Alkaline Single Cell Gel Electrophoresis Research Articles

Copper (II) increases anti-Proliferative activity of thymoquinone in colon cancer cells by increasing genotoxic, apoptotic, and reactive oxygen species generating effects

Thymoquinone is the main active compound derived from the essential oil of the Nigella sativa plant seed. While thymoquinone is an antioxidant, it has been reported in several studies that thymoquinone has dose-dependent pro-oxidant activity with the Fenton reaction in the presence of transition elements such as iron and copper. This study aimed to investigate cytotoxic, apoptotic, genotoxic, and reactive oxygen species (ROS) generating effects of thymoquinone treated with copper in colon cancer cells. HT-29 cells were treated with pro-oxidant-acting doses of thymoquinone alone and together with the non-toxic dose of Copper (II) Sulfate for 24 h. Cytotoxic, apoptotic, genotoxic, and ROS production activities were analyzed by MTT viability test, Acridine Orange/Ethidium Bromide (AO/EB) staining, alkaline single cell gel electrophoresis and H2DCF-DA assay, respectively. Viability results showed that thymoquinone and copper synergistically affect cancer cells, and DNA damage was increased with the synergic effect. The intracellular ROS was increased when thymoquinone and copper were applied together. Applying redox-active copper (II) with thymoquinone increases DNA damage, apoptosis, and cell death by increasing the amount of intracellular ROS through pro-oxidant activity. Treatments targeting copper-related pathways may open new therapeutic avenues for cancer treatment.

Impact of fluazuron on oocyte maturation: May the antiparasitic affect bovine reproduction?

Fluazuron is a novel veterinary pour-on antitick formulation which can be applied simultaneously with bovine reproduction management strategies. Considering the economic importance of the livestock industry in many countries, it is important to know whether antiparasitics such as fluazuron may cause embryonic loss. The aim of this study was to evaluate the toxicological effect of fluazuron on bovine oocytes during in vitro maturation. The best fluazuron concentrations were determined in a preliminary experiment on Chinese hamster ovary (CHO)-K1 cells and further used to compare fluazuron toxicity in both study models. Results of the annexin V and alkaline single cell gel electrophoresis assays demonstrated that fluazuron caused cytotoxicity and genotoxicity in bovine cumulus cells at all the concentrations tested (50, 75 and 100 μg fluazuron/mL). The evaluation of cortical granules and mitochondria distribution showed that cytoplasmic maturation was not affected by fluazuron treatment. However, a decrease in metaphase II + polar body, degenerate oocytes as well as disorganized chromatin in polar body were observed at all concentrations tested. Whereas the fertilization process was not altered by 50 μg/mL fluazuron, the embryo development rate decreased significantly. No significant differences were observed in any of the oxidative stress parameters assessed. This study contributes to a better understanding of fluazuron in bovines, suggesting that the antiparasitic may affect bovine reproduction and might cause embryo loss.

Effect of a phthalimide pesticide on human peripheral blood lymphocytes in vitro: induction of DNA damage

Introduction. Captan is a contact fungicide from the phthalimide class, used in agriculture to combat plant diseases. However, the question of its genotoxicity for humans remains open. Various studies examining the mutagenic activity of captan using different tests have yielded conflicting results. This study aims to investigate the potential of captan to induce DNA damage in human peripheral blood lymphocytes in vitro using single-cell alkaline gel electrophoresis.
 Material and methods. The DNA-damaging effect of captan (0; 2.5; 5.0; 10.0; 12.5 and 25 μg/ml) on lymphocytes of 26 donors was assessed with metabolic activation (+S9) and without it (–S9) using DNA comet analysis.
 Results. In the absence of metabolic activation, captan exhibited a pronounced genotoxic effect on cells. Statistically significant effects of captan was found on lymphocytes of all donors. The level of DNA-damaging effect on lymphocytes from 20 out of 26 donors depended on the concentration. The fold increase in the “%DNA in the tail of comets” indicator compared to the negative control at a concentration of 25 μg/ml varied in the range of 4,3–226 times. In the presence of the S9 mixture, weak but significant effects meeting the criteria for a positive response were detected only in three donors.
 Limitations. The genotoxicity of captan was studied only in vitro.
 Conclusion. Captan induces DNA damage in human peripheral blood lymphocytes in vitro in the absence of metabolic activation. Under conditions of metabolic activation, genotoxic effects were low. The levels of DNA damage in peripheral blood lymphocytes of different donors after exposure to captan varied markedly, indicating the necessity to consider individual sensitivity to genotoxicants when utilizing the DNA comet method to assess the potential mutagenic activity of chemicals.

