Enzymes In Human Hepatocytes Research Articles

Age-dependent changes in cytochrome P450 abundance and composition in human liver.

Cytochrome P450 (CYP) superfamily represents the major drug metabolizing enzymes responsible for metabolizing over 65% of therapeutic drugs, including those for pediatric use. CYP-ontogeny based physiologically-based pharmacokinetic (PBPK) models have emerged as useful tools to mechanistically extrapolate adult pharmacokinetic data to children. However, these models integrate physiological differences in pediatric population including age-dependent differences in the abundances of CYP enzymes. Conventionally, developmental changes in CYP enzymes have been reported using protein abundance and activity data from subcellular fractions such as microsomes, which is prone to high technical variability. Similarly, the available pediatric pharmacokinetic data suffer from the lack of specific CYP substrates, especially in younger children. In the present study, we utilized viable hepatocytes from 50 pediatric (age, day 1- 18 yr) and 8 adult human donors and carried out global proteomics-based quantification of all major hepatic CYP enzymes, including orphan enzymes that have not been studied previously. While CYPs 2B6, 3A5, 4A11, 4F3, and 4V2 did not show significant association with age, all other quantified isoforms either increased or decreased with age. CYPs 1A2, 2C8, 2C18, and 2C19 were absent or barely detected in the neonatal group, while CYP3A7 was the highest in this group. The >1-2 yr age-group showed the highest total abundance of all CYP enzymes. The age-dependent differences in CYP enzymes reported in this study can be used to develop ontogeny-based PBPK models, which in turn can help improve pediatric dose-prediction based on adult dosing, leading to safer drug pharmacology in children. Significance Statement We quantified age-dependent differences in the abundances of hepatic CYP enzymes using a large set of viable pediatric and adult hepatocytes using quantitative global proteomics. We report for the first time the ontogeny in the abundance of CYP enzymes in human hepatocytes, especially, orphan CYPs 20A1, 27A1, 51A1, 7B1, and 8B1 and the CYP4 subfamily of enzymes. Our study provides important data about CYP ontogeny that can be used for the better prediction of pediatric pharmacokinetics using physiologically-based pharmacokinetic modeling.

Evaluation of Human Hepatocyte Drug Metabolism Carrying High-Risk or Protection-Associated Liver Disease Genetic Variants.

Metabolic-dysfunction-associated steatotic liver disease (MASLD), which affects 30 million people in the US and is anticipated to reach over 100 million by 2030, places a significant financial strain on the healthcare system. There is presently no FDA-approved treatment for MASLD despite its public health significance and financial burden. Understanding the connection between point mutations, liver enzymes, and MASLD is important for comprehending drug toxicity in healthy or diseased individuals. Multiple genetic variations have been linked to MASLD susceptibility through genome-wide association studies (GWAS), either increasing MASLD risk or protecting against it, such as PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438. As the impact of genetic variants on the levels of drug-metabolizing cytochrome P450 (CYP) enzymes in human hepatocytes has not been thoroughly investigated, this study aims to describe the analysis of metabolic functions for selected phase I and phase II liver enzymes in human hepatocytes. For this purpose, fresh isolated primary hepatocytes were obtained from healthy liver donors (n = 126), and liquid chromatography-mass spectrometry (LC-MS) was performed. For the cohorts, participants were classified into minor homozygotes and nonminor homozygotes (major homozygotes + heterozygotes) for five gene polymorphisms. For phase I liver enzymes, we found a significant difference in the activity of CYP1A2 in human hepatocytes carrying MBOAT7 (p = 0.011) and of CYP2C8 in human hepatocytes carrying PNPLA3 (p = 0.004). It was also observed that the activity of CYP2C9 was significantly lower in human hepatocytes carrying HSD17B13 (p = 0.001) minor homozygous compared to nonminor homozygous. No significant difference in activity of CYP2E1, CYP2C8, CYP2D6, CYP2E1, CYP3A4, ECOD, FMO, MAO, AO, and CES2 and in any of the phase II liver enzymes between human hepatocytes carrying genetic variants for PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438 were observed. These findings offer a preliminary assessment of the influence of genetic variations on drug-metabolizing cytochrome P450 (CYP) enzymes in healthy human hepatocytes, which may be useful for future drug discovery investigations.

