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Toxicology Letters 215 (2012) 126– 130

Contents lists available at SciVerse ScienceDirect

Toxicology Letters

jou rn al h om epa ge: www.elsev ier .com/ locate / tox le t

nhibition of human and rat 11�-hydroxysteroid dehydrogenases activitiesy bisphenol A

ingjing Guoa,1, Xiaohuan Yuanb,1, Li Qiua, Weiliu Zhua, Chaonan Wangb, Guoxin Huc,anhui Chub, Leping Yea, Yunfei Xud,∗∗, Ren-Shan Gea,∗

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

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.

r t i c l e i n f o

rticle history:eceived 3 September 2012eceived in revised form 2 October 2012ccepted 4 October 2012vailable online 13 October 2012

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 steroidbiosynthesis and metabolism. However, the mechanism is unclear. The objective of the present studyis to investigate the effects of BPA on two isoforms of 11�-hydroxysteroid dehydrogenases (11�-HSD1and 11�-HSD2) in human and rat tissues. Human liver, rat testis microsomes as well as rat adult Ley-dig cells were used for measurement of 11�-HSD1 activity, and human and rat kidney microsomes for

eywords:1�-Hydroxysteroid dehydrogenase1�-HSD11�-HSD2

11�-HSD2 activity. BPA inhibited human and rat 11�-HSD1 activities with the half maximal inhibitoryconcentrations (IC50s) 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 bothhuman and rat 11�-HSD2 activities. At 100 �M, BPA inhibited human and rat enzymes by 51.16% and41.61%, respectively. In conclusion, BPA is an inhibitor for both 11�-HSD1 and 11�-HSD2, with selectivity

.

lucocorticoid metabolism

against the type I enzyme

. Introduction

Many endocrine disruptors interfere with hormone biosyn-hesis or metabolism. A steroid hormone is a small moleculehat binds to its nuclear receptor to act as a hormone. Steroidormones include glucocorticoids, mineralocorticoids, androgens,strogens and progestogens according to their binding to theespective receptors to exert various physiological actions, such asetabolism, inflammation, immune functions, salt and water bal-

nce, sexual behaviors and fertility. One of potential environmental

ndocrine disruptors is bisphenol A (BPA), which has been showno disrupt testosterone biosynthesis (Ye et al., 2011). This led tohe inhibition of testosterone production by Leydig cells. Therefore,

∗ Corresponding author. Tel.: +86 577 88879169.∗∗ Corresponding author.

E-mail addresses: xuyunfeibb@sina.com (Y.F. Xu), r ge@yahoo.com (R.-S. Ge).1 These authors contributed equally to the work.

378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2012.10.002

© 2012 Elsevier Ireland Ltd. All rights reserved.

BPA acts as a potential antiandrogen (Nanjappa et al., 2012). It istrue that prenatal exposure of BPA in the mouse caused reduction ofneonatal serum testosterone level (Tanaka et al., 2006). Gestationalexposure to BPA to rats also caused the reduction of expression ofsteroidogenic acute regulatory protein in the fetal testis (Horstmanet al., 2012). In rodents, postnatal exposure to BPA decreased serumtestosterone production (Akingbemi et al., 2004; D’Cruz et al., 2012;Herath et al., 2004; Nakamura et al., 2010), reduced seminal vesi-cle weights (Takahashi and Oishi, 2001) and daily sperm production(Akingbemi et al., 2004; Herath et al., 2004). Data from an epidemi-ological study indicate that exposure to low environmental levels ofBPA may be associated with a modest reduction in free testosteronelevel of fertile men (Mendiola et al., 2010).

