text:
Influence of ambient air pollution on global DNA methylation in healthy adults: A seasonal follow-up.
ABSTRACT DNA methylation changes are potential pathways of environmentally induced health effects. We investigated whether exposure to ambient concentrations of NO2, PM10, PM2.5 and O3 and traffic parameters were associated with global DNA methylation in blood of healthy adults.
48 non-smoking adults (25 males) with a median age of 39years were sampled in winter and summer. Global DNA methylation in whole blood (% 5-methyl-2'-deoxycytidine, %5mdC) was analyzed with HPLC. Exposure to air pollutants at the home address was assessed using interpolated NO2, PM10, PM2.5 and O3 concentrations for various exposure windows (60- to 1-day moving average exposures and yearly averages) and GIS-based traffic parameters. Associations between pollutants and %5mdC were tested with multiple mixed effects regression models.
Average %5mdC (SD) was 4.30 (0.08) in winter and 4.29 (0.08) in summer. Men had higher %5mdC compared to women both in winter (4.32 vs. 4.26) and summer (4.31 vs. 4.27). When winter and summer data were analyzed together, various NO2, PM10 and PM2.5 moving average exposures were associated with changes in %5mdC (95% CI) ranging from -0.04 (-0.09 to 0.00) to -0.14 (-0.28 to 0.00) per IQR increase in pollutant. NO2, PM10, PM2.5 and O3 moving average exposures were associated with decreased %5mdC (95% CI) varying between -0.01 (-0.03 to 0.00) and -0.17 (-0.27 to -0.06) per IQR increase in pollutant in summer but not in winter.
Decreased global DNA methylation in whole blood was associated with exposure to NO2, PM10, PM2.5 and O3 at the home addresses of non- adults. Most effects were observed for the 5- to 30-day moving average exposures.
- SourceAvailable from: Chiara Scoccianti[Show abstract] [Hide abstract]
ABSTRACT: DNA methylation is a well-characterized epigenetic modification that plays an important role in the regulation of gene expression. There is growing evidence on the involvement of epigenetic mechanisms in disease onset, including cancer. Environmental factors seem to induce changes in DNA methylation affecting human health. However, little is known about basal methylation levels in healthy people and about the correlation between environmental factors and different methylation profiles. We investigated the effect of seasonality on basal methylation by testing methylation levels in the long interspersed nucleotide element-1 (LINE-1) and in two cancer-related genes (RASSF1A and MGMT) of 88 healthy male heavy smokers involved in an Italian randomized study; at enrolment the subjects donated a blood sample collected in different months. Methylation analyses were performed by pyrosequencing. Mean methylation percentage was higher in spring and summer for the LINE1, RASSF1A and MGMT genes (68.26%, 2.35%, and 9.52% respectively) compared with autumn and winter (67.43%, 2.17%, and 8.60% respectively). In particular, LINE-1 was significantly hypomethylated (p = 0.04 or 0.05 depending on the CpG island involved) in autumn and winter compared with spring and summer. Seasonality seems to be a modifier of methylation levels and this observation should be taken into account in future analyses.PLoS ONE 09/2014; 9(9):e106846. · 3.53 Impact Factor
Article: Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues.[Show abstract] [Hide abstract]
ABSTRACT: DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.PLoS ONE 01/2013; 8(11):e79044. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The aim of this review is to comprehensively summarize the recent achievements in the field of toxicogenomics and cancer research regarding genetic-environmental interactions in carcinogenesis and detection of genetic aberrations in cancer genomes by next-generation sequencing technology. Cancer is primarily a genetic disease in which genetic factors and environmental stimuli interact to cause genetic and epigenetic aberrations in human cells. Mutations in the germline act as either high-penetrance alleles that strongly increase the risk of cancer development, or as low-penetrance alleles that mildly change an individual's susceptibility to cancer. Somatic mutations, resulting from either DNA damage induced by exposure to environmental mutagens or from spontaneous errors in DNA replication or repair are involved in the development or progression of the cancer. Induced or spontaneous changes in the epigenome may also drive carcinogenesis. Advances in next-generation sequencing technology provide us opportunities to accurately, economically, and rapidly identify genetic variants, somatic mutations, gene expression profiles, and epigenetic alterations with single-base resolution. Whole genome sequencing, whole exome sequencing, and RNA sequencing of paired cancer and adjacent normal tissue present a comprehensive picture of the cancer genome. These new findings should benefit public health by providing insights in understanding cancer biology, and in improving cancer diagnosis and therapy.Journal of Environmental Science and Health Part C Environmental Carcinogenesis & Ecotoxicology Reviews 04/2014; 32(2):121-58. · 3.23 Impact Factor
Page 1
Influence of ambient air pollution on global DNA methylation in healthy
adults: A seasonal follow-up
Sofie De Prinsa,b,⁎, Gudrun Koppena, Griet Jacobsa, Evi Donsa,c, Els Van de Mieroopd, Vera Nelend,
Frans Fierense, Luc Int Panisa,c, Patrick De Boevera,f, Bianca Coxf, Tim S. Nawrotf, Greet Schoetersa,b
aEnvironmental Risk and Health unit, VITO (Flemish Institute for Technological Research), Boeretang 200, B-2400 Mol, Belgium
bFaculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
cTransportation Research Institute (IMOB), Hasselt University, Wetenschapspark 5 Box 6, B-3590 Diepenbeek, Belgium
dEnvironment and Health Unit, Provincial Institute of Hygiene, Kronenburgstraat 45, B-2000 Antwerp, Belgium
eBelgian Interregional Environment Agency (IRCEL), Kunstlaan 10-11, B-1210 Brussels, Belgium
fCentre for Environmental Sciences, Hasselt University, Agoralaan Gebouw D, B-3590 Diepenbeek, Belgium
a b s t r a c t a r t i c l ei n f o
Article history:
Received 30 April 2013
Accepted 11 July 2013
Available online 3 August 2013
Air pollution
DNA methylation
Human biomonitoring
Background: DNA methylation changes are potential pathways of environmentally induced health effects. We
investigated whether exposure to ambient concentrations of NO2, PM10, PM2.5and O3and traffic parameters
were associated with global DNA methylation in blood of healthy adults.
Methods:48non-smokingadults(25males)with amedianageof39 yearsweresampledinwinterandsummer.
Global DNA methylation in whole blood (% 5-methyl-2′-deoxycytidine, %5mdC) was analyzed with HPLC.
Exposure to air pollutants at the home address was assessed using interpolated NO2, PM10, PM2.5and O3
concentrations for various exposure windows (60- to 1-day moving average exposures and yearly averages)
and GIS-based traffic parameters. Associations between pollutants and %5mdC were tested with multiple
mixed effects regression models.
Results: Average %5mdC (SD) was 4.30 (0.08) in winter and 4.29 (0.08) in summer. Men had higher %5mdC
compared to women both in winter (4.32 vs. 4.26) and summer (4.31 vs. 4.27). When winter and summer data
were analyzed together, various NO2, PM10and PM2.5moving average exposures were associated with changes
in %5mdC (95% CI) ranging from −0.04 (−0.09 to 0.00) to −0.14 (−0.28 to 0.00) per IQR increase in pollutant.
NO2, PM10, PM2.5and O3moving average exposures were associated with decreased %5mdC (95% CI) varying
between −0.01 (−0.03 to 0.00) and −0.17 (−0.27 to −0.06) per IQR increase in pollutant in summer but not
in winter.
Conclusion: Decreased global DNA methylation in whole blood was associated with exposure to NO2, PM10, PM2.5
and O3at the home addresses of non- adults. Most effects were observed for the 5- to 30-day moving average
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Epidemiological and experimental studies described the relation
between air pollution and health. Mortality, cancer, cardiovascular
symptoms, respiratory problems and neurological effects have all been
linked to air quality (Brunekreef, 2007; Calderon-Garciduenas et al.,
2008; Kelly and Fussell, 2011; Pope and Dockery, 2006).
In the last decade, the epigenome is increasingly being suggested as
an important link between environmental exposures and changes in
gene expression. These changes may be an intermediate phenotype in
the exposure–health effect continuum. The most studied forms of
epigenetic changes are DNA methylation and histone modifications
(Jirtle and Skinner, 2007). Methylation of DNA involves the covalent
addition of a methyl group to the carbon-5 position of cytosine, and
occurs at CpG dinucleotides which are clustered in CpG islands. Much
of the CpG islands are located in transposable repetitive elements, of
Environment International 59 (2013) 418–424
Abbreviations:WBC, white blood cells; LINE-1, long interspersed nucleotide elements;
dC, 2′-deoxycytidine; 5mdC, 5-methyl-2′-deoxycytidine; %5mdC, percentage 5mdC; NO2,
nitrogen dioxide; PM10, particulate matter with aerodynamic diameter ≤ 10 μm; PM2.5,
particulate matter with aerodynamic diameter ≤ 2.5 μm; O3, ozone; GIS, geographical in-
BMI, body mass index; BC, black carbon; iNOS, inducible nitric oxide synthase; NO, nitric
oxide; GSH, glutathione; SAM, S-adenosylmethionine; 5mC, 5-methyl-cytosine; 5hmC,
⁎ Corresponding author at: VITO (Flemish Institute for Technological Research),
Boeretang 200, 2400 Mol, Belgium. Tel.: +32 14 33 51 72; fax: +32 14 58 05 23.
