Less Is More: Long-Term in Vitro Exposure to Low Levels of Silver Nanoparticles Provides New Insights for Nanomaterial Evaluation

In view of the vast number of new nanomaterials (NMs) that require testing and the constraints associated with animal models, the majority of studies to elucidate nanotoxicological effects have occurred in vitro, with limited correlation and applicability to in vivo systems and realistic, occupational exposure scenarios. In this study, we developed and implemented a chronic in vitromodel coupled with lower, regulatory dosages in order to provide a more realistic assessment of NM-dependent consequences and illuminate the implications of long-term NM exposure. When keratinocytes were exposed to 50 nm silver nanoparticles (Ag-NPs), we determined that chronically dosed cells operated under augmented stress and modified functionality in comparison to their acute counterparts. Specifically, Ag-NP exposure through a chronic mechanism increased p38 activation, actin disorganization, heightened ki67 expression, and extensive gene modification. Additionally, chronic Ag-NP exposure altered the way in which cells perceived and responded to epidermal growth factor stimulation, indicating a transformation of cell functionality. Most importantly, this study demonstrated that chronic exposure in the pg/mL range to Ag-NPs did not induce a cytotoxic response, but instead activated sustained stress and signaling responses, suggesting that cells are able to cope with prolonged, low levels of Ag-NP exposure. In summary, we demonstrated that through implementation of a chronic dosimetry paradigm, which more closely resembles realistic NM exposure scenarios, it is possible to illuminate long-term cellular consequences, which greatly differ from previously obtained acute assessments

Metal Nanomaterials: Immune Effects and Implications of Physicochemical Properties on Sensitization, Elicitation, and Augmentation of Allergic Disease

Many allergenic metals are being manufactured in nanoparticulate forms. Although the decreased size profile of metal nanomaterials has been consistently associated with more pronounced lung toxicity compared to larger materials, it is unclear if metal-induced immunotoxic effects exhibit a similar size-dependency. The central hypothesis of these studies was that metal nanoparticles cause more pronounced immunomodulatory effects on allergic processes than larger forms of respective metals. Moreover, it was anticipated that, similar to their inflammatory potential in airways, the magnitude of these responses would correlate best with the dose metric of surface area. The first set of studies utilized fine and ultrafine NiO particles to evaluate respiratory toxicity and augmentation of asthmatic responses with respect to different dose metrics. While the degree of pulmonary inflammation caused by NiO was exclusively associated with the administered surface area, NiO-induced augmentation of OVA allergy appeared dependent on multiple parameters, including particle size and dose mass. The second set of studies employed gold in bulk and nanoparticulate (AuNP) forms to study allergic sensitization. AuNP exposure did not cause dermal sensitization or notable respiratory immune effects, however, established contact sensitivity to gold conferred notable immune reactivity upon pulmonary AuNP exposure. Subsequent immune responses were directionally-polarized in a surface area-dependent manner, but alterations in several immune markers appeared more closely related to dose mass or particle size. Collectively, these findings suggest that, unlike their inflammatory potential in the airways, allergic effects caused by metal nanomaterials may involve various physico-chemical properties, and subsequently, implicate multiple dose metrics

Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy

The rapid proliferation of many different engineered nanomaterials (defined as materials designed and produced to have structural features with at least one dimension of 100 nanometers or less) presents a dilemma to regulators regarding hazard identification. The International Life Sciences Institute Research Foundation/Risk Science Institute convened an expert working group to develop a screening strategy for the hazard identification of engineered nanomaterials. The working group report presents the elements of a screening strategy rather than a detailed testing protocol. Based on an evaluation of the limited data currently available, the report presents a broad data gathering strategy applicable to this early stage in the development of a risk assessment process for nanomaterials. Oral, dermal, inhalation, and injection routes of exposure are included recognizing that, depending on use patterns, exposure to nanomaterials may occur by any of these routes. The three key elements of the toxicity screening strategy are: Physicochemical Characteristics, In Vitro Assays (cellular and non-cellular), and In Vivo Assays. There is a strong likelihood that biological activity of nanoparticles will depend on physicochemical parameters not routinely considered in toxicity screening studies. Physicochemical properties that may be important in understanding the toxic effects of test materials include particle size and size distribution, agglomeration state, shape, crystal structure, chemical composition, surface area, surface chemistry, surface charge, and porosity. In vitro techniques allow specific biological and mechanistic pathways to be isolated and tested under controlled conditions, in ways that are not feasible in in vivo tests. Tests are suggested for portal-of-entry toxicity for lungs, skin, and the mucosal membranes, and target organ toxicity for endothelium, blood, spleen, liver, nervous system, heart, and kidney. Non-cellular assessment of nanoparticle durability, protein interactions, complement activation, and pro-oxidant activity is also considered. Tier 1 in vivo assays are proposed for pulmonary, oral, skin and injection exposures, and Tier 2 evaluations for pulmonary exposures are also proposed. Tier 1 evaluations include markers of inflammation, oxidant stress, and cell proliferation in portal-of-entry and selected remote organs and tissues. Tier 2 evaluations for pulmonary exposures could include deposition, translocation, and toxicokinetics and biopersistence studies; effects of multiple exposures; potential effects on the reproductive system, placenta, and fetus; alternative animal models; and mechanistic studies

As nanopartículas de prata induzem citotosicidade e genotoxicidade nas linhas celulares da pele, fígado e sangue?

Doutoramento em BiologiaAs nanopartículas de prata (AgNPs) estão entre as nanopartículas mais utilizadas devido às suas propriedades fisico-químicas e biológicas (como por exemplo a sua eficiente actividade antimicrobiana). Contudo, existe uma crescente preocupação relativamente ao seu potencial impacto no ambiente e na saúde humana. Mais especificamente, ainda não está explicada a influência dos revestimentos das nanopartículas na citotoxicidade, inflamação e potencial genotóxico em células humanas. Neste estudo, as AgNPs revestidas com citrato ou poli(etilenoglicol) (PEG) foram utilizadas para determinar in vitro a influência do revestimento nos perfis de toxicidade das AgNPs em células da pele, fígado e sangue. A linha celular de queratinócitos humanos (HaCaT) foi exposta a AgNPs de 30 nm revestidas com citrato ou PEG (Cit30 ou PEG30) onde foram determinados a viabilidade, stress oxidativo, produção de citoquinas, apoptose e efeito citostático. No geral, as Cit30 foram mais citotóxicas do que as PEG30 demonstrando a influência do revestimento relativamente ao modo da morte celular e à progressão do ciclo celular para as concentrações testadas (IC50 e IC20 para as Cit30, correspondendo a 40 μg/mL e 10 μg/mL, respectivamente). Enquanto que as Cit30 induzem morte por apoptose, as células expostas a PEG30 aparentemente estão numa fase precoce dos mecanismos de apoptose, tal como sugere a análise da expressão génica. As Cit30 foram posteriormente utilizadas para determinar o potencial genotóxico na mesma linha celular, através da determinação do dano no DNA e a indução de micronúcleos (MNi). Foi demonstrado que Cit30 induzem dano no DNA e provocam a ocorrência de MNi, bem como, a redução do Índice de Divisão Nuclear (NDI). Finalmente, e considerando que nesta linha de células a modulação do processo inflamatório é particularmente importante, as células foram expostas a AgNPs de 10 nm com revestimento de citrato ou PEG (Cit10 e PEG10). Os resultados mostraram que Cit10 e PEG10 modularam a resposta inflamatória de forma diferente, onde apenas PEG10 estimulou a indução do Factor Nuclear (NF)- κB. Contudo, ambas as AgNPs não estimularam a produção de citoquinas, mas antes, diminuiram a proteina quimio-atrativa de monócitos -1 (MCP1). A linha celular humana de hepatoma (HepG2) também foi exposta a Cit30 e PEG30 para determinar a influência dos revestimento nos efeitos das AgNPs na viabilidade, apoptose, genes relacionados com a apoptose, ciclo celular e expressão génica de ciclinas. Estas células apresentaram uma sensibilidade similar às AgNPs com ambos os revestimentos, ocorrendo redução de viabilidade e comprometimento do ciclo celular para as concentrações testadas (IC50 e IC20 para as Cit30 que corresponde a 11 μg/mL e 5 μg/mL, respectivamente). A linha celular de macrofágos de ratinho (murganho) (RAW 264.7) foi utilizada para avaliar a citotoxicidade das Cit30, onde a viabilidade, stress oxidativo e as dinamicas do ciclo celular foram determinados. A proliferação e viabilidade das RAW 264.7 apenas decresceu após exposição a concentrações superiores a 75 μg/mL de Cit30, sugerindo uma baixa sensibilidade destas células a baixas concentrações de Cit30. Após exposição de 24 h o conteúdo de ROS (espécies reactivas de oxigénio) diminuiu nas células expostas a 60 μg/mL de Cit30 (IC20) o que pode ter contribuído para a tolerância destas células às AgNPs revestidas de citrato. Contudo, estas células apresentaram o ciclo celular comprometido e foi ainda observado um aumento de células na fase Sub-G1. Este aumento da população Sub-G1 está correlacionado com um aumento da fragmentação do DNA o que sugere um aumento de apoptose nestas células. Assim, e comparando a resposta dos diferentes tipos celulares às AgNPs em termos de viabilidade celular, podemos concluir que os hepatócitos são os mais sensiveis às AgNPs e que os macrófagos parecem ser os mais tolerantes (das mais sensíveis para as mais tolerentes: HepG2>HaCaT>RAW 264.7). Este estudo sugere que o revestimento com PEG pode ser considerado como uma boa alternativa à estabilização das AgNPs com o citrato para a utilização ao nível da indústria e nas aplicações médicas em relação às células da pele humana. Contudo, para os hepatócitos, o potencial citotóxico das AgNPs foi independente dos revestimentos. Para além disso, e considerando que as AgNPs estão presentes num vasto número de produtos de consumo e a sua utilização tem sido provada como útil ao nível industrial e da engenharia biomédica, embora os nossos dados sugiram que no geral PEG-AgNP são menos tóxicas, mais investigação é necessária para determinar as propriedades que conferem menor toxicidade das AgNPs para as diferentes linhas celulares.Silver nanoparticles (AgNPs) are among the most commonly used engineered NPs due to their physicochemical and biological properties (e.g. efficient antimicrobial activity). There is, however, a growing concern about their putative impact on the environment and on human health. In particular, there is no complete understanding of the influence of the coating on the cytotoxic, inflammatory and genotoxic potential of AgNPs to human cells. In this study, AgNPs coated with citrate or poly(ethylene glycol) (PEG) were used to assess in vitro the influence of coating on the AgNPs toxicity profiles on skin, liver and blood cells. Human Keratinocyte cell line (HaCaT) was exposed to 30 nm AgNPs coated with citrate or PEG (Cit30 or PEG30) and assessed for viability, oxidative stress, cytokine production, apoptosis and cytostaticity. Overall Cit30 was more toxic than PEG30 demonstrating the influence of coating regarding the mode of cell death and cell cycle progression for the concentrations tested (IC50 and IC20 for citrate- AgNPs corresponding to 40 μg/mL and 10 μg/mL, respectively). While Cit30 AgNPs clearly induced apoptotic death, cells exposed to PEG30 AgNPs appeared to be at an earlier phase of apoptosis mechanisms, as supported by gene expression analysis. Later these Cit30 were used to assess genotoxic potential on the same cell line, by measuring DNA damage and micronuclei (MNi) induction. It was demonstrated that Cit30 induced DNA damage, and MNi occurrence as well as a reduction of NDI (Nuclear division index). Finally, and considering that in these cell lines the inflammatory modulation is particularly important, cells were exposed to 10 nm (citrate vs. PEG) AgNPs. Data showed that Cit10 and PEG10 differently modulate the inflammatory response, with only PEG10 stimulating the Nuclear factor (NF)-κB induction. However, both AgNPs did not stimulate the release of cytokines but decreased MCP1 (Monocyte chemoattractant protein-1). Human Hepatoma cell line (HepG2) was also exposed to Cit30 and PEG30 to assess the influence of coating on the AgNPs effects on viability, apoptosis, apoptotic related genes, cell cycle and cyclins gene expression. These cells had similar sensitivity to both coatings, and suffered a decrease of viability as well as an impairment of the cells cycle for the concentrations tested (IC50 and IC20 for citrate- AgNPs corresponding to 11 μg/mL and 5 μg/mL, respectively). Mouse macrophage cell line (RAW 264.7) was used to evaluate the cytotoxicity of Cit30, where viability, oxidative stress and cell cycle dynamics were assessed. The proliferation and viability of RAW 264.7 cells only decreased upon exposure to Cit30 at concentrations above 75 μg/mL, suggesting a low sensitivity of RAW cells to lower doses of these AgNPs. After 24 h exposure, ROS content decreased in cells exposed to 60 μg/mL AgNPs (IC20 value) which can have contributed to the high tolerance of these cells to citrate-AgNPs. However, these cells suffered an impairment of the cell cycle and an increase of cells at Sub-G1 phase was observed. This increase of the subG1 population is correlated with an increase of DNA fragmentation which suggests an increase of apoptosis in these cells. Therefore, and taking together the different decreases on cells viability, we conclude that hepatocytes are more sensitive to AgNPs than keratinocytes and that macrophages seem the more tolerant (from the most sensitive for more tolerant: HepG2>HaCaT>RAW 264.7). This study suggests that PEG-coating can be regarded as a good alternative to citrate stabilization of AgNPs used in industrial and medical applications towards human skin cells. However, for hepatocyte cells, AgNPs cytotoxic potential was coating independent Ultimately, and considering that AgNPs are present in a vast number of consumer products and their use has been proved to be helpful in industrial and biomedical engineering, despite our data suggest that overall PEG-AgNP are less toxic, more research is needed to determine the properties that confer less toxicity of AgNPs to different cell lines

