http://informahealthcare.com/iht ISSN: 0895-8378 (print), 1091-7691 (electronic) Inhal Toxicol, 2013; 25(13): 725–734 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2013.844749
RESEARCH ARTICLE
Systemic and vascular effects of circulating diesel exhaust particulate matter Ni Bai and Stephan F. van Eeden
Abstract
Keywords
Objective: Numerous studies have found an association between transiently increased particulate matter air pollution and acute adverse cardiovascular health effects; however, the mechanisms underlying these effects are not clear. Translocation of ultra-fine ambient particulate matter has been proposed to play a key role in these acute side effects. This study was designed to determine the contribution of circulating (translocated) diesel exhaust particles (DEPs) to the systemic and vascular effects. Methods: C57 mice (10-week) received intravenous DEPs via tail vein injection. Following 1-h post-injection, inflammatory cytokines (IL-1b, IL-6 and TNF-a), peripheral blood cell counts, band cell counts, aortic endothelial function and vascular constriction were assessed. Thoracic aortae were isolated, and endothelial function was examined by measuring acetylcholine (ACh) and sodium nitroprusside (SNP)-stimulated vascular relaxation using a wire myograph. In addition, phenylephrine (PE)-stimulated vasoconstriction was also measured. The amount of DEPs deposited and trapped in tissues (the spleen, liver, lungs and heart) were quantified. Results: Acute systemic DEP exposure caused a significant increase in TNF-a, peripheral neutrophil and band cell counts. ACh and SNP-induced relaxation were not affected by acute systemic DEP exposure, neither was PE-stimulated constriction. There was a significantly increased DEP deposition in the spleen as well as in the liver. No significantly increased DEPs were detected in the lung and heart. Conclusion: Here we show that circulating DEPs induce a systemic response characterized by increased TNF-a, peripheral granulocytes, but does not impact endothelial function. Our study also suggests that circulating particles are rapidly removed from the circulation and predominantly sequestered in the spleen and liver.
Diesel exhaust particle, endothelial function, systemic reaction, tissue deposition
Introduction It has been well established that chronic elevation of particulate matter air pollution (PM10) levels are strongly associated with increases in cardiovascular morbidity and mortality, attributable to atherosclerosis, dysrhythmias, ischemic heart disease and stroke (Bai et al., 2007; Brook et al., 2010; Samet et al., 2000; Schwartz, 1999). In addition, evidence from time-series analysis conducted worldwide also indicates the existence of a small, yet consistent association between increased cardiovascular mortality and short-term (1–5 days) elevation of PM10 prior to the cardiovascular events. Having monitored PM10 levels for 300 000 elderly residents in 21 US cities between 1985 and 1999, Burnett and colleagues reported that a 10 mg/m3 increase of PM10 was associated with a significantly increased risk of
Address for correspondence: Dr Stephan van Eeden, MD, PhD, The James Hogg Research Centre, Providence Heart and Lung Institute, St. Paul’s Hospital, University of British Columbia, 1081 Burrard Street, Vancouver, BC V6Z1Y6, Canada. Tel: 1-604-806-8346. Fax: 1-604-8068351. E-mail: [email protected]
History Received 6 June 2013 Revised 4 September 2013 Accepted 10 September 2013 Published online 20 November 2013
hospitalization for myocardial infarction (MI), which was occurred within a few hours after exposure (Burnett et al., 1999; Seaton et al., 1995; Zanobetti & Schwartz, 2005). Another human study showed that hourly changes in PM2.5 concentrations were associated with transiently increased risk of ST segment depression (Lanki et al., 2008). To explain the relative short interval between increases in PM exposure and acute cardiovascular events, Godleski et al. speculated that ultra-fine particles (UFPs, particles with diameter less than 100 nm) were able to enter the blood stream, hence adversely affected the heart and initiated arrhythmias, which could lead to sudden death (Godleski et al., 2000). This hypothesis received its first direct support from a study conducted by Nemmar et al. (2001). Their study demonstrated that inhaled 99mTechnetium-labeled ultra-fine carbon particles were detected in blood one minute after exposure, and remained at peak levels for periods of up to 60 min. These data suggest that inhaled UFPs translocate from the lung directly into the circulation (Choi et al., 2010; Nemmar et al., 2001, 2002; Oberdo¨rster et al., 2002; Peters et al., 2006). Subsequently, several intriguing putative passages by which inhaled UFPs travel from the lung into the circulation and gain access to
20 13
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
The James Hogg Research Centre, Providence Heart and Lung Institute, St. Paul’s Hospital, University of British Columbia, Vancouver, BC, Canada
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
726
N. Bai & S. F. van Eeden
other organs have been proposed. These proposed loci include the fenestrae of sinusoidal endothelial cells (Khandoga et al., 2004), the lung-blood barrier (Hermans & Bernard, 1999), and the alveolar-capillary barrier (Gumbleton, 2001; Shimada et al., 2006). Extra-pulmonary translocation of UFPs provides a plausible explanation for the short interval between exposure to ambient particles and acute cardiovascular events. With the large increase in vehicle traffic, diesel exhaust particles (DEPs) become one of the major components of urban UFPs. Most of DEPs emitted by engines are nano-sized particles with a diameter less than 50 nm (Kittelson, 1998). UFPs deposit in greater numbers and deeply into the lungs, than larger particles do. Due to their larger surface area, compared with bigger particles, DEPs also have greater potential for interactions with biological targets, resulting in a greater inflammatory response (Nemmar et al., 2007; Oberdo¨rster et al., 1996). Both animal and human studies have shown that exposure to DEPs induce endothelial dysfunction (Hansen et al., 2007; Ikeda et al., 1995; Mills et al., 2007; O’Neill et al., 2005). The endothelium serves as an important regulator to maintain vascular tone and vasculature integrity. It does so by producing a number of mediators, in particular, nitric oxide (NO). Endothelium-derived NO protects blood vessels from developing cardiovascular events by suppressing vasoconstriction, attenuating monocyte chemotaxis, preventing the adherence of leukocytes to the endothelium, inhibiting platelet adherence and aggregation. A hallmark of endothelial dysfunction is impairment of acetylcholine (ACh)-stimulated vasorelaxation due to decreased NO production. Endothelial dysfunction is implicated in the pathogenesis of many cardiovascular diseases, including acute coronary syndromes, MI and stroke (Brook et al., 2010; Campen et al., 2005; Vermylen et al., 2005). Studies from others’ and our laboratories have demonstrated that DEPs cause endothelial dysfunction via various mechanisms, including disrupting endothelial vasodilation and endogenous fibrinolysis through reactive oxygen species (Mills et al., 2005), inhibiting endothelial nitric oxide synthase activity, and preventing the release of the vasodilator nitric oxide (Muto et al., 1996). In addition, DEPs and UFPs promote platelet activation and clot formation (Khandoga et al., 2004; Nemmar et al., 2003). Endothelial dysfunction and thrombosis can be the underlying mechanisms attributable to the association between exposure to high levels of PM and the increased incidence of acute cardiovascular events, such as MI (Tofler & Muller, 2006). The secondary inflammatory effect as a result of UFP deposition in the lungs has been well established; however, the direct vascular effects derived from translocated UFPs are not clear. The aim of the present study is to investigate the impact of circulating DEPs (mimic extra-pulmonary translocation) on aortic endothelial function. We hypothesize that circulating DEPs cause direct systemic response, which could lead to endothelial dysfunction. To test our hypothesis, DEPs were injected into the circulation via mouse tail vein. After 1-h post-injection, peripheral blood cell counts, band cell counts and aortic endothelial function were examined. In addition, the amount of DEPs deposited in the tissues (spleen, liver, lung and heart) was assessed.
Inhal Toxicol, 2013; 25(13): 725–734
Materials and methods Diesel exhaust particles preparation Diesel exhaust particles (DEP) (SRM2975) were purchased from US National Institute of Standards and Technology. DEP suspension was sonicated before injection to ensure even distribution and prevent aggregation. Experimental animals All protocols were designed according to the guidelines of the Animal Care Committee of the University of British Columbia. C57 mice were purchased from Jackson Lab (Bar Harbor, ME). These mice were housed in a temperature- and humidity-controlled environment with a 12-h light/dark cycle and free access to water and standard rodent chow. All animals were 10-week-old at the start of the experimental protocol. DEP tail vein injection and sample collections C57 mice were received a tail vein injection of DEP at high dose (0.5 mg/g of body weight) or low dose (0.25 mg/g of body weight) (7.5 or 15 mg DEP suspended in 200 ml saline), and sacrificed at 1-h post-injection. The dose was calculated by assuming a 3-day exposure at 125 (DEP-low) or 250 mg/m3 (DEP-high), and 2% of DEP was translocated to the blood stream. 200 ml saline injection was used as the control. At 1-h post-injection, mice were anesthetized with 10% isoflurane. Upon the loss of all reflexes, blood was collected from inferior vena cava and put into EDTA tubes for cell counts. The thoracic aorta was carefully dissected for vascular function study. The heart, liver, spleen and inflated lung were flash frozen and kept at 80 C. Plasma cytokines Plasma inflammatory factors (IL-1b, IL-6, TNF-a) were measured using Fluorokine Multianalyte Profiling Kits for the Luminex (R&D Systems, Minneapolis, MN) following manufacturer’s instruction. Peripheral differential cell counts White blood cell (WBC) counts and differential leukocyte counts were analyzed using an Abbott Diagnostics Cell-Dyn 3700 instrument (Abbott Park, IL) that was calibrated for analysis of mouse blood cell counts. Peripheral band cell counts Blood smears were fixed with methanol, stained with Wright staining (Sigma Chemicals, St. Louis, MO), coded and examined without knowledge of the experimental groups. A hundred polymorphonuclear leukocytes (PMNs) from randomly selected fields were manually counted and the percentage of band cells was determined. Quantification of DEP in tissue Cryosections (5 mm) of flash frozen heart, liver, spleen and inflated lung were obtained and fixed with cold (4 C) acetone. Blocks were sectioned at 5 mm and stained with hematoxylin and eosin (H&E). Images were captured by a spot digital camera (Microspot, Nikon, Tokyo, Japan), coded
Intravenous DEP exposure and systemic effects
DOI: 10.3109/08958378.2013.844749
and examined without knowledge of the experimental groups. These images were analyzed using a point counting method similar to previously described in more detail (Ling et al., 2011). Briefly, a grid of points was superimposed onto the images captured by Image Pro Plus software (Rockville, MD). The density of the grid and the number of fields were selected to maintain the coefficient of variation of the estimate of the volume below 0.1. The points that contacted with tissue and aggregated DEP were counted. The volume fraction (V/V%) of DEP was obtained by normalizing the total points on aggregated DEP to the total points on tissues.
