Association of NOS2 and NOS3 gene polymorphisms with susceptibility to type 2 diabetes mellitus and diabetic nephropathy in the Chinese Han population.

Inducible nitric oxide synthase (NOS2) and endothelial nitric oxide synthase (NOS3) gene play important roles in the susceptibility to type 2 diabetes mellitus (T2DM). The present study aims to detect the potential association of NOS2 and NOS3 gene polymorphisms with the susceptibility toT2DM and diabetic nephropathy (DN) in the Chinese Han population. Four hundred and ninety T2DM patients and 485 healthy controls were enrolled in this case-control study. The genotypes of NOS2 and NOS3 gene polymorphisms were analyzed by the polymerase chain reaction (PCR)-ligase detection reaction (LDR) method. Our data demonstrated that the NOS2 rs2779248 and NOS2 rs1137933 genetic polymorphisms were significantly associated with the increased susceptibility to T2DM in the heterozygote comparison, dominant model, and allele contrast; and NOS3 rs3918188 genetic polymorphism was significantly associated with the increased susceptibility to T2DM in the homozygote comparison and recessive model. The allele-C and genotype-TC of NOS2 rs2779248, allele-A and genotype-GA of NOS2 rs1137933 and genotype-AA of NOS3 rs3918188 genetic polymorphisms might be the risk factors for increasing the susceptibility to T2DM. And a significant haplotype effect of NOS2 rs10459953/C- rs1137933/G- rs2779248/T was found between T2DM cases and controls. Moreover, NOS3 rs1800783 polymorphism was significantly associated with the increased susceptibility to DN in the heterozygote comparison, recessive model and allele contrast. At last, a positive correlation of family history of diabetes with NOS3 rs11771443 polymorphism was found in DN. These preliminary findings indicate that the NOS2 rs2779248, NOS2 rs1137933, and NOS3 rs3918188 genetic polymorphisms are potentially related to the susceptibility to T2DM, and the rs1800783 polymorphism might be considered as genetic risk factors for diabetic nephropathy, and family history of diabetes was closely associated with rs11771443 polymorphism in DN, and the genetic variants might be used as molecular markers for evaluating the risk of T2DM and diabetic nephropathy. © 2016 IUBMB Life, 68(7):516-525, 2016.

Repression of Biotin-Related Proteins by Benzo[a]Pyrene-Induced Epigenetic Modifications in Human Bronchial Epithelial Cells.

Benzo[a]pyrene (B[a]P) exposure has been associated with the alteration in epigenetic marks that are involved in cancer development. Biotinidase (BTD) and holocarboxylase synthetase (HCS) are 2 major enzymes involved in maintaining the homeostasis of biotinylation, and the deregulation of this pathway has been associated with a number of cancers. However, the link between B[a]P exposure and the dysregulation of BTD/HCS in B[a]P-associated tumorigenesis is unknown. Here we showed that the expression of both BTD and HCS was significantly decreased upon B[a]P treatment in human bronchial epithelial (16HBE) cells. Benzo[a]pyrene exposure led to the global loss of DNA methylation by immunofluorescence, which coincided with the reduction in acetylation levels on histones H3 and H4 in 16HBE cells. Consistent with decreased histone acetylation, histone deacetylases (HDACs) HDAC2 and HDAC3 were significantly upregulated in a dosage-dependent manner. When DNA methylation or HDAC activity was inhibited, we found that the reduction in BTD and HCS was separately regulated through distinct epigenetic mechanisms. Together, our results suggested the potential link between B[a]P toxicity and deregulation of biotin homeostasis pathway in B[a]P-associated cancer development.

Heavy metal-induced metallothionein expression is regulated by specific protein phosphatase 2A complexes.

