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1.
J Nanosci Nanotechnol ; 15(1): 492-503, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26328389

RESUMO

The effect of titanium dioxide nanoparticles (nano-TiO2 Degussa p25) treatment of human lung epithelial cells (BEAS-2B) was examined by analyzing changes in messenger [mRNA] and microRNA [miRNA]. BEAS-2B cells were treated with 0, 3, 10, 30 or 100 µg/ml nano-TiO2 for 1 day (for mRNA analysis) or 3 days (for miRNA analysis). Differentially expressed mRNA and miRNA were analyzed using Affymetrix microarrays and Affymetrix miRNA microarrays, respectively. Although, the tested doses were not cytotoxic, there were alterations in both mRNA and miRNA expression. The expression of mRNA/miRNA changes were examined in MetaCore (GeneGo) and IPA (Ingenuity Pathway Analysis) to delineate associated canonical/signaling pathways. Canonical/signaling pathways altered by nano-TiO2 treatments included: cell cycle regulation, apoptosis, calcium signaling, translation, NRF2-mediated oxidative response, IGF1 signaling, RAS signaling, PI3K/AKT signaling, cytoskeleton remodeling, cell adhesion, BMP signaling, and inflammatory response. Many of the genes in these pathways are known to be regulated by the miRNAs whose expressions were altered by the nano-TiO2 treatment. The miRNA 17-92 cluster and let-7 miRNA family that are involved in lung cancer formation were altered by nano-TiO2 treatment. The miR-17-92 cluster, an oncogenic microRNA cluster, is induced while the tumor suppressor microRNA, let-7 family, is suppressed. The changes of let-7/KRAS signaling pathway was observed in all the doses treated. The observed changes in miRNA expression introduces an additional mechanistic dimension that supports the significance of the observed mRNA expression changes, and demonstrated that the nano-TiO2 in vitro treatment in human lung cells can cause diverse but coordinated pathway alterations associated with changes in in vivo response to tumorigenes.


Assuntos
Expressão Gênica/efeitos dos fármacos , MicroRNAs/metabolismo , Nanopartículas/toxicidade , Mucosa Respiratória/citologia , Transdução de Sinais/efeitos dos fármacos , Titânio/toxicidade , Linhagem Celular , Humanos , MicroRNAs/análise , MicroRNAs/genética
2.
Proteomics ; 11(12): 2406-22, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21595037

RESUMO

Oxidative stress is known to play important roles in engineered nanomaterial-induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial-mediated oxidative stress and toxicity are largely unknown. To identify these toxicity pathways and networks that are associated with exposure to engineered nanomaterials, an integrated proteomic study was conducted using human bronchial epithelial cells, BEAS-2B and nanoscale titanium dioxide. Utilizing 2-DE and MS, we identified 46 proteins that were altered at protein expression levels. The protein changes detected by 2-DE/MS were verified by functional protein assays. These identified proteins include some key proteins involved in cellular stress response, metabolism, adhesion, cytoskeletal dynamics, cell growth, cell death, and cell signaling. The differentially expressed proteins were mapped using Ingenuity Pathway Analyses™ canonical pathways and Ingenuity Pathway Analyses tox lists to create protein-interacting networks and proteomic pathways. Twenty protein canonical pathways and tox lists were generated, and these pathways were compared to signaling pathways generated from genomic analyses of BEAS-2B cells treated with titanium dioxide. There was a significant overlap in the specific pathways and lists generated from the proteomic and the genomic data. In addition, we also analyzed the phosphorylation profiles of protein kinases in titanium dioxide-treated BEAS-2B cells for a better understanding of upstream signaling pathways in response to the titanium dioxide treatment and the induced oxidative stress. In summary, the present study provides the first protein-interacting network maps and novel insights into the biological responses and potential toxicity and detoxification pathways of titanium dioxide.


