Utilization of gene profiling and proteomics to determine mineral pathogenicity in a human mesothelial cell line (LP9/TERT-1)

Our plain-English summary of Utilization of gene profiling and proteomics to determine mineral pathogenicity in a human mesothelial cell line (LP9/TERT-1), by J.M. Hillegass, A. Shukla, M.B. MacPherson, et al (Journal of Toxicology and Environmental Health. Part a, 2010).

The study

• Authors: J.M. Hillegass, A. Shukla, M.B. MacPherson, J.P. Bond, C. Steele, B.T. Mossman
• Journal: Journal of Toxicology and Environmental Health. Part a
• Published: 2010;73(5):423-36
• Cited by: 24 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Identifying and understanding the early molecular events that underscore mineral pathogenicity using in vitro screening tests is imperative, especially given the large number of synthetic and natural fibers and particles being introduced into the environment. The purpose of the work described here was to examine the ability of gene profiling (Affymetrix microarrays) to predict the pathogenicity of various materials in a human mesothelial cell line (LP9/TERT-1) exposed to equal surface area concentrations (15 x 10(6) or 75 x 10(6) microm(2)/cm(2)) of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO(2)), or glass beads for 8 or 24 h. Since crocidolite asbestos caused the greatest number of alterations in gene expression, multiplex analysis (Bio-Plex) of proteins released from LP9/TERT-1 cells exposed to crocidolite asbestos was also assessed to reveal if this approach might also be explored in future assays comparing various mineral types. To verify that LP9/TERT-1 cells were more sensitive than other cell types to asbestos, human ovarian epithelial cells (IOSE) were also utilized in microarray studies. Upon assessing changes in gene expression via microarrays, principal component analysis (PCA) of these data was used to identify patterns of differential gene expression. PCA of microarray data confirmed that LP9/TERT-1 cells were more responsive than IOSE cells to crocidolite asbestos or nonfibrous talc, and that crocidolite asbestos elicited greater responses in both cell types when compared to nonfibrous talc, TiO(2), or glass beads. Bio-Plex analysis demonstrated that asbestos caused an increase in interleukin-13 (IL-13), basic fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF). These responses were generally dose-dependent (bFGF and G-CSF only) and tumor necrosis factor (TNF)-alpha independent (except for G-CSF). Thus, microarray and Bio-Plex analyses are valuable in determining early molecular responses to fibers/particles and may directly contribute to understanding the etiology of diseases caused by them. The number and magnitude of changes in gene expression or "profiles" of secreted proteins may serve as valuable metrics for determining the potential pathogenicity of various mineral types. Hence, alterations in gene expression and cytokine/chemokine changes induced by crocidolite asbestos in LP9/TERT-1 cells may be indicative of its increased potential to cause mesothelioma in comparison to the other nonfibrous materials examined.

In plain English

This laboratory study used human cells, not people. The authors exposed a human mesothelial cell line (LP9/TERT-1, which resembles normal human peritoneal mesothelial cells) and, for comparison, a human ovarian epithelial cell line (IOSE) to crocidolite asbestos, nonfibrous talc, fine titanium dioxide or glass beads. Equal surface-area doses were used, for 8 or 24 hours. The authors applied principal component analysis to previously reported gene-activity (microarray) data. They also measured proteins released by mesothelial cells exposed to crocidolite with or without the signaling protein TNF-alpha. Crocidolite produced the largest gene responses, and mesothelial cells responded more than ovarian cells. Crocidolite raised four proteins the authors focused on. bFGF rose 7-fold at the lower dose and 19-fold at the higher. G-CSF rose 2-fold and 5-fold, but only the higher dose was statistically significant. IL-13 rose significantly. With asbestos alone, VEGF rose but not significantly; asbestos plus TNF-alpha differed significantly from TNF-alpha alone. The authors concluded that crocidolite should be considered the most pathogenic material tested. They said that using gene changes to judge mineral hazard is a hypothesis still being tested.

