Pregnane X receptor mediates steatotic effects of propiconazole and tebuconazole in human liver cell lines

Triazoles are commonly used fungicides which show liver toxicity in rodent studies. While hepatocellular hypertrophy is the most prominent finding, some triazoles have also been reported to cause hepatocellular steatosis. The aim of our study was to elucidate molecular mechanisms of triazole-mediated steatosis. Therefore, we used the two triazoles propiconazole (Pi) and tebuconazole (Te) as test compounds in in vitro assays using the human hepatocarcinoma cell lines HepG2 and HepaRG. Triglyceride accumulation was measured using the Adipored assay and by a gas-chromatographic method. Reporter gene analyses were used to assess the ability of Pi and Te to activate nuclear receptors, which are described as the molecular initiators in the adverse outcome pathway (AOP) for liver steatosis. The expression of steatosis-associated genes was investigated by RT-PCR. Mechanistic analyses of triazole-mediated steatosis were performed using HepaRG subclones that are deficient in different nuclear receptors. Pi and Te both interacted with the constitutive androstane receptor (CAR), the peroxisome proliferator-activated receptor alpha (PPARα), and the pregnane X receptor (PXR). Both compounds induced expression of steatosis-related genes and cellular triglyceride accumulation. The knockout of PXR in HepaRG cells, but not the CAR knockout, abolished triazole-induced triglyceride accumulation, thus underlining the crucial role of PXR in hepatic steatosis resulting from exposure to these fungicides. In conclusion, our findings provide new insight into the molecular mechanisms of steatosis induction by triazole fungicides and identify PXR as a critical mediator of this process.