critical review of the human data on short-term nitrogen ... · on april 30, 1971, setting both the...

(Received 15 June 2009; revised 21 August 2009; accepted 28 August 2009)

ISSN 1040-8444 print/ISSN 1547-6898 online © 2009 Informa UK LtdDOI: 10.3109/10408440903294945 http://www.informahealthcare.com/txc

R E V I E W A R T I C L E

Critical review of the human data on short-term nitrogen dioxide (NO

2) exposures: Evidence for NO

2 no-effect levels

Thomas W. Hesterberg1, William B. Bunn1, Roger O. McClellan2, Ali K. Hamade3, Christopher M. Long3, and Peter A. Valberg3

1Navistar, Inc., Warrenville, Illinois, USA, 2Advisor, Toxicology and Risk Analysis, Albuquerque, New Mexico, USA, and 3Gradient, Cambridge, Massachusetts, USA

AbstractNitrogen dioxide (NO2) is a ubiquitous atmospheric pollutant due to the widespread prevalence of both natural and anthropogenic sources, and it can be a respiratory irritant when inhaled at elevated concentrations. Evidence for health effects of ambient NO2 derives from three types of studies: observational epidemiology, human clinical exposures, and animal toxicology. Our review focuses on the human clinical studies of adverse health effects of short-term NO2 exposures, given the substantial uncertainties and limitations in interpretation of the other lines of evidence. We examined more than 50 experimental studies of humans inhaling NO2, finding notably that the reporting of statistically significant changes in lung function and bronchial sensitivity did not show a consistent trend with increasing NO2 concentrations. Functional changes were generally mild and transient, the reported effects were not uniformly adverse, and they were not usually accompanied by NO2-dependent increases in symptoms. The available human clinical results do not establish a mechanistic pathway leading to adverse health impacts for short-term NO2 exposures at levels typical of maximum 1-h concentrations in the present-day ambi-ent environment (i.e., below 0.2 ppm). Our review of these data indicates that a health-protective, short-term NO2 guideline level for susceptible (and healthy) populations would reflect a policy choice between 0.2 and 0.6 ppm.Extended abstractNitrogen dioxide (NO2) is a ubiquitous atmospheric pollutant due to the widespread prevalence of both natural and anthropogenic sources, and it can be a respiratory irritant when inhaled at elevated concentrations. Natural NO2 sources include volcanic action, forest fires, lightning, and the stratosphere; man-made NO2 emissions derive from fossil fuel combustion and incineration.The current National Ambient Air Quality Standard (NAAQS) for NO2, initially established in 1971, is 0.053 ppm (annual average). Ambient concentrations monitored in urban areas in the United States are ~0.015 ppm, as an annual mean, i.e., below the current NAAQS. Short-term (1-h peak) NO2 concentrations outdoors are not likely to exceed 0.2 ppm, and even 1-h periods exceeding 0.1 ppm are infrequent. Inside homes, 1-h NO2 peaks, typically arising from gas cooking, can range between 0.4 and 1.5 ppm.The health effects evidence of relevance to ambient NO2 derives from three lines of investigation: epidemiol-ogy studies, human clinical studies, and animal toxicology studies. The NO2 epidemiology remains inconsistent and uncertain due to the potential for exposure misclassification, residual confounding, and co-pollutant effects, whereas animal toxicology findings using high levels of NO2 exposure require extrapolation to humans exposed at low ambient NO2 levels. Given the limitations and uncertainties in the other lines of health effects evidence, our review thus focused on clinical studies where human volunteers (including asthmatics, children, and elderly) inhaled NO2 at levels from 0.1 to 3.5 ppm during short-term (½–6-h) exposures, often combined with exercise, and occasionally combined with co-pollutants. We examined the reported biological effects and classified them into (a) lung immune responses and inflammation, (b) lung function changes and airway hyperresponsiveness (AHR), and (c) health effects outside the lungs (extrapulmonary).We examined more than 50 experimental studies of humans inhaling NO2, finding that such clinical data on short-term exposure allowed discrimination of NO2 no-effect levels versus lowest-adverse-effects levels. Our conclusions are summarized by these six points: For lung immune responses and inflammation: (1) healthy sub-jects exposed to NO2 below 1 ppm do not show pulmonary inflammation; (2) at 2 ppm for 4 h, neutrophils and cytokines in lung-lavage fluid can increase, but these changes do not necessarily correlate with significant or sustained changes in lung function; (3) there is no consistent evidence that NO2 concentrations below 2 ppm

Critical Reviews in Toxicology, 2009; 39(9): 743–781Critical Reviews in Toxicology

2009

39

9

743

781

15 June 2009

21 August 2009

28 August 2009

1040-8444

1547-6898

© 2009 Informa UK Ltd

10.3109/10408440903294945

Address for Correspondence: Thomas W. Hesterberg, Navistar, Inc., 4201 Winfield Road, P.O. Box 1488, Warrenville, IL 60555, USA. Phone: (312) 927-2697; Fax: (312) 836-3937; E-mail: [email protected]

TXC

429668

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http://www.informa.com/doifinder/10.3109/10408440903294945

mailto:[email protected]

http://www.informa.com/doifinder/429668

744 T. W. Hesterberg et al.

increase susceptibility to viral infection; (4) for asthmatics and individuals having chronic obstructive pulmonary disease (COPD), NO2-induced lung inflammation is not expected below 0.6 ppm, although one research group reported enhancement of proinflammatory processes at 0.26 ppm. With regard to NO2-induced AHR: (5) studies of responses to specific or nonspecific airway challenges (e.g., ragweed, methacholine) suggest that asthmatic individuals were not affected by NO2 up to about 0.6 ppm, although some sensitive subsets may respond to levels as low as 0.2 ppm. And finally, for extra-pulmonary effects: (6) such effects (e.g., changes in blood chemistry) generally required NO2 concentrations above 1–2 ppm.Overall, our review of data from experiments with humans indicates that a health-protective, short-term-average NO2 guideline level for susceptible populations (and healthy populations) would reflect a policy choice between 0.2 and 0.6 ppm. The available human clinical results do not establish a mechanistic pathway leading to adverse health impacts for short-term NO2 exposures at levels typical of maximum 1-h concentrations in the present-day ambient environment (i.e., below 0.2 ppm).

Keywords: Air pollution; airway challenge; airway hyperresponsiveness; controlled human exposure study; epidemiology; inflammation; lung function; nitrogen dioxide (NO2); air quality standard

Contents

Abstract ......................................................................................................................................................................................... 743

Extended abstract .........................................................................................................................................................................743

Introduction ..................................................................................................................................................................................744

Sources of exposure to NO2 .........................................................................................................................................................747

Review of key epidemiological study findings ...........................................................................................................................748

Review of clinical studies of NO2-induced health effects .........................................................................................................754

Background on pulmonary immune effects ..........................................................................................................................754

Studies of healthy subjects ......................................................................................................................................................759

Inflammatory response .......................................................................................................................................................759

Susceptibility to infection ....................................................................................................................................................767

Studies in susceptible populations .........................................................................................................................................767

Effects of NO2 exposure on lung function and airway responsiveness (AHR) ....................................................................769

NO2-induced AHR to nonspecific challenges....................................................................................................................769

NO2-induced AHR to specific challenges ..........................................................................................................................774

Extrapulmonary effects associated with NO2 exposures ......................................................................................................774

Discussion .....................................................................................................................................................................................775

Conclusions ..................................................................................................................................................................................775

Acknowledgments ........................................................................................................................................................................777

Declaration of interest

References .....................................................................................................................................................................................778

Introduction

Nitrogen dioxide (NO2) is one of six criteria air pollutants

for which the United States Clean Air Act (CAA) directs the US Environmental Protection Agency (US EPA) to set and periodically review both primary (to protect health) and sec-ondary (to protect the public welfare) National Ambient Air Quality Standards (NAAQS). From the language of the CAA, it is clear that the setting of the NAAQS must be informed by the currently available science; however, it is also clear that the ultimate selection of an indicator, averaging time, specific numerical level, and statistical form of a standard represent policy judgments that can only be made by the Administrator of the US EPA. US EPA promulgated the initial NAAQS for NO

2 on April 30, 1971, setting both the primary

(health-based) and secondary (welfare-based) NAAQS for NO

2 at 0.053 ppm (100 µg/m3), as an annual arithmetic

mean. The initial primary standard was based on the review of the NO

2 health effects evidence presented in the US EPA

document “Air Quality Criteria for Nitrogen Oxide” (US EPA, 1971). The key data used in setting the standard were from community epidemiological studies conducted in Chattanooga, TN, purporting to show respiratory effects in children chronically exposed to low levels of NO

2 (Shy et al.,

1970a, 1970b; Pearlman, 1971).The Clean Air Act requires US EPA to periodically review

the NAAQS for criteria pollutants such as NO2 to ensure the

scientific adequacy of the standards. In accord with the CAA requirement for periodic review to ensure scientific adequacy and in response to the August 1977 Clean Air Act amendments, US EPA conducted a scientific review focused on the short-term health effects of NO

2 that was published in the document,

“Health Effects of Short-Term Exposures to Nitrogen Dioxide Air Quality Criteria” (US EPA, 1978). After completion of this document, US EPA (Federal Register, 1978) decided to incor-porate the information on the effects of NO

2 short-term expo-

sures into a more comprehensive revision of the prior 1971

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Human clinical studies of short-term NO2 exposures 745

criteria document, namely the “Air Quality Criteria for Oxides of Nitrogen” (US EPA, 1982a). This criteria document was then used as the basis for the document “Office of Air Quality Planning and Standards NO

2 Staff Paper” (US EPA, 1982b).

This document was relied upon by the US EPA Administrator in reaching the June 19, 1985, final decision that it was appro-priate to retain the NAAQS annual arithmetic average (Federal Register, 1985). In making this determination, US EPA con-cluded that setting an annual standard at 0.053 ppm NO

2 was

protective of both long-term exposure to NO2, as well as short-

term exposures at levels higher than the annual average.In the late 1980s and early 1990s, US EPA again reviewed

the scientific adequacy of the NAAQS for NO2. As part of that

review, McCurdy (1994) concluded, based on an analysis of the relationship between hourly and daily NO

2 levels and

annual average levels, that an annual average standard set at 0.053 ppm NO

2 would be protective of the health effects from

exposures of several hours duration in the range of 0.1–0.2 ppm. This analysis, as well as other available scientific infor-mation on the health and welfare effects of NO

2 and other

oxides of nitrogen, was incorporated into the three-volume document, “Air Quality Criteria for Oxides of Nitrogen” (US EPA, 1993) and subsequently in the Staff Paper, “Review of the National Ambient Air Quality Standard for Nitrogen Dioxide, Assessment of Scientific and Technical Information” (US EPA, 1995). The content of those two documents served as a basis for the US EPA Administrator’s decision to again retain a NAAQS (both primary and secondary) for NO

2 at 0.053

ppm, annual arithmetic average (Federal Register, 1996).In December 2005, US EPA initiated the latest reassess-

ment of the NO2 health effects evidence to support the

review of the NO2 NAAQS. As part of this review process, US

EPA completed two key reports that provide its interpreta-tion of the state of the science on NO

2 health effects and

potential alternatives for a primary (health-based) stand-ard, namely the July 2008 “Integrated Science Assessment for Oxides of Nitrogen—Health Criteria” (US EPA, 2008a) and the November 2008 “Risk and Exposure Assessment to Support the Review of the NO

2 Primary National Ambient Air

Quality Standard” (US EPA, 2008b). Importantly, since the completion of the last NO

2 review in 1996, US EPA modified

the NAAQS review process, replacing the preparation of a Criteria Document with preparation of an Integrated Science Assessment (ISA). As discussed in Peacock (2006), the ISA document is intended to provide “a more concise evaluation, integration and synthesis of the most policy-relevant science, including key science judgments that will be used in conduct-ing the risk and exposure assessments.” In addition, US EPA made the decision to replace the former Staff Paper with both a risk/exposure assessment and a policy assessment, with the latter to be published in the Federal Register as an Advance Notice of Proposed Rulemaking (ANPR) to allow for com-ments by CASAC (Clean Air Scientific Advisory Committee) and the public on the policy options under consideration and for recommendations on alternative policy options.

Although it is unknown to what extent the NAAQS review process will continue to evolve as political administrations

change, one of the key issues to emerge from this latest review of the NO

2 NAAQS involves whether the available

science supports the development of a short-term (i.e., 24-h and/or 1-h averaging time) NO

2 NAAQS and what the aver-

aging time, form, and level of such a short-term standard should be. A short-term NO

2 standard could either replace

or complement a long-term standard. In the NO2 Integrated

Science Assessment (ISA), US EPA (2008a) concluded that “Taken together, recent studies provided scientific evidence that NO

2 is associated with a range of respiratory effects and

provide evidence sufficient to infer a likely causal relation-ship between short-term NO

2 exposure and adverse effects

on the respiratory system.” US EPA (2008a) indicated that this causal determination was predominantly based on epidemiologic studies of short-term ambient NO

2 concen-

trations, continuing: “The greatest weight of evidence comes predominantly from the large body of recent epidemiologic evidence, with supportive evidence from human and ani-mal experimental studies.” Importantly, respiratory mor-bidity from short-term NO

2 exposure was the only health

endpoint for which US EPA concluded there was sufficient health effects evidence to support what they termed either a “causal” or “likely causal” relationship. As such, US EPA’s Risk and Exposure Assessment (US EPA, 2008b) only focused on respiratory morbidity endpoints from short-term NO

2 expo-

sures in its quantitative characterization of NO2 health risks,

relying upon both the NO2 epidemiology and the findings

from controlled human exposure studies.As with the former Criteria Documents and Staff Papers,

the NO2 ISA document (US EPA, 2008a) and the Risk and

Exposure Assessment document (US EPA, 2008b) have considered scientific information from multiple kinds of investigations in assessing the potential health impacts of ambient NO

2 exposure. This has included information from

observational epidemiology studies, human clinical studies, and animal toxicology studies. Although the health effects literature also contains information on highly elevated occupational exposures to NO

2 (e.g., silo-filler’s disease),

these findings are of dubious relevance to lower, ambient NO

2 exposures and were not considered by US EPA (2008a,

2008b). Although US EPA (2008a) relied most heavily on epidemiologic study findings to support its determination of a “likely causal” relationship between short-term NO

2 expo-

sures and respiratory morbidity, it is illustrative to consider how information from different kinds of studies has been used over the decades in setting the NO

2 standard. The first

standard was set in 1971 almost exclusively based on epi-demiological findings. These data soon fell into disfavor as questions were raised concerning the quantification of NO

2

exposures in the epidemiology studies. In the next several reviews, major weight was given to findings from studies in laboratory animal species as a basis for retaining the stand-ard at the level set in 1971.

In general, information from observational epidemiol-ogy studies is of particular value because it is obtained in the species of interest, namely humans, for actual expo-sure conditions, namely ambient NO

2. However, uniform

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shortcomings of observational epidemiology studies involve (a) the adequacy of the assessment of personal exposure of the study population, and (b) the extent to which ambient exposures typically involve exposure to complex mixtures of pollutants and other confounding factors, not just the pollutant of specific interest. When available, studies of volunteers inhaling NO

2 provide valuable human data that

are generally easier to interpret, because exposures are con-trolled and well defined, and are solely to the pollutant(s) of concern. In addition, in human clinical studies, it is possible to simultaneously and precisely measure a range of out-comes with high sensitivity, including both subtle biological responses and functional decrements potentially relevant to health. Given that it is not ethically appropriate to study sub-jects with serious preexisting health conditions, a general shortcoming of human clinical studies involves their limita-tion to small groups of generally healthy adult volunteers who may not be representative of larger populations (e.g., highly susceptible populations). In the absence of adequate data from human subjects, and to extend the utility of any available human data, toxicology studies are frequently conducted with laboratory animal species. Such studies can be conducted with controlled pollutant levels and exposure durations. However, in any study conducted with laboratory animals, there are substantial uncertainties associated with the extrapolation of the observed findings to humans based on interspecies differences. This extrapolation may also be complicated by the need to extrapolate from the high levels of exposure used in laboratory studies to the low-level expo-sures likely to be encountered in the ambient environment.

As discussed in greater detail later, there remain sub-stantial uncertainties and limitations in the available NO

2

epidemiology, and it can be argued that human clinical studies with controlled levels of NO

2 exposure offer the best

opportunity for discerning thresholds for short-term, NO2-

specific health effects. Although US EPA (2008a, 2008b) con-tinues to rely most heavily on the NO

2 epidemiology in its

assessment of NO2 health effects, this idea is not lost on US

EPA authors, who noted in the Introduction to the NO2 ISA

(US EPA, 2008a): “The most compelling evidence of a causal relationship between pollutant exposure and health effects comes from human clinical studies, which evaluate health effects of administered exposure under controlled laboratory conditions.” Importantly, there is now an abundant body of NO

2 human clinical studies addressing a range of respira-

tory health endpoints including airway responsiveness, host defense and immunity, inflammation, and lung function. These studies have been conducted for several subpopula-tions considered to be more susceptible to the adverse health effects of air pollution, including asthmatic children and adults, and elderly subjects with chronic obstructive pulmo-nary disease (COPD). Although human clinical studies are potentially less relevant to the potential effects of long-term exposures, their focus on acute effects from short-duration exposures is especially relevant to the question of whether the available science supports the development of a short-term NAAQS for NO

2, and at what level.

In this paper, we examine and synthesize findings from approximately 50 human clinical studies to assess the human evidence for NO

2 no-effect levels versus lowest-adverse-

effect levels for short-term NO2 exposure. We consider clini-

cal studies where human volunteers (including asthmatics, children, and elderly) inhaled NO

2 at levels from 0.1 to 3 ppm

during short-term (½–6-h) exposures, often combined with exercise, and occasionally combined with co-pollutants. In light of the US EPA (2008a) determination that only for res-piratory morbidity is there sufficient health effects evidence to support either a causal or likely causal relationship with short-term NO

2 exposure, we focus primarily on respira-

tory health endpoints, but we also review findings related to health effects outside the lung (i.e., extrapulmonary effects), given the body of studies reporting associations between air pollution exposures and adverse cardiovascular health effects. We particularly focus on studies examining airway hyperresponsiveness (AHR) among asthmatics, given US EPA’s use of this health endpoint in their quantitative risk assessment (US EPA, 2008b).

In reviewing key findings from the human clinical studies of short-term NO

2 exposures, we consider not only their sta-

tistical significance, but also whether the reported outcomes are relevant to health and are in fact “adverse” in nature. Importantly, the American Thoracic Society (ATS) has pub-lished guidelines on what should be considered a significant outcome of air pollutant-induced health effects (ATS, 1999). Key elements of these guidelines are represented in the fol-lowing statements:

“Healthy persons may sustain transient reductions in pulmonary function associated with air pollution exposure, e.g., reduction of the forced vital capacity (FVC) with exer-cise at times of higher levels of ozone pollution. However, the committee recommends that a small, transient loss of lung function, by itself, should not automatically be designated as adverse. In drawing the distinction between adverse and nonadverse reversible effects, this committee recommended that reversible loss of lung function in combination with the presence of symptoms should be considered as adverse…..This committee considered that any detectable level of permanent lung function loss attributable to air pollution exposure should be considered as adverse.”

As reflected in these statements, ATS (1999) suggests that whereas a permanent loss in lung function is deemed adverse, asymptomatic, transient changes are not. In this review, we will thus evaluate the biological responses reported from the clinical studies using the ATS definition of an adverse health outcome.

As a roadmap for this review, we first briefly address the sources of NO

2 and provide summary data on ambient

concentrations of NO2 to provide perspective for the later

discussion of health responses to NO2. We then review key

epidemiological findings relevant to the adverse health effects of short-term NO

2 exposures, because it is these find-

ings per se that require experimental support. We follow this by a review of the results of clinical studies with human

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subjects exposed for defined periods of time to controlled levels of NO

2. Overall, we critically examine whether human

clinical data on effects of short-term NO2 exposure provide

“plausibility and coherence” to the air pollution epidemiol-ogy studies finding associations between short-term ambi-ent NO

2 concentrations and serious health effects, including

increases in respiratory-related hospital admissions and emergency department (ED) visits.

Sources of exposure to NO2

Due to the widespread prevalence of both natural and anthro-pogenic sources of NO

2, it is a ubiquitous pollutant in both

urban and remote atmospheres. Natural sources include intrusion of stratospheric nitrogen oxides, bacterial and vol-canic actions, forest fires, and lightning. These natural emis-sions are dispersed over the entire earth, thus resulting in relatively small background ambient concentrations that are typically less than 0.0001 ppm (US EPA, 2008a; WHO, 2000). The major source of anthropogenic emissions of nitrogen dioxide is fossil fuel combustion from both stationary and mobile sources (heating, power generation, motor vehicles, etc.). In an internal combustion engine, for example, elevated temperatures drive endothermic reactions between atmos-pheric nitrogen and oxygen in the flame, yielding different species of nitrogen oxides (NO

x), including nitric oxide (NO)

and NO2. Under the Clean Air Act, US EPA has implemented

emissions standards for motor vehicles and electric utilities that have resulted in substantial decreases in nationwide NO

x emissions since the 1970s (Figure 1).

On an annual average basis, ambient NO2 levels across

the United States are generally well below the current annual NAAQS of 0.053 ppm (US EPA, 2008a). Consistent with the trend in nationwide emissions, Figure 2 shows that from 1980 to 2006, there has been a 41% decrease in the nationwide average NO

2 concentration (US EPA, 2008a).

Figure 3 (from US EPA, 2008a) summarizes 2003–2005 ambi-ent NO

2 monitoring data for all US monitors located within

metropolitan statistical areas (MSAs), showing box plots for several different averaging times ranging from 1 h to 1 year. As shown in this figure, in recent years (2003–2005), annual average US concentrations were approximately 0.015 ppm, with a maximum of approximately 0.04 ppm (US EPA, 2008a). Importantly, as reflected in this figure, short-term hourly NO

2 concentrations in US cities rarely exceed 0.2 ppm, and

exceedances of 0.1 ppm are also relatively infrequent.The relationships between concentrations for the differ-

ent averaging times, especially 1-h maximum versus annual average, are noteworthy because these relationships provide insights as to how a standard set for one averaging time may also be protective of health effects observed for an alterna-tive averaging time. As shown in Figure 3, the mean value for 1-h maximum NO

2 measurements is about 30 ppb, or

approximately 2 times the mean value of 15 ppb for annual average concentrations. At the 99th percentile, the 1-h maxi-mum NO

2 concentration is about 70 ppb, again about 2 times

the maximum annual average value of ~35 ppb. As discussed

in US EPA (2008b), US EPA concluded during the prior NO2

NAAQS review that the annual NO2 standard was sufficiently

protective to limit the occurrence of 1-h NO2 concentrations

above 200 ppb. Expanding upon its prior analysis, US EPA (2008b) provides extensive analyses evaluating the protec-tiveness of the current annual average NO

2 standard for

short-term (e.g., 1-h and 24-h) NO2 exposures, and alterna-

tively, the protectiveness of different levels of a 1-h stand-ard (98th percentile) for mean annual NO

2 concentrations.

These analyses demonstrated some variability across loca-tions in the ratio of short-term to annual average NO

2 con-

centrations, particularly for the ratio of 1-h daily maximum concentrations (98th and 99th percentile) to annual average concentrations. Based on these updated analyses, US EPA (2008b) concluded that different levels of control would be needed across locations if an annual average standard was used to also protect against a range of 1-h NO

2 exposures of

health relevance (e.g., 100–300 ppb).Considerable indoor NO

2 exposure may also result from

a variety of common household sources. Indoor sources of

Year1970 1980 1990 2000 2010

Mill

ions

of T

ons

Per

Yea

r

16

18

20

22

24

26

28

Figure 1. US EPA National Emissions Inventory (NEI) NOx Emissions

Estimates (generally excluding fires and dust). Note that in 1996, US EPA refined its methods for estimating emissions. Prior to 1990, NO

x estimates

include emissions from fires, but fires generally contributed only a small percentage of the total NO

x emissions. Data from US EPA (2008d).

Con

cent

ratio

n (p

pm) 0.05

0.04

0.03

0.02

0.01

0.00

0.06National Standard

1980

1981

1982

1983

1984

1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

Figure 2. Nationwide trend in NO2 air quality, 1980–2006, based on

annual arithmetic average concentrations for 87 monitoring sites. The white line shows the mean values, and the upper and lower borders of the blue (shaded) areas represent the 10th and 90th percentile values. From US EPA (2008a).

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NO2 include tobacco burning, wood stoves, candles, and the

use of gas-fired appliances and oil stoves. Indoor concen-trations may sometimes exceed those outdoors, especially with the use of unvented combustion appliances. The aver-age concentration over a period of several days may exceed 0.1 ppm when unvented gas stoves are used for cooking, supplementary heating, or clothes drying (WHO, 2000). Maximum 1-h concentrations in a kitchen or with a room heater may be in the range of 0.4–1.5 ppm (US EPA, 2008a) and can reach concentrations well above those measured in ambient outdoor locations. Therefore, aside from ambient outdoor exposures to NO

2, total personal NO

2 exposure may

be highly impacted by personal activities, which in fact may dominate short-term exposures.

Review of key epidemiological study findings

US EPA’s NO2 Risk and Exposure Assessment (2008b) identi-

fies a handful of epidemiology studies of short-term ambient NO

2 concentrations as constituting “key” studies supporting

a range of potential short-term NO2 standard levels (Table 1).

Even though some of these studies did report positive and statistically significant associations between short-term NO

2

levels and respiratory health outcomes, closer examination of these studies reveals major limitations and large uncer-tainties in their interpretation. The epidemiologic evidence for short-term NO

2 is particularly limited and inconsistent

compared to that available for other criteria air pollutants, with exposure misclassification, residual confounding, and co-pollutant effects potentially complicating its interpreta-tion. Although US EPA relies heavily on the epidemiologic evidence to support its causal determination for respiratory health effects and short-term NO

2 exposure, it acknowledged

in the ISA that “It is difficult to determine from these new studies the extent to which NO

2 is independently associated

with respiratory effects or if NO2 is a marker for the effects

of another traffic-related pollutant or mix of pollutants” (US EPA 2008a).

The key NO2 observational epidemiology highlighted in

US EPA’s Risk and Exposure Assessment includes the time-series studies conducted by Peel et al. (2005) and Tolbert et al. (2007) of respiratory emergency department (ED) visits in the Atlanta metropolitan area, by the New York Department of Health (NYDOH) (2006) and Ito et al. (2007) of asthma ED visits in New York City, and by Jaffe et al. (2003) of asthma ED visits in three Ohio cities. Described as report-ing positive and statistically significant associations with 1-h daily maximum levels of NO

2 close to 0.1 ppm, these

studies are identified by US EPA as key studies relevant to setting the appropriate upper end for the range of possible 1-h daily maximum standard levels. Although each of these studies reported statistically significant positive associations between respiratory-related ED visits and short-term NO

2

concentrations (actually, two associations for 1-h maximum NO

2 concentrations and two associations for mean daily NO

2

concentrations, rather than all for 1-h maximum NO2 levels

as indicated by US EPA, 2008b), notably, each of the studies

also reported similar and oftentimes larger effect estimates for a number of other components of the complex ambient air pollution mixture. In particular, both the Peel et al. (2005) and Tolbert et al. (2007) studies reported positive and statis-tically significant associations between ED visits for all res-piratory diseases and 24-h PM

10 (particulate matter with an

aerodynamic diameter of ≤10 µm), 8-h ozone (O3), 1-h NO

2,

and 1-h carbon monoxide (CO) levels, whereas Peel et al. (2005) reported positive and statistically significant asso-ciations between pediatric asthma ED visits and 24-h PM

10,

1-h NO2, and 1-h CO levels. The Tolbert et al. (2007) study is

an updated analysis of the same Atlanta dataset used in the Peel et al. (2005) study, but with four additional years of fol-low-up. For data analyzed using multi-pollutant models, the 2007 study concluded that PM

10 and ozone were the strong-

est predictors of respiratory ED visits in their Atlanta dataset. Similar to the Atlanta analyses, NYDOH (2006) analyses revealed positive and statistically significant associations for 5 of the 14 pollutants they examined (daily 8-h maximum O

3,

mean daily NO2, mean daily sulfur dioxide [SO

2], mean daily

PM2.5

[particulate matter with an aerodynamic diameter of ≤2.5 µm], and maximum 1-h PM

2.5) and asthma ED visits in

New York City’s Bronx borough. Two- and three-pollutant models indicated O

3, SO

2, and PM

2.5 to be the most robust

predictors. Similarly, Ito et al. (2007) reported positive and statistically significant associations between asthma ED visits in New York City and each of the pollutants (PM

2.5,

O3, NO

2, SO

2, and CO) they included in their models. Even

though US EPA (2008b) highlights Jaffe et al. (2003) as a key US study supporting the linkage between NO

2 and increases

in asthma-related ED visits, this study did not show any statistically significant increases in ED visits with 24-h NO

2

concentrations in their analyses of 5 years of data for two of the three Ohio cities (Cincinnati and Cleveland).

As discussed by US EPA (2008b), the Ito et al. (2007) study is the only one of the five studies that did not show sig-nificant attenuation of NO

2 associations in multi-pollutant

models (note that the Jaffe et al. study did not include any multi-pollutant models). However, although their positive associations between 24-h NO

2 levels and asthma ED visits

for warm season data in New York City did not lose statis-tical significance in two-pollutant models, Ito et al. (2007) did not interpret these findings as necessarily indicating a pollutant-specific NO

2 effect. Instead, because NO

2 was

the pollutant most negatively associated with wind speed, they observed that it may be serving as a good indicator of general local air stagnation, and ultimately concluded that NO

2 “may be a good indicator of more air pollution

from local pollution sources.” Referring to the hypothesis that NO

2 may be a surrogate for ultrafine particles, Ito et al.

(2007) further postulated that “NO2 may also be a marker of

another agent that may not be measured routinely and yet has some potential health effects.” These observations by the study authors are not discussed by US EPA (2008b), nor is it mentioned that each of these four studies reported a number of analyses where either positive NO

2 associations

lacked statistical significance or negative associations were

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Table 1. Summary of ambient NO2 epidemiology studies highlighted in US EPA (2008b) as key studies of relevance to setting a short-term NO

2 standard

level.

Reference, study location, population, and time period Study type/methods NO

2 levels (ppb)

Co-pollutants considered

Statistically significant associations for NO

2

adverse health effectsInterpretations/author’s conclusions

Delfino et al. (2002)Southern California (Alpine, CA)March to April 1996

Daily panel study of 22 asthmatic children (9–19 years of age).Regression analyses performed on binary asthma symptom scores using generalized estimating equations (GEEs); subjects stratified by whether they are on anti-inflammatory medications.

Mean 1-h max of 24, with SD of 10 (range: 8–53); Mean 8-h max of 15, with SD of 7 (range: 6–34)

O3, PM

10, fungi,

pollenOdds ratio (OR) of 1.91 (95% CI: 1.07, 3.39) for risk of asthma symptoms per increase to 90th percentile of exposure for 8-h max NO

2 (lag 0, subjects not

on medication).Odds ratio (OR) of 6.72 (95% CI: 1.73, 26.1) for risk of asthma symptoms from a 90th percentile increase in exposure to 8-h max NO

2 (lag 0) for subjects

reporting a respiratory infection versus those without an infection.None observed between 1-h maximum NO

2 and asthma

episodes.

Authors concluded that “most robust associations” were for lag 0 and 3-day moving averages (lags 0–2) of 8-h maximum and 24-h mean PM

10.

Ito et al. (2007) New York City 1999–2002

Time-series study of daily asthma ED visits Poisson’s Generalized Linear Model used in both single- and multi-pollutant analyses

All-year mean of 31.1 (SD = 8.7)

PM2.5

, O3, SO

2, CO RR of 1.14 (95% CI: 1.09,

1.19) per daily NO2 incre-

ment of 24 ppb for single-pollutant analyses model of all-year data (lag 0).RR of 1.32 (95% CI: 1.23, 1.42) per daily NO

2 increment of

24 ppb for single-pollutant model for warm months only (lag 0).RRs retained statistical significance in multi-pollutant models.

Based on their analyses showing NO

2 to be the

pollutant most nega-tively associated with wind speed, authors observed that it may be serving as a good indicator of general local air stagnation.Ito et al. (2007) postulated that “NO

2 may also be

a marker of another agent that may not be measured routinely and yet has some potential health effects.”

Jaffe et al. (2003) Cincinnati, Cleveland, and Columbus (OH) July 1, 1991, to June 30, 1996

Time-series study of daily asthma ED visits Conducted Poisson regression analyses using a standard gen-eralized additive model (GAM) approach, single-pollutant models only

24-h means (and SDs) of 50 (15) and 48 (16) for Cincinnati and Cleveland, respectively (NO

2 not measured in

Columbus)

O3, PM

10, SO

2None. Authors concluded that

regression results allow for estimation of dose-response for NO

2.US

EPA (2008a) shared this conclusion, observing that findings show no increased NO

2 risk until

minimum concentration of 40 ppb reached.

Linn et al. (2000) Los Angeles 1992–1995

Time-series study of daily hospital admis-sions for cardiop-ulmonary illnesses Used several statistical techniques, includ-ing Poisson regression analyses (single-pollut-ant models only)

Overall mean ± SD of 34 ± 13 (range of 7–91)

CO, PM10

, O3

For analyses of year-round data, a 10 ppb NO

2 incre-

ment in same-day NO2

levels was associated with % increases (SEs) of 1.4% (0.2%) for cardiovascular hospital admissions (HAs), 0.7% (0.3%) for pulmo-nary HAs, 0.4% (0.2%) for abdominal HAs, 1.4% (0.5%) for asthma HAs, and 0.8% (0.4%) for COPD HAs.

Authors cautioned that “We could not distin-guish clearly among CO-, NO

2- and particle-

associated effects.”

Table 1. Continued on next page

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Table 1. Continued.

Reference, study location, population, and time period Study type/methods NO

2 levels (ppb)



2


NYDOH (2006) New York City January 1999 to November 2000

Time-series study of daily asthma ED visits in two NYC communities (mid-town Manhattan and South Bronx)Conducted Poisson regression analyses, including single- and multi-pollutant models

Means of 31 and 36 for the South Bronx and Manhattan, respectively

O3, SO

2, PM

2.5,

Coarse PM, sulfate, elemental carbon, organic carbon, total metals, total alde-hydes, pollen, mold

For analyses of the South Bronx data, observed RR of 1.06 (95% CI: 1.01, 1.10) for a 20 ppb increment in 24-h average NO

2 conc.

(lag 0–4 days). RR retained statistical significant in two-pollutant model with O

3, but lost statisti-

cal significance in other two-pollutant models.Nonsignificant RRs < 1 found for analyses of Manhattan data.

Authors concluded based on two-pollutant and three-pollutant regression models, “O

3 and SO

2, and to

a lesser extent maxi-mum one-hour PM

2.5,

were the most robust pollutants.”Also con-cluded that “the high correlations between pollutants (including components of PM

2.5)

make it difficult in these epidemiologic studies to confidently identify criti-cal compounds.”

Ostro et al. (2001) Central Los Angeles/Pasadena August to November 1993

Panel study of 138 asthmatic African-American children Used generalized estimating equations to conduct time-series analyses of daily data on respiratory symptoms and medication use

For downtown LA: mean 1-h maximum of 79.5 with SD of 43.6 (range of 22–220) For Pasadena: mean 1-h maximum of 68.1 with SD of 31.3 (range of 30–170)

O3, PM

2.5, PM

10,

fungi, pollensOR of 1.12 (95% CI: 1.00, 1.24) and 1.13 (95% CI: 1.04–1.24) for 50 ppb increment in 1-h max. NO

2 and new episodes

of cough and wheeze, respectively (lag = 3 days).OR of 1.08 (95% CI: 1.02, 1.15) for 50 ppb increment in 1-h max. NO

2 and likeli-

hood of wheeze symptoms (lag = 3 days).NO

2 not

associated with use of extra asthma medication.

Significant asso-ciations found between number of co-pollutants and short-ness of breath (PM

10,

PM2.5

, Cladosporium, Alternaria), cough (PM

10,

PM2.5

, Cladosporium, Alternaria), and wheeze (PM

10, Cladosporium,

Alternaria), with PM10

showing the strong-est associations.In the Discussion to their paper, Ostro et al. stress the importance of their findings for particulate matter and spore counts, never once referring to their findings for NO

2.

Peel et al. (2005)Atlanta January 1993 through August 2000

Time-series study of res-piratory ED visits Used Poisson generalized estimating equations to analyze data from 31 Atlanta-area hospitals, included multi-pollutant models

Overall mean ± SD of 45.9 ± 17.3 for 1-h NO

2

PM10

, O3, CO, SO

2,

PM2.5

, coarse PM, 10-100 nm particle count, oxygenated hydrocarbon, PM

2.5

components (water-soluble metals, sul-fate, acidity, organic carbon, elemental carbon)

For 20 ppb increment in 1-h max. NO

2, found RR

of 1.016 (95% CI: 1.006, 1.027) for all respiratory ED visits, 1.019 (95% CI: 1.006, 1.031) for upper respira-tory infection ED visits, and 1.035 (95% CI: 1.006, 1.065) for COPD ED visits (3-day moving lag of 0, 1, and 2 days).For age-specific analyses for pediatric asthma visits (ages 2–18), observed RR for 20 ppb increment in 1-h NO

2 of 1.027 (95% CI:

1.005, 1.050).

Peel et al. (2005) also reported statistically significant associations between ED visits for all respiratory diseases and 24-h PM

10, 8-h

ozone (O3), and 1-h

carbon monoxide (CO) levels, and statistically significant associa-tions between pediatric asthma ED visits and 24-h PM

10 and 1-h CO

levels.Authors reported that “the estimates for NO

2 were generally not

attenuated in multi-pollutant models, while the estimates for the other pollutants showed weaker or no associa-tions in the multi-pollut-ant models.”


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observed between NO2 and a health endpoint (i.e., dimin-

ished health impact with increasing NO2 concentrations).

For example, NYDOH (2006) reported negative (but not statistically significant) associations between daily mean NO

2 levels and asthma ED visits for both single-pollutant

and two-pollutant models of Manhattan ED data (in con-trast to ED data for the Bronx). In fact, each of the studies included a large number of models and tests, increasing the likelihood of positive and “statistically significant” associa-tions arising by chance alone.

Reviewing the actual study publications rather than just reading the brief US EPA (2008a, 2008b) descriptions of these four studies makes it apparent that the study authors them-selves are more convinced in the robustness and importance of their findings for other, non-NO

2 pollutants (e.g., PM

2.5,

PM10

, O3) rather than for NO

2 per se. For example, both Peel

et al. (2005) and Tolbert et al. (2007) concluded that ozone and PM

10 were more consistent predictors than NO

2 of

Atlanta-area respiratory ED visits, yet US EPA relied on find-ings from these studies to conduct their epidemiology-based NO

2 risk assessment. The authors of other “key” studies cited

by US EPA (2008b) have similarly highlighted associations for other pollutants as being of greater importance than their NO

2 findings, including the Ostro et al. (2001) time-series

study of asthma exacerbation in African-American children in Los Angeles. Importantly, Ostro et al. is identified by US EPA (2008b) as one of two key epidemiologic studies report-ing positive and statistically significant NO

2 effect estimates

for some of the highest 1-h NO2 concentrations in the United

States (the Linn et al. [2000] time-series study of air pollution and daily hospital admissions for cardiopulmonary illnesses in metropolitan Los Angeles is the other).

As indicated in US EPA (2008b), Ostro et al. (2001) reported positive and statistically significant associations between 1-h maximum NO

2 concentrations and new episodes of cough

and wheeze in a panel of 138 asthmatic children in central Los Angeles. However, US EPA did not point out that these investigators also reported positive and statistically sig-nificant associations between new episodes of wheeze and cough and 24-h average PM

10, 12-h average PM

2.5, and 24-h

average concentrations of two bioaerosols (Cladosporium and Alternaria), and additionally between 1-h maximum PM

10 and new episodes of cough. In the Discussion to their

paper, Ostro et al. (2001) stressed the importance of their findings for particulate matter and spore counts, never once referring to their findings for NO

2. Linn et al. (2000) include

the caveat: “We could not distinguish clearly among CO-, NO

2- and particle-associated effects.” As mentioned by US

EPA (2008b), neither of these two studies included any eval-uation of multi-pollutant models, thus limiting their utility for assessing the independent association between NO

2 and

respiratory morbidity.One other key study highlighted by US EPA (2008b) in the

NO2 Exposure and Risk Assessment is the Delfino et al. (2002)

panel study of daily asthma symptoms among 22 asthmatic children living in a semirural area in Southern California. US EPA (2008b) characterizes this study, where 98th and 99th percentile 1-h daily maximum NO

2 concentrations were

0.050 and 0.053 ppm, respectively, as providing evidence for NO

2 respiratory effect associations in locales with lower NO

2

levels and thus as being particularly relevant to the question of what is an appropriate lower end of the range of possible short-term NO

2 standard levels. Specifically, US EPA (2008b)

uses it in support of a lower end of 0.05 ppm in the range of

Table 1. Continued.

