Synergistic effects of Rapamycin and Fluorouracil to treat a gastric tumor in a PTEN conditional deletion mouse model.

The tumor suppressor gene phosphatase and tensin homolog (PTEN) in PI3K/Akt/mTOR pathway is essential in inhibiting tumor growth and metastasis. However, whether the mutation of PTEN gene could induce tumorigenesis and impact the treatment of gastric cancer is still unclear. The purpose of the study was to investigate the combined treatment of gastric tumorigenesis using Rapamycin and Fluorouracil (5-Fu) through interfering with the Akt/mTOR pathway in a mouse model with PTEN conditional deletion. Three groups of mice were exposed for 5 days to Rapamycin and 5-Fu separately and together. The gene expression of the Akt/mTOR pathway, the protein expression of caspase-3 and p-Akt, p-S6K and p-4EBP1, and the pathological changes in stomachs were analyzed. Our study demonstrates that the conditional PTEN deletion in the cells of glandular stomach induces hyperplastic gastric tumors in mice. The combined Rapamycin administration with 5-Fu resulted in better outcomes than their separate administration for the treatment of gastric cancer by inhibiting the mTOR signal pathway. Our study indicates that Rapamycin has a synergistic interaction with chemotherapeutic 5-Fu, and demonstrates a potential therapeutic combination treatment on glandular stomach tumor with PTEN functional absence or aberrantly activated Akt/mTOR pathway. It provides important insights into the inhibition of the Akt/mTOR pathway in gastric cancer clinical therapy.

[The Establishment of a Three-color Flow Cytometry Approach for the Scoring of Micronucleated Reticulocytes in Rat Bone Marrow].

OBJECTIVE: To develop and verify a flow cytometric measurement of reticulocytes (RETs) micronucleus in rat bone marrow. METHODS: In our flow cytometric protocol, reticulocytes, leukocytes and DNA were labeled by anti-CD71-fluorescein isothiocyanate (FITC), anti-CD45-phycoerythrin (PE) and DRAQ5, respectively. Sprague-Dawley (SD) rats were assigned to four treatment groups randomly, and were exposed to ethyl methanesulfonate (EMS), cyclophosphamide (CP), ethyl nitrosourea (ENU) and colchicine (COL) respectively. Each treatment group was divided into four subgroups (5 rats per subgroup) according to different exposure dosage. A exposure dose of 0 was used as vehicle control for each group. Rats were administered with testing mutagens by gavage twice with a 24 h interval. Bone marrow from both femurs were collected 24 h after the last administration. The frequency of micronucleated reticulocytes (MN-RETs) and the percentage of reticulocytes (RETs%) were determined by flow cytometric measurement established in this study. And the manual counting method with microscope (by Giemsa staining) was conducted at the same time. RESULTS: A method for detection of reticulocyte micronucleus in bone marrow based on flow cytometry was successfully established. The MN-RETs in rat bone marrow of 20 SD rats treated by vehicle (i.e., background value of MN-RETs) was 0.83‰±0.12‰ by this method. The background value of MN-RETs in manual enumeration method was 1.43‰±0.44‰. It was obvious that the flow cytometric method had lower background value and more stable results. The trend, in which MN-RETs ascended and RETs% descended with increasing dose, can be detected by both methods in rats that exposed to EMS, CP, ENU and COL. Both methods were good to detect the correlation of induced-MN-RETs with four testing mutagens (the correlation coefficients were ranged from 0.834 3 to 0.913 7). CONCLUSION: With its sensitivity, rapidity, easy operation and low background value, the three-color flow cytometric enumerative protocol established in our laboratory can be used as a good substitute for manual micronucleus counting method and used in genotoxicity assessment of chemical substances.

[Optimization ofPig-aGene Mutation in Rats and Assessment of Time-dependent and Dose-response Relationship of N-ethyl-N-nitrosourea].

OBJECTIVES: To optimize the method of Pig-a mutation assay, and to explore the time-dependent and dose-response relationship of N-ethyl-N-nitrosourea (ENU). METHODS: Thirty rats were randomly assigned to 5 groups: treated with PBS (control group)or different doses of ENU (10, 20, 40 and 80 mg/kg) for 3 d by oral gavage. Blood samples were collected at 0 d, 15 d, 30 d, 45 d, 60 d, 75 d and 90 d. After enrichment, erythrocytes were incubated with Anti-CD59-APC and SYTO 13 nucleic acid dye solution. Mutant phenotype erythrocytes (RBCCD59-) and mutant phenotype reticulocytes (RETCD59-) were measured by flow cytometry to analyze mutant frequencies, and the RET percentage was determined as well. RESULTS: The RBCCD59- mutation frequency in 4 ENU groups were significantly increased in a dose- and time-dependent manner. The RETCD59- mutation frequency increased to a stable high level with a slight fluctuation, and decreased at 45 d , with the peak values observed at 30 d. The RETCD59- mutation frequency showed a dose-dependent trend in 4 ENU groups. The RET percentage in all 5 groups declined at 30 d, to a stable low level thereafter, but the trends showed no significant differences by time or group. CONCLUSIONS: The optimized in vivo Pig-a mutation assay could detecte the mutagen, such as ENU, induces mutation in RBC in a time- and dose-dependent manner.

Acute and 28-day sub-acute oral toxicity evaluation of two dietary bamboo charcoal powders in Sprague-Dawley rats.

No data were available on the acute oral toxicity, short-term oral toxicity of vegetable carbon in animals. This study was designed to evaluate the safety of two commercially available dietary bamboo charcoal powders (BCP1 and BCP2). The size distribution of the two powders was determined by a Mastersizer 2000 laser particle size analyzer prior to the in vivo safety studies. For the acute toxicity study, a single dose of 11.24 g/kg body weight of BCP1 and BCP2 was given once orally to healthy Sprague-Dawley (SD) rats. Mortality and clinical symptoms were observed and recorded for the first 30 min after treatment, at 4 h post-administration, and then at least once daily for 14 days after administration. In the repeated dose 28-day oral toxicity study, BCP1 and BCP2 were administered orally at doses of 2.81, 5.62, and 11.24 g/kg body weight for 28 days to SD rats. Animals were sacrificed and organs and blood samples were analyzed. Results showed that both BCP1 and BCP2 were micro-sized and various in size. In the acute toxicity and the repeated dose 28-day oral toxicity studies, BCP caused neither mortality nor visible signs of toxicity in rats. No significant differences were found in the relative organ weights or in biochemical parameters in BCP treated groups compared to a control group. No treatment-related histological changes were observed in the organs of these animals. Based on these data, it is concluded that the median lethal dose (LD50) of BCP for both male and female rats is more than 11.24 g/kg body weight and the no-observed-adverse-effect level (NOAEL) is >11.24 g/kg body weight for 28 days.

[Isolation and Study on the Aflatoxin Genes of Aflatoxin-producing Fungi in Paprika Samples in Chengdu].

OBJECTIVE: To isolate aflatoxin-producing strains from paprika samples and to do a preliminarily study on the relationship between aflatoxin-producing ability and the genes aflR, omt-1 and ver-1. METHODS: Fungi were isolated by traditional culture method. Potential aflatoxin-producing strains were screened by phenotypic traits and multiplex PCR. After these potential aflatoxin-producing strains cultured in the toxigenic culture medium, the levels of aflatoxin B, (AFB1) of the cultures were tested with ELISA method. The phylogenetic tree of aflR, omt-1 and ver-1 was constructed to explore the relationship between these genes and the AFB1-producing capacity. RESULTS: 17 potential aflatoxin-producing fungi were isolated. The ratio of positive toxigenic strains is 64. 71%. 11 isolates were positive in AFB1 detection while existing high sequence homology with AS 3. 4408, 6 isolates were negative in AFB1 detection while existing high sequence homology with Aspergillus oryzae. CONCLUSION: Aspergillus flavus are potential candidates for aflatoxin control. Not all Aspergillus flavus have AFB1-producing capacity, aflR gene had a direct relation to AFB1-producing capacity, while ver-1 and omt-1 were related to the level of AFB1 producing.

Toxic effects of atrazine on reproductive system of male rats.

OBJECTIVE: This study was designed to evaluate the toxic effects of Atrazine (ATZ) on the reproductive system of male rats. METHODS: Male Sprague-Dawley rats were exposed to ATZ by gavage at dosages of 0, 38.5, 77, and 154 mg/kg bw/day for 30 d. The toxic effects of ATZ to rats were assessed through histopathologcal observation, spermatozoa quality evaluation, testicular marker enzyme indicators, antioxidant capacity and reproductive hormone levels. RESULTS: Significant adverse effects on reproductive system were observed in rats exposed to ATZ at different dosages compared with 0 mg/kg group, including an irregular and disordered arrangement of the seminiferous epithelium in 154 mg/kg group; a decreased spermatozoa number and an increased spermatozoa abnormality rate in 77 and 154 mg/kg groups; decreased levels of acid phosphatase (ACP), alkaline phosphatase (AKP), lactic dehydrogenase (LDH), and succinate dehydrogenase (SDH) with the increasing of ATZ concentration; a decreased level of total antioxidant capacity (TAC) in a dose-dependent manner, and a decreased reduced glutathione (GSH) level and an increased malondialdehyde (MDA) content in 154 mg/kg group; and decreased serum levels of testosterone (T) and inhibin-B (INH-B) and an increased serum level of follicle stimulating hormone (FSH) in 77 and 154 mg/kg groups, and an increased serum level of luteinizing hormone (LH) in 154 mg/kg group. CONCLUSION: These results suggested that relatively high doses of ATZ could exert reproductive toxicity of male rats.

[Investigation on levels of polybrominated diphenyl ethers in retail fish and egg products in Shenzhen].

OBJECTIVE: To reveal the levels and distribution of polybrominated diphenyl ethers (PBDEs) in fish and egg products at retail in Shenzhen, and to evaluate the local people's exposure to PBDEs from these food. METHODS: 27 fish and egg samples were collected from supermarket and farmer's market in Shenzhen during August and October in 2008. According to the guideline of USEPA1614 method, the accelerated solvent extraction (ASE) technology was used for the extraction of PBDEs from fish and egg samples. After a series of purification processes including treatments of FMS column chromatography, acidic silica gel, silica gel and Al2O3 column, the levels of eight PBDEs congeners in the samples were determined by isotope dilution high resolution gas chromatography-high resolution mass spectrometry (HRGC-HRMS) method. RESULTS: When BDE-209 was not taken into account, the median concentrations of ΣPBDEs in fish products was 914.7 pg/g wet weight, among which the datas for fresh water fish and sea fish were 328.2 and 1108.8 pg/g wet weight, respectively, showing a statistical significant difference (P < 0.05). BDE-47 was the predominant congener in fresh water fish and sea fish by a contribution proportion of 61% and 57%, respectively. The median concentrations of ΣPBDEs in egg products were 99.8 pg/g wet weight and the predominant congeners are BDE-47 and BDE-99, with a contribution proportion above 70%. BDE-209 was not detected in fresh water fish and the median concentration in sea fish and egg products are 243.7 and 472.6 pg/g wet weight, respectively, which caused the predominant congener changed to BDE-209 in egg products when BDE-209 was take into account. The median dietary intake of PBDEs from fish and egg products among local residents in Shenzhen was estimated as 102 ng/d. CONCLUSION: The level of ΣPBDEs in fish and egg products in Shenzhen is relatively high. The characteristics of PBDEs pollution are quite different between fish and egg products. The level of daily dietary intake of PBDEs from fish and egg products among local residents in Shenzhen is also relatively high.

[Study on protective effect of Tianji soft capsule on blood lipids, internal antioxidant system and vascular endothelial system in hyperlipidemia rats].

