Effects of fine carbonaceous particles containing
high and low unpaired electron spin densities on
lungs of female mice
JOHN E. REPINE, OSCAR K. REISS, NANCY ELKINS, ABDUL R. CHUGHTAI, and
DWIGHT M. SMITH
DENVER, COLO
The negative impacts on human health that accompany inhalation of atmospheric
particles are documented in numerous epidemiologic studies, but the effect of specific
chemical properties of the particles is generally unknown. We developed and employed technology for generating inhalable aerosols of carbonaceous air pollution
particles that have specific physical and chemical properties. We find that inhaling
particles with greater unpaired electron spin (free radical) densities stimulates greater
lung inflammatory and oxidative stress responses. Cultured alveolar macrophages
take up more particles of greater free radical content, develop mitochondrial abnormalities, and release more leukotriene B4 (LTB4) than alveolar macrophages exposed
to lesser free-radical– containing particles in vitro. Mice exposed to high free radical
particles in vivo also develop mitochondrial abnormalities in alveolar macrophages
and increased oxidative stress, which is reflected by increases in lung nitrotyrosine
staining and lung lavage nitrogen oxide levels compared with those of lesser free
radical density. These results provide insight for the unexplained geographic differences and have implications for fossil fuel combustion conditions and the impact of fine
particles on health and disease. (Translational Research 2008;152:185–193)
Abbreviations: Ab ⫽ antibody; A/F ⫽ air/fuel combustion ratio; AM ⫽ alveolar macrophage;
BC ⫽ black carbon; DEP ⫽ diesel exhaust particulate; EPR ⫽ electron paramagnetic resonance; FR ⫽ free radical; high-FR ⫽ high free radical containing; LTB4 ⫽ leukotriene B4; low-FR ⫽
low free radical containing; MPO ⫽ myeloperoxidase; MW ⫽ molecular weight; PAH ⫽ polyaromatic hydrocarbon; PM ⫽ particulate matter; ROS ⫽ reactive oxygen species
N
umerous epidemiologic studies1– 4 indicate that
inhaling fine particulate [particulate matter
(PM) 2.5 to PM 10] pollution is detrimental to
human health, which specifically contributes to cardiovascular, pulmonary, and related diseases that have an
inflammatory and oxidative stress component in their
pathophysiology. Although numerous investigators
speculate that 1 or another physical/chemical property
of inhaled particles underlies their biologic effects,
direct experimental evidence has been elusive. Although many data are available on the effects of organics, metals, particle size, and so on, no direct experimental evidence relates specific chemical properties of
inhaled particles to the initiation of mechanisms that
contribute to the disease processes. Several particle
properties, which include size, surface area, type and
density of surface functional groups,5 surface charge,6
polyaromatic hydrocarbon (PAH) content,7 and un-
From the Webb-Waring Institute for Cancer, Aging, and Antioxidant
Research, University of Colorado Denver Health Sciences Center
(UCDHSC), Denver, Colo, and the Department of Chemistry and
Biochemistry, University of Denver, Denver, Colo.
Reprint requests: John E. Repine, Webb-Waring Institute for Cancer,
Aging and Antioxidant Research, University of Colorado Denver
Health Sciences Center (UCDHSC), Denver, CO 80262; e-mail:
[email protected].
1931-5244/$ – see front matter
© 2008 Mosby, Inc. All rights reserved.
doi:10.1016/j.trsl.2008.08.003
Supported by the Thrasher Research Fund.
Submitted for publication June 5, 2008; revision submitted August
11, 2008; accepted for publication August 19, 2008.
185
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Repine et al
AT A GLANCE COMMENTARY
Background
We are inhaling fine airborne particulates that
have been implicated as contributors to pulmonary
and cardiovascular diseases and hospital admissions. Not all particles are the same, but nonetheless, the nature of inhalable particles and their
respective effects on health have not been investigated.
