Estrogen 2- and 4-Hydroxylase Activity, Catechol Estrogen Formation, and Implications for Estrogen Carcinogenesis in the Hamster Kidney1

(CANCER RESEARCH 45,181-185, January 1985]

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 Kâ€ž,for P-450 multisubstrate monooxygenases catalyze the oxidative estrone was 17.0 MM,and V,â„¢Â«,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 ÃŸ- 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 WÃ¼rzburg,WÃ¼rzburg,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 4Â°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 5Â°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 Â±2.3 MM The microsomes were stored in aliquots (100 /Â¿I)underN2 at -15Â° at a (S.E.) and an apparent Vmaxof 0.5 Â±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 Â¿Â¿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. (20) and Purdy ef al. (22). Briefly, the was also found varying the estrone concentration between 5 incubation mixture consisted of 40 n\ of 10 mM HEPES buffer, pH 7.4, and 25 MMat 150 ng protein per ml. Therefore, all subsequent 201Â¡\of 1.0 M MgCI2, 20 n\ of 6 mM ascorbic acid, 100 units of catechol- ESH assays were carried out at zero-order kinetics with respect O-methyltransferase in 20 n\ of HEPES, 300 n\ of microsomes, 50 Â¡i\of to substrate (50 to 100 MM). S-[3H]adenosyl-L-methionine, and 5 //I of estrogen substrate (50 to 100 //M) in dimethyl sulfoxide. The mixture was preincubated for 5 min at 37Â° Relative Rates of Catechol Estrogen Formation with Var ious Estrogen Substrates by Hamster Kidney Microsomes. with gentle agitation. The enzymatic reaction was initiated by the addition Different stilbene and steroidal estrogens were used as sub of 50 M!of 18 mM NADPH. Aliquots (100 n\) were taken after 5 min just prior to the addition of NADPH and at 10 min. To each aliquot, 1.0 ml of strates for renal ESH activity in castrated hamsters, and these 0.05 M borate buffer, pH 10, was added which contained 20 Â¿ileach of results are summarized in Table 1. Of the estrogens studied, 2-methoxyestradiol (2 mM) and 4-methoxyestradiol (2 mM) as recovery estrone was the most active substrate. Except for /3-dienestrol, standards. The radiolabeled monomethyl ethers were extracted with 7 all of the stilbene estrogens tested exhibited substantial catechol ml of n-hexane. Samples of the n-hexane extract were assessed for formation, which was essentially the same amount found for radioactivity, and the remainder was evaporated under N2 and stored at 17/8-estradiol. Surprisingly, when ethynyl estradici was used as -80Â° for subsequent HPLC analyses. Control incubations containing a substrate, catechol formation was only one-half the level boiled microsomes or lacking either NADPH or substrate were also produced by either 17/3-estradiol or DES and more than 10-fold subjected to the above procedure. less than estrone. As a substrate, estrÃo!revealed relatively low HPLC Analyses of Estrogen Monomethyl Ethers. Separation of the 3H-labeled monomethyl ethers of catechol estrogens was accomplished levels of catechol formation using hamster kidney microsomes. using a Hewlett-Packard Model 1084B liquid Chromatograph equipped with a variable UV detector set at 284 nm. Each sample was dissolved 3.5 in 40 ui of tetrahydrofuran, and a 20-nl aliquot was injected onto a 0.46- x 30-cm Chromegabond diol column (ES Industries, Mariton, NJ). Estro gen samples were eluted isocratically with 4% isopropyl alcohol in n- 2.5 hexane for 5 min, after which a linear gradient from 4 to 8% isopropyl alcohol in n-hexane was run the next 15 min. The flow rate was held constant at 2.0 ml/min, and column temperature was maintained at 40Â°. A fraction collector (SuperRac Model 2211 ; LKB, Rockvilte, MD) was used to collect 0.5-min fractions for determination of radioactivity. After elution of each sample, the column was washed for 10 min with 100% isopropyl alcohol and reequilibrated for 10 min with 4% isopropyl alcohol oÂ«o on in n-hexane before applying the next sample. Retention times of the lis]"Â»" methylated catechol estrogens were compared to the following authentic monomethyl ethers of catechol estrogens provided by Dr. Robert H. 100 200 300 400 Purdy: 2-methoxyestrone; 2-methoxyestradiol; 2-hydroxy-3-methoxyestrone; 2-hydroxy-3-methoxyestradiol; 4-methoxyestrone; and 4-meth PROTEIN (jjg) oxyestradiol. In determining catechol estrogen products of d-equilenin, Chart 1. Linearity of 2- and 4-hydroxylation with estrone at varying hamster kidney microsomalprotein (P)concentrations incubated at 37Â°.Substrateconcen 4-methoxy-d-equilenin and 3-methoxy-4-hydroxy-d-equilenin were pre tration was 50 MM.Inset, Lineweaver-Burk kinetics for hamster kidney ESH. pared from authentic 4-hydroxy-d-equitenin, provided by Dr. R. H. Purdy, Microsomes (150 to 180 jjg) were incubated for 10 min at 37Â°usingestrone as using the ESH assay in the absence of microsomes. The yield was 83:17 substrate (5 to 200 >IM).Points,averageof 2 determinations;oars, S.E.

