UNLABELLEDWe investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage.

METHODSDose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe2+ formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supernatants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2E1 activity.

RESULTSWe first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h. Being similar increased protein expression started to what had been demonstrated with the mRNA level, at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fe2+ formation in human hepatocytes. Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyrin (ZnPP), an antagonist of HO-1 induction.

CONCLUSIONHO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human liver.

Aspartate Aminotransferases ; metabolism ; Bilirubin ; analysis ; Cell Survival ; Cells, Cultured ; Enzyme Induction ; drug effects ; Ethanol ; Gene Expression Regulation, Enzymologic ; drug effects ; Glutathione ; metabolism ; Heme Oxygenase (Decyclizing) ; metabolism ; Heme Oxygenase-1 ; Hepatocytes ; drug effects ; enzymology ; pathology ; Humans ; Iron ; analysis ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Membrane Proteins ; Protoporphyrins ; RNA, Messenger ; biosynthesis ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction