slotkin2 22 06

How Exposure to Common PesticidesCan Damage the Developing Brain:Lessons Learned from Chlorpyrifosand the Organophosphates: 

Theodore Slotkin, Ph.D. Department of Pharmacology andamp; Cancer Biology Integrated Toxicology Program Superfund Basic Research Center Duke University Medical Center Support: NIH ES10356 and 10387 How Exposure to Common Pesticides Can Damage the Developing Brain: Lessons Learned from Chlorpyrifos and the Organophosphates

Why Test Developmental Neurotoxicity?: 

Why Test Developmental Neurotoxicity? 5000 new chemicals/year EPA estimate: 25% neurotoxic High vulnerability of the developing brain Increases in ADHD, learning/cognitive problems? Legal challenges to 10x additional FQPA safety factor

“Draw a Person” — 4.5 y.o.: 

Non-Exposed Exposed Guillette et al, 1998 'Draw a Person' — 4.5 y.o.

Why Testing for Developmental Neurotoxicology is So Difficult: 

Why Testing for Developmental Neurotoxicology is So Difficult Diversity of potential targets and effects in developing brain Choice of morphological endpoints Choice of behavioral endpoints (eg cognitive/hippocampus) Basal behavior vs. challenges Choice of critical periods original: prenatal — now: through weaning importance of adolescence Sex differences - Sex is a 'confounder' (!!!) Cumbersome - costly ...and ineffectual because of false negatives can test only a few each year (eg OPs)

Case Study — Chlorpyrifos: 

Case Study — Chlorpyrifos Developmental neurotoxicity unrelated to mechanisms in adults Incorrect biomarker (cholinesterase inhibition) systemic toxicity from ChE inhibition but fetus/neonate recover from inhibition faster than adult Effects are subtle but widespread Requires multidisciplinary approach to detection Widely used - ubiquitous exposure - OPs = 50% of all insecticide use Not an endocrine disruptor Replaced organochlorines Superfund Site Disposal Problem OPs: nerve gases in warfare/terrorism

Developmental Neurotoxicity of Chlorpyrifos — the Standard View: 

Developmental Neurotoxicity of Chlorpyrifos — the Standard View Chlorpyrifos Chlorpyrifos Oxon CYP450 Irreversible AChE inhibition ACh hyperstimulation andgt; 75% inhibition Acute Toxicity (SLUD) Surrogate: Plasma BuChE Most exposures below SLUD threshold Exposures above allowable limit go undetected

Mechanisms for Disrupted Development: 

Nerve Terminal Receptors Signaling Cascades Nucleus Gene Transcription Replicate Differentiate Grow Die Learn AChE Inhibition: CPF Oxon Direct Actions on Cholinergic Receptors Interaction with Signaling Intermediates Transcription Factor Expression, Function Mechanisms for Disrupted Development

Inhibition of DNA Synthesis — Acute Chlorpyrifos: 

Direct CNS administration of 2 µg: same result Inhibition of DNA Synthesis — Acute Chlorpyrifos

4 Day Rx with Chlorpyrifos: No Lethality, No Growth Inhibition: 

4 Day Rx with Chlorpyrifos: No Lethality, No Growth Inhibition

4 Day Rx with Chlorpyrifos: Inhibition of DNA Synthesis, Delayed Deterioration of Cell Signaling: 

4 Day Rx with Chlorpyrifos: Inhibition of DNA Synthesis, Delayed Deterioration of Cell Signaling

Multiple Mechanisms: A Shifting Target: 

Prenatal Postnatal Birth Neurogenesis and Migration Synaptogenesis Myelination Gliogenesis Multiple Mechanisms: A Shifting Target

Morphology: You’ve Got to Know What to Look For!Glial deficiencies instead of reactive gliosis: 

Morphology: You’ve Got to Know What to Look For! Glial deficiencies instead of reactive gliosis GFAP Cell Ratios

Fetal Chlorpyrifos Exposure: NeurochemistryPersistent Effects on Forebrain Cholinergic Function Below the Threshold for Cholinesterase Inhibition: 

Fetal Chlorpyrifos Exposure: Neurochemistry Persistent Effects on Forebrain Cholinergic Function Below the Threshold for Cholinesterase Inhibition

