OBJECTIVE: To investigate the role of mitochondrial reactive oxygen species (mtROS) on the apoptosis of a mouse liver cell line-AML12 cells after their exposure to paraquat. METHODS: AML12 cells were treated with paraquat at concentrations of 0,25,50,100,200 and 300 μmol/L for 24 h,and cell viability was measured by MTT assay. Apoptosis was assessed by flow cytometry using Annexin V-FITC apoptosis detection kit. DCFH-DA fluorescent probe and MitoSOX were used to determine the level of ROS in whole cell and in mitochondria by flow cytometry,respectively. Mitochondrial membrane potential was evaluated by confocal microscope using JC-1 probe. RESULTS: Paraquat from 25 to 300 μmol/L induced dose-dependent decrease of AML12 cells viability. Apoptosis was significantly induced by paraquat at 300 μmol/L for 24 h. Treatment with 25-100 μmol/L paraquat raised the level of mitochondrial reactive oxygen species. However,200 and 300 μmol/L paraquat decreased the level of mtROS. The increase of ROS level was closely related to the decrease of AML12 cells viability (r=-0.90,P<0.05),and the ROS level was positively correlated with the proportion of the late apoptotic cells (r=0.96,P<0.01). Additionally,300 μmol/L paraquat markedly reduced mitochondrial membrane potential. CONCLUSION: Paraquat could induce apoptosis of AML12 cells via the induction of whole cell ROS which suggest that mtROS may not be directly involved in paraquat-induced liver injury.

OBJECTIVE: To study the effects of glutathione peroxidase 1(GPX1) overexpression on DNA oxidative damage level and phenotype of malignant BERP35T1 cells exposed upon α-particles. METHODS: The eukaryotic expression vector pEGFP-GPX1 was constructed. After PCR,enzyme digestion analysis and sequencing pEGFP-GPX1 and control vector were transfected into BERP35T1 cells with lipofectamine 2000 and G418 screening was used to obtain resistant cell lines. The expression of GPX1 was measured by Western blot. MTT,scratching healing test and anchorage independence growth test were used to analyze the effects of GPX1 overexpression on growth rate,migration and colony forming efficiency of BERP35T1 cells. RESULTS: pEGFP-GPX1 was confirmed by PCR,enzyme digestion analysis and sequencing. The sequence of the target gene GPX1 was entirely correct. The protein expression level of GPX1 in pEGFP-GPX1 transfected group BERP35T1-GPX1-6 was 4.01 times higher than in BERP35T1-pEGFP cells(P<0.05). Compared with BERP35T1 and BERP35T1-pEGFP,the level of growth rate,migration and colony forming efficiency in BERP35T1-GPX1-6 decreased significantly(P<0.05). CONCLUSION: GPX1 overexpression may inhibit the proliferation and metastasis of malignant BERP35T1 cells exposed to α-particles through reducing the level of DNA oxidative damage.

OBJECTIVE: To investigate the effects of epigallocatechin gallate (EGCG) on cisplatin (DDP)-induced cytotoxicity in human embryo kidney (HEK) 293 cells and A549 cells. METHODS: HEK293 cells and A549 cells were cultured in vitro,and effects of DDP and EGCG on HEK293 cells and A549 cells were observed by MTT. The cells were divided into control group,DDP group and DDP+EGCG group. Then,the rates of HEK293 and A549 cell death were investigated by MTT. RESULTS: The IC₅₀ of EGCG on HEK293 cells was 61.6 mg/L. At lower than 40 mg/L,EGCG had no significantly effects on DDP-induced HEK239 cell death. At higher than 40 mg/L,EGCG significantly enhanced DDP-induced HEK239 cell death. The IC₅₀ of EGCG on A549 cells was 33.6 mg/L. At higher than 40 mg/L,EGCG significantly enhanced DDP-induced A549 cell death. CONCLUSION: EGCG had no significant protective effects on DDP-induced HEK239 cell death,but EGCG induced more cytotoxicity on cancer cells than on normal cells. When co-treated with DDP,A549 cell sustained more damage than HEK239 cells.

OBJECTIVE: To investigate the effects of InP/ZnS quantum dots (InP/ZnS QDs) on function of macrophages using in vitro cell model. METHODS: The mouse macrophage RAW264.7 cells were exposed to different concentrations of InP/ZnS QDs. The uptake of InP/ZnS QDs by macrophages was observed under a fluorescence microscope 4 hours later. Effect of InP/ZnS QDs on cell proliferation was assessed by CCK-8 assay,and the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) released by macrophages were determined by ELISA assay. RESULTS: InP/ZnS QDs entered the cells and remained mostly in the cytoplasm in 4 hours. Cells exposed to InP/ZnS QDs showed significantly decreased cell reproduction 24 hours and 48 hours later(P<0.05,P<0.01),and the levels of TNF-α released with or without CpG-ODN stimulationby macrophages significantly increased 24 hours later(P<0.01),but the levels of IL-6 remained unchanged. In addition,InP/ZnS QDs exposure led to increased release of TNF-α by macrophages following re-stimulation with CpG-ODN. CONCLUSION: The results suggest that InP/ZnS QDs could be taken up by macrophages,and could inhibit the cell viability and increase release of TNF-α.

