OBJECTIVE: To investigate the expression of autophagy and accumulation of reactive oxygen species (ROS) after human gastric cancer SGC-7901 cells were treated with vitamin E succinate (VES). METHODS: Human gastric cancer SGC-7901 cells were treated with VES at different doses (0,5,10,15,20 μg/mL) for 24 h. Then,laser confocal microscopy was used to observe fluorescence intensity and distribution of the autophagy marker protein:microtubule-associated protein 1 light chain 3 (LC3). Western blot was used to evaluate the expression levels of LC3 and Beclin-1. Flow cytometry was used to detect the level of intracellular ROS. In addition,cells exposed to 20 μg/mL VES for 24 h with or without antioxidant N-acetyl-L-cysteine (NAC,20 mmol/L),were observed for fluorescence intensity and distribution of LC3. Western blot was used to evaluate levels of LC3 and Beclin-1. Furthermore,cells with BECN-1 knocked-down by RNA interference were treated with 20 μg/mL VES for 24 h. Then,flow cytometry was used to detect the level of intracellular ROS. RESULTS: Compared with the control group,VES treatment increased the amount of intracellular LC3 punctate fluorescence and the number of autophagic vacuoles,and increased the expression of endogenous autophagy marker protein LC3 and Beclin-1. Flow cytometry showed that the level of intracellular ROS was gradually increased. When ROS production was reduced by the antioxidant NAC,the fluorescence intensity of LC3,the expression of LC3 and Beclin-1 were significantly lower compared with VES-treatment alone. After the cells were transfected with BECN-1 siRNA,the level of intracellular ROS was higher compared with VES alone. CONCLUSION: VES induced autophagy and accumulation of ROS in human gastric cancer SGC-7901 cells. In addition,there were interactions between autophagy and ROS level in VES-treated cells.

OBJECTIVE: To construct esophageal epithelial cells which express high levels of DNA methyltransferase 1 (DNMT1). METHODS: Plasmids WV0133,WV0132 and WV0132 were constructed and transfected into esophageal epithelial cells. Transfection of the cells was observed under the inverted fluorescence microscope. Cells that expressed high levels of DNMT1 were selected by puromycin. Expressions of DNMT1 mRNA and protein were determined by using quantitative real-time PCR and Western blot. RESULTS: Compared with the normal cell group, the mRNA expression levels were 1.786 times and 2.721 times in the WV0133- and WV0132-transfected cells, respectively,and their protein expression levels were 2.734 times and 3.100 times,respectively. They were significantly higher than those in the normal cell group (P<0.01). CONCLUSION: An esophageal epithelial cell line which expressed high levels of DNMT1 was successfully constructed. The cell line can be a valuable tool to study mechanisms for esophageal cancer development.

OBJECTIVE: Mutagenic effects of N-(n-butyl)-thiophosphorictriamide (NBPT) in somatic and germ cells of adult male ICR mice were evaluated using the bone marrow chromosome aberration (CA) and dominant lethal mutation (DLM) assays. METHODS: In the CA assay,adult male ICR mice were exposed to NBPT at 250,500 and 1000 mg/kg by a single oral administration. For the 1 000 mg/kg group,bone marrow samples were harvested at 24,48 and 72 h after the treatment and slide were made. For the other groups,bone marrow samples were harvested at 24 h after the treatment. From slides of each mouse,100 metaphase bone marrow cells were observed for chromosome aberration, the cell numbers of chromosome aberration,polyploidy,gap were recorded respectively. In the DLM assay,after mice were exposed to NBPT for successive 5 days,each mated with 2 virgin females for 5 days at predetermined intervals (2 days) for a total 7 rounds. Females of each round were sacrificed on the 14^th day of pregnancy. The total number of implantation,number of corpora lutea,number of stillbirth,live births and fetal absorption were counted and recorded. RESULTS: Chromosome aberration rates for mice after 24 h of exposure to the three doses were 2.4%,3.0% and 2.0%,respectively. Chromosome aberration rates for the 1 000 mg/kg group were less than 4% after the 24,48 and 72 h of exposure. All the observed aberration rates were not significantly different from the vehicle control group (P>0.05). The incidence of tetraploid chromosome and gap for each group showed no correlation with NBPT treatment. In the DLM test,for the 7 batches of mating,pregnancy rates in three NBPT treated groups were 100%. In addition,the average number of maternal implantation,number of corpora lutea,number of live births showed no significant difference compared with the vehicle control group. Preimplantation loss rate increased only in the 4^th round of mating in the 250 mg/kg group. The average number of non-viable fetus decreased in the 6^th round of mating in the 250 mg/kg group. Both showed significant difference compared with the vehicle control (P<0.01). Lethal mutation rates for female mice for each mating round were mostly negative for all the exposed groups. CONCLUSION: Within our experimental conditions, NBPT did not induce bone marrow chromosome aberrations nor dominant lethal mutation.

OBJECTIVE: To investigate whether the pH and serum of cell culture medium would affect the Metridia luciferase (MLuc) assay or not,and to explore a modified assay method for MLuc. METHODS: A recombinant retrovirus vector which expressed MLuc was constructed and then transiently transfected into human cervical cancer cell line HeLa to produce MLuc. Then,the effects of cell culture medium (e.g. buffers combined with reagents) on bioluminescence of MLuc were assayed. RESULTS: Various pH levels of cell culture medium caused great fluctuation of the bioluminescence intensity of MLuc. In addition,serum in the cell culture medium would alter the background of MLuc. When the cell culture medium was replaced by phosphate buffered saline (PBS) supplemented with 0.02% (V/V) NP-40 (pH=7.4),the bioluminescence intensity between them was comparable. However,the background level of MLuc was decreased from 600 to 20. CONCLUSION: Using PBS supplemented with 0.02% NP-40 as an assay buffer for MLuc,the side effects from pH and serum of cell culture medium during MLuc assay would be reduce and application of MLuc in research can be improved.

OBJECTIVE: To investigate the effects of ⁶⁰Co γ rays on testosterone secretion and testicular histopathology of male Beagle dogs. METHODS: 15 male Beagle dogs were subdivided into 3 groups:the control group and the irradiation groups. Irradiation groups were exposed to 0.5 Gy or 5.0 Gy ⁶⁰Co γ ray,dosage rate 0.5 Gy/min. At the 4^th-,7^th-,14^th-,28^th- and 60^th-day after irradiation,the content of serum testosterone was determined by the competitive inhibition immunoassay. And at 60^th day after irradiation,the testicles were obtained for observation and histopathology. RESULTS: During 10-13 days after irradiation,all the animals of the 5.0 Gy γ ray irradiation group died,and hemorrhage spots were found on their testes and epididymises. At 60th day after irradiation,there was no difference statistically between the 0.5 Gy γ ray irradiation group and the control group. The testicle average weight of 0.5 Gy group was 69% of the control group,while there was no statistical difference for testicle/body weight ratio between the 0.5 Gy γ ray irradiation group and the control group. Meanwhile,at 4^th-,7^th-,14^th-,28^th-and 60^th-day after irradiation,there was no statistical difference for serum testosterone content among the 0.5 Gy γ ray irradiation group and blank control group. Spermatogenic cells from the 0.5 Gy and 5.0 Gy γ ray irradiation groups showed necrosis and lysis. In addition,azoospermia generation wasn't found in the testicle of the 5.0 Gy group. CONCLUSION: Acute 0.5 Gy and 5.0 Gy γ ray irradiation induced damage to the reproductive system of male Beagle dogs:the testicle structure, spermatogenic cells and azoospermia generation were severely damaged. However,serum testosterone content did not show much obvious changes,

