Science.gov

Sample records for liver cytochrome p-450

  1. Nerval influences on liver cytochrome P450.

    PubMed

    Klinger, W; Karge, E; Danz, M; Krug, M

    1995-09-01

    In male young adult Wistar rats the influences of nucleus raphe electrocoagulation, spinal cord dissection (cordotomy between C7 and Th1), vagotomy and denervation of liver hilus by phenol on liver cytochrome P450-system (cytochrome P450 concentration, ethylmorphine N-demethylation and ethoxycoumarin O-deethylation activities, hexobarbitone sleeping time) were investigated. In general the influences were small or negligible when compared with sham operated controls, only after vagotomy the depressing effect of sham operation was abolished. In all cases sham operation had a depressing effect until up to five weeks after operation.

  2. KINETICS OF BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P450 ISOENZYMES IN HUMAN LIVER MICROSOMES

    EPA Science Inventory

    Kinetics of Bromodichloromethane Metabolism by
    Cytochrome P450 Isoenzymes in Human Liver Microsomes

    Guangyu Zhao and John W. Allis

    ABSTRACT
    The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have ...

  3. Spaceflight Effects on Cytochrome P450 Content in Mouse Liver

    PubMed Central

    Moskaleva, Natalia; Moysa, Alexander; Novikova, Svetlana; Tikhonova, Olga; Zgoda, Victor; Archakov, Alexander

    2015-01-01

    Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP) superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM). Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine. PMID:26561010

  4. Inducible cytochrome P-450 from rat liver mitochondria

    SciTech Connect

    Raza, H.; Shayiq, F.M.; Avadhani, N.G.

    1987-05-01

    In the present study they have purified US -naphthoflavone (BNF, which induces isotypes similar to 3-MC) and PB induced mitochondrial isoforms. They have been able to purify two isoforms with molecular weights of 54 Kd and 52 Kd from BNF induced mitochondria. Only the 54 KD form, but not the 52 KD species reacts with the polyclonal antibody to microsomal P-450c, though, both show arylhydrocarbon hydroxylase activity in an in vitro system reconstituted with adrenodoxin and adrenodoxin-reductase. Fingerprint analyses, N-terminal sequencing and use of monoclonal antibody probes show that the two mitochondrial isoforms are different from the microsomal P-450c. Further, the 54 Kd mitochondrial isoform is not detected in control mitochondria indicating that it is truly an induced form. Similarly, a PB induced mitochondrial form which exhibits physical, immunochemical and enzymatic properties different from the microsomal P-450b has also been purified.

  5. Cytochromes P450

    PubMed Central

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  6. Metabolism of sesamin by cytochrome P450 in human liver microsomes.

    PubMed

    Yasuda, Kaori; Ikushiro, Shinichi; Kamakura, Masaki; Ohta, Miho; Sakaki, Toshiyuki

    2010-12-01

    Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k(cat)/K(m) value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

  7. Integration and expression of a rabbit liver cytochrome P-450 gene in transgenic Nicotiana tabacum.

    PubMed Central

    Saito, K; Noji, M; Ohmori, S; Imai, Y; Murakoshi, I

    1991-01-01

    Cytochrome P-450 is involved in the oxidative metabolism of a broad range of substrates. We have made a chimeric construct, pSN002, containing the cDNA for rabbit liver cytochrome P-450 (IIC14) under the control of the TR2' promoter for mannopine synthase in the Agrobacterium Ti plasmid. Nicotiana tabacum was transformed with Agrobacterium tumefaciens harboring a cointegrated plasmid pSN002::pGV2260. The presence of mRNA and of the translated protein from the chimeric cytochrome P-450 gene in transgenic plants was confirmed by RNA blot hybridization and by Western blot and immunohistochemical analyses, respectively. The transformants in which the foreign cytochrome P-450 protein is expressed show marked phenotypic changes, notably a tendency rapidly to senesce. We detected 2-propenylpyrrolidine, a degradative metabolite of nicotine alkaloids, in transgenic tobacco showing this pronounced phenotypic change. Such metabolism is likely to be due to the effect of senescence and not directly to the presence of the cytochrome P-450. Images PMID:1714583

  8. Marmoset cytochrome P450 2D8 in livers and small intestines metabolizes typical human P450 2D6 substrates, metoprolol, bufuralol and dextromethorphan.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Hagihira, Yuya; Murayama, Norie; Shimizu, Makiko; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2015-01-01

    1. Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient. 2. A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83-86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat. 3. Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting. 4. By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1'-hydroxylation. 5. These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6.

  9. Isolation and characterization of a constitutive form of rabbit liver microsomal cytochrome P-450.

    PubMed

    Johnson, E F

    1980-01-10

    A heretofore unrecognized form of cytochrome P-450 was purified from rabbit liver microsomes with an average yield and purity similar to that of other highly purified forms of cytochrome P-450. Several properties of this cytochrome are contrasted with those of form 2, the major phenobarbital-inducible cytochrome P-450, form 4, the major 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome, and form 6, a cytochrome that is selectively induced in liver microsomes by 2,3,7,8-tetrachlorodibenzo-p-dioxin during the perinatal period. Thes four forms can be distinguished by virtue of their molecular weights as determined using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, by their respective peptide fingerprints, and by the monospecificity of their antisera. Since the enumerated properties are thought to reflect the primary structure of the cytochromes and since the observed differences are extensive, we suggest that these four forms are not derived from a common protein precursor.

  10. [Activity of cytochromes P-450p and P-450h in liver microsomes and blood corticosteroid levels in experimental animals under the action of physical factors].

    PubMed

    Zolotareva, T A; Gorchakova, G A; Konovalenko, V L; Konovalenko, L N; Grishanova, A Iu; Guliaeva, L F; Liakhovich, V V

    1992-05-01

    In experiments on male Wistar rats it has been found that physical factors applied in medicine (laser radiation of low intensity with wave length 0.89 microns, microwaves of centimeter range of 2450 MHz, and ultrasound of low intensity 880 KHz) changed catalytic activity of liver microsomal and rostenedione 16 alpha- and 6 beta-hydroxylating cytochromes P-450h and P-450p and blood corticosteroids level. Activities of these two steroid-metabolizing cytochromes decreased under ultrasonic skin application on liver region and increased under microwave and laser action. Contents of physiologically inactive form of corticosterone were not changed by the physical factors action while level of active hormone was increased under ultrasonic and microwave action. These findings suggest association of the activity of liver steroid-metabolizing cytochromes P-450 and level of physiologically active form of corticosterone in blood under physical factors skin application on liver region.

  11. Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae.

    PubMed

    Brian, W R; Sari, M A; Iwasaki, M; Shimada, T; Kaminsky, L S; Guengerich, F P

    1990-12-25

    A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is

  12. Purification and characterization of a benzene hydroxylase: A cytochrome P-450 from rat liver mitochondria

    SciTech Connect

    Karaszkiewicz, J.W.

    1989-01-01

    This laboratory previously demonstrated that incubation of ({sup 14}C)benzene with isolated mitochondria resulted in the formation of mtDNA adducts. Since benzene is incapable of spontaneously covalently binding to nuclei acids, it was hypothesized that enzyme(s) present in the organelle metabolized benzene to reactive derivatives. We have purified, to electrophoretic homogeneity, a 52 kDa cytochrome P-450 from liver mitoplasts which metabolizes benzene to phenol. The enzyme has a K{sub M} for benzene of 0.012 mM, and a V{sub MAX} of 22.6 nmol phenol/nmol P-450/10 min, and requires NADPH, adrenodoxin, and adrenodoxin reductase for activity. Activity also can be reconstituted with microsomal cytochrome P-450 reductase. Benzene hydroxylase activity could be inhibited by carbon monoxide and SKF-525A, and by specific inhibitors of microsomal benzene metabolism. The purified enzyme oxidized phenol, forming catechol; aminopyrine N-demethylase activity was also demonstrated. These data confirm that a cytochrome P-450 of mitochondrial origin is involved in benzene metabolism, and indicate a role for the mitochondrion in xenobiotic activation.

  13. cDNA and derived amino acid sequence of ethanol-inducible rabbit liver cytochrome P-450 isozyme 3a (P-450ALC).

    PubMed Central

    Khani, S C; Zaphiropoulos, P G; Fujita, V S; Porter, T D; Koop, D R; Coon, M J

    1987-01-01

    Administration of ethanol to rabbits is known to induce a unique liver microsomal cytochrome P-450, termed isozyme 3a or P-450ALC, which is responsible for the increased oxidation of ethanol and other alcohols and the activation of toxic or carcinogenic compounds such as acetaminophen and N-nitrosodimethylamine. To further characterize this cytochrome P-450 we have identified cDNA clones to isozyme 3a by immunoscreening, DNA hybridization, and hybridization-selection. The cDNA sequence determined from two overlapping clones contains an open reading frame of 1416 nucleotides, and the first 25 amino acids of this reading frame correspond to residues 21-45 of cytochrome P-450 3a. The complete polypeptide, including residues 1 to 20, contains 492 amino acids and has a molecular weight of 56,820. Cytochrome P-450 3a is approximately 55% identical in sequence to P-450 isozymes 1 and 3b and 48% identical to isozyme 2. Hybridization of clone p3a-2 to electrophoretically fractionated rabbit liver poly(A)+ RNA revealed multiple bands, but, with a probe derived from the 3' nontranslated portion of this cDNA, only a 1.9-kilobase band was observed. Treatment of rabbits with imidazole, which increases the content of isozyme 3a, resulted in a transient increase in form 3a mRNA, but this was judged to be insufficient to account for the known 4.5-fold increase in form 3a protein. Genomic DNA analysis indicated that the cytochrome P-450 3a gene does not belong to a large subfamily. Images PMID:3027695

  14. Expression of cytochromes P-450 in rat hepatoma cells. Analysis by monoclonal antibodies specific for cytochromes P-450 from rat liver induced by 3-methylcholanthrene or phenobarbital.

    PubMed

    Wiebel, F J; Park, S S; Kiefer, F; Gelboin, H V

    1984-12-17

    We have studied the expression of aldrin eposidase (AE), 7-ethoxycoumarin-O-deethylase (ECDE), and aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) in nine differentiated or dedifferentiated cell lines derived from H4IIEC3 rat hepatoma cells. The nature of the cytochromes P-450 mediating AE, ECDE and AHH activities was analysed using monoclonal antibodies (MAb) made to the major 3-methylcholanthrene-induced cytochrome P-450 (MAb-MC) or phenobarbital-induced cytochrome P-450 (MAb-PB) from rat liver. The cells were treated with 5 microM dexamethasone for 30 h to increase the levels of the monoxygenase activities. (a) The six differentiated cell lines examined (Faza967, Fao, HF1-4, 2sFou, C2Rev7, and H4IIEC3/G-) contained MAb-PB-sensitive AE comprising 30-75% of the total AE activity. In most of these cell lines MAb-PB also markedly inhibited ECDE; however, the antibody had a considerably weaker effect on AHH. (b) MAb-PB-sensitive AHH, ECDE and AE activities were also observed in untreated and phenobarbital-treated cells. (c) MAb-MC inhibited AHH and ECDE in the two dedifferentiated lines HF1 and H5 by 50-80%. The antibody also inhibited AHH activities in the poorly differentiated line H4IIEC3/T and in the majority of the differentiated lines by 40-65%. MAb-MC-sensitive AHH was found in Fao cells after treatment with benz[a]anthracene but induced AHH in H4IIEC3/T, H4IIEC3/G-, and 2sFou cells 20-30-fold and in Faza967 and Fao cells 3-5-fold. Benz[a]anthracene remained without effect on AHH activity in C2Rev7 cells. The results show that the hepatoma cells examined express to various degrees phenobarbital-inducible cytochrome P-450 and/or 3-methylcholanthrene-inducible cytochrome P-450. These cell lines are versatile tools for studying the regulation of monooxygenase activities and analysing their role in the activation and inactivation of xenobiotics such as carcinogens, drugs and pesticides.

  15. Characterization of benidipine and its enantiomers' metabolism by human liver cytochrome P450 enzymes.

    PubMed

    Yoon, Yune-Jung; Kim, Kwon-Bok; Kim, Hyunmi; Seo, Kyung-Ah; Kim, Ho-Sook; Cha, In-June; Kim, Eun-Young; Liu, Kwang-Hyeon; Shin, Jae-Gook

    2007-09-01

    Benidipine is a dihydropyridine calcium antagonist that has been used clinically as an antihypertensive and antianginal agent. It is used clinically as a racemate, containing the (-)-alpha and (+)-alpha isomers of benidipine. This study was performed to elucidate the metabolism of benidipine and its enantiomers in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of two metabolites, N-desbenzylbenidipine and dehydrobenidipine. The intrinsic clearance (CL(int)) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)-alpha isomer was similar to those from the (+)-alpha isomer (1.9 +/- 0.1 versus 2.3 +/- 2.3 microl/min/pmol P450 and 0.5 +/- 0.2 versus 0.6 +/- 0.6 microl/min/pmol P450, respectively). Correlation analysis between the known P450 enzyme activities and the rate of the formation of benidipine metabolites in the 15 HLMs showed that benidipine metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in liver microsomes and the incubation study of cDNA-expressed enzymes also showed that theN-debenzylation and dehydrogenation of benidipine are mainly mediated by CYP3A4 and CYP3A5. The total CL(int) values of CYP3A4-mediated metabolite formation from (-)-alpha isomer were similar to those from (+)-alpha isomer (17.7 versus 14.4 microl/min/pmol P450, respectively). The total CL(int) values of CYP3A5-mediated metabolite formation from (-)-alpha isomer were also similar to those from (+)-alpha isomer (8.3 versus 11.0 microl/min/pmol P450, respectively). These findings suggest that CYP3A4 and CYP3A5 isoforms are major enzymes contributing to the disposition of benidipine, but stereoselective disposition of benidipine in vivo may be influenced not by stereoselective metabolism but by other factors.

  16. Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

    PubMed

    Haugen, D A; Coon, M J

    1976-12-25

    Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm

  17. The cytochrome p450 homepage.

    PubMed

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  18. Malathion bioactivation in the human liver: the contribution of different cytochrome p450 isoforms.

    PubMed

    Buratti, Franca M; D'Aniello, Alessandra; Volpe, Maria Teresa; Meneguz, Annarita; Testai, Emanuela

    2005-03-01

    Among organophosphorothioate (OPT) pesticides, malathion is considered relatively safe for use in mammals. Its rapid degradation by carboxylesterases competes with the cytochrome P450 (P450)-catalyzed formation of malaoxon, the toxic metabolite. However, impurities in commercial formulations are potent inhibitors of carboxylesterase, allowing a dramatic increase in malaoxon formation. Malathion desulfuration has been characterized in human liver microsomes (HLMs) with a method based on acetylcholinesterase inhibition that is able to detect nanomolar levels of oxon. The active P450 isoforms have been identified by means of a multifaceted strategy, including the use of cDNA-expressed human P450s and correlation, immunoinhibition, and chemical inhibition studies in a panel of phenotyped HLMs. HLMs catalyzed malaoxon formation with a high level of variability (>200-fold). One or two components (K(mapp1) = 53-67 microM; K(mapp2) = 427-1721 microM) were evidenced, depending on the relative specific P450 content. Results from different approaches indicated that, at low malathion concentration, malaoxon formation is catalyzed by CYP1A2 and, to a lesser extent, 2B6, whereas the role of 3A4 is relevant only at high malathion levels. These results are in line with those found with chlorpyrifos, diazinon, azynphos-methyl, and parathion, characterized by the presence of an aromatic ring in the molecule. Since malathion has linear chains as substituents at the thioether sulfur, it can be hypothesized that, independently from the chemical structure, OPTs are bioactivated by the same P450s. These results also suggest that CYP1A2 and 2B6 can be considered as possible metabolic biomarkers of susceptibility to OPT-induced toxic effects at actual human exposure levels.

  19. The role of brain noradrenergic system in the regulation of liver cytochrome P450 expression.

    PubMed

    Sadakierska-Chudy, Anna; Haduch, Anna; Rysz, Marta; Gołembiowska, Krystyna; Daniel, Władysława A

    2013-09-15

    The aim of the present study was to examine the effect of the brain noradrenergic system on the expression of cytochrome P450 in the liver. The experiment was carried out on male Wistar rats. Intracerebroventricular injection of the noradrenergic neurotoxin DSP-4 diminished noradrenaline level in the brain. Simultaneously, significant decreases in the serum concentration of the growth hormone, testosterone and the thyroid hormone thyroxine, as well as an increase in corticosterone level were observed. The concentrations of triiodothyronine and the cytokines interleukine 2 (IL-2) and 6 (IL-6) were not changed by DSP-4. The neurotoxin produced complex changes in the functioning of cytochrome P450. Significant decreases in the activity of liver CYP2C11 (measured as a rate of the 2α- and 16α-hydroxylation of testosterone) and CYP3A (measured as a rate of the 2β- and 6β-hydroxylation of testosterone) were found. In contrast, the activity of CYP1A (measured as a rate of caffeine metabolism) rose, while that of CYP2A (measured as a rate of the 7α-hydroxylation of testosterone), CYP2C6 (measured as a rate of the 7-hydroxylation of warfarin) and CYP2D (the 1'-hydroxylation of bufuralol) remained unchanged. The changes in the activity of CYP1A, CYP2C11 and CYP3A correlated positively with those in CYP protein levels and with the CYP mRNA levels of CYP1A1, CYP2C11 and CYP3A1/2 genes, respectively. The obtained results indicate an important role of the brain noradrenergic system in the neuroendocrine regulation of liver cytochrome P450 expression, which may be of significance in pathological states involving this system, or during pharmacotherapy with drugs affecting noradrenergic transmission.

  20. Inhibition and induction of cytochrome P450 2B1 in rat liver by promazine and chlorpromazine.

    PubMed

    Murray, M

    1992-09-25

    Phenothiazine tranquilizers have been associated with pharmacokinetic drug interactions in man. In this study the in vivo and in vitro effects of the clinically important phenothiazines promazine (PZ) and chlorpromazine (CPZ) on drug oxidations catalysed by specific cytochrome P450 (P450) enzymes were investigated in the rat. In vitro, the two drugs were relatively ineffective inhibitors of constitutive P450 activities, but were inhibitory toward the principal phenobarbital-inducible P450 2B1 and, to a lesser extent, P450 1A1. Administration of PZ and CPZ to male rats did not markedly influence the total microsomal P450 content of the liver. However, the quantitatively important male-specific P450 2C11 was down-regulated by CPZ and concomitant induction of P450 2B1 and associated 7-pentylresorufin O-depentylase activity were noted. A small increase in the activity of microsomal 7-ethylresorufin O-deethylase was also observed following administration of both drugs to rats, suggesting induction of P450 1A1/2. Considered together, it is apparent that the two phenothiazines are preferential inhibitors and inducers of P450 2B1 in rat liver. Drug interactions in humans involving phenothiazines may reflect a combined effect of induction and inhibition processes as well as down-regulation of other P450s, such as that produced by CPZ on P450 2C11.

  1. Cytochrome P450-activated prodrugs

    PubMed Central

    Ortiz de Montellano, Paul R

    2013-01-01

    A prodrug is a compound that has negligible, or lower, activity against a specified pharmacological target than one of its major metabolites. Prodrugs can be used to improve drug delivery or pharmacokinetics, to decrease toxicity, or to target the drug to specific cells or tissues. Ester and phosphate hydrolysis are widely used in prodrug design because of their simplicity, but such approaches are relatively ineffective for targeting drugs to specific sites. The activation of prodrugs by the cytochrome P450 system provides a highly versatile approach to prodrug design that is particularly adaptable for targeting drug activation to the liver, to tumors or to hypoxic tissues. PMID:23360144

  2. Sequential metabolism of sesamin by cytochrome P450 and UDP-glucuronosyltransferase in human liver.

    PubMed

    Yasuda, Kaori; Ikushiro, Shinichi; Kamakura, Masaki; Munetsuna, Eiji; Ohta, Miho; Sakaki, Toshiyuki

    2011-09-01

    Our previous study revealed that CYP2C9 played a central role in sesamin monocatecholization. In this study, we focused on the metabolism of sesamin monocatechol that was further converted into the dicatechol form by cytochrome P450 (P450) or the glucuronide by UDP-glucuronosyltransferase (UGT). Catecholization of sesamin monocatechol enhances its antioxidant activity, whereas glucuronidation strongly reduces its antioxidant activity. In human liver microsomes, the glucuronidation activity was much higher than the catecholization activity toward sesamin monocatechol. In contrast, in rat liver microsomes, catecholization is predominant over glucuronidation. In addition, rat liver produced two isomers of the glucuronide, whereas human liver produced only one glucuronide. These results suggest a significant species-based difference in the metabolism of sesamin between humans and rats. Kinetic studies using recombinant human UGT isoforms identified UGT2B7 as the most important UGT isoform for glucuronidation of sesamin monocatechol. In addition, a good correlation was observed between the glucuronidation activity and UGT2B7-specific activity in in vitro studies using 10 individual human liver microsomes. These results strongly suggest that UGT2B7 plays an important role in glucuronidation of sesamin monocatechol. Interindividual difference among the 10 human liver microsomes is approximately 2-fold. These results, together with our previous results on the metabolism of sesamin by human P450, suggest a small interindividual difference in sesamin metabolism. We observed the methylation activity toward sesamin monocatechol by catechol O-methyl transferase (COMT) in human liver cytosol. On the basis of these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in the human liver.

  3. Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation.

    PubMed

    Hosoi, Yoshio; Uno, Yasuhiro; Murayama, Norie; Fujino, Hideki; Shukuya, Mitsunori; Iwasaki, Kazuhide; Shimizu, Makiko; Utoh, Masahiro; Yamazaki, Hiroshi

    2012-12-15

    Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V(max) and low K(m) values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V(max) and V(max)/K(m) values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation.

  4. Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

    SciTech Connect

    Reed, James R.; Cawley, George F.; Ardoin, Taylor G.; Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W.; Backes, Wayne L.

    2014-06-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

  5. Selective inactivation of rat liver cytochromes P-450 by 21-chlorinated steroids.

    PubMed

    Halpert, J; Jaw, J Y; Cornfield, L J; Balfour, C; Mash, E A

    1989-01-01

    The inactivation by 21-chlorinated steroids of rat liver cytochromes P-450 involved in the hydroxylation of progesterone and androstenedione has been investigated. Preincubation of intact liver microsomes from phenobarbital-treated rats with 21-chloropregnenolone, 21,21-dichloropregnenolone, or 21,21-dichloroprogesterone in the presence of NADPH caused a time-dependent decrease in progesterone 21-hydroxylase and in progesterone or androstenedione 6 beta-hydroxylase activity but had negligible or only minor effects on five other steroid hydroxylases. The compounds differed, however, with regard to the relative rate constants for inactivation of the 21- and 6 beta-hydroxylases. For example, 21,21-dichloroprogesterone and 21,21-dichloropregnenolone inactivated the progesterone 6 beta-hydroxylase at similar rates, but the dichloroprogesterone was a more effective inactivator of the 21-hydroxylase. The results indicate that the introduction of a dichloromethyl group into a substrate bearing a methyl group normally hydroxylated by only one or a few isozymes of cytochrome P-450 may be a rational means of designing isozyme-selective inhibitors but that target and nontarget enzymes may not totally retain the regioselectivity they exhibit towards the underivatized substrate.

  6. The Cytochrome P450 Homepage

    PubMed Central

    2009-01-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 (CYP) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described. PMID:19951895

  7. In vitro inhibition and induction of human liver cytochrome p450 enzymes by milnacipran.

    PubMed

    Paris, Brandy L; Ogilvie, Brian W; Scheinkoenig, Julie A; Ndikum-Moffor, Florence; Gibson, Remi; Parkinson, Andrew

    2009-10-01

    Milnacipran (Savella) inhibits both norepinephrine and serotonin reuptake and is distinguished by a nearly 3-fold greater potency in inhibiting norepinephrine reuptake in vitro compared with serotonin. We evaluated the ability of milnacipran to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, milnacipran did not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19, or 2D6 (IC(50) >or= 100 microM); whereas, a comparator with dual reuptake properties [duloxetine (Cymbalta)] inhibited CYP2D6 (IC(50) = 7 microM) and CYP2B6 (IC(50) = 15 microM) with a relatively high potency. Milnacipran inhibited CYP3A4/5 in a substrate-dependent manner (i.e., midazolam 1'-hydroxylation IC(50) approximately 30 microM; testosterone 6beta-hydroxylation IC(50) approximately 100 microM); whereas, duloxetine inhibited both CYP3A4/5 activities with equal potency (IC(50) = 37 and 38 microM, respectively). Milnacipran produced no time-dependent inhibition (<10%) of P450 activity, whereas duloxetine produced time-dependent inhibition of CYP1A2, 2B6, 2C19, and 3A4/5. To evaluate P450 induction, freshly isolated human hepatocytes (n = 3) were cultured and treated once daily for 3 days with milnacipran (3, 10, and 30 microM), after which microsomal P450 activities were measured. Whereas positive controls (omeprazole, phenobarbital, and rifampin) caused anticipated P450 induction, milnacipran had minimal effect on CYP1A2, 2C8, 2C9, or 2C19 activity. The highest concentration of milnacipran (30 microM; >10 times plasma C(max)) produced 2.6- and 2.2-fold increases in CYP2B6 and CYP3A4/5 activity (making it 26 and 34% as effective as phenobarbital and rifampin, respectively). Given these results, milnacipran is not expected to cause clinically significant P450 inhibition or induction.

  8. Metazoan cytochrome P450 evolution.

    PubMed

    Nelson, D R

    1998-11-01

    There are 37 cytochrome P450 families currently identified in animals. The concept of higher order groupings of P450 families called P450 CLANS is introduced. The mammalian CYP3 and CYP5 families belong to the same clan as insect CYP6 and CYP9. All mitochondrial P450s seem to belong to the same clan. Lack of mitochondrial P450s in C. elegans suggests that mitochondrial P450s probably arose from the mistargeting of a microsomal P450 after the coelomates diverged from acoelomates and pseudocoelomates. Different taxonomic groups appear to have recruited different ancestral P450s for expansion as they evolved, since each major taxon seems to have one large cluster of P450s. In insects, this cluster derives from the ancestor to the CYP4 family. Vertebrates and C. elegans may have used the same ancestor independently to generate the CYP1, 2, 17, and 21 families in vertebrates and a large distinctive clan with 45 genes in C. elegans.

  9. Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450.

    PubMed

    Kokkinakis, D M; Koop, D R; Scarpelli, D G; Coon, M J; Hollenberg, P F

    1985-02-01

    The cis isomer of N-nitroso-2,6-dimethylmorpholine (NNDM), a pancreatic carcinogen for the Syrian golden hamster, is metabolized by hamster liver microsomes to yield N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product. Rabbit liver microsomes catalyze the metabolism of cis-NNDM to HPOP at a rate slower than that observed with hamster microsomes, but significantly faster than that obtained with rat microsomes. Pretreatment of rabbits with phenobarbital results in a 6-fold increase in the cis-NNDM hydroxylase activity of the rabbit microsomes to levels equal to that observed with the hamster; pretreatment of rabbits with other xenobiotics had no effect on the hydroxylation of cis-NNDM. The role of rabbit liver microsomal cytochrome P-450 in the metabolism of the cis isomer of NNDM was studied in the reconstituted system consisting of NADPH:cytochrome P-450 reductase, phospholipid, and cytochrome P-450. Cytochrome P-450LM2, which is induced by pretreatment with phenobarbital, exhibited the highest activity for the metabolism of cis-NNDM. The Vmax for the formation of HPOP was 1.78 nmol/min/nmol cytochrome P-450LM2, and the apparent Km was 360 microM. Cytochrome P-450LM3a also catalyzed the metabolism of NNDM to HPOP at a significant rate (0.25 nmol/min/nmol cytochrome P-450). Of the four other isozymes of cytochrome P-450 (forms 3b, 3c, 4, and 6) tested in the reconstituted system, only forms 3b and 3c exhibited measurable activities (approximately 0.04 nmol of HPOP formed/min/nmol cytochrome P-450). The addition of antibodies to isozyme 2 to microsomes from phenobarbital-treated rabbits resulted in approximately 95% inhibition of the metabolism of NNDM, while the addition of antibodies to LM3a inhibited NNDM metabolism by only 7%. In microsomes from untreated rabbits, inhibition by anti-LM2 and anti-LM3a antibodies was 50 and 64%, respectively. The addition of antibodies to isozyme 3a to microsomes isolated from ethanol-treated rabbits caused

  10. Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline.

    PubMed

    Utoh, Masahiro; Murayama, Norie; Uno, Yasuhiro; Onose, Yui; Hosaka, Shinya; Fujino, Hideki; Shimizu, Makiko; Iwasaki, Kazuhide; Yamazaki, Hiroshi

    2013-12-01

    Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

  11. Expression of cytochromes p450 in fetal, infant, and juvenile liver of cynomolgus macaques.

    PubMed

    Ise, Ryota; Kondo, Satoshi; Kato, Hiroto; Imai, Noritaka; Akiyama, Hideo; Iwasaki, Kazuhide; Yamazaki, Hiroshi; Uno, Yasuhiro

    2011-01-01

    Preclinical data of fetal, infant, and juvenile animals are important for the prediction of drug toxicity in fetuses and children. However, expression of drug-metabolizing enzymes, including cytochromes P450 (CYPs), have not been fully investigated in fetal, infant, or juvenile liver of the cynomolgus macaque, an animal species important for preclinical studies. In this study, hepatic expression of 20 cynomolgus macaque CYPs (mfCYPs) in the CYP1-4 subfamilies that are relevant to drug metabolism was measured in fetuses, infants, and juveniles using DNA microarrays. Expression of most mfCYPs, including those moderately or abundantly expressed in postnatal livers such as mfCYP2A23, mfCYP2A24, mfCYP2B6, mfCYP2C9, mfCYP2C19, mfCYP2C76, mfCYP2D17, mfCYP2E1 mfCYP3A4, and mfCYP3A5, was much less abundant in fetal livers, but increased substantially after birth. In contrast, expression of mfCYP2C8 in fetal livers was not substantially different from postnatal livers. Since human CYP3A7 is expressed more abundantly in fetal livers than in adult livers, mfCYP3A7, an ortholog of human CYP3A7, was analyzed by quantitative polymerase chain reaction. Expression of mfCYP3A7 in fetal livers was much lower than that in postnatal livers, and greatly increased after birth, unlike the expression of human CYP3A7. These results indicate that expression of most mfCYPs examined was low in fetal livers, but increased greatly in postnatal livers, with a few exceptions such as mfCYP2C8.

  12. The cytochrome P450 epoxygenase pathway regulates the hepatic inflammatory response in fatty liver disease.

    PubMed

    Schuck, Robert N; Zha, Weibin; Edin, Matthew L; Gruzdev, Artiom; Vendrov, Kimberly C; Miller, Tricia M; Xu, Zhenghong; Lih, Fred B; DeGraff, Laura M; Tomer, Kenneth B; Jones, H Michael; Makowski, Liza; Huang, Leaf; Poloyac, Samuel M; Zeldin, Darryl C; Lee, Craig R

    2014-01-01

    Fatty liver disease is an emerging public health problem without effective therapies, and chronic hepatic inflammation is a key pathologic mediator in its progression. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory effects. Although promoting the effects of EETs elicits anti-inflammatory and protective effects in the cardiovascular system, the contribution of CYP-derived EETs to the regulation of fatty liver disease-associated inflammation and injury is unknown. Using the atherogenic diet model of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), our studies demonstrated that induction of fatty liver disease significantly and preferentially suppresses hepatic CYP epoxygenase expression and activity, and both hepatic and circulating levels of EETs in mice. Furthermore, mice with targeted disruption of Ephx2 (the gene encoding soluble epoxide hydrolase) exhibited restored hepatic and circulating EET levels and a significantly attenuated induction of hepatic inflammation and injury. Collectively, these data suggest that suppression of hepatic CYP-mediated EET biosynthesis is an important pathological consequence of fatty liver disease-associated inflammation, and that the CYP epoxygenase pathway is a central regulator of the hepatic inflammatory response in NAFLD/NASH. Future studies investigating the utility of therapeutic strategies that promote the effects of CYP-derived EETs in NAFLD/NASH are warranted.

  13. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase.

    PubMed

    Kong, Sandra; Ngo, Suong N T; McKinnon, Ross A; Stupans, Ieva

    2009-07-01

    The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 +/- 0.5 and 2.6 +/- 0.4 nmol/min/mg protein (mean +/- SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 +/- 6.01 microM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species.

  14. Detection of human lung cytochromes P450 that are immunochemically related to cytochrome P450IIE1 and cytochrome P450IIIA.

    PubMed

    Wheeler, C W; Wrighton, S A; Guenthner, T M

    1992-07-07

    We have used monoclonal antibodies that were prepared against and specifically recognize human hepatic cytochromes P450 as probes for solid phase radioimmunoassay and Western immunoblotting to directly demonstrate the presence in human lung microsomes of cytochromes P450 immunochemically related to human liver cytochromes P450IIE1 (CYP2E1) and P450IIIA (CYP3A). The detected levels of these cytochromes are much lower than levels in human liver microsomes, but similar to the levels seen in microsomes from untreated baboon lung. Proteins immunochemically related to two other constitutive hepatic cytochromes P450, cytochrome P450IIC8 (CYP2C8) and cytochrome P450IIC9 (CYP2C9), were not detectable in lung microsomes.

  15. Xenobiotic and Endobiotic Mediated Interactions between the Cytochrome P450 System and the Inflammatory Response In the Liver

    PubMed Central

    Woolbright, Benjamin L.; Jaeschke, Hartmut

    2015-01-01

    The liver is a unique organ in the body as it has significant roles in both metabolism and innate immune clearance. Hepatocytes in the liver carry a nearly complete complement of drug metabolizing enzymes, including numerous cytochrome P450s. While a majority of these enzymes effectively detoxify xenobiotics, or metabolize endobiotics, a sub-portion of these reactions result in accumulation of metabolites that can cause either direct liver injury or indirect liver injury through activation of inflammation. The liver also contains multiple populations of innate immune cells including the resident macrophages (Kupffer cells), a relatively large number of natural killer cells, and blood-derived neutrophils. While these cells are primarily responsible for clearance of pathogens, activation of these immune cells can result in significant tissue injury during periods of inflammation. When activated chronically, these inflammatory bouts can lead to fibrosis, cirrhosis, cancer or death. This Chapter will focus on interactions between how the liver processes xenobiotic and endobiotic compounds through the cytochrome P450 system, and how these processes can result in a response from the innate immune cells of the liver. A number of different clinically relevant diseases, as well as experimental models, are currently available to study mechanisms related to the interplay of innate immunity and cytochrome P450 mediated metabolism. A major focus of the chapter will be to evaluate currently understood mechanisms in the context of these diseases as a way of outlining mechanisms that dictate the interactions between the P450 system and innate immunity. PMID:26233906

  16. Xenobiotic and Endobiotic Mediated Interactions Between the Cytochrome P450 System and the Inflammatory Response in the Liver.

    PubMed

    Woolbright, Benjamin L; Jaeschke, Hartmut

    2015-01-01

    The liver is a unique organ in the body as it has significant roles in both metabolism and innate immune clearance. Hepatocytes in the liver carry a nearly complete complement of drug metabolizing enzymes, including numerous cytochrome P450s. While a majority of these enzymes effectively detoxify xenobiotics, or metabolize endobiotics, a subportion of these reactions result in accumulation of metabolites that can cause either direct liver injury or indirect liver injury through activation of inflammation. The liver also contains multiple populations of innate immune cells including the resident macrophages (Kupffer cells), a relatively large number of natural killer cells, and blood-derived neutrophils. While these cells are primarily responsible for clearance of pathogens, activation of these immune cells can result in significant tissue injury during periods of inflammation. When activated chronically, these inflammatory bouts can lead to fibrosis, cirrhosis, cancer, or death. This chapter will focus on interactions between how the liver processes xenobiotic and endobiotic compounds through the cytochrome P450 system, and how these processes can result in a response from the innate immune cells of the liver. A number of different clinically relevant diseases, as well as experimental models, are currently available to study mechanisms related to the interplay of innate immunity and cytochrome P450-mediated metabolism. A major focus of the chapter will be to evaluate currently understood mechanisms in the context of these diseases, as a way of outlining mechanisms that dictate the interactions between the P450 system and innate immunity.

  17. PROPICONAZOLE-INDUCED CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RAT AND MOUSE LIVER

    EPA Science Inventory

    Conazoles are N-substituted azole antifungal agents used as both pesticides and drugs. Some of these compounds are hepatocarcinogenic in mice and some can induce thyroid tumors in rats. Many of these compounds are able to induce and/or inhibit mammalian hepatic cytochrome P450s t...

  18. Metabolism of melatonin by cytochrome P-450s in rat liver mitochondria and microsomes

    PubMed Central

    Semak, Igor; Korik, Elena; Antonova, Maria; Wortsman, Jacobo; Slominski, Andrzej

    2008-01-01

    In the present study we provide direct evidence for the involvement of rat microsomal cytochrome P450s in melatonin O-demethylation and hydroxylation at two different positions: 2 and 6, as well as generation of N1-acetyl-N2-formyl-5-methoxy-kynuramine (AFMK) and two unknown products. Moreover, we found that mitochondrial cytochrome P450s also converts melatonin into AFMK, N-acetylserotonin (NAS), 2-hydroxymelatonin, 6-hydroxymelatonin and the same two unknown products. Eadie-Hofstee plots for 6-hydroxylation and O-demethylation reactions were curvilinear for all tested fractions, suggestive of involvement of at least two components, one with a high affinity and low capacity, and another with a low affinity and high capacity. Mitochondrial cytochrome P450s exhibited higher affinity (suggesting lower Km value) and higher Vmax for melatonin 6-hydroxylation and O-demethylation for both high-affinity and low-affinity components as compared to microsomal enzymes. The intrinsic clearance for melatonin hydroxylation by high- and low-affinity components displayed the highest values in all tested fractions, indicating that both mitochondrial and microsomal cytochrome P-450s metabolize melatonin principally by 6-hydroxylation, with O-demethylation representing a minor metabolic pathway. PMID:18717775

  19. Metabolism of melatonin by cytochrome P450s in rat liver mitochondria and microsomes.

    PubMed

    Semak, Igor; Korik, Elena; Antonova, Maria; Wortsman, Jacobo; Slominski, Andrzej

    2008-11-01

    In the present study we provide direct evidence for the involvement of rat microsomal cytochrome P450s in melatonin O-demethylation and hydroxylation at two different positions: 2 and 6, as well as generation of N(1)-acetyl-N(2)-formyl-5-methoxy-kynuramine (AFMK) and two unknown products. Moreover, we found that mitochondrial cytochrome P450s also converts melatonin into AFMK, N-acetylserotonin, 2-hydroxymelatonin, 6-hydroxymelatonin and the same two unknown products. Eadie-Hofstee plots for 6-hydroxylation and O-demethylation reactions were curvilinear for all tested fractions, suggestive of involvement of at least two components, one with a high affinity and low capacity, and another with a low affinity and high capacity. Mitochondrial cytochrome P450s exhibited higher affinity (suggesting lower K(m) value) and higher V(max) for melatonin 6-hydroxylation and O-demethylation for both high-affinity and low-affinity components as compared with microsomal enzymes. The intrinsic clearance for melatonin hydroxylation by high- and low-affinity components displayed the highest values in all tested fractions, indicating that both mitochondrial and microsomal cytochrome P450s metabolize melatonin principally by 6-hydroxylation, with O-demethylation representing a minor metabolic pathway.

  20. Cytochromes P450 in Nanodiscs

    PubMed Central

    Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Nanodiscs have proven to be a versatile tool for the study all types of membrane proteins, including receptors, transporters, enzymes and viral antigens. The self-assembled Nanodisc system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native like bilayer environment that maintains functional activity. This system has thus provided a means for studying the extensive collection of membrane bound cytochromes P450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble P450s. These include a plethora of spectroscopic, kinetic and surface based methods. Significant improvements in homogeneity and stability of these preparations open new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of human cytochromes P450 involved in xenobiotic metabolism and in steroid biosynthesis. The experimental methods developed for physico-chemical and functional studies of membrane cytochromes P450 incorporated in Nanodiscs allow for more detailed understanding of the scientific questions along the lines pioneered by Professor Klaus Ruckpaul and his array of colleagues and collaborators. PMID:20685623

  1. Upregulation of cytochromes P450 2B in rat liver by orphenadrine

    PubMed Central

    Murray, Michael; Fiala-Beer, Eva; Sutton, Dylan

    2003-01-01

    The alkylamine drug orphenadrine (ORPH) is an inducer and inhibitor of the microsomal cytochrome P450 (CYP) system in mammals. This study evaluated the selectivity of CYP induction by ORPH in rat liver. Immunoblot analysis indicated that ORPH was a selective inducer of the phenobarbitone (PB)-inducible CYP2B in rat liver. CYP2B protein was increased to ∼14-fold of levels in untreated rat liver. By comparison PB increased CYP2B expression 40-fold. Corresponding increases in the activity of CYP2B-dependent androstenedione 16β-hydroxylation were measured in microsomes from ORPH and PB-induced rats. Northern analysis indicated that CYP2B1/2 mRNA was increased in ORPH-induced rat liver. Consistent with this finding, ORPH was found to activate a PB-responsive enhancer module in constitutive androstane receptor (CAR)-transfected Hep G2 cells. Other alkylamines like troleandomycin impair CYP turnover. We tested whether ORPH induction of CYP2B may include a post-translational component. In PB-pretreated animals ORPH administration delayed the loss of CYP2B after PB withdrawal, but no evidence for altered turnover was found. These studies establish ORPH as a selective inducer of CYP2B in rat liver. Induction appears to be mediated pretranslationally by CAR activation of CYP2B gene transcription. Post-translational stabilisation by an ORPH metabolite does not elicit induction. Induction of CYP2B may influence pharmacokinetic interactions involving ORPH. PMID:12813002

  2. Curcumin Prevents Aflatoxin B1 Hepatoxicity by Inhibition of Cytochrome P450 Isozymes in Chick Liver

    PubMed Central

    Zhang, Ni-Ya; Qi, Ming; Zhao, Ling; Zhu, Ming-Kun; Guo, Jiao; Liu, Jie; Gu, Chang-Qin; Rajput, Shahid Ali; Krumm, Christopher Steven; Qi, De-Sheng; Sun, Lv-Hui

    2016-01-01

    This study was designed to establish if Curcumin (CM) alleviates Aflatoxin B1 (AFB1)-induced hepatotoxic effects and to determine whether alteration of the expression of cytochrome P450 (CYP450) isozymes is involved in the regulation of these effects in chick liver. One-day-old male broilers (n = 120) were divided into four groups and used in a two by two factorial trial in which the main factors included supplementing AFB1 (< 5 vs. 100 μg/kg) and CM (0 vs. 150 mg/kg) in a corn/soybean-based diet. Administration of AFB1 induced liver injury, significantly decreasing albumin and total protein concentrations and increasing alanine aminotransferase and aspartate aminotransferase activities in serum, and induced hepatic histological lesions at week 2. AFB1 also significantly decreased hepatic glutathione peroxidase, catalase, and glutathione levels, while increasing malondialdehyde, 8-hydroxydeoxyguanosine, and exo-AFB1-8,9-epoxide (AFBO)-DNA concentrations. In addition, the mRNA and/or activity of enzymes responsible for the bioactivation of AFB1 into AFBO—including CYP1A1, CYP1A2, CYP2A6, and CYP3A4—were significantly induced in liver microsomes after 2-week exposure to AFB1. These alterations induced by AFB1 were prevented by CM supplementation. Conclusively, dietary CM protected chicks from AFB1-induced liver injury, potentially through the synergistic actions of increased antioxidant capacities and inhibition of the pivotal CYP450 isozyme-mediated activation of AFB1 to toxic AFBO. PMID:27834912

  3. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 CYP4A15.

    PubMed

    Ngo, Suong Ngoc Thi; McKinnon, Ross Allan; Stupans, Ieva

    2006-07-05

    In the present study, the cloning, expression and characterization of hepatic cytochrome P450 (CYP) CYP4A from koala (Phascolarctos cinereus), an obligate eucalyptus feeder, is described. It has been previously reported that microsomal lauric acid hydroxylase activity (a CYP4A marker) and CYP content were higher in koala liver in comparison to that in human, rat or wallaby, species that do not ingest eucalyptus leaves as food [Ngo, S., Kong, S., Kirlich, A., Mckinnon, R.A., Stupans, I., 2000. Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver. Comp. Biochem. Physiol., C 127, 327-334]. A 1544 bp koala liver CYP4A cDNA, designated CYP4A15, was cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CYP4A15 cDNA encodes a protein of 500 amino acids and shares 69% nucleotide and 65% amino acid sequence identity to human CYP4A11. Transfection of the koala CYP4A15 cDNA into Cos-7 cells resulted in the expression of a protein with lauric acid hydroxylase activity. The koala CYP4A15 cDNA-expressed enzyme catalysed lauric acid hydroxylation at the rates of 0.45+/-0.18 nmol/min/mg protein and 4.79+/-1.91 nmol/min/nmol CYP (mean+/-SD, n=3), which were comparable to that of rat CYP4A subfamilies. Total CYP content for koala CYP4A15-expressed protein in Cos-7 cells was 0.094+/-0.001 nmol/mg protein (mean+/-SD, n=3) with negligible CYP content in untransfected Cos-7 cells lysate. Immunoblot analysis, using a sheep anti-rat CYP4A polyclonal antibody, detected multiple CYP4A immunoreactive bands in the liver from all species studied. The koala bands were found to be fainter and less confined but appeared much broader as compared to rat, human and wallaby. Northern blot analysis, utilising the koala CYP4A15 cDNA 417 bp probe, detected a mRNA species of approximately 2.6 kb in the koala liver and a mRNA species of approximately 2.4 kb in other species studied. Relative to the intensity of the beta

  4. Inhibition of Cytochrome P450 by Propolis in Human Liver Microsomes

    PubMed Central

    Ryu, Chang Seon; Oh, Soo Jin; Oh, Jung Min; Lee, Ji-Yoon; Lee, Sang Yoon; Chae, Jung-woo; Kwon, Kwang-il; Kim, Sang Kyum

    2016-01-01

    Although propolis is one of the most popular functional foods for human health, there have been no comprehensive studies of herb-drug interactions through cytochrome P450 (CYP) inhibition. The purpose of this study was to determine the inhibitory effects of propolis on the activities of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 using pooled human liver microsomes (HLMs). Propolis inhibited CYP1A2, CYP2E1 and CYP2C19 with an IC50 value of 6.9, 16.8, and 43.1 μg/mL, respectively, whereas CYP2A6, 2B6, 2C9, 2D6, and 3A4 were unaffected. Based on half-maximal inhibitory concentration shifts between microsomes incubated with and without nicotinamide adenine dinucleotide phosphate, propolis-induced CYP1A2, CYP2C19, and CYP2E1 inhibition was metabolism-independent. To evaluate the interaction potential between propolis and therapeutic drugs, the effects of propolis on metabolism of duloxetine, a serotonin-norepinephrine reuptake inhibitor, were determined in HLMs. CYP1A2 and CYP2D6 are involved in hydroxylation of duloxetine to 4-hydroxy duloxetine, the major metabolite, which was decreased following propolis addition in HLMs. These results raise the possibility of interactions between propolis and therapeutic drugs metabolized by CYP1A2. PMID:27437087

  5. Relative expression of cytochrome P450 isoenzymes in human liver and association with the metabolism of drugs and xenobiotics.

    PubMed Central

    Forrester, L M; Henderson, C J; Glancey, M J; Back, D J; Park, B K; Ball, S E; Kitteringham, N R; McLaren, A W; Miles, J S; Skett, P

    1992-01-01

    Cytochrome P450s play a central role in the metabolism and disposition of an extremely wide range of drugs and chemical carcinogens. Individual differences in the expression of these enzymes may be an important determinant in susceptibility to adverse drug reactions, chemical toxins and mutagens. In this paper, we have measured the relative levels of expression of cytochrome P450 isoenzymes from eight gene families or subfamilies in a panel of twelve human liver samples in order to determine the individuality in their expression and whether any forms are co-regulated. Isoenzymes were identified in most cases on Western blots based on the mobility of authentic recombinant human cytochrome P450 standards. The levels of the following P450 proteins correlated with each other: CYP2A6, CYP2B6 and a protein from the CYP2C gene subfamily, CYP2E1 and a member of the CYP2A gene subfamily, CYP2C8, CYP3A3/A4 and total cytochrome P450 content. Also, the levels of two proteins in the CYP4A gene subfamily were highly correlated. These correlations are consistent with the relative regulation of members of these gene families in rats or mice. In addition, the level of expression of specific isoenzymes has also been compared with the rate of metabolism of a panel of drugs, carcinogens and model P450 substrates. These latter studies demonstrate and confirm that the correlations obtained in this manner represent a powerful approach towards the assignment of the metabolism of substrates by specific human P450 isoenzymes. Images Fig. 1. Fig. 2. Fig. 3. PMID:1736885

  6. Relative expression of cytochrome P450 isoenzymes in human liver and association with the metabolism of drugs and xenobiotics.

    PubMed

    Forrester, L M; Henderson, C J; Glancey, M J; Back, D J; Park, B K; Ball, S E; Kitteringham, N R; McLaren, A W; Miles, J S; Skett, P

    1992-01-15

    Cytochrome P450s play a central role in the metabolism and disposition of an extremely wide range of drugs and chemical carcinogens. Individual differences in the expression of these enzymes may be an important determinant in susceptibility to adverse drug reactions, chemical toxins and mutagens. In this paper, we have measured the relative levels of expression of cytochrome P450 isoenzymes from eight gene families or subfamilies in a panel of twelve human liver samples in order to determine the individuality in their expression and whether any forms are co-regulated. Isoenzymes were identified in most cases on Western blots based on the mobility of authentic recombinant human cytochrome P450 standards. The levels of the following P450 proteins correlated with each other: CYP2A6, CYP2B6 and a protein from the CYP2C gene subfamily, CYP2E1 and a member of the CYP2A gene subfamily, CYP2C8, CYP3A3/A4 and total cytochrome P450 content. Also, the levels of two proteins in the CYP4A gene subfamily were highly correlated. These correlations are consistent with the relative regulation of members of these gene families in rats or mice. In addition, the level of expression of specific isoenzymes has also been compared with the rate of metabolism of a panel of drugs, carcinogens and model P450 substrates. These latter studies demonstrate and confirm that the correlations obtained in this manner represent a powerful approach towards the assignment of the metabolism of substrates by specific human P450 isoenzymes.

  7. Immunochemical evidence for an ethanol-inducible form of liver microsomal cytochrome P-450 in rodents and primates

    SciTech Connect

    Lasker, J.M.; Ardies, C.M.; Bloswick, B.P.; Lieber, C.S.

    1986-05-01

    Polyclonal antibodies against cytochrome P-450-4, a major liver microsomal P-450 isozyme purified from ethanol (E)-treated hamsters, were used to probe for immunochemically-related hemeproteins in other species. Liver microsomes (LM) were obtained from naive and E-treated rats, deermice, hamsters, and baboons. Baboon liver 9000 x g supernatant (S-9) was prepared from needle biopsy samples. LM and S-9 proteins were resolved by SDS-PAGE, then transferred to nylon membranes. Immunodetection was performed on the Western blots using rabbit anti P-450-4 IgG, anti-rabbit IgG-alk. phos., and an appropriate chromagen. Control LM from all species contained a cross-reacting protein of mol. wt. similar to P-450-4 (54k). The amount of this cross-reacting protein as reflected by staining intensity, was much higher in LM from E-treated animals. This protein was also detected in S-9 from E-treated baboons. In contrast, no increase in phenobarbital-inducible P-450-2 related LM protein (assessed using anti P-450-2) was observed after E treatment. Increased P-450-4 related protein in LM from E-treated animals was associated with enhanced oxidation of ethanol and aniline by these LM when compared to controls. In conclusion, LM from rats, deermice, and baboons contain a protein immunochemically homologous to hamster liver P-450-4. As observed in hamsters, the amount of this hepatic protein increases in these other species after E treatment.