Translational Significance
We found that exposure to inhalable particles with
high free radical activity produces greater lung
inflammation and oxidative stress, which could
contribute to the lung and cardiovascular disorders
associated with exposure to airborne particle pollutants, explain individual and geographic variations in these conditions, and produce new guidelines for combustion processes.
paired electron spin (free radical) density,8 are candidates for determining the interaction of inhaled particles with biologic systems based on previous work. It is
only through systematic controlled study of these variables that the biologic effects of specific key properties
can be assessed. Previous research has involved using
such surrogates for inhalation as intratracheal injection
of diesel exhaust particulate (DEP) extracts9,10 and
aqueous suspensions of DEP11 to generate reactive
oxygen species (ROS) such as O2.- and .OH in mice
lungs and human pulmonary artery endothelial cells.12
With in vitro experiments, cell DNA damage by aqueous extracts of PM2.5 has been attributed to semiquinone radicals in the collected particulate.13 The generation of ROS by collected PM also has been correlated
with impaired pulmonary function of Netherlands
school children.14 These experiments did not control
particle properties under physiologic inhalation conditions and thus cannot identify the actual operative variables. Furthermore, any correlations between the biologic response of collected PM and its electron spin
density measurements are specious at best because of
the presence of electron paramagnetic resonance (EPR)
spectrum-distorting impurities, such as metal ions and
sulfur in such material. Oxidative stress biomarkers
increase in mice after a 13-week exposure to carbon
black particles, but chemical properties such as surface
functionalities, PAH profile, and electron spin density,
as well as other chemical properties such as metal
content of the particles, are either unknown or not
reported.15
The free radical content of aerosol particles is measured by EPR spectroscopy. The magnitude of the EPR
signal is a function of the unpaired electron spin density
of the particles, which itself is determined by the characteristics of the fuel and combustion conditions such
as air/fuel combustion ratio (A/F),8 which is a function
of the partial pressure of oxygen. Experiments show
that the unpaired electrons are associated with both the
largely aromatic backbone of the particle structure5 and
the associated PAH molecules,7 many of which are
extractable. In contrast to free radical (FR) in solution,
the unpaired electron (free radical) density of particles
is generally stable over long periods in air, although
treatment with strongly oxidizing species, such as O3,
leads to their diminution. By generating aerosols that
contain soot particles of comparable size and concentration, but with differing spin densities (free radical
concentrations) using different A/F, it has been possible
to associate the origin of lung inflammation and oxidative stress with this particle property.
We developed and employed a technique for generating, from the combustion of pure hydrocarbon fuels,
aerosols that consist of fine black carbon (BC) particles
having specific properties and devoid of metal ions,
which make them appropriate surrogates for airborne
carbonaceous particulate.8,16 We then used these wellcharacterized particles in in vivo studies in which animals (mice in our work) inhale these particles under
controlled environmental conditions where the effects
of varying 1 property can be examined while other
properties are held constant. This technology makes it
possible to identify those particle properties that underlie epidemiologic correlation between airborne particle
exposure to increased mortality and common disease
processes, such as asthma and heart disease. Experts in
this field17,18 have long recognized the need for research programs to identify the harmful components of
inhalable particulate and the mechanism(s) of their
effects. This report presents experimental evidence that
directly links the free radical property of physiologically inhaled particles to a pathophysiologic consequence. Connecting environmental exposure to a serious
human health issue, the results of this research clearly are
translational19,20 in nature.
MATERIALS AND METHODS
Particle generation. For the in vivo experiments,
aerosol atmospheres that contain carbonaceous particles with rigorously controlled chemical and physical
properties are generated with specific A/F ratios from
reagent n-hexane by previously described techniques.8
We employ a special apparatus constructed to allow
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187
Fig 1. Schematic of apparatus used to generate carbon particles, animal exposure chambers, and exposure
controls.
Table I. Properties of typical aerosolized black
carbon particles used for in vivo exposures
Property
Spin density in air,‡ CNI ⫻ 10–6
Average number of particles, p m–3 ⫻ 10–9
Average particle size, ␮m
Low-FR
High-FR
particles* particles†
3.6‡
1.4
0.16
5.7‡
1.1
0.17
Abbreviation: CNI, corrected normalized integral.
