#METABOLOMICS WORKBENCH orlowska_20230727_100007 DATATRACK_ID:4188 STUDY_ID:ST003529 ANALYSIS_ID:AN005797 PROJECT_ID:PR001717 VERSION 1 CREATED_ON October 23, 2024, 2:06 pm #PROJECT PR:PROJECT_TITLE Measurement of itaconic acid in liver of male mice treated with TCDD 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 PR:PROJECT_SUMMARY progression of non-alcoholic fatty liver disease (NAFLD). NAFLD is a spectrum of PR:PROJECT_SUMMARY pathologies that spans simple, reversible, and benign lipid accumulation PR:PROJECT_SUMMARY (hepatic steatosis), to steatosis with inflammation (steatohepatitis) and PR:PROJECT_SUMMARY collagen deposition (fibrosis/cirrhosis) in the absence of excessive alcohol PR:PROJECT_SUMMARY consumption. NAFLD prevalence is projected to increase from ~83 million in 2015 PR:PROJECT_SUMMARY to ~101 million by 2030 in the US alone, while increasing the risk for more PR:PROJECT_SUMMARY complex disorders including Metabolic Syndrome, cardiovascular disease, PR:PROJECT_SUMMARY diabetes, cirrhosis, end-stage liver disease and hepatocellular carcinoma (HCC). PR:PROJECT_SUMMARY The role of AhR-mediated metabolic dysregulation in hepatotoxicity and the PR:PROJECT_SUMMARY etiology of more complex metabolic diseases warrants further investigation. PR:PROJECT_SUMMARY Therofore, in this project on PND28 mice were orally gavaged at the start of the PR:PROJECT_SUMMARY light cycle (zeitgeber [ZT] 0-1) with 0.1 ml sesame oil vehicle or 0.01, 0.03, PR:PROJECT_SUMMARY 0.1, 0.3, 1, 3, 10, and 30 microgram/kg body weight TCDD every 4 days for 28 PR:PROJECT_SUMMARY days for a total of 7 treatments. The first gavage was administered on day 0, PR:PROJECT_SUMMARY with the last gavage administered on day 24 of the 28-day study. On day 28, PR:PROJECT_SUMMARY tissue samples were harvested (ZT 0-3), immediately flash frozen in liquid PR:PROJECT_SUMMARY nitrogen and stored at -80C until analysis. PR:INSTITUTE Michigan State University PR:DEPARTMENT Biochemistry and Molecular Biology PR:LAST_NAME Zacharewski PR:FIRST_NAME Timothy PR:ADDRESS 48824:East Lansing PR:EMAIL tzachare@msu.edu PR:PHONE 517-884-2054 PR:FUNDING_SOURCE NIEHS; Superfund Basic Research Program P42ES004911 #STUDY ST:STUDY_TITLE Measurement of itaconic acid in liver of male mice treated with TCDD 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 ST:STUDY_SUMMARY B12) levels compromising methylmalonyl-CoA mutase (MUT) activity and limiting ST:STUDY_SUMMARY the metabolism of propionyl-CoA to succinyl-CoA using the canonical ST:STUDY_SUMMARY Cbl-dependent carboxylation pathway. More recently, lower Cbl levels have been ST:STUDY_SUMMARY linked to itaconate, a cis-aconitate metabolite produced in large quantities by ST:STUDY_SUMMARY activated macrophages. In the current study, targeted metabolomics analysis of ST:STUDY_SUMMARY hepatic extracts detected a dose-dependent increase in itaconic acid. Itaconate ST:STUDY_SUMMARY can be activated to itaconyl-CoA which can then interact with the ST:STUDY_SUMMARY 5-deoxyadenosyl moiety of AdoCbl to form an uncharacterized adduct that disrupts ST:STUDY_SUMMARY auxiliary repair protein interactions, inactivates AdoCbl, and reduces Cbl ST:STUDY_SUMMARY levels that inhibit MUT activity. 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 5 ST:TOTAL_SUBJECTS 20 ST:NUM_MALES 20 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 56 SU:GENDER male 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 SU:SPECIES_GROUP Mammal SU:ANIMAL_ANIMAL_SUPPLIER Charles Rivers Laboratories #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 M50 XS_082721_022 Sample source:Liver | dose_group:10.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_022.mzML SUBJECT_SAMPLE_FACTORS M76 XS_082721_023 Sample source:Liver | dose_group:10.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_023.mzML SUBJECT_SAMPLE_FACTORS M56 XS_082721_012 Sample source:Liver | dose_group:0.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_012.mzML SUBJECT_SAMPLE_FACTORS M27 XS_082721_026 Sample source:Liver | dose_group:30.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_026.mzML SUBJECT_SAMPLE_FACTORS M57 XS_082721_013 Sample source:Liver | dose_group:0.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_013.mzML SUBJECT_SAMPLE_FACTORS M80 XS_082721_028 Sample source:Liver | dose_group:30.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_028.mzML SUBJECT_SAMPLE_FACTORS M74 XS_082721_020 Sample source:Liver | dose_group:3.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_020.mzML SUBJECT_SAMPLE_FACTORS M78 XS_082721_025 Sample source:Liver | dose_group:10.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_025.mzML SUBJECT_SAMPLE_FACTORS M73 XS_082721_019 Sample source:Liver | dose_group:3.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_019.mzML SUBJECT_SAMPLE_FACTORS M70 XS_082721_015 Sample source:Liver | dose_group:1.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_015.mzML SUBJECT_SAMPLE_FACTORS M44 XS_082721_014 Sample source:Liver | dose_group:1.