Interference of DEHP on the key genes of glucose and lipid metabolism in HepG2 cells / 中国职业医学

OBJECTIVE: To investigate the effects of di-( 2-ethylhexyl) phthalate( DEHP) on the expression of the key genes involved in glucose and lipid metabolism,and explore the toxicity of DEHP on the glucose and lipid metabolism in HepG2 cells cultured in vitro. METHODS: HepG2 cells in logarithmic growth phase were divided into DEHP exposure group and control group. The exposure group was exposed to DEHP with different final concentrations( 5,10,50,100,500 and1 000 μmol / L),and the control group was exposed to dimethyl sulfoxide of corresponding concentrations. After 24 hours of DEHP exposure,real-time fluorescence quantitative polymerase chain reaction( Q-PCR) was applied to detect the level of mRNA transcription of peroxisome proliferators-activated receptor α( PPARα), which is an endogenous marker indicating the success of DEHP exposure. In addition,the level of mRNA transcription of key genes involved in glucose and lipid metabolism were also measured by Q-PCR,including glucose-6-phosphatase( G-6-Pase),phosphoenolpyruvate carboxykinase( PEPCK),stearoyl-coenzyme A desaturase 1( SCD1),fatty acid synthase,sterol regulatory elementbinding protein 1c and acetyl Co A carboxylase 1. P ≤0. 008 was considered as statistical significance. RESULTS: After DEHP exposure,the mRNA transcription level of PPARα was significantly elevated in all exposure groups( P < 0. 008)except for 5 μmol / L DEHP exposure group,which indicated the successful establishment of DEHP exposure model. The mRNA transcription level of G-6-Pase was significantly increased in 100 and 500 μmol / L DEHP exposure groups( P ≤0. 008) when compared with the controls; the PEPCK mRNA transcription level of showed no significant differences between the 6 DEHP exposure groups and their corresponding control groups( P > 0. 008). The mRNA transcription level of SCD1 was significantly down-regulated in 100 μmol / L DEHP exposure group( P < 0. 008) when compared with its control. The mRNA transcription level of other key genes involved in the lipid metabolism were not significantly altered after DEHP exposure( P > 0. 008). CONCLUSION: The effect of DEHP on glucose metabolism was mainly manifested by promoting G-6-Pase gene expression,which is the rate-limiting enzyme for gluconeogenesis. The effect of DEHP on the lipid metabolism of HepG2 cells was limited.

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>

Study on mRNA expression of immune-related genes in patients with allergic dermatitis induced by trichloroethylene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study mRNA expression of immune-related genes (Foxp3, GATA3, CTLA4 and T-bet) in peripheral blood of the patients with allergic dermatitis induced by trichloroethylene (TCE).</p><p><b>METHODS</b>The peripheral blood samples were collected from 8 healthy workers (control group) and 8 patients with allergic dermatitis induced by TCE (case group). Real-time quantitative PCR was applied to detect mRNA expression of immune-related genes (Foxp3, GATA3, CTLA4, T-bet).</p><p><b>RESULTS</b>The mRNA expression levels of Foxp3, GATA3 and CTLA4 genes increased by 115%, 97% and 241% in case group, as compared with control group (P < 0.01). The mRNA expression level of T-bet gene decreased by 47% in case group, as compared with control group (P < 0.01).</p><p><b>CONCLUSION</b>The mRNA expression levels of some immune-related genes changed in patients with allergic dermatitis induced by TCE, those genes may play an important role in TCE-induced allergy.</p>

Pathological analysis and mRNA expression of apoptosis genes in rat kidney tissue after subacute melamine treatment / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>to investigate pathological changes and mRNA expression of apoptosis genes bcl-2, bax and Caspase-3 in rat kidney tissue after rats are administrated with melamine for 28 days.</p><p><b>METHODS</b>10 male SD rats and 10 female SD rats in each group were administrated with three doses of melamine (low dose, middle dose, high dose) by gavage for 28 days. The animals were divided into three experimental groups and one control group. The doses for male rats were 200, 400, 800 mg/kg, but for female rats they were 150, 300, 600 mg/kg. After melamine treatment the animals were sacrificed and the kidneys were taken out for pathological analysis and for detecting mRNA expression of bcl-2, bax and Caspase-3 with fluorescent quantitative PCR assay.</p><p><b>RESULTS</b>the tubular cylinders were observed in three experimental groups. The positive rates of tubular cylinders in three groups (from low dose to high dose) were 11/20, 13/20, 16/20, respectively. Additionally, melamine induced a significant decrease in mRNA expression of bcl-2 at low dose, middle dose or high dose, bcl-2 expression decreased by 20.58% - 49.51% in three groups treated with melamine. Furthermore, bax mRNA levels increased by 44.66% - 300.96% in groups treated with three doses of melamine, and Caspase-3 mRNA levels increased by 64.76% - 360.75% in groups treated with three doses of melamine.</p><p><b>CONCLUSIONS</b>melamine could induce pathological changes of rat kidneys, and it also induces a significant alteration of apoptosis Bcl-2, Bax and Caspase-3 mRNA expression in rat kidney tissue.</p>

