OBJECTIVE: To clarify the genotoxicity induced by acute exposure of ozone with different concentrations on pulmonary cells in rats. METHODS: Thirty-six Wistar rats were randomly divided into control group (filtered air exposure) and ozone exposure group (0.12 ppm, 0.5 ppm, 1.0 ppm, 2.0 ppm, 4.0 ppm) with 6 in each group. After rats were exposed to different concentrations of ozone for 4 h, lung tissues were taken and single cells were isolated. Then, 8-hydroxydeoxyguanosine (8-OHdG) was quantitatively detected by enzyme-linked immunosorbent assay. Comet assay, micronucleus test and DNA- protein cross-linking assay were used to analyze DNA and chromosome damages. RESULTS: Compared with the control group, the content of 8-OHdG in lung tissue was increased significantly from the ozone exposure concentration of 0.12 ppm, reaching the highest value at 0.5 ppm. With the increase of ozone exposure concentration, the tail rate of comets was increased gradually, and there was a significant dose-effect relationship. The cross-linking rate of DNA- protein was increased first and then was decreased with a maximum value at 2.0 ppm group. Although the micronucleus rate of lung cells showed an upward trend, there was no significant difference compared with the control group. CONCLUSION Acute exposure of ozone at low concentrations (0.12 ppm) could lead to DNA damage in the pulmonary cells of rats, while no significant chromosome damage was found even in the group with ozone concentration reached to 4 ppm.
Animals ; Comet Assay ; DNA Damage ; Lung ; cytology ; pathology ; Micronucleus Tests ; Ozone ; adverse effects ; Random Allocation ; Rats ; Rats, Wistar

PURPOSE: The DNA damage response (DDR) is a multi-complex network of signaling pathways involved in DNA damage repair, cell cycle checkpoints, and apoptosis. In the case of biliary tract cancer (BTC), the strategy of DDR targeting has not been evaluated, even though many patients have DNA repair pathway alterations. The purpose of this study was to test the DDR-targeting strategy in BTC using an ataxia-telangiectasia and Rad3-related (ATR) inhibitor. MATERIALS AND METHODS: A total of nine human BTC cell lines were used for evaluating anti-tumor effect of AZD6738 (ATR inhibitor) alone or combination with cytotoxic chemotherapeutic agents through MTT assay, colony-forming assays, cell cycle analyses, and comet assays. We established SNU478-mouse model for in vivo experiments to confirm our findings. RESULTS: Among nine human BTC cell lines, SNU478 and SNU869 were the most sensitive to AZD6738, and showed low expression of both ataxia-telangiectasia mutated (ATM) and p53. AZD6738 blocked p-Chk1 and p-glycoprotein and increased γH2AX, a marker of DNA damage, in sensitive cells. AZD6738 significantly increased apoptosis, G2/M arrest and p21, and decreased CDC2. Combinations of AZD6738 and cytotoxic chemotherapeutic agents exerted synergistic effects in colony-forming assays, cell cycle analyses, and comet assays. In our mouse models, AZD6738 monotherapy decreased tumor growth and the combination with cisplatin showed more potent effects on growth inhibition, decreased Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling than monotherapy with each drug. CONCLUSION: In BTC, DDR targeting strategy using ATR inhibitor demonstrated promising antitumor activity alone or in combination with cytotoxic chemotherapeutic agents. This supports further clinical development of DDR targeting strategy in BTC.
Animals ; Apoptosis ; Ataxia Telangiectasia ; Biliary Tract Neoplasms ; Biliary Tract ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Line ; Cisplatin ; Comet Assay ; DNA Damage ; DNA Repair ; DNA ; Humans ; Mice ; P-Glycoprotein

