BACKGROUND AND AIM: To detect and evaluate the antimutagenicity of ginsenoside Re by TK gene and HGPRT gene mutation tests. MATERIALS AND METHODS: The TK6 human lymphoid cells were exposed to ginsenoside Re at concentrations 12.5, 25, 50,100 μg/ml simultaneously with 5 μg/ml of methyl methanesulfonate (MMS) for 4 h. Determination of mutant frequency of TK locus and HGPRT locus were performed by using the microwell method. RESULTS: The mutant frequencies of TK locus and HGPRT locus after treatment with ginsenoside Re were significantly suppressed compared with the MMS control, in a dose-dependent manner. CONCLUSION: Ginsenoside Re had obvious inhibitory effect on TK gene and HGPRT gene mutation induced by MMS. TK6 could be used for both TK and HGPRT gene mutation tests, and TK gene mutation test was more sensitive than that of HGPRT in detection of ginsenoside Re antimutagenicity.

BACKGROUND AND AIM: To study the differentiation of mouse liver epithelial progenitor cells(LEPCs) to cholangiocytes induced by hairy and enhancer of split 1(Hes1)gene in vitro. MATERIALS AND METHODS: The Hes1 gene was amplified by PCR from mouse tail genomic DNA and subcloned into pEGFP-C1 and pcDNA3.1 vector．LEPCs were transfected and performed in vitro. Cholangiocyte markers, CK19, GGT, HNF6 and HNF1β; hepatocyte markers, GS and BGP; hepatic oval cell marker, Thy-1 were assayed with RT-PCR and Real-time PCR. The fluorescence of the LEPCs tagged with EGFP was observed in vitro. RESULTS: Cholangiocyte markers, CK19, GGT, HNF6 and HNF1β, were up-regulated. Hepatocyte markers GS and BGP, and hepatic oval cell marker Thy-1, were down- regulated. CONCLUSION: It indicated that LEPCs differentiated to cholangiocytes initially in vitro. and Hes1 might play important roles in the differentiation of LEPCs and influence cholangiocyte versus hepatocyte fate determination.

BACKGROUND AND AIM: To investigate the effects of lipid-lowering drugs such as peroxisome proliferators-activated receptor α (PPARα) ligands on human skeletal muscle cells. MATERIALS AND METHODS: WST-1 assay was applied to determine the cytotoxicity of benzafibrate and atorvastatin to human embryo rhabdomyosarcoma (RD) cells. Enzymatic activities of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured by automatic biochemistry analyzer and Hoechest 33342 staining was used to assess apoptosis. RESULTS: Bezafibrate markedly inhibited the proliferation of RD cells, reduced CK activity and caused apoptosis in a dose- and time-dependent manner. Toxicity was enhanced in the co-administration of benzafibrate with atorvastatin. CONCLUSION: Benzafibrate as a PPARα ligand induced obvious cytotoxicity on RD cells and demonstrated synergistic interaction with atorvastain.

BACKGROUND AND AIM: To express the human fascin protein, an important tumor molecular marker, in Pichia pastoris. MATERIALS AND METHODS: The recombinant plasmid pPIC9-fascin was constructed by inserting the full coding DNA sequence of fascin into the secreted expression yeast vector pPIC9. Then pPIC9-fascin was linearized by Mss I and transformed into Pichia pastoris GS115 strain. After identification of transformants by PCR, the positive transformant was cultured and induced by 0.5％ methanol. Silver staining and Western blotting were used to analyz the expression level of fascin in the supernatant. RESULTS: Human fascin protein could be expressed in yeast. Silver staining and western blotting analysis from the supernatant showed the molecular weight of fascin protein expressed by the recombinant GS115 strain was about 69 kD, however, the concentration was less than 40 μg/L . CONCLUSION: Although human fascin could be expressed by Pichia pastoris expression system, the low level was far off from that required for bioengineering.