A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentrationduring the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2µM), estradiol (0.3µM), estriol (0.8µM), estrone (0.2µM), 17α-hydroxyprogesterone (0.1µM), and human growth hormone (0.005µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.

Expression and functional activity of cytochrome P450 enzymes in human hepatocytes with sustainable reproducibility for in vitro phenotyping studies.

Primary human hepatocytes are an essential in vitro tool for evaluating drug metabolism, drug-drug interactions, and hepatotoxicity. This model is considered as the gold standard in matter of DMPK studies in both industrial and academic research. The primary human hepatocytes are used either in suspension or in monolayer, as fresh or frozen cells. However, the use of this model is limited due to the lack of availability, rapid loss of functionality, high cost as well as the variable hepatocyte plating efficiencies in culture and the limited stock of hepatocytes derived from the same origin. Chimeric TK-NOG mice with humanized livers (humanized liver mice) are an attractive platform for drug metabolism and toxicity, which were produced by transplanting human hepatocytes into immunodeficient mice with injured livers. Here, we show that, using humanized mouse liver, in vivo human hepatocyte repopulation was over ~100-fold enabling the continuous and abundant use of human hepatocytes of the same origin and improving their plateability. In our latest cell preparations, hepatocytes isolated from humanized liver mice (Hu-Liver cells) exhibited high purity (ratio of HLA-positive cells: 92±3%), good viability (75±12%), and yield (1.0×108 cells/mouse). Human hepatic drug metabolizing enzymes, transporters, and nuclear receptors genes were expressed in humanized mouse liver. Drug-metabolizing activities in Hu-Liver cells were comparable to or higher than those in primary human hepatocytes. An extensive P450-dependent human drug metabolism was observed in Hu-Liver cells. CYP1A2, CYP2B6, and CYP3A4/5 activities/mRNA in Hu-Liver cells were induced by the hepatocyte exposure to typical human P450 inducers, omeprazole, phenobarbital, and rifampicin, respectively. Finally, Human albumin secretion and CYP3A-mediated drug oxidation activity were maintained over 4-weeks. Altogether, the expression level of pharmacokinetics-related genes, enzyme activity, human-typed drug metabolism, and inducibility of P450 in Hu-Liver cells make from humanized mouse liver a relevant and robust model for in vitro preclinical studies, including drug metabolism, pharmacokinetics, and toxicology studies.

Downregulation of CYP1A2, CYP2B6, and CYP3A4 in Human Hepatocytes by Prolyl Hydroxylase Domain 2 Inhibitors via Hypoxia-Inducible Factor-α Stabilization.

Hypoxia-inducible factor (HIF) is associated with the expression of CYP, but the underlying mechanism remains uncertain. In this study, we investigated the effect of HIF-α stabilization caused by novel prolyl hydroxylase domain (PHD) 2 inhibitors, which are HIF-α stabilizers that mimic hypoxia, on the expressions of CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes. An mRNA expression analysis of human hepatocytes treated with PHD2 inhibitors for 72 hours showed the downregulation of genes encoding CYP1A2, CYP2B6, and CYP3A4. The mRNA repressions were accompanied with an increase in erythropoietin protein, a marker of HIF-α stabilization, indicating that HIF-α stabilization was involved in the downregulation of the CYP isoforms. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors [aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoid X receptor (RXR)] in human hepatocytes treated with the HIF-α stabilizers. As a result, the mRNA level of AhR did not decrease, although ARNT expression was repressed. On the other hand, the mRNA expression levels of CAR, PXR, and RXR were repressed and closely associated with those of CYP2B6 and CYP3A4. Although the underlying mechanism of the downregulation for CYP1A2 remains unclear, the presently reported results suggest that the downregulation of CYP2B6 and CYP3A4 via HIF-α stabilization is caused by a decrease in the expressions of CAR, PXR, and RXR. SIGNIFICANCE STATEMENT: We showed that hypoxia-inducible factor (HIF)-α stabilization downregulates CYP1A2, CYP2B6, and CYP3A4 using prolyl hydroxylase domain 2 inhibitors, which are HIF-α stabilizers, as a new tool to mimic hypoxia in human hepatocytes. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors. Our findings would contribute to a better understanding of the hypoxia-triggered regulatory mechanism of drug-metabolizing enzymes in human hepatocytes.

