Effects Of F-53B On Thyroid Function And Antioxidant Status And Its Mechanism In Zebrafish

In electroplating industry,especially for“hard chrome plating”,perfluorooctane sulfonate?PFOS?is commonly used as an excellent mist suppressant agent.However,due to the regulations on PFOS,some non-PFOS mist suppressants are beginning to appear on the market.In China,6:2 chlorinated polyfluorinated ether sulfonate?F-53B?has been widely used in electroplating industry as a substitute for PFOS.In2009,the annual usage of mist suppressant was 20-30 tons.Furthermore,the lack of specific restrictions on the emissions and disposal of F-53B leads to F-53B eventually entry into the aquatic environment.Given that F-53B was frequently detected in the environment and has a high-bioaccumulation capacity,thus enough attention must be paid to F-53B regarding the potential health concern on aquatic organisms.In this regard,it used zebrafish as a model organism to carry out the following studies.High-sensitivity analysis method for F-53B in zebrafish was established using liquid chromatography-high resolution mass spectrometry?LC-MS/MS?as a detection method..Then,research on the enrichment and metabolic dynamics of F-53B in Zebrafish.Further,the developmental toxicity of zebrafish embryos,thyroid endocrine disrupting effects,and oxidative stress effects and their mechanisms of action were further studied..The results are as follows:?1?Used LC-MS/MS as test method,The Eclipase Plus C₁₈?1.8?m×2.1mm×50mm,Agilent,USA?column was successfully used to detect the content of F-53B in zebrafish.About F-53B and PFOS,the mass spectrometer was operated in a negative electrospray ionization mode with multiple reaction monitoring?MRM?.The fragmentor voltage and collision energy were set at 160 V?100 V?and 30 eV?70 eV?,respectively.The mass-to-charge ratio?m/z?of parent ion and quantitative product ion are 530.8?498.8?and 351.0?79.9?.The correlation coefficients?R²?between the external standard method and the internal standard method were 0.9999 and 0.9997,respectively,indicating that F-53B has a good linear relationship within the linear range of the measurement.According to the results of precision and recovery,the precision?RSD,relative standard deviation?and recovery of the three concentration gradients of low,medium,and high?10,100,500?g/L?were 2.53%,3.83%,1.88%and 86.13±1.28%,90.40±2.86%and 89.67±3.10%,respectively.Because of the RSD is less than 5%and the recovery rate is above 85%,it is consistent with the general requirements of quantitative analysis of biological samples.?2?F-53B is a moderately toxic substance.It showed a time-dependent effect on the acute lethality of zebrafish embryos and larvae.The 72 h-LC₅₀0 of zebrafish embryos and larvae were 18.43 mg/L and 13.09 mg/L,and the corresponding 95%confidence intervals were 15.81 to 22.59 mg/L and 6.59 to 17.43 mg/L,respectively.It showed that the sensitivity to F-53B on larvae stronger than embryos.?3?After exposure to 10ug/L and 100ug/L F-53B,there was a positive correlation between the absorption of F-53B and exposure time in larvae and adult zebrafish?female/male?.During the uptake phase,for larvae,the content of F-53B in the body?wet weight,the same below?reached 2.359 mg/kg and 23.046 mg/kg,respectively.The log₁₀BCF_48h8h values corresponding to the two concentration gradients of 10?g/L and 100?g/L F-53B ranged from 2.659 to 2.693.About the elimination rate constant?k?is between 0.00268 h^-11 and 0.00287 h^-1,then the corresponding half-life is between 241.5 h and 258.6 h.For adult zebrafish,on the uptake phase?0-7d?,the content of F-53B in each tissue was positively correlated with the exposure time,but it did not reach the saturation state;in the depuration phase?7-12 d?,the content of F-53B in each tissue with the elimination of time gradually reduced.At exposure concentrations of 10?g/L and 100?g/L F-53B,the effects of zebrafish on F-53B showed a gender-and tissue-specific difference.Accumulation ability which is male>female.But females consistently expressed as:gonad>liver>gill>brain;and the males showed consistent:liver>gill>gonad>brain.The highest concentrations of F-53B in the low-and high-exposure groups were female gonads?5.42 and 59.19mg/kg,respectively?and male livers?8.98 and 116.24 mg/kg,respectively?.It was inferred that the liver was one of the most important sites of F-53B enrichment in zebrafish,and its higher surface area also showed a higher enrichment potential for F-53B.?4?Detection of GH₃ cell display with T-screen assay,it showed that F-53B enhanced the GH₃ cell proliferation in a dose-dependent fashion,indicating the thyroid receptor agonistic activity of F-53B.The results of experiments on hormones,genes,Western Blot,homologous modeling and molecular docking of zebrafish larvae,it showed that F-53B exposure significantly induced development inhibition.On uptake phase,increased thyroxin?T₄?but not 3,5,3'-triiodothyronine?T₃?levels accompanying a decrease in thyroglobulin?TG?protein level and the transcript levels of most genes involved in the hypothalamic-pituitary-thyroid axis?crh,tsh?,tr?,dio1,dio2,nis,nkx2.1,pax8 and so on?.Interestingly,T₄ levels remained significantly increased,but TG protein levels and the gene transcription were markedly up-regulated after depuration phase.Taking computer simulation of molecular docking,the average root-mean-square deviation?RMSD?of the complex skeleton atoms of zfTTR/F-53B was 2.31?.And the whole system achieves dynamic balance after 10 ns simulation.Furthermore,our molecular docking revealed that F-53B binds to transthyretin?TTR?by forming hydrogen bonds with Lys123 and Lys115,thereby interfering with thyroid hormone binding to TTR,leading to an increase in T₄ levels.Through the quantitative evaluation of zfTTR/F-53B,its binding free energy?G_bindind is-7.36 kcal/mol,and electrostatic energy was the main contributor to the combination of F-53B and zfTTR.Among the residues decomposed such as Leu8,Leu101,Val112,Lys115,Leu125,Pro132,Leu218 and Val229,Leu125 were the most important residue of F-53B binding to zfTTR.Our in vitro,in vivo and in silico studies provided a new set of evidence that F-53B could disturb the thyroid endocrine system under environmentally relevant concentrations and the disrupting effects could not be recovered after depuration.?5?The effect of F-53B on the antioxidant enzyme system of zebrafish larvae and adults,at uptake phase,the content of MDA and GSH decreased,and there was a negative correlation with exposure concentration.The activities of CAT,SOD,CuZn-SOD and GSH-ST were all inhibited,and the inhibitory effect was more pronounced with increasing exposure concentration.GSH-Px activity was induced and was positively correlated with exposure concentration.At depuration phase,The contents of MDA and GSH were up-regulated but still lower than those of the control group;the activities of CAT,SOD,CuZn-SOD,and GSH-ST all recovered,but overall they were still lower than the control group;GSH-Px The induction of activity was down-regulated,but overall it was still higher than the control group.It can be seen that the accumulation of F-53B in the body will have an adverse effect on the anti-oxidation system of zebrafish,affect the function of tissues and the vitality of cells,and cause a variety of subsequent unfavorable environmental effects.Therefore,we must pay enough attention to F-53B.