October 21, 2016
Olivier Taboureau and Karine Audouze
During the past decades, many epidemiological, toxicological and biological studies have been performed to assess the role of environmental chemicals as potential toxicants for diverse human disorders. However, the relationships between diseases based on chemical exposure have been rarely studied by computational biology.
We developed a human environmental disease network (EDN) to explore and suggest novel disease-disease and chemical-disease relationships. The presented scored EDN model is built upon the integration on systems biology and chemical toxicology using chemical contaminants information and their disease relationships from the reported TDDB database.
The resulting human EDN takes into consideration the level of evidence of the toxicant-disease relationships allowing including some degrees of significance in the disease-disease associations. Such network can be used to identify uncharacterized connections between diseases. Examples are discussed with type 2 diabetes (T2D).
Additionally, this computational model allows to confirm already know chemical-disease links (e.g. bisphenol A and behavioral disorders) and also to reveal unexpected associations between chemicals and diseases (e.g. chlordane and olfactory alteration), thus predicting which chemicals may be risk factors to human health.
With the proposed human EDN model, it is possible to explore common biological mechanism between two diseases through chemical exposure helping us to gain insight into disease etiology and comorbidity. Such computational approach is an alternative to animal testing supporting the 3R concept.
Keywords: computational method, environmental contaminants, predictive toxicology, systems biology, human disease network
October 21, 2016
Jennifer Hennen and Brunhilde Blömeke
In vitro approaches address key steps of chemical-induced skin sensitization but there is uncertainty how keratinocytes, which play a crucial role not only regarding xenobiotic metabolism but also skin inflammation, impact on chemicals’ potential and potency of dendritic cell activation. We investigated these aspects by coculturing THP-1 cells, as surrogate dendritic cells, with HaCaT keratinocytes. We tested our HaCaT/THP-1 model with a set of 14 sensitizers, containing 7 prohaptens, and 10 non-sensitizers. Compared to THP-1 alone, coculturing resulted in up to 3.1-fold enhanced maximal CD86 and/or CD54 upregulation on THP-1, resulting in improved concentration-dependency. All 14 sensitizers were found positive for CD86 and/or CD54. Based on the current thresholds of Δmean fluorescence intensity (MFI) ≥ 10 and ΔMFI ≥ 50, respectively, only 1 of 10 non-sensitizers was false-positive. Remarkably, coculture with HaCaT keratinocytes improved the rank correlation of concentrations needed to reach the thresholds for positivity with in vivo data on sensitization potency, especially for CD54 (Spearman r = 0.739, p = 0.006; CD86: r = 0.571, p = 0.041). These promising data suggest that a coculture model has the potential to support the prediction of sensitization potency using in vitro data, helping quantitative risk assessment.
Keywords: inflammatory microenvironment, skin sensitization potency, cross talk, THP-1, HaCaT keratinocytes
October 21, 2016
Andrea Schwab, Annick Meeuwsen, Franziska Ehlicke, Jan Hansmann, Lars Mulder, Anthal Smits, Heike Walles and Linda Kock
There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone which better represents the in vivo situation and enables supply of specific factors to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue (study A). Next, we evaluated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture of 84 days (study B). Finally, we investigated what the optimal defect size is without spontaneous self-healing occurring in this culture system (study C).
It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allows for long-term culture of osteochondral explants, while maintaining cartilage viability, matrixtissue content, structure and mechanical properties up to at least 56 days. Furthermore, it was shown that we can create critical size cartilage defects of different sizes in the model.
The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials for their regenerative potential, evaluation of drug and cell therapies or to study mechanisms of cartilage regeneration, which will undoubtedly reduce the number of animals needed for in vivo testing.
Keywords: ex vivo model, osteochondral biopsy, cartilage repair, critical size defect, replacement
October 11, 2016
Dorothea Döring, Ophelia Nick, Alexander Bauer, Helmut Küchenhoff and Michael H. Erhard
Although the rehoming of laboratory dogs has gained importance, scientific data that evaluate the process are lacking. Therefore, 145 laboratory beagles were tested before leaving the research facility (Test 1). The new owners were surveyed using a standardized telephone interview 1 week (n = 143) and 12 weeks (n = 126) after adoption. The behavior test was repeated with 68 dogs in their new homes 6 weeks after adoption (Test 2). The predictive power of Test 1 as well as the relevance of various factors was analyzed.
We found no significant differences between Tests 1 and 2 regarding the behavior reactions. In contrast, body language scores and heart rates changed significantly, indicating a more relaxed state of the dogs in their new homes.The interviews revealed a significant change toward desired behavior in most dogs within the 11 week period (p < 0.0001). The main behavior problems included separation problems (28%; n = 126), destroying objects (24%), and not being housebroken (39%).Owners of 9 dogs returned the animals, resulting in a rehoming success rate of 94%. The predictive power of Test 1 was low to moderate. Test 1 revealed a significant age effect (P = 0.0066), with younger and older dogs reaching higher scores than dogs approximately 2 years old. Dogs that had been born and reared in the research facility scored higher than dogs that had originally been acquired from a commercial breeder (P = 0.0257). Altogether, the rehoming of laboratory dogs is a valuable alternative to euthanasia.
Keywords: adoption; behavior problem; behavior test; laboratory dog; rehoming
October 11, 2016
Maren Jannasch, Tobias Weigel, Lisa Engelhardt, Judith Wiezoreck, Sabine Gaetzner, Heike Walles, Tobias Schmitz andJan Hansmann
Surgical implantation of a biomaterial triggers foreign-body-induced fibrous encapsulation. Two major mechanisms of this complex physiological process are (I) chemotaxis of fibroblasts from surrounding tissue to the implant region, followed by (II) tissue remodeling. As an alternative to animal studies, we here propose anprocess-aligned in vitrotest platform to investigate the material dependency of fibroblast chemotaxis and tissue remodeling, mediated by material-resident macrophages.
Embedded in a biomimetic three-dimensional collagen hydrogel, chemotaxis of fibroblasts in direction of macrophage-material-incubated cell culture supernatant was analysed by live-cell imaging. A combination of statistical analysis with a complementary parameterized random walk model allowed quantitative and qualitative characterization of the cellular walk process. We thereby identified an increasing macrophage-mediated chemotactic potential ranking from tested biomaterials glass over polytetrafluorethylene to titanium. To address long-term effects of biomaterial-resident macrophages on fibroblasts in a three-dimensional microenvironment, we further studied tissue remodeling by applying macrophage-material-incubated medium on fibrous in vitro tissue models. A high correlation to state of the art in vivo studies to the proposed in vitro tissue model was found. Titanium exhibited significantly lower tissue remodeling capacity compared to polytetrafluorethylene. By this approach we identified a material dependency for both processes chemotaxis and tissue remodeling, strengthening their specific contribution to foreign body reaction.
Keywords: Foreign body reaction, fibroblast chemotaxis, tissue remodeling, in vitro, quantitative characterization
September 30, 2016
Carbonell, Oriol Lopez, Alexander Amberg,
Manuel Pastor and Ferran Sanz
The present study applies a systems biology approach for the in silico predictive modeling of drug toxicity on the basis of high-quality preclinical drug toxicity data with the aim of increasing the mechanistic understanding of toxic effects of compounds at different levels (pathway, cell, tissue, organ). The model development has been carried out using 77 compounds for which gene expression data are available in the LINCS database for primary human hepatocytes treated with the compounds, as well as rodent in vivo hepatotoxicity information is available in the eTOX database. The data from LINCS were used in a systems biology approach to determine the type and number of pathways disturbed by each compound, and to estimate the extent of disturbance (network perturbation elasticity), analyzing the correspondence with the in vivo information from eTOX. Predictive models were developed through this integrative analysis, and their specificity and sensitivity were assessed. The quality of the predictions was determined on the basis of the area under the curve (AUC) of plots of true positive vs. false positive rates (ROC curves). The ROC AUC reached values of up to 0.9 (out of 1.0) for some hepatotoxicity endpoints. Moreover, the most frequently disturbed metabolic pathways were determined across the studied toxicants. They included e.g. mitochondrial beta-oxidation of fatty acids and amino acid metabolism. The process was exemplified by successful predictions on various statins. In conclusion, an entirely new approach linking gene expression alterations to the prediction of complex organ toxicity has been developed and evaluated.
Keywords: systems biology, predictive modeling, drug toxicity, hepatotoxicity, gene regulation
September 29, 2016
John T. Elliott, Matthias Rösslein, Nam Woong Song, Blaza Toman, Agnieszka Kinsner-Ovaskainen, Rawiwan Maniratanachote, Marc L. Salit, Elijah J. Petersen, Fatima Sequeira, Erica L. Romsos, Soo Jin Kim, Jieun Lee, Nadia R. Von Moos, François Rossi, Cordula Hirsch, Harald F. Krug, Wongsakorn Suchaoin and Peter Wick
Design and development of reliable cell-based nanotoxicology assays are important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nano-cytotoxicity MTS cell viability assay. The consensus EC50 values were 22.1 mg/l (95 % confidence intervals 16.9 mg/l to 27.2 mg/l) and 52.6 mg/l (44.1 mg/l to 62.6 mg/l) for the A549 cell line from ATCC for positively charged polystyrene nanoparticles for the serum free and serum conditions, respectively, and were 49.7 µmol/l (47.5 µmol/l to 51.5 µmol/l) and 77.0 µmol/l (54.3 µmol/l to 99.4 µmol/l) for positive chemical control cadmium sulfate for the serum free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nano-cytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.
Keywords: nanotoxicology, MTS assay, interlaboratory comparison, polystyrene nanoparticles, alternatives to animal testing
September 26, 2016
Rachel Tanner and Helen McShane
Tuberculosis (TB) remains a serious global health threat and an improved vaccine is urgently needed. New candidate TB vaccines are tested using preclinical animal models such as mice, guinea pigs, cattle and non-human primates. Animals are routinely infected with virulent Mycobacterium tuberculosis (M.tb) in challenge experiments to evaluate protective efficacy, raising ethical issues regarding the procedure of infection itself, symptoms of disease and humane end-points. We summarise the importance and limitations of animal models in TB vaccine research and review current alternatives and modifications in the context of the NC3Rs framework for replacing, reducing and refining the use of animals for scientific purposes.
Keywords: tuberculosis, vaccine, animal models, M.tb challenge, 3Rs
August 23, 2016; updated October 5, 2016
t4 Workshop Report
David Pamies, Anna Bal-Price, Anton Simeonov, Danilo Tagle, Dave Allen, David Gerhold, Dezhong Yin, Francesca Pistollato, Takashi Inutsuka, Kristie Sullivan, Glyn Stacey, Harry Salem, Marcel Leist, Mardas Daneshian, Mohan C. Vemuri, Richard McFarland, Sandra Coecke, Suzanne C. Fitzpatrick, Uma Lakshmipathy, Amanda Mack, Wen Bo Wang, Yamazaki Daiju, Yuko Sekino, Yasunari Kanda, Lena Smirnova and Thomas Hartung
The first guidance on Good Cell Culture Practice dates back to 2005. This document expands this to aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice which can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance, which will facilitate the generation of reliable data from cell culture systems, but is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered as a first step toward a revised GCCP 2.0.
