Hypoxia-induced modification of poly (ADP-ribose) polymerase and dna polymerase β activity in cerebral cortical nuclei of newborn piglets: role of nitric oxide

Abstract

Previous studies have shown that poly (ADP-ribose) polymerase (PARP) and DNA polymerase β, nuclear enzymes, are associated with cell replication and DNA repair. The present study tests the hypothesis that hypoxia results in increased PARP and DNA polymerase activity in cerebral cortical neuronal nuclei to repair the hypoxia-induced damage to genomic DNA. Studies were conducted in 13 anesthetized and ventilated newborn piglets (age 3–5 days) divided into normoxic ( n=5) and hypoxic ( n=8) groups. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Following isolation of the cortical neuronal nuclei, the activity of PARP and DNA polymerase β was determined. During hypoxia, the tissue ATP level decreased by 73% from 4.12±0.67 μmol/g brain to 1.12±0.34 μmol/g brain, and PCr decreased by 78% from 4.14±0.68–0.90±0.20 μmol/g brain. In hypoxic neuronal nuclei, PARP activity significantly increased from 5.88±0.51 pmol NAD/mg protein/h in normoxic nuclei to 10.04±2.02 ( P=0.001). PARP activity inversely correlated with tissue ATP ( r=0.78) and PCr levels ( r=0.81). Administration of N-nitro- l-arginine prior to hypoxia decreased the hypoxia-induced increase in PARP activity by 67%. Endogenous DNA polymerase β activity increased from 0.96±0.13 in normoxic nuclei to 1.39±0.18 nmol/mg protein/h in hypoxic nuclei ( P<0.005). DNA polymerase β activity in the presence of exogenous template increased from 1.54±0.14 in the normoxic to 2.42±0.26 nmol/mg protein/h in the hypoxic group ( P<0.005). DNA polymerase β activity in the presence or absence of template inversely correlated with the tissue ATP ( r=0.95 and 0.84, respectively) and PCr levels ( r=0.93 and 0.77, respectively). These results demonstrate that the activity of PARP and DNA polymerase β enzymes increase with the increase in degree of cerebral tissue hypoxia. Furthermore, the results demonstrate a direct correlation between the PARP and the DNA polymerase β activity. We conclude that tissue hypoxia results in increased PARP and DNA polymerase β activities indicating activation of DNA repair mechanisms that may result in potential neuronal recovery following hypoxia and the hypoxia-induced increase in PARP activity is NO-mediated.