Detecting the combined toxicity of 30 pesticides and veterinary drugs and their binary and ternary combinations toward CYP3A4 based on fluorescence probe technology

The mixed-use of multiple pesticides and veterinary drugs in agricultural production is becoming more and more common, resulting in the uncertainty of combined toxicity effect, which poses a serious threat to human health. Based on the principle of enzyme inhibition method, the activity of CYP3A4 was directly detected by fluorescent probe NEN (N-ethyl-1,8-naphthalene dicarboxyimide), and a broad-spectrum method for screening the combined toxicity effect of mixed pesticides and veterinary drugs was established. Thirty commonly used pesticides and veterinary drugs and their typical 23 binary and 26 ternary combinations were taken as the research objects, and the combined toxicity effects of pesticide and veterinary drug mixtures on CYP3A4 were detected. The standard mass concentration was determined according to the maximum residue limit of pesticides and veterinary drugs specified in the national food safety standard. The results showed that the pesticide and veterinary drug combinations that showed synergistic effects on the combined toxicity to CYP3A4 under three mass concentration gradients were carbofuran + carbendazim, carbofuran + imidacloprid, acetamiprid + dimethomorph, imidacloprid + carbendazim, cypermethrin + acetamiprid + dimethomorph, carbofuran + acetamiprid + carbendazim, imidacloprid + acetamiprid + carbendazim, imidacloprid + acetamiprid + dimethomorph, chlorpyrifos + acetamiprid + carbendazim and bifenthrin + acetamiprid + carbendazim. When the inhibition rate of a single pesticide or veterinary drug on enzyme activity was high, the combined toxicity effect after mixing with other pesticides and veterinary drugs was antagonistic, while when the inhibition rate of a single pesticide or veterinary drug on enzyme activity was less than 2%, the combined toxicity effect after mixing with others was uncertain. Pesticide and veterinary drug combinations often had a strong synergistic effect on the combined toxicity of CYP3A4 at low concentrations, with the increase of concentration, the combined toxicity effect changes from synergy to antagonism. By analyzing the structure-activity relationship between pesticides and veterinary drugs and CYP3A4, it could be seen that the number of aromatic chlorine groups was positively correlated with the inhibition level on CYP3A4 activity. Pesticides and veterinary drugs, such as chlorothalonil, chlorpyrifos, chlorpyrifos-methyl, prochloraz, with three aromatic chlorine groups or more had the most significant inhibition effect on enzyme activity of CYP3A4, and the inhibition rates were more than 30%. Pesticides and veterinary drugs containing two aromatic chlorine or "strong electron-withdrawing group + one aromatic chlorine" group had a strong inhibitory effect on enzyme activity of CYP3A4, with an inhibition rate of more than 18%, such as difenoconazole, pyridaben and iprodione. Pesticides and veterinary drugs with carbamate bonds alone had little or no toxicity to CYP3A4, while they showed a strong synergistic effect when mixed with other pesticides and veterinary drugs. The detection method established in this study provides a new idea for broad-spectrum screening of combined toxicity of mixed pesticides and veterinary drugs, and the detection results can provide a basis for further risk assessment scheme design of pesticide and veterinary drug mixtures at the cellular and animal levels.