Exposure to the Mycotoxin Zearalenone Impairs Embryonic Development in
Zebrafish
by
Nicole Sidebotham
A PROJECT
submitted to
Oregon State University
University Honors College
in partial fulfillment of
the requirements for the
degree of
Honors Baccalaureate of Science in Biochemistry and Biophysics
(Honors Scholar)
Presented May 14, 2015
Commencement June 2015
AN ABSTRACT OF THE THESIS OF
Nicole Sidebotham for the degree of Honors Baccalaureate of Science in
Biochemistry and Biophysics presented on May 14, 2015. Title: Exposure to the
Mycotoxin Zearalenone Impairs Embryonic Development in Zebrafish.
Abstract approved:
______________________________________________________
A. Morrie Craig
A study was conducted to evaluate the developmental toxicity of three commonly
detected mycotoxins in food (deoxynivalenol, patulin, and zearalenone) using the
zebrafish (Danio rerio) model. Mycotoxin exposures were carried out at five
concentrations (0.0064, 0.064, 0.64, 6.4, and 64 µM). Morphological and photomotor
screenings were conducted at 24 hours post fertilization (hpf) and five days post
fertilization (dpf) to assess the effects of mycotoxin exposure on developing zebrafish
embryos. No significant developmental toxicity was associated with embryonic
exposure to deoxynivalenol or patulin. Incubation with 64 µM zearalenone was
associated with significant incidences of mortality and adverse body morphology
endpoints. Significant hypoactivity was associated with the 6.4 and 64 µM
zearalenone exposures. Subsequently, a shuttle box assay was performed to determine
the effects of incubation with 0.64 µM zearalenone on learning and recall in adult
zebrafish, which were evaluated using active shock avoidance conditioning. Zebrafish
incubated with 0.64 µM zearalenone recollected shock avoidance training at a slower
rate than control fish, suggesting that embryonic zearalenone exposure may lead to
adverse developmental outcomes in adult zebrafish. These results indicate that
zearalenone may represent a developmental hazard, and that additional tests are
warranted to further characterize the impacts of zearalenone exposure.
Key Words: zebrafish, mycotoxins, zearalenone, development
Corresponding e-mail address: Sidebotn@onid.oregonstate.edu
©Copyright by Nicole Sidebotham
May 14, 2015
All Rights Reserved
Exposure to the Mycotoxin Zearalenone Impairs Embryonic Development in
Zebrafish
by
Nicole Sidebotham
A PROJECT
submitted to
Oregon State University
University Honors College
in partial fulfillment of
the requirements for the
degree of
Honors Baccalaureate of Science in Biochemistry and Biophysics
(Honors Scholar)
Presented May 14, 2015
Commencement June 2015
Honors Baccalaureate of Science in Biochemistry and Biophysics project of Nicole
Sidebotham presented on May 14, 2015.
APPROVED:
A. Morrie Craig, Mentor, representing the Department of Biomedical Sciences,
College of Veterinary Medicine
Robert Tanguay, Committee Member, representing the Department of Environmental
and Molecular Toxicology, College of Agricultural Sciences
Jennifer Duringer, Committee Member, representing the Department of
Environmental and Molecular Toxicology, College of Agricultural Sciences
Toni Doolen, Dean, University Honors College
I understand that my project will become part of the permanent collection of Oregon
State University, University Honors College. My signature below authorizes release
of my project to any reader upon request.
Nicole Sidebotham, Author
Introduction
Mycotoxins – secondary metabolites produced by fungi residing in our food
supply – have been associated with a number of adverse health effects. The severity
of disease caused by mycotoxin ingestion is dependent on concentration route and
length of exposure. These compounds are capable of producing both acute and
chronic health effects which range in severity from athlete’s foot to invasive
aspergillosis. Symptoms of mycotoxin exposure include the alteration of blood
protein values, increased sensitivity to bacterial infections, and inhibition of particle
clearance from the lungs (De Boevre et. al., 2015). Both neurological and
developmental effects from mycotoxin exposure have been reported (De Boevre et.
al., 2015); however, the mechanisms that cause these toxic responses have not been
fully developed.
Autism spectrum disorder (ASD) covers a wide range of symptoms and levels
of neurological impairment, including persistent deficits in communication and social
interaction, repetitive behavior patterns, and restricted interests or activities (Geier et.
al., 2010). Individuals on the autism spectrum may be only mildly impaired by their
symptoms (and so be highly functional), or may be severely disabled by their
symptoms and require extensive care and intervention throughout their lives (Geier et.
al., 2010). The prevalence of ASD in the United States and the United Kingdom has
increased greatly in the last few decades (roughly 11.3 out of 1000 people) (MariBauset et. al., 2014); however, the causes for this increase in ASD are still unknown.