*Generated with an air/fuel combustion ratio of 4.3.
†
Generated with an air/fuel combustion ratio of 2.2.
‡
CNI of the EPR signal is a measure of spins per unit mass of
particles and is measured with a reproducibility of ⫾5%.
simultaneous exposure of 2 groups of mice to particles
of differing properties, in this case, particles with different free radical concentrations but similar mass, size,
and concentration (Fig 1). Emitted particles are directed
into 1 of the 20-L smoke chambers until the optical
density, as determined by a laser beam, reaches a predetermined value and the larger particles then settle
until a stable aerosol is maintained. A computerized
TSI Model 3030 Electrical Aerosol Size Analyzer (TSI
Incorporated, Shoreview, Minn), for particle size distribution and number density, and Varian E9 EPR
Spectrometer, for unpaired electron spin density, are
used to characterize the particles. Particles for the EPR
measurements are collected by settling. Typical properties of BC soot particles that comprise the inhalation
aerosols are shown in Table I. High free radical containing (high-FR) particles had greater spin densities
but similar particle concentrations and sizes as low free
radical containing (low-FR) particles.
For the in vitro measurements, the particles also were
generated by n-hexane combustion. For this application, however, they were captured on an inverted Pyrex
funnel (World Kitchen LLC, Greencastle, Pa), transferred from its surface to a glass vessel designed to
allow a slow flow of ozone from an in-house ozone
generator and measurement apparatus, and treated for
various times to yield particles that contained varying
concentrations of FR. The extent of surface oxidation
and spin density were monitored by Fourier transform
infrared and EPR spectroscopies, respectively.
In vitro studies. Cultured NR8383 alveolar macrophages (AMs) (ATCC, Manassas, VA) are grown in
RPMI 1640 medium in 12-well tissue culture dishes
that contain 1 ⫻ 106 cells. The wells contained no
particles or 2 groups of soot particles differing in free
radical density and prepared by ozone oxidation of
otherwise identical n-hexane soots. These particular
macrophages were used in the pilot in vitro studies
because we initially intended to use rats for the entire
study, and we later decided that mice were a better
choice. These soot particles are produced by combustion in the same manner as those used in the in vivo
experiments. A 4-mg/mL carbon suspension is prepared in freshly diluted 50% dimethyl sulfoxide (tissue
culture grade) by vortexing. To 300 ␮L of this suspension is added 150 ␮L of a crude suspension of pulmonary surfactant in 0.9% saline that contained 10
␮mol/mL of organic phosphate and 300 ␮L fetal calf
serum. This suspension is forced 3 times through a #31
hypodermic needle by exerting maximum force by
hand and then placed into an ice bath. It is then treated
with a sonic oscillator 2 times for 20 s. These prepara-
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Repine et al
Fig 2. Electron micrograph of AMs exposed to high-FR particles in
vitro. NR 8383 cultured AMs exposed to high-FR particles for 21 h
shows particles localized in phagolysosomes and damaged mitochondria.
tions, diluted with culture medium to contain 10 ␮g/mL
of the particles, are stable for 48 h at 37°C as measured
by light scattering. The concentration of the particles
was derived from an estimate of the particles in the
human lung fluid after a 24-h exposure to 20 ␮g m⫺3 in
the atmosphere. Particle uptake is determined by light
scattering after 3 or 21 h of incubation in the supernatant following removal of AMs by centrifugation. The
surfactant was obtained by bronchio-alveolar lavages of
rabbit lungs with sterile saline, followed by centrifugation at 4000 ⫻ g for 15 min to remove the cells,
followed by centrifugation of the supernant fraction at
16000 ⫻ g for 45 min. The resulting pellet was suspended in sterile saline, washed 2 times with saline,
suspended in the same, and frozen at ⫺20°. For analysis of leukotriene B4 (LTB4) release, AMs are incubated with the 2 soot preparations for 6 h, and subsequently, LTB4 is released by adding 1 ␮mol/L of
solution of A-23187 in Hams F12 medium without fetal
calf serum. LTB4 concentrations are then determined
with an LTB4 EIA kit (Cayman Chemical Co, Ann
Arbor, Mich), which has a sensitivity of 1 pg/␮L. We
also incubate AMs for 24 h with High-FR particles and
then fix, embed, section, and examine AMs by standard
transmission electron microscopy technique (Fig 2) to
establish the intracellular uptake of the particles and the
effects of particle uptake on cellular structure. No attempts to determine the particle uptake quantitatively
by EM were made, because this task requires extensive
stereological microscopic determination.
