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TOXICOLOGIC PATHOLOGY ISSN:0192-6233 Volume 18, Number I (Part I), 1990

Copyright 1990 by the Society of Toxicologic Pathologists Printed in U.S.A.

Acute Neurotoxicity of Domoic Acid in the Rat*


LEANDER TRYPHONAS, JOHN TRUELOVE, EDWARDO NERA, AND FRANK IVERSON
Toxicology Research Division, Bureau of Chemical Safety, Food Directorate,
Health Protection Branch, Health and Welfare Canada, Ottawa, KiA OL2

ABSTRACT

A recent outbreak of human food poisoning, characterized by severe gastrointestinal and neurologic ab-
normalities, with a fatal outcome in 3 patients, was attributed to the consumption of poisonous mussels
containing domoic acid at an abnormally high concentration. The purpose of the present study was to
determine if domoic acid, a glutamate analogue extracted from poisonous mussel, was neurotoxic to rats.
Groups offemale Sprague-Dawley rats were dosed once intraperitoneally with 0, 1, 2, 4, or 7.5 mg domoic
acid/kg of body weight and observed for a maximum period of 24 hr. Clinically, control rats and rats in the
I mg/kg group were unremarkable. Seventy-five percent of the animals in the 2 mg/kg group had equivocal
transient behavioral signs. One that was given 2 mg/kg and all rats given in excess of 4 mg/kg of body weight
developed unequivocal behavioral and neurologic signs culminating in partial seizures and status epilepticus.
Histopathologically, severely affected rats developed selective encephalopathy characterized by neuronal
degeneration and vacuolation of the neuropil in the limbic and the olfactory systems, and retinopathy
characterized by neuronal hydropic degeneration of the inner nuclear layer and vacuolation of the external
plexiform layer. The results of this study suggest that domoic acid is excitotoxic and causes a characteristic
syndrome with clinical signs and histopathologic lesions similar to those reported for kainic acid.
Keywords. Dendrotoxic lesions; excitotoxins; gliotoxic lesions; hippocampus; neurotoxins; rodent; toxic
encephalopathy; toxic mussels

INTRODUCTION citation when applied iontophoretically and neu-


In the last quarter of 1987, approximately 175 ronal death when higher concentrations are applied
Canadians became moderately to seriously ill after (4, 25, 26). The anions of domoic acid (and quis-
consuming freshly harvested cultivated blue mus- qualic acid) were found to be at least 2 orders of
sels (Mytilus edulis L.) originating in eastern Prince magnitude more potent than L-glutamate, and equal
Edward Island. Clinically, early symptoms included to or stronger than kainate, when tested on rat spinal
nausea, vomiting, and disorientation. Irreversible motoneurons by microelectrophoresis and on frog
neurologic signs developed in 3 persons who even- spinal motoneurons by superfusion of the procaine-
tually died 2-3 weeks later. blocked hemisected spinal cord in vitro (2, 3). Kainic
The agent allegedly responsible for this outbreak acid has been extensively used as a means for se-
was domoic acid, known to be produced by the red lectively lesioning neuronal cell bodies while sparing
alga Chondria armata. The diatom Nitzschia pun- axons en passant (5).
gens, found in waters where mussels are cultivated, To determine the biologic effects and the dose
is now suspected to be the source of domoic acid in response curve of domoic acid present in contam-
this instance (6, 10, 15,20,27). Mussels associated inated toxic mussels, a number ofexperiments were
with the recent outbreak of marine food poisoning carried out in our laboratories using mice, rats, and
were found to contain domoic acid in excess of 800 monkeys (Cynomolgus fascicularis) (10). The pur-
ug/g (11, 15,27). Under normal conditions, domoic pose of the present report is to document the toxicity
acid has not been previously detected in mussels. of domoic acid in the rat using clinical aberrations
Domoic acid is structurally related to kainic acid, and histopathologic lesions as indices of neurotox-
which is a rigid analogue ofthe putative neurotrans- icity.
mitter L-glutamate and which causes neuronal ex- MATERIALS AND METHODS

* Address correspondence to: Dr. Leander Tryphonas, Pa- Animals


thology Section, SirFrederick G. Banting Research Centre, Health
Protection Branch, Tunney's Pasture, Ottawa, Ontario, Canada Seventeen female Sprague-Dawley rats weighing
KIA OU. 180 15 g were obtained from Charles River, S1.

