Determinants of bilirubin neurotoxicity by an in vitro molecular approach

Unconjugated Bilirubin (UCB) is the final product of the heme catabolism. The high serum UCB concentrations in the first days of life of the newborns, due to immature mechanisms for hepatic uptake, conjugation and biliary secretion, is called physiological neonatal jaundice. This common condition is generally a benign and transient phenomenon, but in some cases the hyperbilirubinemia can progress to bilirubin encephalopaties ranging from minimally neurological injury to severe and permanent neurodevelopmental dysfunction. In the present thesis the SH-SY5Y neuroblastoma cell line was used to approach the molecular events associated to bilirubin neurotoxicity and highlight the biochemical and molecular events that are induces in the neurons when get contact with the UCB. Depending on the bilirubin concentration and the time of exposure to UCB, we were able to define experimental setups for the study of bilirubin resistance and bilirubin toxicity. Using the model to study bilirubin resistance, it was demonstrated that the resistance is not entirely achieved by limiting the entrance or increasing extrusion of the pigment from the cell, but rather by enhancing the cellular defensive mechanisms, in particular against the oxidative stress. This was achieved by increasing the intracellular glutathione content via the specific induction of the genes and activity of the System Xc-. Furthermore, the cells exposed to bilirubin over-expressed several additional genes that encode for important antioxidant and detoxifying proteins like Heme Oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. As far as the mechanisms of bilirubin neurotoxicity, we showed that UCB exposure lead to the induction of the intracellular ROS accumulation. Moreover, the data presented report evidences that the bilirubin toxicity could be displayed by a mechanism of excitotoxicity carried out by the cellular release of glutamate. Further studies will be necessary to elucidate the molecular mechanisms by which bilirubin produces neurotoxicity and to understand how the cells avoid the damage. The information presented here could contribute to the identification of targets to avoid the bilirubin damage.