Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver

Abstract

Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s.

Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver

“…The ablation cassette (velocigene cassette [bacterial ␤-galactosidase-polyadenylation signal-loxP (locus of X over P1) site-human ubiquitin C gene Preparation of Microsomes-Microsomes were prepared from Abcb6 ϩ/ϩ , Abcb6 ϩ/Ϫ , and Abcb6 Ϫ/Ϫ mice liver tissues as described (28). Briefly, 0.3-0.5 g of liver tissue was homogenized in homogenization buffer (0.154 M KCl, 0.25 M sucrose in 0.05 M phosphate buffer, pH 7.5) and centrifuged at 17,000 ϫ g for 30 min at 4°C.…”

“…Immunoblotting-Western blot analysis of mitochondrial and microsomal proteins was carried out as previously described (28,29). Polyclonal primary antibodies were used to detect P450 oxidoreductase (catalog no.…”

“…Mass Spectrometry-based P450 Assay-The CYP activities, Cyp3a11 (midazolam to hydroxymidazolam), Cyp2b6 (bupropion to hydroxybupropion), Cyp2e1 (chlorzoxazone to hydroxychlorzoxazone), and Cyp1a2 (melatonin to hydroxymelatonin), were determined in microsomes isolated from mouse liver, using probe substrate metabolism assays as described (28). Briefly, the incubation reaction consisted of 3 M midazolam, 5 M chlorozoxazone, 50 M bupropion, or 30 M melatonin with 0.03 mg of mouse liver microsomes, in a final volume of 200 l of 1ϫ phosphate-buffered saline (pH 7.4).…”

Therapeutic Protein Drug–Drug Interactions: Navigating the Knowledge Gaps–Highlights from the 2012 AAPS NBC Roundtable and IQ Consortium/FDA Workshop

“…IL-6 is an important cytokine in the human liver (14) and one of its primary effects is the secretion of CRP by hepatocytes (15). Supplementary genomic data (5,16) and some clinical studies (2,(17)(18)(19)(20) have all revealed a relationship between CYP3A and CRP or IL-6. Data were presented where CRP changes in 19 selected inflammatory disease clinical studies were compared to CRP changes observed in the tocilizumab-simvastatin study in patients with RA.…”

Human monocyte-derived cells with individual hepatocyte characteristics: a novel tool for personalized in vitro studies

“…On the first glance, primary hepatocytes possess all the required activities and short-term studies can be performed with valid results. 11,12 Yet, primary hepatocytes dedifferentiate in culture dependent on the used culture conditions (plated monolayer, sandwich culture and suspension culture). This dedifferentiation process results in a loss in CYP450 activities and inducibility within few days.…”