Microarray of human P450 with an integrated oxygen sensing film for high-throughput detection of metabolic activities

A microarray chip containing human P450 isoforms was constructed for the parallel assay of their metabolic activities. The chip had microwells that contained vertically integrated P450 and oxygen sensing layers. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). Human P450s (23 types) expressed in E. coli and purified as membrane fractions were immobilized in agarose matrixes on the oxygen sensing layer. The activities of P450s were determined by evaluating the fluorescence intensity enhancement of the oxygen sensor due to the oxygen consumption by the metabolic reaction. By normalizing the responses with the amounts of oxygen sensor and P450 enzymes in microwells, we could obtain fluorescence enhancement patterns that were characteristic to the combination of P450 isoforms and substrate material. The patterns obtained from two psoralen derivatives resembled each other, whereas a structurally different substrate (capsaicin) resulted in a distinct pattern. These results suggest the potential of the microarray to analyze the activities of diverse P450 isoforms in a high-throughput fashion. Furthermore, mechanism-based inactivation (MBI) of P450 could be detected by successively incubating a chip with different substrate solutions and measuring the residual activities.     

Creation of a P450 array toward high-throughput analysis

The rapid metabolism testing of many new chemical entities enables unsuitable candidates to be eliminated from consideration at an early stage of the drug discovery process. We have developed a P450 array toward high-throughput analysis of P450-mediated metabolic reaction. The microsomes containing expressed human P450 enzymes were immobilized on the microassay plate using sol-gel chemistry. A thin-film hydrogel containing microsomes was fabricated using aqueous silicate as a starting material. The TEM image clearly showed that the nanoclusters derived from the silicate formed branched chains, and microsomes were entrapped in the silica network. The different P450 isozymes were immobilized on the microassay plate, and the metabolites by each isozyme were visualized as fluorescent images, which creates opportunity for the inhibitor assays. This method offers several advantages over use of conventional enzyme preparations, including increased storage stability, ease of product isolation from the incubation mixture, and the ability to recover and reuse the enzyme. Because this methodology enabled the development of assay system using P450 that is unstable and involves other enzymes for its function, it can be applicable to various screening assays that require complicated reactions involving many biological components.     

Selective filling of nanowells in nanowell arrays fabricated using polystyrene nanosphere lithography with cytochrome P450 enzymes

This work describes an original and simple technique for protein immobilization into nanowells, fabricated using nanopatterned array fabrication methods, while ensuring the protein retains normal biological activity. Nanosphere lithography was used to fabricate a nanowell array with nanowells 100?nm in diameter with a periodicity of 500?nm. The base of the nanowells was gold and the surrounding material was silicon dioxide. The different surface chemistries of these materials were used to attach two different self-assembled monolayers (SAM) with different affinities for the protein used here, cytochrome P450 (P450). The nanowell SAM, a methyl terminated thiol, had high affinity for the P450. The surrounding SAM, a polyethylene glycol silane, displayed very little affinity toward the P450 isozyme CYP2C9, as demonstrated by x-ray photoelectron spectroscopy and surface plasmon resonance. The regularity of the nanopatterned array was examined by scanning electron microscopy and atomic force microscopy. P450-mediated metabolism experiments of known substrates demonstrated that the nanowell bound P450 enzyme exceeded its normal activity, as compared to P450 solutions, when bound to the methyl terminated self-assembled monolayer. The nanopatterned array chips bearing P450 display long term stability and give reproducible results making them potentially useful for high-throughput screening assays or as nanoelectrode arrays.     

Electrochemiluminescent arrays for cytochrome P450-activated genotoxicity screening. DNA damage from benzo[a]pyrene metabolites

Arrays suitable for genotoxicity screening are reported that generate metabolites from cytochrome P450 enzymes (CYPs) in thin-film spots. Array spots containing DNA, various human cyt P450s, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ were exposed to H2O2 to activate the enzymes. ECL from all spots was visualized simultaneously using a CCD camera. Using benzo[a]pyrene as a test substrate, enzyme activity for producing DNA damage in the arrays was found in the order CYP1B1 > CYP1A2 > CYP1A1 > CYP2E1 > myoglobin, the same as the order of their metabolic activity. Thus, these arrays estimate the relative propensity of different enzymes to produce genotoxic metabolites. This is the first demonstration of ECL arrays for high-throughput in vitro genotoxicity screening.     

