【作者】 卢春凤；

【导师】 邹莉波； 彭双清；

【作者基本信息】 沈阳药科大学 ， 药理学， 2009， 博士

【摘要】 内分泌干扰物(endocrine disruptors,EDs)是我国广泛存在、危害效应十分严重的环境与食品污染物。目前,EDs已成为全世界广泛关注的安全卫生问题之一,特别是其联合暴露的复合效应更是令人担忧。2,3,7,8-四氯二苯并对二噁英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)和多氯联苯类(polychlorinated biphenyls,PCBs)化合物是典型的EDs,由于它们广泛分布并共存于环境中,具有可生物蓄积、难以降解、远距离迁移及高毒等特性,已成为环境毒理学研究的热点。但有关EDs的危险性认识及相关毒理学研究主要关注单一污染物的效应,且研究手段仍主要沿用传统的毒性评价方法,而对于它们联合暴露的复合效应还没有进行系统研究。基于我国多种EDs共存污染的现状,应用新技术开展对多种环境与食品污染物复合毒性效应研究具有十分重要的理论和现实意义。基于核磁共振(nuclear magnetic resonance,NMR)的代谢组学技术是近年来发展起来的新技术,已广泛地应用于毒理学研究。因此,本研究拟将代谢组学技术与传统毒性评价技术相结合,对我国环境污染中危害较严重的内分泌干扰物TCDD与PCBs联合暴露的复合毒性效应进行系统研究,探讨复合效应作用模式及作用机制,寻找复合效应的候选生物标志物,并探讨代谢组学技术在EDs复合效应研究中的应用前景。首先,建立针对EDs毒性研究的代谢组学技术平台。采用2×2析因设计将20只SD大鼠随机分为4组,即对照组、PCBs单独染毒组(Aroclor1254 10 mg/kg)、TCDD单独染毒组(TCDD 10μg/kg)和联合染毒组(TCDD 10μg/kg+Aroclor1254 10 mg/kg),每组5只。灌胃染毒,每天1次,连续3 d。用代谢笼收集各组大鼠染毒前、染毒过程中每天24 h的尿液,对其进行核磁共振氢谱(~1H NMR)分析,建立特征代谢谱,采用主成分分析法(principal component analysis,PCA)对结果进行统计分析。各组间进行的PCA分析结果显示,各样本基本集中分布于得分图的椭圆形(95%置信区内)的4个区域,各染毒组大鼠尿液代谢组轨迹能够明显地与对照组分开,单独与联合染毒组大鼠尿液代谢组轨迹也能明显分开,提示各组大鼠尿液的代谢组都发生了明显的改变。本研究表明,采用代谢组学方法能够很好地表征和描述TCDD与PCBs联合暴露导致的代谢变化,并能对其复合毒性效应进行说明,表明已成功地建立了基于~1H NMR的代谢组学技术平台。代谢组学方法的灵敏性优于传统毒性评价方法,该技术用于EDs复合效应研究具有良好的应用前景。在此基础上,本研究利用所建立的代谢组学技术平台,结合传统毒性评价方法对TCDD与PCBs联合暴露复合毒性效应进行了研究,观察联合暴露后大鼠尿液的代谢模式的变化及其与血液生化指标和组织病理学的相关性,寻找复合效应的候选生物标志物,探讨TCDD与PCBs联合暴露毒性效应机制。SD大鼠每天灌胃染毒1次,连续6 d。用代谢笼收集各组大鼠尿液,测定~1H NMR谱,并进行血清生化指标、组织病理学和组织氧化损伤检测。结果表明,各染毒组大鼠均呈现不同程度的毒性效应,表现为:体重下降、脏器系数与血清生化指标改变、组织病理学改变以及组织发生氧化损伤,这些毒性效应在联合染毒组表现更为明显。运用~1H NMR代谢组学技术研究TCDD与PCBs联合暴露对大鼠尿液代谢产物谱影响的结果表明:各组在不同染毒条件下的代谢模式可明显区分开,自染毒第一天起,各染毒组大鼠的代谢组就明显偏离对照组和染毒前的代谢组成分,且各染毒组的代谢组轨迹不同,提示其毒性作用机制可能不同。大鼠尿液~1H NMR谱改变及其相对位置与其毒性呈较强的对应关系。通过对两种评价方法结果的对比,可发现不同染毒代谢组的改变以及毒性发生发展过程中代谢组的改变均与常规毒性检测指标相符。尿样~1H NMR谱PCA分析结果表明,大鼠尿液中2-酮戊二酸、柠檬酸、琥珀酸、肌酸、乳酸、N-氧三甲胺(trimethylamine-N-oxide,TMAO)、马尿酸、2-羟异戊酸、牛磺酸、二甲胺(dimethylamine,DMA)、肌酐、葡萄糖等代谢产物水平在染毒后发生了改变,且联合暴露组的改变更明显。这些改变了的代谢成分可能成为研究复合毒性效应候选生物标志物。另外,提示联合暴露的毒性效应可能与线粒体功能受损、三羧酸循环的能量代谢异常以及葡萄糖、脂肪和氨基酸代谢紊乱有关。进而对TCDD和PCBs联合暴露复合效应的作用模式及作用机制进行研究。SD大鼠每天灌胃染毒1次,连续12 d。染毒结束后处死大鼠,取血清测定血液生化指标,取组织进行组织病理学检查,同时检测组织脂质过氧化和组织中细胞色素P450 1A1(cytochromeP450 1A1,CYP1A1)、热休克蛋白70(heat shock protein 70,HSP70)的表达情况。结果表明,TCDD与PCBs单独及联合染毒均引起明显的毒性效应,表现为:动物体重明显下降、脏器系数与血清生化指标改变、组织病理学改变以及组织发生氧化损伤、诱导组织CYP1A1和HSP70蛋白高表达,且这些毒性效应在联合染毒组表现更为明显。通过芳烃受体诱导组织CYP1A1高表达,导致氧化损伤在TCDD和PCBs复合毒性效应中可能发挥重要的作用。此外,组织中丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxidedismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxide,GSH-Px)、CYP1A1和HSP70蛋白可能成为TCDD与PCBs复合毒性效应的候选分子生物标志物。析因方差分析结果表明,TCDD与PCBs的复合效应很复杂,在本实验条件下,对于不同的毒性评价终点联合作用模式表现不一,既有相加作用,又有协同和拮抗作用。综合本研究结果,在本实验条件下,可以得到如下结论:①利用代谢组学技术可以区分不同毒性作用机制的代谢表型,代谢组学技术用于EDs复合效应研究具有良好的应用前景。②代谢组学分析结果与常规检测指标具有良好的相关性且具更多优势。③TCDD与PCBs单独及联合暴露均可引起大鼠毒性效应,联合暴露的复合毒性效应更显著。④TCDD与PCBs复合效应的作用模式复杂,主要表现为相加或协同作用。⑤利用代谢组学技术结合传统毒性评价方法筛选出了一些复合效应候选生物标志物。⑥通过芳烃受体诱导组织CYP1A1高表达,导致氧化损伤在TCDD和PCBs复合毒性效应中可能发挥重要的作用。另外,复合毒性效应可能还与线粒体功能受损、三羧酸循环中能量代谢异常以及葡萄糖、脂肪和氨基酸的代谢紊乱有关。更多还原

【Abstract】 Endocrine disruptors(EDs) are widely dispersed environmental and food contaminants in our country that poses a great risk to the public health.They have been one of the widespread current subjects for concern in health safety throughout the world,especially their combined effects of combined exposure.2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) and polychlorinated biphenyls(PCBs) are typical EDs.At present,they are one of the hot spots in the field of environmental toxicology because of their extensive disposition and coexistence in the environment,persistence,bioaccumulation,migration and high toxicity.But most researches on the toxicity and human health hazards of these EDs have focused primarily on toxicology studies conducted on individual compound,and traditional toxicity evaluating techniques remain mainstream,but the possible interactions of combined exposure to EDs have not been investigated systematically.However,considering the facts that