Automatic analysis of slides processed in the Comet assay.

Although arsenic trioxide (ATO) has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by ATO in a human leukemia (HL-60) cell line using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. HL-60 cells were treated with different doses of ATO for 24 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that ATO significantly (P < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner, showing a LD(50) value of 6.4 +/- 0.6 microg/mL. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HL-60 cells associated with ATO exposure. We observed a significant increase (P < 0.05) in comet tail-length, tail arm and tail moment, as well as in percentages of DNA cleavage at all doses tested, showing an evidence of ATO-induced genotoxic damage in HL-60 cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by heavy metals like arsenic. Taken together, our findings suggest that ATO exposure significantly (P < 0.05) reduces cellular viability and induces DNA damage in HL-60 cells as assessed by MTT and alkaline single cell gel electrophoresis assays, respectively.

BackgroundEtoposide is currently one of the most commonly used antitumor drugs. The mechanisms of action proposed for its antitumor activity are based mainly on its interaction with topoisomerase II. Etoposide effects in transformed cells have been described previously. The aim of the present study was to evaluate the genotoxic effects of this drug in non-transformed whole blood cells, such as occurs as collateral damage induced by some chemotherapies.MethodsTo determine etoposide genotoxicity, we employed Comet assay in two alkaline versions. To evaluate single strand breaks and delay repair sites we use pH 12.3 conditions and pH >13 to evidence alkali labile sites. With the purpose to quantified apurinic or apyrimidine (AP) sites we employed a specific restriction enzyme. Etoposide effects were determined on whole blood cells cultured in absence or presence of phytohemagglutinin (PHA) treated during 2 and 24 hours of cultured.ResultsAlkaline (pH > 13) single cell gel electrophoresis (SCGE) assay experiments revealed etoposide-induced increases in DNA damage in phytohemaglutinine (PHA)-stimulated blood and non-stimulated blood cells. When the assay was performed at a less alkaline pH, 12.3, we observed DNA damage in PHA-stimulated blood cells consistent with the existence of alkali labile sites (ALSs). In an effort to elucidate the molecular events underlying this result, we applied exonuclease III (Exo III) in conjunction with a SCGE assay, enabling detection of DNA-AP sites along the genome. More DNA AP-sites were revealed by Exo III and ALSs were recognized by the SCGE assay only in the non-stimulated blood cells treated with etoposide.ConclusionOur results indicate that etoposide induces DNA damage specifically at DNA-AP sites in quiescent blood cells. This effect could be involved in the development of secondary malignancies associated with etoposide chemotherapy.

Single-cell gel electrophoresis or comet assay is a simple and reliable method that reveals DNA damage and repair in individual cells. This method is widely used in the medical and biological fields for genotoxicity testing and human biomonitoring studies. Over the past few years, several computational systems have been proposed to perform comet assay image analysis based on traditional image processing methods rather than up-to-date efficient deep learning techniques. Therefore, this work proposes an automatic segmentation system based on a fully-convolutional neural networks ensemble to process comet assay individual cells. Our database was created from peripheral blood samples and consists of comet cells images related to different levels of DNA damage, randomly divided into training, validation and test sets. The experimental results showed that the proposed segmentation model achieves a competitive performance of 87.5% F1-score, 91.5% precision and 84.4% recall. Compared to free-use comet assay analysis systems that apply common thresholding techniques, our model showed more efficient, robust and reliable results. Our model and database are freely available for scientific and academic purposes.

Garcinia kola (GK) stem bark, Uvaria chamae (UC) root, and Olax subscorpioidea (OS) root are components of various indigenous/traditional anticancer regimens. It is, therefore, possible that they might combat oxidative stress and impair cellular proliferation linked to carcinogenesis. In this study, we investigated the antioxidative, mito-depressive, and DNA-damaging activities of the three plant extracts in order to provide further mechanistic insights into their potential anticancer roles in documented cancer remedies that include them. Antioxidative properties were investigated in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging assays and an animal model of drug (cisplatin)-induced oxidative stress. The Allium cepa assay and the single cell gel electrophoresis (SCGE) assay were used to assess mito-depressive and DNA-damaging activities. GK and OS showed significantly higher antioxidant activities in the DPPH assay than ascorbic acid; OS had the lowest IC50 of the three plants in the NO assay, comparable to that of ascorbic acid. Pretreatment with the extracts produced an ameliorative and protective effect against the cisplatin-induced oxidative stress as shown by inhibition of lipid peroxidation and improved or restored reduced glutathione and superoxide dismutase levels. In the Allium test, the three extracts produced significant decreases in root growth and also significant cytotoxicity as evidenced by decreased mitotic index. Each of the extracts also showed significantly increased tail DNA (%) in the SCGE assay, indicating the significant DNA-damaging effect. Taken together, this study demonstrates the possible chemopreventive and chemotherapeutic potentials of the three study extracts, which may explain the roles of their source plants in traditional remedies in the therapy of cancers.

