#METABOLOMICS WORKBENCH jburgess_20160309_070157 DATATRACK_ID:551 STUDY_ID:ST000365 ANALYSIS_ID:AN000598 VERSION 1 CREATED_ON 08-09-2023 #PROJECT PR:PROJECT_TITLE Zebrafish Metabolomics: Model for Environmental Metal Toxicity PR:PROJECT_SUMMARY This metabolomics seed project will test the hypothesis that zebrafish can PR:PROJECT_SUMMARY provide mechanistic insights into the human health effects of developmental PR:PROJECT_SUMMARY exposure to Cd and Pb. We will use broad spectrum metabolomics of zebrafish PR:PROJECT_SUMMARY larvae after exposure to Cd, Pb, and Cd and Pb compared to controls. Activity PR:PROJECT_SUMMARY observed at 5 days post fertilization is will be used to determine if there is a PR:PROJECT_SUMMARY correlation between biological pathways implicated by these metabolic profiles PR:PROJECT_SUMMARY and cardiovascular, metabolic (obesity), and neurological phenotypes. The PR:PROJECT_SUMMARY behavioral phenotypes have been quantified in zebrafish previously and have been PR:PROJECT_SUMMARY measured in the Newborn Epigenetic STudy (NEST), a NIH-funded project that is PR:PROJECT_SUMMARY investigating how environmental exposures and nutrition, in the womb and during PR:PROJECT_SUMMARY childhood, affect how genes work and how these exposures developed into obesity PR:PROJECT_SUMMARY and other diseases, disorders, and conditions. The results from this study will PR:PROJECT_SUMMARY demonstrate the power of using zebrafish as a model for mechanism discovery in PR:PROJECT_SUMMARY exposure using metabolomics to advance understanding of early life exposure to PR:PROJECT_SUMMARY Cd and Pb and serve as preliminary data for opportunities. PR:INSTITUTE North Carolina State University PR:LAST_NAME Mattingly PR:FIRST_NAME Carolyn PR:ADDRESS 142 David Clark Labs, Raleigh, NC 27695-7617 PR:EMAIL cjmattin@ncsu.edu PR:PHONE 919-515-2024 PR:DOI http://dx.doi.org/10.21228/M8JW2K #STUDY ST:STUDY_TITLE Zebrafish Metabolomics: Model for Environmental Metal Toxicity ST:STUDY_SUMMARY This metabolomics seed project will test the hypothesis that zebrafish can ST:STUDY_SUMMARY provide mechanistic insights into the human health effects of developmental ST:STUDY_SUMMARY exposure to Cd and Pb. We will use broad spectrum metabolomics of zebrafish ST:STUDY_SUMMARY larvae after exposure to Cd, Pb, and Cd and Pb compared to controls. Activity ST:STUDY_SUMMARY observed at 5 days post fertilization is will be used to determine if there is a ST:STUDY_SUMMARY correlation between biological pathways implicated by these metabolic profiles ST:STUDY_SUMMARY and cardiovascular, metabolic (obesity), and neurological phenotypes. The ST:STUDY_SUMMARY behavioral phenotypes have been quantified in zebrafish previously and have been ST:STUDY_SUMMARY measured in the Newborn Epigenetic STudy (NEST), a NIH-funded project that is ST:STUDY_SUMMARY investigating how environmental exposures and nutrition, in the womb and during ST:STUDY_SUMMARY childhood, affect how genes work and how these exposures developed into obesity ST:STUDY_SUMMARY and other