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Journal of Toxicology and Environmental Health, Part A
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Oxidative Injury in The Lungs of Neonatal Rats Following Short-Term
Exposure to Ultrafine Iron and Soot Particles
Cai-Yun Zhong a; Ya-Mei Zhou a; Kevin R. Smith a; Ian M. Kennedy b; Chao-Yin Chen c; Ann E. Aust
d;Kent E. Pinkerton a
a Center for Health and the Environment, University of California, Davis, California, USA b
Department of Mechanical and Aeronautical Engineering, University of California, Davis, California,
USA c Department of Medical Pharmacology, University of California, Davis, California, USA d
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah, USA
Online publication date: 07 April 2010
To cite this Article Zhong, Cai-Yun , Zhou, Ya-Mei , Smith, Kevin R. , Kennedy, Ian M. , Chen, Chao-Yin , Aust, Ann E.
andPinkerton, Kent E.(2010) 'Oxidative Injury in The Lungs of Neonatal Rats Following Short-Term Exposure to
Ultrafine Iron and Soot Particles', Journal of Toxicology and Environmental Health, Part A, 73: 12, 837 — 847
To link to this Article: DOI: 10.1080/15287391003689366
URL: http://dx.doi.org/10.1080/15287391003689366
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837
Journal of Toxicology and Environmental Health, Part A, 73:837–847, 2010
Copyright © Taylor & Francis Group, LLC
ISSN: 1528-7394 print / 1087-2620 online
DOI: 10.1080/15287391003689366
OXIDATIVE INJURY IN THE LUNGS OF NEONATAL RATS FOLLOWING
SHORT-TERM EXPOSURE TO ULTRAFINE IRON AND SOOT PARTICLES
Cai-Yun Zhong1, Ya-Mei Zhou1, Kevin R. Smith1, Ian M. Kennedy2, Chao-Yin Chen3,
Ann E. Aust4, Kent E. Pinkerton1
1Center for Health and the Environment, University of California, Davis, California
2Department of Mechanical and Aeronautical Engineering, University of California,
Davis, California
3Department of Medical Pharmacology, University of California, Davis, California
4Department of Chemistry and Biochemistry, Utah State University, Logan, Utah, USA
Greater risk of adverse effects from particulate matter (PM) has been noted in susceptible
subpopulations, such as children. However, the physicochemical components responsible
for these biological effects are not understood. As critical constituents of PM, transition met-
als were postulated to be involved in a number of pathological processes of the respiratory
system through free radical-medicated damage. The purpose of this study was to examine
whether oxidative injury in the lungs of neonatal rats could be induced by repeated short-
term exposure to iron (Fe) and soot particles. Sprague Dawley rats 10 d of age were exposed
by inhalation to two different concentrations of ultrafine iron particles (30 or 100 mg/m3) in
combination with soot particles adjusted to maintain a total particle concentration of 250 mg/
m3. Exposure at 10 d and again at 23 d of age was for 6 h/d for 3 d. Oxidative stress was
observed at both Fe concentrations in the form of significant elevations in glutathione disul-
fide (GSSG) and GSSG/glutathione (GSH) ratio and a reduction in ferric/reducing antioxidant
power in bronchoalveolar lavage. A significant decrease in cell viability associated with sig-
nificant increases in lactate dehydrogenase (LDH) activity, interleukin-1-beta (IL-1b), and fer-
ritin expression was noted following exposure to particles containing the highest Fe
concentration. Iron from these particles was shown to be bioavailable in an in vitro assay
using the physiologically relevant chelator, citrate. Data indicate that combined Fe and soot
particle exposure induces oxidative injury, cytotoxicity and pro-inflammatory responses in
the lungs of neonatal rats.
As critical constituents of particulate matter
(PM), transition metals were postulated to be
involved in a number of pathological and
physiological processes of the respiratory sys-
tem through free radical-medicated damage
(Valavanidis et al., 2005; Risom et al., 2005;
Sorensen et al., 2005; Zhou et al., 2003).
