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This is to certify that the

thesis entitled

THE DELAYED NEUROTOXIC EFFECTS
OF TRIPHENYL PHOSPHITE (TPP)
IN THE JAPANESE QUAIL

presented by

REJEEV GEORGE VARGHESE

has been accepted towards fulfillment
of the requirements for

M.S. degreein ANIMAL SCIENCE

 

.

Major pro essor

Date 10/ 'q/q"!

07639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

 

LIBRARY
Michigan State
Unlverslty

 

 

 

PLACE ll RETURN BOXtonmovomb Mouflom ywrrocord.
TO AVOID FlNEB mum morbdonddoduo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Vii—7

MSU lsAnNflnn‘lwAcflmEqnl Opponunlly lawman

THE DELAYED NEUROTOXIC EFFECTS OF TRIPHENYL PHOSPHITE (TPP)
IN THE JAPANESE QUAIL

BY

Rejeev George Varghese

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Science

1994

ABSTRACT
THE DELAYED NEUROTOXIC EFFECTS OF TRIPHENYL PHOSPHITE (TPP)
IN THE JAPANESE QUAIL
BY

Rejeev George Varghese

Certain organophosphorus chemicals (OPs) cause a
condition called organophosphorus-induced delayed
neurotoxicity (OPIDN) . Since previous studies indicated
Japanese quail were resistant to Type I OPIDN compounds, the
purpose of this study was toldetermine the sensitivity of this
species to triphenyl phosphite (TPP), a Type II OPIDN
compound. Quail were administered single subcutaneous doses
of TPP at concentrations up to 500 mg/kg. At 24 hrs after
dosing, half of the birds from each dose group were assessed
for whole-brain neurotoxic esterase (NTE) activity. The
remaining birds were observed daily for up to 20 days for the
development of OPIDN clinical signs. Brains from some of
these birds were examined for degeneration. All doses of TPP
resulted in NTE inhibition which ranged from 11 % to 87 % and
clinical signs as soon as 3 days post-dosing. Widespread

degeneration in the brain was also noted.

ACKNOWLEDGEMENTS

I would like to thank my major advisor, Dr. Steve Bursian
for the mentoring, valuable thoughts, guidance and friendship.
Special thanks also go to the members of my guidance
committee, Dr. Duke Tanaka, Dr. Richard Balander and Dr.
Robert Poppenga for their valuable help in my graduate
program. I am.thankful to Dr. Duke Tanaka for helping me with
the research in.neuropathology and.to Dr. Richard Aulerich for
his comments on my manuscript.

Personal appreciation goes to my uncle, Dr. Sam Varghese;
for introducing me into the field of Animal Sciences, for his
valuable help, suggestions and advice. Special thanks go to
my friend Sally Gaff for helping me with the chemical diagrams
and printing.

I am very grateful to my family for their support in my
career pursuits; whose prayers have been my strong foundation.
Above all, I thank the Lord God for His mercy and blessings.
I only hope that I can strive to please Him in all that I do

and through my academic endeavors.

iii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . .

LIST OF FI
INTRODUCTI

LITERATURE

MATERIALS AND METHODS . . . . . . .

RESULTS

DISCUSSION

GIIRE S O O O O O O O O O O O O 0
ON 0 O O O O O O O O O O O O 0

REVIEW . . . . . . . . .
Organophosphorus Compounds
Two Kinds of OPIDN . . . .
History & Incidence . . . .
Chemical Structure 8 Reactivity
Latent Period . . . . . . . . .
Clinical Signs . . . . . . . .
Neuropathological Features . .
Role & Inhibition of Neurotoxic
Age Sensitivity . . . . . . . .
Species Selectivity . . . . . .

Test Species and Husbandry
Treatment . . . . . . . . .
Neurotoxic Esterase Assay .
Clinical Observations . . .
Neuropathological Assessment
Statistical Analysis . . . .
Animal Use and Care . . . . .

Clinical Signs

NTE Assay . . . . . .
Histopathological Assessment

Clinical Signs . . . . . .
NTE Assay . . . . . . . .
Neuropathological Studies .

CONCLUSION . . . . . . . . . . . . . . .

LIST OF RE

FERENCES O O O O O O O O O O 0

iv

ooMoooooooo

o o o o o o o o o 0 ff. 0 o o o o o o

LIST OF TABLES

The effects of TPP and TOTP on the development of
clinical signs characteristic of OPIDN in adult.Japanese
qua i 1 O O O O O O O O O O O O O O O O O O O O O O O 4 3

The effects of TPP and TOTP on whole-brain neurotoxic
esterase (NTE) activity in Japanese quail 24 hours post-
administration . . . . . . . . . . . . . . . . . . . 46

The distribution of degeneration in the forebrain of the
Japanese quai l perfused at various times after TPP
administration 0 O O O O I O O O O O O O O O I O O O 56

LIST OF FIGURES

The chemical structures of tri-o-tolyl phosphate (TOTP),
o-tolyl saligenin phosphate (TSP) and bis(1-methylethyl)
phosphorofluoridate (DFP) . . . . . . . . . . . . . 14

The chemical structure of triphenyl phosphite (TPP), a
Type II organophosphorus compound . . . . . . . . . 17

Darkfield photomicrographs of parasagittal sections
through the forebrain of the Japanese quail illustrating
the dense axonal and terminal degeneration present in the
lateral forebrain.bundle (FPL), caudal neostriatum (NC),
and ectostriatum (E) 9 days after a single subcutaneous
injection of 125 mg TPP/kg body wieght . . . . . . . 49

Photomicrographs illustrating control and TPP-exposed
Fink-Heimer silver impregnated sections through the
caudal neostriatum and ectostriatum . . . . . . . . 51

TPP-induced degeneration in the Japanese quail brain
(sagittal median view) . . . . . . . . . . . . . . . 52

TPP-induced degeneration in the Japanese quail brain
(sagittal lateral view) . . . . . . . . . . . . . . 53

TOTP-induced degeneration in the Japanese quail brain
(sagittal median view) . . . . . . . . . . . . . . . 54

TPP-induced degeneration in the chicken brain (sagittal
median VieW) O O O O O O O O O O O O O O O O O O O O 67

TPP-induced degeneration in the chicken brain (sagittal
lateral view) . . . . . . . . . . . . . . . . . . . 68

vi

INTRODUCTION

Organophosphorus compounds are used extensively in
industry and agriculture as pesticides, flame retardants,
plasticizers, petroleum additives, and intermediates in the
manufacture of pharmaceuticals (Davis and Richardson, 1980;
U.S. Environmental Protection Agency, 1985). Exposure to
certain of these organophosphorus compounds results in a
neurological condition characterized by progressively
developing hindlimb ataxia and paralysis, inhibition of the
enzyme neurotoxic esterase (NTE) and development of axonal
degeneration in both the central (CNS) and peripheral (PNS)
nervous systems (Johnson, 1975a,b, 1982; Davis and Richardson,
1980; Abou-Donia, 1981; Baron, 1981; Abou-Donia and Lapadula,
1990; Tanaka et al., 1990a,b, 1991, 1992a). Since 1978, this
effect has been termed organophosphorus—induced delayed
neurotoxicity or OPIDN (Abou-Donia, 1978). OPIDN's major
visible consequence is the motor dysfunction resulting from
neuropathic lesions, though there are other significant
changes that precede or accompany clinical manifestations.
These changes occur at various levels: changes at the
molecular level are apparent as neurochemical changes, changes
at the cellular level are apparent as neurophysiological

changes, changes at the tissue level are apparent as

1

2
neuropathological changes, and changes at the organism level
are apparent as functional changes, ie neurobehavioral and
neurological alterations. The term "neurotoxic" is a general
term to encompass all of these changes and is adequate enough
to fully define this effect (Abou-Donia, 1981).

The series of poisonings caused by ingestion of tri-o-
tolyl phosphate (TOTP) that occurred at intervals during the
period from 1930 through the 1970s fully depicts the dangers
of human exposure to GP compounds. In most of these cases,
poisoning was from the ingestion of beverages or food
adulterated with TOTP (Senanayake and Johnson, 1982). The
characteristic syndrome was initially called "Ginger Jake
paralysis" because of the consumption of illicit alcoholic
beverages made from extracts of Jamaican ginger contaminated
with TOTP. The victims displayed acute signs of vomiting and
diarrhea which were followed by the onset of muscle pain,
muscle weakness and parathesias. The chronic cases were
characterized by individuals having signs of persistent ataxia
and spasticity.

Recently OPIDN has been divided into 2 categories
designated as Type I and Type II, with each type possessing
distinctive clinical and neurophysiological characteristics
(Abou-Donia and Lapadula, 1990) . Type I is produced by
exposure to organophosphorus compounds such as tri-o-tolyl
phosphate or TOTP (also know as tri-ortho—cresyl phosphate or
TOCP) , bis (I-methylethyl) phosphorofluoridate (DFP) , mipafox,

and leptophos. Features of Type I OPIDN in animals include

3

inhibition of the nervous system enzyme neuropathy target
esterase (NTE) in excess of 70 % and the presence of a
relatively long delay period of 10 - 21 days before the onset
of hindlimb ataxia and paralysis. The accompanying
neuropathology is localized in.peripheral nerves, spinal cord
and brainstem. Type II OPIDN may be produced by exposure to
triphenyl phosphite or tri-ortho-, tri-meta-, or tri-para-
cresyl phosphite. In contrast to Type I 0P compounds, delayed
neurotoxicity induced by Type II compounds is characterized by
NTE inhibition which may be less than 65 - 70 % and by a
shortened delay period of 4 - 7 days before the onset of
ataxia and hindlimb paralysis. Results of recent work by
Tanaka et al. (1990b, 1992a,b) indicate that neuropathology
resulting from exposure to Type II compounds involves not only
the spinal cord and brainstem, but also the midbrain and
forebrain. Type I 0Ps contain a central pentavalent
phosphorus atom while Type II 0Ps contain a central trivalent
phosphorus atom.

To determine the suitability of a species as an OPIDN
model, the pattern of degeneration in the central nervous
system (CNS) is taken into consideration. The chicken is
commonly used as a model to study the pathophysiology of this
syndrome (Cavanagh, 1954) . Several studies using Type I
(Tanaka and Bursian, 1989; Tanaka et al., 1990a) and Type II
(Carrington et a1., 1988a; Konno et a1., 1989; Katoh et a1.,
1990; Tanaka et al., 1991) OPIDN compounds have included a

histopathological examination of nervous system tissue in the

4
chicken. The Fink-Heimer silver impregnation method has been
effective in mapping the total extent of neuropathology
following exposure to Type I and Type II OPIDN compounds in
the chicken (Tanaka and Bursian, 1989; Tanaka et a1., 1990a,b,
1992a,b).

Despite the susceptibility of the chicken to Type I and
Type.II OPIDN, other avian species'have not.displayed the same
sensitivity. For example, the Japanese quail has been shown
to be resistant to the effects of the Type I OPIDN compound
TOTP based on the lack of clinical signs (Francis et a1.,
1980; Bursian et a1., 1983).

