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Online Supplement 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 (10M) Primer Reverse (10M) Cycling Conditions 95°C for 90”, 43°C for 60”, -* -* -* -* -* -* Alu 2l 2l 72°C for 120” for 45 cycles 95°C for 30”, 50°C for 30”, -* -* -* -* -* -* LINE-1 2l 2l 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 1l 1l 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 1l 1l 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 1l 1l 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 1l 1l 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