Reference, study location, population, and time period Study type/methods NO2 levels (ppb)



2


Tolbert et al. (2007)Atlanta January 1993 through December 2004 (an update to the Peel et al., 2005 study, with 4 more years of data and ED data from 10 more hospitals)

Time-series study of cardiorespiratory ED visits Used Poisson generalized linear models to analyze data from 41 Atlanta-area hospitals, included multi-pollutant models

Overall mean ± SD of 43.2 (range: 1.0–181) for 1-h NO

2

PM10

, O3, CO, SO

2,

PM2.5

, coarse PM, 10-100 nm particle count, oxygenated hydrocarbon, PM

2.5

components (water-soluble metals, sulfate, acidity, organic carbon, elemental carbon, total carbon)

For 23 ppb increment in 1-h max. NO

2, found RR

of 1.015 (95% CI: 1.004, 1.025) for combined cardiovascular disease ED visits, and RR of 1.015 (95% CI: 1.004, 1.025) for combined respiratory disease ED visits (3-day moving lag of 0, 1, and 2 days).

In contrast to earlier Peel et al. (2005) analysis, multi-pollutant analyses showed significant attenuation of NO

2 associations,

with NO2 associations

for combined cardiovascular diseases group changing from positive to negative associations. NO

2

associations for combined respiratory diseases group were generally reduced and lost statistical significance.Tolbert et al. concluded that PM

10 and

ozone were the strongest predictors of respiratory ED visits.

Abbreviations: CI = confidence interval; COPD = chronic obstructive pulmonary disease; ED = emergency department; ER = excess risk; OR= odds ratio; RR = relative risk; SD = standard deviation.

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potential alternative 1-h daily maximum standards, despite the fact that Delfino et al. (2002) did not report any positive statistically significant associations between 1-h maximum NO

2 and asthma episodes. Delfino et al. (2002) did report

a statistically significant positive association between 8-h maximum NO

2 levels and asthma episodes in a subgroup of

12 asthmatic children not taking anti-inflammatory medica-tions, although they observed similar associations for 1-h maximum PM

10, 8-h maximum PM

10, 24-h mean PM

10, and

12-h fungal-spore levels, and they ultimately concluded that PM

10 showed the strongest and most robust associations.

Furthermore, it is important to point out that Delfino et al. (2002) reported statistically significant correlations between 8-h maximum NO

2 and every other independent variable

assessed in their study (i.e., 8-h maximum O3, 1-h maximum

PM10

, 24-h mean PM10

, 1-h maximum NO2, 12-h daytime

fungal spores, 24-h pollen, maximum temperature, and 24-h mean relative humidity), with similar findings for 1-h maxi-mum NO

2 (with the exception of 8-h maximum O

3 where the

positive correlation did not achieve statistical significance). Such co-pollutant correlations diminish the plausibility of attributing effects to any one pollutant.

Delfino et al. (2002) pointed to reliance on central-site ambient monitors to represent personal pollutant expo-sures as a major limitation to their analyses, claiming that the possible effects of exposure misclassification were greater for gaseous pollutants than PM. Importantly, more recent research by Delfino et al. (2008) illustrates the poten-tially large impact that exposure misclassification can have on the interpretation of NO

2 epidemiologic associations. In

their panel study of 53 Los Angeles children with asthma, Delfino et al. (2008) observed, in single-pollutant analyses, statistically significant associations between personal, daily exposure to NO

2 (pNO

2) and decrements in lung function

(specifically, measurements of forced expiratory volume in 1 second, or FEV

1), with weaker but still consistent and

statistically significant associations for ambient exposure to daily NO

2. However, in two-pollutant models, Delfino

et al. (2008) observed both personal PM2.5

(pPM2.5

) and pNO

2 exposures to have “largely independent effects” on

FEV1, with both factors confounding the ambient-NO

2 asso-

ciations, such that the ambient-NO2 associations were sub-

stantially reduced and lost statistical significance. Although Delfino et al. (2008) considered the possibility that the associations with pNO

2 may reflect NO

2-specific toxicity,

they ultimately concluded that the low personal NO2 levels

observed in their study were more likely serving as a sur-rogate for other air pollutants. As support for this conclu-sion, they cited human clinical studies of adults with mild asthma that generally observed adverse pulmonary effects of NO

2 per se only at concentrations more than an order of

magnitude higher than the ambient levels measured for their panel.

In reaching their conclusion that personal PM2.5

levels were more likely to represent a causal factor underlying the observed FEV

1 associations than personal NO

2, Delfino

et al. (2008) referred to the growing body of studies that have reported poor (and in some cases, negative) associations between ambient NO

2 concentrations and personal NO

2

exposures. As summarized in Sarnat et al. (2007), a series of panel-based exposure assessment studies conducted in four US cities (Baltimore, Boston, Steubenville, and Atlanta) generally observed weak and statistically insignificant cor-relations between personal and ambient NO

2 (Figure 4).

Only for a Steubenville (Ohio) panel, where subjects were clustered around the ambient monitoring site, were stronger personal-to-ambient NO

2 associations observed (i.e., sub-

ject-specific Spearman’s correlation coefficients generally above 0.50). In contrast, these studies (Sarnat et al., 2001, 2005, 2006) have generally reported much stronger per-sonal-ambient associations for other air pollutants, such as PM

2.5 and particulate sulfates (SO

42−). As observed by Sarnat

et al. (2007), “The findings from these four studies of weak personal-ambient associations for SO

2 and NO

2 suggest

that central-site ambient site measurements may not be good surrogates of their respective personal exposures for many individuals. Moreover, these results raise questions concerning the interpretation of epidemiologic findings showing significant health effects associated with ambient NO

2 concentrations.”

Con

cent

ratio

n (p

pb)

100201 201 129

9080706050403020100

1-h max 1-h 24-h 2 week 1-year

Max9995

7550255

Mean

Figure 3. Ambient concentrations of NO2 measured at all US monitoring sites located within metropolitan statistical areas (MSAs) from 2003 to 2005.

Note definition of box plot symbols at the right-hand edge of the graph. Box plots are shown for both daily maximum 1-h concentrations (1-h max) as well as all hourly average concentrations (1-h). From US EPA (2008a).

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Further, findings from Brook et al. (2007) provide support for the idea that NO

2 could be serving as a surrogate for one

or more infrequently measured hazardous air pollutants rather than just routinely measured criteria air pollutants. Working with a comprehensive ambient measurement data-set that included measurements of PM

2.5, PM

2.5 elemental

constituents, gaseous criteria pollutants (NO2, NO, O

3, CO),

volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs), and particle-phase hopanes and steranes, Brook et al. (2007) observed strong correlations between ambient NO

2 concentrations and concentrations

of a number of gaseous and particulate-phase species. For example, Brook et al. (2007) generally observed strong cor-relations between NO

2 and a variety of VOCs, including 1,3-

butadiene, benzene, toluene, ethylbenzene, xylenes, and acetaldehyde (average correlation coefficients r of .47–.67). Because Brook et al. (2007) observed stronger correlations between NO

2 and particle-bound organic species such as

benzo(e)pyrene and hopanes than between PM2.5

and these species, they suggested that NO

2 could also be acting as a

surrogate of exposure to particle-bound organic species. Brook et al. (2007) questioned the role of ambient NO

2 in the

reported NO2 epidemiologic associations by concluding “The

results support the hypothesis that NO2 is a better indicator

than PM2.5

of a range of other toxic pollutants. This includes VOCs, aldehydes, NO

z [other oxidized nitrogen species, such

as HNO3, peroxyacetyl nitrate, N

2O

5, particle nitrate and gas

and particle phase organic nitrates] and particle-bound organics in motor vehicle exhaust. Thus, overall, the strong effect of NO

2 in Canadian cities could be a result of it being

the best indicator, among the pollutants monitored, of fresh combustion (likely motor vehicles) as well as photochemi-cally processed air.”

US EPA (2008a, 2008b) acknowledges that exposure mis-classification and co-pollutant effects remain potentially significant issues hindering the interpretation of ambient NO

2 epidemiologic studies and place emphasis on indoor

NO2 studies, particularly the Pilotto et al. (2004) Australian

school intervention study, as providing key support for their “causal” interpretation of the respiratory morbidity health effects evidence for short-term NO

2 exposures. However,

although it may be true that indoor NO2 studies are unlikely

to be confounded by other ambient co-pollutants to the same degree as ambient NO

2 epidemiology studies, this

does not mean that confounding by co-pollutants is a nonis-sue for indoor NO

2 studies. For example, the Pilotto et al.

(2004) intervention study examined differences in both respiratory symptoms and lung function among schoolchil-dren with asthma in 18 Australian schools, including 10 that retained their unvented gas heaters (control schools) and 8 that were given replacement vented gas or electric heaters at the beginning of winter (intervention schools). When com-pared to the control (nonintervention) schools, significant reductions were observed in several symptoms associated with asthma (e.g., difficulty breathing during the day, diffi-culty breathing during the night, chest tightness during the day, and daytime asthmatic attacks), but no differences in lung function measures were observed. These differences in respiratory symptoms between the two schools have been attributed to reduced NO

2 levels in the intervention schools

(the intervention schools had mean NO2 levels of approxi-

mately 15 ppb during the 12-week study compared to mean NO

2 levels of 47 ppb in the control schools), but it should be

noted that Pilotto et al. (2004) made no measurements of air pollutants other than indoor NO

2. In other words, evidence

indicates that indoor NO2 concentrations from gas-fired

appliances can be correlated with a mixture of pollutants, in particular other major by-products of natural gas combus-tion, including ultrafine particles, CO, formaldehyde, and a number of gas- and particulate-phase oxidized organic compounds that include PAHs (US EPA, 2008a; Dennekamp et al., 2001), yet Pilotto et al. (2004) did not collect additional measurement data that could have been used to examine potential confounding effects from indoor co-pollutants.

Although of lesser relevance to this analysis of the health-effects evidence for short-term NO

2 exposures than

the short-term epidemiology studies discussed above, it is also important to note that the air pollution cohort studies highlighted by US EPA as providing key evidence in sup-port of PM mortality effects (US EPA, 2008c) have generally reported only inconsistent and equivocal findings for NO

2.

In fact, based on these generally inconsistent findings, US EPA (2008a) concluded that the evidence linking long-term NO

2 exposures with mortality was “inadequate to infer the

presence or absence of a causal relationship.” Although NO2

findings were not provided in the original Dockery et al. (1993) publication of the Harvard Six-Cities Study prospec-tive cohort data, Krewski et al. (2000) reported NO

2 asso-

ciations in their reanalysis of the Harvard Six-Cities Study

Atlanta Baltimore Boston Steubenville

Spe

arm

an’s

Cor

rela

tion

Coe

ffici

ent

–1.0

–0.8

–0.6

–0.4

–0.2

0.0

0.2

0.4

0.6

0.8

1.0

Atl SprAtl FallBalt Sum

Balt WinBos SumBos Win

Steu SumSteu FallOverall Medians

**

** **

Figure 4. Distribution of subject-specific Spearman’s correlation coef-ficients for personal-to-ambient NO

2 by cities studied in the Harvard

panel studies. Triangles represent the median correlation coefficient. **Significance at p < .0001. Adapted from Sarnat et al. (2007).

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data that included a 14–16-year mortality follow-up of over 8000 adults in the six cities (Watertown, MA; Kingston and Harriman, TN; St. Louis, MO; Steubenville, OH; Portage, Wyocena, and Pardeeville, WI; and Topeka, KS). Consistent with the Dockery et al. (1993) findings showing statisti-cally significant positive associations between mortality and nearly all of the pollutants under investigation (e.g., PM

2.5, sulfate, Total Suspended Particulates (TSP), SO

2,

aerosol acidity), Krewski et al. (2000) reported significant associations between long-term NO

2 exposure and both

all-cause mortality (RR= 1.25, 95% CI: 1.07, 1.46, for a 15.8-ppb increment in NO

2 corresponding to the difference in

the means of the cities with the highest and lowest annual NO

2 levels) and mortality from cardiopulmonary disease

(RR= 1.28, 95% CI: 1.04, 1.59, again for a 15.8-ppb NO2

increment). In contrast, in their reanalysis of the American Cancer Society (ACS) prospective cohort study data cover-ing 151 metropolitan statistical areas (MSAs) and approxi-mately 500,000 individuals, Pope et al. (2002) reported a general absence of statistically significant mortality rela-tive risks for NO

2, with in fact, weak, negative associations

for cardiopulmonary mortality and lung cancer mortality. In the most recent reanalysis of NO

2 data representative of

over 400,000 ACS study participants residing in 76 MSAs in 1980, Krewski et al. (2009) reported that NO

2 was not

strongly associated with any cause-of-death category (leass than ±2% per 10 ppb NO

2), including all-cause, car-

diopulmonary, lung cancer, ischemic heart disease (IHD), and all other causes mortality. Importantly, other recent of reanalyses of the Harvard Six-Cities Data (Laden et al., 2006) and ACS cohort data (e.g., Jerrett et al., 2005, 2009; Pope et al., 2009) have focused on pollutants such as PM

2.5

and ozone, without reporting any analyses for NO2.

As a whole, troubling inconsistencies and uncertain-ties thus remain in the NO

2 epidemiology, with studies

of respiratory-related health outcomes reporting a range of both positive and negative associations. Even for those studies reporting positive and statistically significant associations between short-term ambient NO

2 levels and

respiratory morbidity, effect estimates are generally small (e.g., relative risks close to 1.0) and become attenuated in multi-pollutant models. As discussed recently by Boffetta et al. (2008) and Fewell et al. (2007), it is plausible that relative risks on the order of 1.5–2.0 (let alone just above 1.0) can be explained by residual and/or unmeasured confounding. Certainly, residual confounding cannot be readily dismissed for ambient NO

2 epidemiology studies,

given that NO2 levels are often highly correlated with other

co-pollutants such as PM, O3, and CO, and evidence sup-

ports the potential for extensive exposure misclassification in NO

2 studies (Latza et al., 2008). NO

2 epidemiology stud-

ies thus do not offer a clear NO2-specific signal, with both

US EPA (2008a) and air pollution epidemiologists (e.g., Delfino et al., 2008; Ito et al., 2007) raising the possibility that NO

2 may be a surrogate marker for the adverse health

effects of other components of the complex air pollution

mixture, or for the ambient air pollution mixture more generally.

Review of clinical studies of NO2-induced health effects

We now focus on the body of clinical studies that have exam-ined the health effects of NO

2 in human volunteers. Clinical

exposures have been performed on subjects in different age groups and with diverse health backgrounds. Clinical study exposure protocols are generally of short term dura-tion (30 min to 6 h) and have employed NO

2 concentrations

generally much higher (0.1 to 3 ppm) than those typically encountered in outdoor environments. Control exposures generally involve filtered or unfiltered ambient air in which NO

2 concentrations are lower than 0.05 ppm. In this review,

we will refer to control exposures as “clean air” (CA). We sys-tematically review study findings for three broad classes of health outcomes examined in clinical studies of NO

2 health

effects: (1) Immune response and inflammation (also sum-marized in Table 2); (2) lung function and AHR to specific or nonspecific stimuli (also summarized in Tables 3, 4, and 5); and (3) extrapulmonary health effects.

Background on pulmonary immune effectsThe process of acute pulmonary inflammation is an innate immune response that may be initiated by cells already present in all tissues, mainly resident macrophages. Once activated by an injurious agent (infection, xenobiotics, etc.), macrophages undergo activation and release proin-flammatory and vasoactive mediators (tumor necrosis factor [TNF]-α, interleukins, prostaglandins, histamine, etc.). The mediator molecules can alter the blood ves-sels to permit the migration of leukocytes into the lung, mainly neutrophils and eosinophils, which are known as polymorphonuclear cells (PMNs) due to their multilobed nuclei. The neutrophils migrate along a chemotactic gradi-ent created by the local cells to reach the site of injury and release more mediators such as interleukins, proteases, and reactive oxidant species to help combat and limit the injurious agent.

Several reviews of NO2-induced health effects suggest

that NO2 inhalation can trigger proinflammatory responses

and enhance allergic responses in the lung, which can induce lung function changes, promote pulmonary infec-tions, and exacerbate existing lung disease (Krishna and Holgate, 1999; Chauhan et al., 2003; US EPA, 2008a). To better understand the potential mechanisms of action, clinical studies have examined both the inflammatory potential and resistance to infection associated with acute NO

2 exposures. These studies have assessed inflammatory

changes in the respiratory tract by looking at changes in pulmonary PMN levels, proinflammatory cytokines, and prostaglandins. In addition, they have assessed the defense functions of macrophages ex vivo (i.e., collected from the lung and analyzed outside it).

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Table 2. Key clinical studies of NO2-induced immune responses.

Reference Exposed populationNO

2exposure

characteristics

Nature and timing of studied immune responses

Key statistically significant NO2-induced effects Key negative findings

Vagaggini et al. 1996 7 healthy and 8 asthmatic adults and 7 elderly subjects having COPD (mean ages 34, 29, and 58, respectively)

CA, 0.3 ppm, 60 min with exercise

Leukocyte count in sputum and nasal lavage 2 h post exposure

Mild decrease in FEV1 (9%)

2 h after NO2 exposure in

COPD patients. Symptom score showed a mild increase after NO

2

exposure both in normal subjects and in COPD patients.

No change in leukocyte numbers in sputum or nasal lavage fluid. No significant change in FEV

1 in healthy and

asthmatic subjects. No change in symptom frequency in asthmatic subjects.

Gong et al. 2005 6 healthy elderly and 18 elderly hav-ing COPD (mean ages 68 and 72, respectively)

CA, 0.4 ppm, 2 h with exercise

Sputum leukocyte count 22 h post exposure

None. No change in leuko-cyte levels in sputum.

Rubinstein et al. 1991 5 healthy adults (ages 21–36)

CA, 0.6 ppm, 2 h with exercise followed by a SO

2 challenge

BALF and peripheral blood cell counts. Before and 2 h post exposure

Increase in natural killer cells.

No change in BALF or peripheral blood lymphocyte levels. No clinically signifi-cant symptoms.

Frampton et al. 2002 21 healthy adults (mean age 27)

CA, 0.6 or 1.5 ppm, 3 h with exercise

Bronchial and BAL fluids and peripheral blood assessment for cell types, LDH, total protein, and ex vivo macrophage function with virus or respiratory burst activity. 3.5 h post exposure

Decreased hematocrit and total blood lymphocytes and changed blood lymphocyte ratio with sex differences (0.6 and 1.5 ppm). Increased PMN in bronchial lavage (1.5 ppm). Bronchial epithelial cells released more LDH after challenge with virus (1.5 ppm).

No effect on lower airway cells sus-ceptibility to virus. No effects on total protein or albumin in bronchial lavage or BAL fluids. No change in PMN levels at 0.6 ppm NO

2.

Jorres et al. 1995 12 mild asthmat-ics and 8 healthy adults (mean age 27)

CA, 1 ppm, 3 h with exercise

BALF assessment of cell types, prostaglandins, leukotrienes, thrombox-ane, histamine. Before and 60 min post exposure

Small increase in TxB2 in healthy subjects. Slight decrease in FEV

1 (1.4%) and

6-keto-prostaglandin1α (6-keto-PGF1α) Increased thromboxane B2 (TxB2) and prostaglandin D2 (PGD2) in asthmatics.

No change in BALF leukocyte differen-tial. BAL prostag-landin E2 (PGE2), prostaglandin F2α (PGF2α), histamine and leukotriene levels did not change significantly in asth-matics. No change in lung function or BAL cell differential, PGE2), prostaglandin F2α (PGF2α), PGD2 or 6-keto-PGF1α histamine and leukotriene levels in healthy subjects. No change in heart rate.

Azadniv et al. 1998 24 healthy adults (mean age 28)

CA, 2 ppm, 6 h with exercise

BALF and peripheral blood for leukocyte types and macrophage function ex vivo with PMA and influenza virus. Immediately and 18 h post exposure

BALF PMN increased 1% in the leukocyte differential. Small decrease in lym-phocyte subsets in blood (18 h post exposure).

No change in other leukocyte ratios in BALF or in macro-phage function (with PMA or influenza virus activation).


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Table 2. Continued.


2exposure

characteristics


Key statistically significant NO

2-induced effects Key negative findings

Devlin et al. 1999 8 healthy adults (ages 18–35)

2 ppm, 4 h with exercise Bronchial wash and BALF assessment for cell types, IL-6, IL-8, TPA, antitrypsin, and ex vivo macrophage function with virus and in terms of respiratory burst, phagocytosis, and LDH production. 16 h post exposure

PMN, IL-6, IL-8, α1-

antitrypsin and TPA. increased in bronchial wash, Decrease in epithelial cells in BAL, alveolar macrophages show decreased respira-tory burst and decreased ability to phagocytize.

