Thyroid Endocring Disruption Effects Of 2-Ethylhexyl Diphenyl Phosphate On Zebrafish (Danio Rerio)

As an organophosphorus flame retardant used in various industrial products,2-ethylhexyl diphenyl phosphate(EHDPP)was widely detected in various aquatic environments and organisms.Previous in vitro studies have demonstrated that EHDPP can disturb the thyroid endocrine system by affecting the binding of thyroid hormone(TH)to transport proteins.However,no in vivo studies have reported the thyroid endocrine disruption effects of EHDPP and the possible underlying mechanisms remain unknown.In the present study,the model organism zebrafish(Danio rerio)was used as a research model to evaluate the potential thyroid endocrine disrupting effects and trnasfer toxicity of EHDPP through acute exposure and long-term exposure experiments.The main findings are as follows:1)Environmental relevant concentrations of EHDPP induced significant thyroid toxicity in zebrafish embryos/larvae.In this study,zbrafish embryos were exposed to EHDPP(0,0.1,1,10 and 100 μg/L)from the cleavage stage(2 hpf)to 120 h.Chemical analysis revealed accumulation of EHDPP was occurred in the larvae.Moreover,EHDPP exposure inhibited larvae development,with significant reductions in survival,heart rate,hatching rate,and body length,as well as significant increases in malformation rates.Exposure to 1,10 and 100 μg/L EHDPP resulted in significant decreases of thyroxin(T4)and triiodothyronine(T3)levels as well as significant increase of thyroid-stimulating hormone(TSH)levels,indicating severe thyroid endcrine disruption in zebrafish larvae.In this study,down-regulation of genes(nis and tg)related to thyroid hormone synthesis might be responsible for the decrease in T4 and T3.Down-regulation of the deiodinase 1(dio1)and deiodinase 2(dio2)genes played partially role in the decrease in T3.Exposure to EHDPP significantly induced the expression of genes involved in thyroid development(nkx2.1 and pax8),which may be a compensation for the reduced T4 and T3.Moreover,up-regulation of genes related to thyroid hormone secretion(crh,tshβ,trh)might be due to the negative feedback regulation induced by decreased T4 and T3.In addition,the protein expression of transthyretin protein(TTR)were significantly reduced,suggesting that EHDPP may disrupt thyroid hormone homeostasis by affecting TH binding.2)EHDPP induced significant thyroid endocrine disruption and transfer toxicity in zebrafish.In this study,zebrafish(Danio rerio)embryos(2 hpf)were exposed to environmentally relevant concentrations(0,1 and 10 μg/L)of EHDPP for 120 days,and the F1 generation embryos were then collected and cultured in clear water to 120 hpf.In the F0 generation,exposure to EHDPP significantly inhibited the survival rate,body weight,hepatosomatic index(HSI)and gonadosomatic index(GSI)in adult zebrafish.Further analysis revealed that 10 μg/L and 1 μg/L EHDPP decreased serum thyroxin(T4)levels of female and male zebrafish,respectively,which possibly contributed to the growth inhibition in adult zebrafish.Compared with the control group,the number of thyroid follicles in female thyroid gland increased significantly by64.29%,and the area of thyroid follicle increased significantly by 60.35%.These above histological results indictaed that EHDPP is potential to cause thyroid damage in female zebrafish.The decrease of T4 levels in females was accompanied by significant downregulation of the corticotropin-releasing hormone(crh)gene and up-regulation of the uridine diphosphate glucuronosyl transferase(ugt1ab)gene.In addition,the thyroid hormone transporter protein gene(transthyretin,ttr)was significantly downregulated in the 1 μg/L and 10 μg/L group,suggesting that EHDPP might affect the thyroid hormones through interfering the binding of TH to TTR.In male zebrafish,the thyroid hormone receptor genes(thyroid receptor α,trα and thyroid receptor β,trβ)were both downregulated,indicating less activities of TH,which could contributed to and resulted in the body growth inhibition.In F1 larvae,parental exposure to 1 μg/L and 10 μg/L EHDPP resulted in significantly decreased triiodothyronine(T3)and T4 levels in the descendants,respectively,suggesting a maternal transfer of thyroid endocrine disrupting effects by EHDPP.The expressions of TH synthesis(thyroglobulin,tg)and early thyroid development(paired box 8,pax8)related genes were significantly downregulated,which might partially be responsible for the reduced TH levels.The expressions of deiodinase genes(deiodinase 1,dio1 and deiodinase 1,dio2)were both significantly upregulated,which could possibly be a compensatory response to the decrease of T4 levels.