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Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay
Mutation Research 803–804 (2016) 22–26
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay Günter Speit ∗ , Petra Schütz, Julia Bausinger Universität Ulm, Institut für Humangenetik, 89069 Ulm, Germany
a r t i c l e
i n f o
Article history: Received 6 April 2016 Received in revised form 4 May 2016 Accepted 4 May 2016 Available online 5 May 2016 Keywords: Comet assay Mutagens Aphidicolin Polymerases
a b s t r a c t The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15 M). APC was either added for 2 h together with the mutagen or cells were pre-incubated for 30 min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The comet assay is widely used in three main areas of genetic toxicology. These are basic research into mechanisms of DNA damage and DNA repair, genotoxicity testing and biomonitoring (for review, see [1]). The standard alkaline comet assay detects DNAstrand breaks, alkali-labile sites (ALS) and repair incisions during the course of excision repair. The sensitivity of the comet assay in biomonitoring, in particular human biomonitoring of populations exposed to environmental pollutants, may be limited by the fact that environmental chemical mutagens rarely produce DNA strand breaks and ALS. Modifications of the standard alkaline version have been established to improve the specificity and the sensitivity of the assay. The use of lesion-specific enzymes (e.g., formamidopyrimidine glycosylase, Fpg) has added greatly to the value of the comet assay. Since the Fpg protein does not only detect oxidative DNA base damage but also alkylation-induced formamidopyrimidines
∗ Corresponding author. E-mail address: [email protected] (G. Speit). http://dx.doi.org/10.1016/j.mrgentox.2016.05.001 1383-5718/© 2016 Elsevier B.V. All rights reserved.
and abasic sites (AP sites), it might be used to enhance the sensitivity of the assay in human biomonitoring [2,3]. Since induced stable DNA adducts will mainly be detected via incisions during excision repair, this mechanism might be the most relevant for DNA effects measured by the comet assay in (human) biomonitoring. To further improve the sensitivity of the comet assay for the detection of bulky DNA adducts, modifications of the method with polymerase inhibitors have been established for the measurement of transient excision repair-associated breaks [2]. In this approach, polymerase inhibitors (e.g., aphidicolin; APC) are used to diminish repair synthesis, which leads to an accumulation of repair incisions. In this way, the presence of (increased levels of) DNA damage is indirectly indicated in actively repairing cells [4]. This modification of the comet assay has been used to study the contribution of excision repair to the effects measured [5], to establish DNA repair kinetics [6,7] and for measuring individual DNA repair capacity [8,9] although this approach might be limited by the relatively high assay variability [10,11]. However, the APC-modified comet assay did not detect enhanced DNA damage in a population living in a polluted area with a high incidence of lung cancer [12]. Surprisingly, neither the APC-modified comet assay nor the comet assay in combination
G. Speit et al. / Mutation Research 803–804 (2016) 22–26
with the Fpg protein detected enhanced levels of DNA damage in a group of heavy smokers [13]. The APC-modified comet assay has mainly been performed with human blood cells, either whole blood or isolated peripheral blood mononuclear cells (PBMC). It has clearly been demonstrated that APC enhances DNA effects induced by UV and UV-like chemicals such as benzo[a]pyrene (BaP), BPDE or 4-nitroquinoline-1-oxide (4-NQO) [2,4–6]. The enhancing effect was found in whole blood and PBMC, both unstimulated and phytohaemagglutinin (PHA)stimulated. The effect of APC was generally explained by an inhibition of polymerase ␣ which is involved in NER [14]. However, APC also clearly enhanced the effect of MMS, a simple alkylating agent. Since DNA base alkylations are mainly repaired by BER, it was supposed that APC also inhibits polymerases involved in BER. Another surprising aspect of the APC-modified comet assay was our recent finding that no repair activity could be measured in A549 cells (a human lung cancer cell line) exposed to BPDE under conditions where APC induced clear effects in lymphocytes [2,15]. To better understand the biological basis of the APC-modified comet assay and its reliability for measuring DNA repair activity in cultured mammalian cells, we now comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15 M). We used BPDE, because it does not produce frank breaks but specifically induces stable adducts that are solely repaired by NER [15]. In contrast, MMS-induced lesions (alkylations and AP sites) are specifically repaired by BER [6]. APC was either added for 2 h together with the mutagen or it was already added 30 min before mutagen treatment. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC and revealed practical limitations of the use of the APC-modified comet assay.
2. Materials and methods 2.1. Materials BPDE ((±)-anti benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) was purchased from BIU (Grosshansdorf, Germany). If not specifically indicated, all other chemicals used in these experiments were purchased from Sigma (Munich, Germany). Cell culture medium and ingredients were obtained from Invitrogen (Karlsruhe, Germany). Agarose (NEEO) was supplied by Roth (Karlsruhe, Germany) and low melting agarose (LMA, SeaPlaque, “GTG”) was from Biozym (Hameln, Germany).
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Fig. 1. The effect of APC on DNA migration (tail intensity) in the comet assay with A549 cells exposed to BPDE (1 and 2 M) or MMS (80 and 160 M) for 2 h in the absence and presence of APC (15 M). Co, untreated control cultures.
Exponentially growing cells were exposed to (±)-anti BPDE (1 and 2 M) or MMS (80 and 160 M) for 2 h in the absence and presence of APC (3 or 15 M). APC was either added for 2 h together with the mutagen (protocol 1) or it was already added 30 min before mutagen treatment (protocol 2). 2.3. Comet assay The comet assay was performed according to our standard protocol [16]. Aliquots of 10 l cell suspension (about 15 000 cells) were mixed with 120 l low melting point agarose (0.5% in PBS) and added to microscope slides (with frosted ends), which had been covered with a bottom layer of 1.5% agarose. Slides were lysed (pH 10; 4 ◦ C) for at least one hour and then processed using a time of alkali denaturation (at a pH > 13) of 25 min and electrophoresis (0.86 V/cm) of 25 min (HeLa, TK6, V79). Because of higher background migration in controls, alkali denaturation and electrophoresis of 20 min each were used in experiments with A549 cells. Slides were coded and images of 100 randomly selected cells (nucleoids) stained with ethidium bromide were analysed from each slide. Measurements were made by image analysis (Comet Assay IV, Perceptive Instruments, Haverhill, UK) and DNA migration was determined by measuring the “tail intensity” (% tail DNA). 2.4. Statistical analysis Pre-experiments were performed to define the appropriate mutagen concentrations for the comet assay. Three experiments were then independently performed under the same experimental conditions. In experiments which tested the influence of APC on mutagen-induced DNA migration Student’s t-test was applied to define a statistically significant difference (p < 0.05).
