Testing for Organophosphate‐Induced Delayed Polyneuropathy

Angelo Moretto1

1 Università degli Studi di Padova, Padova, Italy
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.5
DOI:  10.1002/0471140856.tx1105s00
Online Posting Date:  May, 2001
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Organophosphorous compounds may cause two distinct types of toxicity: acute cholinergic toxicity and organophosphate‐induced delayed polyneuropathy (OPIDP). The ability of a compound to cause OPIDP is assessed as described by administering the compound to hens and screening the brain, spinal cord, and peripheral nerves for neuropathy target esterase activity to detect OPIDP and acetylcholinesterase activity to rule out the acute toxicity. This assay can also be used as part of a screen for protective agents.

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Table of Contents

  • Basic Protocol 1: Induction and Evaluation of OPIDP in Hens
  • Basic Protocol 2: Determination of Neuropathy Target Esterase Activity
  • Alternate Protocol 1: Determination of the In Vitro Inhibitory Power of a Compound on NTE
  • Reagents and Solutions
  • Commentary
  • Tables
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Basic Protocol 1: Induction and Evaluation of OPIDP in Hens

  • Adult domestic laying hens (>6 months of age, preferably 8 to 14 months), standard size, breeds, and strains
  • Protective compound: e.g. phenylmethylsulfonyl fluoride (PMSF)
  • Organophosphate to be tested
  • Saline or glycerol formal (or other vehicles such as dimethylsulfoxide or corn oil)
  • 20 mg/ml atropine sulfate (e.g., Aldrich) in saline
  • 0.1 mg/ml eserine hemisulfate (e.g., Sigma) in saline
  • 200 mg/ml pyridine‐2‐aldoxime‐1‐methanesulfonate (e.g., Sigma; or other oxime) in saline
  • 50 mg/ml ketamine in saline
  • Halothane
  • Oxygen
  • Heparin
  • 100 mM sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • 4% paraformaldehyde, pH 7.4 (see recipe)
  • 4% formalin: dilute 40% commercial formalin solution to 4% with 100 mM sodium phosphate buffer, pH 7.4
  • Animal house, equipped to rear adult hens under conditions that permit free mobility
  • Gavage needles
  • 21/22‐G needles for intraperitoneal, subcutaneous, and intramuscular injection
  • Vaporizer for anesthetic gases
  • Anesthetizing box (see recipe)
  • Wooden dissecting board appropriate for hens
  • Dissecting equipment including:
  •  Scalpels
  •  Forceps
  •  Bone cutter
  •  Heavy and small scissors
  • Perfusion apparatus: peristaltic pump (50 to 200 ml/min) with connecting tubes and a cannula (a gavage needle can be used)
  • Large (>5 liter) container for the perfusion solution, with faucet on bottom
  • 5‐ to 50‐ml plastic containers with leak‐proof caps to store tissues sampled for histology

Basic Protocol 2: Determination of Neuropathy Target Esterase Activity

  • Adult domestic laying hens (>6 months of age, preferably 8 to 14 months), standard size, breeds, and strains
  • Organophosphate (OP) to be tested, in appropriate solvent (see protocol 1)
  • Tris/EDTA buffer (see recipe), ice‐cold
  • 10 mM mipafox stock solution (see recipe)
  • 10 mM paraoxon stock solution (see recipe)
  • 15 and 30 mg/ml phenyl valerate (PV) stock solutions (see recipe)
  • 0.03% (v/v) Triton X‐100 in double‐distilled water
  • SDS/AAP solution (see recipe)
  • 0.4% (w/v) K 3Fe(CN) 6 in double‐distilled water (stable for months at room temperature)
  • Modified Koenig buffer: 0.7 M glycine/0.05 M triethylamine⋅HCl, pH 6.0 (store up to 2 months at 4°C)
  • 0.35 M perchloric acid/0.36 M sodium acetate
  • Tris/AAP buffer (see recipe)
  • Guillotine
  • Forceps
  • Scalpel
  • Polytron homogenizer (Brinkmann) with PTA 10S and 7S sondes (for brain and spinal cord, a glass‐Teflon Potter‐Elvehjem homogenizer can be used; for the peripheral nerve this might pose some problems and require more time)
  • NOTE: All procedures performed at room temperature unless otherwise specified.

