2019 - Master's Thesis - Fluoride & Bufo Gargarizans

2019 - Master's Thesis - Fluoride & Bufo Gargarizans

Postby admin » Sun Aug 16, 2020 6:10 pm

Bo Xiaoxue - "Effects of fluoride on the development of tadpoles of Bufo gargarizans" Masters Thesis, Shaanxi University, Developmental Biology (2019)
http://gb.oversea.cnki.net/kcms/downloa ... &cflag=pdf

作者】 薄晓雪;

【导师】 吴民耀;

【作者基本信息】 陕西师范大学, 发育生物学, 2019, 硕士

【摘要】 氟(Fluorine)属于卤素家族,它的化学活性很强,通常以化合物的形式广泛地分布于地球上。在自然界的水域中,氟离子的含量通常很低,一般不超过2mg/L。例如:在淡水和海水中,氟离子浓度分别在0.01-0.3 mg F-/L和1.2-1.5 mg F-/L之间。由于人类的活动,如制陶业、含氟农药的使用和沿海工业,导致了水中氟离子浓度的增加。据报道,在许多水生环境中,氟化物浓度已经达到了 2.3-49mg F-/L之间。虽然氟被认为是人和动物健康的一种必需元素,但是过量的氟摄入会对生物造成负效应。肝脏作为以代谢功能为主的器官,不仅在去氧化、储存肝糖原、分泌性蛋白质的合成等方面发挥重要作用,更在有害异物的生物转化过程中起着关键作用。肝脏毒理学研究在污染物毒理研究中占据着重要地位。遗憾的是,到目前为止,人们对于人和动物的肝脏,特别是两栖类动物肝脏对水中氟离子的耐受阈值还不清楚。因此,研究氟化物对两栖类肝脏的影响具有十分重要的理论意义。两栖动物的肢体在胚后发生,其幼体的发育环境具有一定可控性的特点。因此,可通过不同浓度氟离子的水暴露处理,探讨氟化物对肝脏的影响,对于两栖动物生存环境的保护和治理具有一定的应用价值。中华大蟾蜍(Bufogargarizans),由于皮肤的通透性强、对污染物极其敏感,故通常被作为一种水生环境监测物种。本研究以中华大蟾蜍肝脏为研究对象,探讨氟化物对中华大蟾蜍肝脏的毒理分子机制并评估氟化物对两栖动物的影响。本研究内容包括两部分,第一部分,急性暴露实验:我们将Gs37期的中华大蟾蜍幼体暴露于100 mg/L溶液中(24、48和72 h)。首先通过组织病理学方法检测氟化物暴露不同时间后,中华大蟾蜍肝脏结构的变化。其次通过实时定量PCR技术(RT-qPCR)检测氟化物对中华大蟾蜍肝脏中与脂代谢、氧化应激和凋亡相关基因表达的影响。第二部分,慢性暴露实验:我们将Gs26期的中华大蟾蜍幼体暴露于不同浓度的氟离子溶液中(0.5、5、10和50 mg/LNaF),暴露持续时间到Gs42期。通过组织病理学方法检测Gs42期中华大蟾蜍肝脏结构的变化。随后通过RT-qPCR检测Gs42期中华大蟾蜍肝脏中与脂代谢、氧化应激、胆盐合成和分泌相关基因的表达水平。再通过原位杂交技术定位胆盐合成与分泌相关基因(BSEP和HSD3B7)在肝脏中的表达位置。最后运用气相色谱法-质谱法联用(GC-MS)分析研究了氟化物对42期中华大蟾蜍脂肪酸组成和含量的影响。本实验的主要结果和结论如下:1、组织病理学显示,在100 mg/LNaF溶液中暴露72 h造成肝细胞边界消失、肝实质细胞退化、色素细胞数量以及脂滴的量显著增加。结果表明氟化物的急性暴露造成了中华大蟾蜍蝌蚪肝脏的损伤。2、实时定量PCR技术(RT-qPCR)结果显示,在24、48和72 h时,100 mg/L NaF溶液暴露显著下调了脂代谢相关基因,包括乙酰CoA羧化酶(ACC)、脂肪酸延长酶(FAE)和肉碱棕榈酰转移酶(CPT)以及氧化应激相关基因,包括超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GPx)的表达(p<0.05)。但是,在100 mg/LNaF溶液中暴露72 h使得凋亡相关基因,包括bcl-2相关转录因子(BCLAF)、甲状腺受体α和β(TRα、TRβ)的表达显著上调(p<0.05)。结果表明氟化物的急性暴露造成了中华大蟾蜍脂代谢的紊乱、抗氧化能力的减弱以及凋亡的加速。3、组织病理学结果显示,50 mg/L NaF处理造成肝脏内色素细胞数量增多,肝细胞核萎缩,肝脏的胆管扩张。暴露于0.5、5、10和50 mg/L NaF溶液使肝脏中脂滴量减少且脂滴发生了降解和沉积。结果表明氟化物的慢性暴露造成了中华大蟾蜍蝌蚪肝脏的损伤。4、RT-qPCR结果表明,0.5、5、10和50 mg/L NaF溶液处理显著下调了脂肪酸合成相关基因,包括脂肪酸合成酶(FASN)、FAE、线粒体反式-2-烯酰CoA还原酶(MECR)和(17β)羟化类固醇脱氢酶12s(KAR),以及脂肪酸β-氧化的相关基因,包括烯酰CoA水合酶,短链1(ECHS1)、长链3-羟烷基-CoA脱氢酶(HADHA)、固醇载体蛋白2(SCP2)、乙酰CoA酰基转移酶1和2(ACAA1、ACAA2)的表达(p<0.01)。此外,0.5、5、10和50 mg/L NaF溶液暴露明显抑制了氧化应激相关基因,包括SOD、GPx、线粒体钙吸收蛋白1(MICU1)以及胆盐合成基因A-5羟基类固醇脱氢酶,A3β类固醇异构酶(HSD3B7)的表达(p<0.01)。但是0.5、5和50 mg/LNaF溶液造成胆盐分泌基因胆盐外排泵(BSEP)表达水平显著升高(p<0.05)。结果表明氟化物的慢性暴露造成了中华大蟾蜍脂代谢的紊乱、抗氧化能力的减弱以及胆盐分泌的加快、合成的减慢。5、原位杂交结果显示,BSEP在对照组肝细胞核中表达,而暴露于50 mg/LNaF溶液后,BSEP在细胞核和胆管膜上均表达:HSD3B7在对照组和50 mg/LNaF溶液中均在肝细胞核和质膜上表达。这表明氟化物暴露影响BSEP的表达位置,但并不影响HSD3B7的表达位置。6、GC-MS分析结果显示,与对照相比,氟化物的处理造成饱和脂肪酸(SFAs)的含量减少,多不饱和脂肪酸(PUFAs)的含量增加。这表明氟化物暴露能够影响脂肪酸的含量。综上所述,氟化物暴露对中华大蟾蜍蝌蚪的肝脏造成了较严重的结构损伤,引起脂代谢和抗氧化能力等生理生化功能的障碍。本研究将为进一步研究氟化物对人和动物,特别是对两栖动物肝脏的影响研究提供实验参考。对社会大众的环保和生态公共意识的提高具有推进意义,也为政府相关部门的氟离子安全指标的制定提供了实验和理论参考。 还原

