Developmental exposure to the flame retardant, triphenyl phosphate, causes long-lasting neurobehavioral and neurochemical dysfunction.

BACKGROUND: Human exposures to organophosphate flame retardants result from their use as additives in numerous consumer products. These agents are replacements for brominated flame retardants but have not yet faced similar scrutiny for developmental neurotoxicity. We examined a representative organophosphate flame retardant, triphenyl phosphate (TPP) and its potential effects on behavioral development and dopaminergic function. METHODS: Female Sprague-Dawley rats were given low doses of TPP (16 or 32 mg kg-1  day-1 ) via subcutaneous osmotic minipumps, begun preconception and continued into the early postnatal period. Offspring were administered a battery of behavioral tests from adolescence into adulthood, and littermates were used to evaluate dopaminergic synaptic function. RESULTS: Offspring with TPP exposures showed increased latency to begin eating in the novelty-suppressed feeding test, impaired object recognition memory, impaired choice accuracy in the visual signal detection test, and sex-selective effects on locomotor activity in adolescence (males) but not adulthood. Male, but not female, offspring showed marked increases in dopamine utilization in the striatum, evidenced by an increase in the ratio of the primary dopamine metabolite (3,4-dihydroxyphenylacetic acid) relative to dopamine levels. CONCLUSIONS: These results indicate that TPP has adverse effects that are similar in some respects to those of organophosphate pesticides, which were restricted because of their developmental neurotoxicity.

Persistent neurobehavioral and neurochemical anomalies in middle-aged rats after maternal diazinon exposure.

Diazinon is an organophosphate pesticide that has a history of wide use. Developmental exposures to organophosphates lead to neurobehavioral changes that emerge early in life and can persist into adulthood. However, preclinical studies have generally evaluated changes through young adulthood, whereas the persistence or progression of deficits into middle age remain poorly understood. The current study evaluated the effects of maternal diazinon exposure on behavior and neurochemistry in middle age, at 1 year postpartum, comparing the results to our previous studies of outcomes at adolescence and in young adulthood (4 months of age) (Hawkey 2020). Female rats received 0, 0.5 or 1.0 mg/kg/day of diazinon via osmotic minipump throughout gestation and into the postpartum period. The offspring were tested on a battery of locomotor, affective, and cognitive tests at young adulthood and during middle age. Some of the neurobehavioral consequences of developmental DZN seen during adolescence and young adulthood faded with continued aging, whereas other neurobehavioral effects emerged with aging. At middle age, the rats showed few locomotor effects, in contrast to the locomotor hyperactivity that had been observed in adolescence. Notably, though, DZN exposure during development impaired reference memory performance in middle-aged males, an effect that had not been seen in the younger animals. Likewise, middle-aged females exposed to DZN showed deficient attentional accuracy, an effect not seen in young adults. Across adulthood, the continued potential for behavioral defects was associated with altered dopaminergic function, characterized by enhanced dopamine utilization that was regionally-selective (striatum but not frontal/parietal cortex). This study shows that the neurobehavioral impairments from maternal low dose exposure to diazinon not only persist, but may continue to evolve as animals enter middle age.

Is the PentaBDE replacement, tris (1,3-dichloro-2-propyl) phosphate (TDCPP), a developmental neurotoxicant? Studies in PC12 cells.

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 (2,3-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.

Paternal Cannabis Exposure Prior to Mating, but Not Δ9-Tetrahydrocannabinol, Elicits Deficits in Dopaminergic Synaptic Activity in the Offspring.

The legalization and increasing availability of cannabis products raises concerns about the impact on offspring of users, and little has appeared on the potential contribution of paternal use. We administered cannabis extract to male rats prior to mating, with two different 28-day exposures, one where there was a 56-day interval between the end of exposure and mating ("Early Cannabis"), and one just prior to mating ("Late Cannabis"); the extract delivered 4 mg/kg/day of the main psychoactive component, Δ9-tetrahydrocannabinol. We then assessed the impact on dopamine (DA) systems in the offspring from the onset of adolescence (postnatal day 30) through middle age (postnatal day 150), measuring the levels of DA and its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) in various brain regions. Paternal cannabis with either regimen elicited a profound and persistent deficit in DA utilization (DOPAC/DA ratio) in the offspring, indicative of subnormal presynaptic activity. However, the two regimens differed in the underlying mechanism, with Early Cannabis reducing DOPAC whereas Late Cannabis increased DA and elicited a smaller reduction in DOPAC. Effects were restricted to male offspring. The effects of cannabis were not reproduced by equivalent exposure to its Δ9-tetrahydrocannabinol, nor did we see the effects with perinatal exposure to tobacco smoke or some of its fetotoxic contributors (benzo[a]pyrene without or with nicotine). Our studies provide some of the first evidence for adverse effects of paternal cannabis administration on neurodevelopment in the offspring, and reinforce the important consequences of paternal drug use in the preconception period.

Neurobehavioral anomalies in zebrafish after sequential exposures to DDT and chlorpyrifos in adulthood: Do multiple exposures interact?

A sequence of different classes of synthetic insecticides have been used over the past 70 years. Over this period, the widely-used organochlorines were eventually replaced by organophosphates, with dichlorodiphenyltrichloroethane (DDT) and chlorpyrifos (CPF) as the principal prototypes. Considerable research has characterized the risks of DDT and CPF individually, but little is known about the toxicology of transitioning from one class of insecticides to another, as has been commonplace for agricultural and pest control workers. This study used adult zebrafish to investigate neurobehavioral toxicity following 5-week chronic exposure to either DDT or CPF, to or their sequential exposure (DDT for 5 weeks followed by CPF for 5 weeks). At the end of the exposure period, a subset of fish were analyzed for brain cholinesterase activity. Behavioral effects were initially assessed one week following the end of the CPF exposure and again at 14 months of age using a behavioral test battery covering sensorimotor responses, anxiety-like functions, predator avoidance and social attraction. Adult insecticide exposures, individually or sequentially, were found to modulate multiple behavioral features, including startle responsivity, social approach, predator avoidance, locomotor activity and novel location recognition and avoidance. Locomotor activity and startle responsivity were each impacted to a greater degree by the sequential exposures than by individual compounds, with the latter being pronounced at the early (1-week post exposure) time point, but not 3-4 months later in aging. Social approach responses were similarly impaired by the sequential exposure as by CPF-alone at the aging time point. Fleeing responses in the predator test showed flee-enhancing effects of both compounds individually versus controls, and no additive impact of the two following sequential exposure. Each compound was also associated with changes in recognition or avoidance patterns in a novel place recognition task in late adulthood, but sequential exposures did not enhance these phenotypes. The potential for chemical x chemical interactions did not appear related to changes in CPF metabolism to the active oxon, as prior DDT exposure did not affect the cholinesterase inhibition resulting from CPF. This study shows that the effects of chronic adult insecticide exposures may be relevant to behavioral health initially and much later in life, and that the effects of sequential exposures may be unpredictable based on their constituent exposures.

Paternal Δ9-Tetrahydrocannabinol Exposure Prior to Mating Elicits Deficits in Cholinergic Synaptic Function in the Offspring.

Little attention has been paid to the potential impact of paternal marijuana use on offspring brain development. We administered Δ9-tetrahydrocannabinol (THC, 0, 2, or 4 mg/kg/day) to male rats for 28 days. Two days after the last THC treatment, the males were mated to drug-naïve females. We then assessed the impact on development of acetylcholine (ACh) systems in the offspring, encompassing the period from the onset of adolescence (postnatal day 30) through middle age (postnatal day 150), and including brain regions encompassing the majority of ACh terminals and cell bodies. Δ9-Tetrahydrocannabinol produced a dose-dependent deficit in hemicholinium-3 binding, an index of presynaptic ACh activity, superimposed on regionally selective increases in choline acetyltransferase activity, a biomarker for numbers of ACh terminals. The combined effects produced a persistent decrement in the hemicholinium-3/choline acetyltransferase ratio, an index of impulse activity per nerve terminal. At the low THC dose, the decreased presynaptic activity was partially compensated by upregulation of nicotinic ACh receptors, whereas at the high dose, receptors were subnormal, an effect that would exacerbate the presynaptic defect. Superimposed on these effects, either dose of THC also accelerated the age-related decline in nicotinic ACh receptors. Our studies provide evidence for adverse effects of paternal THC administration on neurodevelopment in the offspring and further demonstrate that adverse impacts of drug exposure on brain development are not limited to effects mediated by the embryonic or fetal chemical environment, but rather that vulnerability is engendered by exposures occurring prior to conception, involving the father as well as the mother.

