Abstract: Maternal alcohol consumption is the leading known non-genetic cause of mental retardation.  Prenatal alcohol exposure can cause a range of structural and functional birth defects, which are defined as Fetal Alcohol Spectrum Disorders (FASD).  Growing evidence suggests that excessive cell death in selected cell populations is a major component of the pathogenesis of FASD.  This suggests that a strategy for protecting against ethanol’s teratogenesis by epigenetically regulating the genes involved in the apoptotic pathway is promising for effective intervention and prevention of FASD. We have recently found that treatment with ethanol resulted in a significant decrease in miR-125b expression in neural crest cells (NCCs) and mouse embryos.  We also validated that Bcl-2 antagonist killer 1 (Bak1) and p53-upregulated modulator of apoptosis (PUMA) are the direct targets of miR-125b in NCCs.  In addition, over-expression of miR-125b significantly reduced the ethanol-induced increase in Bak1 and PUMA protein expression, caspase-3 activation, and apoptosis in NCCs, indicating that miR-125b can modulate ethanol-induced apoptosis by the regulation of Bcl-2 and p53 pathways.  Furthermore, microinjection of miR-125b mimic resulted in a significant increase in miR-125b expression and a decrease in the protein expression of Bak1 and PUMA in ethanol-exposed mouse embryos. Up-regulation of miR-125b also significantly reduced ethanol-induced caspase-3 activation and diminished ethanol-induced growth retardation in mouse embryos.  Our studies have also shown that exposure to ethanol resulted in a significant increase in the activities of histone deacetylase (HDAC) and DNA methyltransferase (DNMT), and increased the methylation of Bcl-2 promoter in NCCs.  In addition, ChIP-qPCR assay revealed that ethanol exposure significantly decreased acetyl-histone H3 binding to the Bcl-2 promoter and the expression of Bcl-2.  Supplementing with sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables and a dual epigenetic regulator which can inhibit both DNMTs and HDACs, reversed the ethanol-induced hypermethylation of Bcl-2 promoter and reduction in acetyl-histone H3 binding to the Bcl-2 promoter.  Treatment with SFN also restored the expression of Bcl-2 in ethanol-exposed NCCs.  Furthermore, supplementing with SFN diminished ethanol-induced apoptosis in NCCs and in mouse embryos exposed to ethanol in vivo. These results demonstrate that SFN can epigenetically restore the expression of Bcl-2 and attenuate ethanol-induced apoptosis by decreasing methylation and increasing histone acetylation at the Bcl-2 promoter.  These findings support the potential of dietary consumption of SFN or SFN-rich broccoli sprouts to attenuate ethanol-induced apoptosis and confer in vivo protection against FASD through epigenetic regulation of the expression of anti-apoptotic genes.