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AIRWAY HYPERRESPONSIVENESS CAUSED BY AEROSOL
EXPOSURE TO RESIDUAL OIL FLY ASH LEACHATE IN MICE
Kaoru Hamada, Carroll-Ann Goldsmith, Yasue Suzaki,
Alejandra Goldman, Lester Kobzik
Physiology Program, Department of Environmental Health, Harvard
School of Public Health, Boston, Massachusetts, USA
Particulate air pollution is associated with exacerbation of asthma and other respiratory
disorders. This study sought to further characterize the pulmonary effects of residual oil fly
ash (ROFA), an experimentally useful surrogate for combustion-derived particulates in
ambient air. Mice were exposed to aerosols of the soluble leachate of residual oil fly ash
(ROFA-s). Physiologic testing of airway function ( non invasive plethysmography) showed
increased Penh, an index of airway hyperresponsiveness ( AHR), in a time- and dose-
dependent manner after exposure to ROFA-s. BAL analysis showed a minor influx of neu-
trophils, which was maximal at 12 h after exposure and essentially resolved by the time
point of maximal AHR ( 48 h after exposure). The AHR caused by ROFA-s was repro-
duced by a mixture of its major metal components (Ni, V, Zn, Co, Mn, Cu) but not by any
individual metal alone. Systemic pretreatment of mice with the antioxidant dimethylthio-
urea abrogated ROFA-s-mediated AHR. Analysis of mice of varying ages showed that
ROFA-s had no marked effect on airway responsiveness of 2-wk-old mice, in contrast to
the AHR seen in 3- and 8-wk old mice. ROFA-s-mediated AHR was unchanged in neuro-
kinin 1 receptor knockout mice and in mice treated with an neurokinin antagonist, argu-
ing against a role for this mediator in ROFA-s-mediated effects. Data indicate that ROFA-s
mediates AHR in mice through antioxidant-sensitive mechanisms that require multiple
metal constituents. Maturational differences in susceptibility to ROFA-induced AHR may
be useful for further studies of mechanisms of particle effects.
Elevated levels of air pollution are linked epidemiologically to increased
morbidity from asthma, pneumonia, and other respiratory-tract disorders
(Becker & Soukup, 1998, 1999, Braun-Fahrlander et al., 1992; Gong, 1992;
Nicolai, 1999). In some cases, air pollution components may cause respira-
tory symptoms even in normal subjects (Becker & Soukup, 1998). For ex-
ample, ozone causes airway hyperresponsiveness (AHR) in both human
subjects and animal models, not only in asthmatics but also in normal indi-
viduals (Bhalla, 1999; Shore et al., 2001). Recent epidemiologic studies
identified an important association between levels of particulate matter of
respirable size (PM2.5) and asthma morbidity (Goldsmith & Kobzik, 1999).
Experimental approaches to potential mechanisms for particle effects in-
clude use of well-defined surrogate particles, such as residual oil fly ash
1351
Journal of Toxicology and Environmental Health, Part A, 65:1351–1365, 2002
Copyright© 2002 Taylor & Francis
1528-7394 /02 $12.00 + .00
DOI: 10.1080/0098410029007158 6
Received 3 December 2001; sent for revision 11 January 2002; accepted 1 February 2002.
Supported by NIH grants ES08129, ES00002, and HL19170, and U.S. EPA grants R826779 and
R827353.
Address correspondence to Lester Kobzik, Department of Environmental Health, Harvard School
of Public Health, 665 Huntington Ave., Boston, MA 02115, USA. E-mail: lkobzik@hsph.harvard.edu
(ROFA). Dye et al. (1997) reported that ROFA causes epithelial injury of air-
ways and lungs. The soluble metal fractions of the ROFA play an important
role in the mechanism of this phenomenon (Broeckaert et al., 1997, 1999;
Dreher et al., 1997). ROFA in vitro enhances inflammatory responses (Gold-
smith et al., 1998; Stringer & Kobzik, 1998). In addition, ROFA can potenti-
ate inflammatory processes in vivo in animal models. Prior work from this
laboratory demonstrated that exposure to aerosolized ROFA leachate in-
creases airway hyperresponsiveness in a murine asthma models of very
young mice (Hamada et al., 1999). In these experiments, a control group
exposed to only ROFA leachate (without allergen challenge) did not show
AHR upon methacholine challenge. In contrast, Gavett et al. (1997) reported
that ROFA itself could cause AHR in normal rats. The present study sought
to investigate the effect of aerosolized exposure to ROFA leachate on airway
physiology and pulmonary inflammation in normal mice of varying ages.
