The Influence of Dose of Carcinogen, Emptiness of Stomach,

The experimental study of gastric carcinogenesis in the mouse is complicated by the dualstructure of its stomach—the proximal two-thirds(forestomach) being lined by a stratified squamous epithelium, while the distal third is lined byglandular epithelium. While interest has generallybeen focused on the possibility of inducing tumorsof the glandular mucosa, most of the experimentalresults have, in fact, been concerned with the effects on the forestomach (see reviews : 3, 16, 18,19, 47).

Spontaneous gastric tumors in mice, whetherof the forestomach or the glandular portion, arerare (3, 11, 19, 28, 44, 50), though adenomas havebeen reported in strain I mice and adenocarcinomas in sublines of NHO (28, 46), the sublineBr-s displaying an exceptionally high incidence(45). In C3H and Swiss mice (used in the presentinvestigation), spontaneous papillomas of theforestomach are extremely rare, while carcinomasof that portion or of the glandular portion are apparently never encountered.

The literature on gastric carcinogenesis induced by carcinogens added to the diet includesreferences to the following variations in methodology:

a) The use of different carcinogens, e.g.,1,2,5,6-dibenzanthracene (2, 21—25), 3,4-benzpyrene (4, 7, 18, 29, 81, 49), 20-methylcholanthrene (13, 14, 22—24, 81), 9,10-dimethyl-1,2-benzanthracene (38, 39), 3,4,5,6-dibenzcarbazole(2), 2-acetylaminofluorene (1), p-dimethylaminobenzene-1-azo-naphthalene (27), as well as crudeproducts, e.g., heated fats and other materials(18, 20, 26, 30, 35, 48); of these, acetylaminofluorene and some of the crude products producedonly papillomas, while the others usually yieldedboth papillomas and squamous carcinomas.

* This work was supported in part by grants from the

Damon Runyon Fund (1951—53).

Received for publication January 12, 1955.

b) Tests carried out on different strains of mice,including CSH, C57BL, C57BR, A, DBA, I, CBA,NH, CHI, DB, and certain backcrosses (2, 4, 7,13, 14, 22—24),as well as stock mice (929,31, 35),rats (20, 26, 29), and rabbits (8, 17) ; of these, CSH(7)appearedto bethemostresponsivefortumorproduction in the forestomach and I mice the least(24), though other factors (see below) were not always comparable.

c) The influence of dosage, concentration, andfrequency of administration of the carcinogen.These differed greatly in the various reported investigations.

d) Different media in which the carcinogenswere suspended and the manners of administration. These included simple addition to the diet(18, 26, 27, 29, 81, 85, 49), as oily solution intothe mouth or pharynx (7) or by stomach tube (2),in “associatedcolloids― by stomach tube (37—39),or as oil-water emulsions added to drinking water(4, 22—24); the most striking results were that,whereas the oil-water emulsions stabilized withAerosol OT were carcinogenic to the forestomach(22—24),aswasthecasewithmostothermethods,similar emulsions stabilized with NaOH (or notstabilized at all) induced tumors mainly in theintestine (21, 28).

e) The addition of bile (18) or eugenol (14),injury to the mucosa by surgery (8, 17), doublevagotomy (8), heat (14), or x-rays (37)—all designed to overcome the “mucus barrier― (16) orotherwise to facilitate penetration into the glandular mucosa; none of these methods led to tumorinduction in the glandular mucosa.

That the glandular mucosa of the mousestomach is potentially responsive to carcinogenichydrocarbons was effectively demonstrated bythe production of adenocarcinomas following theirintramural injection or by the introduction intothe submucosa of crystals or threads impregnatedwith such carcinogens (12, 15, 36, 42, 43).

