Free Radical Biology & Medicine, Vol. 35, No. 4, pp. 327–340, 2003
Copyright © 2003 Elsevier Inc.
Printed in the USA. All rights reserved
0891-5849/03/$–see front matter
doi:10.1016/S0891-5849(03)00280-6
Serial Review: Role of Reactive Oxygen and Nitrogen Species (ROS/RNS)
in Lung Injury and Diseases
Guest Editor: Brooke T. Mossman
REACTIVE OXYGEN SPECIES IN PULMONARY INFLAMMATION BY
AMBIENT PARTICULATES
FLORENCE TAO,* BEATRIZ GONZALEZ-FLECHA,*
and
LESTER KOBZIK*†
*Department of Environmental Health, Harvard School of Public Health, Boston, MA, USA; and †Department of Pathology,
Brigham and Women’s Hospital, Boston, MA, USA
(Received 16 December 2002; Revised 14 April 2003; Accepted 17 April 2003)
Abstract—Exposure to ambient air pollution particles (PM) has been associated with increased cardiopulmonary
morbidity and mortality, particularly in individuals with pre-existing disease. Exacerbation of pulmonary inflammation
in susceptible people (e.g., asthmatics, COPD patients) appears to be a central mechanism by which PM exert their
toxicity. Health effects are seen most consistently with PM with aerodynamic diameter ⬍ 2.5 ␮m (PM2.5), although 10
␮m ⬍ PM ⬍ 2.5 ␮m can also be toxic. Through its metal, semi-quinone, lipopolysaccaride, hydrocarbon, and ultrafine
constituents, PM may exert oxidative stress on cells in the lung by presenting or by stimulating the cells to produce
reactive oxygen (ROS). In vivo, PM increase cytokine and chemokine release, lung injury, and neutrophil influx. In vitro
analysis of PM effects on the critical cellular targets, alveolar macrophages, epithelial cells, and neutrophils, demonstrates PM- and oxidant-dependent responses consistent with in vivo data. These effects have been observed with PM
samples collected over years as well as concentrated PM2.5 (CAPs) collected in real time. Oxidative stress mediated by
ROS is an important mechanism of PM-induced lung inflammation. © 2003 Elsevier Inc.
Keywords—Free radicals, Oxidative stress, Ambient air particles, Alveolar macrophages, Epithelial cells, Neutrophils
INTRODUCTION
include exacerbation of pre-existing diseases, such as
asthma and chronic obstructive pulmonary disease
(COPD), and increased hospital admissions for
pneumonia.
The mechanisms by which PM exposure leads to
health effects are under active investigation but remain
undefined. A priori, it is reasonable to expect that different pathways may be identified for different pathophys-
Epidemiologists have linked ambient particulate air pollution (PM) exposure with increased cardiopulmonary
mortality and morbidity [1–3]. The respiratory effects
This article is part of a series of reviews on “Role of Reactive
Oxygen and Nitrogen Species (ROS/RNS) in Lung Injury and Diseases.” The full list of papers may be found on the homepage of the
journal.
Dr. Florence Tao received her Ph.D. in 1999 from McGill University, Montréal, Québec, Canada. She is a Research Associate in the
Physiology Program of the Department of Environmental Health at the
Harvard School of Public Health, Boston, MA. Her research focuses on
the effects of alveolar macrophage-epithelial cell interaction on particle-induced inflammation as well as the effects of ambient particles on
epithelial microbial defense.
Dr. Beatriz González-Flecha obtained her Ph.D. degree at the University of Buenos Aires, Argentina in 1991, and performed postdoctoral studies at the Department of Molecular and Cellular Toxicology,
Harvard School of Public Health from 1992 to 1996. From 1996 to
1997 she was an Instructor of Toxicology at the same Department. She
received the Young Investigator Award of the VIII Biennial Meeting of
the International Society for Free Radical Research, Barcelona, in
1996. She is currently an Assistant Professor of Molecular Biology and
Environmental Health at the Department of Environmental Health,
Harvard School of Public Health, where she investigates the role of
oxygen free radicals as second messengers in proliferation and apoptosis of lung epithelial cells and the oxidant-dependent mechanisms in
response to ambient air particles.
Dr. Lester Kobzik received his M.D. from Tufts University School of
Medicine in Boston, MA. He is Associate Professor of Pathology at
Harvard Medical School and Harvard School of Public Health, and is
a pulmonary pathologist at the Brigham and Women’s Hospital in
Boston. His research investigates lung cell responses to inhaled environmental particles, including the role of scavenger receptors in macrophage interaction with particles and mechanisms of particle-induced
pulmonary inflammation.
Address correspondence to: Dr. Florence Tao, Physiology Program,
Department of Environmental Health, Harvard School of Public
Health, 665 Huntington Avenue, Boston, MA 02115, USA; Tel: (617)
432-4966; Fax: (617) 432-0014; E-Mail: [email protected].
327
328
F. TAO et al.
iologic outcomes. For example, mechanisms for increased cardiac mortality may include effects on the
autonomic nervous system or direct toxicity to myocardium by constituents of PM. In contrast, for pulmonary
inflammatory disorders (e.g., asthma, COPD), PM-mediated enhancement of the inflammation central to these
disorders is a likely pathogenic mechanism. Effects of
PM on pulmonary inflammation have received substantial attention [4,5]. This review will focus on the involvement of reactive oxygen species (ROS) in PM-induced
pulmonary inflammation. The general topic of how ROS
elicit pulmonary inflammation is reviewed elsewhere
[6 – 8]. We will consider here the evidence that (i) PM
have oxidant properties, (ii) PM cause oxidant-dependent
effects in vitro on the critical cells in pulmonary inflammation (alveolar macrophages [AM], epithelial cells
[EC], and polymorphonuclear granulocytes [PMN]), (iii)
PM cause oxidant-dependent proinflammatory effects in
vivo. Although pathways for specific PM health effects
clearly diverge at the organ physiology level (e.g., cardiac arrhythmias [9] vs. COPD exacerbations [10 –13]),
there may be substantial overlap in the initial biochemical mechanism of injury (e.g., oxidative stress by PM
components). Hence, progress in understanding the role
of ROS/RNS in PM effects on pulmonary inflammation
may inform studies of PM effects on the heart and other
organs. The review will emphasize studies of particles
collected from ambient air since such particles are the
toxic agents identified epidemiologically.
OXIDANT PROPERTIES OF PM
PM characteristics
PM are classified as coarse (2.5–10 ␮m aerodynamic
diameter, PM10), fine (0.1 ⱕ 2.5 ␮m aerodynamic diameter, PM2.5), and ultrafine (ⱕ0.1 ␮m aerodynamic diameter). Fine and ultrafine PM are produced by combustion
(motor vehicles and power plants), whereas PM10 are
generated by mechanical processes that produce fugitive
dust from noncombustive sources [14]. PM10 deposit
predominantly in the nose and throat and are cleared by
exhalation or mucociliary clearance and swallowing. Although fine and ultrafine PM can deposit in the extrathoracic airways, depending on air flow rates and diffusion,
they also penetrate into the lower airways and alveoli,
where they may persist for weeks, months, or longer
[15,16]. All PM contain biological material, organic
compounds, hydrocarbons, ions, acid aerosols, reactive
gases, and metals adsorbed or attached to a typically
carbonaceous core. PM10 consist primarily of biological
material (e.g., pollen, bacteria), crustal material, and sea
salt, whereas PM2.5 consist primarily of metals, hydrocarbons, and secondary particles formed by chemical
reactions with gaseous air pollutants. The specific composition of PM in a given location depends on local
geography, climate, season, and sources. In urban areas
of the northeastern United States, aerosol PM2.5 concentrations typically range between 5 and 15 ␮g/m3 and
consist predominantly of sulfur-containing acid particles
in the summer or combustion-product particles in the
winter [17]. By contrast, in São Paulo Brazil, PM10
averaged 82 ⫾ 39 ␮g/m3 (mean ⫾ standard deviation) in
1990 –1991 [18]. PM10 form the highest mass fraction of
ambient particles, whereas ultrafine PM exist in the highest numbers and consequently present the largest bioactive surface area [19].
Mortality and morbidity have been associated with all
three fractions of PM. In studies that examined both
PM10 and PM2.5, some found stronger associations with
PM2.5 than with PM10 [20,21], others found associations
with PM2.5 but not PM10 [22,23], while another found no
specific association with PM2.5 nor PM10 individually
[24]. Similarly, some investigators have seen associations between mortality/morbidity and ultrafine PM exposure [19,25], while others have not [26]. Although
some investigators have implicated PM10 alone in mortality [27], the bulk of evidence indicates that fine and
ultrafine PM are the major bioactive fractions of PM
pollution and so have been the most avidly studied for
health effects.
Many studies of the biological effects of PM tested
particles collected over extended periods of time. For
example, SRM 1649 is an urban air dust sample that was
collected over a 1 year period between 1975 and 1976 in
Washington, DC. Thus, daily and seasonal variations in
PM levels and composition cannot be evaluated with this
sample. In addition, depending on the nature of the
collection filters and recovery methods, these PM may
range broadly in size and may undergo chemical modification during recovery. Recent technological advances
have produced particle concentrators that are capable of
collecting and concentrating ambient PM2.5 (by approximately 30-fold) in real time and with minimal chemical
modification [28,29]. These concentrated ambient particles (CAPs) consist exclusively of particles that are the
most bioactive in size and enable more accurate assessments of the daily and chemical variability of real world
PM. It should be noted that CAPs contain ultrafine PM,
but do not concentrate them.
PM oxidative chemistry
The oxidative capacity of PM is primarily attributed
to its transition metal constituents, which typically include Fe, V, Cr, Mn, Co, Ni, Cu, Zn, and Ti [30,31].
Some of these metals can catalyze Fenton-type reactions
and generate ROS [32]. The oxidative effects of PM in
Oxidative stress by ambient particles
cell-free systems in vitro are summarized in Table 1.
Ghio et al. measured the concentrations of various transition metals and the oxidant capacities of the supernatants of acid-solubilized PM10 (1 mg/ml) from 12 different U.S. cities in a cell-free system (by thiobarbituric
acid-reactive substances of deoxyribose, TBARS) [33].
