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1989 37: 589J Histochem CytochemG B Koelle, N S Thampi, M S Han and E J Olajos

Histochemical demonstration of neurotoxic esterase.  

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The Journal of Histochemistry and Cytochemistry

Copyright © 1989 by The Histochemical Society. Inc.Vol. 37. No. 5, pp. 589-596, 1989

Printedin U.S.A.

Original Article

Histochemical Demonstration of Neurotoxic Esterase’

GEORGE B. KOELLE,2 NAGENDRAN S. THAMPI, MATTHEW S. HAN,

and EUGENE J. OLAJOS3

Department ofPharmacology, Medical School, University ofPennsylvania, Philadelphia, Pennsylvania 19104-6084.

Received for publication August 17, 1988 and in revised form November 14, 1988; accepted November 22, 1988 (8A1462).

We developed a histochemical method for localizing neuro-toxic esterase (NTh), defined as the phenylvalerate (PV)-hydrolyzing esterase that is resistant to 40 �tM paraoxon (A)but inactivated by paraoxon plus 50 �iM impafox (B). NThis considered to be the target enzyme in the production

of organophosphorus ester-induced delayed neurotoxicity(OPIDN). Cryostat sections were incubated in a medium con-taming a-naphthyl valerate and 6-benzamido-4-methoxy-m-toluidine diazonium chloride (fast violet B) after treatmentwith the above-mentioned inhibitors, leading to formationof an aqueous insoluble precipitate at sites of enzymatic ac-tivity. NTh activity was estimated as staining detectable in

IntroductionOrganophosphorus ester-induced delayed neurotoxicity (OPIDN)

is a syndrome produced by certain compounds of that category.

It is characterized by sequential development of axonal degenera-

tion, demyelination, and flaccid paralysis after a latent period of

10-14 days, and is completely unrelated to the anticholinesterase

(anti-ChE) action of such compounds. Several extensive outbreaks

of human OPIDN have been reported, usually resulting from the

contamination of beverages or cooking oil with triorthocresyl phos-

phate. The subject has been reviewed thoroughly by Abou-Donia

(1981) and Zech and Chemnitius (1987).

Although its biochemical basis is still unproven, one ofthe most

generally considered proposals at present is that OPIDN is caused

by alkylphosphorylation and subsequent “aging” (partial dealky-

lation) of neurotoxic esterase (NTh) Uohnson, 1969, 1977). This

enzyme has been defined empirically by Johnson (1977) as the

phenylvalerate (PV)-hydrolyzing esterase that is resistant to 40 p.tM

paraoxon (0,0-diethyl-0-p-nitrophenyl phosphate; E 600) but is in-

activated by paraoxon plus 50 �sM mipafox (N,N-diisopropyl

fluorophosphorodiamide). An additional fraction of PV-hydrolyzing

1 Supported by Contract DAAA15-87-K-0002, Department of the

Army.2 To whom correspondence should be addressed.

3 Present address: Chemical Research, Development and Engineering

Center, US Army, Aberdeen Proving Ground, MD 21010-5423.

A but not in B. In the central nervous system (CNS) ofchicken, NTh appeared to be present primarily in the so-mata of most neurons, but at sites indistinguishable fromthose of the other inhibitor.resistant and -sensitive a-naph.thyl valerate-hydrolyzing esterases. It could not be distin-guished in the CNS of cat, probably because it constitutes

less than 3% ofthe total PV.hydrolyzing activity in the CNSofthat species. (JHistochem Cytochem 37:589-596, 1989)

KEY WORDS: a-Naphthyl valerate; Cat; Central nervous system;

Chicken; Fast violet B; Neurotoxic esterase (NTh); Organophos-

phates; Organophosphorus ester-induced delayed neurotoxicity(OPIDN).

activity is resistant to both these agents but is largely inactivated

by 50 �tM DFP (diisopropyl phosphorofluoridate). As with most

carboxylesterases, the physiological substrate of NTh is unknown.

The enzyme has not yet been purified, isolated, or characterized

(Zech and Chemnitius, 1987).

We have sought to develop a histochemical method for local-

ization ofNTE. Some success has been achieved with tissues of the

central nervous system (CNS) of the chicken but not with those

of the cat, probably for the reasons given below.

