Subscriber access provided by MT ROYAL COLLEGE
Article
Comparative cardiopulmonary effects of particulate matter- and ozone-enhanced smog atmospheres in mice Mehdi Hazari, Kimberly Stratford, Q. Todd Krantz, Charly King, Jonathan Krug, Aimen Farraj, and M. Ian Gilmour Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04880 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 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 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 36
1 2
Environmental Science & Technology
Comparative cardiopulmonary effects of particulate matter- and ozone-enhanced smog atmospheres in mice
3
Mehdi S. Hazari1*, Kimberly M. Stratford2, Q. Todd Krantz3, Charly King3, Jonathan Krug4, Aimen K. Farraj1 and M. Ian Gilmour1
4 5 6 7
8 9 10 11 12 13 14 15 16 17
1
Cardiopulmonary and Immunotoxicology Branch, Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 2
Curriculum in Toxicology, University of North Carolina – Chapel Hill, Chapel Hill, NC, 27599 3
Inhalation Toxicology Facilities Branch, Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 4
Exposure Methods and Measurement Division, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
18 19 20 21 22 23 24 25 26 27 28
*Corresponding author: Mehdi S. Hazari, Environmental Public Health Division, USEPA, 109 Alexander Drive, B105; Research Triangle Park, NC 27711; (Phone: 919-541-4588; Fax: 919-541-0034; email:
[email protected])
29 30 31
Running title: Greater cardiac effects of O3-enhanced smog than PM-enhanced
32
ACS Paragon Plus Environment
Environmental Science & Technology
33 34
Abstract
35
This study was conducted to compare the cardiac effects of particulate matter (PM)-
36
(SA-PM) and ozone(O3)-enhanced (SA-O3) smog atmospheres in mice. Based on our
37
previous findings of filtered diesel exhaust we hypothesized that SA-O3 would cause
38
greater cardiac dysfunction than SA-PM. Radiotelemetered mice were exposed to either
39
SA-PM, SA-O3, or filtered air (FA) for 4 hours. Heart rate (HR) and electrocardiogram
40
were recorded continuously before, during and after exposure. Both SA-PM and SA-O3
41
increased heart rate variability (HRV) but only SA-PM increased HR. Normalization of
42
responses to total hydrocarbons, gas-only hydrocarbons and PM concentration were
43
performed to assess the relative contribution of each phase given the compositional
44
variability. Normalization to PM concentration revealed that SA-O3 was more potent in
45
increasing HRV, arrhythmogenesis, and causing ventilatory changes. However, there
46
were no differences when the responses were normalized to total or gas-phase only
47
hydrocarbons. Thus, this study demonstrates that a single exposure to smog causes
48
cardiac effects in mice. Although the responses of SA-PM and SA-O3 are similar, the
49
latter is more potent in causing electrical disturbances and breathing changes
50
potentially due to the effects of irritant gases, which should therefore be accounted for
51
more rigorously in health assessments.
52
53
54
Abstract art
ACS Paragon Plus Environment
Page 2 of 36
Page 3 of 36
Environmental Science & Technology
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
Introduction
74 75
The association between air pollution exposure and cardiovascular disease is well-
76
established, particularly in people with certain risk factors like high blood pressure or
ACS Paragon Plus Environment
Environmental Science & Technology
77
those with pre-existing conditions1. The picture is less clear for healthy individuals and
78
indeed there are still several unknowns with regards to how air pollution mediates
79
cardiovascular dysfunction in this group, especially when there are no observable
80
symptoms. In addition, nationwide variations in air pollution composition make it difficult
81
to determine exactly which components, or combination of components, drive the
82
response. This is the case for complex air pollution mixtures like smog, which originates
83
as a set of primary pollutants (e.g. nitrogen oxides, volatile organic compounds) that are
84
released from vehicles or industrial sources but thereafter transform after reacting with
85
ultraviolet light to produce secondary pollutants like ozone and certain organic aerosols.
86
Given these uncertainties, research studies need to address the comparative effects of
87
multiple smog atmospheres and focus on determining which compositions, and hence
88
components, cause the most serious health effects.
89 90
The American Heart Association cites several pathways by which air pollution,
91
especially particulate matter (PM), leads to decrements in cardiovascular function2,3.
92
These include elicitation of oxidative stress and inflammation, alteration of vasomotor
93
properties, translocation of certain pollutants into the systemic circulation and
94
modulation of autonomic controls which regulate the heart and vasculature. Some of
95
these may not manifest as overt symptoms, especially in young healthy individuals, but
96
instead as shifts in normal physiological function which render a person susceptible to
97
subsequent stressors or triggers of adverse responses. We previously showed that
98
exposure to a relatively low concentration of diesel exhaust, which contains many of the
99
same pollutants as smog, caused increased arrhythmias and other cardiovascular
ACS Paragon Plus Environment
Page 4 of 36
Page 5 of 36
Environmental Science & Technology
100
effects when healthy rats were challenged with an exercise-like stressor4,5. The insight
101
gained from these and other studies is that exposure to complex air pollution mixtures
102
results in latent autonomic shifts which cause a decrease in cardiac performance during
103
exposure and even up to one day later.
104 105
Although many epidemiological studies point to PM as the main cause of air pollution’s
106
impacts on the cardiovascular system6-8, there are numerous human and rodent studies
107
that implicate ubiquitous gaseous irritants like ozone as well9-11. In fact, our previous
108
studies have shown that cardiovascular dysfunction occurs, sometimes at comparable
109
levels or even after PM is removed from a multipollutant mixture like diesel exhaust12-14.
110
Furthermore, the magnitude of the response is not just determined by the relative levels
111
of PM and ozone but also by the resulting physical and chemical interactions that occur
112
in the mixture. Therefore, the objective of this study was to examine and compare the
113
cardiovascular responses of mice exposed to either a simulated high PM/low ozone
114
(SA-PM) or low PM/high ozone (SA-O3) smog atmosphere. These photo-oxidized
115
mixtures approximated urban and regional evaporative pollutant emissions with similar
116
Air Quality Health Index values. We previously determined that filtered diesel exhaust
117
causes greater acute cardiac effects than whole diesel exhaust, and so we
118
hypothesized that although both smog mixtures would elicit cardiac changes in mice,
119
the atmosphere dominated by gaseous irritants would be more potent, particularly in
120
causing electrical disturbances like arrhythmia.
