本文選題：十溴聯(lián)苯乙烷 + 肝毒性　； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2012年博士論文

【摘要】：【目的】十溴聯(lián)苯乙烷（Decabromodiphenyl ethane,DBDPE）作為多溴聯(lián)苯醚類(lèi)（polybrominated diphenyl ethers，PBDEs）阻燃劑的替代品于上世紀(jì)90年代進(jìn)入市場(chǎng)，由于其高分子量和低脂溶性，在很長(zhǎng)一段時(shí)間內(nèi)被認(rèn)為難以釋放到環(huán)境中、難以被生物降解和利用。2003年，DBDPE首次在淤泥中被檢出，隨后被從室內(nèi)空氣、污水等介質(zhì)和生物體內(nèi)檢出，表明DBDPE與其結(jié)構(gòu)類(lèi)似物十溴聯(lián)苯醚（Decabromodiphenyl ether，BDE-209）一樣，可以進(jìn)入環(huán)境，并可在環(huán)境、食物鏈以及生物體內(nèi)發(fā)生蓄積。相關(guān)研究顯示，DBDPE人群環(huán)境暴露水平持續(xù)增加，人體蓄積水平呈現(xiàn)較快增加趨勢(shì)。因此有必要開(kāi)展DBDPE對(duì)生物體的潛在健康危害研究。目前針對(duì)DBDPE的毒理學(xué)評(píng)價(jià)研究開(kāi)展的比較少。Hardy等開(kāi)展的嚙齒類(lèi)動(dòng)物和水生生物毒性研究表明，DBDPE難以被生物體降解，健康危害風(fēng)險(xiǎn)水平低。Nakari等采用水生生物開(kāi)展的毒性研究表明，DBDPE可被水生生物降解，并對(duì)水生生物具有急性毒性、雌激素樣作用及生殖毒性，但其實(shí)驗(yàn)設(shè)計(jì)存在一定的缺陷，研究中采用甲苯作為溶劑。此外，目前對(duì)DBDPE的代謝和毒作用機(jī)制也尚不清楚，而DBDPE的結(jié)構(gòu)類(lèi)似物PBDEs可作用于機(jī)體內(nèi)分泌系統(tǒng)相關(guān)受體，干擾機(jī)體內(nèi)分泌系統(tǒng)和代謝平衡�；谝验_(kāi)展研究之間的結(jié)果差異和DBDPE結(jié)構(gòu)類(lèi)似物的毒理學(xué)特征、毒作用機(jī)制，結(jié)合肝代謝在溴化阻燃劑（bromoniated flame retardants，BFRs）毒性作用中的關(guān)鍵作用，本研究開(kāi)展了DBDPE的肝毒性和肝代謝機(jī)制研究，以期解決目前研究中存在的問(wèn)題，獲得準(zhǔn)確的實(shí)驗(yàn)數(shù)據(jù)，為開(kāi)展進(jìn)一步研究提供可靠的研究基礎(chǔ)和指明方向。 【內(nèi)容和方法】參考DBDPE結(jié)構(gòu)類(lèi)似物BDE-209的實(shí)驗(yàn)設(shè)計(jì)，本研究首先選用人肝癌細(xì)胞株HepG2細(xì)胞作為實(shí)驗(yàn)對(duì)象；采用0-100mg/L DBDPE作為HepG2細(xì)胞染毒劑量，選擇二甲基亞砜（dimethyl sulfoxide，DMSO）作為DBDPE溶劑配制系列染毒液，DMSO在染毒液中濃度保持0.5%(V/V)；染毒時(shí)間為24h、48h和72h。染毒結(jié)束后，采用噻唑藍(lán)（3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide，MTT）實(shí)驗(yàn)和L-乳酸脫氫酶（L-Lactic dehydrogenase，LDH）實(shí)驗(yàn)分別對(duì)細(xì)胞存活率和細(xì)胞損傷情況進(jìn)行測(cè)定；采用Hoechst33258染料對(duì)染毒后細(xì)胞染色，倒置熒光顯微鏡下觀察、記錄細(xì)胞變化和損傷程度；采用碘化丙啶（Propidium Iodide，PI）染色-流式細(xì)胞儀檢測(cè)方法測(cè)定細(xì)胞凋亡情況；為探究細(xì)胞損傷和細(xì)胞凋亡機(jī)制，本研究測(cè)定了染毒后活性氧自由基（reactiveoxygen species，ROS）含量；為驗(yàn)證ROS與細(xì)胞損傷和細(xì)胞凋亡的關(guān)系，染毒前在細(xì)胞培養(yǎng)基中加入ROS清除劑N-乙酰半胱氨酸（N-acetyl-L-cysteine，NAC），染毒結(jié)束后，采用MTT實(shí)驗(yàn)和PI染色-流式細(xì)胞儀檢測(cè)方法分別測(cè)定細(xì)胞存活率和凋亡情況，分析NAC加入前后ROS生成、細(xì)胞存活率和凋亡變化情況，以驗(yàn)證ROS與細(xì)胞損傷的關(guān)系。 