Comet Tail Length Research Articles

Abstract 164: Alteration of DNA methylation, DNA repair and epithelial-mesenchymal transition in colorectal cancer cell lines by S-adenosylmethionine treatment

Abstract Global DNA hypomethylation can be observed along with the aging of normal cells, and it is also related to tumor initiation and progression. S-adenosylmethionine (SAM) is a universal methyl donor molecule, used as a dietary supplement. SAM is involved in DNA methylation processes, thereby it may have a favorable effect on gene expression of cancer-associated genes through epigenetic modifications, but may also influence DNA folding during repair processes. Our aim was to analyze the effect of SAM treatment on global and promoter-specific DNA methylation level, gene expression, DNA integrity, cell cycle and the proliferation of two different colorectal cancer cell lines (HT-29, SW480). HT-29 and SW480 cells were treated with SAM in different concentrations (0, 0.5, 1 mmol/l) for 48 hours. Global DNA methylation status was analyzed by bisulfite pyrosequencing of long interspersed nuclear element-1 (LINE-1) retrotransposons. Promoter-specific DNA methylation alterations were determined by Reduced Representation Bisulfite Sequencing (RRBS) method. Gene expression changes were detected using Human Transcriptome Array 2.0 (HTA 2.0). DNA integrity analysis was performed with γH2AX ELISA, immunostaining and Comet Assay. Flow cytometry measurement and Sulforhodamine B (SRB) assay were assessed for cell cycle and proliferation determination. Global and promoter-specific DNA methylation alterations, as well as decreased expression (p< 0.05) of genes, that are involved in epithelial-mesenchymal transition were observed after SAM treatment. Increased phosphorylation of H2AX (74.9, 166.5, 200.6 pM) and decreased micronucleus number (1.47, 0.76, 0.45% of cells) were referred to the activation of reparative processes, that was supported by the changes of comet tail lengths. Proportion of cells was decreased in the G0/G1 (48.4, 28.5, 20.4%) phase; however, it was increased in both S (45.7, 61.7, 67.0%) and G2/M (6.0, 10.7, 12.5%) phases. Significant (p< 0.05) decrease of cell proliferation (99.5, 78.0, 70.6%) was also detected with SRB assay. SAM is able to alter the DNA methylation pattern of tumor cells and can induce DNA repair. Activation of these processes can lead to cell cycle arrest, decreased proliferation, and inhibition of epithelial-mesenchymal transition. Tumor cells could be targeted by SAM through different pathways; therefore, it may enhance the effect of chemotherapeutic agents. Citation Format: Sára Zsigrai, Alexandra Kalmár, Krisztina Andrea Szigeti, Zsófia Brigitta Nagy, Barbara K. Barták, Gábor Valcz, Orsolya Galamb, Titanilla Dankó, Zsolt Tulassay, Péter Igaz, Béla Molnár. Alteration of DNA methylation, DNA repair and epithelial-mesenchymal transition in colorectal cancer cell lines by S-adenosylmethionine treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 164.

S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression.

Global DNA hypomethylation is a characteristic feature of colorectal carcinoma (CRC). The tumor inhibitory effect of S-adenosylmethionine (SAM) methyl donor has been described in certain cancers including CRC. However, the molecular impact of SAM treatment on CRC cell lines with distinct genetic features has not been evaluated comprehensively. HT-29 and SW480 cells were treated with 0.5 and 1 mmol/L SAM for 48 h followed by cell proliferation measurements, whole-genome transcriptome and methylome analyses, DNA stability assessments and exome sequencing. SAM reduced cell number and increased senescence by causing S phase arrest, besides, multiple EMT-related genes (e.g., TGFB1) were downregulated in both cell lines. Alteration in the global DNA methylation level was not observed, but certain methylation changes in gene promoters were detected. SAM-induced γ-H2AX elevation could be associated with activated DNA repair pathway showing upregulated gene expression (e.g., HUS1). Remarkable genomic stability elevation, namely, decreased micronucleus number and comet tail length was observed only in SW480 after treatment. SAM has the potential to induce senescence, DNA repair, genome stability and to reduce CRC progression. However, the different therapeutic responses of HT-29 and SW480 to SAM emphasize the importance of the molecular characterization of CRC cases prior to methyl donor supplementation.

