Summary of Study ST003845
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002403. The data can be accessed directly via it's Project DOI: 10.21228/M81C2R This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003845 |
| Study Title | Shift in the urinary metabolome associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin activation of the hepatic aryl hydrocarbon receptor |
| Study Summary | Epidemiological evidence suggests an association between dioxin and dioxin-like compound (DLC) exposure and human liver disease. In rodents, the prototypical DLC, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been shown to induce the progression of reversible hepatic steatosis to steatohepatitis with periportal fibrosis and biliary hyperplasia. Although the effects of TCDD toxicity are mediated by aryl hydrocarbon receptor (AHR) activation, the underlying mechanisms of TCDD-induced liver toxicity are not well understood. In the present study, male C57BL/6NCrl mice were gavaged every 4 days for 28 days with 0.03 - 30 µg/kg TCDD or sesame oil vehicle and evaluated for liver histopathology and gene expression as well as complementary 1-dimensional proton magnetic resonance (1-D 1H NMR) urinary metabolic profiling. Urinary trimethylamine (TMA), trimethylamine N-oxide (TMAO), and 1-methylnicotinamide (1MN) levels were altered by TCDD at doses ≤ 3 µg/kg; other urinary metabolites, like glycolate, urocanate, and 3-hydroxyisovalerate, were only altered at doses that induced moderate to severe steatohepatitis. Hepatic differential gene expression of rate-limiting enzymes of choline, gloxylate, and amino acid metabolism coincided with the altered urinary metabolites. Published single-nuclear RNA-seq (snRNA-seq), AHR ChIP-seq, and AHR knockout gene expression datasets provided further support for hepatic cell-type and AHR-regulated dependency for the affected metabolic pathways. |
| Institute | Michigan State University |
| Department | Biochemistry and Molecular Biology |
| Last Name | Sink |
| First Name | Warren |
| Address | 603 Wilson Rd, East Lansing, Michigan, 48824, USA |
| sinkwarr@msu.edu | |
| Phone | 6162953496 |
| Submit Date | 2025-03-24 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | fid |
| Analysis Type Detail | NMR |
| Release Date | 2025-04-07 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002403 |
| Project DOI: | doi: 10.21228/M81C2R |
| Project Title: | Shift in the urinary metabolome associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin activation of the hepatic aryl hydrocarbon receptor |
| Project Summary: | Epidemiological evidence suggests an association between dioxin and dioxin-like compound (DLC) exposure and human liver disease. In rodents, the prototypical DLC, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been shown to induce the progression of reversible hepatic steatosis to steatohepatitis with periportal fibrosis and biliary hyperplasia. Although the effects of TCDD toxicity are mediated by aryl hydrocarbon receptor (AHR) activation, the underlying mechanisms of TCDD-induced liver toxicity are not well understood. In the present study, male C57BL/6NCrl mice were gavaged every 4 days for 28 days with 0.03 - 30 µg/kg TCDD or sesame oil vehicle and evaluated for liver histopathology and gene expression as well as complementary 1-dimensional proton magnetic resonance (1-D 1H NMR) urinary metabolic profiling. Urinary trimethylamine (TMA), trimethylamine N-oxide (TMAO), and 1-methylnicotinamide (1MN) levels were altered by TCDD at doses ≤ 3 µg/kg; other urinary metabolites, like glycolate, urocanate, and 3-hydroxyisovalerate, were only altered at doses that induced moderate to severe steatohepatitis. Hepatic differential gene expression of rate-limiting enzymes of choline, gloxylate, and amino acid metabolism coincided with the altered urinary metabolites. Published single-nuclear RNA-seq (snRNA-seq), AHR ChIP-seq, and AHR knockout gene expression datasets provided further support for hepatic |
| Institute: | Michigan State University |
| Department: | Biochemistry and Molecular Biology |
| Last Name: | Sink |
| First Name: | Warren |
| Address: | 603 Wilson Rd, East Lansing, Michigan, 48824, USA |
| Email: | sinkwarr@msu.edu |
| Phone: | 6162953496 |
