Human Lung Chromium Toxicity: Role of cytochrome b5

描述（由申请人提供）：支气管上皮是吸入铬（VI）化合物的主要目标，已知铬（VI）化合物会导致严重的肺毒性和致癌性。这些影响背后的机制尚不清楚。在细胞中，铬（VI）暴露导致氧化损伤和基因毒性作用。Cr（VI）的还原可以产生活性物质[例如Cr(V), Cr(IV)，羟基自由基（OH）]，这是其毒性作用所必需的。啮齿类动物和人类在Cr（VI）激活方面存在关键差异，缺乏正常人类支气管上皮Cr（VI）毒性模型。我们的长期目标是了解人体肺中Cr（VI）活化的机制。人类数据表明微粒体酶起着重要作用，其中细胞色素b5 （b5）起着核心作用。最重要的假设是b5对支气管上皮中Cr（VI）的激活有显著作用。细胞和体外方法的整合将用于阐明这一作用。纯化b5在脂质体中的体外使用和电化学研究将检验Cr（VI）还原动力学，以证明b5是近似的电子供体。随着时间的推移，在不同的Cr（VI）浓度下，活性物质的产生及其介导各种形式DNA损伤的能力将被定量地确定。动力学ESR方法将用于区分OH与其他羟基化物质。Cr(V)-DNA复合物的性质将由ESR确定，并确定这种结合对随后DNA损伤的影响。我们将在表达不同水平b5的人支气管上皮细胞中研究Cr（VI）的细胞毒性，以及b5形成的活性物质与随后的DNA损伤之间的联系。铁显著刺激Cr（VI）的活化，但细胞内铁在Cr（VI）毒性中的作用尚未探讨。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。这些研究提供了先前未探索的支气管上皮中Cr（VI）活化机制的详细分析。它们将提供对人体肺部Cr（VI）毒性机制的更深入了解，并将对b5在毒物激活中的作用产生新的见解

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