Ultrafine Carbon Particles Research Articles

The neurological effects of air pollution in children

Environmental exposures in utero and during early life may permanently change the body’s structure, physiology and metabolism, and lead to diseases in adult life [1]. Infants are particularly vulnerable because of their rapid growth and cell differentiation, immaturity of metabolic pathways and development of vital organ systems. The central nervous system has unprotected barriers and a broad time window of conformation, leading to a long period of vulnerability in the developmental process and to susceptibility to any environmental insult [2]. Research conducted among a limited series of pollutants (including lead, mercury and polycyclic aromatic hydrocarbons (PAH)) shows that early-life exposure to chemicals at current environmental levels can be neurotoxic years or even decades after exposure [3]. Traffic-related air pollution, basically urban outdoor pollution, is a global public health problem [4]. Cardiorespiratory effects and mechanisms have been fully investigated [5]. In contrast, little is known regarding neurological effects, with only some preliminary evidence. In rats, ultrafine carbon particles have been found in the olfactory bulb and the cerebrum and cerebellum after inhalation exposure [6]; this finding has been reproduced more recently with manganese particles directly translocated to the olfactory nerve from the nose to the brain [7]. In one study, dogs living in a highly polluted region in Mexico City (Mexico) had an increase in brain inflammation compared with animals living in a less polluted area [8]. The brain tissue of animals from Mexico City had higher levels of nuclear factor-kB activation and nitric oxide production, as well as the principal pro-inflammatory cytokines interleukin (IL)-1 and tumour necrosis factor (TNF)-a, compared with the animals from the nonpolluted area [9]. In a study on human autopsies in Mexico City, exposure to severe air pollution has been associated with increased levels of cyclooxygenase (COX)-2 and accumulation of the 42-amino-acid form of b-amyloid, a cause of neuronal dysfunction [10].

Effect of Inhaled Carbon Ultrafine Particles on Reactive Hyperemia in Healthy Human Subjects

BackgroundUltrafine particles (UFP) may contribute to the cardiovascular effects of exposure to particulate air pollution, partly because of their relatively efficient alveolar deposition and potential to enter the pulmonary vascular space.ObjectivesThis study tested the hypothesis that inhalation of elemental carbon UFP alters systemic vascular function.MethodsSixteen healthy subjects (mean age, 26.9 ± 6.5 years) inhaled air or 50 μg/m3 elemental carbon UFP by mouthpiece for 2 hr, while exercising intermittently. Measurements at preexposure baseline, 0 hr (immediately after exposure), 3.5 hr, 21 hr, and 45 hr included vital signs, venous occlusion plethysmography and reactive hyperemia of the forearm, and venous plasma nitrate and nitrite levels.ResultsPeak forearm blood flow after ischemia increased 3.5 hr after exposure to air but not UFP (change from preexposure baseline, air: 9.31 ± 3.41; UFP: 1.09 ± 2.55 mL/min/100 mL; t-test, p = 0.03). Blood pressure did not change, so minimal resistance after ischemia (mean blood pressure divided by forearm blood flow) decreased with air, but not UFP [change from preexposure baseline, air: −0.48 ± 0.21; UFP: 0.07 ± 0.19 mmHg/mL/min; analysis of variance (ANOVA), p = 0.024]. There was no UFP effect on pre-ischemia forearm blood flow or resistance, or on total forearm blood flow after ischemia. Venous nitrate levels were significantly lower after exposure to carbon UFP compared with air (ANOVA, p = 0.038). There were no differences in venous nitrite levels.ConclusionsInhalation of 50 μg/m3 carbon UFP during intermittent exercise impairs peak forearm blood flow during reactive hyperemia in healthy human subjects.

Deposition, Retention, and Translocation of Ultrafine Particles from the Central Airways and Lung Periphery

Little is known about clearance of ultrafine carbon particles from the different regions of the human lung. These particles may accumulate and present a health hazard because of their high surface area. Technetium Tc 99m ((99m)Tc)-radiolabeled 100-nm-diameter carbon particles were inhaled by healthy nonsmokers, asymptomatic smokers, and by patients with chronic obstructive pulmonary disease (COPD). Using a bolus inhalation technique, particle deposition was targeted either to the airways or to the lung periphery, and retention, clearance, and translocation were measured using retained radiotracer imaging. In vitro studies revealed that mean leaching of soluble (99m)Tc-radiotracer from the carbon particles was 4.1 (2.6 [SD]) % after 24 hours. Cumulative (99m)Tc activity in urine at 24 hours was 1.1 (1.3) % of activity deposited in the lungs. In the lung periphery, particle retention was not affected by smoking or pulmonary disease; retention was 96 (3) % after 24 hours. The small amount of clearance could be attributed to leaching of the (99m)Tc label, suggesting negligible particle clearance. In healthy nonsmokers, retention of particles targeted to the airways was 89 (6) and 75 (10) % after 1.5 and 24 hours, respectively. Radiolabel activity did not accumulate in the liver. Within the limits of detection of our experimental system, most inhaled ultrafine carbon particles are retained in the lung periphery and in the conducting airways without substantial systemic translocation or accumulation in the liver at 48 hours. Repeated exposure may result in significant pulmonary accumulation of ultrafine particles.

