Abstract 2060
Poster Session II, Sunday, 5/2 (poster 224)
Previous studies in hypoxic newborn and fetal brain have shown a laddered pattern of DNA fragmentation as indicative of apoptosis. Studies on DNA excision-repair have also shown that DNA polymerase β carries out this repair function in eucaryotic cells. The present tests the hypothesis that the DNA polymerase β activity will increase allowing repair of hypoxia-induced DNA damage in cerebral cortical neuronal nuclei. Studies were conducted on 8 pregnant guinea pigs divided into normoxic (n=3) and hypoxic (n=5) groups. Hypoxia was induced by exposing the pregnant guinea pigs to an FiO2 of either 0.21 or 0.07 for 1 hour. Fetal cortices were obtained and neuronal nuclei isolated and purified in 2.1 M sucrose gradient. Brain hypoxia was confirmed biochemically by assessing ATP and PCr levels in the fetal cortices. DNA polymerase β activity was measured in a medium containing 250 µg nuclear protein, 50 mM Tris-HCI (pH 8.8), 15% glycerol, 2 mM EGTA, 8 mM MgCl2, 100 mM NaCl, 2 mM ATP, 80 µM dATP, 80 µM dGTP, 80 µM dTTP and 6 µM 3H-CTP in the presence or absence of 20 µg exogenous DNA template extracted from normoxic cortical nuclei. The incubation was carried out at 37°C for 10 min. The activity was corrected for DNA polymerase α by preincubation of nuclei with 10 mM N-ethyl-maleimide at 0°C for 30 min. The reaction was stopped by adding 500 µl of 0.625 N perchloric acid and the incorporation of radiolabeled 3H-CTP into acid insoluble fraction was determined. The DNA polymerase β activity was expressed as nmoles of 3H-CTP incorporated/mg protein/hr. In the hypoxic group, ATP decreased from 4.58 ± 0.17 µmoles/g brain in the normoxic group to 1.03 ± 0.34 µmoles/g brain in the hypoxic group (p<0.001). Similarly, PCr decreased following hypoxia from 3.93 ± 0.3 µmoles/g brain in the normoxic group to 1.10 ± 0.46 µmoles/g brain in the hypoxic group (p<0.001). Endogenous activity of DNA polymerase β following hypoxia increased from 1.03 ± 0.18 nmoles/mg protein/hr in the normoxic group to 1.45 ± 0.17 nmoles/mg protein/hr (p<0.05). Similarly, the activity in the presence of added exogenous template, representing the nucleoplasmic DNA polymerase β was 1.54 ± 0.14 nmoles/mg protein/hr and 2.42 ± 0.26 nmoles/mg protein/hr in the normoxic and hypoxic groups, respectively (p<0.005). We conclude that brain tissue hypoxia results in increased DNA polymerase β activity with or without exogenous template. Since DNA repair pathway (base excision-repair) purifies genomic DNA of damaged nucleotides and abasic sites arising from a variety of exogenous and endogenous insults, we speculate that during hypoxia the increased activity of DNA polymerase β indicates an activation of the DNA repair mechanism that would lead to potential recovery of hypoxic neurons in the immature brain.