2.2. Cell culture and exposure to mutagens and APC 3. Results A549 cells (an epithelial-like human lung adenocarcinoma cell line), HeLa cells (a human transformed epithelial tumour cell line) and V79 cells (a permanent Chinese hamster lung cell line) were cultured in MEM supplemented with 10% FCS, 1% glutamine and 0.5% gentamycin. Cells were maintained in a humidified incubator at 37 ◦ C with 5% CO2 and harvested with 0.15% trypsin and 0.08% EDTA. TK6 cells (a human p53 wild type human B lymphoblastoid cell line) were cultured in RPMI 1640 medium with GlutaMAX, supplemented with 10% fetal calf serum, 1% sodium pyruvate and 0.5% gentamycin. TK6 cells were also kept in a humidified incubator at 37 ◦ C with 5% CO2 . For the experiments, 300.000 cells (A549, HeLa, V79) were seeded into T 12.5 plastic flasks and 1.2 × 106ˆ TK6 cells were seeded into culture tubes about 24 h prior to mutagen exposure.
Fig. 1 shows the effect of APC on BPDE- and MMS-induced DNA effects in the comet assay with A549 cells. In all experiments, two BPDE-concentrations (1 and 2 M) and two MMS-concentrations (80 and 160 M) were tested in the absence and presence of APC (15 M). Cells were exposed for 2 h. The results for A549 cells and BPDE were previously reported in another context [15] and are shown here because the experiments with MMS were performed in parallel. APC did neither clearly enhance BPDE-induced DNA migration nor MMS-induced DNA migration. The tail intensity values were slightly increased in all experiments in the presence of APC. This also applies to the unexposed controls and the net difference in combination with the two mutagens is minimal. We then tested a second protocol where APC was already added 30 min before the
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G. Speit et al. / Mutation Research 803–804 (2016) 22–26
Table 1 Enhancement of mutagen-induced comet assay effects by APC. Cells
Mutagen
APC exposure
Effect
Reference
Human WBC Human WBC (24 h PHA-stimulated) Human WBC Human WBC Human WBC (24 h PHA-stimulated) Human PBMC (24 h PHA-stimulated) Human PBMC (24 h PHA-stimulated) Human WBC (24 h PHA-stimulated) Human PBMC Human PBMC HeLa HeLa HeLa HeLa HeLa MRC5 MRC5 A549 A549 A549 A549 A549 TK6 TK6 TK6 TK6 V79 V79 V79 V79
BPDE BPDE BPDE MMS MMS BPDE BPDE BPDE BPDE MMS UV BPDE BPDE MMS MMS UV B[a]P + S9-mix BPDE (2 h) BPDE BPDE MMS MMS BPDE BPDE MMS MMS BPDE BPDE MMS MMS
1.5 M/15 M; 2 h co-incubation 1.5 M/15 M; 2 h co-incubation 15 M; 30 min pre-incubation 1.5 M/15 M; 2 h co-incubation 1.5 M/15 M; 2 h co-incubation 1.5 M; 30 min pre-incubation 1.5 M; 30 min pre-incubation 1.5 M; 30 min pre-incubation 3 M; 30 min post-incubation 3 M; 30 min post-incubation 15 M; 30 min post-incubation 15 M; 2 h co-incubation 15 M; 30 min pre-incubation 15 M; 2 h co-incubation 15 M; 30 min pre-incubation 15 M; 30 min post-incubation 15 M; 2 h co-incubation 3 M/15 M; co-incubation last 30 min 3 M/15 M; 2 h co-incubation 15 M; 30 min pre-incubation 3 M/15 M; 2 h co-incubation 15 M; 30 min pre-incubation 3 M; 2 h co-incubation 3 M; 30 min pre-incubation 3 M; 2 h co-incubation 3 M; 30 min pre-incubation 15 M; 2 h co-incubation 15 M; 30 min pre-incubation 15 M; 2 h co-incubation 15 M; 30 min pre-incubation
+ + + + + + + + + + + + + – – + + – – + – – ++ ++ ++ ++ – – – –
[2] [2] [2] [2] [2] [9] [11] [11] [6] [6] [4]
[5] [5] [15] [15]
APC, aphidicolin; WBC, whole blood cells; PBMC, peripheral blood mononuclear cells.
Fig. 2. The effect of APC on DNA migration (tail intensity) in the comet assay with A549 cells exposed to BPDE (1 and 2 M) or MMS (80 and 160 M) for 2 h. APC (15 M) was added 30 min before the mutagen. Mean ± SD of three independent experiments. * = p < 0.05 in relationship to the effect in the absence of APC.
mutagen to inhibit polymerases before the damage is induced [7]. Fig. 2 summarizes the results for BPDE and MMS. Interestingly, this protocol caused a significant enhancement of BPDE-induced DNA effects but did not clearly enhance MMS-induced effects. These results suggest that A549 cells are less sensitive towards the action of APC in comparison with the published data with human blood [2]. In contrast to the results obtained with human blood, MMSinduced DNA effects were not enhanced by APC in the comet assay with A549 cells. Next, we utilized HeLa cells for the APC-modified comet assay because an earlier publication had shown that APC enhanced UVinduced DNA migration in the comet assay with HeLa cells [4]. This paper showed comet images of HeLa cells after UV-C irradiation in the absence and presence of APC. Comet tails were only seen if the inhibitor was present. We used two protocols for testing the effect of APC (15 M) on BPDE- and MMS-induced DNA migration. APC
Fig. 3. The effect of APC on DNA migration (tail intensity) in the comet assay with HeLa cells exposed to 1 and 2 M BPDE (A) or 80 and 160 M MMS (B). APC (15 M) was either added for 2 h together with the mutagen (P1) or it was already added 30 min before the mutagen (P2). Co, untreated control cultures. Mean ± SD of three independent experiments. * = p < 0.05; ** = p < 0.01 in relationship to the effect in the absence of APC.
