Mutagenesis, Vol. 18, No. 2, 159-166,
March 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Statistics of the Comet assay: a key to discriminate between genotoxic effects
1 Institut de Pharmacie, Laboratoire de Chimie Bioanalytique, de Toxicologie et de Chimie Physique Appliquée, CP 205/1 and 2 Institut de Pharmacie, Laboratoire de Biochimie Médicale, CP 205/3, Université Libre de Bruxelles, Boulevard du Triomphe, 1050 Bruxelles, Belgium
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Abstract |
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The alkaline Comet assay is a widely used single cell gel electrophoresis technique for the quantification of DNA strand breaks, crosslinks and alkali-labile sites induced by a series of physical and chemical agents. DNA migration in an electric field, supposed proportional to strand breakage, is a proposed estimation of genotoxicity. Breaks are quantified from geometric and fluorescence measurements by image analysis of comet-shaped DNA, often reported parameters being tail DNA and tail moment. Although a variety of statistical approaches have been used in the literature, most of these do not take into account the distribution patterns of comet data. In order to investigate a methodology for statistically demonstrating a comet effect, two different experiments, a reproducibility study and a trend analysis, were undertaken on a murine lymphoma cell line (P388D1) photodynamically stressed after induction of porphyrins with
-aminolaevulinic acid. This treatment results in significant heterogeneity of DNA damage, producing values ranging from 0 to 100% tail DNA in the same sample. The comparison of distribution curves for stressed and non-stressed samples shows that none of the application conditions are verified, either for parametric tests (which require normal distributions), or non-parametric tests (which assume essentially similar distributions). Meaningful statistics (median and 75th percentile) were consequently extracted from repeated experiments and found suitable for comparing stress conditions in an ANOVA and in a trend analysis; the 75th percentile is theoretically more sensitive but tends to more rapidly saturate at extensive stress levels. We conclude that a trend analysis of median comet metrics from repeated experiments at different stress levels is certainly an efficient way to statistically demonstrate a genotoxic effect. Whether the considered comet parameter is tail DNA or tail moment had no influence on the conclusions of our experiments, which were carried up to stress levels leading to a median 70% tail DNA. |
Introduction |
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The Comet assay is a short-term genotoxicity test widely used to reveal a broad spectrum of DNA-damaging agents capable of inducing strand breakage, crosslinks and alkali-labile sites (Singh et al., 1988
; Fairbairn et al., 1995; Anderson,D. et al., 1998; Rojas et al., 1999). This technique has been applied in genotoxicity studies (Fairbairn et al., 1995; Anderson,D. et al., 1998; Marzin, 1999; Rojas et al., 1999; Sasaki et al., 1999), in ecotoxicology (Belpaeme et al., 1998; Cotelle and Ferard, 1999), in biomonitoring (Collins et al., 1997; Kassie et al., 2000; Moller et al., 2000) and in clinical radiobiology (Olive et al., 1990, 1998; Olive, 1999). Its versatility has allowed the investigation of repair mechanisms (Speit and Hartmann, 1995; Alapetite et al., 1997), the detection of apoptosis (Fesus et al., 1991) and the study of alkylating (Monteith and Vanstone, 1995), oxidizing (Gedik et al., 1998) and crosslinking (Pfuhler and Wolf, 1996; Merk and Speit, 1999) agents. A recent review details the many compounds investigated by the Comet assay, which notably include metals, pesticides, opiates, nitrosamines and anticancer drugs (Rojas et al., 1999).
The principle of the test is remarkably simple. The cells are embedded in agarose and lysed, leaving unbroken DNA in a supercoiled state; strand breaks relaxing the supercoiling are revealed on electrophoresis, free loops of DNA extending to the anode to form a comet-shaped structure. These comets can be either classified by visual examination (visual scoring) (Gedik et al., 1992; Anderson,D. et al., 1994) or measured from morphological parameters obtained by image analysis and integration of intensity profiles (Olive et al., 1990; Kent et al., 1995; Bocker et al., 1997; Olive, 1999). Computed parameters include the comet tail length, the proportion of DNA in the comet tail (tail DNA) and derived parameters (moments) intended to combine information from both tail length and tail DNA (Olive et al., 1990; Ashby et al., 1995; Hellman et al., 1995; Kent et al., 1995). Calibration experiments made with ionizing radiation have allowed visual scores and tail DNA to be linearly related to the number of DNA break (Collins, 2002).