Humans are commonly exposed to iron (Fe)
and soot particles from a variety of emission
sources of PM. Iron was found to be the preva-
lent catalytic metal in all size ranges of the
ambient PM present in Los Angeles (Hughes
et al., 1998), and the highest concentration of
Fe in ultrafine particles was found to be in the
size range of 0.056–0.1 μm in 7 Southern
California cities (Cass et al., 2000).
Received 30 October 2009; accepted 1 February 2010.
Cai-Yun Zhong and Ya-Mei Zhou contributed equally to this work.
The authors appreciate Dr. Suzette Smiley-Jewell for her editorial assistance in the preparation of this article. This work was supported
by grants from the Health Effects Institute, U.S. Environmental Protection Agency (R82915 and R832414), National Institutes of Health
(5R01ES012957), and National Institute for Occupational Safety and Health (OH07550). The preliminary observations for portions of
this research were reported in a limited-distribution final report to the Health Effects Institute (Pinkerton et al., 2008).
Address correspondence to Dr. Kent E. Pinkerton, Center for Health and the Environment, Old Davis Road, University of California,
Davis, CA 95616, USA. E-mail: kepinkerton@ucdavis.edu
Downloaded By: [University of California, Davis] At: 16:41 9 April 2010
838 C.-Y. ZHONG ET AL.
Potential sources for transition metals in
the atmosphere are multifarious. Larger parti-
cles (i.e., >2.5 μm) containing metals may be
derived from crustal dusts, while ultrafine parti-
cles are more likely to originate from high-
temperature combustion sources. Iron is a
well-known soot suppressant that might be
emitted into the atmosphere in the form of
ultrafine particles. For example, ferrocene has
been commonly used as fuel additive for soot
suppression. Iron oxide generated from the
decomposition and oxidation of ferrocene is
supersaturated and rapidly nucleates into
numerous nano-sized particles that serve as
sites for carbon deposition. Concentric rings of
carbon laid down on Fe particles form an Fe
and soot particle matrix (Boncyzk, 1991; Ritrievi
et al., 1987). Soot is primarily composed of
elemental carbon and also contains limited
amounts of oxygen, nitrogen, hydrogen, and
polyaromatic hydrocarbons (PAH). The emis-
sion sources of soot include diesel engines, fossil
fuels, and wood smoke. Soot is thermodynam-
ically involved in the reduction of Fe oxide in
the flame at high temperatures (Boncyzk, 1991;
Ritrievi et al., 1987; Zhang & Megaridis, 1996)
and is typically considered to exert little effect
when inhaled alone (Jakab, 1992, 1993; Jakab &
Hemenway, 1994). Since ultrafine particles of
soot have large surface areas and mass ratio,
soot might also act as a carrier for copollutants,
such as transition metals, and might transport
co-pollutants into the respiratory tract. Forma-
tion of these complex particles may influence
deposition and clearance from the lungs, thus,
changing biological potential (Lindenschmidt &
Witschi, 1990; Oberdorster, 2001; Schlesinger,
1995; Sun et al., 1989).
Although the major focus of studies relating
ambient PM and adverse health effects has
been in adults, a number of epidemiological
studies showed that air pollution is also associ-
ated with respiratory morbidity, mortality, and
decrements in pulmonary function and growth
in children (Delfino et al., 2008; Tecer et al.,
2008; Salvi, 2007; Gauderman et al., 2002;
Calderon-Garciduenas et al., 2000; Conceicao
et al., 2001; Horak et al., 2002; Jedrychowski
et al., 1999; Zhang et al., 2002). The response
of a child to particles may be entirely different
from that of an adult, based on differences in
ventilatory rates or maturation of metabolic,
immune, neural, and anatomical systems (Foos
et al., 2008). Furthermore, cellular differentia-
tion, proliferation, physiological function, and
xenobiotic-metabolizing enzymes within the
respiratory system rapidly change during post-
natal growth. Exposure of the respiratory tract
to environmental toxicants during this time has
the potential to significantly affect the matura-
tion, growth, and function of critical elements
compromising this system (Pinkerton & Joad,
2000). However, little is known regarding the
susceptibility and environmental characteristics
that may place children at greater risk from
exposure to PM. A study that is designed to
address the association between adverse effects
and specific chemical composition could pro-
vide meaningful information to better under-
stand the health effects of PM on children
during development. Therefore, the purpose of
this study was to examine the responses of
inhaled Fe and soot particles generated by a dif-
fusion flame system in the respiratory system of
rapidly growing neonatal rats. To do this, Spra-
gue-Dawley rats 10 d and 23 d of age were
exposed by inhalation to 2 different levels of
ultrafine Fe particles (30 μg/m3 and 100 μg/m3) in
combination with soot particles adjusted to main-
tain a total particle concentration of 250 μg/m3.
