#METABOLOMICS WORKBENCH orlowska_20230619_070959 DATATRACK_ID:4097 STUDY_ID:ST002756 ANALYSIS_ID:AN004473 PROJECT_ID:PR001717 VERSION 1 CREATED_ON June 28, 2023, 10:25 am #PROJECT PR:PROJECT_TITLE Prj171_Mm_TCDD_RDDR-28D_Male PR:PROJECT_TYPE targeted metabolomics PR:PROJECT_SUMMARY The aryl hydrocarbon receptor (AhR) is a transcription factor activated by PR:PROJECT_SUMMARY structurally diverse chemicals, endogenous metabolites, and natural products. PR:PROJECT_SUMMARY AhR activation causes the dissociation of chaperone proteins, followed by PR:PROJECT_SUMMARY translocation to the nucleus and dimerization with the AhR nuclear translocator PR:PROJECT_SUMMARY (ARNT). The complex binds dioxin response elements (DREs; 5’-GCGTG-3’) eliciting PR:PROJECT_SUMMARY changes in gene expression. AhR activation by its most potent ligand PR:PROJECT_SUMMARY 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) promotes the development and progression PR:PROJECT_SUMMARY of non-alcoholic fatty liver disease (NAFLD). NAFLD is a spectrum of pathologies PR:PROJECT_SUMMARY that spans simple, reversible, and benign lipid accumulation (hepatic steatosis), PR:PROJECT_SUMMARY to steatosis with inflammation (steatohepatitis) and collagen deposition PR:PROJECT_SUMMARY (fibrosis/cirrhosis) in the absence of excessive alcohol consumption. NAFLD prevalence PR:PROJECT_SUMMARY is projected to increase from ~83 million in 2015 to ~101 million by 2030 in the US PR:PROJECT_SUMMARY alone, while increasing the risk for more complex disorders including Metabolic Syndrome, PR:PROJECT_SUMMARY cardiovascular disease, diabetes, cirrhosis, end-stage liver disease and hepatocellular PR:PROJECT_SUMMARY carcinoma (HCC). The role of AhR-mediated metabolic dysregulation in hepatotoxicity and PR:PROJECT_SUMMARY the etiology of more complex metabolic diseases warrants further investigation. Therofore, PR:PROJECT_SUMMARY in this project on PND28 mice were orally gavaged at the start of the light cycle PR:PROJECT_SUMMARY (zeitgeber [ZT] 0-1) with 0.1 ml sesame oil vehicle or 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and PR:PROJECT_SUMMARY 30 ug/kg body weight TCDD every 4 days for 28 days for a total of 7 treatments. The first PR:PROJECT_SUMMARY gavage was administered on day 0, with the last gavage administered on day 24 of the 28-day PR:PROJECT_SUMMARY study. On day 28, tissue samples were harvested (ZT 0-3), immediately flash frozen in liquid PR:PROJECT_SUMMARY nitrogen and stored at -80°C until analysis. PR:INSTITUTE Michigan State University PR:LAST_NAME Zacharewski PR:FIRST_NAME Timothy PR:DEPARTMENT Biochemistry and Molecular Biology PR:ADDRESS 48824:East Lansing PR:EMAIL tzachare@msu.edu PR:PHONE 517-884-2054 PR:FUNDING_SOURCE NIEHS; Superfund Basic Research Program R01ES029541:P42ES004911 #STUDY ST:STUDY_TITLE Prj171_Mm_TCDD_RDDR-28D_Male ST:STUDY_TYPE chromatograms ST:STUDY_SUMMARY In this study, we tested the hypothesis that the dose-dependent disruption of ST:STUDY_SUMMARY propionyl-CoA metabolism produces toxic intermediates that contribute to TCDD ST:STUDY_SUMMARY hepatotoxicity and progression of steatosis to steatohepatitis with fibrosis. ST:STUDY_SUMMARY Our results suggest TCDD dose-dependently reduced cobalamin (Cbl aka vitamin B12) ST:STUDY_SUMMARY levels compromising methylmalonyl-CoA mutase (MUT) activity and limiting the ST:STUDY_SUMMARY metabolism of propionyl-CoA to succinyl-CoA using the canonical Cbl-dependent ST:STUDY_SUMMARY carboxylation pathway. Consequently, accumulating propionyl-CoA was redirected to ST:STUDY_SUMMARY the alternate Cbl-independent