CAS No. 371-86-8

For more abstracts search PubMed or Toxnet

ACTIVITY: Acaricide, Insecticide (organophosphate)

Note: WHO: Believed to be obsolete or discontinued for use as pesticides.

CAS Name: N,N?-bis(1-methylethyl)phosphorodiamidic fluoride



Wallerian degeneration

Orthograde degeneration, secondary degeneration.

Associated persons:
Augustus Volney WallerDescription:

Degeneration of the distal segment of a peripheral nerve fibre (axon) that has been severed from its nutritive centres (cell body), without local inflammation. The myelin sheath also degenerates, from a distal injury to the same axon, and is transformed into a chain of stainable lipoid droplets (Marchi's stain). This discovery made it possible to trace the course of fibres through the nervous system and demonstrated the importance of the nucleus in the regeneration of nerve fibres. The neurilemma does not degenerate but form a tube that directs the growth of the regenerating axon.


• A. V. Waller:
Experiments on the section of the glossopharyngeal and hypoglossal nerves of the frog, and observations on the alterations produced thereby in the structure of their primitive fibres.
London, Edinburgh and Dublin Philosophical Magazine and Journal of Science, 1850.
Philosophical Transactions of the Royal Society of London, 1850, 140: 423-429.
Edinburgh Medical and Surgical Journal, 1851, 76: 369.

Recherches sur la système nerveux.
Comptes rendus de l’Académie des sciences, Paris, 1851,33: 370-374 and 606-611.

Observations sur les effets de la section des racines spinales et du nerf pneumogastrique au dessus de son ganglion inférieur chez les mammifères.
Comptes rendus hebdomadaires des séances de l’Académie des Sciences, Paris, 1852, 34: 582-587.

Sur la reproduction des nerfs et sur la structure et les fonctions des ganglions spinaux.
Archiv für Anatomie, Physiologie und wissenschaftliche Medicin, Leipzig, 1852:392.

Experience sur les sections des nerfs et les alterations.
Comptes-rendus de la Société de biologie, Paris, 1857, 2 (3): 6.

Research Project: Organophosphate Insecticide Damage to the Mature and Developing Nervous Systems: in Vitro Systems for Detection and Remediation

Principal Investigator: E. Tiffany-Castiglioni

Veterinary Anatomy
Texas A&M University
College Station, Texas 77843

Start Date: 04/2001
End Date: 04/2006
CRIS RPAs: 723  314  711

Excerpts: ... At ten times the concentration of mipafox that causes a 50% inhibition of NTE (5x10-5 M/day) mipafox was found to significantly decrease neurite length in differentiated cells while paraoxon and OPH-hydrolyzed paraoxon at the same concentration did not.

... While some organophosphorus (OP) compounds including paraoxon produce acute toxicity through acetylcholinesterase inhibition, others such as mipafox produce OP-induced delayed neurotoxicity (OPIDN), which is characterized anatomically by Wallerian-type "dying back" neuropathy in the axon and myelin.

... Protein expression of NF200 was shown to be a new biomarker by which the neurotoxic effects of mipafox and paraoxon on SY5Y cells were distinguishable at the molecular level.

... The current study shows that organophosphorus compounds produce not only antiesterase activity but also modifications in protein. Evidence presented suggests that mipafox caused shortening of neurites in differentiated SY5Y cells by a degeneration process, whereas paraoxon inhibited neurite growth in the cells.

Ref: http://www.tard.state.tx.us/index.php?mode=Listing&rl_id=639

... In a study of alkyl phosphate poisoning, Pasi and Leuzinger came to the conclusion that delayed lesions only occur, if at all, after severe cerebral anoxia [176]. As regards anatomical changes in the brain (demyelination), these delayed lesions correspond to those caused by peripheral neuropathy in acute and chronic ortho-tricresyl phosphate poisoning and are confined to fluorine- containing alkyl phosphates - for example, mipafox, DFP, sarin and soman. A synoptic evaluation of 536 civilian cases of alkyl phosphate poisoning made by the above-mentioned authors led them to the conclusion that acute poisoning by civilian alkyl phosphates did not result in delayed lesions. It should be noted, however, that their period of observation of two to three years was inadequate for investigations of delayed lesions beside the scale of Spiegelberg and others [page 40].
Ref: Delayed Toxic Effects of Chemical Warfare Agents. A SIPRI (Stockholm international Peace Research Institute) Monograph. 1975. ISBN 91-85114-29-4. http://projects.sipri.se/cbw/research/cw-delayed.pdf

From Toxline at Toxnet

Journal of Applied Toxicology, Vol. 13, No. 2, pages 143-145, 17 references, 1993

Delayed Neurotoxic Effect of Sarin in Mice after Repeated Inhalation Exposure

Husain K, Vijayaraghavan R, Pant SC, Raza SK, Pandey KS

Abstract: The ability of sarin (107448) to induce delayed neurotoxicity was examined in mice. Female Swiss-albino-mice were exposed to 5mg/m3 sarin vapor 20 minutes/day for 10 days. Other mice were injected subcutaneously with 2.5mg/kg mipafox (371868) daily for 10 days. Mice were observed for clinical signs of toxicity for 14 days starting after the first sarin or mipafox exposure. They were killed on day 14. Brain and spinal cord tissues, and blood platelets were assayed for neurotoxic-esterase (NTE) activity. Spinal cord sections were prepared and examined for histopathological changes. Mice exposed to sarin developed muscular weakness in the limbs and ataxia on day 14. Mipafox exposed mice developed severe ataxia. Both sarin and mipafox inhibited brain, spinal cord, and platelet NTE activity. Sarin was less potent than mipafox. Sarin and mipafox induced spinal cord axonal degeneration. The degree of degeneration was greater in mipafox treated mice. Sarin also caused focal axonal degeneration in the lateral branches of the spinal cord. The authors conclude that sarin seems capable of inducing delayed neurotoxicity in mice following repeat inhalation exposure.

From Toxline at Toxnet

Human and Experimental Toxicology, Vol. 16, No. 2, pages 67-71, 11 references, 1997

The Effects of Multiple Low Doses of Organophosphates on Target Enzymes in Brain and Diaphragm in the Mouse

Williams FM, Charlton C, de Blaquiere GE, Mutch E, Kelly SS, Blain PG

Abstract: The effects of multiple low doses of ecothiopate (513100), paraoxon (311455), and mipafox (371868) on organophosphate target enzymes in the brain and diaphragm were studied in mice. Male albino-mice were injected subcutaneously once with 0 or 110 micromoles per kilogram (micromol/kg) mipafox, 0.5micromol/kg ecothiopate, or 1.5micromol/kg paraoxon or with 0 or 44micromol/kg mipafox, 0.2micromol/kg ecothiopate, or 0.6micromol/kg paraoxon daily for 5 days or 27.5micromol/kg mipafox daily for 8 day s. The mice were killed 3 hours (hr) after the single dose or 3 or 24hr after each of the multiple doses and the brain and diaphragms were removed. The brains were assayed for acetylcholinesterase (AChE) and neuropathy-target-esterase (NTE) activity. The diaphragms were analyzed for AChE activity. The single doses of mipafox, ecothiopate, and paraoxon inhibited diaphragm AChE activity to about the same extent, around 67 to 68%. Mipafox and paraoxon inhibited brain AChE activity by 55 and 73%, respectively. Ecothiopate did not affect brain AChE activity. Only mipafox inhibited brain NTE activity, by 66%. Brain AChE activity was progressively inhibited by 17 to 46% by multiple dosing with 27.5micromol/kg mipafox, by 23 to 49% by multiple injection with 44micromol/kg mipafox, and by 20 to 55% by multiple dosing with paraoxon. Ecothiopate caused a small increase in brain AChE activity. Diaphragm AChE activity was similarly inhibited by mipafox and paraoxon. Ecothiopate also caused a progressive inhibition of diaphragm AChE activity. Brain and diaphragm AChE activity showed some recovery between the daily doses. The extent of inter dose recovery was greater in the case of paraoxon and ecothiopate than with mipafox. Mipafox also produced a progressive inhibition of brain NTE activity, the cumulative inhibitory effect, 74 and 76%, being similar after the two dosing protocols. The authors conclude that exposure to multiple low doses of mipafox, ecothiopate, and paraoxon produces additive inhibition of AChE activity. These results have implications for humans as humans are generally exposed to low levels of organophosphates for extended periods of time.

From Toxline at Toxnet

In Vitro Toxicology. Journal of Molecular and Cellular Toxicology, Vol. 5, No. 3, pages 127-136, 28 references, 1992

Cytotoxic Effects of Organophosphorus Esters and Other Neurotoxic Chemicals on Cultured Cells

Nostrandt AC, Rowles TK, Ehrich M

Abstract: The in-vitro cytotoxicity of organophosphates and other neurotoxic chemicals in a neuronal cell line was examined. Differentiated SY-5Y cells, a human neuroblastoma cell line, were incubated with 0 to 10(-3) molar (M) mipafox (371868), paraoxon (311455), aldicarb (116063), beta,beta-iminodipropionitrile (111944) (IDPN), carbachol (51832), carbaryl (63252), or phenyl-saligenin-phosphate (4081236) (PSP) for 24 hours. Other cells were incubated with the nonneurotoxicants atropine or verapamil for comparison. Effects on viability were determined using the trypan-blue dye exclusion test. SY-5Y cells were incubated with mipafox, paraoxon, aldicarb, IDPN, or carbachol for up to 14 days. In some experiments, atropine was added to the cultures. The cultures were assayed for acetylcholinesterase (AChE) activity after 10 minutes. The cells were analyzed for intracellular calcium (Ca+2) content after 3, 10, 24, and 48 hours. The cultures were examined for histomorphological changes periodically for up to 14 days. A parallel experiment using chicken brain homogenates was performed and compared with the effects on Sy-5Y AChE activity. Only mipafox, carbachol, carbaryl, and PSP significantly decreased cellular viability, with carbaryl and PSP being the most potent. SY-5Y cells exposed to paraoxon, aldicarb, or carbachol became rounded, more refractile, and eventually detached from the culture plate after 3 days incubation. Mipafox induced swelling and blebbing on the neurites after 3 days. The neurites were significantly shorter and thinner on day ten compared to the control cultures. Significant numbers of rounded and detached cells were seen on day 14. No histopathological changes were seen in IDPN treated cells until day ten, at which point they showed changes similar to those induced by mipafox. Carbachol, aldicarb, paraoxon, IDPN, and mipafox inhibited AChE activity to a greater extent in SY-5Y cells than in the chicken brain homogenates, with mipafox, paraoxon, and aldicarb being the most potent. Mipafox, paraoxon, aldicarb, IDPN, and carbachol induced transient increases in Ca+2 content at 4 hours. Peak Ca+2 concentrations occurred at 10 hours, except in the case of paraoxon. Ca+2 concentrations in paraoxon treated cells decreased sharply after 4 hours. Atropine attenuated the increases in Ca+2 concentration induced by the compounds. The authors conclude that SY-5Y cells can be used to assess the cytotoxic effects of neurotoxic chemicals, especially esterase inhibitors.

The reports are available from The National Technology Information Service (NTIS) .
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Details Abstract

Order Number: NTIS/01930011 (4 pages)

2004 - Atomic Crystal Structure of an Organophosphorus Acid Anhydrolase.

Authors: Quiocho FA, Nickitenko A

Baylor Coll. of Medicine, Houston, TX.

Crystal structure
Three dimensional
X ray spectra
Chemical warfare agents
Enzyme inhibitors
X ray crystallography
Opaa(Organophosphorus acid anhydrolase)

Final progress rept. 5 Dec 2000-4 Dec 2003.
The major aim is to determine the three-dimensional atomic structure of an organophosphorus acid anhydrolase (OPAA) by x ray crystallography. This structure is a prerequisite for remodeling the active site, in collaboration with scientists at ECBC and Geo-Centers, Inc. in order to enhance catalytic activity towards fluorinated U-type and other extremely toxic chemical warfare (CW) nerve agents. A decontamination system based on the remodeled OPAA not only provides rapid removal of CW agents, but is also enviromnentally safe and noncorrosive in nature. After overcoming the major difficulty in crystallizing OPAA, we have now determined by MAD and SAD techniques and refined the crystal structure of OPAA to 2.5 A resolution. The structure analysis of OPAA with the bound enzyme inhibitor MIPAFOX (N,N'-di-isopropyl phosphorodiamidic fluoride) is in progress.

Order Number: NTIS/AD-A290 426/6 (17 pages)

1994 - Genetic and Biochemical Manipulation of a Broad-Spectrum Organophosphate Degrading System.

Authors: Wild JR

Texas A and M Research Foundation, College Station.


Recent studies on the plasmid-borne organophosphorus-degrading gene of Pseudomonas diminuta and its enzyme have sought to define both the genetic organization and the protein chemistry involved in this system. The bacterial gene encodes a single, unique enzyme, a phosphotriesterase (organophosphorus anhydrase), which is capable of hydrolyzing a wide spectrum of organophosphorus neurotoxins ranging from insecticides such a parathion, orthene, coumaphos and diazinon to mammalian neurotoxins such as diisopropylfluorophosphate (DFP), sarin, soman and mipafox. The organophosphorus degrading genes (opd) from two different plasmids in the soil bacteria P. diminuta and Flavobacterium have been sequenced andtheir structural organizations are being characterized. The cloned geneshave been expressed in a number of biological systems from bacteria to insect tissue culture, and the enzyme has been purified and characterized from several different sources. The catalytic reaction hasbeen determined to involve [abstract truncated]

Order Number: NTIS/PB94-137155 (7 pages)

1993 - Differential Cytotoxic Sensitivity in Mouse and Human Cell Lines Exposed to Organophosphate Insecticides.

Authors: Veronesi B, Ehrich M

Health Effects Research Lab., Research Triangle Park, NC. Neurotoxicology Div.

Virginia-Maryland Regional Coll. of Veterinary Medicine, Blacksburg, VA.

