DOI: 10.1161/CIRCULATIONAHA.115.018802
Traffic-Related Air Pollution, Blood Pressure, and Adaptive Response of
Mitochondrial Abundance
Running title: Zhong et al.; Air Pollution, Mitochondria, and Blood Pressure
Jia Zhong, MS1*; Akin Cayir, PhD1,2*; Letizia Trevisi, PhD1; Marco Sanchez-Guerra, PhD1;
Xinyi Lin, PhD3; Cheng Peng, MS1; Marie-Abèle Bind, ScD1,4; Diddier Prada, MD, PhD1,5;
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Hannah Laue, MS1; Kasey J.M. Brennan, BS1; Alexandra Dereix, BS1; David Sparrow, DSc6;
Pantel Vokonas, MD6; Joel Schwartz, PhD1; Andrea A. Baccarelli, MD, PhD, FAHA1
1
2
Dept of Environmental Health, Harvard T.H. Chan School of Public Health, Boston,
Bost
Bo
ston
st
onn, MA;
MA;
Vocational Health College, Canakkale Onsekiz Mart University, Çanakkale, Turkey; 3Singapore
In
Institute
nst
stit
itut
it
utee fo
ut
forr Cl
Clinical
lin
inic
i al Sciences, Singapore; 4De
Dept
ept ooff Statistics, Harvardd Un
U
University,
iversity, Cambridge,
MA;; 5Un
M
MA
Unidad
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Inve
Investigación
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Normative
ormativee A
Aging
ging
ng Stu
Study,
udy
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Veterans
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Affairs
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Boston
osto
on Heal
Healthcare
allth
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System
steem
and
an
d the
th
he Dept
Dept of
of Medicine,
Medi
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d ci
di
cine
ne, Boston
ne
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on University
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ve
sity
si
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ch
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oo
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ic
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Boston,
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n, M
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*contributed
*con
*c
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on
trib
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ibut
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uted
ted eequally
qual
qu
ally
al
ly
Address for Correspondence:
Andrea A. Baccarelli, MD, PhD, FAHA
Department of Environmental Health
Harvard T.H. Chan School of Public Health
Bldg 1, Rm G-5, 665 Huntington Ave
Boston, MA 02115
Tel: 617-432-2095
Fax: 617-432-6913
E-mail: [email protected]
Journal Subject Terms: Etiology; Epidemiology
1
DOI: 10.1161/CIRCULATIONAHA.115.018802
Abstract
Background—Exposure to black carbon (BC), a tracer of vehicular-traffic-pollution, is
associated with increased blood pressure (BP). Identifying biological factors that attenuate BC
effects on BP can inform prevention. We evaluated the role of mitochondrial abundance, an
adaptive mechanism compensating for cellular-redox-imbalance, in the BC-BP relationship.
Methods and Result—At one or more visits among 675 older men from the Normative Aging
Study (observations=1,252), we assessed daily BP and ambient BC levels from a stationary
monitor. To determine blood mitochondrial abundance, we used whole blood to analyze
mitochondrial-to-nuclear DNA ratio (mtDNA/nDNA) using quantitative polymerase-chainDownloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
reaction. Every standard deviation (SD) increase in 28-day BC moving average (MA) was
associated with 1.97 mm Hg (95%CI, 1.23-2.72; P<0.0001) and 3.46 mm Hg (95%CI, 2.06-4.87;
P<0.0001) higher diastolic and systolic (SBP) BP, respectively. Positive BC-BP aassociations
ssoc
ss
ocia
oc
iaati
tion
onss
on
existed throughout all time windows. BC MAs (5-day to 28-day) were associated wi
with
th iincreased
ncreased
d
mtDNA/nDNA; every SD increase in 28-day BC MA was associated with 0.12 SD (95%CI,
0.03
03-0
03
-0.20
20;
0; P=0.007)
P=0.0007
07) higher mtDNA/nDNA. High
Hig
gh mtDNA/nDNA
mtDNA/nDNA
A signi
ifi
ficcantly attenuated the
0.03-0.20;
significantly
B
C--SBP associa
iattioon tthroughout
ia
hrou
hr
ough
ou
ghou
gh
outt al
ou
aalll ti
time
me win
inddows
in
do s . T
hee esti
eestimated
sti
tim
matedd ef
mate
effe
fect
fe
ct ooff 28
8-d
-day
yB
C MA oon
n
BC-SBP
association
windows.
The
effect
28-day
BC
SB
P was 1.95
5-fooldd larger
larrgeer for
orr iindividuals
n ivid
nd
id
duaalss att tthe
hee low
west mt
mtDN
DNA
DN
A/nD
nD
DNA qquartile
uarttil
i e midp
dp
pointt (4
4.68
8
SBP
1.95-fold
lowest
mtDNA/nDNA
midpoint
(4.68
mm H
g; 995%CI,
5%CII, 33.03-6.33;
5%CI
.03
03-6
6.33;
3 P
<0.00
0001), compared
comp
co
mpar
mp
a ed to
to the
th
he top
top quartile
quarti
tile
ti
le midpoint
midpo
idpoointt (2
.440 mm H
g;
g;
Hg;
P<0.0001),
(2.40
Hg;
95%CI
81-3 99; P=0.003).
P=0 003)
95%CI, 00.81-3.99;
Conclusions—In older adults, short- to moderate-term ambient BC levels were associated with
increased BP and blood mitochondrial abundance. Our findings indicate that increased blood
mitochondrial abundance is a compensatory response and attenuates the cardiac effects of BC.
Key words: epidemiology; oxidative stress; blood pressure; mitochondria; air pollution
2
DOI: 10.1161/CIRCULATIONAHA.115.018802
The American Heart Association (AHA) has identified particulate matter (PM) pollution as a
primary contributor to cardiovascular morbidity and mortality, which is estimated to account for
~3.7 million premature deaths per year worldwide.1, 2To facilitate cost-efficient abatement,3, 4
recent studies suggested substantial public health benefits could be realized by implementing
control for traffic-related PM pollution.5, 6 Black carbon (BC) – a combustion byproduct that
serves as a proxy for all traffic-related particles – has been associated with adverse
cardiovascular events, particularly due to rapid effects within days after exposure peaks.2, 7, 8In
particular, short- to moderate-term (days to weeks) increases in ambient BC levels are associated
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
with subsequent increased blood pressure (BP), one of the primary intermediate outcomes
Determining
rmi
mini
ning
ni
ng tthe
he
contributing to acute air pollution-related cardiovascular disease (CVD).2, 9 Deter
impact
mpact of ambient BC on BP among vulnerable populations – such as older adults – is key to
developing
deve
elo
lopi
ping
pi
ng targeted
tar
a geete
tedd risk-reduction strategies.
Major ef
effo
efforts
forrtss ha
have
ve bbeen
eenn made
ee
made iin
n identifying
id
den
ntiifying
ngg thee mechanistic
mec
echa
h niist
stic
ic pathways
pat
athhway
ys linking
link
li
nkin
nk
ing trafficin
trafffi
tr
ficcrelated
decades.
Oxidative
has
emerged
ela
l te
ted PM aand
n iincreased
nd
ncreaaseed BP
nc
P ov
over tthe
h ppast
he
ast fe
few
w de
ecaadees. Ox
xida
dati
da
tive
ve sstress
t esss ha
tr
as em
emerge
gedd aass kkey
ge
ey
machinery
mach
ma
chin
ch
iner
in
ery unde
er
un
underlying
nde
derl
rlyi
rl
ying
ing B
BC-associated
C-as
asso
as
soci
so
ciat
ci
ated
at
ed ccardiovascular
ardi
ar
diov
di
ovas
ascu
as
cula
larr ev
la
eevents,
ven
ents
en
ts, du
ts
ddue
uee to tthe
he ppro-inflammatory
ro-inf
inf
nfla
lamm
la
mmat
mm
ator
at
ory an
or
andd
pro-oxidative properties of BC.5 Systemic oxidative stress – which reflects a disturbance in the
redox balance of circulating blood cells – can lead to cellular functional and pathologic changes,
and play a crucial role in different types of CVD.10-12 In response to oxidative stress, cells can
normally recover their redox balance by utilizing the endogenous antioxidant system.13, 14
However, an intensive intracellular reactive oxygen species (ROS) challenge can overwhelm the
antioxidant response and, in compensation, trigger mitochondrial over-production – an important
adaptive response following environmental challenge to reduce cellular oxidative stress.15, 16
Cellular mitochondrial mass is controlled through biogenesis and degradation, but
3
DOI: 10.1161/CIRCULATIONAHA.115.018802
external oxidative stressors can up-regulate the transcriptional/replication machinery of the
mitochondrial genome, resulting in increased mitochondrial abundance. Mitochondrial
abundance can be detected as an increased ratio of mitochondrial DNA to nuclear DNA copy
number (mtDNA/nDNA).15, 17 Elevated mtDNA/nDNA has been shown in peripheral blood cells
following oxidative stress and inflammation.18-20 However, to date, studies on the role of
mitochondrial abundance on pollution-related cardiovascular pathogenesis are absent.
Understanding how ambient BC might alter blood mitochondrial abundance and how this
alteration might impact BC-elicited cardiovascular effects in an aging population can aid the
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
development of preventative strategies.
In this study, we hypothesized that blood mitochondrial abundance is an adaptive
ada
dapt
ptiv
pt
ivee
iv
esponse following ambient BC exposure, which might buffer the impact of ambient BC on BP.
response
We uutilized
tili
ti
lize
li
zedd the
ze
th Normative
No
invessti
tiggat
a e the relationship ooff ambient BC levels at
Aging Study to investigate
m
multiple
ulltiple time windows
wind
wi
ndow
ow
ws (2-day
(2-d
(2
-day
-d
ay to
to 28-day)
28-d
28
-daay)
-d
ay with
wi h BP.
BP
P. We
We then
the
henn examined
exam
amin
am
ined
in
ed the
the
he association
ass
sssoc
ocia
iati
ia
tion
ti
o ooff sh
on
shor
shortortor
t- to
mode
mo
moderate-term
derate-ter
de
erm
er
m ambient
am
ambien
m nt BC levels
lev
vel
e s with
w th
wi
h blood
bloood
od mitochondrial
mitoochon
on
ndriaal abundance,
abun
ab
un
ndanc
n e, aand
nd
d ex
explored
xp re
xplore
redd its rrole
ole ass
an eeffect
ffec
ff
ectt mo
ec
modi
modifier
difi
di
fier
fi
er ffor
or tthe
he rrelationship
elat
el
atio
at
ions
io
nshi
ns
hipp be
hi
betw
between
twee
eenn am
ee
ambi
ambient
bien
bi
entt BC aand
en
nd B
BP.
P. IIn
n ad
addi
addition,
diti
di
tion
ti
on, wee eexplored
on
xp
xplo
plo
lore
redd th
re
thee
correlation between blood mitochondrial abundance and plasma inflammatory markers including
interleukin 6 (IL6), IL8, IL1ȕ, tumor necrosis factor alpha (TNFĮ), TNFȖ, C-reactive protein
(CRP), intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor
(VEGF).
Methods
Study Population
The Normative Aging Study is a prospective cohort of older men established in Eastern
4
DOI: 10.1161/CIRCULATIONAHA.115.018802
Massachusetts by the United States Veterans Administration. The present study included 675
community-dwelling men with available data on ambient BC (at any of the time windows), BP
(systolic or diastolic), and blood mitochondrial abundance (Table 1). Participants were recalled
for visits every three to five years. We considered visits from April 1999 (i.e., the earliest date
with available mitochondrial DNA data) to December 2012, for a total of 1,252 visits (1-4 visits
per participant; average 1.9 visits). The study was approved by the institutional review boards of
all participating institutions. All subjects provided informed consent.
Blood Pressure
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
A physician measured the participant’s resting seated systolic BP (SBP) and diastolic BP (DBP)
in
following
n the morning at each visit using a standard mercury sphygmomanometer, follow
win
ingg a AH
AHAArecommended
ecommended protocol, as previously described.21 The means of the right and left arm
meas
assur
urem
emen
em
ents
en
t w
erre used. Three readings were taken
taake
kenn and the average off tthe
he second and third
measurements
were
readings
ead
dings was used
use
sed for
se
fo
or st
statistical
tat
atis
isti
is
tiica
call an
anal
analysis.
a yssiss.
al
Blood
Bl
loo
ood
d Mito
Mitochondrial
och
chon
ndriall A
Abundance
bun
nda
danc
ncee an
and
d Pl
Plasma
P
asma IInflammatory
asm
nffla
amm
m ator
oryy Ma
or
Markers
M
ark
r er
ers
We m
measured
easu
ea
sure
redd bl
re
bloo
blood
oodd mi
oo
mito
mitochondrial
toch
to
chon
ch
ondr
on
dria
dr
iall ab
ia
abun
abundance
unda
nda
danc
ncee th
nc
thr
through
rou
ough
gh m
mtDNA/nDNA,
tDNA
tD
NA/n
NA
/nDN
/n
DNA
DN
A, a wid
widely
idel
id
ely used
el
ussed bbiomarker
ioma
io
mark
ma
rker
rk
er
representing the mitochondrial DNA copy number versus the nuclear DNA copy number.15, 17 At
every visit, mtDNA copy number was analyzed on whole blood samples, collected after
overnight fasting. We adapted a multiplex quantitative real-time polymerase chain reaction (RTPCR) method with minor modifications.22 To measure mtDNA copy number, we used the
mtDNA 12S ribosomal ribonucleic acid (RNA) TaqMan (Applied Biosystems, Waltham,
Massachusetts) probe (6FAM- 5’ TGCCAGCCACCGCG 3’-MGB). The sequences of primers
used for amplification in mtDNA were mtF805 (5’CCACGGGAAACAGCAGTGATT3’) and
mtR927 (5’CTATTGACTTGGGTTAATCGTGTGA3’). The quantity of mtDNA was corrected
5
DOI: 10.1161/CIRCULATIONAHA.115.018802
by simultaneous measurement of a single copy nuclear Ribonuclease P gene. We used a
commercial kit to quantify nuclear DNA (nDNA) (TaqMan® RNase P Control Reagents Kit,
Applied Biosystems). RT-PCR assays were performed following a published protocol,22 using
Bio-Rad CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, California).
All samples were run in triplicate. The mean of the three measurements was used for statistical
analysis. The within-run and between-run coefficients of variation of this assay were 3.35% and
3.26%, respectively. A laboratory reference DNA sample – which was a pool of 300 test samples
(20 μL taken from each sample, final concentration: 40 ng/μL) – was used to construct standard
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
curves (mtDNA and nDNA R2 •0.99). The standard curves were used to quantify mtDNA and
nDNA copy numbers to standardize the mtDNA/nDNA obtained from all test sam
samples
mpl
ples
es iin
n al
alll
reactions.
eactions.23 We used mtDNA/nDNA in the statistical analysis. A ratio value of 1 indicates that
he mtDNA/nDNA
mtDN
mt
DNA/
DN
A/nD
A/
n NA of the test sample is equal too the
the mtDNA/nDNA in the
the
h reference DNA pool
the
uused
sed
d in the assay.
assaay.