Exposure to estrogens increases the risk of breast and endometrial cancer. It is proposed that the estrogen receptor (ER) may contribute to estrogen carcinogenesis by transduction of the hormonal signal and as a "Trojan horse" concentrating genotoxic estrogen metabolites in the nucleus to complex with DNA, enhancing DNA damage. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in estrogen replacement formulations, autoxidizes to a redox-cycling quinone that has been shown to cause DNA damage. 4-OHEN was found to be an estrogen of nanomolar potency in cell culture using a luciferase reporter assay and, using a chromatin immunoprecipitation assay, was found to activate ERalpha binding to estrogen-responsive genes in MCF-7 cells. DNA damage was measured in cells by comparing ERalpha(+) versus ERalpha(-) cells and 4-OHEN versus menadione, a reactive oxygen species (ROS)-generating, but non-estrogenic, quinone. 4-OHEN selectively induced DNA damage in ERalpha(+) cells, whereas menadione-induced damage was not dependent on cellular ER status. The rate of 4-OHEN-induced DNA damage was significantly enhanced in ERalpha(+) cells, whereas ER status had no effect on the rate of menadione-induced damage. Imaging of ROS induced by 4-OHEN showed accumulation selective for the nucleus of ERalpha(+) cells within 5 min, whereas in ERalpha(-) or menadione-treated cells, no selectivity was observed. These data support ERalpha acting as a Trojan horse concentrating 4-OHEN in the nucleus to accelerate the rate of ROS generation and thereby amplify DNA damage. The Trojan horse mechanism may be of general importance beyond estrogen genotoxins.

Response to challenging dose of X-rays as a predictive assay for molecular epidemiology.

Mechanistic and Pharmacodynamic Studies of Adct-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies

The single cell gel electrophoresis (comet) assay is a simple and effective method for detecting DNA damage in cells with or without the capability of cell division. Methyl methanesulfonate (MMS), as a genotoxic compound that reacts with DNA directly, was confirmed for its DNA damage potential by in vivo comet assay in multiple organs such as liver, kidneys and bone marrow in mice and acetaminophen (APAP), a widely used analgesic drug, was evaluated for whether it possesses DNA damage potential or not. Furthermore, cytotoxicity was verified by hematology and /or blood chemistry simultaneously. Male Crj:CD1(ICR) mice were intraperitoneally once treated with MMS at 50, 100, and 150 mg/kg, and APAP at 12, 60, and 300 mg/kg. These organs were collected at 4 and 24 hr after treatment, and the comet assay was performed concomitantly with hematology and/or blood chemistry. The results showed that MMS induced a significant concentration-dependent increase in the frequency of tailed nuclei (DNA damage), tail moment, % DNA in the tail, and tail length in the liver, kidneys and bone marrow at both time points. With regard to hematology and blood chemistry results, nephrotoxic markers were not changed, but aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased in the 150 mg/kg-treated group, and bone marrow counts (BMC) decreased in all of the treatment groups 24 hr after treatment. These results suggested that DNA damage observed in the kidneys was due to genotoxicity, not nephrotoxicity. The DNA damage was more severe at 4 hr than 24 hr after treatment. This might indicate that the decrease in DNA damage was due to detoxification, repair of the lesions induced by the treatment, or cell turnover, all of which would reduce cellular damage. On the other hand, APAP induced increases in plasma AST and ALT levels in the highest dose group only, and the DNA damage in the liver increased at the same dose. These results suggest that the in vivo comet assay might be used to detect the DNA damage induced by MMS and the subsequent DNA repair in mouse liver, kidneys and bone marrow. APAP at the highest dose induces DNA damage in liver. Blood chemical results may indicate that the DNA damage by APAP treatment was attributable to hepato-cytotoxicity, because DNA damage and hepato-cytotoxicity were detected at the same doses.

The single-cell gel electrophoresis (Comet) assay has been widely used to measure DNA damage in human sperm in a variety of physiological and pathological conditions. We investigated the effects of in vivo radiation, a known genotoxin, on spermatogenic cells of the mouse testis and examined sperm collected from the vas deferens using the neutral Comet assay. Irradiation of differentiating spermatogonia with 0.25-4 Gy X-rays produced a dose-related increase in DNA damage in sperm collected 45 days later. Increases were found when measuring Comet tail length and percentage of tail DNA, but the greatest changes were in tail moment (a product of tail length and tail DNA). Spermatids, spermatocytes, differentiating spermatogonia, and stem cell spermatogonia were also irradiated in vivo with 4 Gy X-rays. DNA damage was indirectly deduced to occur at all stages. The maximum increase was seen in differentiating spermatogonia. DNA damaged cells were, surprisingly, still detected 120 days after stem cell spermatogonia had been irradiated. The distribution of DNA damage among individual sperm cells after irradiation was heterogeneous. This was seen most clearly when changes in the Comet tail length were measured when there were discrete undamaged and damaged populations. After increasing doses of irradiation, an increasing proportion of cells were found in the damaged population. Because a proportion of undamaged sperm cells remains after all but the highest dose, the possibility of normal fertility remains. However, fertilization with a spermatozoa carrying high amounts of DNA damage could lead to effects as diverse as embryonic death and cancer susceptibility in the offspring.

