Search

In recent years, a global increase in allergy incidence following chemical exposure has been observed. While the process of skin sensitization is well characterized through the adverse outcome pathway (AOP) framework, the immunological mechanisms underlying respiratory sensitization remain less well understood. Respiratory sensitizers are classified as substances of very high concern (SVHC) under the European Union (EU) regulation for the registration, evaluation, authorization and restriction of chemicals (REACH), emphasizing the importance of evaluating respiratory tract sensitization as a critical hazard. However, the existing new approach methodologies (NAMs) for the identification of skin sensitizers lack the capacity to differentiate between skin and respiratory sensitizers. Thus, it is imperative to develop physiologically relevant test systems specifically tailored to assess respiratory sensitizers. This study aimed to evaluate the efficacy of ALIsens®, a three-dimensional (3D) in vitro alveolar model designed for the identification of respiratory sensitizers and to determine its ability to correctly identify sensitizers. In this study, we used a range of skin sensitizers and non-sensitizers to define the optimal exposure dose, identify biomarkers, and establish tentative thresholds for correct sensitizer classification. The results demonstrate that ALIsens® is a promising in vitro complex model that could successfully discriminate respiratory sensitizers from skin sensitizers and non-sensitizers. Furthermore, the thymic stromal lymphopoietin receptor (TSLPr) cell surface marker was confirmed as a reliable biomarker for predicting respiratory sensitization hazards. [ABSTRACT FROM AUTHOR]

This report documents the outcomes of the EFSA procurement (OC/EFSA/NIF/2022/01) aimed at identifying in vitro toxicity testing approaches for (novel) proteins in the context of food and feed safety assessment. In the present report, we present an integrated testing strategy for the evaluation of toxicity of novel/toxic proteins. A text‐mining approach was used to create a literature database of toxic outcomes associated with toxic proteins retrieved from the UniProt KB database using the search term "Toxin activity". It was shown that toxic proteins are produced by a relatively limited phylogenetic subset, including, among others, bacteria, insects, serpents, molluscs, and fungi. Toxicological effects of these proteins are generally conserved within phylogenetic groups. Analysis of toxic effects from these proteins was performed using GO term analysis as well as a text‐mining based approach. Relevant tests to address and quantify these toxicity effects were identified and evaluated for their applicability in an in vitro based toxicity testing strategy. A stepwise approach was developed. As a first step, an initial in silico prediction of toxicity is carried out (Step 1). This is followed by a battery of in vitro assays to address the primary mechanisms of toxicity associated with toxic proteins (Step 2). If concern arises in the Step 2 battery of tests, the use of relevant in vitro model systems to explore potential target organ toxicity are required (Step 3). Knowledge gaps have been identified and recommendations are provided in in vitro toxicity testing strategies, in particular for (novel) proteins. Some of these gaps involve the selection and integration of a standardized, relevant in vitro digestion step, reflective of passage through the digestive tract, within the testing strategy, as well as a thorough assessment of the suitability and applicability of in vitro tests and new approach methodologies for regulatory toxicity assessment of (novel) proteins. To accelerate the incorporation of NAMs in the assessment of protein safety, case studies and proof of concept projects are needed to demonstrate the utility and effectiveness of in vitro toxicity testing strategies in the safety assessment of (novel) proteins. [ABSTRACT FROM AUTHOR]

Plenty of new approach methodologies (NAMs) for risk assessment have been developed but only some are included in OECD Test Guidelines (TGs) for regulatory implementation. Nevertheless, NAMs are increasingly applied, e.g. for nanomaterial (NM) risk assessments. The EFSA Guidance on NM risk assessment suggests that NAM‐derived data concerning degradation/dissolution (in relevant biofluids), intestinal uptake/crossing, genotoxicity, cytotoxicity, oxidative stress, (pro‐)inflammatory potential and barrier integrity, for many of which no OECD TGs exist, have to be evaluated first. Consequently, NM risk assessments involve data from non‐guideline studies, requiring time‐consuming and challenging case‐by‐case evaluations. Establishing an OECD TG is a formal process aiming for international use according to the Mutual Acceptance of Data (MAD). However, not every promising NAM can be prioritised for OECD TGs. A qualification, based on an expert opinion, may enable an efficient use of adequate NAMs for a specific context‐of‐use. Furthermore, it supports the optimisation of promising NAMs for regulatory applications. Existing qualification systems operate in the context of e.g., drug development tools (FDA) and research and development into pharmaceuticals (EMA). The NAMS4NANO consortium was tasked to propose a generic framework for a qualification system for chemical risk assessment in the food and feed sector to speed up the regulatory use of NAMs. Here we describe our proposal including the process and evaluation criteria. A detailed test method description, preferably as standard operating procedures (SOPs), describing the set‐up of the NAM including its application and evaluation phase is crucial. Furthermore, the scientific validity, i.e. its reliability and relevance for the context‐of‐use, needs to be demonstrated, for which we suggest a less rigorous process compared to OECD TGs. We propose to initially establish a qualification system for NM risk assessment, aligned with the EFSA framework. This document is an interim version to stipulate a broader discussion among experts and stakeholders. [ABSTRACT FROM AUTHOR]

New Approach Methodologies (NAMs), broadly understood to include in silico, in chemico, in vitro and ex vivo methods, show great potential in advancing risk assessment albeit their regulatory implementation is lagging. The EFSA Guidance on risk assessment of nanomaterials (EFSA Guidance on Nano‐RA) suggests nano‐specific risk assessment is best achieved through Integrated Approaches to Testing and Assessment (IATAs) with NAMs as the first choice to generate new information. Integrating NAMs in risk assessment promises several advantages such as a better human focus, more detailed insights into molecular mechanisms and a higher efficacy. However, applying NAMs to NMs also poses considerable challenges such as issues related to dispersion stability, dosimetry, agglomeration, dissolution, transformations or assay interferences. Significant efforts are being undertaken by standardisation organisations and research projects to establish various NAMs for NMs. Here a thorough review is provided covering NAMs that will be potentially useful for risk assessment of NMs in the food and feed sector. It follows the structure of the EFSA Guidance on Nano‐RA and expands it, where needed, to support decision‐making in selection of NAMs for NM risk assessment. The review begins with an overview on nano‐specific NAM‐frameworks, followed by a description of individual NAMs including those relevant to NM physicochemical characterisation, exposure and hazard assessment covering toxicodynamics and toxicokinetics. The focus is on NAMs concerning NM degradation/dissolution, genotoxicity, cytotoxicity, oxidative stress, (pro‐)inflammation, and barrier integrity as those are important endpoints for initial screening according to the EFSA framework. As a result, in total 267 individual nano‐relevant NAMs, mostly "not validated" (with a few notable exceptions), were included in this review. Validation notwithstanding, NAMs could already prove relevant and reliable for risk assessment of NMs, especially in integrated approaches. [ABSTRACT FROM AUTHOR]

Background: Silica nanoparticles (SiNPs) are among the most widely manufactured and used nanoparticles. Concerns about potential health effects of SiNPs have therefore risen. Using a 3D tri-culture model of the alveolar lung barrier we examined effects of exposure to SiNPs (Si10) and crystalline silica (quartz; Min-U-Sil) in the apical compartment consisting of human alveolar epithelial A549 cells and THP-1-derived macrophages, as well as in the basolateral compartment with Ea.hy926 endothelial cells. Inflammation-related responses were measured by ELISA and gene expression. Results: Exposure to both Si10 and Min-U-Sil induced gene expression and release of CXCL8, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-1β (IL-1β) in a concentration-dependent manner. Cytokine/chemokine expression and protein levels were highest in the apical compartment. Si10 and Min-U-Sil also induced expression of adhesion molecules ICAM-1 and E-selectin in the apical compartment. In the basolateral endothelial compartment we observed marked, but postponed effects on expression of all these genes, but only at the highest particle concentrations. Geneexpressions of heme oxygenase-1 (HO-1) and the metalloproteases (MMP-1 and MMP-9) were less affected. The IL-1 receptor antagonist (IL-1RA), markedly reduced effects of Si10 and Min-U-Sil exposures on gene expression of cytokines and adhesion molecules, as well as cytokine-release in both compartments. Conclusions: Si10 and Min-U-Sil induced gene expression and release of pro-inflammatory cytokines/adhesion molecules at both the epithelial/macrophage and endothelial side of a 3D tri-culture. Responses in the basolateral endothelial cells were only induced at high concentrations, and seemed to be mediated by IL-1α/β released from the apical epithelial cells and macrophages. [ABSTRACT FROM AUTHOR]

