Project description:An estimated 1% or less of the nanoparticles (NPs) deposited in lungs translocate to systemic circulation and enter other organs; however this estimation may not be accurate considering the low sensitivity of the existing in vivo NP detection methods. Moreover, the biological outcomes of such low levels of translocation are not elucidated. The objectives of the present study were to employ a Nano-scale Hyperspectral Microscope to spatially observe and spectrally profile NPs in tissues, and characterize the effects of NPs in blood, liver and heart following pulmonary deposition and subsequent translocation from lungs. Adult female C57BL/6 mice were exposed via intratracheal instillation to 18 and 162 µg per mouse of industrially relevant non-doped titanium dioxide nanoparticles (nano-TiO2). Using the Nano-scale Hyperspectral Microscope translocation to heart and liver was confirmed at both doses and to blood at the highest dose at 24 hours post-exposure time-point. The analysis of biological effects using DNA microarrays, RT-qPCR and ELISA revealed activation of complement cascade and inflammatory process in heart and specific activation of complement factor 3 in blood, potentially suggestive of activation of early innate immune response essential for particle opsonisation and clearance. The liver showed subtle response with changes in the expression of few genes associated with acute phase genes. This study establishes a direct link between particle translocation and systemic effects. An estimated 1% or less of the nanoparticles (NPs) deposited in lungs translocate to systemic circulation and enter other organs; however this estimation may not be accurate considering the low sensitivity of the existing in vivo NP detection methods. Moreover, the biological outcomes of such low levels of translocation are not elucidated. The objectives of the present study were to employ a Nano-scale Hyperspectral Microscope to spatially observe and spectrally profile NPs in tissues, and characterize the effects of NPs in blood, liver and heart following pulmonary deposition and subsequent translocation from lungs. Adult female C57BL/6 mice were exposed via intratracheal instillation to 18 and 162 µg per mouse of industrially relevant non-doped titanium dioxide nanoparticles (nano-TiO2). Using the Nano-scale Hyperspectral Microscope translocation to heart and liver was confirmed at both doses and to blood at the highest dose at 24 hours post-exposure time-point. The analysis of biological effects using DNA microarrays, RT-qPCR and ELISA revealed activation of complement cascade and inflammatory process in heart and specific activation of complement factor 3 in blood, potentially suggestive of activation of early innate immune response essential for particle opsonisation and clearance. The liver showed subtle response with changes in the expression of few genes associated with acute phase genes. This study establishes a direct link between particle translocation and systemic effects. This experiment consists of one dose of nano-TiO2 (162 ug) and one control. There are 2 time points for each treatment and control group, e.g., day 1 and day 28. Each dose or time point has 5-6 biological replicates. There are total 22 samples (arrays)

Project description:Pulmonary exposure to high doses of nanoparticles (NP) leads to well characterized lung toxicity in addition to long-term NP retention. However, pulmonary NP accumulation and toxicity following low dose exposures are not well described. In the present study we sought to: (1) investigate particle retention in mouse lungs following intratracheal instillation of varying doses of nano-sized titanium dioxide (nano-TiO2) and (2) determine the effects of long-term particle accumulation on pulmonary systems. Female C57BL/6 mice were exposed to rutile nano-TiO2 (primary size of 20.6 nm and surface area of 107.7 m2/g) via single intratracheal instillations of 18, 54 and 162 M-BM-5g/mouse and sampled 1, 3 and 28 days post-exposure. The deposition of nano-TiO2 in the lungs was assessed using Nanoscale Hyperspectral Microscope. DNA microarrays, pathway-specific real-time RT-PCR (qPCR) and gene-specific qPCR arrays, and tissue protein analyses were employed to characterize pulmonary responses. Hyperspectral mapping showed dose-dependent retention of nano-TiO2 in the lungs up to 28 days post-exposure time. Retention did not correlate with the extent of inflammatory neutrophil influx into the lungs. DNA microarray analysis showed altered expression of approximately 3000 genes across all treatment groups (M-BM-11.3 fold; p<0.1). Several inflammatory mediators changed in a dose- and time-dependent manner at both the mRNA and protein levels. Although the low dose exposure failed to induce observable inflammation, significant changes in the expression of genes and proteins associated with inflammation were observed. Moreover, diminished (or absent) neutrophil influx in the low and medium dose groups was correlated with negative regulation of genes associated with ion homeostasis and muscle regulation. Gene expression changes for several inflammatory mediators have previously been noted in mice exposed to the same nano-TiO2 via inhalation. Our results suggest that retention of nano-TiO2 in the absence of inflammation and effective clearance can perturb calcium and ion homeostasis, and affect smooth muscle activities over time. This experiment consists of three different dosages of TiO2, e.g., low (18 ug), medium (54 ug) and high (162 ug), and one control. There are 3 time points for each treatment and control group, e.g., day 1, day 3 and day 28. Each dose or time point has 5-6 biological replicates. There are total 65 samples (arrays)

Project description:This experiment investigated the response of mouse pulmonary epithelial cells in culture (FE1 cell line) to 3 different brands of cigarette smoke condensate. The three brands represent a full flavour, regular and king-size light cigarettes. These vary in some key chemical consistuents. Brand #1 has the highest tar content. The cells were exposed to two doses: one at approximately 50% cytotoxicity, and the other at approximately 20%. Both doses were clastogenic. The major pathways affected included xenobiotic metabolism, cell cycle, apoptosis, oxidative stress and inflammation. Although brand #1 exhibited the most numbers of genes that were differentially expressed, there did not appear to be any major differences in the expression profiles between the brands. Although some genes did not reach statistical significance, in general there was consistency in the direction of fold changes for genes across all brands. MutaMouse FE1 cells in culture (derived from lung epithelial cells) were exposed to cigarette smoke condensate (CSC) collected from 3 brands: Brand #1 M-bM-^@M-^S Full Flavor; Brand #3 M-bM-^@M-^S Regular; Brand #5 M-bM-^@M-^S Light King Size. Experiments were performed on 5 replicates per condition (i.e., n=5 per group); one replicate was removed from 4 groups later in the study. Cells were exposed at 70% confluence in 150 mm plates to either 45 M-BM-5g/ml or 90 M-BM-5g/ml CSC alongside cells exposed to 1% DMSO (Sigma-Alderich, Canada) in 1:1 DMEM:F12 (without FBS). Cells were exposed to CSC for 6 hours and then either: (a) immediately harvested, or (b) washed in PBS and cultured for another 4 hours in fresh media. The two time points were called 6 hours or 10 hours (6 hours exposure, 4 hours recovery for the latter). Cells were harvested with TRIzol (Invitrogen) and stored at -80oC. RNA was extracted and labelled, and hybridized to Agilent 22K mouse development microarrays following the manufacturer's protocol (Agilent Technologies Inc., USA). Arrays were washed and scanned on a ScanArray Express (Perkin-Elmer Life Sciences), and data were acquired with ImaGene 5.5 (BioDiscovery Inc.). Biological samples were hybridized in Cy5, and Stratagene Universal Reference RNA was hybridized in Cy3.

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Transcriptomic analysis reveals novel mechanistic insight into murine biological responses to multi-walled carbon nanotubes in lungs and cultured lung epithelia cells

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Transcriptomic analysis reveals novel mechanistic insight into murine biological responses to multi-walled carbon nanotubes in lungs and cultured lung epithelial cells.

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