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Mutagenesis vol. 26 no. 6 pp. 689695, 2011 doi:10.1093/mutage/ger034Advance Access Publication 21 July 2011

The effects on DNA migration of altering parameters in the comet assay protocol suchas agarose density, electrophoresis conditions and durations of the enzyme or thealkaline treatments

Clara Ersson and Lennart Moller*

Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 83Huddinge, Sweden.

*To whom correspondence should be addressed. Tel: 46 8 524 810 75;Fax: 46 8 774 55 38; Email: [email protected] on February 22, 2011; revised on May 13, 2011;accepted on May 17, 2011

The single cell gel electrophoresis (comet assay) isa popular method for measuring DNA migration as anestimate of DNA damage. No standardised comet assayprotocol exists, which make comparisons between studiescomplicated. In a previous inter-laboratory validationstudy of the comet assay, we identified importantparameters in the protocol that might affect DNAmigration. The aim of this study was to assess howdifferent comet assay protocols affect DNA migration. Theresults in this study suggest that (i) there is a significantlinear doseresponse relationship between the agarose gelsdensity and DNA migration and that damaged cells aremore sensitive to the agarose gels density; (ii) incubationwith formamidopyrimidine DNA glycosylase for 10 min isinadequate, whereas 30 min is sufficient; (iii) the typicallyused 20 min of alkaline treatment might be to short whenanalysing samples that contain particular alkali-labile sites(ALS) and (iv) the duration of electrophoresis as well as thestrength of the electric field applied affects the DNAmigration. By using protocol-specific calibration curves, itis possible to reduce the variation in DNA migrationcaused by differences in comet assay protocols. This does,however, not completely remove the impact of thedurations of alkaline treatment and electrophoresis whenanalysing cells containing ALS that are relatively resistantto high alkaline treatment.

Introduction

The single cell gel electrophoresis (comet assay) is a frequentlyused method to measure DNA migration as an estimate ofDNA damage. With the alkaline comet assay (pH . 13), DNAbreaks, i.e. strand breaks and alkali-labile sites (ALS) aremeasured. The increasing use of the comet assay is attributed tothe low number of cells required, its sensitivity in detecting lowlevels of DNA damage and the ability to measure differenttypes of DNA damage by adding lesion-specific enzymes (1).In the formamidopyrimidine DNA glycosylase (FPG) cometassay, the level of 8-oxoguanine as well as other altered purinesis measured by incorporating a digestion with the enzyme FPG(1). FPG recognises and removes specific oxidated purinescreating apurinic (AP) sites. The AP sites are transformed toDNA strand breaks either by the enzymes associated lyase

activity or by the subsequent alkaline treatment. By their abilityto relax DNA supercoiling, DNA breaks allow the DNA tomigrate out of the nucleoids under the influence of an electricfield. A comet-like image is created with a tail consisting ofrelaxed and mainly single-stranded DNA. Several guidelinesfor the comet assay have been published (25), but nostandardised protocol exists and there are considerable differ-ences in the protocols used by different research groups. Thesedifferences negatively affect inter-laboratory comparisons ofresults. In a previous inter-laboratory validation study of thecomet assay, we identified important parameters in the cometassay procedure that might affect the DNA migration, such asthe agarose density, duration of the enzyme treatment, durationof the alkaline treatment and duration of the electrophoresis (6).The duration of the alkaline treatment as well as the duration ofthe electrophoresis have been shown to significantly affect thelevel of DNA migration in c-irradiated cells (7,8). Forchham-mer et al. (7) concluded that differences could be reduced toa large extent by using protocol-specific calibration curves.

In this study, the impact on DNA migration by differentcomet assay protocols was investigated. Key parameters in thecomet assay protocol including agarose density, durations ofenzyme treatment, alkaline treatment and electrophoresis andstrength of the electric field were altered and the impact onDNA migration was assessed.

