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INTRODUCTION |
Epidemiological and pathologic studies have established that
occupational exposure to crystalline silica is associated with the
development of pulmonary silicosis (1, 2) and an increased risk for
lung cancer (2-4). Silica, administered by inhalation or intratracheal
instillation, has been shown to be carcinogenic in rats (4-7).
Intrapleural administration of crystalline silica in rats leads to the
induction of localized malignant histiocytic lymphomas (8). The
pulmonary response to silica, however, was found to vary considerably
in different species, with mice developing silicosis but no lung cancer
and hamsters just showing storage of silica in lung macrophages with no
apparent pathology (4-6). Human evidence is also controversial
regarding the induction of lung cancer in workers exposed to silica
(3). However, based on evidence obtained from studies with laboratory
animals and epidemiological studies in humans, the International Agency
for Research on Cancer has classified crystalline silica as a class 1 human carcinogen (9).
Although crystalline silica is a documented carcinogen, the molecular
mechanisms involved in the silica-induced carcinogenesis are unclear.
Previous studies have shown that silica causes direct DNA damage and
mammalian cell transformation (10, 11). Earlier studies have also
demonstrated that FFSi1 is
capable of generating hydroxyl radicals (·OH) upon reaction with
aqueous media (12-14). Superoxide anion radicals (O
2) may
also be generated (15). The silicon-based free radicals (Si·,
SiO·, and SiOO·) and the associated generation
of H2O2 and ·OH appear to be involved in
the lipid peroxidation and membrane damage (12-15). These radicals are
also associated with silica-induced DNA damage, for example both strand
breakage and hydroxylation of dG residues have been observed (10, 17).
O
2, H2O2, and ·OH are generally
called reactive oxygen species (ROS). For direct ROS generation via a
reaction between silica and aqueous medium in the absence of cells, a
relatively large amount of silica particles is needed to generate
detectable amounts of ROS. However, silica also can generate ROS via
stimulation of cells. In this mechanism of ROS generation, silica
induces ROS production as part of the respiratory burst reaction to
particles. ROS generated by this mechanism may be responsible
for silica-induced activation of the nuclear transcription factor
NF-
B (16). However, the mechanism by which ROS from cells stimulated
by silica activates AP-1 remains to be investigated fully.
Previous studies from our laboratory showed that FFSi causes AP-1
activation both in vitro using cell culture systems and in vivo using transgenic mice (18). AP-1 is a transcription factor that consists of either a Jun-Jun homodimer or a Jun-Fos heterodimer (19). Genes regulated by AP-1 have been reported to play an
important role in neoplastic transformation, tumor progression, and
metastasis (18-22). Blocking
12-O-tetradecanolyphorbol-13-acetate-induced AP-1 activation
has been shown to inhibit neoplastic transformation (23). Inhibition of
AP-1 activity in transformed JB6 RT101 cells causes reversion from the
tumor phenotype (24). Furthermore, a recent study, using transgenic
mice, has demonstrated that AP-1 transactivation is required for tumor
promotion (25). In light of the important role of AP-1 activation in
neoplastic transformation and tumor promotion, we hypothesized that the
silica-induced AP-1 activation may play a critical role in
silica-induced carcinogenesis.
Earlier studies have suggested that FFSi exhibits increased surface
reactivity compared with ASi (12, 13). Because the pulmonary response
to silica differs in the chronic and acute presentation of disease, we
proposed that at least part of the acute response is due to some unique
characteristics of the dust inhaled. Acute silicosis is commonly
associated with sandblasting, rock drilling, tunneling, and silica mill
operations, i.e. operations in which silica particles are
crushed or sheared (2, 13).
Based on the early reports that FFSi was more surface-reactive than
ASi, we proposed that these surface features might be responsible for
the enhanced carcinogenic effects of silica in the lung (17, 18). In
the present study, the cytotoxic and biologic activities of FFSi
versus ASi were investigated by comparing their effects on
the phosphorylation of MAPKs and AP-1 activation. In situ
generation of ROS by cells stimulated with silica was studied using
fluorescence staining and ESR. Finally, the role of ROS in
silica-induced AP-1 activation was evaluated.
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MATERIALS AND METHODS |
Reagents--
SOD, N-acetyl-cysteine, DMPO, sodium
formate, and PVPNO were purchased from Sigma Chemical Co. (St. Louis,
MO). Catalase was purchased from Roche Molecular Biochemicals
(Indianapolis, IN). The spin trap, DMPO, was purified by charcoal
decolorization and vacuum distillation. DMPO solution, thus purified,
did not contain any ESR-detectable impurities. Chelex 100 chelating
resin was purchased from Bio-Rad Laboratories (Richmond, CA). The
phosphate buffer (pH 7.4) was treated with Chelex 100 to remove
transition metal ion contaminants. Eagle's MEM was obtained from
Whittaker Biosciences (Walkersville, MD). FBS, gentamicin, and
L-glutamine were purchased from Life Technologies, Inc.
(Gaithersburg, MD). Luciferase assay substrate was obtained from
Promega (Madison, WI). PhosphoPlus MAPK antibody kits were
purchased from New England BioLabs (Beverley, MA). H2DCFDA and
dihydroethidium were obtained from Molecular Probes (Eugene, OR).
Preparation of Freshly Fractured Silica--
Crystalline silica
was obtained from the Generic Center, Pennsylvania State University
(State College, PA). The detailed method for preparation of the FFSi
has been described elsewhere (14). Briefly, crystalline silica (0.2-10
mm in diameter) was ground for 30 min using a ball grinder equipped
with agate mortar and balls. The ground silica was sieved through a
10-µm mesh filter for 20 min before use. Purity was checked using
x-ray diffraction spectrometry, and diameter was determined by
morphometric analyses, which indicated that fractured silica had a
purity of 99.5% and a mean diameter of 3.7 µm.
Cell Culture--
The mouse JB6/AP/
B cell line, which was
stably transfected with an AP-1 luciferase reporter plasmid (18), was
cultured in Eagle's MEM containing 5% FBS, 2 mM
L-glutamine, and 50 µg of gentamicin/ml.
Assay of AP-1 Activity in Vitro--
A confluent monolayer of
JB6/AP/
B cells was trypsinized, and 5 × 104 viable
cells (suspended in 1 ml of Eagle's MEM supplemented with 5% FBS)
were added to each well of a 24-well plate. Plates were incubated at
37 °C in a humidified atmosphere of 5% CO2. Twelve hours later, cells were cultured in Eagle's MEM supplemented with 0.5% fetal bovine serum for 12-24 h to minimize basal AP-1 activity and then exposed to silica in the same medium to monitor the effects on
AP-1 induction. The cells were extracted with 200 µl of 1× lysis
buffer provided in the luciferase assay kit by the manufacturer. Luciferase activity was measured using a Monolight luminometer, model
3010 (18). The results were expressed as relative AP-1 activity
compared with untreated controls.
Protein Kinase Phosphorylation Assay--
Immunoblots for
phosphorylation of ERKs and p38 kinase were carried out as described in
the protocol of New England BioLabs (Beverly, MA), using
phospho-specific antibodies against phosphorylated sites of ERKs and
p38 kinase. Nonphospho-specific antibodies against ERKs and p38 kinase
proteins provided in each assay kit were used to normalize the
phosphorylation assay by using the same transferred membrane blot.
ESR Measurements--
All ESR measurements were conducted using
a Varian E9 ESR spectrometer and a flat cell assembly. Hyperfine
couplings were measured (to 0.1 G) directly from magnetic field
separation using K3CrO8 and
1,1-diphenyl-2-picrylhydrazyl as reference standards. An EPRDAP 2.0 program was used for data acquisition and analysis. Reactants were
mixed in test tubes in a final volume of 450 µl. The reaction mixture
was then transferred to a flat cell for ESR measurement. The
concentrations given in the figure legends are final concentrations.