Keywords: Good Cell
Culture Practices, in vitro methods,
alternatives to animals, induced pluripotent stem cells
July 27, 2016
Johanna Nyffeler, Christiaan Karreman, Heidrun Leisner, Yong Jun Kim, Gabsang Lee, Tanja Waldmann and Marcel Leist
Migration of neural crest cells (NCCs) is one of the pivotal processes of human fetal development. Malformations arise, if NCC migration and differentiation are impaired genetically or by toxicants. In the currently available test systems for migration inhibition of NCC (MINC), the manual generation of a cell-free space results in extreme operator dependencies, and limits throughput. Therefore, a new test format was established here. The assay avoids scratching by plating cells around a commercially available circular stopper. Removal of this barrier after cell attachment initiates migration. This microwell-based circular migration zone NCC function assay (cMINC) was further optimized for toxicological testing of human pluripotent stem cell (hPSC)-derived NCCs. The challenge of automated image processing to obtain data on viability and migration was addressed by development of a software made generally available for downloading. To optimize the biological system, data on cell proliferation were obtained by labelling of replicating cells, and by careful assessment of cell viability for each experimental sample. The role of cell proliferation as experimental confounder was tested experimentally by performance of the cMINC in the presence of the proliferation-inhibiting drug cytosine arabinoside (AraC), and by a careful evaluation of mitotic events over time. Data from these studies led to an adaptation of the test protocol, so that toxicant exposure was limited to 24 h. Under these conditions, a prediction model was developed that allowed classification of toxicants as either being inactive, leading to unspecific cytotoxicity or specifically inhibiting NC migration at non-cytotoxic concentrations.
Keywords: cell tracking, cell proliferation, high content imaging, developmental toxicity, human stem cells
July 25, 2016; updated September 28, 2016
t4 Workshop Report
Michael Aschner, Sandra Ceccatelli, Mardas Daneshian, Ellen Fritsche, Nina Hasiwa, Thomas Hartung, Helena T. Hogberg, Marcel Leist, Abby Li, William R. Mundy, Stephanie Padilla, Aldert H. Piersma, Anna Bal-Price, Andrea Seiler, Remco H. Westerink, Bastian Zimmer and Pamela J. Lein
There is a paucity of information concerning the developmental neurotoxicity (DNT) hazard posed by industrial and environmental chemicals. New testing approaches will most likely be based on batteries of alternative and complementary (non-animal) tests. As DNT is assumed to result from the modulation of fundamental neurodevelopmental processes (such as neuronal differentiation, precursor cell migration or neuronal network formation) by chemicals, the first generation of alternative DNT tests target these processes. The advantage of such types of assays is that they capture toxicants with multiple targets and modes-of-action. Moreover, the processes modelled by the assays can be linked to toxicity endophenotypes, i.e. alterations in neural connectivity that form the basis for neurofunctional deficits in man. The authors of this review convened in a workshop to define criteria for the selection of positive/negative controls, to prepare recommendations on their use, and to initiate the setup of a directory of reference chemicals. For initial technical optimization of tests, a set of >50 endpoint-specific control compounds was identified. For further test development, an additional "test" set of 33 chemicals considered to act directly as bona fide DNT toxicantsis proposed, and each chemical is annotated to the extent it fulfills these criteria. A tabular compilation of the original literature used to select the test set chemicals provides information on statistical procedures, and toxic/non-toxic doses (both for pups and dams). Suggestions are provided on how to use the >100 compounds (including negative controls) compiled here to address specificity, adversity and use of alternative test systems.
Keywords: neurotoxicity, specificity, test development, AOP, validation
July 21, 2016; updated September 27, 2016
Joakim Ringblom, Elin Törnqvist, Sven Ove Hansson, Christina Rudén and Mattias Öberg
Reducing the number of laboratory animals used and refining experimental procedures to enhance animal welfare are fundamental questions to be considered in connection with animal experimentation. Here, we explored the use of cardinal ethical weights for clinical signs and symptoms in rodents by conducting Trade-Off interviews with members of Swedish Animal Ethics Committees in order to derive such weights for nine typical clinical signs of toxicity. The participants interviewed represent researchers, politically nominated laypersons and representatives of animal welfare organizations. We observed no statistically significant differences between these groups with respect to the magnitude of the ethical weights assigned, even though the political nominees tended to assign lower weights. Hunched posture was considered the most severe clinical sign and body weight loss the least severe. The ethical weights assigned varied considerably between individuals, from none to infinite value, indicating discrepancies in prioritization of reduction and refinement. Cardinal ethical weights may be utilized to include both animal welfare refinement and reduction of animal use in designing as well as in retrospective assessments of animal experiments. Such weights may also be used to estimate ethical costs of animal experiments.