ACKGROUND AND AIM: To study the expression of the key genes in UPR,which are Atf6, Ire1, Perk and Grp78, in normal forelimb and short forelimb formations during mouse embryogenesis. MATERIALS AND METHODS: On gestational day 10 (GD10), the pregnant mice of the treatment group were fed with 80 mg/kg all-trans retinoic acid，and those of the control group received same volume of soybean oil. The forelimbs of all embryos were harvested during GD11-GD18, and the expression levels of Atf6, Ire1, Perk and Grp78 in all samples were measured by real-time quantity reverse transcript polymerase chain reaction (QRT-PCR). RESULTS: Except Ire1 that was not expressed on GD18, the key genes of UPR were all expressed in the normal limbs during GD11- GD18, and there were two expression peaks around GD13 and GD17. In the normal limbs, the expression of Grp78 was highest and that of Ire1 was lowest, and the difference was so obviously. Before GD15, the expressions of Atf6, Ire1, Perk and Grp78 in the abnormal forelimbs were higher than those in the normal limbs, and after GD15, the expression of these four genes in the abnormal forelimbs were lower than those in the normal limbs, and was relatively stable at a low level. In the abnormal limbs, there was a very obvious expression peak of all genes between GD12-GD14; the time course was longer and the expression of this peak was higher than that of the normal limbs. CONCLUSION: During mouse embryogenesis, the expression abundance of key genes of UPR in all-trans retinoic acid-induced short limb malformations was obviously increased during GD11-GD14, and was suppressed during GD16-GD18, so we think UPR may be teratogenic.

BACKGROUND AND AIM: To evaluate the genotoxic and cytotoxic effects of rare earth element praseodymium on the Vicia faba L. root-tip cells. MATERIALS AND METHODS: The primary roots of Vicia faba L. were cultured for 6 hours in various concentrations of PrCl3(64，32，16，8，4，2，1 μg/ml) at 25 ℃, then the roots were restored in double distilled water for 24 h.The root tips of Vicia faba L. were dissected and micronucleus test was performed, mitotic index and frequency of chromosomal aberrations were counted. RESULTS: PrCl3 could cause damage to the root-tip cells of Vicia faba L. attenuated the growth of root-tip with concentration ≥8 μg/ml. Frequency of micronucleus increased in a dose-dependent manner with concentrations of PrCl3 from 1 to 32 μg/ml (r=0.948, P<0.05).Mitosis index rose in a dose-dependent manner when the concentration of PrCl3 increased from 2 to 64 μg/ml (r=-0.789). Frequency of chromosomal aberrations also escalated in a dose-dependent manner when the concentrations of PrCl3 were between 1-8 μg/ml (r=0.992,P<0.05). CONCLUSION: The rare earth element praseodymium has certain genotoxic and cytotoxic effects on the Vicia faba L. root-tip cells.

BACKGROUND AND AIM: Effect of baohuoside-Ⅰ from Cortex Periplocae on apoptosis of human esophageal carcinoma cell Eca-109 and its mechanism were studied. MATERIALS AND METHODS: After treatment with baohuoside-Ⅰ at different concentrations (12.5, 25, 50 μg/ml)for 24 h, 48 h, 72 h, the inhibitory effect on proliferation of Eca-109 cells was analyzed by MTT method.After treatment with baohuoside-Ⅰ under different concentrations(12.5、25、50 μg/ml) for 48 h, cell apoptotic ratio and expression of Eca-109 cells Survivin protein of were measured with flow cytometry (FCM); the ultrastructure was examined by transmission electron microscope; the expression of Survivin mRNA was detected by RT-PCR. RESULTS: Baohuoside-Ⅰ significantly inhibited proliferation of Eca-109 cells, and in concentration- dependent manner(P all<0.05), the IC50 was 24.8 μg/ml. After treatment with different concentrations of Baohuoside-Ⅰfor 48 h, cell apoptosis of Eca-109 cells was induced significantly (the apoptotic ratio is 55.26％ treated with 50 μg/ml of baohuoside-Ⅰ) and showing characteristic ultrastructural changes of apoptosis. Expression levels of Survivin mRNA and protein were decreased significantly(P<0.01). CONCLUSION: Baohuoside-Ⅰ of Cortex Periplocae could inhibit the proliferation and induce apoptosis of Eca-109 cell. This effect was associated with down-regulation of Survivin mRNA expression.

BACKGROUND AND AIM: To investigate the effect of cortex periplocae extract (CPE) on the differentiation of tumor cells K562 in vitro. MATERIALS AND METHODS: We examined the morphological changes of K562 cells after cultured with CPE; and investigated the induction effect of CPE on the production of Hb from K562 cells by using colorimetry. RESULTS: K562 cells morphology were destroyed by CPE. CPE also markedly reduced the production of Hb from K562 cells. CONCLUSION: CPE could kill K562 cells to some extent, but could not induce the differentiation of K562 cells.

BACKGROUND AND AIM: Associations between genetic polymorphisms of CYP1A1 MspⅠor GSTT1 among residents from an electronic waste (e-waste) recycling site in China,and the frequencies of micronucleated binucleated cells (MNed BNC) in peripheral blood lymphocytes were investigated. MATERIALS AND METHODS: We recruited 54 residents from a decade-long e-waste recycling site in southeast China (as exposed group), and another 73 farmers from one village located 50 km away from the e-waste site without other pollutions(as control group). The frequencies of MNed BNC were analyzed by cytokinesis-blocked micronucleus assay. CYP1A1 Msp I genotypes and GSTT1 genotypes were analyzed using polymerase chain reaction -based restriction fragment length polymorphism method and multiple polymerase chain reaction method, respectively. RESULTS: The mean MNed BNC frequency in the exposed group was 4.81‰± 4.14‰, which was approximately four times the mean MNed BNC frequency of the control(1.15‰±1.42‰,P<0.01). After adjusted by age, gender, cigarettes per day and alcohol drinking, we found that the mean frequency of MNed BNC among subjects with homozygous rara allele (aa) genotype at CYP1A1 MspⅠsite was 1.69‰±1.28‰, which was significantly higher than those of subjects with either homozygous wild type or heterozygous genotypes(AA/Aa) (1.06‰±1.44‰, P<0.01)in the control. However, there was no association between the frequency of MNed BNC and GSTT1 genotypes alone or both GSTT1 genotypes and CYP1A1 genotypes. CONCLUSION: The findings suggest that polymorphism of CYP1A1 MspⅠmay be associated with the frequency of MNed BNC among the control subjects. Certain pollutants from the e-waste dismantling site surroundings may affect the health of the local residents.

BACKGROUND AND AIM: To investigate the correlation between red blood C3b receptor (RBC-C3bR) and lymphocytic cell (TC) immune function in tumor patients. MATERIALS AND METHODS: RBC-C3bR, natural killer (NK) cell activity and T lymphocyte subsets of 53 patients with gastric cardia carcinoma, 51 gastric cancer and 30 healthy controls were measured by microzyme garland test , MTT-assay and monoclonal anti-human T lymphocyte antibody. RESULTS: Compared with the control group, RBC-C3bR , NK cell activity, CD3＋, CD4＋,CD4＋/CD8＋ ratio were significantly decreased in tumor patients (F=28.68 ，P<0.01). Positive correlations were observed between RBC-C3bR with both NK cell activity and CD4＋/CD8＋ ratio (gastric cardia carcinoma RBC-C3bR and CD4＋/CD8＋ ratio: r=0.431, P<0.01; RBC-C3bR and NK cell activity：r=0.347,P<0.05; gastric cancer RBC-C3bR and CD4＋/CD8＋ ratio: r=0.429, P<0.01; RBC-C3bR and NK cell activity,r=0.528, P<0.01). CONCLUSION: The immune funtion of erythrocytes was inhibited same as lymphocytes and there was close relationship between them in gastric cardia carcinoma, gastric cancer patients.

BACKGROUND AND AIM: To investigate the relationship between expression of CDC6 and TFF2 and HPV16/18 infection in uterine cervical cancer. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded samples including 50 cases of squamous cell carcinomas, 20 cervical intraepithelial neoplasia (CIN)Ⅱ-Ⅲ and 20 CINⅠ and 20 samples of normal cervical tissues were studied. Using Polymer DetectionSystem For Immuno-Histological Staining(PV-9000) immunohistochemical technique, we examined the expression of CDC6 and TFF2 in these samples. The infections of HPV type 16, 18 DNA were determined by PCR. RESULTS: There were significant differences for CDC6 among carcinomas, CIN and normal cervices(P<0.05). Expression of CDC6 was correlated with tumor grades and lymph node metastasis (all P<0.05). The positive expression of TFF2 in carcinomas was significantly lower than in normal cervical tissue and CIN (P<0.05). The positive rate of TFF2 was associated with age group (P<0.05)but not with tumor grades, clinical stages and lymph node metastasis(all P>0.05).The positive rate of HPV16/18 was significantly different between cervical carcinomas, CIN and normal tissues (P<0.05) but not between tumor grades, clinical stages, age group and lymph node metastasis (all P>0.05). The expression of CDC6 showed positive correlation with that of HPV16/18 (rs=0.386,P<0.05). HPV16/18 and CDC6 both showed negative correlations with TFF2 (rs=-0.500, P< 0.05;rs=-0.356,P<0.05,respectively). CONCLUSION: In tissues of CIN and cervical carcinomas, changes in CDC6 and TFF2 expression may be associated with HPV16/18 infection. Expressions of CDC6 was associated with the malignant level of uterine cervical cancer.