Functional screening of cytochrome P450 activity and uncoupling by capillary electrophoresis

Cytochrome P450s (CYPs) are functionally diverse monooxygenases responsible for oxidation of endogenous and xenobiotic compounds. The function of nonmammalian CYPs are largely unknown and tools for characterization limited. CYPs critical for xenobiotic metabolism are prone to catalytic cycle uncoupling resulting in reactive oxygen species (ROS) generation that is highly dependent on the specific CYP isoform and substrate interaction. This study describes the rapid assessment of the activity and coupling efficiency of CYPs using capillary electrophoresis with UV detection. The coupling efficiency of five zebrafish (Danio rerio) CYP1 isoforms with a series of fluorogenic substrate probes was determined by the rate of NADP(+) formation and compared with fluorescent product turnover rates. In most cases, NADP(+) formation significantly overestimated CYP1 catalytic activity for substrate O-dealkylation suggesting uncoupling. ROS production was confirmed by elevated hydrogen peroxide generation in poorly coupled reactions. Reactions with β-estradiol confirmed that CYP1A, 1C1, and 1C2 have greater catalytic activity and coupling efficiency; CYP1B1 and 1D1 had coupling efficiencies under 4%. This work highlights the wide disparity in uncoupling induced by unproductive substrate binding among different CYP isoforms.     

Temperature derivative spectroscopy to monitor the autoxidation decay of cytochromes P450

Temperature derivative spectroscopy (TDS), a type of relaxation spectroscopy, is a powerful tool to study protein dynamics (Berendzen, J.; Braunstein, D. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 1). We developed the version of temperature derivative spectroscopy to monitor kinetics of autoxidation of cytochromes P450 and applied it to study the properties of the oxy-ferrous complex of a human membrane bound P450, CYP19A1 (aromatase), and that of a bacterial soluble P450, CYP101 when bound with their most common substrates, androstenedione (AD) and camphor, respectively. TDS extends the panel of methods that can be used to monitor heme protein kinetics, providing a rapid measurement technique and enabling measurement of the autoxidation rate over a wide range of temperatures, yielding the activation energy as well as absolute reaction rate in a single experiment.     

Recombinant antibody piezoimmunosensors for the detection of cytochrome P450 1B1

The phase I enzyme known as cytochrome P450 1B1 (CYP1B1) is involved in the metabolism of many endogenous and exogenous compounds, including carcinogens. CYP1B1 is overexpressed in a wide variety of human diseases ranging from diabetes to malignancies, such as invasive breast cancer. Because of its microsomal location in the cell, CYP1B1 could not be measured directly by existing methods but only assessed indirectly via the determination of the catalytic products. We report here a rapid, sensitive piezoimmunosensor for detection of CYP1B1 using single-chain fragment variable antibodies (scFv) as recognition elements and a quartz crystal microbalance (QCM) as the transducer. Three anti-CYP1B1 scFvs (designated B-66, D-23, and L-21) were biotinylated and used to capture and specifically detect CYP1B1 from samples in solution. ScFvs are smaller than most commonly used antibodies and can be coated onto QCM surfaces at much higher density to improve sensor sensitivity and specificity. The scFv-QCM biosensors showed excellent sensitivity (detection limit, 2.2 +/- 0.9 nM) and specificity with a dissociation constant K(d) = (1.54 +/- 0.59) x 10(-7) M. CYP1B1 were quantitatively detected in normal and malignant cell lysates (e.g., human T47D breast cancer cell microsomes). Results demonstrate that an anti-CYP1B1 scFv-QCM immunosensor could be used to detect P450 enzymes in biological samples.     

Direct electron transfer of cytochrome P450 2B4 at electrodes modified with nonionic detergent and colloidal clay nanoparticles

A method for construction of biosensors with membranous cytochrome P450 isoenzymes was developed based on clay/detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated cytochrome P450 2B4 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bioelectrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay-modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between -0.292 and -0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bioelectrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of product formation also indicates the bioelectrocatalysis by CYP2B4.     

Photoregulation of cytochrome P450 activity by using caged compound

Cytochrome P450 (P450) species play an important role in the metabolism of xenobiotics, and assaying the activities of P450 is important for evaluating the toxicity of chemicals in drugs and food. However, the lag time caused by the introduction and mixing of sample solutions can become sources of error as the throughput is heightened by increasing the sample number and decreasing the sample volume. To amend this technological obstacle, we developed a methodology to photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide adenine dinucleotide phosphate (NADP(+)) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH (cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP(+) resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use of caged-G6P enabled assays in isolated microchambers (width, 50 μm; height, 50 μm) by encapsulating necessary ingredients in advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of obtained kinetic constants.     

Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver microsomes

Cytochrome P450 2C9 (CYP2C9), one of the most important phase I drug metabolizing enzymes, could catalyze the reactions that convert diclofenanc into diclofenac 4′-hydroxylation. Evaluation of the inhibitory effects of compounds on CYP2C9 is clinically important because inhibition of CYP2C9 could result in serious drug–drug interactions. The objective of this work was to investigate the effects of curcumin on CYP2C9 in human and cytochrome P450 2C11 (CYP2C11) in rat liver microsomes. The results showed that curcumin inhibited CYP2C9 activity (10?µmol?L^–1 diclofenac) with half-maximal inhibition or a half-maximal inhibitory concentration (IC50) of 15.25?µmol?L^–1 and Ki?=?4.473?µmol?L^–1 in human liver microsomes. Curcumin’s mode of action on CYP2C9 activity was noncompetitive for the substrate diclofenanc and uncompetitive for the cofactor NADPH. In contrast to its potent inhibition of CYP2C9 in human, diclofenanc had lesser effects on CYP2C11 in rat, with an IC50 ≥100?µmol L^–1. The observations imply that curcumin has the inhibitory effects on CYP2C9 activity in human. These in vitro findings suggest that more attention should be paid to special clinical caution when intake of curcumin combined with other drugs in treatment.     

Effects of capsicine on rat cytochrome P450 isoforms CYP1A2, CYP2C19, and CYP3A4

Due to the frequent consumption of capsaicin (CAP) and its current therapeutic application, the correct assessment of this compound is important from a public health standpoint. The purpose of this study was to find out whether CAP affects rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5?mL/kg, which contained phenacetin (15?mg/kg), omeprazole (15?mg/kg), and midazolam (10?mg/kg), was given orally to rats treated for 7?d with oral administration of CAP. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS. The results showed that treatment with multiple doses of CAP had no significant effect on rat CYP1A2. However, CAP had a significant inhibitory effect on CYP2C19 and an inductive effect on CYP3A4. Therefore, caution is needed when CAP is co-administered with some CYP substrates clinically because of potential drug–CAP interactions.     

Microfluidic electrochemical array for detection of reactive metabolites formed by cytochrome P450 enzymes

A novel, simple, rapid microfluidic array using bioelectronically driven cytochrome P450 enzyme catalysis for reactive metabolite screening is reported for the first time. The device incorporates an eight-electrode screen-printed carbon array coated with thin films of DNA, [Ru(bpy)(2)(PVP)(10)](ClO(4)) {RuPVP}, and rat liver microsomes (RLM) as enzyme sources. Catalysis features electron donation to cyt P450 reductase in the RLMs and subsequent cyt P450 reduction while flowing an oxygenated substrate solution past sensor electrodes. Metabolites react with DNA in the film if they are able, and damaged DNA is detected by catalytic square wave voltammetry (SWV) utilizing the RuPVP polymer. The microfluidic device was tested for a set of common pollutants known to form DNA-reactive metabolites. Logarithmic turnover rates based on SWV responses gave excellent correlation with the rodent liver TD(50) toxicity metric, supporting the utility of the device for toxicity screening. The microfluidic array gave much better S/N and reproducibility than single-electrode sensors based on similar principles.     

Activation of phosphorothionate pesticides based on a cytochrome P450 BM-3 (CYP102 A1) mutant for expanded neurotoxin detection in food using acetylcholinesterase biosensors

A novel enzymatic in vitro activation method for phosphorothionates has been developed to allow their detection with acetylcholinesterase (AChE) biosensors. Activation is necessary because this group of insecticides shows nearly no inhibitory effect toward AChE in their pure nonmetabolized form. In contrast, they exert a strong inhibitory effect on AChE after oxidation as it takes place by metabolic activation in higher organisms. Standard chemical methods to oxidize phosphorothionates showed inherent disadvantages that impede their direct use in food analysis. In contrast, a genetically engineered triple mutant of P450 BM-3 (CYP102 A1) could convert the two frequently used insecticides parathion and chlorpyrifos into their oxo variants as was confirmed by GC/MS measurements. The wild-type protein was unable to do so. In the case of chlorpyrifos, the enzymatic activation was as good as the chemical oxidation. In the case of parathion, the P450 activation was more efficient than the oxidation by NBS but neither activation method yielded an AChE inhibition that was as high as with paraoxon. The application of the method to infant food in combination with a disposable AChE biosensor enabled detection of chlorpyrifos and parathion at concentrations down to 20 microg/kg within an overall assay time of 95 min.     

