Mutagenesis, Vol. 17, No. 1, 37-43,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
High throughput Comet assay using 96-well plates
Novartis Pharma AG, Genetic and Experimental Toxicology, WSH2881.5.14, CH-4002 Basel, Switzerland 1 Present address: School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
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Abstract |
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The single-cell gel electrophoresis or Comet assay is becoming established as an industrial genotoxicity screening test. The aim of this study was to increase the throughput of compounds tested and to minimize the amount of test compound needed for an assay. We modified practical aspects of our standard protocol and designed an experimental procedure suitable for use with 96-well plates. By using a suspension culture rather than attached cells, the modified protocol enabled parallel testing of four compounds on a single microplate (10 duplicate concentrations per compound). A significant reduction in work time was achieved by replacing the previously used Trypan blue dye exclusion (TBDE) test by an automated measurement of ATP levels as the concurrent viability test. The rapid and easy to perform ATP test was carried out towards the end of the 3 h treatment. In this way we were able to select for further analysis and slide preparation only those concentrations which induced the desired range of cytotoxicity. The suitability of the modified test conditions and reproducibility of test results was demonstrated by results obtained with standard mutagens and eight drug candidates tested at various concentrations. In each case the results obtained with the standard and the modified protocols were comparable. By introducing the changes to our standard protocol, combined with automated image analysis, we were able to more than double our previous throughput.
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Introduction |
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The single cell gel (Comet) assay is increasingly used in genotoxicity testing in vitro as well as in vivo (Sasaki et al., 2000
; Tice et al., 2000). High sensitivity and specificity combined with easy performance of this test seem to make it well suited to be used as an industrial genotoxicity screening test. Besides the Ames test, pharmaceutical companies currently use the micronucleus test (MNT) in vitro for genotoxicity screening of drug candidates early in drug development. We recently introduced the Comet assay into our routine genotoxicity screening test battery and reported on a comparative study with the Comet assay and the MNT in which 33 drug candidates were tested in V79 Chinese hamster cells (Hartmann et al., 2001a). This study showed a very good correlation between the Comet assay and MNT in the case of compounds with positive calls in the Comet assay. However, several MNT-positive compounds were negative in the Comet assay and it was concluded that this discrepancy might be due to the influence of cytotoxicity on test results in the MNT. For the Comet assay it is considered mandatory to concurrently determine viability of the treated cultures. It was reported that non-genotoxins at cytotoxic concentrations may induce DNA migration in the Comet assay in TK6 human lymphoblastoid cells (Henderson et al., 1998) and rat lymphocytes (Quintana et al., 2000). It was, therefore, recommended that concentrations of test compounds be limited to 
75% viability, at which increased migration values after treatment with non-genotoxins were not detected (Henderson et al., 1998). However, different cell lines may behave differently and using V79 cells, non-genotoxins tested up to highly cytotoxic levels did not lead to positive calls in the Comet assay (Hartmann and Speit, 1997; Hartmann et al., 2001a,b). It is therefore critical to collect more data on cytotoxicity as a potential confounding factor for the Comet assay because it is known that strand break assays such as alkaline elution, alkaline unwinding and sucrose gradients have a potential problem in distinguishing between strand breaks induced by genotoxicity and those due to excessive cytotoxicity (Storer et al., 1996).
The aim of the present study was to significantly reduce the working time and the amount of test compound needed to perform a test. We evaluated whether a cell line growing in suspension is suitable for routine use in the Comet assay. The L5178Y mouse lymphoma cell line is well established for other genotoxicity tests and has been used in the Comet assay to investigate genotoxic effects of chemical mutagens (Miyamae et al., 1997). Moreover, this cell line can be treated in small volumes, e.g. in 96-well plates, which enables the use of minimal amounts of test compounds. With L5178Y cells we investigated whether cytotoxicity is a potential confounding factor, i.e. if cytotoxicity has an influence on DNA migration in the Comet assay. Several compounds with different cytotoxic potential were tested. We were also aiming to replace the time-consuming Trypan blue dye exclusion (TBDE) test previously used for concurrent viability assessment by an automated measurement of ATP levels, which can be performed in 96-wells. For this purpose, several compounds with a range of cytotoxic potentials were used to validate the ATP test and to assess the reproducibility and correlation of test results with TBDE measurements. Furthermore, by obtaining cytotoxicity data prior to preparation of slides for the Comet assay, we were able to select the compound concentrations of interest, i.e. the three or four concentrations per compound out of 10 which resulted in the desired range of cytotoxicity. By closely spacing 10 duplicate concentrations per compound and selecting only the concentrations of interest, we significantly reduced the number of repeat tests as well as the number of slides to be processed in the Comet assay.