727
Figure 1. Significantly increased TNF-_ after high dose DEP exposure. *p50.05.Values are expressed by mean SEM (n ¼ 8).
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Vascular function study Thoracic aortae were carefully cleaned off connective tissue without damaging the endothelium. The vessels were then cut to 2 mm rings and mounted on a wire myograph (Model 610 M; Danish Myo Technology, Denmark). Each vessel was bathed in oxygenated physiological salt solution (PSS) at 37 C for an hour during which the resting tension was gradually increased to 6 mN with three changes of PSS at 10-min intervals followed by stabilizing the vessels at resting tension (6 mN) for 30 min. Thereafter, the vessels were stimulated with 80 mM KCl twice. Vessels were preconstricted with phenylephrine (PE) (1 mM) to achieve a sustained vasoconstriction; thereafter, cumulative concentrations of acetylcholine (ACh; 1 nM–10 mM) and sodium nitroprusside (SNP; 1 nM–10 mM) were applied to evaluate endothelium-dependent or -independent NO-mediated relaxation, respectively. Thereafter, with the same vessel, smooth muscle contractility was assessed by administration of cumulative concentrations of PE (1 nM–10 mM). Furthermore, we also evaluated the effects of ex vivo DEP exposure to isolated mouse thoracic aortae (control nonexposed mice) using a wire myograph. We administrated sonicated DEP suspension directly into the chambers of the wire myograph. Endothelial function was assessed preceded 1 h exposure to DEP at three concentrations (10, 50, 100 mg/ml), respectively. Solutions and chemicals The PSS consisted of the following (in mM): NaCl 119, KCl 4.7, KH2PO4 1.18, NaHCO3 24, MgSO47H2O 1.17, CaCl2 1.6, glucose 5.5 and EDTA 0.026. All reagents were purchased from Sigma (St. Louis, MO). Statistical analysis Results are reported as mean SEM. The statistical significance was evaluated using the unpaired Student t test for simple comparison between two values. The concentration– response curves of the different groups were compared by ANOVA for repeated measurements followed by Bonferroni’s correction. p50.05 was considered to be significant. In all experiments, n equals the number of mice from which samples were obtained.
Results Plasma cytokine levels We observed significantly increased TNF-a level in high DEP exposure group (1.15 0.22 pg/ml versus 1.12 0.19 pg/ml
(A)
(B)
Figure 2. Systemic reaction. (A) WBC counts were not affected by systemic DEP exposure; (B) there was a significant increase in neutrophils after DEP exposure. *p50.05 (control versus low DEP); #p50.01(control versus high DEP). Values are expressed by mean SEM (n ¼ 6–8).
versus 2.13 0.54 pg/ml; control versus DEP low versus DEP high, control versus DEP high, p50.05, Figure 1). IL-1b and IL-6 levels were under detectable levels (the sensitivities of Fluorokine Multianalyte Profiling Kit for cytokines are: IL-1b: 7.82 pg/ml, IL-6: 1.06 pg/ml). Peripheral WBC and band cell counts There were no significant changes in circulating total WBC after either low or high dose of DEP (2.56 0.71 109 versus 3.13 0.23 109 versus 2.35 0.80 109 cells/l; control versus DEP low versus DEP high, p40.05, Figure 2A). Except a significant dose-dependent increase in neutrophil (Figure 2B) and monocytes (DEP low versus DEP high, p50.001), no other significant changes were noted in peripheral cell counts (Table 1). Moreover, we found that the band cell counts were significantly increased in a dosedependent fashion (Figure 3) (EC50 and Emax values are available in the supplement).
728
N. Bai & S. F. van Eeden
Inhal Toxicol, 2013; 25(13): 725–734
Table 1. Differential leukocyte counts were analyzed, and data are expressed by mean SEM (n ¼ 6–8). There are significantly increased PMN counts.
Control DEP-low DEP-high
NEU (% of total WBC)
LYM (% of total WBC)
MONO (% of total WBC)
EOS (% of total WBC)
BASO (% of total WBC)
Platelet (109/l)
7.19 1.87a,b 14.4 1.29 33.70 1.90c
85.5 1.10 83.95 1.20 74.53 7.28
3.10 0.89 1.14 0.12 0.10 0.06d
0.04 0.03 0.06 0.03 0.10 0.07
4.14 1.95 0.43 0.10 13.26 7.43
593.5 136.3 685.2 58.6 598.0 129.3
a
Control versus DEP low: p50.01; bControl versus DEP high: p50.05; cDEP low versus DEP high: p50.05; dDEP low versus DEP high: p50.001.
DEP exposure (Figure 9B). No significant difference of DEP deposition in the lung and heart was found albeit a tend of increase was observed in exposure groups (Figure 9C and D).
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Discussion
Figure 3. Increased band cells after acute systemic DEP exposure. *p50.05; #p50.02. Values are expressed by mean SEM (n ¼ 8).
Vascular function study following intravenous DEP exposure Endothelial function and vasocontractility were assessed at 1h post-injection to evaluate the impact of acute systemic DEP exposure. Neither endothelium-dependent nor independent relaxation was affected by DEP exposure (Figure 4). Similarly, there was no alteration of PE-elicited vasoconstriction (Figure 5). (EC50 and Emax values are available in the supplement). Vascular function study following ex vivo DEP exposure To study the effect of direct DEP exposure in isolated mouse aorta, three different concentrations (10, 50, 100 mg/ml) of DEP suspensions were added into the chambers. After 1-h incubation of DEPs with isolated thoracic aortae, ACh relaxation was assessed. We found that higher concentrations (50 mg/ml and 100 mg/ml) of DEPs attenuated ACh relaxation in a dose-dependent fashion (Figure 6B, C). In addition, DEP exposure did not impair SNP-induced endothelium-independent relaxation (Figure 6D), suggesting DEP exposure caused endothelial dysfunction. PE-elicited vasoconstriction was not affected by DEP (Figure 7). Evaluation of DEP deposition in tissues DEP deposition was significantly increased in the spleen at both low and high DEP exposure groups (Figure 9A). This deposition was significantly greater in liver at only high-dose
Numerous evidence has shown an association between acute PM10 exposure and cardiovascular events and exacerbation (Bhaskaran et al., 2009; Brook et al., 2010; Mustafic et al., 2012; Wichmann et al., 2013), but the mechanisms how transiently increases in ambient particles lead to increased cardiovascular morbidity and mortality is still unclear. One hypothesis is that acute cardiovascular events are resulted from the direct effects of UFPs that translocate from the lung into circulation. Extra-pulmonary translocation of UFPs has been reported by different laboratories (Godleski et al., 2000; Nemmar, 2001; Oberdo¨rster et al., 2004; Shimada et al., 2006), but the significance of these circulating particles on vascular function is unclear. In this study, we injected DEPs into mouse tail vein to mimic extra-pulmonary translocation. DEPs include a subgroup with a large number of UFPs, and approximately 50–90% of the particles in diesel exhaust are in the ultrafine size range with the majority of diesel particles ranging in size from 5 to 50 nm (Kittelson, 1998; Nemmar et al., 2007). These physical characteristics of DEPs offer them the potential to translocate across the gas exchange barrier into the circulation. We used two different doses of DEPs to simulate relative high and low exposure to DEPs assuming 2% of particle translocate into the circulation. This assumption was based on studies by Nemmar and colleagues showing 2% of intratracheal 99mTc-labeled ultrafine carbon particles translocate into the bloodstream (Nemmar et al., 2001). Nemmar and colleagues reported that inhaled UFPs translocated into the circulation rapidly with peak levels up to 60 min after exposure (Nemmar et al., 2001, 2002). In addition, Hansen et al. showed that ACh-induced relaxation was impaired 1 h after injecting DEPs in the peritoneum to ApoE mice (Hansen et al., 2007). A human study found that hourly changes in PM2.5 or outdoor particle concentrations were associated with rapid ischemic responses (Lanki et al., 2008). Based on these studies, we chose to test at 1-h postinjection of DEPs. Some studies also reported that cardiovascular effects were occurred few hours after acute exposure, indicating a potential lag effects which cannot be excluded in this study and need further investigation. We found that circulating DEPs induced a systemic reaction, characterized by increased TNF-a (Figure 1), elevated circulating neutrophils (Figure 2B) and band cells (Figure 3). Increased band cell counts suggest that the
Intravenous DEP exposure and systemic effects
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
DOI: 10.3109/08958378.2013.844749
(A)
(B)
(C)
(D)
729
Figure 4. Concentration-response curves of ACh-induced endothelium-dependent and SNP-induced endothelium-independent relaxation. There was no changes after acute DEP exposure at either low (A, B) or high dose (C, D). Values are expressed by mean SEM (n ¼ 6).
