Effect Modification by DNA Methylation Status
Repetitive Elements Methylation
We observed stronger effects of traffic-related pollutants on fibrinogen and C-reactive protein, among subjects with lower LINE-1 or higher Alu methylation status (Fig. 5). While among subjects with higher Alu methylation, an interquartile range increase in NO2 (14 day moving average) was associated with a 6.4% (3.9% to 8.5%) increase in fibrinogen, we observed only a 2.4% (−0.04% to 5.0%) fibrinogen increase among participants with lower methylation. LINE-1 also increased the effect on fibrinogen of SO4 2−, after 1 to 4 weeks of exposure. The air-pollution effect on ICAM-1 and VCAM-1 was not modified by methylation of repetitive elements status.
The air-pollution effect on fibrinogen was not modified by the mean tissue factor (F3) methylation states. However, when we examined effect modification by position within the promoter region, we observed longer short- and intermediate-term effects on fibrinogen of particle number, black carbon, NO2, and CO among subjects with low F3 methylation status for Position 1 or 5 (Fig. 6). For instance, among the low F3 methylation group for Position 1, an interquartile range increase in NO2 (3-day moving average) was associated with a 6.2% (3.8% to 8.5%) increase in fibrinogen, but only with a 2.9% (0.5% to 5.5%) increase among participants with higher methylation levels. When examining interaction between air pollution and TLR-2 methylation on C-reactive protein, we found greater intermediate-term effects of particle number and NO2, among the low TLR-2 methylation group (Fig. 6). We examined effect modification by ICAM-1 methylation status on ICAM-1 and VCAM-1 levels and did not find any consistent interactions.
As sensitivity analysis, we investigated the epigene-environment interactions using continuous methylation variables; the direction and magnitude of the results did not change. Air pollution and smoking (pack-years or history) were not correlated in our data set. We also examined effect modification by smoking and found no evidence of interactions (results not shown). Because air pollution and methylation are correlated,26 the epigene-air pollution interactions could be the result of a nonlinear effect of air pollution on the blood markers of interest. We checked this possibility by regressing methylation on air pollution and relevant covariates. Because the resulting residuals reflected the methylation variability independent of air pollution, dichotomizing them into low versus high avoids the ambiguity due to effects of air pollution on methylation. When we used them for the interaction term with air pollution, we obtained similar interactions.
Our results suggest that current concentrations of traffic-related air pollutants increase levels of intermediary CVD-related blood markers (fibrinogen, C-reactive protein, ICAM-1, and VCAM-1) after short- and intermediate-term exposures to air pollution (up to one month), and that those responses vary with a person's baseline DNA methylation pattern. Our results support previous findings that showed influence of air pollutants on markers of coagulation, inflammation, and endothelial function.5,7 – 9,23,27
We observed short- and intermediate-term effects of particle number on ICAM-1 and VCAM-1. This short time window is consistent with previous experimental studies. After 1 hour of diesel particles inhalation in a chamber, volunteers had increased ICAM-1 and VCAM-1 levels in their bronchial lining fluid and tissue.27 After concentrated fine-particle exposure, ICAM-1 levels measured in venous blood increased after 4 hours and 22 hours.28 A similar study showed that after hours of diesel particles exposure, healthy subjects had higher VCAM-1 concentrations in the bronchial mucosa.29 For the other traffic-related pollutants, we observed only intermediate-term effects on fibrinogen, ICAM-1, and VCAM-1. Zeka et al5 also found lagged exposure effects of traffic-related particles (1 week) and black carbon (1 month) on fibrinogen. Higher VCAM-1 have been reported after days of black carbon exposures and higher ICAM-1 after weeks of exposure.7,8 A key finding in our study was the associations with secondary pollutants as well as primary pollutants. ICAM-1 and VCAM-1 were also higher after short- and intermediate-term exposure to PM2.5 and sulfates, which is consistent with a previous study reporting similar PM2.5 and sulfate effects (5-day moving average).30 We also found a short-term ozone effect on ICAM-1 and C-reactive protein. A positive association between C-reactive protein and ozone was previously obtained after 3 days of exposure.6
We found the strongest associations with particle number, which is an assessment of fresh traffic emissions. Black carbon is, however, a mix of fresh local emissions and some aged transported traffic particles. In Boston, black carbon shows daily patterns peaking at 6 AM and a lower peak in the afternoon rush hour, suggesting a predominance of local traffic. However, transported black carbon is also important, as its concentrations were higher when the back trajectory of the air mass over Boston originated in New York,31 indicating some secondary contribution as well. NO2 is a secondary pollutant that reflects both local sources and transported pollution. These differences might explain why the time pattern of the effects of particle number appears to be distinct from that of black carbon and NO2. Even though particle number, black carbon, and NO2 are traffic-related pollutants, they may be markers of something more complex, including the freshness of the particles. PM2.5 signals on fibrinogen were weak, which is concordant with previous studies.5,32 Fine particles are too small to easily settle out by gravity and too large to coagulate into larger particles, which makes them able to stay in the atmosphere for weeks and travel thousands of kilometers before returning to Earth. In Boston, sulfate particles and secondary organic aerosols constitute a large fraction of PM2.5 mass, with a lesser role for traffic particles. SO4 2− and O3 (which are other secondary pollutants) were not associated with fibrinogen. Hence, our findings on fibrinogen clearly point toward local traffic particles.