Echinodorus macrophyllus: Acute toxicological evaluation of hydroxycinnamoyl derivatives from SF1 subfractions

Ethnopharmacological relevanceEchinodorus macrophyllus (Kunth.) Micheli (Alismataceae), known as chapéu-de-couro in Brazil, is popularly used to treat inflammatory diseases. We have previously demonstrated a significant reduction in the acute inflammation for the aqueous extract of E. macrophyllus (AEEm) and its ethanolic fraction (Fr20) and described that hydroxycinnamoyl derivatives present in SF1 (Fr20 subfraction) showed higher anti-inflammatory properties by mechanisms that include a reduction of TNF-α, IL-1β, CKCL1/KC, LTB4, and PGE2 levels in exudate. Aim of the studyThis work describes the acute toxicological effect of SF1 subfraction on SW mice treated orally for five days in the air pouch model by evaluating the hematological and biochemical determinations on the blood samples; the relative organ weight and its histopathological analysis; the liver genotoxicity assessment and the activity of liver enzymes from xenobiotic metabolism. Materials and methodsFr20 was earlier fractionated on the Sephadex LH-20 column, yielding mainly four subfractions, including SF1. The SF1 toxicity was evaluated in mice challenged with carrageenan on the air pouch inflammation model and orally treated for five days. The body weight was monitored daily, and the organs were weighed after the euthanasia. Hematological and biochemical determinations were carried out using specific commercial kits and following the protocols provided by the manufacturers. The organs were fixed, sectioned, processed for hematoxylin and eosin staining, and analyzed by light microscopy. Genotoxicity assessment was performed by the alkaline single-cell gel electrophoresis. Livers were processed for ethoxyresorufin-O-deethylase (EROD) and Glutathione S-transferase (GST) assays. ResultsSF1 exhibited low toxicity, as no significant discrepancy was observed in the relative weight of the body organs of mice. Moreover, the daily treatment with SF1 did not alter the number and percentage of red blood cells or hemoglobin concentration in the blood. The treatment with SF1 did not affect the creatinine concentration, but the 25 mg/kg dose reduced the plasma urea level and uric acid, suggesting its use in treating acute renal failure. The parameters analyzed did not present biochemical alterations indicative of liver disease. Regarding serum triglyceride and cholesterol levels, a significant decrease was detected in both parameters in mice treated with SF1. In addition, the histopathological analysis showed that inflammatory focus in the livers seemed more relevant in the control groups than in those treated. There were no significant changes in the renal or splenic tissues of animals treated with SF1. Treatment with SF1 also does not have a genotoxic effect on liver cells. ConclusionTreatment with SF1 showed no toxicity in mice at doses equivalent to those recommended for humans, which provides evidence of the safety of the therapeutic use of this subfraction.

Evaluation of DNA repair capacity in parents of pediatric patients diagnosed with autism spectrum disorder using the comet assay procedure

BackgroundAutism Spectrum Disorder (ASD) is characterized by impairments in social communication, limited repetitive behaviors, impaired language development, and interest or activity patterns, which include a group complex neurodevelopmental syndrome with diverse phenotypes that reveal considerable etiological and clinical heterogeneity and are also considered one of the most heritable disorders (over 90%). Genetic, epigenetic, and environmental factors play a role in the development of ASD. AimThis study was designed to investigate the extent of DNA damage in parents of autistic children by treating peripheral blood mononuclear cells (PBMCs) with bleomycin and hydrogen peroxide (H2O2). MethodsPeripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll method and treated with a specific concentration of bleomycin and H2O2 for 30 min and 5 min, respectively. Then, the degree of DNA damage was analyzed by the alkaline comet assay or single cell gel electrophoresis (SCGE), an effective way to measure DNA fragmentation in eukaryotic cells. ResultsOur findings revealed that there is a significant difference in the increase of DNA damage in parents with affected children compared to the control group, which can indicate the inability of the DNA molecule repair system. Furthermore, our study showed a significant association between fathers' occupational difficulties (exposed to the influence of environmental factors), as well as family marriage, and suffering from ASD in offspring. ConclusionOur results suggested that the influence of environmental factors on parents of autistic children may affect the development of autistic disorder in their offspring. Subsequently, based on our results, investigating the effect of environmental factors on the amount of DNA damage in parents with affected children requires more studies.

