Llular interactions of Libby six-mix with LP9/ TERT-1 cells were examined using SEM. LP9/TERT-1 cells have prominent microvilli and are squamous and contiguous, resembling the normal morphology of mesothelial cells in vivo (Figure 1C). However, treatment with 75?06 m2/cm2 of Libby six-mix caused contraction of the cells around long fibers, cell membrane blebbing, and exudate formation around fibers (Figure 1D). Trypan blue exclusion viability studies demonstrated a doserelated increase in cytotoxicity with increasing concentrations of Libby six-mix, but not glass beads (Figure 1E). Based on these viability studies, we chose the non-toxic Libby six-mix dose of 15?0 6 m 2 /cm 2 with which to carry out microarray studies to avoid assaying dead or dying cells. In contrast, higher surface area concentrations (75?06 m2/cm2) of Libby six-mix and crocidolite asbestos caused approximately 60 and 50-80 decreases [16], respectively, in cell viability in LP9/TERT-1 cells at 24 h.Hillegass et al. Particle and Fibre Toxicology 2010, 7:26 http://www.particleandfibretoxicology.com/content/7/1/Page 3 ofTable 1 Mineral characterizationName Glass beads Libby six-mix Crocidolitea b cChemical Composition SiO2 See references [18-20] Na2Fe2+3Fe3+2Si8O22(OH)Mean S.A. (m2/g)a 3 5Mean Size (m)b 2.06 7.21?.61c 7.40?.Source Polysciences Inc. USGS NIEHS Reference SampleS.A. = surface area. Length ?Pan-RAS-IN-1 web diameter for Libby six-mix and crocidolite asbestos, and diameter for glass beads. See reference [17].Time-related changes in gene expression are observed following exposure of LP9/TERT-1 cells to Libby six-mixGeneChip?Human Genome U133A 2.0 arrays targeting 18,400 human transcripts were used for microarray analyses of LP9/TERT-1 cells treated with 15?06 m2/cm2 Libby six-mix for 8 or 24 h. Exposure to Libby six-mix elicited upregulation of only one gene, SOD2, at 8 h, and 111 gene changes at 24 h. The top 10 genes upregulated or downregulated by Libby six-mix at 8 and 24 h are listed in Table 2. No significant (p < 0.05) alterations in gene expression were observed following exposure to glass beads (non-pathogenic control; 75?06 m2/cm2) at either time point (data not shown). TaqMan?quantitative real time PCR (qRT-PCR) was employed to validate increases in ATF3, IL8, and SOD2 gene PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27872238 expression in HKNM-2 normal human pleural mesothelial cells. Also, since ATF3 and IL8 mRNA expression was increased in LP9/TERT-1 cells treated with 15?0 6 m 2 /cm 2 Libby six-mix and in previous studies using crocidolite asbestos [16], qRT-PCR was used to verify these changes (Table 2). GeneSifter software was utilized to determine the ontology of genes upregulated and downregulated in LP9/TERT-1 cells at 24 h. Overall, 74 genes were upregulated and 37 genes were downregulated at 24 h following exposure to 15?06 m2/cm2 of Libby six-mix (Table 3).SOD2 protein levels and activity are increased in LP9/ TERT-1 cells following Libby six-mix exposuresix-mix (75?06 m2/cm2) and TPA treatment groups (Figure 2B). Both total SOD and SOD2 activity were then examined at 24 h using an assay employing a tetrazolium salt which, upon reduction with superoxide anions generated via xanthine oxidase activity, forms a formazan dye. Since SOD is capable of catalyzing the reduction of superoxide anions to H2O2, this assay measures any changes in formazan dye formation that occurs as a result of increased/decreased SOD production following exposure to the minerals of interest. Total SOD activity was unaffected by expo.