Diquat-Induced Oxidative Damage in BCNU-Pretreated Hepatocytes of Mature and Old Rats

Abstract

The effects of postmaturational aging on the toxicity of diquat, a redox cycling compound, were investigated in hepatocytes that were isolated from mature (6 months) and old (27 months) male Fischer 344 rats and pretreated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The hepatocytes were incubated for 2 hr with 0, 0.5, or 2.0 mM diquat dibromide, and samples were taken at various time points for measurements of glutathione, glutathione disulfide, thiobarbituric acid reactive substances, lactate dehydrogenase leakage, protein sulfhydryl groups, and protein carbonyl groups. Diquat cytotoxicity was intensified in hepatocytes of old rats compared with those of mature rats, and the enhanced toxicity was associated with increased lipid peroxidation and protein carbonyl formation. However, the enhanced toxicity in old rat hepatocytes was also accompanied by a decrease in diquat-induced GSH oxidation and there was no difference in protein sulfhydryl loss. Concentrations of total nonheme iron and low-molecular-weight chelatable Fe 2+, measured with ferene as the chromogen, were several times higher in freshly isolated hepatocytes of old rats than in those of mature rats. We hypothesize that the age-associated enhancement of diquat toxicity could be due to an increased availability of iron for reaction with diquat-generated hydrogen peroxide and for stimulation of lipid and protein oxidation.