Microsomal Cytochrome P-450 Research Articles

Stereostructure-activity mechanism of cyproconazole by cytochrome P450 in rat liver microsomes: A combined experimental and computational study

Cyproconazole (CPZ), representing the chiral triazole fungicides, is widely used in the pharmaceutical and agricultural fields. To clarify its potential adverse effects on the generalized CYP-mediated processes within mammalian, a comparative experimental and computational approach was employed to investigate the CYP-mediated metabolism processes of CPZ stereoisomers in rat liver microsomes (RLMs). The depletion rate of CPZ stereoisomers in vitro incubation system with RLMs followed the order RR-> SS-> SR-> RS-CPZ. The results of kinetic assays were in line with the depletion rate results. Further inhibition assay confirmed the stereoselective metabolism of CPZ stereoisomers by different CYP isoforms. Molecular dynamics (MD) simulation revealed the stereoselective metabolism mechanism. Several hydrogen bonds and π-stacking restrict the position of CPZ isomers in the active cavity of CYPs so that the 4′-nitrogen on the triazole ring can bind closely to the heme of CYP, which results in the metabolism of CPZ isomers. By combining the computational and experimental approaches, the structure-activity relationship of CPZ and CYP was elucidated, and this method can be further applied to predict the degree of uncertainty in the process of xenobiotic biotransformation of triazole fungicides and serve as a basis for risk assessment.

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Futile cycling by human microsomal cytochrome P450 enzymes within intact fission yeast cells

Human cytochrome P450 enzymes (CYPs or P450s) are known to be reduced by their electron transfer partners in the absence of substrate and in turn to reduce other acceptor molecules such as molecular oxygen, thereby creating superoxide anions (O2−•). This process is known as futile cycling. Using our previously established fission yeast expression system we have monitored cells expressing each one of the 50 human microsomal CYPs in the absence of substrate for oxidation of dihydroethidium in living cells by flow cytometry. It was found that 38 of these display a statistically significant increase in O2−• production. More specifically, cells expressing some CYPs were found to be intermediate strength O2−• producers, which means that their effect was comparable to that of treatment with 3 mM H2O2. Cells expressing other CYPs had an even stronger effect, with those expressing CYP2B6, CYP5A1, CYP2A13, CYP51A1, or CYP1A2, respectively, being the strongest producers of O2−•.

An Innovatory GABA Receptor Modulator and Liver Oxidase System Microsomal Cytochrome P450 Activator in Patients with Alcoholism

Molecular docking studies (in Schrodinger and Glide software) showed that the molecule m-Cl-BHU (meta-chlorobenzhydrylurea) is complementary to the benzodiazepine binding site of GABAA receptors. Binding energy was low (-11.14 kcal/mol); m-Cl-BHU interacts with the key amino acids at the α1γ2 interface: Tyr159, Tyr209, and H101 Phe77 with high fit to the dG insertion model: 0.741. Binding of [3H]flunitrazepam with the benzodiazepine site of brain GABAA receptors increased in rats with experimental alcoholism treated with m-Cl-BHU at a dose of 100 mg/kg for 14 days. Changes in pharmacokinetic parameters (T1/2, Clt, MRT, MET, and AUC) of the model substrate antipyrine in saliva were seen in healthy volunteers and male patients with alcoholism using Galodif (m-Cl-BHU) at a dose of 300 mg/day for 21 days. Elimination of antipyrine in patients with alcoholism was increased due to activation of microsomal cytochrome P450 in the liver oxidase system.

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

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.

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

Cytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP–CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP–CPR ET rates are low compared to those of soluble bacterial CYPs.

Towards understanding transfluthrin efficacy in a pyrethroid-resistant strain of the malaria vector Anopheles funestus with special reference to cytochrome P450-mediated detoxification

