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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.
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Ilhas de CpG , Metilação de DNA , Nanopartículas/efeitos adversos , Poli(ADP-Ribose) Polimerases/metabolismo , Dióxido de Silício/efeitos adversos , Linhagem Celular Tumoral , Humanos , Poli(ADP-Ribose) Polimerase-1 , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: To study the potential association of DNA oxidation and DNA methylation, in vitro cultured cells were exposed to different doses of H2O2, 8-oxo-dG formation, cell DNA 5-mC contents were analyzed to explore the time- dose-response relationship of DNA oxidation and DNA methylation. METHODS: A549 cells were exposed to different doses of H2O2, 8-oxo-dG formation and cell genomic DNA 5-mC contents were analyzed by a high-performance liquid chromatography system and high performance capillary electrophoresis (HPCE), respectively. RESULTS: H2O2 induced the formation of 8-oxo-dG and 5-mC in different characteristics, it need at least 10 days for significant changes in the level of DNA methylation, whereas under the same conditions, changes in the level of DNA oxidation cast only 12 hours. H2O2 induced decreased levels of DNA methylation in A549 cells in a dose-dependent manner. In a certain range of time and dose, it showed a negative correlation between DNA oxidation and DNA methylation. CONCLUSION: The study suggests that oxidative DNA could lead to reduced levels of DNA methylation, DNA oxidation may affect the regulation of cellular methylation mechanisms, in the course of chemical mutagenesis, DNA oxidation may be an earlier important molecule event than DNA methylation.
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Dano ao DNA , Metilação de DNA , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo , 8-Hidroxi-2'-Desoxiguanosina , Linhagem Celular , DNA/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , HumanosRESUMO
Natural pyrethrins (NPs), one kind of bio-pesticide, have been widely used in organic agriculture and ecological environment studies. Studies have shown that NPs may affect the metabolism of rat liver and human hepatocytes; nevertheless, the toxic effects of NPs on the liver and the related mechanisms are still incompletely understood. In this research, we utilized three types of human liver cells to investigate the mechanism of NPs' induction of oxidative stress. The results showed that NPs exhibit noteworthy cytotoxic effects on human liver cells. These effects are characterized by the induction of LDH release, mitochondrial collapse, and an increased production of ROS and MDA content, subsequently activating the Kelch-like ECH-associated protein 1/Nuclear factor erythroid 2- related factor 2 (Keap1/Nrf-2) pathway. The ROS inhibitor N-acetyl-L-cysteine (NAC) can alleviate ROS/Nrf2-mediated oxidative stress. In addition, the siRNA knockdown of Nrf-2 exacerbated the injury, including ROS production, and inhibited cell viability. In summary, the ROS-mediated Keap1/Nrf-2 pathway could be an important regulator of NP-induced damage in human liver cells, which further illustrates the hepatotoxicity of NPs and thereby contributes to the scientific basis for further exploration.
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OBJECTIVE: To observe the effect of SiO2 nanoparticles on genome DNA methylation profile in cultured cells. METHODS: 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. RESULTS: 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. CONCLUSION: Genomic DNA methylation levels were decreased in HaCaT cells after short-term exposure to SiO2 nanoparticles.
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Metilação de DNA/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Nanopartículas/toxicidade , Dióxido de Silício/toxicidade , Linhagem Celular , Células Cultivadas , Eletroforese Capilar , Genoma/genética , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Pele/citologiaRESUMO
The increasing applications of silicon dioxide (SiO(2)) nanomaterials have been widely concerned over their biological effects and potential hazard to human health. In this study, we explored the effects of SiO(2) nanoparticles (15, 30, and 100 nm) and their micro-sized counterpart on cultured human epidermal Keratinocyte (HaCaT) cells. Cell viability, cell morphology, reactive oxygen species (ROS), DNA damage (8-OHdG, γH2AX and comet assay) and apoptosis were assessed under control and SiO(2) nanoparticles exposed conditions. As observed in the Cell Counting Kit-8 (CCK-8) assay, exposure to 15, 30 or 100 nm SiO(2) nanoparticles at dosage levels between 0 and 100 µg/ml decreased cell viability in a concentration- and size dependent manner and the IC50 of 24 hour exposure was 19.4 ± 1.3, 27.7 ± 1.5 and 35.9 ± 1.6 µg/ml for 15, 30 and 100 nm SiO(2) nanoparticles, respectively. Morphological examination revealed cell shrinkage and cell wall missing after SiO(2) nanoparticle exposure. Increase in intracellular ROS level and DNA damage as well as apoptosis were also observed in SiO(2) nanoparticle-exposed HaCaT cells. Exposure to SiO(2) nanoparticles results in a concentration- and size-dependent cytotoxicity and DNA damage in cultural HaCaT cells which is closely correlated to increased oxidative stress.