TBARS generation was correlated with overall metal
content as well as with multiple individual metals such as
V, Fe, and Co. Generation of TBARS by Salt Lake City
PM suspensions was completely abolished by dimethylthiourea (DMTU, a •OH scavenger) or deferoxamine
(DFX, a metal chelator with greatest affinity for Fe),
implicating acid-soluble metals and free radicals as mediators of PM-stimulated deoxyribose oxidation. The
same investigators subsequently demonstrated that the
water-soluble and insoluble components of PM collected
from Provo, UT contained metals [34]. Both soluble and
insoluble components (500 ␮g) also increased TBARS
production in vitro that was abolished by DFX, DMTU,
or DMSO (another •OH scavenger). In these studies, the
pH of the samples were not controlled for, which could
have potentially affected metal solubility and possibly
skewed the data [35]. However, Ball et al. showed that
neutral aqueous extracts of 50 mg SRM 1648 (collected
in St. Louis, MO) and SRM 1649 also generated the ROS
malondialdehyde in a DFX-sensitive manner [36], allaying concerns about pH effects on metal solubility and
providing further evidence of the oxidative capacity of
PM metals.
Smith and Aust implicated iron as the main constituent of SRM 1648 and SRM 1649 in mediating oxidantdependent single-strand breaks in DNA in vitro [37]. The
SRMs (100 –500 ␮g/ml) were unable to cause DNA
strand breaks when suspended in neutral saline unless
iron was mobilized from the particles by the chelators
ascorbate or citrate, suggesting that soluble iron was
necessary for the effect. The extent of DNA breakage
was positively associated with the amount of total iron
mobilized from the two PM samples and could be
blocked by DFX treatment of the PM. Prahalad et al. also
showed that SRM 1649 and a PM sample from Dusseldorf, Germany (0.1–1 mg/ml) were able to oxidize dG
oligonucleotides and calf thymus DNA in vitro [38].
However, in these experiments, dG oxidation by PM was
unaffected by diethylenetriamine pentaacetic acid
(DTPA, a metal chelator), DFX, DMSO, catalase, or
superoxide dismutase (SOD) treatment. To rationalize
these enigmatic observations, the authors proposed that
ferryl ions, other active species, or caged/bound •OH had
oxidized the dG.
The controversy between soluble vs. insoluble effects
might be accounted for by a recently proposed alternate
mechanism by which PM2.5 can cause oxidation [14]. In
addition to the typical constituents, PM2.5 collected from
329
five different U.S. cities also contained abundant and
persistent free radicals that were postulated to have been
generated by the combustion of organics such as diesel
fuel [39]. Electron spin resonance spectra identified these
free radicals to likely be members of the family of redox
active semi-quinones. The investigators proposed that
these semi-quinone-like radicals chemisorbed to the particles and presented persistent redox-active surfaces on
the particles. Alternatively, polycyclic aromatic hydrocarbons (PAH) present in PM may be metabolically
converted to redox-active quinones [40].
PM also carry lipopolysaccaride (LPS) [41– 44],
which has been shown to stimulate ROS and reactive
nitrogen species (RNS) production in a number of cell
types [45– 47]. The contribution of LPS to PM-mediated
oxidative stress has not been well characterized, but it
could potentially participate in oxidative stress exerted
by the insoluble component of PM.
Another potential source of oxidative stress could be
the ultrafine subfraction of PM. Wilson et al. tested the
ability of fine (260 nm mean aerodynamic diameter) and
ultrafine (14 nm mean aerodynamic diameter) carbon
black (CB) particles to oxidize DCFH [48]. Ultrafine CB
increased DCFH fluorescence by up to ⱖ 300 units even
at low particle dose (15 ␮g/ml), whereas fine CB had no
effect even at the highest concentration tested (120 ␮g/
ml). Incubating the ultrafine CB particles with transition
metal salts further potentiated DCFH oxidation. Thus,
even without reactive species on its surface, the carbonaceous core of ultrafine PM can cause oxidative stress
merely by presenting a large surface area [49] and
thereby contribute to the total oxidative capacity of PM.
The cell-free in vitro studies demonstrate the oxidative capacity of PM and implicate transition metals,
semi-quinones, LPS, and ultrafine particles as candidate
affectors driving ROS production by PM. The exact
contribution of soluble vs. insoluble components in this
outcome is still under debate, although both components
are active. The potential oxidative capacity of each component may depend on the physical conditions of the
particle’s milieu (e.g., acidic/basic/neutral pH, presence
of ascorbate/citrate).
PM OXIDANT EFFECTS ON CELLS IN VITRO
Toxicological studies of PM are complex because PM
can affect multiple cell types in different ways and activate a cascade of events in vivo. The bulk of in vitro
studies have focused on individual cell types, so do not
provide information about intercellular modulation of
PM effects that occur in whole systems. However, intercellular interaction can affect responses to PM since
AMs cocultured with EC had increased sensitivity and
responsiveness to PM in terms of cytokine secretion [50].
Table 1. PM and CAPs Effects In Vitro
Particle source
12 U.S. cities
Provo, UT
Dose
Bioactive compartment
1 mg/ml
500 ␮g
0.5–1.3 ␮g/mm2
H⫹ soluble
H2O soluble, insoluble
suspension
SRM 1648, SRM 1649
SRM 1649, Dusseldorf
Vermont
Utah Valley
330
Utah Valley, 4 German cities
SRM 1648, Dusseldorf,
Ottawa
50 mg
100–500 ␮g/ml
0.1–1 mg/ml
0.1 ␮g/mm2
H2O soluble
suspension
insoluble
suspension
50 ␮g/105 AM
0.3–1.3 ␮g/mm2
H2O soluble
soluble
soluble 20
suspension
1.3 ␮g/mm2
1–50 ␮g/105 AM
⬎ 84 ␮g/105 AM
soluble 24 h, suspension
suspension
⬎ 1 ␮g/105 AM
ⱖ 9 ␮g/105 AM
SRM 1649
1.2 ␮g/105 AM
Suspension, insoluble
Dusseldorf, Duisberg
42–78 ␮g/10
PMN
H2O soluble
5
DMSO soluble
5 cities
Downey, CA CAPs
Boston, MA CAPs
Boston, MA CAPs
50 ␮g/10 PMN
1.5–15 ␮g/105
⬎ 3.7 ␮g/105
0.8–18 ␮g/105 AM
1.2 ␮g/105 AM
5
suspension
suspension
Suspension, H2O soluble
suspension, insoluble
Effect
TBARS
TBARS
EC IL-8
EC IL-8
EC NF-␬B-DNA binding, IL-6; ICAM-1
TBARS
DNA single strand breaks
dG oligo & DNA oxidation
EC ROS production
EC NF-␬B-DNA binding
EC NF-␬B transcription
AM cytotoxicity
AM phagocytosis
AM oxidative burst
TBARS
EC IL-8, IL-6
EC cytotoxicity
2 AM oxidative burst
AM cytotoxicity
AM oxidative burst
AM TNF-␣
AM IL-6
AM TNF-␣, MIP-2
AM nitrite
ROS in vitro
2 *PMN oxidative burst
ROS in vitro & PMN
2 *PMN oxidative burst
PMN oxidative burst
2RAW/THP-1 GSH:GSSG
RAW/THP-1 HO-1 expression
AM oxidative stress
AM TNF-␣, MIP-2
AM nitrite
Inhibitor
DMTU, DFX
DMSO, DMTU, DFX
DMSO, DMTU, DFX
DFX, SOD, NAC, DMTU
DSX
DFX
DTPA, DFX, DMSO, catalase, SOD
Catalase, DFX
Chelex-100, DFX
Chelex-100
DFX
DFX, DMTU, DMSO
Reference
[33]
[34]
[34,36]
[36]
[37]
[38]
[63]
[54]
[65]
[54,56]
[43]
DFX
DFX, DETA, EDTA
DFX, 1 by DETA, EDTA
[42]
[68]
SOD, catalase
SOD, catalase
DFX
NAC
DFX
DFX, 1 by DETA, EDTA
[30]
[58,59]
[42]
Effects are increases unless otherwise stated. Inhibitors that were tested but had no effect are italicized; agents that potentiated effects are indicated by 1, *PMN ⫽ zymosan-activated PMN.
Oxidative stress by ambient particles
Another concern with the in vitro studies is dosimetry.
A 300 g rat breathing 10 –1000 ␮g/m3 of fine PM for
24 h is estimated to have 0.33–33 ␮g deposited in their
alveoli [51]. At these exposures, the rat AMs would
encounter 0.01–1.1 ␮g/106 AM, assuming 3 ⫻ 107 AMs
per rat [51]. Experimental doses used in the studies
discussed below are at least 2 orders of magnitude higher
than the deposited dose from an ambient PM2.5 exposure
of 20 ␮g/m3 in vivo. For EC, the deposited dose from
inhaling 10 –1000 ␮g/m3 PM2.5 for 24 h would be 0.3–
0.28 ␮g/106 type II cells, assuming homogenous distribution among the approximately 120 ⫻ 106 type II EC in
the rat lung [52]. Tissue culture plates accommodate
approximately 104 EC/mm2 (F. Tao, personal observation) so the predicted deposition of 10 –1000 ␮g/m3
inhaled PM2.5 would be 0.003– 0.3 ␮g/mm2 on the plate.
Airway bifurcations are hot spots for PM deposition with
a median ratio of 9:1 carinal:tubular particle concentration in humans [53]. Even if hot spot deposition is
considered, the exposure of EC in the in vitro studies
would still be at least 10 times higher than in vivo hot
spot exposures at an ambient PM2.5 level of 20 ␮g/m3. A
realistic dose for PMNs is difficult to predict because it
would depend on the extent of neutrophilic inflammation
in the lung.
Since AM-EC coculture enhanced sensitivity to PM
[50], it is conceivable that in vitro effects may become
detectable at lower PM doses if intercellular modulation
is factored into the experimental designs. The currently
available data, however, uses relatively high, and arguably unrealistic, doses of PM in order to measure detectable effects in single cell types, a strategy necessitated by
the lack of effects seen at concentrations that approach
likely real-world exposures (see above). The dosimetry
of each study must be considered when extrapolating
between in vitro mechanisms and in vivo effects.
