A simple fluorescence method for the study of the internalization of particles in cultured cells: Application to asbestos and glass fibres

Our plain-English summary of A simple fluorescence method for the study of the internalization of particles in cultured cells: Application to asbestos and glass fibres, by S.O. Mikalsen, K.R. Modalsli (Toxicology in Vitro, 1994).

The study

• Authors: S.O. Mikalsen, K.R. Modalsli
• Journal: Toxicology in Vitro
• Published: 1994;8(2):243-50
• Read it: PubMed · Publisher

Abstract

A model for the study of internalization of particles in mammalian cells was applied to asbestos and glass fibres. Briefly, a fluorescent fluid-phase endocytic marker, Lucifer Yellow CH (LY), was allowed to be incorporated into the lysosomal compartment of Syrian hamster embryo cells. Mineral fibres that were internalized by the cells subsequently became 'fluorescent', presumably when the fibre-containing endosome fused with the LY-containing lysosomes. This method was compared with differential interference contrast (DIC) optics. Approximately three times as many of the cell-associated fibres were determined to be internalized by the fluorescence method compared with DIC optics. Both fine and coarse glass fibres were internalized as effectively as asbestos fibres. The relative frequency of internalized (i.e. fluorescent) fibres increased until 4 hr after exposure compared with the total number of cell-associated fibres. The frequency of internalized fibres compared with the number of cell-associated fibres was constant over the range of fibre levels studied. A surface modification (octadecyldimethylchlorosilane-derivatization) of amosite fibres that decreased the carcinogenicity of the fibres, decreased slightly the number of internalized fibres relative to the number of cell-associated fibres, but this was not statistically significant. Cytoskeleton-interfering agents significantly decreased the relative number of internalized fibres.

In plain English

This laboratory study tested a simple fluorescence method for detecting mineral fibres taken inside cultured Syrian hamster embryo cells. Cells were preloaded with a fluorescent dye, Lucifer Yellow, which collects in internal compartments called lysosomes. Fibres that were taken up then appeared fluorescent. After 2 hours, the fluorescence method counted on average 30% to 45% of cell-associated fibres as internalized. A standard microscope technique (DIC) counted 8% to 11%. These figures covered anthophyllite asbestos and two glass fibres of very different sizes. The authors reported that the fluorescent share reached its maximum within 4 to 8 hours. At 24 hours it was similar for anthophyllite and lower for glass fibre 100. For amosite asbestos, the share internalized was essentially identical at three doses. Amosite with a chemically modified surface showed a slight decrease that was not statistically significant: 89 of 224 fibres were fluorescent, versus 113 of 236 for unmodified amosite. Three agents that interfere with the cell's internal skeleton each significantly reduced the share of fluorescent fibres. The authors acknowledged it may be questioned whether all fluorescent fibres were truly inside cells, and gave reasons for thinking they were.

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 developing a fluorescence method and comparing it with differential interference contrast (DIC) microscopy
• Who was studied: Early passage Syrian hamster embryo (SHE) cells grown on glass chamber slides; no human subjects
• Exposure: The fibres tested, mostly for 2 hours, were anthophyllite asbestos (0.3 micrograms per square centimetre), glass fibre 100 (0.15), glass fibre 110 (2), amosite (0.1, 0.5 and 1.0) and C-18 surface-modified amosite (0.5). Amosite was also tested together with three agents that interfere with the cytoskeleton: taxol, nocodazole or cytochalasin B.
• Main results:
  • After 2 hours, the mean percentage of cell-associated fibres judged internalized was higher by fluorescence than by DIC. For anthophyllite it was 30 vs 8 (390 fibres). For glass fibre 100 it was 45 vs 11 (273 fibres). For glass fibre 110 it was 36 vs 11 (199 fibres).
  • The authors reported that the number of fluorescent cell-associated fibres reached its maximum within 4 to 8 hours. For anthophyllite, 14/162 fibres (8.7%) were fluorescent at 1 hr, 78/202 (38.5%) at 8 hr and 84/223 (38.9%) at 24 hr. For glass fibre 100, the counts were 19/152 (12.4%) at 1 hr, 106/190 (55.4%) at 8 hr and 89/199 (44.5%) at 24 hr. The decrease for glass fibre 100 from 8 to 24 hr had P = 0.03.
  • For amosite, the relative internalization frequency was essentially identical at 0.1, 0.5 and 1.0 micrograms per square centimetre (125/233, 113/236 and 120/225 fibres fluorescent).
  • For C-18-modified amosite, 89 of 224 cell-associated fibres were fluorescent, versus 113 of 236 for unmodified amosite at the same dose (P = 0.09). The decrease was not statistically significant.
  • With amosite alone, 108 of 221 fibres were fluorescent. The share was significantly lower with taxol, which stabilizes microtubules (78 of 219, P = 0.005). It was also lower with nocodazole, which depolymerizes microtubules (50 of 203, P less than 0.001), and with cytochalasin B, which depolymerizes microfilaments (33 of 203, P less than 0.0001). None of the agents completely stopped the appearance of fluorescent fibres.
• The authors' conclusion: The authors attributed the gap between the DIC and fluorescence counts to the limited focal depth of DIC optics in the flat SHE cells. In such cells, a fibre on the cell surface cannot be told apart from an internalized one. The authors gave several arguments that the fluorescent fibres were internalized.
• Limitations the authors state:
  • The authors noted it may be questioned whether all fluorescent fibres were actually internalized.
  • Occasionally, fibres clearly internalized by DIC showed no fluorescence.
  • The cause of the decrease in fluorescent glass fibre 100 fibres at 24 hours was not known or investigated.
  • The apparently lower attachment of C-18-modified amosite fibres to cells was observed but not measured.

Topics

Medical terms in this study, explained in our Lexicon: Amosite.

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