Chronic ethanol consumption is associated with increased protein oxidation and decreased proteolysis in the liver. We tested the hypothesis that even single-dose treatment with ethanol or bromotrichloromethane causes increased protein oxidation and a distinct proteolytic response in cultured hepatocytes. HepG2 cells were treated for 30 min with ethanol, H(2)O(2) and bromotrichloromethane at various nontoxic concentrations. Protein degradation was measured in living cells using [35S]-methionine labeling. Protein oxidation, and 20S proteasome activity were measured in cell lysates. Oxidized proteins increased immediately after ethanol, H(2)O(2), and bromotrichloromethane exposure, but a further significant increase 24-h after exposure was observed only following ethanol and bromotrichloromethane treatment. All three reagents caused a significant increase of the overall intracellular proteolysis at rather low concentrations, which could be suppressed by the proteasome inhibitor lactacystin. A decline of proteolysis observed at higher-subtoxic-concentrations was not related to decreased proteasome activity. Preincubation with ketoconazole or 4-methylpyrazole completely prevented the ethanol- and bromotrichloromethane-induced but not the H(2)O(2)-induced protein oxidation and proteolysis, suggesting strongly an enzyme-mediated generation of reactive oxygen species. In conclusion single-dose exposure with ethanol or haloalkanes causes increased protein oxidation followed by an increased proteasome-dependent protein degradation in human liver cells.