Alkaline Single Cell Gel Electrophoresis Research Articles

Discovery of organometallic Ruthenium(II)-arene complexes of lidocaine as improved photocytotoxic agents

The new family of Ruthenium(II)-arene complexes of lidocaine of formula [RuII(η6-p-cymene)Cl(LC)](PF6), 1, [RuII(η6-p-cymene) (CCFc) (LC)](PF6), 2, [RuII(η6-p-cymene) (CCFcIP) (LC)](PF6), 3 (LC: lidocaine, FcCCH: ferrocenyl acetylene, CCFcIP: 1′-(phenanthro[9,10-d]imidazole) ferrocenyl-1-acetylene) were prepared and characterized by ESI-MS spectrometry, elemental analysis, IR, 1H and 13C NMR spectroscopy. The photocytotoxicity of 1–3 was studied with visible light (400–700nm) against a panel of human cancer cell lines namely, A-375 (human melanoma), HeLa (human cervical cancer) and MCF-7 (human breast cancer) cancer cells. The photoactivity follows the order 1<2<3 with 2 and 3 having IC50 values in A-375 (melanoma) cells in the low micromolar range. These complexes interact with calf thymus DNA. The photocleaving pUC19 DNA of complexes with visible light (400–700nm) was studied and the results exhibited the active involvement of superoxide and hydroxyl radicals as the reactive oxygen species (ROS) in the DNA photocleavage reactions. These complexes interact with calf thymus DNA via intercalative mode that binding constants vary in the order: 3>2>1. The complexes 2 and 3 were photoactivated in A-375 cells by visible radiation and analyzed by alkaline single-cell gel electrophoresis.

Genotoxicity assessment of acute exposure of 2, 4-dinitroanisole, its metabolites and 2, 4, 6-trinitrotoluene to Daphnia carinata.

The insensitive munition ingredient, 2, 4-dinitroanisole has emerged as an alternative ingredient to 2, 4, 6-trinitro toluene in melt pourable high explosive formulations mainly due to its improved insensitiveness properties. As a result, production of 2, 4-dinitroanisole has increased and as a consequence 2, 4-dinitroanisole has emerged as a potential ingredient to enter the environment and possibly persist in water and soil ecosystems. The present study showed that 2, 4-dinitroanisole, its metabolites (2-amino 4-nitroanisole and 2,4-dinitroanisole) and 2, 4, 6-trinitro toluene were found to induce DNA damages in a freshwater crustacean Daphnia carinata exposed for 48 h and which was investigated by the alkaline single-cell gel electrophoresis (comet assay) method. The value of LC50-48 h of 2, 4-dinitroanisole was determined as 14.87 ± 1.70 (mg L-1) and its metabolites exhibited the similar toxic range although the toxicity of 2, 4, 6-trinitro toluene was seven-fold more toxic (2.32 ± 0.29 mg L-1) than 2, 4-dinitroanisole and its metabolites. Exposure to sub-acute toxicity concentration ranges of 2, 4-dinitroanisole and its metabolites and 2, 4, 6-trinitro toluene showed significant (P < 0.01) DNA damage. The higher concentration of each test chemical exhibited higher tail DNA per cent and increased olive tail moment. The results from this study can be used to identify genotoxic biomarkers for the risk assessment of insensitive munitions exposure in aquatic invertebrates.

Cytotoxic, genotoxic and apoptotic effects of naringenin-oxime relative to naringenin on normal and cancer cell lines

ObjectiveTo assess and compare the cytotoxic, genotoxic, apoptotic and reactive oxygen species (ROS) generating effects of naringenin (NG) and its new derived compound naringenin-oxime (NG-Ox) on MCF-7, HT-29, PC-12 cancer and L-929 normal cell lines. MethodsThe cells were incubated with different doses of NG-Ox and NG (50–1000 μmol/L) for 24 h. The cell viability was assessed based on ATP cell viability assay. Intracellular accumulation of ROS was determined using the fluorescent probes 2′7′-dichlorodihydrofluorescin diacetate. Genotoxic effects were evaluated by alkaline single cell gel electrophoresis assay (comet assay) and, the apoptotic effect was evaluated by acridine orange staining at below the IC50 levels. ResultsBoth NG-Ox and NG exhibited cytotoxic, genotoxic and apoptotic effects and resulted in increased ROS values in a dose-dependent manner. The effects were more pronounced on cancer cell lines. The cytotoxic, genotoxic and apoptotic effects of NG-Ox were higher than that of NG in all cell lines. Significant correlations were observed between cell viability, DNA damage, apoptosis and ROS, in all cell lines exposed to either NG-Ox or NG. ConclusionsThis study showed that both NG-Ox and NG possess cytotoxic, genotoxic and apoptotic activities through the production of ROS on cells, NG-Ox being the more effective one. Therefore, derived compound of NG might be used as antiproliferative agents for the treatment of cancer.