Malaria vector control interventions rely heavily on the application of insecticides against anopheline mosquitoes, in particular the fast-acting pyrethroids that target insect voltage-gated sodium channels (VGSC). Frequent applications of pyrethroids have resulted in resistance development in the major malaria vectors including Anopheles funestus, where resistance is primarily metabolic and driven by the overexpression of microsomal cytochrome P450 monooxygenases (P450s). Here we examined the pattern of cross-resistance of the pyrethroid-resistant An. funestus strain FUMOZ-R towards transfluthrin and multi-halogenated benzyl derivatives, permethrin, cypermethrin and deltamethrin in comparison to the susceptible reference strain FANG. Transfluthrin and two multi-fluorinated derivatives exhibited micromolar potency - comparable to permethrin - to functionally expressed dipteran VGSC in a cell-based cation influx assay. The activity of transfluthrin and its derivatives on VGSC was strongly correlated with their contact efficacy against strain FUMOZ-R, although no such correlation was obtained for the other pyrethroids due to their rapid detoxification by the resistant strain. The low resistance levels for transfluthrin and derivatives in strain FUMOZ-R were only weakly synergized by known P450 inhibitors such as piperonyl butoxide (PBO), triflumizole and 1-aminobenzotriazole (1-ABT). In contrast, deltamethrin toxicity in FUMOZ-R was synergized > 100-fold by all three P450 inhibitors. The biochemical profiling of a range of fluorescent resorufin and coumarin compounds against FANG and FUMOZ-R microsomes identified 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) as a highly sensitive probe substrate for P450 activity. BOMFC was used to develop a fluorescence-based high-throughput screening assay to measure the P450 inhibitory action of potential synergists. Azole fungicides prochloraz and triflumizole were identified as extremely potent nanomolar inhibitors of microsomal P450s, strongly synergizing deltamethrin toxicity in An. funestus. Overall, the present study contributed to the understanding of transfluthrin efficacy at the molecular and organismal level and identified azole compounds with potential to synergize pyrethroid efficacy in malaria vectors.

Инновационный модулятор ГАМК рецепторов — активатор микросомальной цитохром-P450 оксидазной системы печени у больных алкоголизмом

С помощью молекулярного докинга (программа Schrödinger, Glide) показано, что молекула м-Cl-БГМ (мета-хлорбензгидрилмочевина) комплементарна сайту связывания бензодиазепина ГАМКА рецепторов. Энергия связывания низкая (– 11,14 ккал/моль); м-Cl-БГМ взаимодействует с ключевыми аминокислотами на α1γ2 интерфейсе: Tyr159, Tyr209, H101 Phe77 с высокой степенью соответствия модели — dG вставки: 0,741. Связывание [3H]флунитразепама с сайтом бензодиазепина ГАМКА рецепторов головного мозга крыс при экспериментальном алкоголизме, получавших 14 дней м-Cl-БГМ в дозе 100 мг/кг в сутки, увеличивалось. Выявлены изменения фармакокинетических параметров (T1/2, Clt, MRT, МЕТ, AUC) модельного субстрата — антипирина в слюне здоровых добровольцев и больных алкоголизмом мужчин при применении галодифа (м-Cl-БГМ) в дозе 300 мг/сут в течение 21 дня. Элиминация антипирина у больных алкоголизмом была повышена вследствие активации микросомальной цитохром-P450 оксидазной системы печени.

Digital Microfluidics-Enabled Analysis of Individual Variation in Liver Cytochrome P450 Activity.

The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in humans. However, the kinetics of drug clearance via CYPs varies significantly among individuals due to both genetic and external factors, and the enzyme amount and function are also largely impacted by many liver diseases. In this study, we developed a new methodology, based on digital microfluidics (DMF), for rapid determination of individual alterations in CYP activity from human-derived liver samples in biopsy-scale. The assay employs human liver microsomes (HLMs), immobilized on magnetic beads to facilitate determination of the activity of microsomal CYP enzymes in a parallelized system at physiological temperature. The thermal control is achieved with the help of a custom-designed, inkjet-printed microheater array modularly integrated with the DMF platform. The CYP activities are determined with the help of prefluorescent, enzyme-selective model compounds by quantifying the respective fluorescent metabolites based on optical readout in situ. The selectivity and sensitivity of the assay was established for four different CYP model reactions, and the diagnostic concept was validated by determining the interindividual variation in one of the four model reaction activities, that is, ethoxyresorufin O-deethylation (CYP1A1/2), between five donors. Overall, the developed protocol consumes only about 15 μg microsomal protein per assay. It is thus technically adaptable to screening of individual differences in CYP enzyme function from biopsy-scale liver samples in an automated fashion, so as to support tailoring of medical therapies, for example, in the context of liver disease diagnosis.

Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding.

