Pyrrolidine dithiocarbamate augments Hg2+-mediated induction of macrophage cell death via oxidative stress-induced apoptosis and necrosis signaling pathways

Abstract

Exposure to mercury can lead to several injuries in mammals, including immune system dysfunction, and pyrrolidine dithiocarbamate (PDTC), as a metal chelator and antioxidant, has been indicated to increase the cytotoxic effects of toxic metals. However, the toxicological effects and possible mechanisms of mercury in combination with PDTC are mostly unclear. In this study, we showed that PDTC dramatically increase the cytotoxic effect of HgCl2 on cultured murine macrophages (RAW 264.7 cells). PDTC augmented HgCl2-induced cytotoxic effects by facilitating the entry of mercury into the cells. The Hg2+/PDTC complex significantly and rapidly increased the formation of reactive oxygen species (ROS) and decreased intracellular glutathione (GSH) levels in these cells. Flow cytometry analysis showed that the numbers of sub-G1 hypodiploid cells and annexin V-FITC binding cells increased after Hg2+/PDTC complex exposure, and several features of mitochondria-dependent apoptosis were also induced, including mitochondrial membrane depolarization, cytosolic cytochrome c release, poly(ADP-ribose) polymerase (PARP) and caspase 3/7 activation, and DNA fragmentation. Moreover, both apoptotic and necrotic cells were detected using acridine orange/ethidium bromide dual staining. Meanwhile, depleted intracellular ATP levels and increased lactate dehydrogenase (LDH) release were observed, suggesting the induction of necrotic cell death processes. These Hg2+/PDTC complex-induced cytotoxicity-related signals could be reversed by pretreatment with the antioxidant N-acetylcysteine. In conclusion, these results suggest that Hg2+/PDTC complex-induced oxidative stress causes macrophage cell death via both apoptosis and necrosis. These findings imply for the first time that PDTC dramatically increases the uptake and toxicological effects of Hg2+ instead of detoxification.