#METABOLOMICS WORKBENCH sinkwarr_20240911_155653 DATATRACK_ID:5187 STUDY_ID:ST003476 ANALYSIS_ID:AN005711 PROJECT_ID:PR002135 VERSION 1 CREATED_ON September 17, 2024, 10:52 am #PROJECT PR:PROJECT_TITLE Gas chromatography - mass spectrometry (GC-MS) of liver hepatic extracts from PR:PROJECT_TITLE adult male C57BL/6NCrl mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin PR:PROJECT_SUMMARY Epidemiological evidence suggests an association between dioxin and dioxin-like PR:PROJECT_SUMMARY compound (DLC) exposure and human liver disease. The prototypical DLC, PR:PROJECT_SUMMARY 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been shown to induce the PR:PROJECT_SUMMARY progression of reversible hepatic steatosis to steatohepatitis with periportal PR:PROJECT_SUMMARY fibrosis and biliary hyperplasia in C57BL/6NCrl mice. Although the effects of PR:PROJECT_SUMMARY TCDD toxicity are mediated by aryl hydrocarbon receptor (AHR) activation, the PR:PROJECT_SUMMARY underlying mechanisms of TCDD-induced hepatotoxicity are unresolved. In the PR:PROJECT_SUMMARY present study, male C57BL/6NCrl mice were gavaged every 4 days for 28 days with PR:PROJECT_SUMMARY 0.03 - 30 µg/kg TCDD and evaluated for liver histopathology and gene expression PR:PROJECT_SUMMARY as well as complementary 1-dimensional (1-D) 1H NMR urinary metabolic profiling. PR:PROJECT_SUMMARY Urinary trimethylamine (TMA), trimethylamine N-oxide (TMAO), and PR:PROJECT_SUMMARY 1-methylnicotinamide (1MN) levels were altered by TCDD at doses ≤ 3 µg/kg; PR:PROJECT_SUMMARY other urinary metabolites, like glycolate, urocanate, and 3-hydroxyisovalerate, PR:PROJECT_SUMMARY were only altered at doses that induced moderate to severe steatohepatitis. Bulk PR:PROJECT_SUMMARY liver RNA-seq data suggested altered urinary metabolites correlated with hepatic PR:PROJECT_SUMMARY differential gene expression corresponding to specific metabolic pathways. In PR:PROJECT_SUMMARY addition to evaluating whether altered urinary metabolites were liver-dependent, PR:PROJECT_SUMMARY published single-nuclear RNA-seq (snRNA-seq), AHR ChIP-seq, and AHR knockout PR:PROJECT_SUMMARY gene expression datasets provided further support of hepatic cell-type and PR:PROJECT_SUMMARY AHR-regulated dependency, respectively. Overall, TCDD-induced liver effects were PR:PROJECT_SUMMARY preceded by and occurred with changes in urinary metabolite levels due to PR:PROJECT_SUMMARY AHR-mediated changes in hepatic gene expression. PR:INSTITUTE Michigan State University PR:DEPARTMENT Biochemistry and Molecular Biology PR:LABORATORY Dr. Tim Zacharewski's PR:LAST_NAME Sink PR:FIRST_NAME Warren PR:ADDRESS 603 Wilson Rd Rm 212, East Lansing, MI 48823 PR:EMAIL sinkwarr@msu.edu PR:PHONE 6162953496 #STUDY ST:STUDY_TITLE Gas chromatography - mass spectrometry (GC-MS) of liver hepatic extracts from ST:STUDY_TITLE adult male C57BL/6NCrl mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin ST:STUDY_SUMMARY Epidemiological evidence suggests an association between dioxin and dioxin-like ST:STUDY_SUMMARY compound (DLC) exposure and human liver disease. The prototypical DLC, ST:STUDY_SUMMARY 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been shown to induce the ST:STUDY_SUMMARY progression of reversible hepatic steatosis to steatohepatitis with periportal ST:STUDY_SUMMARY fibrosis and biliary hyperplasia in C57BL/6NCrl mice. Although the effects of ST:STUDY_SUMMARY TCDD toxicity are mediated by aryl hydrocarbon receptor (AHR) activation, the ST:STUDY_SUMMARY underlying mechanisms of TCDD-induced hepatotoxicity are unresolved. In the ST:STUDY_SUMMARY present study, male C57BL/6NCrl mice were gavaged every 4 days for 28 days with ST:STUDY_SUMMARY 0.03 - 30 µg/kg TCDD and evaluated for liver histopathology and gene expression ST:STUDY_SUMMARY as well as complementary 1-dimensional (1-D) 1H NMR urinary metabolic profiling. ST:STUDY_SUMMARY Urinary trimethylamine (TMA), trimethylamine N-oxide (TMAO), and ST:STUDY_SUMMARY 1-methylnicotinamide (1MN) levels were altered by TCDD at doses ≤ 3 µg/kg; ST:STUDY_SUMMARY other urinary metabolites, like glycolate, urocanate, and 3-hydroxyisovalerate, ST:STUDY_SUMMARY were only altered at doses that induced moderate to severe steatohepatitis. Bulk ST:STUDY_SUMMARY liver RNA-seq data suggested altered urinary metabolites correlated with hepatic ST:STUDY_SUMMARY differential gene expression corresponding to specific metabolic pathways. In ST:STUDY_SUMMARY addition to evaluating whether altered urinary metabolites were liver-dependent, ST:STUDY_SUMMARY published single-nuclear RNA-seq (snRNA-seq), AHR ChIP-seq, and AHR knockout ST:STUDY_SUMMARY gene expression datasets provided further support of hepatic cell-type and ST:STUDY_SUMMARY AHR-regulated dependency, respectively. Overall, TCDD-induced liver effects were ST:STUDY_SUMMARY preceded by and occurred with changes in urinary metabolite levels due to ST:STUDY_SUMMARY AHR-mediated changes in hepatic gene expression. ST:INSTITUTE Michigan State University ST:DEPARTMENT Biochemistry and Molecular Biology ST:LABORATORY Dr. Tim Zacharewski's ST:LAST_NAME Sink ST:FIRST_NAME Warren ST:ADDRESS 603 Wilson Rd Rm 212, East Lansing, MI 48823 ST:EMAIL sinkwarr@msu.edu ST:PHONE 6162953496 ST:NUM_GROUPS 5 ST:TOTAL_SUBJECTS 25 ST:NUM_MALES 25 ST:STUDY_COMMENTS Adult male C57BL/6NCrl mice (PND 30) were gavaged every 4 days for 28 days with ST:STUDY_COMMENTS 1, 3, 10, or 30 µg/kg TCDD or sesame oil vehicle. Mice were euthanized and ST:STUDY_COMMENTS their liver tissue collected at the conclusion of the TCDD exposure (PND 58). #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE Mice were euthanized and their liver tissue collected on post natal day (PND) SU:AGE_OR_AGE_RANGE 58. SU:WEIGHT_OR_WEIGHT_RANGE 15 - 24 g SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Charles River Laboratories SU:ANIMAL_HOUSING Mice were housed in Innovive Innocages (San Diego, CA) containing ALPHA-dri SU:ANIMAL_HOUSING bedding (Shepherd Specialty Papers, Chicago) at an ambient temperature of 21°C SU:ANIMAL_HOUSING with 30-40% humidity. SU:ANIMAL_LIGHT_CYCLE 12:12 SU:ANIMAL_FEED TEKLAD 8940 #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 0_microg_per_mL A_20231209_002 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_002.CDF SUBJECT_SAMPLE_FACTORS 0.5_microg_per_mL A_20231209_003 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_003.CDF SUBJECT_SAMPLE_FACTORS 1.0625_microg_per_mL A_20231209_004 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_004.CDF SUBJECT_SAMPLE_FACTORS 3.125_microg_per_mL A_20231209_005 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_005.CDF SUBJECT_SAMPLE_FACTORS 6.25_microg_per_mL