Silica-Induced Generation of Extracellular Factor(s) Increases Reactive Oxygen Species in Human Bronchial Epithelial Cells

Alina Deshpande, Padma K. Narayanan,1 and Bruce E. Lehnert,2

Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

Received August 14, 2001; accepted December 4, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic inflammation and production of DNA-damaging reactive oxygen species (ROS) may be involved in silica-induced lung cancer. Studies to date have largely focused on silica-induced production of ROS by lung phagocytes. In this study, we investigated the hypothesis that particulate silica (DQ12) can also induce elevations in intracellular ROS in a cancer-target cell type, i.e., human bronchial epithelial cells (BECs), via an indirect mechanism that involves ROS-inducing extracellular factor(s) that occur upon the interaction of silica with culture medium. The intracellular production of hydrogen peroxide (H2O2) in BECs was assessed by flow cytometry via monitoring dichlorofluorescein (DCF) fluorescence. Culture medium containing 10% human serum was incubated with silica particles in concentrations ranging from 10 to 50 µg/ml, and following incubation for 1 h and removal of the particles, the resulting supernatants were added to BECs. Silica-treated medium induced significant increases in intracellular H2O2 after the medium had been treated with as little as 10 µg/ml of the particles. Further, the level of ROS increases in BECs in response to silica-treated medium was found to be virtually identical to that induced in cells that were directly treated with silica in suspension. Based on enzyme inhibitory studies, the mechanism for this increased generation of intracellular ROS appears to involve both mitochondrial respiration and a NAD(P)H oxidase-like system. Spectrofluorimetric experiments with the antioxidant enzymes superoxide dismutase and catalase showed that superoxide anions (O2•-) and H2O2 are generated in silica-treated medium, but these ROS do not fully account for the induction of the intracellular ROS response. Iron, on the other hand, was found to be crucial to the process. Our collective results suggest silica-aqueous medium interactions can lead to the generation of factor(s) that induce the intracellular production of potentially DNA-damaging ROS in BECs in a manner that does not require direct particle-cell interactions.

Key Words: human bronchial epithelial cells; silica; reactive oxygen species; free radicals; extracellular factors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although silica has been classified as a Group I carcinogen (IARC, 1996Go), the mechanism(s) by which it may cause lung cancer remains unclear. As one contributing mechanism, chronic inflammation brought about by lung macrophages and polymorphonuclear leukocytes is widely thought to be involved in epithelial hypertrophy and/or hyperplasia as contributors to silica's carcinogenic effects (Hext, 1994Go). Additionally, these phagocytic cells also produce reactive oxygen species (ROS) such as superoxide anions (O2•-) and hydrogen peroxide (H2O2) in response to interactions with particulate silica (Zang et al., 2000), which can damage the DNA of lung and airway epithelial cells (Babior, 1984Go), while also contributing to numerous other steps in carcinogenesis such that ROS themselves may be considered as a separate class of carcinogens (Dreher and Junod, 1996Go). Although silica can also lead to increases in intracellular ROS in other lung cell types, including airway epithelial cells (Martin et al., 1997Go), mechanisms by which such stimulation occurs remain an understudied area that is of obvious relevance to silica-induced lung cancer.

In addition to directly activating the production of ROS by phagocytes, interactions of silica with the extracellular fluids lining the conducting airways likely result in local elevations in free radicals given that silica is capable of generating both silica-based hydroxyl radicals (OH) and O2•- in aqueous medium following reduction of dissolved molecular oxygen (Daniel et al., 1993Go). In this regard, Si and Si-O radicals are generated on the surface of silica particles, which in turn generate the highly reactive OH upon coming into contact with water. Trace amounts of iron on to the surface of crystalline silica also contribute to the generation of OH radicals via Fenton reactions. Hence, it is likely that cells lining the respiratory tract may also be exposed to ROS and perhaps other ROS-associated factors via cell-independent, extracellular fluid-dependent mechanisms following the local deposition of inhaled silica. And, in addition to any direct detrimental actions of ROS per se, the generation of the yet other fluid phase ROS-associated factors, e.g., lipid peroxidation products and aldehydes (Cross et al., 1994Go), suggests the possibility that such mediators could contribute to a number of silica-induced airway effects, including changes in airway permeability, the induction of inflammation, the induction of DNA damage, and cell hyperplasia (Saffiotti et al., 1994Go).

