Human Lung Chromium Toxicity: Role of cytochrome b5

描述(申请人提供)：支气管上皮是吸入铬(VI)化合物的主要目标，已知会导致严重的肺部毒性和致癌。这些效应背后的机制尚不清楚。在细胞中，铬(VI)暴露会导致氧化损伤和遗传毒性效应。六价铬的毒性作用需要还原，因为六价铬会产生活性物种[如铬(V)、铬(IV)、羟基自由基(OH)]。啮齿类动物和人类在铬(VI)激活方面存在关键差异，缺乏一个正常的人类支气管上皮铬(VI)毒性模型。我们的长期目标是了解人体肺中铬(VI)活化的机制。人类数据表明，微粒体酶起着重要作用，细胞色素b5(B5)起着中心作用。压倒一切的假设是，b5对支气管上皮中铬(VI)的激活有重要作用。细胞和体外方法的结合将被用来阐明这一作用。纯化的b5在体外用于脂质体和电化学研究，将检测铬(VI)的还原动力学，以证明b5是最接近的电子供体。在不同的铬(VI)浓度下，随着时间的推移，将定量地确定活性物种的生成及其调节各种形式的DNA损伤的能力。动力学ESR方法将被用来区分OH和其他羟化物种。铬(V)-DNA络合物的性质将通过ESR确定，这种结合对后续DNA损伤的影响将被确定。铬(VI)的细胞毒性，以及b5形成的反应物种和随后的DNA损伤之间的联系将在表达不同水平的b5的人支气管上皮细胞中进行检测。铁显着地刺激铬(VI)的激活，但细胞内铁在铬(VI)毒性中的作用尚未被探索。还原的铬(VI)激活、细胞毒性和DNA损伤将在其铁储存已被无毒的胞外铁络合剂耗尽的细胞中进行检测。这些研究详细分析了以前未探索过的支气管上皮中铬(VI)激活的机制。它们将提供对人体肺部铬(VI)毒性潜在机制的更深层次的了解，并将导致对b5在毒物激活中的作用的新的见解。

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