Evaluation of HPRT Gene mutation and Comet Assay in Some Breast Cancer Patients Undergoing Radiotherapy

Introduction: Radiotherapy is a toxic cellular treatment that destroys rapidly dividing cells such as cancer cells. The strategy for HPRT mutation examination in human’s cells was commonly used to establish in vivo history along with mutation rates in children and adults inhabitants subjected to experienced and unfamiliar external mutagens. As just a practical approach to human biosurveillance studies, the comet assay has been introduced over the past years as biomarker to identify ionizing radiation Consequences with Patients undergoing radiotherapy in breast cancer. Materials and Method: This study was carried out on thirty Iraqi women patients with BC patients undergoing radiotherapy about 20-30 Gy locally gamma cells at Al-Amel National Hospital for cancer Management in Baghdad during time 2-13 years, non-smokers and non- alcoholic, aged (35-55 year), with stage (grade) I-III, as well as thirty apparently healthy individuals females collected randomly from population living Baghdad, Old age (35-55) Which are non-alcoholic non-smokers as group of control. Around the ongoing research comet-test and hprt mutation test could be applied to study damages in DNA for genetic two endpoints For patients with cancer throughout radiation therapy. Results: The present study showed significant increase (p<0.05) in the HPRT gene mutation assay for the in breast cancer patients undergoing radiotherapy as compared with the control group. Also there were found that the values of comet tail moment and tail length was increased significantly (p<0.05) in the human lymphocyte in these for breast cancer patients undergoing radiotherapy as compared with the control group. Conclusions: Present results revealed that there is a probability of utilizing human lymphocytes changes as useful biodosimetric markers for the detection of human exposure to ionizing radiation, the data gathered also demonstrated the utility of the HPRT gene mutation and (Alkaline) comet test as just a precise alternative biological marker for routine preventative care of cancer patients receiving radiation therapy.

Changes in the levels of comet parameters before and after fluoxetine therapy in major depression patients.

Major depression belongs to mood disorders and characterized by worthlessness, no interest or happiness in any activity; lasting for atleast two weeks. Etio-pathological changes of major depression include oxidative stress leading to free radical synthesis which causes damage to carbohydrates, proteins, lipids and nucleic acids. Nucleic acid damage can be identified by either single or double strand breaks and for quantitative estimation of the same, neutral or alkaline comet assay is performed. Fluoxetine is the drug of choice for treatment of major depression having antioxidant function. In the current study eighty drug naïve major depression patients were recruited and comet parameters namely total comet length, head diameter and tail length were measured before starting the treatment and after completion of eight week fluoxetine therapy. The levels of comet parameters were higher in females than males suggesting higher prevalence of major depression among females. On categorizing into three age groups, the numbers of major depression patients belonging to 18–30 year age group were higher than 31–40 and 41–50 year age groups. All the parameters of deoxyribonucleic acid damage were reduced after eight week of fluoxetine therapy indicating that fluoxetine has anti-oxidant action along with its antidepressant properties, which cause reversal of oxidative stress induced damage occurring during major depression.

Vernonia calvoana Shows Promise towards the Treatment of Ovarian Cancer.

The treatment for ovarian cancers includes chemotherapies which use drugs such as cisplatin, paclitaxel, carboplatin, platinum, taxanes, or their combination, and other molecular target therapies. However, these current therapies are often accompanied with side effects. Vernonia calvoana (VC) is a valuable edible medicinal plant that is widespread in West Africa. In vitro data in our lab demonstrated that VC crude extract inhibits human ovarian cancer cells in a dose-dependent manner, suggesting its antitumor activity. From the VC crude extract, we have generated 10 fractions and VC fraction 7 (F7) appears to show the highest antitumor activity towards ovarian cancer cells. However, the mechanisms by which VC F7 exerts its antitumor activity in cancer cells remain largely unknown. We hypothesized that VC F7 inhibits cell proliferation and induces DNA damage and cell cycle arrest in ovarian cells through oxidative stress. To test our hypothesis, we extracted and fractionated VC leaves. The effects of VC F7 were tested in OVCAR-3 cells. Viability was assessed by the means of MTS assay. Cell morphology was analyzed by acridine orange and propidium iodide (AO/PI) dye using a fluorescent microscope. Oxidative stress biomarkers were evaluated by the means of lipid peroxidation, catalase, and glutathione peroxidase assays, respectively. The degree of DNA damage was assessed by comet assay. Cell cycle distribution was assessed by flow cytometry. Data generated from the MTS assay demonstrated that VC F7 inhibits the growth of OVCAR-3 cells in a dose-dependent manner, showing a gradual increase in the loss of viability in VC F7-treated cells. Data obtained from the AO/PI dye assessment revealed morphological alterations and exhibited characteristics such as loss of cellular membrane integrity, cell shrinkage, cell membrane damage, organelle breakdown, and detachment from the culture plate. We observed a significant increase (p < 0.05) in the levels of malondialdhyde (MDA) production in treated cells compared to the control. A gradual decrease in both catalase and glutathione peroxidase activities were observed in the treated cells compared to the control. Data obtained from the comet assay showed a significant increase (p < 0.05) in the percentages of DNA cleavage and comet tail length. The results of the flow cytometry analysis indicated VC F7 treatment caused cell cycle arrest at the S-phase checkpoint. Taken together, our results demonstrate that VC F7 exerts its anticancer activity by inhibiting cell proliferation, inducing DNA damage, and causing cell cycle arrest through oxidative stress in OVAR-3 cells. This finding suggests that VC F7 may be a potential alternative dietary agent for the prevention and/or treatment of ovarian cancer.

Polymorphisms in XPC and XPD genes modulate DNA damage in pesticide-exposed agricultural workers of Punjab, North-West India.

The genetic susceptibility of individuals to the genotoxic effect of pesticides may be modulated by variations in genes involved in nucleotide excision repair (NER) pathway and therefore plays an important role in the evaluation of occupational risk. We aimed to evaluate the role of xeroderma pigmentosum complementation group C (XPC) Lys939Gln (A2920C, rs2228001), XPC Ala499Val (C2151T, rs2228000), xeroderma pigmentosum complementation group D (XPD) Asp312Asn (G23591A, rs1799793) and XPD Lys751Gln (A35931C, rs13181) in the modulation of DNA damage. A total of 450 subjects (225 pesticide-exposed agricultural workers and 225 age- and sex-matched controls) from Punjab, North-West India were recruited to study DNA damage by alkaline comet assay. Genotyping was carried out by PCR-RFLP using site-specific restriction enzymes. We found significant elevation in DNA damage parameters in pesticide-exposed agricultural workers as compared to the controls (p < 0.01). Association of comet tail length with XPC 939Gln/Gln (CC), XPD 312Asp/Asn (GA) and XPD 312Asn/Asn (AA) genotypes was observed. Frequency of cells showing DNA migration was significantly higher in exposed workers with variant XPC 939Gln/Gln (CC), XPD 312Asp/Asn (GA) and XPD 312Asn/Asn (AA) genotypes. Mean tail length was significantly increased in agricultural workers carrying XPD 312Asn/Asn (AA) genotype. Elevation in total comet DNA migration was also observed in exposed workers carrying variant XPC 939Lys/Gln (AC), XPC 939Gln/Gln (CC), XPC 499Val/Val (TT) and XPD 312Asn/Asn (AA) genotypes. Our results strongly indicate significant positive association of variant XPC and XPD genotypes with higher pesticide-induced DNA damage in North-West Indian agricultural workers.

Comet assay on one- and two-cell mouse embryos

DNA damage quantified as the comet tail length was assessed using in vitro and in vivo comet assay on one- and two-cell mouse embryos obtained by natural mating. The use of a protocol with three layers of agarose reduces the embryo loss and makes it possible to study a small number of embryos. A significantly lower level of basal, but not induced DNA damage was found in embryos with cleaved zona pellucida compared to embryos with intact zona pellucida. There were no significant differences in the length of the comet's tail between embryos lysed in different lysis solutions, both in cases of basal and induced DNA damage. A significant increase in the comet tail length was detected in one-cell embryos of mice treated with methyl methanesulfonate and etoposide compared to the control. The data show that DNA damage induced in maternal germ cells persists, which can be detected in embryos using the comet assay.

Loss of SIRT1 in diabetes accelerates DNA damage-induced vascular calcification.

