Effects of bisphenol A on OCT4 and SOX2 genes expression in mouse embryonic stem cells / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the effects of bisphenol A (BPA) exposure on toxicity characteristic and OCT4 and SOX2 gene expression of mouse embryonic stem cells (mESC).</p><p><b>METHODS</b>mESC were cultured, and treated with the doses of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L respectively of BPA and DMSO (the solvent control group)for 24 hours, and three groups of cells were treated with the same method. The morphological changes of mESC in the control and exposure groups were observed through an inverted microscope. Cell counting kit 8 (CCK8) was used to detect the effects of BPA on proliferation of mESC, and based on the results, the half inhibitory concentration (IC50) was calculated. Real-time fluorescent quantitative polymerase chain reaction (RT-QPCR) and western blotting were used to detect the expression of OCT4 and SOX2.</p><p><b>RESULTS</b>BPA had certain toxicity on mESC, the treatment of BPA significantly increased cell toxicity in a concentration-dependent manner, and the IC50 was 4.3×10(-4) mol/L, combined with the BPA exposure concentration of the environment and the related literature, eventually taking the five concentrations of 10(-8), 10(-7), 10(-6), 10(-5), 10(-4) mol/L as the experimental groups. The mESC morphology were effected after the treatment of BPA for 24 h, compared with the control group, the number of cells decreased, appearing some floating cells, and the cell cloning became irregular and differentiation in the higher concentration groups. The OCT4 mRNA expression level in the 10(-7) mol/L (1.146 ± 0.087), 10(-6) mol/L (1.156 ± 0.030), 10(-5) mol/L (1.158 ± 0.103) and the 10(-4) mol/L (1.374 ± 0.053) dose group were all significantly higher than the control group (1.000 ± 0.000) (t values were -2.384, -2.953, -3.203, -4.021 respectively, P value all < 0.05). Meanwhile, the SOX2 mRNA expression level in the 10(-4) mol/L (1.113 ± 0.052) were higher than the control group (1.000 ± 0.000) (t value was -2.765, P value < 0.05). Moreover, the OCT4 protein expression level in the 10(-5) mol/L (1.360 ± 0.168) and 10(-4) mol/L (1.602 ± 0.151) were all significantly higher than the control group (1.000 ± 0.000) (t values were -3.538, -4.002 respectively, P value all < 0.05), while no obvious change of the SOX2 protein expression level was detected in all treated groups.</p><p><b>CONCLUSION</b>BPA in a certain dose range could upregulate the expression of OCT4 gene in mouse embryonic stem cells while had no significant effect on the expression of SOX2 gene.</p>

Study on sperm damage caused by trichloroethylene in male rats / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study in vitro sperm damage caused by trichloroethylene in male rats.</p><p><b>METHODS</b>Sperms of Sprague-Dawley (SD) rats were collected 4 hours after being contaminated by trichloroethylene of 0, 2, 4, 6, 8, and 10 mmol/L in vitro. Giemsa staining was performed to observe the morphological changes of sperms, and flow cytometer was used to detect the changes in mitochondrial membrane potential.</p><p><b>RESULTS</b>The sperm motilities in 6, 8, and 10 mmol/L trichloroethylene groups decreased significantly compared with that in control group (P <0.01); the sperm aberration rates in 8 and 10 mmol/L trichloroethylene groups were significantly higher than that in control group (P<0.01). With the increase in exposure dose, the proportion of sperms with reduced mitochondrial membrane potential increased, and there were significant differences in sperm apoptosis rate between the 4, 6, 8, and 10 mmol/L trichloroethylene groups and control group (P<0.01).</p><p><b>CONCLUSION</b>In vitro exposure to trichloroethylene can reduce sperm motility and increase the aberration rate and apoptosis rate of sperms in male SD rats.</p>

A cell-based detection of ciguatoxin using sodium fluorescence probe / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a cell-based detection method of ciguatoxin using fluorescence assay.</p><p><b>METHODS</b>Mouse neuroblastoma N-2A cells were exposed to ouabain and veratridine and different concentrations of standard ciguatoxin samples (P-CTX-1) to establish the curvilinear relationship between the toxin dosage and fluorescence intensity using the sodium fluorescence probe CoroNaTM Green. The toxicity curvilinear relationship was also generated between the toxin dosage and cell survival using CCK-8 method. Based on these standard curves, the presence of ciguatoxin was detected in 33 samples of deep-sea coral fish.</p><p><b>RESULTS</b>A correlation was found between the detection results of cell-based fluorescence assay and cytotoxicity assay, whose detection limit reached 103 g/ml and 1012 g/ml, respectively. The cell-based fluorescent assay sensitivity showed a higher sensitivity than cytotoxicity assay with a 2-4 h reduction of the detection time.</p><p><b>CONCLUSIONS</b>The cell-based fluorescent assay can quickly and sensitively detect ciguatoxin and may serve as a good option for preliminary screening of the toxin.</p>

