Ipso substitution of bisphenol A catalyzed by microsomal cytochrome P450 and enhancement of estrogenic activity

doi:10.1016/j.toxlet.2011.03.010

Toxicology Letters

Volume 203, Issue 1, 30 May 2011, Pages 92-95

https://doi.org/10.1016/j.toxlet.2011.03.010 Get rights and content

Abstract

Bisphenol A (BPA), an industrial chemical with estrogenic activity, was investigated as a substrate for the ipso-metabolism catalyzed by microsomal cytochrome P450 (P450). BPA was expected to be transformed to a quinol via an ipso-addition reaction; however, hydroquinone (HQ) was detected as a metabolite via an ipso-substitution reaction. Isopropenylphenol (IPP) and hydroxycumyl alcohol (HCA) were also produced as eliminated metabolites by C-C bond scission via ipso-substitution. Incorporation of the ¹⁸O atom to HCA from H₂¹⁸O suggested the presence of a carbocation intermediate. Bulkiness of p-substituted group of BPA and/or stability of the eliminated carbocation intermediate may cause ipso-substitution of BPA. CYP3A4 and CYP3A5 showed higher activity for ipso-substitution. CYP2D6*1 also showed the activity; however, the other 9 isozymes did not. IPP showed ER-binding activity in the same degree of BPA. Furthermore, the ER-binding activity of HCA was about a hundred times greater than that of BPA. These results suggested that this new metabolic pathway contributes to the activation of the estrogenic activity of BPA.

Graphical abstract

Highlights

► Bisphenol A is transformed to novel metabolites by P450 via ipso-metabolism. ► The novel metabolites were produced by C–C bond scission. ► CYP3A4 and CYP3A5 showed higher activity for ipso-substitution. ► ER-binding activity of one novel metabolite was 100-fold greater than that of BPA.

Introduction

Bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane) is one of the most widely used industrial chemicals for the production of resins and plastics. This compound is considered to be an endocrine disrupter (Krishman et al., 1993). BPA is widely detected from human urine samples (Calafat et al., 2005); therefore, the clarification of its metabolism has value.

Cytochrome P450 (P450) is an enzyme that catalyzes the oxidation of a wide variety of xenobiotic chemicals, including drugs (Ortiz de Montellano, 2005). BPA is known to be metabolized to 2,2-bis(4-hydroxyphenyl)propanol, o-hydroxybisphenol, or bisphenol-o-quinone as an oxidative product by P450, and the glucuronide is a major conjugated metabolite (Knaak and Sullivan, 1966, Jaeg et al., 2004). Yoshihara et al. (2001) reported that 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) was formed from BPA by an S9 fraction and its estrogenic activity was higher than that of BPA (Okuda et al., 2010).

We have previously demonstrated that the ipso-position metabolism reaction of para-substituted phenols is catalyzed by P450 (Fig. 1). In other words, when a substituent is a member of the alkyl group, a quinol metabolite is formed through an ipso-addition reaction. On the other hand, when a substituent is a carboxyl, acetyl, or hydroxymethyl group, a hydroquinone (HQ) is formed by an ipso-substitution reaction (Ohe et al., 1994, Ohe et al., 1995, Ohe et al., 1997). Estrone and 17β-estradiol, each of which contains a para-alkylphenol moiety, are also metabolized through ipso-addition to the corresponding quinols by P450 (Ohe et al., 2000). Nonylphenol, an endocrine disrupter, is also metabolized to the corresponding quinol, which has no estrogenic activity (Tezuka et al., 2007). In the case of nonylphenol, the ipso-position metabolism by P450 led to metabolic inactivation.

BPA also contains a para-alkylphenol moiety; therefore, it is expected to be metabolized by an ipso-addition reaction. In this study, the new metabolic pathway of BPA catalyzed by rat liver microsomes and human P450 (CYP) is investigated. The estrogenic activity of novel metabolites is also examined.

Section snippets

Chemicals

The sources of the materials used were as follows: BPA; hydroquinone; and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Glucose-6-phosphate (G-6-P) and G-6-P dehydrogenase (G-6-P DHase) were obtained from Roche Diagnostics (Basel, Switzerland). NADP⁺ was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). p-Acetoxybenzophenone and a methylmagnesium iodide solution were obtained from Kanto Chemical Co., Inc.

Metabolism of BPA by rat or human liver microsomes

BPA was incubated with phenobarbital-treated rat liver microsomes or pooled human liver microsomes. We expected that a quinol of BPA would be produced as a metabolite of ipso-addition reaction because BPA contains a para-alkyl group. The metabolite extracts were analyzed by GC-MS after trimethylsilylation, but no ipso-addition product was detected. On the other hand, HQ was detected as a metabolite via an ipso-substitution reaction (Table 1). Trimethylsilylated HQ was identified on the basis of

Discussion

We have already revealed that quinols are produced via an ipso-addition by P450 when the substituents on the para-position of phenols are alkyl groups (Ohe et al., 1997). Therefore, we expected that the ipso-substitution of BPA would not proceed because BPA is a para-alkylated phenol. However, HQ was generated from BPA in both rat and human liver microsome systems (Table 1). This result suggests that ipso-substitution proceeded in the case of BPA in spite of para-alkyl moiety.

When the C–C bond

Conflict of interest statement

None declared.

Acknowledgement

This study was supported in part by the “Open Research Center” Project for Private Universities, a matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science, and Technology), 2007–2011.

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Toxicology Letters

Abstract

Graphical abstract

Highlights

Introduction

Section snippets

Chemicals

Metabolism of BPA by rat or human liver microsomes

Discussion

Conflict of interest statement

Acknowledgement

References (19)

In vitro conversion of environmental estrogenic chemical bisphenol A to DNA binding metabolite(s)

Biochem. Biophys. Res. Commun.

Urinary concentration of bisphenol A and 4-nonylphenol in a human reference population

Environ. Health Perspect.

Characterization of new bisphenol A metabolites produced by CD1 mice liver microsomes and S9 fractions

J. Agric. Food Chem.

Metabolism of bisphenol A in the rat

Toxicol. Appl. Pharmacol.

Degradation pathway of bisphenol A: does ipso substitution apply to phenols containing a quaternary α-carbon structure in the para position?

Appl. Environ. Microbiol.

Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving

Endocrinology

Employment of the human estrogen receptor β ligand-binding domain and co-activator SRC1 nuclear receptor-binding domain for the construction of a yeast two-hybrid detection system for endocrine disrupters

J. Biochem.

Protein measurement with the Folin phenol reagent

J. Biol. Chem.

Application of chemical P450 model systems to studies on drug metabolism. I. Phencyclidine: a multi-functional model substrate

Chem. Pharm. Bull.

Cited by (65)

Less is more: A new perspective for toxicity of emerging contaminants by structures, protein adducts and proteomics

The fate of bisphenol A, bisphenol S, and their respective glucuronide metabolites in ovarian cells

Mn(III)-mediated bisphenol a degradation: Mechanisms and products

Biotransformation of BPA via epoxidation catalyzed by Cytochrome P450

The bisphenol A metabolite MBP causes proteome alterations in male Cyprinodon variegatus fish characteristic of estrogenic endocrine disruption

Urinary bisphenol A and its interaction with CYP17A1 rs743572 are associated with breast cancer risk