Loss of neuropathy target esterase in mice links organophosphate exposure to hyperactivity.

Neuropathy target esterase (NTE) is involved in neural development and is the target for neurodegeneration induced by selected organophosphorus pesticides and chemical warfare agents. We generated mice with disruptions in Nte, the gene encoding NTE. Nte(-/-) mice die after embryonic day 8, and Nte(+/-) mice have lower activity of Nte in the brain and higher mortality when exposed to the Nte-inhibiting compound ethyl octylphosphonofluoridate (EOPF) than do wild-type mice. Nte(+/-) and wild-type mice treated with 1 mg per kg of body weight of EOPF have elevated motor activity, showing that even minor reduction of Nte activity leads to hyperactivity. These studies show that genetic or chemical reduction of Nte activity results in a neurological phenotype of hyperactivity in mammals and indicate that EOPF toxicity occurs directly through inhibition of Nte without the requirement for Nte gain of function or aging.

Each lipase has a unique sensitivity profile for organophosphorus inhibitors.

Lipases sensitive to organophosphorus (OP) inhibitors play critical roles in cell regulation, nutrition, and disease, but little is known on the toxicological aspects in mammals. To help fill this gap, six lipases or lipase-like proteins are assayed for OP sensitivity in vitro under standard conditions (25 degrees C, 15 min incubation). Postheparin serum lipase, lipoprotein lipase (LPL) (two sources), pancreatic lipase, monoacylglycerol (MAG) lipase, cholesterol esterase, and KIAA1363 are considered with 32 OP pesticides and related compounds. Postheparin lipolytic activity in rat serum is inhibited by 14 OPs, including chlorpyrifos oxon (IC50 50-97 nM). LPL (bovine milk and Pseudomonas) generally is less inhibited by the insecticides or activated oxons, but the milk enzyme is very sensitive to six fluorophosphonates and benzodioxaphosphorin oxides (IC50 7-20 nM). Porcine pancreatic lipase is very sensitive to dioctyl 4-nitrophenyl phosphate (IC50 8 nM), MAG lipase of mouse brain to O-4-nitrophenyl methyldodecylphosphinate (IC50 0.6 nM), and cholesterol esterase (bovine pancreas) to all of the classes of OPs tested (IC50 < 10 nM for 17 compounds). KIAA1363 is sensitive to numerous OPs, including two O-4-nitrophenyl compounds (IC50 3-4 nM). In an overview, inhibition of 28 serine hydrolases (including lipases) by eight OPs (chlorpyrifos oxon, diazoxon, paraoxon, dichlorvos, and four nonpesticides) showed that brain acetylcholinesterase is usually less sensitive than butyrylcholinesterase, liver esterase, cholesterol esterase, and KIAA1363. In general, each lipase (like each serine hydrolase) has a different spectrum of OP sensitivity, and individual OPs have unique ranking of potency for inhibition of serine hydrolases.

Blood acylpeptide hydrolase activity is a sensitive marker for exposure to some organophosphate toxicants.

Acylpeptide hydrolase (APH) unblocks N-acetyl peptides. It is a major serine hydrolase in rat blood, brain, and liver detected by derivatization with (3)H-diisopropyl fluorophosphate (DFP) or a biotinylated fluorophosphonate. Although APH does not appear to be a primary target of acute poisoning by organophosphorus (OP) compounds, the inhibitor specificity of this secondary target is largely unknown. This study fills the gap and emphasizes blood APH as a potential marker of OP exposure. The most potent in vitro inhibitors for human erythrocyte and mouse brain APH are DFP (IC(50) 11-17 nM), chlorpyrifos oxon (IC(50) 21-71 nM), dichlorvos (IC(50) 230-560 nM), naled (IC(50) 370-870 nM), and their analogs with modified alkyl substituents. (3)H-diisopropyl fluorophosphate is a potent inhibitor of mouse blood and brain APH in vivo (ED(50) 0.09-0.2 mg/kg and 0.02-0.03 mg/l for ip and vapor exposure, respectively). Mouse blood and brain APH and blood butyrylcholinesterase (BChE) are of similar sensitivity to DFP in vitro and in vivo (ip and vapor exposure), but APH inhibition is much more persistent in vivo (still >80% inhibition after 4 days). The inhibitory potency of OP pesticides in vivo in mice varies from APH selective (dichlorvos, naled, and trichlorfon), to APH and BChE selective (profenofos and tribufos), to ChE selective or nonselective (many commercial insecticides). Sarin administered ip at a lethal dose to guinea pigs inhibits blood acetylcholinesterase and BChE completely but erythrocyte APH only partially. Blood APH activity is therefore a sensitive marker for exposure to some but not all OP pesticides and chemical warfare agents.

