Low micromolar zinc exerts cytotoxic action under H2O2-induced oxidative stress: Excessive increase in intracellular Zn concentration

The ability of zinc to retard oxidative processes has been recognized for many years. However, zinc is cytotoxic under certain oxidative stress. In this study, we investigated the effect of H₂O₂ on intracellular Zn²⁺ concentration of rat thymocytes and its relation to the cytotoxicity. Experiments were cytometrically performed by the use of fluorescent probes, propidium iodide, FluoZin-3-AM, and 5-chloromethylfluorescein diacetate. ZnCl₂ potentiated cytotoxicity of H₂O₂ while TPEN, a chelator for intracellular Zn²⁺, attenuated it. Results suggested an involvement of intracellular Zn²⁺ in the cytotoxicity of H₂O₂. H₂O₂ at concentrations of 30 μM or more (up to 1000 μM) significantly increased intracellular Zn²⁺ concentration. There were two mechanisms. (1) H₂O₂ decreased cellular content of nonprotein thiols, possibly resulting in release of Zn²⁺ from thiols as cellular Zn²⁺ binding sites. (2) H₂O₂ increased membrane Zn²⁺ permeability because external ZnCl₂ application further elevated intracellular Zn²⁺ concentration. Micromolar H₂O₂ may induce excessive elevation of intracellular Zn²⁺ concentration that is harmful to cellular functions. However, the incubation with micromolar ZnCl₂ alone increased cellular content of nonprotein thiols, one of the factors protecting cells against oxidative stress. Though zinc is generally considered to be protective with its antioxidant property, this study reveals the toxic effect of zinc even in micromolar range under oxidative stress induced by H₂O₂.