Abstract
NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.
Highlights
NADPH:Cytochrome P450 reductase (POR) is a ubiquitous microsomal electron transport protein essential for cytochrome P450 (CYP) mediated oxidative metabolism of xenobiotics and physiological processes
In addition to CYPs 2E1 and 2C9, expression of two CYP isoforms involved in bile acid synthesis, CYP7A1 and 8B1, were among the four strongest decreased phase I enzymes with up to 0.28-fold lower expression levels. mRNA expression of two other CYPs 4A11 and 51 involved in fatty acid metabolism and cholesterol biosynthesis, respectively, was decreased, albeit CYP51 was only marginally affected in HepaRG−P450 oxidoreductase (POR)#2 cells
The aim of the present study was an in-depth analysis of effects of deminished POR levels in the HepaRG cells with focus on genes involved in the absorption, distribution, metabolism, and excretion (ADME) and related regulators
Summary
NADPH:Cytochrome P450 reductase (POR) is a ubiquitous microsomal electron transport protein essential for cytochrome P450 (CYP) mediated oxidative metabolism of xenobiotics and physiological processes. This includes biosynthesis of numerous endogenous compounds especially steroids such as cholesterol and bile acids (Riddick et al, 2013). Effects of Por knockout in liver on drug as well as endogenous metabolism were extensively studied in mouse models (Gu et al, 2003; Henderson et al, 2003; Wang et al, 2005; Weng et al, 2005; Finn et al, 2009; Cheng et al, 2014b). In contrast mice are displaying a more hydrophilic complement of bile acid species (Li and Dawson, 2019)
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