Abstract 3524: D-glucose exposure induced DNA damage and apoptosis in MCF-7 cells

Abstract D-glucose is the simple carbohydrate sugar that our bodies rely on to produce ATP energy. It has been shown that sustained high glucose burden is related to the promotion of many long-term health problems to various vital target organs including the kidneys, nervous system, eyes, heart (failure and stroke), erectile dysfunction in men and pregnancy complications in women. Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. Furthermore, the D-glucose paradox as cancer-promoting energy source as well as being toxic to cancer cells is puzzling. Therefore in the present study, we will investigate the mechanisms of glucose-induced toxicity in MCF-7 cells as an in vitro cellular model to simulate diabetic complications in tumor cells using trypan blue exclusion and MTT assays. Mechanisms of DNA damage and apoptosis were tested by alkaline single cell gel electrophoresis (Comet) assay, and flow cytometry analysis using Annexin V FITC/PI and caspase-3 analysis. The MTT assay indicated that low dose (5 mg/mL) of D-glucose slightly increase cell viability upon 2 h of exposure. On the other hand, high doses (10-80 mg/mL) of D-glucose significantly reduced the viability of MCF-7 cells in a dose and time-dependent manner. Similar trends were seen with the trypan blue exclusion test. Data obtained from the comet assay indicated that D-glucose caused DNA damage in MCF-7 cells in a dose-dependent manner. The flow cytometry assessment (Annexin V FITC/PI) showed a strong dose-response relationship between high glucose exposure and Annexin V positive MCF-7 cells undergoing early stage apoptosis. Similarly, a statistically significant and concentration-dependent increase (p &amp;lt;0.05) were recorded for caspase-3 activity in MCF-7 cells undergoing late apoptosis. Based on these in vitro findings, our study provides clear evidence that elevated level of D-glucose induced cytotoxicity to MCF-7 cells via induction of DNA damage, externalization of phosphatidylserine and caspase-3 activation as indicators for the apoptotic mechanisms of D-glucose exposure. This research is supported by a grant from the National Institutes of Health-NIH (Grant No. NIMHD-G12MD007581). Citation Format: Ibrahim O. Farah, Christine K. Tchounwou, Clement G. Yedjou, Paul B. Tchounwou. D-glucose exposure induced DNA damage and apoptosis in MCF-7 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3524.

Exercise and Oxidative Damage in Nucleoid DNA Quantified Using Single Cell Gel Electrophoresis: Present and Future Application.

High intensity exercise can enhance the production of reactive oxygen and nitrogen free radical species, which may cause a number of perturbations to cellular integrity, including deoxyribonucleic acid (DNA) modification. In the absence of adequate DNA repair, it is theoretically possible that several biological disorders may ensue, in addition to premature aging. This striking hypothesis and supposition can only be realized in the presence of sound methodology for the quantification of DNA damage and repair. The alkaline single-cell gel electrophoresis or “comet assay” is a simple and reliable method for measuring the components of DNA stability in eukaryotic cells. The assay is commonly used in research associated with genotoxicology and in human bio-monitoring studies concerned with gene-environment interactions; but is currently less appreciated and under-utilized in the domain of exercise science. No exercise related study for example, has incorporated the comet assay combined with fluorescent in situ hybridization methodology to detect and investigate whole genome, telomeric DNA, or gene region-specific DNA damage and repair in cells. Our laboratory and others have used the comet assay in conjunction with lesion-specific endonucleases to measure DNA strand breaks and oxidized bases to confirm that high intensity exercise can damage and destabilize DNA. Thus, the primary function of this review is to highlight recent advances and innovation with the comet assay, in order to enhance our future understanding of the complex interrelationship between exercise and DNA modification in eukaryotic cells. A brief synopsis of the current literature addressing DNA stability as a function of continuous aerobic exercise is also included.

An in vitro study on the genotoxic effect of substituted furans in cells transfected with human metabolizing enzymes: 2,5-dimethylfuran and furfuryl alcohol.

