Project description:In the present study, we investigate pulmonary transcriptional responses in mice following exposure in situ to ambient air in a heavily polluted urban environment. Mature C57BL/CBA male mice were caged in sheds located in an urban area near two working steel mills and a major highway in Hamilton, Ontario, Canada. Control mice were housed in the same environment but received only high-efficiency particle-filtered air. Whole lung tissues were collected from mice exposed for 3 weeks, 10 weeks or for 10 weeks followed by 6 weeks in the laboratory (16 weeks total). DNA microarrays were used to explore changes in pulmonary gene expression in mice breathing ambient air versus HEPA-filtered air. Transcriptional profiling revealed changes in the expression of genes implicated in the lipid droplet synthesis pathway (plin, dgat2, lpl, s3-12, agpat2), antioxidants (ucp1). We postulate that exposure to particulate matter adsorbed with polycyclic aromatic hydrocarbons triggers lipid droplet synthesis (holding depots for lipids and malformed/excess proteins tagged for degradation) in the lungs, which act to sequester particulates adsorbed with toxic chemicals. Increased lipid droplet synthesis could potentially lead to endogenous/stressor-induced synthesis of reactive oxygen species and activation of antioxidant mechanisms. Further investigation into the stimulation of lipid droplet synthesis in the lung in response to air pollution is warranted in order to better understand these mechanistic changes and the resulting health implications. Mature male C57BL/6 x CBA F1 hybrid mice were exposed to either HEPA-filtered or ambient air in Hamilton, ON, Canada. Animals were exposed starting May 14, 2004 for 3 weeks (3wk), 10 weeks (10wk), or for 10 weeks followed by 6 weeks of recovery (16wk). Each treatment group consisted of five mice, for a total of 30 mice. Total RNA was isolated from a random section of the whole lung using TRIzol reagent (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen). RNA quality was confirmed by UV spectrophotometry and using an Agilent Bioanalyzer. Total RNA (200 ng) from HEPA-filtered air or whole air-exposed mice and Universal Mouse Reference RNA (Stratagene) were used to synthesize double-stranded cDNA and cyanine-labelled cRNA according to the manufacturer's instructions (Agilent Linear Amplification Kits, Agilent Technologies). Biological samples were labelled with Cy5 dye while the commercially available Stratagene mouse reference RNA was labelled with Cy3 dye. At each of the three time points, ambient air-exposed samples and HEPA-filtered samples were hybridized to Agilent microarrays. Arrays were washed, and scanned on an Agilent G2505B scanner. Data were acquired using the Agilent Feature Extraction software version 9.5.3.1.

Project description:The Global Burden of Disease implicates air pollution in mediating as much as 20% of Type 2 Diabetes mellitus globally. This profound impact of air pollution is supported by experimental studies suggesting effects on whole body metabolism and insulin resistance. We explored the effect of chronic inhalational air pollution exposure for 12 weeks, using a whole body inhalation device in mice that allows for daily exposure to concentrated ambient particulate matter <2.5 microns (6 hours/day, 5 days a week) on whole body metabolism and brown adipose tissue (BAT) function, with detailed investigation of differentially methylated regions (DMRs), chromatin accessibility (differential accessible regions or DARs), and differential gene expression (DEGs) levels using a combination of whole genome bisulfite sequencing, ATAC-sequencing, and RNA-sequencing respectively. A significant impact of chronic PM2.5 exposure on BAT function and whole-body metabolism through alterations in key BAT transcriptional programs resulting in increased redox stress, lipid deposition, fibrosis and reduced thermogenic function of BAT was observed. Significant differential methylation with evidence for both hypomethylation and hypermethylation involving target regions corresponding to both circadian and Nrf2 target genes including sites enriched for transcription factor binding sites implicated in redox stress, insulin resistance and BAT function in response to PM2.5. ATAC sequencing revealed widespread chromatin remodeling involving intronic regions and enhancer elements and loss of DARs in Ucp1 and Gclc major regulatory genes associated with thermogenesis and glutathione synthesis. DARs-DEGs interaction analysis pointed to functional pathways linked to diabetes development, fatty acid biosynthesis activation and activation of JAK-STAT, and MAPK in PM-exposed mice. Integrated analysis of DARs, DEGs and DMRs uncovered the Histone de-acetylase Hdac9 and Kdm2b, a Histone demethylase that demethylates 'Lys-4' and 'Lys-36' of histone H3. ChiP studies demonstrated that Hdac9 physically interacts with the BAT transcription factor Prdm16 and rorα in BAT decreasing their expression and providing at least one direct mechanism for our observations. Collectively our results results link DNA methylation with control of chromatin accessibility and gene expression changes involved in the alteration of brown fat metabolism linking chronic air pollution exposure with alterations in BAT and whole-body metabolism

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