Organotypic Human Skin Disease Models for the Assessment of Novel Therapeutic Approaches

PhDComprehensive in vitro modelling of inflammatory human skin conditions is an essential first step in the development and assessment of potential therapeutic approaches. Mouse models or monolayer keratinocyte cultures come with distinct limitations which might be complimented or overcome by the use of human-specific organotypic 3D culture models. Over the course of this thesis, an organotypic culture system, based on patientderived immortalised keratinocyte cell lines on a dermal equivalent collagen 1 gel, was established and used to recapitulate phenotypical features for two hereditary skin diseases, Harlequin ichthyosis and Tylosis with oesophageal cancer. Small molecular compounds, supplied via the medium, or RNA interference were used to modulate disease-specific changes in histology and marker expression of the skin equivalent. Since hyperproliferative skin conditions can be associated with an aberrant wound healing phenotype, the organotypic system was manipulated to obtain a basic in vitro wound healing model. This model displays typical features of re-epithelialisation over time (both normal and disease-specific) which can further be manipulated via shRNAmediated knockdown or the exogenous supply of compounds. In parallel, a non-disease model was used to assess the topical application of novel nanopolymeric drug delivery systems in regard to their ability to penetrate across the permeability barrier. Penetrance profiles for the organotypic model (in dependence of co-application with chemical enhancers) showed a similar pattern as for topical applications performed in parallel on explant skin. In conclusion, a highly adaptable human organotypic keratinocyte culture model was developed and used to recapitulate (and manipulate) skin disease phenotypes and epidermal wound healing in vitro, as well as perform first essential assessments of novel drug delivery systems.European Commission Marie-Curie ITN (FP7) NANODRUG