【Abstract】 Fluorine is a member of the halogen family.Fluoride distributes widely on the earth as fluoride compounds as a consequence of its great chemical reactivity.In the natural water,the content of fluoride ion is usually very low,it is generally no more than 2 mg/L.In unpolluted fresh waters and seawaters,fluoride concentrations range from 0.01 to 0.3 mg F-/L and 1.2 to 1.5 mg F-/L,respectively.However,anthropogenic processes,including ceramics,the use of fluoride containing pesticides,and industries located along the marine coast have led to increased fluoride concentrations in aquatic environments.It was reported that the level of fluoride contaminations ranged from 2.3 to 49 mg F"/L in many aquatic environments.Although fluorine is considered as an essential element for human and animal health,excessive fluoride ingestion can cause negative effects on organisms.Liver,an organ that functions primarily in metabolism,plays important roles not only in deoxidation,storage of liver glycogen,synthesis of secretory proteins and so on but also in the bioconversion processes of xenobiotics.Liver toxicology plays an important role in the study of pollutant toxicology.Unfortunately,to date,the tolerance threshold of fluorine ion in water for liver of people and animals,especially amphibians is not clear.Therefore,it is of great theoretical significance to study the effect of fluoride on amphibian liver.The limbs of amphibians occur at larval stages,and the developmental environment of larvae is controllable.Therefore,the effects of fluoride on the liver can be studied through exposure treatment with different concentrations of fluoride ions,which has certain application values for the protection and governing of the living environment in amphibians.Bufo gargarizans are used as one of important monitoring species in aquatic environments,which are sensitive to aquatic contaminants because its highly permeable skin.The aim of this study was to investigate the molecular mechanism of the toxicological effects of fluoride on the liver of B.gargarizans,thereby evaluating the effect of fluoride on amphibians.This study consists of two parts.The first part is the acute exposure experiment:B.gargarizans at Gs37 were treated with 100 mg/L NaF for 24,48 and 72 h.Histological analysis was conducted to detect morphological alterations of liver in B.gargarizans In addition,real-time quantitative PCR(RT-qPCR)was used to assess the effects of fluoride on expression of lipid metabolism,oxidative stress and apoptosis-related genes in the liver of B.gargarizans.The second part is the chronic exposure experiment:B.gargarizans larvae were exposed to 0.5,5,10 and 50 mg/L NaF from Gs26 to Gs42.Histological analysis was conducted to detect the changes of liver structure in B.gargarizans at Gs42.Subsequently,RT-qPCR was used to detect the expression levels of genes involved in lipid metabolism,oxidative stress,synthesis and secretion of bile acid in the liver of B.gargarizans at Gs42.Furthermore,the locations of synthesis and secretion of bile acid-related genes(BSEP and HSD3B7)in the liver were determined by in situ hybridization.Finally,gas chromatography-mass spectrometer(GC-MS)analysis was conducted to study the effects of fluoride on fatty acid composition and content in B.gargarizans at Gs42.The main results are as follows:1.For B.gargarizans at Gs 37,histological analysis showed that morphological alterations were observed in the liver treated with 100 mg/L NaF for 72 h,including disappearance of cell boundaries,degeneration of hepatic parenchyma cells and significant increase in the number of melanomacrophage centres and the quantity of lipid droplets.Thus,the acute fluoride exposure experiment could cause liver injury in B.gargarizans.2.For B.gargarizans at Gs 37,real-time quantitative PCR(RT-qPCR)results showed that exposure to 100 