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 哪些有毒金属会影响细胞的氧化应激。氧化应激 与许多人类疾病有牵连，包括癌症， 衰老、动脉粥样硬化和纤维化。有一些证据表明有毒物质 金属可以诱导氧化应激，这可能是两者的机制 遗传性和非遗传性增殖性疾病。美国人的能力 有毒和/或致癌金属、铬、镍、镉、铅、铁 和砷，单独或以组合形式在受污染的 在鸡胚胎中，将确定诱导氧化应激的位置 活体内皮细胞和培养内皮细胞。对这些问题的具体假设 研究表明，金属可能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。 这种改变可能是由于直接影响DNA结构而发生的 因此，反式作用因子的DNA识别序列 或通过激活细胞信号以获得更大的丰度和/或 转录因子的活性。我们计划不仅研究可能的 有毒金属诱导的DNA氧化损伤，但也是 这些金属通过以下途径引起基因异常诱导的倾向 细胞信号的改变。拟议研究的具体目标 是：(1)确定有毒金属产生活性物质的能力 培养细胞中氧中间产物(ROI)生化分析 用于活性物种的积累和荧光光谱以 显示细胞内的氧化剂水平。活性氧的诱导 有毒金属造成的过氧化氢和超氧化物等物种可能 可用分光光度和荧光技术进行测定。关卡 的细胞内氧化剂水平可以通过使用可代谢的 罗丹明和二氯二乙酸酯等试剂进入 不可逆的细胞，并通过反应转化为荧光物质 氧气物种。(2)研究DNA氧化损伤的诱导作用 体内和体外培养的正常鸡胚组织中的有毒金属 通过检测猪主动脉内皮细胞DNA单链断裂、DNA- 蛋白质的交联度和8-氧-2‘-脱氧鸟苷的水平。如果是被动的 氧物种由有毒金属在细胞内产生，然后 ROI可能会攻击DNA，导致DNA氧化损伤。DNA链断裂 将通过DNA解旋试验进行分析，DNA-蛋白质交联链 用K+-十二烷基硫酸钠沉淀法和8-氧-2‘-脱氧鸟苷 高效液相色谱与电化学联用 检测(高效液相色谱-电子捕获检测器)。(3)展示金属的含量 转录因子依赖于磷酸化的特异性激活 并在该信令级联中建立速率确定站点。 反式作用因子的激活，它诱导已知的 在氧化应激后表达，即血红素加氧酶和尿激酶- 与纤溶酶原激活物(UPA)一样，将通过电泳法进行检测 流动性分析和对激酶抑制的敏感性将是 下定决心。抑制转录因子激活对血管内皮细胞生长因子的影响 氧化敏感基因的稳态信使核糖核酸水平将在 体内鸡胚组织和培养的正常猪主动脉内皮细胞 细胞。拟议的研究应该提供氧化的证据 有毒金属作用于特定基因表达的途径，以及 洞察金属导致其有毒物质的机制 效果。这反过来可以提供对以下方面的基本见解 旨在防止金属引起氧化应激的策略。

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.