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题名: 渔业产品中新型溴代阻燃剂的生物累积及其细胞毒理学效应
作者: 胡小钟
答辩日期: 2008-03-14
导师: 徐盈
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: PBDEs ; HBCDs ; 饲料 ; 鮰鱼 ; 同位素稀释 ; 基质固相分散 ; 气质联用法 ; 液质联用法 ; 生物累积 ; 细胞凋亡 ; 氧化损伤 ; 复合效应
其他题名: Bioaccumulation of new-style brominated flame retardants in fishery products and their cytotoxicological effects
摘要: 多溴联苯醚(polybrominated diphenyl ethers,PBDEs)和六溴环十二烷(hexabromocyclododecanes,HBCDs)是两类新型溴代阻燃剂,被广泛地应用在电子、电器、化工、交通、纺织、石油等领域中。目前,在越来越多的环境介质中都发现有它们的存在,因此它们被认为是普遍存在的环境污染物。渔业产品(渔业饲料和鱼)与人类生活密切相关,尤其鱼类是人类获取蛋白质的重要来源之一,其卫生安全状况直接关系到人体健康。国外已有许多文献报道,鱼体内容易累积高浓度的持久性有毒污染物PBDEs和HBCDs。但有关PBDEs和HBCDs在鱼体内的浓度分布与生物累积特征的研究报道在我国尚处在起步阶段。此外,关于PBDEs和HBCDs的毒理学研究也是当前国际上研究的一大热点,目前有关PBDEs和HBCDs的毒性及其毒理学机制也还不十分清楚。 本论文选择鮰鱼饲料和鮰鱼为研究对象,建立了渔业饲料及鱼体内PBDEs和HBCDs定性定量检测方法,并用所建立的方法探查了湖北地区鮰鱼饲料中PBDEs和HBCDs的污染水平,研究了PBDEs和HBCDs在鮰鱼中的浓度水平与生物累积。在获得分析结果的基础上,开展体外细胞毒理学试验,对PBDEs和HBCDs单一及复合暴露的细胞毒性进行了评价。获得的主要研究结果如下: 1、建立了渔业产品中PBDEs的GC/MS定性定量检测方法,样品经过索氏提取,固相萃取净化,结合GC/MS法分析可同时测定渔业饲料和鱼体内13种PBDEs同系物(PBDE-28,PBDE-47,PBDE-66,PBDE-100,PBDE-99,PBDE-85,PBDE-154,PBDE-153,PBDE-138,PBDE-183,PBDE-197,PBDE-207和PBDE-209)。研究结果表明,该方法不仅适合于渔业饲料和鱼体内的PBDEs定量分析,而且检测结果显示,我国渔业饲料受到了一定程度的PBDEs污染,主要以PBDE-47,PBDE-154,PBDE-153,PBDE-183为主;配对的渔业饲料与鱼体中PBDEs的浓度之间有显著的相关性(R2 = 0.6162,P<0.001,n=25),说明PBDEs可能借助于渔业饲料的生物传递导致了渔业产品的污染。 2、研究了鮰鱼体内HBCDs异构体的浓度分布与生物累积特征。采用基质固相分散法,同位素稀释定量,HPLC-ESI-MS/MS检测技术,建立了渔业饲料和鱼体中的HBCDs三种非对映异构体,即α-,β-,和γ-HBCD的分析方法。研究结果表明,我国渔业饲料受到了一定程度的HBCDs污染,且主要以异构体α-HBCD为主;配对的渔业饲料与鱼体内中HBCDs的浓度之间有显著的相关性(R2 = 0.8774,P<0.001,n=25),说明HBCDs可能借助于渔业饲料的生物传递导致了渔业产品的污染。 3、以Hep G2细胞为模型,研究了PBDE-209 对Hep G2细胞的毒性作用及其机理。结果表明:PBDE-209能抑制Hep G2的生长,其抑制细胞生长,与促使LDH释放,改变细胞的形态和周期,并诱导细胞凋亡相关联。它诱导了胞内ROS含量增加,而ROS的清除剂NAC可逆转PBDE-209对细胞的存活率影响和所致胞内ROS增加,这表明PBDE-209抑制细胞生长,其作用机理为诱导产生胞内ROS、并介导细胞凋亡。 4、PBDE-47能抑制Hep G2细胞的生长,其抑制细胞生长,与改变细胞的形态,并诱导细胞凋亡相关联。此外,PBDE-47诱导了NOS的活性增加,并诱发细胞胞内外产生NO,而NO的清除剂PTIO可逆转PBDE-47对细胞的存活率和凋亡的影响。这表明PBDE-47抑制细胞生长,其作用机理为诱导产生胞内外NO、并介导细胞凋亡。 5、HBCDs可以抑制Hep G2细胞的生长,其抑制细胞生长,与促使LDH释放,改变细胞的形态,并诱导细胞凋亡相关联。此外,HBCDs诱导了NOS的活性增加,并诱发细胞胞内外产生NO,同时HBCDs也诱导了胞内产生ROS,而ROS的清除剂NAC或NO的清除剂PTIO可逆转HBCDs对细胞的作用,这表明,NO和ROS共同介导了HBCDs诱导的细胞凋亡。 6、PBDE-209,47,183,和HBCDs复合暴露的试验结果表明:它们中两者或三者共存时对细胞的凋亡作用较单一暴露时更加显著;并且具有明显的剂效正相关性;它们这种相互促进细胞凋亡的复合效应属于加合效应。其作用机理为通过氧化应激破坏细胞中酶的抗氧化能力,导致过量NO和ROS产生,从而致使细胞凋亡。
英文摘要: Polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs) belonging to the class of brominated flame retardants (BFRs) have been used industrially in large volumes for flame protection purposes in various commercial products such as electronic equipment, chemical industry, traffic, textiles and petroleum. Currently, concentrations of PBDEs and HBCDs are increasing for many environmental compartments such as both biotic and abiotic samples, and they are considered ubiquitous environmental contaminations. Fishery products including fish feeds and fish are closed related with human living, especially fish is one of the important protein source for human being. Some literatures report that fish can cumulate high concentrations PBDEs and HBCDs. However, there are few reports for distribution and bioaccumulation characteristic of PBDEs and HBCDs in fish in China. At present many literatures are focusing on the toxicological effects of PBDEs and HBCDs, however, it is not still clear about the mechanism of toxicological effects of PBDEs and HBCDs. The important biotic samples, channel catfish feeds and channel catfish are selected in the paper, and the determining methods of PBDEs and HBCDs in the products are established. Using the established methods, the contamination levels of PBDEs and HBCDs in some fish feeds from Hubei province of China are explored, and distribution and bioaccumulation characteristic of PBDEs and HBCDs in fishery products are studied. Based the results of PBDEs and HBCDs in fish products, the paper is designed to determine the anti-proliferative, apoptotic properties of PBDEs, and HBCDs, single and in combination exposed to a human hepatoma Hep G2 line. A method based on gas chromatography-mass spectrometry (GC-MS) has been implemented to determine thirteen PBDE congeners (PBDE-28, PBDE-47, PBDE-66, PBDE-100, PBDE-99, PBDE-85, PBDE-154, PBDE-153, PBDE-138, PBDE-183, PBDE-197, PBDE-207 and