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