[CANCER RESEARCH 37, 3238-3242, September 1977]

Promoting Effect of Bile Acids in Colon Carcinogenesis inGerm-free and Conventional F344 Rats1

Bandaru S. Reddy, K. Watanabe, John H. Weisburger, and Ernst L. Wynder

Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595

SUMMARY

The promoting effect of sodium cholate or sodium cheno-deoxycholate on colon carcinogenesis was studied in female F344 germ-free and conventional rats. Both germ-freeand conventional rats received intrarectal instillations of N-methyl-A/'-nitro-/V-nitrosoguanid¡ne (MNNG) for 2 weeks

(total dose, 8 mg/rat) and then intrarectal doses of sodiumcholate or sodium chenodeoxycholate (20 mg/rat/dose)three times a week for 46 weeks; other groups receivedMNNG for 2 weeks and vehicle thereafter for 46 weeks orbile acids alone for 48 weeks. Sodium cholate or sodiumchenodeoxycholate increased MNNG-induced adenocarci-nomas and adenomas in germ-free rats, whereas in conventional rats these bile acids induced more adenomas. Conventional rats treated with MNNG + sodium cholate orMNNG + sodium chenodeoxycholate had a higher incidence (p < 0.05) of colon tumors than did those givenMNNG alone; germ-free rats that received MNNG + sodiumcholate or MNNG + sodium chenodeoxycholate had ahigher but not significant (p > 0.05) frequency of colontumors than did those given MNNG alone. No tumors weredetected in the colons of germ-free and conventional ratsgiven sodium cholate or sodium chenodeoxycholate alone.It is concluded that sodium cholate or sodium chenodeoxycholate had a promoting effect in colon carcinogenesis inrats evoked by MNNG.

INTRODUCTION

Epidemiological data on the geographic and socioeco-nomic distribution of large bowel cancer, on the migrantpopulation, and on dietary habits of populations with variedrisks suggest that colon cancer incidence is associated withdietary factors, particularly, high intake of fat and meat (1,5, 7, 29). The key question is how a high fat/meat diettranslates into a high risk for colon cancer development inhumans. The suspected effect of dietary fat/meat on coloncarcinogenesis may be related to changes in the activity ofintestinal bacteria and in the composition of bile acids andcholesterol metabolites in colon contents (8). Current concepts are that the bile acids and cholesterol metabolites ofthe lumen of the large bowel help to modify large bowel

carcinogenesis and that these compounds, whether directlyor indirectly, are derived from dietary factors and subsequently are modified by the intestinal bacteria (8, 30).

Studies in metabolic epidemiology have shown a strongassociation between dietary fat intake, fecal secondary bileacids, and cholesterol metabolites and the risk of coloncancer among different populations (8). Recent studies byHill ef al. (9) and Reddy and Wynder (26) indicate that thepatients with colon cancer excrete high levels of fecal secondary bile acids and cholesterol metabolites compared tohealthy controls. Such a relationship is further supported byexperimental studies in which Fischer rats fed a diet high infat excreted more secondary bile acids and cholesterol metabolites and were more susceptible to colon cancer induction by 1,2-dimethylhydrazine than were animals on a dietwith normal amounts of fat (21, 22, 24). The results of Nigroef al. (20) and Chomchai ef al. (2) also support the conceptthat bile salts in the colon have some function in coloncarcinogenesis.

Historically, certain bile acids and neutral sterols are ofinterest due to the fact that they show steric similarity tocarcinogenic polycyclic aromatic hydrocarbons and thathuman gut flora have been suspected to achieve partialaromatization of the steroid ring (3, 6). Certain bile acidscould induce sarcomas but not carcinomas at the injectionsite in experimental animals (4, 11). However, this evidenceof oncogenic effect does not answer the question ofwhether certain bile acids can induce carcinomas in thelarge bowel. The observation that taurodeoxycholic acid orlithocholic acid infused i.r.2 increased the frequency ofMNNG-induced colorectal tumors in conventional rats suggests that the secondary bile acids act as colon tumorpromoters (19). Our recent study, in which the deoxycholicacid increased the number of MNNG-induced colon tumorsin germ-free rats (23), indicates that this bile acid, present inhigh concentrations in stools of human populations consuming a mixed-Western diet (8, 25) had a promoting effectin colon carcinogenesis. Since the evaluation of these primary and secondary bile acids for their promoting activity ina germ-free animal system would ideally indicate whetherfurther modification by the intestinal microflora is requiredfor tumor promotion, we extended our studies to investigatethe colon tumor activity of primary bile acids, cholic acid,and chenodeoxycholic acid in germ-free and conventionalrats.

