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To identify the differential expression of membrane proteins after HaCaT cell was treated with 15 nm silica nanomaterials (SiO2).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.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 (P0. 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.The exposure of 15 nm SiO2 can significantly reduce the cell proliferation and induce a down-regulation of membrane protein expression in HaCaT cells.

To establish a method for identification of the poly(ADP-ribose) binding proteins induced by benzo (a) pyrene.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.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.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.

To study the profile of P66SHc expression and histone modifications in replicatively senescenct cells and oxidative-stress inducing premature senescenct cells.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.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).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.

To observe the effect of SiO2 nanoparticles on genome DNA methylation profile in cultured cells.HaCaT cells were treated with nm-SiO2 at 2.5, 5 and 10 microg/ml and micro-SiO2 at 10 microg/ml for 24h and DAC treatment was given at 10 microg/ml group for 48h. The mC/(mC + C) percent was quantified by high performance capillary electrophoresis (HPCE) assay, and the expression level of mRNA and protein was detected by Real-time Q-PCR and westernblot assay. The activity of DNMTs was determined by DNA Methyltransferase Activity/Inhibition Assay Kit.HPCE assay showed that nm-SiO2-treated cells were decreased in some degree. An average proportion of methylated mC/ (mC + C) was 4.82% in control, 2.7% in 2.5 microg/ml and 2.17% in 10 microg/ml groups, while 3.1% in micro-SiO2 groups, which got the consistent downtrend of genome methylation level during increasing nm-SiO2 dose nanoparticles. The mRNA expression level for DNMT1 decreased gradually with increased dose of nm-SiO2 nanoparticles. The alterations at protein level were similar to those at the mRNA level.Genomic DNA methylation levels were decreased in HaCaT cells after short-term exposure to SiO2 nanoparticles.

To assess the natural variation of hOGG1 gene and the gene-environmental interactions.The hOGG1 codon 326 polymorphism and urinary 8-OHdG levels were investigated in a large sample of healthy individuals (n = 953) from five ethnic populations in China by using PCR-RFLP and HPLC-ECD. Life style parameters were obtained through a questionnaire survey.The allelic frequencies of hOGG1 gene in whole subjects were 0.16 (Ser/Ser), 0.49 (Ser/Cys) and 0.35 (Cys/Cys). The frequency of Ser326Cys was significantly different among the five ethnic populations (P = 0.002). No association was found between the hOGG1 gene polymorphism and other life style parameters except for the association between Ser326Cys and smoking (P = 0.027). A significant increase of urinary 8-OHdG level was observed in Cys326Cys allelic healthy subjects (P = 0.033).There are natural variations of hOGG1 gene in different ethnic populations in China. Smoking is related to the frequencies of Cys/Cys polymorphism, and the repairing of oxidative DNA damage is lower in individuals with the hOGG1 Cys326Cys genotype.

To determine the expression of glutathione transferase omega 1 (GSTO-1) during hormesis induced by low dose trichloroethylene (TCE).Based on the dose-response of hormesis induced by TCE, the total protein and mRNA from four groups was extracted, and expression of GSTO-1 was detected by western blotting and RT-PCR technology.The study showed that increases of GSTO-1 protein expression were induced by TCE at low dose (0.1 mmol/L) TCE, but there was no significant different between the high-dose group and the control (P0.05). RT-PCR analysis presented the expression of GSTO-1 gene in low-dose group was increased, but the opposite effect was observed in the high-dose group.GSTO-1 may be play a key role in hormesis induced by TCE.

Epigenetic regulations of insulin-like growth factor 2 (IGF2) were observed during cellular replicative senescence and premature senescence induced by hydrogen peroxide of human embryonic lung fibroblasts (HEFs).The mRNA level of IGF2 was detected by Q-PCR. The methylation status in the promoter region was observed by methylation-specific PCR. The histone modifications was detected by chromatin immunoprecipitation-Q-PCR assay, including acetylation for H3, H4 and methylation for H3 ( Lys4) and H4 (Lys20).In the process of cellular senescence, the mRNA level of IGF2 increased in both mid-aged and replicative senescent cells, but increased obviously in premature senescent cells compared with that of young cells. In the promotor region from -658 bp to -456 bp, the methylation level for IGF2 was detected only in replicative senescent cells. About the main histone modifications, IGF2 in the region (-856 bp - -634 bp) was H4 acetylation and H3K4 methylation in replicative senescence, while in the region (+9 bp - +145 bp) was H3K4 and H4K20 methylation in replicative senescence and H3K4 methylation in premature senescence.The histone modifications take part in regulating the mRNA expression for IGF2 during cellular senescence and different mechanisms exist between two types of senescence.

To study the effects of hydroquinone (HQ) on expression of ubiquitin-conjugating enzyme Rad6B in human L-02 hepatic cells.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.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 (P0.01), 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.HQ could regulate up the expression of Rad6B in L-02 hepatic cells.

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.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.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.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.

Ciguatoxin may be a terrible coral fish toxin that can result in poisoning in people who eat fish containing ciguatoxin. Being lack of a convenient and accurate detection method for ciguatoxin, Ciguatera fish poisoning have occurred from time to time. To provide references for detection and prevention of ciguatera fish poisoning, in this paper, the current detection methods of ciguatoxin were reviewed.

To study the dioxins (polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans, PCDD/Fs) content and profile in fish from marine and fresh water in a certain water area, and to assess the dioxins intake from fish in the local people.Samples were prepared using advanced technique, confirmation and quantitative analysis of Dibenzo-p-Dioxins and Dibenzo-Furans was performed by isotope dilution HRGC/HRMS using multiple ion detection mode (MID).The average concentration of fish from marine and fresh water was 1.43pg/g wet weight and 3.43pg/g wet weight respectively, and WHO-TEQ concentration was 0.28 and 0.43pg/g wet weight respectively. The congener-specific profile dominated by OCDD and 2,3,7,8-TCDF, and the main contributor of toxicity was 1,2,3,7,8-PeCDD, 2,3,4,7,8-PeCDF, 2,3,7,8-TCDD. The exposure from fish for local people was estimated as 0.63 pg WHO-TEQ/kg bw x day.The level of PCDD/Fs in fresh water fish was higher than that of marine fish with one fold. The average PCDD/Fs concentration was lower than 1.0 pg WHO-TEQ/kg, which is the maximum limit (ML) set up by European Union.

To explore the molecular mechanism of cell response induced by hydroquinone, we investigated the protein profile after treatment with hydroquinone in human embryonic lung fibroblasts.After HQ treatment, cells were lysed in lysis solution, then the supernatant were collected and precipitated with acetone prior to protein concentration determination. The total cellular proteins were separated using two-dimensional gel electrophoresis and visualized by colloidal coomassie blue staining. Digital images were analyzed using Imagemaster 2.0 software. The differentially expressed protein spots were picked and digestion in gel then identified by peptide mass fingerprinting using MALDI-TOF.There were 15 protein spots changed after HQ stimulation. Among them 8 protein spots were identified by PMF including some oxidative stress and cytoskeleton related proteins.Protein profile was altered after HQ stimulation.

Electrospray ionization(ESI) and matrix-assisted laser desorption ionization(MALDI) are two kinds of newly-developed mass spectrometry techniques. The application of mass spectrometry is described in the proteome research such as MS charting, determination of molecular weight, peptide mass fingerprinting(PMF) and peptide sequence validation. The advantages of mass spectrometry are sensitive, accurate, rapid, high-throughput and automated. They have been proved a powerful technique and can be used as an important method for proteome research.