Influence of cytotoxicity and compound precipitation on test results in the alkaline comet assay

Mutation Research 497 (2001) 199–212

Influence of cytotoxicity and compound precipitation on test results in the alkaline comet assay Andreas Hartmann∗ , Evangelos Kiskinis, Ann Fjällman, Willi Suter Novartis Pharma AG, Genetic and Experimental Toxicology, WSH2881.5.14, CH-4002 Basel, Switzerland Received 10 April 2001; received in revised form 19 June 2001; accepted 26 June 2001

Abstract We use the comet assay as part of our genotoxicity screening battery for newly synthesized drug candidates. A dataset of more than 250 tests carried out with 75 drug candidates of various chemical classes was analyzed to elucidate the influence of cytotoxicity and compound precipitation on DNA migration in the comet assay. Using a V79 Chinese hamster cell line, 38 of the compounds were negative and 37 were positive in the comet assay. The reproducibility of test results between repeat experiments was 85%. Data on 72 tests with a negative call in which the compounds were tested up to highly cytotoxic concentrations demonstrated that cytotoxicity, as determined by Trypan blue dye exclusion and occurrence of cells with completely fragmented chromatin, did not lead to false positive test results. The majority (64.2%) of compounds with a positive call induced elevated DNA migration in the absence of excessive cytotoxicity. Compound precipitation was observed in 84 tests. In 88.1% of these cases, the test result at the precipitating concentration did not differ from that found at the highest soluble concentration. Half of the remaining 11.9% of contrary results (most of them weak effects) were not reproducible in the respective repeat experiment, indicating no or only a negligible influence of precipitation on test results. The data indicate that using V79 cells, the comet assay specifically detects genotoxic effects and is not confounded by cytotoxicity or compound precipitation under the conditions used. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Alkaline comet assay; Cytotoxicity; Compound precipitation

1. Introduction The alkaline comet assay was first introduced by Singh et al. [1] is increasingly used in genotoxicity testing and has various applications [2]. Sensitivity and specificity of the test are considered to be very high and guidelines for the conduct of the comet assay have recently been published [3]. However, it has yet not well been established whether cytotoxicity influences DNA migration in ∗ Corresponding author. Tel.: +41-61-32-41951; fax: +41-61-32-41274. E-mail address: [email protected] (A. Hartmann).

the comet assay. It is known that strand break assays, such as alkaline elution, alkaline unwinding or sucrose-gradients have the potential problem to distinguish between strand breaks induced by genotoxicity or excessive cytotoxicity [4]. Similarly, an important issue in genotoxicity testing with the comet assay is the possible influence of cytotoxicity on test results. Dead or dying cells can undergo rapid DNA fragmentation which should be expected to increase DNA migration in the comet assay. It is, therefore, mandatory to perform concurrent viability tests to control for excess cytotoxicity [3]. For the alkaline unwinding technique (which measures a similar end point) it was recommended not to test concentrations of compounds decreasing viability to less that

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70% in order to avoid false positives [4]. However, compounds have been identified which induced DNA degradation due to cytotoxicity but did not affect Trypan blue exclusion, and therefore, led to false positive effects [5,6]. The comet assay provides the advantage over other strand break assays that measurements are made on individual cells. Dead cells can be identified by their distinct morphology as compared to cells exhibiting DNA damage [7–9]. The morphology results from fragmentation of chromatin in such a way that cells with non-detectable cell nuclei (NDCN) are visible in the gels. Scoring NDCN on slides provides an independent parameter for toxicity of a test compound. However, cells exposed to a high concentration of a mutagen may exhibit a similar morphology and, therefore, the distribution pattern between damaged and undamaged cells has also to be taken into consideration [3,10]. We use the comet assay as part of our routine genotoxicity screening battery. In the present study, data on V79 cells treated with 75 compounds of unknown genotoxic potential were used. All compounds were tested with or without metabolic activation (S9), using two parallel cultures in each test. All tests were repeated in an independent test unless a clear positive result was obtained in the first test. Of the 75 compounds tested, 38 were negative and 37 were positive under the test conditions used. Criteria for selection of analyzable concentrations was based on a concurrent viability test (Trypan blue dye exclusion, TBDE) and on scoring NDCN on slides processed in the comet assay. This dataset was used to address the following questions: 1. Effects of cytotoxicity on DNA migration values. Is there any indication that excess cytotoxicity induces elevated DNA fragmentation and, thereby, leads to false positive calls? 2. Influence of precipitating concentrations on test results. It is known that precipitation can have an influence on test results of the chromosome aberration assay [11]. We, therefore, wanted to elucidate whether compound precipitation might pose a potential problem for the comet assay, i.e. is the a possible interference of the precipitate with DNA migration, which may result in a different outcome compared with the highest soluble concentration of a compound.

