130 likes | 290 Views
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)
E N D
Analysis of antioxidantcapacity ofphytocannabinoids toattenuate ethanol-mediateddevelopmentaltoxicity in zebrafish Matthew Perella 4/17/2012 Bio 601
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)
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
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.
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
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
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
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
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
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
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
Resources • References • 2012a. CDC - FASD, Data & Statistics - NCBDDD [Online]. Available: http://www.cdc.gov/ncbddd/fasd/data.html. • 2012b. CDC - FASD, Facts about FASDs - NCBDDD [Online]. Available: http://www.cdc.gov/ncbddd/fasd/facts.html. • AGUADO, T., CARRACEDO, A., JULIEN, B., VELASCO, G., MILMAN, G., MECHOULAM, R., ALVAREZ, L., GUZMÁN, M. & GALVE-ROPERH, I. 2007. Cannabinoids Induce Glioma Stem-like Cell Differentiation and Inhibit Gliomagenesis. Journal of Biological Chemistry, 282, 6854-6862. • AHN, K., MCKINNEY, M. K. & CRAVATT, B. F. 2008. Enzymatic pathways that regulate endocannabinoid signaling in the nervous system. Chem Rev, 108, 1687-707. • ALFONSO-LOECHES, S., PASCUAL-LUCAS, M., BLANCO, A. M., SANCHEZ-VERA, I. & GUERRI, C. 2010. Pivotal role of TLR4 receptors in alcohol-induced neuroinflammation and brain damage. The Journal of Neuroscience, 30, 8285-8295. • 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. • ARENZANA, F. J., CARVAN III, M. J., AIJÓN, J., SÁNCHEZ-GONZÁLEZ, R., ARÉVALO, R. & PORTEROS, A. 2006. Teratogenic effects of ethanol exposure on zebrafish visual system development. Neurotoxicology and Teratology, 28, 342-348. • BARRES, B. A. 2008. The Mystery and Magic of Glia: A Perspective on Their Roles in Health and Disease. Neuron, 60, 430-440. • 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. • 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. • BOWSER, D. N. & KHAKH, B. S. 2007. Vesicular ATP Is the Predominant Cause of Intercellular Calcium Waves in Astrocytes. The Journal of General Physiology, 129, 485-491. • CARVAN III, M. J., LOUCKS, E., WEBER, D. N. & WILLIAMS, F. E. 2004. Ethanol effects on the developing zebrafish: neurobehavior and skeletal morphogenesis. Neurotoxicology and Teratology, 26, 757-768. • CHEN, Y. & BUCK, J. 2000. Cannabinoids Protect Cells from Oxidative Cell Death: A Receptor-Independent Mechanism. Journal of Pharmacology and Experimental Therapeutics, 293, 807-812. • CLARKE, R. B. C. & ADERMARK, L. 2010. Acute ethanol treatment prevents endocannabinoid-mediated long-lasting disinhibition of striatal output. Neuropharmacology, 58, 799-805. • COTA, D., MARSICANO, G., TSCHÖP, M., GRÜBLER, Y., FLACHSKAMM, C., SCHUBERT, M., AUER, D., YASSOURIDIS, A., THÖNE-REINEKE, C., ORTMANN, S., TOMASSONI, F., CERVINO, C., NISOLI, E., LINTHORST, A. C. E., PASQUALI, R., LUTZ, B., STALLA, G. K. & PAGOTTO, U. 2003. The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis. The Journal of Clinical Investigation, 112, 423-431. • CROUZIN, N., DE JESUS FERREIRA, M.