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Some household products have high levels of the antimicrobial 2-n-octyl-4-isothiazolin3-one (OIT). Although the diverse effects of OIT are of concern, information regarding its cellular actions is limited. In a previous study, we found that OIT increased intracellular Zn2+ levels in rat thymocytes. However, because Ca2+ is considered the essential cation that causes cell injury and death, we examined whether Ca2+ and Zn2+ were involved in OIT-induced cytotoxicity and proposed the mechanisms underlying these results. The effects of OIT on the membrane and cellular parameters of rat thymocytes were examined with a flow cytometer and appropriate fluorescent probes. OIT (0.3-3 μM) increased intracellular Zn2+ levels but not intracellular Ca2+ levels. Therefore, the involvement of Zn2+ was studied further. The simultaneous application of 0.3 μM OIT and 3 μM ZnCl2 significantly increased cells with phosphatidylserine-exposed membranes without changing the dead cells. In contrast, applications of 0.3 μM OIT or 3 μM ZnCl2 alone had no effects. The combination of OIT (0.1-1 μM) and ZnCl2 (1-3 μM) significantly decreased the cellular non-protein thiol contents. These changes that were induced by their combination were completely suppressed by adding an intracellular Zn2+ chelator. These results suggested that submicromolar concentrations of OIT induced Zn2+-dependent cytotoxicity in the presence of micromolar concentrations of external Zn2+. Because the threshold of OIT levels that affected cellular parameters in the presence of micromolar concentrations of Zn2+ are much lower than the OIT contents in some household products, the adverse effects of OIT are of great concern.

fundamental toxicological sciences

Zn&lt;sup&gt;2+&lt;/sup&gt;-dependent increase in cells with phosphatidylserine-exposed membranes after treatment with submicromolar concentrations of 2-&lt;i&gt;n&lt;/i&gt;-octyl-4-isothiazolin-3-one in rat thymocytes