2,5-Dimethylfuran (DMF) and furfuryl alcohol (FFA) are two substituted furans that are formed during the processing of foods and have also been used as food flavorings. DMF and FFA are proposed to be bioactivated by human sulfotransferases (SULTs) which are not expressed in conventional cell lines used for genotoxicity testing. Therefore, in addition to the standard V79 cell line, we used a transfected V79 derived cell line co-expressing human cytochrome P450 (CYP) 2E1 and human SULT1A1 to assess the genotoxicity of DMF and FFA. The alkaline single cell gel electrophoresis (SCGE) assay was used to detect DNA damage in the form of single strand breaks and alkali-labile sites after exposure to DMF (0.5h; 0.5, 1, 1.5 or 2mM) or FFA (3h; 1, 3, 6 or 15mM). DMF induced DNA damage in V79 cells in a concentration-dependent manner irrespective of the expression of human CYP2E1 and SULT1A1. Almost no increase in the level of DNA damage was detected after exposure to FFA, except for a weak effect at the highest concentration in the transfected cell line. The results suggest that DNA damage in V79 cells from exposure to DMF detected by the alkaline SCGE assay is independent of human CYP2E1 and SULT1A1, and the genotoxic effect of FFA, as assessed by SCGE, is minimal in V79 cells.

Tousled kinase activator, gallic acid, promotes homologous recombinational repair and suppresses radiation cytotoxicity in salivary gland cells

Accidental or medical radiation exposure of the salivary glands can gravely impact oral health. Previous studies have shown the importance of Tousled-like kinase 1 (TLK1) and its alternate start variant TLK1B in cell survival against genotoxic stresses. Through a high-throughput library screening of natural compounds, the phenolic phytochemical, gallic acid (GA), was identified as a modulator of TLK1/1B. This small molecule possesses anti-oxidant and free radical scavenging properties, but in this study, we report that in vitro it promotes survival of human salivary acinar cells, NS–SV–AC, through repair of ionizing radiation damage. Irradiated cells treated with GA show improved clonogenic survival compared to untreated controls. And, analyses of DNA repair kinetics by alkaline single-cell gel electrophoresis and γ-H2AX foci immunofluorescence indicate rapid resolution of DNA breaks in drug-treated cells. Study of DR-GFP transgene repair indicates GA facilitates homologous recombinational repair to establish a functional GFP gene. In contrast, inactivation of TLK1 or its shRNA knockdown suppressed resolution of radiation-induced DNA tails in NS–SV–AC, and homology directed repair in DR-GFP cells. Consistent with our results in culture, animals treated with GA after exposure to fractionated radiation showed better preservation of salivary function compared to saline-treated animals. Our results suggest that GA-mediated transient modulation of TLK1 activity promotes DNA repair and suppresses radiation cytoxicity in salivary gland cells.

Analysis of possible genotoxicity of the herbicide flurochloridone and its commercial formulations: Endo III and Fpg alkaline comet assays in Chinese hamster ovary (CHO-K1) cells

Cytotoxic and genotoxic effects of flurochloridone (FLC) and its formulations Twin Pack Gold® and Rainbow® were evaluated in CHO-K1 cells. Using the alkaline single-cell gel electrophoresis (SCGE) assay, we observed that FLC (15μg/ml), Twin Pack Gold® or Rainbow® induced primary DNA damage, increasing the frequency of damaged nucleoids. Vitamin E pretreatment did not modify the effect. Decreased cell viability was observed only in Twin Pack Gold®-treated cultures and was significantly ameliorated by vitamin E. Post-treatment of herbicide-damaged CHO-K1 cells with the enzymes Endo III or Fpg did not increase FLC-, Twin Pack Gold®-, or Rainbow®-induced DNA damage. These results demonstrate that neither FLC nor FLC-based formulations induce DNA damage through hydroxyl radical or lipid alkoxyl radical production, and that the induced DNA lesions were not related to oxidative damage at the purine/pyrimidine level. Our observations strongly suggest that the cytotoxic effects observed after Twin Pack Gold® exposure are due to the excipients contained within the technical formulation rather than FLC itself.

Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis

Malathion Induced DNA Damage in Freshwater Fish, Labeo rohita (Hamilton, 1822) Using Alkaline Single Cell Gel Electrophoresis

Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress.

Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium. Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG2) cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet) assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay). The result of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05) increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2 cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05) was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG2) cells.