OBJECTIVE: To study the protective effects of Tianji soft capsule (TJSC) on blood lipids, internal antioxidant system and vascular endothelial system in hyperlipidemia rats. METHODS: Seventy two healthy male rats were divided into six groups. The rats in control group were administered with ordinary diet. The rats in model group were fed with high cholesterol/lipid diet to induce hyperlipidemia. The rats in TJSC and CoQ10 groups were fed with high cholesterol/lipid diet, and treated with TJSC at different doses of 83 (low-dose group, L), 250 (middle-dose group, M), 750 (high-dose group, H) mg/kg, and CoQ10 at the dose of 83 mg/kg, respectively. All animals were put to death after four weeks, effects on lipid level; antioxidant system and endothelial system were evaluated through detection of total cholesterol (TC), triglyceride (TG), very low density lipoprotein (VLDL), atherogenic index (AI), malondidehyde (MDA) and plasma endothelin (ET), HDL/TC ratio, activities of superoxide dismutase (SOD) and nitric oxide (NO). RESULTS: Compared with model group, serum TC, TG, VLDL, AI, MDA and ET reduced and the HDL/TC ratio increased, meanwhile activities of SOD and NO were enhanced. CONCLUSION: TJSC can regulate the lipid metabolism, enhance antioxidant system and protect the vascular endothelia system in hyperlipiemic rats.

[Protective effects of tianji capsule on vascular endothelial cells from oxidative injury induced by hydrogen peroxide in vitro].

OBJECTIVE: To study the protective effects of Tianji capsule (TJ) on vascular endothelial cells from oxidative injury induced by hydrogen peroxide and its possible mechanism of anti-oxidation. METHODS: The effect of TJ on the proliferation of normal human umbilical vascular endothelial cells (HUVECs) as well as its cytotoxicity was evaluated with methylthiazolyl tetrazolium (MTT) assay. After the establishment of oxidative injury model of HUVECs, control, oxidative injury model, TJ and CoQ10 treatment groups were set up. HUVECs were incubated with 37.5, 75, 150 and 300 microg/mL TJ or 100 microg/mL CoQ10 for 24 h, and 0.1 mmol/L H2O2 (final concentration) was added to HUVECs in each groups for 30 min. Then collected the cells for proliferation detection with MTT assay, and the levels of MDA and NO, the activities of SOD, GSH-Px and NOS, as well as the releasing rate of LDH in HUVECs were also determined. RESULTS: No cytotoxicity was observed in HUVECs with less than 400 microg/ mL TJ incubated for 48 h, but increased proliferation rates were noticed. Pretreated with TJ (37.5, 75, 150 and 300 microg/mL), increased proliferation rate, the activities of SOD, GSH-Px, NOS were observed, but the decreased level of MDA and releasing rate of LDH were also found. CONCLUSION: TJ could protect HUVECs against oxidative injury induced by H2O2.

Cytotoxicity and Apoptosis Induced by Chenopodium ambrosioides L. Essential Oil in Human Normal Liver Cell Line L02 via the Endogenous Mitochondrial Pathway Rather Than the Endoplasmic Reticulum Stress.

Chenopodium ambrosioides L. (C. ambrosioides) has been used as dietary condiments and as traditional medicine in South America. The oil of Chenopodium ambrosioides L. (C. ambrosioides) can be used as a natural antioxidant in food processing. It also has analgesic, sedating, and deworming effects, and can be used along with the whole plant for its medical effects: decongestion, as an insecticide, and to offer menstruation pain relief. This study was conducted to investigate the cytotoxicity and apoptosis effects of an essential oil from C. ambrosioides in vitro. The cytotoxicity evaluation of the essential oil from C. ambrosioides on human normal liver cell line L02 was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. AO/EB dual fluorescent staining assay and Annexin V-FITC were used for apoptosis analysis. The changes in mitochondrial membrane potential (MMP) were analyzed with 5,5,6,6'-tetrachloro-1,1,3,3,-tetraethyl-imidacarbocyanine iodide (JC-1) dye under a fluorescence microscope. The level of apoptosis related protein expression was quantified by Western blot. The L02 cells were treated with the essential oil from C. ambrosioides at 24, 48, and 72 h, and the IC50 values were 65.45, 58.03, and 35.47 µg/mL, respectively. The AO/EB staining showed that viable apoptotic cells, non-viable apoptotic cells, and non-viable non-apoptotic cells appeared among the L02 cells under the fluorescence microscope. Cell cycle arrest at the S phase and cell apoptosis increased through flow cytometry in the L02 cells treated with the essential oil. MMP decreased in a concentration-dependent manner, as seen through JC-1 staining under the fluorescence microscope. In the L02 cells as shown by Western blot and qPCR, the amount of the apoptosis-related proteins and the mRNA expression levels of cytochrome C, Bax, Caspase-9, and Caspase-3 increased, Bcl-2 decreased, and Caspase-12, which is expressed in the endoplasmic reticulum, showed no obvious changes in protein amount or mRNA expression level. The essential oil form C. ambrosioides had a cytotoxic effect on L02 cells. It could inhibit L02 cell proliferation, arrest the cell cycle at the S phase, and induce L02 cell apoptosis through the endogenous mitochondrial pathway.