The purpose of the present study was to determine if a
species which is apparently resistant to Type I OPIDN will
also be resistant to Type II OPIDN. Thus, specific objectives
of this study were to: 1) determine if the Japanese quail, a
species which is not sensitive to Type I OPIDN, is sensitive
to the Type II OPIDN compound triphenyl phosphite (TPP) based
on the development of clinical signs, whole-brain NTE
inhibition and the presence of characteristic
neuropathological lesions, and 2) if the Japanese quail was
shown to be sensitive to TPP, to compare the characteristics
of clinical signs, NTE inhibition and neuropathological
lesions in the Japanese quail with those same parameters in

the chicken.

LITERATURE REVIE'

1. ORGANOPNOSPEORUS COMPOUNDS

Organophosphorus (0P) chemicals are used in agriculture
as pesticides to kill undesired insects, worms, weeds and
fungi, and in industry as plasticizers, antioxidants,
stabilizers, plastic extenders, and oil and gasoline additives
(Eto, 1979; Davis and Richardson, 1980; US Environmental
Protection Agency, 1985; Cherniak, 1988). Cresyl phosphates
and other related phosphates also impart flame resistance to
fabrics (Fisher and Van‘Wazer, 1961). While 0P compounds have
been very beneficial in terms of increased food production and
protection of human health (Ecobichon, 1991), they also have
adverse effects on both the peripheral and central nervous
systems.

The most extensively studied 0P effect is the acute
inhibition of acetylcholinesterase (AChE), the enzyme which
hydrolyses the neurotransmitter acetylcholine (ACh). ACh is
released from nerve terminals of postganglionic
parasympathetic nerve fibers, somatic motor nerves innervating
skeletal muscles, preganglionic fibers of the autonomic
nervous system and some neurons in the central nervous system
(Murphy, 1975) . The OP molecule mimics ACh and
phosphorylation of the serine-hydroxyl group at the catalytic
center of the enzyme occurs. The potency of the 0P compound

depends on the degree of enzyme phosphorylation. Inhibition

5

6

of AChE causes ACh to accumulate at the synapses and
neuromuscular junctions causing excessive stimulation of
muscarinic, nicotinic and CNS receptors. Effects of excessive
stimulation of muscarinic receptors, which are present on
smooth muscles, heart, and exocrine gland, are
bronchoconstriction, increased salivation and lacrimation,
vomiting, diarrhea and bradychardia. Effects of excessive
stimulation of nicotinic receptors, located on skeletal
muscles and autonomic ganglia, include fatigue, dyspnea,
pallor, increased blood pressure, hyperglycemia and
tachycardia. Accumulation of ACh at CNS receptors cause
restlessness, anxiety, headaches, confusion and tension
(Murphy, 1975; Barrett and Oehme, 1985). 0P-induced.death may
occur 5 minutes to 24 hours post-exposure, depending on the
chemical and the dose (Abou-Donia and Lapadula, 1990). In
addition to its presence in the nervous system, AChE is also
found in red blood cells. Assessment of AChE activity in red
blood cells is commonly used as an indication of exposure to
and absorption of GP compounds.

In addition to acute inhibition of AChE, some 0?
compounds have delayed neurotoxic properties (Abou-Donia,
1981) . Organophosphorus-induced delayed neurotoxicity
involves permanent locomotor ataxia and subsequent paralysis
that is apparent 10 - 21 days after exposure (Johnson, 1975a).
In addition to the characteristic delay period and
reproducible clinical signs, the delayed neurotoxic 0P

compounds also inhibit the nervous system enzyme neurotoxic

esterase (Johnson, 1982), and induce characteristic
neuropathologic lesions in the peripheral and central nervous
systems (Cavanagh, 1954; Tanaka.et.a1., 1990a,b, 1991, 1992a).
Some animal species are resistant to the effects of typical
OPIDN compounds as are the young of sensitive species. An
adequate explanation for this apparent lack of sensitivity has

yet to be offered.

TWO KINDS OF OPIDN

Soon after tri-o-tolyl phosphate was determined to be the
cause of OPIDN in humans during the "Ginger Jake paralysis”
incident in 1930, it ‘was apparent ‘that. organophosphorus
compounds produced 2 distinct types of delayed neurotoxic
actions (Lillie and Smith, 1932; Smith et a1., 1933; Aird et
a1., 1940). However, only recently have these effects been
defined through various studies (Veronesi et a1., 1986a,b;
Veronesi and.Bvergsten, 1987; Padilla.et.al., 1987; Carrington
and Abou-Donia, 1988a,b; Carrington et a1., 1988a; Abou-Donia
and Brown, 1990). The OPIDN compounds can be assigned to 2
classes (Type I and Type II) based on the following criteria
(Abou-Donia and Lapadula, 1990):

- Chemical structure

- Length of latent period between exposure and

appearance of clinical signs and neuropathologic
lesions

- Morphology and distribution of neuropathologic

lesions
- Inhibition of NTE
- Age sensitivity

- Species specificity

HISTORY E INCIDENCE - Type I OPIDN

Before the 19303, cases of delayed neuropathy occurred
occasionally in tubercular patients being treated with
phosphocreosote, a mixture of esters derived from phosphoric
acid and coal tar phenols (Davis and Richardson, 1980) .
However, the medicant was not realized to be neurotoxic until
a massive outbreak of poisoning in the United States occurred
in 1930 when approximately 20,000 people were affected. This
condition, marked by permanent ataxia and paralysis of the
legs and commonly referred to as "Ginger Jake paralysis", was
caused by a mixture of cresyl phosphates used to extract
ginger for the purpose of flavoring distilled liquors (Smith
et a1., 1930a,b, 1932; Smith and Lillie, 1931; Kidd and
Langworthy, 1933). The symptoms of "Ginger Jake paralysis"
started as a slowly developing paralysis in the legs followed
by tremorsw Even after 6 years, studies conducted on many of
these patients showed little recovery with muscle weakness
being replaced by spasticity, hyperflexia and abnormal
reflexes (Aring, 1942). The historical and epidemiological
aspects of this "Ginger Jake" delayed neuropathy have been

reviewed extensively (Cavanagh, 1973; Johnson, 1975a,b; Hess

et a1., 1978).

Similar cases were reported in European publications
shortly after the US outbreak (Barrett and.0ehme, 1985). This
delayed paralysis, which was found only in women, was linked
to "Apiol", an abortifacient containing between 28 and 35 %
TOTP. During the next 30 years, studies showed that TOTP was
the causative agent in a number of paralytic incidents
(Cavanagh, 1964a) . Fifty eight cases of delayed neurotoxicity
occurred in Durban, South.Africa in 1938 after cooking oil was
contaminated with TOTP. Another incident involved 11 people
who were affected after drinking water from drums which were
used to store cresyl phosphates (Susser and Stein, 1957). In
1942, three employees involved in the manufacture of TOTP
lubricants in Great Britain developed "polyneuritis" after
exposure by inhalation during black-out conditions (Hunter et
a1., 1944). Susser and Stein (1957) quoted Walthard's (1947)
description of the syndrome in 80 men of the Swiss army who
also had been poisoned with TOTP-contaminated cooking oil.

Since the early studies showed that only the ortho isomer
among the symmetrical cresyl phosphates produced a toxic
effect (Smith et a1., 1930a,b, 1932), it became customary for
mixed esters to be prepared from coal-tar stock containing
less than a specified low'amount of ortho-cresol. In spite of
this precaution, further outbreaks of poisoning occurred from
time to time. In 1953, three workers who were engaged in the
laboratory production of a prospective organophosphate

pesticide (mipafox or 1L. N'-di isopropyl

10

phosphordiamidofluoridate) were reported to have developed
clinical signs typical of OPIDN (Bidstrup et a1., 1953).
Laboratory studies concluded that there was no essential
difference between mipafox—induced neurotoxicity and
neurotoxicity caused by some other alkyl phosphoryl esters and
by the triaryl phosphates such as TOTP (Barnes and Denz,
1953).

It was determined that a major outbreak of OPIDN in
Morocco in 1959 was due to TOTP-contaminated lubricating oil
deliberately used to dilute olive oil which was later sold as
cooking oil (Smith and Spalding, 1959; Cavanagh, 1964a;
Metcalf, 1982) . As a result, over 10,000 people were affected
with ataxia and paralysis in this outbreak. Dogs also
displayed characteristic signs of OPIDN after ingesting the
same contaminated substance (Smith and Spalding, 1959) . Other
examples of TOTP-contaminated food products which resulted in
human cases of OPIDN include cooking oil in India (Vora et
a1., 1962; Anon., 1988), flour in the Fiji Islands (Sorokin,
1969), alcohol in Rumania (Vasilescu and Florescu, 1980) and
Morocco (Abou-Donia and Lapadula, 1990) , and sesame oil in Sri
Lanka (Senanayake, 1981).

Unintentional and intentional exposure to GP insecticides
like EPN, omethoate, leptophos, lenthion, trichloronate,
tr ichlorphon , phytosol , tamaron , methamidophos and
chlorpyriphos have resulted in clinical signs characteristic
of OPIDN (Bidstrup et a1., 1953; Fukuhara et a1., 1977;

Hierons and Johnson, 1978; Xintaras et a1 . , 1978; Jedrzejowska

11
et a1., 1980; De Jager et a1., 1981; Senanayake and Johnson,
1982; Vasilescu et a1., 1984; Metcalf et a1., 1985; Lotti and
Morretto, 1986; Abou-Donia and Lapadula, 1990). In addition
to human cases of OPIDN, an incident in Egypt resulted in over
1300 water buffaloes developing OPIDN from exposure to

leptophos (Abou-Donia et a1., 1974).

HISTORY & Increases - Type II OPIDN

As early as 1930, it was apparent that there were 2 types
of OPIDN. Smith and colleagues (Smith et a1., 1930a) were the
first to study the effects of triphenyl phosphite (TPP), the
prototype Type II OPIDN compound, in various animal species.
Since the Type II compounds had not resulted in human cases of
OPIDN, interest in Type II OPIDN faded. It wasn't until the
19808 that the delayed neurotoxicity effects of TPP were again
examined in chickens (Carrington et a1., 1988a,b; Konno et
a1., 1989; Tanaka et a1., 1992a,b) and in rats (Veronesi et
a1. , 1986a; Padilla et a1. , 1987; Veronesi and Dvergsten,
1987). Ironically, TPP has evaded scrutiny, unlike TOTP, and
is still being used in industry and agriculture as a rubber
and plastic stabilizer, a diluent of epoxy resins, a metal
scavenger, an anti-fungal foliar agent and as an insecticidal
synergist though it is known to be an OPIDN-causing compound

(0.8. Environmental Protection Agency, 1985).

12

CHEMICAL STRUCTURE 8 REACTIVITY - TYPO I OPIDN

Type I OPIDN compounds have a pentavalent phosphorus atom
and are derivatives of 'phosphoric, phosphonic, and
phosphoramidic acids and.phosphofluoridates. The pentavalent
phosphorus atom has a tetrahedral configuration. Type I OPIDN
compounds also include sulfur analogs. TOTP and DFP are
examples of Type I compounds (Figure 1) (Stuart and Oehme,
1982).

