【作者】 姜丽平；

【导师】 仲来福；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 前言:2002年瑞典科学家发现,一些富含淀粉类的食品经高温加工处理后可含有一种有毒的、并具潜在致癌性的化学物质丙烯酰胺(Acrylamide, AA)。高温加工过程中天门冬氨酸与葡萄糖发生Maillard反应而生成AA。这一发现引起世界范围对AA的关注。AA不仅具有神经毒性,并且是一种潜在的致癌物,国际癌症研究机构(IARC)将其划分为2A类的致癌物。这表明AA对人类健康存在潜在威胁。流行病学研究至今尚未发现摄入含有AA的食品与患癌症危险的相关性,遗传毒理研究则发现AA诱发DNA损伤和染色体畸变,例如彗星试验显示,AA诱导大鼠甲状腺细胞(PC C13和FRTL5)及人成淋巴细胞(TK6)DNA损伤;大鼠精子细胞的微核试验显示减数分裂微核形成。除了上述体外实验,在小鼠体内实验还观察到了染色体结构的改变、多倍体或微核的形成。在本研究中,我们使用人肝癌细胞系HepG2细胞研究AA的遗传毒性及可能的机制。HepG2细胞来源于人类肝胚细胞瘤,但是它的分化程度较高,保留了较完整的生物转化代谢Ⅰ相酶和Ⅱ相酶的活性。HepG2细胞已经被证实是一种适合的测试物质遗传毒性的系统。肝脏是AA代谢的起始点,当定量地给予动物(口服或腹腔注射)AA后,可在肝脏发现高浓度的AA。方法:试验系统为HepG2细胞系。通过微核试验(MNT)及单细胞凝胶电泳(SCGE)试验检测细胞染色体和DNA损伤情况,评价AA的遗传毒性。为进一步探讨遗传毒性的机制,以2’,7’—二氢二氯荧光素(DCFH)法测定细胞内活性氧水平。通过免疫组化方法测定8-羟脱氧鸟苷(8-OHdG)在细胞内的表达水平。为了了解AA诱导的氧化应激反应,我们用DL-甲硫氨酸磺酰亚胺(DL-BSO)预处理HepG2细胞,致使谷胱甘肽(GSH)耗竭,然后用噻唑蓝(MTT)法检测GSH耗竭时AA对HepG2细胞的毒性的影响(与GSH未耗竭的HepG2细胞比较)。结果: 2.5~20mM的AA作用HepG2细胞1 h后,DNA断裂细胞形成彗星样脱尾,尾长、尾距、尾DNA含量及尾距增大,与未用AA处理细胞比较,差异有明显的统计学意义(P<0.05或0.01),且呈剂量依赖关系。提示AA引起HepG2细胞DNA链断裂程度明显增加,且随AA浓度的增高而增加。HepG2细胞与0.625~2.5mM ?的AA接触24 h后,细胞微核率明显高于未用AA处理细胞(P<0.05或0.01)。与10~40mM?的AA接触1 h后,可引起细胞内ROS水平表达的明显增加。5~20?mM的AA作用于HepG2细胞3h后,细胞内8-OHdG水平的表达增强,与对照组比较,有显著性差别。用40μM?BSO预处理HepG2细胞16h后,MTT方法测定的AA细胞毒性显著性增加,并且呈现剂量关系。结论:AA对HepG2细胞可能具有遗传毒性。AA对HepG2细胞的遗传毒作用,可能是通过细胞内GSH的耗竭,ROS的增高,及8-OHdG形成的增加所造成的氧化性DNA损伤形成。更多还原

【Abstract】 Introduction:In April 2002, scientists in Sweden discovered that large amounts of acrylamide, a potent carcinogen is formed in foods rich in starch that have been heated at high temperatures. It may be formed through the Maillard reaction from amino acids and reducing sugars during drying and baking. This finding raises human health concerns about the risks of AA for the general population.Acrylamide is neurotoxic in humans and laboratory animals, and was classified as“probably carcinogenic to humans”(Group 2A carcinogen) by a working group of the International Agency for Research on Cancer. This might represent a potential threat to public health. But until now, epidemiological studies found no association between consumption of foods containing acrylamide and cancer risk. The relevance of current intake levels in terms of cancer risk still remains an issue under debate. With respect to AA genotoxicity data, AA could induce DNA damage in the PC Cl3 and FRTL5 rat thyroid cell lines, as well as in human lymphoblastoid TK6 cells in the comet assay. In the spermatid micronucleus test, positive result was observed in the rat meiotic cells. In addition to the tests in vitro, induction of structural chromosome aberrations, micronuclei or polyploidy was observed in various studies in mice treated in vivo (dose around 100 mg/kg bw).In the present study, we used human hepatoma G2 (HepG2) cells to investigate the genotoxicity of AA and possible mechanisms. HepG2 cells retain many characteristics of hepatocytes such as the activities of phase I and phase II enzymes and reflect the metabolism of xenobiotics in the human body better than other metabolically incompetent cells. HepG2 cellshave been shown to be a suitable system for genotoxicity testing. The liver is the initial site of acrylamide metabolism following oral or i.p. dosing, and acrylamide is found at high concentrations in the liver.Method: Genotoxicity of AA was assessed by micronucleus test (MNT) and comet assay. To further investigate the mechanism of genotoxicity of AA in HepG2 cells, we measured the intracellular generation of reactive oxygen species (ROS). We analyzed the oxidative DNA damage in AA-treated cells by immunocytochemistry staining of 8-hydroxydeoxyguanosine (8-OHdG). To clarify if the AA-induced oxidative stress is via the depletion of glutathione (GSH), we treatedHepG2 cells with DL-buthionine sulphoximine (BSO) to deplete GSH and MTT assays was performed to determine the cytotoxicity of AA in GSH-depleted and normal control HepG2 cells.Result: The damage to DNA-strand of HepG2 cells significantly increased after exposure to 2.5~20mM AA for 1 h, in a dose-dependent manner. Frequencies of micronuclei significantly increased in HepG2 cells after treatment with 0.625~2.5mM AA for 24 h. ?After exposure to AA for 1 h, intracellular ROS significantly increased. The 8-OHdG was positive in the HepG2 cell treated with AA. With the help of the MTT method described above, we pretreated HepG2 cells with 40μM BSO to deplete GSH for 16 hours ,the cytotoxicity of AA in GSH-depleted significantly increased, in a dose-dependent manner.Conclusion: AA could be genotoxic to HepG2 cell. AA exerts genotoxic effects in HepG2 cells, probably through depletion of GSH,oxidative DNA damage induced by intracellular ROS and increase of 8-OHdG formation.更多还原