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Background: There is limited information about gene-environment interaction on the occurrence and the progression of Alzheimer’s disease. Objective: To explore the effect of environmental low-dose cadmium (Cd) exposure on the progress of Alzheimer’s disease and the underlining mechanism. Methods: We administered 1 mg/L, 10 mg/L cadmium chloride (treated groups), and water (control group) to C57BL/6J and APP/PS1 mice through drinking water, from one week before mating, until the offspring were sacrificed at 6 months of age. The behaviors, Cd level, blood-brain barrier (BBB) leakage, Aβ 1 - 42 deposition, and inflammation expression were evaluated in these mice. Results: Mice of both genotypes had similar blood Cd levels after exposure to the same dose of Cd. The toxic effects of Cd on the two genotypes differed little in terms of neuronal histomorphology and BBB permeability. Cd caused a series of pathological morphological changes in the mouse brains and more fluorescent dye leakage at higher doses. Furthermore, the APP/PS1 mice had more severe damage than the C57BL/6J mice, based on the following five criteria. They are increasing anxiety-like behavior and chaos movement, spatial reference memory damage, Aβ plaque deposition in mouse brains, increasing microglia expression in the brain, and IL-6 higher expression in the cortex and in the serum. Conclusion: Low-dose Cd exposure for 6 months increases Aβ plaque deposition and BBB permeability, exacerbates inflammatory responses, and activates microglia, in APP/PS1 mice. APP/PS1 gene-environmental Cd interaction aggravates the progression of Alzheimer’s disease in mice.

Nano-SiO2 materials play a significant role in the engineered nanomaterials (ENMs) field. The ease of their production as well as their relatively low cost has promoted the wide use of these products in many fields. Nano-SiO2 exposure is known to cause severe DNA damage; however, the underlying mechanisms remain poorly understood. In a previous study, we found that nano-SiO2 exposure regulate the expression of the poly(ADP-ribose) polymerases-1 (PARP-1), a pivotal DNA repair gene, in human HaCaT cells. Here, we employed lentivirus-mediated RNA interference (RNAi) to knock down PAPR-1 expression in HaCaT cells and explored the potential role of PARP-1 in nano-SiO2 induced cytotoxicity. We found that nano-SiO2 treatment of HaCaT cells causes decreased cell viability, increased apoptosis and DNA damage. Nano-SiO2-treated HaCaT cells were also found to have slightly changed cell cycle distribution. Lentivirus-mediated PAPR-1 knockdown partially aggravated cytotoxicity and increased apoptosis induced by nano-SiO2 treatment. Nano-SiO2 had significant toxicity to human HaCaT cells and causes DNA damage. PAPR-1 knock-down cell line appears more sensitive to nano-SiO2 than the control cells in DNA damage. The results suggest that PAPR-1 is involved in protecting cells from damage caused by nano-SiO2.

Alzheimer's disease (AD) is a devastating neurodegenerative disease accompanied by neuropsychiatric symptoms, such as anxiety and depression. The levels of melatonin decrease in brains of AD patients. The potential effect of melatonin on anxiety and depression behaviors in AD and the underlying mechanisms remain unclear. In this study, we treated 10-month-old triple transgenic mice of AD (3xTg-AD) with melatonin (10 mg/kg body weight/day) for 1 month and explored the effects of melatonin on anxiety and depression-like behaviors in 3xTg-AD mice and the protein expression of hippocampal tissues. The behavioral test showed that melatonin ameliorated anxiety and depression-like behaviors of 3xTg-AD mice as measured by open field test, elevated plus maze test, forced swimming test, and tail suspension test. By carrying out two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, we revealed a total of 46 differentially expressed proteins in hippocampus between the wild-type (WT) mice and non-treated 3xTg-AD mice. A total of 21 differentially expressed proteins were revealed in hippocampus between melatonin-treated and non-treated 3xTg-AD mice. Among these differentially expressed proteins, glutathione S-transferase P 1 (GSTP1) (an anxiety-associated protein) and complexin-1 (CPLX1) (a depression-associated protein) were significantly down-regulated in hippocampus of 3xTg-AD mice compared with the WT mice. The expression of these two proteins was modulated by melatonin treatment. Our study suggested that melatonin could be used as a potential candidate drug to improve the neuropsychiatric behaviors in AD via modulating the expression of the proteins (i.e. GSTP1 and CPLX1) involved in anxiety and depression behaviors. © 2017 BioFactors, 43(4):593-611, 2017.

