1. Development of a High Throughput in Vivo System using Zebrafish for the Evaluation of Fetal Alcohol Syndrome Maria S. Sepúlveda, DVM, PhD Associate Professor April 25, 2011 Department of Forestry and Natural Resources and School of Civil Engineering Purdue University

2. Source: Wattendorf et al. 2005. Am Fam. Physician. 72:279-285. Fetal Alcohol Spectrum Disorders  Fetal alcohol spectrum disorders (FASD) are caused by the effects of maternal alcohol consumption during pregnancy  Fetal alcohol syndrome (FAS) is the most clinically recognizable form of FASD:  Pattern of minor facial anomalies  Prenatal and postnatal growth retardation  Functional and/or structural CNS abnormalities

3. Source: Wattendorf et al. 2005. Am Fam. Physician. 72:279-285. Characteristic facial features in a child with fetal alcohol spectrum disorders. Characteristic facial features in children of different ethnicities with fetal alcohol spectrum disorders. Fetal Alcohol Spectrum Disorders FAS

4. Sources: Klein et al. 1995. Therap. Drug Monit. 21: 644; Moore et al. 2003. Clin. Chem. 49:133-136 Fetal Alcohol Spectrum Disorders  The consequences of FASD are lifelong, and the behavioral and learning difficulties are often greater than the degree of neurocognitive impairment  Biomarkers of early diagnosis:  Ethanol is a potent modulator of lipid metabolism  Fatty Acid Ethyl Esters (FAEE): Palmitic, linoleic, and stearic  FAEEs are formed by esterification of ethanol with free fatty acids and trans-esterification of glyceride (> 10,000 ng/g meconium= FAS)  Metabolites in meconium, placenta, and hair

5. Identify novel metabolites indicative of ethanol exposure using a metabolomics approach in zebrafish embryos Zebrafish Metabolomics: Objectives

6. Two strains of ZF: 1. Wild Type strain (AB) ---- an ethanol-sensitive strain 2. Blue Long-Fin strain (BLF)---- an ethanol-resistant strain Four treatment groups: 1.0 mg/dl EtOH (control) 2.100 mg/dl EtOH 3.200 mg/dl EtOH 4.300 mg/dl EtOH ( Dlugos and Rabin,2003) Zebrafish Metabolomics: Methods

7. Zebrafish breeding Embryos collected control100 mg/dl200 mg/dl300 mg/dl 10 embryos per well 2.5 - 3 h 24 h at 28.5 °C 120 embryos Zebrafish Metabolomics: Methods

8. Determined survival rate of embryos Collected embryo samples for metabolomics Hatching Collected larvae for morphological evaluations Remaining embryos were transferred to freshwater and allowed to hatch 72 h at 28.5 °C Zebrafish Metabolomics: Methods

9. Polar phase in methanol Non-polar phase in chloroform 72 samples (40 samples from AB and 32 samples from BLF) Metabolite extraction GCxGC/MS LC-MS/TOF (5 embryos/sample ) Zebrafish Metabolomics: Methods

10. LC-MS Cluster Analysis: BLF strain Control 100 mg/dl EtOH200 mg/dl EtOH300 mg/dl EtOH

11. Control 100 mg/dl EtOH200 mg/dl EtOH 300 mg/dl EtOH LC-MS Cluster Analysis: AB strain

12. HMDB IDClassCommon NameMean. control HMDB02152RetinoidVitamin A-12  HMDB07006GlycerophospholipidCyclic Phosphatidic Acid-10  HMDB12313Glycosphingolipid3-O-Sulfogalactosylceramide (d18:1/16:0)-8  HMDB00816Short chain acyl phosphatePhosphoglycolic acid-8  HMDB09907Long Chain PUFADocosatrienoyl/Palmitic-7  HMDB09846Long Chain PUFALinoleic/Palmitic-6  HMDB09915Long Chain PUFAAdrenic/Stearic-6  HMDB02053PolypeptideHistidylproline diketopiperazine-6  HMDB09681Long Chain PUFADocosahexaenoic/Pentadecanoic-5  HMDB09682Long Chain PUFADocosahexaenoic/Palmitic-5  HMDB08740Long Chain PUFADocosahexaenoic/Eicsoatetraenoic -5  HMDB03073Long Chain Fatty AcidGamma-Linolenic acid0.1  HMDB03764PolypeptideGlutamylalanine0.1  HMDB01991Nucleoside7-Methylxanthine0.1  HMDB00798Fatty Acid (Ester)Ethyl heptanoic acid0.1  HMDB00529Medium Chain Fatty Acid5-Dodecenoic acid0.1  HMDB01474Alcohol (Phenol)3,4-Dihydroxyphenylglycol O-sulfate0.1  HMDB01530Alcohol PhosphateMannitol 1-phosphate0.1  HMDB10736Long Chain Fatty Acid3-Oxooctadecanoic acid3  HMDB00511Medium Chain Fatty AcidCapric acid3  HMDB00745PolypedtideHomocarnosine3  HMDB00318Alcohol (Phenol)3,4-Dihydroxyphenylglycol4  HMDB01931Aromatic AcidGamma-CEHC4  HMDB04874GlycolipidLactosylceramide (d18:1/26:0)5  HMDB00732Amino AcidHydroxykynurenine5  Zebrafish Metabolomics: Results Most Significant Metabolites AB Strain

13. Zebrafish Metabolomics: Conclusions  BLF appears more resistant to the effects of ethanol  Zebrafish embryos exposed to 200-300 mg/dl ethanol responded with alterations in fatty acid profiles  These findings corroborate previous studies with mammalian systems, including humans  Novel metabolites were also identified (Vit. A)  Overall, results support the use of zebrafish as a model for studying FASD

14.  Feng Zhou, Indiana University  Jiri Adamec, University of Nebraska  Amber Hopf, Purdue University)  Ae Polsuwan & Dr. Praneet Damrongphol (Mahidol University, Thailand)  Funding: CTSI, TRAC 1 Acknowledgments