We also observed short- and intermediate-term effects of traffic-related particles, PM2.5, and SO4 2− on ICAM-1 and VCAM-1. These results on adhesion molecules point toward both primary and secondary pollutants. We reported short-term ozone effects on C-reactive protein and ICAM-1. Ozone is a powerful oxidizing agent and is created with high concentration of pollution and daylight UV rays at the Earth's surface. Ozone exposure has been linked to lung dysfunction and respiratory system irritation,33 and the C-reactive protein and ICAM-1 increases indicate acute inflammatory and endothelial responses to short-term exposure.
Our main air pollution findings suggest some plausible biologic mechanisms that could explain some of the exacerbation of CVD morbidity and mortality.2,27 Air pollution exposure may increase systemic cytokine-mediated inflammation and prothrombotic activity. In susceptible people, ultrafine particles were able to provoke alveolar inflammation, with release of mediators capable of increasing blood coagulability.34 Increased plasma viscosity is a potential mechanism explaining why high fibrinogen levels are related to increased CVD risk.3 Similarly, elevated C-reactive protein, ICAM-1, and VCAM-1 levels have been associated with inflammation and cardiovascular risk.10,35 Increase in C-reactive protein may reflect arterial damage from white blood cell invasion and inflammation within the wall due to air pollution exposure, thus inducing cardiovascular events. In normal human arterial tissues, ICAM-1 levels were lower than in atherosclerotic lesions.11 ICAM-1 plays a role in general inflammation and up-regulation in nonendothelial cells, while VCAM-1 may be more involved in distribution in the vascular system.9 This could explain why the ICAM-1 and VCAM-1 signals have slightly different time windows.
We found greater air pollution effects among subjects with high Alu, but low LINE-1, F3, or TLR-2 methylation status. To our knowledge, this is the first study to report evidence of epigene-environment interactions. Our results suggest that DNA methylation may play an important role in inflammatory gene expression and CVD. Knock-out mice for methylation enzymes develop atherosclerotic fatty streaks.36 In the same cohort, LINE-1 hypomethylation was associated with higher VCAM-1 protein levels,19 as well as higher CVD incidence and mortality.20 LINE-1 elements, as retrotransposable sequences, have enhanced activity when they are demethylated, which may induce gene transcription dysfunction, and alterations of the genome by insertion or homologous recombination.37 LINE-1 expression has been identified as a mediator of ischemic heart damage.18 Repetitive elements become activated during oxidative and cellular stress conditions.38 Interestingly, recent studies have reported a higher risk of cardiovascular events among subjects with low LINE-1, but also with high Alu methylation in peripheral blood leukocytes.20,21 Alu and LINE-1 have also showed opposite changes according to season.26 Therefore, LINE-1 hypomethylation and Alu hypermethylation may be distinct responses to oxidative stress and inflammation, and therefore might identify a subset of subjects who have stronger molecular responses to air pollution.
Air pollution effects on fibrinogen were stronger in subjects with lower F3 methylation. F3 expression and fibrinogen increase in response to proinflammatory stimuli, such as those caused by air pollution.4,12 Tissue factor upregulation is found in inflammatory and hypercoagulable states, which are accompanied by some elevations of clotting signals, including fibrin overproduction.39 Tissue factor hypomethylation may participate in pathways leading to increased tissue factor expression and fibrinogen in response to air pollution and therefore reflects a proinflammatory state leading to stronger coagulation responses.
