N-methyl-D-aspartate receptor and neuronal nitric oxide synthase activation mediate bilirubin-induced neurotoxicity Short title: Mediators of bilirubin-induced neurotoxicity

Hyperbilirubinemia may lead to neurotoxicity and neuronal death. Although the mechanisms of nerve cell damage by unconjugated bilirubin (UCB) appear to involve a disruption of the redox status and excitotoxicity, the contribution of nitric oxide (NO) and of N-methyl-D-aspartate (NMDA) glutamate receptors is unclear. Thus, we decided to investigate the role of NO and NMDA glutamate receptors in the pathways of nerve cell demise by UCB. Neurons were incubated with 100 μM UCB, in the presence of 100 μM human serum albumin, for 4 h, at 37oC, alone or in combination with L-NAME (an inhibitor of nitric oxide synthase, nNOS), hemoglobin (a NO scavenger), or MK-801 (a NMDA receptor antagonist). Exposure to UCB leads to increased expression of nNOS and production of both NO and cyclic guanosine 3’,5’-monophosphate (cGMP), together with protein oxidation and depletion of GSH. These events concur for cell dysfunction and death and were counteracted by L-NAME. Moreover, the UCB-induced loss of cell viability was abolished by hemoglobin, whereas the activation of nNOS and production of both NO and cGMP were counteracted by MK-801 resulting in a significant protection from cell dysfunction and death. These results reinforce the involvement of oxidative stress by showing that nerve cell lesion by UCB is mediated by NO and, therefore, counteracted by NO inhibitors or scavengers. Remarkably, our findings strongly suggest that the activation of nNOS and neurotoxicity occur through the engagement of NMDA receptors. These data reveal a role for overstimulation of glutamate receptors in mediating oxidative damage by UCB. Molecular Medicine www.molmed.org U N C O R R E C TE D P R O O F