Comparative Cardiopulmonary Effects of Particulate Matter- And


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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

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Comparative cardiopulmonary effects of particulate matter- and ozone-enhanced smog atmospheres in mice

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Mehdi S. Hazari1*, Kimberly M. Stratford2, Q. Todd Krantz3, Charly King3, Jonathan Krug4, Aimen K. Farraj1 and M. Ian Gilmour1

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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

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*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])

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Running title: Greater cardiac effects of O3-enhanced smog than PM-enhanced

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Abstract

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This study was conducted to compare the cardiac effects of particulate matter (PM)-

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(SA-PM) and ozone(O3)-enhanced (SA-O3) smog atmospheres in mice. Based on our

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previous findings of filtered diesel exhaust we hypothesized that SA-O3 would cause

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greater cardiac dysfunction than SA-PM. Radiotelemetered mice were exposed to either

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SA-PM, SA-O3, or filtered air (FA) for 4 hours. Heart rate (HR) and electrocardiogram

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were recorded continuously before, during and after exposure. Both SA-PM and SA-O3

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increased heart rate variability (HRV) but only SA-PM increased HR. Normalization of

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responses to total hydrocarbons, gas-only hydrocarbons and PM concentration were

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performed to assess the relative contribution of each phase given the compositional

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variability. Normalization to PM concentration revealed that SA-O3 was more potent in

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increasing HRV, arrhythmogenesis, and causing ventilatory changes. However, there

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were no differences when the responses were normalized to total or gas-phase only

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hydrocarbons. Thus, this study demonstrates that a single exposure to smog causes

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cardiac effects in mice. Although the responses of SA-PM and SA-O3 are similar, the

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latter is more potent in causing electrical disturbances and breathing changes

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potentially due to the effects of irritant gases, which should therefore be accounted for

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more rigorously in health assessments.

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Abstract art

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Introduction

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The association between air pollution exposure and cardiovascular disease is well-

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established, particularly in people with certain risk factors like high blood pressure or

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those with pre-existing conditions1. The picture is less clear for healthy individuals and

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indeed there are still several unknowns with regards to how air pollution mediates

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cardiovascular dysfunction in this group, especially when there are no observable

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symptoms. In addition, nationwide variations in air pollution composition make it difficult

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to determine exactly which components, or combination of components, drive the

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response. This is the case for complex air pollution mixtures like smog, which originates

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as a set of primary pollutants (e.g. nitrogen oxides, volatile organic compounds) that are

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released from vehicles or industrial sources but thereafter transform after reacting with

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ultraviolet light to produce secondary pollutants like ozone and certain organic aerosols.

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Given these uncertainties, research studies need to address the comparative effects of

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multiple smog atmospheres and focus on determining which compositions, and hence

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components, cause the most serious health effects.

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The American Heart Association cites several pathways by which air pollution,

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especially particulate matter (PM), leads to decrements in cardiovascular function2,3.

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These include elicitation of oxidative stress and inflammation, alteration of vasomotor

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properties, translocation of certain pollutants into the systemic circulation and

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modulation of autonomic controls which regulate the heart and vasculature. Some of

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these may not manifest as overt symptoms, especially in young healthy individuals, but

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instead as shifts in normal physiological function which render a person susceptible to

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subsequent stressors or triggers of adverse responses. We previously showed that

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exposure to a relatively low concentration of diesel exhaust, which contains many of the

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same pollutants as smog, caused increased arrhythmias and other cardiovascular

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effects when healthy rats were challenged with an exercise-like stressor4,5. The insight

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gained from these and other studies is that exposure to complex air pollution mixtures

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results in latent autonomic shifts which cause a decrease in cardiac performance during

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exposure and even up to one day later.

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Although many epidemiological studies point to PM as the main cause of air pollution’s

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impacts on the cardiovascular system6-8, there are numerous human and rodent studies

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that implicate ubiquitous gaseous irritants like ozone as well9-11. In fact, our previous

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studies have shown that cardiovascular dysfunction occurs, sometimes at comparable

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levels or even after PM is removed from a multipollutant mixture like diesel exhaust12-14.

