Search

The use of the comet assay in large biomonitoring studies may present logistical and technical challenges because of the processing of numerous samples. Proper sample preservation becomes imperative to prevent spurious DNA breakage. Previous research has shown the feasibility of conducting the comet assay on frozen blood samples, highlighting the potential of freezing at - 80 °C in preserving DNA integrity. Nonetheless, this approach presents challenges, including potential DNA damage during freezing and thawing, variability in processing, and the need for standardized protocols. Our objective was to evaluate whether there are comparable results in DNA migration assessed by the comet assay between fresh and frozen blood samples on a larger scale (N = 373). In our findings, elevated DNA migration was evident in frozen samples relative to fresh ones. Additionally, smoking, alcohol consumption, and season were linked to increased DNA damage levels in whole blood cells. Based on our results and available literature, conducting the comet assay on frozen blood samples emerges as a practical and efficient approach for biomonitoring and epidemiological research. This method enables the assessment of DNA damage in large populations over time, with samples, if properly cryopreserved, that may be used for years, possibly even decades. These observations hold significant implications for large-scale human biomonitoring and long-term epidemiological studies, particularly when samples are collected during fieldwork or obtained from biobanks. Continued method optimization and validation efforts are essential to enhance the utility of this approach in environmental and occupational health studies, emphasizing caution when comparing data obtained between fresh and frozen blood samples., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The in vitro micronucleus (MN) assay is a component of most test batteries used in assessing potential genotoxicity. Our previous study adapted metabolically competent HepaRG cells to the high-throughput (HT) flow-cytometry-based MN assay for genotoxicity assessment (Guo et al. in J Toxicol Environ Health A 83:702-717, 2020b, https://doi.org/10.1080/15287394.2020.1822972 ). We also demonstrated that, compared to HepaRG cells grown as two-dimensional (2D) cultures, 3D HepaRG spheroids have increased metabolic capacity and improved sensitivity in detecting DNA damage induced by genotoxicants using the comet assay (Seo et al. in ALTEX 39:583-604, 2022, https://doi.org/10.14573/altex.22011212022 ). In the present study, we have compared the performance of the HT flow-cytometry-based MN assay in HepaRG spheroids and 2D HepaRG cells by testing 34 compounds, including 19 genotoxicants or carcinogens and 15 compounds that show different genotoxic responses in vitro and in vivo. 2D HepaRG cells and spheroids were exposed to the test compounds for 24 h, followed by an additional 3- or 6-day incubation with human epidermal growth factor to stimulate cell division. The results demonstrated that HepaRG spheroids showed generally higher sensitivity in detecting several indirect-acting genotoxicants (require metabolic activation) compared to 2D cultures, with 7,12-dimethylbenzanthracene and N-nitrosodimethylamine inducing higher % MN formation along with having significantly lower benchmark dose values for MN induction in 3D spheroids. These data suggest that 3D HepaRG spheroids can be adapted to the HT flow-cytometry-based MN assay for genotoxicity testing. Our findings also indicate that integration of the MN and comet assays improved the sensitivity for detecting genotoxicants that require metabolic activation. These results suggest that HepaRG spheroids may contribute to New Approach Methodologies for genotoxicity assessment., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Mechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage., (© 2021. The Author(s).)

Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.

The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.

The evaluation of genotoxicity plays an important role within hazard identification and risk assessment of chemicals and consumer products. For genotoxicity assessment, in vitro hepatic cells are often used as they have retained certain level of xenobiotic metabolic activity. However, current protocols are designed for the use on 2D monolayer models that are associated with several limitations due to the lack of numerous biological functions, which results in the loss of many hepatic properties. In this respect, an attractive alternative are three-dimensional (3D) models. The aim of our study was to develop physiologically more relevant 3D cell model (spheroids) from the human hepatocellular carcinoma (HepG2) cell line for genotoxicity testing. The spheroids were prepared by the forced floating method, which had been optimized for the production of a large number of uniform spheroids. The sensitivity of the spheroids to detect genotoxicity was determined by the comet assay after the exposure of spheroids to non-cytotoxic concentrations of model indirect acting genotoxic compounds, namely polycyclic aromatic hydrocarbon (B(a)P), mycotoxin (AFB1), two heterocyclic aromatic amines (PhIP and IQ) and a direct acting etoposide (ET). All five tested compounds concentration dependently induced DNA damage. Higher sensitivity of 3D cell model compared to 2D monolayer culture was noticed particularly for detection of the genotoxicity of the heterocyclic aromatic amines and BaP. Deregulation of mRNA expression (qPCR) by genotoxic compounds revealed that HepG2 cells in 3D express important genes encoding phase I and II metabolic enzymes, as well as DNA damage responsive genes in an inducible form. The newly developed HepG2 3D model shows improved sensitivity for detecting genotoxic compounds compared to 2D cultures and can provide a suitable experimental model for genotoxicity assessment.

Amphibians are constantly exposed to pollutants and the stress of agricultural activities. We selected three anuran amphibian species Dendropsophus minutus, Boana albopunctata, and Physalaemus cuvieri, totaling 309 individuals. We collected tadpoles in 15 permanent ponds: 5 soybean crops, 3 corn crops, and 7 nonagricultural lands. Our study provides the first comparative data on the genotoxicity and mutagenicity of three common amphibian anurans. Dendropsophus minutus was the most vulnerable species compared with B. albopunctata and P. cuvieri for comet assay and micronuclei test. However, the more significant amount of DNA damage seen in D. minutus does not mean that their populations are threatened once such species adapt well to anthropogenic disturbances. Despite, P. cuvieri was less sensitive than the other two species; the DNA damage was significantly higher in soybean crops. Physalaemus cuvieri is a leptodactylidae species that deposit their eggs in foam nests, which are essential to protect eggs from dehydration. Moreover, the foam reduces the contact of eggs with water; thus, P. cuvieri eggs could be less exposed to contaminants present in pounds, compared with D. minutus and B. albopunctata, which deposit their eggs directly in the water. Therefore, this study was sufficiently sensitive to detect genotoxic and mutagenic effects in tadpoles exposed to agroecosystems. We strongly suggest D. minutus in future biomonitoring studies that involve the comparison of anthropized versus not anthropized environments. Overall, we recommend the comet assay and micronucleus test as effective methods for the detection of genotoxic damage in amphibian anurans to the environmental disturbance, especially in agricultural sites.

In vitro genotoxicity testing that employs metabolically active human cells may be better suited for evaluating human in vivo genotoxicity than current bacterial or non-metabolically active mammalian cell systems. In the current study, 28 compounds, known to have different genotoxicity and carcinogenicity modes of action (MoAs), were evaluated over a wide range of concentrations for the ability to induce DNA damage in human HepG2 and HepaRG cells. DNA damage dose-responses in both cell lines were quantified using a combination of high-throughput high-content (HTHC) CometChip technology and benchmark dose (BMD) quantitative approaches. Assays of metabolic activity indicated that differentiated HepaRG cells had much higher levels of cytochromes P450 activity than did HepG2 cells. DNA damage was observed for four and two out of five indirect-acting genotoxic carcinogens in HepaRG and HepG2 cells, respectively. Four out of seven direct-acting carcinogens were positive in both cell lines, with two of the three negatives being genotoxic mainly through aneugenicity. The four chemicals positive in both cell lines generated HTHC Comet data in HepaRG and HepG2 cells with comparable BMD values. All the non-genotoxic compounds, including six non-genotoxic carcinogens, were negative in HepaRG cells; five genotoxic non-carcinogens also were negative. Our results indicate that the HTHC CometChip assay detects a greater proportion of genotoxic carcinogens requiring metabolic activation (i.e., indirect carcinogens) when conducted with HepaRG cells than with HepG2 cells. In addition, BMD genotoxicity potency estimate is useful for quantitatively evaluating CometChip assay data in a scientifically rigorous manner.

The problem of the currently used routine genotoxicity tests is relatively low predictivity of in vitro tests for in vivo genotoxicity and carcinogenicity. An important reason is considered to be inadequate expression of xenobiotic-metabolizing enzymes in indicator cell lines. The aim of our study was to generate metabolically active differentiated hepatic progenies (hDHP) from human adipose tissue-derived mesenchymal stem cells (hASC) for genotoxicity testing. hDHP, generated using a three-step hepatic differentiation procedure, expressed hepatic properties such as glycogen storage and albumin secretion. The results of the comet assay demonstrated comparable sensitivity of hASC and hDHP to detect DNA damage induced by a direct acting genotoxic agent tert-butylhydroperoxide. Exposure to model indirect acting genotoxins benzo(a)pyrene, aflatoxin B 1 , and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine did not induce DNA damage in hASC, while hDHP cells detected DNA damage induced by benzo(a)pyrene and aflatoxin B 1 , indicating their metabolic activity. The gene and protein expression analysis confirmed the presence of key enzymes involved in metabolism of the three genotoxins in hDHP cells. Moreover, the exposure of hDHP to the model pro-carcinogens altered the expression of selected metabolic genes. hDHP were further immortalized with hTERT transfection, resulting in a stable cell line that can be matured to metabolically active hDHP ready for genotoxicity testing in only 2 weeks. The advantage of these immortalized cells is their prolonged replicative life span and consequently limitless supply of hDHP cells. We conclude that hDHP cells have a great potential for the application in the routine genotoxicity testing and are therefore worth further investigations.

Earthworm toxicity assays contribute to ecological risk assessment and consequently standard toxicological endpoints, such as mortality and reproduction, are regularly estimated. These endpoints are not enough to better understand the mechanism of toxic pollutants. We employed an additional endpoint in the earthworm Eisenia andrei to estimate the pollutant-induced stress. In this study, comet assay was used as an additional endpoint to evaluate the genotoxicity of weathered hydrocarbon contaminated soils containing 520 to 1450 mg hydrocarbons kg -1 soil. Results showed that significantly higher DNA damage levels (two to sixfold higher) in earthworms exposed to hydrocarbon impacted soils. Interestingly, hydrocarbons levels in the tested soils were well below site-specific screening guideline values. In order to explore the reasons for observed toxicity, the contaminated soils were leached with rainwater and subjected to earthworm tests, including the comet assay, which showed no DNA damage. Soluble hydrocarbon fractions were not found originally in the soils and hence no hydrocarbons leached out during soil leaching. The soil leachate's Electrical Conductivity (EC) decreased from an average of 1665 ± 147 to 204 ± 20 µS cm -1 . Decreased EC is due to the loss of sodium, magnesium, calcium, and sulphate. The leachate experiment demonstrated that elevated salinity might cause the toxicity and not the weathered hydrocarbons. Soil leaching removed the toxicity, which is substantiated by the comet assay and soil leachate analysis data. The implication is that earthworm comet assay can be included in future eco (geno) toxicology studies to assess accurately the risk of contaminated soils.

The comet assay is a versatile, reliable, cost-efficient, and fast technique for detecting DNA damage and repair in any tissue. It is useable in almost any cell type and applicable to both eukaryotic and prokaryotic organisms. Instead of highlighting one of the numerous specific aspects of the comet assay, the present review aims at giving an overview about the evolution of this widely applicable method from the first description by Ostling and Johanson to the OECD Guideline 489 for the in vivo mammalian comet assay. In addition, methodical aspects and the influence of critical steps of the assay as well as the evaluation of results and improvements of the method are reviewed. Methodical aspects regarding oxidative DNA damage and repair are also addressed. An overview about the most recent works and relevant cutting-edge reviews based on the comet assay with special regard to, e.g., clinical applications, nanoparticles or environmental risk assessment concludes this review. Taken together, the presented overview raises expectations to further decades of successful applications and enhancements of this excellent method.

Objectives: Dental composite materials come into direct contact with oral tissue, especially gingival cells. This study was performed to evaluate possible DNA damage to gingival cells exposed to resin composite dental materials., Materials and Methods: Class V restorations were placed in 30 adult patients using two different composite resins. The epithelial cells of the gingival area along the composite restoration were sampled prior to and after 7, 30, and 180 days following the restoration of the tooth. DNA damage was analysed by comet and micronucleus assays in gingival exfoliated epithelial cells., Results: The results showed significantly higher comet assay parameters (tail length and % DNA in the tail) within periods of 30 and 180 days. The micronucleus test for the same exposure time demonstrated a higher number of cells with micronuclei, karyolysis, and nuclear buds. Results did not reveal any difference between the two composite materials for the same duration of exposure., Conclusion: Based on the results, we can conclude that the use of composite resins causes cellular damage. As dental composite resins remain in intimate contact with oral tissue over a long period of time, further research on their possible genotoxicity is advisable., Clinical Relevance: Long-term exposure of gingival cells to two different composite materials demonstrated certain DNA damage. However, considering the significant decline in micronuclei frequency after 180 days and efficiency in the repair of primary DNA damage, the observed effects could not be indicated as biologically relevant.

The Songhua River, in northeast China, has heavy organic contamination due to domestic sewage and industrial wastewater. Thus, it is important to further determine its genotoxic activity, which is a potential hazard for human health. Short-term genotoxic bioassays using Salmonella, the cytokinesis-block micronucleus cytome assay, and mouse liver cell comet assay were employed to further examine the genotoxic activity of diethyl ether extracts of water samples taken from the Songhua River. Ames test results showed that there were still frame-shift mutagens, both direct and indirect, in water samples at doses of 5.0 or 7.0 L water equivalent/plate. The mutagenicity seems to be less when compared with the results from 2002 to 2003. A dose-response relationship was also obtained between DNA damage in mouse liver cells by comet assay and micronuclei formation by CBMN assay. These results indicate that the water samples showed genotoxic activity with a mutagenic potency. 88 and 104 compounds, respectively, were identified in summer and winter water sample extracts by gas chromatography-mass spectrometry analysis. Four priority pollutants listed by the United States Environmental Protection Agency and six priority pollutants listed by the Chinese Environment Protection Agency were found in summer or winter water samples, respectively. The results indicate that the diethyl ether extracts of surface water samples taken from the Songhua River still show genotoxic activity (≥3.0 L water). The risks of potential carcinogenicity for human health in the Songhua River should be studied further.

A battery of bioassays was used to assess the ecotoxicological risk of soil spiked with a range of phenanthrene levels (0.95, 6.29, 38.5, 58.7, 122, and 303 μg g(-1) dry soil) and aged for 69 days. Multiple species (viz. Brassica rapa, Eisenia feotida, Vibrio fischeri), representing different trophic levels, were used as bioindicator organisms. Among acute toxicity assays tested, the V. fischeri luminescence inhibition assay was the most sensitive indicator of phenanthrene biotoxicity. More than 15 % light inhibition was found at the lowest phenanthrene level (0.95 μg g(-1)). Furthermore, comet assay using E. fetida was applied to assess genotoxicity of phenanthrene. The strong correlation (r (2) ≥ 0.94) between phenanthrene concentration and DNA damage indicated that comet assay is appropriate for testing the genotoxic effects of phenanthrene-contaminated soil. In the light of these results, we conclude that the Microtox test and comet assay are robust and sensitive bioassays to be employed for the risk evaluation of polycyclic aromatic hydrocarbon-contaminated soil.

The comet assay (single cell gel electrophoresis) is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues. The assay depends on the relaxation of supercoiled DNA in agarose-embedded nucleoids (the residual bodies remaining after lysis of cells with detergent and high salt), which allows the DNA to be drawn out towards the anode under electrophoresis, forming comet-like images as seen under fluorescence microscopy. The relative amount of DNA in the comet tail indicates DNA break frequency. The assay has been modified to detect various base alterations, by including digestion of nucleoids with a lesion-specific endonuclease. We describe here recent technical developments, theoretical aspects, limitations as well as advantages of the assay, and modifications to measure cellular antioxidant status and different types of DNA repair. We briefly describe the applications of this method in genotoxicity testing, human biomonitoring, and ecogenotoxicology.

The genotoxic potential of waters in six rivers and reservoirs from Serbia was monitored in different tissues of chub (Squalius cephalus L. 1758) with the alkaline comet assay. The comet assay, or single-cell gel electrophoresis, has a wide application as a simple and sensitive method for evaluating DNA damage in fish exposed to various xenobiotics in the aquatic environment. Three types of cells, erythrocytes, gill cells, and liver cells, were used for assessing DNA damage. Images of randomly selected cells were analyzed with a Leica fluorescence microscope and image analysis by software (Comet Assay IV Image analysis system, PI, UK). Three parameters (tail length-l, tail intensity-i, and Olive tail moment-m) were analyzed on 1,700 nuclei per cell type. The procedure for sum of ranking differences (SRD) was implemented to compare different types of cells and different parameters for estimation of DNA damage. Regarding our nine different estimations of genotoxicity: tail length, intensity, and moment in erythrocytes (rel, rei, rem), liver cells (rll, rli, rlm), and gill cells (rgl, rgi, rgm), the SRD procedure has shown that the Olive tail moment and tail intensity are (almost) equally good parameters; the SRD value was lower for the tail moment and tail intensity than for tail length in the case of all types of cells. The least reliable parameter was rel; close to the borderline case were rei, rll, and rgl (~5 % probability of random ranking).

