Our studies are the first to use robust gene profiling to characterize alterations in gene expression in human mesothelial cells after exposure to Libby six-mix. Prior to initiating microarray studies, we determined a concentration of Libby six-mix that was not overtly toxic to LP9/TERT-1 cells to avoid the induction of gene expression secondary to cell death. This was accomplished by performing dose-response cell viability studies where LP9/TERT-1 cells were exposed to 15×106, 75×106, and 159×106 μm2/cm2 Libby six-mix for 24 h. Results demonstrated that, similar to what we have reported previously for crocidolite asbestos , Libby six-mix caused decreases in cell viability at 24 h that were significant at 75×106 μm2/cm2 and associated with cell membrane blebbing, contraction of the cells, and exudate formation around long fibers. Based upon our toxicity data, the low (15×106 μm2/cm2), nontoxic concentration of Libby six-mix and comparable surface areas of crocidolite and glass beads were chosen for microarray studies. When the top 10 gene changes following Libby six-mix exposure were compared to those reported for LP9/TERT-1 cells following crocidolite asbestos exposure at identical surface area concentrations , numerous similarities were observed. Libby six-mix induced significant upregulation of only one gene at 8 h (SOD2, 4-fold). In contrast, SOD2 (6-fold increase) was one of many genes upregulated at 8 h following crocidolite asbestos exposure . Of the top 10 genes upregulated at 24 h in response to Libby six-mix, 8 were identical to those reported at 24 h following crocidolite asbestos exposure (TFPI2, IL8 C-terminal variant, IL8, PTGS2, PDK4, PHLDA1, ATF3, and SOD2). Similarly, 7 of the top 10 genes downregulated at 24 h in response to Libby six-mix were identical to those reported for crocidolite asbestos (OXTR, C5orf13, C21orf7, CYP24A1, METTL7A, PPL, and PLCL1). Gene ontology classifications for Libby six-mix also exhibited patterns similar to crocidolite asbestos whereby the most changes in expression (either upregulated or downregulated) occurred in genes associated with signal transduction, protein metabolism, cell proliferation, and immune responses .
Several genes upregulated in LP9/TERT-1 cells exposed to Libby six-mix or crocidolite asbestos are associated with inflammation, including the pro-inflammatory cytokine interleukin-8 (IL8). This cytokine is chemotactic for polymorphonuclear neutrophils (PMNs) and can subsequently serve as an activator for these inflammatory cells . IL8 is produced by a wide array of cells including mesothelial cells, and pleural inflammation is known to be initiated by IL8 secretion in response to asbestos exposure [26, 27]. Several studies looking at serum IL8 levels in workers occupationally exposed to asbestos revealed significant increases in this cytokine [28, 29]. Activating transcription factor 3 (ATF3), a member of the cAMP-responsive element-binding (CREB) transcription factor family encoding two isoforms leading to repression or activation of genes, was also upregulated by both Libby six-mix and crocidolite asbestos. Previous work in our laboratory has shown that silencing ATF3 in LP9/TERT-1 mesothelial cells can consequently modulate the production of several well-known asbestos-induced inflammatory cytokines and growth factors including IL-1β, IL-13, granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor-BB (PDGF-BB) . Prostaglandin-endoperoxide synthase 2 (PTGS2; also known as cyclooxygenase 2 or COX2) is an integral enzyme in the biosynthesis of prostenoids, and is implicated in carcinogenesis by modulating inflammation, mitogenesis, cell adhesion, and apoptosis. Exposure of a monocyte-macrophage (J774) cell line to an asbestos-like amphibole fiber known as fluoro-edenite induced a significant increase in PTGS2 expression , and immunohistochemical characterization of human MM samples demonstrated that PTGS2 was highly expressed in these tissues, but not in nonreactive mesothelial tissues from the same individuals . Based upon the upregulation of these genes and others, inflammation induced in an autocrine/paracrine fashion by mesothelial cells may play a critical role in Libby six-mix and crocidolite asbestos-induced cell injury and disease.
