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Effects of Flavonoid Phytochemicals on Cortisol Production and on Activities

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Effects of Flavonoid Phytochemicals on Cortisol Production and on Activities Journal of Steroid Biochemistry & Molecular Biology 80 (2002) 355–363 Effects of flavonoid phytochemicals on cortisol production and on activities of steroidogenic enzymes in human adrenocortical H295R cells Shuji Ohno a, Satoshi Shinoda a, Satoshi Toyoshima a, Hiroyuki Nakazawa b, Tsunehisa Makino c, Shizuo Nakajin a,∗ a Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan b Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan c Department of Obstetrics and Gynecology, School of Medicine, Tokai University, Bohseidai, Isehara City, Kanagawa 259-1143, Japan Received 3 May 2001; accepted 7 December 2001 Abstract Inhibitory effects of flavonoid phytochemicals, flavones, flavonols and isoflavones on cortisol production were examined in human adrenal H295R cells stimulated with di-buthylyl cAMP. In addition, the inhibitory effects of these chemicals on the activity of P450scc, 3␤-HSD type II (3␤-HSD II), P450c17, P450c21 and P45011␤, steroidogenic enzymes involved in cortisol biosynthesis, were examined in the same cells. Exposure to 12.5 ␮M of the flavonoids 6-hydroxyflavone, 4-hydroxyflavone, apigenin, daidzein, genistein and formononetin significantly decreased cortisol production (by 6.3, 69.6, 47.5, 26.6, 13.8 and 11.3%, respectively), and biochanin A significantly decreased cortisol production (by 47.3%) at a concentration of 25 ␮M without any significant cytotoxic effects or changes in cell number. Daidzin, the 7-glucoside of daidzein, did not alter cortisol production by H295R cells at concentrations over 10 ␮g/ml (24 ␮M). Daidzein-induced reduction of cortisol production by H295R cells was not inhibited by the estrogen receptor antagonist ICI 182,780. The flavonoids 6-hydroxyflavone, daidzein, genistein, biochanin A and formononetin strongly and significantly inhibited microsomal 3␤-HSD II activity at concentrations from 1 to 25 ␮M, and I50 values were estimated to be 1.3, 2, 1, 0.5 and 2.7 ␮M, respectively. In addition, these flavonoids significantly inhibited microsomal P450c21 activity at 12.5 and/or 25 ␮M. In addition, 6-hydroxyflavone inhibited activity of microsomal P450c17 and mitochondrial P45011␤ at 12.5 and/or 25 ␮M. Results of Lineweaver–Burk’s plot analysis indicate that daidzein is a competitive inhibitor of the activity of 3␤-HSD II and P450c21. Km and Vmax values of 3␤-HSD II for DHEA were estimated to be 6.6 ␮M and 328 pmol/min mg protein, respectively. Km and Vmax values of P450c21 for progesterone were estimated to be 2.8 ␮M and 16 pmol/min mg protein, respectively. Ki values of 3␤-HSD II and P450c21 for daidzein were estimated to be 2.9 and 33.3 ␮M, respectively. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Adrenocortical H295R cell; Steroidogenesis; Phytoestrogen; Flavonoids; Enzyme inhibition 1. Introduction estrogens to estrogen receptors. Many studies have, there- fore, investigated estrogen receptor binding and subsequent The endocrine system is indispensable in sustaining downstream signaling events. Endocrine-disrupting chem- biological homeostasis. In particular, steroid hormone es- icals are exogenous agents that interfere with synthesis, trogens play an important role in female and male repro- production, transport, binding, action, and elimination of ductive systems and influence growth, differentiation, and the hormones responsible for maintenance of homeostasis, function of many target cells. Recent reports indicate that reproduction, development and/or behavior (White House environmental exposure to endocrine-disrupting chemicals Workshop, January 1997). Inhibition of the biosynthesis or adversely affect human and wildlife reproductive systems production of steroid hormones during fetal, perinatal, and [1]. Many environmental chemicals that are suspected neonatal periods can yield serious irreversible changes in endocrine-disrupting agents display native estrogen-like human and wildlife reproductive systems. structures and exhibit estrogenic activity, and have thus Phytoestrogen flavonoids are plant chemicals that are been dubbed environmental estrogens. Initial estrogenic structurally analogous to estrogen and are known to affect activity is mediated by binding of these environmental estrogenic activity [2]. Leguminous plants generally contain isoflavones, and soybeans are also rich in isoflavones such ∗ Corresponding author. Tel.: +81-3-5498-5775; fax: +81-3-3787-0036. as daidzein, genistein, glycitein and their glucosides, which E-mail address: [email protected] (S. Nakajin). are consumed in traditional diets containing soy-derived 0960-0760/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-0760(02)00021-3 356 S. Ohno et al. / Journal of Steroid Biochemistry & Molecular Biology 80 (2002) 355–363 foods [3,4]. It has been reported that serum concentration were maintained in D-MEM/F-12 medium (1:1 mixture of total phytoestrogens (daidzein and genistein) is greater in of Dulbecco’s-modified Eagle’s and Ham’s