Nitrite enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells by inhibition of myeloperoxidase
Ad M. Knaapen *,
Roel P.F. Schins 1,
Paul J.A. Borm 1 and
Frederik J. van Schooten
Department of Health Risk Analysis and Toxicology, University of Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands and 1 Institut für umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Düsseldorf, Germany
* To whom correspondence should be addressed. Tel: +31 43 3881094; Fax: +31 43 3884146; Email: a.knaapen{at}GRAT.unimaas.nl
 |
Abstract
|
---|
Chronic inhalation of environmental particles is associated with pulmonary carcinogenesis. Although the mechanism has not yet been fully elucidated, influx of inflammatory cells, including neutrophils, is suggested to play a major role in this process. Typically, in the particle-exposed lung, influx of neutrophils is accompanied by an accumulation of nitrite. Previous studies indicated that nitrite may affect the toxicity of neutrophils, involving an interaction with neutrophil-derived myeloperoxidase (MPO). To evaluate the possible consequences of this interaction for inflammation-mediated genotoxicity, we investigated the effect of nitrite on neutrophil-induced DNA damage in pulmonary target cells. Therefore, activated neutrophils were co-cultured with alveolar type II epithelial cells (RLE), and DNA strand breakage was evaluated using single-cell gel electrophoresis (comet assay). In this system, addition of nitrite caused an increase in neutrophil-induced DNA strand breakage in RLE cells, which was associated with an inhibition of MPO activity. Similar results were obtained by co-culturing RLE cells with neutrophils in the presence of the specific MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH). To further investigate the mechanism underlying these observations, in vitro experiments were performed using mixtures of nitrite, MPO and its substrate H2O2. DNA strand breakage by reagent H2O2 was inhibited when it was allowed to react with MPO before addition to the RLE cells. However, when MPO and H2O2 were pre-mixed in the presence of nitrite or 4-ABAH, the inhibitory effect of MPO on resultant DNA damage was reversed. Further studies using catalase indicated that DNA strand breakage by the pre-mixtures of MPO, H2O2 and nitrite was H2O2-specific, suggesting that nitrite prevents consumption of H2O2 by MPO. Collectively, our results show that nitrite enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells. This effect is probably due to an inhibition of MPO activity, which increases the availability of its DNA strand breaking substrate H2O2.
Abbreviations: 4-ABAH, 4-aminobenzoic acid hydrazide; FCS, foetal calf serum; HBSS, Hanks' Balanced Salt Solution; MPO, myeloperoxidase; RLE, rat lung type II epithelial cells; TMB, tetramethylbenzidine reagens.
 |
Introduction
|
---|
Numerous clinical and experimental data have expanded the concept that chronic inflammation is associated with tumour development in a variety of tissues (1). In the lung, inhaled particles such as crystalline silica and those present in cigarette smoke and diesel exhaust are a potent source of inflammation, and it is suggested that pulmonary carcinogenicity upon chronic particle exposure involves an influx and subsequent activation of inflammatory phagocytes (2,3). For instance, we and others have demonstrated that in vivo DNA damage and mutagenicity upon particle exposure is closely related to neutrophil recruitment to the lung (46). One of the properties of phagocytes that provide a possible basis for the link between inflammation and carcinogenesis is their production of DNA damaging and mutagenic reactive oxygen species (7). For neutrophils, H2O2 is a major oxidant that contributes to inflammation-related DNA damage and mutagenesis. It has been hypothesized that H2O2 escapes consumption by neutrophilic myeloperoxidase (MPO) and subsequently diffuses towards the nucleus of neighbouring cells, where it reacts with DNA-bound transition metals to form the highly reactive hydroxyl radical (810). Indeed, we and others have demonstrated that activated neutrophils cause a variety of (mutagenic) DNA modifications that are typical of those induced by H2O2-derived hydroxyl radicals. (5,8,9,1115). It is estimated that under normal circumstances up to 4070% of neutrophil-derived H2O2 is consumed by MPO. MPO is a heme enzyme that is abundantly present within the cytoplasmatic granules of neutrophils. During activation of neutrophils it is released extracellularly, where it reacts with H2O2 to form hypochlorous acid (16,17). Although HOCl is a potent cytotoxic compound, it seems to be less effective in causing cellular DNA strand breakage than H2O2 (8,18). Actually, Schraufstatter et al. (8) showed that addition of MPO inhibits neutrophil-induced DNA strand breakage in target cells. This would suggest that modulation of MPO activity may have profound effects on neutrophil-induced DNA damage and mutagenicity at sites of inflammation.
Although the involvement of neutrophils and epithelial cells has been indicated, alveolar macrophages are considered as the major source of NO· in the particle-exposed lung (19). In aerobic aqueous solutions, NO· readily decomposes to nitrite (
) (20), which accumulates in the particle-exposed lung (21,22). Studies have shown that an interaction between MPO and nitrite could have a profound effect on the toxic capacity of neutrophils (23). On the other hand, recent in vitro studies revealed that in particular circumstances nitrite might act as an inhibitor of MPO, for instance reducing its chlorination activity or its effect on modification of LDL (24,25). However, the consequences of an interaction between nitrite and MPO with respect to neutrophil-related pulmonary genotoxicity remain to be investigated. Therefore, in an attempt to further substantiate the involvement of inflammation-related genotoxic processes in particle-induced carcinogenesis, we investigated the relationship between MPO, nitrite and neutrophil-induced DNA strand breakage in co-cultured pulmonary epithelial type II cells (5). We anticipated a crucial role for neutrophil-derived H2O2, as its availability will largely depend on the presence and activity of MPO. Therefore, single-cell gel electrophoresis was applied to specifically evaluate DNA single-strand breakage in the epithelial cells, as this endpoint closely correlates with the broad spectrum of H2O2-induced DNA modifications (13).
 |
Materials and methods
|
---|
Chemicals
Ham's F12 medium, Hanks' Balanced Salt Solution (HBSS), HEPES buffer, fetal calf serum (FCS) and trypsin were obtained from Gibco (Breda, The Netherlands). Low melting point agarose, 4-aminobenzoic acid hydrazide (4-ABAH), horseradish peroxidase, MPO, penicillin/streptomycin, phenol red, phorbol 12-myristate 13-acetate (PMA), sodium nitrite and Trypan Blue were purchased from Sigma (Zwijndrecht, The Netherlands). Lymphoprep was obtained from Axis-Shields (Oslo, Norway). Tetramethylbenzidine reagens (TMB) was obtained from Bio-Rad (USA). All other chemicals were obtained from Merck (Darmstadt, Germany).
