本文選題：三鹵甲烷 + 一溴二氯甲烷　； 參考：《華中科技大學(xué)》2013年博士論文

【摘要】：一溴二氯甲烷(bromodichloromethane, BDCM)是氯化消毒飲用水中含量最高的一類消毒副產(chǎn)物(disinfection by-products, DBPs)—三鹵甲烷(trihalomethanes, THMs)的一種。除了BDCM, THMs還包括氯仿、二溴一氯甲烷(dibromochloromethane, DBCM)和溴仿。動(dòng)物實(shí)驗(yàn)顯示THMs對(duì)嚙齒類動(dòng)物結(jié)腸、腎臟和肝臟具有致癌作用。其中,兩年致癌實(shí)驗(yàn)證實(shí)BDCM灌胃染毒可誘導(dǎo)F344大鼠大腸腫瘤和腎臟腫瘤及B6C3F1小鼠腎臟腫瘤和肝臟腫瘤。人群流行病學(xué)資料表明含THMs的消毒飲用水可增加人群膀胱癌和大腸癌的發(fā)病率。然而迄今為止,THMs的致癌機(jī)制仍然存在許多未知之處。通常,致癌機(jī)制可分為遺傳毒性機(jī)制和非遺傳毒性機(jī)制兩大類。有研究發(fā)現(xiàn)BDCM經(jīng)谷胱甘肽S轉(zhuǎn)移酶θ-1(glutathione S-transferase-theta, GSTT1-1)代謝活化后才顯示遺傳毒性。而BDCM以致癌劑量染毒嚙齒類動(dòng)物可誘導(dǎo)致癌靶臟器異常的臟器毒性、DNA甲基化和細(xì)胞增生,提示非遺傳毒性機(jī)制可能在BDCM的致癌過程中起重要作用。 盡管在眾多遺傳毒性實(shí)驗(yàn)中THMs顯示了陰性結(jié)果,但也有體外實(shí)驗(yàn)提示THMs須經(jīng)代謝活化方具有遺傳毒性。而來源于人類的代謝酶完全的肝腫瘤細(xì)胞株HepG2是毒理學(xué)實(shí)驗(yàn)中常用的細(xì)胞株,可檢出直接致突變物和間接致突變物。胞質(zhì)分裂阻滯微核法(cytokinesis-block micronucleus test, CBMNT)是一套以檢測染色體斷裂和染色體分離為主的實(shí)驗(yàn)體系。因此本研究以HepG2作為受試細(xì)胞株,應(yīng)用CBMNT檢測四種THMs的遺傳毒性。 在遺傳毒性研究的基礎(chǔ)上,我們選取上述典型溴代THMs—BDCM作為受試物探討該毒物的非遺傳毒性機(jī)制。我們利用動(dòng)物實(shí)驗(yàn)重點(diǎn)研究BDCM在不同染毒時(shí)間點(diǎn)對(duì)雄性F344大鼠不同臟器細(xì)胞增生的影響,并探討E-cadherin介導(dǎo)的細(xì)胞黏連和Wnt信號(hào)轉(zhuǎn)導(dǎo)通路在BDCM誘導(dǎo)的細(xì)胞增生過程中的作用。 本研究分為兩個(gè)部分： 第一部分四種THMs對(duì)HepG2細(xì)胞的誘裂作用 目的：探討四種THMs(氯仿、BDCM、DBCM和溴仿)的誘裂作用。 方法：上述四種THMs分別設(shè)4個(gè)染毒劑量,以苯并(a)芘(benzo(a)pyrene, B(a)P)為陽性對(duì)照,二甲基亞砜(dimathyl sulfoxide, DMSO)為溶劑對(duì)照,應(yīng)用CBMNT檢測四種THMs對(duì)HepG2細(xì)胞微核率和核分裂指數(shù)(nuclei division index, NDI)的影響。 結(jié)果：與溶劑對(duì)照組相比,四種THMs除溴仿外均可使HepG2細(xì)胞的微核率顯著增加(P0.01或P0.05),但誘導(dǎo)微核率顯著性增加的有效濃度高達(dá)10000μmol/L(氯仿和BDCM)和3000μmol/L (DBCM)。 結(jié)論：氯仿、BDCM和DBCM可誘導(dǎo)HepG2細(xì)胞染色體斷裂,具有遺傳毒性。 第二部分BDCM對(duì)雄性F344大鼠不同臟器細(xì)胞增生的機(jī)制探討 目的：通過E-cadherin介導(dǎo)的細(xì)胞黏連和Wnt信號(hào)轉(zhuǎn)導(dǎo)通路探討B(tài)DCM對(duì)雄性F344大鼠不同臟器(結(jié)腸、腎臟和肝臟)細(xì)胞增生的機(jī)制。 方法：將64只雄性F344大鼠隨機(jī)分為兩組,即對(duì)照組和BDCM染毒組(100mg/(kg·d))。以BDCM溶于玉米油灌胃染毒動(dòng)物,每周5天,每天1次。分別于第1、4、8和12周每個(gè)時(shí)間點(diǎn)每組處死8只大鼠,收集結(jié)腸、腎臟和肝臟組織。以蘇木精-伊紅(hematoxylin and eosin, HE)染色觀察大鼠各個(gè)臟器的病理變化,實(shí)時(shí)熒光定量PCR法(real-time PCR, RT-PCR)檢測β-catenin, APC, E-cadherin, c-jun, c-myc和cyclin D1mRNA的表達(dá),以亞硫酸氫鹽測序PCR(bisulfite sequencing PCR, BSP)和免疫組織化學(xué)法分別檢測APC和E-cadherin啟動(dòng)子區(qū)甲基化改變和蛋白水平的表達(dá),同時(shí)應(yīng)用增殖細(xì)胞核抗原(proliferating cell nuclear antigen, PCNA)免疫組織化學(xué)法檢測細(xì)胞增生的改變。 結(jié)果： (1)全身性毒性 染毒期間大鼠未出現(xiàn)異常死亡,除體重下降以外未觀察到與BDCM染毒相關(guān)的全身性毒性反應(yīng)。 (2)結(jié)腸 我們?cè)谌径镜?、4、8和12周四個(gè)時(shí)間點(diǎn)均未觀察到大鼠結(jié)腸出現(xiàn)與BDCM染毒有關(guān)的病理學(xué)改變。但是BDCM染毒12周可誘導(dǎo)粘膜上皮細(xì)胞E-cadherin啟動(dòng)子區(qū)域高甲基化,并降低E-cadherin在mRNA和蛋白水平上的表達(dá)。此外,BDCM染毒12周可誘導(dǎo)結(jié)腸粘膜上皮異常的細(xì)胞增生。 (3)腎臟 BDCM染毒可引起大鼠輕微的腎毒性,在病理學(xué)上表現(xiàn)為在第8和12周皮質(zhì)近端小管上皮細(xì)胞水腫、脂肪變、單個(gè)的異形巨核細(xì)胞及偶爾可見的上皮細(xì)胞脫落壞死。