MATERIALS AND METHODS
Animals
Seven- to 8-wk-old female BALB/
c
mice weighing approximately 20 g
were obtained commercially from Harlan Sprague Dawley (Indianapolis, IN)
and fed a commercial pelletted mouse feed and given water ad libitum. The
mice were housed in an animal facility that was maintained at 22–24°C
with a 12-h dark/light cycle. Two- and 3-wk-old mice weighing approxi-
mately 8–12 g were also purchased with their mother mice and housed as
described (Hamada et al., 1999). Neurokinin receptor 1 knockout mice (8–
10 wk-old, weighing 25 g, BALB/
c
background) were generously provided
by Dr. Norma Gerard (Lu et al., 1997).
Aerosol Exposure to Residual Oil Fly Ash Leachate
A single sample (1 kg) of residual oil fly ash (ROFA), obtained from the
precipitator unit of a local power plant, was generously provided by Dr. John
Godleski (Harvard School of Public Health, Boston). ROFA was suspended
(100 mg/ml in phosphate-buffered saline (PBS), pH 7.4) and sonicated for 10
min. After sitting for 30 min at room temperature, the ROFA suspension was
incubated at 37°C with rotation for 4 h, and then centrifuged at 3000
×
g for
10 min. The supernatant (leacheate) was removed and diluted (10, 50, or 100
mg/ml in PBS) for aerosol exposure. Mice were exposed to a nebulized
aerosol of ROFA-s leachate (ROFA-s) for 30 min within individual compart-
ments of a mouse “pie” chamber (Braintree Scientific, Braintree, MA) using a
Pari IS2 nebulizer (Sun Medical Supply, Kansas City, KS) connected to air
compressor (PulmoAID, DeVilbiss, Somerset, PA) (Rudmann et al., 2000).
Control mice were exposed to PBS alone.
To investigate the time course of both physiological and pathologic
responses, analyses were performed at 6, 12, 24, 48, 72, and 120 h after
exposure to 50 mg/ml of ROFA-s. To examine the dose-response profile for
1352 K. HAMADA ET AL.
ROFA-s, effects of 10, 50, and 100 mg/ml ROFA-s and PBS were compared
using analysis at a time point showing maximal AHR, 48 h after exposure.
Although ROFA was suspended and diluted in PBS (pH 7.4), the final
ROFA-s (50 mg/ml) solution was acidic (pH 5.5).
To evaluate the effect of pH, mice were also exposed to neutralized (pH
7.2 adjusted with NaOH) ROFA-s and acidic (pH 5.5 adjusted with HCl)
PBS. As described, ROFA-s was a supernatant of centrifuged suspension liq-
uid after sonication and incubation; however, to exclude the possible effect
of uncentrifuged or residual fine particles, filtered (0.2 µm) ROFA-s were
also examined.
To study the effects of antioxidant dimethylthiourea (DMTU), mice were
injected intraperitoneally with different doses of DMTU (5, 10, 50, or 100
mg/kg) 30 min before the exposure to ROFA-s (50 mg/ml). Some mice were
treated with DMTU (50 mg/kg) at 12 or 24 h after exposure. DMTU and all
other reagents, unless otherwise specified, were obtained from Sigma Chem-
ical Co. (St. Louis, MO).
Elemental analysis was performed upon ROFA-s using inductively cou-
pled plasma/mass spectrometry (ICP/MS) as previously described (Imrich et
al., 2000). The effects of each individual major metal components of ROFA-s
were evaluated. The same concentrations of nickel sulfate (NiSO
4
, Ni), vana-
dium sulfate (VSO
4
, V), zinc sulfate (ZnSO
4
, Zn), cupric sulfate (CuSO
4
, Cu),
manganese sulfate (MnSO
4
, Mn), and cobalt chloride (CoCl
2
) as in ROFA-s
were dissolved in PBS and adjusted to pH 5.5. Mice were exposed to aero-
sols of each individual metal solution and the reconstituted mixture of all
metal solutions for 30 min and then tested physiologically and pathologi-
cally at 48 h after the exposure.