All experiments were performed at room temperature and under ambient
air except those specifically indicated.
H2O2 Measurements--
JB6 cells (1 × 106) suspended in 1 ml PBS were incubated with or
without silica for 30 min. H2O2 generation was
monitored by measuring the change in fluorescence of scopoletin (0.72 mM) in the presence of horseradish peroxidase (6.6 units/ml). Fluorescence was monitored at an excitation wavelength of
350 nm and an emission wavelength of 460 nm using a Cytofluor multiwell
plate reader series 4000 (PerSeptive Biosystems Inc., Framingham, MA).
H2O2 and O
2 Assay in Intact
Cells--
Dihydroethidium and H2DCFDA are specific dyes used
for staining O
2 and H2O2 produced by
intact cells (26, 27). JB6 cells (2 × 104/well) were
seeded onto a glass coverslip in the bottom of a well of a 24-well
plate for 24 h. The cells were treated with FFSi or ASi in the
presence of dihydroethidium (2 µM) or H2DCFDA (5 µM) for 30 min. The cells were then washed in PBS and
fixed with 10% buffered formalin. The glass coverslip was mounted on a
microscope slide and observed under a Sarastro 2000 (Molecular
Dynamics, Sunnyvale, CA) laser scanning confocal microscope fitted with an argon-ion laser.
Oxygen Consumption Measurements--
Oxygen consumption
measurements were carried out using a Gilson Oxygraph (Model 516, Gilson Medical Electronics, Middleton WI). Cell concentration was
6.25 × 105/ml, and measurements were made over a
period of 10 min.
Statistical Analysis--
Data presented are the means ± S.E. of values compared and analyzed using a one-way ANOVA. Statistical
significance was determined by two-tailed Student's t test
for paired data, and considered significant at p
0.05.
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RESULTS |
Induction of AP-1 Activation by FFSi versus ASi--
Earlier
studies have suggested that FFSi was more surface-reactive than aged
silica (12, 13). To explore the differential effects of FFSi and ASi on
the induction of AP-1 activity, 5 × 104 JB6 cells
were exposed for 24 h to various doses (10-300 µg/ml) of
freshly fractured or fractured silica aged for 12 months. The AP-1
activation induced by FFSi was significantly higher than that of ASi in
JB6 cells (Fig. 1). FFSi induced
significant AP-1 activation at a concentration of 80 µg/ml silica,
reaching a maximum activation at 200 µg/ml. The maximum AP-1
induction by FFSi increased 8-fold compared with controls, whereas the
maximum AP-1 induction by the silica aged for 12 months increased less
than 3-fold compared with controls. These results indicate that FFSi
exhibited a greater effect on AP-1 induction than ASi.

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Fig. 1.
Dose-dependent induction of AP-1
activation by freshly fractured silica versus aged
silica. JB6 cells (5 × 104 in 1 ml of MEM medium
with 5% fetal bovine serum), stably transfected with AP-1 luciferase
reporter plasmid, were seeded into each well of a 24-well plate. After
overnight culture at 37 °C, the cells were cultured in MEM plus
0.1% fetal bovine serum for 24 h. Then the cells were treated for
24 h with various concentrations of freshly fractured silica or
fractured silica aged for 1 year. The AP-1 activity was measured by the
luciferase activity assay as described under "Materials and
Methods." Results, presented as relative AP-1 induction compared with
the untreated control cells, are means and standard errors of eight
assay wells from two independent experiments. *, a significant increase
of freshly fractured silica from aged silica (p 0.05).
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Induction of AP-1 Activity by Freshly Fractured Silica and the
Decay of the Induction with Time after Fracturing--
To further
explore the decay of AP-1 induction by silica with time after
fracturing (aging), the induction of AP-1 activation by FFSi or
fractured silica aged for 2 weeks, 8 weeks, 3 months, 6 months, or 12 months was monitored in the same experiment. JB6 cells (5 × 104) were exposed to 200 µg/ml silica for 24 h, and
the luciferase activity was measured as described under "Material and
Methods." As shown in Fig. 2, the AP-1
induction by silica was decreased with aging. Silica-induced AP-1
activation decreased by 35%, 65%, or 75% after 8 weeks, 6 months, or
12 months of storage, respectively. The half time for the decrease in
the ability of fractured silica to activate AP-1 was proximately 3 months. Fractured silica still produced a 2.5-fold increase in AP-1
induction after 12 months of storage.

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Fig. 2.
Induction of AP-1 activity by freshly
fractured silica and the decay of the induction with time after
fracturing. JB6 cells (5 × 104 cells in 1 ml of
MEM medium containing 5% of fetal bovine serum) were seeded into each
well of a 24-well plate. After overnight culture at 37 °C, cells
were cultured in the same medium plus 0.1% fetal bovine serum for
24 h. The cells were then exposed for 24 h to 200 µg/ml
freshly fractured silica or silica aged for various times after
fracturing. Other experimental conditions were the same as those
described in the legend to Fig. 1. Results, presented as relative AP-1
induction compared with the untreated control cells, are means ± S.E. of eight assay wells from two independent experiments. *, a
significant decrease from cells treated with freshly fractured silica
(p 0.05).
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Phosphorylation of MAPKs Induced by FFSi and the Decline of
Induction with Aging--
Previous studies in our laboratory have
indicated that silica-induced AP-1 activation is mediated through
phosphorylation of MAPKs family members, ERKs and p38 kinases (18). To
investigate the effects of FFSi versus ASi on activation of
MAPKs, phosphorylation of ERKs and p38 kinases was examined. JB6 cells
cultured in MEM medium in 6-well plates were exposed for 30 min to 150 µg/ml freshly fractured silica or fractured silica aged for different
time periods as indicated. Then the cells were lysed by sample buffer
and the phosphorylation of ERKs or p38 kinase was analyzed as described in the protocol of New England BioLabs. Phosphorylation of ERKs or p38
kinase was greater after exposure of the cells to FFSi than ASi (Fig.
3). These results were correlated with
the AP-1 activation induced by FFSi or ASi.

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Fig. 3.
Phosphorylation of ERKs and p38 kinase
induced by FFSi and the decline of induction with aging. JB6
P+ cells were cultured in 5% FBS MEM medium in 6-well
plates until 80% confluent and then cultured in 0.1% FBS MEM medium
for 24 h. Then the cells were exposed for 30 min to 150 µg/ml
freshly fractured silica or fractured silica aged for different time
periods as indicated. The phosphorylated and nonphosphorylated p38
kinase and ERKs proteins in the cell lysate were assayed using a
PhosphoPlus MAPKs kit from New England BioLabs. The phosphorylated
proteins and nonphosphorylated proteins were detected by using the same
transferred membrane blot following a stripping procedure.
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Effects of Antioxidant Reagents on Silica-induced AP-1 Activation
and Phosphorylation of MAPKs--
Because FFSi was more potent than
ASi, we hypothesized that ROS may be involved in silica-induced AP-1
stimulation. To examine this hypothesis, the effects of antioxidants
and other reagents on silica-induced AP-1 activation and
phosphorylation of MAPKs were investigated. JB6 cells (5 × 104) cultured in 24-well plates were pretreated for 1 h with antioxidants and other reagents. Then the cells were exposed to
150 µg/ml FFSi in the presence of the same reagents for 24 h and
the luciferase activity of the cell lysate was tested. The effects of
antioxidants and other reagents on silica-induced AP-1 activation are
shown in Fig. 4. Catalase, a
H2O2-scavenging enzyme, strongly inhibited silica-induced AP-1 activation by 90%. Sodium formate, an ·OH
radical scavenger, had no effect on the induction. SOD, a O
2 radical scavenger that generates H2O2,
increased the AP-1 activation by 70%. Deferoxamine, a metal ion
chelator, had a slight inhibitory effect. N-Acetylcysteine,
a thio-containing antioxidant, or PVPNO, which binds to silanol groups
on the silica surface, significantly inhibited the AP-1 induction.
