How Exposure to Common Pesticides Can Damage the Developing Brain: Lessons Learned from Chlorpyrifos

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How Exposure to Common PesticidesCan Damage the
Developing BrainLessons Learned from
Chlorpyrifosand the Organophosphates
Theodore Slotkin, Ph.D. Department of
Pharmacology Cancer Biology Integrated
Toxicology Program Superfund Basic Research
Center Duke University Medical Center Support
NIH ES10356 and 10387
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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

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

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Case Study Chlorpyrifos

• 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 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

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Developmental Neurotoxicity of Chlorpyrifos the
Standard View
CYP450
Chlorpyrifos
Chlorpyrifos Oxon
Irreversible AChE inhibition
Acute Toxicity (SLUD)
ACh hyperstimulation
gt 75 inhibition
Surrogate Plasma BuChE

• Most exposures below SLUD threshold
• Exposures above allowable limit go undetected

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Mechanisms for Disrupted Development
Direct Actions on Cholinergic Receptors
Interaction with Signaling Intermediates
Signaling Cascades
Nerve Terminal
Nucleus
Transcription Factor Expression, Function
Receptors
??Gene Transcription
AChE Inhibition CPF Oxon
Replicate Differentiate Grow Die Learn
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Inhibition of DNA Synthesis Acute Chlorpyrifos
Direct CNS administration of 2 µg same result
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4 Day Rx with Chlorpyrifos No Lethality, No
Growth Inhibition
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4 Day Rx with Chlorpyrifos Inhibition of DNA
Synthesis, Delayed Deterioration of Cell
Signaling
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Multiple Mechanisms A Shifting Target
Birth
Prenatal Postnatal
Neurogenesis and Migration
Gliogenesis
Synaptogenesis
Myelination
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Morphology Youve Got to Know What to Look
For!Glial deficiencies instead of reactive
gliosis
GFAP
Cell Ratios
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Fetal Chlorpyrifos Exposure NeurochemistryPersi
stent Effects on Forebrain Cholinergic Function
Below the Threshold for Cholinesterase Inhibition
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Chlorpyrifos Treatment Models

• CPF treatments on GD9-12, GD17-20, PN1-4,
  PN11-14
• below threshold for systemic toxicity

Model I Model II
Model III Model IV
GD17-20 Neurogenesis
GD9-12 Neural Tube Stage
PN1-4 Differentiation Early Axonogenesis
PN11-14 Axonogenesis Synaptogenesis
RAT
HUMAN Late 1st/Early 2nd Trimester
Late 2nd/3nd Trimester
Sexual differentiation of the brain
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Model I Gestational Days 9-12Effects on
Serotonin Systems are not Sex-Selectivewhen
Assessed in Adulthood (PN60)
Global upregulation of 5HT synaptic proteins
suggests decreased 5HT activity
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Model II Gestational Days 17-20Massive,
Sex-Selective Effects in Adulthood
Cell signaling assessment Shift to inhibitory
effects of serotonin
note different scale from Model I

• Largest effects in region with 5HT terminal
  projections (striatum)

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

• Cell bodies targeted more than with earlier CPF
  treatment

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Model IV Postnatal Days 11-14Effects and
Sex-Selectivity are Waning Despite Higher Dose
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GD17-20 Chlorpyrifos Exposure Learning
MemorySex-Dependent Effects Belowthe Threshold
for Cholinesterase Inhibition
GD9-12 Not sex-dependent PN1-4
Sex-dependent PN11-14 Not sex-dependent
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PN1-4 Chlorpyrifos ExposureConsequences of
Reduced Serotonergic Function
aka increased risk-taking
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PN1-4 Chlorpyrifos ExposureRadial Arm Maze
Ketanserin ChallengeReveals Abnormal Learning
Mechanisms
Potential cognitive impairment with
antidepressant/antipsychotic Rx
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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

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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/meta
  bolic
• Underlying Mechanism Non-Cholinergic
• Sex selectivity (critical period - sexual
  differentiation of the brain)

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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)

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ConclusionChlorpyrifos damages the developing
brain, BUT

• Other OP Pesticides? Diazinon - yes, but lt
  chlorpyrifos
• NOAEL below threshold for cholinesterase
  inhibition (biomarker?)
• Animal models relevant to human exposure levels?

• Can you tell if youre exposed if youre
  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

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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.
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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

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Cultured Rat Embryo Neural Tube Stage
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NGF-Induced Differentiation of PC12 Cells
Mitosis
NGF
Differentiation
Neurite Extension ACh vs. CA phenotype Susceptible
to apoptosis
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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

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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

• BUT Limitations
• Pharmacokinetics, maternal-fetal, dose-effect
• Cant model cell-cell interactions
• Cant model sex-selective effects

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Morphology Using Non-Mammalian Models - The Sea
Urchin

• Acetylcholine, monoamines used as morphogens
• Neurotransmitters and receptors specified when
  embryonic genome switched on

• Late blastula 1 stage
• Abnormal cell differentiation
• Pigmented cells form an extralarval cap
• Chlorpyrifos oxon ineffective
• non-OP AChEIs ineffective
• Cell signaling target - PKC?

Chlorpyrifos Control
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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

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Behavioral Effects in Adult Zebrafishafter
Embryonic CPF Exposure(Levin et al)
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Identifying the Problems

• Neurotoxicants act in a variety of ways

• Developmental mechanisms different biomarker
  selection

• Alterations not necessarily detectable with
  morphology
• So many new compounds, so little time and money

What are the Solutions?

• Initial high-throughput screens in vitro
  non-mammalian

• mechanism - likely targets - probable critical
  period

• Information determines what to look for in
  mammalian model

• New technologies cDNA/proteomic arrays

• Cannot apply fixed criteria
• MUST include sex differences!

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