The Combined Toxic Effect Of Chlorpyrifos And Difenoconazole On L-02 Cell Line

Background:China has a vast agricultural growing land and the use of pesticides is inevitable.The use of pesticides greatly increases the yield of agricultural products and brings economic benefits.However,long-term or heavy use of pesticides will remain in food and the environment,through diet,contact and other ways into the human body,causing damage to a variety of organs and tissues,and pesticide residues become the focus in food safety work.At the same time,various types of pesticides are used,and they are often mixed and applied alternately.Different toxic substances interact with each other in different ways in the organism.The toxic effects of their compound pollution on organisms will differ from those of single pollution.Combined exposure can more accurately and truly reflect the toxic effects of pollutants,providing data support for environmental toxicology and risk assessment of composite pollutants.Chlorpyrifos and Difenoconazole are two widely used,broad-spectrum pesticides that both exhibit significant hepatotoxicity,but their combined toxic mechanisms of action are unclear and worth exploring.Objective:This topic studies the effects of single and combined exposure of Chlorpyrifos and Difenoconazole on human normal hepatocytes L-02 by cell experiment in vitro,explore the possible differential changes of gene expression in L-02 cells induced by combined exposure of Chlorpyrifos and Difenoconazole.And further studies the related signal path variation,identify the target molecule.To evaluate the toxicity effects of combined exposure of Chlorpyrifos and Difenoconazole,so as to provide scientific basis for evaluating the combined toxicity mechanism of pesticides.Methods and Results:1.Cell titer assay to determine the effects of chlorpyrifos and difenoconazole at different concentrations on L-02 cell viability,morphology and apoptosis after 24 hours of single and combined exposure,and to verify the time-dose response relationship.The results showed that Chlorpyrifos and Difenoconazole alone and in combination significantly changed cell morphology and reduced cell viability after 24 hours exposure to L-02 cells,and the combined exposure induced a significant increase in apoptosis.The IC₅₀of Chlorpyrifos and Difenoconazole on L-02 cells were about 73.75μmol/L and 108.8μmol/L.2.ELISA and other measurements of intracellular reactive oxygen species（ROS）level,the SOD activity,the GSH activity,and changes in malondialdehyde（MDA）content expression levels to study the effects of oxidative stress induced by Chlorpyrifos and Difenoconazole alone and in combination with 24-hour exposure to cells.The experimental results showed that the combined exposure of Chlorpyrifos and Difenoconazole significantly increased ROS production in L-02 cells,decreased intracellular SOD and GSH activities,reduced antioxidant capacity,which in turn aggravated the degree of oxidative damage and induced an increase in cellular MDA content,ultimately leading to oxidative stress effects in L-02 cells.3.Transcriptomic sequencing approach to detect changes in expression levels of differential genes in L-02 cells after individual and combined exposure to Chlorpyrifos and Difenoconazole.To explore the possible regulatory mechanisms of Chlorpyrifos and Difenoconazole alone and in combination with L-02 cell exposure to induce their apoptosis by GO functional enrichment analysis,KEGG pathway enrichment analysis,etc.And the reliability of transcriptome sequencing results was verified by RT-q PCR to provide a scientific basis for assessing the mechanism of combined pesticide exposure toxicity.The results showed that Chlorpyrifos and Difenoconazole alone and in combination induced changes in differential gene expression in L-02 cells,suggesting that combined exposure of Chlorpyrifos and Difenoconazole to L-02 cells activates the endoplasmic reticulum stress signaling pathway.4.Analysis of expression of the proteins PERK,ERO1-Lα,IRE1α,CHOP,and BIP associated with significantly activated endoplasmic reticulum stress signaling pathway,at the gene transcription level and protein level by RT-q PCR and Western blot techniques.T Exploring the mechanism of endoplasmic reticulum stress signaling pathway in injury of L-02 cells.The results showed that the m RNA and protein expression levels of endoplasmic reticulum stress-related factors PERK,ERO1-Lα,IRE1α,CHOP,and BIP showed significant upregulation.5.The mechanism of endoplasmic reticulum stress induced by combined exposure of Chlorpyrifos and Difenoconazole was further verified by adding IRE1αkinase inhibitor Kira6 to detect the corresponding cell viability,intracellular ROS content,SOD viability,GSH viability,intracellular MDA content and other oxidative stress indicators as well as the m RNA and protein expression levels of the corresponding genes of endoplasmic reticulum stress.The results showed that Kira6 significantly reduced the apoptosis rate of L-02 cells induced by combined exposure to Chlorpyrifos and Difenoconazole,alleviated the effects of cellular oxidative stress,and significantly reduced the m RNA and protein expression levels of endoplasmic reticulum stress-related factors IRE1α,CHOP,and BIP in the combined exposure group.Conclusions:The above conclusions suggest that Chlorpyrifos and difenoconazole alone and in combination showed significant cytotoxicity and induced apoptosis in L-02 cells.And the toxicity effect of the combined exposure was more pronounced.Combined exposure of Chlorpyrifos and Difenoconazole significantly increased intracellular ROS levels,reduced intracellular SOD and GSH activities,decreased antioxidant capacity,aggravated oxidative damage,induced an increase in cellular MDA content,and produced oxidative stress effects.Combined exposure to Chlorpyrifos and Difenoconazole induces the expression of key proteins of the IRE1α-dominated signaling pathway,activates the endoplasmic reticulum stress pathway,enhances the effects of oxidative stress,and causes cellular damage.The results of this paper can provide some data support for the toxicological study of the organophosphorus pesticide Chlorpyrifos and the triazole pesticide Difenoconazole.And also provide some reference for the risk assessment of mixed contamination with organophosphorus and triazole pesticide residues...