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2 TRYPHONAS ET AL TOXICOLOGIC PATHOLOGY

TABLE I.-Summary of study design.


Observation
Dose Volume period
(mg/kg) (ml) No. of rats (hr)

1.0 2 4 24
2.0 2 4 6-24
4.0 2 4 4-6
7.5 2 1 2
0.0 2 4 24

Constant, Quebec. They were housed in group banks


in a humidity-, light-, and temperature-controlled
room, and maintained on laboratory chow (Purina
Rodent Laboratory Chow (R) #5001, St. Louis, MO). FIG. 1.- Rat treated with 4 mg domoic acid/kg ip. Emp-
Feed and water were provided ad libitum. ty mastication, salivation, and paw clasping were early
signs of impending seizure.
Chemical
Crude domoic acid (85%) used for the intraperi- increased tone oflimb muscles, and raising the body
toneal injections was isolated from toxic mussel ex- off the ground when walking.
tracts by the Food Research Division ofHealth Pro- Morphologic Evaluation
tection Branch, Health and Welfare Canada, using
repetitive preparative HPLC. Mussel extracts, pre- All rats were killed under deep chloroform an-
pared according to the method suggested for the esthesia by intracardiac perfusion with heparinized
bioassay of paralytic shellfish poison (l0) and con- lactated Ringer's solution, followed by 200 ml of
taining elevated levels of domoic acid, were used as fixative consisting of 2% glutaraldehyde and 1%
the starting material. paraformaldehyde in 0.1 M sodium cacodylate buff-
er, pH 7.2.
A complete necropsy was performed and the fol-
Dosage
lowing tissues were immersed in 10% neutral buff-
Rats were placed individually in clear plastic bins ered formalin for paraffin histologic processing: eyes,
and dosed once intraperitoneally with 0, 1, 2, 4, or optic nerves, olfactory bulbs, brain, and spinal cord
7.5 mg domoic acid/kg of body weight dissolved in with dorsal root ganglia. The brain and the thoracic
physiologic saline. The dose volume was adjusted and lumbar enlargements of the spinal cord were
with physiologic saline so that each rat received 2 sliced into blocks of approximately 2 mm thickness
ml (Table I). and embedded in paraffin. Sections were cut at ap-
proximately 6 and 10 11m thickness and stained with
Clinical Evaluation hematoxylin and eosin, luxol fast blue, Bodian, and
Immediately after the administration of domoic Nissl methods. Selected sections were stained with
acid, the rats were replaced in the clear plastic bins the PAS and Holzer's methods. Selected semithin
and observed continuously for the first 4 hr and sections were stained with toluidin blue.
intermittently for a maximum period of 24 hr. An- Total interneuronal vacuolar area in the 5 affected
imals that developed severe clinical signs were killed and 5 control rats was estimated morphometrically
earlier (Table I). Clinical signs were recorded as they in the hippocampus using a Zeiss lEAS 2 image
appeared. Complex clinical signs were grouped into analysis system interfaced to a Hamamatsu video
minor seizures, status epilepticus, and crab-like camera on a Zeiss Photomicroscope III. Areas were
walking- based on criteria established previously measured from CA3a and CA3b fields of the rostral
(19) and on our own observations. A minor seizure and caudal regions of the hippocampus.
was considered to take place when 1 or more of the RESULTS
following signs occurred intermittently: forelimb
tremor, head nodding, circling, loss of postural con- Clinical Findings
trol, mastication and profuse salivation, praying and Control rats and rats in the 1 mg/kg group were
rearing, scratching, and wet dog shakes. Status epi- unaffected. Three rats in the 2 mg/kg group devel-
lepticus included continuous occurrence of agita- oped transient clinical signs suggestive of central
tion, forelimb tremor, generalized tremor, praying nervous system disturbance. Soon after the intra-
and rearing with falling, and profuse salivation. Crab- peritoneal injection there was a period of inertia
like walking was defined as the combination of se- during which the rats remained motionless. Later,
vere kyphosis, tucked-up abdominal musculature, they developed moderate exploratory behavior, kept
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Vol. 18, No.1 (Part 1), 1990 DOMOIC ACID TOXICITY IN THE RAT 3