1 Supported by USPHS Contract CP-33208 from the National Cancer Insti

tute.Received April 7, 1977; accepted June 13, 1977.

2 The abbreviations used are: i.r., intrarectal; MNNG, W-methyl-A/'-nitro-N-

nitrosoguanidine.

3238 CANCER RESEARCH VOL. 37

Research. on December 29, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

MATERIALS AND METHODS

Inbred weanling female F344 germ-free and conventionalrats were obtained commercially from the Charles RiverBreeding Laboratory, North Wilmington, Mass., and fed adlibitum a steam-sterilized, fortified Purina laboratory chow,501OC. Germ-free rats were housed in plastic cages andmaintained in Trexler flexible plastic isolators, and the conventional animals were maintained in a temperature- andhumidity-controlled clean room. The germ-free status of theanimals was determined at biweekly intervals and also at thetermination of the experiments (28). All cultures from thegerm-free rats were negative for bacteriological contamination.

At 50 days of age, both germ-free and conventional ratswere randomly divided into 6 groups and received i.r. thefollowing compounds in 0.2 ml of 0.9% NaCI solution.Groups 1 and 2 each received 20 mg sodium cholate orsodium chenodeoxycholate, respectively, per animal 3times weekly for 48 weeks. Group 3 received 2 mg MNNGtwice a week for 2 weeks and vehicle thereafter 3 times aweek for 46 weeks. Groups 4 and 5 each received 2 mgMNNG twice a week for 2 weeks followed by 20 mg sodiumcholate or sodium chenodeoxycholate, respectively, per animal 3 times weekly for 46 weeks. Animals in Group 6 weregiven an equal volume of 0.9% NaCI solution. Before i.r.infusion, MNNG was dissolved in 0.9% NaCI solution andsterilized by Millipore filtration, whereas sodium cholate orsodium chenodeoxycholate was dissolved in 0.9% NaCIsolution and steam sterilized. Before sterilization, all solutions were transferred into ampuls and sealed under vacuum.

At the end of 48 weeks, all animals were killed under etheranesthesia, and the organs, including the intestine, wereexamined grossly and histologically for the number andtype of tumors. Tissues were fixed in 10% formalin andembedded in the paraffin. The sections were stained withhematoxylin and eosin.

RESULTS

Table 1 summarizes the tumor incidence in germ-free ratstreated with individual bile acids, MNNG, or MNNG + bile

Bile Acids and Colon Tumor Promotion

acid. No tumors were found in the colon or other organs ofgerm-free rats given cholic acid or chenodeoxycholic acidalone, which suggested that these bile acids were not carcinogenic under our experimental conditions. Animals givenMNNG or MNNG + bile acids had colonie tumors. Animalsthat received MNNG + cholic acid or MNNG + chenodeoxycholic acid had a higher but not significant (p > 0.05)incidence of colon tumors than did those given MNNGalone; the tumors were in the distal part of the large bowel.However, the important finding was that MNNG + cholicacid or MNNG + chenodeoxycholic acid produced colontumors in 50 or 54% of germ-free rats, respectively. Thegroup given MNNG + cholic acid or chenodeoxycholic aciddeveloped more adenocarcinomas (p < 0.05) as well asadenomas (p < 0.05) than did rats given MNNG only. Notumors were detected in any other organ.

Table 2 summarizes the colon tumor incidence in conventional rats. Tumors were not found in any conventional ratsgiven bile acids alone, which again suggested that thesebile acids or their microbially modified metabolites were notcarcinogenic to colon mucosa or other organs. The numberof rats with colon tumors was significantly increased (p <0.025) in rats given MNNG + cholic acid or MNNG + chenodeoxycholic acid compared with the number in the MNNGgroup. The tumor multiplicity for adenocarcinomas wassimilar in the MNNG, MNNG + cholic acid, and MNNG +chenodeoxycholic acid groups. Rats given MNNG + cholicacid or MNNG + chenodeoxycholic acid developed significantly more adenomas than did the MNNG group (p < 0.05).Aside from the colon, none of the other organs showed anytumors. The results also indicate that the conventional ratsgiven MNNG + cholic acid or chenodeoxycholic acid had ahigher incidence of colon tumors than did the germ-freerats treated similarly (Tables 1 and 2).