本研究選擇Wistar大鼠作為染毒對(duì)象，選擇購(gòu)自美國(guó)雅寶公司的商品化DBDPE產(chǎn)品（DBDPE純度≥98.5%）作為染毒化合物，參照已開(kāi)展研究實(shí)驗(yàn)設(shè)計(jì)，設(shè)置染毒劑量為0-1000mg/kg/d，選擇經(jīng)口染毒方式；染毒28天后，測(cè)定Wistar大鼠體重、肝臟重量、臟器系數(shù)和肝臟功能性損傷生化指標(biāo)，探討DBDPE對(duì)肝臟的損傷情況；考慮到細(xì)胞毒性研究中DBDPE可以誘導(dǎo)HepG2細(xì)胞ROS含量增加，對(duì)相關(guān)的谷胱甘肽（Glutathione，GSH）、谷胱甘肽過(guò)氧化物酶（Glutathione peroxidase，GSH-Px）、丙二醛（Malondialdehyde，MDA）、總超氧化物歧化酶（Total superoxide dismutase，T-SOD）等氧化損傷指標(biāo)進(jìn)行了測(cè)定，以期在動(dòng)物水平驗(yàn)證氧化損傷與肝臟毒性的關(guān)系。此外，本研究采用實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)（Real time-Polymerase Chain Reaction，RT-PCR）技術(shù)對(duì)不同染毒劑量組的大鼠肝臟多種相關(guān)的細(xì)胞色素P450酶（cytochrome P450，CYP450）在mRNA水平進(jìn)行了測(cè)定；進(jìn)而采用Western blot實(shí)驗(yàn)對(duì)mRNA水平發(fā)生顯著改變的CYP450酶在蛋白水平進(jìn)行了測(cè)定；采用超高速離心技術(shù)獲得大鼠肝臟微粒體，對(duì)CYP2B酶對(duì)應(yīng)的PROD（pentoxyresorufin O-dealkylation）、CYP3A酶對(duì)應(yīng)的LBD （Luciferin benzylether debenzylase）和尿苷二磷酸葡萄糖醛酸轉(zhuǎn)移酶（Uridinediphosphate-glucuronosyltransferase，UDPGT）活性進(jìn)行了測(cè)定，以分析和推斷DBDPE在肝臟中的代謝情況和作用機(jī)制。 【結(jié)果】在HepG2細(xì)胞毒性研究實(shí)驗(yàn)部分，利用0-100.0mg/L DBDPE對(duì)HepG2細(xì)胞染毒24h、48h和72h，MTT實(shí)驗(yàn)、LDH實(shí)驗(yàn)和細(xì)胞形態(tài)學(xué)觀察實(shí)驗(yàn)表明，DMSO對(duì)細(xì)胞存活率、細(xì)胞損傷程度以及Hoechst33258染色細(xì)胞形態(tài)學(xué)改變的影響與對(duì)照組之間無(wú)顯著性差異；0-6.25mg/L劑量染毒24h、48h和72h后細(xì)胞存活率、細(xì)胞損傷程度以及細(xì)胞形態(tài)學(xué)改變與對(duì)照組相比無(wú)顯著性差異；12.5-100.0mg/L劑量染毒48h和72h可降低細(xì)胞存活率、增加細(xì)胞損傷程度和引起細(xì)胞形態(tài)學(xué)顯著改變，具有明顯的時(shí)間和劑量-反應(yīng)關(guān)系；PI染色-流式細(xì)胞儀檢測(cè)發(fā)現(xiàn)，12.5-100mg/L劑量DBDPE可誘導(dǎo)HepG2細(xì)胞凋亡，存在時(shí)間和劑量-反應(yīng)關(guān)系；研究還發(fā)現(xiàn)，DBDPE可誘導(dǎo)HepG2細(xì)胞ROS生成量增加，通過(guò)NAC驗(yàn)證試驗(yàn)，證實(shí)DBDPE誘導(dǎo)的細(xì)胞凋亡和損傷與ROS有關(guān)。 