2. Materials and methods 2.1. Cell line and test compounds V79 Chinese hamster cells were used for all tests. For each concentration of test chemical, negative or positive control, two cultures were treated for 3 h in the presence or absence of 10% S9 mix of Aroclor 1254-treated male rats. At least two independent tests with or without S9 were performed per test compound; in case of a clear positive call, the respective repeat experiment was skipped. All test compounds were synthesized in-house as drug candidates. The compounds were dissolved either in Eagles minimal essential medium (MEM) or dimethyl sulphoxide (DMSO; end concentration 1%). 2.2. S9 mix preparation The S9 liver homogenate was prepared as described in the literature [12]. At least five, 7- to 9-week-old male Crl:Wist Han (Charles River, Sulzfeld, Germany) were injected with 500 mg/kg Aroclor 1254 and sacrificed 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 (G 6-P) were dissolved in water (Millipore). Then, the following components were added per liter, mixed, and stored on ice: 100 ml Hanks BSS (10× concentrated), 300 ml NADP/G 6-P solution, 292 ml H2 O, 8.00 ml NaHCO3 (4.4%), 100 ml S9 fraction, and 200 ml 0.15 M KCl and then sterilized by filtration. 2.3. Concentration selection and viability test Concentration selection for the comet assay was based on solubility and/or cytotoxicity. At least three concentrations/experiment were analyzed. The spacing between concentrations was 2-fold in the first and 1.5-fold or less in the repeat experiment. For non-cytotoxic compounds, testing was performed either up to precipitating concentrations or, in case of well soluble compounds, 10 mM or 5 mg/ml, whichever was lower. For cytotoxic compounds,

A. Hartmann et al. / Mutation Research 497 (2001) 199–212

concentration selection was based on viability determined by TBDE as performed by Storer et al. [4]. Briefly, cells were rinsed after drug treatment and an aliquot was used for the comet assay. Another aliquot was resuspended in medium and incubated for another 3 h. After this time, cells were pelleted, resuspended in Trypan blue dye and at least 200 cells were scored. In general, treatment of cultures with concentrations resulting in ≤70% relative viability were considered to be too cytotoxic and were not evaluated; however, for the evaluation of the influence of cytotoxicity on

201

DNA migration, concentrations exceeding this limit were analyzed. 2.4. Comet assay The standard procedure originally described by Singh et al. [1] with modifications [13] was used. Regular slides were coated with 1–1.5% agarose and allowed to dry overnight. Following treatment, cells were washed twice with ice-cold PBS and trypsinized with 0.125% trypsin–EDTA for 2 min. Afterwards,

Table 1 Summary of test results of the comet assay: compounds giving contrary results in the respective repeat experimenta Compound code

Test no. −S9

Result

TBD positive cells (%)

NDCN (%)

