Sanjiv Gupta

Induction and decline of hepatic cytochromes P4501A1 and 1A2 in rats exposed to hyperoxia are not paralleled by changes in glutathione S-transferase-α

We investigated the effects of hyperoxia on the activities of hepatic ethoxyresorufin O-deethylase (EROD) (CYP1A1), methoxyresorufin O-demethylase (MROD) (CYP1A2), and glutathione transferase-alpha (GST-alpha), and the status of protein thiols (PSH) in male Sprague-Dawley rats. Twenty-four h of hyperoxia more than doubled EROD and MROD activities, which were increased 7.6- and 3.3-fold, respectively, after 48 h of hyperoxia. The increases in EROD and MROD activities were paralleled by enhanced CYP1A1/1A2 apoproteins contents, as detected by Western analysis. At 60 h of hyperoxia, by which time hyperoxic Sprague-Dawley rats display marked respiratory distress, pulmonary edema, and other markers of pulmonary dysfunction, the activities and levels of hepatic CYP1A1 and 1A2 had declined dramatically and returned to levels observed in air-breathing control animals. Hepatic activities of GST-alpha, as well as PSH status, were not altered significantly in the hyperoxic animals at any time point. The marked induction and subsequent decline of hepatic CYP1A1/1A2 activities in rats exposed to hyperoxia suggest that these enzymes may contribute to the mechanisms of injury and/or to adaptive responses to hyperoxic exposures in vivo.

Rapid decreases in indigenous covalent DNA modifications (I-compounds) of male Fischer-344 rat liver DNA by diquat treatment

I-compounds are indigenously appearing covalent DNA modifications that can be detected by 32P-postlabeling assay in tissues of normal animals without known exposure to any carcinogens or toxins. Although these compounds have not been structurally identified, indirect evidence from earlier work suggested the possibility of involvement of molecular fragments derived from lipid peroxides. Diquat is a herbicide that stimulates lipid peroxidation and massive intrahepatic oxidant stress through redox cycling-mediated generation of reactive oxygen species. In the present study, we examined the effects of diquat on hepatic I-compounds of male Fischer-344 rats. Two groups of rats, approximately 14 weeks and 8 weeks old, were given a hepatotoxic dose (0.1 mmol/kg) of diquat or equal volumes of saline, i.p. Two and 6 h later plasma alanine aminotransferase (ALT) activities were measured and hepatic DNA I-compound levels were examined by nuclease P1-enhanced 32P-postlabeling. Elevated ALT activities were observed in some animals in both groups, at both time points, but considerable inter-animal variation was seen. A total of 15-16 I-compound fractions were measured in control and in diquat-treated animals, but no extra spots indicative of treatment-induced adducts were detected. Despite the qualitative similarities, the quantities of individual I-compounds were markedly decreased at 2 h in diquat-treated animals of both age groups. In 14 week old rats the hepatic I-compound contents were decreased at 2 h by 22-59%, which was statistically significant (ANOVA, P < 0.05) for all of the 9 polar I-compound fractions and none of the non-polar fractions. Eleven I-spots from this group showed significant negative linear correlations (P < 0.05) with ALT values. In 8 week old rats treated with diquat a 22-43% depletion in I-compound contents was statistically significant for 4 of the 7 nonpolar and 2 of the 8 polar adduct fractions, but there was no significant correlation of I-compound contents with ALT values at the 2 h time point. By 6 h most of the I-spot levels had returned to normal or above normal values in both groups of animals. While most I-spots from 14 week old rats did not correlate with ALT levels at 6 h, two I-spots displayed positive correlations in the 8 week group. Overall, the susceptibility to diquat-associated DNA alterations appeared to differ with age.(ABSTRACT TRUNCATED AT 400 WORDS)

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 acute hepatic necrosis by mechanisms that appear to involve increased generation of reactive oxygen species, but the critical targets of the proposed oxidations have not been identified. In the present study the effects of diquat-induced redox stresses on hepatic protein thiol status were determined by derivatization of subcellular fractions with monobromobimane 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 documentation of injury by elevated plasma transaminase activities. Hepatic DNA fragmentation 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 hepatic DNA were not increased by diquat (35.3±6.2 μmol 8-OHdG/mol deoxyguanosine (dG)) over saline-treated controls (28.3±2.6). Plasma NH3 concentrations increased in diquat-treated rats from 49 μM in controls to 170 μM 6 h after treatment with diquat. Hepatic activities of glutamine synthetase (GS) were lower in diquat-treated rats (39.7±13.0 mU/mg protein) than in controls (65.8±13.4, P<0.001), but activities of carbamyl phosphate synthetase-I (CPS-I), were not decreased significantly. The oxidation of proteins to forms reactive with 2,4-dinitrophenylhydrazine (DNPH) was investigated in subcellular fractions by 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 formation in malate dehydrogenase, protein disulfide isomerase, and glutathione transferase. The functional consequences of oxidation of these proteins are not known but the observation of protein carbonyl formation and no measurable loss of protein thiol content are consistent with iron chelate-mediated oxidation in the transformation critical to expression 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 oxidation of DNA may not be responsible.

Diquat- and acetaminophen-induced alterations of biliary efflux of iron in rats

The effects of diquat on the biliary efflux of nonheme iron in rats were studied as a means of examining the possible effects of diquat metabolism on hepatocellular iron metabolism and the association of altered iron metabolism with the initiation of acute hepatic necrosis. Administration of hepatotoxic doses (0.1 mmol/kg) of diquat to male Fischer-344 rats increased biliary iron concentrations from 6 microM to more than 15 microM. However, increases in biliary efflux of iron were not observed during the first 60 min following exposure to diquat, despite the rapid increases in biliary glutathione disulfide concentrations, which increased maximally within 40 min. Biliary efflux of iron was not altered by diquat in Sprague-Dawley rats, which are resistant to hepatic necrosis in response to diquat, despite the marked oxidant stress responses observed in these animals. Conversely, hepatotoxic doses of acetaminophen (1500 mg/kg) caused significant decreases in biliary iron efflux. The rapid decreases in biliary iron caused by acetaminophen and the delay in diquat-induced iron efflux suggested the possibility that some fraction of the biliary iron was being excreted as reversibly formed GS-Fe2+ chelates, with inhibition of export by glutathione disulfide (GSSG) in the case of diquat, or by 3-(glutathion-S-yl)-acetaminophen (GS-AAP) in the case of the acetaminophen-treated animals. However, 50-200 mg/kg doses of acetaminophen showed little effect on biliary iron excretion despite producing biliary GS-AAP conjugate concentrations almost 1000 times the 6 microM concentrations of iron, which would not appear to support the hypothesis of excretion of GS-Fe2+ chelates. The data demonstrate a significant effect of diquat on hepatic iron metabolism in Fischer-344 rats, and the possible importance of this iron redistribution to reactive oxygen-mediated cell damage in vivo is indicated by the absence of similar responses in diquat-treated Sprague-Dawley rats.

Sex Difference in Susceptibility to Hepatic Necrosis caused by Reactive Oxygen Species in Fischer-344 Rats † 1142

Sex Difference in Susceptibility to Hepatic Necrosis caused by Reactive Oxygen Species in Fischer-344 Rats † 1142

DNA FRAGMENTATION CAUSED BY REACTIVE OXYGEN SPECIES IN VIVO WITHOUT INCREASED 8-OH-dG CONTENTS. • 458

Administration of 0.1 mmol/kg of diquat to Fischer-344 rats causes acute hepatic necrosis by mechanisms that appear to involve increased generation of reactive oxygen species (ROS). The oxidation of biological molecules should be pivotal in the expression of the injury, but the specific targets critical to expression of this injury are not known. Hepatic DNA fragmentation (% of total) was increased in diquat-treated animals (6.7±0.3 vs. 24.9±5.1 at 2 h; 4.6±0.3 vs. 57.2±4.1 at 6 h, P<0.001) and oligonucleosomal fragmentation of hepatic DNA was observed. However, 8-OHdG contents in hepatic DNA were not increased by diquat (35.3±6.2 mol 8OHdG/106 mol dG) over controls (28.3±2.6). We assessed protein thiol status by derivatization of subcellular fractions with monobromobimane and separation of the fluorescent derivatives by SDS-PAGE. No differences from saline-treated controls were seen in animals 2 or 6 h after diquat, despite documentation of oxidant stress by contents of glutathione disulfide and of hepatic injury by elevation of plasma transaminase activities. Plasma ammonia concentrations increased in diquat-treated rats from 49±9 in controls to 170±22 μM 6 h after diquat(P<0.002). Hepatic glutamine synthetase activities were lower in diquat-treated rats (39.7±3.0 mU/mg pro) than in controls(65.8±3.4, P<0.001), but activities of carbamyl phosphate synthetase-I, which is a mitochondrial enzyme, were not decreased significantly. Supported by NIH grants GM44263 and GM39338.

246 EVIDENCE FOR A CRITICAL ROLE OF IRON CHELATES IN OXIDATIVE HEPATIC INJURY IN RATS

Diquat (Dq) causes generation of reactive oxygen species in Fisher-344 and Sprague-Dawley (SD) rats, evoking marked liver necrosis in the former, but almost none in the latter strain. To test the hypothesis that iron homeostasis is pivotal in the resistance to injury by reactive oxygen species, we pretreated adult SD rats with FeSO4 ip and gave Dq ip at 30 min. Controls were given normal saline and/or PBS. Blood was drawn 1-24 hr after Dq and ALT measured in plasma. Data from treated rats were compared to controls by two-tailed Fisher exact test No rats killed at 2 hours had abnormal ALTs.We conclude that excess free iron is critical to induce liver injury in Dq-treated rats, but the distribution of the iron needs to be determined Supported by NIH Grant GM44263 and the International Pediatric Research Foundation.