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Pulmonary and cardiovascular effects of acute exposure to concentrated ambient particulate matter in rats
Toxicology Letters 96,97 (1998) 285 – 288
Pulmonary and cardiovascular effects of acute exposure to concentrated ambient particulate matter in rats Terry Gordon *, Christine Nadziejko, Richard Schlesinger, Lung Chi Chen Department of En6ironmental Medicine, New York Uni6ersity School of Medicine, Long Meadow Road, Tuxedo, NY 10987, USA
Abstract 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 mg/m3 PM. The observed pattern of hematological and cardiac changes suggests an activation of the sympathetic stress response. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Particulate matter; Monocrotaline; Respiratory effects; Rat
1. Introduction Both cross-sectional and time-series epidemiologic studies have demonstrated that severe adverse health effects are associated with exposure to ambient particulate matter (PM). A key unanswered question in PM health research is related to the biological plausibility of the association * Corresponding author. Present address: NYU Medical Center, 57 Old Forge Road, Tuxedo, NY 10987, USA. Tel.: +1 914 3514837; fax: + 1 914 3515472; e-mail: [email protected]
between PM and adverse health effects. Little is known regarding the identity of individuals at risk. Moreover, the plausibility of the association between PM and the increases in morbidity and mortality have been severely questioned because these adverse health effects have been observed at very low PM concentrations, often below the current US National Ambient Air Quality Standard (NAAQS). Logic dictates and epidemiology suggests that such small changes in PM concentration are unlikely to affect healthy individuals and that people with compromised health are the
0378-4274/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00084-8
286
T. Gordon et al. / Toxicology Letters 96,97 (1998) 285–288
probable victims. However, there are two very different scenarios in which sensitive subpopulations could suffer adverse health effects from particulate air pollution. In the first scenario, one could suggest that some individuals with pre-existing disease, such as COPD, may suffer much greater effects from inhaled PM than healthy people, perhaps due to increased deposition or exacerbation of lung inflammation. In the second scenario, one could postulate that the small physiological perturbations caused by inhaled PM are significant risk factors for morbidity and mortality in a large population of frail and compromised people. For example, small physiological changes in the cardiovascular system occur when getting out of bed in the morning and are known to result in a significant increase in death rates from heart attacks and strokes in the morning as compared to other times of the day. Similar small pertubations may result from episodic exposure to inhaled PM. We have begun to examine, in animal models, whether the sensitivity of individuals with compromised pulmonary and/or cardiovascular health can account for the increase in morbidity and mortality associated with ambient PM. To examine the possible differences in susceptibility of animal models of compromised health, we have developed an exposure system based upon the use of a centrifugal concentrator to produce concentrations of ambient urban PM \24 h NAAQS of 150 mg/m3. This centrifugal aerosol concentrator was originally designed to increase the sensitivity of a nephelometer. The centrifugal concentrator is similar to those designed by other investigators (Budinsky, 1970; Shutte, 1976) and differs mainly in its ability to concentrate smaller particles. In addition, the centrifugal concentrator was optimized to minimize internal particle losses. The purpose of the present study was to validate the utility of this system for controlled laboratory exposures of rats to concentrated urban PM.
Herman Gerber (Gerber Scientific, Reston, VA), was a gift of the US Navy. A positive displacement vacuum pump (Conde Model c 3CW, Westmoor, Sherrill, NY) was used to draw air from the annular region of the concentrator into the rotating porous inner cylinder. The suction flow rate was controlled by a 3/4 inch gate valve (Fig. 1) located directly downstream from the concentrator. An in-line regenerative blower (Model c DR 068, EG&G Rotron, Saugerties, NY) was used to draw air from the ambient environment through a 1 inch stainless steel tube and introduce the air into the annular region of the centrifugal concentrator. The inlet blower motor was isolated from the blower impeller and a gas tight Teflon lip seal prevented room air from entering the impeller. Calibrated orifice meters were used to monitor inlet and exhaust flow rates. An in-line stainless steel hot wire anemometer (Model c 2011, TSI, St. Paul, MN) was used to monitor flow from the annular region of the centrifugal concentrator to a nose-only animal exposure rig. Potassium iodide and lead oxidecoated honeycomb denuders were placed downstream of the anemometer to remove ozone (95% removal efficiency) and sulfur dioxide (92% removal efficiency). Particle counting with a condensation nuclei counter (Model c 3020, TSI)
2. Material and methods
2.1. Design of exposure system A centrifugal aerosol concentrator, designed by
Fig. 1. The effect of concentrated ambient PM on peripheral blood neutrophils at 3 and 24 h after exposure in monocrotaline-treated rats.
