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Bellavia et al.,
DNA Hypomethylation, Ambient Particulate Matter and Increased Blood Pressure: Findings
from Controlled Human Exposure Experiments
1
Controlled human exposure facility at the Gage Occupational & Environmental Health
Unit, St. Michael’s Hospital, Toronto, Ontario, Canada
We utilized a controlled-human exposure facility that concentrates fine or coarse particles under
controlled conditions, using a high-flow (5,000 L/min) Harvard ambient particle concentrators.
The facility was constructed through an infrastructure grant from the Canadian Foundation for
Innovation to the Southern Ontario Centre for Atmospheric Aerosol Research, University of
Toronto, in collaboration with the Harvard School of Public Health. Fine CAP exposures were
produced with a 3-stage virtual impactor system that concentrated particles with an aerodynamic
diameter between 2.5 and 0.1 µm1. Coarse CAP exposures were produced with a 2-stage virtual
impactor system that concentrated particles between 10.0 and 2.5 µm2. The medical-air control
exposure was carried out using as the inlet air source a gas cylinder of medical-grade air
(Praxair, MSDS no. P-4560-I) with a HEPA filter was inserted in-line downstream of the coarse
concentrator to remove particles prior to delivery to the participant. The medical air was first
humidified to 40% by passing over heated, ozonized, de-mineralized water (Canadian Springs,
Mississauga, ON), then passed through the HEPA filter before delivery to the volunteer.
Protocol for Blood Pressure (BP) measures
For the pre-exposure measure, participants sat inside the exposure chamber with the door open
(mask off) and when resting quietly were asked to place their left forearm on their left leg and
the BP measure was started. Once the exposure was completed, the volunteer removed the mask,
the concentrator pumps were turned off and the door opened, and after a few minutes the BP
measures obtained (post exposure readings). BP was measured using a mercury
sphygmomanometer on the right arm using appropriate cuff sizes. All readings were made to the
2
nearest even digit by rounding up if necessary. Three readings were taken and BP was
calculated from the average of the second and third readings. After each reading, the research
assistant waited at least one minute before proceeding to the next reading. If the participant
moved or an error reading was obtained, the measure was repeated.
DNA methylation analysis by bisulfite pyrosequencing
We developed assays for TLR4, IL6, IL12 and iNOS methylation by locating their promoters
using the Genomatix Software (Genomatix Software Inc, Ann Arbor, MI) and amplified the
sequences shown in Table S1. 500ng DNA (concentration 25 ng/µl) was treated using the EZ
DNA Methylation-Gold™ Kit (Zymo Research, Orange, CA, USA) according to the
manufacturer’s protocol. Final elution was performed with 200µl M-Elution Buffer. A 50 µL
PCR was carried out in 25 µL GoTaq Green Master mix (Promega, Madison, WI, USA), 25 ng
bisulfite-treated genomic DNA. Primer concentrations and PCR cycling conditions for each
assay are also shown in Table S1. PCR products were purified and sequenced by pyrosequencing
as previously described using 0.3 µΜ sequencing primer. Primers for PCR are shown in Table
S2.
REFERENCES
1.
2.
Sioutas C, Koutrakis P, Godleski JJ, Ferguson ST, Kim CS, Burton RM. Fine particle
concentrators for inhalation exposures—effect of particle size and composition. Journal
of Aerosol Science. 1997;28:1057-1071
Demokritou P, Gupta T, Ferguson S, Koutrakis P. Development and laboratory
performance evaluation of a personal cascade impactor. J Air Waste Manag Assoc.
2002;52:1230-1237
3
Figure S1
An experiment at the human exposure facility in Toronto
4
Figure S2. Timeline of the exposure protocol.
Each participant underwent three exposures in randomized order to: a. HEPA-filtered medical air (control); b. fine CAPs; c. coarse
CAPs. Exposures were 130-min long and were preceded and followed by pre- and post-exposure blood sampling, respectively, and
standardized measurements of seated blood pressure.
