Eugenio Vilanova

Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis

The most employed insecticides for indoor and agriculture purposes belong to carbamates, pyrethroid or organophosphates. The chemical structures of these three groups correspond to carbamic, carboxylic and triphosphoric esters. Technical monographs suggest that the hydrolysis of ester bonds of carbamates and pyrethroids plays an important role in the detoxification of these compounds. However, detailed studies about enzymes hydrolysing carbamates and pyrethroids in vertebrates are not available. Certain carbamate hydrolysing activities are associated to serum albumin. Phosphotriesterases, being of an unknown physiological role, hydrolyse (in some cases stereospecifically) organophosphorus insecticides (OP). Phosphotriesterases have been found in a multitude of species, from mammals to bacteria. A phosphotriesterase activity, EDTA-resistant, has been detected in serum albumin. Phosphotriesterases in serum of mammals display polymorphisms. Phosphotriesterases offer applications in therapy of organophosphorus poisonings, in biodegradation and bioremedation of organophosphates. Similar studies should be developed with enzymes hydrolysing pyrethroids and carbamate insecticides. Such studies will improve the knowledge of the detoxification routes in non-target species and will help to design specific and safer carbamate and pyrethroid insecticides.

The role of phosphotriesterases in the detoxication of organophosphorus compounds

The enzymes that hydrolyze organophosphorus compounds are called phosphotriesterases. The presence of phosphotriesterases has been described in a variety of tissues. The physiological role of these enzymes is not known, although a clear correlation exists between the levels of phosphotriesterases and susceptibility of the species to the toxic effects of organophosphorus compounds. Thus, mammals that possess high levels of phosphotriesterases in serum and liver are more tolerant to the toxic effects of these compounds than birds and insects - these being species considered lacking of phosphotriesterases. Because most of these enzymes are not well characterized, they are usually differentiated according to their different patterns of response to activators and/ or inhibitors. Phosphotriesterases usually depend of divalent cations and therefore EDTA usually inhibits them. A peculiar EDTA-resistant phosphotriesterase has been described in serum albumin. The biotechnological and therapeutical applications of phosphotriesterases are currently subject to study.

Anomalous biochemical responses in tests of the delayed neuropathic potential of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved isomers and of some higher O-alkyl homologues.

The interaction with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) in vivo of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved stereoisomers and five higher O-alkyl homologues has been examined along with the ability of these compounds to cause organophosphorus-induced delayed polyneuropathy (OPIDP) in adult hens. For the lower homologues AChE was more sensitive than NTE and it was impossible to achieve high inhibition of NTE in vivo without both prophylaxis and therapy against acute anticholinesterase effects; for then-hexyl homologue high inhibition of NTE could be achieved without obvious anticholinesterase effects and spontaneous reactivation of inhibited AChE was seen as in vitro. The maximum tolerated dose ofl(−) methamidophos or of the ethyl oriso-propyl homologues did not inhibit NTE more than 60%, and surviving birds did not develop OPIDP. Then-propyl,n-butyl andn-hexyl compounds caused typical OPIDP at doses causing a peak of 70–95% inhibition of NTE in brain, spinal cord and sciatic nerve soon after dosing. Racemic methamidophos caused unusually mild OPIDP associated with very high inhibition of NTE at doses estimated to be >8 times the unprotected LD50 and thed-(+) isomer caused OPIDP at about 5−7× LD50. Clinical effects correlated with histopathology in 19 out of 20 examined birds. In contrast to results of many previous studies with organophosphates and phosphonates, all these cases of OPIDP were associated with formation of inhibited NTE which could be reactivated ex vivo by treatment of autopsy tissue with KF solution. It is not clear whether “aging” of inhibited NTE had occurred but with less associated stabilisation of the enzyme-phosphorus bond or whether, even without aging, the unusual N-unsubstituted phosphoramidate caused sufficient disturbance in or near the NTE target to initiate the same degenerative process as that caused typically by generation of “aged” organophosphorylated NTE.

