RESUMO
Assessment of dermal irritation is an essential component of the safety evaluation of medical devices. Reconstructed human epidermis (RhE) models have replaced rabbit skin irritation testing for neat chemicals and their mixtures (OECD Test Guideline 439). However, this guideline cannot be directly applied to the area of medical devices (MD) since their non-toxicity assessment is largely based on the testing of MD extracts that may have very low irritation potential. Therefore, the RhE-methods previously validated with neat chemicals needed to be modified to reflect the needs for detection of low levels of potential irritants. A protocol employing RhE EpiDerm was optimized in 2013 using known irritants and spiked polymers (Casas et al., 2013, TIV). In 2014 and 2015 MatTek In Vitro Life Science Laboratories (IVLSL) and RIVM assessed the transferability of the assay. After the successful transfer and standardization of the protocol, 17 laboratories were trained in the use of the protocol in the preparation for the validation. Laboratories produced data with 98% agreement of predictions for the selected references and controls. We conclude that a modified RhE skin irritation test has the potential to address the skin irritation potential of the medical devices. Standardization and focus on the technical issues is essential for accurate prediction.
Assuntos
Epiderme/efeitos dos fármacos , Equipamentos e Provisões , Irritantes/toxicidade , Polímeros/toxicidade , Testes de Irritação da Pele , Alternativas aos Testes com Animais , Epiderme/metabolismo , Humanos , Interleucina-1alfa/metabolismo , Reprodutibilidade dos TestesAssuntos
Materiais Biocompatíveis/toxicidade , Epiderme/patologia , Irritantes/toxicidade , Polímeros/toxicidade , Testes de Irritação da Pele/métodos , Alternativas aos Testes com Animais , Materiais Biocompatíveis/química , Equipamentos e Provisões , Humanos , Teste de Materiais , Polímeros/químicaRESUMO
A number of studies have shown that induction of pulmonary toxicity by nanoparticles of the same chemical composition depends on particle size, which is likely in part due to differences in lung deposition. Particle size mostly determines whether nanoparticles reach the alveoli, and where they might induce toxicity. For the risk assessment of nanomaterials, there is need for a suitable dose metric that accounts for differences in effects between different sized nanoparticles of the same chemical composition. The aim of the present study is to determine the most suitable dose metric to describe the effects of silver nanoparticles after short-term inhalation. Rats were exposed to different concentrations (ranging from 41 to 1105 µg silver/m(3) air) of 18, 34, 60 and 160 nm silver particles for four consecutive days and sacrificed at 24 h and 7 days after exposure. We observed a concentration-dependent increase in pulmonary toxicity parameters like cell counts and pro-inflammatory cytokines in the bronchoalveolar lavage fluid. All results were analysed using the measured exposure concentrations in air, the measured internal dose in the lung and the estimated alveolar dose. In addition, we analysed the results based on mass, particle number and particle surface area. Our study indicates that using the particle surface area as a dose metric in the alveoli, the dose-response effects of the different silver particle sizes overlap for most pulmonary toxicity parameters. We conclude that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation.
Assuntos
Nanopartículas Metálicas/toxicidade , Pneumonia/induzido quimicamente , Prata/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/análise , Relação Dose-Resposta a Droga , Exposição por Inalação , Pulmão/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Ratos , Ratos Endogâmicos F344 , Prata/metabolismoRESUMO
Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecular interaction of compounds with the neural differentiation process. Within the 11-day differentiation protocol of the assay, embryonic stem cells lost their pluripotency, evidenced by the reduced expression of stem cell markers Pou5F1 and Nanog. Moreover, stem cells differentiated into neural cells, with morphologically visible neural structures together with increased expression of neural differentiation-related genes such as ßIII-tubulin, Map2, Neurogin1, Mapt and Reelin. Valproic acid (VPA) and carbamazepine (CBZ) exposure during hESTn differentiation led to concentration-dependent reduced expression of ßIII-tubulin, Neurogin1 and Reelin. In parallel VPA caused an increased gene expression of Map2 and Mapt which is possibly related to the neural protective effect of VPA. These findings illustrate the added value of gene expression analysis for detecting compound specific effects in hESTn. Our findings were in line with and could explain effects observed in animal studies. This study demonstrates the potential of this assay protocol for mechanistic analysis of specific compound-induced inhibition of human neural cell differentiation.