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Dano ao DNA , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Nanopartículas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Dióxido de Silício/toxicidade , 8-Hidroxi-2'-Desoxiguanosina , Morte Celular/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Histonas/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Queratinócitos/metabolismo , Nanopartículas/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Tamanho da PartículaRESUMO
Background: This study aimed to comprehensively evaluate the toxicity exerted by zinc oxide nanoparticles (ZnO NPs) on rat testis and its effects on fertility and progeny development. Methods: Different concentrations of ZnO NPs were administered by gavage to Sprague Dawley (SD) rats to examine the adverse effects resulting from pre- and post-natal exposure. Systemic distribution of ZnO NPs, developmental performance, sperm parameters, reproductive performance, histopathological examination, and sex hormone levels were determined scheduled in the experimental rats and their male offspring. The comparative in vitro cytotoxicity of the ZnO NPs was determined among C18-4, TM3, and TM4 cells. The toxicity exerted by ZnO NPs on germ cells in vitro and the effects on the expression of cytoskeleton and blood-testis barrier (BTB)-related proteins were also determined. Results: After oral gavage, ZnO NPs mainly accumulated in the liver and testes of rats; 350 mg/kg ZnO NPs adversely affected the epididymal weight, sperm motility, and hormone levels but did not affect the fertility of rats. In addition, 350 mg/kg ZnO NPs significantly reduced the reproductive and developmental performance of offspring male rats. Testicular histopathological and electron microscopic ultrastructure examinations showed more significant abnormal structural changes than those observed in parental rats. The results of in vitro cell experiments further showed that ZnO NPs exerted cytotoxic effects on germ cells, and led to DNA damage, nucleoskeleton and cytoskeleton alterations, and could regulate actin changes through changes in LC3B. Conclusions: It is possible that ZnO NPs act directly on TM4 cells by penetrating the BTB, causing damage to the cytoskeleton and disrupting the dynamic balance of the BTB, thereby destroying the microenvironment necessary for spermatogenesis, which may lead to poor reproduction in rats.
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Background: Research indicates that exposure to polychlorinated biphenyls (PCBs) can cause neurobehavioral impairments in neonates and adults, but the way specific PCBs' congeners impact cognition functions at a low exposure level in a real-life co-exposure system remains poorly understood. This study aimed to investigate the association of PCBs burden with cognition function among elderly adults. Methods: Based on the Weitang Geriatric Diseases study (2014−2015), the current study measured the plasma concentrations of six indicator-PCBs by GC-MS/MS and assessed the cognitive dysfunction (CoD) via an Abbreviated Mental Test in 266 participants (ages 61−90). Sequential logistic regression was used to analyze the effects of PCBs on cognition functions. Female participants aged less than or equal to 80 years were selected, and path analysis was used to determine the direct or indirect impacts of co-exposure PCBs on CoD by structural equation modeling. Results: After sequential adjustments to potential confounding factors and correction by the Bonferroni, no statistically significant correlation between PCBs exposure and CoD was found in participants (p > 0.05). However, in the co-exposure system, after controlling for co-exposures and confounders, exposure to PCB28 had a direct effect on CoD in females aged between 61 and 80, with a factor load of 0.670. Conclusions: After adjusting for the co-exposures and confounders, exposure to PCB28 can directly increase the risk of cognitive impairment in older Chinese females.
