Organophosphate Exposure and Cognition: Novel Mechanisms of … · 2010-09-16 · Organophosphate...
Transcript of Organophosphate Exposure and Cognition: Novel Mechanisms of … · 2010-09-16 · Organophosphate...
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
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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
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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
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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
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Organophosphate ToxicityAcute
Muscarinic (postganglionic parasympathetic) “DUMB-BELS”: diaphoresis and diarrhea, urination, miosis, bradycardia, bronchospasm, emesis, lacrimation, salivation. Nicotinic (neuromuscular junction)- muscle fasciculations, weakness, paralysis, respiratory failure; (CNS)- seizures or CNS depression/coma.
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
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Study Algorithm
Learning and MemoryWater MazeNovel Object RecognitionPrepulse Inhibition5C-SRTTRadial Arm Maze
Neurotrophins (NGF)Western BlotImmunohistochemistry
OP Exposure
Axonal TransportSciatic Nerve-DIC MicroscopyMitochondrial MovementβAPP Immunohistochemistry
OP-FreeWashout
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)
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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.
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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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5C-SRTT-Chlorpyrifos (Every Day Exposure)
Test Session5 10 15 20 25 30 35 40 45
% c
orre
ct
060
70
80
90
100
During Exposure During Washout
VehicleChlorpyrifosVehicleVehicleChlorpyrifosChlorpyrifos
* ***
* * **
* * **** *
****
*** *** ****
Louise Middlemore-Risher
Middlemore-Risher et al., Neurotoxicology and Teratology 32: 415–424, 2010
5C-SRTT-Chlorpyrifos (Every Day Exposure)
Middlemore-Risher et al., Neurotoxicology and Teratology 32: 415–424, 2010
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Test Session5 10 15 20 25 30 35 40 45 50 55 60
% c
orre
ct
0
50
60
70
80
90
100
During Exposure During Washout
VehicleChlorpyrifosVehicleVehicleChlorpyrifosChlorpyrifos
* ** ************************
**** *** *
5C-SRTT-Chlorpyrifos (Alternate Day Exposures)
Middlemore-Risher et al., Neurotoxicology and Teratology 32: 415–424, 2010
5C-SRTT-Chlorpyrifos (Alternate Day Exposures)
Middlemore-Risher et al., Neurotoxicology and Teratology 32: 415–424, 2010
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VehicleTreated
Chlorpyrifos oxon1.0 μM
Rat Cortical Neurons (MitoTracker® Imaging)
Vehicle
Chlorpyrifosoxon
5.0 nM
MitoTracker®
Imaging
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% C
ontr
ol
0
50
100
150
200
250
Mitochondrial Number
020406080
100120
Mitochondrial Length
CPF (μM)0 1 5 10 20
CPF (μM)0 1 5 10 20
% C
ontr
ol
Rat Cortical Neurons(MitoTracker® Imaging)
** *
*
* *
*
# of Mitochondria Moving
% C
ontr
ol
020406080
100120
CPF (μM)0 1 5 10 20
**
*
*
Mitochondrial Length
% C
ontr
ol
0
50
100
150
200
250
Mitochondrial Number
% C
ontr
ol
020406080
100120
CPO (μM)0 1 5 10 20
CPO (μM)0 1 5 10 20
# of Mitochondria Moving
% C
ontr
ol
020406080
100120
CPO (μM)0 1 5 10 20
Rat Cortical Neurons(MitoTracker® Imaging)****
* * * *
* * **
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Fusion
Fission
AnterogradeMovement
RetrogradeMovement
Mitochondrial Morphologyand Movement
Adapted from Detmer and Chan, 2007
CellBody Terminal
Microtubules
Ongoing DFP Studies
C3H7O P F
OH7C3
O
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0
60
120
0 20 100 120
dB
MSEC
0
60
120
0 20 100 120
dB
MSEC
0
60
120
0 20 100 120
dB
MSEC
0
60
120
0 20 100 120
dB
MSEC
Pulse
PulsePrepulse
White Noise
White Noise
Normal Response
InhibitedResponse
“Prepulse Inhibition”
Prepulse Inhibition (PPI)
Restraining Tube
Sound AttenuatingEnclosure
Acoustic (Startle)Source
Motion Sensor
Prepulse InhibitionVehicle Study
Mea
n St
artle
Apl
itude
(Arb
itrar
y U
nits
)
0
200
400
600
800
1000
Startle Stimulus AloneNull StimulusPrepul 5 Prepul 10 Prepul 15
*
*
* +
*#
#
% P
repu
lse
Inhi
bitio
n
0
20
40
60
80
100
75 dB 80 dB 85 dBPrepulse Levels
* *#
% PPI = [100 - (startle amplitude on prepulse-pulse trials ÷ startle amplitude on pulse alone trials) x 100]
Vehicle Study
Star
tle A
mpl
itude
(AU
)
0200400600800
1000120014001600
% P
repu
lse
Inhi
bitio
n
0
20
40
60
80
100
Dose of DFP (mg/kg)
0.0 0.25 0.50 0.75 1.00
0.0 0.25 0.50 0.75 1.00
B
C
% P
repu
lse
Inhi
bitio
n
0102030405060708090
100 VehicleDFP 0.25 mg/kgDFP 0.50 mg/kgDFP 0.75 mg/kgDFP 1.00 mg/kg
75 dB 80 dB 85 dB
A
Prepulse Inhibition Studies
C3H7O P F
OH7C3
O
(Alternate Day Exposures)
N=11-26
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Water Maze Hidden Platform Test
Fig Source: “Fundamental Neuroscience”, Second Edition, Copyright, 2003, Academic Press
Hidden Platform
During Learning After Learning
Day of Testing1 2 3 4 5 6
Dis
tanc
e to
Pla
tform
(cm
)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 VehicleDFP 0.25 mg/kgDFP 0.50 mg/kgDFP 0.75 mg/kgDFP 1.0 mg/kg
*
A
Day of Testing1 2 3 4 5 6
Swim
Spe
ed (c
m/s
ec)
0
10
20
30
40
50B
0.0 0.25 0.50 0.75 1.00
% T
ime
in T
arge
t Qua
dran
t
0
10
20
30
40
Dose of DFP (mg/kg)
*
VehicleDFP 0.25 mg/kgDFP 0.50 mg/kg DFP 0.75 mg/kg DFP 1.00 mg/kg
C
*
Water Maze Studies C3H7O P F
OH7C3
O
N=11-26
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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
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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
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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
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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
**
*
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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
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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.
PharmacologyBao-Ling Adam, Ph.D.Dan BeckLouise Middlemore-RisherChristina WilsonBrandon HallScott Webster
Animal Behavior Patrick CallahanKristy BouchardSamantha WarnerLeah VandenhuerkRosann SchadeMark BeckmanNancy KilleDonna Blessing
Acknowledgements
Terry-Lab Personnel
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Jerry J. Buccafusco, Ph.D.(August 20, 1949 – March 6, 2010)
Regents' Professor, Department of Pharmacology and Toxicology
Director, Alzheimer's Research Center Medical College of Georgia
In Memory of:
Other Acknowledgements
Michael Bartlett, Ph.D.Dale Sickles, Ph.D.Elizabeth Herman, Ph.D.Debra Gearhart, Ph.D.Gary Schwarz, B.S.Jacob Truan, B.S.Adviye Ergul, M.D., Ph.D.NIH/NIEHS ES012241
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