To study the role of neurokinins in the physiological effects of aerosol-
ized exposure to ROFA-s, neurokinin receptor knockout mice were exposed
to ROFA-s (50 mg/ml) and evaluated as described next. In addition, normal
BALB/
c
mice exposed to ROFA-s were treated with neurokinin 1 receptor
antagonist (CP-099,994, 10 mg/kg) by intraperitoneal injection at 30 min
before the exposure (Fahy et al., 1995; Nsa Allogho et al., 1997).
To evaluate maturation effects on AHR caused by ROFA-s exposure, 2-
and 3-wk-old young mice were also exposed to ROFA-s (50 mg/ml) and
evaluated, similarly to their adult counterparts already described.
Physiologic Analysis
Airway responsiveness of mice to increasing concentrations of aerosol-
ized methacholine (Mch) was measured using whole-body plethysmogra-
phy (Buxco, Sharon, CT), as previously reported (Goldsmith et al., 1999;
Hamada et al., 1999). Briefly, each mouse was placed in a chamber and
continuous measurements of box pressure/time wave were calculated via a
connected transducer and associated computer data acquisition system. The
main indicator of airflow obstruction, enhanced pause (Penh), which shows
strong correlation with the airway resistance examined by standard evalua-
ROFA AND AIRWAY RESPONSIVENESS IN MICE 1353
tion methods, was calculated from the box waveform (Hamelman et al.,
1997). After measurement of baseline Penh, aerosolized PBS or Mch in in-
creasing concentrations (3, 6, 12, 25, and 50 mg/ml) were nebulized through
an inlet of the chamber for 1 min, and readings (Penh measurements) were
taken for 9 min after each dose. Penh values for the first 5 min after each
nebulization were averaged and used to compare results across treatment
groups and individual mice.
Pathologic Analysis
After physiologic testing, mice were euthanized with sodium pentobar-
bital (Veterinary Laboratories, Lenexa, KS). The chest wall was opened and
the animals were exsanguinated by cardiac puncture. Serum was prepared
and stored at –20°C. The trachea was cannulated, and bronchoalveolar
lavage (BAL) was performed 5 times with 0.7 ml (empirically adjusted to
0.3 ml for 2-wk-old mice to achieve similar inflation of lungs) of sterile PBS
instilled and harvested gently. Lavage fluid (recovery volume was about
80% of instilled) was collected, centrifuged at 300
×
g for 10 min, and the
cell pellet was resuspended in 0.5 ml PBS. Total cell yield was quantified
with a hemocytometer. BAL differential cell counts were performed on cy-
tocentrifuge slides prepared by centrifugation of samples at 800 rpm for 5
min (Cytospin 2, Shandon, Pittsburgh, PA). These slides were fixed in 95%
methanol and stained with Diff-Quik (VWR, Boston), a modified Wright–
Giemsa stain, and in total 200 cells were counted for each sample by mi-
croscopy. Macrophages, lymphocytes, neutrophils, and eosinophils were
enumerated. in a subset of experiments the lungs from 2-wk-old and adult
mice were removed after euthanasia at 12 h after ROFA-s (50 mg/ml) or
PBS exposure and stored at –70°C. The catalase activity within these tissue
samples was assayed as described by Gonzalez-Flecha et al. (1993).
Statistical Analysis
Data are summarized as means ± standard error. Differences among
groups were evaluated using analysis of variance (ANOVA) with correction
for multiple groups using Statview software (Abacus Concepts, Berkeley,
CA). Statistical significance was accepted when
p
< .05.
RESULTS
Response to Aerosolized ROFA-s
Initial experiments measured the airway responsiveness of normal adult
mice at different time points after an exposure to 50 mg/ml ROFA-s. This
concentration was previously found to amplify airway hyperresponsiveness
(AHR) in “asthmatic” mice (Hamada et al., 1999). Figure 1 shows the time
course of increased Penh in response to methacholine challenge (AHR) in
response to ROFA-s. For clarity, only the data for responses to methacholine
at 6 and 12 mg/ml are shown; proportionally similar results were seen at
higher methacholine concentrations (25, 50 mg/ml; data not shown). Mild
1354 K. HAMADA ET AL.
AHR was seen at 12 h after exposure. The maximum response was seen at
48 h after the exposure (Figure 1) to ROFA-s aerosols. Bronchoalveolar lavage
(BAL) analysis revealed increased neutrophils within the lung and decreased
macrophage recovery (Table 1). In contrast to the physiological response,
neutrophil recruitment was maximal at an earlier time point (12 h).