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Furthermore, the magnitude of the response is not just determined by the relative levels

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of PM and ozone but also by the resulting physical and chemical interactions that occur

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in the mixture. Therefore, the objective of this study was to examine and compare the

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cardiovascular responses of mice exposed to either a simulated high PM/low ozone

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(SA-PM) or low PM/high ozone (SA-O3) smog atmosphere. These photo-oxidized

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mixtures approximated urban and regional evaporative pollutant emissions with similar

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Air Quality Health Index values. We previously determined that filtered diesel exhaust

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causes greater acute cardiac effects than whole diesel exhaust, and so we

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hypothesized that although both smog mixtures would elicit cardiac changes in mice,

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the atmosphere dominated by gaseous irritants would be more potent, particularly in

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causing electrical disturbances like arrhythmia.

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Methods

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Animals - Female C57BL/6 (21 ± 1.1 g) mice between 13 and 15 weeks of age were

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used in this study (Jackson Laboratory - Bar Harbor, ME). Mice were initially housed

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five per cage and maintained on a 12-hr light/dark cycle at approximately 22°C and 50%

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relative humidity in an AAALAC–approved facility. Food (Prolab RMH 3000; PMI

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Nutrition International, St. Louis, MO) and water were provided ad libitum. All protocols

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were approved by the Institutional Animal Care and Use Committee of the U.S.

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Environmental Protection Agency and are in accordance with the National Institutes of

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Health Guides for the Care and Use of Laboratory Animals. Animals were randomly

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assigned to one of the following groups after implantation of radiotelemeters: (1) filtered

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air (FA), (2) SA-PM, or (3) SA-O3.

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Surgical Implantation of Radiotelemeters – Mice were implanted with radiotelemeters as

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previously described15. Animals were anesthetized using inhaled isoflurane (Isothesia,

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Butler Animal Health Supply, Dublin OH). Anesthesia was induced by spontaneous

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breathing of 2.5% isoflurane in pure oxygen at a flow rate of 1 L/min and then

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maintained by 1.5% isoflurane in pure oxygen at a flow rate of 0.5 L/min; all animals

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received the analgesic buprenorphrine (0.03 mg/kg, i.p. manufacturer). Briefly, using

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aseptic technique, each animal was implanted subcutaneously with a radiotelemeter

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(ETA-F10, Data Sciences International, St Paul, MN), the transmitter was placed under

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the skin to the right of the midline on the dorsal side. The two electrode leads were then

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tunneled subcutaneously across the lateral dorsal sides, and the distal portions were

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fixed in positions that approximated those of the lead II of a standard electrocardiogram

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(ECG). Body heat was maintained both during and immediately after the surgery.

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Animals were given food and water post-surgery and were housed individually. All

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animals were allowed 7-10 days to recover from the surgery and reestablish circadian

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rhythms.

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Radiotelemetery Data Acquistion - Radiotelemetry methodology (Data Sciences

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International, Inc., St. Paul, MN) was used to track changes in cardiovascular function

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by monitoring heart rate (HR), and ECG waveforms immediately following telemeter

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implantation, through exposure until 24hours post-exposure. This methodology provided

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continuous monitoring and collection of physiologic data from individual mice to a

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remote receiver. Sixty-second ECG segments were recorded every 15 minutes during

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the pre- and post-exposure periods and every 5 minutes during exposure (baseline and

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hours 1-4); HR was automatically obtained from the waveforms (Dataquest ART

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Software, version 3.01, Data Sciences International, St. Paul, MN, USA). All animals

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were acclimated to the exposure chambers on two separate occasions, even then, an

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increase in HR was always observed when animals were placed in the chamber on the

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day of exposure.

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Electrocardiogram Analysis - ECGAuto software (EMKA Technologies USA, Falls

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Church VA) was used to visualize individual ECG waveforms, analyze and quantify

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ECG segment durations and areas, as well as identify cardiac arrhythmias as previously

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described (Kurhanewicz et al. 2014). Briefly, using ECGAuto, Pwave, QRS complex,

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and T-wave were identified for individual ECG waveforms and compiled into a library.

