Internalization of Libby amphibole asbestos and induction of oxidative stress in murine macrophages

Our plain-English summary of Internalization of Libby amphibole asbestos and induction of oxidative stress in murine macrophages, by D.J. Blake, C.M. Bolin, D.P. Cox, et al (Toxicological Sciences, 2007).

The study

• Authors: D.J. Blake, C.M. Bolin, D.P. Cox, F. Cardozo-Pelaez, J.C. Pfau
• Journal: Toxicological Sciences
• Published: 2007;99(1):277-88
• Cited by: 37 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

The community members of Libby, MT, have experienced significant asbestos exposure and developed numerous asbestos-related diseases including fibrosis and lung cancer due to an asbestos-contaminated vermiculite mine near the community. The form of asbestos in the contaminated vermiculite has been characterized in the amphibole family of fibers. However, the pathogenic effects of these fibers have not been previously characterized. The purpose of this study is to determine the cellular consequences of Libby amphibole exposure in macrophages compared to another well-characterized amphibole fiber; crocidolite asbestos. Our results indicate that Libby asbestos fibers are internalized by macrophages and localize to the cytoplasm and cytoplasmic vacuoles similar to crocidolite fibers. Libby asbestos fiber internalization generates a significant increase in intracellular reactive oxygen species (ROS) as determined by dichlorofluorescein diacetate and dihydroethidine fluorescence indicating that the superoxide anion is the major contributing ROS generated by Libby asbestos. Elevated superoxide levels in macrophages exposed to Libby asbestos coincide with a significant suppression of total superoxide dismutase activity. Both Libby and crocidolite asbestos generate oxidative stress in exposed macrophages by decreasing intracellular glutathione levels. Interestingly crocidolite asbestos, but not Libby asbestos, induces significant DNA damage in macrophages. This study provides evidence that the difference in the level of DNA damage observed between Libby and crocidolite asbestos may be a combined consequence of the distinct chemical compositions of each fiber as well as the activation of separate cellular pathways during asbestos exposure.

In plain English

This laboratory study exposed mouse macrophages to a sample of the amphibole asbestos from the Libby, Montana vermiculite mine. Macrophages are immune cells that clear inhaled particles from the lung. The study compared the Libby fibers with crocidolite asbestos and with wollastonite, a non-asbestos control fiber. Most experiments used a mouse macrophage cell line, and some used macrophages washed from the lungs of mice. Electron microscopy showed that the cells took Libby fibers inside, mostly into small sacs (vacuoles). Libby fibers raised levels of reactive oxygen species, which are chemically reactive molecules; the authors judged these to be mainly superoxide. Libby fibers also lowered the activity of the enzyme superoxide dismutase by 30% at 3 hours. Both Libby fibers and crocidolite lowered glutathione, a protective antioxidant. Crocidolite raised markers of DNA damage, but the Libby fibers did not. After 24 hours, viability compared with unexposed cells was 92% for cells exposed to Libby fibers and 62% for cells exposed to crocidolite. The authors proposed that the difference in DNA damage may reflect the fibers' different chemical make-up and different cell responses. The study used cells in a dish, not people.

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 mouse macrophage cells
• Who was studied: The mouse macrophage cell line RAW264.7, used for most experiments, plus primary alveolar macrophages lavaged from C57BL/6 mice. For the primary cells, results were reported only as 'similar' for reactive oxygen species, with data not shown.
• Exposure: Three fiber samples. Libby amphibole asbestos from the U.S. Geological Survey, labeled '6-mix' because it contains six amphibole fiber types. Crocidolite asbestos. Wollastonite as a non-cytotoxic, non-fibrogenic control. Most experiments used 62.5 micrograms per square centimetre; doses of 6.25 to 62.5 were tested for reactive oxygen species, and 5 was used for electron microscopy.
• Main results:
  • Libby fibers were taken up by the macrophages and were found mostly in cytoplasmic vacuoles or protruding from them; some were free in the cytoplasm or attached to the cell membrane.
  • Libby fibers increased intracellular reactive oxygen species in a dose-dependent way, with the greatest increase at 62.5 micrograms per square centimetre.
  • Crocidolite increased reactive oxygen species after 3 hours, but by less than Libby fibers did. Wollastonite produced no increase.
  • After 24 hours, viability of RAW264.7 cells compared with untreated cells was 92% for Libby asbestos and 62% for crocidolite.
  • Macrophages took in an average of 4.38 (SD 1.06) Libby fibers per cell and 3.28 (SD 1.58) crocidolite fibers per cell, a difference the authors reported as not significant.
  • Macrophages took in significantly fewer wollastonite fibers, an average of 0.88 (SD 0.93) per cell.
  • Pretreatment with superoxide dismutase coupled to PEG significantly lowered the reactive oxygen species produced by Libby fibers. Libby fibers also increased superoxide as measured with the dye DHE.
  • Libby asbestos lowered total superoxide dismutase activity by 30% at 3 hours. Activity was not significantly different from controls at 7, 12 and 24 hours.
  • Crocidolite significantly increased total superoxide dismutase activity by 200% after 24 hours.
  • Both Libby asbestos and crocidolite significantly lowered intracellular glutathione at 7, 12 and 24 hours. Wollastonite did not.
  • Crocidolite significantly raised 8-oxo-dG, a marker of oxidative DNA damage, at 12 and 24 hours.
  • Libby asbestos did not raise 8-oxo-dG at 12 or 24 hours.
  • In the comet assay at 24 hours, crocidolite produced significantly more DNA damage. Libby amphibole did not produce a significant difference.
  • Activity of the DNA repair enzyme Ogg1 was not changed by Libby asbestos or wollastonite. Crocidolite significantly increased it at 12 hours, and it returned to control levels by 24 hours.
• The authors' conclusion: The authors hypothesized that the difference in DNA damage between Libby and crocidolite asbestos may be a combined consequence of the fibers' distinct chemical compositions and of separate cellular pathways activated during exposure.
• Limitations the authors state:
  • The superoxide dismutase assay measured total activity and could not distinguish copper-zinc SOD from mitochondrial manganese SOD.
  • Fiber counting by light microscopy could not tell apart more than five fibers per cell, so five was the upper limit of detection.

Mines named here

Asbestos mines this study is about. Each mine's page says who ran it, when it worked, and what it produced.

• Libby vermiculite mine, Montana

All asbestos mines

Topics

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

Medical terms in this study, explained in our Lexicon: Libby amphiboles, Oxidative Stress, Macrophage, Asbestos Exposure, Fibrosis, Lung Cancer, vermiculite, Amphibole Group.

Related studies

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• Mechanisms of oxidative stress and alterations in gene expression by Libby six-mix in human mesothelial cells — J.M. Hillegass, A. Shukla, et al. (2010)
• Attenuation of oxidative stress and chromosomal aberrations in cultured macrophages and pulmonary cells following self-sustained high temperature synthesis of asbestos — S. La Maestra, R.T. Micale, et al. (2020)
• Functional expression of system x(c)- is upregulated by asbestos but not crystalline silica in murine macrophages — J.C. Pfau, T. Seib, et al. (2012)
• Asbestos Induces Oxidative Stress and Activation of Nrf2 Signaling in Murine Macrophages: Chemopreventive Role of the Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605) — R.A. Pietrofesa, A. Velalopoulou, et al. (2016)
• Oxidative stress-mediated cytotoxicity and apoptosis induction by TiO2 nanofibers in HeLa cells — K.M. Ramkumar, C. Manjula, et al. (2012)

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