Effects of asbestos on initiation of DNA damage, induction of DNA-strand breaks, P53-expression and apoptosis in primary, SV40-transformed and malignant human mesothelial cells

Our plain-English summary of Effects of asbestos on initiation of DNA damage, induction of DNA-strand breaks, P53-expression and apoptosis in primary, SV40-transformed and malignant human mesothelial cells, by B. Burmeister, T. Schwerdtle, I. Poser, et al (Mutation Research, 2004).

The study

• Authors: B. Burmeister, T. Schwerdtle, I. Poser, E. Hoffmann, A. Hartwig, W.U. Müller, A.W. Rettenmeier, N.H. Seemayer, E. Dopp
• Journal: Mutation Research
• Published: 2004;558(1-2):81-92
• Cited by: 30 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Human mesothelial cells (HMC), the progenitor cells of asbestos-induced mesothelioma, are particularly sensitive to the genotoxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in HMC are not well known. The high susceptibility of HMC to simian virus 40 (SV40)-mediated transformation is assumed to play a causative role in the pathogenesis of mesothelioma. The aim of this study was to investigate the asbestos-induced DNA damage in cultured HMC and SV40-transformed HMC (MeT-5A) compared with their malignant counterparts, i.e. human mesothelioma cells (MSTO). The time-dependent initiation of DNA-strand breaks as well as the induction of oxidative DNA base modifications were key factors for investigation. HMC, MeT-5A and MSTO cells were exposed to chrysotile and crocidolite asbestos (3 microg/cm2) during different time periods (1-72 h). DNA damage was investigated by use of the Comet assay and alkaline unwinding, the latter in combination with the Fpg protein. The P53 level was analyzed by immunofluorescence, and measurement of apoptosis was conducted by flow cytometry. We found a significant induction of DNA damage in asbestos-treated HMC already after an exposure time of 1.5 h. This effect could not be observed in treated MeT-5A and MSTO cells. Also, a time-dependent significant increase in DNA-strand breaks was observed by alkaline unwinding in asbestos-treated HMC, but not in treated MeT-5A and MSTO cells. In none of the three cell lines we could detect oxidative DNA damage recognized by the Fpg protein (e.g. 8-oxo-guanine), up to 24 h after exposure to asbestos. In contrast to what was found in HMC, P53 was over-expressed in untreated MeT-5A and MSTO. The induction of apoptosis by asbestos fibers was suppressed in MeT-5A and MSTO cells. Crocidolite fibers induced the higher genotoxic effects and chrysotile the more pronounced apoptotic effects. We conclude that asbestos induces DNA damage in HMC already after a very short exposure time in the absence of 8-oxo-guanine formation. The presence of SV40-Tag in MeT-5A and MSTO cells results in an increased expression of P53, but not in additive genotoxic effects after exposure to asbestos. The deregulation of the apoptotic pathway may lead to proliferation of genomically damaged cells and finally to the development of mesothelioma.

In plain English

This was a laboratory study of cultured human cells, not of people. The authors exposed three kinds of cells to chrysotile or crocidolite asbestos (3 micrograms per square centimetre) for 1 to 72 hours. The cells were normal human mesothelial cells from one patient without cancer, mesothelial cells transformed by simian virus 40 (SV40) (MeT-5A), and mesothelioma cells (MSTO). In normal cells, asbestos significantly increased DNA damage as early as 1.5 hours, and DNA-strand breaks rose over time. No significant increase was seen in MeT-5A or MSTO cells. Neither fiber increased oxidative DNA base damage of the kind recognised by the Fpg protein in any cell type up to 24 hours. Unlike the normal cells, untreated MeT-5A and MSTO cells carried the SV40 T-antigen and had raised P53 levels, and asbestos-induced apoptosis (programmed cell death) was suppressed in them. In normal cells, crocidolite broke DNA strands more strongly than chrysotile weight for weight at shorter exposure times, while chrysotile had more pronounced effects on apoptosis. The authors concluded that SV40 did not add to asbestos's genotoxic effects but deregulated apoptosis. They suggested this may let damaged cells multiply and finally lead to mesothelioma.

