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1. By Quang Nguyen
Research Advisor: Jordan Cocchiaro, Ph.D.
Dept. of Molecular Genetics and Microbiology
Course Director, DukeLEAP: Elizabeth A. Godin, Ph.D.
Duke University, Durham, NC
Director, Duke Center for Science Education: Rochelle Schwartz-Bloom, Ph.D.
Dept. of Pharmacology & Cancer Biology
Duke University Medical Center, Durham, NC
“Every substance is a poison; only dose distinguishes a poison from drug”
~ Paracelce

2. WhyAdultZebrafish?
- Small freshwater teleost fish species
 Easy to care and maintain
- Relatively inexpensive
- Fast sexual maturity timeframe of 2-3 months
- High similarity to metabolic enzyme regulation
These enzymes are important in human drug metabolism
 Popular model organism for genetics, drug toxicity screening and
many other biological sciences research, e.g. study of neurotoxicant of
CuCl2 and CPF on swimming rate(Tilton et al.,2010), or study of ethanol on
swimming behavior(Gerlai et al., 2006)
Photo by Azul (Own work)/FreeUse, via Wikimedia Commons

3. What is Patulin?
- Mycotoxin (fungal toxin) found in moldy fruits and fruit products, esp.
rotting apple and other apple-derived food for young children (Puel et
al., 2010)
- Produced by ~13 fungal strains of Penicillium and Aspergillus genera
+ mostly by Penicillium expansum in pomaceous fruit (Frisvad et al., 2004)
 Certain fungal strains don’t produce PAT
- In 1944, PAT tested for potential antibiotic property against common
cold (Clarke, 2006; Puel et al., 2010)
 Result: Not effective as a treatment.
 Later studies show PAT is rather toxic
PhotobyScottBauer,USDA[Publicdomain],viaWikimediaCommons

4. Toxicity of Patulin
- Small children, 2 – 10 years old consume a higher amount of apple juice
relative to their body weight than older age people (FDA, 2001).
 Particular risk for small children
- Mechanism:
+ Electrophilic PAT covalently binds to sulfhydryl groups on proteins
and amino acids
Inhibiting normal functions of many enzymes, e.g. those in intestine
and the brain (Wu et al., 2012, Puel et al., 2010)
- Organotoxicity in adult mammals, including liver, gastro-intestinal tract
and kidney (McKinley et al., 1982; Speijers et al., 1988)
- Premature death (Becci et al., 1981) and neurotoxicity in rats
+ agitation, tremors, convulsions, and dyspnea (Puel et al., 2010)
PhotobyNeozoon(Ownwork)[CC-BY-SA-3.0viaWikimediaCommons

5. Photo by Roberta F. [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-
sa/3.0)], via Wikimedia Commons
- Trigger local irritation and an acute intoxication (5).
- “Mutagenic, carcinogenic and teratogenic” (Lupescu et al., 2013)
- PAT can interrupt the cell cycle and damage DNA with subsequent suicidal apoptosis of
erythrocytes (Lupescu et al., 2013)
+ Increase cytosolic [Ca2+]  Cells shrinkage  Membrane scrambled
 Eryptosis
* Eryptosis is observed in clinical disorders, such as diabetes, sickle cell
disease, iron deficiency and malaria

6. Why Patulin in Adult Zebrafish?
- U.S. FDA and WHO: maximum allowable limit = 50 μM of PAT in apple juice (FDA, 2001);
however,
- Improper sorting/storing
- PAT nature: Heat resistant, stable in dilute acids (Achachlouei et al., 2007)
- PAT is especially stable in apple juice (Achachlouei et al., 2007)
- Alcoholic fermentation, pasteurization and irradiation will only reduce [PAT] (Achachlouei et al.,
2007).
Apple juice can “occasionally be heavily contaminated” (JECFA, 1995)
+ 60% of examined food products still contained PAT (Yurdun et al., 2001)
- Many fungal metabolites were previously antibiotics have been found to be highly toxic
(Peraica et al.,, 1999)

7. - Zebrafish behavior is not fully examined (Gerlai et al., 2006)
+ Yet, changes in motor behavior is an indication of neural damage (Kyriakatos et al., 2011)
+ Locomotion is fundamental to all other behavioral responses (Tierney, 2011)
- Only 1 study directly looked at developmental nephrotoxicity, but in zebrafish embryos
(Wu et al., 2012)
- Zebrafish is a popular, highly translatable to human application, and relatively less
expensive research tool, but most previous PAT studies were done in mammalian
models.
- In vivo method utilizing mammalian models  expensive and sophisticated
- In vitro method (Achachlouei et al., 2007)  not fully representative of biochemical dynamics.
By this preliminary study, we will better investigate questions:
1. Adult zebrafish = a promising model organism in examining of neurobehavioral toxicity
of PAT and other related chemicals?
+ Quantifiable post-treatment changes in locomotion?
 Potential as an in vivo tool in aiding drug discovery/optimization and genetic as well as
other therapeutic interventions of PAT toxicity and even cancer (Li et al., 2012)?
2. Adult zebrafish = environmental and biological marker of mycotoxin contamination?
Why Patulin in Adult Zebrafish? cont.

