PURPOSE: Alpha-amanitin induces potent oxidative stress and apoptosis, and may play a significant role in the pathogenesis of hepatotoxicity. This study examined the mechanisms of α-amanitin-induced apoptosis in vitro, and whether green tea extract (GTE) offers protection against hepatic damage caused by α-amanitin (AMA) induced apoptosis in vivo. METHODS: The effects of GTE and SIL on the cell viability of cultured murine hepatocytes induced by AMA were evaluated using an MTT assay. Apoptosis was assessed by an analysis of DNA fragmentation and caspase-3. In the in vivo protocol, mice were divided into the following four groups: control group (0.9% saline injection), AMA group (α-amanitin 0.6 mg/kg), AMA+SIL group (α-amanitin and silibinin 50 mg/kg), and AMA+GTE group (α-amanitin and green tea extract 25 mg/kg). After 48 hours of treatment, the hepatic aminotransferase and the extent of hepatonecrosis of each subject was evaluated. RESULTS: In the hepatocytes exposed to AMA and the tested antidotes, the cell viability was significantly lower than the AMA only group. An analysis of DNA fragmentation showed distinctive cleavage of hepatocyte nuclear DNA in the cells exposed to AMA. In addition, the AMA and GTE or SIL groups showed more relief of the cleavage of the nuclear DNA ladder. Similarly, values of caspase-3 in the AMA+GTE and AMA+SIL groups were significantly lower than in the AMA group. The serum AST and ALT levels were significantly higher in the AMA group than in the control and significantly lower in the AMA+GTE group. In addition, AMA+GTE induced a significant decrease in hepatonecrosis compared to the controls when a histologic grading scale was used. CONCLUSION GTE is effective against AMA-induced hepatotoxicity with its apoptosis regulatory properties under in vitro and in vivo conditions.

OBJECTIVE: This study is to compare the effects of green tea polyphenol (GTP) pre-treatment with those of GTP post-treatment on cisplatin (CP)-induced nephrotoxicity in rat. METHODS: Male Sprague-Dawley rats were randomly divided into six groups. Animals in the control group received 0.9% saline (intraperitoneal); animals in the GTP group received 0.9% saline and GTP (0.2% GTP as their sole source of drinking water); the CP group received only CP (7 mg/kg, intraperitoneal); the CP+preGTP group received GTP from two days before CP to four days after CP and the CP+postGTP group received GTP for four days after CP. CP-induced renal toxicity was evaluated by plasma creatinine and blood urea nitrogen (BUN) concentrations; kidney tissue gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (AP) activities and histopathological examinations. RESULTS: High serume creatinine and BUN concentrations were observed in CP treated rats. The GGT and AP activites were lower in kidney of CP treated rats compared to control rats. In addition, treatment with CP resulted in development of a marked tubular necrosis, and tubular dilation in kidney of rats. Pretreatment with GTP resulted in markedly reduced elevation of serum creatinine and BUN amounts and changes of GGT and AP activity in kidney induced by CP. CP-induced histopathological changes, including tubular necrosis and dilation, were ameliorated in GTP pre-treated rats, compared to CP alone or GTP post-treated rats. CONCLUSION: These results demonstrate that GTP might have some protective effect against CP-induced nephrotoxicity in rat, and GTP pre-treatment was more effective than GTP post-treatment on reduction of CP-induced renal dysfunction.
Alkaline Phosphatase ; Animals ; Blood Urea Nitrogen ; Cisplatin ; Creatinine ; Drinking ; gamma-Glutamyltransferase ; Guanosine Triphosphate ; Humans ; Kidney ; Male ; Necrosis ; Plasma ; Rats* ; Rats, Sprague-Dawley ; Tea*