Environmental pollution and an unhealthy lifestyle result in direct exposure to dangerous chemicals that can modify endogenous pathways and induce malignant transformation of human cells. Although the molecular mechanisms of tumorigenesis are still not well understood, epigenetic alteration may be associated with exogenous chemical-induced carcinogenicity. Given the association between nutrition and cancer, nutrient supplementation may reduce aberrant epigenetic modifications induced by chemicals, thus decreasing carcinogenesis. This paper provides an overview of the epigenetic events caused by benzo[a]pyrene, a procarcinogenic and environmental pollutant, and biotin, an essential water-soluble vitamin, and investigates potential connections between them. This paper also discusses the potential inhibitory effect of biotin-related epigenetic modifications on the carcinogenicity of benzo[a]pyrene. The effect of nutritional supplementation on tumorigenesis involving epigenetic modifications is also discussed.

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by a progressive impairment of cognitive functions including spatial learning and memory. Excess copper exposure accelerates the development of AD; however, the potential mechanisms by which copper exacerbates the symptoms of AD remain unknown. In this study, we explored the effects of chronic copper exposure on cognitive function by treating 6 month-old triple AD transgenic (3xTg-AD) mice with 250 ppm copper sulfate in drinking water for 6 months, and identified several potential key molecules involved in the effects of chronic copper exposure on memory by proteomic analysis. The behavioral test showed that chronic copper exposure aggravated memory impairment of 3xTg-AD mice. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry revealed a total of 44 differentially expressed proteins (18 upregulated and 26 down-regulated) in hippocampus between the wild-type (WT) mice and non-exposed 3xTg-AD mice. A total of 40 differentially expressed proteins were revealed (20 upregulated and 20 down-regulated) in hippocampus between copper exposed and non-exposed 3xTg-AD mice. Among these differentially expressed proteins, complexin-1 and complexin-2, two memory associated proteins, were significantly decreased in hippocampus of 3xTg-AD mice compared with the WT mice. Furthermore, the expression of these two proteins was further down-regulated in 3xTg-AD mice when exposed to copper. The abnormal expression of complexin-1 and complexin-2 identified by proteomic analysis was verified by western blot analysis. Taken together, our data showed that chronic copper exposure accelerated memory impairment and altered the expression of proteins in hippocampus in 3xTg-AD mice. The functional analysis on the differentially expressed proteins suggested that complexin-1 and complexin-2 may be the key molecules involved in chronic copper exposure-aggravated memory impairment in AD.

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.

Growing concern has been raised over the potential adverse effects of engineered nanoparticles on human health due to their increasing use in commercial and medical applications. Silica nanoparticles (SiNPs) are one of the most widely used nanoparticles in industry and have been formulated for cellular and non-viral gene delivery in the central nerve system. However, the potential neurotoxicity of SiNPs remains largely unclear. In this study, we investigated the cellular uptake of SiNPs in human SK-N-SH and mouse neuro2a (N2a) neuroblastoma cells treated with 10.0 μg/ml of 15-nm SiNPs for 24 h by transmission electron microscopy. We found that SiNPs were mainly localized in the cytoplasm of the treated cells. The treatment of SiNPs at various concentrations impaired the morphology of SK-N-SH and N2a cells, characterized by increased number of round cells, diminishing of dendrite-like processes and decreased cell density. SiNPs significantly decreased the cell viability, induced cellular apoptosis, and elevated the levels of intracellular reactive oxygen species (ROS) in a dose-dependent manner in both cell lines. Additionally, increased deposit of intracellular β-amyloid 1-42 (Aβ(1-42)) and enhanced phosphorylation of tau at Ser262 and Ser396, two specific pathological hallmarks of Alzheimer's disease (AD), were observed in both cell lines with SiNPs treatment. Concomitantly, the expression of amyloid precursor protein (APP) was up-regulated, while amyloid-β-degrading enzyme neprilysin was down-regulated in SiNP-treated cells. Finally, activity-dependent phosphorylation of glycogen syntheses kinase (GSK)-3β at Ser9 (inactive form) was significantly decreased in SiNP-treated SK-N-SH cells. Taken together, these data demonstrated that exposure to SiNPs induced neurotoxicity and pathological signs of AD. The pre-Alzheimer-like pathology induced by SiNPs might result from the dys-regulated expression of APP/neprilysin and activation of GSK-3β. This is the first study with direct evidence indicating that in addition to neurotoxicity induced by SiNPs, the application of SiNPs might increase the risk of developing AD.

