#METABOLOMICS WORKBENCH epannkuk_20231205_105308 DATATRACK_ID:4490 STUDY_ID:ST003000 ANALYSIS_ID:AN004927 PROJECT_ID:PR001870 VERSION 1 CREATED_ON December 8, 2023, 12:04 pm #PROJECT PR:PROJECT_TITLE Effect of Antibiotic Administration on Metabolomic Radiation Biodosimetry PR:PROJECT_SUMMARY Development of novel biodosimetry assays and medical countermeasures is needed PR:PROJECT_SUMMARY to obtain a level of radiation preparedness in the event of malicious or PR:PROJECT_SUMMARY accidental mass exposures to ionizing radiation (IR). For biodosimetry, PR:PROJECT_SUMMARY metabolic profiling with mass spectrometry (MS) platforms has identified several PR:PROJECT_SUMMARY small molecules in easily accessible biofluids that are promising for dose PR:PROJECT_SUMMARY reconstruction. As our microbiome has profound effects on biofluid metabolite PR:PROJECT_SUMMARY composition, it is of interest how variation in the host microbiome may affect PR:PROJECT_SUMMARY metabolomics based biodosimetry. Here, we chemically ‘knocked out’ the PR:PROJECT_SUMMARY microbiome of male and female C57BL/6 mice (Abx mice) and then irradiated (0, 3, PR:PROJECT_SUMMARY or 8 Gy) them to determine the role of the host microbiome on biofluid radiation PR:PROJECT_SUMMARY signatures (3 d serum). PR:INSTITUTE Georgetown University PR:LAST_NAME Pannkuk PR:FIRST_NAME Evan PR:ADDRESS 3970 Reservoir Rd, NW New Research Build, washington dc, District of Columbia, PR:ADDRESS 20057, USA PR:EMAIL elp44@georgetown.edu PR:PHONE 2026875650 #STUDY ST:STUDY_TITLE Effects of Microbiome Depletion on Radiation Biodosimetry Metabolomics ST:STUDY_SUMMARY Development of novel biodosimetry assays and medical countermeasures is needed ST:STUDY_SUMMARY to obtain a level of radiation preparedness in the event of malicious or ST:STUDY_SUMMARY accidental mass exposures to ionizing radiation (IR). For biodosimetry, ST:STUDY_SUMMARY metabolic profiling with mass spectrometry (MS) platforms has identified several ST:STUDY_SUMMARY small molecules in easily accessible biofluids that are promising for dose ST:STUDY_SUMMARY reconstruction. As our microbiome has profound effects on biofluid metabolite ST:STUDY_SUMMARY composition, it is of interest how variation in the host microbiome may affect ST:STUDY_SUMMARY metabolomics based biodosimetry. Here, we chemically ‘knocked out’ the ST:STUDY_SUMMARY microbiome of male and female C57BL/6 mice (Abx mice) and then irradiated (0, 3, ST:STUDY_SUMMARY or 8 Gy) them to determine the role of the host microbiome on biofluid radiation ST:STUDY_SUMMARY signatures (3 d serum). ST:INSTITUTE Georgetown University ST:LAST_NAME Pannkuk ST:FIRST_NAME Evan ST:ADDRESS 3970 Reservoir Rd, NW New Research Build, washington dc, District of Columbia, ST:ADDRESS 20057, USA ST:EMAIL elp44@georgetown.edu ST:PHONE 2026875650 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Male and female #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 wo_3Gy 7588M Antibiotics:wo | Irradiation:3Gy RAW_FILE_NAME=POS_09; RAW_FILE_NAME=NEG_09 SUBJECT_SAMPLE_FACTORS wo_0Gy 7579M Antibiotics:wo | Irradiation:0Gy RAW_FILE_NAME=POS_10; RAW_FILE_NAME=NEG_10 SUBJECT_SAMPLE_FACTORS w_0Gy 7634F Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_11; RAW_FILE_NAME=NEG_11 SUBJECT_SAMPLE_FACTORS w_3Gy 