Mutagenesis, Vol. 14, No. 5, 497-504,
September 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Processing of DNA damage induced by hydrogen peroxide and methyl methanesulfonate in human lymphocytes: analysis by alkaline single cell gel electrophoresis and cytogenetic methods
Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Roma, Italy
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Abstract |
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The persistence of induced DNA damage in human lymphocytes after mitogen stimulation and its relationship to subsequent cytogenetic alterations were investigated. The analysis of single-strand breaks and alkali-labile sites by single cell gel electrophoresis (SCGE) showed the almost complete repair of damage induced in resting lymphocytes by methyl methanesulfonate (MMS, 140–210 µM) and hydrogen peroxide (H2O2, 25–100 µM) during the first 16 h of culture. On the other hand, DNA damage was shown to persist to a large extent when cells were cultured in the presence of the repair inhibitor cytosine ß-D-arabinofuranoside (Ara-C) (1 µg/ml). Although highly effective in the induction of DNA lesions detectable by SCGE, both agents failed to significantly increase the rate of micronucleus formation in cytokinesis-blocked cells harvested 66 h after treatment. However, when Ara-C was present during the first 16 h of culture, micronuclei were significantly increased at all doses. Conversely, sister chromatid exchange (SCE) rates were increased by chemical treatments to a higher extent in cultures without Ara-C. Delayed treatments, 16 h after mitogen stimulation, led to a significant induction of micronuclei in the case of MMS but not with H2O2. These results suggest that only a minor fraction of DNA damage induced in resting lymphocytes is available for fixation through misreplication, because of its effective repair prior to S phase. However, the processing of damage through recombination pathways can lead to increased SCE rates in treated cells. These features of the processing of DNA damage in human lymphocytes should be taken into account when structural cytogenetic alterations in cultured lymphocytes are used in monitoring human exposure to genotoxic agents.
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Introduction |
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Since its development, the single cell gel electrophoresis (SCGE), or Comet, assay has rapidly become one of the most popular methods in use in genetic toxicology. This methodology allows the detection and quantification at the single cell level of a variety of DNA lesions using alkaline (Singh et al., 1988
; Pfuhler and Wolf, 1996; Miyamae et al., 1997) or neutral incubation conditions (Ostling and Johanson, 1984), eventually coupled with lesion-specific glycosylases (Collins et al., 1993; Dusinska and Collins, 1996), with ease, speed and accuracy. Moreover, it can be successfully applied to almost every cell type, provided that single cell preparations are available. As a consequence, the SCGE assay, especially in its alkaline version, has been applied to address a variety of questions, including DNA repair, mechanisms of mutagenesis, ecogenotoxicology, tissue specificity, etc. (McKelvey-Martin et al., 1993; Fairbain et al., 1995).
One of the fields where alkaline SCGE has been more widely used is biomonitoring of human exposure to genotoxic agents (Betti et al., 1995; Moretti et al., 1996; Andreoli et al., 1997; Collins et al., 1997; Sram et al., 1998). Previous experience shows that SCGE has the potential to become one of the most useful tools for this kind of investigation: the methodology for SCGE is not technically demanding and, therefore, is well suited to the analysis of large population groups. Moreover, it can be applied to biological samples obtained through minimally invasive procedures such as finger pricks (Hellman et al., 1997). In comparison with other methods currently used in human biomonitoring, SCGE seems to be able to identify low level exposures with greater sensitivity (Vodicka et al., 1995; Binkova et al., 1996; Andreoli et al., 1997) as well as subtle effects which may result from impaired physiological status (Hartmann et al. 1994; Betancourt et al., 1995) and lifestyle (Green et al., 1994; Betti et al., 1995). On the other hand, the biological relevance of DNA lesions revealed by SCGE is unclear (Speit et al., 1996; Van Goethem et al., 1997; Vrzoc and Petras, 1997), considering the possibility that such lesions are repaired before their fixation in stable genetic alterations. The repair of DNA damage before fixation may be especially favoured in peripheral lymphocytes, the most common target tissue of biomonitoring studies, because of the considerable time interval which usually elapses between the induction of DNA damage in vivo and its fixation as mutations after in vitro stimulation.