Alveolar macrophages
AMs exposed in vitro to PM have been shown to
undergo intracellular oxidation, become apoptotic, have
impaired phagocytosis, have depressed respiratory burst
responses, and release proinflammatory cytokines (Table
1).
Soukup et al. contrasted the effects of PM from the
Utah Valley that was collected during 3 consecutive
years from 1986 to 1988 on human AMs [54]. In 1986 a
local steel mill was operational; it closed for 1 year in
1987 because of a labor strike and reopened in 1988.
PM10 mass levels in the valley were 1986 ⫽ 1988 ⬎
1987 in parallel with the steel mill’s productivity, but the
distribution of different metals varied among the years
[55]. Extractable transition metals in the PM were lower
in 1987 when the steel mill was closed than in 1986 and
331
1988, when it was open. Only aqueous extracts from
1986 PM (100 ␮g in 100 ␮l) was cytotoxic to 2 ⫻ 105
AMs from healthy volunteers; the resistance of AMs to
an equal mass of 1987 and 1988 PM indicated that high
particle load itself did not cause cytotoxicity. DFX treatment had no effect on 1986 PM toxicity, whereas chelex100 (a chelating resin) prevented cytotoxicity. These
findings with the chelators might reflect different cytotoxic potentials by different metals or reflect nonspecific
biological effects of these chelators unrelated to their
metal-chelating properties. In addition, only PM extracts
(100 ␮g) from 1986 reduced AM phagocytosis of yeast
in vitro. This was prevented by chelex-100, suggesting
that metals mediated the impaired phagocytosis. Shortterm (20 min) exposure to 1986 but not 1987 nor 1988
PM extracts (50 ␮g) elicited a DFX-insensitive oxidative
burst. By contrast, overnight incubation with 1986 and
1988 but not 1987 PM extracts (100 ␮g) inhibited AM
oxidant generation compared with control. Thus, even
though a major source of PM was constant (i.e., the steel
mill), AM function and viability varied according to the
PM sample. The data suggest that AM effects result from
the variable distribution of specific soluble metals rather
than total soluble metal content, since the latter was the
same for1986 and1988 PM even though 1986 PM was
more bioactive than 1988 PM. Differences in the ability
of DFX and chelex-100 to block PM effects also support
the conclusion that metal distribution rather than mass
determines biological outcomes. AM effects were also
independent of total PM mass.
PM also suppressed the respiratory burst of rabbit
AMs resulting from zymosan ingestion [56]. Twentyfour hour preincubation of AMs with suspensions of PM
(1–20 ␮g/105 AMs) collected from four different German cities dose-dependently inhibited the zymosan-induced respiratory burst (ID50 2.8 –3.4 ␮g/105 AMs depending on the city). Inhibition correlated most closely
with particle number, mass, surface area, Sb, and Pb
content. It is unclear whether prior uptake of PM simply
limited the AMs’ ability to subsequently phagacytose
zymosan, which would have also attenuated the respiratory burst. This is reasonable given that the respiratory
burst was associated with particle load and inert particles
were not included in the experimental design to control
for particle preloading of AMs. Although the mechanism
and the specificity of the response to PM are uncertain,
this study demonstrated that PM exposure might be detrimental to host defense by inhibiting endogenous ROS
generation.
Short-term incubation (30 min) with PM (ⱖ1 ␮g/105
AMs) from Dusseldorf and Ottawa but not from St.
Louis elicited oxidative bursts in human AMs [43], demonstrating different capacities of different PM samples to
activate AMs. High doses of PM from St. Louis, Dus-
332
F. TAO et al.
seldorf, and Ottawa (⬎84 ␮g/105 AMs) were cytotoxic
to human and rat AMs in vitro but lower doses of all
three PM samples (ⱖ9 ␮g/105 AMs) stimulated human
and rat AMs to secrete tumor necrosis factor (TNF)-␣
and interleukin (IL)-6. Cytokine secretion from human
AMs was partially dependent on bacterial LPS present in
the PM. Endogenous ROS may be involved in this response since they were shown to mediate LPS induction
of macrophage inflammatory protein-2 by activating nuclear factor (NF)-␬B in rat AMs [57]. By contrast, DFX
pretreatment of PM had no effect on IL-6 responses in
human AMs, indicating that transition metals were not
involved in IL-6 release. The authors did not investigate
which components of the PM samples were involved in
cytotoxicity or in the oxidative burst.
The oxidative burst in hamster AMs exposed to resuspended Boston CAPs (⬃ 0.8 – 4 ␮g/105 AMs) in vitro
appeared to be dose-dependent on the extent of particle
uptake by the AMs [58]. Subsequent studies revealed
that day-to-day variability in CAPs samples (⬃4 –18
␮g/105 AMs) determined the extent of AM oxidation
rather than dose per se [59]. The oxidative capacity of
CAPs was attributed primarily to the component that was
solubilized in buffered saline rather than the washed
insoluble particles. The insoluble particles also stimulated AM oxidation, but the variability among CAPs
samples masked statistical significance. Overnight pretreatment of the CAPs samples with DFX inhibited oxidative stress by both soluble and insoluble components
by 76% and 54% respectively, indicating that the majority of intracellular oxidation was due to metals in the
samples.
SRM 1649 and Boston CAPs suspensions (1.2 ␮g/105
AMs) also stimulated TNF-␣ and macrophage inflammatory protein (MIP)-2 release from rat AMs in vitro
[42]. In this case, bioactivity was associated with the
insoluble pellets for both SRM 1649 and CAPs. Although both PM pellets contained LPS, inhibition with
polymyxin B or recombinant endotoxin neutralizing protein only partially attenuated the TNF-␣ and MIP-2 responses. Polymyxin B also partially inhibited (⬍50%)
TNF-␣ release by RAW 264.7 cells exposed to PM from
central Taiwan [60], corroborating the contribution of
PM endotoxin to cytokine responses. The metal chelators
DFX, diethylenetriamine pentaacetic acid (DETA, relatively specific for Co and Ni), and EDTA (relatively
specific for Ca and Mg) did not affect SRM 1649stimulated TNF-␣ and MIP-2 release, indicating that
metals did not contribute to the non-LPS portion of the
cytokine responses. Nitrite increased in AM supernatants
after SRM 1649 and CAPs exposure, and this response
was also associated with the PM pellets. DFX had no
effect, but DETA and EDTA markedly enhanced nitrite
release by SRM 1649, suggesting that certain PM metals
normally suppress RNS generation.
CAPs from Downey, CA (proximal to a busy highway
that has some of the highest PM concentrations in the
United States) that were collected directly into aqueous
solution dose-dependently (10 –100 ␮g/6.7 ⫻ 105 cells)
decreased the ratio of reduced (GSH) to oxidized glutathione (GSSG) in RAW 264.7 cells [61]. Coarse particles
collected on the same days in the same manner also
decreased GSH:GSSG at high doses (ⱖ50 ug/6.7 ⫻ 105
cells) indicating that both fine and coarse CAPs can
cause intracellular oxidation at appropriate doses. Fine
CAPs (ⱖ25 ␮g/6.7 ⫻ 105 cells) also induced heme
oxygenase-1 (HO-1) expression, a gene that codes for an
enzyme that generates the antioxidant bilirubin from
heme [62]. N-acetylcysteine (NAC) treatment abolished
the HO-1 response to fine CAPs, indicating a role for
thiol oxidation in triggering HO-1 expression. Coarse
particles (ⱖ25 ␮g/6.7 ⫻ 105 cells) could also induce
NAC-sensitive HO-1 expression when their PAH content
was high during autumn and winter. Fine CAPs also
contained a higher fraction of PAH than metals, suggesting that the HO-1 response was associated with oxidative
stress from PAH rather than metals.
Collectively, the data show that PM can exert oxidative stress in AM in vitro. As a result, host defense may
be compromised as AMs undergo apoptosis, have impaired phagocytosis, and have a diminished respiratory
burst. In addition, acute exposure to PM stimulates,
whereas chronic exposure inhibits, RNS formation. Effects have more consistently been associated with PM
components rather than mass, alleviating some concern
about dosimetry in these studies. The LPS component of
PM is partially responsible for cytokine production by
AMs, and this might be mediated by oxidative stress, but
a role for chelatable metal components of PM in cytokine production remains under investigation. Antioxidant
defenses are activated by PM exposure and appear to be
associated with PAH rather than metal components. The
balance between PM-mediated oxidative stress and endogenous antioxidative responses in determining functional outcomes has not been investigated.
Epithelial cells
PM have been shown to stimulate NF-␬B activation,
cytokine upregulation, and cytotoxicity in ECs. Metaldependent oxidative stress appears to be involved in
some of these responses (Table 1).
IL-8 protein and mRNA increased in the human bronchial epithelial cell line (BEAS-2B) and in primary human bronchial EC in response to PM from Provo UT
(⬃0.5–1.3 ␮g/mm2) [34,63]. Stimulating BEAS-2B cells
with Pb, Zn, Fe, or Cu individually at the same concen-
Oxidative stress by ambient particles
trations as were present in Provo PM revealed that Cu
alone could also stimulate IL-8 release. The water-soluble and insoluble components of Provo PM each stimulated IL-8 release from BEAS-2B cells that was completely inhibited by DFX, DMTU, or DMSO [34].
However, if BEAS-2B cells were exposed to whole PM
suspensions, DFX, SOD, and NAC each only partially
attenuated IL-8 release, while DMTU had no effect [63].
These data suggest that while PM-stimulated IL-8 secretion is partly mediated by ROS that may be Cu-dependent, there are additional metal-independent chemical
interactions between the soluble and insoluble components in whole PM suspensions that also result in IL-8
release. IL-6 secretion and surface intercellular adhesion
molecule (ICAM)-1 expression were also upregulated
dose-dependently by Provo PM, but the role of oxidants in
these responses was not investigated. Provo PM and Cu
alone also increased NF-␬B binding to DNA, but again, the
specific involvement of ROS in this relationship was not
tested by these authors. However, Shukla et al. showed that
PM2.5 (0.1 ␮g/mm2) from Vermont exerted oxidative stress
in murine type II cells and that PM activation of NF-␬Bdependent transcription was abolished by catalase [64].
