Christine Nadziejko

Effects of Subchronic Exposures to Concentrated Ambient Particles (CAPs) in Mice: V. CAPs Exacerbate Aortic Plaque Development in Hyperlipidemic Mice

Abstract Recent epidemiological studies suggest that long-term exposure to particulate matter (PM) causes chronic effects on the cardiovascular system that result in cumulative increases cardiovascular morbidity and mortality. Since atherosclerosis is a progressive irreversible condition and an underlying cause of many cardiovascular diseases, we hypothesized that long-term exposure to PM causes adverse cardiovascular effects by exacerbating atherosclerosis. In this study, we exposed C57- and ApoE-deficient (ApoE−/−) and ApoE, LDLr (DK)-deficient mice to concentrated ambient PM2.5 for 6 h/day, 5 days/wk, for up to 5 mo. The overall mean exposure concentration for these groups of animals was 110 μ g/m3. The cross-sectional area of the aorta root of DK mice was examined morphologically using confocal microscopy for the severity of lesion, extent of cellularity, and lipid contents. Aortas from the arch to the iliac bifurcations were also sectioned longitudinally and lesion areas were stained with Sudan IV. All DK mice regardless of exposure had developed extensive lesions in the aortic sinus regions, with lesion areas that covered more than 79% of the total area. In male DK mice, the lesion areas in the aortic sinus regions appeared to be enhanced by concentrated ambient particles (CAPs), with changes approaching statistical significance (p = .06). In addition, plaque cellularity was increased by 28% (p = .014, combined), whereas there were no CAPs-associated changes in the lipid content in these mice. When examining the entire aorta opened longitudinally, both the ApoE−/− and DK mice had prominent areas of severe atherosclerosis covering 40% or more of the lumenal surface. Visual examination of all images suggested that plaques tend to form in clusters concentrating near the aortic arch and the iliac bifurcations. Quantitative measurements showed that CAPs exposure increased the percentage of aortic intimal surface covered by grossly discernible atherosclerotic lesion by 57% in the ApoE−/− mice (p = .03). Changes produced by CAPs in male (10% increase) or female DK mice (8% decrease) were not statistically significant. In this study, we have demonstrated that subchronic exposure to CAPs in mice prone to develop atherosclerotic lesions had a significant impact on the size, severity, and composition of aortic plaque.

Effects of Inhaled Ambient Particulate Matter on Pulmonary Antimicrobial Immune Defense

Respiratory-tract infection, specifically pneumonia, contributes substantially to the increased morbidity and mortality among elderly individuals exposed to airborne particulate matter of <10 µm diameter (PM 10) . These epidemiological findings suggest that PM 10 may act as an immunosuppressive factor that can undermine normal pulmonary antimicrobial defense mechanisms. To investigate whether, and how, compromised pulmonary immunocompetence might contribute to increased mortality, two sets of laboratory studies were performed. The first examined the effects of a single inhalation exposure to concentrated ambient PM 2.5 (CAPS) from New York City air on pulmonary/systemic immunity and on the susceptibility of exposed aged rats to subsequent infection with Streptococcus pneumoniae. The second set of studies determined whether CAPS exposure, at a concentration approximating or somewhat greater than the promulgated 24-h NAAQS of 65 µg/m 3, could exacerbate an ongoing infection. Taken together, results demonstrated that a single exposure of healthy animals to CAPS had little effect on pulmonary immune function or bacterial clearance during subsequent challenge with S. pneumoniae. Alternatively, CAPS exposure of previously infected rats significantly increased bacterial burdens and decreased percentages of lavageable neutrophils and proinflammatory cytokine levels compared to those in infected filtered-air-exposed controls. These studies demonstrate that a single exposure to ambient PM 2.5 compromises a hosts ability to handle ongoing pneumococcal infections and support the epidemiological findings of increased pneumonia-related deaths in ambient PM-exposed elderly individuals.

