title: High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure.
abstract: Our objective was to test neural active compounds in a human developmental neurotoxicity (DNT) model that represents neural tube stages of vulnerability. Previously we showed that 14 days in vitro (DIV 14) was sufficient to generate cryopreserved neuronal cells for post thaw neurite recovery assays. However, short exposure and assessment may not detect toxicants that affect an early neurogenesis continuum, from a mitotic human neural progenitor (hNP) cell population through the course of neurite outgrowth in differentiating neurons. Therefore, we continuously exposed differentiating hNP cells from DIV 0 through DIV 14 to known toxicants and endocrine active compounds in order to assess at DIV 14 effects of these compounds in a human DNT maturation model for neurogenesis.
  
  
  
  The Human DNT continuum (DIV 0 to DIV 14) was determined using immunocytochemistry for SOX1+ (proliferating hNP) and HuC/D+ (post mitotic neurons). The cumulative effects of five compounds was observed on neurite outgrowth in (βIII-tubulin+) and (HuC/D+) cells using high content imaging. All data were analyzed using a one-way ANOVA with a significance threshold of p < 0.05.
  
  
  
  During maturation in vitro, the neural cultures transitioned from uniform hNP cells (DIV 0) to predominantly mature post mitotic neuronal neurons (HuC/D+, 65%; DIV14) but also maintained a smaller population of hNP cells (SOX1+). Using this DNT maturation model system, Bis-1, testosterone, and β-estradiol inhibited neuronal maturation at micromolar levels but were unaffected by acetaminophen. β-estradiol also disrupted neurite extension at 10 μM. Treating cells in this window with Bisphenol A (BPA) significantly inhibited neurite outgrowth and branching in these continuum cultures but only at the highest concentrations tested (10 μM).
  
  
  
  Cumulative effects of neurotoxicant exposure during a maturation continuum altered human neurogenesis at lower exposure levels than observed in acute exposure of static cryopreserved neurite recovery neurons cultures. Unlike prior acute studies, β-estradiol was highly toxic when present throughout the continuum and cytotoxicity was manifested starting early in the continuum via a non-estrogen receptor α (ER α) mechanism. Therefore, the effect of neural developmental neurotoxins can and should be determined during the dynamic process of human neural maturation.
authors.affiliation: Interdisciplinary Toxicology Program, University of Georgia, Athens, GA, 30602, USA.|Regenerative Bioscience Center, University of Georgia, Athens, GA, 30602, USA.
authors.forename: Xian
authors.initials: X
authors.lastname: Wu
authors.affiliation: ArunA Biomedical, Athens, GA, 30602, USA.
authors.forename: Anirban
authors.initials: A
authors.lastname: Majumder
authors.affiliation: Regenerative Bioscience Center, University of Georgia, Athens, GA, 30602, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Webb
authors.affiliation: Interdisciplinary Toxicology Program, University of Georgia, Athens, GA, 30602, USA. sstice@uga.edu.|Regenerative Bioscience Center, University of Georgia, Athens, GA, 30602, USA. sstice@uga.edu.|ArunA Biomedical, Athens, GA, 30602, USA. sstice@uga.edu.
authors.forename: Steven L
authors.initials: SL
authors.lastname: Stice
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007211:Indoles / Q000633:toxicity; D008301:Maleimides / Q000633:toxicity; D057054:Molecular Imaging / Q000379:methods* / Q000592:standards; D058953:Neural Stem Cells / Q000187:drug effects / Q000502:physiology; D016501:Neurites / Q000187:drug effects* / Q000502:physiology; D055495:Neurogenesis / Q000187:drug effects* / Q000502:physiology
chemical_list: D007211:Indoles; D008301:Maleimides; C070515:bisindolylmaleimide I
keywords: Developmental neurotoxicity; Endocrine active compounds; Human neural progenitor; Neurite outgrowth; Neuron maturation