Health effects of concentrated ambient air particulate

Critical Reviews in Toxicology, 2009; 39(10): 865–913

REVIEW ARTICLE

Health effects of concentrated ambient air particulate matter (CAPs) and its components Morton Lippmann, and Lung-Chi Chen

Critical Reviews in Toxicology Downloaded from informahealthcare.com by MT Sinai School of Medicine For personal use only.

Department of Environmental Medicine, New York University School of Medicine, Tuxedo, New York, USA

Abstract 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. Keywords: Accumulation mode PM; CAPs; cardiovascular effects; coarse thoracic PM; fine PM; hepatic system effects; nervous system effects; PMx; pulmonary effects; ultrafine PM concentration

Contents Abstract .............................................................................................................................................................................................. 865 1. Introduction ................................................................................................................................................................................... 867 2. Development of methods for conducting CAPs inhalation studies ......................................................................................... 868 3. Review of studies of the effects of PM and components in humans ........................................................................................ 870 3.1. Introduction ............................................................................................................................................................................ 870 3.2. Human CAPs inhalation studies in Chapel Hill, NC ........................................................................................................... 870 3.3. Human CAPs inhalation studies in Los Angeles, CA .......................................................................................................... 871 3.4. Human CAPs inhalation studies in Toronto, Canada ......................................................................................................... 872 3.5. Human CAPs inhalation study in Edinburgh, Scotland ..................................................................................................... 872 3.6. Other laboratory-based inhalation studies in humans with components of ambient air PM ........................................ 872 3.6.1. Diesel engine exhaust ..................................................................................................................................................... 872 3.7. Short-term responses to ambient air PM inhalation exposures in human panel studies ............................................... 873 3.8. Large population-based studies in humans dealing with responses to PM components .............................................. 876 3.9. Exposures of human volunteers via intratracheal instillation ........................................................................................... 878 4. Review of ambient particulate matter studies in laboratory animals and in vitro .................................................................. 878 4.1. Introduction ............................................................................................................................................................................ 878 4.2. Short-term CAPs inhalation studies ..................................................................................................................................... 879 4.2.1. Boston .............................................................................................................................................................................. 879 Address for Correspondence: Morton Lippmann, Department of Environmental Medicine, NYU School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987, USA. Phone: 845-731-3558; Fax: 845-351-5472; E-mail: [email protected] (Received 20 April 2009; revised 11 August 2009; accepted 31 August 2009) ISSN 1040-8444 print/ISSN 1547-6898 online © 2009 Informa UK Ltd DOI: 10.3109/10408440903300080

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4.2.2. Detroit, MI........................................................................................................................................................................ 881 4.2.3. Grand Rapids, MI ............................................................................................................................................................ 881 4.2.4. New York City .................................................................................................................................................................. 881 4.2.5. Tuxedo, NY....................................................................................................................................................................... 881 4.2.6. Research Triangle Park, NC............................................................................................................................................ 881 4.2.7. Los Angeles, CA ............................................................................................................................................................... 882 4.2.8. The Netherlands .............................................................................................................................................................. 882 4.2.9. Yokohama, Japan............................................................................................................................................................. 882 4.3. Longer-term CAPs inhalation studies .................................................................................................................................. 883 4.3.1. Tuxedo, NY....................................................................................................................................................................... 883 4.3.2. New York City .................................................................................................................................................................. 884 4.3.3. Columbus, OH ................................................................................................................................................................. 885 4.3.4. Los Angeles, CA ............................................................................................................................................................... 885 4.4. Inhalation studies in animals with PM components .......................................................................................................... 885 4.4.1. Diesel exhaust.................................................................................................................................................................. 885 4.4.2. Sidestream smoke ........................................................................................................................................................... 886 4.4.3. Transition metals ............................................................................................................................................................ 886 4.5. Lung instillation studies with PM components ................................................................................................................... 887 4.5.1. Ambient air PM ............................................................................................................................................................... 887 4.5.2. Utah Valley dust............................................................................................................................................................... 888 4.5.3. ROFA................................................................................................................................................................................. 888 4.5.4. Tire dust ........................................................................................................................................................................... 889 4.5.5. Metal oxide nanoparticles .............................................................................................................................................. 889 5.0. Organ system responses ............................................................................................................................................................ 890 5.1. Cardiovascular system ........................................................................................................................................................... 890 5.1.1. Summary of human cardiovascular responses to short-term CAPs exposures ........................................................ 890 5.1.2. Summary of animal cardiovascular responses to short-term CAPs exposures ......................................................... 890 5.1.3. Summary of animal cardiovascular responses to long-term CAPs exposures .......................................................... 891 5.1.4. Short- and long-term inhalation studies in animals with high concentrations of PM components....................... 894 5.1.5. Studies of human cardiovascular responses to ambient air PM................................................................................. 894 5.2. Pulmonary system effects...................................................................................................................................................... 894 5.2.1 Summary of human pulmonary responses to short-term CAPs exposures ............................................................... 894 5.2.2. Summary of animal pulmonary responses to short-term CAPs exposures............................................................... 895 5.2.3. Pulmonary effects of PM collected from ambient air and PM sources ...................................................................... 895 5.2.3.1 Effects of PM associated with specific sources of ambient air pollution on pulmonary responses in animals ....................................................................................................................... 895 5.2.3.2. Utah Valley dust........................................................................................................................................................ 897 5.2.3.3. ROFA.......................................................................................................................................................................... 897 5.2.3.4. Tire dust .................................................................................................................................................................... 898 5.2.3.5. Metal oxide nanoparticles ....................................................................................................................................... 898 5.2.3.6. Bacterial challenge ................................................................................................................................................... 898 5.2.4. Studies of human pulmonary responses to ambient air PM....................................................................................... 898 5.3. Hepatic system effects of long-term CAPs inhalation studies ........................................................................................... 898 5.4. Nervous system effects .......................................................................................................................................................... 899 5.4.1. CAPs inhalation ............................................................................................................................................................... 899 5.4.2. Neurological effects of ROFA after instillation.............................................................................................................. 899 6. Coherence of responses to CAPs exposures in humans and animals in vivo.......................................................................... 899 6.1. Cardiovascular responses...................................................................................................................................................... 900 6.1.1. CAPs exposures in humans and animals ...................................................................................................................... 900 6.1.1.1. Acute effects.............................................................................................................................................................. 900 6.1.1.2. Chronic effects.......................................................................................................................................................... 900 6.1.2. Coherence of responses to CAPs exposures in humans and animals in vivo with responses to PM2.5 in epidemiological studies .................................................................................................................................... 900 6.1.2.1. Acute effects.............................................................................................................................................................. 900 6.1.2.2. Chronic effects.......................................................................................................................................................... 900 6.1.3. Coherence of responses to CAPs exposures in humans and animals with responses to PM2.5 and components in lung inhalation and instillation studies ...................................................................................... 900


Health effects of ambient air CAPs

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6.2. Coherence of pulmonary responses..................................................................................................................................... 901 6.2.1. CAPs Exposures in humans and animals ..................................................................................................................... 901 6.2.1.1. Acute effects.............................................................................................................................................................. 901 6.2.1.2. Chronic effects.......................................................................................................................................................... 901 6.2.2. Coherence of pulmonary responses to CAPs exposures in humans and animals in vivo with responses to PM2.5 in epidemiological studies ..................................................................................................... 901 6.2.2.1. Acute effects.............................................................................................................................................................. 901 6.2.2.2. Chronic effects.......................................................................................................................................................... 902 6.2.3. Coherence of pulmonary responses to CAPs exposures in humans and animals in vivo with responses to PM2.5 using in vivo lung inhalation studies ........................................................................ 902 6.2.3.1. Diesel engine exhaust .............................................................................................................................................. 902 6.3. Coherence in other systems .................................................................................................................................................. 902 7. Discussion of the role and contributions of CAPs studies ......................................................................................................... 902 8. Summary of unresolved issues and conclusions ....................................................................................................................... 904 8.1. Where do PM components fit in the larger picture of PM-associated health effects? ..................................................... 904 8.2. Are there specific source categories that can account, at least in part, for health effects associated with PM2.5 components? ................................................................................................................................... 904 8.3. Addressing research needs relating to health effects of components in ambient air particulate matter ...................... 905 8.4. Biological plausibility............................................................................................................................................................. 906 Acknowledgments............................................................................................................................................................................. 907 Glossary.............................................................................................................................................................................................. 907 References.......................................................................................................................................................................................... 908

1. Introduction Studying the health effects of ambient air pollution has been a challenging endeavor for environmental health scientists for many reasons. Epidemiologists have documented statistically significant associations between the routinely measured mass concentrations of particulate matter (PM) and excess mortality, morbidity, lost function, and lost time at work or school, and these statistically significant associations are usually stronger than those with routinely measured pollutant gases (US EPA, 2004, 2008). Although the relative risks (RRs) for mortality and nonscheduled hospital admissions are small, requiring sophisticated mathematical models for analysis in epidemiological studies, the populations studied and the and the populations at risk are quite large (Pope et al., 2009; Schwartz et al, 2008; Eftim et al., 2008; Miller et al., 2007), resulting in potentially very large public health impacts (e.g. thousands of cases annually in the United States). The bulk of this risk appears to be borne by the elderly or those in poor health, or both. It seems highly unlikely that the effects are caused by nonspecific PM mass. Rather, it is likely that some specific chemical components within the PM mixtures are more potent than other components. Th e situation is complicated by the fact that PM is present in the air over a broad range of chemical compositions and particle sizes. Coarse dust particles with aerodynamic diameters above 10 μm do not normally penetrate beyond the larynx, have not been associated with health effects due to routine air pollution exposures, and are not routinely monitored. Particles with aerodynamic diameters below 10 μm, known as PM10, can

deposit along the conductive airways in the thorax, and nearly all of those with aerodynamic diameters below 2.5 μm penetrate into the gas-exchange region where particle retention times are much larger than for those that deposit on the conductive airways. A mucociliary blanket covering the conductive airways facilitates fairly rapid particle removal to the gastrointestinal tract. Furthermore, the smaller particles, known as fine PM or PM2.5, are chemical mixtures that are quite different from the larger ones. The larger particles are mostly mineral in composition, whereas the PM2.5 is composed largely of primary emissions of diesel engine soot particles and secondary aerosol formed by chemical transformations in the atmosphere from fossil fuel combustion products (both inorganic and organic vapors) and organic vapors from natural biologic processes. Most of these particles initially form as ultrafine PM (UFP), but rapidly aggregate into accumulation mode PM in the 0.1–2.5-μm size range. Suspicion concerning adverse health effects has centered on both fossil fuel combustion products and on inorganic compounds containing metals. Most of the mass of these metals is within the PM2.5. A focus has often been on transition metals, such as iron (Fe), vanadium (V), nickel (Ni), chromium (Cr), copper (Cu), and zinc (Zn), or on carbonaceous compounds, on the basis of their ability to generate reactive oxygen species (ROS) in biological tissues. Most of the evidence pointing to the biological effects of metals, elemental carbon (EC), and organic carbon (OC) has come from studies involving exposures of laboratory animals in vivo, or of cells in vitro. We know of no studies involving exposures of laboratory animals in vivo, or of cells


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in vitro to pure chemicals and their compounds, at doses with environmental relevance, that have been positive. On the other hand, some toxicological studies using high PM mass exposures to diluted tailpipe emissions, especially whole diesel engine exhaust (WDE), or to source-related PM mixtures containing multiple metals, such as residual oil fly ash (ROFA) or coal fly ash (CFA), and to concentrated ambient air particles (CAPs) have produced effects that appear to be related to their relatively low contents of metals and carbonaceous material. However, it has been difficult to determine the roles played by the individual components in the effects observed. Also, many laboratory-based studies have used resuspended dusts at relatively high mass concentrations, and the relevance of the effects observed to human ambient air exposures at much lower PM mass levels is therefore uncertain. Although effects found in high-dose laboratory in vitro exposures have occasionally been suggested to also occur with exposures of ambient air mixtures (e.g., inflammatory indicators in the in vitro exposures to CAPs in the study of Maciejczyk and Chen, 2005), more often effects have not been found (e.g., no abnormal levels of cytokines in human volunteers in the CAPs exposure study of Ghio et al, 2000a). Studies of the effects of relatively low concentrations of airborne PM components in humans have all involved complex mixtures, and are one focus of this critical review. These include those short-term inhalation exposures to (1) CAPs in healthy human volunteers and (2) to diluted WDE, and (3) natural exposures to ambient PM, where data from simultaneous daily and/or seasonal or annual average PM compositional analyses were available for time-series and cross-sectional studies of effects in large human populations. Due to the limitations of statistical power in such natural population studies, the epidemiological analyses have focused more on identifying the contributions to the effects of factors or source-related mixtures than of individual components within the mixtures. Additional information comes from laboratory studies that have involved instillation of particle suspensions into human lungs and subsequent analyses of bronchoalveolar lavage fluid (BALF) samples for particle retention and biomarkers of effects. Studies of the effects of relatively low concentrations of airborne PM components in laboratory animals that involve complex mixtures are another focus of this critical review. These include (1) short-term inhalation exposures to CAPs in mice, rats, and dogs; (2) subchronic inhalation exposures of CAPs to mice and rats; and (3) inhalation and intratracheal lung instillation of components and source-related mixtures. A major objective of this critical review is to combine the analyses of the experimental studies with CAPs, and other ambient air PM components, in humans and other animals, with the associations between ambient air concentrations of PM and its components, to determine the nature and extent of the effects of ambient air PM and its components of major organ systems and their cross-species consistency, and to identify, as possible, the more potent PM components.

It is important to remember that all three particle size ranges are chemically nonspecific pollutant classes, and may originate from, or been derived from, various emission source types. Thus, PM toxicity may well vary, depending on its size distribution, source, and chemical composition. If the PM toxicity could be associated with specific source signatures, then health effects research could be better focused on specific PM components that come from those sources, and specific biological mechanisms could be postulated for further consideration by toxicological studies. PM health effects research is therefore now being increasingly focused on source-apportionment of PM using chemical speciation data, and this review of the CAPs literature emphasizes those CAPs studies that used PM compositional data to identify associations of exposures to PM source categories, or to individual PM components that have been associated with health-related effects. In addition to this Introduction, this critical review paper consists of sections discussing: (2) Development of methods for conducting CAPs inhalation studies, and the advantages and limitations of the available technologies; (3) Studies in humans; (4) Studies in laboratory animals and in vitro; (5) Organ system responses; (6) Concordance of responses to CAPs and other exposures; (7) Summary of the role and contributions of CAPs studies; and (8) Unresolved issues and conclusions.

2. Development of methods for conducting CAPs inhalation studies Laboratory investigators have recognized the need to do studies of health effects attributable to PM air pollution corresponding to those occurring in susceptible groups within natural populations. To do so, they need exposure atmospheres that faithfully represent what people actually breathe, while at the same time exposing them to a sufficient concentration to elicit measurable biological responses. Conducting such studies is inherently challenging because of (1) the extensive temporal and spatial variations in PM composition and particle size distribution; and (2) the lack of knowledge of which air pollutant components are most likely to be influential in causing the observed effects and their temporal patterns. Furthermore, the real-world exposures generally are not “square-wave” exposures as in conventional laboratory-based inhalation exposure studies, but rather are temporally and spatially variable. In addition, there are other variables among human populations, especially biological variables, whose influence on outcome measures can be great: for example, large variations in susceptibility due to age, genetic predisposition, diet, prior exposure, and disease history, as well as ventilation patterns and breathing rates during exposure can influence PM dosimetry. In addition, for many of the health-related measurements, it is not clear when, or with what frequency, they should be made. Finally, because the responses to ambient concentrations of PM are likely to be subtle, or to only occur in small


Health effects of ambient air CAPs fractions of the population, laboratory investigators need to use PM concentrations that are somewhat higher than those occurring in contemporary atmospheres in order to have a reasonable probability of producing measurable responses. In order to generate reasonably representative atmospheres of ambient air PM at elevated concentrations in the size ranges of interest, Sioutas et al. (1995, 1997), Demokritou et al. (2002a, 2002b, 2003), and Gupta et al. (2004) at the Harvard School of Public Health (HSPH), and Sioutas et al. (1999) at the University of Southern California (USC) adapted and refined an inertial PM concentrating technique originally developed for PM air sampling known as virtual impaction (Hounam & Sherwood, 1965; Conner, 1966; Dzubay & Stevens, 1975; Loo et al., 1976, 1988). In it, the incoming atmosphere flows through a nozzle that is coaxial with a tubular inlet that accepts about 10% of the flow emitted by the nozzle. Because of their inertia, particles above a certain aerodynamic size follow the minor airflow stream into the receiving tube and can thereby be conducted into an exposure chamber as a concentrated stream of airborne particulate matter (CAPs). The major flow from the nozzle, which flows around the receiving tube, is devoid of the larger particles, and is discarded. The CAPs remain suspended as individual particles in air containing the original mixture of carrier gases and vapors. In order to concentrate particles smaller than that achievable without modification of original size distribution, Sioutas et al. (1999) and Gupta et al. (2004) developed systems for humidifying the inlet stream to the nozzle, thereby condensing water vapor onto the particles in order to increase their size and momentum before entering the nozzle, and then conducting the CAPs in the minor flow through a diffusion dryer to restore the particles to their original size distribution. Using this approach, a CAPs stream can contain ultrafine PM as well as fine PM. When UFP contribute to the effects of exposures to PM2.5 CAPs, the distinction between the PM concentrators, especially the extent to which they concentrate UFP, may be important. Su et al. (2006) described characterizations made with single-particle spectrometers of ambient air PM entering and leaving the Harvard and USC concentrators that illustrate the differences in performance of these concentrating systems. Gordon et al. (1998a) at New York University (NYU) adapted an aerosol centrifuge to concentrate PM2.5 by a factor of 10, and used it in a series of animal inhalation studies at NYU (Gordon et al., 1998b, 2000; Shukla et al., 2000; Zelikoff et al., 2003). Peer-reviewed papers describing animal and human inhalation studies using PM concentrators to produce CAPs have been published at an ever-increasing pace since Clarke et al. (1999). Not surprisingly, many of the outcomes of the earliest published studies were viewed as having interpretative difficulties. An ideal toxicological study has full control over all exposure-related variables, but with the CAPs studies the exposures are highly dependent on the ambient conditions (i.e., meteorology