The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is degenerated. Recently, we demonstrated that specific CPR mutations in the FMN-domain (FD) may induce a gain in activity for a specific CYP isoform. In the current report, we confirm the CYP isoform dependence of CPR’s degenerated binding by demonstrating that the effect of four of the formerly studied FD mutants are indeed exclusive of a specific CYP isoform, as verified by cytochrome c inhibition studies. Moreover, the nature of CYP’s substrate seems to have a modulating role in the CPR:CYP interaction. In silico molecular dynamics simulations of the FD evidence that mutations induces very subtle structural alterations, influencing the characteristics of residues formerly implicated in the CPR:CYP interaction or in positioning of the FMN moiety. CPR seems therefore to be able to form effective interaction complexes with its structural diverse partners via a combination of specific structural features of the FD, which are functional in a CYP isoform dependent manner, and dependent on the substrate bound.

Kinetics of metabolism of deltamethrin and cis- and trans-permethrin in vitro. Studies using rat and human liver microsomes, isolated rat hepatocytes and rat liver cytosol

The kinetics of metabolism of deltamethrin (DLM) and cis- and trans-permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol. Apparent intrinsic clearance (CLint) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration. However, when apparent CLint values were corrected for nonspecific binding to allow calculation of unbound (i.e., corrected) CLint values, the unbound values did not vary greatly with microsomal protein concentration. Unbound CLint values for metabolism of 0.05-1 μM DLM in rat liver microsomes (CYP and CES enzymes) and cytosol (CES enzymes) were not significantly different from rates of DLM metabolism in isolated rat hepatocytes. This study demonstrates that the nonspecific binding of these highly lipophilic compounds needs to be taken into account in order to obtain accurate estimates of rates of in vitro metabolism of these pyrethroids. While DLM is rapidly metabolised in vitro, the hepatocyte membrane does not appear to represent a barrier to the absorption and hence subsequent hepatic metabolism of this pyrethroid.

A single pretreatment with clofibric acid attenuates carbon tetrachloride-induced necrosis, but not steatosis, in rat liver

The present study investigated whether a single pretreatment with clofibric acid suppresses liver injury in rats after CCl4 intoxication. Rats received a single pretreatment with clofibric acid (100 mg/kg, i.p.) 1 h prior to a CCl4 (1 mL/kg, p.o.) challenge, and were euthanized 24 h after the CCl4 administration. A single pretreatment with clofibric acid effectively suppressed increases in the serum aminotransferase activities and the severity of necrosis following the CCl4 challenge, whereas the pretreatment did not protect against CCl4-induced fatty liver. The clofibric acid pretreatment did not affect blood concentrations of CCl4 in the early stage after CCl4 dosing, or the level of the CCl4 reaching the liver 1 h after the CCl4 challenge. Moreover, the clofibric acid pretreatment did not affect the intensity of the covalent binding of the [14C]CCl4 metabolite to microsomal proteins and lipids. The clofibric acid pretreatment did not alter microsomal cytochrome P450 2E1 activity. Based on these results, we conclude that protection against CCl4-induced hepatocellular necrosis by a clofibric acid pretreatment does not require its repeated administration, and that a single and brief pre-exposure to clofibric acid prior to CCl4 dosing markedly suppresses necrosis without affecting the development and progression of steatosis.

Metabolism of bifenthrin, β-cyfluthrin, λ-cyhalothrin, cyphenothrin and esfenvalerate by rat and human cytochrome P450 and carboxylesterase enzymes

The metabolism of bifenthrin (BIF), β-cyfluthrin (CYFL), λ-cyhalothrin (CYHA), cyphenothrin (CYPH) and esfenvalerate (ESF) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 90, 21 and 15 days and from adult humans. Pyrethroid metabolism was also studied with some human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. All five pyrethroids were metabolised by adult (90 day old) rat hepatic microsomal CYP and CES enzymes and by cytosolic CES enzymes. The pyrethroids were also metabolised by human liver microsomes and cytosol. Some species differences were observed. Pyrethroid metabolism by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds. CYFL, CYHA, CYPH and ESF were metabolised by rat plasma CES enzymes, whereas none of the pyrethroids were metabolised by human plasma. This study demonstrates that the ability of male rats to metabolise these pyrethroids by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90 day old rats. All pyrethroids were metabolised by some of the human expressed CYP enzymes studied and apart from BIF were also metabolised by CES enzymes.

Modulation of Major Human Liver Microsomal Cytochromes P450 by Component Alkaloids of Goldenseal: Time-Dependent Inhibition and Allosteric Effects.

