Najwa Haykal-Coates

TRPA1 and Sympathetic Activation Contribute to Increased Risk of Triggered Cardiac Arrhythmias in Hypertensive Rats Exposed to Diesel Exhaust

Background: Diesel exhaust (DE), which is emitted from on- and off-road sources, is a complex mixture of toxic gaseous and particulate components that leads to triggered adverse cardiovascular effects such as arrhythmias. Objective: We hypothesized that increased risk of triggered arrhythmias 1 day after DE exposure is mediated by airway sensory nerves bearing transient receptor potential (TRP) channels [e.g., transient receptor potential cation channel, member A1 (TRPA1)] that, when activated by noxious chemicals, can cause a centrally mediated autonomic imbalance and heightened risk of arrhythmia. Methods: Spontaneously hypertensive rats implanted with radiotelemeters were whole-body exposed to either 500 μg/m3 (high) or 150 μg/m3 (low) whole DE (wDE) or filtered DE (fDE), or to filtered air (controls), for 4 hr. Arrhythmogenesis was assessed 24 hr later by continuous intravenous infusion of aconitine, an arrhythmogenic drug, while heart rate (HR) and electrocardiogram (ECG) were monitored. Results: Rats exposed to wDE or fDE had slightly higher HRs and increased low-frequency:high-frequency ratios (sympathetic modulation) than did controls; ECG showed prolonged ventricular depolarization and shortened repolarization periods. Rats exposed to wDE developed arrhythmia at lower doses of aconitine than did controls; the dose was even lower in rats exposed to fDE. Pretreatment of low wDE–exposed rats with a TRPA1 antagonist or sympathetic blockade prevented the heightened sensitivity to arrhythmia. Conclusions: These findings suggest that a single exposure to DE increases the sensitivity of the heart to triggered arrhythmias. The gaseous components appear to play an important role in the proarrhythmic response, which may be mediated by activation of TRPA1, and subsequent sympathetic modulation. As such, toxic inhalants may partly exhibit their toxicity by lowering the threshold for secondary triggers, complicating assessment of their risk.

ST Depression, Arrhythmia, Vagal Dominance, and Reduced Cardiac Micro-RNA in Particulate-Exposed Rats

Recently, investigators demonstrated associations between fine particulate matter (PM)-associated metals and adverse health effects. Residual oil fly ash (ROFA), a waste product of fossil fuel combustion from boilers, is rich in the transition metals Fe, Ni, and V, and when released as a fugitive particle, is an important contributor to ambient fine particulate air pollution. We hypothesized that a single-inhalation exposure to transition metal-rich PM will cause concentration-dependent cardiovascular toxicity in spontaneously hypertensive (SH) rats. Rats implanted with telemeters to monitor heart rate and electrocardiogram were exposed once by nose-only inhalation for 4 hours to 3.5 mg/m(3), 1.0 mg/m(3), or 0.45 mg/m(3) of a synthetic PM (dried salt solution), similar in composition to a well-studied ROFA sample consisting of Fe, Ni, and V. Exposure to the highest concentration of PM decreased T-wave amplitude and area, caused ST depression, reduced heart rate (HR), and increased nonconducted P-wave arrhythmias. These changes were accompanied by increased pulmonary inflammation, lung resistance, and vagal tone, as indicated by changes in markers of HR variability (increased root of the mean of squared differences of adjacent RR intervals [RMSSD], low frequency [LF], high frequency [HF], and decreased LF/HF), and attenuated myocardial micro-RNA (RNA segments that suppress translation by targeting messenger RNA) expression. The low and intermediate concentrations of PM had less effect on the inflammatory, HR variability, and micro-RNA endpoints, but still caused significant reductions in HR. In addition, the intermediate concentration caused ST depression and increased QRS area, whereas the low concentration increased the T-wave parameters. Thus, PM-induced cardiac dysfunction is mediated by multiple mechanisms that may be dependent on PM concentration and myocardial vulnerability (this abstract does not reflect the policy of the United States Environmental Protection Agency).

