Characterize Cytochrome P450 Research Articles

Activity-Based Protein Profiling to Probe Relationships between Cytochrome P450 Enzymes and Early-Age Metabolism of Two Polycyclic Aromatic Hydrocarbons (PAHs): Phenanthrene and Retene.

A growing body of literature has linked early-life exposures to polycyclic aromatic hydrocarbons (PAH) with adverse neurodevelopmental effects. Once in the body, metabolism serves as a powerful mediator of PAH toxicity by bioactivating and detoxifying PAH metabolites. Since enzyme expression and activity vary considerably throughout human development, we evaluated infant metabolism of PAHs as a potential contributing factor to PAH susceptibility. We measured and compared rates of phenanthrene and retene (two primary PAH constituents of woodsmoke) metabolism in human hepatic microsomes from individuals ≤21 months of age to a pooled sample (n = 200) consisting primarily of adults. We used activity-based protein profiling (ABPP) to characterize cytochrome P450 enzymes (CYPs) in the same hepatic microsome samples. Once incubated in microsomes, phenanthrene demonstrated rapid depletion. Best-fit models for phenanthrene metabolism demonstrated either 1 or 2 phases, depending on the sample, indicating that multiple enzymes could metabolize phenanthrene. We observed no statistically significant differences in phenanthrene metabolism as a function of age, although samples from the youngest individuals had the slowest phenanthrene metabolism rates. We observed slower rates of retene metabolism compared with phenanthrene also in multiple phases. Rates of retene metabolism increased in an age-dependent manner until adult (pooled) metabolism rates were achieved at ∼12 months. ABPP identified 28 unique CYPs among all samples, and we observed lower amounts of active CYPs in individuals ≤21 months of age compared to the pooled sample. Phenanthrene metabolism correlated to CYPs 1A1, 1A2, 2C8, 4A22, 3A4, and 3A43 and retene metabolism correlated to CYPs 1A1, 1A2, and 2C8 measured by ABPP and vendor-supplied substrate marker activities. These results will aid efforts to determine human health risk and susceptibility to PAHs exposure during early life.

Diet-dependent gene expression highlights the importance of Cytochrome P450 in detoxification of algal secondary metabolites in a marine isopod

Isopods of the genus Idotea have an unusual ability to feed on algae containing high amounts of chemical defense molecules, such as species of the genera Fucus and Ulva. In this study, we compared gene expression patterns of Idotea balthica individuals fed with Fucus vesiculosus to individuals fed with Ulva lactuca. We generated the first-ever transcriptome assembly for this species, and found 3,233 differentially expressed genes across feeding regimes. However, only a handful of biological functions were enriched with regard to differentially expressed genes, the most notable being “alkaloid metabolic process”. Within this category, we found eight differentially expressed cytochrome P450 (CYP) unigenes, all of which had a higher expression in the U. lactuca diet treatment. A phylogenetic analysis showed that the differentially expressed CYP genes are closely related to a CYP gene described from the hepatopancreas of the spiny lobster Panulirus argus, and we hypothesize that these transcripts are involved in metabolite detoxification. This is a first step in the understanding of this algae-grazer interaction, and will form a basis for future work to characterize cytochrome P450 functioning in marine crustaceans.

In vitro and in silico Approaches to Study Cytochrome P450-Mediated Interactions

In vitro and in silico models of drug metabolism are utilized regularly in the drug research and development as tools for assessing pharmacokinetic variability and drug-drug interaction risk. The use of in vitro and in silico predictive approaches offers advantages including guiding rational design of clinical drug-drug interaction studies, minimization of human risk in the clinical trials, as well as cost and time savings due to lesser attrition during compound development process. This article gives a review of some of the current in vitro and in silico methods used to characterize cytochrome P450(CYP)-mediated drug metabolism for estimating pharmacokinetic variability and the magnitude of drug-drug interactions. Examples demonstrating the predictive applicability of specific in vitro and in silico approaches are described. Commonly encountered confounding factors and sources of bias and error in these approaches are presented. With the advent of technological advancement in high throughput screening and computer power, the in vitro and in silico methods are becoming more efficient and reliable and will continue to contribute to the process of drug discovery, development and ultimately safer and more effective pharmacotherapy.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Gene expression and immunochemical localization of major cytochrome P450 drug-metabolizing enzymes in bovine nasal olfactory and respiratory mucosa

