Dose-dependent disruption of hepatic zonation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice: integration of single-nuclei RNA sequencing and spatial transcriptomics

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dose-dependently induces the development of hepatic fat accumulation and inflammation with fibrosis in mice initially in the portal region. Conversely, differential gene and protein expression is first detected in the central region. To further investigate cell-specific and spatially resolved dose-dependent changes in gene expression elicited by TCDD, single-nuclei RNA sequencing and spatial transcriptomics were used for livers of male mice gavaged with TCDD every 4 days for 28 days. The proportion of 11 cell (sub)types across 131,613 nuclei dose-dependently changed with 68% of all portal and central hepatocyte nuclei in control mice being overtaken by macrophages following TCDD treatment. We identified 368 (portal fibroblasts) to 1,339 (macrophages) differentially expressed genes. Spatial analyses revealed initial loss of portal identity that eventually spanned the entire liver lobule with increasing dose. Induction of R-spondin 3 (Rspo3) and pericentral Apc, suggested dysregulation of the Wnt/β-catenin signaling cascade in zonally resolved steatosis. Collectively, the integrated results suggest disruption of zonation contributes to the pattern of TCDD-elicited NAFLD pathologies. Male C57BL/6 mice were received from Charles Rivers Laboratories at postnatal day (PND) 25 and acclimated until PND 28. Mice were randomly assigned to Innocages (Innovive, San Diego, CA) with ALPHA-dri bedding (Shepherd Specialty Papers, Chicago, IL) at 30-40% humidity and a 12-hour light/dark cycle. Mice had free access to Harlan Teklad 22/5 Rodent Diet 8940 (Envigo, Indianapolis, IN) and Aquavive water (Innovive). On PND 28 and every 4 days thereafter, for a total of 7 administrations (24 days), mice were orally gavaged with sesame oil vehicle (Sigma-Aldrich, St. Louis, MO), 0.3, 3, or 30 µg/kg TCDD (AccuStandard, New Haven, CT) between ZT00 and ZT01. On PND 56, 28 days after the initial gavage, mice were euthanized by CO2 asphyxiation and livers were immediately collected, snap frozen in liquid nitrogen and stored at -80°C. All procedures were approved by the Michigan State University Institutional Animal Care and Use Committee. Spatial transcriptomics was performed using the Resolve BioScience Molecular Cartography system. Two biological replicates for each dose group were examined by placing 10 µm thick frozen liver sections in O.C.T compound (Sakura, Torrance, CA) in one of 8 designated custom slide regions. Frozen slides were shipped to Resolve BioSciences for analysis following manufacturer’s instructions (protocol version 3.0).