Olecular events occurring in the human mesothelial cell line, LP9/TERT-1, that may contribute to the toxicity of Libby six-mix. Our laboratory has recently utilized this approach to examine transcriptional alterations in LP9/TERT-1 cells following exposure to crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO2), or glass beads [15,16]. Our ongoing hypothesis for both the previously reported studies and those discussed here is that the number and magnitude of significant gene changes elicited by minerals correlate with their toxicity and pathogenic potential [15,16]. Results of the current studies in LP9/TERT-1 cells appear to support this hypothesis. Since microarray analyses following exposure to Libby six-mix showed that only SOD2 was upregulated at both time points tested, an observation we confirmed in isolates of HKNM-2 normal human pleural mesothelial cells, we conducted a series of experiments to examine both the potential of this amphibole to generate oxidative stress in LP9/TERT-1 cells, and the functional significance of these changes. This was especially relevant since oxidants generated by asbestos fibers or cells after contact with or uptake of asbestos are linked to toxic and biological manifestations in progenitor and effector cells of asbestos-related diseases . Immunoblotting and activity assays confirmed this increase PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28499442 in SOD2 levels, and DCFDA fluorescence staining and flow cytometry revealed a dose- and time-dependent increase in ROS production by LP9/TERT-1 cells exposed to Libby six-mix. Given the role glutathione (GSH) plays as anantioxidant and signaling molecule in the lung, we examined the effects that Libby six-mix and crocidolite asbestos had on GSH and showed that at a toxic concentration (75?0 6 m 2 /cm 2 ), these minerals caused transient decreases in GSH for up to 24 h. These studies are the first to examine the ability of Libby six-mix to elicit transcriptional changes and oxidative stress in human mesothelial cells. These early molecular events may contribute to the toxicity and pre-neoplastic effects of this amphibole in the development of mesotheliomas.ResultsCharacterization and toxicity of mineral preparationsChemical composition, mean surface area (S.A.), and mean size of the glass beads, Libby six-mix and NIEHS crocidolite preparations used in our studies are provided in Table 1 and have been characterized by others [17-20]. The sample of Libby amphibole we used in the studies described here is often referred to as “six-mix” since it includes six different samples collected at the former mine site, and is comprised of a combination of several amphiboles including winchite, richterite and tremolite (approximately 84 , 11 , and 6 of the respirable fraction, respectively), as well as other trace elements not classified in this mineral family . It possesses diverse morphologies including both asbestiform and non-asbestiform structures (cleavage fragments) exhibiting a wide range of aspect ratios . Trace amounts of other elements occur in NIEHS asbestos standards  as well as in Libby six-mix , and the fiber size, length and diameter distributions and proportions of cleavage fragments and nonfibrous particles are different between these preparations. For example, blocky particles and small fragments were a feature of the Libby six-mix preparation when examined by SEM (Figure 1A) as AZD4547MedChemExpress AZD4547 compared to fibrous crocidolite asbestos (Figure 1B). In addition, the morphology and ce.