Tail Moment Research Articles

In vitro Cytotoxicity, Genotoxicity and Apoptoxicity of 8-Hydroxy-3,6-disulphonaphthyl Azohydroxynaphthalenes using Human Lymphocytes: Experimental and Theoretical Profiling

Regulatory agencies require demonstration of non-genotoxicity of new chemical entities prior to their use as pharmaceuticals or additives. Consequently, the in vitro cytotoxicity, genotoxicity and apoptoxicity of five novel monoazo colourants (8-hydroxy-3,6-disulphonaphthyl azohydroxynaphthalenes, 3a-e) on human lymphocytes were evaluated using a cell viability assay, alkaline comet assay, DNA diffusion assay, DFT calculations and molecular docking. The test concentrations of the compounds varied from 0 to 3.4 mM. Relative to negative control, the compounds at concentrations up to 0.5mM induced small dose-dependent reduction (< 20%) in viability of lymphocytes. Statistically significant changes (p<0.05) in DNA damage parameters (percent tail DNA, tail extent moment, olive tail moment) were observed at all concentrations of 3a, 3b while 3c-e showed genotoxicity at 2.8, 0.17 and 3.4 mM respectively. Compounds 3a, 3b, 3d induced apoptosis at concentrations below cytotoxic doses while 3c and 3e were non-apoptoxic at all test concentrations. DFT calculations showed the genotoxicity of 3a-e increased with electrophilicity and ionization potentials of the compounds. Molecular docking of 3a-e with apoptosis-associated proteins revealed binding affinity patterns that were consistent with observed experimental apoptoxicity. The structure-genotoxicity relationships of five novel monoazo compounds, which can be employed in the design of safer congeners, have been elucidated.

The effect of granulocyte colony stimulating factor on genotoxicity in allogeneic peripheral blood stem cell transplantation donors: a prospective case-control study.

Every year, thousands of donors are exposed to granulocyte-colony stimulating factor (G-CSF) for stem cell mobilization in hematopoietic stem cell transplantations (HSCT). Previous studies about the genotoxicity of G-CSF were inconclusive. In this study, the genotoxic effects of G-CSF in peripheral blood stem cell (PBSC) donors were evaluated prospectively by using three different validated and reliable methods for the first time in the literature to the best of our knowledge. Donors of PBSC transplantation (n=36), who received G-CSF were evaluated for genotoxicity by micronucleus test (MNT), nuclear division index (NDI), and comet assay (CA). Genotoxic effects are expected to cause an increase in MNT and CA values and decrease in NDI. Blood samples were collected at three timepoints (TP): before starting G-CSF (TP1), after G-CSF for five days (TP2), and one month after the last dose (TP3). Sixteen controls were included for baseline comparison of genotoxicity tests. CD34 cell counts and hemograms were also analyzed. MNT and CA parameters; comet and tail length, tail DNA%, and tail moment, showed no change in time whereas another CA parameter, Olive`s tail moment (OTM) was increased significantly at TP3 compared to both baseline and TP2 (p=0.002 and p=0.017, respectively). Nuclear division index decreased significantly at TP2 (p < 0.001), then increased above baseline at TP3 (p=0.004). Baseline comparison with controls showed higher MN frequency in donors without statistical significance (p=0.059). Whereas, CA results were significantly higher in controls. CD34 cell count showed moderate positive correlation with white blood cell count at TP2 (Pearson R=0.495, p=0.004). Our results showed the genotoxic effect of G-CSF in healthy donors, in two of the three tests performed, short-term effect in NDI, and long-lasting effect in OTM. So, this study provides novel information for the debate about the genotoxicity of G-CSF and supports the need for further studies with a larger sample size and longer follow-up.

Genotoxic effects of oxyclozanide on hemocytes of Galleria mellonella (Lepidoptera: Pyralidae) larvae

