#METABOLOMICS WORKBENCH sarmas_20200412_165943 DATATRACK_ID:1987 STUDY_ID:ST001362 ANALYSIS_ID:AN002268 PROJECT_ID:PR000932 VERSION 1 CREATED_ON April 15, 2020, 5:32 pm #PROJECT PR:PROJECT_TITLE California mouse fecal metabolite analysis PR:PROJECT_SUMMARY Xenoestrogens are found in plant products, such as genistein (GEN), or PR:PROJECT_SUMMARY industrial chemicals, such as bisphenol A (BPA), present in consumer products PR:PROJECT_SUMMARY that are also pervasive in the environment. Early exposure to such endocrine PR:PROJECT_SUMMARY disrupting chemicals (EDC) may affect neural development by inducing direct PR:PROJECT_SUMMARY neural effects and/or through the microbiome-gut-brain axis. To test this PR:PROJECT_SUMMARY hypothesis, California mice (Peromyscus californicus) offspring were exposed PR:PROJECT_SUMMARY through the maternal diet to GEN (250 mg/kg feed weight) or BPA (5 mg/kg feed PR:PROJECT_SUMMARY weight, low dose- LD and 50 mg/kg, upper dose-UD), and dams were placed on these PR:PROJECT_SUMMARY diets two weeks prior to breeding, throughout gestation, and lactation. Various PR:PROJECT_SUMMARY behaviors, gut microbiome, and fecal metabolome were assessed starting at 90 PR:PROJECT_SUMMARY days of age. The LD but not UD of BPA resulted in individuals spending more time PR:PROJECT_SUMMARY engaging in repetitive behaviors. GEN exposed individuals were more likely to PR:PROJECT_SUMMARY exhibit such behaviors and showed socio-communicative disturbances. BPA and GEN PR:PROJECT_SUMMARY exposed females had increased number of metabolites involved in carbohydrate PR:PROJECT_SUMMARY metabolism and synthesis.. Males exposed to BPA or GEN showed alterations in PR:PROJECT_SUMMARY lysine degradation and phenylalanine and tyrosine metabolism. Current PR:PROJECT_SUMMARY findingsindicate cause for concern that developmental exposure to BPA or GEN PR:PROJECT_SUMMARY might affect the microbiome-gut-brain axis. PR:INSTITUTE University of Missouri PR:DEPARTMENT MU Metabolomics Center PR:LAST_NAME Sarma PR:FIRST_NAME Saurav PR:ADDRESS 1201 Rollins Street, 243 Bond Life Sciences Center, University of Missouri, PR:ADDRESS Columbia, MO, 65201, USA PR:EMAIL sarmas@missouri.edu PR:PHONE 5738825596 #STUDY ST:STUDY_TITLE California mouse fecal metabolite analysis ST:STUDY_TYPE Effect of BPA and genistein exposure on the fecal metabolome ST:STUDY_SUMMARY Xenoestrogens are found in plant products, such as genistein (GEN), or ST:STUDY_SUMMARY industrial chemicals, such as bisphenol A (BPA), present in consumer products ST:STUDY_SUMMARY that are also pervasive in the environment. Early exposure to such endocrine ST:STUDY_SUMMARY disrupting chemicals (EDC) may affect neural development by inducing direct ST:STUDY_SUMMARY neural effects and/or through the microbiome-gut-brain axis. To test this ST:STUDY_SUMMARY hypothesis, California mice (Peromyscus californicus) offspring were exposed ST:STUDY_SUMMARY through the maternal diet to GEN (250 mg/kg feed weight) or BPA (5 mg/kg feed ST:STUDY_SUMMARY weight, low dose- LD and 50 mg/kg, upper dose-UD), and dams were placed on these ST:STUDY_SUMMARY diets two weeks prior to breeding, throughout gestation, and lactation. Various ST:STUDY_SUMMARY behaviors, gut microbiome, and fecal metabolome were assessed starting at 90 ST:STUDY_SUMMARY days of age. The LD but not UD of BPA resulted in individuals spending more time ST:STUDY_SUMMARY engaging in repetitive behaviors. GEN exposed individuals were more likely to ST:STUDY_SUMMARY exhibit such behaviors and showed socio-communicative disturbances. BPA and GEN ST:STUDY_SUMMARY exposed females had increased number of metabolites involved in carbohydrate ST:STUDY_SUMMARY metabolism and synthesis.. Males exposed to BPA or GEN showed alterations in ST:STUDY_SUMMARY lysine degradation and phenylalanine and tyrosine metabolism. Current ST:STUDY_SUMMARY findingsindicate cause for concern that developmental exposure to BPA