Mutagenesis vol.13 no.l pp.89-94, 1998

The ability of the Comet assay to discriminate between genotoxinsand cytotoxins

Leigh Henderson1, Alison Wolfreys, Julia Fedyk, ClareBourner and Sam Windebank

Environmental Safety Laboratory, Unilever Research, Colworth House,Sharnbrook MK44 1LQ, UK

The Comet assay has been used widely in genetic toxicology,radiation biology and medical and environmental research.This assay detects single-strand breaks and alkali-labilesites in DNA and DNA degradation due to necrosis orapoptosis. It may also be modified to detect DNA cross-Unking. Although a considerable number of chemicals havebeen tested in the assay there are many aspects of validationto be considered before the method could be considered toprovide definitive evidence of genotoxic potential. Forexample, very few non-genotoxins have been tested toassess specificity of the Comet assay and there has beenonly one reported study which investigated whether thein vitro Comet assay is prone to false positive responsesdue to cytotoxicity. We have investigated the response ofthe alkaline Comet assay in TK6 human lymphoblastoidcells to cytotoxic damage and genotoxic damage. Severalcompounds which are toxic by different mechanisms weretested in the assay. Cycloheximide and trypsin gave anegative comet response at a highest dose of 5 mg/ml andno toxicity was observed. Sodium lauryl sulphate andpotassium cyanide produced a significant increase in DNAmigration at cell survival levels of =575%. The distributionof damaged cells indicated that cells at various stages ofnecrotic cell death were present Hydrogen peroxide, 4-nitroquinoline oxide, 9-aminoacridine, ethyl methanesulph-onate, iV-nitroso-Af-ethylurea and glyoxal gave a positivecomet response. Mitomycin C was negative at survivallevels of -70%. These results indicate that the maximumconcentration of test substance tested should produce viabil-ities >75% in order to avoid false positive responses dueto cytotoxicity. The assay was able to detect DNA damageinduced by an alkylating agent, an intercalating agent andoxidative damage. The cross-Unking agent mitomycin Cwas not detected if a cut-off point of 75% viabiUty is usedas the criterion of a positive response.

Introduction

The Comet assay promises to be a rapid in vitro screeningassay for detection of genotoxins and also a site of contactassay for in vivo genotoxins. Before it can be used forinvestigation of substances of unknown genotoxic potentialthe assay should be rigorously validated. To date many differentprotocols have been used and the variations encompass use ofdifferent cell types, exposure regimes, use of DNA repairinhibitors, lysis and electrophoresis techniques and scoringcriteria. The effects of varying many of these factors ondetection of genotoxins in the assay are not known. Critical

Ttahlp I. In vitm Pnmrl RtiiHirs in mflmmfllinn rf.lW

Genotoxins Non-genotoxins

Positive in Comet assayEMS (1,6)Cyclophosphamide (2,7)Hydrogen peroxide(5,8,10,13,15)BPDE (6)Benzo[a]pyrene (7)MNNG (7,9,19)UVC (16)X-rays (8,21)Cobalt particles (3)MMS (16)Sodium arsenite (9)Nickel subsulphide (12)Cadmium (9)Styrene oxide (4)Danthron (18)Etopside (18)m-Amsacrine (18)ENNG (20)ENU(20)Aloe-emodin (18)Chlorinated aliphatichydrocarbons (2)

Negative in Comet assayEmodin (18) Tetrachloroethylene (7)

p-Nitrophenol (17)D-Menthol (17)Sodium lauroyl sarcosine(17)

Unknown

Morphine (11)ICI D1694 (14)

Tungsten (3)PCB77 (1)

(1) Belpaeme et al., 1996; (2) Tafazoli and Kirsch-Volders, 1996; (3) Anardet al., 1997; (4) Bastlova et al., 1995; (5) Holz et al., 1995; (6) Leroy et al.,1996; (7) Hartmann and Speit, 1995; (8) Singh et al., 1988; (9) Hartmannand Speit, 1994; (10) Singh et al., 1991; (11) Shafer et al., 1994; (12)Zhuang et al., 1996; (13) Lee et al., 1996; (14) Schober et al., 1994; (15)Collins et al., 1993; (16) Green et al., 1992; (17) Hartmann and Speit,1997; (18) Muller et al., 1996; (19) Slamenova et al., 1991; (20) Fortiniet al., 19%; (21) Singh et al, 1990.