G. Speit et al. / Mutation Research 803–804 (2016) 22–26
Fig. 4. The effect of APC on DNA migration (tail intensity) in the comet assay with TK6 cells exposed to 1 and 2 M BPDE (A) or 80 and 160 M MMS (B). APC (3 M) was either added for 2 h together with the mutagen (P1) or it was already added 30 min before the mutagen (P2). Co, untreated control cultures. Mean ± SD of three independent experiments. * = p < 0.05; ** = p < 0.01 in relationship to the effect in the absence of APC.
was either added for 2 h simultaneously with the mutagen (protocol 1, P1) or cells were pre-incubated for 30 min with APC before the mutagen was added (protocol 2, P2). Both protocols enhanced DNA migration in combination with BPDE (Fig. 3A) but had no effect on MMS-induced DNA migration (Fig. 3B). Interestingly, in the experiments with BPDE, both protocols led to the appearance of “hedgehogs” (i.e., nucleoids with all DNA in the tail and no visible head). The mean percentage of hedgehogs increased with increasing BPDE-concentration from 3% to 12% and from 6% to 21% for the two protocols, respectively. These results suggest a different action of APC on BPDE- and MMS-induced DNA damage and its repair. The same two test protocols (P1 and P2) were also applied to TK6 cells, a human lymphoblastoid cell line which is frequently used in mutation research and genotoxicity testing. Surprisingly, experiments with 15 M APC could not be evaluated because this concentration induced a very high percentage of hedgehogs in combination with both mutagens. Experiments with 3 M APC also induced hedgehogs in combination with both mutagens (between 6% and 25% in the case of BPDE and between 3% and 5% in the case of MMS) in a concentration-related manner. This APC-concentration strongly enhanced BPDE- and MMS-induced DNA migration without a clear difference between the two protocols (Fig. 4). Fig. 5 summarizes results for V79 cells, a rapidly growing Chinese hamster cell line which has been frequently used in genotoxicity testing including the genotoxicity of APC [17]. Interestingly, APC (15 M) did not enhance BPDE- and MMS-induced DNA migration when either applied for 2 h together with the mutagen (P1) or for 30 min before adding the mutagen (P2). Similar to the results
25
Fig. 5. The effect of APC on DNA migration (tail intensity) in the comet assay with V79 cells exposed to 1 and 2 M BPDE (A) or 80 and 160 M MMS (B). APC (15 M) was either added for 2 h together with the mutagen (P1) or it was already added 30 min before the mutagen (P2). Co, untreated control cultures. Mean ± SD of three independent experiments.
obtained with A549 cells, V79 cells seem to be resistant towards APC under the conditions tested here. 4. Discussion Our study confirms that APC is able to enhance mutageninduced comet assay effects. However, these results also show that fundamental differences exist between different cell types. The available data for the APC-modified comet assay are summarized in Table 1. The majority of studies have been performed with human blood. Enhancement of mutagen-induced comet assay effects was reported for whole blood and isolated lymphocytes (PBMC), for unstimulated (non-proliferating) and PHA-stimulated (proliferating) lymphocyte cultures. An APC-concentration of 1.5 M was sufficient to induce an enhancing effect, 15 M APC was a little bit more effective. Pre-incubation with APC for 30 min was not more effective than co-incubation with the mutagen for 2 h [2]. APC enhanced the effect of BPDE, the reactive metabolite of B[a]P that forms stable bulky adducts mainly with guanine. These adducts are repaired by NER and it is assumed that APC leads to an accumulation of transient breaks (incisions) because it inhibits repair synthesis by polymerase ␣ [14]. Surprisingly, MMS-induced DNA migration was also enhanced by APC. MMS induces DNA base alkylations that are mainly repaired by BER. The polymerase mainly involved in BER is polymerase  which is not inhibited by APC. The results reported for blood cells were confirmed in our study by similar experiments with the human lymphoblastoid cell line TK6. Interestingly, TK6 cells were much more sensitive towards the action of APC. A low APC-concentration (3 M) strongly enhanced DNA migration induced by BPDE or MMS and also led to the occurrence of so-called hedgehogs that can be regarded as cells with maximum
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G. Speit et al. / Mutation Research 803–804 (2016) 22–26
damage in the comet assay [18,19]. The enhancement of BPDE- and MMS-induced effects may be explained by the fact that APC not only inactivates polymerase ␣ but also polymerase ␦ and polymerase [20]. The polymerases ␦ and are involved in NER [21,22] but also in long-patch BER [23,24] and there is strong evidence that polymerase ␦ can play a role in the repair of methylation damage [25]. Possibly, inhibition of polymerase ␦ and polymerase by APC is more important for the observed effects in the APC-modified comet assay than inactivation of polymerase ␣. The cell lines tested so far seem differ significantly with regard to their sensitivity towards APC and thus their suitability for measuring DNA excision repair activity by the APC-modified comet assay. The V79 Chinese hamster cells tested here did not show any enhancement of mutagen-induced DNA effects in the presence of APC. Interestingly, APC-resistant Chinese hamster ovary (CHO) cell lines have been described with mutated polymerase ␣ and altered polymerase ␦ and polymerase as a secondary effect [26]. It is unknown, how common such mutants are and whether a genetic defect can explain the APC-insensitivity of the V79 cells used here. HeLa cells and A549 cells seem to possess different sensitivities towards BPDE- and MMS-induced DNA lesions in the APC- modified comet assay. HeLa cells exhibited clear effects of APC in combination with exposure to BPDE but no effects in combination with MMS. Similarly, with A549 cells no enhancing effect of APC was measured after exposure to MMS, whereas a weak positive effect was seen in cells treated with BPDE. APC significantly enhanced BPDE-induced DNA migration in the comet assay only when the cells were pre-incubated with APC. Co-incubation for 2 h only led to marginal enhancement that was not statistically significant. These results with HeLa and A549 cells indicate that APC inhibits NER and long patch BER with different sensitivity and that NER is inhibited more efficiently in these experiments. This difference may be explained by different targets (enzymes) involved or by genetic differences/alterations of the cell lines used. The relative insensitivity of V79, HeLa and A549 cells might be due to enhanced expression of polymerases in rapidly proliferating and transformed cells [27,28]. In any case, the results presented here confirm our concerns [15] that the commonly used APC-modified comet assay may not be universally suited for studying DNA excision repair activities in cultured mammalian cells. The suitability of cells for such a kind of studies needs to be determined on a case-by-case basis. References [1] A. Azqueta, A.R. Collins, The essential comet assay: a comprehensive guide to measuring DNA damage and repair, Arch. Toxicol. 87 (2013) 949–968. [2] G. Speit, P. Schütz, H. Hoffmann, Enhancement of genotoxic effects in the comet assay with human blood samples by aphidicolin, Toxicol. Lett. 153 (2004) 303–310. [3] A. Azqueta, L. Arbillaga, A. Lopez de Cerain, A. Collins, Enhancing the sensitivity of the comet assay as a genotoxicity test, by combining it with bacterial repair enzyme FPG, Mutagenesis 28 (2013) 271–277. [4] C.M. Gedik, S.W. Ewen, A.R. Collins, Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells, Int. J. Radiat. Biol. 62 (1992) 313–320. [5] G. Speit, A. Hartmann, The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test (comet assay), Mutagenesis 10 (1995) 555–559. [6] J. Bausinger, G. Speit, DNA repair capacity of cultured human lymphocytes exposed to mutagens measured by the comet assay and array expression analysis, Mutagenesis 30 (2015) 811–820.
