* Pulmonary Toxicology Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27709;
Pathology Associates, Raleigh, North Carolina;
Harvard School of Public Health, Boston, Massachusetts 02115; and
Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received July 29, 2002; accepted November 6, 2002
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ABSTRACT |
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Key Words: inhaled particulate matter; myocardial injury; Wistar Kyoto rats; bioavailable zinc; cardiovascular disease.
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INTRODUCTION |
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Several pathophysiological mechanisms have been proposed for the induction of PM-induced cardiovascular effects. One involves the concept that metallic substances bound to PM, that are water-soluble and thus bioavailable, pass directly into the pulmonary circulation, migrate to the heart, and induce cardiac injury. Another hypothesis asserts that PM exposure leads to local pulmonary vascular inflammation/microvascular thrombosis and systemic endothelial changes resulting in altered myocardial contractility (Frampton, 2001). Unfortunately, attempts to evaluate fully these proposed mechanisms have been hampered by a variety of problems: (1) Appropriately susceptible animal models that develop myocardial effects at environmentally relevant PM exposures are unavailable. (2) Clinically useful plasma markers of cardiac injury are less sensitive to subtle inflammatory and degenerative changes in the heart. Histological evaluation of cardiac tissue using a variety of staining techniques remains the most accurate method of detecting subtle cardiac toxicity. Unfortunately, these techniques are not used routinely in air pollution studies. (3) Environmental PM contains diverse causative constituents; therefore, without having sufficient knowledge of constituent-specific cardiac tissue effects, it has been difficult to define appropriate PM samples with which to study cardiovascular effects. Based on an understanding of these shortcomings, we retrospectively analyzed cardiac histopathology that included acute and long-term exposures of three rat strains to a combustion emission particle (EPM) sample containing zinc but minimal bioavailable vanadium, nickel, and iron. The EPM sample selected for this study was distinct from other extensively studied residual oil fly ashes (ROFAs) that contain high levels of iron, vanadium, and nickel (Dreher et al., 1997
; Kodavanti et al., 1997
, 1998
, 2002
). Evidently the elemental and organic composition of this EPM was similar to ambient PM composition from select urban locations (Adamson et al, 2000
; Balachandran et al., 2000
; Dye et al., 2001
; Harrison and Yin, 2000
). We hypothesized that exposure of the most susceptible rat strain to this EPM would be associated with time- and dose-dependant cardiac injury.
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MATERIALS AND METHODS |
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Emission PM (EPM) source and composition analysis.
The EPM used in this study was collected in Boston, MA in 1997 in a sterile, 50-ml polyethylene tube from a stack of a power plant burning residual oil (#6). These particles were released into the ambient air from an emission device. Because the elements that readily leach off in the water are likely to be absorbed by lung cells and in circulation within a short period of time after inhalation, the leachable elements of ROFAs have been responsible for the majority of injuries (Dreher et al., 1997; Kodavanti et al., 1997
, 1998
). However, the components not readily leached off may not be bioavailable, especially at distant sites. To better ascertain the role of individual components of EPM, and to relate the composition of this EPM to other residual oil fly ashes and ambient PM studied earlier by us and other investigators (Adamson et al, 2000
, Dye et al., 2001
; Kodavanti et al., 1997
, 1998
, 2000a
), the water-leachable and 1 M HCl-leachable metals and sulfate, as well as total carbon content, were determined and recently reported by our laboratory (Kodavanti et al., 2002b
). In brief, the bulk sample was ground, sieved, and analyzed for size using a TSI Model 3310A Aerodynamic Particle Sizer (TSI, Inc., St. Paul, MN), assuming a log-normal distribution as described earlier (Kodavanti et al., 1998
). The particles were made airborne with a TSI Model 3433 small-scale powder dispenser for size determination. EPM was extracted in double distilled water or 1.0 M HCl as described (Kodavanti et al, 1998
, 2002b
). Supernatants resulting from water and 1.0 M HCl were analyzed for the presence of sulfate (SO4), zinc (Zn), nickel (Ni), vanadium (V), iron (Fe), manganese (Mn), and copper (Cu), which were presumed to be the predominant causative metals of this combustion source emission based on previous studies (Kodavanti et al., 1997
; 1998
). The elemental analysis of the extracts was done using inductively coupled plasma-atomic emission spectroscopy (ICP-AES), as described in Kodavanti et al. (2002b)
.
Nose-only inhalation exposure protocol.
Since this study involved retrospective analysis of cardiac tissues from our recently conducted EPM inhalation study (Kodavanti et al., 2002b), the detailed inhalation exposure protocol and the experimental design are described in that paper. In brief, three rat strains were selected for EPM inhalation exposure. SD rats were evaluated for comparison to a healthy control rat strain that is extensively used in PM studies (Adamson et al, 2000
; Campen et al., 2001
; Dreher et al., 1997
; Dye et al., 2001
; Kodavanti et al., 1997
, 1998
, 1999
). WKY rats were employed for their propensity to develop hypertrophic cardiomyopathy while remaining normotensive (Kuribayashi, 1987
), and SH rats for their predilection to develop hypertension and cardiomyopathy (Kuribayashi, 1987
). SD, WKY, and SH rats were randomized into air control and EPM groups based on body weights (n = 6/group for SD and WKY and 8/group for SH rats). A larger group size (n = 8) was selected for SH rats because these rats have yielded more variable responses to given exposures relative to SD and WKY rats (Kodavanti et al., 2000b
, 2002b
). The selection of exposure concentrations/scenarios was based on the presumption that acute exposures will result in mild to moderate dose-dependent cardiopulmonary injury, and will represent scenarios included in our previous studies with different particles (Kodavanti et al., 2000a
,b
; Schladweiler et al., 2002
) as well as extreme environmental PM episodes. Inclusion of 4 consecutive days of exposure versus episodic exposure over 4 or 16 weeks was to understand the cumulative nature of injury based on our previous study where episodic ROFA exposure over 4 weeks resulted in progressive lung injury while systemic effects occurred only after acute exposures (Kodavanti et al., 2002a
). Rats were exposed by nose-only inhalation to either filtered air or a dry aerosol of EPM at 2, 5, or 10 mg/m3 for 6 h/day on 4 consecutive days, or 10 mg/m3, 6 h/day, 1day/week, for 4 or 16 weeks (Kodavanti et al., 2002b
; Ledbetter et al., 1998
). As reported recently (Kodavanti et al., 2002b
), aerosolized particles were aerodynamically size-separated through a cyclone impactor at
2.5 micrometer and introduced into the mixing chamber. Chamber aerosol concentrations were determined gravimetrically and by a real-time aerosol monitor (RAM-1, GCA Corp., Bedford, MA). Determination of aerosol-size distribution was performed at least once per exposure, using a 7-stage cascade impactor (Intox Products, Albuquerque, NM). Chamber temperature, relative humidity, airflow, and pressure were monitored continuously and maintained at constant levels (Ledbetter et al., 1998
).
Necropsy and histology.
Previous studies have indicated that following an inhalation exposure of 24 days, pulmonary injury and systemic effects remain persistent for at least up to 4 days with regard to inflammation, protein leakage, and pathology (Kodavanti et al., 2002b). Therefore, we decided to evaluate injury/pathology at two days following the last exposure. Two days after the final exposure, rats were weighed and anesthetized with sodium pentobarbital (100150 mg/kg, ip). The trachea was cannulated and the right lung tied; the left lung was inflated with filtered 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.2) at the total lung capacity (28 ml/kg body weight). The heart was removed and cut longitudinally to obtain representative portions of right and left ventricles. After fixation, the lung and heart tissues were embedded in paraffin (Experimental Pathology Laboratory, Research Triangle Park, NC).
Heart and lung sections from SD, WKY, and SH rats were cut at 5 micron, stained with hematoxylin and eosin, and examined microscopically. Based on the EPM-related strain differences in the presence or absence of cardiac lesions, further evaluation using a variety of staining techniques of the cardiac tissues was performed in WKY rats. SD rats were also evaluated further for comparison to a healthy control rat strain that is extensively used in PM studies (Adamson et al., 2000; Campen et al., 2001
; Dreher et al., 1997
; Kodavanti et al., 1997
, 1998
, 1999
). These evaluations were performed for the 16-week-exposure group as this long-term exposure scenario only depicted cardiac pathology. Replicate 5-micron heart sections from WKY and SD rats were stained with periodic acid Schiff (PAS) for the detection of myocardial glycoprotein and polysaccharide, Massons trichrome (MT) for collagen, toluidine blue (TB) for mast cells, phosphotungstic acid hematoxylin (PTAH) for evaluation of myofibrilar loss, von Kossa for calcium deposition, and Movats pentachrome for distinction of myocyte and myofibroblasts. The ApopTag Peroxidase in situ Apoptosis Detection Kit (Intergen, Purchase, NY) was used to detect apoptosis. Histological examinations of the left ventricle, right ventricle, septum, and large coronary vessels were conducted. The lesion severity was assessed based on semiquantitative criteria (Table 1
) previously outlined by Herman et al (1996
, 2000)
and Billingham (1991)
. Mast cells were identified by their large metachromatic, coarse granules in abundant cytoplasm in toluidine blue-stained histologic sections. The number of mast cells from three randomly selected fields per ventricular site was counted. These data were expressed as the mean ± the standard deviation of mast cells/x40 field (0.05 mm2).
