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thesis entitled

NEUROTOXICITV 0F TRIPHENVL PHOSPHINE
IN THE EUROPEAN FERRET

presented by

Stephawée Davu

has been accepted towards fulfillment
of the requirements for

M degree in Wence

Major pro e or

Date 4/31/98

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NEUROTOXICITY OF TRIPHENYL PHOSPHINE
IN THE EUROPEAN FERRET

By

Stephanie Davis

A THESIS

Submitted to
Michigan State University
in partial fiilfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Science

1998

ABSTRACT
NEUROTOXICITY or TRIPI-IENYL PHOSPHINE IN THE EUROPEAN FERRET
By

Stephanie Davis

This study examined the effects of the organophosphorus delayed neurotoxicant
triphenyl phosphine (TPPn) in the European ferret (Mustela putoriusfuro). Adult ferrets
were injected subcutaneously with 250 or 500 mg TPPn/kg body weight, or the diethyl
ether/peanut oil vehicle. Whole-brain acetylcholinesterase (AChE) and neuropathy target
esterase (NTE) activities were measured 24 hours post-dosing. Neither enzyme was
inhibited by TPPn. Clinical signs were assessed over a six day observation period and
ranged from difficulty in holding the head erect beginning at four days post-treatment to
forelimb and hindlimb paralysis which were apparent at six days post-treatment.
Neuropathological damage was assessed by the F ink-Heimer silver impregnation method
in the brains collected at six days post-dosing. Axonal and terminal degeneration occurred
in several forebrain regions including the neocortex, hypothalamus, thalamus, and basal
ganglia. Degeneration in the midbrain was present in superior and inferior colliculi, and
regions of the pontine gray. Brainstem regions that contained axonal and terminal
degeneration were the pons, tegmentum, brainstem nuclei/tracts, and the vestibular nuclear
complex. Degeneration was also found throughout the cerebellum. The results of the
present study indicate that TPPn is a neurotoxicant that produces clinical signs and
pathology consistent with organophosphorus induced delayed neurotoxicity (OPIDN) in

the European ferret without inhibition of NTE and AChE.

For Christopher Jay

iii

ACKNOWLEDGMENTS

I would like to extend my sincere appreciation to my committee members Dr.
Richard Aulerich and Dr. Karen Chou for their guidance throughout my program. I am
also genuinely grateful to Dr. Duke Tanaka Jr. for his excellent technical assistance in
providing me with a working knowledge of neuroanatomy.

I would also like to convey my immense gratitude to my major professor Dr.
Steven Bursian for his excellent insight and critical suggestions in preparing this thesis and
in determining my future as a scientist.

I am especially obliged to Dr. Mark Uebersax for the research support provided to
me throughout my graduate program which guided my project toward successful
completion. My fiiture is forever broadened because he believed in me.

Finally, I would like to thank my family for their constant encouragement and
support in my studies. To my parents, I would like to say thank you for supporting all my
choices, for being real-life teachers, and for your unending love. This paper is in part your~
achievement also. For my precious sister Allison, my rock, who has always been there
believing in me. I admire you more than you can imagine and am so thankful that we are

friends. To my best friend, John, thank you for all of your love and support.

iv

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

be:
LIST OF TABLES ............................................................... vii
LIST OF FIGURES .............................................................. viii
FIGURE ABBREVIATIONS .................................................. x
INTRODUCTION ............................................................... 1
LITERATURE REVIEW ....................................................... 4
Introduction .............................................................. 4
Chemical Structure ...................................................... 5
Historigrl Development ................................................. 5
Inhibition of AChE ...................................................... 5
Recovery of Enzyme Activity ......................................... 7
Acute Sigis of Poisoning ............................................... 8
Introduction to OPIDN ................................................. 9
OPIDN in Humarg ...................................................... 9
Inhibition of NTE ........................................................ IO
NTE and Ogganophosphorus Chemical Reactivity .................. IO
Prophylaxis ............................................................... l l
Qallenge to NTE Threshold ........................................... ll
C_ha_racteristics of OPIDN Type I and Type II Compounds ....... 12
Chemical Structure .......................................... 13
Species Sensitivity ............................................ 13
Age Sensitivity ................................................ 14
Latent Period ................................................. 14
Clinical Signs ................................................. 15
N europathological Changes ............................... 16
Type III OPIDN ......................................................... 20
MATERIALS AND METHODS .............................................. 22

Subjects .................................................................. 22

 

 

 

 

 

Test Chemical ........................................................... 22
Experimental Desigg .................................................... 22
RESULTS ......................................................................... 25
Eagme Activities ........................................................ 25
Clinical Signs ............................................................. 25
Neuropaghology .......................................................... 27
DISCUSSION ..................................................................... 43
AChE Activity ............................................................ 43
NTE Activity .............................................................. 44
Clinical Signs .............................................................. 45
Locus and Extent of CNS Dgegeneration ............................... 47
LIST OF REFERENCES ......................................................... 52

vi

LIST OF TABLES

Table 1 - Identification and physical properties of triphenyl phosphine ........ 21

Table 2 - Whole-brain neuropathy target esterase (NTE) and
acetylcholinesterase (AChE) activities in European ferrets
24hr after exposure to triphenyl phosphine (TPPn) .................. 26

Table 3 - Ferret central nervous system regions which contain
axonal and/or terminal degeneration after administration
of TPPn ..................................................................... 34

vii

Figure 1.

Figure 2.

LIST OF FIGURES

Darkfield photomicrographs illustrating axonal and terminal
degeneration in the primary visual cortex and thalamus.
Under darkfield illumination, CNS regions containing silver
impregnated degenerating elements show up as bright areas
against a light brown or dark yellow background. A. Dense
band of degeneration (arrowheads) located in lamina IV of
the primary visual cortex of a ferret six days after injection
with 500mg TPPn/kg body weight. B. Dense terminal
degeneration located within the mediodorsal (MD), ventral
lateral (VL), and ventromedial (VM) thalamic nuclei of a
ferret six days after injection with 250mg TPPn/kg body
weight. Scale bars = 1 mm ............................................... 28

Photomicrographs illustrating Fink-Heimer silver
impregnated regions showing axonal and terminal
degeneration in the thalamus of a ferret six days after
injection with 250mg TPPn/kg body weight. Panel A
illustrates dense degeneration in the lateral geniculate
nucleus (LGN) on the left side of the photomicrograph.

Note the sharp demarcation between the degeneration in

the LGN and the absence of degeneration in the immediately
adjacent area to the right side of the photomicrograph.

Panel B is a high power view of the caudal part of the
mediodorsal thalamic nucleus. Note the absence of
degeneration and the presence of light brown plump

cell bodies (arrowheads). Panel C shows a high power

view of the ventromedial thalamic nucleus. Note the
presence of fine particulate black axonal and terminal
degeneration as well as a black silver-impregnated pyknotic
degenerating cell body (arrow). Scale bars = 25 um.

Scale bar in B applies to C .............................................. 30

viii

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Photomicrographs through three laminae of the primary
visual cortex showing axonal and terminal degeneration in a
ferret six days after injection with 250mg TPPn/kg body
weight. Panel A is from lamina III and contains almost no
degeneration. Panel B is Item lamina IV and is the site of
termination of degenerating axons from the lateral
geniculate nucleus. Panel C illustrates degenerating axons
passing through lamina VI on their way to terminate in
lamina IV. The black ovoid structures in Panels A and C
are glial cells. Scale bar = 25 um and applies to all panels ........... 32

Line drawings of parasagittal sections depicting the

locations of axonal and terminal degeneration (stippling)

in nuclear regions of the brain in ferrets dosed with 250

or 500 TPPn mg/kg body weight. Drawings are from medial (A)

to lateral (C). Structure abbreviations are found at the

beginning of the text ....................................................... 36

Line drawings of parasagittal sections depicting the

locations of axonal and terminal degeneration (stippling) in

nuclear regions of the brain in ferrets dosed with 250 or

500 TPPn mg/kg body weight. Drawings are fi'om medial (D)

to lateral (F). Structure abbreviations are found at the

beginning of the text ....................................................... 38

Summary schematics in the sagittal plane that compare

qualitatively the extent of degeneration in the brain of the

ferret after a single exposure to DFP, TPP, or TPPn.

The location of degeneration was assessed using a modified
Fink-Heimer silver impregnation technique. Structure

abbreviations are found at the beginning of the text. Stippling
represents areas of degeneration ......................................... 48

ix

FIGURE ABBREVIATIONS

AV
Cb
CM
COn
Cos
CRs
CX
EN
ESs
FF
FTG
ICC
IO
LD
LGN
LL
LP
LRN
MAs

MGN
MMn
NC
NG
PAT
PG
PGL
PGM
PSs
RHs

SCI
SO
SOL
SOM
SPs
SSs
SUB
TGn

anterior ventral thalamic nucleus
cerebellum

centrum medianum thalamic nucleus
cochlear nuclei

coronal sulcus

cruciate sulcus

external cuneate nucleus
entopeduncular nucleus

ectosylvian sulcus

fields of Forel

gigantocellular tegmental field
central nucleus of the inferior colliculus
inferior olivary complex

lateral dorsal thalamic nucleus
lateral geniculate nucleus

lateral lemniscus

lateral posterior thalamic nucleus
lateral reticular nucleus

marginal sulcus

mediodorsal thalamic nucleus
medial geniculate nucleus

medial mammillary nucleus

cuneate nucleus

nucleus gracilis

parataenial nucleus

pontine gray

pontine gray, lateral division
pontine gray, medial division
presylvian sulcus

rhinal sulcus

red nucleus

superior colliculus, intermediate lamina
superior olivary complex

lateral nucleus of the superior olive
medial nucleus of the superior olive
splenial sulcus

suprasylvian sulcus

subthalarnic nucleus

tegmental nuclei

FIGURE ABBREVIATIONS

VA ventral anterior thalamic nucleus

VB ventral basal thalamic nucleus

VCx visual cortex

VL ventral lateral thalamic nucleus

VLV lateral vestibular nucleus, ventral division
VM ventral medial thalamic nucleus

VMN medial vestibular nucleus

VN vestibular nuclear complex

VPL ventral posterior nucleus

VSN superior vestibular nucleus

xi

INTRODUCTION

Exposure to certain organophosphorus compounds (OP’s) in agricultural and
industrial settings can cause neurotoxicity in animals including humans. OP-induced
neurotoxicity is apparent either immediately (acute effects) and/or several days after
(delayed effects) exposure. Reversible acute effects are the result of inhibition of the
nervous system enzyme acetylcholinesterase (AChE) (Davis and Richardson, 1980).
Delayed effects are non-reversible and are commonly known as organophosphorus-
induced delayed neurotoxicity (OPIDN) (Davis and Richardson, 1980).

OPIDN is thought to result from an interaction between a neurotoxic OP
compound and the enzyme neuropathy target esterase (NTE) (Johnson, 1987; Peraica et
al., 1995). Inhibition of whole-brain NTE exceeding 70% is generally associated with
clinical signs and pathology characteristic of OPIDN (Davis and Richardson, 1980; Abou-
Donia and Lapadula, 1990; Richardson, 1992). Although recent studies indicate that the
threshold of NTE inhibition for OPIDN may be less than 70%, depending upon the
species and compound, some degree of NTE inhibition in animals that exhibit OPIDN has
always been reported (Larsen et al., 1986; Veronesi and Dvergsten, 1987; Stumpf et al.,

1989).

Recently, OPIDN has been divided into two subclasses (Type I and Type 11) based
on the following classification criteria (Abou-Donia and Lapadula, 1990). Type I OPIDN
is produced by organic esters of phosphoric acid such as tri-o-tolyl phosphate (TOTP) and
diisopropylfluorophosphate (DFP), while organic esters of phosphorus acid such as
triphenyl phosphite (TPP) cause Type II OPIDN. Differences in species sensitivity to Type
I and Type 11 compounds exist. Chickens and ferrets are sensitive to both Type I and Type
II OPIDN compounds, but rodents and Japanese quail are only sensitive to Type 11
compounds (Abou-Donia and Lapadula, 1990; Tanaka et al., 1990a; Tanaka et al., 1992b;
Varghese et al., 1995). Sensitive species are also susceptible to Type I and Type 11
compounds at different ages. The young of sensitive species are typically not sensitive to
single doses, but may be sensitive to multiple doses of a Type I compound. In contrast, a
single dose of a Type 11 compound can cause OPIDN in both young and adult animals of
sensitive species(Abou-Donia and Lapadula, 1990). Type 1 compounds are typically
associated with a longer delay period before the onset of clinical signs (14-21 days) when
compared to Type II compounds (four to seven days) (Abou-Donia and Lapadula, 1990).
The characteristic clinical signs produced by delayed neurotoxicants vary by type of
compound and species of animal. Clinical signs for Type I OPIDN compounds in the
chicken are similar to clinical signs for Type II compounds, but are distinct in species such
as the cat, ferret, and rat. Neuropathological damage is usually confined to the peripheral
nerves, spinal cord, brainstem, and cerebellum after exposure to a Type I compound, but
additional damage occurs in midbrain and forebrain regions of animals exposed to Type 11
compounds (Veronesi and Padilla, 1985; Abou-Donia and Lapadula, 1990; Tanaka et al.,

1992a)

Recently, a third type of OPIDN, distinct fi'om Types I and II, has been proposed
by Abou-Donia et al. (1996) after examining the neurotoxic characteristics of triphenyl
phosphine (TPPn) in the domestic chicken. It was reported that repeated oral
administration of 500, 2,000, or 5,000 mg/kg body weight triphenyl phosphine produced
clinical signs similar to those caused by other OPIDN-inducing compounds (ataxia and
paralysis), yet whole-brain NTE was not inhibited. Neuropathological examination was
confined to the spinal cord where degeneration was found in both ventral and lateral
tracts.

Previous work in our laboratory has demonstrated that the European ferret is a
sensitive mammalian model for OPIDN (Stumpf et al., 1989). Therefore, it was of interest
to examine the effects of this apparently novel OP in this mammalian species to determine
if clinical signs and neuropathology consistent with OPIDN could be produced without
inhibition of NTE. The specific aims of this study were to:

1. Determine if TPPn inhibits whole-brain NTE in the European ferret

2. Determine clinical signs caused by administration of TPPn and compare them

to clinical signs characteristic of Type I and Type II OPIDN

3. Describe the neuropathological effects of TPPn using the F ink-Heimer silver

impregnation method and compare them to neuropathology resulting from

exposure to Type I and Type II OPIDN compounds

LITERATURE REVIEW

Introduction

Organophosphorus compounds (OP’s) have a variety of industrial and agricultural
uses such as fire retardants for fabrics, modifiers in plastic processing, petroleum additives,
surfactants, chemical intermediates in the manufacture of pharmaceuticals, and insecticides
(Davis and Richardson, 1980; Johnson, 1987; Abou-Donia and Lapadula, 1990). These
compounds are primarily beneficial as insecticides to protect essential agronomic crops
from disease, loss of nutrients, and death. This ultimately leads to higher quality
agronomic crops and thus improved human health (Chambers, 1992).

Unfortunately, organophosphorus compounds are also noted for being potent
neurotoxic agents, teratogens, and possible reproductive toxins (Proctor and Casida,
1975; Davis and Richardson, 1980). The toxic effects of OP’s which have been examined
the most extensively are their elfects on the nervous system. Organophosphorus
compounds cause cellular disruption by altering normal cell function in the central and
peripheral nervous systems through phosphorylation of esterases (acetylcholinesterase
and/or neuropathy target esterase) (Davis and Richardson, 1980; Abou-Donia and

Lapadula, 1990; Peraica et al., 1995)

Chemical Structure

Organophosphorus compounds are organic structures that contain phosphorus-
carbon bonds. The phosphorus atom contains two paired and three unpaired electrons in
its outer shell and is capable of forming either trivalent (pyramidal configuration), or
pentavalent (tetrahedral configuration) compounds (Abou-Donia and Lapadula, 1990). OP
phosphorylation reactions are dependent on the reactivity of the central phosphorus atom,
which is determined by the electrophilic character of the groups attached to the

phosphorus atom.

Historical Development

Gerhard Schrader became known as the “father” of organophosphorus esters in the
late 1930’s and 1940’s because of his insight into the potential uses of OP’s as industrial
chemicals and pesticides (Chambers, 1992). Many OP’s were developed throughout the
1930’s and 1940’s: tabun and satin in 1937, dimefox and schradan in 1941, diisopropyl
phosphorofluoridate (DFP) in 1941, parathion and soman in 1944, and the extensively
used insecticide malathion in 1950. (Chambers, 1992). Organophosphorus compounds
became the insecticides of choice by the 1970’s for fighting pests on a diverse range of
crops including cotton, apples, corn, and stored grain (Racke, 1992). They were also

applied topically to cattle to control cattle ticks (Racke, 1992).

