GSH depletion, protein S-glutathionylation and mitochondrial transmembrane potential hyperpolarization are early events in initiation of cell death induced by a mixture of isothiazolinones in HL60 cells

We recently described that brief exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) induces apoptosis at low concentrations (0.001-0.01%) and necrosis at higher concentrations (0.05-0.1%). In this study, we show that glutathione (GSH) depletion, reactive oxygen species generation, hyperpolarization of mitochondrial transmembrane potential (Delta psi m) and formation of protein-GSH mixed disulphides (S-glutathionylation) are early molecular events that precede the induction of cell death by CM1/M1. When the cells exhibit common signs of apoptosis, they show activation of caspase-9, reduction of A Tin and, more importantly, decreased protein S-glutathionylation. In contrast, necrosis is associated with severe mitochondrial damage and maximal protein S-glutathionylation. CMI/MI-induced cytotoxicity is also accompanied by decreased activity of GSH-related enzymes. Pre-incubation with L-buthionine-(S,R)-sulfoxmine (BSO) clearly switches the mode of cell death from apoptosis to necrosis at 0.01% CMI/MI. Collectively, these results demonstrate that CMI/MI alters the redox status of HL60 cells, and the extent and kinetics of GSH depletion and S-glutathionylation appear to determine whether cells undergo apoptosis or necrosis. We hypothesize that S-glutathionylation of certain thiol groups accompanied by GSH depletion plays a critical role in the molecular mechanism of CMI/MI cytotoxicity. (c) 2006 Elsevier B.V. All rights reserved.