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and PM composition and concentrations). Replication of studies is, by definition, impossible. Also, toxicologists are faced with small number of subjects (animals or humans), whereas the epidemiological studies that have generated the hypotheses being investigated in the CAPs inhalation studies have the ability to include millions of subjects in their analyses. In compensation for these limitations, CAPs investigators have generally had the advantages of being able to artificially increase the concentration of the exposures, to use animal models of susceptible human populations, and to focus their effect assay protocols on the kinds of effects reported in the epidemiological studies. However, in the face of the multiple uncontrollable exposure variables, there is a need for more sophisticated approaches to the analysis of the data generated in CAPs studies. Beginning about 2003, there was increasing recognition of the need for more standardized statistical analyses to support the conclusions related to observed health outcomes. To address the need for standardization, a workshop on CAPs studies was convened by the Health Effects Institute (HEI) in Boston, on May 5, 2004, at a time when only ~15 of the more than 50 papers now in the scientific literature had been published. As summarized by Lippmann et al. (2005), it brought together representatives of most of the laboratories that had conducted animal and/or human inhalation exposure studies with CAPs. Participants agreed that CAPs researchers needed to make serious efforts to harmonize their experimental and analytical protocols to permit the sharing of lessons learned, questions raised, and opportunities for more definitive studies. The HEI Workshop focused on two aspects of dealing with these complexities: sorting out influential particulate matter (PM) components responsible for observed effects, and searching for time-varying responses in continuous outcome data. The need for more complete analyses of PM samples from the CAPs studies was also emphasized, as was obtaining a consistent set of parameters characterizing exposure atmospheres and the ambient PM from which the CAPs is sampled. Standardized outcome measures based on spirometry and response markers in lung bronchoalveolar lavage (BAL) cells and fluids existed, including the appropriate times after exposure to collect samples and measurements, were also discussed. For the then emerging focus on cardiac system responses, many different electrocardiographic (ECG) endpoints had been examined, and there had been too little standardization on markers that are most informative about adverse effects; on when the measurements needed to be made; and on how more comparable measurements could be made. Finally, it was apparent that CAPs studies had already had a significant impact within the air pollution health effects community, especially in regard to cardiovascular system effects, and a follow-up meeting with a greater focus on means to harmonize data collection and analysis was suggested. It was noted that the technology for producing CAPs had been applied to a number of different aerodynamic


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particle size ranges, including fine particulate matter (PM2.5), thoracic coarse particles (PM10-2.5), and UFP (PM0.1). However, there had been very few studies up until then on PM10-2.5 and PM0.1. In regions where the PM10-2.5 is a small fraction of of the PM10, there can be much more PM2.5 than PM10-2.5 in the PM10.The Workshop participants focused their attention on PM2.5 CAPs studies, and identified issues for future collective efforts at harmonization and data sharing. In having only a single-day workshop, the discussion on this topic was quite limited. There was a brief discussion on the reasons for choosing the various species for past studies, that is, rats (many strains had been used, sometimes with no clear basis for strain selection), mice (which can be selected for specific genetic alterations), and dogs. The common theme, however, was appropriate consideration and selection of models to address the research hypotheses and health effects of interest. Many models purported to feature a specific health condition that may not be relevant to the human condition in the context of the CAPs challenge studies, and thus should be characterized and assessed carefully. For example, most rodent emphysema models that exhibit the characteristic remodeling and airspace widening seen in the human condition do not have underlying inflammation. The Workshop also discussed the advantages and limitations of human studies. They noted that there were interesting findings from the limited number of clinical CAPs studies that showed that this line of research can demonstrate that statistically significant responses to short-term CAPs can be found in largely healthy humans. As reported by Lippmann et al. (2005a), these findings, in turn, demonstrate opportunities for CAPs studies to show that comparable effects can occur in animal models, while using exposure concentrations that cannot be justified in humans, and conducting systematic investigations with specific components of the CAPs mixtures. CAPs studies in volunteers were even more limited in the number of observations, and it was recommended that the few groups (US EPA [Devlin, Ghio et al.], USC [Gong, Linn et al.], University of Toronto [Urch, Silverman et al.], and University of Edinburgh/RIVM [Newby, Cassee, Donaldson]) should make efforts to have a number of health outcomes in common. Some specific workshop recommendations were 1. Studies need to have well-defined hypotheses, and statistical analyses should be generic rather than sophisticated. 2. Efforts should be made to harmonize CAPs exposure protocols (particle size ranges, concentration factors, durations, and exposure atmosphere characterization). 3. Efforts should be made to harmonize CAPs effects assays (pulmonary and cardiac function, histology, cellular and molecular alterations, and the times of observation post-exposure).

4. Efforts should be made to harmonize statistical software packages for the studies among the various users/ institutions of concentrator technologies. 5. Collection and analysis of CAPs composition data should consider the usefulness for source apportionment. 6. Additional information could be obtained by combining studies. 7. Access to existing data should be provided.

3. Review of studies of the effects of PM and components in humans 3.1. Introduction This review of human responses is focused on CAPs inhalation studies and their health effects, with an emphasis on studies that identify the particle size ranges and components most closely associated with the observed effects. In doing so, we have grouped studies in the specific geographical areas in which they were performed on the basis that the mixture of ambient air components and size distributions are likely to be at least somewhat different. We also review laboratory-based human inhalation studies involving components of ambient air, such as diluted diesel engine exhaust, in order to provide a basis for evaluating whether the effects produced in such studies can account for the effects seen in the CAPs inhalation studies. We do not review studies involving inhalation of laboratory-generated UFP of specific chemicals, because the laboratory-generated UFP of a single material and ambient air UFP, which is a mixture of UFP from motor vehicles, photochemistry, and SO2 oxidation, are unlikely to have the same effects. We also cover lung instillation studies involving PM suspensions of materials found in ambient air. Finally, we review reported associations between ambient air PM size fractions and components and human health-related responses in natural settings. Th ese include studies of limited numbers of individuals where there is information related to personal exposures and effects (Panel Studies) and larger-population studies that rely on central site air monitoring data and grouped responses. Th is review of epidemiological studies is limited to those that provide a basis for determining whether CAPs study results and results of people in natural settings are consistent, and will provide a basis for the discussions, organized by organ systems in Section 5, with respect to concordance of the CAPs study results with evidence from the epidemiological and other toxicological literature that we present in Section 6. 3.2. Human CAPs inhalation studies in Chapel Hill, NC Ghio et al. (2000a) reported that the 2-h CAPs exposures ranging in mass concentration from 23 to 311 μg/m3 caused neutrophilic inflammation in the lungs and increased fibrinogen levels in the blood. Of the soluble components extracted from the air sampling filters, Fe, As, Se, and SO42− were highly correlated with the PM2.5 mass concentration,


Health effects of ambient air CAPs whereas Ni and Cu were least correlated. In terms of biological responses, a Fe/Se/SO42− factor was associated with increased BALF percentage of polymorphonuclear neutrophils (PMNs), and a Cu/Zn/V factor with increased blood fi brinogen. The increase in plasma fibrinogen correlated with decreases in PMNs and platelets, consistent with a state of systemic inflammation and increased platelet aggregation. Salvi et al. (1999) had previously found, in human volunteers, that diluted diesel exhaust also caused increases in PMNs in airway lavage fluid, as well as in peripheral blood. Devlin et al. (2003) compared the responses of elderly (aged 60–80) healthy adults exposed to CAPs at concentrations ranging from 21 to 80 μg/m3 to those seen in prior Chapel Hill exposures of healthy, young adults (aged 18–40). They reported that heart rate variability (HRV) was significantly decreased immediately after CAPs exposure in the elderly subjects in both time and frequency domains, and that some of the changes persisted into the next day. By contrast, there were no such changes in the young adult group. Samet et al. (2007) presented a summary of a comparison of the effects of CAPs exposures of normal human volunteers by inhalation for 2 h to filtered air (FA) and CAPs in three size ranges in Chapel Hill, NC, the fine CAPs responses reported by Ghio et al. (2000) were compared with those of 14 volunteers exposed to a mean concentration of 89 μg/m3 of coarse thoracic CAPs (PM10-2.5), as described in greater detail by Graff et al. (2009), and 20 exposed to a mean of 47 μg/m3 (151,800 particles/cm3) of UFP CAPs (PM0.16), as described in greater detail by Samet (2009). The responses are summarized in Table 1. There were modestly sized fraction-dependent effects of CAPs exposure on cardiovascular, pulmonary, and hematological parameters in normal adult human subjects. None of the studies showed a significant effect on pulmonary function indices. Changes in BAL markers of inflammation (PMNs and cytokines) were relatively small and unremarkable. Good cohesion in the cardiac endpoints was observed among these studies, with a trend toward changes in cardiac rhythm and blood coagulation (Ghio et al., 2003). 3.3. Human CAPs inhalation studies in Los Angeles, CA Gong et al. (2003) reported that neither healthy nonsmoking adult volunteers nor mild asthmatics exposed for 2 h, with intermittent exercise, to ultrafine CAPs produced significant changes in spirometric indices or hematology as compared to FA. However, both groups had CAPs-related

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decreases in columnar cells in post-exposure induced sputum, slight changes in some mediators of blood coagulability, systemic inflammation, and parasympathetic stimulation of HRV. CAPs exposure decreased systolic blood pressure in asthmatics, and increased it in the healthy normal subjects. Gong et al. (2004a) reported that healthy, elderly nonsmoking adult volunteers and age-matched individuals with chronic obstructive pulmonary disease (COPD) were exposed for 2 h at concentrations of ~200 μg/m3, with intermittent exercise, to PM2.5 CAPs and to FA. There were no significant effects of CAPs exposure on spirometry, symptoms, or induced sputum. There was a significant negative effect on pulse rate immediately after exposure, with the effect being greater in the healthy subjects. Also, peripheral blood basophils increased after CAPs exposure in the healthy, but not in the COPD group. Pre-exposure ectopic heartbeats were more frequent in the COPD group, but diminished after CAPs exposure. HRV was lower after CAPs exposure in healthy subjects, but not in the COPD group. Gong et al. (2004b) exposed healthy, nonsmoking adults and others with mild asthma to PM10-2.5 CAPs at a concentration averaging 157 μg/m3 and to FA. There were no significant effects of PM10-2.5 on lung function. On the other hand, there were small, but significant increases in HR, and decreases in HRV, and they were greater in the healthy subjects. Gong et al. (2005) exposed elderly residents of Los Angeles (6 healthy, mean age = 68; 18 with COPD, mean age = 72) to PM2.5 CAPs (~200 μg/m3), NO2 (400 ppb), both CAPs and NO2, or FA for 2 h with intermittent exercise on separate days. CAPs exposures produced significant changes in maximal mid-expiratory fl ow rate that were greater in normal subjects than in those with COPD, and a reduction in columnar epithelial cells in sputum at 22 h after the exposure. Th ese responses were not affected by co-exposure to NO2. For those exposed to CAPs + NO2, but not for CAPs alone, there were reductions in forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) in proportion to the SO42− content of the CAPs. Gong et al. (2008) exposed healthy and mild asthmatic adult volunteers to PM2.5 CAPs at a concentration averaging 174 μg/m3 and to FA. Data were collected on lung function, symptoms, exhaled NO, ECG, pulse rate, O2 saturation, and inflammatory biomarkers in peripheral blood and induced sputum. The differences between CAPs and FA exposure days were 0.5% reduction in O2

Table 1. Summary of effects of exposure to size-fractionated CAPs on young healthy human volunteers at the US EPA human Studies Facilities*. Pulmonary function BAL fluid cells BAL fluid markers Cardiac endpoints Plasma factors Fine No effect ↑ Total ↓ IL-8 No effect (trend to ↓ HRV) ↑ Fibrinogen ↑ PMN ↑ Monocytes Coarse No effect ↑ PMN ↓ Protein ↓ SDNN 20 h post Trend to ↑ clotting Ultrafine No effect No effect No effect ↓ SDNN 24 h post (ambulatory) ↑ D-dimer *From Samet et al., 2007.

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saturation (p < .01); 2% reduction in FEV1 on the next morning (p < .05); and a transient decrease in low-frequency power in the heart-rate variation (p < .05). There were no differences in these responses between the healthy and asthmatic volunteers. 3.4. Human CAPs inhalation studies in Toronto, Canada Normotensive, nonsmoking, healthy volunteers were exposed to PM2.5 CAPs at a concentration averaging 150 μg/m3 and O3 at 120 ppb for which there were data on PM2.5 composition. Brachial artery diameter (BAD), an index of cardiovascular response, decreased 0.09 mm compared to FA. There were no significant responses in endothelial-dependent flow-mediated dilatation (FMD), endothelial-independent nitroglycerin–mediated dilatation (NMD), or blood pressure (Brook et al., 2002: Urch et al., 2004). The linear regression analyses of change in BAD in relation to PM2.5 components yielded p values of .04 for OC, .05 for EC, .06 for Cd, .09 for K. The p values were between .13 and .17 for Zn, Ca, and Ni, and values were even larger for all of the other measured components. The p value for PM2.5 as a whole was .40. In a follow-up study, there was a significant increase (6 mm Hg) in diastolic blood pressure in those exposed to O3 plus CAPs (p = .013), but it was not possible to determine whether the O3 contributed to the association with CAPs overall, or its measured components. In relation to the PM2.5 components, there was a significant association (p = .009) with OC, whereas the association with PM2.5 mass was not significant (p = .27). In this study, the EC and metals in the CAPs were not significantly associated with BAD constriction or blood pressure (Urch et al., 2005). Fakhri et al. (2009) extended the study to include 50 nonsmoking volunteers, including subjects with asthma. The CAPs mass averaged 122 μg/m3, and the O3 concentration averaged 113 ppb. Although CAPs exposure overall was not associated with HRV changes, HRV varied with O3. The combined exposure increased diastolic BP, and asthmatic status was not a modifying factor. 3.5. Human CAPs inhalation study in Edinburgh, Scotland Mills et al. (2008) exposed healthy and age-matched volunteers with stable coronary heart disease to PM2.5 CAPs and to FA. Data were collected on peripheral vascular vasomotor and fibrinolytic function, and inflammatory variables—including circulating lymphocytes, serum C-reactive protein, and exhaled breath 8-isoprostane and nitrotyrosine—at 6–8 h after exposure. Exhaled breath 8-isoprostane increased after CAPs exposure (p < .05), and there was an increase in blood flow and plasma tissue plasminogen activator (p < .005), there were no significant changes in markers of systemic inflammation, and no effect on vascular function in either group of subjects. In view of the much greater responses seen in their prior exposures of similar volunteers to 300 μm3 of PM in diesel engine exhaust for 1 h (Mills et al., 2005, 2007), they

suggested that the minimal responses to CAPs were due to the particle composition being very low in carbon, with >90% being sea salt. In summary, CAPs exposure, regardless of exposure site, produced a small impact on lung function, with more in individuals with COPD than in healthy subjects. CAPs exposures slightly affected peripheral blood cellularity and chemistry. By far, the most consistent changes were those in cardiac rhythm. 3.6. Other laboratory-based inhalation studies in humans with components of ambient air PM 3.6.1. Diesel engine exhaust Stenfors et al. (2004) exposed young healthy and mild asthmatic Swedish subjects for 2 h to diluted WDE containing DEP at 108 μg/m3 and assessed lung function and airway inflammation. The exposures did not affect FEV1 or FVC, but did produce significant increases in sRaw in both groups, with a greater response in asthmatic subjects. In terms of inflammation, there were significant increases in the healthy subjects, but not in those with mild asthma. In a further study of healthy subjects by the same group (Pourazar et al., 2005), the DEP concentration was 300 μg/m3. The exposure activated redox-sensitive transcription factors, consistent with oxidative stress triggering increased synthesis of proinflammatory cytokines. In archived bronchial biopsies from this inhalation study, Pourazar et al. (2008) showed that the exposure caused significant increases in the expression of epidermal growth factor receptor (EGFR) and phophorylated C-terminal Tyr 1173, whereas Src-related tyrosine (Tyr 416), mitogenactivated protein /extracellular signal-related kinase (MAP/ ERK) kinase (MEK), and ERK pathways were not changed. Bosson et al. (2007) exposed young healthy Swedish subjects to WDE containing 300 μg/m3 of DEP for 1 h, followed 5 h later by 2 h of exposure to 200 ppb of O3. Sputum was collected 18 h after the O3 exposure. The O3 exposure magnified the WDE-induced inflammation. Mills et al. (2005) exposed healthy males undergoing intermittent moderate exercise to FA or diluted whole diesel engine exhaust (WDE) for 1 h (PM mass concentration = 300 μg/m3). When measured 2 h later, there were no differences in forearm blood flow or inflammatory markers associated with either FA or WDE exposure. Injection of a vasodilator did produce a dose-related increase in blood flow, but this could be attenuated with a post-exposure injection of bradykinin (p < .05), whereas the bradykinin injection resulted in an increase in plasma tissue plasminogen activator (p < .001). Tornqvist et al. (2007) performed a follow-up study on 15 healthy males in the same laboratory and measured the effects 24 h after the same exposure protocol used by Mills et al. (2005). At 24 h after the exposures, the WDE exposure had increased cytokine concentrations [tumor necrosis factor alpha (TNFα) and interleukin (IL)-6; both at p < .05], but had decreased acetylcholine (p = .01) and bradykinininduced forearm vasodilatation (p = .08).


Health effects of ambient air CAPs Mills et al. (2007) exposed 20 men with a prior myocardial infarction (MI) to the 300 μg/m3 WDE exposure protocol cited above, and quantified myocardial ischemia by ST-segment analysis during the exposures. At 6 h after the exposures, they assessed vasomotor and fi brinolytic function by intra-arterial agonist infusions. The HR was increased with exposure to both FA and WDE, and the ST-segment depression was greater with WDE exposure (p = .001). WDE exposure did not aggravate preexisting vasomotor dysfunction, but did reduce the acute release of endothelial tissue plasminogen activator (p = .009). Behndig et al. (2006) exposed 15 healthy adults, 7 female and 8 male, to FA or diluted WDE (PM at 100 μg/m3) for 2 h with intermittent exercise. At 18 h post-exposure, they performed bronchoscopies and collected BALF and bronchial biopsy tissues. With these state-of-the-art assays, they were able to find increases in bronchial mucosa PMNs and mast cells, as well as increases in BALF PMNs, IL-8, and myeloperoxidase. Peretz et al. (2008a) exposed healthy young adults in Seattle to WDE containing either 100 or 200 μg/m3 of DEP for 2 h. At 3 h post-exposure for 200 μg/m3, there was a statistically significant increase in HF power, and a decrease in LF/ HF ratio (HF is high frequency, while LF is low frequency), but no effect on time-domain statistics and no effects on HRV at later time points. Peretz et al. (2008b) studied both healthy adults and those with metabolic syndrome (MS) in Seattle subjects who were exposed to WDE containing either 100 or 200 μg/m3 of DEP for 2 h. For the subjects with MS, and for all subjects combined, there was an acute endothelial response and vasoconstriction of a conductive artery. 3.7. Short-term responses to ambient air PM inhalation exposures in human panel studies Peters et al. (2000) followed a panel of patients with implanted cardioverter defibrillators living in eastern Massachusetts, and compared defibrillator discharges with the concentrations of PM10, PM2.5, black carbon (BC) O3, NO2, SO2, and CO. There was at least one discharge in 33 patients and 10 or more in 6 of them. For these six, there were significant associations of discharges with 2- and 3-day lagged exposures to NO2, PM2.5, and CO, but not with BC, O3, or SO2. Peters et al. (2001) followed 772 patients who had a recent MI in the greater Boston area and, in a case-crossover study, and regressed the events with the concentrations of PM10, PM2.5, BC, O3, NO2, SO2, and CO in the hours and days preceding the MI event. There were statistically significant associations with PM10, PM2.5, and BC in the 2 h preceding the MI, and for PM10, and PM2.5 in the previous 24 h, with nearly significant associations of MI with PM10-2.5 and BC. Schwartz (2001) examined the association between blood markers of cardiovascular risk and air pollution in a national sample of the US population, i.e., the third National Health and Nutrition Examination Survey (NHANES III). Specifically, he considered fibrinogen levels and counts of platelets and white blood cells (WBCs) and their correlation

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with PM10, O3, NO2, and SO2 on the day of and the day before the personal examination. PM10 was significantly associated with all three outcomes, and remained significant in multiple-pollutant regression analyses, whereas the gaseous pollutants were not. Pekkanen et al. (2002) studied coronary function of 45 subjects with stable coronary heart disease performing 342 exercise tests in Helsinki, Finland, over a period of 6 months. Levels of PM2.5 days before the test were significantly associated with increased risk of ST-segment depression during the test. For PM2.5, the odds ratio (OR) was 2.8 (95% confidence interval [CI] = 1.4–5.7), whereas for total particle count (PC), it was 3.1 (95% CI = 1.6–6.3). There was no consistent association for PM10-2.5. The associations were stronger for subjects not using beta-blockers. Another panel study is that of Sorensen et al. (2005). They studied 49 students in Copenhagen, and found that their personal exposure to soluble V and Cr was associated with significant increases in oxidative stress and DNA damage (as measured by 8-oxodG concentrations in lymphocytes). Other soluble metals (Fe, Ni, Cu, and Pt) were not. O’Neill et al. (2005) studied the influence of PM2.5, PC, BC, and SO42− on vascular reactivity and endothelial function in 270 residents of greater Boston. They were divided into those with type 1 diabetes, type 2 diabetes, and those with risk factors for diabetes. All four PM metrics were associated with decreased vascular reactivity in those with diabetes, but not in those with risk factors. In terms of PM components, SO42− was associated with both decreased flow-mediated and nitroglycerin-mediated vascular reactivity among diabetics, whereas BC was associated with only decreased flow-mediated vascular reactivity among diabetics. These effects were greater in those with type 2 diabetes. Lanki et al. (2006) studied the influence of ambient air PM2.5 component exposures on exercise-induced ischemia in 45 elderly nonsmokers with stable coronary heart disease in Amsterdam (The Netherlands), Erfurt (Germany), and Helsinki (Finland). Two PM2.5 source classes (traffic and long-range transport) were associated with ST-segment depression during submaximal exercise testing in a clinical laboratory. In a multi-pollutant model, with which the authors were able to separate effects of secondary SO42− from effects of vehicular emissions, only the traffic emissions were significantly associated with the effect. The authors also examined whether potentially toxic transition metals (Fe, Cu, Zn, and V) might be associated with ST-segment depression, given that both these and OC may have the capability to induce oxidative stress in the lung. However, when adjusted for ABS (absorbance, a measure of EC emissions from motor vehicles), none of the metals that were measured were associated with ST-segment depression, whereas the ABS associations remained significant with only slight variation. Chuang et al. (2007) studied a panel of 46 patients with coronary heart disease (CHD) in Taipei, Taiwan. Cardiac


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function was monitored for 1 week using continuous Holter monitors. Th ey had hourly air quality measurements of PM10, PM2.5, and PM2.5 components (SO42−, NO3−, OC, and EC), as well as the gaseous criteria pollutants. HRV was reduced in proportion to elevations in PM2.5, SO42−, and OC, but not with any of the other measured pollutants in single-pollutant models. In multi-pollutant models, only SO42− remained significant. Although the concentrations of transition metals were not measured, they would be more closely correlated with SO42− than with total PM2.5 mass or with OC, suggesting that they may have influenced the reductions in HRV. Yeatts et al. (2007) studied a panel of 12 adult patients with asthma living in or near Chapel Hill, NC, over a 12-week period. They had daily concentration data for PM10-2.5 and PM2.5. Each subject had nine clinic visits, yielding measurements of spirometry, blood biomarkers, and cardiac function by ECG. For each 1 µg/m3 of PM10-2.5, standard deviation of normal beat-to-beat interval (SDNN) decrease (p = .02), circulating eosinophils increased (p = .01), triglycerides increased (p = .02), and very-low density lipoproteins increased (p = .01). There were no significant changes associated with PM2.5, and no lung function changes with either size fraction. Riediker et al. (2004a) studied a panel of nine nonsmoking healthy male highway patrol officers (ages: 23–30) in North Carolina over four late shift tours of duty. Their patrol cars had air samplers that were analyzed for vapor and PM2.5 components each day. PM2.5 components were correlated to cardiac and blood parameters measured 10 and 15 h after the work shift. They reported that in-vehicle PM2.5 mass was associated with changes in cardiac parameters; blood proteins associated with inflammation, hemostasis, and thrombosis; and increased red blood cell volume. In a follow-up study, Riediker et al. (2004b, 2007) used data on PM2.5 components. Those associated with health-related endpoints were Ca (increased uric acid and von Willebrand factor [vWF], and decreased protein C), Cr (increased WBCs and IL-6), Cu (increased blood urea nitrogen, mean cycle length of normal R-R intervals), and S (increased ventricular ectopic beats). Control for the gaseous pollutants had little effect on the effect estimates. Yue et al. (2007) used PM component data that were collected specifically to study of the role of community air pollution on prolonged repolarization and increased levels of inflammation in male coronary artery disease patients in Erfurt, Germany. Particle counts were made in size intervals ranging from 0.01 to 3.0 μm, and air pollutant concentration data were available for O3, NO, NO2, CO, SO2, SO42−, OC, and EC. They identified five specific source categories: (1) airborne soil; (2) UFP from local traffic; (3) secondary PM from local fuel combustion; (4) PM from remote traffic sources; and (5) secondary PM from multiple sources. The health-related parameters measured in their subjects (males, average age = 66) were for ECG, QT interval and T-wave amplitude, and for blood, vWF and C-reactive

protein (CRP). They found that an increase in QT interval and a decrease in T-wave amplitude were associated with traffic-related particles exposure 0–23 h before the ECG recordings. Both traffic-related PM and combustiongenerated PM, at different exposure lag times, were related to an increase in the inflammatory marker vWF. All of the source types showed positive associations with CRP levels above the 90th percentile (8.5 mg/L). This combination of results suggested that traffic-related and combustiongenerated PM had stronger adverse health impacts with regard to cardiac effects, and that PM from other sources induced acute-phase responses. Ibald-Mulli et al. (2001) reported that during a pollution episode in Augsburg, Germany, patients with high plasma viscosity and increased heart rates had elevated systolic blood pressure associated with total suspended particles (TSP). Delfino et al. (2008) measured circulating biomarkers of inflammation, antioxidant activity, and platelet activation to determine their association with UFP and primary combustion PM in elderly subjects with a history of coronary artery disease (CAD) (13 male and 17 female subjects, average age = 86, nonsmokers). They used air quality data from San Gabriel Valley, CA (hourly total particle count; hourly EC and OC; 24-h data for PM0.25, PM0.25-2.5, PM2.5-10, O3, NO2, and CO). They also estimated primary and secondary OC, for both indoor and outdoor microenvironments. The health-related parameters measured in weekly blood samples were CRP, fibrinogen, IL-6, TNFα, vascular cell adhesion molecule (VCAM)-1, intercellular cell adhesion molecule (ICAM)-1, fibrin D-dimer, sP-selectin, myeloperoxidase (MPO), superoxide dismutase (SOD), and glutathione peroxidase (GPx)-1. There were also weekly measurements of exhaled breath NO (eNO), a marker of pulmonary inflammation. There were significant positive associations of IL-6 with PM, which were largely driven by EC, BC, primary OC, and particle count, but not with total OC or secondary OC. Outdoor PM was associated with indoor PM of outdoor origin. Particle number concentrations, and quasi-UFP were more strongly associated with biomarkers than were PM0.18-2.5 and secondary organic aerosol (SOA). The inverse association of SOD with PM was also largely driven by EC, BC, and primary OC. They suggested that inactivation of antioxidant enzymes (SOD, GPx-1) by ROS, reactive nitrogen species (RNS), or electrophiles may be one mechanism of pollutant-induced systemic inflammation and thrombosis. Delfino et al. (2009) extended their study to include 60 elderly volunteers with CAD, to include residents in Los Angeles with greater exposure to traffic sources. They reported that markers of primary combustion (EC, BC, primary OC, CO, NO, but not secondary OC) were positively associated with inflammatory biomarkers and inversely associated with erythrocyte antioxidant enzymes, and that PN (particle number) and PM2.5 were more strongly associated with biomarkers than was PM0.25-2.5. The associations were stronger during cooler periods, when the only


Health effects of ambient air CAPs pollutants that were elevated were primary OC, PN, and NO. They concluded that traffic-related pollutants were associated with systemic inflammation, increased platelet activation, and decreased erythrocyte antioxidant enzyme activity, and that particulate matter components carried by UFP are important. Hoffmann et al. (2009) studied a prospective German cohort of 4814 participants for whom residential PM2.5 was estimated using local measurements, an atmosheric dispersion model, and distance from roadways. They had data on high-sensitivity CRP (hs-CRP) and fibrinogen on the day (in 2000 of their baseline visit), and regressed chronic exposure against sex-stratified variations, controlling for individual level risk factors. They reported that PM2.5 was significantly associated with hs-CRP in men, but not in women. Baccarelli et al. (2006) studied blood coagulation in 1218 normal adults from the Lombardia region of Italy. The prothrombin time (PT) became shorter with increasing ambient air concentrations of PM10, NO2, and CO. Rundell et al. (2007) studied 16 intercollegiate athletes who performed 30 min of exercise while being exposed to ambient air pollution on relatively dirty and relatively clean days in terms of PC concentration. Flow-mediated brachial artery dilation (FMD) and forearm O2 kinetics (FOK) were measured before and after the exercise. There was basal brachial artery vasoconstriction after the high PC (p = .0002), but not low PC. FMD was impaired after high PC (p = .0001), but not after low PC. FOK was reduced after high PC (p = .0006), but not after low PC. Dales et al. (2007) studied 39 healthy adults (aged 18–50, 20 males and 19 females) who sat outside for 2 h at 2 bus stops in Ottawa, Canada, one downtown, and one in an open area. A 30 μg/m3 increase in PM2.5 was associated with a 0.48% decrease in FMD. There were no significant changes associated with other measured air quality parameters (PC, NO2). Rudez et al. (2009) studied 40 healthy adult residents of Rotterdam to determine if there were significant associations between ambient air pollutants (PM10, CO, NO, NO2, and O3), and markers of hemostasis and inflammation in 11–13 blood samples collected over a 1-year period in each subject (platelet aggregation, thrombin generation, fibrinogen, and CRP). Lag times between peak daily exposures and blood sampling were also investigated. The pollutants other than O3 had positive associations with platelet aggregation and thrombin generation, and the associations with PM10 were robust in multi-pollutant models. There were no associations of the pollutants with fibrinogen and CRP. Allen et al. (2008) reported on the associations between PM components (PM2.5 of outdoor origin, PM2.5 of indoor origin, BC, and levoglucosan, a marker for wood smoke) and indices of pulmonary responses (eNO, forced expiratory volume in 1 s [FEV1], forced vital capacity [FVC], peak expiratory flow rate [PEF], and mid-expiratory flow rate [MEF]) in a panel study of residents of Seattle, WA. There

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were significant increases in eNO associated with personal exposure to BC and PM2.5, as well as to outdoor PM2.5, Significant reductions in PEF and MEF were associated with outdoor concentrations of PM2.5, BC, and levoglucosan. Reduced FEV1 was associated only with outdoor levoglucosan. McCreanor et al. (2007) studied 60 adults with mild or moderate asthma who agreed to take two 2-h walks in London, England; one in Hyde Park, and the other along Oxford Street where the vehicular traffic was limited to buses and taxis with diesel engines. There were much higher concentrations of PM2.5, UFP, EC, and NO2 on Oxford Street than in Hyde Park, and this higher level of exposure resulted in significant reductions in FEV1 (p = .04), and FVC (p = .01) overall, with p = .005 at some time points. The effects were greater among the subjects with moderate asthma. There were also increases in biomarkers of neutrophilic inflammation (sputum myeloperoxidase (p = .05), and airway acidification (p = .03). Among the measured air pollutants, the associations were strongest for EC and UFP. Penttinen et al. (2006) studied a panel of 78 adult asthmatics in Helsinki, Finland, who lived within 2 km of a central air-quality monitoring site that provided data on PM2.5, elemental components, and source factors. Their peak expiratory flow rates were significantly reduced in relation to attributed to local sources of PM2.5 (lag 1 day) and to PM2.5 soil sources (lag 3 days). Gent et al. (2009) studied 149 children with physician-diagnosed asthma and symptoms or medication use within the previous 12 months who were living in New Haven, CT, and vicinity. Air sampling filters were collected daily and analyzed for trace elements by x-ray fluorescence (XRF), and BC by light reflectance. Using factor analysis/ source apportionment, they identified six sources of PM2.5. They were motor vehicle, road dust, S (for regional PM2.5), biomass burning, oil combustion, and sea salt. They attributed 42% of the PM2.5 to the motor vehicle source, and 12% to road dust. Increased likelihood of symptoms and inhaler use were largest for 3-day averaged exposures, with a 10% increased likelihood of wheeze per 5 μg/m3 of the motor vehicle source, and a 28% likelihood increase for shortness of breath associated with road dust. There were no associations with increased health outcome risks for PM2.5 per se, or the other source factors. In summary, a broad variety of short-term cardiovascular effects have been significantly associated with peaks in ambient air concentrations of PM2.5 and/or one or more of its chemical components in panel studies. These range from implanted defibrillator discharges associated with 2- or 3-day lagged PM2.5, but not with BC (Peters et al., 2000); MI being associated with PM10, PM2.5, and BC in the preceding 2 h (Peters et al., 2001); PM10, but not gaseous pollutants, being associated with increased WBCs, platelets, and fibrinogen in NHANES III (Schwartz, 2001) and with CRP and fibrinogen (Hoffmann et al., 2009); PM2.5 was associated with platelet aggregation and thrombin