Botanical and other natural products (NPs) are often coconsumed with prescription medications, presenting a risk for cytochrome P450 (P450)-mediated NP-drug interactions. The NP goldenseal (Hydrastis canadensis) has exhibited antimicrobial activities in vitro attributed to isoquinoline alkaloids contained in the plant, primarily berberine, (-)-β-hydrastine, and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alerts with potential to inactivate P450s through formation of metabolic intermediate complexes. Time-dependent inhibition experiments were conducted to evaluate their ability to inhibit major P450 activities in human liver microsomes by using a cocktail of isozyme-specific substrate probes. Berberine inhibited CYP2D6 (dextromethorphan O-demethylation; K I = 2.7 μM, kinact = 0.065 minute-1) and CYP3A4/5 (midazolam 1'-hydroxylation; K I = 14.8 μM, kinact = 0.019 minute-1); (-)-β-hydrastine inhibited CYP2C9 (diclofenac 4'-hydroxylation; K I = 49 μM, kinact = 0.036 minute-1), CYP2D6 (K I > 250 μM, kinact > 0.06 minute-1), and CYP3A4/5 (K I = 28 μM, kinact = 0.056 minute-1); and hydrastinine inhibited CYP2D6 (K I = 37 μM, kinact = 0.049 minute-1) activity. Berberine additionally exhibited allosteric effects on midazolam hydroxylation, showing both positive and negative heterotropic cooperativity. Experiments with recombinant isozymes showed that berberine activated midazolam 1'-hydroxylation by CYP3A5, lowering K m(app), but showed mixed inhibition and negative cooperativity toward this reaction when catalyzed by CYP3A4. Berberine inactivated CYP3A4 at a much faster rate than CYP3A5 and was a noncompetitive inhibitor of midazolam 4-hydroxylation by CYP3A4 but a strong mixed inhibitor of the CYP3A5 catalyzed reaction. These complex kinetics should be considered when extrapolating the risk for NP-drug interactions involving goldenseal. SIGNIFICANCE STATEMENT: Robust kinetic parameters were determined for the reversible and time-dependent inhibition of CYP2C9, CYP2D6, and CYP3A4/5 activities in human liver microsomes by major component isoquinoline alkaloids contained in the botanical natural product goldenseal. The alkaloid berberine also exhibited opposing, isozyme-specific allosteric effects on midazolam hydroxylation mediated by recombinant CYP3A4 (inhibition) and CYP3A5 (activation). These data will inform the development of a physiologically based pharmacokinetic model that can be used to predict potential clinically relevant goldenseal-drug interactions.

Reduction in CYP1A1 and 2B2 activity at low oxygen tension

The Cytochrome P450 (CYP) enzyme family comprises a wide array of monooxygenases involved in the oxidation of endobiotic and xenobiotic molecules. The active site of a CYP enzyme contains an iron protoporphyrin center coordinated to a cysteine thiolate, and then, molecular oxygen is associated with the iron to be converted into dioxygen complex plus substrate. Reduction by CYP reductase expedites hydroxylation of the compound. In this oxidation reaction, insufficient oxygen molecules would affect enzyme catalysis. Nevertheless, biochemical data about CYP kinetics at low oxygen concentrations are not available. In this work, we present the results on the variation in rat liver microsomal CYP Vmax app and Km app under normal and hypoxic conditions. Using alkoxyresorufin molecules as substrates, the Vmax/Km ratios for resorufin production decreased from 426 to 393 for CYP1A1 and from 343 to 202 for CYP2B1 at a low oxygen concentration (4.1 ppm) compared to the ratios observed at a normal oxygen concentration (6.5 ppm). Additionally, the bacterial mutagenicity of 2-aminoanthracene and cyclophosphamide, decreased by 32% and 42%, respectively, at low oxygen concentrations. These results support the hypothesis that low oxygen availability is implicated in the low efficiency of substrate oxidation by CYP.