Plas
Plasma
sma
m inf
inflammatory
nfllamm
nf
matoryy mark
m
markers
a ker
ers ((IL6,
IL6, IL
IL8,
L8, IIL1ȕ,
L1ȕ,, T
TNFĮ,
NFĮ,, TN
TNF
TNFȖ,
FȖ, CRP,
CR
RP, IICAM-1,
CAM--1, and VEG
CA
VEGF)
G F)
weere m
were
measured
easu
ea
sure
redd usin
re
us
using
sin
ingg pr
prev
previously
evio
ious
io
usly
sly ddescribed
escr
es
crib
cr
ibed
ib
ed m
methods.
etho
et
hods
ho
ds.24,, 25
ds
Air Pollution and Weather Data
Daily ambient BC and PM2.5 levels were measured at a Harvard School of Public Health
monitoring site (Boston, MA), 1 km from the clinical examination site, using previously
described methods.21 We evaluated the average pollutant concentrations during different time
windows (2-day, 5-day, 7-day, 14-day, 21-day, and 28-day, before the time of BP measurement
and blood draw). We assigned exposure index to participants based on the visit date. Outdoor air
temperature, relative humidity, dew point temperature, and wind speed were obtained from the
Boston Logan airport weather station. The corresponding moving averages of outdoor apparent
6
DOI: 10.1161/CIRCULATIONAHA.115.018802
temperature and absolute humidity were calculated using previous reported methods.26, 27
Statistical Methods
We adjusted for potential confounders, selected based on literature evidence, i.e., age
(continuous), race (white/non-white), outdoor apparent temperature (continuous), absolute
humidity (continuous), season (winter/spring-fall/summer), highest education (”high
school/college/graduate school), weekday of the visit
(Monday/Tuesday/Wednesday/Thursday/Friday), and the visit date (continuous). To increase
efficiency, we adjusted for major risk factors for increased BP: Body Mass Index (BMI)
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
(continuous), smoking status (in pack years, continuous), physical activity (metabolic equivalent
CRP
RP>1
>100 mg/L
>1
mg/L
of task-hours per week, continuous), alcohol use (<2 drinks/day/•2 drinks/day), CRP>10
yes/no); use of calcium channel blockers, ȕ-blockers, and angiotensin-converting enzyme
(yes/no);
nhiibi
bito
tors
to
rs ((yes/no).
yes/no
ye
noo).
) Due to high cell-type specifi
ici
city
ty of mitochondria con
onte
on
t nts, mtDNA/nDNA in
inhibitors
specificity
contents,
whoole blood mi
igh
ghtt be aaffected
ffec
ff
ecte
ec
teed by ddifferences
iffe
if
fereencees in th
fe
he pr
rop
opor
orrti
tioons
ons off ccell
elll ty
el
ypes.
s. W
adju
just
ju
sted
st
d ffor
or
or
whole
might
the
proportions
types.
Wee ad
adjusted
he percentage
percentaage
pe
g of
of major
majo
or cell types
typ
ypes containing
conttaiinin
in ng mitochondria
mito
ochhonndriaa (i.e.,
(ii.ee., lymphocytes,
lym
ymph
ym
phoccytees, neut
ph
uttro
rophhilss, an
nd
the
neutrophils,
and
plat
pl
atel
at
elet
el
ets)
et
s).
s)
platelets).
Linear relationships were examined using graphical diagnostics and spline regression
models, no deviation from linearity was observed. We utilized the likelihood ratio test to
determine whether it is adequate to model the independent variables or the effect modifier with a
linear trend.
In our dataset, BC moving average at different time windows exhibited different
variability. To compare the magnitude of BC-BP associations across multiple time windows, we
presented the change in BP and mtDNA/nDNA per standard deviation (SD) increase in ambient
BC levels.
7
DOI: 10.1161/CIRCULATIONAHA.115.018802
To account for repeated assessments for many participants, we utilized linear mixed
effect model with random intercept (compound symmetry covariance structure) to examine the
association between ambient BC levels and BP, as well as the association between ambient BC
levels and mtDNA/nDNA:
Yij = ȕ0 + ȕ1BCij + ȕ2X2ij + … + ȕpXpij + bi + İij
(Model 1)
Where Yij was the BP or mtDNA/nDNA for participant i at visit j, ȕ0 was the overall
intercept, bi was the separate random intercept for subject i, and bi ~ N(0, ࠴),İij ~ N(0, ı2). X2ij–
Xpij were the covariates including confounders and risk factors for increased BP, for
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
participant i at visit j. We also examined effect modification by mtDNA/nDNA by including
additionally the main effect of mtDNA/nDNA and an interaction term between mt
mtDN
mtDNA/nDNA
DNA/
DN
A/nD
A/
nDNA
nD
NA
and BC. All analyses were performed using SAS 9.4 (SAS Institute, Cary, NC).
R
Res
Results
esults
su
C
Cohort
oh
hort Characteristics
Chara
r ctterristiicss and
d Ambient
Ambie
ientt BC
ie
BC Levels
Levells
Le
The NAS is a cohort of aging
agi
g ngg men,, between 55 and 100 ye
yyears
ars old at the first visit of the ppresent
resent
study. We acquired data on ambient BC levels, BP, and mtDNA/nDNA among 675 participants.
The baseline characteristics of the participants included in the final analysis are given in Table 1.
All participants were male, and 97.3% were white. Eighty-one percent of the participants were
overweight and 18.7% were diabetics. The study population included 4.3% current smokers and
20.4% heavy alcohol drinkers (• 2 drinks/day) (Table 1). During the study period (April 1999 –
December 2012), the 7-day BC moving average varied between 0.20 μg/m3 to 2.18 μg/m3, with
an average level of 1.02 μg/m3. The baseline average SBP and DBP was 131 mm Hg and 76 mm
Hg, respectively. Participants included or excluded from each analysis are similar in key baseline
characteristics (age, physical activity, alcohol use, diabetes, BMI, smoking status, SBP, and DBP)
8
DOI: 10.1161/CIRCULATIONAHA.115.018802
(data not shown).
Ambient BC Level and BP
Short- to moderate-term ambient BC levels were consistently associated with significantly
increased SBP and DBP (Figure 1). Every SD increase in the 2-day, 5-day, 7-day, 14-day, 21day, and 28-day BC moving average was associated with 1.79 mm Hg (95% confidence interval
(CI), 0.65, 2.92; P=0.002), 2.35 mm Hg (95% CI, 1.14, 3.55; P=0.0001), 2.83 mm Hg (95% CI,
1.57, 4.09; P<0.0001), 3.02 mm Hg (95% CI, 1.68, 4.35; P<0.0001); 3.10 mm Hg (95% CI, 1.71,
4.49; P<0.0001); and 3.46 mm Hg (95% CI, 2.06, 4.87; P<0.0001) increase in SBP, respectively.
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Likewise, every SD increase in the 2-day, 5-day, 7-day, 14-day, 21-day, and 28-day BC moving
average was associated with 0.65 mm Hg (95% CI, 0.05, 1.24; P=0.03), 1.40 mm
m Hg ((95%
95%
95
% CI
CI,
1.11,
0.77, 2.03; P<0.0001), 1.73 mm Hg (95% CI, 1.07, 2.39; P<0.0001), 1.81 mm Hg (95% CI, 1.11
2.52
2.52;
2; P
P<0.0001);
<0.0
<0
.000
.0
0 1);; 1.
00
11.91
91 mm Hg (95% CI, 1.17, 2.
2.65;
.65
65; P<0.0001); and 1.97
97 mm Hg (95% CI, 1.23,
22.72;
.722; P<0.0001)
1) increase
inncre
reas
re
asee in DBP,
as
DBP, respectively
res
e pecctiv
vel
e y (Table
(Tab
ble 22).
).
Amb
Am
Ambient
bi
bient
BC
C Le
Level
evel and
nd Mit
Mitochondrial
ittoccho
h nd
ndriiall Abu
Abundance
bund
bu
n ancce
windows
(2-day,
14-day,
Thee average
Th
aver
av
erag
er
agee ambient
ag
ambi
am
bien
bi
entt BC llevels
en
evel
ev
elss ov
el
oover
ver
er tthe
he ppreceding
rece
re
cedi
ce
ding
di
ng ttime
imee wind
im
wi
ind
ndow
owss (2
-day
day, 55-day,
-da
day, 77-day,
da
-day
day, 14
-day
day,
21-day, and 28-day) were consistently positively associated with blood mitochondrial abundance
(Figure 2). Every SD increase in the 2-day, 5-day, 7-day, 14-day, 21-day, and 28-day BC
moving average was associated with 0.05 SD (95% CI, -0.01, 0.12; P=0.11), 0.12 SD (95% CI,
0.06, 0.19; P=0.0003), 0.14 SD (95% CI, 0.07, 0.21; P=0.0002), 0.15 SD (95% CI, 0.07, 0.23;
P=0.0002), 0.14 SD (95% CI, 0.06, 0.22; P=0.001), and 0.12 SD (95% CI, 0.03, 0.20; P=0.007)
increase in blood mtDNA/nDNA, respectively (Table 3).
