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Matthew Perella 4/17/2012 Bio 601

Analysis of antioxidant capacity of phytocannabinoids to attenuate e thanol-mediated developmental toxicity in zebrafish. Matthew Perella 4/17/2012 Bio 601. Introduction. Investigating antioxidant and receptor induced ability of cannabinoids Fetal Alcohol Syndrome (FAS)

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Matthew Perella 4/17/2012 Bio 601

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  1. Analysis of antioxidantcapacity ofphytocannabinoids toattenuate ethanol-mediateddevelopmentaltoxicity in zebrafish Matthew Perella 4/17/2012 Bio 601

  2. Introduction • Investigating antioxidant and receptor induced ability of cannabinoids • Fetal Alcohol Syndrome (FAS) • Oxidative Stress • Other Factors • Zebrafish Model System • Endocannabinoid division of Nervous System (ECB)

  3. FAS • Developmental defects due to embryonic EtOH exposure • Affects species from mammals to insects • Effects characterized by Fetal Alchol Spectrum Disorders (FASDs) • Delay in development • Cardiac abnormalities • CNS abnormalities • Craniofacial abnormalities • Behavioral and cognitive delay or impairment

  4. Prominence of problem • Annual EtOH abuse = • Economic costs greater than $100 billion1 • 10,000+ lives lost1 • High occurence • CDC: 0.2-2 cases of FASD per 1000 live births in U.S. • Leading cause of Mental retardation in U.S.2 • Limited exposure ≠ no effect • Genetic and Environmental factors yield spectrum of EtOH teratogenicity • Multiple FAS phenotypes • ALI, S., INSTITUTE OF BIOLOGY, L. U., SYLVIUS LABORATORY, LEIDEN, THE NETHERLANDS, CHAMPAGNE, D. L., INSTITUTE OF BIOLOGY, L. U., SYLVIUS LABORATORY, LEIDEN, THE NETHERLANDS, DEPARTMENT OF MEDICAL PHARMACOLOGY, L. A. C. F. D. R., LEIDEN, THE NETHERLANDS, ALIA, A., INSTITUTE OF CHEMISTRY, L. U., LEIDEN, THE NETHERLANDS, RICHARDSON, M. K. & INSTITUTE OF BIOLOGY, L. U., SYLVIUS LABORATORY, LEIDEN, THE NETHERLANDS 2012. Large-Scale Analysis of Acute Ethanol Exposure in Zebrafish Development: A Critical Time Window and Resilience. PLoS ONE, 6. • BILOTTA, J., BARNETT, J. A., HANCOCK, L. & SASZIK, S. 2004. Ethanol exposure alters zebrafish development: A novel model of fetal alcohol syndrome. Neurotoxicology and Teratology, 26, 737-743.

  5. Zebrafish species for FAS model • Simplicity = zebrafiecciency • Non-mammalian • Maternal influence • Relatively small • Transparency • EtOH concentrations • Tools • Dyes • Fluorescent Probes • Multiple Strains and transgenic lines • Negative • Lack of maternal-interaction • Very hard to study in Humans • Multiple phenotypes + variation in individual consumption • Retrospective Studies • Lack of control dose • Exposure duration • Response measures & time • Confounding variables • Unreliable reports • Other drugs

  6. EtOH + Zebrafish • Mechanism • Direct Ethanol teratogenicity • Indirect effects from EtOH metabolism • Effects • Developmental delay • Pericardial/yolk sac oedema • Abnormal Eye Development • Cognitive Defects • Increased Mortality rates • Cause • Modification in sensitivity to oxidative stress, signal transduction pathways, & neurotransmitter systems • Gene expression • Molecular interactions key to development • Antioxidants shown to decrease severity of symptoms

  7. Cannabinoids • Natural class of molecules from Cannabis sativa • THC, CBD, CBN • Lipid messengers • Structural Similarity • Antioxidant, antibacterial, and antifungal properties Images obtained from: THAKUR, G. A., DUCLOS JR, R. I. & MAKRIYANNIS, A. 2005. Natural cannabinoids: Templates for drug discovery. Life Sciences, 78,454-466 And http://en.wikipedia.org/wiki/Vitamin_E

  8. ECB nervous system • Receptors (CB1/CB2), endocannabinoids, enzymes for synthesis and catalysis • “On-demand” production • Pre-synaptic Receptors • CB1 one of most abundant brain receptors • CB2 expressed mainly in periphery and glial cells of immune system • G-protein coupled receptor similar to glutamate and GABA • Interaction with receptors supresses NTMR release at both Inhibitory and excitatory synapses • Potent influence on synaptic transmission • CB2 modulates cytokine release

  9. Developing ECB system • Regulate proliferation, migration, specification and survival of neural progenitors • Dictate phenotypic differentiation of neurons • Dontrolthe establishment of synaptic communication • Shown to affect zebrafish development influencing the hatching process and motility behavior in larvae • CB1/CB2 both characterized in zebrafish • EtOH exposure affects ECB plasticity, change in eCB production, receptor activation, and down regulation of receptor expression

  10. Question and Hypothesis • Will supplementing phytocannabinoids during embryonic EtOH exposure effect the onset of FAS-like developmental defects? • Does inhibiting the role of CB2 receptors play into this? • Antioxidant potential of compounds will partially attenuate the onset of symptoms • Inhibiting CB2 receptors in presence of cannabinoids will result in a difference of the onset of symptoms compared to exposure without the inhibitor

  11. Experimental Protocol • 1.5% EtOH exposure from 6-24 hpf • Cannabidiol • High CB2 Interaction • High antioxidant ability • Shown to attenuate symptoms in rat neuron in vitro without receptor-mediated effects • Vitamin E • Shown to attenuate symptoms due to oxidative stress in other zebrafish toxicity studies • AM 251 • CB2 agonist • Measure body length, eye diameter, intraocular distance, and pericardial edema

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