Mutagenesis vol. 19 no. 4 pp. 313-318,
July 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Parallel evaluation of doxorubicin-induced genetic damage in human lymphocytes and sperm using the comet assay and spectral karyotyping
1Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK, 2GSF Research Center, Institute of Molecular Radiobiology, 85764 Neuherberg, Germany and 3School of Public Health, University of California in Berkeley, Berkeley, CA 94720, USA
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In recent years, two techniques for detecting genetic damage in the whole genome have gained importance: the alkaline comet assay, to detect DNA damage such as strand breaks and alkali-labile sites, and a multicolour FISH method, spectral karyotyping (SKY), to identify chromosomal aberrations simultaneously in all metaphase chromosomes. In the present study, the induction of DNA damage in human sperm and lymphocytes in vitro has been studied employing an anticancer drug, doxorubicin (DX). An increase in DNA damage was observed with the comet assay as the median per cent head DNA of sperm significantly decreased from 82.07 and 85.14% in the untreated control groups to 63.48 and 72.52% at doses of 0.8 µM DX. At 1.6 µM the percentage declined to 60.96% (the corresponding tail moment increased from 4.42 to 12.19). In stimulated lymphocytes, a significant increase was observed in tail moment, from 0.72 and 0.53 in controls to 15.17 and 12.10 at 0.2 µM DX, continuing at the same level to a final concentration of 1.6 µM. Structural aberrations found in the parallel SKY study in stimulated lymphocytes at 0.2 µM DX consisted of 14% chromatid-type and 2% chromosome-type aberrations; none were found in controls. The SKY results correlate very well with the findings of the comet assay in lymphocytes where DNA damage was observed at similar doses. This study is the first reporting use of the comet assay and SKY analysis in parallel after chemical treatment. The potential of the two techniques together is evident, as they represent a set of assays feasible for evaluating damage in human somatic and germ cells after chemical treatment (i) by direct observation of two different end-points, detecting general DNA damage and chromosomal aberrations and (ii) by extrapolation from lymphocytes to sperm, which provides a ‘parallelogram’ approach in human cells.
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Introduction |
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The importance of assessing genotoxicity in human somatic and germ cells will continue to have an important role in the future, particularly with concerns about the detrimental effects on reproduction in terms of male infertility and sperm count (Medical Research Council, 1995
). The single cell gel electrophoresis (comet) assay has already been extensively used to detect DNA damage in the whole genome in single cells as double- and single-strand breaks and alkali-labile sites (Singh et al., 1988; Anderson et al., 1998). However, a combination of the comet assay with a multicolour fluorescence in situ hybridization (FISH) method, spectral karyotyping (SKY), which detects chromosomal aberrations in all human metaphase chromosomes simultaneously (Schröck et al., 1996), allows a different type, yet detailed, assessment of induced damage in the whole genome.
In the comet assay, induced DNA damage is evaluated after single cell gel electrophoresis by measuring the tail moment as the product of per cent tail DNA multiplied by the tail length of the comet and the per cent head DNA. After alkaline lysis, damaged DNA originating from DNA strand breaks and alkali-labile sites thereby pass out of the nuclei moving towards the anode along the electrical field and form comet-like structures (Tice et al., 2000; Olive, 2002). In contrast, with SKY, specific chromosomal damage can be evaluated, for numerical and structural aberrations in all 46 human chromosomes simultaneously by employing FISH with DNA probes labelled with a combination of five different fluorochromes. Thus, each chromosome has a unique fluorescence spectrum, enabling computer software to classify all 24 different human chromosomes (22 pairs plus X and Y) efficiently. The threshold for detecting translocation fragments is 
10–20 Mb (Gray and Collins, 2000; Wang et al., 2002). In contrast to classical methods of chromosome aberration detection, where Giemsa banding (see for example Ronne, 1990) has mainly been used, SKY is able to detect hidden aberrations and uncharacterized marker chromosomes throughout the whole genome by evaluating all chromosomes from their distinct colour (Mohr et al., 2000).
