Analysis of DNA alkylation damage and repair in mammalian cells by the comet assay

The single cell gel electrophoresis (SCGE) or comet assay, whichmeasures DNA strand breaks in individual cells, was used toanalyse DNA damage and repair induced by the SN₁-type alkylatingcarcinogens N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosoureain CHO cells. The comet assay was comparable in sensitivityto the alkaline elution assay. The alkyl-adducts detected asDNA single-strand breaks (ssb) by this technique were completelyrepaired within 24 h after treatment. These data indicate thatlong-lived lesions, such as alkylphosphotriesters, are not convertedinto ssb under the standard SCGE alkaline conditions (pH 13.5).The lesions revealed by the comet assay are mainly apurinic/apyrimidinic(AP) sites and breaks formed as intermediates in the base excisionrepair process of N-alkylpurines. When SCGE was performed atpH 12.5 instead of pH 13.5 a lower level of ssb was detectedand these breaks were completely resealed within 2 h after treatmentThese data suggest that different subsets of lesions are detectedunder different pH conditions. The SCGE combined with inclusionwithin the cells of endo-nuclease III revealed that a high portionof AP sites induced by alkylation damage were not convertedinto ssb by alkali. The level of endonuclease III-sensitivesites decreased as a function of the repair time and by 24 hafter treatment no sites were left on the DNA. The use of thismodified SCGE assay allows the estimation of the total amountof unrepaired AP sites present on DNA. Alkylation-induced ssbas detected by the comet assay should be regarded as an indicatorof repair rate and balance more than a measure of actual DNAdamage. ³To whom correspondence should be addressed     

DNA damage and cytotoxcity induced in mammalian cells by a tetramethylfuroquinolinone derivative

1,4,6,8-Tetramethyl-2H-furo[2,3-h]quinolin-2-one (FQ) is an angelicin isoster characterized by a strong photosensitizing activity. FQ shows a significant antiproliferative activity also in the dark, i.e., without UVA activation. The cytotoxic activity of FQ in the dark was detected in HeLa cells and in normal human lymphocytes; FQ showed notable antiproliferative effects, barely lower in comparison with ellipticine, used as a reference. Similar results were obtained studying the FQ's capacity for forming chromosome aberrations. For both FQ and ellipticine, the chromosomal damage correlated closely with cell killing; when compared with ellipticine at the same levels of survival, FQ appeared to be muchless genotoxic. Using alkaline elution we have investigated the ability of FQ to damage DNA. The formation of equivalent amounts of single-strand breaks (SSB) and DNA-protein cross-links (DPC) was observed; in addition, these lesions appeared to be located at the same sites in DNA. Experiments carried out with neutral elution demonstrated the formation of double-strand breaks (DSB). All these data are consistent with an inhibition of topoisomerase II; this hypothesis was confirmed performing an enzymatic test in vitro using topoisomerase II from Drosophila melanogaster embryos. Environ. Mol. Mutagen. 29:256-264, 1997 © 1997 Wiley-Liss, Inc.     

Radiation-induced DNA damage in canine hemopoietic cells and stromal cells as measured by the comet assay

Stromal cell progenitors (fibroblastoid colony-forming unit; CFU-Fs) are representative of the progenitor cell population of the hemopoietic microenvironment in bone marrow (BM). Previous studies of the radiation dose—effect relationships for colony formation have shown that canine CFU-Fs are relatively radioresistant as characterized by a D₀ value of about 2.4 Gy. In contrast, hemopoietic progenitors are particularly radiosensitive (D₀ values = 0.12-0.60 Gy). In the present study, the alkaline single-cell gel electrophoresis technique for the in situ quantitation of DNA strand breaks and alkalilabile sites was employed. Canine buffy coat cells from BM aspirates and cells harvested from CFU-F colonies or from mixed populations of adherent BM stromal cell (SC) layers were exposed to increasing doses of X-rays, embedded in agarose gel on slides, lysed with detergents, and placed in an electric field. DNA migrating from single cells in the gel was made visible as “comets” by ethidium bromide staining. Immediate DNA damage was much less in cultured stromal cells than in hemopoietic cells in BM aspirates. These results suggest that the observed differences in clonogenic survival could be partly due to differences in the type of the initial DNA damage between stromal cells and hemopoietic cells. © 1996 Wiley-Liss, Inc.     

