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Developmental Neurotoxicology of Pre/Neonatal

Exposure to Domoic Acid & Effects on
Behavioral Outcomes

Melissa Chan
1328440

NBIO 403
Review Paper
Professor Philip Baker
March 14, 2017
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Abstract

Domoic acid (DomA) is a marine biotoxin found in commonly consumed shellfish. At high

concentrations, it is excitotoxic and causes amnesic shellfish poisoning (ASP), a neurological condition

characterized by confusion, temporary anterograde memory loss, seizures, and sometimes death. Historically,

research around DomA neurotoxicity has focused on adult animal models. However, more recent research

indicates that a long-term, low-dose exposure could also have adverse neurodevelopmental and behavioral

outcomes, particularly for children. Specifically, experimental studies with rodent models on pre/neonatal

exposure to low doses of DomA have found effects on the development of normal cognitive processes, such as

attention, adaptations to novel circumstances, anxiety levels, learning, and both working and temporal

memory. Histological analysis has also revealed the hippocampus as the primary site of damage. These

findings have global implications as events of DomA seafood contamination are increasing in scope and

frequency. They are also particularly salient to sensitive subpopulations, such as the Native American

communities along the Washington State coast who are subsistence harvesters of at-risk shellfish. Research in

this field is still relatively new, and the studies presented represent a first step towards addressing chronic, low-

dose DomA exposure as a growing public health concern.

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1. Background

1.1. Introduction

In November of 1987, hospitals in the New Brunswick and Quebec Canadian provinces received a

wave of patients presenting with rapid-onset confusion, disorientation, and temporary anterograde memory

loss (Costa et. al. 411). A total of 250 such cases were identified, with the most severe also including seizures,

coma, and four deaths (Costa et. al. 411). All symptoms were linked to recent consumption of mussels

harvested from the nearby Cardigan River (Grant et. al. 133). Further investigation revealed these mussels

were contaminated with domoic acid, a potent environmental neurotoxin, and the condition was named

amnesic shellfish poisoning (Costa et. al. 411).

This incident was the first to identify the connection between domoic acid (DomA) and the associated

memory and behavioral consequences of amnesic shellfish poisoning (ASP). It catalyzed active research into

the mechanism of DomA neurotoxicity and prompted marine scientists to further investigate the relationship

between ocean health and human illness (Grant et. al. 133). This led to the discovery of harmful algal blooms

(HABs) as a major source of marine biotoxins (Grant et. al. 133). DomA specifically was found to be produced

by the marine plankton Pseudo nitzschia (Costa et. al. 410). Shellfish such as razor clams, crabs, and mussels

become contaminated when they feed on these plankton.

Since then, research conducted by the National Office for Harmful Algal Blooms indicates that HAB

events are becoming more frequent (Grant et. al. 133). Prior to 1972, a retrospective study found no evidence

of DomA-producing blooms along the U.S. coasts (Grant et. al. 133). However, from 1972 to 2006, the coasts

have seen 20 documented incidents of DomA contamination in harvested seafood (Grant et. al. 133).

Additional sampling studies have also revealed the expansion of DomA contamination to ocean waters along

New Zealand, Mexico, and Ireland (Grant et. al. 133).

In response to the 1987 Canadian incident, the U.S. Food and Drug Administration developed

harvesting regulations for at-risk seafood. Harvesting bans are implemented when the human tolerable daily

intake (TDI), currently set at 20 g/g of shellfish tissue, is exceeded (Lefebvre et. al. 223). However,

nearly all the research that these regulations are based on aimed to protect adult consumers from the sudden,
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high-dose toxin exposures that cause ASP. More recent studies have begun to indicate that a long-term, low-

dose exposure could also have adverse neurodevelopmental and behavioral outcomes, particularly for children.

The increasing frequency of HAB events indicates that DomA contamination will become a greater

concern for commercial and recreational consumers as well as the global seafood industry in the near future.

However, contamination is currently a pressing issue for subsistence harvesters who rely on these seafood as

staple parts of their daily diets. Because of cultural traditions, populations like the Native American

communities along the Washington State coast regularly consume at-risk seafood, such as razor clams and

Dungeness crabs. This means that even under current FDA regulations, infants, children, and adults in these

communities are exposed to chronic, low-doses of DomA, thereby making them a vulnerable subpopulation for

adverse outcomes.