Similar results were obtained for the effects of these reagents on
silica-induced phosphorylation of p38 kinase and ERKs. Catalase, a
H2O2 scavenger, inhibited both p38 kinase and
ERKs phosphorylation induced by silica, whereas SOD, which generates
H2O2, enhanced the phosphorylation (Fig. 5, A and B). In
contrast, formate, a ·OH scavenger, was ineffective. These
results suggest that H2O2 might be the mediator
for stimulation of p38 and ERKs signal transduction pathways leading to
AP-1 induction.

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Fig. 4.
Effect of antioxidants and other reagents on
silica-induced AP-1 activation. JB6 cells were seeded into each
well of a 24-well plate. After overnight culture at 37 °C, cells
were incubated in the same medium plus 0.1% fetal bovine serum for
24 h. The cells were then exposed for 24 h to 150 µg/ml
freshly fractured silica and various reagents as indicated. The
concentrations of the reagents used were: catalase, 10,000 units/ml;
sodium formate, 2 mM; SOD, 500 units/ml; deferoxamine, 1 mM; N-acetylcysteine, 1 mM; PVPNO,
50 µg/ml. Other experimental conditions were the same as those
described in the legend to Fig. 1. Results, presented as relative AP-1
induction compared with the untreated control cells, are means ± S.E. of eight assay wells from two independent experiments. *, a
significant decrease from cells treated with freshly fractured silica
(p 0.05). +, a significant increase from cells
treated with freshly fractured silica (p 0.05).
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Fig. 5.
Effect of antioxidant reagents on
silica-induced phosphorylation of p38 kinase and ERKs. JB6
P+ cells were cultured in 5% FBS MEM medium in 6-well
plates until 80% confluent and then cultured in 0.1% FBS MEM medium
for 24 h. The cells were then exposed to 150 µg/ml FFSi in the
presence of various antioxidants or other reagents as indicated. The
concentrations of the reagents used were: catalase, 10,000 units/ml;
sodium formate, 2 mM; SOD, 500 units/ml; PVPNO, 50 µg/ml.
The cells were lysed and phosphorylated, and nonphosphorylated p38
kinase protein (A) and ERKs proteins (B) were
assayed using a PhosphoPlus MAPKs kit from New England BioLabs. The
phosphorylated and nonphosphorylated proteins were analyzed by using
the same transferred membrane blot following a stripping
procedure.
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ROS Generation from JB6 Cells Stimulated by Silica--
To confirm
that ROS are involved in silica-induced AP-1 activation, ESR spin
trapping was used to detect ROS generation during cellular reactions in
the presence of silica (28, 29). Freshly fractured silica or JB6 cells
alone did not generate any detectable radical signal (Fig.
6, a and b).
Whereas freshly ground silica plus JB6 cells generated a spin adduct
signal (Fig. 6c) consisting of a 1:2:2:1 quartet with
hyperfine splitting of aN = aH = 14.8 G, where aN and
aH denote hyperfine splitting of the nitroxyl nitrogen and
-hydrogen, respectively. Based on this splitting and
the 1:2:2:1 line shape, the spectrum was assigned to the DMPO-OH adduct, which is an example of ·OH radical generation. Catalase
significantly decreased this signal, indicating the importance of
H2O2 (Fig. 6d). Addition of SOD
failed to inhibit the signal (Fig. 6e). Addition of
H2O2 increased the signal intensity (Fig.
6f). Because DMPO-OH signal may be generated by a mechanism
other than ·OH trapping (30), spin trapping competition
experiments were performed for verification of ·OH generation.
In these experiments the ·OH radical abstracts a hydrogen atom
from formate, resulting in a decrease of DMPO-OH spin adduct signal
intensity. As shown in Fig. 6g, addition of formate indeed
reduced the signal intensity, showing that ·OH radicals were
generated and trapped in the reaction system. Addition of deferoxamine,
which chelates the metal ions such as Fe(II) to make them less reactive
toward H2O2, also decreased the signal
intensity (Fig. 6h), indicating that a metal-mediated Fenton
or Fenton-like reaction plays a key role in ·OH generation from
the silica-stimulated cells. Addition of
N-acetyl-L-cysteine, a general antioxidant,
decreased the adduct signal (Fig. 6i). Incubation of silica
aged for 1 year with the cells produced only a very week signal (Fig.
6j).

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Fig. 6.
ESR signals generated by DMPO-OH adducts
obtained from JB6 cells or cells treated with silica. ESR spectra
recorded for 1 min from incubation mixtures containing PBS and 100 mM DMPO: (a) freshly fractured silica (150 µg/ml); (b) JB6 cells (1 × 106);
(c) JB6 cells (1 × 106) plus freshly
fractured silica (150 µg/ml); (d) same as c but
with 1000 units/ml catalase; (e) same as c but
with 2000 units/ml SOD; (f) same as c but with 1 mM of H2O2; (g) same as
c but with 50 mM sodium formate; (h)
same as c but with 1 mM deferoxamine;
(i) same as c but with 10 mM
N-acetyl-L-cysteine; (j) JB6 cells
(1 × 106) and 150 µg/ml 1-year-old silica. The
spectrometer settings were: receiver gain, 2.5 × 105;
time constant, 0.3 s; modulation amplitude, 1.0 G; scan time, 3 min; magnetic field, 3340 ± 100 G.
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The above results suggest that H2O2 might
play a key role on silica-induced AP-1 and MAPK activation (Figs. 4 and
5). Next, we measured the H2O2 generation by
JB6 cells upon stimulation with silica. JB6 cells were incubated with
FSi (1 mg/ml) for 30 min, and the generation of
H2O2 was measured as described under "Materials and Methods." As shown in Fig.
7, the generation of H2O2 by JB6 cells treated with FSi was markedly
increased compared with untreated control cells. Catalase substantially
inhibited silica-induced H2O2 generation by JB6
cells. In contrast, SOD had no significant effect. These results are
parallel and comparable with the studies on the effect of catalase and
SOD on silica-induced AP-1 and MAPK activation (Figs. 4 and 5). The
data also provide more evidence for the important role of
H2O2 on silica-induced AP-1 activation.

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Fig. 7.
H2O2 generation from
JB6 cells treated with silica. JB6 cells (1 × 106) were incubated with freshly fractured silica (1 mg/ml)
with or without reagents as indicated for 30 min at 37 °C.
Generation of H2O2 by the cells was monitored
as described under "Materials and Methods." The concentration of
reagents used were: catalase, 2000 units/ml; SOD, 1000 units/ml. Values
are means ± S.E. of three experiments. *, a significant increase
from cells alone (p 0.05). , a significant
decrease from cells plus silica (p 0.05).
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Detection of ROS Generation in Intact Cells--
To further
confirm the ROS generation by JB6 cells stimulated with silica, cells
treated with silica were analyzed by intracellular staining for
H2O2 and O
2. H2DCFDA, a
specific fluorescent dye for H2O2, and
dihydroethidium, a specific fluorescent dye for O
2, were
applied to cells to monitor ROS generation. In the presence of FFSi,
the fluorescent signals of both H2O2 and
O
2 were dramatically increased within 30 min after treatment
of the cells (Fig. 8). In the presence of
silica aged for 1 year, the signals were substantially less intense
than those for the cells treated with FFSi (Fig. 8). Fluorescence was
displayed using a pseudo-color intensity scale where low intensity
sites appear blue, and increasingly high intensity areas are
displayed as green, yellow, red, or
white.