The standards for histological diagnosis of intestinal tumors were as described by Lingeman and Garner (12) and inour previous report (21) (Figs. 1 to 6). Histologically, theadenomas classified in the present study were benign neoplasms of intestinal glandular structure lining, with slight ormoderate atypical epithelial cells, but not invasive into thesubmucosal layer. Adenocarcinomas were malignant neoplasms invading into the submucosal layer and were welldifferentiated. Some of them were signet-ring infiltrating

Table 1Colon tumor incidence in germ-free rats treated i.r. with MNNG and/or bile acids

Animals withcolontumorsTreatmentCA"

C DAMNNGMNNG + CAMNNG + CDANo.

ofrats1010

22o2424No.00

612"

13"%0

0275054Total0

00.27 ±0.07'0.63 ±0.11'1 .08 ±0.20'Tumors/ratAdenocarci-

noma0

00.14 ±0.070.29 ±0.08'0.29 ±0.08'Adenoma0

00.14 ±0.060.34 ±0.06'0.79 ±0.12'

" CA, cholic acid; CDA, chenodeoxycholic acid.6 Of 24 germ-free rats, 2 rats were lost due to technical problems during the 4th week of

the experiment.' Mean ±S.E." Not significantly different from group given MNNG alone by x2 test; p ^ 0.05.' Significantly different from group given MNNG alone by t test; p «0.05.

SEPTEMBER 1977 3239

Research. on December 29, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

B. S. Reddy et al.

Table 2

Colon tumor incidence in conventional rats treated i.r. with M/VA/G and/or bile acids

Animals withcolontumorsTreatmentCA"C

DAMNNGMNNG

+CAMNNG+ CDANo.

ofanimals1212303030No.001120'21'%00376770Total000.55

±0.15"0.87±0.12"1.23±0.20"Tumors/animalAdenocarci-noma000.23

±0.080.24±0.070.27±0.08Adenoma000.32

±0.100.63±0.10"0.96±0.16"

" CA, cholic acid; CDA, chenodeoxycholic acid.* Mean ±S.E.' Significantly different from group given MNNG alone by x2 test; p s 0.025." Significantly different from group given MNNG alone by f test; p «0.05.

: V•

Fig. 1. Histological section of a well-differentiated adenocarcinoma invading into the submucosal layer induced by MNNG in conventional rat.x 90.

Fig. 2. Histological section of a tubular adenoma composed of the mildatypical glandular structure induced by MNNG in conventional rat. x 90.

carcinomas invading into the submucosal and proper muscular layers.

The colon tumors in both germ-free and conventional ratswere localized in the distal half of the colon (from the anusto 12 cm above the anus) and were mainly polypoid orhemispheric and tan. The tumor sizes in all groups rangedfrom 1 to 14 mm in diameter. A large number of adenomaswere smaller than 5 mm in diameter, whereas all of theadenocarcinomas measured over 3 mm in diameter.

Microscopically, the colon tumors induced in germ-free

rats were similar to those induced in conventional rats. Allcarcinomas were polypoid or plaque shaped, and adenomas were pedunculated or sessile types. The adenocarcinomas were mostly well differentiated and exhibited infiltration into the submucosa, whereas some were poorly differentiated adenocarcinomas showing mucous cell infiltrationinto the submucosa. No metastatic lesions were detected.

DISCUSSION

The primary bile acids synthesized by the human liver aretaurine- or glucine-conjugated cholic acid and chenodeoxycholic acid (17, 18). In addition to these primary bile acids,the normal human bile contains secondary bile acids, deox-ycholic acid and lithocholic acid, which result from thecolonie bacterial 7a-dehydroxylation of cholic acid andchenodeoxycholic acid, respectively (17,18). The bile acidspresent in the colon are entirely deconjugated and contain,in addition to small amounts of primary bile acids, a widerange of secondary bile acids, such as deoxycholic acid andlithocholic acid, produced by bacterial oxidoreduction ofthe hydroxyl groups at the C-3, C-7, and C-12 positions togive keto bile acids and those with the /3-hydroxyl group(the inversion products), and by bacterial dehydroxylationat C-7.