Assuntos
Brônquios/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Nanopartículas/toxicidade , Estresse Oxidativo/genética , Mapeamento de Interação de Proteínas , Proteínas Quinases/metabolismo , Transdução de Sinais/genética , Titânio/toxicidade , Apoptose/efeitos dos fármacos , Brônquios/citologia , Brônquios/efeitos dos fármacos , Linhagem Celular , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Eletroforese em Gel Bidimensional , Células Epiteliais/citologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Espectrometria de Massas , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma/análise , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos
3.
J Sex Med ; 6 Suppl 3: 269-78, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19267849

RESUMO

INTRODUCTION: The combination of independent risk factors for erectile dysfunction, obesity, hypertension, and diabetes are collectively manifested in a condition known as metabolic syndrome X (MSX). However, the regulatory mechanisms responsible for the erectile dysfunction (ED) are not fully understood. Clinical studies suggest that a pleiotropic effect of statin's ability to enhance vascular relaxation might be through an impact on nitric oxide signaling or through a regulation of RhoA activation. AIM: We hypothesized that regulatory aspects of short-term statin therapy involve the alteration of the RhoA/Rho-kinase signaling cascade and will reverse the ED seen in a rat model of MSX. MAIN OUTCOME MEASURES: The magnitude and sensitivity of the voltage-dependent maintenance of intracavernosal blood pressure and mean arterial blood pressure. These responses were correlated with tissue protein and mRNA expression levels of RhoA and Rho kinases. METHODS: Erectile function was evaluated by assessing voltage-dependent stimulation of the cavernosal nerve in 16-20 weeks old lean and obese-diabetic Zucker rats treated with 5 mg/kg/day of rosuvastatin intraperitoneally for 3 days. Cavernosal tissue RhoA and Rho-kinases expression levels were evaluated by real-time reverse transcriptase-polymerase chain reaction, Western blot. RESULTS: The voltage-dependent erectile responses were suppressed by >30% in the obese-diabetic Zucker rat. The 3-day treatment with rosuvastatin partially restored the erectile response. The Rho-kinase inhibitor, H-1152, dose dependently increased the erectile responses and shifted the voltage sensitivity with statin treatment. Analysis of protein expression levels suggested elevation of RhoA and Rho kinases in obese-diabetics and statin treatment lowering Rho-kinase II. The RhoA and Rho-kinase II mRNA levels were significantly reduced in the rosuvastatin-treated obese-diabetic animals. CONCLUSIONS: These results support a hypothesis that short-term statin therapy may lower RhoA/Rho-kinase expression levels and improve cavernosal blood pressure response to Rho-kinase inhibition and voltage-stimulation, and reversing an augmented vasoconstricted state associated with diabetes and/or hypertension in MSX.


Assuntos
Anticolesterolemiantes/farmacologia , Anticolesterolemiantes/uso terapêutico , Diabetes Mellitus Experimental/epidemiologia , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Disfunção Erétil , Fluorbenzenos/farmacologia , Fluorbenzenos/uso terapêutico , Impotência Vasculogênica/etiologia , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Síndrome Metabólica/epidemiologia , Óxido Nítrico/metabolismo , Obesidade/epidemiologia , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Quinases Associadas a rho/metabolismo , Animais , Anticolesterolemiantes/administração & dosagem , Western Blotting , Inibidores Enzimáticos/administração & dosagem , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/epidemiologia , Disfunção Erétil/fisiopatologia , Fluorbenzenos/administração & dosagem , Impotência Vasculogênica/enzimologia , Masculino , Inibidores de Fosfodiesterase/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Ratos , Ratos Zucker , Rosuvastatina Cálcica , Sulfonamidas/administração & dosagem
4.
J Appl Physiol (1985) ; 104(5): 1374-80, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18309095