In this work the relationship between the induction of DNA damage in G0 lymphocytes and the production of cytogenetic alterations after stimulation has been investigated. Two model compounds, hydrogen peroxide (H2O2) and methyl methanesulfonate (MMS), producing different spectra of DNA lesions, were used to induce various levels of damage in the DNA of isolated blood lymphocytes. The time course of repair of damage after mitogen stimulation and its inhibition by the nucleoside analogue cytosine ß-D-arabinofuranoside (Ara-C) have been evaluated by SCGE and correlated with the frequencies of sister chromatid exchange (SCE) and micronuclei observed in the same cell samples.
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Materials and methods |
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Chemicals
H2O2 (CAS 7722-84-1, 30% w/w solution in water), MMS (CAS 66-27-3, >99%) and Ara-C (CAS 147-94-4) were purchased from Sigma Chemical Co. (St Louis, MO). Ara-C was dissolved in bidistilled water; MMS was diluted in dimethylsulfoxide (DMSO) immediately before use.
Lymphocyte isolation
Approximately 40 ml of heparinized whole blood were collected by venipuncture from healthy male blood donors (aged 25–38). Lymphocytes were isolated from heparinized whole blood samples, after dilution 1:1 with RPMI 1640 (Gibco BRL, Grand Island, NY) without serum, using Histopaque 1077 (Sigma). After centrifugation at 1300 r.p.m. for 30 min, the buffy coat was collected and the cells washed three times with RPMI 1640 without serum. Viability of cells after isolation, determined using the fluorochrome-mediated viability test (see below), was 
98%.
Chemical treatments
Isolated lymphocytes, resuspended at 1x106 cells/ml, were treated as follows: H2O2 (25–100 µM) was applied in phosphate-buffered saline (PBS) for 5 or 30 min on ice; MMS (140–210 µM) was applied in medium without serum for 2 h at 37°C. At the end of exposure, cells were washed in the same medium used for treatment and then split into two aliquots: one was resuspended in PBS at 2x105 cells/10 µl and immediately used for the SCGE and viability tests; the other was resuspended in complete medium and used to set up cell cultures.
For liquid holding experiments, treated cells were washed in PBS and incubated in medium without serum for 24 h at 37°C before being processed in the SCGE assay.
Fluorochrome-mediated viability test
Cell viability was routinely determined using the fluorescein diacetate (FDA)/ethidium bromide (EtBr) assay according to Strauss (1991). Immediately after treatment, 25 µl of cell sample were mixed with 25 µl of staining solution (30 µg/ml FDA, 8 µg/ml EtBr, in PBS), spread on a microscope slide and covered with a coverslip. Viable cells fluoresced green, whereas dead cells were indicated by orange stained nuclei; erythrocytes do not label. At least 200 cells were scored per data point. In the experiments presented, viability of cells after chemical treatment was always >90%.
SCGE assays
The standard alkaline SCGE, or Comet, assay was performed according to the method developed by Singh and co-workers (Singh et al., 1988), with minor modifications described elsewhere (Andreoli et al., 1997). Briefly, 10 µl of lymphocyte suspension in PBS (2x105 cells) were mixed with 65 µl of 0.7% low melting point agarose (LMA; Bio-Rad, Richmond, CA), layered on a slide previously coated with a layer of 0.5% normal melting point agarose (NMA; Bio-Rad) and covered with another layer of LMA. Then, slides were immersed in lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10, with 10% DMSO and 1% Triton X-100 added fresh) for 1 h at 4°C. After lysis, slides were placed in a horizontal gel electrophoresis tank with fresh alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH 13.5) and left in the solution for 20 min at 4°C. Electrophoresis was conducted at 4°C for 20 min at 25 V (0.8 V/cm) and 300 mA, using a Bio-Rad 300 power supply. Once completed, slides were washed three times with neutralizing solution (0.4 M Tris–HCl, pH 7.5) and stained with EtBr (20 µg/ml).
All experiments were repeated at least twice using blood samples from different donors. Moreover, in all cases preliminary experiments were performed to single out the best experimental conditions.
Cell cultures
For each experimental point, six replicate cultures were established using isolated lymphocytes processed as described above. Cells (5x105 cells/ml) were incubated in RPMI 1640 medium with HEPES and L-glutamine (Gibco BRL), supplemented with 20% heat-inactivated fetal calf serum (HyClone, Logan, UT), 2% phytohemagglutinin (PHA; Gibco BRL), 1% antibiotic solution containing penicillin (5000 IU/ml) and streptomycin (5000 mg/ml) and incubated at 37°C in a humidified atmosphere at 5% CO2 in air. When indicated, the repair inhibitor Ara-C was applied at 1 µg/ml (final concentration) during the first 16 h of incubation and removed by centrifugation.
Two cultures were harvested at different time points (16 or 24 and 48 h) and assayed by SCGE. Two cultures, supplemented with 9 µg/ml 5-bromodeoxyuridine (Sigma), were treated with 10–5 M colchicine (Sigma) 3 h before harvest (72 h), fixed and stained with the fluorochrome plus Giemsa technique (Perry and Wolff, 1974) for the analysis of SCEs and the evaluation of proliferative replication index (PRI). The remaining two cultures were treated with 6 µg/ml cytochalasin B (Sigma) at 44 h and harvested 22 h later by cytocentrifugation (400 r.p.m. for 5 min). The slides were then fixed and stained with Giemsa for the analysis of micronuclei in binucleated lymphocytes following standard procedures (Fenech, 1993).
Analysis of slides
For SCGE assays, slides were examined at 250x magnification with a Leitz DM RB microscope equipped with a 50 W mercury lamp and N2.1 filter block. Slides were analysed by the Casys computerized image analysis system (Synoptics Ltd, Cambridge, UK). DNA damage was quantitated by tail moment measurement, calculated by multiplying the total intensity of the comet tail by the tail length, measured from the centre of the comet head. One hundred cells for each experimental point were scored blind from two slides.
Micronuclei and SCE were scored blind in 1000–2000 binucleated lymphocytes and 25 well-differentiated metaphases, respectively, for each control and treatment dose. The PRI was calculated from the number of first (MI), second (MII) and third (MIII) mitoses over 100 scored metaphases according to the formula PRI = (MI + 2MII + 3MIII)/100 (Lamberti et al., 1983). The nuclear division index (NDI) was calculated on 1000 cells in cultures treated with cytochalasin B according to the formula {[no. mononucleated + (2xno. binucleated) + (3xno. trinucleated) + (4xno. tetranucleated)] / 1000} (Eastmond and Tucker, 1989).
Statistical analysis
The significance of the effect of each treatment dose versus the untreated control was evaluated by the non parametric one-tailed Mann–Whitney U-test, using the cell as the unit of measurement. The effects of chemical treatments under different experimental conditions (i.e. with and without Ara-C) were compared using Student's t-test for paired samples. The statistical significance of cytogenetic end-points in treated versus control cultures was evaluated by the 
2 test and Student's t-test. All analyses were carried out with the SPSS statistical package.
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Results |
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Persistence of DNA damage induced in G0 after mitogen stimulation: analysis by alkaline SCGE
Preliminary experiments to assess the stability of DNA damage induced by H2O2 in human lymphocytes, revealed a sharp decrease in the number of DNA lesions detected by alkaline SCGE during cell growth in vitro. Data in Table I
show average tail moment values of lymphocyte cultures set up from quiescent cells treated with 25–100 µM H2O2 and harvested at different times after stimulation.
