exposure. Specifically, we tested the hypothesis that AhrdCyp1a2(+/+) micewith a poor-affinity AHR and a functioning CYP1A2 enzyme would be mostresistant while Ahrb1Cyp1a2(−/−) mice with a high-affinity AHR and noCYP1A2 enzyme would be most susceptible to PCB-induced developmentalneurotoxicity. Pregnant damswere treated at gestational day 10 and postnatalday 5 (PND5) with the PCB mixture or corn oil vehicle to ensure exposureduring early brain development. Offspring went through a battery ofneurobehavioral tests beginning at PND60. PCB-treated mice with a high-affinity AHR (Ahrb1) had an attenuated startle response compared with PCB-treated Ahrdmice and controls (p<0.01). PCB-treated Ahrb1mice also showedincreased locomotor activity in an open field (p<0.05) and a significantincrease in zone crossings in the Zero Maze (p<0.01). Q-PCR and WesternBlots showed that CYP1A1 mRNA and protein was increased only in Ahrb1

mice, demonstrating AHR-mediated gene expression. Therefore, we concludethat changes in the startle response and increased activity levels aremediated,at least in part, by persistent AHR activation during brain development.

doi:10.1016/j.ntt.2009.04.020

NBTS17

AhR-mediated gene expression in the developing mouse telencephalon

J.M. Gohlkea, P. Stocktonb, S. Sieberc, J. Foleyb, C.J. PortieraaEnvironmental Systems Biology Group, Laboratory of Molecular Toxicology,National Institute of Environmental Health Sciences, Research Triangle Park,NC, USAbLaboratory of Environmental Pathology, National Institute of EnvironmentalHealth Sciences, Research Triangle Park, NC, USAcMicroarray Group, Laboratory of Molecular Toxicology, National Institute ofEnvironmental Health Sciences, Research Triangle Park, NC, USA

We hypothesize that TCDD induced developmental neurotoxicity ismodulated through anAhR dependent interactionwith key regulatory neuronaldifferentiation pathways during telencephalon development. To test thishypothesis we examined global gene expression in both dorsal and ventraltelencephalon tissues in E13.5 AhR−/− and wildtype mice exposed to TCDD orvehicle. Consistent with previous biochemical, pathological and behavioralstudies, our results suggest TCDD initiated changes in gene expression in thedeveloping telencephalon are primarily AhR dependent, as no statisticallysignificant gene expression changes are evident after TCDD exposure in AhR−/−

mice. Based on a gene regulatory network for neuronal specification in thedeveloping telencephalon, the present analysis suggests differentiation ofGABAergic neurons in the ventral forebrain is compromised in TCDD exposedand AhR−/− mice. In addition, our analysis suggests Sox11 may be directlyregulatedbyAhRbased on gene expression and comparative genomics analyses.In conclusion, this analysis supports thehypothesis that AhRhas a specific role inthe normal developmentof the forebrain andprovides amechanistic frameworkfor neurodevelopmental toxicity of chemicals that perturb AhR signaling.

doi:10.1016/j.ntt.2009.04.021

NBTS18

Exposure to pyrethroid insecticides alters expression of genes importantfor dopaminergic neuronal development

Amy Demiccoa, Jason Richardsonb, Keith Coopera, Lori WhiteaaRutgers University, New Brunswick, NJ, USAbUniversity of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA

Recent studies indicate that the developing embryo may be susceptible toexposure to pyrethroids, commonly used residential and agricultural insecti-cides. Data from the Richardson laboratory show that developmental exposureof mice to deltamethrin (a type II pyrethroid) results in increased dopaminetransporter (DAT) expression, in addition to increased expression of thetranscription factors involved in dopaminergic system development (pitx3and nurr1). These mice also displayed increased locomotor activity. We havechosen to use the zebrafish to explore the hypothesis that developmental

pyrethroid exposure alters dopaminergic neuronal development. To investigatethe toxicity of pyrethroids in this model system, zebrafish embryos (3 hpf) wereexposed to pyrethroids and observed individually every 24 h until 144 hpf. Highdoses of pyrethroids caused mortality (LC50 of 55 μg/L for deltamethrin),pericardial edema, body axis curvature, and spasms. Lower doses causedhyperactivity. Ourdata alsodemonstrate that lowdosesofdeltamethrin (1µg/L)causes increased gene expression of DAT (3.4 fold), pitx3 (3.6 fold), nurr1 (3.3fold), and TH (a marker of dopaminergic neurons) (3.2 fold). These changespersisted up to 2 weeks post exposure. To determine whether epigeneticmodification could explain this persistence, expression of themethyltransferasegene dnmt1was examined and our data show that dnmt1mRNAwas increased(2 fold higher) in 72 hpf embryos treatedwith deltamethrin. These data validatethe zebrafish as amodel for studying pyrethroid neurotoxicity, and demonstratethat developmental deltamethrin exposure alters the expression of dopaminer-gic genes in ways that may persist through an epigenetic mechanism.

doi:10.1016/j.ntt.2009.04.022

NBTS19

Profiling developmental toxicity of 387 environmental chemicals usingEPA's Toxicity Reference Database (ToxRefDB)

T.B. Knudsena, M.T. Martina, R.J. Kavlocka, R.S. Judsona, D.J. Dixa, A.V. Singhb

aNCCT, U.S. Environmental Protection Agency, Research Triangle Park, NC, USAbLockheed Martin, Research Triangle Park, NC, USA

EPA's Toxicity Reference Databases (ToxRefDB) was developed by theNational Center for Computational Toxicology inpartnershipwith EPA's Office ofPesticide Programs, to store data derived from in vivo animal toxicity studies[www.epa.gov/ncct/toxrefdb/]. The initial build of ToxRefDB includes sourcedata from guideline prenatal studies on 387 chemicals, mostly pesticides, in ratand rabbit. ToxRefDB data were entered by target-description fields and lowesteffect levels (mLEL,maternal parameters and dLEL, developmental parameters).Of 988 standardized endpoints, 293 (29.7%) were recorded across 751 studies.The distribution of developmental effects was analyzed by target organ system(19 aggregated effects by system), chemical count (387 total, 350 in rat and 317in rabbit), and test species. For 283 chemicals with data in both species, 53chemicals (18.7%) had critical effects on development that were specific (nomaternal toxicity) or sensitive (dLEL < mLEL) to exposure in either species. Ratwas the more sensitive species to developmental effects on an administereddose basis. The species differences may reflect biological factors, withcomplications form study design differences or examination techniques of thefetuses. The primary expressions of developmental toxicity were fetal weightreduction and skeletal defects (rat > rabbit), pregnancy/fetal losses (rabbit >rat), urogenital defects (rat> rabbit), and CNSdefects (rabbit> rat). ToxRefDB isa novel datamodel that provides a structured format for relational assessmentofsource data from guideline (in vivo) prenatal developmental toxicity studies. Itcanbeapplied to large-scale profiling andpredictivemodelingofdevelopmentaltoxicity for environmental chemicals. [This work has been reviewed by EPA andapproved for publication but does not necessarily reflect official Agency policy.]

doi:10.1016/j.ntt.2009.04.023

NBTS20

Scientific consensus statement on environmental agents associated withneurodevelopmental disorders

Steven Gilberta, Elise MillerbaInstitute of Neurotoxicology and Neurological Disorders, Seattle, WA, USAbCollaborative on Health and the Environment, Freeland, WA, USA

In order to document and provide support to prevent neurodevelopmentdisorders, the Scientific Consensus Statement on Environmental AgentsAssociated with Neurodevelopmental Disorders was developed by the Colla-borative on Health and the Environment's Learning and DevelopmentalDisabilities Initiative (posted at http://www.iceh.org/LDDI.html). This state-ment is intended as a guide to scientists, medical professionals, policymakers,

