Results Of Comet Assay Research Articles

Multiple factors influence telomere length and DNA damage in individuals environmentally exposed to a coal-burning power plant

Coal is a mixture of several chemicals, many of which have mutagenic and carcinogenic effects and are a key contributor to the global burden of mortality and disease. Previous studies suggest that coal is related to telomeric shortening in individuals occupationally exposed, however little is known about the effects of mining and burning coal on the telomeres of individuals living nearby. Therefore, the primary objective of this investigation was to assess the impact of proximity to coal power plants and coal mines on the genomic instability of individuals environmentally exposed, while also exploring potential associations with individual characteristics, oxidative stress, inflammatory responses, and the presence of inorganic elements. This study involved 80 men participants from three cities around a thermoelectric power plant and one city unexposed to coal and byproducts. DNA was extracted from peripheral blood samples obtained from each participant, and the telomeres length (TL) was assessed using quantitative real-time polymerase chain reaction (qPCR) methodology. No significant difference was observed between exposed individuals (6227 ± 2884 bp) when compared to the unexposed group (5638 ± 2452 bp). Nevertheless, TL decrease was associated with age and risk for cardiovascular disease; and longer TL was found to be linked with increased concentrations of silicon and phosphorus in blood samples. No correlations were observed between TL with comet assay (visual score), micronucleus test, oxidative stress, and inflammatory results. Additional research is required to ascertain the potential correlation between these changes and the onset of diseases and premature mortality.

Reducing risk of false positives in the in vivo comet assay and improving result reliability

The risk of generating false positive in vivo comet assay results can be increased when procedural bias and/or technical variability is poorly controlled. This has been an ongoing concern since comet was first introduced into regulatory safety testing. But the proprietary nature of regulated studies and the 3Rs have limited the ability to conduct and publish the comparative in vivo studies necessary to determine the effect these factors can have on comet assay results when substances other than well characterized positive control compounds are evaluated in multiple tissues. That changed when Helix3 was asked to repeat for regulatory submission three independent in vivo comet studies with positive results generated by three other laboratories evaluating the effects of three different test substances on the liver, duodenum, and stomach. We repeated each study using the same test substance and experimental design as the original labs but with our standard quality control methods implemented to reduce procedural bias and variability. In every case, we generated negative results that regulatory authorities accepted over the initial positive results due to evidence of high technical variability and procedural bias in the original labs and studies. Meanwhile, the International Workshop on Genotoxicity (IWGT) compared >14 years of Helix3 comet historical control data (HCD) to HCD from 6 other experienced comet laboratories and concluded that our data exhibited the highest overall background % tail DNA levels with the lowest inter-study variability resulting in the highest quality HCD of all the labs evaluated. These case studies and the IWGT report suggest that our enhanced quality control methods and higher (>2 % mean of slide median tail DNA) background levels can effectively mitigate the nuisance factors that can generate false positive in vivo comet assay results. To facilitate a better understanding of the technical parameters that can significantly influence the comet results, we describe our enhanced procedures with justifications and examples.

Investigation of DNA damage and inflammatory marker profile in patients after bariatric surgery

