PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 20, 67-75 (1983)

In Vitro and in Vivo Inhibition of Chicken Brain Neurotoxic Esterase by Leptophos Analogs

JAN HENDRIK REINDERS, LARRY G. HANSEN, ROBERT L. METCALF, AND ROBERT A. METCALF

Departments qf Veterinary Biosciences and Entomology, Institute for Environmental Studies and School of

Medicine, University of Illinois, Urbana, Illinois 61801

Received September 24. 1982; accepted February 17. 1983

Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O-halogenated-phenyl-O- alkyl phenylphosphonates was studied in vitro. The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The p15,, of U-(2,6- dichlorophenyl) O-methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy com- pounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than des- bromoleptophosoxon. Within a like group of chlorinated phenylphosphonates. a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in viva delayed neu- rotoxic potential by the corresponding phosphonothionate exists. In viva inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in viva inhibitor. Ethoxyleptophos and leptophos have about equal in viva esterase inhibitory properties. For des- bromoleptophos and leptophos there is good agreement between the minimum dose causing de- layed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.

INTRODUCTION

Some organophosphorus compounds cause a toxic neuropathy characterized by a delayed onset (2-3 weeks after a single dose), axonal degeneration, and demyelina- tion of the central and peripheral nerve axons, resulting in ataxia, paralysis, and even death. A variety of species, including man, are susceptible to this syndrome, while rodents and some primates are quite insen- sitive. Delayed neurotoxicity is studied ex- tensively and has been reviewed by several authors (l-5). Leptophos [O-(25di-chlo- ro-4-bromophenyl) O-methyl phenylphos- phonothionate] and related phenyl phos- phon(othion)ates are neurotoxic in the laying hen (6-8) the most suitable experi- mental animal for studying the delayed neu- rotoxicity syndrome (9).

Phosphorylation of a rather specific site

in the central nervous system was reasoned to be the first step in the onset of the de- layed neuropathy (10). This site was identi- fied as part of the active site of an esterase-“neurotoxic esterase” (NTE), present in brain tissue-and inhibition of NTE occurred with all known delayed neu- rotoxicants (11-13). Aging of the NTE-in- hibitor complex is a second necessary step (14,15), which apparently triggers the de- generative process characteristic for de- layed neuropathy (5,13). Although most studies use single neurotoxic doses to in- hibit NTE, chronic administration even- tually titrates NTE to the critical level of inhibition (16,17).

Despite the presently unknown phys- iological function of NTE as well as the mysterious course of events leading from NTE inhibition to axonal degeneration, NTE-inhibition studies can be of some pre-

67 0048-3575/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

68 REINDERS ET AL.

dictive value and provide more insight into the mechanism of delayed neurotoxicity. Various authors have contributed to the de- velopment of the in vitro NTE assay (12. 18-25). The in vitro assay has proven to be very convenient as a preliminary assess- ment of neurotoxic potential, compared to the elaborate in vivo testing. In addition to the in vivo testing with a series of phenyl phosphonothionates closely related to lep- tophos, as reported by Metcalf et al. (26), we studied the in vitro NTE inhibition by the oxon analogs of these compounds. Most of the compounds studied also inhib- ited “mipafox-resistant” phenylvalerate es- terase(s) and a procedure (29) was em- ployed to estimate the “true” NTE inhibi- tion. The in vivo NTE inhibition of leptophos, ethoxyleptophos, and des- bromoleptophos are also reported.

MATERIALS AND METHODS

Chemicals. Leptophos and ethoxylep- tophos [O-(2,5-di-Cl-4-Br-phenyl) O-ethyl phenylphosphonothionate] were a gift from Velsicol, Chicago, Illinois. After three re- crystallizations from ethanol, the com- pounds were greater than 99% pure and the mp were found to be 71-72°C and 60-61”C for leptophos and ethoxyleptophos, respec- tively. Mipafox was a gift from Dr. R. J. Richardson, Ann Arbor, Michigan. EPN [0-(CNO,-phenyl) O-ethyl phenylphospho- nothionate] was a gift from Du Pont de Nemours, Wilmington, Delaware, and was recrystallized three times from ethanol to mp 34-35°C. The other phenyl phos- phonothionates were synthesized as pre- viously described by Metcalf et al. (26,27). The phosphonates (leptophos oxon and analogs) were prepared from the corre- sponding phosphonothionate by nitric acid oxidation according to Giang and Hall (28); conversion of leptophos to leptophos oxon has been shown to be essentially complete (Soliman, personal communication). The oxons obtained this way had the same NTE inhibitory and acetylcholinesterase inhibi- tory properties as the oxons synthesized as