The observation that i.r. administration of cholic acid orchenodeoxycholic acid alone produced no tumors in thecolon of either germ-free or conventional rats suggests thatthese bile acids or their metabolites produced by the co-Ionic bacteria were not carcinogenic or at least weaklycarcinogenic to the colon mucosa. This is in line with theobservations of Narisawaef al. (19) in conventional rats andwith our observations in germ-free rats (23).

Our results indicate that the primary bile acids, cholicacid and chenodeoxycholic acid, also produced a promoting activity in the colon of both germ-free and conventionalrats. Cholic acid and chenodeoxycholic acids given i.r. toconventional rats are subject to microbial 7a-dehydroxyla-tion to deoxycholic acid and lithocholic acid, respectively.Conventional rats treated with MNNG + cholic acid or chenodeoxycholic acid showed a higher tumor incidence thandid the germ-free rats treated similarly. This suggests thatthe increase in tumor production with cholic and or chenodeoxycholic acid in conventional rats was probably due to

3240 CANCER RESEARCH VOL. 37

Research. on December 29, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

Bile Acids and Colon Tumor Promotion

production of deoxycholic acid or lithocholic acid by thebacterial 7a-dehydroxylation in the colon of conventionalrats. These secondary bile acids have been shown to have astrong colon tumor-promoting activity in rats (19, 23). Theresults obtained in germ-free rats demonstrate that cholicacid and chenodeoxycholic acid, without being furthermodified by the intestinal bacteria, do act as colon tumorpromoters. This is of interest, since bacterially modifiedsecondary bile acids, but not the primary bile acids, havebeen associated with the incidence of colon cancer (8, 9,26). The above observations stimulate further investigationsof the participation of various bile acids as promoters incolon carcinogenesis.

The mechanism of tumor-promoting activity by variousbile acids, although under investigation, is incompletelydefined. Kawalek and Andrews (10) reported that the bilesalts themselves are not mutagenic in the Ames salmonella-microsome test; however, when they are included in theAmes assay with suboptimal levels of known carcinogens,there is an increase of mutagenicity. The findings of thepresent study and our earlier studies (19, 23), showingcocarcinogenicity of various bile acids to the colon mucosa,suggest that the interactions between these metabolitesand colonie epithelial cells may be relevant to the development of neoplasms. However, it is not clear whether theeffect of various bile acids on colon carcinogenesis is mediated through changes in the mucosal cell kinetics. Thecell renewal system is dynamic and may be influenced bychanges in a number of factors including the compositionof the intestinal microflora (15) and bile acids in the intestine (16). It has been shown that, with proper modificationof the microenvironment of the intestinal tract, it is possibleto alter the cellular kinetics of the mucosa (14). Lipkin (13)demonstrated that, during neoplastic transformation of co-Ionic cells, a similar sequence of changes leading to uncontrolled proliferative activity develops in colon cancer in humans and in rodents given a chemical carcinogen thatinduces colon cancer.

The question may be raised as to the extent to which thisand other studies provide causative explanation to humanlarge bowel cancer. Although a specific carcinogen for thecolon has not yet been identified either in the colon or in thefeces, the experiments thus far in our laboratory indicatethat some of the bile acids present in the lumen of the largebowel have tumor-promoting activity which could result inpart from the dietary habits that increase the production,presence, and excretion of bile acids. The questions thatneed to be answered are: whether the carcinogens thatinitiate colon carcinogenesis are present in the colonielumen or whether they are synthesized by the colonie mucosa, and whether or not the intestinal bacteria are involvedin the production of initiating carcinogens specific to colonfrom the dietary and/or endogenous sources. It is, ofcourse, an item of 1st priority to identify such agents responsible for the initiation of colon carcinogenesis in humans. In relation to this, Hill et al. (8, 9) suggested thatnuclear dehydrogenation of unsaturated bile acids by anuclear-dehydrogenating clostridia in the colon may be animportant factor in colon carcinogenesis. Recently,Varghese ef al. (27) claimed that they have isolated fromhuman fècesan /V-nitroso compound that is potentially a

carcinogenic agent for the colon. Wynder and Reddy (31)suggested that the conversion of cholesterol and dehydro-cholesterol, which are normally found in colonie contentsand mucosa, by electron oxidation to reactive metabolites,which are capable of interacting with a nucleophile and actas carcinogens, may also be an important step in coloncarcinogenesis. Obviously, further studies are warranted toidentify and isolate initiating carcinogens specific to thecolon.

