Subscriber access provided by - Access paid by the | UCSB Libraries
Article
Fine particulate matter constituents, nitric oxide synthase DNA methylation and exhaled nitric oxide Renjie Chen, Liping Qiao, Huichu Li, Yan Zhao, Yunhui Zhang, Wenxi Xu, Cuicui Wang, Hongli Wang, Zhuohui Zhao, Xiaohui Xu, Hui Hu, and Haidong Kan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02527 • Publication Date (Web): 15 Sep 2015 Downloaded from http://pubs.acs.org on September 17, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 34
Environmental Science & Technology
Fine particulate matter constituents, nitric nitric oxide synthase DNA methylation and exhaled nitric oxide
Renjie Chen,†‡¶ Liping Qiao,§¶ Huichu Li,† Yan Zhao,† Yunhui Zhang,† Wenxi Xu,
∥
Cuicui Wang,† Hongli Wang,
§
Zhuohui Zhao,† Xiaohui Xu,⊥ Hui Hu,#
Haidong Kan†‡*
†
School of Public Health, Key Lab of Public Health Safety of the Ministry of
Education, & Key Lab of Health Technology Assessment of the Ministry of Health, Fudan University, Shanghai, China ‡
Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention
(LAP3), Fudan University, Shanghai, China §
State Environmental Protection Key Lab of the Formation and Prevention of
Urban Air Pollution Complex, Shanghai Academy of Environmental Sciences, Shanghai, China ∥
Huangpu District Center for Disease Control and Prevention, Shanghai,
China ⊥
Department of Epidemiology & Biostatistics, Texas A&M School of Public
Health, Texas, USA. #
Department of epidemiology, College of Public Health and Health
Professionals, College of Medicine, University of Florida, Gainesville, Florida, USA. 1
ACS Paragon Plus Environment
Environmental Science & Technology
¶
Page 2 of 34
Renjie Chen and Liping Qiao contributed equally to this work.
*Corresponding
author:
Tel/fax:
+86
(21)
54237908;
e-mail:
[email protected]; mail: P.O. Box 249, 130 Dong-An Road, Shanghai 200032, China.
2
ACS Paragon Plus Environment
Page 3 of 34
Environmental Science & Technology
1
ABSTRACT (199 words)
2
It remains unknown how fine particulate matter (PM2.5) constituents affect
3
differently the fractional concentration of exhaled nitric oxide (FeNO, a
4
biomarker of airway inflammation) and the DNA methylation of its encoding
5
gene (NOS2A). We aimed to investigate the short-term effects of PM2.5
6
constituents on NOS2A methylation and FeNO. We designed a longitudinal
7
study among chronic obstructive pulmonary disease (COPD) patients with 6
8
repeated health measurements in Shanghai, China. We applied linear
9
mixed-effect models to evaluate the associations. We observed that the
10
inverse association between PM2.5 and methylation at position 1 was limited
11
within 24 hours, and the positive association between PM2.5 and FeNO was
12
the strongest at lag 1 day. Organic carbon, element carbon, NO3- and NH4+
13
were robustly and significantly associated with decreased methylation and
14
elevated FeNO. An interquartile range increase in total PM2.5 and the four
15
constituents was associated with decreases of 1.19, 1.63, 1.62, 1.17 and 1.14
16
in
17
13.30%,16.93%, 8.97%, 18.26% and 11.42% in FeNO, respectively. Our
18
results indicated that organic carbon, element carbon, NO3- and NH4+ might be
19
mainly responsible for the effects of PM2.5 on the decreased NOS2A DNA
20
methylation and elevated FeNO in COPD patients.