Piperonyl butoxide: Mode of action analysis for mouse liver tumour formation and human relevance

In a 79 week bioassay the pesticide synergist piperonyl butoxide (PBO) was shown to significantly increase the incidence of hepatocellular adenoma (but not hepatocellular carcinoma) in male CD-1 mice at dietary levels of 100 and 300 mg/kg/day PBO and in female mice at a dietary level of 300 mg/kg/day. As PBO is not a genotoxic agent, a series of investigative studies were undertaken to elucidate the mode of action (MOA) for PBO-induced mouse liver tumour formation. Male CD-1 mice were fed diets to provide intakes of 0 (control), 30, 100 and 300 mg/kg/day PBO and for purposes of comparison 500 ppm sodium phenobarbital (NaPB), a known constitutive androstane receptor (CAR) activator, for 7 and 14 days. Treatment with 100 and 300 mg/kg/day PBO and 500 ppm NaPB increased relative liver weight which was associated with hepatocyte hypertrophy, with hepatocyte replicative DNA synthesis (RDS) being increased after 7 days treatment. The treatment of CD-1 mice with 30−300 mg/kg/day PBO for 14 days resulted in significant dose-dependent increases in hepatic microsomal cytochrome P450 (CYP) content and 7-pentoxyresorufin O-depentylase (PROD) activity and in hepatic Cyp2b10 mRNA levels. In contrast, PBO produced a biphasic effect on markers of activation of the peroxisome proliferator-activated receptor alpha (PPARα), with small increases in microsomal lauric acid 12-hydroxylase activity and hepatic Cyp4a10 mRNA levels being observed in mice given 100 mg/kg/day with PBO, with either no increase or a significant inhibition being observed in mice given 300 mg/kg/day PBO. The hepatic effects of PBO in male CD-1 mice were generally similar to those produced by NaPB and were reversible after the cessation of treatment for 28 days. Studies were also performed in male C57BL/6J (wild type) mice and in hepatic CAR and pregnane X receptor (PXR) knockout mice (CAR KO/PXR KO mice), where in the CAR KO/PXR KO mice PBO had little effect on markers of CAR activation, but produced some increases in markers of PPARα activation. The treatment of male CD-1 mouse hepatocytes for 4 days with 5−50 μM PBO, 10−1000 μM NaPB and 25 ng/mL epidermal growth factor (EGF) resulted in significant increases in hepatocyte RDS. While treatment of hepatocytes from one male and one female human donor with 5−500 μM PBO and 10−1000 μM NaPB for 4 days had no effect on hepatocyte RDS, treatment with EGF resulted in significant increases in RDS in both human hepatocyte preparations. In summary, PBO is predominantly a hepatic CAR activator at carcinogenic dose levels in CD-1 mice, with activation of hepatic CAR resulting in a suppression of the effect of PBO on hepatic PPARα. A robust MOA for PBO-induced mouse liver tumour formation has been established, this MOA being similar to that previously identified for NaPB and some other rodent liver CAR activators. Based on the lack of effect of PBO on RDS in human hepatocytes, it is considered that the MOA for PBO-induced mouse liver tumour formation is qualitatively not plausible for humans.

Metabolism of a New Antiaggregant, Indolinone Derivative.

Cytochrome p450-mediated metabolism of GRS (indolinone antiaggregant) and its effects on activities of cytochrome p450 isoenzymes were studied. Inhibition of 6 isomers of cytochrome p450 in human liver microsomes was studied with the use of specific substrates. It was found that human liver cytochrome p450 enzymes could not induce degradation of GRS and that GRS was not an inductor or inhibitor of cytochrome p450 family members 1A2, 2C9, 2C19, 2D6, 2C8, and 3A4. Hence, clinical use of the prospective antiaggregant would not involve the risk of uncontrolled fluctuations in GRS concentrations in the organism because of interactions between the drugs.

Effects of Thioacetamide—Induced Cirrhosis, after a Drug Washout Period, on the Michaelis–Menten Kinetics of Major Cytochrome P450 Enzymes in the Rat Liver Microsomes