Organophosphate insecticides

Neuroblastoma cell lines were used to examine the differential interspecies response (i.e., species selectivity) to organophosphates (OPs). Baseline activities of the major target esterases, i.e., cholinesterase, carboxylesterase, and neurotoxic esterase, were assayed in mouse and several human neural candidate cell lines. These activities were found to be variable within individual cell lines and among the various tested cell lines. Cytotoxicity data using the neutral red fluorometric assay were collected on both human (SH-SY5Y) and mouse (NB41A3) neuroblastoma clones exposed to a variety of OP insecticides. IC50 data indicated that the tested mouse cell line was consistently more sensitive than the human cell line to equimolar doses of various OP compounds (e.g., mipafox, parathion, paraoxon, DFP, leptophos oxon, fenthion, and fenitrothion). These data suggest that interspecies-selectivity in response to OP-related cytotoxicity is influenced by intercellular differences in metabolism and basel [abstract truncated]

Order Number: NTIS/PB95-126462 (1 8 pages)

1993 - Short-Term Clinical and Neuropathologic Effects of Cholinesterase Inhibitors in Rats.

Authors: Ehrich M, Shell L, Rozum M, Jortner BS

Virginia-Maryland Regional Coll. of Veterinary Medicine, Blacksburg, VA.

Virginia Polytechnic Inst. and State Univ., Blacksburg. Dept. of Statistics.

Supporting Agency: Health Effects Research Lab., Research Triangle Park, NC.

Cholinesterase inhibitors
Nervous system
Organophosphate insecticides

Adult male Long Evans rats were given a single administration of 3 dosage levels of the organophosphorus compounds tri-ortho-tolyl phosphate (TOTP), diisopropyl fluorophosphate (DFP), phenyl saligenin phosphate (PSP), mipafox, malathion, and dichlorvos or the carbamate carbaryl. Acetylcholinesterase and neurotoxic esterase activities were inhibited in a dose-dependent manner, with the highest dosages of all these compounds inhibiting activities of these enzymes in brain by at least 37% and 64%, respectively, at 4 and 48 hours after administration. Rats given the high doses of TOTP (1000 mg/kg), DFP (3 mg/kg), malathion (2000 mg/kg), and carbaryl (160 mg/kg) weighed significantly less than control rats 14 days after administration. A functional observational battery (FOB) was used to screen for neurotoxic effects 1, 2, and 3 weeks after exposure. All 7 test compounds were capable of causing changes in parameters indicative of behavioral and central nervous system excitability. In addition, dose-r [abstract truncated]

Order Number: NTIS/PB93-229508 (10 pages)

1993 - Using Neuroblastoma Cell Lines to Examine Organophosphate Neurotoxicity.

Authors: Veronesi B, Ehrich M

Health Effects Research Lab., Research Triangle Park, NC.

Virginia-Maryland Regional Coll. of Veterinary Medicine, Blacksburg, VA.

Nervous system
Organophosphate insecticides

The paper describes the initial characterization of neuroblastoma cell lines to address several aspects of organophosphate neurotoxicity. Several commercially available human and mouse cell lines (i.e., SY5Y, IMR-32, SK-N-MC, NB41A3) were evaluated for their target esterase activities (i.e., cholinesterase, neurotoxic esterase, carboxylesterase), and of these cells, a human (SY5Y) and mouse (NB41A3) neuroblastoma cell line clone were used to establish an IC50 cytoxicity profile for a variety of organophosphates insecticides (e.g., parathion, paraoxon, diisopropylphosphorofluoridate and mipafox). The human neuroblastoma cell line clone (SY5Y) was further used to distinguish between neuropathy-causing OPs and cholinesterase inhibitors. These initial data support the use of neuroblastoma cell lines as effective test models for organophosphate neurotoxicity. Journal article. Pub. in In vitro Toxicology: A Jnl. of Molecular and Cellular Toxicology, v6 n1 p57-65 1993. Prepared in cooperation with Vi [abstract truncated]

Order Number: NTIS/PB89-106819 (9 pages)

1987 - Triphenyl Phosphite: In vivo and In vitro Inhibition of Rat Neurotoxic Esterase (Journal Version).

Authors: Padilla SS, Grizzle TB, Lyerly D

Health Effects Research Lab., Research Triangle Park, NC.

Northrop Services, Inc., Research Triangle Park, NC.

Toxic substances
Carboxylic ester hydrolases
Triphenyl phosphite

Organophosphorus compounds which, after acute administration, inhibit neurotoxic esterase (NTE) by > or = 65% and undergo a subsequent 'aging' reaction, produce a delayed neuropathy characterized by degeneration of large and long nerve fibers. The present studies examine in detail the NTE-inhibiting properties of triphenyl phosphite (TPP), a plasticizer which produces ataxia and degeneration of the spinal cord in animals. A neurotoxic dosing regimen (1184 mg/kg/week, sc, for 2 weeks) inhibited both brain and spinal cord NTE (< or = 40%) only marginally 4 and 48 hr postdosing. By contrast, TPP was shown in vitro to be a potent inhibitor of rat brain NTE relative to Mipafox or diisopropyl phosphorofluoridate. Preincubation of 10 micromolar TPP in buffer (37 deg C) resulted in a time-dependent loss of TPP's ability to inhibit NTE. In summary, TPP is a powerful NTE inhibitor in vitro, but only a marginal NTE inhibitor after in vivo administration. These results raise questions as to the causal [abstract truncated]

Order Number: NTIS/PB94-137247 (7 pages)

1993 - Relationship of Neuropathy Target Esterase Inhibition to Neuropathology and Ataxia in Hens Given Organophosphorus Esters.

Authors: Ehrich M, Jortner BS, Padilla S

Virginia-Maryland Regional Coll. of Veterinary Medicine, Blacksburg, VA.

Adult White Leghorn hens were acutely exposed to 3 dosages of the following organophosphorus compounds: mipafox, tri-ortho-tolyl phosphate (TOTP), phenyl saligenin phosphate, and diisopropylphosphorofluoridate (DFP). Neuropathy target esterase (NTE) activity was measured in brain and spinal cord 4 or 48 h after exposure. Ataxia was assessed using an 8-point rating scale on days 9 through 21 after administration, and neuropathological examination was conducted on samples collected from perfusion-fixed animals on day 21. Morphological alterations were indicated by lesion scores between 0 (no lesions) and 4 (diffuse involvement of spinal cord tracts and > 25% degeneration of peripheral nerve fibers). Dosages of mipafox, TOTP, phenyl saligenin phosphate, and DFP that were capable of inhibiting NTE > 80% in both brain and spinal cord preceded ataxia which reached maximal levels (scores of 7-8), and development of lesions scored as 4. Hens were notably impaired (ataxia scores of 3-4) 21 days aft [abstract truncated]

Order Number: NTIS/AD-A203 001/3 (9 pages)

1988 - Soman Hydrolyzing and Detoxifying Properties of an Enzyme from a Thermophilic Bacterium,

Authors: Chettur G, DeFrank JJ, Gallo BJ, Hoskin FC, Mainer S

Illinois Inst. of Tech., Chicago.

Gd agent
Organophosphorus acid anhydrolases
Thermophilic bacterium

An enzyme that hydrolyzes soman(1,2,2-trimethylpropyl methylphosphonofluoridate) and two other phosphonofluoridates, but does not hydrolyze DFP (diisopropylphosphorofluoridate), has been partially purified from a rod-shaped spore-forming gram-positive OT (obligate thermophilic) bacterium. The enzyme shows a marked Mn(2+) stimulation, and in this and its substrate preference does not resemble the organophosphorus acid anhydrolase (sometimes termed DFPase) found in squid. Like the squid enzyme, it is not inhibited by mipafox (n,n-diisopropylphosphordiamidofluoridate), is not inactivated by ammonium sulfate, and does hydrolyze the acetylcholinesterase-inhibitory pair of diasteroisomers of soman as well as the relatively non-inhibitory pair, thus detoxifying soman. In these three properties the OT enzyme does not resemble the ubiquitous organophosphorus acid anhydrolase often purified from mammalian and bacterial sources by cold ethanol fractionation. Thus this phosphono-specific OT enzyme may have [abstract truncated]

Order Number: NTIS/PB91-177246 (12 pages)

1990 - Potentiation of Organophosphorus-Induced Delayed Neurotoxicity by Phenylmethylsulfonyl Fluoride.

Authors: Pope CN, Padilla S

Health Effects Research Lab., Research Triangle Park, NC.Neurotoxicology Div.

Northeast Louisiana Univ., Monroe. School of Pharmacy

Nervous system
Organophosphorus compounds
Phenylmethylsulfonyl fluorides
Organophosphorus induced delayed neurotoxicity(OPIDN)

It is well known that pretreatment with the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF) can protect experimental animals from organophosphorus-induced delayed neurotoxicity (OPIDN), presumably by blocking the active site of neurotoxic esterase (NTE) such that binding and 'aging' of the neuropathic OP is thwarted. The authors report here that while PMSF (60 mg/kg, s.c.) given 4 hours before the neuropathic OP mipafox (50 mg/kg, i.m.) completely prevented the clinical expression of OPIDN in hens, the identical PMSF treatment markedly amplified the delayed neurotoxicity (relative to hens treated with the OP only) if administed 4 hours after mipafox (5 or 50 mg/kg, i.m.). Moreover, in a separate experiment using diisopropylphosphorofluoridate (DFP) as the neurotoxicant in place of mipafox, posttreatment with PMSF 4 hours after DFP (0.5 mg/kg) also accentuated the severity of the ataxia. These data indicate that PMSF only protects against OPIDN if given prior to exposure to the neu [abstract truncated]

Order Number: NTIS/PB86-213774 (10 pages)

1986 - In vitro Comparison of Rat and Chicken Brain Neurotoxic Esterase.

Authors: Novak R, Padilla S

Health Effects Research Lab., Research Triangle Park, NC.

Northrop Services, Inc., Research Triangle Park, NC.


A systematic comparison was undertaken to characterize neurotoxic esterase (NTE) from rat and chicken brain in terms of inhibitor sensitivities, pH optima, and molecular weights. Paraoxon titration of phenyl valerate (PV)-hydrolyzing carboxylesterased showed that rat esterases were more sensitive than chicken to paraoxon inhibition at concentrations less than micromole and superimposable with chicken esterases at concentrations of 2.5-1000 micromole. Mipafox titration of the paraoxon-resistant esterases at a fixed paraoxon concentration of 100 micromole (mipafox concentration: 0-1000 micromole) resulted in a mipatox 150 of 7.3 micromole for chicken brain NTE and 11.6 micromole for rat brain NTE. NTE(i.e., paraoxon-resistant, mipafox-sensitive esterase activity) comprised 80% of chicken and 60% of rat brain paraoxon-resistant activity with the specific activity of chicken brain NTE approximately twice that of rat brain NTE. The pH maxima for NTE from both species was similar showing broad, slight [abstract truncated]

Order No. NTIS/PB86-157971 (9 pages)

1985 - Phenylmethylsulfonyl Fluoride Protects Rats from Mipafox-Induced Delayed Neuropathy.

Authors: Veronesi B, Padilla S

Health Effects Research Lab., Research Triangle Park, NC.


Initiation of organophosphorus-induced delayed neuropathy (OPIDN) is thought to consist of two molecular events involving the phosphorylation of the target enzyme, neurotoxic esterase or neuropathy target enzyme (NTE), and a subsequent 'aging' reaction which transforms the inhibited NTE into a charged moiety critical to the neuropathic process. Compounds that inhibit NTE but cannot age because of their chemical structure abort this two-stage initiation process, and when administered before a neurotoxic organophosphorus compound (OP), protect against the neuropathy by blocking NTE's active site (Johnson, 1970). In support of this, the authors report that prior exposure to a non-aging NTE inhibitor, phenylmethylsulfonyl fluoride (PMSF), protects rats from neurological damage after subsequent exposure to a neurotoxic OP, Mipafox. Adult, male Long Evans rats were exposed to either PMSF (250mg/kg, sc) or to Mipafox (15 mg/kg, ip) and a time-course of brain NTE inhibition and recovery was defined. [abstract truncated]

Order Number: NTIS/PB83-209692 ( 7 pages)

1983 - Kinetic Study on the Inhibition of Hen Brain Neurotoxic Esterase by Mipafox.

Authors: Soliman SA, Curley A

Health Effects Research Lab., Research Triangle Park, NC.


A direct method of assaying neurotoxic esterase (NTE) activity, using 4-nitrophenyl valerate, has been described. The technique was used to determine the biomolecular rate (ki), phosphorylation (k2), and affinity (kd) constants for the reaction of hen brain microsomal NTE with mipafox. Results indicate that the new technique for assaying NTE makes detailed kinetic studies of NTE inhibition possible. Journal article, Pub. in Journal of Analytical Toxicology, v6 p4-9 1982.

Order Number: NTIS/PB82-127598 (6 pages)

1982 - Assay of Chicken Brain Neurotoxic Esterase Activity Using Leptophosoxon as the Selective Neurotoxic Inhibitor

Authors: Soliman SA, Curley A

Health Effects Research Lab., Research Triangle Park, NC. Environmental Toxicology Div.

Supporting Agency: Air Force Office of Scientific Research, Bolling AFB, DC.


Hen brain microsomal preparation has phenyl valeratehydrolyzing activity associated with neurotoxic esterase activity. Part of that activity is due to paraoxon-insensitive esterases and a sub-part of this is sensitive to neurotoxic organophosphates, i.e., mipafox and leptophosoxon. This neurotoxic agent sensitive esterase activity is referred to as neurotic esterase (NTE). Because of the commercial unavailability and high toxicity of mipafox, which is usually used as the selective inhibitor for assaying NTE, leptophosoxon was used as an alternative to mipafox. Results indicated that the NTE fraction of hen brain microsomal PV-hydrolyzing activity is the same target for either mipafox or leptophosoxon. The inhibitory effect of leptophosoxon against that fraction was much higher than that of mipafox. The availability of leptophos/ leptophosoxon makes this assay very useful for screening organophosphorus esters for neurotoxic effects.

Order Number: NTIS/AD-A119 217/8 (55 pages)

1982 - In Vitro Studies of Neurotoxic Substances: The Effect of Organophosphates and Acrylamides.

Authors: Nardone RM, Spiegel J, Fedalei A, Krause D, Filipowski RM

Catholic Univ. of America, Washington, DC. Dept. of Biology.