Role of poly (ADP-ribose) polymerase 1 on DNA methylation variation induced by B(a)P in human bronchial epithelial cell / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate DNA methylation variation in human cells induces by B(a)P, and to explore the role of PARP1 during this process.</p><p><b>METHODS</b>The changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B(a)P (1.0, 2.0, 5.0, 10.0, 15.0, 30.0 µmol/L) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP 1 and DNMT 1 were monitored dynamically.</p><p><b>RESULTS</b>The percentage of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP1 cells were separately (4.04 ± 0.08)% and (9.69 ± 0.50)%. After being treated by 5-DAC for 72 hours, mCpG% decreased to (3.15 ± 0.14)% and (6.07 ± 0.54)%. After both being exposed to B(a)P for 72 hours, the mCpG% in 16HBE group (ascending rank) were separately (5.10 ± 0.13), (4.25 ± 0.10), (3.91 ± 0.10), (4.23 ± 0.27), (3.70 ± 0.15), (3.08 ± 0.07); while the figures in 16HBE-shPARP1 group (ascending rank) were respectively (10.63 ± 0.60), (13.08 ± 0.68), (9.75 ± 0.55), (7.32 ± 0.67), (6.90 ± 0.49) and (6.27 ± 0.21). The difference of the results was statistically significant (F values were 61.67 and 60.91, P < 0.01). For 16HBE group, expression of PARP 1 and DNMT 1 were 141.0%, 158.0%, 167.0%, 239.0%, 149.0%, 82.9% and 108.0%, 117.0%, 125.0%, 162.0%, 275.0%, 233.0% comparing with the control group, whose difference also has statistical significance (t values were 11.45, 17.32, 32.24, 33.44, 20.21 and 9.87, P < 0.01). For 16HBE-shPARP1 group, expression of PARP 1 and DNMT 1 were 169.0%, 217.0%, 259.0%, 323.0%, 321.0%, 256.0% and 86.0%, 135.0%, 151.0%, 180.0%, 229.0%, 186.0% comparing with the control group, with statistical significance (t values were 9.06, 15.92, 22.68, 26.23, 37.19 and 21.15, P < 0.01). When the dose of B(a)P reached 5.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE group (ascending rank) were 125.0%, 162.0%, 275.0%, 233.0% times of it in control group, with statistical significance (t values were 12.74, 24.92, 55.11, 59.07, P < 0.01); while the dose of B(a)P reached 2.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE-shPARP1 group were 135.0%, 151.0%, 180.0%, 229.0%, 186.0% of the results in control group, and the differences were statistically significant (t values were 23.82, 40.17, 32.69, 74.85, 46.76, P < 0.01).</p><p><b>CONCLUSION</b>The hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.</p>

The effect of DNA methyltransferase 1 low expression on the global genome DNA methylation status of 16HBE cell / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To construct DNA methyltransferase 1 (DNMT1) low expression 16HBE cell line and observe the variation of cell cycle and global genomic DNA methylation.</p><p><b>METHODS</b>The method of Lenti-virus induced RNA interference was applied to introduce four different shRNA fragment into 16HBE cells. Flow cytometry and 5-mC immunofluorescence methods were used to observe the cell cycle and global DNA methylation status of DNMT1 low expression 16HBE cells.</p><p><b>RESULTS</b>The DNMT1 protein relative expression level of 16HBE-shDNMT1-4 cell line was down regulated about 44% (P < 0.05) compared with the control. No obvious differences of cell cycle and global genome DNA methylation status were observed between the 16HBE and 16HBE-shDNMT1.</p><p><b>CONCLUSION</b>The DNMT1 gene low expression cell is successfully constructed, and there are no obvious changes happened on the cell cycle and global genomic DNA methylation.</p>

Genome DNA hypomethylation in the process of crystalline nickel-induced cell malignant transformation / 中华预防医学杂志

<p><b>OBJECTIVE</b>To observe the effect of crystalline NiS on genome DNA methylation profile in in vitro cultured cells.</p><p><b>METHODS</b>16HBE Cells were treated with crystalline NiS at 0.25, 0.50, 1.00 and 2.00 µg/cm(2) for 24 h and three times at total. DAC treatment was given at 3 µmol/L for 72 h.5-mC immunofluorescence and SssI methyltransferase assay methods were applied to investigate if the hypomethylation of genome DNA involved.</p><p><b>RESULTS</b>The results of 5-mC immunofluorescence showed that the fluorescence intensity of NiS-treated cells were decreased in some degree, and transformed cells were decreased dramatically. By the SssI methylase assay, an average of (81.9 ± 7.3)% methylated CpG were found in negative control cells. By contrast, (77.9 ± 6.2)%, (75.3 ± 6.8)%, (59.5 ± 4.9)%, (67.4 ± 5.1)% methylated CpG were observed in cells treated with NiS for three times at dosage of 0.25, 0.50, 1.00 and 2.00 µg/cm(2) which were abbreviated as NiS0.25, NiS0.50, NiS1.00, NiS2.00 respectively. The ANOVA analysis results showed that there was a significant difference in the 5 groups above (F = 124.95, P < 0.01). The results of Dunnett-t test showed that the methylated CpG of both group NiS1.00 and NiS2.00 were significantly decreased compared with the negative control group (t values were 7.64, 4.89 respectively, P < 0.01). For methylated CpG, (46.2 ± 4.1)% and (43.6% ± 4.3)% were observed in NiS-transformed cells (NSTC1 and NSTC2) which were dramatically decreased compared with the negative control group (t values were 12.79, 13.56 respectively, P < 0.01).</p><p><b>CONCLUSION</b>Genomic DNA methylation levels were decreased during NiS induced malignant transformation.</p>

Relationship between polymerase eta expression and DNA damage-tolerance in human hepatic cells by hydroquinone / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of hydroquinone (HQ) on expression of Polymerase eta (Pol eta) and DNA damage in human hepatic cells (L-02), and to explore the role and possible mechanism of Pol eta involved in the process of DNA damage-tolerance.</p><p><b>METHODS</b>After L-02 hepatic cells were exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L) for 24 h, cell survival rate was detected by MTT assay; DNA impairment was detected by single cell gel electrophoresis (SCGE); Real-time fluorescent quantitative PCR and Western blotting methods were used to measure the expression of Pol eta at the mRNA and protein level in L-02 hepatic cells exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L).</p><p><b>RESULTS</b>MTT assay showed that HQ with concentrations from 0 to 80 micromol/L had little effect on the survival rate of L-02 (P>0.05); whereas the survival rate of the group of 160 micromol/Lwas significantly higher than that of the control (P<0.01) after being treated with HQ for 24 h; the higher dose of HQ presented, the more degrees of DNA damage were produced. It was found that HQ in a low concentration (1-80 micromol/L) could induce the expression of Pol eta which was in proportion to the increasements of HQ concentration; the expression levels of mRNA and protein were reached to the maximum when treated with 80 micromol/L; the expression of Pol eta decreased (the relative quantity values were 2.32 +/- 0.16 and 1.20 respectively) once the concentration of HQ exceeded 160 micromol/L as compared with the group of 80 micromol/L, but it was higher than that of the control.</p><p><b>CONCLUSION</b>This study suggested that Pol eta might involve in the process of DNA damage-tolerance induced by HQ in the hepatic cells.</p>

Effect of hydroquinone on expression of ubiquitin-ligating enzyme Rad18 in human L-02 hepatic cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of hydroquinone (HQ) on expression of ubiquitin-ligating enzyme Rad18 in human hepatic cells (L-02), and to explore the role and possible mechanism of Rad18 involved in toxicity of HQ to hepatic cells.</p><p><b>METHODS</b>After L-02 hepatic cells were exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L) for 24 h, cell survival rate was measured by MTT assay; DNA impairment was evaluated by single cell gel electrophoresis (SCGE); The expression levels of Rad18 mRNA and protein were detected by Real-time fluorescent quantitative polymerase chain reaction (QPCR) technique and Western blot method respectively.</p><p><b>RESULTS</b>HQ with concentration from 0 to 80 micromol/L had little effect on survival rate of L-02 (P > 0.05); Whereas the survival rate in the group of 160 micromol/L was significantly lower than in the control with the significant difference (P < 0.01) after treated with HQ for 24 h; The higher dose of HQ presented, the more degrees of olive tail moment (OTM) were produced and a dose-dependent relationship was shown. HQ in a low concentration (0 to approximately 40 micromol/L) could induce increase in the expression of Rad18 mRNA and protein which was in proportion to the increment of HQ concentration; the expression of Rad18 mRNA was enhanced increasingly, while the expression of Rad18 protein unchanged basically once the concentration of HQ exceeded 40 micromol/L; Besides, there was a positive correlation between OTM and the expression level of Rad18 mRNA (r = 0.919, P < 0.01).</p><p><b>CONCLUSION</b>HQ could regulate up the expression of Rad18 in L-02 hepatic cells.</p>