Inhibition of AChE
OP compounds can affect normal fimctioning of the central nervous system in

animals and humans by interfering with normal transmission of nerve impulses. Specific

neurons in the central and peripheral nervous systems use the neurotransmitter
acetylcholine (ACh). ACh is synthesized by choline acetyltransferase fiom acetyl CoA and
choline in the axon terminal, stored in synaptic vesicles, and released into the synaptic cleft
after a normal nerve impulse. ACh binds to two types of receptors on the postsynaptic
cell (muscarinic and nicotinic) to cause activation of the postsynaptic cell which may be
another neuron, a muscle cell, or a glandular cell (Kandel, I991). Afier a normal nerve
impulse, acetylcholinesterase (AChE) is released from the surface of the postsynaptic
membrane into the synaptic cleft to rapidly hydrolyze ACh to acetate and choline thus
allowing the postsynaptic cell to return to its resting state. AChE is catalytically active
due to the presence of a zone that contains two separate active sites: 1) the anionic site
which attracts, binds, and orients the substrate using electrostatic forces and 2) the
esteratic site which catalyzes the hydrolysis of the substrate. AChE and ACh combine to
form a Michaelis enzyme-substrate complex (Kandel, 1991). The acetyl group from ACh
transfers to the esteratic site (serine) of AChE to form an acetylated enzyme which is then
rapidly hydrolyzed resulting in a re-activation of the enzyme. Choline is then taken up into
the presynaptic terminals for re-synthesis of ACh (Kandel, 1991).

Organophosphorus compounds are effective as insecticides because they target the
nervous system of insects, as well as birds and mammals, through phosphorylation of
AChE at cholinergic nerve endings. This is considered an acute effect because clinical
signs develop shortly (minutes to hours) after exposure. In order to inactivate AChE,
organophosphorus compounds mimic ACh by covalently binding to AChE by nucleophilic
attack forming an OP-AChE complex. This allows phosphorylation to occur at the

esteratic site where the electronegative hydroxyl group of the serine in the esteratic site of

AChE reacts with the central electropositive phosphorus atom of the organophosphorus
compound rendering the phosphate acidic (Timbrell, 1991; Chambers, 1992; Katzung and
Trevor, 1995). Thus, when an OP is bound to AChE and the enzyme is inactivated, ACh
can not be removed effectively from the synaptic cleft after a normal stimulation of the

neuron causing over-stimulation of the postsynaptic cell.‘

Recovery of Enzyme ActiviLy

The reaction between an OP and the active site of AChE results in an inhibited
enzyme that undergoes one of two chemical reactions: regeneration or aging.
Regeneration is the hydrolytic removal of the phosphoryl moiety that returns the enzyme
to its active form (Chambers, 1992). Aging is a stabilization of the OP-AChE complex
through a conformational change resulting from the loss of a second group (dealkylation)
from the phosphorus atom. The aging process permanently alters the electric charge of
the enzyme’s active site preventing dephosphorylation (Johnson, 1987; Chambers, 1992).
In order for AChE to hydrolyze excess ACh after aging has occurred, new enzyme must
be synthesized which takes approximately 20-30 days (Chambers, 1992).

Several chemicals are capable of alleviating symptoms of acute OP
poisoning before the process of aging occurs. The chemical pyridine 2-aldoxime (2-PAM)
is effective in the regeneration of AChE through dephosphorylation of the phosphorylated
enzyme. The nitrogen atom on 2-PAM binds to the anionic site of AChE which facilitates
transfer of the substituted phosphate from the esteratic site of AChE to the oxime. This
regenerates AChE which allows it to hydrolyze remaining ACh (Wilson et al., 1992). The

drug atropine sulfate is also effective in relieving symptoms of acute OP poisoning.

Atropine sulfate competes with ACh for muscarinic binding sites. This reduces excessive
stimulation of muscarinic receptor sites by ACh until new AChE is synthesized (Wilson et
aL,1992)
Acute Signs of Poisoniag

Inhibition of AChE is disruptive at all cholinergic synapses. Cholinergic synapses
include synapses of the central nervous system, the neuromuscular junctions of motor
nerves, sensory nerve endings, pre-ganglionic synapses of both sympathetic and
parasympathetic nerves, postganglionic parasympathetic nerve terminals, and sympathetic
nerve terminals on the sweat glands, blood vessels, and adrenal medulla (Timbrell, 1991).
Inhibition of AChE greater than 50% at these nerve terminals is associated with classical
symptoms of acute OP poisoning (Wilson et al., 1992). Continual stimulation of
muscarinic receptors on smooth muscles causes the following symptoms: tightness in the
chest and wheezing expiration due to bronchoconstriction and increased bronchial
secretions, increased salivation and lacrimation, increased sweating, increased
gastrointestinal tone, peristalsis, nausea, vomiting, abdominal cramps, diarrhea, tenesmus
and involuntary defecation, bradycardia, frequent and involuntary urination, and
constriction of the pupils (Chambers, 1992). Continual stimulation of nicotinic receptors
at motor nerve junctions causes fatigue and weakness of innervated muscles, involuntary
twitching, scattered fasciculations and cramps, dyspnea, and cyanosis (Chambers, 1992).
Continual stimulation of nicotinic receptors at autonomic ganglia causes tachycardia,
pallor, elevation of blood pressure, and hyperglycemia (Chambers, 1992). Accumulation
of ACh in the central nervous system causes tension, anxiety, restlessness, insomnia,

headache, emotional instability, neurosis, apathy, confiision, slurred speech, tremor,

generalized weakness, ataxia, convulsions, depression of respiratory and circulatory
centers, and coma. The cause of death in acute OP poisoning usually results from asphyxia

due to respiratory failure (Chambers, 1992).

Introduction to OPIDN

Another type of neurotoxicity caused by certain organophosphorus compounds is
called organophosphorus-induced delayed neurotoxicity. OPIDN results when there is a
delay between exposure to the organophosphorus compound and the appearance of

characteristic clinical signs (Abou-Donia and Lapadula, 1990).

OPIDN in Him

Since the beginning of the 20th century, over 40,000 cases of OPIDN in humans
have been reported throughout the world (Abou-Donia and Lapadula, 1990). A number
of the early poisonings resulted from a medication for people with tuberculous that was
adulterated with phospho-creosote (Davis and Richardson, 1980). As many as 20,000
peOple were paralyzed in the 1930’s in the US. by an alcoholic drink made from a
Jamaican ginger extract contaminated with tri-ortho-tolyl phosphate (TOTP) (Baron,
1981). Modern concern for potential OP poisoning is related to dangers associated with
occupational exposure. Although the toxicology data for OP’s in humans are very limited,
it is probable that the human may be the most sensitive species since recent studies
indicate that exposure to OP’s may result in damage to higher order brain functions such
as sensorimotor processing and cognitive brain function (Baron, 1981; Tanaka et al.,

1990a)

IO

Inhibition of NTE

OPIDN presumably results from an OP’s interaction with the enzyme neuropathy
target esterase (NTE) (Peraica et al., 1995). NTE is a membrane-bound protein that is
proposed to have a role in lipid metabolism (Abou-Donia and Lapadula, 1990; Lotti et al.,
1993). OPIDN is thought to be induced by an organophosphorus compound through a
two step process: 1) inhibition of NTE by phosphorylation; 2) aging of phosphorylated
NTE (Lotti et al., 1993). The interaction between an OP and NTE (occurring within one
hour of exposure) at the enzyme’s active site creates an NTE-OP complex. The esterase
complex is then phosphorylated creating a stable covalent bond between the active site of
the enzyme and the phosphate moiety which inhibits the catalytic activity of NTE
(Timbrell, 1991; Johnson, 1993; Lotti et al., 1993). Aging involves the loss of an ester
functional group (alkyl or aryl group) on the phosphorus atom of the OP-esterase complex
by cleavage of an ester or amido bond. This leaves the complex negatively charged. The
unbound ester functional group then becomes re-attached to another part of the protein
and is not released into the surrounding region (Johnson, 1993). Thus, the important step
in the development of OPIDN by NTE could be either the liberation of the ester group or
its reattachment to another part of the protein (Johnson, 1993). When NTE is negatively
charged in the phosphorus region, a progressive neuropathy of both the peripheral and

central nervous systems occurs (Timbrell, 1991; Johnson, 1993; Lotti et al., 1993).