E-mail addresses: email@example.com (S. De Prins), firstname.lastname@example.com
(G. Koppen), email@example.com (G. Jacobs), firstname.lastname@example.com (E. Dons),
firstname.lastname@example.com (E. Van de Mieroop), email@example.com
(V. Nelen), firstname.lastname@example.com (F. Fierens), firstname.lastname@example.com (L. Int Panis),
firstname.lastname@example.com (P. De Boever), email@example.com (B. Cox),
firstname.lastname@example.com (T.S. Nawrot), firstname.lastname@example.com (G. Schoeters).
0160-4120/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
Contents lists available at ScienceDirect
Environment International
journal homepage: www.elsevier.com/locate/envint
Page 2
which the long interspersed nucleotide elements (LINE-1) and Alu
sequences are the most common and the best studied in the context
of total DNA methylation (Wu et al., 2011). Aberrant DNA methylation
patterns are studied very intensively in cancer research, but evidence
for the involvement of DNA methylation in other conditions like auto-
immunity, asthma, and neurological diseases is increasing (Portela
and Esteller, 2010; Terry et al., 2011).
Several human epidemiological studies investigated the effects of
environmental pollution on DNA methylation. These studies report on
the association between levels of DNA methylation in white blood
cells (WBC) and environmental exposures including benzene, arsenic,
lead, persistent organic pollutants and air pollutants (Tang et al., 2012;
Terry et al., 2011). In the present study, we investigated whether expo-
sure to ambient concentrations of nitrogen dioxide (NO2), particulate
matterwithaerodynamicdiameter ≤ 10 μm(PM10),particulatematter
with aerodynamic diameter ≤ 2.5 μm (PM2.5) and ozone (O3) and
traffic parameters were associated with global DNA methylation levels
in peripheral blood of healthy adults. These relations were evaluated
in two seasons and for various exposure windows.
2. Material and methods
2.1. Study population
The study population consisted of 48 non-smoking adults not occu-
pationally exposed to air pollutants. Participants were selected among
employees of the study partners. All participants were living and work-
participants were couples. A total of 25 unique home locations were
studied. Two subjects were recruited per residence, with the exception
of two locations where only one person participated in the study. All
subjects were sampled twice, both in winter and summer. Individuals
of the same household were sampled within ten days. The first blood
sampling took place on one of five different days in February and
March 2010 (winter period). In June 2010 participants donated a
second blood sample, on one of four different days (summer period).
Two subjects were sampled in August because of practical reasons. All
blood samplings were scheduled between 14:00 h and 19:00 h. Partic-
ipants filled out questionnaires about their health condition, education,
job, housing conditions, past smoking habits (yes or no, duration),
passive smoking (hours per week spent in the presence of smokers),
food and alcohol consumption, physical activity, medication use and
illness or inflammation (cold, flu, nose, throat or ear infection) in the
week before sampling. Additionally, subjects provided information on
the average travel time to work and the time spent outdoors during
an average week. Cholesterol and vitamin E plasma levels were avail-
able for all subjects.
Ethical approval for the study protocol was granted by the ethical
committee of the University of Antwerp (Reference N° UA A09 22). All
subjects gave written informed consent to participate in the study.
2.2. Quantification of global DNA methylation in whole blood
Quantification of global DNA methylation was done on peripheral
whole blood that was kept in 10 mL sodium heparin vacutainers
(BectonDickinson,FranklinLakes,NJ,USA)at−80 °Cuntilanalysis.Be-
fore DNA extraction, samples were treated with Rnase (Qiagen, Hilden,
Germany) to remove RNA. DNA was extracted from whole blood using
QIAamp DNA Blood Maxi Kits (Qiagen). The DNA concentration was
determined by means of a NanoDrop spectrophotometer (NanoDrop
Technologies, Wilmington, DE, USA). DNA was stored in MilliQ water
at −20 °C until analysis. For DNA methylation analysis, 15 μg DNA
was used (concentration 115 ng/μL). DNA digestion was performed as
described by Rozhon et al. (2008). In short, DNA was incubated
overnight at 37 °C with a mixture of Dnase I and nuclease P1 (both
enzymes from Sigma-Aldrich, St. Louis, MO, USA). Next, nucleotide
monophosphates were dephosphorylated for a further 24 h at 37 °C
with calf intestinealkaline phosphatase (New England Biolabs, Ipswich,
MA, USA). 2′-Deoxycytidine (dC) and 5-methyl-2′-deoxycytidine
(5mdC) concentrations were measured with an Acquity Ultra
Performance Liquid Chromatography (UPLC) system (Waters, Milford,
MA, USA) which was used in high performance LC (HPLC) mode. The
HPLC protocol consisted of isocratic separation using a Nucleosil SA cat-
ion exchange silica 150 × 4.6 mm × 5 μm column (Macherey-Nagel,
Düren, Germany) at a temperature of 30 °C. The acidic mobile phase
consisted of 50 mM ammonium acetate in 15% acetonitrile with a pH
of 4.8. The flow rate was set at 0.5 mL/min and the injected sample
volume was 50 μL. Samples were cooled at 4 °C. UV detection was
performed at 272 nm for dC and at 279 nm for 5mdC. The retention
times for dC and 5mdC were 6 min 30 s and 7 min 42 s respectively.
Chromatograms were analyzed using Empower 2 software (Waters).
Levels of 5mdC and dC were estimated based on the corresponding
standard curve which ranged from 2 to 0.008 μg/mL for 5mdC and
from 20 to 0.08 μg/mL for dC (both standards from MP Biochemicals,
Santa Ana, CA, USA). The relative content of 5mdC was expressed as a
percentage (%5mdC) with respect to the total amount of cytosine
(dC + 5mdC). The technical variation of the assay was less than 3%,
based on six standard replicates included in the analysis.
2.3. Exposure assessment
The participants' home addresses were geocoded to assess air pollu-
tion exposure and traffic-related parameters.
In Belgium, several pollutants including NO2, PM10, PM2.5and O3are
continuously measured by a network of automatic monitoring sites
(http://www.irceline.be). Based on these official monitoring data and
on the coordinates of the home addresses of the participants, daily
24 h average concentrations of NO2, PM10, PM2.5and daily 10:00–
18:00 h averages of O3were calculated on a 4 × 4 km2grid using the
interpolation model RIO. The RIO model was validated earlier by
Janssen et al. (2008). For all pollutants, 60-, 30-, 14-, 10-, 7-, 5-, 3-, 2-
and 1-day moving average exposures were calculated for all subjects
in both sampling campaigns. The moving average is the mean exposure
during the respective time window before blood sampling. The average
Furthermore, the geographical information system (GIS) ArcGIS 9.3
(Esri, Redlands, CA, USA) was used to determine traffic parameters at
allhomeaddresses.Thefollowing parameters were calculated: distance
fromtheresidencetothenearestroadandnearestmajorroad (N10,000
vehicles/day), traffic intensity of light and heavy traffic during 24 h on
the nearest road and nearest major road, total road length in a buffer
with radius 0.1 and 0.3 km, and length of major roads in a buffer with
radius 0.3, 1.0 and 5.0 km.
2.4. Meteorological data
Daily average temperatures (°C) were obtained from the Belgian
Royal Meteorological Institute. Since Flanders is very uniform for
temperature, data from one monitoring station (Retie, Belgium) were
used. The average temperature was calculated for every exposure
window mentioned above.
2.5. Statistical analysis
Statistical analysis was performed using SPSS Statistics 20 (IBM,
Armonk, NY, USA). Results were considered statistically significant if
P-values were lower than 0.05. t-Tests were used to test the effect of
season (paired Student's t-test) and gender (independent samples
Student's t-test) on %5mdC. Pearson correlation was used to correlate
winterand summer%5mdC data.Gender, age, BMI, physical activity, ill-
ness or inflammation in the week before sampling, past smoking habits
S. De Prins et al. / Environment International 59 (2013) 418–424
Page 3
and passive smoking, cholesterol and vitamin E plasma levels, travel
time to work and average time spent outdoors during a seven day peri-
regression. To assess the influence of air pollutants on %5mdC levels a
multiple linear mixed effects regression model was applied where the
variable 'house' was included as a random factor to correct for correla-
tions between subjects living in the same residence. Gender and age
were fixed covariates. Winter and summer data were analyzed sepa-
rately, but data from both sampling periods were also combined in a
model for repeated measures. For NO2, PM10, PM2.5and O3all moving
average exposures were analyzed with the average temperature during
therespective exposure period asanadditional covariate. In therepeat-
ed measures models, an interaction term between season and the
pollutant concentration was included. Exposure effects of NO2, PM10
and PM2.5 were assessed in both seasons, while O3 exposure was
analyzed only in summer since concentrations during winter are very
low. Annual average concentrations of NO2, PM10, PM2.5and O3were
assessed without adjustment for temperature. GIS-based traffic param-
perature during the 30-day moving average exposure window. Results
were expressed as estimated changes in %5mdC per interquartile
range (IQR) increase in pollutant concentrations with the indication of
the 95% confidence interval (CI).
3. Results
The study population (N = 48), all non-smokers,included25males
(52.1%) and the median (P25–P75) age of the participants was 39
(32–48) years. The population characteristics stratified for %5mdC
levels are displayed in Table 1. The average traffic time to work seemed
to be negatively associated with %5mdC. In regression analysis, this
effect was however not significant.
moving averages were higher in winter than in summer for NO2
(28.4 vs. 18.4 μg/m3), PM10(30.3 vs. 20.3 μg/m3) and PM2.5(26.9 vs.
15.2 μg/m3), while O3levels were lower (45.2 vs. 85.1 μg/m3). For all
residences, the PM1024 h European limit value (50 μg/m3on max.
35 days/year) and the O38 h European limit value (120 μg/m3on
max. 25 days/year) were exceeded on 7 to 21 days and 14 to 18 days
in 2010, respectively. The mean PM2.5/PM10ratio in 2010 was 0.79
and was higher in winter (0.89) than in summer (0.75). These ratios
are relatively high when compared with other reports in literature
(Eeftens et al., 2012; Querol et al., 2004; Traversi et al., 2011). This
could be a reflection of the dense traffic and building network in
Flanders, the investigated region. Additionally, we observed higher
ratios during the winter period, which was previously described and is
probably related to building heating (Traversi et al., 2011).
Average %5mdC (SD) was 4.30 (0.08) in winter and 4.29 (0.08) in
summer. The difference in %5mdC between winter and summer was
not significant (Fig. 1A). Winter and summer measurements showed a
positive correlation of 0.22 (P = 0.14, Fig. 1B). Global DNA methylation
od for quantification of %5mdC, which has been in use for over 30 years
amounts of DNA (1–5 μg) are needed. Other techniques that analyze
methylation in repetitive elements such as LINE-1 and Alu require less
DNA (100–500 ng) and are more accessible, but these techniques only
target specific DNA sequences.
In simple regression analyses, male gender, past smokinghabits and
passive smoking, physical activity, plasma cholesterol and average time
spent outdoors during a seven day period were positively linked with
%5mdC while age, BMI, illness or inflammation in the week before sam-
ed with methylation levels. Except for the effect of gender, none of
the tested associations were statistically significant. Men had higher
%5mdC compared to women both in winter (4.32 vs. 4.26, P = 0.03)
and summer (4.31 vs. 4.27, P = 0.04).
Various significant effects of NO2, PM10, PM2.5and O3on %5mdC
were detected (Tables 3 and 4). There were more significant associa-
tions detected with longer-term exposures (≥7 days) compared to
short-term exposures (b7 days), especially for NO2. When winter and
summer data were combined (Table 3), the 60-, 30-, 14-, 10-, 7- and
5-day moving average NO2 exposures were associated with lower
%5mdC. Exposures to PM2.5during the 30-, 14- and 10-day windows
were associated with lower %5mdC. Significant seasonal effect modifi-
cation, as assessed by the interaction between pollutant concentrations
and season,was only observed for PM2.5during the 60-, 30- and 14-day
exposurewindows.Results forPM10weresimilarasthoseforPM2.5,but
Results of per season analysis are given in Table 4. In summer, %5mdC
was significantly influenced by the 30-, 14-, 10-, 7- and 5-day moving
average NO2exposures. In winter, estimates for NO2were borderline
significant, as indicated by the CI range. Effects of O3were observed
for the 14-day, 10-day and 2-day exposure periods in summer. %5mdC
Table 1
Population characteristics. All participants were non-smokers. Data are stratified for
%5mdC (tertiles) based on analysis of %5mdC in samples of the first period (winter).
Data are expressed as median values (P25–P75) or N (%). Except for the variable 'time
spent outdoors', only data from the first period (winter) are displayed.
Characteristic1st tertile2nd tertile 3rd tertile
Gender (males)
Age (years)
BMI (kg/m2)
Cholesterol (mg/dL)
Vitamin E (mg/L)
Past smoking (yes)
Traffic time to work (min)
Physical activity
Average time spent outdoors over a 7 day period (%/day)
5 (10.6)
39 (32–42)
24.9 (21.6–26.3)
172 (152–194)
13.7 (11.9–16.1)
6 (12.8)
60 (30–70)
9 (19.1)
39 (33–52)
25.5 (22.1–28.6)
176 (163–213)
13.6 (12.3–17.1)
0 (0.0)
40 (15–60)
10 (21.3)
42 (32–50)
22.3 (21.0–26.4)
165 (147–202)
13.6 (10.8–16.2)
5 (10.6)
30 (19–60)
9 (19.1)
7 (14.9)
8 (17.0)
8 (17.0)
9 (19.1)
6 (12.8)
3.6 (1.9–4.8)
6.7 (5.5–10.2)
3.8 (2.2–7.4)
10.8 (6.3–19.3)
4.8 (3.9–6.4)
10.0 (5.6–19.9)
Table 2
Environmentalcharacteristics,data are expressedas mean(SD)valuesandmin–max.Winterandsummerdataare basedon 30-daymovingaverageexposuredataatthe home addresses
of the study participants.
Annual (2010)Winter Summer
Mean (SD) Min–maxMean (SD)Min–maxMean (SD) Min–max
Temperature (°C)
Road length in 0.1 km buffer (m)
Traffic density nearest road (vehicles/24 h)
22.8 (5.2)
60.4 (3.0)
21.7 (2.0)
17.1 (1.2)
9.7 (1.1)
0.79 (0.03)
336 (144)
11,642 (18,280)
28.4 (6.5)
45.2 (6.0)
30.3 (5.0)
26.9 (4.3)
1.0 (0.9)
0.89 (0.03)
18.4 (4.7)
85.1 (3.8)
20.3 (2.2)
15.2 (1.6)
14.1 (1.3)
0.75 (0.03)
S. De Prins et al. / Environment International 59 (2013) 418–424
Page 4
showed negative associations with 14-, 10-, 7-, 3- and 2-day PM2.5
moving averages. No significant effects of air pollution parameters on
%5mdC were observed for the sampling campaign during the winter
period. Estimates of annual average pollutant concentration were not
associated with %5mdC (data not displayed). No significant effects of
GIS-variables were observed when analyzed separately or in combina-
tion with PM2.5and temperature (data not displayed).
4. Discussion
Our results describe an association between outdoor air pollution
levels and global DNA hypomethylation in peripheral blood of healthy,
non-elderly adults. These observations are in line with other scientific
reports. Earlier, hypomethylation in repetitive elements was shown to
Table 3
Effectsofmovingaverage NO2andPM2.5exposureson%5mdC.Resultsofmixedmodelre-
gressionanalysisforrepeated measurements.Estimates for PM10were similarbut smaller
than estimates for PM2.5(data not displayed).
Estimateb(95% CI)
−0.05 (−0.10 to −0.01)⁎
−0.06 (−0.11 to −0.02)⁎⁎
−0.06 (−0.09 to −0.02)⁎⁎
−0.05 (−0.08 to −0.02)⁎⁎
−0.05 (−0.09 to −0.02)⁎⁎
−0.04 (−0.09 to 0.00)⁎
−0.01 (−0.04 to 0.02)
−0.01 (−0.04 to 0.02)
0.00 (−0.04 to 0.05)
−0.18 (−0.37 to 0.01)
−0.14 (−0.28 to 0.00)⁎
−0.09 (−0.15 to −0.02)⁎⁎
−0.08 (−0.16 to −0.01)⁎
−0.06 (−0.13 to 0.01)
−0.02 (−0.12 to 0.08)
−0.01 (−0.05 to 0.03)
−0.02 (−0.07 to 0.03)
−0.01 (−0.06 to 0.04)
bEstimated change in %5mdC per IQR increase in pollutant concentration corrected for
was included to correct for correlations between subjects living in the same residence.
cP-value of the interaction between season and pollutant concentration.
⁎ P b 0.05.
⁎⁎ P b 0.01.
Table 4
Effects of moving average NO2, PM2.5and O3exposures on %5mdC in winter and summer.
Results of mixed model regression analysis. Estimates for PM10were similar but smaller
than estimates for PM2.5(data not displayed).
