Functional expression of system x(c)- is upregulated by asbestos but not crystalline silica in murine macrophages

Our plain-English summary of Functional expression of system x(c)- is upregulated by asbestos but not crystalline silica in murine macrophages, by J.C. Pfau, T. Seib, J.J. Overocker, et al (Inhalation Toxicology, 2012).

The study

• Authors: J.C. Pfau, T. Seib, J.J. Overocker, J. Roe, A.S. Ferro
• Journal: Inhalation Toxicology
• Published: 2012;24(8):476-85
• Cited by: 10 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Context: Inhalation of asbestos or silica is associated with chronic and progressive diseases, including fibrosis, cancer, and increased risk of systemic autoimmunity. Because there is a need for treatment options for these diseases, a better understanding of their mechanistic etiologies is essential. While oxidative stress in macrophages is an early consequence of these exposures, it may also serve as a signaling mechanism involved in downstream immune dysregulation. The system x(c)(-) exchange protein is induced by oxidative stress, and exchanges equimolor levels of extracellular cystine for intracellular glutamate. Cystine is subsequently reduced to cysteine, the rate-limiting precursor for glutathione synthesis.

Objective: As the primary transporter responsible for cystine/glutamate exchange on macrophages, system x(c)- was hypothesized to be inducible in response to asbestos and silica, and to increase viability through protection from oxidative stress.

Results: When challenged with amphibole asbestos, but not crystalline silica, RAW 264.7 macrophages increased expression of xCT and the rate of cystine/glutamate exchange in sodium-free conditions. This upregulation was prevented with N-acetylcysteine, implicating oxidative stress. Cystine protected the macrophages from asbestos-induced oxidative stress and cell death, supporting the hypothesis that imported cystine was used for synthesis of cellular antioxidants. System x(c)(-) inhibitors, glutamate and S-4-carboxyphenylglycine ((S)-4-CPG), significantly increased oxidative stress and cell death of asbestos-treated macrophages.

Conclusion: System x(c)(-) plays a critical role in survival of macrophages exposed to asbestos, but not silica. These data demonstrate a very early difference in the cellular response to these silicates that may have important downstream implications in the pathologic outcome of exposure.

In plain English

This was a laboratory study using the RAW 264.7 murine macrophage cell line, treated in culture with Libby amphibole asbestos ("6-Mix", described as a mixture of tremolite, amosite, richterite and other amphiboles), crystalline silica, wollastonite (a non-fibrogenic control fiber), or lipopolysaccharide (LPS), mostly at 35 µg/cm2 for 24 hours. Asbestos and LPS increased surface expression of the xCT subunit of the system xc- transporter (one representative histogram showed 14.2% positive untreated cells versus 67.7% after 6-Mix), while silica and wollastonite did not. Asbestos also raised sodium-independent [3H]-L-glutamate transport dose-dependently at 12 and 24 hours; silica did not. N-acetylcysteine blocked both the rise in reactive oxygen species and the rise in xCT expression. The inhibitor (S)-4-CPG increased asbestos-induced oxidative stress and depleted glutathione. Cystine (2.5 mM) protected cells from asbestos toxicity up to 50 µg/cm2 but did not protect against silica toxicity; added glutamate increased asbestos toxicity. The authors concluded that system xc- is functionally upregulated by amphibole asbestos but not silica, apparently in response to oxidative stress.

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 experimental study in a cultured murine macrophage cell line (flow cytometry, radiolabelled transport, viability, ROS and glutathione assays)
• Who was studied: RAW 264.7 murine macrophages in culture; experiments repeated at least three times, group sizes of n = 3 to n = 8 per assay
• Exposure: Libby amphibole asbestos ("6-Mix": tremolite, amosite, richterite and other amphiboles), crystalline silica, wollastonite, and LPS; mostly 35 µg/cm2 for 24 h, with dose ranges to 100 µg/cm2
• Main results:
  • 6-Mix asbestos and LPS increased xCT expression on RAW cells; silica and wollastonite did not
  • In a representative flow cytometry histogram, untreated cells were 14.2% positive for xCT and 6-Mix-treated cells 67.7% positive
  • CD98 was constitutively expressed on over 90% of RAW cells regardless of treatment
  • Asbestos increased sodium-independent [3H]-L-glutamate transport dose-dependently at 12 and 24 h; silica produced no increase
  • L-glutamate in the culture media of asbestos-treated macrophages was increased, and this was inhibited by (S)-4-CPG
  • N-acetylcysteine (2.5 mM) prevented the asbestos-induced increase in DCFDA fluorescence (ROS) and the increase in system xc- expression
  • The inhibitor (S)-4-CPG increased asbestos-induced ROS, and inhibiting system xc- depleted intracellular glutathione
  • For asbestos-exposed cells, NAC gave the highest level of protection, cystine (2.5 mM) protected up to 50 µg/cm2, and added glutamate increased asbestos toxicity
  • For silica-induced cell death, NAC was protective but cystine was not
  • Blocking system xc- with (S)-4-CPG abolished the protection given by cystine against asbestos-induced cell death
• The authors' conclusion: The authors concluded that system xc- is functionally upregulated in cultured macrophages by amphibole asbestos, apparently in response to oxidative stress, whereas silica did not induce it, suggesting silica-exposed cells use alternate strategies
• Limitations the authors state:
  • The authors state the link between system xc- induction and ROS generation was shown only indirectly
  • No L-glutamine was provided in the experimental media, which the authors say likely contributed to cell death at higher asbestos concentrations despite cystine
  • The authors state that more research is needed on the downstream events affected by system xc-
  • Table 1 summarising silica and asbestos effects is not a complete review of the literature, as the authors note

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: Crystalline, Macrophage, Fibrosis, Oxidative Stress, Glutathione (GSH), Amphibole Asbestos, Silicate.

Related studies

• Internalization of Libby amphibole asbestos and induction of oxidative stress in murine macrophages — D.J. Blake, C.M. Bolin, et al. (2007)
• Synthetic silica fibers of different length, diameter and shape: synthesis and interaction with rat (NR8383) and human (THP-1) macrophages in vitro, including chemotaxis and gene expression profile — N. Białas, N. Rosenkranz, et al. (2024)
• Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52-enriched apoptotic blebs of murine macrophages — D.J. Blake, S.A. Wetzel, et al. (2008)
• Indications for distinct pathogenic mechanisms of asbestos and silica through gene expression profiling of the response of lung epithelial cells — T.N. Perkins, P.M. Peeters, et al. (2015)
• Relative effects of asbestos and wollastonite on alveolar macrophages — W.H. Pailes, D.J. Judy, et al. (1984)
• Characterization of pulmonary responses in mice to asbestos/asbestiform fibers using gene expression profiles — N. Yanamala, E.R. Kisin, et al. (2018)

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