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Histochemical demonstration of neurotoxic esterase.

G B Koelle, N S Thampi, M S Han and E J Olajos J Histochem Cytochem 1989 37: 589 DOI: 10.1177/37.5.2703698 The online version of this article can be found at: http://jhc.sagepub.com/content/37/5/589

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0022-1554/89/$3.30 The Journal of Histochemistry and Copyright 1989 by The Histochemical

Cytochemistry Society.

Vol. 37. Inc.

No.

5, pp.

589-596, 1989 Printedin U.S.A.

Original

Article

Histochemical
GEORGE B. KOELLE,2

Demonstration
NAGENDRAN

of Neurotoxic
MATTHEW S. HAN,

Esterase

S. THAMPI,

and
Department Received

EUGENE

J.

OLAJOS3
Medical August 17, School, 1988 and University in revised ofPennsylvania, form November 14, Philadelphia,
1988;

ofPharmacology, for publication

Pennsylvania November 22,

19104-6084. 1988 (8A1462).

accepted

We developed toxic esterase

for localizing neurophenylvalerate (PV)hydrolyzing esterase that is resistant 40 tM paraoxon to (A) but inactivated by paraoxon plus 0 iM impafox 5 (B). NTh is considered to be the target enzyme in the production

a histochemical (NTh), defined

method as the

A but not in B. In the central nervous system chicken, NTh appeared to be present primarily mata of most neurons, but at sites indistinguishable those of the other inhibitor.resistant and -sensitive

in

(CNS) of the sofrom a-naph.

thyl
guished

valerate-hydrolyzing
in the CNS of

esterases.
cat, probably

of organophosphorus
(OPIDN). Cryostat

ester-induced

delayed

neurotoxicity

It could not be distinbecause it constitutes

activity 37:589-596,
Central (NTh);

taming
m-toluidine

a-naphthyl
the

with tivity.

sections were incubated in a medium convalerate and 6-benzamido-4-methoxydiazonium chloride (fast violet B) after treatment above-mentioned inhibitors, leading to formation

less than 3% ofthe total PV.hydrolyzing ofthat species. (JHistochem Cytochem

KEY WORDS:

in the
1989)

CNS
system;

a-Naphthyl Fast violet B;

valerate; Neurotoxic

Cat;

nervous

Chicken;

esterase

Organophos-

of an aqueous
NTh

insoluble
activity was

precipitate
estimated

at sites
as staining

of enzymatic
detectable

ac- phates;
in(OPIDN).

Organophosphorus

ester-induced

delayed

neurotoxicity

Introduction
Organophosphorus ester-induced produced by certain delayed neurotoxicity compounds of that (OPIDN) category.

activity by 50 tM

is resistant DFP

to (diisopropyl

both

these

agents

but

is largely As with

inactivated most

phosphorofluoridate).

is a syndrome It is characterized tion,

10-14 demyelination, days, and

carboxylesterases, The enzyme has

the not

physiological yet been

substrate purified,

of NTh isolated,

is unknown.

by sequential
and flaccid is completely

development
paralysis unrelated after to

of axonal
a latent the

degeneraperiod

or characterized method for local-

anticholinesterase

(Zech and Chemnitius, 1987). of We have sought to develop ization central of the ofNTE. nervous cat, Some success system (CNS) for the

a histochemical has been of the reasons achieved chicken given

(anti-ChE)
of human

action
OPIDN

of such
have

compounds.
been reported,

Several
usually

extensive
resulting

outbreaks
from

the

with tissues of the but not with those below.

contamination
phate. (1981) The and Zech its

of beverages
subject and biochemical has been

or cooking
reviewed

oil with
thoroughly (1987).

triorthocresyl
by

phos-

probably

Abou-Donia

Chemnitius basis

Although

is still

unproven,

one

ofthe

most

generally
by lation) of

considered
neurotoxic

proposals
and esterase

at present
subsequent (NTh)

is that
aging Uohnson,

OPIDN
(partial 1969, as

is caused
dealky1977). the

Materials and Methods Quantitative. Quantitative

the method ofJohnson
available were Chemical determined and Co studies, tissues (sciatic gc-mass (Milwaukee, (1977). acid

determinations
Phenylvalerate and

alkylphosphorylation

of NTE were conducted was synthesized from

phenol. Product layer was was thin Paraoxon and mipafox

by the

enzyme phenylvalerate

has

been

defined

empirically esterase

by Johnson (1977) that is resistant

commercially This and purity IR spectroscopy, Aldrich

chloride by boiling spectroscopy.

identification chromatography, purchased procured from from Louis, all

point,

(PV)-hydrolyzing

to 40 p.tM

WI)

paraoxon (0,0-diethyl-0-p-nitrophenyl phosphate; activated by paraoxon plus 50 sM mipafox fluorophosphorodiamide). An additional fraction

E 600) but is in(N,N-diisopropyl of PV-hydrolyzing

Ash
MO).