  8. Novel Marmoset Cytochrome P450 2C19 in Livers Efficiently Metabolizes Human P450 2C9 and 2C19 Substrates, S-Warfarin, Tolbutamide, Flurbiprofen, and Omeprazole.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Kawano, Mirai; Shimizu, Makiko; Toda, Akiko; Utoh, Masahiro; Sasaki, Erika; Yamazaki, Hiroshi

    2015-10-01

    The common marmoset (Callithrix jacchus), a small New World monkey, has the potential for use in human drug development due to its evolutionary closeness to humans. Four novel cDNAs, encoding cytochrome P450 (P450) 2C18, 2C19, 2C58, and 2C76, were cloned from marmoset livers to characterize P450 2C molecular properties, including previously reported P450 2C8. The deduced amino acid sequence showed high sequence identities (>86%) with those of human P450 2Cs, except for marmoset P450 2C76, which has a low sequence identity (∼70%) with any human P450 2Cs. Phylogenetic analysis showed that marmoset P450 2Cs were more closely clustered with those of humans and macaques than other species investigated. Quantitative polymerase chain reaction analysis showed that all of the marmoset P450 2C mRNAs were predominantly expressed in liver as opposed to the other tissues tested. Marmoset P450 2C proteins were detected in liver by immunoblotting using antibodies against human P450 2Cs. Among marmoset P450 2Cs heterologously expressed in Escherichia coli, marmoset P450 2C19 efficiently catalyzed human P450 2C substrates, S-warfarin, diclofenac, tolbutamide, flurbiprofen, and omeprazole. Marmoset P450 2C19 had high Vmax and low Km values for S-warfarin 7-hydroxylation that were comparable to those in human liver microsomes, indicating warfarin stereoselectivity similar to findings in humans. Faster in vivo S-warfarin clearance than R-warfarin after intravenous administration of racemic warfarin (0.2 mg/kg) to marmosets was consistent with the in vitro kinetic parameters. These results indicated that marmoset P450 2C enzymes had functional characteristics similar to those of humans, and that P450 2C-dependent metabolic properties are likewise similar between marmosets and humans.

  9. Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

    PubMed Central

    Heinonen, J T; Sidhu, J S; Reilly, M T; Farin, F M; Omiecinski, C J; Eaton, D L; Kavanagh, T J

    1996-01-01

    Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur. Images Figure 1. Figure 2. Figure 3. Figure 3. Figure 4. Figure 4. Figure 5. Figure 6. PMID:8743442

  10. Human Liver Mitochondrial Cytochrome P450 2D6: Individual Variations and Implications in Drug Metabolism

    PubMed Central

    Cook Sangar, Michelle L.; Anandatheerthavarada, Hindupur K.; Tang, Weigang; Prabu, Subbuswamy K.; Martin, Martha V.; Dostalek, Miroslav; Guengerich, F. Peter; Avadhani, Narayan G.

    2009-01-01

    Summary Constitutively expressed human cytochrome P450 2D6 (CYP2D6) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists in the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. Here we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum (ER) and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23-33 and that the positively charged residues at positions 24, 25, 26, 28, and 32 are required for mitochondrial targeting. The importance of the positively charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1′-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism. PMID:19438707

  11. Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke Opisthorchis felineus

    PubMed Central

    Pakharukova, Mariya Y.; Vavilin, Valentin A.; Sripa, Banchob; Laha, Thewarach; Brindley, Paul J.; Mordvinov, Viatcheslav A.

    2015-01-01

    The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke Opisthorchis felineus, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in O. felineus tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in O. felineus. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target. PMID:26625139

  12. Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke Opisthorchis felineus.

    PubMed

    Pakharukova, Mariya Y; Vavilin, Valentin A; Sripa, Banchob; Laha, Thewarach; Brindley, Paul J; Mordvinov, Viatcheslav A

    2015-12-01

    The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke Opisthorchis felineus, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in O. felineus tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in O. felineus. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target.

  13. Evidence for involvement of multiple forms of cytochrome P-450 in aflatoxin B sup 1 metabolism in human liver

    SciTech Connect

    Forrester, L.M.; Wolf, C.R. ); Neal, G.E.; Judah, D.J. )

    1990-11-01

    Liver cancer is a major cause of premature death in many areas of Africa and Asia and its incidence is strongly correlated with exposure to aflatoxin B{sub 1} (AFB{sub 1}). Because AFB{sub 1} requires metabolic activation to achieve a biological response, there is a need for detailed knowledge of the mechanism of activation to assess individual risk. The authors carried out an extensive study using a total of 19 human liver samples to determine the individual variability in the metabolism of the toxin to mutagenic or detoxification products and to identify the specific cytochrome P-450 forms involved in these processes. Metabolism to the toxic 8,9-epoxide or to products mutagenic in the Ames test was found to exhibit very large individual variation. These data demonstrate that, although P450IIIA probably plays an important role in AFB{sub 1} activation, several other cytochrome P-450 forms have the capacity to activate the toxin. Similar considerations apply to detoxifying metabolism to aflatoxin Q{sub 1} and aflatoxin M{sub 1}. The levels of expression of many of the forms of cytochrome P-450 involved in AFB{sub 1} metabolism are known to be highly sensitive to environmental factors. This indicates that such factors will be an important determinant in individual susceptibility to the tumorigenic action of AFB{sub 1}.

  14. Benzene metabolism by human liver microsomes in relation to cytochrome P450 2E1 activity.

    PubMed

    Seaton, M J; Schlosser, P M; Bond, J A; Medinsky, M A

    1994-09-01

    Low levels of benzene from sources including cigarette smoke and automobile emissions are ubiquitous in the environment. Since the toxicity of benzene probably results from oxidative metabolites, an understanding of the profile of biotransformation of low levels of benzene is critical in making a valid risk assessment. To that end, we have investigated metabolism of a low concentration of [14C]benzene (3.4 microM) by microsomes from human, mouse and rat liver. The extent of phase I benzene metabolism by microsomal preparations from 10 human liver samples and single microsomal preparations from both mice and rats was then related to measured activities of cytochrome P450 (CYP) 2E1. Measured CYP 2E1 activities, as determined by hydroxylation of p-nitrophenol, varied 13-fold (0.253-3.266 nmol/min/mg) for human samples. The fraction of benzene metabolized in 16 min ranged from 10% to 59%. Also at 16 min, significant amounts of oxidative metabolites were formed. Phenol was the main metabolite formed by all but two human microsomal preparations. In those samples, both of which had high CYP 2E1 activity, hydroquinone was the major metabolite formed. Both hydroquinone and catechol formation showed a direct correlation with CYP 2E1 activity over the range of activities present. A simulation model was developed based on a mechanism of competitive inhibition between benzene and its oxidized metabolites, and was fit to time-course data for three human liver preparations. Model calculations for initial rates of benzene metabolism ranging from 0.344 to 4.442 nmol/mg/min are directly proportional to measured CYP 2E1 activities. The model predicted the dependence of benzene metabolism on the measured CYP 2E1 activity in human liver samples, as well as in mouse and rat liver samples. These results suggest that differences in measured hepatic CYP 2E1 activity may be a major factor contributing to both interindividual and interspecies variations in hepatic metabolism of benzene

  15. Selective inactivation by 21-chlorinated steroids of rabbit liver and adrenal microsomal cytochromes P-450 involved in progesterone hydroxylation.

    PubMed

    Halpert, J; Jaw, J Y; Balfour, C; Mash, E A; Johnson, E F

    1988-08-01

    The inactivation by 21-chlorinated steroids of rabbit liver cytochromes P-450 involved in the hydroxylation of progesterone has been investigated in intact microsomes encompassing two phenotypes of 21-hydroxylase activity, two phenotypes of 16 alpha-hydroxylase activity, and three phenotypes of 6 beta-hydroxylase activity. In liver microsomes from outbred New Zealand White male rabbits exhibiting a high content of cytochrome P-450 1, 21,21-dichloropregnenolone caused a time- and NADPH-dependent loss of 21-hydroxylase activity. This loss of activity exhibited a number of characteristics of mechanism-based inactivation, including irreversibility, saturation with increasing inhibitor concentrations, and protection by substrate, and was also documented with purified P-450 1 in a reconstituted system. 21,21-Dichloropregnenolone caused no time-dependent loss of 6 beta-hydroxylase activity in microsomes from the New Zealand White rabbits or from control or rifampicin-treated rabbits of the inbred B/J strain. In contrast, in the microsomes from the B/J rabbits, some inactivation of the 16 alpha-hydroxylase was observed (k = 0.04 min-1), regardless of the rifampicin treatment. The other two compounds tested, 21-chloropregnenolone and 21,21-dichloroprogesterone, were less effective than the dichloropregnenolone as inactivators of cytochrome P-450 1. On the other hand, 21,21-dichloroprogesterone, but not 21,21-dichloropregneolone, caused a rapid time-dependent loss of 21-hydroxylase activity in rabbit adrenal microsomes. The results indicate that the introduction of a dichloromethyl group into a substrate bearing a methyl group normally hydroxylated by only one or a few forms of cytochrome P-450 may be a rational means of designing selective inhibitors of the enzyme.

  16. Metabolism of (+)-terpinen-4-ol by cytochrome P450 enzymes in human liver microsomes.

    PubMed

    Haigou, Risa; Miyazawa, Mitsuo

    2012-01-01

    We examined the in vitro metabolism of (+)-terpinen-4-ol by human liver microsomes and recombinant enzymes. The biotransformation of (+)-terpinen-4-ol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Terpinen-4-ol was found to be oxidized to (+)-(1R,2S,4S)-1,2-epoxy-p-menthan-4-ol, (+)-(1S,2R,4S)-1,2-epoxy-p-menthan-4-ol, and (4S)-p-menth-1-en-4,8-diol by human liver microsomal P450 enzymes. The identities of (+)-terpinen-4-ol metabolites were determined through the relative abundance of mass fragments and retention times on GC-MS. Of 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6, and CYP3A4 were found to catalyze the oxidation of (+)-terpinen-4-ol. Based on several lines of evidence, CYP2A6 and CYP3A4 were determined to be major enzymes involved in the oxidation of (+)-terpinen-4-ol by human liver microsomes. First, of the 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6 and CYP3A4 catalyzed oxidation of (+)-terpinen-4-ol. Second, oxidation of (+)-terpinen-4-ol was inhibited by (+)-menthofuran and ketoconazole, inhibitors known to be specific for these enzymes. Finally, there was a good correlation between CYP2A6 and CYP3A4 activities and (+)-terpinen-4-ol oxidation activities in the 10 human liver microsomes.

  17. Cytochrome P450 (CYP450) Tests

    MedlinePlus

    ... By Mayo Clinic Staff Your doctor may use cytochrome P450 (CYP450) tests to help determine how your body processes (metabolizes) a drug. The human body contains P450 enzymes to process medications. Because of inherited (genetic) traits ...

  18. Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

    PubMed Central

    2012-01-01

    Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. Conclusions Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared

  19. Bromopropylate: induction of hepatic cytochromes P450 and absence of covalent binding to DNA in mouse liver.

    PubMed

    Thomas, H; Sagelsdorff, P; Molitor, E; Skripsky, T; Waechter, F

    1994-11-01

    Oral administration of benzilic acid ester-based acaricide bromopropylate at daily doses of 3, 15, 100, and 300 mg/kg body wt to young adult male Tif:MAGf mice for 14 days caused slightly increased liver weights in the high-dose group. A dose-dependent increase of the microsomal cytochrome P450 content was accompanied by elevated ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, pentoxyresorufin O-depentylase, and total testosterone hydroxylase activities. When compared with mice treated in parallel with the model compounds for hepatic xenobiotic metabolizing enzyme induction, phenobarbitone, and 3-methylcholanthrene, the enzyme activity changes observed with bromopropylate largely equalled those expressed in phenobarbitone-treated mice. Immunochemical studies with monoclonal antibodies against rat liver cytochrome P450 isoenzymes of the gene families 1A, 2B, 3A, and 4A confirmed that bromopropylate is a phenobarbitone-type inducer in the mouse liver. Titration of liver microsomal suspensions with bromopropylate yielded Type I substrate binding spectra. The specific amplitude was increased 1.5-fold when microsomes from bromopropylate-treated mice (300 mg/kg body wt) were used instead of control microsomes, indicating the induction of cytochromes P450 catalyzing the oxidative metabolism of the test compound. Single oral administration of 300 mg/kg body wt [14C]bromopropylate to male mice, without or following pretreatment for 14 days with 300 mg/kg body wt unlabeled bromopropylate, gave no indication for DNA binding of the test compound in the liver. This excludes a genotoxic potential via covalent DNA modification. The results suggest that, in analogy to phenobarbitone, bromopropylate acts as a tumor promotor rather than a tumor initiator in the mouse liver.

  20. Selective inhibition by chloramphenicol of pregnenolone-16. cap alpha. -carbonitrile-inducible rat liver cytochrome P-450 isozymes

    SciTech Connect

    Graves, P.E.; Kaminsky, L.S.; Halpert, J.

    1986-03-01

    Pregnenolone-16 ..cap alpha..-carbonitrile (PCN) has been shown to induce, in male rats, cytochrome P-450 isozymes responsible for the formation of R-10-hydroxywarfarin and R-dehydrowarfarin. Antibodies to the major PCN-inducible isozyme (PB/PCN-E) inhibit both activities in microsomal preparations. Recently the authors have shown that PCN treatment of female rats also induces the formation of both R-warfarin metabolites. However, in both sexes chloramphenicol (CAP) treatment selectively inhibits only the rate of formation of the R-dehydrowarfarin. A decrease in microsomal P-450 content occurs after in vivo administration of CAP to PCN-treated rats of both sexes. This is in contrast to the lack of effect of CAP on P-450 levels in phenobarbital-treated rats. Covalent binding of /sup 14/C-CAP to microsomal protein in vitro was increased 3 to 4-fold following PCN treatment. Chromatographic evidences suggests the presence of at least two PCN-induced isozymes of similar molecular weights in both male and female rat liver microsomes. These data are consistent with the multiplicity of PCN-inducible P-450 in rat liver.

  1. Reactive Intermediates in Cytochrome P450 Catalysis*

    PubMed Central

    Krest, Courtney M.; Onderko, Elizabeth L.; Yosca, Timothy H.; Calixto, Julio C.; Karp, Richard F.; Livada, Jovan; Rittle, Jonathan; Green, Michael T.

    2013-01-01

    Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis. PMID:23632017

  2. Induction time course of cytochromes P450 by phenobarbital and 3-methylcholanthrene pretreatment in liver microsomes of Alligator mississippiensis.

    PubMed

    Ertl, R P; Stegeman, J J; Winston, G W

    1998-05-01

    Alligator mississippiensis has at least two classes of inducible hepatic microsomal cytochromes P450 (CYP): (1) those induced by 3-methylcholanthrene (3MC), and (2) those induced by phenobarbital (PB). The rates of induction by these xenobiotic compounds are significantly slower than those reported for mammals. Carbon monoxide binding, western blots, and enzymatic activity measurements indicated that at least 48-72 hr are required to reach full induction. A methoxy-, ethoxy-, pentoxy, and benzyloxyphenoxazone (resorufin) O-dealkylation (MROD, EROD, PROD, and BROD) profile was indicative of substrate selectivity typical of 3MC- and PB-induced P450s. MROD and BROD showed the greatest ability to discriminate between alligator hepatic microsomes induced by 3MC and PB, respectively. This is in contrast to mammals, in which EROD is a biomarker of polycyclic aromatic hydrocarbon exposure because of its ability to discriminate the induction of CYP 1A. In a similar manner, PROD is a highly preferred activity of CYP 2B in mammals; thus, it is used to indicate CYP 2B induction. The induction of P450 by PB is a general phenomenon in mammals and birds. To the best of our knowledge, this is the first report demonstrating PB induction of P450 activities typical of the mammalian CYP 2 family isoforms in alligator or any reptilian liver. The importance of this finding to the evolution of CYP 2 family regulation by PB is heightened by the fact that induction by this xenobiotic is not common to fish and other lower vertebrates (Ertl RP and Winston GW, Comp Biochem Physiol, in press). Although indicating the presence of CYP 1A- and CYP 2B-like isoforms in alligator, it remains to be established how closely related these alligator P450s are to mammalian isoforms.

  3. Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities.

    PubMed

    Kopečná-Zapletalová, Michaela; Krasulová, Kristýna; Anzenbacher, Pavel; Hodek, Petr; Anzenbacherová, Eva

    2017-04-01

    1. The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically. 2. The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with Ki of 35.95 ± 6.96 and 60.56 ± 3.53 μmol/l and CYP3A4 (inhibited by genistein with Ki of 23.25 ± 5.85 μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (Ki of 57.69 ± 2.36 μmol/l) and equol (Ki of 38.47 ± 2.32 μmol/l). 3. Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.

  4. Metabolism of (-)-cis- and (-)-trans-rose oxide by cytochrome P450 enzymes in human liver microsomes.

    PubMed

    Nakahashi, Hiroshi; Yamamura, Yuuki; Usami, Atsushi; Rangsunvigit, Pramoch; Malakul, Pomthong; Miyazawa, Mitsuo

    2015-12-01

    The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes.

  5. Novel extrahepatic cytochrome P450s

    SciTech Connect

    Karlgren, Maria . E-mail: Maria.Karlgren@imm.ki.se; Miura, Shin-ichi; Ingelman-Sundberg, Magnus

    2005-09-01

    The cytochrome P450 enzymes are highly expressed in the liver and are involved in the metabolism of xenobiotics. Because of the initiatives associated with the Human Genome Project, a great progress has recently been seen in the identification and characterization of novel extrahepatic P450s, including CYP2S1, CYP2R1, CYP2U1 and CYP2W1. Like the hepatic enzymes, these P450s may play a role in the tissue-specific metabolism of foreign compounds, but they may also have important endogenous functions. CYP2S1 has been shown to metabolize all-trans retinoic acid and CYP2R1 is a major vitamin D 25-hydroxylase. Regarding their metabolism of xenobiotics, much remains to be established, but CYP2S1 metabolizes naphthalene and it is likely that these P450s are responsible for metabolic activation of several different kinds of xenobiotic chemicals and contribute to extrahepatic toxicity and carcinogenesis.

  6. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    PubMed

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

    2013-09-05

    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

  7. Induction of cytochrome P450IA1 in rat colon and liver by indole-3-carbinol and 5,6-benzoflavone.

    PubMed

    Vang, O; Jensen, M B; Autrup, H

    1990-08-01

    It is known that consumption of cruciferous vegetables protects against the chemical induction of cancer in many organs. It has been suggested that this protection is mediated through an effect on the cytochrome P450 monooxygenase system. This system is responsible for the activation of a number of chemical carcinogens to their ultimate forms. In the present study, the effect of indole-3-carbinol (I3C) and 5,6-benzoflavone (5,6BF) on the expression of cytochrome P450IA1 in rat colon and liver has been investigated. Cytochrome P450IA1 mRNA was induced in colon following a single oral administration of I3C or 5,6BF. A biphasic induction profile was obtained with maxima at 4 and 16 h post-administration. Both inducers caused an approximately 2-fold increase in P450IA1 mRNA at 4 h and a 10-fold increase at 16 h. In contrast, both cytochrome P450IA1 and IA2 mRNAs was increased over the control between 4 and 24 h. The total amount of P450IA mRNAs in liver at 4 and 16 h was increased about 2- and 4-fold respectively by I3C; 5,6BF induced the P450IA mRNAs 4- and 5-fold respectively. The expression of cytochrome P450IA1 and IA2 is induced by I3C and several flavones present in cruciferous vegetables. This suggests that one of the protective effects of cruciferous vegetables in the reduction of chemically induced cancer may be regulation of cytochrome P450s involved in the metabolism of the chemical carcinogens.

  8. Unusual Cytochrome P450 Enzymes and Reactions*

    PubMed Central

    Guengerich, F. Peter; Munro, Andrew W.

    2013-01-01

    Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO3+), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function. PMID:23632016

  9. Cytochrome P450 3A, NADPH cytochrome P450 reductase and cytochrome b5 in the upper airways in horse.

    PubMed

    Tydén, E; Olsén, L; Tallkvist, J; Tjälve, H; Larsson, P

    2008-08-01

    Gene and protein expression as well as catalytic activity of cytochrome P450 (CYP) 3A were studied in the nasal olfactory and respiratory mucosa and the tracheal mucosa of the horse. We also examined the activity of NADPH cytochrome P450 reductase (NADPH P450 reductase), the amount of cytochrome b(5) and the total CYP content in these tissues. Comparative values for the above were obtained using liver as a control. The CYP3A related catalytic activity in the tissues of the upper airways was considerably higher than in the liver. The CYP3A gene and protein expression, on the other hand, was higher in the liver than in the upper airway tissues. Thus, the pattern of CYP3A metabolic activity does not correlate with the CYP3A gene and protein expression. Our results showed that the activity of NADPH P450 reductase and the level of cytochrome b(5) in the relation to the gene and protein expression of CYP3A were higher in the tissues of the upper airways than in the liver. It is concluded that CYP3A related metabolism in horse is not solely dependent on the expression of the enzyme but also on adequate levels of NADPH P450 reductase and cytochrome b(5).

  10. Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase.

    PubMed

    Ged, C; Umbenhauer, D R; Bellew, T M; Bork, R W; Srivastava, P K; Shinriki, N; Lloyd, R S; Guengerich, F P

    1988-09-06

    A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the

  11. Cytochrome P450s and molecular epidemiology

    NASA Astrophysics Data System (ADS)

    Gonzalez, Frank J.; Gelboin, Harry V.

    1993-03-01

    Cytochrome P450 (P450) represent a superfamily of heme-containing monooxygenases that are found throughout the animal and plant kingdoms and in many microorganisms. A number of these enzymes are involved in biosynthetic pathways of steroid synthesis but in mammals the vast majority of P450s function to metabolize foreign chemicals or xenobiotics. In the classical phase I reactions on the latter, a membrane-bound P450 will hydroxylate a compound, usually hydrophobic in nature, and the hydroxyl group will serve as a substrate for the various transferases or phase II enzymes that attach hydrophilic substituents such as glutathione, sulfate or glucuronic acid. Some chemicals, however, are metabolically-activated by P450s to electrophiles capable of reacting with cellular macromolecules. The cellular concentrations of the chemical and P450, reactivity of the active metabolite with nucleic acid and the repairability of the resultant adducts, in addition to the nature of the cell type, likely determines whether a chemical will be toxic and kill the cell or will transform the cell. Immunocorrelative and cDNA-directed expression have been used to define the substrate specificities of numerous human P450s. Levels of expression of different human P450 forms have been measured by both in vivo and in vitro methodologies leading to the realization that a large degree of interindividual differences occur in P450 expression. Reliable procedures for measuring P450 expression in healthy and diseased subjects will lead to prospective and case- cohort studies to determine whether interindividual differences in levels of P450 are associated with susceptibility or resistance to environmentally-based disease.

  12. Site-directed mutation studies of human liver cytochrome P-450 isoenzymes in the CYP2C subfamily.

    PubMed Central

    Veronese, M E; Doecke, C J; Mackenzie, P I; McManus, M E; Miners, J O; Rees, D L; Gasser, R; Meyer, U A; Birkett, D J

    1993-01-01

    Evidence from human studies in vivo and in vitro strongly suggests that the methylhydroxylation of tolbutamide and the 4-hydroxylation of phenytoin, the major pathways in the elimination of these two drugs, are catalysed by the same cytochrome P-450 isoenzyme(s). In the present study we used site-directed mutagenesis and cDNA expression in COS cells to characterize in detail the kinetics of tolbutamide and phenytoin hydroxylations by seven CYP2C proteins (2C8, 2C9 and variants, and 2C10) in order to define the effects of small changes in amino acid sequences and the likely proteins responsible in the metabolism of these two drugs in man. Tolbutamide was hydroxylated to varying extents by all expressed cytochrome P-450 isoenzymes, although activity was much lower for the expressed 2C8 protein. While the apparent Km values for the 2C9/10 isoenzymes (71.6-131.7 microM) were comparable with the range of apparent Km values previously observed in human liver microsomes, the apparent Km for 2C8 (650.5 microM) was appreciably higher. The 2C8 enzyme also showed quite different sulphaphenazole inhibition characteristics. The 4-hydroxylation of phenytoin was also more efficiently catalysed by the 2C9/10 enzymes. These enzymes showed similarities in kinetics of phenytoin hydroxylation and sulphaphenazole inhibition compared with human liver phenytoin hydroxylase. Also of interest was the observation that, among the 2C9 variants, small differences in amino acid composition could appreciably affect both tolbutamide and phenytoin hydroxylations. The amino acid substitution Cys-144-->Arg increased both the rates of tolbutamide and phenytoin hydroxylations, while the Leu-359-->Ile change had a greater effect on phenytoin hydroxylation. We conclude that: (1) although 2C8 and 2C9/10 proteins metabolize tolbutamide. only 2C9/10 proteins play a major role in human liver; (2) 2C9/10 proteins also appear to be chiefly responsible for phenytoin hydroxylation; and (3) subtle differences in

  13. Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes.

    PubMed

    Yoshii, K; Kobayashi, K; Tsumuji, M; Tani, M; Shimada, N; Chiba, K

    2000-01-01

    Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4+/-1.0 microM and 200.5+/-83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r = 0.763, p < 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r = 0.638, p < 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while alpha-naphthoflavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, alpha-naphthoflavone inhibited the chlorpromazine 7-hydroxylase activity to 55-65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited significant activity for the chlorpromazine 7-hydroxylation. The Km values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2.

  14. COMMENTS ON "EFFECT OF PRENATAL EXPOSURE OF DELTAMETHRIN ON THE ONTOGENY OF XENOBIOTIC METABOLIZING CYTOCHROME P450S IN THE BRAIN AND LIVER OF OFFSPRINGS.

    EPA Science Inventory

    Comments on: Effect of prenatal exposure of deltamethrin on the ontogeny of xenobiotic metabolizing cytochrome P450s in the brain and liver of offsprings [Johri et al. Toxicol Appl Pharmacol. 214:279-289, 2006]

    Johri and colleagues recently reported that maternal exposur...

  15. In vivo effect of dried chicory root (Cichorium intybus L.) on xenobiotica metabolising cytochrome P450 enzymes in porcine liver.

    PubMed

    Rasmussen, Martin Krøyer; Zamaratskaia, Galia; Ekstrand, Bo

    2011-01-15

    Cytochrome P450 (CYP) enzymes are widely studied for their involvement in metabolism of drugs and endogenous compounds. In porcine liver, CYP1A2, 2A and 2E1 are important for the metabolism of skatole. Feeding chicory roots to pigs is known to decrease the skatole concentration in plasma and fat. In the present study we investigated the effect of chicory on CYP mRNA and protein expression, as well as their activity. Male pigs were feed dried chicory root for 16 days before liver samples were collected. By the use of RT-PCR and Western blotting we showed that the mRNA and protein expression of CYP1A2 and 2A were increased in chicory fed pigs. The mRNA expression of CYP2E1 was increased, while there was no effect on protein expression. Activity of CYP1A2 and 2A were increased in chicory feed pigs; this was not the case for CYP2E1 activity. In conclusion; oral administration of chicory root for 16 days to pigs increased the mRNA expression of CYP1A2, 2A and 2E1; and the protein expression of CYP1A2 and 2A. The activities of CYP1A2 and 2A were increased.

  16. Separation, purification, and properties of cytochrome P-450 from uninduced rat liver microsomes for the studies of metabolism of environmental chemicals

    SciTech Connect

    Dialameh, G.H. )

    1988-09-01

    This study reports the authors present results on the development of a procedure for purification of multiple forms of cytochrome P-450 from un-induced rat liver microsomes. These cytochromes are catalytically active when reconstituted with NADPH-cytochrome c reductase and lipid and exhibit substrate specificities. The presence of four distinct forms of cytochrome P-450 in uninduced rat liver microsomes which is the result of this research report, compared with the presence of six forms in induced animals represent the importance of genetic control of these enzymes for the metabolism and detoxification of environmental chemicals. These metabolite patterns are not only different for the various species, but also among different individuals. The molecular basis for this are genetic and environmental factors, which exhibit interesting evolutionary aspects.

  17. Suppressive effect of the ethanolic extract of adlay bran on cytochrome P-450 enzymes in rat liver and lungs.

    PubMed

    Yao, Hsien-Tsung; Lin, Jia-Hsuan; Chiang, Meng-Tsan; Chiang, Wenchang; Luo, Mei-Nin; Lii, Chong-Kuei

    2011-04-27

    Adlay ( Coix lachryma-jobi L. var. ma-yuen Stapf) is a grass crop and is reported to protect against various diseases such as cancer. To investigate the effect of the ethanolic extract of adlay bran (ABE) on drug-metabolizing enzymes and glutathione-related antioxidant enzymes in rats, three groups of eight male Sprague-Dawley rats each were fed a control diet or a diet containing 5 or 10% ABE for 4 weeks. Significant decreases in microsomal cytochrome P-450 (CYP) 1A1-catalyzed ethoxyresorufin O-deethylation, CYP2C-catalyzed diclofenac 4-hydroxylation, CYP2D-catalyzed dextromethorphan O-demethylation, and CYP3A-catalyzed testosterone 6β-hydroxylation in the liver and CYP1A1-catalyzed ethoxyresorufin O-deethylation in the lungs of rats fed ABE were observed. Immunoblot analyses also showed decreases of CYP1A1, 1A2, 2C6, 2C11, 2D1, 2E1, 3A1, and 3A2 in the liver and CYP1A1 in the lungs. Furthermore, rats fed the 10% ABE diet had a higher glutathione content and glutathione peroxidase, glutathione reductase, and glutathione S-transferase activities in the lungs, but such an increase was not noted in the liver. Inhibition of various CYP-catalyzed enzyme reactions by ABE in rat and human liver microsomes had also been shown. The results of this study indicate that ABE feeding may suppress CYP enzyme activities and CYP protein expression in the liver and lungs of rats. Moreover, the increase of the antioxidant potential by ABE is tissue-specific.

  18. Expression of Cytochrome P450s in the Liver of Rats Administered with Socheongryong-tang, a Traditional Herbal Formula

    PubMed Central

    Jin, Seong Eun; Ha, Hyekyung; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

    2016-01-01

    Objective: The purpose of this study was to investigate the potential influences of Socheongryong-tang (SCRT) on the messenger ribonucleic acid (mRNA) and protein expression of cytochrome P450 (CYP450) in vivo. Materials and Methods: SCRT was orally administered to either male or female Sprague-Dawley rats once daily at doses of 0, 1000, 2000, or 5000 mg/kg/day for 13 weeks. The mRNA expression of CYP450s (CYP1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2, and 4A1) in liver tissues was measured by reverse transcription polymerase chain reaction. And then, the protein expression of CYP1A1 and CYP2B1/2 in liver tissues was analyzed by the Western blot. Results: We found no significant influence in the mRNA expression of hepatic CYP1A2, 2C11, 2E1, 3A1, 3A2, and 4A1 after repeated administration of SCRT for 13 weeks. By contrast, the mRNA and protein expression of hepatic CYP1A1 was increased by repeated SCRT treatment in male rats, but not in female rats. The mRNA and protein expression of hepatic CYP2B1/2 in both genders was increased by administration of SCRT. Conclusion: A caution is needed when SCRT is co-administered with substrates of CYP2B1/2 for clinical usage. In case of male, an attention is also required when SCRT and drugs metabolized by CYP1A1 are taken together. Our findings provide information regarding the safety and effectiveness of SCRT when combined with conventional drugs. SUMMARY Oral administration of Socheongryong-tang for 13 weeks did not affect the mRNA expression of hepatic CYP1A2, 2C11, 2E1, 3A1, 3A2, and 4A1In male rats, oral administration of Socheongryong-tang for 13 weeks induced the mRNA and protein expression of hepatic CYP1A1 and CYP2B1/2In female rats, oral administration of Socheongryong-tang for 13 weeks induced the mRNA and protein expression of hepatic CYP2B1/2. Abbreviations used: SCRT: Socheongryong-tang, CYP450: Cytochrome P450, HPLC: High performance liquid chromatography, RT-PCR: Reverse transcription polymerase chain reaction. PMID

  19. Comparative modelling of cytochromes P450.

    PubMed

    Kirton, Stewart B; Baxter, Carol A; Sutcliffe, Michael J

    2002-03-31

    The superfamily of enzymes known as the cytochromes P450 (P450s) comprises a wide-ranging class of proteins with diverse functions. They are known, amongst other things, to be involved in the hormonal regulation of metabolism and reproduction, as well as having a major clinical significance through their association with diseases such as cancer, diabetes and hepatitis. Knowledge of the three-dimensional (3D) structure of a protein gives insight into its function. The 3D structures of P450s are therefore of considerable scientific interest. A number of high-resolution structures of P450s have been determined by X-ray crystallography and studies of these structures have provided valuable insights into the mechanism of these enzymes. Only one of these structures is mammalian and as yet there is no structural information on human P450s in the public domain. Until such a structure is solved it is necessary to employ alternative methods to gain structural insight into how human P450s perform their biological function. Here we report on the use of comparative modelling to predict the structure of human P450s based on knowledge of their amino acid sequences plus the 3D structures of other (not human) P450s. As an illustrative example of these techniques we have modelled the structure of P450 2C5 using five bacterial P450 structures as templates. We examine the importance of selecting suitable templates, obtaining a good amino acid sequence alignment, and evaluating the models generated. To improve the quality of the models an iterative cycle of sequence alignment, model building, and model evaluation is employed. The result is a model with excellent stereochemistry, good amino acid side chain environment properties, and a Calpha trace similar to the crystal structure.

  20. Decreased bile-acid synthesis in livers of hepatocyte-conditional NADPH-cytochrome P450 reductase-null mice results in increased bile acids in serum.

    PubMed

    Cheng, Xingguo; Zhang, Youcai; Klaassen, Curtis D

    2014-10-01

    NADPH-cytochrome P450 reductase (Cpr) is essential for the function of microsomal cytochrome P450 monooxygenases (P450), including those P450s involved in bile acid (BA) synthesis. Mice with hepatocyte-specific deletion of NADPH-cytochrome P450 reductase (H-Cpr-null) have been engineered to understand the in vivo function of hepatic P450s in the metabolism of xenobiotics and endogenous compounds. However, the impact of hepatic Cpr on BA homeostasis is not clear. The present study revealed that H-Cpr-null mice had a 60% decrease in total BA concentration in liver, whereas the total BA concentration in serum was almost doubled. The decreased level of cholic acid (CA) in both serum and livers of H-Cpr-null mice is likely due to diminished enzyme activity of Cyp8b1 that is essential for CA biosynthesis. Feedback mechanisms responsible for the reduced liver BA concentrations and/or increased serum BA concentrations in H-Cpr-null mice included the following: 1) enhanced alternative BA synthesis pathway, as evidenced by the fact that classic BA synthesis is diminished but chenodeoxycholic acid still increases in both serum and livers of H-Cpr-null mice; 2) inhibition of farnesoid X receptor activation, which increased the mRNA of Cyp7a1 and 8b1; 3) induction of intestinal BA transporters to facilitate BA absorption from the intestine to the circulation; 4) induction of hepatic multidrug resistance-associated protein transporters to increase BA efflux from the liver to blood; and 5) increased generation of secondary BAs. In summary, the present study reveals an important contribution of the alternative BA synthesis pathway and BA transporters in regulating BA concentrations in H-Cpr-null mice.

  1. Oxidation of tolualdehydes to toluic acids catalyzed by cytochrome P450-dependent aldehyde oxygenase in the mouse liver.

    PubMed

    Watanabe, K; Matsunaga, T; Yamamoto, I; Yashimura, H

    1995-02-01

    Mouse hepatic microsomal enzymes catalyzed the oxidation of o-, m-, and p-tolualdehydes, intermediate metabolites of xylene, to the corresponding toluic acids. Cofactor requirement for the catalytic activity indicates that the microsomes contain NAD- and NADPH-dependent enzymes for this reaction. GC/MS analyses of the carboxylic acids formed by incubation under oxygen-18 gas indicate that the mechanism for this oxidation is an oxygenation and a dehydrogenation for the NADPH- and NAD-dependent reaction. Vmax/Km (nmol/min/mg protein) ratios indicate that the NADPH-dependent activity is more pronounced than the NAD-dependent activity. These results suggest that the NADPH-dependent reaction is mainly responsible for the microsomal oxidation of tolualdehydes. The NADPH-dependent activity was significantly inhibited by SKF 525-A, disulfiram and menadione, inhibitors of cytochrome P450 (P450), suggesting the involvement of P450 in the reaction. In a reconstituted system, P450 MUT-2 (CYP2C29) purified from mouse hepatic microsomes catalyzed the oxidation of o-, m-, and p-tolualdehydes to the carboxylic acids, and the specific activities (nmol/min/nmol P450) were 1.44, 2.81, and 2.32, respectively. Rabbit antibody raised against P450 MUT-2 significantly inhibited the NADPH-dependent oxidation of tolualdehydes to toluic acids by 88% (o-), 63% (m-), and 62% (p-) using mouse hepatic microsomes. The present study demonstrated that a mouse hepatic microsomal aldehyde oxygenase, P450 MUT-2, catalyzed the most of oxidative activity of tolualdehydes to toluic acids in the microsomes.

  2. Identification of the rat liver cytochrome P450 enzymes involved in the metabolism of the calcium channel blocker dipfluzine hydrochloride.

    PubMed

    Guo, Wei; Shi, Xiaowei; Wang, Wei; Zhang, Weili; Li, Junxia

    2014-11-01

    This study aimed to identify the specific cytochrome P450 (CYP450) enzymes involved in the metabolism of dipfluzine hydrochloride using the combination of a chemical inhibition study, a correlation analysis and a panel of recombinant rat CYP450 enzymes. The incubation of Dip with rat liver microsomes yielded four metabolites, which were identified by liquid chromatography-coupled tandem mass spectrometry (LC/MS/MS). The results from the assays involving eight selective inhibitors indicated that CYP3A and CYP2A1 contributed most to the metabolism of Dip, followed by CYP2C11, CYP2E1 and CYP1A2; however, CYP2B1, CYP2C6 and CYP2D1 did not contribute to the formation of the metabolites. The results of the correlation analysis and the assays involving the recombinant CYP450 enzymes further confirmed the above results and concluded that CYP3A2 contributed more than CYP3A1. The results will be valuable in understanding drug-drug interactions when Dip is coadministered with other drugs.

  3. Cytochrome P450 2E1 is responsible for the initiation of 1,2-dichloropropane-induced liver damage.

    PubMed

    Yanagiba, Yukie; Suzuki, Tetsuya; Suda, Megumi; Hojo, Rieko; Gonzalez, Frank J; Nakajima, Tamie; Wang, Rui-Sheng

    2016-09-01

    1,2-Dichloropropane (1,2-DCP), a solvent, which is the main component of the cleaner used in the offset printing companies in Japan, is suspected to be the causative agent of bile duct cancer, which has been recently reported at high incidence in those offset printing workplaces. While there are some reports about the acute toxicity of 1,2-DCP, no information about its metabolism related to toxicity in animals is available. As part of our efforts toward clarifying the role of 1,2-DCP in the development of cancer, we studied the metabolic pathways and the hepatotoxic effect of 1,2-DCP in mice with or without cytochrome P450 2E1 (CYP2E1) activity. In an in vitro reaction system containing liver homogenate, 1,2-DCP was only metabolized by liver tissue of wild-type mice but not by that of cyp2e1-null mice. Furthermore, the kinetics of the solvent in mice revealed a great difference between the two genotypes; 1,2-DCP administration resulted in dose-dependent hepatic damage, as shown biochemically and pathologically, but this effect was only observed in wild-type mice. The nuclear factor κB p52 pathway was involved in the liver response to 1,2-DCP. Our results clearly indicate that the oxidative metabolism of 1,2-DCP in mice is exclusively catalyzed by CYP2E1, and this step is indispensable for the manifestation of the hepatotoxic effect of the solvent.

  4. The impact of individual cytochrome P450 enzymes on oxidative metabolism of benzo[a]pyrene in human livers.

    PubMed

    Šulc, Miroslav; Indra, Radek; Moserová, Michaela; Schmeiser, Heinz H; Frei, Eva; Arlt, Volker M; Stiborová, Marie

    2016-04-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes™ together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b5 , to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP-7,8-dihydrodiol and BaP-9-ol, which are intermediates in BaP-derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP-3-ol, a metabolite that is a 'detoxified' product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP-7,8-dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP-9-ol. BaP-3-ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active.

  5. Hepatocarcinogenic heterocyclic aromatic amines that induce cytochrome P-448 isozymes, mainly cytochrome P-448H (P-450IA2), responsible for mutagenic activation of the carcinogens in rat liver.

    PubMed

    Degawa, M; Tanimura, S; Agatsuma, T; Hashimoto, Y

    1989-06-01

    Male F344 rats were treated with hepatocarcinogenic heterocyclic aromatic amines such as amino acid- and protein-pyrolysate components (Trp P-1, Trp P-2, Glu P-1, Glu P-2, A alpha C, MeA alpha C, IQ and MeIQx) and changes in microsomal cytochrome P-450 isozymes in the livers were examined by means of immuno-Western blotting using anti-rat cytochrome P-450 monoclonal antibodies. The results suggested that all chemicals tested induce cytochrome P-448 isozymes, particularly cytochrome P-448H (P-450IA2), which efficiently mediate mutagenic activation of the carcinogens. This was substantiated by the enzymatic analyses with the substrates showing different characters to rat cytochrome P-450 isozyme-mediated mutagenesis.

  6. Evidence in rat and mouse liver for temporal control of two forms of cytochrome P-450 inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Guenthner, T M; Nebert, D W

    1978-11-15

    In the liver of perinatal rats or mice, the ratio of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced aryl hydrocarbon hydroxylase to total cytochrome P-450 content decreases, whereas the ratio of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced acetanilide 4-hydroxylase to total cytochrome P-450 content increases, between 18 or 19 days and 22 days following conception. The ontogenesis of inducible aryl hydrocarbon hydroxylase corresponds well with increases in a 56000-Mr electrophoretic band; we suggest this band represents the cytochrome P1-450 subunit. The later temporal expression of inducible acetanilide 4-hydroxylase closely parallels 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced increases in size of a 54000-Mr electrophoretic band and a 2--3-nm hypsochromic shift in the Soret peak of the total microsomal reduced cytochrome P-450 . CO complex. We suggest this band represents the cytochrome P-448 subunit. Previous work from this laboratory has shown that this developmental difference is separated by several weeks in rabbit liver, as compared with several day's separation shown in this report with rat or mouse liver. The data here therefore provide evidence in the rodent for temporal control of the expression of different structural gene products regulated by the Ah locus.

  7. Predominant 4-hydroxylation of estradiol by constitutive cytochrome P450s in the female ACI rat liver.

    PubMed

    Wilson, A M; Reed, G A

    2001-02-01

    The ACI rat is extremely sensitive to estrogens as mammary carcinogens, whereas the Sprague-Dawley strain is relatively resistant. Comparison of the disposition and effects of estrogens in these two strains should provide insights into the mechanisms of estrogen carcinogenicity. We have begun this investigation by comparing the metabolism of [(3)H]17beta-estradiol (E2) by liver microsomes prepared from female rats from each strain. Both strains produce estrone (E1) as the major product at E2 concentrations >1 microM, with smaller amounts of 2-hydroxy-E2 formed. As the E2 concentration is decreased, however, aromatic hydroxylation becomes a more dominant pathway for both strains. At starting E2 concentrations as low as 3 nM, Sprague-Dawley liver microsomes produced comparable yields of 2-hydroxy-E2 and E1. In contrast, ACI liver microsomes yielded a profound shift to aromatic hydroxylation as the dominant pathway as E2 concentrations dropped below 1 microM, and this shift reflected the production of 4-hydroxy-E2 as the predominant product. The apparent K(m) for 4-hydroxylation of E2 is <0.8 microM, as opposed to approximately 4 microM for 2-hydroxylation, suggesting that different cytochrome P450s (CYPs) are responsible. Western immunoblotting of the liver microsomal preparations from ACI and Sprague-Dawley rats for CYPs known to catalyze 2- and 4-hydroxylation of E2 revealed that both strains contained comparable amounts of CYP 2B1/2 and 3A1/2, but no detectable amounts of CYP 1B1, the proposed E2 4-hydroxylase. Although this enzyme is not a constitutive CYP in Sprague-Dawley rat liver, its presence in ACI liver could provide a ready explanation for the predominance of 4-hydroxy-E2 as a product. The identity of the estradiol 4-hydroxylase in ACI rat liver and the role of this unique reaction in the heightened sensitivity to E2 carcinogenicity remain to be elucidated.

  8. AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5'-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

    PubMed

    Kim, Ju-Hyun; Kwon, Soon-Sang; Kong, Tae Yeon; Cheong, Jae Chul; Kim, Hee Seung; In, Moon Kyo; Lee, Hye Suk

    2017-03-10

    AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography-tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

  9. Inhibitory effects of seven components of danshen extract on catalytic activity of cytochrome P450 enzyme in human liver microsomes.

    PubMed

    Qiu, Furong; Zhang, Rong; Sun, Jianguo; Jiye, A; Hao, Haiping; Peng, Ying; Ai, Hua; Wang, Guangji

    2008-07-01

    The potential for herb-drug interactions has recently received greater attention worldwide, considering the fact that the use of herbal products becomes more and more widespread. The goal of this work was to examine the potential for the metabolism-based drug interaction arising from seven active components (danshensu, protocatechuic aldehyde, protocatechuic acid, salvianolic acid B, tanshinone I, tanshinone IIA, and cryptotanshinone) of danshen extract. Probe substrates of cytochrome P450 enzymes were incubated in human liver microsomes (HLMs) with or without each component of danshen extract. IC(50) and K(i) values were estimated, and the types of inhibition were determined. Among the seven components of danshen extract, tanshinone I, tanshinone IIA, and cryptotanshinone were potent competitive inhibitors of CYP1A2 (K(i) = 0.48, 1.0, and 0.45 microM, respectively); danshensu was a competitive inhibitor of CYP2C9 (K(i) = 35 microM), and cryptotanshinone was a moderate mixed-type inhibitor of CYP2C9 (K(i) = 8 microM); cryptotanshinone inhibited weakly and in mixed mode against CYP2D6 activity (K(i) = 68 microM), and tanshinone I was a weak inhibitor of CYP2D6 (IC(50) = 120 microM); and protocatechuic aldehyde was a weak inhibitor of CYP3A4 (IC(50) = 130 and 160 microM for midazolam and testosterone, respectively). These findings provided some useful information for safe and effective use of danshen preparations in clinical practice. Our data indicated that it was necessary to study the in vivo interactions between drugs and pharmaceuticals with danshen extract.

  10. The impact of individual cytochrome P450 enzymes on oxidative metabolism of benzo[a]pyrene in human livers

    PubMed Central

    Šulc, Miroslav; Indra, Radek; Moserová, Michaela; Schmeiser, Heinz H.; Frei, Eva; Arlt, Volker M.; White, P.

    2016-01-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes™ together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b5, to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP‐7,8‐dihydrodiol and BaP‐9‐ol, which are intermediates in BaP‐derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP‐3‐ol, a metabolite that is a ‘detoxified’ product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP‐7,8‐dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP‐9‐ol. BaP‐3‐ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active. Environ. Mol. Mutagen. 57:229–235, 2016. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. PMID:26919089

  11. The role of highly purified forms of rat liver cytochrome P-450 in the dimethylation of dimethylnitrosamine and its activation to mutagens.

    PubMed

    Masson, H A; Ioannides, C; Gibson, G G

    1983-06-01

    Highly purified NADPH-cytochrome P-450 reductase and the major phenobarbital (PB) and beta-naphthoflavone (beta NF) forms of cytochrome P-450 were used in reconstituted systems to study the demethylation and subsequent activation of dimethylnitrosamine (DMN) to mutagenic intermediates. Both forms of cytochrome P-450 were active in the demethylation of DMN, cytochrome P-450 from PB-treated animals being more efficient, generating nearly twice as much formaldehyde per nmol of haemoprotein. Neither form of the cytochrome could activate DMN to mutagens in the Ames test. These findings indicate that DMN demethylation does not lead to its activation to mutagenic products.

  12. Determination of cytochrome P450 enzymes involved in the metabolism of (-)-terpinen-4-ol by human liver microsomes.

    PubMed

    Miyazawa, M; Haigou, R

    2011-12-01

    The in vitro metabolism of (-)-terpinen-4-ol was examined in human liver microsomes and recombinant enzymes. The biotransformation of (-)-terpinen-4-ol was investigated by gas chromatography-mass spectrometry. (-)-Terpinen-4-ol was found to be oxidized to (-)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol, major metabolic product by human liver microsomal P450 enzymes. The formation of metabolites of (-)-terpinen-4-ol was determined by relative abundance of mass fragments and retention times on GC. CYP2A6 in human liver microsomes was a major enzyme involved in the oxidation of (-)-terpinen-4-ol by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 had the highest activity for oxidation of (-)-terpinen-4-ol. Second, oxidation of (-)-terpinen-4-ol was inhibited by (+)-menthofuran. Finally, there was a good correlation between CYP2A6 maker activity and (-)-terpinen-4-ol oxidation activities in liver microsomes of 10 human samples. Kinetic analysis showed that the V(max)/K(m) values for (-)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol catalysed by liver microsomes of human sample HH-18 was 2.49 μL/min/nmol. Human recombinant CYP2A6 catalysed (-)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol with V(max) values of 13.9 nmol/min/nmol P450 and apparent K(m) values of 91 μM.

  13. Utility of intersystem extrapolation factors in early reaction phenotyping and the quantitative extrapolation of human liver microsomal intrinsic clearance using recombinant cytochromes P450.

    PubMed

    Chen, Yuan; Liu, Liling; Nguyen, Khanh; Fretland, Adrian J

    2011-03-01

    Reaction phenotyping using recombinant human cytochromes P450 (P450) has great utility in early discovery. However, to fully realize the advantages of using recombinant expressed P450s, the extrapolation of data from recombinant systems to human liver microsomes (HLM) is required. In this study, intersystem extrapolation factors (ISEFs) were established for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 using 11 probe substrates, based on substrate depletion and/or metabolite formation kinetics. The ISEF values for CYP2C9, CYP2D6, and CYP3A4 determined using multiple substrates were similar across substrates. When enzyme kinetics of metabolite formation for CYP1A2, 2C9, 2D6, and 3A4 were used, the ISEFs determined were generally within 2-fold of that determined on the basis of substrate depletion. Validation of ISEFs was conducted using 10 marketed drugs by comparing the extrapolated data with published data. The major isoforms responsible for the metabolism were identified, and the contribution of the predominant P450s was similar to that of previously reported data. In addition, phenotyping data from internal compounds, extrapolated using the rhP450-ISEF method, were comparable to those obtained using an HLM-based inhibition assay approach. Moreover, the intrinsic clearance (CL(int)) calculated from extrapolated rhP450 data correlated well with measured HLM CL(int). The ISEF method established in our laboratory provides a convenient tool in early reaction phenotyping for situations in which the HLM-based inhibition approach is limited by low turnover and/or unavailable metabolite formation. Furthermore, this method allows for quantitative extrapolation of HLM intrinsic clearance from rhP450 phenotyping data simultaneously to obtaining the participating metabolizing enzymes.

