Single Cell Gel Electrophoresis Comet Research Articles

In Vitro Modulation of Genotoxicity and Oxidative Stress by Polyphenol-Rich Fraction of Chinese Ladder Brake (Pteris vittata L.).

Pteris vittata L. is a terrestrial genus growing in moist, shady forests and on hillsides. The plant has considerable ethnomedicinal importance. Investigations have been carried out on chemical profiling and antioxidant compounds from some genera of pteridophytes but studies on the biological properties of P. vittata are lacking. Therefore, the present study investigates antioxidant, antigenotoxic, and antiproliferative potential of the aqueous fraction of P. vittata (PWE). A battery of assays were carried out to assess the antioxidant potential of the PWE. SOS chromotest and DNA nicking assay were used to evaluate the antigenotoxicity of the fraction. The cytotoxic effect of PWE was analyzed using MTT and Neutral Single Cell Gel Electrophoresis comet assay. EC50 of 90.188µg/ml, 80.13µg/ml, 142.836µg/ml, and 12.274µg/ml was obtained in DPPH, superoxide anion scavenging, reducing power and lipid peroxidation assays, respectively. PWE was potent in inhibiting Fenton's reagent-induced nicking of pBR322 plasmid. The fraction significantly inhibited hydrogen peroxide (H2O2) and 4-nitroquinoline-N-oxide (4NQO) induced mutagenicity and a reduction in induction factor was found with increased PWE concentration. GI50 of 147.16µg/ml was obtained in MTT assay in human MCF-7 breast cancer cell line. PWE induced apoptosis as confirmed from confocal microscopy studies. The protective effects can be attributed to the presence of the phytochemicals in PWE. These results will be helpful in the development of functional food characteristics, as well as unravel the benefits of pteridophytes as promoters of health.

Hepatic and renal cellular cytotoxic effects of heparin-coated superparamagnetic Iron oxide nanoparticles

BackgroundSuperparamagnetic iron oxide (SPIO) nanoparticles have been widely used in several biomedical engineering in vivo. Although various surface modifications have been made to these non-biodegradable nanoparticles to make them more biocompatible, their toxic potential still remains a major concern.MethodIn this study, we newly developed unfractionated heparin (UFH)-coated and low molecular weight heparin (LMWH)-coated SPIO nanoparticles through surface modification engineering, which was compared with commercially available dextran-coated SPIO nanoparticles. Their toxicity such as cytotoxicity, single cell gel electrophoresis (SCGE) comet assay, intracellular reactive oxygen species (ROS) content and cellular apoptosis was evaluated to hepatic HepG2 and renal HK-2 cells.ResultsWhen UFH-, LMWH- or dextran-coated SPIO nanoparticles were applied, they did not affect the viability of HepG2 cell. However, HK-2 cells were more sensitive to dextran-coated SPIO nanoparticles than others. In genotoxicity assay using SCGE comet, DNA tail moment values in the groups treated with dextran- and LMWH-coated SPIO nanoparticles significantly increased. However, UFH-coated SPIO nanoparticles was only significantly lowing DNA tail moment value. In addition, UFH-coated SPIO nanoparticles had lower cytotoxicity in HepG2 and HK-2 cells compared to dextran-coated SPIO nanoparticles, especially in terms of apoptosis and intracellular ROS production.ConclusionsCollectively, it is possible that UFH- coated SPIO nanoparticles can be used as alternative negative contrast agents.

EFFECT OF BOILING AND MICROWAVE COOKING ON NUTRITIONAL, ANTI-NUTRITIONAL AND TOXICITY OF WILD EDIBLE PLANTS OF NORTH-EASTERN REGION IN INDIA

