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Dried blood spots extraction and analysis

For blood extraction from dried blood spots(DBS), a 6mm (in diameter) punch was first cut from the center of each blood spot, with Harris Uni-Core punch. Each punch of the sample were ensured to be saturated with blood all the way to the diameter to avoid DBS appearing uneven, or not at least 6mm in diameter. Then each punch was placed in a well of 96-well plate and 200 µl of PBS (0.5% Tween 20) was added to each well for blood extraction. The plate was shaken overnight at 4°C using a rotary plate shaker at approximately 400 rpm. Afterwards, plate was stored at -80°C until further analysis.

Electrochemiluminescence (ECL) tests were conducted using the Mesoscale Discovery MESO QuickPlex SQ 120. Each sample was diluted 2-fold in the diluent supplied with the kit and specified in the company protocol (MSD 2014). For the assay, each well received 25µl sample mixed with 25µl diluent. DBS extractions were measured in singlets. The following analytes were characterized: IL-1β, IL-6, IL-8 and TNF-α. Experiments were performed on two continuous days in our environmental health laboratory by the same trained research assistant to avoid inconsistency.

Due to lack of detection associated with samples found in concentrations below analytical detection limits, 63 DBS samples had reported concentrations of zero pg/ml for IL-1β (46% of total). Since lower limits of detection (LLODs) vary slightly between plates even at the same site, the median LLODs reported for each kit in the Mesoscale company literature (MSD 2014) were used. For these observations with zero pg/ml in concentrations, the values were corrected and expressed as the median LLODs (Skogstrand et al. 2008; Hornung et al. 1990) (median LLOD for IL-1β: 0.04pg/ml; median LLOD for IL-6: 0.06 pg/ml).Levels of all other cytokines, were all found to be above their respective limits of detection.

  1. Hornung, Richard W, and Laurence D Reed. 1990. 'Estimation of average concentration in the presence of nondetectable values', Applied occupational and environmental hygiene, 5: 46-51.
  1. Skogstrand, K., C. K. Ekelund, P. Thorsen, I. Vogel, B. Jacobsson, B. Norgaard-Pedersen, and D. M. Hougaard. 2008. 'Effects of blood sample handling procedures on measurable inflammatory markers in plasma, serum and dried blood spot samples', J Immunol Methods, 336: 78-84.

Table S1.Spearman’s Correlation Matrix among measured ACE-2 pollutants (N = 120)
PM2.5 / PAH / Noise / WSOC / OC / EC / Na / Mg / Al / S / K / Ca / Fe / Ni / Zn / Cu
PM2.5 / 1.00 / 0.72* / 0.58* / 0.21 / 0.71* / 0.69* / 0.08 / 0.25* / 0.36* / 0.39* / 0.43* / 0.29* / 0.42* / -0.20 / 0.34* / 0.01
PAH / 0.72* / 1.00 / 0.61* / 0.02 / 0.60* / 0.73* / -0.15 / 0.26* / 0.33* / 0.37* / 0.31* / 0.27* / 0.51* / -0.08 / 0.27* / 0.01
Noise / 0.58* / 0.61* / 1.00 / -0.03 / 0.51* / 0.67* / -0.09 / 0.22* / 0.23* / 0.28* / 0.31* / 0.16 / 0.23* / -0.06 / 0.27* / 0.12
WSOC / 0.21 / 0.02 / -0.03 / 1.00 / 0.06 / 0.10 / 0.09 / -0.11 / 0.07 / -0.07 / 0.07 / -0.05 / -0.03 / 0.00 / 0.17 / 0.09
OC / 0.71* / 0.60* / 0.51* / 0.06 / 1.00 / 0.65* / -0.07 / 0.25* / 0.30* / 0.32* / 0.36* / 0.32* / 0.37* / 0.00 / 0.35* / 0.13
EC / 0.69* / 0.73* / 0.67* / 0.10 / 0.65* / 1.00 / -0.06 / 0.27 / 0.12 / 0.26 / 0.31* / 0.07 / 0.47* / -0.03 / 0.26 / 0.20
Na / 0.08 / -0.15 / -0.09 / 0.09 / -0.07 / -0.06 / 1.00 / 0.59* / 0.30* / 0.24* / 0.48* / 0.36* / 0.21 / -0.15 / 0.21 / 0.06
Mg / 0.25* / 0.26* / 0.22* / -0.11 / 0.25* / 0.27 / 0.59* / 1.00 / 0.78* / 0.69* / 0.78* / 0.79* / 0.70* / -0.20 / 0.51* / 0.39*
Al / 0.36* / 0.33* / 0.23* / 0.07 / 0.30* / 0.12 / 0.30* / 0.78* / 1.00 / 0.69* / 0.76* / 0.72* / 0.74* / -0.24 / 0.59* / 0.42*
S / 0.39* / 0.37* / 0.28* / -0.07 / 0.32* / 0.26 / 0.24* / 0.69* / 0.69* / 1.00 / 0.66* / 0.74* / 0.75* / -0.10 / 0.51* / 0.31*
K / 0.43* / 0.31* / 0.31* / 0.07 / 0.36* / 0.31* / 0.48* / 0.78* / 0.76* / 0.66* / 1.00 / 0.65* / 0.75* / -0.27* / 0.56* / 0.21*
Ca / 0.29* / 0.27* / 0.16 / -0.05 / 0.32* / 0.07 / 0.36* / 0.79* / 0.72* / 0.74* / 0.65* / 1.00 / 0.71* / -0.10 / 0.58* / 0.26*
Fe / 0.42* / 0.51* / 0.23* / -0.03 / 0.37* / 0.47* / 0.21 / 0.70* / 0.74* / 0.75* / 0.75* / 0.71* / 1.00 / -0.17 / 0.56* / 0.26*
Ni / -0.20 / -0.08 / -0.06 / 0.00 / 0.00 / -0.03 / -0.15 / -0.20 / -0.24 / -0.10 / -0.27* / -0.10 / -0.17 / 1.00 / -0.08 / -0.07
Zn / 0.34* / 0.27* / 0.27* / 0.17 / 0.35* / 0.26 / 0.21 / 0.51* / 0.59* / 0.51* / 0.56* / 0.58* / 0.56* / -0.08 / 1.00 / 0.45*
Cu / 0.01 / 0.01 / 0.12 / 0.09 / 0.13 / 0.20 / 0.06 / 0.39* / 0.42* / 0.31* / 0.21* / 0.26* / 0.26* / -0.07 / 0.45* / 1.00