ACKNOWLEDGMENTS

The expert technical assistance of Kathy Highland, Yves-Marie Louis andChang-ln Choi is gratefully appreciated.

REFERENCES

1. Armstrong, B., and Doll, R. Environmental Factors and Cancer Incidenceand Mortality in Different Countries, with Special Reference to DietaryPractices. Intern. J. Cancer, 75. 617-631, 1975.

2. Chomchai, C., Bhadrachari, N., and Nigro, N. D. The Effect of Bile on theInduction of Experimental Intestinal Tumors in Rats. Diseases ColonRectum, 17: 310-312, 1974.

3. Combs, M. M., Bhatt, T. S., and Croft, C. J. Correlation between Carci-nogenicity and Chemical Structure in Cyclopentaphenanthrene. CancerRes., 33; 832, 1973.

4. Cook, J. W., Kennaway, E. L., and Kennaway, N. M. Production Tumorsin Mice by Deoxycholic Acid. Nature, 145: 625, 1940.

5. Doll, R. The Geographic Distribution of Cancer. Brit. J. Cancer, 23: 1-8,1969.

6. Haddow, A. Chemical Carcinogens and Their Modes of Action. Brit.Med. Bull., 14: 79-92, 1958.

7. Haenszel, W., Berg, J. W., Kurihara, M., and Locke, F. B. Large BowelCancer in Hawaiian Japanese. J. Nati. Cancer Inst., 5Õ:1765-1799,1973.

8. Hill, M. J., Crowther, J. S., Drasar, B. S., Hawksworth, G., Aries, V., andWilliams, R. E. O. Bacteria and Etiology of Cancer of the Large Bowel.Lancet, 1: 95-100, 1971.

9. Hill, M. J., Drasar, B. S., Williams, R. E. O., Meade, T. W., Cox, A. G.,Simpson, J. E. P., and Morson, B. C. Fecal Bile Acids and Clostridia inPatients with Cancer of the Large Bowel. Lancet, J. 535-539, 1975.

10. Kawalek, J. C., and Andrews, A. W. The Effect of Bile Acids on theMetabolism of Benzo(a)pyrene (BaP) and 2-Aminoanthracene (2-AA) toMutagenic Products. Federation Proc., 36: 844, 1977.

11. Lacassagne, A., Buu-Hoi, N. P., and Zajdela. F. Carcinogenic Activity inSitu of Further Steroid Compounds. Nature, 209: 1026-1027, 1966.

12. Lingeman, C. H., and Garner, F. M. Comparative Study of IntestinalAdenocarcinomas of Animal and Man. J. Nati. Cancer Inst., 48. 325-346,1972.

13. Lipkin, M. Biology of Large Bowel Cancer. Present Status and ResearchFrontiers. Cancer, 36: 2319-2324, 1975.

14. Mastromarino, A., and Wilson, R. Increased Intestinal Mucosal Turnoverand Radiosensitivity to Supralethal Wholebody Irradiation Resultingfrom Cholic Acid-induced Alterations of the Intestinal Microecology ofGermfree CFW Mice. Radiation Res., 66: 393-400, 1976.

15. Matsuzawa, T., and Wilson, R. The Intestinal Mucosa of Germfree Miceafter Wholebody X-irradiation with 3 Kiloroentgens. Radiation Res., 25:15-24, 1965.

16. Meslin, J. C., Sacquet, E., and Raibaud, P. Action d'une Flore Micro

bienne qui ne Déconjuquepas les Sels Biliaires sur la Morphologie et leRenouvellement Cellulaire de la Muqueuse de l'Intestin Grêledu Rat.Ann. Biol. Animale Biochim. Biophys., 14: 709-720, 1974.

17. Mosbach, E. H. Hepatic Synthesis of Bile Acids. Arch. Intern. Med., 130:478-487, 1972.

18. Mosbach, E. H., and Salen, G. Bile Acid Biosynthesis. Pathways andRegulation. Am. J. Digest. Diseases, 19: 920-929, 1974.

