Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity》.pdf

The International Journal of Biochemistry Cell Biology 38 (2006) 92–101 Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity Terrence M. Donohue a,b,c,d,∗, Natalia A. Osna a,b , Dahn L. Clemens a,b,d a Liver Study Unit, VA Medical Center, Omaha, NE 68105, USA b Department of Internal Medicine, University of Nebraska College of Medicine, Omaha, NE 68198, USA c Department of Biochemistry/Molecular Biology, University of Nebraska College of Medicine, Omaha, NE 68198, USA d Department of Pathology and Microbiology, University of Nebraska College of Medicine, Omaha, NE 68198, USA Received 14 June 2005; accepted 27 July 2005 Abstract HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the intial ethanol concentration. Compared with unexposed VL- 17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100 mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1+ ) and VA-13 (ADH+ ) c