PURPOSE: Astragalus membranaceus (AM) is an important traditional medicinal herb. Pharmacological research has indicated that AM has various physiological activities such as antioxidant, anti-inflammatory, immunoregulatory, anticancer, hypolipidemic, antihyperglycemic, and hepatoprotective activities. The bioactive substances responsible for the physiological activities in AM, including many antioxidant substances, change during the roasting process. This study investigated and compared the changes in the antioxidant constituents of AM caused by roasting. METHODS: DPPH (1,1-diphenyl-2-picryl hydrazyl) and ABTS⁺ (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) radical scavenging activities and their total phenolic content (TPC) were measured. High-performance liquid chromatography (HPLC) analysis was performed to confirm any changes in the isoflavonoids of roasted AM (R-AM),. The cell viability of UVB-induced HDF (Human dermal fibroblast) cells treated with AM and R-AM extracts was investigated. The comet assay was used to examine the inhibitory effects of R-AM extracts on DNA damage caused by oxidative stress. RESULTS: The DPPH and ABTS⁺ radical scavenging activities were 564.6±20.9 and 108.2±3.1 (IC₅₀ value) respectively, from the 2R-AM. The total phenol content was 47.80±1.40 mg GAE/g from the 1R-AM. The values of calycosin and formononetin, which are the known isoflavonoid constituents of AM, were 778.58±2.72 and 726.80±3.45 µg/g respectively, from the 2R-AM. Treatment of the HDF cells with R-AM (50 ~ 200 µg/mL) did not affect the cell viability. Furthermore, the R-AM extracts effectively protected against UVB-induced DNA damage. CONCLUSION: The findings of this study indicate that R-AM increases its isoflavonoid constituents and protects against UVB-induced DNA damage in HDF cells.
Astragalus membranaceus ; Cell Survival ; Chromatography, Liquid ; Comet Assay ; DNA Damage ; Oxidative Stress ; Phenol ; Plants, Medicinal

BACKGROUND/OBJECTIVES: Glutathione S-transferase (GST) forms a multigene family of phase II detoxification enzymes which are involved in the detoxification of xenobiotics by conjugating substances with glutathione. The aim of this study is to assess the antioxidative status and the degree of DNA damage in the subclinical hypertensive patients in Korea using glutathione S-transferase polymorphisms. SUBJECTS/METHODS: We examined whether DNA damage and antioxidative status show a difference between GSTM1 or GSTT1 genotype in 227 newly diagnosed, untreated (systolic blood pressure (BP) ≥ 130 mmHg or diastolic BP ≥ 85 mmHg) subclinical hypertensive patients and 130 normotensive subjects (systolic BP < 120 mmHg and diastolic BP < 80 mmHg). From the blood of the subjects, the degree of the DNA damage in lymphocyte, the activities of erythrocyte superoxide dismutase, the catalase, and the glutathione peroxidase, the level of glutathione, plasma total radical-trapping antioxidant potential (TRAP), anti-oxidative vitamins, as well as plasma lipid profiles and conjugated diene (CD) were analyzed. RESULTS: Of the 227 subjects studied, 68.3% were GSTM1 null genotype and 66.5% were GSTT1 null genotype. GSTM1 null genotype had an increased risk of hypertension (OR: 2.104, CI: 1.38-3.35), but no significant association in GSTT1 null genotype (OR 0.982, CI: 0.62-1.55). No difference in erythrocyte activities of superoxide dismutase, catalase, or glutathione peroxidase, and plasma TRAP, CD, lipid profiles, and GSH levels were observed between GSTM1 or GSTT1 genotype. Plasma levels of α-tocopherol increased significantly in GSTT1 wild genotype (P < 0.05); however, plasma level of β-carotene increased significantly in GSTT1 null genotype (P < 0.01). DNA damage assessed by the Comet assay was significantly higher in GSTM1 null genotype than wild genotype (P < 0.05). CONCLUSIONS: These results confirm the association between GSTM1 null genotype and risk of hypertension as they suggest that GSTM1 null genotype leads to an increased oxidative stress compared with wild genotype.
Antioxidants ; Blood Pressure ; Catalase ; Comet Assay ; DNA Damage* ; DNA* ; Erythrocytes ; Genotype ; Glutathione Peroxidase ; Glutathione Transferase* ; Glutathione* ; Humans ; Hypertension ; Korea ; Lymphocytes* ; Metabolic Detoxication, Phase II ; Multigene Family ; Oxidative Stress ; Plasma* ; Superoxide Dismutase ; Vitamins ; Xenobiotics

