DNA Comet Assay Research Articles

Inhibitor of protein kinases 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylphenylamino)-1Н-pyrrole-2,5-dione induces DNA damage and apoptosis in human colon carcinoma cells

Background. The heterocyclic scaffolds are in the list of key structural blocks used at synthesis of novel biologically active compounds. Materials and Methods. The present study addressed the evaluation of the mecha­nisms of the DNA damaging and pro-apoptotic actions in vitro of the maleimide derivative 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylphenylamino)-1Н-pyrrole-2,5-dione (MI-1) targeting human colon carcinoma cells of HCT116 line. The Western-blot analysis was used to study changes in apoptosis-associated proteins, DNA comet assay under alkaline conditions was applied for evaluation of the DNA-damaging events, and Barton’s assay with diphenylamine was applied for measuring the level of DNA fragmentation in human colon carcinoma cells treated with MI-1 compound. Results. The results of the Western-blot analysis demonstrated that MI-1 induced the apoptosis in HCT116 cells via mitochondria-dependent pathway. It activated caspase 3 via its cleavage in the treated human colon carcinoma cells. Besides, MI-1 increased the content of mitochondria-specific proteins: endonuclease G (EndoG) and the pro-apoptotic cytosolic protein protease-activating factor 1 (Apaf1). At the same time, MI-1 reduced the level of the anti-apoptotic Bcl-2 protein in HCT116 cells. The DNA comet analysis under alkaline conditions of the targeted human colon carcinoma cells of HCT116 line demonstrated that MI-1 induced DNA single-strand breaks in line with the olive tail moment of 13.2. The results of the colorimetric diphenylamine assay in HCT116 cells have shown that cell treatment with MI-1 increased the content of fragmented DNA to 14.2 %. Conclusions. The anti-proliferative action of MI-1 in human colon carcinoma cells of HCT116 line is associated with apoptosis induction via mitochondria-dependent pathway, as well as the DNA damage through single-strand breaks and DNA fragmentation. These data suggest that the 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylpheny­l­amino)-1Н-pyrrole-2,5-dione (MI-1) might be a promising agent for suppression of growth of colon tumor cells. Keywords: 1Н-pyrrole-2,5-diones, apoptosis, Western-blot assay, comet assay, single-strand breaks, Barton’s assay, DNA fragmentation

Aqueous extract from Taiwanese Agrocybe cylindracea strain B protects DNA against • OH- mediated strand breaks

Water extract from Agrocybe cylindracea strain B (ACE), an edible mushroom produced in Taiwan, has previously been studied for its antioxidative activities. ACE is an efficient antioxidant in vitro. Since oxidative alterations of DNA can be linked to the development of cancer, we decided to study whether ACE protects DNA against oxidative stress. We report here that pUC18 plasmid DNA and HepG2 cells' DNA are damaged by • OH radicals generated from Cu (II) plus H2O2. The DNA damage was quantified by determining the diminution of supercoiled DNA forms and the DNA migration after oxidative attack using agarose gel electrophoresis. The IC50 values of ACE were 69.7 μg/mL and 91.6 μg/mL for cupric-mediated • OH formation by determining the diminution of supercoiled DNA forms and Comet assay, respectively. The antioxidant capacity of ACE was significantly higher than that of Lentinus edodes extract (LEE) and the two known scavengers of hydroxyl radical, mannitol and thiourea. ACE and LEE could also inhibit the oxidative degradation of 2′-deoxyribose caused by • OH radicals generated from metal ions plus H2O2. The malondialdehyde (MDA) inhibition effect of active fractional protein from ACE was better than that of LEE. The major protein components of ACE were different from LEE.

Evaluation of the Effect of p-Tyrosol on the Level of DNA Damage in the DNA Comet Assay In Vivo.

The effect of p-tyrosol on the spontaneous level of DNA damage in the cells of the bone marrow, liver, kidney, and rectum of mice (series I) and on the genotoxic effects of cytostatic drugs with different mechanisms of action in rat testicular cells (series II) was studied by DNA comet assay on C57BL/6 mice. p-Tyrosol was administered in a dose of 40 mg/kg once (series I) or for 5 days before and 5 days after cytostatic exposure (busulfan, paclitaxel, methotrexate; series II). It was found that p-tyrosol reduced spontaneous level of DNA damage in all studied organs. p-Tyrosol exhibited an antigenotoxic effect with respect to the DNA-damaging action of methotrexate and produced no genoprotective effect in case of busulfan and paclitaxel.

Protective Effect of Graviola (Annona muricata) Leaves Extract Against Cadmium-Induced Testicular Toxicity In Male Albino Rats

Graviola (Annona muricata), an edible plant belonging to the Annonaceae family, has a long history in traditional medicine due to its wide range of therapeutic effects on many ailments. This study was planned to assess the protective potentials of Graviola leaves extract (GLE) on cadmium-induced testicular damage in male albino rats. The study was conducted on 32 male adult Wistar rats randomly divided into four groups (n=8/each). A (control group); B (GLE, 100 mg/kg); C (cadmium chloride (CdCl2), 5 mg/kg) and D (GLE, 100 mg/kg + CdCl2 5 mg/kg). All administrations were performed by daily gavage for 8 weeks. Blood, testes, and epididymis were harvested for biochemical and histological investigations. In addition to sperm analysis, sperm DNA comet assay, and apoptotic indices. Results revealed that GLE significantly reversed (p < 0.05) testicular oxidative stress induced by CdCl2, maintained male sex hormones levels, and restored testis architecture via increasing the testicular antioxidant status. GLE co-treatment significantly increased (p < 0.05) sperm count, motility, viability, and DNA integrity along with significantly reducing sperm DNA damage and sperm head abnormality in comparison with CdCl2 group. Additionally, GLE protected germ cells from Cd-induced apoptosis. In conclusion, results of the present study confirmed that GLE has potent antioxidant potentials and cytoprotective capacity against the deleterious effects of CdCl2 on testicular tissue by reducing cellular oxidative stress prompted by the generation of the reactive oxygen species.

Enhancement effects of ethanolic extract of Fagonia cretica on Bisphenol A (BPA)-induced genotoxicity and biochemical changes in rats

BackgroundFagonia cretica L. was considered to be a medical plant that was used for the treatment of different diseases, so the current study was designed to clarify whether Fagonia cretica extract (FCE) can avoid Bisphenol A (BPA)-induced genotoxicity and biochemical alterations in rats. Sixty-three male rats were used in this experiment. These animals were distributed into nine groups (seven rats each): negative control, control of corn oil, positive control that were administrated BPA in corn oil (10 mg/kg. b.w.) for 3 weeks, three protection groups received the same dose of BPA in corn oil at the same period together with FCE (3.3, 4.2 and 5.0 g/kg) daily for 3 weeks, and three therapeutic groups received FCE alone at the same doses for 10 days after cessation of BPA treatment. Genetic and biochemical studies were conducted. Genetic studies involved DNA comet assay, micronucleus test, chromosome examination, and mitotic index analysis. Biochemical studies involved liver function (AST, ALT, ALph, and Bilirubin), kidney function (urea and creatinine contents), protein profile, MDA, and endogenous antioxidative system (SOD, CAT, and ACHE) as well as nucleic acid (DNA and RNA) contents in the liver, kidney, and brain tissues.ResultsThe results demonstrated that the treatment with BPA induced a significant elevation in genetic abnormalities and deleterious effects in biochemical parameters in relation to untreated control. FCE treatment was found to be significantly diminished the massive damage in the genetic constituents and dangerous alterations in biochemical parameters with respect to BPA treatment alone. These enhancements were increased by increasing the dose level of FCE. Moreover, better results were clarified by utilizing FCE as a protective agent than its utilization as a therapeutic agent especially by using the high dose (5.0 g/kg), in which mostly genetic and biochemical alterations were observed to be restored towards natural levels.ConclusionsThese findings clarified a new insight into the protective effect of FCE in minimizing BPA-induced genotoxicity and biochemical changes in rats detecting the capability of such medicinal plant for alleviating the adverse effect of BPA.

