ToxSci Advance Access originally published online on January 11, 2006
Toxicological Sciences 2006 90(2):385-391; doi:10.1093/toxsci/kfj100
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Hypersensitivity of Prediabetic JCR:LA-cp Rats to Fine Airborne Combustion Particle-Induced Direct and Noradrenergic-Mediated Vascular Contraction
* Metabolic and Cardiovascular Diseases Laboratory, Alberta Institute for Human Nutrition, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada; and Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
1 To whom correspondence should be addressed at Metabolic and Cardiovascular Diseases Laboratory, Alberta Institute for Human Nutrition, 4–10 Agriculture Forestry Centre, University of Alberta, Edmonton, AB, T6G 2P5, Canada. E-mail: Jim.Russell{at}ualberta.ca.
Received November 10, 2005; accepted January 4, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Particulate matter with mean aerodynamic diameter
2.5 µm (PM2.5), from diesel exhaust, coal or residual oil burning, and from industrial plants, is a significant component of airborne pollution. Type 2 diabetes is associated with enhanced risk of adverse cardiovascular events following exposure to PM2.5. Particle properties, sources, and pathophysiological mechanisms responsible are unknown. We studied effects of residual oil fly ash (ROFA) from a large U.S. powerplant on vascular function in a prediabetic, hyperinsulinemic model, the JCR:LA-cp rat. Residual oil fly ash leachate (ROFA-L) was studied using aortic rings from young-adult, obese, insulin-resistant rats and lean normal rats in vitro. Contractile response to phenylephrine and relaxant response to acetylcholine were determined in the presence and absence of L-NAME (NG-nitro-L-arginine methyl ester). In a separate series of studies, the direct contractile effects of ROFA-L on repeated exposure were determined. ROFA-L (12.5 µg ml–1) increased phenylephrine-mediated contraction in obese (p < 0.05), but not in lean rat aortae, with the effect being exacerbated by L-NAME, and it reduced acetylcholine-mediated relaxation of both obese and lean aortae (p < 0.0001). Initial exposure of aortae to ROFA-L caused a small contractile response (<0.05 g), which was markedly greater on second exposure in the obese (
0.6 g, p < 0.0001) aortae but marginal in lean (0.1 g) aortae. Our data demonstrate that bioavailable constituents of oil combustion particles enhance noradrenergic-mediated vascular contraction, impair endothelium-mediated relaxation, and induce direct vasocontraction in prediabetic rats. These observations provide the first direct evidence of the causal properties of PM2.5 and identify the pathophysiological role of the early prediabetic state in susceptibility to environmentally induced cardiovascular disease. These are important implications for public health and public policy. Key Words: fine particulate air pollution; insulin resistance; type 2 diabetes; vasculopathy; vasospasm; cardiovascular disease; JCR:LA-cp rat.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The growing pandemic of obesity, the prediabetic metabolic syndrome, and type-2 diabetes is creating a new generation of cardiovascular dysfunction and disease that is increasingly influenced by the external environment (Després et al., 1996
; Uusitupa et al., 1990). Epidemiological studies show a significant association between ambient airborne particulate matter exposure and adverse cardiovascular events within susceptible subpopulations, particularly those with preexisting cardiovascular disease and type 2 diabetes (Pope et al., 2004; Zanobetti and Schwartz, 2001, 2002). The diabetes/cardiovascular–environmental risk relationship has not been widely recognized, yet the environmentally induced disease burden has major implications for public policy and health care.
The identity of causal particulate emission sources, the physicochemical properties of the emissions, and the mechanism(s) by which they affect the cardiovascular system are not well understood (Brook et al., 2004; Weinhold, 2004). Epidemiological studies show statistical associations between specific particulate matter (PM) emission sources, particularly of particles less than 2.5 µm in aerodynamic size (PM2.5) and mortality and morbidity (Clancy et al., 2002; O'Neill et al., 2005; Peters et al., 2004). However, these associations are confounded by geographical location, population demographics, prescription medication usage, and co-pollutant mixture effects, making unequivocal identification of causal emission sources, their physicochemical properties, and mechanisms of injury extremely difficult to determine. Inhaled PM2.5 are taken up and translocated to the circulatory system within 30 min and distributed throughout the body, including the brain (Oberdöster et al., 2004). Following inhalant exposure, metallic constituents of PM appear rapidly in the urine of humans and in a variety of organs in small animals (Esaka et al., 1995; Hauser et al., 1998). Nurkiewicz et al. (2004) have shown that intratracheal infusion of residual oil fly ash (ROFA), a PM2.5, "impairs endothelium-dependent arteriolar dilation" in distant skeletal muscle, indicating rapid and persistent distribution of both soluble and particulate components throughout the vascular system. Pathophysiological studies are now critical to provide mechanistic evidence for the epidemiological association of early and frank diabetes, PM2.5 exposure, and cardiovascular disease. These are essential to establish biological plausibility and provide insight into susceptibility/sensitivity factors for adverse cardiovascular effects.
Chronic insulin resistance and associated hyperinsulinemia result in changes in arterial architecture and vascular dysfunction leading to ischemic damage to the heart and other organs, both in humans and in animal models such as the JCR:LA-cp rat (Russell et al., 1998a). Rats of this strain, homozygous for the autosomal recessive cp gene (cp/cp), are obese and insulin resistant, with hyperinsulinemia and hypertriglyceridemia (Russell et al., 1998b). The cp/cp JCR:LA-cp rats are unique in the spontaneous development of atherosclerosis, vasculopathy, ischemic myocardial lesions, and glomerular sclerosis, all related to the insulin-resistant status (Proctor et al., 2005; Richardson et al., 1998; Russell et al., 1998a). The vasculopathy manifests in male cp/cp rats as defective endothelial function and release of nitric oxide (NO) in arterial vessels, including major conduit arteries (aorta), coronary arteries, and mesenteric resistance arteries (O'Brien and Russell, 1997; O'Brien et al., 1999). Impaired regulation of vascular tone by NO results in a hypercontractile arterial system and is an important contributor to vasospasm and ischemic injury, as seen in the cp/cp rat. The vascular dysfunction is a significant marker for endothelial dysfunction in the presence of hyperinsulinemia and cardiovascular disease (O'Brien and Russell, 1997). The cp/cp rat represents a unique animal model for the study of vascular dysfunction related to extrinsic factors, such as environmental contaminants, that may not affect normal individuals, rat or human.
It is becoming clear that multifactorial diseases, such as cardiovascular disease, are determined by complex interactions between the genome and life-style and environmental factors. Susceptibility to atherosclerosis and vascular dysfunction with progression to vasospasm and myocardial infarction has been proposed to be due to such an interaction (Philip et al., 1999; Sing et al., 2003), and it is evident in the cp/cp rat model. We hypothesize that individuals exhibiting the metabolic syndrome, insulin resistance, or overt type 2 diabetes have vascular dysfunction, making them more susceptible to vasospasm and potential myocardial infarction after exposure to combustion-related air pollution particles. We also propose that vascular status and exposure history are key sensitivity factors that regulate the vascular effects of combustion-related air pollution particles, mediated by their bioavailable constituents. To test these hypotheses, we examined the ability of soluble constituents of ROFA to alter vascular function in aortic rings from rats of the unique insulin-resistant atherosclerosis-prone JCR:LA-cp strain. We show the first experimental evidence that even a prediabetic state leads to a markedly enhanced susceptibility to cardiovascular events on exposure to particulate airborne pollutants.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rats.
Male rats, cp/cp and +/?, were bred and maintained in our established breeding colony of JCR:LA-cp animals at the University of Alberta as in previous studies (Russell et al., 1995
). The strain has recently been rederived and established at Charles River Laboratories Inc. (Wilmington, MA, USA) with the designation Crl:JCR(LA)-Leprcp.). Rats of the strain that are homozygous for the cp gene (cp/cp), are obese and insulin resistant, whereas those that are heterozygous (+/cp) or homozygous normal (+/+) are lean and metabolically normal. The lean rats, collectively designated as +/?, provide a metabolically and pathophysiologically normal control for the obese cp/cp rats. All care of the animals and experimental procedures were in conformity with the guidelines of the Canadian Council on Animal Care and were subject to prior institutional review and approval.
Vascular function.
At 12 weeks of age, rats were anesthetized with isofluorane in 100% oxygen. The chest was opened and the thoracic aorta was excised, trimmed of adhering fat and connective tissue, and cut into 3-mm-long transverse rings. Vascular function of the aortic rings with intact endothelium was studied using an established protocol (Mckendrick et al., 1998). Briefly, the aortic rings were mounted on stainless-steel hooks under 1.5-g resting tension in 10-ml organ baths, bathed at 37°C in Krebs-Henseleit solution, and gassed with 95% O2 and 5% CO2. Tension was recorded isometrically. The tissues were allowed to equilibrate for 45 min before experiments were begun, and during this time the resting tension was readjusted to 1.5 g, as required, and the tissues were washed every 15 min.
The contractile response of endothelium-intact rings of rat aorta to phenylephrine (PE) was measured through concentration-response curves (1 nM to 300 µM PE). The NO-mediated relaxation of the aortic rings was assessed by determining the concentration response to the NO-releasing agent acetylcholine (ACh) (1 nM–300 µM) of rings precontracted with PE to 80% maximal contraction. NG-nitro-L-arginine methyl ester (L–NAME) was added, on each cycle throughout the protocol, to the baths containing two of the eight rings studied from each rat at 10–4 M to inhibit NO synthase activity and to provide a direct measure of NO-mediated effects. The direct NO donor sodium nitroprusside (SNP) (1 nM to 10 µM) was used to determine the vascular smooth muscle response to NO.
Residual oil fly ash.
The ROFA, used in this study has previously been described (Dreher et al., 1997). Fugitive ROFA particles were collected by Southern Research Institute (Birmingham, AL) at a temperature of 204°C on a Teflon-coated glass fiber filter, downstream of the cyclone precipitator of a powerplant burning low sulfur number 6 residual oil. The particles were of respirable size, with a mass median aerodynamic diameter of 1.95 ± 0.18 µm. Washing with saline removed 94% of the initial dry weight, as well as a similar fraction of the metal content, the most abundant elements of which were Mg, Fe, V, and Ni. ROFA was suspended in sterile saline at a concentration of 10 mg ml–1. The suspension was mixed for 10 min at room temperature, centrifuged at 14,000 x g, and filtered through a 0.2-µm Teflon filter. The resulting ROFA leachates (ROFA-L) were used either the day of preparation or frozen overnight at –80°C. Final concentrations in the organ baths were based on the leachate from 1.56 to 12.5 µg of ROFA-L ml–1 of buffer.
Aortae Exposures
Initial Protocol
- Two rings from each rat were assigned to each of the four treatments, control, ROFA-L at 12.5 µg/ml–1, L–NAME, or ROFA-L plus L–NAME.
- Rings were allowed to equilibrate, at a baseline tension of 1.5 g, and the ROFA-L and/or L–NAME was added to the organ bath, after which force of contraction was monitored for 20 minutes
- The contractile dose response to PE was determined
- The rings were washed to remove all agents added and step 2 was repeated
- After monitoring, the rings were precontracted with the EC80 concentration of PE, as calculated for each ring
- The relaxant dose response to ACh was determined
- Steps 4 and 5 were repeated
- The dose response to SNP was measured
Second Protocol
- Four rings from each of five rats in each group were studied
- All rings were subjected to Steps 2–3 above without addition of ROFA-L or L–NAME
- After washing, all rings were precontracted with PE to 80% of maximum contractile response
- Half the rings were used for determination of relaxation response to ACh and half to SNP
- After washing, Steps 2 and 3 of the first protocol above were repeated, with the modification that rings were exposed to ROFA-L only, each ring at one of the concentrations of 1.56, 3.25, 6.26, and 12.5 µg ml–1
- Steps 4–8 above were repeated with only the ROFA-L exposure
Reagents and chemicals.
All reagents and chemicals were obtained from Sigma-Aldrich Canada (Oakville, ON, Canada).
Statistical analysis.
Results are expressed as mean ± SEM and were analyzed using the statistical package SigmaStat (Jandel Scientific, San Rafael, CA), and plotted using SigmaPlot (Jandel) and Prism (Graphpad, San Diego, CA). Data were compared using one-way analysis of variance (ANOVA) followed by multiple comparison tests. Concentration–response curves were analyzed with the program ALLFIT (De Lean et al., 1978), which fits the entire data set to the logistic equation and permits independent testing of differences between individual parameters. A value of p < 0.05 was taken as being statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The direct affect of ROFA-L on PE-mediated contractile force of aortic rings from both cp/cp and +/? rats is shown in Figure 1 and quantified in Table 1. Following exposure only to ROFA-L, aortic rings from cp/cp rats showed a mild baseline contraction and an upward shift of the dose–response curve for PE throughout the range. L-NAME (an inhibitor of nitric oxide synthase and thus of NO synthesis) caused a significant increase in the baseline and maximum contraction. The combination of ROFA-L and L-NAME exposure caused a significant and dramatic synergistic enhancement in ROFA-L baseline contraction and response to PE. Aortae from +/? rats showed lower contraction in response to PE, no baseline contraction in response to exposure to either ROFA-L or L-NAME alone, and less extreme increases in PE-mediated contractility in response to both ROFA-L and L-NAME. There were no significant differences in the EC50 for the response to PE between the treatments of the rings (Fig. 1; numerical data not shown).
