JCM-2373852 Caco-2-HepaRG Cell Culture Media Optimisation & LC-MSMS analysis of APAP metabolism during Caco-2 cell culture

The gut–liver axis is defined by dietary and environmental communication between the gut and the microbiome and the liver and its redox and immune systems, the overactivation of which can lead to hepatic injury. We used media preconditioning to mimic some aspects of the enterohepatic circulation by treating the human Caco-2 intestinal epithelial cell line with 5, 10 and 20 mM paracetamol (N-acetyl-para-aminophenol; APAP) for 24 h, after which cell culture supernatants were transferred to differentiated human hepatic HepaRG cells for a further 24 h. Cell viability was assessed by mitochondrial function and ATP production, while membrane integrity was monitored by cell impedance. Metabolism by Caco-2 cells was determined by liquid chromatography with tandem mass spectrometry. Caco-2 cell viability was not affected by APAP, while cell membrane integrity and tight junctions were maintained and became tighter with in-creasing APAP concentrations, suggesting a reduction in the permeability of the intestinal epithelium. During 24 h incubation, Caco-2 cells metabolised 64–68% of APAP, leaving 32–36% of intact starting compound to be transferred to HepaRG cells. When cultured with Caco-2-preconditioned medium, HepaRG cells also showed no loss of cell viability or membrane integrity, completely in contrast to direct treatment with APAP, which resulted in a rapid loss of cell viability and membrane integrity and, ultimately, cell death. Thus, the pre-metabolism of APAP could mitigate previously observed hepatotoxicity to hepatic tight junctions caused by direct exposure to APAP. These observations could have important implications for the direct exposure of hepatic parenchyma to APAP, administered via the intravenous route. The dataset relates to the upcoming publication Morgan K, Morley SD, Raja AK, Vandeputte M, Samuel K, Waterfall M, Homer NZM, Waterfall M, Hayes PC, Fallowfield JA, Plevris JN (2023). "Metabolism of Acetaminophen by Enteric Epithelial Cells Mitigates Hepatocellular Toxicity In Vitro". Journal of Clinical Medicine (accepted for publication).

肠-肝轴（gut–liver axis）的定义为肠道与微生物群、肝脏与其氧化还原及免疫系统之间的膳食与环境信号交流，其过度激活可引发肝损伤。

本研究采用培养基预条件化策略，通过用5、10及20 mM浓度的对乙酰氨基酚（paracetamol，又名N-乙酰对氨基酚，缩写APAP）处理人类Caco-2肠上皮细胞系24小时，以模拟肠肝循环（enterohepatic circulation）的部分过程；随后将细胞培养上清转移至已分化的人类肝HepaRG细胞中，继续培养24小时。

采用线粒体功能与ATP生成水平评估细胞活力，通过细胞阻抗监测细胞膜完整性；利用液相色谱-串联质谱法检测Caco-2细胞的代谢情况。

结果显示，APAP对Caco-2细胞活力无显著影响，且其细胞膜完整性与紧密连接（tight junctions）得以维持，随APAP浓度升高愈发紧密，提示肠上皮通透性降低。在24小时孵育过程中，Caco-2细胞可代谢64%~68%的APAP，剩余32%~36%的原型化合物被转移至HepaRG细胞。

与直接用APAP处理的结果截然不同，经Caco-2预条件培养基培养的HepaRG细胞未出现细胞活力或细胞膜完整性丧失——直接APAP处理会快速导致细胞活力与细胞膜完整性受损，最终引发细胞死亡。由此可见，APAP的预代谢可缓解此前观察到的直接暴露于APAP所致的肝紧密连接肝细胞毒性（hepatocellular toxicity）。

上述发现对于经静脉途径给药的肝实质（hepatic parenchyma）直接暴露于APAP的情况具有重要参考价值。

本数据集关联即将发表的论文：Morgan K, Morley SD, Raja AK, Vandeputte M, Samuel K, Waterfall M, Homer NZM, Waterfall M, Hayes PC, Fallowfield JA, Plevris JN (2023). "Metabolism of Acetaminophen by Enteric Epithelial Cells Mitigates Hepatocellular Toxicity In Vitro", 发表于《Journal of Clinical Medicine》（已接收待刊）。