BC and BP Association: Effect Modification by Blood Mitochondrial Abundance
The association of ambient BC levels with SBP was modified by blood mtDNA/nDNA across
9
DOI: 10.1161/CIRCULATIONAHA.115.018802
multiple time windows (P=0.05; P=0.04; P=0.03; P=0.01; P=0.01, for 5-day, 7-day, 14-day, 21day, and 28-day, respectively) (Table 4). Ambient BC levels had a relatively smaller impact on
SBP in individuals with higher blood mitochondrial abundance, compared to those with lower
blood mitochondrial abundance (Figure 3 and Supplemental Figure 1). For example, for
individuals whose mtDNA/nDNA was at the midpoint of the lowest quartile, the 28-day BC
moving average was estimated to be associated with 4.68 mm Hg (95%CI, 3.03, 6.33; P<0.0001)
increase in SBP. In comparison, the 28-day BC moving average was estimated to be associated
with 2.40 mm Hg (95%CI, 0.81, 3.99; P=0.003) increase in SBP among individuals whose
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
mtDNA/nDNA was at the midpoint of the top quartile. We did not observe significant effect
modification by blood mitochondrial abundance on 2-day BC-SBP association, aand
nd oon
n th
thee
association between short-term ambient BC levels and DBP (Table 4). In addition, increased
blood
with
bloood mtDNA/nDNA
mtDN
mt
DNA/
DN
A nD
DNA
N was negatively correlated
dw
ith plasma IL6, TNFȖ,
it
TNF
FȖ, CRP, and ICAM-1
levels
P=0.003,
P=0.03,
eveels (P=0.03,
( =0.03, P=0.01,
(P
P=0.0
.0
01,, P
=0.0
=0.0
.003
03,, P
03
=0.003, rrespectively)
=0
especttiv
vely)
y) ((Supplemental
Sup
Su
pple
leme
le
ment
me
ntaal Table
nt
Tab
ble 1).
1).
).
Sensitivity
Analyses
Seens
nsit
i ivityy An
A
allysses
BC iiss the
major
component
particles
the ma
majo
jorr co
jo
comp
mpon
mp
onen
on
entt of ffine
en
inee pa
in
part
rtic
rt
icle
ic
less (P
le
(PM
M2.5); PM
PM2.5 itself
its
tsel
elff is a ccontributor
el
ontr
on
trib
tr
ibut
ib
utor
tor to
to increased
incr
in
crea
cr
ease
ea
sedd BP
se
and enhanced oxidative damage. In sensitivity analysis, we adjusted for the ambient PM2.5 levels
in the matching time window – to rule out the possibility that the observed effect was partially
due to confounding by ambient PM2.5. This adjustment did not affect our conclusion (Tables 2
and 3). In addition, we adjusted for the platelet/lymphocyte and platelet/neutrophil ratios to
minimize the influence from disproportional increase in platelets, and our conclusions remained
the same (data not shown).
Discussion
This study on a cohort of aging male Boston-area residents confirmed that short- to moderate10
DOI: 10.1161/CIRCULATIONAHA.115.018802
term ambient BC levels are associated with increased BP. We showed that short- to moderateterm ambient BC levels also increased peripheral blood mitochondrial abundance in older adults.
Further, individuals with higher blood mitochondrial abundance appeared less susceptible to the
impact of ambient BC levels on BP, compared to those with less blood mitochondrial abundance.
In the United States, the proportion of the population over the age of 65 is projected to
increase from 12.7% in 2000 to 20.3% in 2050.28 The AHA has emphasized that air pollution is
an ubiquitous public health threat, particularly for older people, who are especially susceptible to
air pollution-triggered CVD.2 BC, the byproduct of incomplete combustion of fossil fuels,
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
biofuels, and biomass, is the most effective form of PM (by mass) at absorbing solar energy and
iss considered a potent trigger for systemic oxidative stress upon exposure.29 Incre
Increased
eas
ased
ed B
BP
P related
rela
re
latted
la
to
o ambient BC levels is an alarming physiological change that may represent intermediate
mech
mechanisms
ch
han
anis
isms
is
ms contributing
con
ntr
trib
i uting to acute adverse cardiovascular
cardiov
ovas
ov
ascular events.2, 9
as
Consistent
Consis
i te
tennt w
with
ithh ou
it
ourr pr
prev
previous
evio
ev
ious
io
us study,
stu
t dy
dy, we confirmed
connfirm
med the
the significant
sig
igniifican
ig
ican
ant association
assoociat
atio
tio
ionn between
beetw
twee
eenn BP
ee
BP
and
average
ambient
level
the
days.
an
nd th
the aver
rag
age am
ambien
entt BC
en
C lev
evel iin
ev
n th
he pa
ppast
st 7 da
st
ayss.21 The
The
h present
prese
rese
sent
nt study
stu
t dy presented
preeseent
n ed
da
comp
co
mpre
mp
rehe
re
hens
he
nsiv
ns
ivee an
anal
alys
al
ysis
sis ooff th
thee sh
shor
ortt- tto
or
o mo
mode
dera
de
rate
ra
te-ter
ter
erm
m ca
card
rdio
rd
iova
io
vasc
asc
scul
ular
lar eeffects
ffec
ff
ects
ec
ts ooff BC
BC, ra
rang
ngin
ng
ingg
in
comprehensive
analysis
shortmoderate-term
cardiovascular
ranging
from 2 days to 28 days. The estimated effect size of ambient BC levels increased gradually as the
time window of exposure increased, and the strongest estimated effect was observed for the 28day time window. The BP increase associated with ambient BC levels reflects the physiologic
effect of traffic-related air pollution that may contribute to the pathophysiological changes in the
cardiovascular system – which could be a potential mechanism linking air pollution and cardiac
morbidity and mortality in the aging population. In addition, the observed effect estimates for
ambient BC were increased with a larger magnitude after adjusting for ambient PM2.5 levels,
suggesting that the proportion of BC content in PM2.5 has a stronger effect on BP increases than
11
DOI: 10.1161/CIRCULATIONAHA.115.018802
the actual BC mass.
Recent studies suggest that particle-induced oxidative stress initiates systemic
inflammatory response,30 which is considered a major mechanism underlying the cardiac
responses to air pollution. Mitochondria, the specialized cellular organelles that play a pivotal
role in energy production, are the main immediate target of ROS.11, 12 The role of mitochondrial
biogenesis and mitochondria DNA copy number maintenance in the determination of cell
survival and function under oxidative stress has been increasingly appreciated in recent years.
However, most studies have focused on inflammatory markers such as cytokines, and the role of
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
mitochondria in air pollution-related cardiovascular pathogenesis remains a major gap in current
knowledge. The amount of mitochondrial genomic content within a cell is normally
normal
ally
al
ly controlled,
con
ontr
trol
tr
olle
ol
led,
le
d 31
but upon exposure to environmental stress, mitochondria biogenesis can be enhanced to increase
energy
ener
rgy ssupply
uppl
up
plyy for
pl
fo
or eliminating
e iminating damage to cellular
el
arr ccomponents.
omponents.12, 15Mali
Malik
liik and Czajka suggested
that
hatt mtDNA/nDNA
mtDNA/n
/ DN
DNA
A increases
incrrea
in
easses
ses when
when the
the cell's
celll'l's endogenous
endo
ogeno
ous
us antioxidant
ant
ntio
oxida
xida
dant
nt response
res
e pons
nsee fails
ns
fail
fa
ilss too recover
il
reccov
over
er its
redox
balance.
edo
doxx balanc
ncce.15 For
o example,
exaample
le, exposure
le
expo
possurre to
to air
air pollutants
p lllutan
po
ntss such
suuch as
as benzene
benz
be
nzzen
ne was
waas associated
a soci
as
ciaated
ci
d with
withh
ncr
crea
ease
ea
sess in bblood
se
lood
lo
od m
itoc
it
ocho
oc
hond
ho
ndri
nd
rial
ri
al genomic
gen
enom
omic
om
ic contents.
con
onte
tent
te
ntss.32 S
nt
Systemic
ysste
yste
temi
micc ox
mi
ooxidative
xid
idat
id
ativ
at
ivee st
stre
stress
ress
re
ss sstimulated
timu
ti
mula
late
la
tedd by
te
by tthe
hee
increases
mitochondrial
activation of PI3K/Akt signaling pathway could also result in increased mitochondrial DNA
copy number in leukocytes.20 Mitochondria proliferation and mitochondrial DNA amplification
under oxidative stress was observed even when overall cell division was under arrest.33 The
present study for the first time provides epidemiological evidence showing that ambient BC level
is linked with increased blood mitochondrial abundance, as reflected in elevated mtDNA/nDNA
in peripheral blood.