Stevens

Inc (Detroit,
was

MI).
obtained nerve,

a-Naphthyl
from ileum) were

valerate,
Sigma first

for the
Co with

subsequent
(St. scissors;

histochemical Certain

Chemical minced

Supported To whom US

by

Contract

DAAA15-87-K-0002, should Research, Proving be

Department

of

the

were homogenized in a motor-driven glass-glass homogenizer, in icc-cold buffer (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 0.5ml aliquots final was were concentrations omitted, and added to of: 2 ml (a) of identical (b) blank blank buffer (d) (e), containing inhibitors sM homogsubsequent in zero, a reagent 40 paraoxon, .tM from

Army.
2

correspondence address: Army, Chemical Aberdeen

addressed. Engineering

and
from

(c)40
which

3 Present

Center,

Development and Ground, 21010-5423. MD

paraoxon
enate

plus 50 liM mipafox;

a homogenate

which

589

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590

KOELLE,

THAMPI,

HAN,

OLAJOS

addition tions were

of

phenyl done

valerate in duplicate.

was The

omitted, mixtures

were were

included. incubated

All for

determinaexactly

mm at 37C with shaking. To each tube was then added ml of phenyl 2.5 valerate reagent (9 mg phenyl valerate dissolved 1.0 ml dimethylformain
mide
2.5

(Paraoxon and DFP were prepared as stock solutions in anhydrous acetone and propylene 20 glycol, respectively, and held in desiccators in the refrigerator for a maximum of weeks; 4 mipafox was prepared as an aqueous solution extemporaneously.) After two brief rinses in 0.9% NaCl, slides were
for 1 hr in the pH minute hood, then for a-NV and addition jars allowed jelly or hourly. to dry Permount immersed 1-4 (0.1 ofa-NV the slides After and hr: ml P/B, the were removal, examined. in the 40 in 20 ml following mg; 50 mM was Slides were were (The they They incubation Tris-0.2 somM (What. placed briefly mounted is at room One temperature 8.0, 32 ml; after Coplin and

plus were

30 ml 0.03%
0.3

Triton an icc-water 4 ml mixing,

X-100). acid clear solution 1.0 ml was bath.

After added After (0.05% 0.4%

incubation to each were in 0.5 K3Fc(CN)j color. A standard centrifugation

at 37C tube added M Tris was curve Absorbance and

for the

ml ofcold
in

M pcrchloric

30 mm,dried mix- lution con- ml. 8 pH was

tures taming 9.0); ing

cooled the

in cold,

at rpm 3500 to tubes buffer, added,

EDTA buffer, man

read in

dimethylformamide), filtered were rinsed then precipitate

for 10 mm
after

supernatants

solution added.

2 ml in immediate the

4-aminoantipyrinc development spectrophotometer absorbance

No. 1) into
incubation water in in final kidney, distilled

thorough

result- in fresh directly

solution

of a plum-red at 510 nm. at 510 nm against

overnight.

in a Beckman by plotting

was obtained

glycerin alcohol.) procedure brain, and

after
tical

reaction
with

with
those

aminoantipyrinc
described above.

and

various concentrations of phenol soluble KFe(CN) under conditions idenThis

tions ileum,

ofchickcn

was employed in nine experiments with serial 5ccandtwo each with chicken spinal cord, sciatic nerve,
liver. Serial sections of cat CNS were studied in five

procedures that have been used for loexperiments. In each experiment a total of 16 slides, containing three to calization and Backing,1976; Pcarsc, 1973), the highly six sections each, were stained. sensitive, simultaneous coupling a.zo dye methods appeared to be the most By definition, NTh activity was estimated as staining detectable after promising. This approach was introduced by Menten ct(1944) for localal. pre-incubation in B but not in C; A shows all a-NV-hydrolyzing esterases; ization of alkaline phosphatase, and was first applied to carboxylic acid D shows a-NV-hydrolyzing esterases resistant to all three inhibitors, including csterases by Nachlas and Seligman (1949). Since then many improvements DFP and modifications have been proposed. a-Naphthyl esters generally are employed as substrates, because a-naphthol rapidly forms insoluble precipiHistochemical. tates with the diazonium salts of several amines. The disadvantage of this