The presence of the ortho-methyl group in the aromatic
series may be responsible for the neurotoxic property of these
types of GP chemicals (Casida et a1., 1961; Nomeir and Abou-
Donia, 1984, 1986) . TOTP has to be metabolized to tolyl
saligenin phosphate (TSP) (Figure I) to be neuroactive (Eto et
a1., 1962, 1967; Bleiberg and Johnson, 1965; Johnson, 1975b;
Davis and Richardson, 1980; Abou—Donia, 1984) , while DFP
doesn't have to be metabolized to cause OPIDN (Barrett and
Oehme, 1985). The chemical reactivity of the Type I OPIDN
compounds depends on the phosphorus atom; on its ability to
phosphorylate and on its electrophilic character (0.8.

Environmental Protection Agency, 1985).

CHEMICAL STRUCTURE E REACTIVITY - TYPO II OPIDN

Type II OPIDN compounds have a trivalent phosphorus atom
and are phosphorus acid derivatives (ie. triphosphites and

presumably their sulfur analogs). Triphenyl phosphite is an

13

Figure 1. The chemical structures of tmi-o-tolyl phosphate
(TOTP), o-tolyl saligenin phosphate (TSP), and bis
(l-methylethyl) phosphorofluoridate (DFP). These
chemicals are Type I OPIDN compounds. TSP is the

neurotoxic metabolite of TOTP.

Ca

oils,
cna‘

Trl-o-tolyl phosphate

(TOTP)
C 3
"
0—P-‘O
/ \
0 CH2

o-tolyl saligenin phosphate
(TSP )

CIH3

P——— F

CH3—- CIH— 0/

CH3

Bis(1 -methylethyl) phosphorofluoridate
( DFP )

15
example of a Type II compound (Figure 2). The trivalent
phosphorus atom.has a pyramidal configuration (Abou-Donia and
Lapadula, 1990).

Neurotoxicity of Type II OPIDN compounds does not depend
on metabolism which in turn means that any aryl phosphite can
cause Type II OPIDN; The trivalent phosphorus atom has a pair
of electrons which binds with other atoms, making it very
reactive regardless of other substituents (U.S. Environmental

Protection Agency, 1985).
LATENT PERIOD

Both Type I and Type II OPIDN are characterized by a
delay in the onset of clinical signs. However, the period
between the time of exposure to the OP compound and the time
of onset of clinical signs varies between Type I and Type II
compounds in that the latent period is shorter for Type II
compounds. It takes only 4 - 6 days for the onset for
clinical signs in hens after TPP administration (Carrington
and Abou-Donia, 1988a; Carringtoniet a1., 1988a; Katoh.et a1.,
1990; Tanaka et al., 1992a,b) compared.to»6 - 14 days for TOTP
(Abou-Donia, 1981). Ferrets injected with.DFP started showing
signs around day 14 post-dosing (Tanaka et a1., 1991) and
ferrets dosed with TOTP started showing signs by day 50 post-
dosing (Stumpf et a1., 1989). In contrast, ferrets injected
with TPP started showing signs of ataxia by day 4 post-dosing

and severe paralysis by day 8 post-dosing (Tanaka et a1.,

16

Figure 2. The chemical structure of triphenyl phosphite
(TPP), a Type II organophosphorus compound.

18

1990b).
CLINICAL srsns - Type I OPIDN

The clinical signs of organophosphorus-induced delayed
neurotoxicity are similar in all susceptible species. Smith
and associates (1930a) were the first to report on OPIDN
clinical signs in various species of animals. These initial
reports were followed by detailed descriptions of clinical
signs in humans by Smith and Spalding (1959).

A single injection or oral dose of an OPIDN compound is
sufficient to initiate the irreversible clinical response
(Abou-Donia and Graham, 1979). Low-level dietary
administration (Kinebuchi et a1. , 1977; Abou-Donia and Graham,
1978a,b; Hussain and Oloffa, 1979) and dermal administration
(Abou-Donia and Graham, 1978a,b; Hess et a1., 1978) of TOTP
have also been shown to induce delayed.neurotoxicity4 Some of
the early studies by Smith and colleagues (Smith et a1.,
1930a,b; Smith.and.Lillie, 1931; Smith.et a1., 1932) indicated
that small, individually ineffective doses of TOTP ingested
over a period of time can give rise to ataxia and severe
paralysis, if the total dose ingested approached the minimum
acute dose for paralysis. Smith et al. (1930b) reported that
40 mg TOTP/kg body weight was sufficient to produce OPIDN in
humans. Hopkins (1975) reported calculations by Staehelin
(1941) that suggested as little as 2 mg TOTP/kg body weight

(made from feedstock low in ortho cresol) may cause a

19
neurotoxic response in man.

Ingestion of TOTP by humans may cause some immediate
gastrointestinal distress with nausea, vomiting, and.diarrhea
which may last a few hours to a few days. A latent period of
8 - 18 days generally follows, depending on the size of the
dose and length of exposure. This is followed by a sharp,
cramp-like pain in the calves and numbness and tingling inche
feet and the hands of the patients. This in turn is followed
by increasing weakness of the lower limbs. 'The patient cannot
keep his balance and bilateral footdrop has been reported.
Depending on the severity, there may be weakness at the knee
or hip. Ankle jerks are absent. Knee reflexes may be normal
or occasionally depressed. The patient may experience
weakness in the hands a week or two after the onset of
paralysis in the lower limbs. Wrist-drop and weakness up to
the elbow“may also be seen in some individuals. IMany patients
exhibit sensory loss, conforming roughly to the degree of
motor loss. Sensory loss in the upper limbs usually appears
a few days after it develops in the lower limbs (Susser and
Stein, 1957).

The results reported by Smith and co-workers (1930a) on
the response of hens to TOTP and other neurotoxic OP esters
were remarkably similar to OPIDN clinical signs reported for
humans. Birds treated with TOTP appear normal for a period of
10 - 14 days following acute administration. After this
period, they spend more time in a sitting position. When

exercising, birds begin to display a clumsiness of gait,

20

accompanied by overt weakness, which is a characteristic sign
of ataxia. At 15 - 20 days post-treatment, the birds may
become severely paralyzed in the legs and the wings are
obviously weakened (Bursian et a1., 1983; Calabrese and
Bursian, 1984; Tanaka and Bursian, 1989). At low neurotoxic
doses, the birds experience a slight initial weight loss which
later is reversed. The birds are able to maintain their
health, although the proper use of affected limbs may not be
fully regained.

In contrast, at. higher acute doses, a 'more severe
terminal clinical neurotoxicity is observed. The animal loses
its ability (or desire) to eat and subsequently dies following
a severe‘weight loss and.complete paralysis of the legs (Smith
and Elvove, 1930; Smith.et al., 1930a, 1932; Smith and Lillie,
1931; Lillie and Smith, 1932).

In ferrets treated with DFP, the clinical signs at 21 and
28 days post-treatment ranged from slight hind limb weakness
to severe ataxia or hindlimb paralysis (Tanaka et a1., 1991).
Ferrets exposed to TOTP dermally had diarrhea which was the
only sign of acute toxicity (Stumpf et a1., 1989). The motor
defect was confined to the hind limbs. Occasionally, the
treated animals exhibited hopping movements rather than normal
movement and occasionally animals would fall side to side.
Ferrets treated.orally’with 1000 mg TOTP/kg body’weight showed
detectable clinical signs of ataxia and high-stepping gait by

day 11 post-dosing.

21

cnznxcnr arena - Type II OPIDN

Clinical manifestations of Type II OPIDN have been
studied in various species (Smith et a1., 1933; Roberts et
a1. , 1982; Veronesi et al., 1986a; Tanaka et a1., 1990b,
1992a,b) . TPP administration causes ataxia in chickens, rats,
cats and ferrets. The pattern of clinical signs produced by
TPP are very similar to that produced by neurotoxic phosphoric
acid esters like TOTP and DFP (Abou-Donia, 1981). However,
ataxia and paralysis tend to develop sooner relative to
animals treated with Type I OPIDN compounds (Carrington et
a1., 1988a; Katoh et a1., 1990). Early signs of neurotoxicity
can be observed before 7 days post-dosing.

TPP<did not.produce severe clinical signs typical of Type
II OPIDN in hens when administered orally, but dermal
administration and subcutaneous injections did (Carrington and
Abou-Donia, 1988a; Carrington et a1., 1988a,b; Abou-Donia and
Brown, 1990). Hens developed mild ataxia with a spastic and
awkward gait within a few days after exposure which progressed
to flaccid leg paralysis (Smith et a1., 1933; Carrington and
Abou-Donia, 1988a; Carrington et 31., 1988a, Katoh et a1.,
1990).

Triphenyl phosphite and other phenolic phosphites produce
extensor rigidity in the cat which is not observed with Type
I OPIDN (Smith et a1., 1932, 1933). Cats treated with Type II
compounds developed ataxia and extensor rigidity in both

forelimbs and hindlimbs of relatively long duration (Veronesi

22
et a1., 1986a). Monkeys treated with 2 doses of 1 ml TPP/kg
body weight over a 24-day interval developed ataxia within 12
days of the second dose. After 3 days, extensor rigidity of
the limbs and some retraction of the head developed (Abou-
Donia and Lapadula, 1990).

Rats treated.with Type II OPIDN compounds showed initial
clinical signs of hyper-excitability, some spasticity, and
incoordination with partial flaccid paresis of the extremities
developing later (Smith et a1. , 1933) . Recent studies by
Veronesi and co-workers (Veronesi et al., 1986a; Veronesi and
Dvergsten, 1987) on Long-Evans rats have indicated that.TPP in
multiple doses produced tail-kinking in the proximal 1 inch of
the tail. Hindleg ataxia developed within a week, followed by
paralysis. In addition, rats developed bidirectional circling
behavior following multiple doses of TPP (Veronesi et a1.,
1986a; Padilla et a1., 1987). When placed on their backs, the
dosed rats rolled into a ball whereas the control rats rolled

over to an upright position.

NEUROPATHOLOGICAL FEATURES

Neuropathological features help in determining the
location and severity of somatic and axonal degeneration from
exposure to organophosphorus neurotoxicants. OPIDN compounds
cause histological alterations in the CNS and PNS, with each
class of chemicals inducing characteristic lesions. The

pathological lesions depend on the animal species and the

23

duration of exposure in addition to the chemical.

Type I OPIDN

Degeneration occurs in both the peripheral and the
central nervous systems after a single dose of Type I OPIDN
compounds (Cavanagh, 1954; Bischoff, 1970; Itoh et a1., 1981,
1984; Prentice and Roberts, 1983; Jortner et a1., 1989; Tanaka
and Bursian, 1989; Tanaka et al., 1992b) and is usually
detected at the same time that clinical signs are apparent.
However, some degenerative changes have been detected before
the development of clinical signs (Wilson et a1., 1988; El-
Fawal et a1., 1990). Histological studies on TOTP-exposed
humans showed that lesions were limited to the peripheral
nerves and the anterior horn cells of the spinal cord and
sometimes to the motor cells in the medulla (Smith and Elvove,
1930; Smith et 81., 1930b, 1932; Lillie and Smith, 1932).

