Ethanol and arachidonic acid produce toxicity in hepatocytes from pyrazole-treated rats with high levels of CYP2E1

Ethanol and polyunsaturated fatty acids such as arachidonic acid were shown to be toxic and cause apoptosis in HepG2 cells which express CYP2E1 but no t in control HepG2 cell lines. The goal of the current study was to extend the observations made with the HepG2 cells to non-transformed, intact hepat ocytes. Rats were treated with pyrazole to increase CYP2E1 levels, hepatocy tes were isolated and placed into culture and treated for varying time poin ts with ethanol or arachidonic acid. Comparisons were made to hepatocytes f rom saline-treated rats, with low CYP2E1 content. Incubation with ethanol ( 100 mM) or especially arachidonic acid (60 mu M) resulted in loss of viabil ity of hepatocytes from the pyrazole-treated rats, without any effect on th e hepatocytes from the saline-treated rats. The toxicity appeared to be apo ptotic in nature and was prevented by diallyldisulfide, an inhibitor of CYP 2E1. Toxicity was reduced by trolox, an antioxidant. The treatment with eth anol or arachidonic acid resulted in release of cytochrome c into the cytos ol fraction, and activation of caspase 3 (but not caspase 1) in hepatocytes from the pyrazole-treated rats but not hepatocytes from the saline-treated rats. The activation of caspase 3 was prevented by diallyldisulfide, by tr olox, and by DEVD-fmk. The latter also prevented the toxicity produced by e thanol or arachidonic acid. These results extend previous observations foun d with HepG2 cells expressing CYP2E1 to intact hepatocytes and suggest that release of cytochrome c and activation of caspase 3 play a role in the ove rall pathway by which CYP2E1 contributes towards the hepatotoxic actions of ethanol and polyunsaturated fatty acids.