Browsing by Author "Slotkin, Theodore A"
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Item Open Access Adenylyl Cyclase Cell Signaling as a Target and Underlying Mechanism for Persistent Effects of Early-Life Organophosphate Exposure(2010) Adigun, Abayomi AlexanderOrganophosphates (OPs) are developmental neurotoxicants but also produce lasting effects on metabolism. This dissertation examines the cellular mechanisms underlying metabolic dysfunction after early-life OP exposure. We administered diazinon (DZN) or parathion (PRT) to rats on postnatal days (PN) 1-4 at doses straddling the threshold for cholinesterase inhibition and assessed the longitudinal effects on hepatic and cardiac cell function mediated through the adenylyl cyclase (AC) signaling cascade, which controls neuronal and hormonal inputs that regulate hepatic glucose metabolism and cardiac contractility. Specifically, we investigated if outcomes of metabolic dysfunction are related to hepatic AC dysregulation. In the liver, DZN elicited parallel upregulation of AC activity itself and of the responses to AC stimulants acting at beta-adrenergic receptors (BARs), glucagon receptors, or G-proteins. The effects intensified from adolescence to adulthood. In contrast, PRT elicited upregulation in adolescence that waned by adulthood. Effects on the liver were more substantial than those in the heart and a brain region (cerebellum) that shares similar AC cascade responses. These findings indicate that OPs produce lasting hepatic AC gain-of-function and alter the trajectory of hepatic cell signaling in a manner consistent with the observed emergence of prediabetes-like metabolic dysfunction. Since the effects are unrelated to cholinesterase inhibition, the various OPs differ in their net impact on AC signaling.
We then examined whether OPs directly affect the expression or function of AC signaling elements, using PC12 cells to evaluate effects on transcription of AC pathway genes and on protein function. Whereas different OPs had disparate effects on gene transcription, they had nearly identical effects at the protein level, suggesting that programming occurs post-transcriptionally. We further found that otherwise unrelated developmental toxicants (OPs, dieldrin, nickel) can nevertheless converge on similar outcomes for their impact on the AC pathway, providing a common pathway by which diverse agents can lead to metabolic dysfunction.
The standard view of OPs as developmental toxicants that exclusively target the nervous system requires substantial revision. Through their effects on hepatic cell signaling and other metabolic processes, early-life chemical exposures may play an important role in the worldwide increase in obesity and diabetes.
Item Open Access Adult exposure to insecticides causes persistent behavioral and neurochemical alterations in zebrafish.(Neurotoxicology and teratology, 2020-03) Hawkey, Andrew B; Glazer, Lilah; Dean, Cassandra; Wells, Corinne N; Odamah, Kathryn-Ann; Slotkin, Theodore A; Seidler, Frederic J; Levin, Edward DFarmers 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.Item Open Access Cognitive and Behavioral Impairments Evoked by Low-Level Exposure to Tobacco Smoke Components: Comparison with Nicotine Alone.(Toxicological sciences : an official journal of the Society of Toxicology, 2016-06) Hall, Brandon J; Cauley, Marty; Burke, Dennis A; Kiany, Abtin; Slotkin, Theodore A; Levin, Edward DActive maternal smoking has adverse effects on neurobehavioral development of the offspring, with nicotine (Nic) providing much of the underlying causative mechanism. To determine whether the lower exposures caused by second-hand smoke are deleterious, we administered tobacco smoke extract (TSE) to pregnant rats starting preconception and continued through the second postnatal week, corresponding to all 3 trimesters of fetal brain development. Dosing was adjusted to produce maternal plasma Nic concentrations encountered with second-hand smoke, an order of magnitude below those seen in active smokers. We then compared TSE effects to those of an equivalent dose of Nic alone, and to a 10-fold higher Nic dose. Gestational exposure to TSE and Nic significantly disrupted cognitive and behavioral function in behavioral tests given during adolescence and adulthood (postnatal weeks 4-40), producing hyperactivity, working memory deficits, and impairments in emotional processing, even at the low exposure levels corresponding to second-hand smoke. Although TSE effects were highly correlated with those of Nic, the effects of TSE were much larger than could be attributed to just the Nic in the mixture. Indeed, TSE effects more closely resembled those of the 10-fold higher Nic levels, but still exceeded their magnitude. In combination with our earlier findings, this study thus completes the chain of causation to prove that second-hand smoke exposure causes neurodevelopmental deficits, originating in disruption of neurodifferentiation, leading to miswiring of neuronal circuits, and as shown here, culminating in behavioral dysfunction. As low level exposure to Nic alone produced neurobehavioral teratology, 'harm reduction' Nic products do not abolish the potential for neurodevelopmental damage.Item Open Access Corrigendum to "The Developmental Neurotoxicity of Tobacco Smoke Can Be Mimicked by a Combination of Nicotine and Benzo[a]pyrene: Effects on Cholinergic and Serotonergic Systems".(Toxicological sciences : an official journal of the Society of Toxicology, 2019-03) Slotkin, Theodore A; Skavicus, Samantha; Ko, Ashley; Levin, Edward D; Seidler, Frederic JThe correct units in Tables S3, S5, and S6 are "fmol/mg protein," whereas no units should be specified in Table S4, which presents data as a ratio.Item Open Access Critical developmental periods for effects of low-level tobacco smoke exposure on behavioral performance.(Neurotoxicology, 2018-09) Cauley, Marty; Hall, Brandon J; Abreu-Villaça, Yael; Junaid, Shaqif; White, Hannah; Kiany, Abtin; Slotkin, Theodore A; Levin, Edward DTobacco exposure during development leads to neurobehavioral dysfunction in children, even when exposure is limited to secondhand smoke. We have previously shown in rats that developmental exposure to tobacco smoke extract (TSE), at levels mimicking secondhand smoke, starting preconception and extending throughout gestation, evoked subsequent locomotor hyperactivity and cognitive impairment. These effects were greater than those caused by equivalent exposures to nicotine alone, implying that other agents in tobacco smoke contributed to the adverse behavioral effects. In the present study, we examined the critical developmental windows of vulnerability for these effects, restricting TSE administration (0.2 mg/kg/day nicotine equivalent, or DMSO vehicle, delivered by subcutaneously-implanted pumps) to three distinct 10 day periods: the 10 days preceding mating, the first 10 days of gestation (early gestation), or the second 10 days of gestation (late gestation). The principal behavioral effects revealed a critical developmental window of vulnerability, as well as sex selectivity. Late gestational TSE exposure significantly increased errors in the initial training on the radial-arm maze in female offspring, whereas no effects were seen in males exposed during late gestation, or with either sex in the other exposure windows. In attentional testing with the visual signal detection test, male offspring exposed to TSE during early or late gestation showed hypervigilance during low-motivating conditions. These results demonstrate that gestational TSE exposure causes persistent behavioral effects that are dependent on the developmental window in which exposure occurs. The fact that effects were seen at TSE levels modeling secondhand smoke, emphasizes the need for decreasing involuntary tobacco smoke exposure, particularly during pregnancy.Item Open Access Developmental exposure to the flame retardant, triphenyl phosphate, causes long-lasting neurobehavioral and neurochemical dysfunction.(Birth defects research, 2023-02) Hawkey, Andrew B; Evans, Janequia; Holloway, Zade R; Pippen, Erica; Jarrett, Olivia; Kenou, Bruny; Slotkin, Theodore A; Seidler, Frederic J; Levin, Edward DBackground
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.Item Open Access Developmental Neurotoxicity of Silver and Silver Nanoparticles Modeled In Vitro and In Vivo(2010) Powers, Christina MarieBackground: Silver nanoparticles (AgNPs) act as antimicrobials by releasing monovalent silver (Ag+) and are increasingly used in consumer products, thus elevating exposures in human and environmental populations. Materials and Methods: We evaluated Ag+ in a standard model of neuronal cell replication and differentiation, and then determined whether there were similar effects of the ion in vivo using zebrafish. Next, we compared Ag+ and AgNP exposures in the same two models and incorporated the effects of particle coating, size and composition. Conclusions: This work is the first to show that both Ag+ and AgNPs are developmental neurotoxicants in vitro and in vivo. Moreover, although both the soluble ion and the particles impair measures of neurodevelopment, the outcomes and underlying mechanisms of each toxicant are often wholly distinct. Superimposed on the dichotomies between Ag+ and AgNP exposures are clear effects of particle coating, size and composition that will necessitate evaluation of individual AgNP types when considering potential environmental and human health effects. The results presented here provide hazard identification that can help isolate the models and endpoints necessary for developing a risk assessment framework for the growing use of AgNPs.
Item Open Access Evaluation of the association between maternal smoking, childhood obesity, and metabolic disorders: a national toxicology program workshop review.(Environmental health perspectives, 2013-02) Behl, Mamta; Rao, Deepa; Aagaard, Kjersti; Davidson, Terry L; Levin, Edward D; Slotkin, Theodore A; Srinivasan, Supriya; Wallinga, David; White, Morris F; Walker, Vickie R; Thayer, Kristina A; Holloway, Alison CBackground
An emerging literature suggests that environmental chemicals may play a role in the development of childhood obesity and metabolic disorders, especially when exposure occurs early in life.Objective
Here we assess the association between these health outcomes and exposure to maternal smoking during pregnancy as part of a broader effort to develop a research agenda to better understand the role of environmental chemicals as potential risk factors for obesity and metabolic disorders.Methods
PubMed was searched up to 8 March 2012 for epidemiological and experimental animal studies related to maternal smoking or nicotine exposure during pregnancy and childhood obesity or metabolic disorders at any age. A total of 101 studies-83 in humans and 18 in animals-were identified as the primary literature.Discussion
Current epidemiological data support a positive association between maternal smoking and increased risk of obesity or overweight in offspring. The data strongly suggest a causal relation, although the possibility that the association is attributable to unmeasured residual confounding cannot be completely ruled out. This conclusion is supported by findings from laboratory animals exposed to nicotine during development. The existing literature on human exposures does not support an association between maternal smoking during pregnancy and type 1 diabetes in offspring. Too few human studies have assessed outcomes related to type 2 diabetes or metabolic syndrome to reach conclusions based on patterns of findings. There may be a number of mechanistic pathways important for the development of aberrant metabolic outcomes following perinatal exposure to cigarette smoke, which remain largely unexplored.Conclusions
From a toxicological perspective, the linkages between maternal smoking during pregnancy and childhood overweight/obesity provide proof-of-concept of how early-life exposure to an environmental toxicant can be a risk factor for childhood obesity.Item Open Access Is There a Critical Period for the Developmental Neurotoxicity of Low-Level Tobacco Smoke Exposure?(Toxicological sciences : an official journal of the Society of Toxicology, 2017-01) Slotkin, Theodore A; Stadler, Ashley; Skavicus, Samantha; Card, Jennifer; Ruff, Jonathan; Levin, Edward D; Seidler, Frederic JSecondhand tobacco smoke exposure in pregnancy increases the risk of neurodevelopmental disorders. We evaluated in rats whether there is a critical period during which tobacco smoke extract (TSE) affects the development of acetylcholine and serotonin systems, prominent targets for adverse effects of nicotine and tobacco smoke. We simulated secondhand smoke exposure by administering TSE so as to produce nicotine concentrations one-tenth those in active smoking, with 3 distinct, 10-day windows: premating, early gestation or late gestation. We conducted longitudinal evaluations in multiple brain regions, starting in early adolescence (postnatal day 30) and continued to full adulthood (day 150). TSE exposure in any of the 3 windows impaired presynaptic cholinergic activity, exacerbated by a decrement in nicotinic cholinergic receptor concentrations. Although the adverse effects were seen for all 3 treatment windows, there was a distinct progression, with lowest sensitivity for premating exposure and higher sensitivity for gestational exposures. Serotonin receptors were also reduced by TSE exposure with the same profile: little effect with premating exposure, intermediate effect with early gestational exposure and large effect with late gestational exposure. As serotonergic circuits can offset the neurobehavioral impact of cholinergic deficits, these receptor changes were maladaptive. Thus, there is no single 'critical period' for effects of low-level tobacco smoke but there is differential sensitivity dependent upon the developmental stage at the time of exposure. Our findings reinforce the need to avoid secondhand smoke exposure not only during pregnancy, but also in the period prior to conception, or generally for women of childbearing age.Item Open Access Neurobehavioral anomalies in zebrafish after sequential exposures to DDT and chlorpyrifos in adulthood: Do multiple exposures interact?(Neurotoxicology and teratology, 2021-09) Hawkey, Andrew B; Holloway, Zade; Dean, Cassandra; Koburov, Reese; Slotkin, Theodore A; Seidler, Frederic J; Levin, Edward DA 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.Item Open Access Paternal Cannabis Exposure Prior to Mating, but Not Δ9-Tetrahydrocannabinol, Elicits Deficits in Dopaminergic Synaptic Activity in the Offspring.(Toxicological sciences : an official journal of the Society of Toxicology, 2021-09-30) Slotkin, Theodore A; Levin, Edward D; Seidler, Frederic JThe 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.Item Open Access Paternal Δ9-Tetrahydrocannabinol Exposure Prior to Mating Elicits Deficits in Cholinergic Synaptic Function in the Offspring.(Toxicological sciences : an official journal of the Society of Toxicology, 2020-04) Slotkin, Theodore A; Skavicus, Samantha; Levin, Edward D; Seidler, Frederic JLittle 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.Item Open Access Perinatal diazinon exposure compromises the development of acetylcholine and serotonin systems.(Toxicology, 2019-08) Slotkin, Theodore A; Skavicus, Samantha; Ko, Ashley; Levin, Edward D; Seidler, Frederic JOrganophosphate 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.Item Open Access Persistent neurobehavioral and neurochemical anomalies in middle-aged rats after maternal diazinon exposure.(Toxicology, 2022-04) Hawkey, Andrew B; Pippen, Erica; Kenou, Bruny; Holloway, Zade; Slotkin, Theodore A; Seidler, Frederic J; Levin, Edward DDiazinon 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.Item Open Access The Developmental Neurotoxicity of Tobacco Smoke Can Be Mimicked by a Combination of Nicotine and Benzo[a]Pyrene: Effects on Cholinergic and Serotonergic Systems.(Toxicological sciences : an official journal of the Society of Toxicology, 2019-01) Slotkin, Theodore A; Skavicus, Samantha; Ko, Ashley; Levin, Edward D; Seidler, Frederic JTobacco 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.