The epidemiological association between cancer and exposure to ambient air pollution particles (particles with a 50% cut-off aerodynamic diameter of 10 μm (PM10)) has been related to the ability of PM10 and its constituent nanoparticles (NPs) to cause reactive oxidative species (ROS)-driven DNA damage. However, there are no data on the molecular response to these genotoxic effects.
In order to assess whether PM10, NP and ROS-driven DNA damage induce carcinogenesis pathways, A549 cells were treated with tert-butyl-hyperperoxide (Tbh), urban dust (UD), carbon black (CB), nanoparticulate CB (NPCB), benzo(a)pyrene (BaP) and NPCB coated with BaP for ≤24 h. Single- and double-strand breakage of DNA was determined by comet assay; cell cycle status was analysed using flow cytometry. Nuclear extracts or acid-extracted histones were used for Western blot analysis of p-ser15-p53 (p53 phosphorylated at ser15), p53 binding protein (53BP) 1, phospho-histone H2A.X (p-H2A.X) and phospho-BRCA1 (p-BRCA1).
UD caused both single- and double-strand DNA breaks, while other tested NPs caused only single-strand DNA breaks. NPs significantly altered cell cycle kinetics. Tbh enhanced p-H2A.X after 1 and 6 h (2.1- and 2.2-fold, respectively). NP increased 53BP1 expression at 1 h (2.4–8.7-fold) and p-BRCA1 at 1–6 h. N-acetylcysteine blocked NP-driven p-ser15-p53 response.
In conclusion, nanoparticles and reactive oxidative species induce DNA damage, activating p53 and proteins related to DNA repair, mimicking irradiation-related carcinogenesis pathways.
- DNA damage
- H2A.X histone
- particles with a 50% cut-off aerodynamic diameter of 10 μm
- reactive oxidative species
Increased exposures to particles with a 50% cut-off aerodynamic diameter of 10 μm (PM10) is associated with an increased risk of cardiovascular and respiratory deaths and hospital admissions, as well as lung cancer 1, 2. Direct evidence of DNA damage caused by PM10 has also been confirmed 3. Attention has focused on the PM10 in cities because that is where most deaths occur, where pollution is routinely monitored and hence the associations are best seen. Typical urban PM10 is comprised of ≤50% by mass of combustion-derived nanoparticles (CDNPs; particles <100 nm), which are carbon-centred particles, typically from automobile engine exhausts, with associated compounds including transition metals, ammonium salts of nitrogen, sulphur and chlorine plus geological dust and organic matter 4. Many toxicological studies over the last decade have confirmed that CDNPs readily generate oxidative stress through reactive oxidative species (ROS) and inflammation and nanoparticles (NPs) are seen as the most harmful components of the PM10 mix 5–7. Several investigators have shown that oxidative stress may play a major role in particle-induced DNA damage, which can be prevented by antioxidants and scavengers of ROS 8. However, detailed molecular mechanisms involved in the hallmark cellular responses to genotoxic effects are currently not known, in contrast with other DNA damaging agents, such as ionising radiation (IR).
Oxidative stress is now considered to have an important role in regulating cellular signalling, leading to inflammatory, proliferative and genotoxic effects 9–12. In the case of the latter, a large variety of DNA lesions, including single- and double-strand breaks, and base and sugar damage 13, 14 can all be caused by ROS. Depending on type and severity, acute DNA damage is known to trigger cell cycle arrest, enabling increased DNA repair time, or resulting in cell death 15. Insufficient or erroneous DNA repair may, in the long-term, result in the accumulation of mutations that are well known to contribute to oncogenesis 16. Double-strand DNA breaks (DSBs) induced by X-rays, chemicals or during replication of single-strand breaks (SSBs), and presumably during repair of interstrand crosslinks, are the most harmful due to their dramatic impact on the recombination machinery 17–20.
