(CANCER RESEARCH 45,181-185, January 1985] Estrogen 2- and 4-Hydroxylase Activity, Catechol Estrogen Formation, and Implications for Estrogen Carcinogenesis in the Hamster Kidney1 Sara Antonia Li, John K. Klicka, and Jonathan J. Li2 Medical Research Laboratories, Veterans Administration Medical Center [J. J. L], and Department of Urologie Surgery [S. A. L, J. K. K., J. J. L], University of Minnesota Medical School, Minneapolis, Minnesota 55455 ABSTRACT in the hamster (5, 8, 10). Significant estrogenicity of ethynyl estradiol in the hamster has been demonstrated by the induction Estrogen 2- and 4-hydroxylase (ESH), a microsomal enzyme of progesterone receptor in the untransformed kidney and by which mediates the formation of catechol estrogens, has been the elevation of serum prolactin levels to essentially the same studied in the kidneys of castrated male Syrian hamsters, a extent as produced by either 17/3-estradiol or DES,3 compounds species uniquely susceptible to induction of renal carcinomas by known to be highly carcinogenic at this organ site (8,10). both steroidal and stilbene estrogens. The apparent K„,for P-450 multisubstrate monooxygenases catalyze the oxidative estrone was 17.0 MM,and V,™«,was0.5 pmol per mg protein per metabolism of estrogens, and it has been shown that microsomal min for ESH in renal microsomes derived from castrated ham ESH plays a major role in the metabolism of these hormones (2). sters. Different steroidal estrogen substrates exhibited decreas Other investigators have suggested that the catechol estrogen ing catechol formation with hamster kidney microsomal prepa pathway may potentially be involved in the formation of reactive rations in the following order: estrone > d-equilenin > 17/3- estrogen intermediates (4, 19, 21). Therefore, we have under estradiol > equilin > ethynyl estradiol > estriol. Except for ß- taken to correlate renal ESH and catechol estrogen formation of dienestrol, the stilbene estrogens revealed levels of catechol various steroidal and stilbene estrogen substrates with the car formation that were similar to 170-estradiol. These findings pro cinogenic activity of these compounds, most of which we have vide a rationale for the weak carcinogenic activity of ethynyl reported previously (10), in order to elucidate possible biochem estradiol, estriol, and 0-dienestrol, since they were poor sub ical mechanisms underlying these observations. The present strates for hamster renal ESH and for the relatively potent study also compares rat kidney ESH activity to hamster kidney carcinogenic activity of the distal metabolite of diethylstilbestrol, and assesses the effect of ANF exposure on the activity of this indenestrol B/A, which exhibited substantial levels of o-hydrox- enzyme, as it has been shown to have an inhibitory action on ylation when used as a substrate. Interestingly, ESH activity was estrogen-induced renal tumorigenesis (7). significantly greater in the hamster kidney compared to corre sponding rat tissue, and catechol estrogen formation was found to be 2.5- to 19-fold higher in the hamster kidney compared to MATERIALS AND METHODS the rat, using various steroidal and stilbene estrogen substrates. Chemicals and Reagents. S-[mef/iy/-3HjAdenosyl-L-rnethionine (7.8 Moreover, the finding that a 3.5- to nearly 6-fold decrease, Ci/mmol) was obtained from New England Nuclear, Boston, MA. Cate- compared to untreated levels, in catechol formation in kidneys chol-O-methyltransferase (EC 2.1.1.6), porcine liver (specific activity, but not in livers of a-naphthoflavone-exposed hamsters, depend 2200 units/mg protein), dimethylsulfoxide (Grade 1), and NADPH (type ing on the steroidal or stilbene estrogen substrate used, is III) were provided by Sigma Chemical Co., St. Louis, MO. L-Ascorbic consistent with the belief that the catechol estrogen pathway is acid was supplied by Calbiochem, San Diego, CA. HEPES buffer (0.1 N), pertinent to events leading to estrogen-induced renal tumorigen- pH 7.4, was purchased from Grand Island Biological Co. Laboratories, Grand Island, NY. HPLC grade n-hexane was obtained from Fisher esis in the hamster. Scientific, Chicago, IL. HPLC grade tetrahydrofuran and isopropyl alcohol were purchased from Burdick and Jackson Laboratories, Muskegon, Wl. INTRODUCTION ANF was supplied by Aldrich Chemical Co., Milwaukee, Wl. Indenestrol B and .;-dienestrol were a gift of Dr. Manfred Metzler, Institute for Recent studies in our laboratory strongly indicate that nonhor- Pharmacology and Toxicology, University of Würzburg,Würzburg,Fed monal events may be involved in the tumorigenic activity of both eral Republic of Germany, and the bromo-substituted ethynyl estradiol natural and synthetic estrogens in the hamster kidney and that compounds were generously supplied by Dr. Robert H. Purdy, South the manner in which estrogens are metabolized by P^50 multi- west Biomedicai Foundation, San Antonio, TX. All other estrogens were substrate monooxygenases appears pertinent to the process obtained