Chlorpyrifos Treatment Models: 

GD17-20 Neurogenesis PN1-4 Differentiation Early Axonogenesis GD9-12 Neural Tube Stage PN11-14 Axonogenesis Synaptogenesis CPF treatments on GD9-12, GD17-20, PN1-4, PN11-14 below threshold for systemic toxicity RAT HUMAN Late 1st/Early 2nd Trimester Late 2nd/3nd Trimester Model I Model II Model III Model IV Sexual differentiation of the brain Chlorpyrifos Treatment Models

Model I: Gestational Days 9-12Effects on Serotonin Systems are not Sex-Selectivewhen Assessed in Adulthood (PN60): 

Model I: Gestational Days 9-12 Effects on Serotonin Systems are not Sex-Selective when Assessed in Adulthood (PN60) Global upregulation of 5HT synaptic proteins suggests decreased 5HT activity

Model II: Gestational Days 17-20Massive, Sex-Selective Effects in Adulthood: 

Model II: Gestational Days 17-20 Massive, Sex-Selective Effects in Adulthood Largest effects in region with 5HT terminal projections (striatum) note different scale from Model I Cell signaling assessment: Shift to inhibitory effects of serotonin

Model III: Postnatal Days 1-4Sex-Selective Effects in Adulthood: 

Model III: Postnatal Days 1-4 Sex-Selective Effects in Adulthood Cell bodies targeted more than with earlier CPF treatment

Model IV: Postnatal Days 11-14Effects and Sex-Selectivity are Waning Despite Higher Dose: 

Model IV: Postnatal Days 11-14 Effects and Sex-Selectivity are Waning Despite Higher Dose

GD17-20 Chlorpyrifos Exposure: Learning & MemorySex-Dependent Effects Belowthe Threshold for Cholinesterase Inhibition: 

GD17-20 Chlorpyrifos Exposure: Learning andamp; Memory Sex-Dependent Effects Below the Threshold for Cholinesterase Inhibition GD9-12: Not sex-dependent PN1-4: Sex-dependent PN11-14: Not sex-dependent

PN1-4 Chlorpyrifos ExposureConsequences of Reduced Serotonergic Function: 

PN1-4 Chlorpyrifos Exposure Consequences of Reduced Serotonergic Function aka: increased risk-taking

PN1-4 Chlorpyrifos ExposureRadial Arm Maze — Ketanserin ChallengeReveals “Abnormal” Learning Mechanisms: 

PN1-4 Chlorpyrifos Exposure Radial Arm Maze — Ketanserin Challenge Reveals 'Abnormal' Learning Mechanisms Potential cognitive impairment with antidepressant/antipsychotic Rx

Not Just a Neural Disrupter -Hyperlipidemia and Hyperinsulinemia in Adulthood: 

Not Just a Neural Disrupter - Hyperlipidemia and Hyperinsulinemia in Adulthood Risk factors for cardiovascular disease and diabetes Resembles 'Barker Hypothesis' relating IUGR to adult morbidity

Chlorpyrifos Exposure In Vivo: 

Targeting of Neurons and Glia Subtle morphological changes require foreknowledge/quantitation Altered Synaptic Function: multiple transmitters and behaviors Acetylcholine - learning, memory, cognitive Serotonin - appetite, mood, sleep Abnormal adaptations revealed by 'challenge' Deficits can emerge in adolescence or adulthood Extend beyond neurotoxicity — cardiovascular/metabolic Underlying Mechanism — Non-Cholinergic Sex selectivity (critical period - sexual differentiation of the brain) Chlorpyrifos Exposure In Vivo

Origins of Sex Selectivity: 

Not endocrine disruptor No difference in initial neurotoxic effects (cell damage, etc) Effects emerge at puberty suggests link to sexual differentiation If so, expect critical period in late gestation, early neonatal stages Exposure on GD9-12 - not sex-selective Exposure on GD17-20 or PN1-4 - sex-selective Exposure on PN11-14 - less sex-selective Conclusion: Chlorpyrifos disrupts sexual differentiation of the brain during a critical period ('when' matters as much as 'what' or 'how much' - ignored by standard DNT testing) Origins of Sex Selectivity

