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

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 HepaRG^VC (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 HepaRG^VC cells should be suitable to study the influence of diverse genes on drug metabolism and other hepatic functions.

SUBMITTER: Heintze T

PROVIDER: S-EPMC7806635 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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

Heintze Tamara T Klein Kathrin K Hofmann Ute U Zanger Ulrich M UM

Scientific reports 20210113 1

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 HepaRG<sup>VC</sup> (vector control) cells were phenotypically similar to wild type HepaRG cells and could be differentiated into hepatocyte-like cell ...[more]

PMID: 33441761

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Project description:Membrane cytochrome b5 reductase is a pleiotropic oxidoreductase that uses primarily soluble reduced nicotinamide adenine dinucleotide (NADH) as an electron donor to reduce multiple biological acceptors localized in cellular membranes. Some of the biological acceptors of the reductase and coupled redox proteins might eventually transfer electrons to oxygen to form reactive oxygen species. Additionally, an inefficient electron transfer to redox acceptors can lead to electron uncoupling and superoxide anion formation by the reductase. Many efforts have been made to characterize the involved catalytic domains in the electron transfer from the reduced flavoprotein to its electron acceptors, such as cytochrome b5, through a detailed description of the flavin and NADH-binding sites. This information might help to understand better the processes and modifications involved in reactive oxygen formation by the cytochrome b5 reductase. Nevertheless, more than half a century since this enzyme was first purified, the one-electron transfer process toward potential electron acceptors of the reductase is still only partially understood. New advances in computational analysis of protein structures allow predicting the intramolecular protein dynamics, identifying potential functional sites, or evaluating the effects of microenvironment changes in protein structure and dynamics. We applied this approach to characterize further the roles of amino acid domains within cytochrome b5 reductase structure, part of the catalytic domain, and several sensors and structural domains involved in the interactions with cytochrome b5 and other electron acceptors. The computational analysis results allowed us to rationalize some of the available spectroscopic data regarding ligand-induced conformational changes leading to an increase in the flavin adenine dinucleotide (FAD) solvent-exposed surface, which has been previously correlated with the formation of complexes with electron acceptors.

Project description:Heterocyclic and aromatic amine carcinogens are thought to lead to tumor initiation via the formation of DNA adducts, and bioactivation to arylhydroxylamine metabolites is necessary for reactivity with DNA. Carcinogenic arylhydroxylamine metabolites are cleared by a microsomal, NADH-dependent, oxygen-insensitive reduction pathway in humans, which may be a source of interindividual variability in response to aromatic amine carcinogens. The purpose of this study was to characterize the identity of this reduction pathway in human liver. On the basis of our findings with structurally similar arylhydroxylamine metabolites of therapeutic drugs, we hypothesized that the reductive detoxification of arylhydroxylamine carcinogens was catalyzed by NADH cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5). We found that reduction of the carcinogenic hydroxylamines of the aromatic amine 4-aminobiphenyl (4-ABP; found in cigarette smoke) and the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP; found in grilled meats) was indeed catalyzed by a purified system containing only human b5R and cyt b5. Specific activities were 56-346-fold higher in the purified system as compared to human liver microsomes (HLM), with similar Michaelis-Menten constants (K(m) values) in both systems. The stoichiometry for b5R and cyt b5 that yielded the highest activity in the purified system was also similar to that found in native HLM ( approximately 1:8 to 1:10). Polyclonal antisera to either b5R or cyt b5 significantly inhibited N-hydroxy-4-aminobiphenyl (NHOH-4-ABP) reduction by 95 and 89%, respectively, and immunoreactive cyt b5 protein content in individual HLM was significantly correlated with individual reduction of both NHOH-4-ABP and N-hydroxy-PhIP (NHOH-PhIP). Finally, titration of HLM into the purified b5R/cyt b5 system did not enhance the efficiency of reduction activity. We conclude that b5R and cyt b5 are together solely capable of the reduction of arylhydroxylamine carcinogens, and we further hypothesize that this pathway may be a source of individual variability with respect to cancer susceptibility following 4-ABP or PhIP exposure.

Dataset Information

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

Publications

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

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