Inhibition of human and rat 11β-hydroxysteroid dehydrogenases activities by bisphenol A

Toxicology Letters 215 (2012) 126–130

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Inhibition of human and rat 11␤-hydroxysteroid dehydrogenases activities by bisphenol A Jingjing Guo a,1 , Xiaohuan Yuan b,1 , Li Qiu a , Weiliu Zhu a , Chaonan Wang b , Guoxin Hu c , Yanhui Chu b , Leping Ye a , Yunfei Xu d,∗∗ , Ren-Shan Ge a,∗ a

The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000, PR China Heilongjiang Key Laboratory of Anti-fibrosis Biotherapy, Mudanjiang Medical University, Heilongjiang, PR China c Department of Pharmacology of School of Pharmacy, Wenzhou Medical College, Wenzhou, Zhejiang 325000, PR China d Department of Urology, The Affiliated 10th People’s Hospital of Tongji University, Shanghai, PR China b

h i g h l i g h t s  BPA inhibited 11␤-hydroxysteroid dehydrogenases.  BPA selectively inhibited type I 11␤-HSD.  BPA altered glucocorticoid metabolism.

a r t i c l e

i n f o

Article history: Received 3 September 2012 Received in revised form 2 October 2012 Accepted 4 October 2012 Available online 13 October 2012 Keywords: 11␤-Hydroxysteroid dehydrogenase 11␤-HSD1 11␤-HSD2 Glucocorticoid metabolism

a b s t r a c t Bisphenol A (BPA) is a potential endocrine disruptor. It has been shown that it can interfere with steroid biosynthesis and metabolism. However, the mechanism is unclear. The objective of the present study is to investigate the effects of BPA on two isoforms of 11␤-hydroxysteroid dehydrogenases (11␤-HSD1 and 11␤-HSD2) in human and rat tissues. Human liver, rat testis microsomes as well as rat adult Leydig cells were used for measurement of 11␤-HSD1 activity, and human and rat kidney microsomes for 11␤-HSD2 activity. BPA inhibited human and rat 11␤-HSD1 activities with the half maximal inhibitory concentrations (IC50 s) of 14.81 ± 0.06 ␮M (mean ± SEM) for human and 19.39 ± 0.09 ␮M for rat enzyme, respectively. BPA inhibited rat 11␤-HSD1 activity in intact rat Leydig cells. BPA also weakly inhibited both human and rat 11␤-HSD2 activities. At 100 ␮M, BPA inhibited human and rat enzymes by 51.16% and 41.61%, respectively. In conclusion, BPA is an inhibitor for both 11␤-HSD1 and 11␤-HSD2, with selectivity against the type I enzyme. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Many endocrine disruptors interfere with hormone biosynthesis or metabolism. A steroid hormone is a small molecule that binds to its nuclear receptor to act as a hormone. Steroid hormones include glucocorticoids, mineralocorticoids, androgens, estrogens and progestogens according to their binding to the respective receptors to exert various physiological actions, such as metabolism, inflammation, immune functions, salt and water balance, sexual behaviors and fertility. One of potential environmental endocrine disruptors is bisphenol A (BPA), which has been shown to disrupt testosterone biosynthesis (Ye et al., 2011). This led to the inhibition of testosterone production by Leydig cells. Therefore,

∗ Corresponding author. Tel.: +86 577 88879169. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y.F. Xu), r [email protected] (R.-S. Ge). 1 These authors contributed equally to the work. 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.10.002

BPA acts as a potential antiandrogen (Nanjappa et al., 2012). It is true that prenatal exposure of BPA in the mouse caused reduction of neonatal serum testosterone level (Tanaka et al., 2006). Gestational exposure to BPA to rats also caused the reduction of expression of steroidogenic acute regulatory protein in the fetal testis (Horstman et al., 2012). In rodents, postnatal exposure to BPA decreased serum testosterone production (Akingbemi et al., 2004; D’Cruz et al., 2012; Herath et al., 2004; Nakamura et al., 2010), reduced seminal vesicle weights (Takahashi and Oishi, 2001) and daily sperm production (Akingbemi et al., 2004; Herath et al., 2004). Data from an epidemiological study indicate that exposure to low environmental levels of BPA may be associated with a modest reduction in free testosterone level of fertile men (Mendiola et al., 2010). BPA inhibited several testosterone biosynthetic enzyme activities, especially the 3␤-hydroxysteroid dehydrogenase (Ye et al., 2011). Hydroxysteroid dehydrogenases are a group of steroid oxido-reductases that catalyze the interconversion between hydroxysteroids and ketosteroids. Some of these hydroxysteroid dehydrogenases, including 11␤-hydroxysteroid dehydrogenases