Complex Approach to Evaluating Genotoxicity from Occupational Factors in Coal Mining Industry

The level of cytogenetic damage was analyzed in coal miners (N = 116) and the control sample (N = 169) using an assay for scoring of chromosomal aberrations (CAs) and the DNA comet assay in lymphocytes, as well as the micronucleus test in buccal epithelial cells (MN in BEC). The group of coal miners was characterized by a statistically significantly increase in the main index values of the used test system, compared to the control group. Specifically, the level of chromosomal aberrations in the miner and control groups was 4.69 ± 0.28 and 2.13 ± 0.10%; the proportion of DNA in the comet tail was 4.33 ± 0.38 and 2.16 ± 0.24%; and the micronucleus level was 1.44 ± 0.21 and 0.23 ± 0.12‰, respectively (Р < 0.05). Moreover, in the sample of coal miners, a considerable increase in the levels of additional test indices was observed. For CAs, these parameters included the frequency of single fragments, chromatid-type aberrations, paired fragments, dicentrics without fragments, chromosome exchanges, and chromosome-type aberrations; for the DNA comet assay, these were the comet tail moment and Olive tail moment; for the MN in BEC, the frequencies of binucleated cells, cells with notched nuclei, geminate nuclei, karyorhexis, and apoptotic bodies. Ranking of the results according to major cytogenetic abnormalities made it possible to establish that, in the group of miners, the proportion of individuals with the index values above the background was 68.97% for CAs, 52.13% for the DNA comet assay, and 36.08% for the MN in BEC. The proportion of individuals with cytogenetic damage higher than the background simultaneously in three test systems was 20% of the total sample of miners. To assess the mutagenic effects from occupational factors of coal mining enterprises, it seems reasonable to use a complex of test systems (CA test, DNA comet assay, and MN in BEC).

Synthesis and Cytostatic Activity of New Mepregenol 17-Acetate Derivatives with Respect to Hela Cancer Cell Culture

New mepregenol 17-acetate derivatives with potential cytotoxicity were synthesized. Mepregenol 17-acetate 3-acrylate (III) was shown to exhibit potent cytotoxicity although the ester of N-benzylpyrrolidine-3-carboxylic acid (IV) had weaker activity that was comparable with those of megestrol acetate and progesterone. The IC50 values for III, IV, progesterone, and megestrol acetate were 30, 200, 480, and 130 μMwith respect to native HeLa culture and 9.1, 180, 5.6, and 115 μm for an estradiol-stimulated culture of HeLa, respectively. The cytotoxicities of steroids III and IV correlated with their DNA-damaging effect in a DNA comet assay. Thus, III was promising for further research as an antitumor compound with respect to estradiol-dependent tumors.

Loss of NKX6.3 During Gastric Carcinogenesis Enhances Genomic Instability and Promote Tumor Development

