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Department of Health Risk Analysis and Toxicology, Nutrition and Toxicology Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
1Correspondence: Department of Health Risk Analysis and Toxicology, Maastricht University, PO Box 616, 6200 MD, Maastricht, The Netherlands. E-mail: a.knaapen{at}grat.unimaas.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: DNA adducts • DNA repair • inflammation • lung cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. However, the underlying processes have not yet been fully defined at the biochemical and molecular level. Within the lung, many inflammatory agents may play a part in accelerating carcinogenesis, including inhaled combustion-derived particles, such as cigarette smoke, fine particulate matter (PM), and diesel exhaust particles (3)
. Inhalation of such particles causes a local pulmonary inflammatory response that is characterized by the influx of polymorphonuclear neutrophils (PMNs) into the airways. These inflammatory PMNs are involved in the pathogenesis of various pulmonary diseases, such as chronic obstructive pulmonary disease (COPD) (4)
, fibrosis (5)
, and cancer (6)
.
On entering the lung, neutrophils are activated to release reactive oxygen species (ROS), as well as an array of proteins, such as myeloperoxidase (MPO). MPO catalyzes the conversion of H2O2 into the bactericidal compound hypochlorous acid (HOCl) (7)
. Under physiological conditions, the concentration of HOCl ranges from 10 to 100 µM (8)
, although concentrations up to 200 µM have been reported in some tissues (9)
. Even though HOCl formation has been linked to the induction of DNA damage (10)
, the primary cellular targets for HOCl appear to be proteins (11)
. HOCl-protein interactions result in side-chain modification, backbone fragmentation, and cross-linking (12)
, which may lead ultimately to disturbed enzyme function. In vitro studies indeed have identified HOCl as a potent inhibitor of acetylcholinesterase (13)
and glutathione S-transferase P1–1 (14)
. In addition, HOCl has also been demonstrated to be an inhibitor of DNA strand break repair (15)
, as well as of poly (ADP-ribose) polymerase (PARP) activity, which is involved in base excision repair (16)
.
Epidemiological studies have indicated that neutrophils are involved in pulmonary carcinogenicity by releasing MPO in the cellular environment. For instance, there is evidence that a polymorphism in the promotor region of the MPO gene (–463G
A transition) is associated with a decreased risk of developing lung cancer in smokers (17)
. At present, the most widely accepted explanation for this association is the role of MPO in the bioactivation of inhaled carcinogens, such as polycyclic aromatic hydrocarbons (PAHs). For example, MPO has been demonstrated to enhance the transformation of the prototype PAH benzo[a]pyrene (B[a]P) into DNA-binding metabolites [e.g., B[a]P-diol-epoxide (BPDE)] (18
19
20
21)
. If these helix-distorting DNA lesions escape from specific DNA repair (i.e., nucleotide excision repair), they may cause replication fork blocks, ultimately leading to mutagenesis and carcinogenesis (22
23
24)
.
Recent data have revealed that oxidizing species, such as 4-hydroxynonenal (4-HNE) (25)
, malondialdehyde (MDA) (26)
, and nitric oxide (27)
, are endogenous inhibitors of nucleotide excision repair. A comparable effect of the potent oxidising MPO product HOCl could, thus, provide an additional explanation for the observed association between neutrophils and lung cancer risk in subjects exposed to DNA adduct forming chemicals. In the present study, we investigated the effect of activated neutrophils on the nucleotide excision repair (NER) capacity of pulmonary epithelial cells. We hypothesized that neutrophils, by virtue of their capacity to release HOCl, are potent inhibitors of NER, thereby causing a disturbed removal of promutagenic PAH-DNA adducts. To test this hypothesis, activated neutrophils were cocultured with human epithelial lung cells. We found that neutrophils significantly reduced the nucleotide excision repair capacity of the cocultured epithelial cells, leading to a delayed removal of bulky BPDE-DNA adducts. This effect appeared to be mediated by MPO-derived HOCl. We propose that these effects of neutrophils may provide an additional biological explanation for the observed association between inflammation and lung cancer risk (6)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cocultures of neutrophils and epithelial lung cells
Coincubation experiments were performed as described by Knaapen et al. (28)
. Briefly, neutrophils were freshly isolated from the blood of healthy, nonsmoking volunteers by gradient centrifugation (800 g, 20 min, 4°C), using lymphoprep (Axis-Shields, Oslo, Norway). Lymphocytes were removed, and the remaining erythrocytes were lysed using cold (4°C) lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 10 mM EDTA, pH 7.4). PMNs were washed and resuspended in Hanks’ balanced salt solution (HBSS; Life Technologies, Invitrogen, Breda, The Netherlands) at 10 x 106 cells/ml. This method consistently yielded PMNs with a viability >95%, as assessed by trypan blue dye exclusion (Sigma). During all the stages of the isolation procedure, PMNs and solutions were kept on ice to prevent premature activation.
Neutrophils were cocultured with confluent A549 cells grown in 75 cm2 culture flasks. The final volume of the incubations was always 10 ml with a total neutrophil number of 5 x 106 or 10 x 106 yielding a final neutrophil:A549 ratio of 1:2 and 1:1, respectively. Neutrophils were subsequently activated with phorbol 12-myristate 13-acetate (100 ng/ml; PMA, Sigma) to elicit the respiratory burst. Cells were then coincubated during 1 h in HBSS (37°C, 5% CO2). Control incubations were without PMNs and/or without PMA. After incubation, the neutrophils were removed using two repetitive washings with 10 ml of cold (4°C) HBSS. The A549 cells were harvested by trypsination for analyses of toxicity and NER capacity. Cytotoxicity of PMA and/or PMNs in A549 was tested using trypan blue dye exclusion. All studies were performed at pH 7.4. To investigate the role of MPO, 100 µM of the highly specific MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH, Sigma) (29)
were added to coincubations of A549 with PMA-activated PMNs (1:1 ratio). Coincubations were then performed as described above.
Measurement of MPO activity
MPO activity in the supernatant of the neutrophil-A549 coincubations was assayed according to the method described by Klebanoff et al. (30)
. Therefore, 100 µl of supernatant were mixed with 400 µl MPO assay solution, which comprised 26.9 ml H2O, 3 ml sodium phosphate buffer (0.1 M, pH 7.0), 48 µl guaiacol, and 100 µl H2O2 (0.1 M; all of these chemicals were purchased from Merck, Darmstadt, Germany). The generation of tetra-guaiacol was measured spectrophotometrically (Beckman) at 470 nm. MPO activity was calculated using the formula: U/ml = 
OD/min x 0.7518 and was expressed as mU/ml. One unit is defined as the amount of the enzyme that consumes 1 µmol H2O2/min.
Cell treatment with HOCl
The concentration of the HOCl stock solution (Sigma) was measured spectrophotometrically, immediately before use at 293 nm, using the extinction coefficient of hypochlorite 
293 = 349.2 M–1·cm–1 at pH 10–12. HOCl was diluted in HBSS to final, physiological concentrations ranging from 0 to 200 µM. Confluent A549 cells were washed twice with HBSS of 37°C. Fresh HBSS was then added followed by the addition of HOCl. After 15 min of incubation at 37°C, cells were harvested as described above. To assess prolonged effects of HOCl exposure, cells were treated with HOCl during 15 min, followed by washing and continued incubation in DMEM for up to 24 h. Addition of HOCl did not alter the pH of the reaction mixture. Cytotoxicity of HOCl in A549 was tested using trypan blue dye exclusion.
Repair assay: modified comet assay to assess phenotypically nucleotide excision repair
Phenotypical assessment of NER capacity of the A549 cells was performed using a modified comet-assay developed in our own laboratory (31)
. This assay is based on a method described by Collins et al. (32)
and is specifically designed to test the ability of cell lysates to detect and excise BPDE-DNA adducts in gel-embedded nucleoids. Briefly, nucleoids are prepared by embedding untreated A549 cells in low melting point agarose (Sigma, Zwijndrecht, The Netherlands) on microscopic slides and subsequently lysing these cells overnight in cold (4°C) lysis buffer [2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 0.25 M NaOH, pH 10, 10% dimethyl sulfoxide (DMSO), and 1% Triton X-100]. The resulting nucleoids were then exposed to either 1 µM BPDE (NCI Chemical Carcinogen Reference Standard Repository, Midwest Research Institute, Kansas City, MO, USA) or DMSO during 30 min on ice.
In parallel, cell extracts were prepared from the cocultured A549 cells or the A549 cells treated with HOCl, based on the method developed by Redaelli et al. (33)
, with minor modifications (31)
. In some experiments, lysates were first isolated from untreated A549 cells and then exposed to HOCl in vitro, to investigate the consequences of the direct interaction of HOCl with the repair proteins. Protein concentrations of the cell lysates were determined by the BIO-RAD DC Protein Assay Kit (Bio-Rad, Veenendaal, the Netherlands), using bovine serum albumine (BSA) as external standard, and protein extracts were diluted to 1 mg/ml. To assess the ex vivo repair capacity, each protein extract was finally added onto DMSO-exposed as well as BPDE-exposed gel-embedded nucleoids and incubated during 10 min at 37°C. Subsequently, the slides were processed according to the conventional comet assay as described previously (34)
. Comets were visualized using a Zeiss Axioskop fluorescence microscope and quantified as tail moment. Samples were tested in two independent incubations within each single experiment. On every slide, 50 cells were analyzed randomly using the Comet assay III software program (Perspective Instruments, Haverhill, UK). The increased DNA strand breakage (tail moment) in the BPDE-modified nucleoids vs. the DMSO-treated nucleoids is indicative for the NER capacity of the cell extracts. The final repair capacity was calculated according to Langie et al. (31)
and is expressed as percentage of control.
This sensitive method has the advantage that it can be performed without the use of radioisotopically labeled probes. In addition, instead of using circular plasmids or probes containing a specific DNA lesion, this assay applies whole cell-derived nucleoids that are treated with BPDE, inducing BPDE-DNA adducts that are targets for the repair proteins. These nucleoids retain the normal structure of the mammalian nucleus and are, thus, a more physiological substrate for studying the efficiency of cell lysates to repair eukaryotic DNA. Our assay was validated by showing that lysates obtained from Xeroderma Pigmentosum complementation group A and C deficient fibroblasts (Xpa and Xpc, respectively) were unable to recognize and excise the BPDE-DNA adducts in the nucleoids, whereas fusion of both lysates fully restored the NER capacity (31)
.
32P-postlabeling of BPDE-DNA adducts
As an additional approach to reveal the effects of HOCl on NER capacity in target cells, we studied the influence of HOCl on the kinetics of BPDE adduct removal in proliferating A549 cells. Therefore, A549 cells were exposed to 0.1 µM BPDE during 30 min on ice, followed by treatment with HOCl (100 µM, in HBSS) during 15 min. After treatment with HOCl, the medium was replaced by DMEM (37°C) to allow recovery of the cells. Cells were harvested by trypsination at different time points postexposure (0–4 h) to assess the kinetics of DNA adduct removal using 32P-postlabeling. Standard high salt extraction was used to obtain genomic DNA. 32P-postlabeling was carried out using the nuclease P1 enrichment technique as described by Reddy and Randerath (35)
with some modifications (36)
. In all experiments, three BPDE-DNA standards with known adduct levels (1 adduct/106, 1 adduct/107, and 1 adduct/108 normal nucleotides) were analyzed in parallel for quantification purposes. Quantification was performed using Phosphor-Imaging technology (Fujifilm FLA-3000). Part of the digest was used to determine the final amount of DNA in the assay by HPLC-UV, and the BPDE-DNA adduct levels were corrected accordingly.
ATP measurement
To assess the effects of HOCl on ATP levels in A549 cells, cells were seeded in 24-well plates. At confluency, cells were washed twice with HBSS, followed by the addition of 500 µl of HOCl containing HBSS. After 15 min, cells were washed twice with cold HBSS to remove any residual HOCl. Cells were lysed by adding 1x reporter lysis buffer (125 µl; Promega, Leiden, The Netherlands) to the wells and incubation on ice for 10 min. Cell lysates were centrifuged (13,000 g, 1 min), and supernatants were placed on ice for immediate analysis. Protein concentrations were determined by the Bio-Rad DC protein assay kit. ATP levels in the cells were assessed with a Molecular Probes kit (Molecular Probes Invitrogen, Leiden, The Netherlands) using luciferase and luciferin according to the instructions of the manufacturer. Bioluminescence was measured during 20 s by using a Lumat LB 9507 luminometer (Berthold Technologies, Vilvoorde, Belgium). Concentrations of ATP were determined by comparing the values obtained with a freshly prepared standard curve of ATP and were corrected for protein content.
Measurement of protein carbonyls
Measurement of protein carbonyls was used as an indicator of HOCl-induced oxidation of cellular proteins. Therefore, cells were exposed to HOCl and proteins were extracted by the method used for the repair assay. Briefly, the cell pellets (5x106 cells) were resuspended in 50 µl extraction Buffer A (45 mM HEPES, 0.4 M KCl, 1 mM EDTA, and 10% glycerol, adjusted to pH 7.8 using KOH) (31)
. Since DTT is able to react quickly with 
,ß-unsaturated carbonyl compounds, it was omitted from the original extraction buffer. Before analysis of protein carbonyl levels by ELISA, total protein concentration was determined, using the BIO-RAD DC protein assay kit. To load equal amounts of 20 µg protein in the ELISA procedure, the protein samples were concentrated by mixing a volume containing 20 µg proteins with 0.8 volume of 28% TCA, centrifugation at 10,000 g, and discarding the supernatant. Protein carbonyl concentrations were finally determined by a Zentech PC ELISA test (Zentech Alexis, Lausen, Switzerland), which involves a derivatization reaction of the isolated proteins with dinitrophenylhydrazine and subsequent detection of the derivatized proteins with an antidinitrophenylhydrazine antibody, according to the manufacturer’s instructions. Samples were calibrated against a standard curve constructed using reduced and HOCl-oxidized albumin.
Quantitative real-time polymerase chain reaction (PCR)
Quantitative real-time PCR was applied to assess the effects of HOCl on the expression of relevant NER genes. A549 cells were seeded in 28 cm2 dishes and treated with 100 µM HOCl. After 15 min, HOCl was removed and cells were washed with HBSS, followed by a further incubation of the cells in DMEM for 0 to 24 h. After incubation, A549 cells were lysed in 1 ml Trizol (Life Technologies). Total RNA was isolated and purified using the RNeasy Mini Kit (Qiagen Westburg, Leusden, The Netherlands) in combination with a DNase treatment (Qiagen), according to the manufacturer’s instructions. The quantity and quality of each RNA sample were measured spectrophotometrically. First strand cDNA was generated according to the iScript cDNA synthesis kit protocol (Bio-Rad) using 1 µg total RNA. The following NER genes were tested: XPA, XPC, ERCC-1, XPF (=ERCC-4) and XPG (=ERCC-5), using primers purchased from Operon (Leiden, The Netherlands; see Table 1
for primer sequences). Real time PCR was performed with a MyiQ Single Color real time PCR detection system (Bio-Rad) using SYBR Green Supermix (Bio-Rad), 5 µl diluted cDNA and 0.3 µM primers in a total volume of 25 µl. PCR was conducted in the following manner: denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 45 s. After PCR, a melt curve (60–95°C) was produced for product identification and purity. PCR efficiency of all primer sets, as assessed by the use of cDNA dilution curves, was 
100%. Data were analyzed using the MyiQ Software system (BIO-RAD) and were expressed as relative gene expression (fold increase), using the 2–Ct method and employing both ß-actin and GAPDH as housekeeping genes (37)
.
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Statistical analysis
Data are mean ± SE from three independent experiments, unless stated otherwise. Statistical analysis was performed using SPSS version 11.5 for Windows. ANOVA with post hoc testing (Dunnett’s correction for multiple, two-sided) was applied to test differences between treatments and control. Differences were considered to be statistically significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). In the absence of PMA, neutrophils were unable to inhibit NER in the coincubated A549 cells. The A549 cells coincubated with equal amounts of PMA-activated PMN (1:1 ratio) showed a significant reduction in NER capacity up to 60% (P<0.05). The supernatant of the A549-neutrophil cocultures was analyzed for MPO activity. As shown in Fig. 1B
, MPO activity was only detected in the incubations containing PMA-activated neutrophils. No MPO activity could be detected in the coculture supernatants in the absence of PMA.
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To evaluate the role of MPO in NER inhibition, coculture experiments were performed using the specific MPO-inhibitor 4-ABAH. We applied 4-ABAH because it is known to be one of the most potent inhibitors of MPO, functioning as a suicide substrate that promotes irreversible inactivation of the enzyme and thereby fully inactivating the production of MPO-derived HOCl. Significantly, 4-ABAH does not affect neutrophil-induced NADPH-oxidase and does not inhibit catalase and gluthatione peroxidase, unlike many other MPO inhibitors (29)
. MPO activity was completely inhibited at a concentration of 100 µM 4-ABAH (Fig. 1D
). This MPO inhibition by 4-ABAH resulted in a complete abrogation of the NER inhibition induced by activated PMN (Fig. 1C
). In fact, Fig. 1
indicates a clear inverse relation between neutrophilic MPO activity and NER capacity. Incubation of A549 cells with 4-ABAH alone did not affect NER capacity. In addition, all effects observed in the cocultures were not related to toxicity, because viability of the A549 cells was always >95%.
NER inhibition by PMN is mediated by the MPO product HOCl
The data obtained from the coculture experiments suggest a crucial role of MPO in the NER-inhibitory action of neutrophils. As MPO catalyzes the formation of HOCl, it is likely that the inhibition of NER in the cocultured epithelial cells is mediated by MPO-catalyzed HOCl formation. To investigate the role of HOCl as a mediator of the PMN-induced NER inhibition, A549 cells were exposed to increasing concentrations of HOCl (0–200 µM). These doses can easily be produced in vivo at sites of inflammation and also reflect the amount of HOCl produced in the in vitro cocultures (1:1 ratio with activated neutrophils yields 150 µM HOCl).
Initially, we determined the cytotoxicity of HOCl in the A549 cells and found that the viability of the epithelial cells was >90% after treatment with up to 200 µM of HOCl during 15 min. At higher doses, viability of the A549 cells declined to 40% at 1000 µM HOCl (data not shown). We observed an acute dose-dependent reduction of NER capacity (within 15 min) in the lysates obtained from A549 cells treated with nontoxic, physiological doses of HOCl (0–200 µM), with an inhibition up to 60–80% in cells treated with 100–200 µM HOCl (P<0.05; Fig. 2
A). The IC 50 value for HOCl was 52.9 µM, calculated using the least square fit method by fitting data points from Fig. 2A
to the inhibition formula using the equation Ix = 100 e–k*[x] to investigate the persistency of this inhibiting effect, cells were incubated for 0–24 h in DMEM after an initial acute treatment (15 min) with 100 µM HOCl. The data are shown in Fig. 2B
, indicating that the NER inhibitory effect of acute exposures to HOCl persists for up to 24 h (P<0.05). This effect was also observed in the absence of cytotoxicity.
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HOCl delays the removal of BPDE-DNA adducts in proliferating A549 cells
The results shown in Fig. 2A, B
reflect the NER capacity (i.e., recognition and excision of bulky DNA adducts) of lysates isolated from epithelial cells, after exposure to HOCl. To evaluate the implication of this effect on the kinetics of bulky DNA adduct removal in proliferating epithelial cells, cultured A549 cells were exposed to 0.1 µM BPDE during 30 min followed by HOCl exposure (100 µM during 15 min). Cells were harvested at 0–4 h postexposure, and BPDE-DNA adduct levels were analyzed by 32P-postlabeling. HOCl treatment significantly delays the removal of BPDE adducts from the DNA of BPDE-treated A549 cells, further validating the NER inhibitory effect of HOCl (see Fig. 3
).
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HOCl exposure does not affect intracellular ATP levels in A549 cells
Exposure to HOCl is known to deplete acutely cellular ATP levels in a variety of target cells (38)
. ATP is a crucial cofactor for the NER process. Therefore, we tested the possibility that the effects of HOCl on NER inhibition could be explained by a loss of epithelial cell ATP. However, after a 15 min treatment with concentrations of HOCl for up to 200 µM, no loss of ATP was observed in the A549 cells (Fig. 4
).