8. Hypothesis
Injection of higher dose of
PAT will cause a greater
reduction in locomotor
activity of the adult zebrafish

9. Methodology
1) Anesthetized in tricaine solution
2) Injected fish intraperitoneally using insulin
syringe (5 fish / conc.)
PAT Concentration (μg/μL) PAT Dose (μg)
0.01 0.1
0.1 1
0.5 5
1 10
* Control = 10% v/v DMSO
* DMSO concentration was kept the same in all
treatments.
2) Fish were kept in treatment containers for 24
hours
3) Each was then placed in experimental tank with
grid template beneath for 8-minute video recording

10. Methodology cont.
- Water depths = 3 cm in experimental tanks
- Video camcorder(VIXIA HF M31; Canon, Japan) on a tripod 55
cm above the experimental tanks for horizontal
motions recording.
- Vertical behaviors observed manually 1 m away
from the experimental tanks.
- Recorded videos were blind analyzed for total
number of line(s) crossed.
- Statistical Analysis:
+ One-way ANOVA and Tukey post-hoc test
+ A log-rank (Mantel-Cox)
- All subjects were humanely euthanized in tricaine
solution (250 mg/L)

11. Exp. 1**
Increased locomotor activity as [PAT] increases
 Trend: DMSO < 10 < .1 < 1 < 5
 5 μg PAT = most effective dose
 10 μg PAT = too toxic

12. Exp. 2: PAT Toxicity May Be Dose-dependent
 “Trend:” 5 < DMSO < 1
 1 μg exhibited highest
locomotor activity
 Resembles 5 μg group in
Exp 1
 5 μg PAT resembles 10 μg
group in Exp 1
NOTE: Fish were much smaller

13. Exp. 3: PAT Instability at room temperature
may affect its toxicity
“Trend:” 1 < 5 < DMSO
No consistent significant
pattern observed

14. Survival Analysis
- All survival curves are
significantly different
- DMSO = 100% survived
- Increased [PAT]  Decreased
survival proportion
(Most survived) DMSO > .1 > 1 > 5 > 10 (Least survived)

15. Conclusion
1. Increased concentration of PAT increases the locomotor activity
of injected adult zebrafish
2. 5 μg PAT seemed to be the most effective dose in our experiment
3. 10 μg PAT seemed to be too toxic for adult zebrafish
4. Fish weight or size may play an important role in the toxicity of
PAT as it may affect the actual doses
5. Laboratory form of PAT instability at room temperature may
influence its toxicity
6. DMSO is a safe organic solvent for diluting PAT for IP injection
 Result collected rejects our hypothesis, suggesting that:

16. Limitation & Future Direction
- Limited funding and time constraint
Limited amount of chemical availability and laboratory access
- Subjectivity of manual counting of lines crossed
Computerized quantification analysis instead of manual
observation, e.g. 3D coordinate computation analysis
- Limitation of correlation between lines crossed method
and fish’s activeness
Subsequent confirmatory testing recommended for cellular and
molecular examination

17. Limitation & Future Direction cont.
- Limited number of subjects
Further studies are needed to confirm the possibility of dose
dependency, e.g. EC50 and LC50 of PAT both acute and chronic
intoxication in zebrafish
- Individual variation in subject population
 Individual dose determination based on the weight of each fish
- Highly technical dexterity of IP injection
Computer-controlled injection
Compare results between injection method and fish tank water
immersion method
- Examine ways to reduce PAT toxic effect could be diminished