This, along with evidence from other studies of ROS/RNS
and other experimental particulates in ECs [7], implicate
oxidative stress presented by PM in activating NF-␬B to
increase gene expression of proinflammatory cytokines,
chemokines, and adhesion molecules.
When BEAS-2B cells were exposed to the 1986 –
1988 Utah Valley PM, IL-8 release and mRNA expression were 1988 ⬎ 1986 ⬎ 1987 ⫽ control in a dosedependent manner (0.3–1.3 ␮g/mm2) [65]. IL-6 release
was also dose-dependently 1988 ⬎ 1986 ⬎ 1987 ⬎
control, but the mRNA was similar across all 3 years and
dose-independent. Cytotoxicity was observed only with
the highest concentration (500 ␮g/ml or 1.3 ␮g/mm2) of
1988 PM, so unrelated to PM mass loading. The role of
ROS in these responses was not specifically investigated,
but the authors showed that in vitro DFX-, DMTU-, and
DMSO-sensitive TBARS production ranked 1986 ⬎
1988 ⬎⬎ 1987 PM. This could explain the low IL-8 and
IL-6 secretion with 1987 PM, but cannot fully account
for the greater cytokine responses to 1988 than 1986 PM,
nor the cytotoxicity of 1988 and not 1986 PM. PM 1988
contained double the Cu and half the Zn of 1986 PM,
consistent with the observations discussed earlier that Cu
increased NF-␬B binding to DNA and IL-8 secretion in
the same cell line [63], so variable metal distribution may
be a feasible explanation. Alternatively, nonmetal constituents of PM, such as LPS, might be responsible for
the greater cytokine responses to 1988 than 1986 PM. It
is interesting to note that AMs were more reactive to
1986 PM [54], whereas the ECs were more reactive to
1988 PM.
333
These studies indicate that PM metal-dependent oxidative stress is likely partially responsible for upregulating proinflammatory genes (e.g., IL-6, IL-8, ICAM-1)
via NF-␬B activation in EC. Although ROS cause apoptosis in many cell types [46,66,67], PM-metal dependent oxidative stress has not yet been shown to be
cytotoxic to EC. Like AMs, ECs seem to respond to PM
components rather than mass per se.
Polymorphonuclear granulocytes
PMNs are a potential target for PM health effects
since they are likely present in the lungs of high-risk
groups, e.g., individuals with chronic bronchitis, pulmonary infections. PM effects on PMNs could directly or
indirectly augment ongoing pulmonary inflammation.
Current data regarding PM oxidant effects on PMNs are
summarized in Table 1.
Hitzfeld et al. measured the effect of aqueous and
organic extracts of PM from Dusseldorf and Duisberg,
Germany (42–78 ␮g/105 PMNs) on human peripheral
blood PMN ROS production by luminol chemiluminescence [68]. The organic extracts produced more ROS in
a cell-free in vitro system than the aqueous extracts. In
addition, the organic but not the aqueous extracts stimulated ROS signals in resting PMNs. Pretreatment of
PMNs with either extract inhibited the respiratory burst
due to zymosan uptake, with greater inhibition by the
aqueous than organic extracts. SOD and catalase treatment of the extracts attenuated respiratory burst inhibition, implicating the involvement of extract oxidants in
this response. These findings implicate oxidants in the
organic extracts of PM in stimulating a respiratory burst
in resting PMNs. If the PMNs are already activated by
phagocytosis, exogenous oxidants suppress the phagocytosis-dependent respiratory burst of PMNs.
Prahalad et al. also showed that PM from five different
urban centers stimulated an oxidative burst in resting human peripheral blood PMNs (50 ␮g/105 PMNs) [30]. Bioactivity was attributed to the aqueous-insoluble component
and correlated with Fe, Ti, and Si constituents. DFX pretreatment of PM had no effect on PM-stimulated ROS
generation in the PMNs, further excluding the involvement
of soluble metals in the PMN oxidative burst.
Both these studies suggest that resting PMNs respond
with an oxidative burst to organic oxidants rather than
soluble metals on PM. If the PMNs are preactivated, organic and aqueous PM oxidants suppress PMN oxidative
burst.
OXIDANT-DEPENDENT PM EFFECTS IN VIVO
Intratracheal instillation of PM suspensions into
healthy rodents in vivo has been shown to elicit neutrophil influx into the lungs, increase bronchoalveolar la-
334
F. TAO et al.
Table 2. Effects of Intratracheally Instilled PM In Vivo
PM source
Provo, UT
Dusseldorf, Bochum
Germany
SRM 1649, Provo,
Dusseldorf, Bochum
Utah Valley
Dose
250–500 ␮g/rat
5 mg/rat
10 ␮g/mouse, 5 mg/
rat
1–2.5 mg/rat
Bioactive compartment
H2O soluble ⬎ insoluble PMN influx, BAL protein
suspension
PMN influx, BAL protein
Airway hyperreactivity
suspension
Excess postbacterial infection mortality
H2O soluble
500 ␮g
SRM 1648, SRM 1649, 2.5 mg/rat
Dusseldorf, Ottawa
Effect
suspension, soluble
equimetals
Species Reference
rat
rat
[34]
[70]
mouse, rat [70,71]
BAL cells, protein, LDH
rat
PMN influx
BAL cells, PMN, protein, albumin, fibronectin, human
␣1-antitrypsin, IL-8, TNF-␣, IL-1␤; 2 BAL
tissue factor, fibrinogen
BAL LDH, PMN influx, protein, albumin,
rat
eosinophilia
[69]
[73]
[35]
Effects are increased responses unless otherwise indicated.
vage (BAL) protein, increase cytokine expression in lung
tissue, induce airway hyperresponsiveness to acetylcholine, and compromise host defense (Table 2). None of
these studies tested the effect of metal chelators or antioxidants on these in vivo outcomes. However, the involvement of metals and oxidative stress could be inferred from characterizing the metal content of the PM
samples and the oxidative stress-dependent effects of
these PM by themselves and on cells in vitro.
The animals in these experiments were exposed to
10 –100 times the daily PM mass found in ambient air of
typical U.S. cities. For example, ambient PM2.5 in six
U.S. cities ranged from 18 to 46 ␮g/m3 between 1975
and 1986, with levels stabilizing at 10 –30 ␮g/m3 between 1980 and 1986 [3]. A rat breathing roughly 100
ml/min over 24 h inhales approximately 1.5 m3 of air, or
18 – 40 ␮g of PM per day. Even assuming a high 50%
deposition rate, the lung tissue is exposed to only 9 –20
␮g of PM over the course of a day. The following studies
exposed rats to 1–5 mg of PM in a single instilled bolus
in order to make detectable measurements [35,69,70].
These high experimental doses must be considered when
extrapolating the effects of PM observed in animal models to real-world effects.
In the Provo PM study that had demonstrated oxidative stress by these PM in vitro, some in vivo
responses were also measured [34]. Both soluble and
insoluble components of instilled Provo PM induced
PMN influx and BAL protein in rats, but the soluble
component was more potent (ⱖ250 ␮g/rat for soluble,
ⱖ500 ␮g/rat for insoluble). This soluble ⬎ insoluble
efficacy in vivo was similar to the greater efficacy of
soluble components to generate oxidant-dependent
TBARs and IL-8 release from BEAS-2B cells, suggesting that the in vivo responses were also oxidant
dependent.
Rats (5 mg/250 –300 g rat) and mice (100 ␮g/mouse)
given a single intratracheal instillation of various PM
(SRM 1649, Provo, Dusseldorf, or Bochum, Germany)
and then infected with aerosolized bacteria showed
markedly greater and earlier mortality than cohorts who
were not PM-exposed [70,71]. Inert particles such as
latex beads or TiO2 were not included as controls for
particle mass loading in the rat studies so the excess
mortality may have resulted from the inability of AMs to
further phagocytose bacteria after injesting particles.
However, doses were more reasonable in mice (assuming
an average mouse weighs 25–30 g) and preexposure to
SRM 1648, latex beads, or TiO2 had no effect on mortality from bacterial infection [71]. In this case, excess
mortality appeared to be associated with specific PM
rather than to be an effect of general particle exposure.
Aqueous extracts from Utah Valley PM collected
from 1986 –1988 showed similar effects in healthy rats in
vivo [69]. Twenty-four hours after instilling extracts
from 2.5 mg PM/250 –300 g rat, BAL total cells, total
protein, and lactate dehydrogenase (LDH) were significantly elevated by1986 and 1988 but not by1987 extracts
compared with saline control. The increase in BAL cell
number in 1986 and 1988 extract-treated animals predominantly reflected PMN influx. None of the PM affected airway responsiveness to acetylcholine. By 96 h
postinstillation, only the total cell numbers in BAL from
1986 treated animals remained elevated relative to control. PMNs were still elevated (albeit to a much lesser
degree) and lymphocytes were also increased. BAL total
cells, PMNs, and LDH were also elevated after lowering
the dose of 1986 extract to 1 mg/rat but were similar to
saline control levels with 0.25 mg/rat. BAL total cells at
24 h were correlated with metal dose rather than instilled
mass per se, again implicating particle composition
rather than load in determining biological outcomes.
However, the relevance of these findings to account for
the epidemiological associations between ambient particle exposure and health effects are limited since no
effects were observed with 0.25 mg/rat (at least 10 times
greater than a rat would receive from the ambient air over
Oxidative stress by ambient particles
24 h, see above), whereas the epidemiological associations were seen at much lower ambient exposures.
Since the low dose exposures in these experiments are
still very high by ambient standards, it is still possible
that excessive particle mass overload caused the effects.
This is supported by studies showing that instilling 0.5
mg of ultrafine CB (14 nm mean aerodynamic diameter)
into rats also resulted in lung neutrophilia and increased
BAL LDH [72]. Yet in the same experiments, fine CB
(320 nm mean aerodynamic diameter) instillation did not
have these effects, arguing against simple particle mass
overload as the main predictor of biological effects.