Of the ofcstcrases(Deimling

several

method in the present case that hen brain has been shown is to hydrolyze a-naphthyl valcratc (a-NV) at only one sixth the velocity for PV, and the Quantitative paraoxon-rcsistant, mipafox-sensitive portion constitutes only of the 5% In the CNS ofthe chicken, the proportion ofPV-hydrolyzing activtotal in contrast to50% reported for PV Uohnson, 1975a). Twenty-one diazonium salts, all purchased from Sigma. were tested as ity designated as NTh was found to constitute approximately 6% coupling agents in histochemical experiments with chicken spinal cord orof the total (Table 1). As noted previously with respect to the hubrain similar to those described below. The list, with catalog (1987) numman brain (Lotti and)ohnson, 1980), there was no significant differbets, included: fist black K (7253), fast blue BB (3378), f.st blue RR (0500), ence between various regions. The values obtained here are approxfast bordeaux (1005), fast corinth V (6383), fast dark blue R (0750), fast imately half those reported by Novak and Padilla (1986) and garnet GBC (6504), fastorange GR (3137), fast red AL (5002), fast red B considerably lower than those found byJohnson (1975b) and Reveley (3262), fast red 3GL (0380), fast red (1375), ITR fast red KL (8379), fast et al. (1986); the reasons for these discrepancies are not apparent. red PDC (6876), fast red RC (2256), fast red RI. (1630), fast red TR (6760), The sciatic nerve was found to contain less than half the NTh acfast red violet LB(3381), fast scarlet GG (9379), fast scarlet R(1880), and tivity of the CNS. In the non-neural parenchymatous tissues anafast violet B (1631). The most satisfactory appeared to be the last mentioned, lyzed (ileum, kidney, liver), the PV-hydrolyzing activity two was which is chemically 6-benzamido-4-mcthoxy-m-toluidine diazonium chloride (fast violet B salt; P/B). This is consistent with the rating given for three to times that of the brain but only traces ofNTE activity were
this compound ofacid ofincubation; among 23 tested by Pcarsc Other mounting (1973), variables media. After as a coupling tested included agent fixatives for found. localization phosphatases. and

Results

and

conditions

of fixation;

concentrations

of reagents;

pH;

time

and

tern- of
experi-

Values for NTh in the total PV-hydrolyzing

CNS ofthe activity.

cat ranged

between

only

1-3%

peraturc

29 preliminary

ments with serial sections of chicken spinal cord and seven with chicken brain, the following procedure was adopted. Histochemical The brain and spinal cord ofRhodc Island Red hens weighing 1.5-2.5 Figure 1 illustrates at low magnification kg (decapitated) and cats (sacrificed by means of sodium pentobarbital, employed on the total 50 mg/kg, iv, followed by thoracotomy) were removed and sectioned im- the inhibitors sections ofchicken cervical spinal cord. mediately or after a few days storage at - 70C. Sections were cut at 20 am in a cryostat and placed on slides coated with bovine 2% serum albuincubation for 4 hr resulted in intense mm the previous day. After drying for approximately 1 hr, slides were im-in what appeared to be the penikarya
messed in unbuffered 1% formaldehydc/0.9% NaCI at 5C for higher degree 10 mm and

the pattern In the staining

sequential of staining absence inhibitors, of for a-NV

effects

of

in crossesterase

the
axons

central

gray

matter

(Figure

1A).

of essentially The initial

all neurons in portions of their the white in this restaining were arteries, ad-

rinsed
ing

twice for 10 mm
in contrast

in cold 0.9%
sections; activity.) After drying

NaCI.

(This

distinctly
fixation, limited

improved caused

stainmarked was necesslides

to unfixed of enzyme

longer With this

concentrations of fixation

of formaldehyde,
inhibition

or any concentration times ofincubation required,

for

of glutaraldehyde structural

could sometimes be followed for short distances into matter adjacent to the anterior horns; however, staining
and

gion occurred

was

not in and

marked. the in the

In addition, Virchow-Robin and matter. in marked

marked membranes, spaces

a-NV where surrounding transversely treatment of intensity

esterase they the

the prolonged
sarily compromised.

preservation
temperature,

pia-arachnoid

1 hr at room

herent, were as both throughout (Figure

prc-incubatcd 0.9% NaCI: tM mipafox;

for 20 mm at 37C in the following solutions, containing A, control; B, 40 isM paraoxon; C, 40 iM paraoxon plus 50 D, 40 tM paraoxon plus 50 piM mipafox plus 50laM DFP

longitudinally the white 1B) resulted

frequently Preliminary reduction

cut patterns with paraoxon at the above

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HISTOCHEMISTRY

OF

NEUROTOXIC

ESTERASE

591

Table
Species

1.