When cancer has been diagnosed in a patient, various treatments using multiple combinations of chemotherapeutic agents are possible, depending on the type of cancer. Some of these anticancer agents specifically act on dividing cells during mitosis by inhibiting cell division (reviewed in Stewart et al., 2003), while others act non-specifically (reviewed in Blagosklonny, 2003). As a consequence, they cause apoptosis and/or necrosis, subsequently leading to cell death. Proliferating cells are more likely to die as a result of the damage induced, but cells surviving the exposure may carry mutations and chromosomal aberrations. To evaluate the combination of comet and SKY in human somatic and germ cells, doxorubicin (DX), a well-known anticancer drug, was employed. DX, formerly known as adriamycin, is an anthracycline antibiotic produced by the fungus Streptomyces peucetius. DX treatment at therapeutic doses leads to the induction of CD95-ligand (CD95-L). CD95-L can mediate cell death (apoptosis) by cross-linking the CD95 receptor (CD95) (Friesen et al., 1999). DX is also able to inhibit reverse transcriptase (Tomita and Kuwata, 1976) and RNA synthesis (Li and Yu, 1993). However, at the DNA level, three major mechanisms of action have been identified for DX (Cummings et al., 1991): binding DNA effectively by intercalation of the anthracycline portion; causing DNA damage via the production of free radicals from reactive oxygen species (ROS); stabilizing the topoisomerase II cleavage complex, which is critical for DNA function. As a consequence of these multiple effects, single- and double-strand breaks are introduced into the DNA (Cummings et al., 1991). It was shown that DX induces chromosome aberrations in murine bone marrow cells and in spermatocytes and that chromosome aberrations were sustained in the surviving spermatogonial cells (Au and Hsu, 1980). DX also increased the frequency of meiotic micronuclei in male rats (Laehdetie et al., 1983), which showed impaired fertility after being treated. Decreased weights of the genital organs, an extremely decreased number of sperm, low sperm motility, a low implantation rate and a decreased number of live foetuses were also observed in rats (Imahie et al., 1995).
The current study investigated a combined evaluation of genetic damage with the comet and SKY assays in somatic and germ cells after DX treatment of human lymphocytes and sperm. With the different end-points of both techniques, quantitative information on DNA damage in human lymphocytes and sperm (comet) and the precise number and structure of chromosomal aberrations in human lymphocytes (SKY) were available. Two questions were considered: (i) is it possible to use the results of the amount and type of damage found in lymphocytes in the comet and SKY assays as well as in sperm in the comet assay to extrapolate possible induced chromosomal aberrations in sperm, as chromosomes are normally not accessible in entirely differentiated human germ cells; (ii) is the set of two assays and a subsequent parallel evaluation feasible for the assessment of other DNA-damaging chemicals in humans in the future.
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Material and methods |
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Chemicals
Doxorubicin hydrochloride (CAS no. 25316-40-9), 98% pure, was obtained from Aldrich (Deisenhofen, Germany). A stock solution was prepared by dissolving 1 mg DX in 1 ml of 37°C RPMI medium (RPMI 1640 medium with Glutamax-I; Gibco Life Sciences, Karlsruhe, Germany) and adding 10% DMSO.
Sperm samples and treatment of sperm for the comet assay
The semen sample was obtained from a healthy donor (donor A, age 35, sperm no. 38 000 000 per ml, motility 60%, abnormalities 22%) and stored at –80°C. Approximately 10 000 sperm were mixed with RPMI (total volume 1000 µl) to form a cell suspension and incubated for 1 h at 37°C with various concentrations of DX (0.2, 0.4, 0.8 and 1.6 µM, respectively).