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: principal component analysis of previously reported microarray gene-expression data, plus new Bio-Plex measurements of 27 proteins released into the cell medium
• Who was studied: Human cell lines, not people. The main line was LP9/TERT-1 human mesothelial cells, an immortalized line resembling normal human peritoneal mesothelial cells. The comparison line was IOSE 398 human ovarian epithelial cells, which the authors included essentially as a negative control. Microarrays used 3 independent experiments (n = 3 RNA samples per group); Bio-Plex used n = 3 medium samples per group.
• Exposure: Crocidolite asbestos, nonfibrous talc (MP 10-52), fine titanium dioxide (TiO2) and glass beads, at equal surface-area concentrations of 15 × 10^6 or 75 × 10^6 μm2/cm2 (equivalent to 1 and 5 μg/cm2), for 8 or 24 hours. Not every mineral was tested at every dose and time. For the protein measurements, LP9/TERT-1 cells received crocidolite at either dose, with or without 10 ng/ml TNF-alpha, for 24 hours.
• Main results:
  • In LP9/TERT-1 mesothelial cells, the principal component analysis ranked the response magnitude as follows. Crocidolite at 15 × 10^6 μm2/cm2 for 24 h, or at 75 × 10^6 for 8 h, came first. Crocidolite at 15 × 10^6 for 8 h, or talc at 75 × 10^6 for 8 h, came next. All other minerals came last.
  • In the previously reported counts of genes changed more than 2-fold in LP9/TERT-1 cells, crocidolite at 15 × 10^6 μm2/cm2 changed 29 genes at 8 h and 205 at 24 h. Crocidolite at 75 × 10^6 changed 236 genes at 8 h. Talc at 75 × 10^6 changed 30 genes at 8 h. Glass beads and TiO2 changed 0. In IOSE cells, crocidolite at 75 × 10^6 changed 2 genes at 8 h and 17 at 24 h.
  • The response of LP9/TERT-1 mesothelial cells to crocidolite at 15 × 10^6 μm2/cm2 for 24 h was greater in magnitude than the response of IOSE ovarian cells to crocidolite at 75 × 10^6 μm2/cm2 for 24 h.
  • A 27-protein panel was measured, but the authors focused this article on IL-13, bFGF, G-CSF and VEGF.
  • In LP9/TERT-1 cells, crocidolite at 15 × 10^6 μm2/cm2 caused a 7-fold increase in bFGF protein compared with cells in medium alone.
  • In LP9/TERT-1 cells, crocidolite at 75 × 10^6 μm2/cm2 caused a 19-fold increase in bFGF compared with cells in medium alone. Adding TNF-alpha did not have a significant effect.
  • In LP9/TERT-1 cells, crocidolite at 15 × 10^6 and 75 × 10^6 μm2/cm2 increased G-CSF 2-fold and 5-fold over medium alone. Only the higher dose was statistically significant (p less than 0.05).
  • Combining crocidolite with TNF-alpha significantly increased G-CSF compared with crocidolite alone. However, the combined response did not differ from the sum of the separate responses to asbestos alone and TNF-alpha alone.
  • Both crocidolite doses caused a significant increase in IL-13 production by LP9/TERT-1 cells. This response appeared independent of TNF-alpha.
  • Crocidolite alone increased VEGF in LP9/TERT-1 cells, but the changes were not statistically significant.
  • LP9/TERT-1 cells co-treated with crocidolite and TNF-alpha had VEGF levels significantly different from cells treated with TNF-alpha alone.
  • The protein increases were not necessarily matched by changes in gene activity. Of the four proteins, only VEGF showed a gene-expression change greater than 1.5-fold: a 2.1-fold increase after 15 × 10^6 μm2/cm2 crocidolite for 24 h.
• The authors' conclusion: The authors concluded that crocidolite asbestos caused the largest number of gene changes and released proteins of the materials tested, and should therefore be considered the most pathogenic fiber or particle tested. They also wrote that these changes in LP9/TERT-1 cells may indicate a greater potential to cause mesothelioma than the nonfibrous materials examined.
• Limitations the authors state:
  • The authors state that the idea that the number of gene changes or secreted-protein profiles can measure a mineral's potential pathogenicity is a hypothesis they are still testing.
  • The authors note that VEGF release was more variable than the other proteins examined.

Topics

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

Medical terms in this study, explained in our Lexicon: Mesothelial Cells, Crocidolite, Talc, Fibroblast, Necrosis, Etiology, Cytokine, Mesothelioma.

Related studies

• Gene expression profiles in asbestos-exposed epithelial and mesothelial lung cell lines — P. Nymark, P.M. Lindholm, et al. (2007)
• 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)
• Gene expression profiling and gene copy-number changes in malignant mesothelioma cell lines — C. Zanazzi, R. Hersmus, et al. (2007)
• Continuous exposure to chrysotile asbestos can cause transformation of human mesothelial cells via HMGB1 and TNF-α signaling — F. Qi, G. Okimoto, et al. (2013)
• 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)
• Exosomes from asbestos-exposed cells modulate gene expression in mesothelial cells — P. Munson, Y.W. Lam, et al. (2018)

Browse more research in Journal of Toxicology and Environmental Health. Part a, or return to medical research.