• BMB Reports
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• v.39 no.4
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• pp.452-456
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• 2006

To elaborate the peroxidase activity of cytochrome c in the generation of free radicals from $H_2O_2$, the mechanism of DNA cleavage mediated by the cytochrome c/$H_2O_2$ system was investigated. When plasmid DNA was incubated with cytochrome c and $H_2O_2$, the cleavage of DNA was proportional to the cytochrome c and $H_2O_2$ concentrations. Radical scavengers, such as azide, mannitol, and ethanol, significantly inhibited the cytochrome c/$H_2O_2$ system-mediated DNA cleavage. These results indicated that free radicals might participate in the DNA cleavage by the cytochrome c and $H_2O_2$ system. Incubation of cytochrome c with $H_2O_2$ resulted in a time-dependent release of iron ions from the cytochrome c molecule. During the incubation of deoxyribose with cytochrome c and $H_2O_2$, the damage to deoxyribose increased in a time-dependent manner, suggesting that the released iron ions may participate in a Fenton-like reaction to produce $\cdot$OH radicals that may cause the DNA cleavage. Evidence that the iron-specific chelator, desferoxamine (DFX), prevented the DNA cleavage induced by the cytochrome c/$H_2O_2$ system supports this mechanism. Thus we suggest that DNA cleavage is mediated via the generation of $\cdot$OH by a combination of the peroxidase reaction of cytochrome c and the Fenton-like reaction of free iron ions released from oxidatively damaged cytochrome c in the cytochrome c/$H_2O_2$ system.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.275-289
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• 2002

The objective of this study was to develop an in vitro assessment of sperm fertilizing capacity of bulls and investigate the factors influencing sperm function and characteristics of frozen-thawed bovine spermatozoa. in vitro fertilization (IVF), the evaluation of motility and normal morphology, HOST (hypoosmotic swelling test), Ca-ionophore induced acrosome reaction, luminol and lucigenin-dependent chemiluminescence for the measurement of reactive oxygen species (ROS), the measurement of malondialdehyde formation for the analysis of lipid peroxidation (LPO), and the evaluation of DNA fragmentation using the method of 747-mediated nick end labelling (TUNEL) by flow cytometry were performed in frozen-thawed bovine spermatozoa. Correlations between the rates of fertilization, blastocyst formation after IVF and the values of respective assays were investigated. 1. IVF rate and blastocyst formation rate averaged 64.4% and 34.3% for spermatozoa from high -fertility bull group and averaged 18.5% and 6.2% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. Sperm motility and percentage acrosome reaction averaged 79.0% and 66.2% for spermatozoa from high-fertility bull group and averaged 40.7% and 22.9% for spermatozoa from low-fertility bull group, respectivitely. There were not different between two bull groups. 2. Luminol depenent chemiluminescence, LPO and DNA fragementation averaged 6.4, 2.0 nmol and 2.6% from spermatozoa from high-fertility bull group and averaged 6.5, 3.1 nmol and 7.4% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. There was no significant difference in lucigenin dependent chemiluminescence between two bull groups. 3. Fertilization rate was positively correlated with motility and the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between fertilization rate and the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. 4. Blastocyst formation rate was positively correlated with the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between blastocyst formation rate and motility, the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. In conclusion, these data suggest that ROS significantly impact semen quality. The assays of this study may provide a basis fur improving in vitro assessment of sperm fertilizing capacity.

• YAKHAK HOEJI
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• v.43 no.5
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• pp.623-628
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• 1999

DA-125, a new antitumor agent, was compared with adriamycin, a known DNA intercalator, in terms of inhibitory mechanism of DNA replication by using replicating simian virus 40 (SV40) genome in vivo. In analyzing the SV40 DNA replication intermediates present in cells treated with DA-125, it was not observed to accumulate B-dimers of SV40 DNA which are prominent in adriamycin-treated cells. However, treatment with DA-125 induced dose-dependent formation of DNA-topoisomerase complex which is characteristic of topoisomerase poisons. In addition, DA-125 showed more efficient in inhibiting SV40 DNA replication than adriamycin. Therefore, on the basis of this observation, we suggest that DA-125, a derivative of adriamycin, inhibits DNA replication by blocking topoisomerase activity as a toposomerase poison although adriamycin blocks topoisomerase activity as a DNA intercalator.