Chromium is a potent human mutagen and carcinogen. The capability of chromium to cause cancers has been known for more than a century, and numerous epidemiological studies have been performed to determine its carcinogenicity. In the post-genome era, cancer has been found to relate to epigenetic mutations. However, very few researches have focused on hexavalent chromium (Cr(VI))-induced epigenetic alterations. The present study was designed to investigate whether Cr(VI) would affect the level of a newfound epigenetic modification: histone biotinylation. Histone acetylation and histone biotinylation were studied in detail using human bronchial epithelial (16HBE) cells as an in vitro model after Cr(VI) treatment. Our study showed that Cr(VI) treatment decreased histone acetylation level in 16HBE cells. In addition, low doses of Cr(VI) (≤0.6μM) elevated the level of histone biotinylation. Furthermore, immunoblot analysis of biotinidase (BTD), a major protein which maintains homeostasis of histone biotinylation, showed that the distribution of BTD became less even and more concentrated at the nuclear periphery in cells exposed to Cr(VI). Moreover, Cr(VI)-induced histone deacetylation may take part in the regulation of histone biotinylation. Together, our study provides new insight into the mechanisms of Cr(VI)-induced epigenetic regulation that may contribute to the chemoprevention of Cr(VI)-induced cancers and may have important implications for epigenetic therapy.

Foxa2 is one member of the Foxa subfamily of winged helix/forkhead box (Fox) transcription factors which has been found to play important roles in multiple stages of mammalian life, beginning with early development, continuing during organogenesis, and finally in metabolism and homeostasis in the adult. To explore the involvement of Foxa2 and its epigenetic regulations in cellular senescence, we established the premature senescence model induced by hydrogen peroxide in comparison with replicative senescence. The mRNA level of Foxa2 was downregulated in both replicative and premature senescent cells. We further found the increased DNA methylation level and new methylation at CpG sites in the promoter with 43.6% of methylated CpG islands in premature senescence, while only 5.7% and 17.1% in young cells and replicative senescence separately. Moreover, we noted the alterations of histone modifications including decreased histone H3 acetylation, increased H4 (Lys-20) trimethylation at the Foxa2 CpG islands in the promoter in replicative or premature senescence, while decreased histone H3 (Lys-4) trimethylation across the transcription start regions in cellular senescence. Taken together, epigenetic silencing of Foxa2 is associated with an increased DNA methylation level and histone H4 (Lys-20) trimethylation, decreased histone H3 acetylation and histone H3 (Lys-4) trimethylation, involved in cellular replicative or premature senescence.

