Elsevier

Biochemical Pharmacology

Volume 80, Issue 1, 1 July 2010, Pages 144-149
Biochemical Pharmacology

Induction of the Nrf2-driven antioxidant response by tert-butylhydroquinone prevents ethanol-induced apoptosis in cranial neural crest cells

https://doi.org/10.1016/j.bcp.2010.03.004Get rights and content

Abstract

Previous studies have shown that ethanol exposure causes apoptosis in cranial neural crest cells (NCCs), an ethanol-sensitive cell population implicated in Fetal Alcohol Spectrum Disorders (FASD). Additionally, induction of endogenous antioxidants through activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to prevent oxidative stress and apoptosis in ethanol-exposed mouse embryos. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), an Nrf2 inducer, can protect NCCs against ethanol-induced apoptosis. Ethanol exposure was shown to cause a moderate increase in the protein expression of Nrf2 and its downstream antioxidants in the NCCs. Treatment of NCCs with tBHQ alone significantly increased the protein expression of Nrf2 and its downstream antioxidants and also significantly increased the activities of the antioxidant enzymes. In NCCs exposed to ethanol, the tBHQ-mediated antioxidant response prevented oxidative stress and apoptosis. These results clearly demonstrate that the activation of Nrf2 signaling confers protection against ethanol-induced apoptosis in NCCs.

Introduction

Prenatal alcohol (ethanol) exposure results in a range of structural and functional birth defects. These comprise the Fetal Alcohol Spectrum Disorder (FASD), with full-blown Fetal Alcohol Syndrome (FAS) representing the severe end of the spectrum. Notably, prenatal alcohol exposure is considered to be the leading known cause of mental retardation in the Western world [1], [2].

There are a number of factors that contribute to the genesis of the birth defects that ethanol causes, including the vulnerability of selected cell populations to ethanol-induced cytotoxicity [3], [4], [5], [6], [7]. As shown using rodent and avian animal models, among the vulnerable cell populations are cranial neural crest cells (NCCs) [3], [5], [8], [9], [10]. NCCs are progenitors of multiple cell types, including the skeletal and connective tissues of the face [11], [12], [13]. Ethanol has been shown to diminish NCC populations via the induction of apoptosis, a result that appears to contribute heavily to subsequent facial abnormalities [3], [5], [6], [8], [10].

Substantial evidence supports a critical role for oxidative stress in ethanol-induced apoptosis and subsequent malformations [8], [14], [15], [16], [17]. Reactive oxygen species (ROS) generation has been observed in mouse embryos exposed to ethanol both in vitro and in vivo[15], [17] with various embryonic cell populations, including NCCs, being involved [8], [14], [18], [19]. These findings provided a framework for studies employing exogenous antioxidants to reduce ethanol's teratogenicity. In this regard, superoxide dismutase (SOD) has been shown to diminish ethanol-induced superoxide anion generation, lipid peroxidation and cell death, as well as the incidence of neural tube defects in cultured mouse embryos [17]. In vivo studies have also shown that, in mice, maternal treatment with an SOD and catalase mimetic, EUK-134, reduces ethanol-induced apoptosis in selected cell populations in the developing limb buds and subsequent limb defects [20]. However, while promising for human application, exogenous antioxidants alone are not as effective in reducing ethanol's teratogenicity as desirable.

Another strategy for prenatal protection from ethanol-induced oxidative injury entails chemically mediated upregulation of endogenous antioxidants. In this light, recently, Nrf2 has been demonstrated to be a critical transcription factor that regulates the induction of phase 2 detoxifying and antioxidant genes [21], [22]. Under basal conditions, Nrf2 is anchored mainly in the cytoplasm through binding to Kelch-like ECH-associated protein 1 (Keap1), which in turn facilitates the ubiquitylation and subsequent proteolysis of Nrf2. When challenged by oxidative stress, Nrf2 dissociates from Keap1 and translocates into the nucleus where it forms a heterodimer with its partner, Maf and elicit the antioxidant response by the induction of a battery of gene products, including antioxidant genes and phase 2 detoxification enzymes [23], [24].

