Quantifying expression and metabolic activity of genes regulated by pregnane X receptor in primary human hepatocyte spheroids

Xenoreceptors of the nuclear receptor superfamily, such as pregnane X receptor (PXR), are liver-enriched ligand-activated transcription factors regarded as crucial sensors in xenobiotic exposure and detoxification. PXR controls transcription of many drug-handling genes and influx/efflux transporters, thus playing a crucial role in drug metabolism and excretion. Liver functions have been studied using primary human hepatocytes (PHHs), which, when conventionally cultured, undergo rapid de-differentiation, leaving them unsuitable for long-term studies. Recently, 3D PHHs called spheroids have emerged as an in vitro model that is similar to in vivo hepatocytes regarding phenotype and function and represents the first in vitro model to study the long-term regulation of drug-handling genes by PXR. In this study, we used mathematical modelling to analyze the long-term activation of PXR in 3D PHHs through expression kinetics of three key PXR-regulated drug-metabolizing enzymes, CYP3A4, CYP2C9, and CYP2B6 and the P-glycoprotein efflux transporter encoding gene, MDR1. PXR action in 3D PHHs was induced by the antibiotic rifampicin at two clinically relevant concentrations. The results confirmed that high rifampicin concentrations activated PXR nearly to its full capacity. The analysis indicated the highest PXR-induced transcription rate constant for CYP2B6. The rate constant dictating mRNA degradation associated with activated PXR was highest for CYP3A4. Moreover, we measured the metabolic activity of CYP3A4, CYP2C9, and CYP2B6 and quantified their metabolic rate constants. Metabolic activity rate constant of CYP3A4 was found to be the highest whereas that of CYP2B6 was found to be the lowest among the studied enzymes. Our results provide important insight into the regulation of PXR-target genes in 3D PHHs and show that mRNA expression and metabolic activity data can be combined with quantitative analysis to reveal the long-term action of PXR and its effects on drug-handling genes.Author summary: Pregnane X receptor (PXR) is a ligand-activated transcription factor which is essential for xenobiotic metabolism in the liver and intestine. Drug-handling genes, such as CYP3A4, CYP2C9, and CYP2B6 and the efflux transporters, such as P-glycoprotein encoded by the MDR1 gene, are controlled by PXR to facilitate metabolism and efflux of the majority of currently used drugs. The function of drug-handling genes has principally been studied in conventionally cultured 2D primary human hepatocytes (PHHs) which are not suitable for studying the long-term response to PXR activation due to early de-differentiation changes in the PHH-specific phenotype. There is a need to describe and quantify the processes involved in the long-term responses in the target gene regulation due to PXR activation to better understand drug metabolism regulation. The significance of our research is in quantifying the activation of PXR through pairing quantitative analysis and experimental data obtained from 3D PHH spheroids which allowed us to study the liver functions for long time periods. This work enhances our understanding of the responses to PXR activation at the level of mRNA expression and metabolic activity of drug-handling PXR-controlled genes.