1 Pneumoconiosis Division, School of Medicine, Zhejiang University, Hangzhou 310013, People's Republic of China; and 2 Department of Community, Occupational, and Family Medicine, National University of Singapore, Singapore 119260, Republic of Singapore
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ABSTRACT |
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The main objective of this study was to evaluate the critical role of glutathione (GSH) in silica-induced oxidative stress, cytotoxicity, and genotoxicity in rat alveolar macrophages (AMs). Silica-induced superoxide radical and hydrogen peroxide formation were determined with lucigenin-dependent chemiluminescence and 2',7'-dichlorofluorescin diacetate fluorescence test, respectively. The cytotoxicity of silica was estimated by lactate dehydrogenase leakage, and a comet assay was used for examining silica-induced DNA damage in AMs. The intracellular GSH content was modulated by N-acetylcysteine, a GSH precursor, and buthionine sulfoximine, a specific GSH synthesis inhibitor. It was found that silica led to a dose- and time-dependent decrease in GSH content in AMs. N-acetylcysteine increased intracellular GSH level and protected against silica-induced reactive oxygen species formation, lactate dehydrogenase leakage, and DNA strand breaks in AMs. In contrast, buthionine sulfoximine pretreatment depleted cellular GSH and enhanced the susceptibility of AMs to the cytotoxic and genotoxic effects of silica. It thus appears that GSH plays a critical role in protecting against silica-induced cell injury, most probably through its antioxidant activity.
glutathione; reactive oxygen species; fibrosis; cancer
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INTRODUCTION |
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SILICOSIS is among the oldest pneumoconiosis known to be associated with occupational exposure to crystalline silica. Although the mechanisms of silicosis are still largely unknown, it is well recognized that alveolar macrophages (AMs) are the main target cells, and pulmonary inflammation and subsequent pulmonary fibrosis are the two major pathological events after silica exposure (13, 26).
In recent years, the role of reactive oxygen species (ROS) formation and oxidative stress in silica-induced lung injury have been well established based on the following evidence: 1) production of various species of ROS by silica in cell-free systems measured by electron spin resonance spin trapping (31, 37); 2) silica-induced oxidative damage such as lipid peroxidation and oxidative DNA damage in vitro (12, 32); 3) elevated level of ROS, oxidative damage, and the upregulation of the antioxidant mechanisms in silicotic lungs under in vivo conditions (21, 35); and 4) protective effects of antioxidants against silica-induced cytotoxicity (19, 33). Moreover, a recent study in our laboratory further demonstrated that ROS mediate silica-induced cytotoxicity and genototxicity in AMs (Z. Zhang, Shen, Q.-F. Zhang, and Ong, unpublished data).
Under normal circumstances, there is a delicate balance between ROS formation and antioxidant defenses in lungs. When the generation of ROS is overwhelming, as in the case of silica exposure, or the antioxidant defense mechanism is impaired, an oxidative stress is induced, resulting in cell injury. Glutathione (GSH) is among the most important antioxidants in organisms due to its potent antioxidant capacity, close involvement in many cellular functions, and abundance in tissues or cells (24, 25). It is well known that GSH plays an important role in the antioxidant mechanism in lungs. For instance, it has been noted that the epithelial lining fluid of normal lungs contains very high concentrations of this tripeptide, ~100 times higher than that found in the extracellular fluid of many other tissues (38). Some preliminary studies showed the involvement of GSH in silica-induced lung injury. An in vitro study by Boehme et al. (5) showed that silica treatment led to a decrease in intracellular GSH level in AMs and an increase in GSH level in culture medium, indicating the release of GSH by AMs. GSH content was decreased in silicotic lung tissues in rats in the early stage (15) and increased in the late stage (40). Epidemiologic data also showed that GSH in red blood cells was increased significantly in silicosis patients (6). However, so far there is little or no direct evidence showing the role of GSH in silica-induced cytotoxicity and genotoxicity in AMs. The main objective of the present study was to evaluate the role of GSH on silica-induced oxidative stress, cytotoxicity, and genotoxicity in AMs, the principal target cells of silica. In the present investigation, the intracellular GSH level in AMs was modulated by N-acetylcysteine (NAC), a GSH precursor, and buthionine sulfoximine (BSO), a specific GSH synthesis inhibitor. The data from these experiments provide direct evidence showing that GSH plays a critical role in silica-induced oxidative stress and cell injury.