  14. Cytochrome P450 gene polymorphism and cancer.

    PubMed

    Agundez, Jose A G

    2004-06-01

    Human cytochrome P450 (CYP) enzymes play a key role in the metabolism of drugs and environmental chemicals. Several CYP enzymes metabolically activate procarcinogens to genotoxic intermediates. Phenotyping analyses revealed an association between CYP enzyme activity and the risk to develop several forms of cancer. Research carried out in the last decade demonstrated that several CYP enzymes are polymorphic due to single nucleotide polymorphisms, gene duplications and deletions. As genotyping procedures became available for most human CYP, an impressive number of association studies on CYP polymorphisms and cancer risk were conducted. Here we review the findings obtained in these studies regarding CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP8A1 and CYP21 gene polymorphisms. Consistent evidences for association between CYP polymorphisms and lung, head and neck, and liver cancer were reported. Controversial findings suggest that colorectal and prostate cancers may be associated to CYP polymorphisms, whereas no evidences for a relevant association with breast or bladder cancers were reported. We summarize the available information related to the association of CYP polymorphisms with leukaemia, lymphomas and diverse types of cancer that were investigated only for some CYP genes, including brain, esophagus, stomach, pancreas, pituitary, cervical epithelium, melanoma, ovarian, kidney, anal and vulvar cancers. This review discusses on causes of heterogeneity in the proposed associations, controversial findings on cancer risk, and identifies topics that require further investigation. In addition, some recommendations on study design, in order to obtain more conclusive findings in further studies, are provided.

  15. Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

    PubMed Central

    Zhu, Ge-Jian; Yu, Ying-Nian; Li, Xin; Qian, Yu-Li

    2002-01-01

    AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography (HPLC). RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild typeCYP2C9. However, there were two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 ± 0.109 μmol•min-1 ·g-1 S9 protein or 8.62 ± 2.02 mol•min-1 ·mol-1 CYP, but was undetectable in parental CHL cell. CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL-CYP2C9, efficiently expressing the protein of CYP2C9, was established. PMID:11925616

  16. Purification of the pyrazole-inducible cytochrome P-450 isozyme

    SciTech Connect

    Palakodety, R.; Clejan, L.; Krikun, G.; Feierman, D.; Cederbaum, A.I.

    1987-05-01

    The alcohol dehydrogenase inhibitor, pyrazole, appears to induce a cytochrome P-450 isozyme with properties similar to the ethanol-inducible P-450. The pyrazole-inducible P-450 isozyme was purified from the liver microsomes of rats treated with pyrazole essentially by the procedure of Ryan et al and also by chromatofocussing. The final preparation appeared homogenous by SDS-PAGE with an apparent molecular weight of 52,000, had a specific content of 11 nmoles P-450 per mg protein, showed very high activity of low K/sub m/ dimethylnitrosamine demethylase and produced a type II binding spectrum with dimethylsulfoxide. The enzyme was also active with aniline and aminopyrine as substrates. Pyrazole itself served as an excellent substrate with 4-hydroxy pyrazole being the product. An antibody against the pyrazole-inducible P-450 raised in chickens recognized a protein with mol.wt of about 52,000 in control microsomes. This band was highly enriched in microsomes from rats treated with pyrazole, 4-methyl-pyrazole, ethanol or acetone, but not phenobarbital or 3-methylcholanthrene. In summary, the pyrazole-inducible P-450 has been purified and appears to be identical in its catalytic and immunological properties to the alcohol-inducible P-450.

  17. Reduction of benzyl halides by liver microsomes. Formation of 478 NM-absorbing sigma-alkyl-ferric cytochrome P-450 complexes.

    PubMed

    Mansuy, D; Fontecave, M

    1983-06-15

    The benzyl halides benzyl bromide and 4-nitrobenzyl chloride are reduced anaerobically by NADPH and rat liver microsomes to yield toluene and 4-nitrotoluene, respectively. These reductions and cytochrome P-450-dependent since they are inhibited by CO and metyrapone, and are increased after pretreatment of rats by phenobarbital and 3-methylcholanthrene. During benzyl halide reduction, cytochrome P-450 complexes, which are very unstable to O2 and characterized by a Soret peak at 478 nm, are formed in steady-state concentrations. These concentrations are very dependent on pretreatment of rats and on the nature of the reducing agent (NADPH or dithionite) and the benzyl halide:4-methylbenzyl bromide and benzyl bromide lead to 478 nm absorbing complexes in the presence of NADPH whereas 4-nitrobenzyl chloride and benzyl chloride lead to such completes only in the presence of dithionite. Microsomal reductions of 4-nitrobenzyl chloride and benzyl bromide in D2O lead to partially deuterated 4-nitrotoluene and toluene. From these results, we propose a mechanism for anaerobic microsomal reduction of benzyl halides involving the intermediate formation of sigma-alkyl cytochrome P-450-Fe(III)-CH2Ar complexes which exhibit red-shifted Soret peaks around 478 nm. Toluenes, ArCH3, are formed either by protonation of the sigma-alkyl complexes or by hydrogen abstraction by the intermediate free radical ArCH2.

  18. Monoclonal antibody-directed analysis of cytochrome P-450-dependent monooxygenases and mutagen activation in the livers of DBA/2 and C57BL/6 mice.

    PubMed

    Hietanen, E; Malaveille, C; Friedman, F K; Park, S S; Béréziat, J C; Brun, G; Bartsch, H; Gelboin, H V

    1986-02-01

    Monoclonal antibodies (MAb 1-7-1 and Mab 2-66-3) specific for cytochrome P-450 (cyt. P-450) isozymes inhibited the metabolism of carcinogens, other xenobiotics, and endogenous compounds in two strains of mice. Postmitochondrial liver supernatant (S9) was prepared from untreated, 3-methylcholanthrene-treated, phenobarbital-treated, and pregnenolone 16 alpha-carbonitrile-treated C57BL/6 (B6) and DBA/2 (D2) mice. The modifying effect of two types of MAb to a 3-methylcholanthrene-induced cyt. P-450 and a phenobarbital-induced cyt. P-450 was investigated for: (a) S9-mediated mutagenicity of aflatoxin B1, benzo(a)pyrene 7,8-dihydrodiol, 2-acetylaminofluorene, and N-nitrosomorpholine in Salmonella typhimurium strains; and (b) the activity of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, aminopyrine N-demethylase, and testosterone 6 beta-, 7 alpha-, and 16 beta-hydroxylases. With certain S9s, MAb-1-7-1 inhibited only those cytochrome P-450 isozymes involved predominantly in activity of aryl hydrocarbon hydroxylase, ethoxyresorufin O-deethylase, and ethoxycoumarin O-deethylase and mutagenicity of 2-acetylaminofluorene and benzo(a)pyrene 7,8-dihydrodiol; MAb 2-66-3 inhibited only those involved in aminopyrine N-demethylase and testosterone 6 beta-, 7 alpha, and 16 beta-hydroxylase activity and aflatoxin B1 mutagenicity. Both Mab 1-7-1 and MAb 2-66-3 inhibited cytochrome P-450 isozyme(s) implicated predominantly in testosterone 7 alpha-hydroxylation in S9 from pregnenolone 16 alpha-carbonitrile-treated B6 mice. MAb 1-7-1 did not inhibit N-nitrosomorpholine mutagenicity and MAb 2-66-3 increased it by 2- to 6-fold depending on the source of S9. Using these MAbs, it is thus possible to identify the contribution of the epitope-defined single or class of cyt. P-450 to specific metabolic reactions in S9 from untreated and inducer-treated mice.

  19. Flower colour and cytochromes P450.

    PubMed

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-02-19

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

  20. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes.

    PubMed

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-07-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members' duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes.

  1. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

    PubMed Central

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members’ duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes. PMID:26129850

  2. Cytochrome P450 expression in oesophageal cancer.

    PubMed Central

    Murray, G I; Shaw, D; Weaver, R J; McKay, J A; Ewen, S W; Melvin, W T; Burke, M D

    1994-01-01

    The cytochrome P450 superfamily of enzymes play a central part in the metabolism of carcinogens and anti-cancer drugs. The expression, cellular localisation, and distribution of different forms of P450 and the functionally associated enzymes epoxide hydrolase and glutathione S-transferases have been investigated in oesophageal cancer and non-neoplastic oesophageal tissue using immunohistochemistry. Expression of the different enzymes was confined to epithelial cells in both non-neoplastic samples and tumour samples except the CYP3A was also identified in mast cells and glutathione S-transferase pi was present in chronic inflammatory cells. CYP1A was present in a small percentage of non-neoplastic samples but both CYP2C and CYP3A were absent. Epoxide hydrolase was present in half of the non-neoplastic samples and the different classes of glutathione S-transferase were present in a low number of samples. In carcinomas CYP1A, CYP3A, epoxide hydrolase, and glutathione S-transferase pi were expressed in at least 60% of samples. The expression of glutathione S-transferases alpha and mu were significantly less in adenocarcinoma compared with squamous carcinoma. Images Figure 1 Figure 2 Figure 3 PMID:8200549

  3. Flower colour and cytochromes P450

    PubMed Central

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-01-01

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones. PMID:23297355

  4. The revised human liver cytochrome P450 "Pie": absolute protein quantification of CYP4F and CYP3A enzymes using targeted quantitative proteomics.

    PubMed

    Michaels, Scott; Wang, Michael Zhuo

    2014-08-01

    The CYP4F subfamily of enzymes has been identified recently to be involved in the metabolism of endogenous compounds (arachidonic acid and leukotriene B4), nutrients (vitamins K1 and E), and xenobiotics (pafuramidine and fingolimod). CYP4F2 and CYP4F3B are reported to be expressed in the human liver. However, absolute concentrations of these enzymes in human liver microsomes (HLMs) and their interindividual variability have yet to be determined because of the lack of specific antibodies. Here, an liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based targeted quantitative proteomic approach was employed to determine the absolute protein concentrations of CYP4F2 and CYP4F3B compared with CYP3A in two panels of HLMs (n = 31). As a result, the human hepatic cytochrome P450 (P450) "pie" has been revised to include the contribution of CYP4F enzymes, which amounts to 15% of the total hepatic cytochrome P450 enzymes. CYP4F3B displayed low interindividual variability (3.3-fold) in the HLM panels whereas CYP4F2 displayed large variability (21-fold). However, CYP4F2 variability decreased to 3.4-fold if the two donors with the lowest expression were excluded. In contrast, CYP3A exhibited 29-fold interindividual variability in the same HLM panels. The proposed marker reaction for CYP4F enzymes pafuramidine/DB289 M1 formation did not correlate with CYP4F protein content, suggesting alternate metabolic pathways for DB289 M1 formation in HLMs. In conclusion, CYP4F enzymes are highly expressed in the human liver and their physiologic and pharmacologic roles warrant further investigation.

  5. Differential metabolism of acetanilide versus ethoxycoumarin and benzo[a]pyrene by two 3-methylcholanthrene-inducible forms of rat liver cytochrome P-450.

    PubMed

    Sundheimer, D W; Caveness, M B; Goldstein, J A

    1983-10-15

    The present study compares the catalytic activities of two 3-methylcholanthrene (3-MC) inducible forms of cytochrome P-450. These isozymes (P-448HCB and P-448MC) were isolated from liver microsomes of rats treated with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) and 3-MC, respectively. Catalytic activities of the isozymes were compared in a reconstituted system and by antibody inhibition studies in microsomes. In a reconstituted system, P-448HCB had very little catalytic activity toward benzo[a]pyrene or ethoxycoumarin (substrates metabolized preferentially by P-448MC). In contrast, both isozymes had high turnover numbers for aniline and acetanilide. However, catalytic activities of the purified isozymes were affected dramatically by Emulgen 911, a nonionic detergent. Since nonionic detergents used in the purification of P-450 isozymes cannot be completely removed after purification, residual amounts of detergent probably affect turnover numbers in a reconstituted system. Therefore, specific antibodies to cytochromes P-448MC and P-448HCB were used to examine the contribution of these isozymes to microsomal metabolism. Antibody inhibition studies confirmed that the majority of benzo[a]pyrene and ethoxycoumarin metabolism in 3-MC-induced microsomes was catalyzed by cytochrome P-448MC. In contrast, P-448HCB accounted for the majority of the acetanilide hydroxylase activity in 3-MC- and HCB-induced microsomes. Neither isozyme contributed appreciably to metabolism of these substrates in control microsomes.

  6. Identification of cytochrome p450 enzymes involved in the metabolism of 4'-methyl-alpha-pyrrolidinopropiophenone, a novel scheduled designer drug, in human liver microsomes.

    PubMed

    Springer, Dietmar; Paul, Liane D; Staack, Roland F; Kraemer, Thomas; Maurer, Hans H

    2003-08-01

    4'-Methyl-alpha-pyrrolidinopropiophenone (MPPP) is a new drug of abuse. It is believed to have an abuse potential similar to that of amphetamines. Previous studies with Wistar rats had shown that MPPP was metabolized mainly by hydroxylation in position 4' followed by dehydrogenation to the corresponding carboxylic acid. The aim of the study presented here was to identify the human hepatic cytochrome p450 (p450) enzymes involved in the biotransformation of MPPP to 4'-hydroxymethyl-pyrrolidinopropiophenone. Baculovirus-infected insect cell microsomes and human liver microsomes were used for this purpose. Only CYP2C19 and CYP2D6 catalyzed this hydroxylation. The apparent Km and Vmax values for the latter were 9.8 +/- 2.5 microM and 13.6 +/- 0.7 pmol/min/pmol p450, respectively. CYP2C19 was not saturable over the tested substrate range (2-1000 microM) and interestingly showed a biphasic kinetic profile with apparent Km,1 and Vmax,1 values of 47.2 +/- 12.5 microM and 8.1 +/- 1.4 pmol/min/pmol p450, respectively. Experiments with pooled human liver microsomes also revealed biphasic nonsaturable kinetics with apparent Km,1 and Vmax,1 values of 57.0 +/- 20.9 microM and 199.7 +/- 59.7 pmol/min/mg of protein for the high affinity enzyme, respectively. Incubation of 2 microM MPPP with 3 microM of the CYP2D6-specific inhibitor quinidine resulted in significant (p < 0.01) turnover inhibition (11.8 +/- 1.6% of control). Based on kinetic data corrected for the relative activity factors, CYP2D6 is the enzyme mainly responsible for MPPP hydroxylation, confirmed by CYP2D6 inhibition studies.

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

    PubMed

    Wang, Zhe; Sun, Wei; Huang, Cheng-Ke; Wang, Li; Xia, Meng-Ming; Cui, Xiao; Hu, Guo-Xin; Wang, Zeng-Shou

    2015-04-01

    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.

  8. Simultaneous alterations of brain and plasma serotonin concentrations and liver cytochrome P450 in rats fed on a tryptophan-free diet.

    PubMed

    Kot, Marta; Pilc, Andrzej; Daniel, Władysława A

    2012-10-01

    Our previous study suggested involvement of the brain serotonergic system in the regulation of liver cytochrome P450 (CYP). The aim of the present study was to demonstrate simultaneous responsiveness of liver CYP and the peripheral and brain serotonergic systems to a tryptophan deficient diet during three days and one or three weeks of ingestion. The concentrations of serotonin, noradrenaline, dopamine and their metabolites were measured in blood plasma, the hypothalamus and brain stem of male rats. The enzyme activity and protein levels in the liver were determined for isoforms CYP1A, CYP2A, CYP2B, CYP2C6, CYP2C11, CYP2D and CYP3A. A three-day tryptophan-free diet increased serotonin content in the hypothalamus (but not in the brain stem or plasma). After one week, the level of serotonin was not changed in the brain, but was markedly increased in the plasma. A three week tryptophan restriction significantly reduced the concentration of serotonin in the brain and plasma. Changes in CYP2C6 and CYP2C11 (an increase and a decrease, respectively) were maintained throughout the experiment, while those found in other CYP isoforms varied, which usually resulted in a gradual increase in the enzyme activity within three weeks. The observed alterations in liver CYPs suggest involvement of both central and peripheral serotonin in the regulation of liver CYP expression whose mechanism is discussed. In conclusion, a deficit in tryptophan in the diet may be responsible for very serious food-cytochrome P450 and food-drug metabolism interactions. Interactions of this type may also refer to drugs acting via serotonergic system.

  9. Metabolism of aflatoxin B{sub 1} in Turkey liver microsomes: The relative roles of cytochromes P450 1A5 and 3A37

    SciTech Connect

    Rawal, Sumit; Coulombe, Roger A.

    2011-08-01

    The extreme sensitivity of turkeys to aflatoxin B{sub 1} (AFB{sub 1}) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B{sub 1}-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB{sub 1} to the exo-AFBO and to detoxification products aflatoxin M{sub 1} (AFM{sub 1}) and aflatoxin Q{sub 1} (AFQ{sub 1}), respectively, complicates the kinetic analysis of AFB{sub 1} in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB{sub 1} in TLMs. In control TLMs, AFB{sub 1} was converted to exo-AFBO in addition to AFM{sub 1} and AFQ{sub 1} confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar ({approx} 0.1 {mu}M), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (> 50 {mu}M) AFB{sub 1} concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM{sub 1}, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ{sub 1} following the kinetic Hill equation. At 0.1 {mu}M AFB{sub 1}, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM{sub 1}) compared to 3A37 (0.15: 1, exo-AFBO: AFQ{sub 1}), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB{sub 1} bioactivation and metabolism at environmentally-relevant AFB{sub 1} concentrations in turkey liver. - Graphical abstract: Display Omitted Highlights: > Efficient bioactivation by P450s 1A5 and 3A4

  10. Role of specific cytochrome P450 isoforms in the conversion of phenoxypropoxybiguanide analogs in human liver microsomes to potent antimalarial dihydrotriazines.

    PubMed

    Diaz, Damaris S; Kozar, Michael P; Smith, Kirsten S; Asher, Constance O; Sousa, Jason C; Schiehser, Guy A; Jacobus, David P; Milhous, Wilbur K; Skillman, Donald R; Shearer, Todd W

    2008-02-01

    Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

  11. Cyclosporin A drug interactions. Screening for inducers and inhibitors of cytochrome P-450 (cyclosporin A oxidase) in primary cultures of human hepatocytes and in liver microsomes.

    PubMed

    Pichard, L; Fabre, I; Fabre, G; Domergue, J; Saint Aubert, B; Mourad, G; Maurel, P

    1990-01-01

    In previous papers we demonstrated that cyclosporin A (CsA) was specifically oxidized in rabbit and human liver by cytochrome P-450IIIA. We therefore anticipated that any drug that is an inducer or an inhibitor of this cytochrome should lead to interaction with CsA when given in association with it. In order to confirm this hypothesis, primary cultures of human hepatocytes and human liver microsomes were used to "reproduce" in vitro clinically significant interactions observed between CsA and drugs known either as specific inducers (i.e., rifampicin) or as specific inhibitors (i.e., erythromycin) of P-450IIIA. Our results were in close agreement with the clinical reports. Human hepatocytes maintained in primary cultures for 72 hr in the presence of 50 microM rifampicin exhibited increased levels of P-450IIIA, determined by Western blot using specific antibodies, and concomitant increase in CsA oxidase activity, determined by HPLC analysis of extra and intracellular media. Conversely, these cultures exhibited erythromycin concentration-dependent decreases in CsA oxidase activity when incubated in the presence of 5, 20, and 100 microM erythromycin. In addition, a Lineweaver-Burk analysis of the erythromycin-mediated inhibition of CsA oxidase activity in human liver microsomes revealed competitive inhibition (with Ki of 75 microM) as expected, this macrolide being a specific substrate of P-450IIIA. Using this experimental approach, 59 molecules representative of 17 different therapeutic classes were screened for inducers and inhibitors of CsA oxidase activity. Our results allowed us to elucidate the molecular mechanism of previously observed, but unexplained, drug interactions involving CsA, and to detect drugs that should interfere with CsA metabolism as inducers or inhibitors. Drugs detected as potential inducers of CsA oxidase included: rifampicin, sulfadimidine, phenobarbital, phenytoin, phenylbutazone, dexamethasone, sulfinpyrazone, and carbamazepine. Drugs

  12. Metabolism of methyl tert-butyl ether and other gasoline ethers in mouse liver microsomes lacking cytochrome P450 2E1.

    PubMed

    Hong, J Y; Wang, Y Y; Bondoc, F Y; Yang, C S; Gonzalez, F J; Pan, Z; Cokonis, C D; Hu, W Y; Bao, Z

    1999-03-08

    To reduce the production of pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE) and other ethers such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Metabolism of these gasoline ethers is catalyzed by cytochrome P450 (P450) enzymes. P450 2E1, which metabolizes diethyl ether, was suggested to be an enzyme involved. The present study used 2E1 knock-out mice (2E1-/-) to assess the contribution of 2E1 to the metabolism of MTBE, ETBE and TAME. Liver microsomes prepared from the 2E1 knock-out mice lacked 2E1 activity (assayed as N-nitrosodimethylamine demethylation), but were still active in metabolizing all three gasoline ethers. The levels of ether-metabolizing activity (nmol/min per mg) in the liver microsomes from 7 week old female 2E1 knock-out mice were 0.54+/-0.17 for MTBE, 0.51+/-0.24 for ETBE and 1.14+/-0.25 for TAME at a 1 mM substrate concentration. These activity levels were not significantly different from those of the sex- and age-matched C57BL/6N and 129/Sv mice, which are the parental lineage strains of the 2E1 knock-out mice and are both 2E1+/+. Our results clearly demonstrate that 2E1 plays a negligible role in the metabolism of MTBE, ETBE and TAME in mouse livers.

  13. Chemical proteomic probes for profiling cytochrome P450 activities and drug interactions in vivo

    PubMed Central

    Wright, Aaron T.; Cravatt, Benjamin F.

    2007-01-01

    The cytochrome P450 (P450) superfamily metabolizes many endogenous signaling molecules and drugs. P450 enzymes are regulated by post-translational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here, we describe a chemical proteomic strategy to profile P450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a “clickable” handle provided an activity-based probe that labels multiple P450s both in proteomic extracts and in vivo. This probe was used to record alterations in liver P450 activities triggered by chemical agents, including inducers of P450 expression and direct P450 inhibitors. The chemical proteomic strategy described herein thus offers a versatile method to monitor P450 activities and small molecule interactions in any biological system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class. PMID:17884636

  14. The inhaled glucocorticoid fluticasone propionate efficiently inactivates cytochrome P450 3A5, a predominant lung P450 enzyme

    PubMed Central

    Murai, Takahiro; Reilly, Christopher R.; Ward, Robert M.; Yost, Garold S.

    2010-01-01

    Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5 and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with KI, kinact and partition ratio of 16 μM, 0.027 min-1 and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 μM. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs. PMID:20707410

  15. Role of cytochrome P450 in drug interactions

    PubMed Central

    Bibi, Zakia

    2008-01-01

    Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events. PMID:18928560

  16. Cytochrome P450 1 family and cancers.

    PubMed

    Go, Ryeo-Eun; Hwang, Kyung-A; Choi, Kyung-Chul

    2015-03-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor that dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT). This complex binds to xenobiotics response element (XREs), and then starts the expressions of downstream genes including cytochrome P450 (CYP) 1 family members: CYP1A1, CYP1A2 and CYP1B1. Role of CYP1 family is involved in the metabolism of endogenous hormones, xenobiotics and drug. The expression of CYP1 family is regulated by estradiol (E2) or xenobiotics in diverse cancers. In breast cancers expressing estrogen receptors (ERs), level of CYP1B1 is increased by E2 and reversed by an estrogen receptor antagonist, ICI 182,780 or 4-hydrotamoxifen, which indicates that the expression of CYP1 family in downstream region of AhR is regulated by an activation of ERα. In metabolic pathways, E2 is converted into 4-hydroxyestradiol by CYP1B1, which can be converted into mainly estradiol-3,4-quinone, a potential carcinogen, by peroxidase. Increased expression of CYP1 family indicates the possibility of carcinogenesis by exposure of xenobiotics in endometrial and ovarian cancers. Apart from roles of CYP1 family in relation with ER pathway, CYP1 family is over-expressed in ER independent cancers. CYP1A1 exhibits hydroxylase activity in oxidation of arachidonic acid, which has been transformed to 12(R)-hydrxyeicosatetraenoic (HETEs), a potent activator of AhR activity. On the basis of results, phytoestrogens and dexamethasone are provided as cancer therapy regulating the expression of CYP1 family. Thus, this review focuses on the role(s) of CYP1 family in ER-dependent or ER-independent cancers and the potential for cancer therapy to target CYP1 family in these cancers.

  17. Zonation of hepatic cytochrome P-450 expression and regulation.

    PubMed Central

    Oinonen, T; Lindros, K O

    1998-01-01

    The CYP genes encode enzymes of the cytochrome P-450 superfamily. Cytochrome P-450 (CYP) enzymes are expressed mainly in the liver and are active in mono-oxygenation and hydroxylation of various xenobiotics, including drugs and alcohols, as well as that of endogenous compounds such as steroids, bile acids, prostaglandins, leukotrienes and biogenic amines. In the liver the CYP enzymes are constitutively expressed and commonly also induced by chemicals in a characteristic zonated pattern with high expression prevailing in the downstream perivenous region. In the present review we summarize recent studies, mainly based on rat liver, on the factors regulating this position-dependent expression and induction. Pituitary-dependent signals mediated by growth hormone and thyroid hormone seem to selectively down-regulate the upstream periportal expression of certain CYP forms. It is at present unknown to what extent other hormones that also affect total hepatic CYP activities, i.e. insulin, glucagon, glucocorticoids and gonadal hormones, act zone-specifically. The expression and induction of CYP enzymes in the perivenous region probably have important toxicological implications, since many CYP-activated chemicals cause cell injury primarily in this region of the liver. PMID:9405271

  18. Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation

    PubMed Central

    Kumar, Santosh

    2009-01-01

    Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts. PMID:20064075

  19. Thiomers: Inhibition of cytochrome P450 activity.

    PubMed

    Iqbal, Javed; Sakloetsakun, Duangkamon; Bernkop-Schnürch, Andreas

    2011-08-01

    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 (100kDa)>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.

  20. Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver.

    PubMed

    Kocarek, T A; Reddy, A B

    1996-11-01

    It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of

  1. In vivo and in vitro biotransformation of theobromine by phenobarbital- and 3-methylcholanthrene-inducible cytochrome P-450 monooxygenases in rat liver. Role of thiol compounds.

    PubMed

    Shively, C A; Vesell, E S

    1987-01-01

    A new in vitro method was developed and applied to establish the role of the hepatic cytochrome P-450 monooxygenases in theobromine biotransformation by control and phenobarbital (PB)- and 3-methylcholanthrene (3MC)-induced Sprague-Dawley rats. In vivo theobromine metabolite formation and pharmacokinetic parameters were also determined to serve as a comparison for in vitro studies. In vivo, the major urinary metabolite was 6-amino-5-[N-methylformylamino]-1-methyluracil (3,7DAU) with lesser amounts of 3,7-dimethyluric acid (3,7DMU), 3-methylxanthine, 7-methylxanthine, 7-methyluric acid, and traces of dimethylallantoin (DMA). Following induction with 3MC, but not PB, selective increases occurred in the urinary excretion of 3,7DAU, indicating that a 3MC-inducible cytochrome P-450 isozyme plays a significant role in this metabolic pathway. Both PB and 3MC induction increased slightly urinary elimination of DMA, a minor metabolite. Pharmacokinetic studies after a single oral dose of 5 mg/kg theobromine revealed a marked effect of 3MC treatment on theobromine elimination, as evidenced by a 59% decrease in theobromine t1/2, a 75% decrease in AUC, and a 284% increase in clearance. By contrast, PB had no effect. Fecal 14C elimination accounted for approximately 5% of the administered theobromine dose, and biliary excretion studies revealed the presence of 3,7DMU, DMA, 3,7DAU, and unchanged theobromine. Studies in vitro indicated that 3,7DMU was the major theobromine metabolite produced by liver microsomes. Conversion rates in PB- and 3MC-induced rats were 2- and 11-fold higher, respectively, than in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Catechins in tea suppress the activity of cytochrome P450 1A1 through the aryl hydrocarbon receptor activation pathway in rat livers.

    PubMed

    Fukuda, Itsuko; Nishiumi, Shin; Mukai, Rie; Yoshida, Ken-ichi; Ashida, Hitoshi

    2015-05-01

    Polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) develop various adverse effects through activation of an aryl hydrocarbon receptor (AhR). The suppressive effects of brewed green tea and black tea on 3-methylcholanthrene (MC)-induced AhR activation and its downstream events were examined in the liver of rats. Ad-libitum drinking of green tea and black tea suppressed MC-induced AhR activation and elevation of ethoxyresorufin O-deethylase activity in the liver, whereas the teas themselves did not induce them. Tea showed a suppressive fashion on the expression of cytochrome P450 1A1 (CYP1A1). Tea suppressed the AhR activation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ex vivo. A part of catechins and theaflavins was present in plasma and liver as conjugated and intact forms. The results of this study suggested that active component(s) of tea are incorporated in the liver and suppress the activity of CYP1As through the AhR activation pathway.

  3. Molecular modeling of cytochrome P450 3A4

    NASA Astrophysics Data System (ADS)

    Szklarz, Grazyna D.; Halpert, James R.

    1997-05-01

    The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B' helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6β-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.

  4. Involvement of cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer.

    PubMed Central

    Guengerich, F P; Johnson, W W; Ueng, Y F; Yamazaki, H; Shimada, T

    1996-01-01

    In recent years there has been considerable interest in the effect of variations in activities of xenobiotic-metabolizing enzymes on cancer incidence. This interest has accelerated with the development of methods for analyzing genetic polymorphisms. However, progress in epidemiology has been slow and the contributions of polymorphisms to risks from individual chemicals and mixtures are often controversial. A series of studies is presented to show the complexities encountered with a single chemical, aflatoxin B1 (AFB1). AFB1 is oxidized by human cytochrome P450 enzymes to several products. Only one of these, the 8,9-exo-epoxide, appears to be mutagenic and the others are detoxication products. P450 3A4, which can both activate and detoxicate AFB1, is found in the liver and the small intestine. In the small intestine, the first contact after oral exposure, epoxidation would not lead to liver cancer. The (nonenzymatic) half-life of the epoxide has been determined to be approximately 1 sec at 23 degrees C and neutral pH. Although the half-life is short, AFB1-8,9-exo-epoxide does react with DNA and glutathione S-transferase. Levels of these conjugates have been measured and combined with the rate of hydrolysis in a kinetic model to predict constants for binding of the epoxide with DNA and glutathione S-transferase. A role for epoxide hydrolase in alteration of AFB1 hepatocarcinogenesis has been proposed, although experimental evidence is lacking. Some inhibition of microsome-generated genotoxicity was observed with rat epoxide hydrolase; further information on the extent of contribution of this enzyme to AFB1 metabolism is not yet available. PMID:8781383

  5. Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo

    PubMed Central

    Ueng, Yune-Fang; Chen, Chien-Chih; Chung, Yu-Ting; Liu, Tsung-Yun; Chang, Yu-Ping; Lo, Wei-Sheng; Murayama, Norie; Yamazaki, Hiroshi; Souček, Pavel; Chau, Gar-Yang; Chi, Chin-Wen; Chen, Ruei-Ming; Li, Ding-Tzai

    2011-01-01

    BACKGROUND AND PURPOSE Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo. EXPERIMENTAL APPROACH Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS). KEY RESULTS CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC50 value decreased by 98%). This time-dependent inactivation (kinact 0.044 min−1; KI 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo. CONCLUSIONS AND IMPLICATIONS Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. ‘Poor metabolizers’ carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice. PMID:21418183

  6. Genetics Home Reference: cytochrome P450 oxidoreductase deficiency

    MedlinePlus

    ... hormones, which are needed for normal development and reproduction. The hormonal changes associated with cytochrome P450 oxidoreductase ... which are essential for normal sexual development and reproduction; corticosteroids, which are involved in the body's response ...

  7. De novo sequence analysis of cytochrome P450 1-3 genes expressed in ostrich liver with highest expression of CYP2G19.

    PubMed

    Kawai, Yusuke K; Watanabe, Kensuke P; Ishii, Akihiro; Ohnuma, Aiko; Sawa, Hirofumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2013-09-01

    The cytochrome P450 (CYP) 1-3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1-3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2-10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism.

  8. Use of RNA-seq to determine variation in canine cytochrome P450 mRNA expression between blood, liver, lung, kidney and duodenum in healthy beagles.

    PubMed

    Visser, M; Weber, K; Rincon, G; Merritt, D

    2017-03-19

    RNA sequencing (RNA-seq) is a powerful tool for the evaluation and quantification of transcriptomes and expression patterns in animals, tissues, or pathological conditions. The purpose of this study was to determine the physiologic expression of cytochrome P450 (CYP) mRNA transcripts in whole blood, kidney, duodenum, liver, and lung in healthy, adult male (n = 4) and female (n = 4) beagles via RNA-seq. mRNA expression was above background (transcripts per million) for 45 canine CYPs, with liver, duodenum, and lung expressing a high number of xenobiotic metabolizing CYPs, while prominent endogenous metabolizing CYP expression was present in blood and kidney. The relative expression pattern of CYP2A13, 2B11, 2C21, 2D15, 2E1, 3A12, and 27A1 in liver, lung, and duodenum was verified through qPCR. This is the first global profiling of physiologic CYP mRNA expression in multiple canine tissues, providing a platform for further studies characterizing canine CYPs and changes in gene expression in disease states.

  9. The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s

    PubMed Central

    Nelson, David R.; Goldstone, Jared V.; Stegeman, John J.

    2013-01-01

    The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution. PMID:23297357

  10. Cytochrome P450-2D6 Screening Among Elderly Using Antidepressants (CYSCE)

    ClinicalTrials.gov

    2016-10-24

    Depression; Depressive Disorder; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Intermediate Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant

  11. Effects of Cu(2+) and Pb(2+) on different fish species: liver cytochrome P450-dependent monooxygenase activities and FTIR spectra.

    PubMed

    Henczová, Mária; Deér, Aranka Kiss; Filla, Adrienn; Komlósi, Viktória; Mink, János

    2008-07-01

    The effects of Cu(2+)-sulfate and Pb(2+)-acetate on carp (Cyprinus carpio L.), silver carp (Hypopthalmichtys molitrix V.) and wels (Silurus glanis L.) were studied. The liver microsomal Cyt P450 content, the EROD, ECOD and APND monooxygenase activities were measured. In vivo treatment with 1 mg L(-1) Cu(2+) significantly elevated the activities of these enzymes and Cyt P450 content in silver carp livers. The high-dose Cu(2+) treatment (10 mg L(-1)) on silver carp caused two-fold higher induction in the P450 dependent monooxygenase isoensymes than in wels. Although the 2 mg kg(-1) treatment with Pb(2+) in carp elevated significantly the P450 content, the EROD isoenzyme activities were significantly decreased after 1 day, showing the destructive effect of metal ion on the enzyme system. In vitro, Cu(2+) and Pb(2+) decreased the Cyt P450 content in the carp liver microsomes and the absorption peak shifted to higher wavelength. Fourier Transform Infrared (FTIR) spectroscopy was used to detect the damaging effects of the heavy metals. According to the inhibitory potency to Cu(2+), the most sensitive isoenzyme was the EROD in wels, the least was the silver carp's isoenzyme. The investigated fish P450 isoenzymes showed, that the Cu(2+) was a stronger inhibitor than Pb(2+).

  12. Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry.

    PubMed

    Cieślak, Anna; Kelly, Isabelle; Trottier, Jocelyn; Verreault, Mélanie; Wunsch, Ewa; Milkiewicz, Piotr; Poirier, Guy; Droit, Arnaud; Barbier, Olivier

    2016-11-01

    This study aimed at establishing a sensitive multiple reaction monitoring-mass spectrometry (MRM-MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM-MS analyses were performed using three transitions optimized on one purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM-MS measurement were also determined. The method was applied to liver samples from ten non-cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n = 12; PBC), primary sclerosing cholangitis (n = 10; PSC) or alcoholic liver disease (n = 12; ALD). The established method presented high sensitivity with limit of detection lower than 5 fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM-based CYP3A4 protein quantification in homogenates and microsomes (r = 0.49, p < 0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM-MS quantification of CYP3A4 in homogenates also correlated (r = 0.44; p < 0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion. The established method provides a sensitive tool to evaluate the CYP3A4 protein in human liver homogenates from patients with normal or chronic/severe hepatic injury.

  13. Long-term p-nitrophenol exposure can disturb liver metabolic cytochrome P450 genes together with aryl hydrocarbon receptor in Japanese quail.

    PubMed

    Ahmed, Eman; Nagaoka, Kentaro; Fayez, Mostafa; Samir, Haney; Watanabe, Gen

    2015-08-01

    P-Nitrophenol is a major metabolite of some organophosphorus compounds. It is considered to be one of nitrophenol derivatives of diesel exhaust particles that induce substantial hazards impacts on human and animal health. P-Nitrophenol (PNP) is a persistent organic pollutant. Consequently, bioaccumulation of PNP potentiates toxicity. The objectives of the current study were to assess the potential hepatic toxicity and pathway associated with long-term exposure to PNP. Japanese quails were orally administered different doses of PNP for 75 days. Liver and plasma samples were collected at days 45 (45D), days 60 (60D) and days 75 (75D). Liver histological changes and plasma corticosterone levels were assessed. Basal mRNA level of cytochromes P450 (CYP 450) (CYP1A4, 1A5, 1B1), heme oxygenase (HO-1), and aryl hydrocarbon receptor 1 (AhR1), from the liver of exposed birds and primary hepatocytes cultured for 24 hr with PNP, were analyzed using quantitative real-time PCR. The results revealed various histopathological changes in the liver, such as lymphocytes aggregation and hepatocytes degeneration. Significant increases in corticosterone levels were reported. After 60-days of in vivo exposure, the birds exhibited an overexpression in the liver CYP1A4, 1B1, AhR1, and HO-1. Furthermore, with continuous PNP administration, an overall downregulation of the tested genes was observed. In vitro, although a significant overexpression of CYP1A4, 1B1, and HO-1 was observed, CYP1A5 was downregulated. In conclusion, PNP can interfere with the liver CYP 450 enzymes and modulate HO-1 expression in the in vitro and in vivo experiments. Hence, it could have serious deleterious effects on humans, livestock, and wild animals.

  14. Role of Inflammatory and Oxidative Stress, Cytochrome P450 2E1, and Bile Acid Disturbance in Rat Liver Injury Induced by Isoniazid and Lipopolysaccharide Cotreatment

    PubMed Central

    Hassan, Hozeifa Mohamed; Guo, Hongli; Yousef, Bashir Alsiddig; Guerram, Mounia; Hamdi, Aida Mejda

    2016-01-01

    Isoniazid (INH) remains the core drug in tuberculosis management, but serious hepatotoxicity and potentially fatal liver injury continue to accompany INH consumption. Among numerous theories that have been established to explain INH-induced liver injury, an inflammatory stress theory has recently been widely used to explain the idiosyncrasy. Inflammatory stress usually sensitizes tissues to a drug's toxic consequences. Therefore, the present study was conducted to verify whether bacterial lipopolysaccharide (LPS)-induced inflammation may have a role in enhancing INH hepatotoxicity. While single INH or LPS administration showed no major toxicity signs, INH-LPS cotreatment intensified liver toxicity. Both blood biomarkers and histological evaluations clearly showed positive signs of severe liver damage accompanied by massive necrosis, inflammatory infiltration, and hepatic steatosis. Furthermore, elevated serum levels of bile acid associated with the repression of bile acid synthesis and transport regulatory parameters were observed. Moreover, the principal impact of cytochrome P450 2E1 (CYP2E1) on INH toxicity could be anticipated, as its protein expression showed enormous increases in INH-LPS-cotreated animals. Furthermore, the crucial role of CYP2E1 in the production of reactive oxygen species (ROS) was clearly obvious in the repression of hepatic antioxidant parameters. In summary, these results confirmed that this LPS-induced inflammation model might prove valuable in revealing the hepatotoxic mechanisms of INH and the crucial role played by CYP2E1 in the initiation and propagation of INH-induced liver damage, information which could be very useful to clinicians in understanding the pathogenesis of drug-induced liver injury. PMID:27324775

  15. [Heterologous expression of functionally active human cytochrome P-450s. Cytochrome P-450IIIA4 catalyzes the biotransformation of the anabolic steroid hormone methandrostenolone].

    PubMed

    Krynetskiĭ, E Iu; Kovaleva, I E; Luzikov, V N

    1994-02-01

    The expression of the cytochrome P450IIIA4 gene in the Saccharomyces cerevisiae yeast using the shuttle vector pYeDP1-8/2 has been carried out. The microsomal fraction isolated from the transformed yeast cells was used for biotransformation of the anabolic steroid hormone-methandrostenolone (MA). The microsomal oxidation products were analyzed by HPLC and two-dimensional TLC. It was shown that microsomes of the yeasts expressing human cytochrome P450IIIA4 catalyze the MA conversion into its 6 beta-hydroxy derivative. An identical product is formed via a reaction catalyzed by human liver microsomes. The use of the heterological system of cytochrome P450IIIA4 expression has made it possible to establish its role in MA metabolism. The experimental system simulates the first phase of the drug biotransformation in liver cells.

  16. Selective expression and induction of cytochrome P450PB and P450MC during the development of hereditary hepatitis and hepatoma of LEC rats.

    PubMed

    Sugiyama, T; Suzuki, K; Ookawara, T; Kurosawa, T; Taniguchi, N

    1989-11-01

    The Long-Evans rat with a cinnamon-like coat color (LEC rat) is a mutant strain displaying hereditary hepatitis with severe jaundice. The age related difference in microsomal dealkylation of pentoxyresorufin and ethoxyresorufin was examined. The enzyme activity levels of pentoxyresorufin O-depentylase in LEC rats were decreased to 25% of the levels in control [Long-Evans rats with an agouti coat color (LEA rats)]. In contrast, ethoxyresorufin O-deethylase exhibited a much less marked difference between the strains. In parallel with these strain differences in enzyme activities, a decrease in phenobarbital (PB) inducible P450 isozymes, mainly P450b and P450e, was observed by Western blot analysis. The level of P450PB in LEC rats was more markedly depressed than in the LEA strain. On the other hand, microsomes from uninduced LEC rat liver had more 3-methylcholanthrene (MC) inducible P450MC, mainly P450c and P450d, than microsomes from LEA rat liver and these isozymes in the LEC were markedly induced by 3-methylcholanthrene treatment. The great difference in cytochrome P450PB content of the liver microsomes between LEC and LEA rats and the maintained constitutive levels of hepatic cytochrome P450MC in the LEC rats suggest a possible role of these cytochrome isozymes in the onset of spontaneous hepatitis and hepatoma.

  17. Effect of prenatal exposure of deltamethrin on the ontogeny of xenobiotic metabolizing cytochrome P450s in the brain and liver of offsprings

    SciTech Connect

    Johri, Ashu; Dhawan, Alok; Lakhan Singh, Ram; Parmar, Devendra . E-mail: parmar_devendra@hotmail.com

    2006-08-01

    Prenatal exposure to low doses (0.25 or 0.5 or 1.0 mg/kg, p.o.) of deltamethrin, a type II pyrethroid insecticide, to pregnant dams from gestation days 5 to 21 (GD5-21) produced dose-dependent alterations in the ontogeny of xenobiotic metabolizing cytochrome P450 (CYP) isoforms in brain and liver of the offsprings. RT-PCR analysis revealed dose-dependent increase in the mRNA expression of cerebral and hepatic CYP1A1, 1A2, 2B1, 2B2, and 2E1 isoenzymes in the offsprings exposed prenatally to deltamethrin. Similar increase in the activity of the marker enzymes of these CYP isoforms has indicated that placental transfer of the pyrethroid, a mixed type of CYP inducer, even at these low doses may be sufficient to induce the CYPs in brain and liver of the offsprings. Our data have further revealed persistence in the increase in expression of xenobiotics metabolizing CYPs up to adulthood in brain and liver of the exposed offsprings, suggesting the potential of deltamethrin to imprint the expression of CYPs in brain and liver of the offsprings following its in utero exposure. Furthermore, though the levels of CYPs were several fold lower in brain, almost equal magnitude of induction in cerebral and hepatic CYPs has further suggested that brain CYPs are responsive to the induction by environmental chemicals. The present data indicating alterations in the expression of xenobiotic metabolizing CYPs during development following prenatal exposure to deltamethrin may be of significance as these CYP enzymes are not only involved in the neurobehavioral toxicity of deltamethrin but have a role in regulating the levels of ligands that modulate growth, differentiation, and neuroendocrine functions.

  18. Inhibition on human liver cytochrome P450 3A4 by constituents of fennel (Foeniculum vulgare): identification and characterization of a mechanism-based inactivator.

    PubMed

    Subehan; Zaidi, Syed F H; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2007-12-12

    Fennel, a seed of Foeniculum vulgare, is used as a culinary spice and traditional medicine. The methanolic extract of fennel showed a characteristic of mechanism-based inactivation on erythromycin N-demethylation mediated by human liver microsomal cytochrome P450 3A4 (CYP3A4). The present study was conducted to identify the fennel constituent having the inhibition. Thirteen compounds have been isolated from a methanol extract of fennel and tested for their inhibition on CYP3A4. Among them, 5-methoxypsoralen (5-MOP) showed the strongest inhibition with an IC50 value of 18.3 microM and a mixed type of inhibition. In addition, with the preincubation time of 20 min only 5-MOP showed preincubation time dependency; the IC50 value decreased from 18.3 microM with a preincubation time of 0 min to 4.6 microM with a preincubation time of 20 min. Further investigation on 5-MOP showed the characteristics of time-dependent inhibition, requirement of NADPH, lack of protecting effect of nucleophiles, and recovery of CYP3A4 activity by the competitive inhibitor. This result suggests that the inhibitory activity of CYP3A4 by 5-MOP was a mechanism-based inactivation. The kinetic parameter for mechanism-based inactivation was characterized by a KI value of 15.0 microM and a kinact value of 0.098 min(-1).

  19. High dose of commercial products of kava (Piper methysticum) markedly enhanced hepatic cytochrome P450 1A1 mRNA expression with liver enlargement in rats.

    PubMed

    Yamazaki, Yuko; Hashida, Hiroko; Arita, Anna; Hamaguchi, Keiko; Shimura, Fumio

    2008-12-01

    Commercial products containing the kava plant (Piper methysticum), known to have the anxiolytic activity, are banned in several European countries and Canada because of the suspicion of a potential liver toxicity. In some reports, kava and kavalactones (major constituents of kava) inhibited activities of cytochrome P450 (CYP) isoforms including CYP1A2. On the other hand, a few studies showed that administration of kava to rats moderately increased CYP1A2 proteins in the liver. CYP1A isoforms are likely responsible for the metabolic activation of potent carcinogenic environmental toxins such as aflatoxins, benzo[a]pyrene, and others. On these bases, we have investigated the effects of administration of commercial kava products on gene expression of hepatic CYP1A isoforms in rats. A high dose (equivalent to approximately 380mg kavalactones/kg/day; 100 times of the suggested dosage for human use) of two different types of kava products for 8 days significantly increased liver weights. CYP1A2 mRNA expression was moderately increased (2.8-7.3 fold). More importantly, the high dose of kava markedly enhanced CYP1A1 mRNA expression (75-220 fold) as well as ethoxyresorufin O-deethylase activities and CYP1A1 immunoreactivities. Thus, no observed adverse effect levels of kavalactones would be lower than 380mg/kg/day. When the safety factor of kavalactones is assumed to be 100, a value most often used upon the risk analysis of chemicals and designed to account for interspecies and intraspecies variations, a number of kava product users likely ingest more kavalactones than acceptable daily intakes. Based on overall evidence, we should pay considerable attention to the possibility that kava products induce hepatic CYP1A1 expression in human especially in sensitive individuals.

  20. Cytochrome P450-mediated metabolism of vitamin D

    PubMed Central

    Jones, Glenville; Prosser, David E.; Kaufmann, Martin

    2014-01-01

    The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field. PMID:23564710

  1. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G.

    2010-11-16

    Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

  2. Effects of Eleutheroside B and Eleutheroside E on activity of cytochrome P450 in rat liver microsomes

    PubMed Central

    2014-01-01

    Background Chemicals of herbal products may cause unexpected toxicity or adverse effect by the potential for alteration of the activity of CYP450 when co-administered with other drugs. Eleutherococcus senticosus (ES), has been widely used as a traditional herbal medicine and popular herbal dietary supplements, and often co-administered with many other drugs. The main bioactive constituents of ES were considered to be eleutherosides including eleutheroside B (EB) and eleutheroside E (EE). This study was to investigate the effects of EB and EE on CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in rat liver microsomes in vitro. Method Probe drugs of tolbutamide (TB), dextromethorphan (DM), chlorzoxazone (CLZ) and testosterone (TS) as well as eleutherosides of different concentrations were added to incubation systems of rat liver microsomes in vitro. After incubation, validated HPLC methods were used to quantify relevant metabolites. Results The results suggested that EB and EE exhibited weak inhibition against the activity of CYP2C9 and CYP2E1, but no effects on CYP2D6 and CYP3A4 activity. The IC50 values for EB and EE were calculated to be 193.20 μM and 188.36 μM for CYP2E1, 595.66 μM and 261.82 μM for CYP2C9, respectively. Kinetic analysis showed that inhibitions of CYP2E1 by EB and EE were best fit to mixed-type with Ki value of 183.95 μM and 171.63 μM, respectively. Conclusions These results indicate that EB and EE may inhibit the metabolism of drugs metabolized via CYP2C9 and CYP2E1, and have the potential to increase the toxicity of the drugs. PMID:24383621

  3. Special issue: Cytochrome P450 structure and function: introduction.

    PubMed

    Munro, Andrew W; Leys, David

    2012-05-01

    The 17th International Conference on Cytochrome P450 Biochemistry, Biophysics and Structure was held in Manchester, UK from 26-30 June 2011. This issue of FEBS J. contains review and primary research articles reflecting the breadth of science covered at this conference, and reflecting the impact of P450-related research in fields as diverse as steroid metabolism, plant biochemistry, structural biology and biotechnology.

  4. Enhanced expression of cytochrome P450 in stomach cancer.

    PubMed Central

    Murray, G. I.; Taylor, M. C.; Burke, M. D.; Melvin, W. T.

    1998-01-01

    The cytochromes P450 have a central role in the oxidative activation and detoxification of a wide range of xenobiotics, including many carcinogens and several anti-cancer drugs. Thus the cytochrome P450 enzyme system has important roles in both tumour development and influencing the response of tumours to chemotherapy. Stomach cancer is one of the commonest tumours of the alimentary tract and environmental factors, including dietary factors, have been implicated in the development of this tumour. This type of tumour has a poor prognosis and responds poorly to current therapies. In this study, the presence and cellular localization of several major forms of P450, CYP1A, CYP2E1 and CYP3A have been investigated in stomach cancer and compared with their expression in normal stomach. There was enhanced expression of CYP1A and CYP3A in stomach cancer with CYP1A present in 51% and CYP3A present in 28% of cases. In contrast, no P450 was identified in normal stomach. The presence of CYP1A and CYP3A in stomach cancer provides further evidence for the enhanced expression of specific forms of cytochrome P450 in tumours and may be important therapeutically for the development of anti-cancer drugs that are activated by these forms of P450. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9569036

  5. The Interaction of Microsomal Cytochrome P450 2B4 with its Redox Partners, Cytochrome P450 Reductase and Cytochrome b5

    PubMed Central

    Im, Sang-Choul; Waskell, Lucy

    2010-01-01

    1 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 cytochome 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. PMID:21055385

  6. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of soybean genome sequence allows us to ident...