Adaptation to alkylation damage in DNA measured by the comet assay

The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root tips by the comet assay. The alkaline unwinding/neutral electrophoresis (A/N) and alkaline unwinding/alkaline electrophoresis (A/A) protocols were used for detection of DNA damage. With the A/N comet assay, less DNA damage was seen after conditioning pretreatment with a low dose prior to a high challenging dose of alkylating mutagens as compared to application of the high dose only, whereas a nearly additive effect was seen when the A/A comet assay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease III. The adaptation observed with the A/N comet assay was abolished by pretreatment with the protein synthesis inhibitor cycloheximide. These data suggest that the comet assay is able to detect on molecular level a phenomenon resembling clastogenic adaptation.     

Repair of X-ray induced DNA damage measured by the comet assay in roots of Vicia faba

The comet assay was used to measure DNA damage and repair in nuclei released from 1 cm root ends of Vicia faba after X-ray irradiation. Irradiation induced a linear increase of DNA content in comet tail with doses under various denaturation and electrophoretic conditions. The pH of the electrophoresis solution played the most important role in the detection of DNA damage. After irradiation with 30 Gy of X-rays, most of the DNA damage was removed during the first 20 min, even in the presence of DNA repair inhibitors. This first, rapid phase of DNA repair was not affected by incubation on ice, but was partially blocked by 3-aminobenzamide. When DNA was exposed to alkali (0.3 M NaOH) and electrophoresed at neutral pH, all DNA damage was removed in 2 hr, even in the presence of 3-aminobenzamide. Complete repair was inhibited by incubation on ice (30% of DNA remaining in tail) and partially by aphidicolin (13% DNA remaining in tail). Under alkaline (0.3 M NaOH) pretreatment and electrophoresis, more than 20% of detected DNA damage remained unrepaired after 2 hr of postirradiation incubation with and without 3-aminobenzamide at room temperature. Aphidicolin and incubation on ice inhibited the removal of DNA damage to 33% and 39% DNA, respectively. Moreover, aphidicolin treatment attenuated the first phase of damage removal. Environ. Mol. Mutagen. 32:281–285, 1998 © 1998 Wiley-Liss, Inc.     

DNA damage induced by micro- and nanoparticles--interaction with FPG influences the detection of DNA oxidation in the comet assay

Reliable methods for evaluation of toxicity from particles, such as manufactured nanoparticles, are needed. One promising tool is the comet assay, often used to measure DNA breaks (strand breaks and alkali-labile sites) as well as oxidatively damaged DNA, the latter by addition of specific DNA repair enzymes such as formamidopyrimidine DNA glycosylase (FPG). The aim of this study was to investigate the use of the comet assay for analysis of DNA oxidation by a range of micro- and nanoparticles in the lung cell lines A549 and BEAS-2B and to test the hypothesis that nanoparticles present in the cells during the assay performance may interact with FPG. This was done by investigating the ability of micro- and nanoparticles (stainless steel, subway particles, MnO(2), Ag, CeO(2), Co(3)O(4), Fe(3)O(4), NiO and SiO(2)) to induce DNA breaks, oxidatively damaged DNA (FPG sites, dominantly 8-oxoguanine), intracellular production of reactive oxygen species (ROS) and non-cellular oxidation of the DNA base guanine, as well as by studying interactions of the particles and their released ions with FPG. Several particles caused DNA breaks, but low levels of FPG sites. The ability of FPG to detect DNA oxidation induced by a photosensitiser was however shown. An oxidative capacity of the particles was indicated by increased levels of intracellular ROS, and especially Ag and subway particles caused non-cellular oxidation of guanine. Incubation of FPG with the particles led to less FPG activity, particularly with nanoparticles of Ag but also with CeO(2), Co(3)O(4) and SiO(2). Further investigations of these particles revealed that for Ag, the decreased activity was mainly due to released Ag ions, whereas for CeO(2) and Co(3)O(4), FPG interactions were due to the particles. We conclude that measurement of oxidatively damaged DNA in cells exposed to nanoparticles may be underestimated in the comet assay due to interactions with FPG.     

