Unknown,Transcriptomics,Genomics,Proteomics

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RAW264.7 gene expression after exposure to 1-50ug/ml AF-SWCNT-24 hours

ABSTRACT: Single-walled carbon nanotubes (SWCNTs) are being explored and used for a wide range of applications in industrial and medical sectors, and the increasing exposure of SWCNTs necessitate the studies of their potential environmental and health effects. Considerable efforts have been made to improve the dispersion of SWCNTs by chemical modifications. The present study was designed to determine if the acid functionalization of SWCNTs enhanced the toxicity and more efforts were made to understand the molecular mechanisms. RAW264.7 cells were exposed to 0-100μg/mL of SWCNTs and AF-SWCNTs for 24 hours and cell proliferation, viability, and gene expression profiles were assessed and compared. Results showed that the magnitude of AF-SWCNT-induced cell proliferation inhibition was higher than that of SWCNTs. 10μg/mL of AF-SWCNTs was determined as non-toxic to cells by viability analysis. AF-SWCNTs could enter and aggregate in cell cytoplasm and nuclear areas. Microarray, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assays (ELISA) demonstrated that AF-SWCNTs (1, 10, and 50μg/mL) altered gene and protein expression patterns. The most differentially expressed genes were related to pro-inflammatory cytokines and chemokines (such as CCL-3 and CCL-4), apoptosis (e.g., Caspase-1, -4, and Dffa), protective response (such as Hmox-1and GSTM3), and cell surface molecules such as ICAM-1, Itgb2, indicating a possible involvement of SWCNT in inflammation, anti-apoptotic and DNA fragmentation, carcinogenesis and biased T cell polarization. Furthermore, a substantial amount of down-regulated genes were found related to ribosomal assembly and mitochondrial respiration chain, implicating a possible mechanism of SWCNT-induced oxidative stress and gene expression inhibition. RAW264.7 gene expression after exposure to 1-50ug/ml AF-SWCNT-24 hours. 0ug/ml of AF-SWCNT was used as control sample. The data were obtained from three biological replicates.

ORGANISM(S): Mus musculus

SUBMITTER: Bin Wan

PROVIDER: E-GEOD-20021 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Carbon nanotubes (CNTs) are one of the most attractive nanomaterials that have many potential applications in industrial, medical, and environmental sectors. To investigate the toxic effect of single-walled CNTs (SWCNTs) on the immune system, we examined the influences of SWCNTs (depleted of metal impurities) on viability and global gene expression in murine macrophages. RAW264.7 Cells were exposed to various concentrations of SWCNTs, and cell viability was analyzed at various time points using WST-1 assay. SWCNT cytotoxicity in macrophages was concentration and time dependent, with significant viability loss occurring within 4 hr at 50Î¼g/ml SWCNT exposure. As low as 1Î¼g/ml SWCNTs reduced cell viability at 12 hr, whereas 50Î¼g/ml SWCNT exposure completely abolished cell proliferation. Laser confocal microscopy indicated that SWCNTs could enter and aggregate in cell cytoplasm and nuclear areas. Microarray, real-time reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay revealed that SWCNT altered gene and protein expression pattern. The most differentially expressed genes were related to proinflammatroy cytokines and chemokine, such as CCL-3 and CCL-4, apoptosis (e.g., Caspase-1, and -3), protective response such as Hmox-1and GSTM3) and cell surface molecules such as ICAM-1, Itgb2, indicating a possible involvement of SWCNT in inflammation and pulmonary granuloma formation, carcinogenesis and Th2 polarization of T cells. Furthermore, a substantial amount of down-regulated genes were related to ribosomal assembly and mitochondrial respiration chain, implicating a possible mechanism of SWCNT-induced oxidative stress and gene expression inhibition. The results are a first step in identifying molecular targets for the toxicity of SWCNTs and the elucidating of the molecular mechanisms for the immune modulation ability of SWCNTs. Cells were exposure to 50ug/ml of acid-functionzalized SWCNTs for 24 hours before gene expression profiling, cells without SWCNT exposure were used as control.

Project description:C. jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human foodborne illness. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia). As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC). In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion as evidenced by gentamicin-protection assays. In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC demonstrated Cia secretion as judged by metabolic labeling experiments. DOC was also found to induce the expression of the ciaB gene as judged by B-galactosidase reporter assays and real-time RT-PCR. Microarray analysis revealed that DOC induced the expression of virulence genes (i.e., ciaB, cmeABC, dccR, and tlyA). In summary, we demonstrate that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions. These results provide a foundation to identify genes expressed by C. jejuni in response to in vivo-like culture conditions. Keywords: Stress response For the expression profiling arrays, an indirect comparison of gene expression was performed, where the expression profile of the C. jejuni F38011 cultured in the presence and absence of DOC was measured separately on different slides as described previously (26). Briefly, Cy5 labeled reference DNA from the C. jejuni F38011 strain was mixed with Cy3 labeled test cDNA (C. jejuni F38011 cultured in the presence or absence of DOC) and hybridized to the Campylobacter cDNA array (26) on separate slides. DNA microarrays were scanned using an Axon GenePix 4000B microarray laser scanner (Axon Instruments, Union City, CA) and the data for spot and background intensities were processed using the GenePix 4.0 software. To compensate for any effect of the amount of template and uneven Cy-dye incorporation, data normalization was performed as previously described (26). For the comparison of genes differentially expressed in the presence and absence of DOC,six hybridization measurements were generated per biological experiment (three technical replicate arrays and two replicate features per array).

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RAW264.7 gene expression after exposure to 1-50ug/ml AF-SWCNT-24 hours

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