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EXPERIMENTAL PROCEDURES |
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Chemicals. Standard crystalline silica was obtained from the Institute of Occupational Medicine, Chinese Academy of Preventive Medicine (Beijing, China). Ninety-five percent of the particles were <5 µm in diameter. Lucigenin and 2',7'-dichlorofluorescin diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, OR). NAC, BSO, GSH, o-phthalaldehyde (OPT), penicillin, streptomycin, and ethidium bromide were all from Sigma (St. Louis, MO). RPMI 1640 medium, fetal bovine serum (FBS), low-melting- and normal-melting-point agarose were from GIBCO BRL (Life Technologies, Gaithersburg, MD).
Isolation and primary culture of AMs. Male Sprague-Dawley rats (body weight 220-250 g) were provided by the Animal Center, National University of Singapore. The rats were anesthetized with an intraperitoneal injection of a mixture of fentanyl citrate (0.063 mg/rat), fluanisone (2 mg/rat; both from Jassen), and midazolam (1 mg/rat; Hoffmann-La Roche). AMs were collected by lavaging isolated rat lungs with PBS after the animals were killed by bloodletting in the femoral artery. AMs were washed three times with PBS and cultured in RPMI 1640 medium containing 10% FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin in a 5% CO2 incubator at 37°C.
The stock suspension of silica was prepared in PBS, and the exposure concentration is expressed in micrograms per 106 cells.
The effects of NAC and BSO were examined by pretreating the cells for 12 h in FBS-free medium; silica was then directly added without washing the cells with PBS.
Determination of intracellular GSH content. The determination of intracellular GSH content was conducted according to Hissin and Hilf's (17) method with modification. The stock solution of the fluorescent probe OPT (1 mg/ml) was freshly prepared in methanol. After various designated treatments, AMs were collected with cell scrapers and washed twice with PBS (800 rpm for 5 min at 4°C) and then resuspended in 0.1 M sodium phosphate-5 mM EDTA (pH 8.0). After the total cell number was counted and the cells were thoroughly disrupted by ultrasonication, the cell homogenate (0.75 ml) was mixed with 25% metaphosphoric acid (0.25 ml) to precipitate the proteins. After centrifugation (14,000 rpm for 10 min at 4°C), the supernatant (0.2 ml) was mixed with 0.1 M sodium phosphate-5 mM EDTA (1.7 ml) and OPT (0.1 ml of stock solution, final concentration 50 µg/ml). The fluorescence intensity of OPT was monitored with an excitation wavelength at 350 nm and emission wavelength at 420 nm (Perkin-Elmer LS-5B luminescence spectrometry). A GSH calibration curve was established with standard GSH, and the GSH concentration is expressed in nanomoles per 106 cells.
Determination of lactate dehydrogenase leakage. Activity of lactate dehydrogenase (LDH) in the medium was measured with an Abbott (Chicago, IL) VP biochemical analyzer with a test kit as established earlier in our laboratory (27). Total LDH activity was also detected after the cells were disrupted by ultrasonication. LDH leakage (in percent) was calculated as (LDH activity in medium/total LDH activity) × 100.
Analysis of DNA damage. DNA damage was detected with single-cell gel electrophoresis (or comet assay) according to the method described earlier (41) with modifications. Briefly, fully frosted slides were covered with 0.7% normal-melting agarose as the first layer, a mixture of AMs and 0.7% low-melting agarose as the second layer, and 0.7% low-melting agarose as the third layer. After solidification at 4°C, the slides were immersed in lysing buffer (2.5 mM NaCl, 100 mM Na2EDTA, and 10 mM Tris, pH 10, with freshly added 1% Triton X-100 and 10% DMSO) at 4°C for 1 h. The slides were then placed in an electrophoresis tank filled with freshly prepared electrophoresis solution (300 mM NaOH and 1 mM Na2EDTA, pH 13) for 20 min. Electrophoresis was conducted at 4°C for 20 min (25 V and 0.3 A). The slides were then neutralized in neutralization buffer (0.4 M Tris · HCl, pH 7.5), stained with ethidium bromide, and examined under a fluorescence microscope (Nikon). Images of 100 randomly selected cells from each slide were analyzed. The degree of DNA damage was divided into 5 categories according to the percentage of DNA in the tail: grade 0, no damage, <5%; grade 1, low-level damage, 5-20%; grade 2, medium-level damage, 20-40%; grade 3, high-level damage, 40-95%; and grade 4, total damage, >95%.
Lucigenin-dependent chemiluminescence test. Lucigenin-dependent chemiluminescence (CL) in AMs was determined as described earlier (29). The basic reaction mixture contained 1 × 106 cells and 100 µM lucigenin in 1 ml of PBS with and without the presence of silica. The CL reaction was initiated by the simultaneous addition of lucigenin and silica, and the CL level was monitored as relative light units with a luminometer (Lumi-One, Tampa, FL) for a total period of 10 min at room temperature.
Analysis of DCFH-DA fluorescence in
AMs. The level of hydrogen peroxide
(H2O2)
in AMs was measured with DCFH-DA as a fluorescence probe (28). The
principle of this assay is that DCFH-DA diffuses through the cell
membrane and is enzymatically hydrolyzed by intracellular esterases to
nonfluorescent DCFH. In the presence of ROS (mainly H2O2),
this compound is rapidly oxidized to highly fluorescent dichlorofluorescein (DCF) (4, 22). DCFH-DA was dissolved in absolute
ethanol at a concentration of 5 mM as a stock solution and kept at
70°C in the dark. AMs were incubated in 24-well plates, each
well containing 1.5 × 105
AMs and 2 µM DCFH-DA in 2 ml of culture medium. The reaction was
initiated by the addition of DCFH-DA and incubated at 37°C for up
to 4 h. The fluorescence intensity was measured with a plate reader
(Tecan Spectrafluo-Plus), with an excitation wavelength of 485 nm and
an emission wavelength of 535 nm.
Analysis of data. All data were based on at least three independent experiments and analyzed with one-way ANOVA with Scheffé's test and are presented as means ± SD. A P value < 0.05 was considered as significant.
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RESULTS |
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Effects of silica on intracellular
GSH. Figure
1A shows
the dose-dependent decrease in intracellular GSH when cells were
treated with silica for 4 h. With the highest concentration of silica (100 µg/106 cells), the GSH
level was only one-third of the control value. It was also noted that
the silica-induced decline in intracellular GSH level was time
dependent (Fig. 1B). The GSH
concentration in the control cells remained at a constant level (~3.3
nmol/106 cells), whereas the GSH
level in the cells treated with silica (100 µg/106 cells) decreased
dramatically from 1 h onward.
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Effects of NAC and BSO on intracellular GSH
content. In the present study, AMs were pretreated with
1 mM NAC and 1 mM BSO for 12 h before exposure to silica. Compared with
the control cells, NAC significantly enhanced the intracellular GSH
content of AMs, whereas BSO pretreatment markedly decreased the GSH
level in AMs (Fig. 2). Although the
subsequent silica exposure significantly reduced the GSH level, those
cells with NAC pretreatment contained a higher level of GSH compared
with the cells treated with silica only. On the other hand, BSO
pretreatment further enhanced the extent of GSH depletion caused by
silica exposure because the cells with both BSO and silica treatment
displayed the lowest GSH level among all these groups (Fig. 2).
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Effects of NAC and BSO on silica-induced
cytotoxicity. Figure 3
shows the different effects of NAC and BSO on silica-induced cytotoxicity as measured by the percentage of LDH leakage. After 16 h
of incubation (12 h of pretreatment plus 4 h of silica incubation time), LDH leakage in the control, BSO-only, and NAC-only cells was
5.92 ± 1.70, 8.22 ± 0.66, and 6.82 ± 2.22%, respectively. These data suggest that BSO or NAC pretreatment alone did not cause any
significant damage to the cell. In contrast, NAC pretreatment significantly reduced the LDH leakage induced by silica, whereas the
cells pretreated with BSO showed a marked increase in LDH leakage
compared with those cells exposed to silica only.
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Effects of NAC and BSO on silica-induced
genotoxicity. The effects of NAC and BSO on
silica-induced DNA damage were examined with a comet assay. Figure
4 presents the data showing the different effects of NAC and BSO on the percentage of cells with
grade 3 and
4 damage (cells with >40% DNA in
the tail) caused by silica. NAC or BSO pretreatment alone did not cause
significant DNA damage because >95% of the cells were
grade 0 (data not shown). However, NAC
was able to protect against silica-induced DNA strand breaks. The
percentage of cells with grade 3 and
4 damage decreased from ~55% in
silica-treated AMs to 26% in cells pretreated with NAC. In contrast,
BSO pretreatment tended to enhance the extent of DNA damage induced by
silica. The percentage of cells with grade 3 and 4 damage
increased up to 67%. The above changes were generally consistent with
the pattern observed in the cytotoxicity test (Fig. 3).
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Effects of NAC and BSO on silica-induced
O2· and
H2O2 formation in
AMs.
In this study, a lucigenin-dependent CL test was used for the
determination of O
2· formation and
a DCFH-DA fluorescence test was used for measurement of intracellular H2O2
formation. The results are summarized in Figs.
5 and 6, respectively. As
shown in Figs. 5 and 6, NAC pretreatment did not change the O
2· level either in control cells
or in silica-exposed AMs. In contrast, BSO pretreatment significantly enhanced the O
2· level in both
control cells and silica-treated AMs (Fig. 5). The results in Fig. 6
show that NAC was able to significantly reduce the ROS level in both the control and silica-treated rat AMs. The DCF fluorescence intensity in NAC-pretreated cells decreased nearly 80% compared with that in the
cells treated with silica alone. On the other hand, BSO pretreatment
enhanced the ROS level in control cells as well as in silica-treated
AMs, which is similar to its effect on
O
2· formation (Fig. 5).
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DISCUSSION |
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The involvement of ROS and oxidative damage in silica-induced pulmonary inflammation, fibrosis, and carcinogenesis has been extensively studied (30, 36). It is well known that GSH is the major intracellular antioxidant with multiple biological functions. One of its most important functions is to protect against oxidative damage caused by ROS through enzymatic and nonenzymatic reactions (24, 25). Therefore, it will be of interest to evaluate the effect of GSH on silica toxicity. The present study was thus undertaken to assess the role of intracellular GSH on silica-induced oxidative stress, cytotoxicity, and genotoxicity in primary cultured rat AMs. The intracellular GSH level was modulated with NAC, a GSH precursor, and BSO, a specific GSH synthesis inhibitor. The results obtained show that NAC was able to increase the intracellular GSH content in AMs and suppress silica-induced ROS formation, LDH leakage, and DNA damage. In contrast, BSO pretreatment led to intracellular GSH depletion and enhanced the susceptibility of AMs to silica-induced oxidative stress and cell injury. Therefore, the present study provides convincing evidence supporting the notion that GSH plays an important role in silica-induced oxidative stress, cytotoxicity, and genotoxicity in AMs.
The present study demonstrates that the intracellular GSH level decreased after silica exposure in a dose- and time-dependent manner. Compared with that in the untreated control cells, the GSH content was reduced >70% when AMs were treated with 100 µg silica/106 cells for 4 h (Fig. 1B). This finding is essentially consistent with an earlier report by Boehme et al. (5) that silica exposure leads to GSH release from AMs. An in vivo study also showed that the concentrations of antioxidant molecules, including GSH, decreased in lung tissues after intratracheal instillation of silica dust (15). On the other hand, it is noted that there was a maximal decrease in intracellular GSH in AMs 2 and 4 h after silica exposure (Fig. 1B), which coincides with the dramatic increase in LDH leakage at the same time points after silica treatment. The percentage of LDH leakage in silica-treated cells at 1, 2, and 4 h was 14.6, 30.1, and 75.4%, respectively, whereas the percentage of LDH leakage in the control cells was consistently <12% (Z. Zhang, Shen, Q.-F. Zhang, and Ong, unpublished data). It is believed that there are three possible mechanisms accounting for the decline in cellular GSH in response to silica exposure: 1) via GSH peroxidase (GPx) reaction, 2) via the GSH transferase reaction, and 3) via GSH efflux (5, 14). GPx mainly catalyzes the direct reaction of GSH with ROS such as H2O2 and hydroxyl radical, resulting in the formation of GSSG. Based on our preliminary investigations, the concentration of GSSG in AMs did not change significantly compared with that in the control cells (data not shown), indicating that GPx may not play a major role in silica-induced GSH depletion. Moreover, the close resemblance of the time course of GSH with that of LDH leakage tends to suggest that GSH efflux is an important mechanism accounting for silica-induced intracellular GSH depletion in AMs.