Recently, we reported an association between ROS that are generated extracellularly by ionizing radiation (IR) and the induction of increases in the intracellular production of O2•- and putative H2O2 in human lung fibroblasts via a mechanism that involves the activation of a nonphagocyte NAD(P)H oxidase (Narayanan et al., 1997Go). Based on this observation and the above findings that silica interactions with aqueous fluids can, as with IR, result in the production of extracellular ROS, the present investigation was undertaken to examine the hypothesis that silica can also induce increases in intracellular ROS in the absence of direct particle-cell interactions. Instead of lung fibroblasts, however, we used normal human bronchial epithelial cells (BECs) as "target" cells in our study, given their potential role as target cells in silica-induced lung cancer, particularly bronchogenic carcinoma.

We report that treatment of serum-containing, cell-free medium with particulate silica (DQ12) indeed results in the production of ROS in the medium, and that incubation of BECs with such particle-treated medium results in increases in the metabolic production of supra-basal levels of ROS via mechanisms that involve both NAD(P)H oxidase-like activation and mitochondrial respiration. Further, the elevations in intracellular ROS that are induced by a factor(s) in silica-treated medium are essentially identical to those observed when cells are incubated in silica-containing medium. Our collective results suggest that at least some airway epithelial effects of silica may be mediated by one or more transmissible factors that are generated extracellularly by particle-fluid phase interactions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. Normal human bronchial epithelial cells (BECs) were obtained from Clonetics (BioWhittaker Inc., San Diego, CA) and routinely subcultured in serum-free Bronchial Epithelial Growth Medium (BEGM, Clonetics, San Diego, CA) as recommended by the supplier. Cells used in experiments were from passage 1–3 only; beyond passage 3, their growth rate diminishes with gradual differentiation to a squamous cell phenotype. The BECs were subcultured in 25 cm2 tissue culture flasks (Falcon, Becton and Dickinson Company, France) at an initial seeding density of 2 x 105 cells/flask and incubated at 37°C in a 5% CO2-air mix, humidified atmosphere as described elsewhere (Gadbois and Lehnert, 1997Go).

Silica and extracellular ROS. Silicon dioxide in the form of tertiary quartz sand DQ12 with an average size < 0.5 µm (Messrs. Dorentruper Sand und Thonwerke GmbH of Dorentrup, Westphalia, Germany) was used in the experiments. Prior to use, the DQ12 was placed in Pyrex® glass tubes and heated to 175°C for 10 h to ensure inactivation of any contaminating endotoxins. Silica particles were suspended in Modified Hanks' Balanced Salt Solution containing 10% human serum (MHBSS) at concentrations ranging from 10–50 µg/ml following sonication in plain MHBSS for 2 min to give a uniform suspension. The suspensions were then incubated at 37°C in 5% CO2 for 45 min to 1 h with constant shaking. A negative control consisted of only MHBSS incubated at 37°C without particles. Following incubation, the particles were sedimented from the medium by centrifugation at ~2000 x g for 30 min at room temperature. Supernatants were carefully aspirated from the particle pellets and transferred to sterile tubes (Falcon, Becton and Dickinson Company, Lincoln Park, NJ). It should be noted that when the supernatants from the particle-treated samples were viewed under a light microscope, no particles were evident, thus confirming that the effects observed in this study were not attributable to residual particles.

To confirm the reported (Saffiotti et al., 1994Go; Zay et al., 1995Go) generation of extracellular ROS in aqueous medium, nonfluorescent 2`-7`-dichlorofluorescin (DCFH) prepared from dichlorofluorescein diacetate (DCFH-DA) using 0.1N NaOH to cleave the DA moiety (Rothe and Valet, 1990Go) was added to medium after removal of particles at a final DCFH concentration of 20 µM; oxidation of DCFH with H2O2 and/or peroxidases yields dichlorofluorescein (DCF), which fluoresces green upon 488 nm excitation. Five min thereafter, the samples were analyzed on a Spex Fluorolog 1680 double spectrofluorometer (Spex Industries Inc., Edison, NJ). Excitation was 488 nm with a 1-nm slit, and emission was collected at 528 nm with a 1-nm slit. Negative controls routinely consisted of serum-containing MHBSS treated with DCFH only, and positive controls consisted of medium treated with 100 µM H2O2. A standard curve for DCF fluorescence versus H2O2 concentration was generated by plotting the change in fluorescence intensities from control levels versus different concentrations of externally added H2O2 to otherwise untreated medium. The linear regression equation thus obtained was used to calculate the quantity of H2O2 generated in all samples (expressed in nanomolar quantities; Narayanan et al., 1997Go). Replicate samples were analyzed and fluorescence intensity in terms of counts per second (cps) was determined for each sample.

