Metabolic Activation of Proestrogenic Diphenyl and Related Compounds by Rat Liver Microsomes

298 Journal of Health Science, 49(4) 298–310 (2003) Metabolic Activation of Proestrogenic Diphenyl and Related Compounds by Rat Liver Microsomes Shigeyuki Kitamura,*, a Seigo Sanoh,a Ryuki Kohta,a Tomoharu Suzuki,a Kazumi Sugihara,a Nariaki Fujimoto,b and Shigeru Ohtaa aGraduate School of Biomedical Sciences and bResearch Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi 1–2–3, Minami-ku, Hiroshima 734–8551, Japan (Received April 14, 2003; Accepted April 24, 2003) In this study, liver microsome-mediated activation of diphenyl (DP), diphenylmethane (DPM) and 2,2- diphenylpropane (DPP) to estrogens was demonstrated. These three compounds were negative in estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, they exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the cases of DP and DPM, or of phenobarbital-treated rats in the cases of DP and DPP, in the presence of NADPH. When these compounds were incubated with liver microsomes in the presence of NADPH, monohydroxyl and dihydroxyl derivatives were formed. These hydroxylated metabolites, 4-hydroxydiphenyl, 3-hydroxydiphenyl, 2-hydroxydiphenyl, 4-hydroxydiphenylmethane, 2-(4-hydroxyphenyl)-2-phenylpropane (4-OH-DPP), 4,4′-dihydroxydiphenyl, 4,4′-dihydroxydiphenylmethane and 2,2-bis(4-hydroxyphenyl)propane (bisphenol A), all exhibited estrogenic activity in MCF-7 cells. Binding assay of these hydroxylated compounds with rat uterus estrogen receptor was also positive. These results suggest that the estrogenic activities of DP, DPM and DPP were due to the formation of hydroxylated metabolites by the liver cytochrome P450 system. Key words —–— diphenyl, estrogenic activity, metabolic activation, human breast cancer cell line MCF-7, endo- crine disruption, cytochrome P450 INTRODUCTION exert biological effects. Many so-called xenoestrogens produce a wide variety of toxic ef- Some chemicals in the environment are able to fects in animals. They may be playing a role in the mimic the biological activity of hormones such as increasing incidence of hormonally related cancers sex hormones and thyroid hormone, thereby inter- such as breast cancer and testicular cancer, and other fering with hormone receptor function. These endo- problems of the reproductive system in humans. It crine disrupters include several persistent chlorinated is therefore important to screen environmental con- pesticides, such as kepone, o,p′-dichlorodiphenyl- taminants for estrogenic activity. Their metabolites trichloroethane (o,p′-DDT), dieldrin and methoxy- also need to be identified and screened. We recently chlor, some polychlorinated biphenyl congeners such showed that trans-stilbene, which is the parent com- as polychlorinated diphenyl (PCBs) and dibenzo- pound of diethylstilbestrol, and trans-stilbene ox- dioxins, and industrial compounds used in the plas- ide were not estrogenic, but exhibited a potent es- tics and detergent industries, such as alkylphenols trogenic activity after metabolic activation by a liver and bisphenol A.1–3) Among these compounds, those microsomal oxidation system.4,5) In that report, we which are lipophilic and persistent may be accumu- suggested that the estrogenic activity was due to the lated through the food web, posing a health threat to hydroxylated metabolites, trans-4-hydroxystilbene humans and animals. Endogenous estrogens have and trans-4,4′-dihydroxystilbene, formed by cyto- been shown to have multiple sites of activity and to chrome P450 1A1/2. Furthermore, we demonstrated that styrene oligomers were metabolically activated to estrogens by rat liver microsomes, especially cy- *To whom correspondence should be addressed: Graduate 6) School of Biomedical Sciences, Hiroshima University, Kasumi tochrome P450 2B1. These results demonstrate the 1–2–3, Minami-ku, Hiroshima 734–8551, Japan. Tel.: +81-82- importance of considering proestrogenic potential, 257-5328; Fax: +81-82-257-5329; E-mail: skitamu@hiroshima- when screening for xenoestrogens in the environ- u.ac.jp ment. No. 4 299 Diphenyl (DP) is used as an antifungal agent for Chemical Industry Co. Ltd. (Tokyo, Japan), DPP citrus fruits, and is also used as wrapping paper for (97%) from Aldrich Chemical Co. (Milwaukee, WI, impregnated fruit. Under poor hygienic conditions, U.S.A.), 17β-estradiol (E2; > 98%) from Sigma its production caused the toxicity for workers.7,8) It Chemical Co. (St. Louis, MO, U.S.A.). [17β-2,4,6,7- is known to induce calculi and tumors in the urinary 3H] Estradiol (specific activity = 3.53 GBq/µmol) bladder of rats.9) Diphenylmethane (DPM) is also was purchased from NEN/Perkin Elmer Life Sci- used as a dye carrier and synthetic intermediate, simi- ences (Boston, MA, U.S.A.). larly to DP. DP is metabolized via multiple path- Animals —–— Sprague-Dawley rats (210–230 g, ways, resulting in the production of various deriva- 6 weeks old) were obtained from Japan SLC, Inc. tives, such as conjugates (sulfate and glucuronide) (Shizuoka, Japan). The animals were housed at 22°C of hydroxylated diphenyls. 4-Hydroxydiphenyl, 4,4′- with a 12-hr light/dark cycle, with free access to tap dihydroxydiphenyl and 3,4-dihydroxydiphenyl were water and a standard pellet diet MM-3 (Funabashi identified as urinary metabolites of DP in rats and Farm, Funabashi, Japan). In some experiments, male rabbits.10) Furthermore, 2-hydroxydiphenyl, 2,4′- rats were given intraperitoneal administration of phe- dihydroxydiphenyl and 3-hydroxydiphenyl were nobarbital (80 mg/kg) or 3-MC (25 mg/kg) daily for also identified as in vitro metabolites with liver mi- three days before use. All experiments were con- crosomes of various animals.11) The 2- and 3-hy- ducted in accordance with the Guide for the Care droxylation activities were induced to similar ex- and Use of Laboratory Animals of Hiroshima Uni- tents by 3-methylcholanthrene (MC) pretreatment versity. of the animals. Phenobarbital pretreatment prima- Preparation of Liver Microsomes —–— The livers rily induced 2- and 3-hydroxylation activities, the of male rats were excised from exsanguinated rats latter more dramatically.12) The sulfate conjugate of and immediately perfused with 1.15% KCl. The liv- 4-hydroxydiphenyl is involved in the formation of ers were homogenized in four volumes of the KCl urinary calculi.9) The sulfate conjugate is also re- solution using a Potter-Elvehjem homogenizer. The ported to be a candidate for medicinal treatment of microsomal fraction was obtained from the homo- breast cancer, because it shows a potent inhibitory genate by successive centrifugation at 9000 × g for effect on sulfation of estrogens.13) Ozawa et al.14) re- 20 min and 105000 × g for 60 min. The fraction was ported that 2,5-dihydroxydiphenyl is a cytotoxic me- washed by resuspension in the KCl solution and tabolite. However, the estrogenic activities of DP and resedimentation. The pellets of microsomes were related compounds have not been reported. resuspended in the solution to make 1 ml equivalent In the present study, the estrogenic activities of to 1 g of liver. Protein contents in the liver microso- DP, DPM and 2,2-diphenylpropane (DPP) were ex- mal preparations were determined by the method of amined in the presence or absence of a liver microso- Lowry et al.15) mal oxidase system by employing the estrogen-re- Cell Culture —–— MCF-7 cells were maintained in sponsive element (ERE)-luciferase reporter assay in Minimum Essential medium (MEM; Sigma Chemi- MCF-7 cells. We demonstrate that DP, DPM and cal Co.) containing penicillin and streptomycin with DPP exhibit estrogenic activities after activation to 5% fetal bovine serum (LiGibco/Invitrogen Co., their hydroxylated products by rat liver microsomal Carlsbad, CA, U.S.A.). mixed function oxidase. Assay of Estrogenic Activities of DP and Related Compounds —–— Assay of estrogenic activity was performed according to the previously reported MATERIALS AND METHODS method.6) Briefly, for ERE-luciferase reporter assay using MCF-7 cells, transient transfections were per- Chemicals —–— DP (> 99%), 4-hydroxydiphenyl formed using Transfast™ (Promega Co., Madison, (> 99%), 3-hydroxydiphenyl (> 95%), 2-hydroxy- WI, U.S.A.), with p(ERE)3-SV40-luc and pRL/CMV diphenyl (> 98%), DPM (> 99%), 4-hydroxy- (Promega Co.) as an internal standard. Twenty-four diphenylmethane (> 98%), 4,4′-dihydroxydiphenyl hours after addition of the sample, the luciferase (> 99%), 4,4′-dihydroxydiphenylmethane (> 97%), activity was measured with a Dual Luciferase assay 2-(4-hydroxyphenyl)-2-phenylpropane (4-OH-DPP, kit™ (Promega Co.). > 98%) and 2,2-bis(4-hydroxyphenyl)propane For the assay of the metabolites produced from (bisphenol A, > 99%) were obtained from Tokyo proestrogens, substrates (0.1 µmol) were incubated 300 Vol. 49 (2003) with 0.1 ml of rat liver microsomes in the presence to 100 mg liver wet weight (1.6–2.1 mg protein) in of 1 µmol of NADPH for 30 min in a final volume a final volume of 1 ml of 0.1 M K,Na-phosphate of 1 ml of 0.1 M K,Na-phosphate buffer (pH 7.4). buffer (pH 7.4). The incubation was performed at After the incubation, the mixture was extracted with 37°C for 20 min. After incubation, 0.1 ml of 2 N HCl 5 ml of ethyl acetate and evaporated to dryness. The and 0.1 µmol of 4-hydroxydiphenylmethane or phe- residue was dissolved in 1 ml of ethanol and an ali- nothiazine as an internal standard were added and quot was used for the estrogenic activity assay. The the mixture was extracted with 5 ml of diethyl ether total concentration of the substrate and its metabo- in the case of DP. In the case of DPM or DPP, lites was calculated from the original amount of the 0.1 µmol of phenothiazine or trans-4-hydroxy- substrate. stilbene as an internal standard was added and the Competitive Binding Assay to Estrogen Recep- mixture was extracted with 5 ml of ethyl acetate. The tor —–— Rat uterus was homogenized with extract was evaporated to dryness, the residue was TEGDMo buffer (1 mM disodium EDTA, 1 mM dissolved in 0.1 ml of methanol, and an aliquot (5 µl) dithiothreitol, 10 mM sodium molybdate, 10%(v/v) was analyzed by high-performance liquid chroma- glycerol and 10 mM Tris–HCl, pH 7.4).

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