  7. Development of HepG2-derived cells expressing cytochrome P450s for assessing metabolism-associated drug-induced liver toxicity.

    PubMed

    Xuan, Jiekun; Chen, Si; Ning, Baitang; Tolleson, William H; Guo, Lei

    2016-08-05

    The generation of reactive metabolites from therapeutic agents is one of the major mechanisms of drug-induced liver injury (DILI). In order to evaluate metabolism-related toxicity and improve drug efficacy and safety, we generated a battery of HepG2-derived cell lines that express 14 cytochrome P450s (CYPs) (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 3A7) individually using a lentiviral expression system. The expression/production of a specific CYP in each cell line was confirmed by an increased abundance of the CYP at both mRNA and protein levels. Moreover, the enzymatic activities of representative CYPs in the corresponding cell lines were also measured. Using our CYP-expressed HepG2 cells, the toxicity of three drugs that could induce DILI (amiodarone, chlorpromazine and primaquine) was assessed, and all of them showed altered (increased or decreased) toxicity compared to the toxicity in drug-treated wild-type HepG2 cells. CYP-mediated drug toxicity examined in our cell system is consistent with previous reports, demonstrating the potential of these cells for assessing metabolism-related drug toxicity. This cell system provides a practical in vitro approach for drug metabolism screening and for early detection of drug toxicity. It is also a surrogate enzyme source for the enzymatic characterization of a particular CYP that contributes to drug-induced liver toxicity.

  8. The inhibitory effect of tannic acid on cytochrome P450 enzymes and NADPH-CYP reductase in rat and human liver microsomes.

    PubMed

    Yao, Hsien-Tsung; Chang, Yi-Wei; Lan, Shih-Jung; Yeh, Teng-Kuang

    2008-02-01

    Tannic acid has been shown to decrease mutagenicity and/or carcinogenicity of several amine derivatives and polycyclic aromatic hydrocarbons in rodents. The purpose of this study was to evaluate the effect of tannic acid on cytochrome P450 (CYP)-catalyzed oxidations using rat liver microsomes (RLM) and human liver microsomes (HLM) as the enzyme sources. In RLM, tannic acid showed a non-selective inhibitory effect on 7-methoxyresorufin O-demethylation (MROD), 7-ethoxyresorufin O-deethylation (EROD), tolbutamide hydroxylation, p-nitrophenol hydroxylation and testosterone 6beta-hydroxylation activities with IC(50) values ranged from 14.9 to 27.4 microM. In HLM, tannic acid inhibited EROD, MROD and phenacetin O-deethylation activities with IC(50) values ranged from 5.1 to 7.5 microM, and diclofenac 4-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and testosterone 6beta-hydroxylation with IC(50) values ranged from 20 to 77 microM. In baculovirus-insect cell-expressed human CYP 1A1 and 1A2, the IC(50) values of tannic acid for CYP 1A1- and 1A2-catalyzed EROD activities were 23.1 and 2.3 microM, respectively, indicating that tannic acid preferably inhibited the activity of CYP1A2. Tannic acid inhibited human CYP1A2 non-competitively with a Ki value of 4.8 microM. Tannic acid was also found to inhibit NADPH-CYP reductase in RLM and HLM with IC(50) values of 11.8 and 17.4 microM, respectively. These results suggested that the inhibition of CYP enzyme activities by tannic acid may be partially attributed to its inhibition of NADPH-CYP reductase activity.

  9. Polymorphism in cytochrome P450 2E1 and interaction with other genetic risk factors and susceptibility to alcoholic liver cirrhosis.

    PubMed

    Khan, Anwar Jamal; Ruwali, Munindra; Choudhuri, Gourdas; Mathur, Neeraj; Husain, Qayyum; Parmar, Devendra

    2009-05-12

    The association of polymorphism in cytochrome P450 2E1 (CYP2E1), the major microsomal ethanol metabolizing enzyme and its interaction with genes, involved in detoxification of reactive oxygen species, such as glutathione-S-transferases M1 (GSTM1) and alcohol intake, gamma-aminobutyric acid receptor gamma2 (GABRG2) was studied with the risk to alcoholic cirrhosis in a case-control study. A total of 160 alcoholic cirrhotic and 125 non-alcoholic cirrhotic cases, visiting the OPD facility of Gastroenterology Department of Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGI), Lucknow, India and 250 non-alcoholic and 100 alcoholic controls having no evidence of liver disease were included in the study. PCR-based RFLP methodology was followed for genotyping studies. Our data revealed that the variant genotypes of CYP2E1 5B exhibited significant association with the alcoholic liver cirrhosis when compared to non-alcoholic controls (OR: 4.3; 95%CI: 1.5-12.4; p: 0.003) or non-alcoholic cirrhosis patients (OR: 5.4; 95%CI: 1.2-24.5; p: 0.01) or alcoholic controls (OR: 4.3; 95%CI: 0.95-19.62; p: 0.04). Haplotype approach revealed that haplotype T-A-T was found to be associated with more than 5-fold increase in risk for alcoholic cirrhosis. Likewise, combination of variant genotype of CYP2E1 5B with null genotype of GSTM1, a phase II detoxification enzyme, resulted in several fold increase in risk in alcoholic cirrhotic patients when compared with non-alcoholic controls or non-alcoholic cirrhotic patients. Further, the combination of variant genotype of CYP2E1 5B with GABRG2, significantly increased the risk upto 6.5-fold in alcoholic cirrhotic patients when compared with non-alcoholic controls thereby suggesting the role of gene-gene interaction in alcoholic cirrhosis.

  10. Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide

    PubMed Central

    Stark, Katarina; Dostalek, Miroslav; Guengerich, F. Peter

    2016-01-01

    Summary Cytochrome P450 (P450) 4X1 is one of the so-called “orphan” P450s without assigned biological function. Codon-optimized P450 4X1 and a number of N-terminal modified sequences were expressed in Escherichia coli. Native P450 4X1 showed a characteristic P450 spectrum but low expression in E. coli DH5α cells (<100 nmol P450/L). The highest level of expression (300-450 nmol P450/L culture) was achieved with a bicistronic P450 4X1 construct (N-terminal MAKKTSSKGKL, change of E2A, amino acids 3-44 truncated). Anandamide (arachidonoyl ethanolamide) has emerged as an important signaling molecule in the neurovascular cascade. Recombinant P450 4X1 protein, co-expressed with human NADPH-P450 reductase in E. coli, was found to convert the natural endocannabinoid anandamide to a single monooxygenated product, 14,15-epoxyeicosatrienoic (EET) ethanolamide. A stable anandamide analog (CD-25) was also converted to a monooxygenated product. Arachidonic acid was oxidized more slowly to 14,15- and 8,9-EETs but only in the presence of cytochrome b5. Other fatty acids were investigated as putative substrates but showed only little or minor oxidation. Real-time PCR analysis demonstrated extrahepatic mRNA expression, including several human brain structures (cerebellum, amygdala, and basal ganglia), in addition to expression in human heart, liver, prostate, and breast. The highest mRNA expression levels were detected in amygdala and skin. The ability of P450 4X1 to generate anandamide derivatives and the mRNA distribution pattern suggest a potential role for P450 4X1 in anandamide signaling in the brain. PMID:18549450

  11. Spectroscopic quantitation of cytochrome P-450 in human lung microsomes.

    PubMed

    Wheeler, C W; Guenthner, T M

    1990-01-01

    The cytochrome P-450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide-complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P-450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.

  12. Forster Distances of Ligand-Heme Pairs in Cytochrome P450 3A4

    NASA Astrophysics Data System (ADS)

    Fern, Joel; Guengerich, F. Peter; Marsch, Glenn A.

    2003-04-01

    Cytochrome P450 3A4 is a protein in the human intestine and liver which oxidizes over half of drugs in use today. Cytochrome P450 3A4 has proven resistant to structure determination by NMR or x-ray crystallography. Fluorescence Resonance Energy Transfer (FRET) studies of P450 3A4 can be used to compute distances between fluorophores in the protein, providing information on the structure of the protein. For a ligand to be suitably used as a probe its fluorescence must not be completely quenched by the heme cofactor in P450 3A4. By using quantum yields, fluorescence, and the absorption spectra of six P450 ligands, the following Forster distances between each ligand and the P450 heme moiety were obtained: pyrene 4.6 nm, aflatoxin B2 5.7 nm, alpha-naphthoflavone 3.7 nm, indinavir 2.6 nm, quinidine 3.5 nm, and terfenadine 2.8 nm. Having these distances should yield a better low-resolution cytochrome P450 3A4 structure. Using the Forster distances, FRET experiments on inter-ligand placement in P450 3A4 will be undertaken soon.

  13. Conformational selectivity in cytochrome P450 redox partner interactions

    PubMed Central

    Hollingsworth, Scott A.; Batabyal, Dipanwita; Nguyen, Brian D.; Poulos, Thomas L.

    2016-01-01

    The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein–protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural changes in the P450s that are required for activity. Many P450s now have been shown to experience large open and closed motions. Several structural and spectral studies indicate that the well-studied P450cam adopts the open conformation when its redox partner, putidaredoxin (Pdx), binds, whereas recent NMR studies indicate that this view is incorrect. Given the relevance of this discrepancy to P450 chemistry, it is important to determine whether Pdx favors the open or closed form of P450cam. Here, we have used both computational and experimental isothermal titration calorimetry studies that unequivocally show Pdx favors binding to the open form of P450cam. Analyses of molecular-dynamic trajectories also provide insights into intermediate conformational states that could be relevant to catalysis. PMID:27439869

  14. Interactions among Cytochromes P450 in Microsomal Membranes

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Sineva, Elena V.; Halpert, James R.

    2015-01-01

    The body of evidence of physiologically relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5, and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer, we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states. We also observed the formation of mixed oligomers in CYP3A4/CYP3A5, CYP3A4/CYP2E1, and CYP3A5/CYP2E1 pairs and demonstrated that the association of either CYP3A4 or CYP3A5 with CYP2E1 causes activation of the latter enzyme. Earlier we hypothesized that the intersubunit interface in CYP3A4 oligomers is similar to that observed in the crystallographic dimers of some microsomal drug-metabolizing cytochromes P450 (Davydov, D. R., Davydova, N. Y., Sineva, E. V., Kufareva, I., and Halpert, J. R. (2013) Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone. Biochem. J. 453, 219–230). Here we report the results of intermolecular cross-linking of CYP3A4 oligomers with thiol-reactive bifunctional reagents as well as the luminescence resonance energy transfer measurements of interprobe distances in the oligomers of labeled CYP3A4 single-cysteine mutants. The results provide compelling support for the physiological relevance of the dimer-specific peripheral ligand-binding site observed in certain CYP3A4 structures. According to our interpretation, these results reveal an important general mechanism that regulates the activity and substrate specificity of the cytochrome P450 ensemble through interactions between multiple P450 species. As a result of P450-P450 cross-talk, the catalytic properties of the cytochrome P450 ensemble cannot be predicted by simple summation of the properties of the individual P450 species. PMID:25533469

  15. Cytochrome P450 system expression and DNA adduct formation in the liver of Zacco platypus following waterborne benzo(a)pyrene exposure: implications for biomarker determination.

    PubMed

    Lee, Jin Wuk; Kim, Yong Hwa; Yoon, Seokjoo; Lee, Sung Kyu

    2014-09-01

    Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that causes mutations and tumor formation. Zacco platypus is a sentinel species that is suitable for monitoring aquatic environments. We studied cytochrome P450 system (CYP system) expression and DNA adduct formation in the liver of Z. platypus following waterborne exposure to BaP. The results showed both dose and time dependency. The significant induction levels of CYP system mRNA and protein reached maximums at 2 days and 14 days, respectively, and hepatosomatic index was maximally induced at 4 days during 14 days BaP exposure. DNA adduct formation was significantly induced compared to corresponding controls (t-test, p < 0.01) after 4 days of exposure in 100 μg/L BaP. These results indicate that the only use of mRNA expression level of CYP system as a biomarker make us underestimate prolonged toxicity (4-14 days) of BaP and the only use of protein expression level of CYP system make us underestimate acute toxicity (1-2 days) of BaP. Therefore, we suggests that a combinational use of the mRNA expression level and protein expression level of CYP system, hepatosomatic index is a useful biomarker in risk assessment of waterborne BaP exposure. In addition, DNA adduct formation was a useful biomarker in risk assessment of waterborne BaP exposure at 4 days. CYP1A was a more sensitive biomarker than CYP reductase for BaP exposure when considering both the mRNA and protein level. Furthermore, our results show that Z. platypus is a useful species for assessing the risk of waterborne BaP exposure.

  16. Alcohol dehydrogenase and cytochrome P450 2E1 can be induced by long-term exposure to ethanol in cultured liver HEP-G2 cells.

    PubMed

    Balusikova, Kamila; Kovar, Jan

    2013-09-01

    It has been shown in previous studies that liver HEP-G2 cells (human hepatocellular carcinoma) lose their ability to express active alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Although both are ethanol-inducible enzymes, short-term exposure to ethanol does not cause any changes in expression or activity in cultured HEP-G2 cells. Therefore, we tested the effect of long-term exposure to ethanol on the expression and activity of both ADH and CYP2E1 in these cells. The expression of ADH and CYP2E1 was assessed at the mRNA and/or protein level using real-time PCR and Western blot analysis. Specific colorimetric assays were used for the measurement of ADH and CYP2E1 enzymatic activities. Caco-2 cells (active CYP2E1 and inactive ADH) were used as control cells. Significantly increased protein expression of ADH (about 2.5-fold) as well as CYP2E1 (about 1.6-fold) was found in HEP-G2 cells after long-term (12 mo) exposure to ethanol. The activity of ADH and CYP2E1 was also significantly increased from 12 ± 3 and 6 ± 1 nmol/h/mg of total protein to 191 ± 9 and 57 ± 9 nmol/h/mg of total protein, respectively. We suggest that the loss of activity of ethanol-metabolizing enzymes in cultured HEP-G2 cells is reversible and can be induced by prolonged exposure to ethanol. We are therefore able to reactivate HEP-G2 cells metabolic functions concerning ethanol oxidation just by modification of in vitro culture conditions without necessity of transfection with its side effect - enzyme overexpression.

  17. Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay.

    PubMed

    Liu, Xidong; Hu, Lianghai; Ge, Guangbo; Yang, Bo; Ning, Jing; Sun, Shixin; Yang, Ling; Pors, Klaus; Gu, Jingkai

    2014-08-01

    Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.

  18. Identification of cytochrome P450 enzymes involved in the metabolism of 3',4'-methylenedioxy-alpha-pyrrolidinopropiophenone (MDPPP), a designer drug, in human liver microsomes.

    PubMed

    Springer, D; Staack, R F; Paul, L D; Kraemer, T; Maurer, H H

    2005-03-01

    The metabolism of 3',4'-methylenedioxy-a-pyrrolidinopropiophenone (MDPPP), a novel designer drug, to its demethylenated major metabolite 3',4'-dihydroxy-pyrrolidinopropiophenone (di-HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes. CYP2C19 catalysed the demethylenation with apparent Km and Vmax values of 120.0+/-13.4 microM and 3.2+/-0.1 pmol/min/pmol CYP, respectively (mean+/-standard deviation). CYP2D6 catalysed the demethylenation with apparent Km and Vmax values of 13.5+/-1.5 microM and 1.3+/-0.1 pmol/min/pmol CYP, respectively. HLM exhibited a clear biphasic profile with an apparent Km,1 value of 7.6+/-9.0 and a Vmax,1 value of 11.1+/-3.6 pmol/min/mg protein, respectively. Percentages of intrinsic clearances of MDPPP by specific CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4'-hydroxylation or bufuralol-1'-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively. MDPPP, di-HO-PPP and the standard 4'-methyl-pyrrolidinohexanophenone (MPHP) were separated and analysed by liquid chromatography-mass spectrometry in the selected-ion monitoring (SIM) mode. The CYP2D6-specific chemical inhibitor quinidine (3 microM) significantly (p<0.001) inhibited di-HO-PPP formation by 75.8%+/-1.7% (mean+/-standard error of the mean) in incubation mixtures with HLM and 2 microM MDPPP. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 and CYP2C19 are almost equally responsible for MDPPP demethylenation.

  19. Structural features of cytochrome P450 1A associated with the absence of EROD activity in liver of the of the loricariid catfish Pterygoplichthys sp

    PubMed Central

    Parente, T.E.M.; Rebelo, M.F.; da-Silva, M.L.; Woodin, B.R.; Goldstone, J. V.; Bisch, P.M.; Paumgartten, F.J.R.; Stegeman, J.J.

    2011-01-01

    The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically-related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acids substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrates. PMID:21840383

  20. Effect of antipsychotic drugs on human liver cytochrome P-450 (CYP) isoforms in vitro: preferential inhibition of CYP2D6.

    PubMed

    Shin, J G; Soukhova, N; Flockhart, D A

    1999-09-01

    The ability of antipsychotic drugs to inhibit the catalytic activity of five cytochrome P-450 (CYP) isoforms was compared using in vitro human liver microsomal preparations to evaluate the relative potential of these drugs to inhibit drug metabolism. The apparent kinetic parameters for enzyme inhibition were determined by nonlinear regression analysis of the data. All antipsychotic drugs tested competitively inhibited dextromethorphan O-demethylation, a selective marker for CYP2D6, in a concentration-dependent manner. Thioridazine and perphenazine were the most potent, with IC(50) values (2.7 and 1.5 microM) that were comparable to that of quinidine (0.52 microM). The estimated K(i) values for CYP2D6-catalyzing dextrorphan formation were ranked in the following order: perphenazine (0.8 microM), thioridazine (1.4 microM), chlorpromazine (6.4 microM), haloperidol (7.2 microM), fluphenazine (9.4 microM), risperidone (21.9 microM), clozapine (39.0 microM), and cis-thiothixene (65.0 microM). No remarkable inhibition of other CYP isoforms was observed except for moderate inhibition of CYP1A2-catalyzed phenacetin O-deethylation by fluphenazine (K(i) = 40.2 microM) and perphenazine (K(i) = 65.1). The estimated K(i) values for the inhibition of CYP2C9, 2C19, and 3A were >300 microM in almost all antipsychotics tested. These results suggest that antipsychotic drugs exhibit a striking selectivity for CYP2D6 compared with other CYP isoforms. This may reflect a remarkable commonality of structure between the therapeutic targets for these drugs, the transporters, and metabolic enzymes that distribute and eliminate them. Clinically, coadministration of these medicines with drugs that are primarily metabolized by CYP2D6 may result in significant drug interactions.

  1. Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s

    PubMed Central

    Parvez, Mohammad; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Kgosiemang, Ipeleng Kopano Rosinah; Bamal, Hans Denis; Pagadala, Nataraj Sekhar; Xie, Ting; Yang, Haoran; Chen, Hengye; Theron, Chrispian William; Monyaki, Richie; Raselemane, Seiso Caiphus; Salewe, Vuyani; Mongale, Bogadi Lorato; Matowane, Retshedisitswe Godfrey; Abdalla, Sara Mohamed Hasaan; Booi, Wool Isaac; van Wyk, Mari; Olivier, Dedré; Boucher, Charlotte E.; Nelson, David R.; Tuszynski, Jack A.; Blackburn, Jonathan Michael; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Chen, Wanping; Syed, Khajamohiddin

    2016-01-01

    Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria −42; fungi −19; plant −28; animal −22; plant and animal −1 and common P450 family −1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study’s results offer new understanding of the dynamic structural nature of P450s. PMID:27616185

  2. Cytochrome P450 arachidonic acid metabolism in bovine corneal epithelium

    SciTech Connect

    Masferrer, J.; Schwartzman, M.L.; Abraham, N.G.; Dunn, M.W.; McGiff, J.C.

    1986-03-01

    The presence of the cytochrom P450 system and its involvement in the metabolism of AA was studied in the corneal epithelium. This tissue contains cytochrome P450 as assessed directly by measurement of the carbon monoxide reduced spectrum (specific activity of 161 pmol/10 mg protein) and indirectly by measuring the activity of aryl hydrocarbon hydroxylase (AHH) - a cytochrome P450-dependent enzyme (11-39 pmol 3-OH benzopyrene/mg protein/10 min). When corneal epithelial microsomes were incubated with /sup 14/C-arachidonic acid, 30-50% of the total radioactivity was converted to two peaks, I and II. Further separation using high performance liquid chromatography has shown that each peak contains two metabolites, A,B and C,D. Metabolite formation was dependent on the addition of NADPH (1 mM) and inhibited by carbon monoxide and SKF-525A (100 ..mu..M) suggesting a cytochrome P450-dependent mechanism. Compound C (5-10 ..mu..M) inhibited the activity of corneal epithelial Na-K-ATPase by 30-60%, being 100-fold more potent than ouabain. Compound D (10-100 ng) induced a dose dependent relaxation of the rat caudal artery. Compound D also inhibited corneal Na-K-ATPase activity but less potently than compound C. These compounds may be important to transport processes of ocular epithelia and participate in the control of the ocular circulation and aqueous humor dynamics.

  3. Immunohistochemical localization of cytochrome P450 aromatase in equine gonads.

    PubMed

    Almadhidi, J; Seralini, G E; Fresnel, J; Silberzahn, P; Gaillard, J L

    1995-06-01

    Estrogens are the major steroids produced by equine gonads. To identify the cells responsible for estrogen synthesis, an antiserum against purified equine testicular cytochrome P450 aromatase was produced in rabbits. The reactivity and specificity of the antiserum were assessed by ELISA, immunoblot analysis, and immunoneutralization studies. Immunofluorescence microscopy demonstrated that in the male gonad, cytochrome P450 aromatase (P450arom) was localized in the interstitial tissue, whereas, under the experimental conditions used, the Sertoli and germ cells did not show any specific staining. In the ovary, the granulosa cells of small follicles exhibited faint immunofluorescent staining for P450arom and the granulosa cells of large, viable more follicles showed a high degree of immunoreactivity. In the corpus luteum, all the luteinized cells showed immunoreactivity. No immunoreactivity was detected in other cells of small and large viable follicles. Immunolocalization of P450arom in the equine testicular Leydig cells and in ovarian granulosa and luteinized cells indicates that these cells have the ability to metabolize androgens to estrogens and possibly to catechol estrogens.

  4. Cytochromes P450 in the bioactivation of chemicals.

    PubMed

    Ioannides, Costas; Lewis, David F V

    2004-01-01

    The initial view that the cytochrome P450 enzyme system functions simply in the deactivation of xenobiotics is anachronistic on the face of mounting evidence that this system can also transform many innocuous chemicals to toxic products. However, not all xenobiotic-metabolising cytochrome P450 subfamilies show the same propensity in the bioactivation of chemicals. For example, the CYP2C, 2B and 2D subfamilies play virtually no role in the bioactivation of toxic and carcinogenic chemicals, whereas the CYP1A, 1B and 2E subfamilies are responsible for the bioactivation of the majority of xenobiotics. Electronic and molecular structural features of organic chemicals appear to predispose them to either bioactivation by one cytochrome P450 enzyme or deactivation by another. Consequently, the fate of a chemical in the body is largely dependent on the cytochrome P450 profile at the time of exposure. Any factor that modulates the enzymes involved in the metabolism of a certain chemical will also influence its toxicity and carcinogenicity. For example, many chemical carcinogens bioactivated by CYP1, on repeated administration, selectively induce this family, thus exacerbating their carcinogenicity. CYP1 induction potency by chemicals appears to be determined by a combination of their molecular shape and electron activation. The function of cytochromes P450 in the bioactivation of chemicals is currently being exploited to design systems that can be used clinically to facilitate the metabolic conversion of prodrugs to their biologically-active metabolites in cells that poorly express them, such as tumour cells, in the so-called gene-directed prodrug therapy.

  5. Metabolism of the major Echinacea alkylamide N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide by human recombinant cytochrome P450 enzymes and human liver microsomes.

    PubMed

    Toselli, F; Matthias, A; Bone, K M; Gillam, E M J; Lehmann, R P

    2010-08-01

    Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N-dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N-dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic-metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified.

  6. The rabbit pulmonary cytochrome P450 arachidonic acid metabolic pathway: characterization and significance.

    PubMed Central

    Zeldin, D C; Plitman, J D; Kobayashi, J; Miller, R F; Snapper, J R; Falck, J R; Szarek, J L; Philpot, R M; Capdevila, J H

    1995-01-01

    Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19- and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung. Images PMID:7738183

  7. Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver

    PubMed Central

    Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao

    2015-01-01

    Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. PMID:26400395

  8. Stereo-selective metabolism of methadone by human liver microsomes and cDNA-expressed cytochrome P450s: a reconciliation.

    PubMed

    Chang, Yan; Fang, Wenfang B; Lin, Shen-Nan; Moody, David E

    2011-01-01

    In vitro metabolism of methadone was investigated in cytochrome P450 (CYP) supersomes and phenotyped human liver microsomes (HLMs) to reconcile past findings on CYP involvement in stereo-selective metabolism of methadone. Racaemic methadone was used for incubations; (R)- and (S)-methadone turnover and (R)- and (S)-EDDP formation were determined using chiral liquid chromatography-tandem mass spectrometry. CYP supersome activity for methadone use and EDDP formation ranked CYP2B6 > 3A4 > 2C19 > 2D6 > 2C18, 3A7 > 2C8, 2C9, 3A5. After abundance scaling, CYP3A4, 2B6 and 2C19 accounted for 63-74, 12-32 and 1. 4-14% of respective activity. CYP2B6, 2D6 and 2C18 demonstrated a preference for (S)-EDDP formation; CYP2C19, 3A7 and 2C8 for (R)-EDDP; 3A4 none. Correlation analysis with 15 HLMs supported the involvement of CYP2B6 and 3A. The significant correlation of S/R ratio with CYP2B6 activity confirmed its stereo-selectivity. CYP2C19 and 2D6 inhibitors and monoclonal antibody (mAb) did not inhibit EDDP formation in HLM. Chemical and mAb inhibition of CYP3A in high 3A activity HLM reduced EDDP formation by 60-85%; inhibition of CYP2B6 in 2B6 high-activity HLM reduced (S)-EDDP formation by 80% and (R)-EDDP formation by 55%. Inhibition changed methadone metabolism in a stereo-selective manner. When CYP3A was inhibited, 2B6 mediated (S)-EDDP formation predominated; S/R stereo-selectivity increased. When 2B6 was inhibited (S)-EDDP formation fell and stereo-selectivity decreased. The results confirmed the primary roles of CYPs 3A4 and 2B6 in methadone metabolism; CYP2C8 and 2C9 did not appear involved; 2C19 and 2D6 have minimal roles. CYP2B6 is the primary determinant of stereo-selective metabolism; stereo-selective inhibition might play a role in varied plasma concentrations of the two enantiomers.

  9. Cytochrome P450IA1 induction and localization in endothelium of vertebrate (teleost) heart.

    PubMed

    Stegeman, J J; Miller, M R; Hinton, D E

    1989-11-01

    Previous studies have shown that high levels of cytochrome P450 can occur in cardiac microsomes of vertebrates [Mol. Pharmacol. 21:517-526, (1982)]. Here we identify the dominant cardiac P450 in the marine fish scup as P450E, a teleost representative of P450IA1, and we describe its restricted cellular localization in the heart. Treatment of scup with beta-naphthoflavone produced an unusually strong (10-fold) induction of spectrally measured P450 in cardiac microsomes, with specific content reaching levels (0.5 nmol/mg) similar to those induced in scup liver. Microsomal ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities, catalytic functions of scup P450E, were induced in parallel with P450 content. Similar induction was seen in both atrium and ventricle. Immunoblot analysis with monoclonal antibody 1-12-3, specific to scup P450E and other vertebrate P450IA1 proteins, showed that this hydrocarbon-inducible P450 is the dominant and possibly sole P450 form in heart microsomes of experimentally induced animals. Immunohistochemical analysis of scup heart sections (2-4-microns) with monoclonal antibody 1-12-3 revealed that P450E was detectable only in endothelial cells of the endocardium and of the coronary vasculature. A similar endothelial cell localization of the monoclonal antibody 1-12-3 epitope was observed in heart of rainbow trout, induced with beta-naphthoflavone, indicating a general nature for the endothelial localization of induced cardiac P450. Morphometric analysis showed that endothelium could constitute 8-9% of the volume of teleost heart, from which we calculate that P450IA1 could account for as much as 25% of the endothelial cell microsomal protein. Heart microsomes of untreated animals from contaminated environments also contained high levels of P450E, indicating that induction like that caused by beta-naphthoflavone could occur with chemicals in the environment. Strongly induced P450E (P450IA1) in endothelium could play a critical

  10. mRNA Distribution and Heterologous Expression of Orphan Cytochrome P450 20A1

    PubMed Central

    Stark, Katarina; Wu, Zhong-Liu; Bartleson, Cheryl J.; Guengerich, F. Peter

    2015-01-01

    Cytochrome P450 (P450) 20A1 is one of the so-called “orphan” P450s without assigned biological function. mRNA expression was detected in human liver and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3–4 fold higher in brain and heart than liver. In situ hybridization of rat whole brain sections showed a similar mRNA expression pattern as observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200–280 nmol P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function. PMID:18541694

  11. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  12. QUANTITATIVE EVALUATION OF BROMODICHLOROMETHANE METABOLISM BY RECOMBINANT RAT AND HUMAN CYTOCHROME P450S

    EPA Science Inventory

    ABSTRACT
    We report quantitative estimates of the parameters for metabolism of bromodichloromethane (BDCM) by recombinant preparations of hepatic cytochrome P450s (CYPs) from rat and human. BDCM is a drinking water disinfectant byproduct that has been implicated in liver, kidn...

  13. Key Elements of the Chemistry of Cytochrome P-450: The Oxygen Rebound Mechanism.

    ERIC Educational Resources Information Center

    Groves, John T.

    1985-01-01

    Discusses the structure and function of the liver protein cytochrome P-450, an important catalyst for a variety of detoxification reactions. Diagnostic substracts for this heme-containing monooxygenase, synthetic modes of the active site, and oxidations with synthetic metalloporphyrins are the major topic areas considered. (JN)

  14. Epoxidation Activities of Human Cytochromes P450c17 and P450c21

    PubMed Central

    2015-01-01

    Some cytochrome P450 enzymes epoxidize unsaturated substrates, but this activity has not been described for the steroid hydroxylases. Physiologic steroid substrates, however, lack carbon–carbon double bonds in the parts of the pregnane molecules where steroidogenic hydroxylations occur. Limited data on the reactivity of steroidogenic P450s toward olefinic substrates exist, and the study of occult activities toward alternative substrates is a fundamental aspect of the growing field of combinatorial biosynthesis. We reasoned that human P450c17 (steroid 17-hydroxylase/17,20-lyase, CYP17A1), which 17- and 16α-hydroxylates progesterone, might catalyze the formation of the 16α,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16α,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16α-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products. In contrast, human P450c21 (steroid 21-hydroxylase, CYP21A2) converted 16,17-dehydroprogesterone to the 21-hydroxylated product and only a trace of epoxide. CYP21A2 mutation V359A, which has significant 16α-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3β-ol-20-one) in 4:1 or 12:1 ratios, respectively. Catalase and superoxide dismutase did not prevent epoxide formation. The progesterone epoxide was not a time-dependent, irreversible CYP17A1 inhibitor. Our substrate modification studies have revealed occult epoxidase and 21-hydroxylase activities of CYP17A1, and the fraction of epoxide formed correlated with the 16α-hydroxylase activity of the enzymes. PMID:25386927

  15. Aromatic hydroxylation of salicylic acid and aspirin by human cytochromes P450.

    PubMed

    Bojić, Mirza; Sedgeman, Carl A; Nagy, Leslie D; Guengerich, F Peter

    2015-06-20

    Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids-salicyluric acid and gentisuric acid-and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.

  16. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

    PubMed

    Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2015-10-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation.

  17. Cytochrome P450-derived eicosanoids: the neglected pathway in cancer

    PubMed Central

    Kaipainen, Arja; Greene, Emily R.; Huang, Sui

    2010-01-01

    Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed. PMID:20941528

  18. Human hepatic cytochrome P450-specific metabolism of the organophosphorus pesticides methyl parathion and diazinon.

    PubMed

    Ellison, Corie A; Tian, Yuan; Knaak, James B; Kostyniak, Paul J; Olson, James R

    2012-01-01

    Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (K(m) and V(max)) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (K(m) = 1.25 μM; V(max) = 9.78 nmol · min(-1) · nmol P450(-1)), CYP2C19 (K(m) = 1.03 μM; V(max) = 4.67 nmol · min(-1) · nmol P450(-1)), and CYP1A2 (K(m) = 1.96 μM; V(max) = 5.14 nmol · min(-1) · nmol P450(-1)), and the bioactivation of diazinon was mediated primarily by CYP1A1 (K(m) = 3.05 μM; V(max) = 2.35 nmol · min(-1) · nmol P450(-1)), CYP2C19 (K(m) = 7.74 μM; V(max) = 4.14 nmol · min(-1) · nmol P450(-1)), and CYP2B6 (K(m) = 14.83 μM; V(max) = 5.44 nmol · min(-1) · nmol P450(-1)). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (K(m) = 16.8 μM; V(max) = 1.38 nmol · min(-1) · nmol P450(-1)) and 3A4 (K(m) = 104 μM; V(max) = 5.15 nmol · min(-1) · nmol P450(-1)), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (K(m) = 5.04 μM; V(max) = 5.58 nmol · min(-1) · nmol P450(-1)). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure.

  19. Human Hepatic Cytochrome P450-Specific Metabolism of the Organophosphorus Pesticides Methyl Parathion and Diazinon

    PubMed Central

    Tian, Yuan; Knaak, James B.; Kostyniak, Paul J.; Olson, James R.

    2012-01-01

    Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (Km and Vmax) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (Km = 1.25 μM; Vmax = 9.78 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 1.03 μM; Vmax = 4.67 nmol · min−1 · nmol P450−1), and CYP1A2 (Km = 1.96 μM; Vmax = 5.14 nmol · min−1 · nmol P450−1), and the bioactivation of diazinon was mediated primarily by CYP1A1 (Km = 3.05 μM; Vmax = 2.35 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 7.74 μM; Vmax = 4.14 nmol · min−1 · nmol P450−1), and CYP2B6 (Km = 14.83 μM; Vmax = 5.44 nmol · min−1 · nmol P450−1). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (Km = 16.8 μM; Vmax = 1.38 nmol · min−1 · nmol P450−1) and 3A4 (Km = 104 μM; Vmax = 5.15 nmol · min−1 · nmol P450−1), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (Km = 5.04 μM; Vmax = 5.58 nmol · min−1 · nmol P450−1). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure. PMID:21969518

  20. Induction of hepatic cytochrome P-450 activity in wild cotton rats (Sigmodon hispidus) by phenobarbital and 3-methylcholanthrene

    SciTech Connect

    Elangbam, C.S.; Qualls, C.W.,Jr.; Bauduy, M. )

    1989-05-01

    Wild cotton rats (Sigmodon hispidus) are ubiquitous throughout the Southeast quadrant of the United States, easy to capture, have a generation interval of less than one year and a limited range of movement (less than one hectare). This species may prove to be an excellent model for monitoring environmental contamination. Traditionally, cytochrome P-450 inducing agents are grouped into two classes. One, represented by phenobarbital, induces P-450b and P-450e; the other, represented by 3-methylcholanthrene, induces P-450c and P-450d isoenzymes. The types and amounts of cytochrome P-450 vary among species, organs, health status, sex, and stress of the animal. If the levels of cytochrome P-450 of wild cotton rats are to be used in monitoring environmental pollution, it is necessary to characterize the inducibility and concentration of cytochrome P-450 in this species. This study was designed to determine the concentration and inducibility of cytochrome P-450 in the livers of cotton rats after intraperitoneal (ip) administration of phenobarbital and 3-methylcholanthrene.

  1. Unsaturated fatty acid regulation of cytochrome P450 expression via a CAR-dependent pathway

    PubMed Central

    Finn, Robert D.; Henderson, Colin J.; Scott, Claire L.; Wolf, C. Roland

    2008-01-01

    The liver is responsible for key metabolic functions, including control of normal homoeostasis in response to diet and xenobiotic metabolism/detoxification. We have shown previously that inactivation of the hepatic cytochrome P450 system through conditional deletion of POR (P450 oxidoreductase) induces hepatic steatosis, liver growth and P450 expression. We have exploited a new conditional model of POR deletion to investigate the mechanism underlying these changes. We demonstrate that P450 induction, liver growth and hepatic triacylglycerol (triglyceride) homoeostasis are intimately linked and provide evidence that the observed phenotypes result from hepatic accumulation of unsaturated fatty acids, which mediate these phenotypes by activation of the nuclear receptor CAR (constitutive androstane receptor) and, to a lesser degree, PXR (pregnane X receptor). To our knowledge this is the first direct evidence that P450s play a major role in controlling unsaturated fatty acid homoeostasis via CAR. The regulation of P450s involved in xenobiotic metabolism by this mechanism has potentially significant implications for individual responses to drugs and environmental chemicals. PMID:18778245

  2. Polar bear hepatic cytochrome P450: Immunochemical quantitation, EROD/PROD activity and organochlorines

    SciTech Connect

    Letcher, R.J.; Norstrom, R.J. |

    1994-12-31

    Polar bears (Ursus maritimus) are an ubiquitous mammal atop the arctic marine food chain and bioaccumulate lipophilic environmental contaminants. Antibodies prepared against purified rat liver cytochrome P450-1 Al, -1 A2, -2Bl and -3Al enzymes have been found to cross-react with structurally-related orthologues present in the hepatic microsomes of wild polar bears, immunochemically determined levels of P450-1 A and -2B proteins in polar bear liver relative to liver of untreated rats suggested enzyme induction, probably as a result of exposure to xenobiotic contaminants. Optical density quantitation of the most immunochemically responsive isozymes P450-I Al, -IA2 and -2Bi to polygonal rabbit anti-rat P450-IA/IA2 sera and -2BI antibodies in hepatic microsomes of 13 adult male polar bars from the Resolute Bay area of the Canadian Arctic is presented. Correlations with EROD and PROD catalytic activities and levels of organochlorines, such as polychlorinated biphenyls (PCBs), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene (p,p-DDE) and their methyl sulfone (MeSO2-) metabolites are made to determine if compound-specific enzyme induction linkages exist. Inter-species immunochemical quantitation of isozymic P450 cytochromes can serve as an indicator of exposure to biologically active contaminant.

  3. Differential modulation of cytochrome P-450 1A and P-glycoprotein expression by aryl hydrocarbon receptor agonists and thyroid hormone in Xenopus laevis liver and intestine.

    PubMed

    Colombo, Anita; Bonfanti, Patrizia; Orsi, Federica; Camatini, Marina

    2003-04-10

    Several defence mechanisms, such as cytochrome P450 1A (CYP1A) enzymes and P-glycoprotein (Pgp), may influence the intracellular concentration and consequently the toxicity of xenobiotics. The parallel expression of CYP1A and Pgp has been investigated in mammals and, to a lesser extent in fish, in search for evidence of co-ordinated responses to xenobiotic exposure. The aryl hydrocarbon receptor (AHR) agonists are well known CYP1A inducers but some of them resulted not to have a uniquely defined action on Pgp levels in mammalian and fish species. To the best of our knowledge, no detailed studies have been carried out so far on amphibians Xenopus laevis. For this reason, in this work, the time dependent responses of the hepatic CYP1A and Pgp, to the prototypical CYP1A inducers, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (3MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in X. laevis have been assessed at the protein level and compared. The responsiveness of Xenopus intestinal Pgp to these compounds has also been analysed, as the epithelial cells lining the lumen of intestine represent another preferential site of Pgp expression. In addition, since the thyroid hormone has been demonstrated to down regulate the mdr gene during Xenopus development and in primary culture of Xenopus intestinal epithelial cells, the effects of 3,3',5-triiodo-L-thyronine (T(3)) on CYP1A and Pgp protein levels have been investigated in adult organisms. Western blot evidenced that a single injection of B(a)P (100 mg/kg), 3MC (20 mg/kg), and TCDD (3 microg/kg) elicited a statistically significant induction of hepatic CYP1A at all time points considered (72, 120 and 168 h) which decreased in time. The same trend of liver CYP1A induction was observed in T(3) treated Xenopus (15 microg/kg). Unlike CYP1A induction, the modulation of hepatic and intestinal Pgp expression exhibits an heterogeneous pattern. The basal levels of hepatic and intestinal Pgp were not statistically significant

  4. Cytochrome p450 mRNA expression in the rodent brain: species-, sex-, and region-dependent differences.

    PubMed

    Stamou, Marianna; Wu, Xianai; Kania-Korwel, Izabela; Lehmler, Hans-Joachim; Lein, Pamela J

    2014-02-01

    Cytochrome P450 (P450) enzymes play a critical role in the activation and detoxication of many neurotoxic chemicals. Although research has largely focused on P450-mediated metabolism in the liver, emerging evidence suggests that brain P450s influence neurotoxicity by modulating local metabolite levels. As a first step toward better understanding the relative role of brain P450s in determining neurotoxic outcome, we characterized mRNA expression of specific P450 isoforms in the rodent brain. Adult mice (male and female) and rats (male) were treated with vehicle, phenobarbital, or dexamethasone. Transcripts for CYP2B, CYP3A, CYP1A2, and the orphan CYP4X1 and CYP2S1 were quantified in the liver, hippocampus, cortex, and cerebellum by quantitative (real-time) polymerase chain reaction. These P450s were all detected in the liver with the exception of CYP4X1, which was detected in rat but not mouse liver. P450 expression profiles in the brain varied regionally. With the exception of the hippocampus, there were no sex differences in regional brain P450 expression profiles in mice; however, there were marked species differences. In the liver, phenobarbital induced CYP2B expression in both species. Dexamethasone induced hepatic CYP2B and CYP3A in mice but not rats. In contrast, brain P450s did not respond to these classic hepatic P450 inducers. Our findings demonstrate that P450 mRNA expression in the brain varies by region, regional brain P450 profiles vary between species, and their induction varies from that of hepatic P450s. These novel data will be useful for designing mechanistic studies to examine the relative role of P450-mediated brain metabolism in neurotoxicity.

  5. 2-Diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) revisited: comparative cytochrome P450 inhibition in human liver microsomes by SKF525A, its metabolites, and SKF-acid and SKF-alcohol.

    PubMed

    Franklin, Michael R; Hathaway, Laura B

    2008-12-01

    When incubated with human liver microsomes, 2-diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) undergoes cytochrome P450 (P450)-dependent oxidative N-deethylation to the secondary amine metabolite 2-ethylaminoethyl-2,2-diphenylvalerate (SKF8742). P450-selective inhibitors indicated CYP3As catalyzed this reaction, and the deethylation rate correlated best with the CYP3A activity across a range of human liver microsomes. SKF525A and its metabolite and primary amine analog all inhibited CYP2B6-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A-selective reactions to varying degrees but had little effect on CYP1A2, CYP2A6, and CYP2E1 reactions. Only the inhibition of CYP3A showed major enhancement when the inhibitors were preincubated with NADPH-fortified microsomes, and the extent of metabolic intermediate (MI) complex formation approximated typical CYP3A content. Two "lost with time" SKF525A derivatives devoid of the ethylamine moiety, 2,2-diphenylpropylethanol (SKF-Alcohol) and 2,2-diphenylpropylacetic acid (SKF-Acid) did not form an MI complex and were identified as selective inhibitors of CYP2C9. Although without detectable metabolism, their CYP2C9 inhibition fitted best with a competitive mechanism. Thus, not all the human P450s are inhibited by SKF525A and related compounds, and the mechanisms contributing to those that are inhibited vary with the isoform. P450 MI-complex formation only seems to play a role with CYP3As.

  6. Alteration in the Expression of Cytochrome P450s (CYP1A1, CYP2E1, and CYP3A11) in the Liver of Mouse Induced by Microcystin-LR

    PubMed Central

    Zhang, Bangjun; Liu, Yang; Li, Xiaoyu

    2015-01-01

    Microcystins (MCs) are cyclic heptapeptide toxins and can accumulate in the liver. Cytochrome P450s (CYPs) play an important role in the biotransformation of endogenous substances and xenobiotics in animals. It is unclear if the CYPs are affected by MCs exposure. The objective of this study was to evaluate the effects of microcystin-LR (MCLR) on cytochrome P450 isozymes (CYP1A1, CYP2E1, and CYP3A11) at mRNA level, protein content, and enzyme activity in the liver of mice the received daily, intraperitoneally, 2, 4, and 8 µg/kg body weight of MCLR for seven days. The result showed that MCLR significantly decreased ethoxyresorufin-O-deethylase (EROD) (CYP1A1) and erythromycin N-demthylase (ERND) (CYP3A11) activities and increased aniline hydroxylase (ANH) activity (CYP2E1) in the liver of mice during the period of exposure. Our findings suggest that MCLR exposure may disrupt the function of CYPs in liver, which may be partly attributed to the toxicity of MCLR in mice. PMID:25831226

  7. Regulation of cytochrome P450 (CYP) genes by nuclear receptors.

    PubMed Central

    Honkakoski, P; Negishi, M

    2000-01-01

    Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks. PMID:10749660

  8. Cumene hydroperoxide effected hydroperoxidation by cytochrome P-450.

    PubMed

    Chen, C; Gurka, D P

    1985-04-01

    9-Methylfluorene was found to be oxygenated to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene by cytochrome P-450 in the presence of cumene hydroperoxide. Molecular oxygen is required and carbon monoxide is inhibitory. The reaction is inhibited by SKF-525A and metyrapone. Metyrapone and cumene hydroperoxide also retard the conversion of 9-hydroperoxy-9-methylfluorene to 9-hydroxy-9-methylfluorene. The reaction is different from hydroperoxide-supported oxygenation, since the cumene hydroperoxide appears to act as an effector of the enzyme rather than oxygen donor. It is suggested that substrates with stable radicals can be dioxygenated in this manner.

  9. Cytochromes P450 for terpene functionalisation and metabolic engineering.

    PubMed

    Pateraki, Irini; Heskes, Allison Maree; Hamberger, Björn

    2015-01-01

    Plants have evolved the capacity to produce a striking array of specialised metabolites. Terpenoids are the oldest and most diverse class of such compounds and have attracted interest for industrial and pharmaceutical applications. The development of biotechnological alternatives for their production is the focus of intense research. Photosynthetic systems provide new strategies for autotrophic metabolic engineering. Focusing on cytochromes P450, involved in the functionalisation of the core terpene molecules, this review highlights the latest approaches in this field and looks towards recent discoveries that have the potential to shape the future of terpenoid bioengineering.

  10. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    SciTech Connect

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and {beta}-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from {beta}-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  11. Immunochemical evidence for multiple steroid-inducible hepatic cytochromes P-450 in the rat.

    PubMed Central

    Hostetler, K A; Wrighton, S A; Kremers, P; Guzelian, P S

    1987-01-01

    It has been established that there are glucocorticoid-inducible hepatic cytochromes P-450 in the rat (P-450p), the rabbit (LM3c) and man (HLp) which share extensive structural, functional and regulatory features. We prepared immunochemical probes to P-450p and identified a unique monoclonal antibody, 1G8, that recognizes purified P-450p, but neither purified LM3c nor HLp, on immunoblot analysis. The N-terminal amino acid sequence of purified samples of P-450p was identical with that previously reported for P-450PCN1 [Gonzalez, Nebert, Hardwick & Kasper (1985) J. Biol. Chem. 260, 7435-7441]. Immunoblot analyses of liver microsomes from untreated male rats revealed two 1G8-reactive proteins, whereas liver microsomes from untreated females contained none. Another monoclonal antibody, 13-7-10, reacted specifically with LM3c and HLp, but not with P-450p. A single 13-7-10-reactive microsomal protein was detected in untreated male and female rats, the latter protein exhibiting a greater apparent Mr. 1G8-reactive proteins were induced to the greatest extent by triacetyloleandomycin, followed by dexamethasone, chlordane, pregnenolone-16 alpha-carbonitrile and 2,4,2',4'-tetrachlorobiphenyl. In contrast, 13-7-10-reactive proteins were most strongly induced by dexamethasone, only moderately by triacetyloleandomycin and pregnenolone-16 alpha-carbonitrile, weakly by chlordane and not at all by 2,4,2',4'-tetrachlorobiphenyl. We conclude that the P-450p family in rat liver consists of three or more proteins that are structurally related and yet appear to be under distinct regulatory control. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3311032

  12. Human cytochromes P450 in health and disease

    PubMed Central

    Nebert, Daniel W.; Wikvall, Kjell; Miller, Walter L.

    2013-01-01

    There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases—confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development. PMID:23297354

  13. Regulation of cytochrome P450 expression in Drosophila: Genomic insights

    PubMed Central

    Giraudo, Maeva; Unnithan, G. Chandran; Le Goff, Gaëlle; Feyereisen, René

    2009-01-01

    Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers / induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action. PMID:20582327

  14. Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11

    PubMed Central

    2015-01-01

    Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2–2) for 12-hydroxylation with 12-2H-substituted lauric acid. However, considerable “metabolic switching” to 11-hydroxylation was observed with [12-2H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc.108, 7074–7078] and the use of tritium KIE analysis with [12-3H]lauric acid [Northrop, D. B. (1987) Methods Enzymol.87, 607–625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C–H bond-breaking limit the rate of P450 4A11 ω-oxidation. PMID:25203493

  15. Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.

    PubMed

    Kim, Donghak; Cha, Gun-Su; Nagy, Leslie D; Yun, Chul-Ho; Guengerich, F Peter

    2014-10-07

    Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ω-oxidation.

  16. Effect of dietary eugenol on xenobiotic metabolism and mediation of UDP-glucuronosyltransferase and cytochrome P450 1A1 expression in rat liver.

    PubMed

    Iwano, Hidetomo; Ujita, Wakako; Nishikawa, Miyu; Ishii, Satomi; Inoue, Hiroki; Yokota, Hiroshi

    2014-03-01

    Xenobiotic-metabolizing enzymes (XMEs) play an important role in the elimination and detoxification of xenobiotics and drugs. A variety of natural dietary agents are known to protect against cancer by inducing XME. To elucidate the molecular mechanism of XME induction, we examined the effect of dietary eugenol (4-allyl-1-hydroxy-2-methoxybenzene) on xenobiotic metabolism. In this study, rats were administered dietary eugenol for 4 weeks to investigate the various effects of UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) expression. In rats administered dietary eugenol, expression levels of hepatic CYP1A 1 were reduced to 40% than of the controls, while expression of hepatic UGT1A6, UGT1A7 and UGT2B1 increased to 2-3 times than observed in the controls. Hepatic protein levels of UGT1A6 and 2B1 were also elevated in the eugenol-treated rats. These results suggest that the natural compound eugenol improves the xenobiotic-metabolizing systems that suppress and induce the expression of CYP1A1 and UGT, respectively.

