Induction of the Nrf2-driven antioxidant response by tert-butylhydroquinone prevents ethanol-induced apoptosis in cranial neural crest cells
Graphical abstract
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.
References (47)
- et al.
Fetal alcohol syndrome is now leading cause of mental retardation
Lancet
(1986) - et al.
Differential sensitivity of mouse neural crest cells to ethanol-induced toxicity
Alcohol
(2000) - et al.
The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress
J Biol Chem
(2009) - et al.
The chemical diversity and distribution of glucosinolates and isothiocyanates among plants
Phytochemistry
(2001) - et al.
tBHQ inhibits LPS-induced microglial activation via Nrf2-mediated suppression of p38 phosphorylation
Biochem Biophys Res Commun
(2009) - et al.
Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism
J Nutri
(2001) - et al.
Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element—degradation of Nrf2 by the 26 S proteasome
J Biol Chem
(2003) - et al.
Activation of Nrf2 by arsenite and monomethylarsonous acid is independent of Keap1–C151: enhanced Keap1–Cul3 interaction
Toxicol Appl Pharmacol
(2008) - et al.
PKC delta mediates Nrf2-dependent protection of neuronal cells from NO-induced apoptosis
Biochem Biophys Res Commun
(2009) - et al.
Nrf2, a Cap‘n’Collar transcription factor, regulates induction of the heme oxygenase-1 gene
J Biol Chem
(1999)
Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis
J Biol Chem
Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo
J Biol Chem
Fetal alcohol syndrome
Am J Med Genet C: Semin Med Genet
Increased cell-death and reduced neural crest cell numbers in ethanol-exposed embryos—partial basis for the fetal alcohol syndrome phenotype
Alcohol Clin Exp Res
Selective vulnerability of embryonic cell populations to ethanol-induced apoptosis: implications for alcohol-related birth defects and neurodevelopmental disorder
Alcohol Clin Exp Res
Experimental fetal alcohol syndrome: proposed pathogenic basis for a variety of associated facial and brain anomalies
Am J Med Genet
Alcohol-induced cell death in the embryo
Alcohol Health Res World
Fetal alcohol syndrome—the vulnerability of the developing brain and possible mechanisms of damage
Metab Brain Dis
Free radicals and ethanol-induced cytotoxicity in neural crest cells
Alcohol Clin Exp Res
Fetal alcohol syndrome—embryogenesis in a mouse model
Science
The proliferating field of neural crest stem cells
Dev Dyn
The neural crest and neural crest cells: discovery and significance for theories of embryonic organization
J Biosci
Neural crest stem cells
Adv Exp Med Biol
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