F-12 medium) Japanese (0.16–0.89 ␮M, mean 0.4 ␮M) than Finnish men containing pyridoxine hydrochloride, l-glutamine, and (7–25 nM, mean 12.5 nM) [5], and reach still higher levels 15 mmol/l HEPES supplemented with insulin (6.25 ␮g/ml), in infants who consume large amounts of soy-derived foods transferrin (6.25 ␮g/ml), selenium (6.25 ng/ml), and linoleic (2.2–7 ␮M, mean 3.8 ␮M) [6]. The estrogen-like activity acid (5.35 ␮g/ml). Antibiotics (penicillin: 50 IU/ml and of phytoestrogens has been determined by assaying prolif- streptomycin: 50 ␮g/ml), 1% ITS Plus (Collaborative Re- eration of human breast cancer cells, competitive binding search, Bedford, MA), and 2% ultrose G (Sepracore, assays with estrogen receptor or transient gene expression France) were also added. Cells were maintained as mono- assays [7–13]. However, these reports did not consider the layer cultures in 75 cm2 flasks at 37 ◦C in an atmosphere effects of phytoestrogens on biosynthesis or production of of 5% CO2–95% air. Cells were subcultured onto 24-well steroid hormones. The aim of the present study was to in- dishes for experiments. After 48 h, medium was removed vestigate the inhibitory effects of phytoestrogen flavonoids and cells were treated as described below. on steroidogenesis. The recently developed human adreno- cortical tumor cell line H295R can secrete steroids charac- 2.3. Stimulation of steroid production and analysis teristic of the three adrenocortical zones and, thus, appear to of steroids be pluripotent [14–17]. Therefore, we used adrenocortical H295R cells as a model of human steroidogenic cells to in- Cells subcultured onto 24-well plates were maintained vestigate the inhibitory effects of flavonoid phytochemicals for 24 h in D-MEM/F-12 medium containing 1% ITS Plus, on steroidogenesis. 0.01% bovine serum albumin (Bovuminar®: Intergen, New York) and antibiotics. Medium was then renewed (0.5 ml per well) and phytoestrogen flavonoid chemicals dissolved 2. Materials and methods in ethanol were added. The final concentration of alco- hol solvents in the assay mixture did not exceed 1% and 2.1. Chemicals was confirmed to not obstruct cortisol production. At the same time, dibutyryl cyclic AMP (1 mM) was added to the 5-Hydroxyflavone, 6-hydroxyflavone, 6-methoxyflavone, medium to stimulate steroidogenesis because the H295R 7-hydroxyflavone, 4 -hydroxyflavone, 4 ,5,7-trihydroxyfla- cell line lacks sensitivity to ACTH treatment [15]. To assess vone (apigenin), 7-hydroxy-4 -methoxyisoflavone (for- the effects of phytoestrogen flavonoids, cells were exposed mononetin), and epidermal growth factor (EGF, human for 48 h except as otherwise noted. Cortisol content of each recombinant) were purchased form Funakoshi Co. Ltd. well was determined by radioimmunoassay with the DPC (Tokyo, Japan). Dibutyryl cAMP, 4 ,7-dihydroxyisoflavone cortisol kit (Diagnostic Product Corporation, Los Angels, (daidzein), 4 ,7-dihydroxyisoflavone–7-glucoside (daidzin) CA). Cytotoxicity of each chemical was tested using the 4 ,5,7-trihydroxyisoflavone (genistein), 4 ,5,7-trihydroxyiso- CytoTox96 non-radioactive cytotoxicity assay kit that deter- flavone–7-glucoside (genistin) and 5,7-dihydroxy-4 -metho- mines LDH activity (Promega Corp., Madison, WI). Cells xyisoflavone (biochanin A) were purchased form Wako were washed in phosphate buffered saline and solubilized Pure Chemical Industries Ltd. (Tokyo, Japan). Radioac- in Tris–HCl (50 mM, pH 7.4) containing NaCl (150 mM), 14 tive [4- C] steroids, cholesterol (1887 MBq/mmol), sodium dodecyl sulfate (SDS, 1%), EGTA (5 mM), MgCl2 dehydroepiandrosterone (2.05 GBq/mmol), progesterone (0.5 mM), MnCl2 (0.5 mM) and phenylmethylsulfonylflu- (2.05 GBq/mmol) and deoxycorticosterone (2.22 GBq/mmol) oride (PMSF, 0.2 mM). The protein content of each sam- were purchased from New England Nuclear Corp. (Boston, ple was determined by BCA assay (Pierce Chemical Co., + MA). Non-radioactive steroids, NAD , NADPH, glucose-6- Rockford, IL). phosphate, glucose-6-phosphate dehydrogenase and 3,3,4, 5,7-pentahydroxyflavone (quercetin) were purchased from 2.4. Preparation of H295R mitochondrial Sigma–Aldrich, K.K., Japan (Tokyo, Japan). Estrogen re- and microsomal fractions ceptor antagonist, ICI 182,780 was purchased form Nacalai Tesque, Inc. (Kyoto, Japan). All reagents were of the high- Confluent H295R cells were treated with dibutyryl cAMP est commercially available grade. Adrenodoxin, the redox (1 mM) and EGF (10 ng/ml) for 24 h. Cells were washed partner of cytochrome P450s in adrenal mitochondria, was three times with ice-cold PBS, harvested and collected by purified from bovine adrenal gland following the method of centrifugation at 350 × g for 5 min. All procedures were Kimura et al. [18]. carried out at 4 ◦C or on ice. Cells (1–2.1 g wet weight) were homogenized in a Dounce tissue grinder (Wheaton, 2.2. Cell culture Millville, NJ) in 3 ml of ice-cold 0.25 M sucrose contain- ing 5 mM potassium phosphate (pH 7.4), 0.5 mM EDTA, H295R cells were generously provided by Prof. J.I. Mason 1 mM DTT and 0.1 mM PMSF (buffer A), then centrifuged (University of Edinburgh, Edinburgh, Scotland).
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