Examining Neurobehavioral Toxicity of Patulin in Adult Zebrafish


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  • 70% of human genes have at least 1 homolog identified in the zebrafish genome: * monoamine oxidase (MAO)-A/B (Setiniet al., 2005) * YP3A65 of CYP3A proteins (Chang et al., 2013)
  • Unlike other mycotoxin: aflatoxins, ochratoxin A, zearalenone, trichothecenes and fumonisins (Puel et al., 2010)
  • No studies done in adult zebrafish with PAT that look particularly at behavior, e.g. locomotor.One study used embryos as subject, and in different route of administration. Another study found in mice and rodent, but orally administered. - Another study in rat: agitation (anxiety, nervous excitement, etc.), convulsion (irregular body move), dyspnea (breathing diff.), distention (swell by inside P) of the gastro intestinal tract (Puel et al., 2010) *: may explain why increase in locomotor activity in lower doses PAT is not soluble in water, and must be kept at -20C, cannot be at rm temp for too long. - Ethanol was shown to increase aggressive behavior [3,4] Increase oxidative stress response in mammalian cells (Liu et al, 2007)Organotoxicity in adult mammals, including liver, gastro-intestinal tract and kidney in rats (McKinley et al., 1982; Speijers et al., 1988) Oxidative stress: imbalance between manifestation of reactive oxygen species and a biological system's ability to detoxify reactive intermediates or to repair the resulting damage.  disturb normal redox state of cells  toxic effects by producing peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA It was shown that exposure to PAT may cause organotoxicity, including livers, and kidneys and several other neurotoxicities, as well as damages to the cell cycle and DNA with subsequent apoptosis. In 2013, Lupescu and colleagues also reported that Patulin might stimulate suicidal death of human red blood cells via a process called eryptosis that is also observed in diabetes, sickle cell disease and malaria.
  • US FDA: safety level of PAT in apple juice at 50 μM (μg/L) (van Egmond et al., 2007; Liu et al., 2007)
  • For instance, Tilton et al. (2010) showed that a neurotoxicant, chlorpyrifos (CPF), at .6 μM, decreased swimming rate and AChE muscle activity. Moreover, injection of two neurotoxins – 1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) – reduced travelled distance and speed of zebrafish (Anichtchiket al., 2004).
  • Add some text to this slide! Tell them exactly what you did….
  • Exp1 0 – 8 Outlier removed for 1 ug group (223 lines crossed) After outlier removed (**) DMSO < 10 < .1 < 1 < 5Before outlier removed (*) DMSO < 10 < 1 < .1 < 5 DMSO < 5: p = 0.00385 > 10: p = 0.0301 No significance between .1 and 1
  • Exp 20 – 81 outlier removed for DMSO group (1 line crossed) 1 outlier removed for 5 group (80 lines crossed) Differences in subjects’ size: skinny and smallDoses may have been increased Inversely proportional relationship between PAT dose and fish weight/size FISH SMALLER  DOSE HIGHER  WEIGH FISH TO INJECT FISH WITH EXACT DOSE in future
  • Although PAT is heat resistant and stable in dilute acids, its laboratory form is unstable at room temperature (Cayman Chemical Co.) It is recommended to be stored at -20C (Cayman Chemical Co.) Although soluble in water, it is unstable in polar solvent such as water and methanol and loses its biological potency in alkaline solution (Sigma) DMSO = highly polar organic solvent Thawing and its instability at room temperature Somehow affects its toxicity The importance of manual observation because of the representation of lines crossed What does number of lines crossed mean? How accurately it is? -
  • Concentration increases, more toxic, and more locomotor activity DMSO = 100% safe (not sure its own effect though)
  • 6 major conclusions drawn from the three experiments conducted, using adult zebrafish as model: However, across experimental trials,1 μg group <5 μg. Yet, 5 μg wasn’t always greater than that of DMSOBUT,> DMSO in experiment 1 with statistical significance across time periods. Environmental censors put in streams  high-through put screening tool for drugs  something to block that mechanism Basis mechismIf it is consient in further experiemts, and develop assay using fish to screen for toxicity markers  transenic marker, antibody marker SAME IN FISH  SIMPLE to study prevent with some new drug Moreover, the aqueous solution of PAT was recommended to be stored for no more than one day. As PAT has to be kept at -20oC, thawing/freezing may have changed the drug efficacy.
  • Although we also did observe some statistical significant effects of PAT on locomotion, limited amount of fish tested wasn’t sufficient enough to produce a significant conclusion, especially between .1 μg and 1 μg doses. Thus, further studies are needed to investigate the possibility of dose response. We provided here a starting dose for testing this chemical in the future. Specifically, 5 μg seems to be the maximal effective dose, and 10 μg showed lethality to certain extent in our study. Further studies are needed to reevaluate the effective dose, and later determine the ED50 and/or LD50 of PAT in adult zebrafish. Means of all 3 experiments Further studies STABLE IN SOLUTION  FRESH BATCH OF DRUG and power  reconstitute  Test its stability  EXP 3  control in future experiment Use extract from apple juice concentrates with ethyl acetateUse PAT from apple juice extract Increased locomotion after 24 hours exposure may just be acute toxicityIndividual dose determination  but not generalizable
  • Jordan L. Cocchiaro, PhDPostdoctoral FellowRawls LaboratoryDuke UniversityDept. of Molecular Genetics and MicrobiologyRochelle D. Schwartz-Bloom, Ph.D.Professor of PharmacologyDirector, Duke Center for Science EducationDuke University Medical Centerwebsite: www.rise.duke.eduElizabeth A. Godin, Ph.D.Course Director, LEAPProject Director, RISE
  • Examining Neurobehavioral Toxicity of Patulin in Adult Zebrafish

    1. 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. 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. 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. 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. 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. 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. 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. 8. Hypothesis Injection of higher dose of PAT will cause a greater reduction in locomotor activity of the adult zebrafish
    9. 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. 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. 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. 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. 13. Exp. 3: PAT Instability at room temperature may affect its toxicity “Trend:” 1 < 5 < DMSO No consistent significant pattern observed
    14. 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. 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. 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. 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. 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. 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. 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 Disclaimer: Contents in this PowerPoint in its entirety are exclusively educational. Please do not cite any material referenced. Self-created photos (those without citations) can only be used with author’s permission.