Computer-assisted measurements at the cell level are unavoidably associated with a large amount of more or less dispersed data for which no really satisfying statistical treatment has been proposed (Lovell et al., 1999). On the one hand, some studies tend to consider the comet and not the genotoxic treatment as the comparison unit (Lovell et al., 1999). On the other hand, the proposed statistics may not be appropriate to the data distribution patterns; parametric tests indeed assume Gaussian distributions, whereas non-parametric tests require essentially similar distribution curve shapes (Zar, 1996; Conover, 1999; Statsoft Inc., 1999). The present work investigates whether these conditions are verified in the Comet assay, using data obtained on a murine leukaemia cell line (P388D1) photodynamically stressed after porphyrin induction. Two models are then proposed for the statistical interpretation of results.
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Materials and methods |
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All the work was performed in darkened rooms.
Chemicals
The agaroses, ethidium bromide, phosphate-buffered saline (PBS), cell culture media and reagents were obtained from Invitrogen (Paisley, UK). Tromethamine (Tris) and -aminolaevulinic acid hydrochloride (-ala) were from Sigma-Aldrich (St Louis, MO). Sodium chloride, sodium hydroxide and Triton X-100 were from Merck (Darmstadt, Germany), EDTANa2H2 from Acros Organics (Geel, Belgium) and dimethyl sulphoxide from VWR International (Leuven, Belgium). Unfrosted microscope slides (26x76 mm) were pre-coated by dipping in a 1% solution of normal melting point agarose and dried overnight at room temperature.
Cell culture and irradiation
Murine P388D1 leukaemia cells (ATCC CCL-46) were maintained as a cell suspension in logarithmic growth at 37°C in a humidified atmosphere of 5% CO2 in air, in RPMI 1640 medium supplemented with HEPES (10 mM), penicillin (100 U/ml), streptomycin (100 U/ml) and 9% foetal bovine serum (South-American origin). One day before the experiment, cells were harvested by centrifugation (900 g, 2 min) and suspended at a density of 5x106 cells in 6 ml of fresh medium in 25 cm2 aerated culture flasks. After a 22–26 h stay in the CO2 incubator, 100 µl of a fresh solution of -ala was added up to 300 µM and the flask was maintained in the incubator for 3 h. The stopper was hermetically closed and the flask exposed for 5–60 min to the light from either a typical UVA tanning lamp (total output of an array of four tanning tubes, average fluence rate at the cell level 0.31 mW/cm2; Philips Cleo 15 W) or visible light (total output of three spots, average fluence rate at the cell level 11.9 mW/cm2; Philips Spotone R95, 75 W). The spectra of the lamps, specifications of the photometer (thermopile) and transmission properties of the polyethylene flasks were reported earlier (Duez et al., 2001). During irradiation, the flask temperature was regulated at 4°C with the help of a circulating water bath. Sham controls were prepared in exactly the same way, but without light exposure.
Fluorescence spectroscopy and microscopy (Duez et al., 2001) confirmed that upon -ala incubation (300 µM, 3 h), porphyrins are effectively induced in P388D1 cells; under these conditions, a 30 min photodynamic treatment allows the survival of >80% of the cells for UVA and 100% for visible light (Duez et al., 2001).
Comet assay
The highly alkaline Comet assay introduced by Singh (Singh et al., 1988) was performed with some modifications (Klaude et al., 1996). Immediately after irradiation, the cells were isolated (centrifugation at 900 g, 2 min), washed and resuspended in cold PBS at a density of 
2.5x106 cells/ml. Aliquots of 15 µl of this suspension were mixed with 300 µl of low melting point agarose solution (0.8% in PBS, 37°C), cast on pre-coated slides and laid on ice for 5 min. After overnight lysis at 4°C in a mixture of 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10.0:DMSO:Triton X-100 (89:10:1), the slides were washed twice with the electrophoresis buffer (300 mM NaOH, 1 mM EDTANa2H2, pH > 13), incubated for 40 min and electrophoresed (18°C, 20 min, 0.7 V/cm, 300 mA, 24 slides/run) in a Gibco Horizon 20-25 tank (Invitrogen) connected to a Power Pac 200 unit (Bio-Rad, Hecules, CA). The slides were then washed three times with cold 0.4 M Tris, fixed in –20°C absolute ethanol and dried. After rehydration (100 µl water), staining (200 µl ethidium bromide, 20 µg/ml, 10 min) and washing, comet-shaped structures were measured by epifluorescence microscopy (Axiovert S100 TV; Zeiss, Jena, Germany) using the Komet® 4.0 software (Kinetic Imaging, Liverpool, UK) connected to an Orca-II cooled CCD camera (12 bits, –50°C) (Hamamatsu, Japan). The comet parameters considered in this study were tail DNA and Olive tail moment, the product of tail DNA and the distance between the head and tail centres of gravity.