Figure 5. Concentration–response curves of PE-induced vasoconstriction. There were no alterations following acute DEP exposure at either low (A) or high dose (B). Values are expressed by mean SEM (n ¼ 6).
(A)
neutrophilia is not just demargination of intravascular neutrophils due to stress or cathecholamine effect, but due to stimulation of the bone marrow to release neutrophils (Mukae et al., 2001; Terashima et al., 1997). Our previous studies have shown that this stimulation of the marrow is usually caused by elevated circulation pro-inflammatory mediators (such as IL-6, IL-1b, TNF-a, IL-8) or growth factors (such as GM-CSF and G-CSF) (Ishii et al., 2004; van Eeden et al., 2001). This study suggest that TNF-a plays a key role in the rapid systemic response following translocation of UFP, possibly via bone marrow stimulation. TNF-a could be released from circulating monocytes or tissues macrophages/ hystiocytes (in the liver and spleen) that clear these particles from the circulation. It is well known that inflammatory mediators (such as IL-1b, IL-6, TNF-a and GM-CSF) are able to activate
(B)
vascular endothelium (Bussolino et al., 1989; Kido et al., 2011; Tamagawa et al., 2008). In this study, we hypothesized that intravascular DEP exposure caused systemic inflammation, hence leading to endothelial dysfunction. Endothelial function was tested with ACh (Figure 4), and vasoconstrictive function was also examined by PE (Figure 5). We observed no alteration following DEP exposure. These results are in agreement with another study showing that exposure of rats to DEPs induced both pulmonary and systemic inflammation, but did not modify endothelium-dependent vasodilatation (Robertson et al., 2012). Khandoga et al. showed that intra-arterial UFP exposure to C57 mice increased platelet accumulation and prothrombotic changes in the hepatic microvasculature without inflammatory reactions (Khandoga et al., 2004). This result implies that UFP-induced side effects may be caused directly by
730
N. Bai & S. F. van Eeden (A)
Inhal Toxicol, 2013; 25(13): 725–734
(B)
(C)
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
(D)
Figure 6. Concentration–response curves of ACh-induced vasorelaxation after ex vivo DEP exposure. There was no impaired endothelial function at low (10 mg/ml) concentration (A), but exposure to DEP at higher (50 and 100 mg/ml) concentrations caused endothelial dysfunction (B, C). There was no impairment of SNP-induced endothelium-independent vasorelaxation (D). Values are expressed by mean SEM (n ¼ 6); *p50.05.
(B)
(A)
(C)
Figure 7. Concentration–response curves of PE-induced vasoconstriction after ex vivo DEP exposure. Different concentrations of DEP exposure did not affect aortic constriction (A, B, C). Values are expressed by mean SEM (n ¼ 6).
Intravenous DEP exposure and systemic effects
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
DOI: 10.3109/08958378.2013.844749
(A)
(B)
(C)
(D)
731
Figure 8. Representative photomicrographs of DEP deposition in tissues. (A) DEP aggregates are shown in the spleen; (B) DEPs are shown in the cytoplasm of hepatic cells; (C) DEPs are shown in the cytoplasm of macrophages in the lung; (D) DEP aggregates are shown in the heart. Arrows point to the deposition of DEP aggregates. Magnification: 1000X. Scale bar ¼ 50 mm..
circulating UFPs themselves. To examine the direct effect of circulating DEPs on blood vessels, we exposed similar doses of DEPs to isolated mouse aortae that were not exposed to DEPs. We found that ex vivo DEP exposure caused endothelial dysfunction in a dose-dependent manner (Figure 6A, B, C). That SNP response was not altered (Figure 6D) confirmed that this effect was endothelial dependent. The difference between in vivo and ex vivo responses of blood vessels suggests that circulating DEPs were rapidly cleared from the circulation before they could significantly activate endothelium. To support this concept, we examined DEP deposition in tissues (Figure 8) and show the significant dose-dependent increases in DEP deposition in the spleen (Figure 9A) as well as in the liver after high-dose DEP exposure (Figure 9B). There was also a trend of an increase in particles in the lung and heart (Figure 9C, D). We suspect that endothelial function was not affected by the in vivo exposure to DEP, at least in part, because the majority of DEPs were sieved out and removed from the circulating blood stream by the spleen and liver. In addition, it has been demonstrated that red blood cells can interact and take up DEPs, and this interaction between DEPs and red blood cells may also contribute to increased DEP deposition in spleen (Nemmar & Inuwa, 2008).
The present study is the first to evaluate the amount DEPs trapped and deposited in tissues following acute systemic DEP administration. We demonstrate that DEP aggregates deposit in the spleen after both low and high dose of DEP exposure (Figure 9A), whereas the amount of DEPs significantly increase in the liver only in high DEP group (Figure 9B). These data indicate that the spleen is the primary organ responsible for removing DEPs out of the circulation, and the liver plays an important role when increasing DEPs are in the circulation. Although there are also an increased amount of DEPs deposited in the lung and heart, it is not significantly different from the control (Figure 9C, D). Our data are different from studies conducted by Nemmar et al. (2007), who observed lung inflammation and increased cellular influx in the lung following injection of particles, an effect that could be dose and particle composition dependent. Other study reported that liver and kidneys also served as major organs to clear-off these ambient particles (Nakane, 2012). Since the notion of UFP translocation was proposed a decade ago, many studies have been conducted and reported the possibilities of UFP translocation to the systemic circulation and other organs, including liver, spleen, kidney, heart
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
732
N. Bai & S. F. van Eeden
Inhal Toxicol, 2013; 25(13): 725–734
(A)
(B)
(C)
(D)
Figure 9. DEP deposition in tissues. There were significantly increased DEP deposition in the spleen (A) and liver (B). There was a trend increase in DEP deposition in the lung (C) and the heart (D). *p50.02 (control versus low DEP); #p50.001 (control versus high DEP); **p50.01(control versus high DEP). Values are expressed by mean SEM (n ¼ 8).
or brain (Nemmar et al., 2001; Oberdo¨rster et al., 2004), lymph nodes and bloodstream (Choi et al., 2010; Nemmar et al., 2001, 2002). Nevertheless, this concept is still very controversial (Kreyling et al., 2002; Mills et al., 2006; Mo¨ller et al., 2008), mainly due to technical challenges to trace and quantify translocation. Some of these limitations are also applicable to this study. Because of the size of DEPs, it is difficult to measure or trace small amount of these particles in tissues. In addition, based on previous studies (Kittelson, 1998; Nemmar et al., 2007), more than 50% of DEPs in number are UFPs; therefore, we suspect that a significant number of DEPs seen in the spleen are UFPs, but overall they are likely a mixture of coarse and ultra-fine particles. Moreover, constituents attached to particles may come off during the translocation process. For example, Mills et al. showed no translocation of insoluble, radio-labeled ultrafine carbon particles from the lung into the bloodstream of humans (Mills et al., 2006). They believed that the small amount of radioactivity found in the bloodstream and other organs was attributed entirely to the small amount of leached, soluble radiolabel from the particles. Therefore, quantifying the amount of translocated particles has remained one of the biggest challenges for most researchers. We used stereological methods that are considered as gold standard for obtaining quantitative data on structure at microscopic levels (Weibel et al., 2007). However, circulating DEPs could be internalized into platelets by endocytosis (Chen et al., 2006) and/or leukocytes such as neutrophils and monocytes (Miller et al., 2006). Therefore, we are not able to exclude that certain portion of DEPs is still in the circulation. The vein injection of DEPs may not be the ideal approach to mimic potential UFP translocation from lungs to the circulation, but we think that tail vein injection serves as both
a convenient and reasonable approach to investigate the mechanisms of translocated particles. Similar approach has been used by other studies to investigate direct effects of particles following intravascular administration (Khandoga et al., 2004; Silva et al., 2005). We found the bulk of particles in the spleen suggesting that the majority of particles reached the systemic circulation and were not trapped in the liver following the venous injection. Lastly, the concentrations of DEPs used in this study are higher than EPA standard and most average levels in major North American cities. We selected these concentrations to be able to compare to previous studies and to maximize chances of detecting alterations in vascular function. However, exposure to this high concentration of PM2.5 (4500ug/m3) has recently become realistic in some developing countries, such as China. It is quite possible that at certain times (e.g. rush hours) such high level PM2.5 can be reached along busy routes. In summary, our study demonstrates that circulating DEPs induce a systemic inflammatory response characterized by increased TNF-a, neutrophilia and bone marrow stimulation (increase band cell counts), but do not cause endothelial dysfunction or abnormal vasoconstriction. We speculate that DEPs translocated into the circulation following inhalation are rapidly removed from the circulation by the spleen and liver. As a result, these circulating particles do not significantly contribute to the early vascular dysfunction associated with exposure to ambient particulate matter. Although there is solid evidence for extra-pulmonary translocation of UFPs, this translocation mechanism underlies adverse vascular effects of combustion-derived UFP exposures remains considerably uncertain.
DOI: 10.3109/08958378.2013.844749
Acknowledgements Authors would like to thank David Ngan for measuring inflammatory cytokines for this study.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
References Bai N, Kido T, Kavanagh TJ, et al. (2011). Exposure to diesel exhaust up-regulates iNOS expression in ApoE knockout mice. Toxicol Appl Pharmacol 255:184–92. Bai N, Khazaei M, van Eeden SF, Laher I. (2007). The pharmacology of particulate matter air pollution-induced cardiovascular dysfunction. Pharmacol Ther 113:16–29. Bhaskaran K, Hajat S, Haines A, et al. (2009). Effects of air pollution on the incidence of myocardial infarction. Heart 95:1746–59. Brook RD, Rajagopalan S, Pope III CA, et al. (2010). Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation 121: 2331–78. Burnett RT, Smith-Doiron M, Stieb D, et al. (1999). Effects of particulate and gaseous air pollution on cardiorespiratory hospitalizations. Arch Environ Health 54:130–9. Bussolino F, Wang JM, Defilippi P, et al. (1989). Granulocyte- and granulocyte-macrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate. Nature 337:471–3. Campen MJ, Babu NS, Helms GA, et al. (2005). Nonparticulate components of diesel exhaust promote constriction in coronary arteries from apoE/ mice. Toxicol Sci 88:95–102. Chen J, Tan M, Nemmar A, et al. (2006). Quantification of extrapulmonary translocation of intratracheal-instilled particles in vivo in rats: effect of lipopolysaccharide. Toxicology 222:195–201. Choi HS, Ashitate Y, Lee JH, et al. (2010). Rapid translocation of nanoparticles from the lung airspaces to the body. Nat Biotechnol 28: 1300–3. Godleski JJ, Verrier RL, Koutrakis P, et al. (2000). Mechanisms of morbidity and mortality from exposure to ambient air particles. Res Rep Health Eff Inst 91:5–88. Gumbleton M. (2001). Caveolae as potential macromolecule trafficking compartments within alveolar epithelium. Adv Drug Deliv Rev 49: 281–300. Hansen CS, Sheykhzade M, Møller P, et al. (2007). Diesel exhaust particles induce endothelial dysfunction in apoE/ mice. Toxicol Appl Pharmacol 219:24–32. Hermans C, Bernard A. (1999). Lung epithelium-specific proteins: characteristics and potential applications as markers. Am J Respir Crit Care Med 159:646–78. Ikeda M, Suzuki M, Watarai K, et al. (1995). Impairment of endothelium-dependent relaxation by diesel exhaust particles in rat thoracic aorta. Jpn J Pharmacol 68:183–9. Ishii H, Fujii T, Hogg JC, et al. (2004). Contribution of IL-1 beta and TNF-alpha to the initiation of the peripheral lung response to atmospheric particulates (PM10). Am J Physiol Lung Cell Mol Physiol 287:L176–83. Khandoga A, Stampfl A, Takenaka S, et al. (2004). Ultrafine particles exert prothrombotic but not inflammatory effects on the hepatic microcirculation in healthy mice in vivo. Circulation 109:1320–5. Kido T, Tamagawa E, Bai N, et al. (2011). Particulate matter induces translocation of IL-6 from the lung to the systemic circulation. Am J Respir Cell Mol Biol 44:197–204. Kittelson DB. (1998). Engines and nanoparticles: a review. J Aerosol Sci 29:575–88. Kreyling WG, Semmler M, Erbe F, et al. (2002). Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low. J Toxicol Environ Health A 65:1513–30. Lanki T, Hoek G, Timonen KL, et al. (2008). Hourly variation in fine particle exposure is associated with transiently increased risk of ST segment depression. Occup Environ Med 65:782–6.
Intravenous DEP exposure and systemic effects
733
Ling SH, McDonough JE, Gosselink JV, et al. (2011). Patterns of retention of particulate matter in lung tissues of patients with COPD: potential role in disease progression. Chest 140:1540–9. Mills NL, Amin N, Robinson SD, et al. (2006). Do inhaled carbon nanoparticles translocate directly into the circulation in humans? Am J Respir Crit Care Med 173:426–31. Mills NL, Tornqvist H, Gonzalez MC, et al. (2007). Ischemic and thrombotic effects of dilute diesel-exhaust inhalation in men with coronary heart disease. N Engl J Med 357:1075–82. Mills NL, Tornqvist H, Robinson SD, et al. (2005). Diesel exhaust inhalation causes vascular dysfunction and impaired endogenous fibrinolysis. Circulation 112:3930–6. Mo¨ller W, Felten K, Sommerer K, et al. (2008). Deposition, retention, and translocation of ultrafine particles from the central airways and lung periphery. Am J Respir Crit Care Med 177:426–32. Mukae H, Vincent R, Quinlan K, et al. (2001). The effect of repeated exposure to particulate air pollution (PM10) on the bone marrow. Am J Respir Crit Care Med 163:201–19. Mustafic H, Jabre P, Caussin C, et al. (2012). Main air pollutants and myocardial infarction: a systematic review and meta-analysis. JAMA 307:713–21. Muto E, Hayashi T, Yamada K, et al. (1996). Endothelial-constitutive nitric oxide synthase exists in airways and diesel exhaust particles inhibit the effect of nitric oxide. Life Sci 59:1563–70. Nakane H. (2012). Translocation of particles deposited in the respiratory system: a systematic review and statistical analysis. Environ Health Prev Med 17:263–74. Nemmar A, Al-Maskari S, Ali BH, Al-Amri IS. (2007). Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats. Am J Physiol Lung Cell Mol Physiol 292:L664–70. Nemmar A, Hoet PH, Dinsdale D, et al. (2003). Diesel exhaust particles in lung acutely enhance experimental peripheral thrombosis. Circulation 107:1202–8. Nemmar A, Hoet PH, Vanquickenborne B, et al. (2002). Passage of inhaled particles into the blood circulation in humans. Circulation 105:411–14. Nemmar A, Inuwa IM. (2008). Diesel exhaust particles in blood trigger systemic and pulmonary morphological alterations. Toxicol Lett 176: 20–30. Nemmar A, Nemery B, Hoet PH, et al. (2003b). Pulmonary inflammation and thrombogenicity caused by diesel particles in hamsters: role of histamine. Am J Respir Crit Care Med 168:1366–72. Nemmar A, Vanbilloen H, Hoylaerts MF, et al. (2001). Passage of intratracheally instilled ultrafine particles from the lung into the systemic circulation in hamster. Am J Respir Crit Care Med 164: 1665–8. Oberdorster G. (1996). Significance of particle parameters in the evaluation of exposure-dose-response relationships of inhaled particles. Inhal Toxicol. 8 Suppl: 73–89 (Review). Oberdo¨rster G, Sharp Z, Atudorei V, et al. (2002). Extrapulmonary translocation of ultrafine carbon particle following whole-body inhalation exposure of rats. J Toxicol Environ Health A 65:1531–43. Oberdo¨rster G, Sharp Z, Atudorei V, et al. (2004). Translocation of inhaled ultrafine particles to the brain. Inhal Toxicol 16:437–45. O’Neill MS, Veves A, Zanobetti A, et al. (2005). Diabetes enhances vulnerability to particulate air pollution-associated impairment in vascular reactivity and endothelial function. Circulation 111: 2913–20. Peters A, Veronesi B, Caldero´n-Garciuden˜as L, et al. (2006). Translocation and potential neurological effects of fine and ultrafine particles a critical update. Part Fiber Toxicol 3:13. Robertson S, Gray GA, Duffin R, et al. (2012). Diesel exhaust particulate induces pulmonary and systemic inflammation in rats without impairing endothelial function ex vivo or in vivo. Part Fibre Toxicol 9:9. Samet JM, Dominici F, Curriero FC, et al. (2000). Fine particulate air pollution and mortality in 20 U.S. cities, 1987–1994. N Engl J Med 343:1742–9. Schwartz J. (1999). Air pollution and hospital admissions for heart disease in eight U.S. counties. Epidemiology 10:17–22. Seaton A, MacNee W, Donaldson K, Godden D. (1995). Particulate air pollution and acute health effects. Lancet 345:176–8. Shimada A, Kawamura N, Okajima N, et al. (2006). Translocation pathway of the intratracheally instilled ultrafine particles from the
734
N. Bai & S. F. van Eeden
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
lung into the blood circulation in the mouse. Toxicol Pathol 34: 949–57. Silva VM, Corson N, Elder A, Oberdo¨rster G. (2005). The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP). Toxicol Sci 85:983–9. Tamagawa E, Bai N, Morimoto K, et al. (2008). Particulate matter exposure induces persistent lung inflammation and endothelial dysfunction. Am J Physiol Lung Cell Mol Physiol 295:L79–85. Terashima T, Wiggs B, English D, et al. (1997). Phagocytosis of small carbon particles (PM10) by alveolar macrophages stimulates the release of polymorphonuclear leukocytes from bone marrow. Am J Respir Crit Care Med 155:1441–7. Tofler GH, Muller JE. (2006). Triggering of acute cardiovascular disease and potential preventive strategies. Circulation 114:1863–72. van Eeden SF, Tan WC, Suwa T, et al. (2001). Cytokines involved in the systemic inflammatory response induced by exposure to particulate
Inhal Toxicol, 2013; 25(13): 725–734
matter air pollutants (PM(10)). Am J Respir Crit Care Med 164: 826–30. Vermylen J, Nemmar A, Nemery B, Hoylaerts MF. (2005). Ambient air pollution and acute myocardial infarction. J Thromb Haemost 3: 1955–61. Weibel ER, Hsia CC, Ochs M. (2007). How much is there really? Why stereology is essential in lung morphometry. J Appl Physiol 102: 459–67. Wichmann J, Folke F, Torp-Pedersen C, et al. (2013). Out-of-hospital cardiac arrests and outdoor air pollution exposure in Copenhagen, Denmark. PLoS One 8:e53684. Zanobetti A, Schwartz J. (2005). The effect of particulate air pollution on emergency admissions for myocardial infarction: a multicity case-crossover analysis. Environ Health Perspect 113: 978–82.
RESEARCH ARTICLE
Systemic and vascular effects of circulating diesel exhaust particulate matter Ni Bai and Stephan F. van Eeden
Abstract
Keywords
Objective: Numerous studies have found an association between transiently increased particulate matter air pollution and acute adverse cardiovascular health effects; however, the mechanisms underlying these effects are not clear. Translocation of ultra-fine ambient particulate matter has been proposed to play a key role in these acute side effects. This study was designed to determine the contribution of circulating (translocated) diesel exhaust particles (DEPs) to the systemic and vascular effects. Methods: C57 mice (10-week) received intravenous DEPs via tail vein injection. Following 1-h post-injection, inflammatory cytokines (IL-1b, IL-6 and TNF-a), peripheral blood cell counts, band cell counts, aortic endothelial function and vascular constriction were assessed. Thoracic aortae were isolated, and endothelial function was examined by measuring acetylcholine (ACh) and sodium nitroprusside (SNP)-stimulated vascular relaxation using a wire myograph. In addition, phenylephrine (PE)-stimulated vasoconstriction was also measured. The amount of DEPs deposited and trapped in tissues (the spleen, liver, lungs and heart) were quantified. Results: Acute systemic DEP exposure caused a significant increase in TNF-a, peripheral neutrophil and band cell counts. ACh and SNP-induced relaxation were not affected by acute systemic DEP exposure, neither was PE-stimulated constriction. There was a significantly increased DEP deposition in the spleen as well as in the liver. No significantly increased DEPs were detected in the lung and heart. Conclusion: Here we show that circulating DEPs induce a systemic response characterized by increased TNF-a, peripheral granulocytes, but does not impact endothelial function. Our study also suggests that circulating particles are rapidly removed from the circulation and predominantly sequestered in the spleen and liver.
Diesel exhaust particle, endothelial function, systemic reaction, tissue deposition
Introduction It has been well established that chronic elevation of particulate matter air pollution (PM10) levels are strongly associated with increases in cardiovascular morbidity and mortality, attributable to atherosclerosis, dysrhythmias, ischemic heart disease and stroke (Bai et al., 2007; Brook et al., 2010; Samet et al., 2000; Schwartz, 1999). In addition, evidence from time-series analysis conducted worldwide also indicates the existence of a small, yet consistent association between increased cardiovascular mortality and short-term (1–5 days) elevation of PM10 prior to the cardiovascular events. Having monitored PM10 levels for 300 000 elderly residents in 21 US cities between 1985 and 1999, Burnett and colleagues reported that a 10 mg/m3 increase of PM10 was associated with a significantly increased risk of
Address for correspondence: Dr Stephan van Eeden, MD, PhD, The James Hogg Research Centre, Providence Heart and Lung Institute, St. Paul’s Hospital, University of British Columbia, 1081 Burrard Street, Vancouver, BC V6Z1Y6, Canada. Tel: 1-604-806-8346. Fax: 1-604-8068351. E-mail: [email protected]
History Received 6 June 2013 Revised 4 September 2013 Accepted 10 September 2013 Published online 20 November 2013
hospitalization for myocardial infarction (MI), which was occurred within a few hours after exposure (Burnett et al., 1999; Seaton et al., 1995; Zanobetti & Schwartz, 2005). Another human study showed that hourly changes in PM2.5 concentrations were associated with transiently increased risk of ST segment depression (Lanki et al., 2008). To explain the relative short interval between increases in PM exposure and acute cardiovascular events, Godleski et al. speculated that ultra-fine particles (UFPs, particles with diameter less than 100 nm) were able to enter the blood stream, hence adversely affected the heart and initiated arrhythmias, which could lead to sudden death (Godleski et al., 2000). This hypothesis received its first direct support from a study conducted by Nemmar et al. (2001). Their study demonstrated that inhaled 99mTechnetium-labeled ultra-fine carbon particles were detected in blood one minute after exposure, and remained at peak levels for periods of up to 60 min. These data suggest that inhaled UFPs translocate from the lung directly into the circulation (Choi et al., 2010; Nemmar et al., 2001, 2002; Oberdo¨rster et al., 2002; Peters et al., 2006). Subsequently, several intriguing putative passages by which inhaled UFPs travel from the lung into the circulation and gain access to
20 13
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
The James Hogg Research Centre, Providence Heart and Lung Institute, St. Paul’s Hospital, University of British Columbia, Vancouver, BC, Canada
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
726
N. Bai & S. F. van Eeden
other organs have been proposed. These proposed loci include the fenestrae of sinusoidal endothelial cells (Khandoga et al., 2004), the lung-blood barrier (Hermans & Bernard, 1999), and the alveolar-capillary barrier (Gumbleton, 2001; Shimada et al., 2006). Extra-pulmonary translocation of UFPs provides a plausible explanation for the short interval between exposure to ambient particles and acute cardiovascular events. With the large increase in vehicle traffic, diesel exhaust particles (DEPs) become one of the major components of urban UFPs. Most of DEPs emitted by engines are nano-sized particles with a diameter less than 50 nm (Kittelson, 1998). UFPs deposit in greater numbers and deeply into the lungs, than larger particles do. Due to their larger surface area, compared with bigger particles, DEPs also have greater potential for interactions with biological targets, resulting in a greater inflammatory response (Nemmar et al., 2007; Oberdo¨rster et al., 1996). Both animal and human studies have shown that exposure to DEPs induce endothelial dysfunction (Hansen et al., 2007; Ikeda et al., 1995; Mills et al., 2007; O’Neill et al., 2005). The endothelium serves as an important regulator to maintain vascular tone and vasculature integrity. It does so by producing a number of mediators, in particular, nitric oxide (NO). Endothelium-derived NO protects blood vessels from developing cardiovascular events by suppressing vasoconstriction, attenuating monocyte chemotaxis, preventing the adherence of leukocytes to the endothelium, inhibiting platelet adherence and aggregation. A hallmark of endothelial dysfunction is impairment of acetylcholine (ACh)-stimulated vasorelaxation due to decreased NO production. Endothelial dysfunction is implicated in the pathogenesis of many cardiovascular diseases, including acute coronary syndromes, MI and stroke (Brook et al., 2010; Campen et al., 2005; Vermylen et al., 2005). Studies from others’ and our laboratories have demonstrated that DEPs cause endothelial dysfunction via various mechanisms, including disrupting endothelial vasodilation and endogenous fibrinolysis through reactive oxygen species (Mills et al., 2005), inhibiting endothelial nitric oxide synthase activity, and preventing the release of the vasodilator nitric oxide (Muto et al., 1996). In addition, DEPs and UFPs promote platelet activation and clot formation (Khandoga et al., 2004; Nemmar et al., 2003). Endothelial dysfunction and thrombosis can be the underlying mechanisms attributable to the association between exposure to high levels of PM and the increased incidence of acute cardiovascular events, such as MI (Tofler & Muller, 2006). The secondary inflammatory effect as a result of UFP deposition in the lungs has been well established; however, the direct vascular effects derived from translocated UFPs are not clear. The aim of the present study is to investigate the impact of circulating DEPs (mimic extra-pulmonary translocation) on aortic endothelial function. We hypothesize that circulating DEPs cause direct systemic response, which could lead to endothelial dysfunction. To test our hypothesis, DEPs were injected into the circulation via mouse tail vein. After 1-h post-injection, peripheral blood cell counts, band cell counts and aortic endothelial function were examined. In addition, the amount of DEPs deposited in the tissues (spleen, liver, lung and heart) was assessed.
Inhal Toxicol, 2013; 25(13): 725–734
Materials and methods Diesel exhaust particles preparation Diesel exhaust particles (DEP) (SRM2975) were purchased from US National Institute of Standards and Technology. DEP suspension was sonicated before injection to ensure even distribution and prevent aggregation. Experimental animals All protocols were designed according to the guidelines of the Animal Care Committee of the University of British Columbia. C57 mice were purchased from Jackson Lab (Bar Harbor, ME). These mice were housed in a temperature- and humidity-controlled environment with a 12-h light/dark cycle and free access to water and standard rodent chow. All animals were 10-week-old at the start of the experimental protocol. DEP tail vein injection and sample collections C57 mice were received a tail vein injection of DEP at high dose (0.5 mg/g of body weight) or low dose (0.25 mg/g of body weight) (7.5 or 15 mg DEP suspended in 200 ml saline), and sacrificed at 1-h post-injection. The dose was calculated by assuming a 3-day exposure at 125 (DEP-low) or 250 mg/m3 (DEP-high), and 2% of DEP was translocated to the blood stream. 200 ml saline injection was used as the control. At 1-h post-injection, mice were anesthetized with 10% isoflurane. Upon the loss of all reflexes, blood was collected from inferior vena cava and put into EDTA tubes for cell counts. The thoracic aorta was carefully dissected for vascular function study. The heart, liver, spleen and inflated lung were flash frozen and kept at 80 C. Plasma cytokines Plasma inflammatory factors (IL-1b, IL-6, TNF-a) were measured using Fluorokine Multianalyte Profiling Kits for the Luminex (R&D Systems, Minneapolis, MN) following manufacturer’s instruction. Peripheral differential cell counts White blood cell (WBC) counts and differential leukocyte counts were analyzed using an Abbott Diagnostics Cell-Dyn 3700 instrument (Abbott Park, IL) that was calibrated for analysis of mouse blood cell counts. Peripheral band cell counts Blood smears were fixed with methanol, stained with Wright staining (Sigma Chemicals, St. Louis, MO), coded and examined without knowledge of the experimental groups. A hundred polymorphonuclear leukocytes (PMNs) from randomly selected fields were manually counted and the percentage of band cells was determined. Quantification of DEP in tissue Cryosections (5 mm) of flash frozen heart, liver, spleen and inflated lung were obtained and fixed with cold (4 C) acetone. Blocks were sectioned at 5 mm and stained with hematoxylin and eosin (H&E). Images were captured by a spot digital camera (Microspot, Nikon, Tokyo, Japan), coded
Intravenous DEP exposure and systemic effects
DOI: 10.3109/08958378.2013.844749
and examined without knowledge of the experimental groups. These images were analyzed using a point counting method similar to previously described in more detail (Ling et al., 2011). Briefly, a grid of points was superimposed onto the images captured by Image Pro Plus software (Rockville, MD). The density of the grid and the number of fields were selected to maintain the coefficient of variation of the estimate of the volume below 0.1. The points that contacted with tissue and aggregated DEP were counted. The volume fraction (V/V%) of DEP was obtained by normalizing the total points on aggregated DEP to the total points on tissues.
727
Figure 1. Significantly increased TNF-_ after high dose DEP exposure. *p50.05.Values are expressed by mean SEM (n ¼ 8).
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Vascular function study Thoracic aortae were carefully cleaned off connective tissue without damaging the endothelium. The vessels were then cut to 2 mm rings and mounted on a wire myograph (Model 610 M; Danish Myo Technology, Denmark). Each vessel was bathed in oxygenated physiological salt solution (PSS) at 37 C for an hour during which the resting tension was gradually increased to 6 mN with three changes of PSS at 10-min intervals followed by stabilizing the vessels at resting tension (6 mN) for 30 min. Thereafter, the vessels were stimulated with 80 mM KCl twice. Vessels were preconstricted with phenylephrine (PE) (1 mM) to achieve a sustained vasoconstriction; thereafter, cumulative concentrations of acetylcholine (ACh; 1 nM–10 mM) and sodium nitroprusside (SNP; 1 nM–10 mM) were applied to evaluate endothelium-dependent or -independent NO-mediated relaxation, respectively. Thereafter, with the same vessel, smooth muscle contractility was assessed by administration of cumulative concentrations of PE (1 nM–10 mM). Furthermore, we also evaluated the effects of ex vivo DEP exposure to isolated mouse thoracic aortae (control nonexposed mice) using a wire myograph. We administrated sonicated DEP suspension directly into the chambers of the wire myograph. Endothelial function was assessed preceded 1 h exposure to DEP at three concentrations (10, 50, 100 mg/ml), respectively. Solutions and chemicals The PSS consisted of the following (in mM): NaCl 119, KCl 4.7, KH2PO4 1.18, NaHCO3 24, MgSO47H2O 1.17, CaCl2 1.6, glucose 5.5 and EDTA 0.026. All reagents were purchased from Sigma (St. Louis, MO). Statistical analysis Results are reported as mean SEM. The statistical significance was evaluated using the unpaired Student t test for simple comparison between two values. The concentration– response curves of the different groups were compared by ANOVA for repeated measurements followed by Bonferroni’s correction. p50.05 was considered to be significant. In all experiments, n equals the number of mice from which samples were obtained.
Results Plasma cytokine levels We observed significantly increased TNF-a level in high DEP exposure group (1.15 0.22 pg/ml versus 1.12 0.19 pg/ml
(A)
(B)
Figure 2. Systemic reaction. (A) WBC counts were not affected by systemic DEP exposure; (B) there was a significant increase in neutrophils after DEP exposure. *p50.05 (control versus low DEP); #p50.01(control versus high DEP). Values are expressed by mean SEM (n ¼ 6–8).
versus 2.13 0.54 pg/ml; control versus DEP low versus DEP high, control versus DEP high, p50.05, Figure 1). IL-1b and IL-6 levels were under detectable levels (the sensitivities of Fluorokine Multianalyte Profiling Kit for cytokines are: IL-1b: 7.82 pg/ml, IL-6: 1.06 pg/ml). Peripheral WBC and band cell counts There were no significant changes in circulating total WBC after either low or high dose of DEP (2.56 0.71 109 versus 3.13 0.23 109 versus 2.35 0.80 109 cells/l; control versus DEP low versus DEP high, p40.05, Figure 2A). Except a significant dose-dependent increase in neutrophil (Figure 2B) and monocytes (DEP low versus DEP high, p50.001), no other significant changes were noted in peripheral cell counts (Table 1). Moreover, we found that the band cell counts were significantly increased in a dosedependent fashion (Figure 3) (EC50 and Emax values are available in the supplement).
728
N. Bai & S. F. van Eeden
Inhal Toxicol, 2013; 25(13): 725–734
Table 1. Differential leukocyte counts were analyzed, and data are expressed by mean SEM (n ¼ 6–8). There are significantly increased PMN counts.
Control DEP-low DEP-high
NEU (% of total WBC)
LYM (% of total WBC)
MONO (% of total WBC)
EOS (% of total WBC)
BASO (% of total WBC)
Platelet (109/l)
7.19 1.87a,b 14.4 1.29 33.70 1.90c
85.5 1.10 83.95 1.20 74.53 7.28
3.10 0.89 1.14 0.12 0.10 0.06d
0.04 0.03 0.06 0.03 0.10 0.07
4.14 1.95 0.43 0.10 13.26 7.43
593.5 136.3 685.2 58.6 598.0 129.3
a
Control versus DEP low: p50.01; bControl versus DEP high: p50.05; cDEP low versus DEP high: p50.05; dDEP low versus DEP high: p50.001.
DEP exposure (Figure 9B). No significant difference of DEP deposition in the lung and heart was found albeit a tend of increase was observed in exposure groups (Figure 9C and D).
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Discussion
Figure 3. Increased band cells after acute systemic DEP exposure. *p50.05; #p50.02. Values are expressed by mean SEM (n ¼ 8).
Vascular function study following intravenous DEP exposure Endothelial function and vasocontractility were assessed at 1h post-injection to evaluate the impact of acute systemic DEP exposure. Neither endothelium-dependent nor independent relaxation was affected by DEP exposure (Figure 4). Similarly, there was no alteration of PE-elicited vasoconstriction (Figure 5). (EC50 and Emax values are available in the supplement). Vascular function study following ex vivo DEP exposure To study the effect of direct DEP exposure in isolated mouse aorta, three different concentrations (10, 50, 100 mg/ml) of DEP suspensions were added into the chambers. After 1-h incubation of DEPs with isolated thoracic aortae, ACh relaxation was assessed. We found that higher concentrations (50 mg/ml and 100 mg/ml) of DEPs attenuated ACh relaxation in a dose-dependent fashion (Figure 6B, C). In addition, DEP exposure did not impair SNP-induced endothelium-independent relaxation (Figure 6D), suggesting DEP exposure caused endothelial dysfunction. PE-elicited vasoconstriction was not affected by DEP (Figure 7). Evaluation of DEP deposition in tissues DEP deposition was significantly increased in the spleen at both low and high DEP exposure groups (Figure 9A). This deposition was significantly greater in liver at only high-dose
Numerous evidence has shown an association between acute PM10 exposure and cardiovascular events and exacerbation (Bhaskaran et al., 2009; Brook et al., 2010; Mustafic et al., 2012; Wichmann et al., 2013), but the mechanisms how transiently increases in ambient particles lead to increased cardiovascular morbidity and mortality is still unclear. One hypothesis is that acute cardiovascular events are resulted from the direct effects of UFPs that translocate from the lung into circulation. Extra-pulmonary translocation of UFPs has been reported by different laboratories (Godleski et al., 2000; Nemmar, 2001; Oberdo¨rster et al., 2004; Shimada et al., 2006), but the significance of these circulating particles on vascular function is unclear. In this study, we injected DEPs into mouse tail vein to mimic extra-pulmonary translocation. DEPs include a subgroup with a large number of UFPs, and approximately 50–90% of the particles in diesel exhaust are in the ultrafine size range with the majority of diesel particles ranging in size from 5 to 50 nm (Kittelson, 1998; Nemmar et al., 2007). These physical characteristics of DEPs offer them the potential to translocate across the gas exchange barrier into the circulation. We used two different doses of DEPs to simulate relative high and low exposure to DEPs assuming 2% of particle translocate into the circulation. This assumption was based on studies by Nemmar and colleagues showing 2% of intratracheal 99mTc-labeled ultrafine carbon particles translocate into the bloodstream (Nemmar et al., 2001). Nemmar and colleagues reported that inhaled UFPs translocated into the circulation rapidly with peak levels up to 60 min after exposure (Nemmar et al., 2001, 2002). In addition, Hansen et al. showed that ACh-induced relaxation was impaired 1 h after injecting DEPs in the peritoneum to ApoE mice (Hansen et al., 2007). A human study found that hourly changes in PM2.5 or outdoor particle concentrations were associated with rapid ischemic responses (Lanki et al., 2008). Based on these studies, we chose to test at 1-h postinjection of DEPs. Some studies also reported that cardiovascular effects were occurred few hours after acute exposure, indicating a potential lag effects which cannot be excluded in this study and need further investigation. We found that circulating DEPs induced a systemic reaction, characterized by increased TNF-a (Figure 1), elevated circulating neutrophils (Figure 2B) and band cells (Figure 3). Increased band cell counts suggest that the
Intravenous DEP exposure and systemic effects
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
DOI: 10.3109/08958378.2013.844749
(A)
(B)
(C)
(D)
729
Figure 4. Concentration-response curves of ACh-induced endothelium-dependent and SNP-induced endothelium-independent relaxation. There was no changes after acute DEP exposure at either low (A, B) or high dose (C, D). Values are expressed by mean SEM (n ¼ 6).
