[CANCER RESEARCH 42, 2426-2428, June 1982] 0008-5472/82/0042-OOOOS02.00 Promotive Effects of Steroids and Bile Acids on Hepatocarcinogenesis Initiated by Diethylnitrosamine1

Ross G. Cameron,2 Katsumi Imaida, Hiroyuki Tsuda, and Nobuyuki Ito3

First Department of Pathology, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan

ABSTRACT hepatocarcinogens on the liver in a short period, i.e., 8 weeks (7) or 12 weeks (19). Most of the hepatocarcinogens tested The ability of four steroids and three bile acids to promote enhanced the development of hyperplastic nodules in the liver, the appearance of hyperplastic foci and hyperplastic nodules and this process was enhanced in some cases by partial initiated by diethylnitrosamine (DEN) in the liver of male Fischer hepatectomy during the promotion phase (7). rats was tested and compared with that of the known promoter In this work, we tested for the promoting ability in liver of 2 phénobarbital (PB, 0.05%) as a standard value. Two weeks groups of nonhepatocarcinogens, steroids and bile acids, using after a single dose of 200 mg of DEN per kg, the animals were a short-term assay procedure similar to that described by Ito ef exposed to test chemicals for 10 weeks. At 4 weeks following a/. (7). The promoting effects of the 4 steroids and 3 bile acids DEN, all treatment groups were subjected to partial hepatec- are tentatively compared with those of PB and the hepatocar- tomy. Of the steroids tested at the maximum tolerable doses, cinogen 3'-Me-DAB given in the diet at conventional doses of as determined in preliminary experiments, ethinyl estradici was 0.05 and 0.06%, respectively. the most potent promoter, inducing more hyperplastic nodules than did PB and almost 50% less y-glutamyl transpeptidase- MATERIALS AND METHODS positive foci. However, y-glutamyl transpeptidase-positive foci induced by ethinyl estradiol were twice the average size as Animals and Diet. Male Fischer 344 rats (Charles River Japan, Inc., those induced by PB. The steroids testosterone, cortisone, and Kanagawa, Japan) weighing 120 to 150 g were first kept for 1 week dexamethasone induced more foci than did DEN alone, but few on basal diet (Oriental M, Oriental Yeast Co., Tokyo, Japan) to accli matize them to the 12-hr-light, 12-hr-dark cycle used. All animals were if any hyperplastic nodules. Deoxycholic acid was the most fasted for 18 hr prior to sacrifice. potent promoter of the bile acids tested, inducing 3 to 4 times Chemicals. Chemicals were of the purest grades available commer more hyperplastic nodules than did PB and more y-glutamyl cially and were obtained from the following sources: cortisone, dexa transpeptidase-positive foci, which were 5 times larger on the methasone, lithocholic acid, and deoxycholic acid, from Sigma Chem average than those induced by PB. The other bile acids tested, ical Co., St. Louis, Mo.; ethinyl estradiol, testosterone, 3'-Me-DAB, lithocholic acid and taurine, induced slightly more foci than did and DEN, from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan; DEN alone but no hyperplastic nodules. These findings suggest taurine, from Katayama Chemical Co., Osaka, Japan; and PB, from that ethinyl estradiol and deoxycholic acid are relatively strong Iwaki Seiyaku Co., Tokyo, Japan. promoters of the appearance of preneoplastic lesions in he- Experimental Design. Fifteen animals were allotted to each of Groups 1 to 9 (A-B) and 20 animals to Group 10 (Table 1). The animals patocarcinogenesis. in Groups 1A to 9A and Group 10 were given a single i.p. injection of 200 mg of DEN per kg in 0.9% NaCI solution, a dose known to initiate INTRODUCTION hepatocarcinogenesis (3,14,15). Animals in Groups 1B to 9B received 0.9% NaCI solution i.p. After 2 weeks on basal diet, all groups received The initiation and promotion phases of liver carcinogenesis test chemicals in their diet for 10 weeks at the following doses: ethinyl are being clarified (3). The development of focal proliferations, estradiol, 10 ppm; testosterone, 500 ppm; cortisone, 10 ppm; dexa namely, hyperplastic foci or histochemical y-GT"-positive foci, methasone, 0.5 ppm; deoxycholic acid, 5000 ppm; lithocholic acid, and hyperplastic nodules in "initiated liver" have been referred 5000 ppm; taurine, 5000 ppm; PB, 500 ppm; and 3'-Me-DAB, 600 to as promotion (3). A variety of compounds show promoting ppm. Suitable doses of test chemicals were determined in preliminary ability after exposure to an "initiating" dose of a hepatocar- experiments; the highest doses permitting survival of the most animals cinogen. These chemicals include PB (11), dichlorodiphen- were used. The doses of PB and 3'-Me-DAB chosen were those yltrichloroethane (11), polychlorinated biphenyls (8, 13, 20), conventionally used. For Group 10, basal diet was given continuously 2,3,7,8-tetrachlorodibenzo-p-dioxine (12), 3-(3,5-dichloro- for 12 weeks after exposure to DEN. All groups were subjected to partial hepatectomy (5) in Week 4 after treatment with DEN or 0.9% phenyl)-5,5-dimethyloxazolinedione-2,4 (20), and mestranol NaCI solution to increase the proliferation of hepatocytes during the (22). promotion phase (7, 17). All animals were sacrificed at the end of Recently, several in vivo assays have been developed for Week 12. testing the promoting effects of hepatocarcinogens and non- Histological Examination and Quantitäten of Induced Lesions. At the time of sacrifice, the liver was excised and sections 1 to 2 mm 1 This work was supported in part by Grants-in-Aid 501061 and 501077 for thick were cut with a razor blade. Three sections, obtained from the Cancer Research from the Ministry of Education, Science and Culture and right posterior, right anterior, and caudate lobes, respectively, were Society for Promotion of Pathology, Nagoya, Japan. * Present address: Department of Pathology, Medical Sciences Building, Uni fixed in 10% formalin in phosphate buffer adjusted to pH 7.4, proc versity of Toronto, Toronto, Ontario, Canada M5S 1A8. essed, and stained with hematoxylin and eosin. A total of 4 sections, 3 To whom requests for reprints should be addressed. one each from the right posterior and caudate lobes and 2 from the 4 The abbreviations used are: >~GT. y-glutamyl transpeptidase; PB, phénobar right anterior lobes, were immediately fixed in ice-cold acetone for bital; 3'-Me-DAB, 3'-methyl-4-dimethyl-aminoazobenzene; DEN, diethylnitrosa histochemical examination of y-GJ activity by the method of Ogawa ef mine. Received May 20, 1981; accepted March 10, 1982. al. (9). The numbers, areas, and sizes of y-GT-positive foci were

