Identification, functional characterization, and regulation of a new cytochrome P450 subfamily, the CYP2Ns.
AuthorsOleksiak MF,Wu S,Parker C,Karchner SI,Stegeman JJ,Zeldin DC
JournalThe Journal of biological chemistry
PubMed ID10644680
The screening of liver and heart cDNA libraries from the teleost Fundulus heteroclitus with degenerate oligonucleotide probes to conserved alpha-helical regions in mammalian P450s resulted in the identification of two cDNAs that together represent a novel P450 subfamily, the CYP2Ns. Northern analysis demonstrated that CYP2N1 transcripts are most abundant in ... More
Induction of liver monooxygenases by beta-myrcene.
AuthorsDe-Oliveira AC, Ribeiro-Pinto LF, Otto SS, Gonçalves A, Paumgartten FJ
JournalToxicology
PubMed ID9458003
'Beta-Myrcene (MYR) is an acyclic monoterpene found in the essential oils of a variety of useful plants such as lemongrass (Cymbopogon citratus), hop, verbena, bay and others. MYR and essential oils containing this olefinic monoterpene are widely used as flavoring food additives, as fragrances in cosmetics and as scents in ... More
Enzymatic analysis of cDNA-expressed human CYP1A1, CYP1A2, and CYP1B1 with 7-ethoxyresorufin as substrate.
AuthorsChang TK, Waxman DJ,
JournalMethods Mol Biol
PubMed ID16719376
'Cytochrome P450 (P450) enzymes belonging to the CYP1 family are highly inducible by polycyclic aromatic hydrocarbons and other environmental chemicals and play a major role in the metabolism of many foreign chemicals and endogenous substances. We describe a spectrofluorometric method for determining 7-ethoxyresorufin O-dealkylation catalyzed by CYP1A1, CYP1A2, and CYPB1. ... More
Stereoselective and regioselective hydroxylation of warfarin and selective O-dealkylation of phenoxazone ethers in human placenta.
'The oxidative metabolism of warfarin and a series of phenoxazone ethers was studied in two groups of human placentas which exhibited high or low levels of aryl hydrocarbon hydroxylase (AHH). Warfarin metabolism was stereoselective (mean R/S = 2.48) for the R-enantiomer and regioselective for the 6- and 8- positions in ... More
Methoxyresorufin and benzyloxyresorufin: substrates preferentially metabolized by cytochromes P4501A2 and 2B, respectively, in the rat and mouse.
AuthorsNerurkar PV, Park SS, Thomas PE, Nims RW, Lubet RA
JournalBiochem Pharmacol
PubMed ID8373445
'The cytochrome P450 isozyme specificity for the O-dealkylation of methoxyresorufin (MTR) and benzyloxyresorufin (BZR) in the rat and mouse was investigated. The induction of various alkoxyresorufin O-dealkylation activities was measured in male F344/NCr rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3,4,5,3',4',5'-hexachlorobiphenyl. MTR and ethoxyresorufin (ETR) O-dealkylation activities were induced 30- and 80-fold, ... More
Uroporphyria produced in mice by iron and 5-aminolaevulinic acid does not occur in Cyp1a2(-/-) null mutant mice.
AuthorