Summary of Study ST003476
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 PR002135. The data can be accessed directly via it's Project DOI: 10.21228/M8NG0M 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.
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| Study ID | ST003476 |
| Study Title | Gas chromatography - mass spectrometry (GC-MS) of liver hepatic extracts from adult male C57BL/6NCrl mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin |
| Study Summary | Epidemiological evidence suggests an association between dioxin and dioxin-like compound (DLC) exposure and human liver disease. 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 in C57BL/6NCrl mice. Although the effects of TCDD toxicity are mediated by aryl hydrocarbon receptor (AHR) activation, the underlying mechanisms of TCDD-induced hepatotoxicity are unresolved. In the present study, male C57BL/6NCrl mice were gavaged every 4 days for 28 days with 0.03 - 30 µg/kg TCDD and evaluated for liver histopathology and gene expression as well as complementary 1-dimensional (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. Bulk liver RNA-seq data suggested altered urinary metabolites correlated with hepatic differential gene expression corresponding to specific metabolic pathways. In addition to evaluating whether altered urinary metabolites were liver-dependent, published single-nuclear RNA-seq (snRNA-seq), AHR ChIP-seq, and AHR knockout gene expression datasets provided further support of hepatic cell-type and AHR-regulated dependency, respectively. Overall, TCDD-induced liver effects were preceded by and occurred with changes in urinary metabolite levels due to AHR-mediated changes in hepatic gene expression. |
| Institute | Michigan State University |
| Department | Biochemistry and Molecular Biology |
| Laboratory | Dr. Tim Zacharewski's |
| Last Name | Sink |
| First Name | Warren |
| Address | 603 Wilson Rd Rm 212, East Lansing, MI 48823 |
| sinkwarr@msu.edu | |
| Phone | 6162953496 |
| Submit Date | 2024-09-11 |
| Num Groups | 5 |
| Total Subjects | 25 |
| Num Males | 25 |
| Study Comments | Adult male C57BL/6NCrl mice (PND 30) were gavaged every 4 days for 28 days with 1, 3, 10, or 30 µg/kg TCDD or sesame oil vehicle. Mice were euthanized and their liver tissue collected at the conclusion of the TCDD exposure (PND 58). |
| Raw Data Available | Yes |
| Raw Data File Type(s) | cdf |
| Analysis Type Detail | GC-MS |
| Release Date | 2024-11-01 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002135 |
| Project DOI: | doi: 10.21228/M8NG0M |
| Project Title: | Gas chromatography - mass spectrometry (GC-MS) of liver hepatic extracts from adult male C57BL/6NCrl mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin |
| Project Summary: | Epidemiological evidence suggests an association between dioxin and dioxin-like compound (DLC) exposure and human liver disease. 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 in C57BL/6NCrl mice. Although the effects of TCDD toxicity are mediated by aryl hydrocarbon receptor (AHR) activation, the underlying mechanisms of TCDD-induced hepatotoxicity are unresolved. In the present study, male C57BL/6NCrl mice were gavaged every 4 days for 28 days with 0.03 - 30 µg/kg TCDD and evaluated for liver histopathology and gene expression as well as complementary 1-dimensional (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. Bulk liver RNA-seq data suggested altered urinary metabolites correlated with hepatic differential gene expression corresponding to specific metabolic pathways. In addition to evaluating whether altered urinary metabolites were liver-dependent, published single-nuclear RNA-seq (snRNA-seq), AHR ChIP-seq, and AHR knockout gene expression datasets provided further support of hepatic cell-type and AHR-regulated dependency, respectively. Overall, TCDD-induced liver effects were preceded by and occurred with changes in urinary metabolite levels due to AHR-mediated changes in hepatic gene expression. |
| Institute: | Michigan State University |
| Department: | Biochemistry and Molecular Biology |
| Laboratory: | Dr. Tim Zacharewski's |
| Last Name: | Sink |
| First Name: | Warren |
| Address: | 603 Wilson Rd Rm 212, East Lansing, MI 48823 |
| Email: | sinkwarr@msu.edu |
| Phone: | 6162953496 |
Subject:
