Genistein administration decreases serum corticosterone and testosterone levels in rats

Life Sciences 74 (2003) 733 – 742 www.elsevier.com/locate/lifescie

Genistein administration decreases serum corticosterone and testosterone levels in rats Shuji Ohno a,*, Yonako Nakajima a, Koichi Inoue b, Hiroyuki Nakazawa b, Shizuo Nakajin a a

Department of Biochemistry, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan b Department of Analytical Chemistry, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan Received 29 January 2003; accepted 11 June 2003

Abstract The phytochemical flavonoid genistein has been shown to act as a potent competitive inhibitor of human adrenocortical 3h-hydroxysteroid dehydrogenase and cytochrome P450 21-hydroxylase activities in vitro [J. Steroid Biochem. Molec. Biol. 2002; 80: 355– 363]. In the present study, we evaluated the effects of large amounts of genistein continuously administered to weanling rats, particularly on steroidogenesis at the pubertal stage in vivo. Serum concentrations of free and total genistein were significantly higher in the 40 mg/kg genistein administration group when compared with the control group. In genistein administered rats, adrenal weight was significantly higher. Furthermore, a clear expansion of cells was observed in hematoxylin and eosin stained tissue at the zona fasciculata and zona reticularis of the adrenal cortex. However in the testis, no differences in weights or histologic changes were observed. Serum corticosterone concentration significantly decreased to 50% of control levels by 40 mg/kg genistein administration and testosterone also tended to decrease with this dose of genistein. On the other hand, although serum follicle stimulating hormone was unchanged, adrenocorticotropic hormone and luteinizing hormone levels increased with genistein administration. These results suggest a significant effect of genistein on steroidogenesis in the adrenal gland and testis of rats, and this effect appeared to be more evident on steroid production in adrenals than in testis in vivo. D 2003 Elsevier Inc. All rights reserved. Keywords: Genistein; Isoflavone; Phytochemical; Phytoestrogen; 3h-hydroxysteroid dehydrogenase; Steroidogenersis

* Corresponding author. Tel.: +81-3-5498-5777; fax: +81-3-5498-5776. E-mail address: [email protected] (S. Ohno). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0024-3205(03)00950-0

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Introduction Soybeans contain large amounts of the isoflavones daidzein and genistein and their glycoside forms (Price and Fenwick, 1985; Franke and Custer, 1994; Franke et al., 1995, 1998). These isoflavones have a weak estrogenic activity and are thus called phytoestrogens (Knight and Eden, 1996; Kuiper et al., 1997; Lamartiniere et al., 1998; Setchell and Cassidy, 1999). Among these isoflavones, genistein is well known as a tyrosine kinase inhibitor (Akiyama et al., 1987). Several recent epidemiological studies have reported that Japanese and other individuals of Oriental origin, who tend to have higher daily intake of soy products, showed a significantly lower risk for hormone dependent cancers such as prostate cancer in men and/or breast cancer in women when compared to Westerners (Armstrong and Doll, 1975; Messina et al., 1994; Adlercreutz et al., 1987; Lu et al., 1996, 2000; Kumar et al., 2002). Another study reported that plasma genistein levels in Japanese were higher than those in Westerners (Adlercreutz et al., 1993). Rodent studies also support the hypothesis that soy-isoflavone diets exert a protective effect against prostate cancer-cell growth (Zhang et al., 1997; Makela et al., 1995; Dalu et al., 1998; Landstrom et al., 1998; Bylund et al., 2000; Choi et al., 2000). Furthermore, there is a report that genistein exhibits beneficial effects on bone tissue, protecting against bone loss under estrogen-deficient conditions in mice (Ishimi et al., 2000). Based on these studies, isoflavone began to attract substantial attention from the public. On the other hand, Mesiano et al. reported that genistein and daidzein suppressed cortisol synthesis and stimulated dehydroepiandrosterone (DHEA) production using primary cultured fetal and postnatal adrenocortical cells, and that these phytochemicals did not affect the expression of steroid-metabolizing enzymes while specifically inhibiting cytochrome P-450 21-hydroxylase (P450c21) activity (Mesiano et al., 1999). Experiments using human adult adrenal microsomes (Byrne et al., 1986) and human adrenocortical cartinoma cells (H295R) (Gell et al., 1998) demonstrated that estrogens inhibit cortisol production by specifically inhibiting 3h-hydroxysteroid dehydrogenase coupled with D4–D5 isomerase (3h-HSD) activity. Our previous study found that daidzein and genistein, at relatively high concentrations but exhibiting no cytotoxic effects, strongly reduce cortisol production in H295R cells after stimulation with dibutyryl cAMP (db-cAMP). However, the gluconide forms (daidzin and genistin) have no effect on cortisol production. We further identified that this reduction results in the inhibition of 3hHSD and in a slight inhibition in P450c21 activity (Ohno et al., 2002). Although P450c21 is fundamental enzyme that produces a series of adrenocortical hormones such as cortisol, 3h-HSD, which is strongly inhibited by genistein, is important not only for adrenocortical hormone production but also for sex hormone synthesis. Therefore, in order to evaluate the effects of genistein in vivo, we investigated whether continuous administration of large amounts of genistein to weanling rats would affect steroidogenesis at the pubertal stage.