Adult exposure to insecticides causes persistent behavioral and neurochemical alterations in zebrafish.

Farmers are often chronically exposed to insecticides, which may present health risks including increased risk of neurobehavioral impairment during adulthood and across aging. Experimental animal studies complement epidemiological studies to help determine the cause-and-effect relationship between chronic adult insecticide exposure and behavioral dysfunction. With the zebrafish model, we examined short and long-term neurobehavioral effects of exposure to either an organochlorine insecticide, dichlorodiphenyltrichloroethane (DDT) or an organophosphate insecticide chlorpyrifos (CPF). Adult fish were exposed continuously for either two or 5 weeks (10-30 nM DDT, 0.3-3 µM CPF), with short- and long-term effects assessed at 1-week post-exposure and at 14 months of age respectively. The behavioral test battery included tests of locomotor activity, tap startle, social behavior, anxiety, predator avoidance and learning. Long-term effects on neurochemical indices of cholinergic function were also assessed. Two weeks of DDT exposure had only slight effects on locomotor activity, while a longer five-week exposure led to hypoactivity and increased anxiety-like diving responses and predator avoidance at 1-week post-exposure. When tested at 14 months of age, these fish showed hypoactivity and increased startle responses. Cholinergic function was not found to be significantly altered by DDT. The two-week CPF exposure led to reductions in anxiety-like diving and increases in shoaling responses at the 1-week time point, but these effects did not persist through 14 months of age. Nevertheless, there were persistent decrements in cholinergic presynaptic activity. A five-week CPF exposure led to long-term effects including locomotor hyperactivity and impaired predator avoidance at 14 months of age, although no effects were apparent at the 1-week time point. These studies documented neurobehavioral effects of adult exposure to chronic doses of either organochlorine or organophosphate pesticides that can be characterized in zebrafish. Zebrafish provide a low-cost model that has a variety of advantages for mechanistic studies and may be used to expand our understanding of neurobehavioral toxicity in adulthood, including the potential for such toxicity to influence behavior and development during aging.

Nicotine exposure in adolescence alters the response of serotonin systems to nicotine administered subsequently in adulthood.

Developmental nicotine exposure produces lasting changes in serotonin (5-HT) function. We gave nicotine to adolescent rats (postnatal days, PD, 30-47), simulating plasma levels in smokers, and then examined the subsequent effects of nicotine given again in young adulthood (PD 90-107), focusing on 5-HT(1A) and 5-HT(2) receptors and the 5-HT transporter during nicotine treatment (PD 105) and withdrawal (PD 110, 120, 130), and long-term changes (PD 180). Adolescent nicotine exposure by itself evoked long-term elevations in cerebrocortical binding parameters in males that emerged in young adulthood. Nicotine given in adulthood produced transient elevations in 5-HT receptor expression in both males and females during withdrawal, and persistent upregulation in the male cerebral cortex. In contrast, females showed decrements in cerebrocortical 5-HT receptors by PD 180. Adolescent nicotine exposure altered the responses to nicotine given in adulthood, sensitizing the initial effects and changing both the withdrawal response and long-term actions. Our results thus provide mechanistic evidence that nicotine exposure, during the period in which nearly all smokers begin to use tobacco, reprograms the future response of 5-HT systems to nicotine.

Developmental neurotoxicity of parathion: progressive effects on serotonergic systems in adolescence and adulthood.

