Establishment of a recombinant hepatic cell line stably expressing alcohol dehydrogenase.
Hepatocytes cultured for extended periods of time lose the ability to express alcohol dehydrogenase and thus, the ability to efficiently oxidize ethanol. Therefore, it has been difficult to investigate the effects of chronic ethanol oxidation by hepatocytes in vitro. To circumvent this problem, we have inserted the coding region of an exogenous alcohol dehydrogenase gene into an hepatic cell line. Using the human hepatocellular carcinoma cell line, Hep G2, we have constructed an hepatic cell line that stably expresses alcohol dehydrogenase. These recombinant cells, termed HAD 73.1 cells, express approximately 40% of the alcohol dehydrogenase activity of freshly isolated rat hepatocytes. When the ethanol metabolizing ability of these cells was directly measured, the results indicated that not only were these cells able to metabolize ethanol at approximately 70% of the rate of freshly isolated rat hepatocytes but acetaldehyde concentrations of up to 50 microM were detected in the medium. Furthermore, the level of acetaldehyde produced during ethanol oxidation was augmented by cyanamide, an inhibitor of acetaldehyde oxidation, while the ability of these cells to metabolize ethanol was inhibited by pyrazole, an inhibitor of alcohol dehydrogenase. These results suggest that this in vitro system will be a valuable tool enabling detailed biochemical studies exploring the effects of chronic ethanol oxidation on the liver and the mechanisms of alcohol-induced hepatic cell injury.
Clemens
D L
DL
Liver Study Unit, Department of Veterans Affairs Medical Center, Omaha, Nebraska, USA.
Halgard
C M
CM
Miles
R R
RR
Sorrell
M F
MF
Tuma
D J
DJ
D000079:Acetaldehyde / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008113:Liver Neoplasms; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase; D000079:Acetaldehyde