In part, awareness of the signs and symptoms of ASD and more defined assessment
metrics by pediatricians and psychologists has contributed to an increase in the rate of
diagnosis (Mari-Bauset et. al., 2014). Many proposed single gene causes are not
convincing, but several factors, including those in the mother’s (in utero) and child’s
environment, may dovetail to further explain the increased incidence of ASD. For
example, environmental exposures to compounds such as lead, mercury, PCB’s,
pesticides, and air pollutants have all been associated with an increased risk for
developing ASD (Geier et. al., 2010); however, no study has yet been conducted to
research the potential role of mycotoxins in developmental diseases. Mycotoxins are
ubiquitous in our environment in both food and air, and have been associated with
developmental deficiencies such as neural tube defects in the past (Missmer et. al.,
2006).
This project was designed to evaluate the developmental toxicity of some of
the most predominantly detected mycotoxins in food using the zebrafish (Danio
rerio) model. The zebrafish model is ideal for this study because it allows access to
all developmental stages over a short period of time and on a high-throughput scale
(Truong et. al., 2014). Therefore, the effects of mycotoxins on the development of
zebrafish embryos can be dissected for specific pathologies in a statistically
meaningful way. Further, the zebrafish genome has already been sequenced, so any
genetic disturbances caused by mycotoxin exposure can be illuminated (Truong et.
al., 2014). Many of the developmentally expressed genes in zebrafish are conserved,
and analogous regions in the developing mammalian brain have been identified
(Nishimura et. al., 2015).
In vivo studies yield improved prediction of how an organism will respond to
environmental conditions; however, these studies often require elaborate facilities and
infrastructure (Truong et. al., 2014). The zebrafish model is able to overcome these
limitations, and as a result has become a powerful organism for in vivo toxicity
studies. There is also great similarity in cellular structure, signaling processes,
anatomy, and physiology between zebrafish and other vertebrates, particularly during
early development (Truong et. al., 2014). Zebrafish have also been shown to exhibit
complex neurobehaviors such as startle responses, and can absorb a wide range of
chemicals from the water (Nishimura et. al., 2015).
Mycotoxins Studied and Their Mechanism of Toxicity
Deoxynivalenol
Deoxynivalenol (DON, C15H20O6, MW = 296.32 g/mol) has been shown to
affect the central nervous system through activation of the mitogen-activated protein
kinase signaling pathway and alteration of gene expression for key physiological
functions including the regulation of glycogen, sugar, and lipid metabolism (Ghareeb
et. al., 2015). Increased levels of serotonin and irritation of the gastrointestinal tract
due to DON toxicity result in reduced food intake, thereby decreasing average daily
gain in livestock. Current restrictions on DON intake are 1part per million (ppm) for
humans, 10 ppm for poultry and feedlot cattle, 5 ppm for dogs and cats, and 2 ppm
for dairy cattle (Ghareeb et. al., 2015).
Patulin
Patulin (C7H6O4, MW = 154.12 g/mol) is produced from a variety of molds
and is most commonly found in rotting apples. Patulin in soluble in organic solvents,
and is heat stable (Frizzell et. al., 2014). Patulin has been shown to be primarily toxic
through its affinity to sulfhydroxyl groups, which results in enzyme inhibition
(Frizzell et. al., 2014). Symptoms of patulin toxicity include gastrointestinal
problems, neurotoxicity (convulsions), pulmonary congestion, and edema. Patulin has
also been shown to induce reproductive toxicity and disruption of the endocrine
system in livestock (Frizzell et. al., 2014). Currently, no human or livestock limits
exist for this mycotoxin in food.
Zebrafish embryos have been used to investigate the developmental toxicity of
citrinin and patulin. Gross kidney morphology was not altered in the presence of
citrinin or patulin; however, histological analysis of embryos treated with citrinin
revealed cystic glomerular and tubular legions (Wu et. al., 2012). Disorganized renal
cell arrangement was also found in the group treated with patulin. Lastly, dextran
clearance abilities of embryos from both the citrinin and patulin groups were found to
be significantly reduced.
Zearalenone
Zearalenone (C18H22O5, MW = 318.36 g/mol) is an estrogenic metabolite
which is found in cereal crops such as barley, oats, wheat, and rice, and is produced
by some Fusarium and Gibberella species (Hueza et. al., 2014). Zearalenone has
been shown to permeate through human skin, and its toxicity can cause reproductive
effects including infertility, abortion, and other breeding problems in livestock
(Heuza et. al., 2014). Past work has shown that zearalenone also causes hepatotoxic,
hematoxic, immunotoxic, and genotoxic effects in domestic and laboratory animals
(Zinedine et. al., 2007). The World Health Organization Joint Expert Committee on
Food Additives (JECFA) has established a provisional maximum tolerable daily
intake for zearalenone of 0.5 µg/kg of body weight (ESFA, 2011).
Previous research on the effects of zearalenone in humans has demonstrated
that crops contaminated with zearalenone (in varying concentrations) are associated
with acute and chronic poisoning and carcinoma (Klaric et. al., 2009, Li et. al., 1999).