described by Sanborn (8). Phenylvalerate was synthesized and purified using stan- dard methods. All other chemicals used were analytical or pesticide grade from commercial suppliers.

Animals. While leghorn laying hens (Hy- Line Hybrid from Roth Hatcheries, Wat- seka, Ill.) were obtained at 20 weeks of age. They were allowed 6-10 weeks to age, ac- climate, and come into full egg production prior to any dosing. They were maintained in individual layer cages at 22 t 2°C and on a 14:lO photoperiod. The compound stud- ied for in vivo NTE inhibition was admin- istered orally as crystals in a gelatin capsule 24 hr before decapitation of the bird. After decapitation, brains were removed quickly, freed from blood and meninges, and ho- mogenized in ice-cold buffer (50 m&Z Tris- HCl; 0.2 mM EDTA; pH 8.0). Brain ho- mogenate (1 g brain/IO ml) was centrifuged for 15 min at 87OOg in a Sorvall refrigerated centrifuge. The supernatant was diluted IO- fold and assayed for total paraoxon-insensi- tive phenyl valerate hydrolyzing activity (NTEAPP). After preincubation for 20 min at 37°C in the presence of 40 PM paraoxon with/without 20 pM leptophosoxon as NTE inhibitor (20), incubation was continued for 15 min with phenylvalerate present as a substrate for the esterases (12). Stopping the reaction and measuring the phenol formed followed the procedure given by Sprague et al. (23). NTEAPP activity [ = NTE plus inhibitable residual activity or RA’, as described by Reinders et al. (29)] was expressed as micromoles phenol formed per 15 min per milligram protein. Proteins were estimated according to Low- ry (30), using bovine serum albumin as standard.

In vitro NTE inhibition by a series of compounds was studied using brain homog- enate from control chickens freed of cell debris as described above. The procedure for measuring NTE activity was the same as above, except that the compound to be studied was included in the preincubation mixture in various concentrations in place

LEPTOPHOS ANALOGS’ INHIBITION OF NEUROTOXIC ESTERASE 69

of leptophos oxon. As the compounds were added as acetone solutions, care was taken to have equal acetone concentrations in all incubations: 3.25% during preincubation and 1.63% during phenylvalerate hydro- lysis.

NTE represents only a small proportion of the total phenylvalerate hydrolyzing ac- tivity in the brain (5). In order to assess this activity, irrelevant esterases are inhibited by paraoxon, but at least three distinct types of phenylvalerate hydrolytic enzymes remain: (i) neurotoxic esterase, the largest proportion; (ii) residual or mipafox insensi- tive activity which can be subdivided into (a) a small proportion which is insensitive to leptophos oxon and some analogs, and (b) a slightly greater amount which is inhib- ited by leptophos oxon, but only by high concentration of mipafox or very high con- centrations of 0-(2,6-dichlorophenyl) O- methyl phenylphosphonnate (29). The un- equal inhibition of this residual activity

(RA’) influences the comparison of NTE inhibition by various analogs (see Table 1). By coincubating with the 2,6-dichlorophe- nyl analog, a correction can be made to dis- tinguish inhibition of “true” NTE from inhibition of NTE + RA’ (see Fig. 1).

RESULTS AND DISCUSSION

In vitro NTE inhibition by leptophos analogs. The phosphonothionates lacked in vitro NTE inhibitory properties, as was already found to be true in general for organophosphorus compounds (4) and for leptophos in particular (25).

