The National Environmental Respiratory Center (NERC) experiment in multi-pollutant air quality health research: II. Comparison of responses to diesel and gasoline engine exhausts, hardwood smoke and simulated downwind coal emissions.

The NERC Program conducted identically designed exposure-response studies of the respiratory and cardiovascular responses of rodents exposed by inhalation for up to 6 months to diesel and gasoline exhausts (DE, GE), wood smoke (WS) and simulated downwind coal emissions (CE). Concentrations of the four combustion-derived mixtures ranged from near upper bound plausible to common occupational and environmental hotspot levels. An "exposure effect" statistic was created to compare the strengths of exposure-response relationships and adjustments were made to minimize false positives among the large number of comparisons. All four exposures caused statistically significant effects. No exposure caused overt illness, neutrophilic lung inflammation, increased circulating micronuclei or histopathology of major organs visible by light microscopy. DE and GE caused the greatest lung cytotoxicity. WS elicited the most responses in lung lavage fluid. All exposures reduced oxidant production by unstimulated alveolar macrophages, but only GE suppressed stimulated macrophages. Only DE retarded clearance of bacteria from the lung. DE before antigen challenge suppressed responses of allergic mice. CE tended to amplify allergic responses regardless of exposure order. GE and DE induced oxidant stress and pro-atherosclerotic responses in aorta; WS and CE had no such effects. No overall ranking of toxicity was plausible. The ranking of exposures by number of significant responses varied among the response models, with each of the four causing the most responses for at least one model. Each exposure could also be deemed most or least toxic depending on the exposure metric used for comparison. The database is available for additional analyses.

Health effects of subchronic inhalation exposure to gasoline engine exhaust.

Gasoline engine emissions are a ubiquitous source of exposure to complex mixtures of particulate matter (PM) and non-PM pollutants; yet their health hazards have received little study in comparison with those of diesel emissions. As a component of the National Environmental Respiratory Center (NERC) multipollutant research program, F344 and SHR rats and A/J, C57BL/6, and BALBc mice were exposed 6 h/day, 7 days/week for 1 week to 6 months to exhaust from 1996 General Motors 4.3-L engines burning national average fuel on a simulated urban operating cycle. Exposure groups included whole exhaust diluted 1:10, 1:15, or 1:90, filtered exhaust at the 1:10 dilution, or clean air controls. Evaluations included organ weight, histopathology, hematology, serum chemistry, bronchoalveolar lavage, cardiac electrophysiology, micronuclei in circulating cells, DNA methylation and oxidative injury, clearance of Pseudomonas aeruginosa from the lung, and development of respiratory allergic responses to ovalbumin. Among the 120 outcome variables, only 20 demonstrated significant exposure effects. Several statistically significant effects appeared isolated and were not supported by related variables. The most coherent and consistent effects were those related to increased red blood cells, interpreted as likely to have resulted from exposure to 13-107 ppm carbon monoxide. Other effects supported by multiple variables included mild lung irritation and depression of oxidant production by alveolar macrophages. The lowest exposure level caused no significant effects. Because only 6 of the 20 significant effects appeared to be substantially reversed by PM filtration, the majority of effects were apparently caused by non-PM components of exhaust.

Health effects of subchronic exposure to environmental levels of hardwood smoke.

Hardwood smoke is a contributor to both ambient and indoor air pollution. As part of a general health assessment of multiple anthropogenic source emissions conducted by the National Environmental Respiratory Center, a series of health assays was conducted on rodents exposed to environmentally relevant levels of hardwood smoke. This article summarizes the study design and exposures, and reports findings on general indicators of toxicity, bacterial clearance, cardiac function, and carcinogenic potential. Hardwood smoke was generated from an uncertified wood stove, burning wood of mixed oak species. Animals were exposed to clean air (control) or dilutions of whole emissions based on particulate (30, 100, 300, and 1000 micromg/m3). F344 rats, SHR rats, strain A/J mice, and C57BL/6 mice were exposed by whole-body inhalation 6 h/day, 7 days/wk, for either 1 wk or 6 mo. Effects of exposure on general indicators of toxicity, bacterial clearance, cardiac function, and carcinogenic potential were mild. Exposure-related effects included increases in platelets and decreases in blood urea nitrogen and serum alanine aminotransferase. Several other responses met screening criteria for significant exposure effects but were not consistent between genders or exposure times and were not corroborated by related parameters. Pulmonary histopathology revealed very little accumulation of hardwood smoke particulate matter. Parallel studies demonstrated mild exposure effects on bronchoalveolar lavage parameters and in a mouse model of asthma. In summary, the results reported here show few and only modest health hazards from short-term to subchronic exposures to realistic concentrations of hardwood smoke.