Lesions in the CNS of hens exposed to Type I delayed
neurotoxicants were restricted to the brain stem, cerebellum,
and spinal cord (Tanaka and Bursian, 1989; Tanaka et a1.,
1990a). Moderate to severe axonal degeneration occurred in
the spinal cord of hens exposed to TOTP and DFP (Tanaka et
a1., 1992b; Pope etia1., 1992). Degenerating axons were noted
in the cervical parts of the fasciculus gracilis in the spinal
cord, dorsal and ventral spinocerebellar tracts, and in the
lumbar part of the medial pontine-spinal tract. Moderate

terminal degeneration was also noted in the medial part.of the

24
ventral horn at lumbar cord levels.

In the medulla, moderate terminal and preterminal
degeneration were noted in the lateral vestibular, gracile-
cuneate, external cuneate, and lateral cervical nuclei as a
result of exposure to TOTP or DFP. Degeneration was noted in
lesser amounts in the solitary, inferior olivary, and raphae
nuclei, in the medial and.dorsal vestibular nuclei, and in the
lateral paragigantocellular, gigantocellular, and lateral
reticular nuclei. Fiber degeneration was present in the
medullary portions of the dorsal and ventral spinocerebellar
tracts and in the spinal lemniscus. Axonal degeneration was
also present in nerve fascicles which make up the
intermedullary portions of the glossopharyngeal and vagus
nerves.

Within the cerebellum, mossy fiber degeneration occurred
in the granular layer due to TOTP or DFP exposure. Alternate
parasagittal bands of heavy and light degeneration were also
seen. Small amounts of coarse fiber degeneration were also
noted in the deep cerebellar nuclei (Jortner et a1., 1989).

Microscopic lesions were present in the nervous system of
hens exposed to mipafox, provided that the dose was high
enough to cause greater than 80 % inhibition of NTE (Dyer et
a1., 1992). The hen had a Wallerian-like degeneration. The
fiber breakdown in multiple spinal cord tracts was extensive
and. varied, with. the intensity’ of degeneration. directly
proportional to the mipafox dose. The hen develop a slow,

consistent.and longer lasting neuropathy than the rat (Carboni

25
et a1., 1992), thus being a sensitive species for mipafox-
induced OPIDN, and confirming the previous study by Dyer et
a1. (1992).

TOTP produced histopathological lesions in the spinal
cord and peripheral nerves of cats in a dose-dependant fashion
(Abou-Donia et a1., 1986). Cavanagh (1964b) and Cavanagh and
Patangia (1965) have studied the TOTP-induced lesions in the
cat by light microscopy. In the peripheral nerves, axonal
degeneration occurred initially at the distal ends of the
longest and largest diameter fibers which spread proximally in
a typical "dying-back" process. The areas which were
initially and most severely affected were the large diameter
fibers from annulo-spinal endings of the muscle spindles. The
long pathways in the spinal cord.showed a similar "dying-back"
lesion. Electron microscopy studies by Prineas (1969) showed
that the earliest lesion occurred in the axons and consisted
of accumulation of abnormal membrane-bound vesicles and
tubules. In DFP-treated cats, granular transformation of the
axoplasm was noted in the degenerating axons. All their
neurotubules or neurofilaments were lost and the degenerated
mitochondria were swollen (Bouldin and Cavanagh, 1979).

In ferrets treated with DFP, dense axonal degeneration
was noted in the fasciculus gracilis and dorsal
spinocerebellar tract at cervical levels of the spinal cord
and in the lateral corticospinal tract at lumbar levels
(Tanaka et a1., 1992b). Spinal cord laminae VI - VII had

degenerating terminals, with lumbosacral levels showing severe

26

degeneration. Severe degeneration was also noted in the
ventral motor nucleus of the cervical enlargement at spinal
cord levels. Fasciculus and nucleus gracilis, medial and
dorsal accessory' nuclei of ‘the inferior* olive, inferior
nucleus and lateral reticular formation of the medulla had
dense axonal and terminal degeneration. The cerebellar mossy
fiber degeneration noted in the ferret was similar to that
reported for the hen. It consisted of alternate bands of
light and dark degeneration.

TOTP-induced lesions in the rat were characterized by
giant swellings of myelinated and demyelinated axons, myelin
debris, vacuolated myelin sheaths and hyaline bodies
(Veronesi, 1984). TOTP-induced degeneration was found in the
sensory and motor tracts in a dying-back pattern like in other
species. Within the cervical spinal cord, damage was found
mainly in the dorsal column. In contrast, multiple doses were
needed to produce lesions in the ventrolateral and ventral
columns of the cervical and lumbar cord. Neuropathological
lesions were noted even in the absence of overt ataxia
(Veronesi, 1984). Inui et a1. (1993) studied TOTP-affected
rats and.concluded.that.axonal swelling is.due:to accumulation
of cytoplasmic contents near the node of Ranvier, indicating
paranodal degeneration. Topographical examinations of the
lesions showed that the fasciculus gracilis in the cervical
spinal cord was the major target site (Padilla and Veronesi,
1988).

The nervous system of mipafox-treated rats, like hens,

27

showed microscopic lesions if the dose was high enough to
cause NTE inhibition of 80 % or more. These Wallerian-like
lesions were well developed in the highest dosage group and
were confined to the rostral level of the fasciculus gracilis
in the medulla oblongata (Carboni et a1., 1992; Veronesi et
a1., 1986b). The most prominent lesion in rats was swollen
axons with a single vacuole containing flocculent material
(Dyer et a1., 1992; Carboni et a1., 1992). Quantitatively,
the percentage of affected fibers in the fasciculus was lower
in rats compared to those in hens.

The Type I OPIDN neuropathological studies show that (1)
CNS axonal and terminal degeneration apparently is confined to
the spinal cord, medulla and cerebellum, (2) it takes 1 to 3
weeks post-exposure for the onset of degeneration in different
fiber tracts and nuclei and (3) the delayed onset of OPIDN
clinical signs and the delayed onset of degeneration in many

of the affected CNS fiber systems are mutually consistent.

Type II OPIDN

Pathological lesions are species-dependent and occur in
both the central and peripheral nervous systems (Veronesi and
Dvergsten, 1987; Carrington and Abou-Donia, 1988a; Carrington
et al., 1988a). Exposure to the Type II neurotoxicant TPP
resulted in widespread degeneration in the hen which involved
the spinal cord, cerebellum, medulla, midbrain and forebrain

(Carrington et a1 . , 1988a ; Tanaka et a1 . , 1991; Knoth-Anderson

28

and.Abou-Donia, 1993). Examination showed that the brain stem
(mainly the reticular formation and the cerebellar peduncles) ,
the ventral and lateral tracts of the spinal cord, the gray
matter of the spinal cord and the sciatic nerve contained
swollen and fragmented axons. The damage was more severe in
the lower (thoracic and lumbar) portions of the spinal cord.
Cellular gliosis and decrease in number of motor cells in the
anterior horn were noted in cellular degeneration. These
changes were observed in the medulla, pons, brainstem, and
cerebellar cortex (Smith et a1., 1933). Fatty degeneration,
tigrolysis and cellular necrosis were not significantly
present.

In cats, Smith and co-workers (Smith et a1., 1933)
reported a small amount of neuronal somatic degeneration in
the thalamus and cerebral cortex similar to the findings in
rats. The posterior fasciculi in the cat are not affected by
Type II OPIDN; The medulla and pons, the restiform.bodies and
the brachia conjunctive are the most affected (Smith et a1.,
1933). The spinal ganglia underwent only slight changes.

Degeneration in the same locations was noted in the
studies conducted by Tanaka et al. (1990b, 1992b) in the
brains of ferrets injected with TPP. The Fink-Heimer method
of staining showed widespread axonal and terminal degeneration
in the midbrain, thalamus, and cerebral cortex in addition to
the spinal cord and brainstem. The degeneration was
independent of the individual doses or post-injection survival

periods. TPP exposure also affected a number of sensory and

29

motor systems. Dorsal spinocerebellar tract afferents to the
cerebellum were black and fragmented in appearance which is
characteristic of degenerating axons. The external cuneate
nucleus and the nuclei of the reticular formation also showed
neuronal somatic» degeneration. .Axonal and somatic
degeneration were also seen in the nuclei and tracts of
several sensory systems. ‘The findings of this study show that
CNS damage from TPP exposure is not only in sensorimotor
pathways and nuclei of the brainstemiand spinal cord, but also
may involve other visual, auditory and higher order
sensorimotor areas.

Rats, once considered resistant to the delayed neurotoxic
effects of OPs based on the absence of clinical signs when
dosed with Type I OPs, have been shown to develop lesions in
the spinal cord, brainstem, midbrain, and forebrain after
dosing with Type II OPIDN compounds (Veronesi, 1984; Veronesi
and Padilla, 1985; Padilla and Veronesi, 1985, 1988; Veronesi
et a1., 1986a; Veronesi and Dvergsten, 1987; Tanaka et a1.,
1992b). Smith and co-workers (1933) first reported a small
amount of neuronal somatic degeneration in the thalamus and
cerebral cortex. The CNS of rats exposed to TPP were
thoroughly examined using the Fink-Heimer silver impregnation
method (Lehning et a1., 1990). The studies showed widespread
axonal and terminal degeneration not only in the spinal cord
and brainstem, as is the case with other 0P compounds such as
DFP in ferrets (Tanaka. et a1., 1991), but. also in 'the

midbrain, thalamus and cerebral cortex. But there was only

30
minimal degeneration in.the spinal cord of the rat in contrast

to the ferret.

ROLE E INHIBITION OF NEUROTOXIC ESTERASE

Over the past 50 years, research efforts have increased
in an attempt to develop an understanding of the relationship
between chemical structure, biochemical lesion and
neurotoxicity. All organophosphate esters that induce delayed
neuropathy' are either' direct. esterase inhibitors or are
metabolically converted to inhibitors. Thus, it has been
generally considered that phosphorylation of an esterase is
probably’ needed for' the onset. of delayed, neurotoxicity,
although the phosphorylation doesn't necessarily have to take
place in the nervous system.

The most extensive and successful biochemical
investigations were conducted at the Medical Research Council
Laboratories in Carshalton, England by Barnes and his co-
workers (Barnes and Denz, 1953; Aldridge and Barnes, 1961,
1966) and later by Johnson and his colleagues at the same
institution (Aldridge et a1., 1969; Johnson, 1969a,b,c, 1975b,
1976a,b, 1977; Johnson and Barnes, 1970; Lotti and Johnson,
1978; Clothier and Johnson, 1979). The studies done there
have indicated that delayed neurotoxicity is associated with
the inhibition of a specific membrane-bound protein having
esterase activity. Johnson and his co-workers named this

protein "neurotoxic esterase" (NTE) (Aldridge et a1., 1969).

31

NTE, which is present in various tissues, has no known
biochemical or physiological functions (Abou-Donia, 1981).
Johnson first suggested, followed by others (Johnson,
1969a,b,c, 1977; Clothier’anthohnson, 1979; Abou-Donia, 1981;
Williams and Johnson, 1981; Ehrich et a1., 1985; Carboni et
a1., 1992), that an organophosphorus chemical is considered a
delayed neurotoxicant when approximately 70 - 80 % of the
enzyme is phosphorylated through an irreversible "aging"
reaction. Studies have suggested that aging involves the
formation of a covalently bound, phosphorylated, and
irreversibly inhibited esteratic site on NTE (Johnson, 1982;
Richardson, 1984; Davis et a1., 1985). Aging leads to a
stable, irreversibly'phosphorylated enzyme species which when
hydrolyzed forms a reactivated protein. The aging reaction
may be related to the delayed neurotoxicity induced by certain
OPs. The kinetic relationship of anti-esterase OPs with
hydrolytic enzymes has been extensively reviewed (Cohen and
Osterbaan, 1963; Aldridge and Reiner, 1972).

