Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes.
Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.
Kroese
E D
ED
Division of Toxicology, University of Leiden, The Netherlands.
Bannenberg
G
G
Dogterom
P
P
Noach
A B
AB
Nagelkerke
J F
JF
Meerman
J H
JH
D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005978:Glutathione / Q000378:metabolism*; D006881:Hydroxyacetylaminofluorene / Q000633:toxicity*; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D015227:Lipid Peroxidation / Q000187:drug effects*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D011506:Proteins; D013438:Sulfhydryl Compounds; D006881:Hydroxyacetylaminofluorene; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D005978:Glutathione