[7] M. Klaude, C.M. Gedik, A.R. Collins, DNA damage and repair after low doses of UV-C radiation; comparable rates of repair in rodent and human cells, Int. J. Radiat. Biol. 67 (1995) 501–508. [8] A. Allione, A. Russo, F. Ricceri, K. Vande Loock, S. Guarrera, F. Voglino, M. Kirsch-Volders, G. Matullo, Validation of the nucleotide excision repair comet assay on cryopreserved PBMCs to measure inter-individual variation in DNA repair capacity, Mutagenesis 28 (2013) 65–70. [9] K. Vande Loock, I. Decordier, R. Ciardelli, D. Haumont, M. Kirsch-Volders, An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity, Mutagenesis 25 (2010) 25–32. [10] M. Cipollini, J. He, P. Rossi, F. Baronti, A. Micheli, A.M. Rossi, R. Barale, Can individual repair kinetics of UVC-induced DNA damage in human lymphocytes be assessed through the comet assay? Mutat. Res. 601 (2006) 150–161. [11] G. Speit, C. Leibiger, S. Kuehner, J. Högel, Further investigations on the modified comet assay for measuring aphidicolin-block nucleotide excision repair, Mutagenesis 28 (2013) 145–151. [12] W. Heepchantree, T. Paratasilpin, D. Kangwanpong, A biological evaluation of DNA damage detected by comet assay in healthy populations residing in areas that differ in lung cancer incidence, J. Toxicol. Environ. Health A. 69 (2006) 1071–1082. [13] H. Hoffmann, G. Speit, Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test, Mutat. Res. 581 (2005) 105–114. [14] T. Toyomasu, K. Nakaminami, H. Toshima, T. Mie, K. Watanabe, H. Ito, H. Matsui, W. Mitsuhashi, T. Sassa, H. Oikawa, Cloning of a gene cluster responsible for the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha, Biosci. Biotechnol. Biochem. 68 (2004) 146–152. [15] J. Bausinger, P. Schütz, A.L. Piberger, G. Speit, Further characterization of benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects, Mutagenesis 31 (2016) 161–169. [16] G. Speit, A. Rothfuss, The comet assay: a sensitive genotoxicity test for the detection of DNA damage and repair, Methods Mol. Biol. 920 (2012) 79–90. [17] G. Speit, P. Schütz, The effect of inhibited replication on DNA migration in the comet assay in relation to cytotoxicity and clastogenicity, Mutat. Res. 655 (2008) 22–27. [18] Y. Lorenzo, S. Costa, A.R. Collins, A. Azqueta, The comet assay, DNA, damage, DNA repair and cytotoxicity: hedgehogs are not always dead, Mutagenesis 28 (2013) 427–432. [19] G. Speit, A. Vesely, P. Schütz, R. Linsenmeyer, J. Bausinger, The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay, Mutagenesis 29 (2014) 267–277. [20] R. Mirzayans, K. Dietrich, M.C. Paterson, Aphidicolin and 1-beta-d-arabinofuranosylcytosine strongly inhibit transcriptionally active DNA repair in normal human fibroblasts, Carcinogenesis 14 (1993) 2621–2626. [21] T. Ogi, S. Limsirichaikul, R.M. Overmeer, M. Volker, K. Takenaka, R. Cloney, Y. Nakazawa, A. Niimi, Y. Miki, N.G. Jaspers, L.H. Mullenders, S. Yamashita, M.I. Fousteri, A.R. Lehmann, Three DNA polymerases recruited by different mechanisms, carry out NER repair synthesis in human cells, Mol. Cell. 37 (2010) 714–727. [22] J.A. Marteijn, H. Lans, W. Vermeulen, J.H. Hoeijmakers, Understanding nucleotide excision repair and its roles in cancer and ageing, Nat. Rev. Mol. Cell Biol. 15 (2014) 465–481. [23] M. Stucki, B. Pascucci, E. Parlanti, P. Fortini, S.H. Wilson, U. Hubscher, E. Dogliotti, Mammalian base excision repair by DNA polymerases delta and epsilon, Oncogene 17 (1998) 835–843. [24] Y.J. Kim, D.M. Wilson, Overview of base excision repair biochemistry, Curr. Mol. Pharmacol. 5 (2012) 3–13. [25] M.J. Prindle, L.A. Loeb, DNA polymerase delta in DNA replication and genome maintenance, Environ. Mol. Mutagen. 53 (2012) 666–682. [26] Z. Feher, N.C. Mishra, Aphidicolin-resistant chinese hamster ovary cells possess altered DNA polymerases of the alpha-family, Biochim. Biophys. Acta. 1218 (1994) 35–47. [27] S.W. Wong, A.F. Wahl, P.M. Yuan, N. Arai, B.E. Pearson, K. Arai, D. Korn, M.W. Hunkapiller, T.S. Wang, Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases, EMBO J. 7 (1988) 37–47. [28] A.F. Wahl, A.M. Geis, B.H. Spain, S.W. Wong, D. Korn, T.S. Wang, Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle, Mol. Cell. Biol. 8 (1988) 5016–5025.
