1. Can Co-exposure of Ellagic Acid Mitigate the Adverse Effects of
Aflatoxin B1 on the Visual System of Developing Zebrafish Larvae?
Introduction
 Aflatoxin B1 (AFB1) produced by the mold, Aspergillus flavus is a highly potent carcinogen and
mutagen that creates reactive oxygen species (ROS) in tissues.1, 12
 AFB1 is found on crops after harvest (most abundant in tropical regions) and can be ingested.12
 Ellagic acid (EA) is an antioxidant found in raspberries, strawberries, walnuts and black currants.9
 EA has been found to reduce the generation of ROS and inhibit the mutagenicity of AFB1.8, 9, 10
Purpose
The purpose of this study was to determine if
co-exposure to EA can mitigate the adverse
effects of AFB1 on the visual system of devel-
oping larval zebrafish.
Hypothesis
Larval zebrafish that are co-exposed to EA and
AFB1 will have greater visual ability, as deter-
mined through optokinetic responses, than larval
zebrafish that are exposed solely to AFB1.
Treatments:
 AFB1 at 1.4 μg/mL (AFB)
 AFB1 Vehicle Control (Methanol)
 AFB1 + Low Dose EA at 1.7 μg/mL (A-LEA)11
 AFB1 + Medium Dose EA at 3.4 μg/mL (A-MEA)11
 AFB1 + High Dose EA at 6.8 μg/mL (A-HEA)11
 AFB1 + High EA Vehicle Control
(Methanol + NaOH-HCl) (AHEA VC)
 Low Dose EA (Low EA)
 Medium Dose EA (Med EA)
 High Dose EA (High EA)
 High Dose EA Vehicle Control (NaOH-HCl) (HEA VC)
Subjects were exposed to treatments from 6-54
hours post fertilization (hpf).
Optokinetic Response (OKR):
At 5 days post fertilization (dpf) larvae were
placed in Detain to limit swimming ability and
placed in the apparatus (Fig. 1). The striped
drum was rotated around larvae for 1 minute
and extraocular movements were recorded on
video. Eyes underwent a smooth pursuit of
visual stimulus, then a quick saccade back to
the origin. During film review each subject’s
eye saccades (defined by the quick movement
of both eyes in unison in the opposite direc-
tion of drum rotation) were counted to make
up the OKR score.
Conclusions
 AFB1 did not have a significant negative effect on visual development as
has been observed from past studies in our lab. 6, 11
 There is an observed decline in visual ability in subjects proportional to
the concentration of EA vehicle control (Fig. 3).
 Co-exposures involving medium and high levels of EA significantly im-
paired visual development, and the AFB1+High EA Vehicle Control most
greatly impaired visual ability (Fig. 4).
 These observations, including that increasing EA concentration and EA ve-
hicle controls both negatively impacted visual ability alludes to the idea
that the current method of EA introduction to subjects is flawed and
needs further investigation.
 There was no significant change in average eye diameter in relation to to-
tal length across treatment groups (data not shown). Therefore changes
in visual ability do not appear to be the result of developmental delays.
References
1. Abdulmajeed, N. A. (2010). Therapeutic ability of some plant extracts on aflatoxin B1 induced renal and cardiac damage. Arabian Jour-
nal of Chemistry, 4, 1-10. doi:10.1016/j.arabjc.2010.06.005
2. Bilotta, J. (2000). Effects of abnormal lighting on the development of zebrafish visual behavior. Behavioural Brain Research, 116, 81-87.
3. Bilotta, J., Saszik, S., Givin, C. M., Hardesty, H. R., & Sutherland, S. E. (2002). Effects of embryonic exposure to ethanol on zebrafish visual
function. Neurotoxicology and Teratology, 24, 759-766.
4. Brockerhoff, S. E., Hurley, J. B., Janssen-Bienhold, U., Neuhauss, S. C. F., Driever, W., & Dowling, J. E. (1995). A behavioral screen for iso-
lating zebrafish mutants with visual system defects. Neurobiology, 92, 10545-10549.
5. Easter, S. S., & Nicola, G. N. (1996). The development of vision in the zebrafish (danio rerio). Developmental Biology, 180, 646-663.
