OBJECTIVE: To screen the differential gene expression and related pathways of fetal skeletal malformations induced by deoxynivalenol(DON) in mice，and to explore the mechanism of deformities induced by DON at the molecular level. METHODS：30 pregnant mice were randomly divided into 3 experimental groups and 2 control groups，6 in each group. DON injection was performed from days 7 to 10 of pregnancy by intraperitoneal injection into pregnant mice. At GD 18，all mice were killed under isoflurane anesthesia，and vertebral bone tissue of fetuses were collected. Total RNA of vertebral bone tissues was extracted and cDNA was obtained by RT-PCR. Using whole genome microarray technology，the gene expression profiles and the pathways of the rat vertebral bone tissue were studied. Analysis of the function of differentially expressed genes was performed by using software of gene ontology. Screening signaling pathways of differentially expressed genes was done by DAVID database. The results were verified by fluorescence quantitative PCR(qPCR) technology. RESULTS：Microarray analysis showed that 282 genes，including 148 down-regulated and 134 up-regulated genes，were abnormally expressed in fetal vertebral bones after maternal DON exposure. 153 pathways were related to the differentially expressed genes. The relative quantitative results of qPCR were consistent with the results of gene chip test in Fbn1，Co19a2，Papss2， Pax1，Runx2 and Pthlh genes. CONCLUSION： There were differential gene expressions in deformed fetal skeleton between deoxynivalenol-treated rats and normal rats， involving multiple signaling pathways. The differentially expressed genes and signaling pathways may be related to the molecular mechanisms of deformities induced by DON.

OBJECTIVE: To investigate the effects of digitoxin on proliferation and cell cycle of human lung cancer cell lines NCI-H446 and A549，and to explore its possible mechanisms. METHODS：NCI-H446 and A549 cells were treated with digitoxin at different concentrations(10，20，40，80 and 160 nmol/L). The effect on proliferation of NCI-H446 and A549 cells were detected by MTT assay. Flow cytometry was used to test the cell cycle distribution of NCI-H446 and A549 cells. Protein expressions of Cyclin A1 and P21 in NCI-H446 and A549 cells were evaluated by Western blot. RESULTS：As compared with control group，cell proliferation was inhibited by digitoxin in a dose- and time-dependent manner(P＜0.05)，the IC50 value for digotoxin were 61.26 nmol/L in NCI-H446 and 110.73 nmol/L in A549 at 48 h. After 48 h treatment，the proportion of NCI-H446 and A549 cells in G0/G1 phase was decreased，while the proportion in S phase was increased. Western blot analysis showed that digitoxin could significantly up-regulate the expression of Cyclin A1 and down-regulate the expression of P21 in a dose-dependent way(P<0.05). CONCLUSION： Digitoxin could exert an the anti-proliferative effect on NCI-H446 and A549 cells and induce S phase arrest in vitro. The mechanism may be related to the expressions of proteins associated with cell cycle.

OBJECTIVE: To identify involvement of protein phosphatase 2A B56α in the regulation of cytotoxicity induced by cadmium chloride (CdCl2) and address the underlying molecular mechanism. METHODS：Stable cell lines were generated by infecting HEK cells with lentiviral shRNA targeting B56α subunit. Modified MTT was performed to detect the cytotoxicity induced by CdCl2. Immunoblotting analysis was applied to examine the expressions of B56α，p-JNK and MT in cells treated with different concentrations of CdCl2 or co-treated with SP600125. RESULTS：Immunoblotting results verified that stable cell lines HEK-SHB56α-1 and HEK-SHB56α-2 were successfully established. We found that suppression of PP2A B56α reduced the cytotoxicity induced by CdCl2. In addition，the phosphorylation status of c-Jun N-terminal kinases (JNK) and the expression level of MT were significantly increased in response to CdCl2. Suppression of B56α led to a 2.78 or 1.26-fold(P<0.05) increase of p-JNK in cells treated with CdCl2 for 12 h，and a 1.36 or 1.19-fold (P<0.05) increase of MT. Upon SP600125 treatment，the expression level of p-JNK and MT were reduced by 35%-38% (P<0.05) and 13%-35%(P<0.05)，respectively，while cytotoxicity induced by CdCl2 was enhanced(P<0.05). More-over，the expression of B56α was lowered(P<0.05)，but p-JNK and MT were increased(P<0.05) in a time-dependent manner upon CdCl2 treatment. CONCLUSION：PP2A B56α regulated MT expression via dephosphorylating JNK，and affecting the cytotoxicity induced by CdCl2. Our study demonstrated that PP2A B56α participated in regulating the targets and pathways in response to metallic stress.