Breast cancer resistance protein expression and 5-fluorouracil resistance / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To filtrate breast cancer resistance protein (BCRP)-mediated resistant agents and to investigate clinical relationship between BCRP expression and drug resistance.</p><p><b>METHODS</b>MTT assay was performed to filtrate BCRP-mediated resistant agents with BCRP expression cell model and to detect chemosensitivity of breast cancer tissue specimens to these agents. A high performance liquid chromatography (HPLC) assay was established, and was used to measure the relative dose of intracellular retention resistant agents. RT-PCR and immunohistochemistry (IHC) were employed to investigate the BCRP expression in breast cancer tissue specimens.</p><p><b>RESULTS</b>MTT assay showed that the expression of BCRP increased with the increasing resistance of 5-fluorouracil (5-Fu) (P<0.05, n=3) in the cell model, while HPLC assay indicated that the intracellular retention dose of 5-Fu was significantly correlated with the expression of BCRP (r=-0.897, P<0.05, n=3). A total of 140 breast cancer tissue specimens were collected. BCRP-positive expression was detected in forty-seven specimens by both RT-PCR and IHC. As shown by MTT assay subsequently, the resistance index (RI) of 47 BCRP-positive breast cancer tissue specimens to 5-Fu was 7-12 times as high as that of adjacent normal tissue samples. BCRP expression was related to 5-Fu resistance (R2=0.8124, P<0.01).</p><p><b>CONCLUSION</b>Resistance to 5-Fu can be mediated by BCRP. Clinical chemotherapy for breast cancer patients can be optimized based on BCRP-positive expression.</p>

Study on the relationship between breast cancer resistance protein expression and 5-fluorouracil resistance / 中华预防医学杂志

<p><b>OBJECTIVE</b>To screen breast cancer resistance protein BCRP-mediated resistance agents and to investigate the relations between BCRP expression and drug resistance.</p><p><b>METHODS</b>MT assay was performed to screen BCRP-mediated resistant agents with established BCRP expression cell model. While, the high performance liquid chromatography (HPLC) assay was administrated to measure the related dosage of intracellular retention resistant agents. The BCRP expression was investigated by both real-time RT-PCR and immunohistochemistry (IHC) assay in 140 clinical breast cancer tissue specimens. Chemosensitivity to resistant agents for clinical breast cancer tissue specimens was analyzed by MT assay. The Nonparametric variance statistics method was used to analyze the correlations between clinical breast cancer tissue of BCRP expression and drug resistance.</p><p><b>RESULTS</b>MT assay showed that increasing resistance of 5-fluorouracil (5-Fu) climbed with the increases of the BCRP expressions by 10.58 times (P < 0.05, n = 3) in cell model. HPLC assay also proved that a significant negative correlation between the intracellular retention dose of 5-Fu with different expression of BCRP (r = -0.897, P < 0.05, n = 3). Forty-seven tissue specimens of BCRP-positive expression were rapidly determined by using both real-time RT-PCR and IHC in 140 clinical breast cancer tissue specimens. Subsequently, the resistance index (RI) for 47 BCRP-positive clinical breast cancer tissues to 5-Fu was shown from 7 to 12 times compared with normal cancer-side tissues through MT assay. The statistical correlation between BCRP expression and 5-Fu resistance was observed in clinical breast cancer tissue specimens (R2 = 0.8124, P < 0.01).</p><p><b>CONCLUSION</b>This study results showed that there is a significant relationship between BCRP expression and 5-Fu resistance. Moreover, the results suggest that the chemotherapy scheme could be optimized on BCRP-positive expression breast cancer patients.</p>