NTE Ml Organophosphorus Chemical Reactivity
Inhibition of whole-brain NTE greater than 70% is generally associated with the

clinical signs and neuropathology characteristic of OPIDN (Johnson, 197 7; Abou-Donia

11

and Lapadula, 1990). All organophosphorus compounds that are known to cause OPIDN
are inhibitors of NTE in viva (phosphates, phosphonates, and phosphdramidates), but
some organophosphorus compounds are capable of inhibition of NTE without causing
OPIDN (sulphonylfluorides and phosphinates). Phosphates, phosphonates, and
phosphoramidates are able to induce OPIDN because they are capable of undergoing the
aging reaction due to the presence of either oxygen or amine groups that link the R-group
to the phosphorus atom through labile ester or amido bonds. Phosphinates are not capable
of aging, because they do not contain a labile ester or amido bond and thus do not induce

OPIDN.

Prophylaxis

Administration of a non-aging inhibitor of the phosphinate class (such as phenyl
methanesulfonyl fluoridate or PMSF) before exposure to a neuropathic organophosphorus
compound prevents neuropathy. This occurs because the non-aging compound occupies
the active site on the NTE molecule and prevents an ageable OP compound from binding
to it. Competition for this active site will continue without causing OPIDN until the NTE
critical inhibition level of 70% is reached and aging is occurring (Carrington, 1989). Thus,
the determining factor for whether an OP will be neuropathic or protective is its ability to
undergo an aging reaction.
gralleage to NTE Threahold

Although few scientists would argue that NTE is not involved in the pathogenesis
of OPIDN, many question the enzyme’s role as the target enzyme. This position is

substantiated by the poor correlation of enzyme inhibition induced by certain OP’s with

12

the appearance of OPIDN clinical signs and pathology. For example, tri-2-ethyl-phenyl
phosphate, di-phenyl-2-isopropylphosphate, and the test pesticide Bayer KER-2822 inhibit
whole-brain NTE in excess of 70% and the phosphorylated enzyme does undergo aging,
yet clinical signs and pathology characteristic of OPIDN do not occur (Lotti et al.,1993).
Other OP compounds cause OPIDN clinical signs with concurrent central nervous
system degeneration even though NTE inhibition is well below 70%. For example, rats
exposed to TPP exhibited marked ataxia with spinal cord degeneration despite a whole-
brain NTE inhibition of only 33% (Veronesi and Dvergsten, 1987). Larsen et al. (1986)
reported clinical signs consistent with OPIDN when whole-brain NTE was inhibited by
only 41% in the turkey afier exposure to TOTP. Ferrets dosed with TOTP displayed
clinical signs indicative of OPIDN when whole-brain NTE was inhibited by only 46%

(Stumpfet al., 1989).

Qaracteristics of OPIDN Type I and Type II Compounds

Differences among clinical and neurophysiological responses to organophosphorus
compounds have led to OPIDN being categorized into two distinct classes. These classes
differ in terms of molecular structure of the neuropathic OP, species that are sensitive to
the compound, age of susceptibility, duration before onset of clinical signs, type of clinical
signs, location of affected neural regions, and degree of neuropathy target esterase

inhibition (Abou-Donia and Lapadula, 1990).

13

Chemical Structure

Type I OPIDN compounds are distinguished by a chemical structure that consists
of a pentavalent phosphorus atom characteristic of phosphoric, phosphonic, or
phosphoramidic acid. Examples of Type I OPIDN compounds are TOTP, which is also
known as tri-o-cresyl phosphate (TOCP), and DFP. The presence of an ortho-methyl
group in the aromatic series seems to be essential for aromatic compounds to be
neurotoxic. Type II OPIDN compounds contain a trivalent phosphorus atom and are
derivatives of phosphorus acid and their sulfur analogs. Triphenyl phosphite (TPP) is an

example of a Type II OPIDN compound.

Species Sensitivity

There are differences in species sensitivity to Type I and Type II compounds.
Although chickens are highly sensitive to both Type 1 and Type II OPIDN compounds,
neither rodents nor Japanese quail are sensitive to Type I compounds, but all these species
develop OPIDN in response to a single dose of a Type II compound (Abou-Donia and
Lapadula, 1990). Cats develop OPIDN only after multiple doses of a Type I compound,
but they are sensitive to single doses of a Type II OPIDN compound (Abou-Donia and
Lapadula, 1990). Although ferrets develop OPIDN in response to a single exposure to a
Type 1 compound, OPIDN is more severe after a single exposure to a Type 11 compound

(Stumpf et al., 1989; Tanaka, 1990a).

14

Age Sensitivity

Differences in age sensitivities exist between Type I and Type 11 compounds.
Young animals of sensitive species are not susceptible to single doses, but they may be
affected by multiple doses of a Type 1 compound. A single oral dose of 2 mg/ kg body
weight DFP did not produce OPIDN in young chicks, however, they did develop OPIDN
afier repeated doses of DF P (Johnson and Barnes, 1970). In another study, chicks
younger than 60 days of age did not develop OPIDN afier receiving a single dose of 1.3
mg/ kg body weight DFP, while administration of the same dosage in chickens 60 days
and older produced OPIDN that was more severe with increasing age (Moretto et al,
1991). In contrast, one-week-old chicks developed OPIDN in response to a single dose of
1000 mg/ kg body weight TPP (Abou-Donia and Brown, 1990). Most OPIDN studies
involving ferrets have examined susceptibility only in adult ferrets, but a detailed study on
ferret age susceptibility was conducted using TPP. Ferrets dosed with 1184 mg/ kg body
weight TPP did not develop OPIDN until five weeks of age. Severity of clinical signs and
neuropathology then increased with age and reached adult levels at 10 weeks of age

(Tanaka et al., 1994).

Latent Period

Both Type I and Type II OPIDN are characterized by a delay between time of
exposure and the appearance of clinical signs, but the length of the delay varies by type.
Type II OPIDN has a shorter latent interval then Type I OPIDN. The onset of clinical
signs in the cat' ranges from four to seven days with a Type 11 compound and 14-21 days

with a Type I compound (Smith et al., 1933). Rats exhibit clinical signs characteristic of

15

OPIDN approximately seven days after exposure to a Type 11 compound, but the latent
interval for Type 1 compounds is approximately 14-21 days (Veronesi and Padilla, 1985).
Hens display clinical signs four to six days after exposure to a Type 11 compound and six
to fourteen days after exposure to a Type I compound (Abou-Donia and Lapadula, 1990).
Ferrets display clinical signs four to six days after exposure to a Type 11 compound and
10-14 days after exposure to a Type I compound (Stumpf et al., 1989; Tanaka et al.,

1990a)

Clinical Signs

Clinical signs produced by delayed neurotoxicants vary by type of compound and
species of animal. Clinical signs of ataxia and incoordination produced by Type I OPIDN
compounds in the chicken are similar to clinical signs produced by Type 11 compounds,
but are distinct in species such as the cat, ferret, and rat. Cats treated dermally with
multiple doses of 250 mg/kg body weight TOTP exhibit hind limb ataxia and flaccid
paralysis four to seven days after exposure while cats treated dermally with a single dose
of 1000 mg/ kg body weight TPP develop ataxia after four to twenty-six days and develop
extensor rigidity of both front and hind limbs between eight to thirty days (Smith and
Lillie, 193 l; Abou-Donia et al., 1986). Rats exposed to a single subcutaneous dose of
1000 mg/ kg body weight TOTP do not exhibit any detectable clinical signs, but after
subcutaneous exposure to 1000 mg/ kg body weight TPP they exhibit hyperexcitability,
spasticity, incoordination, circling behavior, tail-kinking, hind limb ataxia, and partial
flaccid paresis of the extremities (Veronesi and Dvergsten, 1987). Ferrets exhibit hind

limb ataxia followed by flaccid hind limb paralysis after a single subcutaneous exposure to

I6

500 and 1000 mg TOTP/ kg body weight and single subcutaneous doses of 2 and 4 mg
DFP/ kg body weight (Tanaka et al. 1991). Single subcutaneous exposure to 500, 1000,
and 2000 mg TPP/ kg body weight in ferrets results in hind limb ataxia leading to extensor
rigidity as well as fore limb ataxia which develops into extensor rigidity (Tanaka et al.,

I 990a).

N europathological Changes

Neuropathological changes occur after exposure to an organophosphorus
compound that induces OPIDN and they are distinct for Type I and Type 11 compounds.
Type I compounds typically affect only the peripheral nerves, spinal cord, brainstem, and
parts of the cerebellum, while Type 11 compounds affect peripheral nerves, spinal cord,
brainstem, cerebellum, midbrain, and forebrain (Abou-Donia and Lapadula, 1990; Tanaka
et al. 1992a).

