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).