Blood samples, culture and treatment of lymphocytes for the comet and SKY assays
Blood was obtained from healthy volunteers (donor A, male, age 35 for comet; donor B, male, age 35, for SKY). As an anticoagulant, 500 µl of heparin (5000 U/ml) was present in 20 ml of whole blood. Heparinized whole blood (0.5 ml each sample) was then added to plastic flasks (25 cm2) containing 3.7 ml of RPMI 1640 medium with Glutamax (Gibco), 750 µl of foetal bovine serum (FBS) (Sigma), 3.7 µl of penicillin (200 000 U/ml) (Gruenenthal, Aachen, Germany) and 18.7 µl of streptomycin (200 mg/ml) (Gruenenthal). The culture medium was supplemented with 130 µl of phytohaemagglutinin-M (PHA-M) (10 mg/ml) (Gibco) to initiate proliferation of T lymphocytes and 40 µl of of 5-bromo-2'-deoxyuridine (BrdU) (0.383 mg/ml) (Serva, Heidelberg, Germany) for cell cycle control. Cultures were incubated for 48 h at 37°C in 5% CO2 in air. For SKY, 48 h after starting the blood culture (10 culture flasks), 20 µl of DX stock solution was added to a 5 ml culture volume within the blood culture flask to give final concentrations of 0.2 and 0.4 µM. For treatment of cycling cells, a further incubation at 37°C with 5% CO2 for 20 h followed (post-treatment). For the comet assay, DX was added to the blood culture flasks (27 culture flasks in total) to yield final concentrations of 0.2, 0.4, 0.8 and 1.6 µM, respectively. In both cases, cells were arrested in metaphase by adding 75 µl of colcemid (10 µg/ml) (Sigma) 3 h before the end of the post-treatment period or culture time, respectively.
Cell viability for lymphocytes and sperm
Cell viability was measured by trypan blue exclusion (Pool-Zobel et al., 1992). It was generally >90%, but always >75%. Cultured and separated lymphocytes were evaluated for cell viability directly after treatment, whereas sperm were assessed after thawing from frozen stock and treatment. Therefore, sperm were checked for functional integrity of their plasma membrane. Trypan blue uptake only takes place after breakdown of the cellular permeability barrier accompanying cell death. Hence, trypan blue is a vital exclusion stain to mark dead cells, which have lost their membrane integrity (Krause et al., 1984; Vigano et al., 1990).
Lymphocyte separation for the comet assay
After culture and treatment, 5 ml of cultured blood was layered on top of 3 ml of Lymphoprep (Axis-Shield, Oslo, Norway) in pointed tubes followed by centrifugation for 20 min at 540 g. Lymphoprep mainly separates lymphocytes and monocytes from granulocytes and erythrocytes. Platelets, however, are removed by the subsequent washing steps. The lymphocyte layer on top of the Lymphoprep layer was transferred to a further tube. Lymphocytes were washed twice with 10 ml of prewarmed (37°C) Hank’s balanced salt solution (HBSS) (Gibco); each wash was followed by a centrifugation at 420 g for 10 min. Finally, the supernatant was removed as carefully as possible without disturbing the pellet. An aliquot of 1 ml of RPMI 1640 with Glutamax (Gibco) was added and the pellet was resuspended.
Metaphase preparation for SKY
After culture and treatment, the cultures were transferred into pointed tubes and washed twice with HBSS at 37°C (Gibco). In order to discard the supernatant, the cell suspension was centrifuged at 190 g for 10 min for each washing step. The subsequent hypotonic treatment was carried out with 1 part HBSS plus 3 parts Millipore water for 7 min at 37°C. After centrifugation for 8 min and removal of the supernatant, the cells were fixed in 3:1 methanol/acetic acid (both from Merck, Darmstadt, Germany). Chromosome preparations were obtained by pipetting 25 µl of cell suspension onto water-covered glass slides, which were then air dried. Ageing for a minimum of 1 week at 37°C in a dry incubator followed. Slides were stored at –20°C until use.