18. References
Achachlouei, B., Zenouz, A., Assadi, Y., & Hesari, J. (2007). Reduction of patulin content in apple juice concentrate using activated carbon
and its effects on several chemical constituents. Journal of Food, Agriculture and Environment, 5(1), 12-16. Retrieved from
http://world-food.net/reduction-of-patulin-content-in-apple-juice-concentrate-using-activated-carbon-and-its-effects-on-several-
chemical-constituents/
Becci, P. J., Hess, F. G., Johnson, W. D., Gallo, M. A., Babish, J. G., Dailey, R. E., & Parent, R. A. (1981). Long-term carcinogenicity and toxicity
studies of patulin in the rat. J. Appl Toxicol 1(5):256-261.
Clarke, M. (2006). The 1944 patulin trial of the british medical research council. Journal of the Royal Society of Medicine, 99(9), 478-480.
FDA. (2001). Patulin in Apple Juice, Apple Juice Concentrates and Apple Juice Products.
http://www.fda.gov/food/foodborneillnesscontaminants/naturaltoxins/ucm212520.htm
Frisvad, J. C., Smedsgaard, J., Larsen, T. O., Samson, R. A., Frisvad, J. C., Smedsgaard, J., . . . Samson, R. A. (2004). Mycotoxins, drugs and other
extrolites produced by species in penicillium subgenus penicillium. Studies in Mycology, 49, 201-241.
Gerlai, R., Lee, V., & Blaser, R. (2006). Effects of acute and chronic ethanol exposure on the behavior of adult zebrafish (danio rerio).
Pharmacology Biochemistry and Behavior, 85(4), 752-761. doi:http://dx.doi.org/10.1016/j.pbb.2006.11.010
JECFA (Joint FAO/WHO Expert Committee on Food additives and Contaminants). Position paper on patulin, 30 session, The Hague, The
Netherlands, 9-13 March, 1995. http://apps.who.int/food-additives-contaminants-jecfa
database/PrintPreview.aspx?chemID=3345.
Kyriakatos, A., Mahmood, R., Ausborn, J., Porres, C. P., Büschges, A., & El Manira, A. (2011). Initiation of locomotion in adult zebrafish. The
Journal of Neuroscience, 31(23), 8422-8431. doi:10.1523/JNEUROSCI.1012-11.2011
Li, Y., Huang, W., Huang, S., Du, J., & Huang, C. (2012). Screening of anti-cancer agent using zebrafish: Comparison with the MTT
assay. Biochemical and Biophysical Research Communications, 422(1), 85-90. doi:
http://dx.doi.org.proxy.lib.duke.edu/10.1016/j.bbrc.2012.04.110

19. References cont.
Lupescu, A., Jilani, K., Zbidah, M., & Lang, F. (2013). Patulin-induced suicidal erythrocyte death. Cellular Physiology and Biochemistry, 32(2),
291-299. Retrieved from http://www.karger.com/DOI/10.1159/000354437
McKinley, E. R., Carlton, W. W., & Boon, G. D. (1982). Patulin mycotoxicosis in the rat: Toxicology, pathology and clinical pathology. Food
and Chemical Toxicology, 20(3), 289-300. doi:http://dx.doi.org/10.1016/S0278-6915(82)80295-0
Peraica, M., Radić, B., Lucić, A., & Pavlović, M. (1999). Toxic effects of mycotoxins in humans. Bull World Organ., 77(9), 754-766. Retrieved
from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2557730/
Puel, O., Galtier, P., & Oswald, I. P. (2010). Biosynthesis and toxicological effects of patulin. Toxins, 2(4), 613-631. doi:10.3390/
toxins2040613
Speijers, G. J. A., Franken, M. A. M., & van Leeuwen, F. X. R. (1988). Subacute toxicity study of patulin in the rat: Effects on the kidney and
the gastro-intestinal tract. Food and Chemical Toxicology, 26(1), 23-30. doi:http://dx.doi.org/10.1016/0278-6915(88)90037-3
Tierney, K. B. (2011). Behavioural assessments of neurotoxic effects and neurodegeneration in zebrafish. Biochimica Et Biophysica Acta
(BBA) - Molecular Basis of Disease, 1812(3), 381-389. doi:http://dx.doi.org/10.1016/j.bbadis.2010.10.011
Tilton, F. A., Bammler, T. K., & Gallagher, E. P. (2011). Swimming impairment and acetylcholinesterase inhibition in zebrafish exposed to
copper or chlorpyrifos separately, or as mixtures. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology,
153(1), 9-16. doi:http://dx.doi.org/10.1016/j.cbpc.2010.07.008
Wu, T., Yang, J., Yu, F., & Liu, B. (2012). Evaluation of nephrotoxic effects of mycotoxins, citrinin and patulin, on zebrafish (danio rerio)
embryos. Food and Chemical Toxicology, 50(12), 4398-4404. doi:http://dx.doi.org.proxy.lib.duke.edu/10.1016/j.fct.2012.07.040
Yurdun, T., Omurtag G. Z., Ersoy, O. (2001). Incidence of patulin in apple juices marketed in Turkey. J Food Prot, 64, 1851-3.

20. Acknowledgement
I would like to express my highest gratitude to Dr. Jordan Cocchiaro
for her enthusiastic and highly supportive mentorship and scientific
guidance. Thank you for boiling my interest in biomedical and biological
sciences research.
I also want to sincerely thank Dr. Rochelle Bloom at the Duke Center
for Science Education and Dr. Elizabeth Godin for allowing me and others
an opportunity to participate in this Duke Launch into Education About
Pharmacology (Duke LEAP) program.
Lastly, I shall not forget to thank all the other staff members and
housekeeping staff members who worked very hard to make this program
go as smoothly as it did.
Thank you, Sigma Xi staffs and judges, for your time and a great
opportunity for us to experience scientific communication
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cite any material referenced. Self-created photos (those without citations) can only be used with
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