or GEN ST:STUDY_SUMMARY might affect the microbiome-gut-brain axis. ST:INSTITUTE University of Missouri ST:DEPARTMENT MU Metabolomics Center ST:LAST_NAME Sarma ST:FIRST_NAME Saurav ST:ADDRESS 1201 Rollins street, 243 Bond Life Science Center, University of Missouri, ST:ADDRESS Columbia, MO 65211, USA ST:EMAIL sarmas@missouri.edu ST:PHONE 3366713357 ST:NUM_GROUPS 8 ST:TOTAL_SUBJECTS 78 ST:NUM_MALES 37 ST:NUM_FEMALES 41 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Peromyscus californicus SU:TAXONOMY_ID 42520 SU:AGE_OR_AGE_RANGE PND90 SU:GENDER Male and female #FACTORS #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 - 1560-M-AIN Exposure:AIN | Gender:M RAW_FILE_NAME=1560-M-AIN SUBJECT_SAMPLE_FACTORS - 1561-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1561-F-AIN SUBJECT_SAMPLE_FACTORS - 1627-M-AIN Exposure:AIN | Gender:M RAW_FILE_NAME=1627-M-AIN SUBJECT_SAMPLE_FACTORS - 1626-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1626-F-AIN SUBJECT_SAMPLE_FACTORS - 1672-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1672-F-AIN SUBJECT_SAMPLE_FACTORS - 1632-M-AIN Exposure:AIN | Gender:M RAW_FILE_NAME=1632-M-AIN SUBJECT_SAMPLE_FACTORS - 1620-M-AIN Exposure:AIN | Gender:M RAW_FILE_NAME=1620-M-AIN SUBJECT_SAMPLE_FACTORS - 1642-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1642-F-AIN SUBJECT_SAMPLE_FACTORS - 1764-M-AIN Exposure:AIN | Gender:M RAW_FILE_NAME=1764-M-AIN SUBJECT_SAMPLE_FACTORS - 1765-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1765-F-AIN SUBJECT_SAMPLE_FACTORS - 1702-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1702-F-AIN SUBJECT_SAMPLE_FACTORS - 1623-M-AIN Exposure:AIN | Gender:M RAW_FILE_NAME=1623-M-AIN SUBJECT_SAMPLE_FACTORS - 1622-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1622-F-AIN SUBJECT_SAMPLE_FACTORS - 1745-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1745-F-AIN SUBJECT_SAMPLE_FACTORS - 1744-M-AIN Exposure:AIN | Gender:M RAW_FILE_NAME=1744-M-AIN SUBJECT_SAMPLE_FACTORS - 1619-M-AIN Exposure:AIN | Gender:M RAW_FILE_NAME=1619-M-AIN SUBJECT_SAMPLE_FACTORS - 1618-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1618-F-AIN SUBJECT_SAMPLE_FACTORS - 1587-F-AIN Exposure:AIN | Gender:F RAW_FILE_NAME=1587-F-AIN SUBJECT_SAMPLE_FACTORS - 1609-M-AIN Exposure:AIN | Gender:M RAW_FILE_NAME=1609-M-AIN SUBJECT_SAMPLE_FACTORS - 1788-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=1788-F-GEN SUBJECT_SAMPLE_FACTORS - 1819-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=1819-M-GEN SUBJECT_SAMPLE_FACTORS - 1975-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=1975-M-GEN SUBJECT_SAMPLE_FACTORS - 1925-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=1925-F-GEN SUBJECT_SAMPLE_FACTORS - 1884-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=1884-M-GEN SUBJECT_SAMPLE_FACTORS - 1912-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=1912-F-GEN SUBJECT_SAMPLE_FACTORS - 1926-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=1926-F-GEN SUBJECT_SAMPLE_FACTORS - 1993-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=1993-M-GEN SUBJECT_SAMPLE_FACTORS - 1940-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=1940-F-GEN SUBJECT_SAMPLE_FACTORS - 1965-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=1965-M-GEN SUBJECT_SAMPLE_FACTORS - 1808-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=1808-M-GEN SUBJECT_SAMPLE_FACTORS - 1911-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=1911-F-GEN SUBJECT_SAMPLE_FACTORS - 1738-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=1738-M-GEN SUBJECT_SAMPLE_FACTORS - 1763-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=1763-F-GEN SUBJECT_SAMPLE_FACTORS - 1829-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=1829-F-GEN SUBJECT_SAMPLE_FACTORS - 1830-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=1830-M-GEN SUBJECT_SAMPLE_FACTORS - 2021-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=2021-M-GEN SUBJECT_SAMPLE_FACTORS - 