The availability of cysteine is one of the speed-limiting factors in
GSH synthesis, and NAC acts as a precursor of GSH to facilitate GSH
synthesis (11). In this study, NAC pretreatment was able to increase
the intracellular GSH level and inhibit silica-induced LDH leakage and
DNA strand breaks (Figs. 2-4), which is consistent with an earlier
study (39) showing that the addition of NAC was responsible for a
decrease in silica-induced cytotoxicity in AMs. Furthermore, the
present study also examined the effects of NAC on silica-induced ROS
formation. It is interesting to note that NAC pretreatment
significantly suppressed
H2O2
formation (Fig. 6), whereas no observable inhibitory effect was found
on the production of O2· in
silica-treated cells (Fig. 5). The differential effects of NAC on
different species of ROS were also observed in an earlier study by
Aruoma et al. (2). They demonstrated that NAC is a potent scavenger of
H2O2 and hydroxyl radicals, whereas the scavenging activity of NAC against
O
2· was found to be rather weak. At present, the antioxidant property of NAC has been well
characterized, and it is known that NAC acts as a potent antioxidant
through the following two mechanisms:
1) as a precursor of GSH to
facilitate intracellular GSH synthesis and
2) as a direct ROS scavenger (2, 11). Therefore, the inhibitory effects of NAC against silica-induced ROS formation, LDH leakage, and DNA strand breaks clearly indicate the
important role of GSH in protection against silica toxicity in AMs.
Some earlier studies (7, 8) in animals and humans used aerosolized GSH
to enhance pulmonary GSH content and to counteract the imbalance of
oxidant-antioxidant in idiopathic pulmonary fibrosis. Based on the in
vitro studies presented here, NAC appears to be more suitable for the
augmentation of pulmonary GSH level in silicosis and other lung
diseases to prevent pulmonary injury and fibrosis.
BSO is a specific inhibitor of -glutamylcysteine synthetase, the key
enzyme in intracellular GSH synthesis. It has been widely used for
depleting intracellular GSH in various cells and tissues (1, 34). In
the present study, BSO pretreatment led to a 70% reduction in GSH
content in control cells and a further decline in GSH level in
silica-treated AMs (Fig. 2). Accordingly, the cells pretreated with BSO
became more susceptible to the toxic effects of silica as shown by the
significantly increased LDH leakage and DNA strand breaks (Figs. 3 and
4). A previous study (23) evaluated the effect of nonprotein sulfhydryl
moieties (including GSH) depletion caused by BSO on silica-induced
inflammation and fibrosis in the mouse lung, and the results suggested
that GSH is able to lessen the potential of silica in eliciting acute lung injury. Results from the present study also showed that
BSO-pretreated AMs generated a higher level of ROS in both the control
and silica-exposed cells (Figs. 5 and 6), indicating that GSH mainly
acts as an ROS scavenger to protect against silica-induced cell injury.
The aggravating effect of BSO on silica-induced oxidative stress,
cytotoxicity, and genotoxicity further supports the notion that GSH
plays a critical role in the toxicity of silica. It seems that
silica-induced ROS formation led to the depletion of GSH and impairment
of the antioxidant system, which, in turn, exacerbates the oxidative damage in silica-exposed cells.
