#METABOLOMICS WORKBENCH dongf01_20230216_122841 DATATRACK_ID:3746 STUDY_ID:ST002481 ANALYSIS_ID:AN004051 PROJECT_ID:PR001603 VERSION 1 CREATED_ON February 17, 2023, 12:51 pm #PROJECT PR:PROJECT_TITLE Urolithin A (UroA) acts at CYP1A1/1B1 substrates leading to enhanced aryl PR:PROJECT_TITLE hydrocarbon receptor (AHR) activity in vivo PR:PROJECT_SUMMARY Many AHR ligands are CYP1A1/1B1 substrates, which can result in the rapid PR:PROJECT_SUMMARY clearance within the intestinal tract and other tissues, limiting both the level PR:PROJECT_SUMMARY and duration of AHR activation. This leads to the hypothesis that there are PR:PROJECT_SUMMARY dietary constituents capable of inhibiting CYP1A1/1B1 increasing the half-live PR:PROJECT_SUMMARY of potent AHR ligands. To test this hypothesis, we examined the ability of PR:PROJECT_SUMMARY urolithin A (UroA) to act at CYP1A1/1B1 substrates leading to enhanced AHR PR:PROJECT_SUMMARY activity in vivo. PR:INSTITUTE Pennsylvania State University PR:LAST_NAME FANGCONG PR:FIRST_NAME DONG PR:ADDRESS 309 Life Sciences Building, State college, PA, 16802, USA PR:EMAIL fxd93@psu.edu PR:PHONE 8146990203 #STUDY ST:STUDY_TITLE Urolithin A (UroA) acts at CYP1A1/1B1 substrates leading to enhanced aryl ST:STUDY_TITLE hydrocarbon receptor (AHR) activity in vivo ST:STUDY_SUMMARY Many AHR ligands are CYP1A1/1B1 substrates, which can result in the rapid ST:STUDY_SUMMARY clearance within the intestinal tract and other tissues, limiting both the level ST:STUDY_SUMMARY and duration of AHR activation. This leads to the hypothesis that there are ST:STUDY_SUMMARY dietary constituents capable of inhibiting CYP1A1/1B1 increasing the half-live ST:STUDY_SUMMARY of potent AHR ligands. To test this hypothesis, we examined the ability of ST:STUDY_SUMMARY urolithin A (UroA) to act at CYP1A1/1B1 substrates leading to enhanced AHR ST:STUDY_SUMMARY activity in vivo. ST:INSTITUTE Pennsylvania State University ST:LAST_NAME DONG ST:FIRST_NAME FANGCONG ST:ADDRESS 309 Life Sciences Building, State college, PA, 16802, USA ST:EMAIL fxd93@psu.edu ST:PHONE 8146990203 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - 1_UroA_0_1 Time (min):0 RAW_FILE_NAME=1_UroA_0_1.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_0_2 Time (min):0 RAW_FILE_NAME=1_UroA_0_2.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_0_3 Time (min):0 RAW_FILE_NAME=1_UroA_0_3.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_10_1 Time (min):10 RAW_FILE_NAME=1_UroA_10_1.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_10_2 Time (min):10 RAW_FILE_NAME=1_UroA_10_2.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_10_3 Time (min):10 RAW_FILE_NAME=1_UroA_10_3.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_20_1 Time (min):20 RAW_FILE_NAME=1_UroA_20_1.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_20_2 Time (min):20 RAW_FILE_NAME=1_UroA_20_2.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_20_3 Time (min):20 RAW_FILE_NAME=1_UroA_20_3.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_40_1 Time (min):40 RAW_FILE_NAME=1_UroA_40_1.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_40_2 Time (min):40 RAW_FILE_NAME=1_UroA_40_2.mzML SUBJECT_SAMPLE_FACTORS - 1_UroA_40_3 Time (min):40 RAW_FILE_NAME=1_UroA_40_3.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_0_1 Time (min):0 RAW_FILE_NAME=2_luciferin CEE_0_1.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_0_2 Time (min):0 RAW_FILE_NAME=2_luciferin CEE_0_2.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_0_3 Time (min):0 RAW_FILE_NAME=2_luciferin CEE_0_3.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_90_1 Time (min):90 RAW_FILE_NAME=2_luciferin CEE_90_1.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_90_2 Time (min):90 RAW_FILE_NAME=2_luciferin CEE_90_2.