Figure 7

Characterization of ethanol metabolizing enzymes in human liver cell cultures. A, western blot analysis of ADH1 and CYP2E1 expression levels in Huh7, BB7, 9–13, 5–15, and FL-Neo cells. Positive controls for ADH included primary human fetal hepatocytes (HFH) [24], and a well differentiated immortalized human liver cell line, HH2 (developed in NF's lab), while controls for CYP2E1 expression included baculovirus expressed CYP2E1 and purified CYP2E1. Western blots were probed with a monoclonal antibody against human ADH, and polyclonal rabbit antiserum against CYP2E1 and Stat1. B, ADH enzyme activity. Huh7 cells were harvested in PBS and whole cell protein extracts prepared via sonication. Conversion of NAD to NADH+ was monitored at a wavelength of 340 nm as described in the Materials and Methods. Purified ADH served as a positive control for ADH activity. C, effect of CYP2E1 and ADH inhibition on ethanol activation of the ISRE. Huh7 cells in 96 well plates were transfected in triplicate with 50 ηg of ISRE-luc and 12 hours later, were treated with 5 mM of the ADH inhibitor 4-MP and 10 mM of the CYP2E1 inhibitor DAS for an additional 12 hours. Cells were also separately exposed to 0.1% DMSO, as an additional control for possible solvent effects. Cells were then treated with 0, 100, or 200 mM ethanol, before luciferase activity was measured by BriteLite assay. Error bars represent standard deviations. The experiments were repeated twice with identical results.