Cytotoxic and genotoxic profiles of the pyrethroid insecticide lambda-cyhalothrin and its microformulation Karate® in CHO-K1 cells.

Lambda-cyhalothrin (LCT) and its microformulation Karate® (25 % a.i.) were analysed for its genotoxicity and cytotoxicity on Chinese hamster ovary (CHO-K1) cells. Cytokinesis-block micronucleus cytome (CBMN-cyt) and alkaline single-cell gel electrophoresis (SCGE) bioassays were selected to test genotoxicity. Neutral red uptake (NRU), succinic dehydrogenase activity (MTT) and apoptogenic induction were employed for estimating cytotoxicity. Both compounds were analysed within a concentration range of 0.1-100µg/mL. Only LCT produced a significant augment in the frequency of micronuclei (MNs) when the cultures were exposed to highest concentrations of 10 and 100µg LCT/mL. A noticeable decrease in NDI was observed for cultures treated with LCT at 10 and 100µg/mL. Karate® induced the inhibition of both the proportion of viable cells and succinic dehydrogenase activity and triggered apoptosis 24h of exposition. Whilst an increased GDI in CHO-K1 cells was observed in the treatments with 1-100µg Karate®/mL, the GDI was not modified in the treatments employing LCT at equivalent doses. SCGE showed that Karate® was more prone to induce genotoxic effects than LCT. Only 50µg/mL of Karate® was able to increase apoptosis. Our results demonstrate the genomic instability and cytotoxic effects induced by this pyrethroid insecticide, confirming that LCT exposure can result in a severe drawback for the ecological equilibrium of the environment.

Oleuropein exhibits anticarcinogen effects against gastric cancer cell lines.

Oleuropein (OLE), the main phenolic compound of the olive fruit and leaves, has many heathful effects. Gastric cancer is the most fatal malignancy in many parts of the world and it is generally related to harmful dietetic factors. The anticarcinogenic role of OLE in gastric cancer has not been studied sufficiently yet. In this study, we aimed to research the cytotoxic, genotoxic and apoptotic effects of OLE on gastric adenocancer (AGS) cells in vitro. A standard cell line derived from gastric adeno cancer (AGS) cells was employed, and its performance following a 24-hour exposure to OLE at various doses was examined. The ATP cell viability assay, 2',7'-dichlorodihydrofluorescein-diacetate assay (H2DCF-DA) and alkaline single cell gel electrophoresis assay (Comet Assay) were used to study the cytotoxicity, production of reactive oxygen species (ROS) and genotoxicity respectively. The induction of apoptosis was discovered using flow cytometry. OLE reduced AGS cells viability about 60% at maximum concentration (500 µmol/L) and also resulted in approximately 100% DNA damage and about 40% apoptosis with necrosis in AGS cells depending on the increased doses. Cell viability was also significantly decreased in relation to increased intracellular reactive oxygen species (ROS) levels (p < 0.05 - 0.001). Oleuropein has shown significant anticarcinogen effects against gastric adenocancer (AGS) cells in vitro. Oleuropein, a nutrient rich in olive and olive oil, seems to be both protective and therapeutic against gastric cancer and may be a new chemotherapeutic agent in the future.

Genotoxicity Evaluation of Vaishvanara Churna - A classical Ayurvedic Formulation

Aims: The current study aimed to evaluate the genotoxicity of the Vaishvanara Churna, a polyherbal formulation by using Peripheral Blood Mononuclear Cells. The study was supported by the study of the content of heavy metals in the ayurvedic formulation. Background: Vaishvanara Churna is a classical ayurvedic formulation that is used for treating conditions, such as flatulence with gurgling sound (Adhmana), abdominal lump (Gulma); Duodenal ulcer (Parinamasula), Rheumatism (Amavata); heart disease (Hrdroga). Objective: The objective is to study the genotoxicity of Vaishvanara Churna on Peripheral Blood Mononuclear Cells using alkaline Single-cell Gel Electrophoresis and correlate the possible association of genotoxicity to the number of heavy metals present in the polyherbal formulation. Methodology: The Alkaline Single Cell Gel Electrophoresis was used to see the effects on the Peripheral Blood Mononuclear cells using 2% Hydrogen Peroxide as a standard oxidizing agent. Observation: Peripheral Blood Mononuclear Cells, isolated from a fresh human blood sample, were treated in vitro with varying concentrations of Vaishvanara Churna (0.2, 0.4, 0.8, 1.6, and 3.2 mg/ml of aqueous extract) using a standard protocol. It was found that none of the assessed concentrations of the ayurvedic formulation was toxic to Peripheral Blood Mononuclear Cells. Conclusion: Single-cell Gel Electrophoresis is a simple technique to evaluate the genotoxicity of any drug to Human Beings, and it can be used as an alternative to animal genotoxicity studies.

Synthesised thymoquinone-oxime induces cytotoxicity, genotoxicity and apoptosis in hepatocellular cancer cells: in vitro study

Hepatocellular carcinoma is the most common primary malignant tumor of the liver, and its incidence is increasing worldwide. There is a need to develop new therapeutic strategies to treat the disease. In this study, we synthesised the oxime derivative of thymoquinone and investigated cytotoxicity, genotoxicity, and apoptosis in hepatocellular cancer cells. The synthesised thymoquinone-oxime structure was confirmed by NMR. After incubating the hepatocellular cancer cell line for 24 h, the cytotoxicity ATP by luminometric, intracellular reactive oxygen species, and intracellular calcium by fluorometric. The mitochondrial membrane potential was determined by flow cytometry. DNA damage by alkaline single-cell gel electrophoresis, and apoptosis damage by acridine orange/ethidium bromide double dye method. Concentrations of thymoquinone-oxime statistically increased cytotoxicity, intracellular reactive oxygen species, intracellular calcium, apoptosis, and DNA damage in a concentration-dependent manner. Mitochondrial membrane potential and glutathione levels are also decreased. These findings show that thymoquinone-oxime has an anti-tumor effect on hepatocellular carcinoma cells.

Antiaging Effects of Limonene in Ageing-Induced HaCaT Cells

Limonene, obtained from citrus oils such as orange, grapefruit, and lemon, is widely used in cosmetics as a scenting agent. It is regarded as safe because it is not mutagenic, carcinogenic, or nephrotoxic to people and has low human toxicity. This study aims to show the beneficial effects of limonene against hydrogen peroxide (H2O2) induced ageing in human keratinocytes (HaCat) cell line. For this purpose, the concentration value at which H2O2 can cause ageing in HaCaT cells was determined as 200 µM by the MTT method. Then, cytotoxic effects of limonene (1, 10, 25, 50, 100, 250 and 500 µg/mL) alone and in aging-induced cells were determined by the MTT method; possible antigenotoxic effects were determined by alkaline single-cell gel electrophoresis (Comet) method. According to the results obtained, limonene, an ingredient in cosmetics, has protective effects against skin aging in HaCaT cells.

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.

The miR-29 family members induce glioblastoma cell apoptosis by targeting cell division cycle 42 in ap53-dependent manner.