Comet assay
DNA strand breaks were measured in the alkaline comet assay using the method described by Anderson et al. (1997a) and Anderson and Plewa (1998) and reviewed by Tice (2000). In brief, each frosted microscope slide (2 slides/culture) was covered with a basic layer of 1% normal melting point agarose (Invitrogen). The slides were dried in a 60°C cabinet overnight and then stored at room temperature. An aliquot of 100 µl of lymphocyte or sperm suspension was mixed with 100 µl of 1% low melting point agarose (Invitrogen) in Eppendorf tubes. Of this suspension, 100 µl each were pipetted on two agarose-coated slides. Slides were covered with coverslips and solidified on ice. After removing the coverslip a third layer of 0.5% low melting point agarose was added, spread using a coverslip and again allowed to solidify on ice for 5 min. The slides were immersed in lysis solution [2.5 M sodium chloride, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, pH set to 10 with NaOH pellets (all five chemicals from Sigma), 10% DMSO (BDH)]. As sperm are resistant to standard lysis solutions, 0.05 mg/ml proteinase K (Sigma) was added (Hughes et al., 1997), followed by incubation at 37°C overnight. The slides were subjected to electrophoresis in a pH 13.5 electrophoresis buffer (1 mM EDTA and 300 mM NaOH). A 20 min incubation prior to electrophoresis allowed the DNA to unwind so that alkali-labile damage could be expressed. Electrophoresis was performed at 4°C for 20 min at 24 V. The amperage should then be constant at 300 mA. Tris buffer (0.4 M, pH 7.5) (Sigma) was used for 5 min to neutralize the alkali buffer then 50 µl of ethidium bromide (20 µg/ml) (Sigma) was added. Coverslips were added to the slides and then analysed within 3 h. Slides were examined at 400x magnification on a fluorescent microscope (Leica, UK) equipped with a BP546/10 excitation filter and a 590 nm barrier filter. From each replicate slide, 25 nuclei were scored (50 nuclei in total per dose). A computerized image analysis system (comet 4.0; Kinetic Imaging, Liverpool, UK) was used. In the comet assay, tail moments and per cent head DNA were measured for lymphocytes and sperm (Anderson et al., 1997a; Anderson and Plewa, 1998).
SKY
The human metaphase spreads on glass slides were incubated for 2.5 min in a pepsin solution [1% pepsin (Sigma) in 10 mM HCl] and for 4 min in 1% formaldehyde in 1x phosphate-buffered saline (PBS), 50 mM MgCl2. Slides were then dehydrated in an ethanol (Merck) series (70, 85 and 100%) and were air dried before being used for SKY. The commercial probe set (Applied Spectral Imaging, Migdal HaEmek, Israel) was denatured according to the company’s protocol. In parallel, the metaphase DNA was denatured in 70% formamide (Sigma) in 2x SSC for 2 min 50 s at 72°C. After dehydration in an ethanol series (cold 70, 85 and 100%) the slides were air dried and the probe set was applied to the metaphase spreads. Hybridization was carried out for 48 h in a moist box at 37°C in the dark. Washing steps and staining with infrared fluorochromes was carried out according to the protocol of ASI. Chromatin was counterstained with DAPI (Sigma) and slides were finally coverslipped with Vectashield, a commercial anti-fading solution (Linaris, Wertheim, Germany). Only first mitotic metaphases of stimulated T lymphocytes were analysed, identified using a cell cycle control assay, where BrdU was incorporated into the DNA during S phase. The BrdU-substituted DNA strands were subject to UV degradation, which led to a different staining pattern of metaphases after one, two or three cell cycles (Tice et al., 1976). Fifty first mitosis metaphases were evaluated in the control and treated groups, respectively.
Statistical analysis
The per cent head DNA and the tail moment values in the comet assay are not normally distributed and violate the requirements for analysis by parametric statistics. Thus, pair-wise comparisons of the treatment group versus the control group were conducted for all doses using a Mann–Whitney U-test (Anderson et al., 2003). Statistics were conducted on median values.