Mechanisms of increased wear resistance of composites based on PTFE doped with fullerene soot

We have studied the effect of small additives of a fullerene soot (FS) on the linear wear intensity I h of poly(tetrafluoroethylele) (PTFE) in sliding friction on steel with water lubrication. The introduction of only 1% FS into PTFE leads to a sharp drop in I h . The investigation of FS samples by means of small-and wide-angle X-ray scattering showed that FS contains C60 fullerene and graphite nanocrystals with an average size of 20–25 nm and ultrafine carbon particles 2–3 nm in size. The presence of dispersed nanoscopic particles probably imparts the FS-doped PTFE samples the properties of nanocomposites. The possible mechanism of microcrack healing in PTFE by nanoparticles representing fullerene fragments is considered.

Inflammatory Response to TiO 2 and Carbonaceous Particles Scales Best with BET Surface Area

In an attempt to identify the proper dose metric for particle toxicity, Wittmaack (2007) reanalyzed our dose–response data (Stoeger et al. 2006) and that of Oberdorster et al. (2005) on acute lung inflammation in rodents after instillation of various particle types. Out of particle BET surface area (SBET), particle number, joint length, and “geometric” surface area, Wittmaack concluded that particle number tends “to work best” as dose metric. We disagree with his conclusion. First, we wonder why Wittmaack (2007) used our data but ignored the data of Oberdorster et al. (2005) for the identification of the best dose metric. Figure 1 shows our dose–response data (in mice) for six different types of ultrafine carbonaceous particles (10–50 nm) and the data of Oberdorster et al. (2005) for fine (~ 250 nm) and ultrafine (~ 20 nm) TiO2 particles; we present the data for rats, which was reanalyzed by Wittmaack, and also the mouse data from Oberdorster et al. (2005). In Figure 1 the inflammatory response after 24 hr is expressed as the ratio of the polymorphonuclear leukocytes (PMNs) to lavaged cells, and the instilled dose is normalized to lung weight, because this facilitates interspecies comparison (Oberdorster et al. 2005). As suggested by Wittmaack (2007), we limit our discussion to the linear response regime [analogous to his Figure 3 (Wittmaack 2007)]. For this data set, the linear correlation coefficient R2 is 0.46, 0.51, 0.67, and 0.72 for particle number, joint length, “geometric” surface area, and SBET, respectively. Particularly, the response to the fine particles, as represented by the red fit line (almost identical to the y-axis in Figure 1A), is not adequately described by particle number (Figure 1A), whereas SBET works well for all particle sizes (Figure 1B). Although we do not suggest SBET as a “universal” dose metric (chemistry, charge, etc., are also relevant), we conclude that for the dose metric examined here, SBET is the most relevant dose parameter. Wittmaack’s preference for particle number appears to be the result of an unsubstantiated restriction of his analysis to our data, which is dominated by particles in a relatively narrow size regime between about 10 and 25 nm. Figure 1 Acute pulmonary inflammatory response (PMNs) to TiO2 [Oberdorster et al. 2005; Figure 4 and Figure S-2 (Supplemental Material available online at http://ehp.niehs.nih.gov/members/2005/7339/supplemental.pdf)] and carbonaceous particles (Stoeger ... Second, all investigated dose parameters (except SBET) depend on accurate determination of the mean particle diameter, , requiring tedious and potentially uncertain single particle analysis. Wittmaack (2007) acknowledged potentially large errors in for particles below about 20 nm [i.e., for four out of our six (carbonaceous) particle types]. Being aware of these limitations, we intentionally reported only a range of observed particle diameters (not ) in our article (Stoeger et al. 2006). Unfortunately, Wittmaack did not discuss his conclusions in light of these methodologic limitations. Especially for the smallest particle type (here spark-generated carbon particles with = 9.8 nm), preferential particle selection is likely to result in an overestimation of . Assuming a 25% sizing error, this yields a systematic error of + 100% in particle number (~ −3), which shifts these data points far away from the linear fit line (see error bars in Figure 1A). In contrast, SBET requires only a single measurement on an aliquot of the administered particles; that is, it is not adversely affected by problems associated with single particle analysis, and it adequately accounts for potentially important particle characteristics such as particle morphology and surface porosity. In summary, we do not agree with the dose–response interpretation of our data by Wittmaack (2007). We conclude that SBET (and not particle number) is the best dose parameter, accounting for 72% (R2 = 0.72) of the observed inflammatory response for both data sets spanning a size range of 10–250 nm.