Oxyclozanide is a salicylanilide derivative anthelmintic drug with a well-known effect on parasites that cause infections in humans and animals. In this study, the effect of oxyclozanide on DNA damage in hemocytes of Galleria mellonella (L.) (Lepidoptera: Pyralidae) larvae, which has been used as a model organism in many fields, was investigated. Hemolymph was collected from the last instar larvae (7th instar) reared on artificial diets containing oxyclozanide at different concentrations (0.003%, 0.03%, 0.3%, and 1.5%) under laboratory conditions and then hemocytes suspension was prepared. Genotoxic damage in hemocytes was determined by the comet assay which enables microscopically detecting DNA damage and is a very sensitive assay in chemical genotoxicity. When compared to the control group, tail length, tail DNA percent, and tail moment values were significantly increased parallel with increasing oxyclozanide concentrations. While the tail length was determined as 5.11 ± 0.46 μm in the control group, it was significantly increased in all tested groups to 13.17 ± 0.53, 27.98 ± 1.08, 98.44 ± 0.77, and 137.67 ± 0.74 μm, respectively. Similarly to tail length, tail DNA percentage and tail moment levels were also significantly increased from 12.86 ± 0.74 to 91.96 ± 0.31 at the highest concentration of oxyclozanide. These results showed that oxyclozanide caused DNA damage in the hemocytes of G. mellonella. It is also known that hemocytes are an important bioindicator in determining the genotoxicity of anthelmintics to be used as insecticides within environmentally friendly limits. It is thought that our results will contribute to the studies in this field.

Cytotoxic and genotoxic effects of nateglinide on human ovarian, prostate, and colon cancer cell lines

Objective: Nateglinide, an oral anti-diabetic medication used to treat type 2 diabetes, activates ATP-dependent potassium channels in pancreatic beta cells and induces insulin secretion. Numerous antidiabetic medicines, particularly metformin, are known to drastically reduce the viability of cancer cells. This study examined the effects of nateglinide on the DNA and viability of human ovarian (A2780), prostate (LNCaP), and colon (Caco-2) cancer cells. Materials and methods: Initially in the study, 1, 10, 100, and 1000 µM doses of nateglinide were administered for 24 hours to A2780, LNCaP, and Caco-2 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to measure cell viability. Using Graphpad Prism 8, the inhibitory logarithmic concentration values (LogIC50) of nateglinide in A2780, LNCaP, and Caco-2 cells were computed based on the results of the MTT experiment. These doses were applied to A2780, LNCaP, and Caco-2 cells for the Comet assay. The Bonferroni-corrected Mann–Whitney U test was used to compare groups, and a value of p&lt;0.05 was considered statistically significant. Results: In A2780 and LNCaP cell lines, only 1000 μM nateglinide concentration decreased cell viability (p&lt;0.05), whereas in Caco-2 cells, all concentrations except 1 μM reduced cell viability (p&lt;0.05). The Comet assay indicated that nateglinide produced DNA damage by increasing the tail lengths and tail moments of A2780, LNCaP, and Caco-2 cells (p&lt;0.05) and reducing the head diameters (p&lt;0.05). Conclusion: According to the findings of this study, nateglinide has cytotoxic effects on human ovarian, prostate and colon cancer cell lines and may possess anticancer properties.

Combined Effect of Pesticides Containing Active Ingredients of Chlorpyrifos and Mancozeb on the DNA Damage of Chlorella Sorokiniana Shihira and Krauss

The intensive use of pesticides in agricultural areas can leave residues of pesticide mixtures on the soil surface. Surface run-off can carry pesticide residues, enter water bodies, and then may affect non-target organisms. Chlorpyrifos and mancozeb are active ingredients commonly contained in pesticides applied in shallot farming. This study aims to evaluate the combined effect of pesticides containing active ingredients of chlorpyrifos and mancozeb on the growth and DNA damage of the microalgae Chlorella sorokiniana. The test organism was exposed to the combined concentration of chlorpyrifos:mancozeb, i.e., 0:0, 20%:20%, 20%:80%, 80%:20%, and 80%:80% of the individual EC50 of each pesticide with sampling time at hours 0, 6, 24, and 48. Microalgae growth was estimated by cell counting method, and DNA damage was analyzed by alkaline comet assay method with parameters, i.e., Tail Intensity (TI%), Head Intensity (HI%), Tail Moment (TM), Olive Tail Moment (OTM), and Tail Factor (TF). The results showed that the combined pesticides inhibited the growth of C. sorokiniana, with the highest growth inhibition being at a combined concentration of 80%:80%. The TM and OTM values of C. sorokiniana increased with the increase of combined concentrations at an exposure period of up to 24 hours. In conclusion, the combined exposure could induce growth inhibition and DNA damage of Chlorella sorokiniana, mainly in the first 24 hours. The TM and OTM can be used as sensitive biomarkers for biomonitoring pesticide pollution.

The first peptide derivatives of dioxybiphenyl-bridged spiro cyclotriphosphazenes: In vitro cytotoxicity activities and DNA damage studies

In this study, we aimed to synthesize new peptide-substituted cyclotriphosphazenes from a series of tyrosine-based peptides and dioxyphenyl-substituted spirocyclotriphosphazenes, and to evaluate their in vitro cytotoxicity and genotoxicity activities. Genotoxicity studies were conducted to understand whether the cytotoxic compounds cause cell death through DNA damage. The structures of the novel series of phosphazenes were characterized by FT-IR, elemental analysis, MS, 1D (31P, 1H, and 13C-APT NMR), and 2D (HETCOR) NMR spectroscopic techniques. In vitro cytotoxic activities were carried out against human breast (MCF-7), ovarian (A2780), prostate (PC-3), colon (Caco-2) cancer cell lines and human normal epithelial cell line (MCF-10A) at different concentrations by using an MTT assay. The compounds showed considerable reductions in cell viability against all human cancer cell lines. Especially, the compounds exhibited notable effects in A2780 cell lines (p < 0.05). The IC50 values of the compounds in the A2780 cell line were calculated to be 1.914 µM for TG, 20.21 µM for TV, 20.45 µM for TA, 4.643 µM for TP, 5.615 µM for BTG, 1.047 µM for BTV, 27.02 µM for BTA, 0.7734 µM for BTP, 21.5 µM for DTG, 1.65 µM for DTV, 2.89 µM for DTA and 4.599 µM for DTP. DNA damage studies of the compounds were conducted by the comet assay method using tail length, tail density, olive tail moment, head length, and head density parameters, and the results showed that the cell death occurred through DNA damage mechanism. In a nutshell, these compounds show promising cytotoxic effects and can be considered powerful candidate molecules for pharmaceutical applications.

Estimation of DNA damage in the roots of Allium cepa exposed to heavy metals using comet assay

Higher plants were used as a bioindicator of environmental toxicity to estimate the severe problems related to the health of living organisms and the environment. Allium cepa plant was used to evaluate the DNA damage caused by heavy metal exposure, the roots of A.cepa plant. They were treated with four concentrations (5, 10, 20 and 25 ppm) for each of the metals Cadmium, zinc, copper and lead. At the same time, concentrations (50, 100, 200 and 500 ppm) were used for the preservative (sodium benzoate). The comet assay, a sensitive and suitable test for assessing DNA damage caused by chemical exposure, was used in this study. The Comet's six characteristics were measured: Head intensity, Head DNA%, Tail length, Tail intensity, Tail DNA% and Tail moment. The results showed that the metals are causing the DNA damage of meristematic cells of the roots of the A. cepa plant, depending on the tail length from most to least effective Cadmium&gt; zinc &gt; sodium benzoate &gt; copper &gt; lead &gt; wastewater. I consider that it is not necessary to write down these values. The results of this study confirm that the meristematic cells of the roots of A. cepa are a suitable model for detecting DNA damage analyzed by the comet assay. Keywords: Toxic metals - Bio-indicator - Single cell gel electrophoresis (SCGE)

Resolving male infertility that is induced by β-and ca channel antagonist drugs: Propranolol/Verapamil using an optimized nanohybrid formula: Experimental and computational studies