Rather, particle size might be more relevant since it
affects deposition location, retention, and available surface area for biological interaction. In this case, the high
doses necessary in the rat studies to induce effects may
reflect the necessity to have enough ultrafine particles in
the sample. In addition, the routes (instillation vs. inhalation) and length of exposure (acute vs. chronic) differ
between the animal models and the epidemiological
studies of humans so direct comparisons are impossible.
The value of these animal instillation studies is to narrow
down the hypotheses of toxicological mechanisms so
that only the most plausible ones will be pursued in
subsequent, more relevant models or experimentally in
humans.
When the water soluble extracts from 500 ␮g of Utah
Valley PM were instilled into healthy, nonsmoking human volunteers, BAL total cells, neutrophils, protein,
albumin, fibronectin, ␣1-antitrypsin, IL-8, TNF-␣, and
IL-1␤ were all elevated by 1986 and 1988 but not 1987
extracts [73]. In contrast, BAL tissue factor and fibrinogen were decreased by 1986 and 1988 but not 1987
extracts. These latter outcomes seemed to be related to
greater coagulation and intracellular fibrinogen of lavaged cells. Since DMTU markedly attenuated while
DFX completely abolished the oxidative capacity of the
three PM extracts to increase the production of TBARS
in vitro, the investigators proposed that the biological
effects of the PM extracts in vivo was at least partly
related to the metal burden and ensuing oxidative stress.
Resting tidal volume in humans is approximately 500
ml. Given a respiratory rate of 10 breaths/minute, minute
ventilation would be 5 l/min [74]. Over 24 h (1440 min),
the average healthy person living in an American city
would inhale 7200 l (or 7.2 m3) of ambient air containing
15 ␮g/m3 of PM, or 108 ␮g of PM. Assuming 40%
deposition [75], exposure would be 43.2 ␮g/d, or about
10% of the 500 ␮g exposure used in the Utah Valley PM
study. If the PM increases to 50 ␮g/m3 and the person
starts exercising such that ventilation increases to 15
l/min, their exposure would become 432 ␮g/d. Thus, the
500 ␮g dose used in the Utah Valley human study can be
relevant to real-world exposures.
335
To better delineate the contribution of metals to the in
vivo effects of PM, Costa and Dreher instilled equimass
(2.5 mg/rat) or equimetal (46 ␮g/rat) doses of various
PM to healthy rats [35]. The equimetal samples were
constructed based on the content and concentration of
individual soluble metals in the PM test samples (Dusseldorf, SRM 1648, SRM 1649, Ottawa). All the equimetal samples induced the same BAL neutrophil and
LDH responses as whole PM suspensions, suggesting
that these responses were completely metal-dependent.
The Ottawa equimetal sample also caused protein and
albumin leak to the same extent as its whole suspension.
However, none of the other equimetal samples increased
BAL protein or albumin, in contrast to their corresponding whole suspensions. The metals of Dusseldorf and
SRM 1649 increased BAL eosinophils like their whole
suspensions, while SRM 1648 metals induced eosinophilia even when the whole suspension didn’t. These
studies confirmed that high doses of metals found in PM
can cause lung injury (cytotoxicity) and inflammation
(PMN influx) in vivo, but replication of these results
using ambient levels that humans are exposed to is still
necessary to confirm the plausibility of these findings.
OXIDANT-DEPENDENT CAPS EFFECTS IN VIVO
CAPs effects have been investigated in healthy and
disease-model animals (Table 3). These studies report a
range of effects, from none at all to acute, albeit variable
and mild, pulmonary inflammation. By using ever more
“realistic” concentrations of PM in more biologically
relevant disease models, these experimental designs reproduce the relatively low incidence of PM health effects
seen epidemiologically (see also below). These studies
have offered important evidence regarding inflammation
and oxidant effects of PM.
In one comparison of healthy vs. bronchitic rats, aerosolized Boston CAPs exposure for 5 h/d over 3 d (205–
733 ␮g/m3) increased peak expiratory flow (PEF) in
bronchitic rats as well as tidal volume in healthy and
bronchitic rats when compared with filtered air exposure
[76]. Twenty-four hours after the final CAPs exposure,
BAL AMs were decreased while BAL lymphocytes,
PMNs, and total protein were increased in healthy and
bronchitic rats. Lung injury was not apparent after CAPs
exposure in either healthy or bronchitic rats. A different
comparison of healthy vs. bronchitic rats showed considerable variability in pulmonary responses measured
immediately after 3 d of 6 h/d of CAPs aerosol (265–
1200 ␮g/m3) collected from Research Triangle Park, NC
[77]. In this study, CAPs had no effect on any of the
inflammatory or lung injury parameters measured in
healthy animals. In bronchitic rats, CAPs had inconsistent effects on BAL protein, albumin, N-acetyl glu-
F. TAO et al.
336
Table 3. Effects of Aerosolized CAPs In Vivo
CAPs source
Dose
Exposure
Boston, MA
205–733 ␮g/m3
Research Triangle Park, NC
Boston, MA
265–1200 ␮g/m
70–150 ␮g/m3
6 h/day ⫻ 2 or 3 d
6 h/day ⫻ 3 d
Tuxedo, NY
300 ␮g/m3
6h
Tuxedo, NY
110–350 ␮g/m3
3h
Chapel Hill, NC
Boston, MA
47–207 ␮g/m
203 ⫾ 147 ␮g/m3
361 ⫾ 267 ␮g/m3
300 ⫾ 60 ␮g/m3
2h
6 h/day ⫻ 3 d
Boston, MA
5 h/day ⫻ 3 d
3
3
5h
Effect
Tidal volume, PEF
BAL lymphocytes, PMN, protein
2 BAL AM
BAL protein, albumin, NAG, PMN
BAL total cells, PMN, AM, lymphocytes
Peripheral blood eosinophils
BAL PMN, blood eosinophils
Lung tissue IL-6, TNF-␣, TNF-␤, TGF␤2, IFN-␥ mRNA
Peripheral blood PMN
BAL PMN, protein, fibrinogen
1 variability in BAL and hematologic
parameters
Lung oxidants
Species Reference
healthy and
bronchitic
rats
[76]
bronchitic rats
young rats
[77]
[78]
old rats
mouse
[64]
hypertensive
rats
humans
dogs
[29]
rats
[79]
[83]
[31]
Effects are increased responses unless otherwise indicated.
taminidase (NAG) activity, and neutrophil counts; some
exposures increased these outcomes while others had no
effect. BAL LDH and total cells were unaffected by
CAPs in healthy and bronchitic rats. Based on the prevailing concept that variable PM characteristics determine variable biological outcomes, the different responses in these two studies of healthy and bronchitic
rats may be partly related to the different geographical
sources of CAPs.
The inflammatory effects of Boston CAPs in young
(4 – 6 weeks) vs. old (⬎17 months) rats has also been
examined [78]. Twenty-four hours after 3 d of 5 h/d of
CAPs (70 –150 ␮g/m3), BAL total cells, PMN, lymphocytes, and AMs were increased in young rats while only
PMNs were increased in old rats. When blood parameters were assessed, CAPs increased peripheral blood
eosinophils in young and old rats. Peripheral blood
PMNs were also increased 3 h after a 3 h Tuxedo, NY
CAPs exposure (110 –350 ␮g/m3) in rats with pulmonary
hypertension [29].
New York CAPs inhalation (300 ␮g/m3 for 6 h) also
increased mRNA of some cytokines (IL-6, TNF-␣,
TNF-␤, transforming growth factor [TGF]-␤2, interferon
[IFN]-␥) but not others (TGF-␤1, TGF-␤3, macrophage
migration inhibitory factor [MIF]) in lung tissues of
normal mice 24 h postexposure [64]. Extrapolating from
in vitro experiments on ECs using VT PM2.5 in the same
report (see above), the authors proposed that the in vivo
cytokine responses were likely associated with PM-derived, oxidant-dependent NF-␬B activation. Though this
seems reasonable, this association can be debatable given
that the different types of experiments were conducted
with different sources of PM.
A recent report by Gurgueira et al. showed that Boston CAPs exposure increased the steady-state concentra-
tion of oxidants in the rat lung and heart. In this study,
CAPs-mediated ROS production was measured noninvasively in intact, anesthetized rats by detecting in situ
organ chemiluminescence (CL) during aerosolized CAPs
inhalation [79]. CL is a low intensity emission in the
visible range mainly due to the decay of excited states of
molecular oxygen (singlet oxygen and excited carbonyls)
[80,81] formed during the termination steps of the chain
reaction of lipid peroxidation [82]. Rats breathing 300 ⫾
60 ␮g/m3 CAPs for 5 h had increased levels of oxidants
in their lungs [79]. These increases were attributed to
inhaled bioactive particles since oxidative stress was also
triggered by metal-containing fine residual oil fly ash
particles, but not by particle-free air nor inert fine CB
aerosols. The CAPs-dependent 2-fold increase in lung
oxidants was accompanied by significant lung edema
(⬃5% increases in the wet/dry ratio, p ⬍ .05) and increased serum levels of LDH (⬃80%, p ⬍ .03), indicating an injurious effect of CAPs at environmentally relevant concentrations. NAC treatment at a dose that
effectively prevented oxidative stress completely abolished CAPs-mediated lung edema, suggesting that ROS
are central mediators of CAPs biological effects in vivo
(B. Gonzalez-Flecha, unpublished results). Data collected on different days during the years 2000 and 2001
showed day-to-day variability in the lung CL response to
CAPs. When regressed to the day-to-day variability in
total mass and metal composition of each day’s CAPs,
the increased lung CL was not associated with the total
CAPs mass concentration, but was strongly correlated
with the CAPs content of Mn, Zn, Fe, and Cu [79].
Healthy human volunteers exposed to CAPs (Chapel
Hill, NC) aerosol (47–207 ␮g/m3) for 2 h also displayed
modest increases in BAL neutrophils, protein, and fibrinogen [83]. In another group of volunteers, short-term
Oxidative stress by ambient particles
exposure to 23.1–311.1 ␮g/m3 of Chapel Hill CAPs
aerosol did not modify lymphocyte phenotype or AM
function [84]. These results in human subjects are particularly noteworthy because the average mass concentration of particles in these experiments approximated
ambient urban air in U.S. cities at the low end [3] and in
sprawling metropolises like São Paulo, Brazil toward the
high end [18]. These studies suggest that the short-term,
high dose outcomes measured in the experimental models can be relevant to the long-term ambient exposures
experienced by humans, particularly during episodic
spikes in PM levels.