Materials and Methods

Quantitative. Quantitative determinations of NTE were conducted by

the method ofJohnson (1977). Phenylvalerate was synthesized from the

commercially available acid chloride and phenol. Product identification

and purity were determined by boiling point, thin layer chromatography,

IR spectroscopy, and gc-mass spectroscopy. Paraoxon was purchased from

Aldrich Chemical Co (Milwaukee, WI) and mipafox was procured from

Ash Stevens Inc (Detroit, MI). a-Naphthyl valerate, for the subsequenthistochemical studies, was obtained from Sigma Chemical Co (St. Louis,

MO).

Certain tissues (sciatic nerve, ileum) were first minced with scissors; all

were homogenized in a motor-driven glass-glass homogenizer, in icc-coldbuffer (50 mM Tris-0.2 mM EDTA, pH 8.0), 1 g wet weight tissue/SO ml.

The homogenate was strained through one layer ofsurgical gauze, and 0.5-

ml aliquots were added to 2 ml of identical buffer containing inhibitors

in final concentrations of: (a) zero, (b) 40 �.tM paraoxon, and (c) 40 �sM

paraoxon plus 50 liM mipafox; a reagent blank (d) from which homog-

enate was omitted, and a homogenate blank (e), from which subsequent

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590 KOELLE, THAMPI, HAN, OLAJOS

addition of phenyl valerate was omitted, were included. All determina-

tions were done in duplicate. The mixtures were incubated for exactly 20

mm at 37C with shaking. To each tube was then added 2.5 ml of phenyl

valerate reagent (9 mg phenyl valerate dissolved in 1.0 ml dimethylforma-

mide plus 30 ml 0.03% Triton X-100). After incubation at 37’C for 30 mm,

2.5 ml ofcold 0.3 M pcrchloric acid was added to each tube and the mix-

tures were cooled in an icc-water bath. After centrifugation at 3500 rpmfor 10 mm in the cold, 4 ml clear supernatants were added to tubes con-

taming 2 ml 4-aminoantipyrinc solution (0.05% in 0.5 M Tris buffer, pH

9.0); after thorough mixing, 1.0 ml 0.4% K3Fc(CN)�j was added, result-

ing in immediate development of a plum-red color. Absorbance was read

in a Beckman spectrophotometer at 510 nm. A standard curve was obtained

by plotting the absorbance at 510 nm against various concentrations of phenolafter reaction with aminoantipyrinc and K�Fe(CN)� under conditions iden-tical with those described above.

Histochemical. Of the several procedures that have been used for lo-calization ofcstcrases(Deimling and Backing, 1976; Pcarsc, 1973), the highlysensitive, simultaneous coupling a.zo dye methods appeared to be the most

promising. This approach was introduced by Menten ct al. (1944) for local-

ization of alkaline phosphatase, and was first applied to carboxylic acidcsterases by Nachlas and Seligman (1949). Since then many improvements

and modifications have been proposed. a-Naphthyl esters are generally em-

ployed as substrates, because a-naphthol rapidly forms insoluble precipi-tates with the diazonium salts of several amines. The disadvantage of this

method in the present case is that hen brain has been shown to hydrolyze

a-naphthyl valcratc (a-NV) at only one sixth the velocity for PV, and theparaoxon-rcsistant, mipafox-sensitive portion constitutes only 5 % of the

total in contrast to 50% reported for PV Uohnson, 1975a).

Twenty-one diazonium salts, all purchased from Sigma. were tested ascoupling agents in histochemical experiments with chicken spinal cord orbrain similar to those described below. The list, with catalog (1987) num-

bets, included: fist black K (7253), fast blue BB (3378), f�.st blue RR (0500),fast bordeaux (1005), fast corinth V (6383), fast dark blue R (0750), fast

garnet GBC (6504), fast orange GR (3137), fast red AL (5002), fast red B

(3262), fast red 3GL (0380), fast red ITR (1375), fast red KL (8379), fast

red PDC (6876), fast red RC (2256), fast red RI. (1630), fast red TR (6760),

fast red violet LB (3381), fast scarlet GG (9379), fast scarlet R (1880), and

fast violet B (1631). The most satisfactory appeared to be the last mentioned,which is chemically 6-benzamido-4-mcthoxy-m-toluidine diazonium chlo-

ride (fast violet B salt; P/B). This is consistent with the rating given forthis compound among 23 tested by Pcarsc (1973), as a coupling agent for

localization ofacid phosphatases. Other variables tested included fixatives

and conditions of fixation; concentrations of reagents; pH; time and tern-peraturc ofincubation; and mounting media. After 29 preliminary experi-

ments with serial sections of chicken spinal cord and seven with chicken

brain, the following procedure was adopted.The brain and spinal cord ofRhodc Island Red hens weighing 1.5-2.5

kg (decapitated) and cats (sacrificed by means of sodium pentobarbital,

50 mg/kg, iv, followed by thoracotomy) were removed and sectioned im-mediately or after a few days storage at - 70’C. Sections were cut at 20