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Table1 using various steroidal and stilbene estrogen substrates. These ESHactivity and relative catechol estrogen formation from various steroidal and data clearly demonstrate that hamster kidney microsomal ESH stilbene estrogens in kidney microsomes of castrated hamsters was markedly more active than corresponding rat kidney in EstrogenEstrone" activity (pmol/mgprotein/min)3.92 respect to nearly all estrogens studied. With estrone as a sub Â±0.76" strate, catechol formation was more than 19-fold greater in the d-EquileninÂ«-Dienestrol" 2.56 Â±0.291.02 hamster kidney compared to the rat (Chart 2). Both ethynyl Â±0.53 estradici and 4-bromoethynyl estradici when used as substrates Hexestrol" 0.79 Â±0.30 showed substantial but lesser differences (4- to 5-fold) in the Indenestrol Ba 0.71 Â±0.13 170-Estradiof1 0.66 Â±0.17 ability of hamster and rat microsomes to form catechols. On the DES8EquÃlin 0.66 Â±0.200.51 other hand, rat kidney microsomes produced 2.5-fold less cate chols than corresponding hamster microsomal preparations us Â±0.05 ing 17/3-estradiol as a substrate. Substantial differences in cat Ãf-Dienestrol 0.46 Â±0.13 Ethynylestradici4-Bromoethynyl 0.31 Â±0.060.1 echol formation were also found between hamster and rat ESH activities with all stilbene estrogen substrates tested (Chart 3). estradici 9 Â±0.04 Catechol formation using hexestrol and a-dienestrol was nearly Estriol 0.04 Â±0.01 5-fold greater with hamster renal microsomes than in rats, 2-Bromoethynyl estradici 0.02 Â±0.00 2,4-DibromoethynylestradiciESH 0.01 Â±0.00 whereas DES showed a 3.5-fold difference. * Estrogens shown to exhibit potent carcinogenicactivity in the hamster kidney Effect of ANF Treatment on Hamster Renal FSH Activity. when animals were exposed to hormone for 8.7 months (3). 6 Mean Â±S.E.of 4 to 9 individualdeterminations. ESH activity and estrogen catechol formation were assessed in liver and kidney microsomes of hamsters fed a diet containing Table2 0.4% ANF for 3 months. No significant differences were observed ESHratios of 2-hydroxy- and 4-hydroxyestrogensformed from hamster and rat in the ability of ESH to form catechols in hepatic microsomes of kidney microsomes ANF-treated hamsters compared to similar microsomes prepared ofSubstrate8Hamster % of [3H]monomethylethers from untreated animals using different estrogen substrates 2-Hydroxyl- (Chart 4A). Interestingly, a dramatic decline in catechol formation ase:4-hydroxyl- was found in ANF-exposed kidney microsomes compared to product92.0 product8.0 ase productratio12:1 microsomes from untreated hamsters. This decrease of 3.4- to kidney 5.6-fold in ESH activity was evident when 170-estradiol, DES, 170-Estradiol Â±0.7* Â±0.7 Estrone 94.6 Â±0.6 5.4 Â±0.6 19:1 and estrone were used as substrates for the enzyme (Chart 48). d-Equilenin 10.6Â±2.0C 89.4 Â±2.0 0.1:1 EthynylestradiciRat 86.0 Â±2.589.9 14.0 Â±2.510.1 6:19:17:1 DISCUSSION kidney Steroidal and stilbene estrogens have been shown to be 170-Estradiol Â±1.5 Â±1.5 Estrone 88.5 Â±2.8 11.5 Â±2.8 carcinogenic in the hamster kidney (9,10). The finding that these d-Equilenin 6.5 Â±0.9C 93.5 Â±0.9 <0.1:1 Ethynyl estradici2-Hydroxy88.8 Â±1.84-Hydroxy11.2 Â±1.8Av. 8:1 " Estrogen substrates (50 ^M) were incubated with either hamster or rat micro 5r somes (150 to 200 iig protein) for 10 min al 37Â°.Results were obtained by comparison with retention times of authentic standards. " Mean Â±S.E.of at least 5 separate determinations. " 2-Methoxy-d-equilenin was not identified from standard and represents the total radioactivity in the other peak. CL oÂ»

Similar low levels of catechol formation were also observed when ^ bromo-substituted ethynyl estradici compounds were used as o substrates, with the possible exception of 4-bromoethynyl estra CL dici which showed slightly higher levels of catechol formation. On the other hand, both the equine estrogens were relatively effective substrates with d-equilenin second to estrone. The relative ratios of 2- to 4-hydroxyestrogen formation of 4 steroidal estrogen substrates are shown in Table 2. These ratios were derived from separation of the monomethyl ethers of cat Lu echol estrogens by HPLC, and their retention times were identical N to those of nonlabeled authentic monomethyl ether standards. Both 17/3-estradiol and estrone produced largely 2-hydroxy prod ucts when used as substrates for ESH with either hamster or rat kidney microsomes. A slight decrease in 2-hydroxylation and concomitant increase in 4-hydroxylation was observed in ham EL. ster kidney microsomes when ethynyl estadiol was the substrate. E, I70-E2 EE2 4BrEE2 2BrEE2 In contrast, d-equilenin produced largely 4-hydroxyestrogen Chart 2. ESH activity in kidneys of castrated hamsters P) and rats (D) using products using renal microsomes prepared from either species. various steroidal estrogens as substrates. Renal microsomes (150 to 200 (/g Comparison of ESH Activities in Both Hamster and Rat protein) were incubated with 50 Â»IMestrone(Â£t),170-estradtol(17$-EÂ¡),ethynyl estradici (EÂ£a),4-bromoethynylestradici (4BrEE2).and2-bromoethynyl estradici Kidney Microsomes. Hamster and rat microsomes were com (2BrEE,)for 10 min at 37Â°Columnsof ESH activity, expressed in pmol/mg protein pared for their ESH activities and ability to produce catechols, (P)/min,mean of at least 5 separate determinations;bars, S.E.