In our studies, superoxide dismutase 2 (SOD2; manganese superoxide dismutase), a mitochondrial antioxidant protein which catalyzes the dismutation of superoxide (O2•-) to hydrogen peroxide (H2O2), was the only gene significantly upregulated by Libby six-mix and crocidolite asbestos at both 8 and 24 h. This increased expression of SOD2 recapitulates previously reported data showing elevated SOD2 expression/activity in human pleural mesothelial cells or tracheal epithelial cells exposed to crocidolite or chrysotile asbestos [23, 32]. The fact that SOD2 expression is upregulated following Libby six-mix and crocidolite or chrysotile asbestos exposures [23, 32], likely represents activation of a defense mechanism to counteract oxidative stress induced by these fibers, specifically through the dismutation of O2•- to H2O2. Increases in steady-state levels of H2O2 can subsequently lead to increases in cell proliferation, invasion, migration, metastasis, and resistance to apoptosis [33–35]. In addition, we have previously shown that transfection of SOD2 into rodent tracheal epithelial cells ameliorates crocidolite asbestos-induced toxicity, suggesting a role of this gene in cell survival from asbestos .
Elevated SOD2 levels have been observed in rodent lungs after inhalation of crocidolite asbestos [37, 38] and in several cancer cell types including gastric , colorectal , breast , and MM . Based upon strong SOD2 immunoreactivity in MMs in contrast to adenocarcinomas , and low SOD2 levels in healthy human pleural mesothelium compared to high endogenous levels in MM lines , SOD2 has been proposed as a diagnostic marker for MM. Moreover, polymorphisms of glutathione-S-transferase M1 (GSTM1) and SOD2 are associated with increased risk of MM, findings contributing to the hypothesis that imbalances between oxidative stress and antioxidant enzymes are features of the pathogenesis of MM . Our results showing that early increases in expression, protein levels, and activity of SOD2 occur in human mesothelial cells after exposure to MM-inducing fibers therefore may be valuable in designing predictive assays for fiber pathogenicity.
Several studies have been conducted recently examining the effects of Libby six-mix and crocidolite asbestos in tandem. For example, gene expression studies in lungs of C57Bl/6 mice instilled intratracheally with Libby six-mix or crocidolite asbestos revealed common gene ontologies related to the plasma membrane, transport channels and signal transduction . Lung fibrosis and collagen deposition were also observed in mice 6 months following exposure to either Libby six-mix or crocidolite asbestos, although the extent of changes observed in Libby six-mix exposed mice was consistently less . A second C57Bl/6 mouse study demonstrated that at 1 week, 1 month, and 3 months post intratracheal instillation of Libby six-mix or crocidolite asbestos at equal weight concentrations, both amphiboles increased the gene expression of Col1A1, Col1A2, and Col1A3, collagen protein deposition, and inflammation . Again, crocidolite asbestos induced a greater response in the majority of these endpoints when compared to Libby six-mix. However, in both these experiments, an equal mass of Libby six-mix was compared to crocidolite asbestos, possibly reflecting lower fiber numbers or different length and diameter ratios of fibers per unit weight.
In vitro experiments using a murine macrophage-like cell line (RAW264.7 cells) and alveolar macrophages lavaged from C57Bl/6 mice and exposed to 62.5 μg/cm2 Libby six-mix for up to 3 h also have shown increases in intracellular ROS levels . This response was subsequently linked to increases in O2•- by demonstrating that Libby six-mix exposure led to increased dihydroethidine (DHE) fluorescence, a probe known to preferentially detect superoxide . These increases in intracellular ROS in murine macrophages resulted in oxidative DNA damage as indicated by increased relative levels of 8-oxo-dG and the percentage of cells in the sub-G1 phase following exposure to crocidolite asbestos, but not Libby six-mix, at an equal weight concentration. Decreased intracellular GSH levels was also found to be a feature of Libby six-mix and crocidolite asbestos toxicity in this cell type . On the basis of these results, the authors suggest that separate cellular responses are induced by these two minerals in vitro. Although it appears from our research that similar mechanisms contribute to the toxicity of these minerals, their overall differences in toxicity and pathogenicity as demonstrated in work cited above, may reflect different numbers, sizes, and proportions of fibers to nonfibrous particles and fragments, as well as the diverse chemical composition of these different amphiboles. In our studies, oxidant generation and GSH depletion occurred only at the highest concentration of Libby six-mix where approximately 60% of cell death occurred at 24 h. The viable cells at this time point may represent those not killed directly by oxidative stress because of intrinsically higher antioxidant defenses or cells exhibiting adaptive responses such as compensatory proliferation in response to this material. DCF fluorescence does not appear to occur coincidentally with cell death as it is often observed in non-apoptotic cells. The molecular parameters governing these responses and their relationship to cell injury and pleural disease demand further examination.