One study determined the incidence of nine different mycotoxins in 37 samples of
cereal and feed randomly collected from households of an endemic nephropathy area
in Croatia in 2007. They determined that zearalenone was the most frequent of the
mycotoxins, contaminating 92% of the samples, with a mean concentration of 318.3
µg/kg using thin-layer chromatography (Klaric et. al., 2009). The levels of
mycotoxins, including zearalenone, were below the permissible limit for animal feed;
however, 29% of the cereals were contaminated with fumonisins, ochratoxin A, or
zearalenone in mass fractions above the permissible limit for humans. The author also
showed that co-occurrence of the mycotoxins varied, and suggested that prolonged
exposure to zearalenone may increase the risk for various chronic diseases (Klaric et.
al., 2009).
Another study used GC-MS, HPLC, and enzyme-linked immunosorbant
assays to determine the concentrations of deoxynivalenol, nivalenol, zearalenone, and
fumonisins in two corn powder samples that were implicated in the human food
poisoning incident in the Guangxi province of China in 1989, and eight wheat and
two barley samples linked to 130,000 cases of gastrointestinal disorder in the Anhui
province in China in 1991 (Li et. al., 1999). Zearalenone was found in all of the corn
and barley samples at a low relative concentration, as well as six of the eight wheat
samples used for this study. The highest level of zearalenone was found in one wheat
samples associated with a case of human gastrointestinal disorder in the Anhui
province (Li et. al., 1999). The author concluded that red-mold infestations that
produced these mycotoxins led to increased incidences of food poisoning in these
provinces in China.
Previous research on the developmental effects of zearalenone on zebrafish
has shown that the effects of early zearalenone exposure are concentration dependent.
In zebrafish larvae exposed to 500 µg/L and above (relatively high concentrations
compared to naturally occurring concentrations in food) defects in heart and eye
development and upward curvature of the body axis were observed (Bakos et. al.,
2014). Another study found that zearalenone metabolites preferentially activated the
zebrafish estrogen receptor (zfER) alpha, which provided specific and convenient in
vitro tools for comparison and assessment of selective activation of zfERs by
chemicals within zebrafish liver cells (Cosnefroy et. al., 2012).
Another study explored continuous, long-term zearalenone exposure as well
as transgenerational effects of parental short term exposure on offspring, and showed
that zebrafish exposure to zearalenone does not affect fertility, hatch, embryo
survival, or gonad morphology of the zebrafish. However, lifelong exposure to
zearalenone for 140 days increased net weight, body length, and condition factor of
female fish at 1000 ng/L (slightly higher that naturally occurring concentrations in
food), and the sex ratio was shifted towards females from 320-1000 ng/L (Schwartz
et. al., 2010). The study also indicated an influence of parental exposure to
zearalenone on the reproductive performance of the offpring. Another group used a
recombinant yeast estrogen screen to show that zearalenone had a strong effect on
induction of plasma vitellogenin and reduced spawning frequency in zebrafish at a
concentration of 3200 ng/L (Schwartz et. al., 2013). The authors concluded that
zearalenone may contribute to the overall increased estrogenic activity detected in the
environment, which poses a threat for wild fish.
The Zebrafish Model
Possible applications of the zebrafish model to aid in modeling human
neuropsychiatric and drug-induced syndromes was investigated recently (Kalueff et.
al., 2014). The study showed that both larval and adult zebrafish can be used to help
increase our understanding of brain function and dysfunction (such as depression,
autism spectrum disorder, psychoses, and drug abuse), and developed a number of
behavior tests to illustrate how common neurological disorders can be modeled and
studied in zebrafish.
Previous research conducted at the Sinnhuber Aquatic Research Laboratory
(SARL) has shown that active avoidance response in adult zebrafish is a very
effective model of learning, and, when combined with automated shuttle box testing,
provides a highly efficient platform for evaluating persistent neurotoxic hazards from
a number of different chemicals (Truong et. al., 2014).
While there have been studies conducted examining possible environmental
factors that contribute to autism spectrum disorder, no study has yet investigated the
potential role the mycotoxins deoxynivalenol, zearalenone, and patulin may play in
this and other developmental diseases. Thus, the purpose of this study was to
determine whether embryonic exposure to deoxynivalenol, patulin, or zearalenone
leads to impaired development in zebrafish. We hypothesized that embryonic
exposure to these mycotoxins would lead to increased incidences of physical
malformations and developmental delays in zebrafish embryos and adult zebrafish.
The photomotor response, touch response, body morphology endpoint, and adult
learning assessments were used in this study in the hope that they would provide
insights into the mechanism of neurodevelopmental toxicity of these mycotoxins.
Materials and Methods
Electrospray ionization (+) mass spectrometry was used to verify the
concentration of the three toxins deoxynivalenol, zearalenone, and patulin (Sulyok
et.al., 2007). Brifly, each of the three mycotoxins was obtained from Sigma-Aldrich
(St. Louis, MO) and diluted to a concentration of 20 mM in DMSO. Mycotoxin
concentrations were checked using a standard curve created using mycotoxin of
known concentration.
