Metal-dependent expression of ferritin and
lactoferrin by respiratory epithelial cells
Andrew J.
Ghio1,
Jacqueline D.
Carter1,
James M.
Samet2,
William
Reed2,
Jacqueline
Quay1,
Lisa A.
Dailey1,
Judy H.
Richards1, and
Robert B.
Devlin1
1 National Health and
Environmental Effects Research Laboratory, Environmental Protection
Agency, Research Triangle Park 27711; and
2 Center for Environmental
Medicine and Lung Biology, University of North Carolina, Chapel Hill,
North Carolina 27599
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ABSTRACT |
Increased
availability of catalytically active metal has been associated with an
oxidative injury. The sequestration of transition metals within
intracellular ferritin confers an antioxidant function to this protein.
Such storage by ferritin requires that the metal be transported across
a cell membrane. We tested the hypothesis that, in response to in vitro
exposures to catalytically active metal, respiratory epithelial cells
increase the production of lactoferrin and ferritin to bind, transport,
and store this metal with their coordination sites fully complexed.
Residual oil fly ash is an emission source air pollution particle with
biological effects that, both in vitro and in vivo, correspond with its
metal content. Cell cultures were exposed to 0-200 µg/ml of oil
fly ash for 2 and 24 h. Concentrations of ferritin and lactoferrin mRNA
were estimated by reverse transcription-polymerase chain reaction, and
concentrations of ferritin and lactoferrin proteins were measured in
parallel. mRNA for ferritin did not change with exposure to oil fly
ash. However, ferritin protein concentrations increased. Although mRNA
for transferrin receptor decreased, mRNA for lactoferrin increased
after incubation with the particle. Similar to changes in mRNA,
transferrin concentration decreased, whereas that of lactoferrin
increased. Deferoxamine, a metal chelator, inhibited these responses,
and exposure of the cells to vanadium compounds alone reproduced
elevations in lactoferrin mRNA. We conclude that increases in ferritin
and lactoferrin expression can be metal dependent. This response can
function to diminish the oxidative stress a metal chelate presents to a
living system.
free radicals; transferrin; deferoxamine; vanadium
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INTRODUCTION |
TRANSITION METALS were selected in molecular evolution
to carry out a wide range of biological functions as a result of their interactions with oxygen and their behavior with donor-acceptor complex
formation (coordination). These metals are essential micronutrients utilized in almost every aspect of normal cell function. As a result of
its abundance in nature, iron has a greater participation in the normal
physiology of a living system relative to other transition metals.
Those same chemical properties that allow these metals to function as
catalysts in the numerous reactions of molecular oxygen also make them
a threat to life via their generation of oxygen-based free
radicals
Although
all living systems depend on transition metals to catalyze homeostatic
and synthetic functions, O
2, H2O2,
and · OH generated by these metals have a capacity to damage biological molecules. Consequently, transition metals must be transported and stored with all coordination sites fully complexed to
prevent formation of these damaging reactive oxygen species (17).
As a result of this oxidative injury, it would be of benefit to the
host to sequester available transition metals. The intracellular sequestration of iron by macrophages can limit its potential to generate free radicals and prevent cellular injury resulting from its
exposure (39). The storage of iron in a chemically less reactive form
within intracellular ferritin confers an antioxidant function to this
protein and, in certain cells, provides cytoprotection in vitro against
oxidants (3, 4, 16).
Ferritin synthesis is known to be regulated by a posttranscriptional
mechanism (5, 7, 19, 24, 27, 33, 42). A specific sequence at the
5'-untranslated end of ferritin mRNA called the iron-responsive
element binds a cubane iron-sulfur cluster, the iron
regulatory protein (IRP), when the IRP exists in the apoprotein form.
Available iron reacts with IRP to alter its conformation. This
decreases the affinity of the protein to mRNA, and it is displaced,
allowing translation of ferritin to proceed. Similar to ferritin,
transferrin-receptor (TfR) mRNA contains five iron-responsive elements
but at the 3'-untranslated region (29, 31, 32, 45). Cells treated
with metal chelators increase TfR synthesis, whereas exposure to iron
salts decreases receptor synthesis. This coordinate regulation of
ferritin and TfR allows the cell to respond rapidly to increased
concentrations of iron by increasing the amount of metal sequestered in
ferritin and decreasing the quantities of iron taken up by the cell.