+S9

12

1 2

(+)b −

2.5 2

12.5 3

14

1 2

(+) −

3 1.5

0 0

20

1 2

+ −

10 15

2 12.5

(+) −

7.3 2.5

11.5 0

26

1 2

27

1 2

(+) −

10 13

13 9.5

34

1 2 1 2

(+) − + −

22 3 3.5 1

11.5 4 2 1.5

1 2

(+) − (+) −

29 9 12 0.5

13.5 2 9.5 4.5

55

1 2

(+) −

N.D. 5.5

9 4.5

57c

1 2

− +

0 1

0 5.5

1 2

(+) − + −

0 0 0 0

0 0 0 0

− +

1.5 2

0 0

48

1 2

65c

1 2

70c

1 2 a

TBD: Trypan blue dye; NDCN: non-detectable cell nuclei. Weak positive effect, i.e. small (but positive) increase only at highest concentration tested. c Precipitation of compound, no cytotoxicity detected. b

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trypsin was stopped by adding media containing 10% fetal calf serum. The cells were resuspended 1:10 in 0.5% low melting agarose (Sea Plaque GTG, FMC, Rockland, USA). A volume of 45 ␮l, of this suspension was spread on a pre-coated slide, covered with a 25 mm × 25 mm coverslip and placed at 4◦ C for 5 min. The coverslip was gently removed and the slide was submerged into 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 buffer (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 2.5 ␮g/ml propidium iodide. To prevent slides from fading and drying out, propidium iodide was dissolved first

in distilled water and then further dissolved 1:5 in Vectashield (Vector Laboratories, CA-Burlingame, USA). 2.5. Evaluation criteria In general, treatment of cultures with concentrations resulting in ≤70% relative viability (as measured by TBDE) were considered to be excessively cytotoxic. Additionally, after processing slides in the comet assay, the occurrence of NDCN was assessed as an indicator of cytotoxicity. These cells were not evaluated with the image analysis but recorded separately. Concentrations resulting in ≥15% NDCN were considered to be excessively cytotoxic and were generally not evaluated. However, for the evaluation of the influence of cytotoxicity on DNA migration, concentrations exceeding these limits were analyzed. Examination of comet assay slides was done with an in-house built fully automatic image analysis system [14]. The parameter used was the tail moment

Table 2 Influence of cytotoxicity on test results in the comet assay: summary of compounds with negative test resultsa Compound code

Test no. −S9

3 3 4 4 6 6 6 6 12 16 16 16 18 20 22 23 24 26 27 28 28 30 30 30

Result

TBD positive cells (%)

NDCN (%)

− − − − − − − − − − − − − − − − − − − − − − − −

0.5 0.5 6.3 4.0 5.0 11.0 7.0 16.0 26.5 43.0 N.D. N.D. 5.0 15.0 N.D. 4.3 10.0 7.0 5.0 4.0 6.0 37.0 1.5 5.0

5.0 8.5 2.5 9.0 3.0 27.5 0 13.5 N.D. 14.0 15.0 25.0 0 12.5 5.0 6.0 0 7.5 1.5 7.5 4.0 4.5 5.0 8.5

TBD positive ≥6%

NDCN ≥5%

+S9

1 2 1 2 1 2 1 2 1 1 2 1 1 2 1 2 1 2 1 1 2 1 1 2

Y Y Y Y Y Y Y Y Y Y

Y Y

Y Y Y

Y Y Y Y Y Y Y Y Y Y

Y Y Y

Y Y

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Table 2 (Continued) Compound code

Test no. −S9

31 31 31 32 32 33 36 39 39 40 41 41 41 41 42 47 47 47 48 48 49 50 50 51 54 54 54 55 55 56 56 57 57 58 58 58 58 59 59 60 60 61 61 63 63 64 64 67 71 71

1 1 2 1 1 1 2 1 1 2 1 2 1 1 2 1 2 2 2 2 1 1 2 1 2 2 2 2 1 1 2 1 2 1 2 1 2 2 3 1 2 1 2

a

TBD positive cells (%)

NDCN (%)