Estimateb(95% CI)
−0.01 (−0.03 to 0.00)
−0.02 (−0.05 to 0.00)
−0.03 (−0.06 to 0.00)
−0.03 (−0.06 to 0.01)
−0.03 (−0.06 to 0.01)
−0.05 (−0.10 to 0.01)
−0.03 (−0.06 to 0.01)
−0.02 (−0.04 to 0.01)
−0.02 (−0.05 to 0.02)
−0.02 (−0.06 to 0.02)
−0.06 (−0.18 to 0.05)
−0.01 (−0.15 to 0.13)
−0.09 (−0.35 to 0.17)
0.00 (−0.19 to 0.19)
0.01 (−0.21 to 0.23)
0.00 (−0.04 to 0.04)
−0.01 (−0.04 to 0.03)
0.00 (−0.01 to 0.01)
−0.01 (−0.03 to 0.00)
−0.01 (−0.03 to 0.00)⁎
−0.03 (−0.05 to −0.01)⁎⁎
−0.04 (−0.07 to −0.02)#
−0.04 (−0.06 to −0.02)#
−0.03 (−0.06 to −0.01)⁎⁎
−0.02 (−0.06 to 0.01)
−0.03 (−0.07 to 0.01)
−0.01 (−0.03 to 0.01)
0.00 (−0.04 to 0.04)
−0.01 (−0.05 to 0.03)
−0.06 (−0.09 to −0.03)⁎⁎
−0.07 (−0.10 to −0.03)#
−0.05 (−0.09 to −0.02)⁎⁎
−0.05 (−0.09 to 0.00)⁎
−0.11 (−0.19 to −0.03)⁎⁎
−0.17 (−0.27 to −0.06)⁎⁎
−0.02 (−0.16 to 0.12)
0.02 (−0.01 to 0.05)
0.02 (0.00 to 0.05)
−0.05 (−0.08 to −0.01)⁎
−0.06 (−0.11 to −0.01)⁎
−0.04 (−0.09 to 0.01)
0.00 (−0.04 to 0.05)
0.31 (−0.05 to 0.68)
−0.26 (−0.52 to 0.00)⁎
0.04 (−0.02 to 0.10)
bEstimated change in %5mdC per IQR increase in pollutant concentration corrected for
was included to correct for correlations between subjects living in the same residence.
⁎ P b 0.05.
⁎⁎ P b 0.01.
#P b 0.001.
Fig. 1. Repeated measurements of %5mdC. A: Comparison between winter and summer
(paired Student's t-test, not significant, the vertical line represents the median and the
bars represent the minimum and maximum values). B: correlation between winter and
summer (Pearson r = 0.22, P = 0.14).
S. De Prins et al. / Environment International 59 (2013) 418–424
Page 5
be associated with exposure to black carbon (BC), PM2.5and sulfate
(SO4) in the Normative Aging Study (Baccarelli et al., 2009; Madrigano
et al., 2011). In this elderly population, various exposure windows
with both short-term (≤7 days) (Baccarelli et al., 2009) and long-term
(N1 month)exposures(Madriganoetal.,2011).Inourstudy,wemostly
observed associations between %5mdC and longer-term exposures
(≥7 days). Some groups investigated the effects of air pollution on
lation of repetitive elements was linked with working in an industrial
estate (Peluso et al., 2012) and exposure to benzene (Bollati et al.,
2007) and PM10(Tarantini et al., 2009). The latter study also observed
lower inducible nitric oxide synthase (iNOS) promoter methylation
after three working days compared to the beginning of the work
week. The enzyme iNOS is responsible for the production of nitric
oxide (NO), which is involved in several inflammatory-related path-
ways (Ricciardolo et al., 2004). Recently, methylation in three NOS
genes was associated with exposure to PM2.5and PM10in a cohort of
children (Breton et al., 2012). Another study which analyzed the 27 K
betweentwo regions with various levels of air pollution.In thepolluted
area methylation was lower in all CpG sites which differed more than
10% from the control region (Rossnerova et al., 2013).
Substantial epidemiological evidence suggests that oxidative stress
(Delfino et al., 2011; Romieu et al., 2008). Increased levels of oxidative
stress deplete antioxidant systems, of which glutathione S-transferases
and their substrate glutathione (GSH) are important elements (Limon-
Pacheco and Gonsebatt, 2009). The production of GSH occurs through
ly linked with the biosynthesis of S-adenosylmethionine (SAM) which is
the most important methyl donor for several methyltransferases. Hence,
the level of SAM is a major determinant for the degree of DNA methyla-
tion (Ji and Hershey, 2012). Whenever an increased synthesis of GSH is
required, production of SAM may be impaired possibly leading to DNA
hypomethylation (Lee et al., 2009). Recently, epidemiological research
showed that the association between BC exposure and DNA hypomethy-
lation was strengthened in the presence of the glutathione S-transferase
mu-1 null genotype (Madrigano et al., 2011). Furthermore, direct oxida-
tive DNA damage such as 8-oxoguanine, 5-hydroxymethyl-cytosine
(5hmC) and bulky DNA adducts could inhibit the binding of methyl-
to the DNA, resulting in decreased DNA methylation (Peluso et al., 2012;
Valinluck et al., 2004). Additionally, a recent hypothesis postulates that
environmental pollutants might affect 5mC and 5hmC by interfering
with metabolic processes through the induction of oxidative stress
(Chia et al., 2011).
We found significantly higher levels of global DNA methylation in
men compared to women. These results are in accordance with other
reports in literature on the effects of gender on global DNA methylation
as well as LINE-1 DNA methylation and gene-specific methylation
(Terry et al., 2011; Zhu et al., 2012). What causes this is not precisely
known. It is suggested that differences in nutrient intake and loss be-
tween men and women, which in turn is related to the bioavailability
of methyl donors, contributes to the gender effect (Hsiung et al., 2007;
dicate a decreased DNA methylation with increasing age. However,
studies are far from consistent in this respect (Terry et al., 2011). In
our study, we did not observe a significant influence of age on %5mdC.
Possible age-related effects might remain undiscovered in our study
because of the relatively small age-range and the small sample size.
Inthis study,allsubjects weresampledintwoseasons.Weobserved
a weak correlation between repeated measurements, which was
however not significant. Earlier, repeated measures of methylation in
repetitive elements in blood samples, that were taken several years
apart, were significantly correlated in the Normative Aging Study
(Bollati et al., 2009). The latter authors used blood cell counts to correct
for effects on DNA methylation. Global methylation profiles are known
to vary between different blood cell types and a shift in blood cell pop-
ulations may affect the blood DNA methylation levels (Wu et al., 2011).
correlation between both measures or to the observation that associa-
tions between pollutants and %5mdC were stronger in summer. Our
questionnaire data on the presence of inflammation and illness in the
week before sampling might reflect changes in differential counts
did howevernotinfluence %5mdCinourstudypopulation.Itwasprevi-
ously described that ambient air pollution can affect white blood cell
composition in patients with chronic pulmonary diseases (Bruske
et al., 2010), diabetics (Jacobs et al., 2010a) and in healthy subjects dur-
ingaerobic exercise (Bos etal.,2013; Jacobs etal., 2010b). Other studies
did however not observe an association between air pollution exposure
and white blood cell composition in healthy adults (Barath et al., 2010;
Mills et al., 2005; Rich et al., 2012).
Our results show associations between air pollutants and %5mdC in
summer but not in winter. Earlier, Nawrot et al. described a stronger
association between PM10and mortality during summer compared to
winter. The investigated region was the same as in our study. The
authors report higher values of PM10in winter (Nawrot et al., 2007). It
was previously described that PM composition shows a large degree of
spatial and temporal variation (Bell, 2012). In Flanders, the air pollution
mixture in the warm season is more dominated by traffic emissions
Air pollution might potentially be more toxic in summer because more
photochemical components are formed (Rager et al., 2011). During sum-
mer, we indeed observed anassociationbetween %5mdC and ozone.Fur-
thermore, seasonal effects may in part be explained by the difference in
time–activity patterns. In summer, more time is spent outdoors, possibly
viouslyshownthat not takingintoaccount time activity patternsoraccu-
(Dons et al., 2011; Dons et al., 2012; Steinle et al., 2013). In our data, we
data on physical activity or time spent outdoors.
Although no significant associations were observed in winter,
estimates for NO2were close to significant. During summer, several
NO2 exposure windows were associated with lower %5mdC. It is
known that NO2is a better surrogate for traffic exposure than PM10or
PM2.5(Health Effects Institute, 2010). Flanders, the investigated region
in our study, is very densely populated and has a dense traffic network.
may be more specifically linked with traffic exposure as opposed to
overall air pollution levels. Further research is needed to elucidate the
role of traffic related exposures in mediating DNA methylation.
We did not detect any significant influences of GIS-based traffic var-
iables on %5mdC. This might be due to the fact that these parameters are
a more robust long-term proxy of air pollution exposure and require
larger populations to detect significant effects. Additionally, since our
study population consisted of couples living in the same house, traffic
parameters were available for only 25 home locations. Because most
couples were sampled on different days, unique modeled exposure
estimations were available for each individual thus obtaining a better
exposure contrast.