[The levels of dioxin in 60 human breast milk samples and the exposure risk factor analysis in Shenzhen].

OBJECTIVE: To investigate the levels of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans (PCDD-Fs) in human breast milk of the mothers who lived in non-directly persistent organic pollutants (POPs) polluted area in Shenzhen, and the correlation of exposure risk factor was analyzed. METHODS: From July to November in 2007, 60 primiparas by vaginal delivery after parturition 3 weeks to 2 months were sampled for breast milk who aged from 20 - 34 years old and has lived in Shenzhen non-directly POPs polluted areas over 5 years. PCDD-Fs were extracted from the frozen-dried samples with accelerated solvent extraction (ASE), cleaned up by fluid management system (FMS) and quantified by high-resolution gas chromatography-high resolution mass spectrometry (HRGC-HRMS) using isotope dilution methodology. TEQ were calculated. The correlation relationship among infant's birth weight and length, participatory's dietary, age, inhabitation period, environment and the body burden of PCDD-Fs in mother was statistically analyzed by SPSS 13.0. RESULTS: The participants aged from 20 - 34 years old (28 years on average) and lived in Shenzhen for 5 - 29 years (10 years on average). The concentration of PCDD-Fs in 60 breast milk samples were 26.957 143 - 669.583 333 pg/g fat (mean: 7.224 817 pg/g fat, median: 84.176 062 pg/g fat), and TEQ-PCDD-Fs in samples were 2.420 793 - 29.014 277 pg/g fat (mean: 8.645 992 pg/g fat, median: 7.751 804 pg/g fat). 2, 3, 4, 7, 8-PeCDF, 1, 2, 3, 7, 8-PeCDD, 2, 3, 7, 8-TCDD were the dominant contributors to the total TEQ, which were 3.691 654 pg/g fat (42.689 378%), 2.478 315 pg/g fat (28.652 356%), and 0.980 995 pg/g fat (11.343 995%) respectively. Significant positive correlations were found among age (r = 0.26, P < 0.05), inhabitation period (r = 0.49, P < 0.05), the consumption of fish (r = 0.37, P < 0.05) and the concentrations of TEQ-PCDD-Fs in the study. CONCLUSION: The levels of dioxin chemicals in the breast milk samples in non-directly POPs polluted areas of Shenzhen are high. Significant positive correlations were found among age, inhabitation period, the consumption of fish and the concentration of PCDD-Fs.

Characterization of Salmonella enterica serotype typhimurium from outpatients of 28 hospitals in Henan province in 2006.

OBJECTIVE: To characterize the diarrheal patients with Salmonella typhimurium (S. typhimurium) infections and to set up the first baseline for S. typhimurium pulsed-field gel electrophoresis (PFGE) patterns in Henan province, thus laying a foundation for comprehensive surveillance of Salmonella in human as well as foods. METHODS: S. typhimurium isolates recovered from outpatients with diarrhea in Henan province from May to October of 2006 were characterized. Antimicrobial susceptibility tests of 8 antimicrobial agents and PFGE were carried out to analyze the S. typhimurium isolates. RESULTS: Twenty-four (0.9%) S. typhimurium isolates were identified from 2661 stool specimens of diarrheal cases. Eighty-eight percent of isolates showed resistance to at least one antimicrobial agent. The resistance to chloramphenicol (79%) was most common. Fifty-eight percent of isolates were resistant to ciprofloxacin. All the 14 ciprofloxacin-resistant isolates were resistant to more than five antimicrobial agents. Thirty-three percent of S. typhimurium isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (R-type ACSSuT). Eight antimicrobia-resistant phenotypes were found among the 24 isolates in 16 PFGE patterns. CONCLUSION: The rate of multidrug-resistant S. typhimurium is relatively high in S. typhimurium PFGE patterns of Henan province. Multidrug-resistant S. typhimurium should be considered a public health threat.

