OBJECTIVE:To observe the effects of 3, 3'-diindolylmethane (DIM) on oxidative stress induced by hydrogen peroxide (H₂O₂) in HaCaT cells. METHODS:HaCaT cells were treated with different concentrations of H₂O₂ to establish the models of oxidative stress. CCK-8 assay was performed to evaluate the inhibitive effects of DIM (1-20 μmol/L) on cells proliferation. The intracellular reactive oxygen species (ROS) levels were measured with flow cytometry. Furthermore, the effects of DIM (0, 5 and 10 μmol/L) on H₂O₂-induced the phosphorylation level of nuclear factor κB (p-NF-κB), the expression of mitogen activated protein kinase (MAPKs) were detected with Western blot. RESULTS: The cell model of oxidative stress was successfully established. The results of CCK-8 assay showed that in the doses of 1-10 μmol/L, DIM did not have obvious influence on cell viability (P>0.05). Flow cytometry results indicated that pre-treatment with DIM (10 μmol/L) could inhibit the level of intracellular ROS (P<0.05). With increasing concentration of DIM, the levels of p-p38-MAPK, p-JNK and p-NF-κB were significantly depressed. CONCLUSION: DIM could protect HaCaT cells from H₂O₂-induced oxidative stress via suppressing production of ROS levels and down-regulating the expression of NF-κB and members of MAPKs. DIM might be used as an effective drug to treat or reduce oxidative stress-mediated injury in skin cells.

OBJECTIVE:To explore whether the DNA copy number changes in 5 gene fragments are specific abnormalities for bladder cancer in Chinese patients. METHODS:Real-time PCR was conducted to gauge DNA copy number alteration of the 5 gene fragments among commercial genome DNA of peripheral blood leucocyte (PBL) derived from Caucasian, genome DNA of PBL from Chinese healthy volunteers, the bladder cancer patients and their corresponding tumor tissues. RESULTS:Comparing with the Caucasian PBL, all the gene fragments revealed copy number changes with decreased CEP63, increased FOSL2, GHR and PAQR6, in the PBL of Chinese healthy volunteers, except ZFAND3. Comparing with the PBL of Chinese healthy volunteers, in the bladder tumor tissue samples, the copy number of CEP63, GHR and PAQR6 increased (all P<0.05);whereas FOSL2 (P=0.41) and ZFAND32 (P=0.062) revealed no statistical difference (all P>0.05). Comparing with the PBL of Chinese healthy volunteers, the copy number of CEP63 and PAQR6 increased but FOSL2 and ZFAND3 decreased in the PBL of bladder cancer patients (all P<0.01). Comparing with the PBL of the bladder cancer patients, gain of copy number was observed with CEP63, FOSL2, GHR and ZFAND3 (all P<0.01), while PAQR6 (P=0.325) exhibited no statistical difference, in the corresponding tumor tissues. CONCLUSION:Copy number variation of CEP63, FOSL2, GHR and PAQR6 was found between Chinese (Han) and Caucasian. Copy number gain of CEP63 and GHR may be involved in carcinogenesis of bladder cancer;while gain of PAQR6 was possibly associated with the genetic susceptibility for this malignancy.

OBJECTIVE:This study was designed to investigate the protective effects and underlying mechanisms of selenium pre-administration on hepatic oxidative injury in rats exposed to cadmium. METHODS:Rats were exposed to cadmium pre-administrated with selenium, then the inhibitory effects of selenium on liver injury, apoptosis and oxidative stress were studied. The measurements of the serum ALT and AST activities were conducted to evaluate liver function. The Bax and Bcl-2 protein levels were measured by Western Blotting and the values of Bax/Bcl-2 were obtained to evaluate hepatic apoptosis. The measurements of hepatic ROS accumulation by DHE stain and hepatic MDA level were conducted to evaluate hepatic oxidative stress. The measurements of hepatic antioxidase SOD, SOD-1, GPx activities in liver and the protein levels of SOD-1, CAT, GP_x-1 and Nrf-2 were obtained to evaluate hepatic antioxidative defense ability. RESULTS:The selenium pre-administration exerted effectively protective effects on cadmium-induced liver injury, supported by the inhibition of increased ALT and AST activity. Secondly, the selenium pre-administration effectively alleviated the liver apoptosis in rats exposed to cadmium, evidenced by the decrease of Bax/Bcl-2 ratio. Thirdly, the selenium pre-administration significantly inhibited the hepatic oxidative stress in rats exposed to cadmium, illustrated by the decrease of hepatic ROS accumulation and MDA level. Finally, the enhancement of antioxidative defense ability might be the key mechanism involved in the protective effects of selenium against cadmium-induced liver injury, supported by the increase of hepatic SOD, GPx activities, and the protein expressions of SOD-1, CAT, and GP_x-1 via Nrf-2 activation. CONCLUSION:Selenium pre-administration had a protective effect against hepatic oxidative injury induced by cadmium exposure and the strengthening of antioxidant defense ability via Nrf-2 activation might contribute to this protective effect.