There are some limitations to our study. The number of participants
was relatively low. Nevertheless, we were able to detect significant
effects of air pollutants on %5mdC. Another limitation is that we did not
conduct personal dosimetry. It has been shown that the use of wearable
air pollution monitors in combination with time activity patterns and
S. De Prins et al. / Environment International 59 (2013) 418–424
Page 6
air pollution exposure (Dons et al., 2011; Steinle et al., 2013) than fixed
measurement stations. Hence, some exposure misclassification cannot
be excluded. A strength of our study is the use of interpolated pollutant
concentrations at the home addresses of the subjects, which provides
more accurate exposure estimations than the nearest monitoring station
as a proximity measure (Jerrett et al., 2005). Moreover, it was shown by
od as in our study – showed a good correlation with individual exposure,
as assessed by carbon load in airway macrophages (Jacobs et al., 2010a).
Another issue that is often mentioned in epidemiological exposure stud-
ies is the strong association between different pollutants. Interpolated
concentrations are mostly proxy estimations for exposure to a more
complex mixture of air pollutants. It is therefore impossible to relate bio-
markers or health effects to a single pollutant (Health Effects Institute,
2010). As mentioned above, no data on blood cell counts was available.
This is a limitation, since blood cell profiles might explain some of the
epigenetic variation. A final limitation is that we did not include gene
specific DNA methylation analysis. As mentioned before, other authors
genes (Breton et al., 2012; Rossnerova et al., 2013; Tarantini et al., 2009).
Targeted analysis can provide more insight into specific mechanisms and
may help with the biological interpretation.
Whether the DNA methylation changes observed here are relevant
for future health outcomes remains to be established. However, it is
creasemutationaleventsand altergeneexpression(Wilsonetal., 2007).
There is strong evidence that DNA hypomethylation is associated
with severaldiseases likecancer, asthma (Terry etal.,2011)and cardio-
vascular conditions (Cash et al., 2011; Castro et al., 2003; Zaina et al.,
2005). Recently, it was also suggested that the DNA methylome may
play a key role in human aging (Mendelsohn and Larrick, 2013). Addi-
tionally, epigenetic marks, once established, can be permanent and
inheritable during cell division, and might even be transmitted onto
future generations when they occur in germ line cells (Jirtle and
Skinner, 2007). Furthermore, the degree of global DNA methylation
of oxidative stressare knowntocontributeto theinitialstageof numer-
ous diseases such as cancer, cardiovascular disease, diabetes, and
neurological disorders (Valko et al., 2007).
In conclusion, we found that decreased global DNA methylation in
healthy non-elderly adults was associated with exposure to ambient
concentrations of NO2, PM10, PM2.5, and O3at the home address. Most
of these effects were observed for the 5- to 30-day moving average
exposures, at concentrations below the current European limit values.
Competing interests
The authors declare that they have no competing interests.
ment, Nature and Energy Department, Environment & Health unit), the
EU-FP 6 INTARESE (Integrated Assessment of Health Risks of Environ-
mental Stressors in Europe) project and VITO PhD scholarships granted
to Sofie De Prins and Evi Dons. The authors would like to thank Karen
Hollanders (VITO) for contributing to the DNA extractions, the field
workers (Provincial Institute of Hygiene and VITO) and all people that
participated in this study.
Baccarelli A, Wright RO, Bollati V, Tarantini L, Litonjua AA, Suh HH, et al. Rapid DNA
methylation changes after exposure to traffic particles. Am J Respir Crit Care Med
Barath S, Mills NL, Lundback M, Tornqvist H, Lucking AJ, Langrish JP, et al. Impaired
vascular function after exposure to diesel exhaust generated at urban transient
running conditions. Part Fibre Toxicol 2010;23:7–19.
Bell ML. Assessment of the health impacts of particulate matter characteristics. Res Rep
Health Eff Inst 2012:5–38.
Bollati V, Baccarelli A, Hou LF, Bonzini M, Fustinoni S, Cavallo D, et al. Changes in
DNA methylation patterns in subjects exposed to low-dose benzene. Cancer Res
Bollati V, Schwartz J, Wright R, Litonjua A, Tarantini L, Suh H, et al. Decline in genomic
DNA methylation through aging in a cohort of elderly subjects. Mech Ageing Dev
Bos I, De Boever P, Vanparijs J, Pattyn N, Panis LI, Meeusen R. Subclinical effects of aerobic
training in urban environment. Med Sci Sports Exerc 2013;45:439–47.
Breton CV, Salam MT, Wang XH, Byun HM, Siegmund KD, Gilliland FD. Particulate matter,
DNA methylation in nitric oxide synthase, and childhood respiratory disease. Environ
Health Perspect 2012;120:1320–6.
Brunekreef B. Health effects of air pollution observed in cohort studies in Europe. J Expo
Sci Environ Epidemiol 2007;17(Suppl. 2):S61–5.
Bruske I, Hampel R, Socher MM, Ruckerl R, Schneider A, Heinrich J, et al. Impact of ambi-
ent air pollution on the differential white blood cell count in patients with chronic
pulmonary disease. Inhal Toxicol 2010;22:245–52.
Calderon-Garciduenas L, Mora-Tiscareno A, Ontiveros E, Gomez-Garza G, Barragan-Mejia
G, Broadway J, et al. Air pollution, cognitive deficits and brain abnormalities: a pilot
study with children and dogs. Brain Cogn 2008;68:117–27.
Cash HL, McGarvey ST, Houseman EA, Marsit CJ, Hawley NL, Lambert-Messerlian GM,
et al. Cardiovascular disease risk factors and DNA methylation at the LINE-1
repeat region in peripheral blood from Samoan islanders. Epigenetics 2011;6:
Castro R, Rivera I, Struys EA, Jansen EEW, Ravasco P, Camilo ME, et al. Increased homocys-
teine and S-adenosylhomocysteine concentrations and DNA hypomethylation in
vascular disease. Clin Chem 2003;49:1292–6.
Chia N, Wang L, Lu XY, Senut MC, Brenner C, Ruden DM. Hypothesis: environmental
regulation of 5-hydroxymethyl-cytosine by oxidative stress. Epigenetics 2011;6:
Delfino RJ, Staimer N, Vaziri ND. Air pollution and circulating biomarkers of oxidative
stress. Air Qual Atmos Health 2011;4:37–52.
Dons E, PanisLI, Van Poppel M, Theunis J,Willems H, Torfs R, et al. Impactof time–activity
patterns on personal exposure to black carbon. Atmos Environ 2011;45:3594–602.
Dons E, Panis LI, Van Poppel M, Theunis J, Wets G. Personal exposure to Black Carbon in
transport microenvironments. Atmos Environ 2012;55:392–8.
Eeftens M, Tsai MY, Ampe C, Anwander B, Beelen R, Bellander T, et al. Spatial variation of
PM2.5, PM10, PM2.5 absorbance and PMcoarse concentrations between and within
20 European study areas and the relationship with NO2 — results of the ESCAPE
project. Atmos Environ 2012;62:303–17.
HEI (Health Effects Institute). A critical review of the literature on emissions, exposure,
and health effects. Special Report, 17. Boston, Massachusetts: Health Effects Institute;
Hsiung DT, Marsit CJ, Houseman EA, Eddy K, Furniss CS, McClean MD, et al. Global DNA
methylation level in whole blood as a biomarker in head and neck squamous cell
carcinoma. Cancer Epidemiol Biomarkers Prev 2007;16:108–14.
Jacobs L, Emmerechts J, Mathieu C, Hoylaerts MF, Fierens F, Hoet PH, et al. Air
pollution-related prothrombotic changes in persons with diabetes. Environ Health
Perspect 2010a;118:191–6.
Jacobs L, Nawrot TS, de Geus B, Meeusen R, Degraeuwe B, Bernard A, et al. Subclinical
responses in healthy cyclists briefly exposed to traffic-related air pollution: an inter-
vention study. Environ Health 2010b;9:64.
Janssen S, Dumont G, Fierens F, Mensink C. Spatial interpolation of air pollution measure-
ments using CORINE land cover data. Atmos Environ 2008;42:4884–903.
Jerrett M, Arain A, Kanaroglou P, Beckerman B, Potoglou D, Sahsuvaroglu T, et al. A review
and evaluation of intraurban air pollution exposure models. J Expo Anal Environ
Epidemiol 2005;15:185–204.
Ji H, Hershey GK. Genetic and epigenetic influence on the response to environmental par-
ticulate matter. J Allergy Clin Immunol 2012;129:33–41.
Jirtle RL, Skinner MK. Environmental epigenomics and disease susceptibility. Nat Rev
Genet 2007;8:253–62.
Kelly FJ, Fussell JC. Air pollution and airway disease. Clin Exp Allergy 2011;41:1059–71.
Kuo KC, Mccune RA, Gehrke CW, Midgett R, Ehrlich M. Quantitative reversed-phase
high-performance liquid-chromatographic determination of major and modified
deoxyribonucleosides in DNA. Nucleic Acids Res 1980;8:4763–76.
Lee DH, Jacobs DR, Porta M. Hypothesis: a unifying mechanism for nutrition and
chemicals as lifelong modulators of DNA hypomethylation. Environ Health Perspect
Limon-Pacheco J, Gonsebatt ME. The role of antioxidants and antioxidant-related enzymes
in protective responses to environmentally induced oxidative stress. Mutat Res
Madrigano J, Baccarelli A, Mittleman MA, Wright RO, Sparrow D, Vokonas PS, et al.
Prolonged exposure to particulate pollution, genes associated with glutathione path-
ways, and DNA methylation in a cohort of older men. Environ Health Perspect
Marshall JD, Brauer M, Frank LD. Healthy neighborhoods: walkability and air pollution.
Environ Health Perspect 2009;117:1752–9.
Mendelsohn AR, Larrick JW. The DNA methylome as a biomarker for epigenetic instability
and human aging. Rejuvenation Res 2013;16:74–7.
Mills NL, Tornqvist H, Robinson SD, Gonzalez M, Darnley K, MacNee W, et al. Diesel ex-
haust inhalation causes vascular dysfunction and impaired endogenous fibrinolysis.
Circulation 2005;112:3930–6.
S. De Prins et al. / Environment International 59 (2013) 418–424
Page 7
Nawrot TS, Torfs R, Fierens F, De Henauw S, Hoet PH, Van Kersschaever G, et al. Stronger
associations between daily mortality and fine particulate air pollution in summer
than in winter: evidence from a heavily polluted region in western Europe.
J Epidemiol Community Health 2007;61:146–9.
Peluso M, Bollati V, Munnia A, Srivatanakul P, Jedpiyawongse A, Sangrajrang S, et al. DNA
methylation differences in exposed workers and nearby residents of the Ma Ta Phut
industrial estate, Rayong, Thailand. Int J Epidemiol 2012;41:1753–60.
Pope CA, Dockery DW. Health effects of fine particulate air pollution: lines that connect.
J Air Waste Manage Assoc 2006;56:709–42.
Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol
of PM10and PM2.5in selected European cities. Atmos Environ 2004;38:6547–55.
Rager JE, Lichtveld K, Ebersviller S, Smeester L, Jaspers I, Sexton KG, et al. A toxicogenomic
comparison of primary and photochemically altered air pollutant mixtures. Environ
Health Perspect 2011;119:1583–9.
Ricciardolo FLM, Sterk PJ, Gaston B, Folkerts G. Nitric oxide in health and disease of the
respiratory system. Physiol Rev 2004;84:731–65.
Rich DQ, Kipen HM, Huang W, Wang GF, Wang YD, Zhu P, et al. Association between
changes in air pollution levels during the Beijing Olympics and biomarkers of inflam-
mation and thrombosis in healthy young adults. JAMA 2012;307:2068–78.
Romieu I, Castro-Giner F, Kunzli N, Sunyer J. Air pollution, oxidative stress and dietary
supplementation: a review. Eur Respir J 2008;31:179–96.
RossnerovaA,TulupovaE,Tabashidze N, SchmuczerovaJ,DostalM,Rossner JrP,etal.Fac-
tors affecting the 27 K DNA methylation pattern in asthmatic and healthy children
from locations with various environments. Mutat Res 2013;741–742:18–26.
Rozhon W, Baubec T, Mayerhofer J, Scheid OM, Jonak C. Rapid quantification of global
DNA methylation by isocratic cation exchange high-performance liquid chromatog-
raphy. Anal Biochem 2008;375:354–60.
Rundell KW, Caviston R, Hollenbach AM, Murphy K. Vehicular air pollution, playgrounds,
and youth athletic fields. Inhal Toxicol 2006;18:541–7.
Steinle S, Reis S, Sabel CE. Quantifying human exposure to air pollution — moving from
static monitoring to spatio-temporally resolved personal exposure assessment.
Sci Total Environ 2013;443C:184–93.
Tang WY, Levin L, Talaska G, Cheung YY, Herbstman J, Tang D, et al. Maternal exposure to
polycyclic aromatic hydrocarbons and 5′-CpG methylation of interferon-gamma in
cord white blood cells. Environ Health Perspect 2012;120:1195–200.
Tarantini L, Bonzini M, Apostoli P, Pegoraro V, Bollati V, Marinelli B, et al. Effects of partic-
ulate matter on genomic DNA methylation content and iNOS promoter methylation.
Environ Health Perspect 2009;117:217–22.
Terry MB, Delgado-Cruzata L, Vin-Raviv N, Wu HC, Santella RM. DNA methylation in
white blood cells — association with risk factors in epidemiologic studies. Epigenetics
Traversi D, Schiliro T, Degan R, Pignata C, Alessandria L, Gilli G. Involvement of
nitro-compounds in the mutagenicity of urban PM2.5and PM10in Turin. Mutat Res
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:
Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxi-
dants in normal physiological functions and human disease. Int J Biochem Cell Biol
Wilson AS, Power BE, Molloy PL. DNA hypomethylation and human diseases. Biochim
Biophys Acta 2007;1775:138–62.
Wu HC, Delgado-Cruzata L, Flom JD, Kappil M, Ferris JS, Liao YY, et al. Global methylation
profiles in DNA from different blood cell types. Epigenetics 2011;6:76–85.
Zaina S, Lindholm MW, Lund G. Nutrition and aberrant DNA methylation patterns in
atherosclerosis: more than just hyperhomocysteinemia? J Nutr 2005;135:5–8.
Zhu ZZ, Hou LF, Bollati V, Tarantini L, Marinelli B, Cantone L, et al. Predictors of global
methylation levels in blood DNA of healthy subjects: a combined analysis. Int J
Epidemiol 2012;41:126–39.
S. De Prins et al. / Environment International 59 (2013) 418–424


text:
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A collection of news and information related to Milk (movie) published by this site and its partners.
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Waste and Recycling Vehicles Companies in St. Kitts & Nevis
Category: Waste & Recycling ×
Subcategory: Waste and Recycling Vehicles ×
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SSI Shredding Systems, Inc.
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SSI Shredding Systems is a leading manufacturer of industrial shredders and size reduction systems. SSI custom designs and manufactures high quality 1-shaft, 2-shaft, and 4-shaft rotary shear shredders and primary waste reducers as well as pre-load transfer station and metals compactors. Our highly qualified staff provide you with complete system design and engineering ...
Surface Active Solutions Ltd
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The Canadian Press - ONLINE EDITION
Comparing the Stanley Cup title won by the Blackhawks, and the NBA title won by the Heat
MIAMI - Comparing the championship runs of the NBA's Miami Heat and the NHL's Chicago Blackhawks:
UNBEATABLE: Both teams had long runs of success on their way to the respective titles, though Miami's was actually unblemished while Chicago's was a bit less shiny. The Heat put together the second-longest winning streak in NBA history during the regular season, 27 consecutive games. The Blackhawks opened the season with a 24-game streak of getting at least one point out of every game — and while that encompassed exactly half of the lockout-shortened season, their longest actual winning streak in that span was just 11 games, with two of those coming in shootouts. Chicago technically lost three times in that 24-game run, all in shootouts, and two of them consecutively.
TITLE TOWNS: Welcome to the Heat and Blackhawk eras in their respective leagues. For Miami, this year's NBA championship was its third in eight years and its second straight. Chicago won the Stanley Cup for the second time in four seasons. Before the 2010 title, the last time the Blackhawks hoisted hockey's biggest prize was 1961. And naysayers could try to take some of the lustre off crowns for both cities, since the Heat title last year and the Blackhawks' crown this season both came in lockout-shortened seasons.
STAR POWER: Maybe something about the winds off Lake Erie helps create superstars. Heat star LeBron James hails from Akron, Ohio, maybe 40 miles or so south of the shores of the great lake — definitely close enough to get some good lake-effect storms in the winter. Blackhawks star Patrick Kane comes from Buffalo, N.Y., situated along the lake's eastern edge. James won the NBA Finals MVP award; Kane was this year's Conn Smythe Award winner, presented to the best player in the entire NHL post-season.
TIES THAT BIND: Florida Panthers general manager Dale Tallon — whose club plays about a half-hour north of the arena that the Heat call home — was the architect of the Blackhawks team that won the Cup in 2010, and some of his moves paid off for Chicago again during this most recent title run. Meanwhile, Heat guard Dwyane Wade hails from Chicago, and remember that the Bulls tried unsuccessfully to sign him, James and Chris Bosh in the summer of 2010, just a few days after the Blackhawks hoisted the Cup.
