anticholinesterases pose risks of acute and chronic...
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Anticholinesterases pose risks of acute andchronic neurotoxicity
The mechanism of this effect and its relationto inhibition of AChE and BChE are activelydebated.
Several anticholinesterases reduce neuriteoutgrowth in tissue culture and may bedevelopmental neurotoxicants
Timing and location of cholinesterase expression in neural development are consistent with morphogenic roles for AChE and BChE
How can anticholinesterases affect development of the nervous system?
Patterns of AChE & BChE expression in rat embryos
Image from Koenigsberger and Brimijoin, 1998
AChE expression on neurite growth cones and cell surfaces
(image from Koenigsberger/Brimijoin et al, 1997)
AChEInactiva-
tion
Pathways of Developmental NeurotoxicityI: Consequences of Inactivating Cholinesterase
ParentChemical(Metabolite
/Speciation)
DelayedResponse
Tissue/Organ Individual
Altered CellStructure
Decreasedneurite
outgrowth
Brain
Loss ofsynaptic
connections
Behavior
Impairedcognitivefunction
MolecularTarget
Alteredsynapticactivity
& receptorabundance
Acute CellularResponse
An ‘Adverse Outcome Pathway’ for one proposed type of developmental neurotoxicity. In this example low chemicalconcentrations interfere with the function of AChE as a morphogen promoting axonal growth. This may occur at chemicalconcentrations lower than those needed to inhibit the enzymatic activity of AChE and could lead to cognitive impairment.
Toxicity Pathway
Adverse Outcome Pathway
N1E.115 neuroblastomacells were stablytransfected with murineAChE cDNA in senseorientation (foroverexpression) orantisense orientation(for under-expression).Neurite outgrowth wasthen examined in culture(Koenigsberger,Brimijoin et al., 1997).
Neurite outgrowth parallels AChE activity in neuroblastoma cells engineered for high or low expression
AChE Enhances Neural Adhesion
(data from Sharma, Bigbee, Brimijoin et al, 2001)
DRG cultures
Correlation between AChE levels andneuronal adhesion
(data from Sharma et al, 2001)
Potential mechanisms for AChE-mediatedcell-substratum adhesion. Tetrameric G4AChE is anchored in the plasma membraneby a 20 kDa protein, which could potentiallysignal adhesive events between AChE andthe extracellular matrix (ECM; A). Throughthis mechanism, AChE could directly activateintracellular signaling pathways. Alternatively,AChE-mediated adhesion could stabilize orfacilitate the binding of other cell adhesionmolecules, e.g., integrins, to their ligands,leading to signal pathway activation (B). Inthis co-receptor role, AChE could alsointeract with the receptor or the ligand.
Model of AChE role in neural adhesion
From Bigbee
AChEBinding
(morphogenicsite)
Pathways of Developmental NeurotoxicityII: Interfering with AChE as “morphogen”
ParentChemical(Metabolite
/Speciation)
CellularResponse
Tissue/Organ Individual
Altered CellStructure
Decreasedneurite
outgrowth
Brain
Loss ofsynaptic
connections
Behavior
Impairedcognitivefunction
MolecularTarget
AlteredIntracellularSignaling
CaMKIMAPK
PI3K GSK3βOthers?
CellularResponse
In this example low chemical concentrations interfere with AChEfunction as a morphogen promoting axonal growth. Interferencemay occur at chemical concentrations lower than needed toinhibit the enzymatic activity of AChE
Toxicity Pathway
Adverse Outcome Pathway
Yang et al (2008) Rat DRG neuronswere treated with varyingconcentrations of CPF or CPFO for24 h in vitro, then fixed andimmunostained for the neuronalantigen PGP9.5. Representativemicrographs of neurons grown in theabsence (A) or presence (B) of CPF(0.1 μM) demonstrate that relative tovehicle controls, neurons treatedwith CPF exhibit shorter axons. CPFand CPFO did not affect the numberof axons per neuron (C), but diddecrease axon length (D).
Neurite outgrowth reduced byChlorpyrifos in concentrations thatdon’t measurably inhibit AChE
AChE-null neurons insensitive to CPF effect
Data from Yang, Lein et al, 2008
Sensitivity to CPF restored by wild type but not serine-deficient AChE
Data from Yang, Lein et al, 2008
Unresolved questions about AChE’s“morphogenic role” as a pathway for developmental neurotoxicity:
1. If the surface structure of AChE is critical for morphogenesis,why can’t a catalytically inactive mutant (i.e., serine-null) function as well?
2. If the key morphogenic feature is catalytic AChE activity why do most agents that block this activity FAIL to cause developmental or morphologic toxicity? And why do others (e.g.,) chlorpyrifos, cause such toxicity at doses NOT associated with measurable inhibition?
3. If AChE activity and AChE surface structure both participate in promotingneural morphogenesis, possibly in collaboration with the related enzyme,BChE, then why are mice genetically null for both AChE and BChE bornwith structurally normal brains???
It is very likely that some anticholinesterasepesticides and related agents cause othertypes of long-term disturbances that we couldnot predict from current understanding oftheir basic mechanisms of action.
20 30 40 50 60 70 80 90 100
0
100
200
300
400
500
600
control
chlorpyrifos
solid lines - males
dashed lines - females
0 5 10 15 20 25
0
10
20
30
40
50
60
70
control
chlorpyrifos
*
*
*
** *
A
B
body
wei
ght,
g
postnatal day
early postnatal
maturing
Male rats exposed to subtoxic 2.5 mg/kg doses of chlorpyrifosduring gestation and lactationexhibit excess weight gain,beginning at puberty.
EXAMPLE: unexpected developmental toxicity from chlorpyrifos
Data from Lassiter & Brimijoin, 2006
DNA-array studies are nowsuggesting that limited exposureto certain insecticides at “subtoxiclevels” during early developmentcan permanently alter the profileof gene expression in the brain
1
RNA metabolism neuron development
23 4 5 6
7 8
9 10 1113 5
2 3
9
8
10
1
5000gene-set size
molecularsignaling
chromosome& DNA binding
circadianclock
protein metab& recycling other
41
1
1
2
2
2 3
3
74
55
5
1
1
23
2
3
Gene pathways--Weanling brain--perinatal Chlorpyrifos
focal adhesion
Unpublished data from Lassiter and Brimijoin
molecular signaling translation,
modification mitochondrial function
2 2 23 37
12
1110
914
4
5
5
56 1 1
inflammatory response
cyclic nucleotide metabolism
2 33 24
41
1
RNA metabolismtransporter function
2
23
34
41
1
collagen
endocytosisexternal stressors
“other”
2
2
334
1
1
0
250
500
21
1 2gene-set size
Gene pathways--Adult brain--perinatal Chlorpyrifos
cell adhesion
Pathway analysis for the adult rat brains exposed to chlorpyrifos GD7-PND21Rank
Table 4
Gene set Pathway Set size % up NTk stat NTk rank NEk stat NEk rank* *Functional Categorymolecular signaling 1GO:0007599 hemostasis 93 73 4.20 56 3.20 12
6GO:0006936 muscle contraction 212 73 5.40 20 2.33 152.51112
GO:0046851GO:0007254
negative regulation of bone remodelingJNK cascade
1056
9070
2.832.69
240262
3.023.44
255
1415
GO:0030155GO:0031098
regulation of cell adhesionstress-activated protein kinase signaling pathway
7057
6368
2.332.33
379.5379.5
3.373.35
67
1718
GO:0016459GO:0030218
myosinerythrocyte differentiation
3823
7174
2.332.33
379.5379.5
3.313.19
913
24GO:0016540 protein autoprocessing 60 45 -1.88 608.5 -3.17 142829
GO:0004930GO:0046777
G-protein coupled receptor activityprotein amino acid autophosphorylation
44459
6746
5.79-1.64
13760.5
1.23-3.07
739.521
46GO:0008601 protein phosphatase type 2A regulator activity 24 63 0.28 1,987 3.10 17
translation, modification 19GO:0006493 protein amino acid O-linked glycosylation 40 68 2.33 379.5 3.13 15
41GO:0005840 ribosome 145 31 -5.90 10 -0.28 1,865
43GO:0003735 structural constituent of ribosome 136 28 -5.80 11 -0.25 1,9064849
GO:0008318GO:0018342
protein prenyltransferase activityprotein prenylation
2223
4548
-0.25-0.03
2,0152,278.5
3.093.03
1924
36GO:0031966 mitochondrial membrane 315 36 -6.19 6 -0.55 1,509mitochondrial function39GO:0005740 mitochondrial envelope 337 38 -6.19 7 -0.44 1,670.544
45
GO:0005743
GO:0019866
mitochondrial inner membrane
organelle inner membrane
275
291
36
37
-5.72
-5.78
15
14
-0.23
-0.20
1,939.5
1,98647GO:0031967 organelle envelope 482 41 -5.18 24 0.20 2,009
inflammatory response 5GO:0001906 cell killing 12 100 3.51 124 3.10 187GO:0050729 positive regulation of inflammatory response 13 77 3.28 151 3.04 22
22GO:0005125 cytokine activity 202 74 5.48 17 1.41 571.526GO:0006954 inflammatory response 237 69 5.41 19 1.34 645
cyclic nucleotide metabolism 13GO:0009187 45 64 2.33 379.5 3.56 121GO:0009975 cyclase activity 27 67 2.05 552.5 3.29 1027GO:0009190 cyclic nucleotide biosynthesis 31 68 1.75 717.5 3.52 3
37GO:0006171 cAMP biosynthesis 19 58 0.74 1,515 3.51 4
cyclic nucleotide metabolism
Fipronil, a pesticide that targets GABAA receptorsinstead of cholinesterase, also causes widespread changes in gene expression that persist into adulthood after limitedperinatal exposure in subtoxic doses.