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Analysis of all experimental ECG waveforms was then based on established libraries.

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The following parameters were determined for each ECG waveform: PR interval (Pstart-

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R), QRS complex duration (Qstart-S), ST segment interval (S-Tend) and QT interval (Qstart-

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Tend). QT interval was corrected for HR using the correction formula for mice QTc =

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QT/(RR/100)1/216. Pre-exposure assessments were measured as the exposure time-

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matched four hours of data from 24 hours before exposure for each animal. Immediately

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post-exposure assessments were the four hours of data taken immediately post-

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exposure. Twenty-four post-exposure assessments were the exposure time-matched

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four hours of data taken 24 hours after exposure.

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Heart Rate Variability Analysis - Heart rate variability (HRV) was calculated as the mean

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of the differences between sequential RRs for the complete set of ECG waveforms

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using ECGAuto. For each 1-min stream of ECG waveforms, mean time between

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successive QRS complex peaks (RR interval), mean HR, and mean HRV-analysis–

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generated time-domain measures were acquired. The time-domain measures included

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standard deviation of the time between normal-to-normal beats (SDNN), and root mean

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squared of successive differences (RMSSD). HRV analysis was also conducted in the

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frequency domain using a Fast-Fourier transform. The spectral power obtained from this

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transformation represents the total harmonic variability for the frequency range being

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analyzed. In this study, the spectrum was divided into low-frequency (LF) and high-

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frequency (HF) regions. The ratio of these two frequency domains (LF/HF) provides an

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estimate of the relative balance between sympathetic (LF) and vagal (HF) activity.

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Whole-Body Plethysmography - Ventilatory function was assessed in awake,

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unrestrained mice using a whole-body plethysmograph (Buxco, Wilmington, NC).

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Assessments were performed one day before exposure, immediately post-exposure

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and 24hrs after exposure. The plethysmograph pressure was monitored using

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Biosystems XA software (Buxco Electronics Inc., Wilmington, NC). Using respiratory-

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induced fluctuations in ambient pressure, ventilatory parameters including tidal volume

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(VT), breathing frequency (f), inspiratory time (Ti), and expiratory time (Te), were

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calculated and recorded on a breath-by-breath basis.

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Photochemical Smog Exposures – A gasoline blend was combined with either α-pinene

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or isoprene to produce a hydrocarbon mixture that was enhanced for particulate matter

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(PM) or ozone (O3) during irradiation, respectively. Thus, photochemical smog

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atmospheres with either high PM2.5 and low O3/nitrogen oxide (NOx) concentrations

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(SA-PM) or low PM2.5 and high O3/NOx (SA-O3) were generated in the Mobile Reaction

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Chamber (MRC). Briefly, SA-PM was artificially generated with 0.491 ppm nitrogen

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oxide (NO), 0.528 ppm NOx, 29.9 ppmC total hydrocarbons (THC), 24 ppmC gasoline

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and 5.3 ppmC α -pinene as the initial conditions, whereas SA-O3 was generated from

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0.794 ppm NO, 0.912 ppm NOx, 12.4 ppmC THC, 5.2 ppmC isoprene and 7.21 ppmC

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gasoline. Each of the initial smog mixtures was then irradiated with ultraviolet light.

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Smog was then transported under vacuum to 0.3 m3 whole body inhalation chambers

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where mice were exposed once for four hours. Continuous gas and aerosol sampling

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for carbon monoxide (CO), O3, NOx, THC and particle mass concentration were

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conducted at both the MRC unit as well as from the inhalation exposure systems. All

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PM was formed as secondary organic aerosol (SOA) from the photochemical reactions

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in the MRC. Particle size distributions and gravimetric mass sampling was measured,

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and filter sampling for gravimetric analysis were conducted for the entire exposure time.

