Bilirubin exerts additional toxic effects in hypoxic cultured neurons from the developing rat brain by the recruitment of glutamate neurotoxicity.
Both hypoxia and bilirubin are common risk factors in newborns, which may act synergistically to produce anatomical and functional disturbances of the CNS. Using primary cultures of neurons from the fetal rat brain, it was recently reported that neuronal apoptosis accounts for the deleterious consequences of these two insults. To investigate the influence of hypoxia, bilirubin, or their combination on the outcome of neuronal cells of the immature brain, and delineate cellular mechanisms involved, 6-d-old cultured neurons were submitted to either hypoxia (6 h), unconjugated bilirubin (0.5 microM), or to combined conditions. Within 96 h, cell viability was reduced by 22.7% and 24.5% by hypoxia and bilirubin, respectively, whereas combined treatments decreased vital score by 34%. Nuclear morphology revealed 13.4% of apoptotic cells after hypoxia, 16.2% after bilirubin, and 22.6% after both treatments. Bilirubin action was specifically blocked by the glutamate receptor antagonist MK-801, which was without effect on the consequences of hypoxia. Temporal changes in [(3)H]leucine incorporation rates as well as beneficial effects of cycloheximide reflected a programmed phenomenon dependent upon synthesis of selective proteins. The presence of bilirubin reduced hypoxia-induced alterations of cell energy metabolism, as reflected by 2-D-[(3)H]deoxyglucose incorporation, raising the question of free radical scavenging. Measurements of intracellular radical generation, however, failed to confirm 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:-methyl-D-aspartate receptors.
Université Henri Poincaré-Nancy 1, 24-30 rue Lionnois, B.P. 3069, 54013 Nancy Cédex, France.
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Grojean
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Lievre
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V
Koziel
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P
Vert
J L
JL
Daval
D000818:Animals; D001663:Bilirubin / Q000633:toxicity*; D001921:Brain / Q000166:cytology / Q000187:drug effects*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D018698:Glutamic Acid / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017470:Receptors, Glutamate / Q000187:drug effects
D018691:Excitatory Amino Acid Antagonists; D017470:Receptors, Glutamate; D018698:Glutamic Acid; D001663:Bilirubin