Background: The maintenance of genomic stability requires correct DNA replication, chromosome segregation, and DNA repair, while defects of these processes result in tumor development or cell death. Here, we investigated whether NKX6.3 plays a role in maintaining the genomic stability of gastric epithelial cells. Methods: qRT-PCR, immunofluorescent, fluorescence in situ hybridization (FISH) and immunoblot were utilized to analyze the copy number alterations (CNAs) and expression levels of CDKN3, DLGAP5, AurkA, TPX2, SPARCL1, and SORBS2 in NKX6.3 depleted cells and gastric cancer tissues. Fluorescence-activated cell sorting was used for cell cycle and DNA repair analysis. DNA fiber assay, BrdU, and EdU assays were performed to analyze DNA replication stress. DNA fragmentation and Comet assays were used for DNA damage analysis. Chromosomal aberrations were analyzed by metaphase spread and mitotic index assays. Xenograft experiments were performed to determine CNAs and expression levels of genes caused by NKX6.3 depletion in vivo. Whole-genome sequencing was performed to analyze CNAs caused by NKX6.3 depletion. Findings: NKX6.3 depleted HFE-145 immortalized gastric epithelial cells induced a rapidly cell cycle with increased S and G2/M phase cell populations, by altering the expression of cell cycle related proteins. During cell cycle, NKX6.3 depletion induced faster fork progression, fork symmetry, longer gap length, mitotic spindle defects, and chromosome mis-segregation. In DNA repair, depletion of NKX6.3 led to an induction of homologous recombination repression. When we investigated whether NKX6.3 was involved in CNAs of gastric epithelial cells, frequently gained or amplified, and lost or deleted broad chromosomal regions were detected in NKX6.3-depleted cells. Hallmark enrichment analyses of NKX6.3-depleted cells revealed gain of CDKN3, DLGAP5, AurkA, and TPX2, and deletion of SORBS2 and SPARCL1. Moreover, NKX6.3-depletion caused mitotic spindle defects and chromosome mis-segregation in vitro and in vivo through the CNA of genes involved in mitotic spindle formation. In 65 human gastric cancers and xenograft mice models for NKX6.3-depleted HFE-145 cells, expression levels of CDKN3, DLGAP5, AurkA, and TPX2 were markedly increased while expression levels of SPARCL1 and SORBS2 were decreased. In NKX6.3-depleted cells, silencing of CDKN3 and DLGAP5 as well as ectopic expression of SPARCL1 and SORBS2 inhibited cell proliferation and colony formation. Interpretation: These results demonstrate that a novel molecular mechanism for gastric carcinogenesis through the induction of genomic instability by abnormal regulation of DNA replication and repair due to NKX6.3-depletion. Thus, we conclude that NKX6.3 may DNA replication and repair regulation for protecting gastric mucosal epithelial cells from genomic instability such as CNAs. Funding Statement: This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017R1D1A1A09000781 to J.H.Y.; 2018R1A2A2A14019713 to W.S.P.). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: Written informed consent was obtained from all subjects. The study was approved by the Institutional Review Board of The Catholic University of Korea, College of Medicine (MC15SISI0015). Animal experiments were performed in mice maintained under pathogen-free conditions based on approval by the Animal Experiment Ethics Committee of the Catholic University of Korea College of Medicine (CUMC-2017-0018-01).

GENOTOXIC AND PROTECTIVE ACTIVITY OF EXTRACTS FROM INULA BRITANNICA (FAM. COMPOSITAE)) IN THE BODY OF LABORATORY MICE

The protective and mutagen-modifying activity of the complex of biologically active substances(BAS) in the extract from the underground part of the plant Inula britannica in the cells of the visceral organs of laboratory mice was studied. Alkaline variation of the comet assay was used to determine genotoxic/antigenotoxic activity. The frequency of single-stranded DNA breaks in the cells of the brain andbone marrow, lungs, heart, kidneys, liver, stomach, spleen was assessed by the following parameters: %of DNA in tail, Olive tail moment, damage index. It was found that extracts of BAS in concentrations of100.0 mg/l and 150.0 mg/l did not show genotoxic activity, the frequency of single-strand breaks in allstudied organs did not statistically significantly exceed the spontaneous level in intact animals. Whenexposed to asymmetric dimethylhydrazine (UDMH), the cells of the studied organs showed a statisticallysignificant increase in the number of single-stranded DNA breaks compared to intact laboratory mice.When combined with UDMH as a positive control, extract of Inula britannica significantly reduces thegenotoxic effect of xenobiotics. The frequency of DNA breaks in animals that simultaneously receivedUDMH and Inula britannica extract was statistically significantly lower compared with mice that received only UDMH. All this suggests the presence of antimutagenic and antigenotoxic activity in theextract of Inula britannica.Key words: Inula britannica, biologically active substances, mutagen, antimutagen, genotoxicity,DNA comet assay.

Influence of butylated hydroxytoluene addition to cryodiluents on freezability and DNA integrity of Boer and Zaraibi buck spermatozoa

Objective: To evaluate the cryoprotective effect of butylated hydroxytoluene on buck frozen semen. Methods: Semen was collected from Boer (n=6) and Zaraibi (n=6) bucks by electroejaculator for 5 weeks. Semen aliquots were diluted at 38 °C in Tris-buffer with egg yolk 15.0% (vol/vol) (Tris- egg yolk extender) or soya lecithin 2.5% (weight/vol) (Tris-soya lecithin extender) supplemented with butylated hydroxytoluene at 0.0 (as the control), 0.5, 1.0, 2.0 and 4.0 mM. Post-thawing motility (at 400x magnification), plasma (hypo-osmotic swelling test), acrosome (Trypan blue/Giemsa dual staining) membranes, DNA (comet assay), and lipid peroxidation (by malondialdehyde concentration) were assessed. Results: Spermatozoa motility was enhanced by butylated hydroxytoluene in Tris-soya lecithin extender at 0.5 mM in the two breeds, and in Tris-egg yolk extender at 1.0 mM in Boer and at 2.0 mM in Zaraibi bucks for up to 3 h post-thawing. Plasma and acrosome membranes and DNA integrity of the two breeds were maximally high with butylated hydroxytoluene at 1.0-2.0 mM in Tris-egg yolk extender and at 0.5-1.0 mM in Tris-soya lecithin extender. Lipid peroxidation was minimal with butylated hydroxytoluene at 1.0-2.0 mM in Tris-egg yolk and soya lecithin extenders in the two breeds. Butylated hydroxytoluene at 4.0 mM deteriorated spermatozoa motility, and plasma and acrosome membranes. Conclusions: The consequence of butylated hydroxytoluene on buck frozen-thawed spermatozoa varies with the levels of supplementation, buck breed, and phospholipid source in the extender. Semen parameters of Boer buck are better in their response to butylated hydroxytoluene than Zaraibi buck. Butylated hydroxytoluene at 1.0 and 2.0 mM in Tris-egg yolk extender, and at 0.5 mM in Tris-soya lecithin extender represents the best concentrations and profitably improves the semen quality of buck semen.

Methodological aspects of DNA-comet assay in vivo in pre-clinical research

Methodological aspects of DNA-comet assay in vivo in pre-clinical research

Gamma-hexalactone flavoring causes DNA lesion and modulates cytokines secretion at non-cytotoxic concentrations

BackgroundThe γ-hexalactone is a flavoring agent for alcoholic beverages, teas, breads, dairy products, coffees, buttery products among others. It presents low molecular weight and exhibits sweet fruity aroma with nuances of nuts. As far as we know, both literature and government regulations have gaps regarding the safe use of the γ-hexalactone. In this context, the main objective of this work was to evaluate the effects of γ-hexalactone through in silico and in vitro approaches.MethodsThe in silico analysis was performed through four free online platforms (admetSAR, Osiris Property Explorer®, pkCSM platform and PreADMET) and consisted of comparative structural analysis with substances present in databases. The computational prediction was performed in the sense of complement and guide the in vitro tests. Regarding in vitro investigations, screening of cytotoxicity (assessed by cell proliferation and viability parameters) in lymphocytes exposed to γ-hexalactone for 72 h were carried out previously to determine non-cytotoxic concentrations. Following this screening, concentrations of 5.15, 0.515, and 0.0515 μM were selected for the study of the respective potentials: genotoxic (assessed by DNA comet assay), chromosomal mutation (analysis of micronucleus frequency) and immunomodulatory (cytokine quantification using ELISA immunoassay). The results of in vitro assays were compared by one-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test, conducted by statistic software.ResultsThe platform PreADMET pointed out that γ-hexalactone is potentially mutagenic and carcinogenic. The comet assay data corroborate with these results demonstrating that γ-hexalactone at 5.15 μM caused lymphocytes DNA damage. In relation to cytokine secretion, the results indicate that lymphocytes were activated by γ-hexalactone at non-cytotoxic concentrations, involving an increase in the IL-1 levels in all tested concentrations, ranging from approximately 56 to 93%. The γ-hexalactone only at 5.15 μM induced increase in the levels of IL-6 (~ 60%), TNF-α (~ 68%) and IFN-γ (~ 29%), but decreased IL-10 (~ 46%) in comparison with the negative control (p < 0.05). No change was observed in total lymphocytes or in cell viability at the concentrations tested.ConclusionsIn summary, the γ-hexalactone demonstrated immunomodulatory and genotoxic effects at non-cytotoxic concentrations in healthy lymphocytes.