factors for conduct of the assay, such as the appropriate toxicitylimits and criteria for a positive response, have not beensystematically investigated. The intra-laboratory and inter-laboratory variability in detecting standard genotoxins in theassay have also not been studied. The sensitivity and specificityof the assay (i.e. ability to detect known genotoxins and abilityto discriminate between genotoxins and non-genotoxins) is notknown. Table I shows a compilation of the substances orexposures which have been assessed in mammalian cells. Theclassifications of positive and negative responses are those ofeach of the authors. Most of the substances investigated aregenotoxins and therefore give no information on the specificityof the assay. To date it would appear that the sensitivity ishigh, with only one substance, emodin, which is genotoxic inother assays reported to be negative in the Comet assay (Mulleret al., 1996). Although it is recognized that the Comet assay

'To whom correspondence should be addressed. Tel: +44 1234 222316; Fax: +44 1234 222122; Email: leigh.henderson@unilever.com

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L.Henderson et al

Table IL Induction of DNA migration by cytotoxins and genotoxins

Chemical Expt No. Dose (mg/ml) Absolute Toxicity(% Trypan Blue exclusion)

Mean Tail Moment Mean Tail Length

Sodium Lauryl Sulphate

Potassium Cyanide

Trypsin

Cycloheximide

Mitomycin C

Hydrogen Peroxide

06.2512.525.037.5020253035400

40005000

040005000

010002000300040005000

010002000300040005000

010002000300040005000

010002000300040005000

00.11.010.0

100.01000.0

010025050010002000

03.46.817.034.001.73.45.16.88.517.00

929386842688888988917593706787857090919088877973778182797791949496939091899092898785868577767285888679897088929192928283858988889392

0.550.720.500.51

11.36**0.440.500.490.290.982.08**0.731.352.52**0.390.681.00**0.680.720.540.731.260.710.640.630.961.030.490.800.560.490.920.56

0.390.690.590.500.470.50-0.300.500.500.631.030.591.040.850.750.811.031.010.901.101.62

13.88"25.88"27.44**29 .33"0.661.120.971.161.57"4.70**

16.53**0.37

22.719.920.120.955.920.018.319.417.419.724.426.936.453.817.422.227.423.730.025.326.831.128.424.225.127.735.434.327.523.223.125.824.621.523.821.120.721.921.920.618.919.119.722.620.321.330.222.824.927.128.527.538.027.865.979.177.276.820.124.024.526.633.244.154.218.4

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The ability of the Comet assay to discriminate between genotoxiiis and cytotoxins

Table H. Continued

Chemical Expt No. Dose (rag/ml) Absolute Toxicity(% Trypan Blue exclusion)

Mean Tail Moment Mean Tail Length

Glyoxal

4-Nitroquinoline oxide 1

Ethyl Methane Sulphonate 1

Ethyl Nitrosourca

9-Aminoacridine

10030010003000

030010003000

00.010.030.10.300.010.030.10.31.001031.61003161000

03604806009600

24036048060096001030100300

929189939298949469637171738486909088839393949591919696966095938694899090977572201

0.220.120.41

15.715.319.3

4.34** 52.70.321.051.78

18.332.237.1

15.91" 82.40.191.30

16.431.5

3.00" 48.26.52*• 70.312.22** 76.10.400.522.94*6.37*'19.22"34.21*'0.120.140.130.210.242.07*'0.227.39*'7.06*4.03*'8.43*'0.413.82*'5.12"7.67*'12.38*'19.51*'0.112.93"5.10*'5.29"10.03*'

23.321.0

' 50.8" 64.6' 71.7' 104.4

13.415.515.516.616.8

> 32.116.2

> 81.91 84.2» 75.9' 88.6

17.8' 56.7• 74.1' 86.8' 81.8' 91.8

13.8' 36.4" 58.4> 59.8

71.7

•P<0.05; **P<0.01

can be used to quantify apoptotic cells (Olive et al, 1993)and it may be used to discriminate between apoptosis andnecrosis (Fairburn et al, 1996), only one group has investigatedwhether cytotoxins may induce DNA migration. Hartmannand Speit (1995, 1997) have studied four cytotoxic compoundsand concluded that the assay is not likely to give false positiveresponses due to cytotoxins. However, the induction of DNAmigration by trypsinizing or scraping fibroblasts has also beenreported (Singh et al, 1991).