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Role of direct NER protein oxidation by HOCl
HOCl is a potent oxidizing molecule that specifically attacks proteins (11)
. Direct oxidative modification of NER proteins could, therefore, explain the acute inhibitory effects of HOCl. To study this possibility, cell extracts were isolated from unexposed A549 cells as described above and then treated in vitro with increasing concentrations of HOCl during 15 min. These modified extracts were subsequently evaluated for their NER capacity (Fig. 5
A). We again observed a dose-dependent NER inhibition in the HOCl exposed extracts, but this inhibitory effect was less pronounced compared to the extracts obtained from HOCl-treated A549 cells (cf., Fig. 2A
). To further reveal direct HOCl-protein interactions as a possible explanation for the observed effects, we analyzed the formation of protein carbonyls in the A549 cells, as this is a sensitive indicator of HOCl-induced protein oxidation in target cells (39)
, which is associated with disturbed enzyme function (40)
. However, HOCl treatment did not significantly increase the protein carbonyl content in A549 cells exposed to NER-inhibiting doses of HOCl (Fig. 5B
).
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Effect of HOCl on expression of NER genes
Figure 2B
indicates that the NER inhibition persisted for up to 24 h, suggesting a disturbance in gene expression and/or de novo synthesis of repair enzymes involved in the recognition and removal of BPDE-DNA lesions. To test this possibility, direct effects of HOCl on the expression of NER genes were studied. The phenotypical repair assay described above reflects the capacity of lysates to perform the recognition and incision phase of the NER process. Therefore, we here focused on genes that are crucially involved in these specific phases of NER, namely XPA and XPC for damage recognition, ERCC-1 and XPF as 5'endonucleases, and XPG as 3'endonuclease. A549 cells were treated during 15 min with 100 µM HOCl followed by a recovery period for up to 24 h in complete medium. As shown in Table 2
, HOCl was found to cause a time-dependent inhibition of XPC mRNA expression, with a maximal effect at 8 h postexposure (50%). No further significant effects of HOCl were found for the expression of ERCC-1, XPA, XPF, and XPG.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In this study, we demonstrated that activated neutrophils are potent inhibitors of the nucleotide excision repair capacity of lung target cells, most likely mediated by the formation of MPO-catalyzed HOCl. Considering the widespread exposure to inhaled DNA-adduct forming carcinogens such as PAHs, as well as the simultaneous presence of neutrophils in the inflamed lung, we propose that this neutrophil-induced reduction of NER may be a significant contributory factor in the development of inflammation-related pulmonary mutagenesis and carcinogenesis.
MPO consumes up to 40–70% of neutrophil-derived H2O2 to generate HOCl (7)
. HOCl is a relatively stable and membrane diffusible molecule that can pass through subcellular compartments and, presumably, can reach the cell nucleus (10)
. As such, it has the potential to interact with many different cellular proteins, including DNA repair proteins. We indeed demonstrated that neutrophil-induced inhibition of NER is mediated by the MPO product HOCl by using coincubations in the presence of the MPO inhibitor 4-ABAH as well as by treating A549 cells with the MPO-derived product HOCl. This effect was persistent for up to 24 h and observed at physiologically relevant concentrations of HOCl, as HOCl levels in the vicinity of activated neutrophils are at or above 100 µM (8)
. In general, these data are in line with other studies showing that NER inhibition can be caused by oxidants such as 4-HNE (25)
and nitric oxide (27)
. Moreover, our data are in accordance with studies by Pero et al. (15)
, who described an HOCl-induced inhibition of unscheduled DNA synthesis (UDS), which can be considered as an indicator of nucleotide excision repair. However, it needs to be stressed that the latter observations were in contrast with other studies that reported an induction of UDS by activated neutrophils, with no apparent role for HOCl (41)
.
The present study showed an HOCl-induced inhibition of the NER capacity after 15 min during treatment. To find an explanation for this profound and acute effect, we initially focused on the possible role of HOCl in depleting ATP in lung epithelial cells (38)
. In the initial steps of the NER pathway, ATP is a crucial cofactor, facilitating the helicase activity of XPB and XPD (24)
. Nevertheless, we did not find any loss of cellular ATP in A549 cells treated with physiological NER-inhibiting doses of HOCl (0–200 µM). This is in contrast with other studies showing a significant depletion of ATP at similar HOCl doses in various other cell types (42
, 43)
and is most likely explained by the relatively high level of antioxidant protection of the A549 cells compared with other (lung) epithelial cell lines (unpublished own observations).