AimsVascular calcification is a recognized predictor of cardiovascular risk in the diabetic patient, with DNA damage and accelerated senescence linked to oxidative stress-associated pathological calcification. Having previously shown that systemic SIRT1 is reduced in diabetes, the aim was to establish whether SIRT1 is protective against a DNA damage-induced senescent and calcified phenotype in diabetic vascular smooth muscle cells (vSMCs).Methods and resultsImmunohistochemistry revealed decreased SIRT1 and increased DNA damage marker expression in diabetic calcified arteries compared to non-diabetic and non-calcified controls, strengthened by findings that vSMCs isolated from diabetic patients show elevated DNA damage and senescence, assessed by the Comet assay and telomere length. Hyperglycaemic conditions were used and induced DNA damage and enhanced senescence in vSMCs in vitro. Using H2O2 as a model of oxidative stress-induced DNA damage, pharmacological activation of SIRT1 reduced H2O2 DNA damage-induced calcification, prevented not only DNA damage, as shown by reduced comet tail length, but also decreased yH2AX foci formation, and attenuated calcification. While Ataxia Telanglectasia Mutated (ATM) expression was reduced following DNA damage, in contrast, SIRT1 activation significantly increased ATM expression, phosphorylating both MRE11 and NBS1, thus allowing formation of the MRN complex and increasing activation of the DNA repair pathway.ConclusionDNA damage-induced calcification is accelerated within a diabetic environment and can be attenuated in vitro by SIRT1 activation. This occurs through enhancement of the MRN repair complex within vSMCs and has therapeutic potential within the diabetic patient.

Combinational treatment of 5-fluorouracil and casticin induces apoptosis in mouse leukemia WEHI-3 cells in vitro.

Leukemia is one of the major diseases causing cancer-related deaths in the young population, and its cure rate is unsatisfying with side effects on patients. Fluorouracil (5-FU) is currently used as an anticancer drug for leukemia patients. Casticin, a natural polymethoxyflavone, exerts anticancer activity against many human cancer cell lines in vitro, but no other reports show 5-FU combined with casticin increased the mouse leukemia cell apoptosis in vitro. Herein, the antileukemia activity of 5-FU combined with casticin in WEHI-3 mouse leukemia cells was investigated in vitro. Treatment of two-drug combination had a higher decrease in cell viability and a higher increase in apoptotic cell death, the level of DNA condensation, and the length of comet tail than that of 5-FU or casticin treatment alone in WEHI-3 cells. In addition, the two-drug combination has a greater production rate of reactive oxygen species but a lower level of Ca2+ release and mitochondrial membrane potential (ΔΨm ) than that of 5-FU alone. Combined drugs also induced higher caspase-3 and caspase-8 activities than that of casticin alone and higher caspase-9 activity than that of 5-FU or casticin alone at 48 hours treatment. Furthermore, 5-FU combined with casticin has a higher expression of Cu/Zn superoxide dismutase (SOD [Cu/Zn]) and lower catalase than that of 5-FU or casticin treatment alone. The combined treatment has higher levels of Bax, Endo G, and cytochrome C of proapoptotic proteins than that of casticin alone and induced lower levels of B-cell lymphoma 2 (BCL-2) and BCL-X of antiapoptotic proteins than that of 5-FU or casticin only. Furthermore, the combined treatment had a higher expression of cleaved poly (ADP-ribose) polymerase (PARP) than that of casticin only. Based on these findings, we may suggest that 5-FU combined with casticin treatment increased apoptotic cell death in WEHI-3 mouse leukemia cells that may undergo mitochondria and caspases signaling pathways in vitro.

Protective effect of catechin on pentachlorophenol-induced cytotoxicity and genotoxicity in isolated human blood cells.

Pentachlorophenol (PCP) is an organochlorine compound that is used as pesticide, biocide, and wood preservative. PCP is highly toxic and carcinogenic. It has been detected in food and several consumable products. The toxicity of PCP is thought to be due to generation of oxidative stress in cells. We examined whether the dietary antioxidant catechin can attenuate or protect human erythrocytes and lymphocytes against PCP-induced cytotoxicity and genotoxicity, respectively. Human erythrocytes were treated with increasing concentrations of catechin (0.05-2.5mM) for 30min followed by addition of 0.75mM PCP and further incubation for 4h at 37°C. Hemolysates were prepared and assayed for various biochemical parameters. Treatment with PCP alone increased the generation of reactive oxygen and nitrogen species, lipid and protein oxidation, and damaged the plasma membrane, when compared to PCP untreated (control) cells. It significantly decreased glutathione level, total sulfhydryl content, and cellular antioxidant power. PCP treatment lowered the activity of antioxidant enzymes and inhibited enzymes of glucose metabolism. However, prior incubation with catechin attenuated the PCP-induced changes in all these parameters in a catechin concentration-dependent manner. Scanning electron microscopy of erythrocytes confirmed these biochemical results. PCP treatment converted the normal discoidal erythrocytes to irregularly contracted cells, acanthocytes, and echinocytes but the presence of catechin inhibited these morphological changes and erythrocytes retained their biconcave shape to a large extent. Genotoxicity was studied in human lymphocytes by single-cell gel electrophoresis (comet assay). It showed strand breaks and longer comet tail length in PCP alone treated cells. The comet tail length was reduced in the catechin +PCP-treated lymphocytes showing that catechin protected cells from PCP-induced DNA damage. These results show that catechin protects human blood cells against PCP-induced oxidative damage.