J. Guo et al. / Toxicology Letters 215 (2012) 126–130

(11␤-HSDs) are also involved in the glucocorticoid metabolism. The metabolic activation of the 11-keto group of cortisone (human) into the 11␤-hydroxyl group of cortisol and the reverse reaction to inactivate cortisol is catalyzed by 11␤-HSD. There are two isoforms, type I (11␤-HSD1) and type II (11␤-HSD2). Both enzymes are microsomal enzymes (Agarwal et al., 1989; Albiston et al., 1994). However, they have different biochemical properties, with 11␤HSD1 as a low-affinity (Km ∼2 ␮M for cortisol) (Monder and White, 1993) and 11␤-HSD2 as a high-affinity enzyme (Km ∼25 nM for cortisol) (White et al., 1997a). 11␤-HSD1 catalyzes both direction, and uses NADP+ as cofactor for oxidase direction and NADPH as cofactor for reductase direction, behaving a primary reductase in the liver and lung. In the lung of later gestational period, the regeneration of active glucocorticoid from inactive one helps the maturation of fetal lung (Suzuki et al., 2003). In the contrast, 11␤-HSD2 catalyzes unidirectional oxidation of cortisol, and uses NAD+ as cofactor. 11␤HSD2 is primarily present in mineralocorticoid targeted tissues such as kidney and colon, this enzyme lowers mineralocorticoid activity by inactivating cortisol and letting aldosterone as the active mineralocorticoid (White et al., 1997a). Mutation of 11␤-HSD2 (the syndrome of apparent mineralocorticoid excess) or significant suppression of its activity by endocrine disruptors causes mineralocorticoid excess including hypokalemia and hypertension (White et al., 1997a). In glucocorticoid target tissues such as placenta, 11␤HSD2 also reduces active maternal cortisol level thus restricting the adverse effects of the glucocorticoid on fetal development. However, the direct inhibition on both 11␤-HSD enzyme activities and the inhibitory selectivity by BPA has not been well studied. The present study was to investigate the direct effects of BPA on both 11␤-HSD isoforms and the mode of inhibition. 2. Materials and methods 2.1. Chemicals and animals [1,2,6,7-3 H] Corticosterone (3 H-CORT) and [1,2,6,7-3 H] cortisol (3 H-cortisol) were purchased from Dupont-New England Nuclear (Boston, MA). 3 H-11Dehydrocorticosterone (3 H-11DHC) and 3 H-cortisone were prepared from labelled 3 H-CORT or 3 H-cortisol as described earlier (Lakshmi and Monder, 1985). Cold CORT, 11DHC, cortisol and cortisone were purchased from Steraloids (Newport, RI). BPA was purchased from Sigma (St. Louis, MO). BPA was prepared using dimethyl sulfonate (DMSO) as a solvent. Sprague Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). Human liver and kidney microsomes were purchased from Gentest (Cat# 452156, Woburn, MA), which were prepared from 50 pooled livers and stored with final concentrations of 20 mg/ml.

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2.4. 11ˇ-HSD1 assay in the microsomes 11␤-HSD1 activity assay tubes contained 25 nM substrate cortisone (for human) or 11DHC (for rat), spiked with 30,000 cpm of their respective 3 H-steriods. Cortisone or 11DHC was used as the substrate to measure 11␤-HSD1 activity. The rat testis microsomes (10 ␮g) or human liver (4 ␮g) microsomes were incubated with substrate, 0.2 mM NADPH and 0.5 mM glucose-6-phosphate (G6P) and various concentrations of BPA at 37 C for 60–90 min. The inhibitory potency of BPA was measured relative to control (only DMSO). BPA was dissolved in DMSO with final DMSO concentration of 0.4%, at which DMSO did not inhibit this enzyme activity. At the end of the reaction, the reaction was stopped by adding 1 ml ice-cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on the thin layer plate in chloroform and methanol (90:10, v/v), and the radioactivity was measured using a scanning radiometer (System AR2000, Bioscan Inc., Washington, DC) as described previously (Ge et al., 1997). The percentage conversion of 11DHC to CORT or cortisone to cortisol was calculated by dividing the radioactive counts identified as 11-OH-steroids by the total counts (see supplementary Fig. 1). 2.5. 11ˇ-HSD1 assay in the intact adult Leydig cells Because intact cells can maintain both 11␤-HSD1 oxidase and reductase activity, its oxidation or reduction was also measured in intact Leydig cells using endogenous NADP+ or NADPH. Because rat Leydig cells have higher 11␤-HSD1 oxidase activity (Ge et al., 1997), the 11␤-HSD1 oxidase assay tubes contained 25 nM CORT spiked with 30,000 cpm of CORT and 0.025 × 106 cells, and the mixture was incubated for 30 min. The 11␤-HSD1 reductase assay tubes contained 25 nM 11DHC spiked with 30,000 cpm of 11DHC and 0.045 × 106 cells, and the mixture was incubated for 120 min. The rest procedure was as described above. The 11␤-HSD1 assays in intact Leydig cells were repeated by four times. 2.6. 11ˇ-HSD2 assay in kidney microsomes 11␤-HSD2 activity assay tubes contained 25 nM (within the range of physiological levels of CORT). [3 H] cortisol or [3 H] CORT were used as substrates to measure either human or rat 11␤-HSD2 oxidase activity. Kidney microsomes were incubated with substrates, NAD+ . The reactions were stopped by adding 1 ml ice-cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin layer plates in chloroform and methanol (90:10), and the radioactivity was measured using a scanning radiometer (System AR2000, Bioscan Inc., Washington, DC). The percentage conversion of CORT to 11DHC and cortisol to cortisone was calculated by dividing the radioactive counts identified as 11DHC (or cortisone) by the total counts associated with both substrate and product. 2.7. Determination of half maximum inhibitory concentrations (IC50 ) and inhibitory mode The IC50 was determined by adding 25 nM substrate and 0.2 mM cofactor and various concentrations of BPA at 250 ␮l reaction buffer (0.1 mM PBS) containing human or rat microsomal protein as described previously (Ye et al., 2011). The mode of inhibition was assayed by adding various concentrations of substrate or cofactor. 2.8. Statistics

2.2. Preparation of microsomal protein Six male adult rat testes were used for preparation of testis microsome to measure 11␤-HSD1 activity, because adult rat testis contains abundant isoform (Ge et al., 1997), and rat kidney for 11␤-HSD2 enzyme because it is primarily located in the kidney (Agarwal et al., 1994). The preparation of microsomes was performed as described previously (Ge et al., 1997). In brief, rat testis or kidney was homogenized in 0.01 mM PBS buffer containing 0.25 M sucrose, and nuclei and large cell debris were removed by centrifugation at 1500 × g for 10 min. The post-nuclear supernatants were centrifuged twice at 105,000 × g, the resultant microsomal pellets were resuspended. Protein contents were measured by Bio-Rad Dye Reagent Concentrate (Cat.# 500-0006). The concentrations of rat testis and kidney microsomes were 20 mg/ml. Microsomes were used for measurement of 11␤-HSD1 or 11␤-HSD2 activities.

2.3. Leydig cell isolation Purified rat Leydig cells were obtained from six 90-day-old Sprague Dawley rats by collagenase digestion of the testes followed by Percoll density centrifugation of the cell suspension, according to the previously described method (Sriraman et al., 2001). Adult Leydig cells were harvested from the Percoll gradient at a band at 1.070 mg/ml. The purity of cell fractions was evaluated by histochemical staining for 3␤-hydroxysteroid dehydrogenase activity with 0.4 mM etiocholanolone as the steroid substrate (Payne et al., 1980). Enrichment of rat Leydig cells was typically more than 95%.

