Effects of Selenium on DNA Binding Activity of Nuclear Factor-κB Induced by Arsenic in Mouse Skin JB6-CI41 Cells in vitro

目的 研究硒对砷诱导小鼠皮肤JB6-CI41细胞核因子κB(nuclear factor κB,NF-κB)的DNA结合活性和细胞凋亡的作用.方法 调整JB6-CI41细胞密度为1×105/孔,分别设立对照(生理盐水)组,不同浓度的亚砷酸(3.125、6.25、12.5 μmol/L)和亚硒酸钠(2.5 μmol/L)单独染毒组,亚硒酸钠(2.5μmol/L)+不同浓度的亚砷酸(3.125、6.25和12.5μmol/L)联合染毒组,NF-κB特异化学抑制剂二乙基二硫代氨基甲酸盐(diethyldithiocarbamate,DTYC,100 μmol/L,提前染毒2 h)+亚砷酸(6.25 μmol/L)联合染毒组.采用凝胶阻滞电泳法(electrophoretic mobility shift assay,EMSA)检测NF-κB的DNA结合活性,采用流式细胞术检测细胞凋亡情况.结果 2.5 μmol/L亚硒酸钠对小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性和细胞凋亡无显著影响(P＞0.05).6.25和12.5 μmol/L的亚砷酸能够增强小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性(P＜0.01),具有明显剂量-效应关系(r=0.942 6,P＜0.01).3.125、6.25和12.5 μmol/L亚砷酸诱导小鼠皮肤JB6-CI41细胞凋亡(P＜0.01),呈现显著剂量-反应关系(r=0.991 8,P＜0.01).NF-κB的DNA结合活性与细胞凋亡率呈显著正相关(r=0.977 5,P＜0.01).2.5 μmol/L亚硒酸钠能抑制6.25和12.5 μmol/L亚砷酸作用下的小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性(P＜0.01),对3.125、6.25和12.5 μmol/L的亚砷酸引起的小鼠皮肤JB6-CI41细胞凋亡率也表现出显著抑制作用(P＜0.01).100 μmol/L的DTYC对6.25μmol/L亚砷酸引起的小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性和细胞凋亡率具有显著抑制作用(P＜0.01).结论 在本实验中,一定剂量(6.25～12.5 μmol/L)的砷能够使小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性显著增高并诱导细胞凋亡,2.5 μmol/L的硒能抑制砷引起的NF-κB的活化和细胞凋亡.     

Effects of Atrazine on Estrogen Receptor α– and G Protein–Coupled Receptor 30–Mediated Signaling and Proliferation in Cancer Cells and Cancer-Associated Fibroblasts

Background: The pesticide atrazine does not bind to or activate the classical estrogen receptor (ER), but it up-regulates the aromatase activity in estrogen-sensitive tumor cells. The G protein estrogen receptor (GPR30/GPER) has been reported to be involved in certain biological responses to endogenous estrogens and environmental compounds exerting estrogen-like activity.Objectives: We aimed to evaluate the potential of atrazine to trigger GPER-mediated signaling in cancer cells and cancer-associated fibroblasts (CAFs).Methods and Results: Using gene reporter assays in diverse types of cancer cells, we found that atrazine did not transactivate endogenous ERα or chimeric proteins that encode the ERα and ERβ hormone binding domains. Conversely, atrazine was able to bind to GPER to induce ERK activation and the expression of estrogen target genes, which, interestingly, appeared to rely on both GPER and ERα expression. As a biological counterpart, atrazine stimulated the proliferation of ovarian cancer cells that depend on GPER and ERα, as evidenced by gene silencing experiments and the use of specific signaling inhibitors. Of note, through GPER, atrazine elicited ERK phosphorylation, gene expression, and migration in CAFs, thus extending its stimulatory role to these main players of the tumor microenvironment.Conclusions: Our results suggest a novel mechanism through which atrazine may exert relevant biological effects in cancer cells and CAFs. On the basis of our data, atrazine should be included among the environmental contaminants that may elicit estrogenic activity through GPER-mediated signaling.Citation: Albanito L, Lappano R, Madeo A, Chimento A, Prossnitz ER, Capello AR, Dolce V, Abonante S, Pezzi V, Maggiolini M. 2015. Effects of atrazine on estrogen receptor α– and G protein–coupled receptor 30–mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts. Environ Health Perspect 123:493–499; http://dx.doi.org/10.1289/ehp.1408586     

Prostaglandin E2 Is Involved in the Increase of Cytochrome P-450 2B1 Expression by α-Tocopheryl Succinate in Primary Rat Hepatocytes in the Presence of Phenobarbital