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These results prompted further experiments to assess the role of excision repair in the removal of such lesions in growing cells. To this end, isolated lymphocytes from two donors were treated with H2O2 (50–100 µM) or with the alkylating agent MMS (140–210 µM). Immediately thereafter, parallel cultures were set up from each treated cell sample using complete medium either with or without the repair inhibitor Ara-C (1 µg/ml). The results of SCGE assays are summarized in Figures 1 and 2
. Average tail moment values of cells harvested at 16 and 48 h (t16 and t48) are shown together with tail moment values measured immediately after chemical treatment (t0). In agreement with the findings from previous experiments, H2O2 produced extensive DNA damage in lymphocytes from both donors (Figure 1), with highly increased comet tail moment values in treated cells analysed immediately after treatment (P <0 .001, U-test). Induced DNA damage was almost undetectable after 16 h or more growth in complete medium. On the other hand, DNA damage persisted to a large extent in cells cultured in the presence of Ara-C: with both donors, average tail moment values of treated cells harvested at 16 h, i.e. at the end of the incubation period in the presence of Ara-C, were still significantly greater (P < 0.005, U-test) than control values. No significant excess damage was observed in cells harvested at 48 h, i.e. after an additional incubation period of 32 h without the DNA repair inhibitor (Figure 1).
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Similar results were obtained with cells treated with MMS (Figure 2
). In this case also, DNA damage persisted to a large extent in cells cultured in the presence of Ara-C. Residual damage, significantly lower than that observed with Ara-C (P < 0.001, t-test), was, however, also detected in cells harvested after 16 h growth without Ara-C. No significant excess damage was seen in cultures harvested at 48 h, either with or without Ara-C.
The apparent, albeit limited, persistence of damage induced by MMS compared with H2O2 points to some differences in the processing of DNA lesions induced by the two agents. This question was investigated in liquid holding experiments. The results of a representative experiment are shown in Figure 3. The extent of damage induced by MMS, as detected by alkaline SCGE, was significantly increased after 24 h of liquid holding (P < 0.01, t0 versus t24, U-test). The reverse result was observed with H2O2, i.e. a significant decrease in tail moment values in cells harvested after 24 h of incubation in serum-free medium (P < 0.01, t0 versus t24, U-test). These results indicate that DNA damage induced by H2O2 is also repaired to some extent in non-growing cells, whereas the processing of DNA lesions induced by MMS leads to single-strand breaks or intermediates labile under alkaline conditions.
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Cytogenetic damage in cultured lymphocytes after chemical treatment in G0 phase
The preliminary observations on the decrease in DNA damage during growth in vitro (Table I
) stimulated an investigation of the consequences of the removal of DNA lesions on delayed effects, such as chromosomal alterations, which arise during replication. To investigate this matter, parallel cultures for micronucleus and SCE analyses were set up from the same cell samples used for SCGE. Figures 4 and 5 summarize the results of the analysis of micronuclei in cytokinesis-blocked cells from two donors, following treatment with H2O2 or MMS before stimulation. Borderline or insignificant increases in micronuclei were observed in cultures set up with cells treated in G0 with both agents, despite the large amount of DNA damage induced by chemical treatments, as shown by the SCGE analysis of t0 samples (cf. Figures 1 and 2). On the other hand, a highly significant increase in micronuclei was observed with both agents when cells were grown in the presence of Ara-C during the first 16 h.
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A converse effect was seen with SCE. Both chemical treatments induced significant increases in SCE (Table II
). On the other hand, exposure to Ara-C during the first 16 h after stimulation resulted in a weak but significant increase in background levels of SCE in untreated cultures (P < 0.05, t-test), with no apparent synergism with the effect induced by chemical treatment. As a consequence, SCE rates in treated cultures were weakly increased with respect to untreated cultures and in only a few cases was statistical significance attained.
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The effect of delayed treatments on micronucleus induction in stimulated lymphocytes
As shown above, treatments of resting lymphocytes with both H2O2 and MMS were almost completely ineffective in inducing micronuclei in cytokinesis-blocked cells harvested after 66 h culture. In view of the rapid decrease in DNA damage following in vitro stimulation, it is conceivable that the lack of induction of clastogenic damage after treatment in G0 may depend on the low levels of DNA damage remaining at the time of DNA replication. This hypothesis implies that delayed induction of DNA damage should be relatively more effective, because a greater proportion of damage should be amenable for fixation during S phase. To check this hypothesis, DNA damage and induction of micronuclei were determined in parallel cultures from the same donor treated with H2O2 or MMS either at G0 or 16 h after stimulation, i.e. in late G1. The results obtained are summarized in Table III
. Comparable high levels of DNA damage were induced by both agents in G0 as well as in G1 cells. With H2O2, both exposure schedules were ineffective in inducing micronuclei. On the other hand, with MMS a significant increase in micronuclei was induced by treatment of cells in G1 but not in G0, in agreement with the mechanism proposed above.
