OXIDATIVE MECHANISMS IN CHROMIUM CARCINOGENESIS

铬（VI）化合物对职业和环境暴露人群构成严重的健康风险。在人类和实验动物中，暴露于铬（VI）会产生肺癌。本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。本研究项目拟验证的假设是：(1)铬的高价+5和+4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，但其重要性低于高价铬的氧化作用；(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损伤具有选择性敏感性。本研究的具体目的是：(1)利用模型高价Cr(V)化合物来测量高价铬直接或以金属为中心氧化DNA的机制。在脱氧核糖的C1′、C3′、C4′和C5′上提取h原子所产生的氧化产物将采用高效液相色谱和气相色谱/质谱法，采用模型二核苷酸糖氧化底物5′，3′-二- o -乙酰基-d （TpT）。鸟嘌呤和胞嘧啶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。与寡核苷酸反应的碱基和序列特异性将通过凝胶电泳确定，以形成直接链断裂和碱不稳定位点。需氧气氛对厌氧气氛的影响将对上述反应进行测定。(2)谷胱甘肽、抗坏血酸和半胱氨酸的配体基自由基在DNA氧化产物形成中的作用将通过这些自由基的特异性（非铬）生成和通过Cr（VI）还原原位形成来探索。EPR将监测自由基的形成和命运。糖和碱氧化产物的测量以及直接链断裂和碱不稳定位点的形成将按照具体目标1的描述进行。(3)测定Cr（VI）在DNA修复缺陷大肠杆菌中的选择性致死率。添加抗坏血酸或调节细胞内谷胱甘肽水平的协同作用将被确定。将质粒转化为敏感的大肠杆菌菌株，以便随后提取和测量碱基和糖氧化产物和突变。提出的研究应深入了解铬（Vl）诱导的DNA损伤对癌症形成至关重要的机制。了解这些机制可以减少暴露人群的风险。

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.