-C., COHEN-SOLAL, C., M'KADMI, C., BERNAD, N., MARTINEZ, J., BARBANEL, G., VIGNES, M. & GUIRAMAND, J. 2011. α-Tocopherol and α-tocopheryl phosphate interact with the cannabinoid system in the rodent hippocampus. Free Radical Biology and Medicine, 51, 1643-1655. • HAMELINK, C., HAMPSON, A., WINK, D. A., EIDEN, L. E. & ESKAY, R. L. 2005. Comparison of Cannabidiol, Antioxidants, and Diuretics in Reversing Binge Ethanol-Induced Neurotoxicity. Journal of Pharmacology and Experimental Therapeutics, 314, 780-788. • HAMPSON, A. J., GRIMALDI, M., AXELROD, J. & WINK, D. 1998. Cannabidiol and (−)Δ9-tetrahydrocannabinol are neuroprotective antioxidants. Proceedings of the National Academy of Sciences, 95, 8268-8273. • HARKANY, T., GUZMAN, M., GALVE-ROPERH, I., BERGHUIS, P., DEVI, L. A. & MACKIE, K. 2007. The emerging functions of endocannabinoid signaling during CNS development. Trends in pharmacological sciences, 28, 83-92. • IRONS, T. D., MACPHAIL, R. C., HUNTER, D. L. & PADILLA, S. 2010. Acute neuroactive drug exposures alter locomotor activity in larval zebrafish. Neurotoxicology and Teratology, 32, 84-90. • JIANG, W., ZHANG, Y., XIAO, L., VAN CLEEMPUT, J., JI, S. P., BAI, G. & ZHANG, X. 2005. Cannabinoids promote embryonic and adult hippocampus neurogenesis and produce anxiolytic- and antidepressant-like effects. J Clin Invest, 115, 3104-16. • KAMRAN, K., YUNUS KHAN, M. & MINHAS, L. 2011. Teratogenic effects of Ethanol Vapour exposure on chick embryos. Journal of Pakistan Medical Association, 61, 328-331. • KASHYAP, B., FREDERICKSON, L. C. & STENKAMP, D. L. 2007. Mechanisms for persistent microphthalmia following ethanol exposure during retinal neurogenesis in zebrafish embryos. Vis Neurosci, 24, 409-21. • KIMMEL, C. B., BALLARD, W. W., KIMMEL, S. R., ULLMANN, B. & SCHILLING, T. F. 1995. Stages of embryonic development of the zebrafish. Developmental Dynamics, 203, 253-310. • LAM, C. S., RASTEGAR, S. & STRÄHLE, U. 2006. Distribution of cannabinoid receptor 1 in the CNS of zebrafish. Neuroscience, 138, 83-95. • LIPNIK-ŠTANGELJ, M. Ethanol Toxicity in the Brain: Alteration of Astroglial Cell Function. • LIPNIK-ŠTANGELJ, M. 2012. Ethanol Toxicity in the Brain: Alteration of Astroglial Cell Function. In: GALLELLI, L. (ed.) Pharmacology.InTech. • MATSUI, J. I., EGANA, A. L., SPONHOLTZ, T. R., ADOLPH, A. R. & DOWLING, J. E. 2006a. Effects of Ethanol on Photoreceptors and Visual Function in Developing Zebrafish. Investigative Ophthalmology & Visual Science, 47, 4589-4597. • MATSUI, J. I., EGANA, A. L., SPONHOLTZ, T. R., ADOLPH, A. R. & DOWLING, J. E. 2006b. Effects of ethanol on photoreceptors and visual function in developing zebrafish. Invest Ophthalmol Vis Sci, 47, 4589-97. • MIGHARINI, B. & CARNEVALI, O. 2009. A novel role for the endocannabinoid system during zebrafish development. Molecular and Cellular Endocrinology, 299, 172-177. • MORANTA, D., ESTEBAN, S. & GARCÍA-SEVILLA, J. A. 2006. Ethanol desensitizes cannabinoid CB1 receptors modulating monoamine synthesis in the rat brain in vivo. Neuroscience Letters, 392, 58-61. • ORTIZ, S., OLIVA, J. M., PÉREZ-RIAL, S., PALOMO, T. & MANZANARES, J. 2004. CHRONIC ETHANOL CONSUMPTION REGULATES CANNABINOID CB1 RECEPTOR GENE EXPRESSION IN SELECTED REGIONS OF RAT BRAIN. Alcohol and Alcoholism, 39, 88-92. • PANIKASHVILI, D., SIMEONIDOU, C., BEN-SHABAT, S., HANUS, L., BREUER, A., MECHOULAM, R. & SHOHAMI, E. 2001. An endogenous cannabinoid (2-AG) is neuroprotective after brain injury. Nature, 413, 527-31. • PARNELL, S. E., SULIK, K. K., DEHART, D. B. & CHEN, S.-Y. 2010. Reduction of ethanol-induced ocular abnormalities in mice through dietary administration of N-acetylcysteine. Alcohol, 44, 699-705. • PERTWEE, R. G. 2008. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: delta9-tetrahydrocannabinol, cannabidiol and delta9-tetrahydrocannabivarin. Br J Pharmacol, 153, 199-215. • QIN, N., NEEPER, M. P., LIU, Y., HUTCHINSON, T. L., LUBIN, M. L. & FLORES, C. M. 2008. TRPV2 is activated by cannabidiol and mediates CGRP release in cultured rat dorsal root ganglion neurons. J Neurosci, 28, 6231-8. • RADWAN, M. M., ELSOHLY, M. A., SLADE, D., AHMED, S. A., KHAN, I. A. & ROSS, S. A. 2009. Biologically Active Cannabinoids from High-Potency Cannabis sativa. Journal of Natural Products, 72, 906-911. • RANG, H. P., DALE, M. M., RITTER, J. M., FLOWER, R. J. & HENDERSON, G. 2011. Rang & Dale's Pharmacology, A Churchill Livingstone Title. • RAO, G. K. & KAMINSKI, N. E. 2006. Induction of intracellular calcium elevation by Δ9-tetrahydrocannabinol in T cells involves TRPC1 channels. Journal of Leukocyte Biology, 79, 202-213. • REIMERS, M. J., FLOCKTON, A. R. & TANGUAY, R. L. 2004a. Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish. Neurotoxicology and Teratology, 26, 769-781. • REIMERS, M. J., HAHN, M. E. & TANGUAY, R. L. 2004b. Two Zebrafish Alcohol Dehydrogenases Share Common Ancestry with Mammalian Class I, II, IV, and V Alcohol Dehydrogenase Genes but Have Distinct Functional Characteristics. Journal of Biological Chemistry, 279, 38303-38312. • REIMERS, M. J., LA DU, J. K., PERIERA, C. B., GIOVANINI, J. & TANGUAY, R. L. 2006. Ethanol-dependent toxicity in zebrafish is partially attenuated by antioxidants. Neurotoxicology and Teratology, 28, 497-508. • RODRIGUEZ-MARTIN, I., DE VELASCO, E. M. F. & RODRIGUEZ, R. E. 2007a. Characterization of cannabinoid-binding sites in zebrafish brain. Neuroscience Letters, 413, 249-254. • RODRIGUEZ-MARTIN, I., HERRERO-TURRION, M. J., DE VELASCO, E. M. F., GONZALEZ-SARMIENTO, R. & RODRIGUEZ, R. E. 2007b. Characterization of two duplicate zebrafish Cb2-like cannabinoid receptors. Gene, 389, 36-44. • ROSEMBERG, D. B., DA ROCHA, R. F., RICO, E. P., ZANOTTO-FILHO, A., DIAS, R. D., BOGO, M. R., BONAN, C. D., MOREIRA, J. C. F., KLAMT, F. & SOUZA, D. O. 2010. Taurine prevents enhancement of acetylcholinesterase activity induced by acute ethanol exposure and decreases the level of markers of oxidative stress in zebrafish brain. Neuroscience, 171, 683-692. • SANTOS-LEDO, A., ARENZANA, F. J., PORTEROS, A., LARA, J., VELASCO, A., AIJÓN, J. & ARÉVALO, R. 2011. Cytoarchitectonic and neurochemical differentiation of the visual system in ethanol-induced cyclopic zebrafish larvae. Neurotoxicology and Teratology, 33, 686-697. • STOKES, A. J., SHIMODA, L. M. N., KOBLAN-HUBERSON, M., ADRA, C. N. & TURNER, H. 2004. A TRPV2–PKA Signaling Module for Transduction of Physical Stimuli in Mast Cells. The Journal of Experimental Medicine, 200, 137-147. • SUGIURA, T., KONDO, S., KISHIMOTO, S., MIYASHITA, T., NAKANE, S., KODAKA, T., SUHARA, Y., TAKAYAMA, H. & WAKU, K. 2000. Evidence That 2-Arachidonoylglycerol but Not N-Palmitoylethanolamine or Anandamide Is the Physiological Ligand for the Cannabinoid CB2 Receptor. Journal of Biological Chemistry, 275, 605-612. • SVÍŽENSKÁ, I., DUBOVÝ, P. & ŠULCOVÁ, A. 2008. Cannabinoid receptors 1 and 2 (CB1 and CB2), their distribution, ligands and functional involvement in nervous system structures — A short review. Pharmacology Biochemistry and Behavior, 90, 501-511. • SYLVAIN, N. J., BREWSTER, D. L. & ALI, D. W. 2010. Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers. Neurotoxicology and Teratology, 32, 472-480. • THAKUR, G. A., DUCLOS JR, R. I. & MAKRIYANNIS, A. 2005. Natural cannabinoids: Templates for drug discovery. Life Sciences, 78, 454-466. • WANG, L., LIU, J., HARVEY-WHITE, J., ZIMMER, A. & KUNOS, G. 2003. Endocannabinoid signaling via cannabinoid receptor 1 is involved in ethanol preference and its age-dependent decline in mice. Proceedings of the National Academy of Sciences, 100, 1393-1398. • WATSON, S., CHAMBERS, D., HOBBS, C., DOHERTY, P. & GRAHAM, A. 2008. The endocannabinoid receptor, CB1, is required for normal axonal growth and fasciculation. Molecular and Cellular Neuroscience, 38, 89-97.