Effects And Mechanisms Of Monocrotophos On The Development Of Zebrafish (Danio Rerio) Embryos

Organophosphorus pesticides have embryotoxicity, they can adversely affect the normal development of embryos. Previous studies have found that monocrotophos (MCP), one kind of organophosphorus, can damage the gonad development of zebrafish embryos, leading to abnormal morphology and arrangement of the germ cell in ovarian. To explore the toxic effect of MCP on zebrafish embryonic development, we statistically analyze the survival rate, hatching rate, heart beat, malformation (including pericardial cyst and curvature of the spine), voluntary movement and growth(including head length and body length) of embryos exposed to MCP at the individual level. Mμl tiple genes are involved in the regμl ation of zebrafish embryos development, we spec u 1 ate that pericardial cysts, curvature of the spine and abnormal gonadal structure may be the res μ 1 t of the impact of MCP on the expression of growth-related genes. Furthermore, exogenous substance can affect gene expression by changing the DNA methylation pattern of the promoter region. Thus, we further investigated the mRNA expression levels of genes involved in the development of heart (Tbx2), muscle(Mef2c) and gonad (Vasa), as well as the DNA methylation pattern of the promoter region of Vasa gene in zebrafish embryos exposed to MCP, exploring the potential mechanisms of MCP on the development of zebrafish embryos at the molecμl ar level.The resμl ts reveal that:(1) Following 96hour exposure to 0.0004,0.004,0.004 and 0.4 mg/L of MCP(purity=99.3%), the survival rate, hatching rate, head length, body length and voluntary movement have no obvious change. However, the heart beat significantly reduced, and the percentage of larva which have pericardial cyst and curvature of the spine increases significantly. The res μ 1 ts above reveal that MCP has embryotoxicity and teratogenicity, can harmf μ l ly influence the development of heart and skeleton in zebrafish embryos.(2) Quantitative real-time PCR was employed to measure the expression of Tbx2 gene and Mef2c gene following 48h,60h exposure, Vasa gene following 48h,60h and 96h exposure to 0.0004,0.004,0.004 and 0.4 mg/L of MCP. The res μ 1 ts demonstrate that 48h MCP exposure significantly reduces Tbx2 gene expression and increases Mef2c gene expression. While neither Tbx2 expression nor Mef2c expression have obvious change after 60h MCP exposure. Our res u 1 ts reveal that MCP can affect Tbx2 and Mef2c gene expression in the key period of the heart development, leading to abnormal heart development. In addition,96h MCP exposure has no effect on Vasa gene expression, but 48h and 60h MCP exposure significantly reduces Vasa gene expression which may have adverse effect on the migration of PGCs.(3) Quantitative real-time PCR was employed to measure the expression of Dnmtl gene and Mecp2 gene following 48h,60h and 96h exposure to 0.0004,0.004, 0.004 and 0.4 mg/L of MCP. Bisμl fite sequencing PCR was employed to measure the DNA methylation state of promoter region of Vasa gene. Our res μ 1 ts demonstrate that 48h,60hpf and 96hpf MCP exposure has no effect on DNA methylation state of the promoter region of Vasa gene, revealing that DNA methylation is not the mechanism through which MCP exposure reduces the expression of Vasa gene. Other res u 1 ts domenstrate that the expression of Dnmtl does not change following exposure to MCP, however, the expression of Mecp2 increases, revealing that the histone deacetylation and methylation may be correlated to the reduced expression of Vasa gene.In summary, our study demonstrates that MCP has embryostoxicity. At the individual level, MCP affect the development of heart and skeleton, leading to pericardial cyst and curvature of the spine; at the molecμl ar level, MCP can influence the Tbx2, Mef2c and Vasa gene expression which may lead to the abnormal development of heart, skeleton and gonad. Further, our study reveals that histone deacetylation and methylation may be the mechanism through which MCP reduces the expression of Vasa instead of DNA methylation. Therefore, this study explores the embryotoxicity and the its mechanism of MCP on zebrafish embryos at individual and molec μ 1 e levels, providing some research ideas for the embryotoxicity study of exogenous compounds.