#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