The leptophosoxon analogs (Table 1) were found to be fair to very potent in vitro NTE inhibitors. From the inhibition curves, interpolated PI,, NTE values are presented in Table 1 and examples of NTE inhibition curves are given in Fig. 1. A com- plicating factor in studying “true” NTE in- hibition is the presence of a “mipafox resistant” component in the “apparent”

TABLE I Neurotoxic Esteruse Inhibition by Leptophos-Andogs

2-mono-Cl CH3 5.05 4.50 4.90 3-mono-Cl CH3 6.05 5.30 5.90 4-mono-Cl CH3 5.60 4.95 5.40 2,3-di-Cl CHx 5.75 4.35 5.50 2,4-di-Cl CH3 5.80 4.20 5.50

CA 6.10 5.80 2.5-di-Cl CH3 6.50 4.60 6.25

C2H5 6.40 6.15 2,6-di-Cl CH3 6.65 <3 6.40 3 ,Cdi-CI CHI 5.60 4.70 5.40 3,5-di-Cl Ch 5.50 5.20 5.45 2,3,4-tri-Cl W 6.20 5.35 6.00 2,3,5-tri-Cl CH, 6.45 5.60 6.20 2.3,6-tri-Cl CH3 6.65 4.40 6.45 2,4,5-tri-Cl CHJ 6.45 5.65 6.20 2,4,6-&i-Cl CJ& 6.60 4.00 6.20

C& 6.30 6.10 3,4,5-tri-Cl CH3 6.15 5.60 5.95 2,3,5,6-tetra-Cl CH3 5.55 3.70 5.15 2,3,4,5,6-penta-Cl CH3 6.00 4.70 5.80

C& 5.80 5.50 2,5-di-Cl-4-Br CHJ 6.80 5.90 6.55

C&I> 6.65 5.80 6.55 2,5-di-Cl-4-1 CHs 6.85 6.40 6.60

REINDERS ET AL.

Leptophos-oxon insensitive

ectivitv

% NTE*einhibition

_--em_ - -

NTE TRUE

- 50 NETRUE

’ Jo 10-8 10-l 10.6 10-s 10-4

concentration (M)

6 Ethoxy-Leptophos-oxon

%NTEAPP-inhibition

RA’

NTETRUE

FIG. 1. Chicken bruin NTE inhibition by leptophosoxon. ethoxyleptophosoxon. desbromolep- tophosoxon; (0) NTEAPP; (0) residual activity (mipafox resistant) measured in the presence of 50 +M

0-(2,6-di-Cl-phenyi) O-methyl phenylphosphonaie; (---) approximated “true” NTE is 90% of the para- oxon-insensitive phenylvalerute hydrolase activity: NTETrUe is 7678Yc of NTEAPQ.

NTE measured by the method using lep- tophos oxon. This component (RAt) was found to be sensitive to several of the com- pounds tested but not to the 2,6-di- chlorophenyl analog (29) which was there- fore used to distinguish “apparent” NTE from “true” NTE (mipafox sensitive). The activity of NTETrUe was determined as NTE-Q’P - RA1 = NTET’““.

A few ethoxyleptophosoxon analogs were studied; they are only slightly less po-

tent NTE inhibitors compared to the corre- sponding methoxy compound, except for EPN and the 0-(2,Cdi-Cl-phenyl) O-ethyl phenylphosphonate. These results suggest that for the O-(phenyl)-phenylphospho- nates, the difference in size between the methoxy and ethoxy group is not enough to cause a major difference in in vitro NTE inhibition. Ohkawa et al. (17) found similar results for a few phenylphosphonates in vitro. Their methods, which were some-

LEPTOPHOS ANALOGS’ INHIBITION OF NEUROTOXIC ESTERASE 71

C 2,5-Di-Cl-Me

% NTE -inhibition

10-T 10-5

concentration M [I

FIG. I. -Continued.

what less accurate because of limited data, resulted in slightly higher PI,, values, how- ever. Of evidently greater importance is the substitution pattern around the 0-phenyl leaving group; analogs with 2,5- or 2,6-di-Cl substitution are especially potent NTE-in- hibitors. One additional Cl or Br on the O- phenyl doesn’t abolish the high pIsONTE values for 2,5- or 2,6-di-Cl-O-phenyl-sub- stituted phenyl phosphonates. A greater number of substituents on the 0-phenyl, as in in the case of tetra-Cl and penta-Cl, re- duce the in vitro NTE inhibitory properties with respect to their 2,5- or 2.6-di-Cl analogs.

