Marked differences in thyroxine (T4) metabolism following in vitro exposure of Wistar rat and human hepatocytes to several reference CAR/PXR nuclear receptor activators

Our study builds upon previous findings (Baze et al., 2024) by investigating species differences in thyroxine (T4) metabolism regulation by CAR/PXR activators using cryopreserved primary Wistar rat hepatocytes (PRH) and human hepatocytes (PHH) in 2D-sandwich over a 7-day treatment period. Daily exposure of PRH to phenobarbital, 5-Pregnen-3β-ol-20-one-16α‑carbonitrile (PCN) or dexamethasone increased T4 clearance over the last 24 h exposure (up to 60 %, 79 % and 67 % over control, respectively) and secretion of T4-glucuronide (T4-G; up to 463, 661 and 545 pmol/10⁶ cells over control, respectively). Effects were concentration-dependent for phenobarbital and PCN and highest at the lowest concentration for dexamethasone, while rifampicin barely affected T4 clearance and T4-G secretion. None of the compounds, at any tested concentration, affected these parameters in PHH. Additionally, mRNA expression data were consistent with the species-specific and concentration-dependent regulation of phase I Cyp/CYP, phase II Ugt/UGT and phase III Mrp2/MRP2 pathways occurring in rat and human liver following CAR/PXR activation. T4-UGT relative activity increased in PRH only, specifically by PCN, dexamethasone and phenobarbital. The comparison of PRH and PHH responses to compounds represents an important step towards using in vitro methods to reduce animal testing. We recommend using relative T4-UGT activity thresholds observed in PRH as benchmarks for defining compound-related effects across species, helping determine the human relevance of thyroid effects in rodents.