All remaining data was collected at the Sinnhuber Aquatic Research
Laboratory (SARL), Oregon State University in Corvallis, Oregon.
Tropical 5D wildtype adult zebrafish were housed at a density of 1000 fish per
100 gallon tank at SARL. Each tank was maintained at 28°C, and a 14 hour light, 10
hour dark photoperiod in fish water. The fish water consisted of reverse osmosis
water supplemented with a commercially available salt from Instant Ocean (Truong
et. al., 2014). Spawning funnels were placed into the tanks one night prior to embryo
collection (Truong et. al., 2014).
Calculation of EC50 for mycotoxins detected in food. A major goal of this
study was to determine the full concentration response to each of the input chemicals
through calculating the No Observed Adverse Effect Level (NOAEL) and the
effective concentration to produce toxic effects in 100% of the fish (EC100) for each
produced endpoint (morphological and behavioral) (Truong et. al., 2013). The
NOAEL and EC100 concentrations were defined in the 5D tropical wild type strain of
zebrafish.
At 4 hours post fertilization, zebrafish embryo chorions were removed
enzymatically via pronase (63.6 mg/ml, Sigma-Aldrich: P5147) and the embryos
were transferred to individual wells of a 96 well plate with 100 uL of mycotoxin
dilutions at the following concentrations: 0.0064 µM, 0.064 µM, 0.64 µM, 6.4 µM,
and 64 µM, as well as a control group incubated with 0 µM zearalenone. All
mycotoxin exposures were initiated 6 hpf with dechorionated embryos, and were
sustained continuously at 28°C until 120 hpf (Truong et.al., 2013).
Embryonic development assessment
Morphological responses. Eighteen different body morphology endpoints
were assessed at five days post fertilization (dpf) from high resolution digital images
as follows: yolk sac edema (YSE), bent body axis (AXIS), eye defect (EYE), snout
malformed (SNOU), jaw malformed (JAW), ear malformed (OTIC), pericardial
edema (PE), brain malformed (BRAI), muscle somite defect (SOMI), caudal fin
defect (CFIN), pectoral fin(s) abnormal (PFIN), circulation slower/faster or less
prevalent than normal (CIRC), pigmentation abnormal (PIG), body trunk length
shorter than normal (TRUN), swim bladder not present or not inflated (SWIM),
notochord curvy or otherwise abnormal (NC), and response to blunt probe gently
touched to head or tail region without characteristic escape response (TR) (Truong et.
al., 2013). Because the morphology endpoints are binary, 0/1 responses for each
chemical-endpoint-concentration-replicate translate to a series (n = 32) of “coinflips”, so the significance threshold was estimated using a binomial test (Truong et.
al., 2014). The imaging system was based on a Nikon Eclipse Ti inverted scope with
a fully motorized, PC-controlled stage. The software imaged 96 wells in less than
three minutes, and automatically uploaded the images to a 100TB server for archiving
and recalled them via barcode identifier for visual scoring of the 18 morphology
endpoints. The system also captured video to assess cardiac endpoints.
Spontaneous tail flexion. Measurement of this variable between 18 and 24 hpf gave
an easily quantifiable and robust indicator of central nervous system deficit (Truong
et. al., 2014). Tail flexions for three minutes from 96 wells were quantified with a
Cognex machine vision camera/ double-telecentric lens system controlled by in-house
software scripts. This system was developed to calculate the difference in binarized
pixels between successive frames via distortion-free imaging of the entire plate.
Photomotor response. Defined as a change in swim activity in response to a sudden
light-dark transition, this test provided a robust measure of central nervous system
deficits by five dpf (Truong et. al., 2014). Four Zebrabox (ViewPoint Life Sciences,
Montreal, Canada) systems tracked zebrafish response digitally in the 96 well plate of
the five dpf larvae. This experiment used a regimen of 10 minutes in visible light
followed by three minutes in dark (IR light) followed by three minutes in visible
light. A test was deemed significant when the incidence of toxicity exceeded the
significance threshold (as defined above) over the control incidence rate.
Adult learning assessment
Behavioral responses. As described above, tropical 5D wildtype zebrafish
were housed in fish water at a temperature of 28°C; embryos were collected and
subjected to the same screening assays at 24 hours and five days (Truong et. al.,
2013). At 120 hpf, 52 control larvae and 61 phenotypically normal larvae incubated
with 0.64 uM zearalenone were reared to adulthood, to approximately three months of
age (Truong et. al. 2014). At this time, all fish were evaluated for adult behavior
testing using the shuttle box assay.
Shuttle box assay. Adult zebrafish were evaluated for the rate at which they
learn an active avoidance response using an automated shuttle box assay (Truong et.
al., 2014). Briefly, the shuttle box hardware consisted of an opaque shuttle box with a
length, width, and depth of 200 x 100 x 90 cm. A central divider was placed with a 10
mm gap between the floor of the box and the bottom of the divider. Water depth in
the shuttle box was 3.5 cm. LED light bars were placed at each end of the shuttle box
to generate the conditioned stimulus. Mild electric shock was used as the
unconditioned stimulus. Stainless steel plates covering each end of the shuttle box
were used to establish an electrical potential to create the shock. Two sensors were
placed to track the motion of the fish through the shuttle box.