Effects of Bisphenol A on Foxl2 gene expression and DNA damage in adult viviparous fish Goodea atripinnis

ABSTRACTBisphenol A (BPA) is an emerging pollutant of global concern. Viviparous fish Goodea atripinnis is endemic to the Central Mexican Plateau where BPA was detected; however, few studies examined the influence of this chemical on native viviparous fish. The effects of BPA (sublethal dose) were determined on DNA integrity and Foxl2 expression in G. atripinnis gonads, and interactions of BPA with FOXL2 protein. Genotoxicity analysis revealed that % comets, at 14 and 28 days and comet tail length (at 14 days) were significantly higher in exposed compared to controls. In general, the % DNA tail was not markedly higher in BPA-treated fish; however, tail moment related to tail length exhibited significant increases in DNA damage. RT-qPCR assays showed Foxl2 overexpression after 14 and 28 days of exposure in females; while in males, Foxl2 was overexpressed after 28 days. In silico analysis demonstrated that BPA interacted with seven residues located in FOXL2 homeodomain. In summary, sublethal BPA doses induced DNA damage and changes in Foxl2 expression in gonadal cells of G. atripinnis, which may adversely affect reproduction in BPA-exposed wild populations. Foxl2 overexpression and BPA-FOXL2 interaction suggested alterations in processes involving Foxl2. Viviparous fish may thus serve as potential non-conventional models for assessing pollutants effects.

Alkaline Comet Assay as a Predictor of DNA Damage in Medical Radiation Workers

The study was aimed to examine the deoxyribonucleic acid (DNA) damage level between medical and non-medical radiation workers using the comet assay method. The radiation-exposed group (29 subjects) and the control group (29 subjects) were chosen from the administration staff. The assessment of single-strand DNA damage hosted by peripheral blood lymphocyte was performed using the alkaline comet assay, the tail length and long-tailed DNA were measured to detect the DNA damage. An independent sample t-test was used to assess the statistical difference between groups. The results showed that comet tail length mean measured in the exposed worker’s group was 25.57±4.4 μm whereas the mean percentage of long-tailed DNA was 6.97±1.53. In the control group, the mean tail length was 23.15±7.57 μm and the long-tailed nucleus percentage was 5.65±1.99. Mean values for the tail length measured were no significantly higher in the exposed group compared to the control group (P=0.15), whereas the percentage of long-tailed nuclei was significantly higher in the exposed group compared to the control group (P=0.007) . The observed single-strand DNA can be measured by using the alkaline comet assay. The DNA damage can be influenced by radiation exposure in medical radiation workers.

Nuclear Damage in Peripheral Erythrocytes of Cyprinus Carpio Exposed to Binary Mixture of Pesticides

The present study was undertaken to examine the DNA damage in peripheral blood erythrocytes of Cyprinus carpio under the binary exposure of bifenthrin and chlorpyrifos by using single cell gel electrophoresis (SCGE). Limited efforts have been made to study the genotoxic effect for long duration period. Therefore, the present investigation was aimed to assess the genotoxicity of pesticide mixture to the freshwater carp, Cyprinus carpio at sub-lethal concentration exposure (33% LC50). At first 96-hr LC50 value of pesticide, the mixture was determined for Cyprinus carpio in a static system and then sub-lethal concentration was calculated and fish was exposed to this sub-lethal concentration of the mixture in glass aquaria for 70 days (five fortnights) at constant laboratory conditions. Peripheral blood erythrocytes were taken on a fortnightly basis for the time-dependent DNA damage assessment in-terms of percentage of damaged cells, genetic damage index and a cumulative tail length of comets. Concentration-dependent increase in the percentage of DNA damaged cells were observed up to a 4th fortnight, followed by a slight decrease in the 5th fortnight. Similarly, statistically significant time-dependent DNA damage was observed in terms of percentage of damaged cells, genetic damage index and a cumulative tail length of comets in treated fish (at 33% of LC50) as compared to control groups. The results supported the use of SCGE for evaluating the toxicity of pollutants which may be used as part of environmental monitoring programs.

Ameliorating potency of Chenopodium album Linn. and vitamin C against mercuric chloride-induced oxidative stress in testes of Sprague Dawley rats

BackgroundMercury has been documented as an industrial risk that posed a serious danger to human health. Mercury exposure results in oxidative stress that may lead to the pathogenesis of male reproductive dysfunction. The present study investigated the ameliorating potential of Chenopodium album L. and vitamin C against mercuric chloride-induced oxidative deterioration of reproductive functions in adult male rats.MethodsGroup 1 (control) received saline. Group 2 received Mercury (0.15 mg/kg b.w, i.p) dissolved in distilled water. Groups 3 and 4 were given oral gavage of vitamin C (200 mg/kg b.w) and the ethanolic extract of C. album (200 mg/kg b.w) respectively, along with Mercury (0.15 mg/kg b.w, i.p). Group 5 was treated only with C. album (200 mg/kg b.w). After 30 days of the treatment, the rats were dissected and their testicular tissue and the cauda epididymis were used for biochemical analysis while blood plasma was used for protein determination.ResultsThe applied dose-treatment of Mercury-induced oxidative stress in the testis and cauda epididymis tissues of the rats was apparent by a noteworthy decrease in total protein, CAT, SOD, POD, and GST values while there was increase in ROS and TBARS levels. Furthermore, Mercury decreases daily sperm production and enhanced sperm DNA damage as noticeable by an increase in the head and tail length of comets and decrease in intact DNA. There was no significant effect on the body weight and the weight of the reproductive tissues. Treatment with C. album significantly ameliorated the total protein, ROS, and TBARS content. Similarly, the level of CAT, SOD, POD, and GST was significantly improved and the daily sperm production was significantly increased. Furthermore, C. album administration significantly protected Mercury-induced sperm DNA damage. The results of the extract treatment group were compared with those of vitamin C in detoxifying the oxidative stress and restoring the sperm parameters.ConclusionC. album showed protection against Mercury-induced oxidative stress by ameliorating antioxidant enzyme activity, daily sperm production, and DNA damage in rat testes. This suggests that C. album could be beneficial against toxicity induced by an environmental toxicant.

Targeting DNA Damage and Repair in Acute Myeloid Leukemia Carrying Internal Tandem Duplication of Fms-like Tyrosine Kinase 3 (FLT3-ITD) - a Mechanistic Study

Objective Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) is one of the most common mutations in acute myeloid leukemia (AML), occurring in nearly 30% of cases. FLT3-ITD involves in-frame duplication of 3-400 base-pairs at the juxta-membrane domain, resulting in ligand-independent activation of FLT3 signaling. Downstream effectors include activation of ERK/STAT5 via SRC kinase, activation of PI3K/AKT, phosphorylation of FOXO3A, down-regulation of equilibrative nucleoside transporter 1 (ENT1) for cytarabine, and induction of reactive oxygen species (ROS) that may lead to increased DNA damage and defective repair. The present study investigated if the latter can be effectively targeted for the treatment of this AML subtype. Methods Primary samples from patients with FLT3-ITD AML, human cell line carrying FLT3-ITD (MOLM-13) as well as mouse B-lymphoid Ba/F3 cells transduced with human FLT3-ITD were used in this study. Traffic Light Reporter (TLR) assay was used to measure fidelity of double-strand breaks (DSB) repair, either via error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Percentage of repair events by HR or NHEJ were quantified by flow cytometry. DNA DSB were examined by γ-H2AX foci staining using confocal microscopy. Single-cell DSB were quantified by neutral comet assay and analysed by OpenComet software. The tail moment was calculated as the length of comet tail multiplied by the tail DNA %. MOLM-13 was transplanted into NOD/SCID/IL2Rg-/- (NSG) mice by tail vein injection. Treatment comprised cytarabine (25mg/kg, i.p., day 1-5) and doxorubicin (1.5mg/kg, i.v., day 1-3), with or without olaparib (25mg/kg, i.p., day 1-5). Comparisons between groups of numerical data were evaluated using Student's t-test. P-values less than 0.05 were considered statistically significant. Results To investigate the link between FLT3-ITD AML and DNA damage response (DDR), expression of critical DDR genes in primary AML samples was examined by real-time quantitative PCR. The panel of genes included apical kinase ATM, ATR and DNA-PKcs; DNA damage mediators BRCA1,BRCA2 and PARP1; downstream response kinase CHEK1 and CHEK2 and effectors TP53. Among them, BRCA2 was significantly down-regulated in FLT3-ITD AML(Wild-type FLT3=18 samples; FLT3-ITD=13 samples; Average dCt 9.7 in WT vs. 10.6 in ITD; p&lt;0.05). Down-regulation of BRCA2 in FLT3-ITD AML was further validated in a microarray database GSE15434 from a multicenter study investigating gene expression profiles of normal karyotype AML( FLT3-WT=148; FL3-ITD=86; ; log2 gene expression 5.61 in WT vs 5.45 in ITD; p&lt;0.001). As BRCA2 is an important protein mediating HR, a DSB DNA repair TLR assay was performed. HR was significantly down-regulated in Ba/F3-FLT3-ITD as compared with parental Ba/F3 line by flow cytometry(1.21% HR repair in control vs. 0.44% in ITD; p&lt;0.05) while NHEJ was unaffected (1.90% NHEJ repair in control vs. 2.38% in ITD; p=0.50). Down-regulation of BRCA2 expression and defective HR in FLT3-ITD AML was reminiscent of BRCA mutant breast and ovarian cancers. Therefore, the effects of poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib were examined. Ba/F3-FLT3-ITD were more sensitive to olaparib than parental line. Olaparib inhibited base excision repair and increased DSB as indicated by increased number of γ-H2AX foci and increased tail moment in Ba/F3-FLT3-ITD cells when compared to parental line. Olaparib-induced DSB in Ba/F3-FLT3-ITD cells was mainly repaired by NHEJ as shown by colocalization of γ-H2AX foci with 53BP1. Combination of chemotherapy (cytarabine and doxorubicin) and olaparib synergistically reduced leukemic cell growth of MOLM-13 in NSG murine xenograft model. Conclusion Results from the present study supported the use of PARPi in the treatment of FLT3-ITD AML. Its therapeutic benefits in combination with chemotherapy would have to be further evaluated. Acknowledgements: Health and Medical Research Fund (Project number: 04152326) and Li Shu Fan Medical Foundation, LKS Faculty of Medicine, University of Hong Kong. Disclosures No relevant conflicts of interest to declare.

Usnic acid induces apoptosis in human gastric cancer cells through ROS generation and DNA damage and causes up-regulation of DNA-PKcs and γ-H2A.X phosphorylation

Usnic acid, a dibenzofuran derivative found in many lichen species, is reported to have anticancer activity against human gastric cancer. We investigated the molecular alterations associated with anticancer effects of usnic acid against human gastric adenocarcinoma AGS and gastric carcinoma SNU-1 cells. Usnic acid (10–25 μM) treatment to these cells caused a significant increase in mitochondrial membrane depolarization and apoptotic cells. Apoptosis induction was accompanied by an increase in the ratio of Bax:Bcl-2 expression and cleaved-PARP. Usnic acid increased the comet tail length and tail DNA in alkaline comet assay indicating DNA double-strand breaks which was also evidenced by an increase in γH2A.X (Ser139) phosphorylation. The expression of DNA damage response proteins including DNA-PKcs, pATM (Ser1981), Chk-2 and p53 were increased. Further, N-acetyl cysteine, a known reactive oxygen species (ROS) scavenger, reversed the effects of usnic acid on expression of DNA damage response proteins and γH2A.X (Ser139) phosphorylation. This reversal was also observed in comet assay in a time and dose-dependent manner suggesting that usnic acid-induced DNA damage was caused by ROS. In addition, the non-toxic concentrations (1–10 μM) of usnic acid inhibited colony forming potential of AGS cells indicating its anti-proliferation activity. More importantly, the concentration of usnic acid that caused significant death in gastric cancer cells, did not show any considerable toxicity to normal human embryonic kidney HEK293 cells, human keratinocyte HaCaT cells and mouse primary gastric cells. Collectively, these results for the first time demonstrated the selective apoptotic effect of usnic acid (10–25 μM) through ROS generation and DNA damage on human gastric cancer cells accompanied with upregulation of γH2A.X (Ser139) phosphorylation, DNA-PKcs and p53.

