* Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 275997310;
Instituto Nacional de Pediatría, Mexico City 14410;
Center for Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill, North Carolina 275997310; and
National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received July 21, 2000; accepted February 9, 2001
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ABSTRACT |
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Key Words: ambient air pollution; dogs; myocardium; mast cells; particulate matter; ozone; apoptotic myocytes and endothelial cells; endothelial injury; Mexico City.
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INTRODUCTION |
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This report describes myocardial changes in dogs chronically exposed to high levels of ambient air pollutants. Mexico City (MC) is a megacity (population 20 million) with severe air pollution problems. The consequences of lifelong daily exposure to atmospheric pollutants on the cardiovascular system of healthy, as well as sick, individuals are of considerable clinical importance. Canines are often the species of choice as an experimental model for the study of pulmonary responses to long-term exposure to air pollutants (Heyder and Takenaka, 1996), and in experimental myocardial ischemia/reperfusion (I/R) models (Albertine et al., 1994; Frangogiannis et al., 1998a
,b
; Hansen, 1995
; Yang et al., 2000
). Chronic exposure of healthy canines to auto exhaust produces electrocardiographic abnormalities (Bloch et al., 1972
), while acute exposures to concentrated urban PM results in heart-rate changes, and animals with surgical coronary occlusion exhibit ST-segment elevations (Godleski et al., 1999
).
The main objective of this study is to identify and characterize the cardiac histopathology in dogs chronically exposed to a polluted environment, and to establish a correlation with pulmonary changes observed earlier in these animals (Calderón-Garcidueñas et al., 2001). The ultimate objective is to provide data to build future hypothesis-driven mechanistic studies that would explain the epidemiological data of increased cardiovascular morbidity and mortality in people exposed to air pollutants.
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MATERIALS AND METHODS |
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Study Areas.
SWMMC, NEMMC, Cuernavaca, Tlaxcala, and Tuxpam are the 5 urban areas selected for this study. Detailed descriptions of the pollutant profile in each geographical location are reported in the accompanying paper (Calderón-Garcidueñas et al., 2001). Briefly, both geographical areas selected in MC are characterized by concentrations of criteria pollutants above the standards. SWMMC`s atmosphere is characterized by average daily maximal O3 concentrations of 0.250 ppm, and an average of 4 ± 1 h/day > 0.08 ppm ozone year-round. PM10 and PM2.5 routinely exceed the U. S. standards (annual NAAQS for PM10 is 50 µg/m3 and PM2.5 is 15 µg/m3) averaging 78 µg/m3 and 21.6 µg/m3, respectively for PM10 and PM2.5 (Cicero-Fernandez et al., 1993
, Edgerton et al., 1999
). NEMMC, on the other hand, is characterized by high PM values. The annual average for PM10 is 235 µg/m3 (Cicero-Fernandez et al., 1993
), and for PM2.5 is 44 µg/m3 (Edgerton et al., 1999
). In addition, O3 and NO2 in NEMMC exceed the U. S. standards on 71.9% and 15.8% days/year, respectively. In contrast, Cuernavaca's atmosphere is characterized by maximal ozone values of 0.330 ppm, with a third of the days in a year with values above the O3 NAAQS. SO2 concentrations exceed the 24-hr primary standard of 0.14 ppm. Total suspended particles have average values of 120 µg/m3 (Oswald, 1999
). Tlaxcala and Tuxpam are in compliance for all major pollutants.
Atmospheric pollutant data.
MC's atmospheric pollutant data were obtained from 2 representative pollutant monitoring stations located in the SW and NE areas, and the available literature. Atmospheric information from the other cities was obtained from the Subsecretaria de Ecología, and the available literature. The data are representative of the air pollution patterns corresponding to the dogs' collection period (19972000).
Necropsy and tissue preparation.
Animals were briefly observed and examined by a veterinarian before they were euthanized according to the recommendations from the Panel on Euthanasia (1993). A clinical respiratory and cardiovascular examination was done. At necropsy, each animal was weighed and its age and nutritional status was noted. Immediately after death, the trachea, extrapulmonary bronchi and lungs, along with the heart, were excised intact from the thoracic cavity. The cardiorespiratory block was inspected for gross lesions and photographed when necessary. In a group of 60 dogs from the 5 selected areas, fresh samples of cardiac tissue were fixed in Carnoy's (Frangogiannis et al., 1999) and EM samples were taken. Fragments of the anterior free walls of the right and left ventricles and the interventricular septum were cut in 2 mm squares, fixed in glutaraldehyde (2.5%), embedded in plastic and conventionally processed for EM.
