【作者】 赵金明；

【导师】 朱惠刚；

【作者基本信息】 复旦大学 ， 劳动卫生与环境卫生学， 2003， 博士

【摘要】 研究背景：常规饮水氯化消毒技术并不能完全有效的消除水体中的藻毒素，而氯化消毒过程中还会产生具有致突变活性的氯化消毒副产物，使氯化消毒副产物与藻毒素在饮水中共存并产生潜在联合作用。目的：研究藻毒素与自来水有机提取物在肿瘤发生过程中的联合作用，并探讨其可能的作用机制。方法：从水体中收集藻毒素和有机提取物样品，在对样品进行化学成份分析的基础上，研究藻毒素在体外条件下对V79(HPRT+/HPRT-)细胞间代谢协同作用和SHE细胞周期及相关基因表达的影响，并运用Ames实验、微核实验、程序外DNA合成试验和V79细胞HPRT基因正向突变实验等配套的致突变筛选实验，对自来水有机提取物进行致突变性检测。在运用SHE细胞体外转化实验研究藻毒素和自来水有机提取物对细胞转化作用的同时，以自来水有机提取物为启动剂，藻毒素为促进剂，构建大鼠肝肿瘤促进模型。收集肝脏标本，进行常规病理形态学检查和分析，同时运用核酸原位杂交、RT-PCR、免疫组化和Western-blot等方法检测肿瘤发生发展过程中肝细胞p-ELK-1、c-fos、 c-jun和GSTPi等基因表达活性的改变，用凝胶移动试验检测肝细胞核蛋白/GEPI、AP-1/DNA结合能力，并对其相互关系进行比较和分析。结果：藻毒素具有极强的急性毒性作用，LD50为106.7mg/kg（89.1~127.6 mg/kg,干藻细胞/体重），肝脏是其主要的靶器官。在体外培养条件下，藻毒素能够抑制V79(HPRT+/HPRT-)细胞间代谢协同作用，并能诱导SHE细胞c-fos和c-jun表达升高，促进细胞增殖。自来水有机提取物具有一定的致突变性，能诱导SHE细胞恶性转化。藻毒素单独作用不能诱导SHE细胞转化，但能促进自来水有机提取物诱导的细胞转化。在实验性大鼠肝癌促进模型研究中，藻毒素和自来水有机提取物共同作用可以诱导动物产生肝细胞增生灶和增生结节。藻毒素单独作用即可以诱导pELK-1、c-fos和c-jun表达升高，使肝细胞核蛋白/GEPI、AP-1/DNA结合能力增强，而相同剂量的藻毒素作用于经自来水有机提取物启动的动物后，其肝细胞p-ELK-1、c-fos和c-jun的表达以及肝细胞核蛋白/GEPI、AP-1/DNA结合能力均较非启动组明显增加（p<0.05）。同时，一次<WP=6>启动剂量的自来水有机提取物单独作用即能显著诱导肝细胞GSTPi基因表达，藻毒素单独作用不能诱导GSTPi基因表达，但能够促进自来水有机提取物诱导产生的GSTPi基因表达，使其较自来水有机提取物单独作用组显著增加（p<0.05）。结论：1. 藻毒素能通过抑制细胞间隙通讯功能和影响MAPK信号通路而促进细胞增殖；2. 藻毒素具有促进实验性大鼠肝癌发生的作用；3. 自来水有机提取物具有致癌启动活性，能诱导实验性大鼠肝癌的发生；4. 藻毒素和自来水有机提取物在实验性大鼠肝癌诱导过程中具有联合作用；5. 藻毒素和自来水有机提取物可能的联合作用机制是：自来水有机提取物作用于细胞后，使细胞实现启动作用而呈自主生长倾向，而藻毒素可通过影响细胞MAPK 信号旁路而促进癌变启动细胞持续分裂，并因此而不断积累恶性转化所需的各个水平的遗传改变，最后恶性转化成癌。更多还原

