Chemosphere 93 (2013) 1189–1193

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New insights for the risk of bisphenol A: Inhibitionof UDP-glucuronosyltransferases (UGTs)

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.06.070

⇑ Corresponding author at: Liaoning Medical University, Jinzhou, Liaoning, China.E-mail address: lyyyjhm@163.com (H.-M. Jiang).

1 These authors contributed equally to this work.

Hua-Mao Jiang a,⇑,1, Zhong-Ze Fang a,b,c,1, Yun-Feng Cao b,1, Cui-Min Hu a,e, Xiao-Yu Sun b, Mo Hong b,Ling Yang d, Guang-Bo Ge d, Yong Liu d, Yan-Yan Zhang b, Qiang Dong a, Ren-Jie Liu a

a Liaoning Medical University, Jinzhou, Liaoning, Chinab Joint Center for Translational Medicine, Dalian Institute of Chemical Physics Chinese Academy of Sciences and The First Affiliated Hospital of Liaoning Medical University, No. 457,Zhongshan Road, Dalian 116023, Chinac Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USAd Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Chinae Department of Microbiology & Immunology, Georgetown University Medical Center, Washington DC, 20057, USA

h i g h l i g h t s

� Stronger inhibition of bisphenol Atowards UGT2B isoforms than UGT1Aisoforms.� Competitive inhibition for UGT2B4,

noncompetitive inhibition for 2B7,2B15, 2B17.� In vitro-in vivo extrapolation (IV-IVE)

was performed.

g r a p h i c a l a b s t r a c t

Bisphenol A (BPA)

Inhibition

UGT2B4, 2B7, 2B15 and 2B17

Toxicology behaviour

1. The metabolism of xenobiotics and endogenous substances2. The exposure of BPA

Influence

a r t i c l e i n f o

Article history:Received 1 April 2013Received in revised form 12 May 2013Accepted 19 June 2013Available online 12 August 2013

Keywords:Bisphenol AUDP-glucuronosyltransferase (UGT)Risk evaluationRecombinant enzymes

a b s t r a c t

Bisphenol A (BPA), the important endocrine-disrupting chemical (EDC), has been reported to be able toinduce various toxicity. The present study aims to understand the toxicity behavior of bisphenol Athrough evaluating the inhibition profile of bisphenol A towards UDP-glucuronosyltransferase (UGT) iso-forms. In vitro recombinant UGTs-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction wasemployed as probe reaction for all the tested UGT isoforms. The results showed that bisphenol A exertedstronger inhibition towards UGT2B isoforms than UGT1A isoforms. Furthermore, the inhibition kinetictype and parameters (Ki) were determined for the inhibition of bisphenol A towards UGT2B4, 2B7,2B15, and 2B17. Bisphenol A exhibited the competitive inhibition towards UGT2B4, and noncompetitiveinhibition towards UGT2B7, 2B15 and 2B17. The inhibition kinetic parameters (Ki) were calculated to be1.1, 32.6, 5.6, and 19.9 lM for UGT2B4, 2B7, 2B15 and 2B17, respectively. In combination with the in vivoconcentration of bisphenol A, the elevation of exposure dose was predicted to increase by 29.1%, 1%, 5.7%,and 1.6% for UGT2B4, 2B7, 2B15, and 2B17, indicating the high influence of bisphenol A towards thein vivo UGT2B isofroms-mediated metabolism of xenobiotics and endogenous substances. All these dataprovide the supporting information for deeper understanding of toxicology of bisphenol A.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Bisphenol A (BPA, 4,40-isopropylidenediphenol), an importantendocrine-disrupting chemical (EDC), has been widely used as akey component in manufacturing polycarbonate plastics and epoxyresins. BPA can be detected in our everyday consumer products,

1190 H.-M. Jiang et al. / Chemosphere 93 (2013) 1189–1193

such as baby bottles, toys, dental sealants, eyeglass lenses, reusablewater bottles, plastic stretch films, consumer electronics, digitalmedia (CDs, DVDs), automobiles, medical equipment, food and bev-erage can linings and glass jar tops (Vandenberg et al., 2007).

The risk of BPA utilization has been speculated in recent years.The exposure of humans to BPA have been correlated with a varietyof diseases, including diabetes (Lang et al., 2008), cardiovascular dis-ease (Melzer et al., 2010), recurrent miscarriages and increasednumbers of premature deliveries in women (Sugiura-Ogasawaraet al., 2005; Cantonwine et al., 2010), and influence of immune sys-tem (Yang et al., 2009). Based on these reported risk of BPA, more andmore government agencies provide a strick regulation towards theutilization of BPA. For example, in Europe, the European food safetyauthority (EFSA) sets the tolerable daily intake (TDI) for BPA to be0.05 mg kg�1 body weight (bw) day�1 (Tyl et al., 2008).