[Gene expression profile of isoniazid liver-injured rat using cDNA microarray].

OBJECTIVE: To investigate the changes of gene expression profile of rat liver tissue by cDNA microarrays. METHODS: Twenty Wistar rats in control group (n = 10) and isoniazid (INH) group (n = 10) were orally administrated with normal saline and 400 mg/kg INH for 14 days, respectively. The differentially expressed genes significantly correlated with liver injury were screened and analyzed. The mechanisms of liver injury caused by INH were specifically analyzed at level of gene expression based on the biological functions of those differentially expressed genes. RESULTS: Thirty-seven differentially expressed genes were found in the rats administrated with INH. Among them, 25 genes were up-regulated, while the other 12 genes were down-regulated. These differentially expressed genes were functionally related to the changes of CYP450-related genes, fatty acid metabolism, and protein metabolism. CONCLUSION: INH can cause the remarkable changes of the gene expression profiles in rat liver cells, which is important for further elucidating the mechanisms of liver injury caused by INH.

[Study on the immunotoxicity of ribosome inhibiting protein of Momordica charantia].

OBJECTIVE: To separate and purify ribosome inhibiting protein (RIP) from Momordica charantia (bitter melon) seeds and to evaluate its acute toxicity and immunotoxicity in animal. METHODS: Ion exchange chromatography and gel filtration chromatography were applied in the separating and purifying of RIP from Momordica charantia seeds. Then the acute toxicity testing of RIP in mice was conducted to obtain its half lethal dose (LD50). Active systemic anaphylaxis(ASA)test in guinea pig and passive cutaneous anaphylaxis test (PCA) in rat were performed to evaluate its immunotoxicity. RESULTS: The LD50 (iv) in mice of RIP was 25.2 mg/kg in ASA, guinea pigs of the higher and lower RIP group all appeared stong allergic responses and most of them died quickly. In PCA, obvious blue dye in skin were observed in SD rats of the RIP group. CONCLUSION: RIP getting from Momordica charantia seeds had a relatively strong immunotoxicity in animals.

Preparation and evaluation of novel solid chlorine dioxide-based disinfectant powder in single-pack.

OBJECTIVE: To prepare and evaluate novel chlorine dioxide-based disinfectant powder in single-pack that is more convenient for use and transportation. METHODS: Orthogonal experiment was performed to determine the recipe of the disinfectant powder. Stability test, suspension quantitative bactericidal test, simulation field trial, and animal toxicity test were carried out to observe its bactericidal and toxicological effects. RESULTS: The orthogonal experiment showed that the type of water solution had no effect on the disinfectant powder and the best ratio of sodium chlorite to solid acid was 1:3. Ten grams of the disinfectant powder was fully dissolved in 20 mL water for 2 min, and diluted to 500 mL in water. After 5-10 min, the concentration of chlorine dioxide (ClO2) solution was 266 mg/L to 276 mg/L. After stored at 54 degrees C for 14 d, the average concentration of ClO2 was decreased by 5.03%. Suspension quantitative bactericidal test showed that the average killing logarithm (KL) value for both Staphylococcus aureus and Escherichia coli in 100 mg/L ClO2 solution for 2 min was over 5.00. in simulation field trial, the average descending KL value for Escherichia coli in the solution containing 100 mg/L ClO2 for 5 min was over 3.00. The mouse acute LD50 in the solution 5 times exceeded 5000 mg/kg. The disinfectant powder was not toxic and irritative to rabbit skin and had no mutagenic effect on mouse marrow polychromatic erythrocytes (PCE). CONCLUSION: The stability and bactericidal efficacy of solid chlorine dioxide-based disinfectant powder in single-pack are good. The solution containing 100 mg/L ClO2 can kill vegetative forms of bacteria. The concentration of ClO2 on the disinfecting surface of objects is 100 mg/L. The disinfectant powder is not toxic and irritative.

