Micro-injection as a tool to detect the effects of bisphenol A, diethyl phthalate, and 17ß-estradiol on ontogenesis of zebrafish (Danio rerio)

Diethyl phthalate (DEP), bisphenol A (BPA), and external estradiol 17β-estradiol (E2) all are endocrine disrupting chemicals (EDCs). Our previous study has found that the development of ceratohyal cartilage (CH) in embryos could be disrupted when the maternal generation was exposed with 8.06 μM DEP, 2.86 μM BPA, and 1.11 μM E2. However, it is still unknown how doses of the residual EDCs in eggs cause abnormal CH development in their offspring. Microinjection is used at the 2-cell stage of embryos to mimic the maternal effect and to observe the toxicities of EDCs in embryos. Results shown that the amounts of DEP, BPA, and E2 were 1.3 × 10^-6 ng, 4.7 × 10^-7 ng, and 1.4 × 10^-7 ng, respectively, inducing the CH angles to become bigger than the control. However, related genes to the migratory pathways of neural crest cells (NCCs) were not influenced upon BPA and E2 treatments. Both sox10 and smad3 gene expressions were up-regulated upon DEP treatment. On the other hand, the CH angles were smaller than the control upon 1.3 × 10^-5, 9.4 × 10^-6, and 1.4 × 10^-6 ng of DEP, BPA, and E2 microinjection, respectively. Furthermore, genes related to migratory NCCs were significantly influenced upon 10⁻⁵ ng of BPA, and 10⁻⁴ ng of DEP treatments on embryos. According to the data, we suggested that 10⁻⁵–10⁻⁷ ng of EDCs in eggs could disrupt CH development as well as significantly increase the mortality on their embryos. The present study raises concern that the responses were highly sensitive in embryos through maternal effects.