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Results |
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The responses of human sperm to DX in the comet assay (per cent head DNA and tail moments) are shown in Table I. A significant decrease in the median per cent head DNA of sperm nuclei from 82.07 (study 1) and 85.14% (study 2) in the untreated control groups to 63.48 (study 1) and 72.52% (study 2), respectively, was observed when treated with 0.8 µM DX. Additionally, at 1.6 µM in the second study the percentage declined further to 60.96%. The corresponding median tail moments increased from 5.38 to 9.40 (study 1, at 0.8 µM) and from 4.42 to 12.19 (study 2, at 1.6 µM). When lymphocytes were treated, significant increases in tail moment of the nuclei were seen (Table II). In contrast to a decrease in per cent head DNA, the tail moment increased relative to the DNA damage. The median tail moments in this study significantly increased from 0.72 (study 1) and 0.53 (study 2) in the untreated control groups to 15.17 (study 1) and 12.10 (study 2) when lymphocytes were exposed to 0.2 µM DX. This significant increase continued to stay at approximately the same level in studies 1 and 2 to a final concentration of 1.6 µM DX (Table II). The same significant DX-induced induction of DNA damage can also be seen when the per cent head DNA was considered, as a decline from 96.11 to 55.30% (study 1) and 97.96 to 59.06% (study 2) was observed.
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In a parallel SKY experiment, structural aberrations found at a dose of 0.2 µM consisted of 14% chromatid aberrations and 2% chromosome aberrations (Table III). Two groups of chromatid aberrations were observed, chromatid breaks (8%) and chromatid exchanges (6%). Both of them resulted from DNA damage induced during the S or G2 phase of the cell cycle. Figure 1 shows seven observed aberrations found after treatment with 0.2 µM DX. Only one chromosome aberration was found: a dicentric chromosome (Figure 1a), which originated from two chromosome breaks during G1/G0-phase of the cell cycles. In Figure 1b and c, two chromatid breaks can be seen. Figure 1d shows an aberration, which could originate from either an isochromatid break or from a terminal deletion. In both cases, the aberrations appear to be similar, however, the mechanisms forming them are different. Figure 1e–g displays chromatid exchanges forming tri-radial and quadri-radial structures. One or even more chromosomes were missing in 14 (28%) of the assessed metaphases. In the control group no chromosome- or chromatid-type aberrations were found, since no colour junctions were observed within the chromosomes, and only three (6%) hypodiploid metaphases were seen. For the treatment with 0.4 µM DX, hardly any metaphases were found on the slides, assuming that there was very little or no stimulation and hence proliferation of T lymphocytes which might be due to cytotoxic effects of DX.
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Discussion |
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DX was the chemical of choice for the induction of DNA damage in human lymphocytes and sperm as it is well known that it interacts with DNA by inducing lesions in a way that is associated with the production of ROS (Cunningham et al., 1984
; Sikic, 1986; Cummings, 1991; Akman et al., 1992). Additionally, DX is thought to interact in a cell cycle-specific manner with nuclear topoisomerase II, essentially affecting DNA function (Cummings, 1991; Wassermann, 1996). The induction of DNA breaks and aberrations by DX was evaluated for the first time with a combination of two techniques, the comet assay and SKY, covering two end-points: firstly, to detect the amount of DNA damage in the whole genome in sperm as well as in lymphocytes; secondly, to assess in detail specific chromosomal aberrations in all metaphase chromosomes of the whole genome in lymphocytes.
For the comet assay, fully mature sperm (thawed aliquot from a frozen semen stock) were treated with DX for 1 h at 37°C in vitro. Sperm nuclei showed a significant decrease in per cent head DNA after being exposed to doses of 0.8 and 1.6 µM DX (Table I). This implies that the damage to the sperm DNA was possibly introduced by generating oxidative free radicals in close vicinity to the target DNA. Mature sperm are incapable of repairing DNA damage as repair is only carried out up to the stage of early spermatids (Sega et al., 1990). DX also showed positive responses in lymphocytes in the comet assay. Stimulated lymphocytes were treated immediately after 48 h culture with various doses of DX for 20 h at 37°C and 5% CO2. Significant increases in tail moments were already observed with the lowest DX dose of 0.2 µM. Significance prevailed at about the same level up to a dose of 1.6 µM (Table II). These positive results are consistent with the results of Anderson et al. (1997b), where previously significantly increased frequencies of DNA damage and chromosome aberrations were observed in human lymphocytes using the comet assay and FISH of a whole chromosome paint probe for chromosome 1.
The initial damage in untreated sperm was almost eight times higher than that of untreated lymphocytes, which was expected according to Anderson et al. (1997a). Because of the high levels of background damage in sperm (15–30%), per cent head DNA is more appropriate for statistical analysis (Hughes et al., 1997) and, therefore, in addition to tail moment, was used as an indicator of induced strand breaks in sperm. Singh et al. (1989) observed a large number of DNA breaks under alkaline conditions using the comet assay on healthy untreated human sperm, however, this was not the case with lymphocytes. Most of the background damage was considered to be alkali-labile sites, which do not represent pre-existing single-strand breaks, suggesting that they represent a functional characteristic of condensed chromatin rather than DNA damage (Singh et al., 1989; McKelvey-Martin et al., 1997). In Figure 2 the data for sperm and lymphocytes (Tables I and II) are compared considering the median tail moment data for lymphocytes and the corresponding median tail moment data for sperm, respectively, for both studies. The plateau effect for lymphocytes and the dose–response characteristics for sperm can be clearly seen. At a dose of 1.6 µM DX the tail moments of both cell types seem to be at a similar level, 12.19 in sperm versus 11.81 and 14.19 in lymphocytes. In a multi-drug in vivo treatment for lymphomas, for instance, cells of an average patient are internally exposed to 2–20 µM DX (after administering 90 mg i.v.). Compared with our in vitro treatment with 0.2–1.6 µM DX, lymphocytes seem to be exposed to a dose 10 times less than in an in vivo treatment and already show significant DNA damage in lymphocytes.
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Our results indicate that sperm reach the significance threshold with higher concentrations of DX. This may be due to the fact that they are not in any cell cycle phase and that they are structurally different, since they have protamine-packed chromatin. In contrast to somatic cells, sperm chromatin is densely packed utilizing protamines (reviewed in Getzenberg et al., 2001
). Lymphocytes, however, seem to be more susceptible to DX during the cell cycle as DX not only produces DNA damage via free radicals but also by inhibiting the topoisomerase II complex affecting DNA function per se (Cummings et al., 1991). In combination with the comet assay, SKY was used with lymphocytes. Our findings show that almost all of the aberrations found by SKY in metaphases are chromatid-type aberrations originating from damage induced during the S and G2 phases of mitosis. In particular, three of the seven chromatid-type aberrations were formed after induction of double-strand breaks within the involved chromosomes, i.e. isochromatid breaks (Figure 1d) and complete symmetrical chromatid exchange (Figure 1g). The other two chromatid exchanges arose from both single- and double-strand breaks (Figure 1e and f). One chromosome-type aberration, a dicentric chromosome, was found, which originated from two double-strand breaks within two chromosomes. These data correspond to the results of Kusyk and Hsu (1976) and Anderson et al. (1997b), that mainly chromatid-type aberrations are found after treatment with DX. Chromosome 6 was involved in four aberrations, albeit of different origin, dic(4;6)(q31;q25) (Figure 1a), +der(6) and del(6)(p22;q14) (not shown), chte(X;6)(p22;q16) (Figure 1e) and chte(6;6)(p21;p21) (Figure 1g), however, there seem to be no hot-spots, since in all aberrations different bands were involved. The relatively high number of aneuploid metaphases (28% hypodiploid, 4% hyperdiploid) may be due to DX-induced damage of the spindle apparatus, resulting in non-disjunction and subsequent chromosome loss (Laehdetie et al., 1983). However, the scoring was carried out on first post-treatment mitoses, therefore damage to the spindle might occur and individual chromosomes were not properly aligned at the metaphase plate. This could contribute to a greater loss of chromosomes during metaphase preparation. Non-specific artefacts during metaphase preparation may also contribute to the observed percentage of hypodiploid metaphases as the 6% observed in the control group may have had this origin.