BACKGROUND AND AIM: To study the effects of acephate on oxidative damage and ovarian function in female rats. MATERIALS AND METHODS: Female Sprague-Dawley rats veceived oral acephate at 0, 11.81, 23.63, 47.25 mg/kg once daily for 30 days. The positive control group was treated with estradiol (0.1mg/kg) by peritoneal injection. The estrous cycle, SOD, MDA, GSH, GST and the histomorphology changes of ovaries were evaluated. RESULTS: In high dosage group the estrous cycle was significantly prolonged compared to negative control group (P<0.05). In the serum, SOD activities of acephate-treated rats were significantly increased compared with that of negative group, but the contents of GSH and GST activities were reduced(P<0.05). The levels of MDA in serum were significantly increased in middle and high dosage groups compared to negative group(P<0.05). In the ovaries, SOD activities of acephate-treated rats were significantly reduced, but GST activities significantly increased compared with those of negative group(P<0.05). In addition, the content of MDA in high dosage group was significantly higher than that of negative group, and the level of GSH in low dosage group was lower than that of negative group(P<0.05). Pathology slices showed increased counts of primordial follicles and primary follicles, while the counts of secondary follicle and mature follicle was decreased in high dosage group. In addition, there were more atretic follicles. CONCLUSION: Acephate had obvious reproductive toxicity on female rats. At the dosage of 47.25mg/kg, it could induce estrous cycle disorders, some pathologic changes in the ovaries, and inhibit the activities of antioxidase and resulting in lipid peroxidation.

BACKGROUND AND AIM: To determine whether the mRNA expressions of hMLH1 and hMSH2 genes involved in mismatch repair are associated with the risk of ESCC. MATERIALS AND METHODS: 112 patients with newly diagnosed, untreated ESCC were recruited in Huai'an county. Esophageal cancer tissues and tissues adjacent to the tumors were collected to determine the mRNA levels of hMLH1 and hMSH2 genes by quantitative real-time reverse transcription-PCR using SYBR Green I with β-actin as an internal control. t-test and logistical regression analysis were used to test the differences of gene expression between cancer tissues and tissues adjacent to the tumors. RESULTS: Compared with tissues adjacent to the tumors, the relative expression level of hMSH2 was significantly lower in esophageal cancer tissues (P=0.010, t-test). The conditional logistic regression analysis showed that the increased risk for esophageal cancer was significantly associated with decreased expression of hMSH2 (OR=1.311, 95％ CI: 1.075~1.599). CONCLUSION: Reduced expression of hMSH2 was significantly associated with risk for ESCC, suggesting this might be important for the early progression of ESCC in Huaian of China.

BACKGROUND AND AIM: To study the combined toxic effects of phoxim (Pho) and methomyl (Met) on female reproductive toxicity. MATERIALS AND METHODS: Twenty-four adult female rats were randomly divided into 4 equal groups (including a control and three treated groups). Daily doses of Pho(29.4 mg/kg), Met(0.34 mg/kg) and Pho＋Met(29.4 mg/kg＋0.34 mg/kg) were given to rats by gastric gavage for 30 successive days. 2×2 factorial analysis was used in the experiment. Vaginal smears of rats were taken to determine estrous cycle. Serum 17β-estradiol(E2) and progesterone(P4) concentrations were measured by radioimmunoassay. The activities of superoxidedismutase (SOD) and glutathione- s-transferase(GST), and the levels of malondialdehyde(MDA) ,glutathione(GSH) and BchE in blood and ovary were measured by spectrophotometry. RESULTS: Significant differences were found in estrous cycle changes, and the concentrations of estradiol (E2) and progesterone (P4) in blood of the Met group. There were significant differences in the levels of SOD,GST ,GSH and the content of MDA in blood and ovary of Pho and Met groups. There were interactions between Pho and Met on the levels of SOD ,GST and the content of GSH in blood and ovary and also the content of MDA and P4 in blood. The joint action was characterized as an antagonistic effect. No interaction was noted between Pho and Met on weight gain in rats, estrous cycle changes,ovary and uterus internal organs coefficient, estradiol(E2) and the levels of BchE in blood ;the joint action was characterized as an additive effect. Ovarian histomorphology showed no obvious changes. Uterine pathology revealed that glands were increased and some were distended. CONCLUSION: Pho and Met could disrupt the reproductive functions of female rats. Markedly increased toxic effect of the pesticide mixtures was found in the antioxidant system. Combined exposure of Pho and Met might produce some additive reproductive toxicities in female rats.