Neonatal exposures to organophosphates that are not acutely symptomatic or that produce little or no cholinesterase inhibition can nevertheless compromise the development and later function of critical neural pathways, including serotonin (5HT) systems that regulate emotional behaviors. We administered parathion to newborn rats on postnatal days (PN) 1-4 at doses spanning the threshold for detectable cholinesterase inhibition (0.1 mg/kg/day) and the first signs of loss of viability (0.2 mg/kg/day). In adolescence (PN30), young adulthood (PN60) and full adulthood (PN100), we measured radioligand binding to 5HT(1A) and 5HT(2) receptors, and to the 5HT transporter in the brain regions comprising all the major 5HT projections and 5HT cell bodies. Parathion caused a biphasic effect over later development with initial, widespread upregulation of 5HT(1A) receptors that peaked in the frontal/parietal cortex by PN60, followed by a diminution of that effect in most regions and emergence of deficits at PN100. There were smaller, but statistically significant changes in 5HT(2) receptors and the 5HT transporter. These findings stand in strong contrast to previous results with neonatal exposure to a different organophosphate, chlorpyrifos, which evoked parallel upregulation of all three 5HT synaptic proteins that persisted from adolescence through full adulthood and that targeted males much more than females. Our results support the view that the various organophosphates have disparate effects on 5HT systems, distinct from their shared property as cholinesterase inhibitors, and the targeting of 5HT function points toward the importance of studying the impact of these agents on 5HT-linked behaviors.

The Developmental Neurotoxicity of Tobacco Smoke Can Be Mimicked by a Combination of Nicotine and Benzo[a]Pyrene: Effects on Cholinergic and Serotonergic Systems.

Tobacco smoke contains polycyclic aromatic hydrocarbons (PAHs) in addition to nicotine. We compared the developmental neurotoxicity of nicotine to that of the PAH archetype, benzo[a]pyrene (BaP), and also evaluated the effects of combined exposure to assess whether PAHs might exacerbate the adverse effects of nicotine. Pregnant rats were treated preconception through the first postnatal week, modeling nicotine concentrations in smokers and a low BaP dose devoid of systemic effects. We conducted evaluations of acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT) systems in brain regions from adolescence through full adulthood. Nicotine or BaP alone impaired indices of ACh presynaptic activity, accompanied by upregulation of nicotinic ACh receptors and 5HT receptors. Combined treatment elicited a greater deficit in ACh presynaptic activity than that seen with either agent alone, and upregulation of nAChRs and 5HT receptors was impaired or absent. The individual effects of nicotine and BaP accounted for only 60% of the combination effects, which thus displayed unique properties. Importantly, the combined nicotine + BaP exposure recapitulated the effects of tobacco smoke, distinct from nicotine. Our results show that the effects of nicotine on development of ACh and 5HT systems are worsened by BaP coexposure, and that combination of the two agents contributes to the greater impact of tobacco smoke on the developing brain. These results have important implications for the relative safety in pregnancy of nicotine-containing products compared with combusted tobacco, both for active maternal smoking and secondhand exposure, and for the effects of such agents in "dirty" environments with high PAH coexposure.

Perinatal diazinon exposure compromises the development of acetylcholine and serotonin systems.

Organophosphate pesticides are developmental neurotoxicants. We gave diazinon via osmotic minipumps implanted into dams prior to conception, with exposure continued into the second postnatal week, at doses (0.5 or 1 mg/kg/day) that did not produce detectable brain cholinesterase inhibition. We evaluated the impact on acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT) systems in brain regions from adolescence through full adulthood. Diazinon produced deficits in presynaptic ACh activity with regional and sex selectivity: cerebrocortical regions and the hippocampus were affected to a greater extent than were the striatum, midbrain or brainstem, and females were more sensitive than males. Diazinon also reduced nicotinic ACh receptors and 5HT1A receptors, with the same regional and sex preferences. These patterns were similar to those of diazinon given in a much more restricted period (postnatal day 1-4) but were of greater magnitude and consistency; this suggests that the brain is vulnerable to diazinon over a wide developmental window. Diazinon's effects differed from those of the related organophosphate, chlorpyrifos, with regard to regional and sex selectivity, and more importantly, to the effects on receptors: chlorpyrifos upregulates nicotinic ACh receptors and 5HT receptors, effects that compensate for the presynaptic ACh deficits. Diazinon can thus be expected to have worse neurodevelopmental outcomes than chlorpyrifos. Further, the disparities between diazinon and chlorpyrifos indicate the problems of predicting the developmental neurotoxicity of organophosphates based on a single compound, and emphasize the inadequacy of cholinesterase inhibition as an index of safety.