Materials and methods

Study design

Cells treated with Ro 19-8022 plus visible light were analysed with the FPGcomet assay, whereas c-irradiated cells and cells treated with hydrogenperoxide (H2O2) were analysed with the alkaline comet assay. Only oneparameter at a time in a standard comet assay protocol was altered: (i) theagarose density, (ii) the duration of the enzyme treatment, (iii) the duration ofthe alkaline treatment, (iv) the duration of the electrophoresis or (v) the strengthof the electric field during electrophoresis. Each experiment was repeated threeto four times. This study did not take into account the effect of these parameterson the detection of cross links.

The selected densities of the agarose gels, the durations of the enzymetreatment, the alkaline treatment and the electrophoresis as well as the strengthsof the electric field were to a large extent based on the protocols used by ourcollaborators in the European Comet Assay Validation group (ECVAG) (6).

FPG was kindly provided by Prof. A.R. Collins (Department of Nutrition,University of Oslo, Norway).

Preparation of cells containing DNA damage

c-Irradiation. Human acute monocytic leukaemia THP1 cells in phosphate-buffered saline (PBS) were irradiated with c-rays at 0, 2.5, 5 and 7.5 Gy aspreviously described (7). The aliquots, predominately containing DNAbreaks, were frozen slowly down to 80C in freezing medium [50% foetalbovine serum (FBS), 40% RPMI 1640 medium and 10% dimethyl sulfoxide(DMSO)].

H2O2 treatment. A549 human type II alveolar epithelial cells were culturedand harvested as previously described (9). The cells were exposed to 200 lMH2O2 in PBS for 5 min on ice. Following exposure, the cells were washed withPBS, isolated by trypsination (5 min) and re-suspended in cell medium. Aftercentrifugation, the cell pellet was suspended in freezing medium (50%

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Dulbeccos modified Eagles medium, 40% FBS and 10% DMSO) and aliquotswere frozen slowly down to 80C in a polystyrene freezing box.

Exposure to photosensitiser and light. A549 cells were treated with 0.6 lMphotosensitiser Ro 19-8022 in PBS and visible light, which predominantlygenerates 8-oxoguanine (10). Cells were irradiated on ice for 5 min froma distance of 33 cm with a 500-W tungsten halogen lamp. Followingirradiation, cells were immediately washed with PBS, isolated by trypsination(5 min) and re-suspended in cell medium. Cells were centrifuged and cellpellets were suspended in freezing medium and frozen slowly down to 80Cin a polystyrene freezing box.

Experimental set-up

Standard comet assay protocol. Cells were embedded in 0.7% low meltingpoint agarose (final concentration) and then lysed (2.5 M NaCl, 0.1 M EDTA,10 mM Tris, pH 10) for a minimum of 1 h at 4C. In the FPG comet assay, thesamples were washed three times for 5 min in buffer (40 mM HEPES, 0.1 MKCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, pH 7.9, 4C) and thenincubated with only buffer or buffer containing FPG for 30 min at 37C. Thisenabled an estimation of both DNA breaks and calculation of net FPG-sensitivesites. After FPG treatment, the samples were placed in an alkaline solution (0.3M NaOH, 1 mM EDTA, pH . 13) for 40 min at 4C. In the alkaline cometassay (where only DNA breaks are measured), the samples were placed in thealkaline solution directly after lysis. Alkaline treatment was followed byelectrophoresis in the same solution for 30 min (1.15 V/cm, 300 mA). TheDNA was stained with ethidium bromide and comets with tails of mainlysingle-stranded DNA could then be seen with a fluorescence microscope. Thecomets were analysed using computerised image analysis (Komet 4.0; KineticImaging Ltd, Bromborough, UK). The magnitude of the DNA tail (% DNA inthe tail) provides information about the level of DNA lesions. One parameter ata time of the comet assay protocol was altered in each experimental set-up.Each experimental set-up was repeated three to four times, and each time, 100cells were scored (50 cells from each of two fields on a microscope slide) andthe mean value reported.