Intracellular ROS analyses. To detect increases in intracellular H2O2, BECs were incubated with 20 µM dichlorofluorescin diacetate (DCFH-DA, Molecular Probes, Eugene, OR) in HBSS + 0.1% Bovine Serum Albumin (BSA) for 30 min at 37°C (Narayanan et al., 1997Go). The assay is based on the ability of DCFH-DA to enter cells through the hydrophobic regions of the cell (Rothe and Valet, 1990Go). Once inside, the dye is cleaved by intracellular esterases to yield 2`-7`-dichlorofluorescin (DCFH). Due to its polar nature, DCFH is trapped intracellularly, where it can be oxidatively modified by intracellular H2O2 and peroxidases to produce fluorescent 2`-7`-dichlorofluorescein (DCF; Robinson et al., 1988Go; Rothe and Valet, 1990Go), as described before. The intensity of fluorescence as measured by emissions at 525 nm via flow cytometry, is proportional to the amount of intracellular H2O2.

Following incubation with the dye, the flasks containing the BECs were washed once with MHBSS. The dye-loaded cells were then exposed to either supernatants from silica-treated MHBSS or untreated MHBSS for 15 min at 37°C in a 5% CO2-air mix incubator. In some experiments, cells were incubated with suspensions of silica, where indicated. Each flask represented a separate sample. Cells were then harvested by trypsinization and resuspended in HBSS with 0.1% BSA at a density of ~1 x 106 cells/ml. As a positive control for putative H2O2 production, a set of BECs was treated in each experiment with 100 µM H2O2, which diffuses into the cell and reacts with the trapped dye, giving green fluorescence following excitation at 488 nm (Carter et al., 1994Go).

Samples were filtered into specialized polystyrene flow cytometric tubes fitted with 35 µm cell strainer caps (Falcon, Becton and Dickinson Company, France) to remove cell aggregates, and the BECs were analyzed for increases in fluorescence with a Becton-Dickinson FACSCalibur® flow cytometer (Becton-Dickinson, San Francisco, CA) using the instrument's standard computer, optics, and electronics. A 15 mW, air-cooled argon-ion laser was used for exciting DCF at 488 nm, and a 530/30 nm band-pass optical filter for DCF detection was placed in the fluorescence collection pathway. Samples were run either in duplicate or triplicate, and fluorescence measurements were collected on a log scale. Data were analyzed by the CellQuest® data analysis software and graphically presented in terms of mean fluorescence intensities in arbitrary units (Narayanan et al., 1997Go). A total of 10,000 events were collected per test sample; mean fluorescence intensity obtained per sample is the average value for fluorescence obtained from n = 10,000.

Statistical analyses. All experiments were performed at least twice on separate days and data presented herein are representative of results obtained from all repetitions. Data are expressed as means ± SEM. Statistical analyses were performed by ANOVA, followed by Tukey's post hoc testing where indicated (Devore and Peck, 1986Go). Probability values (p) < 0.05 were considered to represent significant differences between group values.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ROS in Silica-Treated Culture Medium
Fluorescence analyses of culture medium that had been pretreated with 10 or 50 µg/ml DQ12 showed particle concentration-dependent increases in putative H2O2 levels (Table 1Go). To determine that the fluorescence observed in these experiments was due exclusively to H2O2-dye interactions, 100 units/ml of bovine superoxide dismutase (SOD), 100 units/ml catalase, or both SOD and catalase were added to control and DQ12-containing samples (10 µg/ml) during the 1-h particle-medium incubation period. Catalase effectively inhibited increases in fluorescence intensity in all samples (Table 2Go), while SOD increased the level of putative H2O2 almost 10-fold in the particle-treated supernatants. Thus, the particle-associated increases in fluorescence were specific for H2O2, and the source of the increased levels of H2O2 seen in the DQ12-treated supernatants appeared to involve the generation of O2•- in that SOD markedly increased the oxidation of DCFH in a catalase-inhibitable manner. As well, the results from this experimental series indicate that all of the generated O2•- had not been converted to H2O2 during the 60 min incubation of the particles with medium and the time involved in removing the particles by centrifugation. Accordingly, they were persistently available for conversion to H2O2, and/or, alternatively, that the generation of O2•- per se persisted even after removal of the particles. These possibilities require further study.


