Maria S. Sepúlveda, DVM, PhD Associate Professor April 25, 2011

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

2. 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 Source: Wattendorf et al. 2005. Am Fam. Physician. 72:279-285.

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

4. 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 Sources: Klein et al. 1995. Therap. Drug Monit. 21: 644; Moore et al. 2003. Clin. Chem. 49:133-136

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

6. Zebrafish Metabolomics: Methods Two strains of ZF: 1. Wild Type strain (AB) ---- an ethanol-sensitive strain 2. Blue Long-Fin strain (BLF)---- an ethanol-resistant strain (Dlugos and Rabin,2003) • Four treatment groups: • 0 mg/dl EtOH (control) • 100 mg/dl EtOH • 200 mg/dl EtOH • 300 mg/dl EtOH

7. Zebrafish Metabolomics: Methods Zebrafish breeding Embryos collected 2.5 - 3 h 10 embryos per well 120 embryos 120 embryos 120 embryos 120 embryos control 100 mg/dl 200 mg/dl 300 mg/dl 24 h at 28.5 °C

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

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

10. LC-MS Cluster Analysis: BLF strain Control 300 mg/dl EtOH 200 mg/dl EtOH 100 mg/dl EtOH

11. LC-MS Cluster Analysis: AB strain Control 300 mg/dl EtOH 100 mg/dl EtOH 200 mg/dl EtOH

12. 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. Acknowledgments • Feng Zhou, Indiana University • Jiri Adamec, University of Nebraska • Amber Hopf, Purdue University) • AePolsuwan & Dr. Praneet Damrongphol (Mahidol University, Thailand) • Funding: CTSI, TRAC 1