Possible role of DNA polymerase beta in protecting human bronchial epithelial cells against cytotoxicity of hydroquinone / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.</p><p><b>METHODS</b>DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone (ranged from 10 micromol/L to 120 micromol/L) for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis (SCGE)] were performed respectively to detect the toxicity of hydroquinone.</p><p><b>RESULTS</b>MTT assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups.</p><p><b>CONCLUSIONS</b>Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.</p>

Differential gene expressions in cells with low-dose hydrogen peroxide-induced adaptive response: a study with FDDRT-PCR / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify differentially expressed genes in human embryo lung fibroblasts MRC-5 with adaptive response induced by low concentration of hydrogen peroxide (H(2)O(2)) using fluorescent differential display-RT-PCR (FDDRT-PCR).</p><p><b>METHODS</b>The dose-effect pattern of H2O2 toxicity was determined using MTT assay, and the dose of 0.088, 0.88, 8.8, 88 micro;mol/L was defined as the low concentration, and 1100 micromol/L as the high concentration. Adaptive response model was established in MRC-5 cells verified using LDH release and cell apoptosis analyses. Differentially expressed genes in the cells with exposure to different doses of H(2)O(2) were detected by FDDRT-PCR, and some of the differentially displayed genes were determined using real-time quantitative PCR.</p><p><b>RESULTS</b>Cells challenged with high-concentration H(2)O(2) for 1 h after H(2)O(2) pretreatment at low concentrations for 24 h resulted in lessened toxic effect in comparison with direct high-concentration H(2)O(2) exposure. The adaptive response of the cells was most obvious with H(2)O(2) pretreatment at 0.88 micromol/L. Altogether 60 differentially expressed genes were detected with FDDRT-PCR in different treatment groups, and 5 of them were identified and verified, including 1 unknown gene and 4 known genes (bcl-2, EIF3S5, NDUFS4 and RPS10).</p><p><b>CONCLUSION</b>According to the results of FDDRT-PCR, the genes bcl-2, EIF3S5, NDUFS4 and RPS10 can be involved in H(2)O(2)-induced adaptive response of the MRC-5 cells.</p>

Rapid simultaneous detection of Salmonella and Shigella using modified molecular beacons and real-time PCR / 中华流行病学杂志

<p><b>OBJECTIVE</b>Dual detection of Salmonella and Shigella using modified molecular beacons and real-time PCR was developed. The established method was applied to rapid diagnosis of Salmonella and Shigella' food poisoning, and for routine monitoring programs.</p><p><b>METHODS</b>Two sets of primers were designed based on the core sequence of invA gene and ssaR gene published on GenBank to detect Salmonella, and ipaH gene were selected to detect Shigella. Three corresponding modified molecular beacons labeled with different fluorophors were designed. The molecular beacons and primer sets were tested against numerous strains from 55 different bacterial species. Then the two assays were combined to establish the dual real-time PCR assay, and were applied to the food poisoning diagnosis and surveillance.</p><p><b>RESULTS</b>For the modified molecular beacons-based dual real-time PCR assay, the sensitivity achieved was 69-93 fg/microl, 32-64 CFU/ml or 1-2 CFU/PCR reaction. There was no cross-reaction with other bacteria served as control. The dual real-time PCR assay was used to detect 134 Salmonella strains and 67 Shigella strains but no false signals were observed. 1100 food poisoning samples were tested with 569 Salmonella and 42 were Shigella identified by real time PCR. Among the positive samples, 551 were detected Salmonella and 41 were Shigella by traditional culture method. The overall test could be finished within 2 hours to one day starting from sample preparation.</p><p><b>CONCLUSION</b>The modified molecular beacons-based dual real-time PCR assay was rapid, sensitive, and specific. It could be applied to the rapid diagnosis of Salmonella and Shigella' food poisoning.</p>