beta-oxidation-like pathway resulting in the dose-dependent ST:STUDY_SUMMARY accumulation of acrylyl-CoA, as indicated by the increase in S-(2-carboxyethyl)-L-cysteine, ST:STUDY_SUMMARY a conjugate produced following the spontaneous reaction between the sulfhydryl group ST:STUDY_SUMMARY of cysteine and highly reactive acrylyl-CoA. ST:INSTITUTE Michigan State University ST:DEPARTMENT Biochemistry and Molecular Biology ST:LAST_NAME Zacharewski ST:FIRST_NAME Timothy ST:ADDRESS 48824:East Lansing ST:EMAIL tzachare@msu.edu ST:PHONE 517-884-2054 ST:NUM_GROUPS 6 ST:TOTAL_SUBJECTS 30 ST:NUM_MALES 30 ST:NUM_FEMALES 0 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:SPECIES_GROUP Mammal SU:AGE_OR_AGE_RANGE 56 SU:GENDER male SU:ANIMAL_ANIMAL_SUPPLIER Charles Rivers Laboratories SU:ANIMAL_HOUSING cage_type: Innovive Innocage; bedding_type: ALPHA-dri SU:ANIMAL_LIGHT_CYCLE 12:12 SU:ANIMAL_FEED Harlan Teklad 8940 SU:ANIMAL_WATER Innovive #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS M86 TQSm_042122_021 test_article_name:TCDD | dose_group:1.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_021.mzML SUBJECT_SAMPLE_FACTORS M119 TQSm_042122_036 test_article_name:TCDD | dose_group:30.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_036.mzML SUBJECT_SAMPLE_FACTORS M142 TQSm_042122_038 test_article_name:TCDD | dose_group:30.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_038.mzML SUBJECT_SAMPLE_FACTORS M27 TQSm_042122_012 test_article_name:TCDD | dose_group:0.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_012.mzML SUBJECT_SAMPLE_FACTORS M83 TQSm_042122_016 test_article_name:TCDD | dose_group:0.3 microgram per kilogram RAW_FILE_NAME=TQSm_042122_016.mzML SUBJECT_SAMPLE_FACTORS M123 TQSm_042122_014 test_article_name:TCDD | dose_group:0.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_014.mzML SUBJECT_SAMPLE_FACTORS M107 TQSm_042122_018 test_article_name:TCDD | dose_group:0.3 microgram per kilogram RAW_FILE_NAME=TQSm_042122_018.mzML SUBJECT_SAMPLE_FACTORS M139 TQSm_042122_034 test_article_name:TCDD | dose_group:10.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_034.mzML SUBJECT_SAMPLE_FACTORS M121 TQSm_042122_011 test_article_name:TCDD | dose_group:0.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_011.mzML SUBJECT_SAMPLE_FACTORS M122 TQSm_042122_013 test_article_name:TCDD | dose_group:0.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_013.mzML SUBJECT_SAMPLE_FACTORS M109 TQSm_042122_022 test_article_name:TCDD | dose_group:1.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_022.mzML SUBJECT_SAMPLE_FACTORS M91 TQSm_042122_030 test_article_name:TCDD | dose_group:10.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_030.mzML SUBJECT_SAMPLE_FACTORS M131 TQSm_042122_019 test_article_name:TCDD | dose_group:0.3 microgram per kilogram RAW_FILE_NAME=TQSm_042122_019.mzML SUBJECT_SAMPLE_FACTORS M134 TQSm_042122_024 test_article_name:TCDD | dose_group:1.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_024.mzML SUBJECT_SAMPLE_FACTORS M118 TQSm_042122_035 test_article_name:TCDD | dose_group:30.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_035.mzML SUBJECT_SAMPLE_FACTORS M137 TQSm_042122_029 test_article_name:TCDD | dose_group:3.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_029.mzML SUBJECT_SAMPLE_FACTORS M82 TQSm_042122_015 test_article_name:TCDD | dose_group:0.3 microgram per kilogram RAW_FILE_NAME=TQSm_042122_015.mzML SUBJECT_SAMPLE_FACTORS M85 TQSm_042122_020 test_article_name:TCDD | dose_group:1.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_020.mzML SUBJECT_SAMPLE_FACTORS M143 TQSm_042122_039 test_article_name:TCDD | dose_group:30.