Abstract
 Purpose: Obesity is a significant risk factor in the development of many serious diseases. The most prominent ones among them are diabetes and coronary heart disease. Elevated blood sugar levels in obese individuals lead to increased susceptibility to infections due to the suppression of the immune response. Moreover, fatty skin folds can cause topical infections, ulcers, and delayed/impaired wound healing. Therefore, inflammatory and pro-inflammatory markers in the serum have gained importance in diseases such as diabetes and obesity. Furthermore, in obesity, reactive oxygen species (ROS) lead to DNA damage. 8-oxo-dG, which is the primary product of DNA oxidation, can be detected in the serum, saliva, and urine, making it an ideal biological marker for DNA damage in large population-based studies. The Comet assay analysis is a method used to demonstrate the double-strand breaks in DNA. Induction of γ-H2AX in tissue indicates the initiation of a well-regulated mechanism to reverse double-strand breaks in DNA. Potential benefits of monitoring the genomic health in obesity include creating a sense of urgency for personalized intervention measures and evaluating their progress. DNA damage in obesity is believed to be a reversible condition. Chronic inflammation is an etiological factor known to support DNA damage and neoplastic transformations in cells. Cytokines secreted from adipose tissue, especially TNF-alpha, IL-6, and IL-1β, promote the accumulation of various cells, including neutrophils, macrophages, and dendritic cells, and it indicates the initiation of an inflammatory process. In this project, it was aimed to investigate possible changes in inflammation markers and DNA damage in individuals undergoing bariatric surgery, who were beginning to improve endocrine and metabolic syndrome markers. 
 Materials and methods: For this purpose, blood and urine samples were collected from 45 obese patients, who had undergone bariatric surgery and 45 healthy volunteers matched for age and gender. The levels of inflammatory markers (IL-1β, IL-6, IL-8, and TNF-α) and the DNA damage marker γ-H2AX in serum, as well as the amount of 8-oxo-dG in urine, were determined using ELISA. Additionally, the percentage of DNA damage was determined using the Comet assay analysis. 
 Results: Weight control achieved through bariatric surgery and the subsequent reduction in fat tissue resulted in a significant decrease in the levels of γ-H2AX and 8-oxo-dG, as well as a parallel significant reduction in the percentage of DNA damage in the Comet assay results. The significant decrease in inflammatory markers IL-1β, IL-6, IL-8, and TNF-α levels indicated that bariatric surgery also affects inflammation indirectly. 
 Conclusion: Although there are numerous studies in the literature on individual parameters related to DNA damage in various diseases and obesity, it is believed that the present study determining DNA damage, oxidation, and repair mechanisms simultaneously with inflammatory marker levels serves as a guiding example for comparing genomic health and stability in pre-obese, post-obese, and non-obese individuals.

Hypericum alpestre extract exhibits in vitro and in vivo anticancer properties by regulating the cellular antioxidant system and metabolic pathway of L-arginine.

Conventional treatment methods are not effective enough to fight the rapid increase in cancer cases. The interest is increasing in the investigation of herbal sources for the development of new anticancer therapeutics. This study aims to investigate the antitumor capacity of Hypericum alpestre (H. alpestre) extract in vitro and in vivo, either alone or in combination with the inhibitors of the l-arginine/polyamine/nitric oxide (NO) pathway, and to characterize its active phytochemicals using advanced chromatographic techniques. Our previous reports suggest beneficial effects of the arginase inhibitor NG-hydroxy-nor- l-arginine and NO inhibitor NG-nitro-Larginine methyl ester in the treatment of breast cancer via downregulation of polyamine and NO synthesis. Here, the antitumor properties of H. alpestre and its combinations were explored in vivo, in a rat model of mammary gland carcinogenesis induced by subcutaneous injection of 7,12-dimethylbenz[a]anthracene. The study revealed strong antiradical activity of H. alpestre aerial part extract in chemical (DPPH/ABTS) tests. In the in vitro antioxidant activity test, the H. alpestre extract demonstrated pro-oxidant characteristics in human colorectal (HT29) cells, which were contingent upon the hemostatic condition of the cells. The H. alpestre extract expressed a cytotoxic effect on HT29 and breast cancer (MCF-7) cells measured by the MTT test. According to comet assay results, H. alpestre extract did not exhibit genotoxic activity nor possessed antigenotoxic properties in HT29 cells. Overall, 233 substances have been identified and annotated in H. alpestre extract using the LC-Q-Orbitrap HRMS system. In vivo experiments using rat breast cancer models revealed that the H. alpestre extract activated the antioxidant enzymes in the liver, brain, and tumors. H. alpestre combined with chemotherapeutic agents attenuated cancer-like histological alterations and showed significant reductions in tumor blood vessel area. Thus, either alone or in combination with Nω -OH-nor- l-arginine and Nω -nitro- l-arginine methyl ester, H. alpestre extract exhibits pro- and antioxidant, antiangiogenic, and cytotoxic effects.

Genotoxic effect of Tamiflu and Adamine on DNA content in male albino mice

BackgroundTamiflu (Oseltamivir) and Adamine (Amantadine HCl) are antiviral drugs which are used for prevention and treatment for influenza. The present study was carried out to evaluate the effect of Tamiflu and Adamine on DNA content of hepatocytes of adult male albino mice.ResultsThe results of comet assay showed that treatment with Tamiflu and Adamine significantly increased (P < 0.005) the mean of DNA damage in hepatocytes of the treated mice in time dependent manner compared to control one. However qRT-PCR analysis showed the mRNA expression of BAX in mice liver of all treated groups with Adamine was significantly increased (P < 0.05) after ten days of treatment while the same gene was significantly decreased in Tamiflu treated group after ten days compared to control one, While the treatment of mice with both drugs simultaneously showed a significant increase (P < 0.05) in expression of BCL-2 gene and a decrease in expression of BAX gene after fifteen days of treatment.ConclusionTamiflu and Adamine had produced deleterious impacts on DNA content of hepatocytes of treated mice. Whereas, the results of treatments with Adamine indicated pathogenic impacts more than that induced by Tamiflu.

