The Alkaline Comet Assay: Towards Validation in Biomonitoring of DNA Damaging Exposures

C Basic & Clinical Pharmacology & Toxicology 2006, 98, 336–345.Printed in Denmark . All rights reserved

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ISSN 1742-7835

MiniReview

The Alkaline Comet Assay: Towards Validation inBiomonitoring of DNA Damaging Exposures

Peter Møller

Institute of Public Health, University of Copenhagen, Øster Farimagsgade 5, Building B, 2nd Floor, P.O. Box 2099,DK-1014 Copenhagen K, Denmark

(Received February 21, 2005; Accepted October 26, 2005)

Abstract: Generation of DNA damage is considered to be an important initial event in carcinogenesis. The single cell gelelectrophoresis (comet) assay is a technically simple and fast method that detects genotoxicity in virtually any mammaliancell type without requirement for cell culture. This review discusses the strength of the comet assay in biomonitoring atits present state of validation. The simple version of the alkaline comet assay detects DNA migration caused by strandbreaks, alkaline labile sites, and transient repair sites. By incubation with bacterial glycosylase/endonuclease enzymes,broad classes of oxidative DNA damage, alkylations, and ultraviolet light-induced photoproducts are detected as ad-ditional DNA migration. The most widely measured enzyme sensitive sites have been those detected by formamidopyrimi-dine DNA glycosylase (FPG) and endonuclease III (ENDOIII). Reports from biomonitoring studies show that the basallevel of DNA damage in leukocytes is influenced be a variety of lifestyle and environmental exposures, including exercise,air pollution, sunlight, and diet. Although not all types of carcinogenic exposures should be expected to damage DNA inleukocytes, the comet assay is a valuable method for detection of genotoxic exposure in humans. However, the predictivevalue of the comet assay is unknown because it has not been investigated in prospective cohort studies. Also, it is importantthat the performance of the assay is investigated in multi-laboratory validation trials. As a tool in risk assessment thecomet assay can be used in characterization of hazards.

Human beings are continuously exposed to a variety ofharmful (e.g. genotoxic) and beneficial (e.g. antioxidant)chemicals. Lifestyle may render individuals susceptible tocancer because of hazardous exposures (e.g. smoking andrecreational sun tanning) or insufficient intake of cancerpreventive compounds (e.g. fruits and vegetables). Environ-mentally low-exposure situations are common, and al-though the risk for the individual is low, health effects onpopulation basis can be large because of the high numberof exposed individuals. There is a need for validated assaysdetecting genotoxic end-points in risk assessment. The alka-line single cell gel electrophoresis (comet) assay is widelyused nowadays for detection of genotoxicity, and may beuseful in risk assessment after sufficient validation. The aimof this review is to evaluate the strength of the comet assayat its present stage of validation in biomonitoring.

The comet assay as biomarker of exposure

Biomarkers are measurements that more or less specificallyquantitate exposure, early biological effect, and susceptibil-

Author for correspondence: Peter Møller, Institute of PublicHealth, University of Copenhagen, Øster Farimagsgade 5, BuildingB, 2nd Floor, P.O. Box 2099, DK-1014 Copenhagen K, Denmark(fax π45 35 32 76 86, e-mail p.moller/pubhealth.ku.dk).

ity. Exposure biomarkers encompass: (i) chemicals or meta-bolites thereof, (ii) protein and DNA adducts, includingtypes of DNA lesions detected by the comet assay. In re-lation to cancer, analysis of genotoxicity is distinguishablefrom other exposure biomarkers because it is a measure-ment of the biologically effective dose at the presumed tar-get molecule. The comet assay is an exposure biomarkerassay providing information of the biologically effectivedose. Biomarkers of early biological effect e.g. chromosomeaberrations and micronucleus frequency detect a stage ofcarcinogenesis, which is temporally later than the stage de-tected by exposure biomarkers. Examples of susceptibilitybiomarkers are gene polymorphisms or enzyme activity ofcomponents in metabolism and DNA repair.

Biomarkers must be rigorously validated before they areapplicable as tools in risk assessment of diseases caused byenvironmental agents or other exposures. Rothman et al.

