Toxicological and epidemiological studies of cardiovascular effects of ambient air fine particulate matter (PM2.5) and its chemical components: coherence and public health implications.

http://informahealthcare.com/txc ISSN: 1040-8444 (print), 1547-6898 (electronic) Crit Rev Toxicol, 2014; 44(4): 299–347 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10408444.2013.861796

REVIEW

Toxicological and epidemiological studies of cardiovascular effects of ambient air fine particulate matter (PM2.5) and its chemical components: Coherence and public health implications Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY, USA

Abstract

Keywords

Recent investigations on PM2.5 constituents’ effects in community residents have substantially enhanced our knowledge on the impacts of specific components, especially the HEI-sponsored National Particle Toxicity Component (NPACT) studies at NYU and UW-LRRI that addressed the impact of long-term PM2.5 exposure on cardiovascular disease (CVD) effects. NYU’s mouse inhalation studies at five sites showed substantial variations in aortic plaque progression by geographic region that was coherent with the regional variation in annual IHD mortality in the ACS-II cohort, with both the human and mouse responses being primarily attributable to the coal combustion source category. The UW regressions of associations of CVD events and mortality in the WHI cohort, and of CIMT and CAC progression in the MESA cohort, indicated that SO¼ 4 had stronger associations with CVD-related human responses than OC, EC, or Si. The LRRI’s mice had CVD-related biomarker responses to SO¼ 4 . NYU also identified components most closely associated with daily hospital admissions (OC, EC, Cu from traffic and Ni and V from residual oil). For daily mortality, they were from coal combustion (SO¼ 4 , Se, and As). While the recent NPACT research on PM2.5 components that affect CVD has clearly filled some major knowledge gaps, and helped to define remaining uncertainties, much more knowledge is needed on the effects in other organ systems if we are to identify and characterize the most effective and efficient means for reducing the still considerable adverse health impacts of ambient air PM. More comprehensive speciation data are needed for better definition of human responses.

Annual mortality, aortic plaque, cardiac function, cardiovascular effects, daily mortality, hospital admissions, ischemic heart disease, particle speciation PM2.5

Table of Contents Abstract ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 299 Introduction ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 300 Background and objectives of this critical review ... ... ... ... ... 300 My PM-focused career ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 300 Historical background for ambient air pollution and its effects on health ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 301 Reviews of the more recent literature linking ambient air PM concentrations and health effects in humans ... ... ... ... ... ... ... ... 304 Historic large population-based studies in humans dealing with cardiopulmonary responses to PM and its components ... 305 Recent population-based studies addressing air pollution sources, chemical components, and disease categories 305 Cardiovascular responses in studies using chemical speciation data for individual constituents ... ... ... ... ... ... ... ... ... ... ...305 Cardiovascular responses in studies using chemical speciation data for associations with source categories ... ... ... ... ...306 Pulmonary and other non-CVD responses ... ... ... ... ... ... ... ...307 Human panel studies ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 307 Cardiovascular responses associated with PM major mass components, and pollutant gases ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 307 Cardiovascular responses ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 308

Address for correspondence: Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY, USA. E-mail: [email protected]

History Received 10 December 2012 Revised 21 October 2013 Accepted 30 October 2013 Published online 4 February 2014

Pulmonary responses ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 308 Exposures of human volunteers in clinical settings ... ... ... ... ... ... ... 309 Chapel Hill, NC ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 309 Los Angeles, CA ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 309 Toronto, Canada ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 310 Edinburgh, Scotland ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 310 Laboratory-based inhalation studies in humans with components of ambient air PM2.5 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 310 Diesel engine exhaust ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 310 Effects of ambient air PM inhalation in animals ... ... ... ... ... ... ... ... 311 Historic studies in laboratory animals dealing with cardiopulmonary responses to PM and its components ... ... ... ... ... ... ... ... ... ... ... 311 Short-term CAPs inhalation exposures in laboratory animals ... ... 312 Boston, MA ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 312 Detroit, MI ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 313 Research Triangle Park (RTP), NC ... ... ... ... ... ... ... ... ... ... ... ... ... 314 New York City, NY ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 314 Tuxedo, NY ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 314 Los Angeles, CA ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 314 The Netherlands ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 314 Long-term CAPs inhalation exposures in laboratory animals ... ... 315 Tuxedo, NY ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 315 New York City ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 316 Columbus, OH ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 317 Los Angeles, CA ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 317 Effects of PM source mixture inhalation exposures in laboratory animals ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 317

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Diesel engine exhaust ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 317 Source-related mixtures contributing to ambient air PM ... ... 318 Sidestream cigarette smoke ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 319 Transition metals ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 319 Lung instillation studies with PM components ... ... ... ... ... ... ... ... ... 320 Ambient air PM... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 320 Utah Valley Dust ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 320 In vitro CAPs exposures to PM components ... ... ... ... ... ... ... ... ... ... 321 Addressing a recognized need for additional studies designed to integrate evidence from epidemiological and toxicological effects of ambient air PM2.5 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 321 The Health Effects Institute (HEI) National Particle Component Toxicity (NPACT) program ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 322 The NYU NPACT study ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 322 NYU NPACT epidemiological findings ... ... ... ... ... ... ... ... ... ... ... ... ... 323 Time-series studies of daily hospital admissions and mortality based on PM2.5 chemical constituents, pollutant gases, and source-related mixtures ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 323 Multi-city daily mortality analyses ... ... ... ... ... ... ... ... ... ... ... ...323 Multi-city daily CVD hospitalizations analyses ... ... ... ... ... ...324 Multi-city daily respiratory hospitalizations analyses ... ... ...324 Variations in hospital admissions and distributed lag risks of exposures to PM2.5 in Seattle and Detroit based on daily constituent concentration analyses [supplemental NPACT Study] ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 325 American Cancer Society (ACS) cohort study of annual mortality associations with long-term exposure ... ... ... ... ... ... ... ... ... ... ... 325 Toxicological findings in NYU NPACT study ... ... ... ... ... ... ... ... ... ... 327 Subchronic mouse inhalation exposures ... ... ... ... ... ... ... ... ... ... 327 Cardiac function responses ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 327 Aortic plaque progression ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 328 In vivo aspiration exposures in mice and in vitro exposures of cells to PM10–2.5. PM2.5–0.2, and PM0.2 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 328 Integration and interpretive observations among the NYU NPACT substudy findings ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 328 Roles of PM2.5 constituents on mortality and hospital admissions 329 Roles of PM2.5 constituents on toxicological responses ... ... ... ... ... 330 Overall interpretive observations on the roles of PM2.5 constituents on health-related responses ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 330 Coherence of toxicological responses to PM2.5 exposures ... ... ... 330 Coherence of short-term CVD responses of humans and mice to PM2.5 constituent exposures ... ... ... ... ... ... ... ... ... ... ... ... ... ... 330 Coherence of CVD hospitalization in people with cardiac function changes in ApoE/ mice ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 331 Coherence of annual mortality associations with PM2.5 and its constituents with aortic plaque progression in ApoE/ Mice ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 331 Short-term exposure to PM2.5 and daily mortality and hospital admissions (NYU NPACT only) ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 331 University of Washington (UW) – Lovelace Respiratory Research Institute (LRRI) NPACT study ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 331 Introduction ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 331 Results ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 332 MESA cohort ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...332 WHI cohort ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...332 ApoE/ mice ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...333 HEI NPACT review panel synthesis... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 333 Coherence of the NYU and UW-LRRI NPACT findings with each other and with prior literature’s findings ... ... ... ... ... ... ... ... ... ... ... ... ... 335 Long-term human exposure to PM2.5 components and chronic health effects ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 335 Long-term exposure to PM2.5 components and chronic effects in ApoE/ mice ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 335 PM2.5 components most influential as causal factors for health effects based on the findings of The NPACT studies ... ... ... ... ... ... ... ... 336 Traffic and sulfate in the NYU NPACT study ... ... ... ... ... ... ... ... 336 Traffic and sulfate in the UW-LRRI NPACT study ... ... ... ... ... ... 337 My post-NPACT holistic perspectives on the role of PM2.5 in CVD effects ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 337 What I now know with reasonable certainty ... ... ... ... ... ... ... ... 338 What I consider to be highly uncertain ... ... ... ... ... ... ... ... ... ... 338

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What I consider to be needs to refine the remaining exposureresponse questions ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 338 Implications of findings of the HEI sponsored NPACT studies 338 Implications to further research ... ... ... ... ... ... ... ... ... ... ... ... ...338 Implications of the NPACT studies to the setting of NAAQS and/or control strategies for ambient air PM... ... ... ... ... ... ... ... ... ... ... ... 339 Short-term NAAQS for fine particulate matter ... ... ... ... ... ... ... 339 Long-term NAAQS for fine particulate matter ... ... ... ... ... ... ... 340 Consideration of a NAAQS for coarse thoracic PM (PM10–2.5) and/or ultrafine PM (PM50.15) ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 341 Need for a more comprehensive air quality monitoring program ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 341 Conclusions ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 341 Acknowledgements ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 342 Declaration of interest ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 342 References ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 342

Introduction Background and objectives of this critical review With the recent completion of the Health Effects Institute (HEI) sponsored National Particle Component Toxicity (NPACT) study at New York University (NYU), I have been able to substantially advance my long-term research objective of gaining a more complete understanding of the roles of inhaled particulate matter (PM), and of its numerous chemical components, in causing adverse health effects. My objectives, in this critical review, are to provide my personal perspectives on (1) the evolution of a career focused primarily on inhaled PM in the context of PM air pollution and its effects on health; (2) a relatively concise review of the peer-reviewed literature on ambient air PM and our knowledge of the roles played by its chemical components prior to the initiation of the NPACT program, and of its nature, extent, and limitations; (3) a more detailed review of the objectives and findings of NYU’s NPACT study, as well as of corresponding parts of the University of Washington – Lovelace Respiratory Research Institute (UW-LRRI) NPACT study; and (4) the coherence (Bates, 1992) and implications of the NPACT studies’ findings to public health issues and their resolution. Additional research will be needed to learn more about the sources of, exposures to, and health effects of ambient air PM and its copollutants, and to guide the application of ambient air and emission standards that can better protect public health. My PM-focused career My perspectives on exposures to, and the health effects of, airborne PM have been shaped by my interactions with many mentors and colleagues prior to my role as Director of NYU’S EPA-supported PM Health Effects Research Center, and my subsequent lead role in the HEI-supported NYU NPACT studies. The NPACT program represents the last major stage of my six-decades-long PM research career. My early mentors on PM included Professors Philip Drinker and Leslie Silverman (Harvard), Herbert Stokinger (US Public Health Service [PHS]), Merrill Eisenbud (US Atomic Energy Commission [AEC], and later at NYU), and Norton Nelson and Roy Albert at NYU. In the mid-1950s, as a junior commissioned officer in the US Public Health Service (PHS), I made measurements of dust counts in the Vermont granite sheds, and I designed animal inhalation chambers for use in the PHS occupational health laboratory in Cincinnati, OH


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(Fraser et al., 1959). In the late 1950s and early 1960s, as an industrial hygiene engineer at the AEC’s Health and Safety Laboratory in New York City, I adapted miniature PMcollecting cyclones in order to use them in personal sampling for respirable dust (Lippmann & Harris, 1962), and I characterized environmental exposure to uranium compounds (Lippmann, 1959), and their urinary excretion (Lippmann et al., 1964). In the mid-1960s, my doctoral thesis at NYU defined regional particle deposition within the human respiratory tract (Lippmann & Albert, 1969), described the temporal aspects of tracheobronchial mucociliary particle clearance from the human respiratory tract, and characterized the effects of cigarette smoke on particle clearance (Albert et al., 1969, 1975). In Leikauf et al. (1981, 1984) and Spektor et al. (1989), we described the effects of sulfuric acid droplets on mucociliary particle clearance. With other of my NYU doctoral students, I developed techniques to make hollow airway casts (Schlesinger & Lippmann, 1972, 1976, 1978), and to measure particle and fiber deposition efficiencies within specific airways and their dichotomous branchings (Briant & Lippmann, 1992; Cohen et al., 1988, 1990; Esch et al., 1988; Gurman et al., 1984a,b; Lippmann & Esch 1988; Schlesinger et al., 1977, 1982, Sussman et al., 1991a,b). Since 1980, I have focused more on the effects of PM and its chemical components in studies of human populations and laboratory animals, beginning with sulfuric acid and sulfate (Lippmann, 1989a,b,c, 1993; Lippmann & Thurston, 1996), and extending into subchronic inhalation studies of concentrated ambient air fine PM (CAPs) (Lippmann & Chen, 2009; Lippmann et al., 2005a,b, 2006; Sun et al., 2005). My scientific perspectives on PM in ambient air were broadened by my service on scientific advisory panels with more senior colleagues, most notably Ted Hatch (University of Pittsburgh), Robert Waller (St. Bart’s Hospital, London), David Bates (University of British Columbia), and Sheldon Friedlander (University of California [UCLA], and first Chairman of EPA’s Clean Air Science Advisory Committee [CASAC]). I have also benefited from the perspectives that I gained from my service as Chair of: EPA’s Science Advisory Board (SAB), CASAC, and the SAB Committee for Human Exposure and Indoor Air. I also Chaired the National Institute for Occupational Safety and Health [NIOSH] Board of Scientific Counselors, and the External Scientific Advisory Committee (ESAC) for the Harvard Six-Cities Study. More recently, I chaired the ESACs for the Southern California Children’s Study at USC, the National Environmental Respiratory Center (NERC) Complex Mixtures Study, and the Environmental Protection Agency [EPA]-Sponsored Harvard PM Center. I consider myself to be fortunate to have had the opportunity to address, through the HEI NPACT program, one of the biggest gaps in our collective knowledge about the determinants underlying the significant associations of ambient fine PM (PM2.5) on excess human mortality, morbidity, and quality of life, especially in relation to its highly variable chemical composition, and most notably in terms of an integrated program of toxicology and epidemiology capable of demonstrating their coherence and enhancing the biological plausibility of causality for PM having major public health impacts at concentrations not exceeding current National Ambient Air Quality Standards (NAAQS).

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Historical background for ambient air pollution and its effects on health Smoke generated by coal combustion has been considered a nuisance, and sometimes an intolerable one, for many centuries (Evelyn, 1661). On the other hand, some artists found the effects of coal smoke on light-scatter and reduction of visibility aesthetically interesting. Claude Monet produced over 100 canvases of scenes in London in the 1870s and at the turn of the 19th Century to illustrate the highly variable appearance of the dense smoke and fog (Shanes, 1994). There was also an early widespread belief that the acidic fog was deleterious to health, especially in relation to respiratory disease (Russell, 1924), but quantitative data on exposure variables and health impacts were lacking until Firket (1936) provided an early quantitative estimate of the mortality impact of an air pollution episode in the Meuse Valley in Belgium. Firket (1936) attributed 60 excess deaths out of a population of 6000 to the dense smoke within an atmospheric inversion in 1930 and estimated that a comparable inversion in London could cause 3000 excess deaths. When a particularly dense smoke enveloped London for three days in December 1952 (so severe that it that it halted nearly all surface transportation), the hospitals and morgues were overloaded with people suffering and/or dying of acute bronchitis. A detailed report (Ministry of Health, 1954) estimated that 4000 excess deaths within the 4 weeks following the smoke episode were associated with the very large increase in the concentrations of black smoke (BS) and sulfur dioxide (SO2). While the greatest increase in relative risk (RR) was for bronchitis, the largest number of excess deaths was due to cardiac causes. A later reexamination of the data records for the December 1952 episode that extended the time period out to 4 months concluded that there were 12 000 excess human deaths (Bell et al., 2004). An appendix to the Ministry of Health (1954) report noted that there were 788 excess bronchitis deaths in London in the 4 weeks following the smog episode of 9–11 December 1873. Interestingly, during that earlier episode, prize cattle at the Smithfield livestock show at Earl’s Court in London were severely affected, as reported in The Veterinarian, vol. XLVII January 1874. It stated: ‘‘On Tuesday, the first day of the fog, several of the animals were marked as affected with difficult breathing. No abatement of the fog took place during the day; on the contrary, towards evening it became rather worse, and the majority of the cattle in the Hall showed evidence of suffering from its influence. Sheep and pigs did not, however, experience any ill effect from the state of the atmosphere. During Wednesday night ninety-one cattle were removed from the Hall for slaughter. The postmortem appearance was indicative of bronchitis; the mucous membrane of the smaller bronchial tubes was inflamed, and there was also present the lobular congestion and emphysema which belong to that disease.’’ The Ministry of Health (1954) report noted that, during the December 1952 smog episode, prize cattle at the same venue,


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but not sheep or pigs, were in distress and had to be sacrificed. The differing responses of the cattle and other livestock in both 1873 and 1952 may have been due to much cleaner stalls for the prize cattle, with those stalls having less ammonia vapor to reduce the acidity of the ambient air PM. Concern about adverse health effects of the London smog led Lawther and Waller at St. Bartholomew’s Hospital in London to establish an ongoing laboratory program to monitor pollution exposures based on the method that they had used during the 1952 episode to make daily measurements of BS and airborne acidity. It involved the collection of the smoke particles on a filter paper disc and a measurement of the reduction of the reflectance of white light from the filter. It was calibrated, periodically, against the mass increase of particles on the filter. They also measured the pH change in a scrubbing solution of a parallel sampling stream, and acidity was expressed in terms of SO2 concentration. The measurement of BS and SO2 in London continued for many years, and served as a monitoring system to gauge the effectiveness of the control measures taken to reduce smoke exposures and their health effects. As an example of the utility of daily measurements of BS and SO2, Lawther et al. (1970) conducted a panel study to determine that there was an exposure-related exacerbation of bronchitis. This demonstration of a biological gradient of effect added to the case for causality. The primary control measure in the early years was a ban on the burning of bituminous coal for space heating, and its replacement, initially by anthracite, and later by coke, in order to reduce exposures to the volatile components within the coal and the secondary fine PM with aerodynamic diameters below 2.5 mm (PM2.5), and their components. The efficacy of this control approach was demonstrated when a comparable inversion occurred in December 1962. The excess mortality within 4 weeks was 700, compared to the 4000 in the 1952 episode. The control efforts, which extended to all parts of the United Kingdom (UK), also reduced the annual mortality rates in the UK (Chinn et al., 1981). The most notable early air pollution episode involving a quantitative analysis of human health impacts in the US occurred in 1947 in Donora, PA, a steel-mill town in the steep walls of the Monongahela River Valley. The concentrations of the smoke components and SO2 were not measured, but it was determined that there were 60 excess deaths, and that 43% of the population were affected, with 10% requiring medical assistance (Schrenk et al., 1949). Subsequent analyses of PM collected on air cleaning intake filters in operation during the episode indicated that the PM included unusually high proportions of sulfates and transition metals. In a follow-up study 10 years later, Ciocco & Thompson (1961) reported higher rates of mortality, heart disease, and chronic bronchitis in those having had acute responses in comparison to those that did not, showing that those demonstrating acute responses to the exposures during the episode had longlasting adverse effects. For the epidemiological studies noted above, the investigators’ analyses were limited to crude indices of PM (light reflectance or mass concentration) and SO2 exposures. Most of the more recent studies have relied on gravimetric analyses of mass concentrations of PM that can enter the thorax, i.e.

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particles with aerodynamic diameters less than 10 mm (PM10), or on fine PM, i.e. with aerodynamic diameters less than 2.5 mm (PM2.5), which has been most closely associated with cardiovascular disease (CVD) endpoints, and excess annual mortality. Table 1 summarizes the various CVD effects that have been shown to be significantly associated with PM10, PM2.5, and/or sulfate ion (SO¼ 4 ), a significant mass component of PM2.5. Since we now know ambient air PM is a complex mixture, and that it varies greatly in chemical composition and particle size due to the nature and origins of its sources, studies lacking data on chemical speciation were inherently incapable of identifying PM components that may have had disproportionate health impacts. Ambient air PM includes primary solid particles that are dispersed into the air by mechanical processes that break up solid materials by crushing or grinding. It also includes materials on ground or roadway surfaces that are resuspended by wind action. Particles larger than 2.5 mm in aerodynamic diameter can dominate the mass concentration. Much smaller primary particles, such as soot particles, resulting from incomplete combustion in diesel engines are emitted from exhaust pipes. Power plants burning fossil fuels emit mineral ash particles of non-volatile elements and their oxides as coarse particles. They also emit particles and their aggregates formed by condensation from metals that are volatile at flame temperatures as ultrafine particles smaller than 0.1 mm, and as fine-sized aggregates in the size range extending up to 1 mm. Ultrafine PM is of special concern insofar as such particles (1) can penetrate cellular membranes in the respiratory tract by non-phagocytic mechanisms in lungs (Geiser et al., 2005); and (2) have very large surface areas per unit of mass. Finally, primary pollutants that are emitted as reactive gases undergo chemical reactions within the ambient air, forming secondary particles that remain suspended as fine particles. These secondary particles can be composed of both inorganic and organic chemicals. In consideration of their varying particle size, which largely determines where they deposit within the respiratory tract after inhalation, and their chemical composition, which largely determines whether they are toxic at the deposition sites or at more distant sites in the body after translocation or dissolution into body fluids, the biological effects can vary from negligible to severe. While there is clearly a need for further study the roles of the chemical components of ambient air PM inhalation on adverse health effects, studies that continue to rely on mass concentrations can still be useful in guiding public policy with regard to exposure evaluation, guidelines and standards for PM exposure, and controls to limits exposures. For example, Lepeule et al. (2012) conducted an extended (11 year) follow-up of annual mortality in the Harvard Six-Cities cohort during a period of reduced PM2.5 exposure. They were able to show that the concentration-response function was essentially linear down to a PM2.5 concentration of 8 mg/m3. Crouse et al. (2012) used satellite ground scanning data to estimate ambient air PM2.5 concentrations for a 2.1 million cohort of Canadians for use in a study of annual mortality associated with relative low PM2.5 concentrations (8.7 3.9 mg/m3). Their estimated hazard ratios for 10 mg/m3 increases in PM2.5 were 1.15 for all non-accidental deaths,


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Table 1. CVD effects associated with human exposures to PM2.5, PM10, and/or SO¼ 4.


PM2.5 and PM10 associated CVD effects

Population

Reference

Long-term exposures Annual mortality Annual mortality Annual CVD mortality Annual mortality Annual IHD mortality Fatal and non-fatal CVD events

US cities 6 Cities cohort 6 Cities cohort ACS cohort ACS cohort WHI cohort

Lave & Seskin (1970) Dockery et al. (1993) Lepeule et al. (2012) Pope et al. (1995, 2002) Pope et al. (2004) Miller et al. (2007)

Short-term exposures Daily CVD mortality Daily CVD mortality Daily IHD mortality Daily CVD mortality Daily cerebrovascular mortality Cardiorespiratory morbidity Cardioverter defibrillator discharges Ischemia, ST,- and T-segment depressions S-T segment depression S-T segment depression QT prolongation Intraventricular conduction delay Adverse ventricular repolarization Exercise-induced ischemia HRV HRV HRV Pulse rate, HRV, peripheral basophils HR, HRV HRV HRV (patients with metabolic syndrome) CIMT FMD Flow-mediated dilatation Small artery elasticity Vascular reactivity Fibrinogen, platelet, and WBC counts Plasma fibrinogen Platelets, thrombin, fibrinogen and CRP Plasma tissue plasminogen activator Blood flow WBC SBP and PP SBP and DBP Exhaled breath 8-isoprostane hs-CRP Prothrombin time

100 NMMAPS Counties 9 California counties Canadian cohort Barcelona residents Barcelona residents City of Atlanta Eastern Massachusetts WHI cohort Helsinki cardiac patients Boston residents MESA cohort MESA cohort APACR study Amsterdam, Erfurt, and Helsinki Utah residents Boston residents Chapel Hill elderly Los Angeles elderly Los Angeles asthmatics Taipei, Taiwan CHD patients MESA cohort MESA cohort Ottawa, Canada Type 2 diabetics Type 2 diabetics 270 Boston residents NHANES III cohort London office workers Rotterdam Edinburgh cardiac patients Edinburgh cardiac patients NHANES III cohort MESA cohort Heinz-Nixdorf cohort Edinburgh cardiac patients German cohort Lombardia, Italy

Dominici et al. (2007a) Ostro et al. (2007) Crouse et al. (2012) Perez et al. (2009) Perez et al. (2009) Sarnat et al. (2008) Peters et al. (2000) Zhang et al. (2009) Pekkanen et al. (2002) Gold et al. (2005) Van Hee et al. (2011) Van Hee et al. (2011) Liao et al. (2010) Lanki et al. (2006) Pope et al. (1999) Gold et al. (2000) Devlin et al. (2003) Gong et al. (2004a) Gong et al. (2004b) Chuang et al. (2007) Park et al. (2010) Diez-Roux et al. (2008) Dales et al. (2007) Schneider et al. (2008) Schneider et al. (2008) O’Neill et al. (2005) Schwartz (2001) Pekkanen et al. (2000) Rudez et al. (2009) Mills et al. (2008) Mills et al. (2008) Chen & Schwartz (2008) Auchincloss et al. (2008) Fuks et al. (2011) Mills et al. (2008) Hoffmann et al. (2009) Baccarelli et al. (2006)

CIMT, carotid intimal-medial thickness; WBC, white blood cell count; SPB, systolic blood pressure; PP, pulse pressure; HR, heart rate; HRV, heart rate variability; hs-CRP, high sensitivity C-reactive protein; FMD, flow-mediated brachial artery dilatation.

and 1.31 for ischemic heart disease (IHD) deaths. The biological plausibility of adverse effects occurring at such low levels of chronic PM2.5 exposures has been strengthened by the findings of the NPACT subchronic inhalation studies in a mouse model of atherosclerosis (Chen & Lippmann, 2013), as discussed later in this critical review. Recognizing that some PM components or source-related mixtures were likely to be more toxic than others, epidemiological investigators conducted studies that relied on the ambient air concentrations of other major mass fractions of the PM, including elemental carbon (EC), organic carbon (OC), and markers of traffic. Table 2 summarizes the various CVD effects that have been shown to be significantly associated with EC, OC, nitrate ion (NO 3 ), traffic markers, and/or sulfate ion (SO¼ 4 ). Table 3 summarizes the various CVD effects that have been shown to be significantly

associated with PM constituents present in ambient air in trace concentrations or as source-related mixtures identified by their trace constituents. One of the first studies to go beyond reliance on crude indices of PM concentration in order to examine the associations of PM components with health effects was that of Ozkaynak & Thurston (1987). They used the concentrations of PM2.5 components measured in EPA’s Inhaled Particle Network (IPN), including components that can serve as trace element signatures of source categories. They conducted a cross-sectional study in which they regressed the source strengths against annual mortality rates in US cities. They reported that mortality was significantly, and most strongly, associated with the coal combustion source factor, and was also associated with the traffic and metals industry factors. This pioneering study had a limited impact because it

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Table 2. Associations of commonly measured PM2.5 components (SO¼ 4, NO 3 , EC, and OC) and human CVD health indices in human populations and panels. Constituents or sources associated

Effect


Short-term exposures CVD hosp. admissions Myocardial infarction CVD morbidity Plasma fibrinogen Platelet activation Inflammatory markers DBP SBP and DBP Q-T int., T-wave ampl. S-T segment dep. S-T segment dep. vWF HRV HRV BAD Vascular reactivity IL-6, SOD IL-6 eNO

OC, EC, SO¼ 4 BC Traffic, biomass combustion SB Traffic factor EC, OCpri OC OCpri, OCsec, BC Traffic Traffic, BS OCpri, BC Traffic, combustion effluents SO¼ 4 BC OC, EC SO¼ 4 , BC EC, BC, primary OC EC, PAHs, organic acids OCpri, OCsec, organic acids

Reference Kim et al. (2012) Peters et al. (2001) Sarnat et al. (2008) Pekkanen et al. (2000) Delfino et al. (2008) Delfino et al. (2009) Urch et al. (2005) Delfino et al. (2010a) Yue et al. (2007) Lanki et al. (2006) Delfino et al. (2011) Yue et al. (2007) Chuang et al. (2007) Zanobetti et al. (2010) Urch et al. (2005) O’Neill et al. (2005) Delfino et al. (2008) Delfino et al. (2010b) Delfino et al. (2010b)

BAD, brachial artery diameter; CVD, cardiovascular disease; DBP, diastolic blood pressure; HRV, heart rate variability; OCpri, primary OC; OCsec, secondary OC; SBP, systolic blood pressure; SOD, superoxide dismutase; vWF, von Willebrand factor.

could not account for individual-level variations in personal risk factors in the various communities. It is, therefore, interesting that the recent NPACT study of Thurston et al. (2013), which used more recent speciation data from EPA’s Chemical Speciation Network (CSN), and annual mortality data from the American Cancer Society (ACS) II cohort, and which accounted for personal risk factors at the individual level, came to similar conclusions about the impacts of the major source categories, as will be discussed later in this critical review.