Silica has recently been classified as a confirmed human carcinogen
(20). Silica-elicited DNA damage and changes in cell proliferation are
the two key factors related to its carcinogenicity (30). It has been
proposed that the silica-induced DNA damage is caused by two majors
mechanisms, free radical mediation and direct binding with DNA (30). An
early study showed that asbestos-induced oxidative DNA damage
(8-hydroxydeoxyguanosine formation) was augmented by BSO and
ameliorated by NAC, indicating that GSH modulates the genotoxic effects
of asbestos (18). In the present study, silica-induced DNA strand
breaks were evaluated with a comet assay, and the inhibitory effect of
NAC and the aggravating effect of BSO on silica-induced DNA damage
provide the first evidence showing the protective role of GSH in the
genotoxicity of silica. On the other hand, changes in cell
proliferation, apoptosis, activation of transcription factors, and
induction of oncogene expression are believed to be closely related to
the carcinogenic effect of silica (30). For instance, it has been found
that silica is a potent inducer of nuclear transcription factor nuclear
factor-B, most probably through the production of ROS (9, 10).
Nuclear factor-
B is one of the key molecules controlling many
important functions such as immunity, inflammation, and apoptosis (3).
GSH has been suggested to play an inhibitory role in its activation
(16). Therefore, further studies on the involvement of GSH in
silica-induced DNA damage and changes in cell proliferation will
certainly help to obtain a better understanding of the carcinogenicity
of silica.
In summary, the present study evaluated the critical role of GSH in silica-induced oxidative stress, cytotoxicity, and genotoxicity in cultured rat AMs. It is believed that the understanding of the importance of GSH will be beneficial to the development of preventive or therapeutic agents in control of silica-induced pulmonary fibrosis and carcinogenesis.
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ACKNOWLEDGEMENTS |
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We thank H.Y. Ong and Y. L. Chew for technical assistance.
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FOOTNOTES |
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This study was supported by the Program on Environmental and Occupational Health, China Medical Board (New York, NY) and the Center for Environmental and Occupational Health, National University of Singapore.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C.-N. Ong, Dept. of Community, Occupational, and Family Medicine, National Univ. of Singapore, Singapore 119260, Republic of Singapore (E-mail: cofongcn{at}nus.edu.sg).
Received 16 February 1999; accepted in final form 18 May 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, M. E.
Glutathione: an overview of biosynthesis and modulation.
Chem. Biol. Interact.
111-112:
1-14,
1998.
2.
Aruoma, O. I.,
B. Halliwell,
B. M. Hoey,
and
J. Butler.
The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid.
Free Radic. Biol. Med.
6:
593-597,
1989[Medline].
3.
Baeuerle, P. A.,
and
D. Baltimore.
NF-kappa B: ten years after.
Cell
87:
13-20,
1996[Medline].
4.
Bass, D. A.,
J. W. Parce,
L. R. Dechatelet,
P. Szejda,
M. C. Seeds,
and
M. Thomas.
Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation.
J. Immunol.
130:
1910-1917,
1983
5.
Boehme, D. S.,
K. R. Maples,
and
R. F. Henderson.
Glutathione released by pulmonary alveolar macrophages in response to particles in vitro.
Toxicol. Lett.
60:
53-60,
1992[Medline].
6.
Borm, P. J.,
A. Bast,
E. F. Wouters,
J. J. Slangen,
G. M. Swaen,
and
T. de Boorder.
Red blood cell anti-oxidant parameters in silicosis.
Int. Arch. Occup. Environ. Health
58:
235-244,
1986[Medline].
7.
Borok, Z.,
R. Buhl,
G. J. Grimes,
A. D. Bokser,
R. C. Hubbard,
K. J. Holroyd,
J. H. Roum,
D. B. Czerski,
A. M. Cantin,
and
R. G. Crystal.
Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis.
Lancet
338:
215-216,
1991[Medline].
8.
Buhl, R.,
C. Vogelmeier,
M. Critenden,
R. C. Hubbard,
R. F. Hoyt, Jr.,
E. M. Wilson,
A. M. Cantin,
and
R. G. Crystal.
Augmentation of glutathione in the fluid lining the epithelium of the lower respiratory tract by directly administering glutathione aerosol.
Proc. Natl. Acad. Sci. USA
87:
4063-4067,
1990[Abstract].
9.
Chen, F.,
D. C. Kuh,
S. Sun,
L. J. Gaydos,
and
L. M. Demers.
Essential role of NF-B in silica-induced inflammatory mediator production in macrophages.
