NITRIC-OXIDE PRODUCTION DURING ENDOTOXIC-SHOCK IN CARBON TETRACHLORIDE-TREATED RATS

Earlier studies showed that hepatotoxicant-treated experimental animal s were more susceptible than controls to the lethal effects of bacteri al endotoxin. The exact mechanisms of this effect were not understood. In this paper we showed that nitric oxide ((NO)-N-.) was produced in whole blood and in liver tissues of rats that had been treated with a nonlethal dose of CCl4 (1.3 g/kg) followed by a low dose of lipopolysa ccharide (LPS) (100 mu g/kg). EPR spectroscopy was used in this study to detect nitrosyl-protein complexes. Hemoglobin-nitrosyl complexes we re detected in both whole blood and liver. By performing analyses of E PR spectra obtained from hepatocytes exposed to (NO)-N-., we were able to identify EPR signals attributable to nitrosyl-cytochrome P420 in r at liver. We found that nitrosyl complex formation in red blood cells and liver was inhibited by treatment with N-G-monomethyl-L-arginine, s uggesting enzymatic biosynthesis of (NO)-N-.. A small but significant inhibition of nitrosyl complex formation by gadolinium trichloride pre treatment was found in the liver, suggesting that Kupffer cells were a lso involved in (NO)-N-. biosynthesis, because this treatment decrease d Kupffer cells. There was a synergistic effect of CCl4 and LPS on the serum levels of the hepatic enzymes aspartate aminotransferase, alani ne aminotransferase, lactate dehydrogenase, and sorbitol dehydrogenase , which are indices of parenchymal cell damage. NG-Monomethyl-L-argini ne treatment increased these hepatic enzyme activities, suggesting a p rotective role for (NO)-N-.. EPR resonances at g similar to 2.48, 2.29 , and 1.91, due to low-spin cytochromes P450/ P420 (Fe3+), were decrea sed in the livers of LPS-induced rats that had been previously treated with CCl4, indicating cytochrome P450/P420 destruction or at least a change in the valence state of the cytochrome P450/P420 heme groups to Fe2+ in the presence of (NO)-N-.. Because nitrosyl-cytochrome P450 is not stable, the concomitant detection of nitrosyl-cytochrome P420 (Fe 2+) could account, at least in part, for the decrease of the ferric lo w-spin heme groups. Our novel observations of hepatic nitrosyl species suggest that (NO)-N-. plays an important role during hepatic injury c aused by CCl4 in hosts exposed to endotoxin.