Visual comet scoring revisited: a guide to scoring comet assay slides and obtaining reliable results.

Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20%-25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8%, and 15.2% in training sets I-III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I-III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9%-9.6%) as compared to 1 week (CV = 1.3%-6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18%-20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets.

Anticarcinogenic impact of extracellular vesicles (exosomes) from cord blood stem cells in malignant melanoma: A potential biological treatment.

Incidence of Malignant Melanoma has become the 5th in the UK. To date, the major anticancer therapeutics include cell therapy, immunotherapy, gene therapy and nanotechnology-based strategies. Recently, extracellular vesicles, especially exosomes, have been highlighted for their therapeutic benefits in numerous chronic diseases. Exosomes display multifunctional properties, including inhibition of cancer cell proliferation and initiation of apoptosis. In the present in vitro study, the antitumour effect of cord blood stem cell (CBSC)-derived exosomes was confirmed by the CCK-8 assay (p < 0.05) on CHL-1 melanoma cells and improve the repair mechanism on lymphocytes from melanoma patients. Importantly, no significant effect was observed in healthy lymphocytes when treated with the exosome concentrations at 24, 48 and 72 h. Comet assay results (OTM and %Tail DNA) demonstrated that the optimal exosome concentration showed a significant impact (p < 0.05) in lymphocytes from melanoma patients whilst causing no significant DNA damage in lymphocytes of healthy volunteers was 300 μg/ml. Similarly, the Comet assay results depicted significant DNA damage in a melanoma cell line (CHL-1 cells) treated with CBSC-derived exosomes, both the cytotoxicity of CHL-1 cells treated with CBSC-derived exosomes exhibited a significant time-dependent decrease in cell survival. Sequencing analysis of CBSC exosomes showed the presence of the let-7 family of miRNAs, including let-7a-5p, let-7b-5p, let-7c-5p, let-7d-3p, let-7d-5p and two novel miRNAs. The potency of CBSC exosomes in inhibiting cancer progression in lymphocytes from melanoma patients and CHL-1 cells whilst causing no harm to the healthy lymphocytes makes it a potential candidate as an anticancer therapy.

A pilot biomonitoring study of air pollution in the urban area of Sarajevo, Bosnia and Herzegovina: genotoxicity assessment in buccal cells.

Air pollution, recognized as a human carcinogen, is a significant cause of death in industrial and developing countries, and Bosnia and Herzegovina (B&H) is one of the leading countries for air pollution-caused death rate and has the poorest urban air quality in Europe. Despite a population decrease, urban air pollution in B&H has increased due to traffic pollution and still intensive use of solid fuel for heating and cooking. Human biomonitoring studies, regarding the described air pollution, have not been conducted before, and particularly have not been conducted in the region of Sarajevo. Good health, well-being, and environmental protection are part of the 17 defined Sustainable Development Global Goals. Accordingly, this study aimed to determine baseline levels of DNA damage in a group of Sarajevo citizens and to compare seasonal variations in DNA damage in relation to the reported levels of air pollution. From 33 individuals included in the study, samples were collected in the summer and winter seasons. The buccal micronucleus cytome (BMCyt) assay and comet assay in leucocytes isolated from saliva were performed. Mean values and standard deviations of log-transformed tail intensity (%), tail length (µm), and tail moment results in winter were 1.14 ± 0.23, 2.20 ± 0.14, and 1.03 ± 0.29, respectively, while in the summer season those values were 1.19 ± 0.19, 2.25 ± 0.17, and 1.07 ± 0.25, respectively. No significant differences were found for the comet assay parameters. Nevertheless, BMCyt results showed significant increases in micronuclei (P = .008), binuclear cells (P = .04), karyolysis (P = .0003), condensed chromatin (P = .03), and pyknosis (P = .002) in winter. Although the results of comet and BMCyt assays are not in accordance, this study contributes to the human air pollution biomonitoring in Sarajevo, B&H, and based on the genotoxic effects of air pollution evidenced by the BMCyt biomarker further studies of this kind are necessary.

DNA Oxidative Damage as a Sensitive Genetic Endpoint to Detect the Genotoxicity Induced by Titanium Dioxide Nanoparticles.

