有机磷酸酯阻燃剂在鱼体内的富集、分布和代谢及其机制

发布时间：2017-12-28 22:23

本文关键词：有机磷酸酯阻燃剂在鱼体内的富集、分布和代谢及其机制　出处：《南京大学》2017年博士论文　论文类型：学位论文

【摘要】：有机磷酸酯阻燃剂(Organophosphate Flame Retardants,OPFRs)是一类常用阻燃剂,广泛应用于多种行业,包括纺织、建材、电子以及化工等。OPFRs多以物理添加而非化学键合的方式应用于材料中,极易从终产品中释放出来进入环境。水体是OPFRs的一个重要汇。通过污水管网,大量的OPFRs被排放到水中。越来越多的研究表明,部分OPFRs(如磷酸三苯酯TPHP、磷酸三(1,3-二氯异丙基)酯TDCIPP、磷酸三(丁氧基乙基)酯TBOEP等)对鱼类等水生生物具有明显的发育毒性、心脏毒性、神经毒性和内分泌干扰毒性。污染物在生物体内的累积和代谢过程,是评价其毒性效应和生态风险的重要指标。然而国内外关于OPFRs在鱼体中的累积和代谢研究还很少。本研究以斑马鱼等为受试生物调查考察了 7种典型OPFRs(磷酸三丙酯TPRP、磷酸正三丁酯TNBP、TBOEP、磷酸三(2-氯乙基)酯TCEP、TDCIPP、TPHP和磷酸三对甲苯酯p-TCP)在斑马鱼体内的累积、分布、净化和生物代谢转化过程。并采用模型拟合、理论计算和分子对接,探究不同结构OPFRs在鱼体中的累积和代谢转化机制。主要研究内容和结果如下:(1)结合超声萃取、凝胶渗透色谱仪、固相萃取小柱、气相色谱-质谱联用仪(GC-MS)和液相色谱-质谱联用仪(LC-MS/MS和LC-Q-TOF MS),建立了生物体内有机磷酸酯阻燃剂及其代谢产物的分析方法。(2)通过水相暴露,测定了 7种不同结构的OPFRs在斑马鱼不同组织(脑、肠、腮、肝、卵和肌肉)中的差异富集过程。结果表明相对于其他OPFRs,芳基OPFRs(TPHP和p-TCP)趋向于富集于腮和肠中;烷基(TPRP和TNBP)、烷氧基(TBOEP)和短链氯代烷基(TCEP)OPFRs趋向于富集于卵、脑、肝和肌肉组织;而多氯代(TDCIPP)OPFRs更趋向于累积在肌肉中。TDCIPP、TPHP和p-TCP在卵中的富集量较高,且半衰期长,表明这三种OPFRs有累积于斑马鱼卵中传递到下一代的趋势。通过OPFRs的logKow或鱼组织脂肪含量对生物富集系数作图,发现随着logKow或者组织脂肪含量的增加,OPFRs的富集系数逐渐增加。TPHP和p-TCP的脂肪标化富集系数(BCFlw)最高能达到5474和8909,表明这两种OPFRs从脂肪重量角度评价,具有较大的生物富集潜力。通过测试这7种OPFRs磷酸二酯代谢产物,并运用鱼体生理基础的代谢动力学模型(PBTK模型)发现代谢过程使得TDCIPP、TPHP和TNBP在斑马鱼肝脏中的累积量分别减少了 42.3%、13.7%和3.4%左右,表明代谢过程对OPFRs在斑马鱼体内的富集过程具有重要影响。而扣除代谢影响后剩下的累积量,分别占TDCIPP、TPHP和TNBP在斑马鱼肝脏中的57.7%、86.3%和96.6%,反过来表明,OPFRs在水体和鱼体间的分配作用仍然占富集过程的主导地位。(3)利用高分辨LC-Q-TOF MS筛选了斑马鱼肝脏中OPFRs的代谢产物。共检测出20种代谢产物,分别包括水解二酯产物、羟基化二酯产物、羟基化产物和葡萄糖醛酸结合产物。基于代谢产物提出了 OPFRs在斑马鱼肝脏中的代谢路径,并用分子前线轨道理论解释了代谢的分子基础。分析代谢产物在斑马鱼组织中的分布,发现肝脏和肠道是OPFRs代谢的重要器官,脑、卵和肌肉可能不能代谢OPFRs。水体中检测到的大量二酯和羟基化二酯产物表明,代谢产生的大量二酯和羟基化二酯产物易于排放到水体中,其潜在的环境效应值得我们关注。(4)研究了鱼体肝脏或脑中重要代谢酶活性(包括CYP450酶(ECOD表征)、羧酸酯酶(CESE)、谷胱甘肽转移酶(GST)、乙酰胆碱酯酶(ACHE)和可能存在的磷酸三酯酶(PTE)),随7种OPFRs暴露后的响应变化。结果发现7种OPFRs在设计暴露浓度下对PTE酶活性没有显著影响。但是TPHP和p-TCP对CESE从第3天开始表现出持续的显著性抑制。同源模建和分子对接结果表明,抑制作用可能是TPHP或p-TCP与CESE的Tyr 407(酪氨酸)的疏水相互作用、Glu 404(谷氨酸)的静电相互作用和Arg 371(精氨酸)的氢键相互作用造成。本研究揭示OPFRs在鱼体中的富集、分布和代谢过程及其作用机制,为研究OPFRs在鱼体中的毒性效应提供了数据支持。
[Abstract]:Organophosphate Flame Retardants (OPFRs) is a commonly used flame retardant. It is widely used in many industries, including textile, building materials, electronics and chemical industry. OPFRs is often used in materials by physical, not chemical bonding, and is easily released from the final product to enter the environment. Water body is an important remittance of OPFRs. Through the sewer network, a large number of OPFRs are discharged into the water. More and more studies show that part of OPFRs (such as TPHP, three phosphoric acid phenyl ester phosphate (three 1,3- two chloride isopropyl ester TDCIPP), phosphate (Ding Yangji three ethyl) ester TBOEP) has obvious developmental toxicity, cardiac toxicity, neurotoxicity and endocrine disrupting toxicity to aquatic organisms. The accumulation and metabolism of pollutants in organisms is an important index to evaluate their toxic effects and ecological risks. However, there are few studies on the accumulation and metabolism of OPFRs in the fish body at home and abroad. In this study, zebrafish as test organisms survey of 7 typical OPFRs (three TPRP, phosphate propyl phosphate is three TBOEP, three TNBP, butyl phosphate (2- chlorine ethyl) ester TCEP, TDCIPP, TPHP and p-TCP of three phosphoric acid ester toluene) conversion process in vivo distribution, accumulation, zebrafish and biological purification metabolism. Model fitting, theoretical calculation and molecular docking were used to explore the accumulation and metabolic mechanism of different structures of OPFRs in the fish body. The main research contents and results are as follows: (1) combined with ultrasonic extraction, gel permeation chromatography, solid phase extraction and gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS/MS and LC-Q-TOF MS), an analytical method of organism organic phosphate flame retardant and its metabolites. (2) the differential enrichment