Acute and chronic cardiovascular effects of intermittent hypoxia in C57BL/6J mice.

We investigated the effects of 1) acute hypoxia and 2) 5 wk of chronic intermittent hypoxia (IH) on the systemic and pulmonary circulations of C57BL/6J mice. Mice were chronically instrumented with either femoral artery or right ventricular catheters. In response to acute hypoxia (4 min of 10% O2; n = 6), systemic arterial blood pressure fell (P < 0.005) from 107.7 +/- 2.5 to 84.7 +/- 6.5 mmHg, whereas right ventricular pressure increased (P < 0.005) from 11.7 +/- 0.8 to 14.9 +/- 1.3 mmHg. Another cohort of mice was then exposed to IH for 5 wk (O2 nadir = 5%, 60-s cycles, 12 h/day) and then implanted with catheters. In response to 5 wk of chronic IH, mice (n = 8) increased systemic blood pressure by 7.5 mmHg, left ventricle + septum weight by 32.2 +/- 7.5 x 10(-2) g/100 g body wt (P < 0.015), and right ventricle weight by 19.3 +/- 3.2 x 10(-2) g/100 g body wt (P < 0.001), resulting in a 14% increase in the right ventricle/left ventricle + septum weight (P < 0.005). We conclude that in C57BL/6J mice 1) acute hypoxia causes opposite effects on the pulmonary and systemic circulations, leading to preferential loading of the right heart; and 2) chronic IH in mice results in mild to moderate systemic and pulmonary hypertension, with resultant left- and right-sided ventricular hypertrophy.

Cardiovascular and thermoregulatory effects of inhaled PM-associated transition metals: a potential interaction between nickel and vanadium sulfate.

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.

A model of sleep-disordered breathing in the C57BL/6J mouse.

To investigate the pathophysiological sequelae of sleep-disordered breathing (SDB), we have developed a mouse model in which hypoxia was induced during periods of sleep and was removed in response to arousal or wakefulness. An on-line sleep-wake detection system, based on the frequency and amplitude of electroencephalograph and electromyograph recordings, served to trigger intermittent hypoxia during periods of sleep. In adult male C57BL/6J mice (n = 5), the sleep-wake detection system accurately assessed wakefulness (97.2 +/- 1.1%), non-rapid eye movement (NREM) sleep (96.0 +/- 0.9%) and rapid eye movement (REM) sleep (85.6 +/- 5.0%). After 5 consecutive days of SDB, 554 +/- 29 (SE) hypoxic events were recorded over a 24-h period at a rate of 63.6 +/- 2.6 events/h of sleep and with a duration of 28.2 +/- 0.7 s. The mean nadir of fraction of inspired O(2) (FI(O(2))) on day 5 was 13.2 +/- 0.1%, and 137.1 +/- 13.2 of the events had a nadir FI(O(2)) <10% O(2). Arterial blood gases confirmed that hypoxia of this magnitude lead to a significant degree of hypoxemia. Furthermore, 5 days of SDB were associated with decreases in both NREM and REM sleep during the light phase compared with the 24-h postintervention period. We conclude that our murine model of SDB mimics the rate and magnitude of sleep-induced hypoxia, sleep fragmentation, and reduction in total sleep time found in patients with moderate to severe SDB in the clinical setting.

Cardiovascular and systemic responses to inhaled pollutants in rodents: effects of ozone and particulate matter.

Striking similarities have been observed in a number of extrapulmonary responses of rodents to seemingly disparate ambient pollutants. These responses are often characterized by primary decreases in important indices of cardiac and thermoregulatory function, along with secondary decreases in associated parameters. For example, when rats are exposed to typical experimental concentrations of ozone (O(3), they demonstrate robust and consistent decreases in heart rate (HR) ranging from 50 to 100 beats per minute, whereas core temperature (T(co) often falls 1.5-2.5 degrees C. Other related indices, such as metabolism, minute ventilation, blood pressure, and cardiac output, appear to exhibit similar deficits. The magnitudes of the observed decreases may be modulated by changes in experimental conditions and appear to vary inversely with both ambient temperature and body mass. More recent studies in which both healthy and compromised rats were exposed to either particulate matter or its specific components yielded similar results. The agents studied included representative examples of ambient, combustion, and natural source particles, along with individual or combined exposures to their primary metallic constituents. In addition to the substantial decreases in HR and T(co), similar to those seen with the O(3)-exposed rats, these animals also displayed numerous adverse changes in electrocardiographic waveforms and cardiac rhythm, frequently resulting in fatal outcomes. Although there is only limited experimental evidence that addresses the underlying mechanisms of these responses, there is some indication that they may be related to stimulation of pulmonary irritant receptors and that they may be at least partially mediated via the parasympathetic nervous system.