BPA inhibited several testosterone biosynthetic enzyme activ-ities, especially the 3�-hydroxysteroid dehydrogenase (Ye et al.,

2011). Hydroxysteroid dehydrogenases are a group of steroidoxido-reductases that catalyze the interconversion betweenhydroxysteroids and ketosteroids. Some of these hydroxysteroiddehydrogenases, including 11�-hydroxysteroid dehydrogenases

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11�-HSDs) are also involved in the glucocorticoid metabolism. Theetabolic activation of the 11-keto group of cortisone (human)

nto the 11�-hydroxyl group of cortisol and the reverse reactiono inactivate cortisol is catalyzed by 11�-HSD. There are two iso-orms, type I (11�-HSD1) and type II (11�-HSD2). Both enzymes are

icrosomal enzymes (Agarwal et al., 1989; Albiston et al., 1994).owever, they have different biochemical properties, with 11�-SD1 as a low-affinity (Km ∼2 �M for cortisol) (Monder and White,993) and 11�-HSD2 as a high-affinity enzyme (Km ∼25 nM for cor-isol) (White et al., 1997a). 11�-HSD1 catalyzes both direction, andses NADP+ as cofactor for oxidase direction and NADPH as cofactoror reductase direction, behaving a primary reductase in the livernd lung. In the lung of later gestational period, the regenerationf active glucocorticoid from inactive one helps the maturation ofetal lung (Suzuki et al., 2003). In the contrast, 11�-HSD2 catalyzesnidirectional oxidation of cortisol, and uses NAD+ as cofactor. 11�-SD2 is primarily present in mineralocorticoid targeted tissues

uch as kidney and colon, this enzyme lowers mineralocorticoidctivity by inactivating cortisol and letting aldosterone as the activeineralocorticoid (White et al., 1997a). Mutation of 11�-HSD2

the syndrome of apparent mineralocorticoid excess) or significantuppression of its activity by endocrine disruptors causes mineralo-orticoid excess including hypokalemia and hypertension (Whitet al., 1997a). In glucocorticoid target tissues such as placenta, 11�-SD2 also reduces active maternal cortisol level thus restricting thedverse effects of the glucocorticoid on fetal development. How-ver, the direct inhibition on both 11�-HSD enzyme activities andhe inhibitory selectivity by BPA has not been well studied. Theresent study was to investigate the direct effects of BPA on both1�-HSD isoforms and the mode of inhibition.

. Materials and methods

.1. Chemicals and animals

[1,2,6,7-3H] Corticosterone (3H-CORT) and [1,2,6,7-3H] cortisol (3H-cortisol)ere purchased from Dupont-New England Nuclear (Boston, MA). 3H-11-ehydrocorticosterone (3H-11DHC) and 3H-cortisone were prepared from labelledH-CORT or 3H-cortisol as described earlier (Lakshmi and Monder, 1985). Cold CORT,1DHC, cortisol and cortisone were purchased from Steraloids (Newport, RI). BPAas purchased from Sigma (St. Louis, MO). BPA was prepared using dimethyl sul-

onate (DMSO) as a solvent. Sprague Dawley rats were purchased from Charles Riveraboratories (Wilmington, MA). Human liver and kidney microsomes were pur-hased from Gentest (Cat# 452156, Woburn, MA), which were prepared from 50ooled livers and stored with final concentrations of 20 mg/ml.

.2. Preparation of microsomal protein

Six male adult rat testes were used for preparation of testis microsome to mea-ure 11�-HSD1 activity, because adult rat testis contains abundant isoform (Ge et al.,997), and rat kidney for 11�-HSD2 enzyme because it is primarily located in theidney (Agarwal et al., 1994). The preparation of microsomes was performed asescribed previously (Ge et al., 1997). In brief, rat testis or kidney was homoge-ized in 0.01 mM PBS buffer containing 0.25 M sucrose, and nuclei and large cellebris were removed by centrifugation at 1500 × g for 10 min. The post-nuclearupernatants were centrifuged twice at 105,000 × g, the resultant microsomal pel-ets were resuspended. Protein contents were measured by Bio-Rad Dye Reagentoncentrate (Cat.# 500-0006). The concentrations of rat testis and kidney micro-omes were 20 mg/ml. Microsomes were used for measurement of 11�-HSD1 or1�-HSD2 activities.