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October 2008
In vivo studies. Two groups of 4 friend virus B-type
female mice (Jackson Laboratories, Bar Harbor, Maine)
are exposed simultaneously to particles with a specific
set of properties but that have differing spin densities,
for 4 consecutive days, 6 h per day, at an air flow that
contained about 10 ␮g m⫺3 particles (109 particles
m⫺3). This flow is sufficient to replace the exposure
chamber air 14 times per hour at 22°C at a relative
humidity of 30% ⫾ 3%. The protocol for this procedure
was approved by the Institutional Animal Care and Use
Committee of The University of Denver. For assessing
mitochondrial membrane permeability after particle exposure, AMs are recovered by lung lavage using 0.9%
saline, separated by centrifugation, washed 2 times with
phosphate buffered saline and then stained for 20 min
with a solution of JC-1 (#T3168; Molecular Probes,
Eugene, Ore), which is a dye widely used to determine
changes in mitochondrial membrane permeability21 and
to indicate early stage apoptosis or necrosis.22 AMs
from particle exposed and sham-exposed mice lungs
are treated with 0.3 ␮g/mL of JC-1 for 20 min, analyzed, and compared with controls using fluorocytometry (BD Bioscience, San Jose, Calif) with argon laser
excitation (488 nm) separating emission fluorescence
into the FL-1 (em 530 nm) and FL-2 (em 585 nm)
channels. For analyses of nitrotyrosine fluorescence,
aconitase,23,24 and myeloperoxidase (MPO),25 the mice
are euthanized immediately after the last particle exposure, and the lungs were perfused in situ with 0.9%
saline and inflated with a 0.2% agarose solution. After
the agarose solidifies, 1 lung is immediately fixed in
freshly prepared 10% formaldehyde and the remaining
lungs from each animal are frozen in liquid N2 and
stored at ⫺70°C for aconitase (Aconitase Kit 340;
OXIS Int., Foster City, Calif) and MPO analysis. For
nitrotyrosine staining,26 all reagents must be free of
peroxides. The fixed lungs are washed after 18 –36 h 3
times with 70% ethanol and embedded in paraffin without exposure to commercial preparations of formaldehyde (used in medical histology laboratories). Two
sections from the same mouse are transferred to 1 slide.
One section is stained by the standard procedure,
whereas the other section is stained negatively by preincubating the primary antibody (Ab) with 10 mmol/L
of nitrotyrosine for 1 h. The primary Ab is Anti-Nitrotyrosine (#06-284; Upstate USA Inc., Charlottesville,
VA), and the secondary Ab is Alexa Fluor 488 F(ab’)2
(#A 11070; Molecular Probes, Eugene, Ore), which is
spun at 13000 ⫻ g for 15 min to remove particles prior
to use. The staining with 2o Ab and all subsequent
operations are carried out in subdued light, and the
sections are stored in the dark at 4o. Within 72 h of the
staining, a minimum of 4 random black-and-white photomicrographs are taken from each section and stored
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189
Fig 4. Uptake of high- and low-FR particles by NR8383 cells in
culture. More high-FR particles were taken up than low-FR particles.
Particle uptake was at maximum after 3 h for both high- and low-FR
particles. The values at 3 h are the mean ⫾ SE of 3 determinations,
whereas the values at 21 h are the mean ⫾ standard error of 8
determinations. (Color version of figure is available online.)
protein. Activated cadmium beads are added to the
supernatant to reduce the nitrate to nitrite, which is then
analyzed with the Griess reaction29 (Kit # NB 88;
Oxford Biomedical Research, Oxford, Mich). Total nitrite plus nitrate is a measure of the NOx production.
Statistical analyses of data for nitrotyrosine assessment.
Fig 3. Particle uptake and LTB4 production by AMs exposed to highand low-FR particles in vitro. NR 8383 cultured AMs take up more
(P ⬍ 0.002) high-FR particles (solid bar) than low-FR particles (open
bar) (A). NR 8383 cultured AMs produce more LTB4 (P ⬍ 0.04)
after a 6-h exposure to high-FR particles (solid bar) than low-FR
particles (open bar) (B). Each bar is the mean ⫾ 1 standard error of
the number of determinations shown in the parentheses.
as “tif” files. Photographs were not taken when more
than 1 min was required for focusing, because of
photobleaching. Fluorescent intensity of each micrograph is determined by Threshhold segmentation analysis with the ImageJ version 1.37 software with custom
plug-in and expressed as Threshold_PRCNT.27,28
For lung lavage NOx assessment, the supernatant
fractions after the removal of the cellular elements are
used and stored at ⫺20°C. The supernatant is analyzed
for protein (micro BCA protein assay kit #23235 with
all reagents containing 1% sodium dodecylsulfate;
Pierce Chem. Co., Rockford, Ill). Another aliquot is
treated with ZnSO4 and centrifuged to remove excess
The threshold_percent values of the negative controls
were subtracted from the values of the positive sections, and the difference was evaluated by multivariant
repeated measuring analyses using SAS version 9.1
(SAS Institute, Inc., Cary, NC); for all other analyses,
the software Statmost (Dataxiom Software, Inc., Los
Angeles, Calif) was used to apply the 2-sample t-test.
RESULTS
In vitro analyses. We found that high-FR particles have
greater stimulating effects than low-FR particles (30%
lower in free radical density) in biologic systems in vitro.
Cultured AMs take up more high-FR particles than
low-FR particles (Fig 3, A). Additional separate studies
revealed the uptake of high-FR and low-FR particles by
AMs in culture at 3 and 21 h of incubation (Fig 4).
More high-FR particles were taken up than low-FR
particles. Maximum uptake occurred after 3 h with
continuous agitation. In addition, when measuring
LTB4 release as a general indicator of inflammatory
activity, and because of the potential role of leukotrienes in asthma and pulmonary and cardiovascular conditions,30,31 we find that AMs treated with high-FR
particles release approximately 3 times more LTB4 than
AMs treated with low-FR particles (Fig 3, B). AMs
freshly isolated from mice by lung lavage yield similar
results. However, when treated with low- or high-FR
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Repine et al
particles, lung epithelial cells (A549) with marginal
phagocytic activity produce no measurable LTB4. In
morphologic examinations of AMs exposed to high-FR
particles in vitro, it is evident that many particles are
localized within phagolysosomes and mitochondria,
which are swollen and damaged (see Fig 2).
In vivo analyses. In vivo analyses using BC soot particles of varying unpaired electron spin density reveal
that high-FR particles have potent effects on the lung
compared with low-FR particles. The principal in vivo
findings include measurements of AM mitochondrial
damage, lung nitrotyrosine fluorescence, and lung lavage NOx concentrations. First, the AMs from lungs of
mice exposed to high-FR particles have damaged mitochondria reflected by decreases in JC-1 fluorescence
compared with AMs recovered from lungs of mice
exposed to low-FR particles (Fig 5, B and C). Exposure
to both high- and low-FR particles decrease mitochondrial membrane permeability, but the mitochondrial
damaging effect of high-FR particles exceeds that of
low-FR particles (Fig 5, A–C). The findings are consistent with our in vitro studies, which show mitochondrial damage in AMs exposed to high-FR particles in
vitro (see Fig 2).
Second, exposure to high-FR particles increases lung
nitrotyrosine fluorescence (Fig 6, A, Bottom Panel)
compared with control exposure (Fig 6, A, Top Panel).
Figure 6, B reveals statistically significant increases in
lung nitrotyrosine fluorescence (Threshold_PRCNT)
after exposure of mice to high-FR– containing particles
compared with unexposed mice (P ⫽ 0.01) and mice
exposed to low-FR particles (P ⫽ 0.04). In all experiments, fluorescence increases in this order: sham exposure (control) ⬍ low-FR particle exposure ⬍ high-FR
particle exposure. The increased amount of nitrotyrosine
staining indicates an oxidative response.32,33 Nitrotyrosine
production presumably depends on the interaction of
either peroxidases or the superoxide anion with NO to
produce peroxynitrite, which can then nitrate protein
tyrosine.34 To assess which of these 2 postulated mechanisms might be operative in this instance, we analyzed
mouse lungs for MPO activity but could find none. The
absence of MPO suggests a superoxide anion-dependent process with NO to form protein bound nitrotyrosine and other free radical compounds.33 This mechanism is supported by examination of the aconitase
content of lung homogenates. We also find that aconitase activity is inhibited in lungs of mice exposed to
high-FR particles [0.57-mU/mg protein compared with
control lung values of 1.02-mU/mg protein (P ⫽ 0.02)]
compared with lungs of mice exposed to low-FR particles [0.67-mU/mg protein compared with control lung
values of 1.02-mU/mg protein (P ⫽ 0.05)]. Aconitase
contains a prosthetic Fe–S cluster, which is inhibited by
Translational Research
October 2008
Fig 5. Mitochondrial membrane permeability of AMs recovered from
mice exposed to high-FR and low-FR particles in vivo. Dot plots are
from flowcytometer readings of 10,000 AMs stained with JC-1. AMs
from control unexposed mice (A) fluoresced at 585 nm (right upper
quadrant). AMs from mice exposed to low-FR particles (B) fluoresce
less at 585 nm as reflected by a shift to the lower right quadrant. AMs
from mice exposed to high-FR particles (C) lose nearly all 585-nm
fluorescence as depicted by the nearly complete shift to the lower right
quadrant. Note that this logarithmic plot was obtained directly from the
FACS computer. Expression of these results in terms of percent mean
fluorescence yield the following values: 9.7 ⫾ 2.4 for the controls, 11.3
⫾ 2.7 for the low-FR, and 7.5 ⫾ 0.41 for the high-FR mice, which
indicates an increase in apoptosis caused by the high-FR carbon. (Color
version of figure is available online.)
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191
Fig 6. Photomicrograph of nitrotyrosine stained sections from lungs of mice exposed to air or high-FR particles
in vivo (A) and nitrotyrosine fluorescence (B) as well as nitrite concentrations (C) of mice exposed to high-FR
and low-FR particles in vivo. Unexposed mice show little nitrotyrosine staining (A) compared with lungs of mice
exposed to high-FR particles, which manifest considerably increased nitrotyrosine staining. Nitrotyrosine
fluorescence (B) is the same (P ⫽ ns) for lungs of unexposed mice (open bar) and mice exposed to low-FR
particles (gray bar). In contrast, nitrotyrosine fluorescence of lungs from mice exposed to high-FR particles (solid
bar) is significantly increased compared with lungs from unexposed mice (open bar; P ⬍ 0.01) or mice exposed
to low-FR particles (gray bar; P ⬍ 0.04) in vivo. Lung lavage nitrite concentrations (C) are the same (P ⫽ ns)
in unexposed mice (open bar) and mice exposed to low-FR particles (gray bar). In contrast, nitrite concentrations
in lung lavages from mice exposed to high-FR particles (solid bar) are increased compared with unexposed mice
(open bar; P ⬍ 0.04) and mice exposed to low-FR particles (gray bar; P ⬍ 0.04) in vivo. Each bar is the mean
⫾ 1 standard error of the number of determinations shown in the parentheses.
superoxide anion.23,24 Therefore, our finding suggests
that increased superoxide anions are being generated in
the lung after exposure to high-FR particles.