Materials and methods Chemicals and Animals Genistein, acetonitrile for HPLC and polyethylene glycol 400 (PEG 400) were purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). Dimethyl sulfoxide (DMSO) was purchased from SigmaAldrich Japan K.K (Tokyo, Japan) and h-glucuronidase was purchased from Fluka Chemie AG (Buchs,

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Switzerland). All other chemicals used were of the best commercially available grade. Injectable sodium thiopental was purchased from Tanabe Seiyaku Co. Ltd. (Osaka, Japan). Administration of genistein Male Wister rats (age; 21 days) were obtained from Oriental Yeast Co., Ltd. (Tokyo, Japan) and were given normal food, free access to water and kept under normal light conditions. Thirty rats were randomly divided into three groups (n = 10 each): 1) control (vehicle only), 2) 10 mg/kg genistein treatment or 3) 40 mg/kg genistein treatment. At age 21 days, there were no significant differences in body weight between the three groups. Genistein was dissolved in DMSO and prepared as 10% DMSO/ 90% PEG 400 solutions for administration. High or low dose genistein or vehicle (10% DMSO/90% PEG 400) was subcutaneously administered in a region of the back daily for three weeks. At age 6 weeks, whole blood was collected from the abdominal aorta under thiopental anesthesia, and the testes, prostate and adrenals were collected and weighed. Blood was centrifuged at 1600  g for 15 min at 4 jC and serum was collected and stored at 80 jC until use in hormones level assays. Assay for serum genistein levels Serum genistein levels were measured by the liquid chromatography–mass spectrometry (LC-MS) method. Serum samples were pretreated by the solid phase extraction (SPE) method using SPE cartridges (Shodex SPEC EDS-1, 25 mg/mL). Serum samples (500 AL) were buffered with 20 mM ammonium acetate (pH 6.8) and were subjected to glucuronidase treatment for enzymatic glucuronic acid deconjugation with 0.89 units of glucuronidase at 37 jC for 3 h. Intact and glucuronidase-treated serum samples were diluted with water to a final volume of 1.0 mL and applied to SPE columns, which were equilibrated with 1.0 mL of acetonitrile followed by 1.0 mL of water, and washed with 1.0 mL of water. Retained genistein was then eluted with 3.0 mL of methanol. The eluate was dried under a nitrogen stream and was resuspended in 1.0 mL acetonitrile for genistein concentration determination by LC-MS. LC-MS was performed using a Mightysil RP-18 GP (2  250 mm: 5 Am) reverse-phase column (Kanto Chemical Co., Ltd, Tokyo, Japan) with an Agilent 1100 MSD-SL System linked to an electrospray ionization interface (Agilent Technologies, Palo Alto, CA, USA). Genistein was separated under gradient conditions as follows: 35% acetonitrile (0–15 min), followed by a linear gradient from 35 to 100% (15–20 min), 0.2 mL/min at 40 jC. Electrospray ionization MS was performed at 350 jC with a nitrogen gas stream (12 L/min) at a potential of 3500 V. The m/z 269 ion, which was assigned as the [M–H] of genistein, was detected with a fragmentor voltage of 90 V. Assays for serum hormones levels Serum testosterone concentration was measured using the radioimmunoassay (RIA) kit (DPC total testosterone kit, Diagnostic Product Corporation, CA, USA). Serum corticosterone, adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) concentrations were estimated by the enzyme immunoassay (EIA) kit (corticosterone: Diagnostic Systems Laboratories, Inc., TX, USA; ACTH: Peninsula Laboratories, Inc., CA, USA; LH and FSH: Amersham Biosciences K.K, Tokyo, Japan).