BACKGROUND/OBJECTIVE: This study aims to measure the in vitro antioxidant capacity of Korean diet (KD) with American diet (AD) as a control group and to examine the ex vivo DNA damage reduction effect on human lymphocytes. MATERIALS/METHODS: The KD applied in this study is the standard one-week meals for Koreans (2,000 kcal/day) suggested by 2010 Dietary Reference Intakes for Koreans. The AD, which is the control group, is a one-week menu (2,000 kcal/day) that consists of foods that Americans would commonly take in according to the National Health and Nutrition Examination Survey. The antioxidant capacity of each menu was measured by means of the total phenolic assay and 3 in vitro antioxidant activity assays (2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, trolox equivalent antioxidant capacity (TEAC), Oxygen radical absorbance capacity (ORACROO·)), while the extent of ex vivo lymphocyte DNA damage was measured by means of the comet assay. RESULTS: When measured by means of TEAC assay, the in vitro antioxidant capacity of the KD of the day was higher than that of the AD (P < 0.05) while there was no significant difference in total phenolic contents and DPPH and ORAC assays. The ex vivo lymphocyte DNA damage protective effect of the KD was significantly higher than that of the AD (P < 0.01). As for the one-week menu combining the menus for 7 days, the total phenolic assay (P < 0.05) and in vitro antioxidant capacity (P < 0.001, DPPH; P < 0.01, TEAC) of the KD menu were significantly higher than those of the AD menu. Likewise, the ex vivo DNA damage reduction rate of the Korean seven-day menu was significantly higher than that of the American menu (P < 0.01). CONCLUSION: This study demonstrates that the high antioxidant capacity and DNA damage protective effect of KD, which consists generally of various plant foods, are higher than those of typical AD.
Antioxidants ; Comet Assay ; Diet* ; DNA Damage* ; DNA* ; Humans ; In Vitro Techniques ; Lymphocytes* ; Meals ; Nutrition Surveys ; Oxygen ; Phenol ; Plants ; Recommended Dietary Allowances

PURPOSE: GST (glutathione S-transferase) M1 and T1 gene polymorphisms are known to affect antioxidant levels. This study was carried out to evaluate genetic susceptibility by measuring the effect of DNA damage reduction in the Korean diet by vegetable food according to GST gene polymorphisms using the ex vivo method with human lymphocytes. METHODS: Vegetable foods in the Korean diet based the results of the KNHANES V-2 (2011) were classified into 10 food groups. A total of 84 foods, which constituted more than 1% of the total intake in each food group, were finally designated as a vegetable food in the Korean diet. The Korean diet applied in this study is the standard one-week meals for Koreans (2,000 Kcal/day) suggested by the 2010 Dietary Reference Intakes for Koreans. Ex vivo DNA damage in human lymphocytes was assessed using comet assay. RESULTS: In the Korean food group, the DNA damage protective effect of GSTM1 and GSTT1 was found to be greater in mutant type and wild-type, respectively. and the DNA damage protective effect according to the combined genotype of GSTM1 and GSTT1 was different depending on the food group. On the other hand, in Korean Diet, the DNA damage protective effect appeared to be larger in GSTM1 wild-type than in mutant type and was found to not be affected by GSTT1 genotype. CONCLUSION: These results can be used as basic data to demonstrate the superiority of the antioxidant function of Korean dietary patterns and food groups. Furthermore, it may be a starting point to begin research on customized antioxidant nutrition according to individual genes.
Comet Assay ; Diet* ; DNA Damage* ; DNA* ; Genetic Predisposition to Disease ; Genotype ; Glutathione Transferase* ; Glutathione* ; Hand ; Humans ; Lymphocytes* ; Meals ; Methods ; Plants* ; Recommended Dietary Allowances ; Vegetables