121 122 123
Methods
ACS Paragon Plus Environment
Environmental Science & Technology
124 125
Animals - Female C57BL/6 (21 ± 1.1 g) mice between 13 and 15 weeks of age were
126
used in this study (Jackson Laboratory - Bar Harbor, ME). Mice were initially housed
127
five per cage and maintained on a 12-hr light/dark cycle at approximately 22°C and 50%
128
relative humidity in an AAALAC–approved facility. Food (Prolab RMH 3000; PMI
129
Nutrition International, St. Louis, MO) and water were provided ad libitum. All protocols
130
were approved by the Institutional Animal Care and Use Committee of the U.S.
131
Environmental Protection Agency and are in accordance with the National Institutes of
132
Health Guides for the Care and Use of Laboratory Animals. Animals were randomly
133
assigned to one of the following groups after implantation of radiotelemeters: (1) filtered
134
air (FA), (2) SA-PM, or (3) SA-O3.
135 136
Surgical Implantation of Radiotelemeters – Mice were implanted with radiotelemeters as
137
previously described15. Animals were anesthetized using inhaled isoflurane (Isothesia,
138
Butler Animal Health Supply, Dublin OH). Anesthesia was induced by spontaneous
139
breathing of 2.5% isoflurane in pure oxygen at a flow rate of 1 L/min and then
140
maintained by 1.5% isoflurane in pure oxygen at a flow rate of 0.5 L/min; all animals
141
received the analgesic buprenorphrine (0.03 mg/kg, i.p. manufacturer). Briefly, using
142
aseptic technique, each animal was implanted subcutaneously with a radiotelemeter
143
(ETA-F10, Data Sciences International, St Paul, MN), the transmitter was placed under
144
the skin to the right of the midline on the dorsal side. The two electrode leads were then
145
tunneled subcutaneously across the lateral dorsal sides, and the distal portions were
146
fixed in positions that approximated those of the lead II of a standard electrocardiogram
147
(ECG). Body heat was maintained both during and immediately after the surgery.
ACS Paragon Plus Environment
Page 6 of 36
Page 7 of 36
Environmental Science & Technology
148
Animals were given food and water post-surgery and were housed individually. All
149
animals were allowed 7-10 days to recover from the surgery and reestablish circadian
150
rhythms.
151 152
Radiotelemetery Data Acquistion - Radiotelemetry methodology (Data Sciences
153
International, Inc., St. Paul, MN) was used to track changes in cardiovascular function
154
by monitoring heart rate (HR), and ECG waveforms immediately following telemeter
155
implantation, through exposure until 24hours post-exposure. This methodology provided
156
continuous monitoring and collection of physiologic data from individual mice to a
157
remote receiver. Sixty-second ECG segments were recorded every 15 minutes during
158
the pre- and post-exposure periods and every 5 minutes during exposure (baseline and
159
hours 1-4); HR was automatically obtained from the waveforms (Dataquest ART
160
Software, version 3.01, Data Sciences International, St. Paul, MN, USA). All animals
161
were acclimated to the exposure chambers on two separate occasions, even then, an
162
increase in HR was always observed when animals were placed in the chamber on the
163
day of exposure.
164 165
Electrocardiogram Analysis - ECGAuto software (EMKA Technologies USA, Falls
166
Church VA) was used to visualize individual ECG waveforms, analyze and quantify
167
ECG segment durations and areas, as well as identify cardiac arrhythmias as previously
168
described (Kurhanewicz et al. 2014). Briefly, using ECGAuto, Pwave, QRS complex,
169
and T-wave were identified for individual ECG waveforms and compiled into a library.
170
Analysis of all experimental ECG waveforms was then based on established libraries.
ACS Paragon Plus Environment
Environmental Science & Technology
171
The following parameters were determined for each ECG waveform: PR interval (Pstart-
172
R), QRS complex duration (Qstart-S), ST segment interval (S-Tend) and QT interval (Qstart-
173
Tend). QT interval was corrected for HR using the correction formula for mice QTc =
174
QT/(RR/100)1/216. Pre-exposure assessments were measured as the exposure time-
175
matched four hours of data from 24 hours before exposure for each animal. Immediately
176
post-exposure assessments were the four hours of data taken immediately post-
177
exposure. Twenty-four post-exposure assessments were the exposure time-matched
178
four hours of data taken 24 hours after exposure.
179 180
Heart Rate Variability Analysis - Heart rate variability (HRV) was calculated as the mean
181
of the differences between sequential RRs for the complete set of ECG waveforms
182
using ECGAuto. For each 1-min stream of ECG waveforms, mean time between
183
successive QRS complex peaks (RR interval), mean HR, and mean HRV-analysis–
184
generated time-domain measures were acquired. The time-domain measures included
185
standard deviation of the time between normal-to-normal beats (SDNN), and root mean
186
squared of successive differences (RMSSD). HRV analysis was also conducted in the
187
frequency domain using a Fast-Fourier transform. The spectral power obtained from this
188
transformation represents the total harmonic variability for the frequency range being
189
analyzed. In this study, the spectrum was divided into low-frequency (LF) and high-
190
frequency (HF) regions. The ratio of these two frequency domains (LF/HF) provides an
191
estimate of the relative balance between sympathetic (LF) and vagal (HF) activity.
192
ACS Paragon Plus Environment
Page 8 of 36
Page 9 of 36
Environmental Science & Technology
193
Whole-Body Plethysmography - Ventilatory function was assessed in awake,
194
unrestrained mice using a whole-body plethysmograph (Buxco, Wilmington, NC).
195
Assessments were performed one day before exposure, immediately post-exposure
196
and 24hrs after exposure. The plethysmograph pressure was monitored using
197
Biosystems XA software (Buxco Electronics Inc., Wilmington, NC). Using respiratory-
198
induced fluctuations in ambient pressure, ventilatory parameters including tidal volume
199
(VT), breathing frequency (f), inspiratory time (Ti), and expiratory time (Te), were
200
calculated and recorded on a breath-by-breath basis.
201 202
Photochemical Smog Exposures – A gasoline blend was combined with either α-pinene
203
or isoprene to produce a hydrocarbon mixture that was enhanced for particulate matter
204
(PM) or ozone (O3) during irradiation, respectively. Thus, photochemical smog
205
atmospheres with either high PM2.5 and low O3/nitrogen oxide (NOx) concentrations
206
(SA-PM) or low PM2.5 and high O3/NOx (SA-O3) were generated in the Mobile Reaction
207
Chamber (MRC). Briefly, SA-PM was artificially generated with 0.491 ppm nitrogen
208
oxide (NO), 0.528 ppm NOx, 29.9 ppmC total hydrocarbons (THC), 24 ppmC gasoline
209
and 5.3 ppmC α -pinene as the initial conditions, whereas SA-O3 was generated from
210
0.794 ppm NO, 0.912 ppm NOx, 12.4 ppmC THC, 5.2 ppmC isoprene and 7.21 ppmC
211
gasoline. Each of the initial smog mixtures was then irradiated with ultraviolet light.
