OXIDATIVE MECHANISMS IN CHROMIUM CARCINOGENESIS

铬(VI)化合物对职业和环境暴露的人类群体构成严重的健康威胁。暴露于铬(VI)会导致人类和实验动物患肺癌。这项研究项目的总体目标是阐明铬(VI)化合物作为致癌物的机制。本研究项目要检验的假设是：(1)高价5和4价铬的氧化态是通过DNA-金属直接相互作用导致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铬(VI)的还原形成基于配体的自由基，导致DNA氧化损伤，但不如高价铬氧化那么重要；(3)这些氧化损伤表现在修复缺陷的原核细胞系统中，这些原核细胞系统对体外研究中检测到的DNA损伤选择性敏感。本研究的具体目的是：(1)利用模型高价铬(V)化合物研究高价铬直接氧化或金属中心氧化DNA的机理。脱氧核糖的C_1‘、C_3’、C_4‘和C_5’位的H原子抽提产生的氧化产物将以5‘，3’-二-O-乙酰-d(TPT)为模型二核苷酸糖氧化底物，用高效液相色谱和GC/MS进行测定。将使用d(GPG)和d(CPC)的模型二核苷酸底物来确定鸟嘌呤和胞嘧啶碱氧化产物的形成。与寡核苷酸反应的碱基和序列特异性将通过凝胶电泳法确定，以形成直链断裂和碱不稳定位点。有氧和无氧气氛对上述反应的影响将被确定。(2)基于配体的谷胱甘肽、抗坏血酸和半胱氨酸自由基在DNA氧化产物形成中的作用将通过这些自由基物种的特异性(非铬)生成和它们与铬(VI)的还原原位形成来探讨。EPR将监测自由基的形成和命运。糖和碱氧化产物的测定以及直链断裂和碱不稳定部位的形成将按照特定目标1中的描述进行。(3)将确定铬(VI)在DNA修复缺陷菌株中的选择性致死性。添加抗坏血酸或调节细胞内谷胱甘肽水平的协同效应将被确定。将质粒转化到敏感的大肠杆菌菌株中，以便稍后提取和测定碱基和糖氧化产物和突变。拟议的研究应该能深入了解铬(Vl)诱导的DNA损伤对癌症形成至关重要的机制。了解这些机制可能会降低受感染人群的风险。

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.