Agarose density. To investigate whether the agarose gels density affects thelevel of DNA migration, cells irradiated with different doses of c-radiation (0,2.5, 5 and 7.5 Gy) and cells exposed to H2O2 were analysed with the alkalinecomet assay using different agarose densities (i.e. 0.4, 0.7, 0.9 and 1.3%, offinal agarose concentrations).

Duration of enzyme treatment. To assess how the duration of the enzymetreatment affects DNA migration, cells treated with Ro 19-8022 plus visiblelight were analysed with the FPG comet assay with enzyme treatment for 10, 30and 45 min.

Duration of alkaline treatment. To investigate the impact of the alkalinetreatment, DNA migration was analysed in cells treated with (i) 0 Gy ofc-radiation, (ii) 5 Gy of c-radiation, (iii) H2O2 and (iv) Ro 19-8022 plus visiblelight, with either the alkaline comet assay or the FPG comet assay applying 20,40 and 60 min of alkaline treatment.

Duration of electrophoresis. To investigate how the duration of theelectrophoresis affects the DNA migration, cells irradiated with c-radiation(0, 2.5, 5 and 7.5 Gy) and cells treated with H2O2 were analysed with thealkaline comet assay with 20 and 30 min electrophoresis.

Strength of the electric field during electrophoresis. To assess how the strengthof the electric field affects the DNA migration during electrophoresis, cellsirradiated with c-radiation (0, 2.5, 5 and 7.5 Gy) and cells treated with H2O2were analysed with the alkaline comet assay using different strengths of theelectric field (0.70, 1.15 and 1.6 V/cm).

Statistics

Statistical analyses were performed using PASW Statistics 18.0 (SPSS Inc.,Chicago, IL, USA). Linear doseresponse relationships were analysed withanalysis of variance (ANOVA) and Pearsons correlation coefficients (r) arereported. Differences between doseresponse curves were analysed with two-way ANOVA and whether the slopes of the curves were significantly differentwas assessed. All other comparisons of the impact of different protocol detailswere carried out with Students t-test. Normal distribution was assessed withthe ShapiroWilk test and homogeneity of variances was assessed withLevenes test and it was concluded that parametric tests were appropriate. P-values ,0.05 were considered statistically significant. The unit of variabilitywas the experiment.

Results

Agarose density

Each increase in agarose density caused a significant decreasein DNA migration in cells irradiated with 5 Gy of c-radiationas shown in Figure 1. The linear doseresponse relationship(r 5 0.950) was highly significant (P , 0.001). Nosignificant differences in DNA migration were observed inthe untreated cells (0 Gy of c-irradiation) when comparingdifferent agarose densities with Students t-test. However,the linear doseresponse relationship (r 5 0.709) for theuntreated cells was significant (P , 0.01, ANOVA).The slopes of the two regression curves in Figure 1 weresignificantly different (P , 0.001) from each other.

Duration of enzyme treatment

The duration of the incubation with FPG significantly affectedthe DNA migration (net FPG-sensitive sites) when analysingcells treated with Ro 19-8022 plus visible light. As shown inFigure 2, the DNA migration was significantly increased whenincubation with FPG was performed for both 30 and 45 mincompared to for 10 min (P , 0.05). The DNA migration wasnot significantly affected by prolonging the incubation timewith FPG from 30 to 45 min.

Duration of alkaline treatment

The DNA migration was not significantly affected by theduration of the alkaline treatment in untreated cells (0 Gy ofc-irradiation) (Figure 3A).

Fig. 1. DNA migration in untreated cells, i.e. cells exposed to 0 Gy (filleddiamonds) and cells exposed to 5 Gy (lines) of c-radiation measured by thealkaline comet assay using different agarose densities. Each increase in agarosedensity caused a significantly decreased DNA migration in cells irradiated with5 Gy (Students t-test, where *, ** and *** means P, 0.05, P, 0.01 and P,0.001, respectively). The linear doseresponse relationships were significantboth for untreated cells (r 5 0.71, P , 0.01, ANOVA) and cells exposed to5 Gy of c-radiation (r 5 0.95, P , 0.001, ANOVA). The slopes of the tworegression curves were significantly different (P, 0.001) from each other. Theexperiment was repeated four times. Each filled diamond/line represents themean value of 100 scored cells (50 cells from each of two separate gels).

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The DNA migration increased significantly when theduration of alkaline treatment was increased from 20 to 40min, from 40 to 60 min (P, 0.05) and from 20 to 60 min (P,0.01) in cells irradiated with 5 Gy of c-radiation (Figure 3B).The linear doseresponse relationship (r 5 0.969) was highlysignificant (P , 0.001). The slopes of the regression curves foruntreated cells and cells irradiated with 5 Gy of c-radiationwere significantly different from each other (P , 0.001).

In cells treated with H2O2, the DNA migration significantlyincreased when the duration of alkaline treatment wasprolonged from 20 to 40 min (P , 0.05) and from 20 to 60min (P , 0.001) as shown in Figure 3C. When increasing theduration of alkaline treatment from 40 to 60 min, the DNAmigration was not significantly affected (P 5 0.056). Thelinear doseresponse relationship (r 5 0.954) was highlysignificant (P , 0.001). The slopes of the regression curves foruntreated cells and cells treated with H2O2 were significantlydifferent from each other (P , 0.001).

Both DNA breaks and net FPG-sensitive sites weremeasured with the FPG comet assay in cells treated with Ro19-8022 plus visible light using different durations of thealkaline treatment as shown in Figure 3D. The DNA migration(net FPG-sensitive sites) was not significantly affected byincreasing the alkaline treatment time from 20 to 40 min,whereas a further increase to 60 min caused a significantincrease in DNA migration (P , 0.05). The linear doseresponse relationship (r 5 0.784) was significant (P , 0.01).When measuring the DNA migration without FPG (i.e. theDNA breaks) in cells treated with Ro 19-8022 plus visiblelight, no effect by the duration of alkaline treatment wasobserved when analysed with Students t-test. However, the

linear doseresponse relationship (r 5 0.651) was significant(P , 0.05, ANOVA). The slopes of the regression curves forDNA breaks and net FPG-sensitive sites were not significantlydifferent from each other (Figure 3D).

Fig. 2. DNA migration (DNA breaks 5 filled diamonds and net FPG-sensitivesites 5 lines) in cells treated with Ro 19-8022 plus visible light measured bythe FPG comet assay. The DNA migration was significantly increased whenincubation with FPG was performed for 30 or 45 min compared to for 10 min(P , 0.05, Students t-test). The DNA migration was not significantly affectedby prolonging the incubation with FPG from 30 to 45 min. The experimentwas repeated three times. Each filled diamond/line represents the mean valueof 100 scored cells (50 cells from each of two separate gels).

