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Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles.

ABSTRACT: BACKGROUND: Recently, we used cell-free assays to demonstrate the toxic effects of complex mixtures of organic extracts from urban air particles (PM2.5) collected in four localities of the Czech Republic (Ostrava-Bartovice, Ostrava-Poruba, Karvina and Trebon) which differed in the extent and sources of air pollution. To obtain further insight into the biological mechanisms of action of the extractable organic matter (EOM) from ambient air particles, human embryonic lung fibroblasts (HEL12469) were treated with the same four EOMs to assess changes in the genome-wide expression profiles compared to DMSO treated controls. METHOD: For this purpose, HEL cells were incubated with subtoxic EOM concentrations of 10, 30, and 60 ?g EOM/ml for 24 hours and global gene expression changes were analyzed using human whole genome microarrays (Illumina). The expression of selected genes was verified by quantitative real-time PCR. RESULTS: Dose-dependent increases in the number of significantly deregulated transcripts as well as dose-response relationships in the levels of individual transcripts were observed. The transcriptomic data did not differ substantially between the localities, suggesting that the air pollution originating mainly from various sources may have similar biological effects. This was further confirmed by the analysis of deregulated pathways and by identification of the most contributing gene modulations. The number of significantly deregulated KEGG pathways, as identified by Goeman's global test, varied, depending on the locality, between 12 to 29. The Metabolism of xenobiotics by cytochrome P450 exhibited the strongest upregulation in all 4 localities and CYP1B1 had a major contribution to the upregulation of this pathway. Other important deregulated pathways in all 4 localities were ABC transporters (involved in the translocation of exogenous and endogenous metabolites across membranes and DNA repair), the Wnt and TGF-? signaling pathways (associated particularly with tumor promotion and progression), Steroid hormone biosynthesis (involved in the endocrine-disrupting activity of chemicals), and Glycerolipid metabolism (pathways involving the lipids with a glycerol backbone including lipid signaling molecules). CONCLUSION: The microarray data suggested a prominent role of activation of aryl hydrocarbon receptor-dependent gene expression.

SUBMITTER: Libalova H

PROVIDER: S-EPMC3275518 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles.

Líbalová Helena H Uhlířová Kateřina K Kléma Jiří J Machala Miroslav M Šrám Radim J RJ Ciganek Miroslav M Topinka Jan J

Particle and fibre toxicology 20120112

<h4>Background</h4>Recently, we used cell-free assays to demonstrate the toxic effects of complex mixtures of organic extracts from urban air particles (PM2.5) collected in four localities of the Czech Republic (Ostrava-Bartovice, Ostrava-Poruba, Karvina and Trebon) which differed in the extent and sources of air pollution. To obtain further insight into the biological mechanisms of action of the extractable organic matter (EOM) from ambient air particles, human embryonic lung fibroblasts (HEL12 ...[more]

PMID: 22239852

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Project description:Rationale: Exposure to biomass smoke is believed to increase the risk of developing chronic obstructive pulmonary disease. However, little is known about the mechanisms underlying responses to biomass smoke in human lungs.Objectives: This study had two objectives: first, to quantify "real-life" exposures to particulate matter <2 ?m in diameter (PM2.5) and carbon monoxide (CO) measured during cooking on stoves in rural areas of Nepal in different geographical settings; and second, to assess the effect of biomass smoke extracts on inflammatory responses in ex vivo human lung tissue.Methods: Personal exposures to PM2.5 and indoor near-stove CO concentrations were measured during cooking on a range of stoves in 103 households in 4 different Nepalese villages situated at altitudes between ?100 and 4,000 m above sea level. Inflammatory profiles to smoke extracts collected in the field were assessed by incubating extracts with human lung tissue fragments and subsequent Luminex analysis.Results: In households using traditional cooking stoves, the overall mean personal exposure to PM2.5 during cooking was 276.1 ?g/m3 (standard deviation [SD], 265 ?g/m3), and indoor CO concentration was 16.3 ppm (SD, 19.65 ppm). The overall mean PM2.5 exposure was reduced by 51% (P?=?0.04) in households using biomass fuel in improved cook stoves, and 80% (P?<?0.0001) in households using liquefied petroleum gas. Similarly, the indoor CO concentration was reduced by 72% (P?<?0.001) and 86% (P?<?0.0001) in households using improved cook stoves and liquefied petroleum gas, respectively. Significant increases occurred in 7 of the 17 analytes measured after biomass smoke extract stimulation of human lung tissue (IL-8 [interleukin-8], IL-6, TNF-? [tumor necrosis factor-?], IL-1?, CCL2, CCL3, and CCL13).Conclusions: High levels of real-life exposures to PM2.5 and CO occur during cooking events in rural Nepal. These exposures induce lung inflammation ex vivo, which may partially explain the increased risk of chronic obstructive pulmonary disease in these communities.

