Online) An Open Access

ABSTRACT Chronic toxicity assays using arsenic trioxide were performed on grass carp, Ctenopharyngodon idellus, to evaluate their impact on protein content, cholesterol and enzymes like ACP, ALP, GOT and GPT. The total protein showed a continuous decline from 15 th day (13.16 mg/ml) to 60 th day (12.32 mg/ml) of exposure whereas there was no significant change in the cholesterol content and weight of the fish. Significant decrease in the activities of ACP, ALP, GOT and GPT were recorded with increase in the exposure period. The total protein and enzymes recovered in 60 days of recovery period

Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes

Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1–30 μg/cm2) using an air–liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte- derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs

Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes

Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1–30 μg/cm2) using an air–liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte- derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs

Expert consensus on an in vitro approach to assess pulmonary fibrogenic potential of aerosolized nanomaterials

The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern, such as MWCNTs, in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this test led an international expert group to convene in Washington, DC, USA, to discuss alternative approaches to evaluate the inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure, and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. While fibrosis takes weeks or months to develop in vivo, an in vitro test system may more rapidly predict fibrogenic potential by monitoring pro-fibrotic mediators (e.g., cytokines and growth factors). Therefore, the workshop discussions focused on the necessary specifications related to the development and evaluation of such an in vitro system. Recommendations were made for designing a system using lung-relevant cells co-cultured at the air–liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while considering human-relevant dosimetry and NM life cycle transformations. The workshop discussions provided the fundamental design components of an air–liquid interface in vitro test system that will be subsequently expanded to the development of an alternative testing strategy to predict pulmonary toxicity and to generate data that will enable effective risk assessment of NMs

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances

Molecular Identification of Chironomid Species Based on Its-1 and Its-2 Regions of rDNA

Of all major aquatic invertebrate groups, members of family Chironomidae are most abundant and show a wide range of habitat preferences. The importance of correct identification of Chironomids has been realized in many bioassessment studies mainly because of their worldwide distribution, substrate specificities and predictable responses to various pollutants in the water sources. This study establishes that the sequence data from the Intergenic Spacer Regions (ITS) of ribosomal DNA could be used as molecular markers to distinguish between different Chironomidae species and also to identify them. The need to use molecular approaches, to identify various Chironomidae species, comes from the fact that the rate of misidentifications is fairly high when morphological features are used. A difference of six nucleotides in the sequence data of Chironomus tentans from North America and Europe suggest a low intraspecific variation. A detailed analysis of the ITS-1 and ITS-2 sequence data from seven new species of Chironomids (Thienemanniella xena, Xylatopus par, Tribelos fuscicorne, Robackia demeijerei, Tribelos jucundum, Polypedilum aviceps and Chironomus tentans) along with 15 species obtained from Genbank considered in this study shows a high amount of interspecific variations and also that the European species tend to cluster close to each other when compared to North American ones. The high bootstrap values and short intercluster branches, depicted in the phylogram, might suggest presence of various clusters and rapid divergence of species, respectively within the genus Chironomus. Such phylogenetic analysis could also provide more information on the genetic relatedness among different species

Simulating Hemodynamics in In Vitro Culture Models: Implications on Nano-Biointeractions

Gold nanoparticles (Au-NPs) have demonstrated great potential in the development of a variety of tools with applications ranging from biomedical to military fields. Consequently, there is increasing concern regarding the toxic potential of these nanomaterials. Biodistribution studies demonstrate clearance of Au-NPs from peripheral circulation and bulk localization primarily in the liver and spleen post- intravenous administration. Deposition of Au-NPs in spleen suggests the potential for direct exposure of immune cells to these foreign materials under relatively static conditions. Although much less, due to the Blood Brain Barrier (BBB), Au-NPs appear to also deposit in the brain, suggesting that the resident cells of the brain may also be exposed to Au-NPs. Studies show the toxic potential of Au-NPs in a variety of cell types, however, the overall picture is still inconclusive due to the variation in cell-to-cell responses to these NPs. Additionally, NP aggregation and sedimentation in static in vitro conditions makes it very difficult to achieve uniformly dispersed treatment solutions. Furthermore, static conditions might be physiologically relevant to certain cell types, such as the immune cells in the spleen and lymph nodes; however the `BBB\u27 experiences continuous flow of blood. Therefore, NP research calls for modification of traditional in vitro models to simulate the in vivo conditions. The main aim of this study was to determine the impact of Au-NPs on two model systems; 1) a B-lymphocyte cell line (CH12.LX) which pose as a direct target to NPs in vivo and 2) a co-culture of an astrocytic (C8-D30) and an endothelial cell line (bEnd.3), where endothelial cells shield the astrocytic cell line from direct exposure to NPs. Furthermore, static conditions might be physiologically relevant to certain cell types, such as the immune cells in the spleen and lymph nodes; however the `BBB\u27 experiences continuous flow of blood. Our results demonstrate that treatment with Au-NPs lead to altered B-cell function, in terms of increased antibody expression, but no change in astrocytic and endothelial cell function was observed in terms of the inflammatory cytokine release. This might suggest that Au-NPs might exhibit differential response in different cell types which further emphasizes the need of careful evaluation of NPs before in vivo use. Furthermore, we observed decrease in agglomeration and deposition under flow conditions in comparison to static in vitro conditions suggesting improvement of traditional in vitro models to simulate the in vivo conditions

The effect of shear flow on nanoparticle agglomeration and deposition in <i>in vitro</i> dynamic flow models

<p>Traditional <i>in vitro</i> toxicity experiments typically involve exposure of a mono- or co-culture of cells to nanoparticles (NPs) in static conditions with the assumption of 100% deposition (i.e. dose) of well-dispersed particles. However, cellular dose can be affected by agglomeration and the unique transport kinetics of NPs in biological media. We hypothesize that shear flow can address these issues and achieve more predictable dosage. Here, we compare the behavior of gold NPs with diameters of 5, 10 and 30 nm in static and dynamic <i>in vitro</i> models. We also utilize transport modeling to approximate the shear rate experienced by the cells in dynamic conditions to evaluate physiological relevance. The transport kinetics show that NP behavior is governed by both gravity and diffusion forces in static conditions and only diffusion in dynamic conditions. Our results reveal that dynamic systems are capable of producing a more predictable dose compared to static systems, which has strong implications for improving repeatability in nanotoxicity assessments.</p

Use of EpiAlveolar lung model to predict fibrotic potential of multiwalled carbon nanotubes

Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1–30 μg/cm2) using an air–liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte- derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs