#METABOLOMICS WORKBENCH epannkuk_20210512_073836 DATATRACK_ID:2637 STUDY_ID:ST001792 ANALYSIS_ID:AN002908 PROJECT_ID:000000 VERSION 1 CREATED_ON May 12, 2021, 9:30 am #PROJECT PR:PROJECT_TITLE LDR_mouse_biofluid_metabolomics PR:PROJECT_SUMMARY An important component of ionizing radiation (IR) exposure after a radiological PR:PROJECT_SUMMARY incident may include low-dose rate (LDR) exposures either externally or PR:PROJECT_SUMMARY internally, such as from 137Cs deposition. LDR exposures can have different PR:PROJECT_SUMMARY effects compared to acute high-dose rate exposures from a health and PR:PROJECT_SUMMARY biodosimetry perspective. In this study, a novel irradiation system, VAriable PR:PROJECT_SUMMARY Dose-rate External 137Cs irradiatoR (VADER), was used to expose male and female PR:PROJECT_SUMMARY mice to a variable LDR over a 30-day time span to cumulative doses of 1 (only in PR:PROJECT_SUMMARY males), 2, 2.8, 4.1, 8.8 (only in males), or 9.7 Gy to simulate fall-out type PR:PROJECT_SUMMARY exposures. Urine and serum from mice exposed to an acute dose (~0.8 Gy/min) of PR:PROJECT_SUMMARY x-rays were collected in parallel. Radiation markers were identified by global PR:PROJECT_SUMMARY mass spectrometry based metabolomics and the machine learning algorithm Random PR:PROJECT_SUMMARY Forests. PR:INSTITUTE Georgetown University PR:LAST_NAME Pannkuk PR:FIRST_NAME Evan PR:ADDRESS 3970 Reservoir Rd, NW New Research Building E504 PR:EMAIL elp44@georgetown.edu PR:PHONE 2026875650 #STUDY ST:STUDY_TITLE Effect of external low-dose rate radiation on mouse biofluid metabolomic ST:STUDY_TITLE signatures ST:STUDY_SUMMARY An important component of ionizing radiation (IR) exposure after a radiological ST:STUDY_SUMMARY incident may include low-dose rate (LDR) exposures either externally or ST:STUDY_SUMMARY internally, such as from 137Cs deposition. LDR exposures can have different ST:STUDY_SUMMARY effects compared to acute high-dose rate exposures from a health and ST:STUDY_SUMMARY biodosimetry perspective. In this study, a novel irradiation system, VAriable ST:STUDY_SUMMARY Dose-rate External 137Cs irradiatoR (VADER), was used to expose male and female ST:STUDY_SUMMARY mice to a variable LDR over a 30-day time span to cumulative doses of 1 (only in ST:STUDY_SUMMARY males), 2, 2.8, 4.1, 8.8 (only in males), or 9.7 Gy to simulate fall-out type ST:STUDY_SUMMARY exposures. Urine and serum from mice exposed to an acute dose (~0.8 Gy/min) of ST:STUDY_SUMMARY x-rays were collected in parallel. Radiation markers were identified by global ST:STUDY_SUMMARY mass spectrometry based metabolomics and the machine learning algorithm Random ST:STUDY_SUMMARY Forests. ST:INSTITUTE Georgetown University ST:LAST_NAME Pannkuk ST:FIRST_NAME Evan ST:ADDRESS 3970 Reservoir Rd, NW New Research Building E504 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 #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 114V 12 Factor:LDR | Factor:D5 RAW_FILE_NAME=vader_met_12 