Organophosphate flame retardants (OPFRs) are used as replacements for the commercial PentaBDE mixture that was phased out in 2004. OPFRs are ubiquitous in the environment and detected at high concentrations in residential dust, suggesting widespread human exposure. OPFRs are structurally similar to neurotoxic organophosphate pesticides, raising concerns about exposure and toxicity to humans. This study evaluated the neurotoxicity of tris (1,3-dichloro-2-propyl) phosphate (TDCPP) compared to the organophosphate pesticide, chlorpyrifos (CPF), a known developmental neurotoxicant. We also tested the neurotoxicity of three structurally similar OPFRs, tris (2-chloroethyl) phosphate (TCEP), tris (1-chloropropyl) phosphate (TCPP), and tris (1,3-dibromopropyl) phosphate (TDBPP), and 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), a major component of PentaBDE. Using undifferentiated and differentiating PC12 cells, changes in DNA synthesis, oxidative stress, differentiation into dopaminergic or cholinergic neurophenotypes, cell number, cell growth and neurite growth were assessed. TDCPP displayed concentration-dependent neurotoxicity, often with effects equivalent to or greater than equimolar concentrations of CPF. TDCPP inhibited DNA synthesis, and all OPFRs decreased cell number, and altered neurodifferentiation. Although TDCPP elevated oxidative stress, there was no adverse effect on cell viability or growth. TDCPP and TDBPP promoted differentiation into both neuronal phenotypes, while TCEP and TCPP promoted only the cholinergic phenotype. BDE-47 had no effect on cell number, cell growth or neurite growth. Our results demonstrate that different OPFRs show divergent effects on neurodifferentiation, suggesting the participation of multiple mechanisms of toxicity. Additionally, these data suggest that OPFRs may affect neurodevelopment with similar or greater potency compared to known and suspected neurotoxicants.

Summary 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.

BackgroundIn light of the large number of chemicals that are potential developmental neurotoxicants, there is a need to develop rapid screening techniques.ObjectivesWe 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.ResultsIn 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.ConclusionsThese 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.

BackgroundIn the developing brain, serotonin (5HT) systems are among the most sensitive to disruption by organophosphates.ObjectivesWe 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.ResultsDiazinon 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.ConclusionsOur 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.

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.

BackgroundSilver 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 methodsWe 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.ResultsIn 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.ConclusionsAgNP 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.

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 5HT 1A subtype and upregulation of 5HT 2 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.

BackgroundDevelopmental exposures to organophosphate pesticides are virtually ubiquitous. These agents are neurotoxicants, but recent evidence also points to lasting effects on metabolism.ObjectivesWe 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.MethodsNeonatal 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.ResultsIn 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.ConclusionsNeonatal 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.