Perhaps the most logical approach to the whole

504

and Other Factors on Tumor Induction in the

Forestomach of the Mouse*

I. BERENBLUMANDNECHAMAHARAN

(Department of Experimental Biology, The Weizmann Institute of Science, Rehovoth, Israel)

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BERENBLUM AND HAJL&N—Some Factors Influencing Gastric (Jarcinogenesis 505

RESULTSIn the first series of experiments, duplicate tests

were carried out on Swiss and C3H mice, with 0.5per cent 9,10-dimethyl-1,2-benzanthracene as thecarcinogen. A dose of 0.3 ml. of this solution wasgiven by stomach tube once, 3 times, and 6 times,respectively, at weekly intervals, with no furthertreatment till the end of the experiment (30 weeksfrom the time of the first treatment). The survivors were then killed and their stomachs examined grossly and histologically, as were, also,those mice dying during the course of the experiment.

Tumors of the forestomach were obtained in allsix groups (Table 1), but with differences according to the number of feedings of carcinogen, andwith minor differences in the respective strains:After a single dose of carcinogen, 14/30 Swissmice and 5/25 C3H mice developed papillomas ofthe forestomach, in many cases multiple, and mostof them easily visible to the naked eye. However,none showed evidence of malignancy. The ohserved latent period in these two groups waslonger than in the other groups (see below), notumors being found before the 30th week. Afterthree doses of carcinogen, at weekly intervals, thetumor yields were higher, with 18/25 in Swiss and10/20 in C3H mice; and after six weekly doses ofcarcinogen, the yields were higher still. This correlation is more effectively demonstrated in thecorrected figures (last column of Table 1), whereall deaths during the first 10 weeks are excluded.Even more striking was the increase in the numberof malignant tumors, especially in C3H mice,which rose from zero to twelve as the dose increased from one to six feedings. Regarding thelatent period, the earliest papilloma was observedin a Swiss mouse 4 weeks after the commencementof the experiment (i.e., 1 week after the thirdfeeding) ; the earliest observed malignant tumorwas in a C311 mouse receiving six feedings—thisappeared after 12 weeks (see Figs. 2 and 5).

Two subsidiary groups were included in theabove experiment, in which identical doses of 3,4-benzpyrene and 20-methylcholanthrene were fedto C3H mice at weekly intervals but continuedthrough the experiment (i.e., for 30 weeks). Whilethe total number of tumors was somewhat lowerthan that following six doses of dimethylbenzanthracene, the proportion of malignant tumors washigher, especially with benzpyrene, which pro..duced one papilloma and sixteen carcinomas outof 30 test animals. Thirteen of the mice fed continuously with benzpyrene and ten of those fedcontinuously with methylcholanthrene were tumor-free after 80 weeks, while none were tumor

problem was to cause the carcinogen to passthrough the glandular cells in the process of secretion into the stomach, using certain derivatives ofcarcinogens which have properties similar to certhin basic dyes known to be secreted by thegastric glands (33). No tumors of the glandularmucosa resulted, however, when these compoundswere injected intraperitoneally (32, 34).

In a series of investigations undertaken in ourlaboratories, further attempts were made to studythe factors influencing gastric carcinogenesis in themouse, particularly with the hope of overcomingthe difficulty in inducing tumors of the glandularmucosa, and partly to study the “two-stagemechanism of carcinogenesis― (5) as it affectsthe stomach. The present communication dealswith gastric carcinogenesis per se, while the following communication (6) is concerned with the attempted modification of the separate stages.

The primary objective, in the present experiments, was to determine how far the number offeedings of carcinogen could be reduced for tumorsstill to be elicited, with the use of high concentrations of carcinogen in polyethylene glycol-400,since for the study of the “two-stagemechanism―it was desirable to administer only a single dose,if possible.

MATERIALS AND METHODS

The animals used in these experiments were male mice ofC8H and Swiss strains, about 3 months old, from coloniesbred by brother-to-sister matings in this laboratory. The experimental animals were housed in transparent plastic cagesand kept in air-conditioned rooms at an average temperatureof 21°C. Ten to twenty mice were kept in a cage. The dietwas Purina Laboratory Chow (Ralston Purina Co., U.S.A.)and water ad libitum.

The carcinogens—9,10-dimethyl-1,2.benzanthracene, 20-methylcholanthrene, 3,4-benzpyrene, and 1,2,5,6-dibenzanthracene—obtained from standard commercial firms, weremade up as 0.5 per cent solutions in polyethylene glycol-400and administered to the mice once weekly by stomach tube.These stomach tubes were prepared from polyethylene tubing,of about a 1-mm. bore, as used by Saxen (37), but with oneend closed with Duco cement and a lateral opening made, toreduce to a minimum the danger of mechanical injury to thestomach. (Small acute ulcers, observed in some of the earlytests, were almost entirely eliminated after the technic of passing the tube became, with practice, an easy procedure.)

Before each feeding of carcinogen, the mice were starved for18 hours, receiving only drinking water. The solid food was returned to the cages 2 hours after the treatment. Other modifications (e.g., milk diet for short periods) will be described below in the appropriate section.

At the end of each experiment or, in the case of death of ananimal during the course of the experiment, the stomach ofeach mouse was distended at autopsy with 10 per cent formalinbefore being opened. Paraffin sections of whole stomachs werestained with Harris' hematoxylin and eosin and, in manycases, with Schiff's periodic acid stain.

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506 Cancer Research

free among the survivors that received only sixdoses of dimethylbenzanthracene.

In none of the mice in the above-mentionedeight groups was there any indication of tumorproduction, benign or malignant, of the glandularportion of the stomach. With the exception of occasional localized distension of a group of glandular ducts—by no means consistent in any group orrelated to the type or dose of carcinogen used—noproliferative changes of the glandular mucosacould be discerned.

To test the possibility that undigested food inthe stomach cavity might interfere, to any extent,with the close contact between the carcinogen andthe glandular mucosa, the following method wasdevised for the effective emptying of the stomachprior to the administration of the carcinogen: Themice were kept on a milk diet for 3 days, whichinterval was then followed by the usual 18 hours'starvation (but with drinking water ad libitum).

In the next series, Swiss mice were put on thisregimen for emptying the stomach and then givena single dose of carcinogen (0 3 ml. of a 0.5 percent solution in polyethylene glycol-400). Four

groups were used for the different carcinogens3,4-benzpyrene, 20-methylcholanthrene, 1,2,5,6-dibenzanthracene, and 9,10-dimethyl-1,2-benzanthracene. The previous group of dimethylbenzanthracene (single dose) without milk diet, servedas one control; two other controls consisted of (a)a single dose of dimethylbenzanthracene in liquidparaffin (petrolatum) as solvent instead of polyethylene glycol-400 and (b) no carcinogen at all,but weekly overnight starvation. All the othergroups in this series were also submitted to weekly

overnight starvation, so that they could be usedas controls for other experiments, carried out atth@ same time (see 6).

The results of this series are summarized inTable 2. The following effects were noted : Underthe new conditions (milk diet), benzpyrene,methylcholanthrene, and dimethylcholanthreneelicited strikingly high yields of tumors of the forestomach, while dibenzanthracene proved ineffective; with dimethylbenzanthracene, the tumoryield was 29/41 (71 per cent) when preceded bythe milk diet for 3 days, and 14/30 (47 per cent)without such pretreatment. The last control in

STRAIN TRZATI&ENT

D1@BASwisso@ L1@413A

L@4BA

L1@1BAL @1BA

C3Hd' L@I4BA13P1@V1CA

TABLE1TUMOR

INDUCTION IN TUE FORESTOMACH OF THEMOUSE*Oraladministration (weeldy) of various carcinogens for differentperiods(Each

dose: 0.3 ml. of 0.5 per cent carcinogen in polyethyleneglycol400)0—lOwis.

11—20 was. 21—SOwas.TOTALNo.

DOSES N P Ca N P Ca N P Ca N P CaTOTALPazCENTt1400 400 8140 1614 0

8 610 021 1104 713 56 610 010 0 84 610 414/30

18/2314/2047(54)

72(95)70(100)1

400 000 16 50 20 505/252024)3400 300 3 91 10 9 1

6 000 013 0 79 0 81230 500 501 3 115 13 11630 400 200 4 46 10 4 610/20

20/2017/3010/205063)

100100)57(68)50(68)

* N —negative P —papillomas; Ca —squamous carcinomas of the forestomach. DMBA = 9,1O.dimethyl-1,Q-benzanthracene ; BP S,4-beazpyrene;MCA —20.methylcholanthrene.

t Numbersin parenthesesindicatepercentageofanimalswithtumorsexcludingdeathsin tisefirat1O.weelcperiod.