  17. Effects of contaminated sediment from Cork Harbour, Ireland on the cytochrome P450 system of turbot.

    PubMed

    Kilemade, M; Hartl, M G J; O'Halloran, J; O'Brien, N M; Sheehan, D; Mothersill, C; van Pelt, F N A M

    2009-03-01

    Hatchery-reared juvenile turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to inter-tidal sediments collected from polluted sites in Cork Harbour (Whitegate and Agahda) and a reference site at Ballymacoda Co., Cork, Ireland. The potential of the sediment exposure to induce cytochrome P450 activities and CYP1A1 in the fish was assessed. Chemical analysis revealed that the sediments originating from the reference and harbour sites were contaminated principally with PAHs-the harbour sites having double the levels of those at the reference site. Following 3 weeks exposure to the sediments western blotting demonstrated a strong immunogenic response for CYP1A1 in the liver, but not for gill or intestine. P450 activities were generally significantly higher than those exposed to reference site sediment. Liver was the most responsive tissue with significantly greater P450 activities compared with gill and intestinal tissues.

  18. Purification and immunochemical detections of ?-naphthoflavone- and phenobarbital-induced avian cytochrome P450 enzymes

    USGS Publications Warehouse

    Brown, R.L.; Levi, P.E.; Hodgson, E.; Melancon, M.J.

    1996-01-01

    Livers from mallards (Anas platyrhynchos) were treated with either -naphthoflavone (50 mg/kg) or phenobarbital (70 mg/kg). Purification of induced hepatic cytochrome P450 was accomplished using both DEAE and hydroxyapatite columns, as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis separation. Polyclonal antibodies to these proteins were then produced in young male New Zealand White rabbits. ?-naphthoflavone (?NF)- and phenobarbital(PB)-treated red-winged blackbird, screech owl, European starling and lesser scaup liver microsomes were analyzed in western blots for species cross-reactivity. Although all four of these avian species exhibited cross-reactivity with antibodies to ?NF-induced mallard P450, all but the lesser scaup revealed a protein of higher molecular weight than that of the ?NF-induced mallard. In addition, only the lesser scaup exhibited cross-reactivity with the anti-PB-induced mallard P450 antibodies.

  19. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

    PubMed Central

    Barnes, H J; Arlotto, M P; Waterman, M R

    1991-01-01

    When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. Images PMID:1829523

  20. Cytochrome P450 structure, function and clinical significance: A review.

    PubMed

    Palrasu, Manikandan; Nagini, Siddavaram

    2017-01-25

    The cytochrome P450 (CYP) enzymes are membrane-bound hemoproteins that play a pivotal role in the detoxification of xenobiotics, cellular metabolism and homeostasis. Induction or inhibition of CYP enzymes is a major mechanism that underlies drug-drug interactions. CYP enzymes can be transcriptionally activated by various xenobiotics and endogenous substrates through receptor-dependent mechanisms. CYP enzyme inhibition is a principal mechanism for metabolism-based drug-drug interactions. Many chemotherapeutic drugs can cause drug interactions due to their ability to either inhibit or induce the CYP enzyme system. Predictions based on in silico analyses followed by validation have identified several microRNAs that regulate CYPs. Genetic polymorphisms and epigenetic changes in CYP genes may be responsible for inter-individual and inter-ethnic variations in disease susceptibility and the therapeutic efficacy of drugs. Knowledge about the substrates, inducers, inhibitors of CYP isoforms, and the polymorphisms of CYP enzymes may be used as an aid by clinicians to determine therapeutic strategy, and treatment doses for drugs that are metabolized by CYP gene products. The present review is a comprehensive compilation of cytochrome P450 structure, function, pharmacogenetics, and pharmacoepigenetics and clinical significance.

  1. Identification of the main human cytochrome P450 enzymes involved in safrole 1'-hydroxylation.

    PubMed

    Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw; Chi, Chin-Wen; Ho, Li-Kang

    2004-08-01

    Safrole is a natural plant constituent, found in sassafras oil and certain other essential oils. The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, followed by sulfonation to an unstable sulfate that reacts to form DNA adducts. To identify the main cytochrome P450 (P450) involved in human hepatic safrole 1'-hydroxylation (SOH), we determined the SOH activities of human liver microsomes and Escherichia coli membranes expressing bicistronic human P450s. Human liver (n = 18) microsomal SOH activities were in the range of 3.5-16.9 nmol/min/mg protein with a mean value of 8.7 +/- 0.7 nmol/min/mg protein. In human liver (n = 3) microsomes, the mean K(m) and V(max) values of SOH were 5.7 +/- 1.2 mM and 0.14 +/- 0.03 micromol/min/nmol P450, respectively. The mean intrinsic clearance (V(max)/K(m)) was 25.3 +/- 2.3 microL/min/nmol P450. SOH was sensitive to the inhibition by a CYP2C9 inhibitor, sulfaphenazole, and CYP2E1 inhibitors, 4-methylpyrazole and diethyldithiocarbamate. The liver microsomal SOH activity showed significant correlations with tolbutamide hydroxylation (r = 0.569) and chlorzoxazone hydroxylation (r = 0.770) activities, which were the model reactions catalyzed by CYP2C9 and CYP2E1, respectively. Human CYP2C9 and CYP2E1 showed SOH activities at least 2-fold higher than the other P450s. CYP2E1 showed an intrinsic clearance 3-fold greater than CYP2C9. These results demonstrated that CYP2C9 and CYP2E1 were the main P450s involved in human hepatic SOH.

  2. Cytochrome P450 enzyme mediated herbal drug interactions (Part 1)

    PubMed Central

    Wanwimolruk, Sompon; Prachayasittikul, Virapong

    2014-01-01

    It is well recognized that herbal supplements or herbal medicines are now commonly used. As many patients taking prescription medications are concomitantly using herbal supplements, there is considerable risk for adverse herbal drug interactions. Such interactions can enhance the risk for an individual patient, especially with regard to drugs with a narrow therapeutic index such as warfarin, cyclosporine A and digoxin. Herbal drug interactions can alter pharmacokinetic or/and pharmacodynamic properties of administered drugs. The most common pharmacokinetic interactions usually involve either the inhibition or induction of the metabolism of drugs catalyzed by the important enzymes, cytochrome P450 (CYP). The aim of the present article is to provide an updated review of clinically relevant metabolic CYP-mediated drug interactions between selected herbal supplements and prescription drugs. The commonly used herbal supplements selected include Echinacea, Ginkgo biloba, garlic, St. John's wort, goldenseal, and milk thistle. To date, several significant herbal drug interactions have their origins in the alteration of CYP enzyme activity by various phytochemicals. Numerous herbal drug interactions have been reported. Although the significance of many interactions is uncertain but several interactions, especially those with St. John’s wort, may have critical clinical consequences. St. John’s wort is a source of hyperforin, an active ingredient that has a strong affinity for the pregnane xenobiotic receptor (PXR). As a PXR ligand, hyperforin promotes expression of CYP3A4 enzymes in the small intestine and liver. This in turn causes induction of CYP3A4 and can reduce the oral bioavailability of many drugs making them less effective. The available evidence indicates that, at commonly recommended doses, other selected herbs including Echinacea, Ginkgo biloba, garlic, goldenseal and milk thistle do not act as potent or moderate inhibitors or inducers of CYP enzymes. A good

  3. Cytochrome P450 Expression Profile of the PICM-19H Pig Liver Cell Line: Potential Application to Rapid Liver Toxicity Assays.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In vitro models of the liver are needed to replace animal models for the rapid assessment of drug biotransformation and toxicity. One hepatocellular model, the PICM-19 pig liver stem cell line, may fulfill this need since these cells have many activities associated with xenobiotic phase I and phas...

  4. Cyclosporin A-induced free radical generation is not mediated by cytochrome P-450

    PubMed Central

    Krauskopf, Alexandra; Buetler, Timo M; Nguyen, Nathalie S D; Macé, Katherine; Ruegg, Urs T

    2002-01-01

    Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA). The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS. We show that CsA (1 – 10 μM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7-dichlorofluorescin diacetate. Using cytochrome c as substrate, we show that CsA (10 μM) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. CsA (10 μM) did not uncouple the electron flow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. CsA (10 μM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes. PMID:11861326

  5. Selective Targeting of Heme Protein in Cytochrome P450 and Nitric Oxide Synthase by Diphenyleneiodonium.

    PubMed

    Szilagyi, John T; Mishin, Vladimir; Heck, Diane E; Jan, Yi-Hua; Aleksunes, Lauren M; Richardson, Jason R; Heindel, Ned D; Laskin, Debra L; Laskin, Jeffrey D

    2016-05-01

    Cytochrome P450 (CYP) enzymes mediate mixed-function oxidation reactions important in drug metabolism. The aromatic heterocyclic cation, diphenyleneiodonium (DPI), binds flavin in cytochrome P450 reductase and inhibits CYP-mediated activity. DPI also inhibits CYP by directly interacting with heme. Herein, we report that DPI effectively inhibits a number of CYP-related monooxygenase reactions including NADPH oxidase, a microsomal enzyme activity that generates hydrogen peroxide in the absence of metabolizing substrates. Inhibition of monooxygenase by DPI was time and concentration dependent with IC50's ranging from 0.06 to 1.9 μM. Higher (4.6-23.9 μM), but not lower (0.06-1.9 μM), concentrations of DPI inhibited electron flow via cytochrome P450 reductase, as measured by its ability to reduce cytochrome c and mediate quinone redox cycling. Similar results were observed with inducible nitric oxide synthase (iNOS), an enzyme containing a C-terminal reductase domain homologous to cytochrome P450 reductase that mediates reduction of cytochrome c, and an N-terminal heme-thiolate oxygenase domain mediating nitric oxide production. Significantly greater concentrations of DPI were required to inhibit cytochrome c reduction by iNOS (IC50 = 3.5 µM) than nitric oxide production (IC50 = 0.16 µM). Difference spectra of liver microsomes, recombinant CYPs, and iNOS demonstrated that DPI altered heme-carbon monoxide interactions. In the presence of NADPH, DPI treatment of microsomes and iNOS yielded a type II spectral shift. These data indicate that DPI interacts with both flavin and heme in CYPs and iNOS. Increased sensitivity for inhibition of CYP-mediated metabolism and nitric oxide production by iNOS indicates that DPI targets heme moieties within the enzymes.

  6. Low levels of hepatic cytochrome P-450 in Mexicans with uncomplicated cholelithiasis.

    PubMed

    Martínez-Zedillo, G; Amézcua, J; González Barranco, D

    1994-01-01

    The multienzymatic complex known as cytochrome P-450 represents one of the most important hemoprotein families of the liver. It participates in the metabolism of steroids, fatty acids, prostaglandins and liposoluble vitamins, and also plays a role in the bioactivation of xenobiotic compounds (generates reactive metabolites which produce acute and chronic lesions in liver tissue). This is a report on the low concentrations of total microsomal P-450 (0.093 +/- SD = 0.069 nMoles/mg protein) found in liver biopsies of 19 mexican patients diagnosed as having biliary lithiasis, as compared to an anglosaxon population with the same liver pathology (0.415 +/- 0.105 nMoles/mg protein). These low values are in agreement with the observation of a high incidence in normal mexicans (91.7%) of poor nifedipine metabolizers. Our findings justify an analyses of hepatic RNAm and cDNA of mexican individuals according to ethnic background, diet and environmental contaminants.

  7. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    PubMed Central

    Elenewski, Justin E.; Hackett, John C

    2015-01-01

    The iron(IV)-oxo porphyrin π-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis. PMID:25681906

  8. Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450*

    PubMed Central

    Frank, Daniel J.; Madrona, Yarrow; Ortiz de Montellano, Paul R.

    2014-01-01

    Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58–67 and 100–109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth. PMID:25210044

  9. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    SciTech Connect

    Elenewski, Justin E.; Hackett, John C

    2015-02-14

    The iron(IV)-oxo porphyrin π-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis.

  10. Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita.

    PubMed Central

    Ropp, J D; Gunsalus, I C; Sligar, S G

    1993-01-01

    Cytochrome P-450lin catalyzes the 8-methyl hydroxylation of linalool as the first committed step of its utilization by Pseudomonas incognita as the sole carbon source. By using a polymerase chain reaction-based cloning strategy, a 2.1-kb DNA fragment containing the cytochrome P-450lin gene (linC) was isolated. An open reading frame of 406 amino acids has been identified as that of P-450lin on the basis of amino acid sequence data from peptides of the native protein. Heterologous expression of functional holoprotein is exhibited by Escherichia coli transformed with pUC18 containing the subcloned linC gene under constitutive transcriptional control of the lac promoter. The G+C content of linC was found to be 55% overall and 58% in the third codon position. An optimized amino acid sequence alignment of P-450lin with cytochrome P-450cam shows that the two enzymes have only 25% identity. P-450lin was found to exhibit the expected conservation in the axial cysteine heme ligand-containing peptide and the threonine region postulated to form an O2-binding pocket (T. L. Poulos, B. C. Finzel, and A. J. Howard, J. Mol. Biol. 195:687-700, 1987). The low amino acid sequence identity between P-450lin and all other P-450 sequences has shown that P-450lin is the first member of the CYP111 P-450 gene family. PMID:8376348

  11. Cytochrome P450 Enzyme Metabolites in Lead Discovery and Development

    PubMed Central

    Kandel, Sylvie E.; Wienkers, Larry C.; Lampe, Jed N.

    2015-01-01

    The cytochrome P450 (CYP) enzymes are a versatile superfamily of heme-containing monooxygenases, perhaps best known for their role in the oxidation of xenobiotic compounds. However, due to their unique oxidative chemistry, CYPs are also important in natural product drug discovery and in the generation of active metabolites with unique therapeutic properties. New tools for the analysis and production of CYP metabolites, including microscale analytical technologies and combinatorial biosynthesis, are providing medicinal chemists with the opportunity to use CYPs as a novel platform for lead discovery and development. In this review, we will highlight some of the recent examples of drug leads identified from CYP metabolites and the exciting possibilities of using CYPs as catalysts for future drug discovery. PMID:25797999

  12. Enzyme kinetics of oxidative metabolism: cytochromes P450.

    PubMed

    Korzekwa, Ken

    2014-01-01

    The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 3 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapter 4 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

  13. Personalized Cancer Therapy Considering Cytochrome P450 Variability.

    PubMed

    Preissner, Saskia; Simmaco, Maurizio; Gentile, Giovanna; Preissner, Robert

    2015-01-01

    The individual variability of pharmacokinetics is underestimated and few systematic studies exist in this field. In most cases, this leads to unwanted side effects or toxicity. In polychemotherapy, prodrugs (like ifosfamide), which have to be activated by cytochrome P450 enzymes (CYPs), play an important role. If patients are poor metabolizers for these drugs, the therapy will be ineffective. Furthermore, CYPs and transporters can be (over)expressed in target tissues, which is also not examined and considered in clinical routine. Here, we present a body map showing relevant enzymes in some organs and tissues. Finally, a typical case of a Caucasian chemotherapy patient with breast cancer is presented and discussed regarding a personalized cancer therapy considering the single nucleotide polymorphisms found via genotyping.

  14. Pulmonary oxygen toxicity in rats treated with cytochrome P-450 inducers

    SciTech Connect

    Ebel, R.E.; Barlow, R.L.; Gregory, E.M.

    1987-05-01

    Pulmonary oxygen toxicity is assumed to result from damage caused by superoxide (O/sub 2//sup -/) hydrogen peroxide (H/sub 2/O/sub 2/) and/or hydroxyl radical (OH) produced by the partial reduction of molecular oxygen (O/sub 2/). The microsomal cytochrome P-450 (P-450) monooxygenase system is known to produce O/sub 2//sup -/ and H/sub 2/O/sub 2/. They have studied the influence of monooxygenase induction using phenobarbital (PB) and ..beta..-naphthoflavone (..beta..-NF) on O/sub 2/ toxicity in the rat. PB- or ..beta..-NF induce hepatic P-450 but only ..beta..-NF induces pulmonary P-450. Pulmonary microsomes produced O/sub 2//sup -/ and H/sub 2/O/sub 2/ at rates (expressed per mg microsomal protein) which did not vary as a function of pretreatment. Rats were exposed to 100% O/sub 2/ for up to 3 days. After 3 days of O/sub 2/, lung weights were about 50% above controls regardless of pretreatment. The microsomal monooxygenase enzymes (P-450, b/sub 5/ and NADPH P-450 reductase) were quantified in liver and lung. Lung microsomal P-450 was reduced after 3 days of O/sub 2/ exposure regardless of pretreatment. The protective enzymes (catalase, superoxide dismutase (SOD) and glutathione (GSH) peroxidase) and non-protein sulfhydryl groups (NPSH) were also quantified in lung and liver samples. Lung NPSH and GSH peroxidase were increased after 3 days of O/sub 2/ exposure regardless of pretreatment while SOD was increased in controls and PB- but not ..beta..-NF-treated rats. Three of 14 ..beta..-NF-treated rats died during O/sub 2/ exposure while no animals in the control or PB-treated groups died.

  15. Differential cumene hydroperoxide sensitivity of cytochrome P-450 enzymes IA1 and IIB1 determined by their way of membrane incorporation.

    PubMed

    Balvers, W G; Boersma, M G; Veeger, C; Rietjens, I M

    1992-09-15

    The cytochrome P-450-dependent O-dealkylation of alkoxyresorufins was used to study the effect of cumene hydroperoxide on cytochrome P-450 IIB1 and IA1 in microsomal and reconstituted systems. In liver microsomal systems from respectively phenobarbital and 3-methylcholanthrene pretreated male Wistar rats, cytochrome P-450 IIB1-dependent pentoxyresorufin-O-dealkylation appeared to be more sensitive to cumene hydroperoxide treatment than cytochrome P-450 IA1-dependent ethoxyresorufin-O-dealkylation. This phenomenon was also observed when the cumene hydroperoxide sensitivity of P-450 IIB1 and IA1 was studied in an isosafrole pretreated rat liver microsomal system. The decrease in alkoxy-O-dealkylating activities appeared to proceed by destruction of the cytochrome P-450 component of the enzyme system. Purification and reconstitution of the enzyme system components in a system in which the isolated proteins were not incorporated into a membrane resulted in the disappearance of the difference in sensitivity between the two P-450 enzymes. However, in a reconstituted system with membrane incorporated proteins, again cytochrome P-450 IIB1 expressed a higher sensitivity towards cumene hydroperoxide than cytochrome P-450 IA1. From this it was concluded that the differential cumene hydroperoxide sensitivity of cytochrome P-450 IIB1 and IA1 is not caused by an intrinsic difference in their sensitivity but by a differential effect of membrane incorporation on their cumene hydroperoxide sensitivity.

  16. Inactivation of cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a thiophene-containing anticancer drug.

    PubMed

    Lin, Hsia-lien; Zhang, Haoming; Medower, Christine; Hollenberg, Paul F; Johnson, William W

    2011-02-01

    An investigational anticancer agent that contains a thiophene moiety, 3-[(quinolin-4-ylmethyl)-amino]-N-[4-trifluoromethox)phenyl] thiophene-2-carboxamide (OSI-930), was tested to investigate its ability to modulate the activities of several cytochrome P450 enzymes. Results showed that OSI-930 inactivated purified, recombinant cytochrome P450 (P450) 3A4 in the reconstituted system in a mechanism-based manner. The inactivation was dependent on cytochrome b(5) and required NADPH. Catalase did not protect against the inactivation. No inactivation was observed in studies with human 2B6, 2D6, or 3A5 either in the presence or in the absence of b(5). The inactivation of 3A4 by OSI-930 was time- and concentration-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl)coumarin catalytic activity of 3A4 was characterized by a K(I) of 24 μM and a k(inact) of 0.04 min(-1). This K(I) is significantly greater than the clinical OSI-930 C(max) of 1.7 μM at the maximum tolerated dose, indicating that clinical drug interactions of OSI-930 via this pathway are not likely. Spectral analysis of the inactivated protein indicated that the decrease in the reduced CO spectrum at 450 nm was comparable to the amount of inactivation, thereby suggesting that the inactivation was primarily due to modification of the heme. High-pressure liquid chromatography (HPLC) analysis with detection at 400 nm showed a loss of heme comparable to the activity loss, but a modified heme was not detected. This result suggests either that the heme must have been modified enough so as not to be observed in a HPLC chromatograph or, possibly, that it was destroyed. The partition ratio for the inactivation of P450 3A4 was approximately 23, suggesting that this P450 3A4-mediated pathway occurs with approximately 4% frequency during the metabolism of OSI-930. Modeling studies on the binding of OSI-930 to the active site of the P450 3A4 indicated that OSI-930 would be oriented properly in the active site

  17. Population Pharmacokinetic Model for Docetaxel in Patients with Varying Degrees of Liver Function: Incorporating Cytochrome P450 3A Activity Measurements

    PubMed Central

    Hooker, AC; ten Tije, AJ; Carducci, MA; Weber, J; Garrett-Mayer, E; Gelderblom, H; McGuire, WP; Verweij, J; Karlsson, MO; Baker, SD

    2011-01-01

    The relationship between cytochrome P4503A4 (CYP3A4) activity and docetaxel clearance in patients with varying degrees of liver function (LF) was evaluated. Docetaxel 40, 50, or 75 mg/m2 was administered to 85 patients with advanced cancer; 23 of 77 evaluable patients had abnormalities in liver function tests. Baseline CYP3A activity was assessed using the erythromycin breath test (ERMBT). Pharmacokinetic studies and toxicity assessments were performed during cycle 1 of therapy and population modeling was performed using NONMEM. Docetaxel unbound clearance was lower (317 vs. 470 L/h) and more variable in patients with liver function abnormalities compared to patients with normal LF. Covariates evaluated accounted for 83% of variability on clearance in patients with liver dysfunction, with CYP3A4 activity accounting for 47% of variation; covariates accounted for only 23% of variability in patients with normal LF. The clinical utility of the ERMBT may be in identifying safe docetaxel doses for patients with LF abnormalities. PMID:18183036

  18. Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

    PubMed Central

    Simonov, Alexandr N.; Holien, Jessica K.; Yeung, Joyee Chun In; Nguyen, Ann D.; Corbin, C. Jo; Zheng, Jie; Kuznetsov, Vladimir L.; Auchus, Richard J.; Conley, Alan J.; Bond, Alan M.; Parker, Michael W.; Rodgers, Raymond J.; Martin, Lisandra L.

    2015-01-01

    Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions. PMID:26587646

  19. Cytochrome p450nor, a novel class of mitochondrial cytochrome P450 involved in nitrate respiration in the fungus Fusarium oxysporum.

    PubMed

    Takaya, N; Suzuki, S; Kuwazaki, S; Shoun, H; Maruo, F; Yamaguchi, M; Takeo, K

    1999-12-15

    Fusarium oxysporum, an imperfect filamentous fungus performs nitrate respiration under limited oxygen. In the respiratory system, Cytochrome P450nor (P450nor) is thought to catalyze the last step; reduction of nitric oxide to nitrous oxide. We examined its intracellular localization using enzymatic, spectroscopic, and immunological analyses to show that P450nor is found in both the mitochondria and the cytosol. Translational fusions between the putative mitochondrial targeting signal on the amino terminus of P450nor and Escherichia coli beta-galactosidase resulted in significant beta-galactosidase activity in the mitochondrial fraction of nitrate-respiring cells, suggesting that one of the isoforms of P450nor (P450norA) is in anaerobic mitochondrion of F. oxysporum and acts as nitric oxide reductase. Furthermore, these findings suggest the involvement of P450nor in nitrate respiration in mitochondria.

  20. Aryl hydrocarbon induction of rat cytochrome P-450d results from increased precursor RNA processing.

    PubMed Central

    Silver, G; Krauter, K S

    1990-01-01

    We have previously demonstrated that cytochrome P-450d mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript. Images PMID:2247082

  1. Dechlorination of halocarbons by microsomes and vesicular reconstituted cytochrome P-450 systems under reductive conditions.

    PubMed Central

    Salmon, A G; Nash, J A; Walklin, C M; Freedman, R B

    1985-01-01

    A spectrophotometric assay of the reductive dechlorination of halocarbons was developed and used to determine the kinetic characteristics of dechlorination of a range of haloethanes catalysed by microsomes from rat and rabbit liver. Analysis of the typical reaction of hexachloroethane shows that the reaction is catalysed by cytochrome P-450 and results in the formation of olefinic products as well as less chlorinated alkanes: in other respects the reaction resembles that known to occur with carbon tetrachloride. The dechlorination of haloethanes catalysed by a vesicular reconstituted system of cytochrome P-450 enzymes from rabbit liver was also studied and found to be similar to that catalysed by microsomes: both reductase and a phenobarbital inducible form of cytochrome P-450 were essential. There is no substantial dependence of maximum dechlorination rate on compound structure, suggesting that the reduction of substrate is not the rate limiting step in the overall reaction. The main factor in determining the apparent binding constant to the enzyme is the partition coefficient into the lipid membrane, as assessed by calculated log P values. PMID:3986141

  2. Repurposing Resveratrol and Fluconazole To Modulate Human Cytochrome P450-Mediated Arachidonic Acid Metabolism.

    PubMed

    El-Sherbeni, Ahmed A; El-Kadi, Ayman O S

    2016-04-04

    Cytochrome P450 (P450) enzymes metabolize arachidonic acid (AA) to several biologically active epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs). Repurposing clinically-approved drugs could provide safe and readily available means to control EETs and HETEs levels in humans. Our aim was to determine how to significantly and selectively modulate P450-AA metabolism in humans by clinically-approved drugs. Liquid chromatography-mass spectrometry was used to determine the formation of 15 AA metabolites by human recombinant P450 enzymes, as well as human liver and kidney microsomes. CYP2C19 showed the highest EET-forming activity, while CYP1B1 and CYP2C8 showed the highest midchain HETE-forming activities. CYP1A1 and CYP4 showed the highest subterminal- and 20-HETE-forming activity, respectively. Resveratrol and fluconazole produced the most selective and significant modulation of hepatic P450-AA metabolism, comparable to investigational agents. Monte Carlo simulations showed that 90% of human population would experience a decrease by 6-22%, 16-39%, and 16-35% in 16-, 18-, and 20-HETE formation, respectively, after 2.5 g daily of resveratrol, and by 22-31% and 14-23% in 8,9- and 14,15-EET formation after 50 mg of fluconazole. In conclusion, clinically-approved drugs can provide selective and effective means to modulate P450-AA metabolism, comparable to investigational drugs. Resveratrol and fluconazole are good candidates to be repurposed as new P450-based treatments.

  3. Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

    PubMed Central

    Ito, Michihiro; Sato, Ikuo; Ishizaka, Masumi; Yoshida, Shin-ichiro; Koitabashi, Motoo; Yoshida, Shigenobu

    2013-01-01

    Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (kcat/Km) of 6.4 mM−1 s−1. The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat. PMID:23275503

  4. Inhibition of cytochrome p450 enzymes by enrofloxacin in the sea bass (Dicentrarchus labrax).

    PubMed

    Vaccaro, E; Giorgi, M; Longo, V; Mengozzi, G; Gervasi, P G

    2003-01-10

    Currently, there are no reports on the effects of enrofloxacin (EF), a fluoroquinolone antibiotic, on the cytochrome p450 enzymes in fish, although its use as antimicrobial agent in aquaculture has been put forward. Therefore, the in vivo and in vitro effects of EF on hepatic p450 enzymes of sea bass, a widespread food-producing fish, have been evaluated. Sea bass pretreated with a single dose of EF (3 mg/kg i.p.) or with three daily doses of EF (1 mg/kg i.p.) markedly depressed the microsomal N-demethylation of aminopyrine, erythromycin, the O-deethylation of 7-ethoxycoumarin, ethoxyresorufin and the 6beta-testosterone hydroxylase. In vitro experiments showed that EF at 10 microM inhibited the above-mentioned activities and, in particular, the erythromycin N-demethylase (ERND) and 6beta-testosterone-hydroxylase, likely dependant on a p450 3A isoform. When the nature of ERND inhibition by EF was specifically studied with sea bass liver microsomes, it was found that EF is a potent mechanism-based inhibitor, with K(i) of 3.7 microM and a K(inact) of 0.045 min(-1). An immunoblot analysis with anti p450 3A27 of trout showed that the p450 3A isoform, constitutively expressed in sea bass, is particularly susceptible to inactivation by EF. In vitro experiments with sea bass microsomes have also demonstrated that EF is oxidative deethylated by the p450 system to ciprofloxacin (CF) and that this compound maintains the ability to inactivate the p450 enzymes. The mechanism by which EF or CF inactivate the p450 enzymes has not been studied but an attack of p450 on the cyclopropan ring, present, both in EF and CF structure, with the formation of electrophilic intermediates (i.e. radicals) has been postulated. In conclusion, the EF seems to be a powerful inhibitor of p450s in the sea bass. Therefore, the clinical use of this antibiotic in aquaculture has to be considered with caution.

  5. Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology.

    PubMed

    Girvan, Hazel M; Munro, Andrew W

    2016-04-01

    Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenase enzymes with enormous potential for synthetic biology applications. Across Nature, their substrate range is vast and exceeds that of other enzymes. The range of different chemical transformations performed by P450s is also substantial, and continues to expand through interrogation of the properties of novel P450s and by protein engineering studies. The ability of P450s to introduce oxygen atoms at specific positions on complex molecules makes these enzymes particularly valuable for applications in synthetic biology. This review focuses on the enzymatic properties and reaction mechanisms of P450 enzymes, and on recent studies that highlight their broad applications in the production of oxychemicals. For selected soluble bacterial P450s (notably the high-activity P450-cytochrome P450 reductase enzyme P450 BM3), variants with a multitude of diverse substrate selectivities have been generated both rationally and by random mutagenesis/directed evolution approaches. This highlights the robustness and malleability of the P450 fold, and the capacity of these biocatalysts to oxidise a wide range of chemical scaffolds. This article reviews recent research on the application of wild-type and engineered P450s in the production of important chemicals, including pharmaceuticals and drug metabolites, steroids and antibiotics. In addition, the properties of unusual members of the P450 superfamily that do not follow the canonical P450 catalytic pathway are described.

  6. Inhibition of human cytochrome P450 enzymes by the natural hepatotoxin safrole.

    PubMed

    Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw

    2005-05-01

    The hepatotoxin, safrole is a methylenedioxy phenyl compound, found in sassafras oil and certain other essential oils. Recombinant cytochrome P450 (CYP, P450) and human liver microsomes were studied to investigate the selective inhibitory effects of safrole on human P450 enzymes and the mechanisms of action. Using Escherichia coli-expressed human P450, our results demonstrated that safrole was a non-selective inhibitor of CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 in the IC(50) order CYP2E1 < CYP1A2 < CYP2A6 < CYP3A4 < CYP2D6. Safrole strongly inhibited CYP1A2, CYP2A6, and CYP2E1 activities with IC(50) values less than 20 microM. Safrole caused competitive, non-competitive, and non-competitive inhibition of CYP1A2, CYP2A6 and CYP2E1 activities, respectively. The inhibitor constants were in the order CYP1A2 < CYP2E1 < CYP2A6. In human liver microsomes, 50 microM safrole strongly inhibited 7-ethoxyresorufin O-deethylation, coumarin hydroxylation, and chlorzoxazone hydroxylation activities. These results revealed that safrole was a potent inhibitor of human CYP1A2, CYP2A6, and CYP2E1. With relatively less potency, CYP2D6 and CYP3A4 were also inhibited.

  7. Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase

    PubMed Central

    Bavishi, Krutika; Laursen, Tomas; Martinez, Karen L.; Møller, Birger Lindberg; Della Pia, Eduardo Antonio

    2016-01-01

    Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, “nanodiscs”, and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and −300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s−1. POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products. PMID:27386958

  8. Induction of cytochrome p-450-ia1 in juvenile fish by creosote-contaminated sediment

    SciTech Connect

    Schoor, W.P.; Williams, D.E.; Takahashi, N.

    1991-01-01

    Intact sediment cores, including their surface layers, were used in simulated field exposure tests of juvenile guppies (Poecilia reticulata) to creosote-contaminated sediments. Mixed-function oxygenase activity was induced in the fish after 43 days of exposure to environmentally realistic, sublethal concentrations of creosote-related compounds. An average 50-fold induction in the cytochrome P-450-IA1 was found in the liver in the absence of any histopathological lesions. The possibility that a threshold level for proliferative liver changes was not reached is discussed in the light of the observed biochemical activation.

  9. Human Recombinant Cytochrome P450 Enzymes Display Distinct Hydrogen Peroxide Generating Activities During Substrate Independent NADPH Oxidase Reactions

    PubMed Central

    Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-01-01

    Microsomal enzymes generate H2O2 in the presence of NADPH. In this reaction, referred to as “oxidase” activity, H2O2 is generated directly or indirectly via the formation of superoxide anion. In the presence of redox active transition metals, H2O2 can form highly toxic hydroxyl radicals and, depending on the “oxidase” activity of individual cytochrome P450 isoenzymes, this can compromise cellular functioning and contribute to tissue injury. In the present studies, we compared the initial rates of H2O2 generating activity of microsomal preparations containing various human recombinant cytochromes P450s. In the absence of cytochrome P450s the human recombinant NADPH cytochrome P450 reductase (CPR) generated low, but detectable amounts of H2O2 (∼0.04 nmol H2O2/min/100 units of reductase). Significantly greater activity was detected in preparations containing individual cytochrome P450s coexpressed with CPR (from 6.0 nmol H2O2/min/nmol P450 to 0.2 nmol/min/nmol P450); CYP1A1 was the most active, followed by CYP2D6, CYP3A4, CYP2E1, CYP4A11, CYP1A2, and CYP2C subfamily enzymes. H2O2 generating activity of the cytochrome P450s was independent of the ratio of CYP/CPR. Thus, similar H2O2 generating activity was noted with the same cytochrome P450s (CYP3A4, CYP2E1, and CYP2C9) expressed at or near the ratio of CYP/CPR in human liver microsomes (5–7), and when CPR was present in excess (CYP/CPR = 0.2–0.3). Because CYP3A4/5/7 represent up to 40% of total cytochrome P450 in the liver, these data indicate that these enzymes are the major source of H2O2 in human liver microsomes. PMID:25061110

  10. High-throughput mass spectrometric cytochrome P450 inhibition screening.

    PubMed

    Lim, Kheng B; Ozbal, Can C; Kassel, Daniel B

    2013-01-01

    We describe here a high-throughput assay to support rapid evaluation of drug discovery compounds for possible drug-drug interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of eight concentrations and against a panel of six cytochrome P450 (CYP) enzymes: 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. The method utilizes automated liquid handling for sample preparation, and online solid-phase extraction/tandem mass spectrometry (SPE/MS/MS) for sample analyses. The system is capable of generating two 96-well assay plates in 30 min, and completes the data acquisition and analysis of both plates in about 30 min. Many laboratories that perform the CYP inhibition screening automate only part of the processes leaving a throughput bottleneck within the workflow. The protocols described in this chapter are aimed to streamline the entire process from assay to data acquisition and processing by incorporating automation and utilizing high-precision instrument to maximize throughput and minimize bottleneck.

  11. First principles calculation of the activity of cytochrome P450

    NASA Astrophysics Data System (ADS)

    Segall, M. D.; Payne, M. C.; Ellis, S. W.; Tucker, G. T.; Boyes, R. N.

    1998-04-01

    The cytochrome P450 superfamily of enzymes is of enormous interest in the biological sciences due to the wide range of endogenous and xenobiotic compounds which it metabolises, including many drugs. We describe the use of first principles quantum mechanical modeling techniques, based on density functional theory, to determine the outcome of interactions between an enzyme and a number of compounds. Specifically, we calculate the spin state of an Fe3+ ion present in a haem moiety at the active site of these enzymes. The spin state of this ion indicates if the catalytic reaction will proceed. The computational results obtained compare favorably with experimental data. Only the principle components of the active site of the enzyme are included in the computational models, demonstrating that only a small fragment of the protein needs to be included in the models in order to accurately reproduce this aspect of the enzymes' function. These results open the way for further investigation of this superfamily of enzymes using the methods detailed in this paper.

  12. Cytochrome P450 ω-Hydroxylases in Inflammation and Cancer

    PubMed Central

    Johnson, Amanda L.; Edson, Katheryne Z.; Totah, Rheem A.; Rettie, Allan E.

    2015-01-01

    Cytochrome P450-dependent ω-hydroxylation is a prototypic metabolic reaction of CYP4 family members that is important for the elimination and bioactivation of not only therapeutic drugs, but also endogenous compounds, principally fatty acids. Eicosanoids, derived from arachidonic acid, are key substrates in the latter category. Human CYP4 enzymes, mainly CYP4A11, CYP4F2, and CYP4F3B, hydroxylate arachidonic acid at the omega position to form 20-HETE, which has important effects in tumor progression and on angiogenesis and blood pressure regulation in the vasculature and kidney. CYP4F3A in myeloid tissue catalyzes the ω-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, an inactivation process that is critical for the regulation of the inflammatory response. Here, we review the enzymology, tissue distribution, and substrate selectivity of human CYP4 ω-hydroxylases and their roles as catalysts for the formation and termination of the biological effects of key eicosanoid metabolites in inflammation and cancer progression. PMID:26233909

  13. CYTOCHROME P450 REGULATION: THE INTERPLAY BETWEEN ITS HEME AND APOPROTEIN MOIETIES IN SYNTHESIS, ASSEMBLY, REPAIR AND DISPOSAL123

    PubMed Central

    Correia, Maria Almira; Sinclair, Peter R.; De Matteis, Francesco

    2011-01-01

    Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair and disposal. These less well-appreciated aspects are reviewed herein. PMID:20860521

  14. Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency

    SciTech Connect

    Meier, U.T.; Meyer, U.A.

    1987-12-15

    The metabolism of the anticonvulsant mephenytoin is subject to a genetic polymorphism. In 2-5% of Caucasians and 18-23% of Japanese subjects a specific cytochrome P-450 isozyme, P-450 meph, is functionally deficient or missing. The authors have accumulated evidence that autoimmune antibodies observed in sera of patients with tienilic acid induced hepatitis (anti-liver kidney microsome 2 or anti-LKM2 antibodies) specifically recognize the cytochrome P-450 involved in the mephrenytoin hydroxylation polymorphism. This is demonstrated by immunoinhibition and immunoprecipitation of microsomal (S)-mephenytoin 4-hydroxylation activity and by the recognition by anti-LKM2 antibodies of a single (/sup 125/I)-protein band on immunoblots of human liver microsomes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. The cytochrome P-450 recognized by anti-LKM2 antibodies was immunopurified from microsomes derived from livers of extensive (EM) or poor metabolizers (PM) of (S)-mephenytoin. Comparison of the EM-type cytochrome P-450 to that isolated from PM livers revealed no difference in regard to immuno-cross-reactivity, molecular weight, isoelectric point, relative content in microsomes, two-dimensional tryptic peptide maps, one-dimensional peptide maps with three proteases, amino acid composition, and amino-terminal protein sequence. Finally, the same protein was precipitated from microsomes prepared from the liver biopsy of a subject phenotyped in vivo as a poor metabolizer of mephenytoin. These data strongly suggest that the mephenytoin hydroxylation deficiency is caused by a minor structural change leading to a functionally altered cytochrome P-450 isozyme.

  15. Plant activation of aromatic amines mediated by cytochromes P450 and flavin-containing monooxygenases.

    PubMed

    Chiapella, C; Radovan, R D; Moreno, J A; Casares, L; Barbé, J; Llagostera, M

    2000-10-31

    To know the mechanisms involved in the activation of promutagenic aromatic amines mediated by plants, we used Persea americana S117 system (S117) for the activation of 2-aminofluorene (2-AF) and m-phenylenediamine (m-PDA) in Ames assays. In these assays, the effect of the diphenylene iodonium (DPI), an inhibitor of flavin-containing monooxygenases (FMOs), of the 1-aminobenzotriazole (1-ABT), an inhibitor of cytochromes P450 (cyt-P450s) and of the methimazole, a high-affinity substrate for FMOs, was studied. The efficacy of both inhibitors and of the methimazole was verified to find that they did partially inhibit the mutagenesis of both aromatic amines, activated with rat liver S9. Similarly, both inhibitors and methimazole did produce a significant decrease in 2-AF and m-PDA mutagenesis, when the activation system was S117, indicating that, similar to what occurs in mammalian systems, plant FMOs and cyt-P450s can metabolize aromatic amines to mutagenic product(s). However, the affinity of both FMOs and cyt-P450s of plant for 2-AF and m-PDA was different. Data obtained indicate that the activities of plant FMOs must be the main enzymatic system of m-PDA activation while, in 2-AF activation, plant cyt-P450s have the most relevant activities. In addition, peroxidases of the S117 system must contribute to 2-AF activation and some isoforms of FMOs and/or cyt-P450s of the S117 system, uninhibited by the inhibitors used, must be the responsible for a partial activation of m-PDA.

  16. Molecular genetic analysis of the cytochrome P450-debrisoquine hydroxylase locus and association with cancer susceptibility.

    PubMed Central

    Smith, C A; Moss, J E; Gough, A C; Spurr, N K; Wolf, C R

    1992-01-01

    The cytochrome P450-dependent monooxygenases play a central role in the metabolism of chemical carcinogens. The action of these enzymes can lead to either carcinogen detoxication or activation. Differences in P450 expression in animal models give rise to large differences in susceptibility to chemical carcinogens, so genetic polymorphisms in P450 expression may be expected to be an important factor in individual human susceptibility to cancer. Of particular interest is the genetic polymorphism at the cytochrome P450-debrisoquine/sparteine hydroxylase locus (CYP2D6). Although this is a minor liver P450, its polymorphic expression is associated with the abnormal metabolism of at least 30 therapeutic drugs, including beta-blockers and tricyclic antidepressants. Conflicting reports have been made on the association of this polymorphism with cancer susceptibility. This disagreement may be attributable to limitations of the phenotyping assay used to identify affected individuals (poor metabolizers, PMs). In order to clarify these anomalies, we have developed a simple DNA-based assay with which we can identify the majority of PMs. The assay is centered around the primary gene defect responsible for the polymorphism, a G to A transition at the junction of intron 3/exon 4 which results in a frame-shift in the resultant mRNA. The frequency of this mutation is 70-80% in PMs. We have studied the frequency of mutated alleles in a control population and in a wide range of cancer patients. No association between this polymorphism and lung cancer susceptibility was observed; however, in other populations of cancer patients some very interesting shifts were found in the proportion of PMs and heterozygotes from that in the normal population. PMID:1486838

  17. Transcriptional Regulation of Grape Cytochrome P450 Gene Expression in Response to Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are versatile redox proteins that mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds as plant defense agents against a range of pathogens and insects. To determine if cytochrome P450 monooxygenases are involved in the...

  18. The active site of cytochrome P-450 nifedipine oxidase: a model-building study.

    PubMed

    Ferenczy, G G; Morris, G M

    1989-12-01

    A model of the active site of cytochrome P-450 nifedipine oxidase is built on the basis of sequence homology with cytochrome P-450CAM. Substrates are docked into the binding pocket, and molecular mechanical energy minimization is performed to analyze the forces between the substrates and the enzyme.

  19. The mechanism of cumene hydroperoxide-dependent lipid peroxidation: the function of cytochrome P-450.

    PubMed

    Weiss, R H; Estabrook, R W

    1986-11-15

    The addition of limiting amounts of cumene hydroperoxide to rat liver microsomes resulted in the rapid uptake of molecular oxygen, the formation of thiobarbituric acid reactive products, and the loss of hydroperoxide. The stoichiometry of lipid peroxidation and the yields of 2-phenyl-2-propanol (a major product of the reaction) and acetophenone (a minor product) observed with liver microsomes prepared from untreated rats is greater than that seen with liver microsomes from ciprofibrate-treated rats which, in turn, is greater than that observed with liver microsomes from phenobarbital-treated rats. The Km's and Vmax's of oxygen uptake varied with the type of rat liver microsomes used. Cytochrome P-450 substrates and inhibitors decreased the extents and initial rates of oxygen uptake and thiobarbituric acid reactive product formation. A mechanism is proposed involving the cytochrome P-450-catalyzed homolytic cleavage of the cumene hydroperoxide O-O bond to give the cumyloxyl radical. It is proposed that this oxygen-centered radical abstracts a hydrogen atom from an unsaturated fatty acid associated with a lipid (initiating lipid peroxidation) to give 2-phenyl-2-propanol or that the radical undergoes beta-scission to produce acetophenone and a methyl radical.

  20. Microsomal cytochrome P450 as a target for drug discovery and repurposing.

    PubMed

    El-Sherbeni, Ahmed A; El-Kadi, Ayman O S

    2017-02-01

    Cytochrome P450 (P450) enzymes are ancient electron-transfer-chain system of remarkable biological importance. Microsomal P450 enzymes are the P450 attached to endoplasmic reticulum, which, in humans, are critical for body's defenses against xenobiotics by mediating their metabolism, and cell signaling by mediating arachidonic acid (AA) transformation to several potent bioactive molecules. Only recently, modulating P450-mediated AA metabolism has risen as a promising new drug target. This review presents the therapeutic potential of finding effective, selective and safe treatments targeting P450-mediated AA metabolism, and the several approaches that have been used to find these treatments; among which, our focus was on modulators of P450 activities. We detailed the efforts done to develop new molecular entities designed to modulate P450, and the more recent efforts tried to employ our previous knowledge on drug metabolism to repurpose old drugs with the capacity to alter P450-mediated drug metabolism to target AA metabolism. Because of the long recognition of P450 role in xenobiotic metabolism, several clinically approved agents were identified to alter P450 activity. Repurposing old drugs as P450 modulators can facilitate bringing treatments targeting P450-mediated AA metabolism to clinical trials. However, the capacity of the modulation of P450-derived AA metabolites of clinically approved drugs has to be systematically investigated and validated for their new use in humans.

  1. A three-dimensional model of aromatase cytochrome P450.

    PubMed Central

    Graham-Lorence, S.; Amarneh, B.; White, R. E.; Peterson, J. A.; Simpson, E. R.

    1995-01-01

    P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate. PMID:7549871

  2. [Effects of repeated skin applications of heavy pyrolysis resin on cytochrome P-450 level and glutathione transferase activity in liver microsomes and cytosol in rats in relation to the level of toxic effects of pyrolysis resin on internal organs].

    PubMed

    Kravchenko, M N; Loginov, A S; Petrova, L P; Ausheva, L Kh; Bendikov, E A

    1990-10-01

    Rats received 20 skin applications of heavy pyrolysis resin, containing about 30% of polycyclic aromatic hydrocarbons. The every exposure duration was 4 hours a day. Applications have been carried out daily 5 days a week in the course of 4 weeks. Induction of cytochrome P-450 (P-450) by 79%, significant induction of microsomal (GTm) and cytosol (GTc) glutathione-S-transferase activity (by 46 and 85%, respectively) and small increase of reduced glutathione level (by 9%) also were registered as a result of these exposures. Lipid peroxidation level determined by TBK-reactive products quantity didn't alter significantly. Close correlation between ratio values of P-450/GTm levels and P-450/GTc levels and toxic effect indices of heavy pyrolysis resin on rat immune and endocrine systems.

  3. Cytochrome P450-dependent metabolism of caffeine in Drosophila melanogaster.

    PubMed

    Coelho, Alexandra; Fraichard, Stephane; Le Goff, Gaëlle; Faure, Philippe; Artur, Yves; Ferveur, Jean-François; Heydel, Jean-Marie

    2015-01-01

    Caffeine (1, 3, 7-trimethylxanthine), an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents). A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs) that were highly overexpressed. Flies treated with metyrapone--an inhibitor of CYP enzymes--showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects.

  4. Size-dependent effects of nanoparticles on the activity of cytochrome P450 isoenzymes

    SciTech Connect

    Froehlich, Eleonore; Kueznik, Tatjana; Samberger, Claudia; Roblegg, Eva; Wrighton, Christopher

    2010-02-01

    Nanoparticles are known to be able to interfere with cellular metabolism and to cause cytotoxicity and moreover may interfere with specific cellular functions. Serious effects on the latter include changes in liver cell function. The cytochrome P450 system is expressed in many cells but is especially important in hepatocytes and hormone-producing cells. The interaction of polystyrene nanoparticles with the most important drug-metabolizing cytochrome P450 isoenzymes, CYP3A4, CYP2D6, CYP2C9 and CYP2A1 expressed individually in insect cells (BACULOSOMES) was studied by the cleavage of substrates coupled to a fluorescent dye. The data obtained for individual isoenzymes were compared to metabolism in microsomes isolated from normal liver and from the hepatoma cell line H4-II-E-C3. Small (20-60 nm) carboxyl polystyrene particles but not larger (200 nm) ones reached high intracellular concentrations in the vicinity of the endoplasmic reticulum. These small particles inhibited the enzymatic activity of CYP450 isoenzymes in BACULOSOMES and substrate cleavage in normal liver microsomes. They moreover increased the effect of known inhibitors of the cytochrome P450 system (cimetidine, phenobarbital and paclitaxel). Substrate cleavage by the hepatoma cell line H4-II-E-C3 in contrast was undetectable, making this cell line unsuitable for this type of study. Our results thus demonstrate that nanoparticles can inhibit the metabolism of xenobiotics by the CYP450 system in model systems in vitro. Such inhibition could also potentially occur in vivo and possibly cause adverse effects in persons receiving medication.

  5. A New Marmoset P450 4F12 Enzyme Expressed in Small Intestines and Livers Efficiently Metabolizes Antihistaminic Drug Ebastine.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Yuki, Yukako; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2016-06-01

    Common marmosets (Callithrix jacchus) are attracting attention as animal models in preclinical studies for drug development. However, cytochrome P450s (P450s), major drug-metabolizing enzymes, have not been fully identified and characterized in marmosets. In this study, based on the four novel P450 4F genes found on the marmoset genome, we successfully isolated P450 4F2, 4F3B, 4F11, and 4F12 cDNAs in marmoset livers. Deduced amino acid sequences of the four marmoset P450 4F forms exhibited high sequence identities (87%-93%) to the human and cynomolgus monkey P450 4F homologs. Marmoset P450 4F3B and 4F11 mRNAs were predominantly expressed in livers, whereas marmoset P450 4F2 and 4F12 mRNAs were highly expressed in small intestines and livers. Four marmoset P450 4F proteins heterologously expressed in Escherichia coli catalyzed the ω-hydroxylation of leukotriene B4 In addition, marmoset P450 4F12 effectively catalyzed the hydroxylation of antiallergy drug ebastine, a human P450 2J/4F probe substrate. Ebastine hydroxylation activities by small intestine and liver microsomes from marmosets and cynomolgus monkeys showed greatly higher values than those of humans. Ebastine hydroxylation activities by marmoset and cynomolgus monkey small intestine microsomes were inhibited (approximately 60%) by anti-P450 4F antibodies, unlike human small intestine microsomes, suggesting that contribution of P450 4F enzymes for ebastine hydroxylation in the small intestine might be different between marmosets/cynomolgus monkeys and humans. These results indicated that marmoset P450 4F2, 4F3B, 4F11, and 4F12 were expressed in livers and/or small intestines and were functional in the metabolism of endogenous and exogenous compounds, similar to those of cynomolgus monkeys and humans.

  6. Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

    SciTech Connect

    Osawa, Y.; Coon, M.J.

    1987-08-01

    In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, (1,2,6,7,16,17-3H)progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting (1,7,16-3H)progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of /sup 3/H/sub 2/O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of (1,7,16-/sup 3/H)progesterone and (4-14C)progesterone was employed.