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Statistical analysis Data were analyzed by Student’s t-test. Statistical significance was set at the p < 0.05 level.

Results Changes in serum testosterone level of rats Changes in serum testosterone levels with growth of Wister rats were estimated by testing a small amount of blood taken from the tail. Testosterone concentration at age 21 days was 74.5 F 17.9 pg/mL (n = 9) and that observed at age 6 weeks (43 days) in the control group was 6 times higher than that at age 21 days (455.9 F 89.3 pg/mL; n = 10). This observation confirmed that animals had reached the early stages of puberty. Free and total genistein (free plus glucuronide) concentrations in rat serum Genistein concentrations in rat serum were estimated and expressed as the free genistein concentration, which was associated with intact serum, and the total genistein concentration, which was associated with glucuronidase-treated serum. Free genistein concentration was significantly increased in the 10 mg/kg group when compared with the control group, and was further elevated in the 40 mg/kg administration group. Total genistein concentration also showed a sudden increase in the 40 mg/kg administration group, however, no differences were observed between the 10 mg/kg administration group and the control group. The free genistein serum concentrations were much lower than those of total genistein: about 3.0% in the control group; 5.2% in the 10 mg/kg group; and about 13.7% in the 40 mg/kg group. These results confirm that genistein, when subcutaneously administered, was absorbed and distributed in the blood, and that most of the genistein existed in the glucuronide form (Fig. 1). Effects of genistein administration on body and tissue weights At age 43 days, body weight showed a range of values but no significant differences between the groups were observed (Table 1). Although prostate, testis and adrenal weight varied in each group, adrenal weight was significantly higher in the 40 mg/kg group after data were standardized by individual body weight. Protein content per unit adrenal weight was almost identical in the 40 mg/kg and control groups, indicating that total protein content in the adrenal gland also increased with high-dose genistein treatment (data not shown). However, no significant differences in adrenal weight were observed between the other administration groups, or between any of the administration groups with regard to testis and prostate weight (Table 1). Effects of genistein administration on hormones concentration in rat serum Serum concentrations of corticosterone, which is known to be the main glucocorticoid form in rodents, was clearly reduced in the 40 mg/kg group and testosterone concentration also tended to

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Fig. 1. Serum genistein concentrations obtained from genistein-injected male rats. Genistein concentrations were measured by LC-MS and evaluated as free genistein from intact serum (A) and as total genistein (free plus glucuronide) from glucuronidasetreated serum (B). Details of LC-MS and glucuronidase treatment are described in the Materials and Methods section. Control group was of treated with vehicle only, Genistein 10 and Genistein 40 groups were treated with 10 mg/kg and 40 mg/kg administration, respectively. Values are expressed in ng/mL (mean F S.E.M., n = 10). ** indicates a significant difference with control value p < 0.01.

decrease in this group, but this change was not significant when compared to the control group. In the 10 mg/kg group, no differences were observed in either corticosterone or testosterone concentrations. With regard to tropic hormones, serum LH concentration significantly increased in the 40 mg/kg group and ACTH also tended to increase, but this was not significant when compared to the control group. Serum FSH concentration showed no significant differences in all administration groups (Table 2).