Conclusion:Chlorpyrifos damages the developing brain, BUT:: 

Other OP Pesticides? Diazinon - yes, but andlt; chlorpyrifos NOAEL below threshold for cholinesterase inhibition (biomarker?) Animal models relevant to human exposure levels? Can you tell if you’re exposed if you’re nonsymptomatic? Acute vs. chronic exposure, levels, critical periods Agricultural Workers — US, Third World Superfund Sites — Entry into Water Supplies OPs and warfare/terrorism What safety factors should be applied? 10X 100X 1000X Conclusion: Chlorpyrifos damages the developing brain, BUT:

Dow Agrosciences on Chlorpyrifos (Chlorpyrifos.com): 

Proven Protection and Quality: More than 3,600 studies have been conducted examining critical aspects of chlorpyrifos products as they relate to health and safety. More than US $100 million has been spent examining the uses and impact of chlorpyrifos-containing products on human health and the environment. In terms of human health and safety, no pest control product has been more thoroughly studied. Material Safety Data Sheet Teratology (Birth Defects): Chlorpyrifos did not cause birth defects in laboratory animals. Reproductive Effects: Some evidence of toxicity to the offspring occurred, but only at a dose high enough to produce significant toxicity to the parent animals. Dow Agrosciences on Chlorpyrifos (Chlorpyrifos.com)

How Can We Begin to Test Large Numbers of Chemicals?Alternative Models: 

Rat Embryo Cultures PC12 cells (neurotypic) - C6 cells (gliotypic) Sea Urchin Embryos Zebrafish Embryos How Can We Begin to Test Large Numbers of Chemicals? Alternative Models

Cultured Rat Embryo — Neural Tube Stage: 

Cultured Rat Embryo — Neural Tube Stage

NGF-Induced Differentiation of PC12 Cells: 

NGF Mitosis Differentiation Neurite Extension ACh vs. CA phenotype Susceptible to apoptosis NGF-Induced Differentiation of PC12 Cells

Larger effects on gliotypic cells than on neuronotypic cells: 

Larger effects on gliotypic cells than on neuronotypic cells CPF more potent than CPF oxon Other OPs work, too, but non-OP AChE inhibitor less effective

In Vitro Models — CPF Developmental Neurotoxicity: 

Noncholinergic + Cholinergic Gliotypic and Neuronotypic cells Parallels multiple mechanisms seen in vivo Parallels critical stages of vulnerability High-throughput, rapid screening In Vitro Models — CPF Developmental Neurotoxicity BUT — Limitations: Pharmacokinetics, maternal-fetal, dose-effect Can’t model cell-cell interactions Can’t model sex-selective effects

Morphology Using Non-Mammalian Models - The Sea Urchin: 

Morphology Using Non-Mammalian Models - The Sea Urchin Acetylcholine, monoamines used as morphogens Neurotransmitters and receptors specified when embryonic genome switched on Chlorpyrifos Control Late blastula 1 stage Abnormal cell differentiation Pigmented cells form an extralarval cap Chlorpyrifos oxon ineffective non-OP AChEIs ineffective Cell signaling target - PKC?

Non-Mammalian Models: Zebrafish: 

Development in 3 days Transparent embryo Immobilize in agar Transgenics for specific organs/cells cDNA arrays Can study behavior Can include sex Non-Mammalian Models: Zebrafish

Behavioral Effects in Adult Zebrafishafter Embryonic CPF Exposure(Levin et al): 

Behavioral Effects in Adult Zebrafish after Embryonic CPF Exposure (Levin et al)

Identifying the Problems: 

Identifying the Problems Neurotoxicants act in a variety of ways Developmental mechanisms different: biomarker selection Cannot apply fixed criteria MUST include sex differences! Initial high-throughput screens: in vitro andamp; non-mammalian mechanism - likely targets - probable critical period Information determines what to look for in mammalian model Alterations not necessarily detectable with morphology So many new compounds, so little time and money New technologies: cDNA/proteomic arrays What are the Solutions?

And if we just ignore the problem? Effect of Decreasing IQ by 5 Points: 

And if we just ignore the problem? Effect of Decreasing IQ by 5 Points