Participation of covalent modification of Keap1 in the activation of Nrf2 by tert-butylbenzoquinone, an electrophilic metabolite of butylated hydroxyanisole

Abstract

Butylated hydroxyanisole (BHA) is an antioxidant and class-2B carcinogen. It is biotransformed to tert-butylhydroquinone (TBHQ), which readily auto-oxidizes to the electrophilic metabolite tert-butylbenzoquinone (TBQ). BHA and TBHQ activate Nrf2, a transcription factor that is negatively regulated by Keap1 and plays a role in the initial response to chemicals causing oxidative or electrophilic stress, although, the exact mechanism of Nrf2 activation remains unclear. Here, we examined the role of TBQ in Nrf2 activation. Exposure of RAW264.7 cells to TBQ activated Nrf2 and up-regulated its downstream proteins; under these conditions, TBQ produced cellular reactive oxygen species (ROS). However, while pretreatment with catalase conjugated with polyethylene glycol (PEG-CAT) did not affect the TBQ-induced activation of Nrf2, the ROS generation caused by TBQ was entirely abolished by PEG-CAT, suggesting that ROS is not the dominant factor for TBQ-dependent Nrf2 activation. A click chemistry technique indicated that TBQ chemically modifies Keap1. Furthermore, ultrahigh performance liquid chromatography-tandem mass spectrometry analysis with purified Keap1 revealed that TBQ covalently binds to Keap1 through Cys23, Cys151, Cys226, and Cys368. These results suggest that TBQ derived from BHA activates Nrf2 through electrophilic modification of Keap1 rather than ROS formation.

Introduction

Butylated hydroxyanisole (BHA) is a phenolic antioxidant widely used as a synthetic food and cosmetic additive to preserve oils and fats; it readily undergoes O-dealkylation by cytochrome P450 isozymes to produce tert-butylhydroquinone (TBHQ) (Verhagen et al., 1989) (Fig. 1). This hydroquinone metabolite further auto-oxidizes to tert-butylbenzoquinone (TBQ) and these metabolites are associated with reactive oxygen species (ROS) generation. BHA itself, however, lacks oxygen activating properties (Kahl et al., 1989). We previously reported that redox cycling between 9,10-phenanthraquinone (9,10-PQ) and its two-electron reduction metabolite 9,10-dihydroxyphenanthrene (9,10-PQH₂) is associated with oxidative stress (Taguchi et al., 2007). Based on these reaction sequences, a redox cycle reaction of TBHQ with TBQ, leading to the production of superoxide and hydrogen peroxide, is possible during the metabolic activation of BHA (Fig. 1).

The Keap1/Nrf2 system regulates the expression of genes associated with antioxidants, phase II xenobiotic detoxification enzymes, and phase III transporters (Wild et al., 1999, Ishii et al., 2000, Chanas et al., 2002, Cho et al., 2002, Hayashi et al., 2003). Under basal conditions, Nrf2 undergoes rapid degradation by the ubiquitin–proteasome pathway, resulting in minimal levels in the cytoplasm. However, once cells are exposed to electrophiles or chemicals that cause oxidative stress, the reactive thiol groups of Keap1, the negative regulator of Nrf2, are covalently modified, thereby activating Nrf2 (Itoh et al., 1997, Kobayashi and Yamamoto, 2006).

Several lines of evidence suggest that TBHQ derived from BHA can activate Nrf2. Nrf2 activation and the subsequent up-regulation of its downstream genes during TBHQ exposure are thought to be attributable to oxidative stress (e.g., ROS formation) (Gharavi et al., 2007). Dinkova-Kostova and Wang (2011) recently reported that TBHQ itself does not activate Nrf2; rather, its oxidation product TBQ is the ultimate inducer because of its electrophilic properties (Wang et al., 2010); see also Fig. 1). However, the relative contributions of oxidative stress and covalent modification of TBQ, derived from TBHQ, to Nrf2 activation are not well understood. The purpose of this study is, therefore, to clarify this issue by using RAW264.7 cells (a mouse macrophage cell line) and recombinant Keap1.