Effects of monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and interferon-α2a on P450 enzymes in human hepatocytes in vitro.

Some immunomodulatory agents stimulate the release of cytokines capable of suppressing P450 enzymes and potentially affecting pharmacokinetics of coadministered medications. Cytokines released in response to an immunomodulator in the blood ex vivo can be used to screen for the potential for drug‐drug interactions. Tilsotolimod, an investigational agonist of Toll‐like receptor 9, stimulated the release of macrophage chemoattractant protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), and interferon‐α2a (INF‐α2a) in blood obtained from healthy donors. Although tilsotolimod did not directly affect CYP1A2, CYP2B6, or CYP3A4 expression or activity, the cytokines stimulated by the drug reduced CYP1A2 and CYP2B6 enzyme activities in cultured human hepatocytes. This study sought to identify which cytokines were responsible for tilsotolimod's indirect effects on P450 enzymes in vitro. A 72‐h treatment with recombinant human chemokines MCP‐1 and MIP‐1α did not alter CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP3A4, or signal transducer and activator of transcription 1 (STAT1) mRNA expression or CYP1A2, CYP2B6, or CYP3A4/5 enzyme activity in cocultures of human hepatocytes and Kupffer cells. INF‐α2a, at 2.5 ng/mL but not at the lower concentrations applied to the cells, increased CYP1A2 and STAT1 mRNA by 2.4‐ and 5.2‐fold, respectively, and reduced CYP2B6 enzyme activity to 46% of control. This study established that INF‐α2a, but not MCP‐1 or MIP‐1α, mediated tilsotolimod effects on CYP1A2 and CYP2B6 expression in human hepatocytes.

Expression and inducibility of cytochrome P450s in human hepatocytes isolated from chimeric mice with humanised livers

The evaluation of drug-mediated cytochrome P450 (P450) induction using human hepatocytes is important for predicting drug interactions. In this study, we prepared hepatocytes from chimeric mice with humanised livers (Hu-Liver mice) and evaluated the expression and inducibility of P450s in these hepatocytes.Up to 95% of the Hu-Liver cells stained positive for human leukocyte antigen and the mean viability exceeded 85% (n = 10). Monolayer-cultured Hu-Liver cells displayed a similar morphology to cultures of the corresponding human hepatocytes used as transplantation donors.The mRNA expression levels in Hu-Liver cells of 16 P450 forms belonging to P450 subfamilies 1–4 correlated well with the expression levels of the same enzymes in human hepatocytes. The variations in individual P450 mRNA levels between Hu-Liver cells and the corresponding human hepatocytes were within five-fold for 13 P450 forms.The production of 6β-hydroxytestosterone in Hu-Liver cells was significantly increased (p < .05) following treatment with the CYP3A inducer, rifampicin.Hu-Liver cells have characteristics similar to those of human hepatocytes in terms of mRNA expression levels and the inducibility of the various P450 forms. Thus, Hu-Liver cells can potentially be used for in vitro drug-mediated induction assays of human hepatic P450s.

The effects of drugs with immunosuppressive or immunomodulatory activities on xenobiotics-metabolizing enzymes expression in primary human hepatocytes

In this paper we investigated the effects of several drugs used in transplant medicine, i.e. cyclosporine A, tacrolimus, rapamycin, everolimus, mycophenolate mofetil, fluvastatin and rosuvastatin, on the expression of major drug-metabolizing enzymes in human hepatocytes. Moreover, we tested the ability of these drugs to affect transcriptional activity of glucocorticoid (GR) and aryl hydrocarbon receptor (AhR).We found that most of tested compounds did not induce expression of CYP1A1/1A2/3A4/2A6/2B6/2C9 mRNAs in human hepatocytes. Slight induction was observed for CYP2A6/2C9 mRNAs and CYP2A6 protein in the rapamycin-treated hepatocytes. Decrease of CYP2A6 and CYP2B6 proteins was observed in rosuvastatin-treated cells. Mycophenolate mofetil antagonized the effects of dexamethasone on GR but it potentiated the action of dioxin on AhR. Induction of CYP1A1 mRNA in HepG2 cells by dioxin was modestly antagonized by mycophenolate mofetil, while the induction by benzo[a]pyren or S-omeprazole was significantly potentiated by this drug.In general, tested compounds can be considered safe in the terms of possible drug–drug interaction caused by induction of drug-metabolizing cytochromes P450. Nevertheless, mycophenolate mofetil is of possible concern and its combination with drugs, environmental pollutants or food constituents, which activate AhR, may represent a significant toxicological risk.

Activating and Inhibitory Functions of WNT/β-Catenin in the Induction of Cytochromes P450 by Nuclear Receptors in HepaRG Cells.

The WNT/β-catenin signaling pathway has been identified as an important endogenous regulator of hepatic cytochrome P450 (P450) expression in mouse liver. In particular, it is involved in the regulation of P450 expression in response to exposure to xenobiotic agonists of the nuclear receptors constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and Nrf2. To systematically elucidate the effect of the WNT/β-catenin pathway on the regulation and inducibility of major human P450 enzymes, HepaRG cells were treated with either the WNT/β-catenin signaling pathway agonist, WNT3a, or with small interfering RNA directed against β-catenin, alone or in combination with a panel of activating ligands for AhR [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)], CAR [6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO)], pregnane X receptor (PXR) [rifampicin], and peroxisome proliferator-activated receptor (PPAR) α [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY14,643)]. Assessment of P450 gene expression and enzymatic activity after downregulation or activation of the WNT/β-catenin pathway revealed a requirement of β-catenin in the AhR-, CAR-, and PXR-mediated induction of CYP1A, CYP2B6 and CYP3A4 (for CAR and PXR), and CYP2C8 (for PXR) gene expression. By contrast, activation of the WNT/β-catenin pathway prevented PPARα-mediated induction of CYP1A, CYP2C8, CYP3A4, and CYP4A11 genes, suggesting a dominant-negative role of β-catenin in PPARα-mediated regulation of these genes. Our data indicate a significant effect of the WNT/β-catenin pathway on the regulation of P450 enzymes in human hepatocytes and reveal a novel crosstalk between β-catenin and PPARα signaling pathways in the regulation of P450 expression.

Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5–50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1′-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans.

Anti-CD28 monoclonal antibody-stimulated cytokines released from blood suppress CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes in vitro.

Like most infections and certain inflammatory diseases, some therapeutic proteins cause a cytokine-mediated suppression of hepatic drug-metabolizing enzymes, which may lead to pharmacokinetic interactions with small-molecule drugs. We propose a new in vitro method to evaluate the whole blood-mediated effects of therapeutic proteins on drug-metabolizing enzymes in human hepatocytes cocultured with Kupffer cells. The traditional method involves treating hepatocyte cocultures with the therapeutic protein, which detects hepatocyte- and macrophage-mediated suppression of cytochrome P450 (P450). The new method involves treating whole human blood with a therapeutic protein to stimulate the release of cytokines from peripheral blood mononuclear cells (PBMCs), after which plasma is prepared and added to the hepatocyte coculture to evaluate P450 enzyme expression. In this study, human blood was treated for 24 hours at 37°C with bacterial lipopolysaccharide (LPS) or ANC28.1, an antibody against human T-cell receptor CD28. Cytokines were measured in plasma by sandwich immunoassay with electrochemiluminescense detection. Treatment of human hepatocyte cocultures with LPS or with plasma from LPS-treated blood markedly reduced the expression of CYP1A2, CYP2B6, and CYP3A4. However, treatment of hepatocyte cocultures with ANC28.1 did not suppress P450 expression, but treatment with plasma from ANC28.1-treated blood suppressed CYP1A2, CYP2B6, and CYP3A4 activity and mRNA levels. The results demonstrated that applying plasma from human blood treated with a therapeutic protein to hepatocytes cocultured with Kupffer cells is a suitable method to identify those therapeutic proteins that suppress P450 expression by an indirect mechanism-namely, the release of cytokines from PBMCs.

