| Fluorine is one of the most abundant chemistry elements in the earth. Because of its great reactivity, fluorine cannot be found in nature in its elemental state. It exists either as inorganic fluoride or as organic fluoride compounds. Fluoride is one of the necessary trace elements during animals and humans development. Right amount of fluoride can inhibit dental caries and is beneficial to skeletal development. However, excessive fluorine intake may cause toxic effects, dental and skeletal fluorosis, and nervous system derangement, and endocrine glands damage et al. In recent years, with the development of industry, a mass of fluoride was produced by human activities, such as metallurgy, organic fertilizer processing, and discharge of fluoride wastewater, which cause the increase of the fluoride concentration in the water. Bufo gargarizans is the representative of amphibian in our country. This specie lives mainly in wet areas during embryo and larvae development. Therefore, Bufo gargarizans embryogenesis becomes easier to be exposed to the fluoride.In this paper, we have studied the acute exposure to fluoride on the Bufo gargarizans embryos and the effect of fluoride on the development of embryos at sublethal concentration. Next, we measured the body weight and total length of embryos and distinguished the development stage, and observed through Scanning electron microscopy. Finally, the relative expressions of Dio2, Dio3, TRα and TRβ mRNAs in the embryos of Bufo gargarizans have been analyzed by Real-time PCR. The main results as follows:1. In semi-static toxicity bioassays, Bufo gargarizans embryos (Gosner stage 11-Gosner stage 22) were exposed to six different concentrations of NaF (250-500 mg/L). We calculated the malformation number and death number, and estimated the 216h median effective concentration (EC50) and median lethal concentration (LC50) were 416.37 mg/L and 561.64 mg/L, respectively. The malformation rate and mortality of embryos exposed to 500 mg/L NaF were 66.25% and 47.50%, respectively.2. We examined gross morphological changes by NaF through Entity microscope. The primary malformations were yolk sac edema, curvature body axis and tissue hyperplasia. Further more, we observed the superficial changes of the malformed embryos through Scanning electron microscopy. The result showed that proliferation of epithelium on the outer surface of the tail, cuticularized ciliated cells and changed cellular morphology were observed at the malformed embryos.3. In semi-static toxicity bioassays, Bufo gargarizans embryos (Gosner stage 7-Gosner stage 22) were exposed to six different concentrations of NaF (1-150 mg/L). The morphological data such as body weight and total length were evaluated in order to reveal the effect of fluoride on the embryogenesis. The results showed that the inhibitory effect of fluoride on embryonic development weight and length be doses and time dependent. Body weight and total length were not influenced by 1 mg/L NaF, and significantly reduced at 10-150 mg/L NaF compared to control groups (p<0.05).4. At sublethal concentration, we recorded the development time of embryos when they reached the stage of Gosner 22, and we evaluated the mean stage of embryogenesis during 6-9 days. The effects of NaF in embryonic development had been verified by these two statistical methods. The cumulative developmental hours indicated that the embryogenesis time was significantly retarded by NaF (p<0.05). Stages of embryonic development results showed that the period of embryogenesis were not influenced by 1 mg/L NaF, and significantly delayed at 10-150 mg/L NaF compared to control groups (p<0.05).5. The effect of NaF on Deiodinases genes Dio2 and Dio3 and thyroid hormones receptors TRα and TRβ genes expression during the embryogenesis were examined by Real-time PCR. The results indicated that Dio3 and TRβ mRNA expression appeared to be significantly up-regulated by NaF (p<0.05), and embryos exposed to NaF did not cause any difference (p>0.05) in Dio2 and TRa mRNA expression at Gosner stage 11. Additionally, NaF treatment could significantly up-regulate Dio2, Dio3, TRα and TRfβ expression at Gosner stage 16 (p<0.05). At Gosner stage 18, Dio3 and TRβ mRNA expression appeared to be significantly up-regulated at 100 mg/L NaF -treated groups (p<0.05), and Dio2 and TRα mRNA expression levels, on the other hand, were significantly down-regulated in the 100 and 150 NaF treatment groups compared with the control group (p<0.05). Gosner stage 22 mRNA transcript levels presented these four genes mRNA expression were all significantly down-regulated at NaF -treated groups compared to control groups (p<0.05). The results suggest that NaF may disturb Dio2, Dio3, TRα and TRβ mRNA expression level, and make the negative effects during the development of Bufo gargarizans embryos.This study first researched the toxicity of sodium fluoride on morphology and gene aspects of the developmental of Bufo gargarizans embryos through the traditional toxicology and modern molecular technique. We obtained the relatively complete data and revealed the mechanism of poisoning of sodium fluoride, and offered important reference for the research of amphibians toxicology and environmental protection. |