19. Narisawa, T., Magadia, N. E., Weisburger, J. H., and Wynder, E. L.Promoting Effect of Bile Acids on Colon Carcinogenesis after IntrarectalInstillation of N-Methyl-N'-nitro-N-nitrosoguanidine in Rats. J. Nati. Cancer Inst., 55: 1093-1097, 1974.

20. Nigro, N. D., Bhadrachari, N., and Campbell, R. L. A Rat Model forStudying Colon Cancer. Effect of Cholestyramine on Induced Tumors.Diseases Colon Rectum, 16: 438-443, 1973.

21. Reddy, B. S., Narisawa, R., Vukusich, D., Weisburger, J. H., and Wynder, E. L. Effect of Quality and Quantity of Dietary Fat and Dimethylhy-drazine in Colon Carcinogenesis in Rats. Proc. Soc. Exptl. Biol. Med.,151: 237-239, 1976.

SEPTEMBER 1977 3241

Research. on December 29, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

B. S. Ready et al.

22. Reddy, B. S., Narisawa, R., and Weisburger, J. H. Effect of a Diet withHigh Levels of Protein and Fat on Colon Carcinogenesis in F 344 RatsTreated with 1,2-Dimethylhydrazine. J. Nati. Cancer Inst., 57: 567-569,1976.

23. Reddy, B. S., Narisawa, T., Weisburger, J. H., and Wynder, E. L. Promoting Effect of Sodium Deoxycholate on Adenocarcinomas in GermfreeRats. J. Nati. Cancer Inst., 56. 441-442, 1976.

24. Reddy, B. S., Weisburger, J. H., and Wynder. E. L. Effect of Dietary FatLevel and Dimethylhydrazine on Fecal Acid and Neutral Sterol Excretionand Colon Carcinogenesis in Rats. J. Nati. Cancer Inst., 52. 507-511,1974.

25. Reddy. B. S.. and Wynder. E. L. Large Bowel Carcinogenesis: FecalConstituents of Populations with Diverse Incidence Rates of Colon Cancer. J. Nati. Cancer Inst., 50: 1437-1442, 1973.

26. Reddy, B. S., and Wynder, E. L. Metabolic Epidemiology of Colon

Cancer: Fecal Bile Acids and Neutral Sterols in Colon Cancer Patientsand Patients with Adenomatous Polyps. Cancer, 39: 2533-2539, 1977.

27. Varghese, A. J., Land, P., Furrer, R., and Bruce, W. R. Evidence for theFormation of Mutagenic W-nitroso Compounds in the Human Body.Proc. Am. Assoc. Cancer Res., Õ8:80, 1977.

28. Wagner, M. Determination of Germfree Status. Ann. N. Y. Acad. Sci., 78:89-101, 1959.

29. Wynder, E. L. The Epidemiology of Large Bowel Cancer. Cancer Res.,35: 3388-3394, 1975.

30. Wynder, E. L., and Reddy, B. S. Studies of Large Bowel Cancer: HumanLeads to Experimental Application. J. Nati. Cancer Inst., 50: 1099-1106,

1973.31. Wynder, E. L., and Reddy, B. S. Colon Cancer Prevention: Today's

Challenge to Biomedicai Scientists and Clinical Investigators. Cancer, inpress.

Fig. 3. Histological section of a well-differentiated adenocarcinoma showing invasion into the stalk of polypoid tumor induced by MNNG and cholic acid inconventional rat. x 90.

Fig. 4. Histological section of a tubular adenoma induced by MNNG and cholic acid in conventional rat. x 90.Fig. 5. Histological section of a sessile elevated tumor from the distal colon of MNNG-treated, germ-free rat showing mucous cell carcinoma diffusely

invaded into the submucosal and proper muscular layers, x 90.Fig. 6. Histological section of a tubular adenoma composed of the moderate atypical glandular structure from the polypoid tumor of MNNG-treated, germ-

free rat. x 90.

3242 CANCER RESEARCH VOL. 37

Research. on December 29, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

1977;37:3238-3242. Cancer Res   Bandaru S. Reddy, K. Watanabe, John H. Weisburger, et al.   Germ-free and Conventional F344 RatsPromoting Effect of Bile Acids in Colon Carcinogenesis in

  Updated version

  http://cancerres.aacrjournals.org/content/37/9/3238

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/37/9/3238To request permission to re-use all or part of this article, use this link

Research. on December 29, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from