(1995) have outlined the validation process of biomarkersin a systematic approach that involves four phases. In thefirst phase (laboratory studies) exposure-effect relationshipsare studied in cell cultures and animal experiments. The sec-ond step of biomarker validation is biomonitoring studies(transitional studies), designed to characterize the biomarkerin normal human populations and evaluate exposure-effectrelationships in applied studies of selected populations. Thethird step in the biomarker validation is crucial for the wide

337ALKALINE COMET ASSAYMiniReview

application of the biomarker because it is designed to showassociation between the biomarker and the disease (aetiol-

ogic studies), and it is the incidence of the disease ratherthan the biomarker that is the dependent variable in case-control and prospective cohort studies. The case-controlstudies are easier to perform, but suffer from the drawbackof reverse causality (i.e. the biomarker may be an effect ofdisease rather than predicting development of disease).Prospective cohort studies are best suited for the assessmentof whether a biomarker is predicting a future outcome ofdisease. As the last step of validation (public health appli-

cation) biomarkers are validated as risk assessment tools.Although many laboratory tests for genetic damage have

been developed over the years, remarkably few of these havebeen validated as biomarkers. Validation of biomarkers is alengthy process that takes years of laboratory work and in-ter-laboratory collaboration. Very few biomarkers relatedto research on cancer have been validated in prospectivecohort studies. Probably the finest example of validation isthe association between high chromosome aberration fre-quency and cancer incidence (Hagmar et al. 1998). Vali-dation of biomarkers is required for genetic toxicology teststo be useful in public health assessment and for regulativepurposes.

Development of the comet assay and novel modifications

Important historic events of the development and novelmodifications of the comet assay are outlined in table 1. Itis widely acknowledged that the comet assay was describedas a new method for detection of DNA damage in the late

Table 1.

Highlights of comet assay achievements and novel applicationsA.

Year Event or achievement

1978 Rydberg & Johanson introduce a method for detection of strand breaks in agarose-embedded single cells under alkalineconditions (pHØ12). The amount of single relative to double stranded DNA was measured by staining with acridine orangethat emits green and red light, respectively.

1984 Östling & Johanson describe a modified version of gel-embedded cells with electrophoresis at pH∑9.5. When cells in thismicroelectrophoresis assay were g-irradiated damaged DNA stretched toward the anode while DNA with few strand breaksremained circular.

1988 Singh et al. introduce electroforese at pH�13. This is often regarded as that the original comet assay publication. By October2005 the publication has been cited 1650 times (Web of Science).

1989 Olive describes a different version of the comet assay with electrophoresis under neutral conditions. The images are referred toas ‘‘comets’’. This publication has received surprisingly few citations (44 citations by October 2005, Web of Science)

1991 Gedik et al. describes the enzyme-modified version of the comet assay for detection of base damage. Nucleoids are digested withbacterial enzymes that recognize broad ranges of DNA damage and enzyme sensitive sites are obtained as additional DNAmigration.

1996 Pfuler et al. detect DNA-DNA crosslinks by the comet assay.

1997 Santos et al. use in situ fluorescence hybridization for localization of breaks in specific genes.

2000 International guidelines published for application in genetic toxicology and biomonitoring.

2001 Collins et al. measure DNA repair activity in cell extracts. The incision efficiency of cell extracts from donors is measured ongel-embedded substrate DNA containing specific types of DNA lesions.

2005 The number of publications with comet assay end-points is high (2390 publications using ‘‘comet assay’’ as search term inMedline database, October 2005).

A A detailed description of historic events and references can be read in Møller (2005).

1980s, although detection of DNA damage in gel-embeddedcells had been described before that time (Møller 2005).Two different versions of the comet assay were described bySingh et al. (1988) and Olive (1989). The former version ofthe comet assay has been adopted by most laboratories asillustrated by the number of citations of the original publi-cations (1650 versus 44 citations, Web of Science, October2005).

