OBJECTIVE:To investigate effects and mechanisms of 2, 2', 4, 4'-tetra polybrominated diphenyl ether (BDE-47) on retinoid receptor (RXRα) and estrogen receptors (ERα, ERβ) in MCF-7 cells. METHODS:Stably expressed RXRα in MCF-7 cells at high and low levels were constructed via lentivirus transfection. After exposure of three transfected cell lines to polybrominated diphenyl (BDE-47), cell toxicity was analyzed using the Cell Counting Kit-8(CCK-8) assay. In addition, SOD vitality, MDA content and hOGG1 mRNA expression were analyzed using the TBA, XOD and RT-PCR assays, respectively. Expression levels in mRNA and protein for the three receptors (RXRα, ERα and ERβ) were determined by RT-PCR and Western blot respectively. RESULTS:BDE-47 induced significant cytotoxicity to MCF-7 cells. IC₅₀ of BDE-47 to MCF-7 cells with normal, high and low expressions of RXRα were 23.15, 82.78 and 30.16 μmol/L, respectively. Highest toxicity was observed at 24 h after the exposure to BDE-47. Moreover, BDE-47 induced oxidative damage. Cells with high expression of RXRα receptor had increased toxicity tolerance against BDE-47-induced oxidative damage. BDE-47 also significantly inhibited the expression of RXRα in the three cell lines. For ER, BDE-47 exposure increased the expression of ERα and inhibited the expression of ERβ in normal and low RXRα expression cells, and vice versa in high RXRα expression cells. CONCLUSION:BDE-47 exposure significantly reduced the expression levels in mRNA and protein of RXRα. In addition, the exposure up-regulated ERα and down-regulated ERβ in normal and low RXRα cell lines, but showed opposite effects in the high RXRα cell line. Thus, interference of RXRα expression and breaking the balance of ERα and ERβ may be an important mechanism for disruption of neuroendocrine by BDE-47.

OBJECTIVE:To investigate whether sodium sulfite (Na₂SO₃) induces lipid accumulation by inhibition of very low density lipoprotein (VLDL) assembly and activation of endoplasmic reticulum stress. METHODS:Human diploid liver cells (HL-7702) were cultured in DMEM medium containing 10% fetal bovine serum. Cells were exposed to different concentrations (0.1, 0.5, 2.5, 10 mmol/L) of Na₂SO₃ for 24 h and 48 h, and positive controls were used. VLDL secretion was detected by ELISA and Western blot, mRNA levels of VLDL assembly related factors were detected by quantitative real-time PCR (qPCR) and protein expressions of endoplasmic reticulum stress were detected by Western blot. RESULTS:ELISA and Western blot results showed that after exposure to Na₂SO₃ for 24 h, VLDL contents were significantly increased compared to the controls (P<0.05). After 48 hours, the difference became non-significant (P>0.05). QPCR test results showed that, compared with the negative control group, exposure to 10 mmol/L Na₂SO₃ for 24 h increased the expression of apoB100 mRNA significantly (P<0.05). In addition, the mRNA level of TGH in 10 mmol/L Na₂SO₃ and 2.5 mmol/L Na₂SO₃ groups were significantly increased (P<0.05). After exposure for 48 h, the mRNA levels of TGH in 2.5 mmol/L Na₂SO₃ and 0.5 mmol/L Na₂SO₃ groups were increased significantly (P<0.05). Protein expression of Calnexin was significantly increased (P<0.05) after Na₂SO₃ exposure. CONCLUSION:Na₂SO₃ may increase VLDL assembly and interfere with the correct folding of glycoprotein in hepatocytes.