from either Calbiochem or Sigma. Estrogens, both natural and leading to renal cell transformation (7, 8,10,11). Consistent with synthetic, were routinely assessed for chemical purity by HPLC as this belief is the finding that ethynyl estradiol exhibits poor described previously (10). carcinogenic activity while retaining appreciable hormonal activity Animals and Treatment. Adult castrated male Syrian golden hamsters (LAK x LVG, noninbred strain) weighing 85 to 95 g were purchased from ' This investigation was supported by Grant CA 22008 from the National Cancer Chartes River Lakeview Hamster Colony, Wilmington, MA. Castrated Institute, NIH, Department of Health and Human Services; Grant 1K07 ES00094 young adult male Sprague-Dawley rats (CD) weighing 180 to 190 g were from the National Institute of Environmental Health Sciences, NIH, Department of obtained from the same source. All animals were acclimated at least 1 Health and Human Services; and the General Medical Research Fund of the week on a 12-hr-light, 12-hr-dark cycle prior to use. ANF was mixed with Veterans Administration. Presented in part at the 65th Annual Meeting of the Endocrine Society, San Antonio. TX, June 8 to 10. 1983 (12). standard laboratory rat chow (Ralston-Purina 5012) and prepared in 2 To whom requests for reprints should be addressed, at Medical Research Laboratories. Bldg. 49, Veterans Administration Medical Center, 54th Street and 3 The abbreviations used are: DES, diethylstilbestrol; ESH, estrogen 2- and 4- 48th Avenue South. Minneapolis, MN 55417. hydroxylase; ANF, 7,8-benzoflavone; HPLC, high-performance liquid chromatog- Received May 7,1984; accepted September 27,1984. raphy; HEPES, 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic aokJ. CANCER RESEARCH VOL. 45 JANUARY 1985 181 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1985 American Association for Cancer Research. HAMSTER KIDNEY ESH AND CATECHOL ESTROGENS pellet form by Purina Test Diets, Richmond, IN (12). ANF constituted of the above methoxy-d-equilenin products, respectively, and was con 0.4% of the prepared diet. The pelleted diet was stored at 4°until use. firmed by nuclear magnetic resonance. Fresh diet was provided weekly. No differences in weight gains were Radioactivity and Protein Determinations. Total radioactivity in the observed in animals maintained on ANF diets compared to chow-fed initial aliquots and radioactivity in the collected fractions were measured hamsters. after the addition of 8 ml of Liquifluortoluene (42.5:1000) (New England Microsome Preparation. Kidney microsomes were prepared essen Nuclear). All samples were counted at 5°in a Packard Tri-Carb Model tially by a method described by Holtzman ef al. (3). Briefly, male hamsters 4640 liquid scintillation spectrometer (Packard Instrument Co., Inc., and rats were killed by decapitation, and their kidneys were removed Downers Grove, IL) with a counting efficiency of about 58% for tritium. immediately and rinsed. Thereafter, the tissues were minced, blotted on Protein concentration of the microsomal fractions was determined by the filter paper, weighed, and homogenized in chilled glass:Teflon tissue method of Lowry ef al. (15) using bovine serum albumin as a standard. homogenizers with 150 mM KCI (3 volumes/g):50 mM Tris-HCI (pH 7.4). Kidneys from the same animal were treated as one sample. Tissue homogenates were centrifuged at 9,000 x g for 15 min, and the resultant RESULTS supernatant fractions were then subjected to centrifugation in a Spinco L2-65B ultracentrifuge for 45 min at 165,000 x g. The microsomal Kinetics of Microsomal ESH from Male Hamster Kidney. fractions, free of glycogen, were washed in 1.0 ml of KCI:Tris-HCI Since preliminary results proved that estrone was the most active homogenizing buffer and centrifuged again for 45 min at high speed. The substrate for ESH activity in hamster kidney microsomes, kinetic washed microsomes were then resuspended in 0.5 ml of 50 mM Tris studies were carried out using this hormone. A double reciprocal buffer, pH 7.4, containing 50% glycerol and 0.01% butylated hydroxy- plot of velocity versus substrate concentration yielded a linear toluene and gently homogenized in small conical all-glass tissue grinders. Lineweaver-Burk plot with an apparent Km of 17.0 ±2.3 MM The microsomes were stored in aliquots (100 /¿I)underN2 at -15° at a (S.E.) and an apparent Vmaxof 0.5 ±0.02 pmol per mg protein protein concentration of 12 to 15 mg/ml. Upon thawing, the microsomes per min for kidney microsomes derived from castrated hamsters. were diluted in HEPES buffer, pH 7.4, to a protein concentration of 1.2 to 1.5 mg/ml just prior to use. Liver microsomes from ANF-treated The production of catechol estrogens was linear over 15 min at hamsters were similarly prepared. a microsomal protein concentration of 150 ¿¿gproteinper ml and ESH Assay. ESH activity was determined by a radioenzymatic assay over a range of 20 to 180 ^g protein per ml (Chart 1). Linearity modified from those of Paul ef al.