Utilization of Gene Profiling and Proteomics to Determine Mineral Pathogenicity in a Human Mesothelial Cell Line (LP9/TERT-1)

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 × 10⁶ or 75 × 10⁶ μm²/cm²) of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO₂), 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, TiO₂, 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)-α 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.

Notes

This work was supported by EUROTALC and the Industrial Minerals Association of North America. The authors thank the Vermont Cancer Center DNA Analysis Core Facility (supported in part by grant P30CA22435 from the National Cancer Institute) at the University of Vermont (Burlington) for technical assistance with microarray analysis. We also acknowledge Dr. Pamela Vacek (Department of Medical Biostatistics at the University of Vermont, Burlington) for providing direction in performing statistical analysis and Gary Tomiano (Minteg International, Inc./Specialty Minerals, Inc., Easton, PA) for talc characterization. JMH is also supported by training grant T32 ES007122 from the National Institute of Environmental Health Sciences. Among the authors, BTM and AS conceived of the overall research plan. AS and MBM maintained cell cultures, introduced minerals to cells, collected cell medium, prepared RNA, and performed the microarray analysis. JPB carried out PCA on the acquired microarray data. CS conducted the Bio-Plex analysis. JMH interpreted the data, carried out statistical analysis on the Bio-Plex data, and prepared the article. All authors read and approved the final article.