OBJECTIVE: To develop a detection method for free radical scavenging capacity in biological samples，based on the oxygen radical absorbance capacity (ORAC) method，which was applied to evaluate the biological antioxidant capacity and to classify the extent of environmental pollution and toxic effects. METHODS：Zebrafish was chosen as the experimental animal，to test the hydroperoxyl free radical (ROO·). The azo compound 2，2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was chosen as the oxygen free radical source. Fluorescein (FL) as the fluorescence indicator and Trolox as the quantitative standard. Then fluorescence intensity decay process was recorded after free radicals reacted with FL，and the ability of antioxidant delay the fluorescence intensity recession was assessed to evaluate the effect of chemicals on ORAC. The established method was applied to study the effect of different concentrations of four heavy metals (copper，cadmium，chromium and lead) on ORAC of zebrafish. RESULTS：The established method showed high specificity and good accuracy (between 97% and 108%) and precision (CV less than 5%)；this method also had good reproducibility (CV is 2.92%). The limit of quantitation (LOQ) and the limit of detection (LOD) were 3.03 and 1.00 μmol Trolox/mg protein，respectively，the correlation coefficient(R2) was ≥ 0.99. According to the new established quantitative analysis methods，we determined the physiological normal range (0-50 μmol Trolox/mg protein) and classified the heavy metal contamination degree and toxic effects based on the effect of metals on ORAC of zebrafish. CONCLUSION：The established method may be suitable for evaluating biological antioxidant capacity and classifying environmental pollution and toxic effects. It could be effective in enhancing the inter laboratory data recognition and promoting the application of biomarkers for antioxidant protection in environmental pollution monitor and environmental risk assessment.

OBJECTIVE: To investigate the effect of Axin on the biological behavior of SW480 colon cancer cells and to elucidate its molecular mechanism. METHODS：SW480 colon cancer cells were transfered with pCMV5-HA-Axin and pCMV5-HA and blank control group was desigined. Cell proliferation was detected by MTT assay and colony formation assay，and the cell cycle was analyzed by flow-cytometry. At the same time，Real-time PCR and Western blot were used to assess the expressions of Axin and p53 oncogene in SW480 cells. RESULTS：Over-expression of Axin significantly inhibited the growth of SW480 colon cancer cells. MTT assay revealed over-expression of Axin inhibited the proliferation of SW480 cells(P<0.05). Transfection of Axin increased colon cancer SW480 cell cycle pre G1 ratio，inhibited G1 phase and decreased S ratio(P<0.05). p53 mRNA doubled in SW480 cells with transfered pCMV5-HA-Axin(P<0.05). P53 protein was also doubled in SW480 cells with transfered pCMV5-HA-Axin compared with empty vector groups(P<0.05). CONCLUSION：Over-expression of Axin could significantly inhibit the growth of colon cancer cells，and its mechanism might be related to the activation of the p53 oncogene.

OBJECTIVE: To find the changes of β-catenin and phosphorylated β-catenin (p-β-catenin) Ser675 in esophageal precancerous lesions in Wistar rats induced by methyl benzyl nitrosamine (MBNA). METHODS： Wistar rats received MBNA injections at dose of 3.5 mg/kg 2 times per week. At 10 weeks，the pathological changes of esophageal mucosa were examined. β-catenin mRNA level was measured by quantitative real-time PCR and the expressions of β-catenin protein and p-β-catenin(Ser675) were evaluated by Western blot. The location of β-catenin was detected by immunohistochemistry. RESULTS：Compared with normal group，the level of β-catenin mRNA was significantly decreased，the levels of β-catenin and p-β-catenin (Ser675) protein were significantly increased in treated group. CONCLUSION：The elevated protein levels of β-catenin and p-β-catenin (Ser675) were related with esophageal precancerous lesions in Wistar rats induced by MBNA ，and could be considered as an indicator of esophageal carcinoma.

OBJECTIVE: To explore the potential of human embryonic stem cell-derived hepatocytes (hESC-Heps) for hepatitis B virus (HBV) model. METHODS：Human embryonic stem cells were directly induced to differentiate into hESC-Heps using a step-wised method. Expression of hepatocyte-enriched factors such as hepatocyte nuclear factor 4 alpha (HNF4α) and albumin (ALB)，and HBV functional receptor Na+/taurocholate cotransporting polypeptide (NTCP) receptor in hESC-Heps were examined by immunofluorescence method. Moreover，activation of signal transducer and activator of transcription 2 (STAT2) in hESC-Heps treated with type Ⅲ interferon was examined by Western blotting. RESULTS：Mature hESC-Heps expressed HNF4α，ALB，and NTCP receptor as well as displayed type Ⅲ interferon response specific in primary human hepatocytes. CONCLUSION：hESC-Heps may be a potential model for HBV research.