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Discussion |
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The results obtained indicate that DNA damage induced by H2O2 and MMS in quiescent human lymphocytes decreases to a large extent over the first 16 h after mitogen stimulation. Even though the identity of the molecular lesions induced was not assessed, it is conceivable that the wide specificity of alkaline SCGE covers the entire spectrum of DNA lesions induced by chemical treatment. The disappearance of DNA modifications leading to increased comet tail moment points to the active processing/repair of primary DNA lesions soon after mitogen stimulation. This hypothesis was confirmed by the persistence of DNA damage, measured by SCGE, in cell cultures grown in the presence of the nucleoside analogue Ara-C. Ara-C is believed to act by competing with dCTP, thereby inhibiting DNA synthesis and ligation of the repair patch (Mikita and Beardsley, 1988
). The data presented herein demonstrate that this step plays a key role in the repair of lesions induced by both methylating and oxidizing agents. Differences in the nature and stability of DNA damage induced by H2O2 and MMS were, however, observed. Liquid holding experiments, in particular, showed the disappearance of damage induced by H2O2, on the one hand, and an increase in comet tail moment in cells treated with MMS, on the other. Considering the physiological status of cells in liquid holding, which does not allow de novo synthesis of nucleotides required for repair, the results can be interpreted as evidence for direct rejoining of single-strand breaks induced by H2O2 and for the accumulation of intermediates of repair in the case of MMS.
Whatever the mechanism(s), the results provide evidence for efficient removal of DNA lesions induced by H2O2 and MMS in quiescent (G0) lymphocytes soon after mitogen stimulation. In particular, residual damage was barely detectable by SCGE after 16 h growth in vitro, i.e. at the time corresponding to late G1 phase (Loeb et al., 1968; Fenech and Neville, 1992). This implies that few, if any, primary DNA lesions should be available for fixation through misreplication in the forthcoming S phase. The analysis of cytogenetic end-points in the same treated cell population shed some light on the consequence of the repair of DNA damage in resting cells on delayed genetic effects. A straightforward indication was provided by the analysis of micronuclei in cytokinesis-blocked cells. Here, chemical treatment of unstimulated cells was almost ineffective in inducing micronuclei, unless Ara-C was present during the first 16 h of growth, i.e. over the time interval covering approximately the first G1 phase. Thus, only when DNA damage persisted to a large extent up to the first round of DNA replication were micronuclei effectively induced. This is most likely a result of double-strand breaks originating from the replication of damaged templates (Fenech and Neville, 1992; Surralles et al., 1995). This mechanism may account for the discrepancies between DNA damaging potential and clastogenic activity exerted by some chemicals in human lymphocytes, as recently reported for the benzene metabolite hydroquinone (Andreoli et al., 1999).
A different response was obtained with the analysis of SCE in the same cell populations. In contrast to micronuclei, treatment of quiescent cells with either H2O2 or MMS led to significant increases in the frequency of SCE at the second mitosis. On the other hand, when Ara-C was present during the first 16 h of growth, SCEs were not increased or even decreased. The induction of SCE in cells grown without the repair inhibitor, which showed negligible residual damage at the end of G1 (16 h), may be related to recombinational processes secondary to the repair of DNA lesions induced by chemical treatment (Kaina and Aurich, 1985; Beranek, 1990; Kaina et al., 1993). In the presence of Ara-C, background levels of SCE were slightly increased (Zhang et al., 1988), possibly because of the effect exerted during S phase by the residual amount of inhibitor present in the cells (Gedik and Collins, 1991). SCE in treated cultures were not similarly increased, compared with cultures without Ara-C, because of the inhibitory action of Ara-C on the critical step of ligation during recombination. On the other hand, a small induction of SCE by MMS and H2O2 was indeed also observed in Ara-C-treated cultures. This result can be explained considering that the SCE detected account for the exchanges occurring at both first and second mitoses and that, at least in the case of MMS, exchanges were induced to a similar extent in the first and second cell cycles (Kaina and Aurich, 1985). Thus, the increase in SCE in Ara-C-treated cultures may be due to recombinational events occurring in the second cell cycle, most likely unaffected by previous exposure to the nucleoside analogue. A similar pattern of response, with increased structural aberrations and unchanged SCE rates in the presence of Ara-C, was previously reported for cultured human lymphocytes treated with either MMS or 4-nitroquinoline-1-oxide (Kishi, 1987).