Induction of metallothionein (MT) expression is involved in metal homeostasis and detoxification. To identify the key pathways that regulate metal-induced cytotoxicity, we investigate how phosphorylated metal-responsive transcription factor-1 (MTF-1) contributed to induction of MT expression. Immortal human embryonic kidney cells (HEK cells) were treated with seven kinds of metals including cadmium chloride (CdCl2), zinc sulfate (ZnSO4), copper sulfate(CuSO4), lead acetate (PbAc), nickel sulfate (NiSO4), sodium arsenite (NaAsO2), and potassium bichromate (K2Cr2O7). The MT expression was induced in a dose-response and time-dependent manner upon various metal treatments. A cycle of phosphorylation and dephosphorylation was required for translocation of MTF-1 from cytoplasm to nucleus, leading to the up-regulation of MTs expression. Protein phosphatase 2A (PP2A) participated in regulating MT expression through dephosphorylation of MTF-1. A loss-of-function screen revealed that the specific PP2A complexes containing PR110 were involved in metal-induced MT expression. Suppression of PP2A PR110 in HEK cells resulted in the persistent MTF-1 phosphorylation and the disturbance of MTF-1 nuclear translocation, which was concomitant with a significant decrease of MT expression and enhanced cytotoxicity in HEK cells. Notably, MTF-1 was found in complex with specific PP2A complexes containing the PR110 subunit upon metal exposure. Furthermore, we identify that the dephosphorylation of MTF-1 at residue Thr-254 is directly regulated by PP2A PR110 complexes and responsible for MTF-1 activation. Taken together, these findings delineate a novel pathway that determines cytotoxicity in response to metal treatments and provide new insight into the role of PP2A in cellular stress response.

Gestational Weight Gain and Overweight in Children Aged 3-6 Years.

OBJECTIVE: To determine whether gestational weight gain (GWG) was associated with increased odds of childhood overweight after accounting for pre-pregnancy BMI. METHODS: In a prospective cohort study based on a premarital and perinatal health care system in China, data of 100 612 mother-child pairs were obtained. The main exposure was GWG as both a continuous and categorical variable. The outcome measure was overweight, defined by age- and sex-specific cutoff values for body mass index (BMI) in children aged 3-6 years. RESULTS: A 1-kg increase in maternal GWG was associated with an increase of 0.009 (95% confidence interval [CI]: 0.007-0.010, P < 0.001) in children's mean BMI; in the subgroup of pre-pregnancy overweight/obese mothers, the increase in children's BMI was 0.028 (95% CI, 0.017-0.039, P < 0.001). Excessive GWG played an important role in childhood overweight when adequate GWG was used as the reference, with an odds ratio (OR) of 1.21 (95% CI, 1.12-1.29). The risk was highest (OR 2.22; 95% CI, 1.79-2.76) in the children of mothers who were overweight/obese before pregnancy and gained excessive weight during pregnancy. CONCLUSIONS: Greater maternal GWG was associated with greater offspring BMI, and the risk of overweight was doubled in children whose mothers were overweight/obese before pregnancy and gained excessive weight during pregnancy. As a result, maintenance of appropriate weight gain during pregnancy and prophylaxis of maternal overweight/obesity before pregnancy should be a strategy for preventing childhood overweight/obesity.

[Investigation of trichloroethylene-induced effects on subcellular proteomes in L-02 hepatic cells].

OBJECTIVE: To put the insight into the trichloroethylene (TCE)-induced effect on the differential expression of subcellular proteins in human normal liver cell line (L-02). METHODS: The membrane proteins and nuclear proteins of TCE-treated (8.0 mmol/L) group and controls were extracted by subcellular proteome extraction kit, respectively. The TCE-induced differentially expressions were analyzed by a two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight spectrometry (MALDI-TOF-MS). Bioinformatics analysis was used to reveal the biological processes and predict transmembrane domains of differential expressed proteins. The expression of ATP synthase subunit beta (ATP5B), heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) and far up steam element-binding protein 1 (FUBP1) were measured under TCE treatment by Western blot. RESULTS: After TCE treatment for 24 h in L-02 cells, 14 membrane proteins and 18 nuclear proteins were identified as differential expression. After treated with TCE in concentrations of 0, 2.0, 4.0 and 8.0 mmol/L for 24 h, the relative levels of ATP5B expression were 1.00±0.03, 1.21±0.14, 1.25±0.12 and 1.48±0.17 (F = 8.51, P = 0.007), the relative levels of hnRNP H2 expression were 1.00±0.09, 1.22±0.15, 1.43±0.21, 1.53±0.17 (F = 6.57, P = 0.015), respectively; the relative levels of FUBP1 expression were 1.00±0.11, 0.91±0.07, 0.73±0.04 and 0.67±0.03 (F = 15.81, P = 0.001), respectively, which were consistent with the results in proteomics. The bioinformatics analysis showed that the most dominant biological process were involved in RNA processing (10 proteins, P = 2.46×10(-6)), especially in RNA splicing (9 proteins, P = 1.77×10(-7)). CONCLUSION: The exposure of TCE could alter the expression of membrane proteins and nuclear proteins in L-02 cells. These abnormal expressed proteins involved in RNA splicing would provide novel clues for further understanding of TCE-induced hepatotoxicity.

[Proteomic study on HaCaT cell membrane proteins after exposed to silica nanomaterials].

OBJECTIVE: To identify the differential expression of membrane proteins after HaCaT cell was treated with 15 nm silica nanomaterials (SiO2). METHODS: The HaCaT cells were cultured for 24 h under 15 nm SiO2 in various concentrations (2. 5, 5. 0, 10. 0 mg/L), and ddH2O were used as control. The cell viability were measured with CCK-8 assay. The membrane proteins of SiO2-treated group (10. 0 mg/L) and controls were extracted by ProteoExtract subcellular proteome extraction kit. The differentially expressed membrane proteins were analyzed by a two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS). Bioinformatics analysis was used to reveal the biological functions and predict transmembrane domains of differential expressed proteins. The expression of differential membrane proteins were measured by Western blot analysis. RESULTS: The cell viability was significantly decreased with the increases of 15 nm SiO2 exposure levels. After treatment with 2. 5, 5. 0, 10. 0 mg/L of 15nm SiO2, the cell viability was assessed to (91. 3% ± 6. 1%), (81. 7% ± 7. 0%) and (74. 0% ± 2. 6%) of control level (P < 0. 05), respectively. In the proteomic analysis, a total of 10 proteins were identified as differential expression in the SiO2-treated simples compared with controls. Among these, 7 of these proteins were predicted as membrane proteins with at least one significant transmembrane domain. The most dominant function that the identified proteins involved in was binding and structural molecule activity. The differential expression of G protein-coupled receptor 179 (GPR 179) and L-plastin (LCP1) were verified by Western-blot analysis under 15nm SiO2 exposure in various concentrations. CONCLUSION: The exposure of 15 nm SiO2 can significantly reduce the cell proliferation and induce a down-regulation of membrane protein expression in HaCaT cells.

[Establishment a method for identification of the poly(ADP-ribose) binding proteins induced by benzo(a)pyrene].

OBJECTIVE: To establish a method for identification of the poly(ADP-ribose) binding proteins induced by benzo (a) pyrene. METHODS: Poly (ADP-ribose) binding protein were screened by immunoprecipitation assay and further separated by high performance liquid chromatography (HPLC) and two dimensional electrophoresis, then identified by MALDI-TOF-MS/MS. The proteins sequence were identified by two methods and compared the common binding motif with literature reports. RESULTS: Three poly (ADP-ribose) binding proteins were identified by MALDI-TOF-MS/MS combined with immunoprecipitation assay and HPLC, and twelve poly (ADP-ribose) binding proteins were identified by MALDI-TOF-MS/MS combined with immunoprecipitation assay and two dimensional electrophoresis. Most of them have a common binding motif which was consistent with the reported. CONCLUSION: Combined the immunoprecipitation assay and two dimensional electrophoresis with MALDI-TOF-MS/MS could be used to analyze the poly(ADP-ribose) binding proteins, and these proteins have a common conserved binding motif.

