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Objective To investigate the feasibility of genotoxicity assessment for chemicals via flow cytometry (FCM) and high-content screening (HCS) based on high-throughput screening in vitro micronucleus assays. Methods In reference to the methodology of OECD TG487, the typical positive controls, cyclophosphamide (CP) and mitomycin C (MMC), were selected.And no serum MEM medium was treated as negative control.Dose range of CP was 5-20 mg/L and MMC was 0.25-1.0 mg/L.CHL cells were treated with three concentrations of each chemical for 4 h.High-throughput screening in vitro micronucleus assays based on FCM and HCS were established.The results of the frequency of micronuclei were compared to traditional cytokinesis blocking micronucleus assay in each group with or without metabolic activation. Results The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 1.9%, 7.6%, 10.4% and 5.9%, 11.4%, 16.7%, which were obtained by conventional microscopic scoring.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 2.8%, 2.6%, 7.8% and 3.2%, 3.7%, 5.1%, which were obtained by flow cytometry screening.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately2.8%, 6.2%, 9.1% and 7.9%, 10.1%, 10.2%, which were obtained by high-content screening.Compared with negative controls, the differences of the results were statistically significant(P < 0.05), and there was a dose-response relationship. Conclusion In this study, the results of high-throughput screening assays of FCM and HCS are in accordance to the results of traditional cytokinesis blocking micronucleus assay, indicating that high-throughput screening in vitro micronucleus assays could detect micronucleus formation automatically and improve the efficiency.Therefore, the method could provide data support for using high-throughput screening in vitro micronucleus assays into genotoxicity assessment of chemicals.
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Objective To investigate the permeability of brain microvascular endothelial cells under the condition of high glucose exposure. Methods The bEnd.3 cell line was chosen to detect the value of trans- endothelial electrical resistance (TEER), the activity of alkaline phosphatase (ALP) and γ-glutamyl transferase (γ-GT).Hence, the characteristics of blood-brain barrier in cell model were identified.The permeability of brain microvascular endothelial cells on high glucose exposure was evaluated by cell morphology, cell viability, intracellular lactate dehydrogenase activity and relative expression of ZO-1 and Occludin genes. Results The value of TEER, the activity of ALP and γ-GT increased gradually with increasing incubation time.The observation of cell morphology showed that the number of cells decreased significantly under high glucose exposure, and the adherence was unstable.Cell viability decreased with higher concentration of glucose or longer exposure time under high glucose exposure.The activity of lactate dehydrogenase was also decreased, and there were significant differences among the dose groups (P < 0.05).In addition, the expression levels of tight junction protein ZO-1 and Occludin were further detected.It was found that high glucose exposure inhibited the expression of ZO-1 and Occludin genes in a dose-dependent manner. Conclusion The bEnd.3 cell line has the characteristics of blood-brain barrier.High glucose exposure inhibited the expression of tight junction protein ZO-1 and Occludin. The results might be related to the change of the permeability in brain microvascular endothelial cells
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Objective To investigate the feasibility of genotoxicity assessment for chemicals via flow cytometry (FCM) and high-content screening (HCS) based on high-throughput screening in vitro micronucleus assays. Methods In reference to the methodology of OECD TG487, the typical positive controls, cyclophosphamide (CP) and mitomycin C (MMC), were selected.And no serum MEM medium was treated as negative control.Dose range of CP was 5-20 mg/L and MMC was 0.25-1.0 mg/L.CHL cells were treated with three concentrations of each chemical for 4 h.High-throughput screening in vitro micronucleus assays based on FCM and HCS were established.The results of the frequency of micronuclei were compared to traditional cytokinesis blocking micronucleus assay in each group with or without metabolic activation. Results The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 1.9%, 7.6%, 10.4% and 5.9%, 11.4%, 16.7%, which were obtained by conventional microscopic scoring.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately 2.8%, 2.6%, 7.8% and 3.2%, 3.7%, 5.1%, which were obtained by flow cytometry screening.The frequencies of micronuclei induced by CP and MMC (ascending rank) were separately2.8%, 6.2%, 9.1% and 7.9%, 10.1%, 10.2%, which were obtained by high-content screening.Compared with negative controls, the differences of the results were statistically significant(P < 0.05), and there was a dose-response relationship. Conclusion In this study, the results of high-throughput screening assays of FCM and HCS are in accordance to the results of traditional cytokinesis blocking micronucleus assay, indicating that high-throughput screening in vitro micronucleus assays could detect micronucleus formation automatically and improve the efficiency.Therefore, the method could provide data support for using high-throughput screening in vitro micronucleus assays into genotoxicity assessment of chemicals.
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Objective To investigate the permeability of brain microvascular endothelial cells under the condition of high glucose exposure. Methods The bEnd.3 cell line was chosen to detect the value of trans- endothelial electrical resistance (TEER), the activity of alkaline phosphatase (ALP) and γ-glutamyl transferase (γ-GT).Hence, the characteristics of blood-brain barrier in cell model were identified.The permeability of brain microvascular endothelial cells on high glucose exposure was evaluated by cell morphology, cell viability, intracellular lactate dehydrogenase activity and relative expression of ZO-1 and Occludin genes. Results The value of TEER, the activity of ALP and γ-GT increased gradually with increasing incubation time.The observation of cell morphology showed that the number of cells decreased significantly under high glucose exposure, and the adherence was unstable.Cell viability decreased with higher concentration of glucose or longer exposure time under high glucose exposure.The activity of lactate dehydrogenase was also decreased, and there were significant differences among the dose groups (P < 0.05).In addition, the expression levels of tight junction protein ZO-1 and Occludin were further detected.It was found that high glucose exposure inhibited the expression of ZO-1 and Occludin genes in a dose-dependent manner. Conclusion The bEnd.3 cell line has the characteristics of blood-brain barrier.High glucose exposure inhibited the expression of tight junction protein ZO-1 and Occludin. The results might be related to the change of the permeability in brain microvascular endothelial cells
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Objective We explored the stability of the bacteria strains used in the Ames test to provide a basis for determining the appropriate passage number at which the biological characteristics of the strains would not change. Methods The Salmonella typhimurium (TA97a, TA98, TA100 and TA102 strains) were selected as the experimental strains.The original frozen strains and frozen strains with different passage times were used to compare the biological characteristics and the spontaneously reverting colonies. Results The biological characteristics of four kinds of strains, which were histidine deficiency, lipidpolysaccharide barrier defect, ampicillin resistance, UV sensitivity, and tetracycline resistance, did not change at F1-F6 generation when compared with the F0 generation.However, as for the number of spontaneously reverting colonies, a statistically significant difference (P < 0.05) occurred at F3 generation when compared with F0 generation for the TA97a strain, and a significant difference (P < 0.05) occurred at F4 generation for TA100 and TA102 strains. Conclusion Passage number of strains used in Ames test could affect their spontaneous reversion mutation rate.The passage number should be less than 4 for TA98、TA100、TA102 strains, and less than 3 for TA97a in Ames test.
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<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>
Subject(s)
Humans , Benzo(a)pyrene , Cell Line , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases , Genetics , Metabolism , DNA Damage , DNA Methylation , Epithelial Cells , Metabolism , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases , Genetics , MetabolismABSTRACT
<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>
Subject(s)
Humans , Cell Cycle , Cell Line , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases , Genetics , Metabolism , DNA Methylation , Down-Regulation , Epithelial Cells , Metabolism , RNA Interference , RNA, Small Interfering , GeneticsABSTRACT
<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>