Hepatocarcinogenicity of Dehydroepiandrosterone in the Rat1

(CANCER RESEARCH 52, 2977-2979, May 15, 1992] Advances in Brief Hepatocarcinogenicity of Dehydroepiandrosterone in the Rat1

M. Sambasiva Rao,2 V. Subbarao, Anjana V. Yeldandi, and Janardan K. Reddy

Department of Pathology; Northwestern University Medical School, Chicago, Illinois 60611-3008

Abstract oxidative DNA damage as the most rational mechanism re sponsible for peroxisome proliferator-induced hepatocarcino- Dehydroepiandrosterone, a major secretory steroid hormone of the genesis (10). Others have postulated that cell proliferation may human adrenal gland, possesses mitoinhibitory and anticarcinogenic be responsible for peroxisome proliferator-induced carcinogen- properties. It also induces peroxisome proliferation in the livers of rats icity (11) or that these agents act by exerting a promotional and mice. Because peroxisome proliferators exhibit hepatocarcinogenic effect on the so-called "spontaneously initiated" liver cells (12). potential, it is necessary to examine the long term hepatic effects of dehydroepiandrosterone since this hormone is contemplated for use as a Since DHEA has been shown to be a potent anticarcinogen in potential cancer chemopreventive agent in humans. Dehydroepiandros certain experimental animal tumor model systems, including terone was administered in the diet at a concentration of 0.45% to F-344 its ability to inhibit spontaneous cancer formation (1, 13) and rats for up to 84 weeks. At the termination of the experiment, 14 of 16 also that it was demonstrated to possess a profound antimitotic rats developed hepatocellular carcinomas. Liver tumors induced by de potential (1, 3), it provides a valid tool with which to examine hydroepiandrosterone lacked â€¢¿v-glutamyltranspeptidaseand glutathione the relationship of peroxisome proliferation and liver cancer. S-transferase (placenta! form); these phenotypic properties are identical The results unequivocally demonstrate that the anticarcino to the features exhibited by liver tumors induced by other peroxisome proliferators. Dehydroepiandrosterone was also shown to markedly in genic and antimitotic DHEA is a hepatocarcinogen in the rat, hibit liver cell |3H]thymidine labeling indices, suggesting that cell prolif most likely by virtue of its peroxisome proliferative pleiotropic eration is not a critical feature in liver tumor development with this agent. properties. These results show that although dehydroepiandrosterone exerts anticar cinogenic effects in a variety of tissues, the peroxisome-proliferative Materials and Methods property makes it a hepatocarcinogen. Fifteen-week-old male F-344 rats, weighing 250-275 g, were pur Introduction chased from Charles River Breeding Laboratories (Wilmington, MA). Rats were housed in groups of 3 to 4 in polycarbonate cages containing DHEA,3 a naturally occurring adrenal steroid hormone, has San-i-Cel bedding in a temperature- and humidity-controlled room with been shown to exert a protective role against initiation and a 12-h dark-light cycle. Sixteen rats were fed AIN-76 semipurified diet promotion stages of carcinogenesis in experimental animals (1, (corn oil stripped of vitamin E; US Biochemical Corp., Cleveland, OH) 2). The anticarcinogenic effect of DHEA is attributed primarily containing 0.45% w/w dehydroepiandrosterone acetate (Sigma Chem to the inhibition of glucose-6-phosphate dehydrogenase activity ical Co., St. Louis, MO). Ten rats were maintained on a control diet without DHEA. All rats had free access to food and water. Three resulting in the reduction of cellular NADPH pool (1, 3). DHEA-treated rats were killed between 70 and 75 weeks, and the Decreased NADPH pool leads to low levels of mixed function remaining 13 rats were killed at 84 weeks. Two control rats were killed oxidases, enzymes that participate in the activation of procar- at 70 weeks and the remaining controls were killed at 84 weeks. A cinogens to ultimate carcinogens, and nucleotides that are complete necropsy was performed on all rats. Livers were excised and needed for cell proliferation. Recent studies have shown that examined for gross lesions after serial sectioning. Selected portions of DHEA elicits pleiotropic responses in the rat liver characterized liver were fixed in 10% neutral buffered formalin and processed for by hepatomegaly, peroxisome proliferation, induction of per- routine histolÃ³gica!examination. In addition, some sections of the liver oxisome-associated enzymes, and microsomal enzymes (4-7). were fixed in ethanol-acetic acid and processed for histochemical local These hepatic changes caused by DHEA are very similar to ization of GGT (14). Sections from lungs, pancreas, and kidneys were also processed for light microscopy. Paraffin sections (4-5 firn thick) those induced by certain hepatocarcinogenic xenobiotics re were stained with hematoxylin and eosin, and those containing neo ferred to as peroxisome proliferators (8). Since it is well estab plastic lesions were stained for GST-P by immunoperoxidase method lished that peroxisome proliferators are hepatocarcinogens in (14). To study the effect of DHEA on liver cell proliferation, we have rats and mice (8, 9), the question arises about the possible injected a single dose of [3H]thymidine (1 /jCi/g body weight), l h prior paradox of the well documented anti-cell proliferative and to sacrifice to control rats and rats fed DHEA for 4, 7, or 14 days. antineoplastic properties of DHEA and the implication that it Another group of rats fed DHEA for 24 weeks and age-matched may in fact induce liver tumors by virtue of its peroxisome- controls were also used for ['HJthymidine labeling. Sections of liver proliferative property. Although the mechanism by which per were autoradiographed and the labeled nuclei were counted. Portions oxisome proliferators, which are proved nongenotoxic and non- of liver were also processed for DNA extraction and scintillation mutagenic chemicals, induce liver tumors remains controver counting. sial, we have postulated that oxidative stress and the ensuing