Serine hydrolase targets of organophosphorus toxicants.

Acetylcholinesterase (AChE) is one of several hundred serine hydrolases in people potentially exposed to about 80 organophosphorus (OP) compounds important as insecticides or chemical warfare agents. The toxicology of OPs was interpreted until recently almost solely on the basis of AChE inhibition. It is assumed that each serine hydrolase has a specific function and proposed that every OP compound has a unique inhibitory profile. This review considers the progress in sifting the expanding list of potential serine hydrolase toxicological targets. About 50 serine hydrolase targets have been recognized but only a few studied thoroughly. The toxicological relevance of known secondary OP targets is established mainly from observations with humans (butyrylcholinesterase and neuropathy target esterase-lysophospholipase) and studies with mice (cannabinoid CB1 receptor, carboxylesterase, lysophospholipase and platelet activating factor acetylhydrolase) and hen eggs (arylformamidase or kynurenine formamidase). Pesticides most commonly shown to inhibit these targets in experimental vertebrates are chlorpyrifos and tribufos. Generally the levels of environmental and occupational OP pesticide exposure are well below those causing in vivo inhibition of secondary serine hydrolase targets. Although exposure to OP insecticides is decreasing from stricter regulations and the development of resistant pest strains, it will continue to some degree for decades in the future. Only two OPs are used as pharmaceuticals, i.e. echothiophate as an ophthalmic for treatment of glaucoma and metrifonate as an anthelmintic for Schistosoma (and formerly as a candidate drug for improved cognitive function in Alzheimer's disease). In safety evaluations, knowledge on known OP targets must be balanced against major gaps in current understanding since more than 75% of the serine hydrolases are essentially unknown as to OP targeting and relevance, i.e. it is not clear if they play a role in OP toxicology.

Toxicological and structural features of organophosphorus and organosulfur cannabinoid CB1 receptor ligands.

Potent cannabinoid CB1 receptor ligands include anandamide [N-(2-hydroxyethyl)arachidonamide], Delta9-tetrahydrocannabinol, and 3H-CP 55,940 at the agonist site and selected organophosphorus esters (including some pesticides) and organosulfur compounds at a proposed closely coupled "nucleophilic" site. This study considers the toxicological and structural features of alkylfluorophosphonates, benzodioxaphosphorin oxides, alkanesulfonyl fluorides, and analogs acting at the nucleophilic site. Binding at the agonist site, using3H-CP 55,940 in assays with mouse brain membranes, is inhibited byO-isopropyl dodecylfluorophosphonate (compound 2), dodecanesulfonyl fluoride (compound 14) and dodecylbenzodioxaphosphorin oxide with IC50 values of 2-11 nM. Compounds 2 and 14 are also effectivein vivo, with 84% inhibition of mouse brain CB1 binding 4 h after intraperitoneal dosage at 30 mg/kg. Compound 14-inhibited CB1 in mouse brain requires about 3-4 days for recovery of 50% activity, suggesting covalent derivatization. Delayed toxicity (mortality in 0.3-5 days) from compounds 2, 14, and octanesulfonyl fluoride (18) is more closely associated with in vivo inhibition of brain neuropathy target esterase-lysophospholipase (NTE-LysoPLA) than with that of CB1 or acetylcholinesterase. NTE-LysoPLA inhibited by sulfonyl fluorides 14 and 18 cannot "age," a proposed requirement for NTE phosphorylated by organophosphorus-delayed neurotoxicants. Several octane- and dodecanesulfonamides with N-(2-hydroxyethyl) and other substituents based on anandamide give depressed mobility and recumbent posture in mice, but the effects do not correlate with potency for CB1 inhibition in vitro. Specific toxicological responses are not clearly associated with organophosphorus- or organosulfur-induced inhibition of the proposed CB1 nucleophilic site in mouse brain. On the other hand, the most potent CB1 inhibitors examined here are also NTE-LysoPLA inhibitors and cause delayed toxicity in mice.