Considering the relationship between induction of micronuclei and persistence of DNA damage produced by inhibition of repair, it is expected that the delay in chemical treatment at the end of G1 should similarly lead to a more effective induction of micronuclei. Experiments with MMS confirmed that the point in the cell cycle in which DNA damage is induced plays a critical role in the fixation of primary DNA lesions. In the present work almost identical levels of DNA damage induced just before stimulation or after 16 h of culture had dramatically different consequences in terms of micronucleus induction. The experimental protocol applied did not reveal a similar modulation of the induction of micronuclei by H2O2, possibly because of the faster repair of single-strand breaks (Collins et al., 1995), the prevailing lesion induced by H2O2 treatment (Horvathova et al., 1998).
These results lead to a more general consideration of the significance of chromosomal alterations detected in cultured human lymphocytes, especially with regard to their use as indicators of exposure/early biological effect in human biomonitoring studies. The data presented herein suggest that relatively large amounts of DNA lesions accumulated in circulating lymphocytes, e.g. because of environmental exposures, may escape detection through conventional analyses of clastogenic effects in cultured cells, because of repair of damage after stimulation. This need not imply that such damage is not biologically relevant, because the same lesions can lead to chromosomal alterations when fixed through DNA replication. As shown in this work, this may be obtained in vitro by inhibiting DNA repair or by delaying treatment. Conditions suitable for fixation of damage are expected to occurr in vivo in actively replicating cells. Therefore, negative findings from conventional cytogenetic analyses should be considered with caution in human biomonitoring of chemical exposures. Conversely, unstimulated lymphocytes, because of their long life and low repair capacities, are potentially able to accumulate lesions and may therefore reveal cumulative effects from chronic exposures. The SCGE analysis can detect different DNA lesions in unstimulated cells and, together with a suitable experimental protocol, may provide information as to the identity of the DNA lesions involved.
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Acknowledgments |
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This work was partially supported by the Italian Ministry of Environment (project PR 22-IS) and by Regione Sardegna (project DISIA PI-2/s).
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1 To whom correspondence should be addressed. Tel: +39 06 49902840; Fax: +39 06 49387139; Email: crebelli{at}iss.it
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References |
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Andreoli,C., Leopardi,P. and Crebelli,R. (1997) Detection of DNA damage in human lymphocytes by alkaline single cell gel electrophoresis after exposure to benzene or benzene metabolites. Mutat. Res., 377, 95–104.[Web of Science][Medline]
Andreoli,C., Rossi,S., Leopardi,P. and Crebelli,R. (1999) DNA damage by hydroquinone in human white blood cells: analysis by alkaline single-cell gel electrophoresis. Mutat. Res., 438, 37–45.[Web of Science][Medline]
Beranek,D.T. (1990) Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. Mutat. Res., 231, 11–30.[Web of Science][Medline]
Betancourt,M., Ortiz,R., Gonzales,C., Perez,P., Corts,L., Rodriguez,L. and Villasenor,L. (1995) Assessment of DNA damage in leukocytes from infected and malnourished children by single cell gel electrophoresis/comet assay. Mutat. Res., 331, 65–77.[Web of Science][Medline]
Betti,C., Davini,T., Giannessi,L., Loprieno,N. and Barale,R. (1995) Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects. Mutat. Res., 343, 201–207.[Web of Science][Medline]
Binkova,B., Lewtas,J., Miskova,I., Rossner,P., Cerna,M., Mrackova,G., Peterkova,K., Mumford,J., Meyer,J. and Sram,R.J. (1996) Biomarkers studies in the Northern Bohemia. Environ. Health Perspect., 104, 591–597.
Collins,A.R., Duthie,S.J. and Dobson,V.L. (1993) Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis, 14, 1733–1735.
Collins,A.R., Ai-guo,M. and Duthie,S.J. (1995) The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mutat. Res., 336, 69–77.[Web of Science][Medline]
Collins,A., Dusinska,M., Franklin,M., Somorovska,M., Petrovska,H., Duthie,S., Fillion,L., Panayiotidis,M., Raslova,K. and Vaughan,N. (1997) Comet assay in human biomonitoring studies: reliability, validation and applications. Environ. Mol. Mutagen., 30, 139–146.[Web of Science][Medline]
Dusinska,M. and Collins,A. (1996) Detection of oxidised purines and UV-induced photoproducts in DNA of single cells, by inclusion of lesion-specific enzymes in the comet assay. ATLA Altern. Lab. Anim., 24, 405–411.
Eastmond,D.A. and Tucker,J.D. (1989) Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody. Environ. Mol. Mutagen., 13, 34–43.[Web of Science][Medline]
Fairbairn,D.W., Olive,P.L. and O'Neill,K.L. (1995) The comet assay: a comprehensive review. Mutat. Res., 339, 37–59.[Web of Science][Medline]
Fenech,M. (1993) The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat. Res., 285, 35–44.[Web of Science][Medline]
Fenech,M. and Neville,S. (1992) Conversion of excision-repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes. Environ. Mol. Mutagen., 19, 27–36.[Web of Science][Medline]
Gedik,C.M. and Collins,A.R. (1991) The mode of action of 1-ß-D-arabinofuranosylcytosine in inhibiting DNA repair: new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells. Mutat. Res., 254, 231–237.[Web of Science][Medline]
Green,M.H.L., Lowe,J.E., Waugh,A.P.W., Aldridge,K.E., Cole,J. and Arlett,C.F. (1994) Effect of diet and vitamin C on DNA strand breakage in freshly-isolated human white blood cells. Mutat. Res., 316, 91–102.[Web of Science][Medline]
Hartmann,A., Plapper,U., Raddatz,K., Grunert-Fuchs,M. and Speit,G. (1994) Does physical activity induce DNA damage? Mutagenesis, 9, 269–272.
Hellman,B., Vaghef,H., Friis,L. and Edling,C. (1997) Alkaline single cell electrophoresis of DNA fragments in biomonitoring for genotoxicity: an introductory study on healthy human volunteers. Int. Arch. Occup. Environ. Health, 69, 185–192.[Web of Science][Medline]
Horvathova,E., Slamenova,D., Hlincikova,L., Mandal,T.K., Gabelova,A. and Collins,A.R. (1998) The nature and origin of DNA single-strand breaks determined with the comet assay. Mutat. Res., 409, 163–171.[Web of Science][Medline]
Kaina,B. and Aurich,O. (1985) Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister chromatid exchange induction. Mutat. Res., 149, 451–461.[Web of Science][Medline]
Kaina,B., Fritz,G. and Coquerelle,T. (1993) Contribution of O6-alkylguanine and N-alkylpurines to the formation of sister chromatid exchanges, chromosomal aberrations and gene mutations. Environ. Mol. Mutagen., 22, 283–292.[Web of Science][Medline]
Kishi,K. (1987) Effects of repair inhibition in the G1 phase of clastogen-treated human lymphocytes on the frequencies of chromosome-type and chromatid-type aberrations and sister-chromatid exchanges. Mutat. Res., 176, 105–116.[Web of Science][Medline]
Lamberti,L., Bigatti-Ponzetto,P. and Ardito,G. (1983) Cell kinetics and sister-chromatid exchange frequency in human lymphocytes. Mutat. Res., 120, 193–199.[Web of Science][Medline]
Loeb,L.A., Agarwal,S.S. and Woodside,M. (1968) Induction of DNA polymerase in human lymphocytes by phytohaemagglutinin. Proc. Natl Acad. Sci. USA, 61, 827–834.