.3. Leydig cell isolation

Purified rat Leydig cells were obtained from six 90-day-old Sprague Dawley ratsy collagenase digestion of the testes followed by Percoll density centrifugation ofhe cell suspension, according to the previously described method (Sriraman et al.,

001). Adult Leydig cells were harvested from the Percoll gradient at a band at.070 mg/ml. The purity of cell fractions was evaluated by histochemical stainingor 3�-hydroxysteroid dehydrogenase activity with 0.4 mM etiocholanolone as theteroid substrate (Payne et al., 1980). Enrichment of rat Leydig cells was typicallyore than 95%.

s 215 (2012) 126– 130 127

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 3H-steriods. Cor-tisone or 11DHC was used as the substrate to measure 11�-HSD1 activity. Therat testis microsomes (10 �g) or human liver (4 �g) microsomes were incubatedwith substrate, 0.2 mM NADPH and 0.5 mM glucose-6-phosphate (G6P) and vari-ous concentrations of BPA at 37 C for 60–90 min. The inhibitory potency of BPA wasmeasured relative to control (only DMSO). BPA was dissolved in DMSO with finalDMSO 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. Thesteroids were separated chromatographically on the thin layer plate in chloroformand methanol (90:10, v/v), and the radioactivity was measured using a scanningradiometer (System AR2000, Bioscan Inc., Washington, DC) as described previously(Ge et al., 1997). The percentage conversion of 11DHC to CORT or cortisone to cor-tisol was calculated by dividing the radioactive counts identified as 11-OH-steroidsby 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 endoge-nous NADP+ or NADPH. Because rat Leydig cells have higher 11�-HSD1 oxidaseactivity (Ge et al., 1997), the 11�-HSD1 oxidase assay tubes contained 25 nM CORTspiked with 30,000 cpm of CORT and 0.025 × 106 cells, and the mixture was incu-bated for 30 min. The 11�-HSD1 reductase assay tubes contained 25 nM 11DHCspiked with 30,000 cpm of 11DHC and 0.045 × 106 cells, and the mixture was incu-bated for 120 min. The rest procedure was as described above. The 11�-HSD1 assaysin 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 physiolog-ical levels of CORT). [3H] cortisol or [3H] CORT were used as substrates to measureeither human or rat 11�-HSD2 oxidase activity. Kidney microsomes were incubatedwith 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. Thesteroids were separated chromatographically on thin layer plates in chloroform andmethanol (90:10), and the radioactivity was measured using a scanning radiometer(System AR2000, Bioscan Inc., Washington, DC). The percentage conversion of CORTto 11DHC and cortisol to cortisone was calculated by dividing the radioactive countsidentified as 11DHC (or cortisone) by the total counts associated with both substrateand product.

2.7. Determination of half maximum inhibitory concentrations (IC50) andinhibitory mode

The IC50 was determined by adding 25 nM substrate and 0.2 mM cofactor andvarious concentrations of BPA at 250 �l reaction buffer (0.1 mM PBS) containinghuman or rat microsomal protein as described previously (Ye et al., 2011). The modeof inhibition was assayed by adding various concentrations of substrate or cofactor.

2.8. Statistics

Experiments were repeated four times. Data were subjected to nonlinear regres-sion 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. ANOVAwas used to determine if differences exist. Then a post hoc test using Tukey’s analysiswas used to determine the differences between two groups. All data are expressedas 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 11DHCto CORT (rat) has been shown to be catalyzed in a NADPH-dependent manner by human or rat 11�-HSD1 activity in themicrosomes. BPA significantly inhibited human and rat 11�-HSD1activities with IC50s of 14.81 ± 0.06 �M (mean ± SEM) for humanand 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, BPAinhibited 11�-HSD1 reductase activity in intact rat Leydig cells by82.38%, but the oxidase activity by only 48.84% (Fig. 2). BPA compet-itively inhibited rat 11�-HSD1 reductase activity against substrate

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

Fig. 1. Inhibition of BPA on human and rat 11�-hydroxysteroid dehydrogenase1 (11�-HSD1) reductase activities. 11�-HSD1 assay was performed by incubat-iaM

1rB(

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150

200

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Fig. 3. The mode of BPA on 11�-hydroxysteroid dehydrogenase 1 reduc-

ng 25 nM cortisone (human) or 11DHC (rat) with 0.2 mM NADPH, 0.5 mM G6Pnd microsomes for 60–90 min. IC50s of BPA for both human and rat enzymes.ean ± SEM (n = 4).