PBDE-209) in fish feeds and fish. Soxhlet extraction and a cleanup procedure, which are efficient and convenient sample preparation for determination of PBDEs in fishery products, are used. The established method is suitable for the determination of PBDEs fish feeds at trace levels. PBDEs are detected in 11 of 55 fish feeds and 9 of 80 fish samples. The congeners of PBDEs, PBDE-47, PBDE-154, PBDE-153, and PBDE-183, are dominating in samples. Moderate Pearson correlation coefficients are obtained for PBDEs between fish feed samples and fish samples(R2 = 0.6162;P<0.001;n=25), which shows that PBDEs can be transferred to fish through fish feeds. Distribution and bioaccumulation characteristic of HBCDs in channel catfish are studied in this paper. Isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has firstly been implemented to determine three diastereoisomers α-, β- and γ-HBCD in fish feeds and fish. MSPD is used in this study. The established method is suitable for the determination of HBCDs and its diastereoisomers in fish feeds and fish at trace levels. HBCDs are detected in 8 of 55 fish feeds and in 7 of 80 fish samples. The congener of α-HBCD is dominating in samples. Moderate Pearson correlation coefficients are obtained for HBCDs between fish feed samples and fish samples(R2 = 0.8774;P<0.001;n=25), which shows that HBCDs can be transferred to fish through fish feeds. This study is firstly designed to determine the anti-proliferative, apoptotic properties of PBDE-209, using Hep G2 cells as a model system. The results show that PBDE-209 inhibits the cells viability in time and concentration-dependent characteristics at concentrations (10.0-100.0 mol/L). We find that anti-proliferative effect of PBDE-209 is associated with apoptosis on Hep G2 cells by determinations of lactate dehydrogenase (LDH) release, morphological changes, cell cycle and apoptosis. Mechanism study shows that PBDE-209 can increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine (NAC) partially inhibits the increase of ROS. The mechanism for its hepatoma-inhibitory effects is the induction of cellular apoptosis through ROS generation. This study is also designed to determine the anti-proliferative, apoptotic properties of PBDE-47, using Hep G2 cells. The results show that PBDE-47 inhibits the cells viability in time and concentration-dependent characteristics and induces cell apoptosis. Mechanism study shows PBDE-47 firstly induces the increase of nitric oxide synthase (NOS) activity, leading to the production of NO, and NO induces cell apoptosis since PTIO (NO scavenger) partially reverses the effects of PBDE-47 on Hep G2 cells. The same study is designed to determine the anti-proliferative, apoptotic properties of HBCDs, using Hep G2 cells. The results show that HBCDs inhibits the cells viability in time and concentration-dependent characteristics and induces cell apoptosis. Mechanism study shows that HBCDs firstly induces the increase of NOS activity, leading to the production of NO, which is sufficient to induce ROS formation. N-acetylcyteine (ROS scavenger) or PTIO (NO scavenger) partially reverses the effects of HBCDs on Hep G2 cells. It is concluded that HBCDs induces cell apoptosis by mediation of ROS and NO together. The final section in this paper is designed to determine apoptotic properties of PBDE-209, PBDE-47, PBDE-183 and HBCDs in combination, using Hep G2 cells. The results of the tests of complex exposure show that the two or three presences of the compounds also induce cell apoptosis in concentration-dependent characteristics, and the apoptosis is more marked than one of them alone. It is observed that the two or three presences of them promote cell apoptosis based on results of cell apoptosis rate, but the combined effect of the compounds together is summing effect. Mechanism study shows the compounds destroy antioxidising ability of enzymes in cells though oxidative stress, resulting in superfluous ROS and NO, and inducing cell apoptosis.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12162
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
渔业产品中新型溴代阻燃剂的生物累积及其细胞毒理学效应.胡小钟[d].中国科学院水生生物研究所,2008.20-25
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