The aim of the study was to develop an in vitro model that mimics the alveolar-capillary barrier and that allows assessment of the respiratory sensitizing potential of substances. The 3D in vitro model cultured at the air liquid interface consists of alveolar type II epithelial cells (A549), endothelial cells (EA.hy926), macrophage-like cells (PMA-differentiated THP-1), and dendritic-like cells (non-differentiated THP-1). This alveolar model was exposed apically to nebulized chemical respiratory sensitizers (phthalic anhydride (PA) and trimellitic anhydride (TMA)) or to irritants (methyl salicylate (MeSa) and acrolein (Acr)) at concentrations inducing 25% cytotoxicity. The exposure to respiratory sensitizers induced dendritic-like cell activation and a specific cytokine release pattern, while exposure to irritants did not. In addition, the cell surface marker OX40L was found to identify dendritic-like cell activation by high molecular weight allergens. With this in vitro model we can postulate a set of promising markers that allow the discrimination of chemical respiratory sensitizers from irritants. [ABSTRACT FROM AUTHOR]

To more accurately model inhalation toxicity in vitro, we developed a tetra-culture system that combines lung alveolar epithelial cells, endothelial cells, macrophages, and mast cells in a three-dimensional orientation. We characterized the influence of the added complexity using network perturbation analysis and gene expression data. This allowed us to gain insight into the steady-state profile of the assembled, complete three-dimensional model using all four cell types, and of simpler models of one, two, or three cell types. Gene expression data were analyzed using cause-andeffect biological network models, together with a quantitative network-scoring algorithm, to determine the biological impact of co-culturing the various cell types. In the tetra-culture, macrophages appeared to be the largest contributors to overall network perturbations, promoting high basal levels of oxidative stress and inflammation. This finding led to further optimization of the model using rested macrophages, which decreased the basal inflammatory and cell stress status of the co-culture. We compared transcriptional profiles from publicly available datasets of other in vitro models representing the airways and of healthy human lung tissue with those of our model. We found an increasing correlation between airway models and normal human lung tissue as cell types became more physiologically relevant and the complexity of the system increased. This indicates that the combination of multiple lung-relevant cell types in vitro does indeed increase similarity to the physiological counterpart. [ABSTRACT FROM AUTHOR]

Adverse food reactions (AFR) are a common cause of skin diseases in cats and dogs. The correct diagnosis and management of AFR relies upon clinical nutrition. The reliability of commercial hypoallergenic diets commonly used in AFR has been questioned because studies have shown the presence of proteins not declared on the label ingredients. It is proposed that extensively hydrolysed protein-based diets constitute a reliable nutritional solution. Royal Canin Anallergenic™ Canine and Feline diets are formulated with very low molecular weight feather protein and purified corn starch. Protein gel electrophoresis and thin layer paper chromatography were used to characterize protein hydrolysis in these diets and their hydrolysed raw materials; protein species were identified by mass spectrometry. To detect cross-contaminating protein, species-specific DNA was measured and correlated with ancillary protein content using calibration curves. The only protein components detected in the extensively hydrolysed feather protein raw material were amino acids and small oligopeptides. GBSS-I (Granule-bound starch synthase 1) was detected in the finished diets; this has not been reported as a clinically apparent allergen in dogs or cats. The DNA threshold corresponding to the maximum acceptable level of ancillary protein was not exceeded in 99.9% of more than 2150 product batches tested and no products were released to the market with cross-contaminating proteins. These results demonstrate the extensive level of protein hydrolysis in Royal Canin Anallergenic™ Canine and Feline diets and the absence of cross-contaminating protein, both key requirements for a diet to be used during diagnosis and for management of pets with AFR. [ABSTRACT FROM AUTHOR]