In the present study we have investigated the induction ofcomets by several cytotoxic and genotoxic substances toinvestigate the specificity and sensitivity of the assay and todetermine the appropriate limits of toxicity for the assay. Theresponse of the alkaline Comet assay to various types ofcytotoxins was assessed using sodium lauryl sulphate (SLS),potassium cyanide (KCN), trypsin and cycloheximide. SLSand trypsin exert their toxic effects on the cell membrane,KCN exerts its toxic effect on the cell electron transfer chainand cycloheximide exerts its toxic effect by blocking translationof mRNA and thus inhibiting protein synthesis. The responseof the Comet assay to various classes of genotoxin was also

assessed. The genotoxins tested were hydrogen peroxide,glyoxal and 4-nitroquinoline oxide, which are oxidizing agents,ethyl methanesulphonate and /v'-nitroso-./V-ethylurea, which arealkylating agents, 9-aminoacridine, which is an intercalatingagent, and mitomycin C, which is a cross-linker.

Materials and methods

Test substances

These were hydrogen peroxide, 9-aminoacridine, JV-nitroso-/V-ethylurea, 4-nitroquinoline oxide, SLS, mitomycin C and cycloheximide (Sigma ChemicalCo.), KCN and glyoxal (Aldrich), ethyl methanesulphonate (Koch Light Ltd)and trypsin (Gibco Laboratories). Initial selection of the highest concentrationsfor each agent were based on either an arbitrarily defined upper limit of 5000Hg/ml or, in the case of known genotoxins, a level expected to induce DNAdamage based on consideration of the literature. Preliminary assays wereperformed using the conditions of the Comet assay to determine toxicity ofthe chemicals.

Comet assay

The method used was based on that of Singh et al (1988). Aliquots of 1.0ml cell suspension containing either 2.5 x 10* or 4.0X105 TK6 cells in RPMI1640 medium were dispensed into Eppendorf tubes (two for each dose level)and the cells pelleted by centrifuging at 2000 r.p.m. in a microcentrifuge for

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L.Henderson el aL

Sodium Lauryl SulphatePercentage Survival Tail Moment

10 20 30ug/ml Sodium Lauryl Sulphate

40

Percentage Survival120

TrypsinTail Moment

1412108642

1,000 2,000 3,000ug/ml Trypsin

4,000 5,C

CyclohexamidePercentage Survival Tail Moment

1201412

10

8

6

42

100

80

60

40

20

1412108

6

4

2

1,000 2,000 3,000 4,000ug/ml Cycloheximide

5,0

Potassium CyanidePercentage Survival Tail Moment

120

1,000 2,000 3,000 4,000ug/ml Potassium Cyanide

5,C

Figure 1. Induction of comets by SLS, cycloheximide, trypsin and KCN. • , Survival, test 1; • , tail moment, test 1; O, survival, test 2; Q tail moment, test2; , tail moment doubled, test 1; •••, tail moment doubled, test 2.

2 min. Each cell pellet was resuspended in 1.0 ml fresh RPMI 1640 mediumwithout fetal calf serum containing the test substance at the required dosesand the cells incubated at 4°C for 30 min. Following treatment the cells werepelleted and washed twice in fresh medium and finally resuspended in 0.2 5ml fresh medium without fetal calf serum. Viability of the cell suspensionwas assessed by trypan blue exclusion. Recovery of cells was also measured.The remaining cell suspension was used for preparation of slides.

