The Mechanisms Of Fipronil-induced Toxic Effects On Zebrafish (Danio Rerio) Larvae, And SH-SY5Y And HaCaT Cells

As a kind of phenylpyrazole insecticides,fipronil has been widely used due to its high insecticidalactivity.Nevertheless,the use of fipronil has been restricted by many countries in agriculture because it would cause serious damage to non-target organisms in the environment,especially aquatic organisms.The high toxicity of fipronil to non-target organisms has become an environmental science field research hot topic again.In this paper,through technology of next-generation sequencing,qPCR,and flow cytometry,we studied the acute toxic effects of fipronil on the early stage of development of zebrafish larvae,the effects of oxidative stress and the expression of neurotransmission-associated genes,and DGE profiling of zebrafish larvae induced by fipronil and explored preliminarily relevant transcription factors,as well as the toxic effects of fipronil on human SH-SY5Y and HaCaT cells.We investigated preliminarily some of the mechanisms in toxicity induced by fipronil in combination with phenotypic changes.The following results are obtained in this article:1.The mortality and malformation rate of 75 hpf zebrafish larvae significantly increased after being exposed to fipronil for 96 h.The corresponding LC₅₀ and EC₅₀ were 460?g/L and 340?g/L,respectively.Skeletal development was suppressed resulting in significantly shorter body length of zebrafish larvae.Mortality,malformation rate,and body length changes were in a concentration-dependent relationship with fipronil concentrations.2.At sublethal concentrations,zebrafish larvae showed oxidative stress response to fipronil.The activities of CAT,CES,GST,POD,and SOD enzymes increased in different degrees after induced by fipronil of different concentrations 0.5,5,50,and 100?g/L for 72 h.The activities of the enzymes increased at first and then decreased,and the activities of all these enzymes reached their maximums at the fipronil concentration of 5?g/L.3.At sublethal concentration,fipronil affected the transcription of ache,chat and glrba genes in zebrafish larvae.When the fipronil concentrations were 5,10,50,and 100?g/L,respectively,the relative trascription levels of the genes increased with prolonged exposure time.4.At environmental concentrations,the DGE profiling of 75 hpf zebrafish larvae after induction with fipronil at concentrations of 0.5,5,and 50?g/L for 96 h showed high quality.The numbers of differentially expressed genes induced by three concentrations of fipronil were 58,75,and 98,respectively.The up-regulated genes were 2,4,and 7,and the down-regulated genes were 56,71,and 91,respectively.The number of common differentially expressed genes was 44,and among them 9 belonged to transcription factor AP-1 protein family.12,124,and 23 significantly enriched GO items were obtained at each concentration,that included two GO subgroups of biological processes and molecular function.The co-enrichment of GO items were involved in cell proliferation,adipocyte metabolism,and transcription factor activity.7,6,and 9 significantly enriched pathways were obtained,respectively.HTLV-?infection?ko05166?,Osteoclast differentiation?ko04380?,and Hepatitis B?ko05161?were co-enriched pathways at each concentration.The results of qPCR showed that the relative expression of 15 genes were highly consistent with DGE results,which Pearson's correlation coefficient r=0.97018 and P<0.001.5.The relative expression levels of the transcription factor JDP2,Fosab and MIDN-encoded genes after the zebrafish larvae induced with 50?g/L fipronil for 72 h were tested.Two transcription factors,JDP2and Fosab,regulate the generation and differentiation of osteoclasts,respectively.It was supposed that the significantly down-regulated expression of jdp2b and fosab genes could affect the skeletal growth and development of zebrafish larvae,resulting in zebrafish larvae spine bending or shortened body length.JDP2 can inhibit cell proliferation and the transformation of proto-oncogenes.It is speculated that the down-regulation of jdp2b gene may have carcinogenic risk for zebrafish larvae.MIDN is an important regulatory factor in the pathogenesis of Parkinson's disease,suggesting that the significant downregulation of midn gene induced by fipronil may be a risk of Parkinson's disease in vertebrates such as fish.The relative expression of the midn gene showed a dose-dependent relationship with the environmental concentration of fipronil,which can be a new potential biomarker of zebrafish larvae in response to environmental concentrations of fipronil.6.Cell viability of SH-SY5Y and HaCaT significantly concentration-dependent decreased after induced by fipronil and HaCaT cells were more sensitive to fipronil.Apoptosis rates of both cells increased as the fipronil concentration increased or the exposure time lengthened,indicating that fipronil has a toxic effect on these two human cells,and this toxicity would accumulate over time.