Copyright of Farmacia is the property of Societatea de Stiinte Farmaceutice Romania and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Respiratory sensitization as a consequence of exposure to chemical products has increased over the last decades, leading to an increase of morbidity. The increased use of synthetic compounds resulted in an exponential growth of substances to which we are potentially exposed on a daily basis. Some of them are known to induce respiratory sensitization, meaning that they can trigger the development of allergies. In the past, animal studies provided useful results for the understanding of mechanisms involved in the development of respiratory allergies. However, the mechanistic understanding of the involved cellular effects is still limited. Currently, no in vitro or in vivo models are validated to identify chemical respiratory sensitizers. Nonetheless, chemical respiratory sensitizers elicit a positive response in validated assays for skin sensitization. In this review, we will discuss how these assays could be used for respiratory sensitization and if necessary, what can be learnt from these assays to develop a model to assess the respiratory sensitizing potential of chemicals. In the last decades, much work has been done to study the respiratory toxicity of inhaled compounds especially in developing in vitro assays grown at the air–liquid interface. We will discuss how possibly the tests currently used to investigate general particle toxicity could be transformed to investigate respiratory sensitization. In the present review, we describe the most known mechanism involved in the sensitization process and the experimental in vivo and alternative in vitro models, which are currently available and how to adapt and improve existing models to study respiratory sensitization. [ABSTRACT FROM AUTHOR]

Background: During the last 250 years, the level of exposure to combustion-derived particles raised dramatically in western countries, leading to increased particle loads in the ambient air. Among the environmental particles, diesel exhaust particulate matter (DEPM) plays a special role because of its omnipresence and reported effects on human health. During recent years, a possible link between air pollution and the progression of atherosclerosis is recognized. A central effect of DEPM is their impact on the endothelium, especially of the alveolar barrier. In the present study, a complex 3D tetraculture model of the alveolar barrier was used in a dose-controlled exposure scenario with realistic doses of DEPM to study the response of endothelial cells. Results: Tetracultures were exposed to different doses of DEPM (SRM2975) at the air-liquid-interface. DEPM exposure did not lead to the mRNA expression of relevant markers for endothelial inflammation such as ICAM-1 or E-selectin. In addition, we observed neither a significant change in the expression levels of the genes relevant for antioxidant defense, such as HMOX1 or SOD1, nor the release of pro-inflammatory second messengers, such as IL-6 or IL-8. However, DEPM exposure led to strong nuclear translocation of the transcription factor Nrf2 and significantly altered expression of CYP1A1 mRNA in the endothelial cells of the tetraculture. Conclusion: In the present study, we demonstrated the use of a complex 3D tetraculture system together with a state-of-the-art aerosol exposure equipment to study the effects of in vivo relevant doses of DEPM on endothelial cells in vitro. To the best of our knowledge, this study is the first that focuses on indirect effects of DEPM on endothelial cells of the alveolar barrier in vitro. Exposure to DEPM led to significant activation and nuclear translocation of the transcription factor Nrf2 in endothelial cells. The considerably low doses of DEPM had a low but measurable effect, which is in line with recent data from in vivo studies. [ABSTRACT FROM AUTHOR]

The thyroid hormone (TH) system plays a central role in central physiological processes of many species, including mammals and humans, ranging from growth and cell differentiation, energy metabolism, thermoregulation and phasing of hibernation or annual movements of migratory species, metamorphosis from larvae to adult forms, brain development, reproduction, or the cardiovascular system. Several chemicals are known to be TH-disrupting compounds (THDCs) and have been shown to interact with virtually all elements of TH homeostasis such as feedback mechanisms with the hypothalamus-pituitary axis, TH synthesis, TH storage and release from the thyroid gland, transport protein binding and TH distribution in tissues and organs, cellular TH uptake, intracellular TH metabolism, and TH receptor binding. Therefore, chemicals interfering with the TH homeostasis have the potential to interact with many of these important processes, and especially early-life stage exposure results in permanent alterations of tissue organization and homeostatic regulation of adaptive processes. This is not only of theoretical importance as the reported plasma concentrations of THDCs in human plasma fall well within the range of reported in vitro effect concentrations, and this is of even higher importance as the developing fetus and young children are in a sensitive developmental stage. [ABSTRACT FROM AUTHOR]

Background: The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. Methods: The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. Results: The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. Conclusions: The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were observed for uptake, pro-inflammatory response and changes at the proteome level. The low level of overlap between differentially abundant proteins observed in both Ag NPs and AgNO3 treated co-culture suggests size-dependent responses that cannot only be attributed to soluble Ag. [ABSTRACT FROM AUTHOR]