SUBJECT_SAMPLE_FACTORS 93A 13 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_13 SUBJECT_SAMPLE_FACTORS 63A 14 Factor:HDR | Factor:D1 RAW_FILE_NAME=vader_met_14 SUBJECT_SAMPLE_FACTORS 113V 15 Factor:LDR | Factor:D5 RAW_FILE_NAME=vader_met_15 SUBJECT_SAMPLE_FACTORS 123V 16 Factor:LDR | Factor:D20 RAW_FILE_NAME=vader_met_16 SUBJECT_SAMPLE_FACTORS 159V 19 Factor:Control | Factor:D30 RAW_FILE_NAME=vader_met_19 SUBJECT_SAMPLE_FACTORS 99A 20 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_20 SUBJECT_SAMPLE_FACTORS 153V 21 Factor:Control | Factor:D20 RAW_FILE_NAME=vader_met_21 SUBJECT_SAMPLE_FACTORS 151V 22 Factor:Control | Factor:D20 RAW_FILE_NAME=vader_met_22 SUBJECT_SAMPLE_FACTORS 89A 23 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_23 SUBJECT_SAMPLE_FACTORS 87A 29 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_29 SUBJECT_SAMPLE_FACTORS 90A 30 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_30 SUBJECT_SAMPLE_FACTORS 115V 31 Factor:LDR | Factor:D5 RAW_FILE_NAME=vader_met_31 SUBJECT_SAMPLE_FACTORS 72A 32 Factor:HDR | Factor:D3 RAW_FILE_NAME=vader_met_32 SUBJECT_SAMPLE_FACTORS 186V 33 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_33 SUBJECT_SAMPLE_FACTORS 77A 36 Factor:HDR | Factor:D5 RAW_FILE_NAME=vader_met_36 SUBJECT_SAMPLE_FACTORS 100A 37 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_37 SUBJECT_SAMPLE_FACTORS 172V 38 Factor:LDR | Factor:D2 RAW_FILE_NAME=vader_met_38 SUBJECT_SAMPLE_FACTORS 130V 39 Factor:LDR | Factor:D30 RAW_FILE_NAME=vader_met_39 SUBJECT_SAMPLE_FACTORS 126V 40 Factor:LDR | Factor:D30 RAW_FILE_NAME=vader_met_40 SUBJECT_SAMPLE_FACTORS 75A 46 Factor:HDR | Factor:D3 RAW_FILE_NAME=vader_met_46 SUBJECT_SAMPLE_FACTORS 171V 47 Factor:LDR | Factor:D2 RAW_FILE_NAME=vader_met_47 SUBJECT_SAMPLE_FACTORS 158V 48 Factor:Control | Factor:D30 RAW_FILE_NAME=vader_met_48 SUBJECT_SAMPLE_FACTORS 176V 49 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_49 SUBJECT_SAMPLE_FACTORS 156V 50 Factor:Control | Factor:D30 RAW_FILE_NAME=vader_met_50 SUBJECT_SAMPLE_FACTORS 152V 53 Factor:Control | Factor:D20 RAW_FILE_NAME=vader_met_53 SUBJECT_SAMPLE_FACTORS 70A 54 Factor:HDR | Factor:D2 RAW_FILE_NAME=vader_met_54 SUBJECT_SAMPLE_FACTORS 95A 55 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_55 SUBJECT_SAMPLE_FACTORS 157V 56 Factor:Control | Factor:D30 RAW_FILE_NAME=vader_met_56 SUBJECT_SAMPLE_FACTORS 5V 57 Factor:LDR | Factor:D1 RAW_FILE_NAME=vader_met_57 SUBJECT_SAMPLE_FACTORS 178V 63 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_63 SUBJECT_SAMPLE_FACTORS 67A 64 Factor:HDR | Factor:D2 RAW_FILE_NAME=vader_met_64 SUBJECT_SAMPLE_FACTORS 112V 65 Factor:LDR | Factor:D5 RAW_FILE_NAME=vader_met_65 SUBJECT_SAMPLE_FACTORS 2V 66 Factor:LDR | Factor:D1 RAW_FILE_NAME=vader_met_66 SUBJECT_SAMPLE_FACTORS 96A 67 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_67 SUBJECT_SAMPLE_FACTORS 160V 70 Factor:Control | Factor:D30 RAW_FILE_NAME=vader_met_70 SUBJECT_SAMPLE_FACTORS 97A 71 