Mutagenesis Advance Access originally published online on March 23, 2006
Mutagenesis 2006 21(2):153-158; doi:10.1093/mutage/gel013
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Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair
1Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Health Risk Analysis and Toxicology, Maastricht University, 6200 MD, PO Box 616, Maastricht, The Netherlands and 2Institut für Umweltmedizinische Forschung (IUF), Heinrich-Heine-University, Düsseldorf, Germany
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Abstract |
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There is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacities. Therefore, a modification of the alkaline comet assay was developed to determine the ability of human lymphocyte extracts to perform the initial steps of the nucleotide excision repair (NER) process, i.e. damage recognition and incision. Gel-embedded nucleoids from A549 cells, pre-exposed to 1 µM benzo[a]pyrene-diol-epoxide, were incubated with cell extracts from frozen or freshly isolated lymphocytes. The rate at which incisions are introduced and the subsequent increase in tail moment is indicative for the repair capacity of the extracts. Freshly prepared extracts from lymphocytes of human volunteers (n = 8) showed significant inter-individual variations in their DNA repair capacity, which correlated with the removal of bulky DNA lesions over a period of 48 h determined by 32P-post-labelling (R2 = 0.76, P = 0.005). Repeated measurements revealed a low inter-assay variation (11%). Storage of cell extracts for more than 3 weeks significantly reduced (up to 80%) the capacity to incise the damaged DNA as compared to freshly isolated extracts. This reduction was completely restored by addition of ATP to the extracts before use, as it is required for the incision step of NER. In contrast, extracts freshly prepared from frozen lymphocyte pellets can be used without loss of repair activity. DNA repair deficient XPA–/– and XPC–/– fibroblasts were used to further validate the assay. Although some residual capacity to incise the DNA was observed in these cells, the repair activity was restored to normal wild-type levels when a complementary mixture of both extracts (thereby restoring XPA and XPC deficiency) was used. These results demonstrate that this repair assay can be applied in molecular epidemiological studies to assess inter-individual differences in NER.
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Introduction |
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Epidemiological studies have reported large inter-individual variations in susceptibility to environmental carcinogens and subsequent cancer risk. There is evidence that this differential susceptibility is partly due to genetically determined variations in DNA repair capacity (DRC) (1
). For instance, several studies have described the association between polymorphisms in DNA repair genes and cancer risk (2–4). DNA repair mechanisms have evolved that protect the integrity of the genome, and these can generally be subdivided into base and nucleotide excision repair (BER and NER, respectively). Both pathways function as a result of the joint action of a variety of enzymes (5). However, it is still not completely understood whether polymorphisms in these various DNA repair genes have a significant impact on the phenotypical activity of these repair pathways. Furthermore, by focusing on single nucleotide polymorphisms in DNA repair genes, other factors that could affect DNA repair capacities, for instance dietary modulation (6) or adaptive responses (7,8), are not taken into account. Therefore, there is an increasing need for simple and reliable approaches to phenotypically assess DRCs. Such assays should be well characterized in terms of sensitivity, reproducibility, and both intra- and inter-individual variation.
At present, several approaches to determine DRC have been described (9–13). In general, these assays are largely based on treatment of live cells with damaging agents and subsequent monitoring of damage removal in time. However, such assays have two major limitations as follows: (i) they require freshly isolated cells, or cells that need to be handled in such a way that their survival after cryopreservation is assured; (ii) they are time-consuming. Furthermore, treatment of cells with relatively high levels of DNA damaging agents ex vivo could trigger a cellular response which is not present in vivo. Altogether, these limitations hamper the application of such assays in large molecular epidemiological studies.
Recently, Collins et al. (14) developed an elegant comet assay-based method to measure phenotypical differences in BER that was found to be applicable in molecular epidemiological studies. This alternative approach involves measurement of the capacity of human lymphocyte extracts to perform the initial step of BER, i.e. damage incision, on DNA substrates carrying 8-hydroxydeoxyguanosine (8-OHdG) lesions (14). However, up to now there are to the best of our knowledge only a few comparable comet-based assays to measure NER [e.g. (15)]. Still, many human cancers are directly related to exposure to chemical carcinogens, such as polycyclic aromatic hydrocarbons (PAH), that exert their carcinogenic action through formation of DNA adducts which are generally removed by NER. Therefore, we developed a modified comet assay to phenotypically assess inter-individual differences in NER capacities. This repair assay is based on the capacity of cell extracts to cause incisions in PAH-adduct containing DNA (specifically, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, BPDE–DNA adducts). The assay was validated by comparison with other methodologies, such as assessment of adduct removal, as well as by using DNA repair deficient cell lines. This newly developed assay is reliable, reproducible and can be used on frozen tissues or cells, indicating its applicability in molecular epidemiological studies.
