Organophosphate Exposure and Cognition: Novel Mechanisms of Neurotoxicity Alvin V. Terry, Jr., Ph.D. Professor of Pharmacology and Toxicology Director Small Animal Behavior Core Central Cholinergic Pathways Human Rat Source: “Fundamental Neuroscience”, Second Edition, Copyright, 2003, Academic Press Acetylcholine Appendix A Presentation 3 - Terry RAC-GWVI Meeting Minutes June 28-29, 2010 Page 71 of 214
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Organophosphate Exposure and Cognition: Novel …€¦ · Neurotoxicity (OPIDN)-aggregation and accumulation of cytosketelal proteins, microtubules and neurofilaments (reviewed, Abou-Donia
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Organophosphate Exposure and Cognition: Novel Mechanisms of
Neurotoxicity
Alvin V. Terry, Jr., Ph.D.Professor of Pharmacology and Toxicology
DirectorSmall Animal Behavior Core
Central Cholinergic Pathways
Human
RatSource: “Fundamental Neuroscience”, Second Edition, Copyright, 2003, Academic Press
Acetylcholine
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 71 of 214
Source: “Fundamental Neuroscience”, Second Edition, Copyright, 2003, Academic Press
Acetylcholine (cholinergic) Synapse
Acetylcholine
proNGF NGF
proteolysis
proNGF NGF
p75NTRSortilin
Cell Death Cell Survival
Neuronal Membrane
TrkA
Adapted from Nykjaer et al., Nature 427:843-848, 2004
Nerve Growth FactorSignaling
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 72 of 214
Growth factorKinesinDyneinVesicle
Myelin
Dendrite
ER
Mito
GANode
of Ranvier Terminal
CNS Neuron
Nucleus
“Esters of phosphoric acid”First synthesized in the 1800sInsecticide Potential Recognized in the 1930sFurther developed as “Nerve Agents”by the Germans and British during WWII
Organophosphates R1-O-P-O-R2
O
R3
R1-O-P-O-R2
R3
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 73 of 214
Improvements in Farming ProductivityControl of Vector-Borne Diseases
MalariaYellow FeverTyphusViral Encephalitis
Control of “Nuisance” PestsFlies, Ants, Roaches. Etc
Relatively inexpensive, less persistent than organochlorines
Organophosphate PesticidesAdvantages
R1-O-P-O-R2
O
R3
R1-O-P-O-R2
R3
OH
N-CH2-CH2-O-C-CH3
CH3
CH3
H3C ++
O H3C N-CH2-CH2-OHCH3
CH3
acetylcholinesterase
acetylcholine
choline
acetic acidanionic esteratic
+
HO-C-CH3
OHO-C-CH3
O
R1-O-P-O-R2
O
R3
R1-O-P-O-R2
O
R3organophosphate
++
C3H7O P F
OH7C3
O
Sarin
Soman
DFP
O P F
CH3
O
C(CH3)3
CH3
H
C3H7O P FCH3
O
NH5C2
OP
O
OH5C2
S
ClCl
ClChlorpyrifos
NH5C2
OP
O
OH5C2
O
ClCl
Cl
Chlorpyrifosoxon
CYP 450
NerveAgents
Insecticides
Mechanism of Action
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 74 of 214
Chronic and/or Repeated Low-Level Exposures*Anxiety, depression, psychotic symptoms, deficits in short-term memory, learning, attention, information processing, eye-hand coordination and reaction time, and extrapyramidal symptoms.
* Data primarily from case reports and retrospective epidemiological studies.
Overall ObjectivesDetermine the consequences of repeated, “subthreshold” exposures to representative organophosphates on cognitive function in animal models.
