Activity Of P450s Research Articles

Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.

In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures

Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air–liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

Biochemical mechanisms for metaflumizone resistance in beet armyworm, Spodoptera exigua

The metaflumizone, which belongs to the class of voltage-dependent sodium channel blockers, was registered to control Spodoptera exigua on vegetables in China in 2009. The present study revealed S. exigua has developed high resistance to this novel chemistry insecticide shortly after 2–3years application in Guangdong Province of China. The metabolic mechanisms for metaflumizone resistance in this insect were analysed. The inhibitor of esterases greatly potentiates the toxicity of this chemical against the field resistant populations. The synergism ratio is 5.7 and 3.4-fold for S. exigua collected from Huizhou, Guangdong Province in 2011 and 2012, respectively. The activity of esterases in field populations (HZ12) is also significantly greater than that in the susceptible strain, and further significantly increased by challenge with metaflumizone for 3 generations. However, the inhibitor of P450s or GSTs only has slight synergism on metaflumizone toxicity against resistant populations, and there are no obvious differences in activities of P450s or GSTs between resistant populations and the susceptible strain. These results suggest that esterases might take pivotal role in conferring metabolic resistance to metaflumizone in the field populations of S. exigua, and P450s or GSTs are not involved in this resistance. Moreover, flavin-dependent monooxygenases (FMOs) are discovered to involve in metaflumizone resistance in the field populations of S. exigua. The FMO inhibitor, methimazole, potentiates metaflumizone toxicity in resistant larva of this species substantially. The synergism ratios for methimazole in resistant populations HZ11 and HZ12 were 3.1 and 1.9, respectively. Enzymatic assays also revealed higher FMO activities in resistant populations than in the susceptible strain, and successive selection with metaflumizone further increased the FMO activity in the field resistant population, but not significantly. The higher FMO activities in the older larval stages and in the larval midgut signify the importance of FMO in the detoxification of xenobiotic from food sources. The synergism assay and FMO activity analysis suggest that FMO contributes to metaflumizone detoxification in resistant populations of S. exigua and conferred metaflumizone resistance in S. exigua. A novel mechanism for insecticide resistance by insect was proposed.

Genetic variation in aldo‐keto reductase 1D1 (AKR1D1) affects expression and activity of multiple cytochrome P450s (292.1)

Human liver gene regulatory network analysis identified a cytochrome P450 subnetwork with AKR1D1 as its key regulatory driver. This study assessed the biologic importance of AKR1D1 [key enzyme in synthesis of bile acids, ligand activators of FXR, PXR and CAR, known P450 regulators] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, 2C8, 2C9, 2C19, and 2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. Resequencing AKR1D1 from donor livers identified a 3'‐UTR (rs1872930) SNP that was significantly associated with higher AKR1D1 mRNA. AKR1D1 3'‐UTR‐luciferase reporter studies showed that variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. AKR1D1 3'‐UTR SNP was significantly associated with increased hepatic mRNA of multiple P450s (CYP3A4, 2C8, 2C9, 2C19, and 2B6) and CYP3A4, 2C8, 2C19, and 2B6 activities. After adjusting for multiple testing, association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of P450 network.

Genetic variation in aldo‐keto reductase 1D1 (AKR1D1) affects expression and activity of multiple cytochrome P450s (LB595)

Human liver gene regulatory network analysis identified a cytochrome P450 subnetwork with AKR1D1 as its key regulatory driver. This study assessed the biologic importance of AKR1D1 [key enzyme in synthesis of bile acids, ligand activators of FXR, PXR and CAR, known P450 regulators] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, 2C8, 2C9, 2C19, and 2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. Resequencing AKR1D1 from donor livers identified a 3'‐UTR (rs1872930) SNP that was significantly associated with higher AKR1D1 mRNA. AKR1D1 3'‐UTR‐luciferase reporter studies showed that variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. AKR1D1 3'‐UTR SNP was significantly associated with increased hepatic mRNA of multiple P450s (CYP3A4, 2C8, 2C9, 2C19, and 2B6) and CYP3A4, 2C8, 2C19, and 2B6 activities. After adjusting for multiple testing, association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of P450 network.

Genetic variation in aldo-keto reductase 1D1 (AKR1D1) affects the expression and activity of multiple cytochrome P450s.