OBJECTIVE: Photodynamic therapy (PDT) has been used for superficial neoplasms and its usage has been recently extended to deeper lesions. The purpose of this study was to observe whether or not PDT can cure breast cancer in the solid tumor model, and to define the critical point of laser amount for killing the cancer cells. METHODS: Twenty four BALB/c mouse models with subcutaneous EMT6 mammary carcinomas were prepared. Mice were divided into eight groups depending on the amount of illumination, and the tumor size was between 8 mm and 10 mm. We began by peritoneal infiltration with a photosensitizer 48 hours prior to applying the laser light, and then we applied a non-thermal laser light. The energy was from 350 J/cm2 to 30 J/cm2 to the cancer. RESULTS: Regardless of the tumor size from 8 mm to 10 mm, all mice apparently showed positive results via PDT. We also did not find any recurrence over 90 J/cm2. In all models, the color of the breast cancer lesions began to vary to dark on 2 days post PDT and the tumor regression began simultaneously. Also, we confirmed the complete regression of the breast cancer 21 days after PDT. CONCLUSION: We confirmed that PDT may treat breast cancers that are sized less 10 mm in mouse models. The moderate energy to destruct the breast cancer cells may be 90 J/cm2. Therefore, we can expcect that PDT may be utilized to treat breast cancer, but we need more experience, skills and processing for clinical trials.
Animals ; Breast ; Breast Neoplasms ; Homicide ; Light ; Lighting ; Mice ; Photochemotherapy ; Recurrence ; Triazenes

The aim of this study was to investigate whether green tea extract (GTE) has the protective effects on excess L-arginine induced toxicity in human mesangial cell. Human mesangial cells treated with L-arginine were cultured on Dulbecco's modified eagle medium in the presence and absence of inducible nitric oxide synthase (iNOS) inhibitor and GTE. The cell proliferation was determined by 3 (4,5-dimethylthiazol- 2-yl)-2, 5-diphengltetrqzolium bromide, a tetrazole assay. The iNOS mRNA and its protein expression were detected by reverse transcription polymerase chain reaction and Western blot, respectively. The concentration of nitric oxide (NO) was measured by NO enzyme-linced immuno sorbent assay kit. L-arginine significantly inhibited the proliferation of human mesangial cells, and induced the secretion of NO to the media. NO production by L-arginine was significantly suppressed by GTE and iNOS inhibitor (p<0.01). The expression level of iNOS mRNA and its protein that was significantly increased by L-arginine was decreased by iNOS inhibitor but not by GTE. GTE protected the mesangial cells from the NO-mediated cytotoxicity by scavenging the NO rather than by iNOS gene expression. Therefore, we conclude that GTE has some protective effect for renal cells against oxidative injury possibly by polyphenols contained in GTE.
Antioxidants/metabolism ; Arginine/metabolism/pharmacology/*toxicity ; Cell Line ; Cell Proliferation ; Cell Survival ; Flavonoids/metabolism ; Glomerular Mesangium/cytology/metabolism ; Humans ; Mesangial Cells/*cytology/metabolism ; Nitric Oxide/chemistry/metabolism ; Nitric Oxide Synthase Type II/metabolism ; Phenols/metabolism ; RNA, Messenger/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tea

OBJECTIVE: Green tea polyphenol (GTP) has been shown to have anti-tumor properties in a wide variety of experimental systems. In this study, we evaluated the effects of GTP on the cytotoxic effects of cisplatin in cultured HeLa and SiHa cells. METHODS: The cell lines from Korean Cell Culture Bank were cultured in a RPMI-1640 medium supplemented with a 10% fetal bovine serum, antibiotics streptomycin and penicillin. GTP was extracted from tea leaves (Camellia scinensis) by water extraction and organic solvent fractionation. Cells were seeded at 1 x 10(4) cells/well in RPMI1640 media in triplicate wells on a Nunc Labware 96 well flat bottom microculture plate, with and without GTP (100 microgram/mL) and at different concentrations of cisplatin (0-1000 microgram/mL). After incubating the plates at 37 degrees C in 5% CO2 for 2 days, cell viability was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; thiazolyl blue] assay. RESULTS: The viability of the HeLa cells was decreased to 14% at a 600 microgram/mL concentration of cisplatin, and to 16% at 600 microgram/mL in the SiHa cells as measured by the MTT assay. However, in the HeLa cell, co-cultured with GTP (100 microgram/mL), the cell viability decreased to 68% at 200 microgram/mL of cisplatin and to 17% at 400 microgram/mL of cisplatin. And in the SiHa cell, co-cultured with GTP (100 microgram/mL), the cell viability decreased to 48% at 200 microgram/mL of cisplatin and to 17% at 400 microgram/mL of cisplatin. CONCLUSION: This study showed that cisplatin with GTP seems to have a potentiating effect on Cisplatin cytotoxicity than cisplatin alone.
Anti-Bacterial Agents ; Cell Culture Techniques ; Cell Line* ; Cell Survival ; Cisplatin* ; Guanosine Triphosphate ; HeLa Cells ; Humans ; Penicillins ; Streptomycin ; Tea* ; Uterine Cervical Neoplasms* ; Water