• BMB Reports
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• v.40 no.6
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• pp.895-898
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• 2007

The oxygen dependent regulation of DNA replication is an essential property of proliferating mammalian cells. In human T24 bladder cancer cells, several hours of hypoxia leads to reversible DNA replication arrest and re-entry of oxygen induces a burst of replication initiation. This short communication provides strong evidence that PD184352 initiates DNA replication in living hypoxic cells without elevating the oxygen level. PD184352 releases the regular hypoxic replicon arrest, however, at a low intensity compared to the effect of reoxygenation. Moreover, PD184352 shows no effect on normoxically incubated as well as reoxygenated T24 cells.

• BMB Reports
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• v.47 no.10
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• pp.575-580
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• 2014

In this study, we investigate whether arsenite-induced DNA damage leads to p53-dependent premature senescence using human glioblastoma cells with p53-wild type (U87MG-neo) and p53 deficient (U87MG-E6). A dose dependent relationship between arsenite and reduced cell growth is demonstrated, as well as induced ${\gamma}H2AX$ foci formation in both U87MG-neo and U87MG-E6 cells at low concentrations of arsenite. Senescence was induced by arsenite with senescence-associated ${\beta}$-galactosidase staining. Dimethyl- and trimethyl-lysine 9 of histone H3 (H3DMK9 and H3TMK9) foci formation was accompanied by p21 accumulation only in U87MG-neo but not in U87MG-E6 cells. This suggests that arsenite induces premature senescence as a result of DNA damage with heterochromatin forming through a p53/p21 dependent pathway. p21 and p53 siRNA consistently decreased H3TMK9 foci formation in U87M G-neo but not in U87MG-E6 cells after arsenite treatment. Taken together, arsenite reduces cell growth independently of p53 and induces premature senescence via p53/p21-dependent pathway following DNA damage.

• Molecular & Cellular Toxicology
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• v.1 no.1
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• pp.32-39
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• 2005

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmentally prevalent xenobiotics that exert complex effects on the biological system and characterized as probably carcinogenic materials. Single cell gel electrophoresis assays were performed in order to evaluate DNA damage occurring in the T-and B lymphocytes, spleens (T/B-cell), bone marrow, and livers of mouse exposed to mixture of PAHs (Benzo(a)pyrene, Benzo(e)pyrene, Fluoranthene, Pyrene) at dose of 400, 800, or 1600 mg/kg body weight for 2 days. DNA damage of the cells purified from mice was increased in dose dependent manner. In the blood cells and organs, DNA damage was also discovered to vary directly with PAHs. Especially T-cells had been damaged more than B-cell. Plasma proteomes were separated by 2-dimensional electrophoresis with pH 4-7 ranges of IPG Dry strips and many proteins showed significant up-and -down expressions with the dose dependent manner. Of these, significant 4 spots were identified using matrix-assisted laser desorption/ionization-time of fight (MALDI-TOF) mass spectrometry. Identified proteins were related to energy metabolism and signal transduction.

• Archives of Pharmacal Research
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• v.25 no.1
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• pp.11-24
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• 2002

The antitumor antibiotic (+)-CC-1065 can alkylate N3 of guanine in certain sequences. A previous high-field $^1H$ NMR study on the$(+)-CC-1065d[GCGCAATTG*CGC]_2$ adduct ($^*$ indicates the drug alkylation site) showed that drag modification on N3 of guanine results in protonation of the cross-strand cytosine [Park, H-J.; Hurley, L. H. J. Am. Chem. Soc.1997, 119,629]. In this contribution we describe a further analysis of the NMR data sets together with restrained molecular dynamics. This study provides not only a solution structure of the (+)-CC-1065(N3- guanine) DNA duplex adduct but also new insight into the molecular basis for the sequence- specific interaction between (+)-CC-1065 and N3-guanine in the DNA duplex. On the basis of NOESY data, we propose that the narrow minor groove at the 7T8T step and conformational kinks at the junctions of 16C17A and 18A19T are both related to DNA bending in the drugDNA adduct. Analysis of the one-dimensional $^1H$ NMR (in $H_2O$) data and rMD trajectories strongly suggests that hydrogen bonding linkages between the 8-OH group of the (+)-CC-1065 A-sub-unit and the 9G10C phosphate via a water molecule are present. All the phenomena observed here in the (+)-CC-1065(N3-guanine) adduct at 5'$-AATTG^*$are reminiscent of those obtained from the studies on the (+)-CC-1065(N3-adenine) adduct at $5'-AGTTA^*$, suggesting that (+)-CC-1065 takes advantage of the conformational flexibility of the 5'-TPu step to entrap the bent structure required for the covalent bonding reaction. This study reveals a common molecular basis for (+)-CC-1065 alkylation at both $5'-TTG^*$ and $5'-TTA^*$, which involves a trapping out of sequence-dependent DNA conformational flexibility as well as sequence-dependent general acid and general base catalysis by duplex DNA.