OBJECTIVE: This study was designed to investigate the dose-dependent relationship and the possible mechanisms of renal injury by cadmium in rats. METHODS: Forty male rats were randomly divided into four groups (0,1,3 and 5 mg/kg CdCl₂) groups. The rats were intra-gastrically administrated CdCl₂ daily to investigate the induction of renal injury. Blood urea nitrogen (BUN) and serum creatinine (SCr) were measured to evaluate renal function. Kidney tissues were stained with hematoxylin-eosin (HE) and observed for histopathological changes. Transmission electron microscopye was used to detect ultrastructural abnormalities. Levels of ROS in kidney was detected by DHE and MitoSOX staining. The levels of SOD1,SOD2,GPx-1,CAT,Bax,Bcl-2,GRP78 were measured by Western blotting. RESULTS: After exposure to CdCl₂ for 4 weeks,the relative kidney weights showed no significant difference among the 4 groups. As the dose of CdCl₂ increased,the levels of BUN elevated gradually (r=0.463,P<0.05). Pathological damages to kidneys were progressively aggravated. Swelling of glomerulus and renal tubule,and necrosis of tubular epithelial cells were observed under light microscopy. And it was also observed that the lumina of renal tubules contained necrotic epithelial cellular debris and casts. Interstitial hyperemia and inflammatory infiltration were found as well. The ultrastructure of mitochondria in the epithelium of proximal convoluted tubule showed swelling,deformation and vacuolation in a dose-dependent manner. The level of kidney ROS elevated gradually. Levels of SOD2,GPx-1 and CAT were upregulated (r=0.854,0.975,0.918,P all<0.05) while SOD1 were downregulated (r=-0.987,P<0.05) as the dose of CdCl₂ increased. Meanwhile,levels of the apoptosis-promoting Bax increased (r=0.226,P<0.05) while the anti- apoptotic Bcl-2 decreased (r=-0.85,P<0.05) and the ratio of Bax/Bcl-2 increased (r=0.83,P<0.05). The levels of GRP78 also increased when the dose of CdCl₂ increased (r=0.913,P<0.05). CONCLUSION: The results demonstrate that there was a dose-dependent toxic effect of CdCl₂ on renal structure and function. Moreover,oxidative stress might be the key mechanism involved in the toxicity of cadmium because of increased antioxidase expression and levels of ROS.

OBJECTIVE: To investigate the effect of transgenic rice with the cry1Ab/Ac gene on morphology of intestinal mucosa,cell number of intraepithelial lymphocytes and goblet cells in Cynomolgus monkey. METHODS: Twenty-four cynomolgus monkeys,half male and female,were randomly divided into 3 groups including control group of ordinary rice,low dose group and high dose groups of transgenic rice with the cry1Ab/Ac gene. Monkeys were fed twice a day and each feeding was (100±10) g for 6 months with 4 weeks recovery period. Monkeys were observed for general toxicity,clinical effects,body weight,body temperature,electrocardiogram,hematology,serum biochemistry, urinary biochemistry,organ weight and histopathological examination. HE and PAS staining were done for duodenum, jejunum and lleum. The morphology of intestinal mucosa,cell number of intraepithelial lymphocytes and goblet cells were observed under microscope. RESULTS: After six months,detection of transgenic rice with the cry1Ab/Ac gene were 0, 17.5% and 70%,in control,low dose and high dose groups,respectively. Compared with the control group,no abnormalities in toxicity,etc. were found in the treated groups. No pathological changes were observed in intestinal morphology. Cell number of intraepithelial lymphocytes and goblet cells were not statistically different between control and treated groups. CONCLUSION: Transgenic rice with the cry1Ab/Ac gene had no side effects on small intestine mucosal morphology,cell number of intraepithelial lymphocytes and goblet cells in Cynomolgus monkey.

OBJECTIVE: To investigate the effects of folic acid on N-methyl-N-nitro-N'-nitrosoguanidine (MNNG)-treated esophageal epithelial cells,in expression of DNMT1 activity and to provide a theoretical basis for the prevention and treatment of esophageal cancer. METHODS: Using a 3 factors and 3 levels (3×3) factorial design,esophageal epithelial cells in culture were exposed to different concentrations of folic acid (0.000,0.400,0.800 μg/mL) and MNNG (0,0.750,1.500 μg/mL) for 48,72 and 96 h. DNA methyltransferase activity was detected by ELISA;DNMT1 mRNA expression by RT-PCR;DNMT1 protein expression by Western blot method. RESULTS: Treatment of cells with different concentrations of folic acid and MNNG and in different times,at a fixed MNNG concentration and time factors,and with increasing concentrations of folic acid,the DNMT1 activity, DNMT1 mRNA and protein expression levels all gradually decreased (P<0.01). By fixing folic acid concentrations and time,and with increasing concentrations of MNNG,DNMT1 enzyme activity, DNMT1 mRNA and protein expression levels all increased (P<0.01). By fixing the MNNG and folic acid concentrations with intervention,the extension of time of DNMT1 activity,DNMT1 mRNA and protein expression levels all gradually increased (P<0.01). There were interactions between MNNG concentrations and action time,and folic acid and MNNG concentrations (P<0.01). There were significant differences between different concentrations of folic acid and different action time,between different MNNG concentrations and different time of action (P<0.05). CONCLUSION: Lower folate concentrations had a negative impact on the expression and effect of MNNG on DNMT1 activity in esophageal epithelial cells. The results suggest that folic acid played a role in promoting the occurrence of esophageal carcinoma,and had a protective effect against MNNG toxicity.

