• Proceedings of the Korea Society of Environmental Toocicology Conference
• /
• 2003.10a
• /
• pp.159-159
• /
• 2003

Comet assay is a potential monitoring tool because DNA strand breakage may be produced by a wide range of agents. The comet assay, also called the single-cell gell electrophoresis (SCGE) assay, is rapid and sensitive method for the detection of DNA damage in cells. This study was performed for the identification of DNA damage in the cells from flounders and clams exposed to PAHs. As a control experiments, flounder and clam cells were exposed to $H_2O$$_2$. The cells exposed to $H_2O$$_2$ were displayed a typical nuclei movement DNA damage of cells were significantly increased when the isolated cells from the blood of flounders and the tissue of clams were in vitro exposed to the different concentrations (5, 10, 50, 100 ppb) of five kinds of PAHs (benzo[a]pyrene, pyrene, fluoranthene, anthrancene, and phenanthrene). For the in vivo test, flounders and clams were exposed to the different concentrations of BaP for 4 days. The results showed that DNA strand breakage was effected by the concentration of BaP and the duration of exposure. In high concentration of BaP, the mean tail lengths of nuclei was longer than it In low concentration, while the mean size of head DNA decreased. In this research, both in vitro and in vivo genotoxicity of PAHs could be biomonitored by the comet assay. Especially, clams and flounders seem to be useful as materials for monitoring genotoxic damage by comet assay.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.2
• /
• pp.268-275
• /
• 2004

To investigate the potential application of the single-cell gel electrophoresis (SCGE) assay to carp as an aquatic pollution monitoring technique, gill, liver, and blood cells were isolated from carp exposed to a direct-acting mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or indirect mutagen, $benzo[\alpha]pyrene$ $(B[\alpha]P)$, then the DNA strand breakage was analyzed using the assay. Based on testing 5 different cell isolation methods and 6 electrophoretic conditions, the optimized assay conditions were found to be cell isolation by filter pressing and electrophoresis at a lower voltage and longer running time (at 0.4 V/cm for 40 min). In preliminary experiments, gill and liver cells isolated from carp exposed to MNNG in vitro exhibited DNA damage signals even with 0.5 ppb exposure, which is a much higher dose than previously reported. In the gill cells isolated from carp exposed to 0.01-0.5 ppm MNNG in vivo, significant dose-and time-dependent increases were observed in the tail for 4 days. As such, the linear correlation between the relative damage index (RDI) values and time for each dose based on the initial 48-h exposure appeared to provide effective criteria for the genotoxicity monitoring of direct-acting mutagenic pollution. In contrast, the in vivo exposure of carp to 0.25-1.0 ppm of $B[\alpha]P$ for 7 days resulted in dose-and time-dependent responses in the liver cells, in which 24-h delayed responses for metabolizing activation and gradual repair after 48 h were also observed. Thus, the negative-sloped linear correlation between the RDI and time at each dose based on the initial 48 h appeared to provide more effective criteria for the genotoxicity monitoring of indirect mutagenic pollution.

• Journal of Food Hygiene and Safety
• /
• v.17 no.2
• /
• pp.106-116
• /
• 2002

The 70% ethanol extract of Alpinia officinarum and its major flavonoid, galangin showed strong antioxidative effect on the lipid peroxidation of ethyl linolate with Fenton's reagent and free radical scavenging effect to DPPH radical generation. However, they did not reveal any pro-oxidant effect on bleomycin-Fe(III) dependent DNA degradation. They also showed the protective effect against $H_2O$$_2$, KO$_2$ or UV-induced cytotoxicity in mammalian cells. They also showed the suppressive effect of DNA damage induced by $H_2O$$_2$ or KO$_2$ with dose-dependent manner in single cell gel electrophoresis(SCGE) assay. On the other hand, they have an anticlastogenic effect against adriamycin-induced micronucleated reticulocyte in peripheral blood of mice. These results suggest that the mechanism of inhibition by 70% ethanol extract of Alpinia officinarum and galangin against reactive oxygen species (ROS)-induced genotoxicity or cytotoxicity is due, at least partly, to their antioxidative and free radical scavenging properties without pro-oxidant effect. All these results indicate that 70% ethanol extract of Alpinia officinarum and galangin may be useful for protection against ROS-induced cytotoxicity and DNA damage.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.32 no.5
• /
• pp.739-744
• /
• 2003