  7. tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance

    PubMed Central

    Zhang, Haoming; Lin, Hsia-lien; Walker, Vyvyca J.; Hamdane, Djemel

    2009-01-01

    We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a KI of 0.44 μM and kinact of 0.12 min−1. The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 Å away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ∼20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4. PMID:19720728

  8. Construction of a 3D model of cytochrome P450 2B4.

    PubMed

    Chang, Y T; Stiffelman, O B; Vakser, I A; Loew, G H; Bridges, A; Waskell, L

    1997-02-01

    A three-dimensional structural model of rabbit phenobarbital-inducible cytochrome P450 2B4 (LM2) was constructed by homology modeling techniques previously developed for building and evaluating a 3D model of the cytochrome P450choP isozyme. Four templates with known crystal structures including cytochrome P450cam, terp, BM-3 and eryF were used in multiple sequence alignments and construction of the cytochrome P450 2B4 coordinates. The model was evaluated for its overall quality using available protein analysis programs and found to be satisfactory. The model structure was stable at room temperature during a 140 ps unconstrained full protein molecular dynamics simulation. A putative substrate access channel and binding site were identified. Two different substrates, benzphetamine and androstenedione, that are metabolized by cytochrome P450 2B4 with pronounced product specificity were docked into the putative binding site. Two orientations were found for each substrate that could lead to the observed preferred products. Using a geometric fit method three regions on the surface of the model cytochrome P450 structure were identified as possible sites for interaction with cytochrome b5, a redox partner of P450 2B4. Residues that may interact with the substrates and with cytochrome b5 have been identified and mutagenesis studies are currently in progress.

  9. [Inhibitory action of divalent copper compounds on cumene hydroperoxide oxidative demethylation of N,N-dimethylaniline by cytochrome P-450].

    PubMed

    Kurchenko, V P; Usanov, S A; Metelitsa, D I

    1980-07-01

    The inhibitory action of divalent copper compounds on hydroperoxide-dependent oxidative demethylation of N,N-demethylaniline involving rabbit liver microsomes and highly purified cytochrome P-450 has been studied. CuCl2 is a non-competitive inhibitor, whereas copper tyrosine and lysine complexes are characterized by a mixed type inhibition. The inhibitory action of copper complexes is based on a decrease of cumene hydroperoxide concentration. The reaction results in formation of RO and RO2 radicals destroying cytochrome P-450 CuCl2 (0,001 M) also destroys cytochrome P-450 in the absence of cumene hydroperoxide; the destruction process is characterized by two phases with different rate constants. The nature of the inhibitory action of CuCl2 on N,N-demethylaniline oxidation by hydroperoxides is discussed.

  10. Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

    PubMed Central

    Sanglard, D; Käppeli, O; Fiechter, A

    1984-01-01

    In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells. PMID:6690424

  11. Interaction of Angeli's salt with cytochrome P450 1A2 distal mutants: an optical absorption spectral study.

    PubMed

    Shibata, Y; Sato, H; Sagami, I; Shimizu, T

    1997-11-14

    Angeli's salt, Na2N2O3 or O-N=N+-(OH)(O-) in aqueous solution, is known to release NO- or NO., which relaxes vascular tissue and lowers blood pressure. In the liver, the most abundant heme enzyme is cytochrome P450. In the present study, we studied the effect of rat liver cytochrome P450 1A2 (P450 1A2) in regard to its catalysis of the N=N bond scission of Angeli's salt with optical absorption spectra. Also, we examined the contribution of putative distal amino acids of P450 1A2 to the reaction with the salt. We found that wild-type Fe3+ P450 1A2 markedly enhances the N=N scission of the salt up to 100 fold in terms of absorption spectroscopy. A Fe3+ P450 1A2-NO complex with an absorption peak at 435 nm was formed when the salt was added and the complex was then changed to a 6-coordinated Fe2+-NO complex having a 440-nm peak. Glu318Asp, Glu318Ala and Thr319Ala mutants at the putative distal site of P450 1A2 formed a 5-coordinated Fe2+-NO complex having a 400-nm absorption, that was not formed with the wild type. The Glu318Ala mutant, in particular, did not form the Fe3+-NO complex with the addition of Angeli's salt. The presence of L-Cys, reduced glutathione, catalase or superoxide dismutase markedly stabilized the Fe3+ wild type-NO complex. Thus, our data suggests that the N=N bond of Angeli's salt is cleaved with the P450 1A2 active site and NO- or NO. is released. We discuss mechanisms of redox and ligand changes of the P450 heme.

  12. Catalysis by cytochrome P-450 of an oxidative reaction in xenobiotic aldehyde metabolism: deformylation with olefin formation.

    PubMed Central

    Roberts, E S; Vaz, A D; Coon, M J

    1991-01-01

    As we have briefly described elsewhere, cytochrome P-450 catalyzes the oxidative deformylation of cyclohexane carboxaldehyde to yield cyclohexene and formic acid in a reaction believed to involve a peroxyhemiacetal-like adduct formed between the substrate and molecular oxygen-derived hydrogen peroxide. This reaction is a useful model for the demethylation reactions catalyzed by the steroidogenic P-450s, aromatase, and lanosterol demethylase. In the present study, the cytochrome P-450-catalyzed formation of olefinic products from a series of xenobiotic aldehydes has been demonstrated. Isobutyraldehyde and trimethylacetaldehyde, but not propionaldehyde, are converted to the predicted olefinic products, suggesting a requirement for branching at the alpha carbon. In addition, the four C5 aldehydes of similar hydrophobicity were compared for their ability to undergo the reaction. The straight-chain valeraldehyde gave no olefinic products with five different rabbit liver microsomal P-450 isozymes. However, increasing activity was seen with the other isomers in the order of isovaleraldehyde, 2-methylbutyraldehyde, and trimethylacetaldehyde, with all of the P-450 cytochromes. The catalytic rate with trimethylacetaldehyde is highest with antibiotic-inducible P-450 form 3A6, followed by phenobarbital-inducible form 2B4 and ethanol-inducible form 2E1. Citronellal, a beta-branched aldehyde that is found in many essential oils and is widely used as an odorant and a flavorant, was found to undergo the oxidative deformylation reaction to yield 2,6-dimethyl-1,5-heptadiene, but only with P-450 2B4. The oxidative cleavage reaction with olefin formation appears to be widespread, as judged by the variety of aldehydes that serve as substrates and of P-450 cytochromes that serve as catalysts. PMID:1924356

  13. Effect of age on hepatic cytochrome P450 of Ross 708 broiler chickens.

    PubMed

    Hu, S X

    2013-05-01

    Age has significant impact on hepatic cytochrome P450 (CYP450) systems in animals. Ross 708 broiler chicken is a breed of chicken with fast growth characteristics. Cytochrome P450 in the livers of Ross 708 broiler chicken of different ages has been investigated. The birds were raised under standard husbandry conditions. A certain number of chickens was randomly sampled weekly for liver collection from d 1 to 56 posthatch. The chicken body and liver weights were recorded. The chicken livers were processed for liver microsomes though a multiple-step procedure at low temperature. Total CYP450 content in chicken liver homogenates and liver microsomes was measured using a UV/visible spectroscopic method. The enzymatic activities of CYP450 in the chicken liver microsomes were determined through incubation of CYP450 isoform substrates followed by measurement of formation of their metabolites. The chicken showed an opposite age pattern in hepatic CYP450 content and activities compared with most mammals. The hepatic CYP450 content and activities of chicken at d 1 posthatch were higher than at other ages. The total hepatic CYP450 content in chickens at d 1 posthatch was more than twice the average hepatic value of the chickens at d 7 to 28. This high CYP450 fell quickly in the first week posthatch and slightly rose from d 28 to 56. Hepatic CYP450 activities of CYP1A, 3A, 2C, 2D, and 2H were much higher in the chicken at d 1 posthatch. The differences of these enzymatic activities between d 1 and other ages of chicken were CYP450 isoform dependent. This result suggests that embryonic development of chicken livers has a significant impact on the age profile of hepatic CYP450 content and activities of posthatch chickens.

  14. Reduced Duodenal Cytochrome P450 3A Protein Expression and Catalytic Activity in Patients with Cirrhosis

    PubMed Central

    McConn, Donavon J.; Lin, Yvonne S.; Mathisen, Terri L.; Blough, David K.; Xu, Yang; Hashizume, Takanori; Taylor, Shari L.; Thummel, Kenneth E.; Shuhart, Margaret C.

    2009-01-01

    The small intestine and liver express high levels of cytochrome P450 3A (CYP3A), an enzyme subfamily contributing significantly to drug metabolism. In patients with cirrhosis, reduced metabolism of drugs is typically attributed to decreased liver function, but it is unclear whether intestinal drug metabolism is also compromised. In this study, we compared CYP3A protein expression and in vitro midazolam hydroxylation in duodenal mucosal biopsies from subjects with normal liver function (controls; n=20) and subjects with varying severity of cirrhosis (n=23). Compared to samples from controls, duodenal CYP3A expression and total midazolam hydroxylation was reduced by 47% and 34%, respectively in samples from subjects with cirrhosis. Greater decreases in CYP3A expression were seen in subjects with increasing severity of cirrhosis. Thus, patients with advanced cirrhosis may have increased drug exposure following oral dosing as a result of both impaired liver function and decreased intestinal CYP3A expression and activity. PMID:19212316

  15. Cytochrome P450 enzyme mediated herbal drug interactions (Part 2)

    PubMed Central

    Wanwimolruk, Sompon; Phopin, Kamonrat; Prachayasittikul, Virapong

    2014-01-01

    To date, a number of significant herbal drug interactions have their origins in the alteration of cytochrome P450 (CYP) activity by various phytochemicals. Among the most noteworthy are those involving St. John's wort and drugs metabolized by human CYP3A4 enzyme. This review article is the continued work from our previous article (Part 1) published in this journal (Wanwimolruk and Prachayasittikul, 2014[ref:133]). This article extends the scope of the review to six more herbs and updates information on herbal drug interactions. These include black cohosh, ginseng, grape seed extract, green tea, kava, saw palmetto and some important Chinese medicines are also presented. Even though there have been many studies to determine the effects of herbs and herbal medicines on the activity of CYP, most of them were in vitro and in animal studies. Therefore, the studies are limited in predicting the clinical relevance of herbal drug interactions. It appeared that the majority of the herbal medicines have no clear effects on most of the CYPs examined. For example, the existing clinical trial data imply that black cohosh, ginseng and saw palmetto are unlikely to affect the pharmacokinetics of conventional drugs metabolized by human CYPs. For grape seed extract and green tea, adverse herbal drug interactions are unlikely when they are concomitantly taken with prescription drugs that are CYP substrates. Although there were few clinical studies on potential CYP-mediated interactions produced by kava, present data suggest that kava supplements have the ability to inhibit CYP1A2 and CYP2E1 significantly. Therefore, caution should be taken when patients take kava with CYP1A2 or CYP2E1 substrate drugs as it may enhance their therapeutic and adverse effects. Despite the long use of traditional Chinese herbal medicines, little is known about the potential drug interactions with these herbs. Many popularly used Chinese medicines have been shown in vitro to significantly change the

  16. In vivo oxidative damage in rats is associated with barbiturate response but not other cytochrome P450 inducers.

    PubMed

    Dostalek, Miroslav; Brooks, Joshua D; Hardy, Klarissa D; Milne, Ginger L; Moore, Megan M; Sharma, Sameer; Morrow, Jason D; Guengerich, F Peter

    2007-12-01

    Previously published studies have shown that cytochrome P450 (P450) enzyme systems can produce reactive oxygen species and suggest roles of P450s in oxidative stress. However, most of the studies have been done in vitro, and the potential link between P450 induction and in vivo oxidative damage has not been rigorously explored with validated biomarkers. Male Sprague-Dawley rats were pretreated with typical P450 inducers (beta-naphthoflavone, phenobarbital (PB), Aroclor 1254, isoniazid, pregnenolone 16alpha-carbonitrile, and clofibrate) or the general P450 inhibitor 1-aminobenztriazole; induction of P4501A, -2B, -2E, -3A, and -4A subfamily enzymes was confirmed by immunoblotting and the suppression of P450 by 1-aminobenztriazole using spectral analysis. PB and Aroclor 1254 significantly enhanced malondialdehyde and H2O2 generation and NADPH oxidation in vitro and significantly enhanced formation in vivo, in both liver and plasma. Some of the other treatments changed in vitro parameters but none did in vivo. The PB-mediated increases in liver and plasma F2-isoprostanes could be ablated by 1-aminobenztriazole, implicating the PB-induced P450(s) in the F2-isoprostane elevation. The markers of in vivo oxidative stress were influenced mainly by PB and Aroclor 1254, indicative of an oxidative damage response only to barbiturate-type induction and probably related to 2B subfamily enzymes. These studies define the contribution of P450s to oxidative stress in vivo, in that the phenomenon is relatively restricted and most P450s do not contribute substantially.

  17. Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development

    PubMed Central

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo; Ansong, Charles; Anderson, Lindsey N.; Smith, Jordan N.; Corley, Richard A.

    2016-01-01

    Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography–mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage–dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics. PMID:27084891

  18. Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice

    PubMed Central

    Wang, Xiu Jun; Chamberlain, Mark; Vassieva, Olga; Henderson, Colin J.; Wolf, C. Roland

    2005-01-01

    Cytochrome P450 reductase is the unique electron donor for microsomal cytochrome P450s; these enzymes play a major role in the metabolism of endogenous and xenobiotic compounds. In mice with a liver-specific deletion of cytochrome P450 reductase, hepatic cytochrome P450 activity is ablated, with consequent changes in bile acid and lipid homoeostasis. In order to gain insights into the metabolic changes resulting from this phenotype, we have analysed changes in hepatic mRNA expression using microarray analysis and real-time PCR. In parallel with the perturbations in bile acid levels, changes in the expression of key enzymes involved in cholesterol and lipid homoeostasis were observed in hepatic cytochrome P450 reductase null mice. This was characterized by a reduced expression of Cyp7b1, and elevation of Cyp7a1 and Cyp8b1 expression. The levels of mRNAs for other cytochrome P450 genes, including Cyp2b10, Cyp2c29, Cyp3a11 and Cyp3a16, were increased, demonstrating that endogenous factors play a role in regulating the expression of these proteins and that the increases are due, at least in part, to altered levels of transcripts. In addition, levels of mRNAs encoding genes involved in glycolysis and lipid transport were also increased; the latter may provide an explanation for the increased hepatic lipid content observed in the hepatic null mice. Serum testosterone and oestradiol levels were lowered, accompanied by significantly decreased expression of Hsd3b2 (3β-hydroxy-Δ5-steroid dehydrogenase-2), Hsd3b5 (3β-hydroxy-Δ5-steroid dehydrogenase-5) and Hsd11b1 (11β-hydroxysteroid dehydrogenase type 1), key enzymes in steroid hormone metabolism. These microarray data provide important insights into the control of metabolic pathways by the cytochrome system. PMID:15717863

  19. Propiconazole increases reactive oxygen species levels in mouse hepatic cells in culture and in mouse liver by a cytochrome P450 enzyme mediated process

    EPA Science Inventory

    Propiconazole induces hepatocarcinomas and hepatoadenomas in mice and is a rat liver tumor promoter. Transcriptional, proteomic, metabolomic and biochemical studies of hepatic tissues from mice treated with propiconazole under the conditions of the chronic bioassay indicate that ...

  20. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

    SciTech Connect

    Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.; Gonzalez, F.J. ); Guzelian, P.S. )

    1990-06-01

    Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B{sub 1} to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B{sub 1} to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B{sub 1} to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B{sub 1} as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B{sub 1} activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B{sub 1} in human liver involves the contribution of multiple forms of P450.

  1. Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit.

    PubMed Central

    Bozak, K R; Yu, H; Sirevåg, R; Christoffersen, R E

    1990-01-01

    The ripening of avocado fruit is associated with the expression of a number of mRNAs concomitant with overt changes in texture and flavor. Two overlapping cDNAs for a mRNA that accumulates during ripening were identified. Sequence analysis of these two cDNAs revealed a polypeptide of 471 amino acids with characteristics of a typical P-450: an N-terminal hydrophobic membrane anchor, a conserved heme-binding domain in the C-terminal region, and patches of similarity to various P-450 family members. Further evidence that this polypeptide represents a cytochrome P-450 oxidase comes from the recent isolation and characterization of a cytochrome P-450 from ripe avocado mesocarp [O'Keefe, D. P. & Leto, K. J. (1989) Plant Physiol. 89, 1141-1149]. The N terminus of the predicted polypeptide in the cDNAs is identical to the N terminus of the purified avocado P-450. Gel blot analysis of RNA from fruit at various stages of ripening showed the accumulation of an 1800-nucleotide P-450 mRNA that hybridized to the P-450 cDNA. The P-450 protein predicted by the avocado cDNA sequence shares less than 40% positional identity with any known P-450 gene family. We propose therefore that it be placed in a separate family, P450LXXI, and that the corresponding gene from avocado be named cyp71A1. Images PMID:1692626

  2. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    SciTech Connect

    Kreamer, G.L.; Squibb, K.; Gioeli, D.; Garte, S.J.; Wirgin, I. )

    1991-06-01

    The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of {beta}-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.

  3. Inhibition of cytochrome P450 activity enhances the systemic availability of triclabendazole metabolites in sheep.

    PubMed

    Virkel, G; Lifschitz, A; Sallovitz, J; Ballent, M; Scarcella, S; Lanusse, C

    2009-02-01

    Understanding the disposition kinetics and the pattern of metabolism is critical to optimise the flukicidal activity of triclabendazole (TCBZ) in ruminants. TCBZ is metabolised by both flavin-monooxygenase (FMO) and cytochrome P450 (P450) in the liver. Interference with these metabolic pathways may be useful to increase the systemic availabilities of TCBZ metabolites, which may improve the efficacy against Fasciola hepatica. The plasma disposition of TCBZ metabolites was evaluated following TCBZ co-administration with FMO [methimazole (MTZ)] and P450 [piperonyl butoxyde (PB) and ketoconazole (KTZ)] inhibitors in sheep. Twenty (20) healthy Corriedale x Merino weaned female lambs were randomly allocated into four experimental groups. Animals of each group were treated as follow: Group A, TCBZ alone (5 mg/kg, IV route); Group B, TCBZ (5 mg/kg, IV) + MTZ (3 mg/kg, IV); Group C, TCBZ (5 mg/kg, IV) + PB (30 mg/kg, IV) and Group D, TCBZ (5 mg/kg, IV) + KTZ (10 mg/kg, orally). Blood samples were taken over 240 h post-treatment and analysed by HPLC. TCBZ sulphoxide and sulphone were the main metabolites recovered in plasma. MTZ did not affect TCBZ disposition kinetics. TCBZ sulphoxide Cmax values were significantly increased (P < 0.05) after the TCBZ + PB (62%) and TCBZ + KTZ (37%) treatments compared to those measured in the TCBZ alone treatment. TCBZ sulphoxide plasma AUCs were higher (P < 0.05) in the presence of both PB (99%) and KTZ (41%). Inhibition of TCBZ P450-mediated oxidation in the liver accounted for the increased systemic availability of its active metabolite TCBZ sulphoxide. This work contributes to the search of different strategies to improve the use of this flukicidal drug in ruminants.

  4. ISOLATION OF A CYTOCHROME P-450 STRUCTURAL GENE FROM SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

  5. [Cytochromes P450 and formation of reactive metabolites. Role in hepatotoxicity of drugs].

    PubMed

    Pessayre, D

    1993-01-01

    During the millennia of evolution, animals have been subjected to a relentless biological warfare mounted by the plants that they ingested. By duplication of an ancestral gene, divergent evolution of these 2 genes, and so forth, surviving animals have been endowed with multiple cytochromes P450s which can metabolize (and thus eliminate) a multitude of environmental liposoluble xenobiotics. A disadvantage of this system (fortunately limited by the concomitant installation of several protective systems) is that cytochrome P450 transforms some of these xenobiotics into chemically reactive metabolites. These free radicals or electrophilic metabolites attack tissue constituents, and may lead to mutation, cancer or tissue necrosis. Tissue necrosis affect mainly the liver, whose content in cytochrome P450 is particularly high. Indeed, reactive metabolites are usually extremely unstable, and react mainly in situ, in the same organ that forms them. When the formation of reactive metabolites is extensive, protective mechanisms are overwhelmed, extensive alterations of diverse hepatic constituents occur, and toxic hepatitis ensues. When the formation of reactive metabolites is moderate, severe toxic lesions do not occur. However, the covalent binding of reactive metabolites to hepatic proteins modifies the self of the subject. In some subjects, the presence of this modified self triggers immunization, and leads to immunoallergic hepatitis. The immune response may be directed either against protein (or peptide) epitopes modified by the presence of a reactive metabolite (reaction against modified self) and/or against normal, unmodified, epitopes of proteins (autoimmune reaction against the self, triggered by the modified self). Both metabolic factors, and the HLA phenotype, appear to modulate the likelihood of immunization.

  6. Food Polyphenol Apigenin Inhibits the Cytochrome P450 Monoxygenase Branch of the Arachidonic Acid Cascade.

    PubMed

    Steuck, Maryvonne; Hellhake, Stefan; Schebb, Nils Helge

    2016-11-30

    The product of cytochrome P450 monooxygenase (P450) ω-hydroxylation of arachidonic acid (AA), 20- hydroxyeicosatetraenoic acid (HETE), is a potent vasoconstrictor. Utilizing microsomes as well as individual CYP4 isoforms we demonstrate here that flavonoids can block 20-HETE formation. Apigenin inhibits CYP4F2 with an IC50 value of 4.6 μM and 20-HETE formation in human liver and kidney microsomes at 2.4-9.8 μM. Interestingly, the structurally similar naringenin shows no relevant effect on the formation of 20-HETE. Based on these in vitro data, it is impossible to evaluate if a relevant blockade of 20-HETE formation can result in humans from intake of polyphenols with the diet. However, the potency of apigenin is comparable to those of P450 inhibitors such as ketoconazole. Moreover, an IC50 value in the micromolar range is also described for the inhibition of CYP-mediated drug metabolism leading to food-drug interactions. The modulation of the arachidonic acid cascade by food polyphenols therefore warrants further investigation.

  7. Relevance of cytochrome P450s in plants: also one of Ron Estabrook's research interests.

    PubMed

    Shet, Manjunath S

    2007-01-01

    I worked with Dr. Ronald Estabrook for nearly 10 years at The University of Texas Southwestern Medical Center in Dallas, Texas. In Ron's lab, when I joined I was initially involved in the isolation, purification, and characterization of cytochrome P450s and NADPH-P450(c) reductase(s) from plants, which was his new exploratory project at the time. We developed methods for the isolation, solubilization, and purification of P450s and NADPH-P450(c) reductase from plant tissue microsomes. We carried out number of in vitro experiments to study the involvement P450s and NADPH-P450(c) reductase in the biosynthesis of number of phytoalexins. We successfully isolated, purified, and cloned NADPH-P450(c) reductase from etiolated mung bean (Vigna radiate) seedlings. In addition, a series of studies were undertaken to show that purified mung bean NADPH-P450(c) reductase was able to catalyze P450-supported reactions for mammalian and bacterial P450s. My stay in Ron's lab was very educational and productive. He provided the necessary support and led the way through the maze in different research projects in the lab, which allowed me to understand the roles of P450s in humans, animals, plants, and microorganisms. He liked to teach and discover new things everyday in the lab. He is a great scientist, as well as loving and caring mentor.

  8. Monooxygenation of small hydrocarbons catalyzed by bacterial cytochrome p450s.

    PubMed

    Shoji, Osami; Watanabe, Yoshihito

    2015-01-01

    Cytochrome P450s (P450s) catalyze the NAD(P)H/O2-dependent monooxygenation of less reactive organic molecules under mild conditions. The catalytic activity of bacterial P450s is very high compared with P450s isolated from animals and plants, and the substrate specificity of bacterial P450s is also very high. Accordingly, their catalytic activities toward nonnative substrates are generally low especially toward small hydrocarbons. However, mutagenesis approaches have been very successful for engineering bacterial P450s for the hydroxylation of small hydrocarbons. On the other hand, "decoy" molecules, whose structures are very similar to natural substrates, can be used to trick the substrate recognition of bacterial P450s, allowing the P450s to catalyze oxidation reactions of nonnative substrates without any substitution of amino acid residues in the presence of decoy molecules. Thus, the hydroxylation of small hydrocarbons such as ethane, propane, butane and benzene can be catalyzed by P450BM3, a long-alkyl-chain hydroxylase, using substrate misrecognition of P450s induced by decoy molecules. Furthermore, a number of H2O2-dependent bacterial P450s can catalyze the peroxygenation of a variety of nonnative substrates through a simple substrate-misrecognition trick, in which catalytic activities and enantioselectivity are dependent on the structure of decoy molecules.

  9. Expression of cytochrome P450 genes in CD34+ hematopoietic stem and progenitor cells.

    PubMed

    Soucek, Pavel; Anzenbacher, Pavel; Skoumalová, Ivana; Dvorák, Michal

    2005-10-01

    Expression of major cytochrome P450 forms (P450) was followed in preparation of purified hematopoietic CD34+ stem and progenitor cells. Levels of transcripts as well as mature proteins were traced by quantitative real-time polymerase chain reaction and by Northern and Western blotting. P450 1B1 and P450 2E1 proteins and respective mRNAs were found in all cases. On the other hand, no expression of P450 3A4, P450 3A7, and P450 2C9 was found. The results showed that expression of various P450 enzymes starts at different stages of cell differentiation. Both P450 forms found are known to be connected with cancer cells and with activation of procarcinogens (P450 1B1, polycyclic aromatic hydrocarbons; P450 2E1, nitrosamines, and solvents). Hence, cells at the early stage of differentiation already may be influenced by interaction with xenobiotics. This fact should also be taken into consideration when hematopoietic cell transplant therapy is applied.

  10. Genomewide annotation and comparative genomics of cytochrome P450 monooxygenases (P450s) in the polypore species Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora.

    PubMed

    Syed, Khajamohiddin; Nelson, David R; Riley, Robert; Yadav, Jagjit S

    2013-01-01

    Genomewide annotation of cytochrome P450 monooxygenases (P450s) in three white-rot species of the fungal order Polyporales, namely Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora, revealed a large contingent of P450 genes (P450ome) in their genomes. A total of 199 P450 genes in B. adusta and 209 P450 genes each in Ganoderma sp. and P. brevispora were identified. These P450omes were classified into families and subfamilies as follows: B. adusta (39 families, 86 subfamilies), Ganoderma sp. (41 families, 105 subfamilies) and P. brevispora (42 families, 111 subfamilies). Of note, the B. adusta genome lacked the CYP505 family (P450foxy), a group of P450-CPR fusion proteins. The three polypore species revealed differential enrichment of individual P450 families in their genomes. The largest CYP families in the three genomes were CYP5144 (67 P450s), CYP5359 (46 P450s) and CYP5344 (43 P450s) in B. adusta, Ganoderma sp. and P. brevispora, respectively. Our analyses showed that tandem gene duplications led to expansions in certain P450 families. An estimated 33% (72 P450s), 28% (55 P450s) and 23% (49 P450s) of P450ome genes were duplicated in P. brevispora, B. adusta and Ganoderma sp., respectively. Family-wise comparative analysis revealed that 22 CYP families are common across the three Polypore species. Comparative P450ome analysis with Ganoderma lucidum revealed the presence of 143 orthologs and 56 paralogs in Ganoderma sp. Multiple P450s were found near the characteristic biosynthetic genes for secondary metabolites, namely polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), terpene cyclase and terpene synthase in the three genomes, suggesting a likely role of these P450s in secondary metabolism in these Polyporales. Overall, the three species had a richer P450 diversity both in terms of the P450 genes and P450 subfamilies as compared to the model white-rot and brown-rot polypore species Phanerochaete chrysosporium and Postia placenta.

  11. Metabolism of methyl tert-butyl ether and other gasoline ethers by human liver microsomes and heterologously expressed human cytochromes P450: identification of CYP2A6 as a major catalyst.

    PubMed

    Hong, J Y; Wang, Y Y; Bondoc, F Y; Lee, M; Yang, C S; Hu, W Y; Pan, J

    1999-10-01

    To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Previously, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA) and that cytochrome P450 (CYP) enzymes play a critical role in the metabolism of MTBE. The present study demonstrates that human liver is also active in the oxidative metabolism of ETBE and TAME. A large interindividual variation in metabolizing these gasoline ethers was observed in 15 human liver microsomal samples. The microsomal activities in metabolizing MTBE, ETBE, and TAME were highly correlated among each other (r, 0.91-0. 96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the liver microsomes showed that the highest degree of correlation was with human CYP2A6 (r, 0. 90-0.95), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. Kinetic studies on MTBE metabolism with three human liver microsomes exhibited apparent Km values that ranged from 28 to 89 microM and the V(max) values from 215 to 783 pmol/min/mg, with similar catalytic efficiency values (7.7 to 8.8 microl/min/mg protein). Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that in human liver, CYP2A6 is the major enzyme responsible for the

  12. Cloning and expression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation.

    PubMed Central

    White, P C; New, M I; Dupont, B

    1984-01-01

    We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450C21). Serum from rabbits immunized with purified P-450C21 precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450C21 on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450C21 was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC X dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450C21 serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti-P-450C21 serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, pC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450C21. The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450C21 and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Images PMID:6609358

  13. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans.

    PubMed

    Danielson, P B

    2002-12-01

    Cytochrome p450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the p450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome p450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome p450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome p450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to p450 research. While human cytochrome p450 databases have swelled with a flood of new human sequence variants, however, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome p450 structure/function. This review examines the latest advances in our understanding of human cytochrome p450s.

  14. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

    PubMed Central

    Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

    2016-01-01

    Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

  15. Effect of a New Prokinetic Agent DA-9701 Formulated with Corydalis Tuber and Pharbitidis Semen on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    PubMed Central

    Ji, Hye Young; Liu, Kwang Hyeon; Jeong, Ji Hyeon; Lee, Dae-Young; Shim, Hyun Joo; Son, Miwon; Lee, Hye Suk

    2012-01-01

    DA-9701 is a new botanical drug composed of the extracts of Corydalis tuber and Pharbitidis semen, and it is used as an oral therapy for the treatment of functional dyspepsia in Korea. The inhibitory potentials of DA-9701 and its component herbs, Corydalis tuber and Pharbitidis semen, on the activities of seven major human cytochrome P450 (CYP) enzymes and four UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. DA-9701 and Corydalis tuber extract slightly inhibited UGT1A1-mediated etoposide glucuronidation, with 50% inhibitory concentration (IC50) values of 188 and 290 μg/mL, respectively. DA-9701 inhibited CYP2D6-catalyzed bufuralol 1′-hydroxylation with an inhibition constant (Ki) value of 6.3 μg/mL in a noncompetitive manner. Corydalis tuber extract competitively inhibited CYP2D6-mediated bufuralol 1′-hydroxylation, with a Ki value of 3.7 μg/mL, whereas Pharbitidis semen extract showed no inhibition. The volume in which the dose could be diluted to generate an IC50 equivalent concentration (volume per dose index) value of DA-9701 for inhibition of CYP2D6 activity was 1.16 L/dose, indicating that DA-9701 may not be a potent CYP2D6 inhibitor. Further clinical studies are warranted to evaluate the in vivo extent of the observed in vitro interactions. PMID:22548118

  16. Contribution of carboxylesterase and cytochrome P450 to the bioactivation and detoxification of isocarbophos and its enantiomers in human liver microsomes.

    PubMed

    Zhuang, Xiao-Mei; Wei, Xia; Tan, Yan; Xiao, Wei-Bin; Yang, Hai-Ying; Xie, Jian-Wei; Lu, Chuang; Li, Hua

    2014-07-01

    Organophosphorus pesticides are the most widely used pesticides in modern agricultural systems to ensure good harvests. Isocarbophos (ICP), with a potent acetylcholinesterase inhibitory effect is widely utilized to control a variety of leaf-eating and soil insects. However, the characteristics of the bioactivation and detoxification of ICP in humans remain unclear. In this study, the oxidative metabolism, esterase hydrolysis, and chiral inversion of ICP in human liver microsomes (HLMs) were investigated with the aid of a stereoselective LC/MS/MS method. The depletion of ICP in HLMs was faster in the absence of carboxylesterase inhibitor (BNPP) than in the presence of NADPH and BNPP, with t1/2 of 5.2 and 90 min, respectively. Carboxylesterase was found to be responsible for the hydrolysis of ICP, the major metabolic pathway. CYP3A4, CYP1A2, CYP2D6, CYP2C9, and CYP2C19 were all involved in the secondary metabolism pathway of desulfuration of ICP. Flavin-containing monooxygenase (FMO) did not contribute to the clearance of ICP. The hydrolysis and desulfuration of (±)ICP, (+)ICP, and (-)ICP in HLMs follow Michaelis-Menten kinetics. Individual enantiomers of ICP and its oxidative desulfuration metabolite isocarbophos oxon (ICPO) were found to be inhibitors of acetylcholinesterases at different extents. For example, (±)ICPO is more potent than ICP (IC50 0.031μM vs. 192μM), whereas (+)ICPO is more potent than (-)ICPO (IC50 0.017μM vs. 1.55μM). Given the finding of rapid hydrolysis of ICP and low abundance of oxidative metabolites presence in human liver, the current study highlights that human liver has a greater capacity for detoxification of ICP.

  17. Examination of metabolic pathways and identification of human liver cytochrome P450 isozymes responsible for the metabolism of barnidipine, a calcium channel blocker.

    PubMed

    Teramura, T; Fukunaga, Y; Van Hoogdalem, E J; Watanabe, T; Higuchi, S

    1997-09-01

    1. In a human liver microsomal system, barnidipine was converted into three primary metabolites, an N-debenzylated product (M-1), a hydrolyzed product of the benzyl-pyrrolidine ester (M-3) and an oxidized product of the dihydropyridine ring (M-8). 2. Involvement of CYP3A in the three primary metabolic pathways was revealed by the following studies: (a) inhibition of CYP3A, (b) a correlation study using 10 individual human liver microsomes and (c) cDNA-expression studies. The secondary metabolites, M-2 and M-4 (pyridine forms of M-1 and M-3), were most likely generated from M-8 but were unlikely from M-1 or M-3. Involvement of CYP3A in the secondary pathways of metabolism is also suggested. 3. The possibility of interactions between barnidipine and coadministered drugs was examined in vitro. The formation rate of the primary metabolites was little affected by warfarin, theophylline, phenytoin, diclofenac and amitriptyline at concentrations of 200 microM, but was inhibited by glibenclamide, simvastatin and cyclosporin A. IC50 for the latter drugs was estimated to be > 200, 200 and 20 microM respectively, which was roughly > 200, 6000 and 50 times higher than their respective therapeutic plasma levels, suggesting that interactions with cyclosporin A, a CYP3A inhibitor, are of possible clinical relevance.

  18. Cytochrome P450 dependent metabolism of the new designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP). In vivo studies in Wistar and Dark Agouti rats as well as in vitro studies in human liver microsomes.

    PubMed

    Staack, Roland F; Paul, Liane D; Springer, Dietmar; Kraemer, Thomas; Maurer, Hans H

    2004-01-15

    1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.

  19. Pharmacokinetics of Lipophilic Agents Following Preexposure: Non-Cytochrome P-450 Mediated Mechanisms

    DTIC Science & Technology

    1990-05-30

    FOLLOWING PREEXPOSUR G - AFOSR-87- 185 U’) NON -CYTOCHROME P-450 MEDIATED MECHAN’ICS PE - 61102F (NJ . AUTHOKS) PR - 2312 N TA - A5 Dr Lawrence R Curtis, Dr...Z39- I*. PHARMACOKINETICS OF LIPOPHILIC AGENTS FOLLOWING PREEXPOSURE: NON -CYTOCHROME P-450 MEDIATED MECHANISMS Air Force Grant No. 87-0185 Hillary M...amounts located in non -hepatic tissues. These studies showed that, despite the differences in PDR, the systems involved respond in a dose related manner

  20. Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats.

    PubMed

    Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

    2016-11-01

    The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases.

  1. Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats

    PubMed Central

    Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

    2016-01-01

    The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases. PMID:27882225

  2. Valence tautomerism in synthetic models of cytochrome P450

    PubMed Central

    Das, Pradip Kumar; Samanta, Subhra; McQuarters, Ashley B.; Lehnert, Nicolai

    2016-01-01

    CytP450s have a cysteine-bound heme cofactor that, in its as-isolated resting (oxidized) form, can be conclusively described as a ferric thiolate species. Unlike the native enzyme, most synthetic thiolate-bound ferric porphyrins are unstable in air unless the axial thiolate ligand is sterically protected. Spectroscopic investigations on a series of synthetic mimics of cytP450 indicate that a thiolate-bound ferric porphyrin coexists in organic solutions at room temperature (RT) with a thiyl-radical bound ferrous porphyrin, i.e., its valence tautomer. The ferric thiolate state is favored by greater enthalpy and is air stable. The ferrous thiyl state is favored by entropy, populates at RT, and degrades in air. These ground states can be reversibly interchanged at RT by the addition or removal of water to the apolar medium. It is concluded that hydrogen bonding and local electrostatics protect the resting oxidized cytP450 active site from degradation in air by stabilizing the ferric thiolate ground state in contrast to its synthetic analogs. PMID:27302948

  3. Expression of truncated forms of liver microsomal P450 cytochromes 2B4 and 2E1 in Escherichia coli: influence of NH2-terminal region on localization in cytosol and membranes.

    PubMed Central

    Pernecky, S J; Larson, J R; Philpot, R M; Coon, M J

    1993-01-01

    The currently accepted model for the membrane topology of microsomal cytochrome P450 is that of a largely cytoplasmic domain bound by only one or two transmembrane segments at the NH2 terminus. However, as we have reported previously, P450 2E1 lacking the hydrophobic NH2-terminal signal peptide, like the full-length protein, is located in the inner cell membrane when expressed in Escherichia coli and is active with typical substrates. In the present study, additional variants of alcohol-inducible P450 2E1 as well as truncated forms of phenobarbital-inducible P450 2B4 were similarly expressed to determine the influence of the NH2-terminal region on the membrane-binding properties. After deletion of S1 (the NH2-terminal hydrophobic segment), or both S1 and L1 (the following hydrophilic region, expected to be lumenal or cytosolic), one-third of the resulting P450 2B4 (delta 2-20) and 2B4 (delta 2-27) remained membrane bound. Furthermore, the idea that the first two hydrophobic segments are required for attachment by a hairpin loop is not supported by the finding that after deletion of the S1, L1, and S2 segments about half of the P450 2E1 (delta 3-48) remained membrane bound. Since Na2CO3 treatment of the membrane fraction had no significant effect, the findings are apparently not attributable to a loose attachment or occlusion of the truncated proteins. The replacement of neutral amino acids by positively charged residues in positions 3 and 8 of P450 2E1 (delta 3-29) changed the amount in the cytosol from 35% to 50%, and the deletion of residues 2-20 or 2-27 from P450 2B4, which resulted in positive charges occurring in the NH2-terminal region, changed the amount in the cytosol from 27% to 67%. We conclude that alterations in the NH2-terminal region can change the location of the cytochrome from largely membranous to largely cytosolic and that the first two hydrophobic segments are not uniquely involved in membrane attachment. PMID:8464872

  4. Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450.

    PubMed

    Waxman, D J; Pampori, N A; Ram, P A; Agrawal, A K; Shapiro, B H

    1991-08-01

    Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sex-dependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of the male-specific steroid 2 alpha (or 16 alpha)-hydroxylase P450, designated CYP2C11, was stimulated by GH at both physiological and nonphysiological pulse amplitudes, durations, and frequencies, provided that an interpulse interval of no detectable GH was maintained for at least 2.5 hr. This finding suggests that hepatocytes undergo an obligatory recovery period after stimulation by a GH pulse. This period may be required to reset a GH-activated intracellular signaling pathway or may relate to the short-term absence of GH receptors at the hepatocyte surface after a cycle of GH binding and receptor internalization. These requirements were distinguished from those necessary for the stimulation by GH of normal male growth rates in hypophysectomized rats, indicating that different GH responses and, perhaps, different GH-responsive tissues recognize distinct signaling elements in the sexually dimorphic patterns of circulating GH.

  5. Exploiting the versatility of human cytochrome P450 enzymes: the promise of blue roses from biotechnology.

    PubMed

    Gillam, E M; Guengerich, F P

    2001-12-01

    The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.

  6. Identification of a novel cytochrome P-450 gene from the white rot fungus Phanerochaete chrysosporium.

    PubMed Central

    Kullman, S W; Matsumura, F

    1997-01-01

    A gene fragment belonging to the cytochrome P-450 superfamily has been cloned and identified from stationary cultures of the filamentous fungus Phanerochaete chrysosporium by reverse transcriptase (RT)-PCR. A set of degenerate primers homologous to highly conserved regions of known cytochrome P-450 sequences were used for initial RT-PCRs. Individual PCR products were cloned, sequenced, and identified as those belonging to the cytochrome P-450 superfamily based on amino acid sequence homologies and the presence of the highly conserved heme binding region. The nucleotide sequence of a single cDNA clone indicated the presence of an open reading frame encoding a partial cytochrome P-450 protein of 208 amino acids. Comparisons of the deduced amino acid sequence of the partial protein to other known cytochrome P-450 sequences indicate that it is the first member of a new family of cytochrome P-450s, designated CYP63-1A. Northern blot analysis suggests that CYP63-1A is expressed under both nitrogen-rich and nitrogen-deficient culture conditions and thus not under the same regulatory constraints as the well-studied lignin and manganese peroxidases. Western blot analyses using antibodies raised to the heme binding region of CYP63-1A indicate that the protein has a molecular mass of approximately 44,000 Da. PMID:9212420

  7. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    SciTech Connect

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W. . Patuxent Wildlife Research Center); Woodin, B.R.; Stegeman, J.J. )

    1993-09-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene or phenobarbital. Compared to controls, 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p[prime]-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP1B) were significantly associated with total PCB burdens.

  8. Involvement of Cytochrome P450 in Pentachlorophenol Transformation in a White Rot Fungus Phanerochaete chrysosporium

    PubMed Central

    Ning, Daliang; Wang, Hui

    2012-01-01

    The occurrence of cytochrome P450 and P450-mediated pentachlorophenol oxidation in a white rot fungus Phanerochaete chrysosporium was demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (103±13 pmol P450 per mg protein in the microsomal fraction) by pentachlorophenol. The pentachlorophenol oxidation by the microsomal P450 was NADPH-dependent at a rate of 19.0±1.2 pmol min−1 (mg protein)−1, which led to formation of tetrachlorohydroquinone and was significantly inhibited by piperonyl butoxide (a P450 inhibitor). Tetrachlorohydroquinone was also found in the cultures, while the extracellular ligninases which were reported to be involved in tetrachlorohydroquinone formation were undetectable. The formation of tetrachlorohydroquinone was not detectable in the cultures added with either piperonyl butoxide or cycloheximide (an inhibitor of de novo protein synthesis). These results revealed the pentachlorophenol oxidation by induced P450 in the fungus, and it should be the first time that P450-mediated pentachlorophenol oxidation was demonstrated in a microorganism. Furthermore, the addition of the P450 inhibitor to the cultures led to obvious increase of pentachlorophenol, suggesting that the relationship between P450 and pentachlorophenol methylation is worthy of further research. PMID:23029295

  9. Hepatic cytochrome P450 activity, abundance, and expression throughout human development

    SciTech Connect

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo M.; Ansong, Charles; Anderson, Lindsey N.; Smith, Jordan N.; Corley, Richard A.; Wright, Aaron T.

    2016-07-01

    Cytochrome P450s are Phase I metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes can vary considerably throughout human development, especially when comparing fetal development to neonates, children, and adults. In an effort to develop a more comprehensive understanding of the ontogeny of P450 expression and activity we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. To quantify the functional activity of individual P450s we employ activity-based protein profiling, which uses modified mechanism-based inhibitors of P450s as chemical probes, in tandem with proteomic analyses to quantify activity. Our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. The results were used to distribute P450s into three general classes based upon developmental stage of expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that our ontogeny results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.

  10. Effects of green tea catechins on cytochrome P450 2B6, 2C8, 2C19, 2D6 and 3A activities in human liver and intestinal microsomes.

    PubMed

    Misaka, Shingen; Kawabe, Keisuke; Onoue, Satomi; Werba, José Pablo; Giroli, Monica; Tamaki, Sekihiro; Kan, Toshiyuki; Kimura, Junko; Watanabe, Hiroshi; Yamada, Shizuo

    2013-01-01

    The effects of green tea catechins on the main drug-metabolizing enzymatic system, cytochrome P450 (CYP), have not been fully elucidated. The objective of the present study was to evaluate the effects of green tea extract (GTE, total catechins 86.5%, w/w) and (-)-epigallocatechin-3-gallate (EGCG) on the activities of CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A in vitro, using pooled human liver and intestinal microsomes. Bupropion hydroxylation, amodiaquine N-deethylation, (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation were assessed in the presence or absence of various concentrations of GTE and EGCG to test their effects on CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A activities, respectively. Each metabolite was quantified using UPLC/ESI-MS, and the inhibition kinetics of GTE and EGCG on CYP enzymes was analyzed. In human liver microsomes, IC50 values of GTE were 5.9, 4.5, 48.7, 25.1 and 13.8 µg/mL, for CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A, respectively. ECGC also inhibited these CYP isoforms with properties similar to those of GTE, and produced competitive inhibitions against CYP2B6 and CYP2C8, and noncompetitive inhibition against CYP3A. In human intestinal microsomes, IC50 values of GTE and EGCG for CYP3A were 18.4 µg/mL and 31.1 µM, respectively. EGCG moderately inhibited CYP3A activity in a noncompetitive manner. These results suggest that green tea catechins cause clinically relevant interactions with substrates for CYP2B6 and CYP2C8 in addition to CYP3A.

  11. Main contribution of the cytochrome P450 isoenzyme 1A2 (CYP1A2) to N-demethylation and 5-sulfoxidation of the phenothiazine neuroleptic chlorpromazine in human liver--A comparison with other phenothiazines.

    PubMed

    Wójcikowski, Jacek; Boksa, Jan; Daniel, Władysława A

    2010-10-15

    The aim of the present study was to identify cytochrome P450 (CYP) isoenzymes involved in the 5-sulfoxidation, mono-N-demethylation and di-N-demethylation of the aliphatic-type phenothiazine neuroleptic chlorpromazine in human liver. Experiments were performed in vitro using cDNA-expressed human CYP isoforms (Supersomes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4), liver microsomes from different donors and CYP-selective inhibitors. The obtained results indicate that CYP1A2 is the only CYP isoform that catalyzes the mono-N-demethylation and di-N-demethylation of chlorpromazine (100%) and is the main isoform responsible for chlorpromazine 5-sulfoxidation (64%) at a therapeutic concentration of the drug (10 microM). CYP3A4 contributes to a lesser degree to chlorpromazine 5-sulfoxidation (34%). The role of CYP2B6, CYP2C19 and CYP2D6 in catalyzing of the latter reaction is negligible (0.1-2%). Similar results were obtained at a higher, non-therapeutic concentration of the drug (100 microM); however, the contribution of CYP1A2 to chlorpromazine mono-N-demethylation was noticeably lower (75%), mostly in favour of CYP2C19 and CYP3A4 (about 12% each). The obtained results indicate that the catalysis of chlorpromazine N-demethylation and 5-sulfoxidation in humans exhibits a stricter CYP1A2 preference compared to the previously tested phenothiazines (promazine, perazine, and thioridazine). Hence pharmacokinetic interactions involving chlorpromazine and CYP1A2 substrates and inhibitors are likely to occur. Considering strong dopaminergic D(2), noradrenergic alpha(1) and cholinergic M(1) receptor blocking properties of chlorpromazine and some of its metabolites, as well as their serious side effects, the obtained results may be of pharmacological and clinical importance.

  12. Genetic polymorphism of cytochrome P450 4F2, vitamin E level and histological response in adults and children with nonalcoholic fatty liver disease who participated in PIVENS and TONIC clinical trials.

    PubMed

    Athinarayanan, Shaminie; Wei, Rongrong; Zhang, Min; Bai, Shaochun; Traber, Maret G; Yates, Katherine; Cummings, Oscar W; Molleston, Jean; Liu, Wanqing; Chalasani, Naga

    2014-01-01

    Vitamin E improved liver histology in children and adults with NAFLD who participated in TONIC and PIVENS clinical trials, but with significant inter-individual variability in its efficacy. Cytochrome P450 4F2 (CYP4F2) is the major enzyme metabolizing Vit E, with two common genetic variants (V433M, rs2108622 and W12G, rs3093105) found to alter its activity. We investigated the relationship between CYP4F2 genotypes, α-tocopherol levels and histological improvement in these two trials. V433M and W12G variants were genotyped in TONIC (n = 155) and PIVENS (n = 213) DNA samples. The relationships between CYP4F2 genotypes, plasma α-tocopherol levels at baseline and weeks 48 (w48) and 96 (w96) and histological end points (overall improvement in liver histology and resolution of NASH) were investigated. As a result, the V433M genotype was significantly associated with baseline plasma α-tocopherol in the TONIC trial (p = 0.004), but not in PIVENS. Among those receiving Vit E treatment, CYP4F2 V433M genotype was associated with significantly decreased plasma α-tocopherol levels at w48 (p = 0.003 for PIVENS and p = 0.026 for TONIC) but not at w96. The w96 α-tocopherol level was significantly associated with resolution of NASH (p = 0.006) and overall histology improvement (p = 0.021)in the PIVENS, but not in the TONIC trial. There was no significant association between CYP4F2 genotypes and histological end points in either trial. Our study suggested the a moderate role of CYP4F2 polymorphisms in affecting the pharmacokinetics of Vit E as a therapeutic agent. In addition, there may be age-dependent relationship between CYP4F2 genetic variability and Vit E pharmacokinetics in NAFLD.

  13. Distinct organization of methylcholanthrene- and phenobarbital-inducible cytochrome P-450 genes in the rat.

    PubMed Central

    Sogawa, K; Gotoh, O; Kawajiri, K; Fujii-Kuriyama, Y

    1984-01-01

    The complete nucleotide sequence of the methylcholanthrene-inducible cytochrome P-450c gene was determined by sequence analysis of cloned genomic DNA and the sequence, consisting of 524 amino acids, of the protein was deduced therefrom. The gene for the cytochrome was approximately 6.0 kilobases long and was split into seven exons. Comparison of the gene with that of the phenobarbital-inducible cytochrome P-450e showed that the gene structures for the two types of cytochrome P-450 differ greatly; the location, number, and size of intervening sequences are very dissimilar. However, the sequence homology between the two types of cytochrome suggests that the two genes have evolved from a common ancestor. Images PMID:6089174

  14. Development of GC-MS based cytochrome P450 assay for the investigation of multi-herb interaction.

    PubMed

    Oh, Hyun-A; Lee, Hyunbeom; Kim, Donghak; Jung, Byung Hwa

    2017-02-15

    As drug interactions with cytochrome P450 enzymes become increasingly important in the field of drug discovery, a high-throughput screening method for analysing the effects of a drug is needed. We have developed a simple and rapid simultaneous analytical method using a cocktail approach for measuring the activities of seven cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). Human liver microsomes were used as a source for the seven cytochrome P450 enzymes, and a gas chromatography-mass spectrometry (GC-MS) was used for analysing their activities. Kinetic studies and inhibition assays of CYP enzymes were performed using known substrates and inhibitors for validating and comparing the reaction rates and time-dependent activities between methods using each substrate versus a method using a cocktail solution. The optimized cocktail method was successfully applied to evaluate the effects of the decoction of Socheongryong-tang (SCRT) on cytochrome P450 enzymes. Our cocktail method provides a simultaneous high-throughput activity assay using GC-MS for the first time. This method is applicable for analysing the drug interactions of various plant-derived mixtures.