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_71 SUBJECT_SAMPLE_FACTORS 84A 72 Factor:Control | Factor:D1 RAW_FILE_NAME=vader_met_72 SUBJECT_SAMPLE_FACTORS 122V 73 Factor:LDR | Factor:D20 RAW_FILE_NAME=vader_met_73 SUBJECT_SAMPLE_FACTORS 174V 74 Factor:LDR | Factor:D2 RAW_FILE_NAME=vader_met_74 SUBJECT_SAMPLE_FACTORS 88A 80 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_80 SUBJECT_SAMPLE_FACTORS 173V 81 Factor:LDR | Factor:D2 RAW_FILE_NAME=vader_met_81 SUBJECT_SAMPLE_FACTORS 69A 82 Factor:HDR | Factor:D2 RAW_FILE_NAME=vader_met_82 SUBJECT_SAMPLE_FACTORS 154V 83 Factor:Control | Factor:D20 RAW_FILE_NAME=vader_met_83 SUBJECT_SAMPLE_FACTORS 82A 84 Factor:Control | Factor:D1 RAW_FILE_NAME=vader_met_84 SUBJECT_SAMPLE_FACTORS 68A 87 Factor:HDR | Factor:D2 RAW_FILE_NAME=vader_met_87 SUBJECT_SAMPLE_FACTORS 71A 88 Factor:HDR | Factor:D3 RAW_FILE_NAME=vader_met_88 SUBJECT_SAMPLE_FACTORS 183V 89 Factor:LDR | Factor:D3 RAW_FILE_NAME=vader_met_89 SUBJECT_SAMPLE_FACTORS 3V 90 Factor:LDR | Factor:D1 RAW_FILE_NAME=vader_met_90 SUBJECT_SAMPLE_FACTORS 155V 91 Factor:Control | Factor:D20 RAW_FILE_NAME=vader_met_91 SUBJECT_SAMPLE_FACTORS 175V 97 Factor:LDR | Factor:D2 RAW_FILE_NAME=vader_met_97 SUBJECT_SAMPLE_FACTORS 62A 98 Factor:HDR | Factor:D1 RAW_FILE_NAME=vader_met_98 SUBJECT_SAMPLE_FACTORS 190V 99 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_99 SUBJECT_SAMPLE_FACTORS 141V 100 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_100 SUBJECT_SAMPLE_FACTORS 85A 101 Factor:Control | Factor:D1 RAW_FILE_NAME=vader_met_101 SUBJECT_SAMPLE_FACTORS 177V 104 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_104 SUBJECT_SAMPLE_FACTORS 78A 105 Factor:HDR | Factor:D5 RAW_FILE_NAME=vader_met_105 SUBJECT_SAMPLE_FACTORS 64A 106 Factor:HDR | Factor:D1 RAW_FILE_NAME=vader_met_106 SUBJECT_SAMPLE_FACTORS 184V 107 Factor:LDR | Factor:D3 RAW_FILE_NAME=vader_met_107 SUBJECT_SAMPLE_FACTORS 143V 108 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_108 SUBJECT_SAMPLE_FACTORS 94A 114 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_114 SUBJECT_SAMPLE_FACTORS 4V 115 Factor:LDR | Factor:D1 RAW_FILE_NAME=vader_met_115 SUBJECT_SAMPLE_FACTORS 180V 116 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_116 SUBJECT_SAMPLE_FACTORS 80A 117 Factor:HDR | Factor:D5 RAW_FILE_NAME=vader_met_117 SUBJECT_SAMPLE_FACTORS 121V 118 Factor:LDR | Factor:D20 RAW_FILE_NAME=vader_met_118 SUBJECT_SAMPLE_FACTORS 66A 121 Factor:HDR | Factor:D2 RAW_FILE_NAME=vader_met_121 SUBJECT_SAMPLE_FACTORS 187V 122 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_122 SUBJECT_SAMPLE_FACTORS 86A 123 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_123 SUBJECT_SAMPLE_FACTORS 92A 124 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_124 SUBJECT_SAMPLE_FACTORS 185V 125 Factor:LDR | Factor:D3 RAW_FILE_NAME=vader_met_125 SUBJECT_SAMPLE_FACTORS 179V 131 Factor:Control | Factor:D2 RAW_FILE_NAME=vader_met_131 SUBJECT_SAMPLE_FACTORS 144V 132 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_132 