Effects of nanomaterials on biological barriers, fetal and post-natal, and evaluation of epigenetic toxicity

Beyond the opportunities offered by nanotechnology research, there is a great need of studies aimed at understanding the harmful effects of general exposure to nanomaterials. My Ph.D. project aimed to be part of this evaluation, focusing on the interaction and the induction of possible toxic effects of two fibrous nanomaterials (asbestos and carbon nanotubes) at two critical internal biological barriers: the pleura and the placenta. The work is carried out by using advanced microscopy (\ub5XRM and XRF) based on synchrotron radiation and other microscopes (SEM and AFM), and also conventional molecular analysis (PCR and Sanger sequencing) and advanced spectroscopic measurements (UV-Raman). We conducted biochemical studies by using the advanced X-Ray microscopy and fluorescence (\ub5XRM and XRF) techniques in order to reveal mechanisms of toxicity in human mesothelial (MeT5A) and placental cell lines (BeWo) exposed to carbon nanotubes (raw-SWCNT, purified- and highly purified-SCWCNT) or asbestos (crocidolite fibres). Other microscopes (AFM, atomic force microscopy and SEM, Scanning Electron Microscope) are added in some experiments, to better investigate the morphology and the cell-nanofiber interactions. The results obtained with the combination of microscopic techniques allowed to reveal similar as well as different toxic mechanisms in the two internal barriers. The cells treated with raw-SWCNT and crocidolite fibres compared to the control showed an severe alteration of iron metabolism, which is maximal in the pleural cells and is clearly related to the presence of iron into the fibre. X-ray microscopy images (absorption and phase contrast imaging) confirm that the toxicity of nanomaterials is characterized by membrane damage with vesicle secretion and filipodia formation. In relation to this toxic mechanism quite complex and still unknown we evaluated the presence of intracellular ferritin in treated cells. The results demonstrated that crocidolite and \u201craw\u201d carbon nanotubes increase the amount of intracellular ferritin in both cell models, while purified and highly purified carbon nanotubes give values comparable to control. The stimulation is clearly lower in placental cells, clearly linked to a different or lower uptake of fibres in these cells, suggesting that this barrier is less vulnerable than the pleura. We also investigated the genetic effects and genetic predisposition to toxicity of nanomaterials (nanotoxicogenomic). Since we have to learn from asbestos, one study investigates the possible genetic predisposition to develop mesothelioma after asbestos exposure by looking for BAP1 gene mutations in 30 cases of mesothdelioma. Sanger sequencing of BAP1 gene in the 30 patients identified one non-synonymous variant and two intronic variants. While Sanger sequencing of cDNA revealed no alternative splicing due to the nucleotide change for each mutations. In silico mutation analysis was performed in a predicted protein structure of BAP1 protein without any significant possible effect of the amino acid change about exonic mutations of patient 9. Finally, MLPA (Multiplex ligation-dependent probe amplification) analysis revealed no significant copy number variations at exonic level in all samples. The last aim of molecular studies was to test the feasibility of UV-Raman (IUVS beamline, Elettra Synchrotron of Trieste) spectroscopy to reveal epigenetic changes at DNA level after nanomaterial exposure. An oxidative environment has been created in vitro by using carbon nanotubes (raw-SWCNT), which contain some impurity in metal traces (iron), and free radicals OH\u2022 (derived from H2O2). In this condition the nucleotides (dATP, dCTP, dGTP and dTTP) result in increased susceptibility to oxidative damage. The results demonstrated that UV-Raman spectroscopy is useful to reveal the chemical changes that affect the nitrogenous bases after nanomaterials exposure, providing a \u201cfingerprint\u201d of the oxidative DNA damage