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

Biochim Biophys Acta. 2006 Feb;1763(2):214-25. doi: 10.1016/j.bbamcr.2005.12.012. Epub 2006 Jan 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects*
  • Caspase 9
  • Caspases / analysis
  • Chromatography, High Pressure Liquid
  • Disulfides / analysis
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Flow Cytometry
  • Glucosephosphate Dehydrogenase / analysis
  • Glutathione / analysis
  • Glutathione / deficiency
  • Glutathione / metabolism*
  • Glutathione Peroxidase / analysis
  • Glutathione Reductase / analysis
  • Glutathione Transferase / analysis
  • HL-60 Cells
  • Humans
  • Kinetics
  • Membrane Potentials / drug effects
  • Mitochondria / metabolism*
  • Mitochondria / physiology
  • Necrosis
  • Preservatives, Pharmaceutical / pharmacology*
  • Reactive Oxygen Species / metabolism
  • Spectrophotometry, Ultraviolet
  • Thiazoles / pharmacology*


  • Disulfides
  • Preservatives, Pharmaceutical
  • Reactive Oxygen Species
  • Thiazoles
  • 2-methyl-4-isothiazolin-3-one
  • 5-chloro-2-methyl-4-isothiazolin-3-one
  • Glucosephosphate Dehydrogenase
  • Glutathione Peroxidase
  • Glutathione Reductase
  • Glutathione Transferase
  • CASP9 protein, human
  • Caspase 9
  • Caspases
  • Glutathione