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TABLE 1 Concentrations of Putative Hydrogen Peroxide (nM) in DQ12-Treated Medium
 

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TABLE 2 Effects of Superoxide Dismutase and Catalase on Hydrogen Peroxide Concentrations in Medium Samples Treated with DQ12
 
Induction of Increases in Intracellular ROS by Silica-Treated Culture Medium
BECs were exposed for 15 min to supernatants from medium that had been treated with 10, 25, and 50 µg/ml of DQ12 particles. As illustrated in Figure 1Go, all particle concentrations produced essentially identical increases in intracellular DCF fluorescence relative to the fluorescence intensity levels measured in cells that had been incubated in untreated control medium samples. These findings indicate that the generation of intracellular ROS-inducing factor(s) results from interactions between even relatively low mass concentrations of crystalline silica and aqueous medium and that the ROS-inducing factor(s) activate promptly the intracellular production of ROS in BECs.



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FIG. 1. Relative intracellular DCF fluorescence in BECs exposed to supernatants from serum-containing medium incubated for 60 min with DQ12 at 0, 10, 25, and 50 µg/ml. Cells were exposed to the supernatants for 15 min at 37°C in a 5% CO2-air mix atmosphere prior to the flow cytometric analyses. *Significantly higher than control values.

 
To assess how the increases in intracellular ROS compare to those that may be induced when cells are directly exposed to particulate silica in suspension, BECs were incubated for 15 min with either a 50 µg/ml suspension of DQ12 in MHBSS or supernatants from MHBSS that had been treated with 50 µg/ml DQ12, and their intracellular DCF fluorescences were measured by flow cytometry. As shown in Figure 2Go, exposure of the cells to particles or particle-treated supernatants resulted in equivalent increases in intracellular H2O2 production, thus suggesting the possibility that DQ12-mediated oxidative stress may be primarily mediated via the generation of ROS-inducing factors in the suspension medium that persist even after the removal of the particles from medium versus direct particle-cell interactions per se.



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FIG. 2. Intracellular DCF fluorescence in BECs exposed to either 50 µg/ml DQ12 in suspension or supernatants from DQ12-treated medium (50 µg/ml). Control cell samples consisted of BECs that had been incubated with untreated medium. *Significantly higher than control values.

 
The process of cell activation or apparent cell activation by a factor or factors in supernatants of silica-treated medium hypothetically could occur in at least two different ways: (1) the generation of oxidative species, e.g., H2O2 (Tables 1 and 2GoGo), arising from silica-medium interactions might diffuse or otherwise gain access into the target cells upon exposure and interact with the intracellular dye to increase its fluorescence, and/or (2) the ROS or related factors in silica-treated medium may initiate a signal cascade in the cells that leads to the generation of intracellular ROS.

If the increases in intracellular fluorescence in Figure 1Go were the result of diffusible species like H2O2, then incubation of BECs in medium containing H2O2 should show quantitatively equivalent increases in DCF fluorescence relative to those obtained with BEC in silica-treated medium, and treatment of DQ12-treated medium with catalase predictably should reduce the observed elevations in intracellular ROS. If O2•- was the source of this diffusible H2O2, as suggested by results described earlier, then the addition of SOD to particle-treated medium prior to target cell exposure should enhance levels of intracellular DCF fluorescence by increasing the availability of diffusible H2O2.

A series of experiments were performed to investigate these possibilities. In the first set, we examined how the addition of H2O2 to culture medium can affect the fluorescence intensities of BECs that were preloaded with intracellular DCFH-DA. Not unexpectedly, the BECs showed concentration-dependent increases in intracellular fluorescence following exposure to 12.5, 25, 50, and 100 µM H2O2 (Fig. 3Go). However, the changes in mean fluorescence intensities observed over the 12.5–50 µM H2O2 concentration range were relatively much less than those measured with BECs that had been incubated with DQ12-treated medium, which, based on data shown in Tables 1 and 2GoGo, contained far lesser concentrations of H2O2, e.g., nanomolar.