• Bulletin of the Korean Chemical Society
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• v.31 no.10
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• pp.2873-2876
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• 2010

Excess free iron generates oxidative stress that may contribute to the pathogenesis of various causes of neurodegenerative diseases. Previous studies have shown that one of the primary causes of increased brain iron may be the release of excess iron from intracellular iron storage molecules. In this study, we attempted to characterize the oxidative damage of DNA induced by the reaction of ferritin with $H_2O_2$. When DNA was incubated with ferritin and $H_2O_2$, DNA strand breakage increased in a time-dependent manner. Hydroxyl radical scavengers strongly inhibited the ferritin/$H_2O_2$ system-induced DNA cleavage. We investigated the generation of hydroxyl radical in the reaction of ferritin with $H_2O_2$ using a chromogen, 2,2'-azinobis-(2-ethylbenzthiazoline-6-sulfonate) (ABTS), which reacted with ${\cdot}OH$ to form $ABTS^{+\cdot}$. The initial rate of $ABTS^{+\cdot}$ formation increased as a function of incubation time. These results suggest that DNA strand breakage is mediated in the reaction of ferritin with $H_2O_2$ via the generation of hydroxyl radicals. The iron-specific chelator, deferoxamine, also inhibited DNA cleavage. Spectrophotometric study using a color reagent showed that the release of iron from $H_2O_2$-treated ferritin increased in a time-dependent manner. Ferritin enhanced mutation of the lacZ' gene in the presence of $H_2O_2$ when measured as a loss of $\alpha$-complementation. These results indicate that ferritin/$H_2O_2$ system-mediated DNA cleavage and mutation may be attributable to hydroxyl radical generation via a Fenton-like reaction of free iron ions released from oxidatively damaged ferritin.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.36 no.4
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• pp.346-351
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• 2003

Single-cell gel electrophoresis (comet assay) was used to detect DNA single strand break in blood cells from several marine fish species. Three fish species were collected from Georgia coastal area. Mummichog, Fundulus heteroclitus showed higher DNA damage than sea bass, Lateolabrax japonicus and trout, Oncorhynchus masou masou under the same experimental conditions. Mummichogs had more alkaline-labile sites on their DNA than other fish species. The comet assay with mummichog blood cells at pH 12.5 showed a dose-response curve with the increasing concentrations of hydrogen peroxide. While the isolated leucocytes showed no increase of DNA damage after in vitro exposure to 2-methyl-1,4-naphthoquinone (MNQ), erythrocytes showed dose-dependent DNA damage. These results indicate that the comet assay can be applied successfully as a bioassay using erythrocyte for environmental monitoring.

• The Korean Journal of Zoology
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• v.20 no.1
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• pp.41-48
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• 1977

Dose response of DNA repair synthesis induced by bleomycin was dose-dependent in lower doses, and maximum rate of it at 5 $\\mu$g/ml represents about 15% of total cells analyzed. At higher doses DNA-repair synthesis was reduced and the rate of it remained unchanged even prolonged treatment. Pretreatment with BUdR or IUdR was found to enhance DNA repair synthesis and also to interfere with semiconservative DNA synthesis at higher doses. Time dependence study showed that DNA repair synthesis occurred as long as for 24 hours after removal of bleomycin. These results seem to suggest that bleomycin is not to be an effective chemical in inducing excision repair and that damages induced in DNA by this drug might include not only strand breaks but other types of DNA damage.