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay Günter Speit ∗ , Petra Schütz, Julia Bausinger Universität Ulm, Institut für Humangenetik, 89069 Ulm, Germany
a r t i c l e
i n f o
Article history: Received 6 April 2016 Received in revised form 4 May 2016 Accepted 4 May 2016 Available online 5 May 2016 Keywords: Comet assay Mutagens Aphidicolin Polymerases
a b s t r a c t The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15 M). APC was either added for 2 h together with the mutagen or cells were pre-incubated for 30 min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The comet assay is widely used in three main areas of genetic toxicology. These are basic research into mechanisms of DNA damage and DNA repair, genotoxicity testing and biomonitoring (for review, see [1]). The standard alkaline comet assay detects DNAstrand breaks, alkali-labile sites (ALS) and repair incisions during the course of excision repair. The sensitivity of the comet assay in biomonitoring, in particular human biomonitoring of populations exposed to environmental pollutants, may be limited by the fact that environmental chemical mutagens rarely produce DNA strand breaks and ALS. Modifications of the standard alkaline version have been established to improve the specificity and the sensitivity of the assay. The use of lesion-specific enzymes (e.g., formamidopyrimidine glycosylase, Fpg) has added greatly to the value of the comet assay. Since the Fpg protein does not only detect oxidative DNA base damage but also alkylation-induced formamidopyrimidines
∗ Corresponding author. E-mail address: [email protected] (G. Speit). http://dx.doi.org/10.1016/j.mrgentox.2016.05.001 1383-5718/© 2016 Elsevier B.V. All rights reserved.
and abasic sites (AP sites), it might be used to enhance the sensitivity of the assay in human biomonitoring [2,3]. Since induced stable DNA adducts will mainly be detected via incisions during excision repair, this mechanism might be the most relevant for DNA effects measured by the comet assay in (human) biomonitoring. To further improve the sensitivity of the comet assay for the detection of bulky DNA adducts, modifications of the method with polymerase inhibitors have been established for the measurement of transient excision repair-associated breaks [2]. In this approach, polymerase inhibitors (e.g., aphidicolin; APC) are used to diminish repair synthesis, which leads to an accumulation of repair incisions. In this way, the presence of (increased levels of) DNA damage is indirectly indicated in actively repairing cells [4]. This modification of the comet assay has been used to study the contribution of excision repair to the effects measured [5], to establish DNA repair kinetics [6,7] and for measuring individual DNA repair capacity [8,9] although this approach might be limited by the relatively high assay variability [10,11]. However, the APC-modified comet assay did not detect enhanced DNA damage in a population living in a polluted area with a high incidence of lung cancer [12]. Surprisingly, neither the APC-modified comet assay nor the comet assay in combination
G. Speit et al. / Mutation Research 803–804 (2016) 22–26
with the Fpg protein detected enhanced levels of DNA damage in a group of heavy smokers [13]. The APC-modified comet assay has mainly been performed with human blood cells, either whole blood or isolated peripheral blood mononuclear cells (PBMC). It has clearly been demonstrated that APC enhances DNA effects induced by UV and UV-like chemicals such as benzo[a]pyrene (BaP), BPDE or 4-nitroquinoline-1-oxide (4-NQO) [2,4–6]. The enhancing effect was found in whole blood and PBMC, both unstimulated and phytohaemagglutinin (PHA)stimulated. The effect of APC was generally explained by an inhibition of polymerase ␣ which is involved in NER [14]. However, APC also clearly enhanced the effect of MMS, a simple alkylating agent. Since DNA base alkylations are mainly repaired by BER, it was supposed that APC also inhibits polymerases involved in BER. Another surprising aspect of the APC-modified comet assay was our recent finding that no repair activity could be measured in A549 cells (a human lung cancer cell line) exposed to BPDE under conditions where APC induced clear effects in lymphocytes [2,15]. To better understand the biological basis of the APC-modified comet assay and its reliability for measuring DNA repair activity in cultured mammalian cells, we now comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15 M). We used BPDE, because it does not produce frank breaks but specifically induces stable adducts that are solely repaired by NER [15]. In contrast, MMS-induced lesions (alkylations and AP sites) are specifically repaired by BER [6]. APC was either added for 2 h together with the mutagen or it was already added 30 min before mutagen treatment. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC and revealed practical limitations of the use of the APC-modified comet assay.
2. Materials and methods 2.1. Materials BPDE ((±)-anti benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) was purchased from BIU (Grosshansdorf, Germany). If not specifically indicated, all other chemicals used in these experiments were purchased from Sigma (Munich, Germany). Cell culture medium and ingredients were obtained from Invitrogen (Karlsruhe, Germany). Agarose (NEEO) was supplied by Roth (Karlsruhe, Germany) and low melting agarose (LMA, SeaPlaque, “GTG”) was from Biozym (Hameln, Germany).
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Fig. 1. The effect of APC on DNA migration (tail intensity) in the comet assay with A549 cells exposed to BPDE (1 and 2 M) or MMS (80 and 160 M) for 2 h in the absence and presence of APC (15 M). Co, untreated control cultures.
Exponentially growing cells were exposed to (±)-anti BPDE (1 and 2 M) or MMS (80 and 160 M) for 2 h in the absence and presence of APC (3 or 15 M). APC was either added for 2 h together with the mutagen (protocol 1) or it was already added 30 min before mutagen treatment (protocol 2). 2.3. Comet assay The comet assay was performed according to our standard protocol [16]. Aliquots of 10 l cell suspension (about 15 000 cells) were mixed with 120 l low melting point agarose (0.5% in PBS) and added to microscope slides (with frosted ends), which had been covered with a bottom layer of 1.5% agarose. Slides were lysed (pH 10; 4 ◦ C) for at least one hour and then processed using a time of alkali denaturation (at a pH > 13) of 25 min and electrophoresis (0.86 V/cm) of 25 min (HeLa, TK6, V79). Because of higher background migration in controls, alkali denaturation and electrophoresis of 20 min each were used in experiments with A549 cells. Slides were coded and images of 100 randomly selected cells (nucleoids) stained with ethidium bromide were analysed from each slide. Measurements were made by image analysis (Comet Assay IV, Perceptive Instruments, Haverhill, UK) and DNA migration was determined by measuring the “tail intensity” (% tail DNA). 2.4. Statistical analysis Pre-experiments were performed to define the appropriate mutagen concentrations for the comet assay. Three experiments were then independently performed under the same experimental conditions. In experiments which tested the influence of APC on mutagen-induced DNA migration Student’s t-test was applied to define a statistically significant difference (p < 0.05).
