Activation of p38 MAP kinase by asbestos in rat mesothelial cells is mediated by oxidative stress

Our plain-English summary of Activation of p38 MAP kinase by asbestos in rat mesothelial cells is mediated by oxidative stress, by W.A. Swain, K.J. O'Byrne, S.P. Faux (American Journal of Physiology. Lung Cellular and Molecular Physiology, 2004).

The study

• Authors: W.A. Swain, K.J. O'Byrne, S.P. Faux
• Journal: American Journal of Physiology. Lung Cellular and Molecular Physiology
• Published: 2004;286(4):L859-65
• Cited by: 20 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Asbestos fibers are biopersistent particles that are capable of stimulating chronic inflammatory responses in the pleura of exposed individuals. Exposure of pleural mesothelial cells, the progenitor cell of malignant mesothelioma, to asbestos induces an array of cellular responses. The present studies investigated whether the p38 mitogen-activated protein kinase cascade was induced under asbestos-exposed conditions. p38 plays a vital role in the response to stressful stimuli and enables the cell to enter an inflammatory state characterized by cytokine production. Western blot and in vitro kinase assays showed increases in dual phosphorylation and actual activity of p38 after exposure to fibrous and nonfibrous (milled) crocidolite; in contrast, polystyrene beads and iron (III) oxide had no such effects. In common with other asbestos-induced events, this was shown to be an oxidative stress-sensitive effect, inasmuch as preincubation with N-acetyl-L-cysteine or -tocopherol (vitamin E) ameliorated the effect. The present studies show that p38 activity is important for crocidolite-induced activator protein-1 DNA binding, inasmuch as an inhibitor of p38, SB-203580, reduced this activity. Crocidolite-induced cytotoxicity was also reduced with SB-203580, indicating a role for p38 in asbestos-mediated cell death. Our studies suggest that p38 activity could be a crucial factor in the chronic immune response elicited by asbestos and may represent a target for future pharmacological intervention.

In plain English

This was a laboratory (in vitro) study using a rat pleural mesothelial cell line, 4/4 RM-4, exposed to UICC crocidolite asbestos, to milled (nonfibrous) crocidolite, or to two control particles. Western blotting showed that crocidolite produced dose-dependent increases in p38 MAP kinase dually phosphorylated at Thr180 and Tyr182, with maximum levels reaching 1.5 times control values; increases were significant at 8 and 24 hours but not at 4 hours. Polystyrene beads and iron (III) oxide did not induce p38 phosphorylation at any dose tested. Immune complex kinase assays showed p38 activity increased to nearly double control at 10 and 25 micrograms per square centimetre of crocidolite. Pre-treatment with N-acetyl-L-cysteine (5 mM) returned phosphorylated p38 to untreated control levels, and alpha-tocopherol reduced it to near-control levels. Milled crocidolite yielded over three times as much bioavailable iron as the fibrous sample, and chelation abolished its effect. The p38 inhibitor SB-203580 (250 or 500 nM) reduced crocidolite-induced AP-1 DNA binding and increased cell viability, though not back to control levels.

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 cell-culture experiments (rat mesothelial cell line) using Western blotting, immune complex kinase assays, electrophoretic mobility shift assays and MTT cytotoxicity assays
• Who was studied: A rat pleural mesothelial cell line, 4/4 RM-4, obtained from the European Collection of Animal Cell Cultures; three independent experiments per assay
• Exposure: UICC reference crocidolite asbestos at 5, 10 and 25 micrograms per square centimetre for up to 24 h; milled (nonfibrous) crocidolite; control particles of 0.2 micrometre polystyrene beads and iron (III) oxide; anisomycin as positive control; NAC, alpha-tocopherol and SB-203580 as pre-treatments
• Main results:
  • Crocidolite caused dose-dependent increases in dually phosphorylated p38 in the mesothelial cells, reaching a maximum of 1.5 times control values at 24 h, while native p38 expression was unchanged
  • Polystyrene beads and iron (III) oxide, used at doses that gave responses with fibres, did not increase p38 phosphorylation
  • In the time course, crocidolite significantly increased phosphorylated p38 at 8 and 24 h, but at 4 h levels were like controls
  • In the kinase assay, p38 activity at 5 micrograms per square centimetre crocidolite matched untreated control, while 10 and 25 micrograms per square centimetre nearly doubled it
  • N-acetyl-L-cysteine pre-treatment blocked crocidolite-induced p38 phosphorylation dose-dependently; 5 mM restored untreated control levels
  • Alpha-tocopherol (0.5 and 1 mM) reduced crocidolite-induced p38 phosphorylation towards control levels; 1 mM alpha-tocopherol alone raised dually phosphorylated p38
  • Milled crocidolite contained more bioavailable iron than fibrous crocidolite (milled total 329.14 nmol/mg vs UICC crocidolite 94.27 nmol/mg) and chelation abolished its ability to raise p38 phosphorylation, whereas chelated fibres still gave levels above untreated controls
  • The p38 inhibitor SB-203580 at 250 or 500 nM reduced crocidolite-induced AP-1 DNA binding, while a DMSO vehicle control did not
  • Crocidolite at 25 micrograms per square centimetre reduced cell viability to about 80% in the MTT assay; pre-incubation with 250 or 500 nM SB-203580 significantly increased viability compared with crocidolite alone, but viability did not return to untreated control levels
• The authors' conclusion: The authors concluded that crocidolite activates the p38 arm of the stress-activated protein kinase pathway in a rat mesothelial cell line, upstream of crocidolite-mediated AP-1 DNA binding and cell death, and that further work is needed before p38 could be identified as a clinical target
• Limitations the authors state:
  • The assays used did not show where phosphorylated p38 was located within or between cells; the authors suggested immunofluorescence would be needed
  • The time course could not be extended beyond 24 h because background phosphorylated p38 rose in serum-free controls
  • Chelation may have been incomplete, so residual iron could explain the remaining p38 activation with chelated fibres
  • The MTT assay could not determine the type of cell death involved
  • The effect of milled crocidolite on cell viability was not tested

Topics

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

Medical terms in this study, explained in our Lexicon: Mesothelial Cells, Oxidative Stress, Pleura, Malignant Mesothelioma, Cytokine, Crocidolite.

Related studies

• Patterns of 8-hydroxydeoxyguanosine formation in DNA and indications of oxidative stress in rat and human pleural mesothelial cells after exposure to crocidolite asbestos — H. Fung, Y.W. Kow, et al. (1997)
• Raw single-wall carbon nanotubes induce oxidative stress and activate MAPKs, AP-1, NF-kappaB, and Akt in normal and malignant human mesothelial cells — M. Pacurari, X.J. Yin, et al. (2008)
• Potential role of ferritin heavy chain in oxidative stress and apoptosis in human mesothelial and mesothelioma cells: implications for asbestos-induced oncogenesis — W. Aung, S. Hasegawa, et al. (2007)
• Asbestos inhalation induces tyrosine nitration associated with extracellular signal-regulated kinase 1/2 activation in the rat lung — A. Iwagaki, N. Choe, et al. (2003)
• Role of extracellular signal-regulated protein kinases in apoptosis by asbestos and H2O2 — L.A. Jiménez, C. Zanella, et al. (1997)
• Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells — W. Liu, J.D. Ernst, et al. (2000)

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