Martin K. Johnson

Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential

The assay of neurotoxic esterase (NTE) in brains taken from dosed hens enables potential neurotoxicity of organophosphate pesticides, plasticers, etc. to be assessed. The original assay [Johnson, M. K. Biochem. J. 114, 711–717 (1969)] has been simplified to eliminate centrifugation and transfer steps and both the selectivity and the sensitivity have been increased. The procedures necessary to obtain stable reagent stocks are described.ZusammenfassungDurch Bestimmung der neurotoxischen Esterase (NTE) ist es möglich, im Gehirn von mit phosphororganischen Pflanzenschutzmitteln, Weichmachern und anderen Stoffen behandelten Hühnern die potentielle Neurotoxizität dieser Stoffe zu erfassen. Die ursprüngliche Methode [Johnson, M. K. Biochem. J. 114, 711–717 (1969)] wurde vereinfacht, so daß Zentrifugieren und Transferschritte nicht mehr erforderlich sind. Die Selektivität und Empfindlichkeit der Methode wurde verbessert. Die Herstellung stabiler Reagentienstammlösungen wird beschrieben.

Organophosphorus esters causing delayed neurotoxic effects

Mechanism of ActionEvidence is reviewed that the initial biochemical event leading to delayed neurotoxicity is phosphorylation of the active site of a specific enzyme called Neurotoxic Esterase. This is followed by a bondcleavage (? hydrolytic) leading to formation of a mono-substituted phosphoric acid residue on the protein. The mechanism by which some phosphinates protect hens against neurotoxic compounds is explained.Screening AssayAssay of effects of compounds on Neurotoxic Esterase activity of hen brain in vitro and in vivo provides a quick biochemical screen to supplement the 3-week clinical test. This test provides an estimate of safety margin for compounds which give negative results in the clinical test and are currently used as pesticides, plasticisers, etc. Simplified assay procedures are being developed.Structure/Activity StudiesData is now available for the biochemical and neurotoxic activity of many compounds. This provides a basis for structure/activity predictions; neurotoxicity data published since 1930 has been assessed in this light.ZusammenfassungWirkungsmechanismusDie Beweisführung nimmt an, daß die Phosphorylierung des aktiven Zentrums eines spezifischen Enzyms, “neurotoxische Esterase” genannt, das initiale biochemische Ereignis der zur verzögerten Neurotoxizität führenden Reaktionsfolge ist. Darauf folgt die Spaltung einer Bindung (hydrolytisch?) die einen monosubstituierten Phosphorsäurerest am Protein hinterläßt. — Der Mechanismus, auf dem die Schutzwirkung einiger Phosphonsäureester gegenüber neurotoxischen Substanzen beruht, wird erläutert.Screening-MethodeDie Bestimmung der Wirkung auf die Aktivität der “neurotoxischen Esterase” im Hühnergehirn (in vitro und in vivo) stellt eine schnelle biochemische Probe zur Ergänzung des 3wöchigen klinischen Tests dar. Der Test erlaubt die Abschätzung von Sicherheitsgrenzen für Substanzen, die negative Ergebnisse im klinischen Test erbringen und häufig als Pestizide, Weichmacher usw. verwendet werden. Vereinfachte Bestimmungsmethoden wurden entwickelt.Struktur-Wirkungs-EeziehungenFür viele Verbindungen liegen Daten über die biochemische und neurotoxische Wirkung vor. Diese dienen als Basis für Vorhersagen von Struktur-Wirkungs-Beziehungen. Die seit 1930 veröffentlichten Daten zur Neurotoxizität werden unter diesem Gesichtspunkt behandelt.

Organophosphates and delayed neuropathy—Is NTE alive and well?