2.2. Cell culture and exposure to mutagens and APC 3. Results A549 cells (an epithelial-like human lung adenocarcinoma cell line), HeLa cells (a human transformed epithelial tumour cell line) and V79 cells (a permanent Chinese hamster lung cell line) were cultured in MEM supplemented with 10% FCS, 1% glutamine and 0.5% gentamycin. Cells were maintained in a humidified incubator at 37 ◦ C with 5% CO2 and harvested with 0.15% trypsin and 0.08% EDTA. TK6 cells (a human p53 wild type human B lymphoblastoid cell line) were cultured in RPMI 1640 medium with GlutaMAX, supplemented with 10% fetal calf serum, 1% sodium pyruvate and 0.5% gentamycin. TK6 cells were also kept in a humidified incubator at 37 ◦ C with 5% CO2 . For the experiments, 300.000 cells (A549, HeLa, V79) were seeded into T 12.5 plastic flasks and 1.2 × 106ˆ TK6 cells were seeded into culture tubes about 24 h prior to mutagen exposure.
Fig. 1 shows the effect of APC on BPDE- and MMS-induced DNA effects in the comet assay with A549 cells. In all experiments, two BPDE-concentrations (1 and 2 M) and two MMS-concentrations (80 and 160 M) were tested in the absence and presence of APC (15 M). Cells were exposed for 2 h. The results for A549 cells and BPDE were previously reported in another context [15] and are shown here because the experiments with MMS were performed in parallel. APC did neither clearly enhance BPDE-induced DNA migration nor MMS-induced DNA migration. The tail intensity values were slightly increased in all experiments in the presence of APC. This also applies to the unexposed controls and the net difference in combination with the two mutagens is minimal. We then tested a second protocol where APC was already added 30 min before the
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Table 1 Enhancement of mutagen-induced comet assay effects by APC. Cells
Mutagen
APC exposure
Effect
Reference
Human WBC Human WBC (24 h PHA-stimulated) Human WBC Human WBC Human WBC (24 h PHA-stimulated) Human PBMC (24 h PHA-stimulated) Human PBMC (24 h PHA-stimulated) Human WBC (24 h PHA-stimulated) Human PBMC Human PBMC HeLa HeLa HeLa HeLa HeLa MRC5 MRC5 A549 A549 A549 A549 A549 TK6 TK6 TK6 TK6 V79 V79 V79 V79
BPDE BPDE BPDE MMS MMS BPDE BPDE BPDE BPDE MMS UV BPDE BPDE MMS MMS UV B[a]P + S9-mix BPDE (2 h) BPDE BPDE MMS MMS BPDE BPDE MMS MMS BPDE BPDE MMS MMS
1.5 M/15 M; 2 h co-incubation 1.5 M/15 M; 2 h co-incubation 15 M; 30 min pre-incubation 1.5 M/15 M; 2 h co-incubation 1.5 M/15 M; 2 h co-incubation 1.5 M; 30 min pre-incubation 1.5 M; 30 min pre-incubation 1.5 M; 30 min pre-incubation 3 M; 30 min post-incubation 3 M; 30 min post-incubation 15 M; 30 min post-incubation 15 M; 2 h co-incubation 15 M; 30 min pre-incubation 15 M; 2 h co-incubation 15 M; 30 min pre-incubation 15 M; 30 min post-incubation 15 M; 2 h co-incubation 3 M/15 M; co-incubation last 30 min 3 M/15 M; 2 h co-incubation 15 M; 30 min pre-incubation 3 M/15 M; 2 h co-incubation 15 M; 30 min pre-incubation 3 M; 2 h co-incubation 3 M; 30 min pre-incubation 3 M; 2 h co-incubation 3 M; 30 min pre-incubation 15 M; 2 h co-incubation 15 M; 30 min pre-incubation 15 M; 2 h co-incubation 15 M; 30 min pre-incubation
+ + + + + + + + + + + + + – – + + – – + – – ++ ++ ++ ++ – – – –
[2] [2] [2] [2] [2] [9] [11] [11] [6] [6] [4]
[5] [5] [15] [15]
APC, aphidicolin; WBC, whole blood cells; PBMC, peripheral blood mononuclear cells.
Fig. 2. The effect of APC on DNA migration (tail intensity) in the comet assay with A549 cells exposed to BPDE (1 and 2 M) or MMS (80 and 160 M) for 2 h. APC (15 M) was added 30 min before the mutagen. Mean ± SD of three independent experiments. * = p < 0.05 in relationship to the effect in the absence of APC.
mutagen to inhibit polymerases before the damage is induced [7]. Fig. 2 summarizes the results for BPDE and MMS. Interestingly, this protocol caused a significant enhancement of BPDE-induced DNA effects but did not clearly enhance MMS-induced effects. These results suggest that A549 cells are less sensitive towards the action of APC in comparison with the published data with human blood [2]. In contrast to the results obtained with human blood, MMSinduced DNA effects were not enhanced by APC in the comet assay with A549 cells. Next, we utilized HeLa cells for the APC-modified comet assay because an earlier publication had shown that APC enhanced UVinduced DNA migration in the comet assay with HeLa cells [4]. This paper showed comet images of HeLa cells after UV-C irradiation in the absence and presence of APC. Comet tails were only seen if the inhibitor was present. We used two protocols for testing the effect of APC (15 M) on BPDE- and MMS-induced DNA migration. APC
Fig. 3. The effect of APC on DNA migration (tail intensity) in the comet assay with HeLa cells exposed to 1 and 2 M BPDE (A) or 80 and 160 M MMS (B). APC (15 M) was either added for 2 h together with the mutagen (P1) or it was already added 30 min before the mutagen (P2). Co, untreated control cultures. Mean ± SD of three independent experiments. * = p < 0.05; ** = p < 0.01 in relationship to the effect in the absence of APC.