Fig. 3. DNA migration (DNA breaks5 filled diamonds and net FPG-sensitive sites5 lines) measured with the comet assay with 20, 40 and 60 min of alkalinetreatment. (A) The DNA migration was not significantly affected by the duration ofthe alkaline treatment in untreated cells, i.e. cells irradiated with 0 Gy of c-radiation(Students t-test). The experiment was repeated three times. (B) The DNA migrationincreased significantly when the duration of the alkaline treatment was prolongedfrom 20 to 40 min, from 40 to 60 min (P, 0.05) and from 20 to 60 min (P, 0.01)in cells irradiated with 5 Gy of c-radiation (Students t-test). The linear doseresponse relationship (r 5 0.97) was highly significant (P , 0.001, ANOVA). Theslopes of the regression curves for untreated cells and cells irradiated with 5 Gy ofc-radiation were significantly different from each other (P, 0.001). The experimentwas repeated three times. (C) In cells treated with H2O2, the DNA migrationsignificantly increased when the duration of alkaline treatment was prolonged from20 to 40 min (P, 0.05) and from 20 to 60 min (P, 0.001, Students t-test). Whenincreasing the duration of alkaline treatment from 40 to 60 min, the DNA migrationwas not significantly affected (P5 0.056, Students t-test). The linear doseresponserelationship (r5 0.95) was highly significant (P, 0.001, ANOVA). The slopes ofthe regression curves for untreated cells and cells treated with H2O2 weresignificantly different from each other (P , 0.001). The experiment was repeatedthree times. (D) When analysing the DNA migration (net FPG-sensitive sites) incells treated with Ro 19-8022 plus visible light with the FPG comet assay, the DNAmigration was not significantly affected by increasing the alkaline treatment from 20to 40 min, whereas an increase to 60 min significantly (P , 0.05, Students t-test)affected the DNA migration. The linear doseresponse relationship (r 5 0.78) wassignificant (P , 0.01, ANOVA). The slopes of the regression curves for DNAbreaks and net FPG-sensitive sites were not significantly different from each other.The experiment was repeated four times. Each filled diamond/line represents themean value of 100 scored cells (50 cells from each of two separate gels).

Comet assay protocol affects DNA migration

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Duration of electrophoresis

The DNA migration in cells irradiated with 0, 2.5, 5 and 7.5 Gyof c-radiation measured with the alkaline comet assay with 20or 30 min electrophoresis are shown in Figure 4. The level ofDNA migration was significantly increased when runningelectrophoresis for 30 min compared to 20 min (P , 0.001,two-way ANOVA) and the slopes of the two regression curveswere significantly different from each other (P , 0.001). Thelinear doseresponse relationships (r 5 0.989 for 20 min andr 5 0.992 for 30 min, respectively) were highly significant forboth curves (P , 0.001).

In cells treated with H2O2, the DNA migration wassignificantly increased when the samples had been subject to30 min compared to 20 min electrophoresis (P , 0.01) asshown in Figure 5A. After transforming the DNA migrationinto DNA breaks per 106 base pairs by using the calibrationcurves in Figure 4 as previously described (6), the difference inthe number of breaks detected was not as great, although stillsignificant (P , 0.05) (Figure 5B).

The strength of the electric field during electrophoresis

The DNA migration was significantly affected by the strengthof the electric field during electrophoresis in cells irradiatedwith different doses of c-radiation measured with the alkalinecomet assay (Figure 6). The DNA migration was significantlyincreased (P , 0.001, two-way ANOVA) when comparing1.60 with 1.15 V/cm and when comparing 1.15 with 0.70 V/cm. The linear doseresponse relationships (r 5 0.991, 0.985and 0.993 for 0.70, 1.15 and 1.60 V/cm, respectively) werehighly significant for all curves (P , 0.001). The slopes of the

regression curves were all significantly different from eachother (P , 0.001). Untreated cells were not significantlyaffected by the strength of the electric field, whereas cellsirradiated with 2.5, 5 and 7.5 Gy of c-radiation were

Fig. 4. DNA migration in cells irradiated with different doses of c-radiationmeasured with the alkaline comet assay with 20 or 30 min of electrophoresis.The DNA migration was significantly increased (P , 0.001, two-wayANOVA) when running electrophoresis for 30 min (lines) compared to 20 min(filled diamonds), and the slopes of the two regression curves weresignificantly different from each other. The linear doseresponse relationshipsfor both curves were highly significant (r 5 0.99, P , 0.001, ANOVA). Theexperiment was repeated four times. Each filled diamond/line represents themean value of 100 scored cells (50 cells from each of two separate gels).