NBTS 2009 Abstracts 241

public health advocates, and the general public in advancing their efforts toaddress the important individual and social issues raised by learning anddevelopmental disabilities. This consensus statement outlines the currentscientific understanding of the links between environmental factors andlearning and developmental disabilities. Environmental agents that we areconfident cause learning and developmental disabilities in humans include:alcohol, lead, mercury, PCBs, PBDEs, manganese, arsenic, solvents, PAHs,pesticides and nicotine/environmental tobacco smoke. An overview of theevidence regarding these agents is presented, along with emerging evidenceabout other potential contributors: endocrine disruptors, fluoride and foodadditives. The statement also identifies important research areas that holdpromise of further advancing our understanding of these links. The scientificevidence reviewed in this statement, with a glossary and more than 200references, indicates environmental contaminants are an important cause oflearning and developmental disabilities. The proportion of environmentallyinduced LDDs is a question of profound human, scientific and public policysignificance. Despite some uncertainty, there is sufficient knowledge to takepreventive action to reduce fetal and childhood exposures to environmentalcontaminants. Given the serious consequences of LDDs, a precautionaryapproach is warranted to protect the most vulnerable of our society.

doi:10.1016/j.ntt.2009.04.024

NBTS21

A zebrafish model of the persisting neurobehavioral impairment causedby developmental chlorpyrifos exposure

Damiyon Sledge, Jerry Yen, Terrell Morton, Laura Dishaw, Kathleen Shuler,Susan Donerly, Elwood Linney, Edward LevinDuke University Medical Center, Durham, NC, USA

Developmental exposure of rats to the pesticide chlorpyrifos (CPF) has beenfound to cause persistent neurobehavioral impairment. In a parallel series ofstudies with zebrafish, we are also finding persisting behavioral dysfunction afterdevelopmental CPF exposure. We have developed a battery of measures ofzebrafish behavior, which are reliable and sensitive to toxicant-induced induceddamage. This study determined the critical window of developmental CPFexposure for causing persisting neurobehavioral effects. Tests of learning (3-chamber tank spatial discrimination), stress response (novel tankdiving test) andsensorimotor response (tap startle response and habituation) were conductedwith adult zebrafish after early developmental CPF exposure. The CPF exposurelevel was 100 ng/ml with durations of 0–1, 0–2, 0–3, 0–4 and 0–5 days afterfertilization. In the 3-chamber learning test, the 0–5 day CPF exposure group hadsignificantly lower learning rate. Developmental CPF exposure (0.29 µM0–5DPF)had persisting behavioral effects in zebrafish tested as adults. In the novel tankexploration test, exposed fish showed decreased escape diving response andincreased swimming activity. In the 3-chamber spatial learning test, exposed fishshowed nearly significantly poorer overall accuracy and significantly slowerlearning. In the tactile startle test, CPF exposed fish showeddecreased habituationto startle and a trend toward increased overall startle response. This studydemonstrates the selective long-term behavioral alterations caused by exposureto CPF in zebrafish. The zebrafish model can facilitate the determination of themolecular mechanisms critical for long-term neurobehavioral impairment afterdevelopmental toxicant exposure. (Supported by NIH ES10356)

doi:10.1016/j.ntt.2009.04.025

NBTS22

Down-regulation of DNA methyltransferase expression is associatedwith increased dopaminergic gene expression following developmentalpyrethroid exposure

Jason RichardsonRobert Wood Johnson Medical School, University of Medicine and Dentistry ofNew Jersey, Piscataway, NJ, USAEnvironmental and Occupational Health Sciences Institute, Rutgers University,Piscataway, NJ, USA