Experiments were repeated four times. Data were subjected to nonlinear regression analysis by GraphPad (Version 5, GraphPad Software Inc., San Diego, CA) for IC50 . Lineweaver–Burk plot was used for the analysis of the mode of inhibition. ANOVA was used to determine if differences exist. Then a post hoc test using Tukey’s analysis was used to determine the differences between two groups. All data are expressed as means ± SEM. Differences were regarded as significant at P < 0.05.

3. Results 3.1. The effects of BPA on 11ˇ-HSD1 activity The conversion of cortisone to cortisol (human) or 11DHC to CORT (rat) has been shown to be catalyzed in a NADPHdependent manner by human or rat 11␤-HSD1 activity in the microsomes. BPA significantly inhibited human and rat 11␤-HSD1 activities with IC50 s of 14.81 ± 0.06 ␮M (mean ± SEM) for human and 19.39 ± 0.09 ␮M, respectively (Fig. 1). BPA inhibited both 11␤HSD1 oxidase and reductase activity in intact rat Leydig cells, with the reductase more sensitive to its inhibition. At 100 ␮M, BPA inhibited 11␤-HSD1 reductase activity in intact rat Leydig cells by 82.38%, but the oxidase activity by only 48.84% (Fig. 2). BPA competitively inhibited rat 11␤-HSD1 reductase activity against substrate

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A. 11DHC

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Fig. 1. Inhibition of BPA on human and rat 11␤-hydroxysteroid dehydrogenase 1 (11␤-HSD1) reductase activities. 11␤-HSD1 assay was performed by incubating 25 nM cortisone (human) or 11DHC (rat) with 0.2 mM NADPH, 0.5 mM G6P and microsomes for 60–90 min. IC50 s of BPA for both human and rat enzymes. Mean ± SEM (n = 4).

11DHC (Fig. 3A). However, BPA exerted mixed-type inhibition on rat 11␤-HSD1 reductase activity against cofactor NADPH (Fig. 3B). BPA exerted the same action modes on human 11␤-HSD1 activity (data not shown).

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1/[NADPH], mM 3.2. The effects of BPA on 11ˇ-HSD2 activity The conversion of cortisol to cortisone or CORT to 11DHC has been shown to be catalyzed in a NAD+ -dependent manner by human or rat 11␤-HSD2 in the kidney microsomes. BPA weakly but significantly inhibited human and rat kidney 11␤-HSD2 activities by about 50% (Fig. 4). 4. Discussion Little is understood about the molecular targets of BPA although there is significant accumulation of BPA in human blood and wildlife tissues. In the present study, we demonstrated that BPA is an inhibitor of human and rat 11␤-HSD1 and 11␤-HSD2 activities. Although BPA weakly and significantly inhibited 11␤-HSD2, the selectivity of BPA on 11␤-HSDs is favoring 11␤-HSD1. 11β-HSD1 Oxidation 150

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Fig. 2. Inhibition of BPA on rat intact Leydig cell 11␤-hydroxysteroid dehydrogenase 1 (11␤-HSD1) oxidase and reductase activities. 11␤-HSD1 oxidase assay was performed by incubating 25 nM corticosterone with 0.025 × 106 cells for 30 min. 11␤-HSD1 reductase assay was performed by incubating 25 nM 11DHC with 0.045 × 106 cells for 120 min. Mean ± SEM, n = 4. The conversion rates of 11␤HSD1 oxidase were 32.39 ± 1.38% in PBS control and 30.50 ± 0.45% in ethanol control (CON). The conversion rates of 11␤-HSD1 reductase were 31.40 ± 0.90% in PBS control and 31.17 ± 1.57% in ethanol control (CON). ***Significant differences when compared to control at P < 0.001 for each enzyme; ### Significant difference at P < 0.001 between oxidase and reductase activities after BPA treatment.

Fig. 3. The mode of BPA on 11␤-hydroxysteroid dehydrogenase 1 reductase. Lineweaver–Burk plotting in presence of substrate 11DHC (panel A); Lineweaver–Burk plotting in the presence of NADPH (panel B).