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Disfunção Cognitiva , Poluentes Ambientais , Bifenilos Policlorados , Adulto , Idoso , Idoso de 80 Anos ou mais , China/epidemiologia , Cognição , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/epidemiologia , Poluentes Ambientais/análise , Feminino , Humanos , Recém-Nascido , Pessoa de Meia-Idade , Espectrometria de Massas em TandemRESUMO
Accumulating evidence reveals that exposure to alternative flame retardants (AFRs) results in defective thyroid functions. AFRs are detectable in various environmental media in developed cities in China. However, few studies have reported the contamination levels of AFR in groundwater in rural areas, indicating an urgent need to investigate exposure of AFRs and perform health risk assessment for populations that use groundwater as the main source of drinking water. This study investigated the concentrations of AFRs in groundwater in rural areas of central China. Moreover, Nthy-ori-3-1 cells were used to determine the thyroid cytotoxicities and thyroid-interfering effects of a single AFR as well as the mixtures of AFRs based on the AFR contamination levels in real-world. The results revealed that all classes of AFRs were detectable in rural areas in central China. Dechlorane plus, hexabromocyclododecane, bromophenols (BPs), novel brominated flame retardants (NBFRs) and organophosphate flame retardants (OPFRs) exhibited spatial contamination patterns, with an average concentrations (median) of 157.89 ± 88.61 (185.47) pg/L, 0.09 ± 0.29 (not detectable) ng/L, 5.20 ± 5.92 (3.43) ng/L, 3338.11 ± 3758.78 (2836.72) pg/L, and 79.35 ± 97.19 (53.62) ng/L, respectively. The half maximal effective concentrations (EC50) of BPs, OPFRs, and NBFRs ranged 98.4-4012 µM, 42.0-2506 µM, and 10.1-203.7 µM, respectively. Several AFRs exhibited more cytotoxic effects than did traditional brominated flame retardants. It is intriguing that several single AFRs and mixtures at environmentally-relevant exposure levels promoted the viability of Nthy-ori-3-1 cells. Taken together, our study demonstrates that AFRs are present in the groundwater in rural areas in central China and AFRs exhibit thyroid disrupting effects.
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Retardadores de Chama , Água Subterrânea , China , Monitoramento Ambiental , Retardadores de Chama/análise , Retardadores de Chama/toxicidade , Éteres Difenil Halogenados/análise , Éteres Difenil Halogenados/toxicidade , Organofosfatos , Glândula TireoideRESUMO
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.
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Benzo(a)pireno/efeitos adversos , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Células Epiteliais/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
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.
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DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Células Epiteliais/metabolismo , Ciclo Celular , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Regulação para Baixo , Humanos , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
The increasing amount of nanotechnological products, found in our environment and those applicable in engineering, material sciences and medicine has stimulated a growing interest in examining their long-term impact on genetic and epigenetic processes. We examined here the epigenomic response to nm-SiO(2) particles in human HaCaT cells and methyltransferases (DNMTs) and DNA-binding domain proteins (MBDs) induced by nano-SiO(2) particles. Nm-SiO(2) treatment induced global hypoacetylation implying a global epigenomic response. The levels of DNMT1, DNMT3a and methyl-CpG binding protein 2 (MBD2) were also decreased in a dose dependent manner at mRNA and protein level. Epigenetic changes may have long-term effects on gene expression programming long after the initial signal has been removed, and if these changes remain undetected, it could lead to long-term untoward effects in biological systems. These studies suggest that nanoparticles could cause more subtle epigenetic changes which merit thorough examination of environmental nanoparticles and novel candidate nanomaterials for medical applications.
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Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Nanopartículas/toxicidade , Dióxido de Silício/toxicidade , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/metabolismo , HumanosRESUMO
There is evidence in experimental animals for the urolithiasis and carcinogenicity of melamine, but no evidence for melamine in humans. To evaluate any association between melamine-contaminated powdered formula (MCPF) feeding and urolithiasis, and further the MCPF feeding and oxidative damage to DNA in infants. A cross-sectional study was carried out to assess urolithiasis and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) in four groups of infants according to the type of feeding: (1) Infants receiving over 90% of their intake as MCPF. (2) Infants receiving 50-90% of their intake as MCPF. (3) Infants receiving less than 50% of their intake as MCPF. (4) Infants receiving over 90% of their intake as imported milk powdered formula free of melamine contamination. Groups 1 to 3 are the observation groups, and Group 4 is the reference group. There is a significant correlation between urolithiasis and percentage of MCPF intake. The mean urinary 8-OHdG concentrations for Groups 1, 2, 3, and 4 were: 2.03 +/- 0.52, 1.67 +/- 0.28, 1.90 +/- 0.39, and 1.85 +/- 0.47 micromoles per mole of creatinine, respectively. There were no significant differences in the mean urinary 8-OHdG concentrations among the observation and control groups. There were also no correlation between mean urinary 8-OHdG excretions and percentage of MCPF intake. Our data suggested that melamine exposure were associated with urolithiasis, but it might not cause any increase in oxidative damage of DNA in infants.