G. Speit et al. / Mutation Research 803–804 (2016) 22–26
Fig. 4. The effect of APC on DNA migration (tail intensity) in the comet assay with TK6 cells exposed to 1 and 2 M BPDE (A) or 80 and 160 M MMS (B). APC (3 M) was either added for 2 h together with the mutagen (P1) or it was already added 30 min before the mutagen (P2). Co, untreated control cultures. Mean ± SD of three independent experiments. * = p < 0.05; ** = p < 0.01 in relationship to the effect in the absence of APC.
was either added for 2 h simultaneously with the mutagen (protocol 1, P1) or cells were pre-incubated for 30 min with APC before the mutagen was added (protocol 2, P2). Both protocols enhanced DNA migration in combination with BPDE (Fig. 3A) but had no effect on MMS-induced DNA migration (Fig. 3B). Interestingly, in the experiments with BPDE, both protocols led to the appearance of “hedgehogs” (i.e., nucleoids with all DNA in the tail and no visible head). The mean percentage of hedgehogs increased with increasing BPDE-concentration from 3% to 12% and from 6% to 21% for the two protocols, respectively. These results suggest a different action of APC on BPDE- and MMS-induced DNA damage and its repair. The same two test protocols (P1 and P2) were also applied to TK6 cells, a human lymphoblastoid cell line which is frequently used in mutation research and genotoxicity testing. Surprisingly, experiments with 15 M APC could not be evaluated because this concentration induced a very high percentage of hedgehogs in combination with both mutagens. Experiments with 3 M APC also induced hedgehogs in combination with both mutagens (between 6% and 25% in the case of BPDE and between 3% and 5% in the case of MMS) in a concentration-related manner. This APC-concentration strongly enhanced BPDE- and MMS-induced DNA migration without a clear difference between the two protocols (Fig. 4). Fig. 5 summarizes results for V79 cells, a rapidly growing Chinese hamster cell line which has been frequently used in genotoxicity testing including the genotoxicity of APC [17]. Interestingly, APC (15 M) did not enhance BPDE- and MMS-induced DNA migration when either applied for 2 h together with the mutagen (P1) or for 30 min before adding the mutagen (P2). Similar to the results
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Fig. 5. The effect of APC on DNA migration (tail intensity) in the comet assay with V79 cells exposed to 1 and 2 M BPDE (A) or 80 and 160 M MMS (B). APC (15 M) was either added for 2 h together with the mutagen (P1) or it was already added 30 min before the mutagen (P2). Co, untreated control cultures. Mean ± SD of three independent experiments.
obtained with A549 cells, V79 cells seem to be resistant towards APC under the conditions tested here. 4. Discussion Our study confirms that APC is able to enhance mutageninduced comet assay effects. However, these results also show that fundamental differences exist between different cell types. The available data for the APC-modified comet assay are summarized in Table 1. The majority of studies have been performed with human blood. Enhancement of mutagen-induced comet assay effects was reported for whole blood and isolated lymphocytes (PBMC), for unstimulated (non-proliferating) and PHA-stimulated (proliferating) lymphocyte cultures. An APC-concentration of 1.5 M was sufficient to induce an enhancing effect, 15 M APC was a little bit more effective. Pre-incubation with APC for 30 min was not more effective than co-incubation with the mutagen for 2 h [2]. APC enhanced the effect of BPDE, the reactive metabolite of B[a]P that forms stable bulky adducts mainly with guanine. These adducts are repaired by NER and it is assumed that APC leads to an accumulation of transient breaks (incisions) because it inhibits repair synthesis by polymerase ␣ [14]. Surprisingly, MMS-induced DNA migration was also enhanced by APC. MMS induces DNA base alkylations that are mainly repaired by BER. The polymerase mainly involved in BER is polymerase  which is not inhibited by APC. The results reported for blood cells were confirmed in our study by similar experiments with the human lymphoblastoid cell line TK6. Interestingly, TK6 cells were much more sensitive towards the action of APC. A low APC-concentration (3 M) strongly enhanced DNA migration induced by BPDE or MMS and also led to the occurrence of so-called hedgehogs that can be regarded as cells with maximum
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damage in the comet assay [18,19]. The enhancement of BPDE- and MMS-induced effects may be explained by the fact that APC not only inactivates polymerase ␣ but also polymerase ␦ and polymerase [20]. The polymerases ␦ and are involved in NER [21,22] but also in long-patch BER [23,24] and there is strong evidence that polymerase ␦ can play a role in the repair of methylation damage [25]. Possibly, inhibition of polymerase ␦ and polymerase by APC is more important for the observed effects in the APC-modified comet assay than inactivation of polymerase ␣. The cell lines tested so far seem differ significantly with regard to their sensitivity towards APC and thus their suitability for measuring DNA excision repair activity by the APC-modified comet assay. The V79 Chinese hamster cells tested here did not show any enhancement of mutagen-induced DNA effects in the presence of APC. Interestingly, APC-resistant Chinese hamster ovary (CHO) cell lines have been described with mutated polymerase ␣ and altered polymerase ␦ and polymerase as a secondary effect [26]. It is unknown, how common such mutants are and whether a genetic defect can explain the APC-insensitivity of the V79 cells used here. HeLa cells and A549 cells seem to possess different sensitivities towards BPDE- and MMS-induced DNA lesions in the APC- modified comet assay. HeLa cells exhibited clear effects of APC in combination with exposure to BPDE but no effects in combination with MMS. Similarly, with A549 cells no enhancing effect of APC was measured after exposure to MMS, whereas a weak positive effect was seen in cells treated with BPDE. APC significantly enhanced BPDE-induced DNA migration in the comet assay only when the cells were pre-incubated with APC. Co-incubation for 2 h only led to marginal enhancement that was not statistically significant. These results with HeLa and A549 cells indicate that APC inhibits NER and long patch BER with different sensitivity and that NER is inhibited more efficiently in these experiments. This difference may be explained by different targets (enzymes) involved or by genetic differences/alterations of the cell lines used. The relative insensitivity of V79, HeLa and A549 cells might be due to enhanced expression of polymerases in rapidly proliferating and transformed cells [27,28]. In any case, the results presented here confirm our concerns [15] that the commonly used APC-modified comet assay may not be universally suited for studying DNA excision repair activities in cultured mammalian cells. The suitability of cells for such a kind of studies needs to be determined on a case-by-case basis. References [1] A. Azqueta, A.R. Collins, The essential comet assay: a comprehensive guide to measuring DNA damage and repair, Arch. Toxicol. 87 (2013) 949–968. [2] G. Speit, P. Schütz, H. Hoffmann, Enhancement of genotoxic effects in the comet assay with human blood samples by aphidicolin, Toxicol. Lett. 153 (2004) 303–310. [3] A. Azqueta, L. Arbillaga, A. Lopez de Cerain, A. Collins, Enhancing the sensitivity of the comet assay as a genotoxicity test, by combining it with bacterial repair enzyme FPG, Mutagenesis 28 (2013) 271–277. [4] C.M. Gedik, S.W. Ewen, A.R. Collins, Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells, Int. J. Radiat. Biol. 62 (1992) 313–320. [5] G. Speit, A. Hartmann, The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test (comet assay), Mutagenesis 10 (1995) 555–559. [6] J. Bausinger, G. Speit, DNA repair capacity of cultured human lymphocytes exposed to mutagens measured by the comet assay and array expression analysis, Mutagenesis 30 (2015) 811–820.