Emerging evidence has shown that miR-29 is a promising biomarker and therapeutic target for malignancies. The roles of miR-29a/b/c in glioma pathogenesis remain need further investigation. The expression levels of miR-29a/b/c and CDC42 were systematically analysed, and prognostic significance was evaluated by Kaplan-Meier survival and Cox regression analyses. The roles of miR-29a/b/c in apoptosis and the underlying mechanisms were explored via an alkaline single-cell gel electrophoresis assay, caspase 3/7 activity assays and Western blotting. miR-29a/b/c expression decreased progressively with the elevation of the WHO grade in our 147 human glioma specimens, compared with 20 non-tumour control brain tissues, and decreased miR-29a/b/c expression was associated with more aggressive phenotypes. Kaplan-Meier and Cox regression analyses demonstrated that lower miR-29a/b/c expression was correlated with worse prognosis, which was confirmed by analysis of 198 glioma patients from the CGGA cohort. These all indicate that miR-29a/b/c were independent predictors of prognosis in glioma patients. miR-29a/b/c induced apoptosis in GBM cells by silencing CDC42. Further detailed mechanistic investigation revealed that miR-29a/b/c promoted apoptosis in a p53-dependent manner by suppressing the CDC42/PAK/AKT/MDM2 pathway. miR-29a/b/c are independent predictors of prognosis in glioma patients. They induce glioblastoma cell apoptosis via silencing of CDC42 and suppression of downstream PAK/AKT/MDM2 signalling in a p53-dependent manner.

Protective Effect of Thyme and Chestnut Honeys Enriched with Bee Products against Benzo(a)pyrene-Induced DNA Damage

The aim of the present study was to validate the cytotoxicity, genotoxicity, and preventive potential against benzo(a)pyrene (BaP)-induced DNA damage of nine samples of thyme and chestnut honeys enriched with bee products (royal jelly and propolis, 2-10%). Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (0-250 mg/mL) to select nontoxic concentrations, and DNA damage (0.1-10 μg/mL) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. Treatment with honey samples or royal jelly and propolis did not affect the viability of HepG2 cells up to 100 and 50 mg/mL, respectively. Treatment with 100 μM BaP significantly increased (p ≤ 0.001) the levels of the DNA strand breaks. None of the tested concentrations (0.1-10 μg/mL) of the honey samples (thyme and chestnut), royal jelly, and propolis caused DNA damage per se. All tested samples at all the concentrations used decreased the genotoxic effect of BaP. In addition, all mixtures of thyme or chestnut honeys with royal jelly or propolis showed a greater protective effect against BaP than the samples alone, being the thyme and chestnut honey samples enriched with 10% royal jelly and 10% propolis the most effective (70.4% and 69.4%, respectively). The observed protective effect may be associated with the phenolic content and antioxidant capacity of the studied samples. In conclusion, the thyme and chestnut honey samples enriched with bee products present potential as natural chemoprotective agents against the chemical carcinogen BaP.

Genotoxic potential of different nano-silver halides in cultured human lymphocyte cells

Most antibacterial applications in nanotechnology are carried out using silver nanoparticles (AgNPs). However, there is a dearth of information on the biological effects of AgNPs on human blood cells. In this study, the cytotoxic and genotoxic potentials of ionic silver (Ag+), AgNP, silver bromide (AgBr), silver chloride (AgCl), and silver iodide (AgI) were evaluated through chromosome aberration (CA) test and cytokinesis-blocked micronucleus (CBMN) test in human cultured lymphocytes in vitro. Furthermore, the potential damages that can cause to DNA were evaluated through alkaline single cell gel electrophoresis (Comet) assay on isolated lymphocytes. The results showed that AgNPs exerted cytotoxic effects by reducing the cytokinesis-block proliferation index and mitotic index at 24 and 48 h. AgNPs also increased micronucleus (MN) formation at both exposure times in the cultured cells. Meanwhile, AgCl had no genotoxic effects on the human lymphocyte cultured cells but had a cytotoxic effect at high doses. AgNP, Ag+, AgBr, and AgI caused substantial DNA damage by forming DNA strand breaks. They may also have clastogenic, genotoxic and cytotoxic effects on human lymphocyte cells. Based on the foregoing findings, silver nanomaterials may have genotoxic and cytotoxic potentials on human peripheral lymphocytes in vitro.

Effects of oxidative stress, DNA damage, and inflammation in multiple sclerosis: A clinical perspective

Abstract Multiple sclerosis (MS) is a demyelinating nervous system disease known for its lesions and manifests itself with attacks. According to some theories, inflammation and oxidative stress play an important role in MS. With this study, we aimed to examine the levels of oxidative stress, inflammation and DNA damage in MS patients and to get an idea about the course of the disease from these data. The research comprised patients diagnosed with MS between the ages of 18 and 60. Photometric techniques were used to determine serum native thiol (NT), total thiol (TT), total antioxidant status (TAS), and total oxidant status (TOS) levels. The oxidative stress index (OSI), disulfide (DIS) level, and percentages of DIS/TT, DIS/NT, and NT/TT were determined with mathematical calculations. Inflammation biomarkers tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) were measured by photometric methods with commercially purchased ELISA kits. DNA damage was detected using alkaline single-cell gel electrophoresis. TOS, OSI, and DIS levels, as well as DIS/NT and DIS/TT percentages, IL-1β, IL-6, TNF- α and DNA damage levels were shown to be statistically significantly increased in MS patients than in the healthy control group (p &lt; 0,001), according to the study's findings. Furthermore, TAS, TT, and NT levels were decreased in MS patients. Inflammation occurs as a result of oxidative stress in MS patients and causes DNA damage. Our results show that clinicians should consider oxidative stress, inflammation, and DNA damage when evaluating MS's development.

Oxidative damage and DNA repair in desiccated recalcitrant embryonic axes of Acer pseudoplatanus L.

BackgroundMost plants encounter water stress at one or more different stages of their life cycle. The maintenance of genetic stability is the integral component of desiccation tolerance that defines the storage ability and long-term survival of seeds. Embryonic axes of desiccation-sensitive recalcitrant seeds of Acer pseudoplatnus L. were used to investigate the genotoxic effect of desiccation. Alkaline single-cell gel electrophoresis (comet assay) methodology was optimized and used to provide unique insights into the onset and repair of DNA strand breaks and 8-oxo-7,8-dihydroguanine (8-oxoG) formation during progressive steps of desiccation and rehydration.ResultsThe loss of DNA integrity and impairment of damage repair were significant predictors of the viability of embryonic axes. In contrast to the comet assay, automated electrophoresis failed to detect changes in DNA integrity resulting from desiccation. Notably, no significant correlation was observed between hydroxyl radical (٠OH) production and 8-oxoG formation, although the former is regarded to play a major role in guanine oxidation.ConclusionsThe high-throughput comet assay represents a sensitive tool for monitoring discrete changes in DNA integrity and assessing the viability status in plant germplasm processed for long-term storage.

Cytotoxic effects of Roundup Classic and its components on NE-4C and MC3T3-E1 cell lines determined by biochemical and flow cytometric assays

Cytotoxic effects of the market leading broad-spectrum, synthetic herbicide product Roundup Classic, its active ingredient glyphosate (in a form of its isopropylamine (IPA) salt) and its formulating surfactant polyethoxylated tallowamine (POE-15) were determined on two murine cell lines, a neuroectodermal stem cell-like (NE-4C) and a high alkaline phosphatase activity osteoblastic cell line (MC3T3-E1). Cytotoxicity, genotoxicity, effects on cell viability and cell cycles were examined in five flow cytometry tests, the two former of which were compared by the enzymatic-assay and the alkaline single cell gel electrophoresis (Comet) assay. All of the tests indicated the NE-4C cells being more sensitive, than the MC3T3-E1 cell line to the treatments with the target compounds. Higher sensitivity differences were detected in the viability test by flow cytometry (7–9-fold), than by the MTT assay (1.5–3-fold); in the genotoxicity test by the Comet assay (3.5–403-fold), than by the DNA-damage test (9.3–158-fold); and in the apoptosis test by the Annexin V dead cell kit (1.1–12.7-fold), than by the Caspase 3/7 kit (1–6.5-fold). Cell cycle assays indicated high count of cells (~70%) in the G0/G1 phase for MC3T3-E1 cells, than in NE-4C cell (~40%) after 24 h. The order of the inhibitory potency of the target substances has unequivocally been POE-15 > Roundup Classic > > glyphosate IPA salt.

Does Organic Sprouted Whole Wheat Grain Flourless Bread Decreases DNA Damage in Diabetic Patients?