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The maximum ACh-mediated vascular relaxation in cp/cp rat aortae is significantly reduced below that of the +/? aortae, as has been repeatedly reported. Exposure of the rings to ROFA-L significantly reduced the maximal relaxant response of both cp/cp and +/? aortae to ACh (Fig. 2 and Table 1), and the relaxation was essentially identical in the cp/cp and +/? rings. Addition of L-NAME completely abolished the relaxation, independently of the presence of ROFA-L, confirming the presence of endothelial function (Fig. 2 and Table 1). The dose response to SNP, as shown in Figure 3 and Table 1, did not differ between any of the experimental groups, indicating normal vascular smooth muscle response to NO and NO donors.
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Further studies were performed to identify whether vascular status and/or exposure history regulate sensitivity to ROFA-L–induced contraction in aorta of cp/cp rats. The baseline contractile response to ROFA-L was investigated in a separate experiment (see Second Protocol, Methods) using concentrations from 1.56 to 12.5 µg ml–1 and following multiple exposures to ROFA-L. On initial exposure, there was no significant contractile response to the ROFA-L (Fig. 4a). After determination of the PE dose response, washout of the solutions, and re-exposure of the aortic rings to the ROFA-L, there was a dramatic and highly significant (p < 0.0001) contraction of the cp/cp, but not of the +/? aortic rings (Fig. 4b). No dose–response relationship was observed, and the lowest and highest concentrations of ROFA-L generated similar contractile responses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The striking element of the response to ROFA-L is the baseline contraction, which is exacerbated on a second exposure and by the presence of L-NAME. This contraction, in the absence of the noradrenergic agent PE, only occurred in the aortic rings from the insulin-resistant cp/cp rats and caused the contraction of the vessels to be greater throughout the dose–response curve for PE. In contrast, the PE-mediated contraction of +/? aortic rings was increased only at the higher concentrations of PE, where the increase in contractile force induced by the ROFA-L was similar to that seen in cp/cp rings. This indicates that the ROFA-L increased the sensitivity of cp/cp aortae to low physiological concentrations of a noradrenergic agonist in a way not seen in the normal aorta. Even in the presence of 12.5 µg ml–1 of the ROFA-L, the maximum contraction of +/? aortic rings was less than that of cp/cp rings in the absence of ROFA-L. These differences represent a physiologically significant exacerbation of the vascular dysfunction of the cp/cp arteries. Vasospasm and associated thrombus formation represent an important component of the induction of fatal and nonfatal myocardial infarction and ischemic lesions in both the cp/cp rat (O'Brien et al., 1999
) and the human population (Sing et al., 2003). The ROFA-L–mediated increase in arterial contractility may be expected to exacerbate this process in individuals with insulin resistance and/or other underlying causes of NO deficiency such as eNOS polymorphisms (Philip et al., 1999; Fatini et al., 2004). Radomski et al. (2005) have reported that PM promote aggregation in normal human platelets and intra-arterial thrombus formation in normal healthy rats. Our data would suggest that the prediabetic hyperinsulinemic rat would show more striking effects on the thrombogenic system.
The data from this study do not address the cellular mechanisms underlying the observed adverse effects of ROFA-L. However, key elements of the processes involved are evident. The L-NAME–induced increases in contractile response of cp/cp rings to PE and baseline contraction are due to inhibition of the already impaired NO synthase activity and endothelial secretion of NO in the cp/cp rat. The hypercontractile behavior of the arteries is mediated, in part, through inhibition of NO release by the endothelial cells, indicated by the reduced response to ACh. We have previously reported that the impaired ACh-mediated vascular relaxation in the cp/cp rat is not affected by the cyclo-oxygenase inhibitor meclofenamate, indicating that eicosanoids are not the origin of the impaired relaxation and hypercontractility in this model (O'Brien et al., 1999). The synergistic effects of ROFA-L and L-NAME are consistent with action of an additional non-NO–mediated mechanism. This could involve release of the contractile agent endothelin or some other vascular smooth muscle cell contractile entity. The unimpaired relaxant response of the smooth muscle to the NO donor SNP indicates that the primary ROFA-L–mediated defect in the relaxation of the cp/cp artery does not lie in the medial smooth muscle per se.