OBJECTIVE: The project was based firstly on field surveys to understand plant community ecology around uranium tailings in the North as well as the dominant plant and soil uranium concentrations. Secondly,the characteristics of radioactive nuclide uranium accumulation in the plant was determined in order to further explore phytoremediation technology for uranium-contaminated soil repair. METHODS: To observe the growth condition of mining area of wild plants,to collect common wild herbaceous plant samples in different work places,to detect the content of radioactive uranium using ICP-MS in plants and soil,to screen out plants of strong capability to accumulate uranium,and to explore the methods and technique of phytoremediation technology with contaminated soil. RESULTS: The uranium content of in the underground part of plants samples was higher than that of the above ground part. For each plant,the uranium content from high to low was root,leaf,stem,flowers and fruit respectively. In the No. 3 work place,the uranium content of Polygonum orientale L. root was 64.10 μg/g which was 2.29×higher than that of other plants. At 2 km outside the uranium mine area,the uranium content in edible part (niblet) of collected samples of corn was lower than that in the inedible parts. The uranium enrichment coefficient in edible part of corn (niblet) was 0.000 9,the inedible part of corn was 0.039. CONCLUSION: Plant samples around uranium mine sites showed accumulation of uranium. Enrichment was mainly in plant roots and uranium content in different parts of plant were root > leaf > stem > flower and fruit. Enrichment of plants can be used for phytoremediation of soil pollution,for improvement of ecological environment,and for evaluation of food safety in nuclear accident emergency. Further study on plant enrichment of uranium can have application value and practical significance.

OBJECTIVE: To profile microRNAs expression among workers with trichloroethylene (TCE)-induced medicamentosa-like dermatitis (OMLDT). METHODS: Serum samples from 5 patients with OMLDT and with TCE contacts were collected. The total RNA was extracted then labeled with Hy3 fluorescent. MicroRNAs were hybridized with miRCURY LNA chips and images were acquired using the GenePix 4000B scanner,qPCR method was used to verify the results of miRNA microarray. The results were further analyzed by prediction softwares. RESULTS: A total of 69 microRNAs were differentially expressed between OMLDT and TCE contacts. Among the microRNAs,39 were up-regulated and 30 were down-regulated. Increase of miR-199b-5p was verified using qPCR. Target prediction results showed that the same target genes of miR-409-3p and miR-485-3p had been predicted in all forecasting software. CONCLUSION: The study showed that expression of microRNA in serum of workers with OMLDT was significantly altered,and miR-199b might serve as a candidate biomarker for OMLDT.

OBJECTIVE: To investigate the role of reactive oxygen species (ROS) in isoniazid (INH)-induced DNA damage in L-02 cells and the protection by quercetin. METHODS: L-02 cells were divided into several groups:blank control,INH (10 mmol/L),quercetin low dose (10 mmol/L INH +25 μmol/L quercetin) and high dose (10 mmol/L INH +50 μmol/L quercetin). After cells were treated for 24 hours,DNA damage was detected by using the Comet test;ROS generation and mitochondrial membrane potential were evaluated by application of fluorescent probes DCFH-DA and Rhodamine123. RESULTS: Compared with the blank control group,the percentages of tail DNA,tail length and tail moment (from the Comet assay) of the INH group were significantly increased (P<0.01). Compared with the INH group,the percentages of tail DNA,tail length and tail moment of the low and high dose quercetin groups were significantly reduced (P<0.05 and P<0.01,respectively). The levels of mitochondrial ROS in cells of the INH group were significantly increased over the blank control group (P<0.01);the levels of mitochondrial ROS in the low and high dose quercetin groups were significantly decreased than that of the INH group (P<0.05 and P<0.01,respectively). The mitochondrial membrane potential of the INH group was significantly lower than that of the blank control group (P<0.01). The mitochondrial membrane potential of the low and high dose quercetin groups were significantly higher than that of the INH group (P<0.05 and P<0.01,respectively). CONCLUSION: INH can induce DNA damage in the L-02 cells and the damage may involve ROS-mediated mitochondrial damage. Quercetin has a protective effect against the INH-induction of DNA damage and the effect may be related to its inhibition of ROS-mediated mitochondrial damage.