Information processing and attentionSpatial LearningRecognition MemoryWorking Memory
Determine the consequences of repeated, low-level exposures to representative organophosphates on neurobiological substrates of cognitive function
Cholinergic MarkersNeurotrophinsAxonal Transport
Identify therapeutic targets for drug development
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 75 of 214
Study Algorithm
Learning and MemoryWater MazeNovel Object RecognitionPrepulse Inhibition5C-SRTTRadial Arm Maze
Cholinergic MarkersEnzyme AssaysReceptor AutoradiographyWestern Blot
OP Effects On Axonal Transport?Organophosphate Ester-Induced Delayed Neurotoxicity (OPIDN)-aggregation and accumulation of cytosketelal proteins, microtubules and neurofilaments (reviewed, Abou-Donia 2003)Decreased expression of presynapticcholinergic receptors in the brain (Stone et al., 2000)Disruption of tubulin polymerization & microtubule formation (Prendergast et al., 2007)Inhibited kinesin-dependent microtubule motility (Gearhart et al., 2007)OPs Covalently modify tyrosines on α and βtubulin (Grigoryan et al., 2009)
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 76 of 214
Axo
nal T
rans
port
Qua
ntity
(# v
esic
les/
view
ing
win
dow
)
0
100
200
300
400
500Anterograde Retrograde
-20%*
-20%*
Vehicl
e
Chlorpyri
fos
Vehicl
e
Chlorpyri
fos
Axo
nal T
rans
port
Qua
ntity
(# v
esic
les/
view
ing
win
dow
)
0
100
200
300
400
500Anterograde Retrograde
-30%*
-20%*
Vehicl
e
Chlorpyri
fos
Vehicl
e
Chlorpyri
fos
A B
Time after injection (hours)0 10 20 30 40 50
Axo
nal T
rans
port
Qua
ntity
(# v
esic
les/
view
ing
win
dow
)
250
300
350
400
450
-20%*
-10%*-15%*
Vehicle control
Anterograde
C D
Time after injection (hours)0 10 20 30 40 50
Axo
nal T
rans
port
Qua
ntity
(# v
esic
les/
view
ing
win
dow
)50
100
150
200
250
-33%*
-7%*
-26%*
Vehicle control
Retrograde
Axonal Transport
Chlorpyrifos 18.0 mg/kgN=3-5
Day 14 of WashoutDay 1 of Washout
Single Dose-Temporal Effect Single Dose-Temporal Effect
Terry et al., J. Pharmacol Exp Ther 322: 1117-1128, 2007.
Summary of Previous Chlorpyrifos Studies (repeated Subthreshold exposures)
Impairments in spatial learningImpairments in Prepulse Inhibition of the auditory startle response Decreased expression of cholinergic marker proteins in the brainDecreased expression of neurotrophin-related proteins in the brainImpairments of anterograde and retrograde axonal transport ex vivo
Terry et al., J. Pharmacol Exp Ther 322: 1117-1128, 2007.
Terry et al., J. Pharmacol Exp Ther 305: 375-384, 2003.
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 77 of 214
Recent and Ongoing Chlorpyrifos Studies
NH5C2
OP
O
OH5C2
S
ClCl
Cl
The Rat, Five Choice Serial Reaction Time Task (5C-SRTT)
A H X J A K X O I Y A U B A X
Hit LeverContinuous Performance Task (CPT)
AX Type
Food Magazine
Light Stimulus
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RAC-GWVI Meeting Minutes June 28-29, 2010 Page 78 of 214
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 85 of 214
Sample Trial
Choice Trial
Delay
Spontaneous NovelObject Recognition
15 Min Delay
0.0 0.25 0.50 0.75 1.00
Expl
orat
ion
Tim
e (s
ec)
0
5
10
15
20
25
30
Familar Novel
60 Min Delay
Expl
orat
ion
Tim
e (s
ec)
0
5
10
15
20
25
30
* *
* * * *
0.0 0.25 0.50 0.75 1.00Dose of DFP (mg/kg)
Dose DFP (mg/kg)
d2 In
dex
0.000.050.100.150.200.250.300.35
0.0 0.25 0.50 0.75 1.00
*†
C3H7O P F
OH7C3
O
Spontaneous Novel Object Recognition
N=11-24
d2 index = (novel - familiar)/(novel + familiar)
Averaged Across Delays
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 86 of 214
Treatment Time Point BF HIPP PFC CTX STR(mg/kg)
Vehicle Last Inj Day 12.37±0.78 4.42±0.24 3.83±0.33 3.01±0.15 8.94±0.42DFP 0.25 Last Inj Day 9.33±1.50*** 4.18±0.33 4.16±0.45 3.41±0.09 9.39±0.64DFP 0.50 Last Inj Day 9.88±1.02* 4.74±0.11 3.48±0.08 3.42±0.32 9.83±0.63DFP 0.75 Last Inj Day 8.21±1.11*** 4.69±0.35 3.82±0.37 2.94±0.20 8.34±0.39DFP 1.00 Last Inj Day 6.67±1.35*** 3.86±0.20 3.76±0.28 2.73±0.27 10.00±0.51
Vehicle WO Day 7 9.52±0.60 4.37±0.15 2.75±0.22 3.70±0.19 6.48±0.44DFP 0.25 WO Day 7 7.72±1.24§ 4.29±0.13 2.32±0.16 3.12±0.17 6.59±0.44DFP 0.50 WO Day 7 6.44±1.23** 4.40±0.35 2.58±0.07 3.26±0.12 6.23±0.76DFP 0.75 WO Day 7 8.06±0.94 4.64±0.21 2.21±0.12 3.03±0.09 6.41±0.65DFP 1.00 WO Day 7 7.88±0.61§ 4.85±0.19 2.25±0.10 3.07±0.17 7.37±0.46
Vehicle WO Day 14 9.06±2.22 4.69±0.37 4.95±0.37 2.99±0.13 8.89±0.80DFP 0.25 WO Day 14 8.04±0.60 4.57±0.72 3.78±0.16 2.76±0.26 9.26±1.02DFP 0.50 WO Day 14 8.52±1.66 4.93±0.54 3.97±0.0.63 2.66±0.20 8.27±0.83DFP 0.75 WO Day 14 8.50±2.33 4.74±0.65 3.63±0.29 3.39±0.33 8.01±0.93DFP 1.00 WO Day 14 5.61±1.55** 4.93±0.34 3.65±0.42 2.97±0.38 7.69±0.53
Brain Region
Choline Acetyltransferase (ChAT) Activity
ChAT activity expressed as pmoles of 3H-acetylcholine formed/μg of protein/min (mean ± S.E.M.) N= 5-6. *** p<0.001; **p<0.01; *p<0.05 §p<0.1 versus vehicle control.
