Lipid peroxidation-dependent and -independent protein thiol modifications in isolated rat hepatocytes: differential effects of vitamin E and disulfiram.
Exposure of isolated rat hepatocytes to allyl alcohol (AA), diethyl maleate (DEM) and bromoisovalerylurea (BIU) induced lipid peroxidation, depletion of free protein thiols to about 50% of the control value and cell death. Vitamin E completely prevented lipid peroxidation, protein thiol depletion and cell death. A low concentration (0.1 mM) of the lipophylic disulfide, disulfiram (DSF), also prevented the induction of lipid peroxidation by the hepatotoxins; however, in the presence of DSF, protein thiol depletion and cell death occurred more rapidly. Incubation of cells with a high concentration (10 mM) of DSF alone led to 100% depletion of protein thiols and cell death, which could not be prevented by vitamin E. The level of free protein thiols in cells, decreased to 50% by exposure to AA, DEM and BIU, could be reversed to 75% of the initial level by dithiothreitol (DTT) treatment, indicating that the protein thiols were partially modified into disulfides and partially into other, stable thiol adducts. The 100% depletion of protein thiols by DSF was completely reversed by DTT treatment. The involvement of lipid peroxidation in protein thiol depletion was studied by measuring the effect of a lipid peroxidation product, 4-hydroxynonenal (4-HNE), on protein thiols in a cell free liver fraction. 4-HNE did not induce lipid peroxidation in this system, but protein thiols were depleted to 30% of the initial value, irrespective of the presence of vitamin E. DTT treatment could reverse this for only 25%. Similar, DSF-induced protein thiol depletion could be reversed completely by DTT. We conclude that (at least) two types of protein thiol modifications can occur after exposure of hepatocytes to toxic compounds: one due to interaction of endogeneously generated lipid peroxidation products with protein thiols, which is not reversible by the action of DTT, and one due to a disulfide interchange between disulfides like DSF and protein thiols, which can be reversed by the action of DTT.
Dogterom
P
P
Division of Toxicology, University of Leiden, The Netherlands.
Mulder
G J
GJ
Nagelkerke
J F
JF
D000433:1-Propanol / Q000494:pharmacology; D000818:Animals; D001968:Bromisovalum / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004221:Disulfiram / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D015227:Lipid Peroxidation / Q000187:drug effects; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008298:Maleates / Q000494:pharmacology*; D020005:Propanols; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014508:Urea / Q000031:analogs & derivatives*; D014810:Vitamin E / Q000494:pharmacology*
D008298:Maleates; D020005:Propanols; D011506:Proteins; D013438:Sulfhydryl Compounds; D014810:Vitamin E; C006463:allyl alcohol; D001968:Bromisovalum; D014508:Urea; D000433:1-Propanol; C014476:diethyl maleate; D005978:Glutathione; D004229:Dithiothreitol; D004221:Disulfiram; D019803:Glutathione Disulfide