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Respiratory ailments have plagued occupational and public health communities exposed to World Trade Center (WTC) dust since the September 11, 2001 attack on the Twin Towers in Lower Manhattan. We proposed that these ailments were proposed to be induced by inhalation exposure to WTC particulate matter (WTC PM ), that was released during the collapse of the buildings and its subsequent resuspension during cleanup. We investigated this hypothesis using both an in vitro and an in vivo mouse intranasal (IN) exposure models to identify the inflammatory potential of WTC PM with specific emphasis on respiratory and endothelial tissue responses. The in vitro exposure studies found WTC PM exposure to be positively correlated with cytotoxicity and increased NO 2 - production in both BEAS-2B pulmonary epithelial cells and THP-1 macrophage cells. The in vivo C57BL/6 mouse studies found significant increases in inflammatory markers including increases in polymorphonuclear neutrophil (PMN) influx into nasal and bronchoalveolar lavage fluids (NLF and BALF), as well as increased levels of total protein and cytokine/chemokines levels. Concurrently, NLF, BALF, and serum NO 2 - levels exhibited significant homeostatic temporal deviations as well as temporal myograohic aortic dysfunction in myography studies. Respiratory exposure to- and evidence -based retention of- WTC PM may have contributed to chronic systemic effects in exposed mice that r resembled to observed effects in WTC PM -exposed human populations. Collectively, these findings are reflective of WTC PM exposure and its effect(s) on respiratory and aortic tissues, highlighting potential dysfunctional pathways that may precipitate inflammatory events, while simultaneously altering homeostatic balances. The tight interplay between these balances, when chronically altered, may contribute to- or result in- chronically diseased pathological states., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Epidemiological studies associate inhalation of fine-sized particulate matter (PM 2.5 ) during pregnancy with preterm birth (PTB) and low birth weight (LBW) but disagree over which time frames are most sensitive, or if effects are cumulative., Objectives: Our objective was to provide experimental plausibility for epidemiological observations by testing the hypothesis that exposure to PM 2.5 during discrete periods of pregnancy results in PTB and LBW., Methods: For the first study, timed-pregnant B6C3F1 mice were exposed to concentrated ambient PM 2.5 (CAPs) or filtered air (FA) throughout pregnancy [6 h/d from gestational day (GD) 0.5 through GD16.5]. A follow-up study examined the effects of CAPs exposure during discrete gestational periods (1: GD0.5–5.5; 2: GD6.5–14.5; 3: GD14.5–16.5; 4: GD0.5–16.5) aligning to milestones during human development., Results: In the first experiment, exposure to 160 μg CAPs/m 3 throughout pregnancy decreased gestational term by 0.5 d (∼1.1  wk decrease for humans) and birth weight by 11.4% compared with FA. The follow-up experiment investigated timing of CAPs exposure (mean concentrations at 178, 193, 171, and 173 μg/m 3 for periods 1–4, respectively). Pregnancy was significantly shortened (vs. FA) by ∼0.4d when exposure occurred during gestational periods 2 and 4, and by ∼0.5d if exposure occurred during period 3. Exposure during periods 1, 2, and 4 reduced birth weight by ∼10% compared with FA, and placental weight was reduced (∼8%) on GD17.5 if exposure occurred only during period 3., Conclusions: Adverse PM 2.5 -induced outcomes such as PTB and LBW are dependent upon the periods of maternal exposure. The results of these experimental studies could contribute significantly to air pollution policy decisions in the future. https://doi.org/10.1289/EHP1029.

In this unit, the need for laboratory-based inhalation toxicology studies, the historical background on adverse health effects of airborne toxicants, and the benefits of advance planning for the building of analytic options into the study design to maximize the scientific gains to be derived from the investments in the study are outlined. The following methods are described: (1) the generation and characterization of exposure atmospheres for inhalation exposures in humans and laboratory animals; (2) the delivery and distribution into and within whole-body exposure chambers, head-only exposure chambers, face-masks, and mouthpieces or nasal catheters; (3) options for on-line functional assays during and between exposures; and (4) options for serial non-invasive assays of response. In doing so, a description beyond exposures to single agents and simple mixtures is presented, and included are methods for evaluating biological responses to complex environmental mixtures. It is also emphasized that great care should be taken in the design and execution of such studies so that the scientific returns can be maximized both initially, and in follow-up utilization of archived samples of the exposure atmospheres, excreta, and tissues collected for histology., (Copyright © 2015 John Wiley & Sons, Inc.)

Introduction: The discovery of endothelial progenitor cells (EPCs) may help to explain observed cardiovascular effects associated with inhaled nickel nanoparticle exposures, such as increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone and potentiated atherosclerosis in murine species., Methods: Following an acute whole body inhalation exposure to 500 µg/m(3) of nickel nanoparticles for 5 h, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells (CEPCs), circulating endothelial cells (CECs) and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the exposure., Results and Conclusions: Acute exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation (CEPCs). CECs were significantly elevated indicating that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. These results coincided with a decrease in the mRNA of receptors involved in EPC mobilization and homing. These data provide new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration (OSHA) permissible exposure limit may adversely affect EPCs and exacerbate cardiovascular disease states.

First responders (FRs) present at Ground Zero within the critical first 72 h after the World Trade Center (WTC) collapse have progressively exhibited significant respiratory injury. The majority (>96%) of WTC dusts were >10 μm and no studies have examined potential health effects of this size fraction. This study sought to develop a system to generate and deliver supercoarse (10-53 μm) WTC particles to a rat model in a manner that mimicked FR exposure scenarios. A modified Fishing Line generator was integrated onto an intratracheal inhalation (ITIH) system that allowed for a bypassing of the nasal passages so as to mimic FR exposures. Dust concentrations were measured gravimetrically; particle size distribution was measured via elutriation. Results indicate that the system could produce dusts with 23 μm mass median aerodynamic diameter (MMAD) at levels up to ≥1200 mg/m(3). To validate system utility, F344 rats were exposed for 2 h to ≈100 mg WTC dust/m(3). Exposed rats had significantly increased lung weight and levels of select tracer metals 1 h after exposure. Using this system, it is now possible to conduct relevant inhalation exposures to determine adverse WTC dusts impacts on the respiratory system. Furthermore, this novel integrated Fishing Line-ITIH system could potentially be used in the analyses of a wide spectrum of other dusts/pollutants of sizes previously untested or delivered to the lungs in ways that did not reflect realistic exposure scenarios.

Objectives: Studies have shown that chronic exposure to ambient fine particulate matter (less than 2.5 µm in aerodynamic diameter, PM₂.₅) pollution induces insulin resistance through alterations in inflammatory pathways. It is critical to study how the immune system responds to this stimulant, which has been linked to cardiovascular and autoimmune diseases, but few studies have been focused on such involvement of both neutrophils and monocytes in a timely manner. We hypothesized that the neutrophil was involved in the inflammatory response to air pollution., Methods and Results: C57BL/6 mice were exposed to PM₂.₅ or filtered air (6 hours/day, 5 days/week) for 5, 14, and 21 days, respectively, in Columbus, OH. At the end of each of the exposure periods, we investigated the inflammatory response through flow cytometry, histology, intravital microscopy, and real-time PCR. PM₂.₅-exposed mice demonstrated a significant inflammatory response after 5 days of exposure. In the lung tissue and bronchoalveolar lavage fluid, monocytes/macrophages showed a transient response, while neutrophils showed a cumulative response. In addition, exposure to PM₂.₅ resulted in elevation of the monocyte chemoattractant protein 1 (MCP-1) cytokine, a monocyte/macrophage attractant in blood, at an early stage of exposure., Conclusions: These findings suggest that PM₂.₅ exposure induces the inflammatory responses from both macrophages and neutrophils involvement.

Background: It has been well recognized that toxicity of fine ambient air particulate matter (PM(2.5)) may depend on its chemical constituents, including components such as soluble metals that may theoretically exert distinctive effects. We have recently demonstrated an important effect of PM(2.5) on metabolic function. Since transition metals, such as nickel (Ni), represent an important component of exposure in certain environments, and may significantly influence the toxicity of inhalational exposure, we investigated the effects of Ni as a variable component of ambient PM(2.5) exposure., Methods: Male ApoE knockout mice were exposed to filtered air (FA), fine-sized nickel sulfate particles alone (Ni) at 0.44 μg/m(3), concentrated ambient air PM(2.5) (CAPs) at a mean of 70 μg/m(3), or CAPs+Ni in Tuxedo, NY, 6 hours/day, 5 days/week, for 3 months., Results: Exposure to Ni, irrespective of co-exposure to CAPs, resulted in body weight gain, while exposure to CAPs+Ni significantly enhanced fasting glucose and worsened insulin resistance measures (HOMA-IR), when compared with exposure to CAPs alone. CAPs+Ni exposure induced a significant decrease in phosphorylation of AMP-activated protein kinase (AMPK) α. Exposure to Ni or CAPs+Ni significantly induced microcirculatory dysfunction and increased monocytic cell infiltration into lung and adipose, and decreased uncoupling protein 1 expression at gene and protein levels and several brown adipocyte-specific genes in adipose tissue., Conclusions: Ni exposure has effects on metabolic and inflammatory parameters that are comparable to that of CAPs. Additionally, Ni synergistically exacerbates CAPs-induced adverse effects on some of, but not all of, these parameters, that may be mediated via the AMPK signaling pathway. These findings have important implications for inhaled transition metal toxicity that may exert synergistic effects with other PM(2.5) components.

The total surface area is known to be an effective exposure metric for predicting the lung toxicity of low solubility nanoparticles (NPs). However, if NPs are dissolved quickly enough in the lungs, the mass may be correlated with the toxicity. Recent studies have found that the toxicity of zinc oxide (ZnO) NPs was caused by the release of zinc ions. Thus, we hypothesized that mass could be used as an exposure metric for the toxicity of ZnO NPs. Healthy Sprague-Dawley rats were exposed to a low, moderate, or high dose of 35 and 250 nm ZnO particles or filtered air. Bronchoalveolar lavage fluid was collected to determine lung inflammation, injury and oxidative stress. The lung inflammation induced by ZnO particles according to different concentration metrics, including number, mass and surface area, was compared. The mass concentration was significantly correlated with the percentage of neutrophils (R(2) = 0.84), number of neutrophils (R(2) = 0.84) and total cells (R(2) = 0.73). Similarly, surface area concentration was significantly correlated with the percentage of neutrophils (R(2) = 0.94), number of neutrophils (R(2) = 0.81) and total cells (R(2) = 0.76). There was no correlation between the number and lung inflammation. We found that both mass and surface area were effective as metrics for the toxicity of ZnO NPs, although only surface area was previously indicated to be an effective metric. Our results are also consistent with recent study results that ZnO NPs and released zinc ions may play a role mediating the toxicity of NPs.

We have previously shown that chronic exposure to ambient fine particulate matter (less than 2.5 μm in aerodynamic diameter, PM₂.₅) pollution in conjunction with high-fat diet induces insulin resistance through alterations in inflammatory pathways. In this study, we evaluated the effects of PM₂.₅ exposure over a substantive duration of a rodent's lifespan and focused on the impact of long-term exposure on adipose structure and function. C57BL/6 mice were exposed to PM₂.₅ or filtered air (FA) (6 h/day, 5 days/week) for duration of 10 months in Columbus, OH. At the end of the exposure, PM₂.₅-exposed mice demonstrated insulin resistance (IR) and a decrease in glucose tolerance compared with the FA-exposed group. Although there were no significant differences in circulating cytokines between PM₂.₅- and FA-exposed groups, circulating adiponectin and leptin were significantly decreased in PM₂.₅-exposed group. PM₂.₅ exposure also led to inflammatory response and oxidative stress as evidenced by increase of Nrf2-regulated antioxidant genes. Additionally, PM₂.₅ exposure decreased mitochondrial count in visceral adipose and mitochondrial size in interscapular adipose depots, which were associated with reduction of uncoupling protein 1 (UCP1) expression and downregulation of brown adipocyte-specific gene profiles. These findings suggest that long-term ambient PM₂.₅ exposure induces impaired glucose tolerance, IR, inflammation, and mitochondrial alteration, and thus, it is a risk factor for the development of type 2 diabetes.

Background: Previous studies have reported relationships between adverse respiratory health outcomes and residential proximity to traffic pollution, but have not shown this at a personal exposure level., Objective: We compared, among inner-city children with asthma, the associations of adverse asthma outcome incidences with increased personal exposure to particulate matter mass ≤ 2.5 μm in aerodynamic diameter (PM(2.5)) air pollution versus the diesel-related carbonaceous fraction of PM2.5., Methods: Daily 24-hr personal samples of PM(2.5), including the elemental carbon (EC) fraction, were collected for 40 fifth-grade children with asthma at four South Bronx schools (10 children per school) during approximately 1 month each. Spirometry and symptom scores were recorded several times daily during weekdays., Results: We found elevated same-day relative risks of wheeze [1.45; 95% confidence interval (CI), 1.03-2.04)], shortness of breath (1.41; 95% CI, 1.01-1.99), and total symptoms (1.30; 95% CI, 1.04-1.62) with an increase in personal EC, but not with personal PM(2.5) mass. We found increased risk of cough, wheeze, and total symptoms with increased 1-day lag and 2-day average personal and school-site EC. We found no significant associations with school-site PM(2.5) mass or sulfur. The EC effect estimate was robust to addition of gaseous pollutants., Conclusion: Adverse health associations were strongest with personal measures of EC exposure, suggesting that the diesel "soot" fraction of PM(2.5) is most responsible for pollution-related asthma exacerbations among children living near roadways. Studies that rely on exposure to PM mass may underestimate PM health impacts.

Background: Because associations have been reported between inhaled ambient ultrafine particles and increased risk of cardiopulmonary disease, it has been suggested that inhaled engineered nanoparticles (NPs) may also induce adverse effects on the cardiovascular system., Objective: We examined the long-term cardiovascular effects of inhaled nickel hydroxide NPs (nano-NH) using a sensitive mouse model., Methods: Hyperlipidemic, apoprotein E-deficient (ApoE-/-) mice were exposed to nano-NH at either 0 or 79 μg Ni/m3, via a whole-body inhalation system, for 5 hr/day, 5 days/week, for either 1 week or 5 months. We measured various indicators of oxidative stress and inflammation in the lung and cardiovascular tissue, and we determined plaque formation on the ascending aorta., Results: Inhaled nano-NH induced significant oxidative stress and inflammation in the pulmonary and extrapulmonary organs, indicated by up-regulated mRNA levels of certain antioxidant enzyme and inflammatory cytokine genes; increased mitochondrial DNA damage in the aorta; significant signs of inflammation in bronchoalveolar lavage fluid; changes in lung histopathology; and induction of acute-phase response. In addition, after 5-month exposures, nano-NH exacerbated the progression of atherosclerosis in ApoE-/- mice., Conclusions: This is the first study to report long-term cardiovascular toxicity of an inhaled nanomaterial. Our results clearly demonstrate that long-term exposure to inhaled nano-NH can induce oxidative stress and inflammation, not only in the lung but also in the cardiovascular system, and that this stress and inflammation can ultimately contribute to progression of atherosclerosis in ApoE-/- mice.

Introduction: Particulate matter (PM), specifically nickel (Ni) found on or in PM, has been associated with an increased risk of mortality in human population studies and significant increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone, and potentiated atherosclerosis in murine exposures. Recently, murine inhalation of Ni nanoparticles have been shown to cause pulmonary inflammation that affects cardiovascular tissue and potentiates atherosclerosis. These adverse cardiovascular outcomes may be due to the effects of Ni on endothelial progenitor cells (EPCs), endogenous semi-pluripotent stem cells that aid in endothelial repair. Thus, we hypothesize that Ni nanoparticle exposures decrease cell count and cause impairments in function that may ultimately have significant effects on various cardiovascular diseases, such as, atherosclerosis., Methods: Experiments involving inhaled Ni nanoparticle exposures (2 days/5 h/day at ∼1200 µg/m(3), 3 days/5 h/day at ∼700 µg/m(3), and 5 days/5 h/day at ∼100 µg/m(3)), were performed in order to quantify bone marrow resident EPCs using flow cytometry in C57BL/6 mice. Plasma levels of human stromal cell-derived factor 1α (SDF-1α) and vascular endothelial growth factor (VEGF) were assessed by enzyme-linked immunosorbent assay and in vitro functional assessments of cultured EPCs were conducted., Results and Conclusions: Significant EPC count differences between exposure and control groups for Ni nanoparticle exposures were observed. Differences in EPC tube formation and chemotaxis were also observed for the Ni nanoparticle exposed group. Plasma VEGF and SDF-1α differences were not statistically significant. In conclusion, this study shows that inhalation of Ni nanoparticles results in functionally impaired EPCs and reduced number in the bone marrow, which may lead to enhanced progression of atherosclerosis.

