Developmental toxicity of domoic

Jessica A. Tiedeken a, John S. Ram

Health

Charl

al Un

ity of

ge of

05; ac

ine 2

monkeys [13,32,35,34]. Various doses of DA are charac-

terized by stereotypic scratching, tremors, and tonicclonic

seizures [13,23,32,35]. Along with the behavioral symp-

reported to cause

publication furnished by NOS, in any advertising or sales promotion which

would indicate or imply that NOS approves, recommends, or endorses any

Neurotoxicology and Teratology 27proprietary product or proprietary material mentioned herein or which has

as its purpose any intent to cause directly or indirectly the advertised1. Introduction

Domoic acid (DA), a structural relative to kainic acid and

the neurotransmitter glutamate, activates AMPA and kainite

subtypes of the glutamate receptor family resulting in

excitotoxicity predominantly in brain tissues [24]. Produced

by the diatom genus Pseudo-nitzschia, DA is responsible

for poisonings in marine species as well as humans, known

as amnesic shellfish poison (ASP). Humans become

intoxicated through consumption of shellfish, which accu-

mulate DA by ingesting Pseudo-nitzschia [25]. Birds and

marine mammals receive effective DA doses through

ingestion of contaminated, planktivirous fish [12,17,

29,37]. With prior research focusing on behavioral and

neurological responses [8,23,32], the myriad of effects

exhibited by these organisms following DA exposure are

still being investigated.

Consistent with environmental effects observed in sea

lions [12], symptomatic responses to DA exposure have

been repeatedly documented in mice, rats, and cynomolgus

i Disclaimer notice: The National Ocean Service (NOS) does not

approve, recommend, or endorse any proprietary product or material

mentioned in this publication. No reference shall be made to NOS, or to thisdevelopmental toxicity of DA. Domoic acid was administered by microinjection to fertilized eggs at the 128- to 512-cell stages in

concentrations ranging from 0.12 to 17 mg/kg (DA/egg weight). DA reduced hatching success by 40% at 0.4 mg/kg and by more than 50% at

doses of 1.2 mg/kg and higher. Fifty percent of embryos treated with 1.2 mg/kg DA showed marked tonicclonic type convulsions at 2 days

post fertilization. Four days post fertilization (dpf), all embryos treated with 4.0 mg/kg DA and higher showed a complete absence of touch

response reflexes. Commencing 5 dpf, rapid and constant pectoral fin movements were observed, a response which may be related to the

hallmark effect in rodents of stereotypic scratching. These data indicate that zebrafish show symptoms of developmental DA toxicity as well

as a similar sensitivity comparable to the effects of DA characterized in laboratory rodents.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Domoic acid; Zebrafish; Glutamate; Development; Embryopotent neurotoxin in both adult and developing animals. We have usAbstract

Domoic acid (DA) is a rigid analog of the excitatory amino acid glutamate. It is produced by the diatom genus Pseudo-nitzschia and is a

ed zebrafish (Danio rerio) as a model to investigate and characterize theaMarine Biotoxins Program, Center for Coastal Environmental

219 Fort Johnson Rd.,bDepartment of Cell Biology and Anatomy, Medic

cDepartment of Cell and Developmental Biology and Anatomy, Univers

University of South Carolina Colle

Received 12 April 20

Available onl0892-0362/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ntt.2005.06.013

product to be us

* Corresponding author. Tel.:+1 843 762 8510; fax: +1 843 762 8700.

E-mail address: john.ramsdell@noaa.gov (J.S. Ramsdell).acid in zebrafish (Danio rerio)B

sdell a,*, Ann F. Ramsdell b,c

and Biomolecular Research, NOAA, National Ocean Service,

eston, SC 29412, USA

iversity of South Carolina, Charleston, SC, USA

South Carolina School of Medicine and Program In Womens Studies,

Liberal Arts, Columbia, SC, USA

cepted 12 May 2005

August 2005

(2005) 711 717

www.elsevier.com/locate/neuteratoms, domoic acid exposure has beened or purchased because of NOS publication.prolonged neuroexcitation and extensive degeneration in

brain tissues [24]. The hippocampus exhibits the most

gy andamage, with DA also affecting the septum and olfactory

bulb [6,24,28]. The hippocampal degeneration results in

both learning and memory deficits documented in DA

exposed humans and experimental animals [3,26,30,33].

Most of the published studies are conducted on mature

subjects, while the impact of DA exposure on development

has not been fully investigated. A few researchers have

described developmental effects of DA induced toxicity in

laboratory rodents. Dakshinamurti et al. [9] demonstrated

that intrauterine exposure of 0.6 mg DA/kg dam body

weight to the developing mice offspring induced age related

developmental neurotoxicity, with hippocampal necrosis

observable after just 30 postnatal days. Levin et al. [19] used

a similar exposure which lead to persisting neurobehavioral

effects at lower DA doses (0.3 mg/kg) where overt clinical

signs of toxicity are not present. Xi et al. [38] reported that

neonatal rats were substantially more susceptible to DA than

adults, which was proposed to result from insufficient renal

clearance of toxin, allowing increased bioavailability in the

blood. The maturation of renal function correlates with the

decrease in susceptibility to DA as a function of neonatal

age [10,38]. Neonatal rat exposure to DA, as passed from

the blood to the milk of lactating mother rats, exists, but at

levels appearing to be well below symptomatic doses [21].

Developmental effects in oviparous species including

frog (Xenopus sp), medaka (Oryzias latipes), and zebrafish

(Danio rerio) have been successfully studied through

embryo microinjection of substances ranging from aquatic

pollutants to neuroactive biotoxins to mRNA strands

[22,27]. This approach permits the use of minute amounts

of rare bioactive substances, accurate dose calibration, and

direct entry into specific tissues and cells. Microinjection of

medaka embryos has been used to characterize several

classes of algal toxins including ciguatoxins, brevetoxins,

azaspiracids, and microcystins [7,11,14,15]. Developmental

responses of the embryos are characteristic such that subtle

differences between metabolites of the same toxin can be

distinguished [5,4]. Because several of these classes of algal

toxins have bioaccumulation potential, medaka microinjec-

tion has been used to mimic the maternal transfer of algal

toxins along with characterizing developmental effects.

Domoic acid, in contrast, is rapidly eliminated without

metabolism in urine and hence has little bioaccumulation

potential [21,31]. Despite this rapid elimination, deleterious

developmental effects may still occur in these embryo

stages. By using microinjection, exact doses of DA can be

attributed directly to symptoms, and consistency between

embryos is solidified.

To study early developmental effects of DA, the zebra-

fish embryo (Danio rerio) was chosen as a test subject

because of its characterized rapid development, allowing

increased potential of effects before DA can be eliminated,

and its importance as a genetic study model. This report

provides an initial characterization of the developmental

J.A. Tiedeken et al. / Neurotoxicolo712toxicity of domoic acid in zebrafish embryos and presents a

model comparable to previously described rodent studies.2. Methods

2.1. Zebrafish

Fifty mixed sex wild type zebrafish (Danio rerio)

were obtained from Tideline Aquatics (Hanahan, SC).

Fish were kept on a 14 h light : 10 h dark cycle in an 80

L aquarium. Water quality was maintained with both a

Whisper 30 Power Filter and weekly 10% water changes

of 0.06 g/L Reef Crystalsi salt mix (Aquatic Ecosys-tems, Apopka, FL). Sodium bicarbonate solution was

added to stabilize pH levels between 7.5 and 8.5 at a

water temperature of 28 -C. Zebrafish were fed twicedaily with TetraMin Tropical Fish Flakes (Tetra, Blacks-

burg, VA) and afternoons before breeding, the diet was

supplemented with live Artemia. Trays of marbles with a

plastic plant in the center were placed on the bottom of

the cleaned tank to collect the fertilized eggs (embryos)

in the morning. The embryos were siphoned from the

marbles within the first 2 h of the light cycle, separated

and washed with sterile water.

2.2. Sample preparation and verification

Domoic acid (DA), along with all other reagents, was

purchased from Sigma Chemical (St. Louis, MO). The DA

was resuspended in sterile phosphate buffered saline (PBS)

to a stock concentration of 10 mg/mL. The stock was diluted

to 7.54, 2.38, 0.75, 0.24, and 0.075 Ag/AL in sterile PBS tocreate appropriate doses in 1.4 mg embryo wet weight.

Doses were chosen in half log steps around 4.0 mg/kg, a

common effective DA dose in developmental rodent

exposures.

To determine correct dosing, 2.4 nL samples of 2.38 Ag/AL DA concentrations were collected by microinjection into5 AL of PBS. These samples were taken before (n =4),between (n =2), and after (n =4) the embryo injections to

measure consistency of injection dose. The samples were

then diluted to a concentration of 110 pg/mL (DA/PBS) and

confirmed by domoic acid ELISA (Biosense, Norway).

Analysis of diluted samples produced values averaging

100T27 pg/mL. The variance was insignificant, proving thatthe microinjections were accurate and consistent throughout

the experiment.

2.3. Microinjection

Six hours post fertilization (pf), healthy embryos were

grouped in troughs imbedded in an agarose plate as

described in The Zebrafish Book [36]. The plates, one for

controls and one for DA injections, were filled with 12.5%

Hanks solution [36] until embryos were submerged and

observed using an Olympus S2X9 microscope. A pulled (P-

87; Sutter Instrument Co., Navato, CA) and bevelled (BV-

d Teratology 27 (2005) 71171710; Sutter Instrument Co., Navato, CA) aluminosilicate

filament micropipette (O.D. 1 mm; Sutter Instrument Co.,

embryos had hatched as well as embryos exposed to the

lowest dose (0.12 mg/kg) of DA; 61.5% of embryos

ogy anNavato, CA) was filled with a known DA concentration

using a microloader pipette tip and placed in a micro-

manipulator. A nitrogen gas pico-injector (Harvard Appa-

ratus, PLI-90, Holliston, MA) was calibrated to consistently

produce 2.4 nL of injection material. Starting with the sterile

PBS group (0 Ag DA/g), up to 30 embryos were injected inorder of increasing concentration for each dose. The

embryos were removed from the plates and transferred

individually to wells of a sterile 24-well plate (Corning Life

Sciences, Acton, MA) with 2 mL of sterile 12.5% Hanks

solution, which was gradually replaced with zebrafish

aquarium water in later stages. Twenty-four non-injected

embryos were used as a control for the vehicle-injected

eggs.

2.4. Fish development monitoring

Embryo plates were maintained at 25 -C under a 16 hlight : 8 h dark cycle. A stereomicroscope (Leica MZ 12)

with an ocular micrometer was utilized to conduct obser-

vations on the development of the fish embryos. Embryos

that did not survive 24 h into the experiment were excluded

from the study due to the possibility of outside forces

resulting in mortality. This mortality was constant through-

out all groups, even controls, and was determined to be

unrelated to DA dose. Embryo stages were correlated to

normal development at 28.5 -C using an equation asdescribed by Kimmel et al. [16]. Embryos were observed

daily for viability, hatching, movements, physical abnor-

malities, and heart rate (beats/min) after 2 dpf. Heart rate

was measured (using counter and 30 s timer) in chorion until

hatched where measurements were taken under sedation (5

and 6 dpf). Digital images were captured using a RGB

autoimagecam (MicroImage Video Systems Co. A209,

Boyertown, PA) mounted onto the microscope. Images

were enhanced using Image Pro Plus video frame grabbing

software (Media Cybernetics, Silver Spring, MD). Obser-

vations were concluded 6 dpf followed by euthanization

with a lethal concentration of MS 222 (Ethyl 3-amino-

benzoate methanesulfonate salt, Sigma Chemical, St. Louis,

MO).

2.5. Swimming behavior

In a repeat experiment, hatched larvae were kept to 11

dpf to test reflexive actions to mechanical stimuli. On

day 5 pf the larval zebrafish touch reflexes were tested

by gently placing a polished micropipette against the side

of the tail. Response to this action was noted and in

cases where burst of swimming resulted, duration was

recorded. Those embryos that had not yet hatched 7 dpf

were gently dechorionated using forceps to provide

enough subjects for the swimming study. On days 8

and 11 pf, the behavior of the swimming larvae was

J.A. Tiedeken et al. / Neurotoxicolmeasured in a specially designed well divided into 16

equal areas. A baseline swimming activity was measuredhatched from the 0.4 mg/kg DA treatment (Fig. 1). As the

dose of DA increased, the percentage of hatched embryos

continued to decrease with a low of 25% hatched at 12.6

mg/kg. The 16.8 mg/kg treatment had 30% hatched, an

increase resulting from fewer total viable embryos in the

treatment group at 5 dpf.

Most of the unhatched embryos from the higher DA

treated groups exhibited morphological effects, including

downward curvatures of the spinal column, swelling of the

pericardia, and jaw underdevelopment, which reduced

embryo survival. After dead embryos were removed (24

h post-injection), 100% of the controls, and 75% of the

DA dosed embryos, from 0.12 to 4.0 mg/kg, survived until

the experiment was terminated. In the 12.6 mg/kg DA

treatment group, 61% of the embryos survived, while in

the 16.8 mg/kg group only 43% survived (results not

shown). No critical time point was found to have increased

mortality; however, unhatched embryos past 5 dpf had

depleted yolk resources and were unable to feed while still

in the chorion. This inability to feed caused the embryos toby counting how many partitions were crossed in 1 min,

followed by vibrational stimulated swimming level in the

second minute, and a response swimming level in the

third minute. This testing protocol followed a similar one

that Levin et al. [18] used when testing chlorpyrifos

toxicity on larval zebrafish; however, the temperature was

maintained at 25 -C.

2.6. Analysis

Dunnetts multiple comparisons tests in addition to an

analysis of variance (JMPi statistical software, SASInstitute Inc., Cary, NC) were used to compare the responses

of embryos at each DA treatment dose to the responses of

both the embryos treated only with the vehicle, and the non-

injected embryos. Morphological and behavioral differences

were compared using the images captured with a Sony RGB

camera (DXC-390, Sony Corporation, Japan) and captured

with Image Pro Plus Software (Media Cybernetics, Silver

Spring, MD).

3. Results

3.1. Hatch rate and viability

Zebrafish embryos hatched sporadically between 90 to

120 h post-fertilization at 25 -C, corresponding tocalculated stages from Kimmel et al. [16]. No significant

difference was found between the non-injected controls

and vehicle injected controls in any of the parameters

measured. By day five observations, all viable control

d Teratology 27 (2005) 711717 713become malnourished and more susceptible to fungal

infections, resulting in mortality.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

0.00 0.12 0.40 1.26 4.00 12.60 16.80

Dose Concentration of DA (mg/kg)

Perc

enta

ge o

f via

ble

embr

yos

n=22 n=25

n=26

n=32

n=26

n=28 n=20

Fig. 1. Percent of viable zebrafish embryos hatched 5 days post fertilization (dpf) with respect to treated dose. Total number of embryos viable on 5 dpf is

expressed above group.

J.A. Tiedeken et al. / Neurotoxicology and Teratology 27 (2005) 7117177143.2. Cardiovascular effects

Zebrafish embryos were monitored for heart rate daily

from 2 to 6 dpf. Heart rate increased as the heart beat

became more prominent in both treated and nontreated

embryos between day 2 and 3 from an average of 125T7to 177T8 beats/min, which is a normal developmentalheart rate [16]. No significant differences (95% confi-

dence) in heart rate were observed between control and

DA treated embryos throughout the study (results not

shown).0

10

20

30

40

50

60

70

80

90

100

0.00 0.12 0.40 1.

