#METABOLOMICS WORKBENCH lai_yunjia_20210718_213938 DATATRACK_ID:2752 STUDY_ID:ST001872 ANALYSIS_ID:AN003034 PROJECT_ID:000000 VERSION 1 CREATED_ON July 19, 2021, 7:07 pm #PROJECT PR:PROJECT_TITLE Effects of Acute 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exposure on the Circulating PR:PROJECT_TITLE and Cecal Metabolome Profile PR:PROJECT_TYPE Untargeted metabolomics PR:PROJECT_SUMMARY 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a polyhalogenated planar PR:PROJECT_SUMMARY hydrocarbon belonging to a group of highly toxic and persistent environmental PR:PROJECT_SUMMARY contaminants known as “dioxins”. TCDD is an animal teratogen and carcinogen PR:PROJECT_SUMMARY that is well characterized for causing immunosuppression through activation of PR:PROJECT_SUMMARY Aryl Hydrocarbon Receptor (AHR). In the current study, we investigated the PR:PROJECT_SUMMARY effect of exposure of mice to an acute dose of TCDD on the metabolic profile PR:PROJECT_SUMMARY within the serum and cecal contents to better define the effects of TCDD on host PR:PROJECT_SUMMARY physiology. Our findings demonstrated that within the circulating metabolome PR:PROJECT_SUMMARY following acute TCDD, there was significant dysregulation in the metabolism of PR:PROJECT_SUMMARY bioactive lipids, amino acids, and carbohydrates when compared to the VEH PR:PROJECT_SUMMARY treated mice. These wide-spread changes in metabolite abundance were identified PR:PROJECT_SUMMARY to regulate host immunity via modulating Nuclear Factor-Kappa B (NF-κB) and PR:PROJECT_SUMMARY Extracellular Signal‑Regulated Protein Kinase (ERK1/2) activity, and work as PR:PROJECT_SUMMARY biomarkers for a variety of organ injuries and dysfunctions that follow TCDD PR:PROJECT_SUMMARY exposure. Within the cecal content, of mice exposed to TCDD, we were able to PR:PROJECT_SUMMARY detect changes in inflammatory markers that regulate NF-κB, markers of PR:PROJECT_SUMMARY injury-related inflammation, and changes in lysine degradation, nicotinamide PR:PROJECT_SUMMARY metabolism, and butanoate metabolism, which suggested an immediate suppression PR:PROJECT_SUMMARY microbial metabolism. Collectively, these results demonstrate that acute TCDD PR:PROJECT_SUMMARY exposure results in immediate irregularities in the circulating and intestinal PR:PROJECT_SUMMARY metabolome which likely contributes to TCDD toxicity and can be used as PR:PROJECT_SUMMARY biomarkers for the early detection of individual exposure. PR:INSTITUTE University of South Carolina School of Medicine PR:DEPARTMENT Department of Pathology PR:LABORATORY On behalf of Dr. Mitzi Nagarkatti Lab PR:LAST_NAME Lai PR:FIRST_NAME Yunjia PR:ADDRESS 1104 MHRC, 135 Dauer Dr., Chapel Hill, NC, 27599, USA PR:EMAIL yunjia.lai@outlook.com PR:PHONE 9194805489 PR:FUNDING_SOURCE National Institute of Health: P01AT003961, P20GM103641, R01ES030144, R01AI129788 PR:FUNDING_SOURCE and R01AI123947 PR:PROJECT_COMMENTS This is supporting metabolomics data for the publication in Chemosphere. PR:PUBLICATIONS Chemosphere #STUDY ST:STUDY_TITLE Untargeted LC-MS metabolomics analysis of cecal content of mice treated with ST:STUDY_TITLE TCDD vs. vehicle control (part II) ST:STUDY_SUMMARY Six-week-old female wildtype (WT) C57BL/6 mice were administered a single 100µl ST:STUDY_SUMMARY intraperitoneal injection containing sterile corn oil (VEH group) or an ST:STUDY_SUMMARY intraperitoneal injection of 10µg/kg TCDD suspended within sterile corn oil ST:STUDY_SUMMARY (TCDD group). At the 72h time point following TCDD or VEH exposure, the mice ST:STUDY_SUMMARY were humanely euthanized by an overdose of inhaled isoflurane. During necropsy, ST:STUDY_SUMMARY cecal content and blood serum samples were collected for untargeted metabolomics ST:STUDY_SUMMARY profiling. ST:INSTITUTE University of South Carolina School of Medicine ST:DEPARTMENT Department of Pathology ST:LABORATORY (On behalf of) Mitzi Nagarkatti Lab ST:LAST_NAME Lai ST:FIRST_NAME Yunjia ST:ADDRESS 135 Dauer Drive, Chapel Hill, NC 27599 ST:EMAIL yunjia.lai@outlook.com ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 20 ST:NUM_FEMALES 20 ST:PUBLICATIONS Chemosphere ST:STUDY_TYPE Untargeted metabolomics ST:PHONE 919-480-5489 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 mice (Wild-type) SU:AGE_OR_AGE_RANGE six weeks old SU:GENDER Female #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 - SV1 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV1.raw SUBJECT_SAMPLE_FACTORS - SV2 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV2.raw SUBJECT_SAMPLE_FACTORS - SV3 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV3.raw SUBJECT_SAMPLE_FACTORS - SV4 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV4.raw SUBJECT_SAMPLE_FACTORS - SV5 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV5.raw SUBJECT_SAMPLE_FACTORS - SV6 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV6.raw SUBJECT_SAMPLE_FACTORS - SV7 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV7.raw