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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. - PowerPoint PPT Presentation
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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