Advances in environmental and occupational disorders 2006

In 2006, there continued to be significant contributions to the Journal in the area of environmental and occupational allergy. In addition to very strong clinical observations, there were a number of studies that examined the effect of environmental agents on cellular and molecular processes in allergy, and molecular approaches were also used in investigations of allergen structure and biology. The Journal also received a number of very strong epidemiologic studies examining several aspects of environmental and occupational allergy. Thus, this particular area of our specialty thrives as a result of clinical, mechanistic, epidemiologic, and translational observations. This article reviews a number of these papers presented in the Journal in 2006.

Metal Particles and Extrapulmonary Transport: Oberdörster and Elder Respond

We appreciate the opportunity to address the points raised by Ghio and Bennett in their letter. The pH of our cell-free dissolution studies (Elder et al. 2006) was appropriate because the nasal cavity surface is neutral and does not have airway macrophages and the phagolysosomal pH of nasal epithelial cells is neutral (Johnson 1994). Acidic pH, as in the phagolysosome of alveolar macrophages, dissolves manganese oxide, resulting in increased levels of blood-borne non-particulate Mn. Our neutral pH buffer dissolved only 1.5% of Mn oxide nanoparticles in 24 hr. The dissolution buffer was not “normal saline,” as stated by Ghio and Bennett; it was physiologic saline, a “simulated lung fluid” used for decades in studies of man-made fiber dissolution (Potter and Mattson 1991). It was an oversight on our part to omit the exact composition from our article (Elder et al. 2006), which includes citrate (model organic acid) and glycine (model protein component). Citrate is a stronger metal chelator than glycine. If only soluble Mn translocates, the time required for solubilization would significantly retard the increase of Mn in the olfactory bulb of administered Mn oxide nanoparticles. Instead, we found no difference between the two forms of Mn when we directly compared the translocation rate of the Mn oxide nanoparticles to soluble Mn chloride (Elder et al. 2006), indicating a direct uptake of the Mn oxide nanoparticles by olfactory neuronal structures and subsequent translocation. It is conceivable that subsequent dissolution in the olfactory system occurs. The rapidity of solid nanoparticle transport along neuronal axons is, indeed, remarkable (~ 2.5 mm/hr), as demonstrated earlier by the arrival in the olfactory bulb of 50 nm gold particles within 30 min after intranasal instillation. This and other studies with gold nanoparticles using transmission electron microscopy detection (reviewed by Oberdorster et al. 2005) demonstrate unequivocally that some metal particles are indeed appropriate for demonstrating solid particle transport across epithelial barriers, refuting the absolute statement in the title of Ghio and Bennett’s letter. Ghio and Bennett suggest that a carbon-based particle would be appropriate for studying ultrafine particle transport and translocation. This is true for elemental and organic carbon only if insoluble in vivo. Indeed, study with inhaled ultrafine elemental carbon particles (13C) confirmed their translocation to the olfactory bulb of rats (Oberdorster et al. 2004). In contrast, labeled elemental carbon is problematic, as pointed out by Ghio and Bennett regarding Technegas (99mTc labeled-ultrafine carbon). Recent studies using inhaled Technegas (Mills et al. 2006; Wiebert et al. 2006) showed no translocation in humans, contradicting earlier work (Nemmar et al. 2002). Both leaching of the soluble radiolabel and the inability of the g-camera to detect small amounts (£ 1%) of the deposited dose in extrapulmonary organs are significant limitations with this noninvasive technique, resulting in misinterpretions suggesting either significant particle translocation or lack thereof. Ghio and Bennett cite LeFevre et al. (1982), apparently as evidence that inhaled carbon-based particles do not undergo extrapulmonary transport. However, LeFevre et al. interpreted their findings of high black pigment scores in liver and spleen of coal miners differently—namely, as migration of coal dust in the pneumoconiotic lung into pulmonary lymphatics and then to the systemic circulation, and also as migration of coal mine dust–laden macrophages through the walls of pulmonary blood vessels. Whatever the mechanism, their findings clearly indicate extrapulmonary transport. Heavy silica inhalation exposure has also been found to result in particle accumulation in liver and other extrapulmonary tissues in humans and nonhuman primates (Carmichael et al. 1980; Rosenbruch 1990; Slavin et al. 1985). Ghio and Bennett further state that Decades of research have provided no evidence of an extrapulmonary transport (including via olfactory neuronal pathways) of particles associated with cigarette smoking. How many investigators have tried to find cigarette smoke particles in extrapulmonary tissues? We are aware of only one study in rats in which a 25-min inhalation exposure to 14C cigarette smoke resulted in 0.24–0.83% of retained 14C in the liver within 15 min after the short-term exposure (Chen et al. 1989). Does this indicate extrapulmonary particle transport? Possibly yes. We conclude that the physicochemical characteristics of a nanoparticle—whether metal or not—and the physiologic milieu at the site of deposition in the respiratory tract determine whether extrapulmonary translocation occurs as particle or as solute. A prerequisite for noninvasively measuring this is that the detection method has sufficient sensitivity for identifying the analyte at expected low translocation rates. Regarding our nasal translocation study in rats (Elder et al. 2006), we conclude that inhaled Mn oxide nanoparticles are taken up by the nasal olfactory neuronal pathway as solid particles rather than being slowly dissolved first at neutral pH. For the alveolar region where dissolution is expected to be more rapid, this does not apply.

Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R

Apoptosis and proliferation are important causes of adverse health effects induced by inhaled ultrafine particles. The molecular mechanisms of particle cell interactions mediating these end points are therefore a major topic of current particle toxicology and molecular preventive medicine. Initial studies revealed that ultrafine particles induce apoptosis and proliferation in parallel in rat lung epithelial cells, dependent on time and dosage. With these end points, two antagonistic reactions seem to be induced by the same extracellular stimulus. It was therefore investigated whether proliferation is induced directly by the particles or as a compensation of particle-caused cell death. Experimental conditions excluding compensatory proliferation demonstrated that both end points are induced independently by specific signaling pathways. Events eliciting signaling cascades leading to apoptosis and proliferation were studied with specific inhibitors of membrane receptors. Epidermal growth factor receptor (EGF-R) kinase activity was identified as essential for apoptosis as well as for proliferation. As ultrafine particle-induced proliferation alone was dependent on the activation of beta1-integrins, these membrane receptors are suggested to mediate the specificity of EGF-R signaling concerning the decision as to whether apoptosis or proliferation is triggered. Accordingly, MAP kinase signaling downstream of EGF-R showed comparable specificity with regard to receptor-dependent induction of apoptosis and proliferation. As key mediators of signaling cascades, the activation of extracellular signal-regulated kinases 1 and 2 proved to be specific for proliferation in a beta1-integrin-dependent manner, whereas phosphorylation of c-Jun NH2-terminal kinases 1 and 2 was correlated with the induction of apoptosis.

Inhalation of ultrafine carbon particles triggers biphasic pro-inflammatory response in the mouse lung

High levels of particulate matter in ambient air are associated with increased respiratory and cardiovascular health problems. It has been hypothesised that it is the ultrafine particle fraction (diameter <100 nm) that is largely responsible for these effects. To evaluate the associated mechanisms on a molecular level, the current authors applied an expression profiling approach. Healthy mice were exposed to either ultrafine carbon particles (UFCPs; mass concentration 380 microg x m(-3)) or filtered air for 4 and 24 h. Histology of the lungs did not indicate any pathomorphological changes after inhalation. Examination of the bronchoalveolar lavage fluid revealed a small increase in polymorphonuclear cell number (ranging 0.6-1%) after UFCP inhalation, compared with clean air controls, suggesting a minor inflammatory response. However, DNA microarray profile analysis revealed a clearly biphasic response to particle exposure. After 4 h of inhalation, mainly heat shock proteins were induced, whereas after 24 h, different immunomodulatory proteins (osteopontin, galectin-3 and lipocalin-2) were upregulated in alveolar macrophages and septal cells. In conclusion, these data indicate that inhalation of ultrafine carbon particles triggers a biphasic pro-inflammatory process in the lung, involving the activation of macrophages and the upregulation of immunomodulatory proteins.