The comet assay as a tool for genotoxic studies (Marzin, 1999; Tice et al., 2000) and a method of sensitive and rapid detection of DNA strand breaks in individual cells (Fairbairn et al., 1995) yields reliable results for induced DNA damage in a very short time. Our results demonstrate that the comet assay is useable for the investigation of DNA damage induction in human sperm and lymphocytes. In combination with SKY (Schröck et al., 1996), induced DNA damage can be characterized in detail by means of chromosomal aberrations, as damage to the whole genome concerning all 46 chromosomes can be simultaneously analysed for abnormalities. Both assays combined provide a useful system for genotoxicity evaluation of chemicals in humans. In addition, when using this combination of methods it is possible to extend our knowledge gained from results on diploid lymphocytes and haploid sperm to possible chromosomal aberrations in sperm, as haploid metaphase-like chromosomes are normally not accessible in humans. Thus, diploid chromosomes could serve as a surrogate for non-accessible sperm chromosomes. Although blood from a different donor was used for SKY, which increases the inter-individual variability, SKY proved to assess DX-induced chromosomal aberrations very accurately. In this case no individual correlation can be made, however, it clearly showed the potential to extrapolate from lymphocytes to germ cells. Normally, responses in sperm and lymphocytes occur at the same doses in a dose-related manner in non-cycling cells (Anderson et al., 1997a,b), but in the present study, cycling cells were only used in the lymphocyte comet assay, which might explain why there appears to be a difference in sensitivity with the two cell types. This might also explain the plateau effect observed with doses of 0.2–1.6 µM. Cycling cells were also used to produce parallel results with the SKY assay.
In conclusion, the alkaline comet assay has clearly realized its potential to reproducibly detect single-strand DNA breaks and alkali-labile sites within human sperm and lymphocytes. In proliferating cells DNA strand breaks can consecutively lead to chromatid breaks and thus to chromosomal aberrations, which can be identified simultaneously in all chromosomes by SKY. This provides evidence of the original damage and its downstream appearance within the cell, if cycling cells are used. For certain objectives, the information obtained with the comet assay may not be enough to evaluate chemicals in detail. The solution to this problem may be the combination of both techniques, the comet assay and SKY, to provide damage evaluation for two different end-points at the level of the whole genome in terms of DNA and cytogenetic damage. Information obtained might also be used for extrapolation to human germ cells since it might well be assumed that chromosome damage of a similar type could occur in human sperm over the same dose range. It has been shown previously that damage induced in sperm and lymphocytes in the comet assay occurs in a 1:1 or 1:2 ratio (Anderson et al., 1997a,c). Considering future experiments, a group of subjects should be included as well as other anticancer or DNA-damaging chemicals, like oestrogenic compounds. Such an approach will be extremely valuable not only with respect to mutagenesis and genotoxicity, but could also be of clinical relevance for finding less harmful but more effective compounds.
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Acknowledgements |
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A.B. is a EU Marie-Curie Research Fellow. This research has been supported by a Marie Curie Fellowship of the European Community programme FP5 under contract number QLG4-CT-2002-51611. T.E.S. was a Wellcome Trust Travelling Research Fellow, grant reference no. 062288. E.C. was a Leonardo da Vinci scholar funded by CAEB of the Balearic Islands.
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Notes |
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4To whom correspondence should be addressed. Tel: +44 1274 23 3569; Fax: +44 1274 30 9742; Email: d.anderson1{at}bradford.ac.uk
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References |
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