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The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.The comet assay is commonly used to assess DNA damage. This collection of consensus protocols includes adaptations for a wide range of species and sample types, assay formats and detection of different types of DNA lesions.

Increasing lead contamination in Egyptian ecosystems and high lead concentrations in food items have raised concern for human health and stimulated studies on monitoring ecotoxicological impact of lead-caused genotoxicity. In this work, the alkaline comet assay was modified for monitoring DNA strand breakage in sensitive early life stages of the African catfish Clarias gariepinus. Following exposure to 100, 300, and 500 microg/L lead nitrate, DNA strand breakage was quantified in embryos at 30, 48, 96, 144, and 168 h post-fertilization (PFS). For quantitative analysis, four commonly used parameters (tail % DNA, %TDNA; head % DNA, %HDNA; tail length, TL; tail moment, TM) were analyzed in 96 nuclei (in triplicates) at each sampling point. The parameter %TDNA revealed highest resolution and lowest variation. A strong correlation between lead concentration, time of exposure, and DNA strand breakage was observed. Here, genotoxicity detected by comet assay preceded the manifested malformations assessed with conventional histology. Qualitative evaluation was carried out using five categories are as follows: undamaged (%TDNA 75%) nuclei confirming a dose and time-dependent shift towards increased frequencies of highly and extremely damaged nuclei. A protective capacity provided by a hardened chorion is a an interesting finding in this study as DNA damage in the prehatching stages 30 h-PFS and 48 h-PFS was low in all treatments (qualitative and quantitative analyses). These results clearly show that the comet assay is a sensitive tool for the detection of genotoxicity in vulnerable early life stages of the African catfish and is a method more sensitive than histological parameters for monitoring genotoxic effects. 2008 Wiley Periodicals, Inc.

The effects of lower doses of ionising radiation may not show up for years after exposure and are due to various changes in DNA molecules and chromosomes. Radiation-induced mutations seem to be brought about by the deletion of small pieces of chromosomes during the process of chromosome breakage and repair. Ionising radiation kills rapidly dividing cells, blood lymphocytes among them. The cytogenetical consequences of accidental exposure to g-radiation (radiation dose was 221 mSv) were investigated by using alkaline Comet assay and the analysis of structural chromosomal aberration. Blood samples were collected during one-year period after the incident. By using the Comet assay immediately after accidental exposure a high level of DNA damage was recorded. Although this level was decreasing over a one-year period, it was still elevated compared to normal values of DNA damage for unexposed persons. Immediately after the incident a prevalence of chromosomal over chromatid aberrations was recorded. However, one year after only a few chromatid breaks were recorded. The results confirmed the usefulness of the alkaline Comet assay as a simple and sensitive technique for the biomonitoring of DNA damages, especially in the cases of accidental exposure to ionising radiation.

The alkaline comet assay was employed in the assessment of DNA damage in leukocytes of a worker incidently exposed to gamma radiation (221mSv, Co-60 source). The comet tail lenghts and tail moments were studied. By using the alkaline comet assay immediately after accidental exposure a high level of DNA damage was recordd one day and one week after the radiation incident. Later on, a decrease in both comet parameters was diminished during a one year period, it was still elevated compared to normal values recorded in leukocytes of a healthy, unexposed person.The results, obtained indicate that the alkaline comet assay is a rapid and sensitive microdosimetric technique and is suitable for in vivo human biomonitoring, especially in cases of incidental exposure to ionizing radiation.

The alkaline comet assay was performed to measure DNA integrity in fish hepatocytes. Primary cultures of rainbow trout hepatocytes were exposed to two known genotoxic compounds, hydrogen peroxide (H 2 O 2 ) and benzo[ a ]pyrene (B[ a ]P), and to organic extracts of river sediments. The DNA damage in the form of single-strand breaks was monitored following the formation of DNA comets after alkaline electrophoresis. Exposure of the hepatocytes to H 2 O 2 for 2 hr increased strand breaks in a dose-related manner at the concentration range reported previously in studies with mammalian hepatocytes. B[ a ]P treatment led to a significant increase in strand breaks at the concentrations ranging from 0.1 to 10 μM after 4 hr of exposure. After 48 hr of exposure to B[ a ]P, the level of DNA strand breaks was lower than that of the control. The organic extracts obtained from river sediments significantly increased DNA strand breaks in trout hepatocytes, indicating the presence of genotoxic compounds in the sediment. The results show that the alkaline comet assay is a promising method by which to study the genotoxic potential of xenobiotics found in the aquatic environment.