Human liver gene regulatory (Bayesian) network analysis was previously used to identify a cytochrome P450 (P450) gene subnetwork with Aldo-keto reductase 1D1 (AKR1D1) as a key regulatory driver of this subnetwork. This study assessed the biologic importance of AKR1D1 [a key enzyme in the synthesis of bile acids, ligand activators of farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), known transcriptional regulators of P450s] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. We resequenced AKR1D1 from 98 donor livers and identified a 3'-untranslated region (UTR) (rs1872930) single nucleotide polymorphism (SNP) significantly associated with higher AKR1D1 mRNA expression. AKR1D1 3'-UTR-luciferase reporter studies showed that the variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. Consistent with AKR1D1's putative role as a driver of the P450 subnetwork, the AKR1D1 3'-UTR SNP was significantly associated with increased hepatic mRNA expression of multiple P450s (CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6) and CYP3A4, CYP2C8, CYP2C19, and CYP2B6 activities. After adjusting for multiple testing, the association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA expression and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy and adverse drug events. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of the P450 network.

Transcription profiling of two cytochrome P450 genes potentially involved in acaricide metabolism in citrus red mite Panonychus citri

Acaricide resistance in citrus red mite, Panonychus citri (McGregor), has been a growing problem in the management of this pest globally. As a member of main detoxification enzymes, cytochrome P450 monooxygenases (P450s) play important roles in the development of acaricide resistance in arthropods. In this study, two novel P450 genes (named CYP4CF1 and CYP4CL2) were cloned from P. citri. CYP4CF1 and CYP4CL2 had open reading frames of 1527 and 1758 nucleotides encoding 508 and 585 amino acids, respectively. The putative proteins shared 34% identity with each other. Phylogenetic analysis showed that CYP4CF1 and CYP4CL2 were most closely related to CYP4CF2 and CYP4CL1 from Tetranychus urticae, respectively. The transcriptional activities of CYP4CF1 and CYP4CL2 were also investigated. Quantitative RT-PCR revealed that the expression level of CYP4CF1 was increased after induction by pyridaben, while that of CYP4CL2 increased after induction by abamectin, azocyclotin, pyridaben, and spirodiclofen. The mRNA levels of both CYP4s were significantly higher in larval stages, as well as in a field resistant population (BB) than in laboratory susceptible strain (LS). The identification of this detoxification enzyme activity showed that the activity of P450s was higher than the control after exposure to azocyclotin, pyridaben and spirodiclofen. The results suggested that two CYP4 genes might play distinct but significant roles in larvae of P. citri. The results further indicate that CYP4CF1 is associated with pyridaben metabolism, while CYP4CL2 may be involved in detoxification of abamectin, azocyclotin, pyridaben, and spirodiclofen.

Effect of homomeric P450–P450 complexes on P450 function

Previous studies have shown that the presence of one P450 enzyme can affect the function of another. The goal of the present study was to determine if P450 enzymes are capable of forming homomeric complexes that affect P450 function. To address this problem, the catalytic activities of several P450s were examined in reconstituted systems containing NADPH-POR (cytochrome P450 reductase) and a single P450. CYP2B4 (cytochrome P450 2B4)-, CYP2E1 (cytochrome P450 2E1)- and CYP1A2 (cytochrome P450 1A2)-mediated activities were measured as a function of POR concentration using reconstituted systems containing different concentrations of P450. Although CYP2B4-dependent activities could be explained by a simple Michaelis-Menten interaction between POR and CYP2B4, both CYP2E1 and CYP1A2 activities generally produced a sigmoidal response as a function of [POR]. Interestingly, the non-Michaelis behaviour of CYP1A2 could be converted into a simple mass-action response by increasing the ionic strength of the buffer. Next, physical interactions between CYP1A2 enzymes were demonstrated in reconstituted systems by chemical cross-linking and in cellular systems by BRET (bioluminescence resonance energy transfer). Cross-linking data were consistent with the kinetic responses in that both were similarly modulated by increasing the ionic strength of the surrounding solution. Taken together, these results show that CYP1A2 forms CYP1A2-CYP1A2 complexes that exhibit altered catalytic activity.

Mechanism of intermolecular interactions of microsomal cytochrome P450s CYP17 and CYP21 involved in steroid hormone biosynthesis

Protein-protein interactions play a significant role in regulation of functional activity of cytochrome P450s. The aim of the present study was to elucidate the molecular interactions between steroidogenic enzymes CYP17 and CYP21 localized in endoplasmic reticulum membranes of adrenal cortex and involved in biosynthesis of corticosteroid hormones. In the present work, we demonstrate for the first time the direct interaction at molecular level between highly purified human recombinant cytochrome P450s in a mixed reconstituted system. The dependence of the interaction between CYP17 and CYP21 on concentration of the redox-partner - NADPH-cytochrome P450 reductase - is demonstrated, and it is shown that electrostatic interactions play a crucial role in the interaction between CYP17 and CYP21.