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â€”roo*-tÂ»o-1n with that of Liehr and coworkers (13,14), who reported appre ciable carcinogenic activity of tetrafluoro-DES and reduced carcinogenicity of 2-fluoroestradiol in the hamster kidney. These ENZYMEACTIVITY(pmol/mgP/min) investigators concluded from their findings that the metabolic activation of estradici and DES proceeded via different pathways and that o-catechol formation did not appear to be a significant event in the induction of these carcinomas by stilbene estrogens. However, recent evidence in our laboratory strongly indicates that significant defluorination occurs with fluoro-substituted es- tradiols when exposed to hamster liver microsomes in vitro (6). Enzymatic replacement of fluorine by a hydroxyl group has been demonstrated for a variety of compounds (1). Our finding there fore compromises the use of fluoro-substituted estrogens in assessing the significance of either the hormonal or carcinogenic activities of such substituted estrogenic compounds. Moreover, the observation that both a-dienestrol and hexestrol exhibit potent carcinogenic activity in the hamster (10) also supports TT||ii our contention that steroidal and stilbene estrogens may have carcinogenic intermediates in common, as p-quinone metabolites have not been shown for these stilbene estrogens (16,18). Consistent with the view for a role for the catechol pathway in E, Â«-DIES HEX DES /3-DIES estrogen carcinogenesis is our finding that ESH activity in ham Chart 3. ESH activity in kidneys of castrated hamsters (C3)and rats (D) using ster kidney microsomes was substantially greater than in rat various stilbene estrogens as substrates. Renal microsomes (150 to 200 ^g protein) kidney microsomes, and consequently, more catechol interme were incubated with SO pM ,,-dienestrol (,,-D/ES). hexestrol (HEX). DES, and ÃŸ- dienestrol (ÃŸ-DIES).Estrone (Â£,)at the same concentration is included as a diates were formed in the hamster compared to the rat, a species reference. Columns of ESH activity, expressed in pmol/mg protein (P)/min, mean which is not susceptible to estrogen-induced renal tumorigen- of at least 5 individual determinations: oars. S.E. esis. Moreover, the 3.5- to nearly 6-fold decline in catechol formation found in kidneys but not in livers of ANF-exposed hamsters, using various steroidal and stilbene estrogen sub strates, is also consistent with this view because of the reported inhibitory action of ANF on renal tumor induction by estrogens (7). In this regard, Metzler (17) has provided evidence recently that ANF-treated male hamsters produced smaller amounts of urinary hydroxylated [14C]DES metabolites and increased quan > tities of j3-dienestrol. The data presented herein also suggest a E 4 rationale for the weak carcinogenic activity of ethynyl estradici, estriol, and 0-dienestrol as these estrogens exhibit relatively low levels of o-catechol formation in the hamster kidney when used as substrates. On the other hand, the considerable carcinogenic activity of the more distal DES metabolites, indenestrol B/A, may be related to the levels of o-hydroxylation comparable to those E, E-E, OES found for either DES or 17/8-estradiol. Although the equine Chart 4. Effect of ANF treatment on liver and kidney ESH activity and catechol estrogen, d-equilenin, has a 4-fold-greater ability to produce estrogen formation in castrated male hamsters. ESH activity was assessed in catechols compared to either DES or 17/3-estradiol, the catechol hepatic (A) and renal (B) microsomes of untreated (D) and 0.4% ANF-exposed hamsters for 3.0 months (G). Liver and kidney microsomes (120 to 150 //9 protein) products formed by this hormone were predominately 4-hydrox- were incubated with 50 itu of the following substrates: estrone (Â£,);17/3-estradiol ylated. This finding is in contrast to the other steroidal estrogens (Â£2);ethynyl estradici (Â£-Â£2);andDES. ESH activity is expressed in pmol of catechol estrogen formed/mg protein (P)/min. Columns, mean of 4 to 5 separate determi studied and is in agreement with the result of Purdy ef al. (22) nations; oars, SE. using baboon liver microsomes. It is unlikely, however, that the high levels of 4-hydroxylation necessarily contribute significantly estrogens exhibit substantial ability to form catechols when used to the weak carcinogenicity observed for d-equilenin in the as substrates with renal microsomes derived from hamsters may hamster kidney, since we have found that 4-hydroxyestradiol be pertinent to their carcinogenic activity. For instance, stilbene possesses appreciable carcinogenic activity at this organ site.4 estrogens, such as a-dienestrol and hexestrol, shown previously Similarly, estrone, which is not as potent as either DES or 17/3- to possess potent carcinogenic activity in the hamster kidney, estradiol in effecting transformation of the hamster kidney and undergo o-hydroxylation to about the same extent as either DES yet exhibited the greatest ability to form catechol estrogens in or 17/3-estradiol. These data suggest that both stilbene and this tissue, also indicates that other aspects of estrogen metab steroidal estrogens may elicit renal cell transformation in the olism as well as estrogenic potency contribute importantly to the hamster by metabolic intermediates which arise from a common pathway (Chart 5). This suggestion, however, is not in agreement 4 J. J. Li, S. A. Li, and J. K. Klicka, unpublished observations.