The most widely accepted and extensively used model of DNA damage-induced cell signalling and DNA repair is IR. In this model, signal transduction pathways and participating proteins can be formally divided into sensors, transducers and effectors. Poly (adenosine disphosphate (ADP)-ribose) polymerase, DNA-dependent protein kinase, BRCA1, topoisomerase II binding protein 1, p53 binding protein (53BP) 1, mediator of DNA damage check-point protein (MDC) 1 and H2A.X histone variant are the most likely candidates for sensors. Transducers, such as ataxia/teleangiectasia mutated (ATM) and ataxia/teleangiectasia receptor (ATR) kinases are located immediately downstream from the sensors, playing an important role in the DNA damage check-point by controlling the initial phosphorylation of several key proteins of the overall response, such as p53, Mdm2, BRCA1, Chk2, MDC1, NBS1 and H2A.X, since distinctions between groups of proteins are not absolutely clear and they may be found at different levels downstream of the pathway 21, 22. The present authors have drawn on these mechanisms to derive the hypothesis that exposure to PM10, and specifically the NP component, may result in activation of similar DNA damage response pathways, providing biologically plausible mechanisms for the epidemiological association between PM10 exposure and cancer incidence 1, 2. The present study has examined these end-points in pulmonary epithelial cells treated with urban dust (UD) and various NPs, to determine the expression of DNA damage-inducible genes. Since the IR-induced DNA damage response is highly complex, the key aim of the present study was to first find some clues for the potential of particles to induce DNA response signals using selected parameters.
MATERIALS AND METHODS
All chemicals and reagents used in the present study were obtained from Sigma Chemical (Poole, UK), unless otherwise stated. Cell culture media and reagents were obtained from GIBCO-BRL (Paisley, UK). Tert-butyl-hyperperoxide (Tbh) was prepared in a stock solution of 2 mM in PBS and treatments were carried out at a concentration of 50–75 µM. The thiol antioxidant N-acetyl-l-cysteine (NAC) was stored at -20°C in PBS at a concentration of 0.5 M and used at a final concentration of 5 mM. NAC was added as a pre-treatment to cells 6 h before the addition of NPs.
A549 type II alveolar-like cells derived from human adenocarcinoma expressing wildtype (WT) p53 (ECACC, Porton Down, UK) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (weight (w)/volume (v)) heat-inactivated foetal calf serum, l-glutamine (2 mM) and penicillin-streptomycin solution in 5% CO2 at 37°C. Cells were quiesced overnight in serum-free media and subsequently treated under serum-free conditions. All experiments were carried out with A549 cells between passages 6 and 10.
The model particles and chemicals that were used in the present study are listed in table 1⇓. The rationale for their selection is summarised in the table and was based on the physicochemical properties and/or constituents of particles that are currently considered to be the main driving forces for the induction of oxidative stress, i.e. the particle surface, transition metals or organic constituents such as polycyclic aromatic hydrocarbons 8. Coarse carbon black (CB: Huber 990; H. Haeffner and Co Ltd, Chepstow, UK) had a primary diameter of 260 nm and nanoparticulate CB (NPCB: Printex 90; Degussa, Frankfurt, Germany) had a primary diameter of 14 nm. NPCB coated with benzo(a)pyrene (BaP-NPCB) was prepared as follows: NPCB particles were cleaned by Soxhlet extraction with toluene for 8 h. A total of 2 g of the extracted particles were suspended in 120 mL N-hexane containing 60 mg BaP for 10 min and then filtered under vacuum. Following filtration, the BaP-coated particles were washed once with 10 mL pentane and filtered again under a vacuum, until dry. The resulting BaP content of the coated particles was 26 mg BaP·g−1 Printex 90, as measured by high performance liquid chromatography using a Grom column and fluorescence detection. Standard Reference Material® 1649a, urban dust (UD) was purchased from the National Institute of Standards and Technology (Gaithersburg, US; 23–25). For experiments, particles were suspended in foetal bovine serum-free DMEM at concentration of 100 µg·mL−1 (25 μg·cm−2) and sonicated (Grant Ultrasonic bath XB6; Grant Instruments, Cambridge, England) for 20 min prior to use.
