Mitotic disturbances and micronucleus induction in Syrian hamster embryo fibroblast cells caused by asbestos fibers

Our plain-English summary of Mitotic disturbances and micronucleus induction in Syrian hamster embryo fibroblast cells caused by asbestos fibers, by E. Dopp, J. Saedler, H. Stopper, et al (Environmental Health Perspectives, 1995).

The study

• Authors: E. Dopp, J. Saedler, H. Stopper, D.G. Weiss, D. Schiffmann
• Journal: Environmental Health Perspectives
• Published: 1995;103(3):268-71
• Cited by: 28 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Asbestos and other mineral fibers have long been known to induce lung cancer and mesothelioma. However, the primary mechanisms of fiber-induced carcinogenesis still remain unclear. We investigated the occurrence of mitotic disturbances induced by asbestos (amosite, crocidolite, chrysotile) in an in vitro approach using Syrian hamster embryo (SHE) fibroblast cells. The following endpoints were investigated: micronucleus formation as a result of mitotic disturbances and characterization of the induced micronucleus population by kinetochore staining and visualization of the spindle apparatus. Supravital UV-microscopy was used to analyze changes in interphase chromatin structure, impaired chromatid separation, and blocked cytokinesis. All three asbestos fiber types induced a high frequency of micronucleus formation in SHE cells (> 200/2000 cells) in a dose-dependent manner (0.1-5.0 micrograms/cm2), with a maximum between 48 hr and 66 hr exposure time. At higher concentrations (more than 5.0 micrograms/cm2) the micronucleus formation decreased again as a result of increased toxicity. Kinetochore staining of micronuclei revealed that 48 +/- 2% of asbestos-induced micronuclei reacted positively with CREST (antikinetochore) serum. Furthermore, spindle apparatus deformations occurred in cells with disturbed metaphases and anaphases, while the spindle fiber morphology appeared unchanged. Our results show that asbestos fibers may cause both loss and breakage of chromosomes in the absence of direct interaction with spindle fibers.

In plain English

This was a laboratory experiment on cultured Syrian hamster embryo (SHE) fibroblast cells, not a study of people. The cells were exposed to amosite, crocidolite and Rhodesian chrysotile (UICC standard) asbestos. The authors counted micronuclei, which are small bodies of genetic material outside the main nucleus that can arise from chromosomes or pieces left out when a cell divides. All three fibers increased micronuclei in a dose-dependent way, peaking between 48 and 66 hours. At 66 hours and 5.0 micrograms per square centimeter, crocidolite-treated cells averaged 218.5 and chrysotile-treated cells 210.0 micronuclei per 2000 cells, versus 32.3 and 28.0 in untreated cells. Amosite peaked after 66 hours at 0.25 micrograms per square centimeter, with more than 250 micronuclei per 2000 cells. At very high doses, counts fell because of toxicity. Calcium sulfate, the comparison dust, had no significant effect. Differences between the three fibers were not statistically significant. About half of the micronuclei stained for kinetochores. The authors read this as evidence of both whole-chromosome loss and chromosome breakage. The authors concluded that asbestos fibers may cause both loss and breakage of chromosomes without direct interaction with spindle fibers.

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. It used a micronucleus assay, kinetochore (CREST serum) staining, staining of the spindle apparatus, and supravital UV microscopy of living cells. The article explains that micronuclei originate from chromosome fragments or whole chromosomes or chromatids not incorporated into daughter nuclei when cell division is completed.

• Who was studied: Syrian hamster embryo (SHE) fibroblast cells grown in culture. Micronuclei were counted per 2000 cells, in at least 3 experiments per dose.

• Exposure: Three asbestos fiber types: amosite, crocidolite and Rhodesian chrysotile (UICC standard).

• Average diameters were 0.10, 0.25 and 0.24 micrometers, and average lengths 2.24, 1.71 and 2.50 micrometers, for chrysotile, crocidolite and amosite respectively.

• About 5% of fibers of each type were longer than 5 micrometers.

• Main tested doses were 1.0, 5.0 and 10.0 micrograms per square centimeter for crocidolite and chrysotile. Amosite was also tested at lower doses, with its maximum at 0.25.

• Concentrations up to 184 (chrysotile), 200 (crocidolite) and 120 (amosite) micrograms per square centimeter were also tested.