OBJECTIVE: To explore effect of the chlorpyrifos herbicide (CPF) on growth of Drosophila melanogaster (D. melanogaster). METHODS: Analyzed the median lethal dose (LC₅₀) using Probit after D. melanogaster were treated with 0.5,1.0,1.5,2.0,2.5,3.0 and 4.0 mg/L of CPF for 96 h. According to LC₅₀,weight of D. melanogaster was evaluated after being exposed to 0.04,0.08,0.16 and 0.32 mg/L CPF concentration. The experimental data were analyzed by ANOVA test. RESULTS: The toxicity of CPF to female D. melanogaster (LC₅₀ is 0.447 mg/L) was greater than that for male (LC₅₀ is 0.858 mg/L). There was no statistical significance of the weight changes in female D. melanogaster with different concentrations of CPF at different treatment times (P>0.05). With 0.04~0.32 mg/L CPF,male D. melanogaster had reduction of 0.01 mg on average per day which was significantly different compared with the control group (P<0.05 or P<0.01),and showed clear time-dependent effects. In addition,their weights decreased significantly (ranging between 0.04-0.08 mg) with the treatment of 0.04 mg/L (1/20 of LC₅₀) CPF which showed that male D. melanogaster was extremely sensitive to the toxicity at the low concentration. The weight of male D. melanogaster increased by 0.02 mg at 0.16 mg/L CPF and was significantly different at 72 h and 96 h among each experiment group (P<0.05). And showed clear does-dependent effect. CONCLUSION: CPF had significant impact on the growth of male D. melanogaster as reflected by their weight changes.

OBJECTIVE: The micronucleus test with Vicia faba root tip cells was used to study the genotoxicity of tap water from three northern cities. METHODS: 12 samples:including the source water,chlorinated water and supply tap water,were collected from six waterworks in the northern cities of A,B,C. The samples were used to treat Vicia faba root tip cells directly. Micronuclei frequencies and pollution index (PI) were determined and statistics analyses were performed to evaluate differences in micronuclei rates among different samples. RESULTS: Among the 3 waterworks in city A,the chlorinated water in waterwork # 3 induced higher micronuclei rates than the negative control,and the PI was moderate. Other water samples from city A did not induce micronuclei. In city B,the chlorinated water induced higher micronuclei rate than the negative control,and the PI was slight. The other water samples did not induce micronuclei. However,the PI for the supply tap water was slight. In city C,the micronuclei rates induced by all 3 water samples were not different from that of the negative control. However,the PI for all samples were slight and the order of the PI levels was:chlorinated water > supply tap water > source water. CONCLUSION: Based on the evaluation using micronuclei rates and PI,the mutagenic potential of chlorinated water was the strongest. In addition,the water quality of city A was better than that in cities B and C.

OBJECTIVE: Caenorhabditis elegans were used to investigate the induction of reproductive toxicity of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA),as an alternative models for evaluating reproductive toxicity. METHODS: Three PFOS/PFOA dose (0.1,0.01,0.001 mmol/L) groups and a control group were organized. After synchronization,L4 larvae were exposed for 24 h in 24-well plates,the L1 larvae were exposed for 48 h. The brood size and the generation time of Caenorhabditis elegans were evaluated to determine the induction of reproductive toxicity by PFOS and PFOA. RESULTS: Compared with the control,the high and the medium dose groups with exposure for 24 h showed significant difference in the brood size (P<0.05),and the three PFOA exposure dose groups showed significant difference among them (P<0.05). Generation time showed no significant difference among the exposed and the control groups. Compared with the control group,the high and medium dose groups with exposure for 48 h showed significant difference in the brood size (P<0.05) and the three exposed groups showed significant difference in generation time (P<0.05). CONCLUSION: The results suggested that brood size of Caenorhabditis elegans was a sensitive indicator for evaluating reproductive toxicity of PFOS and PFOA. In addition,generation time measurements was more sensitive using L1 larvae than that of L4 larvae.