Towards in vitro DT/DNT testing: Assaying chemical susceptibility in early differentiating NT2 cells.
Human pluripotent embryonal carcinoma (NT2) cells are increasingly considered as a suitable model for in vitro toxicity testing, e.g. developmental toxicity and neurotoxicity (DT/DNT) studies, as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and permit toxicity testing at different stages of maturation. NT2 cells have recently been reported to show specific changes in dielectric resistance profiles during differentiation which can be observed as early as 24h upon RA-stimulation. These observations suggest altered susceptibility to chemicals at an early stage of differentiation. However, chemical susceptibility of early differentiating NT cells has not yet been studied. To address this question, we have established a cell fitness screening assay based on the analysis of intracellular ATP levels and we applied the assay in a large-scale drug screening experiment in NT2 stem cells and early differentiating NT2 cells. Subsequent analysis of ranked fitness phenotypes revealed 19 chemicals with differential toxicity profile in early differentiating NT2 cells. To evaluate whether any of the identified drugs have previously been associated with DT/DNT, we conducted a literature search on the identified molecules and quantified the fraction of chemicals assigned to the FDA (Food and Drug Administration) pregnancy risk categories (PRC) N, A, B, C, D, and X in the hit list and the small molecule library. While the fractions of the categories N and B were decreased (0.81 and 0.35-fold), the classes C, D and X were increased (1.35, 1.47 and 3.27-fold) in the hit list compared to the chemical library. From these data as well as from the literature review, identifying large fractions of chemicals being directly (∼42%) and indirectly associated with DT/DNT (∼32%), we conclude that our method may be beneficial to systematic in vitro-based primary screening for developmental toxicants and neurotoxicants and we propose cell fitness screening in early differentiating NT2 cells as a strategy for evaluating chemical susceptibility at different stages of differentiation to reduce animal testing in the context of the 3Rs.
Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; Department of Internal Medicine 5, University Medical Centre Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Ann-Katrin
AK
Menzner
Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; Erlangen Graduate School in advanced Optical Technologies (SAOT), Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen, Germany.
Sepideh
S
Abolpour Mofrad
Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; Erlangen Graduate School in advanced Optical Technologies (SAOT), Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen, Germany.
Oliver
O
Friedrich
Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; Erlangen Graduate School in advanced Optical Technologies (SAOT), Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen, Germany. Electronic address: daniel.gilbert@fau.de.
Daniel F
DF
Gilbert
D000255:Adenosine Triphosphate / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D019295:Computational Biology; D016208:Databases, Factual; D054278:Embryonal Carcinoma Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D057166:High-Throughput Screening Assays; D006801:Humans; D058953:Neural Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D055495:Neurogenesis / Q000187:drug effects*; D020258:Neurotoxicity Syndromes / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018570:Risk Assessment; D054852:Small Molecule Libraries; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods*; D014212:Tretinoin / Q000494:pharmacology*
D015415:Biomarkers; D054852:Small Molecule Libraries; D014212:Tretinoin; D000255:Adenosine Triphosphate
Cell viability screening; Chemical susceptibility; Developmental toxicity and neurotoxicity testing; Differentiation; Human pluripotent embryonal carcinoma cells