PURPOSE: A constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG), is known to possess anti-cancer properties. In this study, the time-course of the anticancer effects of EGCG on human ovarian cancer cells were investigated to provide insights into the molecular-level understanding of the growth suppression mechanism involved in EGCG-mediated apoptosis and cell cycle arrest. MATERIALS AND METHODS: Three human ovarian cancer cell lines (p53 negative, SKOV-3 cells; mutant type p53, OVCAR-3 cells; and wild type p53, PA-1 cells) were used. The effect of EGCG treatment was studied via a cell count assay, cell cycle analysis, FACS, Western blot and macroarray assay. RESULTS: EGCG exerts a significant role in suppressing ovarian cancer cell growth, showed dose dependent growth inhibitory effects in each cell line and induced apoptosis and cell cycle arrest. The cell cycle was arrested at the G1 phase by EGCG in SKOV-3 and OVCAR-3 cells. In contrast, the cell cycle was arrested in the G1/S phase in PA-1 cells. EGCG differentially regulated the expression of genes and proteins (Bax, p21, Retinoblastoma, cyclin D1, CDK4 and Bcl-XL) more than 2 fold, showing a possible gene regulatory role for EGCG. The continual expression in p21WAF1 suggests that EGCG acts in the same way with p53 proteins to facilitate apoptosis after EGCG treatment. Bax, PCNA and Bcl-X are also important in EGCG-mediated apoptosis. In contrast, CDK4 and Rb are not important in ovarian cancer cell growth inhibition. CONCLUSION: EGCG can inhibit ovarian cancer cell growth through the induction of apoptosis and cell cycle arrest, as well as in the regulation of cell cycle related proteins. Therefore, EGCG-mediated apoptosis could be applied to an advanced strategy in the development of a potential drug against ovarian cancer.
Apoptosis ; Blotting, Western ; Cell Count ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Line* ; Cyclin D1 ; G1 Phase ; Humans ; Ovarian Neoplasms ; Proliferating Cell Nuclear Antigen ; Retinoblastoma ; Tea*

OBJECTIVE: The aim of this article was investigated whether changes of superoxide dismutase isozymes in the placenta of patients with preeclampsia contribute to radical-induced tissue injury. METHODS: The activities of antioxidant enzymes (superoxide dismutase (SOD), catalase, glutathione peroxidase (GSHPx)) and the contents of thiobarbituric acid reactive substance (TBARS) in the erythrocytes and in the placenta were assayed from 35 women with preclampsia and 35 normotensive pregnant women. RESULTS: The superoxide dismutase (SOD) activities were significantly reduced in the erhtyrocytes and the placenta of patients with pre-eclampsia compared with normotensive pregnant women. The activity of catalase was increased in the erythrocytes of patients with preeclampsia but the statistically significant difference of catalase activity in the placenta and GSHPx activity in both erythrocytes and placenta were not observed. The contents of TBARS were increased significantly in the erythrocytes and placenta of patients with preeclampsia. In preeclamptic placenta, copper and zinc containing superoxide dismutase (CuZn-SOD) was decreased (3.9+/-0.5 vs 5.1+/-0.6 U/mg protein) whereas manganeus containing superoxide dismutase (Mn-SOD) was increased (2.0+/-0.3 vs 2.7+/-0.4 U/mg protein). CONCLUSION: In these results, the decreased CuZn-SOD activity may some roles in increment of TBARS contents in pre-eclamptic placenta and decreased CuZn-SOD activity may be more prone to oxidative stress in the placenta.
Catalase ; Copper ; Erythrocytes ; Female ; Glutathione Peroxidase ; Humans ; Isoenzymes* ; Oxidative Stress ; Placenta* ; Pre-Eclampsia* ; Pregnant Women ; Superoxide Dismutase* ; Superoxides* ; Thiobarbituric Acid Reactive Substances ; Zinc