Induction of Detoxification Enzymes by Quercetin in the Silkworm

Quercetin is one of the most abundant flavonoids and the defense secondary metabolites in plants. In this study, the effect of quercetin on the growth of the silkworm larvae was investigated. Cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and carboxylesterases (COE) were assayed after exposure to different concentrations of quercetin for 3 d (short-term) and 7 d (long-term), respectively. The results showed that the weight gain of the silkworm larvae significantly decreased after the larvae were treated by different concentrations of quercetin except for the treatment with 0.5% quercetin. Activities of P450, GST, and COE were induced by 0.5 or 1% concentration of quercetin. In the midgut, the induction activity of P450s was reached to the highest level (2.3-fold) by 1% quercetin for 7 d, the highest induction activities of GSTs toward CHP and CDNB were 4.1-fold and 2.6-fold of controls by 1% quercetin after 7 d exposure, respectively. For COEs, the highest activity (2.3-fold) was induced by 0.5% quercetin for 7 d. However, P450s in whole body were higher inducible activities in short-term treatment than those in long-term treatment. The responses of eight cytochrome P450 (CYP) genes belonged to CYP6 and CYP9 families and seven GST genes were detected with real-time polymerase chain reaction. In addition, the genes induced by quercetin significantly were confirmed by qRT-PCR. CYP6AB5, CYP6B29, and GSTe8 were identified as inducible genes, of which the highest induction levels were 10.9-fold (0.5% quercetin for 7 d), 6.2-fold (1% quercetin for 7 d), and 7.1-fold (1% quercetin for 7 d), respectively.

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.

Induction of cytochrome P450 family 1 mRNAs and activities in a cell line from the frog Xenopus laevis

Cytochrome P450 family 1 (CYP1) includes four subfamilies of enzymes: CYP1A, CYP1B, CYP1C, and CYP1D. In many vertebrates, CYP1A, 1B, and 1C expression is induced by agonists of the aryl hydrocarbon receptor, including toxic contaminants such as chlorinated dioxins, coplanar chlorinated biphenyls, and polynuclear aromatic hydrocarbons. Assessed at the level of mRNA, protein, or enzyme activity, CYP1s (especially CYP1As) represent potent and popular biomarkers of contaminant exposure in aquatic vertebrates. Alkylated resorufins are synthetic substrates used to detect, quantify, and describe catalytic activities of cytochrome P450s. The ability to oxidize specific resorufin-based substrates can distinguish the catalytic activities of individual CYP1s. Xenopus laevis, the African clawed frog, is the most widely employed amphibian model in aquatic toxicology, yet the number, inducibility, and activities of CYP1s have not been systematically characterized in this species. Here we report the cloning of cDNAs encoding two new CYP1 family members, X. laevis CYP1B and CYP1C, along with an integrated assessment of the induction of alkyloxyuresorufin-O-dealkylase (AROD) activities and mRNA expression of four known X. laevis CYP1s: CYP1A6, CYP1A7, CYP1B, and CYP1C. Using XLK-WG, an X. laevis kidney epithelial cell line, we determined that EROD (ethoxyresorufin substrate) and MROD (methoxyresorufin) were both induced 3000- to 5000-fold following 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) exposure up to 250nM, while BROD (benzyloxyresorufin) and PROD (pentyloxyresorufin) activity was not detectable regardless of TCDD treatment. TCDD induced CYP1A6 and CYP1A7 mRNAs by 2–3 orders of magnitude, while CYP1B and CYP1C were unchanged. The more potent AHR agonist, FICZ (6-formylindolo[3,2-b]carbazole), induced CYP1B up to 10-fold at concentrations between 0.1 and 250nM, while CYP1C induction was less than 3-fold. CYP1B mRNA showed the highest constitutive mRNA expression, 5- to 75-fold greater than the other CYP1 transcripts. Taken together, these results suggest that CYP1A6 and CYP1A7 perform the bulk of EROD and MROD activities we observed in these cells. The ability of each X. laevis CYP1 to catalyze oxidation of individual resorufin substrates remains to be determined. Correlating CYP1 mRNA and induced AROD activity is a significant step toward clarifying the biochemical meaning of these biomarkers and the roles of CYP1 enzymes in X. laevis. The cell culture approach represents an important complement to the long standing use of frog embryos and tadpoles in toxicological studies, providing a well suited model system for determining the molecular mechanisms underlying the regulation of these important biomarkers of contaminant exposure.

