Different Mechanisms for Histone Acetylation by Ethanol and Its Metabolite Acetate in Rat Primary Hepatocytes

Abstract

Ethanol and its major metabolite acetate both induced histone H3 acetylation in primary culture of rat hepatocytes. The acetylation by ethanol was dependent on the reactive oxygen species and mitogen-activated protein kinase pathway, whereas that by acetate was independent of both pathways. Ethanol increased CYP2E1 protein expression but acetate had negligible effect. The level of phospho-H2AX, an indicator of DNA breaks, was elevated by ethanol but not by acetate. Ethanol and acetate differentially activated mRNA expression for different genes, e.g., IL-6, PPARg, c-Fos, Egr-1, and PNPLA3 in hepatocytes. The most striking increase (3-fold) was in PNPLA3 mRNA by ethanol with little change by acetate. It was further shown that acetate inhibited histone deacetylase activity. Taken together, these data establish for the first time that ethanol and acetate exhibit differences in their effects on hepatocytes in gene expression, P-H2AX levels, and the mechanism of histone H3 acetylation. The implications of these differences in the actions of ethanol in liver are discussed.

Binge alcohol alters PNPLA3 levels in liver through epigenetic mechanism involving histone H3 acetylation

“…Hepatocyte preparation and treatments were as described previously (Shukla, Restrepo, et al, 2015). Briefly, hepatocytes from male Sprague-Dawley rats (250–300 g) were isolated using an in situ collagenase perfusion method.…”

“…Western blotting of PNPLA3 for primary rat hepatocytes, mouse, and rat liver tissues was done in whole cell extracts of hepatocyte or liver tissue as described previously (Aroor et al, 2014; Shukla, Aroor, et al, 2015; Shukla, Restrepo, et al, 2015). An equal amount of protein (80 μg) was loaded in each gel lane for western blotting.…”

“…Data for mRNA expression were calculated by the Delta-Delta method with GAPDH as the housekeeping gene (see Shukla, Aroor, et al, 2015; Shukla, Restrepo, et al, 2015). Cycle threshold (CT) values of less than 36 were used.…”

“…Primary rat hepatocytes were collected and treated as described previously (Shukla, Restrepo, et al, 2015). Frozen cells were thawed on ice with the addition of 0.5 mL of a crosslinking solution consisting of 1X PBS/1% formaldehyde at room temperature with tube rotation for 12 min.…”

“…Any involvement of the chromatin modifications on the PNPLA3 transcriptional machinery that orchestrates the PNPLA3-related network has yet to be known (see Sookoian & Pirola, 2014). Histone H3K9 acetylation in chromatin has been shown to increase in vitro in a hepatocyte model and in animal models in vivo of ALD (Aroor, Restrepo, Kharbanda, & Shukla, 2014; Shukla, Aroor, Restrepo, Kharbanda, & Ibdah, 2015; Shukla, Restrepo, Fish, Lim, & Ibdah, 2015). Increases in TG levels after acute, chronic, and acute on chronic exposure to alcohol were reported in rats (Aroor, Jackson, & Shukla, 2011) and mice (Shukla, Aroor, et al, 2015).…”