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Materials and methods |
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Principle of the assay
Gel-embedded nucleoids from A549 cells containing high levels of BPDE–DNA adducts are incubated with cell extracts from frozen or freshly isolated lymphocytes. The principle of the assay is that NER-related enzymes that are present in cell extracts will recognize the BPDE–DNA adducts. Subsequently, the DNA is incised, causing single-strand breaks that can be determined by subsequent single-cell alkaline gel electrophoresis. Damage recognition and incision are the rate limiting steps in NER (5
). Therefore, increased tail moments (TMs) and percentages of fluorescence in the tail are indicative for the NER capacity of the cell extracts.
Cell strains and cell culture
A549 cells (human epithelial lung carcinoma cells) were purchased from the American Tissue Culture Collection (ATCC) and were cultured in T75 flasks in DMEM (Sigma, St Louis) supplemented with 10% heat inactivated fetal calf serum (FCS, Gibco Invitrogen, Scotland, UK) and 1% penicillin/streptomycin. Cells were maintained at 37°C in a 5% CO2 atmosphere.
Xeroderma pigmentosum group A and C fibroblasts (XPA–/– and XPC–/–, respectively), and normal wild-type fibroblasts (WT-fibroblasts) were obtained from the NIGMS Human Genetic Mutant Cell Respiratory (Coriell Institute for Medical Research, Camden, NJ, USA). Fibroblasts were cultured in MEM (Gibco Invitrogen, Scotland, UK) supplemented with 20% uninactivated FCS, 1% penicillin/streptomycin, 0.4% essential and non-essential amino acids, and 0.02% MEM vitamins.
Preparation of BPDE exposed nucleoids from A549 cells
One day prior to performing the repair assay, A549 cells were trypsinized at 80% confluency and diluted to a final concentration of 2 x 106 cells/ml. Aliquots of 25 µl of untreated A549 cells were mixed with 75 µl low melting point agarose [dissolved in phosphate-buffered saline (PBS)] and transferred to microscope slides, which were pre-coated with 1.5% normal electrophoresis grade agarose (Sigma–Aldrich, Germany). Gels were covered with a cover slip and kept at 4°C for 45 min to solidify. Subsequently, cover slips were removed and slides were lysed overnight in cold (4°C) lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 0.25 M NaOH, pH 10, with 10% dimethylsulfoxide (DMSO) and 1% Triton X-100 that were added just before use). The next day, slides were washed for 15 min with PBS. The resulting nucleoids were then exposed to BPDE (1 µM in PBS) (NCI Chemical Carcinogen Reference Standard Repository, Midwest Research Institute, Kansas City, MO, USA) or vehicle control (DMSO, 0.5%) for 30 min. Finally, the slides were washed for 15 min in buffer B (45 mM HEPES, 0.25 mM EDTA, 2% glycerol and 0.3 mg/ml BSA, pH 7.8).
Preparation of protein extracts
The preparation of the cell extracts is based on the method developed by Redaelli et al. (16). It is very simple and sufficient material for several assays can be obtained from lymphocytes of 10 ml human blood. Lymphocytes were isolated from venous blood of human volunteers using a standard density gradient centrifugation method (17). For validation purposes, we also prepared extracts from cultured WT, XPA–/– and XPC–/– fibroblasts. Cell pellets were washed in 4x diluted extraction buffer A (45 mM HEPES, 0.4 M KCl, 1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, adjusted to pH 7.8 using KOH), counted and diluted to a final concentration of 5 x 106 cells/ml. Then, the cell suspensions were centrifuged at 14 000 r.p.m. for 5 min and pellets were either frozen at –20°C for use at a later date or immediately resuspended in 50 µl buffer A per 5 x 106 cells. Resulting aliquots were snap frozen in liquid nitrogen. To each of these aliquots of 50 µl, 15 µl of 1% Triton X-100 in buffer A was added, incubated for 5 min and centrifuged at 11 000 r.p.m. for 5 min at 4°C to remove cell debris. Protein concentrations were determined by the BIO-RAD DC Protein Assay Kit (Veenendaal, The Netherlands) using bovine serum albumin as a standard. Protein extracts from lymphocytes were diluted with 0.23% Triton X-100 in buffer A to a concentration of 2 mg/ml, whereas protein extracts from fibroblasts were diluted to 0.1 mg/ml. Diluted protein extracts were then stored at –80°C until use in the repair assay.