Assuntos
Anticonvulsivantes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Carbamazepina/toxicidade , Células-Tronco Embrionárias/metabolismo , Fibroblastos , Humanos , Camundongos , RNA/biossíntese , Proteína Reelina , Ácido Valproico/toxicidadeRESUMO
To determine relevant endpoints for evaluating developmental immunotoxicity due to juvenile exposure and optimal age of the animals at assessment, a wide range of immunological parameters were assessed in a juvenile toxicity study. Rats were exposed to di-n-octyltin dichloride (DOTC) by gavage from postnatal day (PND) 10 through PND 21 and via the diet after weaning using a benchmark dose (BMD) approach. Immune assessments were performed in male rats on PNDs 21, 42, and 70 and a subset of animals was used to evaluate the T-cell dependent antibody response (TDAR) to Keyhole limpet hemocyanin. Immune effects were more pronounced on PND 21 and 42 and observed at lower doses than developmental effects. The most sensitive immune parameters affected included TDAR parameters and thymocyte subpopulations with lower confidence limits of the benchmark doses (BMDLs) below the overall no-observed-adverse-effect-level (NOAEL) for DOTC reported so far in literature. These findings illustrate the relative sensitivity of the developing immune system for DOTC, the additional value of assessing functional immune parameters, and underscore the relevance of juvenile immunotoxicity testing in view of the risk assessment of chemicals.
Assuntos
Poluentes Ambientais/toxicidade , Compostos Orgânicos de Estanho/toxicidade , Subpopulações de Linfócitos T/efeitos dos fármacos , Fatores Etários , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Citocinas/imunologia , Índices de Eritrócitos/efeitos dos fármacos , Feminino , Hematócrito , Hemocianinas/imunologia , Imunoglobulina G/imunologia , Rim/efeitos dos fármacos , Rim/crescimento & desenvolvimento , Contagem de Leucócitos , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Masculino , Óxido Nítrico/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Wistar , Baço/efeitos dos fármacos , Baço/crescimento & desenvolvimento , Subpopulações de Linfócitos T/imunologia , Timo/efeitos dos fármacos , Timo/crescimento & desenvolvimentoRESUMO
Silver nanoparticles are of interest to be used as antimicrobial agents in wound dressings and coatings in medical devices, but potential adverse effects have been reported in the literature. The most pronounced effect of silver nanoparticles and the role of particle size in determining these effects, also in comparison to silver ions, are largely unknown. Effects of silver nanoparticles of different sizes (20, 80, 113 nm) were compared in in vitro assays for cytotoxicity, inflammation, genotoxicity and developmental toxicity. Silver nanoparticles induced effects in all endpoints studied, but effects on cellular metabolic activity and membrane damage were most pronounced. In all toxicity endpoints studied, silver nanoparticles of 20 nm were more toxic than the larger nanoparticles. In L929 fibroblasts, but not in RAW 264.7 macrophages, 20 nm silver nanoparticles were more cytotoxic than silver ions. Collectively, these results indicate that effects of silver nanoparticles on different toxic endpoints may be the consequence of their ability to inflict cell damage. In addition, the potency of silver in the form of nanoparticles to induce cell damage compared to silver ions is cell type and size-dependent.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Inflamação/patologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Mutagênicos/toxicidade , Tamanho da Partícula , Prata/toxicidade , Animais , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/enzimologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , L-Lactato Desidrogenase/metabolismo , Luz , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/patologia , Nanopartículas Metálicas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Espécies Reativas de Oxigênio/metabolismo , Espalhamento de Radiação , Prata/químicaRESUMO
The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 µM) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Silanos/toxicidade , Triazóis/toxicidade , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/fisiologia , Genes Controladores do Desenvolvimento/efeitos dos fármacos , Camundongos , Silanos/administração & dosagem , Triazóis/administração & dosagemRESUMO
The murine embryonic stem cell test (EST) is an alternative testing method designed to assess potential developmental toxicity of compounds. The implementation of transcriptomics in the EST has been shown to reduce the culture duration and improve endpoint evaluation and is expected to result in an enhanced predictability and definition of the applicability domain. We evaluated the identification of developmental toxicity in the EST using two gene sets ("Van_Dartel_heartdiff_24h" and "EST biomarker genes") defined in our earlier studies. Nonexposed embryonic stem cells (ESC) differentiation cultures were sampled 0, 24, and 48 h after initiation of differentiation. Additionally, cultures exposed to 12 diverse well-characterized positive and negative developmental toxicants were isolated 24 h after the onset of exposure. Inhibition of ESC differentiation was evaluated in parallel by morphological scoring on culture day 10. Transcriptomics analysis was conducted using the Affymetrix Gene Chips platform. We applied principal component analysis on the basis of the two predefined gene sets to define the "differentiation track" that represents ESC differentiation. The significance of derivations in the gene expression-based differentiation track because of compound exposures were evaluated to determine developmental toxicity of tested compounds. We successfully predicted developmental toxicity using transcriptomics for 83% (10/12) and 67% (8/12) of the compounds, respectively, using the two predefined gene sets ("Van_Dartel_heartdiff_24h" and "EST biomarker genes"). Our study suggests that the application of transcriptomics may improve the applicability of the EST for the prediction of the developmental toxicity of chemicals.
Assuntos
Alternativas aos Testes com Animais , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Perfilação da Expressão Gênica , Expressão Gênica/efeitos dos fármacos , Teratogênicos/toxicidade , Animais , Técnicas de Cultura de Células , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Valor Preditivo dos Testes , Testes de Toxicidade/métodos , ToxicogenéticaRESUMO
Genome maintenance is considered a prime longevity assurance mechanism as apparent from many progeroid human syndromes that are caused by genome maintenance defects. The ERCC1 protein is involved in three genome maintenance systems: nucleotide excision repair, interstrand cross-link repair, and homologous recombination. Here we describe in-life and post-mortem observations for a hypomorphic Ercc1 variant, Ercc1(-/Δ7), which is hemizygous for a single truncated Ercc1 allele, encoding a protein lacking the last seven amino acids. Ercc1(-/Δ7) mice were much smaller and median life span was markedly reduced compared to wild-type siblings: 20 and 118 weeks, respectively. Multiple signs and symptoms of aging were found to occur at an accelerated rate in the Ercc1(-/Δ7) mice as compared to wild-type controls, including a decline in weight of both whole body and various organs, numerous histopathological lesions, and immune parameters. Together they define a segmental progeroid phenotype of the Ercc1(-/Δ7) mouse model.
RESUMO
BACKGROUND: Nanoparticle (NP) toxicity testing comes with many challenges. Characterization of the test substance is of crucial importance and in the case of NPs, agglomeration/aggregation state in physiological media needs to be considered. In this study, we have addressed the effect of agglomerated versus single particle suspensions of nano- and submicron sized gold on the inflammatory response in the lung. Rats were exposed to a single dose of 1.6 mg/kg body weight (bw) of spherical gold particles with geometric diameters of 50 nm or 250 nm diluted either by ultrapure water or by adding phosphate buffered saline (PBS). A single dose of 1.6 mg/kg bw DQ12 quartz was used as a positive control for pulmonary inflammation. Extensive characterization of the particle suspensions has been performed by determining the zetapotential, pH, gold concentration and particle size distribution. Primary particle size and particle purity has been verified using transmission electron microscopy (TEM) techniques. Pulmonary inflammation (total cell number, differential cell count and pro-inflammatory cytokines), cell damage (total protein and albumin) and cytotoxicity (alkaline phosphatase and lactate dehydrogenase) were determined in bronchoalveolar lavage fluid (BALF) and acute systemic effects in blood (total cell number, differential cell counts, fibrinogen and C-reactive protein) 3 and 24 hours post exposure. Uptake of gold particles in alveolar macrophages has been determined by TEM. RESULTS: Particles diluted in ultrapure water are well dispersed, while agglomerates are formed when diluting in PBS. The particle size of the 50 nm particles was confirmed, while the 250 nm particles appear to be 200 nm using tracking analysis and 210 nm using TEM. No major differences in pulmonary and systemic toxicity markers were observed after instillation of agglomerated versus single gold particles of different sizes. Both agglomerated as well as single nanoparticles were taken up by macrophages. CONCLUSION: Primary particle size, gold concentration and particle purity are important features to check, since these characteristics may deviate from the manufacturer's description. Suspensions of well dispersed 50 nm and 250 nm particles as well as their agglomerates produced very mild pulmonary inflammation at the same mass based dose. We conclude that single 50 nm gold particles do not pose a greater acute hazard than their agglomerates or slightly larger gold particles when using pulmonary inflammation as a marker for toxicity.