Association of cooking oil fumes exposure and oxidative DNA damage among occupational exposed populations / 中华劳动卫生职业病杂志

<p><b>BACKGROUND</b>Previous investigations indicate that cooks are exposed to polycyclic aromatic hydrocarbons (PAH) from cooking oil fumes (COF). However, Emission of PAH and their carcinogenic potencies from cooking oil fumes sources have not been investigated among cooks.</p><p><b>AIMS</b>To investigate the urinary excretion of a marker for oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG), in different groups of cooks and different exposure groups, and to study the association between 8-OHdG and 1-hydroxypyrene (1-OHP), a biological marker for PAH exposure.</p><p><b>METHODS</b>Urine samples were collected from different groups of cooks (n = 86) and from unexposed controls (n = 36), all are male with similar age and smoking habits. The health status, occupational history, smoking, and alcohol consumption 24 hours prior to sampling was estimated from questionnaires. The urinary samples were frozen for later analyses of 8-OHdG and 1-OHP by high performance liquid chromatography.</p><p><b>RESULTS</b>Excretion in urine of 8-OHdG were similar for controls (mean 1.2 µmol/mol creatinine, n = 36), and for those who had been in the kitchen room with exhaust hood operation (mean 1.5 µmol/mol creatinine, n = 45). COF exposed cooks without exhaust hood operation had increased excretion of 8-OHdG (mean 2.3 µmol/mol creatinine, n = 18). The difference between this group and the unexposed controls was significant. The urinary levels of ln 1-OHP and ln 8-OHdG were still significantly correlated in a multiple regression analysis.</p><p><b>CONCLUSION</b>Results indicate that exposure to PAH or possibly other compounds in COF may cause oxidative DNA damage.</p>

Effect of cadmium chloride on the expression and phosphorylation of mitogen-activated protein kinase in normal rat kidney epithelial cells / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the effect of cadmium chloride on the expression and phosphorylation of mitogen-activated protein kinase (MAPK) in normal rat kidney epithelial (NRK) cells.</p><p><b>METHODS</b>The NRK cells were incubated with cadmium chloride either at respective dose (0, 1, 5, 10, 20, 40 µmol/L) for 24 h or at same dose (10 µmol/L) for respective time (0, 0.5, 1.0, 2.0, 4.0, 8.0 h). Western blotting was applied to test the expression of MAPK in NRK cells (ERK1/2, p38, JNK); and phosphor-specific antibody to detect the phosphorylated MAPK (p-ERK1/2, p-p38, p-JNK).</p><p><b>RESULTS</b>There was no significant difference in the MAPK expression among the groups either treated with different doses or for different time; however, the level of phosphorylated MAPK was comparatively higher than it in control group. There was an obvious expression of p-ERK1/2 at 1.00 ± 0.06 in the group incubated with 10 µmol/L CdCl(2); and the expression in the 20 µmol/L and 40 µmol/L CdCl(2) group was 2.58 ± 0.11, 2.76 ± 0.23 respectively, which was 1.58 and 1.76 times more than it in 10 µmol/L CdCl(2) group. The differences were statistically significant (F = 827.70, P < 0.01). The respective expression of p-p38MAPK in the 20 µmol/L (2.47 ± 0.20)and 40 µmol/L CdCl(2) group (3.73 ± 0.25)was 1.47 and 2.73 times more than it in control group (1.00 ± 0.02), and the differences were also statistically significant (F = 280.06, P < 0.01). There was a dose-effect relationship of the concentration of cadmium in the expression of p-ERK1/2 (r = 0.919, t = 4.69, P = 0.009) and p-p38MAPK (r = 0.945, t = 5.79, P = 0.004). Additionally, phosphorylated MAPK expressed in a time-dependent manner. The expression of p-ERK1/2 was obvious in the group incubated for 1 h (1.26 ± 0.11), and the respective expression in the 4 h group (1.51 ± 0.07) and 8 h group (3.53 ± 0.23) was 1.5 and 3.5 times of it in the control group (1.00 ± 0.02). The differences were statistically significant (F = 427.82, P < 0.001). The expression of p-p38MAPK increased significantly in 1 h group (1.31 ± 0.07); while the respective expression in 4 h group (3.53 ± 0.32) and 8 h group (4.41 ± 0.38) was 3.5 and 4.4 times of it in control group (1.00 ± 0.03). The differences were also statistically significant (F = 280.06, P < 0.001).</p><p><b>CONCLUSION</b>Cadmium chloride could significantly enhance the phosphorylation of MAPK in NRK cells; and it is probably associated with the activation of MAPK.</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>