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Materials and methods |
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Cell line and treatment with test compounds, both test conditions
L5178Y mouse lymphoma cells, grown in RPMI 1640 medium with 10% fetal calf serum at 37°C and 5% CO2, were used. Duplicate cultures (cell density 0.5x106/ml) were treated with a series of concentrations of the test chemicals dissolved in DMSO (end concentration in culture 1%). Duplicate solvent and positive controls were used in every test. The treatment was for 3 h at 37°C in the presence or absence of 10% S9 mix from Aroclor 1254-treated male rats. Generally, two independent tests with and without S9 activation for each chemical were performed. All compounds tested with the exception of the standard mutagens [2-aminoanthracene (2-AA), ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)] were synthesized in house as Novartis drug candidates.
Conditions for the standard Comet assay
For the standard protocol Comet assay the spacing was 2-fold in the first test and 1.5-fold or less in the repeat experiments. The chemicals were initially dissolved in DMSO and one stock sample of the test chemicals was prepared for each different concentration tested. An aliquot of 10 µl of each corresponding stock solution was used to treat 940 µl of L5178Y cells. In addition, 50 µl of S9 mix or 0.15 M KCl was added according to the test conditions, i.e. with or without metabolic activation, respectively. The final volume of the culture medium was 1 ml and each was prepared in 1.5 ml Eppendorf tubes.
Conditions for the high throughput Comet assay (HTC)
For the HTC two stock solutions diluted initially in DMSO and then 100-fold in RPMI medium with a dilution factor of 1.5 between them were prepared. Then two series of five concentrations with a 2-fold spacing were prepared, one from each stock solution, as depicted in Figure 1. Aliquots of 200 µl of `stock 1' and `stock 2' were placed in all wells in columns 3 and 8, respectively. The other wells of the 96-well V-bottomed microplate (Costar 3894) were filled with 100 µl of RPMI medium (containing 1% DMSO) and 1:2 dilutions of the stock solutions using a multi-tip pipette. For the controls 100 µl of either DMSO or standard mutagens pre-diluted in medium were added to wells 1 and 2. Finally, 100 µl of cell suspension (with S9 or 0.15 M KCl) was added to all the wells of the plate and mixed. The total culture volume in each well was 200 µl. The dilution factor between the two stock solutions can be altered (e.g. 1.33, 1.5, 1.8, etc.) so as to achieve the desired spacing of concentrations for each test compound.
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S9 mix preparation
The S9 liver homogenate was prepared as described in the literature (Maron and Ames, 1983
). At least five 7–9-week-old male Crl:Wist Han rats (Charles River, Sulzfeld, Germany) were injected with 500 mg/kg Aroclor 1254 and killed 5 days later. The livers of the animals were homogenized, diluted 1:4 with 0.15 M KCl and centrifuged for 10 min at 9000 g. The supernatant was frozen in small aliquots and stored at –70 to –80°C until use. S9 mix was made in the following way. On the day of treatment, NADP (2.25 g/l) and 5.07 g/l glucose 6-phosphate (G6-P) were dissolved in water (Millipore). Then the following components were added per liter, mixed and stored on ice after sterilization by filtration: 100 ml Hanks BSS (10x concentrated), 300 ml NADP/G6-P solution, 292 ml H2O, 8 ml NaHCO3 (4.4%), 100 ml S9 fraction and 200 ml 0.15 M KCl. The end concentration of S9 fraction in the cultures was 2%.
Cytotoxicity assays and concentration selection
The concentrations of the compounds analysed in the Comet assay were selected on the basis of the solubility and/or cytotoxicity results. At least three concentrations in duplicate per test compound per test were analysed. For non-cytotoxic compounds testing was performed up to 10 mM or precipitating concentrations, whereas for cytotoxic compounds dose selection for further analysis was based on the viability determined either by TBDE or by the ATP cytotoxicity test. At the end of the 3 h treatment cells were centrifuged and resuspended in fresh RPMI medium without test compounds. Trypan blue was diluted 1:5 in medium and 40 µl of this solution was further mixed with 10 µl of the cell suspension for each sample. At least 200 cells were counted per culture. The ATP test (Cytotoxicity and Cell Proliferation Kit; Labsystems, Finland) was performed at the end of the 3 h treatment. The test was carried out according to the instructions of the manufacturer using 100 µl of cell suspension transferred directly from the 96-well incubation microplate into a new microplate (White Cliniplate; Labsystems). The luminescence of the ATP-releasing reaction was followed using a microplate luminometer (Luminoskan Ascent; Labsystems).
