• Environmental Analysis Health and Toxicology
• /
• v.22 no.4
• /
• pp.357-366
• /
• 2007

To investigate the possible enhancement of genotoxicity in stress environment, we examined the of effect of genotoxic material in oxidative stress-induced condition using human tell line. Human lymphoblast cell line, TK6 was treated with hydrogen peroxide ($H_2O_2$) for induction of oxidative stress, and treated with N'-methyl-N'-nitroguanidine (MNNG), af a genetoxic material. We carried out MTS assay to set treatment doses. TK6 was treated with $H_2O_2$ at 6.75 (low dote) or $13.5\;{\mu}M$ (high dose) for 2 h, and treated with MNNG af 0.117 (low dose), 0.234 (middle dose), $0.468\;{\mu}M$ (high dose) for 2 h. As results, a treatment of MNNG induced DNA dam age as dose dependently. And TK6 treated with $H_2O_2$ at low as well as high dose followed by MNNG treatment showed higher DNA damage compared to MNNG alone treated groups. Malondialdehyde, as a marker of lipid peroxidation was increased in $H_2O_2$ and MNNG treated groups. Real-time RT-PCR analyses for expression of several antioxidative enzymes showed that catalase mRNA and glutathione peroxidase 1 mRNA expression were decreased in $H_2O_2$ and MNNG treated groups. Taken together, we conclude that genotoxicity induced by MNNG is enhanced in a condition of oxidative stress induced by $H_2O_2$ and it suggests that it should be associated with induction of lipid peroxidation and decrease of antioxidant enzymes.

• IMMUNE NETWORK
• /
• v.1 no.1
• /
• pp.45-52
• /
• 2001

Background: Many types of cancer become resistant to current chemotherapeutic and radiotherapeutic intervention. To overcome this situation application of gene therapy by the introduction of suicide genes followed by their prodrugs may be promising. A viral enzyme, Herpes simplex thymidine kinase (HSV-tk), which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining prodrug-activating gene therapy and irradiation might result in enhanced antitumor effects. Methods: The HSV-tk gene was cloned into the retroviral vector, pLXSN and established the clones producing retroviruses carrying the HSV-tk gene. The carcinoma cell line, HCT116 and Huh-7 were transduced with high-titer recombinant retroviruses. These cell lines were treated with ganciclovir before or after irradiation for the defining combinational effect of suicide gene therapy and radiotherapy. Results: The titers of cloned PA3 17 amphotropic retroviruses ranged from 4 to 6 X $10^6CFU/ml4$. After selectional periods, the expression of HSV-tk was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). The growth of cells expressing HSV-tk was inhibited as increase of GCV dose after 48 hr and the growth inhibitory effect of GCV was much higher after 72 hr. When the cells transduced with HSV-tk gene were exposed to radiation, the growth inhibitory effect of GCV was significantly increased, as compared with non-transduced parental cells. Conclusions: The results suggest that the addition of HSV-tk gene therapy to standard radiation therapy may improve the effectiveness of treatment for solid tumors.

• Biomedical Science Letters
• /
• v.11 no.2
• /
• pp.165-173
• /
• 2005

A potent demethylating agent, 5-Azacytidine (5-AzaC) has been widely used as in many studies on DNA methylation, regulation of gene expression, and cancer biology. The mechanisms of the demethylating activity were known to be formation of complex between DNA and DNA methyltransferase (MTase), which depletes cellular MTase activity. However, 5-AzaC can also induce hypermethylation of a transgene in a transgenic cell line, G12 cells and it was explained as a result of defense mechanisms to inactivate foreign gene(s) somehow. This finding evoked the question that whether the phenomenon of hypermethylation induced by 5-AzaC is limited to the transgene or it can be occurred in endogenous gene(s). In order to answer the question, mutagenicity test of 5-AzaC and molecular characterization of mutants obtained from the test were performed using an endogenous gene, thymidine kinase (tk) in Chinese hamster V79 cells. When V79 and V79-J3 subclone cells were treated with 1, 2.5 ,5, $10{\mu}M$ of 5-AzaC for 48 hours, their maximum mutant frequencies were revealed as $6\times10^{-3}\;at\;5{\mu}M$(350-fold induction over background) and $8\times10^{-3}\;at\;2.5{\mu}M$ (l,800-fold induction over background) respectively. Since the induction rates were too high to be induced by true mutations, many trifluorothymidine (TFT)-resistant $(TFT^R)$ cells were subjected to Northern blot analysis to check the presence of tk transcripts. Surprisingly, all clones tested possessed the transcripts in a similar level, that implicates the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the gene in spite of unusually high mutation frequency. In addition, it has shown that the TK activity in the pool of 5-AzaC-induced $TFT^R$ cells has about a half of that in spontaneously-induced $TFT^R$ cells or in non-selected parental V79-J3 cells. This result suggests that the mechanism(s) underlying the TFT-resistance between spontaneously occurred and 5-AzaC-induced cells may be different. These findings have shown that the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the tk gene, and 5-AzaC may be induced by one or combined pathways among many drug resistance mechanisms. The exact mechanisms for the 5-AzaC-induced $TFT^R$ phenotype remain to elucidate.