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FIG. 3. Intracellular DCF fluorescence in BECs exposed to 0, 12.5, 25, 50, and 100 µM H2O2 for 15 min at 37°C in a 5% CO2-air mix atmosphere prior to the flow cytometric analyses.

 
In related experiments, we added bovine SOD (100 units/ml) during the incubation of DQ12 (50 µg/ml) with medium and, following centrifugation, we treated BECs with the resulting supernatants. As summarized in Figure 4Go, the addition of SOD during the particle-medium incubation period, which causes marked increases in putative H2O2 in the supernatants (Table 2Go), did not result in corresponding increases in DCF fluorescence. These collective findings, accordingly, suggest that the increases in intracellular ROS in BEC incubated in silica-treated medium were not solely attributable to the diffusion of extracellular H2O2 into the cells.



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FIG. 4. Intracellular DCF fluorescence in BECs exposed to supernatants from DQ12-treated medium (50 µg/ml) that were either untreated or pretreated with bovine superoxide dismutase (SOD, 100 units/ml), catalase (Cat, 100 units/ml), or superoxide dismutase together with catalase. Cells were incubated with the supernatants for 15 min at 37°C in a 5% CO2-air mix atmosphere prior to the flow cytometric analyses. *Significantly higher than values obtained with control, untreated medium. **Significantly lower than values obtained with DQ12-treated medium lacking exogenous superoxide dismutase. ***Significantly higher than values obtained with DQ12-treated medium lacking exogenously added catalase. ****Significantly higher than DQ12-treated medium lacking exogenously added superoxide dismutase.

 
In the same experiments, we also added catalase (100 units/ml) or catalase and SOD (100 units/ml) during the incubation of DQ12 (50 µg/ml) with medium, and we treated BECs with the particle-free supernatants from these samples. Treatment with catalase, which expectedly would diminish intracellular DCF fluorescence by decreasing the availability of extracellular, diffusible H2O2, while also removing H2O2 as a potential stimulator of nonphagocyte NAD(P)H oxidase (Li et al., 2001Go), actually enhanced the intracellular fluorescence of the BECs beyond that measured in BECs incubated with silica-treated medium in the absence of catalase (Fig. 4Go). BECs exposed to control supernatants that had been pretreated with catalase also showed this effect, but substantially less so (data not shown). It should be noted here that the addition of catalase to control medium to which 100 µM H2O2 had been pre-added produced a significant decrease in the level of intracellular fluorescence in BECs when compared to the fluorescence of cells exposed to control medium + 100 µM H2O2 alone (data not shown). Hence, "normal" catalase itself is not an inducer of intracellular ROS in BEC. Inclusion of both catalase and SOD during the incubation of silica with medium still resulted in unexpectedly high increases in intracellular DCF fluorescence, nearly to the same level as that obtained with catalase treatment alone. It should also be pointed out that enzyme treatments in the above experiments were performed during the 1-h incubation of the DQ12 with medium. In order to rule out the possibility that the above results may have been due to direct enzyme-particle interactions in a manner that somehow bestowed an as yet unreported ROS-inducing activity on catalase, the same experiments were performed with the enzyme treatments (SOD and catalase) after the particles had been removed from the medium. Results obtained from these experiments were identical to those just described. Taken together, the results from these experiments further indicate that the elevations in intracellular DCF fluorescence seen in BECs exposed to particle-treated medium is not an ROS-associated, diffusion-mediated process, but, instead, they are consistent with a metabolic origin(s) for the ROS response in the BECs. Moreover, the presence of O2•- and H2O2 in the particle-treated medium by themselves do not appear to account for the induction of the intracellular ROS response in BECs. In fact, the results we obtained using catalase suggest the possibility that the H2O2 that is normally generated in medium treated with silica may actually inhibit the extent of the ROS response, perhaps by rendering one or more factors that are involved in inducing intracellular ROS less active.

