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

1. Drug and Chemical Exposures in Animal Models Related to ASD Theodore Slotkin, Ph.D. Department of Pharmacology & Cancer Biology Integrated Toxicology & Environmental Health Program Duke University Support: NIH ES10356

2. 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

3. 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)

4. Developmental Neurotoxicants - The “Silent Pandemic” LDDI Initiative, 2007 Grandjean & Landrigan, Lancet 2006

5. Why Neuroactive Agents Disrupt Brain Development — Neurotransmitter Signals Control Cell Fate Nerve Terminal Signaling Cascades Nucleus Receptors Gene Transcription Replicate Differentiate Grow Die Learn The same neurotransmitter may be used for multiple decisions

6. Why there is a Critical Period Input After Critical Period Input During Critical Period Change in Cell Differentiation Short-Term Response Elicited Permanent Change in the Response to Stimulation Short-Term, Reversible, Compensatory Adjustments

7. 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

8. Organophosphate Pesticides — 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 • Effects are subtle but widespread • Originally modeled in animals, neurodevelopmental deficits now confirmed in children (inner-city, agricultural populations) • Developmental exposure increases autism risk

9. Chlorpyrifos - Multiple Mechanisms Disrupt Neurodevelopment 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 Critical period in rats: late gestation to early neonatal stage [equivalent - 2nd trimester in human fetus]

10. Chlorpyrifos - Impact on Serotonin Systems = Miswiring Male Female Enhanced neuronal impulse activity (serotonin turnover) Increases in serotonin receptors and transporter BUT….

11. …Impaired Serotonergic Function aka: increased risk-taking, impulsive behavior

12. Chlorpyrifos - Miswiring of Acetylcholine Systems -Serotonin Replaces Acetylcholine for Hippocampal Circuits and Behaviors

13. 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

14. Cerebellum Thinning of cerebellar lobules Thinning of hippocampal CA3 Reactive gliosis Somatosensory cortex - loss of pyramidal cells Critical Period Newborn Rat - PN2-5 = human 2nd trimester Control Terbutaline - 44% decrease in Purkinje cells

15. 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)

16. 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

17. Terbutaline - Impact on Serotonin Systems = Miswiring ≈ Chlorpyrifos Increases in serotonin receptors and transporter Enhanced neuronal impulse activity (serotonin turnover)

18. Terbutaline Followed by Chlorpyrifos Enhanced Effect on Serotonin Turnover

19. 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