Ly Nos2, Ppar, and Dnmt1 presented patterns of changes in transcript levels that reflected opposing responses to ethanol and betaine supplementation. In particular, ethanol feeding increased both the transcript and protein levels of Dnmt1 in heterozygous mice and of Nos2 in pooled genotype samples, each of which were reduced by provision of the methyl donor betaine, whereas the transcript levels of Ppar were reduced by ethanol and maintained at control levels by betaine (Table 3 and Fig. 4). Fifth, the findings of significant methylation changes in the gene body of Nos2 (Table 4) are supported by the novel observation from methylome analysis that the effects of ethanol on mouse chromosomes are confined to gene bodies and are not found in promoter regions (Fig. 3). The significant correlations of these findings with SAM:SAH ratios from all three diet groups supports a potential link between the effects of ethanol and betaine with hepatic methionine metabolism and gene specific methylation. However, since gene expression is regulated by many factors, we cannot infer a specific causative relationship between the methylation pattern of Nos2 (Table 4) at this gene body site and its expression (Table 3), whereas the clinical relevance of this finding may only be established by additional studies in liver biopsies from ASH patients. iNOS is required for the sustained generation of nitric oxide, which, in turn, causes a broad spectrum of injury, including processes of lipid peroxidation, mitochondrial disruption, and DNA damage with pro-apoptotic effects (Davis et al., 2001). Others correlated increased liver Nos2 expression and activity in rats fed high ethanol diets with the severity of steatosis and necroinflammation in association with activation of nuclear factor kappa beta and induction of tumor necrosis factor alpha (Yuan et al., 2006). Furthermore, mitochondrial dysfunction and the histopathology of ASH were ML390 site significantly attenuated in ethanol-fed Nos2 knock-out mice (Venkatraman et al., 2004) and in mice treated with a selective iNOS inhibitor (McKim et al., 2003). Consistent with our present findings, others found more than 40-fold elevation in hepatic Nos2 transcript levels in mice fed an intragastric ethanol and high fat diet, together with more severe histopathology than shown in the present experiment (Xu et al., 2011), whereas betaine supplementation reversed liver histopathology, elevated iNOS levels, and nitric oxide accumulation in an ethanol fed rat model of ASH (Kharbanda et al., 2012).NIH-PA Author H 4065 chemical information manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAlcohol Clin Exp Res. Author manuscript; available in PMC 2015 June 01.Medici et al.PagePPAR functions as a transcription factor to induce the expressions of a series of genes involved in fatty acid transport, mitochondrial fatty acid oxidation, catabolism, and inflammatory responses. Our present data showed a discrepancy between the transcript and protein expressions of Ppar in response to ethanol feeding (Table 3 and Fig. 4) and no changes among diet groups in its DNA methylation according to pyrosequencing (Table 4). Another study showed that Ppar gene expression was both increased and associated with increased DNA methylation in ethanol fed rats (Khachatoorian et al., 2013), while others showed that down-regulation of Ppar gene expression is involved in the development of steatosis associated with ASH (Aoyama et al., 1998). The regulation of Ppar expression is comple.Ly Nos2, Ppar, and Dnmt1 presented patterns of changes in transcript levels that reflected opposing responses to ethanol and betaine supplementation. In particular, ethanol feeding increased both the transcript and protein levels of Dnmt1 in heterozygous mice and of Nos2 in pooled genotype samples, each of which were reduced by provision of the methyl donor betaine, whereas the transcript levels of Ppar were reduced by ethanol and maintained at control levels by betaine (Table 3 and Fig. 4). Fifth, the findings of significant methylation changes in the gene body of Nos2 (Table 4) are supported by the novel observation from methylome analysis that the effects of ethanol on mouse chromosomes are confined to gene bodies and are not found in promoter regions (Fig. 3). The significant correlations of these findings with SAM:SAH ratios from all three diet groups supports a potential link between the effects of ethanol and betaine with hepatic methionine metabolism and gene specific methylation. However, since gene expression is regulated by many factors, we cannot infer a specific causative relationship between the methylation pattern of Nos2 (Table 4) at this gene body site and its expression (Table 3), whereas the clinical relevance of this finding may only be established by additional studies in liver biopsies from ASH patients. iNOS is required for the sustained generation of nitric oxide, which, in turn, causes a broad spectrum of injury, including processes of lipid peroxidation, mitochondrial disruption, and DNA damage with pro-apoptotic effects (Davis et al., 2001). Others correlated increased liver Nos2 expression and activity in rats fed high ethanol diets with the severity of steatosis and necroinflammation in association with activation of nuclear factor kappa beta and induction of tumor necrosis factor alpha (Yuan et al., 2006). Furthermore, mitochondrial dysfunction and the histopathology of ASH were significantly attenuated in ethanol-fed Nos2 knock-out mice (Venkatraman et al., 2004) and in mice treated with a selective iNOS inhibitor (McKim et al., 2003). Consistent with our present findings, others found more than 40-fold elevation in hepatic Nos2 transcript levels in mice fed an intragastric ethanol and high fat diet, together with more severe histopathology than shown in the present experiment (Xu et al., 2011), whereas betaine supplementation reversed liver histopathology, elevated iNOS levels, and nitric oxide accumulation in an ethanol fed rat model of ASH (Kharbanda et al., 2012).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAlcohol Clin Exp Res. Author manuscript; available in PMC 2015 June 01.Medici et al.PagePPAR functions as a transcription factor to induce the expressions of a series of genes involved in fatty acid transport, mitochondrial fatty acid oxidation, catabolism, and inflammatory responses. Our present data showed a discrepancy between the transcript and protein expressions of Ppar in response to ethanol feeding (Table 3 and Fig. 4) and no changes among diet groups in its DNA methylation according to pyrosequencing (Table 4). Another study showed that Ppar gene expression was both increased and associated with increased DNA methylation in ethanol fed rats (Khachatoorian et al., 2013), while others showed that down-regulation of Ppar gene expression is involved in the development of steatosis associated with ASH (Aoyama et al., 1998). The regulation of Ppar expression is comple.