Table 1 Whole body, testis, prostate and adrenal weights of genistein-injected male rats Control Body Weight (g) Testes/Body Weight (mg/g) Prostate/Body Weight (mg/g) Adrenal/Body Weight (mg/g)

215.1 10.52 2.02 0.207

F F F F

5.3 0.10 0.10 0.007

Genistein 10

Genistein 40

225.4 10.09 2.20 0.224

213.6 9.61 1.76 0.259

F F F F

3.6 0.41 0.21 0.016

F F F F

4.8 0.53 0.15 0.016*

Values were obtained from 43-day-old rats. Genistein (10 or 40 mg/kg) or vehicle (control) was subcutaneously injected into rats (n = 10) every day from age 21 days to 42 days. Prostate, testis and adrenal weights are standardized by individual body weight and are expressed as a mean F S.E.M. * Significant difference when compared to control values (P < 0.05).

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Table 2 Serum testosterone, corticosterone, LH, FSH and ACTH concentrations of genistein-injected male rats Control Testosterone (pg/mL) Corticosterone (ng/mL) LH (ng/mL) FSH (ng/mL) ACTH (ng/mL)

469.6 1277.7 27.96 148.9 2.93

F F F F F

63.6 132.3 1.73 19.5 0.22

Genistein 10

Genistein 40

472.3 1251.1 26.63 137.5 1.93

371.2 637.9 36.09 157.5 4.48

F F F F F

72.3 183.6 2.81 11.1 0.21*

F F F F F

59.6 89.1** 2.81* 17.4 1.09

Each value was measured by the methods described in the materials and methods section using sera from 43-day-old rats and are expressed as mean F S.E.M. Genistein (10 or 40 mg/kg) or vehicle (control) was subcutaneously injected into rats (n = 10) every day from age 21 days to 42 days. * and ** significant difference when compared to control values (P < 0.05 and P < 0.01, respectively).

Histological observation of adrenals and testes after genistein administration Adrenal weight, standardized by individual bodyweight, in the 40mg/kg genistein group was significantly increased when compared with that of control group (Table 1), therefore, the effects of

Fig. 2. H – E Stained Adrenal Micrograms. Adrenal tissue was fixed in neutral buffered formalin fixative. Paraffin sections were prepared from the genistein (40 mg/kg) group of at age 6 weeks (A,  400; C,  40), and from control rat (B,  400; D,  40). Cap: capsule; Cor: cortex; Med: medulla; Z.G.: zona glomerulosa; and Z.F.: zona fasciculata.

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genistein on the adrenal and testicular tissues were investigated histologically. The results indicated a clear expansion of cells in hematoxylin and eosin stained tissue at the zona fasciculata and zona reticularis of the adrenal cortex in the 40 mg/kg group (Fig. 2), however, no difference was observed in testicular tissue (data not shown).

Discussion Genistein is also known as phytoestrogen, due to its weak estrogen-like activity (Knight and Eden, 1996; Kuiper et al., 1997; Lamartiniere et al., 1998; Setchell and Cassidy, 1999). Of the various soybeanderived isoflavonoids, daidzein and its gluconide form, daidzin, are the most abundant, followed closely by genistein and its gluconide form, genistin (Nakamura et al., 2000). Nakamura et al. analyzed the isoflavone content of soybeans and estimated that the Japanese daily intake of isoflavonoids from soybeans and soybean-based processed foods was 27.8 mg/day (daidzein: 12.02 mg; glycitein: 2.30 mg; and genistein: 13.48 mg) (Nakamura et al., 2000). We decided to use genistein in the present study in order to investigate the effect of isoflavones in vivo. Oral administration is thought to lead to the highest blood concentrations of genistein because most of the ingested genistein shows entero-hepatic circulation (Adlercreutz et al., 1987). However, we employed subcutaneous administration because dosage can be more strictly regulated and the effects of a single isoflavone can be evaluated. This administration method was slightly modified from that used in a study documenting that genistein exhibits beneficial effects on bone tissue, protecting against bone less in ovariectomized mice (Ishimi et al., 2000). The results of present study showed that the levels of free and total genistein in blood for the 40 mg/kg group were higher when compared to the control group, thus confirming that subcutaneously administrated genistein is absorbed into the blood. Several reports have investigated isoflavone concentration in blood. Adlercreutz et al. reported that the total phytoestrogen (genistein and daidzein) concentration in human serum is greater in Japanese men (0.16–0.89 AM; mean: 0.4 AM) than in Finnish men (7–25 nM; mean: 12.5 nM) (Adlercreutz et al., 1993), and reached higher levels in infants who consumed large amounts of soy-derived foods (2.2–7 AM; mean: 3.8 AM) (Setchell et al., 1997). Recently, Lewis et al., reported that human plasma levels of genistein were 106–356 nM in adult males who consume Trinovin Tab, which contains 40 mg of isoflavones, on a daily basis (Lewis et al., 2002). Plasma genistein concentrations in rat were also reported by Weber et al., who fed the rats a phytoestrogen-rich diet containing about 600 Ag/g isoflavones and found that rats consuming 13.2 mg phytoestrogens per day showed plasma genistein concentrations of 1.48 AM (Weber et al., 2001). In the present study, free and total genistein concentrations in serum were 0.02 and 0.14 AM, respectively, which demonstrated that there were no marked differences with those levels in healthy humans. Genistein and daidzein are known to suppress cortisol synthesis and stimulate DHEA production by inhibiting P450c21 activity without altering tyrosine kinase activity in human adrenal primary cultured cells (Mesiano et al., 1999). Our previous results demonstrated that genistein suppressed cortisol synthesis, and that this was not mediated by estrogen receptors, but by direct inhibition of 3h-HSD activity together with P450c21 activity, though the inhibitory effects on P450c21 were less than those on 3h-HSD (Ohno et al., 2002). The inhibitory effects of genistein on 3h-HSD activity can be supported by the observation that several flavonoids inhibit the 3h-HSD activity of bovine adrenal short-chain alcohol dehydrogenase (Wong and Keung, 1999). It was also shown in a study using human placental