Clinico-hematological, serum biochemical, genotoxic and histopathological effects of trichlorfon in adult cockerels

Trichlorfon, an organophosphorus insecticide, is commonly used in different food crops, public health management, and in veterinary practice to control pests and parasites. Scanty information is available about the mechanisms of trichlorfon toxicity in target and non-target organisms. Therefore, in this study, adult male cockerels were exposed to different concentrations of trichlorfon for 45 days to evaluate its possible toxic effects. Four cockerels were randomly killed from each group at days 15, 30, and 45 for collection of blood and different visceral tissues. After different concentrations of trichlorfon treatments (16 and 20 mg/kg body weight), a dose- and time-dependent significant decrease in feed intake and body mass was recorded. The different blood parameters including red blood cells count, hemoglobin quantity, and hematocrit percent were significantly decreased in comparison with that of control cockerels. Results indicated that trichlorfon elevated the concentration of different liver function tests (ALT, AST, and ALP), kidney function tests (urea and creatinine) and cardiac biomarkers (Cholesterol, CK-Mb, and triglycerides). Results showed that trichlorfon causes higher frequency of formation of micronuclei and erythrocyte with condensed nuclei in exposed cockerels. Moreover, trichlorfon increases the percentile of DNA damage and comet tail length of isolated blood lymphocytes. Histological observation of testicular tissues of trichlorfon-treated cockerels exhibited presence of necrotic spermatids and arrest of process of spermatogenesis, while liver tissues showed congestion, pyknosis, fragmentation, and disintegration of nucleus of hepatocytes. Histopathological analysis of kidneys of trichlorfon-treated cockerels exhibited degeneration of glomeruli, necrosis of tubular epithelium and presence of necrotic epithelial cells in lumen of renal tubules. In conclusion, is reported that trichlorfon causes deleterious effects on clinico-hematological, biochemical, genotoxic, and histopathological parameters in adult cockerels.

DNA damage and adverse neurological outcomes among garlic farmers exposed to organophosphate pesticides

Garlic farmers used organophosphate pesticides to control insects, resulting in an increased risk for adverse health outcomes. Thus, we investigated exposure to organophosphate pesticides, DNA damage, nerve conduction, and neurological symptoms among 134 garlic farmers. They were interviewed, measured nerve conduction, and collected blood and urine for determining DNA damage and dialkylphosphate metabolites. Total dialkylphosphate levels of farmers who began cultivating garlic in October were significantly higher than those of non-farmers and the farmers who began cultivating in November and December. Farmers showed significantly longer comet tail lengths and tail moments than non-farmers. However, their mean compound motor and sensory nerve action potential amplitudes of the median, ulnar, and common peroneal nerves were within normal ranges. Measurement of DNA damage is useful as a biomarker of long-term and low-level exposure to organophosphate pesticides; however, electromyography might be not sufficiently sensitive to detect nerve conduction effects in farmers exposed to pesticides.

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.

Effect of MMP-2 gene silencing on radiation-induced DNA damage in human normal dermal fibroblasts and breast cancer cells

IntroductionDiagnostic and therapeutic ionizing radiation (IR) is one of the well known long term risk factors of breast cancer. Extremely lethal consequences of IR causes double-strand breaks, which are mainly responsible for genomic instability, altered gene expression, and cell death.FindingsThis study evaluated the effect of matrix metalloproteinases-2 (MMP-2) gene silencing using MMP-2 shRNA expression plasmids (pMMP-2) on IR induced cytotoxicity and DNA damage by MTT, dead green, γH2AX and comet assays in human normal dermal fibroblasts (HDFs) and MCF-7 human breast cancer cells. IR has decreased the viability of HDFs and MCF-7 cells with increasing IR (2-10Gy). IR induced DNA damage in both HDFs and MCF-7 cells. However, pMMP-2 transfection has increased the viability of irradiated HDFs (10Gy) and significantly decreased the viability of irradiated MCF-7 cells (10Gy). Further, DNA damage in terms of γH2AX foci decreased with pMMP-2 transfection in irradiated HDFs (10Gy) and increased in irradiated MCF-7 cells (10Gy). In addition, MMP-2 gene silencing using pMMP-2 decreased comet tail length in irradiated HDFs but increased in irradiated MCF-7 cells.ConclusionsThe results conclude that pMMP-2 has protected HDFs and sensitized the MCF-7 cells from IR induced DNA damage. This differential response might be due to IR induced MMP-2 distinctive ROS generation in HDFs and MCF-7 cells.