Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria.
OBJECTIVE
Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets.


METHODS
We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors.


RESULTS
Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P<0.01), as well as disrupted protein mobility (P<0.001). Consistent with increased permeability, cytochrome c was released from the intermembrane space (P<0.01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P<0.01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation.


CONCLUSIONS
UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.
Rodrigues
Cecília M P
CM
Centro de Patogénese Molecular, Faculdade de Farmácia, University of Lisbon, Av. Forças Armadas, 1600-083, Lisbon, Portugal. cmprodrigues@ff.ul.pt
Solá
Susana
S
Brito
Maria A
MA
Brites
Dora
D
Moura
José J G
JJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001663:Bilirubin / Q000378:metabolism* / Q000633:toxicity; D003497:Cyclic N-Oxides; D003574:Cytochrome c Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D008560:Membrane Fluidity / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D013113:Spin Labels
D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D013113:Spin Labels; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl; C055536:7-doxylstearic acid; D001663:Bilirubin