The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment

Cultured hepatocytes have been treated with either DMSO or dimethylnitrosamine(NDMA) for either 1 or 2 h and the cells assessed for DNA-damageusing the single cell alkaline gel electrophoresis assay (cometassay). A strong positive test response was observed producingcomet tails of a length and DNA content not observed in eitherviable or dead control cells. A stronger test response was observedafter a 2 h, as opposed to a 1 h, incubation of hepatocyteswith NDMA. The method of processing the image of the comet isdiscussed and it is proposed that measurement of the lengthof the comet tail should commence at the estimated trailingedge of the cell, rather than at the leading edge or the estimatedcentre of the cell. Using this criterion, many control cellshave no comet tails thereby enabling chemically induced tailsto be more readily assessed. A simplified version of definingthe comet tail moment is proposed. ³To whom correspondence should be addressed     

Assessment of DNA strand breaks induced by bleomycin in barley by the comet assay

Comet assay was applied to study induction and repair of DNA damage produced by bleomycin in barley genome. Experimental conditions were adapted to achieve efficient detection of both DNA single- and double-strand breaks. Substantial increase of the parameter "% of DNA in tail" was observed coupled with almost linear dependence from bleomycin concentration, more pronounced for the induction of DNA double-strand breaks. Data obtained at different recovery periods displayed rapid restoration of breakage, revealing that efficient mechanisms for repair of strand discontinuities induced by bleomycin are functional in barley DNA loop domains.     

Vitamin C modulates DNA damage induced by hydrogen peroxide in human colorectal adenocarcinoma cell lines (HT29) estimated by comet assay in vitro

Introduction

Cancer cells, compared to normal cells, are under increased oxidative stress associated with oncogenic transformation, alterations in metabolic activity, and increased generation of reactive oxygen species.

Material and methods

We investigated the ability of vitamin C to reduce the damage induced by hydrogen peroxide, in human colorectal adenocarcinoma cells in vitro by the comet assay. Additionally, we measured the kinetics and efficacy of the repair of DNA damage after incubation with vitamin C in the presence of H₂O₂.

Results

The obtained results showed that 1 h pre-incubation with vitamin C and exposure to H₂O₂ for the last 10 min of incubation caused a statistically significant (p < 0.05) increase in DNA migration in comet tails in all experimental series. For the 10 µM, 25 µM, 50 µM, 100 µM vitamin C concentrations the levels of DNA damage were as follows: 18.6%, 21.1%, 25.3% and 27.2%, respectively, as compared to the untreated cells (3.26%). However, in comparison with H₂O₂ alone (29.1%), we observed a statistically significant (p < 0.05) decrease of the genotoxic effect in HT29 cells induced by H₂O₂ for the two lowest of concentrations of vitamin C: 10 µM and 25 µM. The HT29 cells were able to achieve effective repair of the damaged DNA within 60 and 120 min after incubation with the tested compounds. All the values obtained in the test were statistically significant (p < 0.05).

Conclusions

Vitamin C caused a weaker DNA damaging effect of hydrogen peroxide and positively influences the level of oxidative DNA damage in HT29 cells (decrease ∼ 30%). We noted that DNA damage was effectively repaired during 120 min postincubation in the tested cells and that oxidative damage was the major type of damage.     

Assessing testicular germ cell DNA damage in the comet assay; introduction of a proof-of-concept

The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell-specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource-efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.     

用彗星试验研究饮用水中有机提取物对小鼠睾丸细胞DNA的损伤作用

目的通过彗星试验探讨饮用水两种不同消毒工艺所产生的非挥发性有机提取物对小鼠睾丸细胞DNA的损伤作用。方法设对照组,低、中和高剂量组共4个组,采用彗星试验检测NZ和XC水厂丰水期和枯水期水源水、自来水水样的非挥发性有机提取物对小鼠睾丸细胞DNA的损伤,并进行比较。结果各剂量组都出现了明显的拖尾现象,NZ与XC水厂的拖尾率比较差异无统计学意义。各剂量组的平均尾长明显高于对照组,NZ水厂低剂量组中,枯水期水源水平均尾长（19.3±2.7）μm高于自来水的（16.0±2.5）μm;高剂量组中,丰水期水源水高于自来水。水源水中,枯水期低剂量组低于丰水期、中剂量组高于丰水期;自来水中,枯水期中剂量组低于丰水期,高剂量组高于丰水期。XC水厂低剂量组中,丰水期的水源水平均尾长（15.5±1.0）μm低于自来水的（24.9±2.6）μm,中剂量组中,枯水期水源水高于自来水。水源水中,丰水期低剂量组低于枯水期,中剂量组和高剂量组丰水期高于枯水期;自来水中,丰水期中剂量组高于枯水期。结论 NZ和XC水厂的丰水期和枯水期有机提取物均含有导致小鼠睾丸细胞DNA损伤的物质;水源水与自来水、丰水期与枯水期表现为不同程度的DNA损伤。     

New measure of DNA repair in the single-cell gel electrophoresis (comet) assay

Since its introduction by Ostling and Johanson [1984; Biochem Biophys Res Commun 123:291-298] and independent modifications by Singh et al. [1990; Exp Cell Res 175:184-191] and Olive et al. [1988; Radiat Res 112:86-94], the comet assay has been widely used in genetic toxicology, environmental biomonitoring, molecular and human epidemiology, and clinical investigations. There are still several issues to be resolved before the comet assay is accepted as a standard assay for detecting DNA damage and repair in a single cell. One of the major issues is the proper quantification of DNA damage/repair. The aim of this article is to develop a new quantitative measure of DNA damage/repair which is represented in the dose-time-response surface. We propose to use the second derivative (2D) of the dose-time-response surface for measuring DNA repair activity. This approach enables us to represent the DNA repair activity of cells exposed to a DNA-damaging agent with a single number by combining all the information of a dose-time-response experiment. The computation procedure includes the application of linear regression. An SAS/AF-based program, "Comet Assay," was developed for this computation and is freely available on the Internet. We considered the response of each of four DNA damage parameters: tail moment, tail length, tail DNA, and tail inertia for constructing the dose-time-response surface. Using data from 25 patients, we observed that 2Ds based on tail moment and tail DNA were highly correlated and that tail inertia might provide information on a somewhat different aspect of DNA damage/repair.     

Evaluation of DNA damage induced by decabromodiphenyl ether (BDE-209) in hemocytes of Dreissena polymorpha using the comet and micronucleus assays