Figure 5. Concentration–response curves of PE-induced vasoconstriction. There were no alterations following acute DEP exposure at either low (A) or high dose (B). Values are expressed by mean SEM (n ¼ 6).
(A)
neutrophilia is not just demargination of intravascular neutrophils due to stress or cathecholamine effect, but due to stimulation of the bone marrow to release neutrophils (Mukae et al., 2001; Terashima et al., 1997). Our previous studies have shown that this stimulation of the marrow is usually caused by elevated circulation pro-inflammatory mediators (such as IL-6, IL-1b, TNF-a, IL-8) or growth factors (such as GM-CSF and G-CSF) (Ishii et al., 2004; van Eeden et al., 2001). This study suggest that TNF-a plays a key role in the rapid systemic response following translocation of UFP, possibly via bone marrow stimulation. TNF-a could be released from circulating monocytes or tissues macrophages/ hystiocytes (in the liver and spleen) that clear these particles from the circulation. It is well known that inflammatory mediators (such as IL-1b, IL-6, TNF-a and GM-CSF) are able to activate
(B)
vascular endothelium (Bussolino et al., 1989; Kido et al., 2011; Tamagawa et al., 2008). In this study, we hypothesized that intravascular DEP exposure caused systemic inflammation, hence leading to endothelial dysfunction. Endothelial function was tested with ACh (Figure 4), and vasoconstrictive function was also examined by PE (Figure 5). We observed no alteration following DEP exposure. These results are in agreement with another study showing that exposure of rats to DEPs induced both pulmonary and systemic inflammation, but did not modify endothelium-dependent vasodilatation (Robertson et al., 2012). Khandoga et al. showed that intra-arterial UFP exposure to C57 mice increased platelet accumulation and prothrombotic changes in the hepatic microvasculature without inflammatory reactions (Khandoga et al., 2004). This result implies that UFP-induced side effects may be caused directly by
730
N. Bai & S. F. van Eeden (A)
Inhal Toxicol, 2013; 25(13): 725–734
(B)
(C)
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
(D)
Figure 6. Concentration–response curves of ACh-induced vasorelaxation after ex vivo DEP exposure. There was no impaired endothelial function at low (10 mg/ml) concentration (A), but exposure to DEP at higher (50 and 100 mg/ml) concentrations caused endothelial dysfunction (B, C). There was no impairment of SNP-induced endothelium-independent vasorelaxation (D). Values are expressed by mean SEM (n ¼ 6); *p50.05.
(B)
(A)
(C)
Figure 7. Concentration–response curves of PE-induced vasoconstriction after ex vivo DEP exposure. Different concentrations of DEP exposure did not affect aortic constriction (A, B, C). Values are expressed by mean SEM (n ¼ 6).
Intravenous DEP exposure and systemic effects
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
DOI: 10.3109/08958378.2013.844749
(A)
(B)
(C)
(D)
731
Figure 8. Representative photomicrographs of DEP deposition in tissues. (A) DEP aggregates are shown in the spleen; (B) DEPs are shown in the cytoplasm of hepatic cells; (C) DEPs are shown in the cytoplasm of macrophages in the lung; (D) DEP aggregates are shown in the heart. Arrows point to the deposition of DEP aggregates. Magnification: 1000X. Scale bar ¼ 50 mm..
circulating UFPs themselves. To examine the direct effect of circulating DEPs on blood vessels, we exposed similar doses of DEPs to isolated mouse aortae that were not exposed to DEPs. We found that ex vivo DEP exposure caused endothelial dysfunction in a dose-dependent manner (Figure 6A, B, C). That SNP response was not altered (Figure 6D) confirmed that this effect was endothelial dependent. The difference between in vivo and ex vivo responses of blood vessels suggests that circulating DEPs were rapidly cleared from the circulation before they could significantly activate endothelium. To support this concept, we examined DEP deposition in tissues (Figure 8) and show the significant dose-dependent increases in DEP deposition in the spleen (Figure 9A) as well as in the liver after high-dose DEP exposure (Figure 9B). There was also a trend of an increase in particles in the lung and heart (Figure 9C, D). We suspect that endothelial function was not affected by the in vivo exposure to DEP, at least in part, because the majority of DEPs were sieved out and removed from the circulating blood stream by the spleen and liver. In addition, it has been demonstrated that red blood cells can interact and take up DEPs, and this interaction between DEPs and red blood cells may also contribute to increased DEP deposition in spleen (Nemmar & Inuwa, 2008).
The present study is the first to evaluate the amount DEPs trapped and deposited in tissues following acute systemic DEP administration. We demonstrate that DEP aggregates deposit in the spleen after both low and high dose of DEP exposure (Figure 9A), whereas the amount of DEPs significantly increase in the liver only in high DEP group (Figure 9B). These data indicate that the spleen is the primary organ responsible for removing DEPs out of the circulation, and the liver plays an important role when increasing DEPs are in the circulation. Although there are also an increased amount of DEPs deposited in the lung and heart, it is not significantly different from the control (Figure 9C, D). Our data are different from studies conducted by Nemmar et al. (2007), who observed lung inflammation and increased cellular influx in the lung following injection of particles, an effect that could be dose and particle composition dependent. Other study reported that liver and kidneys also served as major organs to clear-off these ambient particles (Nakane, 2012). Since the notion of UFP translocation was proposed a decade ago, many studies have been conducted and reported the possibilities of UFP translocation to the systemic circulation and other organs, including liver, spleen, kidney, heart
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
732
N. Bai & S. F. van Eeden
Inhal Toxicol, 2013; 25(13): 725–734
(A)
(B)
(C)
(D)
Figure 9. DEP deposition in tissues. There were significantly increased DEP deposition in the spleen (A) and liver (B). There was a trend increase in DEP deposition in the lung (C) and the heart (D). *p50.02 (control versus low DEP); #p50.001 (control versus high DEP); **p50.01(control versus high DEP). Values are expressed by mean SEM (n ¼ 8).
or brain (Nemmar et al., 2001; Oberdo¨rster et al., 2004), lymph nodes and bloodstream (Choi et al., 2010; Nemmar et al., 2001, 2002). Nevertheless, this concept is still very controversial (Kreyling et al., 2002; Mills et al., 2006; Mo¨ller et al., 2008), mainly due to technical challenges to trace and quantify translocation. Some of these limitations are also applicable to this study. Because of the size of DEPs, it is difficult to measure or trace small amount of these particles in tissues. In addition, based on previous studies (Kittelson, 1998; Nemmar et al., 2007), more than 50% of DEPs in number are UFPs; therefore, we suspect that a significant number of DEPs seen in the spleen are UFPs, but overall they are likely a mixture of coarse and ultra-fine particles. Moreover, constituents attached to particles may come off during the translocation process. For example, Mills et al. showed no translocation of insoluble, radio-labeled ultrafine carbon particles from the lung into the bloodstream of humans (Mills et al., 2006). They believed that the small amount of radioactivity found in the bloodstream and other organs was attributed entirely to the small amount of leached, soluble radiolabel from the particles. Therefore, quantifying the amount of translocated particles has remained one of the biggest challenges for most researchers. We used stereological methods that are considered as gold standard for obtaining quantitative data on structure at microscopic levels (Weibel et al., 2007). However, circulating DEPs could be internalized into platelets by endocytosis (Chen et al., 2006) and/or leukocytes such as neutrophils and monocytes (Miller et al., 2006). Therefore, we are not able to exclude that certain portion of DEPs is still in the circulation. The vein injection of DEPs may not be the ideal approach to mimic potential UFP translocation from lungs to the circulation, but we think that tail vein injection serves as both
a convenient and reasonable approach to investigate the mechanisms of translocated particles. Similar approach has been used by other studies to investigate direct effects of particles following intravascular administration (Khandoga et al., 2004; Silva et al., 2005). We found the bulk of particles in the spleen suggesting that the majority of particles reached the systemic circulation and were not trapped in the liver following the venous injection. Lastly, the concentrations of DEPs used in this study are higher than EPA standard and most average levels in major North American cities. We selected these concentrations to be able to compare to previous studies and to maximize chances of detecting alterations in vascular function. However, exposure to this high concentration of PM2.5 (4500ug/m3) has recently become realistic in some developing countries, such as China. It is quite possible that at certain times (e.g. rush hours) such high level PM2.5 can be reached along busy routes. In summary, our study demonstrates that circulating DEPs induce a systemic inflammatory response characterized by increased TNF-a, neutrophilia and bone marrow stimulation (increase band cell counts), but do not cause endothelial dysfunction or abnormal vasoconstriction. We speculate that DEPs translocated into the circulation following inhalation are rapidly removed from the circulation by the spleen and liver. As a result, these circulating particles do not significantly contribute to the early vascular dysfunction associated with exposure to ambient particulate matter. Although there is solid evidence for extra-pulmonary translocation of UFPs, this translocation mechanism underlies adverse vascular effects of combustion-derived UFP exposures remains considerably uncertain.