Descriptions of comet assay protocols with discussionand recommendations of various technical variations areprovided elsewhere (Olive 2002; Collins 2004). The pro-cedure of the comet assay is shown in fig. 1. In short, singlecell suspensions are embedded in agarose and lysed.Whereas blood samples are single cell suspensions by na-ture, tissues need to be disrupted by mechanical or enzymictreatment. I prefer mechanically tissue disruption in ice-cold buffer because this procedure diminishes the likelihoodof ex vivo generation or repair of DNA lesions. The lysistreatment removes cellular and nuclear membranes and pro-teins. This leaves the gel-embedded DNA in the form ofnucleoids. Following alkaline treatment and electrophoresis,DNA migrates toward the anode in a manner that is de-pendent on the number of lesions in the nucleoids. The ex-tent of migration is visualized in a fluorescence microscopeafter staining of the DNA. Detection of particular sites iscarried out by digestion of the nucleoids with bacterialDNA repair enzymes. In the simple form of the comet as-say, DNA migrates because it contains breaks that mayarise from direct action of genotoxic compounds and transi-ent repair sites. Depending on the pH of the alkaline treat-ment, some DNA lesions are converted to strand breaks;

338 PETER MØLLER MiniReview

Fig. 1. Procedure of the comet assay. Single cell suspensions are obtained by isolation of leukocytes (blood samples) or by mechanicaldisruption of tissue (e.g. by squeezing tissue through a sieve). After embedding in agarose, cells are lysed and the nucleoids are subjected toalkaline unwinding and electrophoresis. After staining, single nucleoids can be view and scored in a fluorescence microscope. Round imagesare nucleoids with little migration (left image), whereas migration results in comet-like appearances (middle images), and highly damagednucleoids will appear as images containing most of the DNA in the tail (right image).


these additional strand breaks are commonly referred to asalkaline labile sites. There has been controversy about whatis being measured by the simple alkaline comet assay. Publi-cations commonly describe the simple alkaline comet assayas detecting strand breaks. This is obviously imprecise be-cause the assay measures DNA migration. However, de-scribing the end-point of the simple alkaline comet assay asDNA migration implies that enzyme-sensitive sites andother novel applications are not related to DNA migration.Mainly due to a lack of specific expression, this publicationuse DNA damage as a term referring to the type of geno-toxicity detected by the simple alkaline comet assay.

The comet assay has been applied in a broad range ofscientific fields, including genetic toxicology, ecotoxicology,DNA repair, and apoptosis. Novel applications of thecomet assay include detection of DNA-DNA crosslinks,and gene-specific DNA damage detected by application offluorescent in situ hybridization methodology in the cometassay (Møller 2005). Also, the comet assay can be modifiedto measure repair activity in cellular extracts as incisionsof gel-embedded DNA substrates carrying oxidative DNAlesions (Collins et al. 2001). The most widely adopted novelmodification has been inclusion of enzymic digestion with

Fig. 2. Examples of DNA lesions detected by ENDOIII: 5,6-dihydroxy-5,6-dihydro-2ø-deoxythymidine (thymidine glycol, dTg), 5,6-dihy-droxy-5,6-dihydro-2ø-deoxyuridine (uracil glycol, dUg), 5-hydroxy-2ø-deoxycytidine (5-OHdC), and 5-hydroxy-2’-deoxyuridine (5-OHdU),and the FPG protein: 7-hydro-8-oxo-2ø-deoxyguanosine (8-oxodG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), and 4,6-diamino-5-formamidopyrimidine (FapyAde).

specific DNA glycosylase/endonuclease enzymes that detectbroad classes of DNA lesions (Collins 2004). It is possibleto detect oxidized pyrimidines and purines by digestion withENDOIII and FPG, respectively. The premutagenic 7-hy-dro-8-oxo-2ø-deoxyguanosine lesion probably is one of themost important lesions detected by the FPG protein; thislesion is also measured by high pressure liquid chromato-graphy coupled to electrochemical detection. Fig. 2 showsthe chemical structure of some of the oxidative DNA lesionsdetected by ENDOIII and FPG enzymes. These enzymeshave by far been the most widely used enzymes. Other en-zymes have been used less frequently, namely the AlkA pro-tein for detection of alkylation damage and T4 endo-nuclease V for UV-induced lesions.

