Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants

Abstract

Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants.

Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes

“…To our knowledge, only one study exists comparing effects of MeHgCl on cell viability and neurite degeneration in a differentiated human neural cell culture model. In the respective study, MeHgCl inhibited neurite growth of differentiating LUHMES cells, whereas MeHgCl did not affect the mature neurites without inducing cell death [25].…”

“…For proliferation, cells were cultured in Advanced Dulbecco's modified Eagle's medium/F12 (Advanced DMEM/F12, Life Technologies GmbH, Darmstadt, Germany) supplemented with 1 × N2 supplement (Life Technologies), 2 mM l-glutamine (Biochrom, Berlin, Germany) and 40 ng/mL recombinant human basic fibroblast growth factor (FGF, R&D Systems, Wiesbaden-Nordenstadt, Germany) at 37 • C in a humidified 95% air, 5% CO 2 atmosphere. In accordance to the published protocol [25,27], cell differentiation was initiated 24 h after seeding the cells at a density of 45,000 cells per cm 2 by replacing the proliferation medium with differentiation medium consisting of Advanced DMEM/F12 containing 1 × N2 supplement, 2 mM l-glutamine, 1 g/mL tetracycline (Sigma-Aldrich), 1 mM dibutyryl cyclic adenosine monophosphate sodium salt (cAMP, Sigma-Aldrich) and 2 ng/mL recombinant human glial cell-derived neurotrophic factor (GDNF, R&D Systems). After 48 h of differentiation, cells were trypsinized and seeded on pre-coated dishes in a defined density (150,000 cells/cm 2 ) in differentiation medium.…”

Mechanisms involved in the regulation of neuropeptide-mediated neurite outgrowth: a minireview

“…veloped (Das et al 2004;Li and Hoff man-Kim 2008;Krug et al 2013). Quantitative assessment of the neurite outgrowth in these assays includes parameters, such as the number of neurites, neurite orientation, and neurite length.…”

Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants_suppl

“…For instance, assays have been developed that assess changes in early neural differentiation Pennings et al, 2012;Theunissen et al, 2012Theunissen et al, , 2013Krug et al, 2013b;Balmer et al, 2014;Waldmann et al, 2014;Rempel et al, 2015;Shinde et al, 2015), neurite outgrowth (Stiegler et al, 2011;Krug et al, 2013a), synaptogenesis (Harrill et al, 2011), gliogenesis (Fritsche et al, 2005) or myelination (Zurich et al, 2000(Zurich et al, , 2002.…”