Received 2/14/92; accepted 3/31/92. Results and Discussion The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Body and liver weights and incidence of liver tumors are accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grant GM23750 and a Veterans Affairs summarized in Table 1. As expected, DHEA treatment caused Merit Review Program. a marked reduction in body weights when compared to controls. 2To whom requests for reprints should be addressed. ' The abbreviations used are: DHEA, dehydroepiandrosterone; GOT, v-g'u- The liver weight: body weight ratios were significantly different tamyl transpeptidase; GST-P, glutathione 5-transferase placenta! form (GST 7- between control and DHEA-fed rats (3.4 g/100 g body weight 7); HCC, hepatocellular carcinoma; NN, neoplastic nodules. versus 6.5 g/100 g body weight), although the differences in 2977

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. DHEA HEPATOCARCINOGENESIS absolute liver weights were not as marked between the two groups. The observed increases in absolute liver weight in animals fed DHEA are attributable, in part, to the presence of tumors. Grossly, livers of 15 of 16 rats treated with DHEA for 70 to 84 weeks showed one or several gray-white lesions, some as large as 25 mm in diameter (Fig. 1). The liver of one DHEA- fed rat that did not contain any gross tumors was infiltrated with leukemic cells. Livers of all the other rats contained altered areas, NN and HCC. Cells in altered areas were larger than adjacent parenchymal cells and exhibited eosinophilic or baso- philic cytoplasm with an increased nuclear cytoplasmic ratio and prominent nucleoli. Cells in NN usually displayed morpho logical features similar to those of cells in altered areas. How ever, in NN the mitotic activity was very prominent, with cells arranged in 2-3-cell-thick plates and causing compression of adjacent parenchyma. HCC were well differentiated with tra- becular, solid sheets or pseudoglandular patterns (Fig. 2). Tu mor cells displayed cytological and nuclear features of low grade hepatocellular carcinomas. Mitotic activity was similar to that seen in NN. Extension of tumors into vascular spaces or me Fig. 2. Well differentiated hepatocellular carcinoma from a rat on DHEA diet. tastasis to lungs was not observed. All types of liver lesions, H & E, x 250. i.e., altered areas, NN and HCC, that were examined for GGT and GST-P lacked these 2 marker enzymes (Fig. 3). Livers from control animals did not show any grossly visible tumors. Histologically, an occasional altered area was present. In 2 control rats there was involvement of liver with acute leukemic infiltration. The results of this study unequivocally demonstrate that DHEA, a compound generally considered anticarcinogenic with inhibitory effects on cell replication, is a hepatocarcinogen in rats. Ninety-four % of rats developed neoplastic lesions includ ing HCC in 88%. No liver tumors were observed in control rats that were fed the same diet without DHEA. The morphological and phenotypic properties of DHEA-induced liver lesions are essentially similar to those induced by classical chemical peroxisome proliferators (7, 14). Lack of GGT and GST-P is

