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Ziram, a dithiocarbamate fungicide, protects various vegetables and fruits against infections by fungus. Recently, there have been increasing anxieties about the risks in the use of dithiocarbamate fungicides. Our previous studies showed that Zn 2+ was a determinant of Ziram cytotoxicity. In addition, Zn 2+ is linked to H 2 O 2 cytotoxicity. Therefore, in this study, we aimed to test the hypothesis that Ziram could augment the cytotoxicity of H 2 O 2 by examining the changes induced by Ziram in some cellular parameters in rat thymic lymphocytes subjected to H 2 O 2 -induced oxidative stress using flow-cytometric methods with fluorescent dyes. Ziram significantly attenuated H 2 O 2 -induced cell death at sublethal concentrations. However, in the cells under oxidative stress elicited by H 2 O 2 , Ziram promoted the changing over from intact cells to living cells with exposed phosphatidylserine (PS) on plasma membranes, whereas it inhibited the transition from PS-exposed living cells to dead cells. Ziram significantly augmented H 2 O 2 actions, including reduction of cellular glutathione levels and elevation of intracellular Zn 2+ concentrations. Conversely, it attenuated H 2 O 2 -induced depolarization of mitochondrial membrane potential. Ziram at sublethal concentrations seems to exhibit promotive and suppressive actions on the process of cell death caused by H 2 O 2 . Ziram increased the number of living cells with exposed PS, a phenomenon characteristic of early stages of apoptosis. Thus, it is concluded that Ziram exhibits pseudo-cytoprotective actions against H 2 O 2 -induced oxidative stress. Ziram at sublethal concentrations exerts promotive and suppressive actions on the process of cell death caused by oxidative stress., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ziram, one of the dithiocarbamate fungicides, is widely applied to agriculture because this agent protects various crops from fungal infections. Risks of dithiocarbamate biocide use are of concern. It was previously reported that Ziram increased the intracellular concentration of Zn 2+ . Therefore, we cytometrically studied the mechanism of Zn 2+ -dependent lethal actions of Ziram on rat lymphocytes at environmentally relevant Zn 2+ levels. Membrane and cellular parameters of rat lymphocytes were estimated by flow-cytometric techniques with appropriate fluorescent probes. The Ziram-induced increase in cell lethality was completely attenuated by Zn 2+ chelators. A significant increase of cell lethality was found on the simultaneous application of Ziram at a sublethal concentration and ZnCl 2 . The combination of Ziram and ZnCl 2 increased the cellular superoxide anion content and decreased the cellular GSH content, which possibly caused the increase in cell lethality. The zinc concentrations under the present experimental conditions were comparable to the environmentally relevant concentrations found in rivers. Therefore, the environmental level of zinc may be critical in estimating the toxicity of Ziram to wild animals.

Dithiocarbamate fungicides are used as alternative antifouling agents to highly toxic organotin antifouling agents, such as tri-n-butyltin and triphenyltin. There are some concerns regarding their environmental and health risks. It has been shown that tri-n-butyltin increases intracellular Zn(2+) levels of mammalian lymphocytes. Therefore, we examined the effects of dithiocarbamate fungicides (Ziram, Thiram, and Zineb) on rat thymic lymphocytes using a flow-cytometric technique to elucidate how these fungicides affect intracellular Zn(2+) levels. We further determined whether the agents increase intracellular Zn(2+) and/or Ca(2+), because both Zn(2+) and Ca(2+) are intracellular signals in lymphocytes, and excessive increases in their intracellular concentrations can have adverse effects. Dithiocarbamate fungicides increased intracellular Zn(2+) levels, without affecting intracellular Ca(2+) levels. Ziram was the most potent compound, increasing intracellular Zn(2+) levels via Zn(2+) influx. Ziram (1μM) greatly decreased the cellular nonprotein thiol content, and Zn(2+) chelators attenuated the Ziram-induced decrease. Ziram increased the population of annexin V-positive cells in a Zn(2+)-dependent manner. Therefore, we propose that dithiocarbamate fungicides induce Zn(2+) influx, resulting in an excessive elevation of intracellular Zn(2+) levels, leading to the induction of apoptosis. This study gives a basic insight into the mechanisms of dithiocarbamate fungicide-induced adverse events., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)