Fig. 5. DNA migration in cells treated with H2O2 analysed with the alkalinecomet assay. (A) There was a significantly increased level of DNA migrationin cells that had been subject to 30 min compared to 20 min electrophoresis(P , 0.01, Students t-test). (B) After transforming the DNA migration intoDNA breaks (lesions per 106 base pairs) by using the calibration curves inFigure 4, the difference in the number of breaks detected was not as great,although still significant (P , 0.05, Students t-test). The experiment wasrepeated four times. Each filled diamond represents the mean value of 100scored cells (50 cells from each of two separate gels).

Fig. 6. DNA migration in cells irradiated with different doses of c-radiationmeasured with the alkaline comet assay using different strengths of the electricfield (0.70, 1.15 and 1.60 V/cm) during electrophoresis. The DNA migrationwas significantly increased (P , 0.001, two-way ANOVA) when comparing1.60 (closed circles) to 1.15 V/cm (lines) and when comparing 1.15 to 0.70 V/cm (filled diamonds) and the slopes of the regression curves were significantlydifferent from each other (P , 0.001). The linear doseresponse relationships(r 5 0.99 for all strengths of the electric fields) were highly significant for allcurves (P , 0.001, ANOVA). The experiment was repeated three times. Eachfilled diamond/line/closed circle represents the mean value of 100 scored cells(50 cells from each of two separate gels).


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significantly affected by each change in the strength of theelectric field (Students t-test).

The strength of the electric field during electrophoresissignificantly affected the DNA migration in cells treated withH2O2 analysed with the alkaline comet assay as shown inFigure 7A. There was no statistically significant difference inDNA migration when using 1.15 V/cm compared to 0.70 V/cm(P 5 0.050). The DNA migration was significantly increasedwhen using 1.60 V/cm compared with 1.15 V/cm (P , 0.05)and 0.70 V/cm (P , 0.01). The linear doseresponserelationship (r 5 0.964) was highly significant (P , 0.001).

After transforming the DNA migration into DNA breaks(lesions per 106 base pairs) by using the calibration curves inFigure 6, no significant difference remained between samplesthat had been subject to 0.70 and 1.15 V/cm or 1.15 and 1.60V/cm (Figure 7B). There was, however, a significant impact ofthe strength on the electric field when comparing 0.70 with1.60 V/cm. The linear doseresponse relationship (r 5 0.720)was significant (P , 0.05).

Discussion

Comet assay is an increasingly used popular method tomeasure DNA damage (1113). No standardised protocolexists and due to considerable differences in protocols, it iscomplicated to compare different studies. The influence onDNA migration by applying different protocols has beendiscussed previously (14). In the present study, the impact onDNA migration by some key parameters in the comet assayprotocol was investigated.

The DNA migration was affected by the density in theagarose gel both in untreated cells and cells exposed to 5 Gyof c-radiation. The damaged cells were more sensitive to

alterations in agarose density, which indicated that thesensitivity of the comet assay could be modified by alteringthe agarose concentration (Figure 1).

The data in the present study suggest that 10 min ofincubation with FPG is inadequate but that 30 min is sufficientfor the enzyme to find all oxidative lesions (Figure 2).Unpublished data from our laboratory suggest that this alsois the case for human 8-oxoguanine-DNA glycosylase1 (hOGG1), the human parallel to FPG (J. Kain, personalcommunication). It is essential that investigators make sure thatthey use enzyme incubation conditions where the saturationlevel is achieved (by using an appropriate concentration of theenzyme and an appropriate duration of the enzyme treatment).