A_20231209_006 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_006.CDF SUBJECT_SAMPLE_FACTORS 12.5_microg_per_mL A_20231209_007 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_007.CDF SUBJECT_SAMPLE_FACTORS 25_microg_per_mL A_20231209_008 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_008.CDF SUBJECT_SAMPLE_FACTORS 100_microg_per_mL A_20231209_009 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_009.CDF SUBJECT_SAMPLE_FACTORS 121 A_20231209_010 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=47.5; RAW_FILE_NAME=A_20231209_010.CDF SUBJECT_SAMPLE_FACTORS 109 A_20231209_011 Sample source:Liver | TCDD_dose_microg_per_kg:1 LiverWeight (gm)=63.5; RAW_FILE_NAME=A_20231209_011.CDF SUBJECT_SAMPLE_FACTORS 138 A_20231209_012 Sample source:Liver | TCDD_dose_microg_per_kg:3 LiverWeight (gm)=51.3; RAW_FILE_NAME=A_20231209_012.CDF SUBJECT_SAMPLE_FACTORS 69 A_20231209_013 Sample source:Liver | TCDD_dose_microg_per_kg:10 LiverWeight (gm)=60.5; RAW_FILE_NAME=A_20231209_013.CDF SUBJECT_SAMPLE_FACTORS 142 A_20231209_014 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=57.5; RAW_FILE_NAME=A_20231209_014.CDF SUBJECT_SAMPLE_FACTORS 122 A_20231209_015 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=52.6; RAW_FILE_NAME=A_20231209_015.CDF SUBJECT_SAMPLE_FACTORS 61 A_20231209_016 Sample source:Liver | TCDD_dose_microg_per_kg:1 LiverWeight (gm)=60.9; RAW_FILE_NAME=A_20231209_016.CDF SUBJECT_SAMPLE_FACTORS 65 A_20231209_017 Sample source:Liver | TCDD_dose_microg_per_kg:3 LiverWeight (gm)=61.9; RAW_FILE_NAME=A_20231209_017.CDF SUBJECT_SAMPLE_FACTORS 139 A_20231209_018 Sample source:Liver | TCDD_dose_microg_per_kg:10 LiverWeight (gm)=62.1; RAW_FILE_NAME=A_20231209_018.CDF SUBJECT_SAMPLE_FACTORS 70 A_20231209_019 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=50; RAW_FILE_NAME=A_20231209_019.CDF SUBJECT_SAMPLE_FACTORS 25 A_20231209_020 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=57.3; RAW_FILE_NAME=A_20231209_020.CDF SUBJECT_SAMPLE_FACTORS 134 A_20231209_021 Sample source:Liver | TCDD_dose_microg_per_kg:1 LiverWeight (gm)=56.5; RAW_FILE_NAME=A_20231209_021.CDF SUBJECT_SAMPLE_FACTORS 66 A_20231209_022 Sample source:Liver | TCDD_dose_microg_per_kg:3 LiverWeight (gm)=58.5; RAW_FILE_NAME=A_20231209_022.CDF SUBJECT_SAMPLE_FACTORS 140 A_20231209_023 Sample source:Liver | TCDD_dose_microg_per_kg:10 LiverWeight (gm)=62.5; RAW_FILE_NAME=A_20231209_023.CDF SUBJECT_SAMPLE_FACTORS 144 A_20231209_024 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=59.2; RAW_FILE_NAME=A_20231209_024.CDF SUBJECT_SAMPLE_FACTORS 49 A_20231209_025 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=47.9; RAW_FILE_NAME=A_20231209_025.CDF SUBJECT_SAMPLE_FACTORS 63 A_20231209_026 Sample source:Liver | TCDD_dose_microg_per_kg:1 LiverWeight (gm)=58.5; RAW_FILE_NAME=A_20231209_026.CDF SUBJECT_SAMPLE_FACTORS 112 A_20231209_027 Sample source:Liver | TCDD_dose_microg_per_kg:3 LiverWeight (gm)=52.3; RAW_FILE_NAME=A_20231209_027.CDF SUBJECT_SAMPLE_FACTORS 67 A_20231209_028 Sample source:Liver | TCDD_dose_microg_per_kg:10 LiverWeight (gm)=61.9; RAW_FILE_NAME=A_20231209_028.CDF SUBJECT_SAMPLE_FACTORS 143 A_20231209_029 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=45.1; RAW_FILE_NAME=A_20231209_029.CDF SUBJECT_SAMPLE_FACTORS 50 A_20231209_030 