Bilirubin induces apoptosis via activation of NMDA receptors in developingrat brain neurons

Increased amounts of bilirubin, the end product of heme degradation, are kn own to be detrimental to the central nervous system, especially in preterm newborns. In an attempt to delineate the cellular mechanisms by which uncon jugated bilirubin exerts its toxic effects on neuronal cells in the develop ing brain, bilirubin (0.25-5 muM) was added to the extracellular medium of 6-day-old primary cultured neurons from the embryonic rat forebrain, and ce ll alterations were studied over the ensuing 96 h. Bilirubin decreased cell viability dose dependently with an ED50 around 1 muM. At the dose of 0.5 m uM, it triggered delayed cell death that affected 24% of the neurons. Nucle ar incorporation of the fluorescent dye DAPI (4,6-diamidino-2-phenylindole) depicted the presence of apoptosis (16%). Apoptosis features were confirme d by DNA fragmentation reflected by a progressive loss of [H-3]thymidine an d sequential changes in macromolecular synthesis, as shown by the time cour se of [H-3]leucine incorporation, as well as by the beneficial effects of c ycloheximide and caspase inhibitors. In parallel, treatments with glutamate receptor antagonists showed that MK-801, but not NBQX, protected neurons a gainst bilirubin neurotoxicity, suggesting a role for NMDA receptors in bil irubin effects. Coupled with previous work about glutamate toxicity in the same culture model, these data support the hypothesis that low levels of fr ee bilirubin may promote programmed neuronal death corresponding to an apop totic process which involves caspase activation and requires the participat ion of NMDA receptors, along with bilirubin-induced inhibition of protein k inase C activity. (C) 2000 Academic Press.