• Preventive Nutrition and Food Science
• /
• v.14 no.1
• /
• pp.21-28
• /
• 2009

In this study, we focused on natural water-soluble antioxidants from Tetraselmis suecica (T. suecica) and Chlorella ellipsoidea (C. ellipsoidea). They were prepared by enzymatic digestion using five carbohydrases (Viscozyme, Celluclast, AMG, Termamyl and Ultraflo) and five proteases (Protamex, Alcalase, Flavourzyme, Neutrase, and Kojizyme), and the potential antioxidant activity of each was assessed. Most enzymatic digests from T. suecica had a higher radical scavenging activity than those from C. ellipsoidea. Among the enzymatic digests, Kojizyme digest from T. suecica exhibited the highest effect on DPPH radical scavenging. Viscozyme (30.2%) and Neutrase (34.6%) digests from T. suecica exhibited higher hydroxyl radical scavenging activity. Kojizyme digest from T. suecica (81.5%) had strong alkyl radical scavenging activity. Neutrase (61.9%) and Kojizyme (61.5%) digest from T. suecica possessed the highest effects on hydrogen peroxide scavenging. Among the tested samples, Neutrase (TN) and Kojizyme (TK) digests from T. suecica showed the highest antioxidant activity (DPPH, alkyl radical, hydrogen peroxide). Therefore, TN and TK digests were selected for use in the further experiments. Those digests showed enhanced cell viability against $H_2O_2$-induced oxidative damage, and relatively good hydrogen peroxide scavenging activity in an African green monkey kidney (Vero) cell line. These results suggested that an enzymatic digestion will be an effective way for the production of a potential water-soluble antioxidant from a microalgae, T. suecica.

• Radiation Oncology Journal
• /
• v.21 no.3
• /
• pp.227-237
• /
• 2003

Purpose: The human chronic myelogenous leukemia cell line, K562, expresses the chimeric bcr-abl oncoprotein, whose deregulated protein tyrosine kinase activity antagonizes via DNA damaging agents. Previous experiments have shown that nanomolar concentrations of herbimycin A (HWA) coupled with X-irradiation have a synergistic effect in inducing apoptosis in the Ph-positive K562 leukemia cell line, but genistein, a PTK inhibitor, is non selective for the radiation-induced apoptosils on $p210^{bcr/abl}$ protected K562 cells. In these experiments, the cytoplasmic signal transduction pathways, the Induction on a number of transcription factors and the differential gene expression in this model were investigated. Materials and Methids: K562 cells in the exponential growth phase were used in this study. The cells were irradiated with 0.5-12 Gy, using a 6 Mev Linac (Clinac 1800, Varian, USA). Immediately after irradiation, the cells were treated with $0.25/muM$ of HMA and $25/muM$ of genistein, and the expressions and the activities of abl kinase, MAPK family, NF- kB, c-fos, c-myc, and thymidine kinase1 (TK1) were examined. The differential gene expressions induced by PTK inhibitors were also investigated. Results: The modulating effects of herbimycin A and genistein on the radiosensitivity of K562 cells were not related to the bcr-abl kinase activity. The signaling responses through the MAPK family of proteins, were not involved either in association with the radiation-induced apoptosis, which is accelerated by HMA, the expression of c-myc was increased. The combined treatment of genistein, with irradiation, enhanced NF- kB activity and the TK1 expression and activity. Conclusion: The effects of HMA and genistein on the radiosensitivity on the K562 cells were not related to the bcr-abl kinase activity in this study, another signaling pathway, besides the WAPK family responses to radiation to K562 cells, was found. Further evaluation using this model will provide valuable information for the optional radiosensitization or radioprotection.

• Proceedings of the Korea Society of Environmental Toocicology Conference
• /
• 2003.05a
• /
• pp.185-185
• /
• 2003

To develope the novel anti-allergic drug, many sophoricoside derivatives were synthesized. Among these derivatives, JSH-II-3, JSH-Ⅵ-3, JSH-Ⅶ-3, and JSH-Ⅷ-3 were selected and subjected to high throughput toxicity screening (HTTS) because they revealed strong IL-5 inhibitory activity and limitation of quantity. Mouse lymphoma thymidine kinase (tk$\^$+/-/) gene assay (MOLY) and single cell gel electrophoresis (Comet) assay in mammalian cells were used as HTTS tool in our laboratory. In MOLY assay, JSH-Ⅶ-3 at 50 ∼ 6 $\mu\textrm{g}$/ml concentrations was not shown significant mutagenic effect in the absence and presence of S-9 metabolic activation system. However, the concentration of ISH-II-3, 38 $\mu\textrm{g}$/ml, induced increased mutation frequency (MF) in the presence of S-9 metabolic activation system. Also in comet assay, DNA damage was not observed in JSH-Ⅵ-3 and JSH-Ⅶ-3, wherase concentration of 32.8 $\mu\textrm{g}$/ml in JSH-II-3 and 13.9 $\mu\textrm{g}$/ml in JSH-Ⅶ-3 were induced DNA damage in the absence of S-9 metabolic activation system. Therefore, we suggest that JSH-Ⅵ-3 and JSH-Ⅶ-3 have no genotoxic effects but JSH-II-3 and JSH-Ⅷ-3 induce some mutagenicity and DNA strand breaks in mouse lymphoma cell line used this study.

• Journal of the Korean GEO-environmental Society
• /
• v.6 no.4
• /
• pp.47-58
• /
• 2005

Development of underground space is conducted for various useful facility and underground structures and deep excavation technology for reclaimed area has been researched and developed. The seepage flow and behavior of phreatic line in reclaimed area was predominated by transient flow caused by tidal action. Also the soil-water characteristic relation is most important factor for transient flow analysis, therefore the research about the soil-water characteristic is strongly required. In this paper, laboratory tests (pressure cell, desiccator, and tensiometer test) and theoretical analysis were performed to investigate the soil-water characteristic such as air-entry value, metric suction, and residual water content. And the feasibility of prediction method for soil-water characteristic are presented by transient seepage analysis and comparison between analysis results and in-situ measured seepage flux in LNG TK-00 storage tank. Based on the result of laboratory and theoretical analysis, Fredlund and Xing's method provide to work out well for reclaimed ground soils. Also, the transient analysis result is more reasonable and effective for design of deep excavation work in coastal and reclaimed ground.