The genotoxicity of nanomaterials has attracted great attention in recent years. As a possible occupational carcinogen, the genotoxic effects and underlying mechanisms of titanium dioxide nanoparticles (TiO2 NPs) have been of particular concern. In this study, the effect of TiO2 NPs (0, 25, 50 and 100 µg/mL) on DNA damage and the role of oxidative stress were investigated using human bronchial epithelial cells (BEAS-2B) as an in vitro model. After detailed characterization, the cytotoxicity of TiO2 NPs was detected. Through transmission electron microscopy (TEM), we found that TiO2 NPs entered the cytoplasm but did not penetrate deep into the nucleus of cells. The intracellular levels of reactive oxygen species (ROS) significantly increased in a dose-dependent manner and the ratios of GSH/GSSG also significantly decreased. The results of the normal comet assay were negative, while the Fpg-modified comet assay that specifically detected DNA oxidative damage was positive. Meanwhile, N-acetyl-L-cysteine (NAC) intervention inhibited the oxidative stress and genotoxicity induced by TiO2 NPs. Therefore, it was suggested that TiO2 NPs could induce cytotoxicity, oxidative stress and DNA oxidative damage in BEAS-2B cells. DNA oxidative damage may be a more sensitive genetic endpoint to detect the genotoxicity of TiO2 NPs.

P-039 Male age is associated with sperm DNA integrity: Selection of high DNA integrity sperm by microfluidics sorting is critical to clinical outcomes in older patients

Abstract Study question Does sperm DNA integrity affect clinical outcomes of ICSI? Summary answer Use of high DNA integrity sperm selected by microfluidics sperm sorting results in lower miscarriage rates in the patients of 39-years old and more. What is known already High sperm DNA damage is associated with decreased normal fertilization, embryo development and pregnancy rates, and an increased miscarriage rate. On the other hand, oocytes from older women have decreased pregnancy rate, and increased miscarriage rate because of possibility of low ability to repair sperm with DNA fragmentation, and dramatical increases of aneuploidy as women age. A microfluidic sperm selection chamber (MSS, ZyMōt™; DxNow) is a device designed to collect sperm with higher chromatin integrity than density gradient centrifugation (DGC). Study design, size, duration Sperm analysis was performed by sperm chromatin dispersion (SCD) test and comet assay in the same sample of 15 cases between October 2020 and February 2021. ICSI outcomes by DGC and MSS were compared with blastocyst development, and pregnancy rates in vitrified-thawed single blastocyst transfers cycle for 518 cases between August 2018 and May 2021. Participants/materials, setting, methods SCD test was optimized as a rapid procedure, with sperm showing a halo deemed normal, and those without a halo abnormal. Comet assay results were analyzed using CometScore 2.0, with comparison of %Tail DNA. ICSI outcomes were analyzed using multiple logistics regressions of male and female ages. Main results and the role of chance We found a positive correlation between male age and sperm DNA fragmentation rates in raw semen using SCD test (r = 0.70) and Comet assay (r = 0.42). Higher DNA integrity sperm could select using MSS than DGC. In this study with ICSI outcomes, 170 of 318 (53.5%) blastocyst transfers resulted in pregnancy, and 49 (28.8%) subsequently miscarried. The data were classified according to less than or more than 39 years old of male age detected by multiple logistics regressions. In patients with ≥39 years of male age, the female age was significantly higher and blastocyst and pregnancy rates were significantly lower, and the miscarriage rate was significantly higher than &amp;lt;39 years of male age. Since sperm DNA fragmentation increased in accordance with male age, we compared MSS and DGC in the patients with male age ≥39 years. There was no significant difference in blastocyst, pregnancy, and miscarriage rates in female age &amp;lt;39 years. While in ≥ 39 years of female age, blastocyst and pregnancy rates in MSS were not significantly different from DGC, but the miscarriage rate in MSS was significantly lower than in DGC (27.3 vs. 57.1%). Limitations, reasons for caution The sample size for each study was small. Analysis of sperm DNA fragmentation and samples in ICSI outcomes were not the same. The retrospective nature of ICSI outcomes in this study does not allow controlling of unknown confounders. Wider implications of the findings Sperm DNA fragmentation depended on male age affected fertility outcomes. However, when male age is higher, masking the effect of male age by female age. In this study, we found out the improvement of ICSI outcome by using high DNA integrity sperm selected by MSS in both ≥39 years. Trial registration number Not applicable

Do cytotoxicity and cell death cause false positive results in the in vitro comet assay?