Metabolic interactions of magnolol with cytochrome P450 enzymes: uncompetitive inhibition of CYP1A and competitive inhibition of CYP2C

Magnolol (MAG; 5,5′-diallyl-2,2′-biphenyldiol) is a major bioactive component of Magnolia officinalis. We investigated the metabolic interactions of MAG with hepatic cytochrome P450 monooxygenase (CYP) through in vitro microsomal metabolism study using human (HLM) and rat liver microsomes (RLM). CYP2C and 3A subfamilies were significantly involved in the metabolism of MAG, while CYP1A subfamily was not in HLM and RLM. The relative contribution of phase I enzymes including CYP to the metabolism of MAG was comparable to that of uridine diphosphate glucuronosyltransferase (UGT) in RLM. Moreover, MAG potently inhibited the metabolic activity of CYP1A (IC₅₀ of 1.62?μM) and 2C (IC₅₀ of 5.56?μM), while weakly CYP3A (IC₅₀ of 35.0?μM) in HLM and RLM. By the construction of Dixon plot, the inhibition type of MAG on CYP activity in RLM was determined as follows: uncompetitive inhibitor for CYP1A (K_i of 1.09–12.0?μM); competitive inhibitor for CYP2C (K_i of 10.0–15.2?μM) and 3A (K_i of 93.7–183?μM). Based on the comparison of the current IC₅₀ and K_i values with a previously reported liver concentration (about 13?μM) of MAG after its seven times oral administration at a dose of 50?mg/kg in rats, it is suggested that MAG could show significant inhibition of CYP1A and 2C, but not CYP3A, in the in vivo rat system. These results could lead to further studies in clinically significant metabolism-mediated MAG–drug interactions.     

Detection of atrazine based on the SPR determination of P450 mRNA levels in Saccharomyces cerevisiae

We describe a novel method for quantification of atrazine based on detection of P450 mRNA levels in Saccharomyces cerevisiae. The selected oligonucleotide probe exhibited specificity against P450 mRNA and was successfully immobilized on the sensor chip. The mRNA was subsequently quantified by RU change using a SPR system. When the cells were disrupted by boiling, mRNA could be measured without further purification at reduced sensitivity. This simple technique permits the detection of atrazine within 15 min. This rapid and highly sensitive method can be used for the detection of atrazine.     

Effect of sodium alterations on hepatic cytochrome P450 3A2 and 2C11 and renal function in rats

Numerous dietary supplements are known to modulate cytochrome P450 (CYP)-mediated metabolism and subsequently alter drug toxicity or efficacy in animals and humans. In the present study we investigated the effect of varying amounts of sodium intake on renal function and the metabolic activity of the hepatic CYP3A2 and CYP2C11 isoforms. Rats were maintained on standard rodent chow or a low-salt rice diet. Within each of these groups rats received either a single intraperitoneal injection of furosemide to initiate salt depletion, or saline. Additional groups included salt supplementation of 500 mg/300 g body weight/day and 1.25 g/300 g body weight/day of sodium chloride solution. Rats receiving the low-salt diet, both with and without a concomitant furosemide administration, had a significant reduction in creatinine clearance without changes in serum creatinine. In addition, urine flow rate was markedly reduced in rats maintained on the low-salt diet. Western blot analysis indicated that neither sodium supplementation nor deprivation altered hepatic microsomal CYP3A2 levels; however, hepatic CYP2C11 levels significantly increased in rats receiving the largest sodium supplement. In vitro metabolic activity of CYP3A2 was unchanged as compared with controls. Activity of CYP2C11 was significantly reduced in both rat groups receiving additional sodium supplements. Acute manipulation of daily sodium intake does alter renal function and specific hepatic CYP isoforms and should be considered when using these rat models.     