Ex vivo repair incubation
Prior to the repair assay, diluted protein extracts were thawed and 4 vols of reaction buffer B (45 mM HEPES, 0.25 mM EDTA, 2% glycerol, 0.3 mg/ml BSA, adjusted to pH 7.8 with KOH) were added. Extracts were kept on ice until use. To assess the ex vivo repair capacity, 50 µl of the protein extract were added to each slide, containing BPDE-exposed gel-embedded nucleoids, and incubated for 10 min at 37°C on a warming plate. After the incubation, slides were immediately put on ice to stop the enzyme reactions. Subsequently, the slides were further processed according to the conventional comet assay as described previously (18). In brief, denaturation of the DNA was performed by immersion of the slides in electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, approximately pH 13) for 20 min, followed by 20 min of electrophoresis at 25 V and 300 mA. The slides were neutralized to pH 7.4 and subsequently dried using 100% ethanol. 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 slide 50 cells were analysed randomly using the Comet assay III software program (Perceptive Instruments, Haverhill, UK). Resulting data were presented as TM or tail intensity (TI) ± standard error. TI never exceeded values of 20% DNA in the tail after an incubation period of 10 min with the extract, indicating that the comet analysis is not saturated. After subtracting background levels (Figure 1, bar A) from all data, the final NER capacity was calculated using the formula:
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DNA isolation and 32P-post-labelling of BPDE–DNA adducts
To study possible inter-individual differences in adduct removal, and to compare adduct removal with the NER capacity assessed with our modified comet assay, lymphocytes from eight volunteers were isolated from 30 ml blood using gradient centrifugation (17
). After isolation, a part of these lymphocytes was used to prepare extracts to determine DRC phenotypically, another part of the lymphocytes were resuspended in RPMI, diluted to a concentration of 1 x 106 cells/ml and exposed to 0.05 µM BPDE. After 30 min of incubation at 37°C, medium containing BPDE was replaced by fresh culture medium. Lymphocytes were harvested after 1, 24 and 48 h of recovery, centrifuged at 300 g and pellets were stored at –20°C until DNA isolation and analysis of BPDE–DNA adducts by 32P-post-labelling.
Standard phenol extraction was used to obtain genomic DNA. 32P-post-labelling was carried out using the nuclease P1 enrichment technique as described by Reddy and Randerath (19) with some modifications (20). Briefly, an aliquot containing 10 µg DNA was digested using micrococcal endonuclease (0.25 U/µl) and spleen phosphodiesterase (2 µg/µl) for 3.5 h at 37°C. Subsequently, DNA-digests were treated with nuclease P1 (2.5 µg/µl) for 30 min at 37°C. To stop the NP1-reaction, 1 M Tris (pH 9.6) was added. BPDE-modified nucleotides were subsequently labelled with [
-32P]ATP (50 µCi/sample; ICN, IN, USA) using T4-polynucleotide kinase (10 U/µl) for 30 min at 37°C. The radiolabelled adducted nucleotide biphosphates were separated on the PEI-cellulose sheets (Machery Nagel, Düren, Germany) by multidirectional thin layer chromatography (TLC). In all experiments two BPDE–DNA standards with known adduct levels (1 adduct/107 and 1 adduct/108 nucleotides) were analysed in parallel for quantification purposes. Quantification was performed using Phosphor-Imaging technology (Fujifilm FLA-3000).
The BPDE–DNA adduct levels were corrected for the amount of DNA in the reaction. Therefore, an aliquot of DNA-digest was diluted and labelled with [-32P]ATP. Nucleotides were separated on a PEI-cellulose sheet by one directional TLC in 0.12 M NaH2PO4 (pH 6). A dAP-standard was analysed along with the other samples for quantification purposes.
Validation by using NER deficient cells
Extracts prepared from NER deficient xeroderma pigmentosum group A (XPA–/–) and group C (XPC–/–) fibroblasts and WT controls were diluted with 0.23% Triton X-100 in buffer A to a concentration of 0.1 mg/ml as described previously. Ex vivo repair incubations were also performed using a complementary mix of the two extracts (1 : 1, v/v). Hypothetically this should restore the NER capacity of the extract. In parallel, pre-exposed nucleoids were also incubated with extracts form WT-fibroblasts, as a control.