OBJECTIVE: To evaluate the inhibitory effects of daidzein on angiogenesis of breast carcinoma，and to study their anti-cancer mechanisms by using the model of 7，12-dimethylbenz[α]anthraxcene (DMBA)-induced breast carcinoma in SD rats. METHODS：The DMBA -treated SD rats were randomly divided into control，10，25，50 and 75 mg/kg daidzein-gavaged groups. The growth of tumors was assessed in each group. The tumor microvessel density (MVD) was determined，and tumor proliferation index and apoptosis index were calculated. Moreover，vascular endothelial growth factor (VEGF)，basic fibroblast growth factor (bFGF) and endostatin levels were measured by ELISA. RESULTS：Compared with the control group，tumor mass was reduced more significantly in the 50 and 75 mg/kg daidzein groups (P<0.05). The apoptosis index of 75 mg/kg daidzein group was increased，and the effect of inhibiting tumor angiogenesis was significant (all P<0.05). Compared with the control group，the VEGF levels of 25，50 and 75 mg/kg daidzein groups were all decreased，in a dose-dependent manner(P<0.05)，meanwhile endostatin levels were increased in the 50 and 75 mg/kg daidzein groups，and the differences were statistically significant (all P<0.05). CONCLUSION： Higher dose of daidzein (≥50 mg/kg) could decrease MVD of mammary tumor and inhibit the growth of breast carcinoma effectively in rats.

OBJECTIVE: To study the effects of norcantharidin (NCTD) on human lung cancer cells，and investigate the mechanisms. METHODS：The growth inhibition of A549 cells treated with 0-240 μmol/L NCTD for 0-72 hours was analyzed by MTT assay. The recovery growth and proliferation of A549 cells treated with 0-120 μmol/L NCTD for 24 h was evaluated by MTT assay. The morphological changes of A549 cells treated with 40，50 and 60 μmol/L NCTD for 0-72 h were examined under inverted microscope. The apoptosis and cell cycle changes of A549 cells treated with 40-60 μmol/L NCTD were detected by flow cytometry. RESULTS：NCTD inhibited the growth of A549 cells in 30-240 μmol/L(P<0.05). Cell growth inhibition could hardly be detected 24 h after 30-240 μmol/L NCTD was removed from culture medium. The vitality of A549 cells was fully recovered in 5 d after 30-60 μmol/L NCTD was removed from culture medium. A549 cells showed G2-M phase block but no obvious apoptosis compared with the control group when treated with 40，50 and 60 μmol/L NCTD for 0-72 h. CONCLUSION： NCTD (40-60 μmol/L) inhibited human lung cancer A549 cell growth mainly by cell G2-M phase block.

OBJECTIVE: To evaluate the diagnostic value of serum autoantibodies against p53 in patients with nasopharyngeal carcinoma. METHODS：The study included 242 patients with NPC and 218 normal controls. Serum levels of autoantibodies against p53 and classical Epstein-Barr virus VCA-IgA were measured by Enzyme-linked immunosorbent assay (ELISA). RESULTS：Serum levels of autoantibodies against p53 were significantly higher in NPC than in normal controls (P<0.01). Measurement of autoantibodies against p53 and VCA-IgA demonstrated a sensitivity/specificity of 40.1% (95%CI：33.9%-46.6%)/95.0% (95%CI：90.9%-97.3%) and 47.5% (95%CI：41.1%-54.0%)/95.4% (95%CI：91.5%-97.7%)，respectively. The combination of autoantibodies against p53 and VCA-IgA yielded an enhanced sensitivity of 69.4% (95%CI：63.1%-75.0%) and a similar specificity of 90.8% (95%CI：86.0%-94.2%). Moreover，detection of autoantibodies against p53 could differentiate early stage NPC patients from normal controls. The positive rate of autoantibodies against p53 was not significantly related to age，gender，T stage，N stage or overall stage (P>0.05). CONCLUSION：Autoantibodies against p53 might be used as a potential biomarker supplementary to VCA-IgA for screening and diagnosis of NPC.

OBJECTIVE: This study aimed to evaluate the expression and prognostic significance of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) protein in colorectal cancer (CRC). METHODS：The expression of UHRF1 in 201 CRC cases and adjacent normal tissues were detected by tissue microarrays and immunohistochemistry (TMA-IHC)，then the relationship between UHRF1 protein expression and clinio-pathological parameters as well as its prognostic value were examined by statistical analysis. RESULTS：Positive expression of UHRF1 was observed in 54.2% (109/201) of CRC tissues，and the frequency in CRC was significantly higher than that in adjacent nomal tissues (P<0.01). Kaplan-Meier survival curves showed that CRC patients with negative expression of UHRF1 had shorter overall survival time (P=0.023). Cox regression analysis indicated that UHRF1 protein expression was an independent prognostic factor (P=0.008). CONCLUSION：UHRF1 might be a biomarker for prognostic evaluation of CRC patients.