7636F Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_12; RAW_FILE_NAME=NEG_12 SUBJECT_SAMPLE_FACTORS wo_0Gy 7614F Antibiotics:wo | Irradiation:0Gy RAW_FILE_NAME=POS_13; RAW_FILE_NAME=NEG_13 SUBJECT_SAMPLE_FACTORS wo_8Gy 7628F Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_14; RAW_FILE_NAME=NEG_14 SUBJECT_SAMPLE_FACTORS w_0Gy 7635F Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_15; RAW_FILE_NAME=NEG_15 SUBJECT_SAMPLE_FACTORS w_8Gy 7642F Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_16; RAW_FILE_NAME=NEG_16 SUBJECT_SAMPLE_FACTORS w_8Gy 7650F Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_17; RAW_FILE_NAME=NEG_17 SUBJECT_SAMPLE_FACTORS w_3Gy 7639F Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_22; RAW_FILE_NAME=NEG_22 SUBJECT_SAMPLE_FACTORS wo_8Gy 7589M Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_23; RAW_FILE_NAME=NEG_23 SUBJECT_SAMPLE_FACTORS w_0Gy 7594M Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_24; RAW_FILE_NAME=NEG_24 SUBJECT_SAMPLE_FACTORS wo_0Gy 7582M Antibiotics:wo | Irradiation:0Gy RAW_FILE_NAME=POS_25; RAW_FILE_NAME=NEG_25 SUBJECT_SAMPLE_FACTORS w_0Gy 7632F Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_26; RAW_FILE_NAME=NEG_26 SUBJECT_SAMPLE_FACTORS w_3Gy 7600M Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_27; RAW_FILE_NAME=NEG_27 SUBJECT_SAMPLE_FACTORS wo_8Gy 7626F Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_28; RAW_FILE_NAME=NEG_28 SUBJECT_SAMPLE_FACTORS w_3Gy 7637F Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_29; RAW_FILE_NAME=NEG_29 SUBJECT_SAMPLE_FACTORS w_0Gy 7596M Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_30; RAW_FILE_NAME=NEG_30 SUBJECT_SAMPLE_FACTORS w_0Gy 7595M Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_31; RAW_FILE_NAME=NEG_31 SUBJECT_SAMPLE_FACTORS w_0Gy 7631F Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_36; RAW_FILE_NAME=NEG_36 SUBJECT_SAMPLE_FACTORS w_8Gy 7607M Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_37; RAW_FILE_NAME=NEG_37 SUBJECT_SAMPLE_FACTORS wo_0Gy 7616F Antibiotics:wo | Irradiation:0Gy RAW_FILE_NAME=POS_38; RAW_FILE_NAME=NEG_38 SUBJECT_SAMPLE_FACTORS wo_0Gy 7581M Antibiotics:wo | Irradiation:0Gy RAW_FILE_NAME=POS_39; RAW_FILE_NAME=NEG_39 SUBJECT_SAMPLE_FACTORS w_8Gy 7604M Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_40; RAW_FILE_NAME=NEG_40 SUBJECT_SAMPLE_FACTORS w_0Gy 7633F Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_41; RAW_FILE_NAME=NEG_41 SUBJECT_SAMPLE_FACTORS w_3Gy 7640F Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_42; RAW_FILE_NAME=NEG_42 SUBJECT_SAMPLE_FACTORS w_8Gy 7608M Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_43; RAW_FILE_NAME=NEG_43 SUBJECT_SAMPLE_FACTORS w_8Gy 7643F Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_44; RAW_FILE_NAME=NEG_44 SUBJECT_SAMPLE_FACTORS wo_3Gy 7623F Antibiotics:wo | Irradiation:3Gy RAW_FILE_NAME=POS_45; RAW_FILE_NAME=NEG_45 SUBJECT_SAMPLE_FACTORS wo_3Gy 7625F Antibiotics:wo | Irradiation:3Gy RAW_FILE_NAME=POS_50; RAW_FILE_NAME=NEG_50 SUBJECT_SAMPLE_FACTORS w_8Gy 7641F Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_51; RAW_FILE_NAME=NEG_51 SUBJECT_SAMPLE_FACTORS w_3Gy 7603M Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_52; RAW_FILE_NAME=NEG_52 SUBJECT_SAMPLE_FACTORS w_8Gy 7644F Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_53; RAW_FILE_NAME=NEG_53 