Ultrafine Particles: Geiser et al. Respond

Nemmar et al. were surprised that we did not cite their study in our article (Geiser et al. 2005) when we referenced state-of-the-art experiments about the translocation of ultrafine particles into secondary organs. We did not cite their human study (Nemmar et al. 2002) because in Figure 2 of their article, they presented clear evidence that a major fraction of the radio-labeled technetium-99 (Tc-99m) came off the Technegas particles. Thus, for methodologic reasons, the fraction of translocated particles could not be determined adequately and was certainly overestimated by Nemmar et al. (2002). This was recently discussed by Kreyling et al. (2004). To briefly illustrate this, we have included Figure 1. Figure 1A shows the original whole-body scintigram published by Nemmar et al. (2002) in which the salivary glands, the thyroid gland, and the urinary bladder are clearly visible, demonstrating that they contain large fractions of the Tc-99m radiolabel. This and the Tc-99m activity in the soft tissue, which shows the contour of the whole body, are clear indications of nonparticulate Tc-99m in the form of pertechnetate. Pertechnetate typically accumulates in these organs, as can be inferred from the Figure 1B, where the same pattern of radiolabel was detected after inhalation of soluble Tc-99m pertechnetate. Figure 1 (A) Gamma camera image after the inhalation of Tc-99m radiolabeled ultrafine carbon particles not controlled for leaching. Reproduced from Nemmar et al. (2002) with permission from Lippincott Williams & Wilkins. (B) Gamma camera image after the ... In the case of inhalation of nonleaching Tc-99m radiolabeled ultrafine carbon particles (Figure 1C), no activity is detectable in these organs or in the soft tissue. Figure 1C shows three images taken from the head (little larynx retention), the thorax (main carbon particle retention in lungs), and the lower abdomen, with a rather faint image of the urinary bladder. A similar pattern has been reported by Brown et al. (2002). In addition, Nemmar et al. are interested in the surface charges of the particles we used for the in vitro studies, because surface charges are likely to be important determinants for the translocation of ultrafine particles as well as for their biologic effects. The polystyrene particles we used for the studies with the macrophages and erythrocytes were either uncharged, amino-modified, or carboxylate-modified (Rothen-Rutishauser B, Gehr P, Schurch S, unpublished data). The surface charges of the gold and titanium particles are not known. We found non-phagocytic uptake of ultrafine particles of all the different materials and surface charges by macrophages and erythrocytes. However, because the aim of our study was not to investigate the effects of surface charges on cellular uptake, we did not measure the actual surface charges of the particles or estimate the total number of particles within cells to quantify particle uptake. We certainly agree with Nemmar et al. on the importance of surface charges for particle-cell interaction, and we hope that we will soon find more literature published on this aspect.

Effects of ultrafine carbon particle inhalation on allergic inflammation of the lung

Epidemiologic studies show that exposure to particulate air pollution is associated with asthma exacerbation. Ultrafine particles (diameter <100 nm) may contribute to these adverse effects. To investigate potential adjuvant activity of inhaled elemental carbon ultrafine particles (EC-UFPs) on allergic airway inflammation. The effects of ultrafine particle inhalation on allergic airway inflammation was analyzed in ovalbumin-sensitized mice and nonsensitized controls. Particle exposure (526 microg/m3, 24 hours) was performed 24, 96, or 168 hours before or 24 or 72 hours after ovalbumin aerosol challenge. Allergic inflammation was analyzed at different time points after allergen challenge by means of bronchoalveolar lavage cell count and cytokine/total protein assays, lung histology, and airway hyperresponsiveness. In sensitized mice, inhalation of ultrafine particles 24 hours before allergen challenge caused a significant increase of bronchoalveolar lavage inflammatory cell infiltrate, protein, IL-4, IL-5, and IL-13 compared with relevant controls. These adjuvant effects were dose- and time-dependent and were still present when particle exposure was performed 4 days before allergen challenge. The adjuvant effect of ultrafine particles was also documented by increased mucus production, peribronchiolar and perivascular inflammation, and enhanced airway hyperresponsiveness. In contrast, particle exposure in sensitized mice after allergen challenge caused only moderate effects, such as a delay of inflammatory infiltrate and a reduction of cytokines in bronchoalveolar lavage fluid. Exposure to ultrafine carbon particles before allergen challenge exerts strong adjuvant effects on the manifestation of allergic airway inflammation. Allergen-sensitized individuals may therefore be more susceptible to detrimental health effects of ultrafine particles.