Possible evolution of alliarinoside biosynthesis from the glucosinolate pathway in Alliaria petiolata

Nitrile formation in plants involves the activity of cytochrome P450s. Hydroxynitrile glucosides are widespread among plants but generally do not occur in glucosinolate producing species. Alliaria petiolata (garlic mustard, Brassicaceae) is the only species known to produce glucosinolates as well as a γ-hydroxynitrile glucoside. Furthermore, A. petiolata has been described to release diffusible cyanide, which indicates the presence of unidentified cyanogenic glucoside(s). Our research on A. petiolata addresses the molecular evolution of P450s. By integrating current knowledge about glucosinolate and hydroxynitrile glucoside biosynthesis in other species and new visions on recurrent evolution of hydroxynitrile glucoside biosynthesis, we propose a pathway for biosynthesis of the γ-hydroxynitrile glucoside, alliarinoside. Homomethionine and the corresponding oxime are suggested as shared intermediates in the biosynthesis of alliarinoside and 2-propenyl glucosinolate. The first committed step in the alliarinoside pathway is envisioned to be catalysed by a P450, which has been recruited to metabolize the oxime. Furthermore, alliarinoside biosynthesis is suggested to involve enzyme activities common to secondary modification of glucosinolates. Thus, we argue that biosynthesis of alliarinoside may be the first known case of a hydroxynitrile glucoside pathway having evolved from the glucosinolate pathway. An intriguing question is whether the proposed hydroxynitrile intermediate may also be converted to novel homomethionine-derived cyanogenic glucoside(s), which could release cyanide. Elucidation of the pathway for biosynthesis of alliarinoside and other putative hydroxynitrile glucosides in A. petiolata is envisioned to offer significant new knowledge on the emerging picture of P450 functional dynamics as a basis for recurrent evolution of pathways for bioactive natural product biosynthesis.

Functional Evaluation of Genetic and Environmental Regulators of P450 mRNA Levels

Variations in the activities of Cytochrome P450s are one of the major factors responsible for inter-individual differences in drug clearance rates, which may cause serious toxicity or inefficacy of therapeutic drugs. Various mRNA level is one of the key factors for different activity of the major P450 genes. Although both genetic and environmental regulators of P450 gene expression have been widely investigated, few studies have evaluated the functional importance of cis- and trans-regulatory factors and environmental factors in the modulation of inter-individual expression variations of the P450 genes. In this study, we measured the mRNA levels of seven major P450 genes (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5) in 96 liver biopsy samples from Chinese population. Both trans-acting (mRNA levels and non-synonymous SNPs of putative regulator genes) and cis-acting (gene copy number and functional SNPs) factors were investigated to identify the determinants of the expression variations of these seven P450 genes. We found that expression variations of most P450 genes, regulator genes and housekeeping genes were positively correlated at the mRNA level. After partial correlation analysis using ACTB and GAPDH expression to eliminate the effect of global regulators, a UPGMA (Unweighted Pair Group Method with Arithmetic Mean) tree was constructed to reveal the effects of specific regulation networks potentially masked by global regulators. Combined with the functional analysis of regulators, our results suggested that expression variation at the mRNA level was mediated by several factors in a gene-specific manner. Cis-acting genetic variants might play key roles in the expression variation of CYP2D6 and CYP3A5, environmental inducers might play key roles in CYP1A1 and CYP1A2 variation and global regulators might play key roles in CYP2C9 variation. In addition, the functions of regulators that play less important roles in controlling expression variation for each P450 gene were determined.

Effects of Ginkgo biloba extract on the pharmacokinetics and pharmacodynamics of tolbutamide in protein-restricted rats

Effects of repeated administration of Ginkgo biloba extract on pharmacokinetics and pharmacodynamics of tolbutamide were examined in rats fed a low-protein diet. Rats were given a low (7% casein) or control (20% casein) protein diet for 21 days and administered Ginkgo biloba extract (100mg/kg per day) for the last 5 days. Tolbutamide was co-administered on the last day. Blood glucose and plasma tolbutamide concentrations were determined over the subsequent 12h and the activity of hepatic cytochrome P450s were determined at 12h after dosing. There were significant decreases in body weight, the ratio of liver to body weight, and plasma albumin concentrations in rats on the low-protein diet compared with controls. The hypoglycaemic effect of tolbutamide was significantly greater and the concentration of the drug in plasma was higher in the former group. The repeated administration of Ginkgo biloba extract had little influence on the hypoglycaemic effect of tolbutamide, but tended to decrease the drug concentration in plasma of control rats, while it reduced significantly the hypoglycaemic action and plasma concentration of tolbutamide in the protein-restricted rats. The effects of Ginkgo biloba extract on the pharmacokinetics and pharmacodynamics of tolbutamide were significantly enhanced in rats on the low-protein diet.

