Using primary organotypic mouse midbrain cultures to examine developmental neurotoxicity of silver nanoparticles across two genetic strains.

Micromass culture systems have been developed as three-dimensional organotypic in vitro alternatives to test developmental toxicity. We have optimized a murine-based embryonic midbrain micromass system in two genetic strains to evaluate neurodevelopmental effects of gold-cored silver nanoparticles (AgNPs) of differing sizes and coatings-20 nm AgCitrate, 110 nm AgCitrate, and 110 nm AgPVP. AgNPs are increasingly used in consumer, commercial, and medical products for their antimicrobial properties and observations of Ag in adult and fetal brain following in vivo exposures to AgNPs have led to concerns about the potential for AgNPs to elicit adverse effects on neurodevelopment and neurological function. Cytotoxicity was assessed at three time points of development by both nominal dose and by dosimetric dose. Ag dosimetry was assessed in cultures and the gold core component of the AgNPs was used as a tracer for determination of uptake of intact AgNPs and silver dissolution from particles in the culture system. Results by both nominal and dosimetric dose show cell death increased significantly in a dose-dependent manner at later time points (days 15 and 22 in vitro) that coincide with differentiation stages of development in both strains. When assessed by dosimetric dose, cultures were more sensitive to smaller particles, despite less uptake of Ag in smaller particles in both strains.

A dynamic in vitro developing testis model reflects structures and functions of testicular development in vivo.

To better define appropriate applications of our 3-dimensional testicular co-culture as a model for reproductive toxicology, we evaluated the ability of the model to capture structural and functional elements that can be targeted by reproductive toxicants. Testicular co-cultures were prepared from postnatal day 5 male rats and cultured with a Matrigel overlay. Following a 2-day acclimation period, we characterized functional pathway dynamics by evaluating morphology, protein expression, testosterone concentrations, and global gene expression at a range of timepoints from experimental days 0-21. Western blotting confirmed expression of Sertoli cell, Leydig cell, and spermatogonial cell-specific protein markers. Testosterone detected in cell culture media indicates active testosterone production. Quantitative pathway analysis identified Gene Ontology biological processes enriched among genes significantly changing over the course of 21 days. Processes enriched among genes significantly increasing through time include general developmental processes (morphogenesis, tissue remodeling, etc.), steroid regulation, Sertoli cell development, immune response, and stress and apoptosis. Processes enriched among genes significantly decreasing over time include several related to male reproductive development (seminiferous tubule development, male gonad development, Leydig cell differentiation, Sertoli cell differentiation), all of which appear to peak in expression between days 1 and 5 before decreasing at later timepoints. This analysis provides a temporal roadmap for specific biological process of interest for reproductive toxicology in the model and anchors the model to sensitive phases of in vivo development, helping to define the relevance of the model for in vivo processes.

Anchoring a dynamic in vitro model of human neuronal differentiation to key processes of early brain development in vivo.

We characterize temporal pathway dynamics of differentiation in an in vitro neurotoxicity model with the aim of informing design and interpretation of toxicological assays. Human neural progenitor cells (hNPCs) were cultured in differentiation conditions up to 21 days. Genes significantly changed through time were identified and grouped according to temporal dynamics. Quantitative pathway analysis identified gene ontology (GO) terms enriched among significantly changed genes and provided a temporal roadmap of pathway trends in vitro. Gene expression in hNPCs was compared with publicly available gene expression data from developing human brain tissue in vivo. Quantitative pathway analysis of significantly changed genes and targeted analysis of specific pathways of interest identified concordance between in vivo and in vitro expression associated with proliferation, migration, differentiation, synapse formation, and neurotransmission. Our analysis anchors gene expression patterns in vitro to sensitive windows of in vivo development, helping to define appropriate applications of the model.

Characterization of 3D embryonic C57BL/6 and A/J mouse midbrain micromass in vitro culture systems for developmental neurotoxicity testing.

In vitro micromass culture systems have been proposed as an alternative method for developmental toxicity assessment to reduce the need for resource-intensive in vivo toxicity testing. In this study, a three-dimensional in vitro embryonic mouse midbrain culture system is characterized in two mouse strains to facilitate gene x environment considerations. Gestational day (GD) 11 C57BL/6 or GD 12 A/J mouse midbrain cells were isolated and cultured in high-density micromass format for 22days in vitro (DIV). Hematoxylin intensity and protein content revealed that neuronal differentiation increases linearly over time in both C57BL/6 and A/J cultures. Protein expression showed time-dependent proliferation markers (PCNA) increased significantly between DIV 4-6 compared to DIV 1. Early and late differentiation markers (e.g. ß-tubulin III and NMDAɛ1) were expressed between DIV 6-8 and DIV 8-15, respectively. Immunohistochemistry and protein expression results for proliferation and differentiation markers were concordant. Protein expression patterns for the two mouse strain micromass systems were similar. This study characterizes a novel method for investigating early neurogenesis and may be used to characterize neurodevelopmental toxicity in vitro. Our findings show how the use of different mouse strains in neurodevelopmental studies may extend test systems for gene and environment interaction studies.

Differential epigenetic effects of chlorpyrifos and arsenic in proliferating and differentiating human neural progenitor cells.

Understanding the underlying temporal and mechanistic responses to neurotoxicant exposures during sensitive periods of neuronal development are critical for assessing the impact of these exposures on developmental processes. To investigate the importance of timing of neurotoxicant exposure for perturbation of epigenetic regulation, we exposed human neuronal progenitor cells (hNPCs) to chlorpyrifos (CP) and sodium arsenite (As; positive control) during proliferation and differentiation. CP or As treatment effects on hNPCs morphology, cell viability, and changes in protein expression levels of neural differentiation and cell stress markers, and histone H3 modifications were examined. Cell viability, proliferation/differentiation status, and epigenetic results suggest that hNPCs cultures respond to CP and As treatment with different degrees of sensitivity. Histone modifications, as measured by changes in histone H3 phosphorylation, acetylation and methylation, varied for each toxicant and growth condition, suggesting that differentiation status can influence the epigenetic effects of CP and As exposures.