title: Mechanisms of cell death pathway activation following drug-induced inhibition of mitochondrial complex I.
abstract: Respiratory complex I inhibition by drugs and other chemicals has been implicated as a frequent mode of mitochondria-mediated cell injury. However, the exact mechanisms leading to the activation of cell death pathways are incompletely understood. This study was designed to explore the relative contributions to cell injury of three distinct consequences of complex I inhibition, i.e., impairment of ATP biosynthesis, increased formation of superoxide and, hence, peroxynitrite, and inhibition of the mitochondrial protein deacetylase, Sirt3, due to imbalance of the NADH/NAD(+) ratio. We used the antiviral drug efavirenz (EFV) to model drug-induced complex I inhibition. Exposure of cultured mouse hepatocytes to EFV resulted in a rapid onset of cell injury, featuring a no-effect level at 30µM EFV and submaximal effects at 50µM EFV. EFV caused a concentration-dependent decrease in cellular ATP levels. Furthermore, EFV resulted in increased formation of peroxynitrite and oxidation of mitochondrial protein thiols, including cyclophilin D (CypD). This was prevented by the superoxide scavenger, Fe-TCP, or the peroxynitrite decomposition catalyst, Fe-TMPyP. Both ferroporphyrins completely protected from EFV-induced cell injury, suggesting that peroxynitrite contributed to the cell injury. Finally, EFV increased the NADH/NAD(+) ratio, inhibited Sirt3 activity, and led to hyperacetylated lysine residues, including those in CypD. However, hepatocytes isolated from Sirt3-null mice were protected against 40µM EFV as compared to their wild-type controls. In conclusion, these data are compatible with the concept that chemical inhibition of complex I activates multiple pathways leading to cell injury; among these, peroxynitrite formation may be the most critical.
authors.affiliation: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, USA; Laboratory of Molecular Genetics, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan. Electronic address: imaizumi@med.u-ryukyu.ac.jp.
authors.forename: Naoki
authors.initials: N
authors.lastname: Imaizumi
authors.affiliation: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, USA.
authors.forename: Kang
authors.initials: K
authors.lastname: Kwang Lee
authors.affiliation: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, USA.
authors.forename: Carmen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, USA.
authors.forename: Urs A
authors.initials: UA
authors.lastname: Boelsterli
mesh_terms: D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D000480:Alkynes; D000818:Animals; D048588:Benzoxazines / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000037:antagonists & inhibitors / Q000378:metabolism; D003521:Cyclopropanes; D065688:Cytochrome P-450 CYP2C9 Inhibitors / Q000494:pharmacology*; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008665:Metalloporphyrins / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000037:antagonists & inhibitors / Q000378:metabolism; D061251:Primary Cell Culture; D056566:Sirtuin 3 / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C492721:5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato-iron(III); D000480:Alkynes; D048588:Benzoxazines; D000081406:Peptidyl-Prolyl Isomerase F; D003521:Cyclopropanes; D065688:Cytochrome P-450 CYP2C9 Inhibitors; D008665:Metalloporphyrins; C000631446:PPIF protein, mouse; C525046:Sirt3 protein, mouse; C473248:iron 5,10,15,20-tetrakis-4-carboxyphenylporphyrin; D009243:NAD; D013481:Superoxides; D030421:Peroxynitrous Acid; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D056566:Sirtuin 3; D021983:Cyclophilins; D042967:Electron Transport Complex I; C098320:efavirenz
keywords: Complex I inhibition; Drug-induced liver injury (DILI); Efavirenz; Mitochondria; Peroxynitrite; Sirt3