OBJECTIVE: We evaluated the effects of atrazine(ATR) exposure in Sprague Dawley rat on the dopaminergic system. METHODS： Healthy adult male SD rats were randomly divided into four groups,control and ATR-dose (50,100,200 mg/kg) groups. Rats were treated orally with ATR for 28 d. The mRNA and protein expression of tyrosine hydroxylase (TH) and nuclear receptor-related factor 1 (Nurr1) were examined in samples of the substantia nigra by fluorescence PCR and immunohistochemistry. We use SPSS 17.0 to analyse the experimental results. RESULTS： qPCR results showed that with 50,100 and 200 mg/kg concentration of ATR exposure,rat substantia nigra TH and Nurr1 mRNA expression was significantly reduced compared with control group (P<0.05),and there was a dose-response relationship. Immunohistochemistry showed that substantia nigra Nurr1 and TH proteins in treated-rats were significantly lower than those among the control group (P<0.05). CONCLUSION： The reduction of Nurr1 and TH suggests that ATR may influence the metabolism of dopamine neurons in dopaminergic neuron injury.

OBJECTIVE: To investigate the clinical value of cytological specimens in the diagnosis and the individualized treatment of non-small cell lung cancer(NSCLC). METHODS: Cytological specimens were stained with HE and with immunocytochemical method to detect expression of an appropriate set of antibodies (TTF-1 NapsinA,CK7,CEA,CD56,Syn,P63,CK5/6,WT-1,E-cadherin),in tumor cells of unknown origin. The epidermal growth factor receptor(EGFR) gene was detected by the amplification refractory mutation system(ARMS) in NSCLC. RESULTS: In 352 patients,345 cases were found cancer cells on the cytological specimens.According to the clinical history and immunocytochemical staining of the 345 malignant cases,335 were NSCLC cytological samples,and the DNA of 302 NSCLC samples were extracted successfully (satisfaction rate at 90.15%). EGFR mutations were detected in 123 of the 302 specimens (40.73%,123/302) and the frequencies of EGFR mutations in exon18,19,20(T790M),21 were 0.99% (3/302) 19.21%(58/302),0.66%(2/302) and 19.87% (60/302),respectively. Higher frequencies of EGFR mutations were detected in exons 18,19 and 21(98.37%,121/123) than in exon 20(P<0.05). EGFR mutations were more frequently detected in women(54.35%,75/138) than in men (29.27%,48/164)(P<0.05),and in non-smokers (51.49%,104/202) than in smokers 19%(19/100)(P<0.05). Mutations were identified in 44.20%(122/276) cases of adenocarcinoma and 4.34%(1/23) in nonadenocarcinoma. Mutation of EGFR gene in adenocarcinoma was higher than that in non-adenocarcinoma(P<0.05). CONCLUSION: The use of cytological specimens in combination with immuno-cytochemical staining and molecular techniques helps in the diagnosis of advanced cancer and individualized treatment option of NSCLC.

OBJECTIVE: To study expressions of miR-143,matrix metalloproteinase-2(MMP-2) and tissue inhibitor of metalloproteinase-1(TIMP-1) in esophageal cancer tissue and normal tissue among Kazaks and to investigate the relationship between their expressions and clinical pathological features. METHODS: Using real time-PCR to detect the expression of miR-143,MMP-2 and TIMP-1 in 30 esophageal cancer specimens. RESULTS: The expression levels of miR-143 in cancer tissue were lower than those of normal tissues(P=0.000).The lower expression of miR-143 was significantly related to the cellular differentiation lymph node metastasis and clinical stage (P=0.042,0.039 0.007).The expression levels of MMP-2,TIMP-1 in cancer tissue were higher than those of normal tissues(P=0.026 0.021).Enhanced expression of MMP-2 was significantly related to lymph node metastasis(r=0.367,P=0.037).The expression of miR-143 in the Kazak's esophageal cancer was negatively related to the expressions of MMP-2 and TIMP-1 (r=-0.442,-0.410; P=0.001,0.003).The expression of MMP-2 positively related to that of TIMP-1(r=0.794,P= 0.000). CONCLUSION: The under-expression of miR-143 and over-expressions of MMP-2 and TIMP-1 may have important roles in carcinogenesis of esophageal cancer in Kazaks.