Two major objectives of the current study, the first was to study theoretically the electronic structure of Chitosan-Sodium alginate, Epigallocatechin-gallate (EGCG) and Sodium alginate-EGCG complex using density-functional theory (DFT) calculations. The binding energy (ΔEbind), the atoms in molecules (AIM) theory and the Natural bond orbital (NBO) analysis of Chitosan-Sodium alginate-EGCG complex have been studied in gas and water phases. The second was to investigate the treatment effect of EGCG in normal form and EGCG-loaded chitosan-sodium alginate nanoparticles (EGCG-CS-SA NPs) on the male infertility that was induced in male albino rats by the oral administration of Propranolol Hydrochloride and Verapamil Hydrochloride. Full characterization of the synthesized EGCG-CS-SA NPs were carried out using different techniques. Then eight groups of rats were classified as follows: control negative (A); Propranolol Hydrochloride (B); Verapamil Hydrochloride (C); EGCG normal (D); EGCG nano (EGCG-CS-SA NPs) (E); Propranolol Hydrochloride + EGCG (F); Propranolol Hydrochloride + EGCG-CS-SA NPs (G); and Verapamil Hydrochloride + EGCG-CS-SA NPs (H). The study registered the body weight gain percentage as well the weights of the liver, testes, and seminal vesicles. Malondialdehyde (MDA), glutathione reduced (GSH), and testosterone levels were assessed. The semen analysis was performed, the deoxyribonucleic acid (DNA) damage was detected, and the histopathological sections were examined. The computational study identified that the interaction between chitosan-sodium alginate and EGCG is covalent and electrostatic interactions in nature in the gas and water phases. The results recorded an increase in body weight gain percentage in Propranolol Hydrochloride + EGCG-CS-SA NPs and Verapamil Hydrochloride + EGCG-CS-SA NPs groups but no significant difference (P› 0.05) in organs weight of experimental groups (B, C, D, E, F, G, and H) as compared to control group. In the treated rats (F, G, and H), the MDA decreased significantly, while the GSH increased.The results showed that the formula improved the negative effects of Propranolol Hydrochloride and Verapamil Hydrochloride administration on serum testosterone level, sperm count, motility, percentage of live sperms, and histological structures in the testes and epididymides. Post-administration of EGCG normal and EGCG-CS-SA NPs to Propranolol Hydrochloride and Verapamil Hydrochloride caused no significant difference (P› 0.05) in tail moment value in testes cells as compared to Propranolol Hydrochloride and verapamil Hydrochloride groups. Finally, the herbal nanohybrid formula EGCG-CS-SA NPs was found to be safe and could be used to treat male infertility caused by antihypertensives.

Fabrication and assessment of potent anticancer nanoconjugates from chitosan nanoparticles, curcumin, and eugenol.

In cancer management and control, the most challenging difficulties are the complications resulting from customized therapies. The constitution of bioactive anticancer nanoconjugates from natural derivatives, e.g., chitosan (Ct), curcumin (Cur), and eugenol (Eug), was investigated for potential alternatives to cancer cells' treatment. Ct was extracted from Erugosquilla massavensis (mantis shrimp); then, Ct nanoparticles (NCt) was fabricated and loaded with Cur and/or Eug using crosslinking emulsion/ionic-gelation protocol and evaluated as anticancer composites against CaCo2 "colorectal adenocarcinoma" and MCF7 "breast adenocarcinoma" cells. Ct had 42.6kDa molecular weight and 90.7% deacetylation percentage. The conjugation of fabricated molecules/composites and their interactions were validated via infrared analysis. The generated nanoparticles (NCt, NCt/Cur, NCt/Eug, and NCt/Cur/Eug composites) had mean particle size diameters of 268.5, 314.9, 296.4, and 364.7nm, respectively; the entire nanoparticles carried positive charges nearby ≥30mV. The scanning imaging of synthesized nanoconjugates (NCt/Cur, NCt/Eug, and NCt/Cur/Eug) emphasized their homogenous distributions and spherical shapes. The cytotoxic assessments of composited nanoconjugates using the MTT assay, toward CaCo2 and MCF7 cells, revealed elevated anti-proliferative and dose-dependent activities of all nanocomposites against treated cells. The combined nanocomposites (NCt/Eug/Cur) emphasized the highest activity against CaCo2 cells (IC50 = 11.13μg/ml), followed by Cur/Eug then NCt/Cur. The exposure of CaCo2 cells to the nanocomposites exhibited serious DNA damages and fragmentation in exposed cancerous cells using the comet assay; the NCt/Eug/Cur nanocomposite was the most forceful with 9.54nm tail length and 77.94 tail moment. The anticancer effectuality of innovatively combined NCt/Cur/Eug nanocomposites is greatly recommended for such biosafe, natural, biocompatible, and powerful anticancer materials, especially for combating colorectal adenocarcinoma cells, with elevated applicability, efficiency, and biosafety.