OBJECTIVE:This study was aimed to investigate molecular mechanisms of acrylamide (AA)-induced hepatotoxicity by examining the effects on mRNA expression, DNA methylation and gene mutations in F344 female rats. METHODS:F344 female rats aged 7 weeks were orally dosed with 0 and 50 μg/mL of AA in drinking water for 28 d. Using qPCR, we detected mRNA expression level of genes which were involved with cell proliferationand and with regulation of DNA methylation in livers. The promoter DNA methylation status of Cdkn1a, Cdkn2a and repetitive sequence Line-1 were examined by COBRA. We also examined the mutation levels of H-ras gene by PCR sequencing. RESULTS:Compared with control, AA decreased mRNA expression levels of Dnmt3a, Cdkn1a, Cdkn2a, Jun and Line-1 in F344 rat liver. No significant change was found in the global DNA methylation and the promoter methylation status of Cdkn1a and Cdkn2a. Moreover, no gene mutation of H-ras was found. CONCLUSION:AA induced expression changes of genes which were involved in DNA methylation regulation and cell proliferation but no alteration in DNA methylation in rat livers. However, no gene mutation was detected

OBJECTIVE:To investigate the induction of DNA damage and telomere length changes in peripheral leukocytes from lead exposed works in comparison with un-exposed controls.METHODS:21 lead exposed workers from a battery plant in the Pearl River Delta Region and 26 non-exposed workers were investigated. Questionnaires were surveyed. Blood samples and peripheral leukocytes were analyzed for blood lead concentrations, telomere length, Comet assay, and cytokinesis-block micronucleus assay. RESULTS:Blood lead concentration of exposed workers (3.08±1.86) mg/mL was significantly higher than that (0.26±0.25) mg/mL of the controls (P<0.05). In addition, tail lengthsand micronuclei frequencies in leukocytes from the exposed workers were significantly higher than that of the controls (P<0.05). However, relative telomere lengths from leukocytes of lead exposed workers were shorter than that of the controls(P<0.05). Correlation analyses indicated that micronucleus frequencies in binucleated cells, rate of micronucleated binucleate cells and rate of nucleoplasmic bridges were positively correlated with work age (P<0.05), and the rank correlation coefficient (rs) were 0.447, 0.432, 0.351 respectively. Work age and blood lead concentration were negatively correlated with relative telomere length of leukocytes (P<0.05), and rs were -0.306, -0.394 respectively. CONCLUSION:Lead exposure induced DNA damage, micronuclei and shorter telomere length in peripheral leukocytes of exposed workers.

OBJECTIVE:To investigate the induction of expression of long non-coding RNA PCA3(LncRNA PCA3) by glycidyl methacrylate (GMA) in malignantly transformed 16HBE cells. METHODS:Malignantly trans-formed 16HBE cells (treated with 8 μg/mL GMA) and solvent control cells (DMSO) of the 30^th generations were harvested. High throughput LncRNA microarray was used to detect differences in expression profile of LncRNAs among the two groups of cells. Changes in LncRNAs were screened through strategies such as fold change and neighboring genes analysis. Real-time fluorescence quantitative PCR (qPCR) and gene chip were applied to measure the expression levels of LncRNA PCA3 and PRUNE2, respectively. RESULTS:Based on the result of LncRNA microarrays, LncRNA PCA3 in GMA-treated cells was up-regulated by 7.17 folds while PRUNE2 was down-regulated by 2.54 folds. QPCR showed that the expression of LncRNA PCA3[(2.67±0.63)×10^-5] in GMA-treated cells was significantly higher than that in the solvent control cells[(1.36±0.44)×10^-5] (P<0.05). On the other hand, the gene chip analyses showed that expression of PRUNE2(10.95) was reduced compared to the solvent control cells(19.46). CONCLUSION:LncRNA PCA3 expression can be considered as one of the stable and specific biomarkers involved in GMA- malignant transformation of 16HBE cells.

OBJECTIVE:Prolactinoma is a common intracranial tumor wihch always leads to serious clinical outcomes. This study aims to confirm the association between the proto-oncogene pokemon and prolactinoma progression, and further explore the molecular mechanisms. METHODS:Tumor tissues were collected from patients with prolactinoma. The primary prolactinoma cells were digested from the tissues and cultured with proper conditions. The expression of pokemon gene was knocked-down by siRNA transfection, and the proliferation of prolactinoma primary cells was analyzed by CCK8 assay. The expression level of pokemon related genes, cyclin-A, p14ARF and p21 in tissues and prolactinoma primary cells were examined by Western blot analysis. RESULTS:Expression of pokemon in prolactinoma primary cell was successfully knocked-down by siRNA transfection. The results showed that siRNA2 showed the highest efficiency of inhibiting the proliferation ability of prolactinoma primary cells. Cyclin-A, p14ARF and p21 were down-regulated by pokemon in both prolactinoma tissues and primary cells. CONCLUSION:Pokemon could improve prolactinoma progression by reducing the expression level of tumor inhibition factors, cyclin-A, p14ARF and p21.

