OXIDATIVE MECHANISMS IN CHROMIUM CARCINOGENESIS

铬致癌的氧化机制

基本信息

批准号：
6525250
负责人：
KENT D SUGDEN
金额：
$ 18.14万
依托单位：
UNIVERSITY OF MONTANA
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2004-07-31
项目状态：
已结题

项目摘要

Chromium(VI) compounds pose a serious health risk to occupationally and environmentally exposed human populations. Exposure to Cr(VI) produces lung carcinomas in humans and laboratory animals. The overall objective of this research project is to elucidate the mechanism by which chromium(VI) compounds act as carcinogens. The hypotheses to be tested in this research project are: (1) that high valent +5 and +4 oxidation states of chromium are the primary intermediates that lead to oxidative DNA damage via direct DNA-metal interactions; (2) that reduction of Cr(VI) by intracellularly important reductants such as glutathione, ascorbate and cysteine form ligand-based radicals leading to oxidative DNA lesions but are of a lesser significance than oxidation by high valent chromium; (3) that these oxidative lesions are manifested in repair-deficient prokaryotic cell systems which are selectively sensitive to the DNA lesions detected in the in vitro studies. The specific aims of the proposed research are: (1) The mechanism of direct- or metal-centered oxidation of DNA by high valent chromium will be measured using model high valent Cr(V) compounds. Oxidation products arising from H-atom abstraction at the C1', C3', C4' and C5' of deoxyribose will be determined by HPLC and GC/MS using the model dinucleotide sugar oxidation substrate, 5',3'-di-O-Acetyl- d(TpT). Formation of guanine and cytosine base oxidation products will be determined using model dinucleotide substrates of d(GpG) and d(CpC). Base- and sequence-specificity of reactions with oligonucleotides will be determined by gel electrophoresis for formation of frank strand breaks and alkali-labile sites. The effect of aerobic vs anaerobic atmospheres will be determined on the above reactions. (2) The role of ligand-based radicals of glutathione, ascorbate and cysteine in the formation of DNA oxidation products will be probed by the specific (non-chromium) generation of these radical species and through their in situ formation by reduction with Cr(VI). The formation and fate of the radicals will be monitored by EPR. Measurement of sugar and base oxidation products as well as the formation of frank strand breaks and alkali-labile sites will be carried out as described in specific aim 1. (3) Selective lethality of Cr(VI) in DNA repair-deficient strains of E. coli will be determined. The synergistic effects of added ascorbate or modulation of intracellular glutathione levels will be determined. Transformation of a plasmid into the sensitive E. coli strains will be carried out for later extraction and measurement of base and sugar oxidation products and mutations. The proposed studies should give insight into the mechanisms of chromium(Vl)-induced DNA damage critical to the formation of cancer. Understanding these mechanisms may allow reduction of risk to exposed human populations.

六价铬化合物对职业和环境暴露人群构成严重的健康风险。人类和实验动物接触 Cr(VI) 会产生肺癌。该研究项目的总体目标是阐明六价铬化合物作为致癌物的机制。本研究项目要测试的假设是：（1）铬的高价+5和+4氧化态是通过直接DNA-金属相互作用导致DNA氧化损伤的主要中间体； (2)细胞内重要的还原剂如谷胱甘肽、抗坏血酸和半胱氨酸对Cr(VI)的还原形成基于配体的自由基，导致DNA氧化损伤，但其重要性不及高价铬的氧化； (3)这些氧化损伤表现在修复缺陷的原核细胞系统中，该系统对体外研究中检测到的DNA损伤选择性敏感。拟议研究的具体目标是：（1）将使用高价 Cr(V) 化合物模型来测量高价铬直接或以金属为中心氧化 DNA 的机制。脱氧核糖 C1'、C3'、C4' 和 C5' 处 H 原子提取产生的氧化产物将使用模型二核苷酸糖氧化底物 5',3'-di-O- 通过 HPLC 和 GC/MS 测定乙酰基-d(TpT)。鸟嘌呤和胞嘧啶碱基氧化产物的形成将使用d(GpG)和d(CpC)的模型二核苷酸底物来确定。寡核苷酸反应的碱基和序列特异性将通过凝胶电泳确定坦率链断裂和碱不稳定位点的形成。有氧与厌氧气氛的影响将根据上述反应来确定。 (2) 谷胱甘肽、抗坏血酸和半胱氨酸的配基自由基在 DNA 氧化产物形成中的作用将通过这些自由基种类的特异性（非铬）生成以及通过用 Cr 还原它们的原位形成来探测（六）。激进分子的形成和命运将受到 EPR 的监控。糖和碱氧化产物的测量以及直链断裂和碱不稳定位点的形成将按照具体目标 1 中的描述进行。 (3) Cr(VI) 在 DNA 修复缺陷菌株中的选择性致死作用大肠杆菌将被确定。将确定添加抗坏血酸或调节细胞内谷胱甘肽水平的协同效应。将质粒转化到敏感的大肠杆菌菌株中，以便随后提取和测量碱基和糖氧化产物和突变。拟议的研究应该深入了解铬 (VI) 诱导的 DNA 损伤的机制，这对癌症的形成至关重要。了解这些机制可能有助于降低暴露人群的风险。