For cell treatments, cells were plated at a density of 0.15×106 cells·well−1 in petri dishes and grown overnight to 80% confluency. The medium was then replaced with serum-free medium for a further 24 h. Treatments were also added for specific times in serum-free medium. For all NP treatments, cells were exposed to 100 µg·mL−1 (25 μg·cm−2) particles in culture medium unless otherwise stated. Cells were treated with 50–75 µM Tbh, 10 µM BaP, 100 µg·mL−1 UD, 100 µg·mL−1 CB, 100 µg·mL−1 NPCB, 100 µg·mL−1 BaP-NPCB and 5 mM NAC for 0.5–24 h. The BaP concentration used was equivalent to that of the NP-bound BaP (i.e. BaP-NPCB; 2.6 µg·mL−1).
Cytotoxicity was assessed by lactate dehydrogenase (LDH) release. Cells were grown to confluency in 96-well flat-bottomed culture plates in media containing 2% FBS (v/v) and exposed to a range of NP concentrations (1–100 µg·mL−1) for the various time intervals. LDH release was measured according to manufacturer’s instructions (Roche Molecular Biochemicals, Mannheim, Germany) using pyurvic acid as a substrate.
DNA strand breakage analysis
DNA strand breakage analysis was performed using the alkaline and neutral modifications of the comet assay, which detect DNA SSB plus alkali-labile sites and DSB, respectively 26, 27. Cells were seeded into 24-well culture dishes (1.2×105 cells·dish−1) and grown for a further 48 h, after which the medium was replaced by serum-free medium for 24 h prior to NP treatment. Following treatment of the cells with NP, the monolayers were rinsed twice with PBS, detached with trypsin-EDTA and immediately suspended in complete culture medium. Cells were then processed for the alkaline comet assay, as described in detail previously 27. For the neutral assay the same method was used with a lysis buffer set at pH 7.5. DNA damage was analysed on an Olympus BX60 fluorescence microscope at 200× magnification using software-assisted determination of tail moments (Comet assay II; Perceptive Instruments Ltd, Suffolk, UK).
Determination of cell viability and proliferation was estimated by flow cytometric quantification of the cellular DNA, using propidium iodide (PI) staining in permeabilised cells 8. Briefly, cellular DNA degradation and cell cycle analysis were performed on cells stained for 30 min with PI (50 mg·mL−1) in tris buffer (100 mM, pH 7.5) containing potassium cyanide (0.1% (w/v)), Nonidet-P40 (0.01% (w/v)), RNase III-A (40 mg·mL−1, 4 KU·mL−1) and NaN3 (0.1% (w/v)). The analysis was performed on an aligned Coulter Epics Profile flow cytometer (Coulter, Hialeah, FL, USA) equipped with an argon laser operating at 488 nm. PI fluorescence was measured in ≥5,000 cells with appropriate bandpass filters. DNA histograms were further analysed by DNA quantification software (MultiCycle; Phoenix Flow Systems Inc., San Diego, CA, USA). Cells were quantified by their relative distribution in the damaged-hypodiploid phase (“early” G0/G1 zone of the DNA fluorescence histograms), diploid phase (G0/G1 zone, pre-DNA synthesis/resting), S-phase (DNA synthesis) and G2/M phase (post-DNA synthesis/mitosis).
Cells specimen processing and nuclei extraction
Samples for protein analysis were prepared at various times from confluent A549 cell. Cells were washed three times in ice-cold PBS, scraped in PBS, pelleted for 15 s at 14,000×g and resuspended in 400 µL of lysis buffer (10 mM hydroxyethyl piperazine ethane sulphonic acid (HEPES), 50 mM KCl, 2 mM MgCl2 , 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.4 mM phenylmethylsulphonyl fluoride (PMSF), 0.2 mM NaF, 0.2 mM sodium orthovanadate, 1 μg·ml−1 leupeptin; pH 7.8) and incubated on ice for 15 min. Following the addition of 10% Nonidet-P40, cells were centrifuged at 14,000×g for 30 s and the supernatant containing cytoplasmic fraction was aspirated and retained. Pelleted nuclei were resuspended in 50 µL of extracting buffer (50 mM HEPES, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT 10% (v/v) sterile glycerol, 0.2 mM NaF, 0.2 mM sodium orthovanadate, 0.66 mM PMSF; pH 7.8), mixed for 20 min on a rotating platform, centrifuged for 5 min at 14,000×g. The supernatant containing nuclear proteins was decanted and saved for further Western blot analysis. A 5 µL aliquot of each sample was stored at -80°C for protein determination by Bicinchoninic Acid Kit for Protein Determination (Sigma-Aldrich, Irvine, Scotland).
Histone acid extraction
The nuclear pellet was further resuspended in 150 µL of distilled H2O, and 2.6 µL of 11.6 M HCl (final concentration 0.2 M) and 1.5 µL of 18 M H2SO4 (final concentration 0.36 M) was added. Eppendorf tubes were placed in a falcon tube and incubated overnight at 4°C on a rotating shaker. Samples were then centrifuged at 13,000×g at 4°C for 10 min, 1.1 mL of ice-cold acetone was added to the protein supernatant containing acid soluble protein and samples were kept overnight in the freezer (-80°C) to precipitate. The samples were then centrifuged at 13,000×g at 4°C for 10 min, the supernatant was removed and 1 mL of ice-cold acetone was added to the pellets, mixed and frozen again for 1 h. After centrifugation at 13,000×g at 4°C for 10 min, the supernatant was decanted, the pellet air-dried, resuspended in 50 µL of dH2O and finally, the protein was measured using Bicinchoninic Acid Kit for Protein Determination (Sigma-Aldrich).
BioRad stock reagent was diluted 1:5 with distilled water and 200 μL of this diluted reagent was added to a 96-well plate, using triplicate groups per test and sample. A 5 μL aliquot of each sample was then added to the appropriate well, mixed and incubated at room temperature for 15 min before being read on a plate reader at 450 nm. Standards of bovine serum albumin solutions were used, ranging 0.025–2 mg·mL−1.
Analysis of phosphorylation status of p53, 53BP1, H2A.X and BRCA1 expression was performed using sequential immunoprecipitations followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and/or Western blotting. Equalised amounts of protein lysate (of nuclear extract or histone extract) were boiled at 90°C for 5 min and then analysed by 5–20% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and incubated in blocking buffer (3–5% nonfat dry milk in PBS or tris-buffered saline) for 2 h before incubation overnight with primary antibody at 4°C. Subsequently, the blots were probed with peroxidise-conjugated secondary antibody for 0.5–2 h and the proteins were detected by ECL plus (Pierce, Rockford, IL, USA).
Data were expressed as mean±sem and were analysed using ANOVA with the Tukey multiple comparison test.
DNA damage in A549 cells
DNA strand breakage was determined by comet assay (fig. 1⇓). NP exposure caused a significant increase in DNA single-strand breaks and alkali-labile sites in A549 cells after 3 h exposure (fig. 1a⇓). Double-strand breaks, detected by neutral comet assay, occurred only in cells treated with UD (fig. 1b⇓). The time-course of UD exposure showed a peak in DNA breakage at 6 h (300% of control; fig. 1c⇓). In comparison, 10-Gy radiation of the cells, which was used as a positive control, caused an immediate damage reaching 600% of control. Cells remained viable by trypan blue dye exclusion and no differences between exposure groups were observed (data not shown).