Nanogap-based electrical PNA chips for the detection of genetic polymorphism of cytochrome P450 2C19

PNA chips for the detection of the genetic polymorphism of Cytochrome P450 2C19 (CYP2C19), a well-known enzyme related to the metabolism of therapeutic drugs, were electrically-interfaced with interdigitated nanogap electrodes (INEs). The average gap distance and effective length of the INEs were about approximately 70 nm and approximately 140/m, respectively. Those INEs having the aspect ratio of about 2000, were prepared by the combination of the photolithography (for the formation of initial electrodes) and the surface-catalyzed chemical deposition (for the gap narrowing), without the e-beam lithography. The PNA probes for the detection of CYP2C19 were immobilized in the gap region of INEs via Schiff base formation. The I-V characteristics clearly showed a sharp increase in the conductance between the nanogap electrodes upon the PNA-DNA hybridization, followed by the adsoprtion of functionalized Au nanoparticles. Four different target DNAs for the diagnosis of CYP2C19 polymorphism were successfully detected and discriminated with the INE-based PNA chips.     

Effect of CYP2C9 genetic polymorphism on the metabolism of flurbiprofen in vitro

CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, and 57 cytochrome P450 2C9 alleles have been previously reported. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for 4′-hydroxyflurbiprofen were determined using recombinant human P450s CYP2C9 microsomes from insect cells Sf21 carrying wild-type CYP2C9*1 and other variants. The results showed that the enzyme activity of most of the variants decreased comparing with the wild type as the previous studies reported, while the enzyme activity of some of them increased, which were not in accordance with the previous researches. Of the 36 tested CYP2C9 allelic isoforms, two variants (CYP2C9*53 and CYP2C9*56) showed a higher intrinsic clearance value than the wild-type protein, especially for CYP2C9*56, exhibited much higher intrinsic clearance (197.3%) relative to wild-type CYP2C9*1, while the remaining 33 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.6 to 83.8%) compared to CYP2C9*1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used non-steroidal anti-inflammatory drug, flurbiprofen (FP). The results indicated that most of the tested rare alleles decreased the catalytic activity of CYP2C9 variants toward FP hydroxylation in vitro. This is the first report of all these rare alleles for FP metabolism providing fundamental data for further clinical studies on CYP2C9 alleles for FP metabolism in vivo.     

An electrochemical microfluidic platform for human P450 drug metabolism profiling

This paper is the first report of a P450-electrode in a microfluidic format. A 30 μL microfluidic cell was made in poly(methyl methacrylate) containing the inlet, outlet, and reaction chamber with two electrode strips, one of which contains the human cytochrome P450 3A4 covalently bound to gold via a 6-hexanethiol and 7-mercaptoheptanoic acid (1:1) self-assembled monolayer. The electrochemical response of the P450-electrode in the microfluidic cell was tested using four drugs that are known substrates of P450 3A4: quinidine, nifedipine, alosetron and ondansetron. Titration experiments allowed the electrochemical measurements of K(M) for the four drugs, with values of 2.9, 29.1, 113.4, and 114.1 mM, respectively. The K(M) values are found to be in good agreement and correctly ranked with respect to the published literature on human liver microsomes and baculosomes: [ondansetron ≈ alosetron > nifedipine > quinidine]. The results presented in this paper represent a step forward for a rapid evaluation of the interaction of P450 and drug, requiring small volumes of new chemical entities to be tested.     

Healable,Transparent, Room‐Temperature Electronic Sensors Based on Carbon Nanotube Network‐Coated Polyelectrolyte Multilayers

Transparent and conductive film based electronics have attracted substantial research interest in various wearable and integrated display devices in recent years. The breakdown of transparent electronics prompts the development of transparent electronics integrated with healability. A healable transparent chemical gas sensor device is assembled from layer‐by‐layer‐assembled transparent healable polyelectrolyte multilayer films by developing effective methods to cast transparent carbon nanotube (CNT) networks on healable substrates. The healable CNT network‐containing film with transparency and superior network structures on self‐healing substrate is obtained by the lateral movement of the underlying self‐healing layer to bring the separated areas of the CNT layer back into contact. The as‐prepared healable transparent film is assembled into healable transparent chemical gas sensor device for flexible, healable gas sensing at room temperature, due to the 1D confined network structure, relatively high carrier mobility, and large surface‐to‐volume ratio. The healable transparent chemical gas sensor demonstrates excellent sensing performance, robust healability, reliable flexibility, and good transparency, providing promising opportunities for developing flexible, healable transparent optoelectronic devices with the reduced raw material consumption, decreased maintenance costs, improved lifetime, and robust functional reliability.