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)

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.

Methods
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.