Cannabinoid CB1 receptor as a target for chlorpyrifos oxon and other organophosphorus pesticides.

Binding of the endocannabinoid anandamide or of Delta(9)-tetrahydrocannabinol to the agonist site of the cannabinoid receptor (CB1) is commonly assayed with [3H]CP 55,940. Potent long-chain alkylfluorophosphonate inhibitors of agonist binding suggest an additional, important and closely-coupled nucleophilic site, possibly undergoing phosphorylation. We find that the CB1 receptor is also sensitive to inhibition in vitro and in vivo by several organophosphorus pesticides and analogs. Binding of [3H]CP 55,940 to mouse brain CB1 receptor in vitro is inhibited 50% by chlorpyrifos oxon at 14 nM, chlorpyrifos methyl oxon at 64 nM and paraoxon, diazoxon and dichlorvos at 1200-4200 nM. Some 15 other organophosphorus pesticides and analogs are less active in vitro. The plant defoliant tribufos inhibits CB1 in vivo, without cholinergic poisoning signs, by 50% at 50 mg/kg intraperitoneally with a recovery half-time of 3-4 days, indicating covalent derivatization. [3H-ethyl]Chlorpyrifos oxon may be suitable for radiolabeling and characterization of this proposed nucleophilic site.

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

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.

Serine hydrolase KIAA1363: toxicological and structural features with emphasis on organophosphate interactions.

Serine hydrolase KIAA1363 is highly expressed in invasive cancer cells and is the major protein in mouse brain diethylphosphorylated by and hydrolyzing low levels of chlorpyrifos oxon (CPO) (the activated metabolite of a major insecticide). It is also the primary CPO-hydrolyzing enzyme in spinal cord, kidney, heart, lung, testis, and muscle but not liver, a pattern of tissue expression confirmed by fluorophosphonate-rhodamine labeling. KIAA1363 gene deletion using homologous recombination reduces CPO binding, hydrolysis, and metabolism 3-29-fold on incubation with brain membranes and homogenates determined with 1 nM [(3)H-ethyl]CPO and the inhibitory potency for residual CPO with butyrylcholinesterase as a biomarker. Studies with knockout mice further show that KIAA1363 partially protects brain AChE and monoacylglycerol lipase from CPO-induced in vivo inhibition. Surprisingly, mouse brain KIAA1363 and AChE are similar in in vitro sensitivity to seven methyl, ethyl, and propyl but not higher alkyl OP insecticides and analogues, prompting structural comparisons of the active sites of KIAA1363 and AChE relative to OP potency and selectivity. Homology modeling based largely on the Archaeoglobus fulgidus esterase crystal structure indicates that KIAA1363 has a catalytic triad of S191, D348, and H378, a GDSAG motif, and an oxyanion hole of H113, G114, G115, and G116. Excellent selectivity for KIAA1363 is achieved on OP structure optimization with long alkyl chain substituents suggesting that KIAA1363 has larger acyl and leaving group pockets than those of AChE. KIAA1363 reactivates faster than AChE presumably due to differences in the uncoupling of the catalytic triad His upon phosphorylation. The structural modeling of KIAA1363 helps us understand OP structure-activity relationships and the toxicological relevance of this detoxifying enzyme.

Platelet-activating factor acetylhydrolase: selective inhibition by potent n-alkyl methylphosphonofluoridates.

Platelet-activating factor (PAF) is a potent endogenous phospholipid modulator of diverse biological activities, including inflammation and shock. PAF levels are primarily regulated by PAF acetylhydrolases (PAF-AHs). These enzymes are candidate secondary targets of organophosphorus (OP) pesticides and related toxicants. Previously known OP inhibitors of other serine hydrolases were tested with PAF-AH from mouse brain and testes of established functional importance compared with the structurally different human plasma enzyme. Several key OP pesticides and their oxon metabolites were very poor inhibitors of mouse brain and human plasma PAF-AH in vitro but moderately active for mouse brain and blood PAF-AH in vivo (e.g., tribufos defoliant and profenofos insecticide, presumably following oxidative bioactivation). OP compounds were then designed for maximum in vitro potency and selectivity for mouse brain PAF-AH vs. acetylcholinesterase (AChE). Lead compounds were found in a series of benzodioxaphosphorin 2-oxides. Ultrahigh potency and selectivity were achieved with n-alkyl methylphosphonofluoridates (long-chain sarin analogs): mouse brain and testes IC50 < or = 5 nM for C(8)-C(18) analogs and 0.1-0.6 nM for C(13) and C(14) compounds; human plasma IC50 < or = 2 nM for C(13)-C(18) analogs. AChE inhibitory potency decreased as chain length increased with maximum brain PAF-AH/AChE selectivity (>3000-fold) for C(13)-C(18) compounds. The toxicity of i.p.-administered PAF (LD50 ca. 0.5 mg/kg) was increased less than 2-fold by pretreatment with tribufos or the C(13)n-alkyl methylphosphonofluoridate. These studies with a mouse model indicate that PAF-AH is not a major secondary target of OP pesticide poisoning. The optimized PAF-AH inhibitors may facilitate investigations on other aspects of PAF metabolism and action.