BACKGROUND AND AIM: To investigate the anti-mutation property of the Periplaneta Americana and Aspongopus Chinensis Dallas mixture. MATERIALS AND METHODS: Broad beans from Qiliqiao village and Xiaoguanyi village were divided into 4 groups, cyclophosphamide group, negative control group, Periplaneta Americana and Aspongopus Chinensis Dallas mixture group, Periplaneta Americana and Aspongopus Chinensis Dallas mixture＋cyclophosphamide group.The purpose was to assess the anti-mutation property of Periplaneta Americana and Aspongopus Chinensis Dallas mixture by means of micronucleus test of Vicia Faba L.root tip cells. RESULTS: Compared with the cyclophosphamide group, the rates of micronucleus in the Periplaneta Americana and Aspongopus Chinensis Dallas mixture＋cyclophosphamide group were lower. The inhibitory rates of Periplaneta Americana and Aspongopus Chinensis Dallas mixture＋cyclophosphamide group were 60.5％(Qiliqiao) and 52.5％(Xiaoguanyi). CONCLUSION: Periplaneta Americana and Aspongopus Chinensis Dallas mixture could effectively inhibit the mutation induced by cyclophosphamide.

BACKGROUND AND AIM: To study the teratogenic effect of fat emulsion of seal oil in rats. MATERIALS AND METHODS: The Wistar rats were divided into three groups each trated with different dosages of test drug(800, 400 and 200 mg/kg), one positive group (cyclophosphamide) and one negative control group (normal saline). On days 15-20 of pregnancy, the drug was injected via tail vein for ten days consecutively. RESULTS: The body weight gains of pregnant rats were significantly lower in the 200 mg/kg group than those of the control group (P<0.05). The embryonic body weights and body lengths of the 400 mg/kg group were significantly less than those of the control group (P<0.05). Compared with negative control group, the number of absorbed embryos was increased significantly in the 800 mg/kg group (P<0.05). The numbers of living embryos, absorbed embryos, dead embryos, the weight, body length and tail length showed no significant differences between the treated groups and the negative control group (P>0.05). No abnormaility was observed in the growth of the skeletal and internal organs in rat fetuses in any groups. CONCLUSION: Seal oil emulsion at 200 mg/kg caused maternal toxicity for pregnant rats, characterized by decreased body weight. Seal oil emulsion at 400 mg/kg led to embryotoxicity. Seal oil emulsion at 800 mg/kg had no teratogenic effects on skeletal and internal organs.

BACKGROUND AND AIM:To study the antimutagenesis and antioxidation properties of aqueous extract of Pulsatilla Chinensis Regel. MATERIALS AND METHODS: Tested the micronucleus rate of bone marrow PCE cell in mice, serum superoxide dismutase(SOD)activity and total anti-oxidative abilities(T-AOC)in mice serum by CP as mutagen and Vit C as control of antioxidant. RESULTS: The aqueous extract of Pulsatilla Chinensis Regel reduced the micronucleus rate of bone marrow PCE cell in mice，and increased SOD activity and T-AOC in mice serum. CONCLUSION: The aqueous extract of Pulsatilla Chinensis Regel showed antimutagenesis and antioxidation activities.The mechanism may lie in eliminating free radicals，interrupting and terminating oxidizing reaction of free radicals,and enhancing the overall anti-oxidative abilitiy.

BACKGROUND AND AIM: To test the safety of Panax japonicus C.A.Mey complex formulation. MATERIALS AND METHODS: Acute toxicity test，bone marrow cell micronucleus test，mice sperm abnormality test，Ames test and 30 d feeding test were used to study the toxicity. Negative control and positive control groups were set up with administration of water and cyclophosphamide in bone marrow cell micronucleus test and mice sperm abnormality test,respectively. Negative control group animals received water in 30 d feeding test. RESULTS: In acute toxicity test, male and female KM mice were fed with 20 g/kg dose of Panax japonicus C.A.Mey complex, no toxic symptoms were observed. The results of bone marrow cell micronucleus test，mice sperm abnormality test and Ames test were negative. In the 30 d feeding test, no obvious toxic effects in clinical, blood examination and biochemical index examinations, organism weight and pathological studies were detected in rats with the dose of 6 g/kg，which is 200 times the clinical prescribed dose. CONCLUSION: Panax japonicus C.A.Mey complex formulation could pass the toxicological assessment using the tests mentioned above.

BACKGROUND AND AIM: To study the acute toxicity and genotoxicity of pearl anti-spot cosmetic slice. MATERIALS AND METHODS: Acute toxicity test, Ames test, micronucleus test of bone marrow PCE cell in mice, sperm abnormality test of mice were used． RESULTS: The oral acute toxicity study revealed that the maximal tolerance dose (MTD) of pearl anti-spot cosmetic slice was greater than 20.0 g/kg in mice. The results of genotoxicity tests were all negative, including Ames test, micronucleus test and sperm shape abnormality test. CONCLUSION: Pearl anti-spot cosmetic slice showed no acute toxicity and genotoxicity in this study. Thus, pearl anti-spot cosmetic slice is safe at the daily recommended dose(2.0 g／person／day)．