| Subject ID: | SU003604 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Age Or Age Range: | Mice were euthanized and their liver tissue collected on post natal day (PND) 58. |
| Weight Or Weight Range: | 15 - 24 g |
| Gender: | Male |
| Animal Animal Supplier: | Charles River Laboratories |
| Animal Housing: | 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. |
| Animal Light Cycle: | 12:12 |
| Animal Feed: | TEKLAD 8940 |
| Species Group: | Mammals |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | TCDD_dose_microg_per_kg |
|---|---|---|---|
| SA383669 | A_20231209_020 | Liver | 0 |
| SA383670 | A_20231211_007 | Liver | 0 |
| SA383671 | A_20231211_005 | Liver | 0 |
| SA383672 | A_20231211_003 | Liver | 0 |
| SA383673 | A_20231209_044 | Liver | 0 |
| SA383674 | A_20231209_042 | Liver | 0 |
| SA383675 | A_20231209_010 | Liver | 0 |
| SA383676 | A_20231209_025 | Liver | 0 |
| SA383677 | A_20231209_015 | Liver | 0 |
| SA383678 | A_20231209_030 | Liver | 0 |
| SA383679 | A_20231209_026 | Liver | 1 |
| SA383680 | A_20231209_011 | Liver | 1 |
| SA383681 | A_20231209_021 | Liver | 1 |
| SA383682 | A_20231209_031 | Liver | 1 |
| SA383683 | A_20231209_016 | Liver | 1 |
| SA383684 | A_20231209_013 | Liver | 10 |
| SA383685 | A_20231209_028 | Liver | 10 |
| SA383686 | A_20231209_018 | Liver | 10 |
| SA383687 | A_20231209_033 | Liver | 10 |
| SA383688 | A_20231209_023 | Liver | 10 |
| SA383689 | A_20231209_032 | Liver | 3 |
| SA383690 | A_20231209_027 | Liver | 3 |
| SA383691 | A_20231209_022 | Liver | 3 |
| SA383692 | A_20231209_017 | Liver | 3 |
| SA383693 | A_20231209_012 | Liver | 3 |
| SA383694 | A_20231209_024 | Liver | 30 |
| SA383695 | A_20231211_008 | Liver | 30 |
| SA383696 | A_20231209_019 | Liver | 30 |
| SA383697 | A_20231209_029 | Liver | 30 |
| SA383698 | A_20231209_014 | Liver | 30 |
| SA383699 | A_20231209_043 | Liver | 30 |
| SA383700 | A_20231209_034 | Liver | 30 |
| SA383701 | A_20231211_006 | Liver | 30 |
| SA383702 | A_20231211_002 | Liver | 30 |
| SA383703 | A_20231211_004 | Liver | 30 |
| SA383704 | A_20231209_004 | Standard | - |
| SA383705 | A_20231209_006 | Standard | - |
| SA383706 | A_20231209_005 | Standard | - |
| SA383707 | A_20231209_007 | Standard | - |
| SA383708 | A_20231209_039 | Standard | - |
| SA383709 | A_20231209_008 | Standard | - |
| SA383710 | A_20231209_041 | Standard | - |
| SA383711 | A_20231209_040 | Standard | - |
| SA383712 | A_20231209_038 | Standard | - |
| SA383713 | A_20231209_037 | Standard | - |
| SA383714 | A_20231209_036 | Standard | - |
| SA383715 | A_20231209_035 | Standard | - |
| SA383716 | A_20231209_009 | Standard | - |
| SA383717 | A_20231209_003 | Standard | - |
| SA383718 | A_20231209_002 | Standard | - |
| Showing results 1 to 50 of 50 |
Collection:
| Collection ID: | CO003597 |
| Collection Summary: | On post-natal day 58 after the mice were repeatedly gavaged every 4 days for 28 days with TCDD, the mice were euthanized, and their livers were collected and immediately snap-frozen. Collected liver was kept frozen until processing. |
| Sample Type: | Liver |
Treatment:
| Treatment ID: | TR003613 |
| Treatment Summary: | Beginning on post-natal day 30, C57BL/6NCrl male mice were orally gavaged every 4 days for 28 days with TCDD or sesame oil vehicle. On post-natal day 58, mice were euthanized, and liver tissue was collected. |
| Treatment Compound: | 2,3,7,8-tetrachlorodibenzo-p-dioxin |
| Treatment Route: | Oral gavage |
| Treatment Dose: | 0, 1, 3, 10, or 30 micrograms per kilogram |
| Treatment Dosevolume: | 0.1 milliliters |
| Treatment Doseduration: | 28 days |
| Treatment Vehicle: | sesame oil |
Sample Preparation:
| Sampleprep ID: | SP003611 |
| Sampleprep Summary: | GC-MS. Extraction, derivatization, and measurement of hepatic metabolites by gas-chromatography mass spectrometry (GC-MS) was performed using protocols adapted from the publicly available Michigan State University Mass Spectrometry and Metabolomics Core website (https://rtsf.natsci.msu.edu/sites/_rtsf/assets/File/MSU_MSMC_004_v1_2_Two_phase_extraction_of_metabolites_from_animal_tissues.pdf). Briefly, ~50 mg of frozen liver tissue (n = 5 per dose) was supplemented with 6 nanomoles of 13C and 15N labeled amino acid internal standards (767964, Sigma-Aldrich) and lysed with a Mixer Mill 300 and a 3 mm stainless steel ball in a solution of methanol/chloroform (1:2 v/v), 