Immune responses to trichloroethylene and skin gene expression profiles in Sprague Dawley rats / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To characterize the immune reaction in SD rats exposed to trichloroethylene (TCE) and to identify the gene expression profiles involved in skin after TCE exposure.</p><p><b>METHODS</b>Fifteen percent of TCE was injected intradermally into the rat back (100 microL/120 g) at intervals of 7 days. Whole blood was collected 24 h after the fifth or seventh intradermic administration of TCE. The percentages of CD4+ and CD8+ of T lymphocytes were measured by a flow cytometer. The concentrations of IFN-gamma and IL-4 in the serum were semi-quantified by ELISA. Total RNAs of skin samples at 3 h or 24 h after the seventh dose of TCE in SD rats were extracted, and gene expression profiles of these tissues were analyszed by rat toxicology U34 array of Affymetrix.</p><p><b>RESULTS</b>Obvious decline of CD4+ in T lymphocytes was observed in the TCE-administer group. No significant concentration differences in IFN-gamma and IL-4 were found between TCE-treated and control rats. Gadd45a and Mel were significantly up regulated in skin tissue 24 h after TCE exposure. The expression regulation of immune response factors was as active as proteins associated with lipid metabolism and synthesis process in these skin samples of SD rats exposed to TCE.</p><p><b>CONCLUSION</b>T-helper type 1 cells mediate immune response can not be elicited in TCE-treated SD rats, but certain immune disorder can be induced.</p>

Differentially express genes in human embryonic lung fibroblasts with damage tolerance induced by low-dose hydroquinone / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the differentially expressed genes in human embryonic lung fibroblasts (HELF) induced by small-dose hydroquinone (HQ) using fluorescence differential display-PCR (DD-PCR).</p><p><b>METHODS</b>According to the dose-effect relation of HQ toxicity we established previously, HQ dose that did not induce observed cell damage or proliferation arrest was defined as low dose (100 pmol/L), and that causing obvious cell damage as the high dose (100 micrommol/L). The cells were then treated with low or high dose of HQ, or exposed to high-dose HQ following pretreatment with low-dose HQ for some time, respectively. Fluorescence DD-PCR was performed and 33 differentially expressed genes were identified in the cells with different treatments, and 8 of the identified genes were amplified, cloned, sequenced and blasted.</p><p><b>RESULTS</b>Seven of the 8 amplified genes were unknown genes, and the left one was identified as a known gene highly homologous to that encoding Homo sapiens Rap1 interacting factor 1 (RIF1).</p><p><b>CONCLUSION</b>Low-dose HQ can induce damage tolerance in HELF, and identification of the differentially expressed genes may provide valuable sight into the mechanism of HQ-induced damage tolerance.</p>

hPARP1 genetic polymorphism in southern Chinese Han and Miao populations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study hPARP1 genetic polymorphism in southern Chinese Han and Miao populations.</p><p><b>METHODS</b>Blood samples from 187 and 210 southern healthy Han and Miao populations were collected. The mutations of exons 12,13,16 and 17 of hPARP1 gene were investigated by PCR-single-strand conformation polymorphism(SSCP).</p><p><b>RESULTS</b>Fragments of 253 bp,313 bp,175 bp,362 bp within exons 12,13,16 and 17 respectively of hPARP1 gene were amplified by multiple PCR. An SSCP variant in exons 12,13,16 and 17 of PARP1 gene in 187 healthy Han and 210 healthy Miao individuals was identified. Seven single-base substitutions compared with the sequence of PARP1 gene were identified: a T to C transition in exon 12 (Phe548Ser), a G to T transition in exon 13 (Ala683Ser), a G to T transition in exon 16 (Asp798Tyr), and a A to G transition in exon 17 (His808Arg).</p><p><b>CONCLUSION</b>There were polymorphism sites in exons 12,13,16,17 of hPARP1 gene in southern Chinese Han and Miao populations; these results may be useful for the establishment of PARP1 genotyping, and these newly described PARP1 alleles would be advantageous indicators for population studies.</p>

Effects of overexpression of human pol-beta on cellular response to DNA damage / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the biological effects of overexpression of the human DNA polymerase (pol-beta) on cellular response to DNA damage.</p><p><b>METHODS</b>The cell strain HLFbeta from the stable overexpression of the human pol-beta was contaminated with methyl methanesulfonate (MMS) for investigating the effects of the pol-beta on the cellular responses to DNA damage on the aspects such as the DNA damage, the cell cycle and the induced mutation rate.</p><p><b>RESULTS</b>The cell HLFbeta from the stable overexpression of the human pol-beta was obtained through the screening. The cellular response to DNA damage of HLFbeta induced by the MMS in the intermediate and high dosage group (ranging from 0.5 to 0.8 mmol/L) was significantly lower than that in the control group. The analysis for the cell cycle distribution showed that both the two types of cells contaminated by MMS had retardation at G(2) phase. In the HLFbeta group, the cells had the obvious G(2) phase retardation and 49.0% of the cells were retarded at G(1) phase as well when the MMS was increased to 0.5 mmol/L while in the control, only 20.1% of the cells were retarded at the G(1) phase when the same dosage of MMS was administered. Moreover, the MMS-induced mutagenesis in HLFbeta was increased from 4.5 x 10(-6) to 8.2 x 10(-6), significantly higher than that in the control group (P < 0.05).</p><p><b>CONCLUSION</b>High Pol-beta level decreases cellular DNA damage induced by MMS. Nevertheless, the overexpression of Pol-beta can also increase error-prone DNA synthesis during DNA repair process.</p>