MATERIALS AND METHODS
Chemicals
Acetylene and ethylene were purchased
from Airgas (Sacramento, CA). Iron pentacarbo-
nyl, reduced glutathione, glutathione disulfide
(GSSG), glutathione reductase, 5,5′-dithio-
bis(2-nitrobenzoic) acid (DTNB), NADPH, 2-
vinylpyridine, ferrous sulfate, ferric chloride,
tripyridytriazine (TPTZ), citrate, and ferrozine
were purchased from Sigma-Aldrich Chemical
Co. (St. Louis, MO).
Iron and Soot Particle Generation System
A diffusion flame system was used to gener-
ate an aerosol of soot and Fe oxide as described
Downloaded By: [University of California, Davis] At: 16:41 9 April 2010
NEONATAL LUNG INJURY BY IRON AND SOOT PARTICLES 839
previously (Yang et al., 2001). Briefly, the fuel
was a mixture of acetylene and ethylene. Con-
centrations of Fe oxide and soot particles in the
post flame gases were controlled indepen-
dently. Iron was introduced by passing ethylene
over liquid Fe pentacarbonyl. The aerosol
emission from the flame was diluted by sec-
ondary air to control the actual particle con-
centration to a level that could be maintained
for the duration of short-term animal exposure
studies. The system was operated to generate
total constant concentrations of aerosol, while
Fe loading varied from 0 to 100 μg/m3 in the
diluted post-flame gases. The two different
concentrations of Fe used in this study were
created by adjusting the flow rate of Fe penta-
carbonyl vapors mixed with acetylene and eth-
ylene fuels. Particles were collected on carbon
grids. The individual size of Fe and soot parti-
cles in the exposure chamber was monitored
using transmission electron microscopy, while
a differential mobility analyzer was used to
measure the size distribution of the aerosol. X-ray
fluorescence was used to measure the mass
concentration of Fe particles (μg/m3). The soot
mass concentration was found by weighing
25-mm Teflon-coated filters (Teflo, Pall, East
Hills, NY) on a microbalance before and after
sample collection. The conditions used in the
diffusion flame system were reproducible in
generating a consistent concentration of Fe
throughout the daily 6-h period of exposure.
Iron Mobilization
The amount of Fe mobilized from Fe and
soot particles was determined as previously
described (Smith et al. 1998), with the follow-
ing modifications. Particles, on filters, were sus-
pended in 50 mM NaCl (1 mg/ml) at a pH of
7.5. The samples were mixed by vortexing for
30 s. Citrate was added to obtain a final con-
centration of 1 mM, and all samples were
placed on an orbital shaker for 24 h. The pH
was readjusted to 7.5 at regular time intervals
throughout the incubation period to prevent
alteration in the rates of Fe mobilization. After
24 h, a 1-ml sample was withdrawn and centri-
fuged at 13,300 × g for 8 min to remove the
particles. The amount of Fe mobilized as the
citrate:Fe complex in the supernatant was
determined, as originally described by Brumby
and Massey (1967) for non-heme Fe determi-
nation, except that ferrozine (0.4%, w/v) was
used instead of 1,10-phenanthroline. This assay
uses ferrozine to quantify both Fe(II) and Fe(III)
as a result of addition of the reductant ascorbate.
The concentration of Fe mobilized by citrate is
reported as nanomoles of Fe per milligram of
particles.