This study was carried out to investigate the antioxidative activity and to identify the active components of hot-water extract of Paeoniajaponica (PJ), which was a main ingredient of a herb mixture preparation recently established as a potent candidate of radioprotector in our laboratory. The water extract was fractionated with CHCl$_3$, EtOAc and n-BuOH. The extract and its fractions showed very low activity in hydroxyl radical scavenging test. In lipid peroxidation test, the extract, EtOAc and water fractions showed moderate inhibition with the ratio above 50%. In DPPH radical scavenging test, the extract, EtOAc and water fraction showed high activity with the ratio above 80%, especially. EtOAc fraction scavenged the radicals as much as synthetic antioxidant (BHA), even at low concentration. It is suggested that mai or partition for antioxidative activity of Paeonia japonica was EtOAc fraction. Subsequently, two active compounds (PJE021-1 and JE024-1) from EtOAc fraction were isolated by using MCI gel and silica gel column chromatography The two compounds inhibited remarkedly the $H_2O$$_2$-induced DNA damage in human peripheral blood lymphocytes, measured by single-cell gel electrophoresis (SCGE). PJE021-1 protected the cells to almost negative control level, dose-dependently. PJE024-1 exhibited a potent inhibition with the ratio of 71% at even low concentration (0.5 $\mu\textrm{g}$/$m\ell$). Finally, their chemical structures were identified as gallic acid (PJE021-1) and (＋)-catechin (PJE024-1), respectively, on the basis of the speculation of spectral and physical data.

• Korean Journal of Environmental Biology
• /
• v.29 no.3
• /
• pp.236-240
• /
• 2011

Mercury known as quicksilver, is the most common cause of heavy metal toxicity. Toxicity caused by excessive mercury exposure is now being recognized as a widespread environmental problem and is continuing to attract a great deal of public concerns. The mercury genotoxicity could be its effect on DNA repair mechanisms, which constitute the defense system designated to protect genome integrity. The objective of this study is to confirm that mercuric chloride inhibits the repair of gamma ray-induced DNA damage. The earthworm of Eisenia fetida was chosen for this study because it is an internationally accepted model species for toxicity testing with a cosmopolitan distribution. Experiments were done to identify the levels of DNA damage and the repair kinetics in the coelomocytes of E. fetida irradiated with 20 Gy gamma rays alone or with gamma rays after 40 mg $kg^{-1}$ $HgCl_2$ treatment by means of the single cell gel electrophoresis assay. The Olive tail moments were measured during 0~96 hours after irradiation. The repair time in the animals treated with the combination of $HgCl_2$ and ionizing radiation was nearly five times longer than that in the animals treated with ionizing radiation alone. Also, E. fetida exposed to mercury showed a statistically lower repair efficiency of gamma ray-induced DNA damage. The results suggest that the mercury could even have deleterious effects on the DNA repair system. Influence of mercury on the DNA repair mechanisms has been confirmed by this study.

• Molecular & Cellular Toxicology
• /
• v.5 no.2
• /
• pp.126-132
• /
• 2009

The usage and types of chemicals are advancing, specializing, large-scaled increasing, and new chemical exposed workers are concerning to occupational disease. The generation of reactive oxygen in the body from carcinogen, mutation and DNA damage in cancer is protected by natural antioxidants (phytochemicals) with antimutagenic effect. There were many reports of ginsenoside Rb$_1$, Rg$_1$ grievances of the genetic mutation to suppress the effect confirm the genetic toxicity test with chromosomal aberration test and the Comet (SCGE) assay confirmed the suppression effect occurring chromosomal DNA damage. We had wanted to evaluate the compatibility and sensitivity between the chromosomal aberration (CA) test and the Comet assay. We used the CA test and Comet assay to evaluate the anti-genotoxicity of ginsenoside Rb$_1$ and Rg$_1$, in CHO-K1 (Chinese hamster ovary fibroblast) cell in vitro, composed negative control (solvent), positive control (benzo[a]pyrene), test group (carcinogen+variety concentration of ginsenoside) group. The positive control was benzo[a]pyrene (50 $\mu$M), well-known carcinogen, and the negative control was the 1 % DMSO solvent. The test group was a variety concentration of ginsenoside Rb$_1$, Rg$_1$ with 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10%. In chromo-somal aberration test, we measured the number of cells with abnormally structured chromosome. In Comet assay, the Olive tail moment (OTM) and Tail length (TL) values were measured. The ratio of cell proliferation was increased 8.3% in 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10% Rb$_1$ treated groups, and increased 10.4% in 10$^{-10}$%, 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1% Rg$_1$ treated groups. In the CA test, the number of chromosomal aberration was decreased all the Rb$_1$ and Rg$_1$ treated groups. In the Comet assay, the OTM values were decreased in all the Rb$_1$ and Rg$_1$ treated groups. To evaluate the compatibility between CA and Comet assay, we compared the reducing ratio of chromosomal abnormalities with its OTM values, it was identified the antimutagenicity of ginsenoside, but it was more sensitive the CA test than the Comet assay. Ginsenoside Rb$_1$ and Rg$_1$ significantly decrease the number of cells with chromosomal aberration, and decrease the extent of DNA migration. Therefore, ginsenoside Rb$_1$, Rg$_1$ are thought as an antioxidant phytochemicals to protect mutagenicity. The in vitro Comet assay seems to be less sensitive than the in vitro chromosomal aberration test.