• Exposure times ranged from 18 to 72 hours.

• Calcium sulfate was used as a negative control.

• Main results:

  • All three fiber types induced micronuclei in SHE cells in a dose-dependent manner.
  • Micronucleus frequency depended on exposure time. It reached a maximum between 48 and 66 hours, with a frequency of up to 15%.
  • Crocidolite-treated cells at 5.0 micrograms per square centimeter for 66 hours had a mean of 218.5 plus or minus 80.3 micronuclei per 2000 cells. Untreated control cells at 66 hours had 32.3 plus or minus 4.2.
  • Chrysotile-treated cells at 5.0 micrograms per square centimeter for 66 hours had a mean of 210.0 plus or minus 56.5 micronuclei per 2000 cells. Untreated control cells at 66 hours had 28.0 plus or minus 4.1.
  • For crocidolite and chrysotile, the difference between asbestos-treated and untreated cells was significant in all cases, with p less than 0.001.
  • Chrysotile and crocidolite raised the micronucleus level sevenfold after 66 hours at 5 micrograms per square centimeter.
  • Amosite raised micronucleus formation ninefold after 66 hours at 0.25 micrograms per square centimeter. Its maximum was more than 250 micronuclei per 2000 cells.
  • The authors described amosite as the most potent fiber. However, the differences in micronucleus formation between the three fiber types were not statistically significant.
  • At the highest concentrations tested, toxicity was very high and micronuclei fell to 24.5 plus or minus 3.7 per 2000 cells.
  • Calcium sulfate (the negative control) did not significantly change micronucleus formation compared with untreated cells.
  • The share of micronuclei that stained positive for kinetochores was:

• chrysotile at 10 micrograms per square centimeter: 48.2 plus or minus 3.7% (220 micronuclei);

• amosite at 5: 47.4 plus or minus 5.9% (133 micronuclei);

• crocidolite at 1: 49.0 plus or minus 1.0% (100 micronuclei).

The control value of 27.0 plus or minus 4.0% was taken from an earlier study by Schiffmann and De Boni.

• The authors interpreted the kinetochore results as evidence that both loss of whole chromosomes and chromosome breakage (clastogenic events) were involved.

• Live-cell UV microscopy of treated cells showed several abnormalities:

• changes in interphase chromatin (budding, compaction, nuclear holes);

• impaired chromatid separation, chromatin bridges and chromosome displacement;

• blocked cytokinesis in numerous cases.

These events were not seen in untreated cells, where more than 50 mitoses were analyzed.

• The authors noted that micronucleus formation also seemed to occur during interphase, as with chromatin budding.
• In amosite-treated cells, about 10% showed a slightly deformed spindle apparatus, although the spindle fibers appeared normal. Deformed spindles occurred only together with disturbed metaphases and anaphases.
• The authors' conclusion: The authors concluded that asbestos fibers may cause both loss and breakage of chromosomes in the absence of direct interaction with spindle fibers. They called for further studies of the direct interaction between asbestos fibers and chromatin or chromosomes.

Topics

Medical terms in this study, explained in our Lexicon: Fibroblast, Mineral Fiber, Lung Cancer, Mesothelioma, Carcinogenesis, Amosite, Crocidolite, Chrysotile.

Related studies

• Kerosene soot genotoxicity: enhanced effect upon co-exposure with chrysotile asbestos in Syrian hamster embryo fibroblasts — M. Lohani, E. Dopp, et al. (2000)
• Mineral fibers induce apoptosis in Syrian hamster embryo fibroblasts — E. Dopp, B. Nebe, et al. (1995)
• In vitro cytotoxicity and transforming potential of industrial carbon dust (fibers and particles) in syrian hamster embryo (SHE) cells — C. Darne, F. Terzetti, et al. (2010)
• Detection of aneuploidy-inducing carcinogens in the Syrian hamster embryo (SHE) cell transformation assay — D.P. Gibson, M.J. Aardema, et al. (1995)
• Asbestos and benzo(a)pyrene synergism in the transformation of Syrian hamster embryo cells — J.A. DiPaolo, A.J. DeMarinis, et al. (1983)
• Characterization of activated proto-oncogenes in chemically transformed Syrian hamster embryo cells — T.M. Gilmer, L.A. Annab, et al. (1988)

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