DOI: 10.3109/08958378.2013.844749
Acknowledgements Authors would like to thank David Ngan for measuring inflammatory cytokines for this study.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
References Bai N, Kido T, Kavanagh TJ, et al. (2011). Exposure to diesel exhaust up-regulates iNOS expression in ApoE knockout mice. Toxicol Appl Pharmacol 255:184–92. Bai N, Khazaei M, van Eeden SF, Laher I. (2007). The pharmacology of particulate matter air pollution-induced cardiovascular dysfunction. Pharmacol Ther 113:16–29. Bhaskaran K, Hajat S, Haines A, et al. (2009). Effects of air pollution on the incidence of myocardial infarction. Heart 95:1746–59. Brook RD, Rajagopalan S, Pope III CA, et al. (2010). Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation 121: 2331–78. Burnett RT, Smith-Doiron M, Stieb D, et al. (1999). Effects of particulate and gaseous air pollution on cardiorespiratory hospitalizations. Arch Environ Health 54:130–9. Bussolino F, Wang JM, Defilippi P, et al. (1989). Granulocyte- and granulocyte-macrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate. Nature 337:471–3. Campen MJ, Babu NS, Helms GA, et al. (2005). Nonparticulate components of diesel exhaust promote constriction in coronary arteries from apoE/ mice. Toxicol Sci 88:95–102. Chen J, Tan M, Nemmar A, et al. (2006). Quantification of extrapulmonary translocation of intratracheal-instilled particles in vivo in rats: effect of lipopolysaccharide. Toxicology 222:195–201. Choi HS, Ashitate Y, Lee JH, et al. (2010). Rapid translocation of nanoparticles from the lung airspaces to the body. Nat Biotechnol 28: 1300–3. Godleski JJ, Verrier RL, Koutrakis P, et al. (2000). Mechanisms of morbidity and mortality from exposure to ambient air particles. Res Rep Health Eff Inst 91:5–88. Gumbleton M. (2001). Caveolae as potential macromolecule trafficking compartments within alveolar epithelium. Adv Drug Deliv Rev 49: 281–300. Hansen CS, Sheykhzade M, Møller P, et al. (2007). Diesel exhaust particles induce endothelial dysfunction in apoE/ mice. Toxicol Appl Pharmacol 219:24–32. Hermans C, Bernard A. (1999). Lung epithelium-specific proteins: characteristics and potential applications as markers. Am J Respir Crit Care Med 159:646–78. Ikeda M, Suzuki M, Watarai K, et al. (1995). Impairment of endothelium-dependent relaxation by diesel exhaust particles in rat thoracic aorta. Jpn J Pharmacol 68:183–9. Ishii H, Fujii T, Hogg JC, et al. (2004). Contribution of IL-1 beta and TNF-alpha to the initiation of the peripheral lung response to atmospheric particulates (PM10). Am J Physiol Lung Cell Mol Physiol 287:L176–83. Khandoga A, Stampfl A, Takenaka S, et al. (2004). Ultrafine particles exert prothrombotic but not inflammatory effects on the hepatic microcirculation in healthy mice in vivo. Circulation 109:1320–5. Kido T, Tamagawa E, Bai N, et al. (2011). Particulate matter induces translocation of IL-6 from the lung to the systemic circulation. Am J Respir Cell Mol Biol 44:197–204. Kittelson DB. (1998). Engines and nanoparticles: a review. J Aerosol Sci 29:575–88. Kreyling WG, Semmler M, Erbe F, et al. (2002). Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low. J Toxicol Environ Health A 65:1513–30. Lanki T, Hoek G, Timonen KL, et al. (2008). Hourly variation in fine particle exposure is associated with transiently increased risk of ST segment depression. Occup Environ Med 65:782–6.
Intravenous DEP exposure and systemic effects
733
Ling SH, McDonough JE, Gosselink JV, et al. (2011). Patterns of retention of particulate matter in lung tissues of patients with COPD: potential role in disease progression. Chest 140:1540–9. Mills NL, Amin N, Robinson SD, et al. (2006). Do inhaled carbon nanoparticles translocate directly into the circulation in humans? Am J Respir Crit Care Med 173:426–31. Mills NL, Tornqvist H, Gonzalez MC, et al. (2007). Ischemic and thrombotic effects of dilute diesel-exhaust inhalation in men with coronary heart disease. N Engl J Med 357:1075–82. Mills NL, Tornqvist H, Robinson SD, et al. (2005). Diesel exhaust inhalation causes vascular dysfunction and impaired endogenous fibrinolysis. Circulation 112:3930–6. Mo¨ller W, Felten K, Sommerer K, et al. (2008). Deposition, retention, and translocation of ultrafine particles from the central airways and lung periphery. Am J Respir Crit Care Med 177:426–32. Mukae H, Vincent R, Quinlan K, et al. (2001). The effect of repeated exposure to particulate air pollution (PM10) on the bone marrow. Am J Respir Crit Care Med 163:201–19. Mustafic H, Jabre P, Caussin C, et al. (2012). Main air pollutants and myocardial infarction: a systematic review and meta-analysis. JAMA 307:713–21. Muto E, Hayashi T, Yamada K, et al. (1996). Endothelial-constitutive nitric oxide synthase exists in airways and diesel exhaust particles inhibit the effect of nitric oxide. Life Sci 59:1563–70. Nakane H. (2012). Translocation of particles deposited in the respiratory system: a systematic review and statistical analysis. Environ Health Prev Med 17:263–74. Nemmar A, Al-Maskari S, Ali BH, Al-Amri IS. (2007). Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats. Am J Physiol Lung Cell Mol Physiol 292:L664–70. Nemmar A, Hoet PH, Dinsdale D, et al. (2003). Diesel exhaust particles in lung acutely enhance experimental peripheral thrombosis. Circulation 107:1202–8. Nemmar A, Hoet PH, Vanquickenborne B, et al. (2002). Passage of inhaled particles into the blood circulation in humans. Circulation 105:411–14. Nemmar A, Inuwa IM. (2008). Diesel exhaust particles in blood trigger systemic and pulmonary morphological alterations. Toxicol Lett 176: 20–30. Nemmar A, Nemery B, Hoet PH, et al. (2003b). Pulmonary inflammation and thrombogenicity caused by diesel particles in hamsters: role of histamine. Am J Respir Crit Care Med 168:1366–72. Nemmar A, Vanbilloen H, Hoylaerts MF, et al. (2001). Passage of intratracheally instilled ultrafine particles from the lung into the systemic circulation in hamster. Am J Respir Crit Care Med 164: 1665–8. Oberdorster G. (1996). Significance of particle parameters in the evaluation of exposure-dose-response relationships of inhaled particles. Inhal Toxicol. 8 Suppl: 73–89 (Review). Oberdo¨rster G, Sharp Z, Atudorei V, et al. (2002). Extrapulmonary translocation of ultrafine carbon particle following whole-body inhalation exposure of rats. J Toxicol Environ Health A 65:1531–43. Oberdo¨rster G, Sharp Z, Atudorei V, et al. (2004). Translocation of inhaled ultrafine particles to the brain. Inhal Toxicol 16:437–45. O’Neill MS, Veves A, Zanobetti A, et al. (2005). Diabetes enhances vulnerability to particulate air pollution-associated impairment in vascular reactivity and endothelial function. Circulation 111: 2913–20. Peters A, Veronesi B, Caldero´n-Garciuden˜as L, et al. (2006). Translocation and potential neurological effects of fine and ultrafine particles a critical update. Part Fiber Toxicol 3:13. Robertson S, Gray GA, Duffin R, et al. (2012). Diesel exhaust particulate induces pulmonary and systemic inflammation in rats without impairing endothelial function ex vivo or in vivo. Part Fibre Toxicol 9:9. Samet JM, Dominici F, Curriero FC, et al. (2000). Fine particulate air pollution and mortality in 20 U.S. cities, 1987–1994. N Engl J Med 343:1742–9. Schwartz J. (1999). Air pollution and hospital admissions for heart disease in eight U.S. counties. Epidemiology 10:17–22. Seaton A, MacNee W, Donaldson K, Godden D. (1995). Particulate air pollution and acute health effects. Lancet 345:176–8. Shimada A, Kawamura N, Okajima N, et al. (2006). Translocation pathway of the intratracheally instilled ultrafine particles from the
734
N. Bai & S. F. van Eeden
Inhalation Toxicology Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
lung into the blood circulation in the mouse. Toxicol Pathol 34: 949–57. Silva VM, Corson N, Elder A, Oberdo¨rster G. (2005). The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP). Toxicol Sci 85:983–9. Tamagawa E, Bai N, Morimoto K, et al. (2008). Particulate matter exposure induces persistent lung inflammation and endothelial dysfunction. Am J Physiol Lung Cell Mol Physiol 295:L79–85. Terashima T, Wiggs B, English D, et al. (1997). Phagocytosis of small carbon particles (PM10) by alveolar macrophages stimulates the release of polymorphonuclear leukocytes from bone marrow. Am J Respir Crit Care Med 155:1441–7. Tofler GH, Muller JE. (2006). Triggering of acute cardiovascular disease and potential preventive strategies. Circulation 114:1863–72. van Eeden SF, Tan WC, Suwa T, et al. (2001). Cytokines involved in the systemic inflammatory response induced by exposure to particulate
Inhal Toxicol, 2013; 25(13): 725–734
matter air pollutants (PM(10)). Am J Respir Crit Care Med 164: 826–30. Vermylen J, Nemmar A, Nemery B, Hoylaerts MF. (2005). Ambient air pollution and acute myocardial infarction. J Thromb Haemost 3: 1955–61. Weibel ER, Hsia CC, Ochs M. (2007). How much is there really? Why stereology is essential in lung morphometry. J Appl Physiol 102: 459–67. Wichmann J, Folke F, Torp-Pedersen C, et al. (2013). Out-of-hospital cardiac arrests and outdoor air pollution exposure in Copenhagen, Denmark. PLoS One 8:e53684. Zanobetti A, Schwartz J. (2005). The effect of particulate air pollution on emergency admissions for myocardial infarction: a multicity case-crossover analysis. Environ Health Perspect 113: 978–82.