CAL; Type 1 compounds produce histopathological lesions in the spinal cord of
cats (Smith and Lillie, 1931; Cavanagh and Patangia, 1964). Degenerating axons are
found in the ascending tracts including the spinocerebellar columns, posterior columns,
and gracile tracts. Descending tracts that contain degeneration are the corticospinal tracts
of the ventral columns (Smith and Lillie, 1931; Cavanagh and Patangia, 1964).

After a single subcutaneous exposure to 1000 mg TPP/kg body weight,
degeneration occurs in many ascending and descending tracts of the spinal cord (Smith et
al., 1933). Degenerating ascending tracts include the spinocerebellar and anterolateral

spinocerebellar tracts. Degenerating descending tracts include the rubrospinal,

17

vestibulospinal, tectospinal, lateral corticospinal, and anterolateral tracts. Degeneration
also occurs in the medulla and pons (Smith et al., 1933).

R_A;_'I’_S_. Neuropathological damage caused by Type I OPIDN compounds is far
less extensive than damage caused by Type II OPHDN compounds. Rats treated with a
single subcutaneous dose of 1000 mg TOTP/kg body weight exhibit degeneration in the
ascending and descending tracts of the spinal cord. Degeneration is confined to the
ventrolateral and ventral columns of the cervical and lumbar cord (Lehning, 1992; Tanaka
et al., l992a).

Rats treated with a single subcutaneous dose of 1000 mg TPP/kg body weight
exhibit degeneration in the spinal cord, as well as forebrain, midbrain, and hindbrain areas
(Veronesi and Dvergsten, 1987; Lehning, 1992; Tanaka et al., l992a). Spinal cord
degeneration includes tracts affected by exposure to TOTP with additional damage in the
small diameter fibers of the gracile fasciculus, and the gray matter in laminae V-IX of the
upper cervical cord. Affected forebrain regions include the transitional cortex, the
neocortex, the septum and hypothalamus, the hippocampal region, and the thalamus.
Midbrain regions affected by a single exposure to TPP in rats include components of the
basal ganglia, superior colliculi, inferior colliculi, oculomotor nucleus, and dorsal raphae
nucleus. Hindbrain regions affected by a single exposure to TPP are the cerebellum, the
vestibular nuclear complex, dorsal cochlear nucleus, and the reticular formation. (Veronesi
and Dvergsten, 1987; Lehning,]992; Tanaka et al., 1992a).

CHICKENS. Chickens that are exposed to a single subcutaneous dose of a Type
I OPIDN (500 mg TOTP/kg body weight or 1 mg DFP/kg body weight) exhibit

degeneration in the spinal cord, medulla, and cerebellum (Tanaka et al., 1990b; Tanaka et

18

al., 1992a). Degenerating axons are found in the gracile fasciculus at cervical levels,
dorsal and ventral spinocerebellar tracts, the medial pontine-spinal tract' at lumbar levels,
and the medial part of the ventral horn at lumbar cord levels. In the medulla, exposure to a
Type I OPIDN results in degeneration in several brainstem nuclei related to the reticular
formation. Degeneration also occurs in medullary fiber tracts. Within the cerebellum,
degeneration occurs in the granular layer of folia I-Vb. (Tanaka et al., 1990b; Tanaka et
al,]992a)

Chickens that are exposed to a single subcutaneous dose of 1000 mg TPP/kg body
weight exhibit degeneration in the same regions affected by Type I OPIDN compounds, as
well as in additional regions throughout the spinal cord, medulla, midbrain, and forebrain
(Tanaka et al., 1992a). Additional spinal cord damage occurs in the spinocerebellar tract
and pontine-spinal tracts at all cord levels, ventral part of the medial longitudinal
fasciculus, lateral and ventral fiJniculi, spinal laminae VII and VIII that extend into the
lateral parts of the ventral horn at the cervicothoracic and lumbosacral levels, and laminae
V,VI, and IX at all cord levels. (Tanaka et al., 1992a). TPP exposure results in additional
degenerated nuclei in the medulla. Degeneration in the cerebellum is more widespread
after exposure to TPP than Type I compounds. There is also degeneration in several
midbrain and forebrain regions not affected by TOTP (Fioroni et al., 1995).

FERRETS. Pathological damage after single subcutaneous exposure to 2 and 4
mg DFP/kg body weight occurs in the spinal cord, brainstem, and cerebellum of the ferret
(Tanaka et al., 1991). Degenerating axons are found in the gracile fasciculus at cervical
levels, dorsal spinocerebellar tract at cervical levels (spinoolivary, spinovestibular, and

spinoreticular pathways), the lateral corticospinal tract at lumbar levels, spinal cord

l9

laminae VI-VII, and ventral motor nucleus of the cervical enlargement. In the medulla,
degeneration is found in the ventral and lateral portions of the medial and dorsal accessory
nuclei of the inferior olive, lateral reticular formation, and inferior vestibular nucleus. In
the cerebellum, after exposure to DFP, degeneration occurs in the white matter and
granule cell layers of folia I-IV of the anterior lobe of the cerebellum (Tanaka et al.,
1991)

Ferrets that are exposed to a single subcutaneous dose of 500, 1000, or 2,000 mg
TPP/ kg body weight exhibit degeneration in the same regions affected by Type I OPIDN
compounds, as well as in additional regions throughout the spinal cord, medulla, midbrain,
and forebrain (Tanaka et al., 1990a). Additional spinal cord damage occurs in the medial
and lateral portions of spinal laminae VI-IX at cervical, thoracic, lumbar, and sacral levels,
and the lateral and ventral funiculi throughout the length of the cord. In the medulla,
degeneration occurs in the external cuneate nucleus, pontine gray, all nuclei of the
reticular formation, red nucleus, and lateral vestibular nucleus. Additional regions of
degeneration are found throughout the thalamus and hypothalamus, as well as forebrain
and brainstem nuclei tracts related to auditory (superior olivary nucleus and inferior
colliculus), visual (lateral geniculate nucleus and visual cortex), and sensorimotor systems
(ventral posterior thalamic nucleus, ventral lateral nucleus). In the cerebellum, additional
degeneration occurs in the granule cell layers of folia I-IX and crura I and II (Tanaka et

al,]990a)

20

Type III OPIDN

Triphenyl phosphine (TPPn) has been proposed to cause a third'type of OPIDN in
the domestic chicken with neurotoxic characteristics distinct from Types I and II (Abou-
Donia et al., 1996). The chemical properties of TPPn are listed in Table I. Abou-Donia et
al. (1996) reported that repeated oral administration of 500, 2,000, or 5,000 mg/kg body
weight TPPn produced OPIDN clinical signs of ataxia, paralysis, and death by four days
post-dosing. Neuropathological examination was confined only to the spinal cord where
degeneration was found in both ventral and lateral tracts. Chicken whole-brain AChE and
NTE activities afier a single oral dose of 500 mg TPPn/ kg body weight (Abou-Donia et

al,]996)

21

Table 1. Identification and physical properties of Triphenyl Phosphine

 

Chemical Family:
Component:
Chemical Description:

Trade names/Synonyms:

Molecular Formula:
Molecular Weight:
Boiling Point:
Melting Point:
Solubility in Water:
Solvent Solubility:

Vapor Pressure:
Vapor Density:
Specific Gravity:
PH:

Reactivity:

Decomposition:

Molecular Structure:

Phosphine compound, aromatic
Triphenyl phosphorus

Clear to off-white, triboluminescent
flakes with a characteristic odor
Triphenylphosphane, OHSZ4360
Triphenylphosphorus

C18-H15-P

262.30

711°F (377°C)

174-178°F (79-81°C)

Insoluble

Soluble in ether, benzene, chloroform,
carbon tetrachloride, acetone, glacial
acetic acid, slightly soluble in ethanol

5 mmHg @ 210°C
9.0

1.132

Slightly basic

Stable under normal temperatures and
pressures

Thermal decomposition products may
include toxic and hazardous oxides of
phosphorus and fumes of phosphine

O."—

 

(Occupational Health Services, Inc., 1995)

MATERIALS AND METHODS

Subjects

Twenty-four adult European ferrets (Mustela putoriusfirro) were used in this
study. The ferrets (642-1348 g) were maintained at the Michigan State University
Experimental Fur Farm. The ferrets were individually housed in cages (72.2 cm L x 61 cm
W x 45.7 cm H) suspended approximately two feet off of the ground in a screen-enclosed
pole-type building. The ferrets were exposed to ambient August temperature and

humidity with a natural photoperiod. Food and water were available ad libitum.

