Reliability of the comet assay in cryopreserved human sperm

BACKGROUND: Although the comet assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the comet assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. METHODS: Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at x400 magnification. RESULTS: The highest correlation was between comet assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. CONCLUSIONS: Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral comet assay.     

The comet assay in human biomonitoring: gene-environment interactions

The comet assay is the method of choice for measuring DNA damage, of various sorts, in human cells such as lymphocytes obtained in the course of population-based studies of environmental and occupational exposure to different genotoxic agents, including radiation, chemicals and oxidative stress. It is noted for its versatility and the breadth of its possible applications. In terms of simplicity, cost, small amount of material required, sensitivity and reliability, the comet assay in its various modifications has few serious competitors. When standardized and validated, the comet assay can provide invaluable information in the areas of hazard identification and risk assessment of environmental and occupational exposure, diseases linked with oxidative stress (e.g. diabetes and cardiovascular disease), nutrition, monitoring the effectiveness of medical treatment and investigating individual variation in response to DNA damage that may reflect genetic or environmental influences. The information obtained could lead to individual advice on lifestyle changes to promote health and especially on relative risks of genotoxic exposure to environmental pollution.     

The effect of potassium diazoacetate on human peripheral lymphocytes, human adenocarcinoma Colon caco-2 cells, and rat primary colon cells in the comet assay.

In previous studies, N-(N'-acetyl-L-propyl)-N-nitrosoglycine (APNG) has been shown to be a potent mutagen in a variety of genotoxicity assays and a carcinogen in a limited cancer study. APNG decomposes to a carboxymethyldiazonium ion, which can also be generated from potassium diazoacetate (KDA). KDA is particularly interesting because it is a stable nitrosated derivative of glycine, one of the most common dietary amino acids. KDA has been shown to produce more O6 carboxymethyl- and O6 methyl-adducts than APNG, so it was anticipated that it might also be a potent genotoxic agent. Thus in the present study KDA has been investigated in the single cell gel electrophoresis (Comet) assay, which primarily measures DNA strand breakage. Since KDA has been shown to be formed in the gut, the genotoxic effects of KDA were investigated in vitro in human adenocarcinoma colon Caco-2 cells, and in rat primary colon cells and compared to responses from human peripheral lymphocytes. KDA induced DNA damage in the three cell types, confirming that KDA is genotoxic in a range of mammalian cells.     

Genotoxicity of some sulfur dyes on tadpoles (Rana hexadactyla) measured using the comet assay.

This report presents the results of a genotoxicity study to evaluate the DNA damage caused by four sulfur dyes used in the textile and tannery industries. Alkaline single-cell gel electrophoresis assay (SCGE) was performed on erythrocytes from Rana hexadactyla tadpoles following whole-body exposure to increasing concentrations of the dyes. The dyes, along with their active ingredients, were Sandopel Basic Black BHLN, Negrosine, Dermapel Black FNI, and Turquoise Blue. The dye-treated tadpoles showed significant DNA damage, measured as mean DNA length:width ratio, when compared with unexposed control animals. Among the four tested dyes Sandopel Basic Black BHLN appears to be highly genotoxic, Dermapel Black FNI was least genotoxic, and Negrosine and Turquoise Blue were moderately toxic to R. hexadactyla tadpoles. The tadpoles showed a significant reduction in DNA damage when placed in dechlorinated tap water after exposure for a 24-hr period to the dye solutions.     

The use of the comet assay in the study of human nutrition and cancer

The influence of diet on carcinogenesis is a hugely complex area; not only is the consumption of major dietary factors such as meat, fat and fruits and vegetables associated with increased or decreased risk of a range of cancers but also an increasing number of specific nutrients such as vitamins, minerals and phytochemicals are being proposed as the next 'superfoods' to combat the development of cancer. As well as epidemiological studies to determine the association of these dietary factors with cancer risk, it is also essential to investigate the underlying mechanisms through which these factors may causally influence carcinogenesis. The comet assay provides a relatively simple, cheap and rapid method to examine DNA damage and repair and is, therefore, an ideal biomarker for the study of the effects of nutrition on cancer. This review focuses on the use of the comet assay in studies involving human subjects or human cell lines, which investigate the effects of various nutrients on biomarkers relevant to carcinogenesis, and discusses the potential of the comet assay and its various modifications for use as cancer-related biomarkers suitable for use in nutritional studies.     

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     

Statistical issues in the use of the comet assay

The comet or single-cell gel electrophoresis assay is now widely used in regulatory, mechanistic and biomonitoring studies using a range of in vitro and in vivo systems. Each of these has issues associated with the experimental design which determine to a large extent the statistical analyses than can be used. A key concept is that the experimental unit is the smallest 'amount' of experimental material that can be randomly assigned to a treatment: the animal for in vivo studies and the culture for in vitro studies. Biomonitoring studies, being observational rather than experimental, are vulnerable to confounding and biases. Critical factors in any statistical analysis include the identification of suitable end points, the choice of measure to represent the distribution of the comet end point in a sample of cells, estimates of variability between experimental units and the identification of the size of effects that could be considered biologically important. Power and sample size calculations can be used in conjunction with this information to identify optimum experimental sizes and provide help in combining the results of statistical analyses with other information to aid interpretation. Interpretation based upon the size of effects and their confidence intervals is preferred to that based solely upon statistical significance tests. Statistical issues associated with the design and subsequent analyses of current validation studies for the comet assay include the identification of acceptable levels of intra- and inter-laboratory repeatability and reproducibility and criteria for dichotomizing results into positive or negative.     

