A link between hyperbilirubinemia, oxidative stress and injury to neocortical synaptosomes.
Cytotoxicity by unconjugated bilirubin involves disturbances of membrane structure, excitotoxicity and cell death. These events were reported to trigger elevated free radicals production and impairment of calcium homeostasis, and to result in loss of cell membrane integrity. Therefore, this study was designed to investigate whether interaction of clinically relevant concentrations of free unconjugated bilirubin with synaptosomal membrane vesicles could be linked to oxidative stress, cytosolic calcium accumulation and perturbation of membrane function. Synaptosomal vesicles were prepared from gerbil cortical brain tissue and incubated with purified bilirubin (<or=1 microM), for 4 h at 37 degrees C. Intracellular concentrations of reactive oxygen species (ROS) and calcium were determined by dichlorofluorescin and BAPTA fluorescent probes, respectively. Membrane protein and lipid oxidation were evaluated by immunocytochemistry and phosphatidylserine exposure by annexin V binding. Levels of reduced and oxidized glutathione (GSH and GSSG, respectively), as well as activities of Mg(2+)-ATPase aminophospholipid translocase (flippase) and Na(+),K(+)-ATPase, were also measured. Our results showed that bilirubin induced oxidative stress, due to a rise in lipid (>or=10%, P<0.05) and protein oxidation (>or=20%, P<0.01), ROS content (approximately 17%, P<0.01), and a decrease in GSH/GSSG ratio (>30%, P<0.01). In addition, synaptosomes exposed to bilirubin exhibited increased externalization of phosphatidylserine (approximately 10%, P<0.05), together with decreased flippase and NA(+),K(+)-ATPase (>or=15%, P<0.05) activities, events that were accompanied by enhanced intracellular calcium levels ( approximately 20%, P<0.01). The data obtained point out that interaction of unconjugated bilirubin with synaptosomal membrane vesicles leads to oxidative injury, loss of membrane asymmetry and functionality, and calcium intrusion, thus potentially contributing to the pathogenesis of encephalopathy by hyperbilirubinemia.
Centro de Patogénese Molecular--UBMBE, Faculdade de Farmácia, University of Lisbon, Av. das Forças Armadas, 1600-083 Lisbon, Portugal. abrito@ff.ul.pt
Maria Alexandra
MA
Brito
Dora
D
Brites
D Allan
DA
Butterfield
D000818:Animals; D001663:Bilirubin / Q000633:toxicity; D015153:Blotting, Western; D001930:Brain Injuries / Q000150:complications / Q000473:pathology*; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005849:Gerbillinae; D005978:Glutathione / Q000378:metabolism; D006932:Hyperbilirubinemia / Q000150:complications / Q000473:pathology / Q000503:physiopathology*; D042541:Intracellular Space / Q000187:drug effects; D015227:Lipid Peroxidation / Q000187:drug effects / Q000502:physiology; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism; D019579:Neocortex / Q000187:drug effects / Q000473:pathology*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D010718:Phosphatidylserines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013574:Synaptosomes / Q000473:pathology*
D008563:Membrane Lipids; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione; D001663:Bilirubin; D002118:Calcium