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Table 1 Numbers, areas, and sizes of y-GT-positive foci and numbers of hyperplastic nodules induced by test compounds fociArea

no. of of hyperplas cmSteroids1A.IB.2A.2B.3A.3B.4A.4B.DENGroup and treatmentDose (ppm)EffectiveratsNo./sq cmY-GT-positive(sq mm/sq cm)Size (sq mm/focus)No. tic nodules/sq

estradiol90.9%—ethinyl ±05.3 ±0.5e00.2 ethinylestradiol*DENNaCI solution -»

-»testosterone0.9% ±03.8 ±0.1rf00.2 testosteroneDENNaCI solution -» ±0.1d00.2 -»cortisone0.9% ±05.6 0.6± cortisoneDENNaCI solution —» ±0.l"012.7 ±02.16-'1.3e1.5"1.8eBile*dexamethasone0.9% -»dexamethasone101050050010100.50.514121514151211137.8NaCI solution

acids5A.SB.6A.6B.7A.7B.DEN ±11.2*0.1 acid0.9%—»deoxycholic ±0.5 3.6± deoxycholicacidDENNaCI solution -» ±3.5 ±0.190.3 0.49±1.0"±1.9e± ±1.1e00.1 acid0.9%•-•lithocholic ±03.9 acidDENNaCI solution -> lithocholic 0.000.1900.0400.0600.0400.480.110.0800.030±±±±±±±±0.06e0.020.01d0.010.2l'0.1190.04d0.013.701.000.60007.20.20000Positive-»taurine0.9% ±09.8'0.591.1"1.4e1.5 ± NaCI solution —»taurine5000500050005000500050004e7e1415121523.8