Types of scoring systems for comets

There are several types of scoring systems in the comet as-say, including continuous (e.g. percent DNA in the tail (%T)and tail length) and categorical (visual scoring in arbitraryunits) measurements, as well as various descriptions of thedistribution of the images. It has been very popular to ex-press DNA damage in terms of the tail moment, which is


Fig. 3. Association between visual score (in arbitrary units) and %T.Data are the mean and S.D. for comparisons reported elsewhere(Collins et al. 1995 & 1997; Duthie et al. 1998; Noroozi et al. 1998;Risom et al. 2003).

the tail length multiplied with %T. In theory, it should bepossible to make direct comparisons of the same comet as-say end-point between laboratories, but this has been muchmore difficult than expected. Recently, the tail length hasbeen critised severely because it is only linear with respectto dose in a narrow interval and it is difficult to picture theappearance of the comets by the size of the value of thetail moment (Collins 2004). I believe that the end-pointsobtained as visual score and %T are superior at the momentbecause they have linear dose-response relationships withknown strand breaking agents over a wide dose range andthey can be compared between laboratories. In addition,there is a reasonably good linearity between visual scoreand %T (fig. 3).

Table 2 shows the results of a literature survey where the%T and visual score are obtained from control groups inbiomonitoring studies (Møller 2006). For comparison with%T, the visual score was recalculated from the original pub-lications to give a range of 0–100 arbitrary units. There isa very good concordance between the two ways of express-ing the level of DNA migration; the mean (S.D.) is 9.5 (5.8)and 10.6 (8.5) for %T and visual score, respectively. This

Table 2.

Level of DNA damage in human leukocytes assessed as %T or visual score (in arbitrary units)A.

%T Arbitrary unitsB Both

No of studies (subjects) 62 (3083) 63 (1988) 121 (4943C)Mean (SD) 9.5 (5.8) 10.6 (8.5) 10.0 (7.2)Median (25–75% quartiles) 8.8 (5.1–14.2) 7.8 (3.9–17.7) 8.6 (4.4–14.5)

A The data represent DNA damage obtained by the simple comet assay. A complete description of the survey is provided in (Møller2006). For comparison between %T and visual score in arbitrary units, the data has been square root transformed because the latterendpoint requires transformation to be normally distributed. There is no difference between the two endpoints (PΩ0.78, Students t-test), i.e. the mean (95% CI) for reversed transformed data is as follows: 8.5 (6.8–10.3) and 8.8 (7.2–10.7) for %T and arbitrary units,respectively.

B Arbitrary units are expressed as the score in the range of 0–100 with five categories. Original references have reported comet scores in3–6 categories. For comparison, the original scores in the 3 and 4 category scoring system have been re-calculated to achieve comparisonwith the 5 category system. The following conversions were used: 0;2;4 (3 categories), 0;1.33;2.67;4 (4 categories), 0;1;2;3;4 (5 categories).The score from one study using a 6-class scoring system was divided by 1.25 as conversion factor.

C The total number of subjects does not add up with the number of subjects in %T and arbitrary units.

means that, as a rule of thumb, the migration of DNA ex-pressed as %T or visual score in leukocytes from healthyhuman beings is 10% or 10 arbitrary units (assuming range0–100 for visual score), respectively (Møller 2006).

Basal level and dynamic range of DNA damage detected bythe comet assay

Reporting comet assay results as visual score or %T haslittle value to people that are not familiar with the method.It is much more informative to express the level of genotox-icity in e.g. number of lesions per cell. Any comet assayend-points can be expressed as the number of lesions percell by calibration with X-rays. The relationship betweenthe yield of strand breaks in eukaryotic cell DNA per Gyhas been investigated by the alkaline sucrose sedimentationtechnique (Kohn et al. 1976; Ahnström & Erixon 1981).The calibration requires a linear dose-response relationshipbetween the X-ray dose and the comet assay end-point ase.g. shown in fig. 4. Although the conditions of X-ray ir-radiations and key analytical procedures (e.g. alkaline con-ditions) are not identical between the methods, it is possibleto estimate the number of lesions per comet assay score.

