Evaluation of two distinct methods to quantify the uptake of crocidolite fibers by mesothelial cells

Our plain-English summary of Evaluation of two distinct methods to quantify the uptake of crocidolite fibers by mesothelial cells, by K. Yamashita, H. Nagai, Y. Kondo, et al (Journal of Clinical Biochemistry and Nutrition, 2013).

The study

• Authors: K. Yamashita, H. Nagai, Y. Kondo, N. Misawa, S. Toyokuni
• Journal: Journal of Clinical Biochemistry and Nutrition
• Published: 2013;53(1):27-35
• Cited by: 8 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Exposure to asbestos fibers increases the risk of mesothelioma in humans. One hypothetical carcinogenic mechanism is that asbestos fibers may directly induce mutations in mesothelial cells. Although the uptake of asbestos fibers by mesothelial cells is recognized, methods for the quantification of the uptake have not been well established. In the present study, we evaluated two distinct methods, using crocidolite fibers and MeT5A mesothelial cells. One method is histological evaluation using the cell-block technique, which allows for the direct cross-sectional observation of cells and fibers. We found the bright field observation with ×1000 magnification (oil-immersion) of the sample with Kernechtrot staining was most suitable for this purpose. The other method is flow cytometric analysis, which permits the evaluation of a much larger number of cells. We observed that the side scatter (SSC) increased with the intracellular fibers, and that the "mean SSC ratio (treated/control)" was useful for quantification. We could collect the cells with abundant internalized crocidolite fibers by sorting. Results of the two methodologies were correlated well in the experiments. The quantities of internalized fibers increased with incubation time and loaded dosage, but they were inversely associated with cellular density in culture.

In plain English

This laboratory study tested two ways to measure crocidolite (blue asbestos) uptake by mesothelial cells in culture. The authors exposed the MeT5A mesothelial cell line to crocidolite, usually at 5 µg/cm2 for 2 hours, and varied exposure time, dose and cell crowding. One method examined thin sections of cell pellets under a microscope, where Kernechtrot staining made fibers inside cells easier to see than routine staining. The other method used flow cytometry, which measures light scattered by cells (side scatter). Electron microscopy showed most fibers were taken up as bundles. The share of cells holding 5 or more bundles or clumps of crocidolite rose from 22.6% after 1 hour to 68.5% after 24 hours. Cells sorted for high side scatter held far more fibers. The mean side-scatter ratio of exposed to control cells rose with dose (r = 0.995 in each of two tests) and fell as cell density rose. The authors reported that the two methods' results correlated well. They noted that flow cytometry is indirect and may count surface-attached fibers, and that detection size limits are both methods' weakest point.

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: laboratory (in vitro) cell culture study comparing two methods, cell-block histology and flow cytometry, for measuring crocidolite uptake by mesothelial cells
• Who was studied: MeT5A mesothelial cell line from the American Type Culture Collection; no humans or animals
• Exposure: UICC crocidolite. The standard condition was 5 µg/cm2 for 2 h. Incubation time (1, 4 and 24 h), dose (1, 3, 5, 10 and 20 µg/cm2) and cell density (20, 30, 50, 70 and 90% confluence) were varied in separate experiments.
• Main results:
  • In cell-block sections, crocidolite fibers inside cells were easier to detect with Kernechtrot staining than with routine HE staining.
  • Transmission electron microscopy showed most crocidolite fibers formed bundles when taken up by the cells, and the fibers were then found within membrane-bound vesicles in the cytoplasm.
  • At 5 µg/cm2, the amount of internalized fibers increased with incubation time. In cell-block counts, the cells including 5 or more bundles of crocidolite fiber or crocidolite in clumps were 22.6% at 1 h (472 cells counted), 38.3% at 4 h (470) and 68.5% at 24 h (559). The mean SSC ratio (treated/control) was 1.277, 1.435 and 2.07 at the same times.
  • After exposure to 20 µg/cm2, cells sorted into the high side scatter gate (P2) contained much more fiber than cells in the normal gate (P1). The cells including 5 or more bundles or crocidolite in clumps were 76.1% of 473 P2 cells versus 10% of 500 P1 cells.
  • The mean SSC ratio (treated/control) rose with applied dose from 1 to 20 µg/cm2 (correlation coefficient r = 0.995, p<0.001, in each of two repeat tests).
  • The mean SSC ratio (treated/control) was inversely associated with cell density from 20 to 90% confluence (r = -0.962, p = 0.0089, in Test 2-1; r = -0.991, p = 0.0011, in Test 2-2).
• The authors' conclusion: The authors concluded that both methods are useful for quantifying crocidolite uptake by mesothelial cells and that their results correlated well. They also concluded that uptake increased with incubation time and dose and was inversely associated with cell density.
• Limitations the authors state:
  • Flow cytometry is an indirect measure, and many other factors can influence the scatter signals.
  • Flow cytometry may not distinguish fibers inside cells from fibers attached to the cell surface.
  • The size limit of detection is the weakest point of both methods.
  • Most of the 'fibers' counted by light microscopy are actually fiber bundles.
  • Cell-block sections can show artifacts, such as fibers transferred onto cells during sectioning.

Topics

Medical terms in this study, explained in our Lexicon: Crocidolite, Mesothelial Cells, Mesothelioma, Carcinogenic.

Related studies

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• Distinct affinity of nuclear proteins to the surface of chrysotile and crocidolite — Y. Kubo, H. Takenaka, et al. (2012)
• DNA damage in bronchial epithelial and mesothelial cells with and without associated crocidolite asbestos fibers — J. Nygren, S. Suhonen, et al. (2004)
• Mesothelial cell proliferation and biopersistence of wollastonite and crocidolite asbestos fibers — J.L. Macdonald, A.B. Kane (1997)
• Effects of crocidolite and chrysotile asbestos on cellular uptake and metabolism of benzo(a)pyrene in hamster tracheal epithelial cells — B.T. Mossman, A. Eastman, et al. (1983)
• A novel method for efficient collection of normal mesothelial cells in vivo — L. Jiang, Y. Yamashita, et al. (2010)

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