Nerve cells

The toxicity of acrylamide, n-methylacrylamide, and crotonamide as well as the organophosphates mipafox, leptophos, paraoxon, EPN, OMPA and DFP were studied in order to see whether or not in vivo-in vitro neurotoxicity correlations could be established. The in vitro systems employed were the mouse neuroblastoma cell line NIE-115 and the chick brain, either as cell aggregate cultures or organ culture. In both the neuroblastoma cell culture and chick brain cell/organ culture systems, acrylamide was the most toxic. The ranking of acrylamide, n-methylacrylamide and crotonamide paralleled the ranking reported in vivo. The end-points which showed this ranking included cell viability and neuron-specific enolase activity and aggregate formation by dissociated brain cells. The organophosphate studies emphasized their effect on neurotoxic esterase activity. A model in vitro test has been developed for the evaluation of neurotoxic esterase effects. The test is based on the hen brain assay test developed by [abstract truncated]


Chem. Res. Toxicol., 19 (2), 334 -339, 2006.

Aging of Mipafox-Inhibited Human Acetylcholinesterase Proceeds by Displacement of Both Isopropylamine Groups to Yield a Phosphate Adduct

Timothy J. Kropp and Rudy J. Richardson*

Toxicology Program, Department of Environmental Health Sciences, University of Michigan, 1420 Washington Heights, Ann Arbor, Michigan 48109-2029

Aging of phosphylated serine esterases, e.g., acetylcholinesterase (AChE) and neuropathy target esterase (NTE), renders the inhibited enzymes refractory to reactivation. This process has been considered to require postinhibitory side group loss from the organophosphorus moiety. Recently, however, it has been shown that the catalytic domain of human NTE inhibited by N,N'-diisopropylphosphorodiamidofluoridate (mipafox, MIP) ages by deprotonation. For mechanistic understanding and biomarker development, it would be important to know the identity of the MIP adduct on target esterases after inhibition and aging occurred. Accordingly, the present study was performed to determine if MIP-inhibited human AChE ages by side group loss or an alternate method, e.g., deprotonation. Diisopropylphosphorofluoridate (DFP), the oxygen analogue of MIP, was used for comparison, because DFP-inhibited AChE is known to age by net loss of an isopropyl group. Kinetics experiments were done with DFP and MIP against AChE to follow the time course of inhibition, reactivation, and aging for each inhibitor. MS studies of tryptic digests from kinetically aged DFP-inhibited AChE revealed a mass shift of 122.8 ± 0.7 Da for the active site peptide (ASP) peak, corresponding to the expected monoisopropylphosphoryl adduct. In contrast, the analogous mass shift for kinetically aged MIP-inhibited AChE was 80.7 ± 0.9 Da, corresponding to a phosphate adduct. Because this finding was unexpected, the identity of the phosphoserine-containing ASP was confirmed by immunoprecipitation followed by MS. The results indicate that aging of MIP-inhibited AChE proceeds by displacement of both isopropylamine groups. Further research will be required to elucidate the detailed mechanism of formation of a phosphate conjugate from MIP-inhibited AChE; however, knowledge of the identity of this adduct will be useful in biomarker studies.

Full text available at Science Direct

Molecular Brain Research . Volume 141, Issue 1 , 18 November 2005, Pages 30-38

Reduction of neuropathy target esterase does not affect neuronal differentiation, but moderate expression induces neuronal differentiation in human neuroblastoma (SK-N-SH) cell line

Ping-An Chang (a, b), Rui Chen (a, b) and Yi-Jun Wu (a)

(a) Laboratory of Molecular Toxicology, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, P.R. China
(b) Graduate School of the Chinese Academy of Sciences, Beijing 100039, P.R. China

Neuropathy target esterase (NTE) is inhibited and aged by organophosphorus compounds that induce delayed neuropathy in human and some sensitive animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neurodifferentiation. However, to date, there is no direct evidence of the relevance of NTE in neurodifferentiation under physiological conditions. In this study, we have investigated a possible role for NTE in the all-trans retinoic acid-induced differentiation of neuroblastoma cells. The functional inactivation of NTE by RNA interference indicated that reduction of NTE does not affect process outgrowth or differentiation of the cells, although moderate expression of NTE by expression of the NTE esterase domain accelerates the elongation of neurite processes. Mipafox, a neurotoxic organophosphate, was shown to block process outgrowth and differentiation in cells that have lowered NTE activity due to RNA interference, suggesting that mipafox may interact with other molecules to exert its effect in this context.


Neurotox Res. 2005;7(3):203-17.

Effects of organophosphorus compounds on ATP production and mitochondrial integrity in cultured cells.

Massicotte C, Knight K, Van der Schyf CJ, Jortner BS, Ehrich M.

Virginia-Maryland Regional College of Veterinary Medicine, 1 Duck Pond Drive, Blacksburg, VA 24061-0442, USA.

Recent studies in vivo and in vitro suggested that mitochondrial dysfunction follows exposure to organophosphorus (OP) esters. As mitochondrial ATP production is important for cellular integrity, ATP production in the presence of OP neurotoxicants was examined in a human neuronal cell line (SH-SY5Y neuroblastoma cells) and primary dorsal root ganglia (DRG) cells isolated from chick embryos and subsequently cultured to achieve maturation with axons. These cell culture systems were chosen to evaluate toxic effects on the mitochondrial respiratory chain associated with exposure to OP compounds that do and do not cause OP-induced delayed neuropathy (OPIDN), a disorder preceded by inhibition of neurotoxic esterase (NTE). Concentration- and time-response studies were done in neuroblastoma cells exposed to phenyl saligenin phosphate (PSP) and mipafox, both compounds that readily induce delayed neuropathy in hens, or paraoxon, which does not. Phenylmethylsulfonyl fluoride (PMSF) was included as a non-neuropathic inhibitor of NTE. Purified neuronal cultures from 9 day-old chick embryo DRG were treated for 12 h with 1 microM PSP, mipafox, or paraoxon. In situ evaluation of ATP production measured by bioluminescence assay demonstrated decreased ATP concentrations both in neuroblastoma cells and chick DRG neurons treated with PSP. Mipafox decreased ATP production in DRG but not in SH-SY5Y cells. This low energy state was present at several levels of the mitochondrial respiratory chain, including Complexes I, II, III, and IV, although Complex I was the most severely affected. Paraoxon and PMSF were not effective at all complexes, and, when effective, required higher concentrations than needed for PSP. Results suggest that mitochondria are an important early target for OP compounds, with exposure resulting in depletion of ATP production. The targeting of neuronal, rather than Schwann cell mitochondria in DRG following exposure to PSP and mipafox was verified by loss of the mitochondrial-specific dye, tetramethylrhodamine, in these cells. No such loss was seen in paraoxon exposed neurons isolated from DRG or in Schwann cells treated with any of the test compounds.

PMID: 15897155 [PubMed - in process]


Biochemistry. 2004 Mar 30;43(12):3716-22.
The mipafox-inhibited catalytic domain of human neuropathy target esterase ages by reversible proton loss.

Kropp TJ, Glynn P, Richardson RJ.

Toxicology Program, Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan 48109-2029, USA.

Aging of organophosphorus (OP)-compound-inhibited neuropathy target esterase (NTE) is the critical event that initiates OP-compound-induced delayed neurotoxicity (OPIDN). Aging has classically been considered to involve side-group loss from phosphylated NTE, rendering the enzyme refractory to reactivation. N,N'-Diisopropylphosphorodiamidofluoridate (mipafox, MIP)-inhibited NTE has been thought to age quickly; however, it can be reactivated under acidic conditions. The present study was undertaken to determine whether MIP-inhibited human recombinant NTE esterase domain (NEST) ages classically by isopropylamine loss. Diisopropylphosphorofluoridate (DFP), the oxygen analogue of MIP, was used for comparison. Kinetic values for DFP against NEST were as follows: k(i) = 17 200 +/- 180 M(-1) min(-1); reactivation t(1/2) approximately 90 min at pH 8.0 and approximately 60 min at pH 5.2; k(4) = 0.108 +/- 0.041 min(-1) at pH 8.0 and 0.181 +/- 0.034 min(-1) at pH 5.2. Kinetic values for MIP against NEST were as follows: k(i) = 1880 +/- 61 M(-1) min(-1); reactivation t(1/2) = 0 min at pH 8.0 and approximately 60 min at pH 5.2; aging was complete at all time points tested at pH 8.0, but no aging occurred at pH 5.2. Mass spectrometry revealed a mass shift of 123.0 +/- 0.6 Da for the active site peptide peak of aged DFP-inhibited NEST, corresponding to a monoisopropyl phosphate adduct. In contrast, the analogous mass shift for aged MIP-inhibited NEST was 162.8 +/- 0.6 Da, corresponding to the intact N,N'-diisopropylphosphorodiamido adduct. Thus, MIP-inhibited NEST does not age by isopropylamine loss. However, because kinetically aged MIP-inhibited NEST yields an intact adduct capable of reversible deprotonation, aging could occur by proton loss. Indeed, MIP-inhibited NEST does not age at pH 5.2 but ages immediately and completely at pH 8.0. Therefore, we conclude that the MIP-NEST conjugate ages by deprotonation rather than classical side-group loss.

PMID: 15035642 [PubMed - indexed for MEDLINE]


Toxicol Appl Pharmacol. 2004 May 1;196(3):319-26.
Lysophospholipase inhibition by organophosphorus toxicants.

Quistad GB, Casida JE.

Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA.

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.

PMID: 15094302 [PubMed - indexed for MEDLINE]


J Toxicol Environ Health A. 2004 Jul 9;67(13):987-1000.
Neurofilament 200 as an indicator of differences between mipafox and paraoxon sensitivity in Sy5Y neuroblastoma cells.

Cho T, Tiffany-Castiglioni E.

Department of Veterinary Anatomy and Public Health and Faculty of Toxicology, Texas A&M University, College Station, Texas 77843-4458, USA.

Organophosphorus (OP) compounds produce potent neurotoxic effects in humans, including organophosphorus-induced delayed neuropathy (OPIDN). This investigation examined the potential for the 200-kD neurofilament protein (NF200) and other neuronal proteins to serve as indicators for neurite damage in a differentiated SY5Y human neuroblastoma cell culture system. Mipafox, which induces OPIDN, increased NF200 protein expression in SY5Y cells differentiated with human recombinant beta-nerve growth factor (NGF, 20 ng/ml) in a concentration-dependent manner, compared to NGF controls, when SY5Y cells were exposed to 0.3 or 30 microM mipafox during the last 5 days of neurite extension (experimental set A). However, mipafox produced little change in NF200 protein expression in SY5Y cells exposed continuously throughout neurite elongation (experimental set B). Paraoxon (up to 30 microM), which does not produce OPIDN, did not produce any change in NF200 expression in set A or set B. The upregulation of NF200 by mipafox may represent a compensatory response to neurite degeneration. Two other neuronal proteins, growth-associated protein 43 (GAP43) and microtubule-associated protein 2ab (MAP2ab), showed no changes in response to OP treatment in NGF-treated cells. Protein expression of NF200 was shown to be an indicator by which the sensitivities of SY5Y cells to mipafox and paraoxon were distinguishable at the molecular level. These results indicate an alternative approach and test system for investigating structure-activity relationships of OPs. Copyright Taylor and Francis Inc.

PMID: 15205030 [PubMed - indexed for MEDLINE]


Toxicol Lett. 2004 Jun 15;151(1):171-81. 

The inhibition of the high sensitive peripheral nerve soluble esterases by mipafox. A new mathematical processing for the kinetics of inhibition of esterases by organophosphorus compounds.

Estevez J, Garcia-Perez AG, Barril J, Pellin M, Vilanova E.

Division de Toxicologia, Universidad Miguel Hernandez de Elche, Avenida de la Universidad, s/n, Elche-Alicante E-03202, Spain.

In the study of organophosphorus (OP) sensitive enzymes, careful discrimination of specific components within a complex multienzymatic mixture is needed. However, standard kinetic analysis gives inconsistent results (i.e., apparently different kinetic constants at different inhibitor concentration) with complex multienzymatic mixtures. A strategy is now presented to obtain consistent kinetic parameters. In the peripheral nerve, soluble carboxylesterases measured with the substrate phenylvalerate (PV) are found with extremely high sensitivity to some inhibitors. Tissue preparations were preincubated with mipafox at nanomolar concentrations (up to 100 nM) for different inhibition times (up to 180 min). Inhibition data were analyzed with model equations of one or two sensitive (exponential) components, with or without resistant components. The most complex model was %act=A1e-k1It+A2e-k2It+AR (step 1). From the curve with the highest mipafox concentration (100 nM), the amplitude for the resistant component was determined as AR=15.1% (step 2). The model equation with a fixed AR value was again applied (step 3) to deduce the second-order inhibition rate constants (k1=2.6 x 10(6) M-1 min-1 and k2=0.28 x 10(6) M-1 min-1), being conserved consistently throughout all mipafox concentrations. Finally, using fixed values of AR, k1, and k2, the amplitudes for the two exponential (sensitive) components (A1 and A2) were re-estimated (A1=50.2% and A2=34.2%). The operational process was internally validated by the close similarity with values obtained by directly fitting with a three-dimensional model equation (activity versus time and inhibitor concentration) to the same inhibition data. Carboxylesterase fractions separated by preparative chromatography showed kinetic properties consistent with the kinetically discriminated components. As practical conclusion, for routine analysis of esterases in toxicological studies, a simplified procedure using the inhibition with mipafox at 30 nM, 1 microM, and 1 mM for 30 min is suggested to discriminate the main esterase components in soluble fraction preparations.

PMID: 15177652 [PubMed - indexed for MEDLINE]


Neurotoxicology. 2003 Dec;24(6):787-96.
Morphological effects of neuropathy-inducing organophosphorus compounds in primary dorsal root ganglia cell cultures.

Massicotte C, Jortner BS, Ehrich M.

Laboratory for Neurotoxicity Studies, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1 Duckpond Drive, Blacksburg, VA 24061-0442, USA.

Chick embryo dorsal root ganglia (DRG) cultures were used to explore early pathological events associated with exposure to neuropathy-inducing organophosphorus (OP) compounds. This approach used an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-day-old chick embryos, and grown for 14 days in minimal essential medium (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then exposed to 1 microM of the OP compounds phenyl saligenin phosphate (PSP) or mipafox, which readily elicit OPIDN in hens, paraoxon, which does not cause OPIDN, or the DMSO vehicle. The medium containing these toxicants was removed after 12 h, and cultures maintained for 4-7 days post-exposure. Morphometric analysis of neurites was performed by inverted microscopy, which demonstrated that neurites of cells treated with mipafox or PSP but not with paraoxon had decreased length-to-diameter ratios at day 4 post-exposure. Ultrastructural alterations of neurons treated with PSP and mipafox included dissolution of microtubules and neurofilaments and degrading mitochondria. Paraoxon-treated and DMSO control neuronal cell cultures did not show such evident ultrastructural changes. This study demonstrates that chick DRG show pathological changes following exposure to neuropathy-inducing OP compounds.

PMID: 14637373 [PubMed - indexed for MEDLINE]


J Toxicol Environ Health A. 2003 Jun 27;66(12):1145-57.

Relative inhibitory potencies of chlorpyrifos oxon, chlorpyrifos methyl oxon, and mipafox for acetylcholinesterase versus neuropathy target esterase.

Kropp T, Richardson R.

Toxicology Program, Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor 48109, USA.

The relative inhibitory potency (RIP) of an organophosphorus (OP) inhibitor against acetylcholinesterase (AChE) versus neuropathy target esterase (NTE) may be defined as the ratio [k(i)(AChE)/k(i)(NTE)], where k(i) is the bimolecular rate constant of inhibition for a given inhibitor against each enzyme. RIPs greater than 1 correlate with the inability of ageable OP inhibitors or their parent compounds to produce OP compound-induced delayed neurotoxicity (OPIDN) at doses below the LD50. The RIP for chlorpyrifos oxon (CPO) is >>1 for enzymes from hen brain homogenate, and the parent compound, chlorpyrifos (CPS), cannot produce OPIDN in hens at sublethal doses. This study was carried out to test the hypothesis that the RIP for the methyl homologue of CPO, chlorpyrifos methyl oxon (CPMO), is >>1 and greater than the RIP for CPO. Mipafox (MIP), an OP compound known to produce OPIDN, was included for comparison. Hen brain microsomes were used as the enzyme source, and k(i) values (mean +/- SE, microM(-1) min(-1)) were determined for AChE and NTE (n = 3 and 4 separate experiments, respectively). The k(i) values for CPO, CPMO, and MIP against AChE were 17.8 +/- 0.3, 10.9 +/- 0.1, and 0.00429 +/- 0.00001, respectively, and for NTE were 0.0993 +/- 0.0049, 0.0582 +/- 0.0013, and 0.00498 +/- 0.00006, respectively. Corresponding RIPs for CPO, CPMO, and MIP were 179 +/- 9, 187 +/- 4, and 0.861 +/- 0.011, respectively. The results demonstrate that RIPs for CPO and CPMO are comparable, markedly different from that for MIP, and >>1, indicating that CPS methyl, like CPS, could not cause OPIDN at sublethal doses.

PMID: 12791540 [PubMed - indexed for MEDLINE]


Toxicol Appl Pharmacol. 2003 Jan 15;186(2):110-8.

Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds.

Hong MS, Hong SJ, Barhoumi R, Burghardt RC, Donnelly KC, Wild JR, Venkatraj V, Tiffany-Castiglioni E.

Department of Chemical Engineering, Texas A&M University, College Station, TX 77845, USA.

Organophosphorus (OP) compounds used as insecticides and chemical warfare agents are known to cause potent neurotoxic effects in humans and animals. Organophosphorus-induced delayed neuropathy (OPIDN) is currently thought to result from inhibition of neurotoxic esterase (NTE), but the actual molecular and cellular events leading to the development of OPIDN have not been characterized. This investigation examined the effects of OP compounds on the SY5Y human neuroblastoma cells at the cellular level to further characterize cellular targets of OP neurotoxicity. Mipafox and paraoxon were used as OP models that respectively do and do not induce OPIDN. Mipafox (0.05 mM) significantly decreased neurite length in SY5Y cells differentiated with nerve growth factor (NGF) while paraoxon at the same concentration had no effect when evaluated after each of three 4-day developmental windows during which cells were treated daily with OP or vehicle. In contrast, paraoxon but not mipafox altered intracellular calcium ion levels ([Ca(2+)](i)), as seen in three types of experiments. First, immediately following the addition of a single high concentration of OP to the culture, paraoxon caused a transient increase in [Ca(2+)](i), while mipafox up to 2 mM had no effect. Paraoxon hydrolysis products could also increase intracellular Ca(2+) levels, although the pattern of rise was different than it appeared immediately after paraoxon administration. Second, repeated low-level paraoxon treatment (0.05 mM/day for 4 days) decreased basal [Ca(2+)](i) in NGF-differentiated cells, though mipafox had no effect. Third, carbachol, a muscarinic acetylcholine receptor agonist, transiently increased [Ca(2+)](i) in differentiated cells, an affect attenuated by 4-day pretreatment with paraoxon (0.05 mM/day), but not by pretreatment with mipafox. These results indicate that the decrease in neurite extension that resulted from mipafox treatment was not caused by a disruption of Ca(2+) homeostasis. The effects of OPs that cause or do not cause OPIDN were clearly distinguishable, not only by their effects on neurite length, but also by their effects on Ca(2+) homeostasis in differentiated SY5Y cells.

PMID: 12639502 [PubMed - indexed for MEDLINE]


Toxicol Lett. 2003 Apr 30;142(1-2):1-10.

Properties of phenyl valerate esterase activities from chicken serum are comparable with soluble esterases of peripheral nerves in relation with organophosphorus compounds inhibition.

Garcia-Perez AG, Barril J, Estevez J, Vilanova E.

Division de Toxicologia, Instituto de Bioingenieria, Universidad Miguel Hernandez de Elche, Avenida del Ferrocarril s/n. E-03202 Alicante, Spain. adolfog@umh.es

Chicken serum, the usual in vivo animal for testing organophosphorus delayed neuropathy, has long been reported not to contain a homologous activity of the neuronal neuropathy target esterase (NTE) activity when it is assayed according to standard methods as the phenyl valerate esterase (PVase) activity, which is resistant to paraoxon and sensitive to mipafox. However, a PVase activity (1000-1500 nmol/min/ml) can be measured in serum that is extremely sensitive to both paraoxon, a non-neuropathic organophosphorus compound and mipafox, a model neuropathy inducer. The inhibition was time progressive in both cases, suggesting a covalent phosphorilating reaction. The fixed time inhibition curves suggest at least two sensitive components. The IC50 for 30 min, at 37 degrees C are 6 and 51 nM for paraoxon and 4 and 110 nM for mipafox, for every sensitive component. When paraoxon was removed from a serum sample pretreated with the inhibitor, the paraoxon sensitive PVase activity was recovered, in spite of showing a time progressive inhibition suggesting that hydrolytic dephosphorylating reaction recovered at a significant rate. The reactivation of the phosphorylated enzyme could explain that the time progressive inhibitions curves for long time with paraoxon tend to reach a plateau depending on the inhibition concentration. However, with mipafox, the curve approached the same maximal inhibitions at all concentrations as expected for a permanent covalent irreversible phosphorylation, which is coherent with the observations that the activity remained inhibited after removing the inhibitor. Data of serum esterases described in this paper showed similar properties to those previously reported for peripheral nerve soluble phenylvalerate esterase: (1) extremely high sensitivity to paraoxon and mipafox; (2) time progressive kinetic with two sensitive components; (3) recovery of activity after removal of paraoxon; and (4) permanent inhibition with mipafox. These properties of serum esterases are very similar to those of soluble fraction of peripheral nerves. So, serum PVases could be considered as appropriate biomarkers, as a mirror for the neural soluble paraoxon and mipafox sensitive soluble esterases that could be used for biomonitoring purpose.

PMID: 12765233 [PubMed - indexed for MEDLINE]


Curr Drug Target CNS Neurol Disord. 2002 Dec;1(6):593-602.

Organophosphate induced delayed polyneuropathy.

Jokanovic M, Stukalov PV, Kosanovic M.

Faculty of Pharmacy, Department of Toxicology, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Yugoslavia. mikatox@hotmail.com

This review discusses the current understanding of organophosphate induced delayed polyneuropathy (OPIDP) with emphasis on molecular mechanisms, pathogenesis and possibilities for prevention/therapy. OPIDP is a rare toxicity caused by certain organophosphorus compounds (OP) characterized by degeneration of some long axons in the central and peripheral nervous system that appear about 2-3 weeks after exposure. The molecular target for OPIDP is considered to be an enzyme in the nervous system known as neuropathy target esterase (NTE). NTE can be inhibited by two types of inhibitors:
a) phosphates, phosphonates, and phosphoramidates, which cause OPIDP when >70% of the enzyme is inhibited, and
b) phosphinates, carbamates, and sulfonyl halides which inhibit NTE and cause either protection from, or promotion, of OPIDP when given before or after a neuropathic OP, respectively.
The ability of a NTE inhibitor to cause OPIDP, besides its affinity for the enzyme, is related to its chemical structure and the residue left attached to the NTE. If such residues undergo the aging reaction i.e. the loss of an alkyl group bound to the enzyme, those OPs usually have a high likelihood of causing OPIDP. Protection from neuropathic doses of OP inhibitors is obtained when NTE is inhibited with nonageable inhibitors. Promotion of OPIDP involves another site besides NTE because it can occur when all NTE is affected. It is now known that this other site is similar to NTE in that it is also sensitive to mipafox but at much higher concentrations. Promotion affects either the progression or expression of OPIDP after the initial biochemical effect on NTE. Some recent observations suggest that development of OPIDP in hens can be influenced by atropine, oximes and methylprednisolone when they are given before or soon after neuropathic OPs.

Publication Types: Review Review, Tutorial

PMID: 12769600 [PubMed - indexed for MEDLINE]


Anal Biochem. 2001 Mar 1;290(1):1-9.

Bioelectrochemical analysis of neuropathy target esterase activity in blood.

Sigolaeva LV, Makower A, Eremenko AV, Makhaeva GF, Malygin VV, Kurochkin IN, Scheller FW.

Faculty of Chemistry, M. V. Lomonosov Moscow State University, Moscow, 119899, Russia. ikur@genebee.msu.su

Bioelectrochemical analysis of neuropathy target esterase (NTE) and its inhibitors is based on the combination of the NTE-catalyzed hydrolysis of phenyl valerate and phenol detection by a tyrosinase carbon-paste electrode. The use of the tyrosinase electrode improves 10-fold the sensitivity of NTE detection in comparison with a spectrophotometric method. The tyrosinase electrode was found to be suitable for measurements in whole human blood where spectrophotometric detection is considerably restricted. The specificity of NTE in blood for mipafox and di-2-propyl phosphorofluoridate was close to that for neuronal NTE. The NTE-like activity in blood was determined to be 0.19 +/- 0.02 nmol/min/mg of protein.

PMID: 11180931 [PubMed - indexed for MEDLINE]


In Vitr Mol Toxicol 2000 Spring;13(1):37-50

Rat cortical neuron cultures: an in vitro model for differentiating mechanisms of chemically induced neurotoxicity.

Schmuck G, Ahr HJ, Schluter G.

BAYER AG, Pharma Research Centre, Wuppertal, Germany. GABRIELE.SCHMUCK.GS@bayer-ag.de

Various structurally unrelated chemicals [2,5 hexandione, acrylamide, organophosphates like mipafox, beta,beta iminodipropionnitrile (IDPN), 3-nitropropionic acid (3-NP), potassium cyanide (KCN), paraquat, and NMDA (N-methyl-D-apartic acid)] are known to cause degenerative damage of the peripheral or central nervous system. Differentiated neuronal cell cultures obtained from fetal rats have been used to differentiate the mechanisms underlying this type of neurotoxicity. Cytotoxicity as measured by a viability assay was not sensitive enough and had to be supplemented by further endpoints covering effects on cytoskeleton and on the energy state of the cells [glucose consumption, mitochondrial membrane potential and adenosine 5'-triphosphate (ATP) concentration]. Compounds like the delayed neurotoxic organophosphates, exert a selective direct effect on cytoskeleton elements in this model at concentrations distinctly below cytotoxic concentrations. Other compounds, like KCN, paraquat, and 3-NP selectively disrupt the balance between energy supply and demand of the neurons either by interacting with mitochondrial respiration or glycolysis. For these compounds cytoskeletal damage seemed to be secondary to the energy depletion. For NMDA, 2,5 hexandione and acrylamide, both mechanisms may contribute to the neuronal damage. In conclusion, primary cortical neuronal cultures of the rat are well suited to detect a neurotoxic potential and to differentiate its underlying mechanisms. Damage of the cytoskeleton may be considered as an endpoint mechanistically related to degenerative neuropathic effects.

PMID: 10900406 [PubMed - indexed for MEDLINE]


Neurosci Lett 1999 Oct 1;273(2):101-4

Localization of [3H]octylphosphonyl-labeled neuropathy target esterase by chicken nervous tissue autoradiography.

Kamijima M, Casida JE.

Department of Environmental Science, Policy and Management, University of California, Berkeley 94720-3112, USA.

Neuropathy target esterase (NTE) undergoes phosphorylation and aging as the initial steps in organophosphorus (OP)-induced delayed neuropathy (OPIDN). Localization of NTE is an important step in characterizing the mechanism of OPIDN. Earlier histochemical immunoreactivity or esterase assays localized NTE in areas of the brain and spinal cord rich in neuronal cell bodies and in the dorsal root ganglion. We use a more direct and quantitative autoradiographic approach of forming phosphorylated and aged [3H]octylphosphonyl-NTE on treatment with the highly potent [octyl-3H]octyl-4H-1,3,2-benzodioxaphosphorin 2-oxide to determine NTE as the labeling site resistant to the non-neuropathic paraoxon and sensitive to the neuropathic mipafox. NTE is observed in the cerebral cortical layer, some layers of the optic tectum, the gray matter of the spinal cord and the sensory neurons of the dorsal root ganglion to a higher extent than in adjacent areas.

PMID: 10505626 [PubMed - indexed for MEDLINE]


Toxicol Lett 1998 Sep 15;98(3):139-46

Organophosphorus neuropathy target esterase inhibitors selectively block outgrowth of neurite-like and cell processes in cultured cells.

Li W, Casida JE.

Department of Environmental Science, Policy and Management, University of California, Berkeley 94720-3112, USA.

