Short-term increases in particulate matter (PM) are associated with heightened morbidity and mortality from cardiovascular causes. Inhalation of PM is known to increase endothelin (ET)-1 levels. Yet, less is known about particle composition-related changes at the molecular level including the endothelinergic system and relationship with cardiovascular function changes. In this work, adult Wistar male rats were exposed for 4 h by nose-only inhalation to clean air, Ottawa urban particles (EHC-93, 48 mg/m3) and water-leached (EHC-93L, 49 mg/m3) particles, to examine the effect of particle compositional changes on oxidative stress, circulating ETs, blood pressure, and heart electrophysiology. Particle deposition in the respiratory compartment was estimated at 85 μg (25 ng/cm2). Lung cell proliferation was low in both treatment groups, indicating absence of acute injury. Inhalation of EHC-93 caused statistically significant elevations (p < 0.05) of oxidative stress markers, ET-1, ET-3, blood pressure, and a decrease of ST-segment duration in the ECG at 1.5 days post-exposure. Leached particles (EHC-93L) caused rapid but transient elevation (p < 0.05) of oxidative stress, ET-1, ET-2, and ET-3 at earlier time points, with no changes in blood pressure or ST-segment. These results demonstrate that inhalation of urban particles at an internal dose inadequate to cause acute lung injury can induce oxidative stress, enhance vasoactive endothelins, leading to vasopressor response, affecting cardiac electrophysiology in Wistar rats, consistent with the cardiovascular impacts of ambient particles in human populations. Change in particle potency after removal of soluble species, notably cadmium, zinc and polar organics suggests that the toxicodynamics of cardiovascular effects can be modified by physicochemical properties of particles.

Ambient particulate matter (PM) exposure is linked to cardiovascular events and death, especially among individuals with heart disease. A model of toxic cardiomyopathy was developed in Spontaneously Hypertensive Heart Failure (SHHF) rats to explore potential mechanisms. Rats were infused with isoproterenol (ISO; 2.5 mg/kg/day subcutaneous [sc]), a beta-adrenergic agonist, for 28 days and subsequently exposed to PM by inhalation. ISO induced tachycardia and hypotension throughout treatment followed by postinfusion decrements in heart rate, contractility, and blood pressures (systolic, diastolic, pulse), and fibrotic cardiomyopathy. Changes in heart rate and heart rate variability (HRV) 17 days after ISO cessation indicated parasympathetic dominance with concomitantly altered ventilation. Rats were subsequently exposed to filtered air or Harvard Particle 12 (HP12) (12 mg/m<SU3</SU)-a metal-rich oil combustion-derived PM-at 18 and 19 days (4 h/day) after ISO infusion via nose-only inhalation to determine if cardio-impaired rats were more responsive to the effects of PM exposure. Inhalation of PM among ISO-pretreated rats significantly increased pulmonary lactate dehydrogenase, serum high-density lipoprotein (HDL) cholesterol, and heart-to-body mass ratio. PM exposure increased the number of ISO-pretreated rats that experienced bradyarrhythmic events, which occurred concomitantly with acute alterations of HRV. PM, however, did not significantly affect mean HRV in the ISO- or saline-pretreated groups. In summary, subchronic ISO treatment elicited some pathophysiologic and histopathological features of heart failure, including cardiomyopathy. The enhanced sensitivity to PM exposure in SHHF rats with ISO-accelerated cardiomyopathy suggests that this model may be useful for elucidating the mechanisms by which PM exposure exacerbates heart disease.</.

Pulmonary C-fibers are stimulated by irritant air pollutants producing apnea, bronchospasm, and decrease in HR. Chemoreflex responses resulting from C-fiber activation are sometimes mediated by TRPV1 and release of substance P. While acrolein has been shown to stimulate C-fibers, the persistence of acrolein effects and the role of C-fibers in these responses are unknown. These experiments were designed to determine the effects of whole-body acrolein exposure and pulmonary chemoreflex response post-acrolein. Rats were exposed to either air or 3 ppm acrolein for 3 h while ventilatory function and HR were measured; 1-day later response to capsaicin challenge was measured in anesthetized rats. Rats experienced apnea and decrease in HR upon exposure to acrolein, which was not affected by either TRPV1 antagonist or NK(1)R antagonist pretreatment. Twenty-four hours later, capsaicin caused apnea and bronchoconstriction in control rats, which was potentiated in rats exposed to acrolein. Pretreatment with TRPV1 antagonist or NK(1)R antagonist prevented potentiation of apneic response and bronchoconstriction 24h post-exposure. These data suggest that although potentiation of pulmonary chemoreflex response 24h post-acrolein is mediated by TRPV1 and release of substance P, cardiopulmonary inhibition during whole-body acrolein exposure is mediated through other mechanisms.

