Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
Received August 14, 2001; accepted December 4, 2001
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ABSTRACT |
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Key Words: human bronchial epithelial cells; silica; reactive oxygen species; free radicals; extracellular factors.
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INTRODUCTION |
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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., 1993). 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., 1994
), 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., 1994
).
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., 1997). 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.
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MATERIALS AND METHODS |
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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 1050 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., 1994; Zay et al., 1995
) 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, 1990
) 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., 1997
). 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., 1997). The assay is based on the ability of DCFH-DA to enter cells through the hydrophobic regions of the cell (Rothe and Valet, 1990
). 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., 1988
; Rothe and Valet, 1990
), 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., 1994
).
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., 1997). 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, 1986). Probability values (p) < 0.05 were considered to represent significant differences between group values.
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RESULTS |
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If the increases in intracellular fluorescence in Figure 1 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. 3). However, the changes in mean fluorescence intensities observed over the 12.550 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 2
, contained far lesser concentrations of H2O2, e.g., nanomolar.
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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., 1992). Figure 5
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., 1993
; Tanaka et al., 1997
). 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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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., 2000; Samuelson et al., 2001
), or that otherwise could underlie a de novo production of cell-derived factors, e.g., cytokines (Thannickal et al., 2000
), that potentially could stimulate increases in intracellular ROS in an autocrine manner in response to silica-treated medium.
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DISCUSSION |
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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 1050 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., 1994), and cells with a defective or nonfunctional complex I have been shown to have increased intracellular ROS (Pitkanen and Robinson, 1996
; Zager, 1996
). 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., 1994; Vallyathan et al., 1988
) 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, 1973
) 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., 1996
).
Recent evidence suggests that nonphagocytic NADPH oxidase can be induced by extracellular H2O2 (Li et al., 2001), 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., 1988
), 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., 1988), 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, 1996
). 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., 1996
; Galle et al., 2001
; Heinloth et al., 2000
). 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, 2000
).
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-B and AP-1 (Abate et al., 1990
; Remacle et al., 1995
; Sen and Packer, 1996
), they have been associated with the increased production of various cytokines like TNF-
and IL-8 (Narayanan et al., 1999
; Remick and Villarete, 1996
), they have been associated with DNA damage (Lehnert and Goodwin, 1997
; Narayanan et al., 1997
), and, as we and others have shown, they can enhance cell proliferation (Iyer and Lehnert, 2000
; Remacle et al., 1995
), all of which can figure into carcinogenesis.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed. Fax: (505) 665-3024. E-mail: lehnert{at}telomere.lanl.gov.
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