环境相关浓度EHDPP长期暴露对斑马鱼的甲状腺内分泌干扰效应

Thyroid Endocrine Disruption of EHDPP at Environmentally Relevant Concentrations on Zebrafish(Danio rerio)

作为一种典型的芳基有机磷阻燃剂,2-乙基己基二苯基磷酸酯(2-ethylhexyl diphenyl phosphate,EHDPP)在各种水生环境和水生生物中有着较高检出率。体外实验表明EHDPP可能通过影响甲状腺激素(thyroid hormones,TH)与转运蛋白结合进而扰乱甲状腺内分泌系统。然而目前尚未有体内实验报道其甲状腺内分泌干扰效应及可能机制。本研究中,斑马鱼胚胎(2 hours post-fertilization,2hpf)暴露在环境相关浓度(0、1和10μg·L^(-1))EHDPP中120 d,并收集F1代胚胎于清水中培养至120 hpf。研究结果表明,在F0代,EHDPP暴露显著降低成鱼存活率、体质量、肝体质常数(hepatosomatic index,HSI)和性腺体质常数(gonadosomatic index,GSI)。进一步分析发现,EHDPP显著降低雌鱼(10μg·L^(-1) EHDPP暴露组)和雄鱼(1μg·L^(-1) EHDPP暴露组)血清中甲状腺素(thyroxin,T4)水平,这可能在一定程度上抑制成鱼的生长。雌鱼T4水平降低伴随着促肾上腺皮质激素释放激素基因(corticotropin-releasing hormone,crh)显著性下调和尿苷二磷酸葡萄糖醛酸转移酶基因(uridine diphosphate glucuronosyl transferase,ugt1ab)显著性上调。此外,甲状腺激素转运蛋白基因(transthyretin,ttr)在1μg·L^(-1)和10μg·L^(-1)暴露组显著下调,表明EHDPP可能通过干扰甲状腺激素与甲状腺激素转运蛋白(transthyretin,TTR)结合从而影响甲状腺激素平衡。雄鱼中,甲状腺激素受体基因(thyroid receptorα,trα和thyroid receptorβ,trβ)显著下调表明较少的甲状腺激素发挥作用从而抑制雄鱼生长。在F1代仔鱼中,亲代暴露于1μg·L^(-1)和10μg·L^(-1) EHDPP分别导致三碘甲状腺原氨酸(triiodothyronin,T3)和T4水平显著降低,表明母体传递的甲状腺内分泌干扰效应。F1代仔鱼TH合成相关基因(thyroglobulin,tg)和早期甲状腺发育相关基因(paired box 8,pax8)显著下调,可能是TH水平降低的部分原因。脱碘酶基因(deiodinase 1,dio1和deiodinase 2,dio2)表达显著上调,可能是T4水平降低的代偿反应。本研究首次在体内实验中证明环境相关浓度EHDPP具有潜在甲状腺内分泌干扰效应,并且这种效应可能传递给F1代仔鱼。

As an aryl organophosphorus flame retardant,2-ethylhexyl diphenyl phosphate(EHDPP)has been 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 this study,zebrafish(Danio rerio)embryos(2 hpf)were exposed to environmentally relevant concentrations(0,1 and 10μg·L^(-1))of EHDPP for 120 d,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 10μg·L^(-1) and 1μg·L^(-1) EHDPP decreased serum thyroxin(T4)levels of female and male zebrafish,respectively,which possibly contributed to the growth inhibition in adult zebrafish.The decrease of T4 levels in females was accompanied by significant down-regulation 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 1μg·L^(-1) and 10μg·L^(-1) 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 receptorα,trαand thyroid receptorβ,trβ)were both downregulated,indicating less activities of TH,which could contribute to and resulte in the body growth inhibition.In F1 larvae,parental exposure to 1μg·L^(-1) and 10μg·L^(-1) 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 2,dio2)were both significantly upregulated,which could possibly be a compensatory response to the decrease of T4 levels.In conclusion,we for the first time demonstrated the potential thyroid disruption effect of EHDPP at environmentally relevant concentrations in vivo,which effect could be transferred to F1 larvae.