Mechanisms of oxidative stress and alterations in gene expression by Libby six-mix in human mesothelial cells

Our plain-English summary of Mechanisms of oxidative stress and alterations in gene expression by Libby six-mix in human mesothelial cells, by J.M. Hillegass, A. Shukla, M.B. MacPherson, et al (Particle and Fibre Toxicology, 2010).

The study

• Authors: J.M. Hillegass, A. Shukla, M.B. MacPherson, S.A. Lathrop, V. Alexeeva, T.N. Perkins, A. van der Vliet, P.M. Vacek, M.E. Gunter, B.T. Mossman
• Journal: Particle and Fibre Toxicology
• Published: 2010;7:26
• Cited by: 21 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Exposures to an amphibole fiber in Libby, Montana cause increases in malignant mesothelioma (MM), a tumor of the pleural and peritoneal cavities with a poor prognosis. Affymetrix microarray/GeneSifter analysis was used to determine alterations in gene expression of a human mesothelial cell line (LP9/TERT-1) by a non-toxic concentration (15×10(6) μm2/cm2) of unprocessed Libby six-mix and negative (glass beads) and positive (crocidolite asbestos) controls. Because manganese superoxide dismutase (MnSOD; SOD2) was the only gene upregulated significantly (p < 0.05) at both 8 and 24 h, we measured SOD protein and activity, oxidative stress and glutathione (GSH) levels to better understand oxidative events after exposure to non-toxic (15×10(6) μm2/cm2) and toxic concentrations (75×10(6) μm2/cm2) of Libby six-mix.

Results: Exposure to 15×10(6) μm2/cm2 Libby six-mix elicited significant (p < 0.05) upregulation of one gene (SOD2; 4-fold) at 8 h and 111 gene changes at 24 h, including a 5-fold increase in SOD2. Increased levels of SOD2 mRNA at 24 h were also confirmed in HKNM-2 normal human pleural mesothelial cells by qRT-PCR. SOD2 protein levels were increased at toxic concentrations (75×10(6) μm2/cm2) of Libby six-mix at 24 h. In addition, levels of copper-zinc superoxide dismutase (Cu/ZnSOD; SOD1) protein were increased at 24 h in all mineral groups. A dose-related increase in SOD2 activity was observed, although total SOD activity remained unchanged. Dichlorodihydrofluorescein diacetate (DCFDA) fluorescence staining and flow cytometry revealed a dose- and time-dependent increase in reactive oxygen species (ROS) production by LP9/TERT-1 cells exposed to Libby six-mix. Both Libby six-mix and crocidolite asbestos at 75×10(6) μm2/cm2 caused transient decreases (p < 0.05) in GSH for up to 24 h and increases in gene expression of heme oxygenase 1 (HO-1) in LP9/TERT-1 and HKNM-2 cells.

Conclusions: Libby six-mix causes multiple gene expression changes in LP9/TERT-1 human mesothelial cells, as well as increases in SOD2, increased production of oxidants, and transient decreases in intracellular GSH. These events are not observed at equal surface area concentrations of nontoxic glass beads. Results support a mechanistic basis for the importance of SOD2 in proliferation and apoptosis of mesothelial cells and its potential use as a biomarker of early responses to mesotheliomagenic minerals.

In plain English

This laboratory study used human cells in dishes, not people. The authors exposed human mesothelial cells (the LP9/TERT-1 line, and sometimes normal HKNM-2 pleural cells) to Libby six-mix. Six-mix is an amphibole mixture from the Libby, Montana vermiculite mine, mostly winchite with richterite and tremolite. Comparisons were glass beads, which the authors called non-pathogenic, given at the higher Libby dose's surface area, and in some tests crocidolite asbestos. The higher dose killed about 60% of cells by 24 hours. At the lower, non-toxic dose, 111 genes changed at 24 hours (74 up, 37 down). Glass beads caused no significant gene changes, though SOD1 protein rose with all treatments, beads included, at 24 hours. In LP9/TERT-1 cells, the antioxidant gene SOD2 rose 4-fold at 8 hours and 5-fold at 24 hours. The higher dose raised SOD2 protein, oxidant levels and HO-1 gene expression. A suggested rise in SOD2 enzyme activity was not statistically significant. After the higher dose, glutathione, a protective molecule, fell and then recovered. The authors concluded SOD2 may be an early marker of response to mesothelioma-causing minerals.