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Dano ao DNA , Estresse Oxidativo/efeitos dos fármacos , Triazinas/toxicidade , Cálculos Urinários/epidemiologia , 8-Hidroxi-2'-Desoxiguanosina , Estudos Transversais , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Exposição Ambiental/análise , Feminino , Contaminação de Alimentos , Humanos , Lactente , Fórmulas Infantis , Recém-Nascido , Masculino , Ultrassonografia , Cálculos Urinários/diagnóstico por imagemRESUMO
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.
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Transformação Celular Neoplásica/induzido quimicamente , Metilação de DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Níquel/efeitos adversos , Brônquios/citologia , Linhagem Celular , Genoma , Humanos , Níquel/químicaRESUMO
Nano silicon dioxide (Nano-SiO2) has been widely used in industries such as the field of biomedical engineering. Despite the existing evidence that Nano-SiO2 exposure could induce oxidative stress and inflammatory responses in multiple organ systems, the carcinogenicity of Nano-SiO2 exposure has rarely been investigated. Thus in this study, two types of human bronchial epithelial cell lines (16HBE and BEAS-2B) were selected as in vitro models to investigate the carcinogenicity of Nano-SiO2. Our results revealed that Nano-SiO2 induces a malignant cellular transformation in human bronchial epithelial cells according to the soft agar colony formation assay. The carcinogenesis induced by Nano-SiO2 was also confirmed in nude mice. By using immunofluorescence assay and high-performance capillary electrophoresis (HPCE), we observed a genome-wide DNA hypomethylation induced by Nano-SiO2. Besides the reduced enzyme activity of total DNMTs upon Nano-SiO2 treatment, altered expression of DNMTs and methyl-CpG binding proteins were observed. Besides, we found that the expression of NRF2 was activated by demethylation of CpG islands within the NRF2 promoter region and the overexpression of NRF2 could alleviate the carcinogenesis induced by Nano-SiO2. Taken together, our results suggested that Nano-SiO2 induces malignant cellular transformation with a global DNA hypomethylation, and the demethylation of NRF2 promoter activates the expression of NRF2, which plays an important role in protecting against the carcinogenesis induced by Nano-SiO2.
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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.
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Benzo(a)pireno/toxicidade , Dano ao DNA , Reparo do DNA , Poli(ADP-Ribose) Polimerases/deficiência , Mucosa Respiratória/efeitos dos fármacos , Brônquios/citologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Relação Dose-Resposta a Droga , Inativação Gênica , Humanos , Modelos Biológicos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Interferência de RNA , Mucosa Respiratória/citologia , Mucosa Respiratória/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
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Hepatócitos/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Proteômica/métodos , Tricloroetileno/farmacologia , Análise de Variância , Sequência de Bases , Western Blotting , Linhagem Celular , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hepatócitos/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas/genética , Proteínas/metabolismo , Proteoma/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
2,2',4,4'-Tetra-bromodiphenyl ether (BDE-47), an important congener among polybrominated diphenyl ether (PBDE) compounds, has been predominantly in environmental samples and human tissue. Thyroid disruption is the most sensitive endpoint effect among a number of health effects of exposure to BDE-47 in animals and humans. However, the detailed underlying mechanisms in humans are not well understood. In the present study, human pregnane X receptor (hPXR)-overexpressing HepG2 cell model and a dual-luciferase reporter assay system were constructed to investigate the role of hPXR in BDE-47-induced alterations of expression of metabolic enzymes and TR in vitro. The results showed that hPXR was significantly activated by BDE-47, and expression levels of both mRNA and protein of the thyroid receptor (TR) isoforms TRα1 and TRß1 were decreased in hPXR-overexpressing HepG2 cells after BDE-47 treatment. However, the increased expression of hepatic microsomal phase I enzyme CYP3A4 and phase II enzymes, UGT1A3 and SULT2A1 were also found. Taken together, the results indicated that BDE-47 was a strong hPXR activator, activation of hPXR played an important role in BDE-47-induced down-regulation of TR, and up-regulations of CYP3A4, UGT1A3, and SULT2A1 participated in the process, which may provide more toxicological evidence on mechanisms of disruption of thyroid hormone induced by BDE-47.