TABLE 2

TUMOR INDUCTION IN THE FORESTOMACH OF THE MOUSE (Swiss d')Single oral administration of different carcinogens

(Milk diet for 3 days prior to carcinogen treatment; dose of carcinogen : 0.8 ml. of 0.5 per cent in polyethylene glycol-400)

0—lOwas. 11—SOwas. 21—SOwas. TOTAL

CARCINOGEN N P Ca N P Ca N P Ca N P Ca TOTAL PERCENTS

BP 2 0 0 0 1 0 1 16 0 8 17 0 17/20 85(90)@4CA 4 0 0 0 0 1 1 18 1 5 18 2 20/25 80(95)DBA 000 100 21 00 22 00 0/22 0(0)DMBA 100 100 10290 12290 29/41 71(73)DMBAf 400 400 8140 16140 14/30 4754D1VJBA@ 800 000 9 71 12 71 8/20 4047

§ 100 500 18 10 24 10 1/25 44)5Numbers inparenthesesindicatepercentageofanimalswithtumorsexcludingdeathsinthefirst1O.weekperiod.tWithoutmilkdietpriortocarcinogen.

@ MilkdietforS days.asabove,but0.3ml.of0.5percentDMBA inliquidparaffin.I Milk diet for S days, no carcinogen feeding, but overnight starvation once a week.

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BERENBLUM AND HARAN—SOme Factors Influencing Gastric Carcinogenesi@ 507

dicated that a papilloma of the forestomach mayvery rarely appear without carcinogenic treatment(it did so, at least, in the group receiving onlyweekly overnight starvation).

Little need be said of the histological features ofthe tumors obtained in these investigations, sincesuch lesions have been fully described in the literature (44). The accompanying illustrations (Figs.1—7)will indicate the appearance of some of thelesions obtained and the rigid criteria used for thediagnosis of a papilloma and squamous carcinoma.The diagnosis of malignancy was only made wherethere was evidence of extensive invasion of theunderlying tissues. All borderline cases were listedas benign.

DISCUSSIONSwiss and C8H mice developed many papil

lomas of the forestomach after a single feeding of9,10-dimethyl-1,2-benzanthracene, 20-methylcholanthrene, or 3,4-benzpyrene. In those receivingsix or more feedings, at weekly intervals, many ofthe tumors were malignant. The results seem allthe more striking, since the experiments onlylasted 30 weeks, and many of the tumors appearedlong before the end—the earliest papilloma beingobserved after only 4 weeks from the commencement of the treatment and the earliest carcinomaafter only 12 weeks. (The true latent periods wereobviously even shorter, since early tumors areonly revealed when animals die by chance beforethe end of the experiment; most of those found atautopsy at the 30th week must have originatedmuch earlier.)

The choice of polyethylene glycol-400 as asolvent represented a compromise in the searchfor a .lipophilic-hydrophilic solvent which wasrelatively nontoxic and in which the carcinogenwould be readily soluble (40). It was noted inprevious work (9, 10, 38, 39) that certain detergents, classed as “association colloids,― such asTriton X-100, are ideally suited from the physicochemical standpoint, in that the dissolved carcinogen remains in clear solution after addition ofwater. Unfortunately, Triton X-100 (and othertypes of “block polymers― possessing similar solu

bility properties), tested by us, proved highlytoxic by mouth. (This no doubt accounts for theuse of very dilute solutions by previous workers[38, 39].) Polyethylene glycol-400, on the contrary,is almost nontoxic by mouth, even when administered undiluted. Like the “association colbids,― it is miscible with water and lipoids and isa good solvent for carcinogens. Unfortunately, onaddition of water, the carcinogen does not remainin solution but produces an opalescent colloidal

suspension. This may explain the poor penetrationof benzpyrene into the glandular mucosa when dissolved in this medium (unpublished observation)in contrast to the fairly good penetration when association colloids are used (9, 10). It may also account for the failure to obtain tumors of glandularmucosa in the present experiments. (However,even with “associationcolboids―as solvent, penetration is less efficient into the glandular than intothe squamous mucosa.)