Aliquots of 85-110 |il 0.75% NMP agarose were added to warmed frostedmicroscope slides (2-4 slides/dose) and a coverslip immediately placed ontop. The slides were held on ice to allow the agarose to solidify. Cellsuspension (10 ul, 10 000 cells) was added to 75 uJ 0.5% LMP agarose, induplicate for each Eppendorf tube. The coverslips were removed from theslides and the slides allowed to warm to room temperature before 75 uJ cellsuspension in agarose was spread onto the slide. A coverslip was placed ontop and the slides placed on ice to allow the agarose to solidify. The coverslipswere then removed and 85 uJ 0.5% LMP agarose were spread onto the slide,a coverslip placed on top and the slides placed on ice to solidify. Thecoverslips were removed and the slides carefully placed in cold freshlyprepared lysis solution (2.5 M NaCl, 100 mM EDTA, 10 raM Tris, 1% sodiumsarcosinate adjusted to pH 10 and supplemented with 1% Triton X-100 and10% DMSO prior to use) and held in the refrigerator for 2 h.

Slides were removed from the lysis solution, rinsed gently in electrophoresisbuffer (1 mM EDTA and 300 mM NaOH, pH 13) and placed in a horizontalgel box containing electrophoresis buffer at a constant temperature of ~18°C.The slides were left for 40 min prior to electrophoresis to allow DNAunwinding and then electrophoresed for 20 min at -25 V and 300 mA. Theywere drained and neutralized with neutralization buffer (pH 7.5). Excessneutralization buffer was drained from the slides and 60 ul ethidium bromidesolution (20 ug/ml) added. Slides were viewed using a Zeiss Axioskopfluorescence microscope with a Green H546 filter and scored using theColourmorph image analysis system (Perceptive Instruments). Data on taillength, tail moment, head intensity, tail intensity, total intensity and total areawere recorded. Fifty cells were scored from each slide. Only cells which hada defined head were scored, i.e. comets of uniform intensity, which areprobably dead cells, were excluded. Most assays were performed at leasttwice. Statistical analysis was performed on the tail moment only. An ANOVAwas performed and, where positive, a t test was used to evaluate die loweststatistically significant concentration.

Results

The induction of DNA migration for all chemicals tested isshown in Table II. The effects of the non-genotoxins SLS,KCN, trypsin and cycloheximide are illustrated in Figure 1.Trypsin and cycloheximide, when tested up to 5000 |i.g/ml,the maximum concentration recommended for mammalian cellgenotoxicity assays, did not affect the viability or increase thetail length or tail moment in TK6 cells. SLS and KCN bothinduced DNA migration. SLS induced a significant increasein tail moment in the first test at 37.5 p.g/ml, when cell survivalequalled 26%, and in the second test at 40 (ig/ml, when cellsurvival was 75%. A significant increase in tail moment wasseen in both tests with 5000 (lg/ml KCN, where survivalequalled 67 and 70%. The distributions of damaged cellsfollowing treatment with SLS and KCN are shown in Figure 2.This indicates that the increase in tail moment is not due solelyto very damaged cells and may represent cells at various stagesof necrotic cell death.

Hydrogen peroxide, glyoxal, 4-nitroquinoline oxide (oxidiz-ing agents), ethyl methanesulphonate, /V-nitroso-N-ethylurea(alkylating agents) and 9-aminoacridine (an intercalating agent)induced large dose-related increases in tail length and tailmoment at cell survivals of >70%. Mitomycin C did notinduce a statistically significant increase in tail moment, eventhough a concentration resulting in a viability of ~70%was tested.

DiscussionComparison of the results for cytotoxins and genotoxinsshowed that cytotoxins are able to induce an increase in DNAmigration, but this was only seen where viability was =£75%.

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No. of Cells

The ability of the Comet assay to discrimiiiate between genotoxiiis and cytotoxms

Sodium lauryl sulphate

30 Concentration (ug/ml)

0-15 15-3 3-4 5 45-6 6-7.5 7.5-9 9-10.5 10.5-12 >UTail Moment

No. of CellsPotassium cyanide

Concentration (ug/ml)

0-15 15-3 3-4.5 4 5-6 6-7 5 7.5-9 9-10.5 10.5-12 >I2Tail Moment

Figure 2. Tail moments induced by SLS (test 2, maximum toxicity 75%) and KCN (test 1, maximum toxicity 67%).

Therefore, to avoid false positive responses with substancesof unknown hazard the viability of the cells used in the in vitroComet assay should exceed 75%. Any increase in tail lengthor tail moment should be dose related.