Cell culture
Rat lung type II epithelial cells (RLE), kindly provided by Dr K.Driscoll (Procter & Gamble, USA), were cultured in Ham's F12 medium, supplemented with 1% 1 M HEPES buffer, 1% penicillin/streptomycin solution and 5% heat-inactivated FCS. Cells were routinely grown in 75 cm2 cell-culture flasks and passaged twice a week. Experiments were always performed between cell passages 40 and 50. For exposures, cells were seeded in 24-well plates, and grown until confluence. All experiments were performed in HBBS (w/o phenol red, with 140 mM NaCl).
Co-culture with neutrophils
Co-culture experiments were performed as described by Knaapen et al. (5). Briefly, neutrophils were freshly isolated from blood of one healthy non-smoking male volunteer using gradient centrifugation (26). Lymphocytes were removed and the remaining erythrocytes were lysed using cold (4°C) lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 10 mM EDTA, pH 7.4). Neutrophils were washed with PBS and finally resuspended in HBSS at 6.5 x 105 cells/ml. Cell viability was assessed using Trypan Blue dye exclusion. Neutrophils were kept on ice to prevent premature activation. Using this protocol, the neutrophil preparation was >95% pure, and the viability always exceeded 98%.
Neutrophils were co-cultured with confluent RLE cells grown in 24-well plates. The final volume of the incubations was always 400 µl with a total neutrophil number of 2.6 x 105, yielding a final neutrophilRLE ratio of 1:1. Cells were co-incubated for 2 h in HBSS (37°C, 5% CO2). Neutrophils were activated with PMA (100 ng/ml) to elicit the respiratory burst. Nitrite or 4-ABAH were added at the indicated concentrations. H2O2 production and MPO activity in supernatants of activated neutrophils were measured as described below. Following incubation, the neutrophils were removed using three repetitive washings with PBS (4°C). The RLE cells were then harvested by trypsination for respective analysis of toxicity and DNA strand breakage. Microscopic differentiation of cytospin preparations from harvested RLE cells showed that washing procedures were efficient (<3% remaining neutrophils). Incubations with reagent H2O2 (25 µM) were used as a positive control. All studies were performed at pH 7.4.
Treatment of cells with oxidants
To investigate possible synergistic DNA damaging effects of H2O2 and nitrite, experiments were performed in which RLE cells were exposed for 2 h to a mixture of reagent H2O2 and nitrite. Furthermore, in order to investigate possible protective effects of MPO, resulting from consumption of genotoxic H2O2, experiments were performed in which H2O2 was allowed to react with purified MPO before addition to the RLE cells. Specifically, the following experiments were performed (Figure 1): H2O2 (25 µM) was mixed with purified MPO (200 mU/ml) in HBSS (pH 7.4) and incubated for 15 min at 37°C. MPO-related removal of H2O2 under these conditions was assayed as described below. The mixture was then transferred to confluent RLE cells grown in 24-well-plates and incubated for a further 30 min at 37°C, followed by evaluation of DNA strand breakage in the RLE cells. In parallel experiments, H2O2 was allowed to react for 15 min with MPO in the presence of either nitrite (100 µM) or 4-ABAH (100 µM), and subsequently transferred to the RLE cells. To assess whether DNA damage in the RLE cells under these conditions was H2O2-dependent, pre-incubations as described above were followed by the addition of catalase for 5 min at a final concentration of 1000 U/ml. Heat-inactivated catalase (5 min at 95°C before addition to the incubations) was used to check for enzyme specificity. Again, the complete mixture was finally transferred to confluent RLE cells and incubated for another 30 min to evaluate DNA strand breakage (Figure 1).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1. Exposure protocol for neutrophil-independent experiments. Hydrogen peroxide (25 µM) was incubated in a test tube in the presence or absence of MPO (200 mU/ml). Moreover, MPO activity was modified by addition of either nitrite (100 µM) or 4-ABAH (100 µM). After 15 min, the various mixtures were either directly transferred to confluent RLE cells (Figure 6a), or incubated with catalase for another 5 min (Figure 6b) before addition to the RLE cells. The RLE cells were then incubated for 30 min with the different pre-mixtures and processed for determination of DNA strand breakage, using the comet assay.
|
|
Hydrogen peroxide measurement
In order to evaluate effects of nitrite on H2O2 production by PMA-activated neutrophils, H2O2 was measured according to the method described by Pick and Keisari (27). Therefore, neutrophils were suspended in HBSS (6.5 x 105 cells/ml) containing 8.5 U/ml horseradish peroxidase and 0.28 mM phenol red, and plated in 24-well plates (final volume was always 400 µl). Cells were activated by PMA (100 ng/ml) and incubated in the presence of nitrite (0200 µM). After incubation (2 h), the reaction was stopped by addition of 5 µl NaOH (1 M). Absorption was measured at 610 nm using a spectrophotometer (Beckman Coulter, USA) and final concentrations were calculated from a standard curve of H2O2. Since neutrophilic MPO cannot compete with the concentration of horseradish peroxidase contained in the reaction medium, H2O2 determined in this experiment is a measure of cumulative H2O2 formation only, and is not influenced by possible consumption by MPO.