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.     

Proposed mode of action for in utero effects of some phthalate esters on the developing male reproductive tract

Phthalate esters containing a straight-chain backbone of 4-6 carbons have demonstrated testicular toxicity and infertility in adult and pre-adolescent rats, mice, hamsters, and ferrets. In recent years, these same phthalates have been shown to interfere with the normal development of the male reproductive tract in rodents and rabbits. The review presented here summarizes studies that provide evidence of a mode of action for these effects. The data indicate that C4-C6 phthalate esters inhibit processes in the Leydig cell, such as the synthesis of testosterone (T) and production of insulin-like factor 3 (insl3), both of which are required for normal development of male genitalia. A proposed secondary effect of reduced androgen production is on Sertoli cells, resulting in failure to proliferate and interference with cell-cell communication (gap-junction intracellular communication) leading to the development of large multinucleate gonocytes. The possibility that phthalates act directly on the Sertoli cells to interfere with intracellular communication is not excluded. The strength, consistency, and plausibility of the proposed mode of action and alternate modes of action are discussed.     

Recommendations for design of the rat comet assay

Although the rodent comet assay is gaining acceptance as a standard technique for evaluating DNA damage in vivo, there is no internationally accepted guideline for its conduct and several aspects of its experimental design have not been optimized. For example, no standard positive control is used, there is no agreement on how tissue toxicity should be measured and sources of experimental variability have not been considered in relation to experimental design. This study showed that methylnitrosourea is a good alternative positive control inducing DNA damage in all tissues examined (stomach, liver, blood and bone marrow) over a dose range of 25-100 mg/kg at both 3 and 24 h after treatment. At the highest dose, significant toxicity was seen in all tissues using the neutral diffusion assay and also by histopathological/haematological analysis, except in the liver where no change was seen even 7 days after dosing. Analyses using control data pooled from several studies showed that, as expected, the greatest variability was seen between tissue preparations from different animals and that different numbers of animals were required to detect the same fold increases in different tissues. Power analyses showed that, preparing three gels for each tissue and scoring 50 nuclei per gel, a group of six animals allows 2-fold increases over control in the liver, bone marrow and stomach and a 3-fold increase in blood to be detected with 80% probability. It is recommended that similar investigations of experimental variability should be performed to determine optimal experimental design in any laboratory using the rodent comet assay.     

Ecotoxicological applications and significance of the comet assay

Application of the single-cell gel electrophoresis or comet assay has revolutionized the field of genetic ecotoxicology or eco-genotoxicology. It is a rapid, sensitive and relatively inexpensive method providing the opportunity to study DNA damage (including oxidative damage), repair and cell death (apoptosis) in different cell types without prior knowledge of karyotype and cell turnover rate. The assay has, however, often attracted criticism for its lack of ecotoxicological relevance. In addition, in contrast to genetic toxicology where rapid technical progress has been made to improve cell- and tissue-specific adoption of the assay, only limited advancement has been made to transfer the methodologies to ecotoxicological studies. While reviewing the recent information available in the literature and underscoring the importance of induced genetic damage in natural species, the aims of this article are to (i) highlight and judiciously analyse the ecotoxicological relevance of the assay; (ii) attempt to correlate the comet response with other relevant biological responses or biomarkers; (iii) identify the technical challenges and various factors affecting its application in order to make it reliable, reproducible and robust; (iv) critically compare the technical developments in genetic toxicology and genetic ecotoxicology and (v) evaluate the future developments with respect to applications of the assay. It is suggested that while complementing other ecotoxicological parameters and further improving the methodologies, the comet assay will continue to play an important role in genetic ecotoxicology to determine induced genetic damage, which has significant consequences for short- and long-term survival of the natural or wild species. Information obtained through integrated studies using simultaneous applications of multiple biomarkers on different wild organisms could also provide an holistic dimension of toxicological impact of environmental contaminants for the protection of human health.     