Animal Exposure
Sprague-Dawley rats at 10 d of postnatal
age were exposed via whole-body inhalation
to two different concentrations of Fe and soot
particles 6 hr/d for 3 d. The average total particle
concentration was maintained at 250 μg/m3.
Iron concentrations were targeted at 30 and
100 μg/m3, respectively. Control animals were
exposed to filtered air only. Due to the capacity
of the particle exposure, eight animals were
included in each group with half male and half
female rats, and two sets of exposure for each
group were performed. Preweanling neonatal
rats were exposed to particles away from the
dam. Exposure to particles was repeated with
the same group of rats at 23 d postnatal age
under identical conditions done at 10 d post-
natal age. Our rationale for using this dosing
scheme based on postnatal age was to subject
the lungs of neonatal animals to particle inhala-
tion during critical periods of lung growth
where robust cell proliferation occurs in the
process of forming new alveoli (postnatal day
10–12) and again during a period of significant
alveolar airspace expansion (postnatal day 23–25).
Following exposure, samples of bronchoalveolar
lavage (BAL) and lung tissues were collected for
analysis. At the termination day, the lungs of
rats were feasible for conducting BAL proce-
dure. Different sets of exposure were done to
allow BAL and lung tissue analyses separately.
Bronchoalveolar Lavage
Preparation of BAL was done following
Harrod’s protocol with some modifications
(Harrod et al., 1998). Within 2 h following the end
of particle exposure at 23 d postnatal age, rats
were anesthetized using sodium pentobarbital,
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840 C.-Y. ZHONG ET AL.
50 mg/kg body weight, ip (Nembutal Cardinal
Health Inc., Sacramento, CA). The trachea was
cannulated, and BAL was performed 4 times
sequentially with phosphate-buffered saline
(Dulbecco’s PBS, Mg2+ and Ca2+-free, pH 7.0,
GibcoBRL, Grand Island, NY), using a volume
equivalent to 28 ml/kg body weight. The 4
aliquots of BAL were centrifuged at 500 × g
for 10 min, supernatant was removed, and
cell pellets were pooled together for deter-
mination of total cell number, viability, and
cell differentials. BAL supernatant was ali-
quoted and stored at −70°C for biochemical
analysis.
BAL Fluid (BALF) Analysis for
Determination of Lung Injury
Cell pellets were resuspended in 500 μl
PBS. Total cell count and viability were deter-
mined by trypan blue exclusion with a
hemocytometer. A minimum of 1 × 105 cells
was used to prepare slides in duplicates by
cytospin of 100 μl cell suspension at 600 rpm
for 5 min. Cells were stained with Hema 3
(Fisher Scientific Company, Swedesboro, NJ)
for the determination of the proportion of
macrophages, lymphocytes, and neutrophils.
Lactate dehydrogenase (LDH) activity was
measured using a colorimetric kit (Sigma-Aldrich,
St. Louis, MO) based on the activity of LDH
released from damaged cells into the BAL
supernatant.
Glutathione
Glutathione (GSH) was measured in BAL
supernatant according to the enzymatic method
proposed by Tieze and colleagues (1969) and
modified by Anderson (1985), using DTNB-
oxidized glutathione reductase recycling assay.
Reduced GSH was oxidized by DTNB to glu-
tathione disulfide (GSSG) with stoichiometric
formation of 5-thio-2-nitrobenzoic acid (TNB).
GSSG was reduced to GSH by the action of
glutathione reductase and NADPH. The rate of
TNB formation was followed at 412 nm and
was proportional to the sum of GSH and GSSG
present. GSSG was determined after GSH was
first derivatized with 2-vinylpyridine.
Antioxidant Power
Antioxidant power was determined in BAL
supernatant by ferric reducing/antioxidant
power (FRAP) assay according to Benzie and
Strain (1999). At low pH, ferric tripyridytrizine
(Fe3+-TPTZ) complex was reduced to the fer-
rous form and was monitored by measuring
the change in absorbance at 593 nm. The change
in absorbance is directly related to the total
reducing power of the electron-donating anti-
oxidants present in the reaction mixture.
Preparation of Lung Homogenate
Twenty-four hours after the last exposure,
rats were anesthetized and the lungs were
removed immediately from the thorax, frozen
in liquid nitrogen, and subsequently homoge-
nized in ice-cold Tris-HCl buffer (25 mM
Tris, 1 mM ethylenediamine tetraacetic acid
[EDTA], 10% glycerol, 1 mM dithiothreitol
[DTT], pH 7.4) with a glass homogenizer. The
homogenate was centrifuged at 10,000 × g for
20 min at 4°C. The supernatant was aliquoted
and stored at −80°C.
Western Blot Analysis for Ferritin
Western blot analysis was used to determine
ferritin levels in the lungs. Briefly, 40 mg protein
of lung homogenate was loaded and separated
on 12% sodium dodecyl sulfate polyacryla-
mide gel electrophoresis (SDS-PAGE) followed
by transfer to a nitrocellulose polyvinylidene fluo-
ride (PDVF) membrane (Bio-Rad, Hercules, CA).
The membrane was blocked for 1 h at 25°C in
Tris-buffered saline containing 0.1% Tween 20
and 5% nonfat dry milk and probed with pri-
mary antibody against human ferritin (rabbit
anti-human ferritin polyclonal antibody, Dako,
Carpinteria, CA) at a dilution of 1:3000. Secon-
dary antibody (horseradish peroxidase-linked
goat anti-rabbit immunoglobulin [Ig] G, Santa
Cruz Biotech. Inc., Santa Cruz, CA) was added at
a dilution of 1:5000. Purified human liver ferritin
(CalBiochem, San Diego, CA) was used as a
positive control. The blots were developed by
enhanced chemiluminescence detection kit (ECL,
AmerSham Pharmacia Biotech, Inc., Piscataway,
NJ) with exposure on autoradiography film.
Downloaded By: [University of California, Davis] At: 16:41 9 April 2010
NEONATAL LUNG INJURY BY IRON AND SOOT PARTICLES 841
Immunoreactive protein bands were quantified
by image densitometry.
ELISA for Proinflammatory Cytokines
Protein levels of proinflammatory cytokines
interleukin (IL)-1b and tumor necrosis factor
(TNF)-a were measured in lung homogenates
by rat enzyme-linked immunosorbent assay
(ELISA) kits according to the manufacturer’s
instructions (R&D System, Minneapolis, MN).
A 1:2 dilution of samples into calibrator diluent
provided in the kit was used for the cytokine
determination. Quantitation of cytokines was
normalized to total protein in the sample.
Statistical Analysis
Student’s t-test was applied to all data with
Staview computer software. All values are pre-
sented as mean ± SE. Differences were consid-
ered significant at p < .05.
RESULTS
Particle Characterization
The mass concentrations of Fe particles
generated in combination with soot under 2
conditions were 30 ± 7 μg/m3and 100 ± 28 μg/m3.
Overall mass concentration of the combined
Fe and soot for all studies was 250 μg/m3. The
majority of Fe particles generated were Fe
oxide in composition with the morphologic
appearance of distinct polyhedrons as deter-
mined by transmission electron microscopy
(TEM) and electron energy loss spectroscopy
(EELS) (Figure 1). EELS analysis of particles
spectra demonstrated a ratio of Fe to oxygen
ranging from 0.5 to 0.7, suggesting the stoichi-
ometric composition of Fe2O3. The average
particle diameter of mixed Fe and soot was 72
nm with a size distribution ranging from 45 to
110 nm. Since Fe pentacarbonyl was present
in the combustion flame, other forms of Fe
may have been produced in smaller amounts
through interaction with soot.
Iron Mobilization From Particles
Incubation of Fe and soot particles in 50 mM
NaCl (pH 7.5), in the absence of a metal
chelator, did not result in mobilization of Fe.