ROFA AND AIRWAY RESPONSIVENESS IN MICE 1355
FIGURE 1. Results of physiological evaluation of airway function in mice exposed to ROFA-s aerosols.
(A) Penh values obtained in response to increasing concentrations of methacholine. Cohorts of mice (n
³
6/group) were evaluated at varying time points after exposures. For clarity, (B) shows Penh values
obtained after ROFA-s in response to two of the methacholine concentrations used. Asterisk indicates sig-
nificant at p < .05 versus preexposure and 120-h time points; double asterisk, p < .05 versus preexposure
and 12-, 72-, and 120-h time points; #, p < .05 versus all other time points; †, p < .05 versus preexpo-
sure; n
³
6/group.
Results of a dose-response analysis using ROFA-s prepared from 10, 50,
and 100 mg/ml ROFA) are shown in Figure 2. The two higher concentrations
produced a similar increase in Penh in response to methacholine challenge,
consistent with airway hyperresponsiveness (AHR). However, a lower con-
centration of ROFA-s (10 mg/ml) did not induce AHR in exposed mice (Fig-
ure 1). Unless otherwise noted, subsequent experiments analyzed effects of
aerosol exposures to 50 mg/ml of ROFA-s at the 48-h time point.
ROFA Component Analysis
Table 2 shows the concentration within ROFA-s of a panel of potentially
toxic metal components of ROFA. To test the effect, if any, of aerosol expo-
1356 K. HAMADA ET AL.
TABLE 1. Summary of Cell Counts and Differentials Obtained After BAL Analysis of 8-wk-old Mice
Exposed for 30 min to Aerosolized ROFA-s (50 mg/ml)
Cell type ROFA-s, 6 h ROFA-s, 12 h ROFA-s, 24 h ROFA-s, 48 h PBS, 48 h
Total cells (
×
10
5
/ml) 0.39 ± 0.02 0.44 ± 0.03 0.40 ± 0.03 0.42 ± 0.03 0.36 ± 0.02
Macrophages (%) 97.39 ± 0.39 91.94 ± 1.25
a
96.94 ± 0.53 97.28 ± 0.82 99.20 ± 0.34
Lymphocytes (%) 1.22 ± 0.19 0.89 ± 0.16 1.00 ± 0.27 0.64 ± 0.16 0.30 ± 0.12
Neutrophils (%) 1.39 ± 0.33 7.10 ± 1.23
a
1.97 ± 0.37 2.08 ± 0.84 0.50 ± 0.22
Eosinophils (%) 0.06 ± 0.04 0.08 ± 0.06 0.08 ± 0.05 ND ND
Note. The data were obtained at the indicated time points after the aerosol exposures; n
³
18/group.
ND, not detected.
a
Significant at p < .05 verus all others.
FIGURE 2. Results of physiological evaluation of airway function in mice exposed to aerosols of ROFA-s
prepared from increasing concentration of ROFA (see methods). Asterisk indicates significant at p < .05
versus 0 and 10 mg/ml, n
³
4/group.
sure to these individual elements, solutions of the metals were prepared at
concentrations identical to those found within ROFA-s (Table 2). Figure 3
summarizes effects on airway responsiveness in mice exposed to the each of
six metal solutions. While no marked effects were seen with the individual
elements, a reconstituted mixture of the panel of six reproduced the AHR
seen with ROFA-s (Figure 3). After exposure to individual metals, only mice
exposed to Ni showed a small increase of neutrophils (2.8 ± 1.0%). Mice
exposed to the mixture of metals showed a low level of BAL neutrophils
(1.3 ± 0.6%), similar to that seen after ROFA-s exposure (Table 1).