Keywords: 3Rs, animal ethics, ethical weights, ethical committees, toxicity testing
7, 2016; updated July 11, 2016
Anita R. Iskandar, Carole Mathis, Florian Martin, Patrice Leroy, Alain Sewer, Shoaib Majeed, Diana Kuehn, Keyur Trivedi, Davide Grandolfo, Maciej Cabanski, Emmanuel Guedj, Celine Merg, Stefan Frentzel, Nikolai V. Ivanov, Manuel C. Peitsch and Julia Hoeng
In vitro toxicology approaches have evolved, from a focus on molecular changes within a cell to understanding of toxicity-related mechanisms in systems that can mimic the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offers a physiologically robust system for studying the effects of exposure through inhalation. Exposure to cigarette smoke (CS) is associated with nasal inflammation; thus the nasal epithelium is relevant for evaluating the pathophysiological impact of CS exposure. The present study investigated further the application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. Aligned with 3Rs strategy, this study aimed to explore the relevance of a human 3-D nasal culture model to assess the toxicological impact of aerosols generated from a candidate modified risk tobacco product (cMRTP), the Tobacco Heating System (THS)2.2, as compared with smoke generated from reference cigarette 3R4F. A series of experimental repetitions where multiple concentrations of THS2.2 aerosol and 3R4F smoke were applied, were conducted to obtain reproducible measurements to understandthe cellular/molecular changes that occur following exposure. In agreement with the Vision and Strategy of the Toxicity Testing in the 21st Century, this study implemented a systems toxicology approach and found that for all tested concentrations, the impact of 3R4F smoke was substantially greater than that of THS2.2 aerosol in terms of cytotoxicity levels, alterations in the tissue morphology, secretion of pro-inflammatory mediators, impaired ciliary function, and increased perturbed transcriptomes and miRNA expression profiles.
Keywords: air-liquid interface, organotypic culture, cigarette smoke, modified risk tobacco product, systems toxicology
June 21, 2016
Antonio Planchart, Carolyn J. Mattingly, David Allen, Patricia Ceger, Warren Casey, David Hinton, Jyotshna Kanungo, Seth W. Kullman, Tamara Tal, Maria Bondesson, Shawn M. Burgess, Con Sullivan, Carol Kim, Mamta Behl, Stephanie Padilla, David M. Reif, Robert L. Tanguay and Jon Hamm
Small freshwater fish models, especially zebrafish,offer advantages over traditional rodent models, including low maintenance and husbandry costs, high fecundity, genetic diversity, physiology similar to that of traditional biomedical models, and reduced animal welfare concerns. The Collaborative Workshop on Aquatic Models and 21st Century Toxicology was held at North Carolina State University on May 5–6, 2014, in Raleigh, North Carolina, USA. Participants discussed the ways in which small fish are being used as models to screen toxicants and understand mechanisms of toxicity. Workshop participants agreed that the lack of standardized protocols is an impediment to broader acceptance of these models, whereas development of standardized protocols, validation, and subsequent regulatory acceptance would facilitate greater usage. Given the advantages and increasing application of small fish models, there was widespread interest in follow-up workshops to review and discuss developments in their use. In this article, we summarize the recommendations formulated by workshop participants to enhance the utility of small fish species in toxicology studies, as well as many of the advances in the field of toxicology that resulted from using small fish species, including advances in developmental toxicology, cardiovascular toxicology, neurotoxicology, and immunotoxicology. We alsoreview many emerging issues that will benefit from using small fish species, especially zebrafish, and new technologies that will enable using these organisms to yield results unprecedented in their information content to better understand how toxicants affect development and health.