Although numerous metal chelates that present an oxidative stress to
the cell are extracellular, ferritin is produced intracellularly. Therefore, sequestration and detoxification by ferritin requires that
the metal be transported across the membrane. To transfer iron across a
membrane, animal cells most frequently use the glycoprotein transferrin
(2, 46). However, with exposure to iron chelates, many cell types
rapidly downregulate TfR expression (29, 31, 32, 45). Consequently,
transferrin-dependent transport of iron across the membrane is unlikely
to contribute to the sequestration of metal and the antioxidative
function of ferritin. An alternative mechanism of iron transport is
required that can function to transfer the metal to intracellular
ferritin after exposure to a chelate. Ideally, this system of transport
should transfer metal to the storage site in a specific manner to
minimize the potential for oxidative stress and cytotoxicity.
Lactoferrin is a monomeric, cationic metal-binding glycoprotein
(molecular mass 76.4 kDa) synthesized by myeloid cells and secretory epithelia (23, 34, 43). It is most commonly found in human
mucosal secretions (e.g., milk, tears, semen, and plasma) and in the
specific granules of polymorphonuclear leukocytes. Lactoferrin rarely
provides metal for the nutritional requirements of the cell except in
the neonate (25), and the function of lactoferrin in iron transport is
assumed to be negligible in the normal healthy state (21, 38). However,
during an inflammatory state, a recognized function of lactoferrin is
the transport of iron across cell membranes to be deposited in the
ferritin of monocytes and macrophages (10, 36, 47, 48). With the use of
specific receptors, both these cells bind lactoferrin, and the
glycoprotein is internalized (9, 36). The metal is then translocated to
cytosolic ferritin.
The location of lactoferrin at sites of interaction between a host and
its external environment suggests a potential role in a detoxification
of metal chelates. We tested the hypothesis that in response to a
catalytically active transition metal, respiratory epithelial cells
increase the in vitro production of both lactoferrin and ferritin to
transport and store this metal with coordination sites fully complexed,
whereas the expression of transferrin is decreased.
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METHODS |
Materials. Respiratory
epithelial cells were exposed to an emission source air pollution
particle. The air pollution particle used in all investigations was a
residual oil fly ash acquired from Southern Research Institute
(Birmingham, AL). It was collected with a Teflon-coated fiberglass
filter downstream from the cyclone of a power plant in Florida that was
burning a low-sulfur no. 6 residual oil (collection temperature
250-300°C). The particle had an average mass median
aerodynamic diameter, from four replicate samples, of 3.60 ± 0.80 µm. Carbon, hydrogen, nitrogen, and mineral ash contents of the oil
fly ash were analyzed. An elemental analysis of carbon and hydrogen was
accomplished with either infrared or thermal conductivity assays
(Galbraith Laboratories, Knoxville, TN). Nitrogen content was measured
with thermal conductivity after acid digest (Galbraith Laboratories).
Mineral ash analysis was performed with inductively coupled plasma
emission spectroscopy (Perkin-Elmer model 40, Norwalk, CT) after acid
digest. There is an abundance of metal compounds included in this
particle, with vanadium, nickel, and iron in the greatest quantities.
Water-soluble concentrations of these specific metals account for 78.9, 97.0, and 80.4%, respectively, of their total concentration in this oil fly ash. Lung injury after human exposure to oil fly ash has occurred predominantly with the cleaning of oil-fired boilers. The
clinical presentation of these individuals has been termed "boilermaker's bronchitis."
Deferoxamine mesylate was obtained from Ciba Pharmaceutical (Summit,
NJ). All other reagents were from Sigma (St. Louis, MO) unless
otherwise specified.
Measurement of oxidative stress. In
vitro oxidant generation by the oil fly ash was measured with
thiobarbituric acid (TBA)-reactive products of deoxyribose. The pentose
sugar 2-deoxy-D-ribose reacts with oxidants to yield a mixture of products. On heating with TBA at a
low pH, these products form a pink chromophore that can be measured by
its absorbance at 532 nm (A532).
This chromophore is indistinguishable from a TBA-malondialdehyde
adduct. The reaction mixture containing 1.0 mM deoxyribose, 1.0 mM
H2O2,
1.0 mM ascorbate, and 200 µg of oil fly ash was incubated in saline
at 37°C for 60 min with agitation and then centrifuged at 1,200 g for 10 min. One milliliter of both
1.0% (wt / vol) TBA and 2.8% (wt / vol) trichloroacetic
acid was added to 1.0 ml of supernatant, heated at 100°C for 10 min, and cooled in ice, and the chromophore concentration was
determined by its A532.
Cell culture. Normal human bronchial
epithelial (NHBE) cells (Clonetics, San Diego, CA) were obtained in
cryopreserved ampules and cultured as per the supplier's instructions.
Cells were grown to 90-100% confluence on plastic 12-well plates
in bronchial epithelial growth medium (Clonetics), which is LHC-9
medium. NHBE cells were passaged two to four times before use in these
experiments.
BEAS-2B cells (S6 subclone, passage
59), obtained through the courtesy of Drs. Curtis
Harris and John Lechner (National Institutes of Health, Bethesda, MD),
were also used in some studies. This is an immortalized line of normal
human bronchial epithelium derived by transfection of primary cells
with SV40 early-region genes. This particular subclone undergoes
squamous differentiation in response to serum (26). Cells were grown to
90-100% confluence on uncoated plastic 12-well plates in
keratinocyte growth medium (Clonetics), which is essentially MCDB 153 medium supplemented with 5 ng/ml of human epidermal growth factor, 5 mg/ml of insulin, 0.5 mg/ml of hydrocortisone, 0.15 mM calcium, bovine
pituitary extract, 0.1 mM ethanolamine, and 0.1 mM
phosphoethanolamine.
RT-PCR. Cells were grown to confluence
in 12-well plates (Costar, Cambridge, MA) and exposed for both 2 and 24 h to the oil fly ash. The supernatant was removed, and the cells were
washed two times with phosphate-buffered saline (Life Technologies,
Grand Island, NY). The cells were lysed with 4 M guanidine thiocyanate (Boehringer Mannheim, Indianapolis, IN), 50 mM sodium citrate, 0.5%
sarkosyl, and 0.01 M dithiothreitol. After the cells were dislodged
from the wells with scrapers (Costar), the lysates were sheared with
four passes through a 22-gauge syringe. RNA was pelleted by
ultracentrifugation through 5.7 M cesium chloride (Boehringer Mannheim)
and 0.1 M EDTA. One hundred nanograms of total RNA were reverse
transcribed (Moloney murine leukemia virus reverse transcriptase, Life
Technologies) (6). The resultant cDNA was amplified (6) for 24, 25, 24, and 36 cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ferritin, TfR, and lactoferrin, respectively, in separate reactions with gene-specific primers. Oligonucleotide sequences were
synthesized with an Applied Biosystems 391 DNA synthesizer (Foster
City, CA) based on sequences published in the GenBank DNA database. The
following sense and antisense sequences were employed: GAPDH,
5'-CCATGGAGAAGGCTGGGG-3' and
5'-CAAATTGTCATGGATGACC-3'; ferritin,
5'-TCGCAGGTGCGCCAGAACTA-3' and
5'-AAGGAAGATTCGGCCACCTC-3'; TfR,
5'-ACGATCACAGCAATAGTCCCATA-3' and
5'-CTCGAGCGGCTGCAGGTTCTTCTG-3'; and lactoferrin,
5'-GAGAAGGAGTGTTCAGTGGT-3' and
5'-ATAGTGAGTTCGTGGCTGTC-3'. Amplification
products were separated on 2% denaturing agarose gel, stained with
ethidium bromide, and photographed under ultraviolet illumination. The
resulting negative (type 55 film, Polaroid, Cambridge, MA) was
quantitated with a BioImage Densitometer (BioImage, Ann Arbor, MI). For
each experimental condition, the integrated optical densities of the
ferritin, TfR, and lactoferrin DNA bands were divided by that of the
GAPDH DNA band (a housekeeping gene unaffected by the addition of oil
fly ash) to correct for variation in the amount of amplifiable cDNA in
each sample.