TBD positive ≥6%

− − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

8.5 7.5 0.5 17.0 20.0 8.0 15.0 14.0 7.0 9.0 10.0 18.0 8.0 14.0 12.5 3.5 8.0 2.8 9.0 0.5 25.0 26.5 4.0 10.0 12.0 5.5 N.D. 2.0 5.5 18.0 6.0 6.0 23 7.0 27.5 8.5 9.0 5.6 4.2 N.D. N.D. 1.0 5.0 10.0 11.0 9.5 13.5 3.0 2.0 3.0

3.0 4.0 9.5 3.0 9.5 0 2.0 2.5 12 4.0 6.0 13 15 7.0 12.5 0 12 10 2.0 5.0 0 11.0 10.5 7.0 1.5 2.0 10.0 16.0 4.5 4.5 2.0 1.0 0 0.5 4.5 8.0 15.5 0 10.5 27.5 13.0 2.5 0.5 8.5 4.5 8.0 17.0 13.5 8.0 5.0

Y Y

NDCN ≥5%

+S9

1 2

1 2 2 1 2

Result

TBD: Trypan blue dye; NDCN: non-detectable cell nuclei.

Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Y

Y Y Y Y Y Y Y Y

Y Y Y Y Y Y Y

Y Y Y

Y Y Y Y Y Y Y Y Y Y Y Y

Y Y Y Y Y

Y Y Y Y Y Y Y

Y Y Y Y

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as described by Olive [15]. The median tail moment of 50 cells per slide was determined and the mean of the two parallel cultures was calculated (i.e. 100 cells per concentration were analyzed). For the determination whether a test compound was positive in the comet assay, no statistics were performed;

instead, descriptive parameters, such as an obvious concentration-related increase in DNA migration, and/or the reproducibility of a specific effect were used. Generally, a positive effect was defined as at least a doubling of the tail moment of the concurrent solvent control.

Table 3 Individual results of some negative compounds tested up to highly cytotoxic concentrationsa Compound code

Test no. −S9

6

Median tail moment

TBD positive cells (%)

NDCN (%)

0 169 339 450 600 EMS, 330

1.3 1.1 1.7 1.2 1.4 4.4

1.5 0.5 0.5 2.5 11.0 1.5

0 0 0.5 0 27.5 0

0 88.9 133.3 200.0 2-AA, 20 ␮M

1.0 1.1 1.4 0.8 6.9

0.5 0 0 16.0 0

0 0 0 13.5 0

+S9

2

6

Concentration (␮g/ml)

2

16

1

0 7.5 15 30 EMS, 312

0.48 0.55 0.40 0.81 1.23

0 0.5 4.5 43 0

0 0 1.5 14 0

30

1

0 7.5 15 37.6 75.3 EMS, 330

1.8 1.6 1.2 1.4 Toxic 10.3

0.5 0 2.5 37.0 50.0 2.0

0 0 2.0 4.5 36.0 0

50

2

0 25.0∗ 75.0∗ 100.0∗ 150.0∗ EMS, 330

1.9 1.1 2.5 1.8 Toxic 6.3

3.0 11.0 12.0 26.5 50.0 2.0

0 6.0 5.5 11.0 11.0 0

55

2

0 20.7 31.1 46.6 EMS, 330

1.6 1.5 1.7 1.7 9.3

1.0 1.0 1.5 2.0 0.5

0 1.5 3.5 16.0 0

a

EMS: ethyl methanesulfonate; 2-AA: 2-amino anthracene; TBD: Trypan blue dye; NDCN: non-detectable cell nuclei.

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3. Results 3.1. Reproducibility In order to determine the reproducibility of individual test results, data on 188 individual experiments (94 tests, 94 repeat experiments) were analyzed. The data show a high degree of reproducibility: in 85% of the cases, the outcome of the repeat experiment was the same as the first test. Table 1 lists the results of 14 tests showing a discrepant result in the respective repeat experiment. However, in nine of these tests, a weak increase of the tail moment was found at the highest concentration only in one of the two experiments. This situation would generally result in a negative call for the compound, i.e. a weak effect at the highest concentration only is not considered to indicate a genotoxic potential. Not considering these weakly positives for the evaluation would increase the reproducibility of test results to 94.2%. 3.2. Influence of cytotoxicity on DNA migration negative compounds To investigate a possible effect of cytotoxicity on DNA migration in the comet assay, the dataset was evaluated for negative compounds showing clearly cytotoxic effects. Definition of a cytotoxic effect was at least a doubling of TBDE and/or NDCN values. Mean values of control cultures under the condition used were ≤3% Trypan-blue positive cells and ≤2.5% NDCN. Table 2 lists 74 tests in which cytotoxic effects occurred in one or both toxicity parameters. It can be seen that even a very high percentage of Trypan-blue positive cells (up to 43%) and/or NDCN values (up to 27.5%) was not associated with a positive call. Interestingly, there was frequently no obvious relationship between TBDE and NDCN values, i.e. a high value in one of the parameters was not necessarily accompanied by a high value of the other parameter. In Table 3, individual data on typical test results are presented. The positive control data is included to demonstrate the validity of the experiment. 3.3. Positive compounds There was no obvious relationship between a positive call (increased DNA migration) and high

Fig. 1. Percentages of Trypan blue dye exclusion positive cells plotted against the percentage of non-detectable cell nuclei. Values from 230 cultures assessed for both parameters.

cytotoxicity as can be see from the scatterplot in Fig. 1. Table 4 lists 81 tests in which increased DNA migration was found in the presence as well as in the absence of cytotoxicity (i.e. more than double the highest TBDE value generally found in solvent controls, which is 3%). In some cases, one of the two experiments for a compound was positive only when high TBDE/NDCN values were found (e.g. compound no. 12, 26, 34 (−S9, see Table 1). On the other hand, the opposite situation was also present (e.g. compound no. 20, see Table 1). Moreover, in cases where no cytotoxicity was observed (the highest concentration resulted in precipitation of the compound) contrary results were seen in the respective repeat experiment. This further demonstrates that contrary results between repeat experiment (as described above) are obviously not related to ‘false’ positive calls which might have resulted from excessive cytotoxicity. 3.4. Precipitation and influence on DNA migration In Table 5, results of 84 individual tests with compounds tested up to precipitating concentrations are shown. In this dataset, positive as well as negative compounds were used to investigate whether discrepant results are obtained at the highest soluble concentration and the next higher, precipitating concentration. In 88% of the tests, results at soluble and precipitating concentrations were the same. In only 10 individual tests, a contrary result was obtained.

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Table 4 Summary of data on positive compoundsa Compound code

Test no. −S9

4 5 5 5 7 7 8 8 9 10 10 10 11 11 11 12 13 13 13 14 18 18 19 19 20 21 21 21 23 23 24 24 25 25 26 27 29 29 33 33 34 34 35 35 35 36 36 37 37 37 38

Result

TBD positive cells (%)

NDCN (%)

Positive only at TBD >6%

(+) + + + + + + + + + + + + + + (+) + + + + + + + + + + + + + + + + + + (+) + + + + + (+) + + + + + + + + + +

1.3 12 3.5 2.5 0 0.5 0.5 1.5 6.5 2.5 1 1.5 2 0 1.5 2.5 1 6.3 2 3 2.5 2 15.5 1.5 10 2 1.5 1 0.8 8.8 20 6 0 10.8 7.3 10 4.8 5 30 4.5 22 3.5 5 20 4.5 3 26 6 15 4 5.5

0 10 9 4 0 0 0 0 8 0 10 0 16.5 0 0 12.5 5 2.5 8 0 14.5 10 12 10.5 2 13.5 13.5 12 0 4.5 6.5 2 2.5 4 11.5 13 15.5 5 13.5 5 11.5 2 13.5 5.5 5 0 3 2.5 15 2.5 1