Keywords: aquatic models, 21st century toxicology, alternatives
June 2, 2016
Amy J. Clippinger, Erin Hill, Rodger Curren and Patricia Bishop
Collaboration between industry and regulators resulted in the development of a decision tree approach using in vitro or ex vivo assays to replace animal tests when determining the eye irritation potential of antimicrobial cleaning products (AMCPs) under the United States Environmental Protection Agency (EPA) Office of Pesticide Programs’ hazard classification and labeling system. A policy document issued by the EPA in 2013 and updated in 2015 describes the alternate testing framework that industry could apply to new registrations of AMCPs and, on a case-by-case basis, to conventional pesticide products. Despite the collaborative effort, the availability of relevant non-animal methods, and the EPA’s change in policy, only a limited number of AMCPs have been registered using the framework. Companies continue to conduct animal tests when registering AMCPs due to various challenges surrounding adoption of the new testing framework; however, recent discussions between industry, regulators, and other interested parties have identified ways these challenges may be overcome. In this article we explore how use of the alternate framework could be expanded through efforts such as increasing international harmonization, more proactively publicizing the framework, and enhancing the training of regulatory reviewers. Not only can these strategies help to increase use of the EPA alternate eye irritation framework, they can also be applied to facilitate the uptake of other alternative approaches to animal testing in the future.
Keywords: non-animal testing strategy, eye hazard classification, EPA, antimicrobial cleaning products, pesticides
May 19, 2016
Ilona J. Kosten, Sander W. Spiekstra, Tanja D. de Gruijl and Susan Gibbs
Here we describe a reconstructed full thickness human oral mucosa (gingiva) equivalent with integrated Langerhans Cells (GE-LC) and use it to compare LC activation and migration from oral versus skin epithelium. The physiologically representative models consist of differentiated reconstructed epithelium (keratinocytes and Langerhans-like cells derived from the MUTZ-3 cell line) on a fibroblast-populated collagen hydrogel which serves as a lamina propria for gingiva and dermis for skin. Topical exposure of GE-LC and the skin equivalent (SE-LC) to sub-toxic concentrations of the allergens cinnamaldehyde, resorcinol and nickel sulphate, resulted in LC migration out of the epithelia. Neutralizing antibody to CXCL12 blocked allergen-induced LC migration in SE-LC but not in GE-LC. Also, gingival fibroblasts secreted very low amounts of CXCL12 compared to skin fibroblasts even when stimulated with rhTNFα or rhIL-1α. Surprisingly, cinnamaldehyde exposure of GE-LC resulted in an increase in MUTZ-3 LC and CD83 mRNA in the hydrogel but did not result in an increase in CD1a+ cells in the collagen hydrogel (as was observed for SE-LC. These results indicate that in gingiva, upon allergen exposure, MUTZ-3 LC migrate in a CXCL12 independent manner from epithelium-to-lamina propria and in doing so mature become CD1a- and increase CD83+ mRNA. These physiologically relevant in vitro models which not only are human but which also resemble specific tissues, may aid in the identification of factors regulating immune stimulation which in turn will aid the development of therapeutic interventions for allergy and inflammation, anti-cancer vaccines as well as improving diagnostics for skin and oral allergy.
Keywords: dendritic cell, skin equivalent, gingiva equivalent, in vitro, allergen
May 15, 2016
Florian Groeber, Lisa Engelhardt, Julia Lange, Szymon Kurdyn, Freia F. Schmid, Christoph Rücker, Stephan Mielke, Heike Walles and Jan Hansmann
Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin & eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research.
Keywords: skin equivalents, alternative to animal testing, vascularization, tissue engineering
May 9, 2016
Joaquín Valdés, Laura Trachsel-Moncho, Ayse Sahaboglu, Dragana Trifunović, María Miranda, Marius Ueffing, François Paquet-Durand and Oliver Schmachtenberg
Diabetic retinopathy (DR) is a major cause of vision loss and one of the most common and debilitating complications of diabetes. Research to prevent DR is hindered by a lack of experimental model systems that faithfully reproduce the disease pathology, in particular for type 2 diabetes, which requires prolonged disease progression in animals to develop some hallmarks of DR. Here, we introduce an alternative in vitro model system for DR, based on serum-free, organotypic rodent retinal explant cultures, which allow physiological and pharmacological manipulation of the retina for up to two weeks under tightly controlled conditions. Retinal explant cultures have the advantage of isolating direct neuronal consequences of diabetic conditions from indirect systemic effects mediated via the retinal vasculature or the immune system. Exposed to conditions emulating type 1 or type 2 diabetes, retinal explants displayed elevated cell death rates among inner retinal neurons as well as photoreceptors, with a particularly strong loss of cone photoreceptors. Our results support a direct impact of diabetic conditions on retinal neurons and may help explain color vision defects observed in DR patients. This serum-free in vitro DR model avoids the animal suffering of established DR models and reduces the overall number of animals needed for such research. It should prove useful to study the mechanisms of neuronal cell death caused by DR and to screen for potential future DR treatments.