[Metabonomic profile of urine from rats administrated with different treatment period of rifampin].

OBJECTIVE: To study the effect of rifampin (RFP) on the metabonomic profile of rat urine and its relationship with traditional toxicity evaluation of blood biochemical indicators and histopathology. METHODS: Thirty-six male Wistar rats were randomly divided into control group, 50 mg/kg RFP group, and 100 mg/kg RFP group, with 12 rats in each group. Rats in each group were given intragastric infusion with a daily dose of 0, 50 mg/kg RFP, and 100 mg/kg RFP for 3, 7, and 14 days, respectively. Then 4 rats in each group were killed on the next day of administration to collect blood samples and liver sample for the determination of blood biochemical indicators and for the pathological analysis of the liver. The urine specimens over 24 hours of each rat were collected before and after each treatment until the rat was killed. 1H nuclear magnetic resonance (1H NMR) spectra of these urine specimens were acquired and subjected to data preprocess and principal component analyses (PCA). RESULTS: The level of serum total bilirubin of the rat administrated with 100 mg/(kg x d) RFP for 7 days was significantly higher than that of control group (P < 0.05). Mild hepatotoxicity to the rat, treated with RFP of higher dosage (100 mg/kg) and longer duration (14 days), was revealed by the traditional histopathological method. The metabonomic spectra of rat urine in different groups differed from each other; a trajectory bias in determination of rat urine by 1H NMR occurred depending on the administration duration. As demonstrated by 1H NMR spectra of urine in rats treated with RFP, the concentration of urinary citrate and 2-oxoglutarate decreased, along with the remarkable increase of the concentrations of urinary taurine and glucose (compared with those of the control group). CONCLUSIONS: Being consistent with the results of traditional toxicity evaluation measurements, metabonomic method is more sensitive. The 1H-NMR metabonomic profile of the rat urine is closely related with the duration of RFP. The hepatic toxicity induced by RFP is related to the reduction of energy metabolism in tricarboxylic acid cycle and the perturbation of glucose and lipid metabolism.

Metallothionein protects against doxorubicin-induced cardiomyopathy through inhibition of superoxide generation and related nitrosative impairment.

Metallothionein (MT) has been shown to be an effective protector against DOX-induced cardiomyopathy, however the involved precise mechanisms are still unknown. The present study was undertaken to clarify whether the inhibition of superoxide generation and related nitrosative damage were involved in the metallothionein attenuation of DOX-induced cardiac injury. MT-I/II null (MT-/-) mice and corresponding wild-type mice (MT+/+) were pretreated with either saline or zinc (300 micromol/kg, s.c., once a day for 2 days) prior to a single dose of DOX (15 mg/kg, i.p.) or equal volume of saline. Animals were sacrificed on the 4th day after DOX administration and samples were collected for further analyses. DOX caused remarkable cardiac damage in both MT+/+ and MT-/- mice as demonstrated by biochemical and histopathological alterations. Zinc pretreatment significantly increased the cardiac MT levels and therefore inhibited the cardiac toxic effects of DOX only in MT+/+ mice, but not in MT-/- mice. Furthermore, elevated formation of superoxide and peroxynitrite were obviously observed after DOX treatment, while these elevation were prevented by MT induction by zinc in MT+/+ mice, but not in MT-/- mice. These findings suggest that metallothionein induction by zinc exhibits protective effects on the cardiac toxicology of DOX, which might be mediated through the prevention of superoxide generation and related nitrosative impairment.

[Metabonomics profile of urine from rats administrated with different treatment period of isoniazid].