The spontaneously hypertensive rat as a model of human cardiovascular disease: evidence of exacerbated cardiopulmonary injury and oxidative stress from inhaled emission particulate matter.

Cardiovascular disease is considered a probable risk factor of particulate matter (PM)-related mortality and morbidity. It was hypothesized that rats with hereditary systemic hypertension and underlying cardiac disease would be more susceptible than healthy normotensive rats to pulmonary injury from inhaled residual oil fly ash (ROFA) PM. Eight spontaneously hypertensive (SH) and eight normotensive Wistar-Kyoto (WKY) rats (12-13 weeks old) were implanted with radiotelemetry transmitters on Day -10 for measurement of electrocardiographic (ECG) waveforms. These and other nonimplanted rats were exposed to filtered air or ROFA (containing leachable toxic levels of metals) on Day 0 by nose-only inhalation (ROFA, 15 mg/m(3) x 6 h/day x 3 days). ECGs were monitored during both exposure and nonexposure periods. At 0 or 18 h post-ROFA exposure, rats were assessed for airway hyperreactivity, pulmonary and cardiac histological lesions, bronchoalveolar lavage fluid (BALF) markers of lung injury, oxidative stress, and cytokine gene expression. Comparisons were made in two areas: (1) underlying cardiopulmonary complications of control SH rats in comparison to control WKY rats; and (2) ROFA-induced cardiopulmonary injury/inflammation and oxidative burden. With respect to the first area, control air-exposed SH rats had higher lung and left ventricular weights when compared to age-matched WKY rats. SH rats had hyporeactive airways to acetylcholine challenge. Lung histology revealed the presence of activated macrophages, neutrophils, and hemorrhage in control SHrats. Consistently, levels of BALF protein, macrophages, neutrophils, and red blood cells were also higher in SH rats. Thiobarbituric acid-reactive material in the BALF of air-exposed SH rats was significantly higher than that of WKY rats. Lung inflammation and lesions were mirrored in the higher basal levels of pulmonary cytokine mRNA expression. Cardiomyopathy and monocytic cell infiltration were apparent in the left ventricle of SH rats, along with increased cytokine expression. ECG demonstrated a depressed ST segment area in SH rats. With regard to the second area of comparison (ROFA-exposed rats), pulmonary histology indicated a slightly exacerbated pulmonary lesions including inflammatory response to ROFA in SH rats compared to WKY rats and ROFA-induced increases in BALF protein and albumin were significantly higher in SH rats than in WKY rats. In addition, ROFA caused an increase in BALF red blood cells in SH rats, indicating increased hemorrhage in the alveolar parenchyma. The number of alveolar macrophages increased more dramatically in SH rats following ROFA exposure, whereas neutrophils increased similarly in both strains. Despite greater pulmonary injury in SH rats, ROFA-induced increases in BALF GSH, ascorbate, and uric acid were attenuated when compared to WKY rats. ROFA inhalation exposure was associated with similar increases in pulmonary mRNA expression of IL-6, cellular fibronectin, and glucose-6-phosphate dehydrogenase (relative to that of beta-actin) in both rat strains. The expression of MIP-2 was increased in WKY but attenuated in SH rats. Thus, SH rats have underlying cardiac and pulmonary complications. When exposed to ROFA, SH rats exhibited exacerbated pulmonary injury, an attenuated antioxidant response, and acute depression in ST segment area of ECG, which is consistent with a greater susceptibility to adverse health effects of fugitive combustion PM. This study shows that the SH rat is a potentially useful model of genetically determined susceptibility with pulmonary and cardiovascular complications.

Cardiac and Thermoregulatory Toxicity of Residual Oil Fly Ash in Cardiopulmonary-Compromised Rats.

