DE 1 STUDY: Diesel exhaust inhalation in asthmatics: Oxidative stress as a mediator of airway reactivity and immune response
The use of diesel engines is increasing because they are more fuel-efficient than gasoline engines, however, diesel engines produce different emissions than gasoline engines. Diesel exhaust is emitted from the tailpipe of both “on- road” diesel engine vehicles (diesel cars, buses and trucks) and “non-road” diesel engines (locomotives, marine vessels and some construction equipment). Diesel exhaust consists of both gaseous and particulate air pollutants. Since people with asthma and allergic diseases appear to be sensitive to air pollution, we wanted to know how diesel exhaust (DE) affected their respiratory and immune systems. Below we present our research findings in this study.
Short-term diesel exhaust inhalation in a controlled human crossover study is associated with changes in DNA methylation of circulating mononuclear cells in asthmatics
Background: Changes in DNA methylation have been associated with traffic-related air pollution in observational studies, but the specific mechanisms and temporal dynamics therein have not been explored in a controlled study of asthmatics. In this study, we investigate short-term effects of diesel exhaust inhalation on DNA methylation levels at CpG sites across the genome in circulating blood in asthmatics.
Methods: A double-blind crossover study of filtered air and diesel exhaust exposures was performed on sixteen non-smoking asthmatic subjects. Blood samples were collected pre-exposure, and then 6 and 30 hours post-exposure. Peripheral blood mononuclear cell DNA methylation was interrogated using the Illumina Infinium HumanMethylation450 Array. Exposure-related changes in DNA methylation were identified. In addition, CpG sites overlapping with Alu or LINE1 repetitive elements and candidate microRNA loci were also analyzed.
Results: DNA methylation at 2827 CpG sites were affected by exposure to diesel exhaust but not filtered air; these sites enriched for genes involved in protein kinase and NFkB pathways. CpG sites with significant changes in response to diesel exhaust exposure primarily became less methylated, with a site residing within GSTP1 being among the significant hits. Diesel exhaust-associated change was also found for CpG sites overlapping with Alu and LINE1 elements as well as for a site within miR-21.
Conclusion: Short-term exposure to diesel exhaust resulted in DNA methylation changes at CpG sites residing in genes involved in inflammation and oxidative stress response, repetitive elements, and microRNA. This provides plausibility for the role of DNA methylation in pathways by which airborne particulate matter impacts gene expression and offers support for including DNA methylation analysis in future efforts to understand the interactions between environmental exposures and biological systems.
Anti-Oxidant N-Acetylcysteine Diminishes Diesel Exhaust-Induced Increased Airway Responsiveness in Person with Airway Hyper-Reactivity
Background: Inhalation of diesel exhaust (DE) at moderate con- centrations causes increased airway responsiveness in asthmatics and increased airway resistance in both healthy and asthmatic sub- jects, but the effect of baseline airway responsiveness and anti- oxidant supplementation on this dynamic is unknown.
Objectives: We aimed to determine if changes in airway responsiveness due to DE are attenuated by thiol anti-oxidant supplementation, particu- larly in those with underlying airway hyper-responsiveness. Meth- ods: Participants took N-acetylcysteine (600 mg) or placebo cap- sules three times daily for 6 days. On the last of these 6 days, par- ticipants were exposed for 2 h to either filtered air (FA) or DE (300 g/m3 of particulate matter smaller than 2.5 microns). Twenty-six non-smokers were studied under each of three experimental con- ditions (filtered air with placebo, diesel exhaust with placebo, and diesel exhaust with N-acetylcysteine) using a randomized, double- blind, crossover design, with a 2-week washout between conditions. Methacholine challenge was performed pre-exposure (baseline air- way responsiveness) and post-exposure (effect of exposure). Re- sults: Anti-oxidant supplementation reduced baseline airway re- sponsiveness in hyper-responsive individuals by 20% (p = 0.001). In hyper-responsive individuals, airway responsiveness increased 42% following DE compared with FA (p = 0.03) and this increase was abrogated with anti-oxidant supplementation (diesel exhaust with N-acetylcysteine vs. filtered air with placebo, p = 0.85).
Conclusions: Anti-oxidant (N-acetylcysteine) supplementation protects against increased airway responsiveness associated with DE in- halation and reduces need for supplement bronchodilators in those with baseline airway hyper-responsiveness. Individuals with vari- ants in genes of oxidative stress metabolism when exposed to DE are protected from increases in airway responsiveness if taking anti-oxidant supplementation.
MicroRNA Expression in Response to Controlled Exposure to Diesel Exhaust: Attenuation by the Antioxidant N-Acetylcysteine in a Randomized Crossover Study
Background: Adverse health effects associated with diesel exhaust (DE) are thought to be mediated in part by oxidative stress, but the detailed mechanisms are largely unknown. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and may respond to exposures such as DE.
Objectives: We profiled peripheral blood cellular miRNAs in participants with mild asthma who were exposed to controlled DE with and without antioxidant supplementation.
Methods: Thirteen participants with asthma underwent controlled inhalation of filtered air and DE in a double-blinded, randomized crossover study of three conditions: a) DE plus placebo (DEP), b) filtered air plus placebo (FAP), or c) DE with N-acetylcysteine supplementation (DEN). Total cellular RNA was extracted from blood drawn before exposure and 6 hr after exposure for miRNA profiling by the NanoString nCounter assay. MiRNAs significantly associated with DEP exposure and a predicted target [nuclear factor (erythroid-derived 2)-like 2 (NRF2)] as well as anti- oxidant enzyme genes were assessed by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) for validation, and we also assessed the ability of N-acetylcysteine supplementation to block the effect of DE on these specific miRNAs. 8-hydroxy-2 ́-deoxyguanosine (8-OHdG) was measured in plasma as a systemic oxidative stress marker.
Results: Expression of miR-21, miR-30e, miR-215, and miR-144 was significantly associated with DEP. The change in miR-144 was validated by RT-qPCR. NRF2 and its downstream antioxidant genes [glutamate cysteine ligase catalytic subunit (GCLC) and NAD(P)H:quinone oxidoreductase 1 (NQO1)] were negatively associated with miR-144 levels. Increases in miR-144 and miR-21 were associated with plasma 8-hydroxydeoxyguanosine 8-OHdG level and were blunted by antioxidant (i.e, DEN).
Conclusions: Systemic miRNAs with plausible biological function are altered by acute moderate- dose DE exposure. Oxidative stress appears to mediate DE-associated changes in miR-144.
Air pollution collectively describes the presence of a complex mixture of particulate matter (PM), organic compounds (e.g. polycyclic aromatic hydrocarbons and endotoxins), gases (e.g. carbon monoxide, sulfur oxides, ground-level ozone, nitrogen oxides) and metals present in indoor and outdoor air, which can cause harm or discomfort to humans or other living organisms. Although the effects of prolonged exposure to traffic-related air pollution (TrAP), specifically, are well characterized with respect to respiratory and cardiovascular outcomes, comparatively little is known about the impact of particulate matter on affective and cognitive processes, neurodegenerative effects and microcirculation. Research using animal models, along with epidemiology, has greatly enhanced our understanding of DE-related cognitive and learning outcomes, and has implicated several important potential mechanisms. However, such models are inherently limited given interspecies differences (animal models), some degree of unavoidable residual confounding (epidemiology), and a bias towards subacute or chronic effects (epidemiology). By largely overcoming these limitations, our in vivo human model, using freshly-generated exhaust that is diluted and aged to reflect real-world conditions, allows us to augment existing knowledge in a manner applicable both to current research on environmental effects on cognition, as well as current public health concerns of great relevance to Canadians.
This study has finished subject enrolment and data analysis is now underway.
IDEA STUDY – THE EFFECTS OF AIR POLLUTION AND INHALED SALBUTAMOL ON LUNG FUNCTION AND ATHLETIC PERFORMANCE IN ASTHMATIC INDIVIDUALS (Collaboration project with Dr. Koehle’s lab)
The detrimental effects of air pollution on cardiovascular and respiratory health are described in a large body of scientific literature. Olympic Games in cities known for high levels of air pollution such as Beijing 2008 and Athens 2000 raised concerns regarding athlete’s health and athletic performance. Ironically, especially athletes, who tend to follow a health-conscious lifestyle, are at risk of cardiorespiratory symptoms and illnesses triggered by air pollution. Physical activity leads to the release of epinephrine. Epinephrine induces a widening of the airways to facilitate the increased minute ventilations required to sustain the physical demands placed by the training loads. The inhalation of increased volumes of polluted air through widened airways, ultimately leads to an impairment in healthy lung function and athletic performance. function and athletic performance.The acute treatment of choice for these asthma-like symptoms is the inhalation of beta-2-agonists (IBA), which mimic epinephrine. While IBAs induce a further widening of the airways and relieve respiratory distress in the short-term, IBA use may increase the chance of pollutants reaching deeper areas in the bronchial tree, where they can cause structural (airway remodeling) and functional (decreased abilities to generate air flow) damage in the long term. The purpose of this proposed study is to investigate short- and long-term effects of IBA-use in the treatment of respiratory symptoms triggered by air pollution exposure in elite athletes.