BmchemscalPharmacology, Vol. 31.No. 6, pp. 1117-1121,1982 00W2952/82/061117-05$03.00/O Printedin GreatBntain. @ 1982Pergamon Press Ltd. EVIDENCE FOR THE EXISTENCE OF NEUROTOXIC ESTERASE IN NEURAL AND LYMPHATIC TISSUE OF THE ADULT HEN*? BENNIE RICHARD DUDEK and RUDY J. RICHARDSONS Toxicology Research Laboratory, Institute of Environmental and Industrial Health, School of Public Health, The University of Michigan, Ann Arbor, MI 48109, U.S.A. (Received 12 January 1981; accepted 31 August 1981) Abstract-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 (Callus 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-‘+ (g wet weight)-’ (mean f 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 (O%), pectoralis muscle (O%), heart (14%), liver (O%), kidney (O%), 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. Certain organophosphorus compounds cause a to neurotoxic and non-neurotoxic inhibitors. Prein- delayed degeneration of long axons in peripheral cubation at an appropriate concentration and time nerves and spinal cord of man, fowl, and certain interval with paraoxon (diethyl p-nitrophenyl phos- other species [1,2]. This condition is referred to as phate), a non-neurotoxic compound, inhibits about delayed neurotoxicity. The hen was used in this study 80% of the total PV esterase activity. If mipafox since this is the animal model commonly used in (N,N’-diisopropyl phosphorodiamidofluoridate), a evaluation of delayed neurotoxicity potential of neurotoxic inhibitor, is included in the preincubation organophosphorus compounds [3]. In species sen- medium, an additional lO-17% of the activity is sitive to a single dose of neurotoxic compounds, inhibited. This decrease in activity upon mipafox there is a latent period of 8-14 days before clinical addition represents NTE activity. signs of ataxia and paralysis and histological evidence This study was undertaken to provide criteria for of distal-to-proximal “dying back” of affected axons identification of NTE activity in tissues of the hen appear. An early biochemical event in this neuro- in the form of complete inhibitor titration curves pathy is the phosphorylation and aging of a particular using the substrate phenyl valerate (PV) [8]. Pre- phenyl valerate (PV) hydrolase in nerve tissue known viously, such curves have been presented for hen as neurotoxic esterase (NTE)Q [4-6]. Inhibition of brain using the less sensitive substrate phenyl phen- hen brain NTE following dosing with neurotoxic ylacetate (PPA) [4] and, more recently, for human organophosphorus compounds is highly correlated brain using PV [9]. It was of interest to rigorously with the production of delayed neurotoxicity [7]. determine whether this enzyme had exclusive local- The NTE assay [5] is based on the differential ization in neural tissue in a given species or whether sensitivity of PV esterases in hen brain homogenate its distribution was more widespread. MATERIALSAND METHODS * This work formed a segment of the Ph.D. dissertation of B. R. D. and was presented in part at the Seventeenth Animals. Spent White Leghorn hens, 1 to 1.5 years Annual Meeting of the Society of Toxicology, San Fran- of age, weighing 1.5 to 2.0 kg were purchased from cisco, CA, March 12-16, 1978. a local supplier. Hens were housed in stainless steel t This research was supported in part by research grants R805339-01, USEPA, and ES01611-01. NIEHS, and bv a wire cages 0.8 X 0.8 X 0.9m with 1.27cm mesh faculty research grant irom the Horace fi. Rackham School stainless wire floors and were kept at least 2 weeks of Graduate Studies, The University of Michigan. before use; birds were maintained in a $ Author to whom correspondence should be sent. temperature-controlled room on a diet of Purina 0 A more precise name that we would like to suggest is Layena laying mash (Ralston Purina Co., St. Louis, neurotoxicant-sensitive esterase (NTSE). MO) and tap water ad lib. A 12-hr light-dark cycle 1117 1118 B. R. DUDEKand R. J. RICHARDSON was maintained throughout the course of the absolute activity remaining at the higher paraoxon experiment. concentrations, yielded an optimum paraoxon con- Chemicals. Paraoxon (diethyl p-nitrophenyl phos- centration, usually 10-100 PM, for use in the mipafox phate) was purchased from the Sigma Chemical Co. titration. (St. Louis, MO) and was purified as described by The mipafox titration curves were generated by Johnson [lo]. Mass spectra of compound purified by including, in the preincubation medium, paraoxdn this method were identical to published spectra [ll]. at the selected optimum concentration, plus mipafox, PV and mipafox were synthesized and purified as dissolved in Tris-EDTA buffer, over the concentra- described by Johnson [lo]. Phenyl valerate was ana- tion range 0.001 to 1024 ,uM. lyzed for impurities using i.r. spectroscopy and by In the cases of lymphocytes and peripheral nerve, assaying for phenol [12]. Both methods showed PV assay volumes were reduced by factors of 20 and 2, to be pure. Purity of mipafox was verified by melting respectively, to accommodate smaller amounts of point: found, 60-61”; published, 61-62” [lo]. All tissue. other chemicals were analytical reagent grade or the For each tissue studied, at least three complete highest grade commercially available; aqueous sol- titration curves were obtained using each inhibitor. utions were prepared using distilled-deionized Data are presented as means ? S.E.M. water. Tissue preparation. The buffer system used RESULTS throughout for tissue homogenization and hom- ogenate dilution was 50 mM Tris-HC1/2 mM EDTA Panels A and B* of Fig. 1 are the neurotoxic and (pH 8.0,25”). Tissues, except for lymphocytes, were non-neurotoxic inhibitor titration curves for hen kept at ice-bath temperature throughout processing, brain PV esterase activity. They serve to illustrate and were blotted before weighing. Homogenizations were generally done in 6.5 vol. of buffer in a glass-Teflon homogenizer with chamber clearance BRAIN: -PARAOXON of 150-230 ,um, using 15 strokes at 2300 ‘pm. *-+MIPAFOX Since peripheral nerve and spinal cord required lengthy manipulation for removal, these tissues were removed from decapitated hens in a 4” cold room. The peripheral nerve used was the endoneurial com- ponent of the ischiatic nerve; tissue was minced with scissors and homogenized using 30-35 strokes in a glass-Teflon homogenizer and passed through a I I I I I ‘-r--i I , nylon screen (333 pm) (TETKO, Inc., Elmsford, a 7 6 5 4 3 2 NY) stretched over the end of a truncated 10 ml -LOG[INHI6ITOR (Ml] plastic syringe to remove residual connective tissue. Gastrocnemius and pectoralis muscles were minced after removal of the thin collagenous sheaths, passed through a tissue press, homogenized, and passed through a nylon screen (333 ,um) to remove 55, residual collagenous material. \ Leucocytes from minced spleen supernatant frac- 1 tions and from peripheral blood were isolated by ggrc40 BRAIN: centrifugation on Ficoll-Paquem (Pharmacia, Pis- ge I-20 TITRATION WITH MIPAFOX “b---. cataway, NJ) [13,14]. Weighed, pelleted cells were 2 PARAOXON = IO0 PM maintained at room temperature until just before use, at which time they were homogenized in buffer 9 6 7 6 5 4 3 with a siliconized glass-Teflon homogenizer (cham- -LOG [MIPAFOX IMI] ber clearance of 100-150 pm). Cells were quantified Fig. 1. (A) Effects of increasing concentrations of paraoxon on the basis of wet weight. Cells obtained by this alone (0) and of increasing concentrations of mipafox in method were checked for identity and purity using the presence of paraoxon (0) on the hydrolysis of phenyl light (Wright’s stain) [15] and electron microscopy. valerate (PV) by hen brain homogenate. The concentration of paraoxon used in the mipafox titration is indicated on This preparation yielded 2 95% lymphocytes. curve B. (B) Effect of mipafox on the paraoxon-insensitive Differential titrations. The NTE assay was done hydrolysis of phenyl valerate by hen brain homogenate; essentially according to the method described pre- 100% = activity remaining that is insensitive to paraoxon viously [5]. Complete paraoxon titration curves were before addition of mipafox. The concentration of paraoxon generated by varying the paraoxon concentration in used in the mipafox titration is indicated on the graph. the preincubation medium, over the concentration Tissue homogenates were preincubated for 20min with range 0.01 to 1000 PM. For tissues that showed very inhibitors before addition of substrate, whereupon incu- high PV esterase activity at low paraoxon concen- bation was continued for an additional 15 min before stop- trations, it was necessary to use an aliquot of the ping the reaction and analyzing for phenol produced is described in Materials and Methods.