Neonatal exposure to low doses of diazinon: long-term effects on neural cell development and acetylcholine systems.

BACKGROUND: The developmental neurotoxicity of organophosphate pesticides involves mechanisms other than their shared property of cholinesterase inhibition. OBJECTIVES: We gave diazinon (DZN) to newborn rats on postnatal days 1-4, using doses (0.5 or 2 mg/kg) spanning the threshold for barely detectable cholinesterase inhibition. METHODS: We then evaluated the lasting effects on indices of neural cell number and size, and on functional markers of acetylcholine (ACh) synapses (choline acetyltransferase, presynaptic high-affinity choline transporter, nicotinic cholinergic receptors) in a variety of brain regions. RESULTS: DZN exposure produced a significant overall increase in cell-packing density in adolescence and adulthood, suggestive of neuronal loss and reactive gliosis; however, some regions (temporal/occipital cortex, striatum) showed evidence of net cell loss, reflecting a greater sensitivity to neurotoxic effects of DZN. Deficits were seen in ACh markers in cerebrocortical areas and the hippocampus, regions enriched in ACh projections. In contrast, there were no significant effects in the midbrain, the major locus for ACh cell bodies. The striatum showed a unique pattern, with robust initial elevations in the ACh markers that regressed in adulthood to normal or subnormal values. CONCLUSIONS: These results indicate that developmental exposures to apparently nontoxic doses of DZN compromise neural cell development and alter ACh synaptic function in adolescence and adulthood. The patterns seen here differ substantially from those seen in earlier work with chlorpyrifos, reinforcing the concept that the various organophosphates have fundamentally different effects on the developmental trajectories of specific neurotransmitter systems, unrelated to their shared action as cholinesterase inhibitors.

Exposure of neonatal rats to parathion elicits sex-selective impairment of acetylcholine systems in brain regions during adolescence and adulthood.

BACKGROUND: Organophosphates elicit developmental neurotoxicity through multiple mechanisms other than their shared property as cholinesterase inhibitors. Accordingly, these agents may differ in their effects on specific brain circuits. OBJECTIVES: We gave parathion to neonatal rats [postnatal days (PNDs) 1-4], at daily doses of 0.1 or 0.2 mg/kg, spanning the threshold for barely detectable cholinesterase inhibition and systemic effects. METHODS: We assessed neurochemical indices related to the function of acetylcholine (ACh) synapses (choline acetyltransferase, presynaptic high-affinity choline transporter, nicotinic cholinergic receptors) in brain regions comprising all the major ACh projections, with determinations carried out from adolescence to adulthood (PNDs 30, 60, and 100). RESULTS: Parathion exposure elicited lasting alterations in ACh markers in the frontal/parietal cortex, temporal/occipital cortex, midbrain, hippocampus, and striatum. In cerebrocortical areas, midbrain, and hippocampus, effects in males were generally greater than in females, whereas in the striatum, females were targeted preferentially. Superimposed on this general pattern, the cerebrocortical effects showed a nonmonotonic dose-response relationship, with regression of the defects at the higher parathion dose; this relationship has been seen also after comparable treatments with chlorpyrifos and diazinon and likely represents the involvement of cholinesterase-related actions that mask or offset the effects of lower doses. CONCLUSIONS: Neonatal exposure to parathion, at doses straddling the threshold for cholinesterase inhibition, compromises indices of ACh synaptic function in adolescence and adulthood. Differences between the effects of parathion compared with chlorpyrifos or diazinon and the non-monotonic dose-effect relationships reinforce the conclusion that various organophosphates diverge in their effects on neurodevelopment, unrelated to their anticholinesterase actions.

Developmental neurotoxicity of perfluorinated chemicals modeled in vitro.