Neuropathy target esterase (NTE) is a membrane-bound protein with high esterase catalytic activity. The physiological function of the protein is not known and the catalytic activity is not essential to health of nerve axons. Nevertheless there is overwhelming evidence that modification of the structure of NTE by covalent binding of some organophosphorus esters initiates an irreversible polyneuropathy: this event can be monitored. The experimental evidence for this conclusion is reviewed and some conceptual objections are resolved. Studies of NTE have generated successful predictions concerning (1) prophylaxis; (2) structure-activity relationships including stereospecificity; (3) the effects of prolonged low-level administration of neurotoxicants; and (4) extrapolations from (a) NTE responses seen after low doses to enzyme and clinical effects seen after high doses, (b) from in vitro to in vivo, and (c) from hen to human responses. The relationship of initiation on NTE to subsequent events in development of neuropathy is considered. Purification of NTE is reaching the point where antibodies may be obtained for neurobiological study. No single rigid protocol can be devised for incorporation of NTE assays into toxicological evaluations. A proposed two-stage procedure requires interpretation of Stage 1 to influence the design of Stage 2.

Neurotoxicity of organophosphorus pesticides: predictions can be based on in vitro studies with hen and human enzymes.

The comparative inhibitory power of organophosphorus esters in vitro against hen brain acetylcholinesterase and neurotoxic esterase correlates with their comparative effects (death or delayed neuropathy) in vivo. Further comparisons of the in vitro effects seen with hen and human enzymes facilitates extrapolations to the human in vivo situation.

Structure-activity relationships for substrates and inhibitors of hen brain neurotoxic esterase

Abstract (1) Neurotoxic esterase is one of several paraoxon-resistant esterases of hen brain. It has previously been assayed with phenyl phenylacetate as substrate by a differential assay using Mipafox as selective inhibitor. The Mipafox-sensitive activity is a greater proportion (55 per cent) of the total when phenyl valerate is used as substrate and this activity behaves as a single enzyme according to several tests. Phenyl esters of other acids and esters of other phenols are less specific substrates and most are hydrolysed slower than phenyl valerate. (2) Neurotoxic esterase does not significantly hydrolyse peptides or amides but some hydrophobic peptides and amides are non-progr essive inhibitors. (3) Inhibition by a range of organophosphorus, carbamic and sulphur-acid esters has been investigated. (4) Neurotoxic esterase is similar to chymotrypsin and trypsin but unlike acetylcholinesterase in the pattern of inhibition by organophosphorus esters. The structure-activity relationships presented give some guidance for design of non-neurotoxic pesticides. (5) More stable and less toxic alternatives to Mipafox as a selective inhibitor of neurotoxic esterase have been found. (6) Benzenesulphonyl fluoride is a selective inhibitor of some of the Mipafox-resistant esterases. (7) All the esterases were inhibited by PCMB(0.1 mM)and by Zn 2+ (0.4 mM).

The delayed neuropathic effects of nerve agents and some other organophosphorus compounds

The in vitro inhibitory potencies of several nerve agents and other organophosphorus compounds against acetylcholinesterase (AChE) and neurotoxic esterase (NTE) have been compared. Although the I50s against AChE were about 0.1–1.0 nM for the nerve agents the I50s against NTE for sarin, soman and tabun were two to four orders of magnitude higher and VX had negligible activity. A series of bis[(ω-phenyl-n-alkyl]phosphorofluoridates inhibited both enzymes at 1.0–100 nM while ω-phenyl-n-alkyl NN-dimethylphosphoramidofluoridates were active at 0.1–10 μM. From the in vitro data it was predicted that nerve agents would cause delayed neuropathy only at doses greatly exceeding the LD50. In hens protected against acute toxicity by pretreatment with physostigmine, atropine and the oxime P2S, delayed neuropathy associated with high inhibition of NTE was found at 30–60 × LD50 for sarin but not at 38 × LD50 for soman or 82 × LD50 for tabun. At the maximum doses tested of the latter two compounds the inhibition of NTE was 55% and 66% respectively. The minimum neuropathic doses were calculated to be about 100–150 × LD50 for soman and tabun.As expected from in vitro data, neuropathy, associated with a high level of inhibition of NTE, was caused by one of the bis-phenylalkyl phosphorofluoridates at doses causing negligible acute toxicity. The required dose was 9X that for DFP although the compound was 300X more active against NTE in vitro suggesting that such compounds are rapidly degraded in vivo. The phenylalkyl NN-dimethylphosphoramidofluoridates produced prolonged acute signs of poisoning but they were not neuropathic at the maximum tolerable doses nor was the NTE greatly inhibited contrary to the prediction from the in vitro data. It is possible that the enantiomer responsible for the inhibition of NTE is preferentially degraded in vivo.Several other phosphoramidofluoridates inhibit NTE in vitro at 1.0–100 μM and a number of bicyclic phosphates were inactive at 23 μM. None of these compounds was tested in vivo.

