Adverse Neurodevelopmental Effects of Dexamethasone Modeled in PC12 Cells: Identifying the Critical Stages and Concentration Thresholds for the Targeting of Cell Acquisition, Differentiation and Viability

Abstract

The use of dexamethasone (DEX) to prevent respiratory distress in preterm infants is suspected to produce neurobehavioral deficits. We used PC12 cells to model the effects of DEX on different stages of neuronal development, utilizing exposures from 24 h up to 11 days and concentrations from 0.01 to 10 mM, simulating subtherapeutic, therapeutic, and high-dose regimens. In undifferentiated cells, even at the lowest concentration, DEX inhibited DNA synthesis and produced a progressive deficit in the number of cells as evaluated by DNA content, whereas cell growth (evaluated by the total protein to DNA ratio) and cell viability (Trypan blue exclusion) were promoted. When cell differentiation was initiated with nerve growth factor, the simultaneous inclusion of DEX still produced a progressive deficit in cell numbers and promoted cell growth and viability while retarding the development of neuritic projections as monitored by the membrane/total protein ratio. Again, even 0.01 mM DEX was effective. We next assessed effects at mid-differentiation by introducing nerve growth factor for 4 days followed by coexposure to DEX. Although effects on cell number, growth, and neurite extension were still detectable, the outcomes were generally less notable. DEX also shifted the fate of PC12 cells away from the cholinergic phenotype and toward the adrenergic phenotype, with the maximum effect achieved at the outset of differentiation. Our results indicate that DEX directly disrupts neuronal cell replication, differentiation, and phenotype at concentrations below those required for the therapy of preterm infants, providing a mechanistic link between glucocorticoid use and neurodevelopmental sequelae.

Dexamethasone-Induced Down-Regulation of Nerve Growth Factor Receptor p75NTR is Mediated by Glucocorticoid Type II Receptor in PC12 Cell Model

“…Dex completely blocked the NGF-induced effect on cell proliferation while partially affected the neurite extension but not the percentage of cells responding to NGF; thus reinforcing recent similar neurotoxic effects in Dex-treated PC12 cells [41], or hyppocampal neurons [42,43]. Since Dex treatment induced additional cellular effects such as downregulation of MAP-2, crucial for NGF-induced neurite outgrowth [23], it also appear plausible that Dex inhibits the expression of proteins necessary for TrkA-mediated neurite outgrowth which would uncouple the TrkA activity from neurite outgrowth.…”

Ameliorating the Developmental Neurotoxicity of Chlorpyrifos: A Mechanisms-Based Approach in PC12 Cells

“…Importantly, each of the mechanisms made a partial contribution to the net outcome for either neural cell replication or differentiation. Cholinergic antagonists, although totally ineffective in preventing the antimitotic effects of CPF in undifferentiated cells (Song et al 1998), were successful once differentiation was under way, in partially reversing the deficits in cell number and the rise in protein/DNA ratio; this is entirely consistent with the transition to a cholinergic phenotype in PC12 cells (Jameson et al 2006a, 2006b; Teng and Greene 1994) and with the increasing role of cholinergic mechanisms in the adverse effects of CPF in vivo as brain development progresses (Slotkin 1999, 2004, 2005; Whitney et al 1995). Nicotine was partially effective in protecting cell replication in undifferentiated cells but likely through antioxidant actions (Qiao et al 2005), so that a similar effect was found with vitamin E. With increasing differentiation, the effects of nicotine acting as a cholinergic agonist offset some of the positive consequences of its antioxidant actions, so that the remaining beneficial effect depended on whether the CPF concentration was low or high.…”

“…In the present study, we examined the effects of CPF with and without each of the ameliorating treatments on both undifferentiated and differentiating PC12 cells with regard to DNA synthesis, indices of cell number and size, and AC signaling. Because each neural cell contains a single nucleus, we measured DNA content to evaluate the number of cells (Winick and Noble 1965) and the protein/DNA ratio as an index of cell size (Abreu-Villaça et al 2005; Jameson et al 2006a; Slotkin et al 2007; Song et al 1998). For AC measurements, we evaluated basal enzymatic activity, the response to global stimulation of G-proteins by fluoride, and maximal enzymatic activity elicited by forskolin, which acts directly on AC, bypassing the need for activation of neurotransmitter receptors or G-proteins (Seamon and Daly 1986).…”

Screening for Developmental Neurotoxicity Using PC12 Cells: Comparisons of Organophosphates with a Carbamate, an Organochlorine, and Divalent Nickel

“…Each neural cell contains only a single nucleus (Winick and Noble 1965), so that the DNA content (micrograms of DNA per culture dish in the present study) reflects the total number of cells (Song et al 1998). Indices of growth were provided by measurements of protein subfractions related to cell size and membrane surface area (Jameson et al 2006a; Thai et al 1996). The total protein/DNA ratio rises with cell enlargement, and the membrane/total protein ratio falls as a consequence of the decreased surface-to-volume ratio.…”

“…On the other hand, with the onset of differentiation, the development of neuritic projections necessitates a rise in the relative contribution of membrane proteins, so that the increase in the membrane/total protein ratio gives an indication of augmented membrane “complexity.” The effects on cell number, size, and cell surface area were compared to those on viability (evaluated by trypan blue exclusion) and lipid peroxidation (evaluated by thiobarbituric acid-reactive species; TBARS). To characterize the catecholaminergic and cholinergic phenotypes, we assessed the ratio of activities of tyrosine hydroxylase (TH) to choline acetyltransferase (ChAT), the respective biosynthetic enzymes for dopamine and acetylcholine (Teng and Greene 1994; Jameson et al 2006a, 2006b). …”