Effects of Chronic Exposure to Air Pollution from Sao Paulo City on Coronary of Swiss Mice,from Birth to Adulthood

Abstract

To explore the hypothesis that air pollution promotes cardiovascular changes, Swiss mice were continuously exposed, since birth, in two open-top chambers (filtered and nonfiltered for airborne particles ≤ 0.3 μm) placed 20 m from a street with heavy traffic in downtown Sao Paulo, twenty-four hours per day for four months. Fine particle (PM_2.5) concentration was determined gravimetrically; hearts were analyzed by morphometry. There was a reduction of the PM_2.5 inside the filtered chamber (filtered = 8.61±0.79 μg/m³, nonfiltered = 18.05±1.25 μg/m³, p < .001). Coronary arteries showed no evidence of luminal narrowing in the exposed group but presented higher collagen content in the adventitia of LV large-sized and RV midsized vessels (p = .001) and elastic fibers in both tunicae adventitia and intima-media of almost all sized arterioles from both ventricles (p = .03 and p = .001, respectively). We concluded that chronic exposure to urban air since birth induces mild but significant vascular structural alterations in normal individuals, presented as coronary arteriolar fibrosis and elastosis. These results might contribute to altered vascular response and ischemic events in the adulthood.

Introduction

Several epidemiological studies associate ambient pollutants, especially those from vehicular sources, with acute cardiopulmonary mortality, hospital admissions, and exacerbations of respiratory and cardiac disease in populations with high cardiovascular and pulmonary risk (Dockery 2001; Pope 2000). Fluctuations of ambient air pollutants were related to ischemic heart disease in predisposed adults aged forty-five years or older (Lin et al. 2003), while transient exposure to traffic-originated airborne pollutants exacerbated exercise-induced myocardial ischemia, impaired endogenous fibrinolytic capacity (Nicholas et al. 2007), and intensified electrocardiographic ischemic alterations in men with coronary heart disease (Mills et al. 2007).

Experimental controlled studies using concentrated ambient particles have shown effects of particulate matter (PM) exposure on cardiovascular function in normal dogs (Godleski et al. 2000; Knuckles and Drehen 2007). Several other authors suggested cardiovascular acute effects secondary to airborne pollutants in normal vessels, such as vasoconstriction in children (Calderón-Garcidueñas et al. 2007) and in adults (Batalha et al. 2002), cardiac edema (Rivero et al. 2005), oxidative stress (Eeden et al. 2001; Gilmour, Donaldson, and MacNee 2002; Kelly 2003), and vascular remodeling (Lemos et al. 2006).

These and other works show plausibility between epidemio-logical human studies and suggest close relation to myocardial findings and coronary endothelial dysfunction, resulting in vascular remodeling and altered repair (Calderón-Garcidueñas et al. 2001; Kodavanti et al. 2007). However, there are no experimental studies associating structural vascular alterations induced by real ambient air continuous exposure from birth to adulthood.

The aim of this study was to verify the effects of ambient air pollution of Sao Paulo City on coronary of healthy nonisogenic Swiss mice, chronically exposed since birth until adulthood. We focused on structural alterations of extracellular matrix (ECM) fibers of the coronary arteriolar wall. This experiment tried to reproduce the closest possible to real-life situation of a human, who is being exposed since birth to air pollution, with diseases manifestation at adulthood.

Materials and Methods

The review board for human and experimental studies of the Clinical Hospital, Medical School of the University of Sao Paulo (CAPPesq-HC-FMUSP) approved this study, research number 803/05.

All animals received humane care in compliance with the “Principles of Laboratory Animal Care” published by the National Institutes of Health (NIH publication 86-23, revised 1985).

Site of Exposure and Air Pollution Monitoring

The exposure site was located in the campus of Medical School of University of Sao Paulo, in downtown São Paulo, 20 m from the roadside, 150 m from a busy traffic crossroad, and 160 m from a monitoring station of the State of Sao Paulo Sanitation Agency (CETESB, Sao Paulo, Brazil). There are no industries or significant biomass burning sources in the surroundings.

The twenty-four–hour concentration of PM_2.5 (fine PM ≤ 2.5 μm) inside the exposure chambers was determined gravimetrically using Harvard Impactors (Air Diagnostics, Harrison, ME) at a flow rate of 10 L.m⁻¹. Impactors were equipped with polycarbonate filters, and results were expressed as μg/m³. NO₂ was determined using the colorimetric method (Lodge 1989) and Black Carbon (BC) concentrations were determined using the light reflectance method (Associação Brasileira de Normas Técnicas [ABNT] ). Hourly concentrations PM₁₀ (PM ≤ 10 μm) (FH62 I-n Beta Attenuation Monitor, Graseby Andersen, Smyrna, GA) and NO₂ (Chemiluminescence 42 Series, Thermo Scientific, Waltham, Massachussetts) were obtained from the monitoring station of the State of Sao Paulo Sanitation Agency (CETESB) located 160 m away from the exposure chambers.