1.2. Mechanism of Domoic Acid Neurotoxicity

DomA is a cyclic amino acid that is structurally similar to kainic acid (KA) (Costa et. al. 413). It

produces exitotoxicity in neural networks by acting as a glutamate agonist, targeting and binding with high

affinity to both KA and AMPA glutamate receptors (Costa et. al. 413). This results in a cyclic release of

glutamate and rapid accumulation of intracellular Ca2+ that promotes glutathione (GSH) efflux, leading to

oxidative stress and neuronal necrosis (Costa et. al. 413).

Neuropathological studies on the four patients who died from ASP in 1987 found neuronal necrosis in

the hippocampus and amygdala where the target glutamate receptors are represented in high density (Costa et.

al. 411). The known roles of these regions in memory, decision making, and emotional reactions both provide

potential explanations for the symptoms of ASP and indicate the potential behavioral complications in children

exposed to chronic, low doses of DomA.

1.3. Pre/Neonatal Sensitivity

Studies with rat models found that fetal and neonatal rat pups were at least two orders of magnitude

more sensitive to DomA toxicity than adults (Costa et. al. 417). This is most likely due to the interaction of

multiple factors, including the greater susceptibility of the developing brain to neuronal necrosis from elevated
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oxidative stress and age-related changes in AMPA/KA glutamate receptor expression (Costa et. al. 413 and

Douchette et. al. 93). Additionally, DomA was detected in fetal rat pup amniotic fluid, as well as in maternal

milk even after it was no longer present in maternal urine (Costa et. al. 412). If these findings are generalizable

to humans, they indicate that infants, particularly those in subsistence harvesting communities, could continue

to be exposed to DomA even after the toxin has cleared the mothers system, thus contributing to a chronic,

low-dose exposure to DomA over a lifetime that begins even before birth (Angus 107).

While the heightened sensitivity of pre/neonatal animal models to DomA has been well established,

the specific effects of this sensitivity to chronic, low-doses of the toxin on neurodevelopment and behavior

have not. Recent research studies have focused on alterations in morphology of the developing hippocampus.

These early explorations have divided the field into prenatal and neonatal studies, both aiming to characterize

how DomA exposure at different developmental time points affects normal cognitive processes.

2. Summary of Current Research

2.1. Prenatal Exposure

In 2005, Levin et. al. used a battery of three behavioral tests to investigate the effects of prenatal

DomA exposure in rats, and found significant changes to response latency, locomotor activity, and working

memory (Levin et. al.). Prenatal exposure was achieved via subcutaneous injection of 23 pregnant rats on

gestation day (GD) 13, known to be a critical developmental time point for the fetal rat hippocampus. The 23

rats were divided into four dose concentration groups: 0, 0.3, 0.6, or 1.2 mg/kg.

After delivery, one male and one female pup from each litter (n = 10 or 12 for each dosing group, n =

46 total) were chosen at random for behavioral testing. Beginning in postnatal week (PNW) 4, pups were

tested on a T-maze to assess spontaneous alternation and latency. Results found no differences in percent

alternation between the 0 mg/kg control and the other three experimental dosing groups. However, there was a

significant increase in response latency when comparing the 1.2 mg/kg group to the 0 mg/kg group within the

second session only.

The pups were then tested on a Figure-8 maze to assess locomotor activity and habitation. Locomotor

activity was plotted as a linear trend to measure habituation, where developmentally-normal pups were
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expected to show an exponential decrease in activity over time. Researchers found this expected habituation

pattern for the 0, 0.3, and 0.6 mg/kg dosing groups, but the 1.2 mg/kg group had an increased rate of

habituation characterized by a higher initial locomotor activity level followed by a greater decline in activity.

Finally, pups were tested on an 8-arm radial maze to assess working memory. Re-entry into an arm

was considered a working memory error, and researchers recorded the number of consecutive entries into

different arms before a re-entry. Previous testing has established that rats normally display sex-related

differences on spatial memory tasks, with the males usually outperforming the females. However, researchers

found that while this was present across all four dosing groups, the male-female accuracy difference

significantly declined in a linear, dose-related manner.