The comet assay is used to measure DNA damage induced by chemical and physical agents. High concentrations of test agents may cause cytotoxicity or cell death, which may give rise to false positive results in the comet assay. Systematic studies on genotoxins and cytotoxins (i.e. non-genotoxic poisons) have attempted to establish a threshold of cytotoxicity or cell death by which DNA damage results measured by the comet assay could be regarded as a false positive result. Thresholds of cytotoxicity/cell death range from 20% to 50% in various publications. Curiously, a survey of the latest literature on comet assay results from cell culture studies suggests that one-third of publications did not assess cytotoxicity or cell death. We recommend that it should be mandatory to include results from at least one type of assay on cytotoxicity, cell death or cell proliferation in publications on comet assay results. A combination of cytotoxicity (or cell death) and proliferation (or colony forming efficiency assay) is preferable in actively proliferating cells because it covers more mechanisms of action. Applying a general threshold of cytotoxicity/cell death to all types of agents may not be applicable; however, 25% compared to the concurrent negative control seems to be a good starting value to avoid false positive comet assay results. Further research is needed to establish a threshold value to distinguish between true and potentially false positive genotoxic effects detected by the comet assay.

Abstract 3730: Folic acid has a cell type- and dose-dependent effect on the genome and the epigenome of colorectal cancer cells

Abstract Synthetic vitamin B9, also known as folic acid (FA), is an extensively used nutritional supplement as well as an adjunctive medication in cancer therapy. However, there is increasing evidence that FA can promote the progression of established colorectal cancers (CRC); therefore, great care is required in the case of its application. In order to gain knowledge about the underlying mechanisms, we analyzed the genomic and the epigenomic effect of different FA supplies on two CRC cell lines with distinct molecular backgrounds. HT-29 and SW480 cells were kept in FA-free media (0 ng/mL) or treated with 100 ng/mL and 10000 ng/mL FA for 72 hours. Firstly, cell proliferation and cell viability alterations were determined with Supforhodamine B and AlamarBlue assays; then, cell cycle analysis was performed using fluorescence-activated cell sorting (FACS). Global DNA methylation level was investigated with the pyrosequencing of long interspersed nuclear element 1 (LINE-1) retrotransposons. Micronucleus scoring performed on DAPI- and anti-γ-H2AX-stained slides, as well as Comet assay, were used to detect the impact of FA on genome integrity. Finally, we analyzed gene expression alterations with Human Transcriptome Array (HTA) 2.0. Our data revealed that 100 ng/mL FA induced significant (p≤0.05) elevation (HT-29100: 128.4±24.9%) of HT-29 cell proliferation compared to the other two circumstances (HT-290: 101.3±13.5%; HT-2910000: 86.1±20.8%). The tendency of cell viability was analogous to the results detected during cell proliferation analyses (HT-290: 91.6±13.3%; HT-29100: 115.8±30.9%; HT-2910000: 64.1±20.2%). The genomic stability of FA-supplemented HT-29 samples was improved in a significant manner (p≤0.05), based on the results of micronucleus scoring (HT-290: 0.56±0.05%; HT-29100: 0.17±0.05%; HT-2910000: 0.25±0.09%) and Comet assay (HT-290: 37.4±3.5%; HT-29100: 31.2±3.4%; HT-2910000: 20.1±3.6%). However, in SW480 cells, remarkable alterations were not detected concerning these parameters. Fundamental differences were observed between the two cell lines in the case of cell cycle (HT-29: G0/1 phase dominance; SW480: S phase dominance) and global DNA methylation analysis (HT-29: 59.1±1.0; SW480: 49.0±0.2), but exposing them to different FA doses did not lead to significant changes. Gene expression alterations were more diverse, as genes involved in carcinogenesis were either up- (HES1, SLC7A11) or downregulated (CCL2) for FA supplementation. We concluded that the effect of FA was considerably influenced by the cell type and the applied FA concentration. Thereby, translating our in vitro results to patient care, we would emphasize the importance of genetic and epigenetic investigations coupled with the choice of proper FA dose upon CRC diagnosis to achieve the best disease outcome. Citation Format: Sára Zsigrai, Alexandra Kalmár, Barbara Kinga Barták, Zsófia Brigitta Nagy, Krisztina Andrea Szigeti, Gábor Valcz, Titanilla Dankó, Anna Sebestyén, Gábor Barna, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Folic acid has a cell type- and dose-dependent effect on the genome and the epigenome of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3730.