The recent widespread production and use of flame retardants, polybrominated diphenyl ethers (PBDEs), is one of the reason of the increasing contamination observed worldwide. At the present, deca-BDE mixture, in which the decabromodiphenyl ether (BDE-209) is the major congener (98%), dominates the EU market. The potential genotoxicity of BDE-209 was examined in the freshwater bivalve zebra mussel (Dreissena polymorpha) by means of Comet assay and micronucleus assay (MN assay). Mussels were exposed in vivo to BDE-209 at nominal concentration of 0.1, 2, and 10 mug/l under laboratory conditions. The assays were performed on the bivalve hemocytes monitoring the levels of DNA strand breaks and the percentage of micronuclei until 168 and 96 hr of exposure, respectively. At the same time, BDE-209 levels were measured daily in mussel soft tissues to evaluate the bioaccumulation. Results of the Comet assay showed a significant increase of DNA damages compared to controls, but a lack of dose/effect relationship probably due to the formation of less-brominated congeners. By contrast, no significant changes in MN frequency from baseline levels were observed. These preliminary results about the potential genotoxicity of this compound in invertebrates indicated a clear BDE-209 capability to induce DNA damage, but no irreversible effects on DNA hemocytes. Furthermore, bioaccumulation of this high-molecular-weight substance and its uptake mechanism in zebra mussel are also discussed.     

A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay

Baseline DNA damage in spermatozoa from fertile and infertilemen was compared using a modified alkali single cell gel electrophoresis(comet) assay. Semen from normozoospermic fertile, normozoospermicinfertile and asthenozoospermic infertile (World Health Organizationcriteria, 1992) samples were studied. No significant differencewas observed in levels of baseline damage between the threegroups. A median value for baseline damage of     

Evaluation of manual and image analysis quantification of DNA damage in the alkaline comet assay

The alkaline comet assay or single cell microgel electrophoresisassay is a sensitive method of detecting DNA strand breaks andalkali labile sites in individual cells. The results of thisassay can be analysed by different methods. In this study wecompared analyses of the same slides by a manual method andby image analysis, post-treatment of clone 707 Friend erythroleukaemiacells with H₂O₂. The parameters which were found to be particularlyuseful were comet area and comet length (measured manually)and percentage tail DNA, tail moment, tail length and tail length/headradius (L/H), measured using image analysis. The manual methodfor comet analysis presented in this paper would appear to providegood and reliable comet data. However, the image analysis cometsystem described offers an alternative analysis method whichavoids the need for photomicrographs and tedious manual analysis.The image analysis parameters: % tail DNA, tail moment, taillength and L/H give good consistent results and for large-scaleanalysis it will, therefore, conceivably be the method of choice. ¹To whom correspondence should be addressed     

Alkaline comet assay study with breast cancer patients: evaluation of baseline and chemotherapy-induced DNA damage in non-target cells

Abstract The sensitivity of the alkaline comet assay for the evaluation of baseline and treatment-induced DNA damage in white blood cells of breast cancer patients receiving adjuvant chemotherapy according to three conventional anthracycline- and cyclophosphamide-containing protocols was investigated. Additionally, baseline DNA damage in cancer patients was compared with the levels of DNA damage recorded in healthy women. Altogether 30 patients with diagnosed breast cancer and 30 female blood donors with no known familial history of breast cancer participated in the study. Alkaline comet assay was performed according to standard protocol and DNA migration in peripheral blood leukocytes was measured by a computer-based image analysis system. For each subject the frequency of “damaged” cells, i.e., long-tailed nuclei (LTN) with tail length exceeding 95th percentile for the considered parameter among controls, is also reported. Breast cancer patients had significantly increased background levels of DNA damage in their peripheral blood leukocytes as compared to healthy women. Prior to the chemotherapy, a majority of patients showed a statistically significant increase in the number of LTN compared to healthy blood donors. Marked interindividual variations in baseline DNA damage among patients were recorded, some of them related to the disease stage and status. The present study confirmed the alkaline comet assay as a sensitive technique able to detect significantly elevated DNA migration in blood cells of patients already one hour after completion of the first cycle of chemotherapy. Administration of antineoplastic drugs in three chemotherapy protocols studied induced a similar increase of primary DNA damage in nontarget cells. The evaluation of the LTN frequencies indicates the best response to the protocol containing cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Our results point to the significance of simultaneous evaluation of DNA migration and frequency of LTN in the same subject and approved the use of alkaline comet assay as a suitable method for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.     