A brain detoxifying enzyme for organophosphorus nerve poisons.

Organophosphorus (OP) insecticides and chemical warfare agents act primarily by inhibiting acetylcholinesterase. There are many secondary targets for OP toxicants as observed for example with the major insecticide chlorpyrifos and its bioactivated metabolite chlorpyrifos oxon (CPO). Therefore, it was surprising that the predominant mouse brain protein labeled in vitro by [(3)H-ethyl]CPO (1 nM) (designated CPO-binding protein or CPO-BP) is not one of these known OP toxicant targets. CPO-BP is a 50-kDa membrane-bound serine hydrolase measured by derivatization with [(3)H]CPO and SDS/PAGE or filtration binding assay. It appears to undergo rapid diethylphosphorylation by [(3)H]CPO followed by either dephosphorylation and reactivation or aging on loss of an ethyl group. CPO and several other OP toxicants potently inhibit CPO-BP in vivo (i.p., 2 h) (50% inhibition at 2-25 mg/kg) and in vitro (50% inhibition at 8-68 nM). Using three chemical labeling reagents, i.e., [(3)H]CPO and the activity-based proteomic probes fluorophosphonate-biotin and fluorophosphonate-rhodamine, mouse brain CPO-BP is identified as serine hydrolase KIAA1363 of unknown function. Brains from KIAA1363(-/-) mice show greatly reduced levels of CPO labeling and hydrolytic metabolism compared to brains from wild-type mice. KIAA1363 therefore is the principal enzyme for metabolizing low levels of CPO in brain and may play a more general role in detoxification of OP nerve poisons.

Lysophospholipase inhibition by organophosphorus toxicants.

Lysophospholipases (LysoPLAs) are a large family of enzymes for removing lysophospholipids from cell membranes. Potent inhibitors are needed to define the importance of LysoPLAs as targets for toxicants and potential therapeutics. This study considers organophosphorus (OP) inhibitors with emphasis on mouse brain total LysoPLA activity relative to the mipafox-sensitive neuropathy target esterase (NTE)-LysoPLA recently established as 17% of the total activity and important in the action of OP delayed toxicants. The most potent inhibitors of total LysoPLA in mouse brain are isopropyl dodecylphosphonofluoridate (also for LysoPLA of Vibrio bacteria), ethyl octylphosphonofluoridate (EOPF), and two alkyl-benzodioxaphosphorin 2-oxides (BDPOs)[(S)-octyl and dodecyl] (IC50 2-8 nM). OP inhibitors acting in vitro and in vivo differentiate a more sensitive portion but not a distinct NTE-LysoPLA compared with total LysoPLA activity. For 10 active inhibitors, NTE-LysoPLA is 17-fold more sensitive than total LysoPLA, but structure-activity comparisons give a good correlation (r(2) = 0.94) of IC50 values, suggesting active site structural similarity or identity. In mice 4 h after intraperitoneal treatment with discriminating doses, EOPF, tribufos (a plant defoliant), and dodecanesulfonyl fluoride inhibit 41-57% of the total brain LysoPLA and 85-99% of the NTE-LysoPLA activity. Total LysoPLA as well as NTE-LysoPLA is decreased in activity in Nte(+/-)-haploinsufficient mice compared to their Nte(+/+) littermates. The lysolecithin level of spinal cord but not brain is elevated significantly following EOPF treatment (3 mg/kg), thereby focusing attention on localized rather than general alterations in lysophospholipid metabolism in OP-induced hyperactivity and toxicity.