Investigation of the antioxidant status and the number of double-stranded DNA breaks in models of brain tumor lesion by metastases of non-small cell lung cancer in vivo

Purpose of the study. Evaluation of the antioxidant status and DNA damage in tissues of subcutaneous xenografts of non-small cell lung cancer and in peritumoral tissues created using the A549 and H1299 cell cultures.Materials and methods. The study included 35 intact male Balb/c Nude immunodeficient mice. Cell lines A549 and H1299 were used as transplantable tumor biomaterial. A CDX model was created in accordance with the protocol for supratentorial injections (Ozawa T., James C. D., 2010) adapted for this experiment. Growth rates were controlled and intracranial xenografts were visualized using a high-resolution micro-CT system. The activity of catalase and superoxide dismutase was determined with non-denaturing electrophoresis in 8 % and 12 % polyacrylamide gel. The concentrations of sulfhydryl groups were determined according to Ellman. The DNA damage in lymphocytes was determined by the comet assay.Results. The experiment resulted in the creation of models of brain tumors characterized by intracranial growth pattern in 100 %. The activity of catalase in the studied lysates of intracranial xenografts, peritumoral tissue and healthy tissues of tumor-bearing animals in all experimental groups increased statistically significantly relative to the healthy tissue of intact animals, and the greatest differences from the control were recorded in the group of animals with implanted H1299 culture at a concentration of 1 × 106 . Superoxide dismutase activity in the studied lysates of intracranial xenografts and peritumoral tissues statistically significantly increased compared to the control sample in all experimental groups. The highest increase in the SOD activity was observed in the tissues of intracranial xenografts with the highest tumor load, which amounted to 28.8 % and 32.9 % of the changes relative to the control sample. A statistically significant increase in the concentration of SH-groups relative to the control sample in tumor tissue lysates was revealed in all experimental groups, and the highest concentration (36.2 ± 0.47) was observed in the group of experimental animals with the highest tumor load. Percentage change in tail moment (DNA damage indicator) in groups O1, O2, O3 and O4 increased statistically significantly compared to the control sample by 55.8 %, 111.8 %, 97.3 % and 170 %, respectively.Conclusions. The observed increase in the activity of the antioxidant defense system, accumulation of oxidative modifications of proteins, and an increase in DNA double-strand breaks in the tissues of intracranial xenografts of non-small cell lung cancer in vivo suggest that the created models reflect processes similar to those in tumors of human non-small cell lung cancer.

Assessing the genotoxicity of oral zinc oxide nanoparticle administration in male rats using micronuclei and comet assay

Zinc oxide nanoparticles (ZnO-NPs) are regularly utilized in the food and fertilizers industries. In our investigation, rats received oral administration of ZnO NPs with a particle size of 30±5 nm once daily at doses of 100, 200, 300, 400, and 600 mg/kg for ten weeks in order to assess the genotoxic effect. Impacts on hematological markers, genotoxic impact, and growth were investigated. The findings showed that ZnO-NPs significantly reduced body weight gain, red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit value (HCT), and platelet count (PLT), while increasing white blood cell (WBC), mean capsular volume (MCV), mean capsular hemoglobin (MCH), and mean capsular hemoglobin concentration (MCHC) in the treated rats. Our results for the comet assay and micronuclei test show a dosage-dependent increase in DNA fragmentation, which was supported by an increase in the percentage of DNA that is tailed, the length and intensity of DNA tails, and the tail moment, especially at the dose of 600 mg/kg. According to the findings, the frequency of micronucleated cells has increased.

Evaluation of the cytotoxic and genotoxic/antigenotoxic effects of resveratrol in human limbal explant cultures.

Resveratrol (RSV) is a natural polyphenol phytoalexin compound and has long been considered to possess antioxidant and anti-inflammatory effects. In order to exploit the protective potential of RSV in anterior segment diseases, we investigated the possible cytotoxic, genotoxic/antigenotoxic effects of human limbal explant cultures to RSV and MMC or H2O2 alone and in combination. A total of 18 limbal explant tissues obtained from three corneal donors were placed on the 12 well tissue culture polystyrene plates and cultured for 14days. Cell growth from limbal explants was observed by inverted phase contrast microscopy. The cytotoxic effects of RSV was studied by neutral red uptake assay. For the assessment of the genotoxic and antigenotoxic effects, basic alkaline technique of comet assay was performed. It was found that the concentrations of RSV up to 100μM did not significantly affect the viability of outgrowth cells of limbal explant during 24h exposure. When compared to negative control, all concentrations of RSV alone caused an increase in DNA strand breakage. Interestingly, 10μg/mL MMC alone caused similar tail intensity and tail moment values with RSV alone. On the other hand, RSV treatment in all doses seemed to decrease the DNA damage induced by either H2O2 or MMC. RSV is an attractive natural compound for the purpose of oxidative stress reduction in ocular surface and can be utilized as a supplement to promote ocular surface regeneration in vivo.