另外,BDCM從第4周起顯著性降低腎臟E-cadherin mRNA的水平,從第8周開始引起E-cadherin蛋白在近端小管上皮細(xì)胞表達(dá)下降。然而與對(duì)照組相比,BDCM染毒組E-cadherin基因啟動(dòng)子區(qū)甲基化水平在整個(gè)實(shí)驗(yàn)期間也未顯示出有統(tǒng)計(jì)學(xué)意義的改變。此外,BDCM可在轉(zhuǎn)錄水平上顯著性增加Wnt通路下游增殖性靶基因c-myc和cyclin D1的表達(dá)。與上述結(jié)果相一致,BDCM在第4、8和12周均可誘導(dǎo)近端小管上皮細(xì)胞異常增生。 (4)肝臟 BDCM染毒12周可引起部分大鼠肝臟出現(xiàn)肝小葉中心區(qū)細(xì)胞水腫和脂肪變?yōu)橹饕∽兊牟±韺W(xué)改變。雖然大鼠肝臟β-catenin和c-myc mRNA在BDCM染毒的第8周顯著性降低,但是二者的表達(dá)在第12周即恢復(fù)至對(duì)照組水平。另外,BDCM對(duì)肝臟APC和E-cadherin基因啟動(dòng)子區(qū)甲基化水平及mRNA和蛋白的表達(dá)均無影響。與對(duì)照組相比,BDCM染毒組在四個(gè)時(shí)間點(diǎn)均未顯示明顯的肝細(xì)胞增生。 結(jié)論：BDCM灌胃染毒可通過抑制E-cadherin mRNA和蛋白的表達(dá)來誘導(dǎo)結(jié)腸粘膜上皮細(xì)胞增生。另外,BDCM通過抑制E-cadherin mRNA和蛋白的表達(dá)和在轉(zhuǎn)錄水平上激活Wnt通路下游基因c-myc和cyclin D1來引起近端小管上皮細(xì)胞異常增生。然而,BDCM未誘導(dǎo)肝細(xì)胞明顯的非遺傳毒性損傷。BDCM在不同臟器可能通過E-cadherin介導(dǎo)的細(xì)胞黏連和Wnt通路影響細(xì)胞增生。這提示非遺傳毒性在BDCM致癌機(jī)制中扮演著十分重要的作用。
[Abstract]:Bromodichloromethane (BDCM) is a kind of disinfection by-products, DBPs, three halomethane (trihalomethanes, THMs) with the highest content of chlorination in drinking water. Apart from BDCM, THMs also includes chloroform, dichloromethane (dibromochloromethane, DBCM) and bromine. Animal experiments show THMs It has a carcinogenic effect on the colon, kidney and liver of rodents. Among them, two years of carcinogenic experiments have confirmed that BDCM gavage can induce colorectal tumors and renal tumors in F344 rats and renal tumors and liver tumors in B6C3F1 mice. Population epidemiological data show that the incidence of bladder and colorectal cancer in population can be increased by the disinfection of drinking water containing THMs. However, so far, there are still a lot of unknown mechanisms for the carcinogenesis of THMs. Generally, the carcinogenic mechanism can be divided into two major categories: genotoxicity and non genotoxicity. Studies have found that BDCM is activated by the metabolic activation of glutathione S transferase (glutathione S-transferase-theta, GSTT1-1). And BDCM is a carcinogenic dose. The toxic rodents can induce abnormal organ toxicity, DNA methylation and cell proliferation, suggesting that the non genetic toxicity mechanism may play an important role in the carcinogenic process of BDCM.
Although THMs shows negative results in many genotoxicity tests, there are also in vitro experiments suggesting that THMs must have genetic toxicity by metabolic activation. The liver tumor cell strain, HepG2, which is derived from human metabolic enzymes, is a common cell strain in toxicological experiments. Direct mutagenicity and indirect mutagenesis can be detected. Cytokinesis resistance can be detected. Cytokinesis-block micronucleus test (CBMNT) is a set of experimental system to detect chromosome breakage and chromosome segregation. Therefore, this study uses HepG2 as a tested cell strain and uses CBMNT to detect the genotoxicity of four THMs.