OBJECTIVE:To study the effect of silver nanoparticles (AgNPs) on proliferation and apoptosis of human lung cancer(A549) and hepatoma cells (HepG2). To discuss the potential reasons for different toxicity of AgNPs in the two cell lines. METHODS:A549 and HepG2 cells were treated with AgNPs at concentrations of 20, 40, 80, 160, 320, 640 μg/mL, respectively for 24 and 48 h. Control cultures did not receive treatment. Cell viability was measured by the MTT assay, and glutathione (GSH) and superoxide dismutase (SOD) detection kit were used to determine the level of GSH and SOD. In addition, some cultures were pretreated with N-acetylcysteine (NAC) prior to incubating with 160 μg/mL AgNPs. Flow cytometry was used to detect apoptotic rates for all cell culture groups. RESULTS:After 24 or 48 h of treatment with all dose groups, the survival rates of A549(except for 20 μg/mL group) and HepG2 cells were significantly lower than that in the control group (P<0.05 or P<0.01). However, the rates of HepG2 cells which were treated with 40-640 μg/mL AgNPs were significantly higher than that of A549 cells (P<0.05 or P<0.01). After 24 h of treatment, the SOD activity of A549 cells which were treated with 40 μg/mL AgNPs group was significantly lower than that of the control group (P<0.05), while the GSH levels of A549 cells which were treated with all dose groups were higher. After 48 h of treatment, SOD activity and GSH levels decreased in the 40-160 μg/mL treatment groups. After 24 or 48 h of treatment, SOD activity and GSH levels were increased in HepG2 cells. After 24 h of treatment, the apoptosis rates of HepG2 cells were significantly higher than that of the control group (P<0.05 or P<0.01), while the apoptosis rate of A549 cells had not changed significantly (P>0.05). After 48 h of treatment, the apoptosis rate of A549 cells (except for 20 μg/mL group) and HepG2 cells (160 μg/mL group) were significantly higher than that of the control group (P<0.05 or P<0.01). However, the apoptosis rate of two cells was efficiently prevented by the use of NAC. CONCLUSION:AgNPs can inhibit cell proliferation and induce apoptosis in A549 and HepG2 cells. The observed effects can be due to differential perturbation of intracellular GSH levels and SOD activity in the two cell lines.

OBJECTIVE:To investigate changes in expression and subcellular localization of prohibitin (PHB) in radiation-induced apoptosis in nasopharyngeal carcinoma cells. METHODS:CNE-1 and CNE-2 cells were cultured in vitro. Their rates of early stage apoptosis after 4 Gy radiation were measured by flow cytometry.Expression of PHB, Bax and P53 proteins were measured by Western blot. In addition, immunofluorescence was used to detect their intracellular locations. RESULTS:At 24 hours after radiation, the apoptosis rates and expression of PHB, Bax and P53 were increased significantly. Before radiation, both PHB and P53 were distributed mainly around the nucleus but much less in the cytoplasm. After radiation, the fluorescence intensity in the cytoplasmic region increased dramatically, while the intensity in the nucleus dropped. It seemed that nuclear export of PHB and P53 were induced by radiation. However, no significant subcellular localization could be found in Bax distribution. CONCLUSION:The radiation-induced apoptosis may be associated with the expression and positional alteration of PHB and P53. Furthermore, PHB and P53 may interact with each other.

