title: Evaluation of mitochondrial function in isolated rat hepatocytes and mitochondria during oxidative stress.
abstract: The majority of toxic agents act either fully or partially via oxidative stress, the liver, specifically the mitochondria in hepatocytes, being the main target. Maintenance of mitochondrial function is essential for the survival and normal performance of hepatocytes, which have a high energy requirement. Therefore, greater understanding of the role of mitochondria in hepatocytes is of fundamental importance. Mitochondrial function can be analysed in several basic models: hepatocytes cultured in vitro; mitochondria in permeabilised hepatocytes; and isolated mitochondria. The aim of our study was to use all of these approaches to evaluate changes in mitochondria exposed in vitro to a potent non-specific peroxidating agent, tert-butylhydroperoxide (tBHP), which is known to induce oxidative stress. A decrease in the mitochondrial membrane potential (MMP) was observed in cultured hepatocytes treated with tBHP, as illustrated by a significant reduction in Rhodamine 123 accumulation and by a decrease in the fluorescence of the JC-1 molecular probe. Respiratory Complex I in the mitochondria of permeabilised hepatocytes showed high sensitivity to tBHP, as documented by high-resolution respirometry. This could be caused by the oxidation of NADH and NADPH by tBHP, followed by the disruption of mitochondrial calcium homeostasis, leading to the collapse of the MMP. A substantial decrease in the MMP, as determined by tetraphenylphosphonium ion-selective electrode measurements, also confirmed the dramatic impact of tBHP-induced oxidative stress on mitochondria. Swelling was observed in isolated mitochondria exposed to tBHP, which could be prevented by cyclosporin A, which is evidence for the role of mitochondrial permeability transition. Our results demonstrate that all of the above-mentioned models can be used for toxicity assessment, and the data obtained are complementary.
authors.affiliation: Department of Physiology, Charles University in Prague, Faculty of Medicine in Hradec Krávlové, Czech Republic. wolff@lfhk.cuni.cz
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Cervinková
authors.forename: Halka
authors.initials: H
authors.lastname: Lotková
authors.forename: Pavla
authors.initials: P
authors.lastname: Kriváková
authors.forename: Tomás
authors.initials: T
authors.lastname: Rousar
authors.forename: Otto
authors.initials: O
authors.lastname: Kucera
authors.forename: Lukás
authors.initials: L
authors.lastname: Tichý
authors.forename: Miroslav
authors.initials: M
authors.lastname: Cervinka
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Drahota
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008365:Manometry; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology*; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D008933:Mitochondrial Swelling / Q000187:drug effects / Q000502:physiology; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress; D010100:Oxygen / Q000032:analysis / Q000378:metabolism; D010101:Oxygen Consumption; D051381:Rats; D017208:Rats, Wistar; D020122:tert-Butylhydroperoxide / Q000633:toxicity
chemical_list: D016877:Oxidants; D020122:tert-Butylhydroperoxide; D010100:Oxygen