Effects of Indralin on Immunohematological Parameters and DNA Damage in Irradiated ICR (CD-1) Outbred Mice

Abstract—Intragastric administration of radioprotector indralin (B-190 drug) to outbred SPF ICR (CD-1) mice 15 minutes before X-ray irradiation at doses of 6 Gy (LD-10/30), 6.5 Gy, and 7 Gy (LD-100/30) increases the 30-day survival of the animals and positively affects the state of the central organs of immunity and hematopoiesis, as well as the number of peripheral blood leukocytes. In addition, a positive effect on the behavioral reactions of the irradiated animals and striated muscles strength was noted. In addition to these secondary features of the indralin positive effect, a significant reduction of radiation-induced DNA damage in spleenocytes was observed using flow cytometry analysis of phosphorylated histone Н2АХ (γН2АХ) (1 h and eight days after X-ray irradiation) and DNA-comet assay (eight days after X-ray irradiation). The abovementioned methods are recommended for experiments investigating the influence of radioprotective drugs on DNA damage induction and repair in the irradiated organism, as well as for initial searching of potential radioprotectors and assessment of their effectiveness level in clinical practice and experiments.

MUS81 Participates in the Progression of Serous Ovarian Cancer Associated With Dysfunctional DNA Repair System.

Objective: Methyl methanesulfonate ultraviolet sensitive gene clone 81 (MUS81) is a structure-specific endonuclease that plays a pivotal role in the DNA repair system of cancer cells. In this study, we aim to elucidate the potential association between the dysfunction of MUS81 and the progression of Serous Ovarian Cancer (SOC).Methods: To investigate the association between MUS81 and prognosis of SOC, immunohistochemistry technology and qPCR were used to analyze the level of MUS81 expression, and transcriptional profile analysis and protein interaction screening chip were used to explore the MUS81 related signal pathways. Random amplified polymorphic DNA (RAPD) analysis, immunofluorescence and comet assays were further performed to evaluate genomic instability and DNA damage status of transduced SOC cells. Experiments both in vitro and in vivo were conducted to verify the impact of MUS81 silencing on chemotherapeutic drug sensitivity of SOC.Results: The overexpression of MUS81 in SOC tissues was related to poor clinical outcomes. The transcriptional chip data showed that MUS81 was involved in multiple pathways associated with DNA repair. Deficiency of MUS81 intensified the genome instability of SOC cells, promoted the emergence of DSBs and restrained the formation of RAD51 foci in SOC cells with exposure to UV. Furthermore, downregulation of MUS81 enhanced the sensitivity to Camptothecin and Olaparib in SOC cell lines and xenograft model.Conclusions: MUS81 is involved in the progression of SOC and inhibition of MUS81 could augment the susceptibility to chemotherapeutic agents. MUS81 might represent a novel molecular target for SOC chemotherapy.

Genotoxic activity of 1,2,3-triazolyl modified furocoumarins and 2,3-dihydrofurocoumarins.