Pulmonary C fibers are stimulated by irritant air pollutants such as acrolein (Lee et al. 1992) producing apnea, bronchospasm, and decreases in heart rate (HR). Lung C fiber-induced chemoreflex activation is mediated by TRPV1 vanilloid receptors and release of substance P (SP). While acrolein has been shown to stimulate C-fibers the persistence of acrolein effects (24hrs) and the role of C-fibers in these responses are unknown. These experiments were designed to determine whether pulmonary C-fiber hyperresponsiveness post-acrolein is mediated by TRPV1 activation and release of SP. Rats were exposed to either air or 3ppm acrolein for 3 hours; 1 day later capsaicin (cap)-induced ventilatory and HR responses were measured in anesthetized rats. Cap (1-8 μg/kg, iv) caused apnea and bronchoconstriction in control rats, that was potentiated in rats exposed to acrolein. Pretreatment with TRPV1-antagonist SB 366791 (2mg/kg, ip) prevented potentiation of this acrolein apneic response but not bronchoconstriction, but pretreatment with NK1R antagonist RP 67580 (3mg/kg, ip) prevented both. Drugs had no effect on controls nor were there any changes in HR. These data suggest that sensitization of pulmonary C-fibers by acrolein involves activation of TRPV1 and release of SP, but does not impact cardiac function. Supported by Curriculum in Toxicology/UNC Grant T32-ES07126. (This abstract does not reflect EPA policy.)

Heart rate variability (HRV) is a measure of cardiac pacing dynamics that has recently garnered a great deal of interest in environmental health studies. While the use of these measures has become popular, much uncertainty remains in the interpretation of results, both in terms of human and animal research. In humans, HRV endpoints, specifically chronic alterations in baseline HRV patterns, have been reasonably well characterized as prognostic indicators of adverse outcomes for a variety of diseases. However, such information is lacking for reversible HRV changes that may be induced by short-term exposures to environmental toxicants. Furthermore, there are minimal substantive data, either acute or chronic, regarding the pathological interpretation or prognostic value of toxicant-induced changes in HRV in rodents. The present report summarizes the physiological and clinical aspects of HRV, the methodological processes for obtaining these endpoints, and previous human and animal studies in the field of environmental health. Furthermore, we include a discussion of important caveats and recommendations for the interpretation of HRV data in animal research.

As computer technology has advanced in the last ten years, the ability to acquire copious amounts of physiological data has become much easier. Our laboratory regularly uses radiotelemetry methodology in rodents to acquire nonstop heart rate (HR) and core temperature (Tco) data while animals are exposed to exogenous substances, including air pollutants such as ozone and particulate matter. Bradycardia and hypothermia are often observed during and following exposure, although it is difficult to pinpoint the response start time and duration using traditional statistical methods. We have developed an approach for analyzing continuously-derived physiological data using functional data analysis (FDA) and subsequent computational techniques (principal component analysis and the Direction-Projection-Permutation hypothesis test) which permit the comparison of data curves. This approach was employed on HR data collected before and after Spontaneously Hypertensive rats (n=35; 8–9/group) were intratracheally instilled with a bolus dose of saline (control) or residual oil fly ash particles (0.83, 3.33, or 8.33 mg/kg). The statistical analysis demonstrated that effects between the control and high dose group persisted for at least 48 hr. The applicability of FDA to data sets having a greater number of repeated measurements than the sample size was established with our rodent HR data and will clearly be useful for statistical analysis of similar data sets.