The consumption of reagent H2O2 upon incubation with purified MPO (200 mU/ml, 15 min) was evaluated by measurement of H2O2, using the method described by Gallati and Pracht (28). Therefore, 75 µl of the H2O2 incubations was mixed with 75 µl TMB containing 8.5 U/ml horseradish peroxidase. After 10 min, the reaction was stopped by addition of 50 µl H2SO4 (1 M). Absorption at 450 nm was measured using a microtiter plate reader (Bio-Rad, USA).
Measurement of myeloperoxidase activity
MPO activity in the supernatants of the neutrophil-RLE coincubations was assayed according to the method as described by Klebanoff et al. (29). Therefore, 100 µl of the supernatant was mixed with 400 µl MPO assay solution, which was made up from 26.9 ml H2O, 3 ml sodium phosphate buffer (0.1 M), 48 µl guaiacol, and 0.4 ml H2O2 (0.1 M). The generation of tetra-guaiacol was measured spectrophotometrically (Beckman) at 470 nm. The MPO activity was then calculated from the formula:
U/ml =
OD/min x 0.752 and was expressed as mU/ml. One unit of the enzyme is defined as the amount that consumes 1 µmol H2O2/min.
Single-cell gel electrophoresis
DNA single strand breakage in RLE cells was determined by single cell gel electrophoresis/alkaline comet assay (30), according to the guidelines proposed by an expert panel (31). Microscope slides were coated with a layer of 1.5% agarose. RLE cells were harvested and suspended in HBSS. Cytotoxicity in RLE cells caused by exposures and/or cell processing was evaluated using Trypan Blue dye exclusion. Subsequently, 25 µl of the cell suspension (2 x 106 cells/ml) was mixed with 75 µl 0.65% low melting point agarose. This mixture was added to the precoated slides and covered with a cover glass. Following solidification (45 min, 4°C), cover glasses were removed and slides were immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-base, 1% sodium lauryl sarcosinate, pH 10; 10% DMSO and 1% Triton X-100) and stored at 4°C. After 18 h, slides were placed in an electrophoresis tank filled with buffer (300 mM NaOH, 1 mM EDTA, pH 13, 4°C) for 30 min. Electrophoresis was conducted at 300 mA and 25 V for 15 min. Subsequently, slides were neutralized by repeated washing (3 x 10 min) with neutralization buffer (0.4 M Tris, pH 7.5). Finally, slides were immersed in ethanol and allowed to dry under air. All steps described were performed in the dark/dimmed light to prevent additional DNA damage. Dried slides were stained with ethidium bromide (10 µg/ml) and comets were visualized using a Zeiss Axioskop fluorescence microscope. Samples were tested in two independent incubations within each single experiment. On every single slide 50 cells were analyzed randomly. Initial series of experiments (see Figures 2 and 4) were analyzed by using a comet score. Therefore, comets (visualized at 1000x magnification) were classified into one out of five categories according to tail length (I, II, III, IV, V, in which I = undamaged cells, no visible tail, and V = head of comet very small, most DNA in tail), according to Collins et al. (32). For final analysis a comet-score of each individual slide was calculated: comet score = sum(class II cells + 2x class III cells + 3x class IV cells + 4x class V cells) (32). All following experiments (Figures 5, 6a and b) were analyzed using a specific software program (Comet assay II, Perceptive Instruments, Haverhill, UK). Comets were visualized using 200x magnification and DNA strand breakage was expressed as tail moment, which is defined as the product of DNA content in the tail and the mean distance of migration in the tail. Data were expressed as mean (± SD) from three independent experiments, unless stated otherwise.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2. (a) Effect of nitrite on neutrophil-induced DNA strand breakage in RLE cells. RLE cells were incubated for 2 h with PMA-activated neutrophils (PMN) (6.5 x 105 neutrophils/ml, 100 ng/ml PMA), at a 1:1 ratio with RLE cells, in the presence of increasing concentrations of nitrite (NO2). DNA strand breakage was specifically assessed in the RLE cells and is expressed as a comet score (see Materials and methods section for details). *P < 0.05 versus control (ctrl, RLE cells without neutrophils, PMA and nitrite). #P < 0.05 versus 0 µM nitrite (n = 3). (b) Distribution of damaged cells over the various comet classes (IIV). See Materials and methods section for a description of this comet analysis. (c) Effect of nitrite on neutrophil-derived MPO activity. Supernatants of the co-incubations of PMA-activated neutrophils (PMN) with RLE cells (Figure 2a) were used to assess the effect of nitrite on MPO activity. No MPO activity was detected in supernatants from RLE cells only (ND, not detectable). MPO activity is expressed as mU/ml. **P < 0.01 versus 0 µM nitrite (n = 3).
|
|
Statistical analysis
All data are expressed as mean ± SD. Statistical analysis was performed using SPPS (version 11.5) for Windows. Differences between experimental groups were analyzed using one-way ANOVA. Multiple comparisons were evaluated using Tukey's method, unless stated otherwise. Correlations between dose (4-ABAH) and effect (MPO activity, DNA strand breakage) were evaluated using Pearson. In all cases, differences were considered statistically significant at P < 0.05.
 |
Results
|
---|
Effect of nitrite on neutrophil-induced DNA strand breakage in RLE cells
PMA-activated neutrophils caused a significant increase in DNA strand breakage in co-cultured RLE cells. Addition of nitrite to these co-cultures led to a dose-dependent enhancement of this effect (Figure 2a). In the absence of PMA, no increased DNA damage was seen in the co-cultured RLE cells. Also, no increased DNA damage was found in the RLE cells upon incubation with a mix of nitrite (100 µM) and PMA (100 ng/ml) only (i.e. no neutrophils added). The distribution of the damaged cells over the various comet classes is shown in Figure 2b. As can be seen in the figure, effects of neutrophils and nitrite on DNA damage in RLE cells are largely reflected by an increase in class IV cells. The effects as shown in Figure 2a were found in the absence of cytotoxicity in the RLE cells (viability was always >96%), as evaluated by Trypan Blue dye exclusion in the same incubations. This aspect was further illustrated by the absence (<5%) of so-called ghost (Class V) cells in our comet assay experiments (Figure 2b). Such ghost cells contain highly damaged DNA and have been suggested to reflect necrotic or apoptotic cells. Also, the pH (7.4) of the incubation medium was not found to be changed upon addition of nitrite.
The supernatants of the RLE-neutrophil co-cultures were also analyzed for MPO activity. As shown in Figure 2b, MPO activity was detected only in the incubations containing neutrophils and its activity was significantly reduced upon addition of physiological levels of nitrite. Reduction of MPO activity was maximal at 100 µM nitrite. At higher nitrite concentrations (up to 500 µM), no further decrease in MPO activity could be observed (data not shown).
Interactions between nitrite and hydrogen peroxide
The effect of nitrite on the production of H2O2 by PMA-activated neutrophils is shown in Figure 3. As can be seen in the figure, the amount of activated neutrophils, used for the co-culture experiments, produce about 30 µM of H2O2 within 2 h. Addition of nitrite (0200 µM) to PMA-activated neutrophils did not appear to affect their H2O2 release. In the absence of PMA, no H2O2 release from the neutrophils could be detected. The presence of nitrite did not interfere with the method used to detect H2O2 production by the neutrophils, since the absorption of 30 µM reagent H2O2 was unaffected by the addition of increasing concentrations of nitrite.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 3. Effect of nitrite on H2O2 production by PMA-activated neutrophils. Neutrophils (PMN, 6.5 x 105 cells/ml) were suspended in HBSS containing nitrite, horseradish peroxidase and phenol red and activated with PMA (100 ng/ml). Following 2 h of incubation, the reaction was stopped with NaOH. Absorption was spectrophotometrically measured at 610 nm and H2O2 concentration was calculated from a standard curve.
|
|
We further evaluated whether nitrite affects direct H2O2-induced DNA strand breakage. Results are shown in Figure 4. Strand breakage was first assessed in RLE cells exposed to 25 µM of reagent H2O2, which equals the amount of H2O2 that was found to accumulate in our co-culture settings. When the RLE cells were exposed to H2O2 in the presence of nitrite, at a level (100 µM) which significantly enhanced neutrophil-induced DNA strand breakage (Figure 2a), the DNA damage did not differ from the damage as induced by H2O2 alone (Figure 4). Nitrite alone did not cause significant DNA strand breakage. All of the above effects were observed in the absence of acute cytotoxicity.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 4. DNA strand breakage by a mixture of H2O2 and nitrite. RLE cells were exposed for 2 h to H2O2, to H2O2 plus nitrite or to nitrite alone. (a) DNA strand breakage was evaluated by the comet assay and expressed as comet score. (b) Distribution of damaged cells over the various comet classes (IIV). See Materials and methods section for a description of this comet analysis. *P < 0.05 versus ctrl (control, without H2O2 or nitrite).
|
|
Effect of 4-ABAH on neutrophil-induced DNA strand breakage in co-cultured RLE cells
In order to further evaluate the role of MPO in neutrophil-induced DNA damage, co-culture experiments as described in Figure 2 were also performed using the specific MPO inhibitor 4-ABAH (Figure 5). MPO activity in the co-culture supernatants was found to be inhibited in a dose-dependent manner by 4-ABAH. At the highest concentration of 4-ABAH, MPO activity was completely inhibited. MPO inhibition by 4-ABAH also resulted in a significant enhancement of neutrophil-induced DNA strand breakage in the RLE cells. In fact, a clear inverse correlation was observed between MPO-activity and DNA strand breakage. In the absence of neutrophils, the combination of 4-ABAH and PMA did not cause DNA strand breakage in the RLE cells.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 5. Effect of 4-ABAH on neutrophilic MPO activity and DNA damaging capacity. Neutrophils were co-cultured with RLE cells at a 1:1 ratio (6.5 x 105 neutrophils/ml) in the presence of PMA (100 ng/ml) and increasing concentrations of 4-ABAH. DNA strand breakage was assessed in the RLE cells by the comet assay and expressed as tail moment. MPO activity in the supernatants of the co-incubations was determined spectrophotometrically and expressed as mU/ml (see Materials and methods section). Bars represent DNA strand breakage (left vertical axis), whereas the solid line represents MPO activity (right vertical axis). The horizontal line indicates the background DNA damage in RLE cells in the absence of neutrophils, PMA and 4-ABAH. MPO activity in the supernatants as well as DNA damage in the RLE cells is significantly correlated with the 4-ABAH dose (Pearson = 0.699, P = 0.03 for MPO activity; Pearson = 0.602, P = 0.004 for DNA strand breakage respectively). *P < 0.05 versus 0 µM 4-ABAH (ANOVA, Dunnett).