To determine the role, if any, of pH changes in the effect of ROFA-s on
airway responsiveness, airway responses after exposure to neutralized (pH
ROFA AND AIRWAY RESPONSIVENESS IN MICE 1357
TABLE 2. Metal Content Data Obtained by ICP/MS
Analysis of ROFA-s (Prepared as 50 mg/ml Solution)
Metal content
of ROFA-s
Metal (µg/ml)
Ni 341.2
V 323.4
Zn 232.3
Co 1 8.33
Mn 15.8
Cu 6.71
Cd 0.61
Fe 0.0
FIGURE 3. Results of physiological evaluation of airway function in mice exposed to aerosolized solu-
tions of single metal components of ROFA, a mixture of all the single components, ROFA-s, or PBS. The
solutions were prepared at the concentrations found in ROFA-s. Data presented was obtained in
response to 25 mg/ml methacholine. Asterisk indicates significant at p < .05 versus all others, n
³
4/
group.
7.2) ROFA-s and filtered ROFA-s were compared to those seen after stan-
dard ROFA-s (50 mg/ml, pH 5.5). Figure 4 shows that a similar degree of
AHR was seen in animals exposed to either acidic or neutralized ROFA-s.
Aerosol exposure to acidic PBS (pH adjusted; pH 5.5) exposure did not per
se cause AHR (Figure 4).
Effects of Antioxidant Treatment
To investigate the role of oxidant stress in ROFA-s effects, the antioxi-
dant dimethythiourea (DMTU) was administered (ip) before exposure to
ROFA-s (50 mg/ml). Figure 5 shows that DMTU pretreatment diminished
AHR in a dose-dependent manner. For clarity, only the results of physiologic
testing using methacholine at 6 and 12 mg/ml are shown. Similar results
were seen with higher concentrations of methacholine (25 or 50 mg/ml,
data not shown). The inhibition was statistically significant at 50 and 100
mg/kg DMTU. As noted earlier, neutrophil numbers in BAL fluid at 48 h
after the exposure to ROFA-s are lower than those seen at 12 h after expo-
sure. At these low levels, it is difficult to detect or interpret downward
decreases. Nevertheless, after DMTU treatment, the level of neutrophils in
BAL samples showed a trend consistent with the diminished AHR seen in
these animals
[
percent neutrophils in BAL fluid: 1.8 ± 0.4 in PBS-treated
group vs. 1.9 ± 0.5, 0.8 ± 0.2, 0.6 ± 0.2, and 1.5 ± 0.2 in DMTU (5, 10,
50, and 100 mg/kg, respectively)-treated groups (mean ± SE)
]
.
1358 K. HAMADA ET AL.
FIGURE 4. Evaluation of the effect of pH on the increased Penh seen with ROFA-s. Physiological eval-
uation of airway function was performed 48 h after exposure of mice to the aerosolized solutions listed
on the x axis. Data presented were obtained in response to 50 mg/ml methacholine. Asterisk indicates
significant at p < .05 versus PBS-exposed groups; n
³
6/group.
Development and Susceptibility to ROFA Effects
To investigate the effect of maturation on ROFA-s mediated AHR, the
effect of ROFA-s aerosols (50 mg/ml) on younger mice (2 and 3 wk old) was
compared to that seen in the 8-wk-old adult mice. Figure 6 shows that
ROFA-s exposures that produce AHR in 8 week old mice have no marked
effect on 2-wk-old mice. The ROFA-s aerosols begin to show an effect on
AHR in 3-wk-old mice. No change in neutrophils was noted in the 2- or 3-
wk-old mice, while the 8-wk-old mice did show a small number of neu-
trophils (see Table 1). Analysis of BAL cellularity in 2-wk-old mice at 6 and
12 h after ROFA-s revealed an initial inflammatory response at 6 h with
rapid return to normal levels of neutrophils by 12 h (BAL PMNs % ± SE, 6
and 12 h after ROFA-s respectively: 23 ± 0.6; 0.5 ± .01;
n
= 5/group).
The potential developmental differences between 2- and 8-wk-old mice
that might affect responses to inhaled irritants include (1) differences in air-
way innervation (Haxhiu-Poskurica et al., 1991) and its regulation of airway
caliber via neurogenic inflammation, and (2) differences in antioxidant
enzyme levels that might modulate ROFA-mediated oxidant stress. To test
the former possibility, mice with a genetic deletion in neurokinin receptor 1
were exposed to ROFA-s (50 mg/ml) prior to evaluation of airway respon-
ROFA AND AIRWAY RESPONSIVENESS IN MICE 1359
FIGURE 5. Evaluation of the effect of varying doses of ip dimethylthiourea on the increased Penh seen
with ROFA-s. Physiological evaluation of airway function was performed 48 h after exposure of mice to
ROFA-s (50 mg/ml). For clarity, data are presented for two of the methacholine concentrations used in
these evaluations. Asterisk indicates significant at p < .05 versus PBS treatment, n = 8/group, except for
100 mg/ml where n = 4/group.