Study on skin sensitization as well as liver and kidney impairment in guinea pigs treated with trichloroethylene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study skin sensitization as well as liver and kidney impairment in guinea pigs treated with trichloroethylene (TCE).</p><p><b>METHODS</b>Guinea pig maximization test (GPMT) was applied in this study, guinea pigs were divided into 3 groups, namely negative control, positive control and TCE treatment. Animals of 3 groups were administrated with olive oil, 2, 4-dinitrochlorobenzene (DNCB), and TCE, respectively, by intradermal injection. The animal skin was observed and blood was collected after various treatment, the liver function tests were conducted, including detection of activities of ALT, AST, LDH and levels of creatinine, uric acid, and urea with automatic biochemical analyzer.</p><p><b>RESULTS</b>Obvious skin impairment was observed in the groups of positive control and TCE treatment, the skin impairment included erythema and edema, the sensitization rate was 100% in positive control and 83.3% in TCE treatment group. Additionally, the activities of ALT, AST and LDH increased significantly in the groups of positive control and TCE treatment when compared with the negative control.</p><p><b>CONCLUSIONS</b>Trichloroethylene is one of the strong hypersensitizing substances, it could induce skin allergic reaction and liver impairment in guinea pigs.</p>

Effects of hydroquinone on expression of human 8-oxo-guanine DNA glycosylase mRNA in human A549 lung adenocarcinoma cell strains / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the effects of hydroquinone (HQ) on reactive oxygen species (ROS) generation, antioxydase activities and the expression of human 8-oxo-guanine DNA glycosylase (hOGG1) mRNA in human A549 lung adenocarcinoma cell strains.</p><p><b>METHODS</b>A549 cells were treated with different concentrations of HQ. Cell survival was determined by methyl thiazolyl tetrazolium (MTT). Changes of ROS were detected by fluorescent probe. The contents of malonaldehyde and activities of antioxydase were determined through colorimetry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the level of hOGG1 mRNA.</p><p><b>RESULTS</b>With the increased concentration of HQ, the findings were as follows. (1) The absorbance value of A549 cell decreased. There was significant difference between 160 micromol/L (0.584+/-0.098) and 320 micromol/L (0.328+/-0.066) of HQ (q=5.56 and 9.07, P<0.05) with the control group (0.989+/-0.150), and the cell survival rate were less than 80%. (2) The ROS in A549 cell increased. 40 micromol/L (39.80+/-4.15) and 80 micromol/L (101.99+/-9.45) had statistical significance (q=10.74 and 30.32, P<0.05) with the control group (5.71+/-0.50). (3) It was found that the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) decreased and malonaldehyde (MDA) increased. Compared with the control group [(25.62+/-0.28) U/mg prot and (38.97+/-2.61) U/mg prot], the activities of SOD and GSH-Px had a significant decrease (q=12.17 and 8.78, P<0.05) in 80 micromol/L [(22.93+/-0.56) U/mg prot and (25.60+/-2.31) U/mg prot]. And MDA had a significant increase (q=10.90 and 15.49, P<0.05) in 40 micromol/L [(1.07+/-0.01) nmol/mg prot] and 80 micromol/L [(1.19+/-0.08) nmol/mg prot] as compared with the control group [(0.77+/-0.04) nmol/mg prot]. The decrease of SOD (r=-0.95, F=20.00, P=0.04) and GSH-Px activities (r=-0.99, F=115.48, P=0.01) and the increase of MDA contents (r=0.96, F=21.31, P=0.04) all had a dose-response relationship. (4) RT-PCR results showed that the expression of hOGG1 mRNA decreased. The significant difference was observed between the expression of hOGG1 mRNA in 80 micromol/L (0.478+/-0.017) (q=11.70, P<0.05) with the control group (0.715+/-0.038).</p><p><b>CONCLUSION</b>This study suggests that HQ could induce oxidative damage and changes of the expression of hOGG1 mRNA in A549 cells.</p>

Molecular epidemiological study on oxidative DNA damage among Hasake ethnic migrants in Shenzhen / 中华流行病学杂志

objective To explore the relationship of migration and oxidative DNA damage by comparative study of oxidative DNA damage effects on people with difierent years of migration among Xinjiang Hasake ethnecity in Shenzhen.Methods Sixty Hasake residents in Shenzhen were selected,and were divided into three groups(n=20)according to the years of migration.Major changes of their life style were investigated.8-hydroxy-2'-deoxyguanosine(8-OH-dG)levels in urine were analyzed,and comet assay of peripheral blood lymphocytes conducted.Results When comparing with the group having a shorter than 1 year of stay,a significant decrease of oliveive tail moment and tail/head length in comet assay in the＞3 years group(P＜0.05)was observed 8-OH-dG level decreased significantly in 1-3 years group (P＜0.05)and＞3 years group(P＜0.01).Conclusion Our results suggested that life style changes which related to migration might reduce DNA damage in Hasake nationalities.

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>