No change in cell numbers or soluble mediators in BALF. No change in macro-phage susceptibility to virus infection. No change in LDH or protein levels in bronchial or BAL fluids.

Balmes et al. 1997 15 healthy adults (mean age 29)

CA, 2 ppm 4 h with exercise

Bronchial and BAL fluid for leukocyte types and total protein. 18 h post exposure

In bronchial lavage increased % neutrophils. In BAL, decrease in % of T-helpers. Decreased total protein in bronchial wash but not BALF.

No change in total leukocytes or cell types in bronchial lavage. No changes in WBC in peripheral blood in non-T-helper lymphocytes. No change in BALF total protein level.

Posin et al. 1978 10 healthy adults CA, 1 ppm or 2 ppm, 2.5–3 h

Peripheral blood assess-ment of RBCs, G6PD, hematocrit and hemo-globin. Immediately after exposure

Increased G6PD and peroxidized RBC lipids in blood. Decreased hemoglobin, hematocrit and RBC acetylcholine esterase with 2 ppm NO

2

exposure.

No changes in blood at 1 ppm NO

2

exposure.

Avissar et al. 2000 21 (NO2) and 25 (O

3)

healthy adults (mean ages 27 and 25, respetively)

CA, 0.6 or 1.5 ppm NO2, 3

or 0.22 ppm O3, 3 h

BALF extracellular glutathione peroxi-dase (eGPx). 3.5 h post exposure and immedi-ately after and 18 h post exposure for O

3

Decrease in eGPx with O3. No change in BALF

eGPx with NO2.

Blomberg et al. 1997 30 healthy adults (mean age 25)

CA, 2 ppm, 4 h with light exercise

Bronchial biopsy and bronchial lavage and BAL fluids for leukocyte types, IL8, and adhesion molecules. 1.5 and 6 h post exposure

In bronchial wash, increased neutrophils (6 h) and IL-8 (1.5 h).

No change in adhe-sion molecules or inflammatory cell levels in bronchial biopsy. No indication of inflammation in BALF. No change in ICAM levels.

Blomberg et al. 1999 12 healthy adults (mean age 26)

CA (1 day), 2 ppm (4 h/day for 4 days with exercise)

Bronchial and BAL fluids assessment for leukocyte types, MPO, and antioxi-dant status.

Decreased neutrophils in bronchial epithelium but increased MPO and neutrophils in bronchial wash after 4 days. Small decrements in FEV

1 and

FVC only after 1st day or exposure.

No change in antioxi-dant status of bron-chial wash or BAL.

Pathmanathanet al. 20038 healthy adults CA (1 day), 2 ppm (4 h/day for 4 days with exercise)

Bronchial biopsy staining for TH2 interleukins and ICAM.

Bronchial biopsy showed increased IL-5, IL-10, IL-13, and ICAM-1 staining.

No changes in IL-6, Gro-a, NF-κB, TNF-α, GM-CSF, or eotaxin.

Helleday et al. 1995 Healthy adults CA, 1.5 or 3.5 ppm, 20 min or 4 h with exercise

Mucociliary clearance. 45 min after short expo-sure and 24 h after long exposure

Reduction in mucocili-ary activity at 45 min post exposure with 3.5 ppm exposure.

No change in muco-ciliary activity at 24 h post exposure to 3.5 ppm NO

2 or with 1.5

ppm NO2 exposure.


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This section reviews clinical studies of healthy individu-als acutely exposed to NO

2 concentrations ranging from 0.3

to 2 ppm and of susceptible subjects (e.g., subjects having asthma, rhinitis, or COPD) exposed to NO

2 at slightly lower

concentrations, ranging from 0.26 to 1 ppm. In allergic and asthmatic individuals, there is inconsistent evidence of NO

2-

induced inflammation and enhancement of the immune responses at the exposure levels used in these studies, as

only half the studies showed proinflammatory responses between 0.26 and 1 ppm and these responses were generally not accompanied by adverse symptoms or changes in respi-ratory function. At NO

2 concentrations greater than 1 ppm,

there is relatively consistent evidence of an NO2-induced

inflammatory response and altered macrophage function in healthy subjects. A summary of studies examining the pul-monary immune effects of NO

2 can be found in Table 2.

Table 2. Continued.


2exposure

characteristics


Key statistically significant NO2-induced effects Key negative findings

Kulle and Clements 1988

152 healthy adults (ages 18–35). (21–23 per group)

CA, 1 or 2 ppm, 2 h/day for 3 consecutive days. Live, attenuated cold-adapted influenza A/Korea/82 reassortant virus was administered intranasally to all subjects after the second day of exposure

Rate of infectivity with virus

None. No significant changes in virus infectivity.

Kleinman et al. 1998 15 healthy adults (mean age 29)

2 ppm, 4 h/day for 4 days with intermittent exercise

BALF assessments of macrophage function, ex vivo, with PMA and zymosan, and respiratory burst activity. 18 h after 3rd day exposure

Increased macrophage res-piratory burst with PMA.

No change in mac-rophage respiratory burst activity with zymosan.

Boushey et al. 1988 9 adult asthmatics (mean age 29)

CA, 0.6 ppm, 2 h with exercise

BALF for IL-1, TNF, and leukocyte types. Before and 2 h post exposure

None. No change in IL-1, TNF levels or leuko-cyte ratios in BALF.

Witten et al. 2005 15 asthmatic adults having HDM allergy (mean age 32)

CA or 0.4 ppm, 3 h with exercise followed by a HDM allergen challenge

Sputum leukocyte count. 6 and 26 h post exposure

Decreased sputum eosinophils at 6 h post exposure.

No change in eosi-nophil levels at 26 h or other leukocyte levels in sputum at 6 or 26 h post exposure.

Solomon et al. 2004 10 adult asthmatics (mean age 33)

CA, 0.4 ppm, 3 h at rest followed by a HDM challenge

Sputum leukocyte count. 6 and 26 h post exposure

None. No change in leukocyte counts in sputum.

Barck et al. 2002 13 mild asthmatics having allergy (mean age 28)

CA, 0.26 ppm, 30 min at rest followed 4 h later with a pollen challenge

Bronchial lavage and BAL fluid for ECP, IL-8, IL-5, ICAM, albumin, and leukocyte types. 19 h post allergen challenge

Enhanced % neutrophils in bronchial wash and BAL. Increased ECP in bronchial fluid.

ECP unchanged in BALF. IL-8, IL-5, ICAM were not differ-ent in any fluids with exposure and there was no difference in albumin levels in bronchial wash or BALF. No changes in asthma symptoms or lung function.

Barck et al. 2005 18 allergic asthmatics (mean age 32)

CA or NO2 exposures at

rest for 15 min on day 1 followed by allergen chal-lenge 4 h post exposure and 2 × 15-min exposures separated by 1 h the fol-lowing day with a similar allergen challenge 3 h post exposure

Sputum and peripheral blood assessment of cells, ECP, and MPO. On the mornings before and after exposure

Increased ECP in sputum and blood after 2 days. Increased MPO in blood after 2 days.

Eosinophil and neutrophil levels were unchanged in sputum and blood. No change in sputum MPO.

Wang et al. 1995 16 adults with seasonalallergic rhinitis (mean age 26)

CA, 0.4 ppm, 6 hat rest followed by a pollen allergen challenge

Nasal lavage fluid assessed for Mast cell tryptase (MCT), ECP, MPO, and IL-8 immediately after exposure

Increased ECP in nasal lavage with NO

2 + allergen

relative to CA + allergen.

No change in MCT, MPO, and IL-8 in nasal lavage fluid. No difference in sneezing frequency or rhinorrhea.

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Table 3. Key clinical studies of NO2-induced lung function responses without a bronchoconstrictor challenge.


2exposure

characteristics

Nature and timing of physiological responses


2-induced

effects Key negative findings

Orehek et al. 1981 7 adult subjects having allergy

CA or 0.11 ppm, 60 min at rest

Before and after exposure

None SRaw

Orehek et al. 1976 20 allergic asthmatics (mean age 26, including teenagers)

CA, 0.11 ppm, 60 min at rest

Before and immedi-ately after exposure

None SRaw, thoracic gas vol-ume (TGV)

Bylin et al. 1988 20 mild asthmatics (mean age 33)

CA, 0.14, 0.27, or 0.52 ppm, 30 min at rest

Before exposure and after 30 min of expo-sure termination

None SRaw, TGV, Raw (airways resistance)

Roger et al. 1990 21 mild asthmatics (mean age 24)

CA, 0.3 ppm CA, 0.15, 0.3, or 0.6 ppm, 3 h


SRaw, FEV1, FVC in pilot

single exposure study (0.3 ppm)

No changes in SRaw, FEV

1, or FVC in exposure

response study

Kim et al. 1991 9 healthy athletes (mean age 21)

CA, 0.18 or 0.3 ppm, 30 min


None FEV1, RT, peak expira-

tory flow rate (PEFR)

Kleinman et al. 1983 31 adult asthmatics CA or 0.2 ppm, 2 h with exercise


None FEV1, FVC, airways

resistance

Jenkins et al. 1999 11 mild asthmatics with atopy (mean age 31)

CA, 0.2 ppm, 6 h or 0.4 ppm, 3 h


None FEV1

Barck et al. 2005 18 allergic asthmatics (mean age 32)

CA, 0.26 ppm exposures at rest for 15 min on day 1 followed by allergen chal-lenge 4 h post exposure and 2 × 15-min exposures separated by 60 minthe following day

Before, during and up to 22 h post exposure

None SRaw, TGV, FEV1

Barck et al. 2002 13 mild asthmatics with allergy (mean age 28)

CA or 0.26 ppm 30 min at rest


None SRaw, TGV, FEV1

Strand et al. 1996 19 mild asthmatics (mean age 32)

CA or 0.26 ppm, 30 min Before, and 30 min, 5 h,7 h, 27 h and7 days post exposure

TGV, immediately after and 7 days post exposure

SRaw

Strand et al. 1997 18 mild asthmatics with pollen allergy (mean age 30)



None SRaw, TGV

Strand et al. 1998 16 mild asthmatics with allergy (mean age 33)



None FEV1

Salome et al. 1996 9 adults and 11 children with asthma requir-ing daily medication (mean ages 36 and 12, respectively)

CA, 0.3 or 0.6 ppm, 60 min at rest

Before and 60 min after exposure

None FEV1, FVC, PEFR

Avol et al. 1988 59 subjects with moder-ate to severe asthma (mean age 30)


Before and up to 2 h after exposure

None SRaw, TGV, FEV1, FVC

Linn et al. 1986 21 mild asthmatics (mean age 24)

CA, 0.3, 1, or 3 ppm, 60 min with exercise


None SRaw, FVC, FEV1

Vagaggini et al. 1996 7 healthy and 8 asth-matic adults and 7 eld-erly subjects with COPD (mean ages 34, 29, and 58, respectivly)

CA or 0.3 ppm, 60 min with exercise

Before, immediately after and 2 h post exposure

Slight decrease in FEV1

in COPD patientsFEV

1 unchanged in

asthmatics and healthy subjects

Avol et al. 1989 34 children with asthma (ages 8–16)

CA or 0.3 ppm, 3 h with exercise

Before and 60 min post exposure

FEV1, PEFR, FVC only for

1st h of 3-h exposureSRaw, TGV, FEV

1, PEFR,

FVC for the 3-h exposure average

Morrow and Utell, 1989 20 healthy and 20 asth-matic adults, 20 healthy and 20 with COPD elderly subjects (mean ages 31, 31, 60, and 61, respectively)



FEV1 decrease in elderly

smokers (7/20)Young, healthy and asth-matic, elderly healthy and COPD, FEV

1


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Studies in healthy subjectsInflammatory responseClinical studies of short-term NO

2 exposure have generally

assessed measures of pulmonary inflammation in one or more of three types of samples: (1) sputum, which is reflec-tive of both lower and upper respiratory tract contents; (2) bronchoalveolar lavage fluid (BALF), which is collected by

injection and withdrawal of a volume of saline or buffer in a segment of the lung, sometimes as two fractions: bronchial wash (BW), which is specific to the bronchial region, and alveolar lavage (AL), which reflects the lower lung segments; and (3) bronchial biopsies, which are more site specific than the previous two samples. Sputum and BALF have been typically assessed by counting and classifying white blood

Table 3. Continued.


2exposure

characteristics

Nature and timing of physiological responses


2-induced

effects Key negative findings

Bauer et al. 1986 15 adults asthmatics (mean age 33)


Before, during, and 30 min and 60 min post exposure

FEV1 decreased during

10 min exercise with NO2

No change in 20 min rest exposure or within 60 min post exposure

Gong et al. 2005 6 healthy and 18 COPD elderly subjects (mean ages 68 and 72, respetively)

CA, 0.4 ppm NO2, 200

µg/m3 concentrated ambient particles (CAPs), or 0.4 ppm NO

2 + 200

µg/m3 CAPs, with exer-cise, 2 h

Before, during, and after exposure

CAPs-related maximum midexpiratory flow (MMEF) and arte-rial oxygen saturation decrease. Decrease in symptoms related to iron in CAPs + NO

2 exposure

FEV1, FVC. 2 bpm

increase in heart rate (significance not reported)

Solomon et al. 2004 10 adult asthmatics (mean age 33)

CA or 0.4 ppm, 3 h at rest Before and hourly after exposure for 6 h

None FEV1

Witten et al. 2005 15 adults with allergy (mean age 32)



None FEV1

Devalia et al. 1994 8 mild asthmatics (mean age 28)

CA or 0.4 ppm, 6 h at rest Before and after exposure

None FEV1, FVC

Wang et al. 1995 16 adults with seasonal allergic rhinitis (mean age 26)

CA or 0.4 ppm, 6 h at rest Before and after exposure

None Nasal airway resistance

Bylin et al. 1985 8 healthy and 8 asth-matic adults (mean ages 25 and 31 resp.)



None SRaw, TGV, Raw

Mohsenin 1987 10 adult asthmatics (mean age 30)



None




None FEV1, FVC, sGaw

Boushey et al. 1988 9 adults asthmatics (mean age 29)


Before and during exposure

None FEV1, FVC,Raw, TGV,

SRaw

Drechsler-Parks et al. 1987

16 elderly nonsmokers (mean age 63)


Before, after, and every 40 min during

None No change in FEV1,

FVC, or MMEF. No change in heart rate

Rubinstein et al. 1990 9 adults with clinically stable asthma



None SRaw, FEV1, FVC

Rubinstein et al. 1991 5 healthy adult non-smokers (ages 21–36)

CA or 0.6 ppm, 2 h, 4 times in 6 days with exercise


None Raw, TGV, SRaw, MMEF

Hackney 1977 20 healthy adults (mean age 29)

CA, 1 or 2 ppm, 2 h Before and after exposure

None FEV1, FVC, other

Jorres et al. 1995 12 adult asthmatics and 8 healthy subjects (mean age 27)

CA or 1 ppm, 3 h with exercise

Before, during, and after exposure

Slight decrease in FEV1

in asthmaticsNo change in asthma symptoms

Blomberg et al. 1999 12 healthy nonsmoking adults (mean age 26)

CA or 2 ppm 4 h/day for 4 days with exercise


FEV1 and FVC decreased

only on day 1 of 4None

Balmes et al. 1997 15 healthy adults (mean age 29)


Tests every 30 min after exercise

None Airway resistance, FVC, FEV

1

Devlin et al. 1999 8 healthy adults (ages 18–35)


Only before and after exposure

Decreased recovery of inhaled NO

2

FEV1, SRaw

Azadniv et al. 1998 24 healthy adults (mean age 28)


Before, and at 2, 4, and 6 h after start of exposure

None Airway resistance, TGV, spirometry

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Table 4. Key studies of NO2-induced AHR to nonspecific bronchoconstrictor challenges.

Reference Exposed population Exposure protocol NO2 exposure level

Timing of physiological response


2-

induced effects Key negative findings

Hazucha et al. 1983 15 healthy and 15 mild asthmatic adults (mean ages 27 and 29, respectively)

NO2 or sham

exposure for 60 min at rest followed 20 min later by a methacholine inhalation challenge

CA, 0.1 ppm Before exposure and after 30 min of exposure termination

None NO2 did not affect

the SRaw response to methacholine challenge. No change in asthma symptoms among exposures

Ahmed et al. 1983b 20 healthy and 20 asthmatic adults

NO2 or sham expo-

sure for 60 min at rest followed immedi-ately by a carbachol challenge

CA, 0.1 ppm Before exposure and immediately after exposure

None No significant change in airway responsiveness to carbachol

Orehek et al. 1976 20 allergic asthmat-ics (mean age 26, includes teenagers)

NO2 or sham expo-

sures at rest followed by a carbachol inhalation challenge, 60 min

CA, 0.11 ppm Before and immedi-ately after exposure

Increased sensitivity to carbachol (~50%) when compared to CA in 13 of 20 sub-jects (as measured by amount of carbachol required to increase SRaw by 100%)

No significant NO2

effect in compari-son to CA when the whole group (n = 20) is considered.


NO2 or sham

exposures at rest for 30 min followed by a histamine inhalation challenge 30 min post exposure

CA, 0.14 ppm Before exposure and after 30 min of expo-sure termination

None TGV, SRaw unchanged as a function of NO

2

exposure (p = .052). No effect on asthma symptoms.


NO2 or sham expo-

sures for 75 min including 3 × 10-min treadmill exercises followed 2 h post exposure with a methacholine challenge

CA, 0.15 ppm Before, during and 2 h post exposure

None No changes in SRaw, FEV

1, or FVC

Kleinman et al. 1983 31 adult asthmatics (mean age 31)

NO2 or sham

exposures with light intermittent exercise for 2 h immediately followed by a metha-choline inhalation challenge


Increased bronchial reactivity (~70%). (as measured by the amount of metha-choline required to cause a 10% drop in FEV

1). (Statistics

performed only in 21 subjects)

Fewer adverse symp-toms with NO

2 rela-

tive to CA exposure.

Jörres and Magnussen 1990

14 adult nonsmok-ing mild asthmatics (mean age 34)

CA, NO2 or SO

2

exposures for 30 min at rest followed by an inhalation challenge of 0.75 ppm SO

2

CA, or 0.25 ppm NO2

or 0.5 ppm SO2

Before and shortly after exposure

Increased bronchial responsiveness (~25%) to 0.75 ppm SO

2 (as measured

by the ventilation rate during exposure required to double SRaw; 38 L/min NO

2

vs. 46 L/min CA)

No change in bron-chial responsiveness with SO

2 relative to

CA.

Jörres and Magnussen 1991

11 adults with mild and stable asthma

NO2 or sham expo-

sure for 30 min (20 at rest then 10 with exercise) followed 60 min later by a methacholine inha-lation challenge

CA, 0.25 ppm Before, immediately after and 1 h post exposure

None No NO2-related

change in bronchial responsiveness.


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Table 4. Continued.




2-


Strand et al. 1996 19 mild asthmatics (mean age 32)

NO2 or sham expo-

sure with intermit-tent exercise with histamine inhalation challenges at 30 min, 5 h, 27 h, and 7 days post exposure

CA, 0.26 ppm Before and at 30 min, 5 h, 27 h, and 7 days post exposure

Increased bronchial responsiveness (~50%) to histamine 5 h post exposure as measured by the amount of histamine required to double SRaw. TGV lower at 20 min and 7 days post exposure. In the week post exposure, increased symptoms in 2 CA and 7 NO

2-

exposed subjects

No change in bron-chial responsiveness at other time points. No exposure related asthma symptoms within 24 h post exposure.


NO2 or sham



Increased sensitivity to histamine (40%) for a doubling of SRaw

No change in asthma symptoms as a func-tion of NO

2 exposure.

Salome et al. 1996 9 adults and 11 children with asthma requiring daily medi-cation (mean ages 36 and 12, respectively)

NO2 or sham

exposures 60 min histamine

CA, or 0.3 ppm NO2,

or 0.3 ppm NO2 +

space heater com-bustion biproducts

Before, during, 1 h and up to a week post exposure

None No change in symptoms or lung function with any exposure.


NO2 or sham

exposures for 75 min including 3 8 10-min treadmill exercises followed 2 h post exposure with a methacholine challenge



1, or FVC.

Avol et al. 1988 59 subjects with moderate to severe asthma (mean age 30)

NO2 or sham expo-

sures for 2 h with alternating 10 min periods of exercise followed by a cold dry air (−17°C) chal-lenge 60 min (n = 59) and 24 h (n = 37) post exposure. Subjects were also exposed to ambient polluted air

CA, 0.3 ppm Before and 1 and 24 h post exposure

None No group differences in asthma symptoms or bronchial reactiv-ity to cold air.