Keywords: retina, diabetes, animal models, photoreceptors, cell death
May 8, 2016
Cédric Pisani, Sébastien Voisin, Karim Arafah, Philippe Durand, Marie-Hélène Perrard, Marie-Roberte Guichaoua, Philippe Bulet and Odette Prat
To measure the testicular toxicity of two fungicides (carbendazim and iprodione), alone or in a mixture, we used a rat ex vivo model of seminiferous tubules, greatly reducing the number of rodents used, in accordance with the 3R rule (Replacement, Reduction, and Refinement). This model allows the representation of puberty, a critical life period with regard to endocrine disruptors. The cellular modifications were followed for three weeks through transcriptomic and proteomic profiling analysis. A quantitative and comparative method was developed to estimate how known pathways were disturbed by each substance. This pathway-driven analysis revealed a strong alteration of steroidogenesis and an impairment of meiosis in all cases, albeit the initial molecular events were different for both substances. The ex vivo cytogenetic analysis confirmed that both fungicides alter the course of the first meiotic prophase. In addition, the mixture of both substances triggered effects greater than the sum of their cumulative effects and compromised future sperm motility after a shorter time of exposure compared with the fungicides tested separately. The alliance of an ex vivo culture with “omics” strategies complemented with a physiological examination is a powerful combination of tools for testing substances, separately or in a mixture, for their testicular toxicity. In particular, proteomics allowed the identification of systematically differentially expressed proteins in the secretomes of exposed cultures, such as FUCO and PEBP1, two proteins linked with the motility and fertilizing ability of spermatozoa, respectively. These proteins may be potential biomarkers of testicular dysfunction and infertility.
Keywords: pesticides, toxicogenomics, spermatogenesis, endocrine disruption, biomarker
April 27, 2016
Miriam N. Jacobs, Annamaria Colacci, Kimmo Louekari, Mirjam Luijten, Betty C. Hakkert, Martin Paparella and Paule Vasseur
Although regulatory requirements for carcinogenicity testing of chemicals vary according to product sector and regulatory jurisdiction, the standard approach starts with a battery of genotoxicity tests. If any of the in vivo genotoxicity tests are positive, a lifetime rodent cancer bioassay may be requested, which allows the detection of non-genotoxic carcinogens (NGTxC). However, under most chemical regulations the cancer bioassay is rarely requested, specific requests to obtain information on non-genotoxic mechanisms of carcinogenicity are few, and there are no OECD approved screening methods. When the in vitro genotoxicity battery is negative, usually no further carcinogenicity testing is requested. Consequently NGTxC might remain unidentified and therefore the risks they may pose to human health will not be managed. In contrast to genotoxic carcinogens NGTxCact through a large variety of specific mechanisms, and a panel of tests covering multiple biological traits will be needed.
The development of an Integrated Approach to Testing and Assessment (IATA) of NGTxC could assist regulatory decision makers. We examine what NGTxC are and discuss chemical regulatory requirements and limitations. With a strong drive to reduce animal testing and costs in mind, it is essential that proper and robust alternatives for animal testing (3Rs) methods for addressing non-genotoxic modes of action are developed and used. Therefore relevant in vitro mechanisms and assays are described and tentatively organized in levels of information, indicating both a possible structure of the future IATA for NGTxC and associated OECD Test Guideline development priorities.
Keywords: non-genotoxic carcinogens, integrated approaches to testing and assessment of chemicals