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_014.mzML SUBJECT_SAMPLE_FACTORS M03 XS_082721_010 Sample source:Liver | dose_group:0.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_010.mzML SUBJECT_SAMPLE_FACTORS M77 XS_082721_024 Sample source:Liver | dose_group:10.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_024.mzML SUBJECT_SAMPLE_FACTORS M72 XS_082721_017 Sample source:Liver | dose_group:1.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_017.mzML SUBJECT_SAMPLE_FACTORS M79 XS_082721_027 Sample source:Liver | dose_group:30.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_027.mzML SUBJECT_SAMPLE_FACTORS M47 XS_082721_018 Sample source:Liver | dose_group:3.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_018.mzML SUBJECT_SAMPLE_FACTORS M71 XS_082721_016 Sample source:Liver | dose_group:1.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_016.mzML SUBJECT_SAMPLE_FACTORS M81 XS_082721_029 Sample source:Liver | dose_group:30.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_029.mzML SUBJECT_SAMPLE_FACTORS M55 XS_082721_011 Sample source:Liver | dose_group:0.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_011.mzML SUBJECT_SAMPLE_FACTORS M75 XS_082721_021 Sample source:Liver | dose_group:3.0 microgram per kilogram test_article_name=TCDD; RAW_FILE_NAME=XS_082721_021.mzML #COLLECTION CO:COLLECTION_SUMMARY Tissue was carefully excised and immediately frozen in liquid nitrogen. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS Snap Frozen CO:STORAGE_VIALS 2 mL screwcap tube #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_ROUTE Oral Gavage Route of Administration TR:TREATMENT_DOSE ['0.0', '0.01', '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 TR:TREATMENT_COMPOUND DTXSID2021315: TCDD #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen liver samples (40 mg) were homogenized (Polytron PT2100, Kinematica) in SP:SAMPLEPREP_SUMMARY acetonitrile:water ratio 8:2, vortexed, shaken for 5 min at 4 C, and centrifuged SP:SAMPLEPREP_SUMMARY at maximum speed (3000 x g). Supernatant was dried under nitrogen and dried SP:SAMPLEPREP_SUMMARY extracts were reconstituted in 200 microliters of a 1:9 methanol:water solution SP:SAMPLEPREP_SUMMARY containing 2% formic acid. SP:PROCESSING_STORAGE_CONDITIONS -80C #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0 min - 100% A, 1.0 min - 100% A, 2.0 min - 80% A, 4.0 min - 1% A, 5.0 min - 1% CH:FLOW_GRADIENT A, 5.01 min - 100% A, 7.0 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 NEGATIVE MS:MS_COMMENTS protocol #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS normalized peak area MS_METABOLITE_DATA_START Samples XS_082721_010 XS_082721_011 XS_082721_012 XS_082721_013 XS_082721_014 XS_082721_015 XS_082721_016 XS_082721_017 XS_082721_018 XS_082721_019 XS_082721_020 XS_082721_021 XS_082721_022 XS_082721_023 XS_082721_024 XS_082721_025 XS_082721_026 XS_082721_027 XS_082721_028 XS_082721_029 Factors Sample source:Liver | dose_group:0.0 microgram per kilogram Sample source:Liver | dose_group:0.0 microgram per kilogram Sample source:Liver | dose_group:0.0 microgram per kilogram Sample source:Liver | dose_group:0.0 microgram per kilogram Sample source:Liver | dose_group:1.0 microgram per kilogram Sample source:Liver | dose_group:1.0 microgram per kilogram Sample source:Liver | dose_group:1.0 microgram per kilogram Sample source:Liver | dose_group:1.0 microgram per kilogram Sample source:Liver | dose_group:3.0 microgram per kilogram Sample source:Liver | dose_group:3.0 microgram per kilogram Sample source:Liver | dose_group:3.0 microgram per kilogram Sample source:Liver | dose_group:3.0 microgram per kilogram Sample source:Liver | dose_group:10.0 microgram per kilogram Sample source:Liver | dose_group:10.0 microgram per kilogram Sample source:Liver | dose_group:10.0 microgram per kilogram Sample source:Liver | dose_group:10.0 microgram per kilogram Sample source:Liver | dose_group:30.0 microgram per kilogram Sample source:Liver | dose_group:30.0 microgram per kilogram Sample source:Liver | dose_group:30.0 microgram per kilogram Sample source:Liver | dose_group:30.0 microgram per kilogram itaconic acid 8.83E-07 2.98E-07 0.000000576 0.000000992 0.000005208 0.000007252 9.50E-06 4.78E-06 4.61E-07 0.000005168 2.48E-06 4.83E-06 0.00001524 4.95E-05 3.45E-05 0.000022464 3.44E-05 0.000033436 4.96E-05 0.000050048 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name XS_082721_010 XS_082721_011 XS_082721_012 XS_082721_013 XS_082721_014 XS_082721_015 XS_082721_016 XS_082721_017 XS_082721_018 XS_082721_019 XS_082721_020 XS_082721_021 XS_082721_022 XS_082721_023 XS_082721_024 XS_082721_025 XS_082721_026 XS_082721_027 XS_082721_028 XS_082721_029 itaconic acid 8.83E-07 2.98E-07 0.000000576 0.000000992 0.000005208 0.000007252 9.50E-06 4.78E-06 4.61E-07 0.000005168 2.48E-06 4.83E-06 0.00001524 4.95E-05 3.45E-05 0.000022464 3.44E-05 0.000033436 4.96E-05 0.000050048 METABOLITES_END #END