TOX-Free Toxicity assessment on neurons and cardiomyocytes by means of FluoRescence Emitting Electrodes

Institutions
  • AG Leist (In vitro Toxikologie und Biomedizin)
Publications
    Cöllen, Eike; Tanaskov, Yaroslav; Holzer, Anna-Katharina; Dipalo, Michele; Schäfer, Jasmin; Kraushaar, Udo; Leist, Marcel (2024): Elements and development processes for test methods in toxicology and human health-relevant life science research Alternatives to Animal Experimentation : ALTEX. Springer. 2024, 41(1), pp. 142-148. ISSN 1868-596X. eISSN 1868-8551. Available under: doi: 10.14573/altex.2401041

Elements and development processes for test methods in toxicology and human health-relevant life science research

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Many laboratory procedures generate data on properties of chemicals, but they cannot be equated with toxicological “test methods”. This apparent discrepancy is not limited to in vitro testing, using animal-free new approach methods (NAM), but also applies to animal-based testing approaches. Here, we give a brief overview of the differences between data generation and the setup or use of a complete test method. While there is excellent literature available on this topic for specialists (GIVIMP guidance; ToxTemp overview), a brief overview and easily-accessible entry point may be useful for a broader community. We provide a single figure to summarize all test method elements and processes required in the development (setup and adaptation) of a test method. The exposure scheme, the endpoint, and the test system are briefly outlined as fundamental elements of any test method. A rationale is provided, why they are not sufficient. We then explain the importance and role of purpose definition (including some information on what is modelled) and the prediction model, aka data interpretation procedure, which depends on the purpose definition, as further essential elements. This connection exemplifies that all fundamental elements are interdependent, and none can be omitted. Finally, discussion is provided on validation as a measure to provide confidence in the reliability, performance, and relevance of a test method. In this sense, validation may be considered a sixth fundamental element for practical use of test methods.

Origin (projects)

    Botham, Philip; Cronin, Mark T. D.; Currie, Richard; Doe, John; Funk-Weyer, Dorothee; Gant, Timothy W.; Leist, Marcel; Marty, Sue; van Ravenzwaay, Bennard; Westmoreland, Carl (2023): Analysis of health concerns not addressed by REACH for low tonnage chemicals and opportunities for new approach methodology Archives of Toxicology. Springer. 2023, 97(12), pp. 3075-3083. ISSN 0340-5761. eISSN 1432-0738. Available under: doi: 10.1007/s00204-023-03601-5

Analysis of health concerns not addressed by REACH for low tonnage chemicals and opportunities for new approach methodology

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In Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) the criterion for deciding the studies that must be performed is the annual tonnage of the chemical manufactured or imported into the EU. The annual tonnage may be considered as a surrogate for levels of human exposure but this does not take into account the physico-chemical properties and use patterns that determine exposure. Chemicals are classified using data from REACH under areas of health concern covering effects on the skin and eye; sensitisation; acute, repeated and prolonged systemic exposure; effects on genetic material; carcinogenicity; and reproduction and development. We analysed the mandated study lists under REACH for each annual tonnage band in terms of the information they provide on each of the areas of health concern. Using the European Chemicals Agency (ECHA) REACH Registration data base of over 20,000 registered substances, we found that only 19% of registered substances have datasets on all areas of health concern. Information limited to acute exposure, sensitisation and genotoxicity was found for 62%. The analysis highlighted the shortfall of information mandated for substances in the lower tonnage bands. Deploying New Approach Methodologies (NAMs) at this lower tonnage band to assess health concerns which are currently not covered by REACH, such as repeat and extended exposure and carcinogenicity, would provide additional information and would be a way for registrants and regulators to gain experience in the use of NAMs. There are currently projects in Europe aiming to develop NAM-based assessment frameworks and they could find their first use in assessing low tonnage chemicals once confidence has been gained by their evaluation with data rich chemicals.

Origin (projects)

    Ückert, Anna-Katharina; Rütschlin, Sina; Gutbier, Simon; Hauer, Isa; Holzer, Anna-Katharina; Meyburg, Birthe; Mix, Ann-Kathrin; Hauck, Christof R.; Böttcher, Thomas; Leist, Marcel (2023): Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration Environment International. Elsevier. 2023, 180, 108229. ISSN 0160-4120. eISSN 1873-6750. Available under: doi: 10.1016/j.envint.2023.108229

Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration

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The causes of nigrostriatal cell death in idiopathic Parkinson’s disease are unknown, but exposure to toxic chemicals may play some role. We followed up here on suggestions that bacterial secondary metabolites might be selectively cytotoxic to dopaminergic neurons. Extracts from Streptomyces venezuelae were found to kill human dopaminergic neurons (LUHMES cells). Utilizing this model system as a bioassay, we identified a bacterial metabolite known as aerugine (C10H11NO2S; 2-[4-(hydroxymethyl)-4,5-dihydro-1,3-thiazol-2-yl]phenol) and confirmed this finding by chemical re-synthesis. This 2-hydroxyphenyl-thiazoline compound was previously shown to be a product of a wide-spread biosynthetic cluster also found in the human microbiome and in several pathogens. Aerugine triggered half-maximal dopaminergic neurotoxicity at 3-4 µM. It was less toxic for other neurons (10-20 µM), and non-toxic (at <100 µM) for common human cell lines. Neurotoxicity was completely prevented by several iron chelators, by distinct anti-oxidants and by a caspase inhibitor. In the Caenorhabditis elegans model organism, general survival was not affected by aerugine concentrations up to 100 µM. When transgenic worms, expressing green fluorescent protein only in their dopamine neurons, were exposed to aerugine, specific neurodegeneration was observed. The toxicant also exerted functional dopaminergic toxicity in nematodes as determined by the “basal slowing response” assay. Thus, our research has unveiled a bacterial metabolite with a remarkably selective toxicity toward human dopaminergic neurons in vitro and for the dopaminergic nervous system of Caenorhabditis elegans in vivo. These findings suggest that microbe-derived environmental chemicals should be further investigated for their role in the pathogenesis of Parkinson's disease.

Origin (projects)

    Suciu, Ilinca; Pamies, David; Peruzzo, Roberta; Wirtz, Petra H.; Pallocca, Giorgia; Hauck, Christof R.; Brunner, Thomas; Hartung, Thomas; Amelio, Ivano; Leist, Marcel (2023): G × E interactions as a basis for toxicological uncertainty Archives of Toxicology. Springer. 2023, 97(7), pp. 2035-2049. ISSN 0340-5761. eISSN 1432-0738. Available under: doi: 10.1007/s00204-023-03500-9

G × E interactions as a basis for toxicological uncertainty

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To transfer toxicological findings from model systems, e.g. animals, to humans, standardized safety factors are applied to account for intra-species and inter-species variabilities. An alternative approach would be to measure and model the actual compound-specific uncertainties. This biological concept assumes that all observed toxicities depend not only on the exposure situation (environment = E), but also on the genetic (G) background of the model (G  ×  E). As a quantitative discipline, toxicology needs to move beyond merely qualitative G  ×  E concepts. Research programs are required that determine the major biological variabilities affecting toxicity and categorize their relative weights and contributions. In a complementary approach, detailed case studies need to explore the role of genetic backgrounds in the adverse effects of defined chemicals. In addition, current understanding of the selection and propagation of adverse outcome pathways (AOP) in different biological environments is very limited. To improve understanding, a particular focus is required on modulatory and counter-regulatory steps. For quantitative approaches to address uncertainties, the concept of “genetic” influence needs a more precise definition. What is usually meant by this term in the context of G  ×  E are the protein functions encoded by the genes. Besides the g ene sequence, the regulation of the gene expression and function should also be accounted for. The widened concept of past and present “ g ene expression” influences is summarized here as G e . Also, the concept of “environment” needs some re-consideration in situations where exposure timing (E t ) is pivotal: prolonged or repeated exposure to the insult (chemical, physical, life style) affects G e . This implies that it changes the model system. The interaction of G e with E t might be denoted as G e  ×  E t . We provide here general explanations and specific examples for this concept and show how it could be applied in the context of New Approach Methodologies (NAM).

Origin (projects)

    Holzer, Anna-Katharina; Dreser, Nadine; Pallocca, Giorgia; Mangerich, Aswin; Stacey, Glyn; Dipalo, Michael; Rovida, Costanza; Wirtz, Petra H.; Hartung, Thomas; Leist, Marcel (2023): Acceptance criteria for new approach methods in toxicology and human health-relevant life science research – part I Alternatives to Animal Experimentation : ALTEX. Springer Spektrum. 2023, 40(4), pp. 706-712. eISSN 1868-596X. Available under: doi: 10.14573/altex.2310021

Acceptance criteria for new approach methods in toxicology and human health-relevant life science research – part I

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Every test procedure, scientific and non-scientific, has inherent uncertainties, even when performed according to a standard operating procedure (SOP). In addition, it is prone to errors, defects, and mistakes introduced by operators, laboratory equipment, or materials used. Adherence to an SOP and comprehensive validation of the test method cannot guarantee that each test run produces data within the acceptable range of variability and with the precision and accuracy determined during the method validation. We illustrate here (part I) why controlling the validity of each test run is an important element of experimental design. The definition and application of acceptance criteria (AC) for the validity of test runs is important for the setup and use of test methods, particularly for the use of new approach methods (NAM) in toxicity testing. AC can be used for decision rules on how to handle data, e.g., to accept the data for further use (AC fulfilled) or to reject the data (AC not fulfilled). The adherence to AC has important requirements and consequences that may seem surprising at first sight: (i) AC depend on a test method’s objectives, e.g., on the types/concentrations of chemicals tested, the regulatory context, the desired throughput; (ii) AC are applied and documented at each test run, while validation of a method (including the definition of AC) is only performed once; (iii) if AC are altered, then the set of data produced by a method can change. AC, if missing, are the blind spot of quality assurance: Test results may not be reliable and comparable. The establishment and uses of AC will be further detailed in part II of this series.