Test Chemicafi
Triphenylphosphine (C6H5)3P was purchased in crystalline fornr (100% pure) from
Aldrich Chemical Company Inc., Milwaukee,WI. The vehicle used consisted of a 1:1 ratio

of diethyl ether and peanut oil.

Experimental Desiga
Twenty-four ferrets were randomly assigned to one of three dose groups. Eight
ferrets were dosed with single subcutaneous injection of TPPn at 250mg/kg body weight

in the dorsum of the neck. Eight ferrets were dosed with single subcutaneous injection of

22

23

TPPn at 500mg/kg body weight in the dorsum of the neck. Eight ferrets were injected
with the diethyl ether/peanut oil vehicle at a volume of 2 kag body weight in the dorsum
of the neck.

Twenty-four hours after dosing, five ferrets from each dose group were killed by
cervical dislocation. The brains were rapidly removed, cut in half in the sagittal plane,
weighed and frozen on dry ice for subsequent NTE and AChE activity evaluation. NTE
activity was determined using the method of Johnson (1977). AChE was assayed
according to the method of Reed et al. (1966). Percent inhibition of both NTE and AChE
activities was calculated as a fiinction of the control activities. NTE and AChE activities
were analyzed statistically using ANOVA. Statements of significance are based on
p<0.05.

The remaining three ferrets per dose group were observed daily for six days for the
onset of clinical signs characteristic of acute toxicity or OPIDN. Evaluation of OPIDN
clinical signs was based on the following criteria: difficulty in holding the head erect, the
presence and degree of front and hind limb ataxia and front and hind limb rigidity. Each
animal was removed fiom the cage and placed on a flat open surface for approximately 10
minutes each day to qualitatively observe clinical signs.

To assess the extent of central nervous system degeneration, the remaining three
ferrets per dose group used for the clinical assessment were perfused for
neuropathological analysis six days after injection. Ferrets were administered a lethal
dose of sodium phenobarbital (150 mg/ml; 3 ml/animal intraperitoneal) and then perfused
transcardially with 10% formalin-saline solution. Just prior to perfiision with fixative, 1 ml

of heparin (168 USP units/ml physiological saline) was injected into the left ventricle.

24

Following perfiision, the brains were removed and placed in a 10% formalin-saline
solution for two days. Brain blocks were obtained and then cryoprotected by immersion
in a 30% sucrose-formalin solution. Afier cyroprotection, frozen sections were cut from
the brains in the parasagittal plane at a thickness of forty um. Every fifth section was
stained using the Fink-Heimer silver method to determine degeneration of cell bodies,
axons, and terminals (Tanaka, 1976). Adjacent sections were stained with cresyl violet to
delineate nuclear boundaries. All silver-impregnated and cresyl violet stained sections
were examined with a light microscope to determine the density and extent of
degeneration present. Selected corresponding Fink-Heimer and cresyl violet stained
sections were photographed at low power with a Wild M400 Photomacroskop.
Degenerated areas were mapped directly onto the photographs using a compound
microscope, and line drawings were traced from the photographs. Nuclei and fiber tracts
were identified according to the atlases of Adrianov and Mering (1959), Berman (1968),
Salazar et al. (1979), and Berman and Jones (1982). The severity of degeneration was

classified as light, moderate, or heavy.

RESULTS

Enzyme Activities

The effects of TPPn on whole-brain NTE and AChE activities 24 hours afier
dosing are presented in Table 2. The administration of 250 mg TPPn/kg body weight and
500 mg TPPn/kg body weight had no significant effect (p > 0.05) on whole-brain NTE

and AChE activities.

Clinical Signs

Ferrets dosed with the vehicle only did not exhibit any clinical signs characteristic
of OPIDN. Clinical signs in dosed ferrets were first apparent four days post-dosing. Both?
groups of dosed ferrets (250 mg/kg body weight TPPn and 500 mg/kg body weight TPPn)
exhibited clinical signs characteristic of OPIDN. These animals had difficulty in holding
their head steady and erect and they displayed slight hind limb ataxia. On the fifth day
post-dosing, both groups of animals exhibited front limb ataxia, extensor rigidity of the
hind limbs and increasing difficulty in maintaining the head erect. The animals moved by
use of the front limbs with the hind limbs being extended toward the rear of the animal.
Animals would often roll from one side to the other during forward locomotion. On the

sixth day post-dosing, animals in both groups were unable to hold their heads erect and

25

26

Table 2. Whole-brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE)
activities in European ferrets 24hr after exposure to triphenylphosphine (TPPn)

 

 

Dose* NTE Activity AChE Activity
Control 1841:1586M 207i10.6**
250 TPPn 1844i83.0 200:130

500 TPPn 1932:453 212:9.8

 

*Doses are expressed as mg TPPn/kg body weight

"Data presented as mean i standard error. NTE activity is expressed as nmoles phenyl
valerate hydrolyzed/ hr lgm brain. AChE activity is expressed as umoles ACh hydrolyzed/
hr / gm brain.

27

both front and hind limbs were extended and rigid. The observed clinical signs were
similar for animals in both the 250 and 500 mg TPPn/kg body weight dose groups.
Neuropatholagy

A broad pattern of terminal and axonal degeneration was similar for all nine
TPPn-exposed ferrets. Degeneration was not present in the brains of control ferrets.
Silver-impregnated degeneration appeared as black debris against a light brown or yellow
background. Fiber tract axonal degeneration appeared as linearly arranged black
fragments. Terminal degeneration in nuclear regions appeared as variably shaped punctate
debris. Artifacts present were silver impregnation of reticular fibers in blood vessels, and
random silver precipitation. Differentiation between degeneration and artifact was
achieved through the recognition of damage within known fiber tracts or nuclei, and the
continuity of degeneration in adjacent sagittal sections. Figures 1, 2, and 3 illustrate CNS
areas containing degeneration and areas that do not contain degeneration for comparative
purposes.

In the forebrain, degenerated areas included the neocortex, the hypothalamus,
basal ganglia, and the thalamus (Table 3, Figure 4 A,B,C). In the neocortex, areas of
degeneration were located in the visual and motor cortices of all TPPn-exposed ferrets
(Table 3, Figure 4 A,B,C). The ferrets had light degeneration in medial portions of the
visual and motor cortices, which progressed to moderate degeneration in the lateral
portions of these cortices (Figure 4 A,B,C). Two basal ganglia components contained
terminal and axonal degeneration. The fields of Forel contained moderate axonal

degeneration (Figure 1B). The entopeduncular nucleus contained light terminal

28

Figure 1. Darkfield photomicrographs illustrating axonal and terminal degeneration in the
primary visual cortex and thalamus. Under darkfield illumination, CNS regions containing
silver impregnated degenerating elements show up as bright areas against a light brown or
dark yellow background. A. Dense band of degeneration (arrowheads) located in lamina
IV of the primary visual cortex of a ferret six days after injection with 500mg TPPn/kg
body weight. B. Dense terminal degeneration located within the mediodorsal (MD),
ventral lateral (VL), and ventromedial (VM) thalamic nuclei of a ferret six days after
injection with 250mg TPPn/kg body weight. Scale bars = 1 mm. Figure abbreviations are

listed at the beginning of the text.

29

 

30

Figure 2. Photomicrographs illustrating Fink-Heimer silver impregnated regions showing
axonal and terminal degeneration in the thalamus of a ferret six days after injection with
250mg TPPn/kg body weight. Panel A illustrates dense degeneration in the lateral
geniculate nucleus (LGN) on the lefl side of the photomicrograph. Note the sharp
demarcation between the degeneration in the LGN and the absence of degeneration in the
immediately adjacent area to the right side of the photomicrograph. Panel B is a high
power view of the caudal part of the mediodorsal thalamic nucleus. Note the absence of
degeneration and the presence of light brown plump cell bodies (arrowheads). Panel C
shows a high power view of the ventromedial thalamic nucleus. Note the presence of fine
particulate black axonal and terminal degeneration as well as a black silver-impregnated
pyknotic degenerating cell body (arrow). Scale bars = 25 um. Scale bar in B applies to C.

Figure abbreviations are listed at the beginning of the text.

 

 

32

Figure 3. Photomicrographs through three laminae of the primary visual cortex showing
axonal and terminal degeneration in a ferret six days after injection with 250mg TPPn/kg
body weight. Panel A is from lamina III and contains almost no degeneration. Panel B is
from lamina IV and is the site of termination of degenerating axons fi'om the lateral
geniculate nucleus. Panel C illustrates degeneration axons passing through lamina VI on
their way to terminate in lamina IV. The black ovoid structures in Panels A and C are glial
cells. Scale bar = 25 um and applies to all panels. Figure abbreviations are listed at the

beginning of the text.

