#METABOLOMICS WORKBENCH dongf01_20230222_074328 DATATRACK_ID:3762 STUDY_ID:ST002491 ANALYSIS_ID:AN004065 PROJECT_ID:PR001608 VERSION 1 CREATED_ON February 22, 2023, 12:50 pm #PROJECT PR:PROJECT_TITLE Tryptophan metabolites that circulate at significant levels in vivo are not PR:PROJECT_TITLE subject to an autoregulatory feedback loop between the AHR and CYP1A1. PR:PROJECT_TYPE MS quantitative study PR:PROJECT_SUMMARY We establish that six tryptophan metabolites are essentially not CYP1A1 PR:PROJECT_SUMMARY substrates and the level of these metabolite in mouse serum are not affected by PR:PROJECT_SUMMARY a lack of CYP1A1 or any other Aryl hydrocarbon Receptor (AHR) dependent PR:PROJECT_SUMMARY metabolic pathway expression in vivo. These results establish that tryptophan PR:PROJECT_SUMMARY metabolites that circulate at significant levels in vivo are not subject to an PR:PROJECT_SUMMARY autoregulatory feedback loop between the AHR and CYP1A1. PR:INSTITUTE The Pennsylvania State University PR:LAST_NAME Dong PR:FIRST_NAME Fancong PR:ADDRESS 309 Life Sciences Building, State college, PA, 16802, USA PR:EMAIL fxd93@psu.edu PR:PHONE 8148651415 #STUDY ST:STUDY_TITLE Six tryptophan metabolites in mouse serum ST:STUDY_SUMMARY We establish that six tryptophan metabolites are essentially not CYP1A1 ST:STUDY_SUMMARY substrates and the level of these metabolite in mouse serum are not affected by ST:STUDY_SUMMARY a lack of CYP1A1 or any other Aryl hydrocarbon Receptor (AHR) dependent ST:STUDY_SUMMARY metabolic pathway expression in vivo. These results establish that tryptophan ST:STUDY_SUMMARY metabolites that circulate at significant levels in vivo are not subject to an ST:STUDY_SUMMARY autoregulatory feedback loop between the AHR and CYP1A1. ST:INSTITUTE Pennsylvania State University ST:LAST_NAME Dong ST:FIRST_NAME Fancong ST:ADDRESS 309 Life Sciences Building, University Park, PA 16802 ST:EMAIL fxd93@psu.edu ST:PHONE 8148651415 #SUBJECT SU:SUBJECT_TYPE Mammal 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_SERUM_HETS1 Genotype:AHR+/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_HETS1.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_HETS2 Genotype:AHR+/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_HETS2.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_HETS3 Genotype:AHR+/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_HETS3.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_HETS4 Genotype:AHR+/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_HETS4.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_HETS5 Genotype:AHR+/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_HETS5.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_HETS6 Genotype:AHR+/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_HETS6.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_KO1 Genotype:AHR-/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_KO1.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_KO2 Genotype:AHR-/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_KO2.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_KO3 Genotype:AHR-/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_KO3.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_KO4 Genotype:AHR-/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_KO4.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_KO5 Genotype:AHR-/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_KO5.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_KO6 Genotype:AHR-/- Treatment=chow diet; RAW_FILE_NAME=1_SERUM_KO6.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT1 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT1.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT2 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT2.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT3 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT3.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT4 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT4.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT5 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT5.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT6 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT6.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT7 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT7.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT8 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT8.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT9 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT9.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT10 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT10.