T. Gordon et al. / Toxicology Letters 96,97 (1998) 285–288
upstream and downstream of the denuders determined that B 5% of the particles were lost in the gas denuders. The concentrated stream of particles was delivered to the nose-only exposure rig (CH Technologies, Westwood, NJ) under slightly positive pressure (0.1 – 0.15 inches of water). A real time aerosol monitor (RAM-1, MIE, Billerica, MA) was used to continuously monitor PM during the 3-h exposure period. The exposure concentration delivered to the animals during the nose-only exposure was determined gravimetrically and the particle size distribution was measured with a piezo-electric cascade impactor (California Measurements).
3. Results and discussion
3.1. Pulmonary hypertension rat model 3.1.1. Systemic response The changes observed following exposure to concentrated ambient PM were generally no different between normal and monocrotalinetreated animals. To date, no deaths or immediately life-threatening health effects have been observed after single or multiple exposure to concentrated ambient PM. Hematological changes were consistently observed at 3 h following exposure to PM. These changes were: (1) evidenced by a consistent increase in circulating blood neutrophils and a decrease in lymphocytes; (2) absent by 24 h post-exposure; and (3) present in both normal and monocrotalinetreated young and aged (6 months) animals after PM exposure. To verify that these peripheral blood changes were a result of particle exposure and not ambient gases or the experimental conditions, additional animals were exposed to air or the output of the centrifugal concentrator’s 10 l/min output, which was scrubbed with a small HEPA filter. No significant differences in circulating blood neutrophil or lymphocyte changes were observed between these two groups, suggesting that the hematological changes were due to inhaled particles. Furthermore, no group differences in the white blood
287
cell differentials were observed between animals exposed to air or the output of the particle concentrator when the inlet to the concentrator was scrubbed with a HEPA filter. This latter experiment has been repeated twice in the last month and should rule out any contribution of the operation of the centrifugal concentrator itself to the observed hematological changes.
3.1.2. Pulmonary response No lung pathology or signs of inflammation were noted in hematoxylin and eosin stained lung sections from animals exposed to air or PM. Examination of biochemical and cellular indices in the lavage fluid of air- and PM-exposed normal animals confirmed this absence of pulmonary injury or inflammation at PM concentrations of 110–350 mg/m3. Similar results were obtained with monocrotaline-treated animals with the exception of one experiment. When monocrotaline-treated animals were exposed to 360 mg/m3 PM, total cell counts, protein and LDH were almost twice the values of air-exposed animals. These changes appeared to be dose-dependent, as the exposure of monocrotaline-treated animals to lower concentrations of concentrated ambient PM did not alter lavage fluid markers of injury and inflammation. 3.1.3. Cardio6ascular response We observed no change in heart rate during exposure to PM, even in animals that had been adapted to the exposure protocol. Small, but persistent increases in heart rate were observed after PM exposure in normal young and monocrotaline-treated rats but not in old rats. In general, this statistically significant increase persisted for 2–6 h after a single 3-h PM exposure. The heart rate response of monocrotalinetreated rats to PM exposure did not appear to differ from normal rats. Interestingly, the 10–20 beats/min increase in heart rate in PM-exposed rats was still seen regardless of the greater overall change in heart rate due to circadian rhythm. When animals were exposed to PM for 6 h/day for 3 days, the magnitude of the increase in heart rate did not change but the increase in
288
T. Gordon et al. / Toxicology Letters 96,97 (1998) 285–288
heart rate was more persistent, particularly on the second and third day of exposure. EKG tracings from all experiments were scanned for arrhythmias which were very rare in normal and in monocrotaline-treated rats. A careful inspection was made on the EKG tracings from 12 monocrotaline-treated rats that were exposed to air or PM for 3 h and monitored during exposure and 18 h after exposure. A total of two atrial arrhythmias were seen, one in each of two PM-exposed animals, which is biologically insignificant.