Measurement of ferritin, transferrin, and lactoferrin
protein concentrations. Cells were grown to confluence
in 12-well plates (Costar) and exposed for both 2 and 24 h to the oil
fly ash. The cells were dislodged from wells with scrapers (Costar)
into the exposure medium (a volume of 1.0 ml for ferritin and
transferrin and 0.2 ml for lactoferrin), and the cell suspension was
disrupted by sonication for 15 s. Ferritin and transferrin protein
concentrations in these cell lysates were measured with commercially
available kits (an enzyme immunoassay and an immunoprecipitin analysis, respectively), controls, and standards from Microgenics (Concord, CA)
and INCSTAR (Stillwater, MN). These assays were modified for use in the
Cobas Fara II centrifugal spectrophotometer (Hoffman-LaRoche, Branchburg, NJ). Lactoferrin was measured with a commercially available
ELISA kit (Calbiochem, La Jolla, CA). As a result of cost, lactoferrin
protein concentrations were measured only at 24 h after exposure.
Statistics. Data are expressed as mean
values ± SE. The minimum number of replicates for all measurements
was six. Differences among multiple groups were compared by one-way
analysis of variance (15). The post hoc test employed was Duncan's
multiple range test. Two-tailed tests of significance were employed.
Significance was assumed at P < 0.05.
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RESULTS |
Organic and mineral analyses of the oil fly ash revealed the presence
of significant concentrations of transition metals (Table 1). Those metals in greatest concentration
were vanadium, iron, and nickel. TBA-reactive products of deoxyribose
verified that the oil fly ash presented an oxidative stress
(A532 0.82 ± 0.03 and 0.04 ± 0.01 for the oil fly ash and saline, respectively). Inclusion of the metal chelator deferoxamine and the free radical scavenger dimethylthiourea (DMTU) in the reaction mixtures (both at a
final concentration of 1.0 mM) diminished the generation of oxidized
products of TBA by oil fly ash, with
A532 values of 0.01 ± 0.01 and
0.09 ± 0.02, respectively. These results implicate metals as that
component of the air pollution particle associated with free radical
production.
Both NHBE and BEAS-2B cells were exposed to 0.0, 5.0, 50.0, and 200.0 µg/ml of oil fly ash for both 2 and 24 h. Neither NHBE nor BEAS-2B
cells demonstrated a significant elevation in lactate dehydrogenase
(LDH) in the cell supernatant after exposure, indicating a lack of
cytotoxicity. Responses for the BEAS-2B cells (both mRNA and proteins)
were comparable to those of the NHBE cells. As a result of the parallel
responses of NHBE and BEAS-2B cells after exposure, data are provided
for NHBE cells only.
Consistent with a posttranscriptional control mechanism, there was no
difference in ferritin mRNA at 2 and 24 h (Fig.
1), but protein in the cell lysate
increased with exposure to the oil fly ash (Fig.
2). To analyze for participation of metals
and catalyzed free radicals in the epithelial response to the oil fly
ash, cells were treated with either deferoxamine or DMTU immediately before the particles were added. Addition of 1.0 mM deferoxamine and
1.0 mM DMTU to incubations of NHBE cells with 200 µg/ml of oil fly
ash did not significantly increase LDH concentrations in the
supernatant. Inclusion of deferoxamine did not significantly change
ferritin mRNA at 24 h. DMTU similarly had no effect on mRNA expression
for ferritin. After exposure to oil fly ash and treatment with
deferoxamine, measurement of ferritin protein demonstrated decreased
concentrations of this storage protein in the cell lysates (Fig.
3). However, addition of DMTU had no effect
on the protein concentration of ferritin.

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Fig. 1.
Effect of oil fly ash on normal human bronchial epithelial (NHBE)
ferritin mRNA levels. NHBE cells were exposed to indicated
concentrations of oil fly ash for 2 and 24 h. Total RNA was extracted,
and levels of ferritin mRNA were measured by RT-PCR with specific
oligonucleotide PCR primers and are expressed as relative optical
densities for each band of ferritin mRNA normalized to corresponding
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
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Fig. 2.
Effect of oil fly ash on ferritin production by NHBE cells. NHBE cells
(n = 6) were exposed to indicated
concentration of oil fly ash for 2 and 24 h. Cells were lysed, and
concentrations of ferritin protein were determined with a commercially
available enzyme immunoassay. * Significant increase relative to
incubation with media (0.0 µg/ml).
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Fig. 3.
Role of metal cations and oxidants in oil fly ash-induced increases in
ferritin production by NHBE cells. NHBE cells
(n = 6) were exposed to oil fly ash
for 24 h in presence or absence of 1.0 mM deferoxamine (DEF) and 1.0 mM
dimethylthiourea (DMTU). Cells were lysed, and concentrations of
ferritin protein were determined with a commercially available enzyme
immunoassay. * Significant increase relative to incubation with
media. ** Significant decrease relative to incubation with ash.
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After 24 h of exposure of NHBE cells to the oil fly ash, there were
decrements in mRNA for TfR (Fig. 4).
Corresponding to this change in mRNA, transferrin concentrations in the
cell lysate were diminished (Fig. 5).
Consistent with a previous investigation (35) demonstrating
transcriptional control of TfR, addition of deferoxamine increased mRNA
for the TfR (Fig. 6). Similarly, transferrin concentrations in the lysates increased with chelation of
the metal (Fig. 7). DMTU had no effect on
either TfR mRNA or transferrin concentration.

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Fig. 4.
Effect of oil fly ash on NHBE transferrin receptor (TfR) mRNA levels.
NHBE cells (n = 6) were exposed to
indicated concentration of oil fly ash for 2 and 24 h. Total RNA was
extracted, and levels of TfR mRNA were measured by RT-PCR with specific
TfR oligonucleotide PCR primers and are expressed as relative optical
densities for each band of TfR mRNA normalized to corresponding GAPDH
mRNA. * Significant decrease relative to incubation with media.
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Fig. 5.
Effect of oil fly ash on transferrin production by NHBE cells. NHBE
cells (n = 6) were exposed to
indicated concentrations of oil fly ash for 2 and 24 h. Cells were
lysed, and concentrations of transferrin protein were determined with a
commercially available immunoprecipitin analysis. * Significant
decrease relative to incubation with media.
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Fig. 6.
Role of metal cations and oxidants in oil fly ash-induced decreases in
TfR mRNA by NHBE cells. NHBE cells (n = 6) were exposed to oil fly ash for 24 h in presence or absence of 1.0 mM DEF and 1.0 mM DMTU. Total RNA was extracted, and levels of TfR mRNA
were measured by RT-PCR with specific TfR oligonucleotide primers and
are expressed as relative optical densities for each band of TfR cDNA
normalized to corresponding GAPDH cDNA. * Significant decrease
relative to incubation with media. ** Significant increase
relative to incubation with ash.
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Fig. 7.
Role of metal cations and oxidants in oil fly ash-induced decreases in
transferrin production by NHBE cells. NHBE cells
(n = 6) were exposed to oil fly ash
for 24 h in presence or absence of 1.0 mM DEF and 1.0 mM DMTU. Cells
were lysed, and concentrations of transferrin protein were determined
with a commercially available immunoprecipitin analysis.
* Significant decrease relative to incubation with media.
** Significant increase relative to incubation with ash.
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In contrast to TfR, there was a significant increase in mRNA for
lactoferrin at 24 h (Fig. 8). The
concentration of lactoferrin increased in the epithelial cell lysate
after 24 h of incubation with air pollution particles (Fig.