This study compares two direct-acting neuropathy target esterase (NTE) inhibitors (mipafox and 2-octyl-4H-1,3,2-benzodioxophosphorin 2-oxide (OBDPO)), a metabolic precursor to an NTE inhibitor (tri-o-cresyl phosphate or TOCP) and a potent acetylcholinesterase inhibitor (chlorpyrifos oxon or CPO) for their effects on outgrowth of neurite-like and cell processes and on viability in differentiated cultured cells (rat adrenal pheochromocytoma (PC-12) and brain glial tumor (C6)). The direct-acting NTE inhibitors block process outgrowth by 50% or more at 50-100 microM for OBDPO and 100-200 microM for mipafox, well below their cytotoxic levels (EC50 values, 445-474 microM for OBDPO and 1021-1613 microM for mipafox). In contrast, the effects on process development for TOCP and CPO parallel their cytotoxicity. These findings suggest that inhibition of neurite-like and cell process outgrowth by OBDPO and mipafox may be associated with NTE inhibition.

PMID: 9788582 [PubMed - indexed for MEDLINE]


Gen Pharmacol 1997 Apr;28(4):567-75

Mipafox differential inhibition assay for heart muscle cholinesterases: substrate specificity and inhibition of three isoenzymes by physostigmine and quinidine.

Chemnitius JM, Haselmeyer KH, Gonska BD, Kreuzer H, Zech R.

Department of Cardiology, Georg-August University, Gottingen, Germany.

1. A differential inhibition assay was developed for the quantitative determination of cholinesterase isoenzymes acetylcholinesterase (AChE; EC, cholinesterase (BChE; EC, and atypical cholinesterase in small samples of left ventricular porcine heart muscle.
2. The assay is based on kinetic analysis of irreversible cholinesterase inhibition by the organophosphorus compound N,N'-di-isopropylphosphorodiamidic fluoride (mipafox). With acetylthiocholine (ASCh) as substrate (1.25 mM), hydrolytic activities (A) of cholinesterase isoenzymes were determined after preincubation (60 min, 25 degrees C) of heart muscle samples with either saline (total activity, A tau), 7 microM mipafox (AM1), or 0.8 mM mipafox (AM2): (BChE) = A tau-AM1, (AChE) = AM1-AM2, (Atypical ChE) = AM2.
3. The mipafox differential inhibition assay was used to determine the substrate hydrolysis patterns of myocardial cholinesterases with ASCh, acetyl-beta-methylthiocholine (A beta MSCh), propionylthiocholine (PSCh), and butyrylthiocholine (BSCh). The substrate specificities of myocardial AChE and BChE resemble those of erythrocyte AChE and serum BChE, respectively. Michaelis constants KM with ASCh were determined to be 0.15 mM for AChE and 1.4 mM for BChE.
4. Atypical cholinesterase, in respect to both substrate specificity and inhibition kinetics, differs from cholinesterase activities of vertebrate tissue and, up to now, could be identified exclusively in heart muscle. The enzyme's Michaelis constant with ASCh was determined to be 4.0 mM. 5. The reversible inhibitory effects of physostigmine (eserine) and quinidine on heart muscle cholinesterases were investigated using the differential inhibition assay. With all three isoenzymes, the inhibition kinetics of both substances were strictly competitive. The physostigmine inhibition of AChE was most pronounced (Ki = 0.22 microM). Quinidine most potently inhibited myocardial BChE (Ki = 35 microM).

PMID: 9147026 [PubMed - indexed for MEDLINE]


Hum Exp Toxicol 1997 Feb;16(2):72-8

Effects of multiple doses of organophosphates on evoked potentials in mouse diaphragm.

Kelly SS, de Blaquiere GE, Williams FM, Blain PG.

Department of Environmental and Occupational Medicine, Medical School, University of Newcastle upon Tyne.

1. Male albino mice were injected s.c. with an organophosphate (mipafox, ecothiopate or paraoxon). Treatments were either a single injection or multiple daily injections with lower doses for 5 or 8 days. At 3 h after injection the activity of brain and diaphragm acetylcholinesterase and of brain neuropathy target esterase (NTE) was measured. Also measured in the diaphragm at 3 h post dose was the duration of spontaneous miniature endplate potentials (eMEPPs), recorded extracellularly.
2. At 7 and 28 days after dosing action potentials and evoked endplate potentials, produced by stimulating the phrenic nerve at 30 Hz, were recorded in diaphragm muscle. The amplitudes, time-course and latencies of these potentials were measured and the variability of latencies (jitter) was calculated.
3. Single doses of mipafox (20 mg/kg), ecothiopate (0.192 mg/kg) or paraoxon (0.415 mg/kg) in the mouse produced ca. 70% inhibition of diaphragm acetylcholinesterase at 3 h after dosing. All three OPs produced a prolongation of the half-decay times of eMEPPs.
4. All three OPs in the above single doses produced increased muscle action potential (postjunctional) jitter but only mipafox produced an increase in endplate potential (prejunctional) jitter. Mipafox in a slightly reduced single dose (17.5 mg/kg) had no effect on prejunctional or postjunctional jitter.
5. Multiple dosing with mipafox (8 mg/kg daily for 5 days) increased both postjunctional and prejunctional jitter at both 7 and 28 days after the end of dosing. After multiple dosing with mipafox (5 mg/kg daily for 5 days) postjunctional (but not prejunctional) jitter was increased. Multiple doses of paraoxon (0.166 mg/kg daily for 5 days) or ecothiopate (0.76 mg/kg daily for 5 days) increased prejunctional and postjunctional jitter.
6. Depending on the dosing regime, all three OPs tested were capable of increasing both prejunctional and postjunctional jitter. Neither ecothiopate nor paraoxon inhibited NTE, so this prejunctional effect is not likely to be related to 'classical' OP-induced delayed neuropathy. The prejunctional effects may be related to long-term inhibition of acetylcholinesterase and the triggering mechanism for increase in prejunctional jitter may involve a relationship between the inhibition of acetylcholinesterase and the time for which it is inhibited. The differences between the time-courses of increases in prejunctional and postjunctional jitter and the differential effects of the different multiple dosing regimes indicate that it is likely that the triggering relationship between enzyme inhibition and time is different for prejunctional and postjunctional effects.

PMID: 9051411 [PubMed - indexed for MEDLINE]


Human and Experimental Toxicology, Vol. 16, No. 2, pages 72-78, 14 references, 1997

Effects of Multiple Doses of Organophosphates on Evoked Potentials in Mouse Diaphragm

Kelly SS, de Blaquiere GE, Williams FM, Blain PG

Abstract: The effects of multiple low doses of ecothiopate (513100), paraoxon (311455), and mipafox (371868) on evoked diaphragm potentials were studied in mice. Male albino-mice were injected subcutaneously once with 0 or 20mg/kg mipafox, 0.192mg/kg ecothiopate, or 0.415mg/kg paraoxon or with 8mg/kg mipafox daily for 5 days or 5mg/kg mipafox daily for 8 days, or 0.076mg/kg ecothiopate or 0.166mg/kg paraoxon daily for 5 days. The mice were killed 3 hours (hr) or 7 or 28 days after injection and their diaphragms removed. Spontaneous miniature endplate potentials (eMEPPs) were recorded externally in animals killed after 3hr. In animals killed after 7 and 28 days post injection, action potentials (APs) and endplate potentials (EPPs) evoked by stimulating the phrenic nerve at 30 hertz were recorded. The APs were used as an indicator of postjunctional jitter and the EPPs as a marker of prejunctional jitter. The single doses of the three compounds significantly prolonged the half decay times of the eMEPPs. Paraoxon was the most effective, followed by ecothiopate and mipafox. The single doses of all three compounds significantly increased the APs. Only mipafox induced an increase in the EPP. Dosing with 8mg/kg mipafox for 5 days increased the AP and EPP at both the 7 and 28 day time points. Dosing with 5mg/kg mipafox for 8 days increased the AP, but not the EPP. Paraoxon and ecothiopate significantly increased the AP and EPP at both time points. The authors conclude that depending on the dosing protocol, mipafox, paraoxon, and ecothiopate increase prejunctional and postjunctional jitter. Since neither ecothiopate nor paraoxon inhibited brain neuropathy target esterase activity in an associated study, the prejunctional effect is not likely to reflect the 'classical' organophosphate induced delayed neuropathy syndrome. The prejunctional effect may be related to long term inhibition of brain and diaphragm acetylcholinesterase since all three compounds are able to inhibit brain and diaphragm acetylcholinesterase activity.


Hum Exp Toxicol 1997 Feb;16(2):67-71

Erratum in:

  • Hum Exp Toxicol 1997 Nov;16(11):690

The effects of multiple low doses of organophosphates on target enzymes in brain and diaphragm in the mouse.

Williams FM, Charlton C, de Blaquiere GE, Mutch E, Kelly SS, Blain PG.

Department of Environmental and Occupational Medicine, Medical School, Newcastle University, UK.

1. Multiple low doses of the direct acting organophosphates, ecothiopate, paraoxon and mipafox produced persistent and additive inhibition of diaphragm acetylcholinesterase. Paraoxon and mipafox had similar effects on brain acetylcholinesterase. There was greater recovery from inhibition between doses for paraoxon and ecothiopate than for mipafox.
2. Ecothiopate did not inhibit brain acetylcholinesterase but there was a small increase in activity.
3. Mipafox also had a cumulative inhibitory effect on brain neuropathy target esterase.
4. These results have particular implication for the use of multiple low doses of organophosphates occupationally by man.

PMID: 9051410 [PubMed - indexed for MEDLINE]


Toxicol Lett 1995 Nov;81(1):45-53

Comparative studies of two organophosphorus compounds in the mouse.

Mutch E, Kelly SS, Blain PG, Williams FM.

Department of Environmental and Occupational Medicine, Medical School, Newcastle University, UK.

A rodent model, the albino mouse, was used to investigate the in vitro and in vivo capacity of 2 organophosphate (OP) compounds, mipafox and ecothiopate, to inhibit enzymes considered to be involved in the mechanisms of OP toxicity. Mipafox and ecothiopate were chosen as model compounds because the former can produce a delayed neuropathy whereas the latter does not. Mipafox (110 mumol/kg, s.c.) inhibited brain acetylcholinesterase (AChE), neuropathy target esterase (NTE) and phenylvalerate hydrolases by 58, 64 and 65%, while diaphragm AChE and phenylvalerate hydrolases were inhibited by 66 and 80%, respectively. In contrast, ecothiopate (0.5 mumol/kg) had no effect on brain NTE or on brain or diaphragm phenylvalerate hydrolases. At the same time, diaphragm AChE was inhibited by 60% while brain AChE activity had increased by 15% of control. Mipafox was a potent inhibitor of AChE and NTE in vitro. Although ecothiopate was a highly potent anti-ChE in vitro, it had no inhibitory effect on NTE

PMID: 8525498 [PubMed - indexed for MEDLINE]


Fundam Appl Toxicol 1995 Jan;24(1):94-101

Comparison of the relative inhibition of acetylcholinesterase and neuropathy target esterase in rats and hens given cholinesterase inhibitors.

Ehrich M, Jortner BS, Padilla S.

Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg 24061.

Inhibition of neuropathy target esterase (NTE, neurotoxic esterase) and acetylcholinesterase (AChE) activities was compared in brain and spinal cords of adult While Leghorn hens and adult male Long Evan rats 4-48 hr after administration of triortho-tolyl phosphate (TOTP po, 50-500 mg/kg to hens; 300-1000 mg/kg to rats), phenyl saligenin phosphate (PSP im 0.1-2.5 mg/kg to hens; 5-24 mg/kg to rats), mipafox (3-30 mg/kg ip to hens and rats), diisopropyl phosphorofluoridate (DFP sc, 0.25-1.0 mg/kg to hens; 1-3 mg/kg to rats), dichlorvos (5-60 mg/kg ip to hens; 600-2000 mg/kg to rats), and carbaryl (300-560 mg/kg ip to hens; 30-170 mg/kg to rats). Inhibitions of NTE and AChE were dose-related after administration of all compounds to both species. Hens and rats given TOTP, PSP, mipafox, and DFP demonstrated delayed neuropathy 3 weeks later, with spinal cord lesions and clinical signs more notable in hens. Ratios of NTE/AChE inhibition in hen spinal cord, averaged over the doses used, were 2.6 after TOTP, 5.2 after PSP, 1.3 after mipafox, and 0.9 after DFP, which contrast with 0.53 after dichlorvos, 1.0 after malathion, and 0.46 after carbaryl. Rat NTE/AChE inhibition ratios were 0.9 after TOTP, 2.6 after PSP, 1.0 after mipafox, 0.62 after DFP, 1.3 after dichlorvos, 2.2 after malathion, and 1.1 after carbaryl. The lower NTE/AChE ratios in rats given dosages of the four organophosphorus compounds that caused delayed neuropathy interferred with survival, an effect that was not a problem in hens.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 7713347 [PubMed - indexed for MEDLINE]


Indian J Physiol Pharmacol 1995 Jan;39(1):47-50

A comparative study of delayed neurotoxicity in hens following repeated administration of organophosphorus compounds.

Husain K, Pant SC, Raza SK, Singh R, Das Gupta S.

Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior.

Hens treated with Mipafox (10 mg/kg, sc), sarin (50 micrograms/kg, sc) or parathion (1 mg/kg, sc) daily for 10 days exhibited severe, moderate and no ataxia respectively on 14th day after the start of exposure. The neurotoxic esterase (NTE) activity was significantly inhibited in the brain, spinal cord and platelets of hens treated with mipafox or sarin whereas no change was noticed with parathion treatment. All three compounds significantly inhibited acetylcholinesterase (AChE) activity in the platelets. Spinal cord of hens treated with mipafox, sarin or parathion showed axonal degeneration heavy, moderate and none respectively. It is concluded that repeated administration of equitoxic doses of mipafox, sarin and parathion to hens are marked, moderate and non-delayed neurotoxic respectively.

PMID: 7705869 [PubMed - indexed for MEDLINE]


J Biochem Toxicol 1994 Jun;9(3):145-52

Partial characterization of neuropathy target esterase and related phenyl valerate esterases from bovine adrenal medulla.

Sogorb MA, Viniegra S, Reig JA, Vilanova E.

Department of Neurochemistry, University of Alicante, Spain.

The mechanism by which organophosphorus-induced delayed polyneuropathy is induced relates to the specific inhibition and subsequent modification ("aging") of a protein known as neuropathy target esterase (NTE), operatively defined as paraoxon-resistant and mipafox-sensitive phenyl valerate (PV) esterase activity. This protein has fundamentally been investigated in hen brain, the latter being the habitually employed OPIDP study model. In the present article, a partial characterization is made of the NTE and other related PV esterases in the bovine adrenal medulla and brain; NTE sensitivity to the neurotoxic organophosphorus compound mipafox is investigated, and its subcellular distribution is studied. The NTE activity of the adrenal medulla was found to be the highest of those among the tissues studied to date (5000 +/- 1400 mU/g tissue; +/- SD, n = 12). This activity represented 93% of the PV esterase activity resistant to 40 microM paraoxon in the particulate fraction of the adrenal medulla and approximately 50% of total PV esterase activity. In the bovine brain, these proportions were 72 and 26%, respectively, i.e., similar to those described in hen brain. The mipafox inhibition curve of PV esterase activity resistant to 40 microM paraoxon in the particulate fraction of the adrenal medulla suggests that NTE activity fundamentally comprises a mipafox-sensitive component with an I50 of 6.39 microM at 30 minutes, which is similar to the value reported in hen brain. NTE activity in the bovine adrenal medulla is almost exclusively limited to the particulate fraction, the microsomal fraction, plasma membrane, and chromaffin granule-enriched fractions being the highest in terms of specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 7983680 [PubMed - indexed for MEDLINE]


Toxicol Lett 1994 Mar;71(1):47-51

In vivo inhibition by mipafox of soluble and particulate forms of organophosphorus neuropathy target esterase (NTE) in hen sciatic nerve.

Carrera V, Diaz-Alejo N, Sogorb MA, Vicedo JL, Vilanova E.

Departamento de Neuroquimica, Universidad de Alicante, Spain.

Neuropathy target esterase (NTE) is a protein suggested to be involved in the initiation mechanism of organophosphorus-induced delayed neuropathy (OPIDP). We previously described two different forms of NTE activity in hen sciatic nerve: a particulate form (P-NTE) representing 40-50% of total NTE activity in sciatic nerve, and a remaining soluble component (S-NTE). In brain tissue on the other hand, more than 90% of NTE activity was recovered as P-NTE. In this work we studied the in vivo inhibition of both NTE forms with different doses of mipafox and the results were compared with sensitivity to mipafox in vitro. The highest dose with no observable neuropathic effects (1.5 mg/kg mipafox p.o.) inhibited 33% P-NTE and 55% S-NTE activity. The difference between P-NTE and S-NTE activity was statistically significant (P < 0.001, n = 9). Higher doses (3 mg/kg) induced neuropathy and inhibited NTE more than 75%, but differences between P- and S-NTE were not significant (P > 0.5). The greater inhibition of S-NTE than P-NTE in vivo contrasts with the observation that S-NTE is less sensitive in vitro.

PMID: 8140588 [PubMed - indexed for MEDLINE]


Neurotoxicology 1994 Summer;15(2):341-8

Acute and subacute organophosphate poisoning in the rat.

De Bleecker J, Lison D, Van Den Abeele K, Willems J, De Reuck J.

Neurology Department, University Hospital, Gent, Belgium.

The intermediate syndrome in organophosphate poisoning is clinically characterized by weakness in the territory of cranial nerves, weakness of respiratory, neck and proximal limb muscles, and depressed deep tendon reflexes. It occurs between the acute cholinergic crisis and the usual onset of organophosphate-induced delayed neurotoxicity. The weakness has been ascribed to muscle fiber necrosis. Fenthion has been the most common cause. This study assesses the occurrence of the necrotizing myopathy in rats in relation to the clinical course and the acetylcholinesterase (AChE) inhibition after poisoning with organophosphates representative for each of the major types of organophosphate-related neurotoxicity. Marked differences are noted in the duration of cholinergic symptoms and of AChE inhibition after either paraoxon and mipafox, or fenthion poisoning. The necrotizing myopathy begins shortly after the initial decline in AChE activity with all organophosphates studied. Maximal muscle involvement occurs within the first 2 days of the poisoning with all organophosphates studied. The myopathy is not aggravated by a further decline in AChE activity in fenthion poisoning. Our data argues against the monophasic necrotizing myopathy being the cause of the intermediate syndrome, and is suggestive of persistent AChE inhibition being involved.

PMID: 7991223 [PubMed - indexed for MEDLINE]


Arch Toxicol 1994;68(7):459-66

Electrophysiological and biochemical effects following single doses of organophosphates in the mouse.

Kelly SS, Mutch E, Williams FM, Blain PG.

Department of Environmental and Occupational Medicine, The Medical School, University of Newcastle upon Tyne, UK.

Single doses of organophosphates (mipafox or ecothiopate) were given subcutaneously to mice. At intervals up to 77 days after dosing animals were killed and muscle action potentials and endplate potentials were recorded intracellularly in mouse phrenic-nerve/hemidiaphragm preparations. Activities of acetylcholinesterase and neuropathy target esterase in brain and acetylcholinesterase in diaphragm were also measured. Mipafox (0.11 mmol/kg), a neurotoxic organophosphate, produced an increase in prejunctional jitter (i.e. the variabilities of the latencies) of endplate potentials. This increase began 14-21 days after administration and lasted more than 23 days. No clinical signs of neuropathy were observed during this study. Mipafox also produced an increase in postjunctional (muscle action potential) jitter. Mipafox inhibited brain and diaphragm acetylcholinesterase and brain neuropathy target esterase. By comparison, a non-neurotoxic organophosphate, ecothiopate (0.5 mumol/kg), was a potent inhibitor of diaphragm acetylcholinesterase and produced a large increase in postjunctional jitter but ecothiopate did not inhibit brain neuropathy target esterase and had no effect on prejunctional jitter. Doses were chosen so that the inhibition of diaphragm acetylcholinesterase by each of the two organophosphates was similar. It is concluded that the neurotoxic organophosphate, mipafox, produced measurable changes in nerve function. These long-term changes may represent a new phenomenon, unrelated to the classical organophosphate induced delayed neuropathy. Alternatively, they may represent a neuropathic process which precedes or is below the threshold for clinical signs.

PMID: 7979963 [PubMed - indexed for MEDLINE]


Journal of the American College of Toxicology, Vol. 12, No. 1, pages 55-68, 35 references, 1993

Short-Term Clinical and Neuropathologic Effects of Cholinesterase Inhibitors in Rats

Ehrich M, Shell L, Rozum M, Jortner BS

Abstract: Short term clinical and neuropathological effects induced by tri-ortho-tolyl-phosphate (78308) (TOTP), diisopropyl-fluorophosphate (55-91-4) (DFP), phenyl-saligenin-phosphate (4081236) (PSP), mipafox (371-86-8), malathion (121755), dichlorvos (62737), and carbaryl (63252) were studied in rats. Male Long-Evans-rats were administered 300 to 1000mg/kg TOTP or 300 to 2000mg/kg malathion orally, injected intramuscularly with 5 to 24mg/kg PSP, injected subcutaneously with 1 to 3mg/kg DFP, or injected intraperitoneally with 3 to 30mg/kg mipafox or dichlorvos or 30 to 160mg/kg carbaryl. The rats were also treated with atropine-sulfate to protect against cholinergic symptoms. Selected rats were killed 4 hours after DFP, PSP, mipafox, dichlorvos, and carbaryl or 48 hours after TOTP and malathion and the brains and spinal cords were removed and assayed for acetylcholinesterase (AChE) and neurotoxic-esterase (NTE) activity. The remaining rats were weighed and evaluated on a functional observational battery (FOB) that measured motor activity and responses to being handled or approached 1, 7, 14, and 21 days after dosing. The rats were then killed and the brains, spinal cords, and tibial nerve branches leading to the gastrocnemius muscle were examined for histopathological changes. The highest doses of all compounds except PSP induced transient cholinergic symptoms and caused 8.3 to 61% mortality within 48 hours. The highest doses of TOTP, DFP, and malathion significantly decreased body weight after 14 days. All compounds caused dose related inhibitions of brain and spinal cord AChE and NTE activity. DFP was the most potent and PSP the least potent. All compounds induced significant changes in FOB parameters related to behavioral and central nervous system excitability 21 days after dosing. Mipafox, PSP, dichlorvos, and carbaryl induced these changes 1 day after dosing. TOTP, DFP, PSP, and mipafox caused mild to moderate myelinated fiber degeneration in the rostral fasciculus gracilis 21 days after dosing. Mipafox was the most potent. DFP also induced Wallerian like degeneration in the tibial nerve branches. Dichlorvos, malathion, and carbaryl did not cause any neurological changes. The authors conclude that some cholinesterase inhibitors cause behavioral changes even after cholinergic signs are no longer evident.


J Neurochem 1993 Dec;61(6):2164-8

Soluble and particulate organophosphorus neuropathy target esterase in brain and sciatic nerve of the hen, cat, rat, and chick.

Tormo N, Gimeno JR, Sogorb MA, Diaz-Alejo N, Vilanova E.

Department of Neurochemistry, Alicante University, Spain.

Considerable evidence exists suggesting that the so-called neuropathy target esterase (NTE) is involved in the mechanisms responsible for organophosphorus-induced delayed polyneuropathy (OPIDP). Earlier studies in the adult hen, the habitually employed experimental model in OPIDP, have shown that most NTE activity in the brain is centered in particulate fractions, whereas approximately 50% of this activity in the sciatic nerve is encountered in soluble form, with the rest being particulate NTE. In the present work, we have studied the particulate and soluble fractional distribution of paraoxon-resistant phenylvalerate esterase activity (B activity), paraoxon- and mipafox-resistant phenylvalerate esterase activity (C activity), and NTE activity (B-C) according to ultracentrifugation criteria (100,000 g for 1 h). To this effect, two sensitive (adult hen and cat) and two scarcely sensitive (rat and chick) models were used. In all four experimental models, the distribution pattern was qualitatively similar: B activity and total NTE were much greater in brain (900-2,300 nmol/min/g of tissue) than in sciatic nerve (50-100 nmol/min/g of tissue). The proportion of soluble NTE in brain was very low (< 2%), whereas its presence in sciatic nerve was substantial (30-50%). The NTE/B ratio in brain was high for the particulate fraction (> 60%) and low in the soluble fraction (7-30%); in sciatic nerve the ratio was about 50% in both fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 8245968 [PubMed - indexed for MEDLINE]


Toxicol Appl Pharmacol 1993 Oct;122(2):165-71

Interactions between neuropathy target esterase and its inhibitors and the development of polyneuropathy.

Lotti M, Moretto A, Capodicasa E, Bertolazzi M, Peraica M, Scapellato ML.

Universita degli Studi di Padova, Istituto di Medicina del Lavoro, Italy.

This paper combines new and old data in order to offer a modified perspective of the mechanism of organophosphate-induced delayed polyneuropathy. Neuropathy target esterase (NTE) is though to be the molecular target and neuropathy to be initiated with a two-step mechanism: progressive inhibition of NTE and aging of the phosphorylated enzyme. When neuropathic organophosphates modify more than 70% of NTE in this way, neuropathy develops 2 weeks later. Other chemicals producing an inhibited NTE, which is incapable of aging, were thought to be not neuropathic. When given before a challenging dose of a neuropathic organophosphate they protect animals from neuropathy. However, recent evidence indicates that aging may not always be essential in causing neuropathy. In fact, mipafox and methamidophos as well as certain classic protective inhibitors such as carbamate and sulfonyl fluoride form an inhibited NTE which apparently does not age and yet produces neuropathy. We propose that all NTE inhibitors may have the potential to cause neuropathy. In analogy with pharmacological models of drug-receptor interactions, NTE inhibitors might have variable intrinsic activities to initiate neuropathy once attached to the protein. Strong neuropathic chemicals require about 70% inhibition of NTE, others 80-90%, and the least potent almost 100%. These differences have been amplified by means of promotion. Different levels of NTE inhibition as caused by different compounds were promoted by the same dose of phenylmethanesulfonyl fluoride to similar degrees of ataxia. Conversely nearly complete NTE inhibitions obtained in chicks with different chemicals were promoted to varying severities of ataxia. Protection from delayed polyneuropathy by the least neuropathic inhibitors can be explained by their weak intrinsic activity: occupying NTE, they prevent the binding of more neuropathic compounds. Methamidophos represents a particular example because it is protective at lower doses and neuropathic at high doses. Moreover, the levels of NTE inhibited by methamidophos which can be promoted to neuropathy are lower than those required for classic protective chemicals and higher than those of classic neuropathic OPs. This suggests that methamidophos has an intermediate position between the most and the least neuropathic NTE inhibitors.

Publication Types:

  • Review
  • Review, Tutorial

PMID: 8211998 [PubMed - indexed for MEDLINE]


Chem Biol Interact 1993 Jun;87(1-3):431-7

Relationship of neuropathy target esterase inhibition to neuropathology and ataxia in hens given organophosphorus esters.

Ehrich M, Jortner BS, Padilla S.

Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg 24061.

Adult White Leghorn hens were acutely exposed to 3 dosages of the following organophosphorus compounds: mipafox, tri-ortho-tolyl phosphate (TOTP), phenyl saligenin phosphate, and diisopropylphosphorofluoridate (DFP). Neuropathy target esterase (NTE) activity was measured in brain and spinal cord 4 or 48 h after exposure. Ataxia was assessed using an 8-point rating scale on days 9 through 21 after administration, and neuropathological examination was conducted on samples collected from perfusion-fixed animals on day 21. Morphological alterations were indicated by lesion scores between 0 (no lesions) and 4 (diffuse involvement of spinal cord tracts and > 25% degeneration of peripheral nerve fibers). Dosages of mipafox (30 mg/kg i.p.), TOTP (500 mg/kg p.o.), phenyl saligenin phosphate (2.5 mg/kg i.m.) and DFP (1 mg/kg s.c.) that were capable of inhibiting NTE > 80% in both brain and spinal cord preceded ataxia which reached maximal levels (scores of 7-8), and development of lesions scored as 4. Hens were notably impaired (ataxia scores of 3-4) 21 days after administration of dosages of mipafox (3 and 6 mg/kg), TOTP (90 mg/kg), phenyl saligenin phosphate (0.1 and 0.2 mg/kg), and DFP (0.4 mg/kg) when spinal cord NTE was inhibited 40-75%. Lesions were, however, only noted in spinal cord and peripheral nerves of hens given TOTP or DFP (scores 1-3). These data indicate that inhibition of spinal cord NTE > 80% was predictive of severe ataxia and extensive pathology in the hen and that less NTE inhibition was indicative of less severe ataxia and a lower score for neuropathological damage.

PMID: 8343999 [PubMed - indexed for MEDLINE]


Chem Biol Interact 1993 Jun;87(1-3):425-30

Reactivation of phosphorodiamidated acetylcholinesterase and neuropathy target esterase by treatment of inhibited enzyme with potassium fluoride.

Milatovic D, Johnson MK.

MRC Toxicology Unit, University of Leicester, UK.

It has been thought that the phosphorus-enzyme bond in inhibited esterases inhibited by such agents as mipafox (N,N'-di-iso-propylphosphorodiamidate) was refractory to reactivating agents either because an 'aging' reaction occurs soon after inhibition or because the bond was intrinsically very strong. We have found that both acetylcholinesterase (AChE) and neuropathy target esterase (NTE) which had been inhibited with either mipafox or with a di-n-butylphosphorodiamidate could be reactivated by prolonged treatment with aqueous potassium fluoride (KF): the reaction proceeded with first-order kinetics. Furthermore there was no time-dependent loss of reactivatability (aging). Di-isopropylphosphoro-butyrylcholinesterase could be fully reactivated by this treatment but after 18 h to allow aging the monoisopropyl phosphoro-enzyme was totally refractory to KF. We conclude that it is likely that the mipafox-enzyme bond in inhibited NTE and AChE is relatively strong but that aging has not occurred. The local disturbance around the active site of NTE caused by attachment of the phosphorodiamidate molecule appears to be sufficient to initiate delayed neuropathy without necessity for an 'aging' reaction.

PMID: 8343998 [PubMed - indexed for MEDLINE]


Chem Biol Interact 1993 Jun;87(1-3):369-81

Biochemical properties and possible toxicological significance of various forms of NTE.

Vilanova E, Barril J, Carrera V.

Department of Neurochemistry, University of Alicante, Spain.

NTE (neuropathy target esterase) is considered to be the target for organophosphorus-induced delayed polyneuropathy and is operationally measured by radiolabelling or by determining its esteratic activity as the paraoxon-resistant mipafox-sensitive phosphorylable site(s). From electrophoresis and density gradient centrifugation using radiolabelling techniques, several phosphorylable sites have been described in hen brain that are paraoxon-resistant mipafox-sensitive; however, only the majority electrophoresis band (155 kDa) shows properties related with the aging reaction. Kinetic criteria have also suggested two components of brain NTE (NTEA and NTEB). Most brain NTE is recovered in the particulate microsomal fraction and only about 1% in soluble fraction. In sciatic nerve about 50%/50% activity is recovered as soluble (S-NTE) or particulate (P-NTE) forms. A similar distribution were observed in hen, cat, rat and young chick. The fixed time inhibition curves show that P-NTE is more sensitive to mipafox, DFP and hexyl-DCP than S-NTE, while the reverse is true for methamidophos. P-NTE fits properly to one sensitive component while S-NTE fits better to two sensitive component models, except in the case of methamidophos. In vivo, significant differences in the inhibition of P- and S-NTE by mipafox were found only when using low non-neuropathic dosing. The possible significance of different NTE forms are discussed.

Publication Types:

  • Review
  • Review, Tutorial

PMID: 8343994 [PubMed - indexed for MEDLINE]


J Appl Toxicol 1993 Mar-Apr;13(2):143-5

Delayed neurotoxic effect of sarin in mice after repeated inhalation exposure.

Husain K, Vijayaraghavan R, Pant SC, Raza SK, Pandey KS.

Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior, India.

Delayed neurotoxicity of sarin in mice after repeated inhalation exposure has been studied. Female mice exposed to atmospheric sarin (5 mg m-3 for 20 min) daily for 10 days developed muscular weakness of the limbs and slight ataxia on the 14th day after the start of the exposure. These changes were accompanied by significant inhibition of neurotoxic esterase (NTE) activity in the brain, spinal cord and platelets. Histopathology of the spinal cord of exposed animals showed focal axonal degeneration. These changes were comparatively less than in animals treated with the neurotoxic organophosphate, mipafox. Results from this study indicate that sarin may induce delayed neurotoxic effects in mice following repeated inhalation exposure.

PMID: 8486913 [PubMed - indexed for MEDLINE]


Toxicology and Applied Pharmacology, Vol. 117, No. 2, pages 218-225, 31 references, 1992

Local Application of Neuropathic Organophosphorus Compounds to Hen Sciatic Nerve: Inhibition of Neuropathy Target Esterase and Peripheral Neurological Impairments

Carrera V, Barril J, Mauricio M, Pellin M, Vilanova E

Abstract: Bioclinical effects induced by local application of neuropathic organophosphates to the sciatic nerve were studied in adult red-hens. Diisopropyl-phosphorofluoridate (55914) (DFP), mipafox (371868), cresylsaligenyl-phosphate (CSP), or phenylsaligenyl-phosphate (4081236) (PSP) were applied locally to 1 or 1.5 centimeter segments of the common trunk of surgically exposed sciatic nerves in one leg of each hen. Doses ranged up to 1790, 17500, 108, or 526 micrograms (microg), respectively. The contralateral leg served as the control. Fifteen minutes later, the treated segments, the adjacent proximal and distal portions, and the most distal segments of the peroneal branch were dissected out and assayed for neuropathy-target-esterase (NTE) activity. DFP and mipafox caused greater than 90% inhibition of NTE activity in the treated sciatic nerve segments. A 40% inhibition of NTE activity was induced by DFP and mipafox in the adjacent proximal and distal segments. Less than 20% NTE inhibition was induced in the terminal segments. PSP and CSP did not significantly affect sciatic NTE activity. Since only DFP and mipafox significantly inhibited NTE activity these were used to investigate clinical symptomatology and histopathological changes induced in the sciatic nerve. Hens with surgically exposed sciatic nerves were treated locally in one or both legs with 27 to 110microg DFP or 18 to 182microg mipafox. Some hens were pretreated with 30mg/kg phenylmethanesulfonyl-fluoride (PMSF) subcutaneously. Hens were observed for clinical signs of toxicity for 15 to 25 days then killed on day 25. Peroneal nerves were removed and examined for histopathological changes. All hens treated in both legs with DFP or mipafox lost the avian retraction reflex. Only birds treated with the maximum DFP or mipafox doses in both legs developed gait abnormalities. Following application to one leg only, hens treated with 110microg DFP showed loss of the avian retraction reflex. No clinical signs of toxicity were seen in birds pretreated with PMSF. DFP or mipafox caused axon swelling, accumulation of endoplasmic reticulum, and intraaxonal and intramyelinal vacuolation. The authors conclude that the peripheral neuropathological effects of locally applied DFP or mipafox appear to be mediated by their effects on NTE.


Res Commun Chem Pathol Pharmacol 1992 Nov;78(2):253-6

Are Wistar rats not susceptible to organophosphate-induced delayed neurotoxicity?

De Bleecker JL, Van Den Abeele KG, Willems JL, De Reuck JL.

Neurology Department, University Hospital, Ghent, Belgium.

Male Wistar rats were sacrificed 12 weeks after single exposure to various organophosphate compounds. Peripheral nerves and skeletal muscles were examined light microscopically for the occurrence of a delayed polyneuropathy. Although unequivocal morphological hallmarks of OPIDN had been demonstrated in other rat strains using similar doses of TOCP or mipafox, we were unable to demonstrate any abnormality with these compounds. Normal findings were also obtained with fenthion, the delayed neuropathic potential of which is debated, and with paraoxon or parathion, which are both highly unlikely to cause OPIDN. These data indicate that the Wistar rat strain is highly likely to be resistant to OPIDN.

PMID: 1335595 [PubMed - indexed for MEDLINE]

• Note from FAN: If this observation is correct it is important when judging the results of other neurotoxic substances tested on Wistar rats.


Comp Biochem Physiol C 1992 Jun;102(2):253-65

Initial characterization of the organophosphate acid anhydrase activity of the chicken, Gallus domesticus.

Westra BD, Landis WG.

Institute of Environmental Toxicology and Chemistry, Huxley College of Environmental Studies, Western Washington University, Bellingham 98225.

1. Supernatant solutions from kidney and liver homogenates of the chicken, Gallus domesticus, were found to hydrolyze the organophosphate (OP) compound diisopropylfluorophosphate (DFP). The activity on DFP as substrate was heat-inactivated and was characterized for temperature and pH optima, enzyme kinetics, and requirements for manganous ion.
2. Gel column chromatography indicated that the DFPase in both tissues is in the range of 82,100 to 93,300 D. This activity is strongly inhibited by N,N'-diisopropylphosphorodia-midofluoridate (mipafox).
3. The chicken has organophosphate acid (OPA) anhydrase activity comparable to other eucaryotic sources in its ability to hydrolyze DFP. Although birds may not have paraoxonase activity comparable to mammalian species, they do not differ significantly in the ability to hydrolyze DFP and probably related compounds.

PMID: 1358539 [PubMed - indexed for MEDLINE]


Mol Pharmacol 1992 Apr;41(4):750-6

Characterization of organophosphate interactions at N-methyl-D-aspartate receptors in brain synaptic membranes.

Johnson PS, Michaelis EK.

Department of Pharmacology and Toxicology, University of Kansas, Lawrence 66045.

Several competitive antagonists of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors are phosphonate analogs of L-glutamic acid. The position of the phosphonate has been shown to be important in the structure-activity relationships of these analogs. To investigate whether other phosphorous-containing compounds had activity at the NMDA receptor, several organophosphates were tested for the ability to inhibit the specific binding to brain synaptic membranes of 3-((+-)-2-carboxypiperazin-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP), a selective antagonist of NMDA receptors. Diisopropylfluorophosphate (DFP), dichlorvos, cyanophos, mipafox, and o-ethyl o-4-nitrophenyl phenylphosphonothioate are relatively potent inhibitors of [3H]CPP binding to synaptic membranes. The inhibition produced by DFP is selective for the NMDA subtype of excitatory amino acid receptors, is irreversible, and can be prevented by preincubation with excess CPP, 2-amino-7-phosphonoheptanoic acid, or L-glutamate. Rat brain synaptic membranes have a population of phosphonate-sensitive [3H]DFP binding sites that are covalently labeled by [3H]DFP. Two protein bands of synaptic membrane proteins subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis are labeled by [3H]DFP in a 2-amino-5-phosphonopentanoic acid-sensitive manner. These proteins have an average molecular size of 47-50 and 32 kDa. Proteins of nearly identical molecular sizes have been shown in other studies to be components of an NMDA receptor complex. These observations are indicative of an interaction between the organophosphates and the NMDA receptor protein complex and suggest that DFP may be another important pharmacological tool that can be used in the elucidation of the molecular structure of the NMDA receptor complex.

PMID: 1533269 [PubMed - indexed for MEDLINE]


Biochem Pharmacol 1992 Feb 18;43(4):823-9

Cholinesterases of heart muscle. Characterization of multiple enzymes using kinetics of irreversible organophosphorus inhibition.

Chemnitius JM, Chemnitius GC, Haselmeyer KH, Kreuzer H, Zech R.

Department of Cardiology, Georg-August-Universitat, Gottingen, Germany.

Cholinesterases of porcine left ventricular heart muscle were characterized with respect to substrate specificity and inhibition kinetics with organophosphorus inhibitors N,N'-di-isopropyl-phosphorodiamidic fluoride (Mipafox), di-isopropylphosphorofluoridate (DFP), and diethyl p-nitro-phenyl phosphate (Paraoxon). Total myocardial choline ester hydrolysing activity (234 nmol/min/g wet wt with 1.5 mM acetylthiocholine, ASCh; 216 nmol/min/g with 30 mM butyrylthiocholine, BSCh) was irreversibly and covalently inhibited by a wide range of inhibitor concentrations and, using weighted least-squares non-linear curve fitting, residual activities as determined with four different substrates in each case were fitted to a sum of up to four exponential functions. Quality of curve fitting as assessed by the sum of squares reached its optimum on the basis of a three component model, thus, indicating the presence of three different enzymes taking part in choline ester hydrolysis. Final classification of heart muscle cholinesterases was obtained according to both substrate hydrolysis patterns with ASCh, BSCh, acetyl-beta-methylthiocholine and propionylthiocholine, and second-order rate constants for the reaction with organophosphorus inhibitors Mipafox, DFP, and Paraoxon. One choline ester-hydrolysing enzyme was identified as acetylcholinesterase (EC, and one as butyrylcholinesterase (EC The third enzyme with relative resistance to organophosphorus inhibition was classified as atypical cholinesterase.

PMID: 1540236 [PubMed - indexed for MEDLINE]


Neurotoxicology 1992 Winter;13(4):745-55

Comparative dose-response studies of organophosphorus ester-induced delayed neuropathy in rats and hens administered mipafox.

Dyer KR, Jortner BS, Shell LG, Ehrich M.

Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State Institute, Blacksburg 24061.

A single injection of mipafox was administered to both Long-Evans hooded rats and White Leghorn hens in dosages which inhibited the activity of brain neurotoxic esterase 30-50%, 60-80%, or greater than 80% four hr after intoxication. All animals were monitored for clinical evidence of organophosphorus induced delayed neuropathy for 21 days, euthanatized, and regions of the nervous system were histologically evaluated. Only hens manifested clinical signs of neuropathy; however, light and electron microscopic lesions were present in the nervous systems of both species. In rats, these lesions were well developed in only the highest dosage group and confined to the rostral level of the fasciculus gracilis in the medulla oblongata. Swollen axons containing a single vacuole filled with flocculent material were the most prominent lesion in rats. Hens manifested more extensive and varied fiber breakdown in multiple spinal cord tracts, with the intensity of degeneration increasing with increasing dosages of mipafox. Both marked Wallerian-like degeneration and swollen axons filled with aggregates of cellular debris were observed in the nervous systems of hens. This study indicates that both rats and hens are susceptible to OPIDN. However, there are qualitative and quantitative differences in both clinical manifestations and histologic appearances between the two species.

PMID: 1302301 [PubMed - indexed for MEDLINE]


Neurotoxicology 1992 Winter;13(4):723-33

Comparative evolution of mipafox-induced delayed neuropathy in rats and hens.

Carboni D, Ehrich M, Dyer K, Jortner BS.

Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg 24061.

Adult male Long-Evans rats and White Leghorn hens were given 30 mg/kg mipafox ip. Administration of this organophosphorus ester resulted in > or = 89% inhibition of brain and spinal cord neurotoxic esterase activity in both species 4 hr after dosing. Our sequential, comparative study of the bilateral mipafox-induced neuropathy in the medulla and cervical spinal cord in hens and rats demonstrated that the rats had well-developed, vacuolar axonopathic lesions in the fasciculus gracilis by post-dosing day 7. Severely affected rats with such lesions were noted through day 21, but not subsequently (days 28 and 35). The hen had a slower developing, but more severe, consistent and longer lasting neuropathy than the rat. In these birds, lesions in the medulla and rostral cervical spinal cord levels were more extensive, involving large regions of both the spinocerebellar tracts and fasciculus gracilis. Neuropathic changes, including myelinated fiber axonopathy and Wallerian-like degeneration, were prominent from days 14 - 35 in hens, and were associated with prominent gliosis in the later stages.

PMID: 1302299 [PubMed - indexed for MEDLINE]


Biochem Int 1991 Aug;24(6):1051-6

Peripheral biochemical marker for organophosphate-induced delayed neurotoxicity.

Husain K.

Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior, India.

Neurotoxicesterase (NTE) activity was assayed in platelets of human and mice as well as in the brain of mice in vitro and in vivo. Mipafox, a well known organophosphate, to induce delayed neurotoxicity, at doses of 5, 10 and 15 mg/kg, subcutaneously, was used to examine the relationship between inhibition of brain and platelet NTE activity in mice. It was observed that the platelet NTE activity of mice was less than in humans. The optimum pH for both brain and platelet NTE of mice, and human platelets, was 8. The results indicate that mipafox produces a dose dependent inhibition of brain and platelet NTE activity in vivo and concentration dependent inhibition in vitro. It can be concluded that assay of platelet NTE can be a useful peripheral biochemical marker for organophosphate-induced delayed neurotoxicity.

PMID: 1781783 [PubMed - indexed for MEDLINE]


Biochem Pharmacol 1991 May 15;41(10):1497-504

Age sensitivity to organophosphate-induced delayed polyneuropathy. Biochemical and toxicological studies in developing chicks.

Moretto A, Capodicasa E, Peraica M, Lotti M.

Universita degli Studi di Padova, Istituto di Medicina del Lavoro, Italy.

Young animals are resistant to organophosphate-induced delayed polyneuropathy (OPIDP). The putative target protein in the nervous system for initiation of OPIDP in the adult hen is an enzyme called Neuropathy Target Esterase (NTE), which is dissected by selective inhibitors among nervous tissue esterases hydrolysing phenyl valerate (PV). We report here that the pool of PV-esterases sensitive to paraoxon was different in peripheral nerves of chicks as compared to that of hens while that of brain and spinal cord was not. NTE activity decreased with age in brain, spinal cord and peripheral nerve, but its sensitivity to several inhibitors remained unchanged. In the adult hen more than 70% inhibition of peripheral nerve NTE by neuropathic OPs is followed by deficit of retrograde axonal transport, axonal degeneration and paralysis. Similar NTE inhibition in 40-day-old or younger chicks however is not followed by changes in retrograde axonal transport nor by OPIDP. Chicks aged 60 to 80 days are only marginally sensitive to a single dose of DFP otherwise clearly neuropathic to hens. In vitro and in vivo phosphorylation by DFP and subsequent aging of brain NTE is similar both in chicks and in hens. The recovery of NTE activity monitored in vivo after inhibition by DFP is faster (half-life of about 3 days) in chick peripheral nerves as compared to chick brain, hen brain and hen peripheral nerve (half-life of about 5 days). It is concluded that the reduced sensitivity to OPIDP in chicks is not due to differences in OP-NTE interactions. The resistance might be explained by a more efficient repair mechanism, as suggested by the faster recovery of peripheral nerve NTE activity.

PMID: 2018554 [PubMed - indexed for MEDLINE]


No Abstract available

J Am Geriatr Soc 1991 Apr;39(4):438

Comment on:

PMID: 2010597 [PubMed - indexed for MEDLINE]


J Toxicol Environ Health 1990 Dec;31(4):261-73

Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride.

Pope CN, Padilla S.

School of Pharmacy, Northeast Louisiana University, Monroe 71209.

It is well known that pretreatment with the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF) can protect experimental animals from organophosphorus-induced delayed neurotoxicity (OPIDN), presumably by blocking the active site of neurotoxic esterase (NTE) such that binding and "aging" of the neuropathic OP is thwarted. We report here that while PMSF (60 mg/kg, sc) given 4 h before the neuropathic organophosphate (OP) mipafox (50 mg/kg, im) completely prevented the clinical expression of OPIDN in hens, the identical PMSF treatment markedly amplified the delayed neurotoxicity (relative to hens treated with OP only) if administered 4 h after mipafox (5 or 50 mg/kg, im). Moreover, in a separate experiment using diisopropylphosphorofluoridate (DFP) as the neurotoxicant in place of mipafox, posttreatment with PMSF 4 h after DFP (0.5 mg/kg) also accentuated the severity of ataxia. These data indicate that PMSF only protects against OPIDN if given prior to exposure to the neurotoxicant; treatment with PMSF after OP exposure critically exacerbates the delayed neurotoxicity from exposure to organophosphorus compounds.

PMID: 2254952 [PubMed - indexed for MEDLINE]


Neurotoxicology 1986 Spring;7(1):207-15

The correlation between neurotoxic esterase inhibition and mipafox-induced neuropathic damage in rats.

Veronesi B, Padilla S, Lyerly D.

The correlation between neuropathic damage and inhibition of neurotoxic esterase or neuropathy target enzyme (NTE) was examined in rats acutely exposed to Mipafox (N, N'-diisopropylphosphorodiamidofluoridate), a neurotoxic organophosphate. Brain and spinal cord NTE activities were measured in Long-Evans male rats 1 hr post-exposure to various dosages of Mipafox (ip, 1-15 mg/kg). These data were correlated with histologically scored cervical cord damage in a separate group of similarly dosed rats sampled 14-21 days post-exposure. Those dosages (greater than or equal to 10 mg/kg) that inhibited mean NTE activity in the spinal cord greater than or equal to 73% and brain greater than or equal to 67% of control values produced severe (greater than or equal to 3) cervical cord pathology in 85% of the rats. In contrast, dosages of Mipafox (less than or equal to 5 mg/kg) which inhibited mean NTE activity in spinal cord less than or equal to 61% and brain less than or equal to 60% produced this degree of cord damage in only 9% of the animals. These data indicate that a critical percentage of NTE inhibition in brain and spinal cord sampled shortly after Mipafox exposure can predict neuropathic damage in rats several weeks later.

PMID: 3714122 [PubMed - indexed for MEDLINE]


Methods Find Exp Clin Pharmacol 1985 Feb;7(2):79-81

Striatal neurochemical changes and motor dysfunction in mipafox-treated animals.

Matin MA, Hussain K.

5 mg/kg mipafox [N,N'-bis(1-methylethyl)phosphordiamidic fluoride] was administered s.c. daily for 60 days in rats. The animals developed motor dysfunction-muscle twitchings, fasciculations and slight ataxia towards the end of the experimental period; the motor dysfunction was accompanied by neurochemical changes in the corpus striatum which included significantly reduced levels of cholinesterase, neurotoxicesterase, dopamine and GABA. The neurochemical imbalance in the corpus striatum may be related to motor dysfunction in mipafox-treated animals.

PMID: 2859406 [PubMed - indexed for MEDLINE]


Toxicol Appl Pharmacol 1985 Jan;77(1):175-80

Neurotoxic esterase in rooster testis.

Lotti M, Wei ET, Spear RC, Becker CE.

Neurotoxic esterase (NTE) is the putative target protein in the nervous system for the initiation of organophosphorus-induced delayed neuropathy. Here it is reported that NTE activity is present in rooster testis. Complete titration of rooster testis phenyl valerate esterases with paraoxon shows that about 15% of the enzymic activity is resistant to paraoxon. NTE activity after complete mipafox titration accounts for 30% of paraoxon-resistant phenyl valerate esterases and corresponds to 7.93 +/- 0.39 nmol/min/mg of protein (mean +/- SD, n = 7). Testis NTE is inhibited in vitro similarly to brain NTE by several organophosphorus compounds. Subcellular fractionation studies of the testis indicate that most NTE activity is particle bound. Testis NTE is also inhibited in vivo by several organophosphorus esters but to a lesser extent than brain NTE. Birds doses with organophosphorus compounds, causing delayed neuropathy, became grossly ataxic, but no testicular pathology was noted by light microscopy in roosters killed 15 days after administration. Serum testosterone levels also measured 15 days after dosing were not different from those of a control group. Recovery of NTE activity was faster in testis than in brain (4 days vs 6 days to recover to 50% of initial activity) in animals that received a high dose of an organophosphorus ester which cause delayed neuropathy.

PMID: 3966239 [PubMed - indexed for MEDLINE]


Biochem Pharmacol 1983 Jun 1;32(11):1693-9

Brain cholinesterases. Differentiation of target enzymes for toxic organophosphorus compounds.

Chemnitius JM, Haselmeyer KH, Zech R.

Cholinesterases in hen brain were characterized with respect to inhibition kinetics and substrate specificity. Three organophosphorus inhibitors were used: diethyl p-nitrophenyl phosphate (Paraoxon, E 600), di-isopropylphosphorofluoridate (DFP), and N,N'-di-isopropylphosphorodiamidic fluoride (Mipafox). The kinetics of irreversible cholinesterase inhibition were studied using two substrates, acetylthiocholine and butyrylthiocholine. The inhibition curves were analysed by the method of iterative elimination of exponential functions. Final classification of the different enzymes was done by combining two inhibitors in sequential inhibition expts. Six cholinesterases were shown to hydrolyse choline esters in hen brain, one was identified as acetylcholinesterase (EC and one as cholinesterase (EC Four enzymes can be classified as intermediate type cholinesterases according to their substrate specificity and to their inhibition constants. The possible role of different brain cholinesterases for the development of atypical symptoms following organophosphate intoxication is discussed.

PMID: 6870909 [PubMed - indexed for MEDLINE]


Neurotoxicology 1983 Winter;4(4):143-55

No Abstract available

Multilevel studies of organophosphate toxicity.

Wilson BW, Ishikawa Y, Chow E, Cisson CM.

PMID: 6686868 [PubMed - indexed for MEDLINE]


Neurotoxicology 1982 Dec;3(4):269-84

No Abstract available

Historical perspective of organophosphorus ester-induced delayed neurotoxicity.

Metcalf RL.

PMID: 6190115 [PubMed - indexed for MEDLINE]


Biochem Pharmacol 1982 Mar 15;31(6):1117-21

Evidence for the existence of neurotoxic esterase in neural and lymphatic tissue of the adult hen.

Dudek BR, Richardson RJ.

Hen brain and spinal cord contain a number of esterases that hydrolyze phenyl valerate (PV). Most of this activity is sensitive to inhibition by micromolar concentrations of paraoxon. Included among the paraoxon-resistant esterases is neurotoxic esterase (NTE), which is inhibited in vivo and in vitro by certain organophosphorus compounds, such as mipafox, which cause delayed neurotoxicity. Since published information on the NTE content of non-neural tissues was heretofore lacking, a comprehensive study was undertaken of the occurrence of this enzyme in tissues of the adult hen (Gallus gallus domesticus), the species of choice in the study of organophosphorus-induced delayed neurotoxicity. Complete differential titration curves of PV esterase activity were obtained by preincubation of each tissue homogenate with a wide range of concentrations of paraoxon, a non-neurotoxic compound, plus or minus mipafox, a neurotoxic compound, followed by PV esterase assay. Brain NTE activity was determined to be 2426 +/- 104 nmoles.min-1.(g wet weight)-1 (mean +/- S.E.M.). Titration of other tissues resulted in the following NTE activities, expressed as percentages of brain NTE activity: spinal cord (21%), peripheral nerve (1.7%), gastrocnemius muscle (0%), pectoralis muscle (0%), heart (4%), liver (0%), kidney (0%), spleen (70%), spleen lymphocytes (26%), and blood lymphocytes (24%). Using an abbreviated procedure, erythrocytes and plasma showed no NTE activity. These results indicate that NTE has limited distribution among the tissues of the adult hen and is present in lymphatic as well as neural tissue.

PMID: 7082366 [PubMed - indexed for MEDLINE]


J Environ Sci Health B 1980;15(2):207-17

Subcellular distribution of neurotoxic esterase activity in lamb and mouse brain.

Soliman SA, El-Sebae AE, Curely A, Ahmed NS.

Brain tissue samples of mice (7.6 g from 25 mouse brains and lamb (25 g) were homogenized and subcellular fractions prepared in order to assay the distribution of neurotoxic esterase (NTE) activity. The specific inhibitor, N,N-diisopropylphosphorodiamidic fluoride (mipafox) was synthesized and purified. Maximum specific activity of NTE was reached in the microsomal fraction (110,000 g) while the enzyme activity in the soluble fraction (110,000 g) was extremely low. This subcellular distribution of NTE activity in mammal brains is an original contribution. Brain microsomal fraction is suggested to be a more reliable source for the highest activity of NTE. The specific activity of NTE of lamb brain was much higher than that of mouse brain. This might help interpretation of the characteristic species variation in susceptibility to NTE inhibitors which are known to be potent delayed neurotoxic agents.

PMID: 7365208 [PubMed - indexed for MEDLINE]

Arch Biochem Biophys 1980 Apr 1;200(2):434-43

Identification and subcellular localization of catalase activity in bovine adrenal medulla and cortex.

Pazoles CJ, Claggett CE, Creutz CE, Pollard HB, Weinbach EC.

PMID: 7436413 [PubMed - indexed for MEDLINE]


J Neurochem 1967 May;14(5):479-88

Effects of bis-(monoisopropylamino)-fluorophosphine oxide (Mipafox) and of starvation on the lipids in the nervous system of the hen.

Joel CD, Moser HW, Majno G, Karnovsky ML.

PMID: 6025618 [PubMed - indexed for MEDLINE]

Br Med J, 1:1068 1953.

Paralysis following poisoning by a new organic phosphorus insecticide (mipafox): report of two cases.

Bidstrup PL et al.

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