OBJECTIVE: To study the effect of different treatment period, of isoniazid (INH) on the metabonomic profile of rat urine and its relationship with traditional toxicity evaluation of blood biochemical indicators and histopathology and to explore the feasibility of metabonomics in the application of drug toxicity. METHODS: Sixty male Wistar rats were orally administrated with 0, 50, 100, 200, and 400 mg x kg(-1) INH for 3, 7, and 14 days, respectively. Rat urine was then collected and its 1H nuclear magnetic resonance (NMR) spectra were acquired. All animals underwent traditional toxicity evaluation. RESULTS: Hepatotoxicity was revealed by traditional toxicity evaluation in rats treated with higher dosage and longer treatment of INH. Time-response relationship existed during the treatment. Time-dependent metabonomics changes conformed with the results of traditional toxicity evaluation. The urine metabonomics showed a trajectory bias from those of the controls or pre-administration, and such bias exaggerated along with the prolongation of treatment, indicating a severer toxic injury. Along with the increase of the concentrations of urinary taurine and glucose and the decrease of the concentrations of urinary citrate and 2-oxoglutarate, the 1H NMR spectra of urine in rats treated with INH also changed. CONCLUSIONS: The metabonomics technique can distinguish the onset and development of toxicity, which helps track and identify biomarkers. The hepatic toxicity induced by INH is related to the injury of mitochondrial function, reduction of energy metabolism in tricarboxylic acid cycle, and perturbations in the metabolism of glucose and lipid. The effect of INH on the rat urine metabonomic profile is related with INH toxicology. Therefore, metabonomics can be recognized as an ideal technique to explore and evaluate the drug toxicities.

[Effect of lycopene on the proliferation of MCF-7 and MDA-MB-231 cells].

OBJECTIVE: To test the effect of lycopene on the proliferation and apoptosis of the estrogen receptor(ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cell lines. METHODS: The cell proliferation was analyzed by the MTT and the H3-TdR incorporation. The effect of lycopene on the cell cycle and apoptosis of the synchronized cells was observed through flow cytometry. RESULTS: Lycopene inhibited the growth and DNA synthesis of both ER-positive MCF-7 and ER-negative MDA-MB-231 cells in a dose-dependent pattern. The maximum inhibition effect appeared after 4 days of exposure to lycopene, with an inhibition rate of 52.6% and 61.9% for the MCF-7 and MDA-MB-231 cells respectively. The flow cytometry analysis found more cells in the G0/G1 phase and less cells in the S and G2/M phase after 24 hours of exposure to lycopene. Lycopene induced apoptosis for the MDA-MB-231 cells, but not for the MCF-7 cells. CONCLUSION: Lycopene inhibits the growth of ER-positive MCF-7 cells through the inhibition of the cell cycle progression. The inhibition of ER- negative MDA-MB-231 cells by lycopene is associated not only with the G1 phase cell cycle-arrest but also the induction of apoptosis.

[Metallothionein inhibited DOX-induced cardiac apoptosis in mice].

OBJECTIVE: To investigate the inhibitive effect of metallothionein (MT) on DOX-induced cardiac apoptosis and the involved possible mechanisms. METHODS: Adult male C57BL mice (6-8 weeks old) were randomly assigned into four groups and given the following treatment: Zinc (ZnSO4, 300 micromol/kg, s.c., once a day for 2 days) or equal volume of physiological saline prior to a single dose of DOX (15 mg/kg, i.p.) or equal volume of saline. Animals were sacrificed on the 4th day after DOX administration and hearts were excised for further studies, including cadmium-hemoglobin affinity assay for MT concentration, ELISA detection of DNA fragmentation and Western blot analysis of Bax and Bcl-2. RESULTS: DOX administration decreased heart weight by 10% and caused remarkable cardiac apoptosis as demonstrated by DNA fragments, while Zinc pretreatment significantly increased the MT levels and therefore inhibited the cardiac apoptotic effect of DOX. Elevated expression of Bax was obviously observed after DOX treatment, while this elevation was prevented by MT induction by Zinc. On the contrary, Bcl-2 protein level was not altered significantly among each group. CONCLUSION: These findings suggest that metallothionein induced by Zinc exhibits protective effects on the cardiac apoptosis of DOX, which might be mediated through the prevention of Bax protein up-regulation by DOX and associated elevation of Bax/Bcl-2 ratio.