Proteins are the major molecular target for HOCl. Of primary importance among the HOCl-mediated protein modifications are tyrosine chlorination, formation of chloramines and carbonyls (39)
, and in some cases cross linking (12)
. This study focused on carbonyls as a biomarker of oxidative HOCl attacks on proteins. In the inflamed lung, for instance, a high correlation between protein carbonyl concentration and myeloperoxidase activity was observed (44)
. Carbonyl groups represent an irreversible protein modification, often leading to the inactivation of these proteins (13
, 14
, 40)
. We, therefore, hypothesized that a loss of function of NER enzymes, originating from direct HOCl-protein interactions, is a possible mechanism explaining the observed acute HOCl-induced inhibition of NER. However, less effective NER inhibition was observed in direct HOCl-treated cell lysates than in lysates obtained from HOCl-exposed whole A549 cells. In addition, we were unable to show protein carbonyls in the A549 cells treated with NER-inhibiting doses of HOCl. Together, this would suggest that direct oxidative inactivation of repair proteins may only partly explain the acute inhibitory effects of HOCl and indicates that other intracellular factors, for instance the formation of protein-DNA cross-links, may be involved in mediating HOCl-induced inhibition of NER in the target cells (45)
.
We demonstrated that HOCl-induced repression of NER capacity persisted for up to 24 h. As a possible explanation for this long-term effect, we addressed the effect of HOCl on the transcription of relevant NER genes. Our repair assay is based on the capacity of cell extracts to perform the initial steps of NER, i.e., damage recognition and incision of DNA containing BPDE adducts. Thus, we only focused on the expression of genes coding for crucial enzymes involved in these specific phases of the NER process, namely ERCC-1, XPA, XPC, XPF, and XPG. Our data showed a significant suppression of XPC gene expression on incubation with HOCl. XPC is crucially involved in damage recognition (24)
. Therefore, although additional studies are needed to assess the significance of this finding, our data indicate that HOCl exposure may affect the ability of cells to recognize DNA lesion-containing DNA.
NER is the most important repair pathway to remove large helix distorting DNA adducts that are produced following inhalation of chemical carcinogens, such as PAHs (23
, 24)
. The formation of these promutagenic PAH-DNA adducts has been implicated as a causal process in lung cancer development in smokers (46)
. As studies with NER-deficient mice showed that effective DNA repair is crucial to prevent the carcinogenic effects of PAH-induced DNA damage (47)
, it can be speculated that processes leading to the suppression of the relevant DNA repair pathways may have a detrimental effect on the cancer susceptibility of individuals exposed to such chemicals. Epidemiological studies have indeed shown that polymorphisms in NER genes, leading to a (hypothetically) decreased NER capacity, are associated with increased risk of lung carcinogenesis (48
, 49)
. Our present results indicate that neutrophilic inflammation, by causing inhibition of NER, might be another important factor determining susceptibility to cancer. For instance, our findings could provide an additional explanation for the association between MPO polymorphisms and risk of pulmonary DNA adduct formation and carcinogenesis in PAH exposed subjects (17
, 21)
. Evidently, although the present study focused on the lung, it needs to be emphasized that our findings may also be of importance for other organs where bulky DNA adduct forming agents and neutrophils are simultaneously present.
In conclusion, in this study we demonstrated that neutrophils are potent inhibitors of NER in human pulmonary epithelial cells. Previous studies have already shown that neutrophils, by releasing MPO, are involved in the carcinogenic process by promoting the activation of precarcinogens such as B[a]P into DNA damaging metabolites. In the present study, we extended these observations by showing that MPO, via generation of HOCl, inhibits the repair of such promutagenic DNA adducts. We propose that these two detrimental effects of activated PMNs at sites of inflammation may contribute synergistically to human inflammation-related carcinogenesis. Therefore, our observations may provide a further explanation for the observed association between neutrophilic inflammation, MPO, and lung cancer risk.
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ACKNOWLEDGMENTS |
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Received for publication January 22, 2007. Accepted for publication March 1, 2007.
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