Exposure Chambers

Exposures were performed from September to December 2003, using two open-top chambers installed on the campus of the Sao Paulo University Medical School. The exposure system employed in this study has been previously used in several studies of our group (Mohallem et al. 2005; Camargo Pires-Neto et al. 2006; Lemos et al. 2006; Mauad et al. 2008, Veras et al. 2008). The two side-by-side exposure chambers consisted of cylindrical aluminum structures, measuring 2.0 m in diameter and 2.15 m in height and covered by a plastic UV film protection. Air entered the chamber at the base of the cylinder and was uniformly distributed throughout the chamber. The air was forced into the chamber and exited at the top, where there was a wide opening. It was a normobaric system and the pressure inside the chambers did not exceed 30 mm H₂O with a flow rate of 20 m³/min. Inside the chambers, animals were kept at the same ambient conditions of temperature, humidity (measured by digital thermography—Soil Control, Tecnologia Instrumentos do Solo, Ltda), day and light cycle, and noise as external ambient.

The filtered chamber had three stages of filters in line. The first eliminated large particles (model JFL-90), the second stage (High Efficiency Particulate Air, HEPA) trapped fine particles (PM < 0.3μm), and the third trappe chemical substances (Al₂O₃ plus KMnO₄). Filters were purchased from Purafil (Sao Paulo, SP, Brazil).

The nonfiltered chamber received ambient air with no filter devices.

Animals and Experimental Groups

Forty-two male, ten-day-old Swiss mice from the animal facility of our medical school were raised in the chambers for four months in 2003 (September–December). They were separated in two groups: F group was raised in the filtered chamber (n = 22), and NF group was raised receiving ambient air in the nonfiltered chamber (n = 20). The animals were housed in poly-propylene cages (maximal of 10 animals/cage) and received the same balanced diet (Nuvital-Nutrients Ltda., Colombo, Brazil) and water ad libitum. After the exposure period, mice were anesthetized with sodium pentobarbital, euthanized by cervical dislocation, and exsanguinated. The whole body and heart weights were obtained from each animal. The animals were also used for data collection for toxicological parameters on the respiratory (upper airways) and reproductive system (Camargo Pires-Neto et al. 2006; Lichtenfels et al. 2007).

Tissue Sampling and Histopathological Studies

The hearts were isolated, fixed in buffered 10% formalin solution, transversely sectioned, and submitted to routine histological procedures (embedded in paraffin wax and cut at 4 μm). The slides were coded for blinded analyses. The sections were stained with hematoxylin and eosin for heart structural analysis, with Picrosirius for collagenous fibers (Mady et al. 1999), and with Weigert’s Resorcin-fuchsin with oxidation for elastic fibers (Caldini et al. 1990).

Morphometric Analysis

Morphometric data were obtained from histological images made in a light microscope (Leica DMR; Leica Microsystems GmbH, Wetzlar, Germany) connected to a video camera coupled to a compatible microcomputer. The images were processed using the Image-Pro Plus 4.1 for Windows software (Media Cybernetics, Silver Spring, MD, USA). All measurements were performed in histological images obtained with the 40x objective lens.

The lumen/wall (L/W) ratio of coronary arterioles was assessed in transversally cut vessels (maximum and minimum diameter variation < 10%). The areas of the lumen and of the vascular wall were determined by point counting technique in Weigert’s Resorcin-fuchsin stained slides (Batalha et al. 2002; Lemos et al. 2006; Rivero et al. 2005). A grid was superimposed to the histological images on the monitor and the points overlying the lumen and those overlying the coronary wall were counted separately. The L/W ratio was calculated as points overlying the lumen/points overlying the arteriolar wall (μm²/μm²). According to the lumen area, coronary arterioles were categorized by tertiles as I small, II median, and III large vessels.

The area occupied by collagenous and elastic fibers in the tunicae adventitia and intima plus media was determined by digital densitometry. We measured together the tunicae intima and media because intima was too thin to be morphometrically analyzed as an isolated tunicae in our study condition. The areas occupied by ECM fibers in arteriolar wall compartments were divided by the perimeter of the outer border of the tunicae media in each studied arteriole. The results were expressed as the amount of collagenous or elastic fibers per unit of perimeter length (μm²/μm).

Statistical Analysis

Comparison between groups was done using either Student’s t test or Wilcoxon/Mann-Whitney U test, depending on the distribution exhibited by variables. The level of significance was set at 5%. Statistical analyses were performed with the SPSS 15.0 (SPSS Inc., Chicago, USA).