BACKGROUND: MPO is an antimicrobial enzyme in the primary granule of neutrophil. MPO exhibits MPO-I, MPO-II, MPO-III using chromatography in human peripheral leukocytes. we investigated MPO isozymes from human leukocytes of peripheral blood. METHODS: Neutrophils were prepared from 5-8mL of peripheral blood in the group of normal persons (normal group) and the group of patients with neutrophilia (patient group). Their cells were adjusted to 2.4X10(7) cells and sonified. We got finally the crude MPO from which native PAGE was performed and treated with diaminobenzine and H2O2. RESULTS: Normal neutrophil MPO had MPO-1 and MPO-2. The expression rates of MPO-1 and that of MPO-2 were respectively 100%, 67.5% in the normal group and 56.6%, 30.2% in the patient group (P=0.000, P=0.000). The expression rate of combined MPO-1 and MPO-2 and that of MPO-1 without MPO-2 were respectively 67.6%, 32.4% in the normal group and 28.3%, 28.3% in the patient group (P=0.000, P=0.000). The expression of MPO-2 without MPO-1 and that of no MPO isozymes were respectively 0.0%, 0.0% in the normal group and 1.9%, 41.5% in the patient group (P=0.000, P=0.000). CONCLUSION: Normal neutrophil MPO expresses MPO-1 and MPO-2 usig native PAGE. The main MPO isozyme is MPO-1. Expressions of MPO isozymes are variable in the patient group.
Humans

Mutans streptococci is the major causative factor in dental caries. Especially, orthodontic patients with fixed appliance are a risk group for dental caries. Because fixed appliances attached on teeth may change the environment of dental plaque, the enamel decalcification or dental caries around the bracket and band is a major side effect of orthodontic treatment. The aim of this study was to search plant extracts that have antimicrobial effect on mutans streptococci. Seed-extract of Casia tora were prepared with ethanol and CHMC-2032, the leaf-extracts from Camellia sinensis extract, was obtained extract, 2 type strains and 20 clinical isolates of mutans streptococci isolated from the interface between orthodontic brackets and tooth surfaces in the orthodontic patients were used in this study. The minimal inhibitory concentration of CHMC-2032 was 5 mg/ml on the S. mutans KCTC 3065, S. sobrinus KCTC 3088, and 8 clinical isolates of S. sobrinus. However, there was no antibacterial effect of seed-extract of C. tora on mutans streptococci. These data suggest that green tea may be more effective than the tea prepared from C. tora in the prevention of enamel decalcification or dental caries around brackets.
Camellia sinensis* ; Camellia* ; Dental Caries ; Dental Enamel ; Dental Plaque ; Ethanol ; Humans ; Orthodontic Brackets* ; Plant Extracts ; Tea ; Tooth*

PURPOSE: Oxidative stress is the well known causative factor for retinal damage. This study investigated the effects of glutathione on reactive oxygen species(ROS) induced injury in human retinal pigment epithelial(HRPE) cells. OBJECTS AND METHODS: HRPE cells (ATCC:CRL-2302) were cultured with DMEM media and exposed to oxidative stress (paraquat, hydrogen peroxide) and/or glutathione modulator[(buthionine sulfoximine (BSO), glutathione (GSH), 2-oxo 4-thiazolidine carboxylic acid (OTC)] for 2 days. The cell viability was determined by measuring the amount of reduced 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). RESULTS: The rate of MTT reduction of HRPE cells decreased by either paraquat or hydrogen peroxide treatment. BSO as a inhibitor of glutathione biosynthesis potentiated paraquat- or hydrogen peroxideinduced HRPE cells injury. On the other hand GSH or OTC reduced the rate of decrement of MTT reduction in HRPE cells by paraquat and hydrogen peroxide. CONCLUSIONS: Glutathione seemed to play some role in prevention of ROS-induced HRPE cells injury and OTC may be used as an agents for prevention of free radical induced HRPE cell injury.
Cell Survival ; Epithelial Cells* ; Glutathione* ; Hand ; Humans* ; Hydrogen ; Hydrogen Peroxide ; Oxidative Stress ; Oxygen* ; Paraquat ; Retinaldehyde*