RESUMO

The purpose of the present study was to determine whether ultrasound is a useful tool to measure the venous characteristics of the lower extremity during a standard venous collecting cuff deflation protocol. To accomplish this, lower extremity pressure-cross-sectional area (CSA) and pressure-volume relationships were measured in eight (25 +/- 1 yr) supine subjects. Popliteal vein CSA was assessed by using high-resolution ultrasound, while calf volume changes were simultaneously assessed by using venous occlusion plethysmography (VOP). Pressure-CSA and pressure-volume relationships were assessed at baseline, during the cold pressor (CP) test, and following sublingual nitroglycerin (NTG) administration. Relationships were modeled with a quadratic regression equation, and beta(1) and beta(2) were used as indexes of venous compliance. Popliteal vein regression parameters beta(1) (8.485 +/- 2.616 vs. 7.638 +/- 2.664, baseline vs. CP; 8.485 +/- 2.616 vs. 7.734 +/- 3.076, baseline vs. NTG; both P > 0.05) and beta(2) (-0.0841 +/- 0.0241 vs. -0.0793 +/- 0.0242, baseline vs. CP; -0.0841 +/- 0.0241 vs. -0.0771 +/- 0.0280, baseline vs. NTG; both P > 0.05) were not affected by CP or NTG. Similarly, calf regression parameters beta(1) and beta(2), obtained with VOP, were not altered during either trial. Intraclass correlations for venous compliance assessed with ultrasound and VOP were 0.92 and 0.97, respectively, indicating acceptable reproducibility. These data suggest that ultrasound is a functional and reproducible tool to assess the venous characteristics of the lower extremity, in addition to VOP. Additionally, popliteal vein and calf compliance were not affected by the CP test or NTG.


Assuntos
Veia Poplítea/diagnóstico por imagem , Veia Poplítea/fisiologia , Adulto , Barorreflexo/efeitos dos fármacos , Barorreflexo/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Temperatura Baixa , Complacência (Medida de Distensibilidade)/efeitos dos fármacos , Endotélio Vascular , Feminino , Força da Mão/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Humanos , Perna (Membro)/irrigação sanguínea , Perna (Membro)/fisiologia , Masculino , Nitroglicerina/farmacologia , Pletismografia , Veia Poplítea/efeitos dos fármacos , Pressão , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 1/fisiologia , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/fisiologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Reprodutibilidade dos Testes , Decúbito Dorsal/fisiologia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Ultrassonografia , Vasodilatadores/farmacologia
5.
Environ Mol Mutagen ; 55(4): 336-42, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24446152

RESUMO

We showed previously that exposure of human lung cells (BEAS-2B) to TiO2 nanoparticles (nano-TiO2 ) produced micronuclei (MN) only when the final concentration of protein in the cell-culture medium was at least 1%. Nanoparticles localize in the liver; thus, we exposed human liver cells (HepG2) to nano-TiO2 and found the same requirement for MN induction. Nano-TiO2 also formed small agglomerates in medium containing as little as 1% protein and caused cellular interaction as measured by side scatter by flow cytometry and DNA damage (comet assay) in HepG2 cells. Nano-TiO2 also increased the activity of the inflammatory factor NFkB but not of AP1 in a reporter-gene HepG2 cell line. Suspension of nano-TiO2 in medium containing 0.1% protein was sufficient for induction of MN by the nanoparticles in either BEAS-2B or HepG2 cells as long the final concentration of protein in the cell-culture medium was at least 1%.


Assuntos
Brônquios/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Células Epiteliais/efeitos dos fármacos , Nanopartículas Metálicas/química , Titânio/farmacologia , Materiais Biocompatíveis/farmacologia , Brônquios/citologia , Brônquios/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular , Células Cultivadas , Ensaio Cometa , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Hep G2 , Humanos , Luciferases/metabolismo , Testes para Micronúcleos
6.
Nanotoxicology ; 7(6): 1111-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22770119

RESUMO

The use of nanoparticles in consumer products increases their prevalence in the environment and the potential risk to human health. Although recent studies have shown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a more detailed view of the underlying mechanisms of this response needs to be established. Here, the effects of nano-TiO2 on the DNA damage response and DNA replication dynamics were investigated in human dermal fibroblasts. Specifically, the relationship between nano-TiO2 and the DNA damage response pathways regulated by ATM/Chk2 and ATR/Chk1 was examined. The results show increased phosphorylation of H2AX, ATM, and Chk2 after exposure. In addition, nano-TiO2 inhibited the overall rate of DNA synthesis and frequency of replicon initiation events in DNA-combed fibres. Taken together, these results demonstrate that exposure to nano-TiO2 activates the ATM/Chk2 DNA damage response pathway.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Titânio/toxicidade , Proteínas Mutadas de Ataxia Telangiectasia/genética , Células Cultivadas , Quinase do Ponto de Checagem 2/genética , Meios de Cultura , Dano ao DNA/fisiologia , Fibroblastos/fisiologia , Histonas/genética , Histonas/metabolismo , Humanos , Nanopartículas Metálicas/química , Microscopia Acústica , Fosforilação , Titânio/química
7.
Toxicol In Vitro ; 27(6): 2013-21, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23872425