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Fig. 8.
Confocal micrograph of O 2 and
H2O2 generation in intact cells treated with
silica. JB6 cells were treated with 150 µg/ml FFSi in the
presence of 2 µM dihydroethidium or 5 µM
H2DCFDA for 30 min. The cells were washed once with PBS and
fixed with 10% buffered formalin. The images were captured with a
laser scanning confocal microscope. The bright greenish yellow
areas in the cells represent oxidized DCFH-DA, and the
bright reddish orange spots represent oxidized
dihydroethidium showing the intracellular localization of
H2O2 and O 2, respectively.
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Increased Oxygen Consumption during the Interaction of JB6 Cells
with Silica--
The above results indicate that O
2 and
H2O2 are generated during the interaction of
JB6 cells with silica. If this is the case, exposure of the cells to
silica should result in an increased rate of oxygen consumption. To
test this hypothesis the rate of oxygen consumption by JB6 cells
(6.25 × 105) in the presence or absence of fresh or
aged silica was monitored using a Gilson Oxygraph. The results are
presented in Fig. 9. Cells alone consumed
molecular oxygen at a steady basal rate, whereas in the presence of
FFSi oxygen consumption increased by 33%. Dismutation of O
2
by SOD resulted in about half the amount of O2 consumption
(data not shown). In contrast, when cells were incubated with
1-year-old ASi, the oxygen consumption was no significantly different
from the basal level. In the presence of PVPNO, the oxygen consumption
by the cells treated with FFSi was significantly decreased.

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Fig. 9.
Oxygen consumption of JB6 cells. Oxygen
consumption by JB6 cells (6.25 × 105) in PBS with or
without silica. Bar 1, cells in PBS; bar 2, cells
treated with 150 µg/ml freshly fractured silica; bar 3,
cells treated with 150 µg/ml fractured silica aged for 1 year;
bar 4, cells treated with freshly fractured silica (150 µg/ml) in the presence of PVPNO (50 µg/ml). Each bar
indicates the mean ± S.E. of three experiments. *, a significant
increase from control (p 0.05). , a significant
decrease from FFSi (p 0.05).
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DISCUSSION |
Crystalline silica is a recently designated carcinogen and is
strongly associated with silicosis, but the molecular and cellular mechanisms involved in the silica-induced pathogenesis are not fully
understood (3, 17). We hypothesize that silica-mediated free radical
reactions may cause a persistent oxidative stress in the lung and play
a key role in the mechanism of silica-induced carcinogenesis. Most of
the studies concerning the role of ROS in silica-induced cytotoxicity
have been limited to lipid peroxidation, DNA damage, and other critical
changes in noncellular systems (10-14). However, our previous study
has shown that silica induces AP-1 activation in cell and animal models
through ERKs and p38 kinase pathways (18). In the present study, we
show that ROS is the mediator for silica-induced AP-1 activation. By
comparing of AP-1 activation induced by FFSi versus ASi, we
found that AP-1 activation induced by FFSi was 4-fold higher than that
of silica fractured and aged for 1 year. The enhanced potency of FFSi
was also exhibited for silica-induced phosphorylation of ERKs and p38
kinase. We also demonstrate that H2O2, formed
during the interaction of silica with the cells, might be a reactive
intermediate responsible for silica-induced AP-1 activation and
phosphorylation of MAPKs.