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT11 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT11.mzML SUBJECT_SAMPLE_FACTORS - 1_SERUM_WT12 Genotype:AHR+/+ Treatment=chow diet; RAW_FILE_NAME=1_SERUM_WT12.mzML SUBJECT_SAMPLE_FACTORS - 1_STD1 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD1.mzML SUBJECT_SAMPLE_FACTORS - 1_STD2 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD2.mzML SUBJECT_SAMPLE_FACTORS - 1_STD3 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD3.mzML SUBJECT_SAMPLE_FACTORS - 1_STD4 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD4.mzML SUBJECT_SAMPLE_FACTORS - 1_STD5 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD5.mzML SUBJECT_SAMPLE_FACTORS - 1_STD6 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD6.mzML SUBJECT_SAMPLE_FACTORS - 1_STD7 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD7.mzML SUBJECT_SAMPLE_FACTORS - 1_STD8 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD8.mzML SUBJECT_SAMPLE_FACTORS - 1_STD9 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD9.mzML SUBJECT_SAMPLE_FACTORS - 1_STD10 Genotype:- Treatment=-; RAW_FILE_NAME=1_STD10.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_C1 Genotype:wild type Treatment=-; RAW_FILE_NAME=2_SERUM_C1.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_C2 Genotype:wild type Treatment=-; RAW_FILE_NAME=2_SERUM_C2.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_C3 Genotype:wild type Treatment=-; RAW_FILE_NAME=2_SERUM_C3.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_C4 Genotype:wild type Treatment=-; RAW_FILE_NAME=2_SERUM_C4.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_C5 Genotype:wild type Treatment=-; RAW_FILE_NAME=2_SERUM_C5.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_C6 Genotype:wild type Treatment=-; RAW_FILE_NAME=2_SERUM_C6.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_PCB1 Genotype:wild type Treatment=PCB; RAW_FILE_NAME=2_SERUM_PCB1.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_PCB2 Genotype:wild type Treatment=PCB; RAW_FILE_NAME=2_SERUM_PCB2.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_PCB3 Genotype:wild type Treatment=PCB; RAW_FILE_NAME=2_SERUM_PCB3.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_PCB4 Genotype:wild type Treatment=PCB; RAW_FILE_NAME=2_SERUM_PCB4.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_PCB5 Genotype:wild type Treatment=PCB; RAW_FILE_NAME=2_SERUM_PCB5.mzML SUBJECT_SAMPLE_FACTORS - 2_SERUM_PCB6 Genotype:wild type Treatment=PCB; RAW_FILE_NAME=2_SERUM_PCB6.mzML SUBJECT_SAMPLE_FACTORS - 2_STD1 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD1.mzML SUBJECT_SAMPLE_FACTORS - 2_STD2 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD2.mzML SUBJECT_SAMPLE_FACTORS - 2_STD3 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD3.mzML SUBJECT_SAMPLE_FACTORS - 2_STD4 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD4.mzML SUBJECT_SAMPLE_FACTORS - 2_STD5 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD5.mzML SUBJECT_SAMPLE_FACTORS - 2_STD6 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD6.mzML SUBJECT_SAMPLE_FACTORS - 2_STD7 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD7.mzML SUBJECT_SAMPLE_FACTORS - 2_STD8 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD8.mzML SUBJECT_SAMPLE_FACTORS - 2_STD9 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD9.mzML SUBJECT_SAMPLE_FACTORS - 2_STD10 Genotype:- Treatment=-; RAW_FILE_NAME=2_STD10.mzML #COLLECTION CO:COLLECTION_SUMMARY Serum samples from mice were collected and stored at -80°C. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY Thawed on ice, serum sample was mixed with 4 volume extraction solvent of TR:TREATMENT_SUMMARY ice-cold methanol containing indole-3-acetic acid-d4 and kynurenic acid-d5. TR:TREATMENT_SUMMARY Mixture was vortexed and incubated at −20 °C for 30 min. Following TR:TREATMENT_SUMMARY centrifugation at 12,000 × g for 15 min at 4℃, supernatant was collected and TR:TREATMENT_SUMMARY subsequently evaporated to dryness (Thermo Scientific, Waltham, MA) and TR:TREATMENT_SUMMARY dissolved in 10% acetonitrile containing 1 µM chlorpropamide. After TR:TREATMENT_SUMMARY centrifugation at 12,000 × g for 15 min at 4℃, supernatants were transferred TR:TREATMENT_SUMMARY to autosampler vials for LC-MS analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY C57BL6/J mice were obtained from Jackson Laboratory (Bar Harbor, ME), bred SP:SAMPLEPREP_SUMMARY in-house and maintained on a chow diet. Ahr+/-, and Ahr-/- mice were kindly SP:SAMPLEPREP_SUMMARY provided by Dr. Christopher Bradfield (University of Wisconsin-Madison) and bred SP:SAMPLEPREP_SUMMARY using female Ahr+/- and male Ahr-/- mice. Mice were housed on corncob bedding in SP:SAMPLEPREP_SUMMARY a temperature- and light-controlled facility and given access to food and water SP:SAMPLEPREP_SUMMARY ad libitum. Mice were also maintained in a pathogen-free facility and treated SP:SAMPLEPREP_SUMMARY humanely with approval from the Animal Care and Use Committee of the SP:SAMPLEPREP_SUMMARY Pennsylvania State University and methods were carried out in accordance with SP:SAMPLEPREP_SUMMARY approved guidelines. Mice in the 8-10-week-old range were utilized in all SP:SAMPLEPREP_SUMMARY experiments. Serum samples obtained from mice exposed to PCB126 for 5 days was SP:SAMPLEPREP_SUMMARY previously described. Briefly, mice were fed dough pills once every 24 h, each SP:SAMPLEPREP_SUMMARY pill contained 24 µg/kg PCB126 for five days and mice were sacrificed on day 6. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu 20AD CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 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 Triple TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Peakview was used for data analysis. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS uM MS_METABOLITE_DATA_START Samples 1_SERUM_HETS1 1_SERUM_HETS2 1_SERUM_HETS3 1_SERUM_HETS4 1_SERUM_HETS5 1_SERUM_HETS6 1_SERUM_KO1 1_SERUM_KO2 1_SERUM_KO3 1_SERUM_KO4 1_SERUM_KO5 1_SERUM_KO6 1_SERUM_WT1 1_SERUM_WT2 1_SERUM_WT3 1_SERUM_WT4 1_SERUM_WT5 1_SERUM_WT6 1_SERUM_WT7 1_SERUM_WT8 1_SERUM_WT9 1_SERUM_WT10 1_SERUM_WT11 1_SERUM_WT12 2_SERUM_C1 2_SERUM_C2 2_SERUM_C3 2_SERUM_C4 2_SERUM_C5 2_SERUM_C6 2_SERUM_PCB1 2_SERUM_PCB2 2_SERUM_PCB3 2_SERUM_PCB4 2_SERUM_PCB5 2_SERUM_PCB6 Factors Genotype:AHR+/- Genotype:AHR+/- Genotype:AHR+/- Genotype:AHR+/- Genotype:AHR+/- Genotype:AHR+/- Genotype:AHR-/- Genotype:AHR-/- Genotype:AHR-/- Genotype:AHR-/- Genotype:AHR-/- Genotype:AHR-/- Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:AHR+/+ Genotype:wild type Genotype:wild type Genotype:wild type Genotype:wild type Genotype:wild type Genotype:wild type Genotype:wild type Genotype:wild type Genotype:wild type Genotype:wild type Genotype:wild type Genotype:wild type indole-3-acetic acid 1.960455514 1.974897872 1.518767404 1.307562929 1.856956662 1.394609033 1.819567365 3.377433617 2.432324848 1.256358826 2.120285681 1.4337051 0.728752789 0.587592435 0.262305577 1.247978711 1.307703414 0.772459002 0.960921935 0.81930508 0.616515286 0.461825736 0.437711931 0.773665704 0.276807571 0.605961507 0.149071024 0.28343965 0.545115104 0.500846648 0.684193848 0.837032681 0.768244282 0.387889582 0.401684743 0.639057516 kynurenic acid 0.092909751 0.453651573 0.235834559 0.093924089 0.252017994 0.193927281 0.136343728 0.223519359 0.103901896 0.024147002 0.141713283 0.162577626 0.232667122 0.243429419 0.149283599 0.310277147 0.642605216 0.200282026 0.303456889 0.382001076 0.234545787 0.156770838 0.16910886 0.211006638 0.472957745 0.374137255 0.224648215 0.239997451 0.337083433 0.358177866 0.193855952 0.564677023 0.27474956 0.262069755 0.379515632 0.177207354 indole-3-carboxaldehyde 0.29560339 0.272965137 0.22588747 0.22753652 0.315921101 0.238673511 0.199274627 0.356932393 0.239285697 0.188152709 0.333867022 0.245502579 0.466049971 0.381134911 0.040096674 0.211974136 0.459856508 0.20388169 0.206206981 0.174777966 0.218447176 0.201761422 0.159401834 0.170609255 0.034198874 0.10580963 0.062210885 0.035346325 0.101742069 0.111878714 0.10846958 0.117954335 0.109903089 0.108018659 0.08646332 0.189473094 indole-3-propionic acid 5.384104776 3.229604038 5.047411215 4.739699503 4.776564171 3.173367316 5.756741535 6.79078572 6.026803428 4.572917793 7.098442672 4.870892037 7.129547893 7.42176927 3.745813354 8.435680809 6.594392466 6.784374538 6.509461745 5.269438034 5.897730019 4.596820172 2.752775029 3.245398418 3.01884928 5.824409675 2.318183347 4.852370791 6.416247455 5.941367956 3.702270108 7.723015918 6.355142351 4.11277743 5.920286859 2.118782552 indole-3-lactic acid 1.51000159 2.425806087 1.784563639 1.303863417 1.651532683 1.495166917 1.609965349 2.212202491 1.622197869 1.609383605 1.793980466 1.804072973 1.35174999 1.298443784 1.411301854 1.410839306 1.924816075 1.070961138 2.286749683 1.777888281 2.211711848 1.315848539 0.952493469 1.439568172 1.684295401 1.865507957 0.978586258 1.524918943 2.034705586 1.304568303 1.54865646 1.756921636 1.805010749 1.760686002 1.734910267 1.608967031 kynurenine 0.749216022 0.802045759 1.29096164 0.751675213 1.222034574 1.169371857 1.491761616 1.538866041 0.911595498 0.57748254 2.507272963 0.971429568 1.061089909 1.071691576 0.841508158 1.46961892 1.377697796 1.237278882 1.165816765 1.0913281 1.005825713 1.540442329 1.141790806 0.682042694 0.910671338 0.846223731 0.614245242 0.683406539 0.684004582 0.764817095 0.694784146 0.93326601 0.774780314 0.606905622 0.725904787 0.556264861 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z Pubchem ID indole-3-acetic acid 130.06 802 kynurenic acid 144.03 3845 indole-3-carboxaldehyde 91.05 10256 indole-3-propionic acid 130.09 3744 indole-3-lactic acid 130.09 92904 kynurenine 146.05 846 METABOLITES_END #END