Fluorescence imaging of reactive oxygen metabolites generated in single macrophage cells (NR8383) upon phagocytosis of natural zeolite (erionite) fibers.

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.

In vitro interaction of zeolite fibers with individual cells (macrophages NR8383): measurement of intracellular oxidative burst.

Inhalation of fibrous minerals such as asbestos and erionite can cause various lung diseases, including cancer. The mechanism by which these fibers induce disease is an area of active research. Interaction of fibers with lung macrophages leads to release of many substances. Among these, reactive oxygen metabolites (which include hydrogen peroxide, superoxide, and possibly hydroxyl radicals) are proposed to cause cellular damage. In this paper, we report a method for observing intracellular hydrogen peroxide release as rat lung-derived macrophages (NR-8383) phagocytize erionite fibers. This is possible by observing the fluorescence of 2',7'-dichlorofluorescein-the intracellular, oxidized form of 5 (and 6)-carboxy-2', 7'-dichlorodihydrofluorescin formed in the presence of newly released hydrogen peroxide. We are able to image the fluorescence within a single cell, thereby allowing us to get information on the spatial distribution of the metabolites.