21
Key words: fine particulate matter; constituent; DNA methylation; airway
22
inflammation
percent
methylation
of
NOS2A,
respectively,
3
ACS Paragon Plus Environment
and
increases
of
Environmental Science & Technology
23
Main text (3973 words)
24
INTRODUCTION
25
A large number of epidemiological studies have reported the hazardous
26
respiratory effects of short-term exposure to fine particulate matter
27
(aerodynamic diameter less than 2.5 micrometer, PM2.5) air pollution, including
28
increased risks of respiratory mortality and hospitalization.1 Elevated
29
inflammation in the respiratory tract is one of the key mechanisms involved in
30
the development of PM2.5-related outcomes.2 As a well-known noninvasive
31
biomarker for assessing airway inflammation, fractional concentration of
32
exhaled nitric oxide (FeNO) has been positively linked to an exposure to PM2.5
33
in recent human studies.2-5 These findings suggest that FeNO is a useful
34
intermediary phenotype in the pathophysiological process of airway
35
inflammation triggered by PM2.5.3
36
The underlying mechanisms linking PM2.5 and FeNO are not fully
37
characterized. There is increasing evidence that epigenetic alternations,
38
typically DNA methylation, can change the expression and function of a gene
39
under exogenous stimuli without changing in its DNA sequence.6 Inducible
40
nitric oxide synthase (iNOS; encoded by nitric oxide synthase isoform 2A
41
[NOS2A], Genbank accession number AF017634) is the major enzyme
42
responsible for nitric oxide synthesis in the respiratory tract.7 Hypomethylation
43
in the promoter of NOS2A gene was previously shown to be inversely related
44
to iNOS.8, 9 Several prior studies have revealed a potential downward influence 4
ACS Paragon Plus Environment
Page 4 of 34
Page 5 of 34
Environmental Science & Technology
45
of PM2.5 on the promoter DNA methylation of NOS2A,3, 10, 11 but the time-lag
46
patterns of PM2.5, NOS2A methylation and FeNO are largely unclear.
47
Furthermore, PM2.5 has a very complex chemical composition, which raises
48
the question of how these constituents affect differently the levels of NOS2A
49
DNA methylation and FeNO. One investigation in the Boston area Normative
50
Aging Study evaluated the association between two components (black carbon
51
and sulfate) and global DNA methylation.6 However, this knowledge remains
52
quite limited. Therefore, we aimed to investigate the short-term effects of PM2.5
53
constituents on the DNA methylation of NOS2A promoter and FeNO in
54
Shanghai, a Chinese city with high PM2.5 levels. We tested these hypotheses
55
in a panel of chronic obstructive pulmonary disease (COPD) patients because
56
respiratory inflammation plays an important role in the development and
57
exacerbation of this disease.
58 59
MATERIALS AND METHODS
60
Design and population. population. This is a longitudinal panel study with repeated
61
measurements on the exposure and health. We initially recruited 30 retired
62
COPD patients from a central urban community in Shanghai with a total area
63
of 1.9 km2. All COPD diagnoses were confirmed by physicians before they
64
were finally included in this study. To reduce the influence of respiratory
65
medication on our results, we only included the stable patients with
66
mild-to-moderate COPD in this study according to the classification of Global 5
ACS Paragon Plus Environment
Environmental Science & Technology
67
Initiative for Chronic Obstructive Lung Disease based on the baseline test of
68
spirometry.12 We excluded those who were current active or passive smokers
69
(living with a current smoker); former smokers (quit smoking for at least 3
70
years); consumed any alcohol; or had severe comorbidities or inflammatory
71
diseases. To reduce the inherent seasonal variations of FeNO and DNA methylation
72 73
13,
six weekly follow-up visits were scheduled from May 27th to July 5th, 2014.
74
To fully capture the variations in the exposure and biomarkers, we arranged
75
health examinations on various weekdays (i.e., Tuesday, Thursday, and
76
Saturday). For each patient, physical examinations were scheduled at the
77
same time (1:30 p.m. to 2:30 p.m.) on the same day of the week in order to
78
control for possible circadian rhythm and day-of-week effects. Data on
79
individual demographic and medical characteristics, such as age, gender,
80
height, weight, education attainment, income, medication use, duration of
81
COPD and chronic comorbidities, were collected during the baseline visit.