Human uridine glucuronosyltransferases (UGTs) are the mem-brane proteins of the endoplasmic reticulum, and involved in theconjugation metabolism of multiple xenobiotics and endogenoussubstances (Fang et al., 2013; Song et al., 2013; Dong et al.,2013). To date, the role of UGTs in the metabolic elimination ofBPA has been detailedly studied. BPA monoglucuronide has beenidentified to be the predominant in vivo metabolite in rats, mon-keys and humans (Pottenger et al., 2000; Kurebayashi et al.,2002; Volkel et al., 2002). The human UGT2B15 was demonstratedto be main UGT isoform involved in the glucuronidation of BPA(Hanioka et al., 2008a,b). Based on these results, some contradic-tions existed that rapid glucuronidation elimination of BPA can sig-nificantly weaken the risk of BPA (Trdan Lusin et al., 2012).

Inhibition of various UGT isoforms by various xenobiotics andendogenous substances have been widely demonstrated in the pre-vious literatures (Schneider et al., 1993; Fang et al., 2013; Lu et al.,2013), which might result in the slower elimination of drugs andpossible metabolic disorders of endogenous substances. In thepresent study, we tried to evaluate the risk of bisphenol A fromthe new insights of UGTs’ inhibition. As previously reported, thein vitro recombinant UGTs-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was used in the present study.

2. Materials and methods

2.1. Chemicals

Bisphenol A (purity P 99%), 4-methylumbelliferone (4-MU),4-methylumbelliferone-b-D-glucuronide (4-MUG), Tris–HCl,7-hydroxycoumarin, and uridine-50-diphosphoglucuronic acid(UDPGA) (trisodium salt) were purchased from Sigma–Aldrich (StLouis, MO). Recombinant human UGT isoforms (UGT1A3, UGT1A4,UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7,UGT2B15 and UGT2B17) expressed in baculovirus-infected insectcells were obtained form BD Gentest Corp. (Woburn, MA, USA). Allother reagents were of HPLC grade or of the highest grade commer-cially available.

2.2. Initial screening of bisphenol A’s inhibition towards recombinantUGTs-catalyzed 4-MU glucuronidation

4-MU, the nonspecific probe substrate for all the UGT isoforms,was used in the present study to initially screen the inhibition poten-tial of ginsenosides as previously reported (Dong et al., 2012). A typ-ical incubation system (total volume = 200 lL) containedrecombinant UGTs, 5 mM UDPGA, 5 mM MgCl2, 50 mM Tris–HCl(pH = 7.4), and 4-MU in the absence or presence of different concen-trations of bisphenol A. Bisphenol A was dissolved in DMSO, and thefinal concentration of DMSO was below 0.5% (v/v). The used incuba-tion time and protein concentration were previously determined to

ensure the reaction rate within the linear range. The 4-MU concen-tration was equally to known Km or S50 values for each UGT isoform(Zhu et al., 2012). The incubation reaction was initiated through add-ing UDPGA to the mixture after 5-min pre-incubation at 37 �C. Thereactions were quenched by adding 100 lL acetonitrile with 7-hydroxycoumarin (100 lM) as internal standard. The mixture wascentrifuged at 20000 � g for 10 min, and an aliquot of supernatantwas transferred to an auto-injector vial for HPLC analysis. The HPLCsystem (Shimadzu, Kyoto, Japan) contained a SCL-10A system con-troller, two LC-10AT pumps, a SIL-10A auto injector, a SPD-10AVPUV detector. Chromatographic separation was carried out using aC18 column (4.6 � 200 mm, 5 lm, Kromasil) at a flow rate of1 mL min�1 and UV detector at 316 nm. The mobile phase consistedof acetonitrile (A) and H2O containing 0.5% (v/v) formic acid (B). Thefollowing gradient condition was used: 0–15 min, 95–40% B; 15–20 min, 10% B; 20–30 min, 95% B.

2.3. Inhibition of bisphenol A towards recombinant UGT1A4-catalyzedtrifluoperazine (TFP) glucuronidation

Due to the low catalytic activity of UGT1A4 towards 4-MU glu-curonidation (Uchaipichat et al., 2008), the UGT1A4-catalyzed TFPglucuronidation was performed to evaluate the inhibition potentialof bisphenol A towards UGT1A4 activity. TFP (40 lM, near its kmvalue) was incubated with recombinant UGT1A4 (0.1 mg mL�1)at 37 �C for 20 min in the absence or presence of bisphenol A(Uchaipichat et al., 2006).