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Compostos de Ouro/toxicidade , Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Fenômenos Químicos , Citocinas/metabolismo , Intubação Intratraqueal , Contagem de Leucócitos , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/patologia , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Neutrófilos/patologia , Tamanho da Partícula , Quartzo/toxicidade , Ratos , Propriedades de Superfície , Testes de ToxicidadeRESUMO
The embryonic stem cell test (EST) has been designed to predict developmental toxicity based upon compound-induced inhibition of embryonic stem cell (ESC) differentiation. The end point scoring, the test duration, and the definition of the predictivity and the applicability domain require improvements to facilitate implementation of the EST into regulatory testing strategies. The use of transcriptomics to study compound-induced differentiation modulation may improve the EST in each of these aspects. ESC differentiation was induced, and cell samples were collected after 0, 24, and 48 h of differentiation. Additionally, samples were collected that were 24 h exposed to one of five developmentally toxic compounds or a nondevelopmentally toxic compound. All samples were hybridized to Affymetrix GeneChips, and analyses revealed that 26 genes were significantly regulated both during ESC differentiation and by exposure to each of the developmentally toxic compounds tested. Using principal component analysis, we defined a "differentiation track" on the basis of this gene list, which represents ESC differentiation. We showed that significant deviation from the differentiation track was in line with the developmental toxic properties of the compounds. The significance of deviation was analyzed using the leave-one-out cross-validation, which showed a favorable prediction of toxicity in the system. Our findings show that gene expression signatures can be used to identify developmental toxicant-induced differentiation modulation. In addition, studying compound-induced effects at an early stage of differentiation combined with transcriptomics leads to increased objectivity in determining differentiation inhibition and to a reduction of the test duration. Furthermore, this approach may improve the predictivity and applicability domain of the EST.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Teratogênicos/toxicidade , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Camundongos , Análise de Componente PrincipalRESUMO
The mouse embryonic stem cell test (EST) was designed to predict embryotoxicity based on the inhibition of the differentiation of embryonic stem cells (ESC) into beating cardiomyocytes in combination with cytotoxicity data in monolayer ESC cultures and 3T3 cells. In the present study, we have tested a diverse group of chemicals in the EST, applying different exposure durations, in an attempt to discriminate between effects on proliferation and differentiation within the EST protocol. Chemicals tested were monobutyl phthalate (MBP), 6-aminonicotinamide (6-AN), 5-fluorouracil (5-FU) and 5-bromo-2'-deoxyuridine (BrdU). We showed that 5-FU and BrdU behaved principally different from MBP and 6-AN. 5-FU and BrdU specifically affected cell proliferation during the first three days of the EST protocol, as shown by EB size, protein concentration and cell cycle stage analysis. In addition, we studied the differentiation state of cells in the EST protocol with time to elucidate the transition of pluripotent ESC to more differentiated cell types. Analysis by flow cytometry of the pluripotency marker SSEA-1 in EST showed that although total SSEA-1 positive cells remained unchanged up to and including day 5, the signal intensity already decreased from day 3 onwards. Furthermore, RT-PCR data showed an upregulation of the mesodermal marker T at day 3, whereas the cardiac muscle marker Myh6 was upregulated from day 5 onwards. These findings confirm that proliferation and differentiation of ESC in the EST are highly intertwined processes. Based on these findings we suggest an amended EST protocol which could more clearly discriminate between proliferation and differentiation effects of chemicals within the same EST differentiation protocol. This proposal includes a cytotoxicity assessment in EB at day 3 of the EST after day 0-3 exposure, and cardiac muscle foci counts after exposure from day 3-10 in the EST.