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) cell culture experiment using gene expression profiling (microarrays) plus protein, enzyme activity, oxidant and glutathione assays
• Who was studied: LP9/TERT-1 human mesothelial cell line; some results confirmed in HKNM-2 normal human pleural mesothelial cells isolated at autopsy; microarrays run on samples from 3 independent experiments
• Exposure: Libby six-mix (Libby amphibole, reported from others' characterization as roughly 84% winchite, 11% richterite and 6% tremolite of the respirable fraction) at a non-toxic 15×10^6 and a toxic 75×10^6 square micrometers of surface area per square centimeter, for 8 and 24 h (glutathione followed to 48 h); doses were set by particle surface area; glass beads (non-pathogenic control) were given at 75×10^6, equal to the highest Libby six-mix dose, including in the microarray experiments run at the lower Libby dose; crocidolite asbestos was compared in some tests at the same surface-area doses as Libby six-mix
• Main results:
  • In LP9/TERT-1 cells at the non-toxic dose, Libby six-mix upregulated only one gene (SOD2) at 8 h and changed 111 genes at 24 h
  • Of the 111 genes changed at 24 h, 74 were upregulated and 37 downregulated
  • SOD2 gene expression in LP9/TERT-1 cells rose 4-fold at 8 h and 5-fold at 24 h
  • Glass beads at the higher surface area dose produced no significant gene expression changes at either time point
  • At 24 h, SOD1 protein in LP9/TERT-1 cells was significantly higher than in the medium control in all treatment groups, including glass beads
  • Oxidant generation and glutathione depletion occurred only at the highest Libby six-mix concentration, where about 60% of cells died at 24 h
  • SOD2 protein in LP9/TERT-1 cells increased at the toxic Libby six-mix dose at 24 h
  • Total SOD activity was unchanged; a dose-related increase in SOD2 activity was suggested but was not statistically significant
  • By fluorescence microscopy, more cells were strongly positive for the oxidant probe at the higher Libby six-mix dose than in control, glass bead or lower-dose groups
  • HO-1 gene expression rose significantly in LP9/TERT-1 cells at the higher Libby six-mix dose, but not with glass beads or the lower dose
  • In HKNM-2 cells, SOD2 expression increased significantly after the higher dose of crocidolite asbestos or Libby six-mix for 24 h
  • Glutathione in LP9/TERT-1 cells exposed to the higher Libby six-mix dose fell from 2 to 8 h, then progressively recovered through 48 h
  • Crocidolite (higher dose) caused significant glutathione depletion at all time points up to and including 24 h
• The authors' conclusion: The authors concluded that their results support a mechanistic role for SOD2 in mesothelial cell proliferation and apoptosis, and its potential use as a biomarker of early responses to minerals that cause mesothelioma
• Limitations the authors state:
  • Too few HKNM-2 normal cells were available to run duplicate untreated control samples
  • Cells surviving the high dose may be those with stronger antioxidant defenses or adaptive responses
  • The link between these cell responses and cell injury and pleural disease needs further study

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: Oxidative Stress, Mesothelial Cells, Amphibole Group, Malignant Mesothelioma, Prognosis, Crocidolite, Superoxide, Glutathione (GSH).

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)
• Internalization of Libby amphibole asbestos and induction of oxidative stress in murine macrophages — D.J. Blake, C.M. Bolin, et al. (2007)
• Exposure to nano-size titanium dioxide causes oxidative damages in human mesothelial cells: The crystal form rather than size of particle contributes to cytotoxicity — K. Hattori, K. Nakadate, et al. (2017)
• 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)
• Expression of genes coding for proteoglycans and Wilms' tumour susceptibility gene 1 (WT1) by variously differentiated benign human mesothelial cells — M. Gulyás, K. Dobra, et al. (1999)
• 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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