N Y N N N N N N Y N N N N N N N N Y N N N N Y N Y N N N N Y Y N N Y Y Y N N Y N Y N Y Y N N Y N Y N N

+S9 1

1 1 2 1 1 1 1 3 1 2 1 2 3 1 1 1 1 2 1 1 2 2 3 1 1 1 2 1 2 1 2 1 2 1 1 1 2 1 2 1 1 1 2 3 1 2 1 1 2 1

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Table 4 (Continued) Compound code

Test no. −S9

38 39 39 42 42 44 44 45 45 46 46 48 48 51 51 52 52 57 62 62 67 67 68 68 69 69 69 70 53 53

2 1 2 1 2 1 1 1 2 1 2 1 1 1 2 1 1 2 1 2 1 2 1 1 1 2 1 2 1 2 a

Result

TBD positive cells (%)

NDCN (%)

Positive only at TBD >6%

+ + + + + + + + + + + + + + + + + + + + + (+) + + + + + + + +

10 20 10 1.5 1.5 10 1.5 1 2 0 1 29 12 18 15.5 16 10 1 4 0 6.5 1 3 0.5 10 8 0 2 0 3

2 5 12 5 7 28 4.5 7.5 0 12.5 3.5 13.5 9.5 9.5 7 9.5 3.5 5.5 6 0 11 1.8 0 1.5 2 2 0 0 3.5 0.5

Y Y Y N N Y N N N N N Y Y Y Y Y Y N N N Y N N N Y Y N N N N

+S9

TBD: Trypan blue dye; NDCN: non-detectable cell nuclei; Y: yes; N: no; (+) weakly positive call; +: positive call.

Table 5 Effect of test compound precipitation on tests resultsa Compound code

Test no. −S9

2 3 4 4 5 7 8 8 14 14 15 15 16

Test result

Result at precipitating concentration

Result at highest soluble concentration

Same result at precipitation/ non-precipitation concentration

− − − (+) + + + + − − − − −

− − − − + + + + − − − − −

− − − (+) + + + + − − − − −

Y Y Y N Y Y Y Y Y Y Y Y Y

+S9

1 1 1 1 1 1 1 1 2 1 1 2 1

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Table 5 (Continued) Compound code

Test no. −S9

17 18 18 18 19 20 22 22 22 22 28 28 33 36 37 37 41 41 42 43 43 44 44 45 46 47 48 49 50 50 54 59 59 61 61 61 62 63 63 63 63 64 64 64 65 65 65 66 66 66 66 67

Result at precipitating concentration

Result at highest soluble concentration

Same result at precipitation/ non-precipitation concentration

− − + + − − − − − − − − + − + + − − + − − + + − − − − − − − − − − − − − − − − − − − − − − + − − − + (+) −

− − + + − − − − − − − − + − + + − − + − − (+) (+) − − − − − − − − − − − − − − − − − − − − − − + − − − − − +

− − + + − − − − − − − − + − − + − − + − − (+) (+) − − − − − − − − − − − − − − − − − − − − − − + − − − (+) + −

Y Y Y Y Y Y Y Y Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N N

+S9 1

1 1 2 1 2 1 2 1 2 1 2 1 1 1 2 1 2 2 1 1 1 1 1 1 1 2 1 1 1 1 1 2 1 1 2 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

Test result

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209

Table 5 (Continued) Compound code

Test no. −S9

67 68 68 69 69 69 70 70 70 70 72 72 73 73 74 74 75 75

Result at precipitating concentration

Result at highest soluble concentration

Same result at precipitation/ non-precipitation concentration

(+) + + + + (+) − + (−) − − − − − − − − −

+ + + + − − − + − − − − − − − − − −

− + + + + + − − + − − − − − − − − −

N Y Y Y N N Y N N Y Y Y Y Y Y Y Y Y

+S9 2

1 1 1 2 1 1 2 1 2 1 2 1 2 2 2 1 1 a

Test result

Y: yes; N: no.

