Combined toxicity of five pesticides and their binary and ternary mixtures on HepG2 cells

To study the combined toxicity of pesticides mixtures in agricultural products, five pesticides with wide application and broad spectrum, including difenoconazole, cypermethrin, dimethomorph, cyhalothrin, acetamiprid, and relevant binary and ternary combinations with high exposure levels in lettuce were selected. Cell Counting Assay Kit-8 method was used to determine the proliferation inhibition toxicity of single pesticide and mixtures on HepG2 hepatocytes. The combined effects were assessed by three prediction models, concentration addition, independent action and combination index, based on the concentration-response curve of individual pesticides. Then the induction effects of two ternary combinations on cell apoptosis were analyzed by High Content Screening. The results showed that the effects of different pesticide combinations on the cell growth inhibitory rate of HepG2 cells were different, all of which had obvious concentration-response relationship. Among them, difenoconazole had a strong inhibition of proliferation toxicity, and the median effective concentration was 24.72 μmol/L. The order of toxicity for the other individual pesticides was ranked as dimethomorph > cyhalothrin > acetamiprid > cypermethrin. The combined toxicity of the pesticide mixtures was determined by the combination index. The results showed that with the increase of pesticide concentrations, the combined effects of all binary combinations changed from antagonism to synergy. While in ternary mixtures, including dimethomorph + difenoconazole + cypermethrin and dimethomorph + cypermethrin + acetamiprid, the combined effects changed from synergy to antagonism when the growth inhibition effect were 60% and 18%, respectively. In addition, the dose reduction index was used to quantify the synergistic effect between pesticide mixtures, indicating that cypermethrin played a greater role in the synergistic effect of the above two ternary combinations. In the measurement of apoptosis toxicity, the apoptotic rate of HepG2 cells significantly increased in the groups treated by individual pesticides in a concentration-dependent manner. The apoptotic rate induced by the ternary combination of dimethomorph + cypermethrin + acetamiprid was significantly increased in concentration between of 20.02-80.10 μmol/L. This work confirmed that the toxicities of proliferation inhibition and apoptosis induction of different pesticides mixtures on HepG2 cells both showed obvious concentration-response relationship, which could provide a scientific basis for toxicity evaluation and risk monitoring of pesticides mixtures. The combination index model as an effective ecotoxicological risk assessment tool can quantitatively describe the interactions of pesticide mixtures under different components and concentrations, which can predict the combined toxicity of pesticide mixtures more accurately compared with the concentration addition model and the independent action model.