Background: Exposure to fine and ultra-fine ambient particles is still a problem of concern in many industrialised parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. A system consisting of four different human cell lines which mimics the cell response of the alveolar surface in vitro was developed to study native aerosol exposure (Vitrocell™ chamber). The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane. Results: The spatial distribution of the cells in the tetraculture was analysed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the transwell. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The cells formed colonies under submerged conditions, which disappeared at the ALI. To evaluate the response to oxidative stress, the dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used together with 2,2'-azobis-2-methylpropanimidamide- dihydrochloride (AAPH) as inducer of oxidative stress. The tetraculture showed less induction of reactive oxygen species (ROS) production after being treated with a positive control compared to the monocultures of EA.hy 926, THP-1 and HMC-1. Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures. The Vitrocell™ aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine NPs in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only. Conclusion: The system can be used in conjunction with a native aerosol exposure system and may finally lead to a more realistic judgement regarding the hazard of new compounds and/or new nano-scaled materials in the future. The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects. [ABSTRACT FROM AUTHOR]

Carotenoid consumption has been linked to a number of beneficial health effects, including the reduction of chronic diseases such as cancer and cardiovascular complications. However, no data are available on their action on the intestinal epithelium, being exposed to the highest concentrations of carotenoids in the human body, and where they could act preventively on intestinal inflammatory diseases such as Crohn's disease and ulcerative colitis. The objective of the present study was to investigate whether lycopene and β-carotene in micelles (M), at concentrations that could be reached via the diet (10–25 μg/ml) could aid in the reduction of TNF-α plus IL-1β-induced inflammation of Caco-2 human epithelial cells. The impact on biomarkers of inflammation, including IL-8, NO and cyclo-oxygenase-2 (through PGE-2α), and the NF-κB and mitogen-activated protein kinase (MAPK) pathways of intracellular signalling cascades were evaluated compared with controls (empty M). Furthermore, proteomic analyses were conducted from total cellular protein extracts. The results revealed that isolated carotenoids had no statistical significant anti-inflammatory effect on the biomarkers observed, or on the regulation of NF-κB and MAPK. Nevertheless, analyses of the proteome suggested that fifteen proteins were significantly (P < 0·05, expression ratio >1·3) differentially regulated following β-carotene exposure, participating mostly in metabolic activities including antioxidant mechanisms, such as glutathione S-transferase A1. Only one protein was differentially regulated by lycopene (profilin-1). To our knowledge, this is the first attempt to investigate pathways involved in the action of carotenoids on the intestinal epithelium. [ABSTRACT FROM PUBLISHER]

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as 'black' AKU chondrocytes, while those coming from the white portion were referred to as 'white' AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both 'white' and 'black' AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in 'black' AKU chondrocytes. J. Cell. Physiol. 227: 3333-3343, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: para-Nonylphenol (p-NP) is a metabolite of alkylphenols widely used in the chemical industry and manufacturing. It accumulates in the environment, where it acts with estrogen-like activity. We previously showed that p-NP acts on human placenta by inducing trophoblast differentiation and apoptosis. Objective: The aim of the present study was to investigate the effect of p-NP on cytokine secretion in human placenta. Methods: In vitro cultures of chorionic villous explants from human placenta in the first trimester of pregnancy were treated with p-NP (10-13, 10-11, and 10-9 M) in 0.1% ethanol as vehicle. Culture medium was collected after 24 hr and assayed by specific immunoassays for the cytokines granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α). Results: p-NP modulated cytokine secretion by inducing the release of GM-CSF, IFN-γ, IL-1β, IL-4, and IL-10, with a maximum effect at 10-11 M. It reduced the release of TNF-α at 10-13 M, whereas levels of IL-2 and IL-5 remained below the detection limit. IL-6 and IL-8 levels were 100- 1,000 times higher than those of other cytokines, and they were not affected by p-NP. We observed significant differences from controls (ethanol alone) only for GM-CSF and IL-10. Conclusion: An unbalanced cytokine network at the maternal-fetal interface may result in implantation failure, pregnancy loss, or other complications. The effects of extremely low doses of p-NP on the placental release of cytokines raise considerable concerns about maternal exposure to this endocrine disruptor during pregnancy. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most frequent primitive malignant tumor of the skeletal system, characterized by an extremely aggressive clinical course that still lacks an effective treatment. Src kinase seems to be involved in the osteosarcoma malignant phenotype. We show that the treatment of human osteosarcoma cell lines with a new pyrazolo[3,4-d]pyrimidine derivative Src inhibitor, namely SI-83, impaired cell viability, with a half-maximal inhibitory concentration of 12 µM in nonstarved cells and a kinetic different from that known for the Src inhibitor PP2. Analysis by terminal deoxynucleotidyl transferase-mediated nick end labeling, Hoechst, and flow cytometric assay showed that SI-83 induced apoptosis in SaOS-2 cells. Moreover, SI-83, by inhibiting Src phosphorylation, decreased in vivo osteosarcoma tumor mass in a mouse model. Finally, SI-83 showed selectivity for osteosarcoma, since it had a far lower effect in primary human osteoblasts. These results show that human osteosarcoma had Src-dependent proliferation and that modulation of Src activity may be a therapeutic target of this new compound with low toxicity for nonneoplastic cells [ABSTRACT FROM AUTHOR]