humans are ubiquitously simultaneously exposed daily to complex mixtures of environmental contaminants in our country, assessment of the mixture effects of multiple pollutants with the new technique is of more theoretical and actual significance.Nuclear magnetic resonance(NMR)-based metabonomic approach is a newly developed technology that has been extensively applied in the studies of toxicology.Therefore,the present study was designed to assess the potential combined toxic effects of combined exposure to TCDD and PCBs with metabonomics combined with conventional toxicologic technologies,so as to explore the possible modes and mechanisms of combined effects and select the possible biomarkers of combined toxicity.The potential application of metabonomics in the combined effects study of EDs was explored.First,the platform,based on metabonomics technique,for EDs toxicity study was established.Twenty Sprague-Dawley rats were subjected to 2×2 factorial experimental design and assigned randomly into four groups of five rats each group,including the control group,the PCBs exposure group,the TCDD exposure group,and the combined-exposure group.Rats were intragastrically administered vehicle(olive oil),PCBs(Aroclor1254,10 mg/kg),TCDD(10μg/kg),or the combination(10μg/kg TCDD+10 mg/kg Aroclor1254),once a day for consecutive 3 days.Rats were housed in metabolism cages and the 24 hour urinary samples of each rat were collected before and after exposure and its hydrogen nuclear magnetic resonance (~1H NMR) spectra were measured,spectral characteristics for each group were summarized and the data were analyzed by principal component analysis(PCA).Based on the results from PCA in all groups,the samples were primarily distributed in four regions of the scoring oval(95% confidence intervals).The urine metabonomic trajectory of all exposure groups animals could obviously separated with that of control animals,and there were separated between the exposure to TCDD or PCBs group and the combined-exposure group,which suggested that all groups had a distinct metabonomic spectra.The results of this partial study suggest that metabonomic techniques can be used to characterize the metabolic consequence combined exposure to TCDD and PCBs,to elucidate the combined toxic effects and mechanism,that the platform based on metabonomics technique has been successfully established.Metabonomics technology can be proved more sensitive than traditional method for toxicity evaluation.Metabonomics is a promising new technology for the combined effects study of EDs.Secondly,using the metabonomics technique combined with conventional toxicologic technologies to investigate the potential combined toxic effects of combined exposure to TCDD and PCBs,to observe the relationship of the metabonomic pattern of rat urine with blood biochemical indices and histopathology and select the possible biomarker candidates of combined toxicity,to explore the toxic effect mechanism of combined exposure.SD rats were intragastrically administered TCDD,PCBs,or the combination daily for consecutive 6 days.The 24 hour urinary samples of each rat were collected before and after exposure and its nuclear magnetic resonance spectra were measured,the serum biochemical analysis,histopathology examination and tissue oxidative damage detection were taken.The results showed that all treatment groups produced different degree toxicities,as characterized by losses in body weight, changes in organ coefficients and serum biochemistry parameters,histological changes and tissue oxidative damage.Most of these effects were more remarkable in the combined-exposure group.The ~1H NMR spectroscopic and pattern recognition methods were used to detect alteration of the urine metabolite expression profile of combined exposure to TCDD and PCBs. Results showed that all treatment groups could be readily distinguished in the PCA