Bilirubin Exerts Additional Toxic Effects in Hypoxic Cultured Neurons from the Developing Rat Brain by the Recruitment of Glutamate Neurotoxicity

Abstract

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 M), 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. Cerebral hypoxia remains a significant risk for neonatal morbidity and mortality, as well as long-term neurologic sequelae such as cerebral palsy, mental retardation, or seizure disorders (1, 2). Although lack of oxygen has been associated with neuronal degeneration (3, 4), it was not clear whether hypoxia itself, without the influence of combined ischemia, kills brain cells. In contrast to studies in intact animals, which are often difficult to interpret due to a number of confounding variables, cell culture seems a useful tool for elucidating cellular and molecular mechanisms involved in clinical disorders such as cerebral hypoxia (5, 6). Indeed, neuronal cells cultured in chemically defined medium have proven to mimic morphologic, electrophysiological, and neurochemical development and differentiation occurring in vivo (7,8), and such a preparation provides the advantage of investigating cellular responses inherent to a single variable. By using cultured neurons from the developing rat forebrain as a model, we have previously demonstrated that transient oxygen deprivation may trigger neuronal death through an apoptotic process that requires specific and time-dependent changes in protein synthesis (9, 10), a conclusion supported by other studies (6, 11).Bilirubin constitutes another potential cause of brain injury (12, 13). Hyperbilirubinemia is still a common concern in neonatology, especially in the care of low birth weight prema- ABSTRACT 507

Reassessment of the Unbound Concentrations of Unconjugated Bilirubin in Relation to Neurotoxicity In Vitro

“…One of the 12 papers selected one was done only at a single B F value of 0.5 M in the absence of albumin (13), and in part duplicated data from another paper by the same group (14) that was performed over a range of B F values. In another selected paper (15), studies were done at B F values below 5 M, but no toxicity was observed at B F values Յ 1 M. In three of the papers selected (13,14,16), the stock UCB solution was markedly supersaturated, so that precipitation and degradation likely occurred; thus, the true threshold for UCB toxicity may have been lower than the calculated B F values.…”

“…In another selected paper (15), studies were done at B F values below 5 M, but no toxicity was observed at B F values Յ 1 M. In three of the papers selected (13,14,16), the stock UCB solution was markedly supersaturated, so that precipitation and degradation likely occurred; thus, the true threshold for UCB toxicity may have been lower than the calculated B F values. Another selected paper studied B F values below 5 M, but there were no studies done at B F values between 383 and 1761 nM, so that the true threshold could not be evaluated (17).…”

“…In the absence of HSA, MTT activity was impaired after 24 h of exposure of embryonic rat hippocampal neurons to 250 nM UCB (p Ͻ 0.05; figure not shown) (25) and of 14-d embryonic rat forebrain neurons to UCB concentrations as low as 400 nM (Fig. 4) (14) or 500 nM (data not shown) (13). The last two studies also demonstrated that exposure to 500 nM UCB for 24 to 96 h caused a large decrease in [ 3 H]thymidine incorporation, accompanied by increases in subsequent [ 3 H]thymidine release and in apoptosis.…”

Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization

“…These investigators performed the majority of analyses of bilirubin-induced apoptosis at a bilirubin: albumin molar ratio of 3, 64 using concentrations at which bilirubin would be anticipated to precipitate from solution. 20 Moreover, as opposed to bilirubin-mediated neuronal cell death, which is triggered by stimulation of the NMDA receptor 66 and prevented by NMDA receptor antagonists, 22 both glutamate and NMDA inhibitors reduce the viability of HT29 adenocarcinoma cells. 67 These disparities in the cellular response may be a result of nonphysiologic concentrations of bilirubin utilized or, alternatively, may reflect distinct mechanisms of bilirubin action in different cell lines.…”

Bilirubin: An Endogenous Product of Heme Degradation with Both Cytotoxic and Cytoprotective Properties: Fig. 1

“…The apoptotic process involves caspase activation and requires the participation of glutamatergic N-methyl-D-aspartate receptors. Moreover, UCB enhanced the effects of hypoxia in these immature neurons by facilitating glutamate-mediated apoptosis (Grojean et al, 2001). On the other hand, UCB inhibited glutamate uptake in cultured rat cortical astrocytes, which play a major role in the transport of synaptically released glutamate (Silva et al, 1999).…”