DOI: 10.5061/dryad.mpg4f4r8f
title: Western blots of liver-expressed genes in C57BL/NCrl mice gavaged every 4 days for 28 days with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

description: 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.
description: Liver lysates (20 μg) from mice gavaged every 4 days for 28 days
 with 1, 3, 10, and 30 µg/kg TCDD or sesame oil vehicle were resolved via
 10% SDS-PAGE gels (Bio-Rad, San Diego, CA, USA) and transferred to
 nitrocellulose membranes (GE Healthcare, Chicago, IL) using the Mini
 Trans-Blot Cell Unit (BioRad) by wet electroblotting (100 V, 45 min). The
 membranes were then blocked with 5% nonfat milk (in Tris-buffered saline
 [TBS]+0.01% Tween) for 1 hour and incubated with primary antibodies:
 anti-FMO3 (1:5000; ab126711; Abcam, Waltham, MA), anti-GLO1 (1:1000;
 MA531148, Life Technologies Corporation, Carlsbad, CA), anti-HAO1 (1:5000;
 ab194790; Abcam, Waltham, MA), anti-AGXT (1:1000; ab178708; Abcam,
 Waltham, MA), and anti-ACTB (1:1000; #4970; Cell Signaling, Danvers, MA)
 overnight at 4°C. Blots were visualized using horseradish peroxidase
 (HRP)-linked secondary antibodies of goat anti-mouse (1:10000;
 Elabscience, Houston, TX) and anti-rabbit (1:1000, Cell Signaling,
 Danvers, MA) and an ECL kit (Millipore Corporation, Billerica, MA).
 Membranes were scanned on a Sapphire Biomolecular Imager (Azure Biosystem,
 Dublin, CA). Protein density values were assessed and calculated using
 ImageJ (v1.53). Protein expression was standardized to ACTB levels per
 sample. NOTE: Each western blot has one biological
 replicate of the TCDD dose-response (0, 1, 3, 10, and 30 µg/kg)
 represented. Lanes were loaded with respect to the protein size ladder
 with the lanes farthest away from the protein size ladder containing liver
 lysates from mice gavaged every 4 days for 28 days with the highest
 concentrations of TCDD. So, the 0 µg/kg TCDD liver lysate lane is always
 immediately next to protein size ladder; the 1 µg/kg TCDD liver lysate
 lane is always immediately next to the 0 µg/kg TCDD lane; the 3 µg/kg TCDD
 liver lysate lane is always immediately next to the 1 µg/kg TCDD lane; the
 10 µg/kg TCDD liver lysate lane is always immediately next to the 3 µg/kg
 TCDD lane; and the 30 µg/kg TCDD liver lysate lane is always immediately
 next to the 10 µg/kg TCDD lane. 
description: # Western blots of liver-expressed genes in C57BL/NCrl mice gavaged every
 4 days for 28 days with 2,3,7,8-tetrachlorodibenzo-*p*-dioxin (TCDD)
 [https://doi.org/10.5061/dryad.mpg4f4r8f](https://doi.org/10.5061/dryad.mpg4f4r8f) The data files in this repository contain raw annotated images of Western blots for ACTB, FMO3, AGXT, HAO1, and GLO1 from liver lysates of mice gavaged every 4 days for 28 with 2,3,7,8-tetrachlorodibenzo-*p*-dioxin (TCDD) or sesame oil vehicle. **Description of data files**. The Western blot data files are arranged in folders named after the blotted protein (e.g., FMO3). Each of these folders has tiff files for each batch of samples (there are no technical replicates deposited, only biological replicates). Each tiff file has a number in its filename (e.g., 28), which corresponds to the name of the csv file containing the densitometry values for each Western blot lane derived from ImageJ. Each tiff file has a copy with the filename "SampleNames", which is a copy of the raw Western blot tiff with ID's above each lane indicating the mouse which is the source of the liver lysate. Furthermore, for each of the proteins detected, there is included at least one copy of a raw Western blot composed with an annotated protein ladder. Each western blot has one biological replicate of the TCDD dose-response (0, 1, 3, 10, and 30 µg/kg) represented. Lanes were loaded with respect to the protein size ladder with the lanes farthest away from the protein size ladder containing liver lysates from mice gavaged every 4 days for 28 days with the highest concentrations of TCDD. So, the 0 µg/kg TCDD liver lysate lane is always immediately next to protein size ladder; the 1 µg/kg TCDD liver lysate lane is always immediately next to the 0 µg/kg TCDD lane; the 3 µg/kg TCDD liver lysate lane is always immediately next to the 1 µg/kg TCDD lane; the 10 µg/kg TCDD liver lysate lane is always immediately next to the 3 µg/kg TCDD lane; and the 30 µg/kg TCDD liver lysate lane is always immediately next to the 10 µg/kg TCDD lane. The csv files have densitometry values for each Western blot lane. The values correspond to the lanes in that the first row value is for a mouse gavaged with 0 µg/kg TCDD, the second row values for a mouse gavaged with 1 µg/kg TCDD, ..., and the final row values is for a mouse gavaged with 30 µg/kg TCDD. In the tab-delimited text file "Prj161_Westerns_ActB_Fmo3_Glo1_Agxt_Hao1.txt", the ID's of the mice are joined with study metadata and per protein densitometry values. **Study Design**. 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-ZT3 for a total of 7 administered doses. On PND 55, urine and feces were collected from individual mice, snap frozen in liquid nitrogen and stored at -80° C. Three days later on PND 58, mice were euthanized by CO2. Whole livers were weighed, snap frozen in liquid nitrogen, and stored at -80° C. **Western Blotting**. Liver lysates (20 μg) from mice gavaged every 4 days for 28 days with 1, 3, 10, and 30 µg/kg TCDD or sesame oil vehicle were resolved via 10% SDS-PAGE gels (Bio-Rad, San Diego, CA, USA) and transferred to nitrocellulose membranes (GE Healthcare, Chicago, IL) using the Mini Trans-Blot Cell Unit (BioRad) by wet electroblotting (100 V, 45 min). The membranes were then blocked with 5% nonfat milk (in Tris-buffered saline [TBS]+0.01% Tween) for 1 hour and incubated with primary antibodies: anti-FMO3 (1:5000; ab126711; Abcam, Waltham, MA), anti-GLO1 (1:1000; MA531148, Life Technologies Corporation, Carlsbad, CA), anti-HAO1 (1:5000; ab194790; Abcam, Waltham, MA), anti-AGXT (1:1000; ab178708; Abcam, Waltham, MA), and anti-ACTB (1:1000; #4970; Cell Signaling, Danvers, MA) overnight at 4°C. Blots were visualized using horseradish peroxidase (HRP)-linked secondary antibodies of goat anti-mouse (1:10000; Elabscience, Houston, TX) and anti-rabbit (1:1000, Cell Signaling, Danvers, MA) and an ECL kit (Millipore Corporation, Billerica, MA). Membranes were scanned on a Sapphire Biomolecular Imager (Azure Biosystem, Dublin, CA). Protein density values were assessed and calculated using ImageJ (v1.53). Protein expression was standardized to ACTB levels per sample.