The heart was cut in the fresh state following the direction of the blood flow using a modified Hudson technique (Hudson, 1965; Silver and Freedom, 1991
). The modifications consisted of incising the atrium parallel to the atrioventricular grove rather than to join the superior and inferior pulmonary veins and to extend the incision to the tip of the appendage, transection of the apices of the ventricles before opening the heart, and keeping the apical mass hinged to the rest of the heart by the posterior epicardium and outer posterior ventricular myocardium (Silver and Freedom, 1991
).
The rationale behind cutting the ventricular apices was based on Drs. Meredith Silver and Robert M. Freedom's (Silver and Freedom, 1991) opinion that this incision permits one to inspect the myocardium and to obtain correctly oriented sections for microscopic study. As part of the Hudson technique, the major blood vessels are opened and explored for thrombus, embolus, parasites, or other pathology. The heart was fixed in formaldehyde for an average of 1 week before taking LM sections. LM blocks from formaldehyde and Carnoy fixed tissues included transmural sections of the ventricular septum, located in the area of maximal thickening, perpendicular to the long axis of the left ventricle, approximately one-third the distance between the aortic valve and the left ventricle (LV) apex; the anterior LV free wall approximately 2 cm lateral to the anterior descending coronary, and from the right ventricle (RV) free wall near the tricuspid valve annulus, and the right atrium.
The rationale behind selecting such sections was based on the need to define the location of potential lesions in anatomically well-defined transverse sections and to keep the conduction system and the coronary vessels intact in case there was any need to go back to the specimen. Paraffin sections 5 µm thick were cut and routinely stained with hematoxylin and eosin. Special stains included Masson's trichrome for collagen staining, Prussian blue for the detection of Fe3+, elastic Verhoeff stain, toluidine blue pH 4.5 for mast cells, and periodic acid Schiff and luxol fast blue (PAS/LFB) for the detection of polysaccharides and mucosubstances containing hexoses or deoxyhexoses. For the Prussian blue reaction, a 4% potassium ferrocyanide solution and a 20% hydrochloric acid solution were employed to treat the sections. The presence of any Fe3+ iron in the heart combines with the ferrocyanide and results in the formation of bright blue ferrocyanide pigment.
The histopathologic severity of the lesions was assessed semiquantitatively from 03 (ranging from 0 for no pathological change to 3 for the most severe pathological change) by observers blinded to the coding of the geographical groups. The parameters evaluated included: the presence of histological elements characteristic of myocyte necrosis or apoptosis, foci of chronic inflammatory cells, the distribution and characteristics of mast cells, vascular changes including PMN margination and microthrombi, endothelial changes, venule and arteriole abnormalities, the distribution of adipocytes, and the characteristics of the small nerves in the epicardial fat.
Myocardial mast cells stained with toluidine blue, from both formaldehyde and Carnoy fixed tissues, were counted blindly in 20 microscopic fields (area 1.32 mm2) in sections from the LV, RV, and the interventricular septum (IVS). Each slide was read from left to right, top to bottom, until the 20 fields per slide were completed. For each dog an average number of mast cells for the 3 anatomical regions was analyzed (LV, RV, and IVS).
The rationale behind staining mast cells with 2 different stains was based on previous reports of 2 populations of mast cells in the canine heart: connective tissue mast cells that stain with toluidine blue regardless of fixation, and a second population that exhibits metachromasia only after fixation in Carnoy (Frangogiannis et al., 1999). Mast cells from control animals exhibit numerous metachromatic granules that occupy most of the cytoplasm and partially obscure the excentric nucleus. Evidence of degranulation implied that few granules could be seen in the cytoplasm, the nucleus was clearly delineated, and metachromatic granules could be seen in the immediate extracellular proximity. Apoptosis was evaluated through analysis of nuclear and cellular morphology using LM and EM, and DNA fragmentation. The terminal deoxynucleotidyl transferase (TdT) labeling assay (TUNEL) was used to assess cells with DNA strand breaks, as described elsewhere (Levin et al., 1999
). All the slides were read blindly with no access to the codes regarding the geographical source of the animals.
Statistics.
Statistics were performed using the Instat program (Graph Pad, San Diego, CA). The following statistical procedures were used: (1) one-way analysis of variance (ANOVA). The Tukey-Kramer multiple comparisons test was used to establish the differences in the myocardial endpoints among the dogs in the different geographical cohorts. (2) Pearson correlation was used to assess the strength of association between the lung perivascular mononuclear cell infiltrates, and myocardial endpoints. Data are expressed as mean values ± SD. Significance was assumed at p < 0.05.