3.1.4. Core temperature response The effects of PM exposure on temperature were more variable than the heart rate results. Rats treated with monocrotaline 4 weeks prior to a single 3-h PM exposure showed a significant and persistent decrease in temperature after PM exposure. Rats treated with monocrotaline 2 weeks prior to a repeated 6-h PM exposure, had a significantly increased temperature after the first exposure but were unchanged from air-exposed rats after PM exposure on the second and third days of exposure. Normal young and 6 month old rats showed no change in temperature after PM exposure.
within a large population, rather than severe changes in a few very sensitive individuals. However, these possibilities are not mutually exclusive. In fact, in one experiment, monocrotaline-treated rats but not normal rats showed signs of pulmonary injury following exposure to a high concentration of PM. This suggests that individuals with pre-existing cardiac or respiratory disease may have greater effects than healthy people. The time course and pattern of the peripheral blood changes (PMN demargination and lymphopenia) are consistent with the activation of the sympatho-adrenal system, i.e. a stress response. While consistent changes in heart rate and hematological parameters were seen in normal and monocrotaline-treated rats, the only change that implied adverse heath consequences (increased lavage fluid protein and LDH) was seen in monocrotaline-treated rats. These data suggest that inhaled PM can cause systemic effects in both normal and monocrotaline-treated rats over a wide range of concentrations but only monocrotaline-treated rats are susceptible to adverse pulmonary effects. Therefore, we hypothesize that inhaled PM causes systemic effects by evoking a stress response which may be well tolerated by healthy individuals, but may cause morbidity and mortality by altering homeostatic processes in those with cardiovascular or pulmonary disease.
4. Conclusions Consistent changes in heart rate and white blood cell differential counts were seen in normal and monocrotaline-treated rats following exposure to concentrated PM. Surprisingly, systemic effects were seen in the absence of pulmonary inflammation or injury. The magnitude of the systemic effects would not be considered lifethreatening or even an adverse health effect if observed in an individual. However, the increased neutrophil number reported in this study may be sufficiently large to be a risk factor for cardiovascular disease and mortality in a large population. Most of our results are consistent with the concept that PM-induced mortality results from small changes in physiological parameters occurring .
Acknowledgements The authors thank Margaret Kransinski and Karen Galdanes for excellent technical assistance and William E. Hill (Walker Muffler, Grass Lake, MI) and Jack Mikhail Wolfson (Harvard School of Public Health, Boston, MA) for providing expert advice in sound abatement and gas denuders, respectively.
References Budinsky, K., 1970. Rotating centrifugal separator with continuous dust removal. Staub 30, 7 – 13. Shutte, A.H., 1976. Filter apparatus. U.S. Patent no. 3262573.
Pulmonary and cardiovascular effects of acute exposure to concentrated ambient particulate matter in rats Terry Gordon *, Christine Nadziejko, Richard Schlesinger, Lung Chi Chen Department of En6ironmental Medicine, New York Uni6ersity School of Medicine, Long Meadow Road, Tuxedo, NY 10987, USA
Abstract 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 mg/m3 PM. The observed pattern of hematological and cardiac changes suggests an activation of the sympathetic stress response. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Particulate matter; Monocrotaline; Respiratory effects; Rat
1. Introduction Both cross-sectional and time-series epidemiologic studies have demonstrated that severe adverse health effects are associated with exposure to ambient particulate matter (PM). A key unanswered question in PM health research is related to the biological plausibility of the association * Corresponding author. Present address: NYU Medical Center, 57 Old Forge Road, Tuxedo, NY 10987, USA. Tel.: +1 914 3514837; fax: + 1 914 3515472; e-mail: [email protected]
between PM and adverse health effects. Little is known regarding the identity of individuals at risk. Moreover, the plausibility of the association between PM and the increases in morbidity and mortality have been severely questioned because these adverse health effects have been observed at very low PM concentrations, often below the current US National Ambient Air Quality Standard (NAAQS). Logic dictates and epidemiology suggests that such small changes in PM concentration are unlikely to affect healthy individuals and that people with compromised health are the
0378-4274/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00084-8
286
T. Gordon et al. / Toxicology Letters 96,97 (1998) 285–288
probable victims. However, there are two very different scenarios in which sensitive subpopulations could suffer adverse health effects from particulate air pollution. In the first scenario, one could suggest that some individuals with pre-existing disease, such as COPD, may suffer much greater effects from inhaled PM than healthy people, perhaps due to increased deposition or exacerbation of lung inflammation. In the second scenario, one could postulate that the small physiological perturbations caused by inhaled PM are significant risk factors for morbidity and mortality in a large population of frail and compromised people. For example, small physiological changes in the cardiovascular system occur when getting out of bed in the morning and are known to result in a significant increase in death rates from heart attacks and strokes in the morning as compared to other times of the day. Similar small pertubations may result from episodic exposure to inhaled PM. We have begun to examine, in animal models, whether the sensitivity of individuals with compromised pulmonary and/or cardiovascular health can account for the increase in morbidity and mortality associated with ambient PM. To examine the possible differences in susceptibility of animal models of compromised health, we have developed an exposure system based upon the use of a centrifugal concentrator to produce concentrations of ambient urban PM \24 h NAAQS of 150 mg/m3. This centrifugal aerosol concentrator was originally designed to increase the sensitivity of a nephelometer. The centrifugal concentrator is similar to those designed by other investigators (Budinsky, 1970; Shutte, 1976) and differs mainly in its ability to concentrate smaller particles. In addition, the centrifugal concentrator was optimized to minimize internal particle losses. The purpose of the present study was to validate the utility of this system for controlled laboratory exposures of rats to concentrated urban PM.