Interleukins-12 and -23 Do Not Alter Expression or Activity of Multiple Cytochrome P450 Enzymes in Cryopreserved Human Hepatocytes

Psoriasis is a T-cell-mediated autoimmune disease involving the skin. Two cytokines, interleukin-12 (IL-12) and IL-23 have been shown to play a pivotal role in the pathogenesis of the disease. Ustekinumab (Stelara) is a therapeutic monoclonal antibody (mAb) targeted against the p40 shared subunit of IL-12 and IL-23. Recently the ability of therapeutic proteins (TP) including mAbs that target either cytokines directly (e.g., Pegasys; peginterferon α-2a) or their respective cell surface receptors [e.g., tocilizumab (Actemra); anti IL-6R] to desuppress cytochrome P450 (P450) enzymes in vitro and in the clinic, has been demonstrated. In the present study the ability of IL-12 and IL-23 to suppress multiple P450 enzymes was investigated in vitro using six separate lots of cultured human hepatocytes. Following exposure of 10 ng/ml IL-12 and IL-23 for 48 hours, either alone or in combination, no change in CYP2B6, 2C9, 2C19, or 3A4 gene expression or functional activity was observed. None of the untreated hepatocyte donors showed appreciable expression of the IL-12 or IL-23 receptors. Similar results were seen with whole human liver samples. Exposure of hepatocytes to IL-12 and/or IL-23, known P450 suppressors (IL-6 and tumor necrosis factor-α) or known P450 inducers (β-naphthoflavone, phenobarbital, and rifampicin) did not appreciably alter the expression of the IL-12 and IL-23 receptors either. Finally, in contrast to the positive control IL-6, expression of the acute phase C-reactive protein was unaltered following IL-12 and/or IL-23 treatment. Together, these data suggest a negligible propensity for IL-12 or IL-23 to directly alter P450 enzymes in human hepatocytes.

Assessment of Remogliflozin Etabonate, a Sodium-Dependent Glucose Co-Transporter-2 Inhibitor, as a Perpetrator of Clinical Drug Interactions: A Study on Drug Transporters and Metabolic Enzymes

Type 2 diabetes mellitus is a chronic disease characterized by progressive deterioration of glycemic control and an increased risk of associated complications. Remogliflozin etabonate is the ester prodrug of remogliflozin, a selective sodium-dependent glucose transporter-2 inhibitor that was under development to treat type 2 diabetes. This work investigated the in vitro inhibition of efflux and uptake transporters and cytochrome P450 enzymes by remogliflozin etabonate, remogliflozin and a number of other metabolites. As well, the ability of remogliflozin to induce cytochrome P450 enzymes in human hepatocytes was examined. Remogliflozin etabonate, remogliflozin and GSK279782 (an active pharmacological metabolite of remogliflozin) were inhibitors of organic anion transporting polypeptide-1B1 with IC 50 values of 5.3, 25 and 14 µM, respectively. Remogliflozin etabonate and remogliflozin were inhibitors of organic cation transporter-1 with IC50 values of 43 and 39 µM, respectively. In contrast, remogliflozin etabonate, remogliflozin, GSK279782, and GSK333081 (a metabolite of remogliflozin) were not inhibitors of P-glycoprotein, a number of other renal transporters, or cytochrome P450 enzymes. Further, three circulating glucuronide metabolites found in human plasma were not inhibitors of cytochrome P450 enzymes or organic anion transporters. Remogliflozin etabonate, but not remogliflozin or GSK279782, activated the pregnane X Receptor in vitro. Further studies demonstrated that remogliflozin (up to 100 µM) did not induce cytochrome P450 3A 4 or 1A1 mRNA in human hepatocytes; however a small increase was noted for CYP2B6 mRNA. Combined with pharmacokinetic data from healthy volunteers and diabetic subjects, these in vitro investigations provide a low drug interaction potential for remogliflozin etabonate, remogliflozin and the associated metabolites to be perpetrators of clinical drug interactions. This information has been used to guide the design of clinical studies with remogliflozin etabonate when given with other co-medications.

