Abstract
Prior studies showed that Toll-like receptor (TLR) signaling pathway genes were upregulated in the liver of rats fed ethanol, but not in rats fed ethanol plus S-adenosylmethionine (SAMe). These results were obtained using a PCR microplate array analysis for TLRs and associated proteins such as proinflammatory cytokines and chemokine mRNA levels. A large number of genes were upregulated by the ethanol diet, but not the ethanol plus SAMe diet. In the present study, using the same experimental rat livers, DNA methylation analysis was done by using an Epitect Methyl DNA Restriction Kit (Qiagen, 335451) (24 genes). The results of all the genes combined show a highly significant increase in methylation in the ethanol-fed group of rats, but not in the dextrose-fed, SAMe-fed or ethanol plus SAMe-fed groups of rats. There was also an increase in DNA methylation in rats with high blood alcohol levels compared to a rat with a low blood alcohol level. The individual genes that were upregulated as indicated by the increased mRNA measured by qPCR correlated positively with the increased methylation of the DNA of the corresponding genes as follows: Cd14, Hspa1a, Irf1, Irak1, Irak2, Map3k7, Myd88, Pparα, Ripk2, Tollip and Traf6.