Evaluation of Oxidative DNA Damage Using an Alkaline Single Cell Gel Electrophoresis (SCGE) Comet Assay, and the Protective Effects of N-Acetylcysteine Amide on Zearalenone-induced Cytotoxicity in Chang Liver Cells

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium that are found in cereals and agricultural products. ZEN has been implicated in mycotoxicosis in farm animals and in humans. The toxic effects of ZEN are well known, but the ability of an alkaline Comet assay to assess ZEN-induced oxidative DNA damage in Chang liver cells has not been established. The first aim of this study was to evaluate the Comet assay for the determination of cytotoxicity and extent of DNA damage induced by ZEN toxin, and the second aim was to investigate the ability of N-acetylcysteine amide (NACA) to protect cells from ZEN-induced toxicity. In the Comet assay, DNA damage was assessed by quantifying the tail extent moment (TEM; arbitrary unit) and tail length (TL; arbitrary unit), which are used as indicators of DNA strand breaks in SCGE. The cytotoxic effects of ZEN in Chang liver cells were mediated by inhibition of cell proliferation and induction of oxidative DNA damage. Increasing the concentration of ZEN increased the extent of DNA damage. The extent of DNA migration, and percentage of cells with tails were significantly increased in a concentration-dependent manner following treatment with ZEN toxin (p < 0.05). Treatment with a low concentration of ZEN toxin (25 microM) induced a relatively low level of DNA damage, compared to treatment of cells with a high concentration of ZEN toxin (250 microM). Oxidative DNA damage appeared to be a key determinant of ZEN-induced toxicity in Chang liver cells. Significant reductions in cytolethality and oxidative DNA damage were observed when cells were pretreated with NACA prior to exposure to any concentration of ZEN. Our data suggest that ZEN induces DNA damage in Chang liver cells, and that the antioxidant activity of NACA may contribute to the reduction of ZEN-induced DNA damage and cytotoxicity via elimination of oxidative stress.

Clastogenicity of Piper cubeba (Piperaceae) seed extract in an in vivo mammalian cell system

The plant Piper cubeba is widely distributed in tropical and subtropical regions and is used medically for various purposes but has not yet been evaluated for genotoxicity. We used male and female Swiss mice and Wistar rats and the comet assay and micronucleus test to investigate the mutagenic potential of a crude extract of P. cubeba seeds. The rodents were administered 0.5 g kg-1, 1.0 g kg-1 and 1.5 g kg-1 of the extract by gavage. For the Swiss mice, peripheral blood was collected 24 h after treatment for the comet assay, and at 48 and 72 h for the micronucleus test. For the Wistar rats, peripheral blood and hepatic cells were collected for the comet assay and bone marrow cells were collected for the micronucleus test 24 h after treatment. At 1.5 g kg-1, the highest dose tested, the extract induced a statistically significant increase in both the mean number of micronucleated polychromatic erythrocytes and the level of DNA damage in the rodent cell types analyzed. Under our experimental conditions, the P. cubeba seed extract was genotoxic in vivo when administered orally to mice and rats.

Estimation of the postmortem interval by determining nuclear DNA degradation from bone marrows and brains in rats / 中国法医学杂志

Objective To detect nuclear DNA degradation of bone marrows and brains in rat cadavers at different temperatures,and develop a new parameter for estimating early postmortem interval(PMI).Methods The brain and bone marrow were taken out for every 4h,during 0~40h after death at 10℃ and 20℃,respectively.And the single cell gel electrophoresis(SCGE) was carried out to detect the nuclear DNA degradation.Linear regression analysis was used to assay the relationship of the comet parameter HeadDNA%,Tail Length(TL) and Olive TailMoment(TM) with PMI.Results Different decline degrees of comet HeadDNA% were found in both brain cells and bone marrow cells after death,the decline of HeadDNA% in brain cells at 20℃ was faster.Compared with degradation in marrow cells,the linear relation between degradation of brain cells and PMI was better.Conclusion with that of comet parameters TL and TM,the perfect linear relationship between HeadDNA% and PMI was also observed.Conclusion Brain tissues are more suitable for PMI estimation by detecting degradation of DNA with SCGE.The HeadDNA% is more valuable for PMI estimation than TL and TM.

Study on cell DNA damage induced by tetramine using comet assay / 中国法医学杂志

Objective To detect DNA damage in rat lymphocytes and brain cells induced by tetramine and study the toxicological mechanism of tetramine.Methods Lymphocytes and brain cells were separated and collected from healthy rats.DNA damages of cells which were exposed to various doses of tetramine for 60 min was detected using the single cell gel electrophoresis (SCGE) or comet assay.Results These are different degree DNA damages of lymphocytes and brain cells exposed from doses 1/20 LD 50 doses of tetramine to 1/2 LD 50 doses of tetramine.The test groups are very significantly statistical different to the control group(P

Arsenic Induces DNA Damage via Reactive Oxygen Species in Human Cells

To elucidate arsenic-induced oxidative DNA damage, the genotoxicity of arsenic in human cells was comparatively studied with single cell gel electrophoresis (SCGE) assay in combination with the observation of the protective effects of dimethyl sulfoxide (DMSO) and catalase. Arsenic, at the concentration of 2.4 μM by coincubation for 24 hours, significantly induced DNA damage in HL60, a human promyelocytic leukemia cell line. In contrast, significant DNA damage was found in human mononucleocytes at the concentration of 4.8 μM or above. The cells were incubated separately with DMSO (12 mM/l), a well-known hydroxyl radical (OH-) scavenger, and catalase (1,300 U/ml), a hydrogen peroxide (H2O2) scavenger, for 6 hours and then further coincubated with various concentrations of arsenic for 24 hours at 37°C and 5% CO2. The findings showed that both DMSO and catalase significantly reduced the arsenic-induced tail moment, a parameter of total damaged DNA, in HL60 and mononucleocytes. Hence our findings indicate that arsenic, with micromolar concentrations, induces typical and various extents of DNA damage in human cells via reactive oxygen species in a dose-dependent manner.

EFFECT OF DIFFERENT LEVELS OF ASCORBIC ACID ON DNA DAMAGE / 营养学报

Objective: The study was to exlpore the nutritional, antioxidative functions and toxicology of ascorbic acid in different levels in vitro. [WT5FZ]Methods: Hela (human transformed epithelial) cells incubated with three levels of ascorbic acid, i.e. 0.1 mmol/L, 0.25 mmol/L and 0.5 mmol/L, were calculated and a single cell gel electrophoresis (SCGE) was used for measuring DNA oxidative damage. [WT5FZ]Results: The results showed that there were no differences in spontaneous DNA damage of Hela cells incubated with three levels of ascorbic acid. However, there was a less DNA oxidative damage induced by H 2O 2 in 0.1 mmol/L and 0.25 mmol/L of ascorbic acid supplemented groups respectively than in control group. In contrast, more serious DNA damage was found in 0.5 mmol/L ascorbic acid supplemented group. [WT5FZ]Conclusion: It is suggested that the higher levels of ascorbic acid might not directly damage DNA; the moderate supplementation of ascorbic acid may increase antioxidative ability of cells; excess ascorbic acid is harmful to DNA and enhances the susceptibility to H 2O 2 potentially.