Examination of extracts from iced-teas on regulatory pathways of drug-metabolizing enzymes in human hepatocytes and cell lines

Examination of extracts from iced-teas on regulatory pathways of drug-metabolizing enzymes in human hepatocytes and cell lines

The Role of Constitutive Androstane Receptor in Oxazaphosphorine-Mediated Induction of Drug-Metabolizing Enzymes in Human Hepatocytes

To investigate the roles of the constitutive androstane receptor (CAR) in cyclophosphamide (CPA)- and ifosfamide (IFO)-mediated induction of hepatic drug-metabolizing enzymes (DME). Induction of DMEs was evaluated using real-time RT-PCR and Western blotting analysis in human primary hepatocyte (HPH) cultures. Activation of CAR, pregnane X receptor (PXR), and aryl hydrocarbon receptor by CPA and IFO was assessed in cell-based reporter assays in HepG2 cells and/or nuclear translocation assays in HPHs. CYP2B6 reporter activity was significantly enhanced by CPA and IFO in HepG2 cells co-transfected with CYP2B6 reporter plasmid and a chemical-responsive human CAR variant (CAR1 + A) construct. Real-time RT-PCR and Western blotting analysis in HPHs showed that both CPA and IFO induced the expressions of CYP2B6 and CYP3A4. Notably, treatment of HPHs with CPA but not IFO resulted in significant nuclear accumulation of CAR, which represents the initial step of CAR activation. Further studies in HPHs demonstrated that selective inhibition of PXR by sulforaphane preferentially repressed IFO- over CPA-mediated induction of CYP2B6. These results provide novel insights into the differential roles of CAR in the regulation of CPA- and IFO-induced DME expression and potential drug-drug interactions.

Markers of Electrophilic Stress Caused by Chemically Reactive Metabolites in Human Hepatocytes

The metabolic activation of a drug to an electrophilic reactive metabolite and its covalent binding to cellular macromolecules is considered to be involved in the occurrence of idiosyncratic drug toxicity (IDT). As a cellular defense system against oxidative and electrophilic stress, phase II enzymes are known to be induced through a Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2/antioxidant response element system. We presumed that it is important for the risk assessment of drug-induced hepatotoxicity and IDTs to observe the biological responses evoked by exposure to reactive metabolites, and then investigated the mRNA induction profiles of phase II enzymes in human hepatocytes after exposure to problematic drugs associated with IDTs, such as ticlopidine, diclofenac, clozapine, and tienilic acid, as well as safe drugs such as levofloxacin and caffeine. According to the results, the problematic drugs exhibited inductive effects on heme oxygenase 1 (HO-1), which contrasted with the safe drugs; therefore, the induction of HO-1 mRNA seems to be correlated with the occurrence of drug toxicity, including IDT caused by electrophilic reactive metabolites. Moreover, glutathione-depletion and cytochrome P450 (P450)-inhibition experiments have shown that the observed HO-1 induction was triggered by the electrophilic reactive metabolites produced from the problematic drugs through P450-mediated metabolic bioactivation. Taken together with our present study, this suggests that HO-1 induction in human hepatocytes would be a good marker of the occurrence of metabolism-based drug-induced hepatotoxicity and IDT caused by the formation of electrophilic reactive metabolites.

Expression and transcriptional activities of nuclear receptors involved in regulation of drug-metabolizing enzymes are not altered by colchicine: Focus on PXR, CAR, and GR in primary human hepatocytes

Recent findings show that colchicine (COL) in submicromolar concentrations downregulates the expression of major drug-metabolizing P450 enzymes in human hepatocytes. Concomitantly, the expression of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) was diminished by COL, whereas expression of glucocorticoid receptor (GR) remained unaltered. A tentative mechanism is perturbation of the GR-PXR/CAR-CYP2/3 signaling cascade, resulting in restricted transcriptional activity of GR receptor by colchicine. In this work we focused on 10-demethylcolchicine (colchiceine; EIN), a structural analogue and a putative metabolite of COL that lacks tubulin-binding activity. We investigated the effects of EIN on the expression of PXR, CAR, and GR receptors in primary cultures of human hepatocytes. In contrast with the effects of COL, EIN does not alter the expression of PXR, CAR, and/or GR receptors mRNAs. In addition, EIN had no effects on transcriptional activities of PXR, CAR, and GR receptors in reporter gene assays using transfected cell lines. Considering that COL and EIN are structurally very close and differ only in their tubulin-binding activity, the data presented imply that the deleterious effects of COL on the GR-PXR/CAR-CYP2/3 cascade are primarily due to perturbation of the microtubule network. Our data support the idea of replacing COL by EIN, which is less toxic and does not interact with xenoreceptors.

