Research On The Cytotoxicity Of Fipronil And Its Metabolites

In 2017,the "poison eggs" incident drew people's attention to fipronil.Fipronil is a phenylpyrazole insecticide and its heavy use has caused the residues in agricultural products.Studies have shown that fipronil and its metabolites are toxic to animals,and the metabolites may be more toxic than the maternal.Current studies mainly focused on animals and plants and it is impossible to know the specific toxicity differences at the cellular level.In this paper,PC12 cells were used as a model to investigate the cytotoxicity of fipronil and its metabolites and thus to explore the differences and reasons of neurotoxicity between fipronil and its metabolites at the micro level,and to provide basis and guidance for toxicity mechanism research and safety evaluation.The metabolism of fipronil and its metabolites in PC12 cells was studied and the differences of cytotoxicity between fipronil and its metabolites in PC12 cells were explored by cell activity assay,lipidomics and amino acid metabolomics.The main conclusions are as follows:UPLC-MS/MS was used to establish the method for the detection of fipronil and its metabolites in PC12 cells.Cell samples were injected and analyzed directly after simple extraction by 0.5% acetic acid acetonitrile with vortex and 10 minutes 4500 rpm centrifugation.The linear range was between 0.1-100 ?g/L.The recoveries of fipronil and its metabolites in PC12 cells were 83.53-114.11%,and the relative standard deviations(RSDs)were less than7.54%..The above results fully verified the method is sensitive,reliable and simple.And this method can provide a basis for subsequent metabolisom research.Degradation and metabolism experiments showed that fipronil and its metabolites were relatively stable during the 72 h of experiment.The main metabolites of fipronil,fipronil sulfide and fipronil desulfinyl are fipronil sulfone,while fipronil sulfone hardly undergoes metabolic transformation.We can conclude that fipronil sulfone is the most stable compounds in mammalian cells and has a persistent effect on cells.Cell viability test showed that the decrease was most obvious in the fipronil desulfinyl group,followed by the fipronil sulfone group.Lipidomics results showed that the content of glycerophospholipids was also different in different groups.Fipronil desulfinyl group and fipronil sulfone group had more differential metabolites and had significant effects on PC,PS and PI.Fipronil and fipronil sulfide group only had a significant effect on PC and PI.Combined with relevant literature,it is known that fipronil and fipronil sulfide mainly affect nerve activity and cell signal transduction,while fipronil desulfinyl and fipronil sulfone also affect mitochondrial apoptosis.These results suggest that the mitochondrial apoptosis process may be responsible for the greater toxicity of fipronil desulfinyl and fipronil sulfone.Metabolomics results showed that tyrosine,arginine and leucine were the common differential metabolites of the four compounds,which were related to neurotransmitter synthesis,apoptosis and energy metabolism.For fipronil desulfinyl,there were other four differential metabolites,phenylalanine,aspartic acid,serine and glutamic acid,which were related to oxidative stress,neuronal damage,phospholipid synthesis and immunity.Fipronil sulfone group had phenylalanine and histidine,which were related to oxidative stress and anti-inflammatory reaction.These results may also be responsible for the high toxicity of flipronil desulfinyl and fipronil sulfone.