|
|
DNA strand breakage by mixtures of H2O2, MPO and nitrite
The effect of nitrite and 4-ABAH on MPO activity and related neutrophil-induced DNA damage was further studied using a neutrophil-independent model in which reagent H2O2 was allowed to react for 15 min with purified MPO (Figure 1). Under these conditions, the original concentration of H2O2 (25 µM) was reduced to 0.6 ± 0.4 µM, because of consumption by MPO. After pre-incubation, the mixture was immediately transferred to the RLE cells and incubated for 30 min to evaluate DNA damage. Results of these experiments are shown in Figure 6a. Hydrogen peroxide caused a significant induction of DNA strand breakage in the RLE cells, whereas DNA damage was abrogated when H2O2 was pre-incubated with the purified MPO. However, upon further addition of either nitrite or 4-ABAH to this mixture, the MPO-related inhibition of H2O2-induced DNA damage was significantly reversed. In comparison with nitrite, this effect seemed to be more pronounced upon addition of 4-ABAH. MPO, 4-ABAH as well as nitrite alone failed to cause DNA strand breakage.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6. (a) Effects of nitrite and 4-ABAH on DNA damaging capacity of mixtures of H2O2 and MPO. H2O2 (25 µM) was pre-incubated with MPO (200 mU/ml) in the presence of nitrite (100 µM) or 4-ABAH (100 µM). After 15 min the mixtures were transferred to confluent RLE cells and incubated for another 30 min. DNA strand breakage was assessed by the comet assay and expressed as tail moment (also see Figure 1). *P < 0.05 versus control. **P < 0.01 versus control. (b) Effect of nitrite-dependent modulation of H2O2 consumption by MPO on DNA damage in RLE cells. Pre-incubations were performed as described in (a). However, before addition of the mixtures to the RLE cells, catalase (Cat, 1000 U/ml), was added and incubated for 5 min to inactivate any possible remaining H2O2. Heat-inactivated catalase (CatHi) was used to assess the specificity of this effect. DNA strand breakage (tail moment) in the RLE cells was determined by the comet assay and expressed as a percentage of control incubations (i.e. RLE cells w/o oxidants) (also see Figure 1). *P < 0.05 versus H2O2; #P < 0.05 versus H2O2/MPO/Cat.
|
|
To verify whether the DNA damaging effects, as shown in Figure 6a, were caused by (unconsumed) H2O2, the pre-incubations were followed by addition of the H2O2 scavenger catalase (Figure 1). The whole mixture was then transferred to RLE cells for DNA damage assessment. Incubation of H2O2 with catalase led to a significant reduction of H2O2-induced DNA damage, whereas heat-inactivated catalase was ineffective (Figure 6b). Similarly, the DNA damaging effect of the premixture of H2O2, MPO and nitrite was reduced if catalase was added prior to incubation with the cells. Again heat inactivated catalase was ineffective.
 |
Discussion
|
---|
It is generally accepted that the capacity of neutrophils to generate a wealth of DNA damaging oxidants provides a biological basis for the link between chronic inflammation and tumour development in the particle-exposed lung (2,3,7). However, although particle-induced neutrophil influx in the lung is often accompanied by an accumulation of the nitric oxide congener nitrite (19,21,22), the possible effects of nitrite on neutrophil-induced DNA damage in lung target cells largely remain to be elucidated.
In the present study, we applied a previously established co-culture system comprised of activated neutrophils and pulmonary type II cells. We specifically applied type II cells, since this is the cell type from which (rat) lung tumours are most probably derived after chronic particle exposure (33). Although our findings may very well have implications for other organs where neutrophil influx is accompanied by an accumulation of nitrite, we specifically focused on the lung epithelium because of several reasons: (i) neutrophils are recognized as crucial effector cells in the pathogenicity of pulmonary inflammation (34,35), (ii) we and others showed that DNA strand breakage and mutagenicity in pulmonary epithelium of (particle-treated) rats was closely related to neutrophil influx (6,15,36), (iii) pulmonary inflammation is characterized by the release of the MPO substrate H2O2 (37) and (iv) mainly by the presence of alveolar macrophages, the lung contains a potent source of nitrite (19,21,22). We demonstrated that nitrite significantly enhances neutrophil-induced DNA strand breakage in co-cultured epithelial cells. This effect was seen at physiologically relevant concentrations of nitrite (<100 µM). For instance, in biological fluids (saliva, gastric fluid) nitrite concentrations up to 200 µM can be found, and in epithelial lining fluids nitrite may reach concentrations that exceed 40 µM (38). It should be emphasized that the increase in DNA damage by PMA-activated neutrophils, as observed in the presence of nitrite, was not caused by a possible effect of nitrite on NADPH-oxidase activity, since the production of H2O2 by PMA-activated neutrophils remained unchanged after addition of nitrite. Moreover, others have shown that the inhibitory effect of nitrite on neutrophilic MPO activity was enhanced upon addition of superoxide dismutase (24), which suggests that the effects of nitrite cannot simply be explained by an inhibition of MPO degranulation by neutrophils. In addition, a synergistic interaction of nitrite with neutrophil-derived H2O2, as well as a possible inhibitory effect of nitrite on target cellular catalase (39) can be excluded in our current study, since we showed that the DNA damaging effects of H2O2 plus nitrite mixture were comparable with H2O2 alone.
To further explore the concept that the neutrophilic DNA strand breaking capacity is increased upon inhibition of MPO activity, co-culture experiments were conducted in the presence of 4-ABAH. We applied 4-ABAH for three reasons: (i) 4-ABAH is known as one of the most potent inhibitors of MPO, (ii) 4-ABAH does not affect neutrophil-induced NADPH-oxidase and (iii) unlike many other MPO inhibitors such as sodium azide, 4-ABAH does not inhibit catalase or glutathione peroxidase (40). As such, we believe that the increase in DNA damage as observed upon 4-ABAH-mediated MPO inhibition can neither be explained by changes in H2O2 production by neutrophils, nor can it be explained by an increased effect of H2O2 due to inhibition of catalase or glutathione peroxidase in the RLE cells. Notably, in contrast to nitrite, 4-ABAH is a suicide substrate that promotes irreversible inactivation of the enzyme (41). This possibly explains why 4-ABAH, unlike nitrite, is able to fully inactivate neutrophil-derived MPO, and subsequently has a more pronounced effect on neutrophil-induced DNA strand breakage.
