Toxicology 275 (2010) 1–9 Contents lists available at ScienceDirect Toxicology journal homepage: www.elsevier.com/locate/toxicol Nimesulide aggravates redox imbalance and calcium dependent mitochondrial permeability transition leading to dysfunction in vitro Brijesh Kumar Singh a,b , Madhulika Tripathi a , Pramod Kumar Pandey b , Poonam Kakkar a,∗ a Herbal Research Section, Indian Institute of Toxicology Research (CSIR) (Formerly-Industrial Toxicology Research Centre), P.O. Box-80, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India b Department of Biotechnology, J.C. Bose Institute of Life Sciences, Bundelkhand University, Jhansi 284128, Uttar Pradesh, India article info Article history: Received 4 March 2010 Received in revised form 4 May 2010 Accepted 4 May 2010 Available online 8 May 2010 Keywords: Nimesulide Mitochondria Ca 2+ overload Reactive oxygen species Membrane permeability transition Redox regulation abstract Nimesulide (selective cyclooxygenase-2 inhibitor) is a nonsteroidal anti-inflammatory drug for the symp- tomatic treatment of painful conditions like osteoarthritis, spondilitis and primary dysmenorrhoea. Nimesulide induced liver damage is a serious side effect of this otherwise popular drug. The mechanism involved in nimesulide induced hepatotoxicity is still not fully elucidated. However, both mitochon- drial dysfunction and oxidative stress have been implicated in contributing to liver injury in susceptible patients. Mitochondria besides being the primary source of energy, act as a hub of signals responsible for initiating cell death, irrespective of the pathway, i.e. apoptosis or necrosis. The present study was aimed to explore the role of compounding stress, i.e. Ca 2+ overload and GSH depletion in nimesulide induced mitochondrial toxicity and dysfunction. Our study showed that, nimesulide (100 M) treatment resulted into rapid depletion of GSH (60%) in isolated rat liver mitochondria and significant Ca 2+ dependent MPT changed. Enhanced ROS generation (DCF fluorescence) was also observed in mitochondria treated with nimesulide. An important finding was that the concentration at which nimesulide oxidized reduced pyri- dine nucleotides (autofluorescence of NAD(P)H), it affected mitochondrial electron flow (MTT activity decreased by 75%) and enhanced mitochondrial depolarization significantly as assessed by Rhodamine 123 fluorescent probe. Therefore, nimesulide was found to aggravate redox imbalance and affect Ca 2+ dependent mitochondrial membrane permeability transition leading to dysfunction and ultimately cell death. © 2010 Elsevier Ireland Ltd. All rights reserved. Abbreviations: BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N ′ ,N ′ -tetraacetic acid; BSA, bovine serum albumin; CCCP, carbonyl cyanide 3-chlorophenyl- hydrazone; CMF, 5 ′ -chloromethylfluorescien; CMFDA, 5 ′ -chloromethylflouroscein diacetate; COX-2, cyclooxygenase subunit-2; CsA, cyclosporineA; DCF, 2 ′ ,7 ′ ,- dichlorohydrofluorescien; DCFH-DA, 2 ′ ,7 ′ ,-dichlorohydrofluorescien diacetate; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol tetraacetic acid; ETC, elec- tron transport chain; GPx, glutathione peroxidase; GR, glutathione reduc- tase; GSH, reduced glutathione; GSSG, oxidized glutathione; HEPES, 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid; MDA, malondialdehyde; MOPS, 3-(N-morpholino)propanesulfonic acid; MPT, mitochondrial membrane per- meability transition; mtSOD, mitochondrial superoxide dismutase; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAPQ1, N-acetyl-p- benzoquinone imine; NEM, N-ethylmaleimide; Nim, nimesulide or (N-(4-nitro- 2-phenoxyphenyl)-methanesulfonamide); NSAID, nonsteroidal anti-inflammatory drug; PUFA, polyunsaturated fatty acids; Rh123, Rhodamine 123; ROS, reactive oxygen species; RuRed, ruthenium red; SODs, superoxide dismutases; TBARS, thiobarbituric acid reactive substances; t-BHP, tert-butyl hydroperoxide; m, mitochondrial membrane potential; mLow, mitochondrial depolarization. ∗ Corresponding author. Tel.: +91 522 2627586x269; fax: +91 522 2628227. E-mail address: poonam kakkar@yahoo.com (P. Kakkar). 1. Introduction In the living cell, mitochondria are at the helm of a number of physiological processes, of which, oxidative phosphorylation, production of reactive oxygen species (ROS), Ca 2+ uptake/release and metabolic pathways are important (Kakkar and Singh, 2007). Recently mitochondria have received considerable attention as a principal target of drug-induced toxicity. It is well known that a large number of natural, commercial, pharmaceutical and envi- ronmental chemicals manifest their toxicity by interfering with mitochondrial bioenergetics. Mitochondrial damage and dysfunc- tion is observed in a number of patho-physiologies including cirrhosis, hepatoma, and chronic liver injuries by alcohol consump- tion, myocardial preconditioning, and ischemia–reperfusion injury (Hoek et al., 2002; Kevin et al., 2003). Mitochondria use approx- imately 90% of the consumed O 2 for oxidative phosphorylation and ATP synthesis, thus it is a probable site for ROS generation. Intra-cellular reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutases (SODs) and a variety of other antioxidant defenses keep ROS in check, which allows cells to function homeostatically and prevent oxida- 0300-483X/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2010.05.001