Bioelectronic Delivery of Electrons to Cytochrome P450 Enzymes

Cytochrome P450s (cyt P450s) are the major oxidative enzymes in human oxidative metabolism of drugs and xenobiotic chemicals. In nature, the iron heme cyt P450s utilize oxygen and electrons delivered from NADPH by a reductase enzyme to oxidize substrates stereo- and regioselectively. Significant research has been directed toward achieving these events electrochemically. This Feature Article discusses the direct electrochemistry of cyt P450s in thin films and the utilization of such films for electrochemically driven biocatalysis. Maintaining and confirming structural integrity and catalytic activity of cyt P450s in films is an essential feature of these efforts. We highlight here our efforts to elucidate the influence of iron heme spin state and secondary structure of human cyt P450s on voltammetric and biocatalytic properties, using methodologies to quantitatively describe the dynamics of these processes in thin films. We also describe the first cyt P450/reductase films that accurately mimic the natural biocatalytic pathway and show how they can be used with voltammetry to elucidate key mechanistic features. Such bioelectronic cyt P450 systems have high value for future drug development, toxicity screening, fundamental investigations, and chemical synthesis systems.

Thiomers: Inhibition of cytochrome P450 activity

The aim of the present study was to investigate the potential of different thiolated polymers (thiomers) on the catalytic activity of CYP450s on one hand and to explore new inhibitors for CYP activity on the other hand. Several thiolated polymers including poly(acrylic acid)–cysteine (PAA–cysteine), chitosan–thioglycolic acid (chitosan–TGA), and thiolated PEG- g-PEI copolymer along with brij® 35, myrj® 52 and the well-established CYPP450 inhibitor verapamil were screened for their CYP3A4 and CYP2A6 inhibitory activity, and their IC 50 values were determined. Both enzyme inhibition assays were performed in 96-well microtiter plates. 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC) and 7-hydroxycoumarin (7-HC) were used as fluorescent substrates in order to determine CYP3A4 and CYP2A6 catalytic activity, respectively. All investigated compounds inhibited CYP3A4 as well as CYP2A6 activity. All tested (thiolated) polymers were found to be more potent inhibitors of CYP3A4 than of CYP2A6 catalytic activity. Apart from verapamil that is a known CYP3A4 inhibitor, brij® 35 and myrj® 52 were explored as potent inhibitors of CYP3A4 and CYP2A6 catalytic activity. Among the tested polymers, the rank order for CYP3A4 inhibition was PAA–cysteine (100 kDa) > brij® 35 > thiolated PEG- g-PEI copolymer (16 kDa) > myrj® 52 > PAA (100 kDa) > PAA–cysteine (450 kDa) > verapamil > PAA (450 kDa) > chitosan–TGA (150 kDa) > chitosan (150 kDa). On the other hand, the rank order of CYP2A6 inhibition was brij® 35 > PAA–cysteine (100 kDa) > chitosan–TGA (150 kDa) > PAA (100 kDa) > thiolated PEG- g-PEI copolymer (16 kDa) > PAA–cysteine (450 kDa) > chitosan (150 kDa) > verapamil > PAA (450 kDa) > myrj® 52. Thus, this study suggests that (thiolated) polymers display a promising potential to inhibit cytochrome P450s activity and might turn out to be potentially valuable tools for improving the oral bioavailability of actively secreted compounds by avoiding intestinal metabolism.

Overexpression of CYP3A4, but not of CYP2D6, Promotes Hypoxic Response and Cell Growth of Hep3B Cells