diseases, disorders, and conditions. The results from this study will ST:STUDY_SUMMARY demonstrate the power of using zebrafish as a model for mechanism discovery in ST:STUDY_SUMMARY exposure using metabolomics to advance understanding of early life exposure to ST:STUDY_SUMMARY Cd and Pb and serve as preliminary data for opportunities. ST:INSTITUTE University of North Carolina ST:DEPARTMENT Discovery Sciences ST:LABORATORY Sumner Lab ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS Eastern Regional Comprehensive Metabolomics Resource Core, UNC Nutrition ST:ADDRESS Research Institute, 500 Laureate Way, Kannapolis, NC, 28081 ST:EMAIL susan_sumner @unc.edu ST:PHONE 704-250-5066 ST:SUBMIT_DATE 2016-03-09 #SUBJECT SU:SUBJECT_TYPE Zebrafish SU:SUBJECT_SPECIES Danio rerio SU:TAXONOMY_ID 7955 SU:SPECIES_GROUP Fish #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - CM_CdPool_1 Metal Exposure:Cd | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_CdPool_2 Metal Exposure:Cd | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_CdPool_3 Metal Exposure:Cd | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_Cdhigh_1 Metal Exposure:Cd | Exposure Level:high (1000 ppb) SUBJECT_SAMPLE_FACTORS - CM_Cdhigh_2 Metal Exposure:Cd | Exposure Level:high (1000 ppb) SUBJECT_SAMPLE_FACTORS - CM_Cdhigh_3 Metal Exposure:Cd | Exposure Level:high (1000 ppb) SUBJECT_SAMPLE_FACTORS - CM_Cdlow_1 Metal Exposure:Cd | Exposure Level:low (10 ppb) SUBJECT_SAMPLE_FACTORS - CM_Cdlow_2 Metal Exposure:Cd | Exposure Level:low (10 ppb) SUBJECT_SAMPLE_FACTORS - CM_Cdlow_3 Metal Exposure:Cd | Exposure Level:low (10 ppb) SUBJECT_SAMPLE_FACTORS - CM_Cdmid_1 Metal Exposure:Cd | Exposure Level:mid (100 ppb) SUBJECT_SAMPLE_FACTORS - CM_Cdmid_2 Metal Exposure:Cd | Exposure Level:mid (100 ppb) SUBJECT_SAMPLE_FACTORS - CM_Cdmid_3 Metal Exposure:Cd | Exposure Level:mid (100 ppb) SUBJECT_SAMPLE_FACTORS - CM_CdPbPool_1 Metal Exposure:Cd+Pb | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_CdPbPool_2 Metal Exposure:Cd+Pb | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_CdPbPool_3-R Metal Exposure:Cd+Pb | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_TotalPool_1 Metal Exposure:CD+Pb | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_TotalPool_2 Metal Exposure:CD+Pb | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_TotalPool_3 Metal Exposure:CD+Pb | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_CdPbhigh_1 Metal Exposure:Cd+Pb | Exposure Level:high (1000 ppb Cd and 1000 ppb Pb) SUBJECT_SAMPLE_FACTORS - CM_CdPbhigh_2 Metal Exposure:Cd+Pb | Exposure Level:high (1000 ppb Cd and 1000 ppb Pb) SUBJECT_SAMPLE_FACTORS - CM_CdPbhigh_3 Metal Exposure:Cd+Pb | Exposure Level:high (1000 ppb Cd and 1000 ppb Pb) SUBJECT_SAMPLE_FACTORS - CM_CdPblow_1 Metal Exposure:Cd+Pb | Exposure Level:low (10 ppb Cd and 10 ppb Pb) SUBJECT_SAMPLE_FACTORS - CM_CdPblow_2 Metal Exposure:Cd+Pb | Exposure Level:low (10 ppb Cd and 10 ppb Pb) SUBJECT_SAMPLE_FACTORS - CM_CdPblow_3 Metal Exposure:Cd+Pb | Exposure Level:low (10 ppb Cd and 10 ppb