Nonselective DNA damage induced by a replication inhibitor results in the selective elimination of extrachromosomal double minutes from human cancer cells

Gene amplification plays a pivotal role in human malignancy. Highly amplified genes frequently localize to extrachromosomal double minutes (dmin), which usually segregate to daughter cells in association with mitotic chromosomes. We and others had shown that treatment with low-dose hydroxyurea (HU) results in the elimination of dmin and reversion of the cancer cell phenotype. HU treatment in early S-phase, when dmin are replicated, results in their detachment from chromosomes at the next M-phase, leading to the appearance of micronuclei enriched in dmin, followed by their elimination. In this article, we examined the effect of low-dose HU on the behavior of dmin in relation to DNA damage induction by simultaneously monitoring LacO-tagged dmin and phosphorylated histone H2AX (gammaH2AX). As expected, treatment with low-dose HU induced numerous gammaH2AX foci throughout the nucleus in early S-phase, and these rarely coincided with dmin. Most chromosomal gammaH2AX foci disappeared by metaphase, whereas, unexpectedly, those that persisted frequently associated with dmin. We found that these dmin aggregated, detached from anaphase chromosomes, and apparently formed micronuclei. Because gammaH2AX foci likely represent DNA double strand breaks (DSBs), the response to DSBs sustained by extrachromosomal dmin appears to be different from that sustained by chromosomal loci, which may explain why DSB-inducing agents cause the selective elimination of dmin.     

Maleic hydrazide induces genotoxic effects but no DNA damage detectable by the comet assay in tobacco and field beans

The plant growth regulator and herbicide maleic hydrazide (MH) induced a high frequency of somatic mutations in leaves of tobacco (Nicotiana tabacum var. xanthi) and a high yield of chromosome aberrations in roots of field beans (Vicia faba, karyotype ACB). In contrast, no significant increase in MH-induced DNA damage, as measured by the Comet assay, could be demonstrated in either plant species. The absence of DNA migration induced by MH was not effected in tobacco by either pH of the MH solution, the sampling time after MH treatment or continuous MH treatment for 14 days. To our knowledge, MH represents the first agent which has proved to be highly mutagenic and clastogenic but does not cause DNA damage as measured by the Comet assay in the same experimental system.     

Aflatoxin exposure and DNA damage in the comet assay in individuals from the Gambia, West Africa.

The single cell gel electrophoresis assay (Comet assay) was used to measure DNA damage in peripheral lymphocytes from a group of individuals from The Gambia in order to determine whether such damage could be associated with increased exposure to aflatoxin in this population. Responses obtained were correlated to responses previously obtained [1] in a cross-sectional study in the same individuals of various cytogenetic alterations [chromosomal aberrations, micronuclei (crest positive and negative staining), and sister chromatid exchanges], and aflatoxin-albumin adducts. Analysis of variance methods were used to assess the effects of smoking, GSTM1 genotype, sex, age, and smoking status. A comparison was also made between The Gambian individuals and a group of healthy, non-smoking volunteers in the United Kingdom where aflatoxin exposure would be expected to be low. From the earlier study [1], it was determined that the levels of the sister chromatid exchanges and micronuclei were higher in The Gambian group than in a European group where aflatoxin exposure was lower, but that there were no correlations between the adduct levels and the cytogenetic abnormalities at the individual level. In the present study, DNA damage as measured in the Comet assay was not significantly higher than in the healthy United Kingdom volunteers. In addition, there were no associations between cytogenetic damage, GSTM1 genotype, age, sex, lifestyle factors (smoking and aflatoxin exposure), and Comet response at the individual level. Comet response was higher in females than males in The Gambia if one outlier was excluded from analysis and not taking into account other sources of variability. It would appear that DNA damage as measured in the Comet assay in peripheral blood lymphocytes is not a sensitive genotoxic marker of aflatoxin exposure in this population.     

Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10(6) bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14-0.33] and 0.31 (95% CI: 0.20-0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.