We have found a linear dose-response relationship ofDNA damage detected by the comet assay in X-ray ir-radiated cells over a range of 0–10 Gy (fig. 4), whereasothers have reported linear dose-response relationships overa range of 0–8 Gy (Collins et al. 1996; Pouget et al. 1999).This corresponds to a dynamic range of 9280–11600 lesions/diploid cell detected by the simple comet assay (assumingthat one Gy generates 1160 strand breaks per diploid cell).The basal level of DNA damage in primary human (di-ploid) lymphocytes has been estimated to contain 1000–1100 lesions detected by the simple alkaline comet assay,1200–1900 ENDO sites, and 600–2000 FPG sites (Møller2006). The dynamic range of enzyme sensitive sites de-pends on the concomitant level of DNA damage in the ex-perimental setting because enzyme sensitive sites are ob-tained by subtraction of samples treated with and withoutenzyme.


The detection limit of the comet assay has been reportedto be 5–10 cGy using X-ray or g-radiation (Tice 1995); thiscorresponds to about 60 additional lesions in diploid cellsor 1–2 extra lesions per chromosome. Using optimized as-say conditions, it is possible to detect doses of ionizing radi-ation as low as 0.6 cGy (Malyapa et al., 1998), which corre-spond to less than 10 lesions per diploid genome. However,it should be recognized that the ultimate goal is not necess-arily the lowest possible limit of detection, but rather assayconditions that enable the widest dose-response range inconjunction with a low limit of detection. This applies toanimal experimental models where the range of doses canbe wide, and biomonitoring studies with concomitant deter-mination of DNA damage and enzyme sensitive sites.

Assay and sample scoring variation

Recently there have been various interesting attempts to in-vestigate the reliability of the comet assay in close detail.This has been done in multi-laboratory trials as wells as insingle laboratory trials. Foremost is the effort by severallaboratories in Europe through the European StandardsCommittee on Oxidative DNA Damage (ESCODD) collab-oration study to establish standardized protocols for deter-mination of oxidative DNA damage and reach agreementon the basal level of oxidative DNA damage (ESCODD2003). In the later phases of the ESCODD project, the FPGversion of the comet assay was included for determinationof oxidative DNA damage in HeLa cells incubated withRo19–8022 that generate oxidative DNA damage withoutstrand breaks. Coded cryopreserved samples were distrib-uted to ESCODD members who analyzed the level of FPGsensitive sites by their own comet assay procedure; this re-vealed that all the laboratories were able to detect differ-ences in coded samples on a qualitative basis, but only halfof the laboratories were able to detect a dose-response re-

Fig. 4. Level of migration in gel-embedded A549 cells X-ray ir-radiated at a dose-rate of 4.59 Gy/min. Circles represent the meanof two experiments. The correlation coefficient (r2) is 0.98 (nΩ8).Detailed description of the experimental procedure is outlined in(Møller et al. 2004a).

lationship (ESCODD 2003). In two different studies therewere 10 times difference in the level of FPG sensitive sitesreported by different laboratories; determined by the coef-ficient of variance (CV), the variation in values obtainedfrom different laboratories were 57 and 66% (ESCODD et

al. 2003 & 2005).In another set of investigations, researchers have deter-

mined the variance in slide scoring. In one study, 19 investi-gators from 7 laboratories scored the same set of slides withcells exposed to H2O2; all but one of the investigators de-tected a dose-response relationship (Garcia et al. 2004).However, the variation in slide scoring was large (fig. 5);CVs ranged from 10% (100 mM H2O2) to 100% (control,estimated from data in figure). The higher reproducibilityin scoring damaged nucleoids is expected if it is assumedthat comet-like images represent events of genotoxic insults.Assuming that the distribution of single nuclei scores persample follows the Poisson distribution, the confidence in-terval will decrease as the number of events increases. Usinga similar approach, we investigated the variation in slidescoring among both experienced and non-experienced in-vestigators. It was found that both experienced and non-experienced investigators were able to distinguish betweenthree slides of X-ray irradiated cells with clear difference inthe average level of migration (Møller et al. 2004a). Themean levels of DNA migration in the three slides were 24arbitrary units (non-irradiated), 160 arbitrary units (lowdose), and 290 arbitrary units (highest dose). The corre-sponding CVs of slide scoring were 20% (high dose), 38%(low dose), and 93% (non-irradiated). Although the vari-ance in slide scoring was large also in this study, it appearedto depend on experience, i.e. the variation was lowestamong the most experienced investigators.

