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Fluorescence Imaging of Reactive Oxygen Metabolites Generated in Single Macrophage Cells (NR8383) upon Phagocytosis of Natural Zeolite (Erionite) Fibers
John F. Long,1 Prabir K. Dutta,2 and Brian D. Hogg2
1Department of Veterinary Biosciences; and 2Department of Chemistry, The Ohio State University, Columbus, Ohio 43210 USA
Abstract
In this paper we address the phenomenon of reactive oxygen metabolite generation subsequent to phagocytosis of mineral fibers by macrophages. Natural erionite fibers were chosen because of their established toxicity. Macrophages (cell line NR8383) were loaded with the dye 5-(and 6)-carboxy-2´,7´-dichlorodihydrofluorescein diacetate and exposed to erionite particles by centrifuging cells and fibers together to effect adherence. Reactive oxygen metabolite generation was examined by monitoring the fluorescence of oxidized dye formed via the reaction with oxygen species produced during phagocytosis. Individual cells were repeatedly scanned for up to 2 hr to monitor the evolution of this fluorescence. It was found that erionite-exposed cells had a mean total fluorescence of three times that of controls during the first 35 min, declining to two times that of controls at 35-60 min and about the same level as that of controls at 60-80 min. Ultrastructural studies of similarly treated aliquots of cells showed marked variation in size and numbers of the phagocytized particles. This study demonstrates that intracellular oxidation can be monitored on a single cell basis over a period of time. Quantitative studies are in progress to establish the relationship between the phagocytized particulate load and the extent of fluorescence. Key words: 5-(and 6)-carboxy-2´,7´-dichlorodihydrofluorescein diacetate, erionite, fluorescence imaging, macrophage, mineral fiber, peroxide. Environ Health Perspect 105:706-711 (1997)
Address correspondence to J.F. Long, Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, Ohio 43210 USA.
We acknowledge the support of the Office of Research, The Ohio State University, for support of this research. We are also grateful to R.J. Helmke for providing the macrophage line. We thank L. Mathes for helpful discussions.