OBJECTIVE:To assess the genetic damage of low dose ionizing radiation(LDIR) occupationally exposed in flaw detection workers. METHODS:We used the Thermo-luminescent Dosimeter(TLD) to measure the individual exposure dose. Thirty-one flaw detection workers and forty-nine people without such exposure were enrolled. The cytokinesis-block micronucleus (CBMN) assay was performed to detect the chromosome damage in peripheral blood lymphocytes. Real-time PCR was used to analyze the mtDNA copy number and supercoiling formation change. Long-PCR was applied to analyze mtDNA integrity. qRT-PCR was applied to analyze mRNA levels of TFAM and PGC-1α. RESULTS:The mean annual effect dose in flaw detection workers was (0.15±0.03) mSv/a, range in (0.12-0.21) mSv/a. The MN and NBUDs rate in flaw detection worker group were obviously elevated compared with controls (P<0.01). In our study, the mtDNA copy number of flaw detection workers(40.04±24.99) was higher than controls (24.86±19.14) (P<0.01), and the supercoiling formation was also evidently changed (P<0.05). The mtDNA integrity of flaw detection workers (95.10±9.54) was decreased compared to controls (313.51±14.58)(P<0.01). The mRNA levels of TFAM and PGC-1α was found to be higher than controls (P<0.01). CONCLUSION:We found all of the annual effect dose of flaw detection workers are conforming the state radiological protection standards. However, the induction of chromosome damage and mtDNA damage caused by LDIR were evident, the health of flaw detection workers are at risk.

OBJECTIVE:To investigate the effects on HCT116 cell proliferation and apoptosis by down-regulating PRPS2 gene expression. METHODS:PRPS2 gene interference vector sh-PRPS2 was constructed. The vector was not the transfected in normal control;the negative vector was transfected in negative control and the RNAi vector was transfected in experimental groups. The vectors were transfected to HCT116 cells after identified by DNA sequencing, and the level of PRPS2 mRNA and protein were detected by RT-PCR and Western blot, respectively. The ability of cell proliferation was evaluated by CCK8 kit. The cell cycle and apoptosis were assessed by flow cytometry. RESULTS:The RNAi expression vector of PRPS2 was successfully constructed as identified by DNA sequencing:sh-PRPS2-1, sh-PRPS2-2 and sh-PRPS2-3. The relative expression levels of PRPS2 mRNA were 0.61±0.03, 0.89±0.02 and 0.27±0.05 in HCT116 cells 72 h after transfected with sh-PRPS2-1, sh-PRPS2-2 and sh-PRPS2-3, respectively, all decreased significantly compared with control (P<0.05). The relative expression levels of PRPS2 protein were 0.37±0.06, 0.84±0.05 and 0.30±0.04, respectively, and also decreased significantly compared with control (P<0.05). 72 h after transfection, the inhibition rate of sh-PRPS2-3 proliferation was 19.8%±2.4%. The apoptosis rate of HCT116 cell was increased to 68.4%±4.6% 72 h post transfection. Sub-G1 population percentage was increased to 12.9%±3.8%. CONCLUSION: Down-regulation of PRPS2 expression level could effectively inhibit cell proliferation as well as promote apoptosis. HCT116 cell was arrested in G0/G1 phase. This study provided a research basis for tumor targeted therapy.