The value of polyethylene glycol-400 as asolvent is that it allows the use of high concentrations of carcinogen to be employed (0.3 ml. of a

0.5 per cent solution, representing 1.66 mg/feeding). This is a larger dose per feeding than everused before and no doubt largely accounts for thehigh tumor incidence and short latent period evenafter a single feeding.

Thus, provided the concentration of carcinogenis high enough, the squamous epithelium of the

forestomach of the mouse is an exceptionallysensitive tissue to carcinogenesis—apparentlyeven more sensitive than the skin. On the otherhand, the hoped-for induction of tumors of theglandular mucosa was not realized, even by additional improvements in technic (i.e., by emptyingthe stomach prior to the administration of thecarcinogen), which merely somewhat enhanced thecarcinogenic action on the squamous epitheliuin.'

The increase in tumor yield with three or sixweekly feedings instead of a single feeding may becontrasted with the results of Shubik and Ritchie(41)onmouseskin,inwhichnosummationorincrease was observed when the carcinogen was applied at weekly intervals but was noted whenmuch longer intervals were allowed. However,their results were concerned with the initiatingphase only and are not, therefore, strictly cornparable.

SUMMARY

1. Swiss and C3H male mice developed papillornas of the forestomach after a single feeding of9,10-dirnethyl-1,2-benzanthracene, 20-rnethylcholanthrene, or 3,4-benzpyrene. After six or morefeedings, many of the tumors were malignant. Theearliest papilboma was observed after 4 weeks fromthe commencement of treatment and the earliestcarcinoma after 12 weeks. The high tumor yieldand short latent period are attributed mainly tothe high concentrations of carcinogen used.

2. When the carcinogen was administered on anempty stomach (following the substitution of milkfor Purina for 3 days, followed by overnight

‘Forfurther evidence of augmentation of carcinogenesiaby prior emptying of the stomach, see the following paper (6).

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508 Cancer Research

starvation), the yields of tumors appeared higherthan when given after only overnight starvation.

3. Unlike the other carcinogens tested, a singledose of 1,2,5,6-dibenzanthracene did not producetumors of the forestomach, under the conditionsof the experiment.

4. No tumors of the glandular mucosa of thestomach were obtained in any of the animals.

REFERENCES1. ARMSTRONG,E. C., and BoNson, C. M. The Carcinogenic

Action of 2-Acetyl-Amino-Fluorene on Various Strains ofMice. J. Path. & Bact., 59: 19—27,1947.

2. . Squamous Carcinoma of the Forestomach andOther ksions in Mice Following Oral Administration of8:4:5:6-Dibenzcarbazole, Brit. J. Cancer, 4:208—11,1950.

8. B@uutnrr, M. K. Avenues of Approach to the GastricCancer Problem. J. Nat. Cancer Inst., 7: 127—57,1946.

4. Bscx, S. The Effect of Feeding Carcinogenic Hydrocarbons Dissolved in Aqueous Soap Solution on the Stomachof CBA Mice. Brit. J. Exper. Path., 27: 155—57,1946.

5. BERENBLUM, I. Carcinogenesis and Tumor Pathogenesis.Adv. Cancer Research, 2: 129-175, 1954.

6. BnamraLuss, I., and HAutaw, N. The Influence of CrotonOil and of Polyethylene Glycol-400 on Carcinogenesis inthe Forestomach of the Mouse. Cancer Research, 16:510-16, 1955.

7. COLLINS, V. J.; G@utDwma,W. U.; and Sm0NO, L. C. Experimental Gastric Tumors in Mice. Cancer Research, 3:20-85, 1948.

8, DENTON, R. W.; SHELDON, P.; and Ivy, A. C. AttemptsTo Produce Gastric Carcinoma Experimentally in aGastric Ulcer. Cancer Research, 10: 684—85,1950.