Three month old zebrafish were used for the experiments. Each adult
zebrafish was subjected to a series of 20 training trials followed by five testing trials
of active avoidance conditioning. For the training trials, a 10 minute acclimation
period was allowed after first introducing the fish to the shuttle box. An eight second
avoidance period then took place in which the side the fish was on at the end of the
acclimation period became dark, while the opposite side received a dim white light.
At the end of the avoidance period, a 16 second shock period began. If the fish never
swam to the light side of the chamber, the entire box received a 20 V shock for the
full 16 seconds. If the fish swam to the light side of the chamber the shock was
terminated. Each trial was followed by a one minute resting period, where no side of
the shuttle box received a shock and the entire box received a dim white light. Then a
new trial began, with the non-shocked side of the shuttle box always being the side
opposite the fish’s location at the end of the last resting period. A humane ‘fault out’
limitation was programmed into the shuttle box control so that eight consecutive trials
of a fish never swimming to the non-shocked side would automatically halt the fish’s
testing. After eight consecutive trials where a shock was never delivered, the task was
considered mastered and the fish’s testing was stopped. After the training trials, the
fish were placed back into their tank for 30 minutes, then subjected to five testing
trials using the same procedure as the training trials.
The 20 training trials were designed to train the fish to swim to the lighted
chamber of the shuttle box against their natural instinct. The five testing trials were
designed to see how well the fish could recall the information they had learned during
the training trials after the 30 minute resting period.
Figure 1 The features of a single shuttle box unit with on-board thru-beam interrupt
detection, microprocessing and pulse width-modulated shock delivery (Truong et. al.,
2014).
Results
Mycotoxin dilution calculations. The concentrations of mycotoxin were
determined to be 20 ± 0.43 mM for zearelenone, 20 ± 0.96 mM for deoxynivalenol,
and 20 ± 0.55 mM for patulin.
Mycotoxin exposure. No significant developmental toxicity was associated
with embryonic exposure to deoxynivalenol or patulin using the 18 morphological
endpoints (figure 2, p < 0.05). Significant hyperactivity was associated with exposure
to 64 µM patulin in the 24 hpf photomotor assay (figure 3, p < 0.02).
Significant mortality was associated with exposure to 64 µM zearalenone by
24 hpf (95% of fish, figure 2). Additional mortality was recorded in the 6.4 µM
exposure group at 120 hpf (65% of fish). Exposure to 64 µM zearalenone was
associated with significant incidences of adverse outcome in the majority of the 120
hpf endpoints (p < 0.05). Significant hypoactivity was associated with 6.4 µM and 64
µM zearalenone exposure in the 24 hpf photomotor assay (figure 3).
Figure 3 The photomotor assay monitored zebrafish response to light
(movement per time) using a Cognex machine vision camera/ doubletelecentric lens system controlled by in-house software scripts. Significant
hypoactivity was seen at 6.4 and 64 µM zearlenone, as demonstrated by the
flat baseline compared to the control group.
Shuttle box assay. Control fish were able to learn a simple active avoidance
task at a significantly higher rate than fish incubated with 0.64 µM zearalenone from
six hpf to five dpf (P < 0.001). Figure 4 shows the ability of the control group and the
zearalenone group to learn active shock avoidance for the 20 training trials. The slope
for the zearalenone group was -0.029, while the slope for the control group was 0.155. This demonstrated that the control group had a greater decrease in seconds
shocked per trial than the zearlenone group, which suggests that the zearalenone
group was not able to learn active shock avoidance at the same rate as the control
group.
Unfortunately, statistically significant data was not obtained for the five
testing trials. Too large of a variation within each group prevented us from seeing a
statistically significant difference between the control fish and fish that had been
incubated with 0.64 µM zearalenone from six hpf to five dpf. The conditioning
protocol of the shuttle box assay could perhaps be changed to yield less variable
results for the five testing trials.
Discussion
Mycotoxins are toxins produced by fungi, and are extremely pervasive in both
the human and animal food supply. We are constantly being exposed to these toxins
in our daily lives; however, this is typically in very minute amounts (De Boevre et.
al., 2015). This project was designed to evaluate the developmental toxicity of some
of the most predominantly detected mycotoxins in food (deoxynivalenol, patulin, and
zearalenone) using the zebrafish model. Deoxynivalenol has been shown to affect
central nervous system development, primarily through activation of the mitogenactivated protein kinase signaling pathway (Ghareeb et. al., 2015). Patulin is a
neurotoxin compound which can cause convulsions, pulomary congestion, and edema
(Frizzell et. al., 2014). Zearalenone is an estrogenic metabolite which can cause
abortion, infertility, and other breeding problems (Hueza et. al., 2014). While the
effects of exposure to these mycotoxins have been studied, their mechanisms of
toxicity have not yet been fully elucidated. The zebrafish model is ideal for this
project because of similarity in cellular structure, signaling processes, anatomy, and
physiology between zebrafish and other vertebrates, particularly during early
development. Therefore, the goal of this study was to provide insight into how
exposure to these mycotoxins affects early stages of development.