Background: The present study aimed to evaluate the potential differences in the biological effects of two types of spherical silver particles of 20 and 200 nm (Ag20 and Ag200), and of PVP-coated silver nanowires (AgNWs) with a diameter of 50 nm and length up to 50 μm, using a complex 3D model representative for the alveolar barrier cultured at air-liquid interface (ALI). The alveolar model was exposed to 0.05, 0.5 and 5 μg/cm2 of test compounds at ALI using a state-of-the-art exposure system (Vitrocell™Cloud System). Endpoints related to the oxidative stress induction, anti-oxidant defence mechanisms, pro-inflammatory responses and cellular death were selected to evaluate the biocompatibility of silver particles and nanowires (AgNMs) and to further ascribe particular biological effects to the different morphologic properties between the three types of AgNMs evaluated. Results: Significant cytotoxic effect was observed for all three types of AgNMs at the highest tested doses. The increased mRNA levels of the pro-apoptotic gene CASP7 suggests that apoptosis may occur after exposure to AgNWs. All three types of AgNMs increased the mRNA level of the anti-oxidant enzyme HMOX-1 and of the metal-binding anti-oxidant metallothioneins (MTs), with AgNWs being the most potent inducer. Even though all types of AgNMs induced the nuclear translocation of NF-kB, only AgNWs increased the mRNA level of pro-inflammatory mediators. The pro-inflammatory response elicited by AgNWs was further confirmed by the increased secretion of the 10 evaluated interleukins. Conclusion: In the current study, we demonstrated that the direct exposure of a complex tetra-culture alveolar model to different types of AgNMs at ALI induces shape- and size-specific biological responses. From the three AgNMs tested, AgNWs were the most potent in inducing biological alterations. Starting from 50 ng/cm2, a dose representative for an acute exposure in a high exposure occupational setting, AgNWs induced prominent changes indicative for a pro-inflammatory response. Even though the acute responses towards a dose representative for a full-lifetime exposure were also evaluated, chronic exposure scenarios at low dose are still unquestionably needed to reveal the human health impact of AgNMs during realistic conditions. [ABSTRACT FROM AUTHOR]

Gene expression profiling via RT-qPCR is a robust technique increasingly used in ecotoxicology. Determination and validation of optimal reference genes is a requirement for initiating RT-qPCR experiments. To our best knowledge, this study is the first attempt of identifying a set of reference genes for the freshwater crustacean Gammarus fossarum. Six candidate genes (Actin, TUB, UB, SDH, Clathrin and GAPDH) were tested in order to determine the most stable ones in different stress conditions and to increase the robustness of RT-qPCR data. SDH and Clathrin appeared as the most stable ones. A validation was performed using G. fossarum samples exposed for 15 days to AgNO3, silver nanoparticles (AgNPs) 40 nm and gold nanoparticles (AuNPs) 40 nm. Effects on HSP90 were evaluated and data normalized using Clathrin and SDH. A down-regulation of HSP90 was observed when G. fossarum were exposed to AuNPs 40 nm whereas no effects were observed when G. fossarum were exposed to AgNPs 40 nm. This study highlights the importance of the preliminary determination of suitable reference genes for RT-qPCR experiments. Additionally, this study allowed, for the first time, the determination of a set of valuable genes that can be used in other RT-qPCR studies using G. fossarum as model organism. [ABSTRACT FROM AUTHOR]