Total

PV-hydrolyzing
Tissue

and

NTh

activities
Number of

oftissues

ofchicken
Total (smolIg
wet

and
PY-esterase

cat
Percent
NTE5

specimens 4 4 anterior posterior 5 5 6 2 2 2 2

weight/mm) 0.33 0.25 0. 18 0.22 0.14 0.75 1.64 0.01 0.25 0. 12 0.52
0.15

NTh/total

Chicken

Cerebral Cerebellum Brainstem, Brainstem, Spinal

cortex

2.99 2.85 1 .97 2.19 1.20 1.84 5.82 5.00 6.17 1.97 1.43 0.68
equivalent to the range.

0. 170 0.188 0. 113 0.148 0.074 0.037 0.002 0.038 0.020 0.042 0.042
0.009

0.044
0.050

5.7 6.6 .8 6.8 6.2 2.0 <0.1 <0.1 <0.1 2.1 2.9 1.3

0.018 0.016 0.010 0.019 0.002 0.004 0.001 0.002 0.003 0.001

cord

Sciatic
Ileum Kidney Liver

nerve

Cat

Brain, whole Brainstem

Spinal
a Mean b Resistant

3 3
2 SEM

cord
-

SEM
to

ld21n

40 sM

(n paraoxon;

1)]#{189}; where

2,

is

inhibited

by

paraoxon

plus

50

sM

mipafox.

sites;

there

was

a distinct

further

reduction

after

treatment

lining with

cells,

heavy

staining

throughout

the

submucosa, muscle; As

and the indicated

scarcely neuin Ta-

paraoxon plus mipafox 1D) resulted in faint The same sequence

(Figure but still is shown

1C). The detectable at higher in Figures is limited.

staining

addition staining. magnification 2A-2D.

pattern

of DFP for For the

after

(Figure detectable
rons of

staining
Auerbachs

ofthe
plexus

circular
were

and longitudinal
faintly stained.

tical regions of the anterior horn noted above, structural integrity

No qualitative change in the

iden- ble 1, these but reasons esterase

sensitive

non-neural minimal

tissues concentrations
(NTh) in

contain
comparison

high

concentrations mipafoxneural with

of PVtissues.

ofparaoxon-resistant, could plus

CNS

PV-esterase

treatment

In the
tions to able in

cat,
any

no consistent and
several ofthe

differences C (paraoxon
regions ofthe

be detected mipafox-treated)
examined.

between 5ccThe prob-

with slides

the

inhibitors

mentioned (see Methods).

could In

be detected other words,

in any of the the

C) appeared

manyB (paraoxon-treated) reason for this

examined

distribution and

of NTh (by definition, be identical with that esterases. The same stem, cerebral where staining progressively in D, but with

Figures 1 and 2, B minus of the other inhibitor-sensitive

is indicated for that less the than

in l#{224}ble as noted, 1; 3% oftotal distribution esterases, a proportion of NTh

NTh in the

activity accat is a be

to account -resistant was found tivity. If it is assumed from that distribution

PV-hydrolyzing as in the would chicken, hardly

was true for the many cortex, and cerebellum of neuronal perikarya

areas of the medulla, brain- not distinct that were studied. In all cases,differential distinguishable. was intense in became it A usually Two as barely examples the fairly detectable are illusnervi stain- Discussion ester- The main the distribution

of the other of so small

lighter in B and C, and was no change in distribution.

trated. As shown in Figure 3, identified hypoglossi ofthe medulla (Yoshikawa, 1968a), ing
ases)

nucleus intense a-NV

of neurons
and less

is detectable
intense staining

in B (paraoxon-resistant
in C (paraoxon and

conclusion ofNTE

to of the

be

drawn CNS The other

from ofthe number

the

present and

study cat (and,

is that appears presum-

mipafox-resistant

in the esterases.