OBJECTIVE：To investigate the genetic characteristics of single nucleotide polymorphisms (SNPs) in serotonin receptor 1B gene (HTR1B) in Chinese Han males. METHODS：SNPs located in HTR1B were identified in 155 cases of target population through sequencing in this study. The genetic characteristics，linkage disequilibrium (LD) of each polymorphism locus，tag SNPs，haplotypes and their blocks were analyzed with HaploView software. RESULTS：Seven SNPs were obtained： rs17273700(-860A>G)，rs1228814(-700G>T)，rs11568817 (-262A >C) and rs130058(-161T>A) in the 5′-flanking region，rs6298(+129A>G)，rs6296(+861G>C) in the coding region and rs6297(+1 180T>C) in the 3′-flanking region. All the allele frequencies were in the Hardy-Weinberg equilibrium (P>0.05). There were high LD between the polymorphisms of -860A>G and -262A>C. +129A>G and +861G>C also showed high LD in this population. Eight major haplotypes (H1-H8) accounting for 94.2% were established，while the frequency of wild type haplotype (H1) was 46.0%.   -860A>G，-700G>T，-161T >A，+129A>G，+1 180T>C were determined as tag SNPs. Two haplotype blocks were constructed and -860A >G，+129A>G were determined as their htSNPs. CONCLUSION：The appropriate tag SNPs and htSNPs in HTR1B are established in Chinese Han males for the first time. It lowered the number of SNP loci in further research，and provided the basis for explorations of their association with diseases.

OBJECTIVE: The aim of the study was to establish a suitable cell model for investigating the role and mechanism of PLK1 overexpression in esophageal cancer through inactivation of PLK1 expression employing lentiviral-mediated inducible shRNA expression system. METHODS：Chemically synthesized PLK1-shRNA oligonucleotides were annealed and ligated into the lentiviral vector pLKO-Tet-On. The ligation products were transformed into the competent E. coli Stbl3 cells. Colony PCR and sequencing analysis were used to identify the positive recombinants. The pLKO-shPLK1-Tet-On construct and packaging plasmids were co-transfected into 293T cells to produce the lentiviral particles. Esophageal cancer cells KYSE510 were infected with the viral supernatant and the stable cell strain was selected with puromycin. Doxcyclin-induced expression efficiency of PLK1-shRNA was determined by qRT-PCR and Western blotting. RESULTS：Colony PCR and sequencing analysis showed that PLK1-shRNA oligos were correctly inserted into the pLKO-Tet-On vector. The stable cell strain KYSE510-shPLK1-Tet-On was obtained through infecting the lentivirus expressing inducible PLK1-shRNA and then selected with puromycin. The results of qRT-PCR and Western blotting indicated that the expression of PLK1 in KYSE510-shPLK1-Tet-On cells could be markedly downregulated by 0.1 μg/mL Dox. CONCLUSION：We successfully constructed a lentivirus-based inducible PLK1-shRNA expression vector and established an esophageal cancer cell line with stable expression of inducible PLK1-shRNA，providing an ideal cell model for further exploring the relationship between aberrant PLK1 expression and development and progression of esophageal cancer.

OBJECTIVE: To investigate the effects of MNNG on methylation and expressions of p16 and FHIT genes in normal esophageal epithelial cells in Kazakhs. METHODS：Kazakh people’s normal esophageal epithelial cells were culture in vitro in medium containing MNNG concentrations of 0.00，0.75，1.50，and 3.00 mg/mL for 24 h. Methylation of p16 and FHIT were analyzed by methylmion specific PCR(MSP)，the mRNA and protein level of p16 and FHIT were measured by real time PCR (RT-PCR) and Western blot. RESULTS：Compared with the control group， p16 and FHIT gene methylation status did not change in treated groups，but the expressions of p16 mRNA and protein level in treated groups were significantly higher than the control group(P<0.05 or P<0.01). The expressions of FHIT mRNA level in 0.75 μg/mL group was significantly decreased compared with the control group(P<0.01)，but in 3.00 μg/mL group FHIT mRNA and protein levels were significantly increased(P<0.05). CONCLUSION：At certain concentration， MNNG could increase the expressions of p16 and FHIT in normal esophageal epithelial cells in Kazakhs.

OBJECTIVE: To investigate the tolerance of various kinds of cell lines to ultraviolet B radiation. METHODS：Cell viability and proliferation were detected by MTT，firefly luciferase activity and cloning efficiency assays after cells including epidermal cells (HaCaT)，melanocytes (A875)，pulmonary adenocarcinoma cells (A549 and H322) and hepatocarcinoma cells (HepG2) of human were irradiated with ultraviolet B at dose of 20 or 50 mJ/cm2. RESULTS：Cell viability including epidermal cells (HaCaT)，melanocytes (A875)，pulmonary adenocarcinoma cells (A549 and H322) and human hepatocarcinoma cells (HepG2)，decreased significantly 1 day after ultraviolet B irradiation at doses of 20 and 50 mJ/cm 2 (P<0.05). The viability of these cells were still lower than untreated control cells 3 days after irradiation. Finally，the proliferative ability of all the examined cells was suppressed after ultraviolet B irradiation at doses of 20 and 50 mJ/cm2(P<0.05)，as shown by cloning efficiency assays. Further firefly luciferase activity assay indicated that the viability of A549 cells were suppressed 5 and 10 days after exposure(P<0.05). CONCLUSION：Ultraviolet B irradiation affected the viability and proliferation of epidermal cell，lung adenocarcinma cell and hepatoma cell. Normal epidermal cell HaCaT possessed the more tolerance than A875，A549，H322 and HepG2 cells which is the source of tumors on ultraviolet B.

OBJECTIVE: To evaluate role of protein Bcl-2 in the oxidative injury induced by radiotherapy or chemotherapy. METHODS：We employed the rat models for radiotherapy and chemotherapy. For each experiment，the rats were divided into four groups：control group，model group，radiotherapy or chemotherapy group，and protection group. The rats for radiotherapy experiment received local radiation of 60Co γ with the accumulative dosage of 47 Gy. The rats for chemotherapy experiment received doxorubicin (2 mg/kg) by intraperitoneal injection every two days. The protection groups received multiple antioxidants (Antitoxin 60 mg/kg+Vit E 20 mg/kg+Tannin 2.5 mg/kg) by gavage every day. These treatments lasted 5 weeks for radiotherapy experiment and 8 weeks for chemotherapy experiment. At the end of the experiments，all of the rats were sacrificed and the specific tissues were fixed to cut sections. The expression of protein Bcl-2 was measured by immunohistochemistry and the positive stainings were quantified by optical density analysis. RESULTS：There was a certain positive expression of protein Bcl-2 in the normal rats. Radiotherapy or chemotherapy treatment significantly inhibited the protein Bcl-2 expression (P<0.05)，in which antioxidant supplementations showed obvious protection (P<0.05). CONCLUSION：Protein Bcl-2 was a key target of oxidative injury induced by radiotherapy and chemotherapy，as well as a potential target of antioxidant supplementation to reduce the side effects of radiotherapy and chemotherapy.

OBJECTIVE: To explore the effects of di(2-ethylhexyl) phthalate (DEHP) on hormonal-related gene expression levels in male rats. METHODS：A total of 40 healthy 5-week-old male Sprague-Dawley rats were randomly divided into four groups with 10 rats in each group. The DEHP doses were 0 mg/kg in control group(corn oil)，100 mg/kg in low dose group，500 mg/kg in medium dose group and 1 500 mg/kg in high dose group. Rats were exposed to DEHP via gavage administration for six consecutive weeks，DEHP administration was once a day and five times a week. After exposure，rats were anesthetized by ether，then sacrificed for further study. The relative gene expression levels of steroidogenic acute regulatory protein (StAR)，3β-hydroxysteroid dehydrogenase(3β-HSD)，17β-hydroxysteroid dehydrogenase (17β-HSD)，estrogen receptors (ERα/β) and gonadotropin releasing hormone (GnRH) were analyzed by real time quantitative RT-PCR. RESULTS：Compared with control group，the relative gene expression levels of StAR and 3β-HSD were increased in medium DEHP dose and high DEHP dose groups (P<0.01 or P<0.05). The relative gene expression levels of 17β-HSD，ERα and ERβ were increased in high DEHP dose group (P<0.01 or P<0.05)，while the relative gene expression levels of GnRH was decreased in medium DEHP dose and high DEHP dose groups(P<0.01 or P<0.05). CONCLUSION：DEHP could cause endocrine disorder and interfere with the synthesis of male hormones in rats，ultimately leading to male reproductive dysfunction.

OBJECTIVE: To explore the pathological damage and ultrastructure changes in female rat ovary when rats were treated with di(2-ethylhexyl) phthalate (DEHP)，to assess the toxicity of DEHP in female reproduction. METHODS：40 female SD rats were randomly divided into control group (corn oil)，DEHP low dose group (100 mg/kg)，DEHP medium dose group (500 mg/kg )，DEHP high dose group (1 500 mg/kg). The rats were treated with DEHP by gavage for 6 weeks. Rat body weight and ovary wet weight and ovary coefficients were calculated，the ovarian pathology was studied after HE staining，the ultrastructure changes in granulose cells were examined through electron microscopy. RESULTS：The ovarian weight and ovary coefficients were increased in DEHP high dose group. Pathological changes showed ovarian follicle damage，loose granular cell structure， follicular atresia after DEHP treatment. We also found mitochondria swollen， mitochondrial cristae decreased， cytoplasmic vacuolation， nuclear chromatin condensation，apoptotic bodies present，cell degeneration and necrosis in DEHP treated groups. CONCLUSION：DEHP could affect follicular development，induce granulose cell apoptosis in female rats，and subsequently causing reproductive toxicity.

OBJECTIVE: To investigate the reproductive toxicity of di(2-ethylhexyl) phthalate (DEHP)， and to assess the expressions of apoptosis genes and oncogenes after DEHP treatment in female SD rats. METHODS：Female SD rats were treated with different doses of DEHP (corn oil group used as control，DEHP treatment including 100 mg/kg，500 mg/kg，1 500 mg/kg) for 6 weeks. The expression levels of apoptosis genes(Bcl-2，Caspase-3，Caspase-8，Caspase-9) and oncogenes (c-fos and k-ras) mRNA were determined by real-time quantitative PCR. RESULTS：Bcl-2 expression level at DEHP 1 500 mg/kg group was significantly decreased in comparison with control group(P<0.01). The expression levels of Caspase-3，Caspase-8 and Caspase-9 mRNA were significantly increased in rat ovarian tissue after DEHP treatment (P<0.05 or P<0.01). c-fos mRNA expression levels showed no significant difference compared with control. k-ras mRNA expression levels increased significantly in DEHP 500 mg/kg and 1 500 mg/kg treatment groups(P<0.01). CONCLUSION：DEHP could activate Caspase apoptosis pathway through the mitochondrial pathway，resulting in apoptosis of ovarian cells，leading to further damage of ovarian function and reproductive endocrine function. DEHP could also activate oncogene expression，therefore DEHP could possess potential carcinogenesis risk.

OBJECTIVE：The genotoxicity of essential oil from Chenopodium ambrosioides L. was evaluated with TK gene mutation test to provide information for its safety. METHODS：The mouse lymphoma cells L5178Y were exposed to essential oil of C. ambrosioides at volume fraction concentrations of 0.002%， 0.004%，0.006% and 0.008% for 3 h. 1% dimethyl sulfoxide (DMSO) and 10 μg/mL cyclophosphamide (CP) were used as solvent and positive controls，respectively. TK gene mutation was assessed with microwell method to detect cytotoxicity and mutation frequency at TK locus in L5178Y cells. RESULTS：Relative survival (RS)，relative suspension growth (RSG) and relative total growth (RTG) decreased when concentration of essential oil from C. ambrosioides increased，with only 29.38%，38.13% and 5.75%，respectively，in 0.008% dose group indicating significant cytotoxicity. Compared with the solvent control mutation frequency (TMF) increased with the rising concentration of essential oil(F=410.5，P=0.00). Multiple comparisons of TMT between the different essential oil groups，positive control group and solvent control group were statistically significant differences (P<0.05). The percentage of small growth mutant (SCM) was more than 95%. CONCLUSION： Essential oil from C. ambrosioides showed strong cytotoxicity and could increase mutation frequency at TK gene locus in L5178Y cells.

OBJECTIVE: To investigate the acute toxicity and genetic damage in experimental animals. METHODS：In the acute toxicity study，mice were intragastrically administered by 21.5，10.0，4.64，2.15 g/kg rabdosia serra teabags groups and calculated the median lethal dose (LD50) by Horn method．In Ames test，set 5 000， 1 000，200，40，8 μg rabdosia serra teabags per plate．In mice bone marrow cell micronucleus and mice sperm abnormality test，2.50，5.00 and 10.0 g/kg rabdosia serra teabags groups．RESULTS：The acute oral toxicity in rabdosia serra teabags was more than 21.5 g/kg．In Ames test with and without S9 metabolic activation，the reverse mutations of typhimurium TA97， TA98，TA100，TA102 were less than double compared with natural mutations，and no dose-response relationship was found．The micronucleus rates and sperm abnormality rates in all doses showed no significant difference from the negative control (P>0.05). CONCLUSION：Under these experimental conditions，rabdosia serra teabags was shown to be a non-toxic substance. It did not cause genetic toxicity.

OBJECTIVE: To study DNA damage induced by di (2-ethylhexyl) phthalate (DEHP) to mice lung cells. METHODS：The negative control group was exposed to saline. Extracted mice lung cells were exposed to different concentrations of DEHP(5，25，125，625 μmol/L) and single cell gel electrophoresis was used to test the DNA breakage degree. RESULTS：Compared with the control group，tail DNA percentage，tail moment and Olive tail moment of 125 μmol/L and 625 μmol/L DEHP-treated groups were increased significantly (P＜0.05). CONCLUSION： DEHP could induce DNA breakage of mice lung cells in vitro.