Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay

Colaianna, Marilena; Ilmjärv, Sten; Peterson, Hedi; Kern, Ilse; Julien, Stéphanie; Baquié, Mathurin; Pallocca, Giorgia; Bosgra, Sieto; Sachinidis, Agapios; Hengstler, Jan G.; Leist, Marcel; Krause, Karl-Heinz

doi:10.1007/s00204-016-1690-2

Cited by 16 publications

(10 citation statements)

References 127 publications

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“…Currently, large efforts are undertaken to establish in vitro tests with the long-term goal to predict human toxicity (Daneshian et al 2016;Frey et al 2014;Ghallab 2015;Kampe et al 2014;Kim et al 2015;Leist et al 2017). For this purpose, in vitro systems with human (Deharde et al 2016;Godoy et al 2013Godoy et al , 2015Godoy et al , 2016Grinberg et al 2014;Luckert et al 2017;Pfeiffer et al 2015) as well as rodent (Arbo et al 2016;Hengstler et al 2000;Hewitt et al 2007;Reif et al 2015) hepatocytes, neuronal cells (Colaianna et al 2017;Krug et al 2013;Rempel et al 2015;Shinde et al 2017;Waldmann et al 2014), kidney cell lines (Gong et al 2016;Liu et al 2016) and cardiomyocytes (Chaudhari et al 2016;Gaspar et al 2014) have been optimized. One challenge of these in vitro approaches is the choice of adequate exposure periods with test compounds.…”

Section: Discussionmentioning

confidence: 99%

Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes

Albrecht

Edlund

et al. 2018

Arch Toxicol

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Primary human hepatocytes (PHHs) remain the gold standard for in vitro testing in the field of pharmacology and toxicology. One crucial parameter influencing the results of in vitro tests is the incubation period with test compounds. It has been suggested that longer incubation periods may be critical for the prediction of repeated dose toxicity. However, a study that systematically analyzes the relationship between incubation period and cytotoxicity in PHHs is not available. To close this gap, 30 compounds were tested in a concentration-dependent manner for cytotoxicity in cultivated cryopreserved PHHs (three donors per compound) for 1, 2 and 7 days. The median of the EC 50 values of all compounds decreased 1.78-fold on day 2 compared to day 1, and 1.89-fold on day 7 compared to day 1. Median values of EC 50 ratios of all compounds at day 2 and day 7 were close to one but for individual compounds the ratio increased up to almost six. Strong correlations were obtained for EC 50 on day 1 and day 7 (R = 0.985; 95% CI 0.960-0.994), day 1 and day 2 (R = 0.964; 95% CI 0.910-0.986), as well as day 2 and day 7 (R = 0.981; 95% CI 0.955-0.992). However, compound specific differences also occurred. Whereas, for example, busulfan showed a relatively strong increase on day 7 compared to day 1, cytotoxicity of acetaminophen did not increase during longer incubation periods. To validate the observed correlations, a publicly available data set, containing data on the cytotoxicity of human hepatocytes cultivated as spheroids for incubation periods of 5 and 14 days, was analyzed. A high correlation coefficient of EC 50 values at day 5 and day 14 was obtained (R = 0.894; 95% CI 0.798-0.945). In conclusion, the median cytotoxicity of the test compounds increased between 1 and 2 days of incubation, with no or only a minimal further increase until day 7. It remains to be studied whether the different results obtained for some individual compounds after longer exposure periods would correspond better to human-repeated dose toxicity. Keywords Hepatotoxicity • Primary human hepatocyte • Incubation period • Cell-titer-blue Abbreviations APAP Acetaminophen ASP Aspirin BOS Bosentan BPR Buspirone Xiaolong Gu and Wiebke Albrecht shared first authorship.

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Section: Discussionmentioning

confidence: 99%

Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes

Albrecht

Edlund

et al. 2018

Arch Toxicol

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“…The potential of using murine embryonic stem cells (mESCs) for in vitro developmental toxicity testing was recognized long time ago (Heuer et al 1993), and the murine ESC-based embryonic stem cell test (EST) became a validated platform for embryotoxicology testing (Genschow et al 2004;Hayess et al 2013). Robust protocols have been developed for neural differentiation of mESC and are in use for DNT (Kuegler et al 2010;Zimmer et al 2011a, b;Theunissen et al 2012;Visan et al 2012;Smirnova et al 2014a, c;Colaianna et al 2016).…”

Section: Non-human Primary Neural Cells Neural Cell Lines and Embrymentioning

confidence: 99%

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities

et al. 2016

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Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

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“…Large libraries of several thousand compounds have been established in the Tox21 program (Huang et al 2016;Richard et al 2016). Examples for smaller libraries are the 150 compounds of the TG-GATEs project for hepatotoxicity (Grinberg et al 2014;Igarashi et al 2015), the 30 compounds of the ESNATS battery (Colaianna et al 2016;Zimmer et al 2014) or the set of about 75 compounds assembled by the National Toxicology Program (NTP) of the USA (Pei et al 2015;Ryan et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Combination of multiple neural crest migration assays to identify environmental toxicants from a proof-of-concept chemical library

et al. 2017

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Many in vitro tests have been developed to screen for potential neurotoxicity. However, only few cell function-based tests have been used for comparative screening, and thus experience is scarce on how to confirm and evaluate screening hits. We addressed these questions for the neural crest cell migration test (cMINC). After an initial screen, a hit follow-up strategy was devised. A library of 75 compounds plus internal controls (NTP80-list), assembled by the National Toxicology Program of the USA (NTP) was used. It contained some known classes of (developmental) neurotoxic compounds. The primary screen yielded 23 confirmed hits, which comprised ten flame retardants, seven pesticides and six drug-like compounds. Comparison of concentration-response curves for migration and viability showed that all hits were specific. The extent to which migration was inhibited was 25-90%, and two organochlorine pesticides (DDT, heptachlor) were most efficient. In the second part of this study, (1) the cMINC assay was repeated under conditions that prevent proliferation; (2) a transwell migration assay was used as a different type of migration assay; (3) cells were traced to assess cell speed. Some toxicants had largely varying effects between assays, but each hit was confirmed in at least one additional test. This comparative study allows an estimate on how confidently the primary hits from a cell function-based screen can be considered as toxicants disturbing a key neurodevelopmental process. Testing of the NTP80-list in more assays will be highly interesting to assemble a test battery and to build prediction models for developmental toxicity.

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Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay

Cited by 16 publications

References 127 publications

Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes

Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities

Combination of multiple neural crest migration assays to identify environmental toxicants from a proof-of-concept chemical library

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