title: Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation.
abstract: The freshwater planarian Dugesia japonica has recently emerged as an animal model for developmental neurotoxicology and found to be sensitive to organophosphorus (OP) pesticides. While previous activity staining of D. japonica, which possess a discrete cholinergic nervous system, has shown acylthiocholine catalysis, it is unknown whether this is accomplished through an acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or a hybrid esterase and how OP exposure affects esterase activity. Here, we show that the majority of D. japonica cholinesterase (DjChE) activity departs from conventional AChE and BChE classifications. Inhibition by classic protonable amine and quaternary reversible inhibitors (ethopropazine, donepezil, tacrine, edrophonium, BW284c51, propidium) shows that DjChE is far less sensitive to these inhibitors than human AChE, suggesting discrete differences in active center and peripheral site recognition and structures. Additionally, we find that different OPs (chlorpyrifos oxon, paraoxon, dichlorvos, diazinon oxon, malaoxon) and carbamylating agents (carbaryl, neostigmine, physostigmine, pyridostigmine) differentially inhibit DjChE activity in vitro. DjChE was most sensitive to diazinon oxon and neostigmine and least sensitive to malaoxon and carbaryl. Diazinon oxon-inhibited DjChE could be reactivated by the quaternary oxime, pralidoxime (2-PAM), and the zwitterionic oxime, RS194B, with RS194B being significantly more potent. Sodium fluoride (NaF) reactivates OP-DjChE faster than 2-PAM. As one of the most ancient true cholinesterases, DjChE provides insight into the evolution of a hybrid enzyme before the separation into distinct AChE and BChE enzymes found in higher vertebrates. The sensitivity of DjChE to OPs and capacity for reactivation validate the use of planarians for OP toxicology studies.
authors.affiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093, USA.
authors.forename: Danielle
authors.identifier: http://orcid.org/0000-0002-9827-1604
authors.initials: D
authors.lastname: Hagstrom
authors.affiliation: Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, 92093, USA.
authors.forename: Hideto
authors.initials: H
authors.lastname: Hirokawa
authors.affiliation: Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, 92093, USA.
authors.forename: Limin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, 92093, USA.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Radic
authors.affiliation: Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, 92093, USA. pwtaylor@ucsd.edu.
authors.forename: Palmer
authors.initials: P
authors.lastname: Taylor
authors.affiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093, USA. emscollins@physics.ucsd.edu.|Department of Physics, University of California, San Diego, La Jolla, CA, 92093, USA. emscollins@physics.ucsd.edu.
authors.forename: Eva-Maria S
authors.initials: ES
authors.lastname: Collins
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D002091:Butyrylcholinesterase / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002801:Cholinesterase Reactivators / Q000494:pharmacology; D002802:Cholinesterases / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D023421:Models, Animal; D009943:Organophosphorus Compounds / Q000633:toxicity*; D010575:Pesticides / Q000633:toxicity*; D010932:Planarians; D013045:Species Specificity
chemical_list: D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D009943:Organophosphorus Compounds; D010575:Pesticides; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase; D002802:Cholinesterases
keywords: Acetylcholinesterase kinetics and inhibition; Fluoride and oxime reactivation; Organophosphorus pesticides; Planarians