Evaluation of the biological response of propofol in zebrafish (Danio rerio): Focusing on biochemical, transcriptional, and molecular level

Propofol, one of the most widely used intravenous anesthetic in clinical practice, has been reported to impair cognitive and memory function. However, the toxicological effects of propofol on aquatic organisms are still poorly understood. This study explored the toxic effects of chronic propofol exposure (0.008, 0.04, and 0.2 mg L−1) on adult zebrafish from biochemical, transcriptional, and molecular level after 7, 14, 21 and 28 days of exposure. Results indicated that the reactive oxygen species (ROS) levels were significantly upregulated during the 28 days exposure period, and excessive ROS caused lipid peroxidation, resulting in increased malondialdehyde (MDA) contents in the zebrafish brain. In order to relieve the oxidative damage induced by the excessive ROS, the activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT)) were significantly activated, and detoxification enzyme (glutathione S-transferase, GST) activities showed an “activation-inhibition” trend. However, the antioxidant enzymes and detoxification enzyme system could not eliminate the excessive ROS in time and thus caused DNA damage in zebrafish brain. The olive tail moment (OTM) values displayed a “dose-response” relationship with propofol concentrations. Meanwhile, the transcription of related genes of Nrf2-Keap1 pathway was activated. Further molecular simulation experiments suggested that propofol could directly combine with SOD/CAT to change the activity of its biological enzyme. These findings indicated that zebrafish could regulate antioxidant capacity to combat oxidative stress at the early exposure stage, but the activity of antioxidant enzymes were significantly inhibited with the increase of propofol exposure time. Our results are of great importance for understanding toxicological effects of propofol on aquatic organisms.

Synergistic Effect of Selenium/Zinc with Sulfasalazine on the Human Colorectal Cancer Cell Line (HT-29)

Introduction: This study aimed at evaluating the anticancer activity of selenium and zinc in human colorectal adenocarcinoma (HT-29) cell line exposed to sulfasalazine (SSZ). Methods: Lipid peroxidation through the thiobarbituric acid reagent, oxygen-free radicals with the dichloro-dihydro-fluorescein diacetate reagent, glutathione (GSH) reserves through the 5,5’-dithiobis-(2-nitrobenzoic acid) reagent, and mitochondrial activity with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reagent were evaluated in the HT-29 cell line. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes and the activity of caspase-3 were determined using an ELISA kit. Genetic toxicity was also assessed by Comet assay. Results and Discussion: Combination therapy of SSZ with zinc or selenium in HT-29 reduced their growth, GSH, and the activity of SOD and GPx enzyme and increased reactive oxygen species and lipid peroxidation and the activity of caspase-3. The amount of tail moment in the comet test in the presence of these substances has also increased, indicating damage to cancer cells' DNA. In general, the best effect of selenium and zinc at concentrations of 100 and 200 μM (p < 0.05) (p < 0.01) was observed. Conclusion: Due to zinc and selenium's mechanism of action in inhibiting cancer cells' growth, these two substances can be used as a supplement to improve the effect of SSZ in treating diseases (e.g., colorectal cancer) and inhibiting drug toxicity.

Gallic acid a flavonoid isolated from Euphorbia hirta antagonizes gamma radiation induced radiotoxicity in lymphocytes in vitro.

The current study was executed to isolate and evaluate gallic acid from Euphorbia hirta for in vitro radioprotective potentials against gamma irradiation caused radiotoxicity in human lymphocytes. The defatted E.hirta plant material was treated to methanol extraction using the soxhlet device. Bioflavonoids were isolated from the E.hirta methanol extract using column chromatography. In human cells exhibited to gamma radiation, separated flavonoid gallic acid was examined for in vitro radioprotective potentials using the micronucleus test, DNA fragmentation assay, superoxide free radical scavenging method, and apoptic assay. The frequency of micronuclei was considerably declined when cells were preprocessed with gallic acid (25g/mL) before being exhibited to 2Gy gamma radiation, as determined by the cytokinesis blocked micronucleus test. Similarly, pre-gamma radiation treatment of human cells with gallic acid led in markedly less DNA injury, as assessed by comet metrics like olive tail moment and percent tail DNA. Gallic acid (25g/mL) given to lymphocytes prior to gamma irradiation considerably decreased the percentage of apoptotic bodies. Gallic acid also considerably lowered the reactive oxygen species concentrations elicited by gamma radiation. Our findings showed that gallic acid protects lymphocytes isolated from human blood from gamma radiation-induced DNA destruction and anti-apoptotic activity, which could be because of inhibition of free radicals formed by gamma radiation as well as the decline of gamma radiation-induced oxidative stress.