Soluble and Participate Forms of the Organophosphorus Neuropathy Target Esterase in Hen Sciatic Nerve

Abstract: Neuropathy target esterase (NTE) is the suggested “target” molecule involved in the initiation of organophosphorus‐induced delayed polyneuropathy. Sciatic nerve NTE was separated into particulate (P‐NTE) and soluble (S‐NTE) fractions by ultracentrifugation at 100,000 g for 1 h in 0.32 M sucrose and compared with the corresponding brain extract. Total sciatic NTE activity was 80–100 nmol/min/g tissue from which 50–60% was recovered in the soluble supernatant fraction and the remaining 40–50% in the pellet fraction. About 90% of brain tissue activity (∼ 1,800 nmol/min/g tissue) was recovered as P‐NTE. A similar distribution was obtained when more drastic centrifugation without sucrose was performed. P‐NTE and S‐NTE were distributed with the membrane and cytosolic markers assayed, respectively, glucose‐6‐phosphatase, Na+,K+‐ATPase, 5′‐nucleotidase, phospholipids, and lactate dehydrogenase. When the pH during the centrifugation was increased from 6.4 to 11, recovered P‐NTE activity decreased from 1,750 to 118 nmol/min/g tissue for brain and from 31 to 12 nmol/min/g for sciatic nerve. However, S‐NTE activity and total nonfractionated control activity were only slightly affected by the same pH treatment. The distribution pattern encountered may be better understood as representing two different proteins than an equilibrium between soluble and membrane‐bound portions of a single protein, with P‐NTE activity depending on a membrane factor from which it is separated through fractionation at high pH. The titration curve corresponding to inhibition by mipafox was studied over the 0.1–200 μM range, in the presence of 40 μM paraoxon, and data obtained were fitted to models of one or two exponential mipafox‐sensitive components plus a resistant component. Mipafox‐resistant activity was 38 and 52% of total paraoxon‐resistant activity for the particulate and soluble fractions, respectively. Particle data suggest that P‐NTE contains mainly one component with I50 of ∼5.4–7.3 μM, this representing >85% of total mipafox‐sensitive activity. However, the soluble fraction data fit better to two sensitive components: high‐ and low‐mipafox‐sensitive components with I50 of 4.9 and 43 μM, representing 35 and 65% of total paraoxon‐resistant activity, respectively.

Model equations for the kinetics of covalent irreversible enzyme inhibition and spontaneous reactivation: Esterases and organophosphorus compounds

Type B carboxylesterases (acetylcholinesterases, neuropathy target esterase, serine peptidases), catalyse the hydrolysis of carboxyl-ester substrates by formation of a covalent acyl-enzyme intermediate and subsequent cleavage and release of the acyl group. Organophosphorus compounds, carbamates, and others exert their mechanism of neurotoxicity by permanent covalent organophosphorylation or carbamylation at the catalytic site of carboxylesterases. Classical kinetic studies converted the exponential kinetic equation to a logarithmic equation for graphic analysis. This process, however, does not allow analysing complex situations. In this paper, kinetic model equations are reviewed and strategies developed for the following cases: (a) single enzyme, with classical linear equation; (b) multi-enzymatic system—discriminating several inhibitor-sensitive and inhibitor-resistant components; (c) ‘ongoing inhibition’—high sensitive enzymes can be significantly inhibited during the substrate reaction time, the model equations need a correction; (d) spontaneous reactivation (de-phosphorylation)—one or several components can be simultaneously inhibited and spontaneously reactivated; (e) spontaneous reactivation from starting time with the enzyme being partly or totally inhibited; (f) aging—single enzyme can be inhibited, spontaneously reactivated and dealkylating reaction (‘aging’) simultaneously occurs; and (g) aging and spontaneous reactivation from starting time with the enzyme being partly or totally inhibited. Analysis of data using the suggested equations allows the deduction of inhibition kinetic constants and the proportions of each of the enzymatic components. Strategies for practical application of the models and for obtaining consistent kinetic parameters, using multi-steps approaches or 3D fitting, are presented.

Biochemical and clinical tests of the delayed neuropathic potential of some O-alkylO-dichlorophenyl phosphoramidate analogues of methamidophos (O,S-dimethyl phosphorothioamidate)

The interaction in vivo of four O-alkyl O-2,5-dichlorophenyl phosphoramidates with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) and their ability to cause delayed polyneuropathy in hens has been examined. Previous studies in vitro (Vilanova, Johnson & Vicedo, Pestic. Biochem. Physiol., 28 (1987) 224) had led to the prediction that these compounds would not be neuropathic but, rather, would be prophylactic agents against organophosphorus-induced delayed polyneuropathy. In vivo the effects of these esters on the enzymes differ in 2 respects from effects in vitro: (i) Relative sensitivity of the enzymes was different: thus greater than 50% of brain NTE remained 24 h after an oral dose of 15 mg/kg of the n-hexyl ester while only 10-30% of AChE remained although NTE was the more sensitive enzyme in vitro; (ii) In no case could the inhibited NTE or AChE in autopsy samples from birds dosed with any of the 4 esters be reactivated by treatment with potassium fluoride in vitro: the inhibited enzymes produced by incubation of tissue with the esters in vitro had been reactivatable. Prophylaxis, with therapy in some cases, was required to prevent acute anticholinesterase poisoning when doses were sufficient to cause high inhibition of neural NTE. Inhibition in brain was typically 5-10% more than in spinal cord and 10-15% more than in sciatic nerve. Unambiguous signs of polyneuropathy (Grade 3 or more on an 8-point scale) were not seen in birds observed up to 3 weeks after doses which caused less than 70% inhibition of NTE in brain and spinal cord or less than 60% inhibition in sciatic nerve of pair-dosed birds assayed 24 h after dosing. Doses of 300, 10, 100 and 65 mg/kg, respectively, of the methyl, ethyl, n-butyl and n-hexyl esters caused greater than 70% inhibition of NTE in all 3 neural tissues and neuropathy in the majority of observed birds. Analysis of consolidated dose/response data from 36 assayed and 51 observed birds showed that effects of Grade 3 or more were produced in about 90% of birds when inhibition of NTE was greater than 90% in brain, greater than 85% in spinal cord or greater than 75% in sciatic nerve.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphotriesterase activity identified in purified serum albumins

Abstract The phosphotriesterase in chicken serum that hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was purified in three chromatographic steps. The activity copurified to apparent homogeneity with albumin monitoring by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS/PAGE) and by SDS-capillary electrophoresis in the purified fractions. Commercial chicken serum albumin was further purified and the phosphotriesterase activity remained associated with albumin. Capillary electrophoresis established a molecular weight of 59 ± 4 kDa for both purified proteins (chicken serum and commercial chicken serum albumin). The purified samples were assayed for hydrolytic activity against several carboxylesters, organophosphates and phosphoramidates. From carboxylesters, only p-nitrophenylbutyrate ( p-NPB) hydrolysing activity was found to copurify with the phosphotriesterase. The purified human, chicken, rabbit and bovine serum albumins and recombinant human serum albumin obtained from commercial sources hydrolysed HDCP and p-NPB. Serum albumin also hydrolysed O-butyl O-2,5-dichlorophenyl phosphoramidate, O-ethyl O-2,5-dichlorophenyl phosphoramidate and O-2,5-dichlorophenyl ethylphosphonoamidate but not other organophosphates and phosphoramidates.