Objective: The target of this appraisal was to explore the impacts of various cooking techniques, for example, boiling and microwave cooking on nutritional, antinutritional, minerals content and in vitro toxicity of ten wild consumable plants viz. Zanthoxylum acanthopodium, Viburnum foetidum, Houttuynia cordata, Sonchus arvensis, Oenanthe linearis, Perilla ocymoides, Clerodendrum colebrookeanum, Solanum gilo, Solanum kurzii, Potentilla lineata, widely consumed by the common individuals of North-Eastern area in India.
 Methods: The proximate parameters like ash, moisture, protein, fat, fibre, carbohydrate, energy content, minerals viz. sodium, calcium, potassium, iron, magnesium, manganese, copper, zinc and antinutritional parameters like oxalate, phytate, tannin, saponin, cyanogenic glycoside content were evaluated in the selected wild edible plants using standard food analysis techniques. In vitro haemolytic toxicity of aqueous extracts (100, 300 and 500µg/cc) of ten palatable wild plants was done with the blood samples were gathered from healthy rat, mixed with Ethylenediaminetetraacetic acid(EDTA) and centrifuged at 5,000 Revolutions Per Minute (rpm) for five minutes. The 10 % erythrocyte suspension was set up in sterile Phosphate buffer saline (PBS, pH 7.4) for haemolytic examination. The genotoxic potential of the concentrates were assessed by a single-cell gel electrophoresis comet test. Cytotoxicity studies were evaluated with fresh goat livers procured from the local market were perfused in PBS (pH 7.4) with collagenase and the liver was then minced in minute pieces and cells were isolated utilizing cell strainer.
 Results: Both cooking medications diminished the congregations of ash, fat, minerals, antinutritional parts and the destructive nature of the consumable plants while the carbohydrate and fiber substance were expanded. The protein focuses in the wild edibles were expanded fundamentally (P<0.05) in the range from 1.26 to 10.45% on microwave cooking while the indistinguishable were exhausted in the range out of 2.20 to11.55% on boiling treatment. The microwave cooking demonstrated lesser misfortunes in minerals in the consumable plants than those cooked by frothing. The microwave cooking also caused the colossal rot (P<0.05) of antinutritional parameters and damaging tendency to a more significant degree than the sputtering medications of the wild edibles.
 Conclusion: Therefore, the outcomes uncovered that microwaving of appealing plants could be prescribed to expand the supporting quality and to diminish the fat, threatening to dietary structure and lethality. The toxicity assessment of the consumable plants at cell and genomic level showed that these are harmless to consume.

RETRACTED: Evaluation of antioxidant activities, toxicity studies and the DNA damage protective effect of various solvent extracts of Litsea cubeba fruits

Litsea cubeba is devoured by the ethnic individuals of Arunachal Pradesh in India as food and has been traditionally used for curing different ailments. The purpose of present study was to investigate the antioxidant activities of fruits of L. cubeba using different solvent extracts, quantification of phenolics, toxicity studies and DNA damage protective activities. The antioxidant activities of fruits using five different solvent extracts completed utilizing different in vitro examines. The quantitation of phenolic and polyphenolic compounds in the methanol extract of the fruits was carried out by HPLC. The in vitro haemolytic examination of plant concentrates were completed on rat erythrocytes. Appraisal of cytotoxicity of eatable fruits was assessed by MTT measure. The genotoxicity of the contemplated plant was tried by the single-cell gel electrophoresis comet measure. The DNA defensive impacts of the aqueous extracts of fruits on rodent lymphocyte DNA lesions were likewise assessed with the comet test. The extract obtained by methanol exhibited the highest antioxidant activity. The HPLC examination of the methanol concentrate of the plant demonstrated the occurrence of different phenolic acids and flavonoids like caffeic acid (145.96μg/100mg DE), syringic acid (125.85 μg/100mg DE), ferulic acid (155.89 μg/100mg DE), apigenin (28.43 μg/100mg DE), kaempferol (53.41 μg/100mg DE) etc. in various amounts. The consequences of haemolytic lethality, cytotoxicity and genotoxicity of fluid concentrates of the edible plant ensure the security at cell and genomic level. The fluid concentrate of the plant fundamentally repressed DNA harm and these information recommend that the watery concentrate of L. cubeba can forestall oxidative DNA harm to rodent lymphocytes, which is likely because of antioxidant constituents in the concentrate. These outcomes demonstrate that L. cubeba can be utilized in dietary applications with a possibility to diminish oxidative pressure.

Nutritive scenario of wild consumable plants of Meghalaya state in India together with heavy metals, anti-nutritional, vitamin and toxicity studies

The present exploration was projected to assess the nutritive potential, minerals content, vitamin quantity, antinutritional properties and toxicity investigations in five wild edible plants for example Agapetes variegata, Ficus virens, Begonia roxburghii, Clerodendrum serratum and Rhododendron arboreum, devoured by the diverse ancestral individuals of Meghalaya State in India as their sustenance. The proximate parameters,minerals,toxic heavy metals and antinutritional components were evaluated using standard food analysis techniques. The water soluble vitamins were carried out by HPLC. The in vitro haemolytic assays of plants were carried out on rat erythrocytes. Assessments of cytotoxicity of edible plants were evaluated by MTT assay. The genotoxicity were tested by the single-cell gel electrophoresis comet assay. The outcomes of examination demonstrated that these plants are rich wellsprings of protein, minerals and vitamins. The estimations of antinutrients and substantial metals in the plants are beneath the lethal levels and furthermore safe to consume.

Wild- edible plants of Meghalaya State in India: Nutritional, minerals, antinutritional, vitamin content and toxicity studies

Aim: The point of the present investigation planned to assess the nutritive value, minerals content, vitamin content, antinutritional properties, and toxicity studies in five wild-consumable plants, for example, Begonia hatacoa, Embelia floribunda, Artemisia vulgaris, Cardamine hirsuta, and Plantago major, devoured by the distinctive innate individuals of Meghalaya State in India as their nourishment. Materials and Methods: The proximate parameters such as protein, fat, fiber, sugar, minerals, and harmful overwhelming metals were assessed in the chose wild-eatable plants utilizing standard sustenance examination procedures. The cyanogenic glycoside, oxalates, tannins, saponins, and phytate substances were done to decide the counter wholesome properties of the considered plants. The quantitations of water-dissolvable vitamins in these plants were completed by high-performance liquid chromatography. The in vitro hemolytic measure of plant concentrates was completed on rodent erythrocytes. Appraisal of cytotoxicity of eatable plants was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide examine. The genotoxicity of the contemplated plants was tried by the single-cell gel electrophoresis comet assay. Results: The consequences of examination demonstrated that these plants are rich wellsprings of protein, sugar minerals, and vitamins, particularly the B gathering of vitamins. The substantial metals lead and chromium were distinguished in exceptionally low sum in all the consumable plants. The largest amount of phytate was found in P. major (0.38% ± 0.03%); oxalate was most astounding in E. floribunda (0.261% ± 0.06%) though tannin was most elevated in P. major (1.04% ± 0.12%). The estimations of antinutrients and overwhelming metals in all above-considered plants are underneath the harmful levels. Conclusion: These wild-eatable plants contribute gigantically to sustenance, nourishment security, well-being, and remedial advantages. The aftereffects of poisonous quality of every single eatable plant ensure the security at cell and genomic level and furthermore safe to expend.

Exogenous Plant-Based Nutraceutical Supplementation and Peripheral Cell Mononuclear DNA Damage Following High Intensity Exercise.

Plant-based nutraceuticals are categorised as nutritional supplements which contain a high concentration of antioxidants with the intention of minimising the deleterious effect of an oxidative insult. The primary aim of this novel study was to determine the effect of exogenous barley-wheat grass juice (BWJ) on indices of exercise-induced oxidative stress. Ten (n = 10) apparently healthy, recreationally trained (V̇O2max 55.9 ± 6 mL·kg−1·min−1), males (age 22 ± 2 years, height 181 ± 6 cm, weight 87 ± 8 kg, body mass index (BMI) 27 ± 1) volunteered to participant in the study. In a randomised, double-blinded, placebo-controlled crossover design, participants consumed either a placebo, a low dose (70 mL per day) of BWJ, or a high dose (140 mL per day) of BWJ for 7-days. Experimental exercise consisted of a standard maximal oxygen uptake test until volitional fatigue. DNA damage, as assessed by the single cell gel electrophoresis comet assay, increased following high intensity exercise across all groups (time × group; p < 0.05, Effect Size (ES) = 0.7), although there was no selective difference for intervention (p > 0.05). There was a main effect for time in lipid hydroperoxide concentration (pooled-group data, pre- vs. post-exercise, p < 0.05, ES = 0.2) demonstrating that exercise increased lipid peroxidation. Superoxide dismutase activity (SOD) increased by 44.7% following BWJ supplementation (pooled group data, pre- vs. post). The ascorbyl free radical (p < 0.05, ES = 0.26), α-tocopherol (p = 0.007, ES = 0.2), and xanthophyll (p = 0.000, ES = 0.5), increased between the pre- and post-exercise time points indicating a main effect of time. This study illustrates that a 7-day supplementation period of a novel plant-derived nutraceutical product is insufficient at attenuating exercise-induced oxidative damage. It is possible that with a larger sample size, and longer supplementation period, this novel plant-based nutraceutical could potentially offer effective prophylaxis against exercise-induced oxidative stress; as such, this justifies the need for further research.

8. Evaluation of Snow Fungus Polysaccharides on benzo[a]pyrene-induced DNA Damage

Snow fungus, Tremella fuciformis, has been demonstrated to have numerous health benefits including purported chemopreventive properties due to free radical-scavenging ability. Protective effects derived from snow fungus polysaccharides are evaluated on Chinese hamster lung fibroblasts (CCL-39) exposed to carcinogen benzo[a]pyrene known to cause free radical formation and oxidative stress to cells.&#x0D; In this experiment, it was hypothesized that the naturally occurring polysaccharides in snow fungus are able to protect against or reduce oxidative stress-induced DNA damage. Polysaccharides were isolated through an alkaline extraction and in-vitro digestion. DNA damage was measured using the single-cell gel electrophoresis comet assay after exposure to benzo[a]pyrene and polysaccharide extract to lung fibroblasts. Results were calculated using the mean and standard deviation data of tail length and area, respectively. Each damaged cell was measured and analyzed through ImageJ Editing Software. The results indicate a promising trend which depict snow fungus polysaccharides yielding lower levels of DNA damage compared to cells exposed to benzo[a]pyrene and compared to the negative control (phosphate buffered saline and Dulbecco’s cell medium). This study suggests polysaccharides from Tremella fuciformis could truly prevent cellular DNA damage by protecting against oxidative stress.

Genotoxicological Assessment of Nebuloside-A a Triterpenoid Saponin Compound on Whole Blood DNA

We investigated the genotoxic effect of nebuloside-A on whole blood DNA by using alkaline Single Cell Gel Electrophoresis Comet assay. Saponins are a widespread class of bioactive compounds produced by many plant species. Nebuloside A was isolated from baby’s-breath (Gypsophila arrostii) by high performance liquid chromatography method. Mass spectra obtained by electrospray ionization-time of flight-mass spectrometry in a negative ion mode to reduce the protonation. Complete nuclear magnetic resonance spectroscopy assignments of nebuloside A was achieved by using 2D-nuclear magnetic resonance spectroscopy techniques, such as double-quantum filtered correlation spectroscopy, heteronuclear single quanthum correlation, heteronuclear multiple-bond correlation, and total correlation spectroscopy. DNA damage was measured in total arbitrary units by visual scoring of comets in different concentrations of nebuloside A and H2O2 treatments. We found that significant dose dependent relationship between nebuloside A and its genotoxic effect.

Evaluation of Cytotoxicity and Genotoxicity ofAcacia aromaLeaf Extracts

Acacia aroma, native plant from San Luis, Argentina, is commonly used as antiseptic and for healing of wounds. The present study was conducted to investigate the in vitro cytotoxicity and genotoxicity of hot aqueous extract (HAE) and ethanolic extract (EE) of A. aroma. The cytotoxic activity was assayed by neutral red uptake assay on Vero cell. Cell treatment with a range from 100 to 5000 μg/mL of HAE and EE showed that 500 μg/mL and 100 μg/mL were the maximum noncytotoxic concentrations, respectively. The CC50 was 658 μg/mL for EE and 1020 μg/mL for HAE. The genotoxicity was tested by the single-cell gel electrophoresis comet assay. The results obtained in the evaluation of DNA cellular damage exposed to varied concentrations of the HAE showed no significant genotoxic effect at range of 1–20 mg/mL. The EE at 20 mg/mL showed moderate genotoxic effect related to the increase of the DNA percentage contained in tail of the comet; DNA was classified in category 2. At concentrations below 5 mg/mL, the results of cytotoxicity and genotoxicity of aqueous and ethanolic extracts of Acacia aroma guarantee the safety at cell and genomic level. However further studies are needed for longer periods including animal models to confirm the findings.

&lt;I&gt;In Vitro&lt;/I&gt; Radiosensitizing Effects of Ultrasmall Gadolinium Based Particles on Tumour Cells

Since radiotherapy is widely used in cancer treatment, it is essential to develop strategies which lower the irradiation burden while increasing efficacy and become efficient even in radio resistant tumors. Our new strategy is relying on the development of solid hybrid nanoparticles based on rare-earth such as gadolinium. In this paper, we then evidenced that gadolinium-based particles can be designed to enter efficiently into the human glioblastoma cell line U87 in quantities that can be tuned by modifying the incubation conditions. These sub-5 nm particles consist in a core of gadolinium oxide, a shell of polysiloxane and are functionalized by diethylenetriaminepentaacetic acid (DTPA). Although photoelectric effect is maximal in the [10-100 keV] range, such particles were found to possess efficient in-vitro radiosensitizing properties at an energy of 660 keV by using the "single-cell gel electrophoresis comet assay," an assay that measures the number of DNA damage that occurs during irradiation. Even more interesting, the particles have been evidenced by MTT assays to be also efficient radiosensitizers at an energy of 6 MeV for doses comprised between 2 and 8 Gy. The properties of the gadolinium-based particles give promising opening to a particle-assisted radio-therapy by using irradiation systems already installed in the majority of hospitals.

Acute effects of benzo[a]pyrene on digestive gland enzymatic biomarkers and DNA damage on mussel Mytilus galloprovincialis

In the present study, mussel ( Mytilus galloprovincialis) digestive gland biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) were investigated. Mussels were exposed to a sublethal dose of B[a]P (75 nM; 19 μg/l per animal) for 24, 48 and 72 h. The following biological responses were measured in the digestive gland tissues: (1) B[a]P hydroxylase (BPH) activity, as phase I biotransformation parameter; (2) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (3) catalase (CAT) activity as potential biomarker of oxidative stress, (4) acetylcholinesterase (AChE) activity as an indication of possible neurotoxicity response. DNA damage was assessed over time using the single cell gel electrophoresis comet assay and the micronuclei test. BPH and GST activities showed an increasing trend over exposure period. CAT activity showed a symmetrical bell shape response with a maximum at 48 h. AChE activity was significantly depressed after 48 and 72 h exposure to B[a]P. Comet assay and micronuclei test in digestive gland cells suggest that B[a]P exposure induced significant DNA damage with a maximum response after 72 h exposure.

Acute effects of benzo[a]pyrene on liver phase I and II enzymes, and DNA damage on sea bream Sparus aurata

In the present study biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated in the liver of Sparus aurata (sea bream). Sexually immature gilthead sea bream were treated by intraperitoneal injection of B[a]P (20 mg kg(-1)) for 6, 12, 24, and 48 h. B[a]P accumulation was quantified in sea bream liver by mean of gas phase chromatography (GPC-MS) after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity, as a phase I biotransformation parameter; (2) liver glutathione S-transferase (GST) activity as a phase II conjugation enzyme. DNA damage was assessed over time using the single-cell gel electrophoresis comet assay. B[a]P bioaccumulation in the liver resulted in a biphasic curve with an increasing uptake up to 5.55 +/- 0.67 microg g(-1) dry weight after only 6 h exposure and 4.67 +/- 0.68 microg g(-1) dry weight after 48 h exposure. EROD activity showed a nonsymmetrical bell-shaped kinetic with a maximum at 24 h and lower but significant activities at 12 and 48 h with respect to control animals. Hepatic GST activities were only significant after 48 h exposure. Comet assay showed an increase in liver cells DNA damage with a maximum after 48 h exposure reaching up to 12.17 % DNA in the tail.