ALS are transformed to DNA strand breaks during alkalinetreatment (15). Typically, 20- or 40-min alkaline treatment isapplied in the comet assay (6). Tice et al. (5) stated that 20-minalkaline unwinding is considered sufficient for most purposes.c-Irradiated cells have previously been shown to be sensitive tothe duration of alkaline treatment (7,8,16), which is inaccordance with the findings in the present study (Figure 3B).Vijayalaxmi et al. (8) speculate that this is due to radiation-induced ALS that are relatively resistant to high alkalinetreatment. In the present study, cells treated with H2O2 were alsoobserved to be sensitive to alkaline treatment (Figure 3C), whichsuggests that these samples also contain ALS that are relativelyresistant to high alkaline treatment. Untreated cells were notsignificantly affected by the duration of the alkaline treatment(Figure 3A). In addition, the significantly different slopes of theuntreated and treated cells indicate that the sensitivity of themethod could be improved by altering the duration of thealkaline treatment. When investigating cells treated with Ro 19-8022 plus visible light, no statistically significant impact onDNA migration (net FPG-sensitive sites) was observed whenincreasing the alkaline treatment from 20 to 40 min (Figure 3D).Taken together, these results suggest that the effect on DNAmigration by the duration of alkaline treatment depends on thetype of DNA damage measured. Typically used durations ofalkaline treatment might be inadequate, provided that one wantsto measure ALS. In order to be able to adjust the comet assayprotocol to the type of damage being investigated, a thoroughinvestigation of how different DNA damages are affected byalkaline treatment is required. In addition, Speit and Hartmann(17) suggested that durations of alkaline treatment andelectrophoresis should be adjusted to obtain valid andreproducible results for each cell type.

The duration of the electrophoresis significantly affected theDNA migration in c-irradiated cells (Figure 4), which supportsprevious findings by Forchhammer et al. (7). Electrophoresis isperformed in the same solution as alkaline treatment. It can bespeculated that the additional 10 min that the samples aresubject to alkaline solution contributes to an increasedconversion of ALS with a relatively high resistance to alkalinetreatment into DNA strand breaks. The significantly steeperslope of the 30-min electrophoresis calibration curve in Figure4 supports this hypothesis. If a sample irradiated with a higherdose of c-radiation contains more ALS, this sample will bemore sensitive to the duration of alkaline treatment. Thirtyminutes compared to 20-min electrophoresis also leads toa significantly increased DNA migration in cells treated withH2O2 (Figure 5A). After transforming the DNA migration intoDNA breaks per 106 base pairs, the impact of the duration ofelectrophoresis was not as great although the difference in thenumber of breaks detected was still significant (Figure 5B). As

Fig. 7. DNA damage in cells treated with H2O2 analysed with the alkalinecomet assay using different strengths of the electric field (0.70, 1.15 and 1.60V/cm). (A) The DNA migration was significantly increased when applying 1.60V/cm when compared with 1.15 V/cm (P , 0.05, Students t-test) and 0.70V/cm (P , 0.01, Students t-test). When comparing 1.15 with 0.70 V/cm, theDNA migration was not significantly affected (P 5 0.050, Students t-test).(B) After transforming the DNA migration into DNA breaks (lesions per 106

base pairs) by using the calibration curves in Figure 6, there were no significantdifferences in DNA breaks when comparing 0.70 and 1.15 V/cm or 1.15 and1.60 V/cm (Students t-test). When comparing 0.70 with 1.60 V/cm, theamounts of DNA breaks were still significantly different (P , 0.05, Studentst-test). The experiment was repeated three times. Each filled diamond representsthe mean value of 100 scored cells (50 cells from each of two separate gels).


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shown in Figure 3, the duration of alkaline treatment affectedboth H2O2-treated cells and c-irradiated cells suggesting thatboth treatments cause formation of ALS that are relativelyresistant to high alkaline treatment. Different proportions ofsuch ALS in the c-irradiated cells and H2O2-treated cells couldbe an explanation to why the impact of the duration ofelectrophoresis was not completely removed by using protocol-specific calibration curves.