We observed greater air pollution effects on C-reactive protein among participants with lower TLR-2 methylation. TLR-2 plays an important role in activation of innate immunity. Several cytokines and interleukins, including IL-6, assist in this immunity process. Compared with knockout mice for TLR-2, wildtype mice had greater PM2.5 effects on IL-6.13 IL-6 rise has been associated with increase in C-reactive protein, which also plays a role in innate immunity as an early defense system against infections. Our findings suggest that TLR-2 hypomethylation may silence the TLR-2 gene and participate in biologic processes enhancing IL-6 and C-reactive protein after air pollution exposure, and therefore induces a proinflammatory state leading to stronger inflammatory responses.
Our results suggest that Alu, LINE-1, F3, and TLR-2 methylation status interact with several air pollutants. While tissue factor methylation status seems to modify the effects of all traffic pollutants on fibrinogen, we observed effect modification by LINE-1 methylation only with black carbon and sulfate. Alu and TLR-2 methylation status interacted with NO2 and particle number. Particle number represents freshly generated ultrafine traffic particles, while black carbon captures both fresh and aged traffic particles. The finding that the freshest and smallest traffic particles interact with Alu, TLR-2, and tissue factor suggests a different mechanism, perhaps movement of the ultrafine particles into the blood stream. The interactions of LINE-1 methylation with sulfates and black carbon, capturing fewer fresh particles, are probably related to a different pathway. A recent study supports this hypothesis.40 After 4 hours of ultrafine particles exposure, mice had increased thrombin formation, related to tissue factor (extrinsic coagulation pathway). Alternatively, the procoagulant chronic effects of particulate matter could be mediated via factor XII activation (intrinsic coagulation pathway).
While Alu methylation state modified the particle-number effect on fibrinogen and C-reactive protein after the first hours and days of exposure, it increased the NO2 effect on fibrinogen after 2 to 4 weeks. Similarly, F3 methylation interacted with all traffic-related pollutants over the first hours or days, while LINE-1 interacted with black carbon or SO4 2− after a longer period (1 to 4 weeks) to modify fibrinogen. These time differences indicate possible distinctions in the properties of air pollutants—especially between primary and secondary pollutants, for which a longer averaging time is required to see an interaction, suggesting different biologic mechanisms.
Strengths and Limitations
We prospectively investigated the role of air pollution from various sources on repeated measures of intermediary CVD-related markers. Our findings were not the result of confounding by CVD risk factors, such as age, seasonality, BMI, temperature, relative humidity, diabetes, statin, and smoking status. Smoking is an important predictor of methylation. However, we found no correlation between air pollution and any smoking variable (category or pack-years). Therefore, we believe that smoking is not confounding the air pollution effects seen in our study. With regard to the possibility that the interaction with methylation is really an interaction with past smoking that produced stable changes in methylation patterns, we examined this question using interaction terms with former and current smoking rather than with methylation. None of these associations explained our results. The observed epigene-air pollution interactions were also not the result of a nonlinear effect of air pollution on the blood markers of interest. Our analysis is, however, limited to 5 methylation variables. Methylation of other genes and histone modifications might be important variables to evaluate. To be confounding variables, they would have to be correlated with Alu, LINE-1, F3, or TLR-2 methylation. There are few data to indicate how likely this is. In addition, DNA methylation is more stable than mRNA or protein expression, and therefore represents a somewhat longer time window in system behavior. We also identified positions within promoter regions of F3 and TLR-2 that may play an epigenetic role in coagulation and inflammation.
We were not able to assess personal air pollution exposure, which is likely to be nondifferentially misclassified. Therefore, exposure misclassification for using ambient monitors presumably attenuates our associations. This attenuation should be larger for short-term exposures and for pollution components that have the greatest spatial variability, which are the traffic pollutants. Exposure assignments for ozone and particularly for sulfates are expected to be fairly accurate and reinforce the null effects found on fibrinogen. Fibrinogen, C-reactive protein, ICAM-1, VCAM-1, and DNA methylation are measured directly from blood. Even though these measurements are likely to be accurate, we also expect some nondifferential misclassification of the underlying biologic function. Our cohort consists of elderly men that may be less susceptible than the population they are representing. Loss to follow-up may also make this study population healthier than the initial population. Furthermore, our results may not be generalizable to the general population if the biologic mechanisms are modified by race, sex, and age.