A test battery, composed of a range of biological assays, was applied to evaluate the ecological health of soil aged for 69 days and spiked with a range of pyrene levels (1.04, 8.99, 41.5, 72.6, 136, and 399 μg g(-1) dry soil; Soxhlet-extracted concentrations after 69 days of aging). Chinese cabbage (Brassica rapa), earthworm (Eisenia fetida), and bacteria (Vibrio fischeri) were used as test organisms to represent different trophic levels. Among the acute ecotoxicity bioassays used, the V. fischeri luminescence inhibition assay was the most sensitive indicator of pyrene toxicity. We observed >8 % light inhibition at the lowest concentration (1.04 μg g(-1)) pyrene, and this inhibition increased to 60 % at 72.6 μg g(-1). The sensitivity ranking for toxicity of the pyrene-contaminated soil in the present study was in the following decreasing order: root elongation of Chinese cabbage < earthworm mortality (14 days) < earthworm mortality (28 days) < luminescence inhibition (15 min) < luminescence inhibition (5 min). In addition, genotoxic effects of pyrene were also evaluated by using comet assay in E. fetida. The strong relationship between DNA damage and soil pyrene levels showed that comet assay is suitable for testing the genotoxicity of pyrene-polluted soil. In addition, tail moment was well correlated with soil pyrene levels (r (2) = 0.99). Thus, tail moment may be the most informative DNA-damage parameter representing the results of comet assay. Based on these results, the earthworm DNA damage assay and Microtox test are rapid and sensitive bioassays and can be used to assess the risk of soil with low to high levels of hydrocarbon pollution. Furthermore, an analysis of the toxic effects at several trophic levels is essential for a more comprehensive understanding of the damage caused by highly contaminated soil.

This study was set up to determine the suitability of the early life stage (ELS) alkaline comet assay for the detection of DNA strand breaks induced by genotoxicants in whole organism. This assay was performed on cells of medaka 2 days posthatch (dph). An efficient procedure for cell dissociation using enzymatic and mechanical digestion was developed. This protocol ensures 80% viability of cells and low DNA damage background. Cells from 2 dph medaka larvae were exposed in vitro to model genotoxicants, hydrogen peroxide, cadmium, and fluoranthene, followed by comet assay analysis. Results show a significant increase in the percentage of DNA damage of dissociated cells by all the tested compounds when compared to controls. The assay was also performed in vivo on medaka larvae (2 dph) exposed for 24 h to waterborne cadmium or fluoranthene. Significant induction of DNA damage levels were observed following larvae exposure to cadmium and fluoranthene at concentrations of 0.1 and 50 μM, respectively. This study demonstrates that cells of embryo life stage medaka respond to known DNA damaging agents and that the ELS comet assay may be a useful biomarker to detect DNA strand breakage in whole body of pluricellular organism induced by a range of agents. This technique may provide a sensitive, nonspecific endpoint of genotoxicity as part of ELS toxicity test.

Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The article reviews studies that have used the comet assay to investigate the toxicity of manufactured nanoparticles. It is shown that at least 46 cellular in vitro studies and several in vivo studies have used the comet assay and that the majority of the nanoparticles tested cause DNA strand breaks or oxidative DNA lesions. This is not surprising considering the sensitivity of the method and the reactivity of many nanomaterials. Interactions between the particles and the assay cannot be totally excluded and need further consideration. It is concluded that studies including several particle types, to enable the assessment of their relative potency, are valuable as are studies focusing both on comet assay end points and mutagenicity. Finally, the article discusses the potential future use of the comet assay in human biomonitoring studies, which could provide valuable information for hazard identification of nanoparticles.

