5). Overall, little interstrain difference in hepatic levels of methionine and SAM, or the effect of alcohol feeding (with the exception of a nonsignificant, yet consistent decrease in SAM) was observed (Fig. 5A,B). Liver SAH (Fig. 5C) and homocysteine (Fig. 5E) levels were elevated as a consequence of alcohol feeding in most strains,

with several strains showing a significant effect. Liver SAM/SAH ratios were decreased (Fig. 5D). Liver injury scores were significantly correlated with SAM/SAH ratio (inverse correlation) and liver homocysteine content only when both control and alcohol-fed groups were considered. Plasma hyperhomocysteinemia has been observed in mice but not rats treated intragastrically with an alcohol-containing diet.21 In addition, hyperhomocysteinemia has been associated with the degree of liver injury.27 We observed that plasma levels of homocysteine are elevated in alcohol-fed mice (Fig. 6A) and that the degree of hyperhomocysteinemia is correlated significantly with both overall liver injury (Fig. 6B) and steatosis (Fig. 6C). These correlations remained significant when only alcohol-fed animals were B-Raf inhibition considered (Supporting Table 2). Homocysteine metabolism is dependent on the concordant action by a number of enzymes in the one-carbon metabolism

pathway. To evaluate the mechanisms of interstrain differences in hyperhomocysteinemia, we evaluated the expression of genes or protein levels of major enzymes responsible for the maintenance of the methyl donor pool in the liver (Fig. 7). It has been previously shown that expression of Bhmt is not affected in alcohol-fed C57BL6 mice.21 However, in our study we did observe changes in Bhmt protein in the liver of alcohol-fed mice of some strains (Fig. 7A). There was a significant decreasing nonlinear relationship between alcohol-induced change

in liver Bhmt and plasma homocysteine (Supporting Table 2). Changes in other regulators of one-carbon metabolism were assessed using gene expression, as messenger RNA (mRNA), protein, and activity levels of these enzymes correlate closely.31 Genes encoding 5-methyltetrahydrofolate-homocysteine methyltransferase (Mtr), an enzyme that catalyzes the final step in methionine biosynthesis, and Mthfr, an enzyme that is involved in homocysteine-methionine transition, were CYTH4 generally down-regulated in alcohol-fed mice, especially in strains that exhibited higher liver injury (Fig. 7B,C). Methionine adenosyltransferase 1 alpha (Mat1a), an enzyme that converts methionine into SAM, was markedly induced in strains with low liver injury (Fig. 7D). Glycine N-methyltransferase (Gnmt), an enzyme that converts SAM to SAH, was also induced in strains that had little liver injury and down-regulated in strains that had the most severe injury (Fig. 8A). Similar trends were observed in the expression of adenosylhomocysteinase (Ahcy) (Fig. 8B), cystathionine-beta-synthase (Cbs) (Fig. 8C), and cystathionase (Cth) (Fig.