Exposure in the tobacco fields: Genetic damage and oxidative stress in tobacco farmers occupationally exposed during harvest and grading seasons

Agricultural workers engaged in tobacco cultivation are constantly exposed to large amounts of harmful agents, such as pesticides and nicotine. Furthermore, most of the flue-cured tobacco leaves are manually graded exposing workers to agents such as tobacco-specific nitrosamines. This study aimed to evaluate genetic damage and oxidative stress in tobacco farmers occupationally exposed during the harvest and grading seasons. We obtained data on DNA damage detected in Comet assay in blood cells and micronucleus experiment with buccal cells from 241 individuals. The serum cotinine levels and nitrates were also evaluated. The Comet Assay results showed a showed an increased visual score for males and females during harvest time and tobacco grading. An increase of micronucleated and binucleated cells was observed in the grading group compared to the control and harvest groups. The oxidative stress measurements showed a clear increase of thiobarbituric acid reactive substances (TBARS) in tobacco farmers during harvest time, and trolox equivalent antioxidant capacity (TEAC) in individuals during harvest and grading time compared to the controls. Significant increases of the cotinine levels were observed during the harvest and grading period (harvest>grading), and nitrates for the grading period compared to the control. In this study, tobacco farmers presented compromised DNA integrity associated with enhanced oxidative stress levels.

In vitro biological and in silico molecular docking and ADME studies of a substituted triazine-coordinated cadmium(II) ion: efficient cytotoxicity, apoptosis, genotoxicity, and nuclease-like activity plus binding affinity towards apoptosis-related proteins.

A cadmium(II) complex containing dppt ligand with the formula [CdCl2(dppt)2], where dppt is 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine was synthesized, elucidated and submitted to in vitro cytotoxicity studies against human breast (MCF-7), glioblastoma (U-87), and lung (A549) cancer cell lines as well as mouse embryo normal cell line (NIH/3T3), in comparison with cisplatin employing MTT assay over 24 and 48h. The complex exhibited the highest cytotoxic effect against MCF-7 cells among the other three cell lines with IC50 values of 8.7 ± 0.5 (24h) and 1.2 ± 0.7µM (48h). Significantly, flow cytometric assessment of the complex-treated MCF-7 and U-87 cells demonstrated a dose-dependent induced apoptotic cell death. The cellular morphological changes were in concord with cytotoxicity and flow cytometric results. The results of comet assay showed that the complex is able to induce DNA damage in MCF-7 cells. These observations are of importance, as sustained damage to cellular DNA could lead to apoptotic cell death. The results of DNA-binding studies indicated that the complex fits into the DNA minor groove and interacts with DNA via a partial intercalation. Moreover, the complex was able to efficiently cleave pUC19 DNA through a hydrolytic mechanism. The binding affinity between the complex and apoptosis-relevant protein targets including APAF1, Bax, Bcl-2, Cas3, Cas7, and Cas9 was evaluated through molecular docking studies. In silico virtual studies revealed the complex's strong affinity towards apoptosis-related proteins; therefore the complex can act as a potential apoptosis inducer. Physicochemical, pharmacokinetics, lipophilicity, drug-likeness, and medicinal chemistry properties of the complex were also predicted through in silico absorption, distribution, metabolism and excretion studies.

Activation of G protein coupled estrogen receptor prevents chemotherapy-induced intestinal mucositis by inhibiting the DNA damage in crypt cell in an extracellular signal-regulated kinase 1- and 2- dependent manner