mg/L NaF for 24,48 and 72 h significantly decreased the relative expression of lipid metabolism-related genes,including acetyl CoA carboxylase(ACC),fatty acid elongase(FAE),and carnitine palmitoyltransferase(CPT)and genes involved in oxidative stress,including superoxide dismutase(SOD)and glutathione peroxidase(GPx)(p<0.05).However,mRNA expression of apoptosis-related genes,including bcl-2-associated transcription factor(BCLAF),thyroid hormone receptors(TRa and TRβ)was significantly upregulated in tadpoles treated with 100 mg F/L NaF for 72 h(p<0.05).This indicated that the acute fluoride exposure could elicit disruption of lipid metabolism,result in impairing of antioxidant capacity and contribute to apoptosis in liver of B.gargarizans.3.For B.gargarizans at Gs 42,histological analysis showed that the increase of number of melanomacrophage centres,atrophy of nucleus and dilation of bile canaliculus were found in the liver of 50 mg/L NaF group.Besides,decrease of quantity,degradation and deposition of lipid droplets were observed in liver of B.gargarizans treated with 0.5,5,10 and 50 mg/L NaF.Thus,the chronic fluoride exposure experiment could result in liver injury in B.gargarizans.4.For B.gargarizans at Gs 42,RT-qPCR results showed that B.gargarizans after exposure to 0.5,5,10 and 50 mg/L NaF had significantly decreased transcript levels of genes involved in fatty acid synthesis,including fatty acid synthase(FASN),FAE,mitochondrial trans-2-enoyl-CoA reductase(MECR)and hydroxy steroid(17-beta)dehydrogenase 12S(KAR)in liver and genes involved in fatty acid P-oxidation,including enoyl-CoA hydratase,short chain 1(ECHS1),long-chain 3-hydroxyacyl-CoA dehydrogenase(HADHA),sterol carrier protein 2(SCP2)and acetyl-CoA acyltransferase(ACAA1 and ACAA2)(p<0.01).Besides,transcript levels of oxidative stress-related genes,including SOD,GPx and mitochondrial calcium uptake 1(MICU1)and hydroxy-delta-5-steroid dehydrogenase,3 beta-and steroid delta-isomerase(HSD3B7)were inhibited in liver of B.gargarizans in 0.5,5,10 and 50 mg/L NaF treatment groups(p<0.01).However,bile salt export pump-like(BSEP)significantly increased at 0.5,5 and 50 mg/L NaF(p<0.05).This indicated that the chronic fluoride exposure could elicit disruption of lipid metabolism,lead to impairing of antioxidant capacity and trigger disturbing of synthesis and secretion of bile acid in liver of B.gargarizans.5.For B.gargarizans at Gs 42,in situ hybridization results showed that in control,BSEP mRNA was expressed in the hepatocyte nucleus,while positive signals for BSEP mRNA were found in the hepatocyte nucleus and on the canalicular membranes in 50 mg/L NaF treatment group.Besides,HSD3B7 mRNA was expressed in the hepatocyte nucleus and on plasma membrane in control and 50 mg/L NaF groups.This indicated that fluoride exposure could affect the location of BSEP mRNA expression,but it did not affect the location of HSD3B7 mRNA expression.6.For B.gargarizans at Gs 42,GC-MS analysis results showed that the content of saturated fatty acids(SFAs)was decreased and the content of polyunsaturated fatty acids(PUFAs)was increased in all fluoride treatment groups compared to untreated controls.This indicated that fluoride exposure could affect the content of fatty acids.In conclusion,fluoride exposure could cause liver injury,elicit disruption of lipid metabolism,and result in impairing of antioxidant capacity in liver of B.gargarizans.The present study will provide experimental references for further studies on the effects of fluoride on liver of humans and animals,especially on amphibians.Also,this study plays roles in enhancing the public awareness of environmental protection and ecology,and provides experimental and theoretical references for relevant government departments to establish the safety index of fluorine ions. 还原
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