chicken

a-NV
same

esterases),
comparisons

but isthmi medial

the
are

distribution
noted in Figure

ofstaining
4, B and

is identical.
C, identified

to that The to be similar ably, PV-hydrolyzing) as

a-NV-hydrolyzing of these

is considerexponential Chemnitius contains 11

the lent pers

nucleus to the

(Yoshikawa, geniculate sciatic nerve no consistent

1968b), nucleus

et al., 1936). Staining of the but variable; here, tween B and C. Non-neural the a-NV same procedure esterase

By means of iterative elimination of superposed which is roughly equiva- able. curves with paraoxon, mipafox, and DFP, of mammals (Ariens Kap-inhibition and Zech (1983a) have demonstrated that hen brain was could extremely be detected stained staining

(not shown) differences tissues

isoenzymes; NTh was identified as numbers 2 and light PV-hydrolyzing 3. approach (Chemnitius and Zech, 1983b) indicated that be- A similar primate brain contains eight such carboxylesterase isoenzymes, 3 is probably studies nervous ofthe system NTh. distribution of carboxylesterhave shown them to be lo-

parenchymatous (not in controls shown) (A),

of the

chicken intense distributed

2 hr

demonstrated and similarly

among which number by histochemical for Earlier marked, ases in the peripheral

nondifferentiable In the liver, hepatic marked glomeruli. staining The

staining in B and C after or 1 cells were stained uniformly. of most ileum tubule cells intense and exhibited

incubation.

The lighter

kidney staining of the

cated largely in the microsomes and endoplasmic reticulum of the perikarya (reviewed by Thomas, 1977), which is consisshowed neuronal tent with the present interpretation. It can be assumed that from of the epithelial these sites NTh and the other carboxylesterases are transported

staining

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592

KOELLE, THAMPI,

HAN,

OLAJOS

iiJ

,;.

.1I

Figure 1. Transverse 2O-tm for 20 minutes at 37#{176}C with plus mipafox plus 50 sM DFR change can be detected in

sections of cervical spinal cord of chicken stained by incubation for 4 hr at 20#{176}Ca solution in containing a-NV plus RIB. Prior treatment the following inhibitors, from lower left, clockwise: (A) none; (B) 40 RM paraoxon; (C) paraoxon tM plus 50 mipafox; (D) paraoxon NTE is identified as staining detectable in B minusthat in C. Although staining is progressively lighter in A through D, no qualitative its distribution. Original magnification x 27. Bar tm. = 500

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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 20 sm. Bar 437.

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594

KOELLE,

THAMPI,

HAN,

OLAJOS

Figure a Similarly stained sections of nucleus nervi hypoglossi ofthe chicken; B and C as in Figure 1. CV, cresyl violet. Original magnification x 27. Bar= 500 m.

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HISTOCHEMISTRY

OF NEUROTOXIC

ESTERASE

595

Figure

4.

Similarly

stained

sections

of the

nucleus

isthmi

of the

chicken;

B and

C as in Figure

1. Original

magnification

x =

100 sm. 100. Bar

throughout the axon and dendrites. Recent evidence axonal rather than perikaryonal NTh is the target tion of OPIDN (Caroldi
(1977)

et

al.,

1984).

kinase indicates that protein site for produc- and Chemnitius, The present valuable
NTh appears to

may

be combined

in a single of the

macromolecule localization
alternative

(Zech of NTE

1987). demonstration
be more consistent

non-selective
with these

AlthoughJohnsons for predicting potential andJohnson, vations have dose the

during

assay for neurotoxicity

NTh has proven of organophosphates

proposals

(Lotti than obser- in a single the

with

that

of the of

direct OPIDN.

role

of the

empirically

defined

1978;Johnson, 1975b, remained unexplained. OPIDN-inducing nervous

2-week

1975c), several critical When hens are given the

overt signs

production

ofcertain central
the

compounds, returns
period

NTh
of

activity normal
motor

system
latent

practically
before

to the that

Acknowledgments of ofDr levelThe assistance

isgratefully dysacknowledged

Nicholas

D.

