Human Lung Chromium Toxicity: Role of cytochrome b5

描述（由申请人提供）：支气管上皮是吸入性铬（VI）化合物的主要靶点，已知铬（VI）化合物会引起严重的肺毒性和致癌作用。这些影响背后的机制尚不清楚。在细胞中，Cr（VI）暴露导致氧化损伤和遗传毒性作用。Cr（VI）的还原可产生反应性物种[例如Cr（V）、Cr（IV）、羟基自由基（OH）]，是其毒性作用所必需的。啮齿类动物和人类在Cr（VI）活化方面存在关键差异，并且缺乏Cr（VI）毒性的正常人支气管上皮模型。我们的长期目标是了解负责人肺中Cr（VI）激活的机制。人类数据表明微粒体酶的重要作用，细胞色素b5（b5）的核心作用。最重要的假设是，b5有助于显着的Cr（VI）激活支气管上皮。细胞和体外方法的整合将被用来阐明这一作用。在体外使用纯化的b5的脂质体和电化学研究将检查Cr（VI）还原的动力学，以证明b5是最接近的电子供体。反应性物种的产生及其介导各种形式的DNA损伤的能力将在不同Cr（VI）浓度下随时间定量测定。将使用动力学ESR方法来区分 OH从其他羟基化物种。Cr（V）-DNA复合物的性质将通过ESR确定，并且将确定这种结合对随后的DNA损伤的影响。铬（VI）的细胞毒性，和反应性物种形成的b5和随后的DNA损伤之间的联系将在人支气管上皮细胞表达不同水平的b5。铁显着刺激Cr（VI）的激活，但细胞内的铁在Cr（VI）毒性的作用尚未探讨。还原性Cr（VI）激活，细胞毒性和DNA损伤将在铁储存已被无毒的细胞外铁螯合剂耗尽的细胞中进行检查。这些研究提供了一个以前未探索的机制，铬（VI）激活支气管上皮细胞的详细分析。他们将提供一个更深入的了解机制的铬（六）毒性在人类肺，并将导致新的见解的作用，b5在毒物活化

DESCRIPTION (provided by applicant): The bronchial epithelium is a primary target of inhaled chromium(VI) compounds, which are known to cause serious pulmonary toxicity and carcinogenesis. The mechanisms underlying these effects are not clear. In cells, Cr(VI) exposure results in oxidative damage and genotoxic effects. The reduction of Cr(VI), which can generate reactive species [e.g. Cr(V), Cr(IV), hydroxyl radical (OH)], is required for its toxic effects. There are key differences between rodents and humans in Cr(VI) activation, and a normal human bronchial epithelium model for Cr(VI) toxicity is lacking. Our long-term objectives are to understand the mechanisms responsible for Cr(VI) activation in human lung. Human data indicate a significant role for microsomal enzymes, with a central role for cytochrome b5 (b5). The overriding hypothesis is that b5 contributes significantly to Cr(VI) activation in bronchial epithelium. An integration of cellular and in vitro approaches will be used to elucidate this role. The in vitro use of purified b5 in liposomes and electrochemical studies will examine the kinetics of Cr(VI) reduction to prove that b5 is the proximate electron donor. The generation of reactive species and their ability to mediate various forms of DNA damage will be determined quantitatively over time at various Cr(VI) concentrations. A kinetic ESR approach will be used to distinguish OH from other hydroxylating species. The nature of Cr(V)-DNA complexes will be determined by ESR, and the effect of this binding on subsequent DNA damage will be determined. The cytotoxicity of Cr(VI), and the link between reactive species formation by b5 and subsequent DNA damage will be examined in human bronchial epithelial cells expressing various levels of b5. Iron markedly stimulates Cr(VI) activation but a role for intracellular iron in Cr(VI) toxicity has not been explored. Reductive Cr(VI) activation, cytotoxicity, and DNA damage will be examined in cells whose iron stores have been depleted by a non-toxic extracellular iron chelator. These studies provide a detailed analysis of a previously unexplored mechanism of Cr(VI) activation in bronchial epithelium. They will provide a deeper understanding of the mechanisms underlying Cr(VI) toxicity in human lung, and will lead to new insights on the role of b5 in toxicant activation