PURPOSE: Fruit and vegetable juices are known to be rich sources of antioxidants, which have beneficial effects on diseases caused by oxidative stress. The purpose of this study was to directly compare the antioxidant activities of fruit and vegetable juices marketed in Korea. METHODS: We analyzed four fruit juices, two vegetable juices, two yellow-green juices, and six mixed vegetable juices. Antioxidant activities were analyzed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) test, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) test, and oxygen radical absorbance capacity (ORAC) assay. Protective effects against DNA damage were determined using an ex vivo comet assay with human lymphocytes. RESULTS: DPPH radical scavenging activities were in the following order: blueberry juice > mixed vegetable C juice > kale juice > mixed vegetable P juice > grape juice. ABTS radical scavenging activities were in the following order: blueberry juice > mixed vegetable C juice > grape juice > mixed vegetable P juice > kale juice. Peroxyl radical scavenging activities as assessed by ORAC assay were in the following order: blueberry juice > kale juice > mixed vegetable C juice > grape juice. Grape or blueberry juice showed strong abilities to prevent DNA damage in lymphocytes, and the difference between them was not significant according to the GSTM1/GSTT1 genotype. CONCLUSION: Antioxidant activities of fruit and vegetable juices and ex vivo DNA protective activity increased in the order of blueberry juice, grape juice, and kale juice, although the rankings were slightly different. Therefore, these juices rich in polyphenols and flavonoids deserve more attention for their high antioxidant capacity.
Antioxidants ; Blueberry Plant ; Brassica ; Comet Assay ; DNA Damage* ; DNA* ; Flavonoids ; Fruit and Vegetable Juices* ; Fruit* ; Genotype ; Humans ; Korea* ; Lymphocytes* ; Oxidative Stress ; Oxygen ; Polyphenols ; Vegetables ; Vitis

PURPOSE: Breast cancer is an important cause of death among women. The development of radioresistance in breast cancer leads to recurrence after radiotherapy. Caffeic acid phenethyl ester (CAPE), a polyphenolic compound of honeybee propolis, is known to have anticancer properties. In this study, we examined whether CAPE enhanced the radiation sensitivity of MDA-MB-231 (estrogen receptor-negative) and T47D (estrogen receptor-positive) cell lines. METHODS: The cytotoxic effect of CAPE on MDA-MB-231 and T47D breast cancer cells was evaluated by performing an 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. To assess clonogenic ability, MDA-MB-231 and T47D cells were treated with CAPE (1 µM) for 72 hours before irradiation, and then, a colony assay was performed. A comet assay was used to determine the number of DNA strand breaks at four different times. RESULTS: CAPE decreased the viability of both cell lines in a dose- and time-dependent manner. In the clonogenic assay, pretreatment of cells with CAPE before irradiation significantly reduced the surviving fraction of MDA-MB-231 cells at doses of 6 and 8 Gy. A reduction in the surviving fraction of T47D cells was observed relative to MDA-MB-231 at lower doses of radiation. Additionally, CAPE maintained radiation-induced DNA damage in T47D cells for a longer period than in MDA-MB-231 cells. CONCLUSION: Our results indicate that CAPE impairs DNA damage repair immediately after irradiation. The induction of radiosensitivity by CAPE in radioresistant breast cancer cells may be caused by prolonged DNA damage.
Breast Neoplasms* ; Breast* ; Cause of Death ; Cell Line ; Comet Assay ; DNA Damage* ; DNA* ; Estrogens* ; Female ; Humans ; Propolis ; Radiation Tolerance* ; Radiation-Sensitizing Agents ; Radiotherapy ; Recurrence

OBJECTIVETo study the pattern of DNA double-strand break (DSB) formation in S-phase cells after thermal damage and explore the mechanisms behind heat sensitivity of S-phase cells and delayed DSBs.