Conceivably, the induction of intracellular ROS by silica-treated medium could involve a process that involves iron, given that treatment of ROS-producing minerals with certain iron chelators like deferroxamine reduces or even blocks the production of O2•-, OH and H2O2 (Kamp et al., 1992Go). Figure 5Go shows the intracellular fluorescence levels of BECs that were exposed to 50 µg/ml DQ12-medium derived supernatants we had pretreated with 1 mM deferroxamine (DFO, Sigma Chemical Co., St. Louis, MO) prior to the 1-h particle-medium incubation period. DFO chelates Fe3+ and prevents Fenton reactions that involve the reduction of iron and the production of OH via O2•- (Deforge et al., 1993Go; Tanaka et al., 1997Go). The addition of deferroxamine reduced levels of intracellular DCF fluorescence to negative control values, suggesting that particle-associated iron or iron present in the medium are fundamentally required for the generation of a factor or factors that stimulate the cells to produce ROS.



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FIG. 5. Intracellular DCF fluorescence in BECs exposed to supernatants from DQ12-treated medium (50 µg/ml) that was either untreated or pretreated with 1 mM deferroxamine (DFO). Treatment with DFO was done during the 60 min particle-medium incubation period. *Significantly higher than values obtained with untreated medium.

 
Cellular Mechanisms Involved in Mediating the Increases in ROS
Major pathways that may be involved in the generation of intracellular ROS (DeForge et al., 1993Go) include: (1) the generation of O2•- radicals during mitochondrial respiration, (2) the generation of O2•- via plasma membrane-associated phagocyte and nonphagocyte NAD(P)H oxidase systems (Deken et al., 2000Go; Geiszt et al., 2000Go; Sundaresan et al., 1995Go), and (3) the hypoxanthine-xanthine oxidase system present within some cell types cells that generates O2•- from molecular oxygen. With all 3 mechanisms, H2O2 (which is measured in our flow cytometric assay) and OH radicals can then be generated from O2•- via superoxide dismutase, spontaneous dismutation, and Fenton reactions (Halliwell and Gutteridge, 1996Go). In order to elucidate the mechanism(s) that may underlie the generation of intracellular ROS by BECs in response to silica-treated medium, cells preloaded with DCFH-DA were treated with inhibitors of the above pathways prior to further incubation with supernatant from DQ12-treated medium, and changes in DCF fluorescence were measured by flow cytometry. Cells exposed to control supernatants were treated in the same manner as above. Figure 6Go shows the percent increase or decrease in DCF fluorescence intensity following pretreatment of BECs with 5 µM diphenylene iodonium (DPI, Molecular Probes, Eugene, OR), a flavoenzyme/NADPH oxidase inhibitor, 1 mM sodium azide (LabChem Inc., Pittsburgh, PA), an inhibitor of mitochondrial respiration (Narayanan et al., 1997Go), or 5 mM allopurinol (Sigma Chemical Co., St. Loius, MO), an inhibitor of the hypoxanthine-xanthine oxidase system (DeForge et al., 1993Go). Cells were treated with these compounds in HBSS + 0.1% BSA for 60 min, and then exposed to either control supernatants or supernatants from DQ12-treated medium (50 µg/ml).



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FIG. 6. Percent change in DCF fluorescence in BECs exposed to supernatants from DQ12-treated medium (50 µg/ml) after the cells were preincubated with diphenylene iodonium (DPI, 5 µM), Allopurinol (5 mM), or sodium azide (Azide, 1 mM) for 1 h prior to being further incubated for 15 min with supernatants from particle-treated medium. *Significantly higher than corresponding values obtained with control, untreated medium (set at 100%). **Significantly lower than values obtained with corresponding control, untreated medium.

 
As shown in Figure 6Go, significant decreases in the DCF fluorescence were obtained with cells treated with DPI and sodium azide following exposure to DQ12 supernatants, suggesting an involvement of an NADPH oxidase-like source or at least a flavoenzyme-inhibitable system that produces O2•-, that undergo dismutation to H2O2, as well as mitochondrial respiration in which the bulk of generated O2•- is locally converted to H2O2 by a Mn-containing superoxide dismutase (Nohl and Hegner, 1978Go). On the other hand, we observed a relatively modest elevation in DCF fluorescence of BECs that had been pretreated with allopurinol and then exposed to the DQ12-treated medium or control medium. Given the apparent involvement of NAD(P)H oxidase and mitochondrial respiration as electron sources for the catalysis of O2 to O2•-, we presently posit that this finding may be explained as a consequence of increased availability of molecular oxygen to the NAD(P)H oxidase system and/or mitochondria due to inhibition of the hypoxanthine-xanthine oxidase system.