Genotoxic Biomonitoring in Children Living near the El Fraile Mine Tailings in Northern Guerrero State, Mexico.

A genotoxic study was conducted with 101 elementary school children (56 girls and 45 boys) in the 6-7, 8-9, and 10-12 age ranges from El Fraile rural community, which is located beside the El Fraile mine tailings in Taxco of Alarcon City, in northern Guerrero State, Mexico. For this, we used the alkaline comet assay in exfoliated buccal mucosa cells, scoring three genotoxic parameters: tail intensity, tail moment, and tail length. Additionally, we detected oxidative DNA damage through urinary 8-OHdG levels by enzyme-linked immunosorbent assay. We also evaluated a control group consisting of 101 children in the same age ranges from Chilpancingo City, Guerrero, who had never lived near mining zones. Genotoxic results showed that there was a significant increase in three genotoxic parameters and urinary 8-OHdG levels in the exposed children group compared with the control group. Analysis of MANOVA revealed that boys aged 8 and 9 years had higher DNA damage than girls from the same exposure group, and Spearman's analysis identified a positive correlation between DNA damage and sex and age. This study provides the first valuable genotoxic data in children living in areas with environmental pollution.

Investigation of Genotoxic Damage in Overweight Individuals Using the Comet Assay

AbstractObjective: Research has revealed that obesity changed the repair mechanism of DNA chain breaks. Also, the increase in body mass index is found out to be associated with genomic instability. In this study, it was aimed to investigate the possible genotoxic damage of overweight individuals.Material and Methods: In the present study the level of genotoxic damage was calculated in Turkish adult peripheral blood samples using Single Cell Gel Electrophoresis assay. The results of possible DNA damage levels belonging to overweight people were compared statistically by SPSS analysis program with the results of normal-weight people.Results: Fifty five volunteers (21 normal weight and 34 overweight); 23 women, mean age=30.13±7.97 and 32 men, mean age=38.13±10.63 participated in the study. Tail moment is an average of 1.21±0.45 in all individuals. Tail moment value was found of overweight people as 1.29±0.46. When this value was compared with the results of individuals with normal weight (1.09±0.40), statistically no significant difference was determined (p&amp;gt;0.05). According to results, no significant difference was found between the increase in body mass index and DNA damage (p&amp;gt;0.05).Conclusion: The present study is the first study which gives information about the level of DNA damage relation with being overweight in adults of Turkey. In the presented study, it was determined that there was no relationship between body mass index and genotoxic damage according to the findings of the comet assay, and we recommended that new studies should be conducted in the future to investigate the level of DNA damage with different genotoxicity tests.

PO03: Evaluation of DNA Damage Induced by the Xoft Axxent Source

<h3>Purpose</h3> To evaluate the level of DNA double strand breaks (DSB) induced by the Xoft Axxent source compared to megavoltage beam qualities for cervical cancer applications. <h3>Materials and Methods</h3> HeLa cells were suspended in DMEM media in 1.5 mL microcentrifuge tubes and irradiated at doses of 0, 2, 4, and 6 Gy using the bare Axxent source (BS) and source in titanium applicator (SIA). Additionally, HeLa cells suspensions in 15 mL centrifuge tubes were irradiated in ⁶⁰Co. Following irradiation, cells were embedded in LMAgarose gel on two well slides and neutral comet assays were run. Cells were stained with SYBR Gold stain, imaged using fluorescence microscopy, and the olive tail moment (OTM) was calculated using CometScore 2.0 software to quantify the number of DSBs. The TOPAS nBio extension was also used to evaluate the damage to a cell nucleus for the three beam qualities. The energy spectrum of the secondary electrons produced in the tubes of cells was scored and the average energies were calculated. Monoenergetic electron sources with these energies were placed in close proximity to a cell with a nucleus with a radius of 4 μm and the multiple nucleus scorers available with the extension were used. The dose rate of the sources was not considered in these simulations. <h3>Results</h3> A significant difference was observed between the BS and SIA at the 6 Gy dose point. A nonsignificant increase in OTM at 2 Gy and 4 Gy was observed between the BS and SIA. Further, a statistically significant difference was observed between the BS and 60Co at all dose points and between the SIA and 60Co at 2 Gy and 4 Gy. A nonsignificant difference was observed between the latter two beam qualities at 6 Gy, where there was a small increase in OTM for the SIA. When comparing to the nBio simulations, there was no significant difference observed between the DNA damage caused by the BS and SIA. The discrepancies between the experiments and simulations indicate the dose rate of the radiation plays a significant role in the damage induced to the DNA strand. <h3>Conclusions</h3> The reduced dose rate of the SIA plays a significant role in the differences observed between the BS and SIA DNA damage. Overall, both Axxent beam qualities provided an advantage of DSB induction compared to megavoltage beam qualities and indicate an advantage of using these sources clinically. Care should be taken to understand the limitations the reduced dose rate has on the resulting biological response to radiation.

