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Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis

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Abstract

Bilirubin is an endogenous antioxidant with cytoprotective properties, and several studies highlight its potential in the treatment of pro-oxidant diseases. We demonstrated that oxidative stress (OS), a key feature in most hepatopathies, induces cholestasis by actin cytoskeleton disarrangement and further endocytic internalization of key canalicular transporters, such as the bile salt export pump (Bsep) and the multidrug resistance-associated protein 2 (Mrp2) . Here, we evaluated the capability of physiological concentrations of unconjugated bilirubin (UB) to limit OS and the impairment in biliary secretory function induced by the model pro-oxidant agent, tert-butylhydroperoxide (tBuOOH). UB fully prevented the formation of reactive oxygen species and membrane lipid peroxidation induced by tBuOOH in isolated rat hepatocytes. In the isolated rat hepatocyte couplet model, UB (17.1 μM) prevented the endocytic internalization of Bsep and Mrp2 and the impairment in their secretory function induced by tBuOOH. UB also prevented actin disarrangement, as evaluated by both plasma membrane bleb formation and actin fluorescent staining. Finally, UB prevented tBuOOH-induced cPKC activation. Experiments in isolated perfused rat livers showed that UB prevents the increase in oxidized glutathione biliary excretion and the drop in bile flow and the biliary excretion of specific Bsep and Mrp2 substrates. We conclude that physiological concentrations of UB are sufficient to prevent the biliary secretory failure induced by OS, by counteracting actin disarrangement and the consequent internalization of canalicular transporters relevant to normal bile formation. This reveals an important role for UB in preserving biliary secretory function under OS conditions.

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Acknowledgments

This work was supported by Grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) to Drs. Aldo D. Mottino and Marcelo G. Roma. We thank Drs. Marcelo Luquita, Mara Ojeda, and Rodrigo Vena for their valuable technical assistance in HPLC determinations, flow cytometric studies, and confocal imaging, respectively.

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The authors declare that they have no conflict of interest.

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Basiglio, C.L., Toledo, F.D., Boaglio, A.C. et al. Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis. Arch Toxicol 88, 501–514 (2014). https://doi.org/10.1007/s00204-013-1143-0

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