On the basis of the study of genotoxicity, we selected the typical brominated THMs - BDCM as the subject matter to explore the non genetic toxicity mechanism of the poison. We used animal experiments to focus on the effect of BDCM on the proliferation of different organ cells in male F344 rats at different time points, and to explore the E-cadherin mediated cell adhesion and Wnt signal. The role of transduction pathway in BDCM induced cell proliferation.
This study is divided into two parts:
The first part is the induction of four kinds of THMs on HepG2 cells.
Objective: To explore the induced action of four THMs (chloroform, BDCM, DBCM and bromoform).
Methods: the four kinds of THMs were treated with 4 poisoned doses respectively, with benzo (a) pyrene (benzo (a) pyrene, B (a) P) as positive control, and two methyl sulfoxide (dimathyl sulfoxide, DMSO) as the solvent control. The effect of CBMNT detection on micronucleus rate and mitotic index of four kinds of cells was applied.
Results: compared with the solvent control group, the micronucleus rate of the four types of THMs except bromoform could significantly increase the micronucleus rate of HepG2 cells (P0.01 or P0.05), but the effective concentration of the induced micronucleus rate was up to 10000 mu mol/L (chloroform and BDCM) and 3000 mu mol/L (DBCM).
Conclusion: chloroform, BDCM and DBCM can induce chromosome breakage in HepG2 cells and have genetic toxicity.
The second part is about the mechanism of BDCM on the proliferation of different organs in male F344 rats.
Objective: To explore the mechanism of BDCM on the proliferation of different organs (colon, kidney and liver) of male F344 rats by E-cadherin mediated cell adhesion and Wnt signal transduction pathway.
Methods: 64 male F344 rats were randomly divided into two groups, that is, the control group and the BDCM (100mg/ (kg. D)). BDCM dissolved in the corn oil for the gavage of the animals, 5 days a week, 1 times a day. The colon, kidney and liver tissues were collected at each time point in the 1,4,8 and 12 weeks respectively, and the colon, kidney and liver were collected. Hematoxylin and eosin, HE) was used to observe the pathological changes of the organs of the rats. The real-time fluorescence quantitative PCR (real-time PCR, RT-PCR) was used to detect the expression of beta -catenin, APC, E-cadherin, c-jun, c-myc and cyclin D1mRNA. The expression of changes and protein levels was changed and the proliferation of cell proliferation was detected by proliferating cell nuclear antigen (PCNA) immunohistochemical method.
Result錛，

本文編號(hào)：1853729