BACKGROUND: The widespread detection of perfluoroalkyl acids and their derivatives in wildlife and humans, and their entry into the immature brain, raise increasing concern about whether these agents might be developmental neurotoxicants. OBJECTIVES: We evaluated perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorooctane sulfonamide (PFOSA), and perfluorobutane sulfonate (PFBS) in undifferentiated and differentiating PC12 cells, a neuronotypic line used to characterize neurotoxicity. METHODS: We assessed inhibition of DNA synthesis, deficits in cell numbers and growth, oxidative stress, reduced cell viability, and shifts in differentiation toward or away from the dopamine (DA) and acetylcholine (ACh) neurotransmitter phenotypes. RESULTS: In general, the rank order of adverse effects was PFOSA > PFOS > PFBS approximately PFOA. However, superimposed on this scheme, the various agents differed in their underlying mechanisms and specific outcomes. Notably, PFOS promoted differentiation into the ACh phenotype at the expense of the DA phenotype, PFBS suppressed differentiation of both phenotypes, PFOSA enhanced differentiation of both, and PFOA had little or no effect on phenotypic specification. CONCLUSIONS: These findings indicate that all perfluorinated chemicals are not the same in their impact on neurodevelopment and that it is unlikely that there is one simple, shared mechanism by which they all produce their effects. Our results reinforce the potential for in vitro models to aid in the rapid and cost-effective screening for comparative effects among different chemicals in the same class and in relation to known developmental neurotoxicants.

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 neurotoxicants target neurodifferentiation into the serotonin phenotype: Chlorpyrifos, diazinon, dieldrin and divalent nickel.

Developmental exposure to organophosphates (OP) produces long-term changes in serotonin (5HT) synaptic function and associated behaviors, but there are disparities among the different OPs. We contrasted effects of chlorpyrifos and diazinon, as well as non-OP neurotoxicants (dieldrin, Ni(2+)) using undifferentiated and differentiating PC12 cells, a well-established neurodevelopmental model. Agents were introduced at 30 microM for 24 or 72 h, treatments devoid of cytotoxicity, and we evaluated the mRNAs encoding the proteins for 5HT biosynthesis, storage and degradation, as well as 5HT receptors. Chlorpyrifos and diazinon both induced tryptophan hydroxylase, the rate-limiting enzyme for 5HT biosynthesis, but chlorpyrifos had a greater effect, and both agents suppressed expression of 5HT transporter genes, effects that would tend to augment extracellular 5HT. However, whereas chlorpyrifos enhanced the expression of most 5HT receptor subtypes, diazinon evoked overall suppression. Dieldrin evoked even stronger induction of tryptophan hydroxylase, and displayed a pattern of receptor effects similar to that of diazinon, even though they come from different pesticide classes. In contrast, Ni(2+) had completely distinct actions, suppressing tryptophan hydroxylase and enhancing the vesicular monoamine transporter, while also reducing 5HT receptor gene expression, effects that would tend to lower net 5HT function. Our findings provide some of the first evidence connecting the direct, initial mechanisms of developmental neurotoxicant action on specific transmitter pathways with their long-term effects on synaptic function and behavior, while also providing support for in vitro test systems as tools for establishing mechanisms and outcomes of related and unrelated neurotoxicants.

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.

Developmental diazinon neurotoxicity in rats: later effects on emotional response.

Developmental exposure to the organophosphorus pesticides chlorpyrifos and diazinon (DZN) alters serotonergic synaptic function at doses below the threshold for cholinesterase inhibition, however there are some indications that the two agents may differ in several important attributes. Previously, we found that low-dose chlorpyrifos exposure in neonatal rats causes lasting changes in emotional response and in the current study we did a comparable evaluation for DZN. Male and female Sprague-Dawley rat pups (N=10-12 of each sex per treatment group) were given 0, 0.5 or 2 mg/(kg day) of DZN s.c. daily on postnatal days (PND) 1-4. These doses bracket the threshold for barely-detectable cholinesterase inhibition. Starting on PND 52, these rats began a battery of tests to assess emotional reactivity. In the elevated plus maze, there was a slight decrease in the time spent in the open arms for DZN-exposed males, while DZN-exposed females were not different from control females. In the novelty-suppressed feeding test, DZN-exposed males had significantly shorter latencies to begin eating than did control males, reducing the values to those normally seen in females. DZN-exposed rats of either sex showed reduced preference for chocolate milk in the anhedonia test that compared the consumption of chocolate milk to water. These findings show that neonatal exposures to DZN at a dose range below the threshold for cholinesterase inhibition nevertheless evokes specific, later alterations in emotional behaviors, particularly in males. The effects show not only some similarities to those of chlorpyrifos but also some differences, in keeping with neurochemical findings comparing the two agents.