DNA Binding and its Degradation by the Neurotransmitter Serotonin and its Structural Analogues Melatonin and Tryptophan: Putative Neurotoxic Mechanism

Over the years, DNA damage has been suggested to be directly linked to mutagenesis, carcinogenesis and ageing. There is ample evidence suggesting that considerable DNA damage may be induced by metabolites produced during normal or aberrant metabolic processes. Herein, we examine the chemical basis of cytotoxicity of some endogenous metabolites. Towards this goal, we study the DNA reactive activities of the neurotransmitter serotonin and its structural analogues tryptophan and melatonin in the presence of copper. Fluorescence spectroscopy reveals a simple mode of interaction of these metabolites with calf thymus DNA and copper ions. The results of agarose gel electrophoresis demonstrate copper mediated strand scission in plasmid pBR322 DNA by these metabolites. Further, the relative DNA binding affinity order was affirmed using molecular docking studies involving the interaction of these metabolites with calf thymus DNA. We employ single cell alkaline gel electrophoresis and determine the relative efficiency of cellular DNA breakage as Serotonin>Melatonin>Tryptophan. We hypothesize that such cellular DNA breakage is mediated by mobilization of copper ions bound to chromatin. Consistent with previous observations that many known antioxidants of plant and animal origin also exhibit prooxidant properties, these metabolites might contribute to oxidative DNA breakage in the presence of redox active metals such as copper. Our findings reveal a putative mechanism by which some endogenous metabolites may cause DNA damage leading to mutations and genetic diseases.

An Evaluation of Root Phytochemicals Derived from Althea officinalis (Marshmallow) and Astragalus membranaceus as Potential Natural Components of UV Protecting Dermatological Formulations.

As lifetime exposure to ultraviolet (UV) radiation has risen, the deleterious effects have also become more apparent. Numerous sunscreen and skincare products have therefore been developed to help reduce the occurrence of sunburn, photoageing, and skin carcinogenesis. This has stimulated research into identifying new natural sources of effective skin protecting compounds. Alkaline single-cell gel electrophoresis (comet assay) was employed to assess aqueous extracts derived from soil or hydroponically glasshouse-grown roots of Althea officinalis (Marshmallow) and Astragalus membranaceus, compared with commercial, field-grown roots. Hydroponically grown root extracts from both plant species were found to significantly reduce UVA-induced DNA damage in cultured human lung and skin fibroblasts, although initial Astragalus experimentation detected some genotoxic effects, indicating that Althea root extracts may be better suited as potential constituents of dermatological formulations. Glasshouse-grown soil and hydroponic Althea root extracts afforded lung fibroblasts with statistically significant protection against UVA irradiation for a greater period of time than the commercial field-grown roots. No significant reduction in DNA damage was observed when total ultraviolet irradiation (including UVB) was employed (data not shown), indicating that the extracted phytochemicals predominantly protected against indirect UVA-induced oxidative stress. Althea phytochemical root extracts may therefore be useful components in dermatological formulations.

Chemical Exposure and Effects in Freshwater Aquatic Species

To protect aquatic ecosystems from stresses induced by toxic chemicals, risk models must be developed, calibrated and verified for feral populations. Although risk models require good estimates of chemical exposure (dose) and quantitative measures of effects (response), few models, if any, have been appropriately calibrated for linking dose and response in situ. It is this lack of knowledge that limits the development of strong cause-effect relationships for aquatic species. Exposure models, using quantitative biomonitoring techniques, have been developed independently from bioassay and/or biomarker systems that are used to quantify stress. The present research integrated these two approaches by comprehensively calibrating the brown bullhead (Ameiurus nebulosus) as an appropriate bioindicator of toxicological stress in aquatic ecosystems. This was achieved by integrating quantitative biomonitoring techniques with a novel, sensitive assay for genotoxicity of polynuclear aromatic hydrocarbons (PAHs). At four sites along the river, chemical exposures and the incidence of dermal papillomas and neoplastic lesions in liver tissue were quantified, along with the measurement of PAH metabolites in bile, quantification of DNA damage in erythrocytes using the alkaline single cell gel electrophoresis (Comet assay), and the quantification of changes in apoptotic processes in dermal and liver tissues of exposed fish. Field studies on metabolite formation and apoptosis were compared to laboratory investigations in order to quantify the link between PAH metabolites and genotoxicity. Bullheads were exposed to Detroit River bottom sediment and environmentally-relevant levels of a major genotoxic PAH, benzo[a]pyrene (B[a]P), and dose-response models of metabolite formation were developed in order to determine the mass of PAH chemicals that are converted to DNA active metabolites. Genotoxicity was assessed in these laboratory populations using the comet assay on erythrocytes and liver cells. We assessed the link between DNA damage and the down-regulation of apoptosis, which is characterized as one of the key initial steps of tumorogenesis. This title belongs to WERF Research Report Series ISBN: 9781843397540 (Print) ISBN: 9781780403922 (eBook)

Total antioxidant status and oxidative stress in recurrent aphthous stomatitis.