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Volatile organic compound (VOC) summa cannisters were periodically collected and

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analyzed by gas chromatography off-line to determine concentrations of various volatile

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organic compounds (VOCs) in the exposure atmosphere. Animals exposed to FA

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received room air, which was transported to the chambers after being HEPA filtered.

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Please refer to Krug et al. in this issue for complete exposure details.

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Statistics - All data are expressed as means ± SEM. Statistical analyses were

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performed using Sigmaplot 13.0 (Systat Software, San Jose, CA) software. The delta

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values (i.e. change during exposure from baseline) of HR, HRV and the ventilatory

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parameters were used and a two-way analysis of variance (ANOVA) for repeated-

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measures was employed with Bonferroni post hoc tests to determine statistical

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differences. Raw ECG and arrhythmia count data were analyzed by two-way ANOVA.

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Raw HR, HRV, ventilatory parameters and arrhythmia counts were also normalized to

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total hydrocarbons (both particle and gas phases), gas-phase hydrocarbons only or PM

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concentration by dividing the physiological parameters by the pollutant concentrations.

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Normalizations were done to compare the relative contribution of these components to

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the physiological response to Smog A versus B and their relative potencies; no

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statistical comparisons were made to FA post-normalization. The statistical significance

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was set at P < 0.05.

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Results

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Exposure characteristics – Table 1 shows the inhalation chamber concentrations of

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PM2.5 (i.e. secondary organic aerosols), NOx, O3, as well as total hydrocarbons in the

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complete smog mixture (particle and gas phases) and in the gas-phase only for both

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SA-PM (enhanced PM2.5) and SA-O3 (enhanced O3). In addition, the respective Air

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Quality Health Indexes (AQHI) and Air Quality Indexes (AQI) along with their color

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codes are also included to provide a health-risk comparison between these

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atmospheres. These indices describe the health risks from polluted air on individual

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days throughout the year, either from independent pollutants as in the case of AQI, or

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from a cumulative PM2.5, O3, and NOx perspective as with AQHI. Refer to Krug et al. in

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this issue for a more comprehensive comparison of SA-PM (MR044) and SA-O3

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(MR059).

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Heart rate – There was no difference in the baseline HR, which was the resting level

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measured in the home cages, between any of the groups (FA = 588.0 ± 19.8 bpm; SA-

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PM = 568.6 ± 7.5 bpm; SA-O3 = 565.9 ± 9.4 bpm). In general, all animals experienced a

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decrease in HR during the exposure; although, SA-PM caused a significant increase in

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HR during Hour1 and less HR decrease over the remaining exposure period when

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compared to FA and SA-O3 (Fig. 1A). However, there was no difference between SA-

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PM and SA-O3 when the responses were normalized to THC (Fig. 1B) or gas-phase

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hydrocarbons only (Fig. 1C). On the other hand, HR decrease was significantly greater

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during SA-O3 when compared to SA-PM after normalization to PM concentration (Fig.

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1D).

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Arrhythmia – None of the animals experienced cardiac arrhythmias during the pre-

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exposure period. Figure 2 shows the raw arrhythmia counts during the four-hour

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exposure. Overall, exposure to SA-O3 increased the incidence of arrhythmias when

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compared to FA (Fig. 2A-D) but there was no difference with respect to SA-PM in the

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denormalized (Fig. 2A) and THC normalized (Fig. 2B) responses. In contrast, SA-O3

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caused significantly more arrhythmias than SA-PM when the responses were

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normalized to gas-phase hydrocarbons (Fig. 2C) or PM concentrations (Fig. 2D).