【Abstract】 INTRODUCTION: Conventional surface water treatment processes are not sufficient to totally remove cyanobacterial toxins, and chronic exposure to cyanobacterial toxins via drinking water may have tumour-promotion effect. Certain disinfections by-products (DBPs) in drinking water produced during the process of water chlorination are found to be mutagenic. Coexistence of DBPs and cyanobacterial toxins in drinking water may lead to potential synergistic effects during carcinogenesis.OBJECTIVE: To investigate the joint actions of cyanobacterial toxins Microcystins (MCs) and organic extractions of water (OEWs) during carcinogenesis, and explore its potential mechanism.METHODS: Cyanobacterial toxins Microcystins (MCs) were isolated and purified from cyanobacterial samples collected during outbreaks of cyanobacterial water bloom. Isomers of MC-LR and MC-YR were quantitatively analyzed with HPLC (High Performance Liquid Chromatography). The effects of MCs on metabolic cooperation in V79 (HPRT+/HPRT-) and on cell cycle and related gene expressions in SHE cells (Syrian Hamster Embryo cell) were investigated in vitro. Organic extractions of water (OEWs) were extracted from tap water sample, and then mutagenicity screening tests including Ames test, micronuclei test (MN), unscheduled DNA synthesis induction (UDS) and HPRT mutation assay were used to detect the mutagenicities of OEWs. SHE cells transformation system in vitro was also used to evaluate the potential carcinogenecities of MCs and OEWs. At the same time, a two-stage hepatocarcinogenesis rat model (Solt-Farber Model) was established using OEWs and MCs applied as initiator and promoter, respectively. The histopathological changes in liver were observed, and expressions of GSTPi and other genes involved in MAPKs (mitogens activated protein kinase) signals pathway such as TCF/ELK-1, c-fos and c-jun during hepatocarcinogenesis were detected by means of immunohistochemical staining, Western-blot, RT-PCR (reverse-transcription PCR) and ISH (in situ Hybridization). DNA binding activity of AP-1 and GEPI binding activity of nuclei proteins were analyzed by EMSA (electrophoretic mobility shift assay) as well. The relationships among expressions of TCF/ELK-1, c-fos, c-jun, and GSTPi were analyzed.<WP=8>RESULTS: The results showed that MCs possessed strong acute toxicity with liver as the target organ, the LD50 was estimated to be 106.7mg/kg (89.1~127.6 mg/kg, mass of dry algal cells / b.w). The inhibited metabolic cooperation in vitro was seen in V79 (HPRT+/HPRT-) treated with MCs. Up-regulated expressions of c-fos and c-jun as well as promoted cell proliferation were also observed in SHE cell co-incubated with MCs in vitro. It was also indicated that OEWs were mutagenic and could induce SHE cell transformation in vitro. MCs alone could not induce SHE cell transformation, but could promote the cell transformation induced by OEWs.The two-stage hepatocarcinogenesis bioassay results showed that OEWs and MCs could induce hyperplastic and precancerous foci as well as nodule in livers of rats. MCs alone could up-regulate the expressions of pELK-1, c-jun, and c-fos, and enhance DNA binding activity of AP-1 and GEPI binding activity of nuclei protein in hepatocytes of rats. Further promoted up-regulation of related gene expressions were also seen in this study when rats were initiated with OEWs before the administration of MCs (p<0.05). It was also indicated that OEWs alone could induce expressions of GSTPi mRNA and protein in livers. While compared to OEWs, MCs alone could not induce the expression of GSTPi, but could promote the expressions of GSTPi mRNA and protein induced by OEWs (p<0.05). CONCLUSIONS: 1. MCs could inhibit cell GJIC (gap junctional intercellular communication) and promote cell proliferation through up-regulation of MAPKs signals pathway.2. MCs possessed tumor-promoting activity during experimental hepatocarcinogenesis in rats.3. OEWs were mutagenic and possessed initiation activity during hepatocarcinogenesis in rats. 4.更多还原