The strength of the electric field significantly affected thelevel of DNA migration in both c-irradiated cells (Figure 6)and H2O2-treated cells (Figure 7A). When using the calibrationcurves in Figure 6 to convert the DNA migration into DNAbreaks (lesions per 106 base pairs) in cells treated with H2O2,the impact of the strength of the electric fields was decreased(Figure 7B). Both high and low strengths of the electric fieldcan cause practical problems. Highly damaged cells could endup outside the methods range of linear detection (3) whena strong electric field is applied together with a sensitiveprotocol. Another problem according to our experience is thatwhen only a small fraction of the DNA is in the comet head,the computer programme might have difficulties to define thecomet head. When using a weak electric field, the DNAfragments might not migrate long enough, i.e. some DNAfragments might not have migrated out from the comet headleading to an underestimation of DNA damage. The signif-icantly different slopes of the regression curves for the differentstrengths of the electric field in Figure 6 indicate that thesensitivity of the assay can be improved by altering the strengthof the electric field.

Forchhammer et al. (7) have shown previously that it ispossible to reduce the variation in DNA migration caused byusing different comet assay protocols by using protocol-specific calibration curves. Observations in previous studiesperformed by ECVAG (6,18) as well as in the present studysupport this finding. In addition to facilitating comparisonsbetween studies that have used different protocols, a real unitsuch as lesions per 106 base pairs is easier to understand forindividuals who are not familiar with the comet assaycompared to primary comet assay end points such as forexample %DNA in tail. In the present study, we observed thatthe transformation of DNA migration into DNA breaks (lesionsper 106 base pairs) by using calibration curves consisting ofc-irradiated cells is not without problems. ALS with a relativelyhigh resistance to alkaline treatment in c-irradiated cells and/orthe cells being investigated could complicate the transforma-tion of DNA migration into DNA breaks. Conversion ofprimary comet assay end points into a real unit by calibrationwith ionising radiation is useful, but the method has someproblems that need to be considered.

Whether results in different studies could be directlycompared when a standardised protocol is used needs to beelucidated. ECVAG is currently performing a study where weuse a reference protocol to enable comparisons betweenlaboratories without the impact of some critical protocoldifferences. One should, however, keep in mind that it might beappropriate to adjust the comet assay protocol to the type ofDNA damage and cell type being investigated.

In conclusion, key parameters in the comet assay protocolincluding (i) agarose density, (ii) duration of enzyme treatment,(iii) duration of alkaline treatment, (iv) duration of electropho-resis and (v) strength of the electric field during electrophoresisaffect DNA migration. By using protocol-specific calibrationcurves to transform the DNA migration into strand breaks per

106 base pairs, it is possible to reduce the variation in DNAdamage caused by using different comet assay protocols.Transformation did not completely remove the variation inDNA migration, which could possibly be explained by thepresence of ALS that are relatively resistant to high alkalinetreatment in cells treated with c-radiation or H2O2. The resultspresented in this paper indicate that the sensitivity of the cometassay can be optimised in relation to the specific performedstudy by altering different parameters in the assay protocolsuch as the agarose concentration, the duration of the alkalinetreatment, the duration of the electrophoresis and the strengthof the electric field.

Funding

Environmental Cancer Risk, Nutrition and Individual Suscep-tibility, a network of excellence operating within the EuropeanUnion 6th Framework Program, Priority 5: Food Quality andSafety (Contract No. 513943); the Swedish Research Council(Vetenskapsradet); Swedish Research Council for Environ-ment, Agricultural Sciences and Spatial Planning (FORMAS).

Acknowledgements

L. Forchhammer (Institute of Public Health, University of Copenhagen,Denmark) is acknowledged for preparing the c-irradiated cells. J. Kain(Department of Biosciences and Nutrition, Karolinska Institutet, Sweden) isacknowledged for blinding the slides. The photosensitiser Ro 19-8022 wasa generous gift from F. Hoffmann-La Roche, Basel, Switzerland. The authorsare members of ECVAG which was created within the ECNIS network ofexcellence in order to validate the comet assay.

Conflict of interest statement: None declared.

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