Monoacylglycerol Lipase Inhibition by Organophosphorus Compounds Leads to Elevation of Brain 2-Arachidonoylglycerol and the Associated Hypomotility in Mice

Toxicology and Applied Pharmacology 211 (2006) 78–83

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Monoacylglycerol lipase inhibition by organophosphorus compounds leads to elevation of brain 2-arachidonoylglycerol and the associated hypomotility in mice

Gary B. Quistad, Rebecka Klintenberg, Pierluigi Caboni, Shannon N. Liang, John E. Casida ⁎

Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, 115 Wellman Hall, Berkeley, CA 94720-3112, USA Received 27 September 2005; revised 24 October 2005; accepted 24 October 2005 Available online 28 November 2005

Abstract

Three components of the cannabinoid system are sensitive to selected organophosphorus (OP) compounds: monoacylglycerol (MAG) lipase that hydrolyzes the major endogenous agonist 2-arachidonoylglycerol (2-AG); fatty acid amide hydrolase (FAAH) that cleaves the agonist anandamide present in smaller amounts; the CB1 receptor itself. This investigation considers which component of the cannabinoid system is the most likely contributor to OP-induced hypomotility in mice. Structure–activity studies by our laboratory and others rule against major involvement of a direct toxicant–CB1 receptor interaction for selected OPs. Attention was therefore focused on the OP sensitivities of MAG lipase and FAAH, assaying 19 structurally diverse OP chemicals (pesticides, their metabolites and designer compounds) for in vitro inhibition of both enzymes. Remarkably high potency and low selectivity is observed with three O-alkyl (C1,C2,C3) alkylphosphonofluoridates (C8,C12) (IC50 0.60–3.0 nM), five S-alkyl (C5,C7, C9) and alkyl (C10,C12) benzodioxaphosphorin oxides (IC50 0.15–5.7 nM) and one OP insecticide metabolite (chlorpyrifos oxon, IC50 34–40 nM). In ip-treated mice, the OPs at 1–30 mg/kg more potently inhibit brain FAAH than MAG lipase, but FAAH inhibition is not correlated with hypomotility. However, the alkylphosphonofluoridate-treated mice show dose-dependent increases in severity of hypomotility, inhibition of MAG lipase activity and elevation of 2-AG. Moderate to severe hypomotility is accompanied by 64 to 86% MAG lipase inhibition and about 6-fold elevation of brain 2-AG level. It therefore appears that OP-induced MAG lipase inhibition leads to elevated 2-AG and the associated hypomotility. © 2005 Elsevier Inc. All rights reserved.

Keywords: 2-arachidonoylglycerol; Cannabinoid system; Fatty acid amide hydrolase; Hypomotility; Monoacylglycerol lipase; Organophosphorus

Introduction endogenous agonists are 2-arachidonoylglycerol (2-AG) and anandamide, and their levels are regulated by monoacylglycerol Organophosphorus (OP) compounds interact with several (MAG) lipase and fatty acid amide hydrolase (FAAH), components of the cannabinoid system (Fig. 1). The principal respectively (Sugiura and Waku, 2000). MAG lipase and FAAH are very sensitive to methyl arachidonylfluorophos- phonate (MAFP) with 50% inhibition at 0.1 to 2 nM (Quistad et Abbreviations: AChE, acetylcholinesterase; 2-AG, 2-arachidonoylglycerol; al., 2001; Saario et al., 2004). Several OPs inhibit agonist BDPO, benzodioxaphosphorin oxide; CB1, cannabinoid receptor; CPO, chlorpyrifos oxon; DFP, diisopropyl fluorophosphate; DMSO, dimethyl binding at the cannabinoid (CB1) receptor, but the site(s) sulfoxide; EOPF, ethyl octylphosphonofluoridate; FAAH, fatty acid amide involved and significance are not known (Martin et al., 2000; hydrolase; IC50, concentration inhibiting 50% of enzyme activity; IDFP, Casida and Quistad, 2004). Most alkylphosphonofluoridates not isopropyl dodecylfluorophosphonate; MAFP, methyl arachidonylfluoropho- only prevent binding at CB1 but also inhibit FAAH to give the sphonate; MAG, monoacylglycerol; MOPF, methyl octylphosphonofluoridate; usual cannabinoid responses in rats (Martin et al., 2000). An NTE-LysoPLA, neuropathy target esterase-lysophospholipase; OG, 1(3)- oleoylglycerol; OP, organophosphorus. exception is methyl octylphosphonofluoridate [n-C8H17P(O) ⁎ Corresponding author. Fax: +1 510 642 6497. (OCH3)F, MOPF], fully potent for cannabinoid pharmacological E-mail address: [email protected] (J.E. Casida). signs but not binding to CB1. The physiological responses to

