OXIDATIVE MECHANISMS IN CHROMIUM CARCINOGENESIS

铬致癌的氧化机制

基本信息

批准号：
6382361
负责人：
KENT D SUGDEN
金额：
$ 20.9万
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.

Chromium（VI）化合物对职业和环境暴露的人口构成严重的健康风险。接触CR（VI）在人类和实验动物中产生肺癌。该研究项目的总体目的是阐明铬（VI）化合物作为致癌物的机制。在该研究项目中要测试的假设是：（1）铬的高瓦伦特+5和+4氧化态是通过直接DNA - 金属相互作用导致氧化DNA损伤的主要中间体；（2）通过细胞内重要的还原剂（例如谷胱甘肽，抗坏血酸，抗坏血酸和半胱氨酸形成基于配体的自由基）减少Cr（VI），导致氧化性DNA病变，但与高瓦伦特铬的氧化相比，具有较低的意义；（3）这些氧化病变表现在缺乏修复的原核生物细胞系统中，这些细胞系统对体外研究中检测到的DNA病变有选择性敏感。拟议的研究的具体目的是：（1）使用高瓦伦特CR（V）化合物来测量高瓦伦特铬对DNA的直接或金属中心氧化的机理。脱氧核糖的C1，C3'，C4'和C5'引起的氧化产物将使用模型的Dinucleotide糖氧化底物，5'，3'-DI-DI-DI-O-O-乙酰基-D（TPT）确定。将使用D（GPG）和D（CPC）模型的Dinucleotide底物确定鸟嘌呤和胞嘧啶碱氧化产物的形成。与寡核苷酸的反应的碱基和序列特异性将由凝胶电泳确定，以形成弗兰克链断裂和碱性含量位点。有氧和厌氧气氛的影响将在上述反应上确定。（2）基于谷胱甘肽，抗坏血酸和半胱氨酸在DNA氧化产物形成中的基于配体的自由基将通过这些自由基物种的特定（非铬）生成以及通过与CR（VI）还原通过其原位形成来探测。自由基的形成和命运将由EPR监测。将按照特定目标1所述进行糖和碱氧化产物的测量以及晶体链断裂和碱性位点的形成。（3）将确定Cr（VI）在DNA修复缺陷型大肠杆菌中的选择性致死性（VI）。将确定添加的抗坏血酸或细胞内谷胱甘肽水平的调节的协同作用。将质粒转化为敏感的大肠杆菌菌株，以便以后提取和测量碱和糖氧化产物和突变。拟议的研究应深入了解铬（VL）诱导的DNA损伤对癌症的形成至关重要。了解这些机制可能会降低暴露于人口的风险。