Subscriber access provided by Washington University | Libraries
Characterization of Natural and Affected Environments
Size-resolved endotoxin and oxidative potential of ambient particles in Beijing and Zürich Yang Yue, Haoxuan Chen, Ari Setyan, Miriam Elser, Maria Dietrich, Jing Li, Ting Zhang, Xiangyu Zhang, Yunhao Zheng, Jing Wang, and Maosheng Yao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01167 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018
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 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 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.
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 24
Environmental Science & Technology
1
Size-resolved endotoxin and oxidative potential of ambient
2
particles in Beijing and Zürich
3 4 5 6 7 8 9
Yang Yue1,2,3,#, Haoxuan Chen1,#, Ari Setyan2,3, Miriam Elser2,3, Maria Dietrich2, Jing Li1, Ting Zhang1, Xiangyu Zhang1, Yunhao Zheng1, Jing Wang2,3,*, Maosheng Yao1,* 1
State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of
10
Environmental Sciences and Engineering, Peking University, Beijing 100871, China
11
2
Institute of Environmental Engineering, ETH Zürich, Zürich 8093, Switzerland
12
3
Laboratory for Advanced Analytical Technologies, Empa, Swiss Federal Laboratories for Materials
13
Science and Technology, Dübendorf 8600, Switzerland
14 15
Revision to
16
Environmental Science and Technology
17
*
18
Maosheng Yao, email:
[email protected];
19
State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of
20
Environmental Sciences and Engineering, Peking University, Beijing 100871, China
Corresponding Authors
21 22
Jing Wang, email:
[email protected] 23
Institute of Environmental Engineering, ETH Zürich, Zürich 8093, Switzerland
24
Laboratory for Advanced Analytical Technologies, Empa, Swiss Federal Laboratories for Materials
25
Science and Technology, Dübendorf 8600, Switzerland
26 27
#
Y.Y. and H.C. contributed equally to this work
28 29
Beijing, China
30
Zürich, Switzerland
31
May 19, 2018
32 33 1
ACS Paragon Plus Environment
Environmental Science & Technology
34
Page 2 of 24
Abstract
35
PM2.5 pollution has become a global health concern, however its size-resolved health impact
36
remains to be poorly elucidated. Here, ambient particulate matter (PM) were collected into thirteen
37
different size ranges (10 nm-18 μm) and the mass, metal, endotoxin distributions and related
38
oxidative potential were investigated in two regions (Zürich, Switzerland and Beijing, China). Results
39
showed that the two regions had remarkably different PM distribution patterns. Swiss urban samples
40
had a mode around 40 nm with 23.3% of total PM mass, while Chinese samples featured two modes
41
around 0.75 and 4.23 μm with 13.8 -18.6% and 13.7-20.4% of total PM mass, respectively. Two peaks
42
for endotoxin at 40-100 nm and 1-4 μm were observed in different regions. For PM-borne metals,
43
Chinese samples had 67.6-100% of total Cd, As and Pb in the size range of 0.1–1 μm, and Swiss
44
samples had similar distributions of Cd and Pb but much lower total metals than Chinese samples.
45
The PM oxidative potential varied greatly with sizes for different regions. Accordingly, the current
46
practice, i.e., sole use of the mass concentration, could lead to inadequate health protection for one
47
region, but unnecessary economic costs for another without achieving extra health benefits.
48 49
Keywords: Particulate matter, Size distribution, Endotoxin, Heavy metal, Oxidative potential
50 51
1 Background:
52 53
Among the air pollutants, ambient particulate matter (PM) is widely studied due to its high risk
54
for respiratory and cardiovascular systems1, 2. Ambient PM contains organic compounds, metals,
55
materials of biological origin, etc. For the PM mediated health effects, the main mechanism in the
56
literature is described to be the oxidative stress, which is responsible for redox status changes3, 4, and
57
subsequent reactions associated with inflammation and apoptosis5-8. Generally, PM10 are classified as
58
inhalable particles, while PM2.5, the fine inhalable particles, can go deep into respiratory bronchioles.
2
ACS Paragon Plus Environment
Page 3 of 24
Environmental Science & Technology
59
Furthermore, the PM1, especially those particles smaller than 200 – 300 nm, can penetrate deep into
60
alveoli and lung epithelial cells, even translocate into blood circulation5,
61
geographical locations, the size distributions of ambient particles down to the nanoscale along with
62
their compositions such as endotoxin and transition metals remain to be poorly elucidated. Their
63
differences, if any, can lead to different levels of size-resolved PM toxicity in different regions.
