Combined Toxic Effects Of Pesticide Residues In Cowpeas On Neuro-2a Cells

According to the first-hand data obtained from routine monitoring in China in recent years,cowpea is one of the typical agricultural products with a low pass rate of pesticide residue monitoring.The main residual pesticides were imidacloprid,acetamiprid,carbendazim,carbofuran,bifenthrin and chlorfenapyr.Moreover,multiple pesticide residues occurred frequently.The mixed coexistence of pesticide residues in fresh foods may induce synergistic,additive and other joint toxic effects,which has attracted increasing attention.Although the actual pesticide residues in cowpeas contain both exceedances and non-exceedances,their potential risk still makes consumers doubt their safety.Therefore,it is of great practical importance to investigate the toxic effects of pesticides typically present in cowpeas by scientific means in order to evaluate the safety of cowpeas for consumption and to guide consumption rationally.In this study,we first explored the differences in toxicity of six pesticides by using neuroblastoma Neuro-2a cells as a model.And then,we established metabolomic and lipidomic approaches to analyze the changes of small molecule metabolites in Neuro-2a cells under different pesticides exposures.In addition,the effects of imidacloprid,acetamiprid,carbendazim and carbofuran on Neuro-2a cell death and redox homeostasis were explored with the help of high content cell imaging system and imaging flow cytometry.The results obtained in this study provide a basis for an in-depth study on the mechanism of pesticide toxicity.The main research contents and results are as follows.The concentration-response curves of the five pesticides except bifenthrin could be plotted based on the results of cell inhibition rate and the corresponding concentrations.Their toxic effects on Neuro-2a cells were determined from strong to weak:carbofuran,chlorfenapyr,carbendazim,acetamiprid and imidacloprid.The CA,IA and CI models were used to analyze the toxic effects of the three typical binary co-exposures,and the CA model was found to be the most accurate.It indicated that the toxic additive effects of any two mixtures of acetamiprid,carbendazim and carbofuran are higher than those of one pesticide alone.The cellular non-targeted metabolomics,non-targeted lipidomics,and 53 amino acid,glycerophospholipid,and sphingolipid targeted method were optimized and established.Among them,the non-targeted lipidomics method was applied by measuring lipid compounds in five types of cells（PC12 cells,MCF-7 cells,Hep G2 cells,RKO-E6 cells and Hela cells）.The correlation coefficients for each compound in the linear range of the targeted method were greater than 0.99,and the recoveries ranged from 82.36%to 96.07%by spiked recovery experiments with the three substances.It indicated that the method is accurate and reliable.Omics techniques helped screened 40 differential metabolites and 14 differential lipids after imidacloprid and acetamiprid exposure at the concentrations of IC₁₀ and IC₂₀.It found that the glycerophospholipid metabolic pathway,sphingolipid metabolic pathway,and GSH metabolic pathway were the three main metabolic pathways affected.The non-targeted omics results together indicated that imidacloprid and acetamiprid cause metabolic abnormalities in different ways when the exposure concentration changes.The targeted omic results showed that imidacloprid exposure mainly induced amino acid and sphingolipid metabolism disorder,while acetamiprid exposure mainly induced amino acid and glycerophospholipid metabolism disorder.High content cell imaging results showed an overall increase in intracellular phospholipid content and an overall decrease in neutral lipid content after imidacloprid and acetamiprid exposure.In addition,the most severe cell necrosis was observed in the ACE20 group among all four exposure groups.The results of lipid hydroperoxide and ROS showed that acetamiprid had a stronger interference than imidacloprid with the lipid peroxidation pathway in Neuro-2a cells.Meanwhile,acetamiprid exhibited stronger toxic effects by more significantly promoted GSH synthesis.The omics results showed that the combination of carbendazim and carbofuran exposure caused more abnormal metabolism in Neuro-2a cells.The potential biomarkers revealed a stronger damage to cell membranes induced by combined exposure.It was also found that carbendazim and carbofuran caused cell death by both cell necrosis and early apoptosis,and it was most significant in the case of combined exposure.In addition,the combined exposure not only disrupted the redox homeostasis in Neuro-2a cells,but also may cause cell death through ferroptosis.