~ 9 am
Pre-exposure
blood samples
~ 10 am
~ 12 pm
Randomized Exposure:
A
– Medical Air (Control)
Pre-exposure
B – fine CAPs
blood pressure
C – coarse CAPs
( ≥2-week washout between exposures)
5
~ 1 pm
Post-exposure
blood pressure
Post-exposure
blood samples
Table S1. Localization of gene promoters, regions amplified, PCR primers concentrations, and cycling conditions
Promoter
Marker Chromosome
Start
End
Amplicon
Start
End
CpGs
Primer
Forward
(10M)
Primer
Reverse
(10M)
Cycling Conditions
95°C for 90”,
43°C for 60”,
-*
-*
-*
-*
-*
-*
Alu
2l
2l
72°C for 120”
for 45 cycles
95°C for 30”,
50°C for 30”,
-*
-*
-*
-*
-*
-*
LINE-1
2l
2l
72°C for 30”
for 45 cycles
Pos1: 120466701
95°C for 60”,
Pos2: 120466694
54°C for 60”,
9
120465960 120466722 120466643 120466751
TLR4
1l
1l
Pos3: 120466685
72°C for 60”
Pos4: 120466677
for 50 cycles
95°C for 60”,
Pos1: 22733847
57°C for 60”,
7
22732791 22733685 22733758 22733893
IL6
1l
1l
Pos2: 22733841
72°C for 60”
for 50 cycles
95°C for 60”,
Pos1: 158750774
60°C for 60”,
5
158750239 158750844 158750649 158750809
IL12
1l
1l
Pos2: 158750754
72°C for 60”
for 50 cycles
95°C for 45”,
60°C for 45”,
72°C 45”
then 4 cycles
decreasing the
Pos1: 26125877
17
26125734 26126409 26125846 26126057
annealing temp by
iNOS
1l
1l
Pos2: 26125884
2°C every cycle;
95°C for 45”,
50°C for 45”,
72°C for 45”
for 35 cycles
*Alu and LINE-1 repetitive elements are sequences with multiple locations across the genome and cannot be assigned to one individual locus.
6
Table S2. Primers for DNA methylation analysis
Marker
Sequence analyzeda
Forward Primer
Reverse Primer
Sequencing Primer
(5’ to 3’)
(5’ to 3’)
(5’ to 3’)
Alu
Biotin TTTTTATTAAAAATAT
AAAAATT
CCCAAACTAAAATAC AATAACTAAAATTA
AATAA
CAAAC
LINE-1
TTTTGAGTTAGGTGTG
GGATATA
Biotin AAAATCAAAAAATT
CCCTTTC
AGTTAGGTGTGGGA
TATAGT
TLR4
BiotinAGGAAGAGAAGATAT
TAGTGTTTT
CCTAACATCATCCTC
ACTACTTCT
ACAACAATAACCCT
ATAAAT
ACA/GTAACTCA/GCTATCACCA/
GTCTAACCA/GAAC
IL6
BiotinTATTTTAGTTTTGAGA
AAGGAGGTG
CAATACTCTAAAACC
CAACAAAAAC
TCCTAATACAAACA
ACCCC
CG/AACCACACG/ACAAAAAC
IL12
BiotinTTGTGTTTAGGTAATG
GGGATG
CACAAATAAAAACC
ACCATTTCTC
CCATTTCTCCAAAA
ACA
TCCA/GAATACCAATCCAATTCT
ACA/GACAT
iNOS
TTAGGGTTAGGTAAA
GGTATTTTTGTTT
BiotinCAATTCTATAAAACC
ACCTAATAATCTTAA
TAAAGGTATTTTTG
TTTTAA
C/TGATTTTC/TGGGTTTTTTTTTA
TTTTG
a
Nucleotides at which DNA methylation was measured are underlined.
7
G/AC/TG/AC/TG/ACCACCA
TTC/TGTGGTGC/TGTC/TGTTTTTT
AAGTC/TGGTTTG
Table S3. Mean blood DNA methylation pre- and post-exposure to Fine Concentrated Air Particles (CAP), Coarse CAPs, and Medical Air
Control (Medical Air)
Alu
Pre-exposure
Post-exposure
Mean
Mean
SD
Fine CAP
p*
SD
Pre-exposure
Post-exposure
Mean
Mean
SD
p*
p **
Coarse
CAPs
vs Control
p **
Fine CAPs
vs Control
Coarse CAP
p*
SD
Pre-exposure
Post-exposure
Mean
Mean
SD
SD
24.16 0.11
23.96 0.11
0.26
23.62 0.10
23.22 0.11
0.05
23.66 0.09
23.68 0.12
0.86
0.0006
0.14
LINE-1 84.41 0.18
84.24 0.19
0.40
84.21 0.17
84.25 0.16
0.84
84.38 0.15
84.09 0.16
0.08
0.94
0.44
TLR4
3.51 0.21
3.62 0.20
0.28
3.62 0.20
3.41 0.22
0.02
3.51 0.20
3.35 0.20
0.05
0.02
0.01
IL-6
44.69 2.05
44.68 2.06
0.75
44.71 2.05
44.73 2.06
0.64
44.74 2.07
44.66 2.07
0.03
0.34
0.57
IL-12
94.11 0.14
94.25 0.14
0.47
94.01 0.15
94.08 0.14
0.70
94.07 0.11
94.27 0.13
0.20
0.42
0.91
iNOS
62.88 0.72
62.88 0.71
0.98
62.67 0.69
62.58 0.68
0.64
62.50 0.70
62.56 0.68
0.75
0.19
0.11
The table reports the mean values of blood DNA methylation pre- and post-exposure to CAPs and to the control group. The table also shows the pvalues associated with the within group differences post-exposure vs baseline (*) and the p-values associated with the post-exposure differences
between exposure to CAPs and the control group (**).