Overall, the Levin et. al. study characterized significant changes in response latency and locomotor

activity for rat pups exposed prenatally to a single 1.2 mg/kg dose of DomA, as well as a dose-related

attenuation of normal sex-related differences in spatial working memory. The researchers results are supported

by a robust sample size for each dosing group. However, while it is explained that the four different dose

concentrations were designed to be below a level that would cause overt clinical symptoms in the pregnant

rats, it is not clear if the chosen concentrations are reflective of low-doses that would be consumed by humans.

Additionally, the researchers do not clearly discuss why they specifically chose the three behavioral tests used

or the translation of their findings to human behavior. Moreover, each test was conducted on both male and

female pups, but only data from the 8-arm radial maze was separated by sex. This leaves the reader unclear as

to whether the significant changes in response latency and locomotor activity on the T-maze and Figure-8 maze

respectively were seen in both or only one sex.

In 2009, Tanemura et. al. expanded on the preliminary findings of Levin et. al. with an additional

battery of four behavioral tests that characterized the effects of low-dose, prenatal DomA exposure at different

developmental time points (Tanemura et. al). Twenty pregnant mice were divided into four groups. Group A

served as control and received saline on all three GDs. Group B received DomA on GD 11.5, representing late

embryonic exposure. Group C received DomA on GD 14.5, representing early fetal exposure. And Group D

received DomA on GD 17.5, representing late fetal period exposure. After delivery, four male pups were

chosen from each litter for behavioral testing at PNW 11.

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First, pups were assessed with three behavioral tests to measure performance in adaptations for novel

circumstances: an open field test (OF), a light/dark transition test (LD), and an elevated plus maze test (EP). In

the OF, researchers found total distance traveled did not differ between Groups A-D. However, time spent in

the central area was significantly prolonged in Group D, indicating that prenatal DomA exposure during the

late fetal period impacted pup response to novel circumstances.

In the LD, latency till first movement from the dark to the brightly lit side, total number of movements

between sides, and time spent on each side were recorded. Researchers found that Group C spent more time in

the brightly lit side while latency till first movement to the brightly lit side was significantly shorter for Groups

C and D. These suggest that DomA exposure during the early and late fetal periods affect pup response to

novel environments.

The EP test measured anxiety levels. Total distance traveled and total time spent on open arms in

comparison to enclosed arms were recorded. Researchers found that mice in Groups B, C, and D had increased

distance traveled and spent more time in the open arms. This indicates that DomA exposure at both the

embryonic and fetal time points reduces anxiety levels.

Following these tests, pups were also tested on a contextual/cued fear conditioning paradigm (FC)

with tone and foot shock to assess impairments in learning and memory. Researchers found that Groups C and

D displayed significantly reduced freezing responses, suggesting that DomA exposure during fetal time points

cause impairments in learning and memory to noxious stimuli.

Finally, after completing all four behavioral tests, pup brains underwent immunohistochemical

analysis. Researchers found reduced immune-reactivity to myelin-associated glycoprotein (MAG), a marker

for myelin, in pup cortex for Groups B and C. They also found increased immune-reactivity for microtubule-

associated protein 2 (MAP2), a marker for neuronal dendrites, in both the cortex and lateral CA3 of the

hippocampus in Groups B, C, and D.

Overall, the Tanemura et. al study showed that neurobehavioral effects were more severe for early and

late fetal exposure to DomA. Their tests characterized significant impairments with adaptations to novel

circumstances and learning/memory deficits, while immunohistochemistry revealed a lack of myelination in

the cortex and over-growth of dendritic processes in the cortex and CA3 region of the hippocampus. The
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experimental design revealed an association between timing of exposure and severity of behavioral outcome.

However, researchers did not explain why they chose to only test male pups, leaving the reader to wonder if

their findings can be generalized to females as well.

Expanding on the morphological discoveries described by Tanemura et. al., a 1993 study by

Dakshinamurti et. al. looked specifically at hippocampal changes in postnatal mice pups after intrauterine

exposure to DomA, and again after a second postnatal injection of DomA (Dakshinamurti et. al). Intrauterine

exposure was delivered via intravenous injection of 0.6 mg/kg DomA or saline through the caudal vein of

pregnant mice at GD 13 (n=5 for each injection type). After delivery, five pups were randomly selected from

each litter for the study (n=50).