How the inhibition and aging of NTE leads to neuronal
damage is still unknown. Only delayed neurotoxic OP compounds
can irreversibly inhibit NTE enzymatic activity while non-
OPIDN compounds don't (Johnson, 1982). Some compounds can
reversibly inhibit NTE, but not result in OPIDN. HDwever,
these compounds can prevent delayed neurotoxicity when given
prior to a neuropathic 0P. Phenylmethyl sulfonyl fluoride
(PMSF) is an example of such a compound. Being a

serine/cysteine protease inhibitor, PMSF protects the animal

32
by a variety of cellular repair and regeneration processes in
many tissues (Johnson, 1970; Baker et a1., 1980; Caroldi et
a1., 1984; Veronesi and Padilla, 1985; Bond and Butler, 1987;
Pope and Padilla, 1990; Pope et a1., 1992). Thus, PMSF
pretreatment actually decreases the incidence of
neuropathologic lesions.

The role of NTE in OPIDN is still questioned since NTE
can be inhibited in non-sensitive species. Rats are less
susceptible to OPIDN compounds (Barnes and Denz, 1953; Abou-
Donia, 1981; Sokmuti et a1., 1988) presumably because of less
NTE activity. In addition, inhibited NTE activity in rats is
reported to return to normal faster than in hens after an OP-
induced suppression of NTE activity (Carrington and Abou-
Donia, 1984). But continuous suppression of NTE activity may
produce more severe neurologic damage in rats (Majno and
Karnovsky, 1961). Despite the equivocal relationship between
NTE inhibition and the development of OPIDN, measuring the
degree of NTE inhibition is valuable because it helps in
assessing potential neurotoxicity of suspect chemicals and
ultimately in evaluating the hazards associated with the use

of such materials (Johnson, 1977; Lotti and Johnson, 1978).

Type I OPIDN

Type I compounds cause NTE inhibition which can persist

for a number of days“ A dose of approximately 1200 mg TOTP/kg

body weight inhibited brain NTE and sciatic nerve NTE

33

completely in chickens and this complete inhibition persisted
for 21 days (Abou-Donia and Lapadula, 1990). A dose of 1 mg
DFP/kg body weight caused 96 % brain NTE inhibition in
chickens initially, followed by a progressive increase in
activity (Tanaka et a1., 1990a). TOTP'at.a dose of 1000 mg/kg
body weight caused significant brain NTE inhibition of over 85
% in pheasants and chickens (Bursian et a1., 1983).
Significant brain NTE inhibition by TOTP was also reported for
turkeys (Larsen et a1., 1986).

There are 2 exceptions to the suggestion.made by Johnson
(1969a,b,c, 1977) that 70 - 80 % of NTE inhibition is needed
for Type I OPIDN clinical signs to develop. NTE can be
inhibited in excess of 70 1; in non-sensitive species, but
OPIDN doesn't develop. For example, TOTP at a dose of 1000
mg/kg body weight caused over 85 % brain NTE inhibition in
Japanese quail and Bobwhite quail but OPIDN clinical signs did
not develop (Bursian et a1., 1983). In contrast, the ferret
is a sensitive species in terms of Type:I OPIDN, but brain.NTE
in this species is inhibited by considerably less than 70 %.
A dermal dose of 1000 mg TOTP/kg body weight in ferrets caused
46 % brain NTE inhibition whereas an oral dose caused 37 %
inhibition (Stumpf et a1., 1989). Ferrets injected with 4 mg
DFP/kg body weight had 42 % brain NTE inhibition (Tanaka et
a1., 1991). In all these cases, animals subsequently

developed clinical signs typical of OPIDN.

34

Typo II OPIDN

Brain NTE must be inhibited by 70 % to produce Type II
OPIDN in chickens (Carrington and Abou-Donia, 1988a). Hens
injected with 1000 mg TPP/kg body weight showed an initial
brain NTE inhibition of 85 % ‘which recovered to 73 %
inhibition over a 21 day period (Tanaka et al., 1992a).

Conversely, brain NTE inhibition of 39 % was reported in
rats following an injection of 1184 mg TPP/kg body weight
(Veronesi et a1., 1986a) while rats treated with 1164 mg
TPP/kg body weight had brain NTE inhibition of 30 % (Padilla

et a1., 1987).

ACE SENSITIVITY

Studies have shown that young animals of certain
sensitive species are resistant to OPIDN (Barnes and Denz,
1953; Smith and Spalding, 1959; Taylor, 1967; Johnson and
Barnes, 1970; Goldstein et a1., 1988; Olson and Bursian, 1988;
Katoh et a1., 1990; Peraica et a1., 1993). A single dose of
50 mg TOTP/kg body weight did not cause delayed neurotoxicity
in very young chicks and the age at which chicks are sensitive
to TOTP was determined to be approximately 60 days (Barnes and
Denz, 1953; Aldridge and Barnes, 1966; Abou-Donia et a1.,
1982). Single doses of DFP ranging from 2 to 5 mg/kg body
weight also did not cause delayed neurotoxicity in 7 to 49-day

old chicks (Johnson and.Barnes, 1970). Studies by Pope.et a1.

35

(1992) and.Peraica.et a1. (1993) showed that 40-day-old.chicks
treated with 1 mg DFP/kg body weight displayed few motor
deficits and the severity of clinical signs increased as the
age at dosing increased. The clinical picture in chicks after
a single dose of 5 mg 2, 2-dichlorovinyl dibutyl phosphate
(DBDCVP)/kg body weight was different from that usually seen
in the hen in that spasticity and complete recovery occurred
in the chicks, but not in adults (Peraica et a1., 1993).

This age susceptibility' tot CPS and. the ability ‘to
withstand neurotoxicant insult by young chickens was earlier
hypothesized by Bondy et a1. (1961), Johnson and Barnes
(1970), Konno and Kinebuchi (1978), Abou-Donia et a1. (1982)
and Katoh et a1. (1990). It is believed that this lack of
sensitivity could be due to a failure in the absorption of the
neurotoxicant (Metcalf, 1984) or it could be due to a faster
elimination rate of the neurotoxicant in young animals. Abou-
Donia (1981) showed that EPN was eliminated 6 times faster in
1-week-old chicks than in adult hens. EPN was noted in the
brain, spinal cord, sciatic nerve, kidneys, and plasma of the
adult hens while only polar breakdown products were seen in
the tissues of chicks. Similarly, leptophos decayed at a
faster rate in 6-month-old chickens than in adult hens. 'Thus,
clinical signs were apparent only in the adult birds (Konno
and Kinebuchi, 1978). Histopathological examination of rats
dosed with OP compounds showed that young animals had fewer
lesions than adults (Johnson and Barnes, 1970).

On the other hand, when TPP was administered to l-week-

36
old chicks, they developed clinical signs characteristic of
OPIDN such as ataxia and paralysis as well as characteristic
lesions in the CNS and PNS (Abou-Donia and Brown, 1990). It
is apparent that the development of Type II OPIDN is not

dependent on the age of the animal like Type I OPIDN is.

SPECIES SELECTIVITY

It was established.at the turn.of this century and quoted
by Roger and Recordier in 1934, that humans are susceptible to
TOTP-induced delayed neurotoxicity (Abou-Donia and Lapadula,
1990) . Later it was determined that not all animals are
susceptible to<0PIDN (Smith.et a1., 1930a). Sensitive.species
include the water buffalo (Abou-Donia et a1., 1974), sheep,
lambs (Draper et a1., 1952; Malone, 1964), pigs (Kruckenberg
et a1., 1973), mallard. ducklings (Herin. et a1., 1978),
pheasants (Johnson, 1975b; Bursian et a1., 1983), turkeys and
chukar partridges (Baron, 1981). Some rat strains, like the
Long-Evans, Sprague-Dawley and Fischer 344, were resistant to
TOTP-induced OPIDN based on lack of clinical signs (Veronesi
and Abou-Donia, 1982; Veronesi, 1984; Somkuti et a1., 1988).
TOTP does not cause any clinical signs in Japanese quail or
Bobwhite quail though NTE inhibition is more than 70 %
(Francis et a1., 1980; Baron, 1981; Bursian et a1., 1983).

The kinetic steps involved in the toxic disposition of
the chemical in the animal may account for the difference in

species susceptibility. These steps are absorption,

37

distribution, metabolic transformation and elimination of the
OP compound from the body. The atypical response by certain
rodents suggests that the difference in species selectivity is
not due to absorption, distribution and metabolism. TOTP is
rapidly absorbed and metabolized in rats to an OP ester which
is neurotoxic in susceptible species (Casida, 1961; Baron et
a1., 1962).

Differential species sensitivity to Type II OPIDN has not
been studied extensively. TPP does cause OPIDN in chickens,
rats, ferrets, cats and monkeys (Carrington et a1., 1988a,b;
Katoh et a1., 1990; Veronesi et al., 1986a; Padilla et a1.,
1987; Tanaka et a1., 1992a,b). IRats are not sensitive to Type
I compounds, but they are to Type II compounds. This is the
rationale for determining if Japanese quail are sensitive to
TPP since they are resistant to the effects of Type I OPIDN

compounds.

MATERIALS AND METHODS

TEST SPECIES AND HUSBANDRY

Adult Japanese quail (Coturnix coturnix japonica) were
housed at the Michigan State University Poultry Science
Teaching and Research Center in cages measuring 53 cm long X
26.5 cm wide X 26.5 cm high in groups of 5 starting one week
prior to the beginning of the trial. Birds were provided with
quail breeder ration and water ad libitum. Temperature and
humidity of the room were ambient and the photoperiod was

maintained at 24 hr light : 0 hr dark.

TREATMENT

Seventy birds were allocated into 4 TPP dose groups of 10
birds each, a negative control group (TOTP) of 10 birds, and
a control group of 20 birds which received the corn oil
vehicle. The TPP solution was prepared by mixing the chemical
(97 % pure, Aldrich Chemical Co., Milwaukee, WI) in corn oil
to give doses of 62.5, 125, 250 and 500 mg/kg body weight in
an injection volume of 1 ml/kg body weight. The solution was
prepared just prior to administration. TPP was injected
subcutaneously over the breast region of each bird after
recording the weight. TOTP (90 % pure, Aldrich Chemical Co.,
Milwaukee, WI) was prepared in a similar fashion at a dose of

500 mg/kg body weight. TOTP was administered orally at 1

38

39
ml/kg body weight. This dose was chosen because it is
routinely used as a neurotoxic dose in chicken OPIDN studies.
There were no restrictions on the intake of feed or water

prior to dosing.

NEUROTOXIC ESTERASE ASSAY

At 24 hr after dosing, half of the birds (5 birds in each
of the 4 TPP groups, 5 TOTP-treated birds and 10 control
birds) were killed by cervical dislocation. The brains were
rapidly removed, weighed and kept frozen at -30°C for
subsequent determination of neurotoxic esterase activity

(Johnson, 1977).