8
Table S4: Mean values of blood pressure pre- and post-exposure to fine Concentrated Air Particles (CAP), coarse CAPs, and medical air
Control (Medical Air)
Fine CAP
p*
Coarse CAP
Pre-exposure
Post-exposure
Mean
SD
Mean
Systolic
107.5
2.6
107.75 2.6
0.74
107.31 2.6
110.28 2.6
Diastolic
63.8
1.8
65.63
0.006
64.74
66.61
SD
1.8
Pre-exposure
Post-exposure
Mean
Mean
SD
1.8
p*
SD
1.8
Pre-exposure
Mean
Post-exposure
p*
Fine Coarse
CAP CAP
vs
vs
Control Control
p **
p **
SD
Mean SD
0.002
108.30 2.6
109.86 2.6
0.0008
0.001
0.03
0.005
67.17
67.99 1.8
0.0007
0.12
0.11
1.8
The table reports the mean values of systolic and diastolic blood pressure pre- and post-exposure to CAPs and to the control group. The table also shows the pvalues associated with the within group differences post-exposure vs baseline (*) and the p-values associated with the post-exposure differences between exposure to
CAPs and the control group (**).
9
Table S5. Effect of controlled exposures to fine concentrated ambient particles (CAPs) and coarse CAPs on blood DNA methylation.*
Mean within-participant differences of DNA methylation in post-exposure samples
after fine or coarse CAPs vs. medical air (control) exposures
Fine CAPs
Coarse CAPs
Stdz β**
(95% CI)
p-value Adj. p-value***
Stdz β*
(95% CI)
p-value Adj. p-value
Alu (%5mC)
(-1.18, -0.32)
(-0.66, 0.10)
-0.74
0.0006
0.03
-0.28
0.14
0.27
LINE-1 (%5mC)
(-0.43, 0.46)
(-0.53, 0.23)
0.01
0.94
0.93
-0.15
0.44
0.56
TLR4 (%5mC)
(-0.38, -0.03)
(-0.46, -0.08)
-0.21
0.02
0.08
-0.27
0.01
0.04
IL-6 (%5mC)
(-0.05, 0.15)
(-0.11, 0.06)
0.05
0.34
0.48
-0.02
0.57
0.65
IL-12 (%5mC)
(-0.58, 0.24)
(-0.34, 0.38)
-0.17
0.42
0.55
0.02
0.91
0.92
iNOS (%5mC)
(-0.74, 0.14)
(-0.72, 0.07)
-0.30
0.19
0.23
-0.32
0.11
0.33
*All mixed models were adjusted for period effect
**Standardized βs and 95% confidence intervals (CIs) expressing the difference in DNA methylation between the exposures and the
control exposures as a fraction of the standard deviation (SD) of DNA methylation. βs indicate the differences of exposure vs. the
control (HEPA-filtered medical air) in the post-exposure blood samples.
***Adjusted p-values for multiple comparison are obtained with a permutation test (see statistical methods)
10
Table S6. Sensitivity analysis. Effect of controlled exposures to fine concentrated ambient particles (CAPs) and coarse CAPs on blood
DNA methylation adjusted for carryover effects.*
Mean within-participant differences of DNA methylation in post-exposure samples
after fine or coarse CAPs vs. medical air (control) exposures
Fine CAPs
Coarse CAPs
95% CI
p-value
95% CI
p-value
Stdz β**
Stdz β**
Alu (%5mC)
(-0.96, -0.15)
(-0.59, 0.17)
-0.55
0.01
-0.21
0.28
LINE-1 (%5mC)
(-0.46,
0.38)
(-0.50,
0.29)
-0.04
0.86
-0.11
0.60
TLR4 (%5mC)
(-0.42,
-0.03)
(-0.49,
-0.12)
-0.22
0.03
-0.31
0.00
IL-6 (%5mC)
(-0.07,
0.12)
(-0.10,
0.07)
0.03
0.57
-0.02
0.68
IL-12 (%5mC)
(-0.52, 0.25)
(-0.33, 0.40)
-0.13
0.50
0.04
0.84
iNOS (%5mC)
(-0.76, 0.13)
(-0.81, 0.00)
-0.32
0.16
-0.41
0.05
*Carryover effect was controlled adjusting for a variable including information on the previous experiment (CAP or medical air) and
time from the last experiment. βs are adjusted for period effect.
**Standardized βs and 95% confidence intervals (CIs) expressing the difference in DNA methylation between the exposures and the
control exposures as a fraction of the standard deviation (SD) of DNA methylation. βs indicate the differences of exposure vs. the
control (HEPA-filtered medical air) in the post-exposure blood samples.
