Development and application of zebrafish liver 3D spheroids for assessing hepatotoxicity.

With the widespread adoption of the 3R principles, three-dimensional (3D) cell models are increasingly employed in toxicology. However, research on 3D cell models in fish remains limited. This study established a 3D zebrafish liver (ZFL) cell model and utilized it to assess the hepatotoxicity of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-Q) and N-Isopropyl-N'-phenyl-1,4-phenylenediamine quinone (IPPD-Q), respectively. ZFL cells were seeded at densities ranging from 1000 to 15,000 cells per well and cultured for 7 days. The 3D ZFL spheroids progressively stabilized in size, and growth kinetics slowed markedly from day 5 onward. Optimal cell viability was observed at a seeding density of 5000 cells/well after 7 days. Compared with 2D ZFL monolayers, 3D ZFL spheroids demonstrated higher urea levels and greater cell viability following 7-day culture. Both IPPD-Q and 6PPD-Q inhibited spheroid growth, upregulated the transcription of apoptosis‑related genes, and increased caspase‑3 activity. No significant effects were observed in 2D monolayers. Furthermore,​ high concentrations of IPPD‑Q and 6PPD‑Q also reduced the fluorescent area in zebrafish larvae, consistent with​ the 3D ZFL spheroid results. The 3D spheroid allows​ extended exposure durations and better recapitulates in vivo functions. This study establishes a novel methodology for assessing the hepatotoxicity of emerging contaminants in fish.