BACKGROUND: Ozone ([O.sub.3]) is a well-documented respiratory oxidant but increasing epidemiological evidence points to extrapulmonary effects, including positive associations between ambient [O.sub.3] concentrations and cardiovascular morbidity and mortality.
OBJECTIVE; With preliminary reports linking [O.sub.3] exposure with changes in heart rate (HR), we investigated the hypothesis that a single inhalation exposure to [O.sub.3] will cause concentration-dependent autonomic modulation of cardiac function in rats.
METHODS: Rats implanted with telemeters to monitor HR and cardiac electrophysiology [electrocardiography (ECG)] were exposed once by whole-body inhalation for 4 hr to 0.2 or 0.8 ppm [O.sub.3] or filtered air. A separate cohort was tested for vulnerability to aconitine-induced arrhythmia 24 hr after exposure.
RESULTS: Exposure to 0.8 ppm [O.sub.3] caused bradycardia, PR prolongation, ST depression, and substantial increases in atrial premature beats, sinoatrial block, and atrioventricular block, accompanied by concurrent increases in several HR variability parameters that were suggestive of increased parasympathetic tone. Low-03 exposure failed to elicit any overt changes in autonomic tone, heart rhythm, or ECG. However, both 0.2 and 0.8 ppm [O.sub.3] increased sensitivity to aconitine-induced arrhythmia formation, suggesting a latent [O.sub.3]-induced alteration in myocardial excitability.
CONCLUSIONS: [O.sub.3] exposure causes several alterations in cardiac electrophysiology that are likely mediated by modulation of autonomic input to the heart. Moreover, exposure to low [O.sub.3] concentrations may cause subclinical effects that manifest only when triggered by a stressor, suggesting that the adverse health effects of ambient levels of air pollutants may be insidious and potentially under-estimated.
KEY WORDS: air pollution, arrhythmia, autonomic, cardiac, electrocardiogram, heart rate variability, inhalation, latent, overt, ozone, rats. Environ Health Perspect 120:348-354 (2012). http://dx.doi. org/10.1289/ehp.l 104244 [Online 2 December 2011]
Ozone ([O.sub.3]) is a major smog-associated oxidant with well-established respiratory effects, including decrements in lung function, airway injury and inflammation, compromised host defense, and asthma exacerbation (Hollingsworth et al. 2007; Mudway and Kelly 2000). Although the lung has understandably been the target organ of interest, recent epidemiological evidence suggests a positive association between inhaled [O.sub.3] and clinical cardiovascular events linked to coronary artery disease, myocardial infarction, and atherosclerosis (Srcbot et al 2009); these effects are largely independent of exposure to other pollutants. In controlled human exposure studies, [O.sub.3] exposure has reduced maximal oxygen uptake (Gong et al 1998) and, in combination with ambient particulate matter (PM), increased diastolic blood pressure (Fakhri et al. 2009) and caused arterial vasoconstriction (Brook et al 2002). Adverse cardiovascular effects, including increased atherosclerotic plaque size (Chuang et al. 2009) and enhanced sensitivity to ischemic injury (Perepu et al. 2010), have also been reported in animal models.
Upon inhalation, [O.sub.3] is thought to oxidate or pcroxidate biological molecules (directly or indirectly) at the surface of the respiratory tract, triggering a pathological cascade characterized by lipid peroxidation, enzyme inactivation, free radical formation, altered membrane permeability, and inflammation (Mustafa 1990). Less is known, however, about the mechanisms mediating [O.sub.3]-induced cardiovascular responses and the potential influence of [O.sub.3]-induced respiratory effects on cardiovascular function. Although preliminary, the available evidence implicates the following mechanisms: vascular oxidative stress, endothelial/vascular dysfunction, inflammation, and altered autonomic tone (Srebotetal. 2009).
Because cardiac impulse formation, propagation, and arrhythmia often result from the modulation of autonomic balance, one of the most conspicuous data gaps in the impact of [O.sub.3] exposure on normal cardiac electrophysiology and heart rate (HR) is the potential contribution of [O.sub.3]-induced modulation of autonomic tone to these effects. Additionally, [O.sub.3] exposure at ambient concentrations may not cause overt functional effects, but rather may produce latent or subclinical effects that appear only when the myocardium or specialized conduction system is further stressed, for example, as a result of cellular calcium loading with aconitine. It is uncertain whether [O.sub.3] exposure elicits such effects. We have previously shown that exposure to PM (Carll et al. 2011; Farraj et al. 2009, 2011; Hazari et al. 2009), diesel exhaust (Hazari et al. 2011), or the irritant acrolein (Hazari et al. 2009) in hypertensive or heart failure rats causes functional cardiac effects, including bradycardia, arrhythmia, increased parasympathetic tone, and/or increased sensitivity to triggered cardiac arrhythmia. The purpose of this study was to examine the concentration-dependent effects of acute [O.sub.3] exposure on HR, heart rhythm, HR variability (HRV; a measure of autonomic tone to the heart), electrocardiography (EGG), and pulmonary and systemic inflammation. In addition, we assessed whether [O.sub.3] exposure increases latent vulnerability to cardiac arrhythmia, hypothesizing that [O.sub.3] acts through rhe autonomic nervous system to prime the heart to react to secondary challenges.
Materials and Methods
Animals. Twelve-week-old male spontaneously hypertensive (SH) rats were obtained from Charles River Laboratory (Raleigh, NC). SH rats were selected because we previously determined (Farraj et al. 2009) that they are more sensitive to the inflammatory and proarrhythmic effects of acute air pollutant exposure [SH rats have higher mean arterial pressure (- 40 mmHg difference), on average, than do control rats with normal blood pressure at 12 weeks of age (El-Mas and Abdel-Rahman 2005)]. Rats were housed in plastic cages (one per cage), maintained on a 12/12-hr light/dark cycle at approximately 22[degrees] C and 50% relative humidity in our Association for Assessment and Accreditation of Laboratory Animal Care-approved facility, and held for a minimum of 1 week before implantation. All protocols were approved by the Institutional Animal Care and Use Committee of the U.S. Environmental Protection Agency (EPA). Rat food (Prolab RMH 3000; PMI Nutrition International, St. Louis, MO) and water were provided ad libitum. All rars were randomized by weight. Animals were treated humanely and with regard for alleviation of suffering.
Experimental design and [O.sub.3] exposure. SH rats were surgically implanted with ECG biopotential telemeters and then exposed via whole-body inhalation to 0.2 or 0.8 ppm [O.sub.3] or filtered air once for 4 hr. ECG, HR, body temperature, and activity were monitored before, during, and after exposure to [O.sub.3] or air. All telemetered rats were sacrificed 1 day after exposure to [O.sub.3] or air. A second cohort of rats (untelemetered) in each exposure group was sacrificed 1 hr after exposure to assess potential immediate inflammatory or toxicity …