In the developing lung, 11␤-HSD1 behaves predominantly as a reductase, utilizing NADPH as a cofactor to catalyze the conversion of inactive cortisone into bioactive cortisol (human) or 11DHC into CORT (rodents). Previous studies have shown that fetal lungs have significant higher enzyme activity of 11␤-HSD1 in the fetal lung towards end of gestation (Suzuki et al., 2003). Importantly, 11␤HSD1 activity was significantly higher than 11␤-HSD2 throughout gestation and after birth, since 11␤-HSD2 does the opposite job as an oxidase (Suzuki et al., 2003). 11␤-HSD1 has been found to be critical for lung development, since the ablation of 11␤-HSD1 gene or inhibition of the enzyme in mice delayed the lung maturity (Hundertmark et al., 2002). This suggests that peripheral rat lung tissue possesses 11␤-HSD1 reductase activity predominantly, thereby increasing tissue glucocorticoid availability by converting the receptor-inactive glucocorticoid to its active forms. Glucocorticoid plays a critical role in the modulation of phosphatidylcholine and surfactant protein synthesis in alveolar type  cells of fetal lung at the late gestation (Rooney et al., 1994). Phosphatidylcholine reduces surface tension of the alveolar wall and prevents atelectasis, and may play an important role in maintaining normal alveolar structure. Surfactant proteins are known to be essential in the host defense system after birth (Mendelson et al., 1998). 11␤-HSD1 is also abundantly present in human and rodent liver (Ge et al., 1997; Agarwal et al., 1995) and acts as reductase to generate active glucocorticoid locally (Agarwal et al., 1995; Ge et al., 1997). The glucocorticoid has been found to profoundly regulate the glucose metabolism in the liver (Andrews and Walker, 1999). Thus, the inhibition of 11␤-HSD1 by BPA may also regulate the glucose metabolism. In rat testis, Leydig cells provide with only sources of 11␤-HSD1, and the activity in the Leydig cells is much higher than that of liver cells per se. 11␤-HSD1 in the Leydig cells has been suggested to regulate testosterone biosynthesis. Thus the interference with 11␤-HSD1 activity could alter testosterone production in this cell type.

J. Guo et al. / Toxicology Letters 215 (2012) 126–130

11β-HSD2 Activity (%)

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Fig. 4. Effects of BPA on human and rat 11␤-hydroxysteroid dehydrogenase 2 (11␤HSD2) activities. 11␤-HSD2 assay was performed by incubating 25 nM cortisol (human) or corticosterone (rat) with 0.2 mM NAD+ and microsomes for 60–90 min. Mean ± SEM (n = 4). The conversion rates of rat 11␤-HSD2 were 13.52 ± 0.12% in PBS control and 15.03 ± 0.73% in ethanol control (CON). The conversion rates of human 11␤-HSD2 were 26.02 ± 0.36% in PBS control and 26.70 ± 0.56% in ethanol control (CON). Panel A, human and panel B rat enzyme. ***Significant differences when compared to control at P < 0.001.

BPA inhibition of human and rat 11␤-HSD1 was competitive for the enzyme substrate (Fig. 3). Therefore, we investigated whether BPA competed with the cofactor NADPH (for 11␤-HSD1 reductase) and examined the effects of different concentrations of cofactors in the presence of varying concentrations of BPA. The results indicated that BPA exerted a mixed-type inhibition of the enzyme with the NADPH. Thus, it is reasonable to infer that when BPA and 11DHC or cortisone are bound the enzyme–substrate–inhibitor complex, the complex cannot form product. When cofactor is supplied, the mixed-type inhibition by BPA may suggest that BPA partially competes with cofactor NADPH in the cofactor binding site of the enzyme. At a higher concentration, BPA also inhibited 11␤-HSD2 activity. The kidney is a mineralocorticoid target tissue. The endogenous ligand for this receptor (MR) is aldosterone. Aldosterone increases sodium and water retention and increases blood pressure after binding to MR. Aldosterone and cortisol have similar affinities for the MR (Arriza et al., 1987). The apparent mineralocorticoid excess (AME) syndrome, in which patients have hypertension and hypokalemia (Edwards et al., 1988; White et al., 1997c), was discovered to be caused by mutational inactivation of the 11␤-HSD2 gene, or by inhibitors of the enzyme (Funder et al., 1988; Stewart et al., 1987; White et al., 1997b). In other words, the role of 11␤HSD2 in kidney is to confer the specificity of aldosterone for the renal MR (Funder et al., 1988; Stewart et al., 1987). In the placenta, 11␤-HSD2 is localized in the syncytiotrophoblast, which is the site of maternal–fetal exchange (Krozowski et al., 1995). The placental