Hydrogen peroxide is a relatively stable compound, which is able to cross cellular membranes. As such in a co-culture model, which allows a close contact between neutrophils and target cells, there will be a constant balance between the consumption of H2O2 by MPO, versus the capture of diffusible H2O2 by the neighbouring target cells (42). Considering that MPO consumes up to 4070% of neutrophil-derived H2O2 to generate HOCl (16,17), a minor inhibition of its activity would most probably lead to a significant increase in the availability of freely diffusible H2O2 that may eventually elicit DNA damage in neighbouring cells. This concept is at least partly confirmed by our co-culture experiments using nitrite and 4-ABAH. Moreover, the data showing an increase in neutrophil-induced DNA damage upon MPO inhibition also provide further support for observations from others who suggested that MPO-derived HOCl is not involved in DNA strand breakage in cells that are exposed to activated neutrophils (8).
To further evaluate inhibition of MPO by nitrite and to investigate whether related enhancement of DNA damage in epithelial cells is caused by a consequential increased availability of H2O2, one could consider applying catalase directly into the co-culture experiments. However by doing this, neutrophil-derived H2O2 would be scavenged by catalase before it could even react with MPO. An alternative approach would be to measure accumulation of H2O2 itself. However, we failed to detect any H2O2 after the co-incubations (data not shown), most probably because diffusible H2O2 will be captured rapidly and consumed intracellularly (42). Considering all of the above aspects, we thus decided to perform neutrophil-independent experiments in which H2O2 was incubated with purified MPO before addition to the RLE cells, and found that MPO caused inhibition of DNA strand breakage because of its complete consumption of H2O2. The inhibiting effect of MPO was reversed by addition of 4-ABAH or (although to a lesser extent) nitrite. This discrepancy is probably explained by the fact that, in contrast to 4-ABAH, nitrite does not fully inhibit the consumption of H2O2 by purified MPO. For instance, others demonstrated that in comparable settings nitrite was able to reduce consumption of H2O2 by MPO up to a maximum of 80% (24). Importantly, our experiments using catalase (Figure 6b) further confirmed that DNA strand breakage by pre-incubations of H2O2, MPO and nitrite was caused by H2O2 that has escaped consumption by MPO. This also indicates that nitrite-induced enhancement of DNA strand breakage by neutrophils, as observed in the co-cultures, is probably caused by inhibition of MPO, and a consequential increased availability of H2O2 that may diffuse into the epithelial cells.
Hydrogen peroxide will generally cause a variety of DNA damages. In the present study we specifically focused on DNA strand breakage using the comet assay, because we previously showed that this method sensitively detects DNA damage upon exposure to low concentrations (<25 µM) of H2O2 (43). Additionally, others showed that DNA strand breakage by H2O2 was well correlated with the sum of a variety of DNA base modifications in H2O2-exposed pulmonary epithelial cells (13), indicating that DNA strand breakage is an integrative measure of the broad spectrum of H2O2-induced DNA modifications. However the results, therefore, do not necessarily reflect the effects of nitrite on alternative (H2O2-independent) forms of neutrophil-mediated DNA damage. For instance, it should be emphasized that MPO has been implicated in the induction of a variety of DNA base lesions, including chlorinated bases (44,45). Furthermore, apart from H2O2, a contribution of other oxidants to comet-detected DNA strand breakage can not be entirely ruled out. For example, one could speculate on a contribution of reactive nitrogen species like NO2· and nitryl chloride (NO2Cl), that are generated during an interaction between MPO and nitrite (23). Nevertheless, although the latter compound has been shown to cause oxidative base damage in naked DNA (46), further studies indicated that it is unlikely to contribute to DNA strand breakage in living cells (18). Actually, it was shown that nitrite might even act as an inhibitor of HOCl-induced strand breakage in naked DNA, possibly via formation of the less reactive NO2Cl (47). Also for NO2·, which is a potent nitrating compound, and which is implicated in the formation of 8-nitroguanine in naked DNA (48), the relevance for DNA damage in living cells is questioned (49).
In conclusion, we showed that nitrite synergistically enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells. Our data suggest that this effect is related to a nitrite-induced inhibition of MPO activity. Further investigations, including application of the MPO inhibitor 4-ABAH, revealed a general concept showing that inhibition of MPO as such can increase the DNA damaging capacity of neutrophils. This effect is probably caused by an increased availability of H2O2 that can freely diffuse into the target cells. Our data indicate that this process may be of specific importance in the pathogenic effects of particle inhalation, as this is characterized by the simultaneous presence of activated neutrophils and nitrite in the lung. Further work is needed to assess the implication of our current observations for in vivo particle-induced mutagenicity and carcinogenicity in relation to neutrophilic pulmonary inflammation.
 |
Acknowledgments
|
---|
A.M.K. is a postdoctoral research fellow of the Netherlands Organisation for Scientific Research (NWO, grant 916.46.092). Part of the study was financially supported by the German Research Foundation (DFG, grant BO-1657/2-1).
Conflict of Interest Statement: None declared.
 |
References
|
---|
- Coussens,L.M. and Werb,Z. (2002) Inflammation and cancer. Nature, 420, 860867.[CrossRef][ISI][Medline]
- Borm,P.J. and Driscoll,K. (1996) Particles, inflammation and respiratory tract carcinogenesis. Toxicol. Lett., 88, 109113.[ISI][Medline]
- Greim,H., Borm,P.J., Schins,R.P., Donaldson,K., Driscoll,K.E., Hartwig,A., Kuempel,E., Oberdörster,G. and Speit,G. (2001) Toxicity of fibers and particles. Inhal. Toxicol., 13, 101119.