9, 10
. For different
64 65
However, air quality is currently being assessed using PM mass concentration standard
66
regardless of locations11-14. Simple comparisons between PM concentration levels, while neglecting
67
its composition and size information, might lead to inadequate health protection for one region, but
68
unnecessary economic costs for another without achieving extra health benefits. Among many other
69
PM components, endotoxin is considered as one of the main causes of respiratory diseases inducing
70
sensitization, allergic asthma and other immune responses15-18. The endotoxin, also called
71
lipopolysaccharides, consists of a lipid and a polysaccharide composed of O-antigen, outer core and
72
inner core joined by a covalent bond. The endotoxin monomers are heterogeneous and tend to form
73
aggregates of varying sizes with apparent hydrodynamic radius of dozens of nanometers and a
74
molecular mass of 1000-7000 kDa19. In the innate immune system, endotoxin can strongly bind to
75
the CD14/TLR4/MD2 protein receptor complex located mainly at the surface of monocytes, dendritic
76
cells and macrophages, and trigger the secretion of pro-inflammatory cytokines and proteins that
77
eventually cause inflammation20, 21. A study on farmers showed that endotoxin exposure was related
78
to bronchial hyper-responsiveness, accelerated lung function decline, chronic obstructive pulmonary
79
disease, asthma-like symptoms and chronic bronchitis16. Recently, Zhong et al. found that exposures
80
to endotoxin significantly increased human systolic and diastolic blood pressure, and concluded that
81
biological PM components contribute significantly to PM-related cardiovascular symptoms22.
82
Accordingly, different size-resolved endotoxin levels could affect the PM toxicity. However, such
83
information for endotoxin as an important biological material of PM is significantly lacking or has not
84
received adequate attention. This situation has hampered a better understanding of the health
3
ACS Paragon Plus Environment
Environmental Science & Technology
85
Page 4 of 24
impacts by PM.
86 87
In this work, we collected ambient PM samples into thirteen different size ranges (10 nm - 18
88
μm) in different environmental settings both from Beijing, China and Zürich, Switzerland using the
89
NanoMOUDI (Micro-Orifice Uniform-Deposit Impactor, Model 122-NR, MSP Corporation, Shoreview,
90
MN, USA). The contents of PM samples including endotoxin and metals in different size ranges were
91
studied. In addition, the oxidative potential per unit of PM mass for different size ranges was also
92
analyzed using dithiothreitol (DTT) method. The rate of consumption of DTT is considered as a
93
quantitative method for the assessment of the oxidative potential of PM, which has been linked to
94
the toxic effects of PM8, 23-25. The composition of PM2.5 such as quinones, copper and manganese
95
were shown to contribute around 92% reactivity; whereas, metals, e.g. lead and nickel, have less
96
than 1% contribution of DTT consumption23. The results from our study can shed new light on size-
97
resolved endotoxin and toxicity of PMs from various locations when assessing their health effects
98
with different levels of air quality. Our results could serve as a reference to re-examine the current
99
practices of assessing air quality in different geographical locations.
100 101
2 Materials and methods:
102
2.1 Sample preparation
103
Our work mainly aimed to study the differences of size-resolved PM oxidative potential and its
104
biological fraction, i.e., endotoxin (which is a strong inflammation agent) of PM from different
105
locations with very different air pollution levels. To do so, we have chosen two cities, Beijing, China
106
and Zürich, Switzerland as examples for bad and good air quality levels, respectively. Four sampling
107
locations were selected in Switzerland and China as shown in Figure S1 (Supporting Information). The
108
sampling site of Swiss Federal Laboratories for Materials Science and Technology (Empa) was close to
109
the NABEL (National Air Pollution Monitoring Network) station at Empa, Zürich, which was
110
representative of a Swiss suburban campus. The sampling site of Oberneunforn is a farm in the 4
ACS Paragon Plus Environment
Page 5 of 24
Environmental Science & Technology
111
village Oberneunforn near Winterthur, Switzerland. The samples were collected in a barn with about
112
50 cows, which had an open roof in the middle and was supplied with fresh straws twice per day.
113
This site represents a farm environment in the rural region and a high concentration of endotoxin is
114
expected. In China, two sampling sites, Peking University campus and Zhongguancun North Str. close
115
to Beijing 4th Ring in Beijing were selected to represent the Beijing urban campus and urban main
116
street, respectively. To effectively identify each sampling site with indication of their environmental
117
settings, they were named as CH-suburban campus (samples from Empa, Federal Laboratories for
118
Materials Science and Technology, Switzerland), CH-farm (samples from Oberneunforn, Switzerland),
119
CN-urban campus (samples from Peking University, Beijing, China) and CN-urban main street
120
(samples from Zhongguancun North Str., Beijing, China). A NanoMOUDI was used to sample both
121
Swiss and Chinese air samples. The NanoMOUDI has thirteen stages with nominal cut points of
122
18000, 10000, 5600, 3200, 1800, 1000, 560, 320, 180, 100, 56, 32, 18 and 10 nm when operated at
123
an inlet flow rate of 30 L/min. During all sampling periods, the air inlet flow was 28.0-28.5 L/min,
124
which was within the allowed range of NanoMOUDI26.
