Anticholinesterases pose risks of acute and chronic neurotoxicity

1. Anticholinesterases pose risks of acute and chronic neurotoxicity Several anticholinesterases reduce neurite outgrowth in tissue culture and may be developmental neurotoxicants The mechanism of this effect and its relation to inhibition of AChE and BChE are actively debated.

2. How can anticholinesterases affect development of the nervous system? Timing and location of cholinesterase expression in neural development are consistent with morphogenic roles for AChE and BChE

3. Patterns of AChE & BChE expression in rat embryos Image from Koenigsberger and Brimijoin, 1998

4. AChE expression on neurite growth cones and cell surfaces (image from Koenigsberger/Brimijoin et al, 1997)

5. Pathways of Developmental NeurotoxicityI: Consequences of Inactivating Cholinesterase Toxicity Pathway Adverse Outcome Pathway Molecular Target Acute Cellular Response Delayed Response Tissue/ Organ Individual Parent Chemical (Metabolite/ Speciation) AChE Inactiva- tion Altered synaptic activity & receptor abundance Altered Cell Structure Decreased neurite outgrowth Brain Loss of synaptic connections Behavior Impaired cognitive function 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.

6. Neurite outgrowth parallels AChE activity in neuroblastoma cells engineered for high or low expression 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).

7. AChE Enhances Neural Adhesion DRG cultures (data from Sharma, Bigbee, Brimijoin et al, 2001)

8. Correlation between AChE levels and neuronal adhesion (data from Sharma et al, 2001)

9. Model of AChE role in neural adhesion 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. From Bigbee

10. Pathways of Developmental NeurotoxicityII: Interfering with AChE as “morphogen” Toxicity Pathway Adverse Outcome Pathway Molecular Target Cellular Response Cellular Response Tissue/ Organ Individual Parent Chemical (Metabolite/ Speciation) AChE Binding (morphogenic site) Altered Intracellular Signaling CaMKI MAPK PI3K GSK3β Others? Altered Cell Structure Decreased neurite outgrowth Brain Loss of synaptic connections Behavior Impaired cognitive function 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

11. Neurite outgrowth reduced by Chlorpyrifos in concentrations that don’t measurably inhibit AChE 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).

12. AChE-null neurons insensitive to CPF effect Data from Yang, Lein et al, 2008

13. Sensitivity to CPF restored by wild type but not serine-deficient AChE Data from Yang, Lein et al, 2008

14. 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 promoting neural morphogenesis, possibly in collaboration with the related enzyme, BChE, then why are mice genetically null for both AChE and BChE born with structurally normal brains???

15. 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.

16. EXAMPLE: unexpected developmental toxicity from chlorpyrifos early postnatal Male rats exposed to subtoxic 2.5 mg/kg doses of chlorpyrifos during gestation and lactation exhibit excess weight gain, beginning at puberty. body weight, g maturing Data from Lassiter & Brimijoin, 2006 postnatal day

17. 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

18. gene-set size 0 500 focal adhesion RNA metabolism neuron development 10 9 8 7 13 5 11 10 3 2 9 1 6 5 4 8 3 2 1 protein metab & recycling other molecular signaling chromosome & DNA binding circadian clock 7 5 2 4 3 5 1 3 5 1 2 4 3 2 2 1 3 2 1 1 Gene pathways--Weanling brain--perinatal Chlorpyrifos Unpublished data from Lassiter and Brimijoin

19. cell adhesion molecular signaling translation, modification mitochondrial function 5 12 5 4 3 3 10 2 2 7 1 2 1 6 1 4 11 5 9 inflammatory response cyclic nucleotide metabolism transporter function RNA metabolism 1 4 4 3 3 2 4 2 3 2 1 1 4 3 1 2 collagen external stressors “other” endocytosis 4 2 3 3 1 2 1 gene-set size 1 2 2 1 Gene pathways--Adult brain--perinatal Chlorpyrifos