BOSTON GIVEAWAYS: Both of these post-seasons will likely be remembered for what Boston lost. Last summer, it was Ray Allen, who turned down more years and money from the Celtics and decided to sign with the Heat. And Allen went on to hit what will go down as the biggest shot in Heat history, the 3-pointer with 5.2 seconds left in regulation of Game 6 that forced overtime and denied the San Antonio Spurs what would have been their fifth NBA title. And during the Stanley Cup final, the Bruins were up by a goal with about a minute remaining in Game 6 — then surrendered two lightning-fast goals to not only lose, but lose in regulation even, a stunning collapse that brought the NHL season to a quick and dramatic close.
LEBRON WATCHED: Not only did LeBron James send a congratulatory tweet to Patrick Kane and the Blackhawks for winning the Stanley Cup, he got into some trouble with his kids over the ending of Chicago's title-clincher. James was watching a movie with his sons when he checked Twitter and got word of Chicago's late rally in Boston. The movie got put on pause so James could see the hockey highlights, and his kids weren't exactly thrilled. Said James, on the similarities between the clubs: "We both had an amazing streak. We both finished first in our conferences. We both were down in series. There's so many similarities to both teams, and to win it, like I said, when I saw them hoisting that trophy up, that Stanley Cup trophy, I was like, 'I know exactly what that feeling is all about.'"
BRING ON BASEBALL (SADLY): Heat fans, Blackhawks fans, take heart. You had a good winter. Entering Tuesday, the Miami Marlins, Chicago Cubs and Chicago White Sox all had one thing in common. They were all in last place. So after the Blackhawks hold their celebration, looks like Miami and Chicago will be parade-free for the next few months.
LUCKY NUMBERS: Heat record in this post-season? 16-7. Blackhawks record in this post-season? 16-7. And with that, the winter season is complete.
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The recent stock market volatility means investors are confused. One article I read stated it nicely:
"The stock market's volatility stems from confusion as to whether the economy is too hot or too cold—and it is worried about the Fed response."
–Bill DeShurko, CFP
Add to this confusion the most recent Chicago Fed report that shows that the economy picked up in September.
"Led by improvements in production-related indicators, the Chicago Fed National Activity Index (CFNAI) rose to +0.47 in September from –0.25 in August. Three of the four broad categories of indicators that make up the index made positive contributions to the index in September, and three of the four categories increased from August."
So what's an investor to do? On the one hand, there is the market drop in October with the return of volatility. And on the other hand, the economy seems to be doing fine in the recent months.
My answer: stay true to your investment approach. Do not change your asset allocation in response to short-term market fluctuations because if there is one thing we know for sure, the future is un-knowable.
Guessing which way the markets are going to move today or next week or next month is a loser's game. Better to set a course and then stay with it during turbulent times. The ride may be rough some times, but you're more likely to reach your destination by staying on course rather than veering off course when the wind rises and the seas broil. Stay the course my friends.


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By WR_LDN at 14:44 on 29/01/13
2 comments... add your comments below
...too old for Dalston, and too young for Wetherspoons?...
A chance to dance on Friday 1st of February 2013
@ White Rabbit 125 Stoke Newington Church Street, London
The frolics start at 8pm
£3 entry and it all goes to a local charity.
Date: Friday, February 01, 2013


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Xbox 360
This page contains How To Train Your Dragon Unlockables for Xbox 360 called "Normal Achievements" and has been posted or updated on Feb 12, 2010 by Tibby. Let us know what you think of this Unlockable by thumbs voting.
- Developer: Etranges Libellules
- Publisher: Activision
- Genre: Action General
- Release Date: Mar 23, 2010
- ESRB: Everyone 10+
Normal Achievements
Complete one of the following achievements to collect their Xbox Gamerscore points:
|How to Unlock:
|All Stars (90 GP)
|Have 4 level 25 dragons.
|All for One (15 GP)
|Win a fight with each default and Legendary dragons in 1 Player Arcade mode.
|Ally or Enemy? (10 GP)
|Import at least one dragon in Arcade mode.
|Belong to the Legend (30 GP)
|Unlock all legendary dragons.
|Brainy (30 GP)
|Perform 6 sequences in a row in the Memory Torch challenge on Expert difficulty.
|Brave (15 GP)
|Win the fourth Tournament always fighting the highest rated opponent.
|Commander (30 GP)
|Win 10 fights in a row in 1 Player Arcade mode on Hard difficulty.
|Conqueror (15 GP)
|Win 100 fights in either 1 Player or 2 Players Arcade mode.
|Cooking Master (15 GP)
|Buy all recipes from the shop.
|Courageous (10 GP)
|Win against the leader of the second Tournament.
|Daring (10 GP)
|Win against the leader of the third Tournament.
|Dragon Doctor (15 GP)
|Have 4 dragons in a great shape in Story mode.
|Duelist (15 GP)
|Win a wild fight.
|Enlightened (15 GP)
|Gather 100 rare ingredients.
|Exceptional Tamer (15 GP)
|Win 20 fights in 1 Player Arcade mode with a team of custom dragons from the Story mode.
|Extreme Competitor (15 GP)
|Earn a gold medal in any challenge on Expert difficulty.
|Fearless (10 GP)
|Win against the leader of the fourth Tournament.
|Final Evolution (30 GP)
|Have a level 25 dragon.
|First Step (5 GP)
|Win a fight in either Story mode or 1 Player Arcade mode.
|Game Over! (30 GP)
|Win all the medals for all difficulty levels in each challenge.
|Glory to Astrid (15 GP)
|Finish the Story mode with Astrid.
|Glory to Hiccup (15 GP)
|Finish the Story mode with Hiccup.
|Great Speaker (15 GP)
|Talk to every person on the island in Story mode.
|In Feathers (15 GP)
|Catch 200 chickens.
|In Great Shape (15 GP)
|Win the second Tournament without taking care of your dragons during it.
|Invulnerable (90 GP)
|Win the final Championship with none of your dragons being KO.
|Legendary Fighter (30 GP)
|Win all wild fights.
|More Wild Meat (15 GP)
|Catch 200 sheep or boars.
|New Champion (10 GP)
|Win against the leader of the first Tournament.
|Outstanding Competitor (10 GP)
|Earn a gold medal in any challenge on Normal difficulty.
|Perfect (15 GP)
|Win 50 fights without being injured in 1 Player Arcade mode.
|Perfectionist (15 GP)
|Have a dragon with 20 points in a characteristic in Story mode.
|Precise (30 GP)
|Recreate 3 dragons in a row without mistakes in the Puzzle Dragon challenge on Expert difficulty.
|Rich Harvest (15 GP)
|Gather 1,000 common ingredients.
|Shepherd Dragon (30 GP)
|Bring back 5 brown sheep in a row in the Flying Shepherd challenge on Expert difficulty.
|Shopping Master (30 GP)
|Spend 10,000 gold at the shop.
|Success (15 GP)
|Earn a gold medal in each challenge.
|They Are Alive! (15 GP)
|Win 20 fights in 1 Player Arcade mode with a team of dragons created with the Dragon Editor.
|To Hit the Bull's Eyes (30 GP)
|Clear 5 checkpoint rings in a row in the Looping Race challenge on Expert difficulty.
|Ultimate Customization (15 GP)
|Unlock all customizations of a dragon family.
|Unstoppable (15 GP)
|Win the first Tournament without being defeated.
|Valiant (15 GP)
|Win the third Tournament always fighting the lowest rated opponent.
|Viking Gold (30 GP)
|Have 10,000 gold in Story mode.
|Viking Inhabitant (30 GP)
|Play more than 20 hours in Story or Arcade mode (primary profile only).
|Weapon Master (15 GP)
|Make a dragon finish all trainings.
|What an Artist! (30 GP)
|Sculpt 10 ice blocks in a row in the Ice Sculpting challenge on Expert difficulty.
|Young Competitor (5 GP)
|Earn a gold medal in any challenge on Novice difficulty.
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In this note, we examine the problem of finding good state assignments when mixed memory type realizations (i.e., including mixtures of delay and trigger flip-flop memories) are permitted. Specifically, we describe situations that take advantage of this extra degree of design freedom, and we indicate how to modify two existing algorithms to determine such mixed realizations systematically.
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Bill Resnick talks with three members of Food Not Bombs, a world-wide organization that provides free food to the hungry and the foot-loose. He also talks with two people from the Iraq Body Count Project whose 125,000 flags are flying near Portland State, each flag representing 5 Iraqis who have died in the war.
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Add some variety to your fitness routine with these upper and lower leg exercises from The American Council on Exercise. We have a large selection of leg exercises that will will help you strengthen and build muscle throughout your legs including your hamstrings, thighs, shin and calf muscles. Start taking advantage of this free information today! If you are looking for full workout routines that incorporate these leg exercises, check out our Workouts section.
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Satisfied with my order
Reviewed By Donnie A (Rayville, LA) —11/13/2014 2:50 PM
I am so satisfied with my CRE Splash Guards, that I am about to place another order and order some for my front wheels.