9, Exw@tu., P.; Enat.u@, P.; Sn@rAi.A,K.; and SJOBL0M, L.Gastric Absorption of 8,4-Benzpyrene, II. The Significance of the Solvent for the Penetration of 8,4-Benzpyrene

into the Stomach Wall. Cancer Research, 11:758-68,1951.10. Eitas@ti@a,P.; SsrrXLX, K.; and EKWALL, P. Gastric Ab

sorption of 8,4-Benzpyrene. I. The Effect of PhysiologicalProcesses on Absorption. Cancer Research, 11:758-57,1951.

11. H@u@m, M. Spontaneous Tumours in Mice. FourthSc. Rep., Imper. Cancer Research Fund, pp. 1—118,1911.

12. HARE, W. V.; STEWART, H. L.; Bxnsrn@, J. G.; andLORENZ, E. Tumors of the Glandular Stomach Inducedin Rats by Intramural Injection of 20-Methylcholanthrene.J. Nat. Cancer Inst., 12:1019—55, 1952.

18. HITCECOCK,C. R. Failure of Bile as Abetting Agent for

20-Methylcholanthrene in Induction of Gastric Tumorsin Mice. J. Nat. Cancer Inst., 12:869—97, 1951,

14, . Failure of Eugenoland Heat To Potentiate GastricTumor Induction by 20-Methylcholanthrene in Mice.Ibid., pp. 728—38,1952.

15. Howss, E. L., and DE OuvEnta, J. R. Early Changes inthe Experimentally Produced Adenomas and Adenocarcinomas of the Stomach. Cancer Research, 8:419-24,1948.

16. Ivy, A. C. Gastric Physiology in Relation to Gastric Cancer. J. Nat. Cancer Inst., 5:318—80, 1945.

17. Ivy, A. C., and Cooxa, A. An Attempt To Produce Malignant Transformation of Gastric Ulcers in Rabbits: APreliminary Report. J. Nat. Cancer Inst., 7:845—48, 1947.

18. Knusy, A. H. M. Attempts To Induce Stomach Tumors. I.The Effect of Cholesterol Heated to 800°C. CancerResearch, 3:519—25, 1943.

19. Kz@mN,A. J., and PALMER, W. L. Experimental GastricCarcinoma. Arch. Path., 29:814—44, 1940.

20. Lei@z, A.; BLICKENSTAFF, D.; and Ivy, A. C. The Carcinogenicity of Fat “Browned―by Heating. Cancer, 3:1044—51, 1950.

21. LORENZ, E., and STEWART,H. L. Intestinal Carcinoma andOther Lesions in Mice Following Oral Administration of1,2,5,6-Dibenzanthracene and 20-Methyicholanthrene.J. Nat. Cancer Inst., 1: 17—40,1940.

22. . Squamous Cell Carcinoma and Other Lesions ofthe Forestomach in Mice, Following Oral Administrationof 20-Methylcholanthrene and 1,2,5,6-Dibenzanthracene(Preliminary Report). ibid., pp. 273—76.

28. . Tumors of the Alimentary Tract Induced inMice by Feeding Olive-Oil Emulsions Containing Carcinogenic Hydrocarbons. Ibid., 7:227—88, 1947.

24. . Tumors of Alimentary Tract in Mice Fed Carcinogenic Hydrocarbons in Mineral-Oil Emulsions. ibid.,9:173—80, 1948.

25. MAGNUS, H. A. The Experimental Production of Malignant Papillomata of the Lung in Mice with 1,2,5,6-Dibenzanthrene. J, Path. & Bact., 49:21—81, 1939.

26. Mosuus, H. P.; Lutsnsr, C. D.; and LIPPINCOTT,S. W,Effects of Feeding Heated Lard to Rats, with a HistologicalDescriptionof the Lesions Observed. J. Nat.Cancer Inst.,4:285—803,1943.

27. Mus@y, A. S., and FIRMINOER, H. I. Precancerous andCancerous Lesions of the Forestomach and DermalSubcutaneousTumours in Rats Fed p-Dimethylaminobenzene-1-Azo-1-Naphthalene. J. Nat. Cancer Inst.,13:57—61, 1952.