Background: Epidemiological studies have reported increased risks of cardiopulmonary-related hospitalization and death in association with exposure to elevated levels of particulate matter (PM) across a wide range of urban areas. In response to these findings, researchers have conducted animal inhalation exposures aimed at reproducing the observed toxicologic effects. However, it is technically difficult to quantitate the actual amount of PM delivered to the lung in such studies, and dose is frequently estimated using default respiration parameters. Consequently, the interpretation of PM-induced effects in rodents exposed via whole-body inhalation is often compromised by the inability to determine deposited dose. To address this problem, we have developed an exposure system that merges the generation of dry, aerosolized particles with whole-body plethysmography (WBP), thus permitting inhalation exposures in the unrestrained rat while simultaneously obtaining data on pulmonary function.Results: This system was validated using an oil combustion-derived particle (HP12) at three nominal concentrations (3, 12, and 13 mg/m(3)) for four consecutive exposure days (6 hr/day); a single 6-hour exposure to 13 mg/m(3) of HP12 was also conducted. These results demonstrated that the system was both reliable and consistent over these exposure protocols, achieving average concentrations that were within 10% of the targeted concentration. In-line filters located on the exhaust outlets of individual WBP chambers showed relative agreement in HP12 mass for each day and were not statistically different when compared to one another (p = 0.16). Temperatures and relative humidities were also similar between chambers during PM and air exposures. Finally, detailed composition analyses of both HP12 filter and bulk samples showed that grinding and aerosolization did not change particle chemistry.Conclusion: The results of this study demonstrate that it is possible to expose rodents to resuspended, dry PM via whole-body inhalation while these animals are maintained in WBP chambers. This new methodology should significantly improve the ability to assess dosimetry under minimally stressful exposure conditions.

A plethora of epidemiological studies have shown that exposure to elevated levels of ambient particulate matter (PM) can lead to adverse health outcomes, including cardiopulmonary-related mortality. Subsequent animal toxicological studies have attempted to mimic these cardiovascular and respiratory responses, in order to better understand underlying mechanisms. However, it is difficult to quantitate the amount of PM deposited in rodent lungs following inhalation exposure, thus making fundamental dose-to-effect assessment and linkages to human responses problematic. To address this need, spontaneously hypertensive rats were exposed to an oil combustion-derived PM (HP12) via inhalation while being maintained in whole-body plethysmograph chambers. Rats were exposed 6 h/day to 13 mg/m(3) of HP12 for 1 or 4 days. Immediately following the last exposure, rats were sacrificed and their tracheas and lung lobes harvested and separated for neutron activation analysis. Total lower respiratory tract deposition ranged from 20-60 microg to 89-139 microg for 1- and 4-day exposures, respectively. Deposition data were compared to default and rat-specific estimates provided by the Multiple Path Particle Deposition (MPPD) model, yielding model predictions that were < 33% of the measured dose. This study suggests that HP12 exposure decreased particle clearance, as the mass of HP12 in the lungs following a 4-day protocol was nearly four times that observed after a 1-day exposure. This work should improve the ability of risk assessors to extrapolate rat-to-human exposure concentrations on the basis of lung burdens and, thus, better relate inhaled doses and resultant toxicological effects.

A consistent association between exposure to high concentrations of ambient particulate matter (PM) and excess cardiopulmonary-related morbidity and mortality has been observed in numerous epidemiological studies, across many different geographical locations. To elicit a similar response in a controlled laboratory setting, spontaneously hypertensive rats were exposed to an oil combustion-derived PM (HP-12) and monitored for changes in pulmonary function and indices of pulmonary injury. Rats were implanted with radiotelemeters to monitor electrocardiogram, heart rate, systemic arterial blood pressure, core temperature, and activity. Animals were divided into four groups and exposed via intratracheal instillation (IT) to suspensions of HP-12 (0.0, 0.83, 3.33, and 8.33 mg/kg; control, low, mid, and high dose, respectively) in saline vehicle. Telemetered rats were monitored continuously for 4-7 days post-IT and pulmonary function was examined using a whole-body plethysmograph system for 6 h/day on post-IT days 1-7. At 24, 96, and 192 h post-IT, bronchoalveolar lavage fluid (BALF) was obtained from subsets of nontelemetered animals in order to assess the impact of HP-12 on biochemical indices of pulmonary inflammation and injury. Immediate dose-related changes in pulmonary function were observed after HP-12 exposure, consisting of decreases in tidal volume (decreasing 12-41%) and increases in breathing frequency (increasing 52-103%), minute ventilation (increasing 12-25%), and enhanced pause (increasing 113-187%). These functional effects were resolved by 7 days post-IT, although some average BALF constituents remained elevated through day 7 for mid- and high-dose groups when compared to those of the saline-treated control group. This study demonstrates significant deficits in pulmonary function, along with significant increases in BALF indices of pulmonary inflammation and injury in SH rats after IT exposure to HP-12.