Pulmonary and cardiovascular effects of acute exposure to concentrated ambient particulate matter in rats

To examine the biological plausibility of the adverse health effects of ambient particulate matter (PM), we have studied the cardio-pulmonary effects of PM in an animal model of pulmonary hypertension. Normal and monocrotaline-treated rats were exposed, nose-only, for 3 h to filtered air or concentrated ambient PM. At 3 h--but not 24 h--post-exposure, the percentage of neutrophils in peripheral blood was significantly elevated in PM-exposed animals while the percentage of lymphocytes was decreased with no change in white blood cell counts. These changes in white blood cell differential occurred in both normal and monocrotaline-treated animals. Small, but consistent changes in heart rate, but not core temperature, were observed after exposure to concentrated ambient PM. Pulmonary injury, as evidenced by increased protein levels in lavage fluid, occurred only in monocrotaline-treated animals exposed to > 360 microg/m3 PM. The observed pattern of hematological and cardiac changes suggests an activation of the sympathetic stress response.

Effects of Subchronic Exposures to Concentrated Ambient Particles (CAPs) in Mice: II. The Design of a CAPs Exposure System for Biometric Telemetry Monitoring

Abstract We modified, assembled, tested, and validated the versatile aerosol concentration enrichment system (VACES) developed by for use in a subchronic experiment that involved exposures of mice in vivo and of respiratory epithelial cells in vitro to concentrated ambient particles (CAPs). Since the labor-intensive nose-only exposure regimen is not an option in a long-term experiment, a whole-body exposure mouse chamber was designed specifically for use with the VACES. The exposure system consists of a stainless-steel (SS) tub with 32 cubicles (1 mouse per cubicle) separated by perforated SS sheets. The tops of these cubicles are covered with perforated plastic sheets to allow telemetry monitoring during the exposure. In each exposure chamber, perforated aluminum tubes are used to distribute CAPs evenly (within 2% difference) throughout the exposure chamber. The exhaust consists of perforated aluminum tubes covered with a urine shield. The modification to the original design of the VACES facilitated the operation of the system in a subchronic study. Mass flow controllers maintain a constant flow rate into the exposure chambers. For a sham control exposure, the identical system is used, except that a HEPA filter at the inlet to the VACES removes 98% of ambient particles. The entire system allows for simultaneous exposure of 64 mice to CAPs, with an equal number of sham-exposed mice as controls. Telemetry receivers have been modified so that 16 mice per group with electrocardiograph (EKG) transmitters can be monitored during exposure. Furthermore, a BioSampler is used to collect CAPs (one sample per day) for the in vitro exposures. In this article, the assessments of flow and particle distribution of the exposure chamber as well as the performance of the system during the subchronic exposure experiment are described.

Effect of Particulate and Gaseous Pollutants on Spontaneous Arrhythmias in Aged Rats

Epidemiology studies suggest that exposure to air pollution increases the frequency of cardiac arrhythmias. A limitation of these studies is that it is difficult to link an increased risk of arrhythmias to a specific air pollutant. Animal exposure studies offer the opportunity to examine the effects of concentrated ambient fine particulate matter (PM), ultrafine PM, and copollutant gases separately. Male Fischer 344 rats, aged 18 mo, with implanted electrocardiograph (ECG) transmitters were used to determine the effects of PM on the frequency of arrhythmias. We found that old F344 rats had many spontaneous arrhythmias. An arrhythmia classification system was developed to quantify arrhythmia frequency. Arrhythmias were broadly grouped into two categories: premature beats and delayed beats. The rats were exposed to concentrated ambient PM (CAPS) or air for 4 h. The rats were exposed twice with a crossover design so each rat could serve as its own control. The CAPS concentrations were 160 μ g/m3 and 200 μ g/m3 for the first and second exposures, respectively. There was a significant increase in the frequency of irregular and delayed beats after exposure to CAPS. The same rats were subsequently exposed to laboratory-generated ultrafine carbon particles, to SO2, or to air with a repeated crossover design. In these experiments there was no significant change in the frequency of any category of spontaneous arrhythmia following exposure to ultrafine carbon or SO2. Thus, this study adds supporting evidence that acute exposure to elevated levels of ambient PM increases the frequency of cardiac arrhythmias.

Effects of subchronic exposures to concentrated ambient particles (CAPs) in mice. III. Acute and chronic effects of CAPs on heart rate, heart-rate fluctuation, and body temperature.

Abstract Normal mice (C57) and mice prone to develop atherosclerosis (ApoE−/−) were implanted with electrocardiograph (EKG), core body temperature, and motion transmitters and were exposed daily for 6 h to Tuxedo, NY, concentrated ambient particles (CAPs) for 5 days/wk during the spring and summer of 2003. Time series of 5-min EKG monitoring and body-temperature measurements were obtained for each animal in the CAPs and filtered air sham exposure groups. Our hypothesis was that chronic exposure could cause cumulative health effects. We used our recently developed nonparametric method to estimate the daily time periods that mean heart rates (HR), body temperature, and physical activity differed significantly between the CAPs and sham exposed groups. CAPs exposure most affected heart rate between 1:30 a.m. and 4:30 a.m. With the response variables being the average heart rate, body temperature, and physical activity, we adopted a two-stage modeling approach to obtain the estimates of chronic and acute effects on the changes of these three response variables. In the first stage, a time-varying model estimated daily crude effects. In the second stage, the true means of the estimated crude effects were modeled with a polynomial function of time for chronic effects, a linear term of daily CAPs exposure concentrations for acute effects, and a random component for unknown noise. A Bayesian framework combined these two stages. There were significant decreasing patterns of HR, body temperature, and physical activity for the ApoE−/− mice over the 5 mo of CAPs exposure, with smaller and nonsignificant changes for the C57 mice. The chronic effect changes of the three response variables for ApoE−/− mice were maximal in the last few weeks. There was also a significant relationship between CAPs exposure concentration and short-term change of heart rate in ApoE−/− mice during exposure. Response variables were also defined for examining fluctuations of 5-min heart rates within long (i.e., 3–6 h) and short time periods (i.e., ∼ 15 min). The results for the ApoE−/− mice showed that heart- rate fluctuation within the longer periods increased to 1.35-fold by the end of exposure experiment, while the heart-rate fluctuation within 15 min decreased to 0.7-fold.

Adaptation to Stress Induced by Restraining Rats and Mice in Nose-Only Inhalation Holders

There are limited data on the efficacy of procedures for adapting rodents to restraint in nose-only holders. We examined: (1) What effect does restraint in nose-only holders have on heart rate and body temperature? (2) Does a gradual increase in the duration of restraint facilitate adaptation? (3) How long does it take for rodents to become fully adapted to nose-only holders? (4) Do rats and mice respond and adapt similarly to restraint in nose only holders? Heart rate and body temperature were monitored as measures of stress using electrocardiograph (ECG) transmitters in male C57Bl/6J mice and Sprague-Dawley rats. In naive animals during the first hour of restraint, heart rate increased by 58 beats per minute (BPM) (18.6%) in rats and by 174 BPM (32.3%) in mice as compared to cage controls. Temperature increased by 2°C in mice and was unchanged in rats compared to cage controls. Heart rate and temperature values remained within normal physiologic values during restraint. In rats, the response to restraint in nose-only holders was the same after 4 days regardless of whether the duration of restraint was increased gradually to 4 h/day or kept constant at 4 h/day. In mice, the group that was gradually adapted had a statistically significant higher heart rate and temperature after 4 days than the fixed-duration adapted group. Rats and mice restrained for 4 h/day every day showed a gradual decrease in heart rate and temperature over time. Full adaptation to restraint required 14 days of fixed-duration daily restraint.