CLINICAL OBSERVATIONS

The remaining birds in each treatment group were observed
daily for development of clinical signs characteristic of
OPIDN beginning 24 hours after dosing and continuing for up to
21 days post-treatment. Assessment of clinical signs was
based on a 4-point scale where 0 indicates no ataxia, 1
indicates mild ataxia, 2 indicates that the bird has
difficulty standing upright and moves primarily on its hocks,
3 indicates that the bird cannot stand, but can sit upright
and moves primarily by wing action and 4 indicates that the
bird is unable to sit upright and cannot move. Moribund birds

were killed to eliminate unnecessary suffering.

4O

NEUROPATHOLOGICAL ASSESSMENT

To determine the extent of degeneration in the central
nervous system, some birds from each dose group were taken at
various times during' the. 21 day’ observation. period for
neuropathological assessment. The birds were anesthetized
deeply with sodium phenobarbital (150 ml/kg body weight) and
perfused transcardially with 10 % formalin-saline solution.
The brains were removed, placed in a 10 % sucrose-formalin
solution for a week, frozen, and cut in the coronal or
sagittal plane at a thickness of 40 um. Every tenth section
was processed using the Fink-Heimer silver impregnation method
for degenerating axons and terminals. Cresyl violet was used
to stain adjacent sections to delineate nuclear boundaries.

Selected adjacent Fink-Heimer and cresyl violet stained
sections were examined carefully using a standard compound
microscope and areas containing axonal and terminal

degeneration were noted.

STATISTICAL ANALYSIS

Mean whole-brain NTE activities of each treatment group
were compared to the mean control whole-brain NTE activity
using Dunnet's test (Gill, 1978). Statements of significance

are based on p < 0.05.

41

ANIMAL USE AND CARE

All animal-related aspects of this study were approved by
the Michigan State University All University Committee on

Animal Use and Care.

RESULTS

CLINICAL SIGNS

The development of clinical signs typical of OPIDN was
assessed beginning on day 1 post-treatment as some birds
started showing signs within the first 24 hours (Table 1).
One of the 5 Japanese quail treated with 62.5 mg TPP/kg body
weight appeared ataxic beginning on day 3 post-dosing. One of
the affected birds was unable to stand by day 6 and used its
wings for movement while the other bird exhibited stiff-legged
movement by day 8 post-dosing.

One bird dosed with 125 mg TPP/kg body weight had
rigidity of the legs beginning at day 3 post-dosing. By day
5, the bird.was moving on its hooks and experienced difficulty
in maintaining its balance. The other 2 affected birds in
this group began showing signs of mild ataxia on days 5 and 6.
One of the these 2 birds later experienced difficulty in
standing. The clinical condition of these 3 birds became more
severe in subsequent days before they were taken for
neuropathological assessment at day 9 post-dosing.

In birds injected with 250 mg TPP/kg body weight, more
extensive clinical signs were initially noted on day 1 post-
dosing in 3 of the birds. By day 5 post-treatment, 4 of the
5 birds were unable to sit or stand.upright and were moving on
their abdomen while the 5th bird could move only by using its

wings. One of these birds died on day 6 displaying severe

42

43

TABLE 1. The effects of TPP and TOTP on the development of
clinical signs' characteristic of OPIDN in adult Japanese
quail.

Days Post-Dosing

IDf Dose 1 2 3 4 s 6 7 8 9 --- 21

471 0 0 0 0 0 o o 0 o --- 0

472 o 0 o 0 0 o 0 o o --- o

473 0 o 0 o o o o o 0 --- o

474 0 0 0 0 o 0 0 0 o --- o

485 Control 0 0 0 o 0 0 o o o —-- o

5801 o o o 0 0 o 0 o 0 --- 0’
5802 0 0 0 0 0 o 0 o 0 --- 0’
5803 0 o 0 0 0 o 0 o 0 —-- 0

5804 0 o o o o 0 0 0 o --- o’
5805 o o 0 o o 0 o o 0 --- o

556 o o 1 1 1 2’

557 o o o o 1 1 1 1’

558 62.5 TPP 0 o 0 0 o o o 0 0 --- o
559 0 0 o o 0 o 0 0 o --- 0
560 o o 0 o 0 o 0 o o -—- o

566 o o 0 o 0 1 2 2 2’

567 o o 1 1 2 2 3 3 3’

568 125 TPP 0 0 0 0 0 0 0 o 0’

569 o o o 0 0 0 o 0 0 --- o

570 0 0 o o 1 1 2 2 2’

571 2 3 4 4 4 4’

572 o o 0 1 2 3’

573 250 TPP 1 2 3 3 4 4’

574 2 3 4 4 4 D

575 o 1 2 2 3 4’

512 o o 1 3 4 4 D

513 3 3 4’

547 500 TPP 3 3 4’

548 1 1 1’

586 1 1 3’

581 o o 0 o o o 0 0 0 --- 0’
582 0 0 o 0 0 0 o 0 0 -—- 0’
583 500 TOTP o 0 o o 0 0 0 o 0 --- 0’
584 0 0 o 0 o 0 0 o o --- o’
585 o 0 0 0 0 0 o 0 0 --- 0’

44

Clinical scores were assigned as follows: 0 = no abuts
1 = mild ataxia; 2 = has difficulty standing

upright, moves on hocks; 3 = cannot stand upright but
can sit upright and moves primarily by wing action; 4 =
unable to sit upright and cannot move.

Doses are expressed as mg TPP or TOTP/kg body weight.
Denotes the bird was perfused on the designated day.
Denotes the bird died.

45
paralysis.

Clinical signs in 4 of the 5 birds treated with 500 mg
TPP/kg body weight were apparent within 24 hours and became
increasingly severe in the subsequent 48 hours. The birds
were unable to sit or stand by day 3 and they were perfused.
The other bird in this group started showing mild ataxia on
day 3 and died on day 6 displaying severe paralysis.

The 5 birds dosed with.500 mg TOTP/kg body weight did not
show clinical signs characteristic of OPIDN during the 21 day
observation period. They were indistinguishable from the

control group in terms of mobility.

NTE ASSAY

The effects of TPP and TOTP on whole-brain neurotoxic
esterase activity in adult Japanese quail 24 hours post-dosing
are presented in Table 2. TPP-induced NTE inhibition ranged
from 11 % (62.5 mg TPP/kg body weight) to 87 % (500 mg TPP/kg
body weight). Birds receiving 500 mg TOTP/kg body weight had

whole-brain NTE activity inhibited by 90 %.

46

TABLE 2. The effects of TPP and. TOTP on. whole-brain
neurotoxic esterase (NTE) activity in Japanese quail 24 hours
post-administration.

Dose N13 Activity” 3 Inhibition
Control 1873 +/- 72 ----
62.5 TPP 1668 +/- 78 11 %
125 TPP 1146 +/- 102”‘ 39 %

250 TPP 512 +/- 47”‘ 73 %

500 TPP 244 +/- 99”‘ 87 %

500 TOTP 194 +/- 29“' 9o %

***

Doses are expressed as mg TPP or TOTP/kg body weight.
NTE activity expressed as nmoles phenyl valerate
hydrolyzed/hr/gram brain. Data expressed as mean +/-
standard error. Sample size equals 5 in the TPP and
TOTP groups and 10 in the control group.

Significantly different from control mean at p < 0.05

47

HISTOPATHOLOGICAL ASSESSMENT

Fink-Heimer silver impregnated degeneration appeared as
black fragmented fibers against a yellow background. There
was no evidence of degeneration in any of the nuclei or fiber
tracts of brains from control birds (Figure 4a,c) . Light
degeneration was noted in the brains of TOTP-treated birds
(Figure 7) and significant histopathological changes were
noted in all the brains from.TPP-treated birds (Figures 3a,b,
4b,d, 5 and 6). .Axons were noted to be swollen and fragmented
in the brain stem. The brains of Japanese quail perfused at
a later stage during the course of clinical observations
showed.more severe and widespread degeneration than brains of

birds perfused at an early stage.

Brainstom

The medulla and cerebellum showed axonal and terminal
degeneration in the spinocerebellar tract, spinal lemniscus,
reticular formation, ventral part of the medial longitudinal
fasciculus and fascicles of cranial nerves IX and X. The
gracile-cuneate, lateral cervical, external cuneate and
lateral paragigantocellular reticular nuclei contained
moderate to heavy terminal degeneration whereas parvocellular
and gigantocellular reticular nuclei had light and scattered
degeneration. Degenerating axons, degenerating terminals and

degenerating cell bodies were noted in the lateral vestibular

48

Figure 3. Darkfield photomicrographs of parasagittal sections
through the forebrain of the Japanese quail
illustrating the dense axonal and terminal
degeneration present in the lateral forebrain
bundle (FPL), caudal neostriatum (NC), and
ectostriatum (E) 9 days after a single subcutaneous
injection of 125 mg TPP/kg body weight. Plate A
illustrates the axonal degeneration passing
dorsally and slightly caudally within the lateral
forebrain bundle to enter the caudal neostriatum.
Plate B illustrates the extremely dense axonal and
terminal degeneration present in the ectostriatum.
The section in Plate A corresponds to Level L3.5
and the section in Plate B corresponds to Level
L5.75 in the pigeon brain atlas of Karten and Hodos
(1967). Abbreviations: E, ectostriatum; FPL,
lateral forebrain bundle; LMD, lamina medullaris
dorsalis; NC, caudal neostriatum; NI, intermediate
neostriatum; PA, paleostriatum augmentatum; PP,
paleostriatum primitivum

 

50

Figure 4. Photomicrographs illustrating control and TPP-

exposed Fink-Heimer silver impregnated sections
through the caudal neostriatum and ectostriatum.
Plate A is from a section through the caudal
neostriatum of a brain from a bird in the control
group. NOte the absence of fragmented fibers of
particulate debris. Plate B illustrates a section
through the neostriatum 7 days after an injection
of 250 mg TPP/kg body weight. Noted the dense
plexus of degenerating axons and terminals. Plate
(2 is a control section through the ectostriatum.
Note the absence of degenerating fibers or
terminals. Plate D illustrates a section through
the ectostriatum 7 days after an injection of 250
mg TPP/kg body weight. Note the large number of
fragmented fibers and punctuate debris. Scale bar
= 50 mm .

51

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nucleus. The cerebellum showed degenerating spinocerebellar
fibers. The deep nuclei in the cerebellum had light to
moderate degeneration. The granule cell layers of foliae I -
VI had heavy mossy fiber degeneration whereas lighter fiber
degeneration was noted in the underlying white matter of
foliae VII - IX.

Moderate to heavy degeneration was noted in several
nuclei of the midbrain and forebrain. Degenerating fibers
extended from the paleostriatum primitivum to the lateral
forebrain bundle and ansa lenticularis. A group of fibers
terminated at the dorso-intermediate posterior thalamic
nucleus and pedunculopontine tegmental nucleus. Some fibers
extended from the lateral spiriform nucleus into the deeper

layers of the optic tectum.

