Participants and study design

Healthy adult (ages 19–39) participants were recruited using advertisements posted to local cycling and university bulletin boards and along cycling routes in the city of Vancouver, Canada. Participants were eligible if they were non-smokers, not diagnosed with or taking a medication for any respiratory or cardiovascular condition, not exposed to environmental tobacco smoke in the home or otherwise exposed to significant respiratory exposures in the workplace. Participants with seasonal allergies were asked to participate at a time of the year when they were asymptomatic. Females were tested during days 1–8 of their follicular phase, while those using monocyclic oral contraceptives were tested on days where they took an active pill to ensure consistent hormonal status. Written informed consent was obtained for each participant after a session given to familiarize each person with the equipment and protocols, prior to commencing the first cycling trial. The Health Canada (certificate #2011–0009) and University of British Columbia Clinical Research (#H10–00902) Ethics Boards approved this study. The study was registered with ClinicalTrials.gov (NCT01708356).

All cycling trials began and ended at the Vancouver General Hospital in central Vancouver, British Columbia during May– November 2010 and May– November 2011. Metro Vancouver has an annual mean particle number concentration of 18,200 particles/cm3 (pt/cm³) (standard deviation = 15,900 pt/cm3) (from 2009 and 2010 data) [30], an annual mean PM 2.5 concentration of 4.3 μg/m3 at the T2:Vancouver- Kitsilano station, and an annual mean PM 10 concentration of 10.5 μg/m3 at the nearest monitoring location (T24: Burnaby North ) [31]. Trials were conducted between 700 and 1600 h, and each participant completed both trials at the same time of day (+/− 1 h), scheduled 2 to 6 weeks apart. Two routes were selected using a recent study that obtained particulate matter exposure measurements from designated bicycle routes in Vancouver [13], with consideration of nearby land-use categories in order to select one predominantly residential use (“Residential”) route and one predominantly commercial or higher density residential (“Downtown”) route. The mid-point of the Downtown ride (Dunsmuir Street at Richards Street, 2011 through-traffic counts) had traffic counts between 7.8 to 9.9 times that of the traffic on a typical section (Ontario Street at 36th Street, based on 2006 traffic counts) along the Residential Route. Route order was assigned randomly. The Downtown route was a 9.7 km loop, and was always traveled in a counter-clockwise direction, with a section of protected bike lane that could be repeated if the cyclist was a faster rider. Most participants repeated the protected bike lane section, resulting in a total elevation gain of 127 m over three major uphill segments (with the most significant segments being 29 m, 26 m, and 26 m in elevation gain) [32]. The Residential route was a 12.0 km loop that was traveled in a clockwise direction for some trials (120 m of elevation gain, from 2 major uphill segments of 49 m and 26 m), or as an out-and-back ride in a counter-clockwise direction, to the far south-east corner of the route before reversing direction (172 m of elevation gain, over 3 major uphill segments of 41 m, 36 m, and 18 m) [32]. A trailing research assistant (also on bike) provided wayfinding and timing directions with the aim of facilitating a 60-min ride, rather than completing a specific distance. Questionnaires prior to the beginning of each trial were used to screen for allergy or cold symptoms, to confirm that each participant had limited alcohol and caffeine consumption, and to confirm that each participant consumed the same meals prior to the trials. Participants were also asked to travel to the study site using the same method of transportation on both trial days.

Exercise and exposure monitoring

Two bicycles of the same model (KHS, Flite 250, Los Angeles, USA), one each of small and large frame size, were used in all trials. A PowerTap Comp (PowerTap, Madison, WI, USA) wiring set which automatically recorded at 3-s intervals during the ride, was installed on the handlebars of each bicycle to measure power output, cadence, and heart rate from a chest-worn heart rate strap. A single PowerTap hub was installed on a wheel that could be transferred between the two bicycles. A P-trak (Ultrafine Particle Counter 8525, TSI Inc., Shoreview, MN, USA), with the tilt sensor removed was mounted into a wire pannier (Swagman Fat Folding Basket, Swagman Racks, Penticton, BC, CAN) by placing a horizontal bar through the handle of the monitor and stabilized using elastic cords and metal bearings, thus allowing the P-trak to remain horizontal despite changing inclines. The P-trak recorded particle number (0.02–1 μm) concentration (PNC) at one-second intervals. A GRIMM Dust Monitor (Model 1.108, GRIMM Technologies, Inc. Douglasville, Georgia, USA) placed into a separate rear pannier measured PM 10 , PM 2.5, and PM 1 concentrations at 6-s intervals. Sampling inlets for both monitors were secured along the top tube of the bicycle, extending to the centre of the handlebars. A GPS logger (DG-100 GPS DataLogger, GlobalSat WorldCom Corporation, New Taipei City, Taiwan) recorded the location of the bicycle at 6-s intervals. To align measurements from all instruments, data collected at intervals longer than 1 s was applied to the closest one second time point until a new data point was available, carried for a maximum of 6 s.