* Correlation significant at = 0.05 (2-tailed).

Table S2. Estimated changes in inflammatory biomarkers relative to baseline
Highway (n=60) / Surface street (n=30) / Clinic (n=30)
0 h post / 3 h post / 8 h post / 0 h post / 3 h post / 8 h post / 0 h post / 3 h post / 8 h post
Hs-CRP
β / 0.09 / 0.04 / -0.05 / -0.1 / -0.1 / -0.03 / -0.05 / -0.1 / -0.1
95% CI / -0.1, 0.3 / -0.2, 0.3 / -0.3, 0.2 / -0.3, 0.1 / -0.4, 0.1 / -0.03, 0.2 / -0.4, 0.3 / -0.5, 0.2 / -0.5, 0.2
% change / 9.3 / 4.5 / -5.1 / -10 / -11.3 / -2.7 / -5 / -12.8 / -12.5
TNF-a
β / -0.1 / -0.08 / -0.08 / 0.07 / -0.06 / 0.15 / 0.05 / -0.001 / 0.06
95% CI / -0.2, 0.04 / -0.2, 0.05 / -0.2, 0.05 / -0.1, 0.3 / -0.3, 0.1 / -0.06, 0.4 / -0.1, 0.2 / -0.2, 0.2 / -0.09, 0.2
% change / -9.4 / -7.8 / -7.7 / 7.6 / -6.1 / 16.5 / 4.7 / -0.1 / 6.1
IL-6
β / 0.3 / -0.005 / 0.3 / 0.1 / 0.08 / 0.5 / -0.1 / -0.06 / 0.3
95% CI / -0.06, 0.6 / -0.4, 0.3 / -0.09, 0.6 / -0.5, 0.7 / -0.5, 0.7 / -0.1, 1.1 / -0.6, 0.3 / -0.5, 0.4 / -0.1, 0.7
% change / 34.3 / -0.5 / 29.9 / 12.3 / 8.5 / 61.6 / -12.2 / -6.1 / 36
IL-8
β / 0.01 / -0.06 / -0.1 / -0.03 / 0.08 / 0.2 / 0.1 / 0.05 / -0.009
95% CI / -0.1, 0.1 / -0.2, 0.07 / -0.2, 0.02 / -0.3, 0.2 / -0.1, 0.3 / -0.04, 0.4 / -0.1, 0.3 / -0.1, 0.3 / -0.2, 0.2
% change / 1.2 / -6.5 / -11.2 / -3.2 / 9 / 23.2 / 10 / 5.4 / -0.9
IL-1β
β / -0.02 / -0.02 / -0.07 / 0.07 / -0.1 / 0.1 / 0.04 / 0.1 / -0.07
95% CI / -0.2, 0.1 / -0.2, 0.1 / -0.2, 0.09 / -0.2, 0.4 / -0.4, 0.2 / -0.2, 0.5 / -0.2, 0.3 / -0.1, 0.3 / -0.3, 0.2
% change / -1.8 / -2.7 / -6.7 / 7.8 / -11.1 / -14.1 / 4.2 / 10.7 / -6.3
sICAM-1
β / 0.01 / 0.01 / -0.007 / 0.03 / -0.1 / 0.0001 / 0.1 / -0.07 / 0.04
95% CI / -0.2, 0.2 / -0.2, 0.2 / -0.2, 0,2 / -0.4. 0.4 / -0.5, 0.3 / -0.4, 0.4 / -0.1, 0.3 / -0.3, 0.2 / -0.2, 0.3
% change / 1.2 / 0.9 / -0.7 / 3 / -12.2 / 0.1 / 10.7 / -6.3 / 4.2
sVCAM-1
Β / 0.05 / 0.07 / 0.06 / -0.2 / -0.1 / -0.09 / 0.07 / 0.1 / -0.02
95% CI / -0.1, 0.2 / -0.09, 0.2 / -0.1, 0.2 / -0.4, 0.03 / -0.4, 0.1 / -0.3, 0.1 / -0.1, 0.2 / -0.07, 0.3 / -0.2, 0.2
% change / 4.7 / 7.3 / 6.3 / -19.1 / -13.1 / -9.1 / 7.9 / 11.1 / -1.6