The modest effects seen with CAPs in animal models
and in humans are consistent with the low incidence of
human health effects observed in the epidemiological
studies. For example, the relative risk for emergency
hospital admission for COPD exacerbation in Minneapolis-St. Paul was 1.54 –1.57 for every 100 ␮g/m3 increase in PM10 [85] or 1.02 for every 10 ␮g/m3 increase
in PM10 in Detroit, MI [10]. Though these risks seem
small, they are significant in a large susceptible population such as patients with mild (nearly 10% globally) or
severe COPD (nearly 1% globally) [86].
The data collectively suggest that CAPs can have
proinflammatory and injurious effects in vivo, but responses are highly variable. The biological variability
appears to arise from compositional variability of different CAPs samples [31] and possibly from the variable
doses of particles delivered as well. Oxidative stress by
transition metals appears to be a mechanism by which
CAPs exert their effects.
The CAPs experiments are an improvement over the
other in vivo PM studies but still have their limitations.
The animals in all the CAPs studies were exposed by
aerosol inhalation, which is a more realistic route of
entry than intratracheal instillation. Doses were still
much higher than typical ambient levels, although offset
by acute rather than chronic exposures. The disease
models described so far have not consistently shown
greater responses to CAPs than healthy animals, unlike
humans in which health effects due to PM exposure are
associated with individuals with preexisting disease. Including parallel “inert” particle controls such as CB to
confirm the specificity of the outcomes to CAPs is difficult because the variability in mass concentration during CAPs exposures precludes exact comparison with
other particles. Despite these limitations, given that
short-term CAPs exposure elicited small yet measurable
biological outcomes, CAPs appear to be a reliable model
to use for toxicological studies of ambient particle exposure. Experiments with longer-term, low dose CAPs exposures would strengthen the conclusions of the existing
data.
337
Table 4. Associations Between Select PM or CAPs Constituents and
In Vitro or In Vivo Effects
Constituent
Effect
V
Fe
Cu
Zn
Co
Al
Mn
Ti
Si
S
LPS
PAH
Ultrafine carbon
TBARS
BAL AM, circulating PMN in vivo
TBARS
DNA strand breaks in vitro
PMN oxidative burst in vitro
BAL PMN, WBC count, circulating
lymphocytes and PMN
Lung CL
EC IL-8 in vitro
BAL PMN
Lung CL
Circulating PMN
Lung CL
TBARS
Total WBC, circulating PMN
Lung CL
PMN oxidative burst in vitro
PMN oxidative burst in vitro
RBC counts, hemoglobin levels
AM TNF-␣, IL-6, MIP-2 in vitro
HO-1 expression
DCFH fluorescence in vitro
Reference
[33]
[31]
[33]
[37]
[30]
[31]
[79]
[63]
[31]
[79]
[31]
[79]
[33]
[31]
[79]
[30]
[30]
[31]
[42,43]
[61]
[48]
SUMMARY AND CONCLUSIONS
PM can exert exogenous oxidative stress on biological
systems through its transition metal, semi-quinone, LPS,
PAH, and ultrafine carbon components (Table 4). Both
water-soluble and insoluble components of PM are oxidatively active. Uptake of PM by phagocytes may also
stimulate an endogenous respiratory burst, but most studies have not made the difficult distinction between exogenous or endogenous sources of oxidants when measuring intracellular oxidation, so the contribution of each
to PM effects is unknown. Bioactivity has generally been
attributed to PM characteristics (i.e., composition, size)
rather than mass. Correlating specific biological effects
with specific metals has been possible albeit difficult due
to the high variability in PM constituents.
In healthy and diseased animals and/or humans, PMmediated oxidative stress has been shown to compromise
host defense (e.g., excess mortality from bacterial infection in rodents) and cause lung injury and inflammation
(e.g., alveolar protein leak, LDH release, and neutrophil
influx). The impaired host defense may arise from increased AM apoptosis and decreased AM and PMN
phagocytosis and respiratory burst, whereas inflammation appears to arise from increased NF-␬B activity,
leading to the upregulation of cytokines and adhesion
molecules in ECs. A role for PM metal-dependent, chelator-sensitive oxidative stress has not yet been solidly
demonstrated for AM cytokine production, even though
oxidative stress mediates AM cytokine responses to mineral dusts [87,88], ROFA [59], and diesel exhaust particles [89] so metal-independent oxidative stress remains a
338
F. TAO et al.
possible cytokine-inducing mechanism of PM. AM cytokine production is partly dependent on LPS found in
PM, and this could potentially be mediated by oxidative
stress.
Most PM studies have focused on ROS mechanisms;
few have examined the involvement of RNS, even
though for example, nitric oxide (•NO) could potentially
contribute to NF-␬B activation in ECs and rodent AMs
by PM. However, •NO production in human AMs is low
or undetectable in response to stimuli that upregulate
cytokines [90,91], so a role for NO in cytokine responses
to PM in humans is less well established (based on
current data). Oxidative stress by ROS and RNS remains
a likely fundamental mechanism of PM toxicity although
many questions about specific components and details of
the cellular targets and cell-particle interactions remain
unanswered.
[13]
[14]
[15]
[16]
[17]
[18]
REFERENCES
[19]
[1] Pope, C. A. 3rd; Bates, D. V.; Raizenne, M. E. Health effects of
particulate air pollution: time for reassessment ? Environ. Health
Perspect. 103:472– 480; 1995.
[2] Pope, C. A. 3rd; Thun, M. J.; Namboodiri, M. M.; Dockery,
D. W.; Evans, J. S.; Speizer, F. E.; Heath, C. W. Jr.. Particulate air
pollution as a predictor of mortality in a prospective study of U.S.
adults. Am. J. Respir. Crit. Care Med. 151:669 – 674; 1995.
[3] Dockery, D. W.; Pope, C. A. 3rd; Xu, X.; Spengler, J. D.; Ware,
J. H.; Fay, M. E.; Ferris, B. G. Jr.; Speizer, F. E. An association
between air pollution and mortality in six U.S. cities. N. Engl.
J. Med. 329:1753–1759; 1993.
[4] Handzel, Z. T. Effects of environmental pollutants on airways,
allergic inflammation, and the immune response. Rev. Environ.
Health. 15:325–336; 2000.
[5] Peden, D. B. Air pollution in asthma: effect of pollutants on
airway inflammation. Ann. Allergy Asthma Immunol. 87(6 Suppl.
3):12–17; 2001.
[6] Folkerts, G.; Kloek, J.; Muijsers, R. B.; Nijkamp, F. P. Reactive
nitrogen and oxygen species in airway inflammation. Eur.
J. Pharmacol. 429:251–262; 2001.
[7] Martin, L. D.; Krunkosky, T. M.; Dye, J. A.; Fischer, B. M.;
Jiang, N. F.; Rochelle, L. G.; Akley, N. J.; Dreher, K. L.; Adler,
K. B. The role of reactive oxygen and nitrogen species in the
response of airway epithelium to particulates. Environ. Health
Perspect. 105:1301–1307; 1997.
[8] Martin, L. D.; Rochelle, L. G.; Fischer, B. M.; Krunkosky, T. M.;
Adler, K. B. Airway epithelium as an effector of inflammation:
molecular regulation of secondary mediators. Eur. Respir. J.
10:2139 –2146; 1997.
[9] Peters, A.; Liu, E.; Verrier, R. L.; Schwartz, J.; Gold, D. R.;
Mittleman, M.; Baliff, J.; Oh, J. A.; Allen, G.; Monahan, K.;
Dockery, D. W. Air pollution and incidence of cardiac arrhythmia. Epidemiology. 11:11–17; 2000.
[10] Schwartz, J. Air pollution and hospital admissions for the elderly
in Detroit, Michigan. Am. J. Respir. Crit. Care Med. 150:648 –
655; 1994.
[11] Lippmann, M.; Ito, K.; Nadas, A.; Burnett, R. T. Association of
particulate matter components with daily mortality and morbidity
in urban populations. Res. Rep. Health Eff. Inst. 5–72, discussion
73– 82; 2000.
[12] Atkinson, R. W.; Anderson, H. R.; Sunyer, J.; Ayres, J.; Baccini,
M.; Vonk, J. M.; Boumghar, A.; Forastiere, F.; Forsberg, B.;
Touloumi, G.; Schwartz, J.; Katsouyanni, K. Acute effects of
particulate air pollution on respiratory admissions: results from
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
APHEA 2 project. Air Pollution and Health: a European Approach. Am. J. Respir. Crit. Care Med. 164:1860 –1866; 2001.
Brauer, M.; Ebelt, S. T.; Fisher, T. V.; Brumm, J.; Petkau, A. J.;
Vedal, S. Exposure of chronic obstructive pulmonary disease
patients to particles: respiratory and cardiovascular health effects.
J. Expo. Anal. Environ. Epidemiol. 11:490 –500; 2001.
Squadrito, G. L.; Cueto, R.; Dellinger, B.; Pryor, W. A. Quinoid
redox cycling as a mechanism for sustained free radical generation by inhaled airborne particulate matter. Free Radic. Biol. Med.
31:1132–1138; 2001.
Lippmann, M.; Yeates, D. B.; Albert, R. E. Deposition, retention,
and clearance of inhaled particles. Br. J. Ind. Med. 37:337–362;
1980.
Brauer, M.; Avila-Casado, C.; Fortoul, T. I.; Vedal, S.; Stevens,
B.; Churg, A. Air pollution and retained particles in the lung.
Environ. Health Perspect. 109:1039 –1043; 2001.
Shore, S.; Kobzik, L.; Long, N. C.; Skornik, W.; Van Staden,
C. J.; Boulet, L.; Rodger, I. W.; Pon, D. J. Increased airway
responsiveness to inhaled methacholine in a rat model of chronic
bronchitis. Am. J. Respir. Crit. Care Med. 151:1931–1938; 1995.