Background: The use of nanoparticles (NPs) in technological applications is rapidly expanding, but the potential health effects associated with NP exposure are still largely unknown. Given epidemiological evidence indicating an association between inhaled ambient ultrafine particles and increased risk of cardiovascular disease morbidity and mortality, it has been suggested that exposure to NPs via inhalation may induce similar cardiovascular responses., Methods: Male C57BL/6 mice were exposed via whole-body inhalation to either filtered air (FA) or nickel hydroxide (NH) NPs (100, 150, or 900 µg/m(3)) for 1, 3, or 5 consecutive days (5 h/day). At 24-h post-exposure, vascular function in response to a vasoconstrictor, phenylephrine (PE), and a vasodilator, acetylcholine (ACh), was measured in the carotid artery., Results: Carotid arteries from mice exposed to all concentrations of NH-NPs showed statistically significant differences in graded doses of PE-induced contractile responses compared with those from FA mice. Similarly, vessels from NH-NP-exposed mice also demonstrated impaired vasorelaxation following graded doses of ACh as compared with FA mice., Conclusions: These results suggest that short-term exposure to NH-NPs can induce acute endothelial disruption and alter vasoconstriction and vasorelaxation. These findings are consistent with other studies assessing vascular tone and function in the aorta, coronary, and mesenteric vessels from mice exposed to motor vehicular exhaust and concentrated ambient particles.

Ambient PM(2.5) (particulate matter with an aerodynamic diameters of less than 2.5 mum) is associated with alterations in the autonomic nervous system and cardiac function, but there are significant response variations. The authors simultaneously studied the effects of concentrated PM(2.5) (CAPs) in Sterling Forest (SF; dominated by long-range transported PM) and at the Mount Sinai School of Medicine (MS; rich in Ni and elemental/organic carbon [EC/OC]) in Manhattan, NY. ApoE(-/-) mice (n = 8/group) were exposed to filtered air or CAPs (average 133 and 123 microg/m(3) in SF and MS, respectively) for 6 h/day, 5 days/week for 6 months. Electrocardiogram (ECG) tracings were monitored using telemetry. At MS, current day CAPs mass was negatively associated with short-term changes in heart rate (HR), and positively with HR variability (HRV). At SF, CAPs mass was positively associated with HR, and negatively with HRV. At MS, HR and HRV changes were associated with PM(2.5) components associated with residual oil combustion > long-range transport > traffic > FeMn > incineration > soil, and fireworks had no associations. At SF, HR and HRV were associated with long-range transport > Ni refinery > soil > residual oil combustion/traffic. At both sites, there were cardiac function associations with PM(2.5), but not EC. At MS, there were associations with Ni and P, whereas at SF, they were with a mixture of long-range transported PM, crustal material, and combustion products. Thus subchronic CAPs exposures at locations with different particle compositions produced different effects on cardiac function in ApoE(-/-) mice.

Environmental tobacco smoke (ETS) and ambient air fine particulate matter (PM(2.5)) are both complex mixtures that have important adverse effects on the cardiovascular system. Although exposures to these complex mixtures have been studied individually, direct comparisons between the two has not been performed. In this study, the authors employed a novel, noninvasive ultrasound biomicroscopy method (UBM) to assess the effects of long-term, low-concentration inhalations of side-stream smoke (SS) and concentrated ambient PM(2.5) (CAPs) on plaque progression. ApoE(-/-) mice (n = 8/group) on high-fat chow (HFC), or normal chow (NC), were exposed to SS (PM = 450 microg/m(3)) or filtered air (FA) for 6 h/day, 5 days/week, for 6 months; CAPs exposure was at 134 microg/m(3) (NC only). Mortality during the SS exposure was greater in the HFC than in the NC, and SS significantly enhanced the effects of diet. No mortality was observed in CAPs-exposed mice. At 4 and 6 months, SS produced the greatest change in plaque area in the left common carotid artery (CCA) in HFC as compared to FA or NC, but not in the brachiocephalic artery. In contrast, CAPs exposure significantly enhanced plaque areas in brachiocephalic and left CCA at 3 and 6 months of exposure. The effect of SS was comparable in magnitude to that produced by CAPs at an average PM(2.5) mass concentration that was only 30% as high. In light of the employment of the same animal model, uniform inhalation exposure protocols, time schedules, a noninvasive monitoring protocol, and a parallel study design, these findings have broad applicability.

Epidemiology studies have reported associations between increased mortality and morbidity with exposure to particulate air pollution, particularly within individuals with preexisting cardiovascular disease (CVD). Clinical and toxicological studies have provided evidence that exposure to ambient air particulate matter (PM) impacts CVD by increasing plaque size. It is unclear whether PM-induced increased plaque size is associated with molecular disease progression. This study examines molecular profiles within plaques recovered from ApoE(-/-) mice exposed to concentrated ambient air particles (CAPs) to determine whether pulmonary deposition of PM contributes to molecular alterations leading to vascular disease progression. Laser capture microdissection was used to recover atherosclerotic plaques from ApoE(-/-) male mice exposed daily for 5 mo to filtered air or CAPs. Alterations in mRNA expression was assessed in microdissected plaques of CAPs-exposed and air controls using the Affymetrix microarray platform. Bioinformatic analysis indicated alterations in 611 genes: 395 genes downregulated and 216 genes upregulated. Gene ontology revealed CAPs-induced changes to inflammation, proliferation, cell cycle, hematological system, and cardiovascular pathways. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) verified microarray data also revealing gene expression alterations undetected by the microarray analysis, i.e., decreased expression of alpha-actin for smooth muscle cells, and increased expression of the macrophage marker Cd68 and of beta-actin. Comparison of CAPs-induced gene expression profiles demonstrated consistency with previously published gene expression profiles in the ApoE(-/-) mouse model and humans associated with plaque progression. These results indicate that exposure to fine PM induces molecular alterations associated with vascular disease progression and provides insight into potential biological pathways responsible for this effect.