Mitochondrial dysfunction is involved in neurotoxicity caused by exposure of various chemicals such as copper. However, the effects of long-term low-dose copper exposure on mitochondrial proteome remain unclear. In this study, we found the treatment of copper (0.13ppm copper sulfate in drinking water) for 12 months caused abnormal expression of a total of 13 mitochondrial proteins (7 up-regulated and 6 down-regulated) as revealed by two-dimensional electrophoresis coupled with mass spectrometry in mouse cortex. Protein functional analysis revealed that these differentially expressed proteins mainly included apoptosis-associated proteins, axon guidance-associated proteins, axonogenesis-associated proteins and mitochondrial respiratory chain complex. Among these differentially expressed mitochondrial proteins, GRP75 (75kDa glucose-regulated protein) and GRP78 (78kDa glucose-regulated protein) were found to be significantly down-regulated as confirmed by Western-blot analysis. The down-regulation of GRP75 was shown to promote apoptosis. The down-regulation of GRP78/BiP could up-regulate endoplasmic reticulum (ER) stress mediators and thus cause apoptosis. Our study suggested that these differentially expressed mitochondrial proteins such as GRP75 and GRP78 could be involved in neurotoxicity caused by long-term low-dose copper exposure and serve as potential molecular targets for the treatment of copper neurotoxicity.

Human exposure to drinking water contaminated with arsenic is a serious global health concern and it predisposes people to cardiovascular diseases, such as hypertension, atherosclerosis, and microvascular diseases. Although accumulating evidence supports a role for angiogenesis responses to arsenic in the pathogenesis of the cardiovascular disease, the detailed molecular mechanism is not well understood. We aimed to determine the role and mechanism of microRNA (miRNA) in arsenic-induced angiogenesis. In our present study, sodium arsenite (NaAsO2) inhibited angiogenesis by decreasing cells proliferation, migration and tube formation in HUVECs. After NaAsO2 treatment, we found the expression of microRNA-425-5p (miR-425-5p) was reduced in vitro and in vivo and over-expression of miR-425-5p reversed the NaAsO2-induced anti-angiogenesis through its direct target cerebral cavernous malformation 3 (CCM3). Furthermore, we showed that NaAsO2 up-regulated CCM3 expression in vitro and in vivo. In addition, we demonstrated that inhibition of Notch and activation of VEGF/p38 signaling were involved in miR-425-5p blocking NaAsO2-induced anti-angiogenesis.

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.

DNA polymerase eta (Polη), the product of the xeroderma pigmentosum variant gene, is required for translesion DNA synthesis, and plays a pivotal role in preventing genome instability after DNA damage induced by genotoxic agents. Studies have previously suggested a link between Polη and susceptibility to hydroquinone (HQ)-induced toxicity. To further address the role of Polη in the response of L-02 cells to HQ, we employed RNA interference to silence Polη expression in L-02 cells and examined the susceptibility of these Polη-deficient cells to the toxic effects of HQ. In this study, cell survival rate was determined using the MTT assay, DNA damage was determined by the Comet assay, apoptosis and cell cycle distribution were determined using flow cytometry, the mRNA expression levels of Polη were determined by real-time PCR, and the protein expression levels of Polη and γ-H2AX were determined by Western blot, γ-H2AX foci were visualized by confocal laser scanning fluorescence microscopy after cells were exposed to HQ at various concentrations for 24 h in vitro. The results showed that stable Polη-knockdown cells were successfully constructed and more than 80% inhibition of Polη expression was confirmed. The results also showed that down-regulation of Polη led to a decrease in cell proliferation and an enhanced susceptibility to HQ-induced cytotoxicity. Polη-deficient cells were 2-fold more sensitive to HQ when compared with nonspecific siRNA control cells. Moreover, Polη-silenced L-02 cells treated with HQ displayed an increased level of DNA double-strand breaks as measured by olive tail moment, and an elevated DNA damage response as indicated by the induction of γ-H2AX. In addition, knockdown of Polη resulted in more enhanced apoptosis and more pronounced S phase arrest following HQ treatment. Together, these results show that Polη plays an important role in the response of L-02 cells to HQ-induced DNA damage.

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.

Hydrogen peroxide (H2O2), 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 H2O2 at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H2O2-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 H2O2. In this study, a human embryo lung fibroblast (MRC-5) cell line was used to model an adaptive response to H2O2, and the relevant differential gene expressions by using fluoro mRNA differential display RT-PCR. The results showed significant suppression of cytotoxicity of H2O2 (1100 μM, 1 h) after pretreatment of the cells with H2O2 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 H2O2 in human cells, at least in this particular cell type. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 478–485, 2014.