analysis diagram without overlap,and just after exposure,the urine metabonomics trajectory biased from that of the controls or pre-exposure.It was well in agreement with the toxic injury differentia of differently treated rats that the metabonomic spectra of urine from these rats could also be distinguished easily from each other.The result of rat urine metabonomic pattern change was in accordance with that of traditional toxicity evaluation.The PCA analysis of urine ~1H NMR data showed that the levels of 2-oxoglutarate,citrate,succinate,creatine,lactate, trimethylamine-N-oxide(TMAO),hippurate,2-hydroxy-isovaleric acid,taurine,dimethylamine (DMA),creatinine and glucose of rats urine changed after exposure,and most of these changes were more conspicuous in the combined-exposure group.These changed metabolites may be considered biomarker candidates of the combined toxicity.Furthermore,the increase of the toxic effects induced by combined exposure to TCDD and PCBs are related to the injury of mitochondrial function,reductions energy metabolism in tricarboxylic acid cycle(TAC) and perturbations in the metabolism of glucose,lipid and amino acidsFinally,the possible modes and mechanisms of combined effects of TCDD and PCBs were investigated.SD rats were intragastrically administered TCDD,PCBs,or the combination daily for consecutive 12 days.After sacrificed rats,serum was used for detecting blood biochemical parameters,the tissues were used to histopathologic study,and tissue lipid peroxidation and the protein expression of cytochrome P450 1A1(CYP1A1),heat shock protein 70(HSP70) were determined.The consequence suggested that all rats of treatment groups showed significant toxicities,as evidenced by significant decreases in body weight,changes in organ coefficients and serum biochemistry parameters,and marked histological changes and tissue oxidative damage,furthermore,high expression of CYP1A1 and HSP70 in tissues.Most of these effects were more conspicuous in the combined-exposure group.These findings indicated that aryl hydrocarbon receptor-mediated the high expression of CYP1A1 elicited oxidative injuries may play an important role in the combined toxicity of TCDD and PCBs,and suggested that malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxide(GSH-Px), CYP1A1 and HSP70 in tissue may be considered molecular biomarker candidates of the combined toxicity.In addition,the result of the two-way analysis of variance revealed that the combined effects of TCDD and PCBs were complicated,being additive,synergistic or antagonistic depending on the selection of toxicity endpoints under the present experimental condition.Based on the results of this study,the following conclusions could be made under the present experimental condition:①The metabonomics spectra of urine could be distinguished readily by different toxic mechanism using metabonomics technique.Metabonomics is a promising new technology for the combined effects study of EDs.②It was in accordance with traditional measurements for evaluating toxicity,but metabonomics method was more sensitive than traditional measurements.③Exposure alone or combined exposure to TCDD and PCBs produced significant toxicities,and the toxic effects of combined exposure were more conspicuous.④The mode of combined effects of TCDD and PCBs were complicated,and showed primarily to be additive or synergistic.⑤Using the metabonomics technique combined with conventional toxicologic technologies had selected the possible biomarkers of combined toxicity.⑥The aryl hydrocarbon receptor-mediated the high expression of CYP1A1 elicited oxidative injuries may play an important role in the combined toxicity of TCDD and PCBs.In addition,the combined toxic effects are also related to the injury of mitochondrial function, reductions energy metabolism in TAC and perturbations in the metabolism of glucose,lipid and amino acids.更多还原