Assessment of phosphamidon‐induced apoptosis in human peripheral blood mononuclear cells: Protective effects of N‐acetylcysteine and curcumin

Abstract

The molecular mechanism for noncholinergic toxicity of phosphamidon, an extensively used organophosphate pesticide, is still not clear. The aim of the present study is to find the possible molecular mechanism of this pesticide to induce apoptosis and the role of different drugs for attenuation of such effects. Human peripheral blood mononuclear cells (PBMC) were incubated with increasing concentrations of phosphamidon (0-20 μM) for 6-24 h. The MTT assay reveals that phosphamidon induces cytotoxicity in a dose-dependent manner. Cellular glutathione (GSH) is depleted in a dose-dependent manner from 55% to 70% at concentrations between 10 and 20 μM. The percentage of cells that bind to Annexin-V, which is a representative of cells either undergoing apoptosis or necrosis during 24 h incubation, increases in a dose-dependent manner. Above 5 μM, significant necrosis of cells was observed. DNA fragmentation assay revealed that at low concentration of phosphamidon (1 μM), no appreciable change in DNA fragmentation was seen; however, distinct fragmentation was observed beyond 2.5 μM. Phosphamidon was found to cause significant depletion of GSH, which correlates well with the percentage of cells undergoing apoptosis. An increasing trend in levels of cytochrome c was observed with increasing concentration of phosphamidon, indicating that the apoptotic effect of phosphamidon is mediated through cytochrome c release. Coadministration of the antioxidants N-acetylcysteine and curcumin attenuated phosphamidon-induced apoptosis. This further supports our hypothesis that oxidative stress, as indicated by GSH depletion, results in the induction of apoptosis by release of cytochrome c.

Ameliorating effect of N-acetylcysteine and curcumin on pesticide-induced oxidative DNA damage in human peripheral blood mononuclear cells

“…The cytotoxicity of all three pesticides as measured by MTT assay was consistent with our previous reports published from this laboratory (Ahmed et al 2009(Ahmed et al , 2010. In our recent studies, we have shown that the pesticides endosulfan, malathion, and phosphamidon caused apoptotic and necrotic cell death in human PBMC in a dose-dependent manner (Ahmed et al 2008(Ahmed et al , 2009(Ahmed et al , 2010.…”

“…In our recent studies, we have shown that the pesticides endosulfan, malathion, and phosphamidon caused apoptotic and necrotic cell death in human PBMC in a dose-dependent manner (Ahmed et al 2008(Ahmed et al , 2009(Ahmed et al , 2010. The aim of the present work was to examine whether oxidative stress plays a role in their cytotoxicity following pesticide exposure.…”

“…In separate wells, cells were also treated with either 20 μM NAC or 25 μM curcumin or both, together with above pesticides to evaluate their protective role. The dose of endosulfan, malathion, phosphamidon, NAC, and curcumin selected in this study is based on our earlier studies (Ahmed et al 2008(Ahmed et al , 2009(Ahmed et al , 2010. The cell viability was determined by MTT assay (Ahmed et al 2009).…”

“…Several pesticides, including endosulfan and malathion, have been shown to cause genotoxicity and immunotoxicity in non-target species making them more susceptible to infections (Das et al 2007). Recently, we have reported that subchronic doses of these pesticides induce apoptosis and depletion of cellular reduced glutathione levels in human peripheral blood mononuclear cells (Ahmed et al 2008(Ahmed et al , 2009(Ahmed et al , 2010.…”

Molecular Mechanisms of Pesticide Toxicity

“…Coadministration of the antioxidants N-acetylcysteine, which enhances GSH synthesis, attenuated phosphamidon-induced apoptosis. This work supports the hypothesis that oxidative stress, as indicated by GSH depletion, results in the induction of apoptosis by release of cytochrome C (Ahmed et al, 2008;Ahmed et al, 2010). .…”