Activation of procarcinogens by human cytochrome P450 enzymes

Enzymatic transformation of most chemical carcinogens is requisite to the formation of electrophiles that cause genotoxicity, and the cytochrome P450 (P450) enzymes are the most prominent enzymes involved in such activation reactions. During the past 15 years the human P450 enzymes have been extensively characterized. Considerable evidence exists that the variation in activity of these enzymes can have important consequences in the actions of drugs. Other studies have been concerned with the activation of procarcinogens by human P450s. Assignments of roles of particular P450s in the metabolism of chemical carcinogens are discussed, along with the current state of evidence for relationships of particular P450s with human cancer.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

The roles of cytochrome b5 in cytochrome P450 reactions

Cytochrome b(5), a 17-kDa hemeprotein associated primarily with the endoplasmic reticulum of eukaryotic cells, has long been known to augment some cytochrome P450 monooxygenase reactions, but the mechanism of stimulation has remained controversial. Studies in recent years have clarified this issue by delineating three pathways by which cytochrome b(5) augments P450 reactions: direct electron transfer of both required electrons from NADH-cytochrome b(5) reductase to P450, in a pathway separate and independent of NADPH-cytochrome P450 reductase; transfer of the second electron to oxyferrous P450 from either cytochrome b(5) reductase or cytochrome P450 reductase; and allosteric stimulation of P450 without electron transfer. Evidence now indicates that each of these pathways is likely to operate in vivo.     

细胞色素P450酶的结构、功能与应用研究进展

细胞色素P450 (cytochrome P450,CYP)酶是广泛存在于微生物、动植物及人体中与膜结合的血红蛋白类酶,具有氧化、环氧化、羟化、去甲基化等多种生物催化活性。CYP酶在药物、类固醇、脂溶性维生素和许多其他类型化学物质的代谢中具有重要作用,其在异源物质的解毒、药物相互作用和内分泌功能等领域的研究是热点问题。本综述对CYP的结构、功能、临床应用与开发前景进行了概述,并对其最新的研究现状和发展前景进行探讨。     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

Psychopharmacological profile of Chamomile (Matricaria recutita L.) essential oil in mice

In this study, the effect of Matricaria recutita L. essential oil (MEO) on the central nervous system (CNS) of mice was investigated using some behavioral methods. Chemical profiling both by GC and GC-MS analyses of the hydrodistilled essential oil of M. recutita revealed α-bisabolol oxide A (28%), α-bisabolol oxide B (17.1%), (Z)-β-Farnesene (15.9%) and α-bisabolol (6.8%) as the main components. Changes induced by MEO (25, 50 and 100 mg/kg) and reference drug caffeine (25 mg/kg) in spontaneous locomotor activities and motor coordinations of mice were investigated by activity cage measurements and Rota-Rod tests, respectively. Open field, social interaction and elevated plus-maze tests were applied to assess the emotional state of the animals. Further, tail-suspension test was performed for evaluating the effect of MEO on depression levels of mice. As a result, at 50 and 100 mg/kg, MEO significantly increased the numbers of spontaneous locomotor activities, exhibited anxiogenic effect in the open field, elevated plus-maze and social interaction tests and decreased the immobility times of animals in tail suspension tests. The falling latencies in Rota-Rod tests did not change. This activity profile of MEO was similar to the typical psychostimulant caffeine. The exact mechanism of action underlying this stimulant-like effect should be clarified with further detailed studies.     

Function of human cytochrome P450s: characterization of the orphans

The human genome has now been established to contain 57 cytochrome P450 genes. The proteins can be grouped into categories of types of substrates, including sterols, fatty acids, eicosanoids, and fat-soluble vitamins. Some P450s have also been demonstrated to have significant roles in the metabolism of drugs and chemicals. In addition to these, at least 13 can be considered to still be without apparent function with endogenous or xenobiotic substrates. The current list includes P450s 2A7, 2S1, 2U1, 2W1, 3A43, 4A22, 4F11, 4F22, 4V2, 4X1, 4Z1, 20A1, and 27C1. Limited information is available about the sites of mRNA expression of some of these orphans. Some strategies for characterization are discussed.     

Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography–mass spectrometry

A sensitive negative ion chemical ionization (NCI) gas chromatographic–mass spectrometric (GC–MS) method was modified for the quantitation of valproic acid (VPA) metabolites generated from in vitro cDNA-expressed human microsomal cytochrome P450 incubations. The use of the inherent soft ionization nature of electron-capture NCI to achieve high sensitivity enabled us to conduct kinetic studies using small amounts of recombinant human P450 enzymes. The assay is based on the selective ion monitoring of the intense [M−181] fragments of pentafluorobenzyl (PFB) esters in the NCI mode, and has the following features: (1) a micro-extraction procedure to isolate VPA metabolites from small incubation volumes (100 μl); (2) a second step derivatization with tert.-butyldimethylsilylating reagents to enhance sensitivity for hydroxylated metabolites; (3) a short run-time (<30 min) while maintaining full separation of 15 VPA metabolites by using a narrow-bore non-polar DB-1 column plus a new temperature gradient; and (4) good reproducibility and accuracy (intra- and inter-assay RSDs <15%, bias <15%) by using seven deuterated derivatives of analytes as internal standards. The derivatives of mono- and diunsaturated metabolites, like the parent drug, produced abundant [M−181]⁻ ions while the hydroxylated metabolites gave an ion at m/z of 273, corresponding to the [M−181]⁻ ion of the tert.-butyldimethylsilyl ethers. In conclusion, the GC–NCI-MS analysis of valproate metabolites provided us with a high resolution and sensitivity necessary to conduct metabolic and kinetic studies of valproic acid in small volume samples typical of the in vitro cDNA-expressed micro-incubation enzymatic systems.     

Xenobiotic metabolizing enzymes in the central nervous system: Contribution of cytochrome P450 enzymes in normal and pathological human brain

The metabolism of xenobiotics in human brain constitutes a field of recent intensive research in relation to the potential implications in the pharmacological effect of drugs acting on the central nervous system. Cytochrome P450 enzymes (CYPs) play a crucial role in these metabolic pathways and the existence of functional CYP monooxygenases in brain is now well established. These enzymes are preferentially localized in the neuronal cells within the microsomal fraction and the inner membrane of mitochondria. Although low, the metabolism in situ could influence individual response to xenobiotics or produce reactive, toxic metabolites causing irreversible damage in the neuronal cells. The abundant presence of CYPs in selective cell populations within different regions of the brain has also suggested a role for these enzymes in brain physiology thus not restricted to xenobiotic-induced neurotoxicity. For instance, CYPs participate in the regulation of neurotransmitters and steroids and brain maintenance of cholesterol homeostasis. Recent advances support an additional role for these enzymes in the pathogenesis of psychiatric and neurodegenerative disorders such as depression, schizophrenia, and Alzheimer's and Parkinson's diseases. The characterization of brain CYP isoforms and their localization, the identification of their substrates and metabolic end-products will allow better understanding of the role of these enzymes in brain physiology, development and diseases.     

Cross-linking of human cytochrome P450 2B6 to NADPH-cytochrome P450 reductase: Identification of a potential site of interaction

The site(s) of interaction between human cytochrome P450 2B6 and NADPH-cytochrome P450 reductase (P450 reductase) have yet to be identified. To investigate this, the cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used to covalently link P450 2B6-P450 reductase. Following digestion with trypsin, the cross-linked peptides were identified by reconstituting the peptides in ¹⁸O-water based on the principle that the cross-linked peptides would be expected to incorporate twice as many ¹⁸O atoms as the non-cross-linked peptides. Subsequent mass spectrometric analyses of the resulting peptides led to the identification of one cross-linked peptide candidate. De novo sequencing of the peptide indicated that it is a complex between residues in the C-helix of the P450 (based upon solved X-ray crystal structures of P450 2B4) and the connecting domain of the P450 reductase. To confirm this experimentally, the P450 2B6 peptide identified through the cross-linking studies was synthesized and peptide competition studies were performed. In the presence of the synthetic peptide, P450 catalytic activity was decreased by up to 60% when compared to competition studies performed using a nonsense peptide. Taken together, these studies indicate that residues in the C-helix of P450 2B6 play a major role in the interaction with the P450 reductase.     

