本文關鍵詞：有機磷酸酯阻燃劑在魚體內(nèi)的富集、分布和代謝及其機制　出處：《南京大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 生物富集 分布 生物代謝 代謝機制 斑馬魚

【摘要】：有機磷酸酯阻燃劑(Organophosphate Flame Retardants,OPFRs)是一類常用阻燃劑,廣泛應用于多種行業(yè),包括紡織、建材、電子以及化工等。OPFRs多以物理添加而非化學鍵合的方式應用于材料中,極易從終產(chǎn)品中釋放出來進入環(huán)境。水體是OPFRs的一個重要匯。通過污水管網(wǎng),大量的OPFRs被排放到水中。越來越多的研究表明,部分OPFRs(如磷酸三苯酯TPHP、磷酸三(1,3-二氯異丙基)酯TDCIPP、磷酸三(丁氧基乙基)酯TBOEP等)對魚類等水生生物具有明顯的發(fā)育毒性、心臟毒性、神經(jīng)毒性和內(nèi)分泌干擾毒性。污染物在生物體內(nèi)的累積和代謝過程,是評價其毒性效應和生態(tài)風險的重要指標。然而國內(nèi)外關于OPFRs在魚體中的累積和代謝研究還很少。本研究以斑馬魚等為受試生物調查考察了 7種典型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和磷酸三對甲苯酯p-TCP)在斑馬魚體內(nèi)的累積、分布、凈化和生物代謝轉化過程。并采用模型擬合、理論計算和分子對接,探究不同結構OPFRs在魚體中的累積和代謝轉化機制。主要研究內(nèi)容和結果如下:(1)結合超聲萃取、凝膠滲透色譜儀、固相萃取小柱、氣相色譜-質譜聯(lián)用儀(GC-MS)和液相色譜-質譜聯(lián)用儀(LC-MS/MS和LC-Q-TOF MS),建立了生物體內(nèi)有機磷酸酯阻燃劑及其代謝產(chǎn)物的分析方法。(2)通過水相暴露,測定了 7種不同結構的OPFRs在斑馬魚不同組織(腦、腸、腮、肝、卵和肌肉)中的差異富集過程。結果表明相對于其他OPFRs,芳基OPFRs(TPHP和p-TCP)趨向于富集于腮和腸中;烷基(TPRP和TNBP)、烷氧基(TBOEP)和短鏈氯代烷基(TCEP)OPFRs趨向于富集于卵、腦、肝和肌肉組織;而多氯代(TDCIPP)OPFRs更趨向于累積在肌肉中。TDCIPP、TPHP和p-TCP在卵中的富集量較高,且半衰期長,表明這三種OPFRs有累積于斑馬魚卵中傳遞到下一代的趨勢。通過OPFRs的logKow或魚組織脂肪含量對生物富集系數(shù)作圖,發(fā)現(xiàn)隨著logKow或者組織脂肪含量的增加,OPFRs的富集系數(shù)逐漸增加。TPHP和p-TCP的脂肪標化富集系數(shù)(BCFlw)最高能達到5474和8909,表明這兩種OPFRs從脂肪重量角度評價,具有較大的生物富集潛力。通過測試這7種OPFRs磷酸二酯代謝產(chǎn)物,并運用魚體生理基礎的代謝動力學模型(PBTK模型)發(fā)現(xiàn)代謝過程使得TDCIPP、TPHP和TNBP在斑馬魚肝臟中的累積量分別減少了 42.3%、13.7%和3.4%左右,表明代謝過程對OPFRs在斑馬魚體內(nèi)的富集過程具有重要影響。而扣除代謝影響后剩下的累積量,分別占TDCIPP、TPHP和TNBP在斑馬魚肝臟中的57.7%、86.3%和96.6%,反過來表明,OPFRs在水體和魚體間的分配作用仍然占富集過程的主導地位。(3)利用高分辨LC-Q-TOF MS篩選了斑馬魚肝臟中OPFRs的代謝產(chǎn)物。共檢測出20種代謝產(chǎn)物,分別包括水解二酯產(chǎn)物、羥基化二酯產(chǎn)物、羥基化產(chǎn)物和葡萄糖醛酸結合產(chǎn)物�；诖x產(chǎn)物提出了 OPFRs在斑馬魚肝臟中的代謝路徑,并用分子前線軌道理論解釋了代謝的分子基礎。分析代謝產(chǎn)物在斑馬魚組織中的分布,發(fā)現(xiàn)肝臟和腸道是OPFRs代謝的重要器官,腦、卵和肌肉可能不能代謝OPFRs。水體中檢測到的大量二酯和羥基化二酯產(chǎn)物表明,代謝產(chǎn)生的大量二酯和羥基化二酯產(chǎn)物易于排放到水體中,其潛在的環(huán)境效應值得我們關注。(4)研究了魚體肝臟或腦中重要代謝酶活性(包括CYP450酶(ECOD表征)、羧酸酯酶(CESE)、谷胱甘肽轉移酶(GST)、乙酰膽堿酯酶(ACHE)和可能存在的磷酸三酯酶(PTE)),隨7種OPFRs暴露后的響應變化。結果發(fā)現(xiàn)7種OPFRs在設計暴露濃度下對PTE酶活性沒有顯著影響。但是TPHP和p-TCP對CESE從第3天開始表現(xiàn)出持續(xù)的顯著性抑制。同源模建和分子對接結果表明,抑制作用可能是TPHP或p-TCP與CESE的Tyr 407(酪氨酸)的疏水相互作用、Glu 404(谷氨酸)的靜電相互作用和Arg 371(精氨酸)的氫鍵相互作用造成。本研究揭示OPFRs在魚體中的富集、分布和代謝過程及其作用機制,為研究OPFRs在魚體中的毒性效應提供了數(shù)據(jù)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.
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：X592;X174

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