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Heart rate variability – Figures 3-5 show the changes in HRV during exposure. There

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were no significant differences in the baseline HRV measures between any of the

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groups (FA – SDNN = 7.4 ± 0.7 msec, RMSSD = 4.0 ± 0.5 msec, LF/HF = 8.4 ± 0.7

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msec2; SA-PM – SDNN = 7.9 ± 0.7 mscec, RMSSD = 5.3 ± 0.6 msec, LF/HF = 7.1 ± 0.6

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msec2; SA-O3 – SDNN = 8.7 ± 0.7 msec, RMSSD = 5.5 ± 0.7 msec, LF/HF = 6.7 ± 0.6

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msec2). SDNN decreased in the first hour of exposure in control animals; this was likely

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due to sympathetic modulation from the stress of handling and placement in the

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chamber. Animals exposed to either SA-PM or SA-O3 had a significant increase in

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SDNN (Fig. 3A) when compared to FA and a similar trend was observed for RMSSD

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(Fig. 4A). Both time-domain measures showed minimal to no effects for either SA-PM or

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SA-O3 when the responses were normalized to THC (Fig. 3B and 4B) or gas-phase

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hydrocarbons only (Fig. 3C and 4C). However, the increase in SDNN and RMSSD

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during SA-O3 exposure was significantly greater than SA-PM when the data were

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normalized to PM concentration (Fig. 3D and 4D). On the other hand, there were no

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significant effects in the frequency-domain measures (i.e. LF/HF) for either smog

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atmosphere with or without normalization of responses (Fig. 5).

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Ventilatory function – Whole-body plethysmography was performed on all animals

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during the baseline period as well as immediately and one day after exposure; results

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shown in Fig. 6 compare the changes in ventilatory parameters from baseline between

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the groups. Baseline values did not differ between the groups (FA – f = 475.1 ± 13.1

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breaths/min, VT = 0.24 ± 0.01 ml, Ti = 0.06 ± 0.002 sec, Te = 0.08 ± 0.002 sec; SA-PM –

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f = 509.8 ± 6.3 breaths/min, VT = 0.24 ± 0.01 ml, Ti = 0.05 ± 0.001 sec, Te = 0.07 ±

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0.001 sec; SA-O3 – f = 499.7 ± 12.1 breaths/min, VT = 0.25 ± 0.01 ml, Ti = 0.06 ± 0.002

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sec, Te = 0.07 ± 0.002 sec). There were no differences in f, VT, Ti or Te between any of

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the groups in the unnormalized results (Fig. 6A). However, normalizing to THC (Fig. 6B)

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or gas-phase only hydrocarbons (Fig. 6C) revealed a significant decrease in f, and

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increase in VT and Ti in SA-PM and SA-O3 immediately after exposure when compared

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to controls. Some of these responses relative to FA persisted one day after exposure

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but there was no difference between SA-PM and SA-O3. In contrast, normalization to

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PM concentrations revealed a significant increase in f, and decrease in VT and Ti

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immediately after exposure in SA-O3-exposed animals when compared to SA-PM (Fig.

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6D); only the changes in f and VT persisted 24hrs later.

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Electrocardiogram – There were no baseline differences in any ECG parameters

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between any of the groups. Changes in ECG during exposure were restricted to a PR

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interval prolongation in mice exposed to SA-PM, which were significantly higher than FA

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and SA-O3 (Table 2).

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Discussion

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The results of this study demonstrate that a single inhalation exposure to atmospheric

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smog causes acute cardiovascular dysfunction in mice, irrespective of whether it is

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comprised predominantly of PM or O3. Our findings also suggest that multipollutant

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mixtures which have a higher irritant gas composition are likely to cause more potent

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acute cardiac effects than those with higher PM/lower gases. Unfortunately, the relative

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toxicity of only the SOA in each of the atmospheres was not assessed, this would have

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provided greater information about their contributions to the differential health effects we

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observed. In either case, it is not possible to firmly conclude whether PM in general

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plays a greater role in causing cardiac effects or not because SOA is not necessarily the

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same as ambient PM. However, in an effort to compare the effects of SA-PM and SA-O3

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relative to their whole compositions, we normalized the data from this study to total

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hydrocarbons, gas-phase hydrocarbons only and PM concentration. Normalization of

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the results showed that there was no difference in heart rate, heart rate variability or

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cardiac arrhythmias between SA-PM and SA-O3 when differences in total hydrocarbons

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or gas-phase only hydrocarbons were taken into account. In contrast, SA-O3 caused

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significantly greater cardiac effects than SA-PM when results were normalized to PM

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concentration indicating that the difference in response was mediated by the gaseous

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components. Thus, these findings point to the complexity of interactions between air

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pollution components and the reactions that determine the composition of the final

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mixture and the resulting cardiovascular response.