Induction of Comets by cytotoxins in this study differs fromresults reported recently by Hartmann and Speit (1997). Theysaw no effects in V79 cells exposed for 2 h to the threecytotoxins p-nitrophenol, D-menthol and sodium lauroyl sarco-sine and no evidence for cells intermediate in damage betweenintact cells and very highly damaged cells. The two studiesdiffer in the cell types used and the exposure time. Possiblythe shorter exposure time used in the present study could resultin cells in the early stages of necrosis being present, whereasthe 2 h exposure time used by Hartmann and Speit may haveresulted in complete loss of damaged cells. Whereas in thepresent study trypsin treatment of lymphoblastoid cells did notinduce DNA migration, Singh et al. (1991) reported thattrypsinization of fibroblasts induced DNA migration (using alower concentration than that used in the present study). Similareffects were induced by scraping. These effects occurred inthe absence of cytotoxicity, as measured by trypan blue

exclusion. Singh et al. hypothesized that these effects mayhave resulted from endonuclease release as a consequence ofmembrane damage or by the activation of toxic oxygen speciesgenerated by activation of NADPH cytochrome P450 reductase.Although not confirmed by our study, this report raises someconcern that Comet formation may reflect indirect DNAdamage arising as a secondary consequence of physical orchemical damage to cell membranes.

In the present study toxicity was assessed by trypan blueexclusion. This may underestimate toxicity, since it is basedon membrane integrity and loss of this function is a late eventof apoptosis (Fairbum et al, 1996). Although cell counts werealso measured, this was not informative in setting toxicitylimits for the assay. The measurement of long-term cell survivalmay provide a more definitive measure of toxicity, but it isless practical for a short-term assay and is not applicableto all cell types, particularly peripheral blood lymphocytes.Although it was not recorded as part of the present study, itwould be prudent to record the frequency of cells which areexcluded from analysis because their morphology suggests theyare apoptotic or necrotic. This may be useful in determining an

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L.Henderson et at

appropriate toxicity limit, as suggested by Tafazoli and Kirsch-Volders (1996).

The Comet assay was able to detect genotoxicity inducedby a range of genotoxins, including an intercalating agent, analkylating agent and an oxidizing agent. This adds to thegrowing database on chemicals which are detectable in theassay. The assay gave a negative result with a cross-linkingagent. Modification of the assay allows detection of cross-linkers based on their ability to retard DNA migration (Pfuhlerand Wolf, 1996). Several studies have compared the sensitivityof the Comet assay to other in vitro mammalian genotoxicitymethods and higher (Tafazoli and Kirsch-Volders, 1996),similar (Hartmann and Speit, 1995; Muller et al, 1996) andlower (Hartmann and Speit, 1994, 1995) sensitivity (measuredas lowest effective dose) for different chemicals and testmethods have been reported. However, further work is requiredto evaluate this method against standard regulatory genetictoxicology assays.

The aim of this study was to detect whether the Cometassay can distinguish genotoxins and cytotoxins and not tooptimize the protocol for genotoxicity testing. Consequently,important parameters, such as exposure time, duration andtemperature, lysis and electrophoresis conditions and incorp-oration of exogenous metabolic capability, were not addressedas part of this study. Treatment of the cells at 4°C was usedto prevent repair. Obviously, this would not be appropriate forsubstances requiring bioactivation and may not be the optimaltemperature for other substances. However, no difference wasseen in the response of cells treated with mitomycin C at 4 or37°C (data not shown).

While this study supports others showing that the methodmay be useful as a rapid in vitro screen for genetic toxicologyscreening, validation will require investigation of protocolfactors affecting detection (especially cell type, lysis andelectrophoresis conditions and exposure times) and modifica-tion of the conditions (exposure temperature and duration)to assay substances requiring metabolic activation. Furtherinvestigations of the specificity and sensitivity of the assay andmeasurement of intra-laboratory and inter-laboratory variabilityare also required.

AcknowledgementsWe are grateful to Andrea Dickens for statistical advice. This work was partlysupported by EU project grant AIR2-CT93-0860.

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Received on June 13, 1997; accepted on October 20, 1997

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