2022-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=2022-F-GEN SUBJECT_SAMPLE_FACTORS - 2040-F-GEN Exposure:GEN | Gender:F RAW_FILE_NAME=2040-F-GEN SUBJECT_SAMPLE_FACTORS - 2041-M-GEN Exposure:GEN | Gender:M RAW_FILE_NAME=2041-M-GEN SUBJECT_SAMPLE_FACTORS - 1800-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=1800-M-UD BPA SUBJECT_SAMPLE_FACTORS - 1827-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=1827-F-UD BPA SUBJECT_SAMPLE_FACTORS - 1682-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=1682-F-UD BPA SUBJECT_SAMPLE_FACTORS - 1747-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=1747-M-UD BPA SUBJECT_SAMPLE_FACTORS - 1651-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=1651-M-UD BPA SUBJECT_SAMPLE_FACTORS - 1652-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=1652-F-UD BPA SUBJECT_SAMPLE_FACTORS - 1689-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=1689-F-UD BPA SUBJECT_SAMPLE_FACTORS - 1690-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=1690-M-UD BPA SUBJECT_SAMPLE_FACTORS - 1614-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=1614-F-UD BPA SUBJECT_SAMPLE_FACTORS - 1615-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=1615-M-UD BPA SUBJECT_SAMPLE_FACTORS - 1612-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=1612-F-UD BPA SUBJECT_SAMPLE_FACTORS - 1613-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=1613-M-UD BPA SUBJECT_SAMPLE_FACTORS - 1606-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=1606-M-UD BPA SUBJECT_SAMPLE_FACTORS - 1607-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=1607-F-UD BPA SUBJECT_SAMPLE_FACTORS - 1688-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=1688-M-UD BPA SUBJECT_SAMPLE_FACTORS - 2004-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=2004-F-UD BPA SUBJECT_SAMPLE_FACTORS - 2037-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=2037-M-UD BPA SUBJECT_SAMPLE_FACTORS - 2038-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=2038-F-UD BPA SUBJECT_SAMPLE_FACTORS - 2006-F-UD BPA Exposure:UD BPA | Gender:F RAW_FILE_NAME=2006-F-UD BPA SUBJECT_SAMPLE_FACTORS - 2028-M-UD BPA Exposure:UD BPA | Gender:M RAW_FILE_NAME=2028-M-UD BPA SUBJECT_SAMPLE_FACTORS - 1991-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=1991-F-LD BPA SUBJECT_SAMPLE_FACTORS - 1992-M-LD BPA Exposure:LD BPA | Gender:M RAW_FILE_NAME=1992-M-LD BPA SUBJECT_SAMPLE_FACTORS - 1848-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=1848-F-LD BPA SUBJECT_SAMPLE_FACTORS - 2008-M-LD BPA Exposure:LD BPA | Gender:M RAW_FILE_NAME=2008-M-LD BPA SUBJECT_SAMPLE_FACTORS - 1913-M-LD BPA Exposure:LD BPA | Gender:M RAW_FILE_NAME=1913-M-LD BPA SUBJECT_SAMPLE_FACTORS - 1914-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=1914-F-LD BPA SUBJECT_SAMPLE_FACTORS - 2010-M-LD BPA Exposure:LD BPA | Gender:M RAW_FILE_NAME=2010-M-LD BPA SUBJECT_SAMPLE_FACTORS - 1791-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=1791-F-LD BPA SUBJECT_SAMPLE_FACTORS - 1854-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=1854-F-LD BPA SUBJECT_SAMPLE_FACTORS - 1910-M-LD BPA Exposure:LD BPA | Gender:M RAW_FILE_NAME=1910-M-LD BPA SUBJECT_SAMPLE_FACTORS - 2019-M-LD BPA Exposure:LD BPA | Gender:M RAW_FILE_NAME=2019-M-LD BPA SUBJECT_SAMPLE_FACTORS - 2001-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=2001-F-LD BPA SUBJECT_SAMPLE_FACTORS - 1953-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=1953-F-LD BPA SUBJECT_SAMPLE_FACTORS - 1923-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=1923-F-LD BPA SUBJECT_SAMPLE_FACTORS - 2005-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=2005-F-LD BPA SUBJECT_SAMPLE_FACTORS - 2056-M-LD BPA Exposure:LD BPA | Gender:M RAW_FILE_NAME=2056-M-LD BPA SUBJECT_SAMPLE_FACTORS - 2009-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=2009-F-LD BPA SUBJECT_SAMPLE_FACTORS - 2049-M-LD BPA Exposure:LD BPA | Gender:M RAW_FILE_NAME=2049-M-LD BPA SUBJECT_SAMPLE_FACTORS - 