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Objective: To investigate the effect of poly-ADP-ribosylation in hexavalent chromium Cr(VI) induced cell damage., Methods: The study object, poly (ADP-ribose) glycohydrolase (PARG) deficient human bronchial epithelial cells (16HBE cells), was constructed previously by our research group. Normal 16HBE cells and PARG-deficient cells were treated with different doses of Cr (VI) for 24 h to compare the differences to Cr (VI) toxicity, meanwhile set up the solvent control group. On this basis, 5.0 µmol/L of Cr (VI) was selected as the exposure dose, after the exposure treatment, total proteins of both cells were extracted for two dimension fluorescence difference gel electrophoresis (2D-DIGE) separation, statistically significant differential protein spots were screened and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS/MS), and further validated by Western blot., Results: After Cr (VI) treatment, the survival rate of PARG-deficient cells was higher than normal 16HBE cells. When the doses reached up to 5.0 µmol/L, the survival rate of 16HBE cells and PARG-deficient cells were respectively (59.67 ± 6.43)% and (82.00 ± 6.25)%, the difference between which was significant (t = -4.32, P < 0.05). 18 protein spots were selected and successfully identified after 2D-DIGE comparison of differential proteins between normal 16HBE cells and PARG-deficient cells before and after exposure. The function of those proteins was involved in the maintenance of cell shape, energy metabolism, DNA damage repair and regulation of gene expression. The differential expression of cofilin-1 was successfully validated by Western blot. The expression level of cofilin-1 in the 16HBE cells increased after Cr (VI) exposure with the relative expression quantity of 1.41 ± 0.04 in treated group and 1.00 ± 0.01 in control group, the difference of which was statistically significant (t = -18.00, P < 0.05), while the expression level in PARG-deficient cells had no statistically significant difference (t = -8.61, P > 0.05)., Conclusion: Most of the identified differential proteins are closely related to tumorigenesis, suggesting that poly-ADP-ribosylation reaction may resist the cytotoxicity of Cr(VI) by inhibiting Cr (VI) induced tumorigenesis, which provides important reference data to clarify the mechanisms of poly-ADP-ribosylation in Cr (VI) induced cell damage.

Objective: To reveal the role of poly-ADP-ribosylation and DNA methylation in carcinogenic process induced induced by Cr (VI), and to discuss the relations between them., Methods: The pre-established Poly (ADP-ribose) glycohydrolase (PARG) deficient cells and 16HBE cells were treated with different concentrations of Cr (VI), and the changes of total genomic DNA methylation level in different groups were detected by methylation immunofluorescent detection, as well as the changes of the activity of methyltransferases. Moreover, RT-PCR and western blotting method were applied to analyze the changes of expression of DNMT1, DNMT3a, DNMT3b and MBD2, upon the protein level., Results: After treated by Cr(VI) for 24 h, the healthy 16HBE cells showed a significant lower level of genomic DNA methylation; however, there was no significant changes (P > 0.05) found in PARG deficient cells by immunofluorescence assay. When the dose of Cr (VI) reached 5.0 µmol/L, the activity of methyltransferases in 16HBE cells and PARG deficient cells (49.33 ± 2.65, 80.05 ± 2.05) decreased by 20% and 50% comparing with contrast group (99.27 ± 1.10, 99.30 ± 0.60) . After treated by Cr (VI) for 24 h, the expression of mRNA and protein level among DNMT1, DNMT3a, DNMT3b and MBD2 decreased significantly in healthy 16HBE cells; and the expression of DNMT1 and DNMT3a decreased in PARG deficiency cells. The relevant expression levels of mRNA of DNMT1 were separately (0.99 ± 0.09), (0.79 ± 0.10), (0.59 ± 0.13) and (0.39 ± 0.02) (F = 247.17, P < 0.01), the expression levels of protein were separately (1.00 ± 0.03), (0.69 ± 0.15), (0.65 ± 0.10) and (0.55 ± 0.13) (F = 214.12, P < 0.01), the expression levels of DNMT3a mRNA were separately (1.00 ± 0.04) , (0.93 ± 0.11) , (0.79 ± 0.07) , (0.59 ± 0.05) (F = 498.16, P < 0.01) , and the expression levels of protein were separately (1.00 ± 0.14) , (0.97 ± 0.11) , (0.79 ± 0.17) , (0.57 ± 0.15) (F = 390.11, P < 0.01) when the dose of Cr (VI) at 0, 0.3, 1.2 and 5.0 µmol/L. However, there were no significant changes of expression found in DNMT3b and MBD2., Conclusion: Poly-ADP-ribosylation could regulate the activity of DNMT3b and MBD2, protect cells against the DNA methylation alteration induced by Cr(VI) and maintain the global genomic DNA methylation level.

Objective: To study the effects of hydroquinone (HQ) on expression of ubiquitin-conjugating enzyme Rad6B in human L-02 hepatic cells., Methods: After L-02 hepatic cells were at the concentrations of 0, 5, 10, 20, 40, 80, and 160 micromol/L HQ for 24h, and the levels of Rad6B mRNA and protein expressions in L-02 hepatic cells were detected by real-time fluorescent quantitative polymerase chain reaction (QPCR) technique and Western blot method respectively., Results: It was found that HQ in the range of 0-160 micromol/L could induce increases in the expressions of Rad6B mRNA and protein, which was in a dose-dependent manner. The levels of Rad6B mRNA and protein expressions in the treated group of 5, 10, 20, 40, 80, and 160 micromol/L were higher than those in the control (P < 0.01), levels of Rad6B mRNA and protein expressions reached the maximun when L-02 hepatic cells were treated by HQ at the concentrations of 160 micromol/L, and the relative quantity values were 4.35 and 0.85, respectively., Conclusion: HQ could regulate up the expression of Rad6B in L-02 hepatic cells.

Objective: To study the effect of hydroquinone (HQ) on expression of XPV mRNA in L-02 hepatic cells and its regularity, in order to explore the possible molecular mechanism of XPV in the process of the toxic effects of HQ to L-02 hepatic cells., Methods: Expression of XPV mRNA in L-02 hepatic cells treated at the dose of 40 micromol/L HQ for 6, 12, 24 and 48h in vitro and its expression in L-02 hepatic cells treated at the concentrations of 0, 5, 10, 20, 40, 80 and 160 micromol/L HQ for 24h was measured by real-time fluorescence quantitative PCR., Results: In the range of 0-24h, the expressions of XPV mRNA in L-02 hepatic cells in treated group (40 micromol/L) and control group increased with increase of time, and expressions of XPV reached the maximum levels at the times of 24h,and reduced the minimum levels at the times of 48h. At the time ranges of 6, 12, 24 and 48h,the expression levels of XPV mRNA in L-02 hepatic cells treated with HQ (40 micromol/L) were more higher than those of the control (0 micromol/L). The expression level of XPV mRNA in L-02 hepatic cells treated by HQ at the concentration of 0-80 micromol/L for 24h increased in a dose-dependent manner.Taken the expression level of the control group as 1, the relative expression levels in various treatment groups were added to 1.20, 2.02, 2.37, 2.67, 4.40 and 2.32 fold respectively. The expressions of XPV mRNA in the group treated with HQ (160 micromol/L) was reduced, but was higher than that of the control., Conclusion: HQ could induce the expression of XPV at mRNA levels in a dose- and time-dependent manner, which indicated that XPV could involved in the response of L-02 hepatic cells of HQ.

Objective: To construct expressing vector of short hairpin RNA (shRNA) in order to inhibit human PARP1 activity., Methods: 2 pairs of 64 base oligos for hairpin RNA expression which targeted PARP1 gene were chemically synthesized and annealed then ligased with pSIREN-RetroQ vector with BamH II and EcoR I . Cut by EcoR t and Bgl II, shRNA and its upstream U6, which have 330 bp, were inserted into the same treated pEGFP-C1 vecter to construct GFP expression plasmids that inhibited hPARP1 protein shRNA plasmid (pEGFP-C1P). Oligos with a scrambled sequence were used as a negative control., Results: Recombinant pEGFP-C1P1, pEGFP-C1P2 and pEGFP-C1N vectors was identified by digestion with EcoR I and Bgl II and confirmed by sequencing analysis with U6 primer. The results demonstrated that the 330 bp had been inserted into the expected site. Furthermore, the insertion sequence was exactly correct., Conclusion: pEGFP-C1-shRNA system has been constructed successfully. This will facilitate the study of PARP1's DNA repairing function.

Objective: To clone the "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants (ECPs)., Methods: According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the "pSIREN-RetroQ-dsRNA" recombinant was obtained. After E. coli DH5alpha was transformed with the "pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoR I and Bgl II , the fragment of"pU6-dsRNA"was purified. And then the "pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of "pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing., Results: The "pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment., Conclusion: The "pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.