title: Bilirubin injury to neurons: contribution of oxidative stress and rescue by glycoursodeoxycholic acid.
abstract: It is well established that high levels of unconjugated bilirubin (UCB) can be toxic to the central nervous system, and oxidative stress is emerging as a relevant event in the mechanisms of UCB encephalopathy. In contrast, the hydrophilic bile acid, ursodeoxycholic acid (UDCA), has been reported as a cytoprotective and antioxidant molecule. In this study, we investigated if exposure of rat neurons in primary culture to clinically relevant concentrations of UCB leads to oxidative injury. The contribution of oxidative stress in UCB neurotoxicity was further investigated by examining whether the reduction of NO production by NAME, an inhibitor of nitric oxide synthase, prevents the disruption of the redox status and neuronal damage. Moreover, we evaluated the ability of glycoursodeoxycholic acid (GUDCA), the most relevant conjugated derivative in the serum of patients treated with UDCA, to abrogate the UCB-induced oxidative damage. Cultured rat neurons were incubated with 50 or 100microM UCB in the presence of 100microM human serum albumin, alone or in combination with 100microM NAME or with 50microM GUDCA, for 4h at 37 degrees C. Protein carbonyls, 4-hydroxy-2-nonenal-protein adducts, intracellular glutathione content and cell death were determined. The results obtained showed that UCB induces protein oxidation and lipid peroxidation, while diminishes the thiol antioxidant defences, events that were correlated with the extent of cell death. Moreover, these events were counteracted by NAME and abrogated in the presence of GUDCA. Collectively, this study shows that oxidative stress is one of the pathways associated with neuronal viability impairment by UCB, and that GUDCA significantly prevents such effects from occurring. These findings corroborate the antioxidant properties of the bile acid and point to a new therapeutic approach for UCB-induced neurotoxicity due to oxidative stress.
authors.affiliation: Centro de Patogénese Molecular/iMed.UL, Faculdade de Farmácia, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal. abrito@ff.ul.pt
authors.forename: Maria A
authors.initials: MA
authors.lastname: Brito
authors.forename: Sílvia
authors.initials: S
authors.lastname: Lima
authors.forename: Adelaide
authors.initials: A
authors.lastname: Fernandes
authors.forename: Ana S
authors.initials: AS
authors.lastname: Falcão
authors.forename: Rui F M
authors.initials: RF
authors.lastname: Silva
authors.forename: D Allan
authors.initials: DA
authors.lastname: Butterfield
authors.forename: Dora
authors.initials: D
authors.lastname: Brites
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D001663:Bilirubin / Q000378:metabolism*; D001921:Brain / Q000196:embryology / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D016923:Cell Death; D002478:Cells, Cultured; D019610:Cytoprotection; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006706:Homeostasis; D015227:Lipid Peroxidation; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009474:Neurons / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018384:Oxidative Stress; D050050:Protein Carbonylation; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000378:metabolism; D014580:Ursodeoxycholic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D000447:Aldehydes; D000975:Antioxidants; D004791:Enzyme Inhibitors; D012709:Serum Albumin; D009569:Nitric Oxide; C024033:glycoursodeoxycholic acid; D014580:Ursodeoxycholic Acid; D019001:Nitric Oxide Synthase; D005978:Glutathione; C027576:4-hydroxy-2-nonenal; D001663:Bilirubin; D019331:NG-Nitroarginine Methyl Ester