OBJECTIVE: We explored the expressions of CD44v6 and Ezrin in colorectal adenoma carcinogenesis and their correlations with clinicopathological factors and prognosis. METHODS：Immunohistochemistry for CD44v6 and Ezrin were performed in normal mucosa(20 cases)，low grade adenoma tissues(40 cases) ，high grades adenoma tissues(20 cases) and colorectal cancer(80 cases). The correlations between expressions of CD44v6 and Ezrin with survival time and clinicopathological factors were evaluated. RESULTS：Expressions of CD44v6 and Ezrin in high grades colorectal adenomas and adenocarcinoma groups were higher than normal mucosa and low grade colorectal adenoma groups(P<0.01). Expressions were higher in lymph node metastasis group，serosal infiltration group，stages III-IV group than no lymph node metastasis group，no serosal infiltration and stages I-II group. CD44v6 and Ezrin were significantly correlated with survival time(Log-rank=18.587，P<0.05；Log-rank=7.804， P<0.05). Cox regression analysis showed that CD44v6 was an independent risk factor for prognosis (HR：7.582，95%CI：1.605-35.815，P=0.011). CONCLUSION：The expressions of CD44v6 and Ezrin were correlated with process of colorectal adenoma-carcinoma transition，invasion and metastasis. It would be valuable to combine CD44v6 and Ezrin to predict malignant transformation of colorectal adenoma and prognosis.

OBJECTIVE: Establish the flow cytometric 96-well microplate-based in vitro micronucleus assay in CHO-K1 cells，and explore the possibility of this method for early genetic toxicity screening during drug discovery. MEHTODS：The test included treatment with and without metabolic activation. For the treatment with metabolic activation，CHO-K1 cells were treated with three different concentrations of cyclophosphamide in the S9 mix medium for 4 h，then incubated with S9-free fresh medium for 20 h. For the treatment without metabolic activation，cells were incubated with three different concentrations of mitomycin C continuously for 24 h. In all cases，after a total of 24 h since initiation of the treatment，cells were processed for microscopic scoring or flow cytometric MN analysis. A flow cytometric method for scoring MN used EMA and SYTOX Green to label the cells in 96-well microplate，and then compared with cytokinesis-block micronucleus assay in cell culture disks based on microscopy. RESULTS：Mitomycin C and cyclophosphamide at different concerntrations caused statistically significant and dose-dependent increasess in micronucleus assay . Non-parametric Spearman's coefficients (rs) is 1.000. CONCLUSION：Similar to literature published， mitomycin C and cyclophosphamide induced positive results in flow cytometric based in vitro micronucleus assay. So the method of flow cytometric 96-well microplate-based in vitro micronucleus assay in CHO-K1 cells was established. The concordance between microscopic scoring and flow cytometric was good，therefore this method is promising for screening and evaluating genetic toxicity of chemicals.

OBJECTIVE: To explore the mechanisms of human sperm apoptosis by hepatitis B virus S protein. METHODS：After the co-incubation of sperms with 0，25，50 and 100 μg/mL HBS for 3 h，the externalization of membrane phosphatidylserine (PS)；Caspase-3，-8，-9 activation and DNA fragmentation of human sperm were assessed. RESULTS：After incubation，the average rates of Annexin V–positive/propidium iodide (PI)-negative sperm，Caspase-3，-8，-9 positive sperm and TUNEL-positive sperm were significantly increased in the test groups as compared to those in the control groups (P<0.05 or P<0.01)，and in a dose-dependent manner. CONCLUSION：HBs exposure could lead to PS externalization，activation of caspases，and DNA fragmentation，resulting in increased apoptosis of sperms and causing sperm dysfunction.