Avol et al. 1989 34 children with asthma (ages 8–16)

NO2 or sham expo-

sures for 3 h with alternating 10 min periods of exercise followed by a cold dry air (−17°C) chal-lenge 60 min post exposure. Subjects were also exposed to ambient polluted air

CA, 0.3 ppm Before and 1 h post exposure

Increase in symp-toms between day 1 and 7 post exposure to NO

2

No group differ-ences in bronchial reactivity to cold air. No NO

2-related

symptoms reported during (p < 0.1) or 24 h (p < 0.091) after study. No effect from ambient air.


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Table 4. Continued.




2-


Morrow and Utell 1989

20 healthy adults, 20 asthmatic adults, 20 elderly healthy and 20 elderly with COPD subjects (mean ages 31, 31, 60, and 61, respectivly)

NO2 or sham expo-

sures with intermit-tent exercise for 4 h followed by an inha-lational challenge with carbachol

CA, 0.3 ppm before and 4 and 24 h after initiation of exposures

Increased bronchial responsiveness in 7/20 elderly subjects who had a history of smoking and in 7/20 asthmatics

No NO2-related

change in bron-chial reactivity in (1) healthy adults, (2) 13/20 asthmatic adults (opposite effect, in fact), (3) elderly subjects with COPD, or in (4) 13/20 elderly health subjects.

Rubinstein et al. 19909 adults with clini-cally stable asthma (mean age 29)

NO2 or sham expo-

sures with intermit-tent exercise for 30 min followed an hour later by an SO

2

inhalation challenge (1.0–4.0 ppm)

CA, 0.3 ppm Before, immediately after and 1 h post exposure

None No group differences in bronchial reactiv-ity to SO

2 as assessed

by FEV1, FVC, or

SRaw measurements.

Bauer et al. 1986 15 adults asthmatics (mean age 33)

NO2 or sham

exposure for 30 min (20 min at rest then 10 min with exercise) followed by a cold air (−11°C) inhalation challenge

CA, 0.3 ppm Before, during, and 30 min and 1 h post exposure

Transient decrease in FEV

1 relative to

CA during exer-cise (−13% and −7%, respectivly). Increased bronchial reactivity to cold air inhalation post exposure.

NO2 did not alter

FEV1 relative to CA

at rest.


NO2 or sham expo-

sures for 60 min with intermittent exercise followed by a cold air inhalation challenge (−16°C)


None No NO2-induced

changes in bron-chial reactivity. No significant difference in respiratory or non respiratory symp-toms during or for a week post exposure.

Bylin et al. 1985 8 healthy and 8 asthmatic adults (mean ages 25 and 31, respectively)

NO2 or sham



Increased bronchial reactivity to hista-mine in asthmatic group.

No change in bron-chial reactivity to histamine in healthy group.

Mohsenin 1987 10 adults asthmatic (mean age 30)

NO2 or sham expo-

sure at rest for 60 min followed by a metha-choline inhalation challenge

CA, 0.5 ppm Before and shortly after exposure

Increased airway reactivity (~50%) to methacholine (dose required to increase Raw 40%)

No change in symptoms with NO

2

relative to CA


NO2 or sham



None TGV, SRaw unchanged as a func-tion of NO

2 exposure.

No effect on asthma symptoms.


NO2 or sham expo-

sures with intermit-tent exercise for 3 h followed by a carbachol inhalation challenge 30 min post exposure

CA, 0.6 ppm Before, during and 30 min post exposure

None No NO2-induced

changes in bron-chial reactivity or lung function. No significant difference in symptoms


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Table 4. Contined.




2-


Salome et al. 1996 9 adults and 11 children with asthma requiring daily medi-cation (mean ages 36 and 12, respectively)

Exposure to CA, NO

2, or NO

2 + space

heater combustion products for 60 min at rest followed by a histamine challenge 60 min after exposure

CA, or 0.6 ppm NO2,

or 0.6 ppm NO2 +

space heater com-bustion by-products

Before, during, 1 h and up to a week post exposure

Increased airway hyperresponsive-ness with histamine (~25%) following 0.6 ppm NO

2 in air

(as assessed by the amount of histamine required for a 20% drop in FEV

1)

No change in airway hyperresponsive-ness following 0.6 ppm NO

2 with the

combustion biprod-ucts. No change in symptoms or lung function with any exposure.

Avol et al. 1988 59 subjects with moderate to severe asthma (mean age 30)

NO2 or sham expo-

sures for 2 h with alternating 10 min periods of exercise followed by a cold dry air (−17°C) chal-lenge 60 min (n = 59) and 24 h (n = 37) post exposure. Subjects were also exposed to ambient polluted air

CA, 0.6 ppm Before and 1 and 24 h post exposure

None No group differences in asthma symptoms or bronchial reactiv-ity to cold air.


NO2 or sham expo-

sures for 75 min including 3 × 10-min treadmill exercises followed 2 h post exposure with a methacholine challenge



1, or FVC.


NO2 or sham expo-

sures for 60 min with intermittent exercise followed by a cold air inhalation challenge (−16°C)


None No NO2-induced

changes in bron-chial reactivity. No significant differ-ence in respiratory or nonrespiratory symptoms during or for a week post exposure.


NO2 or sham expo-

sures with intermit-tent exercise for 3 h followed by a carbachol inhalation challenge 30 min post exposure


Greater drop in FVC in response to carba-chol after NO

2 (3.9%)

relative to CA (1.5%) exposure

No difference in symptoms among exposures.


NO2 or sham expo-

sures for 3 h with intermittent exercise. NO

2 level of 0.05

ppm with a 15-min 2 ppm peaks during each hour


None No NO2-induced

changes in bron-chial reactivity or lung function. No significant difference in symptoms.

Mohsenin 1988 18 health adults (mean age 25)

NO2 or sham expo-

sures for 60 min at rest followed by a methacholine inha-lation challenge

CA, 2.0 ppm Before and within 45 min post exposure

Increased airway hyperresponsiveness to methacholine (~20%) following NO

2 relative to air

(as assessed by the amount of metha-choline required for a 40% drop in specific airway conductance)

No lung func-tion changes with NO

2 without

methacholine.


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cells (leukocytes) and assaying the levels of proinflamma-tory mediators. Biopsies have been generally stained for cellular analysis and molecular expression of proinflamma-tory mediators. Importantly, there are some inconsistencies between studies in terms of the choice of biological sample and the analyzed markers of inflammation, which does not facilitate comparison of all inflammation endpoints. However, the common denominator for all studies is the analysis of changes in leukocyte numbers, which are an inte-gral part of inflammation and may be used for comparison purposes.

Several studies have examined the inflammatory response associated with short-term NO

2 exposure concentrations up

to 1 ppm in healthy subjects. For example, Vagaggini et al. (1996) exposed healthy adult volunteers to NO

2 (0.3 ppm,

1 h) and analyzed sputum and nasal lavage samples for proinflammatory cellular changes. The authors reported no changes in leukocyte numbers in sputum or nasal lavage. However, the authors reported a mild, but statistically signif-icant, increase in symptoms after exposure. Likewise, Gong et al. (2005) reported no changes in leukocyte numbers in sputum samples from healthy elderly volunteers exposed to 0.4 ppm NO

2 for 2 h. At a higher NO

2 exposure concentra-

tion (0.6 ppm, 2 h), Rubinstein et al. (1991) reported elevated levels of natural killer (NK) cells, but no significant eleva-tions in other lymphocyte types in the BALF from healthy adults. However, Frampton et al. (2002) found no changes in NK cells at the same exposure concentration or higher (0.6 and 1.5 ppm NO

2, 3 h) but observed increased PMN levels

in BALF at 1.5 ppm NO2 but not at 0.6 ppm NO

2. Similarly,

Jörres et al. (1995) exposed healthy adults to 1 ppm NO2 (3 h)

and reported no significant changes in BALF leukocytes or vasoactive and proinflammatory prostaglandins except for a small but statistically significant increase in thromboxane B2 (TxB

2), a proinflammatory mediator associated with

bronchoconstriction. Hence, based on the results from these studies, there is little evidence that NO

2 exposure is associ-

ated with pulmonary inflammation below 1 ppm in healthy subjects.

The acute inflammatory response to NO2 concentra-

tions above 1 ppm has also been assessed in healthy sub-jects. Azadniv et al. (1998) exposed healthy volunteers to CA or NO

2 (2.0 ppm, 6 h) with intermittent exercise and

reported results indicative of NO2-induced proinflammatory

responses, including a statistically significant increase (2.2–3.1%) in PMNs in the BALF leukocyte differential count. These results are in agreement with Devlin et al. (1999), who exposed healthy volunteers to CA or NO

2 (2 ppm, 4 h)

with intermittent exercise and also observed NO2-induced

increases in PMNs, in addition to increases in interleukin (IL)-6 and IL-8, both proinflammatory cytokines. These results were consistent with another study where healthy volunteers were repeatedly exposed to NO

2 at 2 ppm, 4 h/

day for 3 days (Balmes et al., 1997). Balmes et al. (1997) col-lected a bronchial wash (BW) and BALF (BW + AL) before and then 18 h after exposure. In the bronchial fraction, the ratio of neutrophils to total leukocytes increased after NO

2

exposure when compared to CA, indicating an inflammatory response that was not seen in BALF. Overall, these studies support a NO

2-induced adverse inflammatory response at

exposure levels around 2 ppm, which may be more obvious in the bronchial region.

NO2-induced pulmonary inflammation was also exam-

ined in a series of studies that targeted specific areas of the lung by collecting and examining endobronchial biop-sies in addition to lung lavage fluid (Blomberg et al., 1997, 1999; Pathmanathan et al., 2003). Blomberg et al. (1997) collected a BW, BALF, and endobronchial biopsy samples from healthy adults following exposure to 2 ppm NO

2 for

4 h. The authors reported significantly increased neutrophils and interleukin-8 (IL-8) levels, indicative of inflammation in BW, but not in BALF, suggesting that inflammation was localized to the bronchial region. Subsequently, Blomberg et al. (1999) exposed healthy adults to CA on 1 day and to 2 ppm NO

2 for 4 h/day on 4 consecutive days. They meas-

ured lung function during and after exposure, collecting BW, BALF, and endobronchial biopsy samples following both the CA and the 4-day NO

2 exposures. As with the single expo-

sure, the authors reported increased neutrophil numbers and this was accompanied by an increase in myeloperoxi-dase (MPO) levels in BW, indicative of neutrophil migration and activation in the airways. Interestingly, this neutrophil migration was not accompanied by changes in intracellular adhesion molecule-1 (ICAM-1) levels. Moreover, small but statistically significant decrements in FEV

1 and forced vital

capacity (FVC) were found after the first exposure to NO2,

but these responses were attenuated with repeated expo-sures. Overall, the results from these studies suggest that

Table 4. Continued.




2-



NO2 or sham

exposures for 60 min with intermittent exercise followed by a cold air inhalation challenge (−16°C)

CA, 3.0 ppm Before and immediately after exposure

None No NO2-induced

changes in bronchial reactivity. No significant difference in respiratory or nonrespiratory symptoms during or for a week post exposure.

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Table 5. Key studies of NO2-induced AHR to specific bronchoconstrictor challenges.

Reference Exposed population Exposure protocol NO2exposure level



2-

induced airway reactivity Key negative findings

Ahmed et al. 1983a 20 adult bronchial asthmatics with ragweed sensitivity (mean age 30)

NO2 or sham

exposure for 60 min at rest followed immediately by an allergen challenge

CA, 0.1 ppm Before exposure, 0 and 24 h post exposure

None No change in SGaw as a function of NO

2

exposure.

10 nonsmoking asth-matics with HDM sensitivity (mean age 28)

NO2 or sham

exposure for 60 min at rest followed immediately by a fixed-dose HDM challenge and a his-tamine challenge 7 and 24 h post exposure

CA, 0.1 ppm Before exposure and 0, 7 and 24 h post exposure

None FEV1 unchanged

with 0.1 ppm NO2.

Orehek et al. 1981 7 adult subjects with grass pollen allergy

NO2 or sham

exposures at rest followed by a grass pollen inhalation challenge, 60 min

CA, 0.11 ppm Before and after exposure

None SRaw did not change significantly.

Jenkins et al. 1999 11 mild asthmatics with atopy (mean age 31), HDM

CA, NO2, O

3, or

NO2 + O

3 exposure

for 6 h with 10 min exercise (32 L/min ventilation) every 40 min followed immediately by a HDM challenge by inhalation

0.2 ppm NO2, 0.1

ppm O3, 0.2 ppm O

2

+ 0.1 ppm O3

FEV1 assessment

before and immedi-ately after exposure

None No change in the amount of allergen required to decrease FEV

1 by 20%.

Strand et al. 1997 18 adult asthmatics with pollen allergy (mean age 30)

NO2 or sham

exposures at rest for 30 min followed 4 h post exposure by a pollen inhalation challenge

CA, 0.26 ppm FEV1 assessments

immediately after (early reaction) or 3–10 h after (late reaction) allergen challenge

Decrease in FEV1

(4%) and PEF (7%) in the late asthma phase

NO2 did not affect

the frequency of late asthmatic reactions. FEV

1 was unchanged

with NO2 in the early

reaction. Asthma symptoms were not affected by NO

2 in

comparison to CA.

Strand et al. 1998 16 mild asthmatics with allergy (mean age 33)

NO2 or sham

exposures at rest for 30 min followed 4 h post exposure by a pollen inhala-tion challenge. This protocol was performed on 4 consecutive days followed by a hista-mine challenge on the 5th day



Decrease in FEV1 in

early phase(−2.5%, NO

2 vs.−0.4%, CA)

No change in FEV1

in late phase. No change in asthma symptoms.NO

2 plus

allergen did not affect histamine responsiveness on the day after the last exposure.

Barck et al. 2002 13 adult mild asth-matics with pollen allergy (mean age 28)

NO2 or sham

exposures at rest for 30 min followed 4 h post exposure by a pollen inhalation challenge



None No change in FEV1,

SRaw, TGV.No change in asthma medicationuse or symptoms post exposure.


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NO2 exposures of 2 ppm are associated with a pulmonary

inflammatory response, but that inflammation may not nec-essarily be correlated with significant or sustained changes in lung function.

Pathmanathan et al. (2003) further assessed the expression of proinflammatory parameters and cytokines in archived bronchial epithelium biopsies from eight healthy adults who participated in the study by Blomberg et al. (1999). In this study, Pathmanathan et al. (2003) assessed the expression of proallergic and proinflammatory mediators, most of which were not assessed by Blomberg et al. (1999). The authors reported statistically significant increases in expression of IL-5, IL-10, and IL-13 in the bronchial epithelium, but not

TNF-α or nuclear factor kappa B (NFκB). These changes were considered by the authors to be suggestive of a proallergic response to NO

2 exposure. The study also reported increased

levels of ICAM-1, which has been linked with enhanced virus infectivity (Bella & Rossmann, 2000). However, in both studies by Blomberg et al. (1997, 1999), ICAM was not detected in tissue samples. Thus, the results are somewhat contradictory despite the fact that Pathmanathan et al. used tissue samples collected in the Blomberg et al. (1999) study. Moreover, Blomberg et al. (1999) collected tissue samples following only 1 day of CA exposure but after 4 days of NO

2

exposures, introducing a timing issue (3 days in the exposure chamber) that may have influenced their findings. Although

Table 5. Continued.

Reference Exposed population Exposure protocol NO2exposure level



2-

induced airway reactivity Key negative findings

Barck et al. 2005 18 adult asthmatics with pollen allergy (mean age 32)

NO2 or sham

exposures at rest for 15 min on day 1 followed by allergen challenge 4 h post exposure and 2 × 15-min expo-sures separated by 60 min the following day with a similar allergen challenge 3 h post exposure


immediately after (early reaction), 3–10 h after (late reaction) or next morning after aller-gen challenge

None No change in symp-toms. No change in FEV

1, TGV or airway

resistance.

Jenkins et al. 1999 11 mild asthmatics with atopy (mean age 31), HDM

CA, NO2, O

3, or NO

2

+ O3 exposure for 3 h

with 10 min exercise (32 l/min ventila-tion) every 40 min followed imme-diately by a HDM challenge

CA or 0.4 ppm NO2,

or0.2 ppm O3, or

0.4 ppm NO2 + 0.2

ppm O3

FEV1 assessment

beforeand immedi-ately after exposure

Increased sensitiv-ity (~40%) to HDM allergen as reflected by amount of HDM required for a 20% decrease in FEV

1 (in

all exposures)

No pulmonary effects of NO

2

without an allergen challenge.

Devalia et al. 1994 8 mild asthmatics with HDM sensitivity (mean age 28)

CA, NO2, SO

2, or

NO2 + SO

2 exposures

for 6 h followed by an HDM inhalation challenge immedi-ately post exposure

CA, or 0.4 ppm NO2,

or 0.2 ppm SO2, or

0.4 ppm NO2 + 0.2

ppm SO2


Increased sensitiv-ity (~60%) to HDM only in the NO

2 +

SO2 exposure (as

reflected by amount of HDM required for a 20% decrease in FEV

1)

No change in sen-sitivity to allergen in terms of FEV

1

changes.

Tunnicliffe et al. 199410 nonsmoking asth-matics with HDM sensitivity (mean age 28)

NO2 or sham expo-

sure for 60 min at rest followed immedi-ately by a fixed-dose HDM challenge and a histamine chal-lenge 7 h and 24 h post exposure

CA, 0.4 ppm Before exposure and 0, 7, and 24 h post exposure

FEV1 decreased with

0.4 ppm NO2 at 7 and

24 h after exposure (−18.6%, NO

2 vs.

−14.6% CA early phase and −8.1%, NO

2 vs. −2.8% CA

late phase)

No pulmonary effects of NO

2without

an allergen challenge.

Witten et al. 2005 15 asthmatic adults with HDM allergy (mean age 32)

NO2 or sham

exposures for 3 h with 30 min of exercise at the beginning of each hour. Followed by HDM challenge to cause 20% FEV

1

decrease

CA, 0.4 ppm Before, each hour up to 6 h post exposure and 26 h post exposure. Also, collected sputum samples at 6 and 26 h post exposure

None No change in sensitivity to allerge In terms of FEV

1

changes.

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these experiments have some limitations, their findings, when taken with others in the same exposure concentration range, contribute to the evidence indicating NO

2-induced

pulmonary inflammation at levels above 1 ppm.Overall, our review thus indicates that there is only incon-

sistent evidence for markers of pulmonary inflammation from short-term NO

2 inhalation exposures at concentrations

below 1 ppm among healthy volunteers. The evidence is more consistent at higher NO

2 concentrations, between 1.5

and 2 ppm. Although more studies are needed to determine a possible threshold for NO

2-induced pulmonary inflam-

mation, it is unlikely that this threshold is below 1 ppm in healthy individuals.

Susceptibility to infectionSeveral clinical studies have examined a role of short-term NO

2 exposure on lung clearance and susceptibility to pulmo-

nary infection. Specifically, Helleday et al. (1995) used fiber optic bronchoscopy to assess the effect of NO

2 (20 min to 1.5

or 3.5 ppm and 4 h to 3.5 ppm) in healthy adults on mucocili-ary clearance, an important pulmonary defense mechanism. The authors assessed mucociliary clearance 45 min after the short exposure (20 min) and 24 h after the long exposure (4 h), reporting a significant reduction in the mucociliary activity 45 min after the 20-min exposure to both 1.5 and 3.5 ppm NO

2, but not 24 h after the 4-h exposure. This suggests that

NO2 levels much higher than ambient concentrations may

transiently slow airway clearance. In an earlier study, Kulle and Clements (1988) exposed 152 healthy subjects to NO

2 (1

and 2 ppm) for 2 h/day on 3 consecutive days, with intrana-sal administration of influenza virus after the second day of NO

2 exposure. The authors reported no significant effect of

NO2 on viral infectivity at 1 or 2 ppm when compared to CA

exposures. This study was repeated 3 times over a period of 3 years in different volunteers, providing robust results show-ing an absence of NO

2 exposure effects on susceptibility to

viral infection by the nasal route. It is noteworthy that both studies (Helleday et al., 1995; Kulle & Clements, 1988) used concentrations that are much higher than those measured in outdoor air. These findings do not support a causal basis for epidemiology findings of NO

2-associated increases in

infectivity rates, and suggest possible confounding by other unknown parameters.

Other studies investigating the immune-compromising role of NO

2 have examined macrophages isolated from BALF

of subjects acutely exposed to NO2 by inhalation. For exam-

ple, Kleinman et al. (1998) isolated macrophages from BALF collected from healthy individuals exposed to CA or NO

2 (2

ppm, 4 h/day, 3 days) by Balmes et al. (1997). The authors reported increased macrophage superoxide free-radical pro-duction in response to NO

2, when compared to CA. Frampton

et al. (2002) found that bronchial cells collected from NO2-

exposed individuals (1.5 ppm, 3 h) and then exposed in vitro to respiratory syncytial virus (RSV) produced significantly more lactate dehydrogenase than similarly treated cells col-lected from CA-exposed individuals, indicating 1.5 ppm NO

2

enhanced RSV-induced epithelial injury. Other pulmonary cell types such as alveolar macrophages collected from the same individuals exposed to NO

2 did not show altered RSV

infectivity as compared to controls, suggesting that although this high level of inhaled NO

2 may be toxic to some cells of

the airways, it does not necessarily compromise the macro-phage defenses against the RSV virus. Azadniv et al. (1998) also found no significant changes in macrophage function or susceptibility to virus infection following NO

2 exposure (2

ppm, 6 h). As discussed in the previous section, Balmes et al., Frampton et al., and Azadniv et al. all observed indicators of pulmonary inflammation. However, these authors did not find consistent evidence for NO

2 exposure concentrations

up to 2 ppm increasing susceptibility to viral infection.Although Devlin et al. (1999) demonstrated that NO

2

exposure did not adversely affect the susceptibility of alveo-lar macrophages to viral infection, the authors reported that acute exposure to 2 ppm for 4 h NO

2 partially compromised

the alveolar macrophage defenses in healthy volunteers when compared to CA exposure. More specifically, alveolar macrophages from BALF of NO

2-exposed subjects showed

a decrease in the ability to phagocytize unopsonized yeast and a decrease in superoxide production, both of which are signs of compromised macrophage defenses.

Changes in lymphocyte levels may also be indicative of immune system compromise. Several important studies examining changes in pulmonary and peripheral blood lymphocyte levels associated with NO

2 exposure were

recently reviewed by Frampton et al. (2002), and results from these studies generally failed to demonstrate statistically significant NO

2-induced effects. Taken together, the results

from studies investigating immune system compromise do not support an NO

2-induced increase in viral infectivity of

the respiratory system, and this appears to hold true at NO2

exposure concentrations as high as 2 ppm.

Studies in susceptible population groupsAsthma is a chronic inflammatory pulmonary disorder char-acterized by reversible obstruction of the airways. In asth-matics, the airways are very sensitive to certain inhaled sub-stances, for example, pollen, cold air, pet dander, allergens generally, and environmental tobacco smoke. The inflam-mation and airway reactivity are mainly mediated by eosi-nophils and their products (e.g., eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil protein X). When the airways react, the muscles around them tighten, causing bronchoconstriction, which can obstruct the flow of air entering and leaving the lungs. This reaction can result in asthma symptoms, which include coughing, wheezing, chest tightness, and shortness of breath. It is hypothesized that NO

2 may lower the threshold for an agent to provoke

an asthma attack, which may have serious consequences if not controlled by medication. Likewise, allergic rhinitis is a collection of symptoms, predominantly in the nose and eyes, that may be caused by inhaled dusts, dander, or plant pollens in individuals who are allergic to these substances.

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COPD is another chronic inflammatory disease charac-terized by symptoms that are similar to those in asthma. However, COPD is a progressive disease, characterized by irreversible airways obstruction, and usually resulting from chronic exposure to lung irritants such as tobacco smoke. It is hypothesized that NO

2 can increase inflammation in

subjects having COPD, potentially accelerating the process of lung injury in those subjects.

Clinical studies have examined NO2-induced effects

in subjects with allergic asthma, rhinitis, and COPD. It is hypothesized that exposure to NO

2 may induce inflam-

mation, or enhance an immune response, and exacerbate symptoms at lower exposure levels in these population groups compared to healthy individuals. We reviewed stud-ies of asthmatic subjects in addition to the smaller set of studies of subjects having rhinitis and COPD. Exposure lev-els to NO

2 in these studies ranged from 0.26 to 1 ppm. These

studies examined various markers of inflammation such as levels of neutrophils, eosinophils, and their degranulation products in sputum, BALF, and blood.

Several NO2 clinical studies provide evidence to support

the enhancement of allergic mediators and proinflamma-tory vasoactive molecules in the lung. For example, levels of select proinflammatory and potentially bronchoconstrictive prostaglandins (TxB

2 and prostaglandin D2 [PGD

2]) were

significantly elevated in BALF from asthmatics exposed to 1 ppm NO

2 for 3 h (PGD

2, 45%; TxB

2, 29%) (Jörres et al., 1995).

Although these findings suggest that this acute exposure to NO

2 can have potentially adverse effects in asthmatics, the

authors reported no simultaneous changes in BALF leuko-cytes. In addition, these changes were only associated with a small, albeit statistically significant, decrease in FEV

1 (1.4%)

relative to CA inhalation, which may diminish the health significance of these findings.

Evidence for NO2-induced enhancement of allergic

reactions also comes from work by Barck et al. (2002). In this study, BW and BALF were collected from subjects with allergic asthma exposed to 0.26 ppm NO

2 or CA for 30 min

and to pollen antigens post exposure. The authors reported significantly increased ECP in BW and increased neu-trophils in BW and BALF compared to controls, suggestive of proinflammatory changes. However, these changes were not associated with lung function deficits, as the authors did not find NO

2-induced changes in lung function or symptom

frequency.In a subsequent experiment, Barck et al. (2005) examined

the response to repeated exposures to 0.26 ppm NO2 (15 min

on day 1 and two 15-min exposures on day 2) in allergic asthmatics. Allergen was inhaled 3–4 h after air or NO

2 expo-

sures on both days. NO2 exposure augmented the allergen-

induced increase in ECP in both sputum and blood and MPO in blood, indicative of neutrophil activation. The authors concluded that several brief exposures to ambient levels of NO

2 can prime circulating eosinophils and enhance the

eosinophilic activity in sputum in response to inhaled aller-gen. The authors suggested that this might be an important mechanism by which air pollutants amplify inflammatory

reactions in the airways. However, the authors reported no changes in neutrophil or eosinophil levels in sputum and blood compared to controls. Moreover, NO

2 + allergen expo-

sure did not significantly change the symptoms and pulmo-nary function responses to allergen when compared to CA + allergen exposure. The overall health significance of the increased ECP and MPO findings is thus unclear, especially given that other studies at comparable NO

2 concentrations

and longer exposure times have also failed to show NO2-

induced elevations in pulmonary leukocyte levels (Solomon et al., 2004; Witten et al., 2005).

To test the inflammatory responses to NO2 in another

subpopulation with an allergic profile, Wang et al. (1995) exposed subjects with allergic rhinitis to NO

2 (0.4 ppm, 6 h)

in conjunction with allergen. The authors then collected a nasal lavage sample and reported increased ECP in the samples. However, no changes were reported in MPO or IL8 with NO

2 + allergen exposure in comparison to CA +

allergen exposure. Interestingly, the authors reported no significant changes in nasal airway resistance or immune cellular changes in response to NO

2 exposure, despite the

increased changes in ECP. This is in agreement with the findings by Barck et al. (2005), who also reported changes in eosinophil degranulation (and ECP), but no changes in airway function.

However, several studies generally found a lack of NO2-

induced inflammation in asthmatics for NO2 exposure

concentrations in the range of 0.3–0.6 ppm. For example, Boushey et al. (1988) exposed asthmatic adults to NO

2 (0.6

ppm, 2 h) and analyzed cellular and cytokine changes in BALF following exposure. The authors reported no signifi-cant changes in IL-1, TNF-α, or lymphocyte populations as a function of NO

2 exposure. Likewise, Vagaggini et al. (1996)

found no changes in leukocyte numbers in sputum from allergic asthmatics exposed to 0.3 ppm NO

2 for 1 h. Solomon

et al. (2004) reported no NO2-induced changes in leukocyte

numbers in sputum samples collected from house dust mite (HDM) allergen-sensitive asthmatics (0.4 ppm, 3 h), whereas Witten et al. (2005) reported similar findings, with the excep-tion of a significant decrease in sputum eosinophils associ-ated with NO

2 exposure. Overall, the results from studies

that examined NO2-induced inflammation among asthmat-

ics are inconsistent for NO2 effects for concentrations in the

range of 0.26–0.6 ppm.Two studies have assessed the effects of exposure to NO

2

in subjects having COPD. Vagaggini et al. (1996) and Gong et al. (2005) exposed subjects having COPD to NO

2 (0.3

ppm, 1 h; 0.4 ppm, 2 h, respectively) and collected sputum samples after exposure. Both studies reported no changes in leukocyte numbers in sputum. In the study by Vagaggini et al. (1996), the authors reported a mild decrease in FEV

1

2 h after NO2 exposure, which was accompanied by a mild,

but statistically significant, increase in symptoms. The no-effect exposure concentrations in these studies (0.3 and 0.4 ppm, respectively) for inflammatory effects were lower than the threshold levels associated with subtle inflammatory changes in healthy subjects (>1 ppm NO

2). Because COPD

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is a chronic and inflammatory disease, the high background level of inflammation may have prevented observation of the subtle changes that have been reported in studies of healthy individuals at higher NO

2 exposure concentrations (Devlin

et al., 1999; Frampton et al., 2002).As discussed previously, we found little evidence for NO

2-

induced proinflammatory and immune changes at NO2

exposure concentrations below 1 ppm in healthy subjects. In addition, although one study (Devlin et al., 1999) sug-gests diminished macrophage response to antigens at NO

2

concentrations greater than 1 ppm in healthy subjects, its results do not support either an NO

2-induced enhancement

of airways viral infectivity or macrophage susceptibility to viral infection. In allergic and asthmatic individuals, the results are mixed for NO

2-induced immune responses in the

0.26–0.6-ppm exposure concentration range, as only half the studies showed proinflammatory and proallergic responses and these effects were rarely accompanied by NO

2-induced

adverse symptoms or changes in respiratory function. In gen-eral, studies of asthmatics show only subtle changes in some markers of inflammation and equivocal evidence of an exac-erbated allergic response to short-term NO

2 exposure. The

health relevance of these changes remains to be determined, especially given that these changes were accompanied with, at most, minor changes in airways function, but no changes in symptom frequency. The two studies that exposed sub-jects having COPD did not indicate that this subpopulation is susceptible to inflammation at NO

2 exposure concentra-

tions less than or equal to 0.4 ppm. Overall, the results from these studies do not provide adequate mechanistic evidence to support NO

2 concentrations below 1 ppm leading to more

serious respiratory morbidity effects, such as hospitaliza-tions and emergency room visits, as would be suggested by a causal interpretation of the epidemiological studies.

Effects of NO2 exposure on lung function and airways hyperresponsiveness (AHR)AHR is characterized by bronchoconstriction with increased sensitivity to certain stimuli. AHR is usually assessed as decrements in pulmonary function (decreased FEV

1) or

increased specific airways resistance (SRaw) following inhalation of an aerosolized specific (e.g., antigen, allergen) or nonspecific (e.g., methacholine, carbachol) bronchoc-onstrictor or stimulus (e.g., exercise, cold air). We reviewed 45 studies examining NO

2-induced AHR with challenge to

stimuli, NO2-induced lung function changes without the

effect of a bronchoconstrictor challenge, or both. The studies that investigated NO

2-induced AHR will be discussed below

based on the nature of challenge (specific or nonspecific), in ascending order of magnitude of exposure concentration. A summary of the surveyed studies without challenge effects can be found in Table 3 (see Figure 5 for a graphical sum-mary), and those with nonspecific and specific challenge effects in Table 4 and Table 5, respectively (both Tables 4 and 5 are illustrated in Figure 6).

In those studies without challenge (see Table 3 and Figure 5), only 7 of the 37 studies reported NO

2-induced

statistically significant changes in lung function for levels between 0.3 and 2 ppm for varying durations (30 min to 6 h), but those changes were usually small (<10%), transient, and not exposure-concentration dependent. For example, Avol et al. (1988) exposed 59 adult asthmatics to 0.3–0.6 ppm NO

2

(2 h) and reported no significant changes in FEV1 or SRaw.

Similarly, Salome et al. (1996) reported no significant NO2-

induced changes at the same concentrations in 20 asthmatic adults and children at rest (1 h). Balmes et al. (1997) also did not find any NO

2-induced changes in FEV

1 or SRaw among

15 healthy adult volunteers exposed to 2 ppm NO2 for 4 h

with exercise. In contrast, Roger et al. (1990) reported NO2-

induced decrements in FEV1 in 21 mild asthmatics after a 3-h

exposure to 0.3 ppm. As a whole, it seems unlikely that NO2

exposure without challenge, even at relatively high levels compared to ambient concentrations, would result in lung functional changes.

NO2-induced AHR to nonspecific challengesNonspecific stimuli such as cold air (lower than −10°C), methacholine, carbachol, and histamine have been com-monly used in clinical inhalation exposures to test for a potentiating or synergistic effect of air pollutants. A list of studies that examined NO

2-induced AHR to nonspecific

challenges is in Table 4. The outcomes from these studies are illustrated in Figure 6.

Orehek et al. (1976) were among the first to test for AHR to assess the acute health effects of NO

2 inhalation. In their

study, Orehek et al. (1976) exposed 20 subjects with slight to moderate asthma to clean air or NO

2 (0.11 ppm, 1 h), followed

by a set concentration of carbachol, a bronchoconstrictor.

0 0.1 0.2 0.3 0.4 0.5 0.6NO2 Exposure Concentration (ppm)

Res

pons

e

+

–

Figure 5. Responses in lung function to inhaled NO2. Each circle repre-

sents a single study. Circle size represents the relative number of subjects who participated in the study. On the y-axis, − represents studies where there was no statistically significant response with NO

2 exposure relative

to CA exposure, and + represents studies where the NO2-exposed group

or a subset therein produced a statistically significant (p < .05) response that was different from the CA-exposed group. Although we have trun-cated the figure to focus on the 0.1–0.6 ppm NO

2 concentration range, it

is important to note that there is a limited number of studies that have examined the effects of NO

2 exposure levels between 1 and 3 ppm. Even

for these higher NO2 concentrations, however, these studies generally

reported mixed findings, with only 3 of 10 group comparisons with CA showing statistically significant responses for NO

2 exposures.

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The aim of the study was to determine whether NO2 exposure

reduced the amount of carbachol needed to induce the same change in SRaw when compared to clean air. The authors found no overall significant effect of NO

2 compared to clean

air in the group as a whole. However, after a more detailed analysis, the authors found a subgroup of individuals (n = 13) who responded with increased resistance (characterized by ≥20% change in SRaw) to NO

2 plus carbachol as compared

to CA + carbachol. When data from these individuals were analyzed separately from nonresponders (n = 7), the authors reported a statistically significant effect of NO

2 on carbachol

response (i.e., less carbachol was required to cause the same change in SRaw).

Given differences in the baseline responses between the responders and nonresponders and the use of suboptimal statistical methods, there are questions regarding the validity of the group comparisons made by Orehek et al. (1976). For example, the group of nonresponders required about half the dose of carbachol to yield a 100% change in SRaw following CA + carbachol control exposures (0.36 ± 0.05 mg for nonre-sponders versus 0.66 ± 0.1 mg for responders). This is con-trary to most other studies, which have reported lower effec-tive challenge doses for asthmatics in comparison to healthy subjects due to the former’s increased pulmonary sensitiv-ity (Morrow & Utell, 1989; Utell et al., 1983; Hazucha et al.,

1983). Similarly, the “responders” had a lower group mean SRaw than nonresponders at baseline before any exposures were performed. This may have facilitated a higher amount of NO

2 and carbachol delivery in responders, thereby affect-

ing larger changes at similar inspired concentrations than in nonresponders. Moreover, the investigators used multiple individual t tests to analyze their data, and did not prop-erly account for multiple comparisons. Notwithstanding the design and statistical issues that weaken this study, the results can be interpreted as suggesting the possibility of a subpopulation that may be hypersensitive to NO

2-induced

carbachol hyperresponsiveness.The study findings by Orehek et al. (1976) prompted

several other clinical studies of acute NO2 exposure concen-

trations below 0.2 ppm. For example, Ahmed et al. (1983b) exposed 20 asthmatics and 20 healthy subjects for 1 h to 0.1 ppm NO

2 or CA. Thirteen of 20 asthmatics experienced non-

significant decreases in the amount of carbachol needed to elicit a 35% decrement in SGaw (specific airways conduct-ance), i.e., the provocative dose 35 (PD35). However, these nonsignificant decreases in PD35 were also reported for 10 healthy individuals. In the remaining subjects, either no NO

2-

induced change or an increase in PD35 was reported. The results from Ahmed et al. (1983b) show considerable varia-tion in NO

2-induced responses, which are not significantly

different from baseline variation in CA-exposed subjects.In another effort to reproduce the Orehek et al. (1976) find-

ings, a randomized double-blind crossover exposure study was conducted by Hazucha et al. (1983). These researchers exposed 15 atopic asthmatics and 15 healthy individuals to 0.1 ppm NO

2 for 1 h, followed by a methacholine challenge.

In agreement with the findings of Ahmed et al. (1983b), and in contrast to the Orehek et al. “responders,” the authors found no NO

2-induced changes in airways resistance. The

authors reported that only 3 of 15 asthmatics showed NO2-

related responses. Moreover, 5 asthmatic and 7 normal subjects showed a decreased (potentially health-favoring) SRaw response to methacholine following NO

2 exposure,

suggesting considerable variability either in response or in the techniques employed. Hence, the results for NO

2-

induced AHR at exposure concentrations around 0.1 ppm show mixed results, with one study (Orehek et al., 1976) sug-gesting effects, but two larger studies (Ahmed et al., 1983b; Hazucha et al., 1983) showing no effects.

Several studies have also examined a potential concen-tration-response effect for NO

2 in the concentration range

including 0.1 ppm, providing information relevant to pos-sible thresholds of effect and mechanisms of action. In one study by Bylin et al. (1988), mild asthmatics (n = 20) were exposed to 0, 0.14, 0.27, and 0.52 ppm NO

2 for 30 min on

separate days over a 4-week period. Bronchial responsive-ness to histamine, measured post exposure, increased only after exposure to 0.27 ppm (14/20 responders, any decrease measured regardless of magnitude), but no significant responses were observed at any other exposure concentra-tion. The authors reported a tendency for increased bron-chial responsiveness with the lower and higher exposure

0 0.1 0.2 0.3 0.4 0.5 0.6NO2 Exposure Concentration (ppm)

Res

pons

e

+

–

Specific ChallengeNonspecific Challenge

Figure 6. Study outcomes of NO2-induced AHR to specific (dark circles)

or nonspecific (light circles) challenges. Each circle represents the overall response from one study. On the y-axis, − represents studies where there was no statistically significant response with NO

2 relative to CA expo-

sure, and + represents studies where the NO2-exposed group or a subset

therein produced a statistically significant (p < .05) response that was dif-ferent from that of the CA-exposed group. The circle size represents the number of subjects who participated in the study. In the case where a subset of an exposed group was analyzed separately from the whole study population because they were found to be “susceptible” (and resulted in “positive” findings), they received a + response with the respective subset size, whereas the sample population not showing a statistically significant response received a negative (−) response. In some cases, circles within the same response type-concentration are staggered vertically for visibil-ity purposes and have no implications for potency of response. Although not shown in this figure, five studies have investigated NO

2-induced AHR

for nonspecific challenges at NO2 concentrations greater than 0.6 ppm,

three that did not report statistically significant responses, and two that did report a statistically significant NO

2-induced AHR response.

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concentrations compared to CA, but the trend was not sig-nificant. A drawback to this study is that most study partici-pants were able to identify low levels of NO

2 by odor, which

may have influenced the results. Moreover, no asthma dete-rioration was noted in subjects over the 4-week duration of the study. Thus, the AHR finding observed in this study at 0.27 ppm may not be adverse to health.

Roger et al. (1990) also examined the concentration-response of NO

2 and reported no NO

2-induced AHR in 21

male volunteers with mild asthma. In this study, subjects were exposed, with exercise, to 0, 0.15, 0.3, and 0.6 ppm NO

2

followed by symptoms measurement and a methacholine challenge. The authors found that NO

2 exposure did not lead

to any significant symptom changes, nor did it significantly alter the methacholine-induced effects. The results from this study suggest no short-term effects of NO

2 on AHR at con-

centrations lower than 0.6 ppm.Kleinman et al. (1983) found some evidence of NO

2-

induced AHR with exposure to 0.2 ppm for 2 h in adult asthmatics. In their study, Kleinman et al. assessed NO

2-

induced bronchial hyperresponsiveness to methacholine in a protocol with intermittent exercise. The authors reported that about 2/3 of subjects (n = 21/31) exhibited changes in the direction of NO

2-induced bronchial hyperrespon-

siveness to methacholine relative to CA. According to the authors, however, the mean changes in AHR did not achieve significance in most statistical comparisons. Of the subjects who showed a methacholine-induced decrement (>10%) in FEV

1 (n = 21), the authors reported a statistically significant

reduction (70%) in the amount of methacholine relative to CA exposure. Interestingly, significantly fewer respiratory or asthma-related symptoms were reported during NO

2 versus

CA exposures, thus potentially diminishing the health sig-nificance of the increased bronchial reactivity to NO

2 in the

exposed asthmatics.The greatest number of studies has examined NO

2-

induced AHR at concentrations between 0.25 and 0.3 ppm. Jörres and Magnussen (1990) exposed mild asthmatics at rest to 0.25 ppm NO

2 for 30 min, followed by exposure to 0.75

ppm sulfur dioxide (SO2). The authors reported no changes

in SRaw as a function of exposure, but lower provocative ventilation with SO

2 was needed following pre-exposure to

NO2 as compared to CA. The authors failed to confirm these

results in a subsequent study on mild and stable asthmatics (Jörres & Magnussen, 1991), where they used methacholine instead of SO

2 as a provocative challenge 1 h after exposure.