�am in a cryostat and placed on slides coated with 2% bovine serum albu-

mm the previous day. After drying for approximately 1 hr, slides were im-

messed in unbuffered 1% formaldehydc/0.9% NaCI at 5C for 10 mm and

rinsed twice for 10 mm in cold 0.9% NaCI. (This distinctly improved stain-ing in contrast to unfixed sections; longer fixation, higher concentrations

of formaldehyde, or any concentration of glutaraldehyde caused markedinhibition of enzyme activity.) With this limited degree of fixation and

the prolonged times ofincubation required, structural preservation was neces-sarily compromised. After drying for 1 hr at room temperature, slides were

prc-incubatcd for 20 mm at 37C in the following solutions, containing

0.9% NaCI: A, control; B, 40 isM paraoxon; C, 40 �iM paraoxon plus 50

�tM mipafox; D, 40 �tM paraoxon plus 50 piM mipafox plus 50 laM DFP

(Paraoxon and DFP were prepared as stock solutions in anhydrous acetone

and propylene glycol, respectively, and held in desiccators in the refrigera-tor for a maximum of 4 weeks; mipafox was prepared as an aqueous solu-

tion extemporaneously.) After two brief rinses in 0.9% NaCl, slides were

dried for 1 hr in the hood, then immersed in the following incubation so-

lution at room temperature for 1-4 hr: P/B, 40 mg; 50 mM Tris-0.2 mM

EDTA buffer, pH 8.0, 32 ml; a-NV (0.1 ml in 20 ml dimethylformamide),

8 ml. One minute after addition ofa-NV the solution was filtered (What.

man No. 1) into Coplin jars and the slides were added. Slides were placed

in fresh incubation solution hourly. After removal, they were rinsed briefly

in distilled water and allowed to dry overnight. They were then mounted

directly in glycerin jelly or Permount and examined. (The precipitate is

soluble in alcohol.)

This final procedure was employed in nine experiments with serial 5cc-

tions ofchickcn brain, and two each with chicken spinal cord, sciatic nerve,

ileum, kidney, and liver. Serial sections of cat CNS were studied in five

experiments. In each experiment a total of 16 slides, containing three to

six sections each, were stained.

By definition, NTh activity was estimated as staining detectable after

pre-incubation in B but not in C; A shows all a-NV-hydrolyzing esterases;

D shows a-NV-hydrolyzing esterases resistant to all three inhibitors, including

DFP

Results

Quantitative

In the CNS ofthe chicken, the proportion ofPV-hydrolyzing activ-ity designated as NTh was found to constitute approximately 6%

of the total (Table 1). As noted previously with respect to the hu-

man brain (Lotti and)ohnson, 1980), there was no significant differ-

ence between various regions. The values obtained here are approx-

imately half those reported by Novak and Padilla (1986) and

considerably lower than those found byJohnson (1975b) and Reveley

et al. (1986); the reasons for these discrepancies are not apparent.

The sciatic nerve was found to contain less than half the NTh ac-

tivity of the CNS. In the non-neural parenchymatous tissues ana-

lyzed (ileum, kidney, liver), the PV-hydrolyzing activity was two

to three times that of the brain but only traces ofNTE activity were

found.

Values for NTh in the CNS ofthe cat ranged between only 1-3%

of total PV-hydrolyzing activity.

Histochemical

Figure 1 illustrates at low magnification the sequential effects of

the inhibitors employed on the total pattern of staining in cross-

sections ofchicken cervical spinal cord. In the absence of inhibitors,

incubation for 4 hr resulted in intense staining for a-NV esterase

in what appeared to be the penikarya of essentially all neurons in

the central gray matter (Figure 1A). The initial portions of their

axons could sometimes be followed for short distances into the white

matter adjacent to the anterior horns; however, staining in this re-

gion was not marked. In addition, marked a-NV esterase staining

occurred in the pia-arachnoid membranes, where they were ad-

herent, and in the Virchow-Robin spaces surrounding the arteries,

as both longitudinally and frequently transversely cut patterns

throughout the white matter. Preliminary treatment with paraoxon

(Figure 1B) resulted in marked reduction of intensity at the above

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HISTOCHEMISTRY OF NEUROTOXIC ESTERASE 591