OBJECTIVE: To explore the expression of hypoxia inducible factor-1α (HIF-1α) in epithelial ovarian cancer and its relationship with clinical pathology，in order to understand the effect of HIF-1α in malignant biological behavior of ovarian epithelial carcinoma clear. METHODS：Original foreign literatures on the correlation between HIF-1α and epithelial ovarian cancer were selected from Cochrane Library，EMbase database，Pubmed，and Chinese original literatures were from CNKI，CBM. All analyses were performed by software STATA 11.0. Relationships between HIF-1α expression and age，pathological type，histological grade，lymph node metastasis，clinical stage were analyzed using pooled odds ratio (OR) with 95% confidence interval (CI). RESULTS：A total of 6 papers including 257 cases of epithelial ovarian cancer were analyzed. Compared with that in normal tissue，total positive rate of HIF-1αin epithelial ovarian cancer was 72.4%，which was increased significantly (OR=0.036，95%CI：0.010-0.135，P=0.000)； HIF-1α was significantly correlated with lymph node metastasis and clinical stage(OR=0.080，95%CI：0.029-0.220，P=0.000； OR=0.258， 95%CI：0.136-0.490， P=0.000). Patients with positive expression of HIF-1α were prone to lymph node metastasis and advanced clinical stage. HIF-1α had no significant relation with age，pathological type and pathological grading (P>0.05). CONCLUSION：The expression of HIF-1α protein increased the risk of epithelial ovarian cancer. HIF-1α could be used as indicator of lymph node metastasis and clinical stage.

OBJECTIVE: To study the toxicological effects of phosgene on pulmonary mitochondria. METHODS：30 SD rats were randomly divided into two groups，with 15 in each group. The rats in control group were exposed to air. The rats in phosgene group were exposed to 10 g/m3 phosgene for 5 min. Two hours after exposure，pulmonary tissues were collected，HE staining and transmission electron microscope were used to identify histological changes. Activities of mitochondrial complexes were determined. RESULTS：Phosgene induced a significant increase of pulmonary wet/dry ratio. After phosgene poisoning，there were profuse neutrophil infiltration and pulmonary alveoli epithelial cell damage. Mitochondria were notably swollen，and even appeared contracted and dissolved. The number of mitochondria was significantly decreased. Mitochondrial ridge was broken and absent. The activities of mitochondrial complex were significantly reduced. CONCLUSION：Mitochondria were main targets of phosgene poisoning，and the results provided basis for the development of new drugs in the treatment of phosgene poisoning.

OBJECTIVE: To investigate the effects of different doses of iron supplementation on lymphocyte DNA damage in rats. METHODS：Forty male Wistar rats were randomly divided into control group，iron deficiency group，10 times-iron as control group and 20 times-iron as control group， containing Fe2+ 0.9，0.3，9 and 18 mg，respectively. All rats were treated with intraperitoneal injection of 0.72 mL iron dextran every other day and the entire study lasted for 6 weeks. The rats in the four groups were provided with deionized water and food without iron，both freely available. After 6 weeks，the level of serum iron was determined by spectrophotomety and the peripheral blood lymphocyte DNA damage was assessed using comet assay. RESULTS：The level of serum iron in the iron deficiency group (53.54 μmol/L) was significantly lower than that of control group (77.62 μmol/L)(P<0.01). In addition，the levels of serum iron in 10 times-iron (104.77 μmol/L) and 20 times-iron groups (205.30 μmol/L) were significantly higher than that of control group(P<0.01). DNA damage assessment indicated that there was no difference between iron deficiency group (25.30 AU) and control group (21.13 AU)(P>0.05). However the DNA damage of 10 times-iron group (82.80 AU) and 20 times-iron group (169.50 AU) were significantly higher than control group (P<0.01) ，being 3.9 times and 8.0 times，respectively，as control group. The results of combined DNA damage induced by 10 μmol/L H2O2 showed that there was no difference between iron deficiency(260.40 AU) group and control group (259.00 AU) (P>0.05). The combined DNA damage of 10 times-iron group (293.80 AU) and 20 times-iron group (308.88 AU) were higher significantly than control group(P<0.01)，which were 1.1 times and 1.2 times，respectively，as control group. CONCLUSION：10 times and 20 times iron supplements could improve the iron nutritional status in body or increase the level of iron load. Iron deficiency could not increase the level of usual DNA damage or combined DNA damage，but iron overload could increase the level of lymphocyte DNA damage and combined DNA damage.

OBJECTIVE: To research the effects of trichloroethylene on the RNA expressions of SET-associated proteins including eEF1A1，eEF1A2 and DDB1 in L-02 cells. METHODS：L-02 cells，being in the logarithmic growth phase，were treated with different concentrations (1.0，2.0，4.0，8.0 mmol/L) of trichloroethylene for 24 h， and DMSO were used as control. The mRNA levels of eEF1A1， eEF1A2 and DDB1 were analyzed with qPCR. RESULTS：The results showed that mRNA levels of DDB1 and eEF1A2 were significantly up-regulated under low concentration of trichloroethylene (1.0 mmol/L) and were significantly down-regulated with increasing concentrations of trichloroethylene. The mRNA levels of eEF1A1 were significantly up-regulated with increasing of trichloroethylene concentration. CONCLUSION：Exposure of trichloroethylene could alter mRNA expression of SET-associated proteins in L-02 cells.