1. Brook RD, Rajagopalan S, Pope CA III, et al.. Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation. 2010;121:2331–2378.
2. Danesh J, Collins R, Appleby P, Peto R. Association of fibrinogen, C-reactive protein, albumin, or leukocyte count with coronary heart disease: meta-analyses of prospective studies. JAMA. 1998;279:1477–1482.
3. Peters A, Doring A, Wichmann HE, Koenig W. Increased plasma viscosity during an air pollution episode: a link to mortality? Lancet. 1997;349:1582–1587.
4. Ruckerl R, Greven S, Ljungman P, et al.. Air pollution and inflammation (interleukin-6, C-reactive protein, fibrinogen) in myocardial infarction survivors. Environ Health Perspect. 2007;115:1072–1080.
5. Zeka A, Sullivan JR, Vokonas PS, Sparrow D, Schwartz J. Inflammatory markers and particulate air pollution: characterizing the pathway to disease. Int J Epidemiol. 2006;35:1347–1354.
6. Chuang KJ, Chan CC, Su TC, Lee CT, Tang CS. The effect of urban air pollution on inflammation, oxidative stress, coagulation, and autonomic dysfunction in young adults. Am J Respir Crit Care Med. 2007;176:370–376.
7. Alexeeff SE, Coull BA, Gryparis A, et al.. Medium-term exposure to traffic-related air pollution and markers of inflammation and endothelial function. Environ Health Perspect. 2011;119:481–486.
8. Madrigano J, Baccarelli A, Wright RO, et al.. Air pollution, obesity, genes and cellular adhesion molecules. Occup Environ Med. 2010;67:312–317.
9. O'Neill MS, Veves A, Sarnat JA, et al.. Air pollution and inflammation in type 2 diabetes: a mechanism for susceptibility. Occup Environ Med. 2007;64:373–379.
10. Ridker PM, MacFadyen J, Libby P, Glynn RJ. Relation of baseline high-sensitivity C-reactive protein level to cardiovascular outcomes with rosuvastatin in the Justification for Use of statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER). Am J Cardiol. 2010;106:204–209.
11. Hwang SJ, Ballantyne CM, Sharrett AR, et al.. Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: the Atherosclerosis Risk in Communities (ARIC) study. Circulation. 1997;96:4219–4225.
12. Gilmour PS, Morrison ER, Vickers MA, et al.. The procoagulant potential of environmental particles (PM10). Occup Environ Med. 2005;62:164–171.
13. Shoenfelt J, Mitkus RJ, Zeisler R, et al.. Involvement of TLR2 and TLR4 in inflammatory immune responses induced by fine and coarse ambient air particulate matter. J Leukoc Biol. 2009;86:303–312.
14. Ljungman P, Bellander T, Schneider A, et al.. Modification of the interleukin-6 response to air pollution by interleukin-6 and fibrinogen polymorphisms. Environ Health Perspect. 2009;117:1373–1379.
15. Stenvinkel P, Karimi M, Johansson S, et al.. Impact of inflammation on epigenetic DNA methylation - a novel risk factor for cardiovascular disease? J Intern Med. 2007;261:488–499.
16. Nakkuntod J, Avihingsanon Y, Mutirangura A, Hirankarn N. Hypomethylation of LINE-1 but not Alu in lymphocyte subsets of systemic lupus erythematosus patients. Clin Chim Acta. 2011;412:1457–1461.
17. Cash HL, Tao L, Yuan JM, et al.. LINE-1 hypomethylation is associated with bladder cancer risk among nonsmoking Chinese. Int J Cancer. 2012;130:1151–1159.
18. Lucchinetti E, Feng J, Silva R, et al.. Inhibition of LINE-1 expression in the heart decreases ischemic damage by activation of Akt/PKB signaling. Physiol Genomics. 2006;25:314–324.
19. Baccarelli A, Tarantini L, Wright RO, et al.. Repetitive element DNA methylation and circulating endothelial and inflammation markers in the VA normative aging study. Epigenetics. 2010;5.