SUBJECT_SAMPLE_FACTORS - SV8 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV8.raw SUBJECT_SAMPLE_FACTORS - SV9 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV9.raw SUBJECT_SAMPLE_FACTORS - SV10 Experimental factor:Vehicle Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=SV10.raw SUBJECT_SAMPLE_FACTORS - ST1 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST1.raw SUBJECT_SAMPLE_FACTORS - ST2 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST2.raw SUBJECT_SAMPLE_FACTORS - ST3 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST3.raw SUBJECT_SAMPLE_FACTORS - ST4 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST4.raw SUBJECT_SAMPLE_FACTORS - ST5 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST5.raw SUBJECT_SAMPLE_FACTORS - ST6 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST6.raw SUBJECT_SAMPLE_FACTORS - ST7 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST7.raw SUBJECT_SAMPLE_FACTORS - ST8 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST8.raw SUBJECT_SAMPLE_FACTORS - ST9 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST9.raw SUBJECT_SAMPLE_FACTORS - ST10 Experimental factor:TCDD Mouse_strain=C57BL/6; Gender=Female; RAW_FILE_NAME=ST10.raw #COLLECTION CO:COLLECTION_SUMMARY Six-week-old female wildtype (WT) C57BL/6 mice acquired from Jackson CO:COLLECTION_SUMMARY Laboratories (Bar Harbor, ME) were housed in an AAALAC-accredited CO:COLLECTION_SUMMARY specific-pathogen-free animal facility located at the grounds of the University CO:COLLECTION_SUMMARY of South Carolina School of Medicine for the entirety of all experiments. Mice CO:COLLECTION_SUMMARY within the facility were housed within polycarbonate cages containing cellulose CO:COLLECTION_SUMMARY fiber chips as bedding in a temperature and humidity-controlled environment. CO:COLLECTION_SUMMARY After a 2-week acclimatization period, the mice were divided randomly into two CO:COLLECTION_SUMMARY groups that would be administered a single 100µl intraperitoneal injection CO:COLLECTION_SUMMARY containing sterile corn oil (VEH group) or an intraperitoneal injection of CO:COLLECTION_SUMMARY 10µg/kg TCDD suspended within sterile corn oil (TCDD group). At the 72h time CO:COLLECTION_SUMMARY point following TCDD or VEH exposure, the mice were humanely euthanized by an CO:COLLECTION_SUMMARY overdose of inhaled isoflurane. Cecal content and blood sera were collected at CO:COLLECTION_SUMMARY the time of necropsy. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY Six-week-old female wildtype (WT) C57BL/6 mice acquired from Jackson TR:TREATMENT_SUMMARY Laboratories (Bar Harbor, ME) were housed in an AAALAC-accredited TR:TREATMENT_SUMMARY specific-pathogen-free animal facility located at the grounds of the University TR:TREATMENT_SUMMARY of South Carolina School of Medicine for the entirety of all experiments. Mice TR:TREATMENT_SUMMARY within the facility were housed within polycarbonate cages containing cellulose TR:TREATMENT_SUMMARY fiber chips as bedding in a temperature and humidity-controlled environment. TR:TREATMENT_SUMMARY After a 2-week acclimatization period, the mice were divided randomly into two TR:TREATMENT_SUMMARY groups that would be administered a single 100µl intraperitoneal injection TR:TREATMENT_SUMMARY containing sterile corn oil (VEH group) or an intraperitoneal injection of TR:TREATMENT_SUMMARY 10µg/kg TCDD suspended within sterile corn oil (TCDD group). At the 72h time TR:TREATMENT_SUMMARY point following TCDD or VEH exposure, the mice were humanely euthanized by an TR:TREATMENT_SUMMARY overdose of inhaled isoflurane. Cecal content and blood sera were collected at TR:TREATMENT_SUMMARY the time of necropsy. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cecal content and serum samples were processed according to previously described SP:SAMPLEPREP_SUMMARY methods for the metabolomic profiling using liquid chromatography-mass SP:SAMPLEPREP_SUMMARY spectrometry (LC-MS) with slight modifications. For cecal content, ~25 mg was SP:SAMPLEPREP_SUMMARY aliquoted into 1.5-mL Eppendorf tubes (Hamburg, Germany) containing ~20 mg acid SP:SAMPLEPREP_SUMMARY washed glass beads (Sigma-Aldrich, St. Louis, MO), extracted into ice-cold SP:SAMPLEPREP_SUMMARY MeOH:water (1:1, v/v) on a Qiagen TissueLyzer at 50 Hz for 10 min (Hilden, SP:SAMPLEPREP_SUMMARY Germany), and centrifuged at 12,000 rpm for 10 min. For blood sera, 20 µL SP:SAMPLEPREP_SUMMARY aliquots were extracted by adding 180 µL cold MeOH, briefly vortexed, and SP:SAMPLEPREP_SUMMARY incubated at -20 ºC for 30 min for protein precipitation. The supernatants of SP:SAMPLEPREP_SUMMARY both matrices were dried in a CentriVap vacuum concentrator (Labconco, MO) and SP:SAMPLEPREP_SUMMARY resuspended in ACN:water (2:98, v/v) upon analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS fullscan *.raw data acquired by Thermo XCalibur software MS:MS_RESULTS_FILE ST001872_AN003034_Results.txt UNITS:m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END