Embryonic exposure to the mycotoxins deoxynivalenol and patulin did not
produce any significant toxic effects in larval zebrafish (p < 0.05). Thus, it appears
that these two compounds do not pose a significant developmental toxicity risk.
However, embryonic exposure to the mycotoxin zearlenone at 6.4 µM and 64 µM
was associated with increased incidences of physical malformations, developmental
delays, and hypoactivity in larval zebrafish (figures 2 and 3). This suggests that
embryonic exposure to zearalenone impairs the ability of zebrafish to develop
properly. In particular, yolk sac edema, curved body axis, a malformed snout, a
malformed jaw, pericardial edema, and pectoral and caudal fin abnormalities were
observed in zebrafish incubated with 64 µM zearalenone from six hpf to five dpf.
Zearalenone is an estrogenic metabolite which is found in cereal crops such as
barley, oats, wheat, and rice and is produced by the fungus Fusarium graminearum
(Hueza et. al., 2014). Zearalenone toxicity has been shown to cause infertility,
abortion, and other breeding problems in mammals (Zinedine et. al., 2007). The
World Health Organization Joint Expert Committee on Food Additives (JECFA)
established a maximum tolerable daily intake for zearalenone of 0.5 µg/kg of body
weight in 2000 (ESFA, 2011). Incidences of zearalenone poisoning have been seen at
a concentration of 318.3 µg/kg in Croatia (Klaric et. al., 2009). Research on
zearalenone toxicity in livestock showed that blastocysts removed at 14 days after
breeding from sows fed 1 mg/kg body weight zearalenone from seven to 10 days after
breeding were fragmented and contained foci of necrosis, suggesting that exposure to
zearalenone had detrimental effects to blastocyst development (Diekman et. al.,
1989). Another study using Sprague-Dawley rats showed that maternal zearalenone
exposure led to dose-related decreases in maternal feed consumption and body weight
gain, as well as a decrease in fetal body weight and delayed fetal development
(Collins et. al., 2006). Fetal viability was also significantly decreased at 8 mg/kg
body weight, leading the authors to believe that maternal exposure to zearalenone
produces both fetotoxic and teratogenic responses in the offspring (Collins et. al.,
2006). Another study using swine oocytes determined that exposure to 3.12 µM
zearalenone during maturation reduced the percentage of oocytes that cleaved and
formed a blastocyst to 12%, as compared to 25% in the control group (Malekinejad
et. al., 2007). Oocyte exposure to zearalenone also led to increased incidences of
aneuploidy, and abnormal spindle formation, suggesting that exposure to zearelenone
may lead to less fertile oocytes and abnormal embryo development in pigs
(Malekinejad et. al., 2007).
Based on these results, zearalenone was chosen for further study to assess its
effects on adult learning. Previous research on the adult learning behavior of
zebrafish determined that aversively motivated operant conditioning, such as shock
avoidance, gives the researcher more experimental control over the application of the
reinforcer than appetitively motivated assays (Blaser et. al., 2014) The shuttle box
assay was designed to determine the learning behavior of adult zebrafish by testing
the ability of three month old individuals to learn an active shock avoidance response.
Previous research at SARL has shown the importance of vehicle choice in accurately
assessing adult learning behavior using the shuttle box assay (Truong et. al., 2014).
This study was the first time that SARL has used the shuttle box assay commercially;
therefore, another goal of this experiment was to test the efficiency of this tool.
Embryonic exposure to the mycotoxin zearalenone at 0.64 µM from six hpf to
five dpf was associated with a significant decrease in the ability of adult zebrafish to
learn a simple active avoidance task, as compared to a control group (figure 4). This
suggests that embryonic exposure to 0.64 µM zearalenone impairs the learning ability
of adult zebrafish, perhaps due to disturbances to the brain during embryonic
development caused by mycotoxin exposure.
During pregnancy, embryos are continuously exposed to contaminants from
the mother’s diet. Maternal exposure to the mycotoxin zearalenone may have
detrimental effects on the developing embryo, such as decreased weight gain, delayed
developmental progression, and physical abnormalities (Chan-Hon-Tong et. al.,
2013); therefore, embryonic exposure to zearalenone may contribute to adverse body
morphologies, developmental delays, and/or hypoactivity. In vitro screenings have
also identified zearalenone as a substrate of the placental BCRP/ABCG2 transporter,
which is responsible for shuttling chemicals across the placenta (Xiao et. al., 2015).