The furocoumarin backbone is a promising platform for chemical modifications aimed at creating new pharmaceutical agents. However, the high level of biological activity of furocoumarins is associated with a number of negative effects. For example, some of the naturally occurring ones and their derivatives can show genotoxic and mutagenic properties as a result of their forming crosslinks with DNA molecules. Therefore, a particularly important area for the chemical modification of natural furocoumarins is to reduce the negative aspects of their bioactivity. By studying a group of 21 compounds-1,2,3-triazolyl modified derivatives of furocoumarin and peucedanin-using the SOS chromotest, the Ames test, and DNA-comet assays, we revealed modifications that can neutralize the structure's genotoxic properties. Theoretical aspects of the interaction of the compound library were studied using molecular modeling and this identified the leading role of the polyaromatic molecular core that takes part in stacking-interactions with the pi-systems of the nitrogenous bases of DNA.

Sirtuins and neuronal DNA damage under experimental chronic cerebral hypoperfusion

Chronic brain hypoperfusion (ChCH) is a risk factor for central nervous system (CNS) diseases, such as chronic brain ischemia, degenerative diseases, and others, in this connection, the study of its pathophysiological mechanisms is an actual problem.Objective. To investigate the relationship between SIRT1/SIRT3 expression and activity and damage of neuronal DNA in conditions of chronic cerebral hypoperfusion in mice.Маterials and methods. The experiments were carried out on male C57Bl mice (6 weeks old, weight 18–20 g), which underwent occlusion of left common carotid artery to model ChCH. On the background of ChCH nicotinamide (NAM, 200 mg/kg, 10 d, i.p.) and resveratrol (RV, 10 mg/kg, 10 d, i.p.) were used. All manipulations were carried out in anesthetized with ketamine (60 mg/kg, i.p.) mice. In 8 weeks, in mice brain tissues the features of neuronal DNA damage were studied by DNA comet assay and SIRT1/SIRT3 gene expression levels by real-time RT-PCR.Results. ChCH modeling was accompanied on the unilateral side by an increase of DNA damage (comets within classes 3 and 4) index of neurons by 6.9 times, and by a decrease of SIRT1 and SIRT3 gene expression by 9.3 and 20.2 times, respectively, compared to control (P &lt; 0.05). Using of NAM and RV treatment resulted in reduction of DNA damage index by 89.6 % and by 92.4 %, respectively, compared to control (P &lt; 0.05), and in the increase of the levels of SIRT1 by 1.7 and 3.5 times, and SIRT3 – by 2.9 and 5.2 times, respectively, in comparison with the ChCH group (P &lt; 0.05).Conclusion. Taken together, these data indicate that modification of SIRT1 activity caused positive effect on ChCH-induced brain injury by attenuating DNA breaks, which was accompanied with up-regulation of SIRT-mediated regulatory pathways.

Effects of Polar Steroids from the Starfish Patiria (=Asterina) pectinifera in Combination with X-Ray Radiation on Colony Formation and Apoptosis Induction of Human Colorectal Carcinoma Cells.

Despite significant advances in the understanding, prevention, and treatment of cancer, the disease continues to affect millions of people worldwide. Chemoradiation therapy is a rational approach that has already proven beneficial for several malignancies. However, the existence of toxicity to normal tissue is a serious limitation of this treatment modality. The aim of the present study is to investigate the ability of polar steroids from starfish Patiria (=Asterina) pectinifera to enhance the efficacy of radiation therapy in colorectal carcinoma cells. The cytotoxic activity of polar steroids and X-ray radiation against DLD-1, HCT 116, and HT-29 cells was determined by an MTS assay. The effect of compounds, X-ray, and their combination on colony formation was studied using the soft agar method. The molecular mechanism of the radiosensitizing activity of asterosaponin P1 was elucidated by western blotting and the DNA comet assay. Polar steroids inhibited colony formation in the tested cells, and to a greater extent in HT-29 cells. Asterosaponin P1 enhanced the efficacy of radiation and, as a result, reduced the number and size of the colonies of colorectal cancer cells. The radiosensitizing activity of asterosaponin P1 was realized by apoptosis induction through the regulation of anti- and pro-apoptotic protein expression followed by caspase activation and DNA degradation.