in apparently purposeless exploratory behavior, rats


in the 2 (one rat), 4, and 7.5 mg/kg groups developed
hunched posture and piloerection of the vibrissae,
reoccuring enophthalmos, occasional wet dog shakes,
waddling gait, and bilateral scratching of an area of
the skin extending from the caudal border of the
scapula to the lateral canthus of the eye by the ip-
silateral hind paw. Further, head nodding, protract-
ed chewing and salivation, rearing and/or "praying"
with forelimb extension and forepaw clasping or
clapping, generalized fine tremor, rigidity of move-
ment giving rise to a "crab-like" walking, loss of
postural control ending in rolling followed by sud-
FIG. 2.- Rat treated with 4 mg domoic acid/kg ip. Lum- den regaining of posture, circling, and occasional
bar kyphosis, raised body (crab-like walking), and partial jumping were superimposed clinical signs that ap-
enophthalmos with squinting appeared intermittently prior peared later (Figs. 1 and 2). Rats in a more advanced
to seizure. stage of disease also leaned against objects, sat on
their hind limbs and tail, and developed intermit-
their ear lobes pulled back, had occasional unilateral tent status epilepticus, gasping, and cyanosis fol-
scratching of the scapular region by the ipsilateral lowed by intervening periods of flaccidity (Fig. 3).
hind paw, and remained withdrawn for varying pe- At this point, severely affected animals were eu-
riods oftime but were fully recovered within 24 hr. thanatized. The specific type and the number of
In addition to becoming withdrawn and engaging signs seen were dose-dependent (Fig. 4).

FIG. 3.-Stages in the evolution of a typical seizure in a rat treated with 4 mg domoic acid/kg ip: A) Pulling back of
the ear lobes, roughened coat, and near normal posture; B) Praying (anterior part of body raised with the head extended,
mouth open, ear lobes pulled back, forelimbs extended, and front paws clasped); C) Sitting on hind legs and tail with
front paws tightly clasped; and D) Loss of balance with early temporary falling.
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4 TRYPHONAS ET AL TOXICOLOGIC PATHOLOGY

1mg Domoic/kg
CHEWING
DEATH
EXPLORING
HEAD JERKS
KYPHOSIS
REARING
SCRATCHING
SEIZURES
WIDE STANCE
WITHDRAWN

20 40
2 mg Domoic/kg
CHEWING
DEATH
EXPLORING
HEAD JERKS
KYPHOSIS
REARING
SCRATCHING
SEIZURES

WIDE STANCE
WITHDRAWN

20 40 60 80 100 120
Time
4mg Domoic/kg
CHEWING
DEATH


EXPLORING
HEAD JERKS


KYPHOSIS
REARING

SCRATCHING
SEIZURES


WIDE STANCE

WITHDRAWN

20 40 60 80 100 120
Time
7.5 mg Domoic/kg
CHEWING
DEATH

EXPLORING

HEAD JERKS
KYPHOSIS


REARING
SCRATCHING


SEIZURES
WIDE STANCE
WITHDRAWN

20 40 60 80 100 120 140


Time
FIG. 4.-Chronologic appearance and evolution ofcJinical signs in treated S-D rats: The time to onset for most cardinal
signs was inversely proportional to dose size. Control rats were unremarkable.

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Vol. 18, No.1 (Part 1), 1990 DOMOIC ACID TOXICITY IN THE RAT 5

TABLE n.-Distribution and frequency of cerebral and retinal domoic-acid-induced damage in Sprague-Dawley rats.
Domoic acid (mg/kg bodyweight)
4
2 7.5 0
Lesioned area (230) (48) (228) (229) (217) (47) (C)

Cerebrum
Amygdaloid complex
Bed. n. stria terminalis +
Medial amygdaloid n. + + +
Posterolat. cort. amyg. n. +
Posteromed. cort. amyg. n. +
Cortex
Cingulum + + + +
Entorrhinal cortex +
Frontoparietal cortex + +
Retrospl. Str. 18 cortex +
Hippocampus
CAl field + + + +
CA3 field + + + + + +
CA4 field + +
Subiculum + +
Tenia tecta (ant. hippoc.) +
Hypothalamus
Arcuate nucleus + + + + +
Lateral hypothalamic area +
Septohypothalamic n. +
Suprachiasmatic n. +
Olfactory system
Anterior olfactory n. + + +
Bed n. access. olf. tract +
Endopyriform n. + + +
Olf. bulb, ext. plex. layer +
Primary olfactory cortex + + + + +
Septum
Lat. septal n. ventralis + +
Eye
Retina
Inner nuclear area + + + +
Outer plexiform layer + + +
( ) rat identification number.

