#METABOLOMICS WORKBENCH epannkuk_20210512_094745_mwtab.txt DATATRACK_ID:2638 STUDY_ID:ST001793 ANALYSIS_ID:AN002909 PROJECT_ID:000000 VERSION 1 CREATED_ON May 12, 2021, 10:01 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 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 18V 1 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_012 SUBJECT_SAMPLE_FACTORS 43V 2 Factor:D30 | Factor:LDR RAW_FILE_NAME=serum_013 SUBJECT_SAMPLE_FACTORS 95A 3 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_014 SUBJECT_SAMPLE_FACTORS 70A 4 Factor:D3 | Factor:HDR RAW_FILE_NAME=serum_015 SUBJECT_SAMPLE_FACTORS 28V 5 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_016 SUBJECT_SAMPLE_FACTORS 50V 6 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_018 SUBJECT_SAMPLE_FACTORS 99A 7 Factor:D5 | Factor:HDR RAW_FILE_NAME=serum_019 SUBJECT_SAMPLE_FACTORS 22V 8 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_020 SUBJECT_SAMPLE_FACTORS 73A 9 Factor:D3 | Factor:HDR RAW_FILE_NAME=serum_021 SUBJECT_SAMPLE_FACTORS 1V 10 Factor:D2 | Factor:LDR RAW_FILE_NAME=serum_022 SUBJECT_SAMPLE_FACTORS 2V 11 Factor:D2 | Factor:LDR RAW_FILE_NAME=serum_028 SUBJECT_SAMPLE_FACTORS 89A 12 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_029 SUBJECT_SAMPLE_FACTORS 54V 13 Factor:D30 | Factor:Control RAW_FILE_NAME=serum_030 SUBJECT_SAMPLE_FACTORS 87A 14 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_031 SUBJECT_SAMPLE_FACTORS 44V 15 Factor:D30 | Factor:LDR RAW_FILE_NAME=serum_032 SUBJECT_SAMPLE_FACTORS 90A 16 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_034 SUBJECT_SAMPLE_FACTORS 37V 17 Factor:D5 | Factor:LDR RAW_FILE_NAME=serum_035 SUBJECT_SAMPLE_FACTORS 64A 18 Factor:D2 | Factor:HDR RAW_FILE_NAME=serum_036 SUBJECT_SAMPLE_FACTORS 32V 19 Factor:D5 | Factor:LDR RAW_FILE_NAME=serum_037 SUBJECT_SAMPLE_FACTORS 45V 20 Factor:D30 | Factor:LDR RAW_FILE_NAME=serum_038 SUBJECT_SAMPLE_FACTORS 74A 21 Factor:D3 | Factor:HDR RAW_FILE_NAME=serum_044 SUBJECT_SAMPLE_FACTORS 62A 22 Factor:D2 | Factor:HDR RAW_FILE_NAME=serum_045 SUBJECT_SAMPLE_FACTORS 94A 23 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_046 SUBJECT_SAMPLE_FACTORS 69A 24 Factor:D3 | Factor:HDR RAW_FILE_NAME=serum_047 SUBJECT_SAMPLE_FACTORS 49V 25 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_048 SUBJECT_SAMPLE_FACTORS 67A 26 Factor:D2 | Factor:HDR RAW_FILE_NAME=serum_050 SUBJECT_SAMPLE_FACTORS 65A 27 Factor:D2 | Factor:HDR RAW_FILE_NAME=serum_051 SUBJECT_SAMPLE_FACTORS 30V 28 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_052 SUBJECT_SAMPLE_FACTORS 98A 29 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_053 SUBJECT_SAMPLE_FACTORS 40V 30 Factor:D30 | Factor:LDR RAW_FILE_NAME=serum_054 SUBJECT_SAMPLE_FACTORS 53V 31 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_060 SUBJECT_SAMPLE_FACTORS 36V 32 Factor:D5 | Factor:LDR RAW_FILE_NAME=serum_061 SUBJECT_SAMPLE_FACTORS 60V 33 Factor:D30 | Factor:Control RAW_FILE_NAME=serum_062 SUBJECT_SAMPLE_FACTORS 93A 35 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_063 SUBJECT_SAMPLE_FACTORS 4V 36 Factor:D2 | Factor:LDR RAW_FILE_NAME=serum_064 SUBJECT_SAMPLE_FACTORS 31V 37 Factor:D5 | Factor:LDR RAW_FILE_NAME=serum_066 SUBJECT_SAMPLE_FACTORS 20V 38 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_067 SUBJECT_SAMPLE_FACTORS 88A 39 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_068 SUBJECT_SAMPLE_FACTORS 83A 40 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_069 SUBJECT_SAMPLE_FACTORS 10V 41 Factor:D3 | Factor:LDR RAW_FILE_NAME=serum_070 SUBJECT_SAMPLE_FACTORS 91A 42 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_076 SUBJECT_SAMPLE_FACTORS 59V 43 Factor:D30 | Factor:Control RAW_FILE_NAME=serum_077 SUBJECT_SAMPLE_FACTORS 101A 44 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_078 SUBJECT_SAMPLE_FACTORS 96A 45 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_079 SUBJECT_SAMPLE_FACTORS 15V 46 Factor:D3 | Factor:LDR RAW_FILE_NAME=serum_080 SUBJECT_SAMPLE_FACTORS 56V 47 Factor:D30 | Factor:Control RAW_FILE_NAME=serum_082 SUBJECT_SAMPLE_FACTORS 17V 48 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_083 SUBJECT_SAMPLE_FACTORS 58V 49 Factor:D30 | Factor:Control RAW_FILE_NAME=serum_084 SUBJECT_SAMPLE_FACTORS 78A 50 Factor:D5 | Factor:HDR RAW_FILE_NAME=serum_085 SUBJECT_SAMPLE_FACTORS 71A 51 Factor:D3 | Factor:HDR RAW_FILE_NAME=serum_086 SUBJECT_SAMPLE_FACTORS 51V 52 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_092 SUBJECT_SAMPLE_FACTORS 11V 53 Factor:D3 | Factor:LDR RAW_FILE_NAME=serum_093 SUBJECT_SAMPLE_FACTORS 76A 54 Factor:D3 | Factor:HDR RAW_FILE_NAME=serum_094 SUBJECT_SAMPLE_FACTORS 12V 55 Factor:D3 | Factor:LDR RAW_FILE_NAME=serum_095 SUBJECT_SAMPLE_FACTORS 16V 56 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_096 SUBJECT_SAMPLE_FACTORS 55V 58 Factor:D30 | Factor:Control RAW_FILE_NAME=serum_098 SUBJECT_SAMPLE_FACTORS 104A 59 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_099 SUBJECT_SAMPLE_FACTORS 82A 60 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_100 SUBJECT_SAMPLE_FACTORS 66A 61 Factor:D2 | Factor:HDR RAW_FILE_NAME=serum_101 SUBJECT_SAMPLE_FACTORS 6V 62 Factor:D2 | Factor:HDR RAW_FILE_NAME=serum_102 SUBJECT_SAMPLE_FACTORS 26V 63 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_108 SUBJECT_SAMPLE_FACTORS 48V 64 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_109 SUBJECT_SAMPLE_FACTORS 80A 65 Factor:D5 | Factor:HDR RAW_FILE_NAME=serum_110 SUBJECT_SAMPLE_FACTORS 103A 66 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_111 SUBJECT_SAMPLE_FACTORS 105A 67 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_112 SUBJECT_SAMPLE_FACTORS 86A 68 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_114 SUBJECT_SAMPLE_FACTORS 25V 69 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_115 SUBJECT_SAMPLE_FACTORS 5V 70 Factor:D2 | Factor:LDR RAW_FILE_NAME=serum_116 SUBJECT_SAMPLE_FACTORS 14V 71 Factor:D3 | Factor:LDR RAW_FILE_NAME=serum_117 SUBJECT_SAMPLE_FACTORS 29V 72 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_118 SUBJECT_SAMPLE_FACTORS 57V 73 Factor:D30 | Factor:Control RAW_FILE_NAME=serum_119 SUBJECT_SAMPLE_FACTORS 79A 74 Factor:D5 | Factor:HDR RAW_FILE_NAME=serum_120 SUBJECT_SAMPLE_FACTORS 41V 75 Factor:D30 | Factor:LDR RAW_FILE_NAME=serum_126 SUBJECT_SAMPLE_FACTORS 46V 76 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_127 SUBJECT_SAMPLE_FACTORS 102A 77 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_128 SUBJECT_SAMPLE_FACTORS 9V 78 Factor:D3 | Factor:HDR RAW_FILE_NAME=serum_129 SUBJECT_SAMPLE_FACTORS 68A 79 Factor:D2 | Factor:HDR RAW_FILE_NAME=serum_130 SUBJECT_SAMPLE_FACTORS 19V 80 Factor:D2 | Factor:Control RAW_FILE_NAME=serum_131 SUBJECT_SAMPLE_FACTORS 63A 81 Factor:D2 | Factor:HDR RAW_FILE_NAME=serum_132 SUBJECT_SAMPLE_FACTORS 13V 82 Factor:D3 | Factor:LDR RAW_FILE_NAME=serum_133 SUBJECT_SAMPLE_FACTORS 61A 83 Factor:D2 | Factor:HDR RAW_FILE_NAME=serum_135 SUBJECT_SAMPLE_FACTORS 27V 84 Factor:D3 | Factor:Control RAW_FILE_NAME=serum_136 SUBJECT_SAMPLE_FACTORS 84A 85 Factor:D5 | Factor:HDR RAW_FILE_NAME=serum_137 SUBJECT_SAMPLE_FACTORS 42V 86 Factor:D30 | Factor:LDR RAW_FILE_NAME=serum_138 SUBJECT_SAMPLE_FACTORS 52V 87 Factor:D5 | Factor:Control RAW_FILE_NAME=serum_139 SUBJECT_SAMPLE_FACTORS 39V 88 Factor:D30 | Factor:LDR RAW_FILE_NAME=serum_140 SUBJECT_SAMPLE_FACTORS 38V 89 Factor:D5 | Factor:LDR RAW_FILE_NAME=serum_141 #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 POSITIVE 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 ST001793_AN002909_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END