Pb) SUBJECT_SAMPLE_FACTORS - CM_CdPBmid_1 Metal Exposure:Cd+Pb | Exposure Level:mid (100 ppb Cd and 100 ppb Pb) SUBJECT_SAMPLE_FACTORS - CM_CdPBmid_2 Metal Exposure:Cd+Pb | Exposure Level:mid (100 ppb Cd and 100 ppb Pb) SUBJECT_SAMPLE_FACTORS - CM_CdPBmid_3 Metal Exposure:Cd+Pb | Exposure Level:mid (100 ppb Cd and 100 ppb Pb) SUBJECT_SAMPLE_FACTORS - CM_NoExpPool_1 Metal Exposure:control | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_NoExpPool_2 Metal Exposure:control | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_NoExpPool_3 Metal Exposure:control | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_NoExp_1 Metal Exposure:control | Exposure Level:No SUBJECT_SAMPLE_FACTORS - CM_NoExp_2 Metal Exposure:control | Exposure Level:No SUBJECT_SAMPLE_FACTORS - CM_NoExp_3 Metal Exposure:control | Exposure Level:No SUBJECT_SAMPLE_FACTORS - CM_PbPool_1 Metal Exposure:Pb | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_PbPool_2 Metal Exposure:Pb | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_PbPool_3-R Metal Exposure:Pb | Exposure Level:- SUBJECT_SAMPLE_FACTORS - CM_Pbhigh_1 Metal Exposure:Pb | Exposure Level:high (1000 ppb) SUBJECT_SAMPLE_FACTORS - CM_Pbhigh_2 Metal Exposure:Pb | Exposure Level:high (1000 ppb) SUBJECT_SAMPLE_FACTORS - CM_Pbhigh_3 Metal Exposure:Pb | Exposure Level:high (1000 ppb) SUBJECT_SAMPLE_FACTORS - CM_Pblow_1 Metal Exposure:Pb | Exposure Level:low (10 ppb) SUBJECT_SAMPLE_FACTORS - CM_Pblow_2 Metal Exposure:Pb | Exposure Level:low (10 ppb) SUBJECT_SAMPLE_FACTORS - CM_Pblow_3 Metal Exposure:Pb | Exposure Level:low (10 ppb) SUBJECT_SAMPLE_FACTORS - CM_Pbmid_1 Metal Exposure:Pb | Exposure Level:mid (100 ppb) SUBJECT_SAMPLE_FACTORS - CM_Pbmid_2 Metal Exposure:Pb | Exposure Level:mid (100 ppb) SUBJECT_SAMPLE_FACTORS - CM_Pbmid_3 Metal Exposure:Pb | Exposure Level:mid (100 ppb) #COLLECTION CO:COLLECTION_SUMMARY The larvae were collected at 48 hours post fertilization (hpf). CO:SAMPLE_TYPE Fish larvae #TREATMENT TR:TREATMENT_SUMMARY Zebrafish larvae were exposured to Cd (10, 100, and 1000 ppb), Pb (10, 100, and TR:TREATMENT_SUMMARY 1000 ppb), and Cd and Pb (10 ppb Cd and 10 ppb Pb, 100 ppb Cd and 100 ppb Pb, TR:TREATMENT_SUMMARY and 1000 ppb Cd and 1000 ppb Pb) and compared to controls. The exposure period TR:TREATMENT_SUMMARY was between 6-48 hours post fertilization (hpf). The larvae were collected at 48 TR:TREATMENT_SUMMARY hpf. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Pools with 40 larvae each were washed and snap frozen before being shipped to SP:SAMPLEPREP_SUMMARY the NIH RTI-RCMRC on dry ice and immediately stored at -80 °C after being SP:SAMPLEPREP_SUMMARY logged in for metabolomics analysis. A total of 30 study samples were SP:SAMPLEPREP_SUMMARY lyophilized overnight for sample preparation. Each sample was mixed with 750 μl SP:SAMPLEPREP_SUMMARY of methanol, vortexed, and centrifuged. 