11
Table S7. Sensitivity analysis. Effect on Systolic and Diastolic Blood Pressure of Controlled Exposures to Fine Concentrated Ambient
Particles (CAP) and Coarse CAP adjusted for carryover effects.*
Mean within-participant differences of systolic and diastolic blood pressure
in post-exposure measurements after fine or coarse CAPs vs. medical air (control)
Fine CAP
Coarse CAP
β**
95% CI
p-value
β**
95% CI
p-value
Systolic BP (mm Hg)
2.27
(0.75 , 3.79)
0.002
1.21
(-0.20 , 2.62)
0.09
Diastolic BP (mm Hg)
1.31
(-0.21 , 2.83)
0.10
0.53
(-0.44 ,1.50)
0.28
* Carryover effect was controlled adjusting for a variable including information on the previous experiment (CAP or medical air) and
time from the last experiment. βs are adjusted for period effect.
** βs and 95% Confidence Intervals (CIs) expressing the difference in Blood Pressure between the CAPs exposures and the control
experiments. βs indicate the difference of exposure groups vs the control group (Medical Air) in the post-exposures measurements.
12
Table S8. Post-exposure proportions of white blood cells composition
Within-participant proportions and differences between CAPs and medical air in post-exposure blood samples
Medical Air (Control)
Fine CAP
Coarse CAP
%
SE
%
% Difference* p-value
%
% Difference
p-value
Neutrophils
59.09
0.004
59.20
0.11
0.54
59.38
0.29
0.36
Eosinophils
1.99
0.001
1.82
-0.16
0.51
1.92
-0.07
0.82
Basophils
0.47
0.001
0.47
<0.01
0.75
0.48
0.01
0.68
Lymphocytes
31.77
0.005
31.47
-0.30
0.30
31.64
-0.13
0.56
Monocytes
6.60
0.001
6.94
0.34
0.16
6.48
-0.12
0.48
*Post-exposure percentage variations for each leukocytes population, comparing post-exposure blood samples (fine CAP or coarse
CAP exposure vs. medical air exposure)
13
Table S9. Post-exposure changes in white blood cells composition, compared to pre-exposure.
Mean within-participant variations in post vs. pre exposure blood samples
Medical Air (Control)
Fine CAP
Coarse CAP
% Difference
SE
p-value
% Difference
SE
p-value
% Difference
SE
p-value
Neutrophils
-0.43
0.004
0.27
0.19
0.004
0.45
1.11
0.004
0.08
Eosinophils
0.02
0.001
0.64
-0.32
0.002
0.34
-0.10
0.002
0.48
Basophils
0.22
0.001
0.44
-0.17
0.001
0.46
-0.12
0.002
0.44
Lymphocytes
0.12
0.005
0.58
0.45
0.007
0.24
-0.91
0.005
0.07
Monocytes
0.10
0.001
0.60
0.19
0.001
0.45
0.09
0.001
0.62
*βs expressing the post-exposure percentage changes for each leukocytes population, compared to pre-exposure.
14
Table S10. Sensitivity analysis. Effect of controlled exposures to fine concentrated ambient particles (CAPs) and coarse CAPs on
blood DNA methylation adjusted for white blood cells count and composition.*
Mean within-participant differences of DNA methylation in post-exposure samples
after fine or coarse CAPs vs. medical air (control) exposures
Fine CAPs
Coarse CAPs
Stdz β**
(95% CI)
p-value
Stdz β**
(95% CI)
p-value
Alu (%5mC)
-0.41
(-0.82, -0.01)
0.05
-0.27
(-0.67, 0.13)
0.19
LINE-1 (%5mC)
-0.13
(-0.56, 0.31)
0.57
-0.34
(-0.76, 0.08)
0.11
TLR4 (%5mC)
-0.18
(-0.36, 0.00)
0.05
-0.21
(-0.40, -0.03)
0.02
IL-6 (%5mC)
0.04
(-0.05, 0.13)
0.34
0.04
(-0.04, 0.13)
0.34
IL-12 (%5mC)
0.04
(-0.36, 0.43)
0.85
0.11
(-0.25, 0.47)
0.54
iNOS (%5mC)
-0.29
(-0.73, 0.16)
0.21
-0.19
(-0.63, 0.24)
0.38
* Results are adjusted for total white blood cells count, % of neutrophils, % of lymphocytes, % of monocytes, % of eosinophils, and
% of basophils.
** Standardized βs and 95% confidence intervals (CIs) expressing the difference in DNA methylation between the CAPs exposures
and the control exposures as a fraction of the standard deviation (SD) of DNA methylation. Βs indicate the differences of exposure vs.
The control (HEPA-filtered medical air) in the post-exposure blood samples.
15