29, 2021. © 1963 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1963;23:1701-1707. Cancer Res   Michael Klein  3-MethylcholanthreneTumorigenesis with 1,2-Benzanthracene, Deoxycholic Acid, and

/J Hybrid Infant Mice to1Susceptibility of Strain B6AF

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