Finally, exposure to high-FR particles also significantly increases lung lavage NOx (total nitrate plus
nitrite) concentrations compared with low-FR particle
exposure, which shows no difference compared with
sham exposure (Fig 6, C). Assessing nitrite concentration provides a measure of the short-lived NO in biologic systems.29 We speculate that high-FR particle
exposure may activate AM mitochondrial inducible
nitric oxide synthetase,35 which is known to occur in
AM mitochondrial damage36 and is associated with an
increased rate of superoxide anion formation.37
DISCUSSION
Our results point to particle unpaired electron spin
density as a property of importance in the mechanism
responsible for the observed alterations in human health
that are linked epidemiologically to fine airborne particulate in select urban areas. It seems that inhaling
high-FR particles damages AM mitochondria and stimulates lung inflammation as well as oxidative stress.
We have shown in other work that as much as half of
the spin density of n-hexane soot particles is associated
with their extractable PAH content.7 In all, 26 distinct
PAH species have been identified as associated with
these particular soot particles, many of which are potentially in free radical form. The breadth of the X-band
EPR signal suggests several contributing species. Furthermore, the same work reveals that an increase in
extractable higher molecular weight (MW) PAH compounds is observed with a decreasing A/F combustion
ratio. Thus, both higher free radical density and higher
MW PAH are associated with particles created by
lower A/F combustion ratios. That is, in the case of
internal combustion engines, it is the “rich” combustion
mixtures (eg, engines under high load) that generate the
most harmful particles. In addition, lower A/F combustion ratios result in decreased particle surface oxidation,
which is also revealed by PAH compounds that are less
oxidized.7 In related work, our studies of soot particle
hydration16,38,39 show that the more oxidized the particles, the more extensively hydrated they are. These
observations and separate experiments indicate that the
more hygroscopic the particles, those with lower free
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October 2008
Repine et al
radical (and PAH) content, are more compatible with
aqueous (physiologic) systems. This conjecture makes
the free radical effects observed in our experiments
even more impressive as, although the particles have
essentially the same number density and size, the interaction of the higher free radical particles with the
aqueous physiologic medium should be relatively less.
Finally, although some investigators focus on the presumed carcinogenic properties of many PAH compounds, the results of our work indicate that it is their
free radical character, which is imparted to the particles
with which they are associated, that underlies the measures of oxidative stress and lung inflammation we have
observed.
This observed mechanism contributes to the currently unexplained detrimental and regional effects of
particulate pollution on human health. As noted, particle free radical density varies with such combustion
conditions as A/F, the FR content of particles generated
in Los Angeles being substantially greater than those in
Denver,8 for example, under otherwise identical formation conditions. At greater partial pressures of oxygen,
open flame combustion speed is greater, which renders
the effective A/F lesser during combustion. Thus, 1
implication of these results is the observed variance of
asthma with geography and altitude40 – 42 in the responses, which are a consequence of particle inhalation.
In addition, the loss of the mitochondrial membrane
integrity of AMs may represent an early stage of apoptosis and/or necrosis.22 Because AMs are the primary
defense against mycobacterial invasion,43 fewer or
damaged AMs may weaken the lung’s defense mechanism. Another potential implication of our findings is
related to the need for developing control mechanisms
for carbonaceous fine-particle (⬍200 nm) emissions
and optimizing the A/F (lean mixtures, low free radical
particles) in fossil fuel combustion. Our findings also
relate to the recent report that links coarse particulate
air pollution to hospital admissions for cardiovascular
and respiratory disease.44
The authors thank Dr. Gary Zerbe of UCHSC for assistance with
the statistical software; John Wennergren of UCHSC for participation
in biologic analyses; Professors G.R. and S.S. Eaton of Denver
University for use of the E9 EPR Spectrometer; Mr. Story Wilson,
Professional Research Assistant, Department of Pathology, UCHSC,
for the image analyses; Mr. Paul Blomquist, Reference Librarian,
Dennison Memorial Library, UCHSC, for help in reference retrievals;
and Ms. Lulu Yip of Denver University for animal care.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
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