PLASMA-MEMBRANE HYDROXYETHYL RADICAL ADDUCTS CAUSE ANTIBODY-DEPENDENTCYTOTOXICITY IN RAT HEPATOCYTES EXPOSED TO ALCOHOL

Background & Aims: We reported previously that patients with alcoholic liver disease (ALD) have circulating immunoglobulins reacting with cy tochrome P4502E1 (CYP2E1) complexed with hydroxyethyl free radicals. T he aim of this study was to investigate whether hydroxyethyl radical a dducts are present on the plasma membranes of ethanol-treated hepatocy tes and their role in antibody-dependent cytotoxicity. Methods: Immuno fluorescence confocal laser microscopy, Western blotting, and antibody -dependent cell-mediated cytotoxicity assay were used. Results: Isolat ed rat hepatocytes incubated in vitro with ethanol or obtained from et hanol-treated animals showed strong surface fluorescence when exposed to rabbit anti-hydroxyethyl radical serum or sera from patients with A LD. No surface fluorescence was evident on control hepatocytes or afte r scavenging hydroxyethyl radicals with 4-pyridyl-1-oxide-t-butyl nitr one. The presence of CYP2E1-hydroxyethyl radical adducts on hepatocyte plasma membranes was shown by Western blot and by immunofluorescence using double staining for human and rabbit anti-CYP2E1 immunoglobulin G. Cytotoxicity was observed in ethanol-treated hepatocytes incubated with immunoglobulin G from patients with ALD and normal human blood mo nonuclear cells. This effect was blocked by preadsorbing the sera with human albumin complexed with hydroxyethyl radicals, which also elimin ated the antibody reaction with the plasma membranes. Conclusions: Hyd roxyethyl radicals bound to CYP2E1 on hepatocyte plasma membranes can target immune reactions triggered by alcohol abuse.