OBJECTIVE: To study the expression of glutathione-s-transferase-π (GST-π) and P-glycoprotein (P-gp) in gastric cardia the cancer in the Chaoshan high risk area and their relationships with clinical pathological characteristics of gastric cardia cancer. METHODS: Immunohistochemical staining was used to detect the expression of GST-π and P-gp in 87 cases of gastric cardia cancer. The correlations between GST-π,P-gp expression and the clinical characteristics of gastric cardia cancer were analyzed. ROC curves of age and maximum tumor diameter were plotted. RESULTS: The positive expression rate of GST-π and P-gp in gastric cardia cancer were 85.05% and 93.1%,respectively. The cutoff points of grouping for age and maximum tumor diameter depended upon the results of ROC curves. The expression of GST-π was related to lymph-vascular space invasion. The expression rate of GST-π of patients with lymph-vascular space invasion was signi?cantly higher than that without the invasion(P<0.05). Expression of P-gp was related to the age of patients and the histological type of gastric cardia cancer. Cancer tissues from elder patients and mucinous adenocarcinomas showed significantly higher rate of expression of P-gp (P<0.05). GST-π expression was positively correlated to P-gp expression (P<0.01). CONCLUSION: GST-π and P-gp expression were high in gastric cardia cancer in the Chaoshan area. Their expression was positively correlated. High GST-π expression was associated with lymph-vascular space invasion. High P-gp expression was associated with elder patients and mucinous adenocarcinoma tissues.

OBJECTIVE: To investigate the expression of miR-34a in different types of cervical lesions from Uyghur and Han women in Xinjiang,and to explore its association with clinicopathologic parameters of cervical carcinoma and its clinical value. METHODS: Quantitative real-time PCR was used to detect miR-34a expression in 58 specimens of cervical carcinoma,60 specimens of cervical intraepithelial neoplasia Ⅱ-Ⅲ level (CINⅡ-Ⅲ level)and 32 specimens of healthy control tissue. Data were used to analyze correlations between the expression levels of miR-34a in cervical lesions to different degrees of Uyghur and Han women and related clinicopathologic parameters of cervical carcinoma. Receiver operating characteristic curve (ROC curve) were used to analyze the ability of potential biomarkers to determine cervical carcinoma. RESULTS: The expression of miR-34a in cervical carcinoma was statistically significantly lower than those in CINⅡ-Ⅲ level and healthy control tissue (P<0.05),but no obvious difference was detected between women with Uyghur and Han ethnicities (P>0.05). The expression of miR-34a was associated with the tumor size and FIGO stage and lymph node metastasis (P<0.01). No significant associations were found between miR-34a level and other clinicopathologic parameters of cervical carcinoma,such as age,differentiation,pathological types (P>0.05). miR-34a yielded a receiver-operator characteristic curve area(AUC) of 0.889 (sensitivity 87.5% and specificity 81.0%) to discriminating cervical carcinoma patients from healthy controls and 0.767 (sensitivity 78.3% and specificity 70.7%) to discriminating patients with cervical carcinoma from CINⅡ-Ⅲ level patients and 0.810 (sensitivity 83.7% and specificity 70.7%) to determining cervical carcinoma from all cases. CONCLUSION: Expression of miR-34a was similar for the different ethnic groups. Down-regulation of miR-34a may be involved in the oncogenesis and development of cervical carcinoma. Therefore,miR-34a may be useful as a novel biomarker for early diagnosis and prognosis monitoring.

OBJECTIVE: To investigate an insulin resistance in BRL-3A cells after their exposure to palmitic acid (PA). METHODS: BRL-3A cells were incubated with PA (0,50,100,200,300,400 μmol/L) for 6,12 or 24 hours. After the exposure,cell viability,basic and insulin-stimulated glucose consumption were assayed. RESULTS: Incubation of cells with high concentrations of PA (200,300,400 μmol/L) significantly decreased survival of cells (P<0.05). Incubation of cells with low concentrations of PA (50,100 μmol/L) for 6 and 12 h had no significant effect on cell viability (P>0.05). The basic and insulin-stimulated glucose consumption in these cells were not altered by different concentration of PA incubation for 6 h (P>0.05). However,24 h after the exposure,all concentrations of PA decreased glucose consumption (P<0.05). At 12 h after the exposure,100 μmol/L of PA decreased basic and insulin-stimulated glucose consumption (P<0.05),and this ability remained unchanged after 72 h of continuing cultured. CONCLUSION: Incubation of BRL-3A cells with 100 μmol/L of PA for 12 h could induce insulin resistance in vitro.