Design of the study
To investigate a methodology for statistically demonstrating a comet effect, two different experiments were undertaken, a reproducibility study and a trend analysis.
In the reproducibility study, nine cell treatments (two controls and seven different photodynamic stresses) were performed on independent samples and analysed in triplicate electrophoresis runs, two slides replicating each treatment in each electrophoresis. Fifty comets were measured on each slide. This design (9 treatmentsx2 slidesx3 electrophoresis runsx50 comets) allowed distributions to be compared, representative statistics (quartiles) to be computed and discrimination between treatments by taking into account the between-electrophoreses reproducibility.
A trend analysis allowed dose–effect relationships (DNA damage versus dose of light) to be investigated and the curves obtained with and without porphyrin induction to be compared. Each treatment (four doses of visible light and UVA, 100 comets measured on two slides) were duplicated in independent experiments and all slides were randomly assigned to electrophoresis runs after a maximum of 2 days in the lysis solution.
Statistical treatment
The normality of distributions was visually appreciated from normal probability plots and objectively evaluated by the Shapiro–Wilk W-test (Shapiro and Wilk, 1965) using the Analyse-it software (Analyse-It Software Ltd, Leeds, UK, http://www.analyse-it.com). Non-parametric comparisons of samples were by the Kruskal–Wallis test (Conover, 1999). Quartiles with their confidence intervals were computed from each distribution according to Aczel (1993) and Conover (1999) and analysed by ANOVA, t-test or regression analysis with the help of the Systat 7.0 software (Systat Software, http://www.systat.com). The variance components, computed according to R.L.Anderson and Bancroft (1952) and Kleinbaum and Kupper (1978), allowed the total precision of the comet measurements to be estimated (Anderson,R.L. and Bancroft, 1952).
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Results |
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Reproducibility study
Normality of comet distributions The Olive tail moment and tail DNA for all the comets obtained in the different treatments and electrophoreses are presented in Figure 1
. Normal probability plots, confirmed by the formal Shapiro–Wilk W-test (Table I), show that these data distributions are generally non-Gaussian (24 of 27), even after logarithmic transformation, which precludes the use of parametric tests.
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Non-parametric tests Accordingly, a comparison of samples was undertaken using the Kruskal–Wallis test, a non-parametric ANOVA based on ranks transformation (Conover, 1999
). Although this method is distribution-free, it theoretically requires similar dispersions and shapes for the compared distributions, a criterion more or less stringent according to the author (Zar, 1996; Conover, 1999). As each sample presents essentially similar distributions across electrophoreses, this test was applied, in a first step, to the sample-by-sample comparison of electrophoreses; the probabilities for these Kruskal–Wallis T statistics are presented in Figure 1
. A close examination of this figure shows that whenever this test demonstrates significant variations between electrophoreses (P < 0.05), these differences are objectively insignificant when compared with the differences between treatments. Our attempts to estimate the within-electrophoresis reproducibility (Mann–Whitney test for each pair of duplicate slides) met with the same problem. These tests are clearly overly sensitive, which is most probably due to the high number of comets compared (nx100). Such classical non-parametric approaches are then unlikely to yield any useful information in comparing treatments and should certainly not be applied to demonstrate a genotoxic effect.
Reduction to statistics
As proposed by Lovell et al. (1999), the unit of comparison should be the treatment and not the comet; each series of measures has then been reduced to a statistic representative of the comet distribution. As a genotoxic stress is usually indicated by an increase in both median values and data dispersions, the median and 75th percentile were retained to characterize each distribution. These statistics notably present a low sensitivity to the few extreme values that can be encountered in comet populations (the so-called ‘apoptotic cells’). Intuitively, if n is sufficiently large (>20), these quartiles should be a reasonably correct approximation of the quartiles of the population and their distribution should approach the normal distribution. This intuition is comforted by the method applied to compute their confidence intervals (Conover, 1999) and has been experimentally verified for a series of samples (data not shown). Methods derived from the general linear model, including the ANOVA, can then be applied to estimate the uncertainties associated with the different sources of variability (Lovell et al., 1999).
