Abstract
Background: Hepatocytes metabolize the vast majority of ingested ethanol. This metabolic activity results in hepatic toxicity and impairs the ability of hepatocytes to replicate. Previous work by our group has shown that ethanol metabolism results in a G2/M cell cycle arrest. The intent of these studies was to discern the roles of acetaldehyde and reactive oxygen, two of the major by-products of ethanol metabolism, in the G2/M cell cycle arrest. Methods: To investigate the role of ethanol metabolites in the cell cycle arrest, VA-13 and VL-17A cells were used. These are recombinant Hep G2 cells that express alcohol dehydrogenase or alcohol dehydrogenase and cytochrome P450 2E1, respectively. Cells were cultured with or without ethanol, lacking or containing the antioxidants N-acetylcysteine (NAC) or trolox, for three days. Cellular accumulation was monitored by the DNA content of the cultures. The accumulation of the cyclin-dependent kinase, Cdc2 in the inactive phosphorylated form (p-Cdc2) and the cyclin-dependent kinase inhibitor p21 were determined by immunoblot analysis. Results: Cultures maintained in the presence of ethanol demonstrated a G2/M cell cycle arrest that was associated with a reduction in DNA content and increased levels of p-Cdc2 and p21, compared with cells cultured in its absence. Inclusion of antioxidants in the ethanol containing media was unable to rescue the cells from the cell cycle arrest or these ethanol metabolism-mediated effects. Additionally, culturing the cells in the presence of acetaldehyde alone resulted in increased levels of p-Cdc2 and p21. Conclusions: Acetaldehyde produced during ethanol oxidation has a major role in the ethanol metabolism-mediated G2/M cell cycle arrest, and the concurrent accumulation of p21 and p-Cdc2. Although reactive oxygen species are thought to have a significant role in ethanol-induced hepatocellular damage, they may have a less important role in the inability of hepatocytes to replace dead or damaged cells.
Highlights
The liver is the primary site of ethanol metabolism
Using the recombinant Hep G2 cell lines, VA-13 that selectively metabolize ethanol via the alcohol dehydrogenase (ADH) pathway and VL-17A cells that express both alcohol dehydrogenase and cytochrome P450 2E1 (CPY2E1), we have investigated the involvement of acetaldehyde and reactive oxygen species in the ethanol metabolism-mediated cell cycle arrest
We have previously shown that ethanol metabolism impairs the replication of recombinant hepatic cells by arresting the cells at the G2/M transition of the cell cycle
Summary
The liver is the primary site of ethanol metabolism. The by-products of this metabolism can result in hepatotoxicity. In many chronic liver diseases, the replication of mature hepatocytes is inhibited In these instances, replacement of dead or damaged hepatocytes occurs by a population of bipotential hepatic cells known as hepatic progenitor cells or oval cells [8,9,10,11]. Impairment of normal hepatocyte replication by ethanol metabolism and activation of hepatic progenitor cells may have a role in the fibrotic scarring characteristics of alcoholic liver disease. Hepatocytes metabolize the vast majority of ingested ethanol This metabolic activity results in hepatic toxicity and impairs the ability of hepatocytes to replicate. Previous work by our group has shown that ethanol metabolism results in a G2/M cell cycle arrest The intent of these studies was to discern the roles of acetaldehyde and reactive oxygen, two of the major by-products of ethanol metabolism, in the G2/M cell cycle arrest.
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