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

Abstract

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.

Comparison of migration disturbance potency of epigallocatechin gallate (EGCG) synthetic analogs and EGCG PEGylated PLGA nanoparticles in rat neurospheres

“…Several studies have shown that EGCG can bind to laminin (Peter et al, 2017;Suzuki and Isemura, 2001) and disturb the adhesion of cells to the extracellular matrix (Bracke et al, 1987;Chen et al, 2003;Suzuki and Isemura, 2001) finally leading to an altered cell migration pattern (Bal-Price et al, 2017;Barenys et al, 2017a;Nyffeler et al, 2017). The structural moieties inducing this altered cell migration seemed to be galloyl/pyrogallol groups because in a previous study, catechins lacking these residues did not disrupt cell adhesion (Barenys et al, 2017a).…”

“…In a previous study using human and rat neurospheres we have shown that EGCG inhibits adhesion and migration of neural progenitor cells (NPCs) and that at higher concentrations it also produces a reduction of the number of migrating young neurons in vitro, without affecting NPC proliferation . An independent research group has recently reproduced these migration alteration effects in a different test model (cMINC) showing that EGCG also inhibits human neural crest cells migration in vitro (Nyffeler et al, 2017). Besides being a freely-available food-supplement, EGCG is also intensively studied at a preclinical level as a potential prenatal treatment to decrease developmental adverse effects induced by toxic substances, i.e.…”

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods

“…UKN2 (cMINC): Pre-differentiated neural crest cells (NCC) (Zimmer et al 2012 ) were seeded to coated multi-well plates with inserted silicon stoppers to create a cell-free area as described earlier (Nyffeler et al 2017a , b ). Cell migration was initiated one day after seeding by removal of the stopper, and test compound was added.…”

Strategies to improve the regulatory assessment of developmental neurotoxicity (DNT) using in vitro methods

“…, ( Mundy et al, 2010 ; Radio et al, 2015 )), migration ( e.g. , ( Nyffeler et al, 2017 )), neurite outgrowth ( e.g. , ( Harrill et al, 2013 , 2015 )), synaptogenesis and neuronal network formation and function ( e.g.…”