title: 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.
abstract: 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 (DeltaPsim) and formation of protein-GSH mixed disulphides (S-glutathionylation) are early molecular events that precede the induction of cell death by CMI/MI. When the cells exhibit common signs of apoptosis, they show activation of caspase-9, reduction of DeltaPsim 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)-sulfoximine (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.
authors.affiliation: Department of Molecular Biology, University of Siena, via Fiorentina 1, 53100 Siena, Italy. distefano@unisi.it
authors.forename: Anna
authors.initials: A
authors.lastname: Di Stefano
authors.forename: Simona
authors.initials: S
authors.lastname: Frosali
authors.forename: Alessandra
authors.initials: A
authors.lastname: Leonini
authors.forename: Anna
authors.initials: A
authors.lastname: Ettorre
authors.forename: Raffaella
authors.initials: R
authors.lastname: Priora
authors.forename: Francesca Cherubini
authors.initials: FC
authors.lastname: Di Simplicio
authors.forename: Paolo
authors.initials: P
authors.lastname: Di Simplicio
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053453:Caspase 9; D020169:Caspases / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis; D005978:Glutathione / Q000032:analysis / Q000172:deficiency / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000032:analysis; D005980:Glutathione Reductase / Q000032:analysis; D005982:Glutathione Transferase / Q000032:analysis; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology; D009336:Necrosis; D011310:Preservatives, Pharmaceutical / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013844:Thiazoles / Q000494:pharmacology*
chemical_list: D004220:Disulfides; D011310:Preservatives, Pharmaceutical; D017382:Reactive Oxygen Species; D013844:Thiazoles; C011506:2-methyl-4-isothiazolin-3-one; C011421:5-chloro-2-methyl-4-isothiazolin-3-one; D005954:Glucosephosphate Dehydrogenase; D005979:Glutathione Peroxidase; D005980:Glutathione Reductase; D005982:Glutathione Transferase; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases; D005978:Glutathione