Asbestos causes DNA strand breaks in cultured pulmonary epithelial cells: role of iron-catalyzed free radicals

Our plain-English summary of Asbestos causes DNA strand breaks in cultured pulmonary epithelial cells: role of iron-catalyzed free radicals, by D.W. Kamp, V.A. Israbian, S.E. Preusen, et al (The American Journal of Physiology, 1995).

The study

• Authors: D.W. Kamp, V.A. Israbian, S.E. Preusen, C.X. Zhang, S.A. Weitzman
• Journal: The American Journal of Physiology
• Published: 1995;268(3 Pt 1):L471-80
• Cited by: 80 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Asbestos causes pulmonary fibrosis and various malignancies by mechanisms that remain uncertain. Reactive oxygen species in part cause asbestos toxicity. However, it is not known whether asbestos-induced free radical production causes alveolar epithelial cell (AEC) cytotoxicity by inducing DNA strand breaks (DNA-SB). We tested the hypothesis that asbestos-induced AEC injury in vitro is due to iron-catalyzed free radical generation, which in turn causes DNA-SB. We found that amosite asbestos damages cultured human pulmonary epithelial-like cells (WI-26 cells) as assessed by 51Cr release and that an iron chelator, phytic acid (500 microM), attenuates these effects. A role for iron causing these effects was supported by the observation that ferric chloride-treated phytic acid did not diminish WI-26 cell injury. Production of hydroxyl radical-like species (.OH) was assessed based upon the .OH-dependent formation of formaldehyde (HCHO) in the presence of dimethyl sulfoxide. A variety of mineral dusts induced significant levels of .OH formation (nmol HCHO at 30 min: carbonyl iron, 85 +/- 21; amosite asbestos, 14 +/- 2; chrysotile asbestos, 7 +/- 1; titanium dioxide, 2.5 +/- 0.5). Phytic acid significantly diminished the asbestos-induced .OH production. DNA damage to AEC was assessed by the alkaline unwinding, ethidium bromide fluorometric technique. Hydrogen peroxide caused dose-dependent DNA-SB in WI-26 cells after a 30-min exposure period [50% effective dose (ED50): 5 microM] that was similar to other cell lines. Amosite asbestos induced dose-dependent DNA-SB in WI-26, A549, and primary isolated rat alveolar type II cells maintained in culture for 7-10 days (alveolar type I-like). Lower doses of amosite (0.5-5 micrograms/ml or 0.25-2.5 micrograms/cm2) caused significant WI-26 cell DNA-SB after prolonged exposure periods (> or = 2 days). Phytic acid ameliorated DNA damage in all three cultured AEC. There was a direct correlation between mineral dust-induced .OH production at 30 min and DNA-SB in WI-26 cells at 4 h (P < 0.0005). These data suggest that mineral dusts can be directly genotoxic to relevant target cells of asbestos, AEC. Furthermore, these results provide additional support for the premise that iron-catalyzed free radicals mediate asbestos-induced pulmonary toxicity.

In plain English

This was a laboratory study of cultured cells, not of people. The authors exposed WI-26 cells from human embryonal lung to amosite asbestos, chrysotile asbestos, titanium dioxide and carbonyl iron, and exposed A549 cells from a human lung carcinoma and cultured rat alveolar cells to amosite. They measured cell injury, hydroxyl radical-like activity (a reactive form of oxygen) and DNA breaks. Amosite injured WI-26 cells after 18 hours, and an iron-binding compound, phytic acid, reduced this injury. Amosite produced more radical activity than chrysotile, while titanium dioxide produced negligible amounts. Amosite caused dose-dependent DNA breaks in all three cell types, and phytic acid reduced them. Chrysotile caused fewer DNA breaks than amosite. With exposures of 48 to 72 hours, lower amosite doses also caused significant DNA breaks in WI-26 cells. Radical production at 30 minutes correlated with DNA breaks at 4 hours (R = 0.97). The authors concluded that certain mineral dusts directly damage DNA in lung alveolar cells. They also concluded that their results support the idea that iron-catalyzed free radicals mediate asbestos-induced lung disease.