Recurrent aphthous stomatitis is an idiopathic, chronic, recurrent inflammatory disease of the oral mucosa. It is thought that oxidative stress caused by systemic inflammation plays a basic role in the etiopathogenesis of recurrent aphthous stomatitis. The aim of this study is to review oxidative status and DNA damage in recurrent aphthous stomatitis. The study included 42 patients with an active recurrent aphthous stomatitis lesion and 39 healthy volunteers with similar demographic characteristics. DNA damage was analyzed using alkaline single cell gel electrophoresis (comet assay). Plasma levels of total antioxidant status and total oxidative status were determined by using an automated measurement method. Oxidative stress index was calculated as total oxidative status/total antioxidant status and × 100. The total oxidative status and oxidative stress index values were significantly higher in the recurrent aphthous stomatitis group compared to the control group, while total antioxidant status values were significantly lower. In the recurrent aphthous stomatitis group, DNA damage was observed to be significantly higher than the control group. In correlation analysis, significant correlation was found between DNA damage and the oxidative stress index and total oxidative status values in the recurrent aphthous stomatitis group. This is the first report in the literature that demonstrates association of recurrent aphthous stomatitis with increased oxidative status.

Evaluation of bioremediation potentiality of ligninolytic Serratia liquefaciens for detoxification of pulp and paper mill effluent

Due to high pollution load and colour contributing substances, pulp and paper mill effluents cause serious aquatic and soil pollution. A lignin-degrading bacterial strain capable of decolourising Azure-B dye was identified as lignin peroxidase (LiP) producing strain LD-5. The strain was isolated from pulp and paper mill effluent contaminated site. Biochemical and 16S rDNA gene sequence analysis suggested that strain LD-5 belonged to the Serratia liquefaciens. The strain LD-5 effectively reduced pollution parameters (colour 72%, lignin 58%, COD 85% and phenol 95%) of real effluent after 144h of treatment at 30°C, pH 7.6 and 120rpm. Extracellular LiP produced by S. liquefaciens during effluent decolourisation was purified to homogeneity using ammonium sulfate (AMS) precipitation and DEAE cellulose column chromatography. The molecular weight of the purified lignin peroxidase was estimated to be ∼28kDa. Optimum pH and temperature for purified lignin peroxidase activity were determined as pH 6.0 and 40°C, respectively. Detoxified effluent was evaluated for residual toxicity by alkaline single cell (comet) gel electrophoresis (SCGE) assay using Saccharomyces cerevisiae MTCC 36 as model organism. The toxicity reduction to treated effluent was 49.4%. These findings suggest significant potential of S. liquefaciens for bioremediation of pulp and paper mill effluent.

Mobilization of Copper ions by Flavonoids in Human Peripheral Lymphocytes Leads to Oxidative DNA Breakage: A Structure Activity Study.

Epidemiological studies have linked dietary consumption of plant polyphenols with lower incidence of various cancers. In particular, flavonoids (present in onion, tomato and other plant sources) induce apoptosis and cytotoxicity in cancer cells. These can therefore be used as lead compounds for the synthesis of novel anticancer drugs with greater bioavailability. In the present study, we examined the chemical basis of cytotoxicity of flavonoids by studying the structure–activity relationship of myricetin (MN), fisetin (FN), quercetin (QN), kaempferol (KL) and galangin (GN). Using single cell alkaline gel electrophoresis (comet assay), we established the relative efficiency of cellular DNA breakage as MN > FN > QN > KL > GN. Also, we determined that the cellular DNA breakage was the result of mobilization of chromatin-bound copper ions and the generation of reactive oxygen species. The relative DNA binding affinity order was further confirmed using molecular docking and thermodynamic studies through the interaction of flavonoids with calf thymus DNA. Our results suggest that novel anti-cancer molecules should have ortho-dihydroxy groups in B-ring and hydroxyl groups at positions 3 and 5 in the A-ring system. Additional hydroxyl groups at other positions further enhance the cellular cytotoxicity of the flavonoids.