[Effect of silicon dioxide nanoparticles on expression and DNA methylation of PARP-1 gene in HaCaT cells].

OBJECTIVE: To study the effect of silicon dioxide nanoparticles on the expression and promoter region CpG islands methylation of (Poly [ADP-ribose] polymerase 1, PARP-1) gene in human HaCaT Cell. METHODS: HaCaT Cells were treated with nm-SiO2at 0, 2.5, 5 and 10 µg/mL and micro-SiO2at 10 µg/ml for 24 h and DAC treatment was given at 10 µg/ml group for 48 h. Real-time PCR and western blot assay was used to detect the expression of PARP-1 mRNA and protein. BSP (Bisulfite Pyrosequence, BSP) assay was used to detect the promoter region CpG islands methylation status of PARP-1 gene. RESULTS: After exposure to nano-SiO2particles, compared to CTRL group, the mRNA and protein expression of PARP-1 in micro-SiO2and 2.5 µg/ml group unchanged, but he mRNA and protein expression of PARP-1 in 5, 10 µg/ml as well as DAC group was down-regulated and there are statistical significance between CTRL group and 5, 10 µg/ml as well as DAC group and the PARP-1 promoter region CpG islands showed methylation. CONCLUSION: nano-SiO2can down-regulate PARP-1 expression in HaCaT Cell and this is associated with the change in the methylation of PARP-1 gene promoter region CpG islands induced by nano-SiO2particles.

Role of poly(ADP-ribose) glycohydrolase silencing in DNA hypomethylation induced by benzo(a)pyrene.

Benzo(a)pyrene (BaP) is a known carcinogen cytotoxic which can trigger extensive cellular responses. Many evidences suggest that inhibitors of poly(ADP-ribose) glycohydrolase (PARG) are potent anticancer drug candidates. However, the role of PARG in BaP carcinogenesis is less understood. Here we used PARG-deficient human bronchial epithelial cell line (shPARG cell) as an in vitro model, and investigated the role of PARG silencing in DNA methylation pattern changed by BaP. Our study shows, BaP treatment decreased global DNA methylation levels in 16HBE cells in a dose-dependent manner, but no dramatic changes were observed in shPARG cells. Further investigation revealed PARG silencing protected DNA methyltransferases (DNMTs) activity from change by BaP exposure. Interestingly, Dnmt1 is PARylated in PARG-null cells after BaP exposure. The results show a role for PARG silencing in DNA hypomethylation induced by BaP that may provide new clue for cancer therapy.

Analysis of trichloroethylene-induced global DNA hypomethylation in hepatic L-02 cells by liquid chromatography-electrospray ionization tandem mass spectrometry.

Trichloroethylene (TCE), a major occupational and environmental pollutant, has been recently associated with aberrant epigenetic changes in experimental animals and cultured cells. TCE is known to cause severe hepatotoxicity; however, the association between epigenetic alterations and TCE-induced hepatotoxicity are not yet well explored. DNA methylation, catalyzed by enzymes known as DNA methyltransferases (DNMT), is a major epigenetic modification that plays a critical role in regulating many cellular processes. In this study, we analyzed the TCE-induced effect on global DNA methylation and DNMT enzymatic activity in human hepatic L-02 cells. A sensitive and quantitative method combined with liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was validated and utilized for assessing the altered DNA methylation in TCE-induced L-02 cells. Quantification was accomplished in multiple reaction monitoring (MRM) mode by monitoring a transition pair of m/z 242.1 (molecular ion)/126.3 (fragment ion) for 5-mdC and m/z 268.1/152.3 for dG. The correlation coefficient of calibration curves between 5-mdC and dG was higher than 0.9990. The intra-day and inter-day relative standard derivation values (RSD) were on the range of 0.53-7.09% and 0.40-2.83%, respectively. We found that TCE exposure was able to significantly decrease the DNA methylation and inhibit DNMT activity in L-02 cells. Our results not only reveal the association between TCE exposure and epigenetic alterations, but also provide an alternative mass spectrometry-based method for rapid and accurate assessment of chemical-induced altered DNA methylation in mammal cells.

Hydrogen peroxide induces adaptive response and differential gene expression in human embryo lung fibroblast cells.

Hydrogen peroxide (H2 O2 ), a substance involved in cellular oxidative stress, has been observed to induce an adaptive response, which is characterized by a protection against the toxic effect of H2 O2 at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H2 O2 -induced adaptive response are limited to animal cells and human tumor cells, and relatively normal human cells have never been observed for an adaptive response to H2 O2 . In this study, a human embryo lung fibroblast (MRC-5) cell line was used to model an adaptive response to H2 O2 , and the relevant differential gene expressions by using fluoro mRNA differential display RT-PCR. The results showed significant suppression of cytotoxicity of H2 O2 (1100 µM, 1 h) after pretreatment of the cells with H2 O2 at lower concentrations (0.088-8.8 µM, 24 h), as indicated by cell survival, lactate dehydrogenase release, and the rate of apoptotic cells. Totally 60 mRNA components were differentially expressed compared to untreated cells, and five of them (sizing 400-600 bp) which demonstrated the greatest increase in expression were cloned and sequenced. They showed identity with known genes, such as BCL-2, eIF3S5, NDUFS4, and RPS10. Real time RT-PCR analysis of the five genes displayed a pattern of differential expression consistent with that by the last method. These five genes may be involved in the induction of adaptive response by H2 O2 in human cells, at least in this particular cell type.

[Investigation of the action mechanisms of poly-ADP-ribosylation in hexavalent chromium induced cell damage].

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.

[Effect of poly-ADP-ribosylation on the alteration of DNA methylation level of human bronchial epithelial cells induced by Cr (VI)].

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.

[The profile of IGF2R gene expression and H3 histone modifications in replicative cell senescence].

OBJECTIVE: To study the profile of IGF2R expression and histone modifications in replicative cell senescence. METHODS: The changes of biological characteristics of young human pulmonary fibroblast (HPF) cells [at population doubling level (PDL) 23] and aging HPF cells (at PDL50) were observed and real-time quantitative PCR was utilized to investigate human IGF2R gene expressions profile during the process of cellular aging (at different PDL). Then chromatinimmunoprecipitation-real time quantitative PCR (CHIP-QPCR) methods were conducted to analyze histone modifications of the regions around the transcriptional start site of IGF2R (H3-Ac, H3K9-tri-Me, H3K9-Ac and H3K4-tri-Me). RESULTS: In contrast to young cells, the aging cells were bigger and less proliferative, their cell cycles arrest, and aging specific beta-galactosidase staining was positive. IGF2R gene expression was in positive correlation with PDL. H3-Ac, H3K9-Ac and H3K4-tri-Me were dominant in the upstream region (-0.6 kb) to the downstream region (+1.2 kb) of transcriptional start site (TSS). While in the downstream of TSS from +1.6 kb to +4.0 kb, H3K9-Ac was declined and H3K9-tri-Me was elevated in turn, but H3K4-tri-Me still prevailed in these areas. CONCLUSION: IGF2R is related to cell replicative senescence and its gene expression is regulated by histone modification of H3. Therefore, epigenetics may play a role in cell senescence.

MicroRNA-152 targets DNA methyltransferase 1 in NiS-transformed cells via a feedback mechanism.