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 and rat lung epithelial cells
• Who was studied: Cultured cells, not people. The cells were SV-40 transformed WI-26 cells (human embryonal lung, type I pneumocyte-like) and A549 cells (human bronchoalveolar carcinoma-derived, type II-like). The study also used alveolar type II cells isolated from adult Sprague-Dawley rats and kept in culture 7 to 10 days so that they became type I-like. The work was done at Northwestern University, Chicago. Each experimental group was run in at least six separate experiments unless noted otherwise.
• Exposure: WI-26 cells were exposed to amosite and chrysotile asbestos (UICC reference standard samples), titanium dioxide (described as a nonfibrogenic particle), carbonyl iron and hydrogen peroxide. A549 and rat alveolar cells were exposed to amosite (50 and 500 micrograms per ml). Asbestos doses ranged from 0.5 to 500 micrograms per ml, with exposure times from 30 minutes to 72 hours. The iron chelator phytic acid (up to 500 micromolar) was used to test the role of iron.
• Main results:
  • In WI-26 cells exposed to amosite (500 micrograms per ml) for 18 hours, phytic acid reduced cell injury in a dose-dependent way. Phytic acid that had been pretreated with ferric chloride did not reduce the injury.
  • Hydroxyl radical-like production at 30 minutes was measured as formaldehyde, in nmol per well. Values were 0.3 for control, 2.5 for titanium dioxide (described as negligible), 14.3 for amosite at 500 micrograms per ml and 6.6 for chrysotile at 500 micrograms per ml. Carbonyl iron gave 84.5 (plus or minus 20.9; n = 6; P less than 0.01 vs. control).
  • Phytic acid (500 micromolar) reduced radical production from both asbestos types. Amosite fell from 14.3 to 9.0 nmol and chrysotile from 6.6 to 0.2 nmol formaldehyde.
  • Hydrogen peroxide caused dose-dependent DNA breaks in WI-26 cells after 30 minutes. The dose causing a 50% reduction in double-stranded DNA was 5 micromolar, lower than the 10 to 50 micromolar seen in other cell lines.
  • WI-26 cells were exposed for 4 hours. Low amosite doses (0.5 to 5 micrograms per ml) caused negligible changes in DNA. Higher doses (50 and 500 micrograms per ml) reduced double-stranded DNA in a dose-dependent manner. At 500 micrograms per ml there was an 80% reduction, with minimal cell injury (51Cr release 12.2%, P = 0.14 vs. control). Titanium dioxide caused negligible changes, and carbonyl iron caused marked reductions similar to amosite.
  • Chrysotile caused significant DNA damage in WI-26 cells at 4 hours, but less than amosite: 62% double-stranded DNA remained with chrysotile versus 17% with amosite.
  • Longer exposures to low amosite doses caused DNA breaks in WI-26 cells. At 24 hours only 50 micrograms per ml was significant (60 vs. 95% double-stranded DNA in controls). At 48 hours 5 micrograms per ml was significant (72 vs. 90%). At 72 hours 0.5 micrograms per ml was significant (62 vs. 90%; P less than 0.05).
  • Amosite (500 micrograms per ml) reduced double-stranded DNA in WI-26 cells at 30 minutes (82 vs. 60%) and at 4 hours (79 vs. 37%). Phytic acid significantly reduced this damage at both times. In A549 cells and rat alveolar cells, amosite (50 and 500 micrograms per ml, 4 hours) also caused dose-dependent DNA damage, which phytic acid significantly reduced.
  • Amosite fibers pretreated with phytic acid and then washed did not reduce DNA breaks in WI-26 or A549 cells over 24 to 72 hours. The authors suggested that either the fibers' iron content alone is insufficient to account for the damage or the phytic acid was washed off.
  • Across mineral dusts, radical production at 30 minutes correlated with DNA damage in WI-26 cells at 4 hours (R = 0.97). The P value is given as 0.005 in the results text and as less than 0.0005 in the figure legend. The authors described this as suggesting a causal relationship.
• The authors' conclusion: The authors concluded that certain mineral dusts are directly genotoxic (damaging to DNA) to alveolar epithelial cells, a relevant target cell of asbestos. They concluded that iron-catalyzed free radicals are important in these changes and that the data further support the notion that such radicals mediate asbestos-induced lung diseases. They added that the DNA-damaging effects depend in part on the cell type exposed.
• Limitations the authors state:
  • The test for hydroxyl radicals was indirect, measuring formaldehyde formed from DMSO.
  • The reduced fluorescence in the DNA test reflects nonspecific DNA lesions, not only strand breaks.
  • Phytic acid could not be applied to the cells for longer than 24 hours, so its effect on the long low-dose exposures could not be tested directly.
  • Radical-like activity appeared without added hydrogen peroxide. The authors suggested the Tris buffer may have mobilized iron from the fibers.
  • The highest asbestos dose mildly quenched the fluorescent signal (about a 14% reduction), which the authors corrected for.
  • Because carbonyl iron colored the solution, its radical levels were estimated indirectly.
  • The source of the iron driving radical formation (the fiber's core structure or surface contaminants) was unclear.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Amosite, Chrysotile.

Medical terms in this study, explained in our Lexicon: Pulmonary Fibrosis, Reactive Oxygen Species (ROS), Amosite, Damages, Hydroxyl Radical, Chrysotile.

Related studies

• Asbestos causes apoptosis in alveolar epithelial cells: role of iron-induced free radicals — A. Aljandali, H. Pollack, et al. (2001)
• Cigarette smoke augments asbestos-induced alveolar epithelial cell injury: role of free radicals — D.W. Kamp, M.J. Greenberger, et al. (1998)
• Iron associated with asbestos bodies is responsible for the formation of single strand breaks in phi X174 RFI DNA — L.G. Lund, M.G. Williams, et al. (1994)
• Iron mobilization from crocidolite asbestos greatly enhances crocidolite-dependent formation of DNA single-strand breaks in phi X174 RFI DNA — L.G. Lund, A.E. Aust (1992)
• Modulation of DNA single-strand breaks by intracellular glutathione in human lung cells exposed to asbestos fibers — A. Puhakka, T. Ollikainen, et al. (2002)
• 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)

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