In addition, we demonstrated that high blood mitochondrial abundance attenuates the
association of ambient BC levels with BP. In in vivo studies, increased mitochondrial
12
DOI: 10.1161/CIRCULATIONAHA.115.018802
biogenesis induced by over-expressing recombinant human mitochondrial transcription factor A
resulted in a 50% reduction in the oxidative damage to proteins following lipopolysaccharide
endotoxin challenge.16 Under the challenge of endogenous or exogenous ROS, increased
mitochondrial DNA copy number in aging tissues is a feedback response that compensates for
insufficient ROS cleavage and upholds the energy metabolism to rescue the cell.12, 34 In our study,
increased blood mitochondria abundance was linked with reduced inflammatory responses.
Collectively, evidence support our hypothesis that blood mitochondrial abundance is an adaptive
process following ambient BC exposure and reduces susceptibility to the cardiovascular effect of
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
BC, possibly by scavenging radical and suppressing inflammation. Further elucidation of the
changes in blood mitochondrial abundance in the process of stress response to rend
nder
nd
er
render
cardiovascular tissue’s survival under oxidative stress is of prime importance for a better
unde
deers
rsta
tand
ta
ndin
nd
ingg off cardio-pathogenesis
in
car
a dio-pathogenesis related to air
air pollution.
pollution.
understanding
Neverthe
heleesss, th
thee st
stre
ress
re
ss res
espo
es
pons
po
nsee off m
ns
itoch
hondr
drria tto
o ooxidative
xid
idattive
id
ive sstress
treess m
ay bbee a doubleay
doub
do
uble
ub
lele
Nevertheless,
stress
response
mitochondria
may
ed
dge
gedd sword.
d. Onn one
one hand,
ha the
thee compensatory
th
comp
mpen
mp
en
nsato
ory increase
incrreaasee in
in blood
blloo
od mitochondrial
mito
mi
ochoondri
riiall proliferation
prollife
life
ferattioon
edged
upp
ppli
lies
li
es energy
ene
nerg
rgy to meet
rg
mee
eett th
thee ne
need
ed ffor
or bblood
lood
lo
od ccell
elll su
el
ssurvival.
urv
rviv
ival
al.12 O
al
On
n th
thee ot
othe
other
herr ha
he
hand
hand,
nd, mi
nd
mito
mitochondria
toch
to
chon
ch
ondr
on
dria
dr
ia aare
re
supplies
the main intracellular source of ROS and play an important role in biological oxidation.11, 12
Excess ROS generated from the increased mitochondria can be pathogenic, initiating a vicious
cycle that leads to chronic oxidative damage and inflammation.11, 12, 15, 35, 36Unfortunately, we
only measured blood mtDNA/nDNA and BP concurrently at each visit; therefore, we are not
able to assess the effect of continuously higher blood mitochondrial abundance on BP due to lack
of temporality. Future studies are warranted to shed light on the cardiovascular effect of
chronically elevated blood mitochondrial abundance due to traffic-related pollution.
This study has several strengths, including its repeated-measure design and relatively
13
DOI: 10.1161/CIRCULATIONAHA.115.018802
large sample size. In addition, we utilized a highly reproducible RT-PCR method to quantify the
mtDNA content from easy-to-obtain blood samples, which has been widely adapted in
epidemiological studies.37 To measure ambient traffic-related PM, we used daily BC monitor
data as a proxy, because variation in short- to moderate-term ambient air pollution was mostly
due to temporal variation rather than spatial variation.38 Measurement error, which is inherent in
air pollution epidemiological studies, cannot be completely avoided due to lack of personal
exposure assessment. However, misclassification is expected to be non-differential and bias
towards the null, because it was unlikely to be associated with participants’ BP status or
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
mitochondrial abundance. To limit confounding, we included in regression models an extensive
ist of covariates. We conducted further analysis to evaluate the sensitivity of ourr rresults
esul
es
ults
ul
ts tto
o
list
covariate specification, and our results were stable and robust. In addition, all BP measures were
cond
duc
ucte
tedd at thee sa
te
ame time of the day to eliminat
atee cconfounding
at
onfounding due to ddiurnal
i rnal variation. While
iu
conducted
same
eliminate
esiidual confounding
confo
f unding
i g due
due to
to unmeasured
unme
un
meas
me
asurred
as
d variables
var
ariaabless is
ar
is possible,
possi
sibl
ibl
blee, chances
cha
hanc
nces
nc
es that
thhat the
the observed
obse
ob
serv
se
rved
rv
ed
residual
as
sso
soci
c ation an
and ef
eff
fect m
odifficaati
t onn rreflected
effle
lect
c ed
d bbias
ias re
esulltiing ffrom
ro
om re
resi
sidu
d al con
nfo
foundi
dinng aree
di
association
effect
modification
resulting
residual
confounding
mini
mi
nimi
ni
mize
mi
zed
ed.
minimized.
The present study has several limitations. Some of the eligible participants were excluded
from analysis due to missing BC data. They were similar in baseline characteristics (including
BP) with participants included in the analysis. Moreover, it is reasonable to assume that the
missingness was independent of ambient BC levels and participants’ mitochondrial abundance
based on study operation. Therefore, selection bias due to informative missingness was unlikely.
In addition, we standardized mtDNA/nDNA measurements to a lab reference DNA sample;
therefore, the absolute value of mtDNA/nDNA from the present study might not be directly
comparable to other studies. Finally, our findings are limited to male older individuals who were
14
DOI: 10.1161/CIRCULATIONAHA.115.018802
residing in a lightly-polluted urban area. Our conclusion might not be generalizable to young
adults, females, or people living in other areas due to differential environmental and
physiological factors.
In summary, our results showed that, in older adults, levels of short- to moderate-term
ambient traffic-related PM were associated with increases in BP and blood mitochondrial
abundance. Individuals with higher blood mitochondrial abundance showed significantly weaker
association of ambient BC with BP. Our study provided a novel mechanistic perspective – the
compensatory increase in blood mitochondrial abundance following exposure to ambient BC
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
might be an adaptive response that attenuates individual susceptibility to post-exposure BP
increase
oxidative
responses
ncrease in older people. Further investigation of the mitochondria-mediated oxid
dat
ativ
ivee re
iv
resp
spon
sp
onsses
on
iss warranted to provide insights into the physiologic effects of traffic-related pollution and may
help
p guide
gui
uide
de targeted
tar
argete
tedd prevention and novel pharmaceutical
te
pharma
maace
ceut
u ical interventions..
Funding
Sources:
This
study
supported
NIH
grants
Fun
Fu
nding Sour
u cees:: Th
hiss stud
dy was su
sup
ppoortedd bby
yN
IH
H gr
gra
antss R01ES015172,
ants
R011ES01551772,, R21ES021895,
R21ES0
S021
S0
2 8995,
R01ES021733,
and
U.S.
R01E
1E
ES0
S021
217733, R21ES021895,
21
R21
21ES
ES02
ES
021895
2
5, R01ES020836,
R01ES0020
R0
2083
8366, R01ES021357,
83
R01ES
01ES
ES02
0213
02
1357
13
57, an
57
nd P30ES000002;
P30E
P3
0ES0
0E
S 00000
002;
2; andd U
.S.
S
Environmental Protection Agency funding (RD-83479801).
(RD-83479801) The Normative Aging Study is
supported by the Cooperative Studies Program/Epidemiology Research and Information Center
of the U.S. Department of Veterans Affairs (VA) and is a component of the Massachusetts
Veterans Epidemiology Research and Information Center, Boston, Massachusetts. David
Sparrow was supported by a VA Research Career Scientist award.
Conflict of Interest Disclosures: None.
