The slow elimination of TCPOBOP in mice leads to prolonged CAR activation and hepatomegaly

Constitutive androstane receptor (CAR, NR1I3), a nuclear receptor superfamily member, plays a pivotal role in liver size regulation. Murine CAR agonist TCPOBOP was reported to induce hepatomegaly in mice, accompanied by hepatocyte hypertrophy and proliferation. However, whether CAR activation-induced hepatomegaly is reversible and the histological changes during reversal process remain elusive. In the current study, C57BL/6 mice were administered TCPOBOP for 5 days and sacrificed at different time points after drug withdrawal. The results showed that TCPOBOP-induced hepatomegaly required a long time to reverse, as evidenced by the liver-to-body weight ratio in the TCPOBOP group remained significantly higher than that in the Vehicle group even 120 days after withdrawal. CTNNB1 and CCND1 staining indicated a reduction in hepatocyte size and proliferating cells. To investigate the involved mechanisms, we measured proteins associated with hepatomegaly and liver regeneration termination. The results suggested that YAP, C-MYC, CTNNB1, FOXM1 and HNF4α changed significantly after TCPOBOP withdrawal and functioned at different stages during reversal. Furthermore, we established a UPLC-MS/MS method to quantify TCPOBOP concentration. The results indicated a long-term hepatic retention of TCPOBOP, which constantly activated CAR and led to sustained hepatomegaly after TCPOBOP withdrawal. Overall, these findings revealed the reversibility of CAR activation-induced hepatomegaly and provided new insights into the safety of CAR as a drug target. Additionally, the results highlighted the importance of considering long-term TCPOBOP accumulation in the liver to avoid potential adverse effects and experimental biases.