We review literature that provides insights on health-related effects observed in laboratory-based inhalation studies in humans and laboratory animals using concentrated ambient air particulate matter (CAPs) in the fine, thoracic coarse, and ultrafine size ranges. The CAPs studies are highly informative on the health effects of ambient air particulate matter (PM) because they represent realistic PM exposure mixtures. When PM components are also analyzed and regressed against the effects, they can sometimes be used to identify influential individual components or source-related mixtures responsible for the effects. Such CAPs inhalation studies are analogous to epidemiological studies of human populations for which both health-related effects were observed and PM composition data were available for multi-pollutant regression analyses or source apportionment. Various acute and chronic health-related effects have occurred in short- and long-term CAPs inhalation studies in the cardiovascular, nervous, hepatic, and pulmonary systems, as well as changes in markers of the metabolic syndrome, and many correspond to effects associated with ambient air PM exposures in epidemiological studies. In addition, many CAPs studies have been conducted in coordination with in vitro studies that have identified biomarkers indicative of the underlying biological mechanisms that account for the responses.

Objective: Fine particulate matter <2.5 microm (PM(2.5)) has been implicated in vasoconstriction and potentiation of hypertension in humans. We investigated the effects of short-term exposure to PM(2.5) in the angiotensin II (AII) infusion model., Methods and Results: Sprague-Dawley rats were exposed to PM(2.5) or filtered air (FA) for 10 weeks. At week 9, minipumps containing AII were implanted and the responses studied over a week. Mean concentration of PM(2.5) inside the chamber was 79.1+/-7.4 microg/m(3). After AII infusion, mean arterial pressure was significantly higher in PM(2.5)-AII versus FA-AII group. Aortic vasoconstriction to phenylephrine was potentiated with exaggerated relaxation to the Rho-kinase (ROCK) inhibitor Y-27632 and increase in ROCK-1 mRNA levels in the PM(2.5)-AII group. Superoxide (O(2).(-)) production in aorta was increased in the PM(2.5)-AII compared to the FA group, inhibitable by apocynin and L-NAME with coordinate upregulation of NAD(P)H oxidase subunits p22(phox) and p47(phox) and depletion of tetrahydrobiopterin. In vitro exposure to ultrafine particles (UFP) and PM(2.5) was associated with an increase in ROCK activity, phosphorylation of myosin light chain, and myosin phosphatase target subunit (MYPT1). Pretreatment with the nonspecific antioxidant N-acetylcysteine and the Rho kinase inhibitors (Fasudil and Y-27632) prevented MLC and MYPT-1 phosphorylation by UFP suggesting a O(2)(.-)-mediated mechanism for PM(2.5) and UFP effects., Conclusions: Short-term air pollution exaggerates hypertension through O(2)(.-)-mediated upregulation of the Rho/ROCK pathway.

Background: Individuals may develop tolerance to the induction of adverse pulmonary effects following repeated exposures to inhaled toxicants. Previously, we demonstrated that genetic background plays an important role in the development of pulmonary tolerance to inhaled zinc oxide (ZnO) in inbred mouse strains, as assessed by polymorphonuclear leukocytes (PMNs), macrophages, and total protein in bronchoalveolar lavage (BAL) phenotypes. The BALB/cByJ (CBy) and DBA/2J (D2) strains were identified as tolerant and non-tolerant, respectively. The present study was designed to identify candidate genes that control the development of pulmonary tolerance to inhaled ZnO., Methods: Genome-wide linkage analyses were performed on a CByD2F2 mouse cohort phenotyped for BAL protein, PMNs, and macrophages following 5 consecutive days of exposure to 1.0 mg/m3 inhaled ZnO for 3 hours/day. A haplotype analysis was carried out to determine the contribution of each quantitative trait locus (QTL) and QTL combination to the overall BAL protein phenotype. Candidate genes were identified within each QTL interval using the positional candidate gene approach., Results: A significant quantitative trait locus (QTL) on chromosome 1, as well as suggestive QTLs on chromosomes 4 and 5, for the BAL protein phenotype, was established. Suggestive QTLs for the BAL PMN and macrophage phenotypes were also identified on chromosomes 1 and 5, respectively. Analysis of specific haplotypes supports the combined effect of three QTLs in the overall protein phenotype. Toll-like receptor 5 (Tlr5) was identified as an interesting candidate gene within the significant QTL for BAL protein on chromosome 1. Wild-derived Tlr5-mutant MOLF/Ei mice were tolerant to BAL protein following repeated ZnO exposure., Conclusion: Genetic background is an important influence in the acquisition of pulmonary tolerance to BAL protein, PMNs, and macrophages following ZnO exposure. Promising candidate genes exist within the identified QTL intervals that would be good targets for additional studies, including Tlr5. The implications of tolerance to health risks in humans are numerous, and this study furthers the understanding of gene-environment interactions that are likely to be important factors from person-to-person in regulating the development of pulmonary tolerance to inhaled toxicants.

