RESUMO
Objective:To study the effects of fluoride exposure on skeletal development of zebrafish larvae and its possible molecular mechanisms.Methods:Six hours post fertilization(6 hpf) wild-type zebrafish embryos were selected and exposed to sodium fluoride [NaF, control group (0 mg/L NaF), low fluoride group (25 mg/L NaF) and high fluoride group (100 mg/L NaF)] for 9 days. Fluorine ion selective electrode was used to detect the overall fluorine content of zebrafish larvaes, and the death and development of zebrafish larvaes were observed and counted. Bone mineralization and chondrogenesis of the zebrafish larvaes were analyzed by alizarin red staining and alcin blue staining, respectively. The expression levels of sry-related-high-mobilty-group box 9a (Sox9a), osteprotegerin (OPG) and receptor-activator of nuclear factor kappa beta ligant (RANKL) were analyzed by real-time quantitative PCR.Results:Compared with control group [(0.12 ± 0.01) μg/140 larvaes], the overall fluorine contents of zebrafish larvaes in low fluoride group [(0.28 ± 0.03) μg/140 larvaes] and high fluoride group [(0.64 ± 0.10) μg/140 larvaes] were significantly higher, and the differences were statistically significant ( P < 0.05). Compared with control group, zebrafish larvaes in high fluoride group had shorter body length, higher swim bladder loss rate and higher spinal curvature rate ( P < 0.05). The alizarin red staining area, integrated optical density (IOD) and the number of mineralized vertebrae were higher in low fluoride group, while the alcin blue staining area of cartilage formation was lower ( P < 0.05). In the high fluoride group, alizarin red staining area, IOD and the number of mineralized vertebrae were lower, while the alcin blue staining area of cartilage formation was higher ( P < 0.05). Compared with control group, the expression levels of OPG mRNA and OPG/RANKL mRNA in low fluoride group were higher ( P < 0.05); the expression level of RANKL mRNA was higher in high fluoride group, while the expression level of OPG/RANKL mRNA was lower ( P < 0.05). Conclusion:A short period of fluoride exposure from zebrafish embryo to zebrafish larvae can cause abnormal bone development of zebrafish larvae, which may be related to endochondral osteogenesis and OPG/RANKL pathway.