Brain Auditory System :

The 3 principal auditory nuclei in the quail brain which
showed degeneration were the lateral mesencephalic nucleus,
pars dorsalis, the thalamic nucleus ovoidalis, and the
neostriatum caudalis (Table 3 and Figure 6). Seven of the 14
quail examined had degeneration in all of the above 3 areas
while 7 Ibirds had. degeneration in 'the thalamic. nucleus
ovoidalis and neostriatum caudalis. Degenerating fibers
originated from.the nucleus ovoidalis, and passed through the
lateral forebrain bundle and dorsally and caudally into the

neostriatum caudalis (Figure 3a). The nucleus ovoidalis

56

 

 

TABLE 3. The distribution of degeneration in the forebrain of
the Japanese quail perfused at various times after TPP
administration.
(Auditory) (visual)

To! Day noso“ MLD 0v no NR ES BSC

Post-dosing‘
556 7 62.5 + + ++ 0 0 O
557 8 62.5 0 + ++ 0 O 0
566 9 125 0 + + 0 0 +
567 9 125 ++ ++ +++ ++ +++ +++
568 9 125 0 + + 0 O O
570 9 125 + ++ +++ + +++ 0
571 7 250 +++ +++ +++ +++ +++ ++
572 7 250 + +++ +++ ++ ++ ++
573 7 250 +++ +++ +++ ++ +++ ++
575 7 250 0 ++ ++ O 0 ++
513 3 500 O +++ ++ ++ + 0
547 3 500 0 +++ ++ ++ - ++
548 3 500 ++ +++ ++ + + O
586 3 500 0 +++ ++ +++ 0 ++

 

The day post-dosing that the bird was perfused.

** Doses are expressed as mg TPP/kg body weight.
+ Light degeneration

++ Moderate degeneration

+++ Heavy degeneration

BSC Brachium of superior colliculus

ES Ectostriatum

MLD Lateral mesencephalic nucleus, pars dorsalis
NC Caudal neostriatum

NR Nucleus rotundus

OV

nucleus ovoidalis

57
contained degenerating cell bodies in the form of black-
impregnated cell somata in addition to degenerating axons and
terminals. The degeneration in the neostriatum caudalis
consisted of degenerating fibers primarily, and in some cases,
degenerating cell somata. In a few cases, degeneration was
noted in the deeper parts of the lateral mesencephalic
nucleus. In one case, axonal degeneration was also noted in
the nucleus angularis, a nucleus equivalent to the mammalian

cochlear nucleus.

Brain Visual System :

Eleven of the 14 quail examined had degeneration in 3
principal nuclei of the visual system, namely, the
ectostriatum, the nucleus rotundus, and. the brachium. of
superior colliculus (Table 3 and Figure 6). Degeneration was
noted in all the 3 areas in 4 birds while 5 birds had
degeneration in only 2 areas. The brachium of superior
colliculus was the sole site of degeneration in 1 bird. The
majority of cases studied showed the nucleus rotundus with
moderate to heavy degeneration. In a few cases, black silver
impregnated cell bodies indicating somatic degeneration were
also present. Degeneration in the ectostriatum, though
inconsistent, encompassed the major portion of the nuclear
region (Figures 3b and 6) . The brachium of the superior
colliculus also showed fiber degeneration which was

particularly evident in the portion of the brachium

58
encompassing the pretectal nucleus before terminating in the
nucleus rotundus. A few degenerating fibers were also noted
within the pretectal nucleus. The optic tectum did not

contain any axonal or somatic degeneration.

DISCUSSION

The data obtained from this study indicate that the
Japanese quail, which.has been reported.to be resistant to the
effects of the Type I OPIDN compound TOTP (Francis et a1.,
1980; Bursian et a1., 1983), is susceptible to the Type II
delayed neurotoxicant TPP. This was apparent in terms of
clinical signs characteristic of OPIDN, whole-brain NTE

inhibition, and central nervous system degeneration.

CLINICAL SIGNS

Observation of clinical signs started from day 1 post-
treatment as signs were evident immediately at the higher
doses (Table 1). There was a positive relationship between
the TPP dose and the severity of the clinical signs exhibited
by the Japanese quail. Birds dosed with 62.5 mg TPP/kg body
weight showed only mild ataxia. Four of the birds were able
to stand upright. The birds in the 125 mg TPP/kg body weight
group were more ataxic. This was evident by the fact that
these birds moved primarily on their books. In the 250 mg
TPP/kg body weight dose group, the birds displayed an
inability to maintain balance. The 500 mg TPP/kg body weight
dose group birds showed signs similar to those in the 250 dose
group, but clinical signs in the former group were evident
earlier.

There was also a positive relationship between the TPP

59

60

dose and the time of onset of clinical signs in that clinical
signs were evident earlier as the dose increased. The onset
of clinical signs occurred at day 3 post-dosing for the 62.5
and 125 TPP birds and as early as day 1 post-dosing for the
250 and 500 TPP birds. The birds in 500 TPP group were unable
to stand on day 1. There was also a positive relationship
between TPP dose and the number of birds affected. The
percentage of birds affected was 40 % in the 62.5 TPP group,
60 % in the 125 TPP group and 100 % in the 250 and 500 TPP
groups.

Day 1 through day 9 seems to be the time period in which
Japanese quail show clinical signs as no bird started to show
signs after that period. This suggests that if an individual
quail is susceptible to TPP, it will show signs prior to day
10.

Previous studies reported that the Japanese quail is not
sensitive to Type I OPIDN compounds like TOTP (Stuart and
Oehme, 1982; Francis et a1., 1980; Bursian et a1., 1983) based
on the absence of clinical signs after doses as high as 1000
mg TOTP/kg body weight. In this study, none of the 5 birds
dosed with 500 mg TOTP/kg body weight showed clinical signs
typical of OPIDN during the 21 day period. Earlier studies
have suggested that differences in absorption, metabolism, and
elimination of the neurotoxicant may play an important role in
species sensitivity to neurotoxic agents (Smith et a1., 1932;
Holmstead et a1., 1973; Whitacre et a1., 1976; Abou-Donia,

1976; Baron, 1981; Chadwick et a1., 1982; Abou-Donia, 1983;

61

Abou-Donia and Nomeir, 1986). Abou-Donia and Nomeir (1986)
concluded that the rates of clearance of the delayed
neurotoxicants EPN and leptophos in insensitive species were
faster than in sensitive species. Lasker et a1. (1982)
studied the in vitro metabolism of the Type I OPIDN compound
EPN and its oxygen-analog EPNO by rat and chicken microsomal
enzymes. The formation of EPNO (active metabolite) and PNP
(p-nitrophenol) (inactive metabolite) from EPN metabolism was
4.6 and 19.4 times higher in the rat than in the chicken,
respectively. In another study with EPN, Abou-Donia et al.
(1983a,b) concluded that the neurotoxicant remained in neural
tissues for a longer time in sensitive species allowing
continuous transfer of the compound and its metabolite to the
site of action. A possible explanation of why the Japanese
quail is resistant to TOTP is that this species may have a
faster rate of clearance of TOTP and/or a greater repair
capability in the insulted physiological system compared to
sensitive species.

Assessment of clinical signs also indicates that the
Japanese quail respond with a shorter delay than the usual
avian model, the domestic hen. In the present study, Japanese
quail in the 2 higher dose groups displayed OPIDN clinical
signs within 24 hours of dosing whereas birds in the lower 2
dose groups showed clinical signs by day 3. Previous studies
have shown that chickens dosed with TPP first started showing
signs between day 5 and day 14 post-dosing (Konno et a1.,

1989; Katoh et a1., 1990; Tanaka et a1., 1992). Mammalian

62

models are similar to the hen in regard to the latent period.
Rats treated with 1184 mg TPP/kg body weight developed hindleg
ataxia within a week followed by paralysis (Veronesi et a1.,
1986a;‘Veronesi and.Dvergsten, 1987). Ferrets.dosed with 500,
1000 and 2000 mg TPP/kg body weight show clinical signs of
ataxia as early as day 4 and paralysis by day 8 (Tanaka et
61., 1990).

Not only is the delay period shorter in Japanese quail,
but the transition from mild ataxia to severe paralysis occurs
more rapidly in Japanese quail than in chickens. Quail
treated with 250 and 500 mg TPP/kg body weight deteriorated
within 2 days after initial signs were observed. Hens treated
with 1000 mg TPP/kg body weight took 7 days to deteriorate
after the onset of clinical signs (Konno et a1., 1989; Tanaka
et 81., 1992).

The types of clinical signs noted in the Japanese quail
are different from those noted in the hen. Once affected by
OPIDN, the hen can stand for a longer period of time whereas
the quail tend to sit. Mortality in the hen is usually noted
much later than in Japanese quail, if at all. Three hens
dosed with 140 mg TPP/kg body weight died at days 16 - 22
post-dosing (Konno et a1. , 1989) , whereas in this study,
Japanese quail died as early as day 6 and 7 post-dosing and
some probably would have died sooner than day 6 or 7 post-
dosing if they weren't taken for neuropathological assessment.
Comparison of TPP-induced neuropathological damage in avian

species have shown differences in the location of lesions in

63

the brains of chickens and Japanese quail. The present study
has indicated lesions in the neostriatum of the Japanese quail
forebrain. Salzen and Parker (1975) have suggested that
damage to the neostriatum causes impairment of fine movements
such as are involved in feeding and visual discrimination.
Thus, TPP-exposed Japanese quail may have trouble locating
feed and eating. Since the metabolism of the Japanese quail
is relatively high, it is possible that any impairment that
affects the bird's ability to eat is going to be more
detrimental to the quail than to the chicken. Thus,
neostriatal damage in the Japanese quail may be contributing
to its early mortality when compared to the chicken. Another
possible explanation may be that the higher metabolic rate of
the Japanese quail might somehow enhance the rapid
distribution of the toxicant to the nervous system.

The development of TPP-induced OPIDN in the Japanese
quail occurs at much lower doses when compared to other
species. A study by Konno et a1. (1989) showed that leg
paralysis could not be produced in adult chickens by a single
dose of 100 or 500 mg TPP/kg body weight which agreed with
earlier study by Smith et a1. (1933). Ferrets were
administered 500 mg TPP/kg body weight in order to produce
clinical signs (Tanaka et a1., 1990). Rats showed clinical
signs after a dose of 1184 mg TPP/kg body weight (Veronesi et
a1., 1986a; Veronesi and Dvergsten, 1987) . However, in
Japanese quail, a single dose of 62.5 mg TPP/kg body weight

caused mild ataxia in 2 of the 5 birds.

64

NTE ASSAY

TPP’ doses as low' as 125 mg/kg‘ body' weight caused
significant inhibition of whole-brain NTE activity in the
Japanese quail (Table 2). These results agree with previous
studies done in the hen and rat (Veronesi et a1., 1986a;
Padilla et a1., 1987; Tanaka et a1., 1992; Knoth-Anderson and
Abou-Donia, 1993) in that TPP inhibits whole-brain NTE
activity. However, the threshold NTE inhibition required to
cause clinical signs may vary among species.

The percent inhibition of NTE is related to the dose of
TPP administered in that higher doses result in greater
inhibition. The percent inhibition is also related to the
severity of clinical signs noted. An 11 % NTE inhibition by
62.5 mg TPP/kg body weight resulted in mild ataxia whereas an
87 % NTE inhibition by 500 mg TPP/kg body weight was related
to severe paralysis which developed very quickly after dosing.