α7-nicotinic acetylcholine receptor
VEH DFP
β-actin
α7-nAChR
VEH DFP
β-actin
α7-nAChR
α7:β-
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
α7:β-
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
***kDa
5543
VEH DFP
β-actin
α7-nAChR
α7:β-
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
BF
HIPP
PFC
kDa
5543
kDa
5543
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 87 of 214
proNGF
BF
VEH DFPkDa
36
proNGFβ-actin
HIPP
VEH DFP
β-actin
PFC
VEH DFP
β-actin
proNGF
proNGF
36
kDa
36
36kDa
36
36
kDa
36
36kDa
36
36
proN
GF:β -
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
proN
GF:β -
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
proN
GF:β-
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
**
p75 neurotrophin receptor (p75NTR)
VEH DFP
β-actin
VEH DFP
β-actin
kDA
7542
kDA
7542
VEH DFP
β-actin
kDA7542
p75NTR
p75NTR
p75NTR
p75:β -
actin
Rat
io
0.0
0.2
0.4
0.6
0.8
VEH DFP
p75:β -
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
**
p75:β -
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
BF
HIPP
PFC
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 88 of 214
mNGF
BF
VEH DFP
mNGFβ-actin
HIPP
VEH DFP
mNGFβ-actin
PFC
VEH DFP
mNGFβ-actin
kDa
3614
kDa
3614
14
36
NG
F:β -
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
NG
F:β-
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
NG
F:β-
actin
Rat
io
0.00.20.40.60.81.0
VEH DFP
*
**
TrkA Receptor
BF
VEH DFP
TrkAβ-actin
HIPP
VEH DFP
TrkAβ-actin
PFC
VEH DFP
TrkAβ-actin
TrkA
:β-a
ctin
Rat
io
0.00.20.40.60.81.0
VEH DFP
TrkA
:β-a
ctin
Rat
io
0.00.20.40.60.81.0
VEH DFP
TrkA
:β-a
ctin
Rat
io
0.00.20.40.60.81.0
VEH DFP
kDa148
36
kDa148
36
kDa148
36
kDa148
36
kDa148
36
kDa148
36
**
*
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 89 of 214
p-TrKA Receptor
BF
VEH DFP
p-TrkAβ-actin
HIPP
VEH DFP
p-TrkAβ-actin
PFC
VEH DFP
p-TrkAβ-actin
pTrk
A:β
-act
in R
atio
0.00.20.40.60.81.0
VEH DFP
pTrk
A:β
-act
in R
atio
0.00.20.40.60.81.0
VEH DFP
pTrk
A:β
-act
in R
atio
0.00.20.40.60.81.0
VEH DFP
kDa148
36
kDa148
36
kDa148
36
kDa148
36
kDa148
36
kDa148
36
*
βAPP Immunostaining
Appendix A Presentation 3 - Terry
RAC-GWVI Meeting Minutes June 28-29, 2010 Page 90 of 214
Summary/Preliminary ConclusionsRepeated, subthreshold exposures to both insecticide and alkylphosphate OPs lead to protracted impairments in the performance of attention and memory-related behavioral tasks in animals.Insecticide and alkylphosphate OPs may have differential effects on specific domains of cognition.The mechanisms underlying OP-related impairments of cognition may involve deleterious effects on mitochondrial morphology and movement, axonal transport, and neurotrophin signaling.