9). Deferoxamine significantly diminished
mRNA for lactoferrin after incubation with oil fly ash (Fig.
10). Corresponding to diminished mRNA for
lactoferrin, the concentration of lactoferrin protein in the lysate
decreased after the addition of deferoxamine to cells exposed to oil
fly ash (Fig. 11). DMTU had no effect on
either lactoferrin mRNA or protein.

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Fig. 8.
Effect of oil fly ash on NHBE lactoferrin mRNA levels.
A: RNA extracts from NHBE cells
(n = 6) exposed to 0 (media control;
a), 5.0 (b), 50.0 (c), and 200.0 (d) µg/ml of oil fly ash for 24 h
were subjected to 36 cycles of RT-PCR with specific lactoferrin
oligonucleotide primers. Arrow, lactoferrin band.
B: relative optical densities for each
band of lactoferrin mRNA normalized to corresponding GAPDH mRNA.
* Significant increase relative to incubation with media.
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Fig. 9.
Effect of oil fly ash on lactoferrin production by NHBE cells. NHBE
cells (n = 6) were exposed to
indicated concentrations of oil fly ash for 24 h. Cells were lysed, and
concentrations of lactoferrin protein were determined with a
commercially available ELISA kit. * Significant increase relative
to incubation with media.
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Fig. 10.
Role of metal cations and oxidants in oil fly ash-induced increases in
lactoferrin (LF) mRNA by NHBE cells. NHBE cells
(n = 6) were exposed to oil fly ash
for 24 h in presence and absence of 1.0 mM DEF and 1.0 mM DMTU. Total
RNA was extracted, and levels of LF mRNA were measured by RT-PCR with
specific lactoferrin oligonucleotide primers and are expressed as
relative optical densities for each band of LF mRNA normalized to
corresponding GAPDH mRNA. * Significant increase relative to
incubation with media. ** Significant decrease relative to
incubation with ash.
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Fig. 11.
Role of metal cations and oxidants in oil fly ash-induced increases in
LF production by NHBE cells. NHBE cells
(n = 6) were exposed to oil fly ash
for 24 h in presence and absence of 1.0 mM DEF and 1.0 mM DMTU. Cells
were lysed, and concentrations of LF protein were determined with
commercially available ELISA kits. * Significant increase
relative to incubation with media. ** Significant decrease
relative to incubation with ash.
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To demonstrate that the effects of the air pollution particle could be
reproduced by exposure to a defined metal salt, NHBE cells were exposed
for 24 h to either medium or a metal solution that included iron (III)
sulfate
[Fe2(SO4)3],
iron (II) sulfate (FeSO4 · 7H2O),
nickel (II) sulfate
(NiSO4 · 6H2O),
vanadium (IV) sulfate oxide
(VOSO4 · 4H2O),
and sodium metavanadate (V)
(NaVO3). There were no changes
in the concentration of LDH in the supernatants. The concentrations of
iron sulfates (2.00 mM) and nickel sulfate (0.75 mM) exceeded those
resulting from oil fly ash exposure but were not associated with an
increase in lactoferrin mRNA. However, in vitro exposure of cells to
that concentration of vanadium (0.75 mM) equivalent to 200 µg/ml of
oil fly ash resulted in elevated lactoferrin mRNA (Fig.
12). The elevation in lactoferrin mRNA
was not specific to one valence state, and both vanadium compounds, vanadium sulfate oxide and sodium metavanadate, increased mRNA.

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Fig. 12.
Effect of vanadium on NHBE LF mRNA levels. NHBE cells
(n = 6) were exposed to either 0.75 mM
vanadium (IV) sulfate oxide or 0.75 mM sodium metavanadate (V)
[vanadium (V)] for 24 h. Total RNA was extracted, and levels of LF
mRNA were measured by RT-PCR with specific LF oligonucleotide primers
and are expressed as relative optical densities for each band of LF
mRNA normalized to corresponding GAPDH mRNA. * Significant
increase relative to incubation with media.