212
Smog was then transported under vacuum to 0.3 m3 whole body inhalation chambers
213
where mice were exposed once for four hours. Continuous gas and aerosol sampling
214
for carbon monoxide (CO), O3, NOx, THC and particle mass concentration were
215
conducted at both the MRC unit as well as from the inhalation exposure systems. All
ACS Paragon Plus Environment
Environmental Science & Technology
216
PM was formed as secondary organic aerosol (SOA) from the photochemical reactions
217
in the MRC. Particle size distributions and gravimetric mass sampling was measured,
218
and filter sampling for gravimetric analysis were conducted for the entire exposure time.
219
Volatile organic compound (VOC) summa cannisters were periodically collected and
220
analyzed by gas chromatography off-line to determine concentrations of various volatile
221
organic compounds (VOCs) in the exposure atmosphere. Animals exposed to FA
222
received room air, which was transported to the chambers after being HEPA filtered.
223
Please refer to Krug et al. in this issue for complete exposure details.
224 225
Statistics - All data are expressed as means ± SEM. Statistical analyses were
226
performed using Sigmaplot 13.0 (Systat Software, San Jose, CA) software. The delta
227
values (i.e. change during exposure from baseline) of HR, HRV and the ventilatory
228
parameters were used and a two-way analysis of variance (ANOVA) for repeated-
229
measures was employed with Bonferroni post hoc tests to determine statistical
230
differences. Raw ECG and arrhythmia count data were analyzed by two-way ANOVA.
231
Raw HR, HRV, ventilatory parameters and arrhythmia counts were also normalized to
232
total hydrocarbons (both particle and gas phases), gas-phase hydrocarbons only or PM
233
concentration by dividing the physiological parameters by the pollutant concentrations.
234
Normalizations were done to compare the relative contribution of these components to
235
the physiological response to Smog A versus B and their relative potencies; no
236
statistical comparisons were made to FA post-normalization. The statistical significance
237
was set at P < 0.05.
238 239
ACS Paragon Plus Environment
Page 10 of 36
Page 11 of 36
240
Environmental Science & Technology
Results
241 242
Exposure characteristics – Table 1 shows the inhalation chamber concentrations of
243
PM2.5 (i.e. secondary organic aerosols), NOx, O3, as well as total hydrocarbons in the
244
complete smog mixture (particle and gas phases) and in the gas-phase only for both
245
SA-PM (enhanced PM2.5) and SA-O3 (enhanced O3). In addition, the respective Air
246
Quality Health Indexes (AQHI) and Air Quality Indexes (AQI) along with their color
247
codes are also included to provide a health-risk comparison between these
248
atmospheres. These indices describe the health risks from polluted air on individual
249
days throughout the year, either from independent pollutants as in the case of AQI, or
250
from a cumulative PM2.5, O3, and NOx perspective as with AQHI. Refer to Krug et al. in
251
this issue for a more comprehensive comparison of SA-PM (MR044) and SA-O3
252
(MR059).
253 254
Heart rate – There was no difference in the baseline HR, which was the resting level
255
measured in the home cages, between any of the groups (FA = 588.0 ± 19.8 bpm; SA-
256
PM = 568.6 ± 7.5 bpm; SA-O3 = 565.9 ± 9.4 bpm). In general, all animals experienced a
257
decrease in HR during the exposure; although, SA-PM caused a significant increase in
258
HR during Hour1 and less HR decrease over the remaining exposure period when
259
compared to FA and SA-O3 (Fig. 1A). However, there was no difference between SA-
260
PM and SA-O3 when the responses were normalized to THC (Fig. 1B) or gas-phase
261
hydrocarbons only (Fig. 1C). On the other hand, HR decrease was significantly greater
ACS Paragon Plus Environment
Environmental Science & Technology
262
during SA-O3 when compared to SA-PM after normalization to PM concentration (Fig.
263
1D).
264 265
Arrhythmia – None of the animals experienced cardiac arrhythmias during the pre-
266
exposure period. Figure 2 shows the raw arrhythmia counts during the four-hour
267
exposure. Overall, exposure to SA-O3 increased the incidence of arrhythmias when
268
compared to FA (Fig. 2A-D) but there was no difference with respect to SA-PM in the
269
denormalized (Fig. 2A) and THC normalized (Fig. 2B) responses. In contrast, SA-O3
270
caused significantly more arrhythmias than SA-PM when the responses were
271
normalized to gas-phase hydrocarbons (Fig. 2C) or PM concentrations (Fig. 2D).
272 273
Heart rate variability – Figures 3-5 show the changes in HRV during exposure. There
274
were no significant differences in the baseline HRV measures between any of the
275
groups (FA – SDNN = 7.4 ± 0.7 msec, RMSSD = 4.0 ± 0.5 msec, LF/HF = 8.4 ± 0.7
276
msec2; SA-PM – SDNN = 7.9 ± 0.7 mscec, RMSSD = 5.3 ± 0.6 msec, LF/HF = 7.1 ± 0.6
277
msec2; SA-O3 – SDNN = 8.7 ± 0.7 msec, RMSSD = 5.5 ± 0.7 msec, LF/HF = 6.7 ± 0.6
278
msec2). SDNN decreased in the first hour of exposure in control animals; this was likely
279
due to sympathetic modulation from the stress of handling and placement in the
280
chamber. Animals exposed to either SA-PM or SA-O3 had a significant increase in
281
SDNN (Fig. 3A) when compared to FA and a similar trend was observed for RMSSD
282
(Fig. 4A). Both time-domain measures showed minimal to no effects for either SA-PM or
283
SA-O3 when the responses were normalized to THC (Fig. 3B and 4B) or gas-phase
284
hydrocarbons only (Fig. 3C and 4C). However, the increase in SDNN and RMSSD
ACS Paragon Plus Environment
Page 12 of 36
Page 13 of 36
Environmental Science & Technology
285
during SA-O3 exposure was significantly greater than SA-PM when the data were
286
normalized to PM concentration (Fig. 3D and 4D). On the other hand, there were no
287
significant effects in the frequency-domain measures (i.e. LF/HF) for either smog
288
atmosphere with or without normalization of responses (Fig. 5).
289 290
Ventilatory function – Whole-body plethysmography was performed on all animals
291
during the baseline period as well as immediately and one day after exposure; results
292
shown in Fig. 6 compare the changes in ventilatory parameters from baseline between
293
the groups. Baseline values did not differ between the groups (FA – f = 475.1 ± 13.1
294
breaths/min, VT = 0.24 ± 0.01 ml, Ti = 0.06 ± 0.002 sec, Te = 0.08 ± 0.002 sec; SA-PM –
295
f = 509.8 ± 6.3 breaths/min, VT = 0.24 ± 0.01 ml, Ti = 0.05 ± 0.001 sec, Te = 0.07 ±
296
0.001 sec; SA-O3 – f = 499.7 ± 12.1 breaths/min, VT = 0.25 ± 0.01 ml, Ti = 0.06 ± 0.002
297
sec, Te = 0.07 ± 0.002 sec). There were no differences in f, VT, Ti or Te between any of
298
the groups in the unnormalized results (Fig. 6A). However, normalizing to THC (Fig. 6B)
299
or gas-phase only hydrocarbons (Fig. 6C) revealed a significant decrease in f, and
300
increase in VT and Ti in SA-PM and SA-O3 immediately after exposure when compared
301
to controls. Some of these responses relative to FA persisted one day after exposure
302
but there was no difference between SA-PM and SA-O3. In contrast, normalization to
303
PM concentrations revealed a significant increase in f, and decrease in VT and Ti
304
immediately after exposure in SA-O3-exposed animals when compared to SA-PM (Fig.
305
6D); only the changes in f and VT persisted 24hrs later.
306
ACS Paragon Plus Environment
Environmental Science & Technology
307
Electrocardiogram – There were no baseline differences in any ECG parameters
308
between any of the groups. Changes in ECG during exposure were restricted to a PR
309
interval prolongation in mice exposed to SA-PM, which were significantly higher than FA
310
and SA-O3 (Table 2).
311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329
ACS Paragon Plus Environment
Page 14 of 36
Page 15 of 36
Environmental Science & Technology
330 331 332
Discussion
333 334
The results of this study demonstrate that a single inhalation exposure to atmospheric
335
smog causes acute cardiovascular dysfunction in mice, irrespective of whether it is
336
comprised predominantly of PM or O3. Our findings also suggest that multipollutant
337
mixtures which have a higher irritant gas composition are likely to cause more potent
338
acute cardiac effects than those with higher PM/lower gases. Unfortunately, the relative
339
toxicity of only the SOA in each of the atmospheres was not assessed, this would have
340
provided greater information about their contributions to the differential health effects we
341
observed. In either case, it is not possible to firmly conclude whether PM in general
342
plays a greater role in causing cardiac effects or not because SOA is not necessarily the
343
same as ambient PM. However, in an effort to compare the effects of SA-PM and SA-O3
344
relative to their whole compositions, we normalized the data from this study to total
345
hydrocarbons, gas-phase hydrocarbons only and PM concentration. Normalization of
346
the results showed that there was no difference in heart rate, heart rate variability or
347
cardiac arrhythmias between SA-PM and SA-O3 when differences in total hydrocarbons
348
or gas-phase only hydrocarbons were taken into account. In contrast, SA-O3 caused
349
significantly greater cardiac effects than SA-PM when results were normalized to PM
350
concentration indicating that the difference in response was mediated by the gaseous
351
components. Thus, these findings point to the complexity of interactions between air
ACS Paragon Plus Environment
Environmental Science & Technology
352
pollution components and the reactions that determine the composition of the final
353
mixture and the resulting cardiovascular response.
354 355
Real-time cardiovascular measurements and responses to exposure as derived here
356
from radiotelemetry often include stress-related effects (e.g. from handling, noise, etc).
357
This was observed in all animals as a transient increase in heart rate upon placement in
358
the exposure chamber, which occurred despite two separate one-hour acclimatizations.
359
In controls, the overall heart rate progressively decreased per hour of exposure as the
360
animals calmed down in the chambers. Similar responses were observed with SA-O3
361
but with a trend of greater decrease in heart rate. Although this result was not
362
statistically significant, it is not entirely surprising that SA-O3 would have this effect given
363
it was rich in irritant gases like O3, NOx and reactive aldehydes, which have the ability to
364
activate airway sensory nerves, elicit autonomic reflexes and modulate parasympathetic
365
function17-19. On the other hand, SA-PM caused a significant increase in heart rate in
366
the first hour of exposure and less decrease (i.e. HR remained elevated above normal)
367
over the remaining exposure period when compared to controls and SA-O3. These
368
disparate effects of SA-PM and SA-O3 are not unusual. We previously showed that
369
exposure to particle-filtered diesel exhaust caused greater decrements in heart rate
370
than whole diesel exhaust while particle-rich air pollution has been shown to increase
371
heart rate13,20,21. Thus, it appears from our data that decreases in heart rate during such
372
exposures are related to the composition and concentration of gases in the complex air
373
pollution mixture. Notice that normalization of the heart rate to total or gas-phase only
374
hydrocarbons did not reveal any difference between SA-PM and SA-O3, yet when the
ACS Paragon Plus Environment
Page 16 of 36
Page 17 of 36
Environmental Science & Technology
375
“effects” of PM were eliminated through normalization SA-O3 caused a significant
376
decrease in heart rate when compared to SA-PM.
377 378
The composition of the smog atmospheres may not have been the only determinant of
379
this response, variations in the smell of SA-PM and SA-O3 could have contributed as
380
well. SA-PM had a very potent smell when compared to SA-O3 and this may have
381
caused an increase in the heart rate. This effect has been demonstrated previously,
382
particularly with burnt or unpleasant smells22, and appears to be mediated by an
383
increase in sympathetic modulation or decrease in heart rate variability. It is likely that in
384
addition to smell, several smog factors could have triggered responses and altered
385
autonomic function simultaneously, particularly given the complexity of the atmosphere
386
(e.g. potent “burnt” smell + chemical airway irritation + secondary activation due to
387
inflammation). The autonomic nervous system controls the heart and vasculature
388
through a dynamic ebb and flow of both parasympathetic and sympathetic influence and
389
tends to lean in the direction of one or the other based on the physiological
390
circumstances. Thus, returning to the issue of composition, the difference in response
391
between a predominantly gaseous mixture and one that is high in particles would
392
depend on the sum of all the factors that impact autonomic function, and hence the
393
resulting direction would be determined by which factor(s) dominates.
