title: Role of intracellular calcium and S-glutathionylation in cell death induced by a mixture of isothiazolinones in HL60 cells.
abstract: Previously we reported 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) shifts the cells into a state of oxidative stress that induces apoptosis and necrosis. In this study, flow cytometric analysis showed that CMI/MI induces early perturbation of calcium homeostasis, increasing cytosolic and mitochondrial calcium and depleting the intracellular endoplasmic reticulum (ER) stores. The calcium chelator BAPTA-AM reduced necrosis and secondary necrosis, the loss of DeltaPsim and S-glutathionylation induced by necrotic doses of CMI/MI, but did not protect against CMI/MI-induced apoptosis, mitochondrial calcium uptake and mitochondrial hyperpolarization. This indicates that increased cytoplasmic calcium does not have a causal role in the induction of apoptosis, while cross-talk between the ER and mitochondria could be responsible for the induction of apoptosis. GSH-OEt pretreatment, which enhances cellular GSH content, reduced S-glutathionylation and cytosolic and mitochondrial calcium levels, thus protecting against both apoptosis and necrosis shifting to apoptosis. Therefore, the degree of GSH depletion, paralleled by the levels of protein S-glutathionylation, may have a causal role in increasing calcium levels. The mitochondrial calcium increase could be responsible for apoptosis, while necrosis is associated with cytoplasmic calcium overload. These findings suggest that S-glutathionylation of specific proteins acts as a molecular linker between calcium and redox signalling.
authors.affiliation: Department of Molecular Biology, University of Siena, via Fiorentina 1, 53100 Siena, Italy.
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: Giuseppe
authors.initials: G
authors.lastname: Di Maio
authors.forename: Sandra
authors.initials: S
authors.lastname: Nuti
authors.forename: Simona
authors.initials: S
authors.lastname: Tavarini
authors.forename: Paolo
authors.initials: P
authors.lastname: Di Simplicio
authors.forename: Anna
authors.initials: A
authors.lastname: Di Stefano
mesh_terms: D002118:Calcium / Q000378:metabolism*; D016923:Cell Death; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism; D013844:Thiazoles / Q000633:toxicity*
chemical_list: D013844:Thiazoles; C011506:2-methyl-4-isothiazolin-3-one; C011421:5-chloro-2-methyl-4-isothiazolin-3-one; D005978:Glutathione; D002118:Calcium