Table 1 . Total PV-hydrolyzing and NTh activities oftissues ofchicken and cat

Species Tissue

Number of

specimensTotal PY-esterase”

(�smolIg wet weight/mm) NTE�5

Percent

NTh/total

Chicken Cerebral cortex 4 2.99 ± 0.33 0. 170 ± 0.044 5.7

Cerebellum 4 2.85 ± 0.25 0.188 ± 0.050 6.6

Brainstem, anterior 5 1 .97 ± 0. 18 0. 113 ± 0.018 �.8

Brainstem, posterior 5 2.19 ± 0.22 0.148 ± 0.016 6.8

Spinal cord 6 1.20 ± 0.14 0.074 ± 0.010 6.2

Sciatic nerve 2 1.84 ± 0.75 0.037 ± 0.019 2.0

Ileum 2 5.82 ± 1.64 0.002 ± 0.002 <0.1

Kidney 2 5.00 ± 0.01 0.038 ± 0.004 <0.1

Liver 2 6.17 ± 0.25 0.020 ± 0.001 <0.1

Cat Brain, whole 3 1.97 ± 0. 12 0.042 ± 0.002 2.1

Brainstem 3 1.43 ± 0.52 0.042 ± 0.003 2.9

Spinal cord 2 0.68 ± 0.15 0.009 ± 0.001 1.3

a Mean ± SEM = l�d21n (n - 1)]#{189}; where n = 2, SEM is equivalent to the range.

b Resistant to 40 sM paraoxon; inhibited by paraoxon plus 50 sM mipafox.

sites; there was a distinct further reduction after treatment with

paraoxon plus mipafox (Figure 1C). The addition of DFP (Figure

1D) resulted in faint but still detectable staining.

The same sequence is shown at higher magnification for iden-

tical regions of the anterior horn in Figures 2A-2D. For the reasons

noted above, structural integrity is limited.

No qualitative change in the staining pattern after treatment

with the inhibitors mentioned could be detected in any of the many

slides examined (see Methods). In other words, the distribution

of NTh (by definition, Figures 1 and 2, B minus C) appeared to

be identical with that of the other inhibitor-sensitive and -resistant

esterases.

The same was true for the many areas of the medulla, brain-

stem, cerebral cortex, and cerebellum that were studied. In all cases,

where staining of neuronal perikarya was intense in A it became

progressively lighter in B and C, and was usually barely detectable

in D, but with no change in distribution. Two examples are illus-

trated. As shown in Figure 3, identified as the nucleus nervi

hypoglossi ofthe medulla (Yoshikawa, 1968a), fairly intense stain-

ing of neurons is detectable in B (paraoxon-resistant a-NV ester-

ases) and less intense staining in C (paraoxon and mipafox-resistant

a-NV esterases), but the distribution ofstaining is identical. The

same comparisons are noted in Figure 4, B and C, identified as

the nucleus isthmi (Yoshikawa, 1968b), which is roughly equiva-

lent to the medial geniculate nucleus of mammals (Ariens Kap-

pers et al., 1936).

Staining of the sciatic nerve (not shown) was extremely light

but variable; here, no consistent differences could be detected be-

tween B and C.

Non-neural parenchymatous tissues of the chicken stained by

the same procedure (not shown) demonstrated intense staining for

a-NV esterase in controls (A), and similarly distributed marked,

nondifferentiable staining in B and C after 1 or 2 hr incubation.

In the liver, hepatic cells were stained uniformly. The kidney showed

marked staining of most tubule cells and lighter staining of the

glomeruli. The ileum exhibited intense staining of the epithelial

lining cells, heavy staining throughout the submucosa, and scarcely

detectable staining ofthe circular and longitudinal muscle; the neu-

rons of Auerbach’s plexus were faintly stained. As indicated in Ta-

ble 1, these non-neural tissues contain high concentrations of PV-

esterase but minimal concentrations ofparaoxon-resistant, mipafox-

sensitive PV-esterase (NTh) in comparison with neural tissues.

In the cat, no consistent differences could be detected between

B (paraoxon-treated) and C (paraoxon plus mipafox-treated) 5cc-

tions in any ofthe several regions ofthe CNS examined. The prob-

able reason for this is indicated in l#{224}ble1; as noted, NTh activity

was found to account for less than 3% oftotal PV-hydrolyzing ac-

tivity. If it is assumed that the distribution of NTh in the cat is

not distinct from that of the other esterases, as in the chicken, a

differential distribution of so small a proportion would hardly be

distinguishable.