This suggests that maternal exposure to zearalenone may have detrimental effects on
the developing embryo, as zearalenone ingested by the mother is able to cross the
placental barrier and exert toxic effects on the embryo.
Research has shown that both larval and adult zebrafish can be used to help
increase our understanding of brain function and disfunction (such as depression,
autism, psychoses, and drug abuse); a number of behavior tests have been developed
to illustrate how common neurological disorders can be molded and studied in
zebrafish (Kalueff et. al., 2014). Based on our results, the effects of embryonic
exposure to zearalenone on the social behavior of zebrafish should be studied in
further tests. Zebrafish are a schooling fish that travel and huddle in large groups, so
social behavior can be easily tested. The SARL group has already developed assays to
test social behavior in zebrafish, making this a logical next step.
Through discovering the mechanisms of toxicity that mycotoxins use to exert
their developmental effects, information can be gained that can be used in more
specific studies to investigate the role of mycotoxins in developmental diseases such
as autism spectrum disorder and other neurological disabilities. Investigating the
mechanisms through which these toxins exert their effects will provide a better
understanding of how autism spectrum disorder and other developmental diseases
come to be. The correlation between embryonic exposure to zearalenone and
increased incidences of physical abnormalities and delayed development suggests that
more careful monitoring of our food supply for mycotoxins is needed to reduce the
environmental hazard that mycotoxins pose to humans and livestock.
Acknowledgements
I would like to acknowledge the Oregon Agricultural Experiment Station
(ORE00871), USDA-NIFA and the Oregon State University DeLoach work
scholarship for providing funding for this project. I would also like to acknowledge
the department of Environmental and Molecular Toxicology at Oregon State
University for granting me the opportunity to complete this research. Lastly, I would
like to express my gratitude for the help and support of all of the personnel at SARL,
including Dr. Robert Tanguay and Dr. Michael Simonich, and everyone at the Craig
lab, especially Dr. A. Morrie Craig and Dr. Jennifer Duringer.
References
Bakos, Katalin, Rober Kovacs et. al. (2013) Developmental toxicity and estrogenic
potency of zearalenone in zebrafish (Danio rerio). Aquatic Toxicology 136,
13-21.
Blaser, R.E., and D.G. Vira (2014). Experiments on learning in zebrafish (Danio
rerio): A promising model of neurocognitive function. Neuroscience and
Behavioral Reviews 42, 224-231.
Chan-Hon-Tong, Anne, Marie-Aline Charles, Anne Forhan, Barbara Heude, and
Veronique Sirot (2013). Exposure to food contaminants during pregnancy.
Science of the Total Environment 27(35), 458-460.
Collins, TF, RL Sprando, TN Black, N Olejnik, RM Eppley, HZ Alam, J Rorie, and
DI Ruggles. Effects of zearalenone on in utero development in rats. Food
Chemical Toxicology 44(9), 1455-1465.
Cosnefroy, Anne, Brion Francois et. al. (2012) Selective Activation of Zebrafish
Estrogen Receptor Subtypes by Chemicals by Using Stable Reporter Gene
Assay Developed in a Zebrafish Liver Cell Line. Toxicological Sciences
125(2), 439-449.
De Boevre, Marthe, Kinga Graniczkowska, and Sarah De Saeger (2015). Metabolism
of modified mycotoxins studied through in vitro and in vivo models: An
overview. Toxicology Letters 233(1), 24-28.
Diekman, MA and GG Long. Blastocyst development on days 10 or 14 after
consumption of zearalenone by sows on days 7 to 10 after breeding (1989).
American Journal of Veterinary Research 50(8), 1224-1227.
European Food Safety Authority Panel on Contaminants in the Food Chain (2011).
Scientific Opinion on the risks for public health related to the presense of
zearalenone in food. ESFA Journal 9(6), 2197-2321.
Frizzell, Caroline, Christopher T. Elliot, and Lisa Connolly (2014). Effects of the
mycotoxin patulin at the levelof nuclear receptor transcriptional activity and
steroidogenesis in vitro. Toxicology Letters 229 (2), 366-373.
Geier, David A., Janet K. Kern, and Mark R. Geier (2010). The biological basis of
autism spectrum disorders: Understanding causation and treatment by clinical
geneticists. ACTA Neurobiologiae Experimentalis 70(2), 209-226.
Ghareeb, Khaled, Wageha A. Awad, Josef Boehm, and Qendrim Zebeli (2015).
Impacts of the feed contaminant deoxynivalenol on the intestine of
monogastric aniamls: poultry and swine. Journal of Applied Toxicology 35(4),
327-337.
Hueza, Isis M., Paulo Cesar F. Raspantini, Leonila Ester R. Raspantini, Andreia O.
Latorre, and Silvana L. Gorniak (2014). Zearalenone, an Estrogenic
Mycotoxin, Is an Immunotoxic Compound. Toxins 6(3), 1080-1095.
Kalueff, Allan V., Adam Michael Stewart and Robert Gerlai (2014). Zebrafish as an
emerging model for studying complex brain disorders. Trends in
Pharmacological Sciences 35(2), 63-75.