Basal EEGs were compared between prenatally exposed pups and control pups at postnatal days

(PND) 10, 20, and 30, which represent the known time frame for hippocampal layer differentiation in mice

pups. Across all measurements, there were no overt signs of clinical seizure, but basal EEGs showed that

exposed pups had significantly increased amplitude and decreased background frequencies at PND 20 and 30

in comparison to controls. The researchers then exposed both the experimental and control pups to a postnatal

dose of 0.6 mg/kg DomA. Control pup EEGs showed occasional spiking while experimental pup EEGs

showed a continuous discharge of spike bursts. Some experimental pups also developed generalized tonic-

clonic motor activity that lasted for 2.5-3 hrs.

Using a different set of control and experimental pups with intrauterine exposure to DomA, the

researchers also characterized relative GABA and glutamate levels, as well as the amount of Ca 2+ influx at

PND 30. Through neurochemical analysis, they found significantly reduced GABA, increased glutamate, and

increased Ca2+ levels, which together support the mechanism of DomA excitotoxicity described previously.

Interestingly, analysis of morphology in the hippocampus revealed no changes when pups were sacrificed on

PND 1, but showed significant granular cell damage in the CA3 and dentate gyrus regions when sacrificed on

PND 14. Additionally, analysis at PND 30 revealed damage to CA4.

Overall, Dakshinamurti et. al. demonstrated significantly altered basal EEGs in mice pups exposed

prenatally to DomA, as well as a lowered seizure threshold to an additional postnatal dose. Histological

changes also suggested that prenatal DomA exposure resulted in hippocampal damage that was progressive
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across PND 1-30. However, in their presentation of data on EEG recordings, the researchers were unclear

about which mice and how many mice were represented in the figures, detracting from the readers confidence

in the study findings.

2.2. Neonatal Exposure

Similarly to the prenatal exposure studies described, recent postnatal exposure studies seek to

characterize the effects on DomA on normal cognitive processes. However, most postnatal research explores

this by using DomA-treated rats as models for negative symptoms of human neuropsychiatric disorders,

namely schizophrenia.

In 2014, Marriott et. al. investigated latent inhibition in adult rats after previous postnatal exposure to

DomA (Marriott et. al.). Latent inhibition (LI) is a normal cognitive process of learned inattention to stimuli

that have shown no consequence in previous experiences. This ability to ignore irrelevant stimuli is disrupted

in schizophrenia, and has been previously modeled by social isolation rearing (SIR). To evaluate the effects of

DomA on LI in comparison to SIR on LI, researchers divided 94 rat pups into four treatment groups: saline-

group housed (SG), saline-single housed (SS), DomA-group housed (DG), and DomA-single housed (DS). All

pups were given daily subcutaneous injections of 20g/kg saline or DomA on PND 8-14. LI was assessed in

adulthood on PND 110 via a conditioned emotional response task, with Test 1 at 48 hrs and Test 2 at 1 week

after conditioning.

Researchers found that male and female rats had opposite results on Test 1. Male SG rats showed

strong LI, while DG and DS rats displayed no LI. However, female SG and SS had no LI while DG and DS

had strong LI. Results from Test 2 differed between sexes, but were also significant from Test 1. Here, male

SG, SS, and DG rats showed no LI while DS rats did. Female rats showed no LI across all four groups.

Overall, Marriott et. al. showed that sex, housing status, and DomA exposure affect LI, but that the

interaction between these factors is unclear and complex. Data from female rats is inconclusive across Tests 1

and 2, but male rats showed in general that postnatal exposure to DomA affects the normal development of LI.

This disruption was maintained across tests for DS, but not DG, suggesting that the effects of DomA exposure

are short-term while the effects of SIR are more long-term. However, the data itself presented very few
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statistically significant points to support these conclusions. The discussion cites apparent trends towards

significance, which could suggest that more data points would be significant if sample size was increased.

Further experiments are needed to demonstrate confidence in these findings.

Finally, in 2012, Robbins et. al. looked at the effects of postnatal DomA exposure on temporal

memory deficits, which are often present in schizophrenia and other neuropsychiatric disorders (Robbins et.

al.). Rat pups were treated with daily injections of either saline or DomA on PND 8-14 (n=20 for each

treatment). At 8-9 months of age, the adult rats were assessed for temporal memory deficits via a recency

discrimination paradigm. Researchers measured the number and duration of exploratory contacts towards the

remote object, as it has been established that rats with no temporal memory deficit display more exploratory

behavior towards the more remote object. Results showed that DomA-exposed females tested with the 96 hrs

delay made significantly fewer exploratory contacts towards the remote object, indicating temporal memory

dysfunction. However, this finding was not replicated in DomA-exposed males.

