Bilirubin exerts additional toxic effects in hypoxic cultured neurons fromthe developing rat brain by the recruitment of glutamate neurotoxicity

Both hypoxia and bilirubin are common risk factors in newborns, which may a ct synergistically to produce anatomical and functional disturbances of the CNS. Using primary cultures of neurons from the fetal rat brain, it was re cently reported that neuronal apoptosis accounts for the deleterious conseq uences of these two insults. To investigate the influence of hypoxia, bilir ubin, or their combination on the outcome of neuronal cells of the immature brain, and delineate cellular mechanisms involved, 6-d-old cultured neuron s were submitted to either hypoxia (6 h), unconjugated bilirubin (0.5 muM), or to combined conditions. Within 96 h, cell viability was reduced by 22.7 % and 24.5% by hypoxia and bilirubin, respectively, whereas combined treatm ents decreased vital score by 34%. Nuclear morphology revealed 13.4% of apo ptotic cells after hypoxia, 16.2% after bilirubin, and 22.6% after both tre atments. Bilirubin action was specifically blocked by the glutamate recepto r antagonist MK-801, which was without effect on the consequences of hypoxi a. Temporal changes in [H-3]leucine incorporation rates as well as benefici al effects of cycloheximide reflected a programed phenomenon dependent upon synthesis of selective proteins. The presence of bilirubin reduced hypoxia -induced alterations of cell energy metabolism, as reflected by 2-D-[H-3]de oxyglucose incorporation, raising the question of free radical scavenging. Measurements of intracellular radical generation, however, failed to confir m the antioxidant role of bilirubin. Taken together, our data suggest that low levels of bilirubin may enhance hypoxia effects in immature neurons by facilitating glutamate-mediated apoptosis through the activation of N-methy l-D-aspartate receptors.