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generation (Rudez et al., 2009). PM2.5 and PC were associated with ST-segment depression (Pekkanen et al., 2002); vascular reactivity was associated with PM2.5, PC, BC, and SO42− in diabetics, especially in those with type 2 diabetes (O’Neill et al., 2005); exercise-induced ischemia was associated with vehicle emissions, especially BC; for patients with CHD, there were significant associations with PM2.5, OC, and SO42−, but only SO42− was significant in a multiple pollutant regression (Chuang et al., 2007); in people with asthma, there were significant decreases in SDNN, and increases in eosinophils, triglycerides, and low-density lipoprotein (LDL) in relation to PM10-2.5 (Yeatts et al., 2007); in young, healthy highway patrol officers, PM2.5, but not gaseous pollutants, was associated with changes in cardiac parameters such as Ca (vWF), Cr (WBCs and IL-6), Cu (R-R intervals), and S (ventricular ectopic beats); and that traffic-related PM were associated with an increased QT interval and decreased T-wave amplitude, with both traffic-related and combustion-generated PM being related to an increase in vWF (Yue et al., 2007); significant associations of IL-6 were reported for EC, BC, primary OC and PC, but not for total OC or secondary OC, and an inverse association of SOD was largely driven by BC, EC, and primary OC. (Delfino et al., 2008, 2009). These various cardiac-related responses, although not necessarily associated with specific PM2.5 components, are certainly consistent with the excess cardiovascular mortality and morbidity in the air pollution health effects literature. In addition, peaks in ambient air PM were associated with a variety of pulmonary effects. Allen et al. (2008) reported that there were significant increases in eNO in Seattle associated with personal exposure to BC and PM2.5, as well as outdoor PM2.5, and significant reductions in PEF and MEF were associated with outdoor concentrations of PM2.5, BC, and levoglucosan, whereas reduced FEV1 was associated only with outdoor levoglucosan. McCreanor et al. (2007) studied adults with mild or moderate asthma who took long walks in Hyde Park, or along Oxford Street, where there were much higher concentrations of PM2.5, UFP, EC, and NO2 that were associated with significant reductions in FEV1 and FVC. Among the measured air pollutants, the associations were strongest for EC and UFP. Penttinen et al. (2006) reported that among adults with asthma, peak expiratory flow rates were significantly reduced in relation to attributed to local sources of PM2.5 (lag 1 day) and to PM2.5 soil sources (lag 3 days). Gent et al. (2009) reported that for children with physician-diagnosed asthma and symptoms or medication use within the previous 12 months living in New Haven, CT, and vicinity, there were six sources of PM2.5 (i.e., motor vehicles, road dust, S for regional PM2.5, biomass burning, oil combustion, and sea salt), with 42% of the PM2.5 attributed to the motor vehicle source, and 12% to road dust. Increased likelihood of symptoms and inhaler use was largest for 3-day averaged exposures, with a 10% increased likelihood of wheeze per 5 μg/m3 of the motor vehicle source, and a 28% likelihood increase for

shortness of breath associated with road dust. There were no associations with increased health outcome risks for PM2.5 per se, or the other source factors. 3.8. Large population-based studies in humans dealing with responses to PM components In the six-city cohort study, Dockery et al. (1993) showed that PM2.5 and SO42− were more closely associated with annual mortality in adults than were PM10, total suspended particles (TSP)–PM10, or the criteria pollutant gases. In the Laden et al. (2000) follow-up study of six cities, they reported that the effects were most closely associated with coal combustion effluents and soil. For the American Cancer Society (ACS) cohort, Pope et al. (1995, 2002) also found that PM2.5 and SO42− were more significantly associated with annual mortality in adults than were the criteria pollutant gases, but did not have other PM metrics for their comparisons. Miller et al. (2007) reported a greater impact of PM2.5 on annual mortality in women in the Women’s Health Initiative (WHI) cohort than in the six-city or ACS cohorts, but did not have any other PM concentration measurements. In another study of members of the WHI cohort, Zhang et al. (2009) studied the associations between PM2.5 and ischemia among 57,908 postmenopausal WHI clinical trial participants from 1993 to 2003. They reported statistically significant associations between PM2.5 and ST- and T-segment abnormalities, which they concluded were indicative of myocardial ischemia. Diez Roux et al. (2008) used data from the Multi-Ethnic Study of Atherosclerosis (MESA) to study the associations of 20 years of exposure to PM10 and PM2.5 with the prevalence of atherosclerosis among 5172 US adults without clinical cardiovascular disease at entry to the study. Long-term PM exposures were estimated on the basis of the modeling of community monitoring data, residential location, and an Environmental Protection Agency (EPA) spatiotemporal model. The subjects’ common carotid intimal-medial thickness (CIMT), coronary artery calcification, and anklebracheal index (ABI) were regressed against PM10 and PM2.5 exposure after controlling for age, sex, ethnicity, socioeconomic factors, diet, smoking, physical activity, blood lipids, diabetes, hypertension, and body mass index (BMI). The only significant association was that CIMT was associated with PM10 and PM2.5. In a cross-sectional study of a large population of office workers in London, England, in the warm season, Pekkanen et al. (2000) reported that there were significant associations of plasma fibrinogen with black smoke and PM10. In the Children’s Health Study in 12 Southern California communities, Gauderman et al. (2004) showed that lung function growth from ages 10 to 18 was significantly lower in proportion to the concentrations of PM2.5, EC, NO2, and acidic vapors. Sarnat et al. (2008) studied the influence of PM2.5 sources (gas engines, diesel engines, wood smoke, resuspended soil, secondary sulfate, secondary nitrate, cement kiln, railroad,


Health effects of ambient air CAPs and metal processing) on cardiorespiratory morbidity in Atlanta, GA, for 4 years (1999–2002). There were clear positive associations between PM2.5 attributed to mobile sources and biomass burning and emergency department (ED) visits for cardiovascular disease. For the summer months, PM2.5 SO42− was significantly associated with respiratory ED visits. The results were similar for different source-apportionment methods. In a Hong Kong, China, sulfur-in-fuel intervention study (Hedley et al., 2002), SO2, Ni, and V fell promptly and substantially after the intervention, whereas other criteria pollutants and metals did not fall (Hedley et al., 2004). Thus, it is possible that the large changes in the three pollutants that fell may account for at least some of the changes in the intervention-related cardiovascular mortality and bronchial hyperreactivity in this study. Another study, also of interest in this context, was that of Lippmann et al. (2006). It noted the high daily mortality associated with PM10 in New York City in the 90-city National Mortality and Morbidity Air Pollution Study (NMMAPS), and showed that in those 60 cities with speciation data, only Ni and V were significantly associated with NMMAPS mortality. The NMMAPS mortality coe fficient for New York City was 3.8× higher than the average, and the Ni in New York City was 9.5× higher than the US average. Dominici et al. (2007) extended this analysis of NMMAPS data in relation to PM2.5 speciation in terms of additional cities and years of data, and confirmed the associations of daily mortality coefficients with Ni and V, but noted that with the exclusion of the New York City data, the overall association was no longer statistically significant. Lipfert et al. (2006) found V and Ni to be significantly associated with long-term mortality, but that the traffic density variable was more robust and had larger explanatory value. Other studies also point to traffic emissions and particular metals as both having significant associations with health endpoints (Janssen et al., 2002; Grahame & Hidy 2004). It is important to recognize that studies that have the capability of examining levels of both metals and of vehicular emissions tend to find both of health importance, and if the exposure to vehicular emissions is of good quality, tend to find little else of health significance (Schwartz et al., 2005; Gold et al., 2005; Ebelt et al., 2005). Janssen et al. (2002), in a study on the influence of air conditioning as a modifier of hospital admission in relation to PM10 concentrations, modeled source contributions to ambient air PM using emissions data, rather than data from measured individual components. Cardiovascular admissions were significantly associated with a number of sources (highway vehicles, oil combustion, coal combustion, and metal processing), but there were no significant associations of the sources with COPD or pneumonia admissions. Ostro et al. (2006) studied the associations between daily mortality and PM2.5 and its major components in

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nine California counties. For cardiovascular causes, they reported significant associations for PM2.5, OC, and EC. There were no significant associations for respiratory causes. Chen and Schwartz (2008) extended the analyses on the NHANES III population beyond the Schwartz (2001) study described earlier. In this study, they examined the associations between annual average concentrations of PM10 and WBC count, and of five components of metabolic syndrome (MS) (insulin resistance, high blood pressure, hypertriglyceridemia, high-density lipoprotein [HDL] cholesterol, and abdominal obesity). They reported a statistically significant association between WBC count and local PM10 levels (p = .035), and a graded association between and WBC count across subpopulations with increasing MS components (trend test p = .15). Although PM10 is not informative about the composition of the inhaled particles, this study is still worthy of a citation because of the support it provides for a role of ambient air PM in contributing to metabolic syndrome. It is also of interest that Schneider et al. (2008) demonstrated that in a panel of people with type 2 diabetes mellitus, there was a decrease in flow-mediated dilatation in brachial artery diameter in relation to PM2.5 exposure during the first day, whereas their small artery elasticity decreased with a delay of 1 and 3 days. High levels of myeloperoxidase led to the strongest effects on endothelial dysfunction. Auchincloss et al. (2008) studied associations between ambient air concentrations of 24-h average PM2.5, SO2, NO2, and CO, averaged over the previous 1, 2, 7, 30, and 60 days, and blood pressure in six US cities as part of the Multi-Ethnic Study of Atherosclerosis (MESA). They found (1) no evidence of strong threshold/nonlinear effects for PM2.5; (2) significant associations of PM2.5 with systolic pressure (SBP) and pulse pressure (PP); (3) associations of PM2.5 with SBP and PP became stronger with averaging time up to 30 days; (4) associations of PM2.5 with SBP and PP became stronger after adjustment for gaseous air pollutants; (5) associations of PM2.5 with traffic exposure indicators were (unexpectedly) significantly negative; (6) associations of PM2.5 with BP were not modified by age, sex, diabetes, smoking, study site, SO2, CO, season, or distance from road; and (7) associations of PM2.5 with BP were stronger for persons on BP medications or having hypertension, during warmer weather, with higher NO2, living 6 μg/ml) within 15 min. HP and LP CAPs (>25 μg/ml) differentially affected the endogenous scavengers, glutathione, and nonprotein sulfhydryl after 1.5 h. Both HP and LP CAPs stimulated the release of proinflammatory cytokines TNFα and IL-6 after 6 h of exposure. Microarray analysis of both HP and LP exposed microglia (75 μg/ml) identified 3200 (HP) and 160 (LP) differentially expressed (up- and down-regulated) genes relative to the medium controls. The results implicate Ni and/or V in production of these effects in that these two metals were much higher in concentration in the HP than the LP CAPs. The biological plausibility for fine and ultrafine PM in ambient air to be translocated, from the lungs to the brain, and to have neurological effects, is supported in a review paper by Peters et al. (2006).

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To investigate the contributions of PM2.5 components to cardiovascular effects, Lippmann et al. (2005c) used the 5 months of daily 6-h source apportionments of Maciejczyk and Chen (2005), the continuous HR data for exposure days (weekdays only) used in Hwang et al. (2005), and the corresponding HRV data used in Chen and Hwang (2005) to determine the source-related PM2.5 components’ associations with HR and HRV. They used HR and HRV data collected on normal (C57) mice and a murine model for atherosclerotic disease (ApoE−/−) (Chen and Hwang, 2005; Hwang et al., 2005). Daily 6-h PM2.5 air samples were also collected and analyzed by x-ray fluorescence (XRF), permitting attribution to major PM2.5 source categories (secondary SO42−, suspended soil, residual oil combustion, and a remainder category, which was largely due to long-range transported motor vehicle traffic). They examined associations between these PM2.5 components and both HR and HRV for three different daily time periods: (1) during exposure; (2) the afternoon following exposure; and (3) late at night. For HR, there were significant transient associations (p ≤ .01) for secondary sulfate during exposure, and for residual oil combustion (predominantly V and Ni) in the afternoon. For HRV, there were comparable associations with suspended soil (predominantly Si, Al, Ca) in the afternoon and for both residual oil combustion and traffic (Br, Fe, elemental carbon), late at night. The biological bases for these various associations and their temporal lags are not known at this time, but may have something to do with the differential solubility of the PM2.5 components at the respiratory epithelia, and their access to cells that release mediators that reach the cardiovascular system. One important parameter that was not addressed in the above study, but that could influence metals’ ability in mediating biological response, is the extent of soluble metal components present in the PM2.5 mass. In a follow-up subchronic PM2.5 CAPs inhalation study of ApoE−/− mice at 85 μg/m3 (Lippmann et al., 2006), there was a dramatic change in cardiac function in the fall months in the ApoE−/− mice. As previously discussed, the 14 days with northwest winds carried more Ni, Cr, and Fe, but less of the other elemental tracers than the 89 days with winds from all other directions, and were associated with significant increases in HR and significant decreases in HRV (Lippmann et al., 2006). V was lower than normal on the 14 days with unusually high levels of Ni, Cr, and Fe in this mouse study. Back trajectory analyses from Sterling Forest for the 14 days with northwest winds led through lightly populated areas to Sudbury, Ontario, which is the location of the largest Ni smelter in North America. At the end of the 6 months of exposure in this study, Sun et al. (2005) compared the mice in the CAPs-exposed subgroup on a high-fat diet (HF) with those exposed to filtered air (FA). For the CAPs-exposed mice, the plaque area in the aorta was 41.5% versus 26.2% in the FA group (p = .001), whereas for the subgroup on a normal diet, CAPs-exposed versus FA-exposed was 19.2% versus 13.2% (p = .15). Lipid


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content in the aortic arch in the HF group versus normal chow (NC) group exposed to CAPs was 30% versus 20% (p = .02). Vasoconstrictor challenges in the thoracic aorta were increased in the CAPs-exposed HF mice versus the FA mice (p = .03), and relaxation in response to acetylcholine was greater (p = .04). In addition, HF mice exposed to CAPs had marked increases in macrophage infiltration, expression of inducible NO synthase, ROS generation, and immunostaining for 3-nitrotyrosine (all with p < .001). Thus the 30-h/week subchronic CAPs exposure of ApoE−/− mice at 85 μg/m3 altered vasomotor tone, induced vascular inflammation, and potentiated atherosclerosis. Results of additional assays on ApoE−/− mice in the same 6-month CAPs exposure study were reported by Sun et al. (2008a). They described the results of in vivo measurements of plaque in the aorta by ultrasound biomicroscopy (UBM) prior to sacrifice, as well as macrophage infiltration (CD68) and tissue factor (TF) expression in the sections of the aorta. UBM-derived plaque areas were 7% larger in the CAPsexposed HF mice than in the FA-exposed controls (p = .04), whereas the comparison among NC mice was not statistically significant (p = .07). Based on immunochemistry, TF expression was increased in the HF mice by CAPs exposure (15% versus 8%, p < .01), as was macrophage infiltration (19% versus 14%, p < .01). In a 10-week, 30 h/week CAPs inhalation exposure study in SD rats conducted in Tuxedo, NY, at an average concentration of 79 μg/m3, Sun et al. (2008b) implanted minipumps for angiotensin II (A-II) after 9 weeks of exposure. Following the infusion, the mean arterial pressure was elevated in the CAPs-exposed rats compared to the FA-exposed rats (p < .001). 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 CAPs-exposed A-II rats. In addition, superoxide levels in the aorta were increased in the CAPs-exposed A-II rats. Based on these findings and some coordinate in vitro PM exposures, they concluded that the CAPs exposure exaggerates hypertension through superoxide-mediated up-regulation of the Rho/ROCK pathway. In a CAPs inhalation study, Tan et al. (2009) exposed four groups of C57BL/6 male mice that had been fed normal chow (NC) or high-fat chow (HFC) to either FA or CAPs for 6 h/day, 5 days/week, for 6 weeks at Tuxedo, NY. After sacrifice, nonalcoholic fatty liver disease (NAFLD) grading and staging were evaluated, stellate-cell activation was detected, and collagen I staining was quantified by morphometric analysis. They also performed in vitro exposures using a reference PM sample (NIST SRM1649a). Wild-type (wt) and Toll-like receptor 4 (TLR4) knockout (TLR4−/−) C57BL/6 mice were sacrificed 24 h after a 500-µg intravenous injection. For the mice exposed to CAPs by inhalation, no significant steatosis was noted for those on a NC diet. Activated stellate cells were detected in both of the HFC-fed groups, but the mean steatohepatitis grade and stage were both significantly higher in the CAPs-exposed group versus the

FA-exposed group. The mean collagen I staining was significantly greater in the HFC group exposed to CAPs compared to the other groups. For the mice exposed by injection, Standard Reference Material (SRM) particles were detected only in Kupffer cells from livers of SRM-injected mice and not in sham-injected mice. In cell culture studies, incubation with 0–200 µg/ml SRM for 24 h induced a dose-dependent increase in proinflammatory cytokine mRNA levels, particularly IL-6 (p = .01). Supernatant analysis confirmed increased IL-6 protein secretion (p = .03). Similarly, PM2.5 exposure (0– 100 µg/ml) increased IL-6 secretion in a dose-dependent manner by wt Kupffer cells (p = .08) and not by TLR4−/− Kupffer cells (p = .29). PM2.5 exposure (0–400 µg/ml, 24 h) did not significantly affect collagen 1A1 mRNA or protein levels in the LX2 stellate cell line. PM2.5 up to 400 µg/ml did not enhance collagen 1A1 levels in wt or TLR4−/− mouse stellate cells. However, collagen 1A1 mRNA levels increased significantly in wt and TLR4−/− stellate cells when incubated with conditioned medium from SRM (200 µg/ml)-exposed RAW cells (p < .01). Thus, direct exposure to PM2.5 activates IL-6 production by Kupffer cells in a TLR4-dependent manner. Therefore, enhanced inflammation and fibrosis in NAFLD via direct activation of Kupffer cells may be caused by inhaled PM that enters the circulation, and exposure to ambient air PM may be a significant risk factor for NAFLD progression. In a study of the effects of CAPs exposure on an obese mouse model, Sun et al. (2009) used male C57BL/6 mice that were fed HF chow for 10 weeks before being exposed to CAPs at Tuxedo, NY, for 6 h/day, 5 days/week for 24 weeks. Compared to the FA-exposed controls, the CAPs-exposed mice had insulin signaling abnormalities that were associated with abnormalities vascular relaxation to insulin and acetylcholine and increased adipose tissue macrophages (F4/80+ cells) in visceral fat expressing higher levels of TNFα/IL-6 and lower IL-10/Mgl1 (macrophage activation marker galactose-N-acetylgalactosamine specific lectin). In coordinate in vitro tests, PM induced cell accumulation in visceral fat and potentiated cell adhesion in the microcirculation. The authors concluded that CAPs exposure exaggerated insulin resistance and visceral inflammation/ adiposity, providing a link between CAPs exposure and type 2 diabetes mellitus and metabolic syndrome. The biological plausibility of ambient air PM contributing to metabolic syndrome in the CAPs-exposed obese mice is enhanced by a report by Chen and Schwartz (2008) on data showing an association of PM10 with white blood cell (WBC) count and metabolic syndrome in the population of the third National Health and Nutrition Examination Survey (NHANES III). 4.3.2. New York City Ying et al. (2009a) exposed ApoE−/− mice on a high-fat diet to FA or PM2.5 CAPs for 6 h/day, 5 days/week, for 4 months in northern Manhattan, at a mean concentration of 173 μg/ m3, to test the hypothesis that exposure to CAPs enhances


Health effects of ambient air CAPs atherosclerosis through induction of vascular reactive oxygen and nitrogen species. They reported that Manhattan CAPs was characterized by higher concentrations of OC and EC than Tuxedo, NY, CAPs. Analysis of vascular responses revealed significantly decreased phenylephrine constriction in CAPs-exposed mice, which was restored by a soluble guanine cyclase inhibitor ODQ (1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one). Vascular relaxation to A23187, but not acetylcholine, was attenuated in CAPsexposed mice. Aortic expression of NADPH oxidase subunits (p47phox and rac1) and inducible nitric oxide synthase (iNOS) were markedly increased, paralleled by increases in superoxide generation and extensive protein nitration in the aorta. The composite plaque area of the thoracic aorta was significantly increased, with pronounced macrophage infiltration and lipid deposition in the CAPs-exposed mice. Thus, CAPs exposure in Manhattan alters vasomotor tone and enhances atherosclerosis through NADPH oxidase– dependent pathways. 4.3.3. Columbus, OH In a follow-up study of the effects of CAPs exposure in Tuxedo, NY, on the Rho/ROCK pathway, Ying et al. (2009b) exposed C57BL/6 male mice that had been fed normal chow (NC) to either FA or CAPs for 6 h/day, 5 days/week, for 12 weeks at Columbus, OH. Following the inhalation exposures, the mice were implanted with minipumps for A-II or vehicle for 14 days. One day after that, they were treated with fasudil, a Rho-kinase inhibitor, or vehicle. The CAPs exposure potentiated A-II–induced hypertension, and this effect was abolished by fasudil treatment. Cardiac and vascular RhoA activation was enhanced by CAPs exposure, along with increased expression of the guanine exchange factors PDZ, RhoGEF, and p115RhoGEF, increased A-II–induced cardiac hypertrophy, and collagen deposition, which were all normalized by fasudil treatment. These findings help to explain the chronic cardiovascular effects of PM2.5 exposures. In a further CAPs inhalation study focused on effects on the liver, Laing et al. (2009) exposed C57BL/6 male mice that had been fed normal chow to either FA or CAPs for 6 h/day, 5 days/week, for 10 weeks at a mean concentration of 76 μg/m3. They also exposed a murine monocytic-macrophage cell line (Sigma-Aldrich RAW264.7) to Columbus PM in vitro at 300 μg/ml. The CAPs inhalation induced endoplasmic reticulum (ER) stress and activation of a unique unfolded protein response (UPR) in the liver. The in vitro exposures demonstrated that macrophage ingestion of PM and the selective activation of the UPR components rely on ROS and Ca signals. In the liver tissue of the CAPs-exposed mice, the selective activation of the UPR components is coordinated with the activation of NF-κB and c-Jun amino-terminal kinase (JNK) and reduced expression of paraoxonase 1 (PON-1) and peroxisome proliferator-activated receptor gamma (PPAR-γ), which favors the development of cardiovascular diseases.