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DISCUSSION |
In vitro, the oil fly ash generated oxidants and therefore has a
potential to present an oxidative stress to the lower respiratory tract
of exposed individuals. Those transition metals in greatest concentration in this emission source air pollution particle (i.e., vanadium, nickel, and iron) can be transported and stored by cells using identical proteins. Transferrin and lactoferrin complex and
transport, whereas ferritin has the capacity to store transition metals
other than iron, including vanadium and nickel (14, 20, 37, 44). This
mechanism of transport and storage results in a regulation of electron
transfer by the metal that either diminishes or inhibits an oxidative
stress associated with the metal (39).
After exposure to the oil fly ash, respiratory epithelial cells
demonstrated no change in ferritin mRNA but demonstrated an increase in
ferritin protein. This is consistent with a posttranscriptional regulation of this storage protein (5, 7, 19, 24, 27, 33, 42). Ferritin
production by respiratory epithelial cells is consistent with previous
investigations showing that metal can induce an increased
expression of this storage protein in numerous and diverse cell types.
The complexation of this metal by deferoxamine inhibited the increased
expression of ferritin by the respiratory epithelial cells. It is
likely that the epithelial cell produces ferritin in an attempt to
sequester the metal in a catalytically less reactive state. The radical
scavenger DMTU had no effect on concentrations of this storage protein,
suggesting that the metal itself, rather than the associated oxidative
stress, is responsible for the increased ferritin expression after oil fly ash exposure. An alternative explanation of these results is that
DMTU did not inhibit radical generation by the particle as a result of
site specificity and that lactoferrin expression can be associated with
an oxidative stress.
Most cells produce transferrin and TfR, reflecting the importance of
this method of iron transport in providing metal to meet the
requirements of the cell. However, exposure to the air pollution particle diminished TfR mRNA and transferrin concentrations. Increases in TfR mRNA after treatment with deferoxamine support previous evidence
of a transcriptional control of this receptor (35). This advocates a
role of this transport system in meeting the nutritional requirements
of the respiratory epithelial cell but does not support a participation
of transferrin in metal detoxification in this specific cell type.
Increases in both lactoferrin mRNA and lactoferrin protein after
exposure to oil fly ash were inhibited by the metal chelator deferoxamine but not by the hydroxyl radical scavenger DMTU. These results support the contention that metals included in the oil fly ash
influenced lactoferrin mRNA and protein. The lactoferrin produced by
bronchial epithelial cells may function to transport metal included in
chelates that present an oxidative stress to the respiratory tract.
After reaction with the metal, the glycoprotein-metal complex may be
endocytosed by cells with lactoferrin receptors, and the metal would be
transported to intracellular ferritin. In the respiratory tract, cells
with lactoferrin receptors include alveolar macrophages. It is likely
that, similar to a number of diverse cells (22, 25, 41), respiratory
epithelial cells have this same receptor because exposure to this metal
chelate results in an increased deposition of iron in ferritin of these cells.
Lactoferrin production by secretory epithelial cells in the respiratory
tract has been previously demonstrated (i.e., the serous acini of the
bronchial glands) (11). Production of this metal-transport glycoprotein
by cultured normal (i.e., untransformed) respiratory epithelial cells
has not been previously described. Cells originating from neoplastic
tissue can reveal lactoferrin mRNA and, with the use of
immunohistochemistry, elevated protein concentrations (18). However,
cancer can be associated with an increased availability of metal, and
the increased expression of lactoferrin in these specific cell lines
(Calu-1, Calu-3, and SK-LU-1) may reflect a response to this metal.
Alternatively, lactoferrin expression may be a characteristic of
undifferentiated epithelial cells.
To transport the metal associated with the particle into a cell and
effect an increased expression of ferritin, transferrin and lactoferrin can both be potentially employed. As a result of
complexation of iron by transferrin and the resultant decrease in the
ability of the coordinated metal to catalyze radical generation, transferrin has been described as a major antioxidant in the lining fluid of the lower respiratory tract. If transferrin is to effectively participate in decreasing the availability of iron to catalyze radical
generation, oxidative stress to the host, and injury after exposure to
the metal, this glycoprotein should increase in concentration. mRNAs
for TfR and transferrin protein both diminished after exposure to the
oil fly ash, which is abundant in metals. Subsequently, transferrin
appears unlikely to contribute to the transport and detoxification of
metal in the lower respiratory tract after exposure to the particle.