• Journal of Nutrition and Health
• /
• v.36 no.2
• /
• pp.125-132
• /
• 2003

The present study was attempted to investigate and compare the antioxidant potency of several well-know flavonoids, antioxidant vitamin and commercially available popular beverages. The antioxidant potency was assessed by the effect on reducing oxidative DNA damage of human lymphocytes. Cellular oxidative DNA damage was measured by SCGE (single-cell gel electrophoresis), also known as comet assay. Lymphocytes were pre-treated for 30 minutes with wide ranges of doses of apigenin, kaempferol, luteolin, myricetin, rutin, quercetin, $\alpha$-tocopherol (10,25,50,100,200,500,1000 $\mu$M) ,green tea extract or grape juice (10,50,100,250,500,1000 $\mu$g/mL) followed by a $H_2O$$_2$(100 $\mu$M) treatment for 5 min as an oxidative stimulus. The physiological function of each antioxidant substance on oxidative DNA damage was analyzed as tail moment (tail length $\times$ percentage migrated DNA in tail) and expressed as relative DNA damage score after adjusting by the level of control treatment. Cells treated with $H_2O$$_2$alone (positive control) had an extensive DNA damage compared with cells treated with phosphate buffered saline (PBS, negative control) or pre-treated with all the tested samples. Of all the six flavonoids, quercetin was the most potent antioxidant showing the lowest $ED_{50}$/ of 8.5 $\mu$g/mL (concentration to produce 50% protection of relative DNA damage). The antoxidant potency of individual flavonoids were ranked as follows in a decreasing order; luteolin (18.4 $\mu$g/mL), myricetin (19.0 $\mu$g/mL) , rutin (22.2 $\mu$g/mL) , apigenin (24,3 $\mu$g/mL) , kaempferol (25.5 $\mu$g/mL). The protective effect of $\alpha$-tocopherol was substantially lower (highest $ED_{50}$value of 55.0 $\mu$g/mL) than all the other flavonoids, while the protective effect was highest in green tea and grape juice with low ED5O value of 7.6 and 5.3, respectively. These results suggest that flavonoids, especially quercetin, and natural compounds from food product, green tea and grape juice, produced powerful anti-oxidative activities, even stronger than $\alpha$-tocopherol. Taken together, supplementation of antioxidants to lymphocytes followed by oxidative stimulus inhibited damage to cellular DNA, supporting a protective effect against oxidative damage induced by reactive oxygen species.

• Korean Journal of Medicinal Crop Science
• /
• v.13 no.4
• /
• pp.199-205
• /
• 2005

This study was carried out to investigate the effect of 70% ethanol extract and each fraction from Rhododendron brachycarpum D. Don leaves on cytotoxicity, anticancer, genotoxicity and immunological activity in vitro bioassay. Cytotoxicity for human normal cells (HEL299 and Chang) of the samples was shown below 35% in 0.5 mg/ml concentration of samples except aqueous fraction by SRB assay. DNA damage on the Chang cell of the samples alone in comet assay was observed very weak damage activity even in high concentration (1 mg/ml) of the samples. The anticancer effect of the samples on human cancer cell lines (A549, AGS, Hep3B, MCF7) was indicated that the cancer cells were inhibited gradually in proportion to the increase of the concentration of the samples by MTT assay. The growth of the Raji and Jurkat cells were hastened by adding butanol fraction among the samples. In the genotoxicity on $H_2O_2-induced$ DNA damage in Chang cells using alkaline comet assay, most of samples were shown a strong protective activity from DNA OTM values.