Ian T. Ryde

Silver Nanoparticles Compromise Neurodevelopment in PC12 Cells: Critical Contributions of Silver Ion, Particle Size, Coating, and Composition

Background Silver exposures are rising because of the increased use of silver nanoparticles (AgNPs) in consumer products. The monovalent silver ion (Ag+) impairs neurodevelopment in PC12 cells and zebrafish. Objectives and methods We compared the effects of AgNPs with Ag+ in PC12 cells for neurodevelopmental end points including cell replication, oxidative stress, cell viability, and differentiation. First, we compared citrate-coated AgNPs (AgNP-Cs) with Ag+, and then we assessed the roles of particle size, coating, and composition by comparing AgNP-C with two different sizes of polyvinylpyrrolidone-coated AgNPs (AgNP-PVPs) or silica nanoparticles. Results In undifferentiated cells, AgNP-C impaired DNA synthesis, but to a lesser extent than an equivalent nominal concentration of Ag+, whereas AgNP-C and Ag+ were equally effective against protein synthesis; there was little or no oxidative stress or loss of viability due to AgNP-C. In contrast, in differentiating cells, AgNP-C evoked robust oxidative stress and impaired differentiation into the acetylcholine phenotype. Although the effects of AgNP-PVP showed similarities to those of AgNP-C, we also found significant differences in potencies and differentiation outcomes that depended both on particle size and coating. None of the effects reflected simple physical attributes of nanoparticles, separate from composition or coating, as equivalent concentrations of silica nanoparticles had no detectable effects. Conclusions AgNP exposure impairs neurodevelopment in PC12 cells. Further, AgNP effects are distinct from those of Ag+ alone and depend on size and coating, indicating that AgNP effects are not due simply to the release of Ag+ into the surrounding environment.

Permanent, Sex-Selective Effects of Prenatal or Adolescent Nicotine Exposure, Separately or Sequentially, in Rat Brain Regions: Indices of Cholinergic and Serotonergic Synaptic Function, Cell Signaling, and Neural Cell Number and Size at 6 Months of Age

Nicotine is a neuroteratogen that disrupts neurodevelopment and synaptic function, with vulnerability extending into adolescence. We assessed the permanence of effects in rats on indices of neural cell number and size, and on acetylcholine and serotonin (5HT) systems, conducting assessments at 6 months of age, after prenatal nicotine exposure, adolescent exposure, or sequential exposure in both periods. For prenatal nicotine, indices of cell number and size showed few abnormalities by 6 months, but there were persistent deficits in cerebrocortical choline acetyltransferase activity and hemicholinium-3 binding to the presynaptic choline transporter, a pattern consistent with cholinergic hypoactivity; these effects were more prominent in males than females. The expression of 5HT receptors also showed permanent effects in males, with suppression of the 5HT1A subtype and upregulation of 5HT2 receptors. In addition, cell signaling through adenylyl cyclase showed heterologous uncoupling of neurotransmitter responses. Nicotine exposure in adolescence produced lasting effects that were similar to those of prenatal nicotine. However, when animals were exposed to prenatal nicotine and received nicotine subsequently in adolescence, the adverse effects then extended to females, whereas the net effect in males was similar to that of prenatal nicotine by itself. Our results indicate that prenatal or adolescent nicotine exposure evoke permanent changes in synaptic function that transcend the recovery of less-sensitive indices of structural damage; further, prenatal exposure sensitizes females to the subsequent adverse effects of adolescent nicotine, thus creating a population that may be especially vulnerable to the lasting behavioral consequences of nicotine intake in adolescence.

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

Background In light of the large number of chemicals that are potential developmental neurotoxicants, there is a need to develop rapid screening techniques. Objectives We exposed undifferentiated and differentiating neuronotypic PC12 cells to different organophosphates (chlorpyrifos, diazinon, parathion), a carbamate (physostigmine), an organochlorine (dieldrin), and a metal (divalent nickel; Ni2+) and examined indices of cell replication and differentiation for both short- and long-term exposures. Results In undifferentiated cells, all the agents inhibited DNA synthesis, with the greatest effect for diazinon, but physostigmine eventually produced the largest deficits in the total number of cells after prolonged exposure. The onset of differentiation intensified the adverse effects on DNA synthesis and changed the rank order in keeping with a shift away from noncholinergic mechanisms and toward cholinergic mechanisms. Differentiation also worsened the effects of each agent on cell number after prolonged exposure, whereas cell growth was not suppressed, nor were there any effects on viability as assessed with trypan blue. Nevertheless, differentiating cells displayed signs of oxidative stress from all of the test compounds except Ni2+, as evidenced by measurements of lipid peroxidation. Finally, all of the toxicants shifted the transmitter fate of the cells away from the cholinergic phenotype and toward the catecholaminergic phenotype. Conclusions These studies point out the feasibility of developing cell-based screening methods that enable the detection of multiple end points that may relate to mechanisms associated with developmental neurotoxicity, revealing some common targets for disparate agents.