The 0-phenyl substituents of the phe- nylphosphonates have a substantial elec- tronic effect (expressed as Cu-) on the reactivity of the leaving group and, thus, on the determination of NTE inhibitory prop- erties (Fig. 2). The u- values used were those of Tribble and Traynham (37) deter- mined from NMR measurements on the substituted phenols in dimethylsulfoxide, Cl, uo- = 0.50. Linear regression analy- sis showed the following correlations be- tween Xu- and ~1,“~~~:

(A) All compounds in Table 1: PI 5o = 0.41 Cu- + 5.61; r = 0.37, n = 18.

(B) All compounds except di-ortho sub- stituted: PI,, = 1.12 Cu- + 5.09; r = 0.70,n = 13.

(C) Di-Cl compounds: PI 5. = 2.75 Za- + 3.75; r = 0.79, n

6. (D) it-i-Cl compounds:

PI,, + 1.31 Xu- + 4.89; r = 0.88, n = 6.

Thus, inhibition of NTE is a direct func- tion of the phosphorylating ability of the P= 0 analogs. However, for the di-ortho substituted phenyl phenylphosphonates, steric parameters are also of major impor- tance. Tribble and Traynham (32) found that 2,6-disubstitution produced major ster- ic perturbations in the ionization of phenols even in dimethylsulfoxide.

Examination of the para substituents on the leaving group may also offer some clues concerning the nature of RA’. In the series: H, Cl, Br, I, the p&e’s for NTE are quite tightly clustered, but the pI,,‘s for RA’ in- crease from 4.60 to 6.40 with increasing size and reactivity of the para substituents (Table 1).

In vivo potency to cause neurotoxicity by the leptophos analogs, as studied by Hol- lingshaus ef al. (7) and Metcalf et al. (26),

72 REINDERS ET AL.

0 23456

3 6.0 -

B 0 w 24 0 .o :: 4 0

0 23

34 0 0

‘; 35

2366

5 z”

% x 5.0 - 0 no ortho-substitution

0 mono-ortho-substitution

0 di-ortho-substitution

7.0 -

4.0’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.6 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.6

Ca-

FIG. 2. Calculated Zu- values of the 0-phenyl lettering grorrp o.f the 0-phenyl O-methyl phenyl-

phosphonates versus the in vitro plroNTE.

correlates well with the pISONTE of the cor- responding O-Me phenylphosphonates, al- though only when the di-Cl and tri-Cl analogs are considered as separate groups (Fig. 3). It should be noted that Hol- lingshaus reported the minimum dose re- sulting in any visually detectable ataxia, which results in lower values than for doses resulting in paralysis, as reported by Met- calf. Both in viva and in vitro, the 2,5- and 2,6-di-Cl-0-phenyl-O-methyl compounds are the most potent in causing delayed neu- rotoxicity and inhibiting NTE. In conclu- sion, it can be said that in the series of O- phenyl-O-methyl phenylphosphon(othi- on)ates, the compounds with chlorine- shielded leaving group-phosphorus bonds are the most potent with respect to NTE inhibition and delayed neurotoxicity. Re- placing the methoxy group by ethoxy re- duces the in vivo delayed neurotoxicity to a much greater extent than NTE inhibition is reduced. It, therefore, became necessary to evaluate NTE inhibition in vivo.