2.4. Kinetic analysis of bisphenol A’s inhibition towards UGT2B4,UGT2B7, UGT2B15, and UGT2B17 activity

The glucuronidation velocity was determined at various con-centrations of 4-MU and bisphenol A. Dixon plot and Linewe-aver–Burk plot were performed to determine the inhibitionkinetic type, and the second plot using the slopes from the Linewe-aver–Burk plot versus the concentrations of bisphenol A.

2.5. In vitro-in vivo extrapolation (IV-IVE)

In vitro-in vivo extrapolation (IV-IVE) was performed using thefollowing equation as previously described (Fang et al., 2013)

AUCi=AUC ¼ 1þ ½I�invivo=Ki

The terms are defined as follows: AUCi/AUC is the predicted ra-tio of in vivo exposure of xenobiotics or endogenous substanceswith or without the co-exposure of bisphenol A. [I]in vivo is thein vivo exposure concentration of bisphenol A, and the Ki valuewas in vitro inhibition kinetic parameters.

3. Results

3.1. The stronger inhibition of bisphenol A towards UGT2B isoformsthan UGT1A isoforms

As shown in Fig. 1, at 100 lM of bisphenol A, the activity ofUGT1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and2B17 was inhibited by 76.7%, 79.5%, 89.1%, 73.7%, 32.4%, 55%,76.8%, 100%, 94.3%, 100%, and 100%, respectively. From these data,we made a conclusion that bisphenol A exhibited stronger inhibi-tion towards UGT2B isoforms than UGT1A isoforms.

3.2. Inhibition kinetic type and parameters of bisphenol A towardsUGT2B isoforms

The inhibition kinetic type and parameters (Ki) were deter-mined for the inhibition of bisphenol A towards UGT2B isoforms.

Fig. 3. Determination of inhibition type and kinetic parameters of bisphenol A towards UGcatalyzed 4-MU glucuronidation; (B) Dixon plot of the inhibition of bisphenol A towardsthe inhibition of bisphenol A towards recombinant UGT2B7-catalyzed 4-MU glucuronconcentrations of bisphenol A.

Fig. 2. Determination of inhibition type and kinetic parameters of bisphenol A towards UGcatalyzed 4-MU glucuronidation; (B) Dixon plot of the inhibition of bisphenol A towardsthe inhibition of bisphenol A towards recombinant UGT2B4-catalyzed 4-MU glucuronconcentrations of bisphenol A.

Fig. 1. Screening of the inhibition potential of bisphenol A towards UGT1A andUGT2B isoforms. 100 lM of bisphenol A was utilized. Triplicate experiments werecarried out.

H.-M. Jiang et al. / Chemosphere 93 (2013) 1189–1193 1191

The concentration-dependent inhibition of bisphenol A towardsUGT2B4, 2B7, 2B15 and 2B17 was observed (Figs. 2A, 3A, 4A and5A). For the inhibition of bisphenol A towards UGT2B4, the inter-section point was located in the second quadrant in Dixon plot(Fig. 2B), and in the vertical axis in Lineweaver–Burk plot(Fig. 2C), indicating the competitive inhibition of bisphenol A to-wards UGT2B4. For the inhibition of bisphenol A towards UGT2B7,2B15, and 2B17, the intersection point was located in the horizon-tal axis in Dixon plot (Figs. 3B, 4B and 5B) and Lineweaver–Burkplot (Figs. 3C, 4C and 5C), suggesting the noncompetitive inhibitionof bisphenol A towards all these three UGT2B isoforms. The inhibi-

T2B7. (A) Dose-dependent inhibition of bisphenol A towards recombinant UGT2B7-recombinant UGT2B7-catalyzed 4-MU glucuronidation; (C) Lineweaver–Burk plot ofidation; (D) Second plot with the slopes from Lineweaver–Burk plot towards the

T2B4. (A) Dose-dependent inhibition of bisphenol A towards recombinant UGT2B4-recombinant UGT2B4-catalyzed 4-MU glucuronidation; (C) Lineweaver–Burk plot ofidation; (D) Second plot with the slopes from Lineweaver–Burk plot towards the

Fig. 4. Determination of inhibition type and kinetic parameters of bisphenol A towards UGT2B15. (A) Dose-dependent inhibition of bisphenol A towards recombinantUGT2B15-catalyzed 4-MU glucuronidation; (B) Dixon plot of the inhibition of bisphenol A towards recombinant UGT2B15-catalyzed 4-MU glucuronidation; (C) Lineweaver–Burk plot of the inhibition of bisphenol A towards recombinant UGT2B15-catalyzed 4-MU glucuronidation; (D) Second plot with the slopes from Lineweaver–Burk plottowards the concentrations of bisphenol A.