Klaric, Maja Segvic, Zdenka Cvetnic et. al. (2009) Co-occurrence of aflatoxins,
ochratoxin A, fumonisins, and zearalenone in cereals and feed, determined by
competitive direct enzyme-linked immunosorbent assay and thin-layer
chromatography. Arhiv Za Higijenu Rada I Toksikolugiju 60(4), 427-434.
Lehner, Andreas F., Jennifer M. Duringer, Charles T. Estill, Thomas Tobin, and A.
Morrie Craig (2011). ESI-Mass spectrometric and HPLC elucidation of a new
ergor alkaloid from perennial ryegrass hay silage associated with bovine
reproductive problems. Toxicology Mechanisms and Methods 21(8), 606-621.
Li, FQ, XY Luo, and T Yoshizawa (1999). Mycotoxins (trichothecenes, zearalenone
and fumonisins) in cereals associated with human red-mold intoxications
stored since 1989 and 1991 in China. Natural Toxins 7(3), 93-97.
Malekinejad, H, EJ Schoevers, IJ Daemen, C Zijlstra, B Colenbrander, J FinkGremmels, and BA Roelen (2007). Exposure of oocytes to the Fusarium
toxins zearalenone and deoxynivalenol causes aneuploidy and abnormal
embryo development in pigs. Biology of Reproduction 77(5), 840-847.
Mari-Bauset, Salvador, Agustin Llopis-Gonzalez, Itziar Zazpe-Garcia, Amelia MariSanchis, and Maria Morales-Suarez-Varela (2014). Nutritional status of
children with Autism Spectrum Disorders (ASDs): A case-control study.
Journal of Autism and Developmental Disorders 45(1), 203-212.
Missmer, Stacey A., Lucina Suarez, Marilyn Felkner, Elaine Wang, Alfred H. Merrill
Jr., Kenneth J. Rothman, and Katherine A. Hendricks. Exposure to
Fumonisins and the Occurance of Neural Tube Defects along the TexasMexico Border. Environmental Health Perspectives 114(2), 237-241.
Nishimura, Yuhei, Soichiro Murakami, et. al.(2015) Zebrafish as a systems
toxicology model for developmental neurotoxicity testing. Congenital
Anomalies 55, 1-16.
Schwartz, Patrick, Karen L. Thorpe et. al. (2010) Short-term exposure to the
environmentally relevant estrogenic mycotoxin zearalenone impairs
reproduction in fish. Science of the Total Environment 409(2), 326-333.
Schwartz, Patrick, Thomas D. Bucheli et. al. (2013) Life-cycle exposure to the
estrogenic mycotoxin zearalenone affects zebrafish (Danio rerio)
development and reproduction. Environmental Toxicology 28(5), 276-289.
Shannon, M., and JW Graef (1996). Lead intoxication in children with pervasive
developmental disorders. Journal of Toxicology- Clinical Toxicology 34(2),
177-181.
Sulyok, Michael, Rudolph Krska, and Rainer Schuhmacher (2007). A liquid
chromatography/tandem mass spectrometric multi-mycotoxin method for the
quantification of 87 analytes and its application to semi-quantitative screening
of moldy food samples. Analytical and Bioanalytical Chemistry 389(5), 15051523.
Truong, Lisa, David Mandrell, Rick Mandrell, Michael Simonich, and Robert
Tanguay (2014) A rapid throughput approach identifies cognitive deficits in
adult zebrafish from developmental exposure to polybrominated flame
retardants. Neurotoxicology 43, 134-142.
Truong, Lisa, et. al. (2014) Multidimensional In Vivo Hazard Assessment Using
Zebrafish. Toxicological Sciences 137(1), 212-233.
Truong, Lisa, Matthew A. DeNardo, Soument Kundu, Terrence J. Collins, and Robert
Tanguay (2013). Zebrafish assays as developmental toxicity indicators in the
green design of TAML oxidation catalysts. Green Chemistry 15, 2339-2343.
Wu, Ting-Shuan, Jiann-Jou Wang Feng-Yih Yu, and Biing-Hui Liu (2012).
Evaluation of nephrotoxic effects of mycotoxins, citrinin and patulin, on
zebrafish (Danio rerio) embryos. Food and Chemical Toxicology 50, 43984404.
Xiao, Jingchen, Qi Wang, Kristin M. Bircsak, Xia Wen, and Lauren M. Aleksunes
(2015). In vitro screening of environmental chemicals identifies zearalenone
as a novel substrate of the placental BCRP/ABCG2 transporter. Toxicology
Research 4, 695-706.
Zinedine, Abdellah, Jose Miguel Soriano, Juan Carlos Molto, and Jordi Manes
(2007). Review on the toxicity, occurrence, metabolism, detoxification,
regulations and intake of zearalenone: An estrogenic mycotoxin. Food and
Chemical Toxicology, 45(1), 1-18.