Gross Findings A common histologic finding in the brain of af-


Rats that developed advanced clinical signs had fected rats included: vacuolation in the neuropil,
unkempt fur and wet hair on the front limbs, around neuronal hyperchromasia and shrinkage with mul-
the mouth, over the ventral aspect of the neck and tiple indentations of the somatic profile, and astro-
abdomen, and at the perineal region due to excessive cytic swelling (Fig. 5). More advanced lesions in-
salivation and incontinence. No lesions were seen cluded neuronal necrosis (Fig. 6).
in treated rats that failed to develop clinical signs. The distribution pattern oflesions in cerebral nu-
Control rats were unremarkable. clei was noticeably different from that seen in struc-
tures possessing a cortical organization. In the for-
mer, lesions were diffuse and consisted of
Histopathologic Findings pronounced vacuolation of the neuropil, moderate
Control rats, as well as rats treated with 1 mg astrocytic swelling, and occasional neuronal hyper-
domoic acid/kg body weight, and 3 of 4 rats given chromasia and shrinkage. In the latter, the lesions
2 mg/kg were unremarkable. One rat given 2 mg/ had a laminar distribution. Thus, lesions ofthe pri-
kg and rats in the 2 higher treatment groups devel- mary olfactory cortex were prevalent in layers II and
oped acute selective encephalopathy and retinopa- III and only occasionally extended to deeper layers.
thy. The spinal cord was unaffected. The regional Lesions of the frontoparietal cortex were confined
distribution and frequency of tissue damage are to the three deepest layers. Their presence was be-
shown in Table II. trayed by a diminished staining of the affected area.

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6 TRYPHONAS ET AL TOXICOLOGIC PATHOLOGY

FIG. 6.-Moderate vacuolation of the neuropil and neu-


ronal necrosis in the arcuate nucleus of a domoic-acid-
treated S-D rat (7.5 mg/kg). H&E. x 100.

Ocular lesions were most often found in the outer


FIG. 5.-Typical domoic-acid-induced CNS changes 4 two-thirds of the inner nuclear layer and the inner
hr post-treatment. Pronounced vacuolation of the neu- half of the external plexiform layer (Table II). In the
ropil, neuronal shrinkage, multiple indentations of so- inner nuclear layer, affected bipolar and horizontal
matic profiles, and astrocytic swelling in the stratum py- cells had undergone hydropic degeneration and/or
ramidale of hippocampus field CA3. H&E. x 600.
necrosis (Fig. 8). Typically affected cells had mas-
sively enlarged cytoplasm which either was vacuous
Lesions in the hippocampus were peculiar in that and edematous or contained a scant amount of ve-
they were limited to the pyramidal cell layer ofCA3 siculated cellular debris. In addition to being en-
and, less often, CAI and CA4 zones, the outer region larged, the nuclei had margination of chromatin,
of the stratum oriens, and the stratum lacunosum were karyorrhectic, or had undergone pyknosis. In
moleculare. Dense microvacuolation and occasion- some instances, pyknotic nuclei were surrounded by
al swollen glial cells were the only lesions seen in a thin clear halo which in tum was bound by a thin
the stratum oriens near the alveus (Fig. 7A) and in membrane. In the outer plexiform layer, there was
the stratum lacunosum moleculare (Fig. 7B). In the moderate microvacuolation which most often oc-
pyramidal cell layer, the neurons and the proximal curred along the inner halfofits width. The vacuoles
end of their dendrites were darkened, had angular were round, occurred singly or in clusters, and only
or indented profiles, and were surrounded by vari- rarely contained a small amount of cellular debris.
ably sized vacuoles which at times were also seen
in the stratum lucidum (Fig. 7C). Amongst the dis-
DISCUSSION
tended vacuolar profiles, and occasionally just out-
side the pyramidal cell layer proper, there were swol- The results of this study indicate that a single
len astrocytes with hydropic cytoplasm and/or intraperitoneal administration ofdomoic acid to rats
nucleus. Hydropic astrocytic nuclei often were only at doses exceeding 2 mg/kg of body weight produces
represented by an ill-defined nuclear envelop. a well defined neurologic syndrome characterized
Unaided light microscopic examination revealed by scratching, "wet dog shakes," empty mastica-
that the severity of hippocampal lesions increased tions, salivation, generalized fine tremor, seizures,
in a rostrocaudal direction. Morphometric analysis and status epilepticus in that order. Its morphologic
offield CA3 changes demonstrated that vacuolation correlates include selective encephalopathy and ret-
of the neuropil was almost twice as extensive in the inopathy. Both the neurologic manifestations and
caudal part than in the rostral end ofthe hippocam- the neuropathologic lesions closely resemble those
pus. Changes in field CA3 of the hippocampus and reported for kainic acid, which is a well studied
in layers II and III of the primary olfactory cortex excitatory amino acid structurally related to domoic
were present in all rats that developed unequivocal acid and L-glutamate and which has a specific type
clinical signs of domoic acid toxicity (Table 11). of selectivity for CNS damage (13).