1DHC (Fig. 3A). However, BPA exerted mixed-type inhibition onat 11�-HSD1 reductase activity against cofactor NADPH (Fig. 3B).PA exerted the same action modes on human 11�-HSD1 activitydata not shown).

.2. The effects of BPA on 11ˇ-HSD2 activity

The conversion of cortisol to cortisone or CORT to 11DHC haseen shown to be catalyzed in a NAD+-dependent manner byuman or rat 11�-HSD2 in the kidney microsomes. BPA weakly butignificantly inhibited human and rat kidney 11�-HSD2 activitiesy about 50% (Fig. 4).

. Discussion

Little is understood about the molecular targets of BPA althoughhere is significant accumulation of BPA in human blood and wild-

ife tissues. In the present study, we demonstrated that BPA is annhibitor of human and rat 11�-HSD1 and 11�-HSD2 activities.lthough BPA weakly and significantly inhibited 11�-HSD2, theelectivity of BPA on 11�-HSDs is favoring 11�-HSD1.

0

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100

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ig. 2. Inhibition of BPA on rat intact Leydig cell 11�-hydroxysteroid dehydro-enase 1 (11�-HSD1) oxidase and reductase activities. 11�-HSD1 oxidase assayas performed by incubating 25 nM corticosterone with 0.025 × 106 cells for

0 min. 11�-HSD1 reductase assay was performed by incubating 25 nM 11DHCith 0.045 × 106 cells for 120 min. Mean ± SEM, n = 4. The conversion rates of 11�-SD1 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 con-rol and 31.17 ± 1.57% in ethanol control (CON). ***Significant differences whenompared to control at P < 0.001 for each enzyme; ###Significant difference at

< 0.001 between oxidase and reductase activities after BPA treatment.

tase. 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 areductase, utilizing NADPH as a cofactor to catalyze the conversionof inactive cortisone into bioactive cortisol (human) or 11DHC intoCORT (rodents). Previous studies have shown that fetal lungs havesignificant higher enzyme activity of 11�-HSD1 in the fetal lungtowards end of gestation (Suzuki et al., 2003). Importantly, 11�-HSD1 activity was significantly higher than 11�-HSD2 throughoutgestation and after birth, since 11�-HSD2 does the opposite jobas an oxidase (Suzuki et al., 2003). 11�-HSD1 has been found tobe critical for lung development, since the ablation of 11�-HSD1gene or inhibition of the enzyme in mice delayed the lung matu-rity (Hundertmark et al., 2002). This suggests that peripheral ratlung tissue possesses 11�-HSD1 reductase activity predominantly,thereby increasing tissue glucocorticoid availability by convertingthe receptor-inactive glucocorticoid to its active forms. Glucocorti-coid plays a critical role in the modulation of phosphatidylcholineand surfactant protein synthesis in alveolar type � cells of fetallung at the late gestation (Rooney et al., 1994). Phosphatidylcholinereduces surface tension of the alveolar wall and prevents atelecta-sis, and may play an important role in maintaining normal alveolarstructure. Surfactant proteins are known to be essential in the hostdefense 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 gen-erate active glucocorticoid locally (Agarwal et al., 1995; Ge et al.,1997). The glucocorticoid has been found to profoundly regulate theglucose metabolism in the liver (Andrews and Walker, 1999). Thus,the inhibition of 11�-HSD1 by BPA may also regulate the glucosemetabolism. In rat testis, Leydig cells provide with only sources of11�-HSD1, and the activity in the Leydig cells is much higher thanthat of liver cells per se. 11�-HSD1 in the Leydig cells has been sug-

gested to regulate testosterone biosynthesis. Thus the interferencewith 11�-HSD1 activity could alter testosterone production in thiscell type.