Developmental neurotoxic effects of chlorpyrifos on acetylcholine and serotonin pathways in an avian model.

The developmental neurotoxicity of organophosphates such as chlorpyrifos (CPF) involves multiple mechanisms that ultimately compromise the function of specific neurotransmitter systems, notably acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT). Studies in mammalian models incorporate both direct effects on brain development and indirect effects mediated through maternal physiology and maternal/neonatal interactions. We examined the effects of CPF in an avian model, which does not share these potential confounds. Chick eggs were injected with CPF (10 or 20 mg/kg) on incubation days 2 and 6 and markers of ACh and 5HT systems were examined at hatching. The higher dose caused a reduction in cholinesterase activity but there was no consistent downregulation of m(2)-muscarinic ACh receptors as would have been expected from ACh hyperstimulation. Both doses evoked significant reductions in the presynaptic high-affinity choline transporter, the rate-limiting factor in ACh biosynthesis, as monitored by binding of hemicholinium-3. Choline acetyltransferase, a constitutive marker for ACh terminals, was unaffected. This suggests that CPF reduces ACh presynaptic activity rather than compromising the development of ACh projections per se. CPF exposure also reduced the expression of cerebrocortical 5HT(1A) receptors. These effects in the chick model recapitulate many of the actions of early gestational CPF exposure in rats, and thus suggest that CPF exerts direct actions on the immature brain to compromise the development of ACh and 5HT pathways.

Persistent cognitive alterations in rats after early postnatal exposure to low doses of the organophosphate pesticide, diazinon.

BACKGROUND: Developmental neurotoxicity of organophosphorous insecticides (OPs) involves multiple mechanisms in addition to cholinesterase inhibition. We have found persisting effects of developmental chlorpyrifos (CPF) and diazinon (DZN) on cholinergic and serotonergic neurotransmitter systems and gene expression as well as behavioral function. Both molecular/neurochemical and behavioral effects of developmental OP exposure have been seen at doses below those which cause appreciable cholinesterase inhibition. OBJECTIVES: We sought to determine if developmental DZN exposure at doses which do not produce significant acetylcholinesterase inhibition cause persisting cognitive deficits. METHODS: Rats were exposed to DZN on postnatal days 1-4 at doses (0.5 and 2 mg/kg/d) that span the threshold for cholinesterase inhibition. They were later examined with a cognitive battery tests similar to that used with CPF. RESULTS: In the T-maze DZN caused significant hyperactivity in the initial trials of the session, but not later. In a longer assessment of locomotor activity no DZN-induced changes were seen over a 1-hour session. Prepulse inhibition was reduced by DZN exposure selectively in males vs. females; DZN eliminated the sex difference present in controls. In the radial maze, the lower but not higher DZN dose significantly impaired spatial learning. This type of nonmonotonic dose-effect function has previously been seen with CPF as well. The lower dose DZN group also showed significantly greater sensitivity to the memory-impairing effects of scopolamine a muscarinic acetylcholine antagonist. CONCLUSIONS: Neonatal DZN exposure below the threshold for appreciable cholinesterase inhibition caused persisting neurocognitive deficits in adulthood. The addition of some inhibition of AChE with a higher dose reversed the cognitive impairment. This non-monotonic dose-effect function has also been seen with neurochemical effects. Some of the DZN effects on cognition resemble those seen earlier for CPF, some differ. Our data suggest that DZN and CPF affect transmitter systems supporting memory function, differently, implying participation of mechanisms other than their common inhibition of cholinesterase.