| Project Comments: | The urine samples were collected by the Zacharewski lab at Michigan State University. The samples were evaluated with 1-dimensional 1H NMR by Dr. Ali Yilmaz at Corewell Health Research Institute. The data upload was done by Warren Sink from the Zacharewski lab. |
Subject:
| Subject ID: | SU003979 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C56BL/6NCrl |
| Age Or Age Range: | post-natal day 58 |
| Weight Or Weight Range: | 16 - 24 grams |
| Gender: | Male |
| Animal Animal Supplier: | Charles River |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Dose |
|---|---|---|
| SA420522 | 8 | 0.03 µg/kg TCDD |
| SA420523 | 12 | 0.03 µg/kg TCDD |
| SA420524 | 16 | 0.03 µg/kg TCDD |
| SA420525 | 26 | 0.03 µg/kg TCDD |
| SA420526 | 34 | 0.03 µg/kg TCDD |
| SA420527 | 38 | 0.1 µg/kg TCDD |
| SA420528 | 31 | 0.1 µg/kg TCDD |
| SA420529 | 29 | 0.1 µg/kg TCDD |
| SA420530 | 19 | 0.1 µg/kg TCDD |
| SA420531 | 3 | 0.1 µg/kg TCDD |
| SA420532 | 5 | 0.3 µg/kg TCDD |
| SA420533 | 11 | 0.3 µg/kg TCDD |
| SA420534 | 18 | 0.3 µg/kg TCDD |
| SA420535 | 21 | 0.3 µg/kg TCDD |
| SA420536 | 30 | 0.3 µg/kg TCDD |
| SA420517 | 40 | 0 µg/kg TCDD |
| SA420518 | 6 | 0 µg/kg TCDD |
| SA420519 | 2 | 0 µg/kg TCDD |
| SA420520 | 14 | 0 µg/kg TCDD |
| SA420521 | 22 | 0 µg/kg TCDD |
| SA420542 | 4 | 10 µg/kg TCDD |
| SA420543 | 9 | 10 µg/kg TCDD |
| SA420544 | 24 | 10 µg/kg TCDD |
| SA420545 | 33 | 10 µg/kg TCDD |
| SA420546 | 17 | 10 µg/kg TCDD |
| SA420537 | 37 | 1 µg/kg TCDD |
| SA420538 | 23 | 1 µg/kg TCDD |
| SA420539 | 32 | 1 µg/kg TCDD |
| SA420540 | 15 | 1 µg/kg TCDD |
| SA420541 | 7 | 1 µg/kg TCDD |
| SA420552 | 10 | 30 µg/kg TCDD |
| SA420553 | 25 | 30 µg/kg TCDD |
| SA420554 | 28 | 30 µg/kg TCDD |
| SA420555 | 35 | 30 µg/kg TCDD |
| SA420556 | 39 | 30 µg/kg TCDD |
| SA420547 | 13 | 3 µg/kg TCDD |
| SA420548 | 20 | 3 µg/kg TCDD |
| SA420549 | 27 | 3 µg/kg TCDD |
| SA420550 | 36 | 3 µg/kg TCDD |
| SA420551 | 1 | 3 µg/kg TCDD |
| Showing results 1 to 40 of 40 |
Collection:
| Collection ID: | CO003972 |
| Collection Summary: | TCDD elicits similar effects of steatohepatitis and hepatotoxicity in both sexes of C57BL/6NCrl mice as previously reported. However, male C57BL/6NCrl mice exhibit heightened sensitivity to TCDD-elicited liver toxicity compared to female C57BL/6NCrl mice (i.e., the TCDD dose-response curve of female C57BL/6NCrl liver toxicity is right-shifted). To observe the range of histological, gene expression, and metabolic differences elicited by TCDD, C57BL/6NCrl males were prioritized. Post-natal day (PND) 25 C57BL/6NCrl males from Charles River Breeding Laboratories (Kingston, NY) were acclimatized for five days prior to treatment. Mice were housed in Innovive Innocages (San Diego, CA) containing ALPHA-dri bedding (Shepherd Specialty Papers, Chicago) at an ambient temperature of 21° C with 30-40 % humidity and a 12 h/12 light/dark cycle. All mice were fed the TEKLAD diet 8940 (Madison, WI) ad libitum. At PND 30, mice were orally gavaged with 0.1 ml sesame oil vehicle (Sigma-Aldrich, St. Louis, MO) or 0.03, 0.1, 0.3, 1, 3, 10, or 30 µg/kg TCDD (AccuStandard, New Haven, CT) every 4 days for 28 days from Zeitgeber (ZT) 0 – 3 for a total of 7 administered doses to induce steady-state levels of TCDD. Moreover, the doses used in the current study were chosen to account for the relatively short study duration as opposite to lifelong cumulative human exposure from diverse AhR ligands, the bioaccumulative nature of halogenated AhR ligands, and different half-lives of TCDD in humans (1−11 years) and mice (8−12 days). The same dose range and treatment regimen has been used in previous studies where we found that mouse hepatic tissue levels were comparable to serum levels following the intentional poisoning of Viktor Yushchenko (1 – 30 μg/kg) and following the accidental exposure from a 1976 chemical plant accident of Seveso women with and without chloracne (0.03 – 0.3 μg/kg). On PND 55, urine and feces were collected from individual mice from ZT 0 – 3, snap-frozen in liquid nitrogen, and stored at -80° C. Three days later, PND 58 mice were euthanized by CO2. Blood, liver, kidney, and epididymal adipose tissues were collected. Whole livers were weighed, snap-frozen in liquid nitrogen, and stored at -80° C. Mice were monitored every day for fluctuations in body weight and changes in chow weight from ZT 0 – 3. Food consumption per day was calculated by dividing the difference in chow weight from the previous day by the number of mice per cage. This study was conducted in accordance with relevant guidelines and regulations. All animal procedures were approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC; PROTO 202100219) and meet the ARRIVE guidelines. |
