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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 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 Nerve Terminal Signaling Cascades Nucleus Receptors Gene Transcription Replicate Differentiate Grow Die Learn The same neurotransmitter may be used for multiple decisions Why there is a Critical Period Input During Critical Period Input After Critical Period Change in Cell Differentiation Short-Term Response Elicited Permanent Change in the Response to Stimulation Short-Term, Reversible, Compensatory Adjustments Apparently Unrelated Agents Can Produce Similar Outcomes — [maybe we shouldn’t focus on common mechanisms?] Correct Connection Miswired Connection Damage or Loss of Input Damage or Loss of Target Mismatched Phenotypes Corollary - exposure to multiple agents can produce additive or synergistic effects - worsened outcome 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 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] Chlorpyrifos - Impact on Serotonin Systems = Miswiring Male Female Chlorpyrifos T reatment on PN1-4 — 1 mg/kg 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 50 male female percent change from control 40 30 Rx x sex, p < 0.00 4 Rx x mea sure, p < 0.005 Rx x mea sure, p < 0.09 * Rx x sex, p < 0.1 Rx x mea sure, p < 0.001 Rx, p < 0.002 Rx x sex, p < 0.0006 ma le: p < 0.00 04 fe male : NS Rx, p < 0.0001 Rx x mea sure, p < 0.006 * * * 20 * 10 * 0 -10 * -20 5HT 1A 5HT 2 5HTT 5HT 1A 5HT 2 5HTT 5HT 1A 5HT 2 5HTT 5HT 1A 5HT 2 5HTT 5HT 1A 5HT 2 5HTT cerebral cortex Enhanced neuronal impulse activity (serotonin turnover) hippocampus striatum midbrain brainstem Increases in serotonin receptors and transporter BUT…. …Impaired Serotonergic Function Plus Maze: CPF (1 mg/kg) Decreases Anxiety in Males Chocolate Milk Preference: CPF (1 mg/kg) Causes Anhedonia 25 7 Control CPF 6 * Milk/Water Preference Percent Time Spent in Open Arms 30 20 15 10 5 Control CPF 5 4 * * 3 2 1 0 0 Male Female aka: increased risk-taking, impulsive behavior Male Female Chlorpyrifos - Miswiring of Acetylcholine Systems Serotonin Replaces Acetylcholine for Hippocampal Circuits and Behaviors 12 Working Memory Errors 10 PN 1-4 Chlorpyrifos 5HT2 Antagonist Drug Challenge 0 mg/kg ketanserin 0.5 mg/kg ketanserin 1.0 mg/kg ketanserin 2.0 mg/kg ketanserin * 8 * * 6 4 2 0 Control Chlorpyrifos 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 Cerebellum Control Terbutaline 44% decrease in Purkinje cells 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 • 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 neuronal impulse 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