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STEROIDAL ESTROGENS

Chart 5. Commonmetabolic pathways for steroidaland stilbene 1â€” Estradici 2,3 qumont 1â€” Estradici 2,3 Â»Â«mi- â€”' 2-Hydroiycstrodiol estrogens in the formation of catechol estrogens. The 4-hydroxylation pathway for steroidalestrogens is not shown but is essentially similar to the 2-hydroxylatkxi pathway indicated. STIL BE NE ESTROGENS

ultimate carcinogenic expression of any given natural or synthetic roles for hormonal and carcinogenic activities. Arch. Toxico)., 55: 110-118, 1984. estrogen. 9. U, J. J., U, S. A., Klicka, J. K., and Metzler, M. Further carcinogenicactivity studies in the Syrian hamster kidney: correlations with catechol estrogen formation. Proc. Am. Assoc. Cancer Res., 25: 83,1984. ACKNOWLEDGMENTS 10. Li. J. J., Li, S. A., Klicka, J. K., Parsons, J. A., and Lam, L. K. T. Relative carcinogenicactivity of various synthetic and natural estrogens in the Syrian We are grateful for the technicalassistance of Thomas Hennesseyand Theresa hamster kidney. Cancer Res., 43: 5200-5204,1983. Tom in these investigations. We are indebted to Dr. Robert H. Purdy, Department 11. U, S. A., Lam, L. K. T., and Li, J. J. Effect of steroid hormone treatment on of Organic Chemistry, Southwest BiomÃ©dicalFoundation,San Antonio, TX, for aryl hydrocarbon hydroxylase activity in the Syrian hamster kidney. Biochem. providing estrogen substrates and standards and for helpful suggestions. Pharmacol.,32: 2847-2850,1983. This paper is dedicated to our friend and colleague. Dr. George M Krause, 12. Li. S. A., and Li, J. J. Changesin estrogen receptor levelsduring DES-induced Director of Research and Develoment, Copley Pharmaceutical.Inc., Boston, MA, hepatocarcmogenesisin the Syrian hamster fed a-naphthoflavone.J. Steroid Biochem., 75: 387-392,1981. for Ns invaluableand unstinting support over the years. 13. Liehr, J. G. 2-Fluoroestradiol. Separation of estrogenicity and carcmogenicity. Mol. Pharmacol.. 23: 278-281,1983. 14. Liehr,J. G., Ballatore,A. M., McLachlan,J. A, and Sirbasku, D. A. Mechanism REFERENCES of diethylstilbestrolcarcinogenicityas studied with the fluorinated analogueÂ£- 3',3",5',5"-tetrafluorodiethylstilbestrol. Cancer Res., 43: 2678-2682,1983. 1. Buening, M. K., Levin, W., Wood, A. W., Chang, R. L., Agranat, I., Rabinovitz, 15. Lowry, 0. H., Rosebrough. N. J., Farr, A. L., and Randall, R. J. Protein M . Buhler, D. R., Mah, H. D., Hernandez,O., Simpson, R. B., Jenna. D. M., measurements with the Folin phenol reagent. J. Bid. Chem., 793: 265-275, Conney, A. H., Miller, E. C., and Miller, J. A. Fluorine substitution as a probe 1951. for the rote of the 6-positton of benzo(a)pyrene in carcmogenesis J. Nati. 16. Metzter, M. Studies on the mechanismof carcinogenicity of diethylstilbestrol: Cancer Inst., 77: 309-314,1983. role of metabolic activation. Food Cosmet. Toxicol., 79: 611-615,1981. 