Organophosphate insecticides target the serotonergic system in developing rat brain regions: disparate effects of diazinon and parathion at doses spanning the threshold for cholinesterase inhibition.

Background In the developing brain, serotonin (5HT) systems are among the most sensitive to disruption by organophosphates. Objectives We exposed neonatal rats to daily doses of diazinon or parathion on postnatal days (PND)1–4 and evaluated 5HT receptors and the 5HT transporter in brainstem and forebrain on PND5, focusing on doses of each agent below the maximum tolerated dose and spanning the threshold for cholinesterase inhibition: 0.5, 1, or 2 mg/kg for diazinon, and 0.02, 0.05, and 0.1 mg/kg for parathion. Results Diazinon evoked up-regulation of 5HT1A and 5HT2 receptor expression even at doses devoid of effects on cholinesterase activity, a pattern similar to that seen earlier for another organophosphate, chlorpyrifos. In contrast, parathion decreased 5HT1A receptors, again at doses below those required for effects on cholinesterase. The two agents also differed in their effects on the 5HT transporter. Diazinon evoked a decrease in the brainstem and an increase in the forebrain, again similar to that seen for chlorpyrifos; this pattern is typical of damage of nerve terminals and reactive sprouting. Parathion had smaller, nonsignificant effects. Conclusions Our results buttress the idea that, in the developing brain, the various organophosphates target specific neurotransmitter systems differently from each other and without the requirement for cholinesterase inhibition, their supposed common mechanism of action.

PCR Based Determination of Mitochondrial DNA Copy Number in Multiple Species

Mitochondrial DNA (mtDNA) copy number is a critical component of overall mitochondrial health. In this chapter, we describe methods for isolation of both mtDNA and nuclear DNA (nucDNA) and measurement of their respective copy numbers using quantitative PCR. Methods differ depending on the species and cell type of the starting material and availability of specific PCR reagents.

Mitochondrial dynamics and autophagy aid in removal of persistent mitochondrial DNA damage in Caenorhabditis elegans.

Mitochondria lack the ability to repair certain helix-distorting lesions that are induced at high levels in mitochondrial DNA (mtDNA) by important environmental genotoxins and endogenous metabolites. These lesions are irreparable and persistent in the short term, but their long-term fate is unknown. We report that removal of such mtDNA damage is detectable by 48 h in Caenorhabditis elegans, and requires mitochondrial fusion, fission and autophagy, providing genetic evidence for a novel mtDNA damage removal pathway. Furthermore, mutations in genes involved in these processes as well as pharmacological inhibition of autophagy exacerbated mtDNA damage-mediated larval arrest, illustrating the in vivo relevance of removal of persistent mtDNA damage. Mutations in genes in these pathways exist in the human population, demonstrating the potential for important gene–environment interactions affecting mitochondrial health after genotoxin exposure.

Exposure of Neonatal Rats to Parathion Elicits Sex-Selective Reprogramming of Metabolism and Alters the Response to a High-Fat Diet in Adulthood

Background Developmental exposures to organophosphate pesticides are virtually ubiquitous. These agents are neurotoxicants, but recent evidence also points to lasting effects on metabolism. Objectives We administered parathion to neonatal rats. In adulthood, we assessed the impact on weight gain, food consumption, and glucose and lipid homeostasis, as well as the interaction with the effects of a high-fat diet. Methods Neonatal rats were given parathion on postnatal days 1–4 using doses (0.1 or 0.2 mg/kg/day) that straddle the threshold for barely detectable cholinesterase inhibition and the first signs of systemic toxicity. In adulthood, animals were either maintained on standard lab chow or switched to a high-fat diet for 7 weeks. Results In male rats on a normal diet, the low-dose parathion exposure caused increased weight gain but also evoked signs of a prediabetic state, with elevated fasting serum glucose and impaired fat metabolism. The higher dose of parathion reversed the weight gain and caused further metabolic defects. Females showed greater sensitivity to metabolic disruption, with weight loss at either parathion dose, and greater imbalances in glucose and lipid metabolism. At 0.1 mg/kg/day parathion, females showed enhanced weight gain on the high-fat diet; This effect was reversed in the 0.2-mg/kg/day parathion group, and was accompanied by even greater deficits in glucose and fat metabolism. Conclusions Neonatal low-dose parathion exposure disrupts glucose and fat homeostasis in a persistent and sex-selective manner. Early-life toxicant exposure to organophosphates or other environmental chemicals may play a role in the increased incidence of obesity and diabetes.