Table 1 Body and liver weights and tumor incidence in dehydroepiandrosterone treated rats' Total no. of Body wt Liver wt Liver wt/100 g Tumor animals Treatment (g) (g) body wt incidence 16 DHEA 285.6 Â±6* 18.5 + 0.5 6.5 Â±0.2C 15(94)c Fig. 3. Hepatocellular carcinoma stained for GST-P by immunoperoxidase 10 control 428.0 Â±10f 14.7 Â±0.6 3.4 Â±0.1 0 stain. No GST-P activity is present, x 250. Â°DHEA was given in diet at a concentration of 0.45% for 70 to 84 weeks. * Mean Â±SEM. c P < 0.001 (Students / test). unique to peroxisome proliferator-induced liver tumors in con trast to genotoxic carcinogen-induced liver tumors in which these markers are consistently expressed (15). This difference in the phenotypic properties of peroxisome proliferator-induced and genotoxic carcinogen-induced lesions has been attributed to possible differences in the mechanism of action of these 2 classes of carcinogens (16). The development of liver tumors in rats treated with this widely regarded anticarcinogenic, DHEA, although it appears surprising, is not totally unexpected because of the ability of DHEA to increase the number of peroxisomes in liver paren chymal cells (4-6). Recent studies from our laboratory have shown that DHEA at a dietary concentration of 0.45% caused a 5-fold increase in the volume density of peroxisomes and a 2- and 12-fold increase in the levels of catalase and peroxisomal bifunctional enzyme mRNAs, respectively (5), in agreement with the suggested mechanism by which peroxisome prolifera tors lead to oxidative stress. Catalase, the enzyme which de Fig. 1. Large solitary liver tumor (2.5 cm) in a rat fed DHEA diet for 84 weeks. grades H2O2, is minimally induced, whereas induction of per- 2978

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. DHEA HEPATOCARCINOGENESIS Table 2 Effect of dehydroepiandrosterone on DNA synthesis in the rat liver" ably elicits its biological effects by interacting with a receptor, I'HJThymidine similar to the mechanism proposed for the transcriptional Liver wt/100 g incorporation Labeled nuclei/1000 activation of ÃŸ-oxidationsystem genes by peroxisome prolifer Group Treatment body wt (dpm/Vg DNA) hepatocyte nuclei ators (17, 18). Thus, these nongenotoxic agents provide impor 1234567DHEAdaysControlsDHEAfor 4 11127 Â±1.316.5 Â±0.18.0 Â±1564 Â±1.64.6 tant tools for elucidating the mechanisms of receptor mediated daysDHEAfor 7 Â±0.28.3 Â±1221 Â±0.61.0 hepatocarcinogenesis (16, 18). daysControlsDHEAfor 14 Â±0.2r4.4 Â±133 Â±0.14.65 Â±0.34.5 Â±O.ld2.1 wkControls7.4Â±0.l'-c5.3for 24 Â±0.2C3.0 Â±437 Â±0.32.4 References Â±0.1135Â± Â±218.0 Â±0.8 " Each group contained 4 to 5 rats. DHEA was mixed in diet at a concentration 1. Schwartz, A. G., Whitcomb. J. M., Nyce, J. W., Lewbart, M. L., and Pashko, of 0.45%. L. L. Dehydroepiandrosterone and structural analogs: a new class of cancer * Mean Â±SEM. chemopreventive agents. Adv. Cancer Res., 51: 391-423, 1988. c P < 0.001 (Student's t test). 2. Boone, C. W., Kellogg, A. J., and Malone, W. E. Identification of candidate dP<0.0\ (Student's t test). cancer chemopreventive agents and their evaluation in animal models and human clinical trials: a review. Cancer Res., 50: 2-9. 1990. 3. Gordon, G. B.. Slum/. L. M., and Talalay. P. Modulation of growth, differentiation and carcinogenesis by dehydroepiandrosterone. Adv. Enzyme oxisomal 0-oxidation system enzymes leads to the generation Regul.. 26: 255-382, 1987. of H2O2 several orders of magnitude higher than that in normal 4. Frenkcl. R. A., Slaughter. C. A., Orth, K., Moomaus. C. R., Hicks, S. H., Snyder, J. M., Bennett. M., Prough, R. A., Putnam, R. S., and Milewich, L. liver (17). Data from several earlier studies have clearly estab Peroxisome proliferation and induction of peroxisomal enzymes in mouse lished that peroxisome proliferators are not capable of causing and rat liver by dehydroepiandrosterone feeding. J. Steroid Biochem., 35: DNA damage directly but they most likely induce DNA damage 333-342, 1990. 5. Rao, M. S., Musunuri, S., and Reddy, J. K. Dehydroepiandrosterone-induced indirectly through receptor-mediated biological actions (16, peroxisome proliferation in the rat liver. Pathobiology, 60: 82-86, 1992. 18). In general, the potency of tumor induction by peroxisome 6. \ .tm.ul.i. J.. Sukuma, M., Ikeda, T.. Fukuda, K.. and Suga, T. Characteristics of dehydroepiandrosterone as a peroxisome proliferator. Biochim. Biophys. proliferators has been correlated well with their capacity to Acta, 1092: 233-243, 1991. induce sustained proliferation of peroxisomes (19). From pre 7. Wu, H., Masset-Brown, J., Tweedie, D. J., Milewich, L., Frenkel, R. A., viously published studies and this present study it is apparent Martin-Wixlrom. C., Estabrook, R. W., and Prough, R. A. Induction of microsomal NADPH-cytochrome P-450 reducÃase and cytochrome P- that DHEA at the dose level used in this study ranks interme 4501VAI (P-450 LAw) by dehydroepiandrosterone in rats: a possible perox diate in potency in inducing peroxisomes when compared to isome proliferator. Cancer Res., 49: 2337-2343, 1989. ciprofibrate and di(2-ethylhexyl)phthalate. 8. Reddy. J. K., and Lalwani, N. D. Carcinogenesis by hepatic peroxisome prolifcrators: evaluation of the risk of hypolipidemic drugs and industrial The results of incorporation of labeled thymidine into liver plasticizers to humans. CRC Crii. Rev. Toxicol., 12: 1-53, 1983. DNA and labeling index of hepatocyte nuclei in DHEA-treated 9. Rao, M. S., and Reddy, J. K. Toxicological implications of peroxisome proliferation. In: R. Meeks, S. Harrison, and J. Bull (eds.), Hcpatotoxicology, and control rats are presented in Table 2. A decrease in thy pp. 621-646. Boca Raton, FL: CRC Press, Inc.. 1991. midine uptake and the number of labeled nuclei is evident in 10. Reddy, J. K., and Rao, M. S. Oxidative DNA damage caused by persistent DHEA-treated rats. peroxisome proliferation: its role in hepatocarcinogenesis. MutÃ¢t.Res.. 214: 63-68, 1989. Liver cell proliferation has been suggested to play a primary 