Cytochrome P450s (P450s) contribute to carcinogenesis by activating procarcinogens and also metabolize anti-cancer drugs. The activity and protein levels of P450s are important in cancer risk and in cancer therapy. In this study, we found that overexpression of CYP3A4 induced growth of a human hepatoma cell line, Hep3B. Overexpression of CYP2D6, by comparison, decreased cell growth. An inhibitor of CYP3A4, ketoconazole, significantly suppressed the growth of Hep3B cells overexpressing CYP3A4, but an inhibitor of CYP2D6, quinidine, did not restore Hep3B cell growth to baseline levels. Overexpression of CYP3A4 increased the production of reactive oxygen species, but this was not the cause of the CYP3A4-induced growth. Previously, we showed that CYP3A4 can produce epoxyeicosatrienoic acids (EETs) from arachidonic acid. The CYP3A4-enhanced cell growth was attenuated by a putative EET receptor antagonist, 14,15-EEZE. CYP3A4 promoted progression of the cell cycle from the G1 to the S phase. CYP3A4 also induced a hypoxic response of Hep3B cells, detected as enhanced erythropoietin gene expression (a typical hypoxic response). The cell growth promoted by CYP3A4 was inhibited by PI3K inhibitor LY294002. These results suggest that CYP3A4 plays an important role in tumor progression, independent of the activation of carcinogens and metabolism of anti-cancer drugs.

Progesterone Receptor Membrane Component 1 Inhibits the Activity of Drug-Metabolizing Cytochromes P450 and Binds to Cytochrome P450 Reductase

Progesterone receptor membrane component 1 (PGRMC1) has been shown to interact with several cytochromes P450 (P450s) and to activate enzymatic activity of P450s involved in sterol biosynthesis. We analyzed the interactions of PGRMC1 with the drug-metabolizing P450s, CYP2C2, CYP2C8, and CYP3A4, in transfected cells. Based on coimmunoprecipitation assays, PGRMC1 bound efficiently to all three P450s, and binding to the catalytic cytoplasmic domain of CYP2C2 was much more efficient than to a chimera containing only the N-terminal transmembrane domain. Down-regulation of PGRMC1 expression levels in human embryonic kidney 293 and HepG2 cell lines stably expressing PGRMC1-specific small interfering RNA had no effect on the endoplasmic reticulum localization and expression levels of P450s, whereas enzymatic activities of CYP2C2, CYP2C8, and CYP3A4 were slightly higher in PGRMC1-deficient cells. Cotransfection of cells with P450s and PGRMC1 resulted in PGRMC1 concentration-dependent inhibition of the P450 activities, and this inhibition was partially reversed by increased expression of the P450 reductase (CPR). In contrast, CYP51 activity was decreased by down-regulation of PGRMC1 and expression of PGRMC1 in the PGRMC1-deficient cells increased CYP51 activity. In cells cotransfected with CPR and PGRMC1, strong binding of CPR to PGRMC1 was observed; however, in the presence of CYP2C2, interaction of PGRMC1 with CPR was significantly reduced, suggesting that CYP2C2 competes with CPR for binding to PGRMC1. These data show that in contrast to sterol synthesizing P450, PGRMC1 is not required for the activities of several drug-metabolizing P450s, and its overexpression inhibits those P450 activities. Furthermore, PGRMC1 binds to CPR, which may influence P450 activity.

Effect of Hepatitis C Virus Infection on the mRNA Expression of Drug Transporters and Cytochrome P450 Enzymes in Chimeric Mice with Humanized Liver

The expression of drug transporters and metabolizing enzymes is a primary determinant of drug disposition. Chimeric mice with humanized liver, including PXB mice, are an available model that is permissive to the in vivo infection of hepatitis C virus (HCV), thus being a promising tool for investigational studies in development of new antiviral molecules. To investigate the potential of HCV infection to alter the pharmacokinetics of small molecule antiviral therapeutic agents in PXB mice, we have comprehensively determined the mRNA expression profiles of human ATP-binding cassette (ABC) transporters, solute carrier (SLC) transporters, and cytochrome P450 (P450) enzymes in the livers of these mice under noninfected and HCV-infected conditions. Infection of PXB mice with HCV resulted in an increase in the mRNA expression levels of a series of interferon-stimulated genes in the liver. For the majority of genes involved in drug disposition, minor differences in the mRNA expression of ABC and SLC transporters as well as P450s between the noninfected and HCV-infected groups were observed. The exceptions were statistically significantly higher expression of multidrug resistance-associated protein 4 and organic anion-transporting polypeptide 2B1 and lower expression of organic cation transporter 1 and CYP2D6 in HCV-infected mice. Furthermore, the enzymatic activities of the major human P450s were, in general, comparable in the two experimental groups. These data suggest that the pharmacokinetic properties of small molecule antiviral therapies in HCV-infected PXB mice are likely to be similar to those in noninfected PXB mice. However, caution is needed in the translation of this relationship to HCV-infected patients as the PXB mouse model does not accurately reflect the pathology of patients with chronic HCV infection.