INDUCTION OF OXIDATIVE STRESS AND ACTIVATION OF TRANSCRIPTION BY METALS

金属诱导氧化应激和激活转录

• 批准号: 6271279
• 负责人: KAREN E WETTERHAHN
• 金额: $ 14.92万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271279
• 关键词: DNA damage; arsenic; biological signal transduction; chemical kinetics; chick embryo; chromium; cytotoxicity; environmental toxicology; free radical oxygen; gel mobility shift assay; gene expression; genetic transcription; heavy metals; high performance liquid chromatography; nucleic acid probes; oxidative stress; spectrometry; tissue /cell culture; transcription factor

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.

项目1的总体目标是了解 有毒金属会影响细胞氧化应激。 氧化应激 与包括癌症在内的许多人类疾病有关 衰老，动脉粥样硬化和纤维化。 有证据表明有毒 金属可以诱导氧化应激，这可能是两者的机制 遗传毒性和非基因毒性增殖性疾病。 能力 有毒和/或致癌金属，铬，镍，镉，铅，铁 和砷，单独和在受污染下发现的组合 位置，诱导氧化应激，将在雏鸡的胚胎中确定 体内和培养的内皮细胞。 这些的特定假设 研究是金属可能导致癌症或其他增殖性疾病 通过改变转录水平上特定基因的表达。 这种改变可能是由于对DNA结构的直接影响而发生的 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。 我们计划不仅检查可能 诱导有毒金属诱导氧化DNA损伤，但也 这些金属的倾向通过 细胞信号的改变。 拟议研究的具体目的 为：（1）确定有毒金属产生反应性的能力 使用生化分析在培养细胞中的氧中间体（ROI） 为了积累反应性物种和荧光光谱 证明细胞内氧化剂水平。 诱导活性氧 物种，例如过氧化氢和超氧化物，有毒金属可以 通过分光光度法和荧光测定技术进行测定。 水平 可以通过使用可代谢来测量细胞内氧化剂水平的水平 进入的试剂，例如进入的试剂 细胞不可逆转，并通过反应性转化为荧光物种 氧。 （2）研究氧化DNA损伤的诱导 通过体内和培养的正常的雏鸡胚胎组织中的有毒金属 猪主动脉内皮细胞通过测量DNA单链断裂，DNA- 蛋白质交联和8-oxo-2'-脱氧鸟苷的水平。 如果反应 氧气是由有毒金属细胞内产生的，然后是 ROI可能会攻击引起氧化DNA病变的DNA。 DNA链断裂 将通过使用DNA放松测定，DNA-蛋白交联，分析 通过使用K+-SDS降水测定法和8-oxo-2'-脱氧鸟苷 使用高性能液相色谱与电化学 检测（HPLC-ECD）。 （3）证明导致的金属水平 转录因子的特异性，磷酸化依赖性激活 并在此信号级联中确定确定位点的速率。 激活跨作用因子，这些因子诱导了已知的基因 在氧化剂应力后表达，即血红素加氧酶和尿激酶 - 像纤溶酶原激活剂（UPA）一样，将通过电泳测定 流动性测定和对激酶抑制的敏感性将是 决定。 抑制转录因子激活对 稳态mRNA水平的氧化剂敏感基因将分析 雏鸡胚胎组织体内和培养的正常猪主动脉内皮 细胞。 拟议的研究应提供氧化的证据。 有毒金属对特定基因表达的作用的途径， 提供有关金属引起其有毒的机制的见解 效果。 反过来，这可以提供基本的见解 旨在防止金属诱导氧化应激的策略。