Cell Cycle Inhibition by Sodium Arsenite in Primary Embryonic Rat Midbrain Neuroepithelial Cells

Abstract

Arsenite (As3+) exposure during development has been associated with neural tube defects and other structural malformations, and with behavioral alterations including altered locomotor activity and operant learning. The molecular mechanisms underlying these effects are uncertain. Because arsenic can cross the placenta and accumulate in the developing neuroepithelium, we examined cell cycling effects of sodium arsenite (As3+ 0, 0.5, 1, 2, and 4 microM) on embryonic primary rat midbrain (gestational day [GD] 12) neuroepithelial cells over 48 h. There was a concentration- and time-dependent As3+-induced reduction in cell viability assessed by neutral red dye uptake assay but minimal apoptosis at concentrations below 4 microM. Morphologically, apoptosis was not apparent until 4 microM at 24 h, which was demonstrated by a marginal but statistically significant increase in cleaved caspase-3/7 activity. Cell cycling effects over several rounds of replication were determined by continuous 5-bromo-2'-deoxyuridine (BrdU) labeling and bivariate flow cytometric Hoechst-Propidium Iodide analysis. We observed a time- and concentration-dependent inhibition of cell cycle progression as early as 12 h after exposure (> or =0.5 microM). In addition, data demonstrated a concentration-dependent increase in cytostasis within all cell cycle phases, a decreased proportion of cells able to reach the second cell cycle, and a reduced cell cycle entry from gap 1 phase (G1). The proportion of affected cells and the severity of the cell cycle perturbation, which ranged from a decreased transition probability to complete cytostasis in all cell cycle phases, were also found to be concentration-dependent. Together, these data support a role for perturbed cell cycle progression in As3+ mediated neurodevelopmental toxicity.

Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation through E2F1-Mediated Increase of Cyclins

“…This decrease in cyclin protein levels indicates a reduction in cellular proliferation. Previous studies have shown in different model organisms that inorganic arsenic treatment inhibits cell cycle progression and induces apoptosis (Sidhu et al, 2006;Wang et al, 2007). Our results were in agreement with the prior observations.…”

High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells

“…Cell cycle status and progression has traditionally been measured using populationbased methods such as flow cytometry (Darzynkiewicz et al, 2006;Sidhu et al, 2006). Flow cytometry is generally dependent on the successful preparation of a single-cell suspension, which is not compatible with high-throughput and high-resolution cell imaging.…”

Adult Hippocampal Neurogenesis and mRNA Expression are Altered by Perinatal Arsenic Exposure in Mice and Restored by Brief Exposure to Enrichment

“…In vitro studies investigating the effects of developmental arsenic exposure in the parts per billion range (up to 4 µM) have shown altered cell cycling including reduced viability, minimal apoptosis, and an increase in caspase 3/7 resulting in inhibition of cell cycle progression in primary embryonic rat midbrain neuroepithelial cells [11]. Other in vitro work with P19 mouse embryonic stem cells (ESC) has demonstrated that low concentrations of arsenic in the ppb range (0.1 up to 1.0 µM sodium arsenite) suppress the differentiation, but not proliferation, of ESC into neurons indicated by reduced Tuj1, neurogenin 1, neurogenin 2, and NeuroD expression in arsenic-treated cells [41].…”

“…Other research has revealed considerable deficits in learning and memory after several methods of arsenic exposure in both rodent models and in humans [2]–[9]. Additionally, developmental arsenic exposure alters cerebellar morphology in the brain and disrupts cell cycle dynamics of neuroepithelial cells in vitro [10], [11]. Recently, we have demonstrated that developmental exposure to arsenic alters components of the hypothalamus-pituitary-adrenal (HPA) axis [12] including increased hypothalamic corticotrophin releasing hormone (CRH), altered corticosterione (CORT) secretion (both at baseline and in response to a stressor), decreased hippocampal 11β-HSD 1, and altered subcellular glucocorticoid receptor (GR) distribution in the hypothalamus.…”