2. Fishman,J. Aromatic hydroxylation of estrogens. Annu. Rev. Physiol.,45:61- 17. Metzter,M. Metabolismof stilbeneestrogensand steroidalestrogens in relation 72, 1983. to carcinogenicity.Arch. Toxicol., 55: 104-109, 1984. 3. Holtzman, J. L., Rumack, B. H., and Erickson, R. R. Spectrophotometric and 18. Metzler, M., and McLachlan, J. A. Oxidative metabolism of the synthetic radiometrie studies on the m vivo binding of phÃ©nobarbitaltohepatic micro- estrogens hexestrol and dienestroiindicatesreactive intermediates.Adv. Exp. somes. Arch. Biochem. Biophys. 773: 710-719,1976. Med. BkÂ».,736.829-837,1981. 4. Homing, E. C., Thenot, J. P., and Helton, E. D. Toxic agents resulting from 19. Nelson, S. D., Mitchell, J. R., Dybing, and Sasame, H. A. Cytochrome P-450- the oxidative metabolism of steroid hormones and drugs. J. Toxicol Environ. mediatedoxidation of 2-hydroxyestrogensto reactive intermediates.Biochem. Health,4:341-361,1978. Biophys. Res. Commun., 70:1157-1165,1976. 5. Kirkman, H. Estrogen-induced tumors of the kidney in Syrian hamsters. Nati. 20. Paul, S. M., Axelrod, J., and Diliberto, E. J., Jr. Catechol estrogen forming Cancer Inst. Monogr., 1:1-59,1959. enzyme of the brain: demonstration of a cytochrome P-450 monooxygenase. 6. Li, J. J., Klicka, J. K., Purdy, R. H., and Li, S. A. Catechol formation from Endocrinology, 70Ã•:1604-1610,1977. fluoro- and bromo-substituted estradioisin hamsterliver microsomes:evidence 21. Purdy, R. H., GokJzieher,J. W., LeQuesne,P. W., Abdel-Baky, S., Durocher, for deiiuormation. In: Abstracts from the Seventh International Congress of C. K., Moore, P. H., Jr., and Rhim,J. S. Active intermediatesin carcmogenesis Endocrinology, p. 975,1984. In: G. L. Meeriamand M. B. Lipsett (eds.), CatecholEstrogens, pp. 123-140. 7. LI.J. J,. and Li. S. A. Inhibitionof estrogen-inducedrenal tumorigenesisin the New York: Raven Press, 1983. Syrian golden hamster by BHA, ÃŸ-anda-naphthoflavone. Proc. Am. Assoc. 22. Purdy, R. H., Moore, P. H., Jr., Williams,J. W., Goldzieher,J. W., and Paul, S. Cancer Res., 22:11,1981. M. Relative rates of 2- and 4-hydroxyestrogen synthesis are dependent on 8. U, J. J., and Li, S. A. Estrogen-induced tumorigenesis in the Syrian hamster: both substrate and tissue. FEBS Lett., 738: 40-44, 1982.

CANCER RESEARCH VOL. 45 JANUARY 1985 185

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1985 American Association for Cancer Research. Estrogen 2- and 4-Hydroxylase Activity, Catechol Estrogen Formation, and Implications for Estrogen Carcinogenesis in the Hamster Kidney

Sara Antonia Li, John K. Klicka and Jonathan J. Li

Cancer Res 1985;45:181-185.

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