Comet assay
The standard procedure originally described by Singh et al. (1988) with modifications (Speit and Hartmann, 1999; Hartmann et al., 2001a) was used. Regular slides were coated with 1% agarose and allowed to dry overnight. Following the 3 h treatment, cells were centrifuged and resuspended in fresh RPMI + 10% fetal calf serum, free of treatment compounds. Aliquots of 15 µl of the cell suspension were resuspended 1:10 in 0.5% low melting point agarose (Sea Plaque GTG; FMC, Rockland, USA). Samples of 45 µl of this suspension were spread on an agarose pre-coated slide, covered with a 25x25 mm coverslip and placed at 4°C for 5 min. The coverslip was gently removed and the slide was submersed in lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, pH 10, 4°C) for at least 1 h. After lysis, the slides were equilibrated for 20 min in a jar containing alkaline solution (300 mM NaOH, 1 mM EDTA, pH > 13, 4°C), transferred into an electrophoresis unit with alkaline buffer and subjected to an electric field of 0.86 V/cm for 20 min at 4°C. Following electrophoresis the microgels were neutralized in 0.4 M Tris (pH 7.5), rinsed with water, dehydrated in 100% ethanol for 2 min and allowed to dry at room temperature. The DNA was stained with 5 µg/ml propidium iodide. To prevent the slides from fading and drying out, propidium iodide was dissolved first in distilled water and then further dissolved 1:5 in Vectashield (Vector Laboratories, Burlingame, USA).
Evaluation criteria
Concentrations resulting in <70% (TBDE) or <50% relative viability (ATP test) were considered too cytotoxic for analysis. The basis for these thresholds was derived from cytotoxicity levels proposed for the alkaline elution assay (Storer et al., 1996). These thresholds were exceeded in the following exceptions: (i) for the determination of a potential influence of cytotoxicity on DNA migration; (ii) in the case of a steep concentration-dependent cytotoxicity. Additionally, after processing slides in the Comet assay the occurrence of non-detectable cell nuclei (NDCN) was assessed as an additional indicator of cytotoxicity. NDCN or clouds are cells exhibiting extensive DNA fragmentation to a point where no clear head/nucleus but only the tail is visible. These cells were not measured by image analysis but recorded separately. Concentrations resulting in 15% NDCN were considered to be too cytotoxic. Cytotoxic doses were analyzed only where the effect of cytotoxicity on DNA migration was to be determined (Table I). Examination of Comet assay slides was done with a modified version of a previously described in-house built fully automatic image analysis system (Frieauff et al., 2001). The modifications will be described elsewhere. The parameter used was tail moment as described by Olive (1989). The median tail moment of 50 cells/slide was determined and the mean of two parallel cultures was calculated (i.e. 100 cells/concentration were analyzed). The median was chosen because of the heterogeneity of data. For determination of whether a test compound was positive in the Comet assay, descriptive parameters, such as an obvious concentration-related increase in DNA migration and/or the reproducibility of a specific effect, were used, rather than a statistical analysis. Generally, a positive effect was defined as at least a doubling of the tail moment over the accompanying solvent control plus a value of 0.2. These criteria are based on extensive historical control data.
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Results |
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Influence of cytotoxicity on Comet assay results
Several compounds were tested up to highly cytotoxic concentrations (sometimes exceeding the limits as given under Materials and methods) as measured by TPDE and NDCN. Table I
lists examples of compounds which were negative in the Comet assay but clearly induced cytotoxicity. The results demonstrate that the tail moments were not increased compared with the concurrent negative control even when TPDE values of <70% relative viability or NDCN values >15% were induced. A more extensive study with 35 compounds is in preparation and will be published elsewhere.
Comparison of TBDE and ATP cytotoxicity tests
Cells were treated with several in-house synthesized drug candidates exhibiting a wide spectrum of cytotoxic potentials. Directly after the end of the 3 h incubation, the TBDE and ATP tests were performed in parallel to measure cytotoxicity. The relative cytotoxicity results for nine compounds are presented in Table II. The respective concurrent negative control values were considered as 100% in both tests and values exceeding the respective control value are given as 100+. The last concentrations with acceptable cytotoxicity levels (i.e. 70% viability for TBDE and >50% ATP levels) are given in bold. It can be seen from the table that these cut-off concentrations either at the respective limit or above it were similar in both tests. This means that, based on the cytotoxicity data of either test, in most cases the same or similar concentrations (a difference of only 1.5- to 2-fold) would have been chosen for analysis in the Comet assay. It is noteworthy that there was no obvious correlation between the two methods regarding the respective limits.
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Comparison between the standard protocol and HTC
In order to validate and standardize the HTC, the effects of three standard mutagens (EMS, MMS and 2-AA) and six in-house synthesized drug candidates with different genotoxic potentials were tested comparatively under both conditions. The results of the tests are given in Table III
. A total of 15 individual experiments were done with the HTC and 13 experiments with the standard protocol. Repeat experiments were performed in all cases (data not shown). The three mutagens showed comparable concentration-related increases in tail moment under both test conditions. Of the six drug candidates tested one (N) was clearly positive under both conditions. One compound (L) was weakly positive throughout all experiments, i.e. a positive effect was found only at the highest analysable concentration. The other four compounds were negative under both test conditions. Therefore, the correlation of results in terms of reproducibility between the standard Comet assay and the HTC was 100%. Generally, dose–response curves are similar for both tests but absolute values for tail moment are lower in the HTC. Furthermore, reproducibility of results between repeat experiments in the HTC was very good, since the outcome of the repeat experiments in all cases was the same as in the first test (data not shown).
