The Toxicological Mechanism Research Of Chlorpyrifos In Zebrafish Embryos

As a typical organophosphorus insecticide, chlorpyrifos(CPF) has been widely used in agricultural production. With the increase of environment pollution events, the complex biological effects of CPF have gradually drawn more attention. In this study, zebrafish embryos and larvae were used for the biology experiments to investigate the environmental biological effects and the mechanisms of CPF at the phenotype, molecular and omics levels. The methods and results are as follows.1. High and low concentration gradients of CPF solution was prepared for acute toxicity test. The low concentration gradient was 0, 0.10, 0.25, 0.50, 0.75 and 1.00 ppm, and the high concentration gradient was 0, 1.00, 2.00, 3.00 and 4.00 ppm. In this study, zebrafish embryos were exposed to all these concentrations of CPF with 3 parallel groups. The results show that, compared with normal control embryos, the incubation was faster in the low concentration gradient CPF treated groups and the hatching rate was proportional to CPF concentration. Moreover, there was no obvious malformation in these groups. When treated with CPF for 60 h, embryos in all low concentration gradient groups were almost hatched and survived well. However, the embryos were almost hatched in high concentration gradient CPF treated groups after 48 h while significant embryonic malformation such as spine malformation and cardiac oedema were observed. The embryos had been found dead when treated with high concentration gradient CPF, and the median lethal dose was closed to 2 ppm at 60 h.2. To determine the endocrine disrupting effects of low concentration chlorpyrifos, flow cytometry was used for cell cycling analysis of HEC-1B. The flow cytometry data showed that CPF could increase cell proliferation like 17β- estradiol, but the effect of CPF was weaker than of E2 in the same concentration. The expression level of related genes had been further analyzed, and the results show that CPF significantly affected the expression of zebrafish related gene VTG and ERα. In addition, the expression level of zebrafish cell proliferation genes c-myc、cyclin D1、c-fos and c-jun were also affected by CPF. The result of acridine orange staining showed that a small amount of apoptotic cells were observed in high power lens in the 0.75, 1.00 mg/L CPF treated groups and the apoptotic related gene expression were disordered in CPF treated groups. To detect why high concentration gradient CPF induced significant embryos death and malformation, 5 neurodevelopment maker genes were investigated in the following study for exploring the developmental neurotoxicity at the level of transcription.3. Bioinformatics technologies such as transcriptomics and proteomics were selected in this study. RNA-seq was used to analyze the transcriptomics of zebrafish, and iTRAQ was used to analyze the proteomics. After exposed to sub-lethal concentration of CPF(0.05 ppm) for 9 days, the zebrafish larvae samples were collected to extract total RNA and protein for omics analysis. Differentially expressed genes, proteins and pathways were analyzed from transcriptomics and proteomics data. Finally, the conjoint analysis of transcriptomics and proteomics were also investigated in this study.Conclusion: The results suggested that the CPF at low concentration had endocrine disrupting effects and increased the proliferation index of cancer cells. The reason why CPF made zebrafish hatch earlier was the estrogen-like effects increased the cell cycling and cell proliferation by stimulating estrogen receptor, and inhibited the cell apoptosis at the same time. CPF at high concentration, which mainly induced neurotoxicity, significantly impacted on the expression of nervous system related genes was neurotoxicity. The results of omics indicated that CPF up-regulated the metabolism and transcript genes and proteins of zebrafish larvae and affected the development. On the other hand, CPF down-regulated immune system and environmental adaptation genes and proteins and induced the low immunity and death. In addition, the differentially expressed genes and proteins from CPF treated groups could bring potential marker for CPF toxicity detection.