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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 Treatment 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
percent change from control
50
male
f emale
40
30
Rx x measure,
p < 0.09
Rx x sex, p < 0.004
Rx x measure,
p < 0.005
*
Rx x sex, p < 0.1
Rx x measure,
p < 0.001
Rx, p < 0.002
Rx x sex,
p < 0.0006
mal e: p < 0.0004
female: NS
Rx, p < 0.0001
Rx x measure,
p < 0.006
*
*
*
20
*
10
*
0
-10
*
-20
5HT1A 5HT2 5HTT 5HT1A 5HT2 5HTT 5HT1A 5HT2 5HTT 5HT1A 5HT2 5HTT 5HT1A 5HT2 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