Effects of Fluoride on Bufo Gargarizans, Rana Chensinensis.

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Effects of Fluoride on Bufo Gargarizans, Rana Chensinensis.

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Wang Ming - "Toxicological Effects of Fluoride on the Growth and Development of Bufo gargarizans and Rana chensinensis" Master's Thesis, Shaanxi University (2016)
http://cdmd.cnki.com.cn/Article/CDMD-10 ... 034822.htm
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氟化物对中华大蟾蜍和中国林蛙生长发育的毒理效应研究
王明
【摘要】:氟是动物发育过程中必需的微量元素之一,适量的氟摄入可以促进动物生长发育,但过多的氟摄入会扰乱机体的正常生理代谢,对机体造成毒性损伤,如氟斑牙、氟骨病以及肝、肾和甲状腺等器官结构和功能的损伤。随着工业化的快速发展,人类生活范围的扩大,生活及工业生产排出的大量污水致使水体中氟浓度持续升高。据报道,污染的水体中氟浓度已普遍高达49 mg/L,在某些地区甚至高至96.8 mg/L。目前,氟化物对两栖类动物的毒性影响研究报道较少。中华大蟾蜍(Bufo gargarizan)和中国林蛙(Rana chensinensis)是我国广泛分布的两栖类物种,其胚胎及幼体对水污染异常敏感。本研究选取这两种蛙为研究对象,探究氟化物对两栖类动物生长发育的影响。本研究将中华大蟾蜍幼体从G26期(后肢芽发育期)至G46期(变态完成期)暴露于不同浓度的氟溶液中(0.68、4.63、21.42、43.16和61.26 mg/L),中国林蛙从胚胎期(G7)开始暴露于氟溶液中直至G46期。采用生物统计学方法、组织病理学方法、分子生物学方法等多种实验手段来检测氟化物的毒理效应。主要研究结果如下:1.慢性暴露实验结束时,对照组、0.68、4.63、21.42和43.16 mg/L氟离子处理组中蟾蜍幼体死亡率分别为2.50%、2.50%、3.33%、11.67%和34.17%。而在氟离子浓度高达61.26 mg/L时,致使幼体死亡率在暴露早期就高达100%。暴露于氟离子浓度为21.42和43.16 mg/L的幼体死亡率呈现时间特异性变化,死亡率在暴露早期急剧上升,随着暴露时间的延长最终趋于稳定状态。该研究结果表明早期幼体比晚期幼体对氟化物毒性更敏感,幼体对氟化物的毒性呈现出相应的驯化和恢复效应。2.蟾蜍幼体暴露于氟离子溶液20天后和40天后,与对照组相比,43.16 mg/L氟离子显著抑制了蟾蜍幼体体重、体长、全长和发育时期的增长。结果表明该浓度的氟离子可致使蟾蜍幼体的生长受到显著抑制。3.在同一时间节点43.16 mg/L氟处理的蟾蜍幼体未能生长至G46期。利用双染色技术检测氟对G46期蟾蜍幼体骨骼发育的影响,结果显示4.63 mg/L氟离子可促进幼体骨骼的发育,而21.42 mg/L氟离子则会抑制幼体骨骼的发育。4.暴露70天后,对照组蟾蜍幼体到达G42期(变态高峰期)的比率高达50%。不同浓度氟离子处理组的蟾蜍幼体变态率均受到影响,其中43.16 mg/L处理组影响最为显著,变态率仅为17.78%;0.68、4.63和21.42 mg/L氟离子中蟾蜍幼体变态率分别为42.22%、41.11%和26.67%。结果显示高浓度氟化物显著抑制蟾蜍幼体的变态发育。5.采用组织病理学方法检测氟对G42期蟾蜍幼体甲状腺结构的损伤。暴露于43.16 mg/L氟离子致使幼体甲状腺滤泡上皮细胞排列松散不规则,并且甲状腺滤泡中出现部分胶质降解。荧光实时定量PCR技术检测不同浓度氟离子处理的G42期幼体各组织中甲状腺系统相关基因(Dio2、Dio3、TRα和TRβ)的表达变化,发现暴露于21.42和43.16 mg/L氟离子引起G42期蟾蜍幼体的肝脏、尾和后肢中Dio2、 Dio3、TRα和TRβ表达水平显著下调。6.林蛙慢性暴露实验中,所有氟离子处理组均呈现出不同程度的胚胎畸形,主要表征为体轴弯曲,水肿和背腹部褶皱等。此外,暴露于19.62和42.41 mg/L氟离子林蛙胚胎的生长发育受到显著抑制。7.慢性暴露15天后,19.62和42.41 mg/L氟离子显著抑制了林蛙幼体体重和体长的增长。暴露30天后,42.41 mg/L氟离子处理组林蛙幼体依然呈现体重和体长的生长受到显著抑制。此外,暴露于42.41 mg/L氟离子林蛙幼体的变态发育也显著延迟。综上所述,氟浓度高于40 mg/L会对中华大蟾蜍和中国林蛙的生长发育造成显著影响,主要表现为生存率降低,胚胎畸形,幼体生长、发育及变态迟滞,甲状腺结构破坏,甲状腺系统相关基因表达水平下调等。我们推测氟化物毒理效应与高浓度氟导致的两栖动物甲状腺分泌系统扰乱相关,为进一步探究氟化物对两栖类生物的毒理效应提供了一定的参考依据。
【学位授予单位】:陕西师范大学
【学位级别】:硕士
【学位授予年份】:2016
【分类号】:X171.5

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【Summary】:Fluorine is one of the necessary trace elements in the process of animal development. An appropriate amount of fluoride intake can promote the growth and development of animals, but excessive fluoride intake will disturb the normal physiological metabolism of the body and cause toxic damage to the body. Bone fluorosis and damage to the structure and function of organs such as the liver, kidneys, and thyroid. With the rapid development of industrialization and the expansion of the scope of human life, a large amount of sewage discharged from domestic and industrial production has caused the concentration of fluorine in water to continue to rise. According to reports, the concentration of fluorine in polluted water is generally as high as 49 mg/L, and even as high as 96.8 mg/L in some areas. At present, there are few reports on the toxic effects of fluoride on amphibians. Bufo gargarizan and Rana chensinensis are widely distributed amphibian species in my country, and their embryos and larvae are extremely sensitive to water pollution. This study selected these two frogs as the research objects to explore the effect of fluoride on the growth and development of amphibians. In this study, the larvae of Chinese giant toads were exposed to different concentrations of fluoride solution (0.68, 4.63, 21.42, 43.16 and 61.26 mg/L) from G26 (hind limb bud development stage) to G46 (metamorphosis completion stage). Exposure to fluoride solution from embryonic stage (G7) until G46 stage. Biostatistics, histopathology, molecular biology and other experimental methods were used to detect the toxicological effects of fluoride. The main research results are as follows:1. At the end of the chronic exposure experiment, the mortality of toad larvae in the control group, 0.68, 4.63, 21.42 and 43.16 mg/L fluoride ion treatment groups were 2.50%, 2.50%, 3.33%, 11.67% and 34.17%, respectively. %. When the fluoride ion concentration was as high as 61.26 mg/L, the mortality of larvae was as high as 100% in the early exposure period. The mortality of larvae exposed to fluoride ion concentrations of 21.42 and 43.16 mg/L showed time-specific changes. The mortality increased sharply in the early stage of exposure, and finally stabilized with the prolongation of exposure time. The results of this study showed that early larvae were more sensitive to fluoride toxicity than late larvae, and larvae exhibited corresponding domestication and recovery effects to fluoride toxicity. 2. After 20 days and 40 days after toad larvae were exposed to fluoride ion solution, compared with the control group, 43.16 mg/L fluoride ion significantly inhibited the growth of toad larvae body weight, body length, full length and developmental period. The results showed that the concentration of fluoride ions could significantly inhibit the growth of toad larvae. 3. At the same time node 43. Toad larvae treated with 16 mg/L fluoride failed to grow to G46 stage. Double staining technique was used to detect the effect of fluoride on the skeletal development of G46 toad larvae. The results showed that 4.63 mg/L fluoride ion can promote the development of larval skeleton, while 21.42 mg/L fluoride ion can inhibit the development of larval skeleton. 4. After 70 days of exposure, the rate of toad larvae reaching the G42 stage (the peak of metamorphosis) in the control group was as high as 50%. The metamorphosis rate of toad larvae in different concentrations of fluoride ion treatment groups were all affected, and the 43.16 mg/L treatment group had the most significant effect, and the metamorphosis rate was only 17.78%; 42.22%, 41.11% and 26.67%. The results showed that high concentration of fluoride significantly inhibited the metamorphosis development of toad larvae. 5. The damage to the thyroid structure of G42 toad larvae by fluorine was detected by histopathological method. Exposure to 43.16 mg/L fluoride ions resulted in loose and irregular arrangement of larval thyroid follicular epithelial cells, and partial glial degradation in thyroid follicles. The expression changes of thyroid system-related genes (Dio2, Dio3, TRα and TRβ) in various tissues of G42 larvae treated with different concentrations of fluoride ions were detected by fluorescence real-time quantitative PCR. The expression levels of Dio2, Dio3, TRα and TRβ were significantly down-regulated in the liver, tail and hindlimb of larvae. 6. In the chronic exposure experiment of Rana sylvaticus, all fluoride ion-treated groups showed different degrees of embryonic malformation, mainly characterized by body axis curvature, edema and dorsal and abdominal folds. In addition, the growth and development of Rana embryos exposed to 19.62 and 42.41 mg/L fluoride ions were significantly inhibited. 7. After 15 days of chronic exposure, 19.62 and 42.41 mg/L fluoride ions significantly inhibited the growth of body weight and body length of Rana sylvatica larvae. After 30 days of exposure, the growth of body weight and body length of the larvae of the 42.41 mg/L fluoride ion treatment group were still significantly inhibited. In addition, the metamorphosis development of Rana larvae exposed to 42.41 mg/L fluoride was also significantly delayed. To sum up, fluoride concentration higher than 40 mg/L would have significant effects on the growth and development of Chinese giant toads and Chinese wood frogs, mainly manifested as decreased survival rate, embryo malformation, delayed growth, development and metamorphosis of larvae, and damage to thyroid structure. , Down-regulation of thyroid system-related gene expression levels, etc. We speculate that the toxicological effects of fluoride are related to the disturbance of the amphibian thyroid secretion system caused by high concentrations of fluoride,
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