Discussion

The main conclusion to be drawn from the present study is that

the distribution ofNTE in the CNS ofthe chicken and cat appears

to be similar to that of the other a-NV-hydrolyzing (and, presum-

ably, PV-hydrolyzing) esterases. The number of these is consider-

able. By means of iterative elimination of superposed exponential

inhibition curves with paraoxon, mipafox, and DFP, Chemnitius

and Zech (1983a) have demonstrated that hen brain contains 11

PV-hydrolyzing isoenzymes; NTh was identified as numbers 2 and

3. A similar approach (Chemnitius and Zech, 1983b) indicated that

primate brain contains eight such carboxylesterase isoenzymes,

among which number 3 is probably NTh.

Earlier histochemical studies ofthe distribution of carboxylester-

ases in the peripheral nervous system have shown them to be lo-

cated largely in the microsomes and endoplasmic reticulum of the

neuronal perikarya (reviewed by Thomas, 1977), which is consis-

tent with the present interpretation. It can be assumed that from

these sites NTh and the other carboxylesterases are transported

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iiJ�

,;�. . � - , .1I�� �

Figure 1. Transverse 2O-�tm sections of cervical spinal cord of chicken stained by incubation for 4 hr at 20#{176}Cin a solution containing a-NV plus RIB. Prior treatmentfor 20 minutes at 37#{176}Cwith the following inhibitors, from lower left, clockwise: (A) none; (B) 40 RM paraoxon; (C) paraoxon plus 50 �tM mipafox; (D) paraoxonplus mipafox plus 50 sM DFR NTE is identified as staining detectable in B minusthat in C. Although staining is progressively lighter in A through D, no qualitativechange can be detected in its distribution. Original magnification x 27. Bar = 500 �tm.

592 KOELLE, THAMPI, HAN, OLAJOS

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.-,�

O

HIS1OCHEMISTRY OF NEUROTOXIC ESTERASE 593

Figure 2. Higher magnification of identical regions of the anterior horn as shown in sections in Figure lA-iD. Original magnification x 437. Bar = 20 �sm.

.�.,

tI

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594 KOELLE, THAMPI, HAN, OLAJOS

Figure a Similarly stained sections of nu-cleus nervi hypoglossi ofthe chicken; B andC as in Figure 1. CV, cresyl violet. Original

magnification x 27. Bar = 500 �m.

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Figure 4. Similarly stained sections of the nucleus isthmi of the chicken; B and C as in Figure 1. Original magnification x 100. Bar = 100 �sm.

HISTOCHEMISTRY OF NEUROTOXIC ESTERASE 595

throughout the axon and dendrites. Recent evidence indicates that

axonal rather than perikaryonal NTh is the target site for produc-

tion of OPIDN (Caroldi et al., 1984).

AlthoughJohnson’s (1977) assay for NTh has proven valuable

for predicting potential neurotoxicity of organophosphates (Lotti

andJohnson, 1978;Johnson, 1975b, 1975c), several critical obser-

vations have remained unexplained. When hens are given a single

dose ofcertain OPIDN-inducing compounds, the NTh activity of

the central nervous system returns practically to the normal level

during the 2-week latent period before overt signs of motor dys-

function appear; furthermore, other compounds that produce near-

total inhibition of NTh do not cause OPIDN (Johnson, 1975b).

It is a.lso notable that OPIDN is produced only by organophosphates

that undergo “aging,” or loss of an alkyl group from the phos-

phorylated enzymatic site. These observations have led to more re-

cent proposals that alkylphosphorylation ofNTE is only the initial

step that culminates in OPIDN, or that it is paralleled by the phos-

phorylation of some other enzyme or macromolecule that is directly

responsible for the production ofOPIDN (Lotti et al., 1984;John.

son, 1980). It has also been suggested that NTh and an essential

protein kinase may be combined in a single macromolecule (Zech

and Chemnitius, 1987).

The present demonstration of the non-selective localization of

NTh appears to be more consistent with these alternative proposals

than with that of the direct role of the empirically defined NTE

in the production of OPIDN.

Acknowledgments

The assistance ofDr Nicholas D. Gonatas in interpreting some ofthe slides

isgratefully acknowledged W� thank CindiPatillofortyping the manuscript.

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