394 395
Although our data do not necessarily demonstrate this (i.e. parasympathetic/
396
sympathetic activation) directly, it is possible that irritant gases drove a predominantly
397
parasympathetic response whereas the PM-rich mixture caused a stress-induced
ACS Paragon Plus Environment
Environmental Science & Technology
398
sympathetic modulation. We observed a greater increase in SDNN and RMSSD, which
399
is indicative of parasympathetic modulation, during SA-O3 exposure when compared to
400
SA-PM. This response was due to the effects of the gas-phase components given the
401
difference in these parameters between the two smog atmospheres became evident
402
when we normalized to PM concentration. As far as PM is concerned, studies have
403
demonstrated, as stated previously, that it not only increases heart rate, blood pressure,
404
low frequency blood pressure variability and noradrenalin release (i.e. stress), but also
405
causes a decrease in heart rate variability3,23. All of these changes point to sympathetic
406
modulation and a perceived increase in cardiac risk. However, we observed an increase
407
in SDNN and RMSSD even with the PM-enhanced SA-PM mixture. This might be
408
explained by the fact that SA-PM also contained irritant gases, some of which were
409
present in high concentrations (see Krug et al. in this issue) and could have opposed
410
the sympathetic modulatory effect of the PM. A lack of response in the LF/HF ratio
411
suggests this parasympathetic-sympathetic push and pull24.
412 413
Comparisons between SA-PM and SA-O3 also included the air quality health index or
414
AQHI, which indicates the health risk of both atmospheres. The equation for this index
415
provides the relative contribution of the PM2.5 and the oxidant gases towards the health
416
metric. In the case of SA-O3, oxidant gases contributed to 97% of the AQHI with a
417
negligible amount coming from PM, once again pointing to the fact that it’s effects were
418
predominantly mediated by irritant gases. In contrast, PM contributed to almost 70% of
419
the AQHI of SA-PM, yet it also had a fairly significant contribution (30%) from the
420
oxidant gases as well (see Krug et al. this issue). These results seem to confirm our
ACS Paragon Plus Environment
Page 18 of 36
Page 19 of 36
Environmental Science & Technology
421
conclusions that although the effects of SA-O3, which included a significant decrease in
422
heart rate and increase in heart rate variability, were almost entirely driven by gases,
423
SA-PM’s effects were likely driven by both. The implications for human health are
424
important, particularly given most of the research over the last decade has focused on
425
PM-driven cardiovascular effects. For example, both an increase of 6 ug/m3 in fine PM
426
and 20 ppb O3 resulted in a significant increase in the risk of out of hospital heart
427
attacks25. Furthermore, exposure to 0.3 ppm O3 caused similar HRV changes in healthy
428
young individuals to what we observed in this study, suggesting gases like O3 may not
429
cause overt symptoms but rather underlying changes.26
430 431
While an increasing or decreasing heart rate or heart rate variability during exposure
432
merely indicates a physiological change, one could potentially infer cardiac dysfunction
433
when it is a deviation from the control response. It represents fluctuations that can
434
normally happen in mammals (e.g. exercise or physical exertion). Sometimes these
435
responses reflect adverse cardiac issues more strongly when observed in conjunction
436
with cardinal signs of dysfunction like arrhythmia. This may be the case for what we
437
observed in the mice exposed to these smog atmospheres. We have shown that
438
gaseous air pollution is more arrhythmogenic than one with a high concentration of PM
439
and the same appears to be the case in this study12,27. SA-O3, particularly when
440
normalized to PM concentration, caused a significantly greater number of sinoatrial (SA)
441
node dysfunction than SA-PM. Furthermore, we found that SA-O3 was more
442
arrhythmogenic than SA-PM even when normalized to gas-phase hydrocarbons
443
suggesting PM had very little effect. This form of arrhythmia or dysrhythmia, which can
ACS Paragon Plus Environment
Environmental Science & Technology
444
increase the risk of escape beats and cardiac insufficiency in humans, is due to an
445
abnormal delay in the firing of the SA node and manifests as blocked p-waves and
446
alternating runs of bradycardia followed by tachycardia.. Interestingly, some of the
447
causes behind this phenomenon include an increase in vagal tone and airway irritation
448
which leads to dyspneic breathing and airway spasm, both of which were observed in
449
our animals28,29.
450 451
Exposure to SA-PM did cause a prolongation of the PR interval, which in some cases is
452
normal but also can indicate a cardiac conduction abnormality. However, in the absence
453
of other effects (e.g. arrhythmia) it would be hard to say that there was any real
454
dysfunction in these mice. Indeed, even changes in breathing impact the heart on a
455
breath-by-breath basis and through this physiological coupling the heart is able to
456
maintain proper function30. Thus, alterations in breathing due to airway irritation and
457
airflow obstruction have the potential to cause adverse cardiac events. Yet, we did not
458
observe any remarkable differences in ventilatory parameters either immediately
459
following the exposure or one day later for either SA-PM or SA-O3. Normalization to
460
total or only gas-phase hydrocarbons did not reveal any differences but SA-O3 caused a
461
significant increase in breathing frequency, and decreases in tidal volume and
462
inspiratory time when the results were normalized to PM concentration. Thus, once
463
again it appears that the gases in SA-O3 caused a rapid shallow breathing in mice
464
possibly due to irritation.
465
ACS Paragon Plus Environment
Page 20 of 36
Page 21 of 36
Environmental Science & Technology
466
In conclusion, the results of this study demonstrate that inhalation exposure to a
467
complex smog atmosphere causes acute cardiac effects in mice, and that the
468
composition of the pollution mixture likely plays a key role in determining the degree of
469
responsiveness. Although the PM-enhanced smog caused a change in cardiac function,
470
it is likely that a single exposure was not enough to elicit worsening symptoms in our
471
relatively young healthy animals. This is particularly true for the relatively low number of
472
cardiac arrhythmias observed in those animals. It is entirely likely however that repeated
473
exposures would have had a more pronounced impact. In contrast, the O3-enhanced
474
smog caused a set of physiological changes which if considered with the increased
475
incidence of arrhythmia suggest an acute irritant gas-mediated autonomic modulation
476
and electrical disturbance. Even though the former is not considered a “toxicological”
477
response, it does reflect a change from the homeostatic normal state of the body. These
478
short-lived reversible effects probably do not pose a serious hazard to the body on their
479
own, but when combined with an additional stressor could predispose the heart to
480
dysfunction, particularly during in the 24 hours following exposure.
