Mipafox (371-86-8) Abstracts. Fluoride Action Network Pesticide Project.

Abstracts
Mipafox
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

Structure:

Wallerian degeneration
Reference

Synonyms:
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.

Bibliography:

• 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

Address:
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) .
Order by: phone at 1-800-553-NTIS (U.S. customers); (703-)605-6000 (other countries); fax at (703)-605-6900; and email at orders@ntis.gov. NTIS is located at 5285 Port Royal Road, Springfield, VA, 22161, USA.

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.

Keywords:
Crystal structure
*Organophosphates
Three dimensional
X ray spectra
Chemical warfare agents
Crystallography
Enzyme inhibitors
Anhydrolases
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.

Keywords:
Genetics
Organophosphates
Pesticides
Pseudomonas
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.

Keywords:
Organophosphate insecticides
Toxicity

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.

Keywords:
Cholinesterase inhibitors
Nervous system
Pathology
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.

Keywords:
Toxicity
Nervous system
Neuroblastoma
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.

Keywords:
Toxic substances
Toxicity
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.

Keywords:
Acids
Gd agent
Hydrolases
Hydrolysis
Detoxification
Organophosphorus acid anhydrolases
Thermophilic bacterium
Soman
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

Keywords:
Nervous system
Organophosphorus compounds
Toxicity
Antidotes
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.

Keywords:
Esterase
Toxicology
Neurotoxicology
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.

Keywords:
Fluorides
Toxicology
Neuropathy
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.

Keywords:
Inhibitors
Esterases
Mipafox
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.

Keywords:
Esterases
Toxicology
Brain
Inhibitors

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.

Keywords:
Toxicity
Nerve cells
Organophosphates
Amides
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]

http://pubs.acs.org/cgi-bin/abstract.cgi/crtoec/2006/19/i02/abs/tx050342o.html

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.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15897155&query_hl=2
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]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15035642
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]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15094302&query_hl=2
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]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15205030&query_hl=2
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]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15177652&query_hl=2
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]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14637373
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]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12791540&dopt=Abstract
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]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12639502&dopt=Abstract
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]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12765233&dopt=Abstract
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]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12769600&dopt=Abstract
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]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11180931&dopt=Abstract
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]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10900406&dopt=Abstract
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]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10505626&dopt=Abstract
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]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9788582&dopt=Abstract
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 inh