Chromatographic Discrimination of Soluble Neuropathy Target Esterase Isoenzymes and Related Phenyl Valerate Esterases from Chicken Brain, Spinal Cord, and Sciatic Nerve

Abstract: Neuropathy target esterase (NTE) activity is operatively defined in this work as the phenyl valerate esterase (PVase) activity resistant to 40 µM paraoxon but sensitive to 250 µM mipafox. Gel filtration chromatography with Sephacryl S‐300 of the soluble fraction from spinal cord showed two PVase peaks containing NTE activity (S‐NTE1 and S‐NTE2). The titration curve corresponding to inhibition by mipafox was studied over the 1–250 µM range, in the presence of 40 µM paraoxon. The data revealed that S‐NTE1 and S‐NTE2 have different sensitivities to mipafox with I50 (30 min) values of 1.7 and 19 µM, respectively. This was similar to the pattern observed in the soluble fraction from sciatic nerve with two components (Vo peak, or S‐NTE1; and 100‐K peak, or S‐NTE2) with different sensitivity to mipafox. However, in the brain soluble fraction, only the high‐molecular‐mass (>700‐kDa) peak or S‐NTE1 was obtained. It showed an I50 of 5.2 µM in the mipafox inhibition curve. The chromatographic profile was different on changing the pH in the subcellular fractionation. When the homogenized tissue was centrifuged at pH 6.8, the Vo peak activity decreased in the soluble fraction from these nerve tissues. This suggests that the Vo peak could be related to materials partly solubilized from membranes at higher pH. The chromatographic pattern and mipafox sensitivity suggest that the different tissues have a different NTE isoform composition. S‐NTE2 should be a different entity than S‐NTE1 and particulate NTE. The potential role of soluble forms in the mechanism of initiation or promotion of neuropathy due to organophosphorus remain unknown.

Purification and Characterization of Naturally Soluble Neuropathy Target Esterase from Chicken Sciatic Nerve by HPLC and Western Blot

Abstract: Neuropathy target esterase (NTE) activity is defined operatively as the paraoxon‐resistant mipafox‐sensitive phenyl valerate esterase activity. A preparation containing a soluble isoform (S‐NTE2) has been obtained from sciatic nerve. It was inhibited by the biotinylated organophosphorous ester S9B [1‐(saligenin cyclic phospho)‐9‐biotinyldiaminononane] in a progressive manner showing a second‐order rate constant of (3.50 ± 0.26) × 106M−1· min−1 with an I50 for 30 min of 6.6 ± 0.4 nM. S‐NTE2 was enriched 218‐fold by gel filtration followed by strong and weak anion‐exchange chromatographies in HPLC. In western blots, this enriched sample showed two bands of endogenous biotinylated polypeptides after treating the blots with streptavidin‐alkaline phosphatase complex. When the sample was treated with S9B, another biotinylated band was observed with a molecular mass of ∼56 kDa, which was not seen when the sample had been pretreated with mipafox before the S9B labeling. It was deduced that this band represents a polypeptide (identified as the S‐NTE2 protein) that is bound by both mipafox and S9B and that should be responsible for the progressive S9B inhibition. It is possible that S‐NTE2 is the target for attack by compounds that promote delayed neuropathy.

A stereospecific phosphotriesterase in hen liver and brain

O-Hexyl, O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral compound that induces delayed neuropathy in hens. This compound is hydrolyzed by a phosphotriesterase known as HDCPase in hen and rat plasma, liver and brain. We studied the stereospecificity of HDCPase in hen tissues and in human and rabbit plasma employing a chromatographic method for analysis and quantification of HDCP stereoisomers. Hen and human plasma HDCPases were not stereospecific. However, rabbit plasma showed a remarkable stereospecificity to S-(-)-HDCP. High levels of stereospecific HDCPase were found in the particulate fraction of hen liver, where S-(-)-HDCP is hydrolyzed faster than R-(+)-HDCP. However, in hen brain the stereospecificity was found in the soluble fraction, where R-(+)-HDCP is hydrolyzed faster than S-(-)-HDCP. It is concluded that liver particulate fraction must be the main tissue responsible for the HDCP stereospecific biotransformation in hens. In an oral administration, the steroisomer R-(+)-HDCP would survive after passing through the liver and would interact with acetylcholinesterase and neuropathy target esterase in the nervous system.