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4.3.4. Los Angeles, CA Kleinman et al. (2007) exposed OVA-treated BALB/c mice to PM2.5 CAPs, PM0.18 CAPs, or FA for 5 days/week, for 2 weeks at 50 and 150 m downwind from a heavily traveled Freeway in Los Angeles. Measurements were made of IL-5, IL-13, immunoglobulin E (IgE), IgG1, and pulmonary infiltration of PMNs and eosinophils. Mice exposed at a distance of 50 m, compared to FA mice, had significant increases in IL-5 and IgG1, whereas those exposed at 150 m did not. The changes at 50 m were significantly associated with the EC and OC in the PM2.5 and PM0.18 CAPs, suggesting that freshly formed carbonaceous PM could exert adjuvant affects and promote the development of allergic airway disease. Araujo et al. (2008) exposed ApoE−/− mice to CAPs for 5 h/day, 3 days/week, for 5 weeks in two size ranges ( SH). The number of macrophages decreased following V and Ni exposure at 6 h, and this decrease was reversed by 24 h in both strains. V caused BALF PMNs to increase only in WKY rats. The Ni-induced increase in BALF PMNs was more dramatic and progressive than that of V, but was similar in both strains. Lung histology similarly revealed more severe and persistent edema, perivascular and peribronchiolar inflammation, and hemorrhage in Ni- than in V-exposed rats. This effect of Ni appeared slightly more severe in SH than in WKY rats. This study showed that inflammatory response to metallic constituents of ROFA is both strain and dose dependent, and that V caused pulmonary injury only in WKY rats, whereas Ni was toxic to both strains. In subsequent studies (Kodavanti et al., 2002b; Wu et al., 2003), however, Zn was found to be the responsible component in a batch of different oil combustion emission particles. Some, or perhaps most, of the differences in biological responses to the metals in the ROFA could be explained by aqueous solubility, and by the effects of solubility on translocation. Wallenborn et al. (2007) measured the elemental content of lungs, plasma, heart, and liver of male WKY rats after IT administration of either saline or 8.3 mg/kg of ROFA from a Boston power plant, and measured tissue concentrations 4 and 24 h after the instillation. Water-soluble metals (V, Ni, Zn, and Mn) were detected in plasma, hearts. and livers at both time points, whereas Al and Si were not. The effects of two ROFA samples of equivalent diameters, but having different metal and SO42− content, on pulmonary responses in SD rats were studied (Gavett et al., 1997). One sample had higher saline-leachable SO42−, Ni, V, and Fe, whereas the other sample had higher Zn. At a dose of 2.5 mg, 4 of 24 rats exposed to high-Zn ROFA suspension or supernatant had died 4 days post IT administration, but none in the high-Ni, -V, and -Fe groups. Pathological indices, such as alveolitis, early fibrotic changes, and perivascular edema, were greater in both high-Zn suspension and supernatant exposed groups than the other ROFA. In surviving rats, exposures to high-Zn ROFA also worsened the baseline pulmonary function parameters and airway hyperresponsiveness (AHR) to acetylcholine as well as BAL PMNs. This study confirmed the finding of an earlier study in guinea pigs that soluble forms of Zn are capable of producing a greater pulmonary response than other sulfated metals in combustion generated particles (Amdur et al., 1978). AHR induced by ROFA and its soluble components was also observed in mice exposed to an aerosolized soluble leachate of ROFA (ROFA-s) was described by Hamada et al. (2002). AHR to acetylcholine challenge occurred in a time- and dose-dependent manner after exposure to ROFA-s with peak at 48 h post inhalation (IH) exposure. AHR was accompanied by an earlier onset of BAL PMNs, which was maximal at 12 h after exposure. The AHR caused by ROFA-s was reproduced by a mixture of

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its major metal components (Ni, V, Zn, Co, Mn, Cu), but not by any individual metal alone. Intraperitoneal pretreatment of mice with the antioxidant dimethylthiourea abrogated ROFA-s–mediated AHR, confirming the role of ROS in metal-induced inflammation. Interestingly, ROFA-s had no effect on AHR of 2-week-old mice, in contrast to the AHR seen in 3- and 8-week-old mice. This study also found that ROFA treatment does not initiate neurogenic inflammation because ROFA-s–mediated AHR was unchanged in neurokinin-1 receptor knockout mice and in mice treated with a neurokinin antagonist. After either IT or IH exposure, of ROFA, lung injury is evident within 24 h of exposure, with a dose-dependent recruitment of PMNs, eosinophils, and monocytes into the airway. The peak of this influx occurs 18–24 h after exposure. The cellular influx persisted 96 h later, and resolution occurs slowly. Inflammatory lung injury after ROFA was accompanied by airway inflammation, an increase in susceptibility to infections, and, at high concentration, noncardiogenic pulmonary edema. ROFA treatment did not initiate neurogenic inflammation, because ROFA-s–mediated AHR was unchanged in neurokinin-1 receptor knockout mice and in mice treated with a neurokinin antagonist. 4.5.4. Tire dust Gottipolu et al. (2008) instilled two kinds of tire dust into the lungs of male WKY rats. TP1 was made from ground tires of recyled styrene butadiene rubber, whereas TP2 was from scrap tires. Elemental analyses were available for both dusts. Tests were done with administered saline, TP1, and TP2. Additional tests were done with soluble Zn, Cu, or both. For TP1 and TP2, there were increases in BAL fluid markers of inflammation and injury (TP2 > TP1) but no effects on cardiac enzymes. Instillation of Zn, Cu, and Zn + Cu decreased the activity of cardiac aconitase, isocitrate dehydrogenase, succinate dehydrogenase, cytochrome c oxidase, and superoxide dismutase, indicative of cardiac oxidative stress. 4.5.5. Metal oxide particles Lu et al. (2009) compared in vitro assays of intrinsic free radical generation, oxidative activity in an extracellular environment, cytotoxicity to A549 lung epithelial cells, hemolysis of healthy human erythrocytes, and inflammation potency in WKY rat lungs. They used nanoparticles of carbon back (CB) and metal oxide nanoparticles (2–30 nm) composed of NiO, CeO2, Co3O4, MgO, SiO2, anatase, rutile, and three kinds of alumina; ZnO as UFP (90–210 nm); and alumina as 0.3-μm microparticles. For assays using equivalent surface areas, only NiO and alumina no. 2 (7 nm) caused significant lung inflammation, whereas 4 of the 13 metal oxides (NiO, CeO2, Co3O4, and CB) caused significant free radical generation (of these, only NiO was inflammogenic), and 3 of 13 (NiO, CeO2, and alumina no. 2) were significantly hemolytic (of these, only NiO was inflammogenic). Thus, in vitro assays cannot be relied on to predict lung inflammation.

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5. Organ system responses


In this section, we first briefly summarize the exposures and responses described in greater detail under Sections 3 and 4 in humans and animals in terms of the effects elicited by specific major organ systems, and their consistency across the studies in various laboratories and airsheds. This approach serves several purposes: 1. To organize, for specific organ systems, the results of laboratory-based CAPs inhalation studies in a consistent manner across species, and to present the findings of human panel and large population studies in a similar format. 2. To examine the literature on other laboratory-based studies of ambient air PM components on responses, and to explore the underlying biological mechanisms that account for the exposure-related effects. 3. To identify critical knowledge gaps that can be filled by further research. Following the discussions by organ system, Section 6 will discuss the overall coherence of the literature on CAPs, epidemiology, and underlying biological mechanisms that account for responses to ambient air PM and its components. 5.1. Cardiovascular system 5.1.1. Summary of human cardiovascular responses to short-term CAPs exposures In Chapel Hill, NC, PM2.5 CAPs exposure caused an elevation in blood fibrinogen that was correlated with a Cu/Zn/V factor. In healthy old adults, CAPs exposures decreased HRV, a response not seen in the young adults. In healthy, young, adults, PM0.18 CAPs decreased HRV and increased D-dimer in the plasma; PM2.5 CAPs increased PMNs and monocytes in BAL cells, decreased IL-8 in BAL fluid, and increased fibrinogen in plasma; whereas PM10-2.5 increased PMN in the BAL cells, decreased protein in the BAL fluid, decreased HRV, and increased the trend for clotting in the plasma (Ghio et al., 2000a; Devlin et al., 2003: Samet et al., 2007). In London, there was an increase in blood fibrinogen associated with ambient air PM10 (Pekkanen et al., 2000). In Helsinki, concentrations of PM2.5 and particle count (PC) were significantly associated with increased risk of ST-segment depression in subjects with stable coronary heart disease. There was no consistent association for PM10-2.5. The associations were stronger for subjects not using beta-blockers (Lanki et al., 2006). In Los Angeles, healthy young adults, as well as patients with asthma or COPD, were exposed to PM10-2.5, PM2.5, or PM0.18. PM0.18 produced only slight changes in mediators of blood coaguability and HRV. Blood pressure decreased in asthmatics and increased in normal subjects. In elderly subjects with COPD, there were reductions in pulse rate and frequency of ectopic beats, but no change in HRV after exposure to PM2.5 CAPs. In mild

asthmatics exposed to PM10-2.5, there were increases in HR, and reductions in HRV. In both healthy normal subjects and mild asthmatics exposed to PM2.5 CAPs, there were reductions in O2 saturation, FEV1 1 day later, and LF HRV in both groups (Gong et al., 2003, 2004a, 2004b, 2008). In Toronto, there was a PM2.5 CAPs–related mean decrease in BAD, but no changes in blood pressure, in one study, whereas in a follow-up study involving most of the same subjects, CAPs exposure produced a significant decrease in diastolic blood pressure. In both studies, the effects were significantly associated with OC. There were suggestive, but not significant, associations with EC and some metals (Cd, K, Zn, Ca, Ni) in the first study (Brook et al., 2002; Urch et al., 2004, 2005). In Edinburgh, healthy and age-matched volunteers with stable coronary heart disease were exposed to PM2.5 CAPs and to FA. After CAPs exposure, there were increases in exhaled breath 8-isoprostane (p < .05), blood flow, and plasma tissue plasminogen activator (p < .005), but there were no significant changes in markers of systemic inflammation, and no effect on vascular function in either group of subjects (Mills et al., 2008). In overall summary, the still quite limited number of human CAPs inhalation studies provide some provocative information on the ability of short-term CAPs inhalations to elicit statistically significant cardiovascular responses at concentrations approximating peak ambient levels in North America and Europe. These include responses to all three size ranges of current interest, in adults covering a wide range of ages and of preexisting health status. In most studies, there was an increase in plasminogen and a decrease in HRV. Furthermore, there is evidence that the chemical composition of the PM affects the responses. The extent to which the differences in PM composition among the different cities can account for the differences in responses in the studies summarized above remains to be determined. 5.1.2. Summary of animal cardiovascular responses to short-term CAPs exposures Summarizing the results of the larger number of shortterm CAPs inhalation studies in animals is more challenging because we are dealing with interspecies differences, genetically altered animal models for human diseases, and differing modes of inhalation that affect the inhaled dose. SD rats exposed to Boston PM2.5 CAPs had significant oxidative stress in the heart, with strong associations of CAPs with the content of Fe, Al, Si, and Ti in the heart. The oxidant stress was associated with increases in the heart water content. In addition, CAPs inhalation led to tissuespecific increases in the activities of SOD and catalase, suggesting that CAPs exposure may also trigger adaptive responses (Clarke et al., 1999). SD rats were exposed to Boston PM2.5 CAPs or FA with and without pretreatment with CPZ, a selective antagonist of vanilloid receptor 1. CPZ prevented the decreased CAPs-induced cardiac oxidative


Health effects of ambient air CAPs stress, lipid peroxidation, edema in the heart, heart rate, and the lengths of the QT, RT, Pdur, and Tpe intervals produced in the rats not pretreated with CPZ (Ghelfi et al., 2008). Dogs previously implanted with balloon occluders and catheters for determining myocardial blood flow underwent 5-min coronary artery occlusions immediately after FA and Boston PM2.5 CAPs exposures. The CAPs exposure, although not affecting HR, increased occlusioninduced peak ST-segment elevation (p = .007), which was correlated (p = .003) with the Si content of the CAPs, as well as with other crustal components, but not with PM2.5 mass, BC, OC, or S. In another study, the dogs underwent 5-min coronary artery occlusions immediately after the FA and CAPs exposures. The CAPs exposure decreased total myocardial blood flow (p < .001), was accompanied with an increase in coronary vascular resistance (p < .001), and the effects were more pronounced in or near the ischemic zone versus more remote myocardium (p < .001). Thus, PM2.5 exacerbates myocardial ischemia (Clarke et al., 2000). WKY male rats were exposed to PM2.5 CAPs or FA in Yokohama, Japan, for 4.5 h/day for 4 days, or for 1 day with FA for 3 days, or to FA for 4 days. Cardiovascular function and mRNA were measured in heart tissue after the end of the 4 days. CAPs exposure caused dose-related up-regulation of P450 (CYP)1B1, HO-1, and endothelin A (ET-A). The up-regulation of ET-A was significantly correlated with that of HO-1, and weakly with the increase in blood pressure (Ito et al., 2008). ApoE−/− mice exposed to PM2.5 CAPs on weekdays for 6 h/days, for 6 months in Tuxedo, NY. Cardiac function parameters were monitored continuously and CAPs composition was determined for each exposure day. Exposures to Ni, Cr, and Fe were much higher on 14 days than on the other 89 exposure days, corresponding to days with unusually high HR and unusually low HRV. In addition, V was lower than normal on the days with high Ni, because the Ni was a distant smelter rather than residual oil combustion, the usual source of elevated Ni. The authors attributed the acute effects on cardiac function to peaks in Ni from a distant point source (Lippmann et al., 2006). In overall summary, inhaled urban PM2.5 CAPs affects cardiac function in mice, rats, and dogs via oxidative stress, and the effects appear to be influenced more by inorganic PM components (transition metals and EC) than by components associated with the OC in secondary aerosols. This conclusion is consistent with that drawn by Mauderly and Chow (2008) in their review on the health effects of organic aerosols. 5.1.3 Summary of animal cardiovascular responses to long-term CAPs exposures In the first in a series of PM2.5 CAPs exposures in Tuxedo, NY, over 5 to 6 months (5 days/week, 6 h/day for ApoE−/− mice), there were acute and chronic effects on cardiac function, as well as increased amounts of, and more invasive, aortic plaque (Lippmann et al., 2005b, 2005c). The associations

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between PM2.5 components and both HR and HRV differed (1) during exposure; (2) the hours following exposure; and (3) late at night. For HR, there were transient associations (p ≤ .01) for secondary SO42− during exposure, and for residual oil combustion (predominantly V and Ni) in the afternoon. For HRV, there were comparable associations with suspended soil (predominantly Si, Al, Ca) in the afternoon and for both residual oil combustion and traffic (Br, Fe, EC) late at night (Lippmann et al., 2005c). In a follow-up 6-month PM2.5 CAPs inhalation study, there was a dramatic change in cardiac function in the fall months. As shown in Figure 1, 14 days with northwest winds carried much more Ni, Cr, and Fe, but much less of the other elemental tracers than the other 89 days, and were associated with significant increases in HR and significant decreases in HRV (Lippmann, 2006). For CAPs-exposed mice on a highfat diet (HF), versus FA-exposed mice, plaque area in the aorta was increased (p = .001), whereas for those on normal chow, the difference between CAPs- and FA-exposed mice was smaller (p = .15). Lipid content in the aortic arch in the HF group versus NC groups exposed to CAPs was increased (p = .02). Vasoconstrictor challenges in the thoracic aorta were increased in the CAPs-exposed HF mice versus the FA mice (p = .03), and relaxation in response to acetylcholine was greater (p = .04). In addition, CAPs-exposed HF mice had marked increases in macrophage infiltration, inducible NO synthase expression, ROS generation, and immunostaining for 3-nitrotyrosine (all with p < .001). Thus, the 30-h/week subchronic CAPs exposure of ApoE−/− mice altered vasomotor tone, induced vascular inflammation, and potentiated atherosclerosis. Additional assays included in vivo measurements of plaque in the aorta by ultrasound biomicroscopy (UBM) prior to sacrifice, as well as macrophage infiltration (CD68) and tissue factor (TF) expression in the sections of the aorta. UBM-derived plaque areas were larger in the CAPs-exposed HF-fed mice than in the FA-exposed controls (p = .04), whereas the comparison among NC-fed mice did not reach statistically significance (p = .07). Based on immunochemistry, TF expression was increased in the HF mice by CAPs exposure (p < .01), as was macrophage infiltration (p < .01) (Sun et al., 2005). In a 10-week CAPs inhalation exposure study in SD rats, in which A-II minipumps were implanted after 9 weeks of exposure, the A-II infusion elevated the mean arterial pressure in the CAPs-exposed versus FA-exposed rats (p < .001). Aortic vasoconstriction to phenylephrine was potentiated, with exaggerated relaxation to the Rhokinase (ROCK) inhibitor Y-27632 and increase in ROCK-1 mRNA levels in the CAPs-exposed A-II rats. In addition, superoxide levels in the aorta were increased in the CAPsexposed A-II rats. Thus, the CAPs exposure exaggerated hypertension through superoxide-mediated up-regulation of the Rho/ROCK pathway (Sun et al., 2008b). In male C57BL/6 mice fed HF chow for 10 weeks to create an obese mouse model, PM2.5 CAPs exposure caused insulin signaling abnormalities that were associated with abnormalities in vascular relaxation to insulin and acetylcholine and


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increased adipose tissue macrophages (F4/80+ cells) in visceral fat expressing higher levels of TNFα/IL-6 and lower IL-10/Mgl1. In coordinate in vitro tests, CAPs induced cell accumulation in visceral fat and potentiated cell adhesion in the microcirculation. Thus, the CAPs exposure exaggerated insulin resistance and visceral inflammation/adiposity, providing a link between CAPs exposure and type 2 diabetes mellitus and metabolic syndrome (Sun et al., 2009). In male ApoE−/− mice exposed for 5 months to PM2.5 CAPs, plaques were quantified for brachiocephalic artery cross-sections (BACs) after 3 and 5 months of exposure using (1) serial ultrasound imaging; (2) H&E histology; and (3) en-face Sudan IV stain. All three methods indicated that CAPs, despite having a much lower PM concentration, caused more plaque development than corresponding exposures to whole diesel exhaust (WDE) or diesel exhaust PM, both containing diesel exhaust particles (DEP) at 436 µg/m3, indicating that some components in ambient PM, not present in WDE, are responsible for the exacerbation of plaque progression by CAPs (Quan et al., 2009). A summary of the plaque progression at 5 months of CAPs and of diesel engine exhaust exposure is presented in Table 2. Male ApoE−/− mice on a HF diet were exposed to FA or PM2.5 CAPs for 4 months in northern Manhattan to test the hypothesis that CAPs exposure enhances atherosclerosis through induction of vascular reactive oxygen and nitrogen species. Manhattan CAPs was characterized by higher concentrations of OC and EC than Tuxedo, NY, CAPs. Analysis of vascular responses revealed significantly decreased phenylephrine constriction in CAPs-exposed mice, which was restored by a soluble guanine cyclase inhibitor ODQ. Vascular relaxation to A23187, but not acetylcholine, was attenuated in CAPs-exposed mice. Aortic expression of NADPH oxidase subunits (p47phox and rac1) and iNOS were markedly increased, paralleled by increases in superoxide generation and extensive protein nitration in the aorta. The composite plaque area of the thoracic aorta was significantly increased, with pronounced macrophage infiltration and lipid deposition in the CAPs-exposed mice. Thus, CAPs exposure in Manhattan altered vasomotor tone and enhances atherosclerosis through NADPH oxidase– dependent pathways (Ying et al., 2009a). In a follow-up study of the effects of PM2.5 CAPs exposure in Tuxedo, NY, on the Rho/Rock pathway, C57BL/6 male mice in Columbus, OH, that were fed normal chow (NC) were exposed for 12 weeks. Following the exposures, the mice were implanted with minipumps for A-II or vehicle for 14 days. One day later, they were treated with fasudil, a Rhokinase inhibitor, or vehicle. CAPs exposure potentiated A-II– induced hypertension, an effect abolished by fasudil treatment. Cardiac and vascular RhoA activation was enhanced by CAPs exposure, along with increased expression of the guanine exchange factors PDZ, RhoGEF, and p115RhoGEF; increased A-II–induced cardiac hypertrophy; and collagen deposition, which all were normalized by fasudil treatment. The authors concluded that these findings help to explain

the chronic cardiovascular effects of PM2.5 exposures (Ying et al., 2009b). A further CAPs inhalation study in Columbus, OH, focused on effects on the liver, but it also has significant implications to cardiovascular disease. C57BL/6 male mice fed normal chow were exposed to either FA or PM2.5 CAPs for 10 weeks. A murine monocytic-macrophage cell line was exposed to CAPs in vitro. The CAPs inhalation induced endoplasmic reticulum (ER) stress and activation of a unique unfolded protein response (UPR) in the liver. The in vitro exposures demonstrated that macrophage ingestion of PM and the selective activation of the UPR components rely on ROS and Ca signals. In the liver tissue of the CAPs-exposed mice, the selective activation of the UPR components is coordinated with the activation of NF-κB and c-Jun amino-terminal kinase (JNK) and reduced expression of paraoxonase 1 (PON-1) and peroxisome proliferator-activated receptor gamma (PPAR-γ), which favors the development of cardiovascular diseases (Laing et al., 2009). ApoE−/− mice were exposed in Los Angeles to CAPs for 5 weeks in two size ranges ( SH). V caused a BALF PMN increase, but only in WKY rats. The Ni-induced increase in BALF neutrophils was greater than that of V, but was similar in both strains. Lung histology revealed more severe and persistent edema, perivascular and peribronchiolar inflammation, and hemorrhage in Ni- than in V-exposed rats. Thus, inflammatory response to metallic constituents of ROFA was both strain and dose dependent. V caused pulmonary injury only in WKY rats, whereas Ni was toxic to both strains. In subsequent studies, however, Zn was found to be the responsible component in a batch of different oil combustion emission PM. ROFA samples of equivalent diameters, but different metal and SO42− content, on pulmonary responses in SD rats were studied. Pathological indices, such as alveolitis, early fibrotic changes, and perivascular edema, were greater in both high-Zn suspension and supernatant exposed groups than the other ROFA. In surviving rats, exposures to high-Zn ROFA also worsened the baseline pulmonary function parameters and airway hyperresponsiveness (AHR) to acetylcholine as well as BAL PMNs. This study confirmed the finding of an earlier study in guinea pigs that soluble forms of Zn are capable of producing a greater pulmonary response than other sulfated metals in combustion generated particles (Amdur et al., 1978; Adamson et al., 2000). Airway hyperreactivity (AHR) induced by ROFA and its soluble components has also been observed in mice exposed to aerosolized soluble leachate of ROFA (ROFA-s). The AHR caused by ROFA-s was reproduced by a mixture of its major metal components (Ni, V, Zn, Co, Mn, Cu), but not by any individual metal alone. After either IT exposure or inhalation of ROFA, lung injury was evident within 24 h, with a dose-dependent recruitment of PMNs, eosinophils, and monocytes into the airway, peaking 18–24 h after exposure. The cellular influx persisted 96 h later, with resolution occurring slowly. Inflammatory lung injury after ROFA was accompanied by an increase in susceptibility to infections, and, at high concentration, noncardiogenic pulmonary edema. 5.2.3.4. Tire dust. Gottipolu et al., (2008) instilled two kinds of tire dust into the lungs of male WKY rats. TP1 was made from ground tires of recyled styrene butadiene rubber, whereas TP2 was from scrap tires. Elemental analyses

were available for both dusts. Tests were done with administered saline, TP1, and TP2. Additional tests were done with soluble Zn, Cu, or both. For TP1 and TP2, there were increases in BAL fluid markers of inflammation and injury (with TP2 > TP1). Similar effects were seen for instilled Zn and Cu. They concluded that the acute pulmonary effects of TP could be due to the metals. 5.2.3.5. Metal oxide nanoparticles. Lu et al. (2009) compared in vitro assays of intrinsic free radical generation, oxidative activity in an extracellular environment, cytotoxicity to A549 lung epithelial cells, hemolysis of healthy human erythrocytes, and inflammation potency in WKY rat lungs. For assays using equivalent surface areas, only NiO and one of four aluminas caused significant lung inflammation, whereas 4 of the 13 metal oxides (NiO, CeO2, Co3O4, and CB) caused significant free radical generation and 3 of 13 (NiO, CeO2, and one of four aluminas) were significantly hemolytic. Thus, in vitro assays cannot be relied on to predict lung inflammation. 5.2.3.6. Bacterial challenge. Zhou and Kobzik (2007) exposed murine primary alveolar macrophages and the murine macrophage cell line J774A.1 to Streptococcus pneumoniae. Boston CAPs increased the binding of bacteria by both cell types. By contrast, CAPs decreased cellular internalization. Soluble CAPs components mediated both the enhanced binding and and decreased internalization. Iron chelation reversed the inhibition of phagocytosis, whereas added iron restored it. 5.2.4. Studies of human pulmonary responses to ambient air PM Several studies in human populations exposed to ambient air PM have shown significant associations with pulmonary effects, including reductions in FEV1 and FVC related to traffic-related PM2.5 (Penttinen et al., 2006; McCreaner et al., 2007; Allen et al., 2008) and ED visits (Sarnat et al., 2008). The coherence of the effects seen in these studies with those seen in the human and animal CAPs inhalation studies is discussed in Section 6.2.1. 5.3. Hepatic system effects of long-term CAPs inhalation studies Male mice fed normal chow (NC) or high fat chow (HFC) inhaled FA or PM2.5 CAPs for 6 weeks at Tuxedo, NY, and nonalcoholic fatty liver disease (NAFLD) and collagen I staining were quantified by morphometric analysis. For the mice exposed to CAPs, no significant steatosis was noted for those on a NC diet. Activated stellate cells were detected in both of the HFC-fed groups, but the mean steatohepatitis grade and stage were both significantly higher in the CAPs-exposed group versus the FA-exposed group, and the mean collagen I staining was significantly greater in the HFC group exposed to CAPs compared to the other groups. For in vitro exposures using a reference PM sample (NIST SRM1649a), wild-type (wt) and TLR4 knockout (TLR4−/−) C57BL/6 mice were exposed, and SRM particles were detected only in Kupffer cells from livers of


Health effects of ambient air CAPs SRM-injected mice. Cell culture studies were done using macrophage (RAW) and stellate (LX2) cell lines. In RAW cells, incubation with SRM for 24 h induced a dosedependent increase in proinflammatory cytokine mRNA levels, particularly IL-6 (p = .01) and IL-6 protein secretion (p = .03). Similarly, PM2.5 exposure increased IL-6 secretion by wt Kupffer cells (p = .08). However, collagen 1A1 mRNA levels increased significantly in wt and TLR4−/− stellate cells when incubated with conditioned medium from SRM-exposed RAW cells (p < .01). Thus, direct exposure to PM2.5 activates IL-6 production by Kupffer cells in a TLR4dependent manner. Therefore, enhanced inflammation and fibrosis in NAFLD via direct activation of Kupffer cells may be caused by inhaled PM that enters the circulation, and PM may be a significant risk factor for NAFLD progression (Tan et al., 2009). In a further CAPs inhalation study focused on effects on the liver, Laing et al. (2009) exposed mice to either FA or CAPs for 10 weeks. They also exposed a murine monocytic-macrophage cell line in vitro. The CAPs inhalation induced endoplasmic reticulum (ER) stress and activation of a unique unfolded protein response (UPR) in the liver. The in vitro exposures demonstrated that macrophage ingestion of PM and the selective activation of the UPR components rely on ROS and Ca signals. In the liver tissue of the CAPs-exposed mice, the selective activation of the UPR components was coordinated with the activation of NF-κB and c-Jun amino-terminal kinase (JNK) and reduced expression of paraoxonase 1 (PON-1) and peroxisome proliferator-activated receptor gamma (PPAR-γ), which favors the development of cardiovascular diseases. 5.4. Nervous system effects 5.4.1. CAPs inhalation ApoE−/− mice were exposed to UFP CAPs at a site 200 m from freeway 110 in central Los Angeles at two different mass concentrations, i.e., 114 and 30 μg/m3. Th ere was a dose-related increase in nuclear translocation of NF-κB and AP-1, which promote inflammation. Increased levels of glial fibrillary acidic protein (GFAP) were also found. Levels of MAP kinases were assayed, and the fraction of JNK present in active form was increased (Kleinman et al., 2008). Male BALB/6 mice were exposed by inhalation to PM2.5 CAPs, UFP CAPs, or FA for 4 h/day, 5 days/week, for 2 weeks at a site 150 m downwind from a heavily traveled freeway in Los Angeles. One and 2 weeks after the last exposure, the mice were challenged with aerosolized ovalbumin (OVA). As compared to FA exposure, both CAPs exposures increased inflammatory indices in the brains of the sensitized mice, and the levels of proinflammatory cytokines IL-1α, TNFα, and NF-κB were increased in the brain tissue (Kleinman et al., 2007). Rats, with and without OVA-induced allergic airway disease, were exposed to PM2.5 CAPs in Grand Rapids, MI. The next day, the brains of the CAPs- and FA-exposed rats were prepared for analyses. CAPs exposure led to a significant increase in norepinephrine in the paraventricular nucleus,

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along with an elevation in serum coricosterone with CAPs + OVA and with OVA presensitization compared to CAPs alone. Thus, CAPs alone or CAPs plus OVA pretreatment can activate the stress axis, which could play a role in aggravating allergic airway disease (Sirivelu et al., 2006). For ApoE−/− mice exposed to PM2.5 CAPs at Tuxedo, NY, for 5 to 6 months, the exposures caused changes in brain cell distribution (Veronesi et al., 2005). The biological plausibility of ambient air PM contributing to changes in brain cell distribution was enhanced by a follow-up study in which in vitro assays of the cellular and genomic responses of immortalized microglia cells (BV2) to CAPs collected during that same study. Two composite samples were applied to the microglia cells; one composed of CAPs from days with high potency (HP) in their stimulation of NF-κB release in human bronchial epithelial cells, and the other from CAPs collected on days with low potency (LP). The LP composites reduced intracellular ATP at doses >250 μg/ml, and depolarized mitochondrial membranes (>6 μg/ml) within 15 min. HP and LP CAPs (>25 μg/ml) differentially affected the endogenous scavengers, glutathione, and nonprotein sulfhydryl after 1.5 h. Both HP and LP CAPs stimulated the release of proinflammatory cytokines TNFα and IL-6 after 6 h of exposure. Microarray analysis of both HP- and LP-exposed microglia (75 μg/ml) identified 3200 (HP) and 160 (LP) differentially expressed (up- and down-regulated) genes relative to the medium controls. The results implicate Ni and/or V in production of these effects in that these two metals were much higher in concentration in the HP than the LP CAPs (Sama et al., 2007). The biological plausibility for fine and ultrafine PM in ambient air to be translocated from the lungs to the brain, and to have neurological effects, is supported in a review paper by Peters et al. (2006). 5.4.2. Neurological effects of ROFA after instillation Zanchi et al. (2008) exposed Wistar rats to ROFA from a Brazilian steel mill (20 μg) or saline by intranasal instillation with and without simultaneous treatment by 150 mg/kg of an antioxidant, NAC, for 30 days. ROFA instillation induced an increase in lipid peroxidation in striatum (p = .03) and cerebellum (p = .03) compared with control, and NAC blocked these effects. In addition, ROFA decreased peripheral walking (p = .006) and exploration (p = .001), and these responses were not blocked by NAC. ROFA treatment was not associated with any changes in emotionality or grooming. Th e ROFA particles had a mean aerodynamic diameter of 1.2 μm, and the content of V, Mn, and Fe were 35 μg/g, 3.9 mg/g, and 44%, respectively.

6. Coherence of responses to CAPs exposures in humans and animals in vivo For short-term responses to PM in ambient air, we can compare short-term exposures of humans and laboratory animals exposed to CAPs by inhalation and to cellular


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responses in vitro to aqueous suspensions of particle mixtures, such as CAPs, ROFA, CFA, and/or to individual components known to occur in ambient air. For chronic effects associated with long-term exposures to ambient air pollution, our most valid comparisons to effects observed in community-based populations are largely limited to those seen in laboratory animals receiving a long-term series of daily CAPs exposures. In this context, it is important to remember that people and animal models can differ substantially in their susceptibilities and sensitivities to PM exposures. For example, Wheeler et al. (2006) showed that, in response to ambient air PM exposures, people with COPD responded with increased HRV, whereas people with recent myocardial infarctions responded with decreased HRV, as did the normal healthy subjects in the CAPs inhalation studies cited in this paper. 6.1. Cardiovascular Responses 6.1.1. CAPs exposures in humans and animals 6.1.1.1. Acute effects. In humans, most PM2.5 CAPs inhalation studies reported increases in plasminogen and decreases in HRV, whereas in dogs and rats, most studies reported increases in one or another index of oxidative stress, with two studies (one in rats and one in dogs) indicating decreased HRV. Variations in responses were attributable to differences in age, preexisting health status, and PM composition. In most of the cases where PM components were identified, the effects were more closely associated with inorganic, rather than with OC. Peretz et al. (2008a) exposed healthy young adults in Seattle to WDE containing either 100 or 200 μg/m3 of DEP for 2 h. At 3 h post-exposure for 200 μg/m3, there was a statistically significant increase in HF power, and a decrease in LF/HF ratio, but no effect on time-domain statistics and no effects on HRV at later time points. In a study by Peretz et al. (2008b), both healthy adults and those with metabolic syndrome (MS) in Seattle who were exposed to WDE containing either 100 or 200 μg/m3 of DEP for 2 h. For the subjects with MS, and for all subjects combined, there was an acute endothelial response and vasoconstriction of a conductive artery. 6.1.1.2. Chronic Effects. There were no chronic CAPs inhalation studies in humans. As indicated in Section 5.1.3, a series of subchronic CAPs inhalation studies have shown PM2.5 CAPs-related chronic, as well as acute, changes in HR and HRV (Hwang et al., 2005; Chen & Hwang, 2005), aortic plaque growth (Chen & Nadziejko, 2005), potentiated atherosclerosis (Sun et al., 2005), tissue factor expression (Sun et al., 2008a), hypertension (Sun et al., 2008b), metabolic syndrome (Sun et al., 2009), and vasomotor tone (Ying et al., 2009). 6.1.2. Coherence of responses to CAPs exposures in humans and animals in vivo with responses to PM2.5 in epidemiological studies 6.1.2.1. Acute effects. Increases in plasminogen in PM2.5 CAPs–exposed humans was seen in the human

populations in relation to ambient air PM2.5 in NHANES III (Schwartz, 2001) and one German panel study (Hoffmann et al., 2009), but not another German panel study (Rudez et al., 2009), that reported associations of PM2.5 with platelet aggregation and thrombin generation. The decreases in HRV seen in human, dog, rat, and mouse PM2.5 CAPS inhalation studies were seen in association with ambient air PM2.5 in Helsinki (Pekkanen et al., 2002), Amsterdam, Helsinki, and Erfurt, Germany (Lanki et al., 2006; Yue et al., 2008), Taipei (Chuang et al., 2007), and North Carolina (Riedeker, 2007). The PM2.5-associated shortterm changes in cardiac wave forms seen by Wellenius et al. (2003, 2004), Chen et al. (2005), Lippmann et al. (2006), and Ghelfi et al. (2008) in CAPs-exposed animals were consistent with the PM2.5-related effects seen by Zhang et al. (2009) in the WHI cohort. 6.1.2.2 chronic effects. PM2.5 CAPs exposure in an obese mouse model for 10 weeks caused insulin signaling abnormalities, providing a link between CAPs exposure and type 2 diabetes mellitus and metabolic syndrome (MS) (Sun et al., 2009). In the NHANES III population, Chen and Schwartz (2008) demonstrated a significant association between WBC count and PM10, and a graded association between WBC count and across subpopulations with increasing MS components. In a Chapel Hill, NC, panel with type 2 diabetes mellitus, there were PM2.5-related decreases in flowmediated dilatation in brachial artery diameter and small artery elasticity, and high levels of myeloperoxidase led to the strongest effects on endothelial dysfunction (Schneider et al., 2008). In greater Boston, four PM metrics were associated with decreased vascular reactivity in diabetics, with SO42− being associated with both decreased flow-mediated and nitroglycerin-mediated vascular reactivity, whereas BC was associated with only decreased flow-mediated vascular reactivity (O’Neill et al., 2005). The other effects produced by long-term PM2.5 CAPs inhalation studies in mice and rats, i.e., increased deposition of plaque in the aorta, vascular inflammation, tissue factor expression, and blood pressure, are consistent with the report of Diez-Roux et al. (2009) on evidence of a significant association of PM2.5 with subclinical atherosclerosis in the MESA study. 6.1.3. Coherence of responses to CAPs exposures in humans and animals with responses to PM2.5 and components in lung inhalation and instillation studies Studies in which collected ambient air PM and specific mixtures containing components of ambient air PM were resuspended for controlled animal inhalation or lung instillation studies have produced results that can help interpret the biological plausibility of fi ndings in the CAPs inhalation studies, and the PM components that are especially influential. In an inhalation study in rats with Ni and V, Ni caused delayed bradycardia, hypothermia, and arhythmogenesis at concentrations >1.2 mg/m3, whereas V alone did not


