RESUMO
The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.
Assuntos
Cobre/toxicidade , Citocinas/metabolismo , Laboratórios/normas , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Testes de Toxicidade/normas , Bioensaio/métodos , Bioensaio/normas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Europa (Continente) , Humanos , Nanopartículas Metálicas/química , Tamanho da Partícula , Reprodutibilidade dos Testes , Prata/química , Propriedades de Superfície , Testes de Toxicidade/métodosRESUMO
Specific information about the particle size distribution, agglomeration state, morphology, and chemical composition of four silica samples, used as additives in food and in personal care products, were achieved with a combination of analytical techniques. The combined use of differential centrifugal sedimentation (DCS), sedimentation field flow fractionation (SdFFF), and scanning and transmission electron microscopy (SEM and TEM) allows to classify the water dispersed samples as "nanomaterials" according to the EC definition. The mechanical stirring and the ultrasound treatment were compared as dispersion methods. The particle surface chemical composition, determined by particle-induced X-ray emission (PIXE) and X-ray photoelectron spectroscopy (XPS), assessed the different levels of purity between the pyrogenic and the precipitated silica and highlighted particle surface chemical composition modifications in the outer shell when dispersed by mechanical stirring. The potential toxic effects of silica on intestinal Caco-2 cells were investigated using MTS assay and by measuring lactate dehydrogenase (LDH) release and caspases 3/7 activity after 24 h of incubation. No or limited decrease of cell viability was observed for all particles regardless of dispersion procedure, suggesting a relative innocuity of these silica samples.
Assuntos
Cosméticos/química , Aditivos Alimentares/química , Nanopartículas/química , Dióxido de Silício/química , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Cosméticos/toxicidade , Aditivos Alimentares/toxicidade , Fracionamento por Campo e Fluxo , Humanos , Nanopartículas/toxicidade , Espectroscopia Fotoeletrônica , Dióxido de Silício/toxicidadeRESUMO
Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.
RESUMO
Prion protein (PrP) is essentially known for its capacity to induce neurodegenerative prion diseases in mammals caused by a conformational change in its normal cellular isoform (PrPC) into an infectious and disease-associated misfolded form, called scrapie isoform (PrPSc). Although its sequence is highly conserved, less information is available on its physiological role under normal conditions. However, increasing evidence supports a role for PrPC in the cellular response to oxidative stress. In the present study, a new link between PrP and senescence is highlighted. The role of PrP in premature senescence induced by copper was investigated. WI-38 human fibroblasts were incubated with copper sulfate (CuSO4) to trigger premature senescence. This induced an increase of PrP mRNA level, an increase of protein abundance of the normal form of PrP and a nuclear localization of the protein. Knockdown of PrP expression using specific small interfering RNA (siRNA) gave rise to appearance of several biomarkers of senescence as a senescent morphology, an increase of senescence associated ß-galactosidase activity and a decrease of the cellular proliferative potential. Overall these data suggest that PrP protects cells against premature senescence induced by copper.
Assuntos
Núcleo Celular/metabolismo , Senescência Celular/efeitos dos fármacos , Sulfato de Cobre/farmacologia , Fibroblastos/metabolismo , Proteínas PrPC/metabolismo , Linhagem Celular , Fibroblastos/patologia , Humanos , RNA Mensageiro/metabolismoRESUMO
Cutaneous Squamous Cell Carcinoma (cSCC) is the second most common type of non-melanoma skin cancer in white-skinned populations. cSCC is associated with sun exposure and aging, which is concomitant with an accumulation of senescent cells in the skin. The involvement of senescent cells in carcinogenesis has been highlighted in several cancer types and an interaction between cSCC cells and senescent cells is proposed, but still little explored. Tumor-associated effects are mostly attributed to the senescence-associated secretory phenotype (SASP). Here, we compared two in vitro models of senescence, namely replicative senescence and UVB-stress induced premature senescence (UVB-SIPS), in human dermal fibroblasts and screened for expression of SASP-related genes in our models. Next, the impact of senescent fibroblasts on three cSCC isogenic cell lines, representing different stages of keratinocyte malignant transformation, was studied. Only a limited impact on cSCC cell lines' growth and migration has been detected with conditioned media collected from senescent fibroblasts and indirect co-cultures. We then investigated the opposite interaction and found that cSCC cell lines maintained in indirect co-cultures with fibroblasts induced and reinforced their senescence state as shown by an increased proportion of cells positive for the senescence-associated ß-galactosidase activity and an increased expression of several SASP-related genes. Moreover, these effects were modulated according to the stage of tumorigenesis of the different cSCC cell lines used. Finally, cSCC cell lines-co-cultures are associated with NF-κB activation in HDFs. Understanding the interplay between tumor cells and their microenvironment may have important influences in cancer research and therapeutic strategies.
Assuntos
Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/patologia , Senescência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/patologia , Neoplasias Cutâneas/patologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem da Célula , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismoRESUMO
In the present work, we indicate that copper is involved in the senescence of human diploid fibroblasts and we describe mechanisms to explain it. Using different techniques, we show for the first time an accumulation of copper in cells during replicative senescence. This accumulation seems to be co-localized with lipofuscin. Second, we observed that an incubation of cells with copper sulfate induced oxidative stress, antioxidant response and premature senescence. Antioxidant molecules reduced the appearance of premature senescence. Third, we found that Nrf2 transcription factor was activated and regulated the expression of genes involved in antioxidant response while p38(MAPK) regulated the appearance of premature senescence.