For each treatment, the computed quartiles were compared by a 2-way ANOVA, considering the slide as the elementary unit of repetition and two random factors, electrophoresis and treatment. A post-hoc t-test was then performed to find the differences between treatments.
Tables II and III present the variance analysis and post-hoc t-tests for, respectively, the medians and 75th percentiles of tail DNA distributions. This analysis demonstrates a statistically significant difference between treatments, no difference between electrophoreses and a significant interaction treatmentxelectrophoresis (P = 0.025 for medians; P = 0.002 for 75th percentiles). Similar results were obtained for the Olive tail moment distributions (data not shown) but for an absence of interaction, which could indicate that this comet parameter depends less on experimental conditions than the tail DNA. Another possibility is that the interaction component disappears because of the lower precision of Olive tail moment statistics (total CV % = 18% for medians and 15% for 75th percentiles), which probably arises from the combination of a second parameter (tail length) in measurements.
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For all quartiles and comet parameters, the post-hoc pairwise comparisons indicate that there is no difference between the treatments control 1, control 2,
-ala and visible light 30 min. All the other treatments differ from the controls and from each other, except for the pairs UVA 30 min versus -ala + visible light 30 min and UVA 30 min versus -ala + visible light 60 min; these latter two comparisons depend on the quartile and comet parameter under consideration, which reflects very similar comet distributions (Figure 1). In the present study, this finding is purely anecdotal as these comparisons make no sense.
Trend analysis
In order to investigate the dose–effect relationships DNA damage versus dose of light, the median and 75th percentile of tail DNA were computed for each comet distribution and plotted as a function of light dose for both UVA and visible light (Figure 2). Similar graphs were obtained for Olive tail moments (data not shown). Table IV presents the slope significance F-test for each regression line and compares the slopes obtained with and without -ala treatment.
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The visible light does not induce any DNA damage in the absence of porphyrins; the determination coefficients and tests for slope significance are largely non-significant (P >> 0.05). Upon porphyrin induction, a significant relationship appears between DNA damage and light dose for both quartiles and comet parameters (P < 0.05). The UVA lamp by itself induced DNA strand breakage in a dose-dependent manner; upon porphyrin induction, the medians show a higher DNA fragmentation with a significant slope increase. Although the 75th percentiles demonstrate a slight similar trend, the slope is not statistically significant, which is probably due to a certain saturation of this statistic at higher break levels.
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Discussion |
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The choice of the most suitable computerized comet parameter is still an issue of active debate and no consensus has been reached (Tice et al., 2000
). We have consequently investigated two often used parameters, the tail DNA and the Olive tail moment, an expression of tail moment little influenced by the difficult to measure tail length parameter. A general recommendation consists in measuring 50–100 comets on between one and three slides (Lovell et al., 1999; Tice et al., 2000), which rapidly generates huge amounts of data. The problem has received much attention (Lovell et al., 1999; Albertini et al., 2000; Tice et al., 2000), but many authors diverge on the methods used for the statistical treatment of these data. Comet samples have notably been compared: (i) by a 
2 test applied after visual classification of tailed and untailed comets (Miyamae et al., 1997), a test unfortunately sensitive to extreme values (Lovell et al., 1999); (ii) by comparing the coefficients of dispersion of the comet populations (the ratio between the range of values and the standard deviation) (Vijayalaxmi et al., 1992), a statistic likewise distorted by extreme values (Lovell et al., 1999); (iii) by parametric tests (t-test or ANOVA), either without considering the non-Gaussian character of distributions (Holz et al., 1995) or after logarithmic transformation of the data (Betti et al., 1994; Churg et al., 1995; Wojewodzka et al., 1998) or only upon checking the eventual normality of distributions (Belpaeme et al., 1998; De Boeck et al., 2000); (iv) by non-parametric tests, including the Mann–Whitney (Anderson,D. et al., 1997; Lehmann et al., 1998), Kolmogorov–Smirnov (Belpaeme et al., 1998; Moretti et al., 1998) and Kruskal–Wallis (Anderson,D. et al., 1997; De Boeck et al., 2000) tests; the Kolmogorov–Smirnov test has also been applied to pooled data from several experiments (Bergqvist et al., 1998), resulting in very high numbers of points which may distort the statistical treatment. Mathematical modelling techniques have assimilated the comet distributions either: (i) to a sum of two normal distributions, allowing the distance between the means to be estimated and compared (Olive and Durand, 1992); (ii) to the 2 distribution, a function with a single parameter used to characterize and compare populations (Bauer et al., 1998). The latter function, however, presents a singularity for low values of the parameter (n < 2) (Bock et al., 1998), precluding a direct comparison of stressed and unstressed samples.