481 482 483 484 485 486
Acknowledgements: We are grateful to Drs. Wayne Cascio, Christopher Gordon and
487
Colette Miller for their careful reviews of this manuscript.
488
Funding: All funding for this study is from U.S. Environmental Protection Agency
489
ACS Paragon Plus Environment
Environmental Science & Technology
490
Disclaimer: This paper has been reviewed and approved for release by the National
491
Health and Environmental Effects Research Laboratory, U.S. Environmental Protection
492
Agency. Approval does not signify that the contents necessarily reflect the views and
493
policies of the U.S. EPA, nor does mention of trade names.
494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509
References
510 511 512
1. Langrish, J.P.; Bosson, J.; Unosson, J.; Muala, A.; Newby, D.E.; Mills, N.L.; Blomberg, A.; Sandström, T. Cardiovascular effects of particulate air
ACS Paragon Plus Environment
Page 22 of 36
Page 23 of 36
Environmental Science & Technology
513
pollution exposure: time course and underlying mechanisms. J Intern. Med.
514
2012, 272(3):224-39.
515 516
2. Brook, R.D.; Urch, B.; Dvonch, J.T.; Bard, R.L.; Speck, M.; Keeler, G.; Morishita,
517
M.; Marsik, F.J.; Kamal, A.S.; Kaciroti, N.; Harkema, J.; Corey, P.; Silverman, F.;
518
Gold, D.R.; Wellenius, G.; Mittleman, M.A.; Rajagopalan, S.; Brook, J.R. Insights
519
into the mechanisms and mediators of the effects of air pollution exposure on
520
blood pressure and vascular function in healthy humans. Hypertension 2009,
521
54(3):659-667.
522 523
3. Brook, R.D.; Rajagopalan, S.; Pope, C.A.; Brook, J.R.; Bhatnagar, A.; Diez-Roux,
524
A.V.; Holguin, F.; Hong, Y.; Luepker, R.V.; Mittleman, M.A.; Peters, A.; Siscovick,
525
D.; Smith, S.C. Jr; Whitsel, L.; Kaufman, J.D. Particulate matter air pollution and
526
cardiovascular disease: An update to the scientific statement from the American
527
Heart Association. Circulation 2010, 121(21), 2331–78.
528 529
4. Hazari, M.S.; Callaway, J.; Winsett, D.W.; Lamb, C.; Haykal-Coates, N.; Krantz,
530
Q.T.; King, C.; Costa, D.L.; Farraj, A.K. Dobutamine "stress" test and latent
531
cardiac susceptibility to inhaled diesel exhaust in normal and hypertensive rats.
532
Environ. Health Perspect. 2012, 120(8):1088-93.
533 534 535
5. Carll, A.P.; Hazari, M.S.; Perez, C.M.; Krantz, Q.T.; King, C.J.; Haykal-Coates, N.; Cascio, W.E.; Costa, D.L.; Farraj, A.K. An autonomic link between inhaled
ACS Paragon Plus Environment
Environmental Science & Technology
536
diesel exhaust and impaired cardiac performance: insight from treadmill
537
and dobutamine challenges in heart failure-prone rats. Toxicol. Sci. 2013,
538
135(2):425-36.
539 540 541
6. Peters, A. Particulate matter and heart disease: evidence from epidemiological studies. Toxicol. App. Pharmacol. 2005, 207(2 Suppl):477–82.
542 543
7. Pope, C.A. 3rd; Burnett, R.T.; Thurston, G.D.; Thun, M.J.; Calle, E.E.; Krewski,
544
D.; Godleski, J.J. Cardiovascular mortality and long-term exposure to particulate
545
air pollution: epidemiological evidence of general pathophysiological pathways of
546
disease. Circulation 2004, 109(1):71-7.
547 548
8. Samet, J.M.; Rappold, A.; Graff, D.; Cascio, W.E.; Berntsen, J.H.; Huang, Y-C.T.;
549
Herbst, M.; Bassett, M.; Montilla, T.; Hazucha, M.J.; Bromberg, P.A.; Devlin, R.B.
550
Concentrated ambient ultrafine particle exposure induces cardiac changes in
551
young healthy volunteers. Am. J. Respir. Crit. Care Med. 2009, 179(11):1034–42.
552 553
9. Ruidavets, J.B.; Cournot, M.; Cassadou, S.; Giroux, M.; Meybeck, M.; Ferrières,
554
J. Ozone air pollution is associated with acute myocardial infarction. Circulation
555
2005, 111(5):563-9.
556 557 558
10. Farraj, A.K.; Hazari, M.S.; Winsett, D.W.; Kulukulualani, A.; Carll, A.P.; HaykalCoates, N.; Lamb, C.M.; Lappi, E.; Terrell, D.; Cascio, W.E.; Costa, D.L. Overt
ACS Paragon Plus Environment
Page 24 of 36
Page 25 of 36
Environmental Science & Technology
559
and latent cardiac effects of ozone inhalation in rats: evidence for autonomic
560
modulation and increased myocardial vulnerability. Environ. Health Perspect.
561
2012, 120(3):348–54.
562 563
11. Jerrett, M.; Burnett, R.T.; Beckerman, B.S.; Turner, M.C.; Krewski, D.; Thurston,
564
G.; Martin, R.V.; van Donkelaar, A.; Hughes, E.; Shi, Y.; Gapstur, S.M.; Thun,
565
M.; Pope, C.A. Spatial Analysis of Air Pollution and Mortality in California. Am. J.
566
Respir. Crit. Care Med. 2013, 188(5):593-600.
567 568
12. Hazari, M.S.; Haykal-Coates, N.; Winsett, D.W.; Krantz, Q.T.; King, C.; Costa,
569
D.L.; Farraj, A.K. TRPA1 and sympathetic activation contribute to increased risk
570
of triggered cardiac arrhythmias in hypertensive rats exposed to diesel exhaust.
571
Environ. Health Perspect. 2012, 119(7):951-7.
