Drug and Chemical Exposures in Animal Models Related to ASD Theodore Slotkin, Ph.D.

Drug and Chemical Exposures in Animal Models Related to ASD

Theodore Slotkin, Ph.D.Department of Pharmacology & Cancer Biology

Integrated Toxicology & Environmental Health ProgramDuke University

Support: NIH ES10356

Main Points

• Why an increase in neurodevelopmental disorders including ASD?

• Why do neuroactive agents produce permanent alterations with developmental exposures?

• Why is there a critical period for these effects?

• Why do apparently unrelated agents produce similar outcomes?

• Example from environmental chemicals: organophosphate pesticides

• Example from prenatal drug exposure: terbutaline

Developmental Neurotoxicity from Environmental Chemical Exposures

5000 new chemicals/year

EPA estimate: 25% neurotoxic

67% of High Production Chemicals Not Tested for Neurotoxicity

High vulnerability of the developing brain

Increases in ADHD, learning/cognitive problems?• 17% of US schoolchildren suffer from neurobehavioral disabilities

• Annual cost: $80-170 billion

• 250% increase in ADHD diagnosis between 1990-1998

• 190% increase in children in special ed for learning disabilities between 1977-1994

• Increase in autistic spectrum disorders from 4/10,000 (1980s) to 30-60 (1990s)

Developmental Neurotoxicants - The “Silent Pandemic”

LDDI Initiative, 2007 Grandjean & Landrigan, Lancet 2006

Why Neuroactive Agents Disrupt Brain Development —Neurotransmitter Signals Control Cell Fate

NerveTerminal

Receptors

SignalingCascades Nucleus

Gene Transcription

Replicate Differentiate Grow Die Learn

The same neurotransmitter may be used for multiple decisions

Why there is a Critical PeriodWhy there is a Critical Period

Change in Cell DifferentiationChange in Cell Differentiation

Permanent Change in the Permanent Change in the Response to StimulationResponse to Stimulation

Input Input AfterAfter Critical Period Critical Period

Short-Term Response ElicitedShort-Term Response Elicited

Short-Term, Reversible, Short-Term, Reversible, Compensatory AdjustmentsCompensatory Adjustments

Input Input DuringDuring Critical Period Critical Period

Apparently Unrelated Agents Can Produce Similar Outcomes —[maybe we shouldn’t focus on common mechanisms?]

Correct Connection Damage or Loss of Input Damage or Loss of Target

Miswired Connection Mismatched Phenotypes

Corollary - exposure to multiple agents can produce additive or synergistic effects - worsened outcome

Organophosphate Pesticides — Chlorpyrifos

Developmental neurotoxicity unrelated to mechanisms in adults

Effects are subtle but widespread

Originally modeled in animals, neurodevelopmental deficits now confirmed in children (inner-city, agricultural populations)

Developmental exposure increases autism risk

• 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

NerveTerminal

Receptors

SignalingCascades Nucleus

Gene Transcription

Replicate Differentiate Grow Die LearnAChE

Inhibition:CPF Oxon

Direct Actions on Cholinergic Receptors

Interaction withSignaling Intermediates

Transcription Factor

Expression, Function

Chlorpyrifos - Multiple Mechanisms Disrupt Neurodevelopment

Critical period in rats: late gestation to early neonatal stage[equivalent - 2nd trimester in human fetus]

Chlorpyrifos - Impact on Serotonin Systems = Miswiring

-20

-10

0

10

20

30

40

50

5HT1A 5HT2 5HTT 5HT1A 5HT2 5HTT 5HT1A 5HT2 5HTT 5HT1A 5HT2 5HTT 5HT1A 5HT2 5HTT

Chlorpyrifos Treatment on PN1-4 — 1 mg/kg

malefemale

cerebralcortex

hippocampus brainstem

ANOVA: Rx, p < 0.0001; Rx x sex, p < 0.0002; Rx x region, p < 0.0001;Rx x measure, p < 0.0003; Rx x region x measure, p < 0.0007

percent change from control

midbrainstriatum

Rx, p < 0.002Rx x sex,p < 0.0006

male: p < 0.0004female: NS

Rx, p < 0.0001Rx x measure,

p < 0.006

Rx x sex, p < 0.004Rx x measure,

p < 0.005

Rx x sex, p < 0.1Rx x measure,

p < 0.001

Rx x measure,p < 0.09

*

**

**

*

*

Male Female

Enhanced neuronalimpulse activity

(serotonin turnover)

Increases in serotonin receptors and transporter

BUT….

…Impaired Serotonergic Function

0

5

10

15

20

25

30

Male Female

Plus Maze: CPF (1 mg/kg)Decreases Anxiety in Males

ControlCPF

*

0

1

2

3

4

5

6

7

Male Female

Chocolate Milk Preference:CPF (1 mg/kg) Causes Anhedonia

ControlCPF

* *

aka: increased risk-taking, impulsive behavior

Chlorpyrifos - Miswiring of Acetylcholine Systems -Serotonin Replaces Acetylcholine for Hippocampal

Circuits and Behaviors

0

2

4

6

8

10

12

Control Chlorpyrifos

PN 1-4 Chlorpyrifos5HT2 Antagonist Drug Challenge0 mg/kg ketanserin0.5 mg/kg ketanserin1.0 mg/kg ketanserin2.0 mg/kg ketanserin

*

*

*

p < 0.0001

Terbutaline Use in Preterm Labor

• Stimulates BARs to inhibit uterine contraction

• Crosses the placenta to stimulate fetal BARs

• Effective for 48 hr max - NOT for maintenance use

• Animal studies from our lab, 1980s-1990s

altered neural cell differentiation

receptor and signaling shifts

permanent changes in responsiveness

• Hadders-Algra 1986 - impaired school performance

• Pitzer 2001 - psychiatric, learning disorders

Control

Terbutaline - 44% decrease in Purkinje cells

CerebellumThinning of cerebellar lobulesThinning of hippocampal CA3Reactive gliosisSomatosensory cortex - loss of

pyramidal cells

Critical Period Newborn Rat - PN2-5 =human 2nd trimester

• Neuroinflammation in cerebral cortex and cerebellum - microglial activation• Morphological changes almost identical to those in postmortem autism samples• Critical period PN2-5• Hyperreactive to novelty, aversive stimuli, sensory input

Decompensation of CVS responses similar to those

in autism

(compare to Ming 2005)

• Continuous terbutaline exposure for 2 weeks: RR=2.0• Male twins with no other affected siblings: RR=4.4

Further increase: BAR polymorphisms (16G, 27E) that prevent desensitization and therefore would enhance terbutaline effects

Terbutaline - Impact on Serotonin Systems = Miswiring ≈ Chlorpyrifos

Enhanced neuronalimpulse activity

(serotonin turnover)

Increases in serotonin receptors and transporter

Terbutaline Followed by Chlorpyrifos

Enhanced Effect on Serotonin Turnover

CONCLUSIONS• Developmental neurotoxicants likely to play an important role in the increased incidence of childhood behavioral disorders including ASD

• Disparate mechanisms and effects converge on common final pathways

— different agents may produce similar outcomes

— different agents may produce additive/synergistic outcomes

• Lasting effects only when exposure occurs in critical periods

• Specific examples with relevance to ASD:

— organophosphate pesticides (ubiquitous exposure)

— terbutaline (use in preterm labor ≈10% US pregnancies)

Neurodevelopmental disorders - CAUSES, not a single ‘cause’

Origins of autism and ASD may not be so distinct from other neurodevelopmental disorders