This suggests a differential effect of NO2 that is dependent on

the challenge type.Several studies performed at the Rancho Los Amigos

Medical Center in Southern California did not show NO2-

induced AHR for NO2 levels as high as 0.6 ppm. These find-

ings are particularly relevant because the studies included relatively large numbers of subjects (34–59 subjects). In one of these studies, Avol et al. (1988) examined the concentra-tion-dependent effect of NO

2 (0, 0.3, or 0.6 ppm, 2 h) with

intermittent exercise on AHR to cold air (−17°C) challenge in 59 adults the authors classified as having moderate to severe

asthma. In all subjects, bronchial reactivity to cold air was measured 1 h post exposure, and at 24 h post exposure in 37 of the subjects. The study found no significant NO

2-induced

changes in AHR to cold air challenge compared to controls. Further analysis, which was restricted to the 20 most severe asthmatics in the study, also did not produce significant responses to NO

2. Interestingly, NO

2-induced AHR in a sub-

set (n = 36) of subjects exposed to ambient Los Angeles air (with lower NO

2 at 0.086 ppm) showed the highest (but not

statistically significant) AHR response in the lung function tests. These results suggest that pollutants other than NO

2 in

the Los Angeles air (e.g., ozone, aldehydes, VOCs) may be more potent inducers of airway hyperreactivity.

In a subsequent study, Avol et al. (1989) exposed 34 asth-matic children (8–16 years old) to 0.3 ppm NO

2 for 3 h with

intermittent exercise. The authors reported no significant NO

2-induced changes in AHR to cold air or NO

2-related

symptoms for 3-h exposures in this highly susceptible popu-lation. The study did find statistically significant, but small and transient, changes in lung function after the first hour of exposure. In addition, the authors reported slight but statistically significant increases in asthma symptoms in the NO

2-exposed group the week after exposure but not the 24 h

immediately after exposure. It is noteworthy that this same group experienced more symptoms during the week before NO

2 exposures, suggesting that the increased symptoms

may be due to variability in the study subjects. The results from these studies by Avol et al. (1988, 1989) complement previously published data from this group (Linn et al., 1986). Linn et al. (1986) exposed 21 adults having mild asthma to 0, 0.3, 1.0, and 3.0 ppm NO

2 (1 h) with intermittent exercise, but

found no significant NO2-induced effects on SRaw or NO

2-

induced AHR to cold air. The authors suggested that because the study participants lived in areas with high outdoor NO

2

levels, their findings may be due to physiological adaptation to NO

2. However, the NO

2 levels in these studies were higher

than the average ambient exposures at the time (0.1 ppm), and adaptation to air pollutants does not usually provide protection at higher exposure levels (Hazucha et al., 1992). Overall, the findings from the Los Angeles area studies sug-gest that individuals with asthma were not affected by NO

2

exposure concentrations as high as 0.6 ppm.In another large study aimed at examining age-specific

and disease-specific effects of NO2 exposure, Morrow and

Utell (1989) exposed 80 subjects to 0.3 ppm NO2 for 4 h in

a double-blind randomized clinical study. The subjects included 20 healthy and 20 asthmatic young adults (21 years old), in addition to 20 healthy elderly subjects and 20 elderly subjects having COPD (60 years old). Lung function assessments and inhalation challenges to carbachol were performed before and 4 and 24 h after initiation of NO

2

exposures. Healthy young subjects showed no significant changes in lung function or AHR in any of the tests. Similarly, the 20 asthmatic subjects as a group showed no significant responses. However, 7/20 asthmatics who had been classi-fied as “responders” in an earlier study by Bauer et al. (1986) showed a statistically significant reduction in FEV

1 and SGaw

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following NO2 exposure when compared to CA. The remain-

ing group of asthmatics (13/20), however, showed greater FEV

1 decrements during CA compared to NO

2 exposure,

suggesting substantial variability in response.In the elderly group, the subjects with COPD showed no

bronchial challenge differences between CA and NO2 expo-

sures, but NO2 exposure significantly decreased FVC when

analyzed using a t test (but not with analysis of variance [ANOVA] accounting for repeated measures). A subgroup analysis of mild versus moderate COPD subjects was insig-nificant for most tests. The healthy elderly subjects showed no significant results for all exposures and tests. However, a subgroup of the healthy elderly cohort who smoked or had a history of smoking (n = 7/20) showed significant differ-ences in FEV

1 responses to NO

2 versus CA. It is worth noting

that lung function changes in the COPD subjects were not examined by changes to the carbachol challenge as in other studies, but rather by changes following a bronchodilator challenge using isoproterenol. Notably, exposure-related symptoms were more frequent and severe with CA expo-sures than with NO

2 exposures. However, no statistical tests

were performed on symptom data. This study suggests that 0.3 ppm NO

2 may pose a risk to a subset of asthmatics and

to elderly smokers who may be exposed to subsequent non-specific challenges.

Lastly, Salome et al. (1996) examined the potential for concentration-dependent (0, 0.3, and 0.6 ppm, 1 h) NO

2-

induced AHR in asthmatic children and adults requiring daily medication use. Interestingly in this study, NO

2 expo-

sures and CA exposures were performed separately and with exposure to combustion by-products from a gas space heater (Vulcan Quasar Flueless, Model 8302 T18) in order to simu-late an indoor setting exposure. Additional NO

2 was added

to NO2 associated with the combustion by-products to result

in NO2 exposure concentrations of either 0.3 or 0.6 ppm. This

made it possible to compare NO2 exposures, not only to CA

exposures, but also to NO2 exposures combined with indoor

sources of air pollutants. The space heater by-products also contained mean concentrations of 0.6 ppm NO and 2 ppm CO. The authors reported NO

2-induced changes in AHR

to histamine challenge following the 0.6-ppm exposure without the by-products, whereas the rest of the exposures showed no NO

2-specific responses. The AHR observed at

0.6 ppm NO2 was not associated with changes in symptoms

or asthma severity based on extended follow-up of up to 7 days post exposure, meaning these findings are of uncertain clinical relevance. The authors suggested that in most of the studies reporting increased AHR with NO

2 exposure, the

findings were close to the limits of reproducibility of AHR tests. This may explain the findings of Salome et al. (1996), where exposure to 0.6 ppm NO

2 with the by-products from

the gas heater had no significant effect on AHR, whereas exposure to 0.6 ppm NO

2 alone required half the amount

of bronchoconstrictor challenge to produce the same effect observed following CA exposure. This study was not cited in the US EPA Integrated Science Assessment for NO

x (US EPA,

2008a). However, the study raises some important questions

concerning AHR test reproducibility and threshold effects, especially in asthmatic children and adults requiring daily medication.

Many of the clinical studies of AHR in NO2-exposed

asthmatics are limited by small numbers of subjects, which affects their statistical power and also contributes to uncer-tainties regarding population representativeness. In an effort to address these limitations, Folinsbee (1992) per-formed a meta-analysis of the results from 19 clinical NO

2

exposure studies assessing AHR in NO2-exposed asthmatics,

all but two of which (the Ahmed et al. [1983a] and Orehek et al. [1981] studies, which used specific challenges, namely ragweed and grass pollen, respectively) relied upon nonspe-cific challenges. Data were included in the analysis only if a subject’s directional change in airway responsiveness was reported. Folinsbee (1992) found statistically significant NO

2-induced AHR to inhaled challenges at relatively low

NO2 concentrations ranging between 0.05 and 0.20 ppm.

This NO2-induced AHR was statistically significant at rest

but not with exercise in asthmatics. In addition, Folinsbee reported an effect threshold level of NO

2 >1 ppm in healthy

subjects, based on statistically significant increases in airway reactivity. However, the methods used by Folinsbee have some important limitations, with the meta-analysis includ-ing studies not sharing standardized protocols, measure-ment methods, or the same bronchoconstrictor stimulus. In addition, responders from different studies were grouped according to exposure concentration and disease status without regard for other personal characteristics. Moreover, responses were not standardized, i.e., a provocative dose method was not established in most studies, making inter-comparison difficult. Lastly, no consideration was placed on whether the changes in airway responsiveness were statisti-cally significant. Given these major limitations, the reliability of the conclusions from this analysis is questionable.

US EPA (2008a) recently performed an updated meta-analysis of the studies included in the Folinsbee analysis. Rather than performing their own independent literature search to identify all relevant studies and specifying formal inclusion and exclusion criteria, US EPA primarily relied upon the earlier Folinsbee (1992) analysis as the source of the studies under consideration. It thus appears that they did conduct their own independent analysis of the quality of the studies and factor this into study selection. Interestingly, US EPA replaced the study by Ahmed et al. (1983a), which used a specific challenge (ragweed), with the Strand et al. (1996) study, which utilized histamine, a nonspecific air-ways challenge. Although US EPA performed this step for the apparent purpose of achieving homogeneity in the type of challenge used, they performed analyses that combined data from studies using different experimental protocols, including different NO

2 exposure techniques (e.g., mouth-

piece versus chamber), different exposure times, different airway challenges (pharmacological agents versus cold air versus sulfur dioxide), different endpoints (airway resist-ance versus airway conductance versus forced expiratory volume), different exposure conditions (exercising versus

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at rest), and different times between exposure to the airway challenge and lung function assessment. Based upon their analyses, which relied upon a statistical test (a sign test) that considered only the frequency of positive responders and not the magnitude of responses, US EPA concluded that short-term exposures to NO

2 at outdoor ambient concentra-

tions (<0.3 ppm) are linked to nonspecific AHR in people with mild asthma. However, it is important to note that US EPA did not consider whether the individual responses were of statistical significance, nor did they consider the magni-tude of change in individual responses. Therefore, the valid-ity of their sign-test conclusions is questionable given that the reported changes in airways responsiveness following NO

2 exposure may be well within the normal variation and

would therefore not constitute an adverse health response.To address some of the major limitations remaining in

the updated US EPA (2008a) meta-analysis, Goodman et al. (2009a) performed a meta-regression analysis of the studies included in US EPA’s analysis, adding back the Ahmed et al. (1983a) study with ragweed and including four additional studies with specific allergen challenges (Barck et al., 2002; Jenkins et al., 1999; Strand et al., 1998; Witten et al., 2005). Goodman et al. (2009a) incorporated these studies into their meta-regression analysis because they represent environ-mentally relevant exposure scenarios for assessing whether NO

2 induces AHR. Meta-regression was used for data analy-

sis rather than a sign test, given that it is a more robust sta-tistical technique that considers the magnitude of response as a function of exposure in determining whether there is evidence of exposure-response relationships. Importantly, with this updated list of studies and improved statistical technique, Goodman et al. (2009a) concluded, “There is no indication from the regression analyses that NO

2 exposure

increases airway responsiveness to either specific or non-

specific airway challenges for individuals with asthma, for concentrations up to at least 0.6 ppm NO

2.” Because the

Goodman et al. (2009a) analysis still shared some of the sig-nificant limitations of US EPA’s recent analysis (e.g., lack of study inclusion and exclusion criteria, heterogeneity among study protocols), Goodman et al. (2009b) conducted a formal meta-analysis of studies assessing airway hyperresponsivess in NO

2-exposed asthmatics that (1) used predetermined

inclusion and exclusion criteria, together with a compre-hensive literature search, to identify studies appropriate for the meta-analysis; and (2) assessed the effects of different study protocols, including exposure during exercise versus at rest; different methods for exposing study subjects to NO

2

(e.g., mouthpiece versus chamber); different exposure times, different airway challenges (pharmacological agents versus cold air versus sulfur dioxide); different endpoints (airway resistance versus airway conductance versus forced expira-tory volume); and different times between exposure to the airway challenge and assessment of lung function. Although the Goodman et al. (2009b) meta-analyses yielded several small effect estimates that achieved statistical significance, Goodman et al. (2009b) did not find any concentration-response associations, and concluded that the observed effect estimates were sufficiently small so as to be of unlikely adversity. Overall, Goodman et al. (2009b) concluded, “The results of our analyses indicate that, to the extent the effects observed are associated with NO

2 exposure, they are suf-

ficiently small such that they do not provide evidence that NO

2 has a significant adverse effect on AHR at concentra-

tions up to 0.6 ppm.”In conclusion, our review has critically examined the

studies included in the Folinsbee meta-analysis and we fur-ther evaluated more recent studies that utilized nonspecific challenges. Some studies are suggestive of effects at NO

2

NO2 Exposure Concentration (ppm)

0

0.5

1

1.5

2

2.5

3

3.5

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Am

ount

of A

irway

Con

stric

tor (

FA/N

O2)

0%

1%

2%

3%

4%

5%

6%

7%

% C

hang

e in

Lun

g Fu

nctio

n

change in [airway constrictor]change in lung function

Kleinman et al. 1983Strand et al. 1997

Bauer et al. 1986

Orehek et al. 1976

Strand et al. 1996

Tunnicliffe et al. 1994Mohsenin 1987

Bylin et al. 1985

Salome et al. 1996Jenkins et al. 1999

Bylin et al. 1988Strand et al. 1998

Jörres & Magnussen 1990

Figure 7. The magnitude of statistically significant NO2-induced changes in AHR. The primary y-axis (left) represents the ratio of the amounts of air-

way constrictor needed to achieve a predetermined study-specific magnitude of change in lung function to a bronchoconstrictor (NO2-induced AHR

relative to CA). Thus, a ratio of 2.0 means that the respective study subjects were twice as sensitive (or hyperreactive) to airways challenge following NO

2 as compared to CA. The secondary y-axis (right) represents the difference in percent change in lung function (FEV

1, FEF) after NO

2 exposure as

compared to after CA exposure. Although not shown in this figure, two studies investigated NO2-induced AHR at NO

2 concentrations greater than 0.6

ppm, reporting statistically significant NO2-induced AHR responses with 1.5 ppm (2.4% decrease in FVC) and 2 ppm (1.25-fold increase in amount of

bronchoconstrictor).

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exposure concentrations between 0.2 and 0.6 ppm; how-ever, the effects are relatively small, and for the majority of studies, NO

2 exposure versus clean air exposure produced

no adverse symptoms or a decrease in adverse symptom frequency. Therefore, the health adversity of the small NO

2-

induced lung function changes (for the most part, only 2–7 % different from baseline or control exposures; see Figure 7) is questionable.

NO2-induced AHR to specific challengesStudies conducted since the meta-analysis by Folinsbee (1992) have mostly examined NO

2 effects on airway respon-

siveness to specific stimuli, based on individuals’ diagnosed allergies. Importantly, results from studies examining NO

2-

induced AHR to specific challenges should be evaluated with caution because the likelihood of elevated levels of both NO

2 and the specific stimulus for a susceptible individual is

likely small. Table 5 summarizes the studies that examined NO

2-induced AHR to specific challenges, while Figure 6

illustrates the outcomes from these studies.Two groups have examined both the effects of NO

2-

induced specific and nonspecific AHR (Ahmed et al., 1983a, 1983b; Orehek et al., 1976, 1981). Only one of the groups, namely Orehek et al. (1976), reported evidence of NO

2-

induced adverse effects and that was in combination with carbachol, a nonspecific airway constrictor, and using unre-liable statistical techniques. Notably, Orehek et al. (1981) failed to confirm their finding with carbachol in their sub-sequent study with a similar design but a specific challenge agent (0.11 ppm NO

2, 1 h, followed by a ragweed challenge).

In contrast to Orehek et al. (1976), Ahmed et al. (1983b) reported no significant effects on AHR to carbachol after 0.1 ppm NO

2 (1 h) exposure in 20 healthy and 20 asthmatic

subjects. This group also exposed 20 adults with a history of bronchial asthma and ragweed sensitivity to 0.1 ppm NO

2

for 1 h (Ahmed et al., 1983a) followed by a ragweed chal-lenge, and reported no NO

2-induced changes in central or

peripheral airway function as assessed by FEV1 and SGaw

measurements, respectively. When taken together, these inconsistent study findings are not highly supportive of NO

2-

specific adverse effects in healthy or asthmatic individuals at exposure concentrations <0.2 ppm.

Also for a 0.1 ppm NO2 exposure concentration, Tunnicliffe

et al. (1994) assessed the NO2-induced effects on AHR in

response to HDM challenge but reported no significant results in patients with mild asthma. However, in the same experiment, exposure to 0.4 ppm NO

2 yielded significant

changes in lung function in response to subsequent HDM challenges (HDM caused FEV

1 decreases of 18.6% for 0.4

ppm NO2 versus 14.6% for CA for early-phase reaction, and

8.1% versus 2.8% for late reaction). Based on their finding, the authors suggested a threshold between 0.1 and 0.4 ppm for NO

2 effects on airway response to specific challenges.

Similarly, Jenkins et al. (1999) exposed mild atopic asthmatic patients to 0.2 ppm NO

2 for 6 h or 0.4 ppm NO

2 for 3 h with

intermittent moderate exercise, followed immediately by a bronchial allergen challenge. The 0.2 ppm NO

2 exposure did

not alter the response to the subsequent allergen challenge. However, with the higher 0.4 ppm NO

2 exposure for a shorter

period of 3 h, the authors reported increased airway respon-siveness to the allergen challenge. In the same study, Jenkins et al. (1999) also found no statistically significant changes in AHR for a 6-h exposure to 0.1 ppm ozone (O

3) + 0.2 ppm

NO2, but significant airway changes with 3-h exposures to

0.2 ppm O3 alone or 0.2 ppm O

3 + 0.4 ppm NO

2. These results

suggest that a threshold concentration, rather than the total amount of inhaled pollutant, may drive pollutant-induced AHR in mild asthmatics.

A series of studies from Sweden (Strand et al., 1997, 1998; Barck et al., 2002, 2005) assessed the proinflammatory effects and AHR to short term NO

2 exposures (0.26 ppm, various

durations). Strand et al. (1997) exposed 18 asthmatics having pollen allergy to 0.26 ppm NO

2 for 30 min (at rest) followed

by an allergen inhalation challenge at 4 h post exposure. NO

2 enhanced the late asthmatic reaction to allergen by

lowering FEV1 (~4%) and peak expiratory flow (PEF) (~7 %)

in comparison to CA even if NO2 did not by itself signifi-

cantly increase the induction of a late asthmatic reaction as compared to CA. Subsequently, Strand et al. (1998) exposed 16 subjects having mild asthma and allergy (birch or grass pollen) for an extended period (4 consecutive days) to the same protocol as in their previous study (Strand et al., 1997). The authors reported a significantly increased asthmatic response after this repeated exposure to NO

2 + allergen as

compared to CA + allergen. The 4-day mean fall in FEV1 after

NO2 was significant at the early (−2.5% NO

2 versus −0.4% CA)

and late phase (−4.4% NO2 versus −1.9% FA) when compared

to sham exposure. However NO2-induced symptoms of

asthma were not significantly increased suggesting unlikely health adversity. The studies by Strand et al. (1997, 1998) suggest that changes in lung function and AHR may occur following NO

2 exposures, and may persist for several days.

However, these observed responses were relatively small, and were not associated with increased asthma symptoms, thus the health relevance of these findings is uncertain.

In an effort to explore the effect of NO2 inhalation on the

allergic and inflammatory response in asthmatics, the same group (Barck et al., 2002, 2005) exposed 31 subjects with mild asthma and allergy at rest to either CA or 0.26 ppm NO

2

(15 or 30 min), followed 4 h later by an allergen inhalation challenge under similar protocols. Subjects were exposed to CA on 1 day and NO

2 on 2 consecutive days. The authors

reported no significant NO2-induced pulmonary function

effects or symptoms in either study, but taken together, reported significantly increased neutrophils, ECP, and MPO in peripheral blood and sputum. The authors suggest that NO

2 can prime eosinophils in sputum and peripheral blood.

However, these increases in PMN degranulation products were only accompanied by an increased number of pul-monary eosinophils (Barck et al., 2002), but no changes in eosinophil numbers in blood or interleukin-5 (IL-5) levels. In addition, the study design did not allow observation of subsequent effects with prolonged exposure to NO

2. In gen-

eral, the findings from this group illustrate inconsistencies

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between NO2-induced changes in lung function, frequency

of adverse symptoms, and proinflammatory/proallergic responses.