Previously, we have demonstrated that in utero exposure of mice to thepyrethroid pesticide deltamethrin (DM) causes persistent hyperactivity,impulsivity, and alterations of dopaminergic system. However, the mechanismbywhich deltamethrin exerts its effects on the dopamine system andwhy theseeffects persist remains unknown. In utero exposure of mice to DM (3 mg/kg)resulted in persistent (10–11 months) increased expression of the dopaminetransporter (DAT; 82%). This increasewas accompanied by increased expressionof the nuclear transcription factors Nurr1 (68%) and Pitx3 (34%), which havebeen demonstrated to regulate DAT expression. Because of the persistence ofthese effects, we sought to determine whether epigenetic alterations wereresponsible. Indeed, DNA methyltransferase (DNMT) 1, 3a, and 3b expressionwas reduced by 45, 51, and 74%, respectively. To explore mechanisms by whichDMmayalter DNMTexpression,we exposedMES23.5 dopaminergic cells to DM(10nM) and observed increasedDAT (34%),Nurr1 (71%), and Pitx3 (98%)mRNAexpression. These increases were accompanied by decreased DNMTexpression.Because DM causes delay of closing of sodium channels which can result inpersistent depolarization anddepolarizationhas beendemonstrated to decreaseDNMT expression, we depolarized cells with KCl for 24 h. Depolarizationincreased expression of DAT, Nurr1, and Pitx3 by 50%, 46%, and 46%, respectively.Taken in concert, these data suggest that persistent depolarization anddecreased DNMT expression following developmental DM exposure mayprovide a plausible mechanism for the persistence of DM's effects on thedopaminergic system. Supported by NIEHS R01ES015991.

doi:10.1016/j.ntt.2009.04.026

NBTS23

Late-emerging cognitive impairment in male rats after neonatalparathion exposure

Olga Timofeeva, Liwei Yang, Frederic Seidler, Theodore Slotkin, Edward LevinDuke University Medical Center, Durham, NC, USA

Developmental exposure of rats to the organophosphate (OP) pesticideschlorpyrifos, diazinon and parathion has been found in our studies and others tocause persistent neurobehavioral effects in juvenile and young adults. The currentstudy was conducted to determine the persisting effects of early postnatalexposure on cognitive function in older adult and aged rats. On PND 1–4, weadministered parathion to male and female Sprague–Dawley rat pups at dosesnear the threshold for overt signs of systemic toxicity and spanning the thresholdfor barely-detectable cholinesterase inhibition (0, 0.1 or 0.2 mg/kg/day). The ratsbegan training on the 16-arm radialmaze startingwhen theywere 4 or 14monthsof age. In a series of studies we have found that at doses, which cause minimalcholinesterase inhibition chlorpyrifos, diazinonandparathion causequite differenttypes of persisting effects on cognitive function. Brief postnatal exposure to a lowdoseof parathion, causes a slight improvement in accuracy in the radial-armmaze,when the offspring begin training in young adulthood. However, during the agingprocess neurocognitive impairment becomes apparent in males. When trainingbegins in later adulthood, males exposed to low doses of parathion during earlypostnatal development showasignificant impairment in radial-armmaze learningrelative to age matched male controls. The normal sex-difference in accuracy onthis spatial task is eliminated by brief postnatal low-dose parathion exposure. Thiseffect was seen more than a year after the brief period of low-level postnatalexposure. (Supported by NIH ES10356)

doi:10.1016/j.ntt.2009.04.027

NBTS24

Development of a robotic surrogate for measurement of young children'sexposure to environmental toxins

Stuart Shalata,b, Adam Stamblerc, Marta Hernandezb, Kathleen BlackbaEnvironmental and Occupational Health Sciences Institute, Rutgers University,Piscataway, NJ, USAbRobert Wood Johnson Medical School, University of Medicine and Dentistry ofNew Jersey, Piscataway, NJ, USAcRutgers, The State University of New Jersey, New Brunswick, NJ, USA

NBTS 2009 Abstracts242