125 126
All aerosol samples were collected onto clean aluminum foils. A total of one hundred and thirty
127
aluminum foil samples and twenty blanks were collected during June 2017 - September 2017 (SI,
128
Table S1). The Swiss farm samples were collected in January of 2017. During all sampling periods,
129
there was no rainfall or strong wind. All the samples were stored at -20 °C until analysis. The
130
meteorological data, from Swiss NABEL and Beijing Municipal Environmental Monitoring Center, for
131
each sampling site during the sampling periods were recorded for further analysis (SI, Table S1). The
132
extraction from filter samples followed the procedures reported in previous studies25, 27, 28. Each filter
133
sample was first cut into 12 equal pieces, and 5 pieces of them were deposited into 2 mL of pyrogen-
134
free water (Associates of Cape Cod, Inc., USA) with 0.05% Tween-20, in pyrogen-free centrifuge tubes,
135
and then subjected to vortex mixing for 120 min at 1200 rpm. In this procedure, water soluble
136
components, including endotoxin and soluble metals, as well as some parts of insoluble particles can
5
ACS Paragon Plus Environment
Environmental Science & Technology
Page 6 of 24
137
be extracted from the samples. During the process, all filter pieces were submerged in the elution
138
fluid. Each eluate was centrifuged for 10 min at 3500 rpm, to remove insoluble particles and filter
139
fragments that may interfere with the following analyses. The supernatants were then transferred to
140
pyrogen-free tubes for further tests. Blank aluminum foils were brought to site without sampling and
141
used as negative controls.
142 143
2.2 Sample analysis
144
Endotoxin test: For the endotoxin, all samples were analyzed using Limulus amebocyte lysate
145
(LAL) (Associates of Cape Cod Inc., East Falmouth, MA) according to the manufacturer’s instructions.
146
Endotoxin concentrations were reported as units (EU) referring to standard endotoxin, LPS from E.
147
coli. Briefly, equal volume of samples or standard solution and LAL reagent were incubated at 37 °C
148
and reacted to a gel-clot product, which had specific absorption at 405 nm. The absorption of 405
149
nm was recorded every 30 seconds for 60 min during the measurement. The detection limit for
150
endotoxin was 0.005 EU/mL. As the LAL method is very sensitive to endotoxin, all consumables were
151
pyrogen-free in the test. Spike control, positive control, negative control and blank samples were also
152
included to control the quality of the endotoxin test.
153 154
Metal analysis: Three hundred microliter extract of each sample in pyrogen-free water with
155
0.05% Tween-20 was diluted to 3 mL by pyrogen-free water. 0.1 mL 65% HNO3 (Titrisol, Merck,
156
Germany) was added to 3 mL of each sample to prepare acid solution for metal analysis. Target
157
metals concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS,
158
Thermo X series, Winsford, UK). A mixture standard solution of different elements was used in the
159
measurement (10 μg/mL, Agilent, USA). Rh and Re were used as the internal standards to control the
160
quality of measurements.
161 162
Oxidant generation test: The oxidant generation capacity of each sample was measured with
6
ACS Paragon Plus Environment
Page 7 of 24
Environmental Science & Technology
163
the dithiothreitol (DTT) method. One hundred microliter extract of each sample in pyrogen-free
164
water with 0.05% Tween-20 was incubated with 0.5 mM DTT solution in K2HPO4-KH2PO4 buffer at pH
165
7.4 and 37 °C for 30 min. The 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, 1mM) was used to titrate
166
the remaining DTT in the reaction mixture. The reaction product had specific absorbance at 412 nm.
167
As a standard oxidant, 1,4-naphthoquinone (1,4-NQ) can react with DTT and was used to calculate
168
the normalized index of oxidant generation (NIOG)25, 29, 30. The NIOG calculation is shown below
169
based on the index of oxidation generation (IOG) of 1,4-NQ:
= ,
170
,
171
=
172
∗ 100/(& ∗ ').
173 174
Here, T is the reaction time (min), M is the sample mass (μg), Absblank is the absorption of reaction
175
with pyrogen-free water with 0.05% Tween-20, Abssample is the absorption of the product after the
176
catalytic redox reaction.
177 178
PM characterization: The ambient PM samples were characterized by scanning electron
179
microscopy (SEM, Phenom ProX, Eindhoven, The Netherlands). PM samples on aluminum foils from
180
each stage of the NanoMOUDI were mounted on aluminum stubs and were analyzed in secondary
181
electron detector (SED) mode in 10 kV or 15 kV. All images were acquired at 5 000-times or 10 000-
182
times magnification.
183
2.3 Statistical analysis
184
All data were analyzed by GraphPad Prism version 6.01 for Windows (GraphPad Software Inc.,
185
San Diego, CA, USA). All samples were analyzed in triplicates. All the data were first tested for the
186
normality using D'Agostino & Pearson omnibus normality test. The size-resolved distribution of PM,
187
endotoxin and NIOG for different sampling sites were analyzed by two-way ANOVA and Tukey's
7
ACS Paragon Plus Environment
Environmental Science & Technology
Page 8 of 24
188
multiple comparisons test. Both PM size and sampling site factors were tested based on
189
homogeneous variance. Graphs were prepared with GraphPad Prism. For all sample analysis,
190
negative controls were used to prevent false positives. Values with p-value