在DBDPE對(duì)Wistar大鼠染毒動(dòng)物毒性研究中，采用0-1000mg/kg劑量DBDPE對(duì)Wistar大鼠連續(xù)經(jīng)口染毒28天后發(fā)現(xiàn)，DBDPE染毒后Wistar大鼠體重、肝臟重量、臟器系數(shù)等指標(biāo)與對(duì)照組相比，無(wú)顯著性差異；血清學(xué)檢測(cè)發(fā)現(xiàn)，較高劑量組DBDPE可誘導(dǎo)雄性Wistar大鼠乳酸脫氫酶（lactate dehydrogenase，LDH）、谷丙轉(zhuǎn)氨酶（Glutamic pyruvic transaminase，ALT）、谷草轉(zhuǎn)氨酶（Aspartatetransaminase，AST）、膽汁酸（Total bile acid，TBA）、總膽紅素（total bilirubin，TBA）和葡萄糖（Glucose，Glu）的顯著改變，部分劑量組還可誘導(dǎo)γ-谷氨酰轉(zhuǎn)移酶（glutamyl transpeptidase，GGT）、血漿總蛋白（Total protein，TP）、甘油三酯（Triglyceride，TG）、尿素氮（urea nitrogen，UN）和肌酐（Creatinine，Cr）的顯著改變；此外，較高劑量組DBDPE還可誘導(dǎo)雌性Wistar大鼠堿性磷酸酶（Alkaline phosphatase，ALP）、AST和Glu的顯著改變，部分劑量組還可誘導(dǎo)TBA、Cr和TG的顯著改變。此外，中高DBDPE染毒劑量組GSH水平與對(duì)照組存在顯著性差異，結(jié)合DBDPE可致HepG2細(xì)胞ROS生成量增加，提示DBDPE可能致肝臟發(fā)生氧化損傷。上述結(jié)果表明， DBDPE對(duì)大鼠具有肝毒性，可引起肝損傷，還可影響大鼠膽汁排泄等功能和正常糖等的代謝，且相關(guān)研究結(jié)果提示DBDPE可能干擾肝臟脂肪和蛋白的代謝，分析DBDPE可能具有一定的內(nèi)分泌干擾作用，可能通過(guò)干擾機(jī)體內(nèi)分泌途徑，啟動(dòng)機(jī)體某些信號(hào)通路，干擾機(jī)體正常代謝功能；還可能通過(guò)氧化損傷等作用引起肝損傷，進(jìn)而引起代謝功能受損。 DBDPE對(duì)Wistar大鼠染毒后，肝CYP450代謝酶mRNA、蛋白和酶活性檢測(cè)發(fā)現(xiàn)，染毒組CYP1A1/2mRNA與對(duì)照組相比無(wú)顯著性差異，提示DBDPE可能具有較低或無(wú)二VA英樣毒性作用；CYP2B1和CYP3A1/3mRNA與對(duì)照組相比無(wú)顯著性差異；雄性大鼠CYP2B2、CYP3A2在三個(gè)水平上，較高劑量組與對(duì)照組相比存在顯著差異；雌性大鼠CYP2B2、CYP3A2在三個(gè)水平上，個(gè)別劑量組與對(duì)照組相比存在顯著差異；UDPGT活性檢測(cè)表明，較高劑量組DBDPE對(duì)雄性大鼠UDPGT活性具有誘導(dǎo)作用，并具有一定的劑量反應(yīng)關(guān)系；僅500mg/kg.d劑量組DBDPE對(duì)雌性大鼠UDPGT活性的影響與對(duì)照組存在顯著性差異。據(jù)此推測(cè)DBDPE對(duì)Wistar大鼠代謝酶的影響存在一定的性別差異，對(duì)雄性Wistar大鼠影響較大；分析認(rèn)為DBDPE可能通過(guò)激活組成型雄烷受體（constitutiveandrostane receptor，CAR）和孕烷X受體（pregnane xenobiotic receptor，PXR）信號(hào)通路，進(jìn)而誘導(dǎo)肝臟Ⅰ相和Ⅱ相代謝酶對(duì)DBDPE進(jìn)行代謝以及干擾Wistar大鼠內(nèi)分泌系統(tǒng)，影響Wistar大鼠體內(nèi)正常代謝穩(wěn)態(tài)，發(fā)揮毒性作用。 【結(jié)論】DBDPE具有一定的肝毒性，ROS和氧化損傷分別在肝細(xì)胞毒性和大鼠肝損傷中發(fā)揮重要作用；DBDPE可能具有較低或無(wú)二VA英樣毒性作用；DBDPE可通過(guò)CAR和PXR信號(hào)通路誘導(dǎo)大鼠肝代謝酶活性；DBDPE還可能通過(guò)CAR/PXR信號(hào)通路干擾內(nèi)源性活性物質(zhì)的動(dòng)態(tài)平衡，具有一定的內(nèi)分泌干擾活性；DBDPE對(duì)大鼠的毒性作用具有一定的性別差異，雄性大鼠較雌性大鼠敏感。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R114

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