一溴二氯甲烷的微核效应及对细胞增生的机制探讨

发布时间：2018-05-06 20:18

  本文选题：三卤甲烷 + 一溴二氯甲烷　； 参考：《华中科技大学》2013年博士论文

【摘要】：一溴二氯甲烷(bromodichloromethane, BDCM)是氯化消毒饮用水中含量最高的一类消毒副产物(disinfection by-products, DBPs)—三卤甲烷(trihalomethanes, THMs)的一种。除了BDCM, THMs还包括氯仿、二溴一氯甲烷(dibromochloromethane, DBCM)和溴仿。动物实验显示THMs对啮齿类动物结肠、肾脏和肝脏具有致癌作用。其中,两年致癌实验证实BDCM灌胃染毒可诱导F344大鼠大肠肿瘤和肾脏肿瘤及B6C3F1小鼠肾脏肿瘤和肝脏肿瘤。人群流行病学资料表明含THMs的消毒饮用水可增加人群膀胱癌和大肠癌的发病率。然而迄今为止,THMs的致癌机制仍然存在许多未知之处。通常,致癌机制可分为遗传毒性机制和非遗传毒性机制两大类。有研究发现BDCM经谷胱甘肽S转移酶θ-1(glutathione S-transferase-theta, GSTT1-1)代谢活化后才显示遗传毒性。而BDCM以致癌剂量染毒啮齿类动物可诱导致癌靶脏器异常的脏器毒性、DNA甲基化和细胞增生,提示非遗传毒性机制可能在BDCM的致癌过程中起重要作用。 尽管在众多遗传毒性实验中THMs显示了阴性结果,但也有体外实验提示THMs须经代谢活化方具有遗传毒性。而来源于人类的代谢酶完全的肝肿瘤细胞株HepG2是毒理学实验中常用的细胞株,可检出直接致突变物和间接致突变物。胞质分裂阻滞微核法(cytokinesis-block micronucleus test, CBMNT)是一套以检测染色体断裂和染色体分离为主的实验体系。因此本研究以HepG2作为受试细胞株,应用CBMNT检测四种THMs的遗传毒性。 在遗传毒性研究的基础上,我们选取上述典型溴代THMs—BDCM作为受试物探讨该毒物的非遗传毒性机制。我们利用动物实验重点研究BDCM在不同染毒时间点对雄性F344大鼠不同脏器细胞增生的影响,并探讨E-cadherin介导的细胞黏连和Wnt信号转导通路在BDCM诱导的细胞增生过程中的作用。 本研究分为两个部分： 第一部分四种THMs对HepG2细胞的诱裂作用 目的：探讨四种THMs(氯仿、BDCM、DBCM和溴仿)的诱裂作用。 方法：上述四种THMs分别设4个染毒剂量,以苯并(a)芘(benzo(a)pyrene, B(a)P)为阳性对照,二甲基亚砜(dimathyl sulfoxide, DMSO)为溶剂对照,应用CBMNT检测四种THMs对HepG2细胞微核率和核分裂指数(nuclei division index, NDI)的影响。 结果：与溶剂对照组相比,四种THMs除溴仿外均可使HepG2细胞的微核率显著增加(P0.01或P0.05),但诱导微核率显著性增加的有效浓度高达10000μmol/L(氯仿和BDCM)和3000μmol/L (DBCM)。 结论：氯仿、BDCM和DBCM可诱导HepG2细胞染色体断裂,具有遗传毒性。 第二部分BDCM对雄性F344大鼠不同脏器细胞增生的机制探讨 目的：通过E-cadherin介导的细胞黏连和Wnt信号转导通路探讨BDCM对雄性F344大鼠不同脏器(结肠、肾脏和肝脏)细胞增生的机制。 方法：将64只雄性F344大鼠随机分为两组,即对照组和BDCM染毒组(100mg/(kg·d))。以BDCM溶于玉米油灌胃染毒动物,每周5天,每天1次。分别于第1、4、8和12周每个时间点每组处死8只大鼠,收集结肠、肾脏和肝脏组织。以苏木精-伊红(hematoxylin and eosin, HE)染色观察大鼠各个脏器的病理变化,实时荧光定量PCR法(real-time PCR, RT-PCR)检测β-catenin, APC, E-cadherin, c-jun, c-myc和cyclin D1mRNA的表达,以亚硫酸氢盐测序PCR(bisulfite sequencing PCR, BSP)和免疫组织化学法分别检测APC和E-cadherin启动子区甲基化改变和蛋白水平的表达,同时应用增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)免疫组织化学法检测细胞增生的改变。 结果： (1)全身性毒性 染毒期间大鼠未出现异常死亡,除体重下降以外未观察到与BDCM染毒相关的全身性毒性反应。 (2)结肠 我们在染毒第1、4、8和12周四个时间点均未观察到大鼠结肠出现与BDCM染毒有关的病理学改变。但是BDCM染毒12周可诱导粘膜上皮细胞E-cadherin启动子区域高甲基化,并降低E-cadherin在mRNA和蛋白水平上的表达。此外,BDCM染毒12周可诱导结肠粘膜上皮异常的细胞增生。 (3)肾脏 BDCM染毒可引起大鼠轻微的肾毒性,在病理学上表现为在第8和12周皮质近端小管上皮细胞水肿、脂肪变、单个的异形巨核细胞及偶尔可见的上皮细胞脱落坏死。另外,BDCM从第4周起显著性降低肾脏E-cadherin mRNA的水平,从第8周开始引起E-cadherin蛋白在近端小管上皮细胞表达下降。然而与对照组相比,BDCM染毒组E-cadherin基因启动子区甲基化水平在整个实验期间也未显示出有统计学意义的改变。此外,BDCM可在转录水平上显著性增加Wnt通路下游增殖性靶基因c-myc和cyclin D1的表达。与上述结果相一致,BDCM在第4、8和12周均可诱导近端小管上皮细胞异常增生。 (4)肝脏 BDCM染毒12周可引起部分大鼠肝脏出现肝小叶中心区细胞水肿和脂肪变为主要病变的病理学改变。虽然大鼠肝脏β-catenin和c-myc mRNA在BDCM染毒的第8周显著性降低,但是二者的表达在第12周即恢复至对照组水平。另外,BDCM对肝脏APC和E-cadherin基因启动子区甲基化水平及mRNA和蛋白的表达均无影响。与对照组相比,BDCM染毒组在四个时间点均未显示明显的肝细胞增生。 结论：BDCM灌胃染毒可通过抑制E-cadherin mRNA和蛋白的表达来诱导结肠粘膜上皮细胞增生。另外,BDCM通过抑制E-cadherin mRNA和蛋白的表达和在转录水平上激活Wnt通路下游基因c-myc和cyclin D1来引起近端小管上皮细胞异常增生。然而,BDCM未诱导肝细胞明显的非遗传毒性损伤。BDCM在不同脏器可能通过E-cadherin介导的细胞黏连和Wnt通路影响细胞增生。这提示非遗传毒性在BDCM致癌机制中扮演着十分重要的作用。
[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锛，

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