Low concentration of hydroquinone-induced adaptive response in hPARP-1 protein normal and deficient cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate whether or not adaptive response of hPARP-1 protein normal and deficient cells is induced by low dose of hydroquinone (HQ), and to analyze the relationship between the adaptive response and micronuclei formation, and cell cycle alteration in human embryo lung fibroblasts (HLF), so as to elucidate the mechanism of adaptive response.</p><p><b>METHODS</b>HLF, HLFC and HLFP cells pretreated with low concentration were retreated by high concentration of HQ. Cellular viability, the rate of micronuclei and abnormal nuclei, cell cycle and DNA strand break were determined.</p><p><b>RESULTS</b>The tolerance to 80.0 micromol/L concentration of HQ was enhanced when HLF, HLFC and HLFP cells were pretreated with HQ from 0.001 - 0.050 micromol/L. There were varying degrees of micronuclei and abnormal nuclei in three cells pretreated with low concentration of HQ and then retreated with high concentration of HQ; the cell numbers of G1, G2, S phase in cell cycle were obviously different. When compared with only high attack dose, the micronuclei rate and abnormal nuclei rate of HLF, HLFC and HLFP decreased by pretreatment with HQ at high concentration (P < 0.05), meanwhile increased by pretreatment with HQ at low concentration (P < 0.05). HLF, HLFC and HLFP showed blockage in G2 phase when pretreated with HQ at 0 approximately 0.05 micromol/L, but HLFP showed blockage in G1 phase, and in S phase at 1.0 and 2.0 micromol/L.</p><p><b>CONCLUSION</b>The level of adaptive response of hPARP-1 protein deficient cells was lower than normal cell, suggesting that hPARP-1 protein may play an important role in the adaptive response of cells, which may be related with the regulation of cell cycle.</p>

Study on differential proteomic expression in human liver cells stimulated by trichloroethylene with proteomics / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the differential proteomic expression in human liver cells L-02 induced by different dosages of trichloroethylene (TCE).</p><p><b>METHODS</b>Human liver cells L-02 were treated with different concentrations of TCE and the solvent control (dimethylsulfoxide). The total cellular proteins were separated using 2DE and visualized with silver staining after TCE treatment. The images were analyzed with Image Master 2D Platinum 5.0 analysis software. The differentially expressed protein spots were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS).</p><p><b>RESULTS</b>Fifteen protein spots with significant difference were found, and went upward or downward or disappeared after the stimulation of TCE with different dosages, which indicated that TCE induced the change of the proteomic expression in the liver cells. The mass spectrum identification and the IPI human database retrieval were used for identifying 9 proteins related to the L-02 Liver cells induced by TCE.</p><p><b>CONCLUSION</b>The result provides an insight to TCE-related molecular mechanism and which might be useful for further study of the TCE-associated proteins and molecular markers.</p>

Two-dimensional electrophoresis and mass spectrometry analysis on effects of trichloroethylene on protein of L-02 liver cells / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the effects of trichloroethylene (TCE) on the protein in L-02 cells in vitro.</p><p><b>METHODS</b>Thiazolyl blue and Trypan blue tests were used to investigate the cytotoxicity of TCE to L-02 liver cell. The 2-D electrophoresis was used to analyse the expression of proteins in L-02 liver cells. The differentially expressed protein spots were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS).</p><p><b>RESULTS</b>When the concentration of TCE exceeded 30 micromol/L, there was distinct cytotoxicity to L-02 cell (P < 0.05). Selected 40 micromol/L to treat L-02 liver cells and analyze the differential proteome expression, the results showed that the expression level of 37 protein spots was up-regulated and 15 protein spots was down-regulated. And 15 proteins were identified by MALDI-TOF-TOF-MS.</p><p><b>CONCLUSION</b>TCE can change the proteome expression of L-02 liver cell. It should provide the fundamental information to identify proteins related to TCE in further study.</p>