The two methods of metal transport employed by respiratory epithelial
cells (i.e., transferrin- and lactoferrin-dependent transfer) responded
in a diametrical fashion to metal availability. This may indicate
separate functions of these two transport systems that, in the
respiratory epithelial cell, may not overlap. TfR mRNA and transferrin
concentrations increased with the depletion of iron in the environment.
This supports the widely accepted role of increased transferrin
production by the cell in meeting its nutritional requirements.
However, after an increase in catalytically active metal, with exposure
to the emission source air pollution particle, the reverse situation
was evident, with TfR mRNA and transferrin concentrations decreasing,
whereas lactoferrin mRNA and protein were elevated. This supports a
role for lactoferrin, but not for transferrin, transport in the
detoxification of metal by the cell.
Such a balance between the two metal transport systems supports
previous investigations demonstrating that
1) iron depletion increases
transferrin and 2) iron excess
increases lactoferrin while decreasing transferrin. Although the data
supporting the association between transferrin release and iron
depletion are well recognized, those suggesting the relationship
between lactoferrin production and metal excess are not. In exposures
to microbial agents, the replication of which is dependent on a
mobilization and availability of host iron, the two systems of binding
iron also respond in a dissimilar fashion. Serum transferrin
concentrations diminished after bacterial infection, whereas
lactoferrin concentrations in the immediate environment were elevated
(8). Exposure of the human lung to aerosolized microbials similarly
decreased lavage transferrin or increased lactoferrin, again supporting
disparate functions of the two systems of transport (30). With a direct challenge (ingestion) of humans to iron, transferrin concentrations in
the serum do not increase to meet an in vivo oxidative stress, whereas
lactoferrin levels do (13). Finally, in human injury associated with
elevations in catalytically active iron, such as rheumatoid arthritis,
the local concentrations of lactoferrin in the synovial fluid, but not
of transferrin, increased in concentration (1). It has been proposed
that these differences between transferrin and lactoferrin may reflect
disparities in cell handling of the two iron-binding proteins (40).
Transferrin increases metal that the cell can use to meet nutritional
needs (12), whereas lactoferrin elevates iron in a different
intracellular pool (e.g., that associated with ferritin) (47).
The oil fly ash used in this investigation includes several transition
metals that support electron transport and oxidant generation.
Prominent among these are vanadium, iron, and nickel. It is not
possible to attribute the effects of this air pollution particle on
ferritin, transferrin, and lactoferrin mRNA and protein concentrations
on a single transition metal. Deferoxamine complexes metal cations
other than iron (28). Changes in an effect after treatment with this
metal chelator do not directly implicate iron. However, increases in
lactoferrin mRNA after incubation of respiratory epithelial cells with
vanadium compounds verify that exposure to at least one of these
transition metals can reproduce an effect of the oil fly ash on these
cells.
We conclude that increases in ferritin and lactoferrin expression after
exposure of respiratory epithelial cells to an emission source air
pollution particle were metal dependent. These two proteins may
function in a coordinated manner to transport and store transition
metals in a chemically less reactive form. These responses can function
to diminish the oxidative stress a metal chelate presents and therefore
detoxify it.
 |
ACKNOWLEDGEMENTS |
This report has been reviewed by the National Health and
Environmental Effects Research Laboratory, United States Environmental Protection Agency, and approved for publication. Approval does not
signify that the contents necessarily reflect the views and policies of
the Agency nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
 |
FOOTNOTES |
Address for reprint requests: A. J. Ghio, National Health and
Environmental Effects Research Laboratory, Environmental Protection
Agency, Research Triangle Park, NC 27711.
Received 2 September 1997; accepted in final form 6 February 1998.
 |
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