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) experiment on cultured human cells
• Who was studied: Three cell types. Primary human mesothelial cells (HMC) were obtained from the ascites fluid of one noncancerous patient at the University Hospital Essen, Germany. SV40-transformed human mesothelial cells (MeT-5A) were bought from ATCC. Human mesothelioma cells (MSTO-211H) were bought from the German Collection of Microorganisms and Cell Cultures.
• Exposure: UICC standard crocidolite and chrysotile asbestos fibers at 3 micrograms per square centimetre, for exposure times of 1 to 72 hours. X-rays, visible light and cisplatin were used as positive controls.
• Main results:
  • In normal HMC, the Comet assay showed a significant increase in DNA damage after 3 to 72 hours of asbestos exposure (P less than 0.001). For example, at 3 hours the mean was 16.0 plus or minus 6.5 in untreated controls, 24.0 plus or minus 8.0 with crocidolite, and 21.0 plus or minus 7.0 with chrysotile.
  • In normal HMC, a statistically significant increase in DNA damage was already seen after 1.5 hours of exposure (P less than 0.001).
  • The Comet assay found no significant difference between untreated and asbestos-treated cells in MeT-5A cells (1 to 6 hours) or in MSTO cells (1 to 48 hours, data not shown).
  • By alkaline unwinding, HMC treated with asbestos for 1 to 24 hours showed a significant, time-dependent increase in DNA-strand breaks. MSTO cells showed only very few strand breaks after 24 hours, and asbestos-treated MeT-5A cells showed none.
  • Background (steady-state) levels of Fpg-sensitive sites differed between the cell types. None were present in MSTO cells, while HMC and MeT-5A cells had about 0.5 Fpg-sensitive sites per million base pairs.
  • Neither crocidolite nor chrysotile induced Fpg-sensitive sites (oxidative DNA base damage such as 8-oxo-guanine) above these levels in any of the three cell types up to 24 hours. Visible light, used as a positive control, did produce such sites in all three.
  • Untreated HMC showed no SV40-Tag and no increased P53. Untreated MeT-5A and MSTO cells expressed SV40-Tag in their nuclei and showed accumulation of P53.
  • In HMC, chrysotile significantly increased the percentage of apoptotic (AnnexinV-positive) cells (P less than 0.01). The increase with crocidolite depended on exposure time (3 to 24 hours).
  • In MeT-5A cells, crocidolite did not significantly increase the percentage of apoptotic cells, while chrysotile (12 to 72 hours) did (P less than or equal to 0.05). In MSTO cells, a significant increase was seen after chrysotile treatment for 48 hours.
  • In crocidolite-exposed HMC, the share of early apoptotic cells fell from 35.5 plus or minus 11.2 percent in untreated controls to 3.3 plus or minus 0.9 percent at 72 hours, while late apoptotic cells rose to up to 97%. In MSTO cells, the early apoptotic share was lower and did not decrease with exposure time.
  • Both fibers induced DNA-strand breaks in normal HMC. Crocidolite had stronger effects on a per weight basis at shorter exposure times.
  • The authors reported that chrysotile produced the more pronounced apoptotic effects.
• The authors' conclusion: The authors concluded that asbestos induces DNA damage in normal human mesothelial cells after a very short exposure, without 8-oxo-guanine formation. They also concluded that SV40-Tag affects the extent of DNA damage but does not produce additive genotoxic effects with asbestos, and that it deregulates the apoptotic pathway. They suggested this deregulation may lead to proliferation of genomically damaged cells and finally to mesothelioma.
• Limitations the authors state:
  • The authors stated it cannot be excluded that their version of the Comet assay is less sensitive for detecting asbestos-induced DNA damage, although the alkaline-unwinding method gave similar results.
  • The authors noted that their findings on oxidative DNA damage apply to the low-exposure conditions they studied. Positive results in other studies usually came from longer exposure times, higher fiber concentrations or different cell lines.

Topics

Medical terms in this study, explained in our Lexicon: Apoptosis, SV40, Mesothelial Cells, Mesothelioma, Chrysotile, Crocidolite, Comet Assay.

Related studies

• Modulation of DNA single-strand breaks by intracellular glutathione in human lung cells exposed to asbestos fibers — A. Puhakka, T. Ollikainen, et al. (2002)
• DNA breakage in asbestos-treated normal and transformed (TSV40) rat pleural mesothelial cells — V. Levresse, A. Renier, et al. (2000)
• SV40 oncoproteins enhance asbestos-induced DNA double-strand breaks and abrogate senescence in murine mesothelial cells — J.R. Pietruska, A.B. Kane (2007)
• Asbestos causes DNA strand breaks in cultured pulmonary epithelial cells: role of iron-catalyzed free radicals — D.W. Kamp, V.A. Israbian, et al. (1995)
• Increased incidence of DNA double-strand breaks and anti-ds DNA antibodies in blood of workers occupationally exposed to asbestos — B. Marczynski, A.B. Czuppon, et al. (1994)
• Effect of simian virus large T antigen expression on cell cycle control and apoptosis in rat pleural mesothelial cells exposed to DNA damaging agents — V. Levresse, S. Moritz, et al. (1998)

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