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RESULTS |
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Gross Cardiac Morphology
No gross abnormalities were seen in the pericardium, large blood vessels, and hearts of animals from MC, Tlaxcala, and Tuxpam. A polypoid visceral pericardial mass, with a brownish smooth surface (2 x 1.7 x 0.4 cm), and a 0.5 cm stalk was present on the left ventricular anterior surface of a well-nourished Cuernavaca dog. No evidence of intercurrent disease was seen in any of the hearts, with the exception mentioned above. A summary of the major histopathological light and electron microscopy cardiac findings in young animals (< 4 years) from SWMMC and NEMMC, older SWMMC dogs (> 5 years), and young (< 4 years) Cuernavaca, Tlaxcala, and Tuxpam dogs is shown in Table 1.
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EM examination of myocardial fibers showed agglomerates of mitochondria in a paranuclear location beside lipofuscine in various stages of formation, and glycogen (Fig. 5A). Capillaries frequently showed endothelial cells with numerous transport vesicles, both luminal and abluminal, irregular luminal surfaces, and numerous filopodia (Figs. 5B and 5C
). The elongated filopodia mostly arose at the level of the zonula occludentes (Fig. 5C
). Platelet aggregates and fibrin depositionidentified by its 22 nm periodicitypartially degranulated PMN in close contact with the endothelium, RBC fragments, and fragments of unidentified cells were observed in myocardial capillaries (Figs. 5D and 6
). EM showed myocardial mast cells with few granules in the midst of an abundant and dilated rough endoplasmic reticulum (RER). The pericardial mass in a Cuernavaca dog showed abundant fibroblasts and blood vessels and foci of extramedullary hematopoiesis. At the stalk, an old hemorrhagic area, with abundant hemosiderin-laden macrophages could be seen.
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DISCUSSION |
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The crucial pathology in these dogs seems to be lung epithelial and endothelial damage (Finkelstein et al., 1997; Krishnaswamy et al., 1999
; Saffiotti, 1996
; Simon and Paine, 1995
) induced by air pollutants, which presumably results in the production of cytokines and growth factors that give rise and perpetuate a chronic inflammatory cascade. The observation of ultrafine PM in type I and II alveolar cells, interstitial macrophages, endothelial cells, and intravascular macrophage-like cells (Calderón-Garcidueñas et al., 2001
), supports previous work reporting penetration of ultrafine PM into lung tissue and the inflammatory reactions it evokes (Churg et al., 1998
; Oberdorster et al., 1995
).
A significant association was seen between the lung epithelial and endothelial pathology and perivascular chronic inflammatory infiltrates, with the presence of myocardial foci of mononuclear cells and degranulated mast cells (r = 0.7; p < 0.0001), as well as with myocardial adipocyte islands (r = 0.42; p < 0.01) (Calderón-Garcidueñas et al., 2001).
Hearts obtained from dogs living in MC and Cuernavaca showed degranulated mast cells, and apoptotic myocytes, endothelial and immune effector cells. There was also a discrete infiltration of the myocardium by mononuclear cells surrounding partially degranulated mast cells and mature fat cells, and islands of adipocytes without inflammation described in human hearts as "metaplastic fat" and "fat dissociation syndrome" (Fontaine et al., 1999). In addition, the presence of degranulated mast cells in the epineurium and endoneurium of small epicardial nerves was also noted. The findings of vascular myocardial PMN margination and microthrombi formation, capillary fibrin deposition and the ultramicroscopic capillary alterations suggest endothelial injury and could be related to endothelial dysfunction in these dogs (Bevilacqua and Gimbrone, 1987
; Chen and Manning, 1995
; Luscher and Barton, 1997
). PMN adherent to endothelial cells can damage them, resulting in the capillary leak that is central to the evolution of a systemic inflammatory response (Hansen, 1995
; Ricevuti et al., 1991
). Indeed, we observed indirect evidence of capillary leakage in both lungs and myocardium of MC and Cuernavaca dogs; red blood cells (RBC) were seen in alveolar spaces, in BAL macrophages, and in macrophages around capillaries in the myocardium. Apoptotic endothelial cells in exposed animals are potentially important since the apoptotic cells may circulate as procoagulant bodies and lead to a significant increase in the expression of phosphatidylserine and thrombin formation (Bombeli et al., 1997
).
Cardiac mast cells likely play a crucial role in the pathology seen in exposed canines. Degranulated mast cells were commonly observed in MC and Cuernavaca dogs, and the phagocytosis of mast cell granules may explain the inflammatory infiltrate surrounding degranulated mast cells (Baggiolini et al., 1982). Cultured cardiomyocytes from neonatal rats incubated with mast cell granules for 24 h cause an apoptotic death in 70% of cardiomyocytes (Hara et al., 1999
). Cardiac mast cells store large amounts of histamine that once released, may increase sinus rate and ventricular automaticity, and give rise to severe tachyarrhythmias (Genovese and Spadaro, 1997
). Additionally, mast cells have been implicated in the process of endogenous fibrinolysis (Sillaber et al., 1999
), and in the pathogenesis of coronary artery spasm, atheroesclerosis, myocardial ischemia, and idiopathic dilated cardiomyopathy as well as being contributive to changes in heart rate (Marone et al., 1999
; Martin et al., 1993
, Patella et al., 1998
).
The presence of degranulated mast cells in epicardial nerves raises a number of questions about the interaction between mast cells and peripheral nerves in the context of a chronic inflammatory state. The "cross-talk" between mast cells, lymphocytes, neurons, and glia constitutes a neuroimmune axis that has been implicated in a number of neurodegenerative and inflammatory diseases (Basbaum and Levine, 1991; Purcell and Atterwill, 1995
). Mast cells and nerve growth factor are involved in neuroimmune interactions and tissue inflammation (Leon et al., 1994
), and mast cell accumulation and degranulation occur within the endoneurium of injured peripheral nerves, accounting for the microvascular changes observed after the nerve lesion (Zochodne et al., 1994
).
Two recent observations are relevant to our mast cell findings. First, in the experimental canine myocardial ischemia/reperfusion (I/R) model, resident mast cells degranulate and release preformed tumor necrosis factor- (TNF-
) initiating a cytokine cascade responsible for myocyte ICAM-1 induction and subsequent PMN-induced injury (Frangogiannis et al., 1998a
). Second, the role of TNF-
in mediating myocardial inflammation, cell growth, differentiation, and apoptosis (Pulkki, 1997
; Sack et al., 2000
). TNF-
injected in vivo in guinea pigs causes in vitro myocardial depression and reduces cardiac responsiveness to norepinephrine (Heard et al., 1992
). Further, picomolar levels of TNF-
influence the movement of small hydrophilic molecules across the myocardial microvascular barrier in vivo and induces a prolonged decrease in cardiac performance (Hansen et al., 1994
).
TUNEL positive myocytes in the context of air pollution could represent apoptotic cells or cells with increasing activity for DNA repair, a possibility suggested for hearts with dilated cardiomyopathies (Kanoh et al., 1999). Progressive loss of individual myocytes by apoptosis is observed in chronic heart failure (Rayment et al., 1999
). The finding of TUNEL positive myocardial cells closely associated with the presence of myocardial fat, however, raises the possibility that myocardial cells are being replaced by adipocytes. Fat myocardial infiltration is seen in arrhythmogenic idiopathic cardiomyopathies (Lobo et al., 1992
), is associated with sudden deaths in young people (Larsson et al., 1999
) and remains a puzzling finding at autopsy, both in adults (Anversa et al., 1997; Fontaine et al., 1999
) and children (Carter and Variend, 1992
).
This study has several limitations. Although the study was done on clinically healthy dogs, we had no control over the exposures of these animals to their local environments, their genetic background is unknown, and we had no access to their nutritional history. Therefore, the possibility that some of the myocardial findings could be related to any of the above mentioned factors cannot be ruled out. There is a difference in sample size of dogs obtained from polluted and nonpolluted cities. Cities in compliance are also smaller cities and the number of available dogs consequently reduced. However, due to the substantial difference in pathology between these groups, the observed gradient of the key findings, and given that the cardiac alterations such as the ones we described are not seen in dogs used as controls in the numerous heart studies available in the literature, we believe the number of control animals is sufficient to draw the conclusions described here. Heart morphometric studies will be necessary in the future; in this study we focused on a semiquantitative analysis.
Most importantly however, the myocardial findings should be seen in the context of the concomitant lung pathology and with the understanding that the changes are discrete, distinct, and follow a gradient from low to highly polluted areas. Furthermore, some of the ultrastructural endothelial changes we described are also observed after the in vivo administration of proinflammatory cytokines in live rodents (Fujita et al., 1991; Rhodin et al., 1999
), and in vitro after the administration of oxidized human low density lipoprotein to umbilical cord endothelial cells (Chow et al., 1998
). Thus, this study provides the pathophysiological base for further studies including chamber-controlled exposures with purebred animals and experimental models in which specific mechanistic hypotheses can be tested. We also hope it will stimulate the forensic study of the cardiorespiratory system in young people dying suddenly in different urban environments.
In summary, the histopathology we observed in both the lungs and hearts of dogs chronically exposed to high levels of ambient pollution are of sufficient magnitude and clinical significance to warrant concern that similar histopathology may be occurring in humans residing in large metropolitan areas such as MC. If so, these alterations in pulmonary and cardiac tissues could provide an insight into the biological plausibility and underlying pathophysiological mechanisms responsible for the observed association between human morbidity/mortality and air pollutants.
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ACKNOWLEDGMENTS |
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NOTES |
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The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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