As a perspective, strikingly few publications contain dataon the assay variation of the comet assay; however thosethat report assay variation typically obtain CVs of 20–50%in cryopreserved assay control samples (Holz et al. 1995;Hellman et al. 1999; De Boeck et al. 2000; Møller et al.

2002, 2003 & 2004b; Speit et al. 2003; Møller 2005; Avogbe

Fig. 5. Variation in the scoring of slides with nuclei exposed tovarious concentrations of H2O2. The squares and whiskers (meanand S.D.) represent the migration in arbitrary units of 19 investi-gators that scored the same slides. The diamonds represent the coef-ficient of variation of the data shown in squares. The data are ob-tained from fig. 3 in Garcia et al. (2004).


et al., 2005; Lee et al. 2005). The CVs obtained in controlgroups of biomonitoring studies is in the range of 36% (95%confidence interval 27–46%) (Møller et al. 2000). This sug-gests that most laboratories have similar assay variation.Considering the large differences obtained in the slide scor-ing exercises, an important contribution to the assay vari-ation could be differences in slide scoring. An assessmentof the contribution of intra-assay and inter-assay variationindicated that approximately two-third of the variance wasattributed to inter-assay variation (Møller et al. 2004b).These data underscore the importance of validation trialsand highlights the importance of developing standards thatcan be used to calibrate assays.

Application in biomonitoring studies

The comet assay has been increasingly popular as genotox-icity test in biomonitoring studies of occupational and en-vironmental exposures. The exposure levels of carcinogensthat human beings encounter in these studies are lower thanthe doses of genotoxic compounds in animal experimentalmodel systems. Elevated levels of DNA damage have beenobserved in leukocytes of persons in putatively high-ex-posure circumstances due to occupation or treatment withantineoplastic agents, although data from occupationalstudies are conflicting (Møller et al. 2000). The maximaleffect ratios of DNA damage in leukocytes have differedconsiderably following various treatments with antineoplas-tic alkylating agents. Moreover, it appears that a similarrange of fold-induction of DNA damage is observed by ex-posure to antineoplastic agents and exposure to occu-pational and environmental agents (Møller et al. 2000). As-suming that patients are exposed to high doses of genotoxiccompounds in chemotherapy, and antineoplastic agents arehighly reactive, studies of patients receiving chemotherapyshould yield higher levels of DNA damage than observedin studies of occupational or environmental exposures.Since this is not the case, it suggests that the comet assayresults presently is not suitable for determination of dose-response relationships in biomonitoring studies, i.e. it is notpossible to produce risk estimates by the value of the DNAdamage detected by the comet assay. However, it is possiblethat the discrepancy in fold-induction between chemo-therapy and occupational exposures is due to the use ofsuboptimal comet assay end-points that cannot be com-pared. The use of either %T or visual score in arbitraryunits could ease the comparison between the studies. Thefar-reaching implication of this lack of clear-cut dose-re-sponse relationships is that the comet assay may not be astrong tool in risk assessment, unless for the purpose forhazard characterization. In this respect, it is important toremember that the comet assay is a reliable tool for assess-ment of exposure. E.g., we have found dose-dependent re-lationships between air pollution and the level of FPG sitesin mononuclear blood cells of people exposed to the rela-tively clean air of Copenhagen, Denmark (Vinzents et al.

2005). A more pronounced effect in FPG sites was observed

among people living in Cotonou, Benin, which is highly air-polluted because of high traffic intensity of old vehicles andpoor gasoline (Avogbe et al. 2005).