572 573
13. Lamb, C.M.; Hazari, M.S.; Haykal-Coates, N.; Carll, A.P.; Krantz, Q.T.; King, C.;
574
Winsett, D.W.; Cascio, W.E.; Costa, D.L.; Farraj, A.K. Divergent
575
electrocardiographic responses to whole and particle-free diesel exhaust
576
inhalation in spontaneously hypertensive rats. Toxicol. Sci. 2012, 125(2):558-68.
577 578
14. Carll, A.P.; Hazari, M.S.; Perez, C.M.; Krantz, Q.T.; King, C.J.; Winsett, D.W.;
579
Costa, D.L.; Farraj, A.K. Whole and particle-free diesel exhausts differentially
580
affect cardiac electrophysiology, blood pressure, and autonomic balance in heart
ACS Paragon Plus Environment
Environmental Science & Technology
581
failure-prone rats. Toxicol. Sci. 2012, 128(2):490-9.
582 583
15. Kurhanewicz, N.; McIntosh-Kastrinsky, R.; Tong, H.; Walsh, L.; Farraj, A.K.;
584
Hazari, M.S. Ozone co-exposure modifies cardiac responses to fine and ultrafine
585
ambient particulate matter in mice: concordance of electrocardiogram and
586
mechanical responses. Part. Fibre Toxicol. 2014, Oct 16;11:54.
587 588 589
16. Mitchell, G.F.; Jeron, A.; Koren, G. Measurement of heart rate and Q-T interval in the conscious mouse. Am. J Physiol. 1998, 274(3) Pt 2.
590 591 592
17. Widdicombe, J.; Lee, L.Y. Airway reflexes, autonomic function, and cardiovascular responses. Environ. Health Perspect. 2001, 109:579–584.
593 594 595
18. Taylor-Clark, T.E.; Undem, B.J. Ozone activates airway nerves via the selective stimulation of TRPA1 ion channels. J. Physiol. 2006, 588:423–433.
596 597
19. Kurhanewicz, N.; McIntosh-Kastrinsky, R.; Tong, H.; Ledbetter, A.; Walsh, L.;
598
Farraj, A.; Hazari, M.S. TRPA1 mediates changes in heart rate variability and
599
cardiac mechanical function in mice exposed to acrolein. Toxicol. Appl.
600
Pharmacol. 2016, 324:51-60.
601 602 603
20. Peters, A.; Perz, S.; Dfiring, A.; Stieber, J.; Koenig, W.; Wichmann, H.E. Increases in Heart Rate during an Air Pollution Episode. Am. J. Epidemiol. 1999,
ACS Paragon Plus Environment
Page 26 of 36
Page 27 of 36
604
Environmental Science & Technology
150:1094-1099.
605 606
21. Unosson, J.; Blomberg, A.; Sandström, T.; Muala, A.; Boman, C.; Nyström, R.;
607
Westerholm, R.; Mills, N.L.; Newby, D.E.; Langrish, J.P.; Bosson, J.A. Exposure
608
to wood smoke increases arterial stiffness and decreases heart rate variability in
609
humans. Part. Fibre Toxicol. 2013, 6;10-20.
610 611 612
22. Glass, S.T.; Lingg, E.; Heuberger, E. Do ambient urban odors evoke basic emotions? Front. Psychol. 2014, 5:340.
613 614
23. Ying, Z.; Xu, X.; Bai, Y.; Zhong, J.; Chen, M.; Liang, Y.; Zhao, J.; Liu,
615
D.; Morishita, M.; Sun, Q.; Spino, C.; Brook, R.D.; Harkema, J.R.; Rajagopalan,
616
S. Long-term exposure to concentrated ambient PM2.5 increases mouse blood
617
pressure through abnormal activation of the sympathetic nervous system: a role
618
for hypothalamic inflammation. Environ. Health Perspect. 2014, 122(1):79-86.
619 620 621
24. Billman, G.E. The LF/HF ratio does not accurately measure cardiac sympathovagal balance. Front. Physiol. 2013, 4:26.
622 623 624
25. Ensor, K.B.; Raun, L.H.; Persse, D. A case-crossover analysis of out-of-hospital cardiac arrest and air pollution. Circulation 2013 127:1192-1199.
625
ACS Paragon Plus Environment
Environmental Science & Technology
626
Page 28 of 36
26. Devlin, R.B.; Duncan, K.E.; Jardim, M.; Schmitt, M.T.; Rappold, A.G.; Diaz-
627
Sanchez, D. Controlled exposure of healthy young volunteers to ozone causes
628
cardiovascular effects. Circulation 2012 126(1):104-111.
629 630 631
27. Hazari, M.S.; Haykal-Coates, N.; Winsett, D.W.; Costa, D.L.; Farraj, A.K. A single
632
exposure to particulate or gaseous air pollution increases the risk of aconitine-
633
induced cardiac arrhythmia in hypertensive rats. Toxicol.
634
Sci. 2009 Dec,112(2):532-42.
635 636
28. Benditt, D.G.; Sakaguchi, S.; Lurie, K.G.; Lu, F. Sinus node dysfunction. In
637
Cardiovascular Medicine, Eds. James T. Willerson, Hein J. J. Wellens, Jay N.
638
Cohn, David R. Holmes Jr. Springer, London 2007.
639 640 641
29. Haqqani, H.M.; Kalman, J.M. Aging and sinoatrial dysfunction. Circulation 2007, 115:1178-1179.
642 643
30. Ben-Tal, A.; Shamailov, S.S.; Paton, J.F.R. Evaluating the physiological
644
significance of respiratory sinus arrhythmia: looking beyond ventilation–perfusion
645
efficiency. J. Physiol. 2012, 590:1980-2008.
