title: Phenylenediamine induced hepatocyte cytotoxicity redox. Cycling mediated oxidative stress without oxygen activation.
abstract: Muscle necrosis induced by various phenylenediamine derivatives has been correlated with their autoxidation rate. However, a more detailed investigation of the cytotoxic mechanism using a model system of isolated hepatocytes and 2,3,5,6-tetramethylphenylenediamine (DD) shows little oxygen activation as indicated by the absence of cyanide resistant respiration, lipid peroxidation and lack of cytoprotection by iron chelators, superoxide dismutase mimics and xanthine oxidase inhibitors. Cytotoxicity was however attributed to oxidative stress as GSH was not only rapidly oxidized to GSSG but mixed protein disulfide formation also occurred. Furthermore, the disulfide reductant dithiothreitol added some time after DD restored protein thiols and prevented further cytotoxicity. This oxidative stress was attributed to a futile two electron redox cycle involving oxidation of DD to the corresponding diimine by the mitochondrial electron transport chain and rereduction by DT diaphorase. Evidence suggesting this was that both diimine accumulation and the ensuing cytotoxicity were markedly increased by inactivating hepatocyte DT diaphorase but were prevented by a subtoxic concentration of the mitochondrial respiratory inhibitor cyanide. Furthermore, addition of NADH generating substrates such as lactate, sorbitol, xylitol or ethanol prevented DD induced GSH oxidation and cytotoxicity. This suggests that DD undergoes intracellular redox cycling without oxygen activation until the hepatocyte is unable to maintain redox homeostasis and mixed protein disulfide cytotoxicity ensues.
authors.affiliation: Faculty of Pharmacy, University of Toronto, Ont., Canada.
authors.forename: C
authors.initials: C
authors.lastname: Sood
authors.forename: S
authors.initials: S
authors.lastname: Khan
authors.forename: P J
authors.initials: PJ
authors.lastname: O'Brien
mesh_terms: D000818:Animals; D016923:Cell Death; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D005978:Glutathione / Q000032:analysis; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D009243:NAD / Q000032:analysis; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D010655:Phenylenediamines / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015219:Substrate Cycling
chemical_list: D010655:Phenylenediamines; D009243:NAD; D016660:NAD(P)H Dehydrogenase (Quinone); D005978:Glutathione