Incubation of Fe and soot particles in the phys-
iologically relevant chelator citrate resulted in
Fe mobilization with levels of 37.7 ± 0.9 nmol
Fe/mg particles after 24 h. Incubation of blank
filters under identical conditions resulted in no
detectable mobilization of Fe.
Cytotoxicity Assessment
To assess the cytotoxicity of inhaled Fe and
soot particles, total cell number, cell viability,
cell differential, and LDH activity in BAL were
used for the determination of acute pulmonary
injury (Table 1). There were no significant dif-
ferences in total cell number or the cell differ-
ential between exposure groups. However,
exposure to 100 μg/m3 Fe in combination with
soot particles resulted in significant decrease in
cell viability and increase in LDH activity. No
marked changes were observed in animals
exposed to particles containing 30 μg/m3 Fe
combined with soot particles.
Ferritin
Intracellular ferritin levels in homogenized
lung tissues were measured using Western blot-
ting following Fe and soot particles exposure. A
significant increase in ferritin expression was
noted (2.5-fold) in the lungs of animals exposed
to particles containing 100 μg/m3 Fe (Figure 2).
No significant difference was present in rats
exposed to particles containing 30 μg/m3 Fe.
FIGURE 1. Photomicrograph showing carbon support film,
graphitic carbon, and iron oxide. Bar = 100 nm.
Downloaded By: [University of California, Davis] At: 16:41 9 April 2010
842 C.-Y. ZHONG ET AL.
Oxidative Stress
Reduced glutathione (GSH), oxidized glu-
tathione (GSSG), the ratio of GSSG/(GSH+GSSG)
or glutathione redox ratio (GRR), and ferric/
reducing antioxidant power (FRAP) were used
as markers of oxidative stress. There were no
significant differences in GSH levels between
exposure groups. However, exposure to both
30 and 100 μg/m3 Fe-containing particles
resulted in a significant elevation in GSSG and
GRR (Figure 3) as well as a significant reduction
in antioxidant power (Figure 4).
Proinflammatory Cytokines
Proinflammatory cytokines IL-1b and TNF-a
in lung tissues were measured as markers of
inflammatory response. Exposure to particles
containing 100 μg/m3 Fe was associated with a
significant increase in IL-1b (Figure 5). No sig-
nificant difference in TNF-a levels was found
between groups.
TABLE 1. Changes in BAL Fluid Parameters Following Exposure to Iron and Soot Particles in Neonatal Rats
Group LDH (units/L) Total cells/ml × 105Cell viability (%) Macrophages (%) Lymphocytes (%) Neutrophils (%)
Control 4.18 ± 1.47 8.23 ± 2.72 94 ± 2.2 98 ± 0.9 1.34 ± 0.89 0.52 ± 0.46
Exposure 1 5.09 ± 0.92 7.38 ± 2.73 92 ± 2.6 98 ± 0.6 0.99 ± 0.58 0.80 ± 0.57
Control 4.09 ± 1.83 16.93 ± 10.16 81 ± 6.1 99 ± 0.5 0.56 ± 0.44 0.26 ± 0.33
Exposure 2 6.96 ± 2.60* 12.74 ± 7.76 66 ± 17.0* 99 ± 0.2 0.41 ± 0.23 0.27 ± 0.17
Note.Exposure 1: iron = 30 μg/m3 with addition of soot to maintain total mass concentration as 250 μg/m3. Exposure 2: iron = 100 μg/m3
with addition of soot to maintain total mass concentration as 250 μg/m3. Values indicate mean ± SE of eight rats per group. Asterisk
indicates significant difference from controls at p < .05.
FIGURE 2. Western blot analysis of ferritin expression in rat lung
homogenates. (A) Exposure to particle mixture with 30 μg /m3 of
iron. (B) Exposure to particle mixture with 100 μg /m3 of iron.
Lanes 1 to 4: control animals; lanes 5 to 8: exposure animals;
lane 9: positive control with purified human liver ferritin. (C) Rela-
tive band intensity of ferritin expression by imaging densitometry.
Asterisk indicates significant difference from control (p < .05).