- Nehls,P., Seiler,F., Rehn,B., Greferath,R. and Bruch J. (1997) Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure. Environ. Health Perspect., 105, (suppl. 5), 12911296.[Medline]
- Knaapen,A.M., Seiler,F., Schilderman,P.A., Nehls,P., Bruch,J., Schins,R.P. and Borm,P.J. (1999) Neutrophils cause oxidative DNA damage in alveolar epithelial cells. Free Radic. Biol. Med., 27, 234240.[CrossRef][ISI][Medline]
- Driscoll,K.E., Deyo,L.C., Carter,J.M., Howard,B.W., Hassenbein,D.G. and Bertram,T.A. (1997) Effects of particle exposure and particle-elicited inflammatory cells on mutation in rat alveolar epithelial cells. Carcinogenesis, 18, 423430.[Abstract]
- Wiseman,H. and Halliwell,B. (1996) Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem. J., 313, 1729.[ISI][Medline]
- Schraufstatter,I., Hyslop,P.A., Jackson,J.H. and Cochrane,C.G. (1988) Oxidant-induced DNA damage of target cells. J. Clin. Invest., 82, 10401050.[ISI][Medline]
- Jackson,J.H., Gajewski,E., Schraufstatter,I.U., Hyslop,P.A., Fuciarelli,A.F., Cochrane,C.G. and Dizdaroglu,M. (1989) Damage to the bases in DNA induced by stimulated human neutrophils. J. Clin. Invest., 84, 16441649.[ISI][Medline]
- Henle,E.S. and Linn,S. (1997) Formation, prevention, and repair of DNA damage by iron/hydrogen peroxide. J. Biol. Chem., 272, 1909519098.[Free Full Text]
- Shacter,E., Beecham,E.J., Covey,J.M., Kohn,K.W. and Potter,M. (1988) Activated neutrophils induce prolonged DNA damage in neighboring cells. Carcinogenesis, 9, 22972304.[Abstract]
- Dizdaroglu,M., Olinski,R., Doroshow,J.H. and Akman,S.A. (1993) Modification of DNA bases in chromatin of intact target human cells by activated human polymorphonuclear leukocytes. Cancer Res., 53, 12691272.[Abstract]
- Spencer,J.P., Jenner,A., Chimel,K., Aruoma,O.I., Cross,C.E., Wu,R. and Halliwell,B. (1995) DNA strand breakage and base modification induced by hydrogen peroxide treatment of human respiratory tract epithelial cells. FEBS Lett., 374, 233236.[CrossRef][ISI][Medline]
- Kim,H.W., Murakami,A., Williams,M.V. and Ohigashi,H. (2003) Mutagenicity of reactive oxygen and nitrogen species as detected by co-culture of activated inflammatory leukocytes and AS52 cells. Carcinogenesis, 24, 235241.[Abstract/Free Full Text]
- Auten,R.L., Whorton,M.H. and Nicholas Mason,S. (2002) Blocking neutrophil influx reduces DNA damage in hyperoxia-exposed newborn rat lung. Am. J. Respir. Cell Mol. Biol., 26, 391397.[Abstract/Free Full Text]
- Hampton,M.B., Kettle,A.J. and Winterbourn,C.C. (1998) Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood, 92, 30073017.[Free Full Text]
- Klebanoff,S.J. (1999) Myeloperoxidase. Proc. Assoc. Am. Physicians, 111, 383389.[ISI][Medline]
- Spencer,J.P., Whiteman,M., Jenner,A. and Halliwell,B. (2000) Nitrite-induced deamination and hypochlorite-induced oxidation of DNA in intact human respiratory tract epithelial cells. Free Radic. Biol. Med., 28, 10391050.[CrossRef][ISI][Medline]
- Huffman,L.J., Prugh,D.J., Millecchia,L., Schuller,K.C., Cantrell,S. and Porter,D.W. (2003) Nitric oxide production by rat bronchoalveolar macrophages or polymorphonuclear leukocytes following intratracheal instillation of lipopolysaccharide or silica. J. Biosci., 28, 2937.[ISI][Medline]
- Ignarro,L.J., Fukuto,J.M., Griscavage,J.M., Rogers,N.E. and Byrns,R.E. (1993) Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: comparison with enzymatically formed nitric oxide from L-arginine. Proc. Natl Acad. Sci. USA, 90, 81038107.[Abstract/Free Full Text]
- Porter,D.W., Millecchia,L., Robinson,V.A., Hubbs,A., Willard,P., Pack,D., Ramsey,D., McLaurin,J., Khan,A., Landsittel,D., Teass,A., and Castranova,V. (2002) Enhanced nitric oxide and reactive oxygen species production and damage after inhalation of silica. Am. J. Physiol. Lung Cell Mol. Physiol., 283, L485493.[Abstract/Free Full Text]
- Nelin,L.D., Krenz,G.S., Chicoine,L.G., Dawson,C.A. and Schapira,R.M. (2002) L-Arginine uptake and metabolism following in vivo silica exposure in rat lungs. Am. J. Respir. Cell Mol. Biol., 26, 34855.[Abstract/Free Full Text]
- Eiserich,J.P., Hristova,M., Cross,C.E., Jones,A.D., Freeman,B.A., Halliwell,B. and van der Vliet,A. (1998) Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature, 391, 393397.[CrossRef][ISI][Medline]
- van Dalen,C.J., Winterbourn,C.C., Senthilmohan,R. and Kettle,A.J. (2000) Nitrite as a substrate and inhibitor of myeloperoxidase. Implications for nitration and hypochlorous acid production at sites of inflammation. J. Biol. Chem., 275, 1163811644.[Abstract/Free Full Text]
- Carr,A.C. and Frei,B. (2001) The nitric oxide congener nitrite inhibits myeloperoxidase/H2O2/Cl-mediated modification of low density lipoprotein. J. Biol. Chem., 276, 18221828.[Abstract/Free Full Text]
- Boyum,A. (1976) Isolation of lymphocytes, granulocytes and macrophages. Scand. J. Immunol., Suppl. 5, 915.[ISI][Medline]
- Pick,E. and Keisari,Y. (1980) A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J. Immunol. Methods, 38, 161170.[CrossRef][ISI][Medline]
- Gallati,H. and Pracht,I. (1985) Horseradish peroxidase: kinetic studies and optimization of peroxidase activity determination using substrates H2O2 and 3,3',5,5'-tetramethylbenzidine. J. Clin. Chem. Clin. Biochem., 23, 453460.[ISI][Medline]
- Klebanoff,S.J., Waltersdorph,A.M. and Rosen,H. (1984) Antimicrobial activity of myeloperoxidase. Meth. Enzymol., 105, 399403.[ISI][Medline]
- Singh,N.P., McCoy,M.T., Tice,R.R. and Schneider,E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res., 175, 184191.[ISI][Medline]
- Tice,R.R., Agurell,E., Anderson,D., Burlinson,B., Hartmann,A., Kobayashi,H., Miyamae,Y., Rojas,E., Ryu,J.C. and Sasaki,Y.F. (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen., 35, 206221.[CrossRef][ISI][Medline]
- Collins,A.R., Ma,A.G. and Duthie,S. J. (1995) The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mutat. Res., 336, 6977.[CrossRef][ISI][Medline]
- Johnson,N.F., Smith,D.M., Sebring,R. and Holland,L.M. (1987) Silica-induced alveolar cell tumors in rats. Am. J. Ind. Med., 11, 93107.[ISI][Medline]
- Sibille,Y. and Marchandise,F.X. (1993) Pulmonary immune cells in health and disease: polymorphonuclear neutrophils. Eur. Respir. J., 6, 15291543.[Abstract]
- Duffin,R., Gilmour,P.S., Schins,R.P., Clouter,A., Guy,K., Brown,D.M., MacNee,W., Borm,P.J., Donaldson,K. and Stone,V. (2001) Aluminium lactate treatment of DQ12 quartz inhibits its ability to cause inflammation, chemokine expression, and nuclear factor-kappaB activation. Toxicol. Appl. Pharmacol., 176, 1017.[CrossRef][ISI][Medline]
- Knaapen,A.M., Albrecht,C., Becker,A., Hohr,D., Winzer,A., Haenen,G.R., Borm,P.J. and Schins,R.P. (2002) DNA damage in lung epithelial cells isolated from rats exposed to quartz: role of surface reactivity and neutrophilic inflammation. Carcinogenesis, 23, 11111120.[Abstract/Free Full Text]
- Dekhuijzen,P.N., Aben,K.K, Dekker,I., Aarts,L.P., Wielders,P.L., van Herwaarden,C.L. and Bast,A. (1996) Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care. Med., 154, 813816.[Abstract]
- Green,L.C., Wagner,D.A, Glogowski,J., Skipper,P.L., Wishnok,J.S. and Tannenbaum,S.R. (1982) Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem., 126, 131138.[CrossRef][ISI][Medline]
- Titov,V.Y. and Petrenko,Y.M. (2003) Nitrite-catalase interaction as an important element of nitrite toxicity. Biochemistry (Mosc), 68, 627633.[CrossRef][ISI][Medline]
- Kettle,A.J., Gedye,C.A., Hampton,M.B. and Winterbourn,C.C. (1995) Inhibition of myeloperoxidase by benzoic acid hydrazides. Biochem. J., 308, 559563.[ISI][Medline]
- Burner,U., Obinger,C., Paumann,M., Furtmuller,P.G. and Kettle,A.J. (1999) Transient and steady-state kinetics of the oxidation of substituted benzoic acid hydrazides by myeloperoxidase. J. Biol. Chem., 274, 94949502.[Abstract/Free Full Text]
- Test,S.T. and Weiss,S.J. (1984) Quantitative and temporal characterization of the extracellular H2O2 pool generated by human neutrophils. J. Biol. Chem., 259, 399405.[Abstract/Free Full Text]
- Schins,R.P., Knaapen,A.M., Cakmak,G.D., Shi,T., Weishaupt,C. and Borm,P.J. (2002) Oxidant-induced DNA damage by quartz in alveolar epithelial cells. Mutat. Res., 517, 7786.[ISI][Medline]
- Jiang,Q., Blount,B.C. and Ames,B.N. (2003) 5-Chlorouracil, a marker of DNA damage from hypochlorous acid during inflammation. A gas chromatography-mass spectrometry assay. J. Biol. Chem., 278, 3283432840.[Abstract/Free Full Text]
- Henderson,J.P., Byun,J., Takeshita,J. and Heinecke,J.W. (2003) Phagocytes produce 5-chlorouracil and 5-bromouracil, two mutagenic products of myeloperoxidase, in human inflammatory tissue. J. Biol. Chem., 278, 2352223528.[Abstract/Free Full Text]
- Whiteman,M., Spencer,J.P., Jenner,A. and Halliwell,B. (1999) Hypochlorous acid-induced DNA base modification: potentiation by nitrite: biomarkers of DNA damage by reactive oxygen species. Biochem. Biophys. Res. Commun., 257, 572576.[CrossRef][ISI][Medline]
- Whiteman,M., Hooper,D.C., Scott,G.S., Koprowski,H. and Halliwell,B. (2002) Inhibition of hypochlorous acid-induced cellular toxicity by nitrite. Proc. Natl Acad. Sci. USA, 99, 1206112066.[Abstract/Free Full Text]
- Byun,J., Henderson,J.P., Mueller,D.M. and Heinecke,J.W. (1999) 8-Nitro-2'-deoxyguanosine, a specific marker of oxidation by reactive nitrogen species, is generated by the myeloperoxidase-hydrogen peroxide-nitrite system of activated human phagocytes. Biochemistry, 38, 25902600.[CrossRef][ISI][Medline]
- Tuo,J., Liu,L., Poulsen,H.E., Weimann,A., Svendsen,O. and Loft,S. (2000) Importance of guanine nitration and hydroxylation in DNA in vitro and in vivo. Free Radic. Biol. Med., 29,147155.[CrossRef][ISI][Medline]
Received March 22, 2005;
revised April 22, 2005;
accepted April 26, 2005.