RESUMO

Silver nanoparticles (Ag NP) have been shown to generate reactive oxygen species; however, the association between physicochemical characteristics of nanoparticles and cellular stress responses elicited by exposure has not been elucidated. Here, we examined three key stress-responsive pathways activated by Nrf-2/ARE, NFκB, and AP1 during exposure to Ag NP of two distinct sizes (10 and 75 nm) and coatings (citrate and polyvinylpyrrolidone), as well as silver nitrate (AgNO3), and CeO2 nanoparticles. The in vitro assays assessed the cellular response in a battery of stable luciferase-reporter HepG2 cell lines. We further assessed the impact of Ag NP and AgNO3 exposure on cellular redox status by measuring glutathione depletion. Lastly, we determined intracellular Ag concentration by inductively coupled plasma mass spectroscopy (ICP-MS) and re-analyzed reporter-gene data using these values to estimate the relative potencies of the Ag NPs and AgNO3. Our results show activation of all three stress response pathways, with Nrf-2/ARE displaying the strongest response elicited by each Ag NP and AgNO3 evaluated here. The smaller (10-nm) Ag NPs were more potent than the larger (75-nm) Ag NPs in each stress-response pathway, and citrate-coated Ag NPs had higher intracellular silver concentrations compared with both PVP-coated Ag NP and AgNO3. The cellular stress response profiles after Ag NP exposure were similar to that of AgNO3, suggesting that the oxidative stress and inflammatory effects of Ag NP are likely due to the cytotoxicity of silver ions.


Assuntos
Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Transporte Biológico , Sobrevivência Celular , Cério/toxicidade , Ácido Cítrico/química , Genes Reporter , Glutationa/metabolismo , Células Hep G2 , Humanos , Luciferases/genética , Nanopartículas Metálicas/química , Fator 2 Relacionado a NF-E2/genética , NF-kappa B/genética , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Povidona/química , Proteína de Replicação C/genética , Prata/química , Nitrato de Prata/toxicidade
8.
ACS Nano ; 7(3): 1929-42, 2013 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-23387956

RESUMO

The widespread use of titanium dioxide (TiO2) nanoparticles in consumer products increases the probability of exposure to humans and the environment. Although TiO2 nanoparticles have been shown to induce DNA damage (comet assay) and chromosome damage (micronucleus assay, MN) in vitro, no study has systematically assessed the influence of medium composition on the physicochemical characteristics and genotoxicity of TiO2 nanoparticles. We assessed TiO2 nanoparticle agglomeration, cellular interaction, induction of genotoxicity, and influence on cell cycle in human lung epithelial cells using three different nanoparticle-treatment media: keratinocyte growth medium (KGM) plus 0.1% bovine serum albumin (KB); a synthetic broncheoalveolar lavage fluid containing PBS, 0.6% bovine serum albumin and 0.001% surfactant (DM); or KGM with 10% fetal bovine serum (KF). The comet assay showed that TiO2 nanoparticles induced similar amounts of DNA damage in all three media, independent of the amount of agglomeration, cellular interaction, or cell-cycle changes measured by flow cytometry. In contrast, TiO2 nanoparticles induced MN only in KF, which is the medium that facilitated the lowest amount of agglomeration, the greatest amount of nanoparticle cellular interaction, and the highest population of cells accumulating in S phase. These results with TiO2 nanoparticles in KF demonstrate an association between medium composition, particle uptake, and nanoparticle interaction with cells, leading to chromosomal damage as measured by the MN assay.