Cell cycle status
Cell cycle status was assessed at 3, 6 and 24 h by flow cytometry (table 2⇓, fig. 2⇓). All experiments were expressed as mean values of cell cycle distribution and compared with corresponding controls. Ambient particles and oxidative stress altered cell cycle kinetics. Specifically, UD caused significant G0/G1 arrest of exposed cells, whereas NPCB and BaP exposure resulted in significant G2 block of A549 cells. Tbh-exposed cells accumulated significantly more cells at early G0/G1 phase.
p53 phosphorylation at ser15
Immunoprecipitation followed by immunoblot was used to detect p53 protein phosphorylated at ser15 in cells exposed to particles and BaP for different times ranging 0.5–24 h (figs 3a⇓ and 3c⇓). The results show that p53 phosphorylation on ser15 occurred in response to all tested NPs but not with CB or BaP-NPCB. The highest response was achieved after 1-h exposure to Tbh (9.6-fold increase), NPCB (7.3-fold increase), UD (5.2-fold increase) and BaP (3.3-fold increase; fig. 3c⇓). Pre-treatment of cells with NAC for 6 h blocked ser15 p53 phosphorylation in treated cells (fig. 3a⇓), indicating an oxidative stress-driven mechanism.
53BP1 expression in A549 cells increased significantly after 1 h exposure (figs 3b⇑ and 3d⇑): Tbh, 2.4-fold; UD, 7.1-fold; CB, 7.8-fold; NPCB, 6.8-fold; BaP, 8.7-fold; and BaP-NPCB, 7.0-fold, respectively. Pre-treatment of cells with NAC did not block 53BP1 expression (data not shown).
H2A.X histone phosphorylated on serine 139-γH2A.X
Expression of γH2A.X was detected in response to 75 µM Tbh after 1 and 6 h (2.1- and 2.3-fold, respectively) and BaP-NPCB after 1 h (1.2-fold; figs 4a⇓ and 4b⇓). Further A549 cells were exposed to 50 µM Tbh and PM10 for 24 h. Expression of γH2A.X was detected in response to UD and NPCB (1.46- and 1.27-fold, respectively; figs 4c⇓ and 4d⇓). Pre-treatment of cells with 2 mM N-acetylcysteine 0% medium 6 h before the addition of particles did not show conclusive data (data not shown).
BRCA1 phosphorylation and 53BP1 expression
BRCA1 phosphorylation in A549 cells, increased after 1 h exposure of Tbh, UD and CB at 1 h and after 6 h exposure of all tested agents, including CB (fig. 5⇓). Pre-treatment of cells with NAC (6 h) blocked BRCA1 phosphorylation in treated cells (figs 5a⇓ and 5c⇓). However, the NAC effect delayed BRCA1 phosphorylation expression; while in the nonNAC-treated cells the protein expression waned at 6 and 24 h, the NAC-treated cells began to show protein at these later time points (figs 5b⇓ and 5d⇓). A similar pattern of protein expression was seen for ser15-p53 and p53 throughout the time-course (data not shown).
Summary of DNA damage and responses in A549 cells to particles in relation to relevant physicochemical properties and constituents are shown in table 3⇓.
The genetic instability driving tumourigenesis is fuelled by DNA damage and by errors made by the DNA machinery 28. DNA damage has been well documented following exposure to IR 29–32 and chemotherapy 33–35. It is widely accepted that such exposures elicit DNA damage response, including ATM/ATR-dependent phosphorylation of proteins and molecules taking part in damage sensing, regulating cell cycle and maintaining DNA integrity 30, 35, 36. Recent studies show that PM10 causes DNA damage through oxidative stress 37, 38 and CDNP are most likely to be the harmful components 5. Oxidative stress, arising from the surfaces of the inhaled particles and reactions involving their associated metals and organic constituents, elicits pulmonary inflammation that is considered to drive local as well as systemic (e.g. cardiovascular) effects 39, 40. Moreover, NPs may be more potent at causing oxidative stress-driven DNA damage due to their greater surface area. Despite such evidence, there are no existing data on how cells sense, and/or react to PM10 and/or NP-induced DNA damage. The present authors hypothesised that PM10, specifically its NP component, would have signalling pathways similar to IR. If this is the case, long-term exposure to particles may be a consequence of similar errors in DNA replication, resulting in carcinogenesis, and hence may provide an explanation for increased cancer incidence in polluted areas 1.
