The slow elimination of 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene in mice leads to prolonged constitutive androstane receptor activation and hepatomegaly

Constitutive androstane receptor (CAR, NR1I3), a nuclear receptor superfamily member, plays a pivotal role in liver size regulation. Murine CAR agonist 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (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 the 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 remaining significantly higher than that in the vehicle group even 120 days after withdrawal. β-catenin and cyclin D1 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 yes-associated protein, C-MYC, β-catenin, forkhead box M1, and hepatocyte nuclear factor 4α changed significantly after TCPOBOP withdrawal and functioned at different stages during reversal. Furthermore, we established an ultra performance liquid chromatography-tandem mass spectrometry 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. SIGNIFICANCE STATEMENT: This study demonstrated that hepatic retention of 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) is the fundamental cause of constant constitutive androstane receptor (CAR) activation and sustained hepatomegaly after drug withdrawal, which provided new data for the safety of CAR as a drug target and offered novel insights for CAR manipulation on liver diseases. Notably, when using TCPOBOP as a murine CAR agonist, attention should be paid to its hepatic accumulation and retention to avoid potential experimental biases and adverse effects.