| Sample Type: | Urine |
Treatment:
| Treatment ID: | TR003988 |
| Treatment Summary: | TCDD elicits similar effects of steatohepatitis and hepatotoxicity in both sexes of C57BL/6NCrl mice as previously reported. However, male C57BL/6NCrl mice exhibit heightened sensitivity to TCDD-elicited liver toxicity compared to female C57BL/6NCrl mice (i.e., the TCDD dose-response curve of female C57BL/6NCrl liver toxicity is right-shifted). To observe the range of histological, gene expression, and metabolic differences elicited by TCDD, C57BL/6NCrl males were prioritized. Post-natal day (PND) 25 C57BL/6NCrl males from Charles River Breeding Laboratories (Kingston, NY) were acclimatized for five days prior to treatment. Mice were housed in Innovive Innocages (San Diego, CA) containing ALPHA-dri bedding (Shepherd Specialty Papers, Chicago) at an ambient temperature of 21° C with 30-40 % humidity and a 12 h/12 light/dark cycle. All mice were fed the TEKLAD diet 8940 (Madison, WI) ad libitum. At PND 30, mice were orally gavaged with 0.1 ml sesame oil vehicle (Sigma-Aldrich, St. Louis, MO) or 0.03, 0.1, 0.3, 1, 3, 10, or 30 µg/kg TCDD (AccuStandard, New Haven, CT) every 4 days for 28 days from Zeitgeber (ZT) 0 – 3 for a total of 7 administered doses to induce steady-state levels of TCDD. Moreover, the doses used in the current study were chosen to account for the relatively short study duration as opposite to lifelong cumulative human exposure from diverse AhR ligands, the bioaccumulative nature of halogenated AhR ligands, and different half-lives of TCDD in humans (1−11 years) and mice (8−12 days). |
Sample Preparation:
| Sampleprep ID: | SP003985 |
| Sampleprep Summary: | Urine samples (500 µl) in a 1.5 mL Eppendorf tube were centrifuged at 12,000 g for 10 minutes at 4° C to remove debris. The supernatant (300 µL) was transferred to a clean 1.5 mL Eppendorf tube containing 35 μL of D2O and 15 μL of buffer (11.667 mM DSS [disodium-2,2-dimethyl-2-silapentane-5-sulphonate], 730 mM imidazole, and 0.47 % NaN3 in H2O). Samples (350 μL) were then transferred to a standard 3 mm thin-walled glass NMR tube for 1H NMR spectral analysis. All 1H NMR spectra were randomly collected on a Bruker Ascend HD 600 MHz spectrometer equipped with a 5 mm TCI cryoprobe and acquired at 25° C using the modified version of the first transient of the Bruker noesy-presaturation pulse sequence providing a high degree of quantitative accuracy. Spectra were collected with 128 transients and 16 steady-state scans using a 5-second acquisition time and a 5.1-second recycle delay. Prior to spectral analysis, all FIDs were zero-filled to 128K data points, and line broadened by 0.5 Hz. The methyl singlet produced by a known quantity of DSS (1000 μM) was used as an internal standard for chemical shift referencing (set to 0 ppm) and for quantification. All 1H NMR spectra were processed and analyzed using Chenomx NMR Suite Professional software package version 8.3 (Chenomx Inc., Edmonton, CA). Prior to statistical analysis, all NMR spectra were manually inspected for technical faults. Analysis identified 107 unique metabolites in the mouse urine samples. |
Analysis:
| Analysis ID: | AN006319 |
| Analysis Type: | NMR |
| Num Factors: | 8 |
| Num Metabolites: | 111 |
| Units: | micromolar |
NMR:
| NMR ID: | NM000313 |
| Analysis ID: | AN006319 |
| Instrument Name: | 600 HHz Avance III HD |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| Spectrometer Frequency: | 600 MHz |
| NMR Probe: | TCI- croprobe |
| NMR Solvent: | Water- D2O (90/10) |
| NMR Tube Size: | 3.00 mm |
| Shimming Method: | Topshim 3d |
| Pulse Sequence: | 1dnoesy |
| Water Suppression: | presat |
| Receiver Gain: | 80.6 |
| Number Of Scans: | 128 |
| Acquisition Time: | 5.3 sec |
| Spectral Width: | 20.8 |
| Line Broadening: | no |
| Apodization: | Exponential |
| Baseline Correction Method: | BXR-spline |
| Chemical Shift Ref Std: | 0.0 ppm (DSS) |