Alternate Protocol 1: Determination of the In Vitro Inhibitory Power of a Compound on NTE

  • Test compound (putative inhibitor)
  • Acetone
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Literature Cited

Literature Cited
   Abou‐Donia, M.B. and Graham, D.G. 1979. Delayed neurotoxicity of O‐ethyl‐O‐4‐nitrophenyl phenylphosphonothioate: Toxic effects of a single oral dose on the nervous system of hens. Toxicol. Appl. Pharmacol. 48:57‐66.
   Barril, J.B., Vilanova, E., and Pellin, M.C. 1988. Sciatic nerve neuropathy target esterase: Methods of assay, proximo‐distal distribution and regeneration. Toxicology 49:107‐114.
   Bickford, A.A. and Sprague, G.L. 1983. The significance of background neurologic lesions in acute delayed neurotoxicity studies: A comparison of neurohistopathological lesions induced in commercial hens by tri‐o‐tolyl phosphate (TOCP) with those observed in negative control hens. Neurotoxicology 4:283‐310.
   Bouldin, T.W. and Cavanagh, J.B. 1979a. Organophosphorus neuropathy. I. A teased‐fiber study of the spatio‐temporal spread of axonal degeneration. Am. J. Pathol. 94:241‐252.
   Bouldin, T.W. and Cavanagh, J.B. 1979b. Organophosphorous neuropathy. II. A fine‐structural study of the early stages of axonal degeneration. Am. J. Pathol. 94:253‐270.
   Caroldi, S. and Lotti, M. 1982. Neurotoxic esterase in peripheral nerve: Assay, inhibition and rate of resynthesis. Toxicol. Appl.Pharmacol. 62:498‐501.
   Correll, L. and Ehrich, M. 1991. A microassay method for neurotoxic esterase determinations. Fundam. Appl. Toxicol. 16:110‐116.
   Escudero, M.A., Sogorb, M.A., and Vilanova, E. 1996. An automatable microassay for phenyl valerate esterase activities sensitive to organophosphorus compounds. Toxicol. Lett. 89:241‐247.
   Johnson, M.K. 1969. The delayed neurotoxic effects of some organophosphorus compounds: Identification of the phosphorylation site as an esterase. Biochem. J. 114:711‐717.
   Johnson, M.K. 1977. Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Arch. Toxicol. 37:113‐115.
   Johnson, M.K. 1982. The target for initiation of delayed neurotoxicity by organophosphorus esters: Biochemical studies and toxicological applications. In Reviews in Biochemical Toxicology (E. Hodgson, J.R. Bend, and R.M. Philpot, eds.) vol. 4:141‐212. Elsevier, New York.
   Johnson, M.K. and Barnes, J.M. 1970. Age and the sensitivity of chicks to the delayed neurotoxic effects of some organophosphorus compounds. Biochem. Pharmacol. 19:3045‐3047.
   Jortner, B.S. and Ehrich, M. 1987. Neuropathological effecs of phenyl saligenin phosphate in chickens. Neurotoxicology 8:303‐314.
   Kayyali, U.S., Moore, T.B., Randall, J.C., and Richardson, R.J. 1991. Neurotoxic esterase (NTE) assay: Optimized conditions based on detergent‐induced shifts in the phenol/4‐aminoantipyrine chromophore spectrum. J. Anal. Toxicol. 15:86‐89.
   Koch, T. 1973. Anatomy of the Chicken and Domestic Birds. Iowa State University Press, Ames, Iowa.
   Lotti, M. 1992. The pathogenesis of organophosphate delayed polyneuropathy. Crit. Rev. Toxicol. 21:465‐487.
   Lotti, M. and Johnson, M.K. 1980. Repeated small doses of a neurotoxic organophosphate. Monitoring of neurotoxic esterase in brain and spinal cord. Arch. Toxicol. 45:263‐271.
   Lotti, M., Caroldi, S., Moretto, A., Johnson, M.K., Fish, C.J., Gopinath, C., and Roberts, N.L. 1987. Central‐peripheral delayed neuropathy caused by diisopropyl phosphorofluoridate (DFP): Segregation of peripheral nerve and spinal cord effects using biochemical, clinical and morphological criteria. Toxicol. Appl. Pharmacol. 88:87‐96.
   Lotti, M., Caroldi, S., Capodicasa, E., and Moretto, A. 1991. Promotion of organophosphate‐induced delayed polyneuropathy by phenylmethanesulfonyl fluoride. Toxicol. Appl. Pharmacol. 108:234‐241.