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Vol. 18, No. 1 (Part 1), 1990 DOMOIC ACID TOXICITY IN THE RAT 7

FIG. 8.-Retina of treated SoD rat (7.5 mg/kg). Mod-


erate hydropic degeneration and nuclear pyknosis in cells
of the inner nuclear layer and variably sized dilated pro-
files in the external plexiform layer. Toluidine blue. x 250.

limbic systems than was the case with domoic acid


(19). In the retina, kainic acid has a predilection for
the inner plexiform and the inner half of the inner
nuclear layers (13, 23). In contrast, domoic acid
caused severe edema and necrosis of the somata and
processes ofcells in the outer two-thirds of the inner
nuclear layer and vacuolation in the external plex-
iform layer. The reason for such differences is not
readily apparent, as neither the operant neurotrans-
mitter nor the distribution of domoic acid receptors
in the retina have been determined. However, do-
moic-acid-induced lesions seem to occur at the first
level of synapses of the visual pathway. The occur-
rence oflesions at this predominantly glutamatergic
site lends support to Olney's view that development
of lesions is mediated by glutamate (9, 16, 17).
In addition, the extent of damage reported in tar-
get sites common to both compounds was greater
in animals treated with kainic acid (17-19,24). Fur-
thermore, it was reported that kainic-acid-induced
lesions in the olfactory cortex and parts of the amyg-
daloid complex become more severe with the pas-
sage of time after treatment (19, 24). These differ-
FIG. 7.-Hippocampus ofa treated S-D rat (4 mg/kg). ences are difficult to reconcile at this time especially
A) Dilated profiles in the stratum pyramidale and dense because domoic acid is known to be at least 2 orders
microvacuolation and occasional swollen glial cells in the of magnitude more potent than L-glutamate and
outer zone of the stratum oriens in field CA I; B) Dense
equal to or stronger than kainate (I , 8). It is not clear
dilated profiles in the stratum lacunosum moleculare ; and
C) Dilat ed profiles are prominent in the strata pyramidal e whether these differences are due to drug specificity,
and lucidum of field CA3. H&E. x 250. to drug potency, or to the relatively shorter exposure
time allowed domoic acid animals in the present
experiment. Present scarcity ofdomoic acid has pre-
Despite the close qualitative similarity with kai- vented us from studying the effects ofa wider dosage
nic-acid-induced lesions reported in the literature, range. However, given the proven higher potency
there were notable differences in the distribution of of domoic acid and until further mechanistic ex-
target sites in the brain and the eyes between do- periments are carried out , it is reasonable to assume
moic-acid- and kainic-acid-treated rats. In the brain, that our rather short post-treatment period is the
kainic acid is known to damage more areas of the most likely reason for the apparent discrepancy.
cerebral cortex and more nuclei in the olfactory and The exact mechanism by which domoic acid ex-

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8 TRYPHONAS ET AL TOXICOWGIC PATHOLOGY

erts its deleterious effecton the neuron is not known. ferences in target site distribution and severity of
However, given the similarity with kainic-acid-in- damage between domoic acid and kainic acid tox-
duced lesions, the close structural resemblance to icity are compound specific or are due to differences
kainic acid, and the previously published results on in dosage and length ofexposure. It is also necessary
kainic acid toxicity (13, 19), it would be reasonable to establish whether domoic acid utilizes kainic acid
to suggest that the operant mechanism by which post-synaptic receptors or makes use of distinct re-
domoic acid is causing cerebral and ocular damage ceptors. Finally, it is important to ascertain the ex-
is like that of kainic acid. Originally, kainic acid tent of eNS damage produced by extracts of toxic
toxicity was thought to be mediated by an excess of mussels containing domoic acid.
synaptic glutamate. Presently, action of kainic acid
is hypothesized to be at a site on the synaptic mem-
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