Origin (projects)

    Kranaster, Petra; Blum, Jonathan; Dold, Jeremias E.G.A.; Wittmann, Valentin; Leist, Marcel (2023): Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells Journal of Neurochemistry. Wiley. 2023, 164(4), pp. 481-498. eISSN 1471-4159. Available under: doi: 10.1111/jnc.15737

Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells

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Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non-protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non-toxic inhibitor of protein glycosylation, and of N-butyl-deoxynojirimycin (NBdNM) a well-tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid-Sia) or sialylated N-glycosylated proteins (protein-Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of 'target-specific MGE', we found that in dopaminergic or sensory neurons > 60% of Sia is lipid bound, and thus polysialic acid-neural cell adhesion molecule (PSA-NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell-specific sialylation processes for separate classes of membrane constituents.

Origin (projects)

    Neuhaus, Winfried; Reininger-Gutmann, Birgit; Rinner, Beate; Plasenzotti, Roberto; Wilflingseder, Doris; De Kock, Joery; Hartung, Thomas; Pallocca, Giorgia; Rovida, Costanza; Leist, Marcel (2022): The Current Status and Work of Three Rs Centres and Platforms in Europe Alternatives to Laboratory Animals : ATLA. Sage. 2022, 50(6), pp. 381-413. ISSN 0261-1929. eISSN 2632-3559. Available under: doi: 10.1177/02611929221140909

The Current Status and Work of Three Rs Centres and Platforms in Europe

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The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Origin (projects)

    Holzer, Anna-Katharina; Karreman, Christiaan; Suciu, Ilinca; Furmanowsky, Lara-Seline; Wohlfarth, Harald; Loser, Dominik; Dirks, Wilhelm G.; Pardo González, Emilio; Leist, Marcel (2022): Generation of Human Nociceptor-Enriched Sensory Neurons for the Study of Pain-Related Dysfunctions Stem Cells Translational Medicine. Oxford University Press (OUP). 2022, 11(7), pp. 727-741. ISSN 2157-6564. eISSN 2157-6580. Available under: doi: 10.1093/stcltm/szac031

Generation of Human Nociceptor-Enriched Sensory Neurons for the Study of Pain-Related Dysfunctions

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In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9000 cells were used to establish the "fraction of reactive cells" in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.

Origin (projects)

    Pallocca, Giorgia; Leist, Marcel (2022): On the usefulness of animals as a model system (part II) : Considering benefits within distinct use domains Alternatives to Animal Experimentation : ALTEX. Springer Spektrum. 2022, 39(3), pp. 531-539. ISSN 1868-596X. eISSN 1868-8551. Available under: doi: 10.14573/altex.2207111

On the usefulness of animals as a model system (part II) : Considering benefits within distinct use domains

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In many countries, animal experiments can only be performed when their necessity has been demonstrated in a legal document. As the usefulness of animals in research is also a significant societal and political issue, criteria to structure debates and evaluations are needed. Here, background information is given on laboratory animal studies. Moreover, parameters that may be considered in judging their usefulness are suggested. The discussion is strictly focused on animals used as tools/test systems/models to provide information on humans. In this context, general features and performance characteristics of models are discussed. Examples are given for well-recognized criteria (e.g., robustness, relevance, predictivity) to judge the usefulness of predictive models. The main hypothesis put forward here is that a benefits evaluation (usefulness metrics) is only possible within sharply circumscribed "use domains". Examples are given for the research fields of drug and vaccine research, toxicology, disease pathogenesis, and basic biological research. Efficacy, safety, and quality studies are highlighted as "use domains" within the field of drug discovery and production. A further separation into individual diseases, drug targets or symptoms is suggested for, e.g., efficacy studies or pathophysiology. Finally, an outlook is given on the evaluation of model advantages and disadvantages to arrive at their "net benefit". Moreover, the need to compare the net benefits of animal models versus that of their alternatives is highlighted.

Origin (projects)

Funding sources
Name Finanzierungstyp Kategorie Project no.
ERC Drittmittel Forschungsförderprogramm 575/21
Further information
Period: 01.06.2021 – 31.05.2024