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/genéticaRESUMO
Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Alterations in GJC are associated with carcinogenesis, but the mechanisms involved are unknown. Chloral hydrate (CH), a by-product of chlorine disinfection of water, is carcinogenic in mice, and we demonstrated that CH reduced GJC in a rat liver epithelial cell line (Clone 9). To examine the mechanism(s) by which CH inhibits GJC, Clone 9 cells treated with CH were examined using Western blot, real-time polymerase chain reaction, immunocytochemical, and dye-communication techniques. Treatment with CH (0.15 mM for 24 h) resulted in a dose-dependent inhibition of GJC as measured by Lucifer yellow dye transfer. Western blot analysis demonstrated expression of connexin (Cx) 43 and 26 in control cells and reduced expression of Cx 43 but not Cx 26 protein from 0.1 to 1 mM CH. CH treatment from 2.5 to 5 mM caused an apparent increase in expression of both connexins that was concomitant with a reduction in mRNA expression for both connexins. Similarly, with immunocytochemistry, a dose-dependent decrease in Cx 43 staining at sites of cellcell contact was apparent in CH (0.55 mM)-treated cultures, whereas no Cx 26 staining was observed. Thus, Clone 9 cells contain two types of connexins but only one type of plasma membrane channel. Understanding of the regulation of connexin may shed light on mechanisms responsible for inhibition of GJC by chemical carcinogens.
Assuntos
Comunicação Celular/efeitos dos fármacos , Hidrato de Cloral/toxicidade , Células Epiteliais/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Fígado/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Linhagem Celular , Conexina 26 , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Células Epiteliais/metabolismo , Junções Comunicantes/fisiologia , Humanos , Fígado/citologia , Fígado/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Triadimefon, propiconazole, and myclobutanil are conazoles, an important class of agricultural fungicides. Triadimefon and propiconazole are mouse liver tumorigens, while myclobutanil is not. As part of a coordinated study to understand the molecular determinants of conazole tumorigenicity, we analyzed the microRNA expression levels in control and conazole-treated mice after 90 d of administration in feed. MicroRNAs (miRNAs) are small noncoding RNAs composed of approximately 19-24 nucleotides in length, and have been shown to interact with mRNA (usually 3' UTR) to suppress its expression. MicroRNAs play a key role in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Groups of mice were fed either control diet or diet containing 1800 ppm triadimefon, 2500 ppm propiconazole, or 2000 ppm myclobutanil. MicroRNA was isolated from livers and analyzed using Superarray whole mouse genome miRNA PCR arrays from SABioscience. Data were analyzed using the significance analysis of microarrays (SAM) procedure. We identified those miRNAs whose expression was either increased or decreased relative to untreated controls with q < or = 0.01. The tumorigenic conazoles induced many more changes in miRNA expression than the nontumorigenic conazole. A group of 19 miRNAs was identified whose expression was significantly altered in both triadimefon- and propiconazole-treated animals but not in myclobutanil-treated animals. All but one of the altered miRNAs were downregulated compared to controls. This pattern of altered miRNA expression may represent a signature for tumorigenic conazole exposure in mouse liver after 90 d of treatment.
Assuntos
Carcinógenos , Neoplasias Hepáticas Experimentais/patologia , MicroRNAs/análise , Triazóis/toxicidade , Animais , Testes de Carcinogenicidade , Carcinógenos/toxicidade , Regulação para Baixo , Fungicidas Industriais/toxicidade , Regulação Neoplásica da Expressão Gênica , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , Modelos Biológicos , Método de Monte Carlo , Nitrilas/toxicidade , Reação em Cadeia da PolimeraseRESUMO
Nano-TiO2 and nano-CeO2 are among the most widely used engineered nanoparticles (NPs). We investigated a variety of endpoints to assess the toxicity of eight of these NPs to induce potentially adverse health effects in an In Vitro human respiratory epithelial cell model. These endpoints include cytotoxicity, reactive oxygen species (ROS)/reactive nitrogen species (RNS) production, 8-hydroxy-2_-deoxyguanosine (8-oxo-dG), endogenous DNA adducts, Apurinic/apyrimidinic (AP) sites, 4-Hrdoxynonenal (4-HNE) protein adducts, Malondialdehyde (MDA) protein adducts, and genomics analysis on altered signaling pathways. Our results indicated that cytotoxicity assays are relatively insensitive, and we detected changes in other endpoints at concentrations much lower than those inducing cytotoxicity. Among the ROS-related endpoints, 8-oxo-dG is relatively more sensitive than other assays, and nano-TiO2 induced more 8-oxo-dG formation than nano-CeO2. Finally, there are many signaling pathways changes at concentrations at which no cytotoxicity was observed. These alterations in signaling pathways correlated well with In Vitro toxicity that was observed at higher concentrations, and with in vivo adverse outcome pathways caused by nano-TiO2 and nano-CeO2 in experimental animals.
