Liver organoids uncover tire-derived 6-PPDQ-induced hepatotoxicity: A preliminary application of environmental toxicology and safety assessment

The novel pollutant, N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) leaked out of the tire and has attracted extensive concerns due to its high lethal toxicity of salmon. However, the potential hepatotoxicity of 6-PPDQ exposure and its mechanisms are unknown. As a novel 3D cell culture, liver organoids (LOs) are more similar to real organ invitro in structure and function, which showed great potential for toxicity assessment. Herein, stable LOs were generated and their applicability on hepatotoxicity assessment was evaluated with four hepatotoxic compounds. The negative effect of 6-PPDQ was explored in LOs, live/dead staining visually demonstrated the damage to the liver, and the changes of ATP, LDH, ALT, and AST effectively reflected its hepatotoxicity. Meanwhile, machine learning-based quantitative assessments of LOs morphology changes provided objective data on area, circularity, and luminance changes, enabling sensitive detection of 6-PPDQ-induced hepatotoxicity. Furthermore, transcriptomic analysis revealed that the pathways related to DNA replication and repairment, cancers, and inflammation were significantly involved in the process of 6-PPDQ-induced liver injury; Disease enrichment analysis highlighted an increased risk of chronic liver diseases, and biliary atresia were validated by Cholyl-Lys-Fluorescein (CLF). Moreover, molecular docking analysis identified potential molecular targets of 6-PPDQ, including Slc6a9, Yes1, and Nos2. This study underscored the potential of LOs for toxicological studies and highlighted the toxic effects of 6-PPDQ on the liver, suggesting the need for further investigations to understand its long-term impact on human health.