SUBCELLULAR DISTRIBUTION OF MARKER ENZYMES AND OF NEUROTOXIC ESTERASE IN ADULT HEN BRAIN

Neurotoxic esterase (NTE) is now regarded as the site of the primary biochemical lesion in the delayed neuronal degeneration produced by certain organophosphorus esters. Since hens are the species of choice in studies of this neuropathy the subcellular distribution of NTE and marker enzymes in adult hen brain was carried out. Up to 70%, of NTE was recovered in a microsomal fraction (P3) which was also enriched in 5′‐nucleotidase (5′‐ribonucleotide phosphohydrolase EC 3.1.3.5), a plasma membrane marker. The protein content of this fraction (31% of the parent homogenate) is double that of equivalent mammalian brain fractions. The LDH distribution suggests that the P3 fraction contained many small synaptosomes. Subfractionation of microsomes by rate and equilibrium centrifugation on sucrose density gradients segregated the RNA but failed to separate the NTE. 5′‐nucleotidase and glucose‐6‐phosphatase (D‐glucose‐6‐phosphate phosphohydrolase EC 3.1.3.9) from each other. NTE was considerably concentrated (2–5 times) in subfractions of the P2 fraction, which are believed to be enriched in synaptosomal membranes. A similar localization of NTE and AChE was found in subfractions of P2 from neonatal chick brain. Axon fragments contained a significant amount of NTE which was not associated with the myelin. Nuclear and mitochondrial fractions were low in NTE. Microsomes could be partitioned in biphasic aqueous polymer systems, but with little enrichment of NTE. The possible association of NTE with synaptosomal membranes suggests that early events in organophosphorus neuropathy may occur at the axonal (? synaptic) surface.

Clinical and toxicological investigations of a case of delayed neuropathy in man after acute poisoning by an organophosphorus pesticide

Progressive neuropathy developed in a man during 2–8 weeks after acute poisoning by a pesticide said to contain trichlorphon. The neuropathy was typical of that caused by organophosphorus esters in the delay and in the maintenance of normal conduction velocity in surviving nerve fibres. A sample alleged to be typical of the ingested material was not more active against hen brain neurotoxic esterase (NTE) than was pure trichlorphon. Delayed neuropathy has never been produced in hens by a single dose of trichlorphon. This incident and studies of human brain in vitro suggest that the ratio neurotoxicity/lethality for trichlorphon is higher in man than in the hen. Suggestion is made of laboratory tests to improve neurotoxicity screening.

Metabolism of chloroethanol in the rat

When chloroethanol is given orally to rats, liver GSH is rapidly depleted and S-carboxymethylglutathione is formed. Release of 36chloride ion from [36Cl]-chloroethanol has been studied in vitro and shown to require stoichiometric amounts of GSH (1 mole) and NAD (2 moles). S-carboxymethyl-glutathione has been identified as the product in vitro. Ethanol inhibits the depletion of GSH in vivo and release of 36chloride in vitro. Chloroethanol is readily dehydrogenated in vitro by purified yeast and horse liver alcohol dehydrogenases. The route from chloroethanol to S-carboxymethyl-glutathione has been studied in vitro. S-carboxymethyl-glutathione is degraded by kidney homogenate to glycine, glutamic acid and S-carboxymethylcysteine. Subsequent metabolism of the latter compound is discussed. It is suggested that the toxicity of chloroethanol is due to its conversion to chloroacetaldehyde in vivo.

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.