Table 6 Examples of individual results of compounds tested up to precipitating concentrationsa Compound code

Test no. (3 h) −S9

Concentration (␮g/ml)

Tail moment

TBD positive cells (%)

NDCN (%)

+S9

4

0 2.5 10 20b

7

1.1 2.3 1.8 1.1

1.8 1.0 1.0 1.0

0 0 0 0

0 30 67 100b

2.1 3.0 4.3 9.8

1.5 1.0 0.5 0.5

0 0 0 0

37

1

0 42 83 166b

1.4 1.0 1.6 8.2

2.0 2.0 1.0 5.0

0 0 1.0 7.0

37

2

0 100 150 200 250b

1.6 3.4 7.4 7.0 13.6

1.5 1.5 2.0 3.0 4.0

0.5 0 0.2 1.0 2.5

66

1

0 46.3 69.4 86.5 104.0b

2.0 2.1 3.6 4.3 1.4

0.8 0.8 0.8 0.8 0.8

0 0 0 0 0

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Table 6 (Continued) Compound code

Test no. (3 h) −S9

67

TBD positive cells (%)

NDCN (%)

0 46.3 69.4 86.5 104.0b

1.7 2.6 3.6 3.5 3.1

0.3 0.3 0.3 0.3 0.3

1

0 13.1 19.7 29.6b

1.8 3.7 5.7 4.8

0 6.0 3.0 10.0

0 0 0 2.0

2

0 15.0 22.0 28.0 35.0 44.4b

2.4 4.1 6.0 3.9 5.7 3.3

1.0 1.0 3.5 2.0 8.0 7.5

0.5 0 0 1.5 2.0 2.0

0 28.9 43.3 65.0b

1.7 2.2 4.2 2.5

0 0 0 1.0

0 0.5 0.5 5.5

1

0 14.6 26.4 47.5

1.4 1.9 1.8 1.4

4.5 1.0 0.5 1.5

0 1.0 0 0

2

0 75.0 100.0b 154.0b

2.1 3.8 4.7 5.1

1.0 0 2.0 2.0

0 0.5 1.0 0

1

0 26.4 47.5 85.5 154.0b

1.3 2.5 2.2 3.1 2.5

2.0 2.5 1.0 0 1.5

0 0 1.5 0 0.5

2

0 50 75 100 154.0b

2.5 4.1 3.1 4.4 3.2

2.5 3.5 2.0 1.5 3.0

0 4.0 0.5 1.0 0

1

0 41.3 62.0 93.1 140.0b

4.5 2.7 2.6 3.1 10.2

1.5 0.8 1.5 2.3 8.5

0 0 0 1 1.5

2

0 78.1 93.1 116.1 140.0b 0

3.3 2.8 3.0 4.7 Toxic 1.6

1.0 7.5 7.0 3.0 1.5 1.0

0 4.0 7.5 13.5 21.0 0

1

70

Tail moment

+S9 2

69

Concentration (␮g/ml)

1

0 0 0 0 0

A. Hartmann et al. / Mutation Research 497 (2001) 199–212

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Table 6 (Continued) Compound code

Test no. (3 h) −S9

b

Tail moment

TBD positive cells (%)

NDCN (%)

+S9

2

a

Concentration (␮g/ml)

62.0 93.0 115.0 140.0b

2.6 3.4 4.5 5.8

1.0 0.8 2.8 6.5

0 7.0 7.5 11.0

0 62.0 93.0 115.0 140.0b

1.7 1.7 2.0 3.0 3.8

2.5 2.5 0.5 1.5 1.0

0 0 0 0 1.8

Positive concentrations are in bold. Precipitating concentration.

Table 7 Historical control data for the comet assaya Treatment condition

No. of experiments

Average tail moment ± S.D.