SUBJECT_SAMPLE_FACTORS wo_8Gy 7592M Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_54; RAW_FILE_NAME=NEG_54 SUBJECT_SAMPLE_FACTORS wo_3Gy 7586M Antibiotics:wo | Irradiation:3Gy RAW_FILE_NAME=POS_55; RAW_FILE_NAME=NEG_55 SUBJECT_SAMPLE_FACTORS wo_3Gy 7622F Antibiotics:wo | Irradiation:3Gy RAW_FILE_NAME=POS_56; RAW_FILE_NAME=NEG_56 SUBJECT_SAMPLE_FACTORS wo_8Gy 7630F Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_57; RAW_FILE_NAME=NEG_57 SUBJECT_SAMPLE_FACTORS wo_3Gy 7587M Antibiotics:wo | Irradiation:3Gy RAW_FILE_NAME=POS_58; RAW_FILE_NAME=NEG_58 SUBJECT_SAMPLE_FACTORS w_3Gy 7599M Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_59; RAW_FILE_NAME=NEG_59 SUBJECT_SAMPLE_FACTORS wo_0Gy 7619F Antibiotics:wo | Irradiation:0Gy RAW_FILE_NAME=POS_64; RAW_FILE_NAME=NEG_64 SUBJECT_SAMPLE_FACTORS w_8Gy 7605M Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_65; RAW_FILE_NAME=NEG_65 SUBJECT_SAMPLE_FACTORS w_3Gy 7638F Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_66; RAW_FILE_NAME=NEG_66 SUBJECT_SAMPLE_FACTORS wo_0Gy 7583M Antibiotics:wo | Irradiation:0Gy RAW_FILE_NAME=POS_67; RAW_FILE_NAME=NEG_67 SUBJECT_SAMPLE_FACTORS wo_8Gy 7591M Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_68; RAW_FILE_NAME=NEG_68 SUBJECT_SAMPLE_FACTORS wo_8Gy 7627F Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_69; RAW_FILE_NAME=NEG_69 SUBJECT_SAMPLE_FACTORS wo_0Gy 7618F Antibiotics:wo | Irradiation:0Gy RAW_FILE_NAME=POS_70; RAW_FILE_NAME=NEG_70 SUBJECT_SAMPLE_FACTORS w_3Gy 7602M Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_71; RAW_FILE_NAME=NEG_71 SUBJECT_SAMPLE_FACTORS w_0Gy 7598M Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_72; RAW_FILE_NAME=NEG_72 SUBJECT_SAMPLE_FACTORS wo_0Gy 7617F Antibiotics:wo | Irradiation:0Gy RAW_FILE_NAME=POS_73; RAW_FILE_NAME=NEG_73 SUBJECT_SAMPLE_FACTORS w_8Gy 7606M Antibiotics:w | Irradiation:8Gy RAW_FILE_NAME=POS_78; RAW_FILE_NAME=NEG_78 SUBJECT_SAMPLE_FACTORS w_0Gy 7597M Antibiotics:w | Irradiation:0Gy RAW_FILE_NAME=POS_79; RAW_FILE_NAME=NEG_79 SUBJECT_SAMPLE_FACTORS wo_8Gy 7590M Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_80; RAW_FILE_NAME=NEG_80 SUBJECT_SAMPLE_FACTORS wo_3Gy 7621F Antibiotics:wo | Irradiation:3Gy RAW_FILE_NAME=POS_81; RAW_FILE_NAME=NEG_81 SUBJECT_SAMPLE_FACTORS wo_8Gy 7629F Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_82; RAW_FILE_NAME=NEG_82 SUBJECT_SAMPLE_FACTORS w_3Gy 7601M Antibiotics:w | Irradiation:3Gy RAW_FILE_NAME=POS_83; RAW_FILE_NAME=NEG_83 SUBJECT_SAMPLE_FACTORS wo_3Gy 7585M Antibiotics:wo | Irradiation:3Gy RAW_FILE_NAME=POS_84; RAW_FILE_NAME=NEG_84 SUBJECT_SAMPLE_FACTORS wo_8Gy 7593M Antibiotics:wo | Irradiation:8Gy RAW_FILE_NAME=POS_85; RAW_FILE_NAME=NEG_85 SUBJECT_SAMPLE_FACTORS wo_3Gy 7624F Antibiotics:wo | Irradiation:3Gy RAW_FILE_NAME=POS_86; RAW_FILE_NAME=NEG_86 #COLLECTION CO:COLLECTION_SUMMARY On day 3, mice were euthanized by CO2 inhalation, blood was collected by cardiac CO:COLLECTION_SUMMARY puncture, and tissues were collected. Tissues were flash frozen. Serum samples CO:COLLECTION_SUMMARY were prepared using BD Microtainer Tube (REF 365967) with ~100 µL of whole CO:COLLECTION_SUMMARY blood added to each tube, kept at room temperature for 30 min, then spun at CO:COLLECTION_SUMMARY 1300× g at 4 °C for 10 min. All tissues and biofluids were stored at −80 °C CO:COLLECTION_SUMMARY until analysis. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY We used 8 week old male and female C57BL/6 mice that were purchased from Charles TR:TREATMENT_SUMMARY River Laboratories (Frederick, MD) and were assigned to sham, 3 Gy, or 8 Gy TR:TREATMENT_SUMMARY cohorts. A control group (Abx-con) did not receive antibiotics in drinking water TR:TREATMENT_SUMMARY and a treatment group (Abx) received broad-spectrum antibiotics (n=10 per group, TR:TREATMENT_SUMMARY half male half female) (S1 Fig). Mice were provided with deionized water ad TR:TREATMENT_SUMMARY libitum either with or without the antibiotic cocktail (enrofloxacin [0.575 TR:TREATMENT_SUMMARY mg/ml] and ampicillin [1 mg/ml]) for 8 days prior to irradiation [24]. Mice were TR:TREATMENT_SUMMARY irradiated in an acrylic, 12-slot mouse pie cage (MPC-1, Braintree Scientific, TR:TREATMENT_SUMMARY Braintree, MA) on top of a specimen turntable (XD1905-0000, Precision X-Ray Inc, TR:TREATMENT_SUMMARY Branford, CT) and 0, 3 or 8 Gy X-ray irradiated (1.67 Gy/min; X-Rad 320, TR:TREATMENT_SUMMARY Precision X-Ray Inc.; filter, 0.75 mm tin/ 0.25 mm copper/1.5 mm aluminum). For TR:TREATMENT_SUMMARY Abx-con mice, spot urines (>100 µL) were collected 1 d prior to irradiation and TR:TREATMENT_SUMMARY at 1 and 3 d post-irradiation (fecal samples collected 1 d prior to irradiation TR:TREATMENT_SUMMARY and at 3 d post-irradiation). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY On day 3, mice were euthanized by CO2 inhalation, blood was collected by cardiac SP:SAMPLEPREP_SUMMARY puncture, and tissues were collected. Tissues were flash frozen. Serum samples SP:SAMPLEPREP_SUMMARY were prepared using BD Microtainer Tube (REF 365967) with ~100 µL of whole SP:SAMPLEPREP_SUMMARY blood added to each tube, kept at room temperature for 30 min, then spun at SP:SAMPLEPREP_SUMMARY 1300× g at 4 °C for 10 min. All tissues and biofluids were stored at −80 °C SP:SAMPLEPREP_SUMMARY until analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The LC and MS conditions for serum was as follows: LC solvent A (water/0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid [FA]), solvent B (acetonitrile/0.1% FA), and solvent C CH:CHROMATOGRAPHY_SUMMARY (isopropanol/0.1% FA). Operating conditions for ESI were, capillary voltage 3 CH:CHROMATOGRAPHY_SUMMARY kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow 600 CH:CHROMATOGRAPHY_SUMMARY L/Hr. The gradient for serum was: 4 min 98% A 2% B, 4 min 40% A 60% B, 1.5 min CH:CHROMATOGRAPHY_SUMMARY 2% A 98% B, 2 min 11.8% B 88.2% C, 0.5 min 50% A 50% B, and 1 min 98% A 2% B at CH:CHROMATOGRAPHY_SUMMARY a flow rate of 0.5 ml/min, column temp 60 °C (S3 Fig). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (50 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:SOLVENT_C 100% isopropanol; 0.1% formic acid CH:FLOW_GRADIENT 4 min 98% A 2% B, 4 min 40% A 60% B, 1.5 min 2% A 98% B, 2 min 11.8% B 88.2% C, CH:FLOW_GRADIENT 0.5 min 50% A 50% B, and 1 min 98% A 2% B CH:FLOW_RATE 0.5 ml/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Operating conditions for ESI were, capillary voltage 3 kV, cone voltage 30 V, MS:MS_COMMENTS desolvation temperature 500°C, desolvation gas flow 600 L/Hr. MS:MS_RESULTS_FILE ST003000_AN004927_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END