Mercury (Hg) is one of the major aquatic contaminants even though emissions have been reduced over the years. Despite the relative abundance of investigations carried out on Hg toxicity, there is a scarcity of studies on its DNA damaging effects in fish under realistic exposure conditions. This study assessed the Hg genotoxicity in Golden grey mullets (Liza aurata) at Laranjo basin, a particularly contaminated area of Ria de Aveiro (Portugal) well known for its Hg contamination gradient. (1) Fish were seasonally caught at Laranjo basin and at a reference site (S. Jacinto), and (2) animals from the reference site were transplanted and caged (at bottom and surface), for 3 days, in two different locations within Laranjo basin. Using the comet assay, blood was analyzed for genetic damage and apoptotic cell frequency. The seasonal survey showed greater DNA damage in the Hg-contaminated area for all sampling seasons excluding winter. The temporal variation pattern of DNA lesions was: summer approximately autumn > winter > spring. Fish caged at Laranjo also exhibited greater DNA damage than those caged at the reference site, highlighting the importance of gill uptake on the toxicity of this metal. No increased susceptibility to apoptosis was detected in either wild or caged fish, indicating that mercury damages DNA of blood cells by a nonapoptotic mechanism. Both L. aurata and the comet assay proved to be sensitive and suitable for genotoxicity biomonitoring in mercury-contaminated coastal systems.

Malathion [S-(1,2-dicarboethoxyethyl) O, O-dimethyl phosphorodithioate] is a widely used organophosphorus insecticide throughout the world. However, limited efforts have made to study its genotoxic effect in different fish tissues. The present investigation was aimed to assess the genotoxic potential of the pesticide to the freshwater teleost fish Channa punctatus at sublethal concentrations using the micronucleus test and comet assay. Initially, the 96-h LC50 value of commercial-grade malathion (50% EC) was determined as 5.93 ppm in a semistatic system. Based on LC50, three test concentrations (viz. sublethal I, sublethal II, and sublethal III) were determined to be 1.48, 0.74, and 0.59 ppm, respectively, and the fish specimens were exposed to these concentrations. Tissue samplings were done on days 0, 1, 3, 7, 15, 22 and 29 of malathion exposure for assessment of the induction of micronuclei (MN) frequency and DNA damage. The MN formation in the peripheral blood cells was found to be significantly higher (p < 0.05) in the treated specimens at all sampling intervals compared to the control. The MN frequency reached maximum on days 3 and 7 at sublethal I and II concentrations, respectively, followed by a nonlinear decline with the progression of the experiment. Similarly, significant effects (p < 0.05) of both concentration and time of exposure were observed on DNA damage in the gill, kidney, and lymphocytes. All of the tissues exhibited a concentration-dependent increase in DNA damage up to day 3, followed by a nonlinear decrease with the duration of exposure. A comparison of the extent of DNA damage among the tissues showed the sensitivity of gill tissue to malathion.

Genotoxic effects induced in vitro by the third generation mobile communication standard UMTS have recently been described by Schwarz et al. (Int Arch Occup Environ Health 81:755-767, 2008). These findings which may have considerable significance for environmental health have been commented upon by Lerchl (Int Arch Occup Environ Health in press, 2008) (this issue). These comments which are invalid in part have to be set right. Although some of his minor points are correct the objected inconsistencies are largely based on the author's incomplete and superficial consideration of published data in the field. Moreover, the statistical points being made cannot cast doubts on the validity of the experimental data reported by Schwarz et al. and may not change the principal conclusion of in vitro genotoxic action of UMTS signals.

Tamoxifen (TAM) is a non-steroidal anti-estrogen used widely in the treatment and chemoprevention of breast cancer. TAM treatment can lead to DNA damage, but the mechanism of this process is not fully understood and the experimental data are often inconclusive. We compared the DNA-damaging potential of TAM in normal human peripheral blood lymphocytes and MCF-7 breast cancer cells by using the comet assay. In order to assess whether oxidative DNA damage may contribute to TAM-induced lesions, we employed two DNA repair enzymes: endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg). The kinetics of repair of DNA damage was also measured. In order to evaluate the involvement of free radicals in the genotoxicity of TAM we pre-treated the cells with nitrone spin traps: DMPO and POBN. The use of common antioxidants: vitamin C, amifostine and genistein, helped to assess the contribution of free radicals. TAM damaged DNA in both normal and cancer cells, inducing mainly DNA strand breaks but not alkali-labile sites. The drug at 5 and 10 microM induced DNA double strand breaks (DSBs) in lymphocytes and at 10 microM in MCF-7 cells. We observed complete repair of DSBs in cancer cells by contrast with incomplete repair of these lesions in lymphocytes. In both types of cells TAM induced oxidized purines and pyrimidines. Incubation of the cells with nitrone spin traps and antioxidants decreased, with exception of amifostine in MCF-7 cells, the extents of DNA damage in both kinds of cells, but the results were more distinct in cancer cells. Our results indicate that TAM can be genotoxic for normal and cancer cells by free radicals generation. It seems to have a higher genotoxic potential for normal cells, which can be the result of incomplete repair of DNA DSBs. Free radicals scavengers can modulate TAM-induced DNA damage interfering with its antitumour activity in cancer cells.