  15. 4-Alkyl radical extrusion in the cytochrome P-450-catalyzed oxidation of 4-alkyl-1,4-dihydropyridines

    SciTech Connect

    Lee, J.S.; Jacobsen, N.E.; Ortiz de Montellano, P.R. )

    1988-10-04

    Rat liver microsomal cytochrome P-450 oxidizes the 4-methyl, 4-ethyl (DDEP), and 4-isopropyl derivatives of 3,5-bis(carbethoxy)-2,6-dimethyl-1,4,-dihydropyridine to mixtures of the corresponding 4-alkyl and 4-dealkyl pyridines. A fraction of the total microsomal enzyme is destroyed in the process. The 4-dealkyl to 4-alkyl pyridine metabolite ratio, the extent of cytochrome P-450 destruction, and the rate of spin-trapped radical accumulation are correlated in a linear inverse manner with the homolytic or heterolytic bond energies of the 4-alkyl groups of the 4-alkyl-1,4-dihydropyridines. No isotope effects are observed on the pyridine matabolite ratio, the destruction of cytochrome P-450, or the formation of ethyl radicals when (4-{sup 2}H)DDEP is used instead of DDEP. N-Methyl- and N-ethyl-DDEP undergo N-dealkylation rather than aromatization but N-phenyl-DDEP is oxidized to a mixture of the 4-ethyl and 4-deethyl N-phenylpyridinium metabolites. In contrast to the absence of an isotope effect in the oxidation of DDEP, the 4-deethyl to 4-ethyl N-phenylpyridinium metabolite ratio increases 6-fold when N-phenyl(4-{sup 2}H)DDEP is used. The results support the hypothesis that cytochrome P-450 catalyzes the oxidation of dihydropyridines to radical cations and show that the radical cations decay to nonradical products by multiple, substituent-dependent, mechanisms.

  16. Cumene hydroperoxide supported demethylation of N,N-dimethylaniline by cytochrome P-450 from adrenal cortex mitochondria.

    PubMed

    Akhrem, A A; Khatyleva SYu; Shkumatov, V M; Chashchin, V L; Kiselev, P A

    1982-01-01

    The interaction of highly purified cytochrome P-450 from bovine adrenal cortex mitochondria (cytochrome P-450scc) with N,N-dimethylaniline (DMA), aniline, N-dimethylcyclohexylamine and cumene hydroperoxide (CHP) has been investigated. The formation of complexes between cytochrome P-450scc and the above listed compounds could be demonstrated. The reaction of oxidative demethylation of DMA by cumene hydroperoxide involving cytochrome P-450scc has been carried out at 37 degrees C; the mechanism of this process is discussed. Incubation of cytochrome P-450scc with negatively charged phospholipids, phosphatidylglycerol (PG), and phosphatidylinosite (PI) exerts an inhibiting effect on the reaction of oxidative demethylation. The interaction of cytochrome P-450scc with CHP is accompanied by hemoprotein destruction in a complex biphasic way. The process of oxidative demethylation of DMA in the system of cytochrome P-450scc-CHP has been concluded to have a predominantly radical character.

  17. A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity.

    PubMed

    Zvelebil, M J; Wolf, C R; Sternberg, M J

    1991-02-01

    A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.

  18. The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1)

    PubMed Central

    Yoshimoto, Francis K.; Auchus, Richard J.

    2014-01-01

    The steroid hydroxylation and carbon-carbon bond cleavage activities of cytochrome P450 17A1 (CYP17A1) are responsible for the production of glucocorticoids and androgens, respectively. The inhibition of androgen synthesis is an important strategy to treat androgen-dependent prostate cancer. We discuss the different enzymatic activities towards the various substrates of CYP17A1, demonstrating its promiscuity. Additionally, a novel interhelical interaction is proposed between the F-G loop and the B′-helix to explain the 16α-hydroxylase activity of human CYP17A1 with progesterone as the substrate. The techniques used by biochemists to study this important enzyme are also summarized. PMID:25482340

  19. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu-g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than five-fold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O-dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p, p'-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

  20. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O- dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black- crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross- reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p'-DDE were detected in embryos from Cat Island. Cytochrome P450- associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

  1. Simultaneous pharmacokinetics evaluation of human cytochrome P450 probes, caffeine, warfarin, omeprazole, metoprolol and midazolam, in common marmosets (Callithrix jacchus).

    PubMed

    Uehara, Shotaro; Inoue, Takashi; Utoh, Masahiro; Toda, Akiko; Shimizu, Makiko; Uno, Yasuhiro; Sasaki, Erika; Yamazaki, Hiroshi

    2016-01-01

    1. Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) composite, after single intravenous and oral administrations at doses of 0.20 and 1.0 mg kg(-1), respectively, to four male common marmosets were investigated. 2. The plasma concentrations of caffeine and warfarin decreased slowly in a monophasic manner but those of omeprazole, metoprolol and midazolam decreased extensively after intravenous and oral administrations, in a manner that approximated those as reported for pharmacokinetics in humans. 3. Bioavailabilities were ∼100% for caffeine and warfarin, but <25% for omeprazole and metoprolol. Bioavailability of midazolam was 4% in marmosets, presumably because of contribution of marmoset P450 3A4 expressed in small intestine and liver, with a high catalytic efficiency for midazolam 1'-hydroxylation as evident in the recombinant system. 4. These results suggest that common marmosets, despite their rapid clearance of some human P450 probe substrates, could be an experimental model for humans and that marmoset P450s have functional characteristics that differ from those of human and/or cynomolgus monkey P450s in some aspects, indicating their importance in modeling in P450-dependent drug metabolism studies in marmosets and of further studies.

  2. HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Fletke, Kyle J.

    2007-01-01

    Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among…

  3. Diversity in mechanisms of substrate oxidation by cytochrome P450 2D6. Lack of an allosteric role of NADPH-cytochrome P450 reductase in catalytic regioselectivity.

    PubMed

    Hanna, I H; Krauser, J A; Cai, H; Kim, M S; Guengerich, F P

    2001-10-26

    Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Differences in the regioselectivity of oxidation products formed in systems containing NADPH-P450 reductase/NADPH and the model oxidant cumene hydroperoxide have been proposed by others to be due to an allosteric influence of the reductase on P450 2D6 (Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L.-Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. K. (1997) Biochemistry 36, 4461-4470). We examined the differences in the formation of oxidation products of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, metoprolol, and bufuralol between reductase-, cumene hydroperoxide-, and iodosylbenzene-supported systems. Catalytic regioselectivity was not influenced by the presence of the reductase in any of the systems supported by model oxidants, ruling out allosteric influences. The presence of the reductase had little effect on the affinity of P450 2D6 for any of these three substrates. The addition of the reaction remnants of the model oxidants (cumyl alcohol and iodobenzene) to the reductase-supported system did not affect reaction patterns, arguing against steric influences of these products on catalytic regioselectivity. Label from H(2)18O was quantitatively incorporated into 1'-hydroxybufuralol in the iodosylbenzene- but not in the reductase- or cumene hydroperoxide-supported reactions. We conclude that the P450 systems utilizing NADPH-P450 reductase, cumene hydroperoxide, and iodosylbenzene use similar but distinct chemical mechanisms. These differences are the basis for the variable product distributions, not an allosteric influence of the reductase.

  4. Evaluation of cytochrome P450{sub BS{beta}} reactivity against polycyclic aromatic hydrocarbons and drugs

    SciTech Connect

    Torres, Eduardo; Hayen, Heiko; Niemeyer, Christof M.; E-mail: christof.niemeyer@uni-dortmund.de

    2007-03-30

    The oxidation of 10 polycyclic aromatic hydrocarbons (PAH) by cytochrome P450{sub BS{beta}} using three different electron acceptors is reported. Three PAH were found to be substrates for the oxidation by P450{sub BS{beta}}, namely anthracene, 9-methyl-anthracene and azulene. The respective oxidation products were identified by reversed-phase high-performance liquid chromatography coupled to electrospray ionization-mass spectrometry. In addition, 10 drug-like compounds were investigated for their effects on the catalytic activity of P450{sub BS{beta}} by carrying out inhibition studies. The stability of P450{sub BS{beta}} against hydrogen peroxide, cumene, and ter-butyl hydroperoxide was determined. Overall, the results of this study suggested that the P450{sub BS{beta}} enzyme represents a powerful catalyst in terms of the catalytic activity and operational stability.

  5. Effects of 6-paradol, an unsaturated ketone from gingers, on cytochrome P450-mediated drug metabolism.

    PubMed

    Kim, Hyeong Jun; Kim, In Sook; Rehman, Shaheed Ur; Ha, Sang Keun; Nakamura, Katsunori; Yoo, Hye Hyun

    2017-02-20

    Paradols are unsaturated ketones produced by biotransformation of shogaols in gingers. Among them, 6-paradol has been investigated as a new drug candidate due to its anti-inflammatory, apoptotic, and neuroprotective activities. In this study, the inhibitory effects of 6-paradol on the activities of cytochrome P450 (CYP) enzymes were investigated with human liver microsomes and recombinant CYP isozymes. 6-Paradol showed concentration-dependent inhibitory effects on CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 isozymes, with IC50 values ranging from 3.8 to 21.4µM in recombinant CYP isozymes. However, the inhibition was not potentiated following pre-incubation, indicating that 6-paradol is not a mechanism-based inhibitor. These results suggest that pharmacokinetic drug-drug interactions might occur with 6-paradol, which must be considered in the process of new drug development.

  6. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2.

    PubMed

    Reed, James R; dela Cruz, Albert Leo N; Lomnicki, Slawo M; Backes, Wayne L

    2015-12-01

    Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductase and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2-CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2.

  7. Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

    SciTech Connect

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle; Totah, Rheem A.

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k{sub cat} {approx} 25 min{sup -1}). Black-Right-Pointing-Pointer Reduction is a direct hydride transfer from R-NADP{sup 2}H to the carbonyl moiety. Black-Right-Pointing-Pointer P450 domain variants enhance reduction through potential allosteric/redox interactions. Black-Right-Pointing-Pointer Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k{sub cat} of {approx}25 min{sup -1} was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP{sup 2}H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP{sup 2}H but not D{sub 2}O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.

  8. Natural variation in the expression of cytochrome P-450 and dimethylnitrosamine demethylase in Drosophila

    SciTech Connect

    Waters, L.C.; Simms, S.I.; Nix, C.E.

    1984-09-28

    Electrophoresis of Drosophila microsomes resolves two major heme-containing protein bands with apparent molecular weights of 59,290 (band a) and 55,750 (band b). The hemoproteins in these two bands can account for most of the cytochrome P-450 in the organism. Band a is present in all strains examined: band b is not. Dimethylnitrosamine demethylase, a P-450 enzyme, is a component of band b. 22 references, 2 figures, 1 table.

  9. Toxaphene detoxification and acclimation in Daphnia magna: do cytochrome P-450 enzymes play a role?

    PubMed

    Kashian, Donna R

    2004-01-01

    Toxaphene is a persistent environmental contaminant that has been shown to alter male production in Daphnia magna and to induce P-450 activity in mammals. Cytochrome P-450-mediated metabolism may lead to xenobiotic detoxification resulting in acclimation. To determine if D. magna acclimate to toxaphene via P-450 pathways, chronic and acute toxicity tests were conducted with D. magna exposed to toxaphene in the presence and absence of piperonyl butoxide (PBO), an inhibitor of cytochrome P-450 enzymes. Toxaphene exposure increased male production in acute but not chronic assays, indicating that D. magna may acclimate to chronic toxaphene exposure. Upon co-administration of toxaphene and PBO in chronic tests, D. magna exhibited a decline in growth rate, fecundity and survival. The observed toxaphene acclimation in chronic tests, along with its increased toxicity in the presence of a P-450 suppressor, suggests that P-450 enzymes may contribute to detoxification and subsequent acclimation of D. magna to chronic toxaphene exposure. Additional chronic toxicity tests indicated that toxaphene acclimation occurs between 7 and 12 days following initial exposure, at which time sex determination is no longer affected. Thus, sublethal toxaphene toxicity effects such as reproductive impairments may be detectable with acute but not chronic tests, potentially due to the upregulation of P-450 isozymes.

  10. High-throughput fluorescence assay of cytochrome P450 3A4

    PubMed Central

    Cheng, Qian; Sohl, Christal D; Guengerich, F Peter

    2013-01-01

    Cytochrome P450 mono-oxygenases (P450s) are the principal enzymes involved in the oxidative metabolism of drugs and other xenobiotics. In this protocol, we describe a fluorescence-based, high-throughput assay for measuring the activity of P450 3A4, one of the key enzymes involved in drug metabolism. The assay involves the oxidative debenzylation of a substituted coumarin, yielding an increase in fluorescence on reaction. The entire procedure can be accomplished in 1 h or less. PMID:19661996

  11. Cytochrome P450 Family 1 Inhibitors and Structure-Activity Relationships

    PubMed Central

    Liu, Jiawang; Sridhar, Jayalakshmi; Foroozesh, Maryam

    2014-01-01

    With the widespread use of O-alkoxyresorufin dealkylation assays since the 1990’s, thousands of inhibitors of cytochrome P450 family 1 enzymes (P450s 1A1, 1A2, and 1B1) have been identified and studied. Generally, planar polycyclic molecules such as polycyclic aromatic hydrocarbons, stilbenoids, and flavonoids are considered to potentially be effective inhibitors of these enzymes. However, the details of structure-activity relationships and selectivity of these inhibitors are still ambiguous. In this review, we thoroughly discuss the selectivity of many representative P450 family 1 inhibitors reported in the past 20 years through a meta-analysis. PMID:24287985

  12. Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1

    PubMed Central

    Wu, Jun; Chen, Ruohong; Zhang, Caihui; Li, Kangbai; Xu, Weiying; Wang, Lijuan; Chen, Qingmei; Mu, Peiqiang; Jiang, Jun; Wen, Jikai; Deng, Yiqun

    2016-01-01

    Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1. PMID:27626447

  13. Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter-system extrapolation factors.

    PubMed

    Crewe, H K; Barter, Z E; Yeo, K Rowland; Rostami-Hodjegan, A

    2011-09-01

    The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.

  14. Cytochrome P450 of wood-rotting basidiomycetes and biotechnological applications.

    PubMed

    Ichinose, Hirofumi

    2013-01-01

    Wood-rotting basidiomycetes possess superior metabolic functions to degrade woody biomass, and these activities are indispensable for the carbon cycle of the biosphere. As well as basic studies of the biochemistry of basidiomycetes, many researchers have been focusing on utilizing basidiomycetes and/or their enzymes in the biotechnology sector; therefore, the unique activities of their extracellular and intracellular enzymes have been widely demonstrated. A rich history of applied study has established that basidiomycetes are capable of metabolizing a series of endogeneous and exogeneous compounds using cytochrome P450s (P450s). Recently, whole genome sequence analyses have revealed large-scale divergences in basidiomycetous P450s. The tremendous variation in P450s implies that basidiomycetes have vigorously diversified monooxygenase functions to acquire metabolic adaptations such as lignin degradation, secondary metabolite production, and xenobiotics detoxification. However, fungal P450s discovered from genome projects are often categorized into novel families and subfamilies, making it difficult to predict catalytic functions by sequence comparison. Experimental screening therefore remains essential to elucidate the catalytic potential of individual P450s, even in this postgenomic era. This paper archives the known metabolic capabilities of basidiomycetes, focusing on their P450s, outlines the molecular diversity of basidiomycetous P450s, and introduces new functions revealed by functionomic studies using a recently developed, rapid, functional screening system.

  15. Cancer Activation and Polymorphisms of Human Cytochrome P450 1B1

    PubMed Central

    Chun, Young-Jin; Kim, Donghak

    2016-01-01

    Human cytochrome P450 enzymes (P450s, CYPs) are major oxidative catalysts that metabolize various xenobiotic and endogenous compounds. Many carcinogens induce cancer only after metabolic activation and P450 enzymes play an important role in this phenomenon. P450 1B1 mediates bioactivation of many procarcinogenic chemicals and carcinogenic estrogen. It catalyzes the oxidation reaction of polycyclic aromatic carbons, heterocyclic and aromatic amines, and the 4-hydroxylation reaction of 17β-estradiol. Enhanced expression of P450 1B1 promotes cancer cell proliferation and metastasis. There are at least 25 polymorphic variants of P450 1B1 and some of these have been reported to be associated with eye diseases. In addition, P450 1B1 polymorphisms can greatly affect the metabolic activation of many procarcinogenic compounds. It is necessary to understand the relationship between metabolic activation of such substances and P450 1B1 polymorphisms in order to develop rational strategies for the prevention of its toxic effect on human health. PMID:27123158

  16. Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis

    PubMed Central

    2016-01-01

    Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products. PMID:27119279

  17. Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803.

    PubMed

    Wlodarczyk, Artur; Gnanasekaran, Thiyagarajan; Nielsen, Agnieszka Zygadlo; Zulu, Nodumo Nokolunga; Mellor, Silas Busck; Luckner, Manja; Thøfner, Jens Frederik Bang; Olsen, Carl Erik; Mottawie, Mohammed Saddik; Burow, Meike; Pribil, Mathias; Feussner, Ivo; Møller, Birger Lindberg; Jensen, Poul Erik

    2016-01-01

    Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66mg/L of p-hydroxyphenylacetaldoxime and 5mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.

  18. Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

    PubMed

    Ponnamperuma, K; Croteau, R

    1996-05-01

    Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

  19. 21-Aminosteroids prevent the down-regulation of hepatic cytochrome P450 induced by hypoxia and inflammation in conscious rabbits

    PubMed Central

    Galal, Ahmed; du Souich, Patrick

    1999-01-01

    This study was conducted to assess whether a 21-aminosteroid, U74389G, could prevent the down-regulation of hepatic cytochrome P450 (P450) induced by acute moderate hypoxia or an inflammatory reaction.The rabbits of two groups (n=6 per group) were subjected to acute moderate hypoxia (PaO2≈35 mmHg), one pre-treated with U74389G (3 mg kg−1 i.v. every 6 h, for 48 h). The rabbits of two other groups received 5 ml of turpentine s.c., one of them being pre-treated with U74389G (3 mg kg−1 i.v. every 6 h, for 72 h). The kinetics of theophylline (2.5 mg kg−1) were assessed to evaluate the activity of the P450. Once the rabbits were sacrificed, the P450 content and the amount of thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, were estimated in the liver.Compared with control rabbits, hypoxia and inflammation increased theophylline plasma concentrations, as a result of a decrease in theophylline systemic clearance (P<0.05). Both experimental conditions reduced hepatic content of P450 by 40–50% (P<0.05) and increased the amount of hepatic TBARS by around 50% (P<0.05). Pre-treatment with U74389G prevented the hypoxia- and inflammation-induced decrease in theophylline systemic clearance, the down-regulation of hepatic P450, and the increase in liver TBARS.It is concluded that in the rabbit, U74389G prevents hepatic P450 depression produced by acute moderate hypoxia and a turpentine-induced inflammatory reaction, possibly by eliciting a radical quenching antioxidant activity. PMID:10510447

  20. Key Residues Controlling Phenacetin Metabolism By Human Cytochrome P450 2A Enzymes

    SciTech Connect

    DeVore, N.M.; Smith, B.D.; Urban, M.J.; Scott, E.E.

    2009-05-14

    Although the human lung cytochrome P450 2A13 (CYP2A13) and its liver counterpart cytochrome P450 2A6 (CYP2A6) are 94% identical in amino acid sequence, they metabolize a number of substrates with substantially different efficiencies. To determine differences in binding for a diverse set of cytochrome P450 2A ligands, we have measured the spectral binding affinities (K{sub D}) for nicotine, phenethyl isothiocyanate (PEITC), coumarin, 2{prime}-methoxyacetophenone (MAP), and 8-methoxypsoralen. The differences in the K{sub D} values for CYP2A6 versus CYP2A13 ranged from 74-fold for 2{prime}-methoxyacetophenone to 1.1-fold for coumarin, with CYP2A13 demonstrating the higher affinity. To identify active site amino acids responsible for the differences in binding of MAP, PEITC, and coumarin, 10 CYP2A13 mutant proteins were generated in which individual amino acids from the CYP2A6 active site were substituted into CYP2A13 at the corresponding position. Titrations revealed that substitutions at positions 208, 300, and 301 individually had the largest effects on ligand binding. The collective relevance of these amino acids to differential ligand selectivity was verified by evaluating binding to CYP2A6 mutant enzymes that incorporate several of the CYP2A13 amino acids at these positions. Inclusion of four CYP2A13 amino acids resulted in a CYP2A6 mutant protein (I208S/I300F/G301A/S369G) with binding affinities for MAP and PEITC much more similar to those observed for CYP2A13 than to those for CYP2A6 without altering coumarin binding. The structure-based quantitative structure-activity relationship analysis using COMBINE successfully modeled the observed mutant-ligand trends and emphasized steric roles for active site residues including four substituted amino acids and an adjacent conserved Leu{sup 370}.

  1. A molecular model for the interaction between vorozole and other non-steroidal inhibitors and human cytochrome P450 19 (P450 aromatase).

    PubMed

    Koymans, L M; Moereels, H; Vanden Bossche, H

    1995-06-01

    In a previous study (Vanden Bossche et al., Breast Cancer Res. Treat. 30 (1994) 43) the interaction between (+)-S-vorozole and the I-helix of cytochrome P450 19 (P450 aromatase) has been reported. In the present study we extended the "I-helix model" by incorporating the C-terminus of P450 aromatase. The crystal structures of P450 101 (P450 cam), 102 (P450 BM-3) and 108 (P450 terp) reveal that the C-terminus is structurally conserved and forms part of their respective substrate binding pocket. Furthermore, the present study is extended to the interaction between P450 aromatase and its natural substrate androstenedione and the non-steroidal inhibitors (-)-R-vorozole, (-)-S-fadrozole, R-liarozole and (-)-R-aminoglutethimide. It is found that (+)-S-vorozole, (-)-S-fadrozole and R-liarozole bind in a comparable way to P450 aromatase and interact with both the I-helix (Glu302 and Asp309) and C-terminus (Ser478 and His480). The weak activity of (-)-R-aminoglutethimide might be attributed to a lack of interaction with the C-terminus.

  2. Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis.

    PubMed

    Domanski, T L; Halpert, J R

    2001-06-01

    Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

  3. The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

    SciTech Connect

    Abdulla, Dalya; Goralski, Kerry B.; Renton, Kenneth W. . E-mail: Ken.Renton@dal.ca

    2006-10-01

    It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo, an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 {mu}g of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.

  4. Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition

    SciTech Connect

    Ishihara, Yasuhiro; Shiba, Dai; Shimamoto, Norio . E-mail: n-shimamoto@kph.bunri-u.ac.jp

    2006-07-15

    Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as {alpha}-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.

  5. Updates on cytochrome P450-mediated cardiovascular drug interactions.

    PubMed

    Cheng, Judy W M; Frishman, William H; Aronow, Wilbert S

    2009-01-01

    Cytochrome P (CYP) 450 is a superfamily of hemoproteins that play an important role in the metabolism of steroid hormones, fatty acids, and many medications. Many agents used for management of cardiovascular diseases are substrates, inhibitors, or inducers of CYP450 enzymes.When two agents that are substrates, inhibitors, or inducers of CYP450 are administered together, drug interactions with significant clinical consequences may occur. This review discusses CYP450-mediated cardiovascular drug interactions as well as noncardiovascular drug interactions that produced significant cardiovascular side effects. The principles in predicting drug interactions are also discussed.

  6. Cytochrome P450 CYP1B1 activity in renal cell carcinoma.

    PubMed

    McFadyen, M C E; Melvin, W T; Murray, G I

    2004-08-31

    Renal cell carcinoma (RCC) is the most common malignancy of the kidney and has a poor prognosis due to its late presentation and resistance to current anticancer drugs. One mechanism of drug resistance, which is potentially amenable to therapeutic intervention, is based on studies in our laboratory. CYP1B1 is a cytochrome P450 enzyme overexpressed in a variety of malignant tumours. Our studies are now elucidating a functional role for CYP1B1 in drug resistance. Cytochrome P450 reductase (P450R) is required for optimal metabolic activity of CYP1B1. Both CYP1B1 and P450R can catalyse the biotransformation of anticancer drugs at the site of the tumour. In this investigation, we determined the expression of CYP1B1 and P450R in samples of normal kidney and RCC (11 paired normal and tumour and a further 15 tumour samples). The O-deethylation of ethoxyresorufin to resorufin was used to measure CYP1B1 activity in RCC. Cytochrome P450 reductase activity was determined by following the reduction of cytochrome c at 550 nm. The key finding of this study was the presence of active CYP1B1 in 70% of RCC. Coincubation with the CYP1B1 inhibitor alpha-naphthoflavone (10 nM) inhibited this activity. No corresponding CYP1B1 activity was detected in any of the normal tissue examined (n=11). Measurable levels of active P450R were determined in all normal (n=11) and tumour samples (n=26). The presence of detectable CYP1B1, which is capable of metabolising anticancer drugs in tumour cells, highlights a novel target for therapeutic intervention.

  7. Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450

    SciTech Connect

    Marinello, A.J.; Bansal, S.K.; Paul, B.; Koser, P.L.; Love, J.; Struck, R.F.; Gurtoo, H.L.

    1984-10-01

    The hepatic cytochrome P-450-mediated metabolism and metabolic activation of (chloroethyl-3H)cyclophosphamide (( chloroethyl-3H)CP) and (4-14C)cyclophosphamide (( 4-14C)CP) were investigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of (14C)acrolein, a metabolite of (4-14C)CP, were also investigated. The metabolism of (chloroethyl-3H)CP and (4-14C)CP to polar metabolites was found to depend on the presence of NADPH and showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reductase. Km and Vmax values were essentially similar. The patterns of inhibition by microsomal mixed-function oxidase inhibitors, anti-cytochrome P-450 antibody, and heat denaturation of the cytochrome P-450 were essentially similar, with subtle differences between (4-14C)CP and (chloroethyl-3H)CP metabolism. The in vitro metabolic activation of CP in the reconstituted system demonstrated predominant binding of (chloroethyl-3H)CP to nucleic acids and almost exclusive binding of (4-14C)CP to proteins. Gel electrophoresis-fluorography of the proteins in the reconstituted system treated with (4-14C)CP demonstrated localization of the 14C label in the cytochrome P-450 region. To examine this association further, hepatic microsomes were modified with (14C)acrolein in the presence and the absence of NADPH. The results confirmed covalent association between (14C)acrolein and cytochrome P-450 in the microsomes and also demonstrated further metabolism of (14C)acrolein, apparently to an epoxide, which is capable of binding covalently to proteins. The results of these investigations not only confirm the significance of primary metabolism but also emphasize the potential role of the secondary metabolism of cyclophosphamide in some of its toxic manifestations.

  8. Appetite suppressant drugs as inhibitors of human cytochromes P450: in vitro inhibition of P450-2D6 by D- and L-fenfluramine, but not phentermine.

    PubMed

    von Moltke, L L; Greenblatt, D J; Ciraulo, D A; Grassi, J M; Granda, B W; Duan, S X; Harmatz, J S; Shader, R I

    1998-08-01

    The activity of D-fenfluramine, L-fenfluramine, and phentermine as inhibitors of five human cytochromes P450 was evaluated using human liver microsomes in vitro. All three compounds produced negligible inhibition of P450-1A2, -2C9, -2E1, and -3A. Phentermine also did not inhibit P450-2D6. However, D- and L-fenfluramine significantly inhibited P450-2D6 activity as measured by dextromethorphan O-demethylation, with mean 50% inhibitory concentrations (15.1 microM) within one order of magnitude of that for fluoxetine (2.7 microM). Findings from the in vitro assay are consistent with clinical studies showing significant inhibition of desipramine clearance by coadministration of fenfluramine.

  9. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 FAMILY

    EPA Science Inventory

    The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures ...

  10. Third international symposium: Cytochrome P450 biodiversity. Final report, January 1, 1995--December 31, 1995

    SciTech Connect

    Loper, J.C.

    1997-03-01

    The Symposium was held on October 8-12, 1995 at the Marine Biological Laboratory in Woods Hole Massachusetts. Other international symposia promote cytochrome P450 research but have a primary focus on mammalian systems. This symposium is exclusively devoted to research in other organisms, and major topics reflect the distribution and dominance of non-mammalian species in the biosphere. The five sessions focused on basic mechanism, regulation, biodiversity, host-parasite interactions, and practical applications. 170 Scientists contributed 38 oral presentations and 91 posters, with a truly international composition of the symposium. Practical applications were a recurring feature, linking reports on mechanism and regulation to studies on the engineering of substrate specificity, microorganisms to degrade halogenated hydrocarbons and herbicides, and the production of in vitro P450 electrochemical bioreactors. At the time of the symposium there were 477 cytochrome P450 sequences in the database. Expansion of the known plant P450 genes was reported, with 20 new plant P450 families added in the last 3 years. Of these only 5 families have a physiological function associated with them. A growing number of identified invertebrate P450s was documented, where in insects, the forms identified are primarily involved in inducible xenobiotic metabolism and detoxification of toxic plant substances.

  11. Plant cytochrome P450s: nomenclature and involvement in natural product biosynthesis.

    PubMed

    Rasool, Saiema; Mohamed, Rozi

    2016-09-01

    Cytochrome P450s constitute the largest family of enzymatic proteins in plants acting on various endogenous and xenobiotic molecules. They are monooxygenases that insert one oxygen atom into inert hydrophobic molecules to make them more reactive and hydro-soluble. Besides for physiological functions, the extremely versatile cytochrome P450 biocatalysts are highly demanded in the fields of biotechnology, medicine, and phytoremediation. The nature of reactions catalyzed by P450s is irreversible, which makes these enzymes attractions in the evolution of plant metabolic pathways. P450s are prime targets in metabolic engineering approaches for improving plant defense against insects and pathogens and for production of secondary metabolites such as the anti-neoplastic drugs taxol or indole alkaloids. The emerging examples of P450 involvement in natural product synthesis in traditional medicinal plant species are becoming increasingly interesting, as they provide new alternatives to modern medicines. In view of the divergent roles of P450s, we review their classification and nomenclature, functions and evolution, role in biosynthesis of secondary metabolites, and use as tools in pharmacology.

  12. Characterization of maize cytochrome P450 monooxygenases induced in response to safeners and bacterial pathogens.

    PubMed

    Persans, M W; Wang, J; Schuler, M A

    2001-02-01

    Plants use a diverse array of cytochrome P450 monooxygenases in their biosynthetic and detoxification pathways. To determine the extent to which various maize P450s are induced in response to chemical inducers, such as naphthalic anhydride (NA), triasulfuron (T), phenobarbital, and bacterial pathogens (Erwinia stuartii, Acidovorax avenae), we have analyzed the response patterns of seven P450 transcripts after treatment of seedlings with these inducers. Each of these P450 transcripts has distinct developmental, tissue-specific, and chemical cues regulating their expression even when they encode P450s within the same biosynthetic pathway. Most notably, the CYP71C1 and CYP71C3 transcripts, encoding P450s in the DIMBOA biosynthetic pathway, are induced to the same level in response to wounding and NA treatment of younger seedlings and differentially in response to NA/T treatment of younger seedlings and NA and NA/T treatment of older seedlings. NA and T induce expression of both CYP92A1 and CYP72A5 transcripts in older seedling shoots, whereas phenobarbital induces CYP92A1 expression in older seedling shoots and highly induces CYP72A5 expression in young and older seedling roots. Expressed sequence tag (EST) 6c06b11 transcripts, encoding an undefined P450 activity, are highly induced in seedling shoots infected with bacterial pathogens.

  13. Characterization of Maize Cytochrome P450 Monooxygenases Induced in Response to Safeners and Bacterial Pathogens1

    PubMed Central

    Persans, Michael W.; Wang, Jian; Schuler, Mary A.

    2001-01-01

    Plants use a diverse array of cytochrome P450 monooxygenases in their biosynthetic and detoxification pathways. To determine the extent to which various maize P450s are induced in response to chemical inducers, such as naphthalic anhydride (NA), triasulfuron (T), phenobarbital, and bacterial pathogens (Erwinia stuartii, Acidovorax avenae), we have analyzed the response patterns of seven P450 transcripts after treatment of seedlings with these inducers. Each of these P450 transcripts has distinct developmental, tissue-specific, and chemical cues regulating their expression even when they encode P450s within the same biosynthetic pathway. Most notably, the CYP71C1 and CYP71C3 transcripts, encoding P450s in the DIMBOA biosynthetic pathway, are induced to the same level in response to wounding and NA treatment of younger seedlings and differentially in response to NA/T treatment of younger seedlings and NA and NA/T treatment of older seedlings. NA and T induce expression of both CYP92A1 and CYP72A5 transcripts in older seedling shoots, whereas phenobarbital induces CYP92A1 expression in older seedling shoots and highly induces CYP72A5 expression in young and older seedling roots. Expressed sequence tag (EST) 6c06b11 transcripts, encoding an undefined P450 activity, are highly induced in seedling shoots infected with bacterial pathogens. PMID:11161067

  14. Cytochrome P450 107U1 is required for sporulation and antibiotic production in Streptomyces coelicolor

    PubMed Central

    Tian, Zhenghua; Cheng, Qian; Yoshimoto, Francis K.; Lei, Li; Lamb, David C.; Guengerich, F. Peter

    2013-01-01

    The filamentous bacterium Streptomyces coelicolor has a complex life cycle involving the formation of hair-like aerial mycelia on the colony surface, which differentiate into chains of spores. Genes required for the initiation of aerial mycelium formation have been termed ‘bld’ (bald), describing the smooth, undifferentiated colonies of mutant strains. We report the identification of a new bld gene designated as sco3099 and biochemical analysis of its encoded enzyme, cytochrome P450 (P450, or CYP) 107U1. Deletion of sco3099 resulted in a mutant defective in aerial hyphae sporulation and sensitive to heat shock, indicating that P450 107U1 plays a key role in growth and development of S. coelicolor. This is the first P450 reported to participate in a sporulation process in Streptomycetes. The substrate and catalytic properties of P450 107U1 were further investigated in mass spectrometry-based metabolomic studies. Glycocholic acid (from the medium) was identified as a substrate of P450 107U1 and was oxidized to glyco-7-oxo-deoxycholic acid. Although this reaction is apparently not relevant to the observed sporulation deficiency, it suggests that P450 107U1 might exert its physiological function by oxidizing other steroid-like molecules. PMID:23357279

  15. Effects of 2-acetylaminofluorene, dietary fats and antioxidants on nuclear envelope cytochrome P-450

    SciTech Connect

    Carubelli, R.; Graham, S.A.; Griffin, M.J.; McCay, P.B.

    1986-05-01

    The authors reported a marked loss of cytochrome P-450 in hepatic nuclear envelope (NE) but not in microsomes of male Sprague-Dawley rats fed a semipurified diet containing 0.05% w/w 2-acetylaminofluorene (AAF) for 3 weeks. This may reflect loss of NE capacity to detoxify AAF metabolites generated by microsomal P-450. They are now investigating if dietary effects such as progressive decrease in the incidence of AAF-induced tumors in rats fed high polyunsaturated fat diet (HPUF) vs. high saturated fat diet (HSF) vs. low fat diet (LF), and the anticarcinogenic activity of butylated hydroxytoluene (BHT; 0.3% w/w) correlate with preservation of NE P-450. Rats fed AAF HSF (25.6% w/w corn oil) showed marked loss of NE P-450 after 3 weeks; BHT protected against this loss. Rats fed AAF in HSF (25.6% w/w; 18 parts beef tallow + 2 parts corn oil), on the other hand, experienced a marked drop in NE P-450 after 9 weeks; BHT protected against this loss. Comparison of NE P-450 levels in control rats fed HPUF or HSF for 3 weeks with those of rats fed a semipurified diet with 10% fat or Purina chow (ca. 5% fat), support the prediction of an inverse correlation between the levels of dietary fat and the NE P-450 content. Studies on AAF and BHT effects using LF (2% w/w corn oil) are in progress.

  16. Function and membrane topology of wild-type and mutated cytochrome P-450c21.

    PubMed Central

    Hu, M C; Hsu, L C; Hsu, N C; Chung, B C

    1996-01-01

    We have studied membrane topology of cytochrome P-450c21 (P450c21) using the approaches of mutagenesis and protease digestion. P450c21 is located at the cytoplasm with an N-terminal hydrophobic domain integrated into microsomal membranes. When this hydrophobic domain was replaced by a secretory signal peptide, P450c21 was translocated into the lumen and lost enzymic activity. No other topogenic sequence was detected in the bulk of the P450c21 peptide. A mutant protein with Pro-30 replaced by Leu (L30) corresponding to the mutation found in the diseased state was created. L30 protein lost 90% of enzymic activity, while a double mutant (L30R32) with an additional Leu-32 to Arg mutation had slightly higher residual enzymic activity. Apart from lower activity, L30 was also present in the cell at a lower level than wild-type P450c21. This lower level is probably due to increased degradation, as L30 is synthesized at a normal rate. Both L30 and L30R32 proteins, however, were integrated into membranes normally. Therefore the Pro-30 --> Leu mutation did not affect membrane integration, but affected the abundance and enzymic activity of P450c21. PMID:8645225

  17. Male specific expression of a cytochrome P450 (Cyp312a1) in Drosophila melanogaster.

    PubMed

    Kasai, Shinji; Tomita, Takashi

    2003-01-24

    Using cDNA array techniques, the transcriptional levels of Drosophila cytochrome P450 (P450) genes were compared between male and female flies. Of the 86 P450s, 15 isoforms were picked up and the levels of transcription were confirmed by the real time quantitative RT-PCR. Cyp315a1 and Cyp302a1, which had been reported as P450s involved in the ecdysteroid biosynthesis, were included in the 15 isoforms and expression of these P450s was 8.6- and 7.9-fold higher in females than in males, respectively. In addition, we confirmed that expression of Cyp312a1 was 82-fold higher in adult males than females. This gene expression was observed mostly in the abdomen and its transcription level gradually increased from pupal stage and peaked in the 5-day-old adult. Furthermore, the male specific expression of Cyp312a1 was universally observed in three Drosophila strains originated from the USA (Oregon R), China (Canton S), and Japan (HKJ), suggesting possible involvement of this P450 in significant endogenous catalytic reaction(s). This is the first report of a P450 enzyme being predominantly expressed in male Drosophila.

  18. Electrocatalytically driven omega-hydroxylation of fatty acids using cytochrome P450 4A1.

    PubMed Central

    Faulkner, K M; Shet, M S; Fisher, C W; Estabrook, R W

    1995-01-01

    The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein. In general these electrons are transferred from NADPH to the P450 via an FMN- and FAD-containing flavoprotein (NADPH-P450 reductase). The present paper shows that NADPH can be replaced by an electrochemically generated reductant [cobalt(II) sepulchrate trichloride] for the electrocatalytically driven omega-hydroxylation of lauric acid. Results are presented illustrating the use of purified recombinant proteins containing P450 4A1, such as the fusion protein (rFP450 [mRat4A1/mRatOR]L1) or a system reconstituted with purified P450 4A1 plus purified NADPH-P450 reductase. Rates of formation of 12-hydroxydodecanoic acid by the electrochemical method are comparable to those obtained using NADPH as electron donor. These results suggest the practicality of developing electrocatalytically dependent bioreactors containing different P450s as catalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals. PMID:7644480

  19. Accumulation of Mitochondrial P450MT2, NH2-terminal Truncated Cytochrome P4501A1 in Rat Brain during Chronic Treatment with β-Naphthoflavone

    PubMed Central

    Boopathi, Ettickan; Anandatheerthavarada, Hindupur K.; Bhagwat, Shripad V.; Biswas, Gopa; Fang, Ji-Kang; Avadhani, Narayan G.

    2013-01-01

    The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to β-na-phoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH2 termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin + adrenodoxin reductase electron donor system and exhibited high erythromycin N-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited high N-de-methylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals. PMID:10915793

  20. Engineering of daidzein 3’-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450

    PubMed Central

    2012-01-01

    A cytochrome P450 (CYP) enzyme, 3’-daidzein hydroxylase, CYP105D7 (3’-DH), responsible for daidzein hydroxylation at the 3’-position, was recently reported. CYP105D7 (3’-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3’-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8 μM-1 min-1, which was about 24-fold higher than that of the 3’-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3’-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type. PMID:22697884

  1. Cytochromes P450 and species differences in xenobiotic metabolism and activation of carcinogen.

    PubMed Central

    Lewis, D F; Ioannides, C; Parke, D V

    1998-01-01

    The importance of cytochrome P450 isoforms to species differences in the metabolism of foreign compounds and activation of procarcinogens has been identified. The possible range of P450 isozymes in significant variations in toxicity exhibited by experimental rodent species may have a relevance to chemical risk assessment, especially as human P450s are likely to show changes in the way they metabolize xenobiotics. Consequently, in the safety evaluation of chemicals, we should be cautious in extrapolating results from experimental animal models to humans. This paper focuses on examples in which species differences in P450s lead to significant alterations in carcinogenic response, and includes a discussion of the current procedures for toxicity screening, with an emphasis on short-term tests. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9755138

  2. Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support.

    PubMed

    Tan, Cheau Yuaan; Hirakawa, Hidehiko; Suzuki, Risa; Haga, Tomoaki; Iwata, Fumiya; Nagamune, Teruyuki

    2016-11-21

    Bacterial cytochrome P450s (P450s), which catalyze regio- and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost-effective industrial manufacturing. However, immobilization of P450s is difficult because electron-transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein-mediated co-immobilization of a bacterial P450, and its electron-transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co-immobilized enzymes catalyzed monooxygenation because the electron-transfer protein fused to PCNA via a single peptide linker retained its electron-transport function.

  3. Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

    PubMed Central

    Di Nardo, Giovanna; Gilardi, Gianfranco

    2012-01-01

    Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes. The progress made in the improvement of its catalytic performance towards drugs, the substitution of the costly NADPH cofactor and its immobilization and scale-up of the process for industrial application are reported. PMID:23443101

  4. Pyrethroid Activity-Based Probes for Profiling Cytochrome P450 Activities Associated with Insecticide Interactions

    SciTech Connect

    Ismail, Hanafy M.; O'Neill, Paul M.; Hong, David; Finn, Robert; Henderson, Colin; Wright, Aaron T.; Cravatt, Benjamin; Hemingway, Janet; Paine, Mark J.

    2014-01-18

    Pyrethroid insecticides are used to control a diverse spectrum of diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid metabolizing and non-metabolizing mosquito P450s, as well as rodent microsomes to measure labeling specificity, plus CPR and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using a deltamethrin mimetic PyABP we were able to profile active enzymes in rat liver microsomes and identify pyrethroid metabolizing enzymes in the target tissue. The most reactive enzyme was a P450, CYP2C11, which is known to metabolize deltamethrin. Furthermore, several other pyrethroid metabolizers were identified (CYPs 2C6, 3A4, 2C13 and 2D1) along with related detoxification enzymes, notably UDP-g’s 2B1 - 5, suggesting a network of associated pyrethroid metabolizing enzymes, or ‘pyrethrome’. Considering the central role that P450s play in metabolizing insecticides, we anticipate that PyABPs will aid the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of new tools for disease control.

  5. Engineering Macaca fascicularis cytochrome P450 2C20 to reduce animal testing for new drugs.

    PubMed

    Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Di Nardo, Giovanna; Gilardi, Gianfranco

    2012-12-01

    In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called "molecular Lego", a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K(M) values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 ± 0.2, 5.9 ± 2.3, 3.0 ± 0.5 μM for paclitaxel and 1.2 ± 0.2, 1.6±0.2 and 1.4 ± 0.2 μM for amodiaquine, respectively. The data obtained not only show that the engineering of M. fascicularis did not affect its catalytic properties but also are consistent with K(M) values measured for the microsomal human P450 2C8 and therefore show the feasibility of developing alternative in vitro animal tests.

  6. A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

    PubMed Central

    Sansom, Mark S. P.; Mulholland, Adrian J.

    2014-01-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

  7. The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism.

    PubMed

    Slominski, Andrzej; Semak, Igor; Zjawiony, Jordan; Wortsman, Jacobo; Li, Wei; Szczesniewski, Andre; Tuckey, Robert C

    2005-08-01

    We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20-hydroxycholecalciferol and 20,22-dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)-hydroxyvitamin D3. P450scc did not metabolize 25-hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non-P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized.

  8. The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism

    PubMed Central

    Slominski, Andrzej; Semak, Igor; Zjawiony, Jordan; Wortsman, Jacobo; Li, Wei; Szczesniewski, Andre; Tuckey, Robert C.

    2008-01-01

    We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20-hydroxycholecalciferol and 20,22-dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)-hydroxyvitamin D3. P450scc did not metabolize 25-hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non-P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized. PMID:16098191

  9. Orphans in the Human Cytochrome P450 Superfamily: Approaches to Discovering Functions and Relevance in Pharmacology

    PubMed Central

    Cheng, Qian

    2011-01-01

    As a result of technical advances in recombinant DNA technology and nucleotide sequencing, entire genome sequences have become available in the past decade and offer potential in understanding diseases. However, a central problem in the biochemical sciences is that the functions of only a fraction of the genes/proteins are known, and this is also an issue in pharmacology. This review is focused on issues related to the functions of cytochrome P450 (P450) enzymes. P450 functions can be categorized in several groups: 1) Some P450s have critical roles in the metabolism of endogenous substrates (e.g., sterols and fat-soluble vitamins). 2) Some P450s are not generally critical to normal physiology but function in relatively nonselective protection from the many xenobiotic chemicals to which mammals (including humans) are exposed in their diets [as well as more anthropomorphic chemicals (e.g., drugs, pesticides)]. 3) Some P450s have not been extensively studied and are termed “orphans” here. With regard to elucidation of any physiological functions of the orphan P450s, the major subject of this review, it is clear that simple trial-and-error approaches with individual substrate candidates will not be very productive in addressing questions about function. A series of liquid chromatography/mass spectrometry/informatics approaches are discussed, along with some successes with both human and bacterial P450s. Current information on what are still considered “orphan” P450s is presented. The potential for application of some of these approaches to other enzyme systems is also discussed. PMID:21737533

  10. A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

    PubMed

    Miyakawa, Kazuhisa; Albee, Ryan; Letzig, Lynda G; Lehner, Andreas F; Scott, Michael A; Buchweitz, John P; James, Laura P; Ganey, Patricia E; Roth, Robert A

    2015-08-01

    Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

  11. Placental expression and molecular characterization of aromatase cytochrome P450 in the spotted hyena (Crocuta crocuta).

    PubMed

    Conley, A J; Corbin, C J; Browne, P; Mapes, S M; Place, N J; Hughes, A L; Glickman, S E

    2007-07-01

    At birth, the external genitalia of female spotted hyenas (Crocuta crocuta) are the most masculinized of any known mammal, but are still sexually differentiated. Placental aromatase cytochrome P450 (P450arom) is an important route of androgen metabolism protecting human female fetuses from virilization in utero. Therefore, placental P450arom expression was examined in spotted hyenas to determine levels during genital differentiation, and to compare molecular characteristics between the hyena and human placental enzymes. Hyena placental P450arom activity was determined at gestational days (GD) 31, 35, 45, 65 and 95 (term, 110), and the relative sensitivity of hyena and human placental enzyme to inhibition by the specific inhibitor, Letrozole, was also examined. Expression of hyena P450arom in placenta was localized by immuno-histochemistry, and a full-length cDNA was cloned for phylogenetic analysis. Aromatase activity increased from GD31 to a peak at 45 and 65, apparently decreasing later in gestation. This activity was more sensitive to inhibition by Letrozole than was human placental aromatase activity. Expression of P450arom was localized to syncytiotrophoblast and giant cells of mid-gestation placentas. The coding sequence of hyena P450arom was 94% and 86% identical to the canine and human enzymes respectively, as reflected by phylogenetic analyses. These data demonstrate for the first time that hyena placental aromatase activity is comparable to that of human placentas when genital differentiation is in progress. This suggests that even in female spotted hyenas clitoral differentiation is likely protected from virilization by placental androgen metabolism. Decreased placental aromatase activity in late gestation may be equally important in allowing androgen to program behaviors at birth. Although hyena P450arom is closely related to the canine enzyme, both placental anatomy and P450arom expression differ. Other hyaenids and carnivores must be investigated to

  12. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

    SciTech Connect

    Pechurskaya, Tatiana A. . E-mail: usanov@iboch.bas-net.by

    2007-02-16

    The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.

  13. Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.

    PubMed

    Chowdhury, Goutam; Calcutt, M Wade; Nagy, Leslie D; Guengerich, F Peter

    2012-12-18

    Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine [N,N-dimethylnitrosamine (DMN)], a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥8. The KIE was not attenuated in noncompetitive intermolecular experiments with rat liver microsomes {(D)V = 12.5; (D)(V/K) = 10.9 [nomenclature of Northrop, D. B. (1982) Methods Enzymol. 87, 607-625]} but was with purified human P450 2E1 [(D)V = 3.3; (D)(V/K) = 3.7], indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine [N,N-diethylnitrosamine (DEN)], the intrinsic KIE was slightly lower and was not expressed [e.g., (D)(V/K) = 1.2] in noncompetitive intermolecular experiments. The same general pattern of KIEs was also seen in the (D)(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH(3)NH(2), CH(3)CH(2)NH(2), and NO(2)(-)). Experiments with deuterated N-nitroso-N-methyl-N-ethylamine demonstrated that the lower KIEs associated with ethyl versus methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 [Chowdhury, G., et al. (2010) J. Biol. Chem. 285, 8031-8044]. These same features (no lag phase for HCO(2)H formation and a lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has a lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkyl nitrosamine oxidation appears to be shared by a number of P450s.

  14. Oxidation of Methyl and Ethyl Nitrosamines by Cytochromes P450 2E1 and 2B1

    PubMed Central

    Chowdhury, Goutam; Calcutt, M. Wade; Nagy, Leslie D.; Guengerich, F. Peter

    2012-01-01

    Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine (N,N-dimethylnitrosamine, DMN), a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥ 8. The KIE was not attenuated in non-competitive intermolecular experiments with rat liver microsomes (DV 12.5, D(V/K) 10.9, nomenclature of Northrop, D.B. (1982) Methods Enzymol. 87, 607–625) but was with purified human P450 2E1 (DV 3.3, D(V/K) 3.7), indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine (N,N-diethylnitrosamine, DEN), the intrinsic KIE was slightly lower and was not expressed (e.g., D(V/K) 1.2) in non-competitive intermolecular experiments. The same general pattern of KIEs was also seen in the D(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH3NH2, CH3CH2NH2, and NO2−). Experiments with deuterated N-nitroso-N-methyl,N-ethylamine demonstrated that the lower KIEs associated for ethyl compared to methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 (Chowdhury, G. et al. (2010) J. Biol. Chem. 285, 8031–8044). These same features (no lag phase for HCO2H formation, lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkylnitrosamine oxidation appears to be shared by a number of P450s. PMID:23186213

  15. Characterization of feline cytochrome P450 2B6.

    PubMed

    Okamatsu, Gaku; Komatsu, Tetsuya; Ono, Yuka; Inoue, Hiroki; Uchide, Tsuyoshi; Onaga, Takenori; Endoh, Daiji; Kitazawa, Takio; Hiraga, Takeo; Uno, Yasuhiro; Teraoka, Hiroki

    2017-02-01

    1. Little is known about drug metabolism in carnivores. Although the domestic cat (Felis catus) is an obligate carnivore and is the most common companion animal, usage and dosage of many drugs are determined according to information obtained from humans and dogs. We determined the complete cDNA sequence of CYP2B6 from the feline lung. 2. Feline CYP2B6 consists of 494 deduced amino acids, showing highest identity with the dog CYP2B ortholog, followed by those of horse, pig, primate and human. 3. Feline CYP2B6 transcripts were expressed predominantly in the lung and slightly in the small intestine but not in the liver without significant sex-dependent differences. Western blot analysis with an anti-human CYP2B6 antibody confirmed the presence of CYP2B protein in the lung but not in the liver. 4. Feline CYP2B6 proteins heterologously expressed in Escherichia coli metabolized several substrates specific to human CYP2B6, including 7-ethoxy-4-(trifluoromethyl) coumarin (EFC). The metabolic activity was strongly inhibited by medetomidine and atipamezole, potent inhibitors of canine CYP2B11 (now officially CYP2B6) as well as by ticlopidine and sertraline, inhibitors selective to human CYP2B6. 5. The results suggest that feline CYP2B6 is a functional CYP2B ortholog that plays a role in the local defense mechanism in the cat respiratory system and intestine.