OBJECTIVE:To study the effects of carboplatin combined with TRAIL (TNF- related apoptosis inducing ligand) on proliferation and apoptosis of human lung cancer A549 cells. METHODS:After treated by TRAIL, carboplatin or combination of two drugs, the proliferation of A549 was measured by MTS and apoptosis rate was evaluated by flow cytometry. The morphology was investigated by microscopy. RT-PCR and Western blot were used to examine the gene and protein expressions of DR4, DR5, Survivin and XIAP. RESULTS:Carboplatin or TRAIL alone or combined inhibited the proliferation of A549 cells in concentration-dependent (20-80 μg/mL) manner. Carboplatin combined with TRAIL exhibited a stronger inhibitory effect and induced apoptosis than when used alone. Carboplatin could down-regulate the protein expression level of Survivin and XIAP and increase the DR5 protein expression level, but have no effect on DR4 expression. CONCLUSION:Combination of carboplatin and TRAIL could inhibit proliferation and apoptosis via influencing the expressions of Survivin, XIAP and DR5 in A549 cells.

OBJECTIVE:To explore the presence of G protein-coupled estrogen receptor (GPER) in the Kunming mouse gubernaculum testis. To explore the rapid effects of diethylstilbestrol (DES) on cultured mouse gubernaculum testis cells via ERK signaling pathway. METHODS:The expression of GPER was assessed by immunohistochemical staining with the development of mouse gubernaculums testis (GD17, GD19, PD0, PD3, PD7, PD14 and PD21). Subcellular localization of GPER was identified by Immunoelectron Microscopy. Cells subcultured from mouse gubernaculum testis were divided into three groups randomly, that is, control group(DES 1.5×10^-8 mol/L), ICI 182780-DES group (pretreated with 1 nmol/L estrogen receptor inhibitor ICI 182780 90 min), and G15-DES group (pretreated with 1 nmol/L GPER inhibitor G15 90 min). p-ERK1/2 was detected in the cells with 1.5×10^-8 mol/L DES in different groups by Western blot. RESULTS:The immunohistochemical staining showed that GPER was expressed in the development of gubernaculums testis, mainly in the unconsolidated mesenchymal tissue of inner gubernaculum testis, and GPER staining was strong both on the membrane and in the cytoplasm. The subcellular localization of GPER protein was mainly in the mesh-like network of cytoplasm, the rough endoplasmic reticulum. G15 could reverse the DES-induced inhibitory effect of p-ERK1/2, but ICI182780 didn't have the same effect. CONCLUSION:GPER was expressed in membrane structure of gubernaculums testis cells in different stages of development. The nongenetic effects of DES on gubernaculums testis cells were likely to be partly mediated by GPER. Therefore, our data provide new insight into the role of EEs in the etiology of male reproductive system and will help develop better approaches for the prevention and therapy of male reproductive malformation.

OBJECTIVE:To study the effects of atrazine(ATR) on apoptosis of dopaminergic neurons. METHODS:The 7-8 months old SD male rats were randomly divided into four groups, control group and ATR-dose groups (10, 50, 100 mg/kg). The rats were gavaged for 90 days then sacrificed. Midbrain nigra was collected and stored frozen in -80 ℃. We used transmission electron microscope to examine the apoptosis of dopaminergic neurons, Real-time PCR to measure apoptotic genes Bax, Bcl-2 and Caspase-3 mRNA expression levels and Western blot to evaluate apoptotic proteins Bax and Caspase-3 expression levels. RESULTS:Compared with the control group, with increasing dose, dopaminergic neurons developed mitochondrial swelling, vacuoles and mitochondrial autophagy. Compared with the control group, the Bax mRNA and protein levels were significantly increased (P<0.05), whilst the Bcl-2 mRNA and protein levels of each group were significantly lower than control group(P<0.05). The expression of Capase-3 mRNA level in 10 mg/kg group was significantly higher than control group (P<0.05) and in the 50 and 100 mg/kg groups were significantly lower than control group (P<0.05). The expressions of Caspase-3 protein levels were increased with increasing does in the medium and high dose groups(P<0.05). CONCLUSION:Atrazine, through mitochondrial apoptotic signal could cause the death of neurons.

