The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA. The mechanism of formation of comets (under neutral or alkaline conditions) is best understood by analogy with nucleoids, in which relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break releases that loop to extend into a halo-or, in the case of the comet assay, to be pulled towards the anode under the electrophoretic field. A consideration of the simple physics underlying electrophoresis leads to a better understanding of the assay. The sensitivity of the assay is only fully appreciated when it is calibrated: between one hundred and several thousand breaks per cell can be determined. By including lesion-specific enzymes in the assay, its range and sensitivity are greatly increased, but it is important to bear in mind that their specificity is not absolute. Different approaches to quantitation of the comet assay are discussed. Arguments are presented against trying to apply the comet assay to the study of apoptosis. Finally, some of the advantages and disadvantages of using the comet assay on lymphocyte samples collected in human studies are rehearsed.

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Oxidized bases in DNA can be measured directly by high-performance liquid chromatography (HPLC). 7, 465-469 (1996)

Oxidized bases in DNA can be measured directly by high-performance liquid chromatography (HPLC). 7,8-Dihydro-8-oxo-guanine (8-OHgua), as the most abundant oxidation product, is often regarded as an indicator of oxidative stress. Estimates of endogenous 8-OHgua levels in human lymphocyte DNA are between 2 and 8 for every 10(5) unaltered bases--a high frequency in view of the potential mutagenicity of this base alteration and of the presence of an effective base excision repair pathway in eukaryotic cells. An alternative approach to the measurement of oxidized bases makes use of repair endonucleases with appropriate lesion specificities--endonuclease III, for oxidized pyrimidines and formamidopyrimidine glycosylase for 8-OHgua. These enzymes introduce breaks at sites of damage in DNA. The comet assay (single cell gel electrophoresis) can then be used to detect the DNA breaks. This modified comet assay, like other enzyme-linked DNA breakage assays, gives a value for endogenous oxidative base damage that is more than 10-fold lower than most estimated from HPLC. It is possible that HPLC-based estimates are artificially high because oxidation of guanine occurs during isolation, storage, or hydrolysis of DNA. Using a revised DNA isolation procedure designed to decrease in vitro oxidation, we have obtained results for 8-OHgua concentrations in human lymphocytes that are closure to the figures obtained by the comet assay. It is still an open question whether 8-OHgua, measured by HPLC or by the comet assay, is a valid marker for oxidative damage.

We investigated association between polymorphisms in DNA repair genes and the capacity to repair DNA damage induced by gamma-irradiation and by base oxidation in a healthy population. Irradiation-specific DNA repair rates were significantly decreased in individuals with XRCC1 Arg399Gln homozygous variant genotype (0.45 +/- 0.47 SSB/10(9) Da) than in those with wild-type genotype (1.10 +/- 0.70 SSB/10(9) Da, P=0.0006, Mann-Witney U-test). The capacity to repair oxidative DNA damage was significantly decreased among individuals with hOGG1 Ser326Cys homozygous variant genotype (0.37 +/- 0.28 SSB/10(9) Da) compared to those with wild-type genotype (0.83 +/- 0.79 SSB/10(9) Da, P=0.008, Mann-Witney U-test). Investigation of genotype combinations showed that the increasing number of variant alleles for both XRCC1 Arg399Gln and APE1 Asn148Glu polymorphisms resulted in a significant decrease of irradiation-specific repair rates (P=0.008, Kruskal-Wallis test). Irradiation-specific DNA repair rates also decreased with increasing number of variant alleles in XRCC1 Arg399Gln in combination with variant alleles for two other XRCC1 polymorphisms, Arg194Trp and Arg280His (P=0.002 and P=0.005, respectively; Kruskal-Wallis test). In a binary combination variant alleles of hOGG1 Ser326Cys and APE1 Asn148Glu polymorphisms were associated with a significant decrease in the capacity to repair DNA oxidative damage (P=0.018, Kruskal-Wallis test). In summary, XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms seem to exert the predominant modulating effect on irradiation-specific DNA repair capacity and the capacity to repair DNA oxidative damage, respectively.

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Workers employed in tire plants are exposed to a variety of xenobiotics, such as 1,3-butadiene (BD), soots containing polycyclic aromatic hydrocarbons, and other organic chemicals (e.g., styrene). In the present study, we investigated markers of genotoxicity [chromosomal aberrations (CAs) and single-strand breaks (SSBs)] in a cohort of 110 tire plant workers engaged in jobs with different levels of xenobiotic exposure in relation to various polymorphisms in genes coding for biotransformation enzymes (CYP1A1, CYP2E1, EPHX1, GSTM1, GSTP1, and GSTT1) and in genes involved in DNA repair (XPD exon 23, XPG exon 15, XPC exon 15, XRCC1 exon 10, and XRCC3 exon 7). In addition, the expression of CYP2E1, a gene playing a key role in BD metabolism, was determined by real-time PCR in peripheral blood lymphocytes, and the capacity of lymphocytes to repair gamma-ray-induced SSBs and to convert 8-oxoguanine in HeLa cell DNA into SSBs was assessed using in vitro assays. No positive associations were detected between the CA frequency or SSB induction and levels of workplace exposure; however, a nonsignificant twofold higher irradiation-specific DNA repair rate was found among highly exposed workers. In evaluations conducted with the markers of individual susceptibility, workers with low-EPHX1-activity genotypes exhibited a significantly higher CA frequency as compared to those with medium and high-EPHX1-activity genotypes (P = 0.050). CA frequencies were significantly lower in individuals homozygous for the XPD exon 23 variant allele in comparison to those with the wild-type CC genotype (P = 0.003). Interestingly, CAs were higher in individuals with higher CYP2E1 expression levels, but the association was nonsignificant (P = 0.097). The results from this study suggest the importance of evaluating markers of individual susceptibility, since they may modulate genotoxic effects induced by occupational exposure to xenobiotics.

The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations.