Cell Gel Electrophoresis Comet Assay 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.

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.

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.

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.

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.