The pathogenesis of myopathies occurring in critically ill patients (critical illness myopathy, CIM) is poorly understood. Both local and systemic responses to sepsis and other severe insults to the body are presumed to be involved but the precise mechanisms by which muscle function is impaired are far from clear. To elucidate such mechanisms we investigated the effects of blood serum fractions (5 kDa to 100 kDa molecular weight cut-off, MWCO) from patients with CIM and from control persons on membrane and contractile functions in intact mammalian single skeletal muscle fibres and chemically skinned fibre bundles. In intact fibres, resting membrane potentials were less negative when exposed to CIM serum fractions compared with control serum fractions. Half-width and maximum rise time of action potentials (AP) were smaller in CIM serum low MWCO fractions vs. control serum. Peak amplitudes of fast inward sodium currents (I(Na)) were increased by low MWCO-CIM fractions compared with control sera fractions. Additionally, voltage dependent inactivation of I(Na) was shifted towards more positive potentials by high MWCO fractions of CIM sera. In skinned fibres, pCa-force relations were similar in CIM and control serum fractions but peak force of Ca2+ induced force transients was decreased by low MWCO-CIM vs. control serum fractions. Our results (i) provide the first evidence that serum from CIM patients affects membrane excitability and the excitation-contraction coupling process at the level of the sarcoplasmic reticulum Ca2+ release of mammalian muscle fibres and (ii) also show that even control serum fractions "per se" alter the response to important physiological membrane and contractility parameters compared with physiological saline.

During the last few years, there has been increasing interest in buccal epithelial cells for cytogenetic evaluation of different materials. In the present study, the use of these cells and peripheral lymphocytes for cytogenetic evaluation of chlorhexidine digluconate (CHX) with comet assay (single cell gel electrophoresis, or SCGE) is reported. This technique detects DNA strand breaks in individual cells in alkaline conditions. Thirteen volunteers were requested to rinse their mouths with 0.12% CHX solution for 18 days. Buccal epithelial cells and peripheral blood lymphocytes were obtained from all participants at baseline and the end of the experimental period. One hundred cells per subject were analysed for the DNA damage. A statistical increase was observed in the damaged buccal and blood cells after the CHX application. The mean grade of damage in buccal cells was statistically different from that in blood cells. Due to minimal absorption of chlorhexidine into the tissues and low concentrations of free chlorhexidine in the oral cavity, the DNA damage produced by chlorhexidine in lymphocytes was lower than in buccal epithelial cells. As chlorhexidine does not accumulate in the body, the frequencies of DNA damage could be transient. Detected DNA damage after CHX use might be the indication of an earlier effect, before DNA repair begins, and could be reversible.

Recent in vivo and in vitro data of patients analyzed for genetic susceptibility to radiation during cancer therapy have shown structural changes in the chromosomes to be prevalent both in the patients being treated and in their immediate family members. As structural changes in chromosomes frequently lead to activation of proto-oncogenes and elimination of tumor-suppressor genes, they represent important mechanisms for the initiation of DNA repair processes and tumorigenesis. With the exception of rare genetic syndromes such as AT (Ataxia telangiectasia) or NBS (Nijmegen Breakage Syndrome), the background for the inheritance of genetic susceptibility to radiation is unknown. Recently, a large-scale genetic screen of mouse mutants has been established within the German Human Genome Project (Hrabè de Angelis and Balling 1998). The goal of this ENU (ENU: ethylnitrosourea) mutagenesis screen is the generation of mutant mice that will serve as animal models for human diseases and genetic susceptibility. In order to fully utilize the potential of a genetic screen of this magnitude, in which exploration for genes responsible for genomic instability and radiation sensitivity is to occur, it is necessary to establish a simple assay system that is amenable to automation. Hence, we are using the single-cell gel electrophoresis (comet assay) to detect mouse mutants that display a genetic susceptibility to ionizing radiation. We have established the analysis parameters in the comet assay which are currently used to detect radiation-sensitive mouse mutants and to control the variance within the mouse population in the ENU screen. The assay can be used to isolate genes that are responsible for DNA repair and radiation sensitivity in mouse and human.