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18
DOI: 10.1161/CIRCULATIONAHA.115.018802
Clinical Perspective
Traffic-related pollution (black carbon, BC) is a pervasive environmental threat, particularly for
older individuals, who are especially susceptible to pollution-triggered cardiovascular morbidity
and mortality. Systemic oxidative stress is a key mechanism linking BC pollution and
cardiovascular events, and the role of mitochondria abundance in compensating for cellularredox-imbalance under oxidative stress has been increasingly appreciated. However, the
response of mitochondria in the peripheral blood upon ambient BC exposure remains unknown.
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
We utilized the Normative Aging Study to address a mechanistic question about cardiovascular
mitochondrial
effects of air pollution: how does short- to moderate-term ambient BC alter bloodd m
itoc
it
ocho
oc
hond
ho
ndri
nd
rial
ri
abundance; and how this alteration might impact BC-elicited cardiovascular effects. Our results
indicated
that,
ambient
ndiica
cate
tedd th
te
that
at, in
at
n oolder
l er adults, levels of short- too mo
ld
moderate-term ambien
nt BC
B were associated with
increased
blood
mitochondrial
with
ncrreased bloodd pr
ppressure
esssu
sure
ree ((BP)
BP)) and
BP
and bl
bloo
oodd mi
oo
mito
tocchond
to
ndrial
nd
al aabundance.
bund
bu
ndan
nd
ance
cee. In aaddition,
dditio
io
on, iindividuals
ndiv
nd
ivid
iv
id
dua
uals
ls w
ith
it
lower
larger
effect
owe
werr bloodd mitochondrial
m to
mi
ochhonndrrial abundance
abun
ab
undaanc
un
nce were
we e estimated
esstimatted
d to
to exhibit
ex
xhi
hibi
biit 1.95-fold
1..955-fooldd la
arg
rger
e eff
ffeect off BC
ff
increased
blood
mitochondrial
abundance
wiith ssystolic
with
yssto
ysto
toli
licc BP
li
BP, su
ssuggesting
ugg
gges
gg
esti
es
ting
ti
ng tthat
hatt in
ha
incr
crea
cr
ease
ea
sedd bl
se
bloo
oodd mi
oo
mito
toch
to
chon
ch
ondr
on
dria
dr
iall ab
ia
abun
unda
nda
danc
ncee bu
nc
bbuffered
uff
ffer
ff
ered
er
ed tthe
he eeffect
ffec
ff
ectt off
ec
ambient BC on BP. The impact of our study is in the investigation of compensatory increase in
blood mitochondrial abundance as an adaptive response to reduce the cardiovascular effects of
ambient BC. The present study provides insights into the physiologic effects of traffic-related
pollution and may aid targeted risk-reduction and novel pharmaceutical interventions. Because of
the central role of oxidative stress in the pathogenesis of cardiovascular diseases, the study
results may provide novel perspective to the cardiovascular effect of other environmental risk
factors.
19
DOI: 10.1161/CIRCULATIONAHA.115.018802
Table 1. Baseline characteristics, black carbon (BC) levels, blood mitochondrial DNA to
nuclear DNA copy number ratio (mtDNA/nDNA), and blood pressure (BP) in the Normative
Aging Study (N=675), 1999-2012.
Characteristic
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Age
55-69 years
70-79 years
80-89 years
> 90 years
Physical Activity
<12 MET-hrs/wk
12-29 MET-hrs/wk
•30 MET-hrs/wk
Alcohol Use
<2 drinks/day
•2 drinks/day
Diabetes
No
Yes
Yees
Racce
Race
N n-white
No
Non-white
Whit
Wh
itee
it
White
BMII
<25 kg/m2
•25 kg/m2
Smoking Status
Never
Current
Former
Education
”High School
College
Graduate School
N (%)
BC
(μg/m3)
Mean (SD)
SBP
(mm Hg)
Mean (SD)
DBP
(mm Hg)
Mean (SD)
mtDNA/nDNA*
Mean (SD)
215 (31.8)
340 (50.4)
117 (17.3)
3 (0.4)
1.29 (0.40)
1.23 (0.41)
1.17 (0.42)
0.74 (--)
129.8 (14.9)
131.0 (17.7)
132.3 (20.8)
122.7 (15.3)
79.2 (8.3)
74.8 (9.6)
72.4 (10.0)
77.0 (6.6)
1.04 (0.24)
1.00 (0.24)
1.01 (0.34)
1.08 (0.09)
434 (64.3)
146 (21.6)
95 (14.1)
1.25 (0.41)
1.22 (0.41)
1.21 (0.42)
131.1 (17.7)
127.8 (15.8)
134.2 (18.2)
75.7 (9.6)
75.6 (10.1)
76.5 (9.0)
1.01 (0.27)
1.01 (0.22)
1.02 (0.29)
537 (79.6)
138 (20.4)
1.24 (0.41)
1.20 (0.42)
130.3 (17.4)
132.7 (17.5)
76.1 (9.7)
74.8 (9.3)
11.02
.0
02 (0
(0.2
(0.26)
.26)
.2
6)
11.00
.00
000 ((0.25)
0.25
25))
25
549 (81.3)
126 (18.7)
1.25 (0.41)
1.19 (0.41)
1130.1
30.1 (17.1)
1133.9
33.9 (18.3)
76.22 ((9.5)
9.5)
74.3
3 ((10.0)
10.0)
1.01 (0.27)
1.01 (0.22)
18 ((2.7)
2.7)
2.
6657
57 (9
(97.
7.3)
7.
3)
(97.3)
1.
.322 (0.4
.47)
7
1.32
(0.47)
1.
.233 (0
0.4
.41))
1.23
(0.41)
1128.7
28
8.7 ((13.5)
13..5)
1130.8
30
0.8
8 ((17.5)
17..5)
.5)
75.4
4 ((9.5)
9.55)
75.8
75
8 ((9.6)
9.6)
6)
00.65
. 5 (0
.6
((0.44)
.444)
11.02
.022 ((0.25)
0.255)
130 (19.3)
545 (80.7)
1.26 (0.41)
1.23 (0.41)
130.6 (19.0)
130.8 (17.1)
73.9 (9.0)
76.3 (9.7)
1.04 (0.32)
1.01 (0.24)
199 (29.4)
29 (4.3)
447 (66.2)
1.24 (0.41)
1.21 (0.41)
1.24 (0.41)
130.7 (17.4)
132.2 (15.7)
130.7 (17.6)
76.5 (9.4)
76.0 (6.6)
75.5 (9.9)
1.02 (0.24)
1.02 (0.31)
1.01 (0.27)
177 (26.2)
337 (49.9)
161 (23.9)
1.26 (0.40)
1.23 (0.41)
1.21 (0.43)
131.3 (18.4)
130.6 (17.5)
130.5 (16.1)
75.5 (9.8)
75.7 (9.6)
76.4 (9.4)
1.00 (0.23)
1.01 (0.28)
1.04 (0.25)
N indicates the number of participants; SBP and DBP indicate systolic and diastolic BP, respectively; SD indicates
standard deviation; MET indicates metabolic equivalent of task; BMI indicates body mass index.
* mtDNA/nDNA measurements reflects the average cellular mitochondrial abundance, calculated based on the ratio
of copy number estimates of a mitochondrial gene to those of a nuclear gene. The ratio was scaled to a standard
DNA sample presenting the relative mitochondrial content within a cell.
20
DOI: 10.1161/CIRCULATIONAHA.115.018802
Table 2. Association of ambient black carbon (BC) levels with blood pressure (BP).
Time Window
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
P
N
n
0.002
0.0001
<0.0001
<0.0001
<0.0001
<0.0001
656
657
657
654
649
650
1243
1252
1252
1234
1206
1211
0.03
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
656
657
657
654
649
650
0
1243
1252
1252
1234
1206
11211
2111
21
Sensitivity Analysis Adjusting for PM2.5‡
2-day
3.72
2.27 to 5.16 <0.0001
5-day
4.03
22.58
.58 to 5.47 <0.000
<0.0001
001
00
2.85
7-dayy
4.29
2.85 to 5.73 <0.0001
114-day
4-d
-day
ay
4.366
22.90
.900 to
to 55.82
.882 <
<0.0001
0.00
00001
00
221-day
1--day
4.199
22.68
.688 to
to 5.
