DNA Comet Assay Research Articles

Effect of CAT gene polymorphism on sensitivity of human lymphocytes to genotoxic effect of organochlorine pesticide in vitro

Introduction. Over recent decades, toxicogenetic studies have focused on the issues of genome instability under the action of genotoxicants, taking into account biomarkers of sensitivity. The question about the genotoxic potential of chlorpyrifos remains open, since both positive and negative effects have been revealed in various tests. The aim of the study is the investigation of sensitivity of donor peripheral blood lymphocytes to chlorpyrifos in vitro and evaluation of the contribution of polymorphism of antioxidant defense system genes (CAT (rs1001179), SOD2 (rs4880)) to the response of human cells to the action of genotoxicant. Materials and methods. The DNA damaging effect of chlorpyrifos was assessed on lymphocytes from fifty two donors using DNA-comet assay with metabolic activation (+S9) and without it (–S9). The study of cytotoxic effects of chlorpyrifos on human lymphocytes was carried out using an automatic fluorescent cell analyzer ADAMII LS. Results. Chlorpyrifos had a pronounced cytotoxic effect on lymphocytes in most donors in the absence of metabolic activation system. With increasing concentration of the pesticide in the medium and time of cultivation, the viability of lymphocytes decreased, and the proportion of cells in late apoptosis and necrosis increased. Positive genotoxic effects were found on the cells of 33 donors (-S9). In the presence of the S9, mild but statistically significant effects were detected only on cells from 2 donors. % DNA values in the comet tail after exposure to the pesticide varied for cells from different donors. In the absence of metabolic activation, a statistically significant increase in the level of DNA damage was found in cells of individuals with genotype AA (homozygote for the minor allele) for the CAT G262A catalase gene (rs1001179), compared with homozygote for the dominant GG allele. Limitations. The genotoxicity of chlorpyryfos was studied in vitro. Conclusion. The results of the study shown cytotoxic and genotoxic effects of chlorpyrifos. The sensitivity of lymphocytes from different donors to the pesticide was found to be significantly different. The association of the level of DNA damage under exposure of chlorpyrifos in vitro with the G262A polymorphism of the catalase gene was found. The research also confirms the possibility of using a model test-system with peripheral blood lymphocytes to assess the potential genetic risk for humans and to study the contribution of gene polymorphism to individual sensitivity to the action of genotoxicants.

Analysis of the Restoration of DNA Integrity in S-Phase and non-S-Phase Cells after the Treatment with Methyl Methanesulfonate by DNA Comet Assay

Analysis of the Restoration of DNA Integrity in S-Phase and non-S-Phase Cells after the Treatment with Methyl Methanesulfonate by DNA Comet Assay

Evaluation of the Effect of Dibornol on the Level of DNA Damage in the DNA Comet Test In Vivo.

In male C57BL/6 mice (8-12 weeks) and male Wistar rats (12 weeks), the effect of dibornol (2,6-diisobornyl-4-methylphenol) on the level of spontaneous DNA damage in cells of the bone marrow, liver, kidneys, and rectum of mice (series I) and genotoxic effects in rat testicular cells after administration of cytostatic drugs with different mechanisms of action (series II) were studied using the DNA comet assay. In series I, dibornol was intragastrically administered to mice once at doses of 200, 400, and 2000 mg/kg; in series II, dibornol was intragastrically administered to rats at a dose of 10 mg/kg for 5 days before and 5 days after the cytostatic treatment (methotrexate, doxorubicin). It was found that dibornol in all studied doses did not produce the genotoxic (carcinogenic) effect and reduced the level of spontaneous DNA damage in the bone marrow. After combined administration of cytostatic drugs (doxorubicin, methotrexate) and dibornol, the level of DNA breaks was reduced to 38.5 and 49% of the control, respectively.

Labilization of the DNA structure in peripheral blood lymphocytes of COVID-19 patients

Introduction. Available data indicate the SARS-CoV-2 coronavirus to be potent of impairing DNA repair processes and cause oxidative stress, which can lead to the accumulation of DNA damage in human cells. However, the DNA-damaging effect of the virus has not yet been sufficiently studied. The purpose of the research was to study the ability of SARS-CoV-2 to cause DNA damage in human peripheral blood lymphocytes. Materials and methods. One hundred forty COVID-19 patients and 24 donors of the control group are included in the study. The level of DNA fragmentation in lymphocytes was determined by alkaline DNA-comet assay. Statistical differences between the mean medians of the «%DNA in the comet tail» (tail DNA%) were assessed using Student’s t-test. The Jeffers test was used to compare the proportions of cells with different levels of DNA-damage. Statistical differences between groups were assessed using the Mann-Whitney test. Results. In the COVID-19 patients, an increase in the level of breaks and alkali-labile sites in DNA was revealed when compared to controls (p = 0.025). In the group of patients infected with SARS-CoV-2, the proportion of comets with DNA damage of up to 5% decreased (p = 0.009), while the proportion of comets containing more than 10% DNA tail increased (p = 0.000). The number of atypical comets compared to the control increased by 3.7 and 5.9 times with mild and moderate severity of the disease, respectively (r = 0.993; p = 0.001). In the association with diseases – coronary heart disease (CHD) and diabetes mellitus type II (DM type 2), the level of DNA fragmentation in lymphocytes statistically significantly increased compared to the group of patients without these diseases. Limitations. A limitation is the lack of data on DNA-structure damage in severe COVID-19 disease. Conclusion. SARS-CoV-2 infection leads to labilization of the DNA structure in human peripheral blood lymphocytes. The level of DNA damage depends on the severity of COVID-19 and the presence of comorbid diseases: CHD and DM type 2. The results of the study are important for understanding the mechanisms of action of the virus on human immunocompetent cells.

Various investigations of ameliorative role of Ashwagandha seeds (Withania somnifera) against amoxicillin toxicity.

Several studies showed the adverse effects of amoxicillin on various body organs. So, this research has been designed to evaluate the modulatory role of Ashwagandha seed extract (ASE) against amoxicillin (AM) toxicity. Rats treated with AM (90mg/kg), protected by ASE doses (100, 200 and 300mg/kg), and treated by ASE at the same three doses. At the end of the experimental period, DNA comet assay, cytogenetic examinations, sperm-shape analysis, evaluation of the malondialdehyde (MDA) percentages, histopathological examinations, and biophysical tests (modulus, relaxation time, permittivity, entropy, and internal energy change of brain) were documented. The results confirmed that AM treatment induced significant elevation of DNA damage, cytogenetic aberrations, and MDA content in brain, liver, and testis tissues and sperm-shape anomalies. ASE treatment significantly minimized the genetic changes, sperm-shape anomalies, and MDA generation. These enhancements were more pronounced by protective ASE and increased by increasing the dose level. In histopathological examinations, AM treatment caused neurotoxicity in brain tissue. ASE treatment, partially, minimized these damages and the positive effects of therapeutic ASE were more noticeable. Biophysical parameters showed that therapeutic ASE was better for relaxation time, permittivity, and free energy change. Protective and therapeutic ASE were able to recover entropy and internal energy changes in variant degrees.

Host-parasite interaction in severe and uncomplicated malaria infection in ghanaian children.

During malarial infection, both parasites and host red blood cells (RBCs) come under severe oxidative stress due to the production of free radicals. The host system responds in protecting the RBCs against the oxidative damage caused by these free radicals by producing antioxidants. In this study, we investigated the antioxidant enzyme; superoxide dismutase (SOD) activity and cytokine interactions with parasitaemia in Ghanaian children with severe and uncomplicated malaria. One hundred and fifty participants aged 0-12 years were administered with structured questionnaires. Active case finding approach was used in participating hospitals to identify and interview cases before treatment was applied. Blood samples were taken from each participant and used to quantify malaria parasitaemia, measure haematological parameters and SOD activity. Cytokine levels were measured by commercial ELISA kits. DNA comet assay was used to evaluate the extent of parasite DNA damage due to oxidative stress. Seventy - Nine (79) and Twenty- Six (26) participants who were positive with malaria parasites were categorized as severe (56.75 × 103 ± 57.69 parasites/µl) and uncomplicated malaria (5.87 × 103 ± 2.87 parasites/µl) respectively, showing significant difference in parasitaemia (p < 0.0001). Significant negative correlation was found between parasitaemia and SOD activity levels among severe malaria study participants (p = 0.0428). Difference in cytokine levels (IL-10) amongst the control, uncomplicated and severe malaria groups was significant (p < 0.0001). The IFN-γ/IL-10 /TNF-α/IL-10 ratio differed significantly between the malaria infected and non- malaria infected study participants. DNA comet assay revealed damage to Plasmodium parasite DNA. Critical roles played by SOD activity and cytokines as anti-parasitic defense during P. falciparum malaria infection in children were established.

Amelioration of gold nanoparticles mediated through Ocimum oil extracts induces reactive oxygen species and mitochondrial instability against MCF-7 breast carcinoma.

Phytomedicines are potential immunity-boosting components with effective anticystic properties, minimal side effects, and biomedical applications, making them valuable for combating various diseases. India is renowned globally for Ayurveda, an ancient treatment methodology known for its holistic approach in identifying the root cause of diseases. Tulsi (Ocimum sanctum) is a common household medicine in India. While essential oils from plants like Tulsi have long been recognized for their medicinal properties, there is a gap in understanding their potential in synthesizing gold nanoparticles (AuNPs) and their efficacy against breast carcinoma, particularly in the context of immunosuppressive conditions. We investigated the potential application of essential oils isolated from O. sanctum in the synthesis of AuNPs and their efficacy against MCF-7 breast carcinoma. Gas chromatography-mass spectroscopy identified compounds with potential anticancer effects against breast cancer cells. Synthesised AuNPs displayed high hemocompatibility and antimicrobial activity against nosocomial Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, and Bacillus subtilis strains. Os-AuNPs induced chromosomal instability and mitotic arrest in the G2/M cell cycle phase. Subsequent fluorescence and cell cytometry studies demonstrated the systemic release of ROS, depolarisation of mitochondrial membrane potential, and production of apoptotic bodies. DNA damage and comet assays confirmed the anticancer potential of synthesised AuNPs. This study illuminates the potential of O. sanctum-derived AuNPs in breast carcinoma treatment, paving the way for future AuNP-based therapies in biomedicine.

Mechanisms related to carbon nanotubes genotoxicity in human cell lines of respiratory origin

Potential genotoxicity and carcinogenicity of carbon nanotubes (CNT), as well as the underlying mechanisms, remains a pressing topic. The study aimed to evaluate and compare the genotoxic effect and mechanisms of DNA damage under exposure to different types of CNT.Immortalized human cell lines of respiratory origin BEAS-2B, A549, MRC5-SV40 were exposed to three types of CNT: MWCNT Taunit-M, pristine and purified SWCNT TUBALL™ at concentrations in the range of 0.0006–200 μg/ml. Data on the CNT content in the workplace air were used to calculate the lower concentration limit. The genotoxic potential of CNTs was investigated at non-cytotoxic concentrations using a DNA comet assay. We explored reactive oxygen species (ROS) formation, direct genetic material damage, and expression of a profibrotic factor TGFB1 as mechanisms related to genotoxicity upon CNT exposure.An increase in the number of unstable DNA regions was observed at a subtoxic concentration of CNT (20 μg/ml), with no genotoxic effects at concentrations corresponding to industrial exposures being found.While the three test articles of CNTs exhibited comparable genotoxic potential, their mechanisms appeared to differ. MWCNTs were found to penetrate the nucleus of respiratory cells, potentially interacting directly with genetic material, as well as to enhance ROS production and TGFB1 gene expression. For A549 and MRC5-SV40, genotoxicity depended mainly on MWCNT concentration, while for BEAS-2B – on ROS production. Mechanisms of SWCNT genotoxicity were not so obvious. Oxidative stress and increased expression of profibrotic factors could not fully explain DNA damage under SWCNT exposure, and other mechanisms might be involved.

Investigation on photo-isomeric impurity of Roxadustat: Structure conformation, physicochemical characterization, interconversion feasibility and in vitro toxicological evaluation

This research article describes the comprehensive characterization and toxicological activities of a photo-isomeric impurity (PI) of an antianemic drug Roxadustat (ROX). The PI, which has the same molecular mass as that of the drug, possesses strikingly distinct structural features compared to the drug. A detailed investigation on structural as well as toxicological aspects of this impurity is warranted due to likelihood of formation of this impurity because of routine light exposure of the API, and due to alerts concerning to its carcinogenicity, mutagenicity, and teratogenicity based on in silico assessments reported in the literature. The molecular structure of the PI was confirmed by single crystal XRD providing the first evidence for the unambiguous structure determination. Complete physicochemical characterization of the impurity was carried out using powder X-ray diffraction, Fourier Transform Infrared Spectroscopy, Differential Scanning Calorimetry, and Thermogravimetric Analysis establishing phase identification and phase purity of the impurity. The feasibility of interconversion of API (ROX) to the impurity PI and vice versa was evaluated using the RP-HPLC and Triple Quadrupole LC-MS techniques for both the solution state as well as solid state. The in vitro toxicological activity of the PI was assessed through cytotoxicity assays, DNA fragmentation assays, morphological analysis, and COMET assay. These structural, synthetic and toxicological studies contribute to the overall understanding of drug stability, safety, and pharmaceutical formulation development of ROX.

Synergistic antitumor efficacy of rMV-Hu191 and Olaparib in pancreatic cancer by generating oxidative DNA damage and ROS-dependent apoptosis

BackgroundMalignancies with BRCA1/2 deficiencies are particularly sensitive to PARP inhibitors. Thus, combining PARP inhibitors with agents that impair DNA damage repair to treat BRCA1/2 wild-type PDAC could broaden the clinical use of these promising PARP inhibitors. Here we examined the synergism and mechanism of oncolytic measles virus (rMV-Hu191) with a PARP inhibitor (Olaparib) in vitro and in vivo. MethodsThe cell viability assay, cell cycle analysis, colony formation assay, TCID 50 method, western blotting, flow cytometry, DNA comet assay, Mice bearing PDAC xenografts, IF, IHC and TUNEL assay were performed to explore the antitumor efficacy and underlying mechanisms. ResultsIn this study, we explored the antitumor activities of rMV-Hu191 and Olaparib in two PDAC cell lines harboring wild-type BRCA1/2 genes. Compared to monotherapy, the combination of rMV-Hu191 and Olaparib was able to synergistically cause growth arrest, apoptotic cell death and DNA damage, accompanying with excessive oxidative stress. Mechanistically, the data indicated that the observed synergy depended on the oxidative DNA damage and ROS-dependent apoptosis generating by rMV-Hu191 combined with Olaparib in human PDAC cells. Tumor inhibition and prolonged survival of PDAC mice xenografts in vivo confirmed the synergism of combinational treatment with trivial side-effects. ConclusionsOur findings firstly suggested that combination treatment with rMV-Hu191 and Olaparib had a profound and synergistic therapeutic effect against human PDAC through synthetic lethality. In conclusion, we recommend combining oncolytic rMV-Hu191 with a PARP inhibitor (Olaparib) as a novel therapeutic strategy and provided a potential mechanism for advanced PDAC regardless of BRCA mutation status.

STUDY OF CYTOCOMPATIBILITY OF DIFFERENT SUPPLEMENTS IN GEL COMPOSITIONS USED FOR THE TREATMENT OF PERIODONTAL DISEASES IN ORTHODONTIC PATIENTS VIA EXPLORATION OF MECHANISMS OF THEIR CYTOPATHIC AND CYTOPROTECTIVE EFFECTS IN CULTURED HUMAN AND MOUSE CELLS

Introduction. A search continues for effective means which may reduce the overload of harmful factors, eliminate the inflammatory process, and reduce stress on the periodontal tissues during the active period of orthodontic treatment. We developed and patented the gel composition (GC) Benzidaflaziverdine prepared based on Proteflazid® (flavonoids) and benzydamine hydrochloride (BH) T-Sept® for the local treatment of the periodontal tissues in the form of a periodontal dressing in the orthodontic patients.&#x0D; The aim of this study was to evaluate the cytocompatibility of different combinations of components in gel composition based on flavonoid complex and benzydamine hydrochloride (Benzidaflaziverdine) used for the treatment of periodontal diseases in orthodontic patients. For this, mechanisms of their cytopathic and cytoprotective effects will be explored using cultured human and mouse cells.&#x0D; Methods. We studied the effect of different supplements used in GC Benzidaflaziverdine on the viability of pseudonormal human keratinocytes of the HaCaT line and mouse fibroblasts of the BALB-3T3 line, and mouse macrophages of the J774.2 line. Various methods of cell survival assessment were used: MTT-assay, staining of cells with fluorescent dyes Hoechst 33342 and Propidium iodide (PI), as well as a test for the genotoxic effects on cells (DNA comet assay). The antioxidant properties of the developed GC variants were evaluated using DPPH (1,1-diphenyl-2-picrylhydrazyl), Merck (Dam-stadt, Germany), and DCFDA-H2 (2’,7’-dichlorodihydrofluorescein diacetate).&#x0D; Results. We demonstrated that the Sample containing gel base and BH in the form of a solution (Tantum Verde®) possessed weak prooxidant properties. While the Sample contained gel base, powdered BH (T-Sept®) and Sample containing gel base and powdered BH (T-Sept® and Proteflazid®) possessed pronounced antioxidant properties.&#x0D; Conclusions. Tests with DPPH and DCFDA dyes were used to confirm the hypothesis regarding the cytoprotective effect of the patented gel composition Benzidaflaziverdine for local application in the form of a periodontal bandage due to the antioxidant activity of the flavonoid complex, which reaches the maximum level at the 2nd hour of exposure. This gel composition can be recommended for use in clinical periodontology for medical support of orthodontic patients before and during the active phase of orthodontic treatment.

Validation of Simple In vitro alkaline DNA Comet assay for detection and evaluation of Genotoxicity and Biosafety of inactivated veterinary vaccines on Peripheral blood cell lymphocytes

Validation of Simple In vitro alkaline DNA Comet assay for detection and evaluation of Genotoxicity and Biosafety of inactivated veterinary vaccines on Peripheral blood cell lymphocytes

Assessment of the effect of cell transplantation on DNA repair in rat hepatocytes exposed to carbon tetrachloride (CCl4) by DNA comet assay

Introduction. The effect of carbon tetrachloride (freon-10, asordin, hladon-10) is an organochlorine compound with the chemical formula CCl4 and the subsequent transplantation of fetal liver cells (FLC) on DNA degradation and repair in rat hepatocytes by means of alkaline single-cell gel electrophoresis (DNA comet assay) was assessed. Material and methods. Acute toxic damage to the rat liver was simulated by a single oral administration to female Wistar rats of CCl4 in an oil solution at a dose of 3000 mg/kg. As a protective agent, a suspension of FLC of E19 rat fetuses was used. Quantitative assessment of the degree of damage to the nuclear DNA of liver cells was performed by DNA comet assay on days 1, 3, 5, 7 and 16 of the experiment. Results. Intravenous injections of fetal liver cells 6 h after exposure to CCl4 induces DNA repair processes in rat hepatocytes in 5-7 days and led to a decrease in the intensity of nuclear DNA damage. The trend toward a decrease in the number of undamaged hepatocytes continued on the 16th day of the experiment, and, therewith, the enhancement of reparative processes after FLC injection revealed itself in in a significant decrease in the number of hepatocytes with a high intensity of nuclear DNA damage. Limitations. To prevent unwanted death of animals in the group, studies were limited to a dose of 3000 mg/kg of CCl4 in oil solution. Conclusion. The method of alkaline single-cell gel electrophoresis (DNA comet assay) allowed quantitative assessment of the degrees of genome damage and repair. The obtained positive results suggest that FLC exert a protective effect of the structure of the DNA of rat liver role after acute exposure to CCl4.

Combined Radiomodifying Effect of Fucoidan from the Brown Alga Saccharina cichorioides and Pacificusoside D from the Starfish Solaster pacificus in the Model of 3D Melanoma Cells.

Cancer is one of the main causes of human mortality worldwide. Despite the advances in the diagnostics, surgery, radiotherapy, and chemotherapy, the search for more effective treatment regimens and drug combinations are relevant. This work aimed to assess the radiomodifying effect and molecular mechanism of action of fucoidan from the brown alga Saccharina cichorioides (ScF) and product of its autohydrolysis (ScF_AH) in combination with pacificusoside D from the starfish Solaster pacificus (SpD) on the model of viability and invasion of three-dimension (3D) human melanoma cells SK-MEL-2. The cytotoxicity of ScF (IC50 JB6 Cl41 > 800 µg/mL; IC50 SK-MEL-2 = 685.7 µg/mL), ScF_AH (IC50 JB6 Cl41/SK-MEL-2 > 800 µg/mL), SpD (IC50 JB6 Cl41 = 22 µM; IC50 SK-MEL-2 = 5.5 µM), and X-ray (ID50 JB6 Cl41 = 11.7 Gy; ID50 SK-MEL-2 = 6.7 Gy) was determined using MTS assay. The efficiency of two-component treatment of 3D SK-MEL-2 cells was revealed for ScF in combination with SpD or X-ray but not for the combination of fucoidan derivative ScF_AH with SpD or X-ray. The pre-treatment of spheroids with ScF, followed by cell irradiation with X-ray and treatment with SpD (three-component treatment) at low non-toxic concentrations, led to significant inhibition of the spheroids' viability and invasion and appeared to be the most effective therapeutic scheme for SK-MEL-2 cells. The molecular mechanism of radiomodifying effect of ScF with SpD was associated with the activation of the initiator and effector caspases, which in turn caused the DNA degradation in SK-MEL-2 cells as determined by the Western blotting and DNA comet assays. Thus, the combination of fucoidan from brown algae and triterpene glycoside from starfish with radiotherapy might contribute to the development of highly effective method for melanoma therapy.

Estimation of the applied doses in irradiated anchovy and bluefish for shelf-life extension using image analysis in combination with DNA comet assay

Estimation of the applied doses in irradiated anchovy and bluefish for shelf-life extension using image analysis in combination with DNA comet assay

Genotoxic effects of antiepileptic drugs. Literature review

Based on the selected criteria data from studies of the genotoxic activity of antiepileptic drugs in eukaryotic test systems in vitro and in vivo, performed by DNA comet assay, chromosomal aberrations and micronuclei assays, published in the period 19952022, were selected and summarized.&#x0D; Among the 20 drugs reviewed, for one drug (N03AA05 Benzobarbital), there are no data on studies of genotoxic activity; for 7 drugs information is presented only as a summary on the FDA and EMA websites without primary data and information on experimental designs. Among the remaining 12 drugs, only three drugs (phenobarbital, valproic acid and levetiracetam) have information on in vivo studies, both by DNA damage assay and by cytogenetic methods. Based on known publications, it is impossible to draw reasonable conclusions about the genotoxic potential of individual drugs. The available data are fragmentary, incomplete and contradictory. It remains to state the facts of detection of genotoxic effects in individual drugs in separate studies. In general, there is no doubt about the potential genotoxic hazard of this group the drugs in.&#x0D; Additional studies are needed to clarify the data on the genotoxicity of antiepileptic drugs including beyond the standard protocols. In the course of their implementation, one should take into account the possible tissue-specific manifestation of antiepileptics genotoxicity, as indicated by the facts of genotoxic effects detection in tissue cells that are not targets in classical genotoxic studies. The expediency of objectifying approaches when choosing a drug for safe therapy, taking into account information about its genotoxicity, is emphasized, and the prospects for possible studies on antigenotoxic prophylaxis in patients with epilepsy are pointed out.

Alpha-actnin-4 (ACTN4) selectively affects the DNA double-strand breaks repair in non-small lung carcinoma cells

BackgroundACTN4 is an actin-binding protein involved in many cellular processes, including cancer development. High ACTN4 expression is often associated with a poor prognosis. However, it has been identified as a positive marker for platinum-based adjuvant chemotherapy for non-small cell lung cancer (NSCLC). The goal of our study was to investigate the involvement of ACTN4 in the NSCLC cells’ response to the genotoxic drugs.ResultsWe generated H1299 cells with the ACTN4 gene knock-out (ACTN4 KO), using the CRISPR/Cas9 system. The resistance of the cells to the cisplatin and etoposide was analyzed with the MTT assay. We were also able to estimate the efficiency of DNA repair through the DNA comet assay and gamma-H2AX staining. Possible ACTN4 effects on the non-homologous end joining (NHEJ) and homologous recombination (HR) were investigated using pathway-specific reporter plasmids and through the immunostaining of the key proteins. We found that the H1299 cells with the ACTN4 gene knock-out did not show cisplatin-resistance, but did display a higher resistance to the topoisomerase II inhibitors etoposide and doxorubicin, suggesting that ACTN4 might be somehow involved in the repair of DNA strand breaks. Indeed, the H1299 ACTN4 KO cells repaired etoposide- and doxorubicin-induced DNA breaks more effectively than the control cells. Moreover, the ACTN4 gene knock-out enhanced NHEJ and suppressed HR efficiency. Supporting the data, the depletion of ACTN4 resulted in the faster assembly of the 53BP1 foci with a lower number of the phospho-BRCA1 foci after the etoposide treatment.ConclusionsThus, we are the first to demonstrate that ACTN4 may influence the resistance of cancer cells to the topoisomerase II inhibitors, and affect the efficiency of the DNA double strand breaks repair. We hypothesize that ACTN4 interferes with the assembly of the NHEJ and HR complexes, and hence regulates balance between these DNA repair pathways.

RETSAT associates with DDX39B to promote fork restarting and resistance to gemcitabine based chemotherapy in pancreatic ductal adenocarcinoma

BackgroundSevere hypoxia is a prominent character of pancreatic ductal adenocarcinoma (PDAC) microenvironment. In the process of gemcitabine based chemotherapy, PDAC cells are insulted from replication stresses co-induced by hypoxia and gemcitabine. However, PDAC cells get outstanding abilities to resist to such harsh conditions and keep proliferating, causing a major obstacle for current therapy. RETSAT (Retinol Saturase) is defined as a hypoxia convergent gene recently, with high expression in PDAC hypoxic sectors. This study aimed to explore the roles of RETSAT in replication stress resistance and hypoxia adaptation in PDAC cells, and decipher the underlying mechanism.MethodsThe expression of RETSAT was examined in TCGA (The Cancer Genome Atlas), human pancreatic cancer microarray, clinical specimens and cell lines. Functions of RETSAT were studied by means of DNA fiber assay and comet assay in monolayer cultured PDAC cell lines, three dimensional spheroids, patient derived organoids and cell derived xenograft mouse models. Mechanism was investigated by using iPOND (isolate proteins on nascent DNA) combined with mass spectrometry, immunoprecipitation and immunoblotting.ResultsFirst, we found the converse relationship of RETSAT expression and PDAC chemotherapy. That is, PDAC patients with high RETSAT expression correlated with poor survival, while ones holding low RETSAT expression were benefitted more in Gemcitabine based chemotherapy. Second, we identified RETSAT as a novel replication fork associated protein. HIF-1α signaling promotes RETSAT expression under hypoxia. Functionally, RETSAT promoted fork restarting under replication stress and maintained genomic stability. Third, we uncovered the interaction of RETSAT and R-loop unwinding helicase DDX39B. RETSAT detained DDX39B on forks to resolve R-loops, through which avoided fork damage and CHK1 initiated apoptosis. Targeting DDX39B using chemical CCT018159 sensitized PDAC cells and organoids to gemcitabine induced apoptosis, highlighting the synergetic application of CCT018159 and gemcitabine in PDAC chemotherapy.ConclusionsThis study identified RETSAT as a novel replication fork protein, which functions through interacting with DDX39B mediated R-loop clearance to promote fork restarting, leading to cellular resistance to replication stresses co-induced by tumor environmental hypoxia and gemcitabine in pancreatic ductal adenocarcinoma.

Assessment of DNA damage in somatic and germ cells of animals living with increased radiation background and their offspring

Purpose The aim of this work is to assess DNA damage in the somatic and germ cells in root voles living for a long time under conditions of an increased radiation background and to examine the of manifestation of long-term consequences in their offspring. Materials and methods Using the DNA comet assay (neutral version), we assessed the proportion of cells with DNA damage in the cells of the thyroid, bone marrow and testicular in root voles (Microtus oeconomus Pall.) that lived under conditions of increased radiation background (exposure dose rate − 0.50–20 μSv/h; Komi Republic, Russia) and in their offspring (F1–F3) that were reproduced in a vivarium with a normal radiation background. Results In animals caught in a radioactively contaminated area, the level of DNA fragmentation in the thyroid gland, bone marrow and testicular remained within the range of values of control animals. The studies that we continued on the offspring of irradiated root voles that were developing in the vivarium under normal radiation background allowed us to identify an increase in the level of DNA DSBs in the thyroid gland in the F1 generation, in the bone marrow and testicular cells in the F2 generation. The modifying effect of urethane showed a similarity in the response of somatic cells in voles that lived for a long time in a radioactively contaminated area and in their offspring that developed with a normal radiation background. The effect of urethane was more conspicuous in thyroid cells that, than in bone marrow cells. Conclusion The data obtained on voles from the experimental site indicate adaptation to habitat conditions in a radioactively polluted environment. The provocative effect of urethane made it possible to reveal different response of organs with different proliferative activity. Long-term habitation of voles under conditions of an increased radiation background led to genome instability in their offspring.

1,2,4] Triazolo [3,4-a]isoquinoline chalcone derivative exhibits anticancer activity via induction of oxidative stress, DNA damage, and apoptosis in Ehrlich solid carcinoma-bearing mice

Despite the advances made in cancer therapeutics, their adverse effects remain a major concern, putting safer therapeutic options in high demand. Since chalcones, a group of flavonoids and isoflavonoids, act as promising anticancer agents, we aimed to evaluate the in vivo anticancer activity of a synthetic isoquinoline chalcone (CHE) in a mice model with Ehrlich solid carcinoma. Our in vivo pilot experiments revealed that the maximum tolerated body weight-adjusted CHE dose was 428 mg/kg. Female BALB/c mice were inoculated with Ehrlich ascites carcinoma cells and randomly assigned to three different CHE doses administered intraperitoneally (IP; 107, 214, and 321 mg/kg) twice a week for two consecutive weeks. A group injected with doxorubicin (DOX; 4 mg/kg IP) was used as a positive control. We found that in CHE-treated groups: (1) tumor weight was significantly decreased; (2) the total antioxidant concentration was substantially depleted in tumor tissues, resulting in elevated oxidative stress and DNA damage evidenced through DNA fragmentation and comet assays; (3) pro-apoptotic genes p53 and Bax, assessed via qPCR, were significantly upregulated. Interestingly, CHE treatment reduced immunohistochemical staining of the proliferative marker ki67, whereas BAX was increased. Notably, histopathological examination indicated that unlike DOX, CHE treatment had minimal toxicity on the liver and kidney. In conclusion, CHE exerts antitumor activity via induction of oxidative stress and DNA damage that lead to apoptosis, making CHE a promising candidate for solid tumor therapy.