Assuntos
Células Epiteliais , Titânio , Animais , Humanos , Pulmão , Espécies Reativas de Oxigênio , Titânio/toxicidadeRESUMO
BACKGROUND: Administration of amplitude modulated 27·12â¯MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. METHODS: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. FINDINGS: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2â¯T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. INTERPRETATION: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Cálcio/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Magnetoterapia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/patologia , Magnetoterapia/métodos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Especificidade de Órgãos , RNA Interferente Pequeno/genética , Radiometria , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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 MicronúcleosRESUMO
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/toxicidadeRESUMO
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 BovinaRESUMO
Macrophages, when entering inflamed tissue, encounter low oxygen tension due to the impairment of blood supply and/or the massive infiltration of cells that consume oxygen. Previously, we showed that such macrophages release more bacteriotoxic hydrogen peroxide (H2O2) when exposed in vitro to low oxygen than when cultured at usual ambient oxygen conditions. In this study, we use this low-oxygen, inflammatory macrophage model to test the macrophages response to low-frequency magnetic fields. Low-frequency fields are clinically used for bone and wound healing and are emerging as therapy for inflammatory diseases. The acceptance of these non-invasive therapies is slow due to the lack of knowledge of the cellular targets for magnetic fields. One possible target is biologically relevant ions. The Ion Parametric Resonance (IPR) concept predicts that specific externally applied AC and DC magnetic fields will resonate with the cyclotron motion of ions. This concept is supported by experimental evidence, especially on a neuronal cell line. Using our macrophage model, we tested AC and DC magnetic fields at the amplitude ratio and frequency predicted by the IPR model for resonance with hydrogen, magnesium and manganese ions. Under these conditions, we found a significant increase in H2O2 release compared to control cells. Magnetic field exposure conditions in which parameters differed from the predictions of the IPR model showed no, or a smaller difference, with respect to the control cultures. These data indicate that magnetic fields can enhance H2O2 release of inflammatory macrophages, which is consistent with the predictions of the IPR model.
RESUMO
People in industrialized nations live in an environment of ubiquitous electromagnetic field (EMF) exposure, both natural and anthropogenic. The intensity, variety, and geographic distribution of anthropogenic EMF exposures have grown dramatically since the mid 20th century, with many uses serving, and in close proximity to, human populations, such as electric power distribution, radio and television transmission, and more recently, personal cell phone communication units and transmitting towers. Thus, it is reasonable to ask if this EMF exposure could cause alterations in the physiology of developing organisms, since they are generally assumed to be the most sensitive to chemical stressors. In this report, we review work published beginning in the late 1980s. Initial reports indicated that exposure of chicken eggs during embryonic development to power-line electric fields of 50 and 60 Hz, at 10 V/m in air (which is frequently in locations inhabited by humans), could cause the brain tissues of the hatched chickens to respond differently in a particular test. More recently, an anecdotal report of human sensitivity to EMF has appeared that shows a health-related influence of prior exposure history to particular power-line frequencies in chemically sensitized individuals. These reports open the question of whether the ambient electromagnetic environment can leave an imprint on developing organisms and if such imprint changes have the potential for health consequences.