The modulation of cytochrome P-450 2B1 expression by α-tocopheryl succinate and whether prostaglandin E₂ is involved in this modulation in primary rat hepatocytes in the presence of phenobarbital were investigated. A primary rat hepatocyte culture model that faithfully reproduces the phenobarbital response observed in vivo was used. Intracellular α-tocopherol content was dose dependently increased by α-tocopheryl succinate incubation. Hepatocytes were demonstrated to have prostaglandin E₂-synthesizing capability. α-Tocopheryl succinate inhibited prostaglandin E₂ synthesis by hepatocytes and increased cytochrome P-450 2B1 expression in the presence of phenobarbital; however, it had little effect on intracellular cAMP level. To mimic the exogenous source of prostaglandin E₂ from nonparenchymal cells, various concentrations of prostaglandin E₂ were added to the cell culture. High doses of exogenous prostaglandin E₂ (100 and 1,000 nM) inhibited the cytochrome P-450 2B1 expression in the presence of phenobarbital compared with low doses (1 and 10 nM); however, the presence of high doses of prostaglandin E₂ had no effect on intracellular cAMP level. Forskolin significantly increased intracellular cAMP level and inhibited cytochrome P-450 2B1 expression in the presence of phenobarbital. The results of this study indicate that α-tocopheryl succinate increases cytochrome P-450 2B1 expression via its inhibition of prostaglandin E₂ synthesis in the presence of phenobarbital; however, changes in intracellular cAMP level are not related to cytochrome P-450 2B1 expression.     

The citrus flavonoid hesperidin induces p53 and inhibits NF-κB activation in order to trigger apoptosis in NALM-6 cells: involvement of PPARγ-dependent mechanism

Background  

Hesperidin, a flavanone present in citrus fruits, has been identified as a potent anticancer agent because of its proapoptotic and antiproliferative characteristics in some tumor cells. However, the precise mechanisms of action are not entirely understood.     

Alcohol-fermented soybean increases the expression of receptor-interacting protein 2 and IκB kinase β in mouse peritoneal macrophages

Soybean is a useful component of traditional Korean medicine with well-documented health-promoting effects. We investigated the effects of alcohol-fermented soybean (AFS) on immune function. When AFS treatment was used in combination with recombinant interferon-γ (rIFN-γ), there was a marked cooperative induction of nitric oxide (NO) and tumor necrosis factor (TNF)-α production in mouse peritoneal macrophages. AFS increased the expression of inducible NO synthase mRNA and protein in rIFN-γ-primed macrophages. Treating macrophages with pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-κB (NF-κB), decreased the synergistic effects of AFS. In addition, AFS in combination with rIFN-γ increased the phosphorylation of p38 and c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. However, AFS had no effect on phosphorylation of mitogen-activated protein kinases by itself. The p38 inhibitor SB203580 or the JNK inhibitor SP600125 inhibited the AFS-induced NO and TNF-α production. When AFS was used in combination with rIFN-γ, there was a co-operative activation of NF-κB and receptor-interacting protein 2 (Rip2)/IκB kinase (IKK)-β. Our results indicate that AFS increases the production of NO and TNF-α through the activation of Rip2/IKK-β in rIFN-γ-primed macrophages.     

The APE1 Asp/Asp Genotype and the Combination of APE1 Asp/Asp and hOGG1-Cys Variants Are Associated With Increased p53 Mutation in Non–Small Cell Lung Cancer

Background

The hOGG1 Ser326Cys polymorphism is associated with lung cancer risk, but there are limited data regarding an association between the APE1 Asp148Glu polymorphism and lung cancer. Biological evidence shows that the hOGG1-Cys allele results in less DNA repair activity; however, this is not associated with p53 mutation in lung cancer. Therefore, we investigated whether an interaction between hOGG1 and APE1 is associated with the frequency of p53 mutation in lung cancer.

Methods

We studied 217 Taiwanese adults with primary lung cancer. DNA polymorphisms of hOGG1 and APE1 were determined by polymerase chain reaction (PCR)-based restriction fragment length polymorphism. Mutations in p53 exons 5–8 were detected by direct sequencing. Multiple logistic regression was used to estimate odds ratios (ORs) and 95% CIs for the risk of p53 mutation associated with polymorphisms of hOGG1 and APE1 in lung cancer.

Results

As expected, no association between hOGG1 polymorphism and p53 mutation was observed in this population. However, a higher risk of p53 mutation was found in participants with the APE1 Asp/Asp genotype than in those with the APE1-Glu allele (OR, 2.15; 95% CI, 1.19–3.87; P = 0.011). The risk of p53 mutation was also higher in participants with APE1 Asp/Asp plus hOGG1-Cys than in those with APE1-Glu plus hOGG1 Ser/Ser (OR, 3.72; 95% CI, 1.33–10.40; P = 0.012).