controls8A.88.9A.9B.10.DENand negative —PBa0.9% .3 ±0.5e<0.01h28.0 ±<0.1*22.6 PBaDENNaCI solution -» ±7.5e9.9 -.3'-Me-DAB0.9% ±31.7 ±4.1e0.1 3.6e± 3'-Me-DABDENNaCI solution -» ±2.2 -* basal diet8500500600600111013131914.4 ±2.6e5.4e9.8e0.91 ±0.10.09<0.031.260.320.04±h±±±0.03e0.27e0.12e0.022.108.811.21.2±1.0e±0.7±0.7 a Reported previously (1). " Mean ±S.D. 0 Significantly more than with DEN alone (Group 10); p < 0.001. " Significantly more than with DEN alone (Group 10); p < 0.01. e Deoxycholic acid (5000 ppm) in the diet was somewhat toxic, and many rats in Groups 5A and 5B died after partial hepatectomy. ' Significantly more than with DEN alone (Group 10); p < 0.05. 9 Six of 7 livers had y-GT-positive foci. Only 3 of 7 livers had hyperplastic nodules. h Two rats in this group had only one focus, each 0.03 sq mm. measured in y-GT-stained sections, as described previously (21 ), using nodules than did PB and 65% more y-GT-positive foci than did a color video image processor (VIP-21C, Olympus-lkegami Tsushin PB which were 5 times the average size of those with PB. The Co., Tokyo, Japan). Numbers of hyperplastic nodules were counted in other bile acids, lithocholic acid and taurine, induced fewer sections stained with hematoxylin and eosin. The criteria for the rec foci than did PB and no hyperplastic nodules. It is noteworthy ognition of hyperplastic nodules were as described previously (2, 3, 16). Statistical analyses were done by Student's ( test. that deoxycholic acid without DEN (Group 5B) induced a few y-GT-positive foci and nodules.

RESULTS DISCUSSION

As shown in Table 1, exposure of animals to a single dose of This study shows clearly that, following an initial exposure to DEN and partial hepatectomy 4 weeks later (Group 10) resulted DEN, induction of hyperplastic foci and nodules in the liver was in the appearance of a few y-GT-positive foci but no hyperplas considerably enhanced by the sex steroid ethinyl estradiol and tic nodules in the liver. All 9 animals in Groups 1A to 9A the bile acid deoxycholic acid in hepatectomized rats. Ethinyl exposed to a test chemical after DEN had significantly more estradiol has been reported to increase the incidence of foci than did animals treated with DEN alone (Group 10). "benign liver cell tumors" in both male and female rats in long- Deoxycholic acid (Group 5A) and 3'-Me-DAB with DEN (Group term experiments (6). A previous study has shown that another 9A) and without DEN (Group 9B) caused the greatest in sex steroid, mestranol, also increases the number of y-GT- creases. These 3 groups also had the largest average sizes positive foci in DEN-initiated liver (22). Sumi ef al. (17) found and total areas of y-GT-positive foci and the most hyperplastic that diethylstilbestrol, a potent synthetic estrogen, is an initiator nodules. Of the steroids, ethinyl estradici (Group 1A) induced and promoter of liver carcinogenesis. Taper (18) also reported the most foci and hyperplastic nodules (2). that a synthetic estradiol promotes induction of liver tumors in When tentatively compared to the promoting effect of PB as rats pretreated with A/-nitrosomorpholine. They found that ex a standard value following DEN, ethinyl estradiol (Group 1A) posure of 9 castrated male rats to diethylstilbestrol for 14 induced 76% more hyperplastic nodules than did PB and 50% months induced neoplastic (hyperplastic) nodules in 3 rats and less foci of twice the average size than PB after DEN treatment hepatocellular carcinoma in 1 rat. (Group 8A). The other steroids, testosterone, cortisone, and The mechanism of the promoting actions of these steroid dexamethasone, induced fewer foci than did ethinyl estradiol hormones in liver is unknown. Cyproterone acetate, a synthetic and fewer hyperplastic nodules or no nodules (dexametha steroid, has been shown to increase tritiated thymidine uptake sone). Deoxycholic acid induced 3 to 4 times more hyperplastic in DEN-initiated foci to a much higher level than in the surround-

