Biological risks for neurological abnormalities associated with hyperbilirubinemia.

Unconjugated bilirubin (UCB) injury to glial cells leads to the secretion of glutamate and elicits a typical inflammatory response. Release of pro-inflammatory cytokines may influence gliogenesis and neurogenesis, and lead to deficits in learning and memory. Glutamate metabolism dysregulation and overexpression of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta are consistent with schizophrenia neuropathology. Recently, an increased prevalence of schizophrenia was reported in individuals with Gilbert's syndrome and among those who have had elevated levels of UCB in the neonatal life. In this review, we explore the reactivity of astrocytes, neurons and microglia to UCB, the cascade of events implicated in the immunostimulant effects of UCB, as well as the role of each nerve cell type and maturation state in the neuropathology of UCB. Identification of the signaling events promoted by UCB will be relevant for developing novel therapies that might reduce the risk of brain injury and disabilities.

Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin.

Nerve cell injury by unconjugated bilirubin (UCB) has been implicated in brain damage during neonatal hyperbilirubinemia, particularly in the preterm newborn. Recently, it was shown that UCB is a substrate for the multidrug resistance-associated protein 1 (Mrp1), an ATP-dependent efflux pump, which may decrease UCB intracellular levels. To obtain a further insight into the role of Mrp1 in the increased vulnerability of immature cells to UCB, we evaluated the mRNA and the protein levels of Mrp1 throughout differentiation in primary cultures of rat neurons and astrocytes. Furthermore, in order to provide supportive evidence for the role of Mrp1 in the protection of nerve cells from UCB-induced effects, we evaluated cell susceptibility to UCB when Mrp1 was inhibited with MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl) ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid). The results are the first to demonstrate that Mrp1 is expressed in neurons and that both mRNA and protein levels of Mrp1 increase with cell differentiation. Additionally, inhibition of Mrp1 was associated with an increase in UCB toxic effects, namely cell death, cell dysfunction, and secretion of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, as well as of glutamate. These results point to a novel role of Mrp1 in the susceptibility of premature babies to UCB encephalopathy, and provide a startup point for the development of a new therapeutic strategy.

Dissociated primary nerve cell cultures as models for assessment of neurotoxicity.

Exogenous and endogenous neurotoxins may have poisoning effects on living organisms. Neurotoxic signs can result from human intoxication by substances present in natural ecosystems as pollutants, such as inorganic mercury, cadmium, manganese and lead, or by abnormal accumulation of endogenous compounds, as bilirubin. Dissociated primary nerve cell cultures are powerful models that can be used to evaluate the responses of target cells at the cellular and molecular levels to the deleterious effects of neurotoxic substances. Primary cultures of nerve cells are prepared from either fetal (neurons) or 2-day-old (macroglia and microglia) rat brains, cultured with specific media. Cells can then be used to evaluate the neurotoxic effects of a particular substance. By using cells with different days-in-culture it is possible to mimic and evaluate developmental-related modifications. These modifications can comprise morphological changes, cell death by necrosis (release of lactate dehydrogenase, LDH) and apoptosis (nuclear fragmentation), altered neurotransmission (impaired uptake or increased release of glutamate), neuroinflammation (enhanced cytokine production) and the generation of oxidative damage (formation of reactive oxygen species and disruption of glutathione metabolism). Here we describe the methods for nerve cell cultures, as well as some of the procedures that can be used to assess neuronal and glial cytotoxicity induced by different neurotoxins.