Oxymetazoline Inhibits Proinflammatory Reactions: Effect on Arachidonic Acid-Derived Metabolites

The nasal decongestant oxymetazoline effectively reduces rhinitis symptoms. We hypothesized that oxymetazoline affects arachidonic acid-derived metabolites concerning inflammatory and oxidative stress-dependent reactions. The ability of oxymetazoline to model pro- and anti-inflammatory and oxidative stress responses was evaluated in cell-free systems, including 5-lipoxygenase (5-LO) as proinflammatory, 15-lipoxygenase (15-LO) as anti-inflammatory enzymes, and oxidation of methionine by agglomerates of ultrafine carbon particles (UCPs), indicating oxidative stress. In a cellular approach using canine alveolar macrophages (AMs), the impact of oxymetazoline on phospholipase A(2) (PLA(2)) activity, respiratory burst and synthesis of prostaglandin E(2) (PGE(2)), 15(S)-hydroxy-eicosatetraenoic acid (15-HETE), leukotriene B(4) (LTB(4)), and 8-isoprostane was measured in the absence and presence of UCP or opsonized zymosan as particulate stimulants. In cell-free systems, oxymetazoline (0.4-1 mM) inhibited 5-LO but not 15-LO activity and did not alter UCP-induced oxidation of methionine. In AMs, oxymetazoline induced PLA(2) activity and 15-HETE at 1 mM, enhanced PGE(2) at 0.1 mM, strongly inhibited LTB(4) and respiratory burst at 0.4/0.1 mM (p < 0.05), but did not affect 8-isoprostane formation. In contrast, oxymetazoline did not alter UCP-induced PLA(2) activity and PGE(2) and 15-HETE formation in AMs but inhibited UCP-induced LTB(4) formation and respiratory burst at 0.1 mM and 8-isoprostane formation at 0.001 mM (p < 0.05). In opsonized zymosan-stimulated AMs, oxymetazoline inhibited LTB(4) formation and respiratory burst at 0.1 mM (p < 0.05). In conclusion, in canine AMs, oxymetazoline suppressed proinflammatory reactions including 5-LO activity, LTB(4) formation, and respiratory burst and prevented particle-induced oxidative stress, whereas PLA(2) activity and synthesis of immune-modulating PGE(2) and 15-HETE were not affected.

Instillation of Six Different Ultrafine Carbon Particles Indicates a Surface Area Threshold Dose for Acute Lung Inflammation in Mice

Increased levels of particulate air pollution are associated with increased respiratory and cardiovascular mortality and morbidity. Some epidemiologic and toxicologic research suggests ultrafine particles (UFPs) (< 100 nm) to be more harmful per unit mass than larger particles. Our study was aimed at a quantitative comparison of acute adverse effects of different types of carbonaceous UFPs at a dose range that causes a moderate inflammatory response in lungs. We used six different particle types (primary particle size 10–50 nm, specific surface area 30–800 m2/g, and organic content 1–20%): PrintexG, Printex90, flame soot particles with different organic content (SootL, SootH), spark-generated ultrafine carbon particles (ufCP), and the reference diesel exhaust particles (DEP) SRM1650a. Mice were instilled with 5, 20, and 50 μg of each particle type, and bronchoalveolar lavage was analyzed 24 hr after instillation for inflammatory cells and the level of proinflammatory cytokines. At respective mass-doses, particle-caused detrimental effects ranked in the following order: ufCP > SootL ≥ SootH > Printex90 > PrintexG > DEP. Relating the inflammatory effects to the particle characteristics—organic content, primary particle size, or specific surface area—demonstrates the most obvious dose response for particle surface area. Our study suggests that the surface area measurement developed by Brunauer, Emmett, and Teller is a valuable reference unit for the assessment of causative health effects for carbonaceous UFPs. Additionally, we demonstrated the existence of a threshold for the particle surface area at an instilled dose of approximately 20 cm2, below which no acute proinflammatory responses could be detected in mice.

Aggregates of ultrafine particles impair phagocytosis of microorganisms by human alveolar macrophages

We investigated whether exposure of alveolar macrophages to aggregates of ultrafine carbon particles affected subsequent phagocytosis of microorganisms. Human alveolar macrophages were obtained by bronchoalveolar lavage and exposed to aggregates of ultrafine carbon particles or diesel exhaust particles (DEP) for 20 h before measurements of phagocytosis. The particle loads were estimated to be comparable to those of air pollution exposure with established health effects in humans. Phagocytotic activity was measured as attachment and ingestion of four different test particles (amorphous silica particles, yeast cells from Candida albicans, and Cryptococcus neoformans opsonized with specific IgG or fresh serum) that bind to scavenger, mannose, Fc, and complement receptors, respectively. Carbon preloading significantly impaired the attachment and ingestion process ( P < 0.01 ) for all particles, except for yeast cells from C. neoformans opsonized with specific IgG. On the average, the accumulated attachment decreased by 30% and the ingested fraction decreased by 10%. Loading of alveolar macrophages with either aggregates of ultrafine DEP or carbon particles impaired the phagocytosis of silica test particles in a similar way. Exposure of human alveolar macrophages to aggregates of carbon or DEP, in concentrations relevant to human environmental exposures, caused significant impairment of phagocytosis of silica particles and microorganisms. The inhibitory effect on particle phagocytosis mediated by four different receptors suggests that air pollution particles cause a general inhibition of macrophage phagocytosis. Such an effect may contribute to increased susceptibility to infections and, for example, result in more exacerbations of asthma and chronic obstructive pulmonary disease.