Recent epidemiological studies have reported a positive association between levels of ambient particulate matter (PM) and daily morbidity and mortality due to respiratory or cardiovascular causes; however, toxicological evidence supporting these findings is limited. The present study compared cardiac and thermoregulatory responses to intratracheal instillations of residual oil fly ash (ROFA) in normal and cardiopulmonary-compromised male Sprague-Dawley rats. Animals (n = 64) were implanted with radiotelemetry transmitters capable of continuously monitoring heart rate, core body temperature, and electrocardiographic waveforms. Comparisons of ROFA toxicity were conducted between (1) healthy rats and rats with cardiopulmonary stress or disease, including (2) rats exposed to an ambient temperature of 10° C, (3) rats preexposed to ozone to induce pulmonary inflammation, and (4) rats pretreated with monocrotaline (MCT) to induce pulmonary hypertension and vasculitis. Animals from each regimen were instilled with 1 of 4 doses of ROFA (0, 0.25, 1.0, 2.5 mg), and telemetry data were acquired for 96 h following ROFA instillation. Dose-related hypothermia and bradycardia were observed in healthy animals following exposure to ROFA; the magnitude and duration of these responses were potentiated in all compromised models. Delayed hypothermic and bradycardic responses occurred in healthy animals receiving 2.5 mg ROFA up to 48 h following instillation. These delayed responses were exacerbated in the MCT-and 10°C-exposure models, but attenuated in the 03-preexposed group. Additional observed effects of ROFA included induction of cardiac arrhythmias and increased mortality. These results demonstrate a distinct cardiac component to ROFA toxicity that agrees with epidemiological findings of PM-related excess cardiovascular mortality. Furthermore, the dose-related hypothermia and bradycardia observed in rodents from this study may confound the interpretation of results from similar air pollution toxicology studies.

Lung injury from intratracheal and inhalation exposures to residual oil fly ash in a rat model of monocrotaline-induced pulmonary hypertension.

A rat model of monocrotaline (MCT)-induced pulmonary injury/hypertension has been recently used in particulate matter (PM) health effects studies, however, results have been equivocal. Neither the mechanism by which mortality occurs in this model nor the variation in response due to differences in PM exposure protocols (i.e., a bolus dose delivered intratracheally versus a similar cumulative dose inhaled over three days) have been fully investigated. Sprague Dawley rats (SD, 60 d old; 250-300 g) were injected with either saline (healthy) or MCT, 60 mg/kg, i.p. (to induce pulmonary injury/hypertension). Ten days later they were exposed to residual oil fly ash (ROFA), either intratracheally (IT; saline, 0.83 or 3.33 mg/kg) or by nose-only inhalation (15 mg/m3 x 6 h/d x 3 d). Lung histology, pulmonary cytokine gene expression (0 and 18 h postinhalation), and bronchoalveolar lavage fluid (BALF) markers of injury were analyzed (24 and 96 h post-IT; or 18 h post-inhalation). Data comparisons examined three primary aspects, 1) ROFA IT versus inhalation effects in healthy rats; 2) pulmonary injury caused by MCT; and 3) exacerbation of ROFA effects in MCT rats. In the first aspect, pulmonary histological lesions following ROFA inhalation in healthy rats were characterized by edema, inflammatory cell infiltration, and thickening of alveolar walls. Increases in BALF markers of lung injury and inflammation were apparent in ROFA-IT or nose-only exposed healthy rats. Increased IL-6, and MIP-2 expression were also apparent in healthy rats following ROFA inhalation. In regards to the second aspect, MCT rats exposed to saline or air showed perivascular inflammatory cell infiltrates, increased presence of large macrophages, and alveolar thickening. Consistently, BALF protein, and inflammatory markers (macrophage and neutrophil counts) were elevated indicating pulmonary injury. In regards to the third aspect, 58% of MCT rats exposed to ROFA IT died within 96 h regardless of the dose. No mortality was observed using the inhalation protocol. ROFA inhalation in MCT rats caused exacerbation of lung lesions such as increased edema, alveolar wall thickening, and inflammatory cell infiltration. This exacerbation was also evident in terms of additive or more than additive increases in BALF neutrophils, macrophages and eosinophils. IL-6 but not MIP-2 expression was more than additive in MCT rats, and persisted over 18 h following ROFA. IL-10 and cellular fibronectin expression was only increased in MCT rats exposed to ROFA. In summary, only the bolus IT ROFA caused mortality in the rat model of lung injury/hypertension. Exacerbation of histological lesions and cytokine mRNA expression were most reflective of increased ROFA susceptibility in this model.

Cardiac arrhythmia induction after exposure to residual oil fly ash particles in a rodent model of pulmonary hypertension.