82
Study subjects were asked to record any use of medications, acute
83
exacerbation of COPD, and whether they went out of the central urban areas
84
during the study period. The Institutional Review Board in the School of Public
85
Health at Fudan University approved the study protocol. We obtained written
86
consent forms from all subjects.
87
FeNO measurements. measurements. All physical examinations were conducted at the
88
Community Health Center. We measured FeNO levels using a portable NIOX 6
ACS Paragon Plus Environment
Page 6 of 34
Page 7 of 34
Environmental Science & Technology
89
MINO machine (Aerocrine AB, Solna, Sweden) according to standardized
90
procedures by the American Thoracic Society and the European Respiratory
91
Society. A maximum of 5 repeated tests with 5 minutes rest between testes
92
were allowed for subjects who could not complete the test at the first time.
93
Foods, beverages, and intense exercises were not allowed at least within one
94
hour before the FeNO measurements.
95
Collection of buccal samples. Buccal samples were collected directly after the
96
FeNO test. Specifically, the subjects were asked to rinse the mouth using
97
purified water. They were provided with two tooth-brushes and instructed to
98
brush the teeth using the first one. Then, they were instructed to moderately
99
brush the bilateral buccal mucosa 20 times at each side with the second
100
toothbrush. The brush was then placed in a container that was filled with 10-ml
101
phosphate buffer solution (Recipe: NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4
102
10 mmol/L and KH2PO4 4.2mmol/L). At last, patients swished liquid (10 ml)
103
throughout their mouths and expelled the fluid into the container. Buccal-cell
104
suspensions were immediately centrifuged at 1,000 r/min in the laboratory,
105
and the pellets were stored frozen at –80°C until used for DNA extraction.
106
DNA methylation. Genomic DNA was extracted using the QIAmp DNA Mini Kit
107
(Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
108
Purified DNA was quantified using a ND1000 spectrophotometer (Nanodrop,
109
Wilmington, DE, USA) and about 300 ng DNA was bisulfite modified using the
110
EZ Methylation Gold-Kit (Zymo Research, Orange, CA, USA). Final elution 7
ACS Paragon Plus Environment
Environmental Science & Technology
111
was performed with a 10 μl M-Elution Buffer.
112
We examined three CpG loci located in NOS2A according to previous
113
studies. To be specific, positions 1 and 2 were in a non-CpG island region of
114
the promoter because the promoter was previously shown to be inversely
115
related to iNOS.9,
116
between exon 1 and exon 2.10 We performed DNA methylation analyses using
117
a bisulfite-PCR and pyrosequencing assay. We evaluated the levels of DNA
118
methylation at 2 loci (position 1 and 2) in the non-CpG island regions of
119
NOS2A promoter, which have been negatively associated with PM2.5 exposure
120
in previous studies.10, 14 The detailed method, location of the gene promoter,
121
amplified regions, and CpG sites that were evaluated have been published
122
previously.10 Methylation level of each CpG dinucleotide was expressed as
123
methylated cytosines over the sum of methylated and unmethylated cytosines,
124
i.e., the percentage of 5-methylcytosine (%5mC). Each sample was tested in
125
triplicate and the average was used for statistical analysis. Ten controls were
126
included in every pyrosequencing run. To ensure that pyrosequencing was
127
sequencing the correct pattern, two wells were filled with oligonucleotide with
128
known sequence. To verify bisulfite conversion efficiency, built-in controls were
129
used in every assay. Moreover, a human unmethylated (0%) standard and fully
130
methylated (100%) standard were used as sample controls.
131
Environmental data. Real-time concentrations of PM2.5 and its constituents
132
were measured by a fixed-site monitor located on the rooftop of a five-story
10
Position 3 was located in a non-CpG island region
8
ACS Paragon Plus Environment
Page 8 of 34
Page 9 of 34
Environmental Science & Technology
133
building (about 15 m high above the ground) at Shanghai Academy of
134
Environmental Sciences, which was about 4 km away from the community
135
where the subjects resided. Both the sites were not in direct vicinity of major
136
sources of air pollution including main roads. The mass concentration of PM2.5
137
was measured by an online particulate monitor (FH 62 C14 series, Thermo
138
Fisher Scientific Inc.) using beta attenuation techniques equipped with a
139
verified PM2.5 cyclone. The carbonaceous concentrations [i.e., organic carbon
140
(OC) and elemental carbon (EC)] in PM2.5 were measured by a
141
semicontinuous OC/EC analyzer (model RT-4, Sunset Laboratory Inc.)
142
equipped with a PM2.5 cyclone and an upstream parallel-plate organic denuder
143
(Sunset Laboratory Inc.). The concentrations of 8 major water-soluble
144
inorganic ions in PM2.5 (Cl−, NO3−, SO42−, NH4+, Na+, K+, Mg2+, Ca2+) were
145
measured by a commercial instrument for online monitoring of aerosols and
146
gases (MARGA, model ADI 2080, Applikon Analytical B.V.). The principle and
147
operation of this instrument has been given in detail elsewhere15,
148
obtained daily mean temperature and mean relative humidity from Shanghai
149
Meteorological Bureau to allow for controlling for the confounding effects of
150
weather conditions.
151
Statistical analyses. Before statistical analyses, FeNO levels were natural
152
log-transformed because they were not normally distributed, but such
153
transformation was not required for the NOS2A promoter methylation because
154
the data were normally distributed. Environmental and individual data were 9
ACS Paragon Plus Environment
16.
We
Environmental Science & Technology
155
merged by the time of physical examinations (rounded to the integer hour).
156
We applied the linear mixed-effect model to evaluate the FeNO-PM2.5
157
association and methylation-PM2.5 association.11 In the basic model, PM2.5 and
158
its components were incorporated as the fixed-effect terms one at a time; a
159
random intercept for each patient was added to account for correlations among
160
multiple repeated measurements collected per person. We also included
161
several covariates as fixed-effect terms: (1) an indicator variable of “week” of
162
physical examinations to exclude any unknown weekly time trends in the levels
163
of FeNO and DNA methylation; (2) an indicator variable of “day of the week” to
164
exclude any variations of the response variables in one week; (3) the moving
165
average of mean temperature and relative humidity on the present day and
166
previous 3 days to control for the confounding effects of weather conditions 17;
167
and (4) individual characteristics including age, gender, body mass index,
168
education, duration of COPD and chronic comorbidities.
169
In addition to the above basic single-constituent model, we also built a
170
“constituent-PM2.5 adjustment model” after controlling for the confounding
171
effects of total PM2.5 mass, as well as a “constituent-residual model” after
172
accounting for the collinearity between a constituent and total PM2.5.18 For the
173
third model, we firstly obtained the residual of each constituent by establishing
174
a linear regression model between total PM2.5 and a constituent, and then
175
introduced the residual into the basic model replacing the individual
176
constituents. The constituent residual can be interpreted as a crude measure 10
ACS Paragon Plus Environment
Page 10 of 34
Page 11 of 34
Environmental Science & Technology
177
of the “independent” contribution of each constituent to the effects of PM2.5
178
after excluding its collinearity of the remaining constituents.19
179
In order to fully investigate the time-lag patterns for the effects of PM2.5, we
180
examined the above models using multiple periods preceding the time of
181
physical examinations, i.e., single lags of 0–6 hour (h), 7–12 h, 13–24 h, 0–24 h
182
(0 d), 25–48 h (1 d), 2 d and 3–7 d.
183
As an additional analysis, we included a multiplicative interaction term for
184
PM2.5 or its constituent and methylation in single-pollutant model or each
185
single-constituent models to examine whether the association between PM2.5
186
(or its constituents) and FeNO can be modified by NOS2A methylation.3
187
The statistical tests were two-sided, and values of P