Reviewed By Kenneth B (Soldotna, AK) Reviewed for a 2002 Ford F-150 —11/9/2014 8:57 PM
Had to call via phone to get a deal, still spendy for flaps. Shipping took almost 2 weeks. Purchased two pairs for my truck. Was surprise that the items were made of plastic instead of rubber. Hope a rock doesn't break or crack them, especially in below zero weather. Fit was good but you do have to take off the rear tires to mount. Color is good.


text:
Rick Hanson PhD
Apr 09, 2014
Feel Whole
When I look back on mistakes I've made – like dumping my anger on someone, making assumptions in haste, partying too much, losing my nerve, being afraid to speak from my heart – in all cases a part of me had taken over. You know what I mean. The parts of us that have a partial view, are driven by one aim, clamp down on other parts, really want to have a particular experience or to eat/drink/smoke a particular molecule, yammer away critically, or hold onto resentments toward others.
The mega part – the big boss – is of course the inner executive, the decision-maker and driver …
Wildmind Meditation News
Apr 01, 2014
Treating chronic pain with meditation
Brian Steiner, The Atlantic: In some cases, the holistic practice could replace narcotics. Integrating meditation into regular treatment could significantly cut healthcare costs.
Sarah Kehoe tried Aleve for her back pain. She tried stretching. She tried yoga. She tried forgetting about it. She tried pain patches. She tried acupuncture. A shot of painkillers into her back. Prescription anti-inflammatory pain patches. Opiates. Surgery. Physical therapy. Heat and compresses. Ignoring it again. Steroids. More opiates. Acupuncture again. She couldn't sit, stand up straight, lie down on her back. She was weak, had lost muscle tone. She fainted on the subway. Sarah Kehoe, an otherwise healthy …
Wildmind Meditation News
Mar 20, 2014
Mindfulness: Is it a fad or a powerful life-changing coping skill? A look at the science.
Amanda Mascarelli, The Washington Post: Imagine this scenario: You come home from work tired and frazzled, and your little kids are running wild. Perhaps this doesn't require much imagination. People in such situations might find solace in a popular meditative practice called mindfulness.
With mindfulness, you train your mind to focus on the present and respond with reason before emotion. It's about taking a pause and guiding yourself to become "aware enough in the moment so that before you react, you're aware of how you're responding to a situation," says Ronald Epstein, a professor of family medicine at the University of Rochester Medical …
Rick Hanson PhD
Mar 20, 2014
Developing a "Buddha Brain" through gratitude
A "buddha brain" is one that knows how to be deeply happy, loving, and wise. We develop ourselves in this way by cultivating wholesome qualities and uprooting unwholesome ones. In a sense, we plant flowers and pull weeds in the garden of the mind – which means that we are gradually changing the brain for the better.
Gratitude is a powerful tool in this "garden" since what you rest your attention upon is what will shape your brain the most. That's because "neurons that fire together, wire together." Gratitude shifts your attention away from resentment, regret, and guilt – and therefore stops you from building up the neural substrates of these …
Wildmind Meditation News
Feb 28, 2014
5 ways meditation gives your brain a boost
Dan Harris, ABC News: I never in a million years thought I'd be the type of person who meditates. I've had an aversion to all things airy-fairy since age five, when my parents – recovering hippies – sent me to a yoga class for kids. The teacher, who disapproved of the jeans I was wearing, made me strip down and do sun salutations in my tighty-whities.
But then, a few years ago, I heard about an explosion of scientific research suggesting that meditation has an extraordinary range of health benefits. In particular, I found the neuroscience compelling. Studies say you can sculpt your …
Wildmind Meditation News
Feb 26, 2014
Should we be mindful of mindfulness?
David Derbyshire,
The Observer: It has been prescribed by the NHS for depression since 2004 but recently mindfulness has spawned a whole industry of evening classes and smartphone apps. What is the evidence that the practice – part meditation, part CBT – works?
At just after 6.15pm in a brightly lit conference room in Oxford, 22 grown men and women are lying on the floor trying hard to focus on their left knee. From across the room a lilting, calm voice has already invited the group to explore their feet and ankles with "gentle curiosity" and is heading up through the body. "When your mind …
Wildmind Meditation News
Feb 11, 2014
Meditation helps pinpoint neurological differences between two types of love
Bill Hathaway, Yale News: These findings won't appear on any Hallmark card, but romantic love tends to activate the same reward areas of the brain as cocaine, research has shown.
Now Yale School of Medicine researchers studying meditators have found that a more selfless variety of love — a deep and genuine wish for the happiness of others without expectation of reward — actually turns off the same reward areas that light up when lovers see each other.
"When we truly, selflessly wish for the well-being of others, we're not getting that same rush of excitement that comes with, say, a tweet from our romantic love interest, because it's not about us …
Wildmind Meditation News
Aug 30, 2013
What happens to the brain when you meditate (and how it benefits you)
Bell Beth Cooper, Lifehacker: Ever since my dad tried to convince me to meditate when I was about 12, I've been fairly skeptical of this practice. It always seemed so vague and hard to understand that I just decided it wasn't for me. More recently, I've actually found how simple (not easy, but simple) meditation can be and what huge benefit it can have for my day to day happiness.
As an adult, I first started my meditation practice with just two minutes per day. Two minutes! I got that idea from Leo Babauta's Zen Habits blog, where he points out how starting with …
Rick Hanson PhD
Apr 10, 2013
Hug the monkey
Your brain evolved in three stages (to simplify a complex process):
Reptile – Brainstem, focused on avoiding harm
Mammal – Limbic system, focused on approaching rewards
Primate – Cortex, focused on attaching to "us"
The first post in this series – pet the lizard – was about how to soothe the most ancient structures of the brain, the ones that manage the first emotion of all: fear. The next one – feed the mouse – addressed how to help early mammalian neural systems feel rewarded and fulfilled. This JOT is about weaving the sense of being included and loved into the primate …
Jan 16, 2013
The neuroscience of mindfulness
We live in a world filled with material wealth, live longer and healthier lives, and yet anxiety, stress, unhappiness, and depression have never been more common. What are the driving forces behind these interlinked global epidemics? Professor Mark Williams examines the neuroscience of mindfulness in this short video.


text:
Slapp Symphony - Ice Palace
Artist: Slapp Symphony
Producer: Slapp Symphony
Album: Drown 'em in Beats; Save 'em with Rhymes
Avg Rating: 3.7 ( 10 votes )
If there's a narcotic that can make crack cocaine look innocuous, methamphetamine is it—and few locales have been hit as hard by the drug as Hawaii, where treatment facilities can are powerless to keep pace with ever-rising abuse rates. Here to raise consciousness regarding the dire situation in their local streets are Salt Lake, Hi. beat crew Slapp Symphony and fellow Aloha State native Slo-Mo with new single Ice Palace. Led off with a sampled news report featuring some alarming statistics, Let-T-Let and Westbrook's chilly, piano-led beat sets an ominous mood as the Fortilive rhymesayer describes speed's effects on his community in general, and his friends and classmates in particular: "My coastline lookin' like a ghetto out of the South, old classmates' teeth fallin' out of they mouth / B*tches that I used to dig smokin' ice in front of they kids, pregnant with another in the crib." Sobering, incisive and defiantly real, this downbeat jam is just one of many quality tunes set for inclusion on Slapp Symphony's Drown 'em in Beats; Save 'em with Rhymes LP, released on Black Friday and available now online.
Written by richard on 12/8/11
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Slapp Symphony Music Group
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Animals at the EDGE
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saving the world's rarest creatures
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EDGE (Evolutionarily Distinct and Globally Endangered) species are evolutionarily unique, which means if they don't survive, there will be no similar species left on Earth. But all hope is not lost. EDGE researchers are working hard to locate, create awareness of, and ensure a safe future for the... Read More »
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Tap Paradise Cove Answers for iPhone - iPodHome / iPhone - iPod /
Game Questions & Answers (Q&A) service lets you ask questions about video games for game consoles or PC games. So ask your Tap Paradise Cove question for iPhone - iPod and get answers from real gamers or answer questions and share your insights and experience with the rest of the gaming community.
- Developer: Pocket Gems, Inc.
- Publisher: Pocket Gems, Inc.
- Genre: Simulation General
- Release Date: Mar 7, 2012
- ESRB: Not Set
Question & Answers
Why can't I buy Cat Ebony
Why friends were able to buy Ebony with money and I'm only given the choice of buying it with rubies?
Answer from: Senne68
I have THE Same problem. Maybe it's because I got the quest again after it dissapeared. I was not finishend in time, the quest dissapeared and came back after a few days. I thought to finish it this time but I won't buy rubies.
Answer from: Ket
I have the same problem.. I mean they could at least set the ruby-price low.. Did u find à salution?
Answer from: Mars
Thank you two for the replies. No I haven't found any solutions yet and time is running out.
Unanswered Questions
From: Moo
Posted on: 10-20-2014
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Posted on: 09-20-2014
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Tap Paradise Cove - Expand your colony Trailer
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