   Lotti, M., Moretto, A., Bertolazzi, M., Peraica, M., and Fioroni, F. 1995. Organophosphate polyneuropathy and neuropathy target esterase: Studies with methamidophos and its resolved optical isomers. Arch.Toxicol. 69:330‐336.
   Moretto, A., Lotti, M., and Spencer, P.S. 1989. In vivo and in vitro regional differential sensitivity of neuropathy target esterase to di‐n‐butyl‐2,2‐dichlorovinyl phosphate.. Arch. Toxicol. 63:469‐473.
   Moretto, A., Capodicasa, E., and Lotti, M. 1992. Clinical expression of organophosphate‐induced delayed polyneuropathy in rats. Toxicol. Lett. 63:97‐102.
   Moretto, A. and Lotti, M. 1993. Promotion of peripheral axonopathies by certain esterase inhibitors. Toxicol. Ind. Health 9:1037‐46.
   Moretto, A., Bertolazz, M., and Lotti, M. 1994. The phosphorothioic acid O‐(2‐chloro‐2,3,3‐trifluorocyclobutyl) O‐ethyl S‐propyl ester exacerbates organophosphate polyneuropathy without inhibition of neuropathy target esterase. Toxicol. Appl. Pharmacol. 129:133‐137.
   Organization for Economic Cooperation and Development (OECD). 1995a. Delayed neurotoxicity of organophosphorus substances: Following acute exposure. In OECD Guideline for Testing of Chemicals, 418, adopted 27.07.95.
   Organization for Economic Cooperation and Development (OECD). 1995b. Delayed neurotoxicity of organophosphorus substances: 28‐day repeated dose study. In OECD Guideline for Testing of Chemicals, 419, adopted 27.07.95.
   Padilla, S. and Veronesi, B. 1988. Biochemical and morphological validation of a rodent model of organophosphorus induced neuropathy. Toxicol. Ind. Health 4:361‐371.
   Peraica, M., Capodicasa, E., Moretto, A., and Lotti, M. 1993. Organophosphate polyneuropathy in chicks. Biochem. Pharmacol. 45:131‐135.
   Pope, C.N. and Padilla, S. 1990. Potentiation of organophosphorus‐induced delayed neurotoxicity by phenylmethylsulfonyl fluoride. Toxicol. Environ. Health 31:261‐273.
   Prentice, D.E.c and Majeed, S.K. 1983. A subchronic study (90 day) using multiple dose levels of tri‐ortho‐cresyl phosphate (TOCP): Some neuropathological observations in the domestic hen. Neurotoxicology 4:277‐282.
   Prentice, D.E. and Roberts, N.L. 1983. Acute delayed neurotoxicity in hens dosed with tri‐ortho‐cresyl phosphate (TOCP): Correlation between clinical ataxia and neuropathological findings. Neurotoxicology 4:271‐276.
   Roberts, N.L., Fairley, C., and Phillips, C. 1983. Screening acute delayed and subchronic neurotoxicity studies in the hen: Measurements and evaluations of clinical signs following administration of TOCP. Neurotoxicology 4:263‐270.
   Sprague, G.L., Sandvik, L.L., Bickford, A.A., and Castles, T.R. 1980. Evaluation of a sensitive grading system for assessing acute and subchronic delayed neurotoxicity in hens. Life Sci. 27:2523‐2528.
   Vilanova, E., Barril, J., Carrera, V., and Pellin, M.C. 1990. Soluble and particulate forms of organophosphorus neuropathy target esterase in hen sciatic nerve. J. Neurochem. 55:1258‐1265.
Key References
   Caroldi and Lotti, 1982. See .
  Description of the original method for measuring NTE activity in peripheral nerves.
   Johnson, 1977. See .
  Describes the original method (but contains a typographical error on page 114, last paragraph, line 3, where the concentration of tissue should be 6.6 mg and not 0.6 mg).
   Johnson, 1982. See .
  Extensive review on OPIDP, including a description of the method for measuring NTE activity in brain and spinal cord tissue.
   Lotti 1992. See .
  Most recent extensive review on OPIDP.
   Lotti et al., 1987. See .
  Describes the procedures for clinical observation.
   Prentice and Roberts, 1983. See .
  Describes and scores clinical signs and morphological lesions.
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