Neutral Phosphate Esters

Some organophosphorus compounds produce neurologic dysfunctions, known as OPIDN, after a delay period that is accompanied by neuropathic damage in the central and peripheral nervous systems. This group of chemicals may be divided into two classes, Type I and II, based on chemical structure, species selectivity, age sensitivity, the length of latent period, clinical signs, morphology and distribution of neuropathologic lesions, protection with phenylmethyl sulfonyl fluoride, inhibition of neurotoxic esterase, and effect on catecholamine secretion from bovine adrenome-dullary chromaffin cells. The importance of this effect is underlined by the fact that incidents involving more than 40,000 cases of OPIDN in humans have been documented from 1899 to 1989. Most of these compounds are direct or indirect inhibitors of AChE, and produce acute cholinergic effects. Neurologic deficits are characterized by three phases: progressive, stationary, and improvement. Prognosis of OPIDN depends on the extent of damage of the nervous system. Improvement or even recovery of functions may follow mild cases, whereas severe toxicity results in long-lasting neurologic dysfunctions reflecting spinal cord damage. Recent studies have shown that delayed neurotoxic organophosphorus compounds interact with Ca2+/calmodulin kinase II (CaM kinase II), an enzyme responsible for the endogenous phosphorylation of cytoskeletal proteins, i.e. microtubules, neurofilaments, and MAP-2. This leads to an increased activity of CaM kinase II and enhanced phosphorylation of cytoskeletal elements, and eventually in the disassembly of cytoskeletal proteins. The dissociation of cytoskeletal proteins causes increased fast axonal transport in the treated animals resulting in the accumulation of altered cytoskeletal elements in the distal portions of the axon. Abnormal tubulin and neurofilaments are transformed into filamentous polymers and undergo condensation and dissolution. Concomitantly, proliferated endoplasmic reticulum and accumulated mitochondria degenerate and release Ca2+ ions. This leads to Ca2(+)-activated proteolysis of the cytoskeleton and interruption of ionic balance across the axonal membrane resulting in the uptake of water and axonal swelling, which subsequently degenerates. A similar mechanism may cause secondary myelin degeneration.

Evidence 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 Assay. Assay 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 Studies. Data 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.

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.