McKelvey-Martin,V.J., Green,M.H.L., Schmezer,P., Pool-Zobel,B.L., De Meo,M.P. and Collins,A. (1993) The single cell gel electrophoresis assay (Comet assay): a European review. Mutat. Res., 288, 47–63.[Web of Science][Medline]
Miyamae,Y., Iwasaki,K., Kinae,N., Tsuda,S., Murakami,M., Tanaka,M. and Sasaki,Y.F. (1997) Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis (Comet) assay. 2. Relationship between DNA migration and alkaline condition. Mutat. Res., 393, 107–113.[Web of Science][Medline]
Mikita,T. and Beardsley,G.P. (1988) Functional consequences of the arabinosyl–cytosine structural lesion in DNA. Biochemistry, 27, 4698–4705.[Medline]
Moretti,M., Villarini,M., Scassellati-Sforzolini,G., Monarca,S., Libraro,M., Fatigoni,C., Donato,F., Leonardis,C. and Perego,L. (1996) Biological monitoring of genotoxic hazard in workers of the rubber industry. Environ Health Perspect., 104 (suppl. 3), 543–545.
Ostling,O. and Johanson,K.J. (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun., 123, 291–298.[Web of Science][Medline]
Perry,P. and Wolff,S. (1974) New Giemsa method for the differential staining of sister chromatids. Nature (Lond.), 251, 156–158.[Medline]
Pfuhler,S. and Wolf,H.U. (1996) Detection of DNA-crosslinking agents with the alkaline comet assay. Environ. Mol. Mutagen., 27, 196–201.[Web of Science][Medline]
Singh,N.P., McCoy,M.T., Tice,R.R. and Schneider,E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell, Res., 175, 184–191.[Web of Science][Medline]
Speit,G., Hanelt,S., Helbig,R., Seidel,A. and Hartmann,A. (1996) Detection of DNA effects in human cells with the comet assay and their relevance for mutagenesis. Toxicol. Lett., 88, 91–98.[Web of Science][Medline]
Sram,R.J., Podrazilova,K., Dejmek,J., Mrackova,G. and Pilcik,T. (1998) Single cell gel electrophoresis assay: sensitivity of peripheral white blood cells in human population studies. Mutagenesis, 13, 99–103.
Strauss,G.H.S. (1991) Non-random cell killing in cryopreservation: implications for performance of the battery of leucocyte tests (BLT) I. Toxic and immunotoxic effects. Mutat. Res., 252, 1–15.[Web of Science][Medline]
Surralles,J., Xamena,N., Creus,A. and Marcos,R. (1995) The suitability of the micronucleus assay in human lymphocytes as a new biomarker of excision repair. Mutat. Res., 342, 43–59.[Web of Science][Medline]
Van Goethem,F., Lison,D. and Kirsch-Volders,M. (1997) Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt-tungsten carbide. Mutat. Res., 392, 31–43.[Web of Science][Medline]
Vodicka,P., Bastlova,T., Vodickova,L., Peterkova,K., Lambert,B. and Hemminki,K. (1995) Biomarkers of styrene exposure in lamination workers: levels of O6-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes. Carcinogenesis, 16, 1473–1481.
Vrzoc,M. and Petras,M.L. (1997) Comparison of alkaline single cell gel (Comet) and peripheral blood micronucleus assays in detecting DNA damage caused by direct and indirect acting mutagens. Mutat. Res., 381, 31–40.[Web of Science][Medline]
Zhang,S., Huang,J., Chen,P. and Li,C. (1988) Sister chromatid exchange and cell cycle patterns of normal human bone marrow cells after in vitro exposure to cytostatic drugs. Cancer Genet. Cytogenet., 31, 157–163.[Web of Science][Medline]
Received on February 16, 1999; accepted on April 29, 1999.
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