Project description:Atmospheric particulate matter (PM) is a recognized risk factor for the global burden of disease in human populations. We are presenting here the application of toxicogenomics in the evaluation of the toxic effects of organic content of atmospheric particle matter (PM), from urban and rural environments (city of Barcelona and village of La Pobla, NE Spain), using the developing zebrafish embryo. The main goal is to identify the metabolic pathways involved in the adverse effects observed in zebrafish embryos exposed to PM organic content from urban and rural environments, also allowing the selection of genes of interest that are differentially expressed. The relevance of particle size to the PM toxicity is also addressed. Indeed, the zebrafish embryos were exposed to PM of aerodynamic diameter larger than 7.2 Î¼m and smaller than 0.5 Î¼m (PM10 and PM0.5, respectively). PM0.5 concentrated biological and toxic activities linked to organic substances. Transcriptomic analyses showed strong induction of the AhR signalling pathway (a.k.a. dioxin-like activity) for embryos exposed to both rural and urban extracts, correlating with the concentrations of PAHs. Urban extracts, with strong contribution of traffic emissions, specifically de-regulated oxidative stress-related genes, as well as pancreatic and eye-lens specific genes, two organs known to be affected by air pollution in humans. Exposure to rural extracts, with high contribution of wood burning emissions, affected genes implicated in basic cellular functions, in agreement with their strong embryotoxicity. Extracts from rural and urban samples elicited both common and specific transcriptome responses, suggesting different potentially adverse outcomes depending on PM source and composition. The authors thank the financial support of the Spanish Ministry (project TEA-PARTICLE, grant number CGL2011-29621) and the Portuguese Foundation for Science and Technology for the doctoral grant of Sofia R. Mesquita (SFRH/BD/80710/2011) funded by the Program POPH-QREN through the Portuguese Ministry of Education and Science and the European Social Fund, and support through project PEst-C/MAR/LA0015/2013. The PM filter samples used in the present study were PM10 and PM0.5 from the urban site - total of 4 samples from 2 sampling months; and PM0.5 from the rural site - total of 2 samples from 2 sampling months (PM10 samples from the rural site were not tested due to their very low concentration in organic compounds, and insignificant biological activity, previously measured). Sampling occurred during the cold periods of the year 2012/2013. The organic fraction of PM samples was extracted by sonication using a mixture of dichloromethane:methanol. Then, the extracts were filtered, evaporated and re-dissolved in Dimethyl sulfoxide (DMSO). Zebrafish fertilized eggs were exposed to PM organic extracts (0.2% DMSO) from the urban and rural sites from 24 to 120h post-fertilization. Total RNA was isolated from whole embryos (pools of 20 individuals), using Trizol reagent protocol (Invitrogen Life Technologies, Carlsberg, CA). The microarray study was performed using the Agilent Two-colour D. rerio Oligo Microarray v3 platform, following the Agilent Microarray â?? Based Gene Expression Analysis protocol. Three biological replicates were performed for each PM sample and controls. Biological replicates, a technical replicate and a self-to-self, were simultaneously amplified and labelled using Cyanine 3 (Cy3) and Cyanine 5 (Cy5) dye. After cRNA purification (RNeasy Kit, Quiagen GmbH, Hilden, Germany), the cRNA concentration and labelling were quantified in the NanoDrop spectrophotometer, obtaining incorporation rates in the range of 15-20pmol of cyanide dye/Âµl cRNA and yield values > 0.825ug, as recommended. cRNA was fragmented and hybridized in 4x44K arrays, for 17h at 65oC. After the hybridization the array slides were washed, and immediately scanned using Microarray Scanner Agilent G2505C system. Data was extracted using Agilent Feature Extraction Software v10.5.1.1, and the quality of microarray data was evaluated using the Quality Control report provided by Agilent Software. Based on the microarray data analysis, 22 primers were selected and designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA). Genes were quantified by real-time PCR to validate the microarray data, and a subset of 13 genes were further selected by their robust behaviour in the validation assays, allowing to differentiate the toxic potential of PM from urban and rural sources.

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Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles.

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Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles.

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