  16. Cytochrome P450, CYP93A1, as a defense marker in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CYP93A1 is a cytochrome P450 that is involved in the synthesis of the phytoalexin glyceollin in soybean (Glycine max L. Merr). The gene encoding CYP93A1 has been used as a defense marker in soybean cell cultures, however, little is known regarding how this gene is expressed in the intact plant. To f...

  17. EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2

    EPA Science Inventory

    EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2. T M Ross1, B P Anderson1, G Zhao2, R A Pegram1 and J W Allis1. 1U.S. EPA, ORD, NHEERL, Research Triangle Park, NC; 2University of North Carolina, Chapel Hill, NC.
    Sponsor: H Barton

    Bromodichlorometh...

  18. INCREASED BLOOD PRESSURE IN MICE LACKING CYTOCHROME P450 2J5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cytochrome P450 (CYP) enzymes participate in a wide range of biochemical functions including metabolism of arachidonic acid and steroid hormones. Mouse CYP2J5 is abundant in the kidney where its products, the cis-epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. To d...

  19. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    EPA Science Inventory

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  20. Screening and identification of novel cytochrome P450s in ticks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450s are the major phase I drug metabolizing enzymes found in most species, including those belonging to the phylum Arthropoda. Much of the work within the area of xenobiotic metabolism in this phylum has centered on mosquito species such as Anopheles gambiae due to their role as vectors...

  1. Aryl Hydroxylation of the Herbicide Diclofop by a Wheat Cytochrome P-450 Monooxygenase 1

    PubMed Central

    Zimmerlin, Alfred; Durst, Francis

    1992-01-01

    Wheat (Triticum aestivum L. cv Etoile de Choisy) microsomes catalyzed the cytochrome P-450-dependent oxidation of the herbicide diclofop to three hydroxy-diclofop isomers. Hydroxylation was predominant at carbon 4, with migration of chlorine to carbon 5 (67%) and carbon 3 (25%). The 2,4-dichloro-5-hydroxy isomer was identified as a minor reaction product (8%). Substrate-specificity studies showed that the activity was not inhibited or was weakly inhibited by a range of xenobiotic or physiological cytochrome P-450 substrates, with the exception of lauric acid. Wheat microsomes also catalyze the metabolism of the herbicides chlorsulfuron, chlortoluron, and 2,4-dichlorophenoxyacetic acid and of the model substrate ethoxycoumarin, as well as the hydroxylation of the endogenous substrates cinnamic and lauric acids. Treatments of wheat seedlings with phenobarbital or the safener naphthalic acid anhydride enhanced the cytochrome P-450 content of the microsomes and all related activities except that of cinnamic acid 4-hydroxylase, which was reduced. The stimulation patterns of diclofop aryl hydroxylase and lauric acid hydroxylase were similar, in contrast with the other activities tested. Lauric acid inhibited competitively (Ki = 9 μm) the oxidation of diclofop and reciprocally. The similarity of diclofop aryl hydroxylase and lauric acid hydroxylase was further investigated by alternative substrate kinetics, autocatalytic inactivation, and computer-aided molecular modelisation studies, and the results suggest that both reactions are catalyzed by the same cytochrome P-450 isozyme. PMID:16653070

  2. PRIMARY STRUCTURE OF THE CYTOCHROME P450 LANOSTEROL 14A-DEMETHYLASE GENE FROM CANDIDA TROPICALIS

    EPA Science Inventory

    We report the nucleotide sequence of the gene and flanking DNA for the cytochrome P450 lanosterol 14 alpha-demethylase (14DM) from the yeast Candida tropicalis ATCC750. An open reading frame (ORF) of 528 codons encoding a 60.9-kD protein is identified. This ORF includes a charact...

  3. Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

    SciTech Connect

    Arnold, Frances H.

    2012-02-27

    The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical

  4. Update on allele nomenclature for human cytochromes P450 and the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Database.

    PubMed

    Sim, Sarah C; Ingelman-Sundberg, Magnus

    2013-01-01

    Interindividual variability in xenobiotic metabolism and drug response is extensive and genetic factors play an important role in this variation. A majority of clinically used drugs are substrates for the cytochrome P450 (CYP) enzyme system and interindividual variability in expression and function of these enzymes is a major factor for explaining individual susceptibility for adverse drug reactions and drug response. Because of the existence of many polymorphic CYP genes, for many of which the number of allelic variants is continually increasing, a universal and official nomenclature system is important. Since 1999, all functionally relevant polymorphic CYP alleles are named and published on the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Web site (http://www.cypalleles.ki.se). Currently, the database covers nomenclature of more than 660 alleles in a total of 30 genes that includes 29 CYPs as well as the cytochrome P450 oxidoreductase (POR) gene. On the CYP-allele Web site, each gene has its own Webpage, which lists the alleles with their nucleotide changes, their functional consequences, and links to publications identifying or characterizing the alleles. CYP2D6, CYP2C9, CYP2C19, and CYP3A4 are the most important CYPs in terms of drug metabolism, which is also reflected in their corresponding highest number of Webpage hits at the CYP-allele Web site.The main advantage of the CYP-allele database is that it offers a rapid online publication of CYP-alleles and their effects and provides an overview of peer-reviewed data to the scientific community. Here, we provide an update of the CYP-allele database and the associated nomenclature.

  5. Natural variation in the expression of cytochrome P-450 and dimethylnitrosamine demethylase in Drosophila

    SciTech Connect

    Waters, L.C.; Simms, S.I.; Nix, C.E.

    1984-09-28

    Electrophoresis of Drosophila microsomes resolves two major hemecontaining protein bands with apparent molecular weights of 59,290 (band a) and 55,750 (band b). The hemoproteins in these two bands can account for most of the cytochrome P-450 in the organism. Band a is present in all strains examined: band b is not. Dimethylnitrosamine demethylase, a P-450 enzyme, is a component of band b. Numerous studies have shown that P-450-attributed activities of Drosophila are genotype dependent. Drosophila, therefore, represents a unique system for studying the genetics of, and the molecular mechanisms that regulate, the expression of constitutive levels of P-450 isozymes. Here we explore the molecular basis for the large differences in P-450 expression between strains. Microsomal proteins from several wild-type strains were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Significant differences in the protein bands that contained P-450 were observed between strains with high or low mixed-function oxidase activity. 22 references, 2 figures, 1 table.

  6. Conformational Plasticity and Structure/Function Relationships in Cytochromes P450

    PubMed Central

    Kazanis, Sophia; Dang, Marina

    2010-01-01

    Abstract The cytochrome P450s are a superfamily of enzymes that are found in all kingdoms of living organisms, and typically catalyze the oxidative addition of atomic oxygen to an unactivated C-C or C-H bond. Over 8000 nonredundant sequences of putative and confirmed P450 enzymes have been identified, but three-dimensional structures have been determined for only a small fraction of these. While all P450 enzymes for which structures have been determined share a common global fold, the flexibility and modularity of structure around the active site account for the ability of P450 enzymes to accommodate a vast number of structurally dissimilar substrates and support a wide range of selective oxidations. In this review, known P450 structures are compared, and some structural criteria for prediction of substrate selectivity and reaction type are suggested. The importance of dynamic processes such as redox-dependent and effector-induced conformational changes in determining catalytic competence and regio- and stereoselectivity is discussed, and noncrystallographic methods for characterizing P450 structures and dynamics, in particular, mass spectrometry and nuclear magnetic resonance spectroscopy are reviewed. Antioxid. Redox Signal. 13, 1273–1296. PMID:20446763

  7. Characterization of a novel ACTH inducible cytochrome P-450 from rat adrenal microsomes

    SciTech Connect

    Otto, S.A.; Marcus, C.M.; Jefcoate, C.R. )

    1990-02-26

    In rat adrenal cortex 7,12 dimethylbenz(a)anthracene (DMBA) causes massive necrosis that is dependent of ACTH. This is related to an ACTH inducible adrenal microsomal cytochrome P-450 that catalyzes hydrocarbon metabolism. Rat adrenal microsomes, catalyze the formation of DMBA 3,4 diol a precursor of the bay region reactive electrophile DMBA 3,4 diol 1,2 oxide. Both DMBA metabolism and a 57Kd protein have disappeared from microsomes 30 days after hypophysectomy, but are restored by 14 days treatment with ACTH. Dexamethasone which fully suppresses ACTH only partially suppresses this activity. The 57 Kd protein was partially purified to a single major band in one step from solubilized microsomes by h.p.l.c. chromatography using detergent elution from a novel column that mimics phospholipid membranes. This preparation exhibits a specific content of 2 nm P-450/mg protein and a turnover number of 1,500pm DMBA/nm P-450/minutes. A polyclonal antisera raised against this preparation provides a single western blot corresponding to the 57Kd ACTH sensitive protein. This antibody did not blot microsomal P-450 c21, nor did selected antibodies from known families react with this adrenal P-450 protein, suggesting substantial sequence differences from known P-450's.

  8. Crystal Structure of Human Cytochrome P450 2D6 with Prinomastat Bound*

    PubMed Central

    Wang, An; Savas, Uzen; Hsu, Mei-Hui; Stout, C. David; Johnson, Eric F.

    2012-01-01

    Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice. This study determined the structure of P450 2D6 complexed with a substrate and potent inhibitor, prinomastat, to 2.85 Å resolution by x-ray crystallography. Prinomastat binding is well defined by electron density maps with its pyridyl nitrogen bound to the heme iron. The structure of ligand-bound P450 2D6 differs significantly from the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition of the structures reveals significant differences for β sheet 1, helices A, F, F′, G″, G, and H as well as the helix B-C loop. The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly-218, which is followed by a well defined helix F′ that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6. PMID:22308038

  9. Direct Observation of an Oxepin from a Bacterial Cytochrome P450-Catalyzed Oxidation.

    PubMed

    Stok, Jeanette E; Chow, Sharon; Krenske, Elizabeth H; Farfan Soto, Clementina; Matyas, Csongor; Poirier, Raymond A; Williams, Craig M; De Voss, James J

    2016-03-18

    The cytochromes P450 are hemoproteins that catalyze a range of oxidative C-H functionalization reactions, including aliphatic and aromatic hydroxylation. These transformations are important in a range of biological contexts, including biosynthesis and xenobiotic biodegradation. Much work has been carried out on the mechanism of aliphatic hydroxylation, implicating hydrogen atom abstraction, but aromatic hydroxylation is postulated to proceed differently. One mechanism invokes as the key intermediate an arene oxide (and/or its oxepin tautomer). Conclusive isolation of this intermediate has remained elusive and, currently, direct formation of phenols from a Meisenheimer intermediate is believed to be favored. We report here the identification of a P450 [P450cam (CYP101A1) and P450cin (CYP176A1)]-generated arene oxide as a product of in vitro oxidation of tert-butylbenzene. Computations (CBS-QB3) predict that the arene oxide and oxepin have similar stabilities to other arene oxides/oxepins implicated (but not detected) in P450-mediated transformations, suggesting that arene oxides can be unstable terminal products of P450-catalyzed aromatic oxidation that can explain the origin of some observed metabolites.

  10. Genome-wide identification and expression analyses of cytochrome P450 genes in mulberry (Morus notabilis).

    PubMed

    Ma, Bi; Luo, Yiwei; Jia, Ling; Qi, Xiwu; Zeng, Qiwei; Xiang, Zhonghuai; He, Ningjia

    2014-09-01

    Cytochrome P450s play critical roles in the biosynthesis of physiologically important compounds in plants. These compounds often act as defense toxins to prevent herbivory. In the present study, a total of 174 P450 genes of mulberry (Morus notabilis C.K.Schn) were identified based on bioinformatics analyses. These mulberry P450 genes were divided into nine clans and 47 families and were found to be expressed in a tissue-preferential manner. These genes were compared to the P450 genes in Arabidopsis thaliana. Families CYP80, CYP92, CYP728, CYP733, CYP736, and CYP749 were found to exist in mulberry, and they may play important roles in the biosynthesis of mulberry secondary metabolites. Analyses of the functional and metabolic pathways of these genes indicated that mulberry P450 genes may participate in the metabolism of lipids, other secondary metabolites, xenobiotics, amino acids, cofactors, vitamins, terpenoids, and polyketides. These results provide a foundation for understanding of the structures and biological functions of mulberry P450 genes.

  11. Dendroctonus armandi (Curculionidae: Scolytinae) cytochrome P450s display tissue specificity and responses to host terpenoids.

    PubMed

    Dai, Lulu; Ma, Mingyuan; Gao, Guanqun; Chen, Hui

    2016-11-01

    Bark beetles oxidize the defensive allelochemicals of their host trees both to detoxify them and convert them into components of their pheromone systems which were catalyzed by cytochrome P450 enzymes (CYPs) and occur in different tissues of the insect. We study P450 genes in the Chinese white pine beetle (Dendroctonus armandi), and some bio-information analysis was done for the full-length deduced amino acid sequences. The tissue specificity of these P450 genes was determined in three tissues (antenna, gut and reproductive organs). Differential expression of the P450 genes was observed between sexes, and within these significant differences exposed to stimuli (α-pinene (1:1 racemic mix), (S)-(-)-α-pinene, (S)-(-)-β-pinene, (+)-3-carene, (±)-limonene and turpentine oil) at 24h. Increased expression of P450 genes suggested that they play a role in the detoxification of monoterpenes released by the host trees. The different transcript accumulation patterns of these bark beetle P450 genes provided insight into ecological interactions of D. armandi with its host pine.

  12. Interindividual Variability in Cytochrome P450–Mediated Drug Metabolism

    PubMed Central

    Tracy, Timothy S.; Chaudhry, Amarjit S.; Prasad, Bhagwat; Thummel, Kenneth E.; Schuetz, Erin G.; Zhong, Xiao-bo; Tien, Yun-Chen; Pan, Xian; Shireman, Laura M.; Tay-Sontheimer, Jessica; Lin, Yvonne S.

    2016-01-01

    The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug–drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals. PMID:26681736

  13. Involvement of cytochrome P450 in host-plant utilization by Sonoran Desert Drosophila.

    PubMed Central

    Frank, M R; Fogleman, J C

    1992-01-01

    The four Drosophila species endemic to the Sonoran Desert (Drosophila mettleri, Drosophila mojavensis, Drosophila nigrospiracula, and Drosophila pachea) utilize necrotic cactus tissue or soil soaked by rot exudate as breeding substrates. Each Drosophila species uses a different cactus species as its primary host. D. pachea is limited to senita cactus by a biochemical dependency on unusual sterols available only in that cactus. For the other Drosophila species, no such chemical dependencies exist to explain the relationships with their primary host plants. Each cactus species has a different array of allelochemicals that have detrimental effects on non-resident fly species. We have hypothesized that the desert fly-cactus associations are due, in part, to differences between the fly species in their allelochemical detoxication enzymes, the cytochrome P450 system. To test whether P450s are involved in the detoxication of cactus allelochemicals, several experiments were done. (i) The effect of a specific P450 inhibitor, piperonyl butoxide, on larval survival through eclosion on each cactus substrate was investigated. (ii) In vitro metabolism of cactus alkaloids was determined for each Drosophila species. The effects of specific inducers and inhibitors were included in these experiments. (iii) The basal and induced content of cytochrome P450 in each species was determined. The results support the hypothesis that P450 enzymes are involved in host-plant utilization by these Sonoran Desert Drosophila species. Images PMID:1465429

  14. Screening of bacterial cytochrome P450s responsible for regiospecific hydroxylation of (iso)flavonoids.

    PubMed

    Pandey, Bishnu Prasad; Lee, Nahum; Choi, Kwon-Young; Jung, Eunok; Jeong, Da-Hye; Kim, Byung-Gee

    2011-04-07

    Screening of cytochrome P450 monoxygenases responsible for the regiospecific hydroxylation of flavones, isoflavones and chalcones was attempted using a P450 library constructed from Streptomyces avermitilis MA4680, Bacillus and Nocardia farcinica IFM10152 strains. As electron transfer redox partners with the P450s in Escherichia coli system, putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida were used. Among the 50 soluble P450s in the library screened, three cytochrome P450s, i.e. CYP107Y1, CYP125A2 and CYP107P2 from S. avermitilis MA4680 showed good hydroxylation activities towards flavones and isoflavones. However, low product yields prevented us from identifying complete structure of the products. By using S. avermitilis MA4680 as their expression host, further analysis identified that CYP107Y1(SAV2377), CYP125A2(SAV5841) and CYP107P2(SAV4539) showed good regiospecific hydroxylation activities towards genistein (4',5,7-trihydroxyisoflavone), chrysin (5,7-dihydroxyisoflavone) and apigenin (4',5,7-dihydroxyisoflavone) to produce 3',4',5,7,-tetrahydroxyisoflavone, B-ring hydroxylated 5,7-dihydroxyflavone and 3',4',5,7,-tetrahydroxyflavone, respectively. Analyses of the reaction products were performed using HPLC, ESI-MS-MS and GC-MS and 1H NMR.

  15. Photoaffinity ligands in the study of cytochrome p450 active site structure.

    PubMed

    Gartner, Carlos Augusto

    2003-04-01

    While photoaffinity ligands have been widely used to probe the structures of many receptors and nucleic acid binding proteins, their effective use in the study of cytochrome p450 structure is less established. Nevertheless, significant advances in this field have been made since the technique was first applied to p450cam in 1979. In several cases, especially studies involving p450s of the 1A and 2B families, peptides covalently modified with photoaffinity ligands have been isolated and characterized. Some of these peptides were predicted by molecular modeling to line substrate binding regions of the enzymes. Other data obtained from such studies were more difficult to reconcile with theory. This review addresses the status of photoaffinity labeling as a tool for studying cytochrome p450 structure. In addition, potential future directions in this field are discussed, including the development of heme-directed agents and validation of their effectiveness as photoaffinity ligands using sperm whale myoglobin as a test protein. The potential for hydroxyaromatic compounds to serve as photoactivated probes of active site nucleophiles is also discussed. This class of compounds and its derivatives has long been known in the fields of photochemistry and photophysics to be precursors of reactive radicals and quinone methides that are likely to serve as effective active site probes of the p450s.

  16. Steroid hydroxylations: A paradigm for cytochrome P450 catalyzed mammalian monooxygenation reactions

    SciTech Connect

    Estabrook, Ronald W. . E-mail: Ronald.estabrook@utsouthwestern.edu

    2005-12-09

    The present article reviews the history of research on the hydroxylation of steroid hormones as catalyzed by enzymes present in mammalian tissues. The report describes how studies of steroid hormone synthesis have played a central role in the discovery of the monooxygenase functions of the cytochrome P450s. Studies of steroid hydroxylation reactions can be credited with showing that: (a) the adrenal mitochondrial enzyme catalyzing the 11{beta}-hydroxylation of deoxycorticosterone was the first mammalian enzyme shown by O{sup 18} studies to be an oxygenase; (b) the adrenal microsomal enzyme catalyzing the 21-hydroxylation of steroids was the first mammalian enzyme to show experimentally the proposed 1:1:1 stoichiometry (substrate:oxygen:reduced pyridine nucleotide) of a monooxygenase reaction; (c) application of the photochemical action spectrum technique for reversal of carbon monoxide inhibition of the 21-hydroxylation of 17{alpha}-OH progesterone was the first demonstration that cytochrome P450 was an oxygenase; (d) spectrophotometric studies of the binding of 17{alpha}-OH progesterone to bovine adrenal microsomal P450 revealed the first step in the cyclic reaction scheme of P450, as it catalyzes the 'activation' of oxygen in a monooxygenase reaction; (e) purified adrenodoxin was shown to function as an electron transport component of the adrenal mitochondrial monooxygenase system required for the activity of the 11{beta}-hydroxylase reaction. Adrenodoxin was the first iron-sulfur protein isolated and purified from mammalian tissues and the first soluble protein identified as a reductase of a P450; (f) fractionation of adrenal mitochondrial P450 and incubation with adrenodoxin and a cytosolic (flavoprotein) fraction were the first demonstration of the reconstitution of a mammalian P450 monooxygenase reaction.

  17. Time-dependent expression of cytochrome p450 epoxygenases during human prenatal development

    PubMed Central

    Cizkova, Katerina; Konieczna, Anna; Erdosova, Bela; Ehrmann, Jiri

    2014-01-01

    There is growing evidence that some members of cytochrome P450 enzymes contribute to regulation of normal prenatal development. CYP epoxygenases (CYP2C and CYP2J subfamilies) convert arachidonic acid into four regioisomeric epoxyeicosatrienoic acids (EETs), biologically active molecules involved in mitogenesis and cell signaling. Almost nothing is known about localization of their expression in tissues during human prenatal development. The spatio-temporal expression pattern of CYP2C8, CYP2C9, CYP2C19 and CYP2J2 in human embryonic/fetal intestines, liver, and kidney was investigated by immunohistochemical method. CYP epoxygenases are expressed already in early stages of development in these embryonic/fetal tissues (as early as 7th week of IUD in the intestines, 5th week of IUD in the liver, and 6th week of IUD in the kidney). In kidney, CYP epoxygenases are expressed in the metanephrogenic blastema (but not in the uninduced mesenchyme) and in the tubular system. In the intestines, diverse CYP epoxygenases distribution along crypt-villus axis could suggest role in cell differentiation. Moreover, we detected higher CYP2J2 level in these organs than in adult tissue samples. PMID:24492490

  18. Investigation of the Regulatory Effects of Saccharin on Cytochrome P450s in Male ICR Mice.

    PubMed

    Jo, Jun Hyeon; Kim, Sunjoo; Jeon, Tae Won; Jeong, Tae Cheon; Lee, Sangkyu

    2017-01-01

    Saccharin, the first artificial sweetener, was discovered in 1879 that do not have any calories and is approximately 200~700 times sweeter than sugar. Saccharin was the most common domestically produced sweetener in Korea in 2010, and it has been used as an alternative to sugar in many products. The interaction between artificial sweeteners and drugs may affect the drug metabolism in patients with diabetes, cancer, and liver damage, this interaction has not been clarified thus far. Here, we examined the effects of the potential saccharin-drug interaction on the activities of 5 cytochrome P450 (CYPs) in male ICR mice; further, we examined the effects of saccharin (4,000 mg/kg) on the pharmacokinetics of bupropion after pretreatment of mice with saccharin for 7 days and after concomitant administration of bupropion and saccharin. Our results showed saccharin did not have a significant effect on the 5 CYPs in the S9 fractions obtained from the liver of mice. In addition, we observed no differences in the pharmacokinetic parameters of bupropion between the control group and the groups pretreated with saccharin and that receiving concomitant administration of saccharin. Thus, our results showed that saccharin is safe and the risk of saccharin-drug interaction is very low.

  19. Investigation of the Regulatory Effects of Saccharin on Cytochrome P450s in Male ICR Mice

    PubMed Central

    Jo, Jun Hyeon; Kim, Sunjoo; Jeon, Tae Won; Jeong, Tae Cheon; Lee, Sangkyu

    2017-01-01

    Saccharin, the first artificial sweetener, was discovered in 1879 that do not have any calories and is approximately 200~700 times sweeter than sugar. Saccharin was the most common domestically produced sweetener in Korea in 2010, and it has been used as an alternative to sugar in many products. The interaction between artificial sweeteners and drugs may affect the drug metabolism in patients with diabetes, cancer, and liver damage, this interaction has not been clarified thus far. Here, we examined the effects of the potential saccharin-drug interaction on the activities of 5 cytochrome P450 (CYPs) in male ICR mice; further, we examined the effects of saccharin (4,000 mg/kg) on the pharmacokinetics of bupropion after pretreatment of mice with saccharin for 7 days and after concomitant administration of bupropion and saccharin. Our results showed saccharin did not have a significant effect on the 5 CYPs in the S9 fractions obtained from the liver of mice. In addition, we observed no differences in the pharmacokinetic parameters of bupropion between the control group and the groups pretreated with saccharin and that receiving concomitant administration of saccharin. Thus, our results showed that saccharin is safe and the risk of saccharin-drug interaction is very low. PMID:28133510

  20. Time-dependent expression of cytochrome p450 epoxygenases during human prenatal development.

    PubMed

    Cizkova, Katerina; Konieczna, Anna; Erdosova, Bela; Ehrmann, Jiri

    2014-01-01

    There is growing evidence that some members of cytochrome P450 enzymes contribute to regulation of normal prenatal development. CYP epoxygenases (CYP2C and CYP2J subfamilies) convert arachidonic acid into four regioisomeric epoxyeicosatrienoic acids (EETs), biologically active molecules involved in mitogenesis and cell signaling. Almost nothing is known about localization of their expression in tissues during human prenatal development. The spatio-temporal expression pattern of CYP2C8, CYP2C9, CYP2C19 and CYP2J2 in human embryonic/fetal intestines, liver, and kidney was investigated by immunohistochemical method. CYP epoxygenases are expressed already in early stages of development in these embryonic/fetal tissues (as early as 7th week of IUD in the intestines, 5th week of IUD in the liver, and 6th week of IUD in the kidney). In kidney, CYP epoxygenases are expressed in the metanephrogenic blastema (but not in the uninduced mesenchyme) and in the tubular system. In the intestines, diverse CYP epoxygenases distribution along crypt-villus axis could suggest role in cell differentiation. Moreover, we detected higher CYP2J2 level in these organs than in adult tissue samples.

  1. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  2. Engineering of a hybrid biotransformation system for cytochrome P450sca-2 in Escherichia coli.

    PubMed

    Ba, Lina; Li, Pan; Zhang, Hui; Duan, Yan; Lin, Zhanglin

    2013-07-01

    P450sca-2 is an industrially important enzyme that stereoselectively converts mevastatin into pravastatin. However, little information or engineering efforts have been reported for this enzyme or its redox partner. In this study, we successfully reconstituted the P450sca-2 activity in Escherichia coli by co-expression with putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from the Pseudomonas putida cytochrome P450cam system. With an HPLC-based screening assay, random mutagenesis was applied to yield a mutant (R8-5C) with a pravastatin yield of the whole-cell biotransformation 4.1-fold that of the wild type. P450sca-2 wild-type and R8-5C were characterized in terms of mevastatin binding and hydroxylation, electron transfer, and circular dichroism spectroscopy. R8-5C showed an active P450 expression level that was 3.8-fold that of the wild type, with relatively smaller changes in the apparent k(cat)/K(M) with respect to the substrate mevastatin (1.3-fold) or Pdx (1.5-fold) compared with the wild type. Thus, the increase in the pravastatin yield of the whole-cell biotransformation primarily came from the improved active P450 expression, which has resulted largely from better heme incorporation, although none of the six mutations of R8-5C are located near the heme active site. These results will facilitate further engineering of this P450sca-2 system and provide useful clues for improving other hybrid P450 systems.

  3. Spectral characterization and chiral interactions of plant microsomal cytochrome P450 with metolachlor and herbicide safeners.

    PubMed

    Liu, Huijun

    2010-01-01

    The content and spectral characteristics of cytochrome P450 (Cyt P450) and cytochrome b(5) (Cyt b(5)) extracted from shoots of etiolated maize and rice seedlings were studied by using ultraviolet (UV) difference spectrophotometry. The results showed that fenclorim, rac-metolachlor and S-metolachlor may induce the same P450 isoenzyme with lambda(max) at 453 nm, while naphthalic anhydride (NA) induced another one with lambda(max) at 447 nm. The microsomal Cyt P450 and Cyt b(5) content of maize seedlings was higher than that of rice, and the Cyt b(5) content was higher than that of Cyt P450. Maize and rice microsomal Cyt P450 and Cyt b(5) were induced at different levels by the four chemicals, with the order as follows: NA > fenclorim > rac-metolachlor > S-metolachlor with p < 0.05. When induced by NA, fenclorim, rac-metolachlor and S-metolachlor, the maize Cyt P450 content was, respectively, 5.63-, 3.30-, 3.02- and 2.48-fold that of the control, the rice Cyt P450 content was 8.54-, 2.20-, 1.91- and 1.33-fold that of the control, the maize Cyt b(5) content was 9.89-, 5.49-, 4.69- and 3.40-fold that of the control, and the rice Cyt b(5) content was 7.76-, 4.56-, 2.60- and 1.82-fold that of the control. An enantio-difference existed when rac- and S-metolachlor combined with plant Cyt P450. The interaction of microsomal Cyt P450 with S-metolachlor is higher than that with rac-metolachlor, which may be one of the reasons why S-metolachlor is superior at killing weeds compared with rac-metolachlor. These results will help to develop an understanding of the tolerance for and selectivity of rac- and S-metolachlor.

  4. Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14 alpha-sterol demethylase purified from Candida albicans.

    PubMed Central

    Hitchcock, C A; Dickinson, K; Brown, S B; Evans, E G; Adams, D J

    1990-01-01

    The interaction of azole antifungal antibiotics with purified Candida albicans cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) was measured spectrophotometrically and by inhibition of enzyme activity. Ketoconazole and ICI 153066 (a triazole derivative) formed low-spin complexes with the ferric cytochrome and induced type II difference spectra. These spectra are indicative of an interaction between the azole moiety and the sixth co-ordination position of P-450DM haem. Both azoles inhibited the binding of CO to the sodium dithionite-reduced ferrous cytochrome, and inhibited reconstituted P-450DM activity by binding to the cytochrome with a one-to-one stoichiometry. Similarly, total inhibition of enzyme activity occurred when equimolar amounts of clotrimazole, miconazole or fluconazole were added to reconstituted P-450DM. These results correlated with the inhibition of P-450DM in broken cell preparations, confirming that all five azoles are potent inhibitors of ergosterol biosynthesis in C. albicans. PMID:2180400

  5. Occurrence of a barbiturate-inducible catalytically self-sufficient 119,000 dalton cytochrome P-450 monooxygenase in bacilli.

    PubMed

    Fulco, A J; Ruettinger, R T

    1987-05-04

    In a recent publication (Narhi, L.O. and Fulco, A.J.[1986] J. Biol. Chem. 261, 7160-7169) we described the characterization of a catalytically self-sufficient 119,000 Dalton cytochrome P-450 fatty acid monooxygenase (P-450BM-3) induced by barbiturates in Bacillus megaterium ATCC 14581. We have now examined cell-free preparations from 12 distinct strains of B. megaterium and from one or two strains each of B. alvei, B. brevis, B. cereus, B. licheniformis, B. macerans, B. pumilis and B. subtilis for the presence of this inducible enzyme. Using Western blot analyses in combination with assays for fatty acid hydroxylase activity and cytochrome P-450, we were able to show that 11 of the 12 B. megaterium strains contained not only a strongly pentobarbital-inducible fatty acid monooxygenase identical to or polymorphic with P-450BM-3 but also significant levels of two smaller P-450 cytochromes that were the same as or similar to cytochromes P-450BM-1 and P-450BM-2 originally found in ATCC 14581. Unlike the 119,000 Dalton P-450, however, the two smaller P-450s were generally easily detectable in cultures grown to stationary phase in the absence of barbiturates and, with some exceptions, were not strongly induced by pentobarbital. None of the non-megaterium species of Bacillus tested exhibited significant levels of either fatty acid monooxygenase activity or cytochrome P-450. The one strain of B. megaterium that lacked inducible P-450BM-3 was also negative for BM-1 and BM-2. However, this strain (ATCC 13368) did contain a small but significant level of another P-450 cytochrome that others have identified as the oxygenase component of a steroid 15-beta-hydroxylase system. Our evidence suggests that the BM series of P-450 cytochromes is encoded by chromosomal (rather than by plasmid) DNA.

  6. Different structure of the complexes of two cytochrome P-450 isozymes with acetanilide by 1H-NMR relaxation and spectrophotometry.

    PubMed

    Woldman YaYu; Weiner, L M; Lyakhovich, V V

    1993-05-28

    The functional and spectral characteristics of the interaction of acetanilide with phenobarbital- and methylcholanthrene- induced rat liver microsomes, as well as with corresponding major isozymes (cytochromes P-450b and P-450c) have been compared. The magnitude of the reverse 1st type binding spectra proved to be negatively correlated with the acetanilide oxidation on isozymes under study. The data on paramagnetic relaxation of acetanilide protons in the presence of P-450 have shown the structure of the enzyme-substrate complex to be different for two isozymes, acetanilide molecule being closer to Fe ion in the active site in the case of P-450c, which is active towards acetanilide oxidation. For the P-450c-acetanilide complex the group oxidized (phenyl) is the closest to Fe ion.

  7. Ab Initio Electronic Structure Calculations of Cytochrome P450 -- Ligand Interactions

    NASA Astrophysics Data System (ADS)

    Segall, M. D.; Payne, M. C.; Ellis, S. W.; Tucker, G. T.

    1997-03-01

    The Cytochrome P450 superfamily of enzymes are of great interest in pharmacology as they participate in an enormous range of physiological processes including drug deactivation and xenobiotic detoxification. We apply ab initio electronic structure calculations to model the interactions of the haem molecule at the P450 active site with substrate and inhibitor ligands. These calculations, based on density function theory, were performed with the CETEP code which uses a plane wave basis set and pseudopotentials to perform efficient LDA, GGA and spin dependent calculations. A change in the spin state of the haem iron atom is observed on binding of a substrate molecule, consistent with the accepted reaction mechanism.

  8. Mechanistic aspects of CYP74 allene oxide synthases and related cytochrome P450 enzymes

    PubMed Central

    Brash, Alan R.

    2009-01-01

    The existence of CYP5, CYP8A, and the CYP74 enzymes specialized for reaction with fatty acid peroxide substrates presents opportunities for a “different look” at the catalytic cycle of the cytochrome P450s. This review considers how the properties of the peroxide-metabolizing enzymes are distinctive, and how they tie in with those of the conventional monooxygenase enzymes. Some unusual reactions of each class have parallels in the other. As new enzyme reactions and new P450 structures emerge there will be possibilities for finding their special properties and edging this knowledge into the big picture. PMID:19747698

  9. Protection against chemical-induced lung injury by inhibition of pulmonary cytochrome P-450

    SciTech Connect

    Verschoyle, R.D.; Dinsdale, D. )

    1990-04-01

    Protection afforded by trialkyl phosphorothionates against the lung injury caused by trialkyl phosphorothiolates probably results from the inhibition by the P{double bond}S moiety of the thionates, of one or more pulmonary cytochrome P-450 isozymes. The aromatic hydrocarbons p-xylene and pseudocumene also protect against this injury and inhibit some P-450 isozymes, but by a different mechanism. OOS-Trimethylphosphorothionate and p-xylene were compared as protective agents against the effect of OOS-trimethylphosphorothiolate and two other lung toxins ipomeanol and 1-nitronaphthalene that are known to be activated by cytochrome P-450. The effects of these protective compounds, in vivo, on pulmonary cytochrome P-450 activity were also determined. Both compounds inhibited pentoxyresorufin O-deethylase activity, but not ethoxyresorufin O-deethylase. The phosphorothionate was most effective against lung injury caused by the phosphorothiolates and 1-nitronaphthalene, whereas p-xylene was much more effective against ipomeanol. {beta}-Naphthoflavone, which induces pulmonary ethoxyresorufin O-deethylase activity, did not protect against phosphorothiolate or 1-nitronaphthalene injury, and it was only marginally effective in decreasing the toxicity or ipomeanol.

  10. Role of Protein–Protein Interactions in Cytochrome P450-Mediated Drug Metabolism and Toxicity

    PubMed Central

    2015-01-01

    Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein–protein interactions play a critical role in this process. Historically, the study of CYP–protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein–protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein–protein interactions with CYP enzymes. PMID:25133307

  11. Role of protein-protein interactions in cytochrome P450-mediated drug metabolism and toxicity.

    PubMed

    Kandel, Sylvie E; Lampe, Jed N

    2014-09-15

    Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein-protein interactions play a critical role in this process. Historically, the study of CYP-protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein-protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein-protein interactions with CYP enzymes.

  12. Interaction of epicatechins derived from green tea with rat hepatic cytochrome P-450.

    PubMed

    Wang, Z Y; Das, M; Bickers, D R; Mukhtar, H

    1988-01-01

    Green tea has been used for generations in China and Asia as an antipyretic and diuretic. Prior studies have shown that extracts of green tea inhibit the mutagenicity of polycyclic aromatic hydrocarbons and aflatoxin B1. In this study, we investigated the interaction of certain flavonoid components of green tea epicatechin derivatives including (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin-3-gallate (EGCG) with rat hepatic microsomal cytochrome P-450 (P-450). The addition of EC, EGC, ECG, and EGCG to hepatic microsomes prepared from phenobarbital (PB)-treated rats resulted in spectral changes characterized by absorbance maxima at 420 nm and minima at 380 nm, typical of modified Type II (reverse Type I) binding. Of the epicatechin derivatives, EGCG and ECG showed greater spectral change with oxidized P-450 and time- and concentration-dependent inhibition of the binding of carbon monoxide to dithionite-reduced cytochrome P-450. The addition of EC, EGC, ECG, and EGCG to microsomes prepared from control, PB- or 3-methylcholanthrene-treated rats resulted in a dose-dependent inhibition of cytochrome P-450-dependent aryl hydrocarbon hydroxylase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase activities. EGCG was the most potent in this regard. Green tea polyphenols and epicatechin derivatives also significantly inhibited NADPH-cytochrome c reductase activity. An examination of the structure activity relationship of epicatechin derivatives suggests that the inhibitory effect on the microsomal enzyme system may be due to the galloyl groups or hydroxyl groups on the molecule. Our data indicate that these extracts of green tea may have potential as anticarcinogens.

  13. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    EPA Science Inventory

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  14. Transcriptional Regulation of the Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression in Response to Xylella fastidiosa Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are a group of versatile redox proteins that mediate the biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds which act as plant defense agents. To determine if cytochrome P450 monooxygenases are involved in defense response to...

  15. Selective Usage of Transcription Initiation and Polyadenylation Sites in Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are versatile redox proteins that mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds as plant defense agents against a range of pathogens and insects. To determine if cytochrome P450 monooxygenases are involved in the...

  16. [Overexpression, homology modeling and coenzyme docking studies of the cytochrome P450nor2 from Cylindrocarpon tonkinense].

    PubMed

    Li, N; Zhang, Y Z; Li, D D; Niu, Y H; Liu, J; Li, S X; Yuan, Y Z; Chen, S L; Geng, H; Liu, D L

    2016-01-01

    Cytochrome P450nor catalyzes an unusual reaction that transfers electrons from NADP/NADPH to bound heme directly. To improve the expression level of P450nor2 from Cylindrocarpon tonkinense (C.P450nor2), Escherichia coli system was utilized to substitute the yeast system we constructed for expression of the P450nor2 gene, and the protein was purified in soluble form using Ni(+)-NTA affinity chromatography. In contrast to P450nor from Fusarium oxysporum (F.P450nor) and P450nor1 from Cylindrocarpon tonkinense (C.P450nor1), C.P450nor2 shows a dual specificity for using NADH or NADPH as electron donors. The present study developed a computational approach in order to illustrate the coenzyme specificity of C.P450nor2 for NADH and NADPH. This study involved homology modeling of C.P450nor2 and docking analyses of NADH and NADPH into the crystal structure of F.P450nor and the predictive model of C.P450nor2, respectively. The results suggested that C.P450nor2 and F.P450nor have different coenzyme specificity for NADH and NADPH; whilst the space around the B'-helix of the C.P450nor2, especially the Ser79 and Gly81, play a crucial role for the specificity of C.P450nor2. In the absence of the experimental structure of C.P450nor2, we hope that our model will be useful to provide rational explanation on coenzyme specificity of C.P450nor2.

  17. Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b/sub 5/ and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

    SciTech Connect

    Klasing, S.A.; Mora, M.I.; Wilson, W.C.; Fahey, G.C. Jr.; Garst, J.E.

    1985-09-01

    Experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b/sub 5/, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose, or 6.0% arenaceous flour. Chicks fed 12.0% wood molasses had a higher cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic, had a higher cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.

  18. In Silico Docking of Ligands to Drug Oxidation Enzymes Cytochrome P450 3A4 and Cytochrome P450 1A2.

    NASA Astrophysics Data System (ADS)

    Smith, David; Guglielmon, Jonathan; Glenn, Marsch; Peter, Guengerich F.

    2009-03-01

    Cytochrome P450 3A4 (CYP3A4) and Cytochrome P450 1A2 (CYP1A2) oxidize most drugs in humans. Protein modeling toolkits from OpenEye Scientific Software were used to examine the interaction of drug substrates with CYP3A4 and CYP1A2. Conformers and partial atomic charges were generated for each drug molecule. User-defined volumes were defined around CYP3A4 and CYP1A2 active sites. Ligands were docked assuming protein and substrates as rigid bodies. To assess rigid docking accuracy, x-ray diffraction coordinates of CYP3A4-erythromycin and CYP3A4-metyrapone complexes were obtained. Rigid re-docking of erythromycin and metyrapone into CYP3A4 yielded poses similar to the crystal structures. Rigid docking revealed two other energetically-favorable CYP3A4-metyrapone poses. The best poses were obtained by using all the Open Eye scoring functions. Optimization of protein-ligand interactions within 5-10 Angstroms of the docked ligand was then performed using the Merck Molecular Force Field in which the protein was assumed to be flexible and the ligand to be rigid. Nearby protein residues pulled slightly closer to the substrate, reducing the volume of the active site.

  19. Prometryn and humic acid induce Cytochrome P450 1A expression in Danio rerio (zebrafish).

    PubMed

    Zhao, Qian; Shi, Feng; Zhu, Lin

    2017-01-01

    Humic acid (HA) is a major component of dissolved organic matter, is ubiquitous in the aquatic environment and influences the biological toxicity of organic pollutants. In this study, we investigated the cytochrome P450 1A (CYP 1A) mRNA expression and ethoxyresorufin-O-deethylase (EROD) activity in the gills and liver of zebrafish following exposure to the s-triazine herbicide prometryn with or without HA present. Prometryn induced both CYP 1A mRNA expression and EROD activity. The CYP 1A mRNA expression of zebrafish that were exposed to a combination of prometryn and HA was increased compared to those exposed to prometryn alone. A likely cause for CYP 1A induction is the impact of special components of HA, functioning as aryl hydrocarbon receptor (AHR) agonists. In combination with HA, these increase prometryn levels in tissues. Similar results for EROD activity were evident. In our time course study, CYP 1A mRNA expression reached maximum values during 24h. This revealed CYP 1A mRNA transcription as a comparatively sensitive toxicity index. In a recovery experiment, we found a faster decrease of CYP 1A mRNA expression to control levels (CK) in gills compared to liver tissue. Following exposure to HA, CYP 1A mRNA expression in liver tissue displayed a faster decrease to CK levels. HA induced enhanced metabolic rates for prometryn. In contrast, recovery regularity of CYP 1A expression in gills was independent of the presence of HA. This result indicates different detoxification mechanisms for HA in liver and gills.

  20. Cytochrome P450-2E1 promotes fast food-mediated hepatic fibrosis

    PubMed Central

    Abdelmegeed, Mohamed A.; Choi, Youngshim; Godlewski, Grzegorz; Ha, Seung-Kwon; Banerjee, Atrayee; Jang, Sehwan; Song, Byoung-Joon

    2017-01-01

    Cytochrome P450-2E1 (CYP2E1) increases oxidative stress. High hepatic cholesterol causes non-alcoholic steatohepatitis (NASH) and fibrosis. Thus, we aimed to study the role of CYP2E1 in promoting liver fibrosis by high cholesterol-containing fast-food (FF). Male wild-type (WT) and Cyp2e1-null mice were fed standard chow or FF for 2, 12, and 24 weeks. Various parameters of liver fibrosis and potential mechanisms such as oxidative and endoplasmic reticulum (ER) stress, inflammation, and insulin resistance (IR) were studied. Indirect calorimetry was also used to determine metabolic parameters. Liver histology showed that only WT fed FF (WT-FF) developed NASH and fibrosis. Hepatic levels of fibrosis protein markers were significantly increased in WT-FF. The nitroxidative stress marker iNOS, but not CYP2E1, was significantly elevated only in FF-fed WT. Serum endotoxin, TLR-4 levels, and inflammatory markers were highest in WT-FF. FAS, PPAR-α, PPAR-γ, and CB1-R were markedly altered in WT-FF. Electron microscopy and immunoblot analyses showed significantly higher levels of ER stress in FF-fed WT. Indirect calorimetry showed that Cyp2e1-null-mice fed FF exhibited consistently higher total energy expenditure (TEE) than their corresponding WT. These results demonstrate that CYP2E1 is important in fast food-mediated liver fibrosis by promoting nitroxidative and ER stress, endotoxemia, inflammation, IR, and low TEE. PMID:28051126

  1. Cytochrome P450 monooxygenases involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium.

    PubMed

    Chigu, Nomathemba Loice; Hirosue, Sinji; Nakamura, Chie; Teramoto, Hiroshi; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2010-08-01

    Cytochrome P450 monooxygenases (P450s) involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium were identified by comprehensive screening of both catalytic potentials and transcriptomic profiling. Functional screening of P. chrysosporium P450s (PcCYPs) revealed that 14 PcCYP species catalyze stepwise conversion of anthracene to anthraquinone via intermediate formation of anthrone. Moreover, transcriptomic profiling explored using a complementary DNA microarray system demonstrated that 12 PcCYPs are up-regulated in response to exogenous addition of anthracene. Among the up-regulated PcCYPs, five species showed catalytic activity against anthracene. Based upon both catalytic and transcriptional properties, these five species are most likely to play major roles in anthracene metabolic processes in vivo. Thus, the combination of functional screening and a microarray system may provide a novel strategy for obtaining a thorough understanding of the catalytic functions and biological impacts of PcCYPs.

  2. Enzymatic metabolism of ergosterol by cytochrome p450scc to biologically active 17alpha,24-dihydroxyergosterol.

    PubMed

    Slominski, Andrzej; Semak, Igor; Zjawiony, Jordan; Wortsman, Jacobo; Gandy, Michael N; Li, Jinghu; Zbytek, Blazej; Li, Wei; Tuckey, Robert C

    2005-08-01

    We demonstrate the metabolism of ergosterol by cytochrome P450scc in either a reconstituted system or isolated adrenal mitochondria. The major reaction product was identified as 17alpha,24-dihydroxyergosterol. Purified P450scc also generated hydroxyergosterol as a minor product, which is probably an intermediate in the synthesis of 17alpha,24-dihydroxyergosterol. In contrast to cholesterol and 7-dehydrocholesterol, cleavage of the ergosterol side chain was not observed. NMR analysis clearly located one hydroxyl group to C24, with evidence that the second hydroxyl group is at C17. 17alpha,24-Dihydroxyergosterol inhibited cell proliferation of HaCaT keratinocytes and melanoma cells. Thus, in comparison with cholesterol and 7-dehydrocholesterol, the 24-methyl group and the C22-C23 double bond of ergosterol prevent side chain cleavage by P450scc and change the enzyme's hydroxylase activity from C22 and C20, to C24 and C17, generating bioactive product.

  3. Molecular Regulation of the Induction of Cytochrome P-450E in the Estuarine Fish Fundulus Heteroclitus.

    DTIC Science & Technology

    1989-02-01

    that Fundulus microsomes contained no free labeled amino acid at 2 or 23 hr after injection, and that specific radioactivity was higher at 2 than at...Label" Fundulus 169 10 LIST OF FIGURES Page Chapter 2: Fig. 1: Immunoblot of MAb 1-12-3 and PAb 119p with scup P-450s 59 Fig. 2: Titration of...in Fundulus liver homogenate and liver microsome fractions 173 Fig. 5-3: [3H] (amino acid ) incorporation into Fundulus P-450E 174 Fig. 5-4: [14C] (heme

  4. Short-term fasting alters cytochrome P450-mediated drug metabolism in humans.

    PubMed

    Lammers, Laureen A; Achterbergh, Roos; de Vries, Emmely M; van Nierop, F Samuel; Klümpen, Heinz-Josef; Soeters, Maarten R; Boelen, Anita; Romijn, Johannes A; Mathôt, Ron A A

    2015-06-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug metabolism. In a randomized crossover study design, nine healthy subjects ingested a cocktail consisting of five P450-specific probe drugs [caffeine (CYP1A2), S-warfarin (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6), and midazolam (CYP3A4)] on two occasions (control study after an overnight fast and after 36 h of fasting). Blood samples were drawn for pharmacokinetic analysis using nonlinear mixed effects modeling. In addition, we studied in Wistar rats the effects of short-term fasting on hepatic mRNA expression of P450 isoforms corresponding with the five studied P450 enzymes in humans. In the healthy subjects, short-term fasting increased oral caffeine clearance by 20% (P = 0.03) and decreased oral S-warfarin clearance by 25% (P < 0.001). In rats, short-term fasting increased mRNA expression of the orthologs of human CYP1A2, CYP2C19, CYP2D6, and CYP3A4 (P < 0.05), and decreased the mRNA expression of the ortholog of CYP2C9 (P < 0.001) compared with the postabsorptive state. These results demonstrate that short-term fasting alters cytochrome P450-mediated drug metabolism in a nonuniform pattern. Therefore, short-term fasting is another factor affecting cytochrome P450-mediated drug metabolism in humans.

  5. Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

    PubMed Central

    Pallan, Pradeep S.; Wang, Chunxue; Lei, Li; Yoshimoto, Francis K.; Auchus, Richard J.; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 m−1 s−1) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 m−1 s−1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. PMID:25855791

  6. Characterization of arachidonic acid metabolism by rat cytochrome P450 enzymes: the involvement of CYP1As.

    PubMed

    El-Sherbeni, Ahmed A; El-Kadi, Ayman O S

    2014-09-01

    Cytochrome P450 (P450) enzymes mediate arachidonic acid (AA) oxidation to several biologically active metabolites. Our aims in this study were to characterize AA metabolism by different recombinant rat P450 enzymes and to identify new targets for modulating P450-AA metabolism in vivo. A liquid chromatography-mass spectrometry method was developed and validated for the simultaneous measurements of AA and 15 of its P450 metabolites. CYP1A1, CYP1A2, CYP2B1, CYP2C6, and CYP2C11 were found to metabolize AA with high catalytic activity, and CYP2A1, CYP2C13, CYP2D1, CYP2E1, and CYP3A1 had lower activity. CYP1A1 and CYP1A2 produced ω-1→4 hydroxyeicosatetraenoic acids (HETEs) as 88.7 and 62.7%, respectively, of the total metabolites formed. CYP2C11 produced epoxyeicosatrienoic acids (EETs) as 61.3%, and CYP2C6 produced midchain HETEs and EETs as 48.3 and 29.4%, respectively, of the total metabolites formed. The formation of CYP1A1, CYP1A2, CYP2C6, and CYP2C11 major metabolites followed an atypical kinetic profile of substrate inhibition. CYP1As inhibition by α-naphthoflavone or anti-CYP1As antibodies significantly reduced ω-1→4 HETE formation in the lungs and liver, whereas CYP1As induction by 3-methylcholanthrene resulted in a si