Cardiovascular Responses in Unrestrained WKY Rats to Inhaled Ultrafine Carbon Particles

Based on epidemiologic observations, the issue of adverse health effects of inhaled ultrafine particles (UFP) is currently under intensive discussion. We therefore examined cardiovascular effects of UFP in a controlled animal exposure on young, healthy WKY rats. Short-term exposure (24 h) to carbon UFPs (38 nm, 180 μg m−3), generated by spark discharging, induced a mild but consistent increase in heart rate (18 bpm, 4.8%), which was associated with a significant decrease in heart-rate variability during particle inhalation. The timing and the transient character of these responses point to a particle induced alteration of cardiac autonomic balance, mediated by a pulmonary receptor activation. After 24 h of inhalation exposure, bronchoalveolar lavage revealed significant but low-grade pulmonary inflammation (clean air 1.9% vs. UFPs 6.9% polymorphonuclear cells) and on histopathology sporadic accumulation of particle-laden macrophages was found in the alveolar region. There was no evidence of an inflammation-mediated increase in blood coagulability, as UFP inhalation did not induce any significant changes in plasma fibrinogen or factor VIIa levels and there were no prothrombotic changes in the lung or the heart at both the protein and mRNA level. Histological analysis revealed no signs of cardiac inflammation or cardiomyopathy. This study therefore provides toxicological evidence for UFP-associated pulmonary and cardiac effects in healthy rats. Our findings suggest that the observed changes are mediated by an altered sympatho-vagal balance in response to UFP inhalation, but do not support the concept of an inflammation-mediated prothrombotic state by UFP.

MARCO expression on pediatric alveolar macrophages.

Phagocytosis of unopsonized pollutant particles by alveolar macrophages (AM) occurs via the macrophage receptor with collagenous structure (MARCO). In a previous study, we demonstrated that the laser scanning cytometer (LSC) can be used to characterize receptor expression in pediatric AM. Since we have demonstrated the presence of ultrafine carbon particles within AM obtained from young infants, we sought to determine MARCO expression by pediatric AM using the LSC, hypothesizing that MARCO expression, and hence the ability to phagocytize pollutant particles, is mature in early infancy. AM from 17 healthy children undergoing elective surgery, between 0.1 and 14.1 years of age, were obtained by bronchoalveolar lavage. Cytocentrifuged AM were dual immunostained with monoclonal antibodies to the panmacrophage marker CD68, and either MARCO or an appropriate isotypic control. Expression of MARCO was quantified using LSC and adjusted for cell area. Intensity of immunostaining was compared to each child's own isotypic control. MARCO was present on 98% of pediatric AM. There was no association between age and either intensity of MARCO expression or the proportion of cells expressing MARCO. We conclude that there is no developmental immaturity in the major receptor responsible for unopsonized particle uptake in healthy young children.

Ultrafine particle deposition in subjects with asthma.

Ambient air particles in the ultrafine size range (diameter < 100 nm) may contribute to the health effects of particulate matter. However, there are few data on ultrafine particle deposition during spontaneous breathing, and none in people with asthma. Sixteen subjects with mild to moderate asthma were exposed for 2 hr, by mouthpiece, to ultrafine carbon particles with a count median diameter (CMD) of 23 nm and a geometric standard deviation of 1.6. Deposition was measured during spontaneous breathing at rest (minute ventilation, 13.3 +/- 2.0 L/min) and exercise (minute ventilation, 41.9 +/- 9.0 L/min). The mean +/- SD fractional deposition was 0.76 +/- 0.05 by particle number and 0.69 +/- 0.07 by particle mass concentration. The number deposition fraction increased as particle size decreased, reaching 0.84 +/- 0.03 for the smallest particles (midpoint CMD = 8.7 nm). No differences between sexes were observed. The deposition fraction increased during exercise to 0.86 +/- 0.04 and 0.79 +/- 0.05 by particle number and mass concentration, respectively, and reached 0.93 +/- 0.02 for the smallest particles. Experimental deposition data exceeded model predictions during exercise. The deposition at rest was greater in these subjects with asthma than in previously studied healthy subjects (0.76 +/- 0.05 vs. 0.65 +/- 0.10, p < 0.001). The efficient respiratory deposition of ultrafine particles increases further in subjects with asthma. Key words: air pollution, asthma, deposition, dosimetry, inhalation, ultrafine particles.