Assuntos
Éteres Difenil Halogenados/toxicidade , Receptores de Esteroides/efeitos dos fármacos , Receptores alfa dos Hormônios Tireóideos/efeitos dos fármacos , Receptores beta dos Hormônios Tireóideos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP3A/biossíntese , Indução Enzimática/efeitos dos fármacos , Glucuronosiltransferase/biossíntese , Células Hep G2 , Humanos , Luciferases/genética , Receptor de Pregnano X , Receptores de Esteroides/fisiologia , Sulfotransferases/biossíntese , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genéticaRESUMO
Nano silicon dioxide (nano-SiO2) is becoming more and more widely applied in the fields of industry. The potential toxic effects of nano-SiO2 and its hazard to human health are drawing more attention. The mRNA expression of poly(ADP-ribose) polymerases-1(PARP-1), a pivotal repair gene, has been decreased by nano-SiO2 exposure. However, the effect of epigenetic modification on nano-SiO2-induced low PARP-1 expression has not been reported. In this study, HaCaT cells with or without DNA methyltransferase 1(DNMT1) knock down were incubated with nano-SiO2 and then further treated with DNMT inhibitor, 5-aza-2-deoxycytidine (DAC), which is a kind of key epigenetic modification reagents. Real-time Q-PCR and western blotting were used to examine the mRNA and protein expression of PARP-1. For promoter methylation status of PARP-1, methylation-specific PCR (MSP) and Bisulfite sequencing assay were performed. Results showed a dramatic decrease of PARP-1 expression on mRNA and protein level and a simultaneously obvious increase in the level of PARP-1 methylation in nano-SiO2-treated cells compared to the control group. Further, the expression and promoter methylation of PARP-1 in HaCaT cells were restored following DNMT1 knock down, suggesting that the effects of PARP-1 promoter hypermethylation are mediated at least in part by DNMT1. Taken together, methylation of PARP-1 promoter might be involved in the regulation of nano-SiO2-induced decrease of PARP-1 expression.
Assuntos
Metilação de DNA , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Nanopartículas , Poli(ADP-Ribose) Polimerases/genética , RNA Mensageiro/genética , Dióxido de Silício/toxicidade , Western Blotting , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Epigênese Genética , Técnicas de Silenciamento de Genes , Vetores Genéticos , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Lentivirus/genética , Plasmídeos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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.
Assuntos
Modelos Animais de Doenças , Proteoma/efeitos dos fármacos , Resinas Sintéticas/toxicidade , Triazinas/toxicidade , Cálculos da Bexiga Urinária/induzido quimicamente , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Masculino , Proteoma/química , Proteoma/metabolismo , Ratos , Ratos Sprague-Dawley , Resinas Sintéticas/química , Resinas Sintéticas/metabolismo , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Espectrometria de Massas em Tandem , Triazinas/química , Triazinas/metabolismo , Cálculos da Bexiga Urinária/química , Cálculos da Bexiga Urinária/metabolismo , Difração de Raios XRESUMO
Formaldehyde (FA), a volatile organic compound, is a ubiquitous air pollutant that is classified as 'Carcinogenic to humans (Group 1)' by IARC (2006). As a well-recognized human carcinogen, its carcinogenic mechanisms are still poorly understood. Previous studies have emphasized on genetic changes. However, little is known about the epigenetic mechanisms of FA exposure. In this study, We not only characterized the epigenomic response to long-term low-dose FA exposure in 16HBE cells, but also examined the expression of DNA methyltransferases (DNMTs) and the methyl-CpG-binding protein DNA-binding domain protein 2 (MBD2). Each week the 16HBE cells were treated with 10 µM FA for 24 h (h). After 24 weeks (W) of exposure to FA, the level of genomic DNA methylation gradually decreased in a time-related manner. Moreover, our results showed that FA exposure down-regulated the expression of DNMT3a and DNMT3b at both mRNA and protein level, and up-regulated the levels of DNMT1 and MBD2 at both mRNA and protein level. Our study indicated that long-term FA exposure could disrupt genomic DNA methylation, which may be one of the possible underlying carcinogenic mechanisms of FA.