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_039.mzML SUBJECT_SAMPLE_FACTORS M75 TQSm_042122_010 test_article_name:TCDD | dose_group:0.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_010.mzML SUBJECT_SAMPLE_FACTORS M94 TQSm_042122_037 test_article_name:TCDD | dose_group:30.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_037.mzML SUBJECT_SAMPLE_FACTORS M112 TQSm_042122_027 test_article_name:TCDD | dose_group:3.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_027.mzML SUBJECT_SAMPLE_FACTORS M106 TQSm_042122_017 test_article_name:TCDD | dose_group:0.3 microgram per kilogram RAW_FILE_NAME=TQSm_042122_017.mzML SUBJECT_SAMPLE_FACTORS M88 TQSm_042122_025 test_article_name:TCDD | dose_group:3.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_025.mzML SUBJECT_SAMPLE_FACTORS M116 TQSm_042122_033 test_article_name:TCDD | dose_group:10.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_033.mzML SUBJECT_SAMPLE_FACTORS M92 TQSm_042122_031 test_article_name:TCDD | dose_group:10.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_031.mzML SUBJECT_SAMPLE_FACTORS M89 TQSm_042122_026 test_article_name:TCDD | dose_group:3.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_026.mzML SUBJECT_SAMPLE_FACTORS M115 TQSm_042122_032 test_article_name:TCDD | dose_group:10.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_032.mzML SUBJECT_SAMPLE_FACTORS M110 TQSm_042122_023 test_article_name:TCDD | dose_group:1.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_023.mzML SUBJECT_SAMPLE_FACTORS M113 TQSm_042122_028 test_article_name:TCDD | dose_group:3.0 microgram per kilogram RAW_FILE_NAME=TQSm_042122_028.mzML #COLLECTION CO:COLLECTION_SUMMARY Samples were collected by necropsy and flash frozen in liquid nitrogen CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS Snap Frozen #TREATMENT TR:TREATMENT_SUMMARY Mice were orally gavaged with 0.1 mL of the test article in vehicle to achieve TR:TREATMENT_SUMMARY the expected dosage every 4 days for 28 days for a total of 7 administrations. TR:TREATMENT_COMPOUND DTXSID4051378: TCDD TR:TREATMENT_ROUTE Oral Gavage Route of Administration TR:TREATMENT_DOSE ['0.0', '0.03', '0.1', '0.3', '1.0', '10.0', '3.0', '30.0'] TR:TREATMENT_DOSEVOLUME 0.1 mL TR:TREATMENT_VEHICLE DTXSID9033971: Sesame Oil TR:ANIMAL_FASTING ad libitum TR:ANIMAL_ENDP_EUTHANASIA Carbon dioxide asphyxiation TR:ANIMAL_ENDP_TISSUE_COLL_LIST Liver #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Flash frozen liver samples (about 25 mg) were extracted using HPLC-grade methanol SP:SAMPLEPREP_SUMMARY and water (5:3 ratio)containing 13C-,15N-labeled amino acid (Sigma; 767964) SP:SAMPLEPREP_SUMMARY internal standards. HPLC-grade chloroform (methanol:water:chloroform ratio 5:3:5) SP:SAMPLEPREP_SUMMARY was added, vortexed, shaken for 15 min at 4 Celsius, and centrifuged at maximum SP:SAMPLEPREP_SUMMARY speed (3000 x g) to achieve phase separation. The methanol:water phase containing SP:SAMPLEPREP_SUMMARY the polar metabolites was transferred, dried under nitrogen gas at room SP:SAMPLEPREP_SUMMARY temperature. Untargeted extractions were reconstituted with 400 ul of 10 mM SP:SAMPLEPREP_SUMMARY tributylamine and 15 mM acetic acid in 97:3 water:methanol for analysis. SP:PROCESSING_STORAGE_CONDITIONS -80C SP:SAMPLE_SPIKING 13C,15N-methionine #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The mobile phases were 10mM PFHA in water (mobile phase A) and acetonitrile CH:CHROMATOGRAPHY_SUMMARY (mobile phase B) using the following gradient: 0 min - 100% A, 1.0 min - 100% A, CH:CHROMATOGRAPHY_SUMMARY 6.0 