28. McP&sx, E.,and WARREN, S.The PathologyofGastricCarcinoma in Mice. J. Nat. Cancer Inst., 7:809—11, 1947.

Fio. 1.—Mouse 87-17 C8H c?': One feeding of 0.8 ml. of0.5 per cent DMBA. Squamous papilloma of the forestomach,fourd at 30th week, X85.

Fio. 2.—Mouse 84-4 Swiss c@@:Three weekly feedings ofDMBA (dose as above). Highly keratinizing papilloma of forestomach,found at 4th week ofexperiment(i.e.,1 week afterlast feeding). This was the earliest tumor of the forestomachever observed. X35.

Fm. 3.—Mouse 34A-4 Swiss o'@:Three weekly feedings ofDMBA. A highly malignant carcinoma of the forestomach,extensively invading the liver (left) and other organs. Foundat 20th week, X4.

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FIG. 4.—Mouse 39-3 C3H o@: Six weekly feedings ofDMBA. Squamous carcinoma of the forestomach, showing invasion of muscle. Found at 20th week. X 150.

FiG.5.—Mouse39-2C311@ :SixweeklyfeedingsofDMBA.Squamous carcinoma of the forestomach, showing early invasion across the basement membrane of the glandular mucosa.Found at 12th week. This was the earliest malignant tumor ofthe forestomach in the series. X 150

FIG. 6.—Mouse IV.5 C3H cr: 28 weekly feedings of 0.3 ml.of 0.5 per cent BP. A carcinoma of the forestomach, with onlysmall foci of keratinization, growing as islets in a fairly cellularstroma. Found at 29th week. X 150.

FIG. 7.—(same as FIG. 6). Illustrates invasion of same tu.mor (Fig. 6) within the glandular mucosa. X 150.

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BERENBLUM AND HA1@—Some Factors Influencing Gastric Carcinogenesi@ 509

40. SnvXii, K. Fluorescence Studies on Carcinogens Dissolved in Polyethylene Glycols. Ada path. et microbioLScandinav., 26:223—83,1949.

41. Snurnx, P., and Rrvcsuz, A. C. Epidermal Carcinogenesiain the Mouse Induced by One, Two, or Three Applicationsof 9,10-Dimethyl-1,2-Benzanthracene Followed by Repeated Applications of Croton Oil. A New Hypothesis ofthe Mechanism of Carcinogenesis. Cancer Research, 13:843-46, 1953.

42. STEWART, H. L., and HARE, W. V. Variation in Susceptibiity of the Fundic and Pyloric Portions of the Glandular Stomach of the Rat to Induction of Neoplasia by 20-Methylcholanthrene. Acts Union internat. contre cancer,7:176—77, 1950.

43. STEWART,H. L., and LORENZ,E. Adenocarcinoma of thePylonc Stomach and Other Gastric Neoplasms in MiceInduced with Carcinogenic Hydrocarbons. J. Nat. Canocr Inst., 3: 175—89,1942.

44. . Morbid Anatomy, Histopathology, and Histopathogenesis of Forestomach Carcinoma in Mice FedCarcinogenic Hydrocarbons in Oil Emulsions. ibid., 10:147—66,1949.

45. STRONG,L. C. Genetic Analysis of the Induction of Tumorsby Methylcholanthrene. IX. Induced and SpontaneousAdenocarcinomas of the Somach in Mice. ,J. Nat. CancerInst., 5:839—56, 1945.

46. SmoNo, L. C.; CoLusrs, V. J.; and Duiwm, E. A. AGenetic Analysis of the Induction of Tumors by Methyl..cholanthrene. IV. The Probable Remote Induction ofVarious Types of Gastric Lesions. Cancer Research, 3:21—28,1943.

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2In “Erratum,―authors were changed to: E Saxen,P. Ekwall, and K. Setklli.

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1955;15:504-509. Cancer Res   I. Berenblum and Nechama Haran  the Mouseand Other Factors on Tumor Induction in the Forestomach of The Influence of Dose of Carcinogen, Emptiness of Stomach,

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