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Discussion |
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The Comet assay has become well established as a specific and sensitive genotoxicity test in vitro as well as in vivo and has various applications (Rojas et al., 1999
). It is now being used in the pharmaceutical industry as part of a genotoxicity screening battery (Hartmann et al., 2001a) and is a promising test that may be used as the second in vivo test for regulatory purposes (Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment, 2000; Hartmann and Suter, 2001). By introducing changes to the standard in vitro protocol we were able to increase the number of compounds tested and to significantly reduce the amount of test compound needed to run an assay (to 5 mg/test). The changes introduced included use of the L5178Y mouse lymphoma cell line, which can be treated in small volumes in 96-well plates, and replacement of the time-consuming TBDE by an automated ATP cytotoxicity test. The usefulness and validity of the HTC protocol was demonstrated by highly correlated concentration–response curves for standard mutagens between the standard and modified tests. Furthermore, test results with candidate drugs of various genotoxic potentials were highly reproducible between the two test conditions.
The comparison between the TBDE and ATP cytotoxicity results shows that using cut-off points of <70% viability (TBDE) or <50% reduction in ATP levels, the same range of compound concentrations were selected for further analysis in the Comet assay. These levels are in agreement with previously proposed cut-off levels (i.e. 
70% viability by TBDE or 50% by ATP levels) used for the alkaline unwinding test (Storer et al., 1996). This comparative investigation used several measurements of cytotoxicity and demonstrated that the TBDE test and the ATP level test are well correlated. Moreover, other investigators concluded that determination of ATP levels may be the more accurate parameter for cytotoxicity, because ATP levels are an indicator of the functional integrity of living cells, as any cell injury or cell death will lead to a corresponding decrease in cytoplasmic ATP content (Crouch et al., 1993). In the case of the TBDE, any cell that is damaged or dead and has a ruptured membrane will allow uptake of the dye and hence will be considered dead. However, according to Elia et al. (1993) there are certain compounds that induce acute cytotoxicity without having an immediate effect on membrane integrity. Moreover, previous comparisons between the two tests (Elia et al., 1994) have shown that there is a better correlation between ATP levels taken directly after the end of the 3 h incubation period and TBDE measurements made after a 3 h recovery incubation (in fresh culture medium), further supporting this statement. Any other differences may also arise from the greater sensitivity and subjectivity of the ATP test as a consistent indicator of cytotoxicity relative to the TBDE test.
The major advantage of the ATP test is that it can be performed directly in the wells of a microplate. It is also faster, because as an automated test procedure, the tedious microscopic evaluation of Trypan blue stained cells can be omitted. We performed the test towards the end of the 3 h incubation period and the cytotoxicity data obtained were used for selection of doses which do not show excessive cytotoxicity. Comet slides were then prepared only from these compound concentrations. As treatment of the L5178Y cell line in the HTC was carried out in 96-well plates, a simple transfer of cell suspensions into a new plate and subsequent running of the ATP test saved significant time and laboratory work.
Similar to earlier studies carried out with V79 cells (Hartmann and Speit, 1997; Hartmann et al., 2001b), excessive cytotoxicity did not result in positive effects in the Comet assay under the conditions used. For other cell types cytotoxicity was reported as a possible confounding effect, inducing elevated DNA migration presumably by extensive DNA fragmentation upon cell death (Henderson et al., 1998; Quintana et al., 2000). However, the Comet assay has the advantage that dead or dying cells can be identified on microscope slides by their morphology. Such cells exhibit extensive DNA fragmentation and are without a visible nucleus (Tice et al., 2000). In the present study occurrence of these cells was recorded separately and used as a measure of cytotoxicity, but these cells were not included in measurements by the image analysis system.
In conclusion, by introducing the HTC and including the ATP cytotoxicity test in combination with automated slide analysis we were able to increase our throughput by >2-fold. The HTC as described in this study can be applied early in drug development or elsewhere when limited quantities of test material are available and rapid analysis of genotoxic potential is required.
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Acknowledgments |
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We wish to thank Dr A.Elhajouji for critical discussions and M.E.Schneider and C.Blaser (Novartis Pharma AG) for their valuable technical support.
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Notes |
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2 To whom correspondence should be addressed. Tel: +41 61 32 41951; Fax: +41 61 32 41274; Email: andreas.hartmann{at}pharma.novartis.com
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Received on May 10, 2001; accepted on August 13, 2001.
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