Download Developmental Neurotoxicity from Environmental Chemical Exposures

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