Herman Gerber (Gerber Scientific, Reston, VA), was a gift of the US Navy. A positive displacement vacuum pump (Conde Model c 3CW, Westmoor, Sherrill, NY) was used to draw air from the annular region of the concentrator into the rotating porous inner cylinder. The suction flow rate was controlled by a 3/4 inch gate valve (Fig. 1) located directly downstream from the concentrator. An in-line regenerative blower (Model c DR 068, EG&G Rotron, Saugerties, NY) was used to draw air from the ambient environment through a 1 inch stainless steel tube and introduce the air into the annular region of the centrifugal concentrator. The inlet blower motor was isolated from the blower impeller and a gas tight Teflon lip seal prevented room air from entering the impeller. Calibrated orifice meters were used to monitor inlet and exhaust flow rates. An in-line stainless steel hot wire anemometer (Model c 2011, TSI, St. Paul, MN) was used to monitor flow from the annular region of the centrifugal concentrator to a nose-only animal exposure rig. Potassium iodide and lead oxidecoated honeycomb denuders were placed downstream of the anemometer to remove ozone (95% removal efficiency) and sulfur dioxide (92% removal efficiency). Particle counting with a condensation nuclei counter (Model c 3020, TSI)
2. Material and methods
2.1. Design of exposure system A centrifugal aerosol concentrator, designed by
Fig. 1. The effect of concentrated ambient PM on peripheral blood neutrophils at 3 and 24 h after exposure in monocrotaline-treated rats.
T. Gordon et al. / Toxicology Letters 96,97 (1998) 285–288
upstream and downstream of the denuders determined that B 5% of the particles were lost in the gas denuders. The concentrated stream of particles was delivered to the nose-only exposure rig (CH Technologies, Westwood, NJ) under slightly positive pressure (0.1 – 0.15 inches of water). A real time aerosol monitor (RAM-1, MIE, Billerica, MA) was used to continuously monitor PM during the 3-h exposure period. The exposure concentration delivered to the animals during the nose-only exposure was determined gravimetrically and the particle size distribution was measured with a piezo-electric cascade impactor (California Measurements).
3. Results and discussion
3.1. Pulmonary hypertension rat model 3.1.1. Systemic response The changes observed following exposure to concentrated ambient PM were generally no different between normal and monocrotalinetreated animals. To date, no deaths or immediately life-threatening health effects have been observed after single or multiple exposure to concentrated ambient PM. Hematological changes were consistently observed at 3 h following exposure to PM. These changes were: (1) evidenced by a consistent increase in circulating blood neutrophils and a decrease in lymphocytes; (2) absent by 24 h post-exposure; and (3) present in both normal and monocrotalinetreated young and aged (6 months) animals after PM exposure. To verify that these peripheral blood changes were a result of particle exposure and not ambient gases or the experimental conditions, additional animals were exposed to air or the output of the centrifugal concentrator’s 10 l/min output, which was scrubbed with a small HEPA filter. No significant differences in circulating blood neutrophil or lymphocyte changes were observed between these two groups, suggesting that the hematological changes were due to inhaled particles. Furthermore, no group differences in the white blood
287
cell differentials were observed between animals exposed to air or the output of the particle concentrator when the inlet to the concentrator was scrubbed with a HEPA filter. This latter experiment has been repeated twice in the last month and should rule out any contribution of the operation of the centrifugal concentrator itself to the observed hematological changes.