FIGURE 3. Glutathione redox status in bronchoalveolar lavage
fluid (BALF). GSH = reduced glutathione; GSSG = glutathione
disulfide; GRR = GSSG/(GSH+GSSG), glutathione redox ratio.
Data are presented as mean ± SE (n = 8 per group). Asterisk
indicates significant difference from control (p < .05).
Downloaded By: [University of California, Davis] At: 16:41 9 April 2010
NEONATAL LUNG INJURY BY IRON AND SOOT PARTICLES 843
DISCUSSION
Exposure to environmental toxicants during
periods of rapid lung development has the poten-
tial to significantly affect overall growth and func-
tion of the respiratory system (Pinkerton & Joad,
2000; Pinkerton et al., 2004). Children may be
especially susceptible to air pollutants since
the relative deposition of respirable particles is
increased compared with adolescents and adults
(Bennett & Zeman, 1998). The effects of ambient
PM have been studied and characterized to some
extent in adults, but little is known about effects
in the developing lung. The present study pro-
vides new insights that exposure to ultrafine Fe
and soot particles results in lung injury, oxidative
stress, and inflammatory response in a dose-
dependent pattern in neonatal rats.
Although 24-h average PM2.5 concentrations
above 65 mg/m3 are relatively rare in the eastern
United States, those concentrations are more
prevalent in California, reaching over 150 mg/m3
(24-h average) in winter in some cities (Ostro,
1995). In the United States, the proportion of Fe
may be as high as 16% as measured in Phoenix
(U.S. EPA, 2002). Therefore, the particle concen-
tration and compositions used in the present
study are environmentally relevant.
Iron is essential for metabolic processes.
However, increased availability of Fe may medi-
ate the production of reactive oxygen species
(ROS) via the Fenton reaction and may induce
oxidative injury and cellular toxicity. Ferritin is
an Fe storage protein in the cytoplasm of cells
and responsible for the regulation of intracellular
Fe (Crichton & Charloteauz-Wauters, 1987;
Harrison & Arosio, 1996). Increased synthesis
of ferritin reflects an increase in the storage
capacity of free Fe (Cermak et al., 1993) and is
indicative of bioavailable Fe following expo-
sure to Fe-containing particulate (Fang & Aust,
1997). The current study revealed that a signif-
icant induction of ferritin occurred in lungs of
neonatal rats exposed to Fe-containing parti-
cles at 100 μg/m3, but not at 30 μg/m3, in com-
bination with soot particles. The amount of Fe
mobilized from Fe and soot particles by citrate
in the present study is approximately 2.2-fold
greater than that reported for diesel exhaust
particulate (Aust et al., 2002), and the amount
of Fe mobilized from Utah coal fly ash with a
diameter less than 2.5 μm was approximately
1.5-fold higher than from Fe and soot particles
(Smith et al., 1998). Thus, the mobilization of
Fe from Fe and soot particles is mid-range for
what has been reported for other particles.
Smith et al. (1998) demonstrated a direct
correlation between the amount of ferritin
FIGURE 4. Changes in ferric/reducing antioxidant power
(FRAP) in BALF. Data are presented as mean ± SE (n = 8 per
group). Asterisk indicates significant difference from control
(p< .05).
FIGURE 5. Effect of iron and soot exposure on protein levels of
cytokines (A) IL-1b and (B) TNF-a in lung homogenates. Values
are mean ± SE (n = 6 per group): Asterisk indicates significant
difference from control (p < .05).
