Chemico-Biological Interactions 172 (2008) 39–47 Available online at www.sciencedirect.com Tetramethylphenylenediamine-induced hepatocyte cytotoxicity caused by lysosomal labilisation and redox cycling with oxygen activation Jalal Pourahmad a , Peter J. O’Brien b,∗ , Katie Chan b , Ataollah Shakouri a a Faculty of Pharmacy & Pharmaceutical Sciences Research Center, Shaheed Beheshti Medical University, Tehran, PO BOX 14155-6153, Iran b Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, Ont. M5S 3M2, Canada Received 11 September 2007; received in revised form 4 December 2007; accepted 5 December 2007 Available online 15 December 2007 Abstract It has already been reported that in vivo muscle necrosis induced by various phenylenediamine derivatives correlated with their in vitro autoxidation rate [9]. Now in a more detailed investigation of the cytotoxic mechanism of a ring-methylated phenylenediamine known as tetramethylphenylenediamine or durenediamine (DD) towards isolated rat hepatocytes has been carried out. Cytotoxicity was preceded by ROS formation which was markedly increased by inactivating DT-diaphorase or catalase but were prevented by a subtoxic concentration of the mitochondrial respiratory inhibitor cyanide. This suggests that ROS generation could be attributed to a futile two-electron redox cycle involving oxidation of phenylenediamine to the corresponding diimine by the mitochondrial electron transfer chain and re-reduction by the DT-diaphorase. Endocytosis inhibitors, lysosomotropic agents or lysosomal protease inhibitors also prevented DD-induced cytotoxicity sug- gesting that DD-induced ROS caused lysosomal damage and protease activation in hepatocytes. Furthermore preincubation with deferoxamine (a ferric iron chelator) or addition of antioxidants, catalase or ROS scavengers (mannitol, tempol or dimethylsulfox- ide) prevented DD cytotoxicity. These results suggest that H 2 O 2 reacts with lysosomal Fe 2+ to form “ROS” which causes lysosomal lipid peroxidation, membrane disruption, protease release and cell death. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Hepatocyte; Phenylenediamine; Cytotoxicity; Oxidative stress; Cytochrome oxidase; Lysosomes; Proteolysis Abbreviations: DD, durenediamine (2,3,5,6-tetramethylphenylen- ediamine); TMPD, N, N,N ′ ,N ′ -tetramethylphenylenediamine; GSH, glutathione; GSSG, oxidized glutathione; ROS, reactive oxygen species; rpm, rotations per minute; S.E., standard error; S.E.M., stan- dard error of the mean; SOD, superoxide dismutase; ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; DCF, dichlorofluorescein; HEPES, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid. ∗ Corresponding author. Tel.: +1 416 978 2716; fax: +1 416 978 8511. E-mail address: peter.obrien@utoronto.ca (P.J. O’Brien). 1. Introduction Phenylenediamines are present in hair dye con- stituents [1], photographic developing agents [2], rubber industry vulcanizing agents [3] and as industrial antioxidants [4]. Rats given subcutaneous injections of N,N,N ′ ,N ′ -tetramethylphenylenediamine (TMPD) developed degeneration and dissolution of skeletal mus- cle fibers; necrotic lesions on the tongue; cytoplasmic vacuolation in cardiac muscle and lesions in the gastroc- nemius [5,6] and was also cytotoxic towards cultured 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2007.12.002