Abstract

The present work describes a two-stage approach to analyzing combustion-generated samples for their potential to produce oxidant stress. This approach is illustrated with the two commonly encountered transition metals, copper and iron. First, their abilities to generate hydroxyl radical were measured in a cell-free, phosphate-buffered saline solution containing ascorbate and/or citrate. Second, their abilities to induce heme oxygenase-1 in cultured human epidermal keratinocytes were assessed in cell culture. Combustion-generated copper oxide nanoparticles were active in both assays and were found to be soluble in culture medium. Depletion of glutathione in the cells or loading the cells with ascorbate greatly increased heme oxygenase-1 induction in the presence of copper. By contrast, iron oxide nanoparticles were active in the phosphate-buffered saline but not in cell culture, and they aggregated in culture medium. Soluble salts of copper and iron exhibited the same contrast in activities as the respective combustion-generated particles. The results suggest that the capability of combustion-generated environmental samples to produce oxidant stress can be screened effectively in a two step process, first in phosphate-buffered saline with ascorbate and subsequently in epithelial cell culture for those exhibiting activity initially. The results also point to an unanticipated interaction in cells of oxidant stress-generating metals with an antioxidant (ascorbate) that is usually missing in culture medium formulations. Thus, ascorbate supplementation of cultured human cells is likely to improve their ability to model the in vivo effects of particulate matter containing copper and other redox-active metals.