Conclusions

These results suggest that the APE1 Asp/Asp genotype and the combination of the APE1 Asp/Asp and hOGG1-Cys variants are associated with increased risk of p53 mutation in non–small cell lung cancer.Key words: hOGG1 Ser326Cys, APE1 Asp148Glu, p53 mutation, NSCLC     

Saponins from the Roots of Platycodon grandiflorum Suppresses TGFβ1-Induced Epithelial-Mesenchymal Transition Via Repression of PI3K/Akt,ERK1/2 and Smad2/3 Pathway in Human Lung Carcinoma A549 Cells

Transforming growth factor β (TGFβ) is a multifunctional cytokine that induces growth arrest, tissue fibrosis, and epithelial-mesenchymal transition (EMT) through activation of Smad and non-Smad signaling pathways. EMT is the differentiation switch by which polarized epithelial cells differentiate into contractile and motile mesenchymal cells. Our previous studies have shown that saponins from the roots of Platycodon grandiflorum (CKS) have antiinflammatory, antioxidant, antimetastatic, and hepatoprotective effects. In this study, we investigated the inhibitory effect of CKS on TGFβ1-induced alterations characteristic of EMT in human lung carcinoma A549 cells. We found that CKS-treated cells displayed inhibited TGFβ1-mediated E-cadherin downregulation and Vimentin upregulation and also retained epithelial morphology. Furthermore, TGFβ1-increased Snail expression, a repressor of E-cadherin and an inducer of the EMT, was reduced by CKS. CKS inhibited TGFβ1-induced phosphorylation of Akt, ERK1/2, and glycogen synthase kinase-3β (GSK-3β). Inhibition of PI3K/Akt and ERK1/2 also blocked TGFβ1-induced GSK-3β phosphorylation and Snail activation. Furthermore, TGFβ1-increased Snail expression was reduced by selective inhibitors of Akt and ERK1/2. Moreover, CKS treatment attenuated TGFβ1-induced Smad2/3 phosphorylation and upregulated Smad7 expression. These results indicate that pretreatment with the CKS inhibits the TGFβ1-induced EMT through PI3K/Akt, ERK1/2, GSK-3β and Smad2/3 in human lung carcinoma cells.     

Polymorphism of Type I–F CRISPR/Cas system in Escherichia coli of phylogenetic group B2 and its application in genotyping

E. coli of phylogenetic group B2 is responsible for many extraintestinal infections, posing a great threat to health. The relatively polymorphic nature of CRISPR in phylogenetically related E. coli strains makes them potential markers for bacterial typing and evolutionary studies. In the current work, we investigated the occurrence and diversity of CRISPR/Cas system and explored its potential for genotyping. Type I–F CRISPR/Cas systems were found in 413 of 1190 strains of E. coli and exhibited the clustering within certain CCs and STs. And CRISPR spacer contents correlated well with MLST types. The divergence analysis of CRISPR showed stronger discriminatory power than MLST, and CRISPR polymorphism was instrumental for differentiating highly closely related strains. The timeline of spacer acquisition and deletion provided important information for inferring the evolution model between distinct serotypes. Identical spacer sequences were shared by strains with the same H-antigen type but not strains with the same O-antigen type. The homology between spacers and antibiotic-resistant plasmids demonstrated the role of Type I–F system in limiting the acquisition of antimicrobial resistance. Collectively, our data presents the dynamic nature of Type I–F CRISPR in E. coli of phylogenetic group B2 and provides new insights into the application of CRISPR-based typing in the species.     

Chrysophyllum albidum fruit peel attenuates nociceptive pain and inflammatory response in rodents by inhibition of pro-inflammatory cytokines and COX-2 expression through suppression of NF-κB activation