Results

Inside the filtered chamber, the efficiency of the filters were 32 % of NO₂, 100% of BC, and 52% of PM_2.5 when compared to the nonfiltered chamber (p < .001, Table 1). Mean values of PM₁₀ during the period were 28.28±1.71 μg/m³ in the external ambient. PM_2.5 measured in the experimental chambers comprised 0.6 of PM₁₀ (Mauad et al. 2008). External PM_2.5 (18.53±2.25 μg/m³) was similar to PM_2.5 measured inside the nonfiltered chamber (18.05±1.25 μg/m³). External NO₂ annual means were 60 μg/m³, which is similar to that measured inside the nonfiltered chamber.

No mice died or became sick during the experiments.

There was no difference in body and heart weight of mice between the groups (F: 45.11±1.05g and 0.21±0.007g; NF: 43.38±1.22g and 0.22±0.012g; body and heart weight, respectively; M±SE).

The coronary L/W ratio presented no differences between groups even after vascular categorization—I (100–499 μm²): 0.35±0.03 versus 0.61±0.12; II (500–999 μm²): 1.23±0.25 versus 1.28±0.45; III (≥ 1000 μm²): 1.68±0.22 versus 1.85±0.18; F versus NF, respectively.

Pollution induced increment of the ECM content in all layers of coronary wall of both ventricles of animals from NF group when compared to those from F group (Figures 1, 2, and 3).

The adventitia layer expressed higher collagen fibers content in all coronary arterioles of NF group when compared to F group (p ≤ .02). After categorization, LV large-sized and RV midsized arterioles expressed this increment significantly (p = .001 and p = .05, respectively) in NF group (Figure 1).

Elastin content in the adventitia was greater in vessels of all sized categories of both ventricles of NF group (p ≤ .01)), except for RV midsized arterioles (Figure 2).

In tunicae intima plus media, NF group also expressed increment of elastic fibers content in all arteriolar-sized categories of both ventricles (p ≤ .001), except for small vessels (Figure 3).

Figure 4 shows the structural aspects in the coronary arteries of experimental mice. Animals of NF group presented higher collagen (Figure 4B) and elastic (Figure 4D) content when compared to those of F group (Figures 4A and 4C, respectively).

Discussion

Chronic exposure to ambient air pollution in the city of Sao Paulo from birth to adulthood in healthy mice induced fibrosis and elastosis of coronary arterioles.

We tried to mimic as close as possible real exposition to ambient air. Exposure chambers really reflected the environmental atmosphere of São Paulo (Table 1). We chose the four-month-old Swiss mice because this strain is nonisogenic, and this age (3–6 months) reflects an adult sexually mature human individual of about thirty to forty years old. This group consists of mice that are past development but not yet affected by senescence (Harrison 2008). Cardiovascular events in big cities are prevalent above the forties (American Heart Association Statistics Committee and Stroke Statistics Subcommittee 2007).

At the site of exposition, in the last five years, PM₁₀ presented ambient fluctuations around 50 μg/m³ and has been characterized mainly from vehicular sources (CETESB 2003–2008). During this experiment, it was 28.28±1.71μg/m³ (M±SD) due to sazonality and recent public control of pollution from vehicular sources in São Paulo City. Characterization of PM_2.5 (PM ≤ 2.5μm) mass collected at the monitoring station and on the rooftop of the Medical School, 25 m above ground level, has shown that approximately 67% of the PM_2.5 mass is traffic related, with a black carbon/organic carbon ratio ranging between 40% and 70% throughout the day. Elemental analyses in PM_2.5 filters collected at the exposure site confirmed that vehicular emissions and crusted resuspension are the major PM_2.5 components at this site (Mauad et al. 2008). The filtering of the clean chamber proved to be efficient particularly to particles (Table 1). Clean ambient was free of BC and presented less than 50% of PM_2.5. In addition, nonfiltered chamber corresponded to external air as both presented similar pollutants (Table 1). Therefore, historical and recent data show that vehicular sources are the major components of PM_2.5 mass at this exposure site and that the exposure chambers are selective for particles filtering.

Experimental models of acute exposure to concentrated selective air pollutants are designed to characterize the effects of a specific component of atmospheric pollution. However, real ambient air constantly presents newly reactive pollutants, a process that modifies physical and chemical characteristics of suspended particles and gases, depending on changes of emissions (fluctuations of traffic, for instance), season, photochemistry, and weather conditions (Becker et al. 2005; Carvalho-Oliveira et al. 2005). Therefore real environmental exposure protocols may be of use in detecting the effects of such complex mixtures. On the other hand, the design of this experimental exposure has limitations. When moving an inhalation chamber to the streets, the amount of air pollution monitoring equipment that can be operated is limited, and thus, we only measured particles and NO₂ inside the chambers. Other air hazardous pollutants such as polycyclic aromatic hydrocarbons, oxidant gases, and other particle characteristics (particle number, for instance) were not quantified. Therefore it is safer to consider PM_2.5 and NO₂ as surrogates of a complex mixture of particle and gaseous pollutants, respectively, as we consider that other hazardous effects by other pollutants are also important.