Trichloroethylene (TCE) is a common chemical pollutant that exists in air, soil, and drinking water. TCE exposure is known to cause severe hepatotoxicity; however, the mechanisms underlying TCE hepatotoxicity remain poorly understood. In a previous proteomics study, we found that TCE exposure up-regulated the expression of the inhibitor 2 of protein phosphatase 2A (I2PP2A), a potent and specific endogenous inhibitor of protein phosphatase (PP) 2A, in human hepatic L-02 cells. Here, we employed lentivirus-mediated RNA interference (RNAi) to knock down I2PP2A expression in L-02 cells and explored the potential role of I2PP2A in TCE-induced cytotoxicity. We found that TCE treatment of L-02 cells causes decreased cell viability, increased apoptosis and elevated I2PP2A mRNA and protein levels. TCE-treated L-02 cells were also found to have significantly reduced PP2A activity. Lentivirus-mediated I2PP2A knockdown partially prevented the decrease in viability and increased apoptosis induced by TCE treatment. Knockdown of I2PP2A in TCE-treated L-02 cells also suppressed the inhibition of PP2A activity and prevented caspase-3 activation. These data for the first time demonstrate that the up-regulation of I2PP2A could mediate, at least in part, TCE-induced liver cell toxicity through the inhibition of PP2A activity and caspase-3-mediated pathway, and suggest that I2PP2A may play a crucial role in mediating TCE hepatotoxicity.

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.

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.

The aim of this study is to investigate epigenetic mechanism of ABCG2 induced drug-resistance. It is not only expatiate for drug-resistance regulation mechanism in all-round, but also to provide scientific experimental basis for selecting target to reverse its drug-resistance. Apply methylation-specific PCR (MSP) to have tested methylation of ABCG2 promoter region −359 to −353 specific positions in breast cancer tissues and paired adjacent tissue of 22 cases, and test their methylation positions with MSP products for sequencing; and adopt fluorescent quantitation RT-PCR to test expression level DNMT1, DNMT3A, DNMT3B and ABCG2; to make analysis on relationship between them with statistical spearman correlation. Specific positions of ABCG2 gene promoter region of 18 cases among the 22 cases with breast cancer (18/22, 82%) existed high methylation (P

Hexavalent chromium (Cr(VI)), a commonly used industrial metal, is a well-known mutagen and carcinogen, and occupational exposure can induce a broad spectrum of adverse health effects, including cancers. Although Cr(VI)-induced DNA damage is thought to be the primary mechanism of chromate genotoxicity and mutagenicity, there is an increasing number of reports showing that epigenetic mechanisms of gene regulation might be a central target of Cr(VI) toxicity. Epigenetic changes, such as changes in phosphorylation, altered DNA methylation status, histone acetylation and signaling pathways, have been observed after chromium exposure. Nevertheless, to better demonstrate the roles of epigenetic modifications in Cr(VI)-induced carcinogenesis, more work needs to be carried out. This study is aimed to investigate changes in biotinidase (BTD) and holocarboxylase synthetase (HCS), two major proteins which maintain homeostasis of the newfound epigenetic modification: histone biotinylation, in cells exposed to Cr(VI). The data showed that Cr(VI) decreased BTD expression at the transcriptional level in human bronchial epithelial cells (16HBE). In addition, using the epigenetic modifiers, 5-Aza-2'-deoxycytidine (Aza) and Trichostatin A (TSA), we found that modifications of histone acetylation reversed the inhibition of BTD, suggesting that Cr(VI) may cause down regulation of BTD by modifications of histone acetylation.

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.

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 large samples (n = 953) of healthy individuals from five Chinese ethnic populations by using PCR-RFLP and HPLC-ECD. Life-style parameters under study were obtained through a questionnaire. The allelic frequencies of the hOGG1 gene in the Chinese populations were found to be 0.16 (Ser/Ser), 0.49 (Ser/Cys) and 0.35 (Cys/Cys), respectively. The frequencies of Ser326Cys polymorphism were significantly different among the five Chinese 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). These results suggest that there are natural variations of hOGG1 gene among Chinese ethnic populations. Smoking relates to Cys/Cys polymorphism frequencies, and oxidative DNA damage is repaired less in individuals with the hOGG1 Cys326Cys genotype.

Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. To uncover the molecular mechanisms of nickel sulfide (NiS)-induced cell transformation, we investigated epigenetic alterations in a set of DNA repair genes. The silencing of the O(6)-methylguanine DNA methyltransferase (MGMT) gene locus and upregulation of DNA methyltransferase 1 (DNMT1) expression was specifically detected in NiS-transformed human bronchial epithelial (16HBE) cells. In addition, we noted epigenetic alterations including DNA hypermethylation, reduced histone H4 acetylation and a decrease in the ratio of Lys-9 acetylated/methylated histone H3 at the MGMT CpG island in NiS-transformed 16HBE cells. Meanwhile, we identified concurrent binding of methyl-CpG-binding protein 2, methylated DNA-binding domain protein 2 and DNMT1 to the CpG island of the MGMT promoter, demonstrating that these components collaborate to maintain MGMT methylation in NiS-transformed cells. Moreover, depletion of DNMT1 by introduction of a small hairpin RNA construct into NiS-transformed cells resulted in a 30% inhibition of cell proliferation and led to increased MGMT gene expression by reversion of the epigenetic modifications at the MGMT promoter region. MGMT suppression and hypermethylation at the CpG island of the MGMT promoter occurred 6 days after NiS treatment, indicating that epigenetic modifications of MGMT might be an early event in tumorigenesis. Taken together, these observations demonstrate that epigenetic silencing of MGMT is associated with DNA hypermethylation, histone modifications and DNMT1 upregulation, which contribute to NiS-induced malignant transformation.

Polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/Fs) concentrations in 31 fish samples from 19 commonly consumed freshwater and saltwater species from the Pearl River Delta Area were analyzed. The PCDD/Fs dietary intake from fish for the local population was evaluated to provide a database for setting the national PCDD/F limits in fish for the People’s Republic of China. The median concentration from the 31 fish samples was 1.27 pg/g wet weight for the total of PCDD/Fs, and the median WHO-TEQ was 0.26 pg/g wet weight, and ranged from 0.063 to 1.30 pg WHO-TEQ/g wet weight. The dominant contributors to the WHO-TEQ were 1,2,3,7,8-PeCDD and 2,3,4,7,8-PeCDF, which accounted for 38% and 28%, respectively. The dietary intake of PCDD/Fs from fish for local people was estimated to be 0.47 pg WHO-TEQ/kg bw · day. In view of the findings, the dietary of PCDD/Fs from other foods of animal origins in China should be studied in more detail as soon as possible in order that standards can be put forward to protect human health.

Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was upregulated and hippocampal neuronal apoptosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway.

Histone modifications are major post-translational mechanisms responsible for regulation of gene transcription involved in cellular senescence. By using immunofluorescence and Western blot, we showed that the global acetylated levels of histone H3 and H4 were significantly reduced in both replicative and premature senescence of human embryonic lung fibroblasts. However the whole trimethylated level of histone H4 lysine 20 was higher in senescent cells. The alterations in the mRNA and protein levels of histone acetyltransferases (HATs), histone methyltransferase (HMT), and histone deacetylases (HDACs) indicate that differential expression exists between replicative and premature senescent cells. Meanwhile, the reduced activity of HDACs was accompanied by cellular senescence. By employing the quantitative chromatin immunoprecipitation assay in detecting specific histone modifications in senescence-related genes including p53 and p16, it was demonstrated that the mRNA expression of p53 was associated with increased H4 acetylation in replicative senescence and increased H4 acetylation and trimethylation of histone H3 at lysine 4 (H3K4me3) in premature senescence. Both acetylation and trimethylation of H3 were involved in replicative senescence, while the acetylation of histone H3 and H4 was predominant in premature senescence, contributing to the mRNA expression of p16. In summary, the global hypoacetylation of histone H3 and H4 and the hypertrimethylation of histone H4 lysine 20 account for epigenetic characteristics in senescence, controlled by HATs, HMT, and HDACs differentially between replicative and premature senescence. Taken together, these findings suggest that the specific histone modifications are involved in regulating the expression of genes related to senescence of human embryonic lung fibroblasts.