While further investigations are obviously required to more firmly identify the metabolic sources of the ROS response, what is evident from our study is that the intracellular ROS response occurs within minutes upon incubation of the BECs with particle-treated medium. Hence, the response is likely unrelated to transcriptional and translational processes that are required to up-regulate NAD(P)H oxidase function in some cell types (Cifuentes et al., 2000Go; Samuelson et al., 2001Go), or that otherwise could underlie a de novo production of cell-derived factors, e.g., cytokines (Thannickal et al., 2000Go), that potentially could stimulate increases in intracellular ROS in an autocrine manner in response to silica-treated medium.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous investigators have attributed several cellular effects, e.g., lipid peroxidation and DNA damage, of several types of particulate minerals to their surface-related free radical activity in the context of direct particle-cell contacts (Donaldson et al., 1996Go; Mossman et al., 1996Go; Saffiotti et al., 1994Go; Zhang et al., 1998Go). In part, such effects are widely thought to be mediated by O2•--driven, iron-catalyzed Haber-Weiss (Fenton) reactions that generate OH (Castranova, 1994Go; Mossman and Churg, 1998Go; Saffiotti et al., 1994Go). As well, the uptake of particles like silica by alveolar macrophages and other cell types including lung epithelial cells, causes the cellular production of ROS, specifically O2•- and H2O2, (Cho et al., 1999Go; Ding et al., 2001Go; Gercken et al., 1996Go; Lim et al., 1997Go; Martin et al., 1997Go; Vallyathan et al., 1988Go) as yet other sources of potentially damaging oxidative stress.

In this investigation, we have obtained evidence that suggests a "new" mechanism by which silica can stimulate the metabolic production of potentially damaging intracellular ROS in cancer-relevant, normal human bronchial epithelial cells (BECs) via a process that does not involve direct particle-cell interactions per se. Instead, we have found that particulate silica can lead to the induction of increases in cellular ROS production by one or more extracellular, fluid-phase factors that are generated by silica in serum-containing medium, which shares at least some features with physiological fluids like airway lining fluid. Of significance: (1) the silica-associated ROS-inducing factor(s) appeared to be maximally generated in a mass concentration-independent manner over relatively low DQ12 concentrations ranging from 10–50 µg/ml, and (2) the levels of induction of intracellular ROS by the ROS-inducing factor(s) in particle-treated medium were virtually identical to those measured when cells were directly incubated with suspensions of particulate DQ12, at least over the relatively short incubation period we used in our study, i.e., 15 min. Further, by using inhibitors of cellular pathways that potentially could contribute to the supra-basal production of ROS by BECs in response to particle-free medium that had been pretreated with particulate DQ12, we have additionally obtained evidence that the cellular sources of ROS involve as the mitochondrial electron transport system and activation of an NADPH oxidase-like system. With regard to the latter and for the sake of balance, however, it should be noted that the flavoenzyme inhibitor DPI can also potentially inhibit complex I of the mitochondrial electron transport system (Majander et al., 1994Go), and cells with a defective or nonfunctional complex I have been shown to have increased intracellular ROS (Pitkanen and Robinson, 1996Go; Zager, 1996Go). Even so, this possibility seems unlikely given that we used a relatively low concentration of DPI (5 µM) in our studies and given the finding that inhibition of mitochondrial respiration by sodium azide lessened but did not eliminate the intracellular ROS response.

While we have as yet to identify the responsible molecular specie(s) that mediate the increases in intracellular ROS, our results suggest that the cellular ROS response to some extent involves the occurrence of O2•- that are generated in aqueous medium treated with silica (6; Saffiotti et al., 1994Go; Vallyathan et al., 1988Go) inasmuch as the addition of superoxide dismutase during the incubation of the silica with medium partially inhibited the intracellular ROS response in BEC. DQ12-associated iron (Robock, 1973Go) and/or iron in the medium also appeared to play a fundamentally decisive role in the process in that the addition of the iron chelator deferroximine during the incubation of silica with medium totally inhibited intracellular ROS response. Of potential relevance to the in vivo condition, human lung fluid contains chelatable redox active iron, and this iron has the potential to act as a catalyst and generate ROS (Gutteridge et al., 1996Go).

