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烷基有机磷阻燃剂在鱼类体内代谢转化过程的研究
Alternative TitleStudy on metabolic process of alkyl organophosphorus flame retardants in fish
侯瑞
Subtype博士
Thesis Advisor王子健 ; 许宜平
2018-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
Keyword有机磷阻燃剂(opfrs),代谢过程,磷酸二酯(daps),基于生理的 毒代动力学模型(pbtk),体外代谢,细胞色素 P450(cyp)酶
Abstract

      随着多溴联苯醚的禁用,有机磷阻燃剂(OPFRs)在生活中被广泛用作阻燃剂,通常在各个环境基质中都有大量检出,成为一种新型有机污染物。众多研究表明,各类 OPFRs均具有生殖发育毒性、神经毒性、内分泌干扰效应和致癌性,其环境危害逐渐受到人们关注。 OPFRs在生物体内的积累和代谢过程,是研究其毒性机制和评价其生态风险的基础,但是这方面的研究工作仍旧比较缺乏,尤其是对于烷基有机磷阻燃剂( Alkyl-OPFRs)。基于此,本研究以稀有鮈鲫作为模式生物,通过体外代谢实验和活体暴露研究相结合的方法,研究了典型  Alkyl-OPFRs在稀有鮈鲫体内的代谢路径,分析了体外肝微粒体的代谢动力学过程及酶促作用机制,建立了Alkyl-OPFRs在稀有鮈鲫体内的代谢动力学模型,解释了主要的代谢产物的体内组织分布和消除过程;本研究进而调查了北京市河流中主要淡水鱼体内的OPFRs和磷酸二酯代谢产物(DAPs)的分布情况。研究成果包含以下几个方面:
(1)对样品前处理后,利用 UPLC-QTOF-MS对三种Alkyl-OPFRs在稀有鮈鲫肝脏中的代谢产物进行初步定性,筛选出了的主要代谢产物;包括 DAPs、羟基化产物( OH-OPFRs)和葡萄糖醛苷结合产物。本研究推断了其在鱼体内的主要代谢路径,即主要通过脱烷基化和羟基化进行I相代谢,并由葡萄糖醛苷结合进行II相代谢。
(2)Alkyl-OPFRs在鱼肝微粒体可被迅速代谢,其代谢符合 Michaelis-Menten模型,其中TBOEP和TNBP的清除速率(CLint)分别为   3.9和 3.1μl/min/mg  protein;但其无法被肠道微粒体代谢,说明肝脏是鱼体内代谢  Alkyl-OPFRs的主要器官。在鱼肝微粒体测定中,定量发现磷酸双(2-丁氧基乙基)羟乙基酯(BBOEHEP)和双(2-丁氧基乙基)3-羟基-2-丁氧基乙基磷酸酯(3-OH-TBOEP)是   TBOEP的最主要代谢产物,而3-羟基丁基磷酸酯(3-OH-TNBP)是 TNBP的主要代谢产物。通过   CYP酶抑制实验,证实CYP3A4是催化鱼肝微粒体中 TBOEP和  TNBP脱烷基和羟基化代谢过程的重要  CYP亚酶。
(3)通过活体暴露研究,发现  TBOEP和  TNBP主要分布在稀有鮈鲫的肝脏和肾脏组织中。本研究建立了两种化合物的基于生理的毒代动力学(PBTK)模型,描述了其体内代谢转化过程,其中肾脏具有最高的富集和分配系数。肝脏、肠道和肾脏是代谢产物主要分布的组织,BBOEP和 DNBP分别是 TBOEP和TNBP在体内各组织富集最多的代谢产物。虽然BBOEHEP是体外代谢最主要的代谢物,但其在体内为代谢中间产物,其在体内富集比BBOEP和3-OH-TBOEP低。通过计算代谢产物母体稳定因子(MPCF),发现与母体化合物相比主要代谢产物的富集能力有限。
(4)本研究调查了8种OPFRs及其4种DAPs代谢产物在中国北京周边地区不同摄食习性的三种淡水鱼类(麦穗鱼、鲫鱼和泥鳅)中的分布。研究发现,野生淡水鱼积累了相对较高的OPFRs(264.7-1973ng/g  lw),其中TNBP、TCEP、TCIPP和TEHP是主要富集的  OPFRs。而  DAPs在野生淡水鱼体内也有较高的富集,富集量为其母体化合物的0.10-1.12倍。与实验室研究相似,调查发现肝脏是野生淡水鱼类体内 OPFRs和   DAPs中生物积累最多的组织。这项研究强调了 DAPs作为OPFRs暴露的代谢产物的重要性,需要进一步的研究来验证这些代谢产物的毒性机制及其生态风险。

