十溴联苯乙烷肝毒性及肝代谢机制初步研究

发布时间：2018-06-27 20:55

本文选题：十溴联苯乙烷 + 肝毒性　；参考：《中国人民解放军军事医学科学院》2012年博士论文

【摘要】：【目的】十溴联苯乙烷（Decabromodiphenyl ethane,DBDPE）作为多溴联苯醚类（polybrominated diphenyl ethers，PBDEs）阻燃剂的替代品于上世纪90年代进入市场，由于其高分子量和低脂溶性，在很长一段时间内被认为难以释放到环境中、难以被生物降解和利用。2003年，DBDPE首次在淤泥中被检出，随后被从室内空气、污水等介质和生物体内检出，表明DBDPE与其结构类似物十溴联苯醚（Decabromodiphenyl ether，BDE-209）一样，可以进入环境，并可在环境、食物链以及生物体内发生蓄积。相关研究显示，DBDPE人群环境暴露水平持续增加，人体蓄积水平呈现较快增加趋势。因此有必要开展DBDPE对生物体的潜在健康危害研究。目前针对DBDPE的毒理学评价研究开展的比较少。Hardy等开展的啮齿类动物和水生生物毒性研究表明，DBDPE难以被生物体降解，健康危害风险水平低。Nakari等采用水生生物开展的毒性研究表明，DBDPE可被水生生物降解，并对水生生物具有急性毒性、雌激素样作用及生殖毒性，但其实验设计存在一定的缺陷，研究中采用甲苯作为溶剂。此外，目前对DBDPE的代谢和毒作用机制也尚不清楚，而DBDPE的结构类似物PBDEs可作用于机体内分泌系统相关受体，干扰机体内分泌系统和代谢平衡。基于已开展研究之间的结果差异和DBDPE结构类似物的毒理学特征、毒作用机制，结合肝代谢在溴化阻燃剂（bromoniated flame retardants，BFRs）毒性作用中的关键作用，本研究开展了DBDPE的肝毒性和肝代谢机制研究，以期解决目前研究中存在的问题，获得准确的实验数据，为开展进一步研究提供可靠的研究基础和指明方向。【内容和方法】参考DBDPE结构类似物BDE-209的实验设计，本研究首先选用人肝癌细胞株HepG2细胞作为实验对象；采用0-100mg/L DBDPE作为HepG2细胞染毒剂量，选择二甲基亚砜（dimethyl sulfoxide，DMSO）作为DBDPE溶剂配制系列染毒液，DMSO在染毒液中浓度保持0.5%(V/V)；染毒时间为24h、48h和72h。染毒结束后，采用噻唑蓝（3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide，MTT）实验和L-乳酸脱氢酶（L-Lactic dehydrogenase，LDH）实验分别对细胞存活率和细胞损伤情况进行测定；采用Hoechst33258染料对染毒后细胞染色，倒置荧光显微镜下观察、记录细胞变化和损伤程度；采用碘化丙啶（Propidium Iodide，PI）染色-流式细胞仪检测方法测定细胞凋亡情况；为探究细胞损伤和细胞凋亡机制，本研究测定了染毒后活性氧自由基（reactiveoxygen species，ROS）含量；为验证ROS与细胞损伤和细胞凋亡的关系，染毒前在细胞培养基中加入ROS清除剂N-乙酰半胱氨酸（N-acetyl-L-cysteine，NAC），染毒结束后，采用MTT实验和PI染色-流式细胞仪检测方法分别测定细胞存活率和凋亡情况，分析NAC加入前后ROS生成、细胞存活率和凋亡变化情况，以验证ROS与细胞损伤的关系。本研究选择Wistar大鼠作为染毒对象，选择购自美国雅宝公司的商品化DBDPE产品（DBDPE纯度≥98.5%）作为染毒化合物，参照已开展研究实验设计，设置染毒剂量为0-1000mg/kg/d，选择经口染毒方式；染毒28天后，测定Wistar大鼠体重、肝脏重量、脏器系数和肝脏功能性损伤生化指标，探讨DBDPE对肝脏的损伤情况；考虑到细胞毒性研究中DBDPE可以诱导HepG2细胞ROS含量增加，对相关的谷胱甘肽（Glutathione，GSH）、谷胱甘肽过氧化物酶（Glutathione peroxidase，GSH-Px）、丙二醛（Malondialdehyde，MDA）、总超氧化物歧化酶（Total superoxide dismutase，T-SOD）等氧化损伤指标进行了测定，以期在动物水平验证氧化损伤与肝脏毒性的关系。此外，本研究采用实时荧光定量聚合酶链式反应（Real time-Polymerase Chain Reaction，RT-PCR）技术对不同染毒剂量组的大鼠肝脏多种相关的细胞色素P450酶（cytochrome P450，CYP450）在mRNA水平进行了测定；进而采用Western blot实验对mRNA水平发生显著改变的CYP450酶在蛋白水平进行了测定；采用超高速离心技术获得大鼠肝脏微粒体，对CYP2B酶对应的PROD（pentoxyresorufin O-dealkylation）、CYP3A酶对应的LBD （Luciferin benzylether debenzylase）和尿苷二磷酸葡萄糖醛酸转移酶（Uridinediphosphate-glucuronosyltransferase，UDPGT）活性进行了测定，以分析和推断DBDPE在肝脏中的代谢情况和作用机制。