Saldiva, P. H.; Lichtenfels, A. J.; Paiva, P. S.; Barone, I. A.;
Martins, M. A.; Massad, E.; Pereira, J. C.; Xavier, V. P.; Singer,
J. M.; Bohm, G. M. Association between air pollution and mortality due to respiratory diseases in children in São Paulo, Brazil:
a preliminary report. Environ. Res. 65:218 –225; 1994.
Oberdorster, G. Pulmonary effects of inhaled ultrafine particles.
Int. Arch. Occup. Environ. Health. 74:1– 8; 2001.
Schwartz, J.; Dockery, D. W.; Neas, L. M. Is daily mortality
associated specifically with fine particles? J. Air Waste Manag.
Assoc. 46:927–939; 1996.
Cifuentes, L. A.; Vega, J.; Kopfer, K.; Lave, L. B. Effect of the
fine fraction of particulate matter versus the coarse mass and other
pollutants on daily mortality in Santiago, Chile. J. Air Waste
Manage. Assoc. 50:1287–1298; 2000.
Anderson, H. R.; Bremner, S. A.; Atkinson, R. W.; Harrison,
R. M.; Walters, S. Particulate matter and daily mortality and
hospital admissions in the west midlands conurbation of the
United Kingdom: associations with fine and coarse particles,
black smoke and sulphate. Occup. Environ. Med. 58:504 –510;
2001.
Pope, C. A. 3rd; Burnett, R. T.; Thun, M. J.; Calle, E. E.; Krewski,
D.; Ito, K.; Thurston, G. D. Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution.
JAMA. 287:1132–1141; 2002.
Lipfert, F. W.; Morris, S. C.; Wyzga, R. E. Daily mortality in the
Philadelphia metropolitan area and size-classified particulate matter. J. Air Waste Manage. Assoc. 50:1501–1513; 2000.
Wichmann, H. E.; Spix, C.; Tuch, T.; Wolke, G.; Peters, A.;
Heinrich, J.; Kreyling, W. G.; Heyder, J. Daily mortality and fine
and ultrafine particles in erfurt, germany part I: role of particle
number and particle mass. Res. Rep. Health Eff. Inst. 5– 86,
discussion 87–94; 2000.
Osunsanya, T.; Prescott, G.; Seaton, A. Acute respiratory effects
of particles: mass or number? Occup. Environ. Med. 58:154 –159;
2001.
Ostro, B. D.; Hurley, S.; Lipsett, M. J. Air pollution and daily
mortality in the Coachella Valley, California: a study of PM10
dominated by coarse particles. Environ. Res. 81:231–238; 1999.
Sioutas, C.; Koutrakis, P.; Burton, R. M. A technique to expose
animals to concentrated fine ambient aerosols. Environ. Health
Perspect. 103:172–177; 1995.
Gordon, T.; Nadziejko, C.; Schlesinger, R.; Chen, L. C. Pulmonary and cardiovascular effects of acute exposure to concentrated
ambient particulate matter in rats. Toxicol. Lett. 96 –97:285–288;
1998.
Prahalad, A. K.; Soukup, J. M.; Inmon, J.; Willis, R.; Ghio, A. J.;
Becker, S.; Gallagher, J. E. Ambient air particles: effects on
cellular oxidant radical generation in relation to particulate elemental chemistry. Toxicol. Appl. Pharmacol. 158:81–91; 1999.
Clarke, R. W.; Coull, B.; Reinisch, U.; Catalano, P.; Killingsworth, C. R.; Koutrakis, P.; Kavouras, I.; Murthy, G. G.; Law-
Oxidative stress by ambient particles
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
rence, J.; Lovett, E.; Wolfson, J. M.; Verrier, R. L.; Godleski, J. J.
Inhaled concentrated ambient particles are associated with hematologic and bronchoalveolar lavage changes in canines. Environ.
Health Perspect. 108:1179 –1187; 2000.
Stohs, S. J.; Bagchi, D. Oxidative mechanisms in the toxicity of
metal ions. Free Radic. Biol. Med. 18:321–336; 1995.
Ghio, A. J.; Stonehuerner, J.; Pritchard, R. J.; Piantadosi, C. A.;
Quigley, D. R.; Dreher, K. L.; Costa, D. L. Humic-like substances
in air pollution particulates correlate with concentrations of transition
metals and oxidant generation. Inhal. Toxicol. 8:479 – 494; 1996.
Ghio, A. J.; Stonehuerner, J.; Dailey, L. A.; Carter, J. D. Metals
associated with both the water-soluble and insoluble fractions of
an ambient air pollution particle catalyze an oxidative stress.
Inhal. Toxicol. 11:37– 49; 1999.
Costa, D. L.; Dreher, K. L. Bioavailable transition metals in
particulate matter mediate cardiopulmonary injury in healthy and
compromised animal models. Environ. Health Perspect. 105:
1053–1060; 1997.
Ball, J. C.; Straccia, A. M.; Young, W. C.; Aust, A. E. The
formation of reactive oxygen species catalyzed by neutral, aqueous extracts of NIST ambient particulate matter and diesel engine
particles. J. Air Waste Manage. Assoc. 50:1897–1903; 2000.
Smith, K. R.; Aust, A. E. Mobilization of iron from urban particulates leads to generation of reactive oxygen species in vitro
and induction of ferritin synthesis in human lung epithelial cells.
Chem. Res. Toxicol. 10:828 – 834; 1997.
Prahalad, A. K.; Inmon, J.; Dailey, L. A.; Madden, M. C.; Ghio,
A. J.; Gallagher, J. E. Air pollution particles mediated oxidative
DNA base damage in a cell free system and in human airway
epithelial cells in relation to particulate metal content and bioreactivity. Chem. Res. Toxicol. 14:879 – 887; 2001.
Ross, M. M.; Chedekel, M. R.; Risby, T. H. Electron paramagnetic resonance spectrometry of diesel particulate matter. Environ. Int. 7:325–329; 1982.
Bolton, J. L.; Trush, M. A.; Penning, T. M.; Dryhurst, G.; Monks,
T. J. Role of quinones in toxicology. Chem. Res. Toxicol. 13:135–
160; 2000.
Ning, Y.; Imrich, A.; Goldsmith, C. A.; Qin, G.; Kobzik, L.
Alveolar macrophage cytokine production in response to air particles in vitro: role of endotoxin. J. Toxicol. Environ. Health A.
59:165–180; 2000.
Imrich, A.; Ning, Y.; Kobzik, L. Insoluble components of concentrated air particles mediate alveolar macrophage responses in
vitro. Toxicol. Appl. Pharmacol. 167:140 –150; 2000.
Becker, S.; Soukup, J. M.; Gilmour, M. I.; Devlin, R. B. Stimulation of human and rat alveolar macrophages by urban air particulates: effects on oxidant radical generation and cytokine production. Toxicol. Appl. Pharmacol. 141:637– 648; 1996.
Dong, W.; Lewtas, J.; Luster, M. I. Role of endotoxin in tumor
necrosis factor alpha expression from alveolar macrophages
treated with urban air particles. Exp. Lung Res. 22:577–592;
1996.
Chamulitrat, W.; Skrepnik, N. V.; Spitzer, J. J. Endotoxin-induced oxidative stress in the rat small intestine: role of nitric
oxide. Shock. 5:217–222; 1996.
Rollet-Labelle, E.; Grange, M. J.; Elbim, C.; Marquetty, C.;
Gougerot-Pocidalo, M. A.; Pasquier, C. Hydroxyl radical as a
potential intracellular mediator of polymorphonuclear neutrophil
apoptosis. Free Radic. Biol. Med. 24:563–572; 1998.
Elder, A. C.; Finkelstein, J.; Johnston, C.; Gelein, R.; Oberdorster,
G. Induction of adaptation to inhaled lipopolysaccharide in young
and old rats and mice. Inhal. Toxicol. 12:225–243; 2000.
Wilson, M. R.; Lightbody, J. H.; Donaldson, K.; Sales, J.; Stone,
V. Interactions between ultrafine particles and transition metals in
vivo and in vitro. Toxicol. Appl. Pharmacol. 184:172–179; 2002.
Duffin, R.; Tran, C. L.; Clouter, A.; Brown, D. M.; MacNee, W.;
Stone, V.; Donaldson, K. The importance of surface area and
specific reactivity in the acute pulmonary inflammatory response
to particles. Ann. Occup. Hyg. 46:242–245; 2002.
Tao, F.; Kobzik, L. Lung macrophage-epithelial cell interactions
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
339
amplify particle-mediated cytokine release. Am. J. Respir. Cell
Mol. Biol. 26:499 –505; 2002.
Oberdorster, G.; Yu, C. P. Lung dosimetry— considerations for
noninhalation studies. Exp. Lung Res. 25:1– 6; 1999.
Crapo, J. D.; Barry, B. E.; Foscue, H. A.; Shelburne, J. Structural
and biochemical changes in rat lungs occurring during exposures
to lethal and adaptive doses of oxygen. Am. Rev. Respir. Dis.
122:123–143; 1980.
Churg, A.; Vedal, S. Carinal and tubular airway particle concentrations in the large airways of non-smokers in the general population: evidence for high particle concentration at airway carinas.
Occup. Environ. Med. 53:553–558; 1996.
Soukup, J. M.; Ghio, A. J.; Becker, S. Soluble components of
Utah Valley particulate pollution alter alveolar macrophage function in vivo and in vitro. Inhal. Toxicol. 12:401– 414; 2000.
Pope, C. A. 3rd. Respiratory disease associated with community
air pollution and a steel mill, Utah Valley. Am. J. Public Health.
79:623– 628; 1989.
Labedzka, M.; Gulyas, H.; Gercken, G. Zymosan-induced chemiluminescence of alveolar macrophages: depression by inorganic dust
constituents. Arch. Environ. Contam. Toxicol. 21:51–57; 1991.
Shi, M. M.; Chong, I.; Godleski, J. J.; Paulauskis, J. D. Regulation
of macrophage inflammatory protein-2 gene expression by oxidative stress in rat alveolar macrophages. Immunology. 97:309 –
315; 1999.
Goldsmith, C. A.; Frevert, C.; Imrich, A.; Sioutas, C.; Kobzik, L.
Alveolar macrophage interaction with air pollution particulates.