Recent epidemiological studies have reported a positive association between exposure to ambient concentrations of particulate matter (PM) and the incidence of cardiopulmonary-related morbidity and mortality. The present study examined the effects of fugitive residual oil fly ash (ROFA) PM on cardiac arrhythmia induction in healthy and cardiopulmonary-compromised rodents. Male Sprague-Dawley rats were implanted with radiotelemetry transmitters capable of monitoring the electrocardiogram and were subjected to one of two treatment regimens. Rats in the first treatment regimen (n = 16) served as normal control animals whereas rats in the second treatment regimen (n = 16) were injected with monocrotaline (MCT, 60 mg/kg, ip) to induce pulmonary vascular inflammation and hypertension and served as a model of cardiopulmonary disease. Rats within each treatment regimen were equally divided into four dose groups (0.0, 0.25, 1.0, 2.5 mg ROFA), instilled intratracheally, and monitored for 96 h. In the animals in the first treatment regimen, ROFA instillation caused dose-related increases in the incidence and duration of serious arrhythmic events that appeared to be associated with impaired atrioventricular conduction and myocardial hypoxia. There were no lethalities in the normal animals following ROFA instillation. The frequency and severity of arrhythmias were greatly exacerbated in the MCT-treated animals in the second treatment regimen and were accompanied by one, three, and two deaths in the low-, medium-, and high-dose groups, respectively. The results of the present study demonstrate substantial cardiac effects in normal and compromised rats after exposure to ROFA PM and implicate both conductive and hypoxemic arrhythmogenic mechanisms in the observed cardiac-related lethalities. These results support previous epidemiological studies that suggest a link between preexisting cardiopulmonary disease and potentiation of adverse health effects following exposure to anthropogenic particulates.

Impact of the hypothermic response in inhalation toxicology studies.

Previous studies from this laboratory showed that the decreases in Tco and associated functional parameters often observed in rodents following exposure to xenobiotic agents are capable of modulating the subsequent toxic response and that the magnitude of this induced hypothermic response may itself be modified by a number of experimental conditions. A moderate hypothermic response, characterized by a temperature drop of approximately 2 degrees C, appears to afford the optimal protection. Studies in which exposures occur through inhalation of harmful gases or particles present a special set of problems. In such studies, the dose of the toxic agent to which the animal is exposed is a function of the concentration of the agent in the atmosphere and the minute ventilation of the animal. Although ambient concentrations is generally held constant in laboratory studies, minute ventilation varies directly with metabolism, and both of these parameters may change significantly across experimental conditions. Thus, at low Tas, metabolism and minute ventilation are relatively high and uptake of inhalable toxic agents is increased. However, the development of the hypothermic response during the exposure entails a directly correlated reduction in these parameters and, presumably, in dose. For the most part, inhalation toxicological studies are conducted using resting animals or exercising humans. Animals are sometimes concurrently exposed to CO2 to simulate the increased ventilation of exercise and more closely mimic human studies. The experimental protocols employed in the above inhalation studies permitted examination of (1) the impact of species, size, handling stress, and changes in Ta on both the induced hypothermic response and the concomitant pulmonary toxicity; (2) the additive impact of exercise stress on O3 toxicity; and (3) the toxicity of ambient-derived particulate matter in normal rats and in rats with preexisting pulmonary inflammation. The results of these studies demonstrate that the magnitude of the induced hypothermic response is directly proportional to the uptake of the toxic agent by the lung and inversely proportional to the mass of the animal and the ambient temperature at which the exposure is conducted. The hypothermic response is sensitive to a number of experimental stresses including handling and changes in cage conditions. Exercise attenuates the hypothermic response, whereas CO2-stimulated increases in ventilation employed as an exercise surrogate may potentiate the response. Toxic exposures conducted in animals with lung disease or compromised pulmonary function may induce a severe hypothermic response while comparable exposures in normal animals produce only mild or moderate responses. In general, the development of the hypothermic response in the presence of ambient pollutants serves to decrease the minute ventilation of the animal and therefore limits the uptake and dose of the airborne toxicant. The results of these inhalation studies support our previous conclusions concerning the impact of the hypothermic response on toxicity and emphasize the need to monitor and incorporate these changes in functional parameters into analyses of toxicological data. Furthermore, because humans do not demonstrate a robust hypothermic response following exposure to toxic agents, extrapolation of the results obtained from animal studies and comparisons with data from human studies are considerably more complicated.