Solvent, 3 h, without S9 Solvent, 3 h, with S9 EMS, 5 mM 2-AA, 23 ␮M

172 124 172 124

1.41 1.54 4.56 5.33

a

± ± ± ±

0.4 0.6 1.4 3.2

Minimum

Maximum

0.5 0.9 1.2 1.5

4.5 2.1 10.3 12.8

EMS: ethyl methanesulfonate, 2-AA: 2-amino anthracene.

However, 5 of the 10 contrary test results were not confirmed in the respective repeat experiment which might have been to closer dose-spacing in the repeat experiment. Therefore, the reproducibility of test results with compounds tested in precipitating concentrations is in the same range as the overall reproducibility of test results. Furthermore, the results show that precipitation does not create artificial conditions leading to high percentages of positive calls. Table 6 lists several individual test results. Historical control data is presented in Table 7.

4. Discussion In the present study, comet assay data on 75 in-house synthesized new drug candidates was used to further elucidate the ability of the comet assay to specifically detect genotoxic effects and to investigate a possible influence of test compound precipitation on test results. The results strongly support earlier data demonstrating that in V79 Chinese hamster cells or human leukocytes extensive DNA fragmentation induced by cytotoxicity does not lead to elevated DNA

migration values (i.e. false positive calls) in the comet assay [9]. However, different cell lines may behave differently and it was reported that non-genotoxins at cytotoxic concentrations may induce DNA migration in the comet assay in TK6 human lymphoblastoid cells [10] or rat lymphocytes [16]. It was, therefore, recommended to limit concentrations of test compounds to ≥75% viability, where no longer increased migration values after treatment with non-genotoxins was detected [10]. However, the present data show that using V79 cells, excessive cytotoxicity did not result in a positive call. The potential problem of possible false positive calls in the comet assay due to excessive cytotoxicity arises from extensive DNA fragmentation upon cell death. The comet assay has the advantage that dead or dying cells can be identified on microscope slides by their morphology. Such cells exhibit completely fragmented chromatin without a visible nucleus [3]. The present data show that scoring NDCN provides a valuable parameter to obtain information about cytotoxicity. However, NDCN should not be used as the sole parameter for cytotoxicity because complete fragmentation of chromatin of a cell can result from

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two different mechanisms: cell death or genotoxic action. As a result, a typical pattern of a cytotoxic non-genotoxin should, therefore, be the occurrence of NDCN among several undamaged cells. By setting our evaluation parameters in a way that NDCN are not analyzed by the automated image analysis, we found no elevated DNA migration values even when a high number of NDCN was present. Based on the results of this evaluation, our concentration-selection strategy of using two parameters of cytotoxicity (a toxic concentration defined as to induce viability by TBDE ≥70% or NDCN ≤15%) proved to be very useful. Interestingly, there was no obvious relationship between TBDE and NDCN values, i.e. a high value in one of the parameters was not necessarily accompanied by a high value of the other parameter. The specificity of the comet assay for detecting genotoxicity was also demonstrated in a previous comparative investigation, evaluating results with 33 compounds tested in the comet assay and the micronucleus test (MNT) showed that compounds being positive in the comet assay were also positive in the MNT [17]. On the other hand, several MNT-positive compounds came out negative in the comet assay. We assumed that the main reason for the higher proportion of positive results in the MNT compared to the comet assay may be the influence of cytotoxicity on test results. The compounds were tested up to high cytotoxic concentrations in both tests. It is known that at low levels of cell survival, mechanisms other than genotoxicity can lead to chromosome damage related to cytotoxicity [18]. The present evaluation further demonstrates that the comet assay is not prone to false positive calls due to cytotoxicity under the test conditions and with the cell line used. References [1] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191. [2] E. Rojas, M.C. Lopez, M. Valverde, Single cell electrophoresis: methodology and applications, J. Chromatogr. B 722 (1999) 225–254. [3] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J.-C. Ryu, Y.F. Sasaki, The single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35 (2000) 206–221.

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