Cytogenetic status and oxidative DNA-damage induced by atorvastatin in human peripheral blood lymphocytes: Standard and Fpg-modified comet assay

Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test

Background: Recently the “comet assay” or “single-cell gel electrophoresis assay” has been established as a sensitive method for the detection of DNA damage and repair. Most of the software now available to quantify various parameters for DNA damage requires the interaction of a human observer. In this report, we describe an automated analysis system that is based on self-developed software and hardware and needs minimal human interaction. Methods: The image analysis is divided into two parts: 1) automatic cell recognition and comet classification and 2) quantification of desired comet parameters. Image preprocessing, segmentation, and feature classification were developed with algorithms based on mathematical morphology. To enhance evaluation speed, we have introduced parallel processing of data under the Windows NT operating system (Microsoft Corporation, Redmond, WA). Use of an analogue real-time autofocus unit (Böcker et al.: Phys Med Biol 1997;42:1981–1992) allows for faster analysis. Results: Our recognition software shows a sensitivity of 95.2% and a specificity of 92.7% when tested on test samples from routine work with DNA damage by low-dose radiation (0–2 Gy). The parallel hardware and software concept enables us to analyze 100 comets on one slide in less than 15 min. Conclusions: A comparison of measurements made on the same samples by manual and automated analysis systems revealed that there are no significant differences. The slope of the dose-response curves and the repair kinetics are very similar and demonstrate that automatic comet assay analysis is possible. Cytometry 35:134–144, 1999. © 1999 Wiley-Liss, Inc.

Screening methods to indicate the genotoxic potential of individual chemicals or environmental mixtures rely mainly on short-term bacterial tests. Differences in the genotoxic response of prokaryotic and eukaryotic cells necessitate the development of nonbacterial screening assays. A promising approach for this purpose could be the comet (single-cell gel electrophoresis) assay performed with fish cells in vitro. In the present study, we evaluated the comet assay with two different fish cell lines from rainbow trout (Oncorhyhnchus mykiss), the fibroblast-like RTG-2 cell line established from gonad tissue, and the epitheloid RTL-W1 cell line established from liver tissue. The cells were exposed in vitro during 2 hr to the genotoxins, 4-nitroquinoline-1-oxide (NQO), and benzo(a)pyrene (BaP), as well as to environmental samples. The LOEC values for NQO were similar in both cell lines, whereas for BaP, the RTL-W1 cells were found to be more sensitive than the RTG-2 cells. The slopes of the concentration-response curves of the two test compounds differed between the two cell lines, with RTG-2 cells showing a steeper slope for NQO, and RTL-W1 cells showing a steeper slope for BaP. When exposed to environmental samples from a remediation site, the RTL-W1 cell line, but not the RTG-2 cell line, indicated a genotoxic potential of the samples. The differences in the genotoxic response pattern of the two cell lines could be only partly explained in relation to metabolic enzymes, cytochrome P4501A, glutathione-S-transferase, and xenobiotic reductase. The findings of this study demonstrate that the comet assay with fish cell lines is suitable as in vitro screening assay in environmental genotoxicity testing, but the choice of test cell line may be critical.

beta-Propiolactone (BPL) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are two direct alkylating agents that induce multiple genetic lesions and tumors in the rodent stomach. We measured the kinetics of the induction of DNA damage by using the single-cell gel electrophoresis assay (SCGE) and the induction of gene mutations by using the MutaMouse model in the glandular stomach mucosa of mice exposed to a single oral administration of BPL or MNNG. The aims were to determine the optimal sampling time and to investigate the cause-effect relationship between DNA damage and gene mutations. The induction of comets, evaluated in individual cells with the tail moment, was analyzed 1, 2, 4, 24, and 72 hr after a single oral administration of 25 mg/kg BPL or 20 mg/kg MNNG. The effects of both compounds were most intense at the earlier sampling times (1-2 hr), tailing off 4 hr after treatment and becoming undetectable at 72 hr. The lacZ mutant frequency (MF) was measured 3, 7, 14, 28, and 50 days after a single oral administration of 150 mg/kg BPL or 100 mg/kg MNNG, and 3 and 14 days after a single administration of 25 mg/kg BPL or 20 mg/kg MNNG. The MF was strongly enhanced at the highest doses and all sampling times, the most marked effects being observed 14 days (11.1-fold) and 28 days (19.0-fold) after BPL and MNNG administration, respectively. At the lowest doses, only a small increase in MF ( approximately 2.5- to 3.5-fold) was found at both sampling times. Primary DNA damage detected with SCGE shortly after treatment (1-2 hr) was rapidly (3 days) transformed into stable gene mutations that remained detectable for 50 days. These results illustrate the ability and complementarity of the SCGE and MutaMouse models to assess the genotoxicity of direct alkylating agents in the mouse gastric mucosa in vivo.

Genotoxic effect of ozone in human peripheral blood leukocytes