Gestational exposure to methylmercury alters the developmental pattern of trk-like immunoreactivity in the rat brain and results in cortical dysmorphology

Nerve growth factor signal transduction mediated through the trk receptor has been implicated in neuronal growth, differentiation, and survival. In this study, we examined the effects of gestational exposure to the developmental neurotoxicant methylmercury (CH3Hg) on the ontogeny of trk-immunoreactivity (IR). Long-Evans dams were dosed on gestational days 6-15 (p.o.) with 0, 1, or 2 mg/kg CH3Hg dissolved in saline. Pups were sacrificed and perfused with buffered paraformaldehyde on postnatal days (PND) 1, 4, 10, 21 and 85. The brains were sectioned sagitally, Nissl-stained or stained immunohistochemically for trk receptors or glial fibrillary acidic protein (GFAP), and examined throughout the medial to lateral extent of the brain. The greatest density of IR in neural cell bodies was seen in the olfactory bulb, hippocampus, cerebral, and cerebellar cortex, striatum, septum, nucleus basalis, inferior colliculus, pons, and brain stem nuclei. trk IR was not limited to nerve cell bodies, with prominent axonal and dendritic staining in the brainstem, neocortex, hippocampus, cerebellum, and olfactory tract. The regional pattern of trk IR varied in an age-dependent manner. In controls, trk-like IR appeared to peak in most regions between PND4-10 and decreased dramatically after PND21. This age-related difference in trk IR was supported by western blot analysis of PND10 and adult neocortex. This reduced and more adult-like pattern of trk IR was apparent on PND21 with some persistent trk-like IR in the olfactory bulb, hippocampus, neocortex, cerebellum and basal forebrain. In contrast to the normal regional patterns of trk IR, CH3Hg produced a dose-related decrease in trk-like IR in the absence of overt maternal toxicity or neonatal toxicity. CH3Hg-induced decreases in trk-like IR were especially apparent during the early postnatal period when trk IR was the greatest. The effects of CH3Hg exposure were restricted regionally, with the largest decrease in trk-like IR apparent in cortical regions, basal forebrain nuclei, and brain stem nuclei. Subsequent to the effects of CH3Hg on cortical trk-like IR were alterations in the development of cortical laminae on PND10 and 21 of neocortex. These alterations were characterized by quantifiable decreases in cell density, cell size and the widths of the layers of posterior neocortex. Not all of the CH3Hg-induced effects were characterized by decreased trk-like IR. Robust increases in trk IR in glial cells in the corpus callosum and brain stem were observed coincident with increased GFAP IR in cells of similar morphology. The present results localize the cellular and regional ontogeny of trk and suggest that developmental exposure to CH3Hg alters the normal ontogeny of this trophic factor receptor which may be associated with the developmental neurotoxicity of this chemical.

Increased Nonconducted P-Wave Arrhythmias after a Single Oil Fly Ash Inhalation Exposure in Hypertensive Rats

Background Exposure to combustion-derived fine particulate matter (PM) is associated with increased cardiovascular morbidity and mortality especially in individuals with cardiovascular disease, including hypertension. PM inhalation causes several adverse changes in cardiac function that are reflected in the electrocardiogram (ECG), including altered cardiac rhythm, myocardial ischemia, and reduced heart rate variability (HRV). The sensitivity and reliability of ECG-derived parameters as indicators of the cardiovascular toxicity of PM in rats are unclear. Objective We hypothesized that spontaneously hypertensive (SH) rats are more susceptible to the development of PM-induced arrhythmia, altered ECG morphology, and reduced HRV than are Wistar Kyoto (WKY) rats, a related strain with normal blood pressure. Methods We exposed rats once by nose-only inhalation for 4 hr to residual oil fly ash (ROFA), an emission source particle rich in transition metals, or to air and then sacrificed them 1 or 48 hr later. Results ROFA-exposed SH rats developed nonconducted P-wave arrhythmias but no changes in ECG morphology or HRV. We found no ECG effects in ROFA-exposed WKY rats. ROFA-exposed SH rats also had greater pulmonary injury, neutrophil infiltration, and serum C-reactive protein than did ROFA-exposed WKY rats. Conclusions These results suggest that cardiac arrhythmias may be an early sensitive indicator of the propensity for PM inhalation to modify cardiovascular function.