process of 7 different structures of OPFRs in different tissues of zebrafish (brain, intestines, cheeks, liver, eggs and muscles) was measured by water exposure. The results show that compared with other OPFRs aryl OPFRs (TPHP and p-TCP) tend to be enriched in the gills and intestine; alkyl (TPRP and TNBP), alkoxy (TBOEP) and short chain chlorinated alkyl (TCEP) OPFRs tend to be enriched in eggs, brain, liver and muscle tissue; and polychloro (TDCIPP) OPFRs tends to be accumulated in the muscle. The concentration of TDCIPP, TPHP and p-TCP in the eggs was high and the half-life was long, indicating that these three kinds of OPFRs were accumulated in the zebrafish eggs and transferred to the next generation. Bioconcentration coefficient was plotted by OPFRs logKow or fish tissue fat content. It was found that with the increase of logKow or tissue fat content, the enrichment factor of OPFRs increased. The fat normalization enrichment coefficient (BCFlw) of TPHP and p-TCP can reach 5474 and 8909, indicating that these two OPFRs have larger bioaccumulation potential from the angle of fat weight. Through the test of the 7 OPFRs two phosphate ester metabolites, the metabolic kinetics model and using the physiological basis of the fish (PBTK model) found that accumulation in zebrafish liver in the metabolic process of TDCIPP, TPHP and TNBP which were reduced by 42.3%, 13.7% and 3.4%, indicating that the metabolic process of OPFRs has important influence in the enrichment process in zebrafish. The accumulation of TDCIPP and TPHP in the liver of zebrafish accounted for 57.7%, 86.3% and 96.6% of the total accumulation of TNBP, respectively, which in turn indicated that the distribution of OPFRs between water body and fish body still dominated the enrichment process. (3) a high resolution LC-Q-TOF MS was used to screen the metabolites of OPFRs in the liver of zebrafish. 20 kinds of metabolites were detected, including two ester hydrolysates, hydroxylated two ester products, hydroxylation products and glucuronic acid binding products. The metabolic pathway of OPFRs in the liver of zebrafish was proposed based on the metabolites, and the molecular basis of metabolism was explained by the theory of Molecular Frontier orbit. The distribution of metabolites in zebrafish tissues was analyzed. It was found that the liver and intestines were important organs of OPFRs metabolism, and the brain, eggs and muscles could not metabolize OPFRs. A large number of two esters and hydroxylated two ester products detected in water indicate that a large number of two ester and hydroxyl two ester products produced by metabolism are easy to be discharged into the water body. Its potential environmental effects deserve our attention. (4) we studied the important metabolic enzyme activities in fish liver or brain, including CYP450 enzyme (ECOD characterization), carboxylesterase (CESE), glutathione transferase (GST), acetylcholinesterase (ACHE) and possible phosphatase three esterase (PTE), along with the response of 7 OPFRs exposed. The results showed that the 7 kinds of OPFRs had no significant effect on the activity of PTE enzyme at the design exposure concentration. However, TPHP and p-TCP showed persistent significant inhibition of CESE from third days. Homology modeling and molecular docking results indicate that inhibition may be caused by hydrophobic interaction of TPHP or p-TCP with CESE 407 Tyr (tyrosine), electrostatic interaction of Glu 404 (glutamic acid) and hydrogen bond interaction of Arg 371 (arginine). This study revealed the enrichment, distribution and metabolism of OPFRs in the fish body and its mechanism of action, which provided data support for the study of the toxic effects of OPFRs in the fish body.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X592;X174

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