Inhibitory effects of phthalimide derivatives on the activity of the hepatic cytochrome P450 monooxygenases CYP2C9 and CYP2C19

Affecting hepatic cytochrome (CYP) activity is one of the major concerns in drug–drug interaction. Thus the testing of drug candidates on their impact on these enzymes is an essential step in early drug discovery. We tested a collection of 480 in-house phthalimide derivatives against different CYP450s using a high throughput inhibition assay. In initial tests with the isoform CYP2C19 about 57.5% of the tested phthalimide derivatives showed significantly enhanced inhibitory effects against this enzyme. In addition similar patterns of phthalimide inhibition for CYP2C9 and CYP2C19 were found, whereas the unrelated isoforms CYP2D6 and CYP3A4 were not specifically affected. Also less than 10% of randomly chosen substances inhibited CYP2C9. Analyses of structure-function relationships revealed that the substituent at the nitrogen atom in the isoindole ring is of crucial impact for the activity of CYP2C9/19.     

Spectroelectrochemistry of cytochrome P450cam

The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis.     

Differential effect of phenobarbital and beta-naphthoflavone on the mRNAs coding for cytochrome P450 and NADPH cytochrome P450 reductase

The induction in rat liver of a specific variant(s) of cytochrome P450 (PB-P450) by phenobarbital and its repression by β-naphthoflavone occur through corresponding changes in the levels of mRNA coding for the protein(s). The level of translatable mRNA coding for NADPH-cytochrome P450 reductase in rat liver increases on treatment with phenobarbital but not β-naphthoflavone.     

Assessment of the genetic variation of chamomile (Matricaria chamomilla L.) populations using phytochemical,morphological and ISSR markers

Inter simple sequence repeat (ISSR) markers were used to assess the genetic diversity of 23 populations of chamomile. Ten primers were applied, producing 193 polymorphic bands. The polymorphic bands were analysed using Simple Matching (SM) similarity index and UPGMA cluster analysis. The resulting dendrogram consisted of four major groups which were in accordance to the geographical regions from which the populations had originated. Populations from North-western (NW), South-Eastern (SE), Central (C) Iran and Europe tended to be classified separately into groups. The highest gene diversity was observed in populations from the Central regions of Iran (0.25). Principal coordinate analysis (PCoA) confirmed the results of cluster analysis, while morphological classification was not related to molecular results. The population from Hungary had the greatest yield of essential oil (0.88 gr/dw), whereas for Iranian ones the greatest and the lowest essential oil yields belonged to NW (Aza = 0.6 and Ard = 0.7) and C (Isf 4, Isf 5 and Isf 6 = 0.44) regions, respectively. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the chamomile germplasm.     

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.     

Measurement of immunoreactive cytochrome P450 2E1 in human leucocytes

We describe initial results on a Western blotting method, using a ployclonal antibody and chemiluminescence detection, for the measurement of cytochrome P450 2E1 in human lymphocytes. The method has been used to study the levels of 2E1 in lymphocytes isolated from 5 ml blood samples collected from a small group of well-controlled type 1 diabetics and healthy individuals. The described method offers increased sensitivity compared with a previously published method and does not need in vitro culturing of the lymphocytes prior to 2E1 measurement. The apparent molecular weight of the lymphocyte P450 2E1 was 55 kDa. There was approximately a six-fold difference in expression levels of 2E1 detected by this immunochemical technique across the study population.     

Measurement of immunoreactive cytochrome P450 2E1 in human leucocytes

We describe initial results on a Western blotting method, using a ployclonal antibody and chemiluminescence detection, for the measurement of cytochrome P450 2E1 in human lymphocytes. The method has been used to study the levels of 2E1 in lymphocytes isolated from 5 ml blood samples collected from a small group of well-controlled type 1 diabetics and healthy individuals. The described method offers increased sensitivity compared with a previously published method and does not need in vitro culturing of the lymphocytes prior to 2E1 measurement. The apparent molecular weight of the lymphocyte P450 2E1 was 55 kDa. There was approximately a six-fold difference in expression levels of 2E1 detected by this immunochemical technique across the study population.     