Assuntos
Aberrações Cromossômicas , Dano ao DNA , Nanopartículas Metálicas/toxicidade , Mutagênicos/toxicidade , Titânio/toxicidade , Animais , Bovinos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Meios de Cultura/química , Humanos , Nanopartículas Metálicas/ultraestrutura , Testes para Micronúcleos , Soroalbumina Bovina
9.
Nanotoxicology ; 5(4): 546-56, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21142840

RESUMO

Six TiO2 and two CeO2 nanomaterials with dry sizes ranging from 6-410 nm were tested for their ability to cause DNA centered free radicals in vitro in the concentration range of 10-3,000 ug/ml. All eight of the nanomaterials significantly increased the adduction of the spin trap agent 5,5-dimethyl-1-pyroline N-oxide (DMPO) to DNA as measured by the experimental technique of immuno-spin trapping. The eight nanomaterials differed considerably in their potency, slope, and active concentration. The largest increase in DNA nitrone adducts was caused by a TiO2 nanomaterial (25 nm, anatase) from Alfa Aesar. Some nanomaterials that increased the amount of DNA nitrone adducts at the lowest exposure concentrations (100 ug/ml) were Degussa TiO2 (31 nm), Alfa Aesar TiO2 (25 nm, anatase) and Nanoamor CeO2 (8 nm, cerianite). At exposure concentrations of 10 or 30 ug/ml, no nanomaterials showed significant in vitro formation of DNA nitrone adducts.


Assuntos
Cério/toxicidade , Adutos de DNA/análise , DNA/efeitos dos fármacos , Nanoestruturas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Titânio/toxicidade , Análise de Variância , Animais , Bovinos , Óxidos N-Cíclicos , Relação Dose-Resposta a Droga , Óxidos de Nitrogênio , Tamanho da Partícula , Detecção de Spin
10.
Am J Physiol Heart Circ Physiol ; 294(6): H2535-9, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18408124

RESUMO

Cardiovascular events are more common in the winter months, possibly because of hemodynamic alterations in response to cold exposure. The purpose of this study was to determine the effect of acute facial cooling on central aortic pressure, arterial stiffness, and wave reflection. Twelve healthy subjects (age 23 +/- 3 yr; 6 men, 6 women) underwent supine measurements of carotid-femoral pulse wave velocity (PWV), brachial artery blood pressure, and central aortic pressure (via the synthesis of a central aortic pressure waveform by radial artery applanation tonometry and generalized transfer function) during a control trial (supine rest) and a facial cooling trial (0 degrees C gel pack). Aortic augmentation index (AI), an index of wave reflection, was calculated from the aortic pressure waveform. Measurements were made at baseline, 2 min, and 7 min during each trial. Facial cooling increased (P < 0.05) peripheral and central diastolic and systolic pressures. Central systolic pressure increased more than peripheral systolic pressure (22 +/- 3 vs. 15 +/- 2 mmHg; P < 0.05), resulting in decreased pulse pressure amplification ratio. Facial cooling resulted in a robust increase in AI and a modest increase in PWV (AI: -1.4 +/- 3.8 vs. 21.2 +/- 3.0 and 19.9 +/- 3.6%; PWV: 5.6 +/- 0.2 vs. 6.5 +/- 0.3 and 6.2 +/- 0.2 m/s; P < 0.05). Change in mean arterial pressure but not PWV predicted the change in AI, suggesting that facial cooling may increase AI independent of aortic PWV. Facial cooling and the resulting peripheral vasoconstriction are associated with an increase in wave reflection and augmentation of central systolic pressure, potentially explaining ischemia and cardiovascular events in the cold.


Assuntos
Artérias/fisiologia , Pressão Sanguínea , Temperatura Baixa , Temperatura Cutânea , Adulto , Aorta/fisiologia , Artéria Braquial/fisiologia , Artérias Carótidas/fisiologia , Complacência (Medida de Distensibilidade) , Feminino , Artéria Femoral/fisiologia , Testa , Humanos , Masculino , Manometria , Decúbito Dorsal , Sístole , Vasoconstrição
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