#METABOLOMICS WORKBENCH cwalker_bcm_20200127_072952 DATATRACK_ID:1909 STUDY_ID:ST001309 ANALYSIS_ID:AN002180 PROJECT_ID:PR000890 VERSION 1 CREATED_ON January 29, 2020, 2:04 pm #PROJECT PR:PROJECT_TITLE Metabolomic profiling after early-life exposure to an endocrine disrupting PR:PROJECT_TITLE chemical in the liver. PR:PROJECT_TYPE Targeted MS analysis PR:PROJECT_SUMMARY Metabolic profiling in the liver (240 days post-natal) after early-life exposure PR:PROJECT_SUMMARY to an endocrine disrupting chemical. PR:INSTITUTE Baylor College of Medicine PR:LAST_NAME Walker PR:FIRST_NAME Cheryl PR:ADDRESS 1 Baylor Plaza, Houston, TX, 77030, USA PR:EMAIL Cheryl.walker@bcm.edu PR:PHONE 713-798-8219 #STUDY ST:STUDY_TITLE Metabolite expression in liver after early life exposure to an endocrine ST:STUDY_TITLE disruptor at 240 days postnatal (part-I) ST:STUDY_TYPE Metabolite expression after chemical exposure versus control. ST:STUDY_SUMMARY Our early-life environment has a profound influence on developing organs that ST:STUDY_SUMMARY impact metabolic function and determines disease susceptibility across the ST:STUDY_SUMMARY life-course. Using a rat model for exposure to an endocrine disrupting chemical ST:STUDY_SUMMARY (EDC), we show that early-life exposure causes metabolic dysfunction in ST:STUDY_SUMMARY adulthood and reprograms histone marks in the developing liver to accelerate ST:STUDY_SUMMARY acquisition of an adult epigenomic signature. This epigenomic reprogramming ST:STUDY_SUMMARY persists long after the initial exposure, but many reprogrammed genes remain ST:STUDY_SUMMARY transcriptionally silent with their impact on metabolism not revealed until a ST:STUDY_SUMMARY later life exposure to a Western-style diet. Diet-dependent metabolic disruption ST:STUDY_SUMMARY was largely driven by reprogramming of the Early Growth Response 1 (EGR1) ST:STUDY_SUMMARY transcriptome and production of metabolites in pathways linked to cholesterol, ST:STUDY_SUMMARY lipid and one-carbon metabolism. These findings demonstrate the importance of ST:STUDY_SUMMARY epigenome: environment interactions, which early in life accelerate epigenomic ST:STUDY_SUMMARY aging, and later in adulthood unlock metabolically restricted epigenetic ST:STUDY_SUMMARY reprogramming to drive metabolic dysfunction. ST:INSTITUTE Baylor College of Medicine ST:DEPARTMENT Molecular and Cellular Biology ST:LABORATORY Center for Precision Environmental Health ST:LAST_NAME Walker ST:FIRST_NAME Cheryl ST:ADDRESS 1 Baylor Plaza, Houston, TX, 77030, USA ST:EMAIL Cheryl.walker@bcm.edu ST:PHONE 713-798-8219 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 10 ST:NUM_MALES 10 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 SU:GENOTYPE_STRAIN Sprague Dawley SU:AGE_OR_AGE_RANGE 240 days SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Harlan SU:ANIMAL_HOUSING polycarbonate-free caging SU:ANIMAL_LIGHT_CYCLE 14-hr light and 10-hr dark SU:ANIMAL_FEED Phytoestrogen Reduced II 18-5 (Ziegler Bros, Inc) or D09100301 (Research Diets, SU:ANIMAL_FEED Inc) #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - HFD-VEH13 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH15 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH18 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH12 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-VEH14 Treatment:vehicle Day of Liver Harvest Post-Birth=240; BPA_exposure=0 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA12 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA13 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA14 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA16 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. SUBJECT_SAMPLE_FACTORS - HFD-BPA18 Treatment:BPA Day of Liver Harvest Post-Birth=240; BPA_exposure=50 ug/kg post natal days 1, 3, and 5; Diet=phytoestrogen-reduced a diet for first 180 days post birth then diet high in fat (40% kcal), fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days. #COLLECTION CO:COLLECTION_SUMMARY Liver tissue was harvested on post-natal day 240 after challenge with CO:COLLECTION_SUMMARY Western-style diet.Tissue was snap-frozen in liquid nitrogen. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Neonatal rats were treated with vehicle (sesame oil) or bisphenol A (BPA; 50 TR:TREATMENT_SUMMARY µg/kg dissolved in sesame oil) orally via pipette tip on post-natal days 1, 3, TR:TREATMENT_SUMMARY and 5. Littermates were randomly assigned to the treatment groups. BPA was TR:TREATMENT_SUMMARY obtained from the National Institute of Environmental Health Sciences (NIEHS). TR:TREATMENT_SUMMARY The dose and route of administration recapitulates human exposure to BPA. At day TR:TREATMENT_SUMMARY 180, adult rats in both treatment groups were fed a diet high in fat (40% kcal), TR:TREATMENT_SUMMARY fructose (20% kcal) and cholesterol (2%) (Western-style diet) for 60 days TR:TREATMENT_SUMMARY (D09100301, Research Diets, Inc). Rats were fasted overnight prior to tissue TR:TREATMENT_SUMMARY collection. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from crushed liver samples and a mouse liver pool was SP:SAMPLEPREP_SUMMARY used for quality control. Twenty-five mg of crushed liver was used for the SP:SAMPLEPREP_SUMMARY metabolic extraction. The extraction step started with the addition of 750 µL SP:SAMPLEPREP_SUMMARY ice-cold methanol:water (4:1) containing 20 µL spiked internal standards to SP:SAMPLEPREP_SUMMARY each tissue sample. Ice-cold chloroform and water were added in a 3:1 ratio for SP:SAMPLEPREP_SUMMARY a final proportion of 1:4:3:1 water:methanol:chloroform:water. The organic SP:SAMPLEPREP_SUMMARY (methanol and chloroform) and aqueous layers were mixed, dried and resuspended SP:SAMPLEPREP_SUMMARY with 50:50 methanol: water. The extract was deproteinized using a 3kDa molecular SP:SAMPLEPREP_SUMMARY filter (Amicon ultracel-3K Membrane; Millipore Corporation, Billerica, MA) and SP:SAMPLEPREP_SUMMARY the filtrate was dried under vacuum (Genevac EZ-2plus; Gardiner, Stone Ridge, SP:SAMPLEPREP_SUMMARY NY). Prior to mass spectrometry, the dried extracts were re-suspended in SP:SAMPLEPREP_SUMMARY identical volumes of injection solvent composed of 1:1 water: methanol and were SP:SAMPLEPREP_SUMMARY subjected to liquid chromatography-mass spectrometry. Fifty µl of sample was SP:SAMPLEPREP_SUMMARY used for preparation. Internal standards were spiked into the samples. Then it SP:SAMPLEPREP_SUMMARY was processed through a 3 kDa filter. After that, 50 µl of sample was diluted SP:SAMPLEPREP_SUMMARY with 450 µl solvent (methanol: water = 50:50 v/v) and subjected to LC/MS SP:SAMPLEPREP_SUMMARY analysis. The injection volume was 10 µl. For internal standards, SP:SAMPLEPREP_SUMMARY high-performance liquid chromatography (HPLC)-grade acetonitrile, methanol, and SP:SAMPLEPREP_SUMMARY water were procured from Burdick & Jackson (Morristown, NJ). Mass SP:SAMPLEPREP_SUMMARY spectrometry-grade formic acid was purchased from Sigma-Aldrich (St Louis, MO). SP:SAMPLEPREP_SUMMARY Calibration solution containing multiple calibrants in a solution of SP:SAMPLEPREP_SUMMARY acetonitrile, trifluroacetic acid, and water was purchased from Agilent SP:SAMPLEPREP_SUMMARY Technologies (Santa Clara, CA). Metabolites and internal standards, including SP:SAMPLEPREP_SUMMARY N-acetyl Aspartic acid-d3, Tryptophan-15N2, Sarcosine-d3, Glutamic acid-d5, SP:SAMPLEPREP_SUMMARY Thymine-d4, Gibberellic acid, Trans-Zeatine, Jasmonic acid, 15N Anthranilic SP:SAMPLEPREP_SUMMARY acid, and Testosterone-d3, were purchased from Sigma-Aldrich (St. Louis, MO). SP:SAMPLEPREP_SUMMARY Three LC- MS methods were used to separate metabolites. Method A: In ESI SP:SAMPLEPREP_SUMMARY positive mode the HPLC column was waters X-bridge amide 3.5 µm, 4.6 x 100 mm SP:SAMPLEPREP_SUMMARY (Waters, Milford, MA). Mobile phase A and B were 0.1% formic acid in water and SP:SAMPLEPREP_SUMMARY acetonitrile, respectively. Gradient flow: 0-3 min 85% B; 3-12 min 30% B, 12-15 SP:SAMPLEPREP_SUMMARY min 2% B, 16 min 95%B, followed by re-equilibration till the end of the gradient SP:SAMPLEPREP_SUMMARY 23 min to the initial starting condition of 85% B. Flow rate of the solvents SP:SAMPLEPREP_SUMMARY used for the analysis is 0.3 ml/min. Injection volume was 10 µL. Method B: In SP:SAMPLEPREP_SUMMARY ESI negative mode the HPLC column was waters X-bridge amide 3.5 µm, 4.6 x 100 SP:SAMPLEPREP_SUMMARY mm (Waters, Milford, MA). Mobile phase A and B were 20 mM ammonium acetate in SP:SAMPLEPREP_SUMMARY water with pH 9.0 and 100% acetonitrile, respectively. Gradient flow: 0-3 min SP:SAMPLEPREP_SUMMARY 85% B, 3-12 min 30% B, 12-15 min 2% B, 15-16 min 85% B followed by SP:SAMPLEPREP_SUMMARY re-equilibration till the end of the gradient 23 min to the initial starting SP:SAMPLEPREP_SUMMARY condition of 85% B. Flow rate of the solvents used for analysis is 0.3 ml/min. SP:SAMPLEPREP_SUMMARY Injection volume was 10 µL. Method C: In ESI positive mode the HPLC column was SP:SAMPLEPREP_SUMMARY Luna 3 µM NH2 100 A0 Chromatography column (Phenomenex, Torrance, CA). Mobile SP:SAMPLEPREP_SUMMARY phase A and B were 20 mM ammonium acetate in water with pH 9.0 and 100% SP:SAMPLEPREP_SUMMARY acetonitrile, respectively. Gradient flow: 0-3 min 85% B, 3-12 min 30% B, 12-15 SP:SAMPLEPREP_SUMMARY min 2% B, 15-16 min 85% B followed by re-equilibration till the end of the SP:SAMPLEPREP_SUMMARY gradient 23 min to the initial starting condition of 85% B. Flow rate of the SP:SAMPLEPREP_SUMMARY solvents used for analysis is 0.3 ml/min. Injection volume was 10 µL. For data SP:SAMPLEPREP_SUMMARY acquisition through LC/MS analysis, 10 µL of suspended samples were injected SP:SAMPLEPREP_SUMMARY and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent SP:SAMPLEPREP_SUMMARY Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, SP:SAMPLEPREP_SUMMARY Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were SP:SAMPLEPREP_SUMMARY as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; SP:SAMPLEPREP_SUMMARY Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V SP:SAMPLEPREP_SUMMARY positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V SP:SAMPLEPREP_SUMMARY negative. Approximately 8–11 data points were acquired per detected SP:SAMPLEPREP_SUMMARY metabolite. The data acquired using Agilent mass hunter software and data was SP:SAMPLEPREP_SUMMARY analyzed using mass hunter quantitative analysis software. SP:SAMPLEPREP_PROTOCOL_FILENAME Targeted.MS.method.pdf;unbiased.liver.MS.method.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 6495 QQQ CH:COLUMN_NAME Phenomenex Luna NH2 (150 x 2.1mm, 3um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS For data acquisition through LC/MS analysis, 10 µL of suspended samples were MS:MS_COMMENTS injected and analyzed using a 6495 triple quadrupole mass spectrometer (Agilent MS:MS_COMMENTS Technologies, Santa Clara, CA) coupled to a HPLC system (Agilent Technologies, MS:MS_COMMENTS Santa Clara, CA) via Multiple reaction monitoring (MRM). Source parameters were MS:MS_COMMENTS as follows: Gas temperature- 250°C; Gas flow- 14 l/min; Nebulizer - 20psi; MS:MS_COMMENTS Sheath gas temperature - 350°C; Sheath gas flow- 12 l/min; Capillary - 3000 V MS:MS_COMMENTS positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V MS:MS_COMMENTS negative. Approximately 8–11 data points were acquired per detected MS:MS_COMMENTS metabolite. The data acquired using Agilent mass hunter software and data was MS:MS_COMMENTS analyzed using mass hunter quantitative analysis software. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak intensity MS_METABOLITE_DATA_START Samples HFD-VEH13 HFD-VEH15 HFD-VEH18 HFD-VEH12 HFD-VEH14 HFD-BPA12 HFD-BPA13 HFD-BPA14 HFD-BPA16 HFD-BPA18 Factors Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:BPA Treatment:BPA Treatment:BPA Treatment:BPA Treatment:BPA sarcosine 82232 91277 78257 101557 91955 106474 88174 70692 87759 71497 Pyroglutamic acid 82904 73567 57574 79710 84946 68715 27441 39083 72938 93465 Aminophosphovaleric acid 3245336 3174061 3145381 3193194 2755589 3423002 2927230 3279544 3178523 3275461 Ornithine 31197 31619 36095 45113 34389 37190 37371 38194 34917 43299 Adenine 31805 23146 30875 23848 31585 26243 24518 27080 28892 40759 Methionine 22106 28798 24529 27843 23091 40874 29258 29280 29993 35779 Amino Adipic acid 42286 48564 38893 35716 39454 37611 54342 52660 48307 49213 2 Methyl glutamic acid 42286 48582 38889 35711 39454 37589 54342 52658 48307 49213 S-methyl-5-thioadenosine 169205 129590 167623 161135 146399 91783 106588 98121 124853 99833 Glutathione, Reduced (GSH) 543637 619758 350443 459494 316885 550001 305554 211249 390110 247938 L-Arginine (internal standard) 199504.4141 163161.8882 187917.7179 177633.5492 171738.9751 161561.0927 150378.6499 158129.4978 167299.1757 183638.8857 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name sarcosine Pyroglutamic acid Aminophosphovaleric acid Ornithine Adenine Methionine Amino Adipic acid 2 Methyl glutamic acid S-methyl-5-thioadenosine Glutathione, Reduced (GSH) L-Arginine (internal standard) METABOLITES_END #END