min - 35% A, 6.01 min - 10% A, 7.0 min - 10% A, 7.01 min - 100% A, 9.0 min - CH:CHROMATOGRAPHY_SUMMARY 100% A CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters TQS CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water;10mM perfluoroheptanoic acid CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0 min - 100% A, 1.0 min - 100% A, 6.0 min - 35% A, 6.01 min - 10% A, 7.0 min - CH:FLOW_GRADIENT 10% A, 7.01 min - 100% A, 9.0 min - 100% A CH:FLOW_RATE 0.3 ml/minute CH:COLUMN_TEMPERATURE - #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-TQ-S MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS - #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area, normalized MS_METABOLITE_DATA_START Samples TQSm_042122_010 TQSm_042122_011 TQSm_042122_013 TQSm_042122_014 TQSm_042122_015 TQSm_042122_016 TQSm_042122_017 TQSm_042122_018 TQSm_042122_019 TQSm_042122_020 TQSm_042122_021 TQSm_042122_022 TQSm_042122_023 TQSm_042122_024 TQSm_042122_025 TQSm_042122_026 TQSm_042122_027 TQSm_042122_028 TQSm_042122_029 TQSm_042122_030 TQSm_042122_031 TQSm_042122_032 TQSm_042122_033 TQSm_042122_034 TQSm_042122_035 TQSm_042122_036 TQSm_042122_037 TQSm_042122_038 TQSm_042122_039 Factors test_article_name:TCDD | dose_group:0.0 microgram per kilogram test_article_name:TCDD | dose_group:0.0 microgram per kilogram test_article_name:TCDD | dose_group:0.0 microgram per kilogram test_article_name:TCDD | dose_group:0.0 microgram per kilogram test_article_name:TCDD | dose_group:0.3 microgram per kilogram test_article_name:TCDD | dose_group:0.3 microgram per kilogram test_article_name:TCDD | dose_group:0.3 microgram per kilogram test_article_name:TCDD | dose_group:0.3 microgram per kilogram test_article_name:TCDD | dose_group:0.3 microgram per kilogram test_article_name:TCDD | dose_group:1.0 microgram per kilogram test_article_name:TCDD | dose_group:1.0 microgram per kilogram test_article_name:TCDD | dose_group:1.0 microgram per kilogram test_article_name:TCDD | dose_group:1.0 microgram per kilogram test_article_name:TCDD | dose_group:1.0 microgram per kilogram test_article_name:TCDD | dose_group:3.0 microgram per kilogram test_article_name:TCDD | dose_group:3.0 microgram per kilogram test_article_name:TCDD | dose_group:3.0 microgram per kilogram test_article_name:TCDD | dose_group:3.0 microgram per kilogram test_article_name:TCDD | dose_group:3.0 microgram per kilogram test_article_name:TCDD | dose_group:10.0 microgram per kilogram test_article_name:TCDD | dose_group:10.0 microgram per kilogram test_article_name:TCDD | dose_group:10.0 microgram per kilogram test_article_name:TCDD | dose_group:10.0 microgram per kilogram test_article_name:TCDD | dose_group:10.0 microgram per kilogram test_article_name:TCDD | dose_group:30.0 microgram per kilogram test_article_name:TCDD | dose_group:30.0 microgram per kilogram test_article_name:TCDD | dose_group:30.0 microgram per kilogram test_article_name:TCDD | dose_group:30.0 microgram per kilogram test_article_name:TCDD | dose_group:30.0 microgram per kilogram S-(2-carboxyethyl)-L-cysteine 0.000868122 0.000620638 0.000652392 0.001085993 0.000830808 0.000514378 0.000479138 0.0007233 0.001996125 0.00112917 0.000834018 0.000673156 0.000976508 0.001210419 0.000901465 0.000747007 0.000759907 0.000885868 0.001525694 0.000517435 0.001884607 0.001874886 0.002140891 0.000606476 0.019529131 0.011745653 0.010382331 0.015825954 0.007211339 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name DTXSID inchi_key Average Mass elution_time S-(2-carboxyethyl)-L-cysteine DTXSID40193329 FBPINGSGHKXIQA-BYPYZUCNSA-N 193.22 2.6 METABOLITES_END #END