INDUCTION OF OXIDATIVE STRESS AND ACTIVATION OF TRANSCRIPTION BY METALS

项目1的总体目标是通过以下方式了解该机制： 有毒金属影响细胞氧化应激。 氧化应激 与许多人类疾病有关，包括癌症， 老化、动脉粥样硬化和纤维化。 有证据表明有毒物质 金属可以诱导氧化应激，这可能是两者的机制 遗传毒性和非遗传毒性增殖性疾病。 的能力 有毒和/或致癌金属、铬、镍、镉、铅、铁 和砷，单独和组合发现，在污染 将在鸡胚中确定诱导氧化应激的位点， 体内和培养的内皮细胞。 具体的假设是 研究表明，金属可能会导致癌症或其他增殖性疾病， 通过在转录水平上改变特定基因的表达。 这种改变可能来自对DNA结构的直接影响， 因此反式作用因子的DNA识别序列 或通过激活细胞信号传导以获得更大的丰度和/或 转录因子的活性。 我们计划不仅检查 有毒金属对DNA氧化损伤的诱导， 这些金属的倾向，导致异常基因诱导，通过 改变细胞信号。 拟议研究的具体目标 （1）确定有毒金属产生反应的能力 使用生物化学分析的培养细胞中的氧中间体（ROI） 对于活性物质的积累和荧光光谱， 显示细胞内氧化剂水平。 活性氧诱导 有毒金属对过氧化氢和超氧化物等物种的影响， 通过分光光度计和荧光技术进行测定。 水平 细胞内氧化剂水平的变化可以通过使用可代谢的 试剂如罗丹明和二氯芴二乙酸酯， 细胞不可逆地转化为荧光物质， 氧物种。 (2)研究氧化性DNA损伤的诱导 在体内和培养的正常鸡胚组织中的毒性金属 猪主动脉内皮细胞DNA单链断裂，DNA- 蛋白质交联和8-氧代-2 '-脱氧鸟苷水平。 如果反应性 有毒金属在细胞内产生氧， ROI可以攻击DNA，导致氧化DNA损伤。 DNA链断裂 将通过使用DNA解旋测定、DNA-蛋白质交联 通过使用K+-SDS沉淀分析和8-氧代-2 '-脱氧鸟苷， 采用高效液相色谱法和电化学 检测（HPLC-ECD）。 (3)证明金属的含量会导致 转录因子的特异性磷酸化依赖性激活 并在这个信号级联中建立速率决定位点。 激活反式作用因子，诱导已知的基因， 在氧化应激后表达，即，血红素加氧酶和尿激酶- 如纤溶酶原激活剂（uPA），将通过电泳分析 迁移率测定和对激酶抑制的敏感性将是 测定 抑制转录因子活化对 将测定氧化剂敏感基因的稳态mRNA水平， 体内鸡胚组织和培养的正常猪主动脉内皮细胞 细胞 拟议的研究应提供证据， 毒性金属对特定基因表达的作用途径，以及 深入了解金属导致其毒性的机制， 方面的影响. 这反过来又可以提供基本的见解， 旨在防止金属诱导氧化应激的策略。

The overall objective of Project 1 is to understand the mechanism by which toxic metals effect cellular oxidative stress. Oxidative stress has been implicated in a number of human diseases, including cancer, aging, atherosclerosis, and fibrosis. There is some evidence that toxic metals can induce oxidative stress, which may be the mechanism for both genotoxic and non-genotoxic proliferative disease. The ability of the toxic and/or carcinogenic metals, chromium, nickel, cadmium, lead, iron and arsenic, individually and in combinations found at contaminated sites, to induce oxidative stress will be determined in chick embryo in vivo and cultured endothelial cells. The specific hypothesis for these studies is that metals may cause cancer or other proliferative diseases by altering expression of specific genes at the level of transcription. This alteration may occur either from a direct effect on DNA structure and consequently the DNA recognition sequences for trans-acting factors or through activation of cell signaling for a greater abundance and/or activity of transcription factors. We plan to not only examine possible induction of oxidative DNA damage by toxic metals, but also the propensity of these metals to cause aberrant gene induction through alteration of cell signaling. The specific aims of the proposed research are to: (1) Determine the ability of toxic metals to produce reactive oxygen intermediates (ROIs) in cultured cells using biochemical analysis for accumulation of reactive species and fluorescence spectroscopy to demonstrate intracellular oxidant levels. Induction of reactive oxygen species, such as hydrogen peroxide and superoxide, by toxic metals can be assayed by spectrophotometric and fluorometric techniques. The level of intracellular oxidant levels can be measured by use of metabolizable reagents such as rhodamine and dichlorofluoresin diacetate which enter cells irreversibly and are converted to fluorescent species by reactive oxygen species. (2) Investigate the induction of oxidative DNA damage by toxic metals in chick embryo tissues in vivo and in cultured normal pig aorta endothelial cells by measuring DNA single strand breaks, DNA- protein cross-links and levels of 8-oxo-2'-deoxyguanosine. If reactive oxygen species are produced intracellularly by toxic metals, then the ROIs may attack DNA causing oxidative DNA lesions. DNA strand breakage will be analyzed by using a DNA unwinding assay, DNA-protein cross-links by using a K+-SDS precipitation assay and 8-oxo-2'-deoxyguanosine by using high performance liquid chromatography with electrochemical detection (HPLC-ECD). (3) Demonstrate the levels of metals that cause specific, phosphorylation-dependent activation of transcription factors and establish the rate determining sites in this signaling cascade. Activation of trans-acting factors, which induce genes known to be expressed following oxidant-stress, i.e., heme oxygenase, and urokinase- like plasminogen activator (uPA), will be assayed by electrophoretic mobility assays and the sensitivity to kinase inhibition will be determined. The effect of inhibiting transcription factor activation on steady state mRNA levels of oxidant-sensitive genes will be assayed in chick embryo tissues in vivo and cultured normal pig aorta endothelial cells. The proposed studies should provide evidence for oxidative pathways for toxic metal action on expression of specific genes, and provide insight into the mechanism by which metals cause their toxic effects. This, in turn, could provide fundamental insights into strategies designed to prevent metal induction of oxidative stress.