From the present study, we can conclude that a number of the proposed approaches may not be entirely valid. The investigated genotoxic treatment results in significant heterogeneity of DNA damage among cells so that the comet distributions, either in terms of tail DNA or of Olive tail moment are practically never Gaussian, even after logarithmic transformation; their comparison by a non-parametric test allows easy detection of significant, but objectively unimportant, differences (Kruskal–Wallis tests, Figure 1). We demonstrate in the present paper that the reduction of data to representative non-parametric statistics (essentially the medians) is an effective tool to compare samples, either by analysis of repeated experiments or by trend analysis. The definite loss of information due to these techniques can be compensated for by a box plot representation of data, an easy visual comparison of samples without the bias induced by three-dimensional graphs, and, to a certain extent, by the examination of 75th percentiles. The data distribution curves should, however, be systematically investigated for an eventual bimodal distribution, such information being lost by graphical and statistical data summarizing.
Although a slight interaction treatmentxelectrophoresis was found for tail DNA, there is no apparent influence of the electrophoresis parameter; to avoid any bias, random assignment of samples to electrophoresis runs is recommended.
The trend analysis appears to be a powerful tool, notably to prove the absence of effect (e.g. no effect from visible light irradiation) and to demonstrate weakly genotoxic activities (e.g. -ala photodynamic treatment with visible light irradiation). Its application may, however, be limited by cytotoxicity possibly induced at the higher dosages of genotoxic agents. In order to determine if the use of the median or 75th percentile affects the sensitivity of the comet procedure, we compared the slopes of the relationships comet metric vesus dose of light, described in Table I; a statistical parameter will effectively be considered more sensitive if small changes in light dosage cause significantly larger changes in the response function (Causon, 1997). For the treatments visible light, porphyrins + visible light and porphyrins + UVA there is no significant difference between the slopes of the median and 75th percentile response curves (P > 0.05 for both tail DNA and Olive tail moment); for the treatment UVA there is a significantly higher slope for the 75th percentile curve (P < 0.05 for tail DNA and P < 0.01 for Olive tail moment). From these data, we can conclude that the 75th percentile offers at least equal, and in some cases higher, sensitivity as the median; however, this statistical parameter tends to saturate for higher stress levels (Table I).
Although the tail DNA has been reported as the best comet parameter, increasing linearly with stress intensity up to 75% (Collins et al., 1996), the tail moments are designed to take into account differences in DNA migration that may be due to the nature and extent of DNA relaxation (Olive et al., 1990; Hellman et al., 1995; Kent et al., 1995). In all our studies (up to stress levels leading to a median 70% tail DNA), both tail DNA and Olive tail moment essentially reach the same conclusions and no obvious differences have been seen between the two parameters with regard to the investigated statistics. However, depending on the mechanism of action of the tested genotoxic agent, these parameters could lead to different conclusions and it might be cautious to consider both.
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Conclusion |
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Porphyrin photo-excitation
The photodynamic effect of
-ala-induced porphyrins, under both UVA and visible light, leads to high DNA strand breakage. These findings confirm the previously reported DNA-damaging effect of induced porphyrins; as shown by HPLC with amperometric detection (Duez et al., 2001), their photooxidation provokes the appearance of a highly mutagenic oxidized adduct, 8-oxo-7,8-dihydro-2'-deoxyguanosine. The possible implications of these findings with regard to human solar carcinogenesis have been fully discussed (Duez et al., 2001).
Comet data treatment
The dispersed data obtained in the Comet assay can be efficiently analysed by the proposed statistical treatments, leading to reliable conclusions on observed trends. These, however, require multiple repeats of experiments and/or investigation of dose–effect relationships, resulting in higher workloads and probably in a need for automation of data acquisition.
As these statistical methods lead to a certain loss of information, further promising data treatments are currently being investigated in our laboratory, including quantile regression methods and mathematical modelling of combined comet profiles.
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Acknowledgments |
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We are grateful to Professor M.Kirsch-Volders and to Mrs M.De Boeck for introducing us to the Comet assay technique. We thank Mrs M.Bette for her skilful technical help and enthusiasm.
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Notes |
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3 To whom correspondence should be addressed at: Tel: +32 2 650 5242; Fax: +32 2 650 5187; Email, pduez{at}ulb.ac.be
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Received on July 4, 2002; revised on October 28, 2002; accepted on November 4, 2002.
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