Recent epidemiological studies suggest that long-term exposure to particulate matter (PM) causes chronic effects on the cardiovascular system that result in cumulative increases cardiovascular morbidity and mortality. Since atherosclerosis is a progressive irreversible condition and an underlying cause of many cardiovascular diseases, we hypothesized that long-term exposure to PM causes adverse cardiovascular effects by exacerbating atherosclerosis. In this study, we exposed C57- and ApoE-deficient (ApoE-/-) and ApoE, LDLr (DK)-deficient mice to concentrated ambient PM2.5 for 6 h/5 days/wk, for up to 5 mo. The overall mean exposure concentration for these groups of animals was 110 microg/m3. The cross-sectional area of the aorta root of DK mice was examined morphologically using confocal microscopy for the severity of lesion, extent of cellularity, and lipid contents. Aortas from the arch to the iliac bifurcations were also sectioned longitudinally and lesion areas were stained with Sudan IV. All DK mice regardless of exposure had developed extensive lesions in the aortic sinus regions, with lesion areas that covered more than 79% of the total area. In male DK mice, the lesion areas in the aortic sinus regions appeared to be enhanced by concentrated ambient particles (CAPs), with changes approaching statistical significance (p = .06). In addition, plaque cellularity was increased by 28% (p = .014, combined), whereas there were no CAPs-associated changes in the lipid content in these mice. When examining the entire aorta opened longitudinally, both the ApoE-/- and DK mice had prominent areas of severe atherosclerosis covering 40% or more of the lumenal surface. Visual examination of all images suggested that plaques tend to form in clusters concentrating near the aortic arch and and the iliac bifurcations. Quantitative measurements showed that CAPs exposure increased the percentage of aortic intimal surface covered by grossly discernible atherosclerotic lesion by 57% in the ApoE-/- mice (p = .03). Changes produced by CAPs in male (10% increase) or female DK mice (8% increase) were not statistically significant. In this study, we have demonstrated that subchronic exposure to CAPs in mice prone to develop atherosclerotic lesions had a significant impact on the size, severity, and composition of aortic plaque.

This subchronic (6-mo) inhalation study of the effects of concentrated ambient air fine particulate matter (PM2.5) in normal mice (C57) and a murine model of humans with an advanced level of aortic plaque (ApoE-/- or ApoE-/- LDLr-/-) was designed to determine the presence and extent of a variety of health-related responses. The animals were exposed for 6 h/day, 5 day/wk during the spring and summer of 2003 to concentrations that were elevated 10-fold in Tuxedo, NY, a regional background site that is upwind and approximately 50 km west-northwest of New York City. The average PM2.5 concentration during exposure was 110 microgram/m3, and the long-term average was 19.7 microg/m3. There were substantial daily variations in concentration, and we sought evidence both for the influence of peak exposures on acute responses and for the cumulative effects of the prolonged series of exposures. Acute responses were characterized in terms of: (1) short-term electrocardiographic (EKG), core body temperature, and physical activity differences between PM and sham-exposed mice; and (2) in vitro toxicity of a simultaneously collected PM2.5 sample to lung epithelial cells. Cumulative responses to PM2.5 were characterized in terms of changes in heart rate, heart-rate variability, heart-rate variance, aortic plaque density, genetic marker expression, and brain cell distributions. There were no significant changes in the normal mice. The nature and extent of the exposure-related responses that were seen in the ApoE-/- as well as ApoE-/- LDLr-/- mice are described in the articles that follow in this special issue of Inhalation Toxicology.

Normal mice (C57) and mice prone to develop atherosclerosis (ApoE-/-) were implanted with electrocardiograph (EKG), core body temperature, and motion transmitters were exposed daily for 6 h to Tuxedo, NY, concentrated ambient particles (CAPs) for 5 day/wk during the spring and summer of 2003. The series of 5-min EKG monitoring and body-temperature measurements were obtained for each animal in the CAPs and filtered air sham exposure groups. Our hypothesis was that chronic exposure could cause cumulative health effects. We used our recently developed nonparametric method to estimate the daily time periods that mean heart rates (HR), body temperature, and physical activity differed significantly between the CAPs and sham exposed group. CAPs exposure most affected heart rate between 1:30 a.m. and 4:30 a.m. With the response variables being the average heart rate, body temperature, and physical activity, we adopted a two-stage modeling approach to obtain the estimates of chronic and acute effects on the changes of these three response variables. In the first stage, a time-varying model estimated daily crude effects. In the second stage, the true means of the estimated crude effects were modeled with a polynominal function of time for chronic effects, a linear term of daily CAPs exposure concentrations for acute effects, and a random component for unknown noise. A Bayesian framework combined these two stages. There were significant decreasing patterns of HR, body temperature, and physical activity for the ApoE-/- mice over the 5 mo of CAPs exposure, with smaller and nonsignificant changes for the C57 mice. The chronic effect changes of the three response variables for ApoE-/- mice were maximal in the last few weeks. There was also a significant relationship between CAPs exposure concentration and short-term changes of heart rate in ApoE-/- mice during exposure. Response variables were also defined for examining fluctuations of 5-min heart rates within long (i.e., 3-6 h) and short time periods (i.e., approximately 15 min). The results for the ApoE-/- mice showed that heart-rate fluctuation within the longer periods increased to 1.35-fold by the end of exposure experiment, while the heart-rate fluctuation within 15 min decreased to 0.7-fold.

We modified, assembled, tested, and validated the versatile aerosol concentration enrichment system (VACES) developed by Sioutas et al. (1999) for use in a subchronic experiment that involved exposure of mice in vivo and of respiratory epithelial cells in vitro to concentrated ambient particles (CAPs). Since the labor-intensive nose-only exposure regimen is not an option in a long-term experiment, a whole-body exposure mouse chamber was designed specifically for use with the VACES. The exposure system concsists of a stainless-steel (SS) tub with 32 cubicles (1 mouse per cubicle) separated by perforated SS sheets. The tops of these cubicles are covered with perforated plastic sheets to allow telemetry monitoring during the exposure. In each exposure chamber, perforated aluminum tubes are used to distribute CAPs evenly (within 2% difference) throughout the exposure chamber. The exhaust consists of perforated aluminum tubes covered with a urine shield. The modification to the original design of the VACES facilitated the operation of the system in a subchronic study. Mass flow controllers maintain a constant flow rate into the exposure chambers. For a sham control exposure, the identical system is used, except that a HEPA filter at the inlet to the VACES removes 98% of ambient particles. The entire system allow for simultaneous exposure of 64 mice to CAPs, with an equal number of sham-expose mice as controls. Telemetry receives have been modified so that 16 mice per group with electrocardiograph (EKG) transmitters can be monitored during exposure. Furthermore, a BioSampler is used to collect CAPs (one sample per day) for the in vitro exposures. In this article, the assessments of flow and particle distribution of the exposure chamber as well as the performance of the system during the subchronic exposure experiment are described.