Chemotherapy-induced intestinal mucositis (CIM) is a common adverse reaction to antineoplastic treatment with few appropriate, specific interventions. We aimed to identify the role of the G protein coupled estrogen receptor (GPER) in CIM and its mechanism. Adult male C57BL/6 mice were intraperitoneally injected with 5-fluorouracil to establish the CIM model. The selective GPER agonist G-1 significantly inhibited weight loss and histological damage in CIM mice and restored mucosal barrier dysfunction, including improving the expression of ZO-1, increasing the number of goblet cells, and decreasing mucosal permeability. Moreover, G-1 treatment did not alter the antitumor effect of 5-fluorouracil. In the CIM model, G-1 therapy reduced the expression of proapoptotic protein and cyclin D1 and cyclin B1, reversed the changes in the number of TUNEL+ cells, Ki67+ and bromodeoxyuridine+ cells in crypts. The selective GPER antagonist G15 eliminated all of the above effects caused by G-1 on CIM, and application of G15 alone increased the severity of CIM. GPER was predominantly expressed in ileal crypts, and G-1 inhibited the DNA damage induced by 5-fluorouracil in vivo and vitro, as confirmed by the decrease in the number of γH2AX+ cells in the crypts and the comet assay results. Referring to the data from GEO dataset we verified GPER activation restored ERK1/2 activity in CIM and 5-fluorouracil-treated IEC-6 cells. Once the effects of G-1 on ERK1/2 activity were abolished with the ERK1/2 inhibitor PD0325901, the effects of G-1 on DNA damage both in vivo and in vitro were eliminated. Correspondingly, all of the manifestations of G-1 protection against CIM were inhibited by PD0325901, such as body weight and histological changes, the mucosal barrier, the apoptosis and proliferation of crypt cells. In conclusion, GPER activation prevents CIM by inhibiting crypt cell DNA damage in an ERK1/2-dependent manner, suggesting GPER might be a target preventing CIM.

SPERM DNA FRAGMENTATION AND FUNCTIONAL FEATURES OF HUMAN SPERM SELECTED BY MICROFLUIDIC SORTING FOR CLINICAL USE

High sperm DNA damage is associated with decreased normal fertilization, embryo development and pregnancy rates, and an increased miscarriage rate. Thus, selecting sperm with minimal DNA damage is important for assisted reproductive technology. A microfluidic sperm selection chamber (MSS, ZyMōt™; DxNow) is a device designed to collect sperm with high motility and chromatin integrity, which has recently been clinically used for ICSI/IVF. Here we aimed to comprehensively evaluate MSS compared to density-gradient centrifugation (DGC) in terms of (1) sperm DNA fragmentation by sperm chromatin dispersion (SCD) test and comet assay, (2) sperm motility and morphological features of motile sperm, and (3) blastocyst development after ICSI and clinical outcomes after vitrified-thawed single blastocyst transfers (vSBT). Sperm analysis was performed in 23 cases between October 2020 and February 2021. SCD test was optimized as a rapid procedure (≤30 min), with sperm showing a halo deemed normal, and those without a halo abnormal. Comet assay results were analyzed using CometScore 2.0, with comparison of %Tail DNA, Tail Length (TL), and Tail Moment (TM). Sperm motility parameters were investigated using the Sperm Motility Analysis System (SMAS, Dietect). To assess motile spermatozoa characteristics, middle piece morphology and active mitochondrial distribution were labeled by MitoTracker-Red. The reactive oxygen species (ROS) level was labeled using CM-H2DCFDA. ICSI outcomes (normal fertilization, blastocyst development, and pregnancy rates) were compared between DGC and MSS for 620 cycles between September 2019 and December 2020. In SCD test, MSS yielded a significantly lower abnormality rate than for raw sperm and DGC in all cases. The comet assay showed that %Tail DNA, TL, and TM were significantly lower with MSS than for raw sperm or DGC. Sperm motility rates were significantly higher with DGC and MSS than for raw sperm. Sperm motility analysis demonstrated significantly lower straightness, linearity, and beat-cross frequency (Hz) with MSS compared to DGC. The morphological abnormality rate of the sperm midpiece was significantly higher with MSS than DGC. Abnormal sperm midpieces exhibited significantly high ROS activity around mitochondria. Fertilization after ICSI and blastocyst rates were similar between MSS and DGC, while the vitrified blastocyst rate was significantly higher with MSS than DGC. Clinical pregnancy and miscarriage rates after vSBT did not significantly different between MSS and DGC. MSS-selected sperm had high DNA integrity and were homogeneous. The sperm had a higher motility rate, but the characteristic sperm motility pattern was meandering motion. Additionally, MSS did not exclude sperm that were morphologically abnormal in the middle piece with high ROS levels. MSS-ICSI yielded an improved vitrified blastocyst rate, and MSS is an effective device for sperm collection for ICSI.