646 647
TABLE 1. Concentrations of criteria pollutants and air quality indexes PM2.5 3 (mg/m )
NOx/AQI (ppm)
O3/AQI (ppm)
AQI
AQI
AQI
Total hydrocarbons (ppmc)
ACS Paragon Plus Environment
Gas-phase 3 (mgC/m )
AQHI
Page 29 of 36
648 649 650 651 652 653 654 655
Environmental Science & Technology
Filtered air
0.009 37 (green)
ND -
0.002 1 (green)
ND
ND
500 (brown)
0.380 154 (orange)
0.190 287(red)
5.5
3.847
92.5
SA-O3
0.0523 142 (orange)
0.647 199 (red)
0.448 >300 (purple)
6.1
3.125
102.6
ND – none detected Green – AQ is satisfactory – little or no risk Yellow – AQ is acceptable – moderate health concern for a very small number of people who are unusually sensitive to air pollution Orange – Sensitive people may experience health effects Red – Everyone may experience health effects; sensitive people at greater risk Purple – Everyone may experience serious health effects Brown – Health warnings at emergency conditions; entire population will be affected
656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673
TABLE 2. Electrocardiographic parameters
Air Baseline
PR interval (msec)
QRS duration (msec)
QTc (msec)
36.1 ± 0.5
14.7 ± 1.9
94.2 ± 3.0
ACS Paragon Plus Environment
Environmental Science & Technology
Page 30 of 36
Hour 1 Hour 2 Hour 3 Hour 4 24hrs Post-exp
33.4 ± 1.8 35.7 ± 1.4 35.9 ± 0.6 37.5 ± 1.0 36.0 ± 0.7
13.9 ± 0.5 14.6 ± 0.4 14.8 ± 0.4 15.3 ± 0.4 16.3 ± 0.3
92.7 ± 1.5 92.9 ± 1.5 94.9 ± 3.0 94.5 ± 2.0 94.6 ± 4.0
SA-PM Baseline Hour 1 Hour 2 Hour 3 Hour 4 24hrs Post-exp
32.8 ± 0.6 40.7 ± 1.0*♦ 42.3 ± 1.1*♦ 43.3 ± 0.9*♦ 42.8 ± 1.2*♦ 33.4 ± 0.5
14.6 ± 0.3 13.3 ± 0.2 13.4 ± 0.4 13.7 ± 0.2 13.6 ± 0.3 14.4 ± 0.2
88.2 ± 1.5 91.0 ± 4.6 93.3 ± 4.1 91.7 ± 2.6 99.3 ± 7.0 81.5 ± 1.4
SA-O3 Baseline Hour 1 Hour 2 Hour 3 Hour 4 24hrs Post-exp
33.6 ± 0.9 34.4 ± 1.1 36.3 ± 0.8 36.6 ± 0.7 37.1 ± 0.7 34.2 ± 0.6
13.4 ± 1.1 11.8 ± 2.2 11.0 ± 2.4 9.6 ± 3.2 10.1 ± 2.8 14.4 ± 0.8
89.9 ± 1.8 95.9 ± 3.4 91.5 ± 3.6 92.7 ± 4.9 92.4 ± 2.9 88.9 ± 0.8
674 675 676 677 678 679 680 681 682 683 684 685
ACS Paragon Plus Environment
Page 31 of 36
Environmental Science & Technology
Figure 1. Exposure to smog alters heart rate responses in mice. In general, mice experienced a steady decrease in HR (from baseline) over the first two hours of exposure, which then leveled off in hours three and four. Mice exposed to SA-PM experienced a significant increase in HR during the first hour and had less decrease in HR thereafter when compared to FA and SA-O3 (A.). However, there was no difference between SA-PM and SA-O3 when the change in HR during exposure was normalized to total hydrocarbons (B.) or gas-phase hydrocarbons only (C.). In contrast, decrease in HR during SA-O3 exposure was significantly greater than SA-PM when responses were normalized to PM concentration (D.). significantly different from FA; • significantly different from SA-O3; ǂ significantly different from SA-PM. p < 0.05. 324x249mm (96 x 96 DPI)
ACS Paragon Plus Environment
Environmental Science & Technology
Figure 2. Exposure to O3-enhanced smog increases cardiac arrhythmias in mice. Overall, exposure to SA-O3 caused a significant increase in the number of cardiac arrhythmias in mice when compared to FA. There was no difference between SA-O3 and SA-PM in the denormalized (A.) and THC-normalized (B.) responses; however, SA-O3 caused significantly more arrhythmias than SA-PM when the responses were normalized to gas-phase hydrocarbons (C.) or PM concentration (D.). significantly different from FA; • significantly different from SA-PM. p < 0.05. 324x348mm (96 x 96 DPI)
ACS Paragon Plus Environment
Page 32 of 36
Page 33 of 36
Environmental Science & Technology
Figure 3. Exposure to smog increases SDNN in mice. SDNN decreased during the first hour of FA exposure probably due to stress. On the other hand, mice exposed to either SA-PM or SA-O3 experienced a significant increase in SDNN (A.) when compared to FA. There was no difference in SDNN between SA-PM and SA-O3 in the denormalized (A.), THC- (B.) or gas-phase only hydrocarbon (C.) normalized responses. In contrast, SDNN was significantly increased during SA-O3 exposure when compared to SA-PM when responses were normalized to PM concentration (D.). significantly different from FA; • significantly different from SA-PM. p < 0.05. 324x291mm (96 x 96 DPI)
ACS Paragon Plus Environment
Environmental Science & Technology
Figure 4. Exposure to O3-enhanced smog increases RMSSD in mice. There was no effect of SA-PM or SA-O3 on RMSSD when compared to FA although a trend of increase was observed for both (A.). There was no difference in RMSSD between SA-PM and SA-O3 in the THC- (B.) or gas-phase only hydrocarbon (C.) normalized responses. On the other hand, RMSSD was significantly increased during SA-O3 exposure when compared to SA-PM when responses were normalized to PM concentration (D.). significantly different from FA; • significantly different from SA-PM. p < 0.05. 324x255mm (96 x 96 DPI)
ACS Paragon Plus Environment
Page 34 of 36
Page 35 of 36
Environmental Science & Technology
Figure 5. Change in LF/HF during smog exposure. There were no significant effects of SA-PM or SA-O3 on LF/HF when compared to FA or when either smog atmosphere was compared to the other (A.). Normalization of the responses to THC- (B.), gas-phase only hydrocarbons (C.), or PM concentrations (D.) did not reveal any significant difference between SA-PM and SA-O3 except in (D.) where a trend of increase was observed in SA-O3-exposed mice. 324x257mm (96 x 96 DPI)
ACS Paragon Plus Environment
Environmental Science & Technology
Figure 6. O3-enhanced smog alters breathing in mice immediately after exposure. There was no effect of SA-PM (filled circles) or SA-O3 (open triangles) on any ventilatory parameters when compared to FA (open circles) (A.) and there were no differences observed between SA-PM and SA-O3 in the THC- (B.) or gasphase only hydrocarbon (C.) normalized responses. However, f was increased, and VT and Ti were significantly decreased after SA-O3 exposure when compared to SA-PM when responses were normalized to PM concentration (D.). significantly different from FA; • significantly different from SA-PM. p < 0.05. 324x354mm (96 x 96 DPI)
ACS Paragon Plus Environment
Page 36 of 36
