Anticholinesterases pose risks of acute and chronic neurotoxicity

Anticholinesterases pose risks of acute and chronic neurotoxicity

The mechanism of this effect and its relationto inhibition of AChE and BChE are actively debated.

Several anticholinesterases reduce neurite outgrowth in tissue culture and may be developmental 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?

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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

Parent Chemical (Metabolite/Speciation)

DelayedResponse

Tissue/Organ Individual

Altered Cell Structure

Decreased neurite

outgrowth

Brain

Loss of synaptic

connections

Behavior

Impaired cognitive function

MolecularTarget

Altered synaptic activity

& receptorabundance

Acute CellularResponse

An ‘Adverse Outcome Pathway’ for one proposed type of developmental neurotoxicity. In this example low chemical concentrations interfere with the function of AChE as a morphogen promoting axonal growth. This may occur at chemical concentrations lower than those needed to inhibit the enzymatic activity of AChE and could lead to cognitive impairment.

Toxicity Pathway

Adverse Outcome Pathway



N1E.115 neuroblastoma cells were stably transfected with murine AChE cDNA in sense orientation (for overexpression) or antisense orientation (for under-expression). Neurite outgrowth was then 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 and neuronal adhesion

(data from Sharma et al, 2001)



Potential mechanisms for AChE-mediated cell-substratum adhesion. Tetrameric G4 AChE is anchored in the plasma membrane by a 20 kDa protein, which could potentially signal adhesive events between AChE and the extracellular matrix (ECM; A). Through this mechanism, AChE could directly activate intracellular signaling pathways. Alternatively, AChE-mediated adhesion could stabilize or facilitate the binding of other cell adhesion molecules, e.g., integrins, to their ligands, leading to signal pathway activation (B). In this co-receptor role, AChE could also interact with the receptor or the ligand.

Model of AChE role in neural adhesion

From Bigbee

AChEBinding

(morphogenic site)

Pathways of Developmental NeurotoxicityII: Interfering with AChE as “morphogen”

Parent Chemical (Metabolite/Speciation)

CellularResponse

Tissue/Organ Individual

Altered Cell Structure

Decreased neurite

outgrowth

Brain

Loss of synaptic

connections

Behavior

Impaired cognitive function

MolecularTarget

Altered IntracellularSignaling

CaMKI MAPKPI3K GSK3β

Others?

CellularResponse

In this example low chemical concentrations interfere with AChE function as a morphogen promoting axonal growth. Interference may occur at chemical concentrations lower than needed to inhibit the enzymatic activity of AChE

Toxicity Pathway

Adverse Outcome Pathway



Yang et al (2008) Rat DRG neurons were treated with varying concentrations of CPF or CPFO for 24 h in vitro, then fixed and immunostained for the neuronal antigen PGP9.5. Representative micrographs of neurons grown in the absence (A) or presence (B) of CPF (0.1 μM) demonstrate that relative to vehicle controls, neurons treated with CPF exhibit shorter axons. CPF and CPFO did not affect the number of axons per neuron (C), but did decrease axon length (D).

Neurite outgrowth reduced by Chlorpyrifos in concentrations that don’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 anticholinesterase pesticides and related agents cause other types of long-term disturbances that we could not predict from current understanding of their basic mechanisms of action.

20 30 40 50 60 70 80 90 100

0

100

200

300

400

500

600

controlchlorpyrifos

solid lines - malesdashed lines - females

0 5 10 15 20 250

10

20

30

40

50

60

70

controlchlorpyrifos

**

*

** *

A

B

bo

dy

wei

gh

t, 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 now suggesting that limited exposure to certain insecticides at “subtoxic levels” during early development can permanently alter the profile of gene expression in the brain

1

RNA metabolism neuron development

23 4 5 6

7 8

910 11

13 52 3

9

8

10

1

5000

gene-set size

molecularsignaling

chromosome& DNA binding

circadianclock

protein metab& recycling other

41

1

1

2

2

2 3

3

74

55

5

1

1

2

3

2

3

Gene pathways--Weanling brain--perinatal Chlorpyrifos

focal adhesion

Unpublished data from Lassiter and Brimijoin

molecular signaling translation,

modification mitochondrial function

22 23 3

7

12

1110

91

4

4

5

5

5

6 1 1

inflammatory response

cyclic nucleotide metabolism

2 33 24

4

1

1

RNA metabolismtransporter function

2

2

3

3

4

41

1

collagen

endocytosis

external stressors“other”

2

2

334

1

1

0

25

0

50

0

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 Category

molecular signaling 1GO:0007599 hemostasis 93 73 4.20 56 3.20 12

6GO:0006936 muscle contraction 212 73 5.40 20 2.33 152.5

11

12

GO:0046851

GO:0007254

negative regulation of bone remodeling

JNK cascade

10

56

90

70

2.83

2.69

240

262

3.02

3.44

25

5

14

15

GO:0030155

GO:0031098

regulation of cell adhesion

stress-activated protein kinase signaling pathway

70

57

63

68

2.33

2.33

379.5

379.5

3.37

3.35

6

7

17

18

GO:0016459

GO:0030218

myosin

erythrocyte differentiation

38

23

71

74

2.33

2.33

379.5

379.5

3.31

3.19

9

13

24GO:0016540 protein autoprocessing 60 45 -1.88 608.5 -3.17 14

28

29

GO:0004930

GO:0046777

G-protein coupled receptor activity

protein amino acid autophosphorylation

444

59

67

46

5.79

-1.64

13

760.5

1.23

-3.07

739.5

21

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 1,906

48

49

GO:0008318

GO:0018342

protein prenyltransferase activity

protein prenylation

22

23

45

48

-0.25

-0.03

2,015

2,278.5

3.09

3.03

19

24

36GO:0031966 mitochondrial membrane 315 36 -6.19 6 -0.55 1,509mitochondrial function

39GO:0005740 mitochondrial envelope 337 38 -6.19 7 -0.44 1,670.5

44

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

1,986

47GO: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 18

7GO:0050729 positive regulation of inflammatory response 13 77 3.28 151 3.04 22

22GO:0005125 cytokine activity 202 74 5.48 17 1.41 571.5

26GO:0006954 inflammatory response 237 69 5.41 19 1.34 645

cyclic nucleotide metabolism 13GO:0009187 45 64 2.33 379.5 3.56 1

21GO:0009975 cyclase activity 27 67 2.05 552.5 3.29 10

27GO: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

12

3

4 5 6 7

1

23

5 8 10

11 1314 15

17

ribosomal functioncell adhesion& communication

DNA repair proteasome

phosphatase activity other

12

1

2

3

3 41 2 3

13 4

56

1 2

1 2 3 4 5 6

1

2

0 500

gene-set size

Gene pathways--Weanling brain--perinatal Fipronil

neuron structure/function

1 23 4

58

6

9

immune function

1

2 3 57

molecular signaling

1

23 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 have common mechanisms of acute toxicity but probably have multiple mechanisms of long-term toxicity in the nervous and endocrine systems. Understanding these issues should be a current research 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

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Anticholinesterases pose risks of acute and chronic neurotoxicity