Divergent Electrocardiographic Responses to Whole and Particle-Free Diesel Exhaust Inhalation in Spontaneously Hypertensive Rats

Diesel exhaust (DE) is a major contributor to traffic-related fine particulate matter (PM)(2.5). Although inroads have been made in understanding the mechanisms of PM-related health effects, DEs complex mixture of PM, gases, and volatile organics makes it difficult to determine how the constituents contribute to DEs effects. We hypothesized that exposure to particle-filtered DE (fDE; gases alone) will elicit less cardiac effects than whole DE (wDE; particles plus gases). In addition, we hypothesized that spontaneously hypertensive (SH) rats will be more sensitive to the electrocardiographic effects of DE exposure than Wistar Kyoto rats (WKY; background strain with normal blood pressure). SH and WKY rats, implanted with telemeters to monitor electrocardiogram and heart rate (HR), were exposed once for 4 h to 150 μg/m(3) or 500 μg/m(3) of wDE (gases plus PM) or fDE (gases alone) DE, or filtered air. Exposure to fDE, but not wDE, caused immediate electrocardiographic alterations in cardiac repolarization (ST depression) and atrioventricular conduction block (PR prolongation) as well as bradycardia in SH rats. Exposure to wDE, but not fDE, caused postexposure ST depression and increased sensitivity to the pulmonary C fiber agonist capsaicin in SH rats. The only notable effect of DE exposure in WKY rats was a decrease in HR. Taken together, hypertension may predispose to the potential cardiac effects of DE and components of DE may have divergent effects with some eliciting immediate irritant effects (e.g., gases), whereas others (e.g., PM) trigger delayed effects potentially via separate mechanisms.

Particulate matter inhalation exacerbates cardiopulmonary injury in a rat model of isoproterenol-induced cardiomyopathy.

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 β-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/m3)—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.

Continuous Electrocardiogram Reveals Differences in the Short-Term Cardiotoxic Response of Wistar-Kyoto and Spontaneously Hypertensive Rats to Doxorubicin

Electrocardiography (ECG) is one of the standard technologies used to monitor and assess cardiac function, and provide insight into the mechanisms driving myocardial pathology. Increased understanding of the effects of cardiovascular disease on rat ECG may help make ECG assessments in rat toxicology studies routine, thus facilitating continuous measurement of functional decrements associated with cardiotoxicant exposure. These studies seek to test the hypothesis that hypertensive rats are more susceptible to the short-term cardiotoxic effects of doxorubicin (DOX) when compared with normotensive rats with respect to continuously measured ECG endpoints. Male Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats surgically implanted with radiotelemeters were treated once a week for three weeks with either vehicle, 1.25 (low), 2.5 (medium), or 5 (high) mg/kg DOX (i.p.). ECG, heart rate (HR), and core body temperature (T(co)) were continuously monitored during the 1-week baseline and throughout the experimental period until rats were sacrificed 24 h after the third injection. DOX prevented normal body weight gain in both strains and significantly decreased diurnal HR and T(co) of high DOX SH rats. In the ECG, SH rats had prolonged baseline PR intervals and QT(c) when compared with WKY rats. All DOX-treated WKY rats subsequently developed PR interval prolongation; however only those treated with high DOX had increased QT(c). DOX caused an increase in ST interval in SH rats, and resulted in ECG morphology changes. The number of arrhythmias due to DOX was increased in both strains. In conclusion, ECG analysis can reveal underlying cardiovascular disease as a risk factor in the hearts response to toxicant-induced injury in the rat; and be a valuable tool to evaluate baseline vulnerability and assess cardiotoxicity.