Despite tremendous advancement in the characterization of nasal enzyme expression, knowledge of the role of the nasal mucosa in the metabolism of xenobiotics is still inadequate, primarily due to the limited availability of in vitro models for nasal metabolism screening studies. An extensive knowledge of the oxidative and conjugative metabolizing capacity of the cattle (Bos taurus) olfactory and respiratory mucosa can aid in efficient use of these tissues for pre-clinical investigations of the biotransformation and toxicity of therapeutic agents following nasal administration or inhalation. Cows are also exposed to a variety of airborne pollutants and pesticides during their lifetime, the metabolism of which can have profound toxicological and ecological consequences. The aim of the present study was to characterize cytochrome P450 (CYP) enzyme expression in the bovine nasal mucosa. Amplification of the specific genes through RT-PCR confirmed expression of several CYP enzymes in bovine hepatic and nasal tissues. The results demonstrate that bovine nasal olfactory and respiratory mucosal and liver tissues express similar populations, families, and distributions of CYP enzymes, as has been previously reported with other species, including humans. Bovine ex vivo tissues can serve as a readily available reference tissue to elucidate preclinical toxico-kinetic effects resulting from exposure to substances in the environment or following drug administration.

Inhibition of cytochrome P450 enzymes involved in ketamine metabolism by use of liver microsomes and specific cytochrome P450 enzymes from horses, dogs, and humans

To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies. Human, canine, and equine liver microsomes and human single CYP3A4 and CYP2C9 and their canine orthologs. Chemical inhibitors selective for human CYP enzymes and anti-CYP antibodies were incubated with racemic ketamine and liver microsomes or specific CYPs. Ketamine N-demethylation to norketamine was determined via enantioselective capillary electrophoresis. The general CYP inhibitor 1-aminobenzotriazole almost completely blocked ketamine metabolism in human and canine liver microsomes but not in equine microsomes. Chemical inhibition of norketamine formation was dependent on inhibitor concentration in most circumstances. For all 3 species, inhibitors of CYP3A4, CYP2A6, CYP2C19, CYP2B6, and CYP2C9 diminished N-demethylation of ketamine. Anti-CYP3A4, anti-CYP2C9, and anti-CYP2B6 antibodies also inhibited ketamine N-demethylation. Chemical inhibition was strongest with inhibitors of CYP2A6 and CYP2C19 in canine and equine microsomes and with the CYP3A4 inhibitor in human microsomes. No significant contribution of CYP2D6 to ketamine biotransformation was observed. Although the human CYP2C9 inhibitor blocked ketamine N-demethylation completely in the canine ortholog CYP2C21, a strong inhibition was also obtained by the chemical inhibitors of CYP2C19 and CYP2B6. Ketamine N-demethylation was stereoselective in single human CYP3A4 and canine CYP2C21 enzymes. Human-specific inhibitors of CYP2A6, CYP2C19, CYP3A4, CYP2B6, and CYP2C9 diminished ketamine N-demethylation in dogs and horses. To address drug-drug interactions in these animal species, investigations with single CYPs are needed.

Development of an optimized procedure for the preparation of rat intestinal microsomes: comparison of hepatic and intestinal microsomal cytochrome P450 enzyme activities in two rat strains

The objective of this study was to characterize cytochrome P450 (CYP) activities in both intestinal and hepatic microsomes from Wistar and Sprague–Dawley rats.Specific probes for measuring CYP activities were selected using rat recombinant CYP.The intestinal microsome preparation was optimized getting a more relevant and reproducible abundance of CYPs to measure CYP activities.Testosterone, propranolol, diclofenac, and midazolam were determined as specific substrates of rat CYP2C11, CYP2D2, CYP2C6, and CYP3A, respectively. Ethoxyresorufin and pentoxyresorufin were not specific substrates of CYP1A2 and CYP2B1, respectively. Hepatic and intestinal microsomes expressed active CYP1A1, CYP1A2, CYP2B1, and CYP3A2. Only liver expressed active CYP2C6, CYP2C11, and CYP2D2. Wistar liver expressed more active CYP1A and CYP3A2, but less active CYP2B1 than Wistar intestine. Sprague–Dawley liver expressed more active CYP2B1 and CYP3A2, but less active CYP1A than Sprague–Dawley intestine.In conclusion, CYP activities were qualitatively equivalent but not quantitatively in both strains.

Evidence for cytochrome P450 2E1 catalytic activity and expression in rat blood lymphocytes

Studies initiated to characterize cytochrome P450 2E1(CYP2E1) in freshly isolated rat blood lymphocytes revealed significant mRNA of CYP2E1 in control blood lymphocytes. RT-PCR studies have shown that as observed in liver, acute treatment of ethanol (single oral dose of 0.8 ml/kg b.wt , i.p), resulted in increase in the mRNA expression of CYP2E1 in freshly isolated rat blood lymphocytes. Western blotting studies using polyclonal antibody raised against rat liver CYP2E1 demonstrated significant immunoreactivity, comigrating with the liver isoenzyme, in freshly isolated control rat blood lymphocytes. Similar to that seen in liver, pretreatment of ethanol was found to produce an increase in the CYP2E1 isoenzyme in the blood lymphocytes. Blood lymphocytes were also found to catalyze the CYP dependent N-demethylation of N-nitrosodimethylamine (NDMA), which like in liver increased 2–3 fold following pretreatment of rats with known CYP2E1 inducers. Kinetic studies have further shown significant increase in the apparent Vmax and the affinity towards the substrate in rat blood lymphocytes indicating that as observed in liver, the increase in mRNA and protein expression following exposure to CYP2E1 inducers is associated with the increased catalytic activity of CYP2E1 in freshly isolated rat blood lymphocytes. The data indicating similarities of the blood lymphocyte CYP2E1 with the liver enzyme suggest that lymphocyte CYP2E1 levels in freshly isolated rat blood lymphocytes could be used to monitor tissue enzyme levels.

Selectivity of 7-alkoxycoumarins as probe substrates for rat hepatic cytochrome P450 forms is influenced by the substitution pattern on the coumarin nucleus

The O-dealkylation of 7-alkoxycoumarins is widely used as an assay to characterize cytochrome P450 (CYP) activity. These substrates can also undergo oxidative attack at additional sites on the coumarin nucleus, which may influence their apparent selectivity for particular CYP forms.Accordingly, the effect of blockade of these additional sites was investigated on the selectivity towards rat hepatic CYP forms, with emphasis on the CYP1A and 2B forms.Blockade of the 3-/4- and 6-positions resulted in substrates for which the CYP1A1/2 selectivity of the unsubstituted 7-alkoxycoumarins was altered to a CYP2B selectivity; this was achieved with little overall change in the molecular dimensions of the substrate. Limited analysis of other inducible CYP forms indicated at most only small effects of structure modification on activity.The findings suggest that the sensitivity of probe substrates for CYP forms may be limited by the occurrence of competing side reactions of the substrate, and that better probes may be derived by blocking the sites of these side reactions.

In vitro metabolism of a new neuroprotective agent, KR-31543 in the human liver microsomes: identification of human cytochrome P450.

KR-31543, (2S,3R,4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl)amino]-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran, is a new neuroprotective agent for preventing ischemia-reperfusion damage. This study was performed to identify the metabolic pathway of KR-31543 in human liver microsomes and to characterize cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-31543. Human liver microsomal incubation of KR-31543 in the presence of NADPH resulted in the formation of two metabolites, M1 and M2. M1 was identified as N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl)amine on the basis of LC/MS/MS analysis with a synthesized authentic standard, and M2 was suggested to be hydroxy-KR-31543. Correlation analysis between the known CYP enzyme activities and the rates of the formation of M1 and M2 in the 12 human liver microsomes have showed significant correlations with testosterone 6beta-hydroxylase activity (a marker of CYP3A4). Ketoconazole, a selective inhibitor of CYP3A4, and anti-CYP3A4 monoclonal antibodies potently inhibited both N-hydrolysis and hydroxylation of KR-31543 in human liver microsomes. These results provide evidence that CYP3A4 is the major isozyme responsible for the metabolism of KR-31543 to M1 and M2.

Role of human cytochrome P450 3A4 in the metabolism of DA–8159, a new erectogenic

1. The purpose of this paper was to characterize cytochrome P450 (CYP) enzymes involved in N-dealkylation of a new oral erectogenic, DA-8159 to DA-8164, a major circulating active metabolite, in human liver microsomes and to investigate the inhibitory potential of DA-8159 on CYP enzymes.2. CYP3A4 was identified as the major enzyme responsible for DA-8159 N-dealkylation to DA-8164 based on correlation analysis and specific CYP inhibitor and antibody-mediated inhibition study in human liver microsomes, and DA-8159 metabolism in cDNA expressed CYP enzymes. There is the possibility of drug-drug interactions when prescribing DA-8159 concomitantly with known inhibitors or inducers of CYP3A4.3. DA-8159 was found to be only a very weak inhibitor of eight major CYPs (1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4), the largest inhibition occurring against CYP2D6 (IC50 67.7 μM) in human liver microsomes. Drug–drug interactions would not be predicted on the basis of DA-8159 inhibiting the metabolism of coadministered drugs.