Rodents often demonstrate a profound depression in physiological function following acute exposure to toxic xenobiotic agents. This effect, termed the hypothermic response, is primarily characterized by significant decreases in core temperature and heart rate and is generally accompanied by similar deficits in other important functional parameters. This response appears to be remarkably consistent across a wide variety of toxic agents and exposure regimens; however, the magnitude and duration of the induced effects may be modulated by changes in dose, animal mass, and environmental conditions. While the initiating stimulus and underlying mechanism(s) remains elusive, this response may represent an inherent reflexive pattern that is unique to the rodent and serves to attenuate the induced toxicity. Given that rodents are the primary animal species used in toxicological studies, it is important to consider this hypothermic response and its modulatory factors when interpreting the results of such studies and extrapolating those results to man.

Particulate matter air pollution has been associated with cardiopulmonary morbidity and mortality in many recent epidemiological studies. Previous toxicological research has demonstrated profound cardiac and thermoregulatory changes in rats following exposure to residual oil fly ash (ROFA), a combustion-derived particulate. The response to ROFA appeared biphasic, consisting of both immediate (0-6 h) and delayed (24-96 h) bradycardia and hypothermia. Other studies have demonstrated that much of the pulmonary toxicity of ROFA was caused by its constitutive transition metals, namely, Fe, Ni, and V. This study examined the contributions of these metals to the observed cardiac and thermoregulatory changes caused by ROFA in conscious, unrestrained rats. Prior to exposure, each animal was surgically implanted with a radiotelemetry device capable of continuously monitoring heart rate, electrocardiographic, and core temperature data. Individual metals were intratracheally instilled in healthy rats (n = 4 per metal species) and in rats with monocrotaline (MCT; 60 mg/kg)-induced pulmonary hypertension (n = 10 per metal species); combinations of metals were instilled in MCT-treated rats only (n = 6 per combination of metal species). Metals were administered in doses equivalent to those found in the highest dose of ROFA used in previous studies, that is, 105 microg Fe(2)(SO(4))(3), 263 microg NiSO(4), and 245 microg VSO(4). Healthy and MCT-treated rats demonstrated similar responses to metals. Fe caused little response, whereas V caused marked bradycardia, arrhythmogenesis, and hypothermia immediately following instillation and lasting approximately 6 h. Ni caused no immediate response, but induced a delayed bradycardia, arrhythmogenesis, and hypothermia that began approximately 24 h after instillation and lasted for several days. When instilled in combination, Ni appeared to exacerbate the immediate effects of V, whereas Fe attenuated them. These data suggest that the biphasic response to instilled ROFA may result from a summation of the temporally different effects of V and Ni.

Recent epidemiological studies have shown an association between daily morbidity and mortality and ambient particulate matter (PM) air pollution. It has been proposed that bioavailable metal constituents of PM are responsible for many of the reported adverse health effects. Studies of instilled residual oil fly ash (ROFA) demonstrated immediate and delayed responses, consisting of bradycardia, hypothermia, and arrhythmogenesis in conscious, unrestrained rats. Further investigation of instilled ROFA-associated transition metals showed that vanadium (V) induced the immediate responses, while nickel (Ni) was responsible for the delayed effects. Furthermore, Ni potentiated the immediate effects caused by V when administered concomitantly. The present study examined the responses to these metals in a whole-body inhalation exposure. To ensure valid dosimetric comparisons with instillation studies, 4 target exposure concentrations ranging from 0.3-2.4 mg/m(3) were used to incorporate estimates of total inhalation dose derived using different ventilatory parameters. Rats were implanted with radiotelemetry transmitters to continuously acquire heart rate (HR), core temperature (T-CO), and electrocardiographic data throughout the exposure. Animals were exposed to aerosolized Ni, V, or Ni + V for 6 h per day x 4 days, after which serum and bronchoalveolar lavage samples were taken. Even at the highest concentration, V failed to induce any significant change in HR or T-CO. Ni caused delayed bradycardia, hypothermia, and arrhythmogenesis at concentrations > 1.2 mg/m(3). When combined, Ni and V produced observable delayed effects at 0.5 mg/m(3) and potentiated responses at 1.3 mg/m(3), greater than were produced by the highest concentration of Ni (2.1 mg/m(3)) alone. These results indicate a possible synergistic relationship between inhaled Ni and V, and provide insight into potential interactions regarding the toxicity of PM-associated metals.