[Role of poly (ADP-ribose) polymerase 1 on DNA methylation variation induced by B(a)P in human bronchial epithelial cell].

OBJECTIVE: To investigate DNA methylation variation in human cells induces by B(a)P, and to explore the role of PARP1 during this process. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[The effect of DNA methyltransferase 1 low expression on the global genome DNA methylation status of 16HBE cell].

OBJECTIVE: To construct DNA methyltransferase 1 (DNMT1) low expression 16HBE cell line and observe the variation of cell cycle and global genomic DNA methylation. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Genome DNA hypomethylation in the process of crystalline nickel-induced cell malignant transformation].

OBJECTIVE: To observe the effect of crystalline NiS on genome DNA methylation profile in in vitro cultured cells. METHODS: 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. RESULTS: 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). CONCLUSION: Genomic DNA methylation levels were decreased during NiS induced malignant transformation.

An investigation of hormesis of trichloroethylene in L-02 liver cells by differential proteomic analysis.

Hormesis is the dose-response pattern of the biological responses to toxic chemicals, characterized by low-dose stimulation and high-dose inhibition. Although it is known that some cell types exhibit an adaptive response to low levels of cytotoxic agents, its molecular mechanism is still unclear and it has yet to be established whether this is a universal phenomenon that occurs in all cell types in response to exposure to every chemical. Trichloroethylene (TCE) is an organic solvent widely used and is released into the atmosphere from industrial degreasing operations. Acute (short-term) and chronic (long-term) inhalation exposure to trichloroethylene can affect the human health. In order to elucidate a cell-survival adaptive response of L-02 liver cells exposed to low dose of TCE, CCK-8 assay was used to assess cytotoxicity, and examined the possible mechanisms of hormesis by proteomics technology. We found that exposure of L-02 liver cells to low level of TCE resulted in adaptation to further exposure to higher level, about 1,000 protein-spots were obtained by two-dimensional electrophoresis (2-DE) and five protein spots were identified by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. Our results suggest that a relationship may exist between identified proteins and TCE-induced hormesis, which are very useful for further study of the mechanism and risk assessment of TCE.

Effect of PARP-1 deficiency on DNA damage and repair in human bronchial epithelial cells exposed to Benzo(a)pyrene.

Benzo[a]pyrene is a ubiquitously distributed environmental pollutant known to cause DNA damage, whereas PARP-1 is a nuclear enzyme that is activated by damaged DNA and plays an important role in base excision repair and genomic stability. Here, 16HBE and its PAPR1-deficient cells were exposed to BaP, and the DNA damage level and repair ability of both cell lines were measured by alkaline comet assay. The results showed that cell viability of both cell lines decreased in a dose-dependent manner when exposed to BaP, but there was no significant difference between two cell lines. Comet assay showed that BaP caused DNA damage in both cell lines at an obvious dose- and time-dependent manner. Compare with 16HBE, the PARP1-deficient cells were more sensitive to the damage caused by BaP. The results of DNA repair experiment showed that both cell lines can recover from the damage in a time-dependent pattern. The relative repair percentage of PARP1-deficient cells were generally lower than that of 16HBE at all exposed concentrations at the early stage of repair, but tended to be closer between two cell lines at the later period. According to results, we came to the conclusion that PARP1-deficient cells were more sensitive to BaP in contrast to normal 16HBE; DNA repair capacity in PARP1-deficient cells decreased significantly at the early stage of repair, but increased to the equivalent level of normal 16HBE in the later period. PARP-1 plays an important role in early repair of DNA damage caused by BaP in 16HBE notwithstanding the main repair work is taken by NER pathway.

Proteome of melamine urinary bladder stones and implication for stone formation.

Melamine can cause urinary stones related to nephropathy of the kidney and hyperplasia or carcinoma of the bladder, but the mechanism of stone formation is not well understood. In this study, male rats were administered melamine for thirteen weeks to establish melamine bladder stone models and the stones were analysed by Fourier transform infrared (FTIR) spectroscopy, powder X-ray diffraction (XRD), energy dispersive X-ray (EDX) spectroscopy, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and western blot, respectively, for the composition and proteome, and to explore the implication of proteins for stone formation. The results showed bladder stones were composed of predominant melamine and a few amount of proteins. The proteins had a wide range of molecular weights and 1051 proteins were identified. Gene Ontology (GO) classification of the identified proteins showed most proteins were from injured cells, involved in various metabolic processes and had binding functions. Of the identified proteins, there were a few inflammatory proteins and urinary proteins. Physicochemical characteristics of the identified proteins showed that 67.1% proteins' isoelectric points (pI) value was below 7.0, 91.1% proteins' grand average of hydropathicity (GRAVY) scores were below 0 and nearly half of the proteins were stable. Our data indicated proteins might play an important role in melamine bladder stone formation.