REDOX STRESS AND HEPATIC DNA FRAGMENTATION INDUCED BY DIQUAT IN-VIVO ARE NOT ACCOMPANIED BY INCREASED 8-HYDROXYDEOXYGUANOSINE CONTENTS

Administration of 0.1 mmol/kg of diquat to Fischer-344 rats causes acu te hepatic necrosis by mechanisms that appear to involve increased gen eration of reactive oxygen species, but the critical targets of the pr oposed oxidations have not been identified. In the present study the e ffects of diquat-induced redox stresses on hepatic protein thiol statu s were determined by derivatization of subcellular fractions with mono bromobimane and separation of the fluorescent derivatives by SDS-PAGE. No differences in hepatic thiol status were seen in animals 2 or 6 h after diquat, relative to saline-treated controls, despite documentati on of injury by elevated plasma transaminase activities. Hepatic DNA f ragmentation was increased in diquat-treated animals (24.9+/-5.1 vs 6. 7+/-0.3% (controls) at 2 h; 57.2+/-4.1 vs 4.6+/-0.3% (controls) at 6 h , P <0.001). However, 8-hydroxydeoxyguanosine (8-OHdG) contents in hep atic DNA were not increased by diquat (35.3+/-6.2 mu mol 8-OHdG/mol de oxyguanosine (dG)) over saline-treated controls (28.3+/-2.6). Plasma N H3 concentrations increased in diquat-treated rats from 49 mu M in con trols to 170 mu M 6 h after treatment with diquat. Hepatic activities of glutamine synthetase (GS) were lower in diquat-treated rats (39.7+/ -3.0 mU/mg protein) than in controls (65.8+/-3.4, P <0.001), but activ ities of carbamyl phosphate synthetase-I (CPS-I), were not decreased s ignificantly. The oxidation of proteins to forms reactive with 2,4-din itrophenylhydrazine (DNPH) was investigated in subcellular fractions b y Western blot analyses with a monoclonal antibody to DNP-derivatized bovine serum albumin (BSA). N-terminal sequencing of bands exhibiting reactivity with anti-DNP-BSA antibodies indicated protein carbonyl for mation in malate dehydrogenase, protein disulfide isomerase, and gluta thione transferase. The functional consequences of oxidation of these proteins are not known but the observation of protein carbonyl formati on and no measurable loss of protein thiol content are consistent with iron chelate-mediated oxidation in the transformation critical to exp ression of tissue damage. The time course data are consistent with DNA fragmentation as a mechanism contributing to the development of cell injury, but the absence of increases in 8-OHdG indicates that direct o xidation of DNA may not be responsible.