| Pesticides are chemicals used in agriculture to protect crops.Exposure of food and drinking water contaminated with pesticide residues to organisms can cause acute poisoning or chronic health effects.Pesticides in the environment are usually found in combination.Although the residue of individual pesticide complies with maximum residue limits(MRLs),the toxicological impact of pesticide mixtures on human health is largely unknown,and the risks associated with the exposure of multiple pesticides have become a continuing challenge in human health assessment.In this paper,based on human hepatocellular carcinoma HepG2 cells,the combined exposure experiment was carried out with difenoconazole,cypermethrin and triazophos,which were widely used in agricultural products with strong broad spectrum,as the research object.Then,we used the predictive models of Concentration Addition(CA),Independent Action(IA)and Combination Index(CI)to evaluate the combined effect of the mixtures and explore the combined action mode of pesticide mixtures.At the same time,molecular docking was used to explore the interaction of pesticides and their mixtures on the metabolic level of cytochrome enzyme.The main research contents and results are as follows:This study first established the toxicity of individual pesticides and their mixtures to HepG2 cells.HepG2 cells were induced to cytotoxicity after exposure to individual pesticides and mixtures for 24 h,resulting in a decline in cell viability.The toxicity of most pesticides and mixtures to HepG2 cells was positively correlated with the exposure concentration,among which the toxicity of difenoconazole was the strongest,and the half maximal inhibitory concentration was 24.72 μM.The order of toxicity for the other individual pesticides was ranked as cypermethrin(127.88 μM)> triazophos(136.99 μM).Compared with CA and IA models,CI model could predict the combined toxicity of the mixture more accurately.The combined toxicity of pesticide mixtures was determined based on CI index.It was found that the combined effects of all binary combinations changed from antagonism to synergy with the increase of pesticide concentrations.At the same time,the dose reduction index showed that triazophos played a greater role in the synergistic effect of difenoconazole + triazophos and cypermethrin +triazophos.In this study,oxidative damage,apoptosis induction and DNA synthesis were used as observation indexes to study the toxicity mechanism of individual pesticides and their mixtures on HepG2 cells.Difenoconazole,cypermethrin,triazophos and their mixtures induced cell apoptosis in a concentration-dependent manner.At a concentration of 10～30 μM,pesticide mixtures increased ROS levels,damaged cells by destroying cell membranes and producing large amounts of LDH,and induced the formation of large amounts of DNA fragments.Besides,the activities of Caspase-3,Caspase-7 and PARP were significantly enhanced at the concentration of 30 μM,and their cleavage was induced.At the same time,DNA synthesis was significantly inhibited and cell cycle was blocked.Specifically,mixtures of difenoconazole showed higher toxicity than difenoconazole,which means that pesticide mixtures carry higher health risks.Finally,we investigated the effect of individual pesticides and their mixtures on CYP3A4 enzyme activity.Difenoconazole and its mixtures showed significant inhibitory effect on CYP3A4 enzyme activity in a concentration-dependent manner,and the inhibitory rate of CYP3A4 enzyme activity reached 97% at 50 μM concentration.There was no significant difference in the inhibition of CYP3A4 enzyme activity between the mixtures and difenoconazole.Molecular docking verified the strong binding effect of difenoconazole and CYP3A4 protein,and revealed the interaction pattern between them.The results of this paper can provide data support for the toxicological study of pesticides mixtures,providing scientific basis for the risk monitoring of pesticides mixtures,which is of great significance for food safety. |
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