2007-F-LD BPA Exposure:LD BPA | Gender:F RAW_FILE_NAME=2007-F-LD BPA #COLLECTION CO:COLLECTION_SUMMARY Fecal boli were collected at PND 90, snap frozen in LN2, stored in -80 freezer CO:COLLECTION_SUMMARY until processing CO:SAMPLE_TYPE Feces CO:COLLECTION_METHOD Snap freezen in LN2 right after excretion CO:COLLECTION_LOCATION Animal Science Research Center, University of Missouri, Columbia CO:COLLECTION_FREQUENCY 1 time from each animal CO:VOLUMEORAMOUNT_COLLECTED 3-4 boli CO:STORAGE_CONDITIONS -20℃ CO:COLLECTION_VIALS 2mL vials CO:STORAGE_VIALS 2mL vials #TREATMENT TR:TREATMENT_SUMMARY 2 weeks before breeding and during gestation, dams were fed estrogen-free AIN TR:TREATMENT_SUMMARY 93G or AIN 93G diet containing LD BPA, UD BPA or genistein untill one male and TR:TREATMENT_SUMMARY one female pups weaned. F1 pups fed on AIN 93G diet after weaning. TR:TREATMENT diet TR:TREATMENT_ROUTE oral TR:TREATMENT_DOSE Genistein: 250 mg/kg feed weight; BPA: 5 mg/kg feed weight for LD BPA and 50 TR:TREATMENT_DOSE mg/kg feed weight for UD BPA group TR:TREATMENT_DOSEDURATION 2 weeks before breeding and untill one male and one female pups weaned. TR:TREATMENT_VEHICLE AIN control diet TR:ANIMAL_ENDP_TISSUE_COLL_LIST Fecal TR:TREATMENT_COMPOUND Genistein and Bisphenol A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fecal samples were snap frozen in liqud nitrogen and no further preparation SP:PROCESSING_METHOD Fecal samples were lyophilized for 24 h before weighing SP:PROCESSING_STORAGE_CONDITIONS -20℃ SP:EXTRACTION_METHOD For LCMS analysis lyophilized fecal sample was extracted with 80% Methanol SP:EXTRACTION_METHOD containg 18 µg/ml of umbelliferon, sonicated and vortexed 20 seconds each, SP:EXTRACTION_METHOD shaken at 140 rpmfor 2 h. For GCMS-polar analysis samples were extracted with SP:EXTRACTION_METHOD water, containing 25 μg/ml ribitol, vortex for 20 seconds, sonicated for 20 SP:EXTRACTION_METHOD seconds, incubated at 50C for 1h. For GCMS-nonpolar samples were extracted with SP:EXTRACTION_METHOD chloroform, containing 10 μl/ml docosanol, vortex for 20 seconds, sonicated for SP:EXTRACTION_METHOD 20 second, incubated at 50C for 1h. SP:EXTRACT_ENRICHMENT Solution were centrifuged at 3000g for 40 min then extracted 0.5 mL of SP:EXTRACT_ENRICHMENT supernatant for LCMS, 1 mL each of supernatant was collected for GCMS-polar and SP:EXTRACT_ENRICHMENT GCMS-nonpolar analysis. SP:EXTRACT_STORAGE -20℃ SP:SAMPLE_RESUSPENSION For GCMS analysis extracts dried under nitrogen flow and storred at -20C then SP:SAMPLE_RESUSPENSION resuspended in 25 μl of pyridine containing 15 mg/ml methoxyamine hydrochloride SP:SAMPLE_DERIVATIZATION GCMS samples were derivatized with 25 μl MSTFA SP:SAMPLE_DERIVATIZATION (N-methyl-N-(trimethyl-silyl)trifluoroacetamide) + 1% TMCS SP:SAMPLE_DERIVATIZATION (chlorotrimethylsilane) SP:SAMPLE_SPIKING Umbelliferon, ribitol and docosanol were used as internal standard for LCMS, SP:SAMPLE_SPIKING GCMS-polar and GCMS-nonpolar respectively. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 1:1 split for GCMS-nonpolar injection with 1uL injection volume. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 6890N CH:COLUMN_NAME DB-5MS (J&W Scientific) 60m CH:FLOW_RATE 1mL/Min of Helium gas CH:COLUMN_TEMPERATURE variable between 80-315 CH:COLUMN_PRESSURE variable CH:INTERNAL_STANDARD Docosanol CH:RETENTION_TIME 9 to 61 min CH:SAMPLE_INJECTION 1:1 split injection with 1uL injection volume CH:ANALYTICAL_TIME 61 min #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5973 MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Agilent Chemstation software for data acquisition and AMDIS and MetIdea for data MS:MS_COMMENTS analysis MS:HELIUM_FLOW 1mL/min MS:ION_SOURCE_TEMPERATURE 230 MS:IONIZATION_ENERGY 70ev MS:REAGENT_GAS Helium MS:SOURCE_TEMPERATURE 230 MS:DATAFORMAT .d MS:MS_RESULTS_FILE ST001362_AN002268_Results.txt UNITS:intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END