Interferon‐inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication†

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Comparative Effects of Fibrates on Drug Metabolizing Enzymes in Human Hepatocytes

The induction potential of different fibric acid derivatives on human drug metabolizing enzymes was evaluated to help assess the role of enzyme induction on pharmacokinetic drug interactions. Effects of gemfibrozil, fenofibric acid, and clofibric acid on expression levels of cytochromes P450 (CYPs) 3A4 and 2C8 and UDP-glucuronyltransferase (UGT) 1A1 were evaluated in primary human hepatocyte cultures. The potential for these fibrates to activate human pregnane X receptor (PXR) also was studied in a cell-based PXR reporter gene assay. All three fibrates caused increases in mRNA levels of CYP3A4 (2- to 5-fold), CYP2C8 (2- to 6-fold), and UGT1A1 (2- to 3-fold). On average, the effects on CYP3A4 were less than (< or =30% of rifampin), while those on CYP2C8 and UGT1A1 were comparable to or slightly higher than (up to 200% of rifampin) the corresponding effects observed with rifampin (10 microM). Consistent with the mRNA results, all fibrates caused moderate (approximately 2- to 3-fold) increases in CYP3A4 activity (measured by testosterone 6beta hydroxylase), as compared to about a 10-fold increase by rifampin. Significant increases (3- to 6-fold) in amodiaquine N-deethylase (a functional probe for CYP2C8 activity) also were observed with clofibric acid, fenofibric acid, and rifampin, in agreement with the mRNA finding. However, in contrast to the mRNA induction, marked decreases (>60%) in CYP2C8 activity were obtained with gemfibrozil treatment. Consistent with this finding, co-incubation of amodiaquine with gemfibrozil, but not with fenofibric acid, clofibric acid, or rifampin, in human liver microsomes or hepatocytes resulted in significantly decreased amodiaquine N-deethylase activity (IC50 = 80 microM for gemfibrozil, >500 microM for fenofibric or clofibric acid, and >50 microM for rifampin). Similar to rifampin, all three fibrates caused a modest change in the glucuronidation of chrysin, a nonspecific substrate of UGTs. No significant activation on human pregnane X receptor (PXR) was observed with the three fibrates in a PXR reporter gene assay. In human hepatocytes, both fenofibric acid and clofibric acid are inducers of CYP3A4 and CYP2C8. Gemfibrozil is also an inducer of CYP3A4, but acts as both an inducer and an inhibitor of CYP2C8. In this system, all fibrates are weak inducers of UGT1A1. The enzyme inducing effects of fibrates appear to be mediated via a mechanism(s) other than PXR activation. These results suggest that fibrates may have potential to cause various pharmacokinetic drug interactions via their differential effects on enzyme induction and/or inhibition.

Analysis of CYP mRNA expression by branched DNA technology

Publisher Summary The branched DNA (bDNA) signal amplification assay provides a sensitive and relatively straightforward method for quantifying the levels of specific mRNAs. The cytochrome P450 (CYP) induction studies performed in laboratory with the bDNA signal amplification assay provided a reliable alternative to traditional techniques, such as enzyme analysis and immunoblotting, to detect the induction of CYP enzymes in human hepatocytes. The protocol developed that allows human hepatocytes cultured in one 60-mm petri dish to be analyzed for changes in expression of up to 30 genes, in duplicate. The assay is suitable for the measurement of any mRNA molecule, in cultured cells or tissue, for which a specific probe set can be developed. In laboratory, bDNA assay probe sets were developed to study the expression and regulation (inducibility) of the major human cytochrome P450 enzymes (CYPs), UDP-glucuronosyltransferases (UGTs), and sulfonotransferases (SULTs).