In the present study, all the NP test agents caused DNA damage after 3 h exposure. The most profound damage, DSBs, were observed only with UD. DSBs caused by UD treatment occurred after only 1 h and were found to persist, probably due to the continuous presence of the particles in the cell culture. The decline in DSBs observed at 24 h might reflect either repair or loss of cells that have undergone apoptosis following severe DNA damage. Metal-dependent oxidative stress might be a major mechanism for the strand breakage caused by UD, which, in contrast to the other dusts, is relatively rich in various metals 41. DSBs induced by metal exposure have been investigated previously. Ha et al. 17 found that Cr(VI) exposure of normal human fibroblasts lead to DSBs formation in the S-phase but not in G1 synchronised cells, indicating S-phase-dependency. Xie et al. 36 suggest that, in human bronchial cells, lead chromate clastogenesis is mediated by the extracellular dissolution of the particles and not their internalisation. These findings have important implications for the understanding of the physicochemical mechanism of particulate chromates.
In the present study, SSBs were observed with NP, Tbh and BaP, but not with coarse CB. The ability of Tbh or BaP to induce DSBs is a topic of ongoing debate. The present data are consistent with findings of DNA breaks in Balb-c cells exposed to PM10 by Alfaro-Moreno et al. 42 and Buschini et al. 43, who found that NP fraction of airborne particulate generally caused the most DNA damage in human lung fibroblasts. In the present study, exposure of A549 cells to UD resulted in transient arrest of cell cycle passage. After 6–24 hours of exposure, UD caused G0/G1cell arrest; Tbh exposure led to G0/G1cell arrest and S-phase accumulation; while NPCB and BaP caused transient G2/M block.
Although DNA damage by ambient air particles has been investigated by several researchers 36, data on the molecular responses to this damage are rather scarce in comparison with investigations using IR. Johnson et al. 44 demonstrated that exposure of A549 cells to asbestos fibres induced a dose-dependent increase in the G2 phase cell numbers. Aneuploidy was associated with an increase in the protein levels of genes such as p53, Cip1 and Gadd45, which are induced by DNA damage. Okayasu et al. 45 showed early DSBs formation by asbestos. They found that 24-h exposure of xrs-5 cells to asbestos fibres resulted in lower cell survival accompanied by a cell growth delay, as well as a higher DNA DSBs induction in this mutant cell line 45. Neonatal rats exposed to NP soot and iron particles show a significantly reduced cell proliferation rate in the proximal alveolar region, suggesting that exposure to airborne particles during early neonatal life may have significant direct effects on lung growth by altering cell division 46. The combustion by-product, BaP, is a prevalent airborne environmental mutagen and a constituent of cigarette smoke. In a study reported by Binková et al. 47, the cell cycle of diploid lung fibroblasts was altered after 12–24 h of exposure to 0.1 μM dibenzo(a,1)pyrene, resulting in an ∼24% increase in S-phase. Enhanced benzo(a)pyrene-diol-epoxide (BPDE)-DNA adducts in response to BPDE resulted in G2/M retardation or apoptosis 48. Consistent with the present results, subsequent elevation in the proportion of G2/M was reported by Zhu and Gooderham 49. Increased DNA ploidy induced resistance to confluence-initiated cell death and the morphological change was accompanied by substantial changes in growth pattern, indicating that selection of carcinogen-induced transformants, under prolonged confluence culture, may be the pivotal mechanism of neoplastic disease development.