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. R. G. Cameron et al. ing normal liver (10). Therefore, one way in which steroids 6. International Agency for Research on Cancer. IARC Monographs on the Evaluation of Carcinogenic Risk for Chemicals to Humans. Sex Hormones II, might act as promoters is by differential stimulation of DNA Vol. 21, pp. 233-255. Lyon, France: International Agency for Research on synthesis and growth of preneoplastic lesions. Cancer, 1979. Bile acids such as deoxycholic acid, although known to 7. Ito, N.. Tatematsu, M., Nakanisni, K., Hasegawa, R., Takano, T., Imaida, K., and Ogiso, T. The effects of various chemicals on the development of promote colon carcinogenesis (see Ref. 14 for reference), have hyperplastic liver nodules in hepatectomized rats treated with /V-nitrosodi- not yet been tested for ability to promote carcinogenesis in the ethylamine or N-2-fluorenylacetamide. Gann, 7: 832-842, 1980. liver. The strong promoting activity of the bile acid deoxycholic 8. Kimura, N. T.. Kanematsu, T.. and Baba, T. Polychlorinated biphenyl(s) as a promoter in experimental hepatocarcinogenesis in rats. Z. Krebsforsch., acid in the liver is of considerable interest, particularly because 87. 257-266, 1976. this compound is an intrinsic chemical. Deoxycholic acid may 9. Ogawa, K., Soll. D. B., and Farber, E. Phenotypic diversity as an early function as an "intrinsic promoter" of liver carcinogenesis property of putative preneoplastic hepatocyte populations in liver carcino genesis. Cancer Res.. 40: 725-733, 1980. under certain pathological conditions. Some estrogenic com 10. Ohde, G., Schuppler, J., Schulte-Hermann, R., and Keiger. H. Proliferation pounds and anabolic androgenic steroids are known to de of rat liver cells in preneoplastic nodules after stimulation of liver growth by xenobiotic inducers. Arch. Toxicol. Suppl.. 2. 451-455, 1979. crease biliary excretion which, in turn, may lead to increased 11. Peraino, C., Fry, R. J. M., Staffeldt, E., and Christopher. J. P. Comparative amounts of relevant bile acids and ethinyl estradici in the liver enhancing effects of phénobarbital,amobarbital, diphenylhydantoin and or other organs. Therefore, the indirect actions of these hor dichlorodiphenyltrichloroethane on 2-acetylaminofluorene-induced hepatic tumorigenesis in the rat. Cancer Res., 35. 2884-2890, 1975. mones may be involved in their promotion of the lesions (see 12. Pilot, H. C., Goldsworthy, T., Campbell, H. A., and Poland, A. Quantitative Ref. 4 for reference). evaluation of the promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxine of he The comparison of the promoting abilities of test chemicals patocarcinogenesis from diethylnitrosamine. Cancer Res., 40: 3616-3620, with that of PB or 3'-Me-DAB in this work merits some com 1980. 13. Preston, B. D., Van Miller, J. P., Moore, R. W., and Allen, J. R. Promoting ments. This tentative comparison was made using the highest effects of polychlorinated biphenyls (Aroclor 1254) and polychlorinated dibenzofuran-free Aroclor 1254 on diethylnitrosamine-induced tumorigene or close to the highest doses of test compounds and the sis in the rat. J. Nati. Cancer Inst., 66. 509-515, 1981. standard dose of the known promoter PB or the potent hepa- 14. Reddy, B. S., Weisburger, J., and Wynder, E. L. Colon cancer: bile salts as tocarcinogen 3'-Me-DAB. Therefore, it could be useful in prac tumor promoters. In: T. J. Slaga, A. Sivak, and R. K. Boutwell (eds.), Carcinogenesis, Vol. 2, pp. 453-464. New York: Raven Press. 1978. tical comparison of the potencies of test compounds. 15. Solt, D., and Farber, E. New principle for the analysis of chemical carcino The facts that estrogenic hormones are promoters in the liver genesis. Nature (Lond.), 263. 701-703, 1976. as well as in endocrine organs (6) and that deoxycholic acid is 16. 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Ross G. Cameron, Katsumi Imaida, Hiroyuki Tsuda, et al.

Cancer Res 1982;42:2426-2428.

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