Oxidative Stress And Enantioselectiyity Of Organophosphorus And Pyrethroid Pesticides In PC12Cells

Previous studies have demonstrated the occurrence of enantioselectivity of chiral pesticides in environmental fate, endocrine disruption, reproductive toxicity and immune toxicity. However, research on the molecular mechanisms of enantioselective toxicity of chiral pesticides has been limited. Therefore, it is important to investigate the environmental risk of the chiral pesticides, and explore the toxic mechanism in-depth. In this study, we used the rat pheochromocytoma PC12cell line as an in vitro model to evaluate the involvement of the oxidative stress pathway in enantioselective toxicity of several common used chiral pesticides, represented by bifenthrin (BF) and profenofos (PFF). Both the underlying mechanisms and enantioselectivity of toxicities were investigated using molecular biology methods. The major results were listed as follows:(1) In this part, cis-BF was chosen to evaluate the involvement of the oxidative stress in enantioselective cytotoxicity of cis-BF. Following exposure of cells to cis-BF and its enantiomers, a significant reduction in cell survival and superoxide dismutase (SOD), as well as increased production of lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), was observed in1S-cis-BF, while1R-cis-BF exhibited these effects to a lesser degree. These results demonstrated that enantiomer-specific cis-BF-induced oxidative stress is possibly the mechanism of cis-BF-induced enantioselective cytotoxicity.(2) Based on the result that cis-BF could enantioselectively induce oxidative stress, the expression patterns of different genes encoding heat shock protein and antioxidant enzymes were investigated by real-time quantitative PCR in PC12cells after exposure to cis-BF and its enantiomers. The results showed that exposure to1S-cis-BF resulted in increased transcription of HSP90、HSP70、HSP60. Cu-Zn-superoxide dismutase (Cu-ZnSOD)、Mn-superoxide dismutase (MnSOD)、 catalase (CAT) and glutathione-s-transferase (GST) at a concentration of5×10-6mol L-1and the above, while exposure to1R-cis-BF and rac-cis-BF exhibited these effects to lesser degrees. In addition, induction of antioxidant enzyme gene expression produced by1S-cis-BF might occur, at least in part through activation of p38MAPK and ERK, while increase of stress protein response produced by1S-cis-BF might occur through p38MAPK signaling pathway.(3) The role of oxidative stress in enantiomer-specific, profenofos (PFF)-induced cytotoxicity and genotoxicity was investigated using PC12cells. The results demonstrated that PFF enantioselectively induced inhibition of cell viability and DNA damage in PC12cells. A concentration-and time-dependent significant induction of reactive oxygen species, malondialdehyde and gene expression encoding antioxidant enzyme (Cu-ZnSOD, GST and CAT) and stress protein (HSP70and HSP90), was observed in (-)-PFF, while (+)-PFF and rac-PFF exhibited these effects to lesser degrees. Pre-treatment with vitamin E (6×10mol L-1) caused a significant attenuation in the toxic effect; reversing subsequent PFF-induced elevation of ROS and MDA levels, further strengthening the involvement of oxidative stress in PFF mediated toxicity. In addition, the results also showed that PFF-dependent ROS accumulation, MDA release and oxidative stress gene expression preceded the loss of cell viability and induction of DNA damage, and already significantly changed at concentrations which are not yet cytotoxic or genotoxic. These results indicate that oxidative stress may contribute to PFF induced toxicity and was not a consequence of it.(4) The primary objective of this part is to compare the ability of acephate (ACE), methamidophos (MET), chloramidophos (CHL), malathion (MAT) and malaoxon (MAO) to induce cytotoxicity, DNA damage and oxidative stress at5mg L-1～80mg L-1in PC12cells. The results demonstrated that MET、MAT and MAO caused significant inhibition of cell viability and increased DNA damage in PC12cells at40mg L-1. MAO was more toxic than the other organophosphorous (OP) compounds. ACE、 MET、MAT and MAO increased the levels of intracellular ROS and MDA, and decreased the activity of SOD、CAT and GSH at20mg L-1and40mg L-1to different degrees. Pre-treatment with vitamin E (6×10-4mol L-1) caused a significant attenuation in the cytotoxic and genotoxic effect; reversing subsequent OP-induced elevation of peroxidation products and the decline of anti-oxidant enzyme activities. These results indicate that oxidative damage contributes to the OP-induced cytotoxicity and DNA damage.Overall, results of our study suggest that enantiomer-specific chiral pesticide-induced oxidative stress is possibly the mechanism of chiral pesticide-induced enantioselective cytotoxicity and genotoxicity.1S-cis-BF and (-)-PFF are the major contributors. This paper provided a scientific basis for screening out the monomer of chiral pesticides which both has high pesticide effect to target organism and has low toxicity to non-target organism, and provided a theoretical reference for researching and developing the environment-friendly pesticides.