Reviews of the more recent literature linking ambient air PM concentrations and health effects in humans The more recent literature that is reviewed here is limited to those papers that provide background and/or data for understanding the role of PM chemical composition on healthrelated biological responses. I have not included studies that failed to find associations between PM2.5 components or source-related mixtures because of the many reasons that could have accounted for such results, including (1) inadequate statistical power due to limited population size; (2) exposure measurement limitations due analytical sensitivities; (3) exposure measurement errors due to spatial and temporal variations of concentrations within communities not reflected at monitoring sites; and (4) studies being conducted at times and/or places where the concentrations of the most toxic components were inadequate to elicit measurable responses.

Table 3. Associations of PM2.5 components (including trace elements) and human CVD health indices in human populations, panels, and CAPs exposure studies. Effect

Constituents or sources associated

Reference

Long-term exposures Annual mortality Annual mortality Annual mortality Annual mortality Annual CVD mortality Annual CVD mortality Annual IHD mortality Annual IHD mortality

Coal comb., traffic, metals industry Coal combustion, soil factors Traffic factor, Ni, V Traffic factor, SO¼ 4 , Mn, Cl, As, EC, Ni Ni, V EC, OC, SO¼ 4 , Cu, Fe, Mn, V, Zn K, OC, SO¼ 4 , NO3 , Fe, Si, Zn, EC S, Ni, V

Ozkaynak & Thurston (1979) Laden et al. (2000) Lipfert et al. (2006) Lipfert et al. (2009) Hedley et al. (2002, 2004) Ostro et al. (2007) Ostro et al. (2010) Cahill et al. (2011a)

Short-term exposures Daily mortality Daily mortality Daily mortality CVD mortality CVD mortality CVD hosp. admissions CVD hosp. admissions CVD hosp. admissions CVD hosp. admissions CVD ED visits Stroke hosp. admiss. HR HRV Plasma fibrinogen PMNs and platelets WBCs and IL-6 BUN, R-R interval Uric acid and vWF Oxidative stress

SO¼ 4 , Fe, Ni, Zn Ni, V Al, Ni, SO¼ 4 , As Se, SO¼ 4 , EC, OC, Si Traffic, oil comb., SO¼ 4 , NO3 , soil, road dust Traffic, oil and coal comb., metals ind. Ni, V, EC Se, Zn, OC, EC, SO¼ 4 , Ni, V Transition metals Traffic, biomass burning BC, Ni, S Ni Oil combustion, long-range transport Cu/Zn/V factor Fe, Se, SO¼ 4 factor Cr Cu Ca V, Cr

Burnett et al. (2000) Lippmann et al. (2006) Franklin et al. (2008) Ito et al. (2011) Ostro et al. (2011) Janssen et al. (2002) Bell et al. (2009) Ito et al. (2011) Suh et al. (2011) Sarnat et al. (2008) Mostofsky et al. (2012) Hsu et al. (2011) de Hartag et al. (2009) Ghio et al. (2000) Ghio et al. (2000) Riediker et al. (2004b); Riediker, (2007) Riediker et al. (2004b); Riediker, (2007) Riediker et al. (2004b); Riediker, (2007) Sorensen et al. (2005)

BUN, blood urea nitrogen; CVD, cardiovascular disease; ED, emergency department; HR, heart rate HRV, heart rate variability; PMN, polymorphonuclear neutrophils; vWF, von Willebrand factor; WBC, white blood cells.

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Historic large population-based studies in humans dealing with cardiopulmonary responses to PM and its components A prior review of the peer-reviewed literature on large population-based studies in humans, dealing with responses to inhaled PM components, was presented in this Journal by Lippmann & Chen (2009), and the following paragraph is a brief listing and summary of the papers that were cited therein. This is followed by a section that summarizes the findings of more recent studies that have been able to generate more information on the influence of additional indices of PM exposures and more specific indices of response. The literature has been essentially limited to cardiovascular and pulmonary responses since very few studies have characterized responses to PM exposures in other organ systems. A broad variety of cardiovascular and pulmonary health effects have been significantly associated with long-term average mass-based concentrations of ambient air PM in terms of one or more of its particle size fractions and/or of its major mass components, such as black smoke (BS) and SO¼ 4 in a variety of large population studies. Cohort studies include the thorough studies of morbidity and longevity in relation to ambient air concentrations of PM10, PM2.5, and at least some of their PM constituents and associated gaseous criteria pollutants, as in the Harvard six-city cohort (Dockery et al., 1993; Laden et al., 2000), the American Cancer Society (ACS) cohort (Pope et al., 1995, 2002, 2004), US Military Veterans (Lipfert et al., 2006), the National Health and Nutrition Examination Survey (NHANES) III (Chen & Schwartz, 2008), MESA (Diez-Roux et al., 2008), WHI (Zhang et al., 2009), the Southern California Children’s Health Study (Gauderman et al., 2004), and Medicare hospital admissions study (Bell et al., 2009). Applications of recently available CSN speciation data were used in the studies relating them to available daily mortality data from publicly available mortality data and the NMMAPS study (Dominici et al., 2007a; Lippmann et al., 2006). The results of these various studies continue to implicate PM2.5 as a useful index of excess mortality risk, and some of them, having access to speciation data, implicate transition metals (Bell et al., 2009; Burnett et al., 2000; Franklin et al., 2008; Hedley et al., 2002, 2004; Lipfert et al., 2006; Lippmann et al., 2006) and traffic markers (Ebelt et al., 2005; Gold et al., 2005; Ostro et al., 2007, 2008; Pekkanen et al., 2000; Schwartz et al., 2005), or both metals and traffic markers (Janssen et al., 2002) within the PM2.5 as being especially likely to be causal factors for the associations. There were also studies of the effects of air quality interventions and their health consequences, e.g. Hong Kong S-in-fuel intervention (Hedley et al., 2002, 2004), and of the significantly lower community rates of mortality and hospital admissions during a 14-month strike at a steel mill in Utah than in the preceding and following years (Pope, 1989, 1991; Pope et al., 1992). The metals contents on air sampling filters were lower during the strike than in the preceding and following years, corresponding to in vitro and in vivo toxicity of the metals extracts from the filters (Dye et al., 1997, 1999, 2001; Frampton et al., 1999; Ghio & Devlin, 2001).


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The studies cited above have demonstrated that: (1) the largest public health impacts are more closely associated with PM2.5 than with other particle size fractions; (2) cardiovascular responses account for much more of the public health burden than respiratory responses; (3) much of the quite considerable variability in cardiovascular response from site-to-site, season-to-season, and day-to-day is related to differences in the chemical composition of the PM mixture. Recent population-based studies addressing air pollution sources, chemical components, and disease categories Most of the more recent studies, described below in the first section that follows, have focused on cardiovascular responses, and have often gone well beyond the earlier studies in terms of the more complex aspects of the relationships between PM exposures and responses, with greater attention being given to (1) chemical composition within particle size ranges, and the variations associated with different source strengths; (2) response characterization in terms of organ system affected, disease categories, acute and chronic responses, time-lags between exposures and responses, and host factors affecting responsiveness; and (3) the possible roles of gaseous co-pollutants on responses to PM exposures. The second section below summarizes those studies reporting both cardiovascular and pulmonary effects, which is followed by a brief section summarizing studies that described associations of ambient air components on pulmonary and other non-cardiovascular effects. Cardiovascular responses in studies using chemical speciation data for individual constituents Bell (2012) showed that concentrations of PM2.5 components vary across counties and regions of the US as well as over seasons, and that daily PM2.5 total mass concentrations and hospitalizations for cardiovascular and respiratory disease also vary over season and region. There were (1) statistically significant increases in CVD admissions associated with same-day PM2.5 total mass in spring and fall, and that they were largest in winter; (2) regional differences that were greatest across the group of 108 northeastern US counties; (3) increases in respiratory hospitalizations that were most pronounced on the second day. Only EC, which made up the largest fraction of PM2.5 mass, explained the variation. For the remaining components studied, Ni and V were associated with largest effect estimates for both cardiovascular and respiratory hospitalizations. She also noted that PM10 mass associations with total non-accidental mortality were also larger in regions and seasons with higher fractions of V, and particularly of Ni. It is notable that EC, a mass constituent with concentrations near or above 1 mg/m3, was implicated as a cause of respiratory hospitalizations, while nickel (Ni) and vanadium (V), which are trace constituents typically below 10 ng/m3, had the largest effect estimates for both respiratory and cardiovascular hospital admissions. Cahill et al. (2011b) described a substantial drop in annual IHD mortality in Bakersfield, CA, at the southern end of the 400 mile-long Central Valley, between 1989 and 1991.


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That drop in IHD mortality was associated with the replacement, by natural gas, of crude oil combustion for generating steam to enhance heavy petroleum recovery at the local oil refinery in 1990. Measured concentrations of fine particle sulfur (S), Ni, V, zinc (Zn), and lead (Pb) at Bakersfield in 1974–1976, in ng/m3, averaged 1685 for S, 38 for Ni, 19 for V, 61 for Zn, and 1714 for Pb, and these concentrations were much lower in the more northern parts of the Valley. By 2009, the corresponding concentrations in 2009 in Bakersfield were 505, 2.3, 0.2, 32 and 9.4 ng/m3. Although there is relatively little variation, within the Central Valley, of climate, elevation, or population demography, the IHD mortality in 1989– 1991 was 60% higher in the area around Bakersfield than in areas further north within the Valley, while in 2003–2007, the IHD mortality around Bakersfield had dropped by 30%. The mortality reductions, which were associated with the substantial reductions in the concentrations of one or more of the trace metal components, suggest causality, given the lack of any other known exposures that varied so greatly over that time span. This association is consistent with the other studies listed in Table 3 that found excess mortality and morbidity being associated with trace concentrations of the transition metals. Ito et al. (2011) studied the associations of PM2.5 and its constituents (EC, OC, Ni, V, Zn, selenium (Se), bromine (Br), þ SO¼ 4 , NO3 , and sodium ion (Na ), and also with NO2, SO2, and CO with daily excess cardiovascular hospitalizations and mortality in New York City (NYC) for those over 40 years of age for 2000 through 2006. There were statistically significant excess risks for CVD hospitalizations with 0-d lag in the colder 6 months for PM2.5, EC, OC, Ni, Zn, silicon (Si), Se, Br, NO 3 , NO2, SO2, and CO, but none for longer lags. In the warmer 6 months, the only constituents associated with statistically significant risks were EC, OC, NO2, and CO. For daily mortality, the only PM constituents with statistically significant excess risks were PM2.5 (0 and 1 d), EC (1 d), OC (0 and 1 d), SO¼ 4 (0 and 1 d), Si (0 and 1 d), Br (0 and 1 d), and NO 3 (1 d) for warmer days, with none for the colder days. Mostofsky et al. (2012) used data on 18 constituents and data from 1060 patients admitted to a Boston medical center with ischemic stroke in 2003–2008 and illustrated several options for modeling the association between constituents and stroke incidence, while accounting for the impact of PM2.5. Although the different methods yielded results with different interpretations, the relative rankings of the association between constituents and ischemic stroke were fairly consistent across models. The strongest associations were for EC, and the second strongest were for Ni, whose average concentration was only 2.3 ng/m3, representing only 0.02% of PM2.5. The association with Ni at such a low concentration may seem unlikely from a toxicological perspective, but the fact that it also has been associated with CVD effects in the other cited studies suggests that it, or the other pollutants emitted with it from fossil fuel combustion sources, can be causal factors. de Hartag et al. (2009) measured SDNN and high frequency (HF) power of heart rate variability (HRV) in female subjects with coronary heart disease (CHD) biweekly in Amsterdam (11 subjects), Erfurt (4), and Helsinki (21) over 6 months in relation to their exposures to PM2.5, S, V, Zn, Ca, Cl, Fe, Cu, BC, and their source categories (local traffic,

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long-range transport, oil combustion, industry, crystal, and salt), and their medication usage. Beta-blockers were used by 13 of the 39 subjects. There were no significant associations of SDNN or HF for those subjects using beta-blockers. For those not using them, there were significant depressions of SDNN with BC and V (lag 1-d), S (lags 2- and 3-d), local traffic and long-range transport (3-d lags), and oil combustion (lag 1). For HF, there were with 2- and 3-d lags for S, and a 3d lag for the oil combustion source. Ostro et al. (2010) studied the associations between annual mortality and long-term exposure to PM2.5 [concentration inter-quartile range (IQR) ¼ 6.1 mg/m3] and some of its constituents EC [IQR ¼ 0.16 mg/m3], OC [IQR ¼ 1.0 mg/m3], SO¼ [IQR ¼ 1.3 mg/m3], NO [IQR ¼ 3.6 mg/m3], Fe 4 3 3 [IQR ¼ 0.06 mg/m ], potassium (K) [IQR ¼ 0.05 mg/m3], Si [IQR ¼ 0.05 mg/m3], and Zn [IQR ¼ 0.01 mg/m3]) among a prospective cohort of 7888 active and retired female teachers in California. There were significant IHD mortality risks in single pollutant models for all the measured pollutants, with the highest risks being for K, OC, NO 3, and SO¼ . For pulmonary mortality, there were significant 4 risks only for OC, SO¼ 4 , and Si. For all but EC, the risks were higher for average exposure over the 3 years preceding death than those for the last year only. Cardiovascular responses in studies using chemical speciation data for associations with source categories Ostro et al. (2011) studied the effects of PM2.5 sources on daily total and CVD mortality in a case-crossover study of Barcelona, Spain for 2003 through 2007, using speciation data collected every sixth day to do factor analyses to determine the contributions of source categories to PM2.5 mass and to the risks. The statistically significant percentage increases for cardiovascular mortality, for 2-d lags were traffic (10.3%), PM2.5 secondary sulfate (7.2%), road dust (6.7%), minerals (6.6%), secondary nitrate (5.5%), and fuel oil combustion (4.6%). Auchincloss et al. (2008) studied associations among ambient air concentrations of 24-h average PM2.5, SO2, nitrogen dioxide (NO2), and carbon monoxide (CO), averaged over the previous 1, 2, 7, 30, and 60 d, and blood pressure (BP) 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 increasing averaging time up to 30 d; (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 (to the authors 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 a roadway; 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 5300 m from a major road, or surrounded by a high road density. Lanki et al. (2006) found an influence of ambient air PM2.5 component exposures in on exercise-induced ischemia in 45


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elderly non-smokers with stable CHD in Amsterdam, Erfurt, and Helsinki. The traffic and long-range transport sources were associated with ST-segment depression during submaximal exercise testing. In a multi-pollutant model, only the traffic source was significantly associated with the effect. Average daily hospital admissions, for Medicare patients in Atlanta, GA, were examined by Suh et al. (2011) in relation to the concentrations of groups of PM2.5 constituents. There were significant increases in CVD admissions associated with (1) the transition metal oxides group (for those with 490% of the concentrations above the lower limit of detection, i.e. copper (Cu), manganese (Mn), Zn, titanium (Ti), and iron (Fe); and (2) the alkane group. In terms of specific disease categories, the transition metals were significantly associated with admissions for IHD, congestive heart failure (CHF), and atrial fibrillation (afib). The alkane group was associated with respiratory-related admissions. In contrast, the aromatic group and the microcrystalline oxide group (arsenic [As], Br, Se, Pb, and Si) were associated with decreased CVD- and respiratoryrelated hospital admissions. Sarnat et al. (2008) studied the influence of PM2.5 source factors (gasoline powered engines, diesel powered engines, wood smoke, resuspended soil, secondary sulfate, secondary nitrate, cement kiln, railroad, 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 for emergency department (ED) visits for CVD. For the summer months, SO¼ 4 was significantly associated with respiratory ED visits. The results were similar when different sourceapportionment methods were used. Son et al. (2012) studied the associations of 14 months of 1-h average concentrations of PM2.5, EC, OC, and of some of its ionic components (Mg, Na, K, Ca, Cl, ammonium ion [NH4þ], SO¼ 4 , and NO3 ) with total, cardiovascular, and respiratory mortality in Seoul, S. Korea. Of 92 deaths per d, 22.4 were classified as cardiovascular and 5.4 as respiratory. For cardiovascular mortality, there were nearly significant associations (p50.1) for an interquartile change in NH4þ, SO¼ 4 , and NO3 , while for respiratory mortality, the only components with p50.1 were Cl and magnesium (Mg). Kim et al. (2012) examined the temporal lag structure of the associations of CVD and respiratory hospitalizations in Denver, CO for 2003–2007 for PM2.5 and for its major mass components, i.e. EC, OC, SO¼ 4 , and NO3 . For IHD, the only significant associations were for EC and OC concentrations at 0-d lag, and for EC at both 0- and 1-d lags. For CHF, the only significant associations were for OC concentrations at lag days 2 and 3. For cerebrovascular disease, there were significant negative associations with PM2.5 for lag days 1 through 4. For asthma, there were significant associations with PM2.5 for lag days 4 through 12; with EC for lag days 2 through 11; with OC and SO¼ 4 for lag days 4 through 11; and with NO3 for lag days 5 through 12. For pneumonia, there were significant negative associations with OC for lag days 5 through 11. In summary, the PM2.5 constituents most closely associated with adverse health effects varied considerably from study-tostudy, and significant associations were also seen in those studies that included gaseous criteria pollutants as well as PM2.5 constituents. However, before concluding that PM2.5


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composition is not important, one must consider the limitations of each study in terms of their variations of (1) study designs and statistical powers; (2) atmospheric compositions from cityto-city, within each city, and over time; (3) the detection limits for many of the trace constituents that were measured; and (4) the choices of constituents to include in their analyses. It does seem clear that some major mass PM2.5 components (SO¼ 4 , EC, and OC), as well as some trace constituents (Ni, V, Cu, Zn) need to be considered as likely causal factors or at least as markers for influential source categories. Pulmonary and other non-CVD responses Patel et al. (2009) studied the associations of 3-month averages of PM2.5 and the Ni, V, Zn, and EC within the PM2.5 on longitudinal reports of symptoms in a birth cohort in Manhattan and the Bronx (in NYC) who lived near EPA speciation sites. Symptom reports for the prior 3 months were collected every 3 months from 3 to 24 months of age. About 90% of the children were on Medicaid, and 30% were reported to have or might have asthma based on a doctor’s questionnaire entry at 24 months. Symptoms of wheeze and cough were significantly associated with Ni, V, and Zn, whereas traffic-related EC was associated with only cough, and PM2.5 was not associated with either symptom. In contrast, when Patel et al. (2010) studied symptoms among 249 adolescent students in a panel study in NYC and its suburbs in relation to peaks in PM2.5, NO2, and BC, there were significant associations with BC and NO2, but not with PM2.5, and they were largest among urban dwellers and asthmatic subjects. Ebisu & Bell (2012) studied the associations of PM2.5 constituents with low birth weight in the northeastern and mid-Atlantic US regions for 2000 through 2007. There were statistically significant excess risks for IQR concentrations ranges of Ni (5.7%), Ti (5.0%), aluminum (Al) (4.9%), and EC (4.7%). Human panel studies A thorough review of the peer-reviewed literature on human panel studies dealing with responses to PM2.5 constituents was presented by Lippmann & Chen (2009), and the following is a brief listing and summary of the papers that were cited therein.

Cardiovascular responses associated with PM major mass components, and pollutant gases A broad variety of short-term CVD effects have been significantly associated with peaks in ambient air concentrations of PM2.5, its major mass components, and/or one or more gaseous pollutants in human panel studies. These range from implanted defibrillator discharges associated with 2- or 3-d lagged PM2.5, but not with BC (Peters et al., 2000). Myocardial infarction (MI) was associated with PM10, PM2.5, and BC in the preceding 2 h (Peters et al., 2001). PM10, but not gaseous pollutants, was associated with increased white blood cells (WBCs), platelets, and fibrinogen in the third National Health and Nutrition Examination Survey (NHANES III) (Schwartz, 2001), and with C-reactive protein (CRP) and fibrinogen (Hoffmann et al., 2009). PM2.5 was


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associated with platelet aggregation and thrombin generation (Rudez et al., 2009). PM2.5 and particle number count (PC) were associated with ST-segment depression (Pekkanen et al., 2002). Vascular reactivity was associated with PM2.5, PC, BC, and SO¼ 4 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, ¼ OC, and SO¼ 4 , but only SO4 was significant in a multiple pollutant regression (Chuang et al., 2007). In people with asthma, there were significant decreases in the standard deviation of normal-to-normal beat (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 (von Willebrand factor [vWF]), Cr (WBCs and interleukin six (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 superoxide dismutase (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 CVD mortality and morbidity in the air pollution health effects literature. Cardiovascular responses Sorensen et al. (2005) studied 49 students in Copenhagen, and reported that their personal exposure to soluble V and Cr, but not Fe, Ni, Cu, and Pt, were associated with significant increases in oxidative stress and DNA damage (as measured by 8-oxodG concentrations in lymphocytes (LYMs)). 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. PM2.5 components were correlated to cardiac and blood parameters measured 10 and 15 h after the work shift. In-vehicle PM2.5 mass was associated with changes in cardiac parameters; blood proteins associated with inflammation, hemostasis, thrombosis, and increased red blood cell (RBC) volume. Using data on PM2.5 components, Riediker (2007) and Riediker et al. (2004b) found that calcium (Ca) was associated with increased uric acid and vWF, and with decreased protein C; Cr with increased WBCs and IL-6; Cu with increased blood urea nitrogen, mean cycle length of normal R-R intervals; and S with increased ventricular ectopic beats. Anderson et al. (2010) studied the activation of implanted cardioverter defibrillators in elderly patients in London, UK, in relation to peak concentrations of PC, PM10, BC, SO2, NOx, CO, and SO¼ 4 . The only significant association of defibrillator discharges with pollutant concentration was for SO¼ 4 with 0- and 1-d lags. Delfino et al. (2011) studied electrographic ST-segment depression in relation to traffic-related aerosols in 38 elderly

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subjects (27 male and 11 female) with coronary artery disease in Los Angeles, CA. They found significant positive associations of combustion-related particulate and gaseous pollutants with averaging times ranging from 1 h to 4 d. The strongest effects were associated with primary OC and PC. Delfino et al. (2010a) studied traffic-related air pollution and blood pressure in elderly subjects in Los Angeles, CA (38 males and 26 females) with coronary artery disease in relation to their hourly outdoor and residential exposures to PC, PM2.5, BC, EC, OC, O3, NO2, and CO. They found significant associations of systolic and diastolic blood pressure (DBP) with PC, PM2.5, BC, and OC for averaging times of 3–7 d, with the greatest increases for OC, and greater for primary OC than for secondary OC. Also, the effects were greater with exercise and among those with higher body mass indices (BMIs). Delfino et al. (2010b) studied traffic-related air pollution and airway inflammation, indexed by eNO, and systemic inflammation, indexed by IL-6, in elderly subjects with coronary artery disease in Los Angeles, CA (34 males and 26 females) in relation to their exposures to PM2.5-0.25, PM0.25, primary OC, O3, PAHs and hopanes, secondary OC, water soluble OC and n-alkanoic acids, O3, NOx, and CO. They found significant associations of eNO with secondary OC, PM2.5-0.25, and O3. They also found significant associations of eNO with primary OC, PM0.25, CO, and NOx. Strak et al. (2012) studied responses to ambient air pollution in 31 healthy young adults (21 female and 10 male) at five different locations (underground railroad station, two traffic sites, a farm, and an urban background site) in the Netherlands. Measurements of O3, NO2, NOx, PM10, PM2.5, PC, PM2.5 light absorbance, EC, OC, trace metals and ions in PM2.5 and PM10-2.5, endotoxin, and oxidative potential (OP) were made at each site. Lung function measurements before and at three times after 5 h of exposure with intermittent light exercise included FVC, FEV1, forced expiratory volume between 25 and 75% of vital capacity (FEF25-75), PEF, and eNO. For eNO in single pollutant regressions, there were significant associations with PC, NOx, and with light absorbance, EC, OC, OP, Fe, Cu, V, and soluble Ni in PM2.5 samples. The most consistent associations in twopollutant models for eNO, seen immediately, and at 2 h after exposure, were for PC and Fe. For single pollutant models, there were significant associations for FVC and FEV1, but not for FEF25-75 and PEF, with NOx, PC, absorbance, EC, Fe, Cu, and soluble Ni. For two-pollutant models immediately after exposure, there were consistent associations for PC, NO2, and soluble Ni. The Lippmann & Chen (2009) review of human responses in CAPs inhalation studies emphasized the particle size ranges and components in ambient air that were most closely associated with the observed effects. There have also been controlled exposures to ambient air pollutants in human volunteers in clinical laboratories, as described in the next section. Pulmonary responses Peaks in ambient air PM were associated with a variety of pulmonary effects. Allen et al. (2008) reported that there were


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significant increases in exhaled nitric oxide (eNO) in Seattle associated with personal exposure to BC and PM2.5, as well as outdoor PM2.5, and significant reductions in peak expiratory flowrate (PEF) were associated with outdoor concentrations of PM2.5, BC, and levoglucosan, whereas reduced forced expiratory volume in 1 s (FEV1) was associated only with outdoor levoglucosan. McCreanor et al. (2007) studied adults in London, England, 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, PC, EC, and NO2 that were associated with significant reductions in FEV1 and forced vital capacity (FVC). Among the measured air pollutants, the associations were strongest for EC and PC. Penttinen et al. (2006) reported that among adults with asthma, PEF was significantly reduced in relation to attributed to local sources of PM2.5 (lag 1 d) and to PM2.5 soil sources (lag 3 d). Gent et al. (2009) reported that for children with physiciandiagnosed 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 as a marker 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-d averaged exposures, with a 10% increased likelihood of wheeze per 5 mg/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.


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inflammation and increased platelet aggregation. Salvi et al. (1999) had previously found that, in human volunteers, 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 mg/m3 to those seen in prior Chapel Hill exposures of healthy, young adults (aged 18–40). They reported that 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. In contrast, there were no such changes in the young adult group. Sama 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 mg/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 mg/m3 (151 800 particles/cm3) of UFP CAPs. The PM was described in greater detail by Samet et al. (2009). 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. Some cardiac endpoint changes were observed in each of these studies, with a trend toward changes in cardiac rhythm and blood coagulation (Ghio et al., 2003).

Exposures of human volunteers in clinical settings The laboratory-based studies involving CAPs exposures are grouped by specific geographical areas, on the basis that the mixtures of ambient air components and PM size distributions were expected to vary among airsheds. As demonstrated in the sections that follow describing data collected during the NYU NPACT study, this expectation was shown to have a demonstrable validity. In addition, laboratory-based human inhalation studies involving components of ambient air, such as diluted diesel engine exhaust, are also reviewed in order to provide a basis for evaluating whether the effects produced in such studies can account for the effects associated with PM2.5 inhalation. Chapel Hill, NC Ghio et al. (2000) reported that the 2-h CAPs exposures, ranging in mass concentration from 23 to 311 mg/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 SO¼ 4 were highly correlated with the PM2.5 mass concentration, whereas Ni and Cu were least correlated. In terms of biological responses, a Fe/Se/SO¼ 4 factor was associated with increased BALF percentage of polymorphonuclear neutrophils (PMNs), and a Cu/Zn/V factor with increased blood fibrinogen. The increase in plasma fibrinogen correlated with decreases in PMNs and platelets, consistent with a state of systemic

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 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 g/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.

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Gong et al. (2004b) exposed healthy, non-smoking adults and others with mild asthma to PM10-2.5 CAPs at a concentration averaging 157 mg/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 heart rate (HR), and decreases in HRV, and they were greater in the healthy subjects.


Toronto, Canada Normotensive, non-smoking, healthy volunteers were exposed to PM2.5 CAPs at a concentration averaging 150 mg/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 endothelialdependent flow-mediated dilatation (FMD), endothelial-independent nitroglycerin–mediated dilatation (NMD), or blood pressure (BP) (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 0.04 for OC, 0.05 for EC, 0.06 for cadmium (Cd), and 0.09 for K. The p values were between 0.13 and 0.17 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 0.40. In a follow-up study, there was a significant increase (6 mm Hg) in DBP in those exposed to O3 plus CAPs (p ¼ 0.013), 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 ¼ 0.009) with OC, whereas the association with PM2.5 mass was not significant (p ¼ 0.27). In this study, the EC and metals in the CAPs were not significantly associated with BAD constriction or BP (Urch et al., 2005). Fakhi et al. (2009) extended the study in Toronto to include 50 non-smoking volunteers, including subjects with asthma. The CAPs mass averaged 122 mg/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. Edinburgh, Scotland Mills et al. (2008) exposed healthy and age-matched volunteers with stable CHD to PM2.5 CAPs at 300 mg/m3 and to FA in Edinburgh, Scotland. Data were collected on peripheral vascular vasomotor and fibrinolytic function, and inflammatory variables - including circulating LYMs, serum CRP, and exhaled breath 8-isoprostane and nitrotyrosine - at 6–8 h after exposure. Exhaled breath 8-isoprostane increased after CAPs exposure (p50.05), and there was an increase in blood flow and plasma tissue plasminogen activator (p50.005). 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 mg/m3 of PM in diesel engine exhaust for 1 h (Mills et al., 2007), they suggested that the minimal responses to CAPs were due to the particle composition being very low in carbon, with 490% being sea salt.

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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.

Laboratory-based inhalation studies in humans with components of ambient air PM2.5 Diesel engine exhaust Stenfors et al. (2004) exposed young healthy and mild asthmatic Swedish subjects for 2 h to diluted whole diesel engine exhaust (WDE) containing diesel exhaust particles (DEP) at 108 mg/m3 and assessed lung function and airway inflammation. The exposures did not affect FEV1 or FVC, but did produce significant increases in specific airway resistance (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 (Porazar et al., 2005), the DEP concentration was 300 mg/m3. The exposure activated redox-sensitive transcription factors, consistent with oxidative stress triggering, increased synthesis of pro-inflammatory cytokines. In archived bronchial biopsies from this inhalation study, Porazar et al. (2008) showed that the exposure caused significant increases in the expression of epidermal growth factor receptor (EGFR) and phosphorylated C-terminal Tyr 1173, whereas Src-related tyrosine (Tyr 416), mitogen-activated 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 mg/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 WDE for 1 h (PM mass concentration ¼ 300 mg/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 (p50.05), whereas the bradykinin injection resulted in an increase in plasma tissue plasminogen activator (p50.001). 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 (TNF) and IL-6; both at p50.05], but had decreased acetylcholine (p ¼ 0.01) and bradykinin- induced forearm vasodilatation (p ¼ 0.08). Mills et al. (2007) exposed 20 men with a prior MI to the 300 mg/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


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and fibrinolytic 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 ¼ 0.001). WDE exposure did not aggravate preexisting vasomotor dysfunction, but did reduce the acute release of endothelial tissue plasminogen activator (p ¼ 0.009). Behndig et al. (2006) exposed 15 healthy adults, 7 female and 8 male, to FA or diluted WDE (PM at 100 mg/m3) for 2 h with intermittent exercise. At 18 h post-exposure, they performed bronchoscopies and collected BALF and bronchial biopsy tissues. They were able to find increases in bronchial mucosa PMNs and mast cells, as well as increases in bronchoalveolar lavage fluid (BALF) PMNs, IL-8, and myeloperoxidase. Peretz et al. (2008a) exposed healthy young adults in Seattle to WDE containing either 100 or 200 mg/m3 of DEP for 2 h. At 3 h post-exposure for 200 mg/m3, there was a statistically significant increase in HF power, and a decrease in low frequency (LF) to HF ratio, but no effect on timedomain 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 mg/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. In a recent review paper, Ghio et al. (2012) noted that short-term inhalation exposures of healthy adult volunteers to WDE and DEP have resulted in pro-inflammatory lung and systemic responses, but only at DEP concentrations of 300 mg/ m3 and higher, but that there was a lack of such a response in asthmatic subjects. On the other hand, such controlled human exposure studies of cardiovascular effects have also shown that diesel exhaust has a capacity to precipitate coronary artery disease. In addition, there is a relationship between WDE and DEP exposure and vascular endpoints; these effects in WDE may be diminished with removal of DEP. Many extra-pulmonary health effects of diesel exhaust exposure, including systemic inflammation, pro-thrombotic changes, and cardiovascular disease, are considered consequent to proinflammatory events and inflammation in the lung. In summary, short-term inhalation exposures to WDE and DEP can produce human health effects of concern, but it is not clear that they are likely to do so at DEP concentrations likely to be present in ambient air.

Effects of ambient air PM inhalation in animals As noted in the Introduction, deaths among show cattle in London were associated with the killer smogs of 1873 and 1952, but nearly all subsequent reports of toxicological studies of animal responses to ambient air PM and/or its components have been based on much lower exposure levels that produced lesser responses in laboratory animals. The literature review that follows is restricted to laboratory animal studies that were focused on exposures with direct relevance to effects of ambient air PM, or its components, and at doses that produced effects that are similar to those associated with effects seen in human populations and/or in


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members of panels being monitored for nature and extent of their exposures and exposure-response relationships. The literature reviewed below is discussed in terms of (1) effects of short-term CAPs inhalation exposures in laboratory animals; (2) effects of long-term CAPs inhalation exposures in laboratory animals; (3) effects of PM component inhalation exposures in laboratory animals; and (4) lung instillation studies with PM components. The animal exposure studies that indicated effects associated with CVD are summarized in Table 4. The toxicological studies reviewed in this section are playing an important role in our increasing appreciation of the roles played by PM constituents as health stressors in terms of (1) reinforcing the epidemiological work, thereby adding to the causality argument for both short- and long-term exposures and responses; and (2) providing greater insights into exposure-response relationships and biological mechanisms.

Historic studies in laboratory animals dealing with cardiopulmonary responses to PM and its components Amdur et al. (1978) conducted toxicological research to determine the roles of the PM components collected during the Donora smog episode on the pulmonary system and demonstrated that aerosol acidity and zinc compounds were related to the excess mortality and pulmonary effects that had been observed. More recent toxicological studies that have investigated the effects of ambient air PM mixtures have been reviewed by Lippmann & Chen (2009). In these studies, laboratory animals have been exposed by inhalation to concentrated ambient fine PM2.5 (CAPs), and samples of the exposure atmospheres have been analyzed to determine the concentrations of the PM constituents. The earliest of these studies (Clarke et al., 1999, 2000) were limited to short-term exposures and acute effects, while subsequent studies at NYU involved daily exposures extending over multiple months, such as the first of a series of 6-month studies in Tuxedo, NY that were described by Lippmann et al. (2005a,b, 2006). In the literature as a whole, there is considerable evidence that some individual PM constituents have much greater impacts on health-related indices than other constituents of the ambient air pollution mixture, as reviewed by Lippmann & Chen (2009). The findings of some key studies for studies that involve PM exposures of human populations, laboratory animals, and cells in vitro are summarized below. In each case, sufficient data were available on PM composition to demonstrate that some of the constituents were much more influential than were other constituents. Studies of laboratory animal responses to ambient air PM in inhalation studies have helped to identify the particle size ranges and components most closely associated with the observed effects. As for studies of human responses, the studies are grouped according to 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. In addition, laboratory-based animal inhalation studies involving components and/or source-related

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Table 4. Associations of PM2.5 components and CVD effects in CAPs exposure studies in laboratory animals.


Effects

Species

Location

Components

Reference

CVD effects of short-term inhalation exposures to PM2.5 HR Rats HR, Cardiac oxidative stress Rats Lipid peroxidation, Edema Rats QT, RT, Pdur, and Tpe intervals Rats Blood flow Dogs Cardiac vascular resistance Dogs

and PM2.5 components Boston Boston Boston Boston Boston Boston

Wellenius et al. (2004) Ghelfi et al. (2008) Ghelfi et al. (2008) Ghelfi et al. (2008) Bartoli et al. (2009) Bartoli et al. (2009)

CVD effects of short-term inhalation exposures to PM2.5 WBC an lymphocyte counts Dogs Small pulmonary arteries Rats Oxidative stress Rats S-T segment elevation Rats HR and HRV Rats HR and HRV Mice

constituents and sources Boston Al/Si factor Boston Si, Pb, SO¼ 4 , EC Boston Fe, Al, Si, Ti Boston Si, Al, Fe, Ti Boston Al, Si, Fe Tuxedo, NY Ni

Clarke et al. (2000) Batalha et al. (2002) Gurgueira et al. (2002) Wellenius et al. (2003) Rhoden et al. (2005) Lippmann et al. (2006)

CVD effects of long-term inhalation exposures to PM2.5 Vasomotor tone Mice Vascular inflammation Mice Potentiated atherosclerosis Mice Macrophage infiltration Mice Tissue factor expression Mice Aortic vasoconstriction Rats Non-alcoholic fatty liver Mice Glucose metabolism Mice Insulin resistance Obese mice Insulin resistance Mice Insulin resistance Mice Altered vasomotor tone Mice Enhanced atherosclerosis Mice Potentiated hypertension Mice Allergic airway disease Mice

Tuxedo, NY Tuxedo, NY Tuxedo, NY Tuxedo, NY Tuxedo, NY Tuxedo, NY Tuxedo, NY Columbus, OH Tuxedo, NY Tuxedo, NY Columbus, OH Tuxedo, NY Tuxedo, NY Columbus, OH Los Angeles, CA

mixtures in ambient air, such as diluted WDE are also reviewed in order to provide a basis for evaluating whether the exposures in such studies can account for the effects seen. For studies in animals, we are not limited to short-term exposures and acute responses, and the effects produced by longer-term inhalation exposures can be compared with the effects of chronic exposure in the epidemiological studies. The review that follows deals first with short-term inhalation exposures and the acute effects that they produce. It then deals with longer-term inhalation exposures, changes in responses during the course of daily exposures, and the cumulative changes resulting from the exposures. I made no attempt, in this critical review, to combine the results of the toxicological studies using meta-analytical techniques because there has been very little commonality in study design; animal species, strain, and ages; exposure atmospheres; durations of exposures; or responses to the exposures or times at which responses were measured. I have also summarized the results of some studies involving exposures to PM mixtures that are commonly found in community air and their effects following inhalation exposures, as well as some involving intra-tracheal (IT) instillation in which differentiation between the influences of the particles’ chemical constituents were investigated.

Short-term CAPs inhalation exposures in laboratory animals The discussion of the studies reviewed below is organized by the location of the laboratory in which they were conducted in

Sun et al. (2005) Sun et al. (2005) Sun et al. (2005) Sun et al. (2008a) Sun et al. (2008a) Sun et al. (2008b) Tan et al. (2009) Zheng et al. (2013) Sun et al. (2009) Xu et al. (2011b) Xu et al. (2012) Ying et al. (2009a) Ying et al. (2009a) Ying et al. (2009b) Kleinman et al. (2007)

consideration of the wide variety of ambient air PM compositions in different airsheds. Boston, MA Clarke et al. (1999) exposed normal Sprague-Dawley (SD) rats, with or without chronic bronchitis, induced by preexposure to 250 ppm SO2 for 5 h/d, 5 d/week for 6 weeks, to FA or PM2.5 CAPs at daily concentrations of 206, 733, and 607 mg/m3 in Boston, MA, for 5 h/d on three consecutive days. Pulmonary function was measured after the final exposure, and BAL was performed 1 d after the last exposure. Bronchitic rats exposed to CAPs had increased tidal volumes and PEFs, pulmonary inflammation, as well as increased numbers of BAL PMNs and LYMs, and total lavage protein, with lesser effects for the non-bronchitic rats. In order to determine if PM2.5 inhalation can induce cardiopulmonary effects, Clarke et al. (2000) investigated pulmonary inflammatory and hematological responses of canines after exposure to Boston PM2.5 CAPs at 203– 360 mg/m3. For pulmonary inflammatory studies, normal dogs were exposed in pairs to either CAPs or FA (paired studies) for 6 h/d on three consecutive days. For hematological studies, dogs were exposed for 6 h/d for three consecutive days, with one receiving CAPs while the other was simultaneously exposed to FA; crossover of exposure took place the following week (crossover studies). No statistical differences in biologic responses were found when all CAPs and all FA exposures were compared. However, the variability in biologic response was



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considerably higher with CAPs exposure. Subsequent exploratory graphical analyses and mixed linear regression analyses suggested associations between CAPs constituents and biologic responses. Factor analysis was applied to the compositional data from paired and crossover experiments to determine elements consistently associated with each other in CAPs samples. In paired experiments, four factors were identified; in crossover studies, a total of six factors (V/Ni, S, Al/Si, Br, Na/Cl, and Cr) were observed. Of these, only the Al/Si factor was significantly associated with responses, i.e. increased BAL PMN percentage, total peripheral WBC counts, circulating PMNs, and circulating LYMs. Increased PMNs and BALs were associated with decreased LYMs. Saldiva et al. (2002) studied the effects of CAPs inhalation on lung inflammation. They exposed normal and bronchitic rats to Boston PM2.5 CAPs or FA for 5 h/d for 3 d, with the mean CAPs concentrations varying from 126 to 481 mg/m3. The CAPs produced significant pulmonary inflammation. Some inorganic CAPs components (Si, V, Pb, and Br) were significantly associated with increases in PMNs in BALF and lung tissue. Rhoden et al. (2005) exposed adult male SD rats to CAPs at 700 mg/m3 for 5 h and measured oxidative stress and cardiac function immediately after the exposure. HR was increased for 30 min after exposure, whereas SDNN had a delayed increase. The intraperitoneal injection of the anti-oxidant N-acetylcystine (NAC) prevented lung inflammation. Pretreatment of rats with 5 mg/kg atenolol immediately before inhalation exposure to CAPs effectively prevented CAPsdependent increased accumulation of oxidized lipids. Likewise, vagal blockage by intravenous administration of 0.3 mg/kg glycopyrrolate prevented CAPs-induced increases in heart TBARS levels. The results regression analyses by Rhoden et al. (2004) showed strong associations between increases in thiobarbituric reactive substances (TBARS) accumulation in the heart and the CAPs content of Al, Si, and Fe, and between BALF PMN count and Cr, Zn, and Na, and that both atenolol and glycopyrrolate prevented CAPsinduced cardiac oxidative stress. They concluded that CAPs exposure increases cardiac oxidants via autonomic signals and the resulting oxidative stress is associated with significant functional alterations in the heart. The results are interesting, in the context of demonstrating that a soil-related cluster of elements were associated with neutrophil accumulation, while Cr and Zn were associated with oxidative stress. Gurgueira et al. (2002) exposed adult SD rats to Boston CAPs at 300 mg/m3 for 5 h and showed significant oxidative stress in the lung and heart, but not in the liver. The increase in the lung concentrations of reactive oxygen species (ROS) upon exposure to CAPs was rapid, indicating an almost immediate effect of PM, or PM components, on the intracellular sources of free radicals. Furthermore, the transient nature of these increases points to a reversible interaction of PM components with cellular targets. Both observations are compatible with Fenton-type reactions catalyzed by transition metals, redox-cycling processes, or biochemical changes triggered by non-covalent binding to membrane receptors. Using single-component regression analyses, increases in chemiluminescence (an index of oxidant load) showed strong associations with the CAPs content of Fe, Mn, Cu, and Zn in

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the lung, and with Fe, Al, Si, and Ti in the heart. The oxidant stress imposed by 5-h exposure to CAPs was associated with slight, but significant, increases in the lung and heart water content, and with increased serum levels of lactate dehydrogenase (LDH), indicating mild damage to both tissues. In addition, CAPs inhalation also led to tissue-specific increases in the activities of the antioxidant enzymes. Batalha et al. (2002) exposed normal rats and rats with SO2induced bronchitis to PM2.5 CAPs in Boston for 5 h/d for three consecutive days (median CAPs ¼ 183 mg/m3) and studied the effects of the exposures on the morphology of their small pulmonary arteries. Increases in CAPs exposures and the CAPs contents of Si, Pb, SO¼ 4 EC, and (L/W) ratios. As demonstrated by Gurgueira et al. (2002), oxidative stress can be induced by exposure to high levels of metals in the CAPs and could be responsible for changes in cardiac parameters. In vivo CAPs exposure may also trigger adaptive responses. Wellenius et al. (2003) exposed dogs by inhalation via a tracheostomy to FA or Boston PM CAPs at a mean concentration of 286 mg/m3 for 6 h. The dogs were previously implanted with balloon occluders around the left anterior descending coronary artery and catheters for determining myocardial blood flow. The dogs underwent 5-min coronary artery occlusions immediately after the FA and CAPs exposures. The CAPs exposure did not affect HR, but did increase occlusion-induced peak ST-segment elevation (p ¼ 0.007), and the elevation was correlated (p ¼ 0.003) with the Si content of the CAPs, as well as with other crustal components, but not with PM2.5 mass, BC, OC, or S. Wellenius et al. (2004) exposed SD rats that had thermocoagulation-induced MI to PM2.5 CAPs (320–350 mg/m3) for 1 h, with and without co-exposure to 35 ppm CO. CO exposure reduced the ventricular premature beat frequency (p ¼ 0.012), but CAPs alone had no such effect. However, CAPs exposure increased HR during exposure (p ¼ 0.025) in a mass concentration-related manner (p ¼ 0.032), but not in relation to particle count. Particle component concentrations were available for BC, OC, EC, and 21 elements, but there were no significant associations of effects with the PM2.5 components. Bartoli et al. (2009) exposed dogs via tracheostomy to FA or Boston PM CAPs at a mean concentration of 349 mg/m3 for 5 h. The dogs were previously implanted with balloon occluders around the left anterior descending coronary artery and catheters for determining myocardial blood flow. The dogs underwent 5-min coronary artery occlusions immediately after the FA and CAPs exposures. The CAPs exposure decreased total myocardial blood flow (p50.001) was accompanied with an increase in coronary vascular resistance (p50.001), and the effects were more pronounced in or near the ischemic zone versus more remote myocardium (p50.001). They concluded that PM2.5 exacerbates myocardial ischemia. Detroit, MI Morishita et al. (2004) exposed normal and allergic Brown Norway rats to PM2.5 CAPs for 10 h at concentrations ranging from 300 to 650 mg/m3 in a mobile laboratory in Detroit. The allergic rats had, compared to the normal rats,

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increased pulmonary retention of lanthanum (La), V, Mn, and S, as well as increased lung inflammation. Using source-apportionment analyses, Morishita et al. (2006) concluded that the pattern of the airway responses was likely to be associated with the local combustions sources, including refineries and incinerators, and was independent of SO¼ 4 and total mass of PM2.5. These studies demonstrated a pattern of rat pulmonary and systemic effects that were not linked to high mass, but rather appeared to be dependent on CAPs chemical composition.


Research Triangle Park (RTP), NC Kodavanti et al. (2000) performed a series of 2- and 3-d, 6-h exposures to FA, PM2.5 CAPs, and aerosolized residual oil flyash (ROFA) of Sprague-Dawley (SD) rats in Research Triangle Park (RTP), NC, using rats with and without SO2induced bronchitis. The CAPs concentrations ranged from 475 to 907 mg/m3, and the ROFA concentration was 1 mg/m3. The CAPs exposures produced some CAPs concentrationrelated pulmonary injury in the bronchitic rats, but not in the healthy rats, and the FA and ROFA did not produce measurable effects in either group of rats. The concentrations of leachable SO¼ 4 and metals in the CAPs were not associated with the effects. An in vivo inhalation study by Kodavanti et al. (2005), utilizing two different rat strains, made a number of findings. They exposed two different strains of rats (spontaneously hypertensive [SH] and Wistar-Kyoto [WKY]) to CAPs from ambient air in RTP, at concentrations ranging from 144 to 1765 mg/m3, with Zn, Cu, and Al being enriched several-fold on low mass days. The CAPs were drawn from an area in reasonably close proximity to a major freeway near the intersection with another major road, suggesting that the effects seen might have been related to vehicular and PM2.5 mass. They demonstrated a pattern of rat strain-specific pulmonary and systemic effects that were not linked to high mass, but rather were dependent on CAP chemical composition. New York City, NY Shukla et al. (2000) examined exposure of mice to CAPs produced using an air centrifuge to expose murine C10 alveolar cells to (1) PM in NYC and (2) ultrafine carbon black (uCB). Twenty-four hours after a 6- exposure to the CAPs, lavaged lung tissues of the mice showed significant increases in steady-state messenger RNA (mRNA) levels of nuclear factor kappa B (NF-kB)-responsive cytokines and IL-6. Both the PM and the uCB increased activation of oxidantdependent NFkB in the murine cells. FA had no effect on such activation. There were no effects with 1-d exposures (4 h/d), but for 2-d exposures effects were found, suggesting that the effects took longer than 4 h to become manifest. However, biological effects did not correlate with CAP mass. On days with lower mass, when effects were observed, concentrations of Zn, Al, and Cu were enriched several-fold, but OC was increased to a lesser extent. The authors stated that these studies demonstrated a pattern of rat strain-specific pulmonary and systemic effects that were not linked to high

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mass, but rather appeared to be dependent on CAPs chemical composition. Tuxedo, NY Lippmann et al. (2006) exposed ApoE/ mice to PM2.5 CAPs on weekdays for 6 h/d, for 6 months, at an average mass concentration of 85 mg/m3, and cardiac function was monitored continuously over the 6 months and CAPs composition was determined for each exposure day. Most of the results of this study are described in the following subsection on long-term animal inhalation exposures. However, this was also a time-series study of short-term responses as well as being a chronic effects study. 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 source of high Ni was a distant Ni smelter rather than residual oil combustion, the usual source of elevated Ni concentrations. The authors attributed the acute effects on cardiac function to peaks in Ni. Los Angeles, CA Campbell et al. (2005) exposed male BALB/6 mice to PM2.5 CAPs (mean ¼ 442 mg/m3), ultrafine (0.18 mm) CAPs (mean ¼ 283 mg/m3), or FA for 4 h/d, 5 d/week, for 2 weeks at a site 150 m downwind from a heavily traveled freeway in Los Angeles. One- and two-weeks after the last exposure, the mice were challenged with aerosolized OVA. As compared to FA exposure, both CAPs exposures increased inflammatory indices in the brains of the sensitized mice, and the levels of pro-inflammatory cytokines ILa, and TNFa and NF-kB were increased in the brain tissue. Kleinman et al. (2008) exposed ApoE/ mice to UFP CAPs for 5 h at a site 200 m from Freeway 110 in central Los Angeles at two different mass concentrations, i.e. 114 and 30 mg/m3. There was a dose-related increase in nuclear translocation of NF-kB and activator protein (AP)-1, which promotes inflammation. Increased levels of glial fibrillary acidic protein (GFAP) were also found. Levels of MAP kinases were assayed, and the fraction of c-Jun aminoterminal kinase (JNK) present in active form was increased. The Netherlands Kooter et al. (2006) exposed spontaneously hypertensive SH rats for 2 d to PM2.5 CAPs at a city background location (concentration range ¼ 400–3600 mg/m3) and to PM2.5 and UFP at a location in The Netherlands dominated by traffic (concentration range ¼ 260–556 mg/m3). SO¼ and 4 , NO3 þ NH4 accounted for a mean of 56% of the PM2.5, but only 17% of the UFP þ PM2.5. Unambiguous uptake of PM2.5 by macrophages was seen only in the UFP þ PM2.5 group. Marginally significant effects were seen in both exposure groups in terms of heme oxygenase (HO)-1 and malindialdehyde in blood samples. The HO-1 response was nonmonotonic, with an optimum at 600 mg/m3 for PM. Both exposure groups had significant decreases in WBCs.

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Long-term CAPs inhalation exposures in laboratory animals


Tuxedo, NY A series of longer-term PM2.5 CAPs inhalation studies were conducted at NYU’s laboratory in Sterling Forest (Tuxedo, NY). Because these studies collected both long-term electrocardiographic (ECG) data, as well as simultaneous data on PM2.5 composition, such studies had sufficient statistical power to identify possible causal components of ambient PM2.5 on short-term changes in cardiac function. In addition, the NYU subchronic CAPs inhalation studies involved a series of measurements that were used to determine both acute and cumulative effects of daily inhalation exposures to PM2.5 CAPs in a mouse model of atherosclerosis. The results of the first of these studies, involving 5–6 months of warmseason daily exposures (5 d/week, 6 h/d to an average CAPs concentration of 110 mg/m3) were described in a special issue of Inhalation Toxicology (Chen & Hwang, 2005; Chen & Nadziejko, 2005; Gunnison & Chen, 2005; Hwang et al., 2005; Lippmann et al., 2005a,b,c; Maciejczyk & Chen, 2005; Veronesi et al., 2005). These papers documented CAPs exposure-associated acute and chronic effects on cardiac function, increased amounts of, and more invasive, aortic plaque, and changes in brain cell distribution and in gene expression markers, as well as data on the effects of daily CAPs exposures in vitro on NF-kB activation. The biological plausibility of ambient air PM contributing to changes in brain cell distribution was enhanced by a followup study by Sama et al. (2007), in which they conducted in vitro assays of the cellular and genomic responses of immortalized microglia cells (BV2) to CAPs collected during the study described by Veronesi et al. (2005). Two composite samples were applied to the microglia cells; one composed of CAPs from days with high potency (HP) in their stimulation of NF-kB release in human bronchial epithelial cells, and the other from CAPs collected on days with low potency (LP). The LP composites reduced intracellular adenosine triphosphate (ATP) at doses 425 mg/ml, and depolarized mitochondrial membranes (46 mg/ml) within 15 min. HP and LP CAPs (425 mg/ml) differentially affected the endogenous scavengers, glutathione, and non-protein sulfhydryl after 1.5 h. Both HP and LP CAPs stimulated the release of pro-inflammatory cytokines TNFa and IL-6 after 6 h of exposure. Microarray analysis of both HP and LP exposed microglia (75 mg/ml) identified 3200 (HP) and 160 (LP) differentially expressed (up- and down-regulated) genes relative to the medium controls. The results implicated 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). 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 & 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 & Hwang (2005) to


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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 & 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 SO¼ 4 , 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 0.01) 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, and Ca) in the afternoon and for both residual oil combustion and traffic (Br, Fe, and EC), 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 mg/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 d with northwest winds carried more Ni, Cr, and Fe, but less of the other elemental tracers than the 89 d 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 d with unusually high levels of Ni, Cr, and Fe in this mouse study. Back trajectory analyses from Sterling Forest for the 14 d 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 HF diet with those exposed to FA. For the CAPs-exposed mice, the plaque area in the aorta was 41.5% versus 26.2% in the FA group (p ¼ 0.001), whereas for the subgroup on a normal diet, CAPs-exposed versus FA-exposed was 19.2% versus 13.2% (p ¼ 0.15). Lipid content in the aortic arch in the HF group versus normal chow (NC) group exposed to CAPs was 30% versus 20% (p ¼ 0.02). Vasoconstrictor challenges in the thoracic aorta were increased in the CAPsexposed HF mice versus the FA mice (p ¼ 0.03), and relaxation in response to acetylcholine was greater (p ¼ 0.04). 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 p50.001). Thus, the 30-h/week subchronic CAPs exposure of ApoE/ mice at 85 mg/m3 altered vasomotor tone, induced vascular inflammation, and potentiated atherosclerosis.


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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 ¼ 0.04), whereas the comparison among NC mice was not statistically significant (p ¼ 0.07). Based on immunochemistry, TF expression was increased in the HF mice by CAPs exposure (15% versus 8%, p50.01), as was macrophage infiltration (19% versus 14%, p50.01). In a 10-week, 30 h/week CAPs inhalation exposure study in SD rats conducted in Tuxedo, NY, at an average concentration of 79 mg/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 (p50.001). 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 HF chow (HFC) to either FA or CAPs at 85 mg/m3 for 6 h/d, 5 d/week, for 6 weeks at Tuxedo, NY. After sacrifice, non-alcoholic 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-mg 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 mg/ml SRM for 24 h induced a dosedependent increase in pro-inflammatory cytokine mRNA levels, particularly IL-6 (p ¼ 0.01). Supernatant analysis confirmed increased IL-6 protein secretion (p ¼ 0.03). Similarly, PM2.5 exposure (0–100 mg/ml) increased IL-6 secretion in a dose-dependent manner by wt Kupffer cells (p ¼ 0.08) and not by TLR4/ Kupffer cells (p ¼ 0.29). PM2.5 exposure (0–400 mg/ml, 24 h) did not significantly affect collagen 1A1 mRNA or protein levels in the LX2

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stellate cell line. PM2.5 up to 400 mg/ml did not enhance collagen 1A1 levels in wild type or TLR4/ mouse stellate cells. However, collagen 1A1 mRNA levels increased significantly in wild type and TLR4/ stellate cells when incubated with conditioned medium from SRM (200 mg/ml)exposed RAW cells (p50.01). 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 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 73 mg/m3 at Tuxedo, NY, for 6 h/d, 5 d/week, for 24 weeks. Compared to the FA-exposed controls, the CAPs-exposed mice had insulin signaling abnormalities that were associated with abnormalities in vascular relaxation to insulin and acetylcholine and increased adipose tissue macrophages (F4/80þ cells) in visceral fat expressing higher levels of TNFa/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 micro- circulation. 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 (ms). The biological plausibility of ambient air PM contributing to ms in the CAPs-exposed obese mice is enhanced by a report by Chen & Schwartz (2008) on data showing an association of PM10 with WBC count and ms in NHANES III. New York City Ying et al. (2009a) exposed ApoE/ mice on a HF diet to FA or PM2.5 CAPs for 6 h/d, 5 d/week, for 4 months in northern Manhattan, at a mean concentration of 173 mg/m3, to test the hypothesis that exposure to CAPs enhances 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 CAPs- exposed 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 CAPsexposed mice. Thus, CAPs exposure in Manhattan altered vasomotor tone and enhances atherosclerosis through NADPH oxidase–dependent pathways.

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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 at 74 mg/m3 for 6 h/d, 5 d/week, for 12 weeks at Columbus, OH. Following the inhalation exposures, the mice were implanted with minipumps for A-II or vehicle for 14 d. 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 p115Rho- GEF, 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. (2010) exposed C57BL/6 male mice that had been fed normal chow to either FA or CAPs for 6 h/d, 5 d/week, for 10 weeks at a mean concentration of 76 mg/m3. They also exposed a murine monocytic-macrophage cell line (Sigma-Aldrich RAW264.7) to Columbus PM in vitro at 300 mg/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-kB and c-Jun amino-terminal kinase (JNK) and reduced expression of paraoxonase 1 (PON-1) and peroxisome proliferator-activated receptor gamma (PPARgamma), which favors the development of cardiovascular diseases. In a more recent study, Zheng et al. (2013) exposed C57BL/6 mice were exposed to Columbus, OH CAPs for 6 h/d, 5 d/week at a mean concentration of 75 mg/m3 for 3 or 10 weeks. Significant effects were seen in mice exposed for 10 weeks, in terms of a non-alcoholic steatohepatitus (NASHlike phenotype, characterized by hepatic steotosis, inflammation, and fibrosis. They displayed impaired hepatic glycogen storage, glucose intolerance, and insulin resistance. Los Angeles, CA Kleinman et al. (2007) exposed ovalbumin (OVA) pre-treated BALB/c mice to PM2.5 CAPs at 300–400 mg/m3, PM0.18 CAPs at 200–300 mg/m3, or FA for 5 d/week, for 2 weeks, at 50 and 150 m (meters) 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.


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Araujo et al. (2008) exposed ApoE/ mice to CAPs for 5 h/d, 3 d/week, for 5 weeks in two size ranges (50.18 and 52.5 mm) at a site in Los Angeles that was 300 m from the 110 Freeway. The UFP-exposed mice had greater early atherosclerotic lesions than the mice exposed to PM2.5 CAPs or FA, and UFP exposure also inhibited the anti-inflammatory capacity of plasma high-density lipoprotein and produced greater oxidative stress as evidenced by a significant increase in hepatic malonaldehyde levels and up-regulation of Nrf2regulated antioxidant genes.

Effects of PM source mixture inhalation exposures in laboratory animals This section summarizes some studies that used PM components found in ambient air that have been known to produce significant health-related effects at concentrations that are relevant to current or recent human exposures. Each subsection begins with studies conducted at PM concentrations occurring in occupational environments, and then describe some studies conducted using complex mixtures somewhat higher PM concentrations (up to about 1 mg/m3), on the basis that they may be relevant to our subsequent comparisons of results with those of the CAPs studies. Diesel engine exhaust In a study that compared the effects of diluted WDE with PM2.5 CAPs, Quan et al. (2010) exposed male ApoE/ mice 5.2 h/d, 5 d/week, for 5 months to five different exposure atmospheres: (1) FA; (2) Tuxedo, NY, PM2.5 CAPs (mean ¼ 110 mg/m3); (3) WDE, containing DEP at 436 mg/m3; (4) diesel exhaust gases (DEG, equivalent to gases in WDE; and (5) CAPs þ DEG. Atherosclerotic plaques were quantified for brachiocephalic artery cross-sections (BACs), after 3 and 5 months of exposure using (1) serial ultrasound imaging; (2) hematoxylin and eosin (H&E) histology; and (3) en-face Sudan IV stain. All three methods indicated that: (1) DEG did not exacerbate progression. (2) There were no interactive effects between DEG and CAPs. (3) CAPs, despite having a much lower PM concentration, caused more plaque progression than WDE, indicating that some components in ambient PM, not present in WDE, are most responsible for the exacerbation of plaque progression by CAPs. Campen et al. (2003) examined the cardiac effects of diesel exhaust exposure in SH rats. They were exposed to 0, 30, 100, 300, and 1000 mg DEP/m3) for 6 h/d for 7 d. Control rats displayed a reduced daytime HR from the beginning of the protocol, whereas exposed rats maintained a significantly elevated HRs. This difference persisted during the evenings of the exposure period but was not observed at any time during the pre-or post-exposure periods. The PQ interval, an index of atrioventricular node sensitivity, was significantly prolonged among exposed animals in a concentration-dependent manner. Increased HR with prolongation of the PQ interval may represent a substrate for ventricular arrhythmias. Anselme et al. (2007) exposed adult male WKY rats with and without IHD to 500 mg/m3 of DEP for 3 h. They were evaluated for ventricular arrhythmia during and after the


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exposure by ECG telemetry. There was an immediate decrease in RMSSD (root mean square of the standard deviation of normal to normal beat) in both groups of rats, and an immediate increase in premature ventricular beats in the IHD rats only. The changes in HRV were over after 2.5 h, whereas the pro-arrythmic effects in the IHD rats persisted for 5 h after the end of DEP exposure. Saxena et al. (2009) exposed WKY and SH rats to 0, 500, or 2000 mg/m3 of DEP for 4 h/d, 5 d/week, for 4 weeks to determine pulmonary retention of EC. While SH rats had 50% higher minute volumes, they had 16% less EC at the end of the exposures at 500 mg/m3, and 32% less at 2000 mg/m3 than the WKY rats. For the same exposures, Gottipolu et al. (2009) noted neutrophilic influx in the lung lavage in both strains, with only minimal changes in injury markers. This study provided evidence that WDE produced a hypertensive-like cardiac gene expression pattern associated with mitochondrial oxidative stress in the WKY rats, but not in the SH rats. Hazari et al. (2011) exposed SH rats to either 150 or 500 mg/m3 of DEP for 4 h to WDE or the same concentration of DEG, and assessed arrhythmogenesis 24 h later by continuous intravenous infusion of aconitine, an arrhythmogenic drug, while HR and electrocardiogram (ECG) were monitored. Rats exposed to WDE or DEG had slightly higher HRs and increased LF:HF ratios (sympathetic modulation) than did controls; ECG showed prolonged ventricular depolarization and shortened repolarization periods. Rats exposed to WDE developed arrhythmia at lower doses of aconitine than did controls; the dose was even lower in rats exposed to DEG. Pretreatment of low WDE–exposed rats with a TRPA1 antagonist or sympathetic blockade prevented the heightened sensitivity to arrhythmia. Lamb et al. (2012) extended this line of research and exposed both SH and WKY rats to WDE containing either 150 or 500 mg/m3 of DEP for 4 h or to DEG (the filtered stream having the same gas concentrations). Exposure to WDE, but not DEG, caused post-exposure ST depression and increased sensitivity to the pulmonary C fiber agonist capsaicin in SH rats, while exposure to DEG caused immediate ECG alteration in cardiac repolarization (ST depression) and atrioventricular conduction block (PR prolongation) as well as bradycardia in SH rats. For WKY rats, the only notable effect of WDE exposure was a decrease in HR. Hazari et al. (2012) continued this line of research and exposed both SH and WKY rats to WDE to 150 mg/m3 of DEP for 4 h. Increasing doses of dobutamine, a b1-adrenergic agonist, were administered to conscious unrestrained rats 24 h later to elicit the cardiac response observed during exercise while HR and ECG were monitored. The WDE exposure potentiated the HR response of WKY and SH rats during dobutamine challenge, and prevented HR recovery at rest. During peak challenge, WDE-exposed SH rats had lower overall HR variability when compared with controls, in addition to transient ST depression. All WDE-exposed animals also had increased arrhythmias. Carll et al. (2012) exposed SH rats to WDE containing 150 mg/m3 of DEP for 4 h or to DEG having the same gas concentrations and measured HR and HRV. WDE increased BP and decreased HRV. DEG and WDE decreased HR in the 4 h after exposure. Thus, WDE and DEG differentially alter

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CV and autonomic physiology and may increase risk through divergent pathways. Carll et al. (2013) extended their research to a 4-h exposure to 500 mg/m3 of DEP in WDE and the associated DEG. The WDE increased HRV during exposure. In the 4 h post-exposure, WDE increased cardiac output, left ventricular volume, stoke volume, HRV, and atrioventricular block arrhythmias, ST and T amplitudes, ST area, T-peak to T-end duration, but not HR. Changes in HRV were correlated with bradyarrythmia frequency, repolarization, and echocardiographic parameters. At 24 h post-exposure, WDE exposed rats had increased serum CRP and pulmonary eosinophils. Source-related mixtures contributing to ambient air PM The National Environmental Respiratory Center (NERC) at the Lovelace Respiratory Research Institute (LRRI) laboratory in Albuquerque, NM investigated responses to acute (7 d) and subchronic (6 h/d, for 6-months) inhalation exposures of rodents (F-344 and SHR rats, A/J, C57BL/6, and BALB/C mice) to graded dose levels of laboratory-generated sourcerelated mixtures of combustion effluents, with maximum PM concentrations up to 1000 mg/m3, and comprehensive analyses of PM and gaseous component concentrations in the exposure chambers. In a paper reporting on the health effects of 6-months of inhalation exposure to simulated downwind coal combustion emissions generated in a laboratory setting, Seilkop et al. (2012) reported that only 17 of the 270 species-genderoutcome-responses were affected by the exposures, with only three being strongly related to PM. Greater responses had been seen in the previous 6-month studies involving diesel engine, gasoline engine and hardwood smoke emissions. As described by Godleski et al. (2011a), diluted and aged effluents from real electric utility power plants in the Toxicological Evaluation of Realistic Emission Source Aerosols (TERESA) study. The emissions were drawn directly from the discharge stacks of three different US power plants, and photochemically aged in passing through ductwork in a mobile laboratory before being delivered to Sprague-Dawley (SD) rats for 6 h in the exposure chambers, with mass concentrations ranging from 44 to 257 mg/m3, with trace elements being present at very low concentrations. Each plant had a different coal source and emission controls. The exposures were to either effluent or FA. Four different aerosols were delivered to the exposure chambers. They were: primary PM (P), secondary oxidized PM (PO), PO þ secondary organic PM (POS), and neutralized POS (PONS). The most robust outcomes were for the PO scenario (increased respiratory frequency with decreases in inspiratory and expiratory time); and the PONS scenario (decreased PEF and expiratory flow at 50%). The PONS findings were most strongly associated with ammonium, neutralized sulfate and EC in univariate analyses, but only with EC in multivariate analyses. In terms of cellular responses at 24 h post-exposure, Godleski et al. (2011b) noted that the POS and PONS scenarios produced significant increases in BAL total cells and macrophages at two plants, while the P and PONS scenarios were associated with BAL neutrophils, and BAL neutrophils wee associated with Zn. At one of the power


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plants, Wellenius et al. (2011) exposed rats with acute MI. They found that POS exposure (PM mass ¼ 219 mg/m3, sulfate ¼ 173 mg/m3, acidic sulfate ¼ 133 mg/m3, OC ¼ 51 mg/ m3) was associated with increased PVB frequency and decreased respiratory expiratory time and end-expiratory pause, but not with changes in HR, HRV, or ECG intervals. They concluded that the POS exposure scenario may be associated with increased risk of ventricular arrhythmias in susceptible animals. In the first of a series of reports of the final results of the NERC program, Seilkop et al. (2012) described data-mining results of the multiple assays of exposure and pro-atherosclerotic vascular responses in ApoE/ mice to freshly generated WDE, whole gasoline engine emissions (WGE), hardwood combustion smoke (WS), and downwind coal combustion effluents (CCE). Assays of the chamber atmospheres were performed in order to determine, if possible, which of the over 700 chemical constituents were most closely associated with biological responses. The response indicators of atherosclerosis that they selected were vascular endothellin-1 (ET-1), vascular endothellin growth factor (VEGF), matrix metalloptoteinase-3 (MMP-3), MMP-7, MMP-9, tissue inhibitor of MMP-2 (TIMP2), heme-oxygenase-1 (HO-1), and TBARS. They reported that 2 or 3 of the indicators typically explained most of the variation in response, although their rankings differed among the responses, with SO2, NH3, NOx, and CO being the exposure variables that were most predictive for responses. It is notable that these vapor concentrations were more predictive of these responses than were the PM constituents. I cannot provide a definitive explanation of the more predictive association of gas-phase pollutants in laboratorybased inhalation exposures of fresh motor vehicle exhaust aerosols, wood smoke, and simulated downwind coal combustion aerosol. In contrast, the findings of most epidemiological studies of populations exposed to ambient air pollution mixtures have shown that the associations are stronger for PM2.5 than for criteria pollutant gases. I can only speculate that (1) the ambient air PM2.5 is more toxic than the particles used in the laboratory inhalation studies; and/or (2) the vapors in the fresh motor-vehicle exhaust are more reactive than the vapors in ambient air. Since human exposures are generally further downwind of the effluent sources, and the concentrations of the reactive vapors are likely to dissipate faster than those of PM components, it is possible that responses to the exposures of PM constituents could become more influential in terms of human responses. In the most recent subchronic inhalation exposure study at LRRI, conducted as part of the Health Effects Institute (HEI)’s National Particle Component Toxicity (NPACT) program, there were once again subchronic (6 h/d, 50 d) inhalation exposures, but with different PM2.5 and pollutant vapor components. They were (1) diluted mixed motor vehicle (tailpipe) whole engine emissions (MVE), with 50 mg/m3 of PM2.5 from a spark ignition engine and 250 mg/m3 from a diesel engine; (2) FA), i.e. particle free diluted mixed motor vehicle engine emissions (MVEG); (3) 300 mg/m3 of resuspended road dust; (4) 300 mg/m3 of SO¼ (5) 300 mg/m3 of NO (6) MVEG þ SO¼ 4; 3; 4; (7) MVEG þ NO3 ; and (8) MVEG þ road dust. The results


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of these exposures are discussed in the section later in this Critical Review on the HEI-sponsored NPACT program, including the observation that the effects of the component mixtures were often greater than additive. Sidestream cigarette smoke Chen et al. (2009) exposed ApoE/ mice to sidestream smoke (SS) as a surrogate for environmental tobacco smoke (ETS) at a PM concentration of 450 mg/m3 for 6 h/d, 5 d/week, for 6 months. The mice were exposed to SS or FA, 6 h per d, 5 d per week for 6 months. The SS exposure significantly enhanced atherosclerosis lesion progression and reduced survival. Using UBM, SS significantly exacerbated plaque progression by 4 months after starting of exposure. Effects seen during SS exposure (1) activity, body temperature, and HR increased; (2) SDNN (standard deviation of normal to normal beat), RMSSD decreased; and (3) the effects of SS exposure, varied across different time segments of the day. These results were comparable in kind and magnitude to those produced in a similar mouse model exposed to the same exposure regimen for PM2.5 CAPs at an average concentration of only 134 mg/m3. Transition metals Gavett et al. (1997) showed that the transition metals and SO¼ 4 within ROFA determines airway hyper-reactivity and lung injury in rats. Campen et al. (2001) examined responses to Ni and V in conscious rats by whole-body inhalation exposure. The authors tried to ensure valid dosimetric comparisons with prior instillation studies, by using concentrations of V and Ni ranging from 0.3 to 2.4 mg/m3. The concentrations used incorporated estimates of total inhalation dose derived using different ventilatory parameters. HR, core temperature, and ECG data were measured continuously throughout the exposure. The rats were exposed to aerosolized Ni, V, or Ni þ V for 6 h per day for 4 d, after which serum and bronchoalveolar lavage samples were taken. Whereas Ni caused delayed bradycardia, hypothermia, and arrhythmogenesis at concentrations 41.2 mg/m3, V failed to induce any significant change in HR or core temperature, even at the highest concentration. When combined, Ni and V produced observable delayed bradycardia and hypothermia at 0.5 mg/m3 and potentiated these responses at 1.3 mg/m3 to a greater degree than were produced by the highest concentration of Ni (2.1 mg/m3) alone. The results are suggestive of a possible synergistic relationship between inhaled Ni and V, albeit these studies were performed at metal concentrations orders of magnitude greater than their typical ambient concentrations. In a study using dogs with preexisting cardiovascular disease, Muggenburg et al. (2000) evaluated the effects of shortterm inhalation exposure (oral inhalation for 3 h on each of three successive days) to aerosols of transition metals. HR and the ECG readings were studied in conscious beagle dogs (selected for having preexisting cardiovascular disease) that inhaled respirable particles of oxide and sulfate forms of transition metals (Mn, Ni, V, Fe, Cu oxides, and Ni and V sulfates) at concentrations of 0.05 mg/m3. No significant

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effects of exposure to the transition metal aerosols were observed. Hamada et al. (2002) exposed female BALB/c mice for 30 min to an aerosol nebulized from a solution of PM components dissolved from ROFA that had been collected at a Boston power plant. Penh, an index of airway hyperresponsiveness (AHR), was increased in a time- and doserelated manner, peaking at 48 h post-exposure. PMNs in BAL peaked at 12 h post-exposure. A simulated ROFA extract, containing the same concentrations of Ni, V, Zn, Co, Mn, and Cu as the ROFA, produced the same AHR response, but the summed responses to each metal separately did not.


Lung instillation studies with PM components Besides using CAPs inhalation studies, which can be expensive and time consuming, studies using collected urban PM for IT instillation and inspiration (IA) exposures to healthy and compromised animals have also produced interesting information concerning influential PM components and their health-related effects. Although there are many issues such as extrapolation and dosimetry that need to be addressed when IT is used in a toxicological study, the results of IT of ambient PM collected from different geographical areas can be used to support the hypothesis that PM composition is one of the most relevant parameters affecting ambient PM-associated health effects. Similarly, IT delivery of well-defined components of ambient air PM, in studies of comparative toxicity, can also be informative for the identification of particularly influential components, even when the exposures deliver dosages at concentrations considerably greater than those received via inhalation. Ambient air PM Instillation in rats of Ottawa PM extracts at 2.5 mg induced pronounced biphasic hypothermia, a severe drop in HR, and increased arrhythmias (Watkinson et al., 2000a,b) that were not seen with a comparable instilled dose of Mt. St. Helens volcanic ash. The results of this study showed that urban sites with high contributions from vehicles and industry were most toxic. This study also showed that the biological effects differ as a function of site and season. The analysis based on chemical class indicated that PM containing metal oxides, transition metals (Pb, Mn, Cu, Se, Zn, and As), EC, OC, and hopanes/steranes were the most important predictors of cytotoxic and inflammatory responses. The analysis also indicated that SO¼ 4 , secondary organic aerosols, meat cooking, and vegetative detritus were not correlated with the biological responses. On the other hand, in an analysis based on the source apportionment, the most toxic samples were from the sites during seasons with the largest contributions of diesel and gasoline emissions, whereas wood burning was only weakly correlated with toxicity end points. The analysis also indicated that SO¼ 4 , secondary organic aerosols, meat cooking, and vegetative detritus were not correlated with the biological responses. This study supports the concept that specific constituents and/or sources of PM affect its toxicity. Adamson et al. (2000) sought to determine which component(s) of the urban air PM from Toronto could account for its pulmonary toxicity. They did an aqueous extraction of the

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whole dust and instilled the extract and equivalent concentrations of the soluble metals within it into the lungs of mice. Three days later, in comparison to IT saline, only the whole extract and the Zn solution produced significant increases in inflammatory cells and protein in BALF. With 28 d of exposure, the Zn solution produced focal necrosis of type 1 alveolar cells, and focal fibrosis was seen at 4 weeks. Gerlofs-Nijland et al. (2007) collected PM2.5 and PM10-2.5 CAPs from six European cities with contrasting traffic profiles, PM composition, and in vitro analyses, and exposed SH rats IT with 3 or 10 mg PM/kg. They assessed changes in biochemical markers, cell differentials, and histopathological changes in the lungs and blood 24 h later. They reported doserelated adverse effects with both PM2.5 and PM10-2.5 CAPs that were mainly related to cytotoxicity, inflammation, and blood viscosity. There was a trend toward greater toxicity with increasing traffic levels. They selected component markers for traffic-related PM sources, i.e. polynuclear aromatic hydrocarbons (PAHs), Zn, Cu, Ba, and K. There was no correlation of any of the effect markers with combustion-exhaust-related PAHs, except for an increase of LYMs associated with PM2.5 CAPs (p ¼ 0.04). There was a significant correlation between PM2.5 Zn and BALF protein (p ¼ 0.01) and LDH (p ¼ 0.03), and PM2.5 K with total BALF cells and PMNs. In pathological assays, there were significant associations of PM2.5 K with alveolar inflammatory foci, and ascorbate with PM2.5 Cu (p ¼ 0.02) and Ba (p ¼ 0.01). For the PM10-2.5 CAPs, there were significant correlations of BALF protein with Cu (p ¼ 0.02) and with Ba (p ¼ 0.05), and alveolitis with Cu (p ¼ 0.04). They concluded that the effects were attributable to components derived from brake wear (Cu and Ba), tire wear (Zn), and wood smoke (K). Using projection-to-latentsurface (PLS) techniques, the largest contributors of effects were diesel and gasoline engine emissions, whereas wood burning was only weakly correlated with toxicity end points. The PLS analysis also indicated that SO¼ 4 secondary organic aerosols, meat cooking, and vegetative detritus were not correlated with the biological responses. Utah Valley Dust Some of the most convincing evidence to demonstrate that the lung dose of bioavailable transition metals, not just instilled PM mass, was the primary determinant of the acute inflammatory response was derived from a series of studies using ambient PM10 collected in the Utah Valley (Dye et al., 2001; Frampton et al., 1999, Ghio & Devlin, 2001). Frampton et al. (1999) showed that the extract of PM10 collected during the strike (having the lowest metal content, specifically soluble Fe, Cu, Pb, and Zn) showed no apparent cytotoxicity, minimal induction of cytokines, and lowest oxidant generation ability compared to extracts from PM10 (collected before and after the strike) having higher metal content. These experiments indicate (a) that instillation of ambient air particles, albeit at a very high concentration, can produce cardiovascular effects; and (b) that exposures of equal mass dose to particle mixtures of differing composition did not produce the same cardiovascular effects, suggesting that PM composition rather than just mass was responsible for the observed effects. To investigate the dose, time course, and the roles of specific


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metals, Dye et al. (2001) exposed SD rats, by IT, with equivalent masses of aqueous extracts of the same Utah ambient PM10, described above, at 0, 0.83, 3.3, 8.3, or 16 mg extract/kg body weight in 0.3 ml saline. Twenty-four hours after IT, rats exposed to extracts of PM10 collected when the plant was open developed significant pulmonary injury and PMN inflammation. Additionally, 50% of rats exposed to these extracts had increased airway responsiveness to acetylcholine, compared to 17% and 25% of rats exposed to saline or to the extracts of PM10 collected when the plant was closed. By 96 h, these effects were largely resolved, except for increases in lung lavage fluid PMNs and LYMs in rats exposed to PM10 extracts from prior to the plant closing. Analogous effects were observed with lung histologic assessment. Chemical analysis of extract solutions demonstrated that extracts of PM10 collected when the plant was open contained more SO¼ 4 , cationic salts (e.g. Ca, K, Mg), and certain metals (e.g. Cu, Zn, Fe, Pb, As, Mn, Ni). The qualitative coherence among these human epidemiological, clinical, and animal toxicological studies clearly showed that soluble metals could be the most important components related to PM exposure-related health outcomes. Molinelli et al. (2002) exposed human bronchial epithelial cells to extracts from Utah Valley sampling filters, with and without ion-exchange extractions to remove metal ions. The untreated extracts showed a concentration-dependent increase in the inflammatory mediator IL-8, whereas the chelated extract did not.

In vitro CAPs exposures to PM components Daily PM2.5 samples, collected over 6-months in a Biosampler impinger, were analyzed for PM constituents, and aliquots were used to expose BEAS-2B lung cells followed by assays for NFkB activity. The only significant association found was for the residual oil combustion source, which accounted for only 2% of the PM2.5 mass (Maciejczyk & Chen, 2005). Further analyses of the roles of individual PM2.5 constituents showed significant associations of Ni (averaging 38 ng/m3), Ba (13 ng/m3), Mn (9 ng/m3), and Fe (500 ng/m3) (Maciejczyk et al., 2010). The composite PM samples from the first NYU 6-month study of high NFkB activity in lung cells, and low NFkB activity were applied in a dose-response study to microglial cells, and the only elements having significant dose-related associations were Ni and V (Sama et al., 2007).

Addressing a recognized need for additional studies designed to integrate evidence from epidemiological and toxicological effects of ambient air PM2.5 There is a growing recognition of the need for a more focused program of research on the determinants of toxicity attributable to ambient air PM, as recommended recently by Kelly & Fussell (2012). The epidemiological studies that were reviewed above showed associations between ambient air concentrations measured at central monitoring sites and indices of mortality and morbidity for populations residing in the communities around the monitoring sites. Such studies have notable limitations that limit their utility for identifying


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the PM2.5 constituents that are most influential in causing health effects. On the exposure side of the exposure-response relationship, the principal limitations are (1) the variable representativeness of the concentrations of the concentrations measured at the central site for the population as a whole; and (2) the variations in the ability of the various constituents to penetrate into the microenvironments where the people spend most of their time. On the response side of the exposureresponse relationship, the principle limitations are (1) the highly variable susceptibility within the populations due to variations in age, diet, and ethnic distributions; (2) preexisting diseases affecting health status and access to health care services; (3) other health stressors such as poverty, exposures to cigarette smoke and other indoor pollutants, and occupational exposures to toxicants. The clinical studies that were reviewed above have their own advantages and limitations in terms of informing our understanding of exposure-response relationships among the general population. The advantages include (1) generally much better characterization of the exposure environment; (2) much more detailed characterization of personal risk factors for the individuals in the panel; (3) high-quality characterization of a broad range of functional and biomarker responses, including biomarkers of both exposure and effects that can enable exploration of underlying biological mechanisms. On the other hand, there are ethical concerns that impose some significant study limitations in terms of levels and durations of exposure, and of use of invasive assays of responses. In almost all cases, these include (1) restriction to acute exposures and short-term responses; (2) exposure concentrations that, by design, are unlikely to elicit adverse health effects among the volunteer subjects; and (3) use of single PM components or CAPs without the co-presence of ambient air gases and vapors that might potentiate the effects of the exposures to PM and its chemical components. The in vivo toxicological studies that were reviewed above have advantages that include those of the clinical studies, as well as other advantages, which include (1) the option to use animal species with well-defined genetic characteristics in order to limit response variability; (2) ability to control other environmental variables that can affect responses, such as diet, housing climate between challenge exposures; (3) ability to conduct repetitive in vivo assays over extended time periods to determine the presence of response progression during and after further exposure; and (4) ability to perform histological assays of responses in a variety of organ systems and cells. Such in vivo toxicological studies also have had some significant limitations in terms of the relevance of their results to human risk assessment. These include (1) most of these studies have used challenge atmospheres at concentrations much higher, generally orders of magnitude higher, than those encountered in recent decades by human populations. When very high concentrations are used, there can be concern about the relevance of the effects found to human health concerns; (2) the responses in the animal models selected for the study are likely to differ from those of humans similarly exposed, due to differences in genetic factors, metabolic factors, organ sizes and structures, particle retention and translocation differences, etc.

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The Health Effects Institute (HEI) National Particle Component Toxicity (NPACT) program In recognition of the need to foster better integration of epidemiological and toxicological findings on the health effects of ambient air PM2.5, the Health Effects Institute (HEI) issued a request for interdisciplinary research proposals (RFP) in 2005. Their Research Committee selected two research teams to engage in 4-year programs of study to constitute the HEI’s National PArticle Component Toxicity (NPACT) program. One of these programs was conducted at the New York University (NYU) School of Medicine campus in Sterling Forest (Tuxedo, NY), and the other was a consortium of the University of Washington (UW) in Seattle, WA, for epidemiological studies, and the Lovelace Respiratory Research Institute (LRRI) in Albuquerque, NM, for toxicological studies. The HEI released two reports (HEI Report # 177 on the NYU Study by Lippmann et al. (2013), and #178 on the UW-LRRI Study) in October 2013 that describe these two research programs and their findings, along with critical peer commentaries of the HEI NPACT Review Panel.

The NYU NPACT study The NYU NPACT study was designed to explore the associations between the various constituents of ambient air particulate matter (PM) and indices of health status. It focused on the roles of inhalation exposures to the various chemical constituents within the fine PM size fraction (PM2.5) and its sources on CVD responses in humans and laboratory animals. The overall concept of this NPACT study is illustrated in Figure 1, and its findings and discussions of their implications are described in detail in the HEI final report (HEI report no. 177, 2013). The studies were conducted on a national scale on the basis of substantial differences of source strengths and PM chemical composition within the US. Using CSN network data, the NYU NPACT team determined the extent of the variation of some major source contributions to ambient air PM2.5, as illustrated in Figure 2.

Figure 1. Integrated approach adopted for the NYU NPACT program.

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The NYU team (Ito et al., 2013) conducted time-series studies of short-term responses to the inhalation of ambient air PM2.5 for the populations residing in 150 US Metropolitan Statistical Areas (MSAs). In order to determine the influence of gaseous criteria pollutants (CO, NO2, and SO2), which were measured at only 64 of the CSN sites, the influences of simultaneous exposures to these gases and PM2.5 and its constituents, were determined, in these 64 US MSAs, in terms of daily variations in mortality and hospital admissions by cause. In addition, Chen and Lippmann (2013) conducted time-series studies of cardiac function in a mouse model of atherosclerosis (ApoE/) exposed to concentrated ambient PM2.5 (CAPs) for 6 h/day, 5 d/week over six months at five sites (four US cities and a regional upwind site to the northwest of NYC). The urban sites were Mount Sinai Medical Center (MS) in NYC, University of Washington in Seattle (SEA), Michigan State University in East Lansing (EL), and The University of California in Irvine (IR). The cumulative PM2.5 exposures of the mice over 6 months were similar in magnitude to those of the urban residents of the MSAs being studied in the time-series studies, as well as those studied as part of their study of the roles of PM2.5 constituents and their sources on annual mortality rates of the American Cancer Society Cohort II (ACS-II) by Thurston et al. (2013). This provided a basis for relating the chronic effects (aortic plaque progression) of the CAPs and CAPsconstituent exposures in the mice to the longevity reductions associated with long-term average PM2.5 constituent concentrations in the ACS-II cohort communities having CSN data. NYU’s epidemiological time-series and ACS cohort studies were limited to the PM2.5 particle size fraction since they depended on concentration data generated by EPA’s CSN. The same particle size limitation applied to the 6-month long CAPs inhalation studies in the ApoE/ mice, since PM larger than 2.5 mm in aerodynamic diameter do not penetrate to the lower respiratory tract in mice, and were, therefore, removed prior to the entry of the CAPs into the exposure chamber. These considerations limited the scope of three of their four substudies to the PM2.5 subfraction of the ambient air PM. The design of the NYU NPACT substudies was based, in part, on established knowledge demonstrating that health effects in humans have been associated with both larger and smaller particle size ranges. PM with aerodynamic diameters between 2.5 and 10 mm (PM10–2.5) penetrate beyond the upper respiratory tract and are efficiently deposited on the tracheobronchial airways within the human thorax, where they can cause airway irritation and exacerbate asthma. The ultrafine particles within PM2.5 (those smaller than 0.1– 0.2 mm), which contribute little mass to PM2.5, have also been of concern as causes of health effects on the basis that they dominate the number concentration and can cross epithelial members that are barriers to the penetration of larger particles. In order to begin to characterize the relative toxicities of the PM10–2.5, PM2.5, and PM0.2 particle size fractions, which differ in chemical composition and deposition sites as well as in size range, they collected high-volume samples of all three size fractions at the same five sites studied in the mouse inhalation studies. These samples were used in in vitro cellular exposures and in vivo lung aspiration


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Figure 2. Illustrations of PM2.5 source impacts on ambient air concentrations in ACS-II cohort communities.

exposures of short-term responses in order to study their associations with specific elemental constituents. The discussion that follows summarizes the key findings for each of the four NYU NPACT substudies, with special emphasis on the associations of CVD-related responses with specific elemental constituents and/or source categories, and then compares and contrasts the associations in the short- and long-term responses across substudies, while taking into account the species and endpoint differences in the various substudies. Table 5 provides a summary of some of the key NYU NPACT epidemiological studies’ findings to facilitate comparisons and integration with those of the earlier literature on the same issues that were presented in Tables 1–3. Table 6 provides a summary of some of the key NYU NPACT toxicological studies’ findings to facilitate comparisons and integration with those of the earlier literature on the same issues that were presented in Table 4. Table 7 summarizes the findings of the NYU NPACT studies by Gordon and Lippmann (2013) involving in vivo aspiration exposures in mice and in vitro exposures of vascular cells to PM10–2.5. PM2.5–0.2, and PM0.2

NYU NPACT epidemiological findings Time-series studies of daily hospital admissions and mortality based on PM2.5 chemical constituents, pollutant gases, and source-related mixtures Multi-city daily mortality analyses In addition to examining PM2.5 mass, its chemical constituents, and gaseous pollutants for their associations with the health outcomes, the NYU NPACT study also developed sourceoriented exposure indices by conducting factor analysis on the chemical constituents and gaseous pollutants in the 64 cities where measurements of both categories were available. In the

first stage of the analysis, Poisson time-series analysis regression models was fitted in each of the 150 (PM2.5 mass concentrations) and 64 (PM2.5, its constituents, gaseous pollutants, and factor analysis-derived source indices) cities to estimate percent excess risk for each pollutant at lag 0, 1, 2, and 3 d, adjusting for seasonal cycle, temporal trends, day of week, and immediate and delayed temperature effects. The risk estimates from the individual cities at each lag were then combined using a random effects model. The city-to-city variation in PM2.5 mass risk estimates across cities was modeled, in the second-stage analysis, as a function of cityspecific characteristics such as the average levels of PM2.5 chemical constituents, gaseous pollutants, land-use, traffic density, and port berth volume (i.e. indicator of emissions from marine vessels). The following bullets describe a qualitative summary of the findings, but see the HEI report for detailed method descriptions and summary tables of results that allow an assessment of consistency of associations with the health outcomes across individual constituents, season, and lag days. In the combined risk estimates from multiple cities, many more ambient air constituents were significantly associated with all-cause daily mortality in only the warm season (NO 3 , EC, Pb, and V) than in the cold season (OC), while some showed associations in both seasons (SO2, Cu, K, OC, and Si). Among the six source-related factor scores examined, the traffic, soil, and coal combustion factors showed significant associations with all-cause mortality in an all-year analysis. The results of the second-stage analysis to examine the influence of specific ambient PM2.5 air constituents on the risks associated with PM2.5 mass are shown in Figure 3. For example, for CVD hospitalizations, the cities with average high Cu concentrations tend to have higher PM2.5 risk estimates for CVD hospitalizations. Additional

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Table 5. Associations of PM components with health effects: CVD effects in humans constituents and/or sources associated with effects.


Short-term exposures Multi-city daily mortality Warm season NO 3 , SO2, EC, OC, Pb, Si, V Cold season SO2, OC, Si Both seasons SO2, Cu, K, OC, Si, and traffic, soil, coal combustion source factors Second stage analysis SO¼ 4 , V, seaport berth volume within 60 miles, sum of road lengths Multi-city daily CVD hospitalizations Warm season NO 3 , Na Cold season PM2.5, NO2, SO2, CO, Cu, EC, Fe, OC, SO¼ 4 , Se, Si, Zn, V Both seasons Traffic, Salt Second stage analysis Cu, Ni, V, SO2, extent of land-use development Long-term exposures American Cancer Society (ACS) cohort annual mortality All cause mortality Coal combustion source factor IHD mortality Coal combustion source factor, and to a lesser extent, traffic and salt factors Lung cancer mortal Coal combustion source factor

Table 6. CVD effects in mice constituents and/or sources most closely associated with effects. Short-term cardiac function responses in vivo (HR, SDNN, RMSSD) Mount Sinai4Sterling Forest (with 0 and 1-d lags) E. Lansing4Irvine4Seattle (with 2-d lags) Mount Sinai BC, Al, Mg, Na, Ni, P, V, Residual Oil Combustion, Secondary sulfate Sterling Forest OC, Cr, Cu, K, Mn, Pb, Zn, Secondary sulfate, Upwind Ni Refinery, Soil Seattle Soil, residual oil combustion, salt 5-Site regressions Ni (r2 ¼ 0.96), Al (r2 ¼ 0.81), EC (r2 ¼ 0.79), P (r2 ¼ 0.77), S (r2 ¼ 0.65), V (r2 ¼ 0.35) Aortic plaque progression over 6 months of daily CAPs inhalation exposures Plaque volume (Biomicroscopy): Mount Sinai ¼ Sterling Forest4E. Lansing. None @ Irvine & Seattle Plaque surface area (Visual Inspection @ Irvine and E. Lansing only): E. Lansing4Irvine

analyses that took land use regressions into account showed that, SO¼ 4 , V, the seaport berth volume within 60 miles, and the sum of road lengths were also significant predictors in explaining the variation of PM2.5 all-cause daily mortality risk estimates. The SO¼ 4 , V, the seaport berth volume within 60 miles are consistent with the site location being adjacent to the Port of Seattle Residual Oil combustion source, while the sum of the road lengths is consistent with the known influence of the Traffic source. Multi-city daily CVD hospitalizations analyses

In contrast to all-cause daily mortality, the first-stage analyses showed that the individual pollutants’ associations with CVD hospitalizations occurred mostly at lag 0d in the cold season. The pollutants associated with CVD hospitalizations at lag 0-d in the cold season were PM2.5, NO2, SO2, CO, Cu, EC, Fe, OC, SO¼ 4 , Se, Si, and Zn. V showed associations at lags 1- and 3-d, and also a nearly significant association at lag 0-d. Several pollutants with lag 0-d associations (NO2, SO2, CO, EC, and OC) also showed associations at lag 3-d. In the warm season, NO 3 (lag 0-d) and Na (lag 2-d) showed associations. The reasons for the differences in the associations of components with total daily mortality and CVD hospital admissions are not known at this time. The mortality events are fewer, precluding our ability to demonstrate CVD and respiratory causes separately, and respiratory mortality may be more closely associated with different PM2.5 components than CVD mortality. It is interesting that there were similar, and significant associations of PM2.5 mass

and SO¼ 4 concentrations with total daily mortality, as they were for annual mortality in our ACS cohort study of longterm mortality, and these similarities are consistent with the results of a large number of historic studies that showed that both PM2.5 mass and SO¼ 4 concentration measurements were significantly associated with excess mortality (Lippmann & Thurston, 1996). It is also notable that SO¼ 4, which had the strongest association with total daily mortality, was not even positively associated with CVD hospitalization. Among the six factor scores examined, only the traffic and salt factors showed significant associations in allyear analysis. In the second-stage analysis, Cu, V, Ni, Fe, NO2, and the extent of land-use development, were important positive predictors of the variation of PM2.5 CVD hospitalizations risk estimates across cities. The associations with Cu, Fe, land-use development, and NO2 are consistent with the influence of the Traffic source, while the associations with V and Ni are most likely due to their emissions from the Port of Seattle merchant ships.

Multi-city daily respiratory hospitalizations analyses These results are based on analyses of daily (as opposed to every-third-day or every-sixth-day in the nationwide speciation network) concentrations of PM2.5 components (see Appendix F on the HEI Web site and Zhou et al., 2011) through the HEI supplemental funding. The daily data allowed us to examine the effects of distributed lags for these city-specific data sets. Sub-categories of CVD and respiratory hospitalizations were examined in this analysis.



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Figure 3. The influence of specific constituents on the associations of PM2.5 mass concentrations on total daily mortality (left panel) and CVD hospitalizations (right panel) in the second-stage analysis in 150 US communities. From Ito et al. (2013).

In contrast to the combined estimates from the multi-city mortality analysis, Seattle mortality analysis showed associations between multiple PM2.5 components (Al, K, Si, Zn, EC, CO, and NO2) in the cold season. Consistent with the multi-city analysis, Detroit mortality analysis showed associations with PM2.5 mass and S in the warm month. Generally consistent with the multi-city results, the associations between CVD hospitalizations with PM2.5 components or gaseous pollutants were limited in the cold season both in Seattle (Fe, Ni, and V) and Detroit (K, S, EC, and NO2), and they were mainly driven by IHD hospitalizations. The multi-day risk estimates computed using distributed lag models in these two cities were generally larger than the individual day effect estimates, suggesting that risk estimates for individual lags may underestimate the effects. In contrast to CVD hospitalizations, the first-stage analyses showed that respiratory hospitalizations were associated with the pollutants in both the warm season (As, OC, and SO¼ 4 ) and in the cold season (Cu, EC, and Si), with PM2.5, CO, and K in both seasons. Among the six factor scores examined, only the traffic factor showed significant associations with respiratory hospitalizations.

Variations in hospital admissions and distributed lag risks of exposures to PM2.5 in Seattle and Detroit based on daily constituent concentration analyses [supplemental NPACT Study] These results, obtained through the HEI supplemental funding, are based on analyses of daily (as opposed to every-third-day or every-sixth-day in the nationwide speciation network) concentrations of PM2.5 components (see

Appendix F on the HEI Final Report on their Web site and Zhou et al., 2011). Having daily data allowed us to examine the effects of distributed lags for these city-specific data sets. Sub-categories of CVD and respiratory hospitalizations were examined in this analysis. In contrast to the combined estimates from the multi-city mortality analysis, Seattle mortality analysis showed associations between multiple PM2.5 components (Al, K, Si, Zn, EC, CO, and NO2) in the cold season. Consistent with the multi-city analysis, Detroit mortality analysis showed associations with PM2.5 mass and S in the warm months. Generally consistent with the multi-city results, the associations between CVD hospitalizations with PM2.5 components or gaseous pollutants were limited in the cold season both in Seattle (Fe, Ni, and V) and Detroit (K, S, EC, and NO2), and they were mainly driven by IHD hospitalizations. The residual oil combustion effluents in Seattle were attributable to the seaport operations, while the coal combustion effluents in Detroit were attributable to power plant combustion, both of which were relatively independent of season. The multi-day risk estimates computed using distributed lag models in these two cities were generally larger than the individual day effect estimates, suggesting that risk estimates for individual lags may underestimate the effects.

American Cancer Society (ACS) cohort study of annual mortality associations with long-term exposure

A factor analysis of the 2000–2005 nationwide EPA CSN data for PM2.5 identified major elemental groupings, interpretable as being associated with specific PM2.5

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source categories, at many locations around the US, including 167 sites in 102 metropolitan statistical areas (MSA’s). The major source categories identified and their key elements were metals industry (Pb, Zn); soil (Ca, Si); motor vehicles (OC, EC, NO2); steel industry (Fe, Mn); coal combustion (As, Se); oil combustion (V, Ni); and, salt (Na, Cl). Inspection of spatial plots of the factor analysis scores (i.e. source-related impacts) indicated that motor vehicle impacts were highest in Southern California; soil impacts were highest in the Southwest; steel impacts were highest in cities with steel works (e.g. Detroit, MI and Birmingham, AL); coal combustion impacts were highest in the Ohio River Valley region (e.g. Pittsburgh, PA); and residual oil burning impacts were highest in cities with wintertime residual fuel oil burning (e.g. New York City [NYC]) or having deep water ports (e.g. Los Angeles and Long Beach, CA; Savannah, GA, and Newark, NJ-NYC, NY), consistent with impacts by emissions from oceangoing ships burning highly polluting ‘‘bunker fuel’’. The analysis reveals the same major U.S. source factors and spatial distribution as those previously reported for the 1979–1983 IP Network (Ozkaynak & Thurston, 1987), albeit at lower levels than in the earlier period, indicating a qualitative consistency in the spatial representativeness of these results over the past thirty years. Individual risk factor data for approximately 446 000 adults collected by the ACS cohort from 100 MSAs across the US were linked with fine PM2.5 mass, trace element, and source factor exposure data throughout the US. The strongest PM2.5, trace element, and source factor associations were found for IHD and lung cancer deaths (LCD), consistent with past ACS analyses (Pope et al., 1995, 2002). All cause mortality (ACM) analyses indicated that PM2.5 and the trace elements associated with coal combustion (i.e. Se, As, and S) where most significantly associated with increased risk of death. The source-specific PM2.5 source mass apportionment associations with ACM deaths were largely consistent with results derived when considering individual trace constituents, as the most consistent source-related ACM associations were with the coal combustion-related source factor and coal combustion PM2.5 mass contributions. As shown in Figure 4, the inclusion of contextual variables did not change the rankings or statistical significance of the various source-related contributions to annual IHD mortality, meaning that they did not confound the associations. IQR’s were used to normalize the exposure variable the various RR’s to make them more comparable (so the effect size is not dependent on the relative concentrations of the source impacts). The IQR for each is provided on the figure, so the reader can convert (for example) to a per mg/m3 effect size. While PM2.5 pollution from most industrial and fossil fuel combustion categories had RR estimates above 1.0 for IHD deaths, coal combustion PM2.5, and its correlated trace elements (e.g. As, Se, S) were most strongly and consistently associated with IHD mortality across all the various model specifications considered. Traffic PM2.5

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and a key elemental tracer (EC) were also associated with IHD in some models. PM2.5 mass originating from windblown soil or from biomass burning (e.g. wood combustion) was generally not associated with increased risk of IHD mortality in this cohort, while associations with other sources were more equivocal across models. Respiratory mortality was most significantly associated with long-term exposure to secondary OC, but not with any specific source. Only the coal burning source factor and two of its associated trace elements (Se, S) were significantly associated with increased risk of death from lung cancer. Source-specific PM2.5 mass associations with health impacts are consistent with, but more easily interpreted than, result when considering individual trace constituents coming from a variety of sources. Overall, modeling results from NYU’s main analyses indicated that long-term exposures to PM2.5 from one key source, and its elemental tracers, were most explanatory of the PM2.5–mortality associations found in past ACS cohort studies that were limited to PM mass and sulfate concentrations. In particular, the coal combustion source factor was most consistently associated with increased risk of IHD mortality across models considered (i.e. models with and without random effects, and with and without contextual variables). Meanwhile, the soil or wood combustion source factors were consistently not associated with any causes of mortality across models considered. The traffic and salt source factors, and especially their respective tracers, EC and Cl, also showed significant associations with IHD mortality in some models. Associations between PM2.5 and lung cancer mortality were also displayed by the coal combustion source factor and its tracers, Se and S, but not by other components of PM2.5. This is a particularly important finding in light of the major health benefits associated with reductions in annual mortality that have been attributed to reductions of long-term PM2.5 exposures through the implementation of the Clean Air Act. In a supplemental NYU NPACT analysis, the total RR impacts (TRIs) based on the source-related factors (and especially with secondary PM2.5 constituents also included) were generally larger than the TRI based on PM2.5 mass alone, suggesting that the source-specific information allows for a more accurate exposure and risk estimate, and that past estimates using non-specific PM2.5 mass have provided underestimates of the total PM2.5 mortality effect. The TRIs including the secondary PM2.5 constituents (SO¼ 4 , NO3 , OC) tended to be somewhat larger than the directly comparable TRI excluding the secondary PM2.5 constituents, providing evidence consistent with a contribution by secondary PM2.5 constituents to PM2.5 associations with mortality. Furthermore, the evidence from the TRI analysis of an association for mortality with any of the three gaseous pollutants examined over and above that associated with PM2.5 mass (and its constituents) was generally weak, supporting the hypothesis that the chronic health effects of ambient air pollution are primarily due to constituents of PM2.5. However, such inferences are somewhat constrained at this time, due to the still limited number of MSAs having sufficient data on the ambient air concentrations of both PM2.5 constituents and criteria pollutant gases.

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Figure 4. Influence of source-related contributions to PM2.5 mass on annual all cause mortality in ACS II cohort members in 100 US communities with (closed circles) and without contextual variables (open circles). From Thurston et al. (2013).

Toxicological findings in NYU NPACT study Subchronic mouse inhalation exposures There were substantial PM2.5 mass mean concentration differences in the exposure chambers at the five sites, with two ‘‘cleaner’’ sites (SEA @ 61 mg/m3, and EL @ 68 mg/m3), and three sites having about twice as much PM2.5 mass (MS @ 123 mg/m3, IR @ 138 mg/m3, and SF @ 136 mg/m3). There were some much greater mean PM2.5 constituent concentration differences within the exposure chambers at the five sites. For example, S ranged from 3.8 mg/m3 @ SEA to 11.3 mg/m3 @ SF; BC ranged from 0.53 mg/m3 at EL to 2.67 mg/m3 @ MS; Fe from 0.30 mg/m3 @ EL to 1.88 mg/m3 at MS; Mn from 14.2 ng/m3 @ SF to 107 ng/m3 @ MS; Zn from 51.3 ng/m3 @ EL to 760 ng/m3 @ MS; Cu from 5.4 ng/m3 @ EL to 100.5 ng/m3 @ IR; Se from 6.0 ng/m3 @ SEA to 24 ng/m3 @ IR; V from 17.1 ng/m3 @ SF to 45.7 ng/m3 @ IR; and Ni from 6.6 ng/m3 @ EL to 69.9 ng/m3 @ MS. The substantial variation in constituent concentrations at these five sites made it possible to explore their individual contributions to a range of health effects. Cardiac function responses In terms of CAPs concentrations examined for three cardiac function indices (HR, SDNN, and MSRRD); three different lag-days; and four different times-of-day, there were 56 statistically significant differences in cardiac function indices between CAPS- and FA-exposed mice at MS and 38 such responses at SF, while there were only six at the EL site, only five at IR, and only three at the SEA site. The statistically significant responses that were tabulated could be either positive (þ) or negative () for day- and time-interval, as functional cardiac change could be accelerated or retarded depending on applied dose. We do not consider bi-directional physiological changes to necessarily represent either ‘‘adverse’’ or ‘‘beneficial’’ effects per se. For example, inhalation exposure to irritants such as cigarette smoke or

sulfuric acid produces accelerations in tracheobronchial mucociliary particle clearance at low levels of exposure, and slowing of clearance at higher exposure levels (Leikauf et al., 1984; Lippmann & Schlesinger, 1984). Many, if not most, such changes may be adaptive, and may only contribute to cumulative ‘‘adverse’’ effects upon long-term exposure. As noted, there were much larger numbers of statistically significant functional changes at the sites in the northeastern US (MS and SF) than at the other sites, suggesting that components of the northeastern regional secondary aerosol, which were absent at IR, and SEA, and much lower at EL, were the most efficacious at producing short-term cardiac function responses. It is also notable that the CAPs mass concentration at IR was about twice that at EL and SEA. We could have controlled for PM2.5 mass concentration, but chose not to because our hypothesis was that responses would be affected more by the concentrations of PM2.5 components than by total mass. At the same time, since we felt that it was premature to focus on any one component or source-related mixture, we relied on regression analyses for multiple components to identify the most influential components. The concentration of CAPs was included in our model analysis, and the changes seen in HR/HRV were expressed as change in beats/minute per mg/m3. The statistically significant functional changes at the IR, EL and SEA sites were mostly at the 2-d lag. In contrast, the much larger numbers of functional changes at the MS and SF sites were at 0 - and 1-d lags. This is consistent with the hypothesis that higher levels of exposure result in significant differences in responses being seen at earlier times after exposure. There were nearly equal numbers of functional changes during the four time periods of the exposure day at all five sites, albeit with many fewer ‘‘hits’’ at EL, SEA, and IR than at MS and SF. The number of constituents having statistically significant (p 0.05) associations with functional responses was not in proportion to CAPs mass concentration. For example, MS with CAPs ¼ 123 mg/m3 had 56 significant functional


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responses (39þ & 17) between CAPs- and FA-exposed mice, while IR with CAPs ¼ 138 mg/m3 had just five such responses (2þ & 3). In terms of individual PM2.5 constituents, there were many more functional change ‘‘hits’’ at MS than at SF for BC, Al, Mg, Na, Ni, P, and V, while there were many more functional change ‘‘hits’’ at SF than at MS for OC, Cr, Cu, K, Mn, Pb, and Zn. There were highly correlated groupings of elements at MS and SF, the sites showing the most exposure-response relationships for cardiac function responses. Groupings included Al, Si, and Ti, which are derived from soil; and Br, Se, P, and S, which are derived from coal combustion. At MS, Ni was closely associated with V, S, and EC, which are derived from residual oil combustion (Peltier & Lippmann, 2010), while at SF, Ni was associated with Cr and Fe, which, along with Ni, have been associated with stack effluents from the distant upwind Sudbury, ONT., Ni smelter (Lippmann et al., 2006). For 21 single component concentrations examined for three cardiac function indices for three lag-days and four times-of-day, there were about three times as many significant differences in cardiac function indices between CAPS- and FA-exposed mice at MS and SF than at the EL and SEA sites, and more than two times as many at the IR site. As shown in Table 8, 5-site regressions of mean component concentrations with number of positive and significant responses in terms of indices of cardiac function (HR, SDNN, and RSSMD), Ni (r2 ¼ 0.96), Al (r2 ¼ 0.81), EC (r2 ¼ 0.79), P (r2 ¼ 0.77), S (r2 ¼ 0.65), and V (r2 ¼ 0.35) indicated positive exposure-response relationships. Others had negative and less consistent exposure-response relationships; these were Se (r2 ¼ 0.19), K (r2 ¼ 0.13), and Zn (r2 ¼ 0.27). Some elemental concentrations (Cu, Si, Fe, Mg, Mn, and Pb) showed less consistent and fewer exposure-response relationships with the cardiac function indices, with both positive and negative associations for different lag-days and times-of-day. There were strong associations of cardiac function effects (changes in HR, SDNN, and RSSMD) for at least one source category at each of the five sites. The most influential source category differed at each site, with the strongest signals for soil and a moderate signal for residual oil combustion in SEA, strong signals for residual oil combustion and secondary sulfate at MS, with strong signals for secondary sulfate and a moderate signal for a distant upwind Ni-refinery effluent at SF. The sources associated with Ni sources were either the first or second strongest at MS, SF, and SEA, which had the highest Ni concentrations, and were less influential at EL, which had a much lower Ni concentration. Two source categories that are not generally considered as likely causal factors were among those associated with cardiac function changes, i.e. soil in SEA and SF, and salt in SEA. Aortic plaque progression Table 9 summarizes the long-term plaque progression in the brachiocephalic (BA) artery in terms of plaque volume, as measured by UBM. It varied by site location, with substantial plaque volume progression at MS and SF in NY (with the highest secondary sulfate and Ni concentrations). There was less, but still statistically significant plaque volume progression at EL, but none in SEA and IR.

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In supplemental measurements of plaque surface area by visual inspection in mice sacrificed after 2, 4, and 6 months of CAPs or FA exposure at IR, there was a progressive rise in plaque surface area over time. Comparable measurements were made for mice sacrificed after 6 months of exposure at EL, and the plaque surface areas attributable to CAPs exposure were greater than those for the IR mice. Unfortunately, such assays were not performed at the ends of earlier CAPs inhalation studies at MS, SF, and SEA.

In vivo aspiration exposures in mice and in vitro exposures of cells to PM10–2.5. PM2.5–0.2, and PM0.2 Particle size range, season and site, were significant factors influencing intra-cellular ROS levels in vitro. PM10–2.5 elicited the greatest PMN response in FVB/N mice, regardless of site. In vitro ROS production did not predict in vivo lung inflammation. Metals associated with the traffic, coal combustion, and residual oil combustion sources were significantly correlated with in vitro responses, while components associated with traffic and endotoxin showed significant associations with the in vivo responses. Specific elements that were highly correlated (p50.001) with in vitro ROS production in vascular endothelial cells were Cu, Sb, K, Sr, V, Fe, Co, Be, Ti, Ca, Sc, Mg, Ni, and P. Specific elements that were highly correlated (p50.001) with ROS production in airway epithelial cells were: Cu, Sb, V, Co, Be, and Ni. Specific elements that were strongly correlated (p50.01) with mRNA levels in airway epithelial cells were: Cu, K, Ni, Sr, and V for CSF-2; As, Cr, Cu, Mn, Sb, Sn, Sr, Ti and Tl for HO-1; Ca, Cr, Cu, S, Sb, Sc, Sr, Fe, Mn, Ni, Yi, Ti and V for IL-6; Cu, Mn, and V for IL-8; and As, Cu, Cr, Fe, K, Mn, Sr, and Ti for VEGFa. Specific elements that were highly correlated (p50.01) with mRNA levels in vascular endothelial cells were: Cu, Sb, Fe, Co, Ti, Be, Ni, Mn, and Cr for HMOX-1; Sr, Co, Be, Mn, and Tl for ICAM-1; Co, Be, Ni, Pb, and Tl for IL-8; Co, Be, Ni, Pb, and Tl for TXNRD1; and K, Co, Be, Ni, and Pb, Tl for VEGFa. Some PM samples that were aspirated into the lungs of ApoE/ mice induced significant effects on spontaneous in vivo beat frequency. These included winter samples from Manhattan in the fine and ultrafine particle size ranges, which had unusually high concentrations of S and Zn.

Integration and interpretive observations among the NYU NPACT substudy findings Opportunities for integration of responses to PM2.5 and its constituents in human and animals in vivo were limited to cardiovascular effects, since no data were generated on respiratory system responses or lung cancer in these toxicological studies. In other subchronic inhalation studies in mice, conducted in collaboration with colleagues at other research laboratories, data were generated on the cumulative effects of prolonged exposures to CAPs on other organs (brain and liver) and on fat metabolism and metabolic syndrome, but, for such effects, there are no comparable data for human populations.


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Table 7. In vivo aspiration exposures in mice and in vitro exposures of vascular cells to PM10–2.5. PM2.5–0.2, and PM0.2. Metals associated with the traffic, coal combustion, and residual oil combustion sources were significantly correlated with in vitro responses, while components associated with traffic and endotoxin showed significant associations with the in vivo responses Specific elements that were highly correlated (p50.001) with in vitro ROS production in vascular endothelial cells were Cu, Sb, K, Sr, V, Fe, Co, Be, Ti, Ca, Sc, Mg, Ni, and P Specific elements that were highly correlated (p50.01) with mRNA levels in vascular endothelial cells were Cu, Sb, Fe, Co, Ti, Be, Ni, Mn, and Cr for HMOX-1; Sr, Co, Be, Mn, and Tl for ICAM-1; Co, Be, Ni, Pb, and Tl for IL-8; Co, Be, Mn, Pb, and Tl for TXNRD1; and K, Co, Be, Ni, and Pb for VEGFa Some PM samples that were aspirated into the lungs of ApoE/ mice induced significant effects on spontaneous in vivo cardiomyocyte beat frequency. These included winter samples from Mount Sinai in the fine and ultrafine particle size ranges, which had unusually high concentrations of S and Zn Table 8. Regressions of PM2.5 constituent concentrations with changes in cardiac function indices at the five sites with subchronic CAPs inhalation exposures (MS, SF, SEA, EL, and IR). Pollutant Ni Al EC P S V Mg Zn CAPs Se Pb Ca K Mn OC Ti Na Br Fe Si Cr Cu

Slope

r-Square

Correlation rank

0.91 7.18 18.60 2.18 183.35 0.34 13.54 15.09 0.74 0.15 6.11 10.62 1.86 1.36 48.85 0.51 23.57 0.16 15.24 4.52 0.12 0.14

0.96 0.81 0.79 0.77 0.65 0.35 0.30 0.27 0.23 0.19 0.18 0.18 0.13 0.11 0.08 0.08 0.06 0.03 0.02 0.02 0.02 0.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Table 9. % of Long-term plaque progression in the brachiocephalic (BA) artery in terms of plaque volume, as measured by ultrasound biomicroscopy (UBM) was greater for ApoE ¼ mice exposed to CAPs for 6-months at the three sites with PM2.5 from coal combustion (MS, SF, and EL) than at the two sites without (SEA and IR). Site

Air sham control

CAPs exposure

p Value

MS SF SEA EL IR

32.2 5.9 23.3 8.8 47.0 6.6 23.4 4.5 29.0 3.1

40.2 9.0 34.2 8.8 46.4 4.7 27.8 4.5 29.4 4.4

0.03 0.02 0.80 0.03 0.77

Roles of PM2.5 constituents on mortality and hospital admissions The PM2.5 components that were significantly associated with excess risks varied substantially between short- and long-term effects, and within each with season, city, and disease category.

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One of the most remarkable findings of the NYU NPACT study was that the PM2.5 components that that were most closely associated with the short-term health effects were different from those associated with the long-term effects, with transition metals and traffic markers being associated with daily mortality and hospital admissions, and coal combustion components being associated with annual rates of IHD and lung cancer mortality. Another notable finding was that within the short-term effects, there were differences in the PM2.5 constituents associated with total daily mortality and CVD hospital admissions. In the second stage time-series analyses, Cu, Ni, and V were significantly associated with CVD hospital admissions while SO¼ 4 , Se, As, OC, and EC were not. In contrast, total daily mortality was significantly associated with SO¼ 4 , with positive, if not significant associations with Se and As. This suggests that the coal combustion source may be responsible for excess short-term mortality as well as excess annual mortality. However, the comparison may be inappropriate, since the coefficient for CVD daily mortality could not be determined due to the study’s limited sample size. When there were associations of excess risks of multiple PM2.5 constituents that were present at relatively low mass concentrations, the magnitudes of the risks, per IQR for the measured constituents, were usually similar to those of total PM2.5, or those of S and EC, which were present at relatively large mass concentrations. This helped to explain why these most commonly measured indices of PM pollution (PM2.5 S, and EC) have often been useful markers of short-term public health risks. To the extent that specific constituents present at relatively low concentrations are most causal for specific health endpoints, the implication is that the concentrations of total PM2.5, S and EC, as well as the specific constituents, often rise and fall together due to meteorological factors. When there were no associations of excess risks of both total PM2.5 and multiple PM2.5 constituents on the magnitudes of the risks, per IQR, it suggests that causal PM2.5 constituents were too low, at those times and places, to cause measurable effects. The epidemiological associations are often driven by responses to elevated concentrations on a relatively few days with little or no measurable response on a majority of days. The specific PM2.5 constituents that were most often significantly associated with excess risks were markers of fossil fuel combustion, e.g. EC from traffic, Se from coal combustion, V from residual oil combustion, and K from biomass combustion and a variety of other sources. The frequent association of K with excess morbidity and mortality was unexpected, and needs further investigation of (1) its possible role in causing the effects or (2) as a marker of more toxic co-pollutants. The ambient air K concentrations appeared to be too high, in most cities, to be primarily attributable to wood burning, and may be due to sources associated with soil PM. PM2.5 components that are not considered to be markers of combustion were also frequently significantly associated with hospital admissions and/or daily mortality. These included Al and Si (associated with Soil), and Na (associated with Salt).


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The largest single day excess risks are typically for hospital admissions associated with EC, and to a lesser extent with OC, at 0-day lag, with little or no excess at lag days 1 and 2. This suggests short-term responses to components or surface coatings of carbonaceous particles. The largest distributed-lag excess risks were typically for hospital admissions associated with the inorganic elements, with relatively little decrease in excess risk over several later lag days. These components appear to be more closely associated with persistent CVD responses. The excess CVD risks were mostly attributed to IHD and heart failure, while the excess respiratory disease risks were most often attributed to COPD and/or acute respiratory failure. Characterizing risks in terms of total CVD or total respiratory categories tends to obscure the effects related to more specific disease subcategories.

Roles of PM2.5 constituents on toxicological responses The PM2.5 components that were significantly associated with excess risks in the mice also varied substantially between short- and long-term effects, and with site. As for the epidemiological findings of the NYU NPACT study, the PM2.5 components that that were most closely associated with the short-term health effects were different from those associated with the long-term effects, with transition metals and traffic markers being associated with daily changes in cardiac function, and coal combustion components being associated with aortic plaque progression, suggesting that the coal combustion source may be responsible for a range of chronic health effects When there were no associations of excess short-term risks of both total PM2.5 and multiple PM2.5 constituents on the magnitudes of the risks, per IQR, it suggests that causal PM2.5 constituents were too low, at those times and places, to cause measurable effects. The specific PM2.5 constituents that were most often significantly associated with excess short-term cardiac function changes were markers of fossil fuel combustion, e.g. Ni and V from residual oil combustion, S from coal combustion, EC from traffic, and Al from soil. We cannot be sure if the effects were due to the elemental markers of the sources, to other source constituents, or to the combined effects of multiple components of the ambient air mixture.

Overall interpretive observations on the roles of PM2.5 constituents on health-related responses For both our epidemiological and toxicological studies, there were PM2.5 components and source-related mixtures that were significantly associated with measured indices of health status that were stronger than those for PM2.5 mass concentration. There were also other component concentrations that were less strongly associated with these indices of health status, or had negative associations. The positive associations could have been due to causality, or perhaps to ambient air concentration correlations with a causal agent. The generally non-significant negative associations could have been due to health benefits arising from the exposures to those agents, but were likely due

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to random influences, limited statistical power, analytical uncertainties, and exposure measurement errors. Thus, while these substantial studies could not, and did not, establish causality for specific PM2.5 components, they were able to identify components that warrant further, more detailed, and better focused study as contributory factors affecting healthrelated outcomes and their underlying biological mechanisms.

Coherence of toxicological responses to PM2.5 exposures The 5-site regressions of mean component concentrations with the most significant responses in terms of indices of cardiac function in ApoE/ mice (HR, SDNN, and RSSMD) were Ni (r ¼ 0.98), S (r ¼ 0.88), and V (r ¼ 0.66), and these elements were among those most closely associated with ROS production and with mRNA levels in vascular endothelial and airway epithelial cells in vitro. The winter samples from Manhattan in PM2.5 and PM0.2 particle size ranges that were aspirated into the lungs of mice induced significant effects on spontaneous in vivo HR in cardiomyocytes. Such samples are enriched in residual oil combustion effluent. These results are consistent with the finding that MS CAPs affected cardiac function during our 6-month CAPs inhalation exposure study to a much greater extent than at other sites that had much lower Ni concentrations. Coherence of short-term CVD responses of humans and mice to PM2.5 constituent exposures The observation that among the CVD daily mortality and hospitalizations associated with human exposures to ambient air PM2.5, the largest excess risks were for IHD, is coherent with the observations of (1) acute cardiac function changes in mice exposed 5-d each week by inhalation to PM2.5 CAPs; (2) increased HR in mice exposed in vivo by aspiration of Manhattan fine and ultrafine PM; and (3) increased ROS production and mRNA levels in endothelial and epithelial cells in vitro that were exposed to fine and ultrafine PM. Transition metals were PM2.5 constituents that were closely associated with the cardiac responses seen in the NYU human time-series studies, as well as in their studies of mice exposed to PM2.5 in vivo, and cells exposed to PM2.5 in vitro. The association of short-term variations of cardiac function in mice with the residual oil combustion source in Seattle is coherent with the findings of the NYU NPACT supplemental study that compared daily mortality and hospitalization rates in Seattle and Detroit. Zhou et al. (2011) described significant associations of excess daily mortality and hospital admissions with daily variations of PM2.5 components for residents of Seattle that were not found for residents of Detroit, a considerably larger city with much higher annual average PM2.5 concentrations (15.1 versus 9.1 mg/m3), and one with much higher percentages of minority groups, lower income groups, and elderly residents, such population groupings being generally considered to be especially susceptible population subgroups for air pollutionrelated health effects.


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Coherence of CVD hospitalization in people with cardiac function changes in ApoE/ mice Any direct comparison can be challenged on the basis of (1) species difference and (2) severity of response, i.e. modest functional response versus a clearly adverse health effect. On the other hand, we were able to detect functional responses in an animal model of atherosclerosis. On this basis, an integrated consideration of these two very different short-term responses to PM2.5 exposures has some justification in terms of identifying PM2.5 constituents responsible for short-term cardiovascular responses. For other short-term human responses seen in NYU’s time-series analyses, such as hospital admissions for respiratory diseases, or for total mortality, there were no measurable clinical responses in the mice. NYU’s comparison between cardiac function change in mice and IHD hospitalization in humans was limited to the comparisons of the PM2.5 constituents in regard to possible causality. In this regard, they showed that the PM2.5 constituents most closely associated with cardiac function changes in the mice for five different sites were Ni, EC, S, and V. They also showed that the PM2.5 constituents most closely associated with hospital admissions in 64 US MSAs, lagged 3 d for IHD, were EC, OC, V, and Ni. For the cold season in Seattle, there were significant excess distributed lag risks for IHD hospital admissions for Al, Fe, Ni, S, Si, V, Zn, and EC. The component correspondence for the humans and mice was close, but not perfect. Common to all three analyses are EC, Ni, and V. The influences of the other components that showed up in these three different analyses is likely due to the differences in the PM2.5 mixtures in the MSAs that were studied, varying from 150 or 64 in the national time-series analysis, to 5 in the mouse inhalation studies, and to one season in one city in the daily time-series study in Seattle. It seemed reasonable to conclude that, for short-term effects related to IHD (1) the effluents of residual oil combustion (Ni, V, S, and EC) are particularly influential; (2) other combustion source categories that have generated tailpipe EC and S are also important; and (3) the presence of other constituents of PM2.5 may exacerbate the effects of the most influential constituents. In a previous study relating HR in COPD patients to coarse and fine PM constituents in New York City (NYC) and Seattle, the only component that had a significant association was Ni, but only in the PM2.5 fraction in NYC, a city with notably high concentrations of Ni, V, S, and EC (Hsu et al., 2011). Coherence of annual mortality associations with PM2.5 and its constituents with aortic plaque progression in ApoE/ Mice The NYU analyses of the ACS cohort data identified coal combustion, and to a lesser extent, traffic as the source categories that were most closely associated with excess annual IHD mortality. In addition, strong associations were also found for LCD, consistent with past ACS analyses (Pope et al., 2002). Long-term plaque volume progression in the bracheocephalic artery of CAPs-exposed ApoE/ mice exposed to CAPs versus those exposed to FA varied by site location, with a significant difference in plaque at 6 months in mice at MS (40 for CAPs versus 32% for FA) and SF (34 versus 23%) and

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at EL where Ni concentrations were the lowest of the five sites (28 versus 23%). There was no progression at all in SEA or at IR (where sulfate was next to lowest, but PM2.5 mass was highest). Since the PM2.5 mass concentration in EL was only about half of that in IR, and the contribution of the Traffic source to local PM2.5 was much lower in EL, it appears that the PM2.5 attributable to coal combustion was more influential on plaque volume progression in the mice than those from all other sources, including traffic and residual oil combustion. Supplemental analysis of plaque surface area coverage, by visual inspection in mice after 2, 4, and 6 months of exposure at IR, provided evidence of plaque area progression, but the extent of progression at 6 months was less than for mice exposed at EL.

Short-term exposure to PM2.5 and daily mortality and hospital admissions (NYU NPACT only) There were generally close associations of daily mortality and hospital admission rates with exposure to traffic-related effluents (EC, OC, and road dust), and with one or more transition elements (Fe, Ni, Zn, V, Cu, Cr) in the most of the prior short-term exposure studies listed in Table 3. In Table 5, summary of the short-term mortality and hospital admissions findings of the NYU NPACT study indicates that the only significant associations were (1) with the traffic factor and with constituents associated with traffic, such as EC, OC, Cu, and Pb, and with those associated with re-suspended road dust (Si and Fe). However, in the secondstage analyses, which identified the PM2.5 constituents that have the greatest influence on the associations of PM2.5 mass concentrations with short-term health effects, and account for pollutant concentration correlations, the pollutants and other variables that most closely influence the cardiovascular hospital admissions include residual oil combustion components (Ni, V, S, and seaport berth volume), as well as trafficrelated factors (Cu and road lengths), with SO¼ 4 showing a non-significant negative association. There was little evidence for an association of hospital admissions with markers for coal combustion effluents (SO¼ 4 , As, and Se). On the other hand, for the second stage analysis for daily mortality, SO¼ 4 was the most influential constituent, and Se and As showed positive, albeit not significant associations. In sum, these findings suggest that, even for short-term peak exposures, there are differences in the components causing hospital admissions (OC and transition metals) and daily mortality (coal combustion effluents).

University of Washington (UW) – Lovelace Respiratory Research Institute (LRRI) NPACT study Introduction The UW part of the UW-LRRI NPACT study (Vedal et al., 2013) was limited to the cardiovascular effects of chronic exposures of ambient air PM2.5 in two human cohorts by the UW investigators; i.e. the Multi-Ethnic Study of Atherosclerosis (MESA), which was focused on subclinical measures (Carotid Intima-Media Thickness [CIMT] and Coronary Artery Calcium [CAC]), and the Women’s Health Initiative (WHI), which was focused on cardiovascular events


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(CVD mortality, hospital admissions, and other events, such as MI and coronary revascularization). The MESA study was conducted in six US cities, i.e. New York City; Baltimore, MD; Winston-Salem, NC; Chicago, IL; St. Paul, MN; and Los Angeles, CA. The WHI was conducted in 45 US cities; 8 in the Northeast, 11 in the Southeast, 13 in the Midwest, 3 in the Southwest, 4 in California, and 6 in the Northwest. For both cohorts, the exposures at the individual cohort member level were estimated using a national spatial model using 2009 chemical concentration data from the US EPA’s Chemical Speciation Network (CSN) for population centers and the IMPROVE network for ‘‘background’’ sites. For the MESA cohort, a more complex spatio-temporal model was developed that utilized 2-weeklong UW sampling measurements at 3–7 sites in each region, as well at 50 home sites in each of the two seasons on a rotating basis. The primary measurements were the concentrations of EC, OC, S, and Si, on the basis of them being major components of overall PM2.5 mass. These four PM2.5 components were also considered to represent major PM2.5 sources, with EC representing primary emissions from fossil fuel and biomass combustion, OC representing secondary organic particles in addition to primary fossil fuel emissions, S representing secondary inorganic particles and Si representing soil-derived particles. The concentrations of selected PM2.5 constituents that contributed much smaller amounts to PM2.5 mass were considered to be of secondary interest (Ni, V, Cu) and were investigated for their associations with measured indicators of health-related responses, along with SO¼ 4 , NO3 , SO2, and NO2. The LRRI part of the UW-LRRI NPACT study was focused on the cardiovascular responses in an animal model of human atherosclerosis (ApoE/ mice). The mice were studied in groups of 8–10. They were fed HF chow and were exposed by inhalation for 6 h/d for 50 d to 100 and 300 mg/m3 of selected PM2.5 components found in ambient air. The eight different exposure atmospheres consisted of (1) diluted mixed motor vehicle (tailpipe) emissions (MVE), containing 50 mg/m3 of PM2.5 from a spark ignition engine and 250 mg/m3 from a diesel engine, as well as the pollutant vapors in the exhaust stream; (2) filtered (particle free) diluted mixed motor vehicle tailpipe emission gases (MVEG); (3) 300 mg/m3 of resuspended road dust; (4) 300 mg/m3 of 3 ¼ SO¼ of NO 4 ; (5) 300 mg/m 3 ; (6) MVEG þ SO4 ; (7) MVEG þ NO3 ; and (8) MVEG þ road dust. The differences in cardiovascular biomarker responses that were measured were: lipid peroxidation (TBARS); plaque growth; plaque inflammation; vascular gelatinase activity; NO pathway components; MMP expression; vasoconstriction; and oxidized LDL. The selection of the eight different pollutant mixtures provided a basis for determining the effects of specific PM2.5 components that account for most of the PM2.5 mass (traffic and stationary fossil fuel combustion), and the extent to which fossil fuel combustion-related gases and vapors may enhance the toxicity of the airborne PM. Results MESA cohort For CIMT, there was a statistically significant association for PM2.5 mass concentration that was similar to those for EC and

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Si, with substantially larger effects for SO¼ 4 and OC when using their spatiotemporal exposure model to account for differences in exposure within each community. The associations they found when using their national exposure model were essentially the same for SO¼ 4 , OC, and Si, and there was no association for EC. In their analyses of the associations of other measured PM2.5 components on CIMT, the only one approaching significance was Cu. Based on the UW source apportionment, none of the four PM2.5 components of primary interest in the epidemiologic analyses seemed to be influenced largely by MVE. Of these components, EC has traditionally been used as a marker of exposure to MVE (specifically diesel exhaust). The source apportionment indicated that their EC measure reflected a complex mix of sources, although the diesel exhaust/brake wear-like feature contributed to EC to some degree in every MESA city, with contributions ranging from 6% to 36%, depending on the city. To the extent that EC was an indicator of MVE exposure, the MESA cohort study did not find much support for a role for MVE in atherosclerosis or in cardiovascular events. Other potential markers of exposure to MVE were NO2 and Cu. The source apportionment indicated that the diesel exhaust/brake wear-like feature also contributed to NO2, arguably to a greater extent than to EC, with contributions ranging from 1% to 46% across the MESA cities. Because NO2 (along with NO 3 ) was of secondary interest, their health analyses of NO2 were completed only in the MESA cohort and then only using the spatiotemporal model exposure predictions. In those analyses, UW found little evidence that NO2 was associated with their endpoints. To the extent that exposure to MVE was reflected by either EC or NO2, and these are not particularly good markers of MVE exposure, the epidemiologic studies found little evidence to support a role for MVE. Cu, however, might be a better marker of exposure to MVE than either EC or NO2, with contributions from the source apportionment ranging from 32% to 57%. The UW epidemiologic findings for Cu, although limited in scope, suggested that exposure to MVE could be important in the development of atherosclerosis. For CAC, there were no associations for any of the PM components for either exposure model, except for OC when using the national exposure model. WHI cohort For cardiovascular disease (CVD) events and CHD events, there were statistically significant and comparable hazard ratios (HRs) for PM2.5 and SO¼ 4 , but none for EC, OC, and Si. For MIs, only SO¼ 4 had a statistically significant HR. For coronary revascularization, SO¼ 4 had a greater statistically significant HR than PM2.5, there was no association for EC, OC, or Si. For stroke, there were statistically significant and comparable HRs for PM2.5, OC, and SO¼ 4 , but none for EC, and Si. Using 2-pollutant models for OC and SO¼ 4 , there were significant and higher HRs for SO¼ 4 than for OC for CVD events, CHD, MI, and coronary revascularization. In contrast, there were significant and higher HRs for OC than for SO¼ 4 for CVD and for stroke. For CVD deaths, there were statistically significant and comparable HRs only for OC. For atherosclerotic CVD deaths, there were statistically significant HRs for OC, EC,

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and SO¼ 4 exposure, which were similar in HR magnitude to that for the non-significant HR for PM2.5. There was no association for Si. For stroke deaths, the only significant HR was for OC.


ApoE/ mice For vascular (aortic) lipid peroxidation, there were significant effects (in decreasing order of potency) for whole MVE, SO¼ 4 with MVEG, NO 3 with MVEG, and road dust with MVEG. For plaque inflammation, there were strong associations for ¼ whole MVE, SO¼ 4 , SO4 with MVEG, and NO3 with MVEG, and a smaller association with MVEG alone. For vasoconstriction, there was a strong association for SO¼ 4 , and lesser associations for whole MVE and for SO¼ 4 with MVEG. For oxidized LDL, there was a strong association for whole MVE, but none for the other PM2.5 mixtures. For MMP expression, the only PM2.5 mixture to have a significant association was SO¼ 4 with MVEG, while for vascular gelatinase activity, the only PM2.5 mixture to have a significant association was whole MVE. It is interesting that SO¼ 4 alone had very strong associations with vasoconstriction, while NO 3 alone did not. SO¼ and NO , when combined with MVEG, had a strong 4 3 associations with plaque inflammation. Neither of these ions alone was significantly associated with TBARS, while when mixed with MVEG, both mixtures were strongly associated with TBARS. In summary, these studies demonstrated that (1) subchronic exposure to vehicle-related mixed emissions resulted in statistically significant increases in lipid peroxidation, circulating oxLP, vascular MMP expression and activity, and enhanced vasoconstriction in ApoE/ mice; and ¼ (2) exposure to NO 3 , SO4 , and road dust alone did not appear to drive any of the statistically significant effects observed in the cardiovascular system.

HEI NPACT review panel synthesis In this section, I have included substantial parts of the conclusions of the HEI’s Review Panel’s Executive Summary (HEI NPACT Review Panel, 2013). SO¼ 4 (measured as elemental S) is well captured by the CSN. S concentrations are typically well above detection limits, are measured with relatively high certainty, and have relatively low spatial variability. Therefore, exposure measurement error associated with SO¼ 4 is expected to be low. Se, As, V, and Ni, which are key components for identifying coalburning and fuel-oil combustion, are often below the limit of detection in the CSN database. The low concentrations of those pollutants, which have been decreasing over the past decades, hinder assessment of how they might be linked to health impacts. However, as reported by the Lippmann team in the current and prior studies, in some locations, notably New York City (NYC) concentrations of V and Ni are sufficiently high that it has been possible to identify associations of these elements with health outcomes. However, new local regulations in NYC that address fuels used for residential heating are expected to reduce concentrations of Ni and V in ambient air. For their epidemiologic analyses, the two NPACT teams adopted somewhat different philosophies on the use of source apportionment to link health outcomes to PM components.


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The Lippmann team relied heavily on a source apportionment approach that they had developed previously to link source categories directly to health outcomes in their epidemiologic analyses, whereas the Vedal team used source apportionment to assist in the interpretation of their health effects estimates and to support their focus on OC, EC, Si, and S as markers of specific sources in their analyses of health outcomes. The Panel noted that all current source apportionment approaches introduce uncertainty. For some approaches, those potential errors can be quite large. In their analyses using an approach based on factor analysis methods that they had developed previously, the Lippmann team found differences among locations in terms of which components contributed to similar source categories, providing indications that source emissions vary spatially, that the factor analytic approaches are sensitive to measurement uncertainties, that there are temporal variations in the composition of the emissions, and that other factors may add uncertainty to this approach. How their results might differ from those obtained using a different source apportionment technique and what the effect would have been of including measurement uncertainties and MDLs in the analyses remain unknown. Furthermore, it is not apparent which chemical components drive the associations between source categories and key health outcomes in the Lippmann report, which is different from determining which components are contained in the source categories that they identified. It was reassuring, however, that the Lippmann team came to consistent interpretations when they did include individual components in their analyses. The UW team applied positive matrix factorization (PMF), a widely used source apportionment approach, to support their focus on EC, OC, Si, and S as key components in their analyses of health outcomes. The Panel thought that their approach was defensible. The PMF factors they identified were reasonably consistent with what was expected in terms of sources and were also generally consistent with the source apportionment results of the Lippmann team. However, it would be of interest to compare the PMF results of the UW team directly with the source apportionment results of the Lippmann team in those cities that the two studies had in common. The Panel thought that the question of how (or whether) to use source apportionment to identify which PM components have strong associations with adverse health outcomes is an important one. It is generally preferable to use both source categories and component concentrations directly in the health analyses, if the study design permits, with a focus on examining consistencies and differences between the two approaches. When source apportionment results are used for health analyses, researchers should recognize, discuss and, if possible, address the uncertainties introduced by this method. When associations of PM2.5 components and health outcomes are analyzed in single-pollutant models, potential interactions or high correlations between components could affect the analysis and lead to misidentification of which pollutants may be most strongly associated with the observed human and animal health effects. Furthermore, other constituents of inhaled atmospheres - such as gaseous pollutants -might complicate assessment of which associations may be causally related. The Lippmann team attempted to address these issues by employing source apportionment in all their


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studies, two-pollutant models in time-series analyses in which they controlled for PM2.5 mass, and a TRI approach in their cohort study. The Vedal team made simple comparisons between the results for individual components and those for PM2.5 mass in their epidemiologic study and carried out sensitivity analyses involving two-pollutant models. Although the Panel appreciated the efforts of the investigators, they concluded that any future research using PM component data needs to more directly address appropriate analyses for multipollutant atmospheres in the statistical design. The Panel discussed the main findings in terms of what sources and PM components the teams found to play a role in the health outcomes they assessed, looking for consistency across the epidemiologic and toxicological studies within and across the two main NPACT studies. The Lippmann team’s time-series study (the Ito study) identified a fairly large number of PM components associated with daily hospitalizations due to cardiovascular disease (CVD) and daily all-cause and CVD mortality. Source categories attributed to primary vehicle exhaust and secondary SO¼ 4 were found to be important in some of these associations. The long-term American Cancer Society (ACS) cohort study (the Thurston study) also identified a number of PM components that could explain some of the mortality associations, including EC and S. However, OC, Si, and K (a marker for biomass combustion) were not associated with mortality in the cohort study. Source categories attributed to coal combustion and traffic pollution were found to be important in the associations with long-term effects, whereas little evidence was found for associations with source categories attributed to crustal sources or biomass combustion. There was minimal overlap between the PM2.5 components associated with short-term responses and those associated with long-term responses. Results for metals varied, but many effect estimates were highly uncertain (i.e. the confidence intervals were large), possibly due to the limited number of measurements above the limit of detection for metallic components in many cities. The Vedal epidemiologic study focused primarily on EC as a marker of vehicle exhaust, on OC as a marker of secondary organic aerosol and combustion emissions, on Si as a marker of crustal PM, and on S as a marker of secondary PM. Results suggested that OC and S were associated with several of the endpoints studied, but EC and Si were not. The Panel agreed with the investigators that this suggests that trafficrelated pollution and secondary PM could be playing a role in PM toxicity. The Lippmann team’s animal inhalation study (the Chen study) showed that a large number of components were positively or negatively associated with acute changes in HR and HRV in mice. When the investigators tried to rank these components, they concluded that Ni, Al, EC, P, and S had stronger associations with the cardiac endpoints than did PM2.5 mass. Effects of CAPs exposures on plaque progression in mice were primarily seen at Tuxedo, NY, Manhattan, NY, and East Lansing, MI, where the investigators deemed pollution mixtures to be more influenced by coal-fired power plant emissions than at Irvine, CA, and Seattle, WA. The Lippmann teams’ in vitro and in vivo study of PM collected on filters (the Gordon study) found that PM size and

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composition (determined by location and season) played a complex role in PM toxicity. The Panel noted that no size classes or components could be ruled out. The toxicological study conducted at LRRI used laboratory-generated atmospheres based on MVE and MVE gases combined with non-vehicular PM. Several combinations of particles and gases were found to affect different biologic markers in aortic tissues. The whole MVE mixture produced the largest changes, with MVE gases producing smaller and fewer changes. Fewer effects were observed with primary ¼ NO 3 and SO4 particles, and none with fine road dust particles. Combining non-vehicular PM with MVE gases increased the effects over non-vehicular PM alone, but generally did not exceed the effects of MVE by itself. Thus there was little evidence of a more-than-additive effect when exposure atmospheres were combined. The results support the role of both particulate and gaseous components in the induction of various cardiovascular outcomes, but whether there are important particle–gas interactions remains unclear and requires further research. In reflecting on the main NPACT findings, the Panel concluded that both the Lippmann and Vedal studies found that S and SO¼ 4 (markers primarily of coal and oil combustion) were associated with adverse health outcomes in their epidemiological and toxicological evaluations. Both studies support associations of health effects with trafficrelated pollutants, although their relative importance remains unclear. The less consistent findings for traffic-related components in the Vedal epidemiological study should be regarded with caution - not because the results suggest either a lack or a presence of adverse health effects, but because exposure to traffic-related pollutants is likely to vary across a metropolitan area and thus is subject to more uncertainty than exposure to pollutants from other source categories. On the other hand, there were only small differences in EC concentrations measured at roadside locations compared with urban background locations, indicating either spatial homogeneity in concentrations or, as noted above, potentially high measurement error for EC due to the 2-week sampling protocol. The degree to which the results observed for secondary SO¼ 4 particles were more consistent because SO¼ 4 concentrations were more accurately estimated in the Lippmann and Vedal studies (due to their spatial homogeneity) than concentrations of other pollutants remains an open question. With regard to the association of health effects with EC compared with those associated with OC, the differences in findings between the Lippmann and Vedal studies were surprising to the Panel. In typical urban environments, mobile sources are expected to be the major source of EC and an important contributor to OC. It is noteworthy that these studies report such prominent differences between the results for EC and OC, given the strong correlation between the two in many cities. Again, these differences may be due to the stronger spatial gradients between cities for OC than for EC, the exposure models and study designs, and the difficulties involved in measuring OC and EC. The Lippmann and Vedal studies suggest that further efforts to characterize EC, OC, and metals (i.e. combustion- and traffic-related components) should be a priority. Additional analytic efforts to lower the


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detection limits of some components would also be beneficial, as would be the collection of daily measurements. In summary, the Panel concluded that - except for the fairly consistent associations of many of the health outcomes with S and SO¼ 4 , which may, in part, be due to better exposure assessment, associations with other components were mixed, and linkages to sources were not definitive. Overall, this comprehensive and ambitious research program has shown that research on the toxicity of PM components is not likely to easily identify a single culprit PM component or source category or to identify a unique set of biomarkers that could be reliably used to monitor exposure. More work remains to be done to refine statistical methods for simultaneous modeling of multiple pollutants; to improve the representation of spatial contrasts in component concentrations, especially within cities; and to improve source identification and attribution. Further toxicological studies are needed to connect particle components with physiologic mechanisms; to study the relative toxicity of particles and gaseous pollutants; to study atmospheric aging of complex mixtures to better reflect real-world conditions; and to provide more insight into the role of PM2.5 components in causing tissue injury and dysfunction. Such enhanced understanding of exposure and health will be needed before it can be concluded that regulations targeting specific sources or components of PM2.5, while not targeting other components or sources, will protect public health more effectively than continuing to follow the current practice of targeting PM2.5 mass as a whole.

Coherence of the NYU and UW-LRRI NPACT findings with each other and with prior literature’s findings Long-term human exposure to PM2.5 components and chronic health effects The associations of IHD annual mortality rates with exposure to coal combustion effluents in the NYU NPACT study of the ACS cohort are broadly coherent with the associations of annual PM2.5 exposure and CVD events with OC and SO¼ 4 in the UW’s WHI cohort. The association with SO¼ 4 with annual mortality in the WHI cohort is most likely related to the strong correlations of SO¼ 4 with Se and As (which are tracers of coal combustion as was noted in the UW-LRRI NPACT Final Report). It was also notable that using 2-pollutant models for OC and SO¼ 4 , there were significant and higher HRs for SO¼ 4 than for OC for overall CVD events, as well as for the subcategories of CHD, MI, and coronary revascularization. In contrast, there were significant and higher HRs for OC, than for SO¼ 4 , for the cerebrovascular disease and stroke subcategories, suggesting that OC exposure may be a more important risk factor for cerebrovascular effects than for CVD effects. In the earlier literature, SO¼ 4 , most likely acting as a surrogate measure of exposure to coal combustion effluents, had significant associations with annual mortality (Dockery et al., 1993; Lave & Seskin, 1970; Ozkaynak & Thurston, 1987; Ostro et al., 2007, 2010; Pope et al., 1995, 2002). The traffic source, as indexed by OC and Cu, was strongly associated with annual CVD mortality in the UW’s WHI cohort, but only marginally associated with annual IHD


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mortality in the NYU’s ACS cohort. However, there were no consistent associations, in the either the NYU or UW NPACT studies of annual mortality rates with exposures to other source-related factors, such as those associated with the soil or residual oil combustion sources of PM2.5. As indicated in Table 3, associations of annual mortality rates with traffic were also found by Lipfert et al. (2006) and Ostro et al. (2010), while associations with residual oil components with long-term mortality were reported by Hedley et al. (2002) and Cahill et al. (2011a) in studies in which there were much higher concentrations of residual oil combustion effluent concentrations than were present in the ACS and WHI cohorts in the years that their analyses covered. Long-term exposure to PM2.5 components and chronic effects in ApoE/ mice In the NYU NPACT study, ApoE/ mice were exposed to CAPs for 6 h/d, 5 d/week for 6 months at: Mount Sinai (MS) in New York City, Sterling Forest (SF) in Tuxedo, NY, and in E. Lansing (EL), MI, where coal combustion effluent accounts for significant fractions of the ambient air PM, had significant aortic plaque progression, while ApoE/ mice exposed to CAPs for 6 h/d, 5 d/week for 6 months in Seattle (SEA), WA, and in Irvine (IR), CA, where there is no such coal effluent exposure, did not. While the number of sites was small, it is notable that the PM2.5 mass concentrations in the exposure chambers were about twice as high in MS, SF, and IR as those in EL and SEA, yet IR had no plaque progression, while EL did. In the LRRI part of the UW-LRRI NPACT study, ApoE/ mice were exposed to laboratory generated PM2.5 components and component mixtures for 6 h/d for 50 d at PM2.5 concentrations that were 3–6 times higher than those for the mice in the NYU NPACT study, although the number of days of exposure were fewer than those in the NYU study. There was significant plaque growth for road dust þ MVEG, but not for any of the other seven components and mixtures, including the whole motor vehicle exhaust. The explanation might lie in the combination of resuspended inorganic oxide dusts and reactive gases being more effective in stimulating plaque progression than the combination of carbonaceous particles and gases in the whole motor vehicle emissions. For plaque inflammation, there were significant associations for whole ¼ MVE, MVEG, SO¼ 4 , SO4 þ MVEG, and for NO3 þ MVEG. There were also significant associations of vasoconstriction ¼ with whole MVE, SO¼ 4 , and SO4 þ MVEG. ¼ It is noteworthy that SO4 when mixed with MVEG was strongly associated with plaque inflammation in the LRRI mice. Sulfate is mostly attributable of coal combustion, MVEG is attributable to traffic, as is a large fraction of the NO 3 . The strength of the association of sulfate with IHD mortality in humans and with aortic plaque progression in mice in the NYU NPACT studies could have been due to the coal combustion effluent mixture, or to SO¼ 4 itself. The LRRI mice plaque inflammation responses to SO¼ 4 suggest that, even in the absence of a biological mechanism for such an effect, SO¼ 4 may not only be a marker for coal combustion as a causal factor for long-term CVD effects, but may also be a causal factor by itself.


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Taken together, it is not clear whether there is coherence of the NYU and LRRI results with respect to effects on plaque in the mouse aorta. There were differences in the experimental protocols. The NYU mice ate normal chow, while the LRRI mice ate high-fat (HF) chow. Both NYU and LRRI used 6 h/d exposures, but the NYU exposures extended over 6 months, as compared to the LRRI exposures that extended over 50 d. There were relatively high concentrations of SO¼ 4 in the CAPs at NYU’s MS, SF, and EL sites, along with other components of fossil fuel combustion. In contrast, the SO¼ 4 -alone exposure at LRRI lacked other ambient air constituents. The freshlygenerated MVE, and the SO¼ 4 þ MVEG mixture in the LRRI study, may have been more acidic due to the presence of organic monolayers on the ultrafine sulfuric acid droplets, whose neutralization by ammonia is retarded by organic surface layers. Strong acid may account for some of the vascular effects attributable to the exposures. The relatively high concentrations of organics in the MVEG mixtures in the LRRI NPACT study were not present in the CAPs studies at NYU. The LRRI study with SO¼ 4 alone was the first to demonstrate that a subchronic inhalation exposure to pure SO¼ 4 (at 300 mg/ m3) could cause significant effects on aortic contraction, and that co-exposure to MVEG could substantially enhance plaque progression. The association of SO¼ 4 with cardiac function and plaque progression in the CAPs-exposed NYU mice could have been due to the co-presence of SO¼ 4 and other ambient air pollutants. As noted by Campen in the LRRI part of the UWLRRI NPACT Final Report, good arguments have been made that SO¼ 4 is a relatively non-toxic component of PM (Grahame & Schlesinger, 2005). The LRRI toxicological study findings, however, showed effects of SO¼ 4 both alone and in combination with the MVE-containing mixtures. Of all the pollutant atmospheres, an atmosphere of pure SO¼ 4 caused the most substantial changes in aortic vasoreactivity. Changes in aortic vasoreactivity were also noted for SO¼ 4 combined with MVEG, though to a lesser extent. There may also be a suggestion that the atmosphere of pure SO¼ 4 increased plaque area and plaque inflammation. Other than these effects, SO¼ 4 had effects only when combined with mixtures containing MVE. In light of these findings, it is possible that SO¼ 4 itself, rather than its presence in a complex mixture or some other compounds in the mix, is responsible for the associations found in the UW and other epidemiological studies.

PM2.5 components most influential as causal factors for health effects based on the findings of The NPACT studies Sulfate and Traffic [as indexed by OC and Cu] were more closely associated with chronic CVD responses than other source categories or individual PM2.5 components in both NPACT studies, but there were some differences in their relative influence between the two NPACT groups. For shortterm CVD responses, measured only by the NYU NPACT, Ni and V were also influential. The specific associations for chronic effects are summarized below for each NPACT. Traffic and sulfate in the NYU NPACT study The Traffic source was significantly associated with total daily all-year mortality, as were two markers of the Traffic source,

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i.e. Cu and OC. The traffic and salt sources were the only source categories that were significantly associated with CVD hospital admissions In terms of daily CVD mortality, the second stage analysis showed significant associations for SO¼ 4 and V, but not for EC, OC, or Cu, which are markers for Traffic. Traffic was more closely associated with hospital admissions for respiratory diseases than with hospital admissions for either total CVD or for IHD. Cu, Ni, V, NO2, Fe, Na, and Zn were significantly associated with the second stage daily CVD hospital admissions, but OC, EC, SO¼ 4 , As and Se were not. In terms of chronic effects in humans, the traffic source was less clearly associated with excess annual mortality than the coal combustion source. However, the traffic source was more closely associated with annual mortality than the PM2.5 sources other than coal combustion. In the ApoE/ mice, the traffic source category was considerably less closely associated with cardiac function in CAPs exposure than the residual oil combustion source, and EC was the only significantly associated individual component that was a traffic marker. In terms of aortic plaque progression in the mice exposed to CAPs at the five sites for 6 months, there was significant progression at MS, SF, and EL, but not at IR and SEA. As a source of PM2.5 mass concentration, traffic was substantial at MS, IR, and SEA, but minor at SF and EL. In contrast, SO¼ 4 was a substantial fraction of PM2.5 at MS, SEA, and EL, but minor at SEA and EL. These differences strongly suggest that coal and residual oil combustion, which accounts for most of the SO¼ 4 , are more influential in plaque progression than the traffic source. This interpretation is consistent with the different influences on plaque progression of SF CAPs, sidestream cigarette smoke, and diesel engine exhaust in prior NYU subchronic inhalation studies in ApoE/ mice. As reported by Lippmann & Chen (2009), the CAPs produced more plaque progression with a PM2.5 concentration of 110 mg/m3 than did either sidestream smoke or diesel exhaust with PM2.5 concentrations of 400 mg/m3. Furthermore, the sidestream cigarette smoke and diesel engine exhaust contained OC and volatile organic vapors not present in the CAPs, but they lacked the transition and heavy metals present in the CAPs. To the extent that the traffic source did affect either shortor long-term health effects in the NYU NPACT studies, it was not at all clear whether the effects could be attributed to PM2.5 constituents or gas phase pollutants, although the results of the TRI supplement to NYU’s ACS cohort study did reduce the likelihood that the effects are attributable to gaseous criteria pollutants. To the extent that PM2.5 constituent elements were causal, NYU’s analyses lacked the power to distinguish between tailpipe emissions of transition metals, brake wear emissions (e.g. Cu), and resuspended road dust containing metal oxides. The NYU studies lacked the power to determine what role, if any, was played by the OC within the PM2.5. In terms of chemical analyses of PM2.5, they were limited to total OC, which is generally dominated by aged secondary OC. Thus, whether the freshly generated primary OC, a focus of the studies by Delfino et al. (2009, 2010a,b, 2011) was causal for the health endpoints could not be determined. In terms of ultrafine PM, which contained the largest number concentration of airborne particles, there were no particle number


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counts, and only ultrafine mass concentration data for the in vitro cellular toxicity analyses, and the in vivo analyses of toxicity follow exposures by lung aspiration. In these analyses, the ultrafine samples generally produced more toxicity, per unit mass, than did the other PM2.5 components that generally dominated the PM2.5 mass. In view of these considerations, it is unlikely that ultrafine particles could have accounted for all the short- or long-term health effects that were closely associated with the mass concentrations of PM2.5 constituents. One limitation in the interpretation of the role of traffic was the reliance on central site monitors within the metropolitan areas as an index of individual-level exposure, which introduces differential measurement error across the various sources. Traffic markers, such as EC, can display significant variation on scales of 50–500 m within cities (Henderson et al., 2007) while elevated pollution sources (e.g. power plants with tall stacks) are more spatially homogeneous, potentially biasing the effect estimates of localized sources, such as traffic, towards the null. Another potential limitation is the use of PM2.5 data from 2000 to 2004 collected at the end of the follow-up period. This could introduce exposure misclassification if the PM2.5 levels and constituents in the studied metropolitan areas have changed over time. However, previous work in this cohort has shown the PM2.5 levels in different time-periods to be correlated, with lower levels at the end of the follow-up period, but indicating that areas with the highest exposures in the 1980s are still the most highly exposed in the 2000 (Pope et al., 2002). Similarly, it has been shown by Thurston et al. that the constituents that make-up the PM2.5 exposure today are also correlated with, albeit lower than, the levels in 1980 (Thurston et al., 2011). Despite this limitation, the PM2.5– IHD relationship in this analysis is consistent with past results (e.g. Krewski et al., 2009). Traffic and sulfate in the UW-LRRI NPACT study LRRI found that the MVE exposure caused the most consistent effects across all endpoints. The UW studies had no direct correlate of MVE. Based on their source apportionment, none of the four PM2.5 components of primary interest in the epidemiologic analyses seemed to be influenced largely by MVE. Of these components, EC, a traditional marker of exposure to MVE (specifically diesel exhaust), reflected a complex mix of sources. The diesel exhaust/brake wear-like feature contributed to EC to some degree in every MESA city, with contributions ranging from 6% to 36%, depending on the city. To the extent that EC was an indicator of MVE exposure, the UW studies did not find much support for a role for MVE in atherosclerosis or in cardiovascular events. Predicted exposure to OC in the UW study was associated with CIMT and cardiovascular events, especially cardiovascular deaths. Their source apportionment indicated that a secondary aerosol-like contribution was prominent in all six MESA cities (with contributions ranging from 26% to 48%), a biomass-like contribution in four of the cities (contributions from 15% to 45%; and a diesel exhaust/brakewear-like contribution in five of the six cities (with contributions ranging from 3% to 23%).