【结果】在HepG2细胞毒性研究实验部分，利用0-100.0mg/L DBDPE对HepG2细胞染毒24h、48h和72h，MTT实验、LDH实验和细胞形态学观察实验表明，DMSO对细胞存活率、细胞损伤程度以及Hoechst33258染色细胞形态学改变的影响与对照组之间无显著性差异；0-6.25mg/L剂量染毒24h、48h和72h后细胞存活率、细胞损伤程度以及细胞形态学改变与对照组相比无显著性差异；12.5-100.0mg/L剂量染毒48h和72h可降低细胞存活率、增加细胞损伤程度和引起细胞形态学显著改变，具有明显的时间和剂量-反应关系；PI染色-流式细胞仪检测发现，12.5-100mg/L剂量DBDPE可诱导HepG2细胞凋亡，存在时间和剂量-反应关系；研究还发现，DBDPE可诱导HepG2细胞ROS生成量增加，通过NAC验证试验，证实DBDPE诱导的细胞凋亡和损伤与ROS有关。在DBDPE对Wistar大鼠染毒动物毒性研究中，采用0-1000mg/kg剂量DBDPE对Wistar大鼠连续经口染毒28天后发现，DBDPE染毒后Wistar大鼠体重、肝脏重量、脏器系数等指标与对照组相比，无显著性差异；血清学检测发现，较高剂量组DBDPE可诱导雄性Wistar大鼠乳酸脱氢酶（lactate dehydrogenase，LDH）、谷丙转氨酶（Glutamic pyruvic transaminase，ALT）、谷草转氨酶（Aspartatetransaminase，AST）、胆汁酸（Total bile acid，TBA）、总胆红素（total bilirubin，TBA）和葡萄糖（Glucose，Glu）的显著改变，部分剂量组还可诱导γ-谷氨酰转移酶（glutamyl transpeptidase，GGT）、血浆总蛋白（Total protein，TP）、甘油三酯（Triglyceride，TG）、尿素氮（urea nitrogen，UN）和肌酐（Creatinine，Cr）的显著改变；此外，较高剂量组DBDPE还可诱导雌性Wistar大鼠碱性磷酸酶（Alkaline phosphatase，ALP）、AST和Glu的显著改变，部分剂量组还可诱导TBA、Cr和TG的显著改变。此外，中高DBDPE染毒剂量组GSH水平与对照组存在显著性差异，结合DBDPE可致HepG2细胞ROS生成量增加，提示DBDPE可能致肝脏发生氧化损伤。上述结果表明， DBDPE对大鼠具有肝毒性，可引起肝损伤，还可影响大鼠胆汁排泄等功能和正常糖等的代谢，且相关研究结果提示DBDPE可能干扰肝脏脂肪和蛋白的代谢，分析DBDPE可能具有一定的内分泌干扰作用，可能通过干扰机体内分泌途径，启动机体某些信号通路，干扰机体正常代谢功能；还可能通过氧化损伤等作用引起肝损伤，进而引起代谢功能受损。 DBDPE对Wistar大鼠染毒后，肝CYP450代谢酶mRNA、蛋白和酶活性检测发现，染毒组CYP1A1/2mRNA与对照组相比无显著性差异，提示DBDPE可能具有较低或无二VA英样毒性作用；CYP2B1和CYP3A1/3mRNA与对照组相比无显著性差异；雄性大鼠CYP2B2、CYP3A2在三个水平上，较高剂量组与对照组相比存在显著差异；雌性大鼠CYP2B2、CYP3A2在三个水平上，个别剂量组与对照组相比存在显著差异；UDPGT活性检测表明，较高剂量组DBDPE对雄性大鼠UDPGT活性具有诱导作用，并具有一定的剂量反应关系；仅500mg/kg.d剂量组DBDPE对雌性大鼠UDPGT活性的影响与对照组存在显著性差异。据此推测DBDPE对Wistar大鼠代谢酶的影响存在一定的性别差异，对雄性Wistar大鼠影响较大；分析认为DBDPE可能通过激活组成型雄烷受体（constitutiveandrostane receptor，CAR）和孕烷X受体（pregnane xenobiotic receptor，PXR）信号通路，进而诱导肝脏Ⅰ相和Ⅱ相代谢酶对DBDPE进行代谢以及干扰Wistar大鼠内分泌系统，影响Wistar大鼠体内正常代谢稳态，发挥毒性作用。【结论】DBDPE具有一定的肝毒性，ROS和氧化损伤分别在肝细胞毒性和大鼠肝损伤中发挥重要作用；DBDPE可能具有较低或无二VA英样毒性作用；DBDPE可通过CAR和PXR信号通路诱导大鼠肝代谢酶活性；DBDPE还可能通过CAR/PXR信号通路干扰内源性活性物质的动态平衡，具有一定的内分泌干扰活性；DBDPE对大鼠的毒性作用具有一定的性别差异，雄性大鼠较雌性大鼠敏感。