 

 

34

Table 3. Ferret central nervous system regions which contain axonal and/or terminal

degeneration after administration of TPPn

Forebrain
Neocortex
VCx

SMCx

Septum and Hypothalamus

MMn

Thalamus
MD
VM
VA
VL
LGN
MGN
CM
PAT
VPL
SUB

Basal Ganglia
EN

FF

Midbrain
SCI
ICC
PGL
PGM

Brainstem
Pons
LL

Tegmentum
Rn

TGn

Brainstem Nuclei/Tracts
F TG

ii

+++
+++
+++
+++
+++
++

++

+++
+++

++

++
+++
++
++

++
++

35

Table 3 Continued

 

CX ++

SOM ++

SOL ++

COn ++
Vestibular Nuclear Complex

VMN 4+

VSN ++

VLV ++
Cerebellum

Granular cell layer + to +++

Representation of degeneration density: + = light, ++ = moderate, +++ = dense
Structure abbreviations are found at the beginning of the text

36

Figure 4. Line drawings of parasagittal sections depicting the locations of axonal and
terminal degeneration (stippling) in nuclear regions of the brain in ferrets dosed with 250
or 500 TPPn mg/kg body weight. Drawings are from media] (A) to lateral (C). Structure

abbreviations are found at the beginning of the text.

38

Figure 5. Line drawings of parasagittal sections depicting the locations of axonal and
terminal degeneration (stippling) in nuclear regions of the brain in ferrets dosed with 250
or 500 TPPn mg/kg body weight. Drawings are from medial (D) to lateral (F). Structure

abbreviations are found at the beginning of the text.

39

 

 

/.~‘ ’ , ow I A-A “3‘1“..‘y‘ “'3

 
 
      

' ("Ewe W o t. s

.5
o

I,-.‘ .‘. '
' "’1'- .
r‘..:..:".‘" —" I I v.
‘- . .

4O

degeneration (Figure 4C). In the hypothalamus, degeneration was detected in the medial,
lateral and posterior parts of the medial mamillary nucleus (Table 3, Figure 4 A).

Several thalamic nuclei also contained degeneration (Figures 4 and 5). Moderate
terminal degeneration was found in the medial portion of the mediodorsal thalamic
nucleus. This progressed to dense degeneration in the lateral portions of the nucleus
(Figure 4 AB). Dense terminal degeneration was found in the ventral anterior, ventral
lateral, and ventral medial thalamic nuclei, as well as in the lateral geniculate and medial
geniculate nuclei (Figures 4, and 5 C,D,E). The central medial thalamic nucleus also
contained moderate terminal degeneration. A band of degenerating axons in the thalamus
was found oriented in the rostral-caudal direction. It was bounded by the central medial
thalamic nucleus on the dorsal side and fields of F orel on the ventral side (Figure 4 B).
The parataenial nucleus contained moderate terminal degeneration. The ventral posterior
lateral thalamic nucleus contained dense terminal degeneration. Finally, the subthalamic
nucleus contained dense terminal degeneration.

Moderate numbers of degenerating axons and terminals were found throughout the
midbrain in the areas of the intermediate lamina of the superior colliculus, central nucleus
of the inferior colliculus, lateral and medial paragigantocellular reticular nuclei,
tegmentum, and pons (Figure 4). A small region of the intermediate lamina of the superior
colliculus showed moderate degeneration. As more lateral areas of this nucleus were
examined, the intermediate lamina of the superior colliculus contained increasing numbers
of degenerating terminals. A band of degenerated axons was found in the nridbrain region
which was on the rostral border by the centrum medianum thalarrric and mediodorsal

thalamic nuclei and on the caudal border by the superior colliculus (Figure 4 AB) The

41

central nucleus of the inferior colliculus contained dense terminal degeneration in medial
regions of the nucleus that became less dense in more lateral regions. Both the medial and
lateral paragigantocellular reticular nuclei contained moderate terminal degeneration
(Figure 4 AB). The tegmentum of the midbrain contained degeneration in two separate
nuclei. The red nucleus contained light terminal degeneration in medial portions of the
nucleus and became more degenerated in lateral areas of the nucleus (Figure 4 AB). The
tegmental nucleus contained moderate terminal degeneration (Figure 4 A). A moderately
dense band of degenerating axons projected in a rostral-caudal direction at the rostral end
by the red nucleus and the caudal end by the tegmental nucleus. The other band of
degenerated axons was oriented in the rostral-caudal direction and was bordered by the
fields of Forel on the rostral side and the red nucleus on the caudal side. The lateral
lemniscus in the pons contained moderate axonal degeneration. This fiber tract formed a
band of degenerated axons that was oriented in the dorsal-ventral direction bordered on
the dorsal end by the inferior colliculus and the ventral end by the superior olive (Figure
4C).

In the hindbrain, both terminal and axonal degeneration could be found in the
vestibular nuclear complex, cerebellum, and several brainstem nuclei and tracts (Figures 4
and 5). Within the vestibular nuclear complex, three nuclei contained degeneration. The
medial vestibular nucleus (Figure 4 B), the superior vestibular, and the ventral division of
the lateral vestibular nucleus contained moderate terminal degeneration (Figure 4 C).
Several nuclei and fiber tracts within the brainstem displayed damage in the form of axonal
and terminal degeneration. The gigantocellular tegmental field contained a broad band of

light degenerating axons that extended from the rostral end of the tegmental nucleus to the.

42

caudal end of the external cuneate nucleus (Figure 4 A). The cochlear nucleus contained
moderate terminal degeneration (Figure 5 D). The external cuneate nucleus, which is
located on the caudal side of the medial vestibular nucleus, contained moderate terminal
degeneration. The lateral nucleus of the superior olive contained moderate terminal
degeneration that projected from the caudal end of the nucleus to form the lateral
lemniscus (Figure 4 C). The cerebellum contained varying degrees of degeneration
ranging from light in medial sections to heavy in more lateral sections. Within the
cerebellum, axonal degeneration was detected in the white matter and granule cell layer

underlying folia I-XI (Figures 4 and 5 A,B,C,D,E).

DISCUSSION

AChE Activity

Anticholinesterase activity depends largely on the chemical structure of the OP
compound that comes into contact with AChE. Inhibition of AChE is due to the
phosphorylation of the esteratic site (serine hydroxyl group) of AChE. OP
phosphorylation reactions are dependent on the reactivity of the central phosphorus atom,
which is determined by the electrophilic character of the groups attached to the
phosphorus atom (Timbrell, 1991; Chambers, 1992; Katzung and Trevor, 1995). The
presence of electronegative groups causes the phosphorus atom to become positively
charged making it more susceptible to nucleophilic attack (Eto, 1979; Davis and
Richardson, 1980). Triphenyl phosphine has no electronegative side groups so it would
not be expected that TPPn would inhibit AChE. Inhibition of AChE by an OP compound
is also dependent on the lability, stretching fiequencies, and hydrolysis rates of the
phosphorus-oxygen bond (Eto, 1979). TPPn, however, does not contain any phosphorus-

oxygen bonds thus making it an ineffective AChE inhibitor.

43

44

NTE Activity

Approximately 70% inhibition of whole-brain NTE activity after administration of
a single dose of an OP compound is thought to be required for subsequent expression of
OPIDN clinical signs and neuropathy (Richardson, 1992; Johnson, 1993). Several recent
studies contradict the 70% inhibition requirement in that OPIDN occurred when NTE
activities were inhibited by less than 70%.

Stumpf et al. (1989) reported OPIDN signs in ferrets dosed with various
concentrations of TOTP with no more than 47% NTE inhibition, while Larsen et al.
(1986) reported OPIDN clinical signs in TOTP-dosed turkeys with NTE inhibition as low
as 41%. Varghese et al. (1995) reported OPIDN clinical signs in quail after administration
of TPP at doses which resulted in minimal NTE inhibition (11%). Rats exposed to
multiple doses of TPP exhibited OPIDN clinical signs with NTE inhibition of only 33%
(Veronesi et al., 1986). Recently, Abou-Donia et al. (1996) reported OPIDN-like clinical
signs in chickens after exposure to triphenyl phosphine despite the absence of any NTE
inhibition.