Chlorpyrifos oxon potentiates diacylglycerol-induced extracellular signal-regulated kinase (ERK 44/42) activation, possibly by diacylglycerol lipase inhibition.

Chlorpyrifos oxon (CPO) activates extracellular signal-regulated kinase (ERK 44/42) in Chinese hamster ovary (CHOK1) cells but the mechanism is not defined. This study tests the hypothesis that diacylglycerol (DAG) is the secondary messenger responsible for CPO-induced ERK 44/42 activation. It is known that DAG is sequentially hydrolyzed by DAG lipase and monoacylglycerol (MAG) lipase, both of which are organophosphate sensitive. Inhibition of these enzymes might therefore lead to the accumulation of DAG and MAG, of which only DAG is a secondary messenger. The experiments show that treatment of CHOK1 cells with CPO significantly inhibits DAG/MAG lipase activity and elevates cellular DAG levels. Pretreatment of CHOK1 cells with CPO or a carbamate known to be a DAG lipase inhibitor, followed by treatment with a cell-permeable DAG (1,2-dihexanoyl-sn-glycerol), results in synergistic activation of ERK 44/42. CPO-potentiated DAG-induced ERK 44/42 activation is both time and concentration dependent. This activation is blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase, suggesting that these enzymes are important in CPO/DAG cellular signaling. Activation by a stable DAG analogue (phorbol ester) was not altered by CPO, suggesting that DAG metabolism is the probable target for CPO-potentiated DAG-induced ERK 44/42 activation. These observations support the hypothesis that CPO potentiates DAG signaling in CHOK1 cells by inhibiting a CPO-sensitive DAG lipase, thereby providing a potential mechanism of toxicity not associated with acetylcholinesterase inhibition.

Selective inhibitors of fatty acid amide hydrolase relative to neuropathy target esterase and acetylcholinesterase: toxicological implications.

Fatty acid amide hydrolase (FAAH) plays an important role in nerve function by regulating the action of endocannabinoids (e.g., anandamide) and hydrolyzing a sleep-inducing factor (oleamide). Several organophosphorus pesticides and related compounds are shown in this study to be more potent in vivo inhibitors of mouse brain FAAH than neuropathy target esterase (NTE), raising the question of the potential toxicological relevance of FAAH inhibition. These FAAH-selective compounds include tribufos and (R)-octylbenzodioxaphosphorin oxide with delayed neurotoxic effects in mice and hens plus several organophosphorus pesticides (e.g., fenthion) implicated as delayed neurotoxicants in humans. The search for a highly potent and selective inhibitor for FAAH relative to NTE for use as a toxicological probe culminated in the discovery that octylsulfonyl fluoride inhibits FAAH by 50% at 2 nM in vitro and 0.2 mg/kg in vivo and NTE is at least 100-fold less sensitive in each case. More generally, the studies revealed 12 selective in vitro inhibitors for FAAH (mostly octylsulfonyl and octylphosphonyl derivatives) and 9 for NTE (mostly benzodioxaphosphorin oxides and organophosphorus fluoridates). The overall in vivo findings with 16 compounds indicate the expected association of AChE inhibition with acute or cholinergic syndrome and >70% brain NTE inhibition with delayed neurotoxic action. Surprisingly, 75-99% brain FAAH inhibition does not lead to any overt neurotoxicity or change in behavior (other than potentiation of exogenous anandamide action). Thus, FAAH inhibition in mouse brain does not appear to be a primary target for organophosphorus pesticide-induced neurotoxic action (cholinergic or intermediate syndrome or delayed neurotoxicity).

Evidence that mouse brain neuropathy target esterase is a lysophospholipase.