[Abstract]:[Objective] ten brominated diphenyl ethane (Decabromodiphenyl ethane, DBDPE), as a substitute for polybrominated diphenyl ethers (polybrominated diphenyl ethers, PBDEs), entered the market in the 90s. Due to its high molecular weight and low fat solubility, it is considered difficult to be released into the environment for a long time and is difficult to be biodegraded and difficult to be biodegraded. Using.2003, DBDPE was detected in silt for the first time, and then detected from indoor air, sewage and other media and organisms, indicating that DBDPE and its structural analogues, ten brominated diphenyl ethers (Decabromodiphenyl ether, BDE-209), can enter the environment and accumulate in the environment, food chain and living organisms. Related studies show DBDPE people. The exposure level of the group environment continues to increase, and the accumulation level of human body is increasing rapidly. Therefore, it is necessary to carry out the research on the potential health hazards of DBDPE to the organism. At present, the study of rodents and aquatic biotoxicology conducted on the toxicological evaluation of DBDPE has shown that DBDPE is difficult to be degraded by organisms. The toxicity study of aquatic organisms, such as low levels of health hazard risk and low.Nakari, shows that DBDPE can be degraded by aquatic organisms and has acute toxicity, estrogen like action and reproductive toxicity to aquatic organisms, but the experimental design has some defects. In the study, toluene is used as a solvent. In addition, the metabolism and toxicity of DBDPE are present. The mechanism is still unclear, and the structural analogues of DBDPE, PBDEs, can act on the body's endocrine system related receptors, interfere with the body's endocrine system and metabolic balance. Based on the differences between the results of the studies and the toxicological characteristics of the DBDPE structural analogues, the mechanism of toxic action, and the combination of liver metabolism in the brominated flame retardant (bromoniated flame re) Tardants, BFRs) the key role of toxicity, this study carried out the study of hepatotoxicity and liver metabolism mechanism of DBDPE, in order to solve the problems existing in the present study, to obtain accurate experimental data, and to provide a reliable research basis and direction for further research.