It was previously discussed by Varghese et al. (1995) that apparent subthreshold
inhibition of NTE with subsequent development of OPIDN may be the result of
measurements being taken after the threshold value has been reached and the enzyme
actively is beginning to recover due to de novo synthesis. However, it is unlikely that
synthesis of NTE in amounts sufficient to bring activity up to control levels could have
taken place in the 24h period between dosing and harvesting of brains for NTE analysis in
the present study. Thus, it is apparent that threshold inhibition (in excess of 70%) of

NTE is not essential for the development of OPIDN, and the degree of NTE inhibition

45

after exposure to a neuropathic organophosphate compound does not appear to be an
unequivocal indicator of the compound’s ability to produce OPIDN. It’ must also be
reasoned, therefore, that NTE may not be the target esterase in the development of OP-
induced neurotoxicity but rather an esterase whose loss in activity is initiated by some
second messenger system after CNS degeneration has begun. It is also possible that
another (yet unknown) function of NTE, and not its esterase activity, is involved in the
initiation of axonal degeneration (Carrington, 1989). It would be of value to measure
other esterase activities after exposure to neurotoxic OP’s to assess their behavior in
relation to NTE activity.

These data also emphasize the erroneous classification of neuropathic OP’s into
Type 1 and Type II categories based on the degree of NTE inhibition. As previously
demonstrated, many neuropathic OP compounds exhibit variability in the degree of NTE
inhibition before the presentation of OPIDN in different susceptible species. Although the
chicken has consistently been susceptible to OPIDN only when NTE is inhibited by at least-
70% with both Type I and Type II OPIDN compounds, exposure to TPPn resulted in
OPIDN clinical signs without NTE inhibition (Abou-Donia et al., 1996). TPPn, therefore,
is an OP compound that produces OPIDN-like signs by some other mechanism than

inhibition of NTE in both the chicken and the ferret.

Clinical Sigr_rs
OPIDN clinical signs observed in the ferret after exposure to TPPn were

somewhat similar to clinical signs found after exposure to Type I OPIDN compounds, and

closely resembled clinical signs observed following exposure to the Type 11 compound

46

TPP. For example, subcutaneous injection in ferrets administered 250, 500, and 1000 mg
TOTP/kg body weight exhibited ataxia and flaccid paralysis of the hind limbs (Stumpf et
al., 1989). DFP, when administered subcutaneously at doses of 2 or 4 mg DFP/kg body
weight, produced ataxia and paralysis of only the hind limbs in ferrets (Tanaka et al.,
1991). Yet, ferrets subcutaneously exposed to 500, 1000, and 2000 mg TPP/kg body
weight exhibited ataxia, rigidity, and extensor rigidity of both fore- and hind limbs (Tanaka
et al., I990a). In the present study, single subcutaneous doses of TPPn resulted in clinical
signs of ataxia, paralysis, and extensor rigidity of both fore- and hind limbs in ferrets
similar to that observed in ferrets after exposure to TPP.

Differences in neuropathological damage patterns between Type I OPIDN
compounds and TPPn in the ferret may be due to differences in species selectivity or
dosage of chemical administered. When comparing Type I degeneration patterns with
TPPn degeneration patterns in the ferret, Type I OPIDN degeneration is confined to a
smaller number of brain regions. Type I OPIDN compounds would, therefore, be
expected to result in less motor control difficulties than TPPn, which is responsible for
widespread brain degeneration in the ferret.

The mechanism by which TPP causes deterioration of motor coordination in the
ferret is the same as that causing lack of motor control after exposure to TPPn in the
ferret. The TPPn-induced axonal and somatic degeneration found in several nuclei and
regions of the central nervous system involved in motor control (cerebellar cortex,
vestibular nuclei, ventral medial thalamic nuclei, primary cortex) effectively accounts for
the severe sensory and equilibrium deficits present in the affected animals. However, since

pathological examination has not yet involved the spinal cord or peripheral nerves, it is

47

possible that degeneration in these structures may have also contributed to the rapid onset
and severity of observed clinical signs.
Locus and Extent of CNS Degeneraaion

The data reported in this investigation show for the first time the distribution of
brain degeneration in the ferret after exposure to TPPn. Exposure to this compound
results in both axonal and terminal degeneration in many brain regions. The pattern of
degeneration caused by TPPn is more extensive than degeneration patterns induced by
Type I OPIDN compounds, yet very similar to patterns induced by TPP in the ferret.

Figure 6 is a comparison of axonal and terminal degeneration in the ferret central
nervous system after exposure to three different OPIDN compounds. Degeneration
patterns caused by TPP and TPPn are similar, while the degeneration pattern for DFP is
distinct. DF P degeneration in the ferret is much less widespread than degeneration
patterns for both TPP and TPPn. TPP- and TPPn-induced degeneration is extensive in
midbrain, forebrain, thalamic, and cerebral cortical areas involved in processing of visual,
auditory, and sensorimotor information. Both TPPn and TPP appear to cause comparable
damage to the spinal cord and sciatic nerves (ventral, lateral, and ventral-lateral tracts) in
the chicken, although TPPn-induced spinal cord and nerve degeneration has not yet been
examined in the ferret (Tanaka et al., l992b; Abou-Donia et al., 1996).
Neuropathological damage caused by DFP in the ferret is restricted to the brainstem and
cerebellum (Figure 6).

Exposure to TPP in ferrets results in degeneration patterns characteristic of Type I

degeneration in the brainstem and cerebellar regions, as well as additional degenerated

48

Figure 6. Summary schematics in the sagittal plane which compare qualitatively the extent
of degeneration in the brain of the ferret after a single exposure to DFP, TPP, or TPPn.
The location of degeneration was assessed using a modified F ink-Heimer silver
impregnation technique. Stippling represents areas of degeneration. Figure abbreviations

are listed at the beginning of the text.

 

 

 

 

 

 

 

 

Triphenyl Phosphine

50

areas throughout the medulla, midbrain, and forebrain (Figure 6).

Exposure of ferrets to TPPn results in degeneration patterns similar to TPP-
exposed ferret brains with additional damage occurring in the medial mammilary nucleus
of the hypothalamus, the ventral medial thalamic nucleus and medial geniculate nucleus of
the thalamus, and the superior colliculus of the midbrain. Yet, TPPn- induced
degeneration was not detected in the inferior olivary nucleus which does contain axonal
degeneration after exposure to TPP in the ferret (Figure 6).

Similar CNS regions are affected by all neurotoxic OP’s in the hen, ferret, and rat.
Both Type I and Type II OPIDN compounds cause pathogenic damage to tracts of the
spinal cord, some brainstem nuclei, and rostral portions of the cerebellum. These CNS
regions, therefore, are critical factors in assessing the neuropathic potential of an OP
regardless of Type I or Type 11 classification. The mechanisms underlying resistance and
susceptibility to OP’s by different neural systems or fiber tracts are not yet understood,
although it is clear that certain areas of the CNS seem to be affected preferentially and
selectively among susceptible species. This phenomena may be partially resolved by
examining the susceptibility of neurons before and after maturation of a neuron cell.

The results of this study indicate that damage in the ferret brain caused by TPPn is
much more widespread than damage caused by Type I compounds. The location and
extent of axonal and terminal degeneration in the ferret after exposure to Type I OPIDN
compounds does not involve rrridbrain and forebrain regions, and the extent of cerebellar
damage is minimal when compared to the effects of TPPn. This strongly suggests that the
pathogenic mechanism of Type I degeneration in the ferret are distinct from mechanisms

involved in TPPn degeneration. These differences, however, could be more discretely

51

defined by additionally obtaining patterns of TPPn-degeneration in the hen and rat to
compare to Type I OPIDN compounds within the same species.

Although neuropathological damage after exposure to TPPn is more severe and
widespread than damage after exposure to a Type 1 compound (DFP), damage is very
similar to that caused by Type 11 compounds in the ferret. The neural damage caused by
both TPP and TPPn is widespread, affecting regions not only associated with basic
sensorimotor activities, but those involved in higher order integrative and cognitive
functions as well. The absence of significant additional degenerated brain regions by TPPn
suggests that the mechanisms by which TPP and TPPn cause neurological damage are
similar and distinct from Type I OPIDN compounds. It would be of interest to obtain
neuropathological damage patterns at set time intervals after exposure to an OP in order
to obtain a chronological map of damage. This would indicate the order that brain regions
are affected by an OP and thus, hopefiilly, reveal its mechanism of action.

In conclusion, results based on clinical signs and neuropathology indicate that
TPPn induces OPIDN in the ferret despite the absence of NTE inhibition. The pattern of
degeneration, as well as observed clinical signs caused by TPPn, closely resembles those
of TPP in the ferret. The clinical signs and neuropathology caused by TPPn in the ferret
are not inconsistent with Type II OPIDN despite the lack of NTE involvement and does

not necessitate the creation of a Type III OPIDN.

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