PurposeThioacetamide (TAA) is used to induce liver cirrhosis in rodents. However, the drug itself directly inhibits cytochrome P450 (CYP450) enzymes. The purpose of this study was to determine the effects of TAA‐induced liver cirrhosis, after a washout period to eliminate TAA, on the Michaelis‐Menten (MM) kinetics and protein contents of the major CYP450 enzymes CYP2D, CYP2E1, and CYP3A in rat liver microsomes.MethodsAdult, male Sprague‐Dawley rats were treated for 10 weeks with either TAA (200 mg/kg ip, 3 days per week) or vehicle (n = 7/each group). After a 10‐day washout period, animals were euthanized, and the liver was collected after cardiac perfusion with cold saline. Liver microsomes (MC) were prepared by differential centrifugation. The activities of the liver microsomal CYP2D (dextromethorphan O‐demethylation), CYP2E1 (chlorzoxazone 6‐hydroxylation) and CYP3A (midazolam 1′‐hydroxylation) were estimated by measurement of the metabolites by LC‐MS/MS. The microsomal CYP450 reductase (CPR) activity was analyzed spectrophotometry. Furthermore, the protein expressions of the isoenzymes were determined using Western blot analysis. The MM kinetic parameters maximum velocity (Vmax) and MM constant (Km) were determined using nonlinear regression analysis. Unpaired t‐test was used for statistical analysis of data, with a p value of < 0.05 as significant.ResultsWhereas the MM kinetics of dextromethorphan demethylation (CYP2D) were best fit by a two‐enzyme equation, those of chlorzoxazone hydroxylation (CYP2E1) and midazolam 1′‐hydroxylation (CYP3A) were best fit by a single‐enzyme equation. Cirrhosis resulted in significant reductions in the Vmax values for all the tested microsomal activities. The microsomal high‐ and low‐capacity Vmax values for CYP2D demethylase activity were significantly reduced by 74% (p value < 0.0001) and 51% (p < 0.05), respectively, in the TAA group. This was associated with a significant (p < 0.001) reduction (71%) in the protein contents of CYP2D in the TAA group. However, Km values were not affected by cirrhosis. For CYP2E1, cirrhosis caused a significant reduction (52%) in the Vmax value (p < 0.0001) for the chlorzoxazone hydroxylation activity. However, the Km values and protein contents of CYP2E1 remained unchanged. The Vmax and Km values of microsomal CYP3A hydroxylase activity, resulting in 1′‐hydroxylation of midazolam, were significantly reduced by 71% (p < 0.001) and 46% (p < 0.05), respectively, which was associated with a 30% reduction (p < 0.05) in the protein contents of CYP3A. Cirrhotic animals also showed a 31% decline (p < 0.05) in the microsomal CPR activity.ConclusionsTAA‐induced cirrhosis in rats resulted in a decreased liver microsomal activity of CPR and major CYP450 enzymes (CYP2D, CYP2E1, and CYP3A). Except for CYP2E1, the decreased isoenzyme activities were also associated with decreases in the protein concentrations of the isoenzymes. However, the cirrhosis‐induced decrease in the CYP2E1 activity, which was in the absence of a change in its protein levels, is possibly due to a decrease in the microsomal CPR activity.Support or Funding InformationFunding: Chapman University School of Pharmacy

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches – formerly indicated to be involved in CPR:CYP interactions – or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

Biosensor selection of small compounds, modulating a complex formation between cytochrome P450s and NADPH-dependent P450 oxidoreductase

The study of the effect of low-molecular-weight compounds (substrates, endogenous metabolites, drugs and xenobiotics) on the kinetic and equilibrium parameters of functionally significant binary protein-protein interactions (PPIs) is of both fundamental and clinical importance. The surface plasmon resonance (SPR) is the method of the first choice for studying PPIs. Earlier, SPR analysis revealed the modulating effect of steroidal substrates on the affinity of interactions between steroidogenic microsomal cytochromes P450 (CYP) and their redox partner (cytochrome b5). In this work, we have shown the suitability of the experimental approach for assessing the selective effect of the cofactor NADPH on the interaction between cytochromes CYP3A4 or CYP2E1 with NADPH-dependent P450 oxidoreductase (CPR). Experiments have shown that the CYP3A4/CPR complex is not modulated by NADPH, while the dissociation rate of the CYP2E1/CPR complex in the presence of NADPH significantly decreased: the koff values in the absence and presence of NADPH were (3.6 ± 0.2) • 10-3 s-1 and (3.8 ± 0.2) • 10-4 s-1, respectively. Thus, in the presence of NADPH, an increase in the affinity of CYP2E1/CPR complex formation by approximately one order of magnitude was observed, while NADPH did not affect the kon value of this complex. Co-injection of NADPH at the CYP2E1/CPR complex preformed in the absence of NADPH had minor influence on the koff values (<10%). This suggests a stabilizing role of NADPH for the CYP2E1/CPR complex formation. Thus, the use of our approach made it possible to assess the effect of the main electron supplier for the microsomal cytochrome P450 monooxygenase system on the kinetic rate constants of CYP/CPR complexes.