[content and method] referring to the experimental design of the DBDPE structure analogues BDE-209, this study first selected human hepatoma cell line HepG2 cells as the experimental object. 0-100mg/L DBDPE was used as the dose of HepG2 cells, and two methyl sulfoxide (dimethyl sulfoxide, DMSO) was selected as DBDPE solvent to prepare a series of dye solution, and DMSO in the toxic liquid. After the exposure time was 0.5% (V/V); after the exposure time was 24h, 48h and 72h. were finished, the test of thiazolium (3- (4,5) -dimethylthiahiazo (-z-y1) -3,5-di-phenytetrazoliumromide, MTT) and L- lactate dehydrogenase (L-Lactic) experiment were used to determine the cell survival rate and cell damage, respectively. The cell apoptosis and the cell apoptosis were measured by Propidium Iodide (PI) staining and flow cytometry, and the mechanism of cell damage and apoptosis was investigated. The reactive oxygen free radicals (reactiveoxygen specie) were measured in this study. S, ROS) content; to verify the relationship between ROS and cell damage and cell apoptosis, N- acetylcysteine (N-acetyl-L-cysteine, NAC) was added to the cell culture medium before exposure to N- acetylcysteine (N-acetyl-L-cysteine, NAC). The cell survival rate and apoptosis were measured by MTT test and PI staining flow cytometry, and the ROS before and after NAC addition was analyzed. Generation, cell viability and apoptosis were examined to verify the relationship between ROS and cell injury.
In this study, Wistar rats were selected as infected subjects and selected from the commercialized DBDPE product (DBDPE > 98.5%) purchased from the American company of Ya Bao as a toxic compound. According to the research experiment design, the dosage was set to 0-1000mg/kg/d, the way of oral exposure was selected, and the weight of Wistar rats, liver weight and organs were measured after 28 days of infection. Coefficient and biochemical indexes of liver functional injury to investigate the damage of DBDPE to the liver. Considering that DBDPE can induce the increase of ROS content in HepG2 cells in the study of cytotoxicity, related glutathione (Glutathione, GSH), glutathione peroxidase (Glutathione peroxidase, GSH-Px), malondialdehyde (Malondialdehyde, MDA), and total hyper oxidation The Total superoxide dismutase (T-SOD) and other oxidative damage indexes were measured in order to verify the relationship between oxidative damage and liver toxicity at animal level. In addition, the real-time fluorescent quantitative polymerase chain reaction (Real time-Polymerase Chain Reaction, RT-PCR) technique was used in the rat liver of different dose groups. A variety of related cytochrome P450 enzymes (cytochrome P450, CYP450) were measured at the mRNA level, and then the CYP450 enzyme in the Western blot test, which significantly changed the level of mRNA, was measured at the protein level; the rat liver microsomes were obtained by ultra high speed centrifugation, and PROD (pentoxyresorufin) for CYP2B enzyme was obtained. Ylation), the activity of the LBD (Luciferin benzylether debenzylase) and the uridine two phosphate glucuronotransferase (Uridinediphosphate-glucuronosyltransferase, UDPGT) corresponding to the CYP3A enzyme was measured to analyze and infer the metabolism and mechanism of DBDPE in the liver.
[results] in the experimental part of HepG2 cytotoxicity study, the effects of 0-100.0mg/L DBDPE on 24h, 48h and 72h, MTT, LDH and cell morphology showed that the effect of DMSO on cell survival, the degree of cell damage and the morphological changes of Hoechst33258 staining cells had no significant difference between the control group and the control group; 0 There was no significant difference in cell survival rate, degree of cell damage and morphological changes after -6.25mg/L dose of 24h, 48h and 72h, compared with the control group. 12.5-100.0mg/L dose of 48h and 72h can reduce cell survival rate, increase the degree of cell damage and cause morphological changes of cells, and have obvious time and dose reaction. PI staining flow cytometry showed that 12.5-100mg/L dose DBDPE could induce apoptosis of HepG2 cells, and there was time and dose response relationship. The study also found that DBDPE could induce the increase of ROS production in HepG2 cells, and confirmed the apoptosis and damage induced by DBDPE induced by NAC, and that the apoptosis and injury induced by DBDPE were related to ROS.