Biochem. Biophys. Res. Commun.
214:
985-992,
1995[Medline].
10.
Chen, F.,
Y. Lu,
L. M. Demers,
Y. Rojanasakul,
X. Shi,
V. Vallyathan,
and
V. Castranova.
Role of hydroxyl radical in silica-induced NF-kappa B activation in macrophages.
Ann. Clin. Lab. Sci.
28:
1-13,
1998[Abstract].
11.
Cotgreave, I. A.
N-acetylcysteine: pharmacological considerations and experimental and clinical applications.
Adv. Pharmacol.
38:
205-227,
1997[Medline].
12.
Daniel, L. N.,
Y. Mao,
T. C. Wang,
C. J. Markey,
X. Shi,
and
U. Saffiotti.
DNA strand breakage, thymine glycol production, and hydroxyl radical generation induced by different samples of crystalline silica in vitro.
Environ. Res.
71:
60-73,
1995[Medline].
13.
Davis, G. S.
Pathogenesis of silicosis: current concepts and hypothesis.
Lung
164:
139-154,
1986[Medline].
14.
Deneke, S. M.,
and
B. L. Fanburg.
Regulation of glutathione.
Am. J. Physiol.
257 (Lung Cell. Mol. Physiol. 1):
L163-L173,
1989
15.
Ghio, A. J.,
R. H. Jaskot,
and
G. E. Hatch.
Lung injury after silica instillation is associated with an accumulation of iron in rats.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L686-L692,
1994
16.
Ginn-Pease, M. E.,
and
R. L. Whisler.
Optimal NF kappa B mediated transcriptional responses in Jurkat T cells exposed to oxidative stress are dependent on intracellular glutathione and costimulatory signals.
Biochem. Biophys. Res. Commun.
226:
695-702,
1996[Medline].
17.
Hissin, P. J.,
and
R. A. Hilf.
Fluorometric method for determination of oxidized and reduced glutathione in tissues.
Anal. Biochem.
74:
214-226,
1976[Medline].
18.
Howden, P. J.,
and
S. P. Faux.
Glutathione modulates the formation of 8-hydroxydeoxyguanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA100 induced by mineral fibres.
Carcinogenesis
17:
2275-2277,
1996[Abstract].
19.
Huang, S. H.,
S. Leonard,
X. Shi,
M. R. Goins,
and
V. Vallyathan.
Antioxidant activity of lazaroid (U-75412E) and its protective effects against crystalline silica-induced cytotoxicity.
Free Radic. Biol. Med.
24:
529-536,
1998[Medline].
20.
International Agency for Research on Cancer.
IARC Working Group in the evaluation of carcinogenic risks to humans: silica, some silicates, coal dust, and para-aramid fibrils.
IARC Monogr. Eval. Carcinog. Risks Hum.
68:
41-242,
1997[Medline].
21.
Janssen, Y. M.,
J. P. Marsh,
M. P. Absher,
D. Hemenway,
K. P. M. Vace,
K. O. Leslie,
P. J. Born,
and
B. T. Mossman.
Expression of antioxidant enzymes in rat lung after inhibition of asbestos or silica.
J. Biol. Chem.
267:
10625-10630,
1992
22.
LeBel, C. P.,
H. Ischiropoulos,
and
S. C. Bondy.
Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress.
Chem. Res. Toxicol.
5:
227-231,
1992[Medline].
23.
Lombard-Gillooky, K.,
and
A. K. Hubbard.
Modulation of silica-induced lung injury by reducing lung non-protein sulfhydryls with buthionine sulfoximine.
Toxicol. Lett.
66:
305-315,
1993[Medline].
24.
Meister, A.
Glutathione, ascorbate, and cellular protection.
Cancer Res.
54:
1968s-1975s,
1994.
25.
Meister, A.,
and
M. E. Anderson.
Glutathione.
Annu. Rev. Biochem.
52:
711-760,
1983[Medline].
26.
Mossman, B. T.,
and
A. Churg.
Mechanisms in the pathogenesis of asbestosis and silicosis.
Am. J. Respir. Crit. Care Med.
157:
1666-1680,
1998
27.
Shen, H. M.,
C. N. Ong,
and
C. Y. Shi.
Involvement of reactive oxygen species in aflatoxin B1-induced cell injury in cultured rat hepatocytes.
Toxicology
99:
115-123,
1995[Medline].
28.
Shen, H. M.,
C. Y. Shi,
Y. Shen,
and
C. N. Ong.
Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B1.
Free Radic. Biol. Med.
21:
139-146,
1996[Medline].
29.
Shen, H. M.,
C. F. Yang,
and
C. N. Ong.
Induction of oxidative stress and apoptosis in sodium selenite-treated human hepatoma cells (HepG2).
Int. J. Cancer.
81:
820-828,
1999[Medline].
30.
Shi, X.,
V. Castranova,
B. Halliwell,
and
V. Vallyathan.
Reactive oxygen species and silica-induced carcinogenesis.
J. Toxicol. Environ. Health Part B Crit. Rev.
1:
181-197,
1998[Medline].
31.
Shi, X.,
N. S. Dalal,
and
V. Vallyathan.
ESR evidence for hydroxyl radical generation in aqueous suspension of quartz particles and its possible significance to lipid peroxidation in silicosis.
J. Toxicol. Environ. Health
23:
237-245,
1988.
32.
Shi, X.,
Y. Mao,
L. N. Daniel,
U. Saffiotti,
N. S. Dalal,
and
V. Vallyathan.
Silica radical-induced DNA damage and lipid peroxidation.
Environ. Health Perspect.
102, Suppl. 10:
149-154,
1994[Medline].
33.
Shi, X.,
Y. Mao,
U. Saffiotti,
L. Wang,
Y. Rojanasakul,
S. S. Leonard,
and
V. Vallyathan.
Antioxidant activity of tetrandrine and its inhibition of quartz-induced lipid peroxidation.
J. Toxicol. Environ. Health
46:
233-248,
1995[Medline].
34.
Thanislass, J.,
M. Raveendran,
and
H. Devaraj.
Buthionine sulfoximine-induced glutathione depletion, its effect on antioxidant, lipid peroxidation and calcium homeostasis in the lung.
Biochem. Pharmacol.
50:
229-234,
1995[Medline].
35.
Vallythan, V.,
S. Leonard,
P. Kuppusamy,
D. Pack,
M. Chzhan,
S. P. Sanders,
and
J. Zweir.
Oxidative stress in silicosis: evidence for the enhanced clearance of free radicals from whole lungs.
Mol. Cell. Biochem.
168:
125-132,
1997[Medline].
36.
Vallyathan, V.,
and
X. Shi.
The role of oxygen free radicals in occupational and environmental lung diseases.
Environ. Health Perspect.
105, Suppl. 1:
165-177,
1997[Medline].
37.
Vallyathan, V.,
X. Shi,
N. S. Dalal,
W. Irr,
and
V. Castranova.
Generation of free radicals from freshly fractured silica dust. Potential role in acute silica-induced lung injury.
Am. Rev. Respir. Dis.
138:
1213-1219,
1988[Medline].
38.
Van Klaveren, R. J.,
M. Demedts,
and
B. Nemery.
Cellular glutathione turnover in vitro, with emphasis on type II pneumocytes.
Eur. Respir. J.
10:
1392-1400,
1997
39.
Voisin, C.,
C. Aerts,
and
B. Wallaert.
Prevention of in vitro oxidant-mediated alveolar macrophage injury by cellular glutathione and precursors.
Bull. Eur. Physiopath. Respir.
23:
309-313,
1987[Medline].
40.
Yamano, Y.,
J. Kagawa,
T. Hanaoka,
T. Takahashi,
H. Kasai,
S. Tsugane,
and
S. Watanabe.
Oxidative DNA damage induced by silica in vivo.
Environ. Res.
69:
102-107,
1995[Medline].
41.
Yang, C. F.,
H. M. Shen,
and
C. N. Ong.
Protective effect of ebselen against hydrogen peroxide-induced cytotoxicity and DNA damage in HepG2 cells.
Biochem. Pharmacol.
57:
273-279,
1999[Medline].