Modulation of DNA single-strand breaks by intracellular glutathione in human lung cells exposed to asbestos fibers

Our plain-English summary of Modulation of DNA single-strand breaks by intracellular glutathione in human lung cells exposed to asbestos fibers, by A. Puhakka, T. Ollikainen, Y. Soini, et al (Mutation Research, 2002).

The study

• Authors: A. Puhakka, T. Ollikainen, Y. Soini, K. Kahlos, M. Säily, P. Koistinen, P. Pääkkö, K. Linnainmaa, V.L. Kinnula
• Journal: Mutation Research
• Published: 2002;514(1-2):7-17
• Cited by: 31 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

We investigated the role of glutathione and nitric oxide synthase (NOS) in fiber-induced cell and DNA toxicity using alkaline (pH 13) single-cell gel electrophoresis (the Comet assay). Transformed cultured human pleural mesothelial (MeT-5A) cells and alveolar epithelial cells (A549) were exposed to crocidolite asbestos fibers (1-10 microg/cm(2)) in the presence of buthionine sulfoximine (BSO) or L-arginine-methyl ester (L-NAME). BSO inhibits gamma-glutamylcysteine synthetase (gamma-GCS) and causes glutathione depletion, and L-NAME inhibits nitric oxide generation. Studies were also conducted to assess the expression of the heavy and light subunits of gamma-GCS in human pleural mesothelium and bronchial epithelium in vivo and the induction of inducible NOS (iNOS) by asbestos fibers. Asbestos fibers caused DNA single-strand breaks, and the process was significantly enhanced by BSO (69% compared to the non-treated cells). A549 cells had a 3.5-fold glutathione content compared to MeT-5A cells, which was consistent with the higher resistance of these cells against oxidants and fibers. Flow cytometry of iNOS showed no change of iNOS by the fibers in either cell type in vitro. L-NAME had no effects on the DNA single-strand breaks in the Comet assay, either. Studies on lung biopsies showed that the immunoreactivities of both gamma-GCS subunits were very low in healthy human mesothelium in vivo. We conclude that glutathione may play an essential role in protecting intact cells against fiber-induced oxidative DNA alterations, and low gamma-GCS reactivity in pleural mesothelium may be associated with the high sensitivity of mesothelial cells to fiber-induced toxicity.

In plain English

This laboratory study exposed two cultured human cell lines, MeT-5A pleural mesothelial cells and A549 alveolar epithelial cells, to crocidolite asbestos fibers. Doses ranged from 1 to 10 micrograms per square centimetre, and DNA single-strand breaks were measured with the Comet assay. Some cells were first treated with BSO, which lowers the antioxidant glutathione, or L-NAME, which blocks nitric oxide production. Crocidolite significantly increased DNA breaks in MeT-5A cells. Lowering glutathione with BSO significantly increased the breaks further; the abstract gives 69% 'compared to the non-treated cells' without defining those cells. A549 cells contained more glutathione than MeT-5A cells (90.1 versus 23.5 nmol/mg protein), and fibers caused marginal cell injury only in MeT-5A cells. L-NAME had no significant effect on DNA breaks, and the fibers did not change iNOS levels. Healthy pleural mesothelium tissue showed no or very weak staining for gamma-GCS, the enzyme that limits glutathione production. Bronchial epithelium from five lung carcinoma patients showed weak to moderate staining. The authors concluded that low glutathione, together with the fibers' oxidant effects, may play a crucial role in fiber-induced DNA strand breaks.

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) study of cultured human cell lines, with immunohistochemical staining of human lung tissue samples
• Who was studied: Two cultured human cell lines: transformed MeT-5A pleural mesothelial cells and A549 alveolar epithelial cells. Also six samples of peripheral lung tissue with non-neoplastic, non-inflamed pleura (healthy controls), and bronchial epithelium from non-neoplastic areas of five lung carcinoma patients, in Finland.
• Exposure: Crocidolite asbestos fibers at 1 to 10 micrograms per square centimetre (lower doses for MeT-5A than A549), alone or after pre-treatment with BSO (glutathione depletion) or L-NAME (nitric oxide inhibition). Hydrogen peroxide was used as a comparison and positive control.
• Main results:
  • In MeT-5A mesothelial cells, crocidolite significantly increased the mean tail extent moment, a marker of DNA strand breaks.
  • Glutathione depletion with BSO significantly enhanced fiber-induced DNA single-strand breaks. The abstract gives a figure of 69% 'compared to the non-treated cells' and does not specify which cells are meant by non-treated.
  • Glutathione content was 23.5 nmol/mg protein in MeT-5A cells and 90.1 nmol/mg protein in A549 cells (n = 4).
  • Crocidolite at 1 to 10 micrograms per square centimetre caused only marginal cell injury, and only in MeT-5A cells.
  • L-NAME, which blocks nitric oxide production, had no significant effect on DNA breaks in either cell line.
  • Crocidolite exposure did not change iNOS expression in either cell line.
  • Healthy pleural mesothelium was negative or very weakly positive for both gamma-GCS subunits in all cases. Bronchial epithelium showed weak to moderate staining in all five cases.
• The authors' conclusion: The authors concluded that low intracellular glutathione, combined with the oxidant effects of the fibers, may have a crucial role in DNA strand breaks during prolonged asbestos exposure. They also concluded that low gamma-GCS in human mesothelium may affect fiber-induced DNA alterations in these cells.
• Limitations the authors state:
  • Results from cultured cells cannot be directly applied to the living body. Nitric oxide may still cause DNA damage in vivo.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Glutathione (GSH), Comet Assay, Crocidolite, Mesothelium, Epithelium, A549 Cells, MeT-5A Cells, Mesothelial Cells.

Related studies

• Regulation of nitric oxide synthase induction by iron and glutathione in asbestos-treated human lung epithelial cells — S.H. Park, A.E. Aust (1998)
• Role of alphavbeta5 integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells — P. Pande, T.A. Mosleh, et al. (2006)
• 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 — B. Burmeister, T. Schwerdtle, et al. (2004)
• In vitro sensitivity of normal human mesothelial and malignant mesothelioma cell lines to four new chemotherapeutic agents — T. Ollikainen, A. Knuuttila, et al. (2000)
• Modulation of cell and DNA damage by poly(ADP)ribose polymerase in lung cells exposed to H(2)O(2) or asbestos fibres — T. Ollikainen, A. Puhakka, et al. (2000)
• Cytotoxicity of oxidants and asbestos fibers in cultured human mesothelial cells — V.L. Kinnula, K. Aalto, et al. (1994)

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