EURL ECVAM Literature Review Series on Advanced Non-Animal Models for Respiratory Diseases, Breast Cancer and Neurodegenerative Disorders.

In vivo models are used in biomedical research to reproduce human disease and develop new drugs. However, they do not mimic the disease as it occurs in humans, and their use has failed to identify novel therapies effective for many highly prevalent non-communicable diseases, such as Alzheimer's disease. Indeed, the clinical failure rate in drug development remains very high, with an overall likelihood of approval from Phase I of about 9.6%. On the other hand, human-based models, advanced imaging techniques and human epidemiological studies may increase our understanding of disease aetiology and pathogenesis and enable the advance of safe and effective therapies. Particularly when human tissues are used, they may produce faster, cheaper results, more predictive for humans, whilst yielding greater comprehensions of human biochemical processes. A first effort to collect existing knowledge about non-animal models of highly prevalent human diseases was made by the Joint Research Centre of the European Commission. The final aim was to identify and share information on the capabilities and limits of human-based models at different levels: scientific communities, universities and secondary schools, national committees for animal welfare and the public at large.

Is Cadmium Toxicity Tissue-Specific? Toxicogenomics Studies Reveal Common and Specific Pathways in Pulmonary, Hepatic, and Neuronal Cell Models.

Several harmful modifications in different tissues-organs, leading to relevant diseases (e.g., liver and lung diseases, neurodegeneration) are reported after exposure to cadmium (Cd), a wide environmental contaminant. This arises the question whether any common molecular signatures and/or Cd-induced modifications might represent the building block in initiating or contributing to address the cells towards different pathological conditions. To unravel possible mechanisms of Cd tissue-specificity, we have analyzed transcriptomics data from cell models representative of three major Cd targets: pulmonary (A549), hepatic (HepG2), and neuronal (SH-SY-5Y) cells. Further, we compared common features to identify any non-specific molecular signatures. The functional analysis of dysregulated genes (gene ontology and KEGG) shows GO terms related to metabolic processes significantly enriched only in HepG2 cells. GO terms in common in the three cell models are related to metal ions stress response and detoxification processes. Results from KEGG analysis show that only one specific pathway is dysregulated in a significant way in all cell models: the mineral absorption pathway. Our data clearly indicate how the molecular mimicry of Cd and its ability to cause a general metal ions dyshomeostasis represent the initial common feature leading to different molecular signatures and alterations, possibly responsible for different pathological conditions.

The CAM Model-Q&A with Experts.

The chick chorioallantoic membrane (CAM), as an extraembryonic tissue layer generated by the fusion of the chorion with the vascularized allantoic membrane, is easily accessible for manipulation. Indeed, grafting tumor cells on the CAM lets xenografts/ovografts develop in a few days for further investigations. Thus, the CAM model represents an alternative test system that is a simple, fast, and low-cost tool to study tumor growth, drug response, or angiogenesis in vivo. Recently, a new era for the CAM model in immune-oncology-based drug discovery has been opened up. Although there are many advantages offering extraordinary and unique applications in cancer research, it has also disadvantages and limitations. This review will discuss the pros and cons with experts in the field.

Progress towards an OECD reporting framework for transcriptomics and metabolomics in regulatory toxicology.

Omics methodologies are widely used in toxicological research to understand modes and mechanisms of toxicity. Increasingly, these methodologies are being applied to questions of regulatory interest such as molecular point-of-departure derivation and chemical grouping/read-across. Despite its value, widespread regulatory acceptance of omics data has not yet occurred. Barriers to the routine application of omics data in regulatory decision making have been: 1) lack of transparency for data processing methods used to convert raw data into an interpretable list of observations; and 2) lack of standardization in reporting to ensure that omics data, associated metadata and the methodologies used to generate results are available for review by stakeholders, including regulators. Thus, in 2017, the Organisation for Economic Co-operation and Development (OECD) Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) launched a project to develop guidance for the reporting of omics data aimed at fostering further regulatory use. Here, we report on the ongoing development of the first formal reporting framework describing the processing and analysis of both transcriptomic and metabolomic data for regulatory toxicology. We introduce the modular structure, content, harmonization and strategy for trialling this reporting framework prior to its publication by the OECD.

Heme catabolism by tumor-associated macrophages controls metastasis formation.

Although the pathological significance of tumor-associated macrophage (TAM) heterogeneity is still poorly understood, TAM reprogramming is viewed as a promising anticancer therapy. Here we show that a distinct subset of TAMs (F4/80hiCD115hiC3aRhiCD88hi), endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), plays a critical role in shaping a prometastatic tumor microenvironment favoring immunosuppression, angiogenesis and epithelial-to-mesenchymal transition. This population originates from F4/80+HO-1+ bone marrow (BM) precursors, accumulates in the blood of tumor bearers and preferentially localizes at the invasive margin through a mechanism dependent on the activation of Nrf2 and coordinated by the NF-κB1-CSF1R-C3aR axis. Inhibition of F4/80+HO-1+ TAM recruitment or myeloid-specific deletion of HO-1 blocks metastasis formation and improves anticancer immunotherapy. Relative expression of HO-1 in peripheral monocyte subsets, as well as in tumor lesions, discriminates survival among metastatic melanoma patients. Overall, these results identify a distinct cancer-induced HO-1+ myeloid subgroup as a new antimetastatic target and prognostic blood marker.

In vitro and bioinformatics mechanistic-based approach for cadmium carcinogenicity understanding.

Cadmium is a toxic metal able to enter the cells through channels and transport pathways dedicated to essential ions, leading, among others, to the dysregulation of divalent ions homeostasis. Despite its recognized human carcinogenicity, the mechanisms are still under investigation. A powerful tool for mechanistic studies of carcinogenesis is the Cell Transformation Assay (CTA). We have isolated and characterized by whole genome microarray and bioinformatics analysis of differentially expressed genes (DEGs) cadmium-transformed cells from different foci (F1, F2, and F3) at the end of CTA (6 weeks). The systematic analysis of up- and down-regulated transcripts and the comparison of DEGs in transformed cells evidence different functional targets and the complex picture of cadmium-induced transformation. Only 34 in common DEGs are found in cells from all foci, and among these, only 4 genes are jointly up-regulated (Ccl2, Ccl5, IL6 and Spp1), all responsible for cytokines/chemokines coding. Most in common DEGs are down-regulated, suggesting that the switching-off of specific functions plays a major role in this process. In addition, the comparison of dysregulated pathways immediately after cadmium treatment with those in transformed cells provides a valuable means to the comprehension of the overall process.

Neuron-Like Cells Generated from Human Umbilical Cord Lining-Derived Mesenchymal Stem Cells as a New In Vitro Model for Neuronal Toxicity Screening: Using Magnetite Nanoparticles as an Example.

The wide employment of iron nanoparticles in environmental and occupational settings underlines their potential to enter the brain. Human cell-based systems are recommended as relevant models to reduce uncertainty and to improve prediction of human toxicity. This study aimed at demonstrating the in vitro differentiation of the human umbilical cord lining-derived-mesenchymal stem cells (hCL-MSCs) into neuron-like cells (hNLCs) and the benefit of using them as an ideal primary cell source of human origin for the neuronal toxicity of Fe3O4NPs (magnetite-nanoparticles). Neuron-like phenotype was confirmed by: live morphology; Nissl body staining; protein expression of different neuronal-specific markers (immunofluorescent staining), at different maturation stages (i.e., day-3-early and day-8-full differentiated), namely ß-tubulin III, MAP-2, enolase (NSE), glial protein, and almost no nestin and SOX-2 expression. Synaptic makers (SYN, GAP43, and PSD95) were also expressed. Fe3O4NPs determined a concentration- and time-dependent reduction of hNLCs viability (by ATP and the Trypan Blue test). Cell density decreased (20-50%) and apoptotic effects were detected at ≥10 µg/mL in both types of differentiated hNLCs. Three-day-differentiated hNLCs were more susceptible (toxicity appeared early and lasted for up to 48 h) than 8-day-differentiated cells (delayed effects). The study demonstrated that (i) hCL-MSCs easily differentiated into neuronal-like cells; (ii) the hNCLs susceptibility to Fe3O4NPs; and (iii) human primary cultures of neurons are new in vitro model for NP evaluation.

Investigating cell type specific mechanisms contributing to acute oral toxicity.

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of Integrated Approaches to Testing and Assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evaluation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 98 were identified with target organ specific effects, of which 93% were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% as non-toxic. This analysis will help to prioritise the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.

Human 3D Cultures as Models for Evaluating Magnetic Nanoparticle CNS Cytotoxicity after Short- and Repeated Long-Term Exposure.