Trichloroethylene (TCE) is an effective solvent for a variety of organic materials. Since the wide use of TCE as industrial degreasing of metals, adhesive paint and polyvinyl chloride production, TCE has turned into an environmental and occupational toxicant. Exposure to TCE could cause severe hepatotoxicity; however, the toxic mechanisms of TCE remain poorly understood. Recently, we reported that SET protein mediated TCE-induced cytotoxicity in L-02 cells. Here, we further identified the proteins related to SET-mediated hepatic cytotoxicity of TCE using the techniques of DIGE (differential gel electrophoresis) and MALDI-TOF-MS/MS. Among the 20 differential proteins identified, 8 were found to be modulated by SET in TCE-induced cytotoxicity and three of them (cofilin-1, peroxiredoxin-2 and S100-A11) were validated by Western-blot analysis. The functional analysis revealed that most of the identified SET-modulated proteins are apoptosis-associated proteins. These data indicated that these proteins may be involved in SET-mediated hepatic cytotoxicity of TCE in L-02 cells.

It has been previously reported that Bisphenol A (BPA) can disturb the development of mammary structure and increase the risk of breast cancer in experimental animals. In this study, an in vitro model of human embryonic stem cell (hESC) differentiation into mammary epithelial cells was applied to investigate the effect of low dose BPA on the early stages of mammogenesis. A newly established hESC line was directionally differentiated into mammary epithelial cells by a well-established three-dimensional (3D) culture system. The differentiated mammary epithelial cells were characterized by immunofluorescence and western blotting assay, and were called induced differentiated mammary epithelial cells (iDMECs) based on these data. The hESCs were treated with low doses of BPA range 10 −9 –10 −6 M during the differentiation process, with DMSO as the solvent control and 17-β-estrodiol (E2) as the estrogen-positive control. Our results showed that low dose BPA and E2 could influence the mammosphere area of iDMECs and upregulate the expression level of Oct4 and Nanog proteins, while only BPA could downregulate the expression of E-cadherin protein. Taken together, this study provides some insights into the effects of low dose BPA on the early differentiation stage of mammary epithelial cells and suggests an easier canceration status of iDMECs under the effect of low dose BPA during its early differentiation stage.

Lead (Pb) contamination in the air is a severe global problem, most notably in China. Removal of Pb from polluted air remains a significant challenge. It is unclear what potential effects silica nanoparticles (SiNPs) exposure can have on atmospheric Pb. Here we first characterized the features of SiNPs by measuring the particle size, zeta potential and the specific surface area of SiO(2) particles using a Nicomp 380/ZLS submicron particle sizer, the Brunauer-Emmett-Teller (BET) method and transmission electronic microscopy (TEM). We measured the content of the metal Pb adsorbed by SiNPs exposed to two Pb polluted electric battery plants using inductively coupled plasma mass spectrometry (ICP-MS). It is found that SiNPs exposed to two Pb polluted electric battery plants absorb more atmospheric Pb compared to either blank control or micro-sized SiO(2) particles in a time-dependent manner. This is the first study demonstrating that SiNPs exposure can absorb atmospheric Pb in the polluted environment. These novel findings indicate that SiNPs have potential to serve as a significant adsorbent of Pb from industrial pollution, implicating a potentially novel application of SiNPs in the treatment of environmental heavy metal pollution.

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.

