Abstract 400

Redox cycling metabolism of diquat catalyzes generation of reactive oxygen species and the acute Hepatic necrosis caused in male Fischer-344 rats by diquat had been studied as a mode of oxidant mechanisms of cell killing in vivo. At equal doses of diquat, female F344 rats sustained lower levels of hepatic damage than did male rats, as estimated by plasma ALT activities. Equal doses of diquat elicited greater increases in biliary GSSG efflux in male rats than in females, indicating greater hepatic diquat levels or rates of redox cycling metabolism in males. However, different doses of diquat that gave comparable increases in biliary GSSG efflux were more hepatoxic to male F344 rats than to females, suggesting fundamental differences in susceptibilities to redox stresses in males and females. Hepatic activities of glutathione peroxidase dismutase were similar and glutathione reductase and glutathione S-transferase activities were higher in the more susceptible male rats. Previous studies in male rats have implicated iron chelate-catalyzed redox reactions as mechanisms critical to the expression of acute cell death in vivo. However, diquat-treated female rats showed higher levels of DNPH-reactive "protein carbonyls" in hepatic homogenates and in bile than did males, both at identical doses and at similar plasma ALT activities. Differences in the specific proteins affected in male and female rats were observed. A consistent finding on H&E-stained sections of liver from diquat-treated female was a diffuse pattern of marked, centrilobular necrosis with karyorrhexis and karyolysis, with some hepatocytes exhibiting cell shrinkage and chromatin fragmentation, characteristics of apoptosis. In contrast, lesions from similarly treated male rats were less extensive, despite higher plasma ALT activities, and where multifocal and predominantly midzonal. Fragmentation of hepatic DNA, assessed by detergent treatment and centrifugal separation of non-sedimented DNA from intact chromatin, was increased from 5% in control animals to more than 50% in some diquat-treated animals. TUNEL analyses showed extensive centrilobular reactivities in liver sections from diquat-treated female rats, whereas sections from similarly treated males showed predominantly midzonal and much lower TUNEL reactivities, whether compared by equal doses of diquat, elevations of plasma ALT activities, or biochemical assessment of hepatic DNA fragmentation. Hepatic DNA fragmentation in female rats was elevated with 2 h, before increases in plasma ALT activities, and by doses of diquat that did not increase plasma ALT activities. In the present, hepatic necrosis in diquat-treated F344 rats is most closely associated with DNA fragmentation, and the data suggest that DNA fragmentation may contribute significantly to the early mechanisms responsible for the initiation and expression of hepatic necrosis as well as apoptosis. Supported by GM44263 from the NIH.