Dose Concentrat

Perc

enta

ge o

f Em

bryo

s

n=22

n=26

n=32

n=22 n=26

n=29

n=25

Fig. 2. Percent of viable embryos convulsing ( ) 2 dpf; no convulsions were obs

touch response (swimming when gently prodded) ( ) on 5 dpf; touch response w

viable on 2 dpf and 5 dpf is expressed above the group.3.3. Neurotoxic effects

Neurotoxic effects, resembling tonicclonic convul-

sions, were evident in zebrafish embryos 2 dpf. Con-

vulsions were marked as a whole body contraction with a

shuddering motion. Each contraction lasted approximately

35 s, with the most frequent contractions occurring at

the 1.26 mg/kg dose. These convulsions were present in

37% percent of the embryos treated with 0.4 mg/kg and

52% of the embryos treated with 1.26 mg/kg DA. This

response then decreased to 29% in the 4.0 mg/kg26 4.00 12.60 16.80

ion of DA (mg/kg)

Touch response (5dpf)Convulsions (2dpf)

n=26 n=28 n=20

n=33

n=27

n=30 n=21

erved at doses below 0.4 mg/kg. Percent of hatched embryos that exhibited

as not observed at doses 4.0 mg/kg and higher. Total number of embryos

treatment and was only present in 13% of all higher

treatments (Fig. 2). This decrease in seizure activity

correlates to an absence of overall movement from the

embryos in higher doses.

3.4. Behavioral response

3.4.1. Automatism

The primary behavioral response observed in all DA

treated embryos was rapid and constant pectoral fin

movements, commencing at 5 dpf (Fig. 3). In contrast,

the control larvae exhibited sporadic pectoral fin move-

ments that were used to right themselves in a vertical

position. Embryos treated with 1.26 and 4.0 mg/kg DA

exhibited a hyperactive pectoral fin movement, even when

still in chorion, which was insufficient to right those

hatched into a vertical position. Those embryos injected

with high doses (12.6 and 16.8 mg/kg) also exhibited the

hyperactive pectoral fin behavior while the rest of the body

elicited no movements.

3.4.3. Swimming behavior

A similar lack of response in higher DA treatments was

noted in the swimming behavior study. Larvae at doses of

1.2 mg/kg and higher exhibited an inability to swim or

move. The few larvae that did show motility at treatments

above 1.2 mg/kg, exhibited tail paralysis and relied on

pectoral fin movements and body convulsions to produce

subtle motions. Subsequently, these larvae were unable to

cross well divisions and were excluded from the swimming

study. Once again there was no significant difference

between the 0.12 mg/kg DA dose and the controls; however,

the 0.4 mg/kg dose did show a significant decrease

( p =0.02) in motion, as measured by divisions crossed,

while still able to exhibit some normal swimming behaviors.

There was a significant increase of motion between the

baseline observation and the vibrational stimulated obser-

vation across all groups ( p =0.001), with the 0.4 mg/kg

embryos still exhibiting reduced motion.

4. Discussion

entra

n=32

a stere

J.A. Tiedeken et al. / Neurotoxicology and Teratology 27 (2005) 711717 7153.4.2. Touch response

The natural reflex of the larvae is to escape when

touched, a commonly tested sensory response. Control

larvae were able to sense and react to the probe, often before

being touched, whereas doses of 4.0 mg/kg and higher

elicited no movement, even when fish were touched

multiple times (Fig. 2). Those fish in the 0.4 mg/kg and

1.2 mg/kg treatment groups responded to the touch, but did

not swim as long (avg 0.75 s) as the controls. The average

duration of swimming response, (2.6T0.5 s) was notsignificantly different between the controls and 0.12 mg/

kg treatment larvae.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

0.00 0.12 0.40

Dose Conc

Perc

enta

ge o

f Em

bryo

s

5 dpf6 dpf

n=22

n=25

n=26

n=25

Fig. 3. Percent of zebrafish larvae exhibiting incessant pectoral fin motions,day ( ), and maintaining these levels throughout the rest of the experiment. Total n

one number present indicates the number that remained constant.1.26 4.00 12.60 16.80

tion of DA (mg/kg)

n=28

n=20

otypic response. This motion first appeared 5 dpf ( ), increasing on the 6thThis report provides the first examination of observable

DA toxicity on zebrafish development. These toxic effects

include reduced hatching, which may relate to the spinal

deformities observed. These deformities were prevalent in

all embryos that had not hatched by 5 dpf, however the

precise mechanism effect of DA is unresolved. It is possible

that DA may inhibit the hatching gland or directly cause the

downward spinal curvatures that may inhibit the embryo

from pushing out of the chorion. The other pronounced

effects of DA on the zebrafish embryos include uncontrolled

pectoral fin motions and tonicclonic like convulsions.

n=26 n=27 n=18umber of embryos viable on 5 and 6 dpf is expressed above the group; only

block gene expression. Zebrafish offer additional opportu-

nities for future toxicity studies with an increasing battery of

gy anAll of the zebrafish embryos and larvae (6 dpf) treated

with DA exhibited some rapid and constant pectoral fin

movements. These movements were observable in 100% of

embryos at 4.0 mg/kg and higher DA treatments, including

those still in the chorion. While possibly relating to typical

motions larvae use to position themselves, this particular

pectoral fin motion seemed to be uncontrolled by the

individual when compared to the control fish. An uncon-

trolled response, or automatism, observed in rodents treated

with DA is stereotypic scratching [32], which is comprised

of repetitive flexionextension of the hindlimbs directed

toward the head and neck [10]. This demonstrates that both

zebrafish and rodents exhibit a similar automatism in

appendages when dosed with DA.

Around the prim-22 zebrafish developmental stage (2

dpf), the DA treated embryos exhibited frequent tonic

clonic type convulsions. Baraban et al. [2] have recently

described clonus-like convulsions in zebrafish. Barabans

study also relates neurological patterns in zebrafish seizures

with those described in rodents and links the seizures to

glutamate receptors. As an analog of glutamate, domoic acid

has been shown to elicit a definitive convulsion response in

rodents [8]. These continuous tonicclonic convulsions are

also observed early in developmental rats, between 014

postnatal days (PND) [10]. Convulsions observed in this

study closely match those described by Baraban et al. [2]

and can be related to tonicclonic convulsions expressed in

postnatal rats.

The dose dependency of these convulsions and autom-

atisms also correlates well to developmental toxicity in the

rat [10,38]. Neonatal rats show an increased (40-fold)

sensitivity to domoic acid induced automatisms and seizures

at PND 05 [38] which decreases nearly 10-fold in

sensitivity between PND 0 (0.12 mg/kg) to PND 22 (1.06

mg/kg) [10]. The lowest observable effect level of toxicity

observed in zebrafish by egg microinjection occurs at 0.4

mg/kg, closely relating to effects observed by intraperitoneal

injection of PND 14 rats at that dose [10]. These

developmental effects indicate that zebrafish can exhibit a

rodent-comparable toxic response, both in types of

responses and dose dependency to domoic acid.

While physical symptoms are relatively easy to classify,

toxin exposure during development may have effects on

processes with symptoms manifesting later in life. Charac-

teristically, the effects are time sensitive and dependent on a

given developmental process as well as the expression of

potential targets (i.e., receptors and other signaling pro-

cesses) for toxicity. The midgestational period in rodents

(PND-13) has been examined for domoic acid toxicity by

various researchers [9,38]. Studies by Dakshinamurti et al.

[9] have indicated that exposure to sublethal doses of

domoic acid leads to changes in hippocampal structure by

PND-10 in the offspring. A recent related study has

indicated that persistent neurobehavioral responses occur

J.A. Tiedeken et al. / Neurotoxicolo716as the rodents mature to juveniles and adults, demonstrated

by maze trials [38]. Challenges of these adults with aneurobehavioral tests, along with the use of morpholino

oligonucleotides to characterize toxicity.

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Developmental toxicity of domoic acid in zebrafish (Danio rerio)IntroductionMethodsZebrafishSample preparation and verificationMicroinjectionFish development monitoringSwimming behaviorAnalysis

ResultsHatch rate and viabilityCardiovascular effectsNeurotoxic effectsBehavioral responseAutomatismTouch responseSwimming behavior

DiscussionReferences