Background Nanometer silicon dioxide (nano-SiO2) has a wide variety of applications in material sciences, engineering and medicine; however, the potential cell biological and proteomic effects of nano-SiO2 exposure and the toxic mechanisms remain far from clear. Results Here, we evaluated the effects of amorphous nano-SiO2 (15-nm, 30-nm SiO2). on cellular viability, cell cycle, apoptosis and protein expression in HaCaT cells by using biochemical and morphological analysis, two-dimensional differential gel electrophoresis (2D-DIGE) as well as mass spectrometry (MS). We found that the cellular viability of HaCaT cells was significantly decreased in a dose-dependent manner after the treatment of nano-SiO2 and micro-sized SiO2 particles. The IC50 value (50% concentration of inhibition) was associated with the size of SiO2 particles. Exposure to nano-SiO2 and micro-sized SiO2 particles also induced apoptosis in HaCaT cells in a dose-dependent manner. Furthermore, the smaller SiO2 particle size was, the higher apoptotic rate the cells underwent. The proteomic analysis revealed that 16 differentially expressed proteins were induced by SiO2 exposure, and that the expression levels of the differentially expressed proteins were associated with the particle size. The 16 proteins were identified by MALDI-TOF-TOF-MS analysis and could be classified into 5 categories according to their functions. They include oxidative stress-associated proteins; cytoskeleton-associated proteins; molecular chaperones; energy metabolism-associated proteins; apoptosis and tumor-associated proteins. Conclusions These results showed that nano-SiO2 exposure exerted toxic effects and altered protein expression in HaCaT cells. The data indicated the alterations of the proteins, such as the proteins associated with oxidative stress and apoptosis, could be involved in the toxic mechanisms of nano-SiO2 exposure.

Chinese hamster ovary cells were incubated with radioactive amino acids, the DNA was isolated by standard proteinase K/phenol/chloroform extraction and residual amino acids complexed to the DNA were examined as an index of metal induced DNA-protein crosslinks. Using this method, both chromate and nickel caused residual histidine and cysteine to be complexed with the DNA isolated from metal-treated cells. In the case of chromate, a number of amino acids were studied and Tyr, Thr and Cys were found to be complexed to DNA at a level (above the untreated control) that was statistically significant. Stability studies indicated that some of the chromate-induced DNA-protein complexes were mediated by direct participation of chromium(III), whereas others that were resistant to dissociation by EDTA and mercaptoethanol did not seem to involve direct chromium(III) participation. A significant portion of the cysteine complexed to DNA by chromate was believed to involve glutathione since treatment of cells with cycloheximide did not decrease chromate-induced cysteine-DNA crosslinks. In the case of nickel, most of the stable DNA-protein crosslinks did not involve direct metal participation and were probably oxidatively mediated by Ni(II)/Ni(III) redox cycling. These findings present new methodology for analysis of DNA-protein crosslinks by examination of residual amino acids associated with the DNA. This method should be highly sensitive and will yield important information about the mechanism of metal-induced DNA-protein crosslinks.

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.

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.

DNA methylation is considered to play an essential role in cellular senescence. To uncover the mechanism underlying cellular senescence, we established the model of premature senescence induced by hydrogen peroxide (H(2)O(2)) in human embryonic lung fibroblasts and investigated the changes of genome methylation, DNA methyltransferases (DNMTs) and DNA-binding domain proteins (MBDs) in comparison with those observed during normal replicative senescence. We found that premature senescence triggered by H(2)O(2) exhibited distinct morphological characteristics and proliferative capacity which were similar to those of replicative senescence. The genome methylation level decreased gradually during the premature as well as replicative senescence, which was associated with the reduction in the expression of DNMT1, reflecting global hypomethylation as a distinct feature of senescent cells. The levels of DNMT3b and methyl-CpG binding protein 2 (MeCP2) increased in both mid-aged and replicative senescent cells, while DNMT3a and MBD2 were upregulated in the mid-aged cells. Only DNMT3b was elevated in the cells in the premature senescence persistence status. Additionally, the expression for DNMTs, MBD2 and MeCP2 was increased rapidly upon H(2)O(2) treatment. These results indicate that H(2)O(2)-induced premature senescence share some features of replicative senescence, such as basic biological characteristics and global hypomethylation while there are slight differences in the profile of methylation-associated enzyme expression. Oxidative damage may hence be a causative factor in epigenetic alteration partly responsible for cellular senescence.