Short/long-term cryopreservation prior to comet assay of whole-blood leukocytes and in vitro-cultured lung fibroblasts

Single-cell gel electrophoresis (comet assay) is a valuable test that can be used in ecotoxicological, epidemiological, and biomonitoring contexts. We assessed the effects of short- (without cryopreservation) and long-term (with cryopreservation) storage of DMEM-cultivated human peripheral blood leukocytes (HPBLs) and a human lung fibroblast cell line (FLECH-104) on comet assay results. Samples were stored for 6 or 24 h at room temperature (23°С) or 4 °C and frozen at −80 °C or −196 °C for 1, 2, or 4 weeks. Short-term storage led to significant increases in the comet tail intensity (TI) and Olive tail moment (OTM) in HPBL and FLECH-104 samples. Freezing FLECH-104 samples at −80°С and −196°С resulted in TI mean increases, with no differences in OTM. All frozen HPBL samples did not exhibit significant increases in TI or OTM, and instead exhibited a slight decrease in TI versus the control at both −80 °C and −196 °C. Increased frequency of highly damaged cells was observed in FLECH-104 and HPBL cultures during both short-term storage and after freezing, which may indicate a significant destructive effect. Therefore, freezing of cell cultures and whole blood according to our protocol is not recommended.

Evaluation of soils under the influence of coal mining and a thermoelectric plant in the city of Candiota and vicinity, Brazil

Coal burning generates gases, particles, and condensation by-products that are harmful to soil, water, and to the atmosphere. The aim of this study was to characterize and identify the cytotoxic and mutagenic potential of soil samples from the cities of Aceguá, Bagé, Candiota and Pinheiro Machado, near a large coal-fired power plant. Our study describes soil characteristics and contributes to the evaluation of the genotoxic activity of coal mining and burning, using the Comet Assay and Micronucleus test in V79 cells, as well as mutagenicity assays with Salmonella typhimurium strains. Comet Assay results show that the winter soil samples of Candiota and Pinheiro Machado induced a significant increase of the Damage Index for cells, as well as for the Aceguá summer sample. The micronucleus test did not detect differences between cities and seasons. A component analysis indicates associations between results obtained in Comet Assay and Ti and phenanthene concentrations for Pinheiro Machado during the winter, and Al for Aceguá during the summer and Zn during the winter. Results of Salmonella/microsome assays were negative, only Candiota and Pinheiro Machado samples showed a statistical increase of his + colonies in TA102. Our work describes biological data on these cells exposed to coal-contaminated soil, confirming the sensitivity of the Comet Assay in V79 cells and Salmonella/microsome assay for the evaluation of the effects of complex mixtures. These findings help to understand the spatial distribution of contaminants in the local soil related to a power plant, which is important for planning public safety actions.

Heating and convection associated with alkaline electrophoresis.

Alkaline version of the single-cell gel electrophoresis (Comet assay) is widely used in toxicological, environmental, and monitoring studies to assess the DNA damage levels in individual cells. The change in the temperature of the electrophoretic solution is one of the reasons leading to interlaboratory variation of Comet assay results. In this work, changes of surface temperature of the solution during electrophoresis were studied using technique of real-time thermal imaging. It has been found that the electrophoresis is accompanied by nonuniform temperature rise in different areas of the electrophoretic chamber. The maximum of heating was observed in the central region of the chamber, where temperature increased by an average of 7°C. The minimum temperature rise in other parts of the chamber was about 5°C. After removing the solution, the temperature on the surface of slides was higher than that on the surface of the solution. We believe that (1) nonuniform heating of the electrophoretic solution and convection could be the reasons responsible for the variability of results both in inter- and intralaboratory studies; (2) the spatial distribution of heating of the solution depends on the size and configuration of the electrophoretic chambers used.

Antigenotoksična svojstva ekstrakta jagode obogaćenog antocijaninom (cv. Romina) na oštećenja DNK izazvana H2O2 u leukocitima periferne krvi čoveka

Strawberry fruit Fragaria × ananassa Duchesne, Rosaceae (cv. Romina), rich in anthocyanin polyphenols, has been demonstrated to have favorable effects on health due to its antioxidant properties. The present study investigated the antigenotoxic potential of anthocyanin enriched fraction of Romina strawberry methanolic extract (ACY) against DNA damage on human peripheral blood leukocytes, induced by hydrogen peroxide in vitro. Five concentrations of the ACY extract were used in all experiments (2.5, 5, 10, 15, 20 µg/ml). The results of the alkaline comet assay showed no genotoxic effect of the ACY. After the pre-exposure of the leukocytes to the ACY, and subsequent incubation with H2O2, a decreased number of DNA damaged cells was recorded in all the tested concentrations, compared to controls. In the posttreatment, there was a concentration-dependent DNA damage reduction, while a statistically significant decrease was achieved with 15 and 20 µg/ml concentrations. The results indicate that ACY is efficient in oxidative DNA damage reduction, and it is more potent as a post-applicative than a protective agent.