300 μl of the supernatant were used for SP:SAMPLEPREP_SUMMARY the study samples, phenotypic pools were created using 350 μl of each phenotype SP:SAMPLEPREP_SUMMARY and a total study pool was created by mixing 35 μl from each sample. Three SP:SAMPLEPREP_SUMMARY aliquots were created from each pool for a total of 15 pooled samples. All SP:SAMPLEPREP_SUMMARY samples were dried in vacuum overnight. Each sample was reconstituted with 250 SP:SAMPLEPREP_SUMMARY μl of 0.5 mM phosphate buffer (pH 7.5) and 25 μl of d-DSS/Chenomx as an SP:SAMPLEPREP_SUMMARY internal standard. The tubes were vortexed for 2 min on a multi-tube vortexer SP:SAMPLEPREP_SUMMARY and centrifuged at 16,000 rcf for 10 min. A 200 µl aliquot of the supernatant SP:SAMPLEPREP_SUMMARY was transferred into pre-labeled 3 mm (4”) NMR tubes for data acquisition on a SP:SAMPLEPREP_SUMMARY 700 MHz spectrometer. 1H NMR spectra of zebrafish extraction samples were SP:SAMPLEPREP_SUMMARY acquired on a Bruker 700 MHz NMR spectrometer (located at David H. Murdock SP:SAMPLEPREP_SUMMARY Research Institute, Kannapolis, NC, USA) using a 5 mm cryogenically cooled ATMA SP:SAMPLEPREP_SUMMARY inverse probe and ambient temperature of 25 ℃. A 1D NOESY presaturation pulse SP:SAMPLEPREP_SUMMARY sequence (noesypr1d, [recycle delay (RD)-90°-t1-90°-tm-90°-acquire free SP:SAMPLEPREP_SUMMARY induction decay (FID) was used for data acquisition. For each sample 32 SP:SAMPLEPREP_SUMMARY transients were collected into 65k data points using a spectral width of 17.17 SP:SAMPLEPREP_SUMMARY ppm, 2 s relaxation delay, 100 ms mixing time, and an acquisition time of 3.9 s SP:SAMPLEPREP_SUMMARY per FID. The water resonance was suppressed using resonance irradiation during SP:SAMPLEPREP_SUMMARY the relaxation delay and mixing time. NMR spectra were processed using TopSpin SP:SAMPLEPREP_SUMMARY 3.2 software (Bruker-Biospin, Germany). Spectra were zero filled, and Fourier SP:SAMPLEPREP_SUMMARY transformed after exponential multiplication with line broadening factor of 0.5. SP:SAMPLEPREP_SUMMARY Phase and baseline of the spectra were manually corrected for each spectrum. SP:SAMPLEPREP_SUMMARY Spectra were referenced internally to the DSS-d6 signal. The quality of each NMR SP:SAMPLEPREP_SUMMARY spectrum was assessed for the level of noise and alignment of identified SP:SAMPLEPREP_SUMMARY markers. Spectra were assessed for missing data and underwent quality checks. #CHROMATOGRAPHY #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker Avance III NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D 1H NM:SPECTROMETER_FREQUENCY 700 MHz #NMR_BINNED_DATA NMR_BINNED_DATA_START Bin range(ppm) CM_CdPool_1 CM_CdPool_2 CM_CdPool_3 CM_Cdhigh_1 CM_Cdhigh_2 CM_Cdhigh_3 CM_Cdlow_1 CM_Cdlow_2 CM_Cdlow_3 CM_Cdmid_1 CM_Cdmid_2 CM_Cdmid_3 CM_CdPbPool_1 CM_CdPbPool_2 CM_CdPbPool_3-R CM_TotalPool_1 CM_TotalPool_2 CM_TotalPool_3 CM_CdPbhigh_1 CM_CdPbhigh_2 CM_CdPbhigh_3 CM_CdPblow_1 CM_CdPblow_2 CM_CdPblow_3 CM_CdPBmid_1 CM_CdPBmid_2 CM_CdPBmid_3 CM_NoExpPool_1 CM_NoExpPool_2 CM_NoExpPool_3 CM_NoExp_1 CM_NoExp_2 CM_NoExp_3 CM_PbPool_1 CM_PbPool_2 CM_PbPool_3-R CM_Pbhigh_1 CM_Pbhigh_2 CM_Pbhigh_3 CM_Pblow_1 CM_Pblow_2 CM_Pblow_3 CM_Pbmid_1 CM_Pbmid_2 CM_Pbmid_3 NMR_BINNED_DATA_END #END