OBJECTIVE:To investigate the expression of silent information regulator 1(SIRT1) in bladder urothelial cell carcinoma (BUC), and analyze its clinical significance. METHODS:The expression of SIRT1 protein was assessed by immunohistochemistry in bladder sections of 19 normal bladders and 72 patients with BUC. We analyzed the relationship of SIRT1 and the pathological grade and clinical stage of BUC. RUSULTS:SIRT1 was observed in normal bladder samples and BUC．Normal bladder showed negative or weakly positive expression of SIRT1(31.58%), however BUC samples showed moderately or strongly positive expression(77.78%). Furthermore, the expression level of SIRT1 increased with tumor pathological grade and clinical stage. SIRT1 positive expression rates showed significant difference between various pathology grades (P<0.05). CONCLUSION:SIRT1 was high expressed in BUC, and was related to its pathological grade and clinical stage, making it a useful tumor marker and a possible indicator for estimating the invasiveness and recurrence of BUC．Blocking SIRT1 expression and its functions may become targeted therapy of bladder cancer treatment．

OBJECTIVE:In this study, the toxicokinetics and toxicodynamics of chlorpyifos were assessed in rats following one single subcutaneous exposure. METHODS:Adult female SD rats, randomly divided into three dose groups (69.75, 139.5, 279 mg/kg) and one control group (0 mg/kg), received one subcutaneous injection. Blood and cerebral cortex samples were collected on 3, 6, 12, 24, 48 and 72 hours in each group, and 0-24, 0-48, 0-72 h urine samples were collected continuously. The indicators included chlorpyrifos (CPF) in serum, 3, 5, 6-trichloro-2-pyridyol (TCP) in urine and serum, and acetylcholinesterase (AChE) in serum and cortex. RESULTS:With the increase in the exposure dose, the concentration of serum CPF, serum TCP, urine TCP and inhibition of serum and cortex AChE increased. The peak concentration of serum CPF and TCP were 6 and 12-24 h, respectively. Maximum inhibition of serum and cortex AChE occured at 24 and 24-48 h, respectively. Half-life(t_1/2) of serum CPF was 29-48 h, and 0-72 h area under the curve (AUC) was 31-110 h·μmol/L in each group. t_1/2 of serum TCP was 28-54 h, and 0-72 h AUC value was 897-3 450 h·μmol/L. Serum and cortex AChE activity demonstrated a positive correlation (P<0.01), and serum AChE activity had higher inhibition (P<0.01). There was a dose-response relationship between serum CPF and AChE, fitting by DoseResp curve. CONCLUSION:External exposure dose of CPF was correlated with CPF, TCP and AChE activity. There was a dose-effect relationship between CPF internal level and serum AChE activity. Excretion of TCP in urine can be used as a biomarker of exposure. Serum AChE activity was an effective biomarker of cortex AChE activity.

OBJECTIVE:To study influences of nm23-H1 gene transfection on the expressions of adhesion molecules in lung cancer cell line L9981. METHODS:Cell adhesion experiment and Boyden room were used to evaluate the change of cell adhesion and invasion. Semi-RT-PCR and flow cytometry were used to detect the mRNA and protein expressions of E-cadherin, integrin β1 and integrin β3 betwteen transfected and non-transfected cell lines. RESULTS: nm23-H1 cell line in vitro adhesion and invasion were all down-regulated. mRNA of E-cadherin in the transfected cell line was stronger than that of non-transfected cell line, but mRNAs of integrin β1 and integrin β3 of transfected cell line were weaker than that of non-transfected cell line. Flow cytometry showed similar results as above. CONCLUSION: nm23-H1 could reverse lung cancer malignant phenotype. nm23-H1 could up-regulate E-cadherin expression, while down-regulate expressions of integrins β1 and β3, thus inhibit the metastasis of lung cancer cells.