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=52; RAW_FILE_NAME=A_20231209_030.CDF SUBJECT_SAMPLE_FACTORS 62 A_20231209_031 Sample source:Liver | TCDD_dose_microg_per_kg:1 LiverWeight (gm)=48.6; RAW_FILE_NAME=A_20231209_031.CDF SUBJECT_SAMPLE_FACTORS 137 A_20231209_032 Sample source:Liver | TCDD_dose_microg_per_kg:3 LiverWeight (gm)=52.7; RAW_FILE_NAME=A_20231209_032.CDF SUBJECT_SAMPLE_FACTORS 115 A_20231209_033 Sample source:Liver | TCDD_dose_microg_per_kg:10 LiverWeight (gm)=64; RAW_FILE_NAME=A_20231209_033.CDF SUBJECT_SAMPLE_FACTORS 71 A_20231209_034 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=64.8; RAW_FILE_NAME=A_20231209_034.CDF SUBJECT_SAMPLE_FACTORS OA_0_microg_per_mL A_20231209_035 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_035.CDF SUBJECT_SAMPLE_FACTORS OA_1.0625_microg_per_mL A_20231209_036 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_036.CDF SUBJECT_SAMPLE_FACTORS OA_3.125_microg_per_mL A_20231209_037 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_037.CDF SUBJECT_SAMPLE_FACTORS OA_6.25_microg_per_mL A_20231209_038 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_038.CDF SUBJECT_SAMPLE_FACTORS OA_12.5_microg_per_mL A_20231209_039 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_039.CDF SUBJECT_SAMPLE_FACTORS OA_25_microg_per_mL A_20231209_040 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_040.CDF SUBJECT_SAMPLE_FACTORS OA_100_microg_per_mL A_20231209_041 Sample source:Standard | TCDD_dose_microg_per_kg:- LiverWeight (gm)=-; RAW_FILE_NAME=A_20231209_041.CDF SUBJECT_SAMPLE_FACTORS 121 A_20231209_042 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=47.5; RAW_FILE_NAME=A_20231209_042.CDF SUBJECT_SAMPLE_FACTORS 142 A_20231209_043 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=57.5; RAW_FILE_NAME=A_20231209_043.CDF SUBJECT_SAMPLE_FACTORS 122 A_20231209_044 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=52.6; RAW_FILE_NAME=A_20231209_044.CDF SUBJECT_SAMPLE_FACTORS 70 A_20231211_002 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=50; RAW_FILE_NAME=A_20231211_002.CDF SUBJECT_SAMPLE_FACTORS 25 A_20231211_003 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=57.3; RAW_FILE_NAME=A_20231211_003.CDF SUBJECT_SAMPLE_FACTORS 144 A_20231211_004 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=59.2; RAW_FILE_NAME=A_20231211_004.CDF SUBJECT_SAMPLE_FACTORS 49 A_20231211_005 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=47.9; RAW_FILE_NAME=A_20231211_005.CDF SUBJECT_SAMPLE_FACTORS 143 A_20231211_006 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=45.1; RAW_FILE_NAME=A_20231211_006.CDF SUBJECT_SAMPLE_FACTORS 50 A_20231211_007 Sample source:Liver | TCDD_dose_microg_per_kg:0 LiverWeight (gm)=52; RAW_FILE_NAME=A_20231211_007.CDF SUBJECT_SAMPLE_FACTORS 71 A_20231211_008 Sample source:Liver | TCDD_dose_microg_per_kg:30 LiverWeight (gm)=64.8; RAW_FILE_NAME=A_20231211_008.CDF #COLLECTION CO:COLLECTION_SUMMARY On post-natal day 58 after the mice were repeatedly gavaged every 4 days for 28 CO:COLLECTION_SUMMARY days with TCDD, the mice were euthanized, and their livers were collected and CO:COLLECTION_SUMMARY immediately snap-frozen. Collected liver was kept frozen until processing. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Beginning on post-natal day 30, C57BL/6NCrl male mice were orally gavaged every TR:TREATMENT_SUMMARY 4 days for 28 days with TCDD or sesame oil vehicle. On post-natal day 58, mice TR:TREATMENT_SUMMARY were euthanized, and liver tissue was collected. TR:TREATMENT_COMPOUND 2,3,7,8-tetrachlorodibenzo-p-dioxin TR:TREATMENT_ROUTE Oral gavage TR:TREATMENT_DOSE 0, 1, 3, 10, or 30 micrograms per kilogram TR:TREATMENT_DOSEVOLUME 0.1 milliliters TR:TREATMENT_DOSEDURATION 28 days TR:TREATMENT_VEHICLE sesame oil #SAMPLEPREP SP:SAMPLEPREP_SUMMARY GC-MS. Extraction, derivatization, and measurement of hepatic metabolites by SP:SAMPLEPREP_SUMMARY gas-chromatography mass spectrometry (GC-MS) was performed using protocols SP:SAMPLEPREP_SUMMARY adapted from the publicly available Michigan State University Mass Spectrometry SP:SAMPLEPREP_SUMMARY and Metabolomics Core website SP:SAMPLEPREP_SUMMARY (https://rtsf.natsci.msu.edu/sites/_rtsf/assets/File/MSU_MSMC_004_v1_2_Two_phase_extraction_of_metabolites_from_animal_tissues.pdf). SP:SAMPLEPREP_SUMMARY Briefly, ~50 mg of frozen liver tissue (n = 5 per dose) was supplemented with 6 SP:SAMPLEPREP_SUMMARY nanomoles of 13C and 15N labeled amino acid internal standards (767964, SP:SAMPLEPREP_SUMMARY Sigma-Aldrich) and lysed with a Mixer Mill 300 and a 3 mm stainless steel ball SP:SAMPLEPREP_SUMMARY in a solution of methanol/chloroform (1:2 v/v), 1% formic acid, and 0.01% BHT. SP:SAMPLEPREP_SUMMARY Subsequently, the samples were sonicated for 15 minutes. The mixture was twice SP:SAMPLEPREP_SUMMARY supplemented with Milli-Q water, vortexed, centrifuged, and the resulting SP:SAMPLEPREP_SUMMARY aqueous supernatant was transferred to a separate Eppendorf tube and made basic SP:SAMPLEPREP_SUMMARY by the addition of NaOH. The samples were evaporated to dryness by a SpeedVac SP:SAMPLEPREP_SUMMARY without heat and stored at -20° C. For derivatization, the dried samples were SP:SAMPLEPREP_SUMMARY set at 60°C overnight with 100 µL of 40 mg/mL of methoxyamine HCl dissolved in SP:SAMPLEPREP_SUMMARY pyridine and incubated overnight with 100 µL of SP:SAMPLEPREP_SUMMARY N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) containing 1% SP:SAMPLEPREP_SUMMARY tert-butyldimethylsilyl chloride. SP:SAMPLEPREP_PROTOCOL_FILENAME Protocol_MSU_MSMC_004_version_1.2.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Derivatized samples (1 µL) were injected with a 1:10 split at 230 °C and CH:CHROMATOGRAPHY_SUMMARY analyzed on an Agilent 7890A GC/ single quadrupole mass spectrometer with 5975C CH:CHROMATOGRAPHY_SUMMARY inert XL MSD. The carrier gas was helium, which flowed at 1.0 mL/min. Separation CH:CHROMATOGRAPHY_SUMMARY was achieved on an Agilent J&W VF-5ms (30 m x 0.25 mm x 0.25 µm) (Agilent, CH:CHROMATOGRAPHY_SUMMARY Santa Clara, CA) using the following temperature profile: initially set at 40 CH:CHROMATOGRAPHY_SUMMARY °C, ramped to 80 °C at 20 °C/min, immediately ramped to 250 °C at 5 °C/min CH:CHROMATOGRAPHY_SUMMARY and held for 1 minute, and ramped to 320 °C at 50 °C/min and held for 5 CH:CHROMATOGRAPHY_SUMMARY minute. The mass spectrometer was operated at an electron ionization of 70 eV CH:CHROMATOGRAPHY_SUMMARY and a scan range of 50-600 m/z. Glycolic acid (PHR1427, Sigma), hydroxyproline CH:CHROMATOGRAPHY_SUMMARY (PHR1939, Sigma), oxalic acid (PHR3291, Sigma), and urea (PHR1406, Sigma) were CH:CHROMATOGRAPHY_SUMMARY used as external standards to calculate metabolite levels per