Age, sex, and many environmental exposures have beenreported to affect the level of DNA damage detected by thecomet assay (Møller et al. 2000). Environmental exposuresinclude antioxidants, exercise, sunlight, air pollution; theseexposures affect the level of DNA damage in appliedstudies, but they are virtually never important determinantsin cross-sectional studies. Several studies have describedseasonal variation of the level of DNA damage by thecomet assay (Møller et al. 2002; Sørensen et al. 2003; Tsili-migaki et al. 2003). It has been argued that the seasonalvariation is due to sunlight exposure, but the contributionof other seasonal changes in environmental exposures can-not be ruled out. Seasonal variation is an issue that needsto be investigated in further detail; especially the mechan-ism of the effect remains to be elucidated in more detail. Itshould be pointed out that the seasonal variation is mainlyobserved for DNA damage detected by the simple alkalinecomet assay, whereas similar effects are less investigated forenzymic sensitive sites or non-excisting. Presently, the re-ports of single exposures and the effect of age and sexshould be considered as potential confounding factors, butit is very important that future studies assess the magnitudeof effects of these factors. An assessment of the contri-bution of single exposures or interaction between exposuresis only possible in large investigations. Alternatively, re-analysis of published data in joined databases may serve asplatform for elucidation for the effect of lifestyle factors.As exemplified by determination of DNA damage (table 2),it is possible to include data from 4943 patients in a jointdatabase.

There are publications of many intervention studies in-vestigating the effect of antioxidants and antioxidant-richfood products by the comet assay. It is not possible to per-form a formal meta-analysis because of differences in studydesigns and treatment. However, a critical survey of the re-ports of antioxidant supplementation trials indicates thatprotective effects were more convincing in short-termstudies (i.e. studies lasting less that 24 hr) than in the long-term studies. Unfortunately, many of the long-term studieshave sequential study design that cannot control for periodeffects. We have devised a small scoring system for evalu-ation of the study design in which studies could obtainscores from zero (weak design) to three (strong design):points were given for studies with a placebo group, paralleldesign, and inclusion of sampling after the interventionperiod. This assessment showed that studies reporting oxi-dative DNA damage-lowering effect had poorer design thanstudies showing null effect, and this could not be explainedby differences in the statistical power to detect differencebetween two groups (Møller & Loft 2002). In addition,DNA damage detected by the simple comet assay usuallyshowed no effect of antioxidant supplementation, whereasthere was a tendency that protective effects in terms ofENDOIII and FPG sensitive sites are observed following


antioxidant supplementation in male subjects (Møller &Loft 2004). These antioxidant intervention studies havebeen conducted in healthy subjects. The conclusions cannoteasily be extrapolated to the whole population because ofdifferences in the antioxidant status, and the aging processmay render subjects more susceptible to oxidative stress. Itis possible that the effect of antioxidants is observed mainlyin oxidatively stressed subjects.

Need for inter-laboratory validation studies

There is progress on agreement of experimental proceduresin the comet assay, yet there is need for validation andstandardization of the comet assay in inter-laboratory col-laborations (ESCODD et al. 2005). The framework used bythe multi-laboratory micronucleus (HUMN) project is aninteresting set-up because the assays share the same prob-lems and advantages such as ease of slide scoring and thefeasibility of scoring large number of nuclei per sample.This worldwide collaborative study was recently startedwith the objectives to compare baseline values of mi-cronuclei frequency in lymphocytes and exofoliated cellsfrom different laboratories, establish standard protocols forthe micronucleus assay, and initiate a prospective cohortstudy with micronucleus assay data from each laboratory(Fenech et al. 1999). There is a certain degree of subjectivityin scoring of micronuclei that may explain the large vari-ation in the baseline levels between different laboratoriesover the world. In order to overcome this problem, membersof the HUMN project initiated an inter-laboratory slide-scoring exercise based on detailed instructions for scoringcells and included a set of reference illustrations for differ-ent types of micronuclei. This effort indicated that all lab-oratories were able to detect a dose-response relationship ofcoded samples of cells exposed to ionizing radiation, yetwith large variation in micronucleus frequency between lab-oratories (Fenech et al. 2003). As for the comet assay, it isonly detection of FPG sensitive sites that has been investi-gated in a multi-laboratory validation trial, whereas manylaboratories have done their own validation of other end-points by determination of dose-response relationships ofgenotoxic agents and optimization of experimental pro-cedures to the particular need of the research. Importantproblems needed to be solved can be summarized as fol-lows: (i) differences in baseline levels of DNA lesions, proto-cols, and scoring methods, (ii) conflicting reports of the ef-fect of age, sex, season, and smoking.