Long-term exposure to fine particulate air pollution (PM2.5) has been associated increased risk of death from cardiopulmonary diseases. Cardiac function parameters have also been affected by ambient particulate matter (PM) exposure, including heart-rate variability (HRV), a measure of autonomic function that has been recognized as a well-defined, quantitative indicator of autonomic dysfunction. However, the role of HRV in ambient PM-induced cardiovascular effect is not fully understood. In an accompanying article, we report significant decreasing patterns of heart rate (HR), body temperature, and physical activity for mice lacking apoliproprotein (ApoE-/-) over 5 mo of exposure to concentrated ambient PM (CAPs), with smaller and nonsignificant change for C57 mice. In this article, we report the effects of subchronic CAPs exposure on HRV parameters that are sensitive to cardiac sympathetic and parasympathetic nerve activity. The standard deviation of normal to normal beat intervals (SDNN) and the square root of the mean squared differences of successive RR intervals (RMSSD) in the late afternoon and overnight for the ApoE-/- mice showed a gradual increase for the first 6 wk, a decline for about 12 more wk, and a slight turn upward at the end of the study period. For C57 mice, there were no chronic effect changes of SDNN or RMSSD in the late afternoon, an a slight increase after 6 wk for the overnight period. The response patterns of ApoE-/- mice indicated a perturbation of the homeostatic function in the cardiovascular system (initial enhancement and late depression of the HRV parameters). Our results complement the findings in human panel and controlled CAPs exposure studies in demonstrating that increased levels of particle pollution are able to perturb cardiac autonomic function, which may lead to adverse cardiovascular outcomes.

This study reports that subchronic exposure of Tuxedo, NY concentrated ambient particulates (CAPs) produces neuropathological damage in the brains of Apo E-deficient mice (Apo E-/-). These genetically modified mice are characterized by elevated levels of oxidative stress (OS) in the brain. Microscopic examination of coronal sections of the brain, immunocytochemically stained for dopamineric neurons, indicated that neurons from the substantia nigral nucleus compacta were significantly reduced by 29% in CAPs-exposed Apo E-/- mice relative to air-exposed Apo E-/- controls. In addition, statistically significant increases (p < .05) in immunocytochemically stained astrocytes were noted. The dopaminergic neurons of the nucleus compact are specifically targeted in Parkinson's disease. The present study expands the systems affected by particulate matter to include the brain, and supports an environmental role for the development of neurodegeneration in OS-susceptible individuals.

The purpose of this exploratory study within the integrated subchronic inhalation exposure study (Lippmann et al., 2005) was to identify genes in heart and lung tissue that changed in expression level as a result of subchronic exposure to concentrated ambient particles (CAPs). Identification of CAPs exposure-related changes in gene expression could serve in the formulation of mechanistic hypotheses and/or to suggest possible biomarkers of exposure. In this exploratory study undertaken here, tissues from multiple replicates of ApoE/low-density-lipoprotein double knockout (DK) mice were examined for relative exposure-related changes in gene expression. Due to limited resources, the number of replicates was three for each tissue (lung and heart) of each exposure condition (CAPs or air control). A rigorous comparison of exposure versus control data using the "significance analysis of microarrays" (SAM) method indicated that only one gene was differentially expressed at a significant level. However, when using a less restrictive, nonstatistical analytical treatment of the data, several genes that might be involved in PM-related heart or lung pathology, and/or the circadian rhythm of physiological processes, were identified. A more comprehensive study is required to mre definitively assess differences in gene expression in heart and lung resulting from exposure to CAPs.

Striking regional differences in the prevalence of coal workers' pneumoconiosis (CWP) have been observed but not fully understood. This study investigated the early biological responses of primary lung cells to treatment with coal dusts from various seams. High-density oligoarray technology (GeneChip, Affymetrix, Santa Clara, CA) was used to compile gene expression profiles of primary human bronchial epithelial cells to low concentrations (2 microg/cm(2)) of coals for 6 h or 24 h of treatment. Data showed that a total of 1050 out of 12,000 genes on the chip were altered by 2 coal dusts. The coal from the Pennsylvania (PA) coal-mine region with a high prevalence of CWP altered 908 genes, many more than the coal from Utah (UT) with a low prevalence of CWP, which affected 356 genes. Many genes decreased their expression levels in response to the PA coal at 6 h and/or 24 h of treatment. For example, transferrin receptor, a gene known to control cellular iron uptake, was downregulated in the cells treated with the iron-containing PA coal in order to protect cells from iron overload. The UT coal without bioavailable iron had no such effect. The downregulation patterns of genes were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). This study is one of the first in profiling gene expressions of primary bronchial epithelial cells treated with coals from various seams, which may set stages for future studies on specific genes.