20. Table 4 Pathway analysis for the adult rat brains exposed to chlorpyrifos GD7-PND21 Gene set Pathway Rank Set size % up Functional Category NTk stat NTk rank NEk stat * NEk rank * molecular signaling GO:0007599 hemostasis 1 93 73 4.20 56 3.20 12 73 5.40 20 2.33 152.5 GO:0006936 muscle contraction 6 212 3.02 25 90 2.83 240 negative regulation of bone remodeling 10 11 GO:0046851 3.44 5 70 2.69 262 JNK cascade 56 12 GO:0007254 3.37 6 63 2.33 379.5 regulation of cell adhesion 70 14 GO:0030155 3.35 7 68 2.33 379.5 stress-activated protein kinase signaling pathway 57 15 GO:0031098 3.31 9 71 2.33 379.5 myosin 38 17 GO:0016459 3.19 13 74 2.33 379.5 erythrocyte differentiation 23 18 GO:0030218 -3.17 14 45 -1.88 608.5 protein autoprocessing 60 24 GO:0016540 1.23 739.5 67 5.79 13 G-protein coupled receptor activity 444 28 GO:0004930 -3.07 21 46 -1.64 760.5 protein amino acid autophosphorylation 59 29 GO:0046777 3.10 17 63 0.28 1,987 protein phosphatase type 2A regulator activity 24 46 GO:0008601 translation, modification 68 2.33 379.5 3.13 15 GO:0006493 protein amino acid O-linked glycosylation 19 40 -0.28 1,865 31 -5.90 10 ribosome 145 41 GO:0005840 -0.25 1,906 28 -5.80 11 structural constituent of ribosome 136 43 GO:0003735 3.09 19 45 -0.25 2,015 protein prenyltransferase activity 22 48 GO:0008318 3.03 24 48 -0.03 2,278.5 protein prenylation 23 49 GO:0018342 -0.55 1,509 36 -6.19 6 mitochondrial membrane 315 mitochondrial function 36 GO:0031966 -0.44 1,670.5 38 -6.19 7 mitochondrial envelope 337 39 GO:0005740 -0.23 1,939.5 36 -5.72 15 mitochondrial inner membrane 275 44 GO:0005743 -0.20 1,986 37 -5.78 14 organelle inner membrane 291 45 GO:0019866 0.20 2,009 41 -5.18 24 organelle envelope 482 47 GO:0031967 100 3.51 124 3.10 18 inflammatory response GO:0001906 cell killing 5 12 3.04 22 77 3.28 151 positive regulation of inflammatory response 13 7 GO:0050729 1.41 571.5 74 5.48 17 cytokine activity 202 22 GO:0005125 1.34 645 69 5.41 19 inflammatory response 237 26 GO:0006954 cyclic nucleotide metabolism 64 2.33 379.5 3.56 1 GO:0009187 cyclic nucleotide metabolism 13 45 3.29 10 67 2.05 552.5 cyclase activity 27 21 GO:0009975 3.52 3 68 1.75 717.5 cyclic nucleotide biosynthesis 31 27 GO:0009190 3.51 4 58 0.74 1,515 cAMP biosynthesis 19 37 GO:0006171

21. Fipronil, a pesticide that targets GABAA receptors instead of cholinesterase, also causes widespread changes in gene expression that persist into adulthood after limited perinatal exposure in subtoxic doses.

22. 6 5 3 4 2 6 1 5 2 1 1 3 4 2 3 1 0 500 mitochondrial function neuron development 6 7 5 4 17 1 3 15 14 2 13 11 8 10 5 3 2 1 cell adhesion & communication transcription & RNA metabolism ribosomal function 3 other phosphatase activity DNA repair proteasome 4 2 3 2 1 1 gene-set size 2 1 Gene pathways--Weanling brain--perinatal Fipronil

23. immune function neuron structure/function molecular signaling 1 7 10 11 6 5 4 3 9 5 2 2 5 3 3 4 8 1 2 1 6 RNA polymerase external stressors steroid synthesis mitochondrial function 6 5 4 3 3 2 2 1 1 1 3 2 2 1 oxidoreductase protein folding lysosomal function other 2 1 1 2 3 2 2 gene-set size 1 1 0 500 Gene pathways--Adult brain--perinatal Fipronil

24. 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.

25. Gene pathways--Weanling brain--perinatal Chlorpyrifos