In the current study, p53 phosphorylation was observed at ser15, which occurred at early time-points in response to nearly all the tested NPs, peaking at 1 h. NAC blocked p53 activation at early time points, suggesting the involvement of oxidative stress. Other investigators 50, 51 have shown involvement of p53 in the differential regulation of p21 mitogen-activated protein kinase and retinoblastoma in cellular response to oxidative stress and oxidative stress-induced apoptosis. Although the “genome guardian” role of p53 has mainly been studied with reference to apoptosis, recent findings 52 suggest that p53 is also activated in the early stages of DNA damage promoting signalling events, which leads to repair of damaged cells. Hammond et al. 53 reported that p53 ser15 and histone H2A.X were both phosphorylated in response to hypoxia in an ATR-dependent manner, and in response to reoxygenation-induced DNA damage in an ATM-dependent manner. Again, in these studies phosphorylation was inhibited by NAC, indicating a ROS-driven pathway. In the present study, phosphorylation of BRCA1, subsequently blocked by NAC pre-treatment, phosphorylation of H2A.X and expression of 53BP1 was observed within early time points (figs 3⇑–⇑5). Previously, the functional interaction between histone H2A.X, ATR-interacting protein, as well as the BRCT-motif-containing molecules 53BP1, MDC1, and BRCA1 were exclusively studied in IR and/or chemotherapy models 22. 53BP1 was phosphorylated in response to DNA damage and rapidly relocalised to presumptive sites of DNA damage along with the phosphorylated histone 2A variant, γ-H2A.X. Fernandez-Capetillo et al. 54 reported that mice lacking either H2A.X or 53BP1, but not Chk2, manifest a G2/M check-point defect close to that observed in ATM(−/−) cells after exposure to low, but not high, doses of IR. Moreover, H2A.X regulated the ability of 53BP1 to efficiently accumulate into IR-induced foci. Morales et al. 55 found that 53BP1 deficient mice were sensitive to γ-IR, and cells from these animals exhibit chromosomal abnormalities consistent with defects in DNA repair. These animals were growth-retarded and showed various immune deficiencies, including a specific reduction in thymus size and T-cell count. The increased chromosomal instability and tumour susceptibility apparent in mutant mice deficient in both p53 and either histone H2A.X or proteins that contribute to the nonhomologous end-joining mechanism of DNA repair, indicate that DNA damage check-points play a pivotal role in tumour suppression. Albino et al. 56 proposed a H2A.X phosphorylation-based test to be applied for testing potential carcinogens in products. Further studies are needed to assess the direct or indirect link between oxidative stress-driven DNA damage, ATM/ATR dependent signalling pathway and transcription factors. Since different particles may have different effects, further investigation is also needed into the effect of NP exposure and oxidative stress on ATM-dependent and independent gene expression changes, as well as on focus formation by DNA checkpoint signalling and repair factors to NP.
In conclusion, in the present study the expression of DNA damage-inducible genes, which previously have been shown to be involved in responses to IR treatment, was determined. Since the IR-induced DNA damage response is highly complexed, selected parameters were focused on in order to identify possible similarities in DNA damage response signals by particles. This is schematically shown in figure 6⇓.
Indeed, the present data provide some first clues for the potential of air pollution particles, specifically their nanoparticulate components, to act on similar signalling pathways in pulmonary epithelial cells. Current findings justify further investigation on the elucidation of the role(s) that particles play in disruption of mechanisms that regulate cell-cycle check-points, DNA repair and apoptosis, and which are known to culminate in genomic instability. Such information, improved by exploration of particulate matter interactions should shed light on the well-documented relationship between exposure to ambient particulate matter and cancer.
This project was funded by the European Respiratory Society, Fellowship No. LTRF2003-014, awarded to R.M. Mroz, and, in part, by the German Research Council (DFG-SFB503), which was awarded to R.P.F. Schins and H. Li.
Statement of interest
- Received January 18, 2007.
- Accepted November 13, 2007.
- © ERS Journals Ltd