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Results |
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Optimal incubation time and protein concentration
For all the repair assay experiments, 10 min was chosen as the optimal incubation time for the extracts, since longer incubation periods induced higher background levels, causing a reduction of the sensitivity of the assay (TMBPDE–/Extract– = 0.26 ± 0.09 and 0.61 ± 0.16 for 10 and 20 min of incubation, respectively).
In general, the incision activity of an extract is dependent on its protein concentration. We observed that high protein concentrations caused a lower relative difference between the total (BPDE+/Extract+, bar D in Figure 1) and non-specific damage recognition (BPDE–/Extract+, bar C in Figure 1), thus resulting in a decreased sensitivity of the assay to detect NER capacity (Table I). Therefore, we determined the protein concentration showing the largest difference between BPDE–/Extract+ and BPDE+/Extract+ values. To this end, protein extracts from lymphocytes and fibroblasts (as described above) were diluted in buffer A to various concentrations ranging from 0.1 to 5 mg/ml. For protein extracts prepared from human lymphocytes, the optimal protein concentration was found to be 2 mg/ml (Table I). At this protein concentration, the non-specific damage recognition resulted in a TMBPDE–/Extract+as low as 1.40 and a relative difference with TMBPDE+/Extract+ of 3.00. Similar results were obtained by using TI as a read-out, although the relative difference between BPDE–/Extract+ and BPDE+/Extract+ values was lower.
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Protein extracts from fibroblasts showed much higher recognition and incision activity as compared to lymphocyte extracts (Table I). Protein concentrations as low as 0.5 mg/ml already resulted in TMBPDE+/Extract+ of 6.25 ± 0.06. By comparison, in the case of lymphocytes, such levels of TMBPDE+/Extract+ were only observed at 3 mg/ml, suggesting that fibroblasts have a
6-fold higher repair activity. The optimal protein concentration for fibroblasts was as low as 0.1 mg/ml, showing the lowest recognition of the non-specific damage and relative differences of 6.42 and 5.27 for TM and TI, respectively. No dilutions of protein extracts lower than 2 mg/ml (for lymphocytes) or 0.1 mg/ml (for fibroblasts) were tested to prevent the TMBPDE–/Extract+ from becoming too close to the background levels.
Effect of long-term cryopreservation
A second optimization step was performed to evaluate the possibility to analyse frozen materials at a later date. We showed that protein extracts will lose their capacity to incise the damaged DNA after long-term storage at –80°C. Aliquots of the same sample thawed at intervals less than a month generated lower TM values; up to 80% reduction was observed compared to freshly isolated extracts (Figure 2). This reduction was completely restored by addition of 2.5 mM ATP to the extracts before use. This is a first indication for the validity of our assay, as ATP is required for the incision step of NER (5). In contrast, no decline in repair activity was observed after use of extracts freshly prepared from lymphocyte pellets that were stored at –20°C for up to 40 days. Addition of ATP to these extracts did not further increase NER capacity.
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Inter-individual variations in NER capacity and assay reproducibility
To evaluate the reproducibility of the repair assay, lymphocytes from four volunteers were frozen as cell pellets at –20°C. After 1 month of storage, protein extracts were prepared and used in the repair assay. The results were plotted as DRCs and showed a clear variation in NER capacities between the different individuals (Figure 3). Six months later, thus after 7 months of storage of the lymphocytes, the experiment was repeated to check the reproducibility of the assay. The resulting data showed the same pattern as in the first experiment and the overall variance between the two experiments was 11.1% ± 4.4.
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Correlation between repair capacity and BPDE–DNA adduct removal
As a second validation step, BPDE–DNA adduct removal as studied by 32P-post-labelling and NER capacity were evaluated in parallel. If the repair assay is actually measuring individual NER capacities, there should be a correlation between repair capacity as detected in our assay and BPDE–DNA adduct removal. Therefore, lymphocytes from eight volunteers were isolated. Part of it was frozen as lymphocyte pellets at –20°C to assess the repair capacity by the modified comet assay. The remaining part was resuspended in RPMI and exposed to 0.05 µM BPDE for 30 min. After acute exposure to BPDE, DNA adduct levels were determined after 1, 24 and 48 h of recovery. The results are presented as regression lines through the data points (Figure 4A). A significant decline of BPDE–DNA adducts in time was observed (P = 0.015). Subsequently, the slopes of the individual regression lines were plotted against the logarithmically transformed NER capacity, as detected by our repair assay. When the repair capacity was calculated by using TM values, a significant linear correlation was observed between the DRCs and BPDE-adduct removing capacities (R2 = 0.763, Figure 4B). Similar results were obtained by using TI as a read-out of the repair capacities (R2 = 0.529, P = 0.041).