Fig. 5. Determination of inhibition type and kinetic parameters of bisphenol A towards UGT2B17. (A) Dose-dependent inhibition of bisphenol A towards recombinantUGT2B17-catalyzed 4-MU glucuronidation; (B) Dixon plot of the inhibition of bisphenol A towards recombinant UGT2B17-catalyzed 4-MU glucuronidation; (C) Lineweaver–Burk plot of the inhibition of bisphenol A towards recombinant UGT2B17-catalyzed 4-MU glucuronidation; (D) Second plot with the slopes from Lineweaver–Burk plottowards the concentrations of bisphenol A.

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tion kinetic parameters (Ki) were determined using the plot withslopes from Lineweaver–Burk plot versus the concentrations ofbisphenol A, and the values were calculated to be 1.1, 32.6, 5.6,and 19.9 lM for UGT2B4, 2B7, 2B15 and 2B17, respectively(Figs. 2D, 3D, 4D and 5D).

4. Discussion

Previous studies have shown that bisphenol A inhibits the activ-ity of UGT1A6 when using both 4-methylumbelliferone (4-MU) and

serotonin as probe substrates, which might contribute to the hor-mone disruption and reproductive toxicity of bisphenol A (Haniokaet al., 2008a,b). However, the complete inhibition profile of bisphe-nol A towards all UGT isoforms remains unclear. The present studytried to evaluate the inhibitory potential of bisphenol towards vari-ous important UGT isoforms in the liver and intestine using in vitroincubation system. Compared with the inhibition of the isoformsin UGT1A family, bisphenol A exhibited stronger inhibitory capabil-ity towards the isoforms in UGT2B family. UGT2B4 can catalyze theglucuronidation conjugation of various important molecules,

H.-M. Jiang et al. / Chemosphere 93 (2013) 1189–1193 1193

including bile acids, 5a-reduced androgens, catecholesterogens, andphenolic and monoterpenoid compounds (Pillot et al., 1993; Lev-esque et al., 1999; King et al., 2000). Previous studies have shownthat UGT2B4 plays a key role for the glucuronidation of 6a-hydrox-ylated bile acid hyodeoxycholic acid (HDCA) in human liver (Pillotet al., 1993). The decreased UGT2B4 activity might induce the path-ophysiological accumulation of bile acids. Additionally, the de-creased activity of UGT2B4 can significantly increase the risk ofcancer (Dura et al., 2012). UGT2B7 has been arguably the mostimportant UGT isoform in human, and been demonstrated to be in-volved in the glucuronidation elimination of many important com-pounds, including nonsteroidal anti-inflammatory drugs (NSAIDs),ethanol and opioids (Miners et al., 2010; Al Saabi et al., 2013).UGT2B15 was the main enzyme involved in the metabolic elimina-tion of bisphenol A, and the inhibition of UGT2B15 might increasethe exposure of bisphenol A which might induce stronger toxicity(Partosch et al., 2013). UGT2B17 has been demonstrated to be in-volved in the metabolism of many important endogenous sub-stances, such as testosterone (Okano et al., 2013).

Furthermore, we aims to evaluate whether the in vivo concen-tration of bisphenol A can induce the serious toxicity results dueto the inhibition of these UGT2B isoforms. The in vivo exposureof bisphenol A was reported to be 0.24–38.53 lg L�1 (0.001–0.17 lM) in maternal urine, and 0.42–73.50 lg L�1 (0.002–0.32 lM) in the urine of the child (Perera et al., 2012). Due to thebig difference among the individuals, we used the maximum con-centration of bisphenol A (0.32 lM). The in vivo concentration [I]/in vitro kinetic parameters (Ki) was calculated to be 29.1%, 1%,5.7%, and 1.6% for UGT2B4, 2B7, 2B15, and 2B17.

In conclusion, bisphenol A exhibited stronger inhibition to-wards UGT2B isoforms than UGT1A isoforms. Among the UGT2Bisoforms, the inhibition capability orders were as followed: UGT2-B4 > UGT2B15 > UGT2B17 > UGT2B7. All these information washelpful for understanding of the toxicity of bisphenol A.

Acknowledgement

This work was supported by 973 Program of China(2013CB531800 & 2009CB522808), the National Natural ScienceFoundation of China (No. 81202586, 81001473, 81102507 &81273590).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2013.06.070.

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