| 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 TR?)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. |
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