METHODSFlow cytometry was used to analyze the cell cycle arrest in H1299 cells exposed to thermal damage, and EdU incorporation assay was employed to evaluate the DNA replication capacity of the cells. The cells synchronized in S phase were obtained by serum starvation and DSBs were observed dynamically using neutral comet assay. Trypan blue dye exclusion technique was used to analyze the cell viability after thermal damage. Western blotting (WB) was used to detect the phosphorylation of ATM and DNA binding RAD18.

RESULTSThe percentage of S-phase cells increased significantly after exposure of the cells to 45 degrees celsius; for 1 h (P<0.01). The time-dependent variation pattern of EdU incorporation was similar to that of S-phase cell fraction. The comet tail began to appear only after incubation of the cells at 37 degrees celsius; for some time and the Olive tail moment (OTM) increased with prolonged incubation. Cell death remained low until 7.5 h after heat exposure of the S-phase cells and then increased rapidly. The phosphorylation of ATM first increased but then decreased drastically. In cells with heat exposure, DNA binding RAD18 was attenuated obviously compared that in non-exposed cells.

CONCLUSIONThermal damage causes cell cycle arrest in S phase, and delayed fatal DSBs occur in the arrested cells due to persistent replication and DNA damage repair suppression, which are the possible cause of heat sensitivity of S-phase cells.

Ataxia Telangiectasia Mutated Proteins ; metabolism ; Cell Cycle Checkpoints ; Cell Line ; Cell Survival ; Comet Assay ; DNA Breaks, Double-Stranded ; DNA Repair ; DNA Replication ; DNA-Binding Proteins ; metabolism ; Hot Temperature ; Humans ; Phosphorylation ; S Phase ; Ubiquitin-Protein Ligases

OBJECTIVETo analyze changes in gene amplification in the mitochondrial genome and in the ID4 gene promoter methylation region in patients with chronic aplastic anemia (CAA) suffering from Kidney (Shen) yin deficiency or Kidney yang deficiency.

METHODSBone marrow and oral epithelium samples were collected from CAA patients with Kidney yin deficiency or Kidney yang deficiency (20 cases). Bone marrow samples were collected from 20 healthy volunteers. The mitochondrial genome was amplified by polymerase chain reaction (PCR), and PCR products were used for sequencing and analysis.

RESULTSHigher mutational rates were observed in the ND1-2, ND4-6, and CYTB genes in CAA patients suffering from Kidney yin deficiency. Moreover, the ID4 gene was unmethylated in bone marrow samples from healthy individuals, but was methylated in some CAA patients suffering from Kidney yin deficiency (positive rate, 60%) and Kidney yang deficiency (positive rate, 55%).

CONCLUSIONSThese data supported that gene mutations can alter the expression of respiratory chain enzyme complexes in CAA patients, resulting in energy metabolism impairment and promoting the physiological and pathological processes of hematopoietic failure. Functional impairment of the mitochondrial respiration chain induced by gene mutation may be an important reason for hematopoietic failure in patients with CAA. This change is closely related to maternal inheritance and Kidney yin deficiency. Finally, these data supported the assertion that it is easy to treat disease in patients suffering from yang deficiency and difficult to treat disease in patients suffering from yin deficiency.

Adult ; Anemia, Aplastic ; genetics ; Base Sequence ; Biopsy ; Bone Marrow ; pathology ; Case-Control Studies ; Child ; Chronic Disease ; DNA Methylation ; genetics ; DNA, Mitochondrial ; genetics ; Electrophoresis, Agar Gel ; Female ; Genome, Mitochondrial ; genetics ; Humans ; Inhibitor of Differentiation Proteins ; genetics ; Kidney ; pathology ; Male ; Middle Aged ; Mutation ; genetics ; Polymorphism, Genetic ; Promoter Regions, Genetic ; genetics ; Yin Deficiency ; genetics ; Young Adult