Since nanoparticles (NPs) can translocate to the brain and impact the highly vulnerable central nervous system (CNS), novel in vitro tools for the assessment of NP-induced neurotoxicity are advocated. In this study, two types of CNS spheroids have been developed from human D384 astrocyte- and SH-SY5Y neuronal-like cells, and optimized in combination with standard assays (viability readout and cell morphology) to test neurotoxic effects caused by Fe3O4NPs, as NP-model, after short- (24­48 h; 1­100µg/ml) and long-term repeated exposure (30days; 0.1­25µg/ml). Short-term exposure of 3D-spheroids to Fe3O4NP induced cytotoxicity at 10 µg/mL in astrocytes and 25 µg/mL neurons. After long-term repeated dose regimen, spheroids showed concentration- and time-dependent cell mortality at 10 µg/mL for D384 and 0.5 µg/mL for SH-SY5Y, indicating a higher susceptibility of neurons than astrocytes. Both spheroid types displayed cell disaggregation after the first week of treatment at ≥0.1 µg/mL and becoming considerably evident at higher concentrations and over time. Recreating the 3D-spatial environment of the CNS allows cells to behave in vitro more closely to the in vivo situations, therefore providing a model that can be used as a stand-alone test or as a part of integrated testing strategies. These models could drive an improvement in the species-relevant predictivity of toxicity testing.

Rotenone exerts developmental neurotoxicity in a human brain spheroid model.

Growing concern suggests that some chemicals exert (developmental) neurotoxicity (DNT and NT) and are linked to the increase in incidence of autism, attention deficit and hyperactivity disorders. The high cost of routine tests for DNT and NT assessment make it difficult to test the high numbers of existing chemicals. Thus, more cost effective neurodevelopmental models are needed. The use of induced pluripotent stem cells (iPSC) in combination with the emerging human 3D tissue culture platforms, present a novel tool to predict and study human toxicity. By combining these technologies, we generated multicellular brain spheroids (BrainSpheres) from human iPSC. The model has previously shown to be reproducible and recapitulates several neurodevelopmental features. Our results indicate, rotenone's toxic potency varies depending on the differentiation status of the cells, showing higher reactive oxygen species (ROS) and higher mitochondrial dysfunction during early than later differentiation stages. Immuno-fluorescence morphology analysis after rotenone exposure indicated dopaminergic-neuron selective toxicity at non-cytotoxic concentrations (1 µM), while astrocytes and other neuronal cell types were affected at (general) cytotoxic concentrations (25 µM). Omics analysis showed changes in key pathways necessary for brain development, indicating rotenone as a developmental neurotoxicant and show a possible link between previously shown effects on neurite outgrowth and presently observed effects on Ca2+ reabsorption, synaptogenesis and PPAR pathway disruption. In conclusion, our BrainSpheres model has shown to be a reproducible and novel tool to study neurotoxicity and developmental neurotoxicity. Results presented here support the idea that rotenone can potentially be a developmental neurotoxicant.

Toxicogenomics applied to in vitro Cell Transformation Assay reveals mechanisms of early response to cadmium.

Cadmium is a well recognized carcinogen, primarily released into the environment by anthropogenic activities. In the effort to understand the early events responsible for cadmium carcinogenesis, we have used an in vitro biological system (the Cell Transformation Assay, CTA), that has been shown to closely model some key stages of the conversion of normal cells into malignant ones. Cadmium-triggered early responses in CTA were analysed through microarray-based toxicogenomics. Metallothioneins represent the earliest cell response, together with Slc30a1 encoding for a ZnT-1 zinc exporter. Other genes were found to be up-regulated in the first 24 h following Cd administration: phospatidylinositol-4-phospate 5-kinase alpha (Pip5k1a), glutathione S-transferase (Gstα 1-3), Gdf15 and aldolase. However, after the exposure, a number of genes expressing zinc proteins were found to be down-regulated, among which were many olfactory receptors (ORs) coding genes. Cd administration also promoted massive Zn release inside the cell that could be related to moonlighting activities of regulated genes (proteins). On the whole our data suggest that, despite the early involvement of defence mechanisms (metallothionein and GST), Cd-triggered Zn release, as well as Cd interference with different proteins, may lead to gene expression alterations which later induce metabolic changes, directing the cells towards uncontrolled growth.

Air-Liquid Interface In Vitro Models for Respiratory Toxicology Research: Consensus Workshop and Recommendations.

In vitro air-liquid interface (ALI) cell culture models can potentially be used to assess inhalation toxicology endpoints and are usually considered, in terms of relevancy, between classic (i.e., submerged) in vitro models and animal-based models. In some situations that need to be clearly defined, ALI methods may represent a complement or an alternative option to in vivo experimentations or classic in vitro methods. However, it is clear that many different approaches exist and that only very limited validation studies have been carried out to date. This means comparison of data from different methods is difficult and available methods are currently not suitable for use in regulatory assessments. This is despite inhalation toxicology being a priority area for many governmental organizations. In this setting, a 1-day workshop on ALI in vitro models for respiratory toxicology research was organized in Paris in March 2016 to assess the situation and to discuss what might be possible in terms of validation studies. The workshop was attended by major parties in Europe and brought together more than 60 representatives from various academic, commercial, and regulatory organizations. Following plenary, oral, and poster presentations, an expert panel was convened to lead a discussion on possible approaches to validation studies for ALI inhalation models. A series of recommendations were made and the outcomes of the workshop are reported.

