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