The highest exposure concentration used to examine NO

2-specific AHR to specific challenges was 0.4 ppm.

Solomon et al. (2004) and Witten et al. (2005) both exposed house dust mite-sensitive asthmatic subjects for 3 h to CA or 0.4 ppm NO

2, but reported no significant NO

2-induced effect

on AHR to HDM allergen. Similarly, Devalia et al. (1994) did not show NO

2-induced AHR in subjects with asthma.

Devalia et al. (1994) studied the effect of 6-h exposures to CA, 0.4 ppm NO

2, 0.2 ppm SO

2, and the two gases in combi-

nation on AHR to inhaled HDM allergen in HDM-sensitive asthmatics. Compared with CA, neither SO

2 nor NO

2 (or

their combination) significantly altered FEV1 or FVC, but

the authors found significant effects on HDM-induced AHR to HDM after exposure to NO

2 and SO

2 together in mixture.

The results from this study suggest that 0.4 ppm NO2 expo-

sure, by itself, is not sufficient to cause AHR to HDM in these asthmatic subpopulations. The results from these studies are in contrast with those from both Jenkins et al. (1999) and Tunnicliffe et al. (1994) who reported NO

2-specific AHR at

a 0.4 ppm concentration. Overall, results from these studies suggest mixed effects in asthmatics for NO

2 exposure con-

centrations as high as 0.4 ppm.To our knowledge, the short-term studies examining

NO2-induced AHR to specific stimuli are limited to a narrow

concentration range between 0.1 and 0.4 ppm in subjects having asthma. Our survey of these studies indicates that in only some did NO

2 elicit significant changes in airways

responsiveness. However, the changes were generally small and manifested mainly at exposure concentrations above 0.25 ppm NO

2. These mixed results indicate that there may

be a subpopulation of asthmatics that is susceptible to NO2-

induced AHR at NO2 concentrations between 0.2 and 0.4

ppm as indicated by Jenkins et al. (1999). However, when such effects are indeed observed, the symptoms generally do not rise to the category of “adverse” by ATS guidelines (ATS, 1999). The small magnitude of NO

2-induced pulmo-

nary changes and the transient nature of such changes in the absence of symptoms, raising questions regarding the health adversity and public health relevance of these findings.

Extrapulmonary effects associated with NO2 exposureClinical studies have focused primarily on the pulmonary effects of NO

2, with only a few studies examining possible

health outcomes in other organ systems. Some studies that have collected peripheral blood samples provide some lim-ited evidence of changes in blood chemistry following short-term NO

2 exposure, but only at NO

2 concentrations that are

much higher than those usually encountered in ambient air (see our Figure 3). For example, Barck et al. (2005) reported an increase in ECP (an eosinophil degranulation product) in blood with short-term exposures to 0.26 ppm NO

2 in sub-

jects having asthma. At higher exposure levels of 0.6 and 1.5 ppm, Frampton et al. (2002) reported decreased hematocrit and lymphocyte levels, but such effects were not observed

in a similar study by Rubinstein et al. (1991) for 0.6-ppm exposures. At a higher NO

2 exposure concentration of 2

ppm (but not 1 ppm), Posin et al. (1978) reported changes in RBC chemistry with decreased hematocrit and hemoglobin levels.

Other studies have monitored and reported physiologi-cal measurements during NO

2 exposures (Drechsler-Parks

et al., 1987; Gong et al., 2005; Kim et al., 1991; Jörres et al., 1995). For example, Jörres et al. found no significant NO

2-

induced changes in AHR (1 ppm NO2, 3 h), whereas Gong

et al. (2005) reported an absence of NO2-induced changes in

blood pressure (0.4 ppm NO2, 2 h). Although findings from

these studies do not indicate NO2-associated extrapulmo-

nary physiological changes, it is important to note that they were not specifically designed to examine them.

Overall, the few studies examining the extrapulmonary effects of NO

2 exposure have mostly utilized exposure con-

centrations much higher than those occurring in outdoor air. Although the study results are mixed with respect to cellular changes in blood, there is no evidence of sig-nificant changes in cardiovascular function in the limited number of studies that examined them. Therefore, there is only limited human clinical evidence for NO

2-induced

extrapulmonary effects, and more research is warranted at exposure concentrations that are relevant to environmen-tal exposures.

Discussion

Our review of human volunteer studies on NO2 suggests

that short-term exposure to NO2 concentrations well above

those usually encountered in ambient outdoor atmospheres (see our Figure 3) generally have mild and reversible effects on lung function. Greater sensitivity has generally been observed in individuals with some degree of preexisting lung disease, such as asthma. These individuals appear responsive to lower NO

2 concentrations than healthy volun-

teers, as indicated by NO2-induced AHR after specific and

nonspecific challenges. However, a collective examination of the concentration-response data for studies that showed statistically significant NO

2-induced AHR (see Figure 7)

did not show a concentration-dependent effect for these outcomes. In fact, the surveyed studies provide only weak evidence of pulmonary effects at exposures in the range of 0.2 to 0.6 ppm NO

2.

Our review indicates that in only some of the studies did NO

2 elicit significant changes in airways responsiveness (see

Figure 6). However, these changes were generally small (~5%) (see Figure 7), and when observed, the associated symptoms generally did not rise to the level of “adverse” by ATS guide-lines. Moreover, there is no exposure-dependent increase in studies showing statistically significant NO

2-induced AHR

(see Figure 7). The small magnitude of NO2-induced pulmo-

nary changes, and the transient nature of such changes in the absence of symptoms, suggest that some of the observed NO

2-induced changes are of minimal adversity to health and

may be of little public health relevance.

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Although a threshold of effect cannot be determined with certainty, it can be inferred with some confidence that no adverse effects occur for short-term exposures below 0.2 ppm NO

2. Although one older study (Orehek et al., 1976)

identified a subpopulation of “responders” to 0.11 ppm NO2

and calculated a significant difference in those individuals’ responses compared to those of nonresponders, all other studies have reported no statistically significant effects in that concentration range (Hazucha et al., 1983; Ahmed et al., 1983a, 1983b; Tunnicliffe et al., 1994; Orehek et al., 1981; Bylin et al., 1988; Roger et al., 1990). Indeed, most studies of short-term exposures to NO

2 suggest a likely threshold for

health effects in the 0.2–0.6-ppm concentration range for susceptible populations.

The two studies that used a 0.2 ppm NO2 exposure level

have contrasting outcomes (Kleinman et al., 1983; Jenkins et al., 1999). Kleinman found some evidence of NO

2-induced

AHR, whereas Jenkins failed to show such effects at 0.2 ppm NO

2 but did observe effects at 0.4 ppm NO

2. This variation in

outcomes among studies may be possibly due to a difference in the type of airway challenge used. Kleinman et al. (1983) used methacholine whereas Jenkins et al. (1999) used house dust mite (HDM) inhalation challenges. Moreover, the NO

2-

induced changes in bronchial reactivity reported in a sub-set of study participants by Kleinman et al. (1983) were not associated with increased asthma symptom frequency. To the contrary, the authors reported more adverse symptoms with sham exposures, suggesting that these changes were not health-adverse effects of NO

2 exposure.

The most recent studies of individuals with pulmonary susceptibilities such as asthma have focused on examin-ing NO

2-induced effects at concentrations above 0.25 ppm

(Salome et al., 1996; Barck et al., 2002, 2005). However, even in studies where statistically significant changes in lung function and bronchial sensitivity were reported, the health relevance of these results remains unclear, because these changes have been mild and transient in the majority of studies. In addition, these changes are usually not accom-panied by NO

2-dependent increases in adverse health

symptoms.We attempted to identify a possible threshold for adverse,

NO2-induced effects on pulmonary function by relying on

studies demonstrating an exposure-response relationship for NO

2. In some studies, there was an absence of NO

2-

induced health effects at all exposure concentrations tested. For example, Roger et al. (1990) examined methacholine-induced airway reactivity in 21 mild asthmatics after 0.15, 0.30, or 0.6 ppm NO

2 exposures for 75 min and reported no

significant effects. Likewise, Avol et al. (1988) followed 0.3 and 0.6 ppm NO

2 exposure (120 min) with cold air and found

no significant effects in 59 subjects whom the authors classi-fied as having moderate to severe asthma. One study (Bylin et al., 1988) on postexposure histamine-induced changes in lung function in 20 mild asthmatics reported significant effects only at 0.27 ppm but not at lower and higher con-centrations (0.14 or 0.48 ppm NO

2, 30 min). Overall, these

studies, which were specifically designed to test for both an

exposure concentration-dependent effect and a threshold of NO

2-induced AHR, were not conclusive.

Some evidence to support a threshold comes from Jenkins et al. (1999) and Tunnicliffe et al. (1994); both groups reported NO

2-induced AHR at 0.4 ppm but not at 0.2 ppm or

0.1 ppm. When taken together, these two studies suggest a threshold above 0.2 ppm NO

2. Salome et al. (1996) reported

NO2-induced AHR at 0.6 ppm but not at 0.3 ppm, suggesting

a threshold above 0.3 ppm NO2. Taken together, data from

controlled studies of short-term NO2 exposures suggest that

a threshold for NO2-induced changes in lung function is

unlikely to be below 0.2 ppm because most studies with sig-nificant outcomes used exposure concentrations between 0.26 and 0.6 ppm NO

2. The possibility of a higher threshold

for adverse effects is difficult to examine because no studies on AHR were performed at concentrations above 0.6 ppm NO

2. This limits our assessments and conclusions to the few

concentration-response studies available, in addition to a collective examination of single-concentration exposure studies.

Despite the differences in challenge type, both sets of studies using specific and nonspecific challenges appear to show statistically significant NO

2-associated pulmonary

changes mostly in the 0.25–0.3-ppm range. As discussed previously, these pulmonary changes do not increase in a concentration-dependent manner. It is possible that this increased likelihood for effects is simply a result of hav-ing a larger number of studies performed in that range. Nevertheless, this suggests that a certain number of subjects in these studies are more susceptible than others.

It is possible that physiological changes and immune responses corresponding to larger and more persistent effects may evade detection due to inadequate response assessment. For example, experimental designs may intro-duce factors that mask a potential NO

2 effect. It has been

suggested by Folinsbee (1992) that exercise may diminish the chances of observing a significant effect of NO

2, i.e., that

incorporating exercise into studies examining the potential for NO

2-induced hyperreactivity may underestimate the

effect of NO2. Two arguments to that effect include a reduc-

tion in the relative uptake of NO2 at tracheobronchial sites

with exercise and a masking of an NO2 effect by exercise-

induced bronchoconstriction. However, increased ventila-tion during exercise results in a higher total delivered dose of NO

2, with exercise having been shown to increase the

total percentage of inhaled NO2 that is absorbed (Bauer

et al., 1986). A significant portion of inhaled NO2 is removed

in the nasopharynx; thus, as breathing becomes more oral with exercise, increased delivery to the lower respiratory tract can be expected (Miller et al., 1989). However, this does not diminish the amount of inhaled gas reaching the tra-cheobronchial sites; rather this may increase such delivery given the higher intake with a higher inspired volume, with only a change in the ratio of tracheobronchial-to-alveolar distribution.

Bronchoconstriction and increased airway resistance occur as a result of exercise, and this has been hypothesized

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to diminish the significance of responses to NO2 in stud-

ies combining these two parameters. It is possible that the large effect of exercise on SRaw may mask the much smaller effects of NO

2. However the results from several studies

showing NO2-induced effects with exercise do not support

this hypothesis (Kleinman et al., 1983; Bauer et al., 1986; Strand et al., 1996; Bylin et al., 1988; Frampton et al., 1991; Jenkins et al., 1999). For example, Bauer et al. (1986) demon-strated NO

2-induced AHR to cold air increases with exercise

in asthmatic volunteers (0.3 ppm NO2, 30 min). In addition,

many studies conducted at rest have reported a lack of sig-nificant NO

2-induced AHR at NO

2 concentrations ranging

from 0.1 ppm to higher than 0.3 ppm (Ahmed et al., 1983a, 1983b; Hazucha et al., 1983; Bylin et al., 1988; Salome et al., 1996; Barck et al., 2002, 2005), and this further weakens sup-port for the hypothesis suggesting that exercise masks NO

2-

induced AHR.The mechanisms of NO

2-induced pulmonary effects

remain uncertain. Elucidating a mechanism (or mecha-nisms) of action for NO

2 may help explain the variations in

clinical study outcomes. In addition, an improved mecha-nistic understanding can help focus research efforts on spe-cific health endpoints and assist with the development of acceptable exposure levels. The hypotheses for mechanisms of action involve NO

2-stimulated release of proinflammatory

mediators, proallergenic responses, or mediation of airway smooth muscle contraction. In a review of such potential mechanisms, Krishna and Holgate (1999) suggest that inflammatory pathways underlie the potentiating effects of NO

2 on AHR responses to inhaled aeroallergens in aller-

gic airways disease. This hypothesis is based on increased levels of proinflammatory mediators in the lung, namely neutrophils and eosinophils, which potentiate the early and later asthma responses and thereby augment allergic airway disease.

In some of the studies addressed in this review, increases in PMN or allergic mediators such as eosinophils or ECP protein have been reported, but without any NO

2-induced

changes in AHR to a specific challenge (e.g., at 0.26 ppm: Barck et al., 2002, 2005; at 0.4 ppm: Wang et al., 1995). Other studies at similar concentrations (e.g., 0.4 ppm, 3 h: Solomon et al., 2004; Witten et al., 2005) have not shown changes in either lung function or inflammation in response to NO

2

exposure. This absence of effect or discordance between pulmonary biochemical responses and lung function at lower NO

2 concentrations calls into question the validity of

Krishna and Holgate’s hypothesis that ambient NO2 concen-

trations cause changes in AHR via inflammatory responses and also with the respective methods used to assess inflam-mation and lung function.

Instead, at high NO2 concentrations, there may be coor-

dinated changes in NO2-induced pulmonary inflammation

and lung function. This is supported by Jörres et al. (1995) who reported lung function decrements and increased proinflammatory prostaglandins in the lung following 1 ppm NO

2 inhalation by asthmatic subjects. Additional evidence is

found in the study by Blomberg et al. (1999) who reported

transient decrements in lung function and persistent airway neutrophilia with 2 ppm NO

2 exposure (4 h/day for 4 succes-

sive days). However, questions remain about the timing of these effects, because the inflammation persisted whereas the lung function effects dissipated, suggesting differences in the adaptation of biological systems to NO

2 inhalation.

NO2-induced AHR and lung function changes may also

occur independently of inflammation. The mechanisms of action for NO

2 may be similar to autonomic nervous system

modulators that have also been shown to cause AHR in response to chemical challenges. For example, Vatrella et al. (2001) demonstrated that low nonbronchoconstrictor doses of inhaled propranolol, a sympathetic antagonist, consid-erably increased AHR to methacholine in asthmatics and rhinitics (but not healthy individuals). The parasympathetic nervous system is the dominant neuronal pathway in the control of airway smooth muscle tone. Cholinergic agonists such as methacholine and carbachol stimulate the para-sympathetic branch of the autonomic nervous system and have bronchoconstrictor effects. Similarly to propranolol-induced AHR, NO

2 concentrations that were not sufficient to

affect lung function changes independently may instead be capable of inducing AHR by lowering the threshold of action for the challenge chemicals. This may explain why, in most studies, NO

2-induced AHR was more likely to be observed at

levels much lower than those inducing inflammation.Although it can be generally argued that findings from

human clinical studies of small groups of relatively healthy adult volunteers may not be representative of the responses of larger populations (e.g., potentially including highly sus-ceptible populations), it is important to note that several aspects of the NO

2 clinical studies likely serve to increase their

likelihood of detecting adverse responses and thus of identi-fying NO

2 threshold levels that are protective for the general

population. In particular, it is likely that subject recruitment, study designs, and exposure protocols employed in clinical studies of NO

2-induced AHR contribute to their increased

likelihood of detecting adverse responses. This likelihood stems from two key considerations: (1) The subjects from these studies include not only healthy volunteers but also individuals varying in disease status, ranging from mild asthmatics to moderate asthmatics requiring daily medica-tion (note that the Avol et al., 1988, study specifically clas-sified some of its volunteers as having “severe” asthma), as well as COPD patients; and (2) the hypothetical setting that is inherent to these experimental designs may not represent a typical everyday exposure scenario given that it requires a susceptible individual and high concentrations of both NO

2 and airway bronchoconstrictor or stimulus in a tandem

sequence. This setting, plus use of sensitive laboratory tests and instruments, is more likely to result in the detection of potentially adverse NO

2-induced effects.

Conclusions

Epidemiology studies have frequently reported associations between increments in low ambient levels of NO

2 (i.e., for

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0.01 ppm NO2 increments) and adverse health outcomes.

Some studies have suggested that no exposure threshold exists for NO

2-induced health outcomes. A valid interpreta-

tion of these associations is very difficult to achieve at the present time, however, because of issues regarding exposure misclassification, unmeasured co-pollutants, and residual confounding, such as from highly correlated co-pollutants. There is a striking discordance between these positive epi-demiological associations and the results from controlled clinical NO

2 exposure. Clinical studies using NO

2 exposure

concentrations that are 10-fold or even higher than the epidemiological, ambient NO

2 levels have predominantly

reported a lack of health-adverse outcomes in healthy sub-jects, as well as in lung-compromised subjects. Moreover, clinical studies do not suggest meaningful mechanistic pathways that would explain the associations in epidemiol-ogy studies at ambient NO

2 levels. Because the clinical study

outcomes fail to support the epidemiological associations, this strengthens the potential role for confounding by meas-ured or unmeasured with co-pollutants, or by other unmeas-ured parameters, in the epidemiologic findings.

Our review found that the populations most suscepti-ble to NO

2-induced pulmonary effects appear to be those

with asthma or allergic diseases. However, adverse effects in the lung were not likely to occur, even at high NO

2 expo-

sure concentrations, without a coexposure to specific or nonspecific stimuli such as bronchoconstrictor agents or pollen allergens. NO

2-induced adverse pulmonary effects

in healthy individuals were generally not observed below NO

2 concentrations of 1 ppm. The observed pulmonary

effects in asthmatics were predominantly observed at NO2

exposure concentrations ranging from 0.2 to 0.6 ppm and were generally small and transient. The small magnitude of NO

2-induced pulmonary changes (for the most part,

only 2–7% different from baseline or control exposures), and the transient nature of these changes in the absence of symptoms, diminish the likelihood of a causal basis to the adverse health outcomes reported by epidemiology studies and raise questions regarding the clinical significance of these findings.

Abundant clinical data on NO2-induced health effects

are available for short-term NO2 exposures, and these

controlled exposure studies, particularly those for NO2-

induced AHR in asthmatic populations, should serve as a key resource in the NO

2 standard-setting process. Because

these studies specifically preselect individuals who have a known significant response to the challenge chemical suggests that the outcomes from these experiments are representative of those of a highly susceptible population, and this idea must be considered when weighing alterna-tive air-quality standard levels. Our review of these clinical studies indicates that such a standard for short-term NO

2

exposure, set between 0.2 and 0.6 ppm, can be anticipated to be protective of both potentially susceptible populations and healthier populations.

In commenting on the relevance of the human-volunteer study data to the setting of the NAAQS, we recognize that

the selection of a specific numerical standard (matched to the indicator, averaging time, and statistical form) is a policy judgment reserved for the US EPA Administrator. Our analy-sis indicates that a policy choice within the range of 0.2 and 0.6 ppm can be anticipated to be protective of the public health, including that of potentially sensitive subpopula-tions, for short-term NO

2 exposures. As an alternative, the

Administrator could make a policy judgment based on these data and knowledge of the relationship between the annual average NO

2 and 1-h maximum NO

2 concentrations that the

current annual standard of 0.053 ppm would be sufficiently protective of the health effects from short-term NO

2 expo-

sures in the range of 0.2–0.6 ppm.

Acknowledgments

Many thanks to Dr. Sonja Sax of Gradient for her editorial review of the manuscript during its preparation.

Declaration of interest: This review was prepared by T. W. Hesterberg and W. B. Bunn in the course of their employ-ment with Navistar, Inc., and by R. O. McClellan, Advisor in Toxicology and Risk Analysis, and A. K. Hamade, C. M. Long, and P. A. Valberg of Gradient Corporation, as consultants to Navistar, Inc. Gradient provides consulting services to a variety of parties, including industry (e.g., utilities, engine manufacturers), governmental agencies, regulators, and law firms. R.O. McClellan has also served as a consultant to the American Petroleum Institute on matters related to the set-ting of National Ambient Air Quality Standards. The scien-tific analyses, content, and writing of this paper are the sole responsibility of the authors.

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