Gao Yue, Cai Chenghui, Zheng Wanyu, Fan Shicheng, Zhang Yifei, Zhou Yanying, Li Huilin, Yang Jie, Guan Shaoxing, Bi Huichang
NMPA Key Laboratory for Research and Evaluation of Drug Metabolism & Guangdong Provincial Key Laboratory of New Drug Screening & Guangdong-Hongkong-Macao Joint Laboratory for New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China.
Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.
Drug Metab Dispos. 2025 Jun;53(6):100092. doi: 10.1016/j.dmd.2025.100092. Epub 2025 Jun 6.
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.
组成型雄烷受体（CAR，NR1I3）是核受体超家族成员，在肝脏大小调节中起关键作用。据报道，小鼠CAR激动剂1,4-双[2-(3,5-二氯吡啶氧基)]苯（TCPOBOP）可诱导小鼠肝脏肿大，伴有肝细胞肥大和增殖。然而，CAR激活诱导的肝脏肿大是否可逆以及逆转过程中的组织学变化仍不清楚。在本研究中，给C57BL/6小鼠连续5天给予TCPOBOP，并在停药后的不同时间点处死。结果表明，TCPOBOP诱导的肝脏肿大需要很长时间才能逆转，停药120天后，TCPOBOP组的肝重与体重比仍显著高于溶剂对照组。β-连环蛋白和细胞周期蛋白D1染色显示肝细胞大小和增殖细胞减少。为了研究其中涉及的机制，我们检测了与肝脏肿大和肝脏再生终止相关的蛋白质。结果表明，Yes相关蛋白、C-MYC、β-连环蛋白、叉头框M1和肝细胞核因子4α在TCPOBOP停药后发生了显著变化，并在逆转过程的不同阶段发挥作用。此外，我们建立了一种超高效液相色谱-串联质谱法来定量TCPOBOP浓度。结果表明TCPOBOP在肝脏中长期滞留，持续激活CAR，并导致停药后持续的肝脏肿大。总体而言，这些发现揭示了CAR激活诱导的肝脏肿大的可逆性，并为CAR作为药物靶点的安全性提供了新的见解。此外这些结果强调了考虑TCPOBOP在肝脏中长期蓄积以避免潜在不良反应和实验偏差的重要性。重要声明：本研究表明，1,4-双[2-(3,5-二氯吡啶氧基)]苯（TCPOBOP）在肝脏中的滞留是停药后持续激活组成型雄烷受体（CAR）和持续肝脏肿大的根本原因，这为CAR作为药物靶点的安全性提供了新的数据，并为CAR在肝脏疾病中的调控提供了新的见解。值得注意的是，当使用TCPOBOP作为小鼠CAR激动剂时，应注意其在肝脏中的蓄积和滞留，以避免潜在的实验偏差和不良反应。