Generation of Ultrafine Particles by Spark Discharging

Ultrafine carbon, metal, and metal oxide particles were generated with a commercially available spark generator designed for the production of carbon particles. Aerosols with number concentrations up to 107 cm−3 were produced at flow rates up to 150 lpm. Lognormal size distributions with modal diameters in the range of 18–150 nm and geometric standard deviations of about 1.5 were obtained. The chemical composition, size, number concentration, morphology, and surface area of the particles were varied, and the generation of particles with fixed characteristics could be maintained over many hours. The particle characteristics, however, could not be varied independently. For a certain chemical composition only size and number concentration were variable; morphology and surface area were fixed regardless of particle size. The particles grow by coagulation of primary particles formed by nucleation. The coagulated particles can either stick together and maintain their identity or fuse together and lose their ide...

Effect of Particulate and Gaseous Pollutants on Spontaneous Arrhythmias in Aged Rats

Epidemiology studies suggest that exposure to air pollution increases the frequency of cardiac arrhythmias. A limitation of these studies is that it is difficult to link an increased risk of arrhythmias to a specific air pollutant. Animal exposure studies offer the opportunity to examine the effects of concentrated ambient fine particulate matter (PM), ultrafine PM, and copollutant gases separately. Male Fischer 344 rats, aged 18 mo, with implanted electrocardiograph (ECG) transmitters were used to determine the effects of PM on the frequency of arrhythmias. We found that old F344 rats had many spontaneous arrhythmias. An arrhythmia classification system was developed to quantify arrhythmia frequency. Arrhythmias were broadly grouped into two categories: premature beats and delayed beats. The rats were exposed to concentrated ambient PM (CAPS) or air for 4 h. The rats were exposed twice with a crossover design so each rat could serve as its own control. The CAPS concentrations were 160 μ g/m3 and 200 μ g/m3 for the first and second exposures, respectively. There was a significant increase in the frequency of irregular and delayed beats after exposure to CAPS. The same rats were subsequently exposed to laboratory-generated ultrafine carbon particles, to SO2, or to air with a repeated crossover design. In these experiments there was no significant change in the frequency of any category of spontaneous arrhythmia following exposure to ultrafine carbon or SO2. Thus, this study adds supporting evidence that acute exposure to elevated levels of ambient PM increases the frequency of cardiac arrhythmias.

Systemic Effects of Inhaled Ultrafine Particles in Two Compromised, Aged Rat Strains

Epidemiological studies associate morbidity and mortality with exposure to particulate air pollution in elderly individuals with existing cardiopulmonary disease. These associations led to the hypothesis that inhaled particles can exert adverse effects outside of the lung, particularly on the cardiovascular system. We tested this hypothesis by examining the pulmonary and peripheral effects of inhaled ultrafine carbon particles in old rats that were injected with endotoxin (lipopolysaccharide, LPS) to model systemic gram-negative bacterial infection. Fischer 344 rats (23 mo) and spontaneously hypertensive (SH) rats (11–14 mo) were injected with LPS (2 mg/kg, ip) immediately before being exposed to inhaled ultrafine carbon particles for 6 h (150 μg/m3, CMD = 36 nm). Controls were injected with sterile saline or were sham exposed. Twenty-four hours after LPS injection, bronchoalveolar lavage (BAL) fluid, cells, and blood were obtained to assess endpoints of inflammation, oxidant stress, coagulability, and the acute-phase response. LPS did not cause an influx of neutrophils (PMNs) into the alveolar space, but did increase the number and percentage of circulating PMNs and the concentration of plasma fibrinogen in both rat strains. Inhaled ultrafine particles did not induce lung inflammation in either rat strain. In both strains, ultrafine particles (UFP) were found to decrease the number of blood PMNs, increase the intracellular oxidation of a fluorescent dye (DCFD) in blood PMNs, and affect plasma thrombin–anti-thrombin (TAT) complex and fibrinogen levels. UFP were also found to interact with ip LPS with respect to plasma TAT complex levels and blood PMN DCFD oxidation. Differences between the two rat strains were also found for TAT complex levels, BAL cell reactive oxygen species release, and DCFD oxidation in both BAL macrophages and blood PMNs. These results suggest that inhaled ultrafine carbon particles inhaled at concentrations mimicking high episodic increases in urban air can exert extrapulmonary effects in old rats and that they can change the systemic response to an inflammatory stimulus.