OBJECTIVE:To study the cytotoxicity of CHL cells after treatment with nanometer copper-oxide. METHODS:CHL cells in different densities (1×10⁵, 5×10⁴, 2.5×10⁴and1.25×10⁴/mL) were monitored continuously by RTCA and their whole growth curves were evaluated. Then, the cytotoxicity of CHL cells treated by nanometer copper-oxide in different doses (0, 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.0313 mg/mL) was real-time monitored, and the accurate IC₅₀ value of the continuous time and the curve of IC₅₀ in continuous time were studied. RESULTS:Growth curves in cultures with different cell densities were measured, and cultures with 5×10⁴/mL inoculated cell number was found the best for toxic effect test. The starting time of the toxic effect after treatment with nano-copper oxide was about 4-5 h. At 6 h after treatment with nanometer copper-oxide, the IC₅₀ value was bigger with a large volatility, while it presented a dramatic decline after 6 h. The value of IC₅₀ after 12, 24, 36 and 48 h was 0.1570, 0.0511, 0.0429, 0.0168 mg/mL, respectively. CONCLUSION:RTCA was useful for detecting cytotoxicity of nanometer copper-oxide, considering the unique characteristics of nanomaterials. In addition, cytotoxicity at 4-18 h after treatment should be investigated further in the future.

OBJECTIVE:To establish the method of isolation and culture of mouse placental pericytes and to use them for screening of placental vascular toxicants. METHODS:Placental pericytes were isolated from day 18.5 pregnant mice by mechanical separation and tissue digestion, and cultured in vitro. Immunocytochemistry tests revealed the cell characteristics and purity of placental pericytes. Cell proliferation was measured by using the Pomega MTS Cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 6.25-100.00 μmol/L lead acetate and cell toxicity of each group was determined by MTS assay. RESULTS:Mouse placenta pericytes were successfully isolated by mechanical and tissue digestion method and could grow well on culture plates. The pericytes exhibited typical polygonal morphology and positive staining for α-SMA. After 6.25-100.00 μmol/L lead acetate treatment for 24 h, a gradual decline in cell viability was observed, and the difference was statistically significant. CONCLUSION:High purity placental pericytes from mice can be successfully collected by mechanical separation and tissue digestion, and can be used to screen placental vascular toxicants.

OBJECTIVE:To investigate the effects of pro-anthocyanidin on inhibiting inflammatory mediator secretion in LPS activation of BV2 microglia. METHODS:Neuroinflammation model was established by LPS-activated BV2 microglia.BV2 microglial were pretreated with different concentrations of pro-anthocyanidin (0.1, 0.5, 1.0, 5.0 μg/mL) and stimulated with LPS (1.0 μg/mL) for 24 h. Cell viability was assessed by MTT assay. Griess method was utilized to detect the content of NO. The secretion level of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 was examined using specific ELISA kits. RESULTS:Within a certain dosage ranges of pro-anthocyanidin and LPS, there was no negative effect on cell viability (P>0.05). LPS (1.0 μg/mL) significantly increased the release of inflammatory mediator nitric oxide (NO) as well as the cytokines TNF-α, IL-1β and IL-6(P<0.05). Compared with the LPS group, different concentrations of pro-anthocyanidin (0.1, 0.5, 1.0, 5.0 μg/mL) attenuated the release of NO, TNF-α, IL-1β and IL-6 in cells that were cultured in conditioned media from LPS-induced BV2 microglia. The effect was dose-dependent (P<0.05). CONCLUSION:We showed that pro-anthocyanidin has a protective effect on inflammatory responses of LPS-activated BV2 microglia in vitro.

OBJECTIVE:We evaluated the combined effects of paraquat and maneb on expression of cognition- and memory-related genes in hippocampus of Sprague Dawley pups. METHODS:Pregnant SD rats were randomly divided into three groups:control (physiological saline) and PQ-MB treatment (PQ 10 mg/kg+MB 30 mg/kg and PQ 15 mg/kg +MB 45 mg/kg). They were treated twice a week with PQ-MB from day 5 after pregnancy till ablactation. Pups at 3-month old and their tissues were evaluated using Morris water maze, optical microscopy, electron microscopy, fluorescence PCR and Western blot for changes in behavior, morphology of hippocampus, microstructure of hippocampal neurons, mRNA and protein levels of CREB, P-CREB and BDNF. We use ANOVA to analyze the experimental data. RESULTS:Morris water maze showed that the exposed groups had obvious complex trajectories and a significantly reduced number of shuttle (P<0.05) compared to the control group. Both optical and electron microscopic investigations showed obvious morphological changes in hippocampus of high-dose exposed pups. There was a significant reduction in mRNA expressions among all the treated groups (P<0.05) and significant decrease in hippocampus CREB, P-CREB and BDNF protein levels(P<0.05). CONCLUSION:Combined exposure to paraquat and maneb in utero may impact cognition and memory of Sprague Dawley rat pups via down-regulating the expression of related genes in hippocampus.