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_90_3 Time (min):90 RAW_FILE_NAME=2_luciferin CEE_90_3.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_ICZ_90_1 Time (min):90 RAW_FILE_NAME=2_luciferin CEE_ICZ_90_1.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_ICZ_90_2 Time (min):90 RAW_FILE_NAME=2_luciferin CEE_ICZ_90_2.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_ICZ_90_3 Time (min):90 RAW_FILE_NAME=2_luciferin CEE_ICZ_90_3.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_without NADPH_90_1 Time (min):90 RAW_FILE_NAME=2_luciferin CEE_without NADPH_90_1.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_without NADPH_90_2 Time (min):90 RAW_FILE_NAME=2_luciferin CEE_without NADPH_90_2.mzML SUBJECT_SAMPLE_FACTORS - 2_luciferin CEE_without NADPH_90_3 Time (min):90 RAW_FILE_NAME=2_luciferin CEE_without NADPH_90_3.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_0_1 Time (min):0 RAW_FILE_NAME=3_ICZ_0_1.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_0_2 Time (min):0 RAW_FILE_NAME=3_ICZ_0_2.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_0_3 Time (min):0 RAW_FILE_NAME=3_ICZ_0_3.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_120_1 Time (min):120 RAW_FILE_NAME=3_ICZ_120_1.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_120_2 Time (min):120 RAW_FILE_NAME=3_ICZ_120_2.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_120_3 Time (min):120 RAW_FILE_NAME=3_ICZ_120_3.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_UroA_120_1 Time (min):120 RAW_FILE_NAME=3_ICZ_UroA_120_1.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_UroA_120_2 Time (min):120 RAW_FILE_NAME=3_ICZ_UroA_120_2.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_UroA_120_3 Time (min):120 RAW_FILE_NAME=3_ICZ_UroA_120_3.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_UroA_without NADPH_120_1 Time (min):120 RAW_FILE_NAME=3_ICZ_UroA_without NADPH_120_1.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_UroA_without NADPH_120_2 Time (min):120 RAW_FILE_NAME=3_ICZ_UroA_without NADPH_120_2.mzML SUBJECT_SAMPLE_FACTORS - 3_ICZ_UroA_without NADPH_120_3 Time (min):120 RAW_FILE_NAME=3_ICZ_UroA_without NADPH_120_3.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_0_1 Time (min):0 RAW_FILE_NAME=4_UroA_0_1.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_0_2 Time (min):0 RAW_FILE_NAME=4_UroA_0_2.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_0_3 Time (min):0 RAW_FILE_NAME=4_UroA_0_3.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_90_1 Time (min):90 RAW_FILE_NAME=4_UroA_90_1.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_90_2 Time (min):90 RAW_FILE_NAME=4_UroA_90_2.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_90_3 Time (min):90 RAW_FILE_NAME=4_UroA_90_3.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_ICZ_90_1 Time (min):90 RAW_FILE_NAME=4_UroA_ICZ_90_1.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_ICZ_90_2 Time (min):90 RAW_FILE_NAME=4_UroA_ICZ_90_2.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_ICZ_90_3 Time (min):90 RAW_FILE_NAME=4_UroA_ICZ_90_3.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_ICZ_without NADPH_90_1 Time (min):90 RAW_FILE_NAME=4_UroA_ICZ_without NADPH_90_1.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_ICZ_without NADPH_90_2 Time (min):90 RAW_FILE_NAME=4_UroA_ICZ_without NADPH_90_2.mzML SUBJECT_SAMPLE_FACTORS - 4_UroA_ICZ_without NADPH_90_3 Time (min):90 RAW_FILE_NAME=4_UroA_ICZ_without NADPH_90_3.mzML SUBJECT_SAMPLE_FACTORS - 5_Purified diet_1 Time (min):120 RAW_FILE_NAME=5_Purified diet_1.mzML SUBJECT_SAMPLE_FACTORS - 5_Purified diet_2 Time (min):120 RAW_FILE_NAME=5_Purified diet_2.mzML SUBJECT_SAMPLE_FACTORS - 5_Purified diet_3 Time (min):120 RAW_FILE_NAME=5_Purified diet_3.mzML SUBJECT_SAMPLE_FACTORS - 5_Purified diet_UroA_1 Time (min):120 RAW_FILE_NAME=5_Purified diet_UroA_1.mzML SUBJECT_SAMPLE_FACTORS - 5_Purified diet_UroA_2 Time (min):120 RAW_FILE_NAME=5_Purified diet_UroA_2.mzML SUBJECT_SAMPLE_FACTORS - 5_Purified diet_UroA_3 Time (min):120 RAW_FILE_NAME=5_Purified diet_UroA_3.mzML #COLLECTION CO:COLLECTION_SUMMARY 40 µg microsomes isolated from Hepa 1 cells treated with 5 nM TCDD for 24 h, or CO:COLLECTION_SUMMARY lung microsomes (100 µg) from mice on the semi-purified diet, were incubated CO:COLLECTION_SUMMARY with various compounds and incubation times as indicated. The reactions were CO:COLLECTION_SUMMARY carried out at 37°C in an incubator in the presence of NADPH regeneration CO:COLLECTION_SUMMARY system and terminated by addition of 800 µL ice cold 100% methanol (v/v) CO:COLLECTION_SUMMARY containing 1 µM chlorpropamide as internal standard. CO:SAMPLE_TYPE Hepa 1 cells #TREATMENT TR:TREATMENT_SUMMARY Then mixtures were kept in -20°C for 30 mins followed by centrifugation at TR:TREATMENT_SUMMARY 12,000 × g for 20 min at 4°C. The supernatants were dried in a under vacuum TR:TREATMENT_SUMMARY and reconstituted in 60 μL 50% methanol (v/v). After centrifugation, TR:TREATMENT_SUMMARY supernatants were transferred to autosampler vials for LC-MS/MS analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 40 µg microsomes isolated from Hepa 1 cells treated with 5 nM TCDD for 24 h, or SP:SAMPLEPREP_SUMMARY lung microsomes (100 µg) from mice on the semi-purified diet, were incubated SP:SAMPLEPREP_SUMMARY with various compounds and incubation times as indicated. The reactions were SP:SAMPLEPREP_SUMMARY carried out at 37°C in an incubator in the presence of NADPH regeneration SP:SAMPLEPREP_SUMMARY system and terminated by addition of 800 µL ice cold 100% methanol (v/v) SP:SAMPLEPREP_SUMMARY containing 1 µM chlorpropamide as internal standard. Then mixtures were kept in SP:SAMPLEPREP_SUMMARY -20°C for 30 mins followed by centrifugation at 12,000 × g for 20 min at 4°C. SP:SAMPLEPREP_SUMMARY The supernatants were dried in a under vacuum and reconstituted in 60 μL 50% SP:SAMPLEPREP_SUMMARY methanol (v/v). After centrifugation, supernatants were transferred to SP:SAMPLEPREP_SUMMARY autosampler vials for LC-MS/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu 20AD CH:COLUMN_NAME BEH C18 column (2.1 × 100 mm × 1.7 µm particle size) CH:SOLVENT_A 100% wate; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT The initial condition were 97% A and 3 % B, increasing to 45% B at 10 min, 75% B CH:FLOW_GRADIENT at 12 min where it was held at 75% B until 17.5 min before returning to the CH:FLOW_GRADIENT initial conditions. CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 55 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5600 TripleTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS The capillary voltage was set at 5.5 kV in positive ion mode with a declustering MS:MS_COMMENTS potential of 80 V. The mass spectrometer was operated with a 100 ms TOF scan MS:MS_COMMENTS from 50 to 500 m/z, and 16 MS/MS product ion scans (100 ms) per duty cycle using MS:MS_COMMENTS a collision energy of 50 V with a 20 V spread. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS 1 MS_METABOLITE_DATA_START Samples 1_UroA_0_1 1_UroA_0_2 1_UroA_0_3 1_UroA_10_1 1_UroA_10_2 1_UroA_10_3 1_UroA_20_1 1_UroA_20_2 1_UroA_20_3 1_UroA_40_1 1_UroA_40_2 1_UroA_40_3 2_luciferin CEE_0_1 2_luciferin CEE_0_2 2_luciferin CEE_0_3 2_luciferin CEE_90_1 2_luciferin CEE_90_2 2_luciferin CEE_90_3 2_luciferin CEE_ICZ_90_1 2_luciferin CEE_ICZ_90_2 2_luciferin CEE_ICZ_90_3 2_luciferin CEE_without NADPH_90_1 2_luciferin CEE_without NADPH_90_2 2_luciferin CEE_without NADPH_90_3 3_ICZ_0_1 3_ICZ_0_2 3_ICZ_0_3 3_ICZ_120_1 3_ICZ_120_2 3_ICZ_120_3 3_ICZ_UroA_120_1 3_ICZ_UroA_120_2 3_ICZ_UroA_120_3 3_ICZ_UroA_without NADPH_120_1 3_ICZ_UroA_without NADPH_120_2 3_ICZ_UroA_without NADPH_120_3 4_UroA_0_1 4_UroA_0_2 4_UroA_0_3 4_UroA_90_1 4_UroA_90_2 4_UroA_90_3 4_UroA_ICZ_90_1 4_UroA_ICZ_90_2 4_UroA_ICZ_90_3 4_UroA_ICZ_without NADPH_90_1 4_UroA_ICZ_without NADPH_90_2 4_UroA_ICZ_without NADPH_90_3 5_Purified diet_1 5_Purified diet_2 5_Purified diet_3 5_Purified diet_UroA_1 5_Purified diet_UroA_2 5_Purified diet_UroA_3 Factors Time (min):0 Time (min):0 Time (min):0 Time (min):10 Time (min):10 Time (min):10 Time (min):20 Time (min):20 Time (min):20 Time (min):40 Time (min):40 Time (min):40 Time (min):0 Time (min):0 Time (min):0 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):0 Time (min):0 Time (min):0 Time (min):120 Time (min):120 Time (min):120 Time (min):120 Time (min):120 Time (min):120 Time (min):120 Time (min):120 Time (min):120 Time (min):0 Time (min):0 Time (min):0 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):90 Time (min):120 Time (min):120 Time (min):120 Time (min):120 Time (min):120 Time (min):120 UroA na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na 0.158083 0.178372 0.183419 0.037646 0.039954 0.040886 0.136355 0.154577 0.147173 0.174648 0.168737 0.167822 na na na na na na UroC 0.000101 0.000126 0.000157 0.000408 0.000479 0.000567 0.0008 0.000386 0.000791 0.001141 0.001242 0.001239 na na na na na na na na na na na na na na na na na na na na na na na na 0 0 0 0.007407 0.008378 0.008824 0.006565 0.00435 0.00455 0 0 0 104.4 126.2 115.4 282.3 170.8 201.3 luciferin na na na na na na na na na na na na 0 0 0 0.012122975 0.010322912 0.009197956 0.003110829 0.002186291 0.001973642 0 0 0 na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na 2,8-dihydroxyICZ na na na na na na na na na na na na na na na na na na na na na na na na 0 0 0 0.0135 0.0241 0.0211 0.0107 0.0104 0.00805 0 0 0 na na na na na na na na na na na na na na na na na na MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 1_UroA_0_1 1_UroA_0_2 1_UroA_0_3 1_UroA_10_1 1_UroA_10_2 1_UroA_10_3 1_UroA_20_1 1_UroA_20_2 1_UroA_20_3 1_UroA_40_1 1_UroA_40_2 1_UroA_40_3 2_luciferin CEE_0_1 2_luciferin CEE_0_2 2_luciferin CEE_0_3 2_luciferin CEE_90_1 2_luciferin CEE_90_2 2_luciferin CEE_90_3 2_luciferin CEE_ICZ_90_1 2_luciferin CEE_ICZ_90_2 2_luciferin CEE_ICZ_90_3 2_luciferin CEE_without NADPH_90_1 2_luciferin CEE_without NADPH_90_2 2_luciferin CEE_without NADPH_90_3 3_ICZ_0_1 3_ICZ_0_2 3_ICZ_0_3 3_ICZ_120_1 3_ICZ_120_2 3_ICZ_120_3 3_ICZ_UroA_120_1 3_ICZ_UroA_120_2 3_ICZ_UroA_120_3 3_ICZ_UroA_without NADPH_120_1 3_ICZ_UroA_without NADPH_120_2 3_ICZ_UroA_without NADPH_120_3 4_UroA_0_1 4_UroA_0_2 4_UroA_0_3 4_UroA_90_1 4_UroA_90_2 4_UroA_90_3 4_UroA_ICZ_90_1 4_UroA_ICZ_90_2 4_UroA_ICZ_90_3 4_UroA_ICZ_without NADPH_90_1 4_UroA_ICZ_without NADPH_90_2 4_UroA_ICZ_without NADPH_90_3 5_Purified diet_1 5_Purified diet_2 5_Purified diet_3 5_Purified diet_UroA_1 5_Purified diet_UroA_2 5_Purified diet_UroA_3 UroA na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na 0.158083 0.178372 0.183419 0.037646 0.039954 0.040886 0.136355 0.154577 0.147173 0.174648 0.168737 0.167822 na na na na na na UroC 0.000101 0.000126 0.000157 0.000408 0.000479 0.000567 0.0008 0.000386 0.000791 0.001141 0.001242 0.001239 na na na na na na na na na na na na na na na na na na na na na na na na 0 0 0 0.007407 0.008378 0.008824 0.006565 0.00435 0.00455 0 0 0 104.4 134.8 115.4 282.3 170.8 201.3 luciferin na na na na na na na na na na na na 0 0 0 0.012122975 0.010322912 0.009197956 0.003110829 0.002186291 0.001973642 0 0 0 na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na 2,8-dihydroxyICZ na na na na na na na na na na na na na na na na na na na na na na na na 0 0 0 0.0135 0.0241 0.0211 0.0107 0.0104 0.00805 0 0 0 na na na na na na na na na na na na na na na na na na METABOLITES_END #END