OBJECTIVE:To evaluate the acute toxicity and mutagenicity of potassium 2-(1-hydroxypentyl)-benzoate(dl-PHPB). METHODS:KM mice were intravenously treated with dl-PHPB at the doses of 256.0、286.3、320.0、357.8、400.0 and 447.2 mg/kg. We observed the clinical signs of toxicity and calculated the median lethal dose (LD₅₀) and LD₅₀ 95% confidence interval. Mutagenicity of dl-PHPB was studied by Ames test, bone marrow micronucleus study in male mice and in vitro CHL cell mammalian chromosome aberration assay. In Ames test, the tester strains used were Salmonella typhimurium TA97, TA98, TA100 and TA102, mutagenicity was evaluated with the plate incorporation method beginning at 0.5, 5, 50, 500 and 5 000 μg per plate in the absence or presence of S9 metabolic activation. In bone marrow micronucleus study, KM male mice were intravenously treated with dl-PHPB at the dosse of 210、70 and 23.3 mg/kg. Bone marrow cells were harvested from mice at 24 hours after dosing. 1 000 PCEs per animal were examined microscopically for the presence of micronucleated polychromatic erythrocytes(MNPCEs). The in vitro CHL cell mammalian chromosome aberration study on dl-PHPB consisted of the premilinary toxicity assay and chromosome aberration assay, with and without S9 metabolic activation system, and chromosomal aberration percentage per dose were analyzed. RESULTS:In the acute toxicity study, all mice after dosing developed transient toxic symptoms such as tachypnea, sautonomic activity decreased, prostration, then the dying state were observed at dosage of ≥320.0 mg/kg, mice appeared respiratory depression, cyanosis, loss of righting reflex, and died during 2 to 30 minute after administration. The mortality rate of mice treated with dl-PHPB from 256.0 to 447.2 mg/kg were 0, 0, 10%, 30%, 70%, 100%, respectively. The survival mice returned to normal after 30 min. In the 14-days observation period, there were no abnormal clinical signs in the surviving animals and the general autopsy showed no abnormality in major organs. The intravenous median lethal dose of dl-PHPB was 373.3 mg/kg in mice, LD₅₀ 95% confidence interval was 355.6 to 392.0 mg/kg. In the Ames assay, no positive mutagenic response was observed. In bone marrow micronucleus study, a single bolus intravenous administration of dl-PHPB at dosages of 23.3, 70 and 210 mg/kg didn't induce a significant increase in the number of MNPCEs in the bone marrow, MNPCEs were less than 4‰ at three dosages. In CHL chromosome aberration assay, dl-PHPB achieved a chromosome aberration of less than 5% in the presence and absence of S9 mixture. CONCLUSION:The intravenous median lethal dose of dl-PHPB was 373.3 mg/kg in mice, and dl-PHPB was not mutagenic in these 3 screening mutation assays.

OBJECTIVE:To evaluate the acute toxicity and mutagenicity of bamboo charcoal powder (BCP). METHODS:Oral acute toxicity test, Ames test, rat bone marrow micronucleus test and in vivo comet assay were conducted according to the OECD standards. Dose designed for oral acute toxicity test was 20 mL/kg. Doses were designed at 0.008, 0.04, 0.2, 1, 5 mg/plate of Ames test, at 2.81, 5.62, 11.24 g/kg of micronucleus test and at 2.81, 5.62, 11.24 g/kg of in vivo comet assay. RESULTS:The mean volume diameter of BCP was 16.596 μm. The LD₅₀ of BCP in rats was > 11.24 g/kg in this experimental condition. In the three mutagenicity tests, no mutagenic effect was observed in any BCP-treated group compared to the control group. CONCLUSION:BCP is a micro-sized mixture, and was non-toxic according to acute toxicity classification, and showed no mutagenic effect in this experimental design.

OBJECTIVE:To separate and refine the chemical components from n-butanol extract of Ipomoea stolonifera (BE-IS) in Chaoshan area, and investigate their anti-inflammatory activities in vivo. METHODS:The chemical components from BE-IS were separated by different kinds of chromatography and identified by spectrum analysis methods. Then anti-inflammatory activity was evaluated in croton oil-induced ear edema model and carrageenan-induced paw edema model. RESULTS:Scopoletin, umbelliferone, esculetin, hesperetin and curcumin were extracted from BE-IS. Compared to the control group, compounds from BE-IS could inhibit the edema induced by croton oil or carrageenan to different degree, and the difference was statistically significant (P<0.01 or P<0.05). CONCLUSION: The chemical components from BE-IS have anti-inflammatoty activities, which explained why Ipomoea stolonifera possess strong anti-inflammatoty activity.