55.70
70 <0.00
<0.0001
001
<0.0001
228-day
8--day
44.32
.322
.3
22.78
.788 to
to 55.86
.86
86 <
0 00001
0.
606
610
610
600
600
585
5
583
83
1048
1058
1052
11021
0211
02
9844
9855
2-day
5-day
7-day
14-day
21-day
28-day
606
610
610
600
585
583
1048
1058
1052
1021
984
985
SBP
(mm Hg)
2-day
5-day
7-day
14-day
21-day
28-day
DBP
(mm Hg)
2-day
5-day
7-day
14-day
21-day
28-day
SBP
(mm
mm
m Hg
Hg))
DBP
(mm Hg)
Estimate*
95% CI
Original Model†
1.79
0.65 to 2.92
2.35
1.14 to 3.55
2.83
1.57 to 4.09
3.02
1.68 to 4.35
3.10
1.71 to 4.49
3.46
2.06 to 4.87
0.65
1.40
1.73
1.81
1.91
1.97
0.05 to 1.24
0.77 to 2.03
1.07 to 2.39
1.11 to 2.52
1.17 to 2.65
1.23 to 2.72
11.64
64
2.40
2.59
2.61
2.54
2.49
00.89
89 to 2.38
2 38
1.66 to 3.14
1.85 to 3.33
1.86 to 3.36
1.77 to 3.31
1.71 to 3.26
<0.0001
<0 0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
SBP and DBP indicate systolic and diastolic BP, respectively; N and n indicate the number of participants and
observations, respectively; PM2.5 indicates particulate matter up to 2.5 micrometers in size.
* Estimate presents the increase in BP (mm Hg) per standard deviation (SD) increase in BC.
† Results were adjusted for: apparent temperature and absolute humidity at the matching time window; race;
percentage of major cell types (lymphocytes, neutrophils, and platelets); age; weekday; date of visit; body mass
index; C-reactive protein>10 mg/L; smoking; alcohol use; season; physical activity; education level; use of calcium
channel blockers, ȕ-blockers, and angiotensin-converting enzyme inhibitor.
‡ Results were adjusted for all the covariates listed above, and PM2.5 at the matching time window.
21
DOI: 10.1161/CIRCULATIONAHA.115.018802
Table 3. Association of ambient black carbon (BC) levels with blood mitochondrial DNA to
nuclear DNA copy number ratio (mtDNA/nDNA).
Time Window
2-day
5-day
7-day
14-day
21-day
28-day
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
2-day
5-day
7-day
14-day
21-day
28-day
Estimate*
P
N
n
0.11
0.0003
0.0002
0.0002
0.001
0.007
656
657
657
654
649
650
1241
1250
1250
1232
1204
1209
Sensitivity Analysis Adjusting for PM2.5‡
0.12
0.04 to 0.20
0.005
0.17
0.09 to 0.25
<0.0001
0.15
0.07 to 0.23
0.0002
0.14
0.06 to 0.23
0.001
0.12
0.03 to 0.21
0.008
0.09
0.00 to 0.17
0.05
606
610
610
600
5
585
583
1046
1056
1050
1019
10
19
9822
98
983
0.05
0.12
0.14
0.15
0.14
0.12
95% CI
Original Model†
-0.01 to 0.12
0.06 to 0.19
0.07 to 0.21
0.07 to 0.23
0.06 to 0.22
0.03 to 0.20
N and n indicate the number of participants and observations, respectively; PM2.5 indicates particulate matter up to
2.5 micrometers in ssize.
ize.
iz
* Estimate
in BC.
Essti
tim
mate presents
mat
pre
reseents the
the increase in standard deviation (SD)
(SD
D) for
fo
or mtDNA/nDNA
A per
per SD
D increase
in
Results
apparent
temperature
matching
window;
race;
† Re
Res
sults weree adjusted
adjuste
teed for:: app
pparent te
pp
emp
mperature and absolute
absollutte humidity
humid
idit
ityy at the
he m
atchin
ng time w
indow; rac
in
ce;
e
major
cell
visit;
ppercentage
perc
e cen
e tage of majo
or ce
elll ttypes
y es (lymphocytes,
yp
(ly
lymp
mphhocy
mp
cytees, neutrophils,
cy
neu
utropphiils, and
an
nd platelets);
plat
atel
at
elets)
el
s); age;
s)
age; weekday;
weekd
kday
kd
a ; datee ooff vi
ay
isi
sit;
t;; bbody
odyy ma
od
mass
sss
index;
protein>10
mg/L;
alcohol
activity;
level;
ndeex; C-reactive pr
rottein>
>10
0 mg/L
L; smoking; alc
cohol use;
use; season;
seaason; pphysical
hyssiccal ac
hy
ctivvity; eeducation
duucattio
on leve
el;
l uuse
se off calcium
m
channel
blockers,
ȕ-blockers,
inhibitor.
han
ann
nel blocke
ers
rs, ȕ-bllockeerss, andd angiotensin-converting
ang
ngiotens
ng
nsin--coonverrtin
ns
ng enzyme
en
nzy
ymee in
nhib
bitoor.
at th
tthee ma
matc
matching
t hi
hing
ng time
tim
me wi
window.
ind
ndow
ow.
ow
‡ Results
adjusted
covariates
above
Resu
Re
sult
su
ltss were
lt
were adj
juste
us ed for
fo al
alll the co
covari
riat
ri
ates llisted
at
iste
is
teed ab
abov
ovve an
andd PM2.5
2 at
22
DOI: 10.1161/CIRCULATIONAHA.115.018802
Table 4. Effect modification by blood mitochondrial DNA to nuclear DNA copy number ratio
(mtDNA/nDNA), on the association of ambient black carbon (BC) with blood pressure (BP).
2-day
5-day
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
7-day
1414
4day
daay
21day
28day
mtDNA/nDNA
Q1 Midpoint
Q2 Midpoint
Q3 Midpoint
Q4 Midpoint
Pinteraction
Q1 Midpoint
Q2 Midpoint
Q3 Midpoint
Q4 Midpoint
Pinteraction
Q1
Q Midpoint
p
Q2 Midpoint
Q3 Midpoint
Q4 Midpoint
Pintera
interaction
acti
c on
ct
Q1 Midpoint
Mid
dpoint
Q2
2 Midpoint
Mid
idpo
p in
intt
Q3 Midpoint
Mid
dpoin
nt
Q4 Midpoint
Mid
dpoin
nt
Pinteraction
Q1 Midpoint
Q2 Midpoint
Q3 Midpoint
Q4 Midpoint
Pinteraction
Q1 Midpoint
Q2 Midpoint
Q3 Midpoint
Q4 Midpoint
Pinteraction
SBP (mm Hg)
Estimate
95% CI
2.36
0.99 to 3.73
1.98
0.82 to 3.15
1.65
0.49 to 2.80
1.09
-0.38 to 2.56
3.19
2.69
2.24
1.51
1.71 to 4.66
1.43 to 3.95
1.02 to 3.46
0.04 to 2.99
3.76
3.25
2.79
2.03
2.23 to 5.3
1.92 to 4.57
1.51 to 4.06
0.55 to 3.51
4.01
3.48
3.
48
3.
3.02
2.26
2
2.39 to 5.62
5.662
22.07
.00 to 44.89
.07
.899
11.68
.668 to
o 44.37
.377
00.74
.774 to 33.78
.788
4.22
3.62
3.08
2.19
2.56 to 5.88
2.15 to 5.08
1.68 to 4.48
0.62 to 3.76
4.68
4.00
3.40
2.40
3.03 to 6.33
2.54 to 5.46
1.99 to 4.80
0.81 to 3.99
P
0.001
0.001
0.01
0.15
0.14
<0.0001
<0.0001
0.0003
0.04
0.05
<0.0001
<0.0001
<0.0001
0.01
0.04
<0.0001
<0
<0.0001
0.0
.000
0011
00
<0
<0.0001
0.0
.000
001
00
00.004
.00044
00.03
.03
03
<0.0001
<0.0001
<0.0001
0.01
0.01
<0.0001
<0.0001
<0.0001
0.003
0.01
DBP (mm Hg)
Estimate
95% CI
0.80
0.08 to 1.51
0.67
0.06 to 1.28
0.56
-0.05 to 1.17
0.38
-0.40 to 1.15
1.50
1.44
1.39
1.31
0.73 to 2.27
0.79 to 2.10
0.76 to 2.03
0.54 to 2.08
1.93
1.82
1.72
1.56
1.13 to 2.73
1.13 too 2.