The study of the influence of exogenous factors on Saccharomyces cerevisiae DNA spheroplast

The possibility of fixing genotoxic changes in Saccharomyces cerevisiae yeast under the influence of physicochemical factors was evaluated using the DNA-comet assay. Peripheral blood leukocytes of mice subjected to similar effects were used as an object of comparison. The data obtained showed that under the action of both the alkylating agent and X-ray irradiation, the changes were more pronounced in experiments with leukocytes of the blood of mice. At a methyl-methane sulphonate concentration of 10 mM, the comet tail length in mouse peripheral blood leukocytes was about 40 μm, while the yeast spheroplasts tail length of a comet was 0.16 μm. The DNA content in the tail of the comet did not exceed 5 % for yeast after treatment with an alkylating agent at a concentration of 40 mM, which is 10 times less than in peripheral blood leukocytes of mice under similar conditions. Under the action of X-ray radiation, the length of the comet’s tail and the DNA content in it for spheroplasts of yeast also differed significantly from the leukocytes of the peripheral blood of the mouse. Thus, the comet test allows to register a significant increase in the level of DNA damage in yeast spheroplasts under the action of physicochemical factors in relatively high doses.

Propolis Potentiates Methotrexate Anticancer Mechanism and Reduces its Toxic Effects

Egyptian propolis is a powerful antioxidant and free radical scavenger produced by bees. The current study was designed to characterize Egyptian propolis, investigate its anticancer effect in vitro and its protective role against methotrexate (MTX) toxicity in Ehrlich ascites carcinoma (EAC) experimental model. Our results revealed a high content of total phenolics, flavonoids and dihydroflavonols in propolis ethanolic extract (PEE). PEE prompted cytotoxic effects in cancer cell lines and antitumor effects against EAC mice model by reducing tumor volume, count of viable tumor cells with a significant elevation in the life span as well as the mean survival time of mice. The hepatic and renal biochemical and toxicity parameters of EAC-bearing mice treated with MTX were improved by PEE. Also, it elevates the expression of Bax, caspase-3 and cytochrome-C and reduces the Bcl2 expression in EAC cells. Moreover, PEE with MTX induced cell cycle arrest at the G0/G1 phase. Interestingly, the combination of PEE with MTX showed potent apoptosis as shown by DNA fragmentation gel, comet assay and dihydrofolate reductase level (DHFR). These findings demonstrate that Egyptian propolis extract had high chemical diversity and different antioxidant effects. Also, it optimizes the antitumor potential of MTX and declined its toxic effects.

Genotoxic Assessment of the Metabolite M-11 of Mepanipyrim, the Active Ingredient in the Plant Protection Product FRUPICA SC

The VKM Panel for plant protection products considered Frupica SC in a meeting on 25.11.2010, and found the active ingredient problematic with regard to carcinogenic effects and possible genotoxicity. M11 is a metabolite of mepanipyrim which is the active ingredient the plant protection product Frupica SC. The Norwegian Food Safety Authority has asked the applicant for further assessment of the genotoxic potential of the metabolite M11. The applicant has submitted a rat liver in vivo Comet assay of the metabolite, and the panel has been requested to consider if the genotoxic properties of mepanipyrim and the metabolite M11 is adequately documented.&#x0D; The metabolite M11 caused positive findings in in vitro studies for bacterial mutation and chromosomal aberrations. Three in vivo studies (Micronucleus, unscheduled DNA synthesis and Comet assay) did not show evidence of genotoxicity. Based on the documentation available, VKMs Panel on Plant Protection Products concludes that mepanipyrim and the metabolite M11 should not be considered genotoxic in vivo. The lack of demonstrated in vivo genotoxicity makes it likely that mepanipyrim induces liver tumors in rats and mice by a mechanism that involves a threshold below which tumors are not expected to develop. This conclusion is strengthened by the finding of a promoter-like behavior of mepanipyrim for induction of gamma-glutamyl-transpeptidase positive foci in rat liver.