Breathing ambient air of the city of Sao Paulo downtown produced structural alterations of coronary arterioles. In this study, mice from NF group presented structural alteration of ECM in coronary artery wall of both ventricles, but there was no alteration in L/W ratio. Although the ECM increment was not significantly present in all sized coronary arteries, almost all of them presented a trend to it (Figures 1, 2, 3, and 4). Both elastin and collagen increment predominated in LV and affected all large-sized arteries (Figure 1, 2, and 3). Elastin fibers augmented in both intima-media and adventitia layers of almost all sized arteries of ventricles (Figures 2 and 3), while collagen increased predominantly in midsized (RV) and large-sized arteries (LV; Figure 1).

Patency of small coronary arteries is responsible for endocardial nourishment, and their obstruction leads to subendocardial ischemic events or even necrosis. Midsized and large-sized vessels transport oxygen to myocardial transmural wall and, if not, induce transmural infarct. Both situations may lead to sudden death and cardiovascular morbidity, although ischemia due to larger arteries is linked to a greater area of necrosis, which may lead to the worst effects such as cardiogenic shock due to severe congestive heart failure, ventricular fibrillation, and death. Therefore, it would be interesting to diagnose which artery size is more affected by pollution. Recently, several myocardial infarcts have been diagnose in nonpredisposed individuals with normal or quite normal coronaries (Romagnoli and Lanza 2007). These findings are associated with altered functional vascular response to aggressors as pollutants (Godleski et al. 2000; Calderón-Garcidueñas et al. 2001). Thus, mild alterations of vascular wall may favor this hypothesis.

Vascular reactivity has been demonstrated in literature linked to acute peak of ambient air pollution or artificially exposition to high levels of pollutants. Similarly, Lemos et al. (2006) showed altered L/W ratio after four months of ambient air exposure of Sao Paulo City in 2001. Although their work was much similar to ours, pollutant levels during 2001 were higher than in 2003 (CETESB 2001), which could have favored vascular reactivity significance. In addition, there was no checking of previous peak of pollution in the previous days of data collection in that work, which could justify a more intense lung response to pollutants.

The imbalance between production and breakdown of ECM proteins depends on internal and external stimuli. Several mediators may promote phenotypic differentiation, migration, and collagen synthesis of adventitial fibroblasts from arteries and consequent vascular remodeling.

On theoretical bases, favoring matrix degradation are proteases such as metalloproteinases, which can be exacerbated during inflammation (Nugent et al. 2007). The oxidant air pollutant ozone, but not NO₂, also fragments elastin and increases its susceptibility to proteolysis of the lungs, which is reduced by antioxidants such as ascorbate and EDTA (Winters, Burnette-Vick, and Johnson 1994). On the other hand, other cytokine networks may enhance ECM content such as vascular endothelial growing factor (VEGF; Infanger et al. 2008), the vasoconstrictor urotensin II (Zhang et al. 2008), and alpha-1 antitrypsin, which sufficiently limits elastase activity to guide arterial response to injury (Waugh et al. 2001). Granulocyte–Monocyte Colony Stimulating Factor (GM-CSF) is a prerequisite for the maintenance of structural integrity of the vessel wall because it affects the production of fiber assembly/organization of the vascular collagenous matrix such as the elastic system production and integrity of elastic lamellae (Weissen-Plenz et al. 2008).

Resistance artery narrowing and stiffening are key elements in the pathogenesis of vascular narrowing and ischemia and have a greater collagen component than elastin, which correspond to a minor contribution. However, for all parameters of collagen, elastin and fenestrae area, and internal elastic lamina thickness, elastin organization might be a key determinant of intrinsic elastic properties (Briones et al. 2003; González et al. 2005). The increased deposition of elastic and collagen in the adventitia in early stages (postnatal) may contribute to less effective elastase digestion and lower susceptibility of elastic fiber to enzymatic degradation. These data suggested that abnormal elastic fiber deposition might increase resistance artery stiffness at an early age, which might participate in vessel narrowing in later life (González et al. 2006). In this regard, Pourageaud, Crabos, and Fresion (1997) suggested that the elastic modulus (intrinsic elastic properties, resistance) is increased in coronary of SHR (spontaneous hypertensive rats). The functional alteration is accompanied by an increase in the relative proportion of collagen, the component with a high elastic modulus in the vascular wall.

We conclude that chronic exposure to ambient air pollution of Sao Paulo City, Brazil, from birth to adulthood, induces fibrosis and elastosis of coronary arterioles in healthy Swiss mice.

Comprehensive and systematic approaches are needed to identify molecular networks implicated in the health effects of urban airborne and its constituents.