A wide range of natural and synthetic small molecules with diverse chemical backgrounds are potent inducers of Nrf2 activity [25], [26], [27]. Among these Nrf2 inducers are isothiocyanates, 1,2-dithiole-3-thiones (D3T) and tert-butylhydroquinone (tBHQ) [27], [28], [29]. Of these, tBHQ, which is approved for human use, is of particular interest. It is a metabolite of the widely used food antioxidant butylated hydroxyanisole that increases Nrf2 protein stability through inhibition of the Keap1-mediated ubiquitination [30], [31], [32]. It has been suggested that tBHQ directly acts on the thiol group of Keap1 by a C151-dependent mechanism [33].

Using an in vivo FASD model, recent studies have shown that maternal ethanol treatment increases both Nrf2 protein levels and Nrf2–ARE binding in mouse embryos. Ethanol exposure also resulted in a moderate increase in the mRNA expression of Nrf2 downstream target detoxifying and antioxidant genes as well as an increase in the expression of antioxidant proteins. Pretreatment with the Nrf2 inducer, D3T, significantly increased Nrf2 protein levels and Nrf2–ARE binding, and strongly induced the mRNA expression of Nrf2 downstream target genes. In addition, maternal D3T pretreatment resulted in a significant decrease in ethanol-induced ROS generation and apoptosis in the embryos [15]. These results demonstrate that Nrf2 signaling is involved in the induction of an antioxidant response in ethanol-exposed mouse embryos.

While the studies of intact embryos have contributed significantly to our base of knowledge regarding Nrf2 activation in mouse embryos following ethanol exposure, for a more complete understanding of the role Nrf2 signaling in ethanol-induced teratogenesis, studies focused on vulnerable cell populations are needed. To this end, the current study employed cultured NCCs to elucidate the molecular mechanisms involved in ethanol-induced Nrf2 activation in NCCs, and to determine whether the Nrf2 inducer, tBHQ, can provide protection against ethanol-induced oxidative stress and apoptosis in NCCs.

Section snippets

Animal care

C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) were mated for 2 h early in the light cycle. The time of vaginal-plug detection was considered 0 days, 0 h of gestation (GD 0:0). Mice were maintained on an ad libitum diet of breeder chow and water. Pregnant mice were killed on GD 10.5. The embryos were removed from the uterus and processed for NCC culture as described below. All protocols used in this study were approved by the University of North Carolina at Chapel Hill Institutional

Ethanol exposure and treatment with tBHQ significantly increased Nrf2 protein expression in NCCs

To determine whether ethanol exposure and treatment with tBHQ can induce Nrf2 expression in NCCs, Nrf2 protein level was examined in NCCs that were exposed to ethanol or tBHQ alone, or in combination. Western blot analysis revealed an increase in the level of Nrf2 protein in NCCs exposed to ethanol or tBHQ alone. As illustrated in Fig. 1, NCCs treated with 10 μM tBHQ alone showed a 2.8-fold increase in Nrf2 protein expression as compared to control cultures. Exposure to 100 mM ethanol for 24 h

Discussion

The results of this study show that in vitro exposure of a vulnerable embryonic cell type, NCCs, to teratogenic ethanol concentrations increases the protein expression of Nrf2. It also increases the protein expression and catalytic activity of Nrf2's downstream antioxidants, SOD and catalase. These results provide support for the hypothesis that Nrf2 signaling is involved in the induction of antioxidant response in ethanol-exposed mouse embryos. Importantly, they also, demonstrate that Nrf2

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work is supported by NIH grants AA017446 (S.-Y.C), AA013908 (S.-Y.C.), AA11605 (K.K.S) and AA012974 (K.K.S) from the National Institute on Alcohol Abuse and Alcoholism. The study sponsor was not involved in a study design, in the collection, analysis, and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication.

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