PROTEINS BUT NOT NUCLEIC-ACIDS ARE MOLECULAR TARGETS FOR THE FREE-RADICAL ATTACK DURING REOXYGENATION OF RAT HEPATOCYTES

Isolated rat hepatocytes generate large amounts of reactive oxygen spe cies and suffer a significant cell injury during postanoxic reoxygenat ion. The aim of this study was to determine whether oxidation of prote ins and nucleic acids occurs during reoxygenation and whether their da mage is related to the development of hepatocyte injury. Isolated perf used rat hepatocytes were exposed sequentially to 1 h of aerobic contr ol, 2.5 h of anoxia, and 2 h of reoxygenation. Protein oxidation was d etermined by measuring the hepatocyte protein carbonyl content. DNA an d RNA oxidation was assessed by measuring the 8-hydroxydeoxyguanosine and 8-hydroxyguanosine adducts, respectively. The control preanoxic ca rbonyl content was 6.48 +/- 1.03 nmol/mg protein. The preanoxic 8-8-hy droxydeoxyguanosine and 8-hydroxyguanosine levels were 4.76 +/- 1.22 p mol/ml and 14.19 +/- 2.17 pmol/ml, respectively. During anoxia, protei n and nucleic acid oxidation did not change significantly. With reoxyg enation, the protein carbonyl content increased significantly within 3 0 min, reaching a value of 10.25 +/- 1.58 nmol/mg. The nucleic acid ox idation level remained stable. Perfusion with 100 mu M of Deferoxamine during reoxygenation abolished protein oxidation. These results indic ate that in rat hepatocytes during the early phases of reoxygenation: (1) the protein oxidation level increased significantly above the prea noxic aerobic values; (2) DNA and RNA oxidation does not appear to occ ur, and (3) free metal-mediated free radical reactions are involved in the oxidative protein damage. (C) 1997 Elsevier Science Inc.