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Repair capacity of XP–/– cells, as measured by the repair assay
As a final validation step, the new repair assay was tested using extracts prepared from NER deficient XPA–/– and XPC–/– fibroblasts. Results were presented as percentages of the DRC of XPA–/– cell extracts (Figure 5). Each extract (XPA–/–, XPC–/–) on its own showed residual incising capacity. However, upon using a complementary mix of the two extracts (1 : 1), thereby reconstituting NER (both XPA and XPC enzymes are present), repair capacity was restored to the normal WT levels. This indicates that our assay indeed specifically measures NER.
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Discussion |
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During the past decades, many techniques have been developed to detect differences in DRC (9
–13). However, a major drawback of these assays is the need to carry out prolonged incubations. Even more important is that all of these approaches require freshly isolated cell material or cells that have been stored in such a way that they survive cryopreservation. This dramatically reduces their applicability in molecular epidemiological or intervention studies.
Recently Collins et al. (14) have adapted the comet assay to measure in vitro BER capacity of cell extracts. We have used this as a basis to develop an assay that assesses NER. In our assay we focus on repair of bulky pre-mutagenic BPDE–DNA adducts, which are specifically repaired by NER. The method has a high sensitivity of detecting strand breaks (21), the method is quick and robust and is economical on material; only 2–3 ml human blood is needed to obtain sufficient material to perform one assay. Moreover, lymphocytes can be frozen as cell pellets without loss of repair activity. We think that this repair assay is highly suitable for molecular epidemiological applications and intervention studies.
Optimization experiments showed that the DNA-incising activity is dependent on the extract's protein concentration and that optimal concentrations should be assessed for each cell type (Table I). Extracts were found to lose their activity during long-term storage at –80°C. Addition of ATP to the extracts before use restored their repair activity completely (Figure 2). Interestingly, in contrast to BER, the incision step of NER is ATP dependent. These results are a first indication that the predominant activity measured by this assay is the incising activity of NER enzymes. A further validation was provided by the close correlation between the removal of BPDE–DNA adducts, specifically involving NER, and the corresponding DRC of lymphocytes from eight individuals (Figure 4B).
To further confirm our findings, the repair assay was also performed using extracts from XPA–/– and XPC–/– fibroblasts (Figure 5). Although it is generally accepted that XPA is essential for the recognition and incision of damaged DNA by the NER pathway, the extracts from XPA deficient fibroblasts showed some residual capacity to incise the BPDE-treated DNA in our assay. It is not yet known whether these incisions are made by NER-related enzymes in absence of XPA, or by other pathways that recognize BPDE-damaged sites (22). Nonetheless, the capacity to recognize and incise BPDE–DNA adducts is reduced in extracts of XPC and XPA deficient cells, whereas it is restored by using a mixture of both extracts, indicating the validity of our assay.
In conclusion, we developed and validated an assay to phenotypically assess NER. Our assay is able to assess inter-individual differences in DRC and has a high reproducibility. Moreover, the assay is versatile, as it can be readily adapted to measure excision capacities of extracts of human cells or tissues on nucleoids containing lesions other than BPDE–DNA adducts, e.g. substrates exposed to UV, X-rays or MMS (23). The technique could still be fine-tuned by using nuclear cell extracts to improve the sensitivity even further (23), but this would decrease its applicability in field studies, as it is more time consuming. On the other hand, many tissue banks have stored human samples as total white blood cells (WBC) rather than lymphocytes. Theoretically, it is possible to use protein extracts from total WBC to assess inter-individual differences in DNA repair. However, differences between WBC subpopulations (e.g. lymphocytes, granulocytes and monocytes) need to be assessed first to correctly interpret the results obtained from total WBC extracts. Finally, this modified comet assay provides a powerful tool to further substantiate the role of DRC in the development of cancer and to provide answers to important research questions on DNA repair and human health.
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Acknowledgments |
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We are thankful to Dr Andrew R. Collins (Department of Nutrition, University of Oslo, Oslo, Norway) for providing us with the protocol on the base excision in vitro repair assay. A.M.K. was supported by a postdoctoral fellowship from the Netherlands Organisation for Scientific Research (NWO, grant 916.46.092).
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Notes |
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* To whom correspondence should be addressed. Tel: +31 43 388 1104; Fax: +31 43 388 4146; Email: R.Godschalk{at}GRAT.unimaas.nl
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