1% formic acid, and 0.01% BHT. Subsequently, the samples were sonicated for 15 minutes. The mixture was twice supplemented with Milli-Q water, vortexed, centrifuged, and the resulting aqueous supernatant was transferred to a separate Eppendorf tube and made basic by the addition of NaOH. The samples were evaporated to dryness by a SpeedVac without heat and stored at -20° C. For derivatization, the dried samples were set at 60°C overnight with 100 µL of 40 mg/mL of methoxyamine HCl dissolved in pyridine and incubated overnight with 100 µL of N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) containing 1% tert-butyldimethylsilyl chloride. |
| Sampleprep Protocol Filename: | Protocol_MSU_MSMC_004_version_1.2.pdf |
Combined analysis:
| Analysis ID | AN005711 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 7890A |
| Column | Agilent J&W VF-5ms (30 m x 0.25 mm, 0.25 um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent 7890A |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH004332 |
| Chromatography Summary: | Derivatized samples (1 µL) were injected with a 1:10 split at 230 °C and analyzed on an Agilent 7890A GC/ single quadrupole mass spectrometer with 5975C inert XL MSD. The carrier gas was helium, which flowed at 1.0 mL/min. Separation was achieved on an Agilent J&W VF-5ms (30 m x 0.25 mm x 0.25 µm) (Agilent, Santa Clara, CA) using the following temperature profile: initially set at 40 °C, ramped to 80 °C at 20 °C/min, immediately ramped to 250 °C at 5 °C/min and held for 1 minute, and ramped to 320 °C at 50 °C/min and held for 5 minute. The mass spectrometer was operated at an electron ionization of 70 eV and a scan range of 50-600 m/z. Glycolic acid (PHR1427, Sigma), hydroxyproline (PHR1939, Sigma), oxalic acid (PHR3291, Sigma), and urea (PHR1406, Sigma) were used as external standards to calculate metabolite levels per sample. Quantification of the derivatized metabolites and internal standard were calculated using the peak area from the extracted ion chromatogram. The SIM ion used for derivatized glycolic acid, hydroxyproline, oxalic acid, and urea were 247, 416, 261, and 231 m/z, respectively. Derivatized glycolic acid, hydroxyproline, and urea were normalized to labeled glycine, while oxalic acid was normalized to labeled alanine. Oxalic acid and alanine were quantified by separate runs in the SIM mode, while the other compounds were quantified from runs in the scan mode. Additionally, hydroxyproline was normalized to labeled proline. After calculating concentrations via a linear calibration curve, concentrations were normalized to the liver weight per sample from which metabolites were extracted. |
| Instrument Name: | Agilent 7890A |
| Column Name: | Agilent J&W VF-5ms (30 m x 0.25 mm, 0.25 um) |
| Column Temperature: | initially set at 40 °C, ramped to 80 °C at 20 °C/min, immediately ramped to 250 °C at 5 °C/min and held for 1 minute, and ramped to 320 °C at 50 °C/min and held for 5 minute |
| Flow Gradient: | - |
| Flow Rate: | 1.0 mL/min |
| Solvent A: | - |
| Solvent B: | - |
| Chromatography Type: | GC |
MS:
| MS ID: | MS005434 |
| Analysis ID: | AN005711 |
| Instrument Name: | Agilent 7890A |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | The mass spectrometer was operated at an electron ionization of 70 eV and a scan range of 50-600 m/z. Glycolic acid (PHR1427, Sigma), hydroxyproline (PHR1939, Sigma), oxalic acid (PHR3291, Sigma), and urea (PHR1406, Sigma) were used as external standards to calculate metabolite levels per sample. Quantification of the derivatized metabolites and internal standard were calculated using the peak area from the extracted ion chromatogram. The SIM ion used for derivatized glycolic acid, hydroxyproline, oxalic acid, and urea were 247, 416, 261, and 231 m/z, respectively. Derivatized glycolic acid, hydroxyproline, and urea were normalized to labeled glycine, while oxalic acid was normalized to labeled alanine. Oxalic acid and alanine were quantified by separate runs in the SIM mode, while the other compounds were quantified from runs in the scan mode. Additionally, hydroxyproline was normalized to labeled proline. After calculating concentrations via a linear calibration curve, concentrations were normalized to the liver weight per sample from which metabolites were extracted. |
| Ion Mode: | POSITIVE |
