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

Our plain-English summary of Fluorescence imaging of reactive oxygen metabolites generated in single macrophage cells (NR8383) upon phagocytosis of natural zeolite (erionite) fibers, by J.F. Long, P.K. Dutta, B.D. Hogg (Environmental Health Perspectives, 1997).

The study

• Authors: J.F. Long, P.K. Dutta, B.D. Hogg
• Journal: Environmental Health Perspectives
• Published: 1997;105(7):706-11
• Cited by: 19 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This laboratory study exposed cells from a rat lung macrophage cell line (NR8383) to a natural erionite sample from Pine Valley, Nevada. Erionite is a naturally occurring zeolite mineral fiber. The authors used a dye that glows when oxidized to track reactive oxygen metabolites inside single cells, scanning the same cells repeatedly for about 90 minutes. Erionite-exposed cells had mean fluorescence more than three times that of unexposed control cells at 20-40 minutes and more than twice at 40-60 minutes. By 60-80 minutes the two groups were relatively indistinguishable. Exposed cells varied much more than controls; some held five or six particles and others one or none. A test on whole cell populations also detected released oxygen metabolites. A dead-cell stain showed no increase over 80 minutes, and electron microscopy found fibers inside lysosomes. The authors concluded the method allows study inside cells of changes associated with mineral fiber exposure. They noted they could not scan during the first 10 minutes and that particle size and number varied between cells.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: In vitro laboratory experiment on cultured cells (cell culture), using laser fluorescence imaging of single cells, a cytochrome c assay, a propidium iodide viability test and electron microscopy
• Who was studied: Cells from a rat lung alveolar macrophage cell line (NR8383), obtained from the University of Texas (San Antonio) and studied at The Ohio State University. The number of cells imaged is not stated.
• Exposure: A naturally occurring erionite (natural zeolite) mineral sample (#27091, Pine Valley, Nevada), with particles ranging from colloidal size to fibers about 10-15 micrometers long. The sample was settled to remove the larger particles and was added at about 1.4 x 10^6 particles/ml for imaging. Control cells received buffer only. Zymosan (heat-killed yeast particles) was used to show that the cells could produce an oxidative burst.
• Main results:
  • For the 20-40 min period, erionite-exposed cells had a mean cellular fluorescence more than three times that of control cells.
  • During the 40-60 min period, erionite-exposed cells had a mean cellular fluorescence still more than twice that of control cells.
  • Only during the 60-80 min period did the mean fluorescence of erionite-exposed cells decline to a level relatively indistinguishable from that of controls.
  • Fluorescence varied markedly among erionite-exposed cells and less among control cells. The authors suggested this may relate to differences in particle size and number: some cells showed five or six particles within lysosomes, while others had as few as one or none.
  • In the cytochrome c assay on cell populations after 1 hour, Table 1 lists cytochrome c released as 1.05 for erionite-exposed cells, 1.08 for zymosan-exposed cells and 0.07 for buffer controls. The text states that the erionite and zymosan increases were each equivalent to 3.2 nmol reduced cytochrome.
  • Erionite-exposed cells showed no increase in propidium iodide (dead-cell stain) fluorescence during the 80 minutes tested.
  • By electron microscopy, 20 minutes after contact the fibers were characteristically inside the cells within lysosomes. Lysosomal membranes ranged from intact-appearing to visibly interrupted. No specific change was noted in other membranes or organelles during the 90-minute time frame.
• The authors' conclusion: The authors concluded that the evidence suggests the increased fluorescence indicates where in the cell reactive oxygen metabolite generation occurs. They stated that the method provides an opportunity for intracellular study of the pathophysiologic changes associated with cellular exposure to mineral fibers.
• Limitations the authors state:
  • The design did not allow cells to be scanned during the first 10 minutes after fiber contact, so the authors stated they might be missing the early part of the oxidative burst.
  • Exposure was not uniform: single cells interacted with fibers of variable size and number. The authors said uniform fiber-cell exposure would be needed to refine the study.
  • The fluorescent foci inside cells could not be identified as specific cell structures from the images.

Topics

Medical terms in this study, explained in our Lexicon: Macrophage, Phagocytosis, Zeolite, erionite, Mineral Fiber.

Related studies

• In vitro interaction of zeolite fibers with individual cells (macrophages NR8383): measurement of intracellular oxidative burst — B.D. Hogg, P.K. Dutta, et al. (1996)
• Production of reactive oxygen intermediates by human macrophages exposed to soot particles and asbestos fibers and increase in NF-kappa B p50/p105 mRNA — R. Oettinger, K. Drumm, et al. (1999)
• Iron and reactive oxygen species in the asbestos-induced tumor necrosis factor-alpha response from alveolar macrophages — P.P. Simeonova, M.I. Luster (1995)
• Cellular mechanisms of reactive oxygen metabolite generation from human polymorphonuclear leukocytes induced by crocidolite asbestos — T. Ishizaki, E. Yano, et al. (1997)
• Analysis of the biological and chemical reactivity of zeolite-based aluminosilicate fibers and particulates — E. Fach, W.J. Waldman, et al. (2002)
• Proteomic Profiling of Alveolar Macrophages Identifies Loss of Lysosomal Content as an Indicator of Nanofiber-Induced Frustrated Phagocytosis — T. Stobernack, A. Vennemann, et al. (2026)

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