Fig. 1. Some components of the cannabinoid system.

MOPF are unexplained but suggested due to alternate receptor cides, their metabolites and designer compounds) elevates 2- subtypes or another mechanism (Martin et al., 2000). AG in brain, thereby resulting in OP-induced hypomotility MAG lipase is increasingly viewed as a major factor in the in a mouse model. cannabinoid system (Hohmann et al., 2005), along with FAAH (McKinney and Cravatt, 2005). MAG lipase is unique Materials and methods among the lipases in using only monoacylglycerols as substrates (Karlsson et al., 2000), and the highest levels of Chemicals. Caution: some of the compounds have high acute toxicity, and MAG lipase mRNA occur in brain regions where CB1 others are delayed neurotoxicants (Casida and Quistad, 2004; Wu and receptor is expressed (Dinh et al., 2002). MAG lipase occurs Casida, 1996). They were used under careful containment conditions. The in both cytosol and cellular membranes. Rat brain MAG OPs examined were of diverse chemical structures represented by lipase is a 33-kDa protein with 92% amino acid sequence alkylphosphonofluoridates, benzodioxaphosphorin oxide (BDPO) analogs, commercial pesticides and activated metabolites. Sources for the pesticides, identity compared to mouse adipocyte MAG lipase and is their metabolites and other candidate inhibitors (N95% purity) were as related to esterases, lysophospholipases and haloperoxidases follows: 2-AG and diisopropyl fluorophosphate (DFP) from Sigma Chemical (Dinh et al., 2002; Karlsson et al., 1997). (St. Louis, MO); chlorpyrifos oxon (CPO), paraoxon, dichlorvos, chlorpyr- Cannabinoid pharmacological effects include hypomotility, ifos and tribufos from Chem Service (West Chester, PA); profenofos from also induced by injection of 2-AG and anandamide (Mechoulam Syngenta (Greensboro, NC). The other OPs were synthesized in this laboratory: isopropyl dodecylfluorophosphonate (IDFP) and decanesulfonyl et al., 1995; Quistad et al., 2001; Sulcova et al., 1998). The fluoride (Segall et al., 2003a); ethyl octylphosphonofluoridate (EOPF) (Wu relative contribution for MAG lipase and 2-AG compared to and Casida, 1995); octanesulfonyl fluoride (Wu and Casida, 1996); stearyl- FAAH and anandamide is of considerable current interest. MAG BDPO and diazoxon (Quistad et al., 2001); other BDPOs (Wu and Casida, lipase plays the major role in vivo in modulating the 1992, 1994). MOPF was prepared by our previous method for its ethyl pharmacological activity of 2-AG as shown with FAAH- analog EOPF (Wu and Casida, 1995). Glycerol coupled via carbodiimide to [14C]oleic acid (PerkinElmer Life Sciences, Boston, MA) gave 1(3)-[14C] deficient mice which hydrolyze 2-AG at the same rate as wild- oleoylglycerol (OG) and reacted with heptadecanoyl chloride (Sigma) gave type mice (Lichtman et al., 2002). RNA interference-mediated 1(3)-heptadecanoylglycerol. silencing of MAG