Developmental toxicity of E804 in zebrafish larvae (n = 3 replicates, N = 3 biological replicates, 90 embryos per replicate). (A) Lethal rate at 96 hpf (F = 52.3, df = 35) (B) Malformation rates at 96 hpf (F = 74.4, df = 35). (C) Hatching rates at 72 hpf (F = 7.68, df = 35). (D) Body length at 96 hpf (F = 56.72, df = 59). (E) The phenotypic changes of embryos at 24, 48, 72, and 96 hpf. The values are expressed as mean ± SD (n = 3). *Represents p-value less than 0.05 and **represents p-value less than 0.01. df means the degrees of freedom, F means F-distribution.

Effects of E804 on developing heart (n = 3 replicates, 10 per replicate). (A) Phenotypes of larvae of Tg(cmlc2:EGFP) lines. SV-BA distance is indicated by white row. (B) Heart rates at 72 hpf (F = 37.63, df = 29). (C) Pericardial area at 72 hpf (F = 24.62, df = 49). (D) SV-BA distance at 72 hpf (F = 19.53, df = 49). The values are expressed as mean ± SD (n = 3). *Represents p-value less than 0.05 and **represents p-value less than 0.01.

Effects of E804 on angiogenesis (n = 3 replicates, 10 embryos per replicate). (A) Phenotype of larvae of Tg(fli-1:EGFP) lines. The ISVs were counted and analyzed in white box regions. (B) The number of the ISVs at 48 hpf (F = 91.86, df = 49). (C) The total length of the ISVs at 48 hpf (F = 53.5, df = 49). The values are expressed as mean ± SD (n = 3). *Represents p-value less than 0.05 and **represents p-value less than 0.01.

Effects of E804 on developing liver (n = 3 replicates, 10 embryos per replicate). (A) Phenotypes of larvae of Tg(L-FABP : EGFP) lines. (B) Liver area at 96 hpf (F = 24.62, df = 49). (C) Liver intensity at 96 hpf (F = 125, df = 54). The values are expressed as mean ± SD (n = 3). *Represents p-value less than 0.05 and **represents p-value less than 0.01.

Effects of E804 on the macrophage and neutrophil (n = 3 replicates, 10 embryos per replicate). (A) Phenotypes of larvae of Tg(Lyz : EGFP) lines. The total number of macrophage and neutrophil was counted in white box regions. (B) Total number of macrophage and neutrophil in larvae at 72 hpf (F = 14.12, df = 49). The values are expressed as mean ± SD (n = 3). **Represents p-value less than 0.01.

Effects of E804 on the ROS and apoptosis (n = 3 replicates, 10 embryos per replicate). (A) Fluorescence micrographs of ROS generation in zebrafish larvae at 72 hpf. (B) Quantitative analysis of ROS generation (F = 26.87, df = 49). (C) AO staining of the apoptotic cells. (White arrows indicate apoptotic cells in heart). (D) Quantitative analysis of the apoptotic cells (F = 47.21, df = 49). The values are expressed as mean ± SD (n = 3). **Represents p–value less than 0.01.

Effects of E804 on activities of SOD and the content of MDA in zebrafish at 72 hpf (n = 3 replicates, 50 embryos per replicate). (A) SOD activities (F = 14.55, df = 14). (B) MDA content (F = 27.18, df = 19). The values are expressed as mean ± SD. *Represents p-value less than 0.05 and **represents p-value less than 0.01.

Effects of E804 on gene expression at 96 hpf (n = 3, 30 embryos per replicate). The mRNA expression level of L-FABP, scn5Lab, BCL2, Bax, Caspase 3 (A), PPAR-α, SOD, TNF-α, and NRF-2 (B). The values are expressed as mean ± SD (F = 35.97, df = 14). *Represents p-value less than 0.05 and **represents p-value less than 0.01.