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RESULTS |
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Histological examination of EPM-exposed WKY rat hearts stained by the Movats Pentachrome and Massons Trichrome stains revealed increased collagen deposition/myocardial fibrosis within degenerative foci (Fig. 3A). Fibrotic changes were not apparent in control clean-air-exposed WKY rats. In addition, heart sections stained with toluidine blue to identify mast cell numbers and granulation demonstrated no increase in the number of mast cells among ventricular sites in EPM-exposed WKY or SD rats. Conversely, decreased numbers of granulated mast cells were noted in EPM-exposed WKY rats (Figs. 3C
and 3D
, Table 2
), suggesting mast cell degranulation and its possible involvement in the process of myocardial degeneration/inflammation. Presence of mast cells within degenerating foci was also apparent in selected hematoxylin and eosin-stained heart tissue sections of WKY rats (Fig. 3B
), but a granulation pattern was not clearly visible. To determine if myocardial degeneration was associated with increased apoptotic cardiomyocytes and/or inflammatory cells, TUNEL staining was carried out for the heart tissue sections from control and EPM-exposed WKY and SD rats. No increase in apoptosis was detected in cardiomyocytes from EPM-exposed WKY and SD rats over air-exposed WKY rats. Finally, no calcium deposition, as determined by von Kossa staining, was noted in hearts from either EPM- or air-exposed WKY rats.
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DISCUSSION |
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Among the three rat strains evaluated, only WKY rats demonstrated marked EPM-induced pathology. Whether this susceptibility is related to systemic or local myocardial changes is unclear. Strain predilection for development of cardiac disease has previously been described in WKY and SH rats. Normotensive, and therefore often used as negative controls to the SH, WKY rats develop spontaneous cardiomyopathy in the absence of alterations in blood pressure (Kuribayashi, 1987; Leenen and Yuan, 1998
). Under the experimental conditions outlined in this study, EPM-induced inflammatory cardiac disease may have developed as a result of this predilection. In contrast, SD and SH rats developed indistinguishable incidences of myocardial disease seen in a comparison of the two exposure groups. The SH rats, although they are desirable as models of human cardiomyopathy, develop spontaneous inflammatory myocardial lesions and myocardial degeneration. Because the basal inflammatory disease was expressed almost identically in the air- and EPM-exposed groups, a clear separation of the potential contribution of EPM exposure from the onset or severity of myocardial inflammation in these rats was likely made impossible. Thus, the usefulness of the SH rat for this type of study may be limited. The current findings suggest, therefore, that the WKY rat may be a highly sensitive and desirable model system with which to study incipient, PM-induced morphological lesions in the myocardium.
There has been limited research on the cardiotoxicity of inhaled pollutants. Most of the studies have focused on cardiophysiological changes (Campen et al., 2001; Watkinson et al., 1998
; Wellenius et al., 2002
). However, in the field of cardiotoxicology, histopathology and molecular techniques have proven highly effective in the study of cardiotoxic agents such as doxorubicin and monoxidil (Herman et al., 1996
, 2000
). Histopathology has been considered the most accurate method of detecting subtle cardiotoxicty, as analysis of clinical plasma markers is often insensitive to small changes. We applied a panel of histochemical stain techniques classically used in cardiotoxic studies to confirm the cardiac injury that occurred in WKY rats after long-term EPM exposure. The use of different staining techniques allowed us to more fully define the extent, severity, and character of the cardiac injury and the possible role of mast cell degranulation. Based on this finding, it could be postulated that the lesions in WKY rats developed over time as a result of long-term EPM exposure, since fibrosis, myocyte degeneration, and active inflammation were all apparent within the same foci.
It is noteworthy that cardiac lesions were detected only after 16-week episodic exposures to EPM of 10 mg/m3, 6 h/day, 1 day/week, but not after 4-week exposures (1 day/week for 4 weeks or 4 consecutive days), suggesting that long-term exposure was necessary for detectable histological changes. Although histologic evidence of cardiac injury was not discerned at shorter durations or lower exposure concentrations, biochemical and molecular changes are likely. In addition, it is possible that high-concentration exposures might have resulted in injury that is detectable in a short time. Based on the present observation, several hypotheses can be proposed about the time course of injury, the dose response, the mechanism, and the possible causative constituent responsible for cardiotoxicty of inhaled EPM. These possibilities are currently being investigated by our laboratory to identify and evaluate potential mechanisms of EPM-induced cardiac injury in Wistar Kyoto rats.