Neuropathy target esterase (NTE) is inhibited by several organophosphorus (OP) pesticides, chemical warfare agents, lubricants, and plasticizers, leading to OP-induced delayed neuropathy in people (>30,000 cases of human paralysis) and hens (the best animal model for this demyelinating disease). The active site region of NTE as a recombinant protein preferentially hydrolyzes lysolecithin, suggesting that this enzyme may be a type of lysophospholipase (LysoPLA) with lysolecithin as its physiological substrate. This hypothesis is tested here in mouse brain by replacing the phenyl valerate substrate of the standard NTE assay with lysolecithin for an "NTE-LysoPLA" assay with four important findings. First, NTE-LysoPLA activity, as the NTE activity, is 41-45% lower in Nte-haploinsufficient transgenic mice than in their wild-type littermates. Second, the potency of six delayed neurotoxicants or toxicants as in vitro inhibitors varies from IC50 0.02 to 13,000 nM and is essentially the same for NTE-LysoPLA and NTE (r2 = 0.98). Third, the same six delayed toxicants administered i.p. to mice at multiple doses inhibit brain NTE-LysoPLA and NTE to the same extent (r2 = 0.90). Finally, their in vivo inhibition of brain NTE-LysoPLA generally correlates with delayed toxicity. Therefore, OP-induced delayed toxicity in mice, and possibly the hyperactivity associated with NTE deficiency, may be due to NTE-LysoPLA inhibition, leading to localized accumulation of lysolecithin, a known demyelinating agent and receptor-mediated signal transducer. This mouse model has some features in common with OP-induced delayed neuropathy in hens and people but differs in the neuropathological signs and apparently the requirement for NTE aging.

Major intermediates in organophosphate synthesis (PCl3, POCl3, PSCl3, and their diethyl esters) are anticholinesterase agents directly or on activation.

Three phosphotrichlorides [phosphorus trichloride (PCl(3)), phosphorus oxychloride (POCl(3)), and thiophosphoryl chloride (PSCl(3))] with an annual U.S. production of >500,000,000 pounds and their diethyl esters are intermediates in the production of organophosphorus pesticides, plastics, flame retardants, and hydraulic fluids. They are classified as highly toxic to mammals based on acute oral and inhalation data with rats. This study considers their mechanisms of toxicity. PCl(3) and POCl(3) inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) from several species with in vitro IC(50) values of 5-36 and 88-1200 microM, respectively; PSCl(3) is a less potent inhibitor. These phosphotrichlorides have high vapor toxicity to houseflies with in vivo inhibition of brain AChE activity correlating with mortality. PCl(3) and POCl(3) produce cholinergic poisoning signs on ip administration to mice, and all three phosphotrichlorides give marked in vivo inhibition of serum BChE but not brain AChE activity. PCl(3) is a direct acting AChE inhibitor. Our earlier proposed activation of POCl(3) is confirmed here by preparing pure Cl(2)P(O)OH and its potassium and dicyclohexylamine salts that reproduce the action of POCl(3) as in vitro AChE inhibitors and toxicants in mice. PSCl(3) on hydrolysis yields Cl(2)P(O)SH [which oxidizes with peracid to Cl(2)P(O)SOH] as the proposed activation product. Vapors of (EtO)(2)PCl, (EtO)(2)P(O)Cl, and (EtO)(2)P(S)Cl are lethal to houseflies as in vivo AChE inhibitors, the first two acting directly and the last one on oxidative activation to (EtO)(2)P(O)Cl (possibly by P450) or (EtO)(2)P(O)SCl (a phosphorylating agent in a peracid oxidation system). Thus PCl(3), (EtO)(2)PCl, and (EtO)(2)P(O)Cl act directly as AChE inhibitors whereas POCl(3) and PSCl(3) undergo hydrolytic activation and (EtO)(2)P(S)Cl undergoes oxidative activation. In contrast, the toxicity to mice of phosphofluorides [FP(O)Cl(2), F(Cl)P(O)OH, and F(2)P(O)OH; studied as model compounds for comparison] may be due to liberating fluoride ion.

Arachidonylsulfonyl derivatives as cannabinoid CB1 receptor and fatty acid amide hydrolase inhibitors.

Arachidonylsulfonyl fluoride (3), reported here for the first time, is similar in potency to its known methyl arachidonylfluorophosphonate (2) analogue as an inhibitor of mouse brain fatty acid amide hydrolase activity (IC(50) 0.1 nM) and cannabinoid CB1 agonist [3H]CP 55,940 binding (IC(50) 304-530 nM). Interestingly, 3 is much more selective than 2 as an inhibitor for fatty acid amide hydrolase relative to acetylcholinesterase, butyrylcholinesterase and neuropathy target esterase. N-(2-Hydroxyethyl)arachidonylsulfonamide (4) is at least 2500-fold less potent than N-(2-hydroxyethyl)arachidonamide (anandamide) (1) at the CB1 agonist site.