Environ. Health Perspect. 105:1191–1195; 1997.
Goldsmith, C. A.; Imrich, A.; Danaee, H.; Ning, Y. Y.; Kobzik, L.
Analysis of air pollution particulate-mediated oxidant stress in alveolar macrophages. J. Toxicol. Environ. Health A. 54:529 –545; 1998.
Huang, S. L.; Cheng, W. L.; Lee, C. T.; Huang, H. C.; Chan,
C. C. Contribution of endotoxin in macrophage cytokine response
to ambient particles in vitro. J. Toxicol. Environ. Health A 65:
1261–1272; 2002.
Li, N.; Kim, S.; Wang, M.; Froines, J.; Sioutas, C.; Nel, A. Use of
a stratified oxidative stress model to study the biological effects of
ambient concentrated and diesel exhaust particulate matter. Inhal.
Toxicol. 14:459 – 486; 2002.
Maines, M. D. The heme oxygenase system: a regulator of second
messenger gases. Annu. Rev. Pharmacol. Toxicol. 37:517–554;
1997.
Kennedy, T.; Ghio, A. J.; Reed, W.; Samet, J.; Zagorski, J.; Quay,
J.; Carter, J.; Dailey, L.; Hoidal, J. R.; Devlin, R. B. Copperdependent inflammation and nuclear factor-kappaB activation by
particulate air pollution. Am. J. Respir. Cell Mol. Biol. 19:366 –
378; 1998.
Shukla, A.; Timblin, C.; BeruBe, K.; Gordon, T.; McKinney, W.;
Driscoll, K.; Vacek, P.; Mossman, B. T. Inhaled particulate matter
causes expression of nuclear factor (NF)-kappaB-related genes
and oxidant-dependent NF-kappaB activation in vitro. Am. J.
Respir. Cell Mol. Biol. 23:182–187; 2000.
Frampton, M. W.; Ghio, A. J.; Samet, J. M.; Carson, J. L.; Carter,
J. D.; Devlin, R. B. Effects of aqueous extracts of PM10 filters
from the Utah Valley on human airway epithelial cells. Am. J.
Physiol. 277:L960 –L967; 1999.
Gansauge, S.; Gansauge, F.; Gause, H.; Poch, B.; Schoenberg,
M. H.; Beger, H. G. The induction of apoptosis in proliferating
human fibroblasts by oxygen radicals is associated with a p53and p21WAF1CIP1 induction. FEBS Lett. 404:6 –10; 1997.
Karbowski, M.; Kurono, C.; Wozniak, M.; Ostrowski, M.; Teranishi, M.; Nishizawa, Y.; Usukura, J.; Soji, T.; Wakabayashi, T.
Free radical-induced megamitochondria formation and apoptosis.
Free Radic. Biol. Med. 26:396 – 409; 1999.
Hitzfeld, B.; Friedrichs, K. H.; Ring, J.; Behrendt, H. Airborne
particulate matter modulates the production of reactive oxygen
species in human polymorphonuclear granulocytes. Toxicology
120:185–195; 1997.
Dye, J. A.; Lehmann, J. R.; McGee, J. K.; Winsett, D. W.;
Ledbetter, A. D.; Everitt, J. I.; Ghio, A. J.; Costa, D. L. Acute
pulmonary toxicity of particulate matter filter extracts in rats:
340
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
F. TAO et al.
coherence with epidemiologic studies in Utah Valley residents.
Environ. Health Perspect. 109:395– 403; 2001.
Pritchard, R. J.; Ghio, A. J.; Lehmann, J. R.; Winsett, D. W.;
Tepper, J. S.; Park, P.; Gilmour, M. I.; Dreher, K. L.; Costa, D. L.
Oxidant generation and lung injury after particulate air pollutant
exposure increase with the concentrations of associated metals.
Inhal. Toxicol. 8:457– 477; 1996.
Hatch, G. E.; Boykin, E.; Graham, J. A.; Lewtas, J.; Pott, F.;
Loud, K.; Mumford, J. L. Inhalable particles and pulmonary host
defense: in vivo and in vitro effects of ambient air and combustion
particles. Environ. Res. 36:67– 80; 1985.
Brown, D. M.; Stone, V.; Findlay, P.; MacNee, W.; Donaldson,
K. Increased inflammation and intracellular calcium caused by
ultrafine carbon black is independent of transition metals or other
soluble components. Occup. Environ. Med. 57:685– 691; 2000.
Ghio, A. J.; Devlin, R. B. Inflammatory lung injury after bronchial instillation of air pollution particles. Am. J. Respir. Crit.
Care Med. 164:704 –708; 2001.
Vander, A.; Sherman, J.; Luciano, D. Respiration. In: Human
physiology: the mechanisms of body function. New York:
McGraw-Hill; 2001:476 – 477.
Lazaridis, M.; Broday, D. M.; Hov, O.; Georgopoulos, P. G.
Integrated exposure and dose modeling and analysis system. 3.
Deposition of inhaled particles in the human respiratory tract .
Environ. Sci. Technol. 35:3727–3734; 2001.
Clarke, R. W.; Catalano, P. J.; Koutrakis, P.; Murthy, G. G.;
Sioutas, C.; Paulauskis, J.; Coull, B.; Ferguson, S.; Godleski, J. J.
Urban air particulate inhalation alters pulmonary function and
induces pulmonary inflammation in a rodent model of chronic
bronchitis. Inhal. Toxicol. 11:637– 656; 1999.
Kodavanti, U. P.; Mebane, R.; Ledbetter, A.; Krantz, T.; McGee,
J.; Jackson, M. C.; Walsh, L.; Hilliard, H.; Chen, B. Y.; Richards,
J.; Costa, D. L. Variable pulmonary responses from exposure to
concentrated ambient air particles in a rat model of bronchitis.
Toxicol. Sci. 54:441– 451; 2000.
Clarke, R. W.; Catalano, P.; Coull, B.; Koutrakis, P.; Krishna
Murthy, G. G.; Rice, T.; Godleski, J. J. Age-related responses in
rats to concentrated urban air particles (CAPs). Inhal. Toxicol.
12:73– 84; 2000.
Gurgueira, S. A.; Lawrence, J.; Coull, B.; Krishna Murthy, G. G.;
Gonzalez-Flecha, B. Rapid increases in the steady-state concentration of reactive oxygen species in the lungs and heart after
particulate air pollution inhalation. Environ. Health Perspect.
110:749 –755; 2002.
Boveris, A.; Cadenas, E.; Reiter, R.; Filipkowski, M.; Nakase, Y.;
Chance, B. Organ chemiluminescence: noninvasive assay for oxidative radical reactions. Proc. Natl. Acad. Sci. USA 77:347–351;
1980.
Cadenas, E.; Sies, H. Low-level chemiluminescence as an indicator of singlet molecular oxygen in biological systems. Methods
Enzymol. 105:221–231; 1984.
Halliwell, B.; Gutteridge, J. M. Role of free radicals and catalytic
metal ions in human disease: an overview. Methods Enzymol.
186:1– 85; 1990.
Ghio, A. J.; Kim, C.; Devlin, R. B. Concentrated ambient air
particles induce mild pulmonary inflammation in healthy human
volunteers. Am. J. Respir. Crit. Care Med. 162:981–988; 2000.
Harder, S. D.; Soukup, J. M.; Ghio, A. J.; Devlin, R. B.; Becker,
S. Inhalation of PM2.5 does not modulate host defense or immune
parameters in blood or lung of normal human subjects. Environ.
Health Perspect. 109:599 – 604; 2001.
Schwartz, J. PM10, ozone, and hospital admissions for the elderly
in Minneapolis-St. Paul, Minnesota. Arch. Environ. Health 49:
366 –374; 1994.
Gulsvik, A. The global burden and impact of chronic obstructive
[87]
[88]
[89]
[90]
[91]
pulmonary disease worldwide. Monaldi Arch. Chest Dis. 56:261–
264; 2001.
Simeonova, P. P.; Luster, M. I. Iron and reactive oxygen species in
the asbestos-induced tumor necrosis factor-alpha response from alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 12:676 – 683;
1995.
Gossart, S.; Cambon, C.; Orfila, C.; Seguelas, M. H.; Lepert, J. C.;
Rami, J.; Carre, P.; Pipy, B. Reactive oxygen intermediates as
regulators of TNF-alpha production in rat lung inflammation
induced by silica. J. Immunol. 156:1540 –1548; 1996.
Nel, A. E.; Diaz-Sanchez, D.; Li, N. The role of particulate
pollutants in pulmonary inflammation and asthma: evidence for
the involvement of organic chemicals and oxidative stress. Curr.
Opin. Pulm. Med. 7:20 –26; 2001.
Thomassen, M. J.; Kavuru, M. S. Human alveolar macrophages
and monocytes as a source and target for nitric oxide. Int. Immunopharmacol. 1:1479 –1490; 2001.
Weinberg, J. B. Nitric oxide production and nitric oxide synthase
type 2 expression by human mononuclear phagocytes: a review.
Mol. Med. 4:557–591; 1998.
ABBREVIATIONS
AM—alveolar macrophage
BAL— bronchoalveolar lavage
CAPs— concentrated ambient particles
CB— carbon black
CL— chemiluminescence
DETA— diethylenetriamine pentaacetic acid
DFX— deferoxamine
DMTU— dimethylthiourea
EC— epithelial cell
ICAM-1—intercellular adhesion molecule 1
IFN-␥—interferon gamma
IL-6 —interleukin-6
IL-8 —interleukin-8
LDH—lactate dehydrogenase
LPS—lipopolysaccaride
NAC—N-acetylcysteine
NAG—N-acetyl glutaminidase
NF-␬B—nuclear factor kappa B
•
NO—nitric oxide
PEF—peak expiratory flow
PM—ambient air pollution particles
PMN—polymorphonuclear granulocyte
RNS—reactive nitrogen species
ROFA—residual oil fly ash
ROS—reactive oxygen species
SOD—superoxide dismutase
SRM—standard reference material
TBARS—thiobarbituric acid-reactive substances of deoxyribose
TGF-␤—transforming growth factor beta
TNF-␣—tumor necrosis factor alpha
TNF-␤—tumor necrosis factor beta