Respiratory-tract infection, specifically pneumonia, contributes substantially to the increased morbidity and mortality among elderly individuals exposed to airborne particulate matter of <10 microm diameter (PM(10)). These epidemiological findings suggest that PM(10) may act as an immunosuppressive factor that can undermine normal pulmonary antimicrobial defense mechanisms. To investigate whether, and how, compromised pulmonary immunocompetence might contribute to increased mortality, two sets of laboratory studies were performed. The first examined the effects of a single inhalation exposure to concentrated ambient PM(2.5) (CAPS) from New York City air on pulmonary/systemic immunity and on the susceptibility of exposed aged rats to subsequent infection with Streptococcus pneumoniae. The second set of studies determined whether CAPS exposure, at a concentration approximating or somewhat greater than the promulgated 24-h NAAQS of 65 microg/m(3), could exacerbate an ongoing infection. Taken together, results demonstrated that a single exposure of healthy animals to CAPS had little effect on pulmonary immune function or bacterial clearance during subsequent challenge with S. pneumoniae. Alternatively, CAPS exposure of previously infected rats significantly increased bacterial burdens and decreased percentages of lavageable neutrophils and proinflammatory cytokine levels compared to those in infected filtered-air-exposed controls. These studies demonstrate that a single exposure to ambient PM(2.5) compromises a host's ability to handle ongoing pneumococcal infections and support the epidemiological findings of increased pneumonia-related deaths in ambient PM-exposed elderly individuals.

The predominant exposure route for nickel compounds is by inhalation, and several studies have indicated the correlation between nickel exposure and respiratory cancers. The tumor-promoting effects of nickel compounds are thought to be associated with their transactivation of transcription factors. We have investigated the possible activation of activator protein-1 (AP-1) and nuclear factor KB (NF-kappaB) in mouse C141 epidermal cells and fibroblasts 3T3 and B82, and human bronchoepithelial BEAS-2B cells in response to nickel compound exposure. Our results show that NF-kappaB activity is induced by nickel exposure in 3T3 and BEAS-2B cells. Conversely, similar nickel treatment of these cells did not induce AP-1 activity, suggesting that nickel tumorigenesis occurs through NF-kappaB and not AP-1. We also investigated the role of NF-kappaB in the induction of Cap43 by nickel compounds using dominant negative mutant Ikappabeta kinase b-KM BEAS-2B transfectants.

After repeated exposures, many individuals develop tolerance to the adverse health effects of inhaled pollutants. Pulmonary tolerance can be characterized as the ability of the lung to withstand the adverse actions of a toxic compound after repeated exposures. To determine whether genetic background is important to the development of pulmonary tolerance to inhaled pollutants, 11 inbred strains of mice were exposed once (1x) or for 5 consecutive days (5x) to 1.0 mg/m(3) of zinc oxide (ZnO). Development of pulmonary tolerance was assessed by measuring polymorphonuclear leukocyte and protein levels in bronchoalveolar lavage fluid and comparing the responses of the 1x and 5x groups. Significant interstrain variation in polymorphonuclear leukocyte and protein responses was observed between the groups with 1x and 5x exposures, which indicates that genetic background has an important role in the development of pulmonary tolerance. The BALB/cByJ strain and the DBA/2J strain were the most tolerant and nontolerant, respectively. The CByD2F1/J offspring were uniformly nontolerant. The development of tolerance was also investigated in BALB/cByJ and DBA/2J mice after 1x and 5x exposure to ozone and aerosolized endotoxin. Discordance in the phenotypic pattern of pulmonary tolerance among strains after exposure to ZnO, ozone, and endotoxin suggested that different mechanisms may be responsible for the development of pulmonary tolerance to these agents.

Significant respiratory-tract exposure to insoluble aluminum compounds, such as alumina (aluminum oxide, Al(2)O(3)), can occur in occupational settings, yet little is known about the temporal pattern of pulmonary clearance of these materials from the lungs with repeated exposures, and potential subsequent translocation to other organs. This study evaluated the clearance pattern of alumina from the lungs of rats, and burdens in selected extrapulmonary organs (brain, bone, liver, spleen, kidney). Rats were instilled with alumina once weekly for 20 wk. Quantification of retention was performed by measuring aluminum burdens in the lungs and extrapulmonary organs during the exposure period, and then weekly for an additional 19 wk after the exposures ended. Lung burdens of aluminum were found to steadily increase during exposure. Clearance of the material following the end of the exposure regime was extremely slow; only approximately 9% of the amount in the lungs following the 20 weekly exposures was cleared by the end of the postexposure period. This study supports the concept of gradual accumulation and long-term retention of aluminum within the respiratory tract of individuals repeatedly exposed to alumina in occupational settings.

Epidemiologic studies have provided strong evidence that episodic exposure to ambient particulate matter is associated with increases in morbidity and mortality. These adverse effects have been demonstrated at concentrations far below the National Ambient Air Quality Standard (NAAQS), and thus, the biological plausibility of these effects has been questioned. For the purpose of exposing test animals to relevant and reproducible exposure concentrations of ambient particulate matter (PM), we have developed a simple and inexpensive concentrator system that can concentrate ambient particles 10-fold. A high-volume blower is used to deliver ambient air to the inlet manifold of a centrifugal concentrator and the entrained particles travel along a concentric annulus formed by a stationary solid outer cylinder and a porous inner cylinder rotating at high speed (up to 12,500 rpm). Suction applied at one end of the porous shaft causes the dispersion medium (air) to pass through the porous cylinder and into the shaft. Since the rotational velocity of airborne particles is comparable to that of the rotating cylinder near its surface, the particles move radially outward due to the centrifugal force, in addition to their motion laterally along the cylinder and inward due to the suction of air into the rotating porous cylinder. The particles reach their highest concentration near the outlet manifold, where they enter the exposure chamber under positive pressure ( approximately 0.4 cm H2O). Except for coarse particle loss due to impaction and diffusional loss of ultrafine particles in the concentrator, the increase in particle concentration is the ratio of the flow rates for the inlet air and the air delivered to the exposure chamber. We have used the centrifugal concentrator to deliver concentrated ambient urban PM to a nose-only exposure chamber and examined the concentrating effect across ambient particle sizes.