Effects of gestational methylmercury exposure on immunoreactivity of specific isoforms of PKC and enzyme activity during post-natal development of the rat brain

Protein kinase C (PKC)-mediated phosphorylation has been implicated in neuronal growth and differentiation [R.S. Turner, R.L. Mazzei, G.J. Raynor, P.R. Girard, J.F. Kuo, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3143-3147.]. We examined effects of gestational exposure to the neurotoxicant, methylmercury (CH3Hg), on the developmental profile of immunoreactivity (IR) for alpha, beta, gamma and epsilon PKC isoforms and cytosolic PKC activity. Long-Evans dams were dosed on gestational days (GD)6-15 (p.o.) with 0, 1, or 2 mg kg-1 day-1 CH3Hg dissolved in saline. Pups were sacrificed and perfused with buffered paraformaldehyde on post-natal days (PND) 1, 4, 10, 21, 45 and 85. The brains were sectioned sagittally, stained immunohistochemically, and examined throughout the medial to lateral extent. IR in neuronal cell bodies for PKC isoforms alpha, beta, gamma, and epsilon was densest in the olfactory bulb, hippocampus, shell of the inferior colliculus, pons, cerebral, piriform, and cerebellar cortex, whereas axonal staining was prominent in the brainstem, internal capsule, corpus callosum, anterior commissure, fornix and olfactory tract. In controls, the PKC alpha and epsilon IR was highest on PND1-4, decreased dramatically by PND10, and decreased further by PND21. In the neonate, the regional and cellular distributions of alpha and epsilon IR were similar. The PKC gamma IR was greater at post-weaning ages (PND21-85) with the greatest regional density apparent in the hippocampus, cortex, and cerebellum. Only the highest dose of CH3Hg (2 mg kg-1 day-1; GD6-15) produced a persistent decrease in regional alpha and epsilon, but not beta or gamma IR during the post-natal period. These regional and time-dependent changes in PKC isoforms were complemented by the examination of PKC activity in cortex, olfactory bulb, cerebellum and brainstem. Cytosolic PKC activity increased from PND1 to 10 in cortex, olfactory bulb, and cerebellum. On PND21, PKC activity decreased in the cortex and olfactory bulb, but remained high in the cerebellum. By contrast, PKC activity in the brainstem was highest on PND1 and 4 and decreased dramatically by PND21. CH3Hg (2 mg kg-1 day-1) significantly decreased PKC activity on PND1 and 4 in the cortex. The present results characterize the cellular and regional ontogeny of PKC isoenzymes alpha, beta, gamma and epsilon, and indicate that developmental exposure to CH3Hg can alter the ontogeny of specific isoforms and regional PKC activity.

Overt and Latent Cardiac Effects of Ozone Inhalation in Rats: Evidence for Autonomic Modulation and Increased Myocardial Vulnerability

Background: Ozone (O3) is a well-documented respiratory oxidant, but increasing epidemiological evidence points to extrapulmonary effects, including positive associations between ambient O3 concentrations and cardiovascular morbidity and mortality. Objective: With preliminary reports linking O3 exposure with changes in heart rate (HR), we investigated the hypothesis that a single inhalation exposure to O3 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 O3 or filtered air. A separate cohort was tested for vulnerability to aconitine-induced arrhythmia 24 hr after exposure. Results: Exposure to 0.8 ppm O3 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-O3 exposure failed to elicit any overt changes in autonomic tone, heart rhythm, or ECG. However, both 0.2 and 0.8 ppm O3 increased sensitivity to aconitine-induced arrhythmia formation, suggesting a latent O3-induced alteration in myocardial excitability. Conclusions: O3 exposure causes several alterations in cardiac electrophysiology that are likely mediated by modulation of autonomic input to the heart. Moreover, exposure to low O3 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 underestimated.

Potentiation of pulmonary reflex response to capsaicin 24 h following whole-body acrolein exposure is mediated by TRPV1

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.