Chrysophyllum abidum fruit is a seasonal fruit commonly eaten as snacks with abundant health promoting phytochemicals in the fruit peels. The fruit peels have been reported to be rich in anti-inflammatory eleagnine, myricetin rhamnoside, quercetin, linoleic acid and oleic acid. We hypothesized that the anti-inflammatory effect of the peel extract involve suppression of pro-inflammatory cytokines, cyclooxygenase-2 and nuclear factor-kappa B (NF-κB). Hence, this study was designed to assess the anti-nociceptive and anti-inflammatory effects of fruit peel extract of Chrysophyllum albidum in animal models of nociception and inflammation. The anti-nociceptive activity of CAPEE (100 and 400 mg/kg) was evaluated in acetic acid-induced writhing and formalin-induced paw licking in mice. Formalin-induced paw edema and carrageenan-induced air pouch models of inflammation were used to evaluate the anti-inflammatory activity. CAPEE (100 and 400 mg/kg) significantly reduced abdominal writhing and paw licking in acetic acid and formalin tests in mice, respectively. CAPEE demonstrated significant inhibition of paw edema at 24 h (41.0% and 55.7%) and 72 h (52.3% and 86.6%) after formalin injection. CAPEE suppressed inflammatory responses in carrageenan-induced air pouch by reducing exudates, inflammatory cells infiltration, nitrites and myeloperoxidase activity. There was significant inhibition of tumor necrosis factor-alpha, interleukin-6 levels and reduced immunopositive expression of COX-2 and NF-κB. In conclusion, CAPEE has anti-nociceptive and anti-inflammatory potentials via mechanisms associated with inhibition of pro-inflammatory cytokines and cyclooxygenase-2 (COX-2) expression through suppression of nuclear factor kappa B (NF-κB) activation, and has potential as a functional food ingredient.     

Effects of Laser Printer–Emitted Engineered Nanoparticles on Cytotoxicity,Chemokine Expression,Reactive Oxygen Species,DNA Methylation,and DNA Damage: A Comprehensive in Vitro Analysis in Human Small Airway Epithelial Cells,Macrophages, and Lymphoblasts

Background

Engineered nanomaterials (ENMs) incorporated into toner formulations of printing equipment become airborne during consumer use. Although information on the complex physicochemical and toxicological properties of both toner powders and printer-emitted particles (PEPs) continues to grow, most toxicological studies have not used the actual PEPs but rather have primarily used raw toner powders, which are not representative of current exposures experienced at the consumer level during printing.

Objectives

We assessed the biological responses of a panel of human cell lines to PEPs.

Methods

Three physiologically relevant cell lines—small airway epithelial cells (SAECs), macrophages (THP-1 cells), and lymphoblasts (TK6 cells)—were exposed to PEPs at a wide range of doses (0.5–100 μg/mL) corresponding to human inhalation exposure durations at the consumer level of 8 hr or more. Following treatment, toxicological parameters reflecting distinct mechanisms were evaluated.

Results

PEPs caused significant membrane integrity damage, an increase in reactive oxygen species (ROS) production, and an increase in pro-inflammatory cytokine release in different cell lines at doses equivalent to exposure durations from 7.8 to 1,500 hr. Furthermore, there were differences in methylation patterns that, although not statistically significant, demonstrate the potential effects of PEPs on the overall epigenome following exposure.

Conclusions

The in vitro findings obtained in this study suggest that laser printer–emitted engineered nanoparticles may be deleterious to lung cells and provide preliminary evidence of epigenetic modifications that might translate to pulmonary disorders.

Citation

Pirela SV, Miousse IR, Lu X, Castranova V, Thomas T, Qian Y, Bello D, Kobzik L, Koturbash I, Demokritou P. 2016. Effects of laser printer–emitted engineered nanoparticles on cytotoxicity, chemokine expression, reactive oxygen species, DNA methylation, and DNA damage: a comprehensive in vitro analysis in human small airway epithelial cells, macrophages, and lymphoblasts. Environ Health Perspect 124:210–219; http://dx.doi.org/10.1289/ehp.1409582     

Understanding the complex associations between patient–provider relationships,self-care behaviours,and health-related quality of life in type 2 diabetes: A structural equation modeling approach

Objectives: To assess associations between perceptions of the patient–provider relationship (PPR), BMI, adherence to diet and exercise in a rural population with type 2 diabetes and determine how these variables relate to HRQL.Methods: A model of the hypothesized relationships between the constructs was proposed and tested using structural equation modeling (SEM) and data collected as part of a controlled study to improve care for individuals with type 2 diabetes in rural health regions.Results: In the final model, positive perceptions of the PPR had a direct impact on adherence to diet (β = 0.23; p <0.05), exercise (β = 0.13; p <0.05) and diabetes management attitudes (β = 0.33; p <0.05). The direct path from management attitudes to exercise was also significant (β = 0.12; p <0.05). Direct predictors of HRQL included management attitudes (β = 0.16; p <0.05), exercise adherence (β = 0.14, p <0.05) and BMI (β = −0.23; p <0.05). Exercise adherence predicted BMI, whereas adherence to diet did not. The final model had an acceptable fit with the measured data (χ² = 30.6 (26, N = 372), p = 0.25; RMSEA = 0.02; TLI = 0.98; SRMR = 0.02).Conclusion: Patient–provider relationship and exercise adherence appeared to be key constructs in the model. HRQL in people with type 2 diabetes was positively associated with exercise adherence, which was related to a positive PPR. Adherence to diet was also related to a positive PPR, but diet adherence had no association with HRQL.