Recent evidence suggests that nonphagocytic NADPH oxidase can be induced by extracellular H2O2 (Li et al., 2001Go), which was readily produced in our DQ12-treated medium samples. Yet, the generation of H2O2 by silica-medium interactions evidently is not importantly involved in the induction of intracellular ROS in our cell system. If anything, the inclusion of catalase during the incubation of DQ12 with medium actually resulted in the stimulation of higher levels of intracellular ROS than those observed with BECs incubated with particle-medium preparations that contained no antioxidants other than those already present in the serum-containing medium. And, since catalase disproportionates H2O2 and O2•- concentrations and suppresses the formation of OH from silica (Vallyathan et al., 1988Go), such a finding additionally suggests that hydroxyl radicals generated from H2O2-Fe2+ interactions also do not contribute to the induction of intracellular ROS, either directly or indirectly by possibly modifying other constituents in medium that in turn may activate the cellular production of ROS.

Given the chemical complexity of serum-containing medium coupled with the complex physicochemical surface characteristics of silica that can depend on variables such as age since fracturing (Vallyathan et al., 1988Go), it is not currently possible to comment definitively about the chemical nature of the intracellular ROS-inducing factor(s) that are generated in silica-treated medium without further study. What is clear is that iron appears to be required for the process in a manner that does not appear to require major roles for O2•- and H2O2. One process that may pertain to our findings is iron-stimulated lipid peroxidation, which is known to occur in the presence of oxygen by an as yet to be understood mechanism, even in the presence of catalase and OH scavengers (Halliwell and Gutteridge, 1996Go). Conceivably, lipid hydroperoxides so formed, or perhaps lipid peroxides present in serum due to normal peroxidation, in turn could react with Fe2+ to form reactive alkoxyl and peroxy radicals. However speculative, observations have been reported that some oxidized lipoproteins and downstream products of lipid peroxidation such as 4-hydroxynonenol can induce NAD(P)H oxidase-mediated increases in intracellular ROS (Dianzani et al., 1996Go; Galle et al., 2001Go; Heinloth et al., 2000Go). Along the same line, oxidative changes in some proteins as a possible collateral effect of lipid peroxidation can also result in oxidized forms that activate NAD(P)H oxidase (Moraga and Janciauskiene, 2000Go).

The potential significance of our findings merits some discussion. Prior mechanistic models of silica-induced diseases have primarily focused on cellular effects and related downstream events that occur upon direct particle-cell interactions at the cell surface or after the particles have been endocytosed. The results presented herein suggest that the current mechanistic paradigm for silica-induced effects in cells may require revision, while additionally adding to the complexity of the modeling of silica dose-response relationships for the purpose of risk assessment. At least for the induction of intracellular ROS as one experimental endpoint in cancer-relevant airway epithelial cells, measurable responses can occur in the absence of any direct silica-cell interactions via one or more fluid phase, transmissible factors. Assuming for now that our findings extend to the in vivo condition, our results suggest that some responses to silica in the respiratory track potentially may occur at cellular sites located some distance from those where free particles are deposited or are otherwise present for interaction with airway fluids.

Finally, and from a biological perspective, several consequences of intracellular ROS responses like those observed in our investigation are noteworthy. In this regard, supra-basal levels of intracellular ROS have been shown to regulate numerous transcription factors, e.g., NF-{kappa}B and AP-1 (Abate et al., 1990Go; Remacle et al., 1995Go; Sen and Packer, 1996Go), they have been associated with the increased production of various cytokines like TNF-{alpha} and IL-8 (Narayanan et al., 1999Go; Remick and Villarete, 1996Go), they have been associated with DNA damage (Lehnert and Goodwin, 1997Go; Narayanan et al., 1997Go), and, as we and others have shown, they can enhance cell proliferation (Iyer and Lehnert, 2000Go; Remacle et al., 1995Go), all of which can figure into carcinogenesis.


    ACKNOWLEDGMENTS
 
This study was supported by the U.S. Department of Energy, the Los Alamos National Laboratory Flow Cytometry Resource (NIH Grant p41-RR01315) and by the NIH/NCI (CA82598).


    NOTES
 
1 Present address: Safety Assessment, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406. Back

2 To whom correspondence should be addressed. Fax: (505) 665-3024. E-mail: lehnert{at}telomere.lanl.gov. Back


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