11. Butterworth, B. E., Loury, D. J., Smith-Oliver T., and Cattley, R. S. The role in tumorigenicity of peroxisome proliferators (11). The potential role of chemically induced hyperplasia in the carcinogenic activity of the hypolipidemic carcinogens. Toxicol. Ind. Health, 3: 129-146, 1987. results of the present study show that liver cell proliferation in 12. Kraupp-Grasl. B., Huer, W., Taper, H., and Schulte-Herman, R. Increased vivo is inhibited by DHEA which tends to negate the hypothesis susceptibility of aged rats to hepatocarcinogenesis by the peroxisome prolif that cell proliferation is the basis for peroxisome proliferator erator nafenopin and the possible involvement of altered liver foci occurring spontaneously. Cancer Res., 51: 666-671, 1991. carcinogenesis. DHEA was shown to inhibit DNA synthesis in 13. Schwartz, A. G. Inhibition of spontaneous breast cancer formation in female both in vitro and in vivo experiments (1, 3). Finally, the non- C3HAVY/3 mice by long term treatment with dchydroepiandrosterone. genotoxic nature of peroxisome proliferators prompted the Cancer Res., 39: 1129-1132. 1979. 14. Rao, M. S., Tatematsu, M.. Subbarao. V., Ito, N., and Reddy, J. K. Analysis suggestion that peroxisome proliferators act as promoters of of peroxisome proliferator-induced prcneoplastic and neoplastic lesions of spontanesouly initiated liver lesions by virtue of the mitogenic rat liver for placenta! form of glutathione .V-transferase and -y-glutamyltrans- peptidase. Cancer Res., 46: 5287-5290, 1986. potential of these chemicals (12). The fact that DHEA does not 15. Farber, E. Pre-cancerous steps in carcinogenesis: their physiological adaptive induce cell proliferation in liver suggests that promotion of the nature. Biochim. Biophys. Acta, 738: 171-180. 1984. so-called spontaneously initiated cells may not be the mecha 16. Rao, M. S., and Reddy, J. K. Peroxisome proliferation and hepatocarcino nism in DHEA-induced hepatocarcinogenesis. It is worth not genesis. Carcinogenesis (Lond.). 8: 631-636, 1987. 17. Reddy, J. K., Goel, S. K., Nemali. M. R., Carrino. J., Laffler, T. A., Reddy, ing that DHEA has been shown indeed to inhibit the sponta M. K., Sperbcck, S. J., Ozumi, T., Hashimoto. T.. Lalwani, N. D., and Rao, M. S. Transcriptional regulation of peroxisomal fatty acyl-COA oxidase and neous breast cancer formation in mice (13). These data strongly enoyl-Coa hydratase/3hydroxyacyl-Coa dehydrogenase in rat liver by per support our view that enormous increases in the free radical- oxisome proliferators. Proc. Nati. Acad. Sci. USA, 83: 747-751, 1986. 18. Isseman. I., and Green. S. Activation of a member of the steroid hormone generating peroxisomal enzymes can cause initiation albeit receptor superfamily by peroxisome proliferators. Nature (Lond)., 347:645- slowly and that such cells can then escape from or become 650, 1990. resistant to the peroxisome proliferators (20). If normal endog 19. Reddy. J. K., Reddy. M. K.. Usman, M. L, and Lalwani. N. D., and Rao, M. S. Comparison of hepatic peroxisome proliferative effect and its implication enous levels of oxidative stress are accepted as the reason for for hcpatocarcinogenicity of phthalate esters, di(2-ethylhexyl)phthalate, and the presumed spontaneous initiation (12, 21), then why would di(2-ethylhexyl)adipate with a hypolipidemic drug. Environ. Health Per- not the well documented remarkable increases in the levels of spect., 65:317-327, 1986. 20. Yokoyama, Y., Tsuchida, S, Hatayama. I., Satoh. K., Narita, T.. Rao, M. S., hydrogen peroxide-generating peroxisomal fatty acyl-CoA ox Reddy, J. K.. Yamada, J.. Suga, T.. and Sato, K. Loss of peroxisomal enzyme idase in livers be responsible for increased rates of initiation expression in preneoplastic and neoplastic lesions induced by peroxisome proliferators in rat livers. Carcinogenesis (Lond.). 13: 265-269, 1992. and tumorigenesis in animals with peroxisome proliferator- 21. Loeb. L. A. Endogenous carcinogenesis: molecular oncology into the twenty- induced chronic and sustained oxidative stress? DHEA presum first centuryâ€”Presidential Address. Cancer Res., 49: 5489-5496. 1989.

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M. Sambasiva Rao, V. Subbarao, Anjana V. Yeldandi, et al.

Cancer Res 1992;52:2977-2979.

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