In vivo NTE inhibition by leptophos, eth-

oxyleptophos, and desbromoleptophos. Inhibition of NTE in chicken brain was

studied 24 hr after an oral dose with lep- tophos and analogs. In a time-dependent study, Ohkawa et al. (17) showed the in vivo NTE inhibition being maximal l-2 days after oral dosing with leptophos. The in vivo NTEAPP inhibition by leptophos and analogs is dose-dependent (Fig. 4). Ethox- yleptophos and leptophos have equal NTEAPP inhibitory properties in vivo. Des- bromoleptophos is much more potent. NTEAPP is inhibited greater than 70% by leptophos, ethoxyleptophos, and des- bromoleptophos at doses of 450.398 and 40 kmol/kg as calculated from Fig. 4. The min- imum neurotoxic dose from laying hens is approximately: 480 (200 mg), 3500 (1500 mg), and 75 (21 mg) pmol/kg for leptophos, ethoxyleptophos, and desbromoleptophos, respectively (7,8,27,33). Considering 7690% NTE inhibition is required for clini- cal delayed neuropathy (341, there is rea- sonable agreement between in vivo NTEAPP inhibition and in vivo delayed neu-

LEPTOPHOS ANALOGS’ INHIBITION OF NEUROTOXIC ESTERASE 73

X=S in viva

X=0 in vitro -

M,Tri-Cl

I I I . E 5.5 6 6.5

in vitro p15DNTE H=Hollingshans atal. 1979 M=Metcalf etaI. 1982

FIG. 3. In vivo neurotoxicity of O-phenyl O-methyl phenylphosphonothionates, as reported by Hol- lingshaus et al., (7) (H) and Metcalf er al., (26) (M) versus in vitro ~15~~~ of the corresponding oxons. Delayed neurotoxicity potentiul is expressed (IS the minimum nerrrotoxic dose (H) und the

minimum dose to paralysis times duys to proceed to ptrrcrlysis CM).

rotoxic potency for leptophos and des- bromoleptophos.

In vivo NTE inhibition by leptophos and analogs reported by other authors (12, 17, 21) included limited dose-response rela- tionships; their data, however, fit very well into the curves obtained in this study.

The observed 70% inhibition of NTEAPP by only about 350 mg/kg of ethoxylep- tophos seems at odds with the requirement of about 1200-1500 mg/kg of this compound to produce neuropathy (27,7). Several pos- sibilities must be evaluated and these in- clude:

(A) Inadequate in vivo sampling. G. L. Sprague (personal communication) has found a high degree of variation in the in vivo inhibition of NTE among individual hens given the same oral dose of neurotoxi- cant.

(B) Absorption of a single dose of the compound has reached saturation at about 300-400 mg/kg so that the critical amount never reaches the receptor.

(C) Spinal cord NTE may be the critical factor and inhibition of cord NTE lags be- hind that of brain (35).

(D) NTEAPP is a poor monitor for NTEtrUe. This seems unlikely for ethox-

% NTEAPP-inhibition 1OOr in vim

. Leqtophos

0 Ethoxy-Lsptophos

ADesbromo-LeptoPhas

10.5 10-4

dose [-oles/kgj

FIG. 4. In vivo chicken bruin NTE*PP inhibition by

Ieptophos, ethoxyleptophos. and desbromol~~ptopphos, measured 24 hr ufter an oral dose. Control NTEAPP

activity was 679 -C 143 (n = 4) prnol phenol pro- ducedll5 minlmg protein.

74 REINDERS ET AL.

yleptophos, since the I,,,‘s measured in vitro were rather similar.

(E) Inhibition of NTEr’“” has not been followed by an efficient aging process. This seems doubtful, since the same phosphory- lated species is probably produced from EPN oxon which ages with a half-life of 10 min (5).

These possibilities, especially variability and pharmacokinetics, must be evaluated carefully before rejecting assay of NTJ.We as a monitor for the response of dosed hens.

Although some progress has been made in understanding the biochemical mecha- nism underlying delayed neurotoxicity [as recently reviewed by Johnson (S)], still much has to be revealed in order to make any reliable structure-based predictions on the neurotoxic potential of chemicals.

ACKNOWLEDGMENTS

This work was supported by the Research Board of the University of Illinois and by the U.S. Department of Agriculture Pesticide Impact Assessment Program. North Central Region.

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