SUBJECT_SAMPLE_FACTORS 189V 133 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_133 SUBJECT_SAMPLE_FACTORS 124V 134 Factor:LDR | Factor:D20 RAW_FILE_NAME=vader_met_134 SUBJECT_SAMPLE_FACTORS 33V 135 Factor:Control | Factor:D1 RAW_FILE_NAME=vader_met_135 SUBJECT_SAMPLE_FACTORS 73A 138 Factor:HDR | Factor:D3 RAW_FILE_NAME=vader_met_138 SUBJECT_SAMPLE_FACTORS 81A 139 Factor:Control | Factor:D1 RAW_FILE_NAME=vader_met_139 SUBJECT_SAMPLE_FACTORS 128V 140 Factor:LDR | Factor:D30 RAW_FILE_NAME=vader_met_140 SUBJECT_SAMPLE_FACTORS 127V 141 Factor:LDR | Factor:D30 RAW_FILE_NAME=vader_met_141 SUBJECT_SAMPLE_FACTORS 111V 142 Factor:LDR | Factor:D5 RAW_FILE_NAME=vader_met_142 SUBJECT_SAMPLE_FACTORS 65A 148 Factor:HDR | Factor:D1 RAW_FILE_NAME=vader_met_148 SUBJECT_SAMPLE_FACTORS 1V 149 Factor:LDR | Factor:D1 RAW_FILE_NAME=vader_met_149 SUBJECT_SAMPLE_FACTORS 181V 150 Factor:LDR | Factor:D3 RAW_FILE_NAME=vader_met_150 SUBJECT_SAMPLE_FACTORS 142V 151 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_151 SUBJECT_SAMPLE_FACTORS 182V 152 Factor:LDR | Factor:D3 RAW_FILE_NAME=vader_met_152 SUBJECT_SAMPLE_FACTORS 188V 155 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_155 SUBJECT_SAMPLE_FACTORS 145V 156 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_156 SUBJECT_SAMPLE_FACTORS 83A 157 Factor:Control | Factor:D1 RAW_FILE_NAME=vader_met_157 SUBJECT_SAMPLE_FACTORS 76A 158 Factor:HDR | Factor:D5 RAW_FILE_NAME=vader_met_158 SUBJECT_SAMPLE_FACTORS 129V 159 Factor:LDR | Factor:D30 RAW_FILE_NAME=vader_met_159 SUBJECT_SAMPLE_FACTORS 34V 165 Factor:Control | Factor:D1 RAW_FILE_NAME=vader_met_165 SUBJECT_SAMPLE_FACTORS 61A 166 Factor:HDR | Factor:D1 RAW_FILE_NAME=vader_met_166 SUBJECT_SAMPLE_FACTORS 125V 167 Factor:LDR | Factor:D20 RAW_FILE_NAME=vader_met_167 SUBJECT_SAMPLE_FACTORS 91A 168 Factor:Control | Factor:D3 RAW_FILE_NAME=vader_met_168 SUBJECT_SAMPLE_FACTORS 32V 169 Factor:Control | Factor:D1 RAW_FILE_NAME=vader_met_169 SUBJECT_SAMPLE_FACTORS 79A 172 Factor:HDR | Factor:D5 RAW_FILE_NAME=vader_met_172 SUBJECT_SAMPLE_FACTORS 35V 173 Factor:Control | Factor:D1 RAW_FILE_NAME=vader_met_173 SUBJECT_SAMPLE_FACTORS 98A 174 Factor:Control | Factor:D5 RAW_FILE_NAME=vader_met_174 SUBJECT_SAMPLE_FACTORS 74A 175 Factor:HDR | Factor:D3 RAW_FILE_NAME=vader_met_175 #COLLECTION CO:COLLECTION_SUMMARY Serum was collected after irradiation CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY The VADER was designed to deliver controlled dose rates in the range 0.1 – 1 TR:TREATMENT_SUMMARY Gy/day to a cohort of up to 15 mice. The VADER uses ~0.5 Ci of retired 137Cs TR:TREATMENT_SUMMARY brachytherapy seeds that are arranged in two platters placed above and below a TR:TREATMENT_SUMMARY “mouse hotel”. The platters can be placed ~0.5 – 60 cm above and below the TR:TREATMENT_SUMMARY mouse hotel allowing implementation of time-variable dose rates. Offline TR:TREATMENT_SUMMARY dosimetry of the VADER was performed annually using a NIST