3.1.2. Pulmonary response No lung pathology or signs of inflammation were noted in hematoxylin and eosin stained lung sections from animals exposed to air or PM. Examination of biochemical and cellular indices in the lavage fluid of air- and PM-exposed normal animals confirmed this absence of pulmonary injury or inflammation at PM concentrations of 110–350 mg/m3. Similar results were obtained with monocrotaline-treated animals with the exception of one experiment. When monocrotaline-treated animals were exposed to 360 mg/m3 PM, total cell counts, protein and LDH were almost twice the values of air-exposed animals. These changes appeared to be dose-dependent, as the exposure of monocrotaline-treated animals to lower concentrations of concentrated ambient PM did not alter lavage fluid markers of injury and inflammation. 3.1.3. Cardio6ascular response We observed no change in heart rate during exposure to PM, even in animals that had been adapted to the exposure protocol. Small, but persistent increases in heart rate were observed after PM exposure in normal young and monocrotaline-treated rats but not in old rats. In general, this statistically significant increase persisted for 2–6 h after a single 3-h PM exposure. The heart rate response of monocrotalinetreated rats to PM exposure did not appear to differ from normal rats. Interestingly, the 10–20 beats/min increase in heart rate in PM-exposed rats was still seen regardless of the greater overall change in heart rate due to circadian rhythm. When animals were exposed to PM for 6 h/day for 3 days, the magnitude of the increase in heart rate did not change but the increase in
288
T. Gordon et al. / Toxicology Letters 96,97 (1998) 285–288
heart rate was more persistent, particularly on the second and third day of exposure. EKG tracings from all experiments were scanned for arrhythmias which were very rare in normal and in monocrotaline-treated rats. A careful inspection was made on the EKG tracings from 12 monocrotaline-treated rats that were exposed to air or PM for 3 h and monitored during exposure and 18 h after exposure. A total of two atrial arrhythmias were seen, one in each of two PM-exposed animals, which is biologically insignificant.
3.1.4. Core temperature response The effects of PM exposure on temperature were more variable than the heart rate results. Rats treated with monocrotaline 4 weeks prior to a single 3-h PM exposure showed a significant and persistent decrease in temperature after PM exposure. Rats treated with monocrotaline 2 weeks prior to a repeated 6-h PM exposure, had a significantly increased temperature after the first exposure but were unchanged from air-exposed rats after PM exposure on the second and third days of exposure. Normal young and 6 month old rats showed no change in temperature after PM exposure.
within a large population, rather than severe changes in a few very sensitive individuals. However, these possibilities are not mutually exclusive. In fact, in one experiment, monocrotaline-treated rats but not normal rats showed signs of pulmonary injury following exposure to a high concentration of PM. This suggests that individuals with pre-existing cardiac or respiratory disease may have greater effects than healthy people. The time course and pattern of the peripheral blood changes (PMN demargination and lymphopenia) are consistent with the activation of the sympatho-adrenal system, i.e. a stress response. While consistent changes in heart rate and hematological parameters were seen in normal and monocrotaline-treated rats, the only change that implied adverse heath consequences (increased lavage fluid protein and LDH) was seen in monocrotaline-treated rats. These data suggest that inhaled PM can cause systemic effects in both normal and monocrotaline-treated rats over a wide range of concentrations but only monocrotaline-treated rats are susceptible to adverse pulmonary effects. Therefore, we hypothesize that inhaled PM causes systemic effects by evoking a stress response which may be well tolerated by healthy individuals, but may cause morbidity and mortality by altering homeostatic processes in those with cardiovascular or pulmonary disease.
4. Conclusions Consistent changes in heart rate and white blood cell differential counts were seen in normal and monocrotaline-treated rats following exposure to concentrated PM. Surprisingly, systemic effects were seen in the absence of pulmonary inflammation or injury. The magnitude of the systemic effects would not be considered lifethreatening or even an adverse health effect if observed in an individual. However, the increased neutrophil number reported in this study may be sufficiently large to be a risk factor for cardiovascular disease and mortality in a large population. Most of our results are consistent with the concept that PM-induced mortality results from small changes in physiological parameters occurring .
Acknowledgements The authors thank Margaret Kransinski and Karen Galdanes for excellent technical assistance and William E. Hill (Walker Muffler, Grass Lake, MI) and Jack Mikhail Wolfson (Harvard School of Public Health, Boston, MA) for providing expert advice in sound abatement and gas denuders, respectively.
References Budinsky, K., 1970. Rotating centrifugal separator with continuous dust removal. Staub 30, 7 – 13. Shutte, A.H., 1976. Filter apparatus. U.S. Patent no. 3262573.