neuron development mitochondrial function
transcription &RNA metabolism
1 2 34 5 6 7
12
35 8 10
11 13 14 1517
ribosomal functioncell adhesion& communication
DNA repair proteasome
phosphatase activity other
121
2
3
3 41 2 3
13 4
56
1 2
1 2 3 4 5 6
12
0 500
gene-set size
Gene pathways--Weanling brain--perinatal Fipronil
neuron structure/function
1 23 4 5 8
6
9
immune function
1
2 3 57
molecular signaling
12 3 54 6
steroid synthesis
1 2 3
external stressors
1 2 3
RNA polymerase
1
2
protein folding
1 2
oxidoreductase
1
2
lysosomal function
1 2
other
132
mitochondrial function
1 2 3 4 5 6
10 11
gene-set size
0 500
Gene pathways--Adult brain--perinatal Fipronil
Conclusion
Anticholinesterases may havecommon mechanisms of acutetoxicity but probably have multiplemechanisms of long-term toxicityin the nervous and endocrinesystems. Understanding theseissues should be a currentresearch priority.
Numbered pathways in each functional category correspond to the following gene ontologies (GO). RNA metabolism: 1) RNA catabolism
(6401); 2) mRNA metabolism (16071); 3) mRNA processing (6397); 4) RNA metabolism (16070); 5) RNA processing (6396); 6) nuclear mRNA
splicing (398); 7) deaminase activity (19239); 8) mRNA catabolism (6402); 9) RNA splicing (8380); 10) RNA binding (3723); 11) RNA splicing
factor activity (31202); 12) ribonucleoprotein binding (43021); 13) ribonucleoprotein complex (30529). Neuron development: 1) cell projection
biogenesis (30031); 2) regulated secretory pathway (45055); 3) neurotransmitter secretion (7269); 4) neuron remodeling (16322); 5) hindbrain
development (30902); 6) tissue regeneration (42246); 7) focal adhesion (KEGG 04510); 8) secretory pathway (45045); 9) secretion (46903); 10)
synapse (45202). Molecular signaling: 1) diacylglycerol binding (199992); 2) G-protein signaling (7189); 3) phosphatase binding (19902); 4)
protein phosphatase activity (8138); 5) adenylyl cyclase activation (7190); 6) protein phosphatase binding (19903); 7 MAP kinase kinase (4709).
Chromosome/DNA binding:1) telomerase-dependent telomere maintenance (7004); 2) chromosome (5694); 3) structure-specific DNA binding
(43566); 4) double strand DNA binding (3690); 5) chromosome organization & biogenesis (7001). Circadian clock: 1) casein kinase I activity
(4681); 2) casein kinase activity (4680); 3) circadian rhythm (KEGG 4710). Protein metabolism:1) serine endopeptidase (4252); 2) early
endosomes (5769). Other: 1) mitochondrial transport (6839); 2) anagen (42640); 3) O-methyltransferase (8171); 4) response to other organism
(51707); 5) glycerolipid biosynthesis (45017).
Gene pathways--Weanling brain--perinatal Chlorpyrifos