Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells

Tamara Heintze,Ute Hofmann,Kathrin Klein,Ulrich M Zanger

doi:10.1038/s41598-020-79952-1

Tamara Heintze, Ute Hofmann + Show 2 more

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https://doi.org/10.1038/s41598-020-79952-1

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Abstract

HepaRG cells are increasingly accepted as model for human drug metabolism and other hepatic functions. We used lentiviral transduction of undifferentiated HepaRG cells to deliver Cas9 and two alternative sgRNAs targeted at NADPH:cytochrome P450 oxidoreductase (POR), the obligate electron donor for microsomal cytochromes P450 (CYP). Cas9-expressing HepaRGVC (vector control) cells were phenotypically similar to wild type HepaRG cells and could be differentiated into hepatocyte-like cells by DMSO. Genetic POR-knockout resulted in phenotypic POR knockdown of up to 90% at mRNA, protein, and activity levels. LC–MS/MS measurement of seven CYP-activities showed differential effects of POR-knockdown with CYP2C8 being least and CYP2C9 being most affected. Further studies on cytochrome b5 (CYB5), an alternative NADH-dependent electron donor indicated particularly strong support of CYP2C8-dependent amodiaquine N-deethylation by CYB5 and this was confirmed by genetic CYB5 single- and POR/CYB5 double-knockout. POR-knockdown also affected CYP expression on mRNA and protein level, with CYP1A2 being induced severalfold, while CYP2C9 was strongly downregulated. In summary our results show that POR/NADPH- and CYB5/NADH-electron transport systems influence human drug metabolizing CYPs differentially and differently than mouse Cyps. Our Cas9-expressing HepaRGVC cells should be suitable to study the influence of diverse genes on drug metabolism and other hepatic functions.

Highlights

Application of genome editing technologies, in particular CRISPR/Cas[9] to study human hepatic cytochrome P450 (CYP)-dependent drug metabolism and drug transport functions has been hampered by the limitations of the few cell models that reliably reflect relevant liver functions[1, 2]
As P450 oxidoreductase (POR)-knockout effects on cytochromes P450 (CYP)-activities may depend on CYP genotype, we determined major alleles for the CYPs included in our study (Table 1)
While previously described genotypes for CYPs 2C9 (*2/*2), 2C19 (*1/*1), 2D6 (*2/*9) and 3A5 (*3/*3) were verified[49], we found that HepaRG cells are homozygous for CYP2C8*3 and heterozygous for CYP2B6*6 (Table 1)

Summary

Introduction

Application of genome editing technologies, in particular CRISPR/Cas[9] to study human hepatic cytochrome P450 (CYP)-dependent drug metabolism and drug transport functions has been hampered by the limitations of the few cell models that reliably reflect relevant liver functions[1, 2]. As shown by genome-wide gene expression profiling studies, HepaRG cells are more similar to primary hepatocytes and human liver tissue than any other human liver cell line[5]. HepaRG cells demonstrate stable and functional expression of several CYP enzymes as well as phase 2 enzymes, drug transporters, and liver-specific transcription factors including dedicated ligand-activated nuclear receptors and are widely accepted as a highly useful model to study various aspects of drug metabolism, transport and its regulation[6,7,8,9,10,11,12]. Phenotype of variant alleles Not known[31] Higher inducibility Decreased function Increased in vitro function[59] Decreased function No variant allele detected Decreased function (*9) No variant allele detected Splicing defect, severely decreased expression

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