J. Guo et al. / Toxicology Letter

0

50

100

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ity

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A. Human

0

50

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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 PBScontrol and 15.03 ± 0.73% in ethanol control (CON). The conversion rates of human1(c

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1�-HSD2 were 26.02 ± 0.36% in PBS control and 26.70 ± 0.56% in ethanol controlCON). Panel A, human and panel B rat enzyme. ***Significant differences whenompared to control at P < 0.001.

BPA inhibition of human and rat 11�-HSD1 was competitive forhe enzyme substrate (Fig. 3). Therefore, we investigated whetherPA competed with the cofactor NADPH (for 11�-HSD1 reductase)nd examined the effects of different concentrations of cofactors inhe presence of varying concentrations of BPA. The results indicatedhat BPA exerted a mixed-type inhibition of the enzyme with theADPH. Thus, it is reasonable to infer that when BPA and 11DHCr cortisone are bound the enzyme–substrate–inhibitor complex,he complex cannot form product. When cofactor is supplied, the

ixed-type inhibition by BPA may suggest that BPA partially com-etes with cofactor NADPH in the cofactor binding site of thenzyme.

At a higher concentration, BPA also inhibited 11�-HSD2 activity.he kidney is a mineralocorticoid target tissue. The endogenousigand for this receptor (MR) is aldosterone. Aldosterone increasesodium and water retention and increases blood pressure afterinding to MR. Aldosterone and cortisol have similar affinitiesor the MR (Arriza et al., 1987). The apparent mineralocorticoidxcess (AME) syndrome, in which patients have hypertension andypokalemia (Edwards et al., 1988; White et al., 1997c), was dis-overed to be caused by mutational inactivation of the 11�-HSD2ene, or by inhibitors of the enzyme (Funder et al., 1988; Stewartt al., 1987; White et al., 1997b). In other words, the role of 11�-

SD2 in kidney is to confer the specificity of aldosterone for the

enal MR (Funder et al., 1988; Stewart et al., 1987). In the placenta,1�-HSD2 is localized in the syncytiotrophoblast, which is the sitef maternal–fetal exchange (Krozowski et al., 1995). The placental

s 215 (2012) 126– 130 129

11�-HSD2 plays a key role in pregnancy maintenance and fetalmaturation. Thus, placental 11�-HSD2 is the critical enzyme thatprotects the fetus from overexposure to maternal cortisol, whichmay check fetal development and cause cardiovascular, metabolicand neuropsychiatric disorders (Doyle et al., 2003; Seckl, 2004;Seckl and Holmes, 2007). Thus, at higher level, BPA caused affectfetal development or water and salt metabolism.

These observations are relevant to public health, because BPAoccurs in the environment at comparable levels. Data from an epi-demiological study indicates that exposure to low environmentallevels of BPA may be associated with a modest reduction in freetestosterone level of fertile men (Mendiola et al., 2010). Childrenmay be more susceptible to BPA exposure than adults. A recentstudy found much higher concentrations of BPA in samples of infantthan those of adults (Edginton and Ritter, 2009). Neonatal fed withliquid formula is among the most exposed because 13 �g per kg ofbody weight per day of BPA can be ingested from polycarbonatebottle (Ackerman et al., 2010). The mean urinary concentrationof BPA was 30.3 �g/l (131 �M) in premature infants with inten-sive therapeutic medical interventions, which was one order ofmagnitude higher than that among the general population (Calafatet al., 2009). This concentration of BPA had significantly inhibitoryeffect on human 11�-HSD1 activity (Fig. 1). Although the exactconcentrations of BPA in liver or Leydig cells are varied, rat BPApharmacokinetics data showed that BPA is eliminated with 18 hafter oral administration (Pottenger et al., 2000).

In conclusion, BPA is an inhibitor of both 11�-HSD1 and 2enzyme activities with selectivity against type I enzyme. However,the relation of this result to effects in the whole animal, a futurestudy of measuring in vivo 11�-HSD1 and 11�-HSD2 activities willbe performed to address its usefulness to risk assessment.

Conflict of interest statement

None.

Acknowledgments

We thank Hongzhi Li for technical assistance. The study waspartially supported by NSFC (81102150, 81070329 and 81102150).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2012.10.002.

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