20. Baccarelli A, Wright R, Bollati V, et al.. Ischemic heart disease and stroke in relation to blood DNA methylation. Epidemiology. 2010;21:819–828.
21. Kim M, Long TI, Arakawa K, Wang R, Yu MC, Laird PW. DNA methylation as a biomarker for cardiovascular disease risk. PLoS One. 2010;5:e9692.
22. Zou B, Chim CS, Zeng H, et al.. Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers. Gastroenterology. 2006;131:1835–1843.
23. Peters A, Frohlich M, Doring A, et al.. Particulate air pollution is associated with an acute phase response in men; results from the MONICA-Augsburg Study. Eur Heart J. 2001;22:1198–1204.
24. Zanobetti A, Schwartz J, Samoli E, et al.. The temporal pattern of respiratory and heart disease mortality in response to air pollution. Environ Health Perspect. 2003;111:1188–1193.
25. Panasevich S, Leander K, Rosenlund M, et al.. Associations of long- and short-term air pollution exposure with markers of inflammation and coagulation in a population sample. Occup Environ Med. 2009;66:747–753.
26. Baccarelli A, Wright RO, Bollati V, et al.. Rapid DNA methylation changes after exposure to traffic particles. Am J Respir Crit Care Med. 2009;179:572–578.
27. Salvi S, Blomberg A, Rudell B, et al.. Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers. Am J Respir Crit Care Med. 1999;159:702–709.
28. Holgate ST, Devlin RB, Wilson SJ, Frew AJ. Health effects of acute exposure to air pollution. Part II: Healthy subjects exposed to concentrated ambient particles. Res Rep Health Eff Inst. 2003;:31,50; discussion 51–67.
29. Stenfors N, Nordenhall C, Salvi SS, et al.. Different airway inflammatory responses in asthmatic and healthy humans exposed to diesel. Eur Respir J. 2004;23:82–86.
30. Wilker EH, Alexeeff SE, Suh H, Vokonas PS, Baccarelli A, Schwartz J. Ambient pollutants, polymorphisms associated with microRNA processing and adhesion molecules: the Normative Aging Study. Environ Health. 2011;10:45.
31. Park SK, O'Neill MS, Vokonas PS, Sparrow D, Schwartz J. Effects of air pollution on heart rate variability: the VA normative aging study. Environ Health Perspect. 2005;113:304–309.
32. Thompson AM, Zanobetti A, Silverman F, et al.. Baseline Repeated Measures from Controlled Human Exposure Studies: Associations between Ambient Air Pollution Exposure and the Systemic Inflammatory Biomarkers IL-6 and Fibrinogen. Environ Health Perspect. 2010;118:120–124.
33. Keller R, Baltzer P, Keller-Wossidlo H, Gamp R, Ragaz A, Schaub T. Acute effects of the natural atmospheric ozone exposure on lung function of clinically normal smokers and non-smokers. Schweiz Med Wochenschr. 1990;120:1724–1730.
34. Seaton A, MacNee W, Donaldson K, Godden D. Particulate air pollution and acute health effects. Lancet. 1995;345:176–178.
35. Luc G, Arveiler D, Evans A, et al.. Circulating soluble adhesion molecules ICAM-1 and VCAM-1 and incident coronary heart disease: the PRIME Study. Atherosclerosis. 2003;170:169–176.
36. Hiltunen MO, Yla-Herttuala S. DNA methylation, smooth muscle cells, and atherogenesis. Arterioscler Thromb Vasc Biol. 2003;23:1750–1753.
37. Ostertag EM, Kazazian HH Jr. Biology of mammalian L1 retrotransposons. Annu Rev Genet. 2001;35:501–538.
38. Valinluck V, Tsai HH, Rogstad DK, Burdzy A, Bird A, Sowers LC. Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acids Res. 2004;32:4100–4108.
39. Chu AJ. Role of tissue factor in thrombosis. Coagulation-inflammation-thrombosis circuit. Front Biosci. 2006;11:256–271.
40. Kilinc E, van Oerle R, Borissoff JI, et al.. Factor XII Activation is Essential to Sustain the Procoagulant Effects of Particulate Matter. J Thromb Haemost. 2011;9:1359–1367.
We thank the Normative Aging Study participants, Tania Kotlov, and Johanna Lepeule.
Supplemental Digital Content
© 2012 Lippincott Williams & Wilkins, Inc.