Overall, Robbins et. al. associated postnatal DomA exposure and delayed temporal memory deficits in

adult rats. However, the male-female sex differences in their results was even more extreme than that of the

Marriott et. al. study. This suggests a significant difference between male and female rat developmental time

lines that warrants further investigation before further experimentation.

3. Analysis & Contextualization

3.1. Implications

The studies summarized present preliminary findings on the effects of pre/neonatal DomA exposure

on the development of normal cognitive processes, such as attention, adaptations to novel circumstances,

anxiety levels, learning, and both working and temporal memory. Parallel histology analyses corroborate

previous studies showing localization of morphological changes in hippocampal areas CA1, CA3, and the

dentate gyrus. These findings indicate that pre/neonatal exposures to DomA can result in persisting behavioral

effects without overt clinical symptoms of toxicity. Considering this, the established heightened sensitivity of

pre/neonatal animal models to DomA suggests that the current FDA TDI level of 20 g/g of shellfish

tissue is not protective enough for sensitive subpopulations. Developed with adult animal models and normed
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on the standard 60 kg white male, this TDI level is not representative of children or Native American

communities, let alone children in Native American communities. Further research in both animal models and

epidemiological studies is necessary to develop a revised TDI.

3.2. Evaluation of Current Methods & Models

While these studies produced both statistically and clinically significant results, issues with the use of

rodents as model organisms, as well as the study designs detract from the translation of the results to human

populations. First, it is known that rodents have an inherent male-female performance difference on a variety

of behavioral tests, most likely due to different developmental time lines. Consequently, the Levin et. al.,

Marriott et. al., and Robbins et. al. studies all described significant sex-related differences in test results. One

of the goals with pre/neonatal DomA exposure research is to characterize the developmental time frame of

greatest vulnerability for exposure. Tanemura et. al. attempted to accomplish this. However, since the sex-

related developmental differences in rats are not well understood, the application of the groups results to

human developmental timelines cannot confidently be paralleled.

Second, the route of exposure used in these studies does not accurately represent methods of human

environmental exposure. All studies used either intraperitoneal, intravenous, or subcutaneous injections to

administer DomA, and all pregnant rodents in prenatal studies received only a single dose. These models

therefore represent the effects of acute, low-dose pre/neonatal exposure via injection, while all human

exposures to DomA are via oral consumption of contaminated shellfish. This is problematic both because the

relationship between DomA concentration and toxicity depends on exposure route, and because the current

public health concern is with chronic, low-dose exposure.

3.3. Future Directions

I believe that research into the neurodevelopmental and behavioral effects of chronic, low-dose DomA

exposure is important as both an immediate local and future global concern. Currently, the National Institute of

Environmental Health Science is conducting the first human epidemiological study on chronic, low-dose

DomA exposure with three Native American tribes along the Washington State coast for which DomA
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exposure is a pressing public health concern. As a collaborator on the basic sciences component of this

initiative, the research lab I am involved with studies the effects of chronic, low-dose prenatal exposure, and is

the first to use nonhuman primates with an oral exposure route. Our maternal generation is exposed before,

during, and after pregnancy to replicate a subsistence consumer environment. Doses are designed to represent

concentrations present in commonly consumed shellfish below the current TDI level. Using an animal model

with much greater developmental similarity to humans, along with a representative oral exposure route, our

study aims to further characterize the neurodevelopmental and behavioral effects of chronic, low-dose DomA

exposure with greater confidence in the application of our findings to human populations.

In conclusion, research on the effects of long-term, low-dose DomA exposure on pre/neonatal

neurodevelopment and behavior is still relatively new. Experiments with animal models have begun to link

exposure with progressive morphological changes and impairments of cognitive processes. However, further

studies are needed to fully characterize these in the context of human populations. Ultimately, the studies

described and future directions proposed represent the first step to addressing a public health concern that

currently disproportionally impacts subpopulations in our local Native American communities, and may

become a growing concern for our global community in the near future.
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