6. Fadeyi, S. (2014). Inhibition of the adverse effects of aflatoxin B1 on the visual development of zebrafish, Danio rerio, by co-exposure to
ellagic acid. Undergraduate thesis, Washington College Chestertown, MD.
7. Fleisch, V. C., & Neuhauss, S. C. F. (2006). Visual bevahior in zebrafish. Zebrafish, 3(2), 1-11.
8. Lee, W., Ou, H., Hsu, W., Chou, M., Tseng, J., Hsu, S., . . . Sheu, W. H. (2010). Ellagic acid inhibits oxidized LDL-mediated LOX-1 expression,
ROS generation, and inflammation in human endothelial cells. Journal of Vascular Surgery, 52(5), 1290-1300. doi:10.1016/
j.jvs.2010.04.085
9. Loarca-Piña, G., Kuzmicky, P. A., González de Mejía, E., Kado, N. Y., & Hsieh, D. P. H. (1996). Antimutagenicity of ellagic acid against
aflatoxin B1 in the salmonella microsuspension assay. Mutation Research, 360, 15-21.
10.Mandal, S., Ahuja, A., Shivapurkar, N. M., Cheng, S., Groopman, J. D., & Stoner, G. D. (1987). Inhibition of aflatoxin B1 mutagenesis
in salmonella typhimurium and DNA damage in cultured rat and human tracheobronchial tissues by ellagic acid. Carcinogenesis, 8(11),
1651-1656.
11.McGlumphy, E. J. (2009). Embryonic toxicity of alfatoxin B1 on zebrafish, Danio rerio, development and visual behaviors. Undergraduate
thesis, Washington College Chestertown, MD.
12.Peraica, M., Radic, B., Lucic, A., & Pavlovic, M. (1999). Toxic effects of myotoxins in humans. Bulletin of the World Health Organization,
77(9), 754-766.
Acknowledgments
Thank you to Dr. Martin Connaughton for guiding and mentoring me through this re-
search and for also stepping back and letting me make my own mistakes and discover-
ies. Thank you to Dr. Shaun Ramsey and his students for helping me with electronics, to
Dr. Mike Kerchner for alternative perspectives, to Dr. Rick Locker for acid-base advice,
and to the rest of Toll’s professors for guidance and the occasional supplies. Funding for
this study was provided by the Hodson Science Fellowship and the Department of Biol-
ogy at Washington College. Funding for my travels to the 2015 Annual Meeting was
provided by The Louise and Rodney Layton.
Results
Figure 3. Larvae exposed solely to EA had no change in visual
ability in comparison to controls. Mean ± one standard error number
of saccades (OKR score) of subjects within control, EA treatment and EA ve-
hicle control groups.
Figure 4. Larvae exposed to AFB1 did not show a decrease in
visual ability. Larvae co-exposed to AFB1 and medium and high
doses of EA had worse visual ability than larvae exposed solely
to AFB1. Mean ± one standard error number of saccades (OKR score) of
subjects within AFB1 and AFB1 + EA treatment groups. Letters above bars in-
dicate significant differences between groups.
Joshua Rogers and Dr. Martin Connaughton
Department of Biology, Washington College
Statistics: Collective data were examined
statistically using a one-way ANOVA and a
post-hoc multiple comparisons test by
GraphPad Software.
Fig 5. 5 days post fertiliza-
tion (dpf) larval Zebrafish
(Danio rerio) under mag-
nification. Eye Diameter
was measured as a per-
centage of total length
(red lines).
Methods
Subjects:
Zebrafish were mated and fertilized eggs
were incubated at a density of 20 eggs in
20 mL of spring water in 100 mL beakers.
Camera
Petri Dish
Belt
MotorLight Table
Striped Drum
Figure 2. Larvae respond to the rotating striped drum visual
stimulus rather than to rotating solid drums or motor vibra-
tions. Mean ± one standard error number of saccades (OKR score) of sub-
jects introduced to a rotating solid white drum, a rotating solid black drum, a
rotating striped drum, and a striped drum disconnected from a running mo-
tor. Letters above bars indicate significant differences between groups.
Eye Diameter: Subjects were imaged and
eye diameter was measured as a percentage
of total length (Fig. 5).
Figure 1. The spinning drum apparatus.
Abstract No.
376