[7] M. Klaude, C.M. Gedik, A.R. Collins, DNA damage and repair after low doses of UV-C radiation; comparable rates of repair in rodent and human cells, Int. J. Radiat. Biol. 67 (1995) 501–508. [8] A. Allione, A. Russo, F. Ricceri, K. Vande Loock, S. Guarrera, F. Voglino, M. Kirsch-Volders, G. Matullo, Validation of the nucleotide excision repair comet assay on cryopreserved PBMCs to measure inter-individual variation in DNA repair capacity, Mutagenesis 28 (2013) 65–70. [9] K. Vande Loock, I. Decordier, R. Ciardelli, D. Haumont, M. Kirsch-Volders, An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity, Mutagenesis 25 (2010) 25–32. [10] M. Cipollini, J. He, P. Rossi, F. Baronti, A. Micheli, A.M. Rossi, R. Barale, Can individual repair kinetics of UVC-induced DNA damage in human lymphocytes be assessed through the comet assay? Mutat. Res. 601 (2006) 150–161. [11] G. Speit, C. Leibiger, S. Kuehner, J. Högel, Further investigations on the modified comet assay for measuring aphidicolin-block nucleotide excision repair, Mutagenesis 28 (2013) 145–151. [12] W. Heepchantree, T. Paratasilpin, D. Kangwanpong, A biological evaluation of DNA damage detected by comet assay in healthy populations residing in areas that differ in lung cancer incidence, J. Toxicol. Environ. Health A. 69 (2006) 1071–1082. [13] H. Hoffmann, G. Speit, Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test, Mutat. Res. 581 (2005) 105–114. [14] T. Toyomasu, K. Nakaminami, H. Toshima, T. Mie, K. Watanabe, H. Ito, H. Matsui, W. Mitsuhashi, T. Sassa, H. Oikawa, Cloning of a gene cluster responsible for the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha, Biosci. Biotechnol. Biochem. 68 (2004) 146–152. [15] J. Bausinger, P. Schütz, A.L. Piberger, G. Speit, Further characterization of benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects, Mutagenesis 31 (2016) 161–169. [16] G. Speit, A. Rothfuss, The comet assay: a sensitive genotoxicity test for the detection of DNA damage and repair, Methods Mol. Biol. 920 (2012) 79–90. [17] G. Speit, P. Schütz, The effect of inhibited replication on DNA migration in the comet assay in relation to cytotoxicity and clastogenicity, Mutat. Res. 655 (2008) 22–27. [18] Y. Lorenzo, S. Costa, A.R. Collins, A. Azqueta, The comet assay, DNA, damage, DNA repair and cytotoxicity: hedgehogs are not always dead, Mutagenesis 28 (2013) 427–432. [19] G. Speit, A. Vesely, P. Schütz, R. Linsenmeyer, J. Bausinger, The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay, Mutagenesis 29 (2014) 267–277. [20] R. Mirzayans, K. Dietrich, M.C. Paterson, Aphidicolin and 1-beta-d-arabinofuranosylcytosine strongly inhibit transcriptionally active DNA repair in normal human fibroblasts, Carcinogenesis 14 (1993) 2621–2626. [21] T. Ogi, S. Limsirichaikul, R.M. Overmeer, M. Volker, K. Takenaka, R. Cloney, Y. Nakazawa, A. Niimi, Y. Miki, N.G. Jaspers, L.H. Mullenders, S. Yamashita, M.I. Fousteri, A.R. Lehmann, Three DNA polymerases recruited by different mechanisms, carry out NER repair synthesis in human cells, Mol. Cell. 37 (2010) 714–727. [22] J.A. Marteijn, H. Lans, W. Vermeulen, J.H. Hoeijmakers, Understanding nucleotide excision repair and its roles in cancer and ageing, Nat. Rev. Mol. Cell Biol. 15 (2014) 465–481. [23] M. Stucki, B. Pascucci, E. Parlanti, P. Fortini, S.H. Wilson, U. Hubscher, E. Dogliotti, Mammalian base excision repair by DNA polymerases delta and epsilon, Oncogene 17 (1998) 835–843. [24] Y.J. Kim, D.M. Wilson, Overview of base excision repair biochemistry, Curr. Mol. Pharmacol. 5 (2012) 3–13. [25] M.J. Prindle, L.A. Loeb, DNA polymerase delta in DNA replication and genome maintenance, Environ. Mol. Mutagen. 53 (2012) 666–682. [26] Z. Feher, N.C. Mishra, Aphidicolin-resistant chinese hamster ovary cells possess altered DNA polymerases of the alpha-family, Biochim. Biophys. Acta. 1218 (1994) 35–47. [27] S.W. Wong, A.F. Wahl, P.M. Yuan, N. Arai, B.E. Pearson, K. Arai, D. Korn, M.W. Hunkapiller, T.S. Wang, Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases, EMBO J. 7 (1988) 37–47. [28] A.F. Wahl, A.M. Geis, B.H. Spain, S.W. Wong, D. Korn, T.S. Wang, Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle, Mol. Cell. Biol. 8 (1988) 5016–5025.