The results are consistent with, and provide a mechanistic basis for, observational findings in other animal models and humans. The particle-free ROFA-L, represents bioavailable constituents, and the bath concentrations were equivalent to those in plasma of rats following pulmonary exposures to ROFA (Costa and Dreher, 1997; Kodavanti et al., 2003; Wellenius et al., 2002). The absence of a dose response in Figure 4 indicates that the concentrations used were in excess of those required to cause significant vascular effects in the cp/cp rat. The concentrations that the aortic rings were exposed to are not greater than those resulting from pulmonary exposures that have been shown to cause vascular effects in both humans and other animal models (Costa and Dreher, 1997; K. L. Dreher, unpublished observations; Goldberg et al., 2005; Wellenius et al., 2002). Concentrations of ROFA in this range have been shown to induce alterations in heart function in cardiac compromised metabolically normal rats (Costa and Dreher, 1997; Rivero et al., 2005). Among firefighters, coronary heart disease is responsible for 45% of on-duty deaths associated with stressful events (Kales et al., 2003). This effect is seen in firefighters, who are occupationally exposed to high levels of particulates, but not in police officers or EMS technicians, who are exposed to less severe conditions. Heart rate variability in humans, an index of cardiac autonomic function, has also been shown to be associated with PM2.5 exposure (Magari et al., 2002; Pristipino et al., 2000). Similarly, O'Neill et al. (2005) have shown an inverse relationship between ambient PM2.5 and both flow- and nitroglycerine-mediated vascular reactivity in people with type 2 diabetes. This unique observation provides a direct link between PM2.5 and the pathophysiology of CVD in humans. The authors emphasize, as we have here, the need for studies in appropriate animal models to isolate the pathways involved.
Consistent with the reports on human exposure, PM2.5 ROFA at 3.4 mg m–3, caused cardiac arrhythmias in Sprague-Dawley rats with a prior induced myocardial infarct (Wellenius et al., 2002). Similarly, Rivero et al. (2005) found that tracheal instillation of PM2.5 at 50 and 100 µg caused alterations in cardiac function in normal rats. Further, Kodavanti et al. (2003) have shown that pulmonary exposure of WKY rats to PM2.5 ROFA, at 10 mg m–3, resulted in myocardial lesions entirely similar to those that develop spontaneously in the cp/cp rats of the JCR:LA-cp strain (Richardson et al., 1998). These lesions are consistent with stage 2 and stage 4 lesions seen in the cp/cp rats, representing the early phase of cell lysis following ischemic damage and stable mature lesions after collagen deposition, respectively. Collectively, previous observations are consistent with the induction of vasospasm leading to focal myocardial ischemia and infarct following pulmonary deposition of ROFA. However, none of the studies to date has enabled identification of potential mechanisms leading to enhanced susceptibility to cardiovascular events on exposure to particulate contaminants in the diabetic subpopulation. Here we report, for the first time, that the vasculopathy of the prediabetic insulin-resistant state can lead to a dramatic exacerbation of the propensity to vasospasm on exposure to ROFA. We propose that a similar heterogeneity of sensitivity, due to an interaction between the metabolic syndrome (and early type 2 diabetes) and airborne particulate pollution, exists within the human population.
In conclusion, it is clear that bioavailable constituents of fine particulate air pollution derived from the combustion of heavy oil can alter vascular function and induce hyper-reactivity preferentially in aortae from prediabetic insulin-resistant rats. The direct contractile effect of ROFA-L is probably mediated through the vascular smooth muscle cells, which are known to be hyperactive (Absher et al., 1999; O'Brien and Russell, 1997; O'Brien et al., 1999) and may be due to transition metals (such as Fe, V, and Zn) contained in particles derived from heavy oil combustion (Dreher et al., 1997). In addition, bioavailable constituents of particles derived from the combustion of heavy oil can preferentially sensitize cp/cp rat arteries to noradrenergic agonists, and this may be expected to lead to hypercontractility in vivo in response to both hormonal and neurogenic stimuli. We emphasize that the effects of the bioavailable constituents of ROFA are not only influenced by the underlying status of the vascular system but also by the recent exposure history. These results support the argument of Sing and co-authors (2003), which proposes a complex multifactoral underlying basis for the etiology of coronary vascular disease (CVD). Finally, this is the first study to demonstrate enhanced vascular susceptibility to specific emission particles in a common disease state. The results provide confirmation of the epidemiological studies of O'Neill et al. (2005) and others that reported association between type 2 diabetes and enhanced risk for adverse cardiovascular events after exposure to fine particulate air pollution. There are large numbers of individuals with insulin-resistant status in the population, and there is evidence of subgroups with genetic and/or disease-related susceptibility to vascular dysfunction (Gupta et al., 2002). In this context, the demonstration of a direct link between the prediabetic insulin-resistant state and cardiovascular sensitivity to PM2.5 contributes to the growing weight of evidence that fine-particulate air pollution represents a significant public health risk. Our findings and the cp/cp rat model provide the basis for much more extensive studies to identify the underlying mechanisms and potential preventative treatments, as well as to provide guidance for public policy on airborne particulate pollution.
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ACKNOWLEDGMENTS |
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S. D. Proctor was supported by the National Health and Medical Research Council of Australia. The information described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policy of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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