Gonatas

in interpreting the

some

ofthe

slides

W thank

CindiPatillofortyping

manuscript.

function

appear;

furthermore, of NTh do that OPIDN

other

compounds

produce

near-

total inhibition It is a.lso notable

OPIDN (Johnson, 1975b). Literature Cited only by organophosphates Abou-Donia MD (1981): Organophosphorus ester.induced delayed neuthat undergo aging, or loss of an alkyl group from the phos-rotoxicity. Annu Rev Pharmacol Toxicol 21:511 phorylated enzymatic site. These observations have led to more reAriens Kappers CV, Huber CG, Crosby EC (1936): The comparative anatcent proposals that alkylphosphorylation ofNTE is only the initial omy of the nervous system ofvertebrates including man. 2.Vol New York, step that culminates in OPIDN, or that it is paralleled by the phos- Macmillan, 1170 phorylation of some other enzyme or macromolecule that is directly Caroldi 5, Lotti M, Masutti A (1984): Intra-arterial injection of diisopropylresponsible for the production ofOPIDN (Lotti et al., 1984;John. tluorophosphate or phenylmcthanesulfonyl fluoride produces unilateral neuor protection, respectively, in hens. Biochem Pharmacol 33:3213 son, 1980). It has also been suggested that NTh and an essential ropathy

not cause is produced

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596

KOELLE,

THAMPI,

HAN,

OLAJOS

Chemnitius 3M, Zech R (1983a): Inhibition of brain carboxylcstcrases by neurotoxic and non-neurotoxic organophosphorus compounds. Mol Pharmacol 23:717 Arch ChemnitiusJM,
acterization Deimling of OV,

Lotti Mjohnson (1978): MK Neurotoxicity predictions can be based onstudies in vitro Toxicol 41:215
Menten ML,JungeJ, phosphatase MM, Cancer for alkaline Nachlas j NatI

oforganophosphorus
with hen and human

pesticides:
enzymes.

Zech
multiple Bocking

R (1983b):
forms. A

Carboxylesterases

in primate 15:1019 in histochemistry

brain:

chartest and ultraesterase.

Green (1944): MHA coupling

histochemical

azo

dye

mt

J Biochem
Esterases

the in

kidney.

J Biol

Chcm 153:471 demonstration of

histochemistry. Johnson Nature J ohnson Johnson hibitors

Johnson phosphorus 287:105

Histochem

(1976): 8:215 J

Seligman AM The Inst 9:415

(1949): histochemical

MK (1980): MK (1977):

Neurotoxicity:

mechanisms

explored

and

exploited.

Novak R, Padilla S (1986): Anin vitro comparison neurotoxic esterase. Fundam Appl Toxicol
Pearse AGE (1973): Histochemistry.

ofrat

and

chicken

brain

6:464
Theoretical and applied. Vol 2. 3rd

Improved
for delayed

assay

of neurotoxic
potential.

esterase
Arch

for screening
Toxicol 37:113

organophosphatcs

neurotoxicity

MK (1975a): Structure-activity of hen brain neurotoxic

relationships cstcrasc. Biochem neuropathy

ed. Edinburgh, Churchill Livingstonc, 761 for substrates and inPharmacol 24:797 RcveleyJW, Sabourin TD, Moore MT. Goss
organoToxicol rotoxic 3:289

LB(1986):

Distribution

of ncuphos-

MK (1975b): The
esters: mechanism

delayed
and

caused by some
CRC Crit Rev

challenge.

esterase activicy in the brain of control phorofluoridate-treatedhens: in vivo and in v#{252}m exposure. Histochemic der Enzyme

and diisopropyl Toxicol Len 31:45 Nervensystem.

Johnson MK (1975c): effects: mechanism icol 34:259

Organophosphorus

ofaction

esters causing delayed neurotoxic and structure/activity relationships. Arch

Toxsity

Thomas (1977): E Stuttgart, Yoshikawa T(1968a):

of Tokyo

in peripheren

Gustav
Press, F25 Atlas

Fischer

Verlag,

47,

62
animals. Tokyo,

Atlas ofthc brains

ofdomcstic

Univer-

Johnson MK(1969): The delayed neurotoxic effrct compounds identification of the phosphorylation chem J 114:711
Lotti M, Becker CE, Aminoff

ofsome

organophosphorus

site as an esterase. polyncuropanervous

BioYoshikawa T(1968b): ofthc brains ofdomestic animals. Tokyo, Univer-

thy:
Lotti

pathogenesis
M,Johnson 34:747

and

MJ (1984): Organophosphate prevention. Neurology 34:658 esterase in human

sity of Tokyo Press, zch R, ChcmnitiusJM

ropathy. Prog

F17,F18
(1987):

Neurotoxicant oforganophosphorus
29:193

sensitive

esterase. ester-induced

Enzymol. delayed ncu-

MK (1980): Neurotoxic

tissue.

ogy and pathophysiology

Ncurobiol

J Neurochem

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