For Type I compounds, it is generally assumed that NTE
must be inhibited by at least 70 % for clinical signs and
neuropathy to develop. In this study, OPIDN developed when
NTE inhibition was only 11 %. Similar effects in rats have
been reported in that TPP caused NTE inhibition of
approximately 40 %, yet clinical signs and neuropathy
developed (Veronesi et al., 1986a; Padilla et a1., 1987).
Conversely, TOTP caused threshold inhibition of NTE in
Japanese quail yet did not cause OPIDN. The latter agrees

with a previous study by Bursian et a1. (1983). In that

65

study, Japanese quail were administered TOTP at doses of 125,
250, 500 and 1000 mg/kg body weight, doses sufficient.totcause
threshold inhibition of whole-brain NTE, yet clinical signs
did not develop. The reason why TOTP causes significant NTE
inhibition yet doesn't cause clinical signs is unknown.

Johnson (1976) stated that the NTE assay can be used as
a tool to determine the neurotoxic potential of OPIDN
compounds in that there is a relationship between NTE
inhibition in excess of 70 % and the development of clinical
signs. The present study indicates that these rules do not

apply to Japanese quail.

NEUROPATHOLOGICAL STUDIES

TPP doses as low as 62.5 mg/kg body weight caused
histopathological changes in the CNS of Japanese quail. The
fact that.TPP induces CNS damage in.quail agrees with.previous
studies on the effects of TPP on the CNS of chickens, rats,
and ferrets (Veronesi et al., 1986a; Padilla et a1., 1987;
Veronesi and Dvergsten, 1987; Carrington et a1., 1988; Tanaka
et a1. , 1990, 1992) , although the minimum dose needed to cause
degeneration varied among species.

The present study shows that the Japanese quail is very
sensitive to TPP which is indicated by the presence of
widespread lesions in the cerebellum, medulla, midbrain and
forebrain (Figures 5 and 6) . The spinocerebellar tract,

spinal lemniscus, reticular formation, ventral part of the

66

medial longitudinal fasciculus and fascicles of cranial nerves
IX and X had axonal and terminal degeneration. Moderate to
severe degeneration was noted in the gracile-cuneate, lateral
cervical, external cuneate and lateral paragigantocellular
reticular nuclei. Parvocellular and gigantocellular reticular
nuclei had light and scattered degeneration. The nucleus
cerebellaris internus in the cerebellum showed. moderate
degeneration. Degenerating fibers were noted in the forebrain
and midbrain, in the paleostriatum primitivum, lateral
forebrain bundle, ansa lenticularis, dorso-intermediate
posterior thalamic nucleus, pedunculopontine tegmental
nucleus, and lateral spiriform nucleus. The brain auditory
and visual systems in the Japanese quail were also affected.
Lesions were noted in the lateral mesencephalic nucleus, pars
dorsalis, thalamic nucleus ovoidalis, neostriatum caudalis,
thalamic nucleus rotundus and ectostriatum.

The distribution of neuropathy in hens dosed with TPP
(Figures 8 and 9) is similar to that noted in TPP-dosed
Japanese quail. Slight to moderate degeneration was noted in
hens dosed with 1000 mg TPP/kg body weight (Carrington.et a1.,
1988a; Tanaka et a1., 1992). Moderate degeneration was noted
in the medulla in the lateral vestibular, gracile-cuneate,
external cuneate and lateral cervical nuclei. Less
degeneration was noted in the solitary, inferior olivary and
raphae nuclei, in the medial and dorsal vestibular nuclei, and
in the lateral paragigantocellular, gigantocellular, and

lateral reticular nuclei. In the cerebellum, mossy fiber

67

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69

degeneration was noted in the granular layer of foliae I - Vb,
especially in foliae IV and V. The degenerating fibers were
noted as alternating parasagittal bands of heavy and light
degeneration. The deep cerebellar nuclei also had small
amounts of coarse fiber degeneration. unlike the Japanese
quail, no lesions were found in the auditory and visual
systems of the chicken (Figures 8 and 9).

Despite the similarity in neuropathological changes,
neuropathy in the Japanese quail was detected earlier and at
lower doses than in the hen. Swollen axons are present in the
brainstem of hens dosed with 1000 mg TPP/kg body weight at day
7 post-dosing (Carrington et a1. , 1988) whereas Japanese quail
dosed with 250 and 500 mg TPP/kg body weight in the present
study showed degeneration on day 3 post-dosing. Some birds
dosed with 500 mg TPP/kg body weight showed clinical signs as
early as.day'1 post-dosing; .Although birds were not taken for
perfusion until day 3, it can be speculated that degeneration
is present in the CNS of the Japanese quail on day 1 post-
dosing. The earlier onset of TPP-induced neuropathological
lesions in Japanese quail in this study may be due to a more
rapid distribution of TPP in the Japanese quail.

Differences in the location of degeneration may
contribute to the severity of clinical signs in different
species. Studies by Karten (1967, 1968) and Boord (1969)
showed that the pathway of the auditory system in birds begins
at the nucleus angularis and continues through the lateral

mesencephalic nucleus, pars dorsalis and nucleus ovoidalis to

70

terminate in the caudal neostriatum. Studies on TPP exposure
in the chicken by Tanaka et a1. (1992) showed that TPP caused
limited degeneration in the forebrain, which included
descending motor pathways originating in the basal ganglia and
projecting to several midbrain and medullary nuclei. The
midbrain lateral mesencephalic nucleus pars dorsalis contains
degeneration whereas neither the nucleus ovoidalis nor the
caudal neostriatum show any degeneration (Figures 8 and 9).
The results of the present study in Japanese quail document
for the first time that TPP exposure results in degeneration
of the forebrain auditory and visual systems in an avian
species. Degeneration was noted especially in the thalamic
nucleus and neostriatum of the auditory pathway (Figure 6).
The neostriatum is crucial for simultaneous visual
discrimination learning, feeding behavior and bodily movements
(Salzen and Parker, 1975).

As mentioned previously, fine adjustments involved in
feeding and preening, like neck, limb, and bill movements, are
controlled by sensorimotor coordinating circuits in the
neostriatum. Damage to the neostriatum may cause difficulty
and inaccuracy in pecking. In the present study, lesions were
noted in the neostriatum region of the Japanese quail
forebrain whereas no such lesions were noted in TPP-exposed
chickens (Tanaka et a1., 1992) . It can be suggested that
lesions in the neostriatum contributed to mortality of TPP-
exposed. Japanese: quail as ‘the birds face difficulty in

locating and consuming feed.

71

Degeneration has been noted in the forebrain and midbrain
motor nuclei and pathways of TPP-exposed mammals. Previous
studies in mammals have reported extensive degeneration in the
thalamus and cerebral cortex following exposure to TPP (Tanaka
et a1., 1990, 1992a) and the organophosphate compound soman
(McLeod et a1., 1984; Churchill et a1., 1985; Pazdernik et
a1., 1985; Lallement et a1., 1993). Tanaka et a1. (1990)
reported widespread degeneration in ferrets dosed with 1000 mg
TPP/kg body weight. Lesions were noted in the external
cuneate nucleus, pontine gray, nucleus of the reticular
formation, red nucleus, superior olivary nucleus, inferior
colliculus, ventral lateral nucleus and the lateral geniculate
nucleus.

Rats, like the Japanese quail, though resistant to TOTP-
induced neuropathy, are sensitive to TPP (Lehning et a1. ,
1990; Tanaka et a1., 1992b). Large amounts of CNS
degeneration were noted in rats administered 1184 mg TPP/kg
body weight. Degeneration was noted in the sensorimotor
cerebral cortex, auditory cortex, medial geniculate thalamic
nucleus, ventral medial thalamic nucleus, substantia nigra,
pars compacta and the deeper layers of the superior
colliculus.

Though the Japanese quail is sensitive to TPP, the
present study confirms the results from previous studies
(Francis et a1., 1980; Bursian et a1., 1982) indicating that
this species is resistant to TOTP-induced neuropathy.

Widespread degeneration was absent in the brain of the

72
Japanese quail administered TOTP (Figure 7). Moderate
degeneration was noted in nucleus cerebellaris internus in the
cerebellum and gracile-cuneate nucleus in the medulla.
Lighter amounts of lesions were noted in the medullary nucleus
supraspinalis and inferior olivary nucleus.

Unlike the Japanese quail, the domestic chicken is very
sensitive to TOTP. Neuropathological lesions were confined to
the medulla and cerebellum in hens dosed with 500 mg TOTP/kg
body weight (Tanaka and Bursian, 1989). Moderate amounts of
degeneration were noted in the lateral vestibular, gracile,
external cuneate and lateral cervical nuclei. Lesser amounts
of degeneration were noted in the solitary nucleus, inferior
olivary nucleus, and.raphae nucleus, in the medial, descending
and lateral 'vestibular' nuclei, and. in. ‘the lateral
paragigantocellular, gigantocellular, and lateral reticular
nuclei. Medullary portions of the dorsal and ventral
spinocerebellar tracts and spinal lemniscus had fiber
degeneration. Moderate amounts of degeneration were also
noted in the deep cerebellar nuclei and granular layers of
cerebellar folia I - V in the cerebellum.

Rats appear to be somewhat more resistant to TOTP-induced
neuropathy than the Japanese quail. Rats dosed with 1160 mg
TOTP/kg body weight had no neuropathological lesions in the
brainstem region (Veronesi and Dvergsten, 1987). However,
degeneration was noted in the fasciculus gracilis at the
cervical level and dorsolateral columns at the lumbar levels

in the spinal cord. The rat is an example of a mammalian

73
species similar to the Japanese quail in terms of resistance
to Type I OPIDN.

The present histological examinations are consistent with
previous studies which indicate that TPP causes OPIDN. The
results indicate that TPP causes degeneration in the auditory
and visual areas of the brain in addition to the forebrain,
midbrain, cerebellum and medulla. The distribution of
neuropathological lesions, however, indicates that the

biochemical activity of TPP differs among species.

CONCLUSION

This study demonstrated that TPP is neurotoxic in the
Japanese quail based on the development of clinical signs,
whole-brain NTE inhibition and the presence of
neuropathological lesions. The Japanese quail may serve as an
excellent model for the study of TPP neurotoxic effects in
avian species. The Japanese quail is perhaps a better model
for Type II OPIDN studies than the hen as it appears to be
very sensitive to TPP as indicated by the early onset of
clinical signs, lower threshold dose for NTE inhibition, and
the degeneration of many motor and sensory nuclei and
pathways. It should also be noted that the Japanese quail is
not a suitable model for Type I OPIDN as it is resistant to
neurotoxic effects of TOTP indicated by the absence of
clinical signs and widespread degeneration. The Japanese
quail is similar to the rat in regard to sensitivity to Type
I and Type II OPIDN. The distinctly different clinical,
biochemical and neuropathological profiles produced by Type I
and Type II OPIDN compounds in Japanese quail suggest that
different mechanisms may be responsible for their
neuropathological effects. The Japanese quail is an excellent
avian model for studying the differences in'Type I and Type II
OPIDN mechanisms. The neuropathological lesions caused by TPP
in. Japanese quail are «different from those in. the hen
suggesting further that the neurotoxic effects of the same

chemical vary among species.

74

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