Development of a High Throughput Development of a High Throughput in Vivo System using Zebrafish for in Vivo System using Zebrafish for

the Evaluation of Fetal Alcohol the Evaluation of Fetal Alcohol SyndromeSyndrome

Maria S. Sepúlveda, DVM, PhDAssociate Professor

April 25, 2011

Department of Forestry and Natural Resources and School of Civil Engineering

Purdue University

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

Fetal Alcohol Spectrum DisordersFetal 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.

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 DisordersFetal Alcohol Spectrum Disorders

FAS

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

Fetal Alcohol Spectrum DisordersFetal 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

Identify novel metabolites indicative of ethanol

exposure using a metabolomics approach in

zebrafish embryos

Zebrafish Metabolomics: ObjectivesZebrafish Metabolomics: Objectives

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: MethodsZebrafish Metabolomics: Methods

Zebrafish breeding

Embryos collected

control 100 mg/dl 200 mg/dl 300 mg/dl

10 embryos per well

2.5 - 3 h

24 h at 28.5 °C

120 embryos 120 embryos 120 embryos120 embryos

Zebrafish Metabolomics: MethodsZebrafish Metabolomics: Methods

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: MethodsZebrafish Metabolomics: Methods

Polar phase in methanol

Non-polar phasein chloroform

72 samples (40 samples from AB and 32 samples from BLF)

Metabolite extraction

GCxGC/MS LC-MS/TOF

(5 embryos/sample)

Zebrafish Metabolomics: MethodsZebrafish Metabolomics: Methods

LC-MS Cluster Analysis: BLF strain

Control 100 mg/dl EtOH200 mg/dl EtOH300 mg/dl EtOH

Control 100 mg/dl EtOH 200 mg/dl EtOH300 mg/dl EtOH

LC-MS Cluster Analysis: AB strain

HMDB ID Class Common Name Mean. control

HMDB02152 Retinoid Vitamin A -12 HMDB07006 Glycerophospholipid Cyclic Phosphatidic Acid -10 HMDB12313 Glycosphingolipid 3-O-Sulfogalactosylceramide (d18:1/16:0) -8 HMDB00816 Short chain acyl phosphate Phosphoglycolic acid -8 HMDB09907 Long Chain PUFA Docosatrienoyl/Palmitic -7 HMDB09846 Long Chain PUFA Linoleic/Palmitic -6 HMDB09915 Long Chain PUFA Adrenic/Stearic -6 HMDB02053 Polypeptide Histidylproline diketopiperazine -6 HMDB09681 Long Chain PUFA Docosahexaenoic/Pentadecanoic -5 HMDB09682 Long Chain PUFA Docosahexaenoic/Palmitic -5 HMDB08740 Long Chain PUFA Docosahexaenoic/Eicsoatetraenoic -5 HMDB03073 Long Chain Fatty Acid Gamma-Linolenic acid 0.1 HMDB03764 Polypeptide Glutamylalanine 0.1 HMDB01991 Nucleoside 7-Methylxanthine 0.1 HMDB00798 Fatty Acid (Ester) Ethyl heptanoic acid 0.1 HMDB00529 Medium Chain Fatty Acid 5-Dodecenoic acid 0.1 HMDB01474 Alcohol (Phenol) 3,4-Dihydroxyphenylglycol O-sulfate 0.1 HMDB01530 Alcohol Phosphate Mannitol 1-phosphate 0.1 HMDB10736 Long Chain Fatty Acid 3-Oxooctadecanoic acid 3 HMDB00511 Medium Chain Fatty Acid Capric acid 3 HMDB00745 Polypedtide Homocarnosine 3 HMDB00318 Alcohol (Phenol) 3,4-Dihydroxyphenylglycol 4 HMDB01931 Aromatic Acid Gamma-CEHC 4 HMDB04874 Glycolipid Lactosylceramide (d18:1/26:0) 5 HMDB00732 Amino Acid Hydroxykynurenine 5

Zebrafish Metabolomics: ResultsZebrafish Metabolomics: ResultsMost Significant Metabolites AB StrainMost Significant Metabolites AB Strain

Zebrafish Metabolomics: ConclusionsZebrafish 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

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

AcknowledgmentsAcknowledgments