Spectroscopic characterization of cytochrome P450 Compound I

The cytochrome P450 protein-bound porphyrin complex with the iron-coordinated active oxygen atom as Fe(IV)O is called Compound I (Cpd I). Cpd I is the intermediate species proposed to hydroxylate directly the inert carbon–hydrogen bonds of P450 substrates. In the natural reaction cycle of cytochrome P450 Cpd I has not yet been detected, presumably because it is very short-lived. A great variety of experimental approaches has been applied to produce Cpd I artificially aiming to characterize its electronic structure with spectroscopic techniques. In spite of these attempts, none of the spectroscopic studies of the last decades proved capable of univocally identifying the electronic state of P450 Cpd I. Very recently, however, Rittle and Green [9] have shown that Cpd I of CYP119, the thermophillic P450 from Sulfolobus acidocaldarius, is univocally a Fe(IV)O–porphyrin radical with the ferryl iron spin (S = 1) antiferromagnetically coupled to the porphyrin radical spin (S′ = 1/2) yielding a S_tot = 1/2 ground state very similar to Cpd I of chloroperoxidase from Caldariomyces fumago. In this mini-review the efforts to characterize Cpd I of cytochrome P450 by spectroscopic methods are summarized.     

Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase‐null mice

Previous studies have demonstrated that the NADH‐dependent cytochrome b₅ electron transfer pathway can support some cytochrome P450 monooxygenases in vitro in the absence of their normal redox partner, NADPH‐cytochrome P450 oxidoreductase. However, the ability of this pathway to support P450 activity in whole cells and in vivo remains unresolved. To address this question, liver microsomes and hepatocytes were prepared from hepatic cytochrome P450 oxidoreductase‐null mice and chlorzoxazone hydroxylation, a reaction catalyzed primarily by cytochrome P450 2E1, was evaluated. As expected, NADPH‐supported chlorzoxazone hydroxylation was absent in liver microsomes from oxidoreductase‐null mice, whereas NADH‐supported activity was about twofold higher than that found in normal (wild‐type) liver microsomes. This greater activity in oxidoreductase‐null microsomes could be attributed to the fourfold higher level of CYP2E1 and 1.4‐fold higher level of cytochrome b₅. Chlorzoxazone hydroxylation in hepatocytes from oxidoreductase‐null mice was about 5% of that in hepatocytes from wild‐type mice and matched the results obtained with wild‐type microsomes, where activity obtained with NADH was about 5% of that obtained when both NADH and NADPH were included in the reaction mixture. These results argue that the cytochrome b₅ electron transfer pathway can support a low but measurable level of CYP2E1 activity under physiological conditions. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:357–363, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20299     

An inhibition study of beauvericin on human and rat cytochrome P450 enzymes and its pharmacokinetics in rats

Beauvericin is a secondary metabolite natural product from microorganisms and has been shown to have a new potential antifungal activity. In this study, the metabolism and inhibition of beauvericin in human liver microsomes (HLM) and rat liver microsomes (RLM) were investigated. The apparent K_m and V_max of beauvericin in HLM were determined by substrate depletion approach and its inhibitory effects on cytochromes P450 (CYP) activities were evaluated using probe substrates, with IC₅₀ and the (K_i) values were 1.2 μM (0.5 μM) and 1.3 μM (1.9 μM), respectively for CYP3A4/5 (midazolam) and CYP2C19 (mephenytoin). Similarly, beauvericin was also a potent inhibitor for CYP3A1/2 (IC₅₀: 1.3 μM) in RLM. Furthermore, the pharmacokinetics of beauvericin in the rat were studied after p.o administration alone and co-administration with ketoconazole, which indicated a pharmacodynamic function may play a role in the synergistic effect on antifungal activity.