Establishment of a longitudinally tractable mouse model of cholestatic liver injury via drinking water administration of MDA

Background & Aims Longitudinal animal models are essential for understanding the temporal dynamics of liver injury and recovery. While drinking water-based administration is ideal for sustained exposure in high feasibility, available compounds are limited, with thioacetamide (TAA) being the primary option. Here, we aimed to establish a novel drinking water-induced mouse model of cholestatic liver injury using 4,4’-methylenedianiline (MDA), and to characterize its pathological trajectory in comparison to the TAA model. Methods Mice were administered MDA via drinking water for 28 Days. To elucidate the early events that give rise to chronic pathological divergence, we conducted a multi-layered analysis comprising plasma biochemical assays, immune cell profiling by flow cytometry, and hepatic transcriptomics at five time points during the early phase. The MDA model was evaluated against the established TAA model. Results MDA administration induced sustained ALT elevation, peribiliary fibrosis, and spatially irregular focal hepatocellular necrosis, distinguishing it from the centrilobular injury observed with TAA. Additionally, the MDA model showed significant elevations in ALP, TBIL, and TCHO, indicating cholestatic liver dysfunction. Early-phase analyses revealed model-specific differences in immunological and molecular responses, including increased CD8⁺ T cell populations and enrichment of fibrinolysis-related gene expression in MDA-DW mice. Conclusions We present the MDA-DW model as a novel, longitudinally tractable liver injury model that complements existing systems by capturing alternative spatial, immunological, and transcriptional patterns of injury. This model offers a valuable platform for dissecting the temporal dynamics of liver disease progression in experimental settings. Significance Statement We developed a cost-effective, non-invasive mouse model of cholestatic liver injury using drinking-water administration of 4,4’-methylenedianiline (MDA). This model exhibits periportal-predominant damage, peribiliary fibrosis, and spatially irregular focal hepatocellular necrosis, distinct from conventional centrilobular models. Early-phase multi-omics analysis revealed immunological and transcriptomic differences, including increased CD8⁺ T cells and activation of fibrinolysis-related pathways. The low mortality rate and ease of implementation enable long-term studies and cross-sectional comparisons across time points or interventions. This study provides not only a practical model for investigating chronic liver injury, but also a rich time-series, multi-view dataset, offering a valuable resource for advancing research on liver pathophysiology and toxicological mechanisms.