Drinking-Water 4,4′-Methylenedianiline Establishes a Longitudinally Tractable Mouse Model of Cholestatic Liver Injury

Abstract

Longitudinal studies of liver injury require models that can be maintained with simple administration, low procedure-related stress, and acceptable survival. Drinking-water thioacetamide (TAA-DW) is useful for this purpose, but it mainly produces centrilobular hepatocellular injury. Few drinking-water models are available for cholestatic liver injury. Here, we tested whether 4,4′-methylenedianiline (MDA), a known bile-duct-damaging hepatotoxicant, can be used to build a practical drinking-water model of cholestatic liver injury in mice. Male C57BL/6JJcl mice received MDA in drinking water for up to 28 days. A route- and administration-schedule-matched TAA-DW model was used as a reference. Drinking-water MDA (MDA-DW) caused sustained ALT elevation without observed mortality under the tested regimen. Compared with TAA-DW, MDA-DW produced stronger cholestatic biochemical features, including increased ALP, TBIL, and TCHO. Histology showed bile duct hyperplasia with degeneration, periportal/peribiliary fibrosis, and scattered focal hepatocellular necrosis. Whole-slide image analysis supported the distinct lesion distribution between MDA-DW and TAA-DW. Early time-resolved multimodal profiling showed model-specific responses: MDA-DW showed ALT elevation around Days 2–3, increased CD8-positive T cells on Day 8, and enrichment of fibrinolysis-related gene sets, whereas TAA-DW showed a sharp early injury peak and transient eosinophil increase. Impact Statement: This study expands the range of drinking-water liver injury models by providing a low-mortality MDA-DW model with cholestatic liver injury. The model offers a simple platform for time-resolved studies of liver injury progression, tissue remodeling, and model-specific inflammatory responses.