OBJECTIVE:To study some physiological effects of strontium on Chinese cabbage and rape. METHODS:The Hoagland solutions of different strontium concentrations(0, 1, 2, 3, 4 mmol/L) were used to culture Chinese cabbage and rape. After 4 weeks, leaves were used to analyze chlorophyll content and activities of antioxidant enzymes, including malondialdehyde (MDA), catalase (CAT), peroxidase (POD) and total superoxide dismutase (T-SOD). RESULTS:Compared with controls, the content of MDA in treated Chinese cabbage decreased from the peak of 2 mmol/L of strontium (P<0.05), while there was no significant difference between groups in rape (F=2.798, P>0.05). Also, no significant difference was found between the two vegetables (F=0.057, P<0.05). The activity of CAT, POD and T-SOD decreased first and then increased significantly (P<0.05). Chlorophyll content for the 14 d, 21 d, 28 d increased significantly over that of 7 d (P<0.05). CONCLUSION:Strontium has strong influence on MDA, antioxidant enzyme and chlorlphyll of Chinese cabbage and rape.

OBJECTIVE: To detect the expression of Caveolin-1(Cav-1) and E-cadherin(E-cad) in esophageal carcinoma TE1 cell line and to explore the involvement of Cav-1 in metastasis of esophageal carcinoma. METHODS:Small interfering RNAs Cav-1 siRNA1 and Cav-1 siRNA2 which targeted Cav-1 and negative control siRNA were individually transfected into esophageal carcinoma TE1 cell line. Non-transfected cells were used as blank control. Expressions of Cav-1 and E-cad were detected in cell extracts using Western blot. Migration and invasion ability of cells were evaluated using the Transwell assay. RESULTS:The Western blot analysis showed that Cav-1 siRNAs effectively suppressed the expression of Cav-1 and up-regulated the expression of E-cad in TE1 cells. The transwell assay showed that the migration and invasion ability of Cav-1 siRNA treated group were significantly weakened compared with the blank control group (P<0.05). CONCLUSION:Cav-1 may affect metastasis of esophageal carcinoma by up-regulating the expression of E-cad.

OBJECTIVE:To investigate the expression of PFTAIRE protein kinase 1(PFTK1) in human non-small cell lung cancer (NSCLC) and to analyze its clinical significance. METHODS:Immunohistochemistry was used to detect the protein expression of PFTK1 in 119 cases of NSCLC. The sample rate was tested by chi-square test. Kaplan-Metier method was used to calculate survival rates and survival rates were tested by Log-Rank test. RESULTS:In NSCLC tissues, PFTK1 existed mainly in the cytoplasm and nucleus. In the 119 cases, PFTK1 expression was higher in cancer than in adjacent normal alveolar epithelium tissues, and the ratio was 63.9% (76/119). PFTK1 expression was significantly correlated with both different T (primary tumor) stage and lymph node metastasis (P<0.05), whereas it was not correlated with age, sex, clinical stage, pathological grade, pathology type, tumor size and N (regional lymph node)stage (P>0.05). In patients with lung squamous cell carcinoma, if stratification factor N stage was controlled, the survival analysis showed that when the expression level of PFTK1 was higher the overall survival time of patients was shorter(P<0.05). CONCLUSION:PFTK1 was overexpressed in NSCLC, was closely associated with T stage and lymph node metastasis, and affected prognosis of patients. PFTK1 may become a potential biomarker for NSCLC.