Assessment of Protective Effects of Carvacrol on Haloperidol-Induced Oxidative Stress and Genotoxicity in Human Peripheral Blood Lymphocytes.

Haloperidol is a first-generation antipsychotic drug that has several indications in a wide range of mental conditions. The extensive prescription of haloperidol is correlated with some less-known adverse effects such as genotoxicity. Carvacrol is a monoterpenoid mainly found in oregano and thyme. It has the potential to scavenge free radicals in addition to increasing antioxidant defense enzyme activities and glutathione levels. In this study, we attempted to explore the possible potential of haloperidol in inducing genotoxicity in human peripheral lymphocytes as well as the protective role of carvacrol against this effect. The lymphocytes were divided into separate groups as follows: control group (cosolvent and NS); carvacrol group (5 μM); haloperidol group (25, 50, and 100 ng/ml); haloperidol (25, 50, and 100 ng/ml) + carvacrol (5 μM); positive control (0.8 μg/ml Cisplatin). After 24 hours of treatment, we conducted a cytokinesis-Block micronucleus test and an alkaline comet assay in order to determine genetic damage. Additionally, we measured glutathione and MDA levels as the biomarkers associated with oxidative stress. Significant increases in the levels of genotoxicity biomarkers (micronucleus frequency, DNA percentage in tail and tail moment) were observed in haloperidol-treated cells. The result of our oxidative stress tests also demonstrated that haloperidol had the potential to induce oxidative stress via reducing the levels of glutathione and increasing lipid peroxidation. Treatment with carvacrol significantly decreased the genotoxic events. It can be presumed that the induction of oxidative stress by haloperidol is the critical event associated with haloperidol-mediated genotoxicity. Therefore, using carvacrol as a natural antioxidant protected human lymphocytes against haloperidol genetic damage.

Oxidative stress and DNA damage in earthworms induced by methyl tertiary-butyl ether in natural soils.

Adverse effects of methyl tertiary-butyl ether (MTBE) have been noticed at different trophic levels by international researchers. However, there was unclear evidence about its effects on oxidative stress and DNA damage in earthworms. In this study, earthworms were cultivated in various doses of MTBE (0.0mg/kg, 10.0mg/kg, 30.0mg/kg, and 60.0mg/kg) contaminated agricultural soil for 7days, 14days, 21days, and 28days, respectively. The result showed that the reactive oxygen species (ROS) content of earthworms significantly increased in MTBE treatment groups compared to the control group. In MTBE treatment groups, the activities of superoxide dismutase, catalase, peroxidase, and glutathione S-transferase were significantly activated at the exposure of 7days, which increased by 36.3-78.9%, 51.8-97.3%, 36.5-61.9%, and 12.0-54.8%, respectively. Then, the activities of these defense enzymes showed various changes following the changes in exposure times and MTBE concentrations. Especially in the 60.0mgkg-1 group, both antioxidant enzymes and GST were still significantly activated at the exposure of 14days and then significantly inhibited at the exposure of 28days. The analysis of olive tail moment showed significant DNA damage in the 10.0mgkg-1 group at the exposure of 28days, and this damage in 30.0mg/kg and 60.0mg/kg groups was found at the exposure of 7days. This result was consistent with the malondialdehyde accumulation in earthworms. Additionally, the analysis of IBRv2 showed the effects of MTBE treatments on earthworms in dose- and time-dependent manners. This study helps better to understand the effects of MTBE on soil invertebrate animals and provide theoretical support for soil protection in governing MTBE application.