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Background Lead is widely distributed. Lead exposure interferes with early life development in zebrafish, but the mechanisms by which lead exposure affects skeletal development and cardiac development are not clear as yet. Objective To investigate the molecular mechanisms of bone development and cardiac development toxicity induced by lead acetate exposure. Methods Zebrafish embryos were exposed to different concentrations of lead acetate (0, 6, 12, 24, and 48 μmol·L−1) for 3 h post-fertilization (3 hpf) until 5 d post-fertilization (5 dpf). The malformation phenotypes of 5 dpf were counted, and the mRNA expressions of spinal development-related genes (bmp2b, bmp4, bmp9, runx2a, runx2b) and heart development-related genes (nkx2.5, myh6, myh7) were detected by quantitative PCR (qPCR). Expressions of genes of development-related regulatory pathways including Wnt/β-catenin pathway (wnt5a, wnt8a, wnt10a, β-catenin) and TGF-β pathway (tgf-β1, tgf-β2) as well as key molecule eph of Eph-Ephrin signaling were analyzed. Results At 5 dpf, the zebrafish in the lead acetate treated groups showed deformed phenotypes including spinal curvature and pericardial sac edema compared to the control group. In the lead acetate groups at 24 and 48 μmol·L−1, the spinal curvature deformity rates reached 26.47% and 71.52% (P<0.01) respectively. The qPCR results revealed that the expression levels of spinal development-related genes bmp2b, bmp4, bmp9, runx2a, and runx2b were downregulated in the 48 μmol·L−1 exposure group compared to the control group by 82.8%, 58.0%, 88.7%, 85.5%, and 69.2%, respectively (P<0.05 or P<0.01); the expression levels of heart development-related genes myh6, myh7, and nkx2.5 were down-regulated by 63.7%, 58.9%, and 55.2%, respectively (P<0.01); the expression levels of wnt8a and β-catenin in the Wnt/β-catenin pathway were down-regulated by 71.5% and 47.3% (P < 0.05 or P < 0.01), respectively; the expression level of tgf- β1 in the TGF-β pathway was down-regulated by 67.5% (P<0.01); the expression level of eph was down-regulated by 86.9% (P<0.01). Conclusion Lead acetate exerts developmental toxic effects on zebrafish heart and bone by down-regulating the expressions of genes related to spinal development and heart development, as well as inhibiting development-related Wnt/β-catenin and TGF-β pathways and Eph-Ephrin signaling, causing malformed phenotypes such as spinal curvature and pericardial sac edema.
RESUMO
Chlorella can produce large amounts of lipids and therefore has great potential for biodiesel production. In this study, Chlorella protothecoides was hydrolyzed by several kinds of extracellular bacterial proteases produced by Pseudoalteromonas sp. ZB23-2, B27-3 and JS4-1 before lipid extraction. Hydrolysates with high antioxidant activity were obtained. The scavenging activities of 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide, and hydroxyl free radicals reached 33.47 ± 0.68%, 46.81 ± 2.38%, and 7.35 ± 0.37 µmol·TE/µmol, respectively. Likewise, proteolysis reduced biomass, which resulted in a reduction in lipid leaching reagents by 35.34-45.49%. Compared to the commonly used Kates and Paradis method (171.77 ± 2.50 mg/g), the modified ethanol lipid extraction combined with JS4-1 enzyme pretreatment (291.06 ± 1.70 mg/g) and acetone-ethanol lipid extraction combined with B27-3 protease pretreatment (277.20 ± 3.30 mg/g) resulted in a larger and more diverse lipid extraction. Protease pretreatment combined with less toxic solvents for lipid extraction improved microalgal biorefinery and reduced environmental pollution.