Nickel (Ni) compounds are well-recognized human carcinogens, yet the molecular mechanisms by which they cause human cancer are still not well understood. MicroRNAs (miRNAs), which are small non-coding RNAs, are involved in diverse biological functions and carcinogenesis. In previous study, we identified upregulation of DNA methyltransferase 1 (DNMT1) expression in nickel sulfide (NiS)-transformed human bronchial epithelial (16HBE) cells. Here, we investigated whether some miRNAs are aberrantly expressed and targets DNMT1 in NiS-transformed cells. Our results showed that the expression of miRNA-152 (miR-152) was specifically downregulated in NiS-transformed cells via promoter DNA hypermethylation, whereas ectopic expression of miR-152 in NiS-transformed cells resulted in a marked reduction of DNMT1 expression. Further experiments revealed that miR-152 directly downregulated DNMT1 expression by targeting the 3' untranslated regions of its transcript. Interestingly, treatment of DNMT inhibitor, 5-aza-2-deoxycytidine, or depletion of DNMT1 led to increased miR-152 expression by reversion of promoter hypermethylation, DNMT1 and MeCP2 binding to miR-152 promoter in NiS-transformed cells. Moreover, inhibition of miR-152 expression in 16HBE cells could increase DNMT1 expression and result in an increase in DNA methylation, DNMT1 and MeCP2 binding to miR-152 promoter, indicating an interaction between miR-152 and DNMT1 is regulated by a double-negative circuit. Furthermore, ectopic expression of miR-152 in NiS-transformed cells led to a significant decrease of cell growth. Conversely, inhibition of miR-152 expression in 16HBE cells significantly increased cell growth. Taken together, these observations demonstrate a crucial functional crosstalk between miR-152 and the DNMT1 via a feedback loop involved in NiS-induced malignant transformation.

Identification of serum biomarkers for occupational medicamentosa-like dermatitis induced by trichloroethylene using mass spectrometry.

Occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) is an autoimmune disease and it has become a serious occupational health hazard. In the present study, we collected fasting blood samples from patients with OMLDT (n=18) and healthy volunteers (n=33) to explore serum peptidome patterns. Peptides in sera were purified using weak cation exchange magnetic beads (MB-WCX), and analyzed by matrix-assisted laser desorption ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) and ClinProTools bioinformatics software. The intensities of thirty protein/peptide peaks were significantly different between the healthy control and OMLDT patients. A pattern of three peaks (m/z 2106.3, 2134.5, and 3263.67) was selected for supervised neural network (SNN) model building to separate the OMLDT patients from the healthy controls with a sensitivity of 95.5% and a specificity of 73.8%. Furthermore, two peptide peaks of m/z 4091.61 and 4281.69 were identified as fragments of ATP-binding cassette transporter family A member 12 (ABCA12), and cationic trypsinogen (PRRS1), respectively. Our findings not only show that specific proteomic fingerprints in the sera of OMLDT patients can be served as a differentiated tool of OMLDT patients with high sensitivity and high specificity, but also reveal the novel correlation between OMLDT with ABC transports and PRRS1, which will be of potential value for clinical and mechanistic studies of OMLDT.

Role of poly(ADP-ribose) glycohydrolase in the regulation of cell fate in response to benzo(a)pyrene.

Poly(ADP-ribosyl)ation is a crucial regulator of cell fate in response to genotoxic stress. Poly(ADP-ribosyl)ation plays important roles in multiple cellular processes, including DNA repair, chromosomal stability, chromatin function, apoptosis, and transcriptional regulation. Poly(ADP-ribose) (PAR) degradation is carried out mainly by poly(ADP-ribose) glycohydrolase (PARG) enzymes. Benzo(a)pyrene (BaP) is a known human carcinogen. Previous studies in our laboratory demonstrated that exposure to BaP caused a concentration-dependent DNA damage in human bronchial epithelial (16HBE) cells. The role of PARG in the regulation of DNA damage induced by BaP is still unclear. To gain insight into the function of PARG and PAR in response to BaP, we used lentiviral gene silencing to generate 16HBE cell lines with stably suppressed PARG, and determined parameters of cell death and cell cycle following BaP exposure. We found that PARG was partially dependent on PAR synthesis, PARG depletion led to PAR accumulation. BaP-induced cell death was regulated by PARG, the absence of which was beneficial for undamaged cells. Our results further suggested that PARG probably has influence on ATM/p53 pathway and metabolic activation of BaP. Experimental evidences provided from this study suggest significant preventive properties of PAR accumulation in the toxicity caused by BaP.