Abbreviations: high sensitive C-reactive-protein (hs-CRP), interleukin -1β (IL- 1β), interleukin-6 (IL-6), Interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), soluble intracellular adhesive molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1)

Table S3. Estimated changes in exhaled nitric oxide (eNO) and lung function (FEV1, FVC) relative to baseline
eNO / FEV1 / FVC
β / 95% CI / p value / % change / β / 95% CI / p value / % change / β / 95% CI / p value / % change
Highway (n=60)
0-hour post commute / 0.06 / 0.11,0.01 / 0.01 / 6.4 / -0.02 / -0.03,-0.001 / 0.03 / -1.9 / -0.023 / -0.04,-0.008 / 0.002 / -2.2
1- hour post commute / 0.10 / 0.15, 0.06 / <.0001 / 10.9 / -0.007 / -0.02,0.01 / 0.4 / -0.7 / -0.017 / -0.03,-0.002 / 0.02 / -1.7
2- hour post commute / 0.09 / 0.14, 0.05 / 0.0002 / 9.7 / -0.004 / -0.02,0.01 / 0.6 / -0.4 / -0.022 / -0.04,-0.006 / 0.004 / -2.2
3- hour post commute / 0.09 / 0.14, 0.04 / 0.0002 / 9.6 / -0.007 / -0.02,0.01 / 0.4 / -0.8 / -0.022 / -0.04,-0.006 / 0.005 / -2.1
8- hour post commute / -0.003 / 0.04, -0.05 / 0.89 / -0.3 / 0.001 / -0.01,0.02 / 0.8 / 0.1 / -0.018 / -0.03,-0.003 / 0.01 / -1.8
Surface street (n=30)
0-hour post commute / 0.07 / 0.13, 0.003 / 0.04 / 7.2 / -0.009 / -0.03,0.01 / 0.4 / -1.0 / -0.02 / -0.04,0.03 / 0.10 / -1.8
1- hour post commute / 0.11 / 0.18, 0.04 / 0.002 / 12.2 / 0.00001 / -0.03,0.02 / 0.9 / 0 / -0.02 / -0.04,0.002 / 0.08 / -2.1
2- hour post commute / 0.09 / 0.16, 0.02 / 0.01 / 9.8 / -0.005 / -0.03,0.2 / 0.7 / -0.5 / -0.01 / -0.03,0.01 / 0.2 / -1.4
3- hour post commute / 0.13 / 0.20, 0.05 / 0.0007 / 13.9 / -0.003 / -0.03,0.02 / 0.8 / -0.3 / -0.02 / -0.04,0.02 / 0.06 / -2.3
8- hour post commute / 0.02 / 0.09, -0.05 / 0.6 / 2.2 / -0.007 / -0.03,0.02 / 0.6 / -0.7 / -0.03 / -0.5,-0.004 / 0.02 / -2.9
Clinic (n=30)
0-hour post commute / 0.15 / 0.22, 0.08 / <.0001 / 16.3 / 0.02 / -0.01,0.04 / 0.2 / 1.8 / -0.01 / -0.03,0.004 / 0.1 / -1.5
1- hour post commute / 0.12 / 0.20, 0.05 / 0.001 / 13.4 / 0.002 / -0.03,0.03 / 0.8 / 0.2 / -0.01 / -0.03,0.004 / 0.1 / -1.7
2- hour post commute / 0.10 / 0.18, 0.03 / 0.009 / 11.1 / 0.02 / -0.008,0.05 / 0.1 / 2.3 / -0.01 / -0.03,0.0 / 0.3 / -1.2
3- hour post commute / 0.13 / 0.21, 0.06 / 0.0009 / 14.6 / 0.0002 / -0.03,0.03 / 0.9 / 0 / -0.03 / -0.05,-0.007 / 0.01 / -3.0
8- hour post commute / 0.08 / 0.15, 0 / 0.05 / 8.2 / 0.002 / -0.02,0.03 / 0.8 / 0.2 / -0.02 / -0.04,0.001 / 0.06 / -2.2

Abbreviations: eNO = exhaled nitric oxide; FVC = forced expiratory vital capacity; FEV1 = forced expiratory volume in 1 second

FIGURE LEGENDS

Figure S1. (A) Average percent change from baseline in FVC and (B) Average percent change from baseline in FEV1, by exposure scenario.

Error bars indicate upper and lower confidence intervales. Abbreviations: FVC = forced expiratory vital capacity; FEV1 = forced expiratory volume in 1 second

Figure S2. Average percent change from baseline in exhaled NO levels,by exposure scenario.

Error bars indicate upper and lower confidence interval. Abbreviations: NO = nitric oxide

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