Downloaded By: [University of California, Davis] At: 16:41 9 April 2010
844 C.-Y. ZHONG ET AL.
induced in human lung epithelial (A549) cells
treated with coal fly ash and the amount of Fe
mobilized from these particles by citrate in the
absence of cells. However, mobilization of
Fe by citrate did not correlate with total Fe
content of coal fly ash (Smith et al., 1998) or
urban PM (Smith & Aust, 1997). Thus, simply
determining the amount of Fe present in parti-
cles is not likely to allow prediction of bioavail-
ability of Fe. The amount of Fe mobilized from
particles is more likely due to factors including
the form of the Fe or the surface area of the
particles. Bioavailable Fe in urban PM (Smith &
Aust, 1997), crocidolite asbestos (Lund & Aust,
1992), and silicates (Hardy & Aust, 1995) was
demonstrated to be responsible for catalyzing
the formation of ROS. In the present study,
the significant induction of ferritin in animals
exposed to particles containing 100 μg/m3 Fe
suggests that at least part of the Fe from ultrafine
Fe and soot particles deposited in the lungs of
neonatal rats is bioavailable. Possible mecha-
nisms accounting for bioavailable Fe follow-
ing particle exposure may be due to potential
interaction between Fe and soot particles lead-
ing to reduction of Fe oxide, or enhancement
of particle deposition in the lungs and uptake
by cells. In support of this possible mechanism
is our previous study, which demonstrated that
although exposure to Fe or soot particle alone
did not induce biological effects, significant
oxidative responses in rats exposed to a mix-
ture of these particles were observed, illustrat-
ing a synergistic interaction between Fe and
soot particles (Zhou et al., 2003).
Previously Zhou et al. (2003) reported that
exposure of adult rats to the combination of Fe
and soot particles induced pulmonary oxida-
tive stress and IL-1b elevation, effects not asso-
ciated with decrease in cell viability and increase
in LDH activity. Data from our present study
revealed the vulnerability of developing lung to
the exposure of environmentally relevant con-
centrations of these particles, as evidenced by
the induction of oxidative stress, IL-1b elevation,
and lung injury. Oxidative stress was observed
in neonatal rats exposed to both 30 and
100 μg/m3of Fe in combination with soot par-
ticles, as demonstrated by a significant increase
in oxidized glutathione (GSSG) and elevation
in GRR, associated with a decrease in antioxidant
power. Furthermore, animals exposed to 100 μg/
m3 Fe in the presence of soot demonstrated
lung injury in the form of decreased cell via-
bility and increased LDH activity, as well as
elevation of proinflammatory cytokine IL-1b.
It is postulated that higher Fe concentration
(100 μg/m3) in combination with soot leads to
greater bioavailability of Fe, resulting in severe
cellular responses including oxidative injury
and inflammatory response. Indeed, Smith et al.
(2000) reported a direct relationship, above a
threshold level of bioavailable Fe, between levels
of the proinflammatory cytokine interleukin-8
(IL-8) and bioavailable Fe in A549 cells treated
with coal fly ash. Induction of this cytokine was
inhibited by antioxidants, suggesting a role of
ROS. In addition, pretreatment of coal fly ash
with the metal chelator to remove mobilizable
Fe prior to treatment of A549 cells resulted in
attenuation of IL-8 production to levels similar
to those in untreated cells, suggesting a role of
Fe in increased cytokine production. The obser-
vations in our current study, that exposure to
Fe and soot particles induced elevation in
proinflammatory cytokine IL-1b, but not in
TNF-a, are consistent with the results of previ-
ous Fe-containing particle studies (Broeckaert
et al., 1997; Dreher et al., 1997; Kodavanti
et al., 1997; Zhou et al., 2003). It is notewor-
thy that although Fe and soot particle exposure
resulted in decreased in cell viability and
increased LDH activity in BAL fluid, although
no significant change in inflammatory cell
count was found. A possible explanation for
this observation is that exposure to Fe and soot
particles induced cell death of local bronchoal-
veolar cells and LDH elevation in the absence
of the recruitment of inflammatory cells at the
examination time point in our study; the
recruitment of inflammatory cells might be
influenced by the concentrations of exposures
as well as the phases of inflammatory responses.
In conclusion, these findings suggest that
exposure to the mixture of Fe and soot parti-
cles induces oxidative injury and inflamma-
tory response in the lungs of neonatal rats in
a dose-dependent pattern. The responses
Downloaded By: [University of California, Davis] At: 16:41 9 April 2010
NEONATAL LUNG INJURY BY IRON AND SOOT PARTICLES 845
observed from the present study are associ-
ated with the bioavailable Fe from inhaled
particle mixtures.
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