Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria

Abstract

Background/Aims: 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. q

Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization

“…That bilirubin is capable of directly causing depolarization of isolated mitochondria, as has previously been proposed by Rodrigues et al, 64 is supported by our light-scattering and rhodamine fluorescence data. Our finding that bilirubin-induced large-amplitude swelling is not blocked by cyclosporin A, an established inhibitor of the mitochondrial PTP, 58,77 in conjunction with data from Rodrigues et al indicating that bilirubin alters mitochondrial lipid polarity, 23,78 further suggests that bilirubin is capable of exerting a direct effect on mitochondrial membrane integrity.…”

Cytochrome P450 2A5 and bilirubin: Mechanisms of gene regulation and cytoprotection

“…BR causes mitochondrial dysfunction with collapse of inner mitochondrial membrane potential, a diminished activity of cytochrome c oxidase, and the release of cytochrome c to the cytosol, which is an initiation of apoptosis via mitochondrial intrinsic pathway (Rodrigues et al, 2002;Ostrow et al, 2004;Brites and Brito, 2012). BR may also induce apoptosis via mitochondrial extrinsic pathway by activation of caspase-8, suggesting that BR interaction with a death receptors (e.g.…”

“…Many reports have shown BR disrupts the functions of mitochondria at the membrane level and also induce apoptosis via both intrinsic and extrinsic apoptotic pathway (Rodrigues et al, 2002;Seubert et al, 2002;Ostrow et al, 2004;Vaz et al, 2011;Brites and Brito, 2012). BR has been also reported to induce expression of regulatory genes and proteins for unfolded protein response (UPR), which is a key regulatory defense mechanism against ER stress (Calligaris et al, 2009;Oakes and Bend, 2010).…”

Bilirubin: An Endogenous Product of Heme Degradation with Both Cytotoxic and Cytoprotective Properties: Fig. 1

“…UCB increased lipid polarity and fluidity, as well as protein mobility, resulting in an increased permeability of this membrane and release of cytochrome c (Rodrigues et al, 2002b,c). Ursodeoxycholic acid, a mitochondrial membrane-stabilizing agent that prevents the changes in mitochondrial transmembrane potential, almost completely abolished the UCB-induced membrane perturbation in isolated mitochondria (Rodrigues et al, 2002b) and inhibited the UCB-mediated apoptosis of neurons and astrocytes (Silva et al, 2001). Rodrigues et al (2002d) reported that the apoptotic effect of UCB in primary cultures of rat neurons and astrocytes decreases as a function of age-in-culture of these cells, thus confirming the earlier findings of Amit and Brenner (1993) in primary cultures of fetal rat glial cells.…”