Dinuclear manganese(II) complexes of hexaazamacrocycles bearing N-benzoylated pendant separated by aromatic spacers: Antibacterial, DNA interaction, cytotoxic and molecular docking studies

Three new homodinuclear manganese(II) complexes of the type [Mn2L1–3(ClO4)(H2O)](ClO4)3 (1–3) have been synthesized via cyclocondensation of terephthalaldehyde with three different benzoylated pendants in the presence of manganese(II) perchlorate and characterized by spectroscopic methods. Cyclic voltammetric investigation of complexes (1–3) depict two quasi-reversible one electron reduction processes in the cathodic potential region (E1pc=–0.73 to–0.83V, E2pc=–1.31 to −1.40V) and two quasi-reversible one electron oxidation processes in the anodic potential region (E1pa=1.03 to 1.10V, E2pa=1.69 to 1.77V). Electronic absorption spectra of the complexes suggested tetrahedral geometry around the central metal ion. The observed low magnetic moment values (μeff, 5.60–5.68 B.M.) of the complexes indicate the presence of an antiferromagnetic spin–exchange interaction between two metal centers, which was also supported by the broad EPR signal. All the compounds were tested for antibacterial activity against Gram (–ve) and Gram (+ve) bacterial strains. The binding studies of complexes with CT-DNA suggested minor-groove mode of interaction. Molecular docking studies were carried out in order to find the binding affinity of complexes with DNA and protein EGFR Kinase. The complexes are stabilized by additional electrostatic and van der Waals interaction with the DNA, and support minor groove mode of binding. The cleavage activity of complexes on pBR322 plasmid DNA displays efficient activity through a mechanistic pathway involving hydroxyl radicals. The cytotoxicity of complexes 2 and 3 have been tested against human liver adenocarcinoma (HepG2) cell line. Nuclear-chromatin cleavage has also been observed with propidium iodide (PI) staining and alkaline single-cell gel electrophoresis (comet assay) techniques.

Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay.

The food processing contaminants 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), 5‐hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N‐hydroxy‐PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild‐type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N‐hydroxy‐PhIP and HMF in vivo. Environ. Mol. Mutagen. 56:709–714, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

Effects of 5-Fluorouracil, Etoposide and CdCl2 in Aquatic Oligochaeta Limnodrilus udekemianus Claparede (Tubificidae) Measured by Comet Assay.

Genotoxicity of 5-fluorouracil (5-FU), etoposide (ET) and cadmium chloride (CdCl2) was evaluated in Limnodrilus udekemianus, cosmopolitan tubificid species, by alkaline single-cell gel electrophoresis (comet assay). Groups of 50 individuals were exposed in vivo in water-only short-term (96 h) tests to 5-FU (0.004, 0.04, 0.4, 4 and 40 μM), ET (0.004. 0.04, 0.4 and 4 μM) and CdCl2 (0.004, 0.04, 0.4, 4 and 40 μM). Mortality of worms was observed only for CdCl2 (4 and 40 μM). Cell viability lower than 70 % was detected for 5-FU (0.4, 4 and 40 μM), ET (4 μM) and CdCl2 (0.4 and 4 μM). All tested substances induced significant increase of DNA damage except 0.004 μM of ET. L. udekemianus being sensitive to all tested substances indicates that it can be used in ecogenotoxicology studies. Concern should be raised to cytostatics, especially to 5-FU, since concentration of 0.004 μM induced DNA damage is similar to ones detected in wastewaters.

Mobilization of copper ions in human peripheral lymphocytes by catechins leading to oxidative DNA breakage: A structure activity study

Epidemiological studies suggest that dietary consumption of plant polyphenols is related to a lower incidence of various cancers. Among these compounds catechins (present in green tea and other beverages) are considered to be potent inducers of apoptosis and cytotoxicity to cancer cells. Thus these compounds can be used as leads to synthesize novel anticancer drugs with greater bioavailability. In view of this in this paper we have examined the chemical basis of cytotoxicity of catechins by studying the structure–activity relationship between catechin (C), epicatechin (EC), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG). Using single cell alkaline gel electrophoresis (comet assay) we have established the relative efficiency of cellular DNA breakage as EGCG>EGC>EC>C. We also show that cellular DNA breakage is the result of mobilization of copper ions bound to chromatin and the generation of reactive oxygen species. Further the relative DNA binding affinity order was confirmed using molecular docking and thermodynamic studies by studying the interaction of catechins with calf thymus DNA. The results suggest that the synthesis of any novel anti cancer molecule based on the structure of catechins should have as many galloyl moieties as possible resulting in an increased number of hydroxyl groups that may facilitate the binding of the molecule to cellular DNA.