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Real-time cardiovascular measurements and responses to exposure as derived here

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from radiotelemetry often include stress-related effects (e.g. from handling, noise, etc).

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This was observed in all animals as a transient increase in heart rate upon placement in

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the exposure chamber, which occurred despite two separate one-hour acclimatizations.

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In controls, the overall heart rate progressively decreased per hour of exposure as the

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animals calmed down in the chambers. Similar responses were observed with SA-O3

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but with a trend of greater decrease in heart rate. Although this result was not

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statistically significant, it is not entirely surprising that SA-O3 would have this effect given

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it was rich in irritant gases like O3, NOx and reactive aldehydes, which have the ability to

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activate airway sensory nerves, elicit autonomic reflexes and modulate parasympathetic

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function17-19. On the other hand, SA-PM caused a significant increase in heart rate in

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the first hour of exposure and less decrease (i.e. HR remained elevated above normal)

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over the remaining exposure period when compared to controls and SA-O3. These

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disparate effects of SA-PM and SA-O3 are not unusual. We previously showed that

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exposure to particle-filtered diesel exhaust caused greater decrements in heart rate

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than whole diesel exhaust while particle-rich air pollution has been shown to increase

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heart rate13,20,21. Thus, it appears from our data that decreases in heart rate during such

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exposures are related to the composition and concentration of gases in the complex air

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pollution mixture. Notice that normalization of the heart rate to total or gas-phase only

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hydrocarbons did not reveal any difference between SA-PM and SA-O3, yet when the

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“effects” of PM were eliminated through normalization SA-O3 caused a significant

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decrease in heart rate when compared to SA-PM.

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The composition of the smog atmospheres may not have been the only determinant of

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this response, variations in the smell of SA-PM and SA-O3 could have contributed as

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well. SA-PM had a very potent smell when compared to SA-O3 and this may have

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caused an increase in the heart rate. This effect has been demonstrated previously,

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particularly with burnt or unpleasant smells22, and appears to be mediated by an

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increase in sympathetic modulation or decrease in heart rate variability. It is likely that in

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addition to smell, several smog factors could have triggered responses and altered

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autonomic function simultaneously, particularly given the complexity of the atmosphere

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(e.g. potent “burnt” smell + chemical airway irritation + secondary activation due to

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inflammation). The autonomic nervous system controls the heart and vasculature

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through a dynamic ebb and flow of both parasympathetic and sympathetic influence and

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tends to lean in the direction of one or the other based on the physiological

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circumstances. Thus, returning to the issue of composition, the difference in response

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between a predominantly gaseous mixture and one that is high in particles would

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depend on the sum of all the factors that impact autonomic function, and hence the

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resulting direction would be determined by which factor(s) dominates.

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Although our data do not necessarily demonstrate this (i.e. parasympathetic/

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sympathetic activation) directly, it is possible that irritant gases drove a predominantly

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parasympathetic response whereas the PM-rich mixture caused a stress-induced

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sympathetic modulation. We observed a greater increase in SDNN and RMSSD, which

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is indicative of parasympathetic modulation, during SA-O3 exposure when compared to

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SA-PM. This response was due to the effects of the gas-phase components given the

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difference in these parameters between the two smog atmospheres became evident

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when we normalized to PM concentration. As far as PM is concerned, studies have

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demonstrated, as stated previously, that it not only increases heart rate, blood pressure,

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low frequency blood pressure variability and noradrenalin release (i.e. stress), but also

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causes a decrease in heart rate variability3,23. All of these changes point to sympathetic

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modulation and a perceived increase in cardiac risk. However, we observed an increase

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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

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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

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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

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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

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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

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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)

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AQHI

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648 649 650 651 652 653 654 655

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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

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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

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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)

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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)

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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)

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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)

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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)

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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)

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