1360
FIGURE 6. Comparison of the effect of ROFA-s on airway responsiveness (Penh) in mice of varying age (2, 3, and 8 wk old). Data presented was
obtained 48 h after exposure to a 50-mg/ml solution. Asterisk indicates significant at p < .05 versus PBS, n
³
6/group.
siveness. The airway responsiveness of these mice was compared to wild-
type (BALB/
c
) mice exposed to ROFA-s or PBS. Normal mice were also pre-
treated with neurokinin 1 receptor antagonist (CP-099,994) prior to ROFA-s
exposure. Figure 7 shows that ROFA-s produced a similar level of AHR in
wild-type, NK1 receptor knockout mice, or mice treated with the NK1
receptor antagonist. To evaluate antioxidant status, catalase activity was
measured 12 h after exposure to ROFA-s or PBS in lung tissues from young
(2-wk-old) and adult (8-wk-old) mice (Figure 8). These assays showed simi-
lar levels of catalase activity after both treatments in the two age groups.
DISCUSSION
This study used aerosol exposures to a leachate of ROFA to investigate
mechanisms of respiratory effects of particulate air pollution. The major
findings include: (1) striking developmental differences in susceptibility to
ROFA-s; (2) a discordance between the time course of pulmonary inflam-
mation and AHR; and (3) a requirement for interaction between component
metals to induce AHR. Abrogation of ROFA-s effects by sytemic treatment
with the antioxidant DMTU supports a role for oxidant-mediated pathways
in the generation of AHR by ROFA.
ROFA AND AIRWAY RESPONSIVENESS IN MICE 1361
FIGURE 7. Evaluation of the effect of ROFA-s (50 mg/ml) on mice with a genetic deletion of neurokinin
1 receptor (–/–) or wild-type (+/+) mice treated ip with a neurokinin 1 antagonist. Data presented was
obtained in response to 50 mg/ml methacholine. Asterisk indicates significant at p < .05 versus all other
groups; n
³
4/group.
Particulate air pollution is associated with cardiopulmonary diseases
including bronchial asthma (Dockery et al., 1993; Goldsmith & Kobzik,
1999). Previous studies have found that ROFA causes airway or pulmonary
epithelial injury, and the mechanism(s) of this injury has been considered to
be associated with the harmful effect of active oxygen species from transi-
tion metal components of ROFA (Broeckaert et al., 1999; Dreher et al.,
1997; Dye et al., 1997). The soluble fraction of ROFA acts to increase AHR
in a mouse model of asthma (Hamada et al., 1999). It has been reported to
produce airway hyperresponsiveness in normal adult rats and lung inflam-
mation in mice (Broeckaert et al., 1999; Gavett et al., 1997), but this labo-
ratory observed an absence of AHR in very young mice exposed to ROFA
leachate aerosols (Hamada et al., 1999). Hence this study sought to more
rigorously characterize the effects of aerosol exposure in young and adult
mice.
In adult mice, aerosolized exposure to ROFA leachate increased airway
responsiveness as described in the text. The time courses of the physiologi-
cal and pathological (inflammatory cell recruitment) changes were note-
worthy. Although mild and transient recruitment of neutrophils was seen at
12 h after the exposure as the maximum in the lavage fluid, a physiological
response was not prominent at this point. Rather, AHR became more pro-
nounced and maximal in the 24–48 h after the exposure, after the decline
of inflammatory cell numbers in the BAL analyses. Data indicate that oxy-
gen radical species from aerosolized ROFA leachate play a pivotal role
since pretreatment of antioxidant dimethylthiourea (DMTU) abrogated
these responses. The antioxidant
N
-acetylcysteine also abrogated ROFA-
mediated inflammatory responses in vitro (Stringer & Kobzik, 1998).