Validation status of the comet assay as a biomarker inbiomonitoring

Animal experimental models are pivotal in the first phaseof the validation process (laboratory studies) because thelarge rodent carcinogen databases provide unique oppor-tunities to compare the performance of the biomarker withother analytical methods of genotoxicity. There has beenpublished many studies of exposure to genotoxic com-

pounds, but an outstanding Japanese study that investi-gated the generation of DNA damage by exposure to 208rodent carcinogens and non-carcinogens in eight organs ofmice and rats indicates that the comet assay is a goodscreen-test for in vivo genotoxicity (Sasaki et al. 2000). Byfar, detection of DNA damage has been the preferred end-point investigated by comet assay in animal experimentalstudies, although the action mechanism will not be eluci-dated by detection of DNA damage only. This is unfortu-nate because the novel applications encompassing enzymicdetection of DNA damage, gene-specific lesions, and cross-links facilitate a much more detailed understanding of themechanism of genotoxicity. There is no study like the Ja-panese for these novel applications of the comet assay. Rela-tively few laboratories have adopted the enzyme-modifiedversion of the comet assay for detection of oxidative DNAdamage, yet it has been validated in the ECSODD studywhere approximately half of the laboratories detected adose-response effect. Also, validations reported by individ-ual research groups have indicated that the enzymic detec-tion of oxidative DNA damage is reliable on dose-responsebasis after exposure to ionizing radiation and Ro19-8022(Collins et al. 1996; Pouget et al. 1999; Risom et al. 2003).

The second step of biomarker evaluation has shown thatgenotoxic effects can be measured by the comet assay inleukocytes and various non-lymphatic tissues of human. Inaddition, there is extensive knowledge of factors affectingthe basal level of DNA damage in healthy human beings.The potential of the comet assay to detect DNA damage inleukocytes has been investigated in cancer patients receivingtherapy with antineoplastic alkylating agents. The aggre-gated data from biomonitoring studies indicate that thecomet assay is a useful assay for estimation of exposure.Considering that the comet assay is a reliable technique fordetermination of DNA damage in various animal organs,analysis of DNA damage in other cells than leukocytes isinfrequent in biomonitoring studies, most likely reflectingthe difficulty in obtaining human biopsy material. Thenumber of investigations of DNA damage in non-lymphatictissues of human beings is expanding slowly, and may forma new direction of comet assay applications in the near fu-ture.

The third step in the biomarker validation (aetiologic

studies) is the application in case-control studies where theincidence of the disease rather than the biomarker is themeasured parameter. The association between the effect ofa biomarker and disease can be expressed as the odds ratioby this approach. Reports from case-control studies con-taining comet assay endpoints indicate progress in this stepof the validation process. At the moment, though, the ma-jority of the case-control studies are better characterized asapplied studies. There are some case-control studies thathave shown increased odds ratio for adenocarcinoma of theesophagus (Olliver et al. 2005), bladder cancer (Schabath et

al. 2004), breast cancer (Smith et al. 2003), and thyroid can-cer (Sigurdson et al. 2005) among patients with high levelof DNA damage. However, it should be stressed that bio-


marker-based case-control studies are likely to be associatedwith reverse causality, and the results from prospective co-hort studies may turn out to be less exciting. Most likely,the prospective cohort studies with biobank material do nothave cryopreserved leukocytes in medium suitable foranalysis of DNA damage by the comet assay. It is essentialthat samples are collected and stored in a way that is appro-priate for later analysis by the assay. In case a multi-labora-tory project is initiated, it will be possible to prospectivelycollect data from biomonitoring studies into a large data-base as is being done in the HUMN project. Probably thismeans that information from prospective cohort studies in-volving the comet assay is not an issue in the near future.It will take a number of years before the comet assay willreach the final step in the validation process (public health

application), assuming that it performs well in prospectivecohort studies.

AcknowledgementsThe study was supported by a grant from Else og Mog-

ens Wedell-Wedellsborgs Fond.

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Documents

The Alkaline Comet Assay: Towards Validation in Biomonitoring of DNA Damaging Exposures