Mycobacterium immunogenum Causes Hypersensitivity Pneumonitis-Like Pathology in Mice

A surprising number of cases of hypersensitivity pneumonitis have been observed at work sites employing automotive machinists. Because hypersensitivity pneumonitis is not typically associated with exposure to metalworking fluid aerosols, this study examined whether Mycobacterium immunogenum (M. immunogenum), a rapidly growing mycobacterium isolated from several affected work sites, could induce hypersensitivity pneumonitis in mice. Hypersensitivity pneumonitis-like histologic changes occurred in mice treated with heat-killed and lysed M. immunogenum. These lung lesions were characterized by peribronchial and perivascular lymphohistiocytic inflammation and noncaseating granulomas in the parenchyma. The pathologic changes observed in mice instilled with M. immunogenum-contaminated used metalworking fluid were indistinguishable from those observed with M. immunogenum alone. The role of genetic factors in M. immunogenum-induced lung lesions was examined by comparison of the response of eight inbred strains of mice. The observed immunologic changes in the lung were significantly greater in C57Bl/6, 129, and BALB/c mice than in the other strains, suggesting that genetic factor(s) contribute to the susceptibility of workers exposed to M. immunogenum-contaminated metalworking fluid aerosols. Thus, these studies provide indirect evidence that M. immunogenum is an unrecognized class of microorganisms capable of causing hypersensitivity pneumonitis and plays a role in the outbreaks of hypersensitivity pneumonitis in automotive plants.

Immediate effects of particulate air pollutants on heart rate and respiratory rate in hypertensive rats

Time-series studies have shown that the lag time between elevated particulate air pollution (PM) and increases in cardiovascular-related hospital admissions and death is very short-1 d or less. If PM does cause serious cardiovascular effects shortly after exposure, one would expect to see some physiological change during exposure. In this study, spontaneously hypertensive rats (SHRs) with surgically implanted blood pressure transmitters were exposed to concentrated ambient PM (CAPS) for 4 h to determine whether CAPS inhalation causes immediate effects. The rats were also exposed to sulfuric acid aerosols because acid is one of the components of PM that could potentially activate irritant receptors and cause effects during exposure. Exposure to CAPS caused a striking decrease in respiratory rate that was apparent soon after the start of exposure and stopped when exposure to CAPS ceased. The decrease in respiratory rate was accompanied by a decrease in heart rate. Exposure of the same rats to fine-particle-size sulfuric acid aerosol also caused a significant decrease in respiratory rate similar to the effects of CAPS. Ultrafine acid had the opposite effect on respiratory rate compared to CAPS. Because acids have been shown to evoke sensory irritant responses in rodents, the similarity between the effects of fine acid aerosol and CAPS suggests that CAPS activates airway-irritant receptors during exposure.

Most of the lipid in purulent sputum is bound to mucus glycoprotein.

Mucus glycoprotein (mucin) is the principal biochemical constituent of sputum. Appreciable quantities of lipid, DNA, and nonmucin proteins are also present, particularly in purulent sputum. Previous studies have shown that purified mucin from respiratory tract secretions contains non-covalently bound lipid. However, it is not known whether lipids in purulent sputum are associated only with mucin or with nonmucin proteins and DNA as well. Purulent sputum was obtained from cystic fibrosis patients. Tracheal aspirates were obtained from noncystic patients with purulent secretions due to Pseudomonas species, as well as from noninfected, noncystic patients who had mucoid airway secretions. The lipid content of unfractionated airway secretions (sputum or tracheal aspirates), gel filtration-purified mucin, and nonmucin components of the airway secretions was analyzed. The purified mucin from all three groups had a significantly higher content of solvent-extractable lipid as compared to unfractionated airway secretions. The nonmucin fractions contained only small amounts of lipid. Density gradient centrifugation verified that the lipid recovered in the purified mucin fraction was complexed with the glycoprotein. The results of this study indicate that most of the lipids in purulent sputum are associated with mucin.