Study of cytoskeletal changes induced by okadaic acid in HL-7702 liver cells and development of a fluorimetric microplate assay for detecting diarrhetic shellfish poisoning.

Diarrhetic shellfish poisoning (DSP) is a gastrointestinal illness with symptoms such as diarrhea, nausea, vomiting, headache, chills and moderate to severe abdominal pain. DSP has been recognized as a worldwide public health problem, causing great concern to the shellfish industry. Accumulation of DSP in shellfish is an unpredictable phenomenon that necessitates the implementation of a widespread collection and thorough monitoring program for mollusk toxicity. Therefore, development of accurate analytical protocols for the rapid determination of toxicity levels would be necessary. In this study we investigated cytoskeletal changes induced by okadaic acid in HL-7702 Liver Cells and developed a new cytotoxicity assay for detection and quantitation of DSP. This assay is based on fluorometric of F-actin depolymerization induced by okadaic acid (OA) compounds in HL-7702 liver cell line. The measurable range of OA was 2.5 ∼ 40 nmol/L. The detection limit of the F-actin assay for OA was 2.01 µg/100 g muscles in shellfish extracts. The performance of this assay has been evaluated by comparative analysis of shellfish samples by the fluorescent assay, mouse bioassay, and ELISA assay. Comparison of the results by all three methods revealed excellent consistency, the results of fluorescent assay were in significant correlation with ELISA assay (R(2) = 0.830). Examination of F-actin assay is very convenient, rapid, and sensitive, which can be used to quantify the amount of OA in shellfish samples.

[Effects of bisphenol A on OCT4 and SOX2 genes expression in mouse embryonic stem cells].

OBJECTIVE: To explore the effects of bisphenol A (BPA) exposure on toxicity characteristic and OCT4 and SOX2 gene expression of mouse embryonic stem cells (mESC). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Profile of P66SHC expression and histone modifications in replicative cell senescence and oxidative-stress induced premature senescence].

OBJECTIVE: To study the profile of P66SHc expression and histone modifications in replicatively senescenct cells and oxidative-stress inducing premature senescenct cells. METHODS: HPF cells were continuously cultured and subcultured in vitro to build replicative cellular model. HPF cells were treated with 200 pmol/L H2 O2 four times to build oxidative-stress inducing premature senescenct model. Comparative Q-PCR was utilized to investigate target gene (P66SHC, EP300, HDAC1) expressions respectively in H2O2 treated groups and normal cell groups. Then CHIP-QPCR was conducted to analyze histone modifications of P66SHC between young cells and aging cells. RESULTS: P66SHC expression was positive correlation with H2O2 doses and population doubling level (PDL) (R = 0.909, P = 0.000; R = 0.743, P = 0.006), while EP300 was negative correlation with H2O2 (R = - 0.922, P = 0.000) and both EP300 and HDAC1 were negative with PDL (R = -0.709, P = 0.010, R = -0.599, P = 0.040). H3 histone modifications were declined in P66SHc gene regulating region. H3-Ac, H3K9-Ac and H3K4-tri-Me were dominant in the upstream region of transcriptional site (-3.0 kb) and alternative promotor (+3.8 kb). CONCLUSION: P66SHC, EP300 and HDAC1 probably play a role in cellular replicative senescence and oxidative-stress inducing premature senescence. Besides, histone modification could regulate P66SHC gene expression.