sample. CH:CHROMATOGRAPHY_SUMMARY Quantification of the derivatized metabolites and internal standard were CH:CHROMATOGRAPHY_SUMMARY calculated using the peak area from the extracted ion chromatogram. The SIM ion CH:CHROMATOGRAPHY_SUMMARY used for derivatized glycolic acid, hydroxyproline, oxalic acid, and urea were CH:CHROMATOGRAPHY_SUMMARY 247, 416, 261, and 231 m/z, respectively. Derivatized glycolic acid, CH:CHROMATOGRAPHY_SUMMARY hydroxyproline, and urea were normalized to labeled glycine, while oxalic acid CH:CHROMATOGRAPHY_SUMMARY was normalized to labeled alanine. Oxalic acid and alanine were quantified by CH:CHROMATOGRAPHY_SUMMARY separate runs in the SIM mode, while the other compounds were quantified from CH:CHROMATOGRAPHY_SUMMARY runs in the scan mode. Additionally, hydroxyproline was normalized to labeled CH:CHROMATOGRAPHY_SUMMARY proline. After calculating concentrations via a linear calibration curve, CH:CHROMATOGRAPHY_SUMMARY concentrations were normalized to the liver weight per sample from which CH:CHROMATOGRAPHY_SUMMARY metabolites were extracted. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent J&W VF-5ms (30 m x 0.25 mm, 0.25 um) CH:SOLVENT_A - CH:SOLVENT_B - CH:FLOW_GRADIENT - CH:FLOW_RATE 1.0 mL/min CH:COLUMN_TEMPERATURE initially set at 40 °C, ramped to 80 °C at 20 °C/min, immediately ramped to CH:COLUMN_TEMPERATURE 250 °C at 5 °C/min and held for 1 minute, and ramped to 320 °C at 50 °C/min CH:COLUMN_TEMPERATURE and held for 5 minute #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7890A MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometer was operated at an electron ionization of 70 eV and a scan MS:MS_COMMENTS range of 50-600 m/z. Glycolic acid (PHR1427, Sigma), hydroxyproline (PHR1939, MS:MS_COMMENTS Sigma), oxalic acid (PHR3291, Sigma), and urea (PHR1406, Sigma) were used as MS:MS_COMMENTS external standards to calculate metabolite levels per sample. Quantification of MS:MS_COMMENTS the derivatized metabolites and internal standard were calculated using the peak MS:MS_COMMENTS area from the extracted ion chromatogram. The SIM ion used for derivatized MS:MS_COMMENTS glycolic acid, hydroxyproline, oxalic acid, and urea were 247, 416, 261, and 231 MS:MS_COMMENTS m/z, respectively. Derivatized glycolic acid, hydroxyproline, and urea were MS:MS_COMMENTS normalized to labeled glycine, while oxalic acid was normalized to labeled MS:MS_COMMENTS alanine. Oxalic acid and alanine were quantified by separate runs in the SIM MS:MS_COMMENTS mode, while the other compounds were quantified from runs in the scan mode. MS:MS_COMMENTS Additionally, hydroxyproline was normalized to labeled proline. After MS:MS_COMMENTS calculating concentrations via a linear calibration curve, concentrations were MS:MS_COMMENTS normalized to the liver weight per sample from which metabolites were extracted. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples A_20231209_002 A_20231209_003 A_20231209_004 A_20231209_005 A_20231209_006 A_20231209_007 A_20231209_008 A_20231209_009 A_20231209_010 A_20231209_011 A_20231209_012 A_20231209_013 A_20231209_014 A_20231209_015 A_20231209_016 A_20231209_017 A_20231209_018 A_20231209_019 A_20231209_020 A_20231209_021 A_20231209_022 A_20231209_023 A_20231209_024 A_20231209_025 A_20231209_026 A_20231209_027 A_20231209_028 A_20231209_029 A_20231209_030 A_20231209_031 A_20231209_032 A_20231209_033 A_20231209_034 A_20231209_035 A_20231209_036 A_20231209_037 A_20231209_038 A_20231209_039 A_20231209_040 A_20231209_041 A_20231209_042 A_20231209_043 A_20231209_044 A_20231211_002 A_20231211_003 A_20231211_004 A_20231211_005 A_20231211_006 A_20231211_007 A_20231211_008 Factors Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:1 Sample source:Liver | TCDD_dose_microg_per_kg:3 Sample source:Liver | TCDD_dose_microg_per_kg:10 Sample source:Liver | TCDD_dose_microg_per_kg:30 Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:1 Sample source:Liver | TCDD_dose_microg_per_kg:3 Sample source:Liver | TCDD_dose_microg_per_kg:10 Sample source:Liver | TCDD_dose_microg_per_kg:30 Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:1 Sample source:Liver | TCDD_dose_microg_per_kg:3 Sample source:Liver | TCDD_dose_microg_per_kg:10 Sample source:Liver | TCDD_dose_microg_per_kg:30 Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:1 Sample source:Liver | TCDD_dose_microg_per_kg:3 Sample source:Liver | TCDD_dose_microg_per_kg:10 Sample source:Liver | TCDD_dose_microg_per_kg:30 Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:1 Sample source:Liver | TCDD_dose_microg_per_kg:3 Sample source:Liver | TCDD_dose_microg_per_kg:10 Sample source:Liver | TCDD_dose_microg_per_kg:30 Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Standard | TCDD_dose_microg_per_kg:- Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:30 Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:30 Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:30 Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:30 Sample source:Liver | TCDD_dose_microg_per_kg:0 Sample source:Liver | TCDD_dose_microg_per_kg:30 Glycolic acid 154.31 2782.56 6100.65 14708.72 30314.03 58564.08 126035.16 466362.59 7516.63 15756.52 8993.94 16514.97 17483.3 17253.31 21753 28440.92 23331.72 15309.62 12264.3 20563.94 15789.61 24289.63 20250.18 12854.19 14505.19 15820.03 26939.17 14642.09 17115.87 14631.94 17901.79 25321.17 22654.21 Hydroxyproline 4.11 1320.95 2945.73 18389.81 16587.78 33132.08 77884.5 57507.85 1924.46 1909.27 1239.62 1616.85 225.83 2002.58 1483.19 1893.34 2010.18 236.89 1684.29 2610.03 1959.76 2976.41 1381.51 2164.74 149.08 1327.22 2412.81 4106.97 1714.81 1433.46 906.17 451.68 993.08 Proline 51.04 67.09 91.31 89.46 4.3 88.32 95.79 119.95 31.34 5.11 56.09 109.65 31.9 92.86 88.7 77.19 79.13 62.89 63.12 68.99 31.94 108.14 76.59 153.26 73.15 194.5 99.36 106.14 Glycine 4685.7 5977.24 7064.7 8130.46 8176.02 7504.43 7486.53 8936.79 4877.3 5022.28 4593.25 5103.91 7648.14 5552.83 4964.2 4148.02 6589.01 7974.07 7596.97 6212.32 6851.3 8747.16 6451.73 5391.08 5776.29 4848.25 8342.57 8620.69 4837.1 4199.38 5819.57 7247.9 9351.74 Oxalic acid 755.14 1198.01 1645.4 2280.13 3034.15 5338.76 20193.95 4009.84 2417.68 3141.65 2910.47 3492.71 2811.47 2503.47 2116.87 3056.83 4237.2 Alanine 6703.17 7468.5 6983.63 8088.2 6342.34 5662.59 6301.69 3371.81 4163.76 3485.26 4344.42 3234.21 3341.48 2730.13 4454.67 2892.86 4483.42 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z Pubchem ID KEGG ID Glycolic acid 18.3 247 757 C00160 Hydroxyproline 32.05 416 5810 C01157 Proline 24.75 261 145742 C00148 Glycine 19.6 221 750 C00037 Oxalic acid 19.3 261 971 C00209 Alanine 18.93 264 5950 C00041 METABOLITES_END #END