traceable 10x6-6 TR:TREATMENT_SUMMARY ionization chamber (Radcal Corp., Monrovia, CA). Dose uniformity across the TR:TREATMENT_SUMMARY surface was measured using EBT3 film (Ashland, Covington, KY, USA) and the TR:TREATMENT_SUMMARY variation was 15% across the hotel. A lead and high-density concrete brick TR:TREATMENT_SUMMARY shield ensured minimal radiation doses to occupationally exposed personnel TR:TREATMENT_SUMMARY (operators) inside (< 0.1 mGy/wk) and outside the room (< 0.02 mGy/wk). The TR:TREATMENT_SUMMARY mouse hotel consists of an acrylic box (35 x 35 x 12 cm) allowing housing of ≤ TR:TREATMENT_SUMMARY 15 mice with bedding material and food/water ad libitum. Temperature (20 – TR:TREATMENT_SUMMARY 25°C), humidity (40 – 60%), airflow and lighting were fully controlled to TR:TREATMENT_SUMMARY required animal care standards (temperature/humidity sensor, HWg HTemp, TruePath TR:TREATMENT_SUMMARY Technologies Victor, NY). Environmental controls and monitoring were integrated TR:TREATMENT_SUMMARY into the mouse hotel for easy replacement in case of radiation damage. Mice were TR:TREATMENT_SUMMARY monitored in real time using a 180° fisheye ELP USB camera (Amazon). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were prepared and analyzed as previously described.18, 19 Briefly, serum SP:SAMPLEPREP_SUMMARY (5 μl) was deproteinized (195 μl 66% cold acetonitrile [ACN]) with internal SP:SAMPLEPREP_SUMMARY standards (2 μM debrisoquine [M+H]+ = 176.1188; 30 μM 4-nitrobenzoic acid SP:SAMPLEPREP_SUMMARY [M-H]- = 166.0141), vortexed, incubated on ice (10 min), and centrifuged for 10 SP:SAMPLEPREP_SUMMARY min (max speed, 4 °C). SP:PROCESSING_STORAGE_CONDITIONS -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Mobile phases consisted of the following: solvent A (water/0.1% formic acid CH:CHROMATOGRAPHY_SUMMARY [FA]), solvent B (ACN/0.1% FA), solvent C (isopropanol [IPA]/ACN (90:10)/0.1% CH:CHROMATOGRAPHY_SUMMARY FA). The gradient for urine was (solvent A and B) 4.0 min 5% B, 4.0 min 20% B, CH:CHROMATOGRAPHY_SUMMARY 5.1 min 95% B, and 1.9 min 5% B at a flow rate of 0.5 ml/min. The gradient for CH:CHROMATOGRAPHY_SUMMARY serum was (solvent A, B, and C) 4.0 min 98:2 A:B, 4.0 min 40:60 A:B, 1.5 min CH:CHROMATOGRAPHY_SUMMARY 2:98 A:B, 2.0 min 2:98 A:C, 0.5 min 50:50 A:C, and 1.0 min 98:2 A:B at a flow CH:CHROMATOGRAPHY_SUMMARY rate of 0.5 ml/min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C18 (50 x 2.1mm, 1.7 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 S QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Negative and positive electrospray ionization (ESI) data-independent modes were MS:MS_COMMENTS used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- = MS:MS_COMMENTS 554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage MS:MS_COMMENTS 2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow MS:MS_COMMENTS 1000 L/Hr. MS:MS_RESULTS_FILE ST001792_AN002908_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END