OBJECTIVE: To identify a proper qPCR method for examining expression of methylation-related genes in mouse early embryonic cells. METHODSNormal qPCR,isothermal pre-amplification-based qPCR,and PCR pre-amplification-based qPCR methods were applied in quantitative assay of methylation-related genes (TET1,TET2,TET3 and DNMT3A) in mouse early embryonic cells. RESULTS: The sensitivity of these three methods decreased from isothermal pre-amplification-based qPCR method,PCR pre-amplification-based qPCR method to normal qPCR method. The former two methods detected expression of methylation-related genes in a few mouse embryonic cells within a proper PCR cycle number but the PCR pre-amplification-based qPCR method has lower cost than isothermal pre-amplification-based qPCR method. Therefore,we used PCR pre-amplification-based qPCR method to examine methylation-related genes in mouse oocytes and early embryonic cells after 22 h fertilization. The results showed that the expression level of TET1 mRNA was very low and not detectable,the expression of TET2 decreased,and the expression of TET3 and DNMT3A significantly increased (P<0.01) in this process,which was consistent with reported results. CONCLUSION: The sensitivity of isothermal pre-amplification-based qPCR method was the highest among the three qPCR methods. However,the PCR pre-amplification-based qPCR method is the most suitable one for examining multi-gene expression in a few cells in daily practice due to the simple procedure relatively high sensitivity and low expenditure.

OBJECTIVE: To observe the dynamic histological changes in dimethylbenzanthracene(DMBA)-induced cancer in the Chinese hamster's buccal pouch mucosa cancer experimental animal model. METHODS: 60 male Chinese hamsters were randomly divided into three groups;experimental group(24 hamsters),solvent control group (12 hamsters) and control group(24 hamsters). In the solvent control group,the site was only coated with acetone solution. The buccal pouch mucosas were treated with 0.5% DMBA 3 times a week for 15 weeks. For the experimental and the control groups,6 hamsters were randomly selected and killed after 6 weeks,9 weeks,12 weeks and 15 weeks. For the solvent control group,3 hamsters were killed each time. The morphological and histological changes of buccal pouch mucosa were dynamic observed under naked eye and light microscope. RESULTS: Compared with the control group,a series of abnormal changes was consecutively observed in the experimental group;hyperemia,incrassation,festering Canker leukoplakia Cauliflower shape vegetations. The stages of histological changes had the simple hyperplasia(6^th-9^th week),the epithelial dysplasia of different degrees(9^th-12^th week),carcinoma in situ and squamous cell carcinomas (12^th-15^th week). CONCLUSION: Our experimental model shows similar changes to that of human oral squamous cell carcinoma. Therefore,the model can be used to study oral squamous cell carcinoma in multi-stage and multi-step dynamic development.

OBJECTIVE: To evaluate the toxicity and safety of tobacco seed oil in mice. METHODS: Mice were orally exposed to tobacco seed oil for 14 and 30 days according to Care Food Inspection and Evaluation of Technical Specifications. Three genetic toxicology tests were used to evaluate mutagenic effects. In addition thirty day feeding test was conducted to detect sub-chronic toxicity. RESULTS: There was no evidence of toxicity or death in mice within 14 day,which indicated the median lethal dose (LD₅₀) was more than 21 500 mg/kg. In the Ames test,the revertant colonies numbers in each dose group were two times less than the numbers of spontaneous revertant colonies,and no dose- response relationship occurred. Four bacterial strains did not show positive results with or without S9 activation. In the mouse bone marrow cell micronucleus test,the rates from the exposed groups were similar to that of the negative control group (P>0.05). In the mouse bone marrow cell chromosome aberration test,the rates from the exposed groups were no significantly difference with the negative control group. After feeding for 30 days,there was no poisoning and death in the exposed mice. There was no significant difference from the control group in body weight,food utilization,organ-body ratios,and blood biochemical indexes. Histopathologic examination showed that fat vacuoles were observed in the liver tissue of high-dose group,but the other main organs of mice in the other groups had no pathological changes. CONCLUSION: Tobacco seed oil was non-toxic and did not show potentially mutagenic effects and sub-chronic toxicity within the text doses.

OBJECTIVE: To access the safety of Panaxnotoginseng as a health food through assessing its acute toxicity and mutagenicity. METHODS: Oral acute toxicity test,mouse bone marrow micronucleus test,mouse sperm malformation test,Ames test were performed according to the national standard procedures for toxicological assessment of foods. RESULTS: The MTD of Panaxnotoginseng in mouse was more than 15 g/kg,the polychromatic eryrocyte micronucleus rates,sperm abnormality rates at all doses (10.00,5.00,2.50 g/kg) were not significantly different from the solvent control. In the presence or absence of S9 or not,the number of revertant colonies in six dose groups (2,8,40,200,1 000,5 000 μg/plate) did not exceed 2- fold of the spontaneous revertant colony number,nor was there a dose-response relationship,the results of Ames test was negative. CONCLUSION: Panaxnotoginseng did not show acute toxicity and mutagenicity under our study conditions.