2.51
51
1.06 to 2.
2.39
39
0.79 to 2.33
22.00
.00
11.89
1.8
.889
11.80
.80
11.64
.64
64
1.15 to 2.85
1.15
15 tto
o 2.
22.63
633
11.09
.009
09 to 22.50
.550
00.84
. 4 to 22.44
.8
.444
2.17
2.05
1.94
1.76
1.29 to 3.05
1.27 to 2.82
1.20 to 2.68
0.92 to 2.59
2.21
2.10
1.99
1.82
1.34 to 3.09
1.33 to 2.87
1.25 to 2.74
0.98 to 2.66
P
0.03
0.03
0.07
0.34
0.35
0.0001
<0.0001
<0.0001
0.001
0.67
<0.0001
<
<0.0001
0.00
0.
0001
00
<0.0001
<
0 00
0.
0001
01
<0.0001
0.39
<0.0001
<
<0.0001
0.00
0.
0001
00
<
<0.0001
0.00
0001
00
<
<0.0001
0.00001
00.40
0.
40
<0.0001
1
<0.0001
<0.0001
<0.0001
0.33
<0.0001
<0.0001
<0.0001
<0.0001
0.36
N and n indicate the number of participants and observations, respectively; SBP and DBP indicate systolic and
diastolic BP, respectively.
Estimate represents that per standard deviation (SD) increase in mtDNA/nDNA, the lessened increase in BP (mm
Hg) per SD increase in BC.
Results were adjusted for: apparent temperature and absolute humidity at the matching time window; race;
percentage of major cell types (lymphocytes, neutrophils, and platelets); age; weekday; date of visit; body mass
index; C-reactive protein>10 mg/L; smoking; alcohol use; season; physical activity; education level; use of calcium
channel blockers, ȕ-blockers, and angiotensin-converting enzyme inhibitor.
23
DOI: 10.1161/CIRCULATIONAHA.115.018802
Figure Legends:
Figure 1. Association of ambient black carbon (BC) levels with blood pressure (BP), Normative
Aging Study, 1999-2012. Estimates represent the increase in BP per standard deviation increase
in BC. Results were adjusted for: apparent temperature and absolute humidity at the matching
time window; race; percentage of major cell types (lymphocytes, neutrophils, and platelets); age;
weekday; date of visit; body mass index; C-reactive protein>10 mg/L; smoking; alcohol use;
season; physical activity; education level; use of calcium channel blockers, ȕ-blockers, and
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
angiotensin-converting enzyme inhibitor.
Figure 2. Association of ambient black carbon (BC) levels with blood mitochondrial DNA to
nuclear
(mtDNA/nDNA),
Normative
Study,
nucl
lea
earr DN
DNA
A copy
py number ratio (mtDNA/nDNA
A), N
ormative Aging S
tudy
tu
d , 1999-2012.
Estimates
E
stiimates represent
repres
esen
es
nt the
th increase
incr
in
crea
cr
eaase in standard
sta
tand
ndardd deviation
deeviaatio
on (SD)
(SD) fo
for mtDNA/nDNA
m DN
mt
DNA
A/nnDNA
NA per
er SD
SD increase
incr
incr
crea
easee
ea
inn BC.
apparent
BC.
C Results
Resul
ults
ul
t were
ts
werre adjusted
ad
dju
usted
d for:
for
o : ap
appa
paareent
n temperature
temper
mp ratturee and
andd absolute
ab
bso
s lu
lute
te humidity
hum
umidit
ityy at the
it
he matching
maatcchingg
time
window;
major
ime win
indo
in
dow;
do
w;; rrace;
ace;
ac
e; ppercentage
erce
er
cent
ce
ntag
nt
agee of ma
ag
majo
jorr ce
jo
cell
ll types
types
yppes (lymphocytes,
(ly
lymp
mpho
mp
hocy
ho
cyte
tess, neutrophils,
te
neu
euttro
roph
phil
ph
ilss, aand
il
nd pplatelets);
late
la
tele
te
lets
le
ts);
ts
); aage;
ge;;
ge
weekday; date of visit; body mass index; C-reactive protein>10 mg/L; smoking; alcohol use;
season; physical activity; education level; use of calcium channel blockers, ȕ-blockers, and
angiotensin-converting enzyme inhibitor.
Figure 3. Association of ambient black carbon (BC) levels with systolic blood pressure (SBP) at
different blood mitochondrial DNA to nuclear DNA copy number ratio (mtDNA/nDNA) levels,
Normative Aging Study, 1999-2012. Results were adjusted for: apparent temperature and
absolute humidity at the matching time window; race; percentage of major cell types
24
DOI: 10.1161/CIRCULATIONAHA.115.018802
(lymphocytes, neutrophils, and platelets); age; weekday; date of visit; body mass index; Creactive protein>10 mg/L; smoking; alcohol use; season; physical activity; education level; use
of calcium channel blockers, ȕ-blockers, and angiotensin-converting enzyme inhibitor.
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
25
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Figure 1
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Figure 2
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Figure 3
Traffic-Related Air Pollution, Blood Pressure, and Adaptive Response of Mitochondrial
Abundance
Jia Zhong, Akin Cayir, Letizia Trevisi, Marco Sanchez-Guerra, Xinyi Lin, Cheng Peng, Marie-Abèle
Bind, Diddier Prada, Hannah Laue, Kasey J.M. Brennan, Alexandra Dereix, David Sparrow, Pantel
Vokonas, Joel Schwartz and Andrea A. Baccarelli
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Circulation. published online December 11, 2015;
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2015 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circ.ahajournals.org/content/early/2015/12/11/CIRCULATIONAHA.115.018802
Data Supplement (unedited) at:
http://circ.ahajournals.org/content/suppl/2015/12/11/CIRCULATIONAHA.115.018802.DC1
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SUPPLEMENTAL MATERIAL
S1
Supplemental Table 1: Correlation between whole
blood mitochondrial DNA to nuclear DNA copy
number ratio (mtDNA/nDNA) and plasma
inflammatory markers.
Correlation Coefficient
P*
IL6
-0.07
0.03
IL8
-0.03
0.32
IL1β
-0.05
0.10
TNFα
-0.06
0.09
TNFγ
-0.08
0.01
CRP
-0.09
0.003
ICAM-1
-0.06
0.03
VEGF
-0.06
0.07
IL indicates interleukin; TNF indicates tumor
necrosis factor; CRP indicates C-reactive protein;
ICAM-1 intercellular adhesion molecule 1; VEGF
indicates vascular endothelial growth factor.
*P value represents Prob > |r| under H0: Rho
(Spearman's rank correlation coefficient) = 0
S2
Supplemental Figure 1: Association of 28-day ambient black carbon (BC) moving average with
systolic blood pressure (SBP) at different blood mitochondrial DNA to nuclear DNA copy
number ratio (mtDNA/nDNA) levels, Normative Aging Study, 1999-2012.
Q1, Q2, Q3, and Q4 indicate the first, second, third, and fourth quartiles, respectively. The
association of BC on BP is modified by mtDNA/nDNA ratio, as indicated by the different slopes.
The four lines in the figure represent the estimated relationship between BC and BP when the
mtDNA/nDNA ratio is at the midpoint of a given quartile. If there was no effect modification,
the four lines would be the same. Results were adjusted for: apparent temperature and absolute
humidity at the matching time window; race; percentage of major cell types (lymphocytes,
neutrophils, and platelets); age; weekday; date of visit; body mass index; C-reactive protein>10
mg/L; smoking; alcohol use; season; physical activity; education level; use of calcium channel
blockers, β-blockers, and angiotensin-converting enzyme inhibitor.
S3