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The LRRI study did not include atmospheres of secondary organic aerosols or biomass emissions, so these effects could not be assessed experimentally. The separate effects of OC could not be addressed directly by the LRRI study. The diesel exhaust/brake wear-like feature from the UW source apportionment was a larger contributor to Cu than to the other components studied. For Cu, the contributions of the diesel exhaust/brake wear-like feature ranged from 32% to 57% across the MESA cities. Analyses of Cu were completed only in the MESA cohort. In these relatively limited exposure and health analyses, Cu was associated with both CIMT and the presence of CAC. Thus, the UW epidemiological studies provided, at best, mixed support for the primary finding from the LRRI toxicological study about MVE. To the extent that exposure to MVE was reflected by either EC or NO2, which are not particularly good markers of MVE exposure, they found little evidence to support a role for MVE. Cu, however, might be a better marker of exposure to MVE than either EC or NO2. The UW epidemiologic findings for Cu, although limited in scope, suggested that exposure to MVE could be important in the development of atherosclerosis. Predicted exposure to S (as a marker for SO¼ 4 ) in the MESA cohort study was associated with CIMT and with cardiovascular events. This association might indicate that SO¼ 4 was the component directly (and possibly causally) responsible for the observed cardiovascular associations, but UW considered to be at least as likely that either SO¼ 4 was exerting its effects in combination with other pollutants in the pollutant mix or that other pollutants in the mix were solely responsible for the effects. The LRRI toxicological study findings, however, showed effects of SO¼ 4 both alone and in combination with the MVEcontaining mixtures. Of all the pollutant atmospheres, an atmosphere of pure SO¼ 4 caused the most substantial changes in aortic vasoreactivity. Changes in aortic vasoreactivity were also noted for SO¼ 4 combined with MVEG, although to a lesser extent. Other than these effects, SO¼ 4 had strong effects only when combined with mixtures containing MVE. In order to attempt to provide an epidemiologic counterpart to the toxicological observations that SO¼ 4 in combination with MVE produced effects on some endpoints, UW performed an exploratory analysis that assessed whether the SO¼ 4 associations in the MESA cohort were modified by exposure to traffic emissions using the distance to a major roadway and predicted outdoor NO2 concentration at a MESA participant’s home address as indicators of exposure to traffic emissions. They assessed modifications of the SO¼ 4 associations with CIMT and CAC by including traffic-SO¼ 4 interaction terms in the health effect models. Possibly because of the uncertainty in specifying both traffic exposure and ¼ exposure to SO¼ 4 , they found little evidence that the SO4 effect was modified by traffic exposures and was unable to find support in the epidemiologic study results for the effects of the SO¼ 4 –MVE mixture seen in the LRRI results.

My post-NPACT holistic perspectives on the role of PM2.5 in CVD effects In this section, I provide my perspective on the lessons learned from the NPACT studies, and those from the other

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literature cited in this critical review, to formulate summary statements on current knowledge and on knowledge gaps concerning the role of PM2.5 and its components on initiating and potentiating CVD effects.


What I now know with reasonable certainty PM2.5 exposures lead to excess CVD mortality and morbidity. Within the CVD effects, the greatest are for IHD. The greatest financial benefits of PM2.5 exposure controls result from reductions in annual IHD mortality. The chronic IHD effects in both humans and animals are more closely associated with secondary SO¼ 4 than with PM2.5 mass or any of the other measured PM2.5 components. CVD effects coefficients are greater in the northeastern US than in other US regions, at least in terms of annual mortality in humans, and in cardiac function in ApoE/ mice. The largest source of secondary SO¼ 4 in most of the eastern US is coal combustion, a second SO¼ 4 source in seaport cities is residual oil combustion, and the largest SO¼ 4 source in other US cities is exhaust from motor vehicles. Residual oil combustion and exhaust from motor vehicles are also the largest sources of trace concentrations of nickel (Ni) and vanadium (V). SO¼ 4 may itself have adverse IHD effects, based on the LRRI studies in ApoE/ mice, and these effects are likely to be potentiated by simultaneous exposure to motor-vehicle exhaust gases (MVEG) and vapors and, based on the NYU studies in ApoE/ mice, by trace metals emitted during fossil fuel combustion. The source category with the second strongest association with CVD health effects is traffic. The components of PM2.5 attributable to traffic that are most likely to account for its association with CVD are elemental carbon (EC) and organic carbon (OC, i.e. both primary and/or secondary organic compounds) emitted from tailpipes, and copper (Cu) from brake wear. The associations of adverse CVD effects with Cu, Ni, and V demonstrate that low concentrations of trace metals can be influential on health-related responses. The PM2.5 components most closely associated with acute responses differ from those most closely associated with chronic responses. Studies limited to speciation of PM2.5 components making relatively large mass concentrations (SO¼ 4 , NO3 , OC, EC, Si, Al, and Fe) contribute less to our understanding than those including trace concentrations. What I consider to be highly uncertain The roles of EC and OC exposures as causal and/or contributory factors in CVD effects. Whether effects associated with OC are mainly due to primary OC and less to aged OC. Whether measurement technique differences for the thermal assays distinctions between EC and OC account for the differences in associations of EC and OC with health-related endpoints in the NYU and UW-LRRI NPACT studies. Whether either EC or OC should be considered to be causal factors in CVD effects, or are more useful as markers of other, more causal, components emitted by traffic sources.

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What I consider to be needs to refine the remaining exposure-response questions The most influential exposure agents. Spatial and temporal variations in population exposures, and their contributions to exposure assessment measurement errors in population-based epidemiological studies. More detailed characterization of individual and population-based exposures to PM2.5 components. Utility of traditional markers of population exposures for epidemiological studies, e.g. PM2.5, SO¼ 4 , BS, OC, EC, NO2, Si, Al, and Fe. Adequacy of current speciation networks for trace elements used for epidemiological studies. Useful indices of adverse health impacts for epidemiological studies. Biological mechanisms underlying acute responses and disease progression. Implications of findings of the HEI sponsored NPACT studies Implications to further research The findings of both the NPACT programs supported the hypothesis that specific PM2.5 components and sources are more closely associated with the chronic health effects that have been attributed to PM2.5 mass concentrations than are other constituents and sources. This was particularly the case for IHD-related annual mortality, which accounts for a major part of the overall health impact, and the major part of the tabulated benefits of ambient air pollution control. Furthermore, the clear association of coal combustion effluents with excess annual mortality in the NYU study of the ACS cohort was coherent with aortic plaque progression in NYU’s study of the mice exposed to PM2.5 CAPs at the three sites with coal combustion effluents in the ambient air, but not at the two sites lacking coal combustion effluents. The UW WHI cohort study did not perform source apportionment analyses that could have demonstrated whether or not the excess annual mortality that they associated with long-term PM2.5 exposure was attributable to coal combustion effluents, but it is reasonable to speculate that the strong association of chronic effects with SO¼ 4 , which were demonstrated in the WHI and MESA cohorts, could have been due to coal combustion effluents that are the major source of SO¼ 4 in the eastern and Midwestern regions of the US. Significant associations between annual mortality rates and annual average SO¼ 4 concentrations have been reported over the past half-century (e.g. Dockery et al., 1993; Lave & Seskin, 1970; Pope et al., 1995, 2004). There was also coherence of the results of the UW’s CIMT findings in the MESA cohort with the findings of the biomarker assays in the LRRI’s in mice exposed for 50 d to laboratorygenerated atmospheres of SO¼ 4 and other chemical components and component mixtures. However, it must be noted that the concentrations in the LRRI mouse exposures to SO¼ 4 were much higher than those in ambient air and in the NYU 6-month CAPs exposures of mice. On the other hand, there was more hydrogen ion associated with the ambient air SO¼ 4 in the NYU study than in ammonium sulfate used in the LRRI study.


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To the extent that coal combustion effluents indexed by SO¼ 4 are driving the excess annual mortality and other chronic effects associated with exposures to ambient air PM2.5, a uniform annual PM2.5 NAAQS provides different degrees of public health protection in different regions, with less protection in the regions downwind of coal-fired power plants. This implication is of sufficient importance and magnitude to warrant further research to investigate its impact on health and the nature and urgency of further emission controls on specific source classes. The results from NYU’s NPACT toxicological and epidemiological studies were coherent across species for both the short-term and long-term effects, but the putative causal components differed between short- and long-term effects. The chemical species most closely associated with short-term effects were EC, OC, and transition metals, with much weaker associations with the coal combustion source, a source that was so closely associated with the long-term effects in the ACS cohort and in the mice exposed subchronically to CAPs. The NYU NPACT subchronic CAPs exposure studies were limited to five sites, with none of them being in the southeastern, the great plains, the southwestern, or the western mountain states of the US nor in other countries with still different PM2.5 compositions and different mixtures of co-pollutant gases. Comparable studies should be carried out at such additional sites to allow comparisons of results and the data, in order to be able to identify the most influential constituents on both short- and long-term health-related responses. On the epidemiological side, we could do a more thorough evaluation of the shortterm mortality and morbidity attributable to ambient air PM if there was a more extensive speciation site network with continuous, or at least daily, monitoring at multiple sites within the larger communities. The design of future studies should also take into consideration the prior findings of the broad range of subchronic CAPs rodent inhalation studies performed by NYU and Ohio State University investigators that go beyond CVD effects. These results of these studies, showing chronic responses in the nervous system (Sama et al., 2007; Veronesi et al., 2005), liver (Laing et al., 2010; Tan et al., 2009; Zheng et al., 2013), atherosclerosis (Sun et al., 2008a), hypertension (Sun et al., 2008b), metabolic syndrome (Sun et al., 2009), insulin resistance and mitochondrial dysfunction (Xu et al., 2012), cardiac remodeling (Ying et al., 2009a,b), oxidative stress and altered gene expression in adipose tissue (Xu et al., 2011a), obesity and diabetes (Xu et al., 2011b), etc., can help to guide the design of future toxicological and epidemiological studies of the effects of peak exposures on short-term responses, and long-term exposures and cumulative effects, especially for intervention studies of the health benefits of specific source controls, and especially if the epidemiological investigators had access to more chemical speciation data. Future epidemiological studies will greatly benefit from a more complete PM speciation and gaseous pollutant database that has been available up to now if they are to succeed in identifying (1) the most causal constituents and (2) mortality and morbidity incidence for ICD codes other than CVD and respiratory disease. Ideally, they should include studies of communities having a large range of PM composition, and


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multiple years of co-located PM speciation and gaseous pollutant data. Findings from NPACT studies, and from additional such studies, can also aid in the design of future controlled rodent inhalation studies that are focused on additional health endpoints as well as better definitions of the roles of specific PM constituents and their interactions of gaseous air pollutants and PM constituents that are added or subtracted from controlled exposures to PM mixtures. When more such information becomes available, it may become possible to build realistic multi-pollutant models that can account for the observed associations of exposures and effects in humans and animals. We currently lack adequate knowledge and understanding of the effects of complex mixtures, and of the interactions among the constituents of the mixtures.

Implications of the NPACT studies to the setting of NAAQS and/or control strategies for ambient air PM In the absence of a distinction based on the chemical composition of the PM2.5, EPA’s selections of 24 h as an averaging time or a short-term PM2.5 NAAQS, and an annual average for long-term PM2.5 NAAQS were reasonable choices. However, the NYU NPACT study showed that the associations of PM2.5 and source-related PM2.5 mixtures and constituents with health-related effects differed between short- and long-term responses, with both short- and longterm responses being very much dependent on chemical composition. Recognition that the chronic health effects of PM2.5 differ from the short-term health effects in the extent of their of adversity and cost-benefit implications will make the future selections of short-term and annual NAAQS for PM2.5 mass and/or their components a more complex task. Short-term NAAQS for fine particulate matter If the revision of the short-term NAAQS is to be limited to mass concentration, then it is important to recognize that there were stronger and closer associations of short-term responses to ambient air PM2.5 mass in the northeastern US than in other geographic regions in (1) NYU’s time-series studies of daily IHD hospitalization and mortality in 64 US SMSAs; (2) NYU’s time-series analysis of cardiac function in ApoE/ mice at five US sites (two in the northeast, one in the midwest, and two on the west coast). These results are consistent with the findings on regional variations in daily mortality attributable to PM2.5 in the NMMAPS study (Dominici et al., 2007b). The geographic differences were most stark in terms of the cardiac function in the ApoE/ mice, where the responses were much greater in the mice in both MS in New York City (NYC) and in Sterling Forest (SF) State Park in Tuxedo, NY, which is generally upwind of NYC, than they were in EL, SEA, and IR. There were some common PM2.5 constituents at both NY sites in that both contain relatively high concentrations of long-range transported regional aerosol, composed largely of secondary PM resulting from chemical reactions of gaseous precursors originating from fossil fuel combustion in upwind coal-fired power plants and from traffic throughout the eastern


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megalopolos. Also, both MS and SF had much higher concentrations of Ni and S than any of the other locations, while MS had high concentrations of EC and V as well. In a previous study relating HR in COPD patients to coarse and fine PM components in NYC and Seattle, the only constituent that had a significant association with HR was Ni, but only for the Ni PM2.5 fraction in NYC (Hsu et al., 2011). NYU’s 64-cities time-series studies in human populations showed that adverse acute health effects were occurring in cities that did not exceed, or barely exceeded, the current 24-h PM2.5 NAAQS (35 mg/m3) that was retained in December 2012 (Ito et al., 2013). It is still based on PM2.5 mass concentration. Our supplemental time-series study based on daily measurements in Seattle (Zhou et al., 2011) demonstrated that adverse short-term effects were occurring in a city would meet even the more stringent concentration limit that was recently under consideration for a revised 24-h PM2.5 NAAQS (25 mg/m3). Therefore, consideration should be given to either replacing the current mass-based short-term primary (health-based) PM2.5 NAAQS with a limit based on our increasing knowledge of exposure to constituents most closely associated with the short-term health risks, i.e. those associated with the traffic and residual oil combustion sources, or on shifting the focus on controls to more stringent emission limits on these sources. If future fine PM NAAQS are to recognize that chemical composition matters in terms of health-related responses, then the results of the NPACT studies and those of prior and contemporary studies having speciation data should greatly help in guiding future NAAQS selections. While the NYU NPACT research could not determine the extent to which individual PM2.5 constituents causally contributed to adverse short-term health effects, they did show that some of them were much more closely associated with the measured effects than were others. Among the individual PM2.5 constituents that were most closely associated with short-term IHD effects in both humans and mice were Ni, V, Cu, EC, and S, while total OC, Al, As, Ca, Fe, K, P, Se, Si, Ti, and Zn were less consistently associated with such effects, and Pb was not associated with any of the tabulated effects. The NYU NPACT findings differed somewhat from those reported by Suh et al. (2011) for patients in Atlanta, GA for CVD admissions, which were significantly associated with a group of transition metals (Cu, Mn, Zn, Ti, and Fe). Suh et al. (2011) showed that this group of metals was significantly associated with admissions for IHD, congestive heart failure, and atrial fibrillation. In contrast, their microcrystalline oxide group (As, Br, Se, Pb, and Si) was associated with decreased CVD-related hospital admissions. The differences between the NYU NPACT study findings and those of Suh et al. (2011) may be due to the fact that, for at least some constituents, associations are likely due to close correlation of the measured constituent’s airborne concentration(s) with the concentrations of other constituents that are more causal, and which come from the same source(s). Both these studies showed associations of CVD admissions for Medicare patients with Cu. Suh et al. (2011) did not show associations with Ni and V, which were not present at elevated concentrations in Atlanta. For both the NPACT study results,

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and for the Suh et al. study, there was a lack of consistently increased CVD effects associated with As, Se, Pb, OC, and Si, which have often served as markers of the coal combustion and traffic sources. S, which is present in ambient air as sulfate ion, is unlikely to have any inherent toxicity in isolation, but originates from the S content within coal, residual oil, and motor vehicle fuels, and may interact with other constituents (COMEAP, 2000). OC, which is inconsistently associated with effects, is a very broad category of organic compounds. As demonstrated by Delfino et al. (2008, 2009) OC includes some primary OC species that are likely to be toxic, as well as many secondary OC species having little or no acute toxicity. Total OC, as measured in the CSN, has little predictive power with respect to short-term health effects. Long-term NAAQS for fine particulate matter If, as for a short-term NAAQS revision, the long-term NAAQS is to be limited to mass concentration, then it would be important to note that there were significant associations of annual IHD mortality rates with the long-term mean concentrations for ambient air PM2.5 mass in the US in NYU’s ACS cohort that involved people living in 100 US SMSAs that did not exceed the then current (as of November 2012) annual PM2.5 NAAQS concentration limit of 15 mg/m3, nor to the more stringent value of 12 mg/m3 that was adopted in December 2012. If, however, future PM2.5 NAAQS are to recognize that chemical composition matters in terms of health-related responses, then the results of the NPACT studies, and those of prior and contemporary studies having speciation data, should greatly help in guiding future NAAQS selections based on annual average fine particle concentrations. NYU’s NPACT analyses showed that the risks of longterm exposures were primarily attributable to the PM2.5 constituents arising from effluents of the coal combustion and, to a lesser extent, traffic sources. We have long known that the exposure to coal combustion derived PM2.5 is largely confined to the eastern half of the US, while exposure to high concentrations of traffic-derived PM2.5 is largely limited to residents of the largest cities and those living very close to major traffic arteries. Since little of the chronic effects excess was attributable to other nationwide PM2.5 sources, the benefits of the more stringent annual mass-based PM2.5 NAAQS adopted in December 2012 (12 mg/m3) can be questioned. Controls focused on emissions from coal combustion and traffic sources, and, in coastal regions impacted by marine transport sources, on emissions of SO¼ 4 , Ni, and V from residual oil combustion, may be more effective in reducing adverse chronic health effects. While NYU’s NPACT research could not determine the extent to which individual PM2.5 constituents causally contributed to adverse long-term health effects, it did show that one PM2.5 source, specifically coal combustion, appeared to be much more closely associated with the measured longterm effects (annual mortality in humans and aortic plaque progression in mice) than any of the others, and appears to have a higher toxicity per unit mass than other components for these endpoints.

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Consideration of a NAAQS for coarse thoracic PM (PM10–2.5) and/or ultrafine PM (PM50.15) While the NPACT epidemiological studies and NYU’s subchronic mouse inhalation study were limited to the effects of PM2.5 exposures, some of NYU’s short-term in vitro and in vivo results suggested that a NAAQS for coarse thoracic PM (PM10-2.5), along with a program devoted to the acquisition of PM10-2.5 speciation, could, if implemented, provide a more robust knowledge base for future rounds of reviews focused on a PM10-2.5 NAAQS. Although the NYU NPACT studies did not compare the toxicity of urban versus rural coarse PM, an issue complicating previous consideration of a PM10-2.5 NAAQS, their findings did demonstrate that different sources of coarse PM, as would occur at urban versus rural sites in the US, produce different degrees of toxicity. Some of their in vitro and in vivo results also suggested that PM in sizes below 0.1–0.2 mm might also warrant a separate nation-wide monitoring program, at least for initial research purposes. Need for a more comprehensive air quality monitoring program The NPACT research programs at NYU and UW-LRRI have clearly demonstrated that PM mass concentrations in ambient air provide relatively crude indices of health risks, and that the risks vary considerably with particle size, chemical composition, and season. The NYU NPACT has also shown that the PM constituents that are most closely associated with acute effects differ from those that are most closely associated with chronic effects. Furthermore, both NYU and UW have benefited from their access to the currently available CSN, but found that these data are inadequate for definitive determinations of the roles of specific PM constituents as causal factors for the effects. The CSN deficiencies include (1) too few monitoring sites for adequate characterization of spatial distributions of PM2.5 constituent concentrations and of gaseous criteria pollutants; (2) too little PM2.5 temporal and spatial resolution (limited currently to 24-h concentrations every third or sixth day at only one site in most cities); no discrimination for OC PM2.5 constituents; (3) no measurements of biogenic components; (4) insufficient numbers of speciation sites with co-located gaseous air pollutant measurements, which limited the statistical power of the NYU time-series study of daily mortality and hospital admissions; (5) no measurements of the chemical constituents of PM10–2.5 or PM50.2. A more robust monitoring network will make it possible to determine whether some of the associations of effects with PM constituents are causal, or are likely to be due other coconstituents whose concentrations are closely correlated with the monitored species. The data generated by such an enhanced CSN would enable EPA to establish better-targeted PM NAAQS and control strategies, and thereby reduce the burden of adverse health effects.

Conclusions Substantial increments of knowledge have been provided by the recently completed HEI-sponsored NPACT projects, and


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by several other recent investigations of the roles of ambient air PM2.5 constituents and/or their source-related mixtures on health-related biological responses in community residents. New insights have also been gained by the NPACT studies on laboratory animals that have been exposed to CAPs or PM2.5 components and complex mixtures of PM2.5 components and engine exhaust vapors that were designed to complement the epidemiological studies in order to (1) establish interspecies coherence of responses; (2) explore the underlying biological mechanisms for the responses; and (3) identify promising lines of inquiry for further toxicological and epidemiological studies. One extremely important increment of knowledge coming out of the NPACT program is in the realm of the effects of long-term PM2.5 exposure on chronic health effects, especially for the exposures that vary in multiple component concentrations on a national scale for population cohorts with extensive data on other personal risk factors (NYU’s ACS-II cohort, and UW’s WHI and MESA cohorts). NYU’s subchronic CAPs mouse inhalation studies at five sites provided data on substantial variations in aortic plaque progression by geographic region that was coherent with the regional variation in annual IHD mortality in the ACS-II cohort, with both the human and mouse responses being primarily attributable to the coal combustion source category. While the UW cohort regressions of associations of CVD events and mortality in the WHI cohort, and of CIMT and CAC progression in the MESA cohort were limited to the ambient air concentrations of OC, EC, Si, and SO¼ 4 , and did not extend to utilization of their source apportionment analyses, they did show that, of the four PM2.5 components of primary focus, there were statistically significant associ¼ ations for only OC and SO¼ 4 , with SO4 having the stronger associations with CVD-related human responses, and OC having the stronger associations with cerebrovascular responses. The LRRI’s mice had CVD-related biomarker responses to SO¼ 4 that were exacerbated by co-exposure to MVEG. The UW source apportionment description noted that the ambient air SO¼ 4 concentrations were highly correlated with the concentrations of Se and As. Thus, it is reasonable to assume that, on a national scale, SO¼ 4 can serve as a surrogate index of the coal combustion source and that the UW-LRRI findings are consistent with the NYU attribution of most of the chronic CVD effects to the coal combustion source. Another extremely important increment of knowledge, coming out of the NYU NPACT program, is that the sourcerelated component that is dominant for the chronic CVD effects (coal combustion) differs from the components that dominate acute health effects in both humans (hospital admissions) and mice (cardiac function). The components most closely associated with these acute effects are OC, EC, and Cu from the traffic source, and Ni and V from the residual oil source. For daily mortality in humans, there appears to be a role for coal combustion, as indexed by SO¼ 4 , Se, and As. The major PM2.5 components that appear to be least associated with health effects are soil-related minerals and NO 3 , except insofar as such PM components serve as carriers of reactive vapors, enabling them to penetrate more deeply into respiratory tract airways. This was most clearly demonstrated in the LRRI subchronic inhalation studies in


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mice in which mixtures of MVEG and SO¼ 4 , NO3 , and road dust produced greater effects on CVD biomarkers than did SO¼ 4 , NO3 , and road dust alone. These new findings and insights provide further support for the hypothesis that some PM2.5 components are considerably more toxic than others, but also illustrate the remaining uncertainties concerning the adequacy of PM2.5 mass as an index of the health risks of ambient air PM2.5 pollution exposures, and especially so for an annual average NAAQS where the variation in the contributions of coal combustion effluents to PM2.5 mass is so extreme. In contrast, the contribution of the traffic source to PM2.5 is far more uniform, and more closely correlated with that of PM2.5 mass. While the recent research into the roles of PM2.5 components on CVD has clearly filled some major knowledge gaps, and helped to define remaining uncertainties, much more knowledge is needed if we are to identify and characterize the most effective and efficient means for reducing the still considerable adverse health impacts of ambient air PM. The recent research, discussed herein, being focused on CVD-related effects of PM2.5 and its components, has done little to better define the risks in the respiratory tract, the nervous system, the liver, diabetes, etc. Likewise, the risks associated with PM10-2.5, which appears to be especially important in the respiratory tract, and those with PM50.2, remain poorly defined. Advances in characterization of health risks associated these other organ systems will require more epidemiological studies, which, in turn will require an expanded chemical speciation, network as described above, as well as more subchronic toxicological studies with comprehensive analysis of the exposure, biomarker variables, organ and tissue responses to specific PM2.5 constituents and mixtures, and the underlying biological mechanisms.

Acknowledgements NYU’s NPACT program involved my extensive collaboration with colleagues at NYU and at other research laboratories. I especially acknowledge the roles of Dr. Kazuhiko Ito (PI for the NPACT time-series epidemiology), Dr. George Thurston (PI for the NPACT annual mortality epidemiology), Dr. LungChi Chen (PI for the NPACT subchronic mouse inhalation exposures), and Dr. Terry Gordon (PI for the NPACT in vitro and in vivo lung aspiration exposures). I would also like to acknowledge the contributions made by the numerous CRT peer reviewers, and by Dr. Sverre Vedal. Their questions for clarifications and suggestions for additional information stimulated me to re-think the content of this paper and to thereby improve it.

Declaration of interest The affiliation of the author is as shown on the cover page. The research that is the cornerstone of this paper was funded by the Health Effects Institute (HEI), which supported the performance of the recent NPACT studies; the Environmental Protection Agency (EPA Grant R827351), which supported NYU’s PM2.5 Center Program; and the National Institute of Environmental Health Sciences (NIEHS – Grant ES00260),

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which has provided Center Program support for the author’s research since 1964. The author received no external funding for preparation of this paper. The author has frequently offered advice to government agencies on the setting of environmental and occupational standards for airborne materials. The author has not offered expert testimony in legal proceedings during the past 5 years on scientific matters that are the subject of this paper. The data summaries, their synthesis, and the interpretations of the data in this Critical Review are exclusively those of the author, and do not necessarily represent those of the HEI, EPA or NIEHS.

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Toxicological and epidemiological studies on effects of airborne fibers: coherence and public [corrected] health implications.

Correction to: Lippmann, Toxicological and epidemiological studies on effects of airborne fibers: Coherence and public health implications.

The influence of air quality model resolution on health impact assessment for fine particulate matter and its components.

Role of microbial and chemical composition in toxicological properties of indoor and outdoor air particulate matter.

Associations of Annual Ambient Fine Particulate Matter Mass and Components with Mitochondrial DNA Abundance.

Obesity and the cardiovascular health effects of fine particulate air pollution.

Spatial association between ambient fine particulate matter and incident hypertension.

Air particulate matter and cardiovascular disease: the epidemiological, biomedical and clinical evidence.

Ambient air concentrations exceeded health-based standards for fine particulate matter and benzene during the Deepwater Horizon oil spill.

Ambient Particulate Matter Air Pollution Exposure and Mortality in the NIH-AARP Diet and Health Cohort.

Spatial variable selection methods for investigating acute health effects of fine particulate matter components.

Health Outcomes of Exposure to Biological and Chemical Components of Inhalable and Respirable Particulate Matter.

Global chemical composition of ambient fine particulate matter for exposure assessment.

Characterization of fine particulate matter in ambient air by combining TEM and multiple spectroscopic techniques--NMR, FTIR and Raman spectroscopy.

Associations of particulate matter and its components with emergency room visits for cardiovascular and respiratory diseases.

Physicochemical characterization of ambient air particulate matter in Tabriz, Iran.

A Source Apportionment of U.S. Fine Particulate Matter Air Pollution.

Characteristics and oxidative stress on rats and traffic policemen of ambient fine particulate matter from Shenyang.

An investigation into the relationship between the major chemical components of particulate matter in urban air.

Ambient Fine Particulate Matter and Mortality among Survivors of Myocardial Infarction: Population-Based Cohort Study.

The association between ambient fine particulate matter and incident adenocarcinoma subtype of lung cancer.

Ambient Fine Particulate Matter, Nitrogen Dioxide, and Hypertensive Disorders of Pregnancy in New York City.

Controlled exposure of humans with metabolic syndrome to concentrated ultrafine ambient particulate matter causes cardiovascular effects.

Pulmonary function and ambient particulate matter: epidemiological evidence from NHANES I.