Other Abstract

      Organophosphate flame retardants (OPFRs) are one class of flame retardants that have  a  variety  of  applications  in  a  wide  range  of  products  after  the  phased  out Polybrominated diphenyl  ethers (PBDEs).  Several toxicological  studies have  shown that exposure to OPFRs  can potentially cause adverse reproductive  effects, endocrine disruptive   effects   and   systemic   effects    in   fish,   birds,   rodents   and   humans.
      Understanding  the  accumulation  and metabolism  of  OPFRs  in  the  body  can  give insight into the toxicology and  the ecological risk of OPFRs, especially for the Alkyl-OPFRs. Therefore, in this study, the rare minnow  (Gobiocypris rarus) was selected as a  model  fish  to investigate  the  metabolic  process  of  Alkyl-OPFRs  and  the tissue distribution and elimination of their primary metabolites. A  pilot field investigation of tissue distribution profile of  OPFRs and di-alkyl phosphate (DAPs) were  also studied in  dominant freshwater  fish collected  from  bodies of  water  in Beijing,  China.  The major results are as follows.
(1)  The  metabolic  process   of  Alkyl-OPFRs  in  liver  of   rare  minnow  were investigated  using  a  liquid  chromatography  coupled  to  a  quadrupoletime-of-flight mass   spectrometer    (UPLC-QTOF-MS),    after   cleanup    procedure.   The    major metabolites  of the  Alkyl-OPFRs were  DAPs,  the products  of  hydroxylation and  of glucuronide conjugation.  The phase  I metabolism  pathways of  Alkyl-OPFRs in  fish involving O-dealkylation and hydroxylation, and  the phase II metabolism pathways is the glucuronide conjugation.
(2) The  rapid metabolism  of Alkyl-OPFRs in  fish liver  microsomes were both best fitted  to  the Michaelis–Menten  model (at  administrated concentrations  ranging from  0.5  to  200  μM),  with a CLint  (intrinsic clearance)  of  3.9  and  3.1  μl/min/mg protein,   respectively   for   TBOEP   and    TNBP.  But   no   significant   (P >   0.05) biotransformation was  observed for  these compounds  in intestinal  microsomes. The results suggests  that the fish  liver make  a primary contribution to  the metabolism  of Alkyl-OPFRs. In   fish   liver   microsomes   assay,   bis(2-butoxyethyl)   hydroxyethyl phosphate (BBOEHEP)  and  bis(2-utoxyethyl) 3-hydroxyl-2-butoxyethyl  phosphate (3-OH-TBOEP)  were  the   most  abundant  metabolites   of  TBOEP,  and  dibutyl-3-hydroxybutyl    phosphate   (3-OH-TNBP)    was    the   predominant    metabolite    of TNBP.  CYP3A4 was the  significant  CYP450 isoforms catalyzing  the  formation of the hydroxylated and DAPs metabolites.
(3) After exposure, TBOEP and TNBP could  be mainly accumulated in liver and kidney  of rare  minnow.  A  physiologically based  toxicokinetic  (PBTK) model  was estabolished to  discribe the  metabolic process  of TBOEP and  TNBP. The  estimated partition coefficients between kidney and  blood were higher than those between other tissues and blood. The  metabolites were also accumulated more in  liver, intestine and kidney. BBOEP  and DNBP  were the  most aboundant  metabolites in  various tissues for  TBOEP and  TNBP,  respectively.  BBOEHEP,  the major  in  vitro  metabolite of TBOEP , was  lower accumulated than  BBOEP and 3-OH-TBOEP in  tissues of   fish.This  may because  BBOEHEP  was the metabolic  intermediates  of TBEOP  in  fish.Metabolite parent  concentration factor  (MPCF) were  calculated for  the metabolites.All the  metabolites reached  lower  accmulations than  their parents,  characterized by the low MPCF.
(4) The accumulation and distribution  of 8 OPFRs and their 4  DAPs metabolites were first investigated  in whole-body samples and various  tissues of three freshwater fish species (topmouth gudgeon,  crucian carp and loach) with different  feeding habits from locations around  Beijing, China. Accumulation  of OPFRs was relatively  higher in freshwater fish across all sampling locations (264.7-1973 ng/g lw), in which  TNBP,TCEP, TCIPP  and TEHP  were the  main  contributors to  the total  OPFR levels.  The DAPs concentrations  were observed  to be  relatively large in  fish, which  were 0.10-1.12  times of  OPFRs  concentrations. With  respect  to their  distribution  in  different tissues, both the parent OPFRs and  DAPs were found at relatively higher levels in the liver   than  in   other   tissues   (muscle,  intestine,   kidney   and   ovary).  This   study emphasizes  the  importance  of  monitoring  DAPs  in  biota,   and  future  study  need improve  the  understanding  of  the  toxicology  and  ecological  risk of    this  OPFRs metabolites.

Pages134
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/41459
Collection环境水质学国家重点实验室
Recommended Citation
GB/T 7714
侯瑞. 烷基有机磷阻燃剂在鱼类体内代谢转化过程的研究[D]. 北京. 中国科学院生态环境研究中心,2018.
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