Whether generation of these oxidants requires or is derived from the tran-
1362 K. HAMADA ET AL.
FIGURE 8. Measurement of catalase activity in 2- or 8-wk-old mice exposed to control PBS or ROFA-s
aerosol shows no difference in this antioxidant among these groups; n = 3/group.
sient neutrophilia observed remains undetermined. We have previously ob-
served robust neutrophil influx into the lungs (induced by lipopolysaccha-
ride
[
LPS
]
aerosols) without development of AHR (Goldsmith et al., 1999).
There have been reports that soluble metal components of the ROFA
leachate produce varying airway and lung injury (Broeckaert et al., 1999;
Dreher et al., 1997; Kodavanti et al., 1998). The effects of vanadium (V),
nickel (Ni), and iron (Fe) were considered to be important. The potential
role of iron in oxidant injury is widely recognized. However, our ROFA
leachate did not include iron (Table 1). Although aerosolized exposure to
single soluble metal solutions (even acidic) failed to cause AHR, mixed
metal solution could reproduce the same response produced by ROFA
leachate. This indicates that interactive effects of metal constituents are
required for the responses observed in this study. it is possible that these
interactive effects derive from synergistic, antagonistic, or additive effects
among the five metals studied.
Neurogenic inflammation in both human and experimental animal
models plays an important role on airway hyperresponsiveness, including
asthma (Barnes, 1996). A physiological response without detectable patho-
logical changes, especially cellular inflammation, may reflect neurogenic
inflammation. In these studies, aerosolized exposure to ROFA leachate in-
duce relatively transient recruitment of neutrophils, with only a few inflam-
matory cells remaining at the time when the mice showed maximal AHR to
methacholine challenge. Neurokinin receptor 1 knockout mice were used
to investigate one pathway by which neurokinin, a key factor in neurogenic
inflammation (Baluk, 1997; Barnes, 1996), might mediate the observed
AHR. However, the neuokinin receptor 1 knockout mice revealed the same
physiological response to ROFA-s as their wild-type counterparts. More-
over, treatment with a neurokinin antagonist (Fahy et al., 1995; Nsa Allogho
et al., 1997) also did not decrease the AHR response. Hence the data do
not support a mechanistic role for neurokinin receptor 1. The potential role
of other receptor subtypes or other mediators, such as endotoxin (Ning et
al., 2000), was not directly addressed in these experiments. However,
ROFA-s had undetectable levels of endotoxin when tested by
Limulus
assay
(data not shown), and LPS aerosols sufficient to cause neutrophilia did not
cause AHR in other experiments (Goldsmith et al., 1999). Other limitations
of this study include the differences in composition between ROFA and
urban air particles and the more general concern that experimental studies
(including this one) use toxicologic doses in their attempts to identify mech-
anistic pathways.
It was noteworthy that there was a different physiological and inflam-
matory response to aerosolized exposure to ROFA leachate seen in mice of
varying age. In adult mice, ROFA leachate induced AHR. In contrast, AHR
was not seen in 2-wk-old young mice, despite a robust and earlier neu-
trophilia. This laboratory has reported that 2-wk-old mice can show AHR
after inhalation of aerosolized allergen (Hamada et al., 1999). Hence, the
ROFA AND AIRWAY RESPONSIVENESS IN MICE 1363
absence of AHR to ROFA-s in the 2-wk-old mice in this study does not re-
flect a global inability to manifest or detect AHR. Since oxygen radical
species are operative in the mechanisms of AHR by ROFA-s, the possibility
that young mice might have relatively high activity of oxygen radical scav-
engers such as catalase in comparison with adult mice was investigated.
However, catalase activity in the lung tissue of both young and adult was
the same. Other potential antioxidants (e.g., superoxide dismutase, ascor-
bic acid, or glutathione) remain to be investigated. Hence, the mecha-
nism(s) of the maturation-based differences in susceptibility to ROFA-s re-
main undetermined. The relative resistance of young mice to ROFA effects
contrasts with other experimental and epidemiologic (Bhalla, 1999; Gold-
smith & Kobzik, 1999; Shore et al., 2001) observations suggesting greater,
not diminished, susceptibility of young subjects to inhaled pollutants. Future
studies of the maturational transition from resistant to susceptible pheno-
type may provide insights into mechanisms of particle effects on the lung.
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