The observation of cardiac lesions resulting from EPM exposure is significant, because PM air pollution has been consistently linked to poor cardiac health and increased cardiac-related deaths (reviewed in Dockery, 2001; Morris, 2001
; Peters et al., 2001
; Schwartz, 2001
; U.S. EPA, 1996
). These and other recent epidemiological studies, together with overt public concern, were responsible for EPAs new, more stringent National Ambient Air Quality Standard for PM (2.5 micrometer, 60 microgram/m3, 24-h average, and 15 microgram/m3 annual average) imposed in 1997. This decision was based primarily on consistent epidemiological associations; however, providing causality has been difficult, because animal studies using ambient PM exposure conditions have often led to none or minimal increases in cardiopulmonary morbidity or mortality. Moreover, the use of higher-than-ambient concentrations of combustion and ambient-derived PM has been criticized as being irrelevant. Therefore, the search for detectable pulmonary and cardiovascular effects at lower levels of PM in healthy and susceptible animal species, especially cardiopulmonary-compromised models, has been continued (Campen et al., 2001
; Kodavanti et al., 1999
, 2000b
, 2002a
, b
; Wellenius et al., 2002
). Susceptible animal models are particularly useful, because epidemiological studies show that PM exerts its greatest impact on this population (Peters et al., 2001
; Pope, 2001
).
The EPM concentration used in this study (10 mg/m3, 6 h/day, 1 day/week, 16 weeks), while actually high, may be "translated" over the entire period of 16 weeks to 357 microgram/m3 continuous exposure. Based on the differences in the morphology and physiology of the respiratory tract of rats and humans, it is argued that for a given PM sample of respirable size, humans may require lower concentration than rats to achieve the same deposition dose (Vincent et al., 1997). Thus, higher concentrations of PM may be needed to achieve an effect in the rat, whereas humans may experience the same effect at lower ambient PM levels. However, these assumptions have not been experimentally proven for all types of particles. Particle deposition can vary with size, composition, and physical characteristics. A protracted exposure of this degree is not likely to be encountered, but the episodic nature of the single weekly exposure allowed us to test the potential for direct cardiac injury/pathology in the context of an ambient-like PM to assess a range of susceptible animal models. With this scenario, we were able to detect significant focal myocardial degeneration, fibrosis, and inflammation in WKY rats. Thus, these findings are consistent with recent epidemiological associations of increases in cardiac morbidity following exposure to ambient PM in an industrialized urban environment (Dockery, 2001
; Peters et al., 2001
). Furthermore, the WKY rat may prove to be a useful animal model with which to investigate the mechanism by which PM exposure results in cardiac lesions.
We have at present no direct mechanistic data. One of the possible explanations for these findings is that EPM exposure stimulated systemic endothelin release and microvascular thrombosis resulting in myocardial injury (Vincent et al., 2001). No significant elevation of fibrinogen or changes in white blood cells were noted in rats exposed to EPM (Kodavanti et al., 2002b
), suggesting that these processes may not be significant in WKY rat cardiac disease induced by EPM. Of greater interest might be the possible direct role of zinc on cardiac tissues. Among water-leachable bioavailable metals, zinc is the only predominant metal in this EPM. Bioavailable metals are easily translocated to the circulation and may impact the heart. Moreover, occupational exposure to zinc has been blamed for causation of myocardial effects associated with pulmonary and systemic inflammation, collectively referred to as "zinc-fume fever" (Cire, 1983
). The possibility of zinc being one of the causative constituents of this EPM for cardiac injury is currently being investigated.
Heavy metals have previously been shown to induce in vivo vascular endothelial growth factor (VEGF) expression in the heart (Levy et al., 1995). While zinc has been shown to activate MAP kinases via activation of epidermal growth factor (EGF) receptors and contributes to the inflammatory response in the lung (Huang et al., 2002
), leachable zinc may possibly reach the coronary arteries and similarly induce activation of EGF and VEGF receptors. This activation through tyrosine kinase phosphorylation and MAP kinase cell signaling may cause increased inflammatory cytokine gene expressions (Fournier et al., 1999
; Huang et al., 2002
) in the heart, which in turn stimulate a cascade of inflammatory events. The presence of active inflammatory foci within the myocardium of EPM-exposed WKY rats was most likely reflective of the activation of cytokine genes.
In summary, this investigation demonstrated for the first time that ambient-like EPM can cause histologically discernible myocardial injury, inflammation, and degeneration in WKY rats, following long-term episodic inhalation at a concentration that does not cause significant lung injury.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Pulmonary Toxicology Branch, MD B143-02, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, 109 T. W. Alexander Drive, Research Triangle Park, NC 27709. Fax: (919) 541-0026. E mail kodavanti.urmila{at}epa.gov.
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