Disrupting Effects Of Fluoride On Hypothalamic-pituitary- Thyroid Axis In Zebrafish(Danio Rerio)

A lot of researchs show that high fluoride can inhibit the growth of fish growth, but its mechanism has not been clarified. Endocrine regulation is very important to the body’s growth and development. The hypothalamus-pituitary-thyroid (HPT) axis is one of the members of the endocrine system of endocrine regulation. To investigate whether fluoride affect normal growth and development of fish by influencing the fish HPT axis, two months health zebrafish were chosen and randomly divided into 3 groups:the control group (0 mg/L),40 mg/L and 80 mg/L fluoride exposure group, respectively. The growth and development status of the zebrafish, thyroid and liver histopathology, homone levels, and mRNA expression of thyroid-related genes were detected by HE staining, ELIS A, and real-time fluorescent quantitative PCR after exposure to fluoride for 45 d and 90 d. The results were as follows:1. Study on growth parameters showed that growth and development, body weight, weight gain rate, specific weight gain rate and fat full were increased in fish exposed to 40 mg/L fluoride, while were decreased in male and female fish exposed to 80 mg/L fluoride for 45 d. Body length and growth rate were decreased with the concentration increasing. Except of male fish fat full, all anther indexes were decreased with the concentration increasing after exposure to Fluoride for 90 days.2. Thyroid histopathology results:Fluoride exposure caused zebrafish thyroid follicular lesions, including follicular swelling, epithelial cells hyperplasia, and in and out fold, colloid atrophy or foamy and so on. Thyroid follicular lesions became more severe with the increase of exposure time and exposure dose. Liver in fish exposed to 40 mg/L fluoride for 45 days, occured lipoid degeneratiaon, blood clots. The more severe lesions could be found in liver with the increase of exposure concentration. After exposure to fluoride for 90 d, more fat and blood clots were found in female fish liver. A large number of liver cells disrupted and the more nucleus gathered in male fish exposed to 80 mg/L fluoride.3. The thyroid hormones T3 and T4 ELIS A results:Fluoride exposure affected the zebrafish levels of T3 and T4, and T3/T4 ratio. And the influence degree varied according to the exposure time, concentration and gender. After exposed to fluoride for 45 d, fluoride improved the levels of T3 and T4 and decreased T3/T4 ratio. After expsosure to fluoride for 90 d, T3/T4 ratio and T3 level were decreased in 80 mg/L females, while T3/T4 ratio and T3 level were higher in the other groups, and all the T4 levels were lower than that in the control group.4. After fluoride exposure, the results of the CRH, TSH, TG, NIS, TTR, DIO1, DIO2 and TRα、TRβ and UGTlab mRNA expressions showed:expect to CRH and TRa, other eight gene expression were significantly lower in female zebrafish exposed to 40 mg/L fluoride for 45 d; except to TG and NIS, TTR and TRa, six other gene expression were significantly lowered in female fish exposed to 80 mg/L fluoride; After exposure to fluoride for 45 d, except CRH, NIS and DIO1, other 7 gene expression were significantly higher in male zebrafish exposed to 40 mg/L fluoride. Besides UGT1ab and TTR gene, other eight gene expressions were significantly elevated in male zebrafish exposed to 80 mg/L fluoride. After exposure to fluoride for 90 d, The gene expressions of DIO1, DIO2 in female fish exposed to 40 mg/L, the levels of TG, DIO1, DIO2 in female fish exposed to 80 mg/L fluoride and the expressions of TRa, TRβ in all fluoride-exposed female fish were significantly enhanced, whereas the expressions of TTR, TRa in female fish exposed to 40 mg/L fluoride, and the levels of UGTlab mRNA expression were significantly lower than those in control fish. The gene expressions of TSH, TG, DIO1, TTR, TRa in male fish exposed to 40 mg/L fluoride and the mRNA levels of TG, DIO1, TRa in male fish exposed to 80 mg/L fluoride were significantly higher compared with those in the control group, while the gene expressions of CRH in male fish exposed to 40 mg/L fluoride and the mRNA levels of CRH, DIO2, TTR, UGT1ab in male fish exposed to 80 mg/L fluoride were significantly decreased.The results showed that the fluoride exposure affected their growth and development by influencing the histopathology of zebrafish thyroid and liver, thyroid hormone levels. With the increase of exposure concentration and exposure time, the effects gradually increased. Fluoride altered 10 gene expression of all zebrafish HPT axis with the exposure concentration, time, and gender. Fluoride interfered with the content of thyroid hormone in the body and, in turn, affected the body’s growth and development. All above results provide basic information for clarifying the mechanism of fluoride affecting growth in fish.