The results from the present study show that silica-induced AP-1
activation involves ROS-mediated reactions. A major role of
H2O2 in silica-induced AP-1 activation is
supported by the following observations: (a) catalase, whose
function is to remove H2O2, blocked the AP-1
activation and phosphorylation of MAPKs; (b) SOD, which
converts O
2 to H2O2, enhanced AP-1
activation; (c) sodium formate, a scavenger of ·OH
radical, did not exhibit any effect; and (d) treatment of
cells with freshly fractured silica resulted in a 9.5-fold increase in
H2O2 production. As for ROS generation, it may
be noted that our earlier studies (12-15) have shown that FFSi
generates silicon-based radicals (Si·, SiO·,
and SiOO·). These silicon radicals can generate ROS upon
reaction with aqueous medium. The present study shows that silica is
also able to generate ROS via stimulation of cells and that this ROS
production occurs at a lower concentration of silica than in
noncellular systems. During these processes, molecular oxygen was
consumed to generate O
2 radical, which produced
H2O2 by dismutation.
H2O2 generated the ·OH radical via a
Fenton or Fenton-like reaction. The following experimental observations
support the above pathway of ROS generation from silica-stimulated
cells: (a) Oxygen consumption assay showed that
silica-stimulated cells consumed molecular oxygen; (b) both O
2 and H2O2 were generated as measured
by fluorescence staining of silica-exposed cells; (c) ESR
spin trapping studies showed that ·OH radicals were generated;
(d) catalase inhibited the signal, whereas
H2O2 enhanced it; (e) addition of
SOD, whose function is to remove O
2, did not substantially
alter ·OH generation; and (f) addition of
deferoxamine, which chelates metal ions such as Fe(II) to make them
less reactive toward H2O2, reduced ·OH generation.
The signal transduction pathways leading to AP-1 activation and the
possible involvement of ROS were also investigated in the present
study. It is well known that stress-related signals, such as UV
radiation or ROS, induce the activation of MAPK pathways. ERKs, JNKs,
and p38 are important signal transduction pathways involved in AP-1
activation, and AP-1 is one of the downstream targets of these three
MAPK members. An earlier study has shown that FFSi caused
phosphorylation of ERKs and p38, but not JNKs (18). The results
obtained from the present study show that FFSi caused phosphorylation
of both p38 and MAPKs to a greater degree than that induced by ASi.
Moreover, catalase inhibited the silica-induced phosphorylation of ERKs
and p38 kinase, suggesting that H2O2 is
required in the phosphorylation process. These results further suggest
that H2O2 plays a key role in AP-1 activation.
It should be noted that PVPNO significantly inhibited silica-induced
AP-1 activation and phosphorylation of MAPKs. SiOH groups on the silica
surface have been proposed to be involved in silica-induced cellular
damage. Chemical modification of the silica surface can be used to
reduce toxicity in vitro and fibrosis in vivo
(31). It is known that when silica particles are exposed to water,
surface silicon-oxygen bonds (Si-O) are hydrated, resulting in the
formation of SiOH groups. PVPNO is able to bind to SiOH groups. It has
been reported that PVPNO inhibits silica-induced toxicity and decreases or delays the development of silicosis in experimental animals and
humans. It also blocks the interaction of the silica surface with
phosphate groups of DNA in vitro. It has been reported that PVPNO inhibits silica-induced ROS generation in cells (31, 32).
The present study used mouse epidermal cells as an in vitro
model system. These epidermal cells were employed, because they exhibit a stronger although qualitatively similar response compared with bronchial epithelial cells (18). Furthermore, in our previous studies, we have shown that in vivo responses in transgenic
mice were qualitatively similar to studies in epidermal cells (18).
Our studies show that FFSi particles are much more potent in inducing
AP-1 activation than are ASi. Upon stimulation by silica, JB6 cells are
able to generate a whole spectrum of ROS. Compared with a direct
reaction between silica with aqueous medium, the yield of ROS
generation from silica-stimulated cells is much higher. Among the ROS
produced, H2O2 appears to be the species
responsible for silica-induced AP-1 activation. Our novel findings
suggest that ROS may play a key role in silica-induced oncogene
stimulation. Because oncogene stimulation is thought to be involved in
carcinogenesis, there is a need to study further the mechanisms
involved in this process.