Increased proteolysis after single-dose exposure with hepatotoxins in HepG2 cells.
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.
Pirlich
Matthias
M
Department of Gastroenterology and Hepatology, University Hospital Charité, Humboldt-University Berlin, Germany.
Müller
Cornelia
C
Sandig
Grit
G
Jakstadt
Manuela
M
Sitte
Nicolle
N
Lochs
Herbert
H
Grune
Tilman
T
D001975:Bromotrichloromethane / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000633:toxicity*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D016133:Polymerase Chain Reaction; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D017382:Reactive Oxygen Species
D017931:DNA Primers; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D011506:Proteins; D017382:Reactive Oxygen Species; D000431:Ethanol; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D001975:Bromotrichloromethane