Human Lung Chromium Toxicity: Role of cytochrome b5

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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