State-of-the-art and new options to assess T cell activation by skin sensitizers: Cosmetics Europe Workshop.

Significant progress has been made in the development and validation of non-animal test methods for skin sensitization assessment. At present, three of the four key events of the Adverse Outcome Pathway (AOP) are assessable by OECD-accepted in vitro methods. The fourth key event describes the immunological response in the draining lymph node where activated dendritic cells present major histocompatibility complex-bound chemically modified peptides to naive T cells, thereby priming the proliferation of antigen-specific T cells. Despite substantial efforts, modelling and assessing this adaptive immune response to sensitizers with in vitro T cell assays still represents a challenge. The Cosmetics Europe Skin Tolerance Task Force organized a workshop, bringing together academic researchers, method developers, industry representatives and regulatory stakeholders to review the scientific status of T cell-based assays, foster a mutual scientific understanding and conceive new options to assess T cell activation. Participants agreed that current T cell assays have come a long way in predicting immunogenicity, but that further investment and collaboration is required to simplify assays, optimize their sensitivity, better define human donor-to-donor variability and evaluate their value to predict sensitizer potency. Furthermore, the potential role of T cell assays in AOP-based testing strategies and subsequent safety assessment concepts for cosmetic ingredients was discussed. It was agreed that it is currently difficult to anticipate uses of T cell assay data for safety assessment and concluded that experience from case studies on real-life risk assessment scenarios is needed to further consider the usefulness of assessing the fourth AOP key event.

Human Co-culture Model of Neurons and Astrocytes to Test Acute Cytotoxicity of Neurotoxic Compounds.

Alternative methods and their use in planning and conducting toxicology experiments have become essential for modern toxicologists, thus reducing or replacing living animals. Although in vitro human co-culture models allow the establishment of biologically relevant cell-cell interactions that recapitulate the tissue microenvironment and better mimic its physiology, the number of publications is limited specifically addressing this scientific area and utilizing this test method which could provide an additional valuable model in toxicological studies. In the present study, an in vitro model based on central nervous system (CNS) cell co-cultures was implemented using a transwell system combining human neuronal cells (SH-SY5Y cell line) and glial cells, namely astrocytes (D384 cell line), to investigate neuroprotection of D384 on SH-SY5Y and vice versa. The model was applied to test acute (24-48 hours) cytotoxicity of 3 different neurotoxicants: (1) methyl mercury (1-2.5 µM), (2) Fe3O4 nanoparticles (1-100 µg/mL), and (3) methylglyoxal (0.5-1 mM). Data were compared to mono-cultures evaluating the mitochondrial function and cell morphology. The results clearly showed that all compounds tested affected the mitochondrial activity and cell morphology in both mono-culture and co-culture conditions. However, astrocytes, when cultured together with neurons, diminish the neurotoxicant-induced cytotoxic effects that occurred in neurons cultured alone, and astrocytes become more resistant in the presence of neurons. This human CNS co-culture system seems a suitable cell model to feed high-throughput acute screening platforms and to evaluate both human neuronal and astrocytic toxicity and neuroprotective effects of new and emerging materials (eg, nanomaterials) and new products with improved sensitivity due to the functional neuron-astrocyte metabolic interactions.

The challenge of the application of 'omics technologies in chemicals risk assessment: Background and outlook.

This survey by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) highlights that 'omics technologies are generally not yet applied to meet standard information requirements during regulatory hazard assessment. While they are used within weight-of-evidence approaches to investigate substances' modes-of-action, consistent approaches for the generation, processing and interpretation of 'omics data are not applied. To date, no 'omics technology has been standardised or validated. Best practices for performing 'omics studies for regulatory purposes (e.g., microarrays for transcriptome profiling) remain to be established. Therefore, three frameworks for (i) establishing a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) 'omics data processing; and (iii) quantitative WoE approaches to interpret 'omics data have been developed, that are presented in this journal supplement. Application of the frameworks will enable between-study comparison of results, which will facilitate the regulatory applicability of 'omics data. The frameworks do not constitute prescriptive protocols precluding any other data analysis method, but provide a baseline for analysis that can be applied to all data allowing ready cross-comparison. Data analysis that does not follow the frameworks can be justified and the resulting data can be compared with the Framework-based common analysis output.