Health effects of ambient air CAPs induce any significant changes. However, when combined, Ni and V produced observable delayed bradycardia and hypothermia at 0.5 mg/m3, suggesting a synergistic relationship at high metal concentrations (Campen et al., 2001). However, these effects at very high concentrations of Ni and V do not necessarily support the associations of Ni with acute cardiac function changes at the very much lower Ni concentrations in the CAPs study in mice. IT studies involving lung exposures to dusts from the Utah Valley before, during, and after the strike, and ROFA dusts have been particularly informative because of wellestablished human health effects associated with the inhalation of these dusts. As discussed earlier in this review, the effects of ROFA and Utah Valley dusts have been attributed largely to their metals contents. This is consistent with the results of the intervention study in Hong Kong in which both monthly cardiovascular (and pulmonary) mortality rates dropped substantially after a mandated switch to low-S fuels and the associated step-function reduction in airborne Ni, V, and SO2 (without any corresponding reduction in other metals or gaseous criteria pollutants) (Hedley et al., 2002, 2004). It is also consistent with daily peaks of Ni (but not V) and increased HR and decreased HRV in an atherosclerotic mouse model during the course of a 6-month CAPs inhalation study (Lippmann et al., 2006). On the other hand, there are clearly other PM2.5 components that confer cardiovascular toxicity. For example, Ottawa PM extracts instilled into rat lungs induced pronounced biphasic hypothermia, a severe drop in heart rate, and increased arrhythmias that were greater than those with the ROFA particles. No such effects were seen with a comparable instilled dose of Mt. St. Helens volcanic ash (Watkinson et al., 2002a, 2002b). Furthermore, PM2.5 and PM10-2.5 CAPs collected from six European cities with contrasting traffic profiles, PM composition, and in vitro analyses were instilled into spontaneously hypertensive (SH) rats. PM2.5 and PM10-2.5 CAPs dose–related effects included blood viscosity, with a trend toward greater toxicity with increasing traffic levels. However, there was no correlation of any of the effect markers with combustionexhaust-related PAHs except for an increase of lymphocytes associated with PM2.5 CAPs (Gerlofs-Nijland et al., 2007). An important role for metals is also evident in a study in which two tire dusts were instilled into the lungs of rats. TP1 was made from ground tires of recycled styrene butadiene rubber, whereas TP2 was from scrap tires. Tests were done with administered saline, TP1, TP2, soluble Zn, Cu, or both. At very higher concentrations, the exposures induced cardiac oxidative stress (Gottipolu et al., 2008). Aside from metals, there has been a considerable focus on motor vehicle exhaust as a source category that could account for the adverse health effects associated with PM2.5, and especially the soot in the exhaust from diesel engines. For acute responses, the most direct laboratorybased comparison of the effects of PM2.5 in ambient air and in DEP can be found in papers by Cassee et al. (2002, 2005). They were able to produce acute increases in blood fibrinogen with both CAPs and concentrated DEP, but it took

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far higher concentrations of DEP than CAPs to do so. In terms of acute human responses, the situation is less clear. Mills et al. (2005) found acute exposure to DEP for 1 h at 300 μg/m3 did produce blood fibrinogen responses, whereas their CAPs exposures (Mills et al., 2008) did not. However, they noted that their CAPs aerosol (in Edinburgh, Scotland) was nearly all sea salt. Human CAPs exposures in Chapel Hill, Los Angeles, and Toronto, at concentrations well below 300 μg/m3, did produce cardiac system responses. These findings are consistent with the associations between hospital admissions for cardiovascular diseases for Medicare patients in 106 US counties as reported by Bell et al. (2009) in which the effects were most closely associated with Ni, V, and EC in the PM2.5. Recent studies in which the cumulative effects associated with subchronic inhalation exposures to ambient air PM2.5 CAPs were directly compared in the same animal models and exposure durations to those of other complex toxicant mixtures, such as diluted diesel engine exhaust (Quan et al., 2009) and sidestream cigarette smoke (Chen et al., 2009), were especially informative. As shown in Table 2, they showed that eastern US regional CAPs was considerably more atherogenic than either diesel exhaust or sidestream smoke on the basis of PM mass inhaled, even without consideration of the gaseous toxicants associated with the sidestream smoke and diesel exhaust particles. Overall, it appears that the cardiovascular effects of ambient air PM2.5 are greatly influenced, if not dominated, by their metals contents, especially the transition metals, and that Ni is likely to be a key component. 6.2. Coherence of Pulmonary Responses 6.2.1. CAPs exposures in humans and animals 6.2.1.1. Acute effects. In humans, there were very few CAPs inhalation studies with information on component concentrations, but there was evidence in a study in Chapel Hill in which the association with oxidant stress in terms of neutrophilic inflammation included an association of oxidative stress with a Fe/Se/SO42− factor in the PM2.5, but with very small changes in pulmonary function. In rats and dogs, there were many studies providing evidence for CAPs exposures being associated with pulmonary inflammation, increases in WBCs, and lavage protein, with greater effects in bronchitic animals. These effects were usually associated with inorganic metal components, especially transition metals. 6.2.1.2. Chronic effects. There were no chronic CAPs inhalation studies, and none of the long-term CAPs inhalation studies in mice and rats produced any chronic pulmonary effects. 6.2.2. Coherence of pulmonary responses to CAPs exposures in humans and animals in vivo with responses to PM2.5 in epidemiological studies 6.2.2.1. Acute effects. The increases in lung inflammation in CAPs-exposed humans and animals were seen in asthmatic


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walkers in London exposed to diesel exhaust, in terms of PMNs in BAL (McCreanor et al., 2007), in residents in Seattle exposed to wood smoke, in terms of exhaled NO (Allen et al., 2008), and in elderly residents in San Gabriel Valley in California, in terms of an increase in IL-6 in association with EC, BC, and/or PC (Delfino et al., 2008). 6.2.2.2. Chronic effects. There were no chronic pulmonary effects seen in the subchronic CAPs exposure studies to compare to (1) the reduction in effects seen in the Hong Kong, China, S-in-fuel intervention study (Hedley et al., 2002, 2004); (2) the association of annual average Ni and V with NMMAPS average city mortality (Lippmann et al., 2006); (3) Ni, V, and traffic being significantly associated with long-term mortality in the Veterans’ cohort study (Lipfert et al., 2006); or (4) the influence of PM2.5 sources (gas engines, diesel engines, wood smoke, resuspended soil, secondary sulfate, secondary nitrate, cement kiln, railroad, and metal processing) on cardiorespiratory morbidity was studied in Atlanta, GA. For the summer months, PM2.5 SO42− was significantly associated with respiratory ED visits (Sarnat et al., 2008). In these epidemiological studies, the PM2.5-associated excess mortality was most closely related to inorganic components associated with fossil fuel combustion. 6.2.3. Coherence of pulmonary responses to CAPs exposures in humans and animals in vivo with responses to PM2.5 using in vivo lung inhalation studies 6.2.3.1. Diesel engine exhaust. Stenfors et al. (2004) exposed young healthy and mild asthmatic Swedish subjects for 2 h to diluted WDE containing DEP at 108 μg/m3 and assessed lung function and airway inflammation. The exposures did not affect FEV1 or FVC, but produced significant increases in sRaw in both groups, with a greater response in asthmatic subjects. In a further study of healthy subjects by Pourazar et al. (2005), a DEP concentration of 300 μg/m3 activated redox-sensitive transcription factors, and in archived bronchial biopsies from this inhalation study, Pourazar et al. (2008) showed that the exposure caused significant increases in the expression of EGFR and phophorylated C-terminal Tyr 1173. Bosson et al. (2007) exposed young healthy Swedish subjects to WDE containing 300 μg/m3 of DEP for 1 h, which was followed 5 h later by 2 h of exposure to 200 ppb of O3. Sputum was collected 18 h after the O3 exposure. Th e O3 exposure magnified the WDE-induced inflammation. Healthy males undergoing intermittent moderate exercise in Edinburgh were exposed to 300 μg/m3 of WDE for 1 h. Healthy adults, seven female and eight male, were exposed to FA or diluted WDE. At 18 h post exposure, they performed bronchoscopies and BALF and bronchial biopsy tissues were collected. There were increases in bronchial mucosa PMNs and mast cells, as well as increases in BALF PMNs, IL-8, and myeloperoxidase (Behndig et al., 2006). Salvi et al (1999) found, in human volunteers, that diluted WDE caused increases in PMNs in airway lavage fluid, as well as in peripheral blood.

There were no corresponding studies of pulmonary response to WDE in laboratory animals. In summary, the responses to WDE in humans at 100– 300 μg/m3, summarized above, are similar to those seen in humans exposed to CAPs or PM2.5 in ambient air in US studies at much lower PM2.5 mass concentrations. 6.3. Coherence in other systems The lack of reports on PM-associated responses in the hepatic and nervous systems in short-term CAPs inhalation studies and in panel and larger population studies related to ambient air concentrations precludes an examination of concordance in these systems.

7. Discussion of the role and contributions of CAPs studies The pace at which CAPs studies have been undertaken, especially for subchronic inhalation studies, has grown substantially in recent years, and the results have significantly expanded our ability to appreciate the impacts of air pollution on public health, especially in regard to the environmental relevance of cardiovascular and nervous system effects. In summary, short-term exposures of human volunteers and laboratory animals at concentrations from normal to near the upper bound of current ambient PM levels have been associated with statistically significant changes in HR, HRV, abnormal heartbeats, arrhythmias, and in flow changes in brachial arteries, and where compositional data were available, were most closely associated with the inorganic components, i.e., EC and trace metals. Subchronic PM2.5 CAPs exposures at long-term mean concentrations approximating the current annual PM2.5 NAAQS of 15 μg/m3 have produced a series of remarkable findings. Th ese include persistent changes in HR, HRV; enhancement of aortic plaque size; changes in brain cell distribution and function; fatty liver deposits; and progression of the metabolic syndrome. The enhancement of aortic plaque size is consistent with the epidemiological fi nding of PM-related progression of carotid intima-media thickening (Kunzli et al., 2005), a commonly used surrogate for atherosclerosis. The findings of significant hepatic and nervous system effects in the subchronic CAPs inhalation studies in animals were unanticipated, and such effects have not yet been found in studies of human populations. The lack of such findings in humans may simply be due to the absence of inquiry rather than to the lack of effects, and future studies of human populations should now be designed to look for such effects. Finding adverse effects in these organ systems with CAPs exposure, however, does imply that PM inhalation is capable of producing systemic inflammation and possible mechanisms for the development of adverse cardiopulmonary outcomes. The results of the studies that focused on cardiopulmonary outcomes, although still well short of accounting for the


Health effects of ambient air CAPs natural history of PM-related diseases, do provide biological plausibility for excess cardiovascular mortality and morbidity that was previously lacking. However, it should be noted that almost all of the subchronic CAPs inhalation studies performed to date have been done in New York and Ohio, and their results may not represent those to be found in studies in other communities with quite different PM compositions where such studies are now underway (Seattle, WA), or where planned studies are yet to be performed by New York University (Ann Arbor, MI, and Anaheim, CA) as part of the ongoing HEI NPACT project. The biological plausibility of community air PM2.5 contributing to premature mortality and excess morbidity in the United States at current ambient levels has been further enhanced by the results of recent subchronic CAPs inhalation studies in which direct comparisons, using the same animal models and the same exposure systems and exposure durations, were made between the effects of CAPs and those of sidestream cigarette smoke (Chen et al., 2009) and of fresh diluted Diesel engine exhaust (Quan et al., 2009). In both of these head-to-head comparisons, the CAPs, despite having lower PM2.5 mass concentrations, and much lower EC, OC, and organic vapor concentrations, produced greater health-related effects than wellstudied mixtures that are commonly accepted as being highly toxic. The biological plausibility of ambient air PM contributing to metabolic syndrome in CAPs-exposed obese mice (Sun et al., 2009) is enhanced by a report by Chen and Schwartz (2008) on data showing an association of PM10 with white blood cell (WBC) count and metabolic syndrome in the NHANES III population. It is also of interest that Schneider et al. (2008) demonstrated that in a panel of people with type 2 diabetes mellitus, there was a decrease in flow-mediated dilatation in brachial artery diameter in relation to PM2.5 exposure during the first day, whereas their small artery elasticity decreased with a delay of 1 and 3 days. High levels of myeloperoxidase led to the strongest effects on endothelial dysfunction Now that a growing body of well-designed and wellexecuted CAPs inhalation studies have demonstrated that PM2.5-associated effects of public health concern and regulatory interest are highly plausible, the question remains as to why the effects are taking place at such low PM2.5 mass concentrations. The limited body of CAPs and epidemiologic studies in which compositional data were available indicate that some specific PM2.5 components, such as EC and Ni, are more closely associated with the effects than other components. However, we still don’t know the extent to which these components account for the effects observed. For example, do ionic Ni and Ni in insoluble forms have the same effects? If so, is the amount of the Ni in ionic form dependent on the pH of the ambient aerosol? For EC, which adsorbs organic vapors, are the effects associated with EC due to insoluble core particles, or to a surface layer of adsorbed OC? If so, does the surface activity of the EC particles change with time-

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of-day as freshly formed reactive compounds deposit on the particle surfaces and react with other atmospheric chemicals? To answer these questions, we will need to develop a better understanding of atmospheric acid formation and subsequent neutralization by alkalinity in the particles and by atmospheric ammonia, and of the dynamics of photochemistry in community air. At this point in time, it seems highly likely that at least some of the toxicity associated with ambient air PM2.5 and UFP is due to particle surface coatings of reactive chemicals that were freshly formed in the atmosphere. Thus, although we now know that the effects are real, we are still at an early stage in our quest to know which PM components are especially influential in causing some specific health-related responses and which genetic, preexisting conditions, past occupational and environmental exposure history, and activity patterns predispose individuals to adverse health effects in relation to current ambient air PM exposures. In any case, the technology for conducting inhalation exposure studies of CAPs in human volunteers and laboratory animals has been well developed and refined, as have the techniques for quantitative analyses of the PM components that were inhaled, and for sampling and analysis of biomarkers of exposure, susceptibility, and effects in those exposed. Thus, it is now possible to greatly enhance our appreciation and understanding of the health effects of exposure to ambient air PM. In conjunction with the simultaneous advances in the sophistication in statistical modeling, use of biomarkers, and their applications in recent epidemiological studies of PM effects, there is less justification for questioning the biological plausibility of a significant burden of adverse health effects occurring as a result of contemporary exposures to PM in ambient air. Conclusions about causality of any specific PM components, or of source-related mixtures, must be tentative at this time. What the insights provided by the studies reviewed here do provide is a better basis for the design of future studies whose results may be more definitive. Because of their potential for oxidative activity and the production of ROS, OC and transition metals have long been suspected to be major components of ambient PM that produce adverse health effects. The importance of transition metals in producing adverse health effects was confirmed in the pseudo-intervention study of Utah Valley, which indicated that reduction in metals in PM associated with year-long closure of a local steel mill was associated with improved health conditions in the local population. The role of metals (e.g., V, Fe, Cu, Zn, and Ni) was further confirmed by later studies using human clinical, as well as animal and in vitro toxicology studies. At this point in time, based on recent advances in knowledge cited herein, the PM components in community air that seem likely to be most potent are the EC and trace metals that are emitted from fossil fuel combustion sources, with the greatest risks applying to the cardiovascular, nervous, pulmonary, and hepatic organ systems.


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Other than access to adequate research funding, there is no obvious impediment to further progress in animal and human CAPs exposure studies. On the other hand, further progress in panel and epidemiological studies is currently limited by the paucity of speciation data, as discussed by Lippmann et al. (2009) and EPA (2009). Finally, in consideration of the recent increase in epidemiological and toxicological research that has considered the influence of PM2.5 composition on health-related responses, it seems ever clearer that spatial and temporal differences in PM2.5 composition do exist, and for the same ambient level of PM2.5 mass, the health implications of exposure to ambient air depend on the PM composition. Therefore, the mass-based ambient standards may provide a different level of protection at different places, and at different times at the same place, depending on the source mix contributing to the ambient mass concentration.