In the study of the toxicity of DBDPE to Wistar rats, the 0-1000mg/kg dose of DBDPE was used for 28 days after continuous oral exposure to Wistar rats. There was no significant difference in weight, liver weight, organ coefficient and other indexes of Wistar rats after DBDPE exposure. The serological test found that the higher dose group DBDPE could induce male Wistar. Lactate dehydrogenase (LDH), Glutamic pyruvic transaminase (ALT), cereal grass transaminase (Aspartatetransaminase, AST), bile acid (Total bile acid), total bilirubin and grape sugar, and a partial dose group can also induce gamma glutamyl transferase (gamma glutamyl transferase) Utamyl transpeptidase, GGT), the significant changes in plasma total protein (Total protein, TP), triglyceride (Triglyceride, TG), urea nitrogen (urea nitrogen, UN) and creatinine. It can also induce significant changes in TBA, Cr and TG. In addition, there is a significant difference between the GSH level of the medium high DBDPE dose group and the control group, and the combination of DBDPE can increase the ROS production of HepG2 cells, suggesting that DBDPE may cause oxidative damage in the liver. The results show that DBDPE has liver toxicity in rats, can cause liver damage, and can also affect rat bile. The metabolism of Juice Excretion and normal sugar, and the related results suggest that DBDPE may interfere with the metabolism of liver fat and protein, and the analysis of DBDPE may have certain endocrine disrupting effects. It may disturb the body's endocrine pathway, start some signal pathways and interfere the normal metabolic function of the body; it may also be oxidized by oxidation. Injury and other effects cause liver injury, which can lead to impaired metabolic function.
The detection of CYP450 metabolic enzyme mRNA, protein and enzyme activity of liver CYP450 after exposure to DBDPE in Wistar rats showed that there was no significant difference between the CYP1A1/2mRNA and the control group, suggesting that DBDPE might have lower or no two VA like toxicity, CYP2B1 and CYP3A1/3mRNA had no significant difference compared with the control group, and the male rats CYP2B2, CYP3A2 were three On the level, there was significant difference in the high dose group compared with the control group; the female rats CYP2B2, CYP3A2 at three levels, the individual dose group had significant difference compared with the control group; the UDPGT activity test showed that the higher dose group DBDPE had the induction of UDPGT activity in the male rats, and had a certain dose response relationship; only 500mg/. The effect of kg.d dose group DBDPE on the activity of UDPGT in female rats was significantly different from that of the control group. Accordingly, it was suggested that there was a certain gender difference in the effect of DBDPE on the metabolic enzymes of Wistar rats, which had great influence on the male Wistar rats. It was suggested that DBDPE may be activated by the activation of the constituent male alkane receptor (constitutiveandrostane receptor, CAR). The X receptor (pregnane xenobiotic receptor, PXR) signaling pathway induces the metabolism of DBDPE and the metabolic enzymes of phase I and II phase of the liver and interferes with the endocrine system of Wistar rats, which affects the normal metabolic homeostasis in Wistar rats and plays a toxic role.
[Conclusion] DBDPE has a certain liver toxicity, ROS and oxidative damage play an important role in hepatotoxicity and rat liver injury, respectively; DBDPE may have low or no two VA like toxic effects; DBDPE can induce rat liver metabolic enzyme activity through CAR and PXR signaling pathways; DBDPE may also interfere with the CAR/PXR signaling pathway. The dynamic balance of the source active substance has certain endocrine disrupting activity, and the toxic effect of DBDPE on rats has a certain sex difference, and the male rats are more sensitive than the female rats.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R114

【参考文献】