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11␤-HSD2 plays a key role in pregnancy maintenance and fetal maturation. Thus, placental 11␤-HSD2 is the critical enzyme that protects the fetus from overexposure to maternal cortisol, which may check fetal development and cause cardiovascular, metabolic and neuropsychiatric disorders (Doyle et al., 2003; Seckl, 2004; Seckl and Holmes, 2007). Thus, at higher level, BPA caused affect fetal development or water and salt metabolism. These observations are relevant to public health, because BPA occurs in the environment at comparable levels. Data from an epidemiological study indicates that exposure to low environmental levels of BPA may be associated with a modest reduction in free testosterone level of fertile men (Mendiola et al., 2010). Children may be more susceptible to BPA exposure than adults. A recent study found much higher concentrations of BPA in samples of infant than those of adults (Edginton and Ritter, 2009). Neonatal fed with liquid formula is among the most exposed because 13 ␮g per kg of body weight per day of BPA can be ingested from polycarbonate bottle (Ackerman et al., 2010). The mean urinary concentration of BPA was 30.3 ␮g/l (131 ␮M) in premature infants with intensive therapeutic medical interventions, which was one order of magnitude higher than that among the general population (Calafat et al., 2009). This concentration of BPA had significantly inhibitory effect on human 11␤-HSD1 activity (Fig. 1). Although the exact concentrations of BPA in liver or Leydig cells are varied, rat BPA pharmacokinetics data showed that BPA is eliminated with 18 h after oral administration (Pottenger et al., 2000). In conclusion, BPA is an inhibitor of both 11␤-HSD1 and 2 enzyme activities with selectivity against type I enzyme. However, the relation of this result to effects in the whole animal, a future study of measuring in vivo 11␤-HSD1 and 11␤-HSD2 activities will be performed to address its usefulness to risk assessment. Conflict of interest statement None. Acknowledgments We thank Hongzhi Li for technical assistance. The study was partially supported by NSFC (81102150, 81070329 and 81102150). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet. 2012.10.002. References Ackerman, L.K., Noonan, G.O., Heiserman, W.M., Roach, J.A., Limm, W., Begley, T.H., 2010. Determination of bisphenol A in U.S. infant formulas: updated methods and concentrations. Journal of Agricultural and Food Chemistry 58, 2307–2313. Agarwal, A.K., Monder, C., Eckstein, B., White, P.C., 1989. Cloning and expression of rat cDNA encoding corticosteroid 11␤-dehydrogenase. Journal of Biological Chemistry 264, 18939–18943. Agarwal, A.K., Mune, T., Monder, C., White, P.C., 1994. NAD(+)-dependent isoform of 11 beta-hydroxysteroid dehydrogenase. Cloning and characterization of cDNA from sheep kidney. Journal of Biological Chemistry 269, 25959–25962. Agarwal, A.K., Rogerson, F.M., Mune, T., White, P.C., 1995. Analysis of the human gene encoding the kidney isozyme of 11 beta-hydroxysteroid dehydrogenase. Journal of Steroid Biochemistry and Molecular Biology 55, 473–479. Akingbemi, B.T., Sottas, C.M., Koulova, A.I., Klinefelter, G.R., Hardy, M.P., 2004. Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells. Endocrinology 145, 592–603. Albiston, A.L., Obeyesekere, V.R., Smith, R.E., Krozowski, Z.S., 1994. Cloning and tissue distribution of the human 11 beta-hydroxysteroid dehydrogenase type 2 enzyme. Molecular and Cellular Endocrinology 105, R11–R17. Andrews, R.C., Walker, B.R., 1999. Glucocorticoids and insulin resistance: old hormones, new targets. Clinical Science (London) 96, 513–523.

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