Developmental neurotoxicity of low dose diazinon exposure of neonatal rats : Effects on serotonin systems in adolescence and adulthood

The developmental neurotoxicity of organophosphate pesticides targets serotonin (5HT) systems, which are involved in emotional and appetitive behaviors. We exposed neonatal rats to daily doses of diazinon on postnatal days 1-4, using doses (0.5 or 2mg/kg) spanning the threshold for barely-detectable cholinesterase inhibition. We then evaluated the effects on 5HT(1A) and 5HT(2) receptors, and on the 5HT transporter in cerebral cortical regions and the brainstem in adolescence through adulthood. Diazinon evoked a lasting deficit in 5HT(1A) receptors in males only, whereas it caused a small but significant increase in 5HT transporters in females; neither effect showed a significant regional selectivity. This pattern differed substantially from that seen in earlier work with another organophosphate, chlorpyrifos, which at pharmacodynamically similar doses spanning the threshold for cholinesterase inhibition, evoked a much more substantial, global upregulation of 5HT receptor expression; with chlorpyrifos, effects on receptors were seen in females, albeit to a lesser extent than in males, and were also regionally distinct. The effects of diazinon were nonmonotonic, showing larger alterations at the lower dose, likely reflecting positive trophic effects of cholinergic stimulation once the threshold for cholinesterase inhibition is exceeded. Our results reinforce the idea that different organophosphates have fundamentally distinct effects on the developmental trajectories of specific neurotransmitter systems, unrelated to their shared action as cholinesterase inhibitors. The effects on 5HT circuits expand the scope of behavioral endpoints that need to be considered in evaluating the developmental neurotoxicity of organophosphates.

Silver Impairs Neurodevelopment: Studies in PC12 Cells

Background Exposure to silver is increasing because of silver nanoparticles in consumer products. Objectives and methods Many biological effects of silver entail actions of Ag+ (monovalent silver ions), so we used neuronotypic PC12 cells to evaluate the potential for silver to act as a developmental neurotoxicant, using chlorpyrifos (CPF), a pesticide known to evoke developmental neurotoxicity, as a positive control for comparison. Results In undifferentiated cells, a 1-hr exposure to 10 μM Ag+ inhibited DNA synthesis more potently than did 50 μM CPF; it also impaired protein synthesis but to a lesser extent than its effect on DNA synthesis, indicating a preferential effect on cell replication. Longer exposures led to oxidative stress, loss of viability, and reduced numbers of cells. With the onset of cell differentiation, exposure to 10 μM Ag+ evoked even greater inhibition of DNA synthesis and more oxidative stress, selectively impaired neurite formation without suppressing overall cell growth, and preferentially suppressed development into the acetylcholine phenotype in favor of the dopamine phenotype. Lowering the exposure to 1 μM Ag+ reduced the net effect on undifferentiated cells. However, in differentiating cells, the lower concentration produced an entirely different pattern, enhancing cell numbers by suppressing ongoing cell death and impairing differentiation in parallel for both neurotransmitter phenotypes. Conclusions Our results show that silver has the potential to evoke developmental neurotoxicity even more potently than known neurotoxicants, such as CPF, and that the spectrum of effects is likely to be substantially different at lower exposures that do not show signs of outright toxicity.

Ameliorating the developmental neurotoxicity of chlorpyrifos: a mechanisms-based approach in PC12 cells.

Background Organophosphate developmental neurotoxicity involves multiple mechanisms converging on neural cell replication and differentiation. Objectives We evaluated mechanisms contributing to the adverse effects of chlorpyrifos (CPF) on DNA synthesis, cell number and size, and cell signaling mediated by adenylyl cyclase (AC) in PC12 cells, a neuronotypic cell line that recapitulates the essential features of developing mammalian neurons. Results In undifferentiated cells, cholinergic receptor antagonists had little or no protective effect against the antimitotic actions of CPF; however, when nerve growth factor was used to evoke differentiation, the antagonists showed partial protection against deficits in cell loss and alteration in cell size elicited by CPF, but were ineffective in preventing the deterioration of AC signaling. Nicotine, which stimulates nicotinic acetylcholine receptors but also possesses a mixture of prooxidant/antioxidant activity, had adverse effects by itself but also protected undifferentiated cells from the actions of CPF and had mixed additive/protective effects on cell number in differentiating cells. The antioxidant vitamin E also protected both undifferentiated and differentiating cells from many of the adverse effects of CPF but worsened the impact on AC signaling. Theophylline, which prevents the breakdown of cyclic AMP, was the only agent that restored AC signaling to normal or supranormal levels but did so at further cost to cell replication. Conclusions Our results show definitive contributions of cholinergic hyperstimulation, oxidative stress, and interference with AC signaling in the developmental neurotoxicity of CPF and point to the potential use of this information to design treatments to ameliorate these adverse effects.