Cryotrapped Reaction Intermediates of Cytochrome P450 Studied by Radiolytic Reduction with Phosphorus-32

Unstable reaction intermediates of the cytochrome P450 catalytic cycle have been prepared at cryogenic temperatures using radiolytic one-electron reduction of the oxy-P450 CYP101 complex. Since a rate-limiting step in the catalytic cycle of the enzyme is the reduction of the ferrous oxygenated heme protein, subsequent reaction intermediates do not normally accumulate. Using (60)Co gamma-irradiation, the primary reduced oxy-P450 species at 77 K has been identified as a superoxo- or hydroperoxo-Fe(3+)-heme complex (Davydov, R., Macdonald, I. D. G., Makris, T. M., Sligar, S. G., and Hoffman, B. M. (1999) J. Am. Chem. Soc. 121, 10654-10655). The electronic absorption spectroscopy is an essential tool to characterize cytochrome P450 intermediates and complements paramagnetic methods, which are blind to important diamagnetic or antiferromagnetically coupled states. We report a method of trapping unstable states of redox enzymes using phosphorus-32 as an internal source of electrons. We determine the UV-visible optical spectra of the reduced oxygenated state of CYP101 and show that the primary intermediate, a hydroperoxo-P450, is stable below 180 K and converts smoothly to the product complex at approximately 195 K. In the course of the thermal annealing, no spectral changes indicating the presence of oxoferryl species (the so-called compound I type spectrum) was observed.

Interplay between CYP3A-mediated metabolism and polarized efflux of terfenadine and its metabolites in intestinal epithelial Caco-2 (TC7) cell monolayers.

To further characterize cytochrome P450 (CYP) and P-glycoprotein (Pgp) expression in monolayers of the Caco-2 cell clone TC7, a cell culture model of the human intestinal epithelium. To study the interplay between CYP3A and Pgp as barriers to intestinal drug absorption in TC7 cells using terfenadine and its metabolites as substrates. mRNA expression of eight CYPs and Pgp was investigated in TC7 and parental Caco-2 (Caco-2p) cell monolayers using RT-PCR. The CYP3A kinetics was determined in microsomes from both cell lines. The transport, metabolism and efflux of terfenadine and its metabolites were investigated in TC7 monolayers. Both TC7 and Caco-2p cells expressed mRNA for Pgp and several important CYPs. However, mRNA for CYP3A4 was detectable anly from TC7 cells. The relative affinity of CYP3A for terfenadine metabolism in the two cell lines was comparable, but the maximum reaction rate in the TC7 cells was 8-fold higher. The rate of transport of terfenadine and its metabolites hydroxy-terfenadine (HO-T) and azacyclonol across TC7 monolayers was 7.1-, 3.5- and 2.1-fold higher, respectively, in the basolateral to apical direction than it was in the apical to basolateral (AP-BL) direction. Inhibition studies indicated that the efflux was mediated by Pgp. Ketoconazole increased the AP-BL transport terfenadine dramatically by inhibiting both terfenadine metabolism and Pgp efflux. Cell culture models such as TC7 provide qualitative information on drug interactions involving intestinal CYP3A and Pgp.

Polychlorinated biphenyls: a new type of inducer of cytochrome P-448 in the liver.

The CO-difference spectrum of microsomes from rats treated with the polychlorinated biphenyls mixture, Aroclor 1254, has an absorption maximum at 448 nm. With ethylisocyananide as the ligand for reduced microsomes, Aroclor 1254 treatment causes a shift in the 455-nm peak to 453 nm and increases the ratio of absorbance of 455 nm to that at 430 nm from 0.53, obtained with untreated rats, to 1.24. These findings are similar to those seen in rats treated with the polycyclic hydrocarbon, 3-methylcholanthrene, but differ from those that characterize cytochrome P-450 in control or phenobarbital-treated rats. Aroclor 1254 treatment results in a tripling of cytochrome P-448 content and a 10-fold increase in benzo-[a]pyrene hydroxylation. However-unlike 3-methylcholanthrene, but like the phenobarbital type of inducing agents-Aroclor 1254 treatment causes a significant enhancement of ethylmorphine N-demethylase. These data suggest that Aroclor 1254-induced cytochrome P-448 may be catalytically different from the 3-methylcholanthrene-induced P-448 or that the hemoprotein(s) induced by Aroclor 1254 may be a mixture of cytochromes P-448 and P-450 exhibiting catalytic properties of both cytochromes.