Article

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

Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA.
Toxicology and Applied Pharmacology (Impact Factor: 3.71). 07/2005; 205(2):149-56. DOI: 10.1016/j.taap.2004.09.018
Source: PubMed

ABSTRACT

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.

Full-text preview

Available from: berkeley.edu
  • Source
    • "Diisopropyl phosphonofluoridate and phenylmethanesulfonyl fluoride were from Sigma-Aldrich (St. Louis, MO). Compounds available from previous or analogous syntheses in this laboratory were: S-and R-octyl- BDPO, 2-methylphenyl-BDPO, mipafox (Wu and Casida, 1992); isopropyl n-dodecylphosphonofluoridate, n-dodecanesulfonyl fluoride (Segall et al., 2003a); n-octanesulfonyl fluoride (Segall et al., 2003b); ethyl n-octylphosphonofluoridate (Wu and Casida, 1995); dipentyl-dichlorvos, phenyl benzylcarbamate (Wu and Casida, 1996); and dipentyl-chlorpyrifos oxon (Quistad et al., 2005a). Oksana Lockridge provided the fluorophosphonate (FP)-biotin probe (Schopfer et al., 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography–mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.
    Full-text · Article · Nov 2008 · Toxicology and Applied Pharmacology
  • Source
    • "Diisopropyl phosphonofluoridate and phenylmethanesulfonyl fluoride were from Sigma-Aldrich (St. Louis, MO). Compounds available from previous or analogous syntheses in this laboratory were: S-and R-octyl- BDPO, 2-methylphenyl-BDPO, mipafox (Wu and Casida, 1992); isopropyl n-dodecylphosphonofluoridate, n-dodecanesulfonyl fluoride (Segall et al., 2003a); n-octanesulfonyl fluoride (Segall et al., 2003b); ethyl n-octylphosphonofluoridate (Wu and Casida, 1995); dipentyl-dichlorvos, phenyl benzylcarbamate (Wu and Casida, 1996); and dipentyl-chlorpyrifos oxon (Quistad et al., 2005a). Oksana Lockridge provided the fluorophosphonate (FP)-biotin probe (Schopfer et al., 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC50 values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined.
    Full-text · Article · Nov 2007 · Toxicology and Applied Pharmacology
  • Source
    • "Moreover profenofos and its metabolites have been determined in a case of fatal poisoning [14]. Furthermore, Quistad et al. [15] reported that profenofos became more active inhibitor of mouse brain and blood platelet-activating factor acetylhydrolase (PAF-AH) in vivo than in vitro presumably following the oxidative bioactivation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Biotransformations of profenofos were studied in vitro. Two metabolites, desthiopropylprofenofos and hydroxyprofenofos, were detected by LC–MS after incubation of profenofos with human liver homogenates and different mammalian liver microsomes. The rank order of desthiopropylprofenofos formation in liver microsomes based on intrinsic clearance (Vmax/Km) was mouse > human > rat, while for profenofos hydroxylation it was mouse > rat > human. In view of the ratio between desthiopropylation and hydroxylation intrinsic clearance rates, human liver microsomes were most active in profenofos bioactivation. The interspecies differences and interindividual variation were within range of the default uncertainty/safety factors for chemical risk assessment. CYP3A4, CYP2B6 and CYP2C19 were identified as profenofos-oxidizing enzymes in human liver on the basis of recombinant expressed enzymes and correlation with CYP model activities. The rank order of CYPs in profenofos activation was CYP3A4 > CYP2B6 > CYP2C19, whereas it was the contrary for profenofos hydroxylation. Profenofos inhibited relatively potently several human liver microsomal activities: the lowest IC50 values were about 3 μM for CYP1A1/2 and CYP2B-associated activities. Profenofos is extensively metabolized by liver microsomal CYP enzymes and its interaction potential with several CYP activities is considerable.
    Full-text · Article · Mar 2007 · Pesticide Biochemistry and Physiology
Show more