Physiological measurements

All measurements were completed within one hour prior to the beginning of each bicycle ride, and were repeated approximately 15 min after the cyclists’ return and completed within 90 min of ride termination. Tests were administered in uniform order (endothelial function, followed by spirometry, followed by bloodwork) before and after each ride, with exceptions noted and replicated for a given subject when possible. For a given individual, tests for the second trial were performed within one hour of the same time of day as they were performed in the first trial. Endothelial function was measured using the EndoPAT 2000 device (Itamar Medical Ltd., Caesarea, Israel) to measure RHI, following the five-minute occlusion procedure recommended in the user manual. Lung function was measured with a KoKo spirometer (nSpire Health, Longmont, Colorado, USA), following American Thoracic Society standards [33]. Five milliliters of blood were collected, centrifuged, and frozen prior to analysis for CRP (Dimension Vista® System Flex® reagent cartridge for high sensitivity CRP, Siemens Healthcare Diagnostics Products GmbH 2009), IL-6 (Quantikine® ELISA Human IL-6 Immunoassay D6050, R&D Systems, Inc. Minneapolis, MN, USA) and 8-OHdG (Highly Sensitive 8-OHdG Check ELISA method, Japan Institute for the Control of Aging, Nikken Seil Co. Ltd., Fukuroi, Shizuoka, Japan). Increased RHI indicates improved endothelial function and increases in spirometric measures indicate improved lung function, whereas increases in any of the blood measures indicate increased oxidative stress and/or inflammation.

The first 15 participants completed an abbreviated indoor cycling test after the one-hour ride. Minute ventilation was recorded while cycling indoors at the mean heart rate recorded during the ride along each outdoor route. All indoor cycling tests were completed on an adjustable Velotron Dynafit Pro cycle ergometer (Racermate Inc., Seattle WA). The remaining 23 participants completed a step-wise submaximal exercise test, in increments of 20 watts every two minutes for females and 30 watts every two minutes for males. Heart rate using a Polar HR sensor strap (Polar s810i, Polar Electro, Finland), and minute ventilation (\( {\dot{\mathrm{V}}}_{\mathrm{E}} \)) using a respirometer (Spirolab II, Medical International Research, Rome, Italy) were recorded during the second minute at every resistance level throughout the heart rate range experienced by each participant. Heart rate data from each participant’s submaximal test results were used to estimate a subject-specific HR-\( {\dot{\mathrm{V}}}_{\mathrm{E}} \) relationship, which was then used to estimate instantaneous and mean \( {\dot{\mathrm{V}}}_{\mathrm{E}} \) for each outdoor trial for each participant; \( {\dot{\mathrm{V}}}_{\mathrm{E}} \) was estimated at each data point during outdoor cycling trials to calculate the total volume of inhaled air, and therefore PM intake values.

Statistical analysis

Only participants who completed trials on both routes were included in the data analysis. Out of 76 exposure trials, 6 trials had missing exposure data. Health measures were analyzed in the group results only when complete pairs of pre and post measurements were available; incomplete pairs were excluded. Data were analyzed by paired t-tests comparing the two trials for each participant, and in mixed effects regression models. Fixed variables (e.g. bivariate variables comparing the Residential and Downtown routes, or continuous variables such as air pollution exposure or intake values), and random variables (participants) were modeled in R [34] using the lme4 package [35] to predict changes in clinical measures. Both exposure and pollutant intake were considered in analyses.

Intake was estimated for each participant by summing the product of each 1-s pollutant concentration (in pt/cm3 or μg/m3) by the volume of air inhaled each second derived from the heart rate data (the mean \( {\dot{\mathrm{V}}}_{\mathrm{E}} \) of the entire trial, converted to L/s). As high correlations were measured between the PM 10 , PM 2.5 , and PM 1 measurements, (Pearson product-moment correlation coefficients of 0.96 to 0.99) only results for the models that include PM 2.5 and PNC concentrations are presented. Pollutant concentration distributions within each ride were right-skewed and summarized by the geometric mean (GM). Four mixed-effects models (with participant included as a random effect) were built in order to evaluate the effect of route differences (model 1), the effects of pollutant exposure (model 2) or intake (model 3) and the effect of pollutant intake while including route in the same model (model 4):

1) Change in clinical measurement = ß Route + participant. 2) Change in clinical measurement = ß GM exposure + participant. 3) Change in clinical measurement = ß Pollutant intake + participant. 4) Change in clinical measurement = ß Route + ß Pollutant intake + participant

Effect modification was explored by modeling body mass index (BMI: lower and upper half, stratified at the median), age (younger half and older half, stratified at the median), and sex (female or male) variables with the Route variable scaled to the Residential route.