8. Summary of unresolved issues and conclusions 8.1. Where do PM components fit in the larger picture of PM-associated health effects? There is clearly emerging evidence that the inhalation of some components of ambient air PM are associated with adverse health effects at concentrations near or not much higher than current ambient PM2.5 mass concentrations. These components include EC, Ni, V, and Pb, and suggestive evidence exists for others, such as Al, Zn, and OC. There is also a rapidly growing literature implicating motor vehicle–related pollution in human health effects, as indexed by proximity to major roadways, and by measured concentrations of OC, NO2, and UFP. However, there are also metals in motor vehicle exhaust and resuspended road dust whose role, if any, in causing traffic-related human health effects at contemporary ambient air concentrations is unresolved, with a suggestion that the metals in resuspended road dust may be important, as discussed in the next section. Furthermore, there is some evidence that adverse health effects are significantly associated with aerosol acidity originating from fossil fuel combustion, which could be due to its irritancy, or to its role in solubilizing metals within the particles. 8.2. Are there any specific source categories that can account, at least in part, for health effects associated with PM2.5 components? It is known that ROFA, which is a mixture that is presumed to be similar in composition to the fly ash emitted by power plants burning residual oil, and which is notably high in the content of Ni and V, as compared to other metals, and Utah Valley dust, which is a mixture enriched in steel mill emissions, were more toxic than other source-related mixtures that have been tested in laboratory animals in vivo, or in cells in vitro. For acute pulmonary system responses, it appears, from such tests, that V and Zn may play prominent roles, and that the effects may depend on interactions among the

metals. For acute cardiovascular effects, Ni appears to play a more important role. By contrast, other source-related mixtures, such as coal combustion effluents, that are notable for their content of Se, Fe, and Mn, and resuspended soil, which contains more refractory metals, have been found to be less acutely toxic. Recent research has suggested that traffic-generated PM can account for pulmonary effects, with Gent et al. (2009) showing that the motor vehicle source was associated with a 10%/5 μg/m3 increase in wheeze. In asthmatic children, the road dust source was associated with a 28% increase in shortness of breath. This response is consistent with the findings reported by Gottipolu et al. (2008) based on the instillation of two kinds of tire dust into the lungs of male WKY rats in relation to the elemental composition. There were increases in BAL fl uid markers of inflammation and injury, and similar effects were seen for instilled Zn and Cu. Thus, the acute pulmonary effects of tire dust could be due to the metals. Janssen et al. (2002) modeled source contributions to ambient air PM using emissions data. Cardiovascular admissions were significantly associated with a number of sources (highway vehicles, oil combustion, coal combustion, and metal processing), but there were no significant associations of the sources with COPD or pneumonia admissions. Aside from metals, there has been a considerable focus on motor vehicle exhaust as a source category that could account for the adverse health effects associated with PM2.5, and especially the soot in the exhaust from diesel engines. For acute responses, the most direct laboratorybased comparison of the effects of PM2.5 in ambient air and in DEP can be found in papers by Cassee et al. (2002, 2005). They were able to produce acute pulmonary changes and increases in blood fibrinogen with both CAPs and concentrated DEP, but it took far higher concentrations of DEP than CAPs to do so. In terms of acute human responses, the situation is less clear. Mills et al. (2005) found acute exposure to DEP for 1 h at 300 μg/m3 did produce acute pulmonary and blood fibrinogen responses, whereas their CAPs exposures (Mills et al., 2008) did not. However, they noted that their CAPs (in Edinburgh, Scotland) was nearly all sea salt. Human CAPs exposures in Chapel Hill, Los Angeles, and Toronto, at concentrations well below 300 μg/m3, did produce pulmonary and cardiac system responses. The recent studies in which the cumulative effects associated with subchronic inhalation exposures to ambient air PM2.5 CAPs were directly compared in the same animal models and exposure durations to those of other complex toxicant mixtures, such as diluted diesel engine exhaust (Quan et al., 2009) and sidestream cigarette smoke (Chen et al., 2009), were especially informative. As shown in Table 2, eastern US regional CAPs was considerably more potent in terms of aortic plaque progression than the PM in either diesel exhaust or sidestream smoke on the basis of PM mass inhaled, even without consideration of the gaseous toxicants

Health effects of ambient air CAPs


associated with the sidestream smoke and diesel exhaust particles. In summary, tailpipe emissions, although likely to play some role in causing the cardiovascular and pulmonary effects associated with ambient air PM2.5, may require especially high concentrations, and may not play a dominant role in the effects in the population as a whole, whereas toxic metals from power plants, and possibly from resuspended road dust, are worthy of increased concern. 8.3. Addressing research needs relating to health effects of components in ambient air particulate matter There are many reasons why past research has not resolved the roles that PM components may play in the health-related effects of ambient air PM. These are (1) concentrations of components in ambient air PM generally range from a few µg/m3 in some nonvolatile metals to less than 10 ng/m3 for transition metals that are known to generate ROS, raising the issue of biological plausibility; (2) epidemiologic research opportunities have been limited because of the paucity of data on the concentrations of PM components. Even now, when PM2.5 speciation data have been available since 2000 for many US cities, they are mostly limited to every third or sixth day, which severely limits their use for studying acute responses (Lippmann, 2009); (3) few toxicologists or clinical researchers have had the resources needed to perform subchronic CAPs inhalation studies that include speciation data on the PM in the exposure samples; (4) controlled exposures to pure compounds at concentrations of environmental relevance have been uniformly negative, even when sensitive animal models were used; (5) a lack of studies defining the relationship between personal exposure and ambient air levels for most metal species; (6) most controlled clinical and laboratory animal exposure studies have been limited to one or a few days, which may not be sufficient to elicit responses of concern; and (7) in vitro studies of the biological mechanisms underlying the effects seen in controlled laboratory-based exposures and in populations exposed to ambient air PM have all been done at doses of carbonaceous and elemental PM that are at least several orders of magnitude larger than those in the in vivo studies in animals or in human populations. The subchronic inhalation studies in New York, Ohio, and California suggest that CAPs studies overall, and those with elevated concentrations of Ni and EC in particular, can yield evidence that current levels of ambient air concentrations produce health effects of interest in terms of public health. Furthermore, there are many toxicology studies of ROFA cited earlier that are buttressed by epidemiological studies (Hedley et al., 2002, 2004; Janssen et al., 2002; Lipfert et al., 2006), suggesting a line of continuity between both types of studies with regard to damage from the combination of V and Ni. There are also some studies showing oxidative stress and DNA damage associated with V but not with Ni (Sorensen et al., 2005). If the inhalation

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of Ni, or Ni in combination with V, at current, relatively low, ambient air concentrations, does appreciably affect cardiac function and mortality in humans, one may wonder why the effects of such exposures have not previously been recognized. One reason may be that the increment in cardiovascular mortality that they may have produced is a relatively small part of the very large cardiovascular mortality. Also, it is possible that other PM2.5 components may account for the more numerous studies, not having speciation data, showing associations of PM2.5 mass with health effects because of the influence of other components alone or because the other components potentiate the effects of Ni. Also, the statistically significant transient and progressive changes that Ni produced in cardiovascular function in the ApoE−/− mice were relatively subtle, required advanced analytical techniques for their detection, and are unlikely to be detected in the kinds of short-term exposure studies that have previously been undertaken in laboratory animals. In addition, the exact physical and chemical characteristics of Ni-bearing ambient PM have not been determined. Based on the previous work at NYU, the fact that the potency of ultrafine Zn particles with a thin coating of sulfuric acid is much greater than uncoated acid particles (Amdur & Chen 1989) raises the likelihood that a specific form of a metal that has not been reproduced in the laboratory could be responsible for the observed biological effects. Many studies have used ROFA as a surrogate for ambient PM in various in vivo and in vitro experiments. ROFA contains many soluble metals, and because it is clear, from this review, that they interact with each other, chemically and biologically, it should not be surprising that there are inconsistent and confusing results. Although ROFA was useful in providing plausible evidence that metals are important in eliciting adverse cardiopulmonary effects, it should not be the focus of future studies. The experimental in vitro design of Maciejczyk and Chen (2005), in which CAPs were collected in a Biosampler impinger simultaneously with a series of daily CAPs inhalations, provided a sound basis for parallel daily in vitro assays. Performing such assays in parallel with future animal inhalation studies and/or human clinical studies could provide opportunities for gaining a better understanding of the source profile that may contribute to the adverse effects seen in animals and humans. Much of the remaining skepticism concerning the biological plausibility of the premature mortality and increased morbidity associated with ambient air PM2.5 has been due to the paucity of exposure-response data in laboratory studies involving PM2.5 inhalation, and the heretofore seemingly impossible task of identifying any specific causal components. The subchronic CAPs inhalation studies that were performed in Sterling Forest (Tuxedo, NY) (Lippmann et al., 2005b, 2006; Sun et al., 2005) helped to establish such plausibility, and have also developed a


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mechanistic base for the initiation and progression of effects attributable to the long-range transported aerosol in the northeastern United States (Sun et al., 2005). The consistency, in these analyses and reexaminations of available data lead to the conclusions that (1) Ni is a particularly influential component of ambient PM2.5 in terms of cardiac responses to the inhalation of ambient air PM2.5; (2) further research is needed on the specific influences of both Ni and V, which are both generally most closely associated with residual oil combustion effluents, on both acute and chronic respiratory and cardiovascular health effects; and (3) further research is also needed on the currently unknown impacts of other toxic metals in ambient air. 8.4. Biological plausibility For most biological scientists today, biological plausibility needs to be based on a clear understanding of the underlying biological mechanisms that can account for the empirical observations arising from laboratory-based exposures or epidemiological studies of populations in the real world. Until recently, mechanistic understanding, based on in vitro studies, has been quite sparse, and the evidence for causal relationships between exposures to ambient air PM and adverse health effects has been more indirect. Of necessity, in the absence of much knowledge on underlying mechanisms, we have had to rely the consistency of significant associations between indices of PM exposures and specific responses, their stability over time and space, and the coherence of each specific response with other responses that one would expect to see if the first was really reliable. In part, this situation was necessitated by the fact that we have had to rely on exposure indices based on particle mass, and have seldom had access to the concentrations of the causal components within the PM, and their temporal concentration variation. Also, as noted above, in vitro studies of the biological mechanisms underlying the effects seen in controlled laboratory-based exposures and in populations exposed to ambient air PM have all been done at doses of carbonaceous and elemental PM that are at least several orders of magnitude larger than those in the in vivo studies in animals or in human populations. We have only recently begun to have opportunities to frame the questions about underlying biological mechanisms in conjunction with observational studies of exposure-response relationships. Numerous examples can be found in the descriptions of the subchronic CAPs inhalation studies in this critical review. For example: 1. Sun et al. (2005) demonstrated that the in vivo CAPs exposure increase of plaque in the aorta was associated with lipid content; responses to vasoconstrictor challenge; relaxation in response to acetylcholone; increases in macrophage infiltration; expression of inducible NO-synthase; ROS generation; and immunostaining for 3-nitrotyrosine.

2. Sun et al. (2008a) demonstrated that the CAPs exposure also increased tissue factor expression in the aorta. 3. Sama et al. (2007) demonstrated that CAPs that altered brain cell distributions in vivo caused changes in immortalized microglial cells in vitro, intracellular ATP, depolarization of mitochondrial membranes, glutathione, nonprotein sulfhydryl, TNFα, IL-6, and gene regulation. Th e responses were dose related, with the primary determinant being the capacity of CAPs to activate NF-κB in human bronchial epithelial cells. 4. Sun et al. (2008b) demonstrated that, after CAPs exposure, infusion of angiotensis II increased mean arterial pressure, potentiated aortic vasoconstriction to phenylephrine, and exaggerated relaxation of a Rhokinase inhibitor, and increased superoxide levels in the aorta. 5. Sun et al. (2009) demonstrated that the CAPs exposure in an obese mouse model had insulin signaling abnormalities that were associated with abnormalities vascular relaxation to insulin and acetylcholine and increased adipose tissue macrophages (F4/80+ cells) in visceral fat expressing higher levels of TNFα/IL-6 and lower IL-10. In coordinate in vitro tests, PM induced cell accumulation in visceral fat and potentiated cell adhesion in the microcirculation, supporting a link between CAPs exposure and type 2 diabetes mellitus and metabolic syndrome. 6. In association with their CAPs inhalation study of the influence of CAPs exposure on nonalcoholic fatty liver disease (NAFLD), Tan et al. (2009) performed in vitro exposures using a reference PM sample (NIST SRM1649a). For the mice exposed by injection, SRM particles were detected only in Kupffer cells from livers of SRM-injected mice and not in sham-injected mice. Cell culture studies were done using macrophage (RAW) and stellate (LX2) cell lines. Direct exposure to PM2.5 activated IL-6 production by Kupffer cells in a TLR4-dependent manner. Th ey concluded that exposure to ambient air PM may be a significant risk factor for NAFLD progression. 7. In conjunction with the CAPs inhalation study focused on effects on the liver, Laing et al. (2009) also exposed a murine monocytic-macrophage cell line to PM in vitro, demonstrating that macrophage ingestion of PM and the selective activation of the UPR components rely on ROS and Ca signals. In the liver tissue of the CAPs-exposed mice, the selective activation of the UPR components is coordinated with the activation of NF-κB and c-Jun amino-terminal kinase (JNK) and reduced expression of paraoxonase 1 (PON-1) and peroxisome proliferatoractivated receptor gamma (PPAR-γ), which favors the development of cardiovascular diseases. These examples identify some of the biological pathways that are activated by particles taken as a result of inhalation exposure and the cells that participate in the responses.

Health effects of ambient air CAPs Future studies can utilize pure materials as well as ambient air PM of mixed composition to obtain more information on the roles of specific PM components and their interactions and combined effects, and thereby identify those components most in need of control in order to reduce the health impacts of airborne PM.


Acknowledgments The extensive literature summarized in this critical review includes a considerable number of papers that were described previously in a review of metal toxicology in Inhalation Toxicology (Chen & Lippmann, 2009), as well as descriptive material on a 2004 CAPs Workshop (Lippmann et al., 2005). We also acknowledge the presubmission review comments and suggestions made by our friends and colleagues: John Bachmann, William F. McDonnell Jr., and Terry Gordon. Declaration of interest: The authors acknowledge the support they received from a Center Grant (ES 00260) from the National Institute of Environmental Health Sciences (NIEHS), a research grant from NIEHS (R01 ES015495), and a research grant from the Health Effects Institute. One of the authors, M.L., is currently serving on a US EPA Clean Air Scientific Advisory Committee Panel on Particulate Matter that is reviewing the science, including research findings on CAPs, under-girding potential revision of the National Ambient Air Quality Standards for Particulate Matter. The authors prepared this review during the normal course of their emplyment as noted on the cover page, and have sole responsibility for the writing and content of the paper.

Glossary 8-oxodG 8-hydropxydeoxyguanosine A-II angiotensin II ABS absorbance (of light by BC on a sampling filter) AHR airway hyperresponsiveness ApoE−/− apolipoprotein A deficient (knockout) mouse BAD brachial artery diameter BAL bronchoalveolar lavage BALF bronchoalveolar lavage fluid BC black carbon, a.k.a. soot, measured as light absorbance by, or reflectance of, a sampling filter BP blood pressure Br bromine CA California CAPs concentrated ambient air particles/concentrated ambient air particulate matter Cd cadmium CDP concentrated diesel particles CFA coal fly ash CHD coronary heart disease CI confidence interval CO carbon monoxide COPD chronic obstructive pulmonary disease

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CPZ capsazepine Cr chromium CRP C-reactive protein Cu copper CYP cytochrome P450 isoenzyme DEG Diesel exhaust gases DEP Diesel exhaust particles EC elemental carbon ECG electrocardiogram or electrocardiographic ED emergency department eNO exhaled nitric oxide eNOS endothelial nitric oxide synthase EPA Environmental Protection Agency EST environmental tobacco smoke ET-A endothelin A FA filtered air Fe iron forced expiratory volume in 1 s FEV1 FMD flow-mediated dilatation FVC forced vital capacity GFAP glial fibrillary acidic protein GPx-1 glutathione peroxidase 1 HDL high-density lipoprotein HEI Health Effects Institute HO-1 heme oxygenase-1 HR heart rate HRV heart rate variability HSPH Harvard School of Public Health IgE immunoglobulin E IgG1 immunoglobulin G1 IL-1 interleuken-1 IL-13 interleuken-13 is a cytokine secreted by many cell types, but especially T-helper type 2 (Th2) cells[1], that is an important mediator of allergic inflammation and disease. IL-5 interleuken-5 IL-6 interleuken-6 is an interleukin that acts as both a proinflammatory and anti-inflammatory cytokine. It is secreted by T cells and macrophages to stimulate immune response to trauma, especially burns or other tissue damage leading to inflammation. IL-8 interleuken-8 is a chemokine (ability to induce directed chemotaxis in nearby responsive cells) produced by macrophages and other cell types such as epithelial cells. iNOS inducible nitric oxide synthase IT intratracheal JNK c-Jun amino-terminal kinase K potassium LDH lactate dehydrogenase LYM lymphocyte MA Massachusetts MCT monocrotaline MEF mid-expiratory flow rate MESA Multi-Ethnic Study of Atherosclerosis MI myocardial infarction MMAD mass median aerodynamic diameter

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mRNA MS NAC NADPH phosphate NAFLD NC NF-κB NHANES III Ni NIST Technology NMD NMMAPS NO2 NY NYC NYU O3 OC OVA P450 1B1 PAH PC Pdur PEF PM PM0.18 PM10 PM10-2.5 PM2.5 PMN PON-1 PP PPAR-γ gamma ppb Pt QT interval R-R interval RBC RMSSD ROFA ROS RR RTP RTp

messenger RNA metabolic syndrome N-Acetylcystine nicotinamide adenine

dinucleotide

nonalcoholic fatty liver disease North Carolina nuclear factor kappa B Third National Health and Nutritional Examination Survey nickel National Institute of Science and nitroglycerin-mediated dilatation National Morbidity and Mortality Air Pollution Study nitric dioxide New York New York City New York University ozone organic carbon ovalbumin cytochrome P4501B1 isoenzyme polynuclear aromatic hydrocarbon total particle counts P duration, time interval between the beginning and the end of the P-wave peak expiratory flow rate particulate matter PM with diameters