Genotoxicity of Enflurane in Human Peripheral Blood Lymphocytes Studied in vivo by Single Cell Gel Electrophoresis

Background: The alkaline single cell gel electrophoresis comet assay was applied to study the genotoxic properties of enflurane on the human peripheral blood lymphocytes (PBL) of cancer patients before and during anesthesia as compared to an non-cancer control group. Method: The cancer group consisted of 24 patients (aged 15-77 years), while the control group consisted of 14 trauma individuals (aged 20-81 years). After anesthesia induction (thiopental 4 mg/kg and vecuronium 0.1 mg/kg), it was maintained by enflurane inhalation; 1-2 minimal alveolar concentration in oxygen - nitrous oxide mixture. Venous blood samples were obtained before the induction of anesthesia, and after 60 and 120 min of anesthesia. The comet assay detects DNA damage, such as strand breaks and alkaline labile sites induced directly by genotoxic agents, and DNA degradation due to cell death. Fifty cells from each sample were examined and Olive tail moments (OTM) were calculated using Komet software. Results: OTM values were no different between controls and patients before anesthesia. However, the OTMs of blood sampled from cancer patients at 60 (7.97 1.83) and 120 min (7.86 2.05), and from trauma patients at 120 min (8.04 1.32) of anesthesia were significantly increased. Conclusions: In immunocompromised cancer patients, we suggest the existence of a higher risk of an association DNA damage and enflurane exposure.

Phototoxicity to diuretics and antidiabetics in the cultured keratinocyte cell line HaCaT: evaluation by clonogenic assay and single cell gel electrophoresis Comet assay)

Potential phototoxicity has been described for a number of drugs and chemical substances. Psoralens, chlorpromazines and fluoroquinolones have been described as inducing photomutagenicity and photocarcinogenicity in vitro and in vivo. We wanted to investigate oral antidiabetics and diuretics for potential phototoxicity and possible DNA damage in the HaCaT cell line. : The oral antidiabetics tolbutamide, glibenclamide and glipizide, and the diuretics bendroflumethiazide, butizide, furosemide, hydrochlorothiazide and trichlormethiazide were dissolved in DMSO to final concentrations of 1 mM, 0.1 mM, and 0.01 mM, incubated together with the cells, and exposed to UVA1 (23 or 48 J/cm2). Cell survival was evaluated in a clonogenic assay and phototoxic DNA damage was investigated by single cell gel electrophoresis (comet assay). To investigate possible inhibiting effects of antioxidants, L-ascorbic acid and alpha-tocopherol were added at a final concentration of 1 mM 24 h before treatment with the drugs. Bendroflumethiazide, furosemide, hydrochlorothiazide, trichlormethiazide and tolbutamide induced dose-dependent phototoxicity in the clonogenic assay. Cells incubated with bendroflumethiazide, tolbutamide and glibenclamide and irradiated with UVA1 demonstrated increased oxidative DNA damage, revealed as alkali-labile sites in the comet assay. Pretreatment with L-ascorbic acid or alpha-tocopherol suppressed the UVA-induced DNA damage in cells incubated with 1 mM bendroflumethiazide, furosemide, glibenclamide, glipizide, tolbutamide or trichloromethiazide. Several oral antidiabetics and diuretics show phototoxic effects in the HaCaT cell line. Inhibiting effects of antioxidants point towards involvement of reactive oxygen species in phototoxic DNA damage, suggesting a link between the phototoxic and photocancerogenic potential of the sulfonamide-derived oral antidiabetic and diuretic drugs. Excessive exposure to UV light may be deleterious for patients treated with oral antidiabetic and diuretic drugs.