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microsomes that isoflavonoids, as well as other compounds that possess a phenolic B ring in the 3 position of the pyran ring, preferentially inhibit 3h-HSD and/or 17h-HSD activities (Le Bail et al., 2000). 3h-HSD is an essential enzyme in steroidogenesis, and is required not only for adrenocortical hormone production, but also for sex hormone production. If genistein affects sex hormone production both in vitro and in vivo, then it is possible that they have some impact in reproduction and differentiation stages. In fact, an in vivo study recently clarified that when a high-phytoestrogen diet was fed to SD rats, plasma testosterone concentration was suppressed (Weber et al., 2001). We therefore administered large daily amounts of genistein to male rats immediately after weaning in order to determine the effects of genistein on the production of testosterone and corticosterone, which is the main rat glucocorticoid, during puberty. The results obtained in the present study showed that serum corticosterone concentration significantly decreased in the 40 mg/kg group. It is believed that the increase in serum ACTH concentration was accompanied by a decrease in corticosterone concentration through a feedback effect. It can also be inferred that released ACTH from the anterior pituitary gland led to increased total protein content in the adrenal gland as well as increased adrenal gland weight overall, which was observed as cell expansion in the zona fasciculata and the zona reticularis areas on HE-stained micrograms. In contrast, the weight of the testes did not change at all. Testosterone concentration tended to decrease, but this was not significant, while serum LH concentration increased significantly. These phenomena can also be explained by the feedback effects of the testosterone-LH relationship, similar to the events observed in the corticosterone-ACTH relationship. However, genistein did not affect FSH at all. FSH does not generally target Leydig cells, which are involved in androgen production, but does target Sertoli cells, which are involved in spermatogenesis. In the present report, we focused on serum testosterone and corticosterone concentrations. However, our results suggest that determining testosterone concentration in testicular tissue as well as in serum might provide valuable insight into the effects of phytoestrogens, such as genistein, in vivo. Our results suggest that large amounts of genistein effect steroidogenesis in the adrenals and testes, but exert a greater effect on the adrenal glands. Furthermore, excessive intake of genistein may not only exhibit beneficial effects, such as lowering the risk of cancer and/or protecting against osteoporosis, but may also affect hormonal balance. Therefore, we are very interested in further investigating human cases in more detail, particularly the correlations between isoflavone concentration and several steroid hormone levels in blood between Japanese and Western population groups.

Conclusion In conclusion, administration of large amounts of genistein affects hormone production in the adrenal glands and testis of rats in vivo, particularly influencing serum corticosterone and LH concentrations, and this effect was more evident in the adrenal gland than in the testes.

Acknowledgements This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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