DNA-damaging effect of cyclophosphamide on human blood cells in vivo and in vitro studied with the single-cell gel test (comet assay)

The single-cell gel (SCG) test was used to study the induction and persistence of DNA damage by cy-clophosphamide (CP) in human blood cells after treatment in vitro and in vivo. S9-mix-activated CP (from 0.1 mM upward) induced DNA effects in a concentration-dependent manner in the in-vitro SCG test. Blood cells from various donors showed considerable intra- and interindividual variability. Incubation of CP-treated blood samples at 37°C caused a rapid decrease in DNA effects, but DNA migration was still significantly increased 1 hr after the end of the CP treatment. Comparative studies with the in vitro sister chromatid exchange (SCE) test were performed that demonstrated that much lower CP concentrations (about 100 times) were required for a significant induction of SCEs. A group of 11 patients who suffered from vasculitis/collagen disease and were treated with low CP doses (50–200 mg/day) exhibited an elevated level of DNA damage in the SCG test with peripheral blood cells, compared with a group of 11 control persons or 5 patients without chemotherapy. Increases in DNA damage were variable and not clearly related to the CP dose. SCE tests could successfully be performed with 5 out of the 11 CP-treated patients; all showed significantly increased SCE frequencies. For six patients no result could be obtained with the SCE test due to a failure of lymphocyte proliferation. Three multiple sclerosis patients who received high doses of CP were investigated with the SCG test before, during, and after the treatment. The results indicate that CP-induced DNA effects that are detectable with the SCG test persist in vivo for a period of several days, but for less than 2 weeks. The results of our study provide information with regard to the use of the SCG test in human monitoring. The advantages and limitations of the test are discussed. © 1995 Wiley-Liss, Inc.     

Correlation of apoptosis with comet formation induced by tea polyphenols in human leukemia cells.

Induction of apoptosis is an important approach to cancer control. Apart from morphological changes in cells, apoptosis is characterized by fragmentation of nuclear DNA. The characteristic DNA ladder formation that is observed on gel electrophoresis does not reflect the DNA breakdown in individual cells; contributions from small subpopulations are usually overlooked. On the other hand, alkaline comet assay as measured by single cell gel electrophoresis accurately measures DNA fragmentation at a single cell level. The comet assay was originally developed as a cytogenetic test to measure the genotoxicity of various chemicals. However, the comet image generated by an apoptotic cell is different from that obtained with a cell treated for a short time with a genotoxic agent. In the present study using human leukemic cells, typical apoptotic features such as morphological characteristics, FACS analysis, caspase activation, and expression of apoptosis-related genes as induced by tea polyphenols have been found to correlate with the comet tail formation. It is apparent from the high degree of correlation observed between the comet tail moment and each parameter of apoptosis that the comet assay can accurately reflect the measure of DNA fragmentation and, hence, can be used to detect a cell undergoing apoptosis.     

Detection and assay of some human proteins by immunoconcentration technique.

For detection and semi-quantitative assays of some human proteins by immunoconcentration techniques, specific antibodies covalently bound to nylon microparticles and then captured in the form of small dots on glass microfibre disc, were used. The discs were placed on a water absorbant material in a simple plastic device. For the technique, antibodies labeled with peroxidase were also applied. The use of several chromogenic substrates for peroxidase was checked in the technique.     

Detection of ghost cells in the standard alkaline comet assay is not a good measure of apoptosis

The single cell gel electrophoresis assay, or Comet assay, is a powerful tool for measurement of DNA strands breaks, oxidative damage, and alkali labile sites, and the assay was recently modified to detect DNA cross-links. It has also been proposed as a measure of apoptosis since apoptotic cells are suspected to result in total migration of the DNA from the nucleus into the tail. Cells with this appearance are called ghost cells, clouds, hedgehogs, or NDCN (nondetectable cell nuclei). The aim of this study was to determine if ghost cells can be used to measure apoptosis in the standard alkaline comet assay. To answer this question, we made use of two cell lines: CTLL-2 cells that can enter apoptosis upon addition of apoptosis stimuli or IL-2 deprivation, and CTLL-2 bcl2 cells that are protected from apoptosis due to the overexpression of the apoptosis inhibitor gene bcl2. The two cell lines were treated with cytotoxins (nongenotoxic apoptosis inducers, nongenotoxic necrotic agents) or genotoxins. They were also subjected to growth factor withdrawal, which induced apoptosis in the CTLL-2 cell line. The level of apoptosis was measured by the Annexin V-FITC method in parallel with performing the Comet assay. The results obtained in the two cell lines suggest that apoptotic or necrotic death does not correlate well with the detection of ghost cells, presumably because these cells are lost upon electrophoresis. A variant of the alkaline Comet assay that was performed without electrophoresis (halo method) was able to efficiently detect cells undergoing apoptosis, but it was unable to clearly distinguish between apoptosis and genotoxic damage.     

Detection of DNA-crosslinking agents with the alkaline comet assay

The single cell gel electrophoresis, or comet assay, under alkaline conditions is a sensitive, simple and rapid method for the detection of DNA damage at the individual cell level. Its applicability as an indicator for the DNA crosslinking potency of a test substance was investigated in human white blood cells by combined treatment with the DNA damaging agent methyl methanesulphonate (MMS) for 2 hr at 37°C. The known crosslinking agents cisplatinum, mitomycin C and formaldehyde, and the formaldehyde releasers diazolidinyl urea and dimethylol urea, were shown to reduce MMS-induced DNA migration in the comet assay in a concentration-dependent manner. Two other protocols, adding MMS to the cells before or after treatment with a crosslinking agent, were carried out and achieved similar results. The results of this study indicate that the comet assay is a useful tool for the detection of crosslinking agents. Advantages and limitations of this method compared to the alkaline elution technique are discussed. © 1996 Wiley-Liss, Inc.