Project description:Gene expression microarrays were used to compare gene alterations induced by exposure to equitoxic doses of crocidolite asbestos and cristobalite silica in an isolate of normal human bronchial epithelial cells. Primary human bronchial epithelial cells (NHBE) were exposed to crocidolite asbestos, cristobalite silica, and saline as control for 24 hours. Total RNA was extracted from cell cultures, 3 separate cultures were used for each condition. The isolated RNA was used to analyze mRNA expression via Affymetix GeneChip Human U133A 2.0 arrays.
Project description:Gene expression microarrays were used to compare gene alterations induced by exposure to equitoxic doses of crocidolite asbestos and cristobalite silica in an isolate of normal human bronchial epithelial cells.
Project description:Asbestos is the general term for a family of naturally occurring fibrous silicate minerals having commercial importance. They have been used widely for construction and for a number of industrial applications until early 70’s. It is now well known that workers exposed to asbestos are at high risk of developing many lung diseases including asbestosis, malignant mesothelioma, bronchial adenocarcinoma; squamous cell carcinoma of the respiratory epithelium and large/ small cell lung carcinoma. However, the disease causing potential and related processes associated with various asbestos/asbestiform fiber exposures are still largely unknown. In this study, we exposed C57BL6 female mice to asbestos (crocidolite, tremolite), asbestiform fibers (erionite) and a low pathogenicity mineral fiber (wollastonite) to assess pulmonary toxicity responses along with gene expression profiles in the lungs following 7 days post pharyngeal aspiration.
Project description:Human mesothelial cells (LP9/TERT-1) were exposed to low and high (15 and 75 μm2/cm2 dish) equal surface area concentrations of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO2), or glass beads for 8 or 24 h. RNA was then isolated for Affymetrix microarrays, GeneSifter analysis and QRT-PCR. Gene changes by asbestos were concentration- and time-dependent. At low nontoxic concentrations, asbestos caused significant changes in mRNA expression of 29 genes at 8 h and 205 genes at 24 h, whereas changes in mRNA levels of 236 genes occurred in cells exposed to high concentrations of asbestos for 8 h. Human primary pleural mesothelial cells also showed the same patterns of increased gene expression by asbestos. Nonfibrous talc at low concentrations in LP9/TERT-1 mesothelial cells caused increased expression of 1 gene Activating Transcription Factor 3 (ATF3) at 8 h and no changes at 24 h, whereas expression levels of 30 genes were elevated at 8 h at high talc concentrations. Fine TiO2 or glass beads caused no changes in gene expression. In human ovarian epithelial (IOSE) cells, asbestos at high concentrations elevated expression of 2 genes (NR4A2, MIP2) at 8 h and 16 genes at 24 h that were distinct from those elevated in mesothelial cells. Since ATF3 was the most highly expressed gene by asbestos, its functional importance in cytokine production by LP9/TERT-1 cells was assessed using siRNA approaches. Results reveal that ATF3 modulates production of inflammatory cytokines (IL-1β, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB) in human mesothelial cells. Microarrays were performed on samples from 3 independent experiments. All cell types, time points, and mineral types and concentrations were included in all 3 experiments. For each experiment, n=3 dishes were pooled into one sample per treatment group. Each of the pooled samples was analyzed on a separate array, i.e., n=3 arrays per condition (3 independent biological replicates). We tested the hypothesis that alteration in gene expression in human cells correlate with mineral pathogenicity. We used GeneSifter program to analyze our data and pairwise analysis showed that number of gene changes correlate with toxicity of pathogenic minerals. While non-pathogenic minerals glass beads and fine TiO2 treatment to cell resulted in no gene change, crocidolite asbestos caused maximum number of gene changes followed by talc.
Project description:The goal was to assess global gene expression changes in primary human bronchial epithelial cells exposed to environmental tobacco smoke (ETS) condensate. ETS-C was standardized by HPLC analysis and two timepoints of exposure in two different donor bronchial epithelial cell populations were assessed. These findings demonstrate that even short exposure (4.5 h) to ETS is sufficient to induce a stress response, as reflected by decreased antioxidant levels, induced HSP family members, and modulation of the family of glutathione metabolism enzymes in primary human lung cells. Upon longer exposures (48 h) with ETS-condensate, bronchial epithelial cells arrest at the G2/M phase of the cell cycle. Taken together, these data support a stress-induced state in primary human bronchial epithelial cells that culminates in cell cycle arrest. Keywords: time course, comparative, stress response
Project description:Human mesothelial cells (LP9/TERT-1) were exposed to low and high (15 and 75 μm2/cm2 dish) equal surface area concentrations of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO2), or glass beads for 8 or 24 h. RNA was then isolated for Affymetrix microarrays, GeneSifter analysis and QRT-PCR. Gene changes by asbestos were concentration- and time-dependent. At low nontoxic concentrations, asbestos caused significant changes in mRNA expression of 29 genes at 8 h and 205 genes at 24 h, whereas changes in mRNA levels of 236 genes occurred in cells exposed to high concentrations of asbestos for 8 h. Human primary pleural mesothelial cells also showed the same patterns of increased gene expression by asbestos. Nonfibrous talc at low concentrations in LP9/TERT-1 mesothelial cells caused increased expression of 1 gene Activating Transcription Factor 3 (ATF3) at 8 h and no changes at 24 h, whereas expression levels of 30 genes were elevated at 8 h at high talc concentrations. Fine TiO2 or glass beads caused no changes in gene expression. In human ovarian epithelial (IOSE) cells, asbestos at high concentrations elevated expression of 2 genes (NR4A2, MIP2) at 8 h and 16 genes at 24 h that were distinct from those elevated in mesothelial cells. Since ATF3 was the most highly expressed gene by asbestos, its functional importance in cytokine production by LP9/TERT-1 cells was assessed using siRNA approaches. Results reveal that ATF3 modulates production of inflammatory cytokines (IL-1β, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB) in human mesothelial cells.
Project description:This study is the first to show transfer of regulatory bacterial sRNAs from bacterial OMVs to host cells. Demonstration of transfer of bacterial sRNA from Pseudomonas aeruginosa OMVs to host cells in two cell types. RNA isolated from PA14 OMV exposed primary human bronchial epithelial cells or CFBE41o- bronchial epithelial cells as well as unexposed control cells was analyzed by small RNA-Seq. Primary cell exposures were performed on two donors and exposures of CFBE41o- cells were done in triplicate.