Abstract Diabetes is one of the main health concerns, especially in developed countries. During the last few decades, the percentage of diabetic persons is constantly increasing. Although the genetic factors have a strong influence in the development of diabetes, environmental influence (physical inactivity, inadequate nutrition leading to obesity) also have an important impact. The main objective of this investigation was to evaluate the possible influence of organic sprouted whole wheat grain flourless bread Tonus® (product of Trivit, Becej, Serbia) on DNA damage at various stages of progression through type 2 diabetes mellitus (T2DM). In addition to control (non-diabetic) subjects we analyzed obese, pre-diabetic and diabetic patients, for a total of four experimental groups. All subjects used to eat Mediterranean diet for at least two years before being included in our study. In each of four groups we had five persons practicing the Mediterranean diet but instead of bread they consumed flourless bread Tonus®. The DNA damage was evaluated on peripheral blood mononuclear cells by alkaline single cell gel electrophoresis (Comet) assay at the very beginning (before starting the Tonus® bread diet), and after exactly the three months of consumption of Tonus® bread. Statistical analysis revealed that only in patients with type 2 diabetes, Tonus® bread intake led to decreased DNA damage compared to the level of DNA damage of these patients before they started Tonus® bread diet. We assume that decrease of body weight and hyperinsulinemia caused by Tonus® bread in the diet might be one of the main causes of decreased DNA damage.

DNA polymerase β may be involved in protecting human bronchial epithelial cells from the toxic effects induced by methyl tert-butyl ether exposure.

Methyl tert-butyl ether (MTBE), a widely used gasoline additive and a ubiquitous environmental pollutant in many countries and regions, can cause various kinds of toxic effects on human health. However, the molecular mechanism underlying its toxic effects remains elusive. The present study aimed to explore the cytotoxicity, DNA damage and oxidative damage effects of MTBE on human bronchial epithelial cells (16HBE) and the possible role of DNA polymerase β (pol-β) in this process. RNA interference (RNAi) was used to obtain pol-β gene knocked-down cells (pol-β-). CCK-8 assay was adopted to analyze the cell viability. Alkaline single-cell gel electrophoresis (SCGE) was performed to detect the DNA damage effects of MTBE. The enzyme activity of GSH-Px, SOD, CAT and the level of MDA were assessed. The data indicated that when treated with MTBE at the concentration exceeding 50 μmol/L and for the time exceeding 24 h, the pol-β- exhibited significantly decreased cell viability and increased DNA damage effects, as compared to the control (P < 0.05). Furthermore, there was significant difference in the levels of GSH-pX, SOD, CAT and MDA between the pol-β- and the control (P < 0.05). Our investigation suggests that MTBE can cause obvious cytotoxicity, DNA damage and oxidative damage effects on 16HBE cells. DNA polymerase β may be involved in protecting 16HBE cells from the toxic effects induced by MTBE exposure. These findings provide a novel insight into the molecular mechanism underlying the toxic effects of MTBE on human cells.

Indigenous Fish-Based Assessment of Genotoxic Potentials of the Helong Reservoir in Guangzhou, China.

The present study was conducted to assess the genotoxic potential of water from the Helong Reservoir, which was designated as a strategic drinking water source by the Guangdong Provincial Government of China in October 2016. Four kinds of common indigenous fish samples (Labeo rohita, Cirrhinus molitorella, red tilapia, and Oreochromis niloticus) were collected at 6 sampling sites during the period from July to November 2020. Fish from the clean drinking water source of the upper reaches of the Liuxihe Reservoir in Guangzhou were collected as the control. Both the alkaline single cell gel electrophoresis assay and the micronucleus test were used to detect DNA damage and the micronucleus rate in erythrocytes of fish samples, respectively. The results indicated that there was a significant increase in comet tail length, Olive tail moment, and micronucleus rates of all fish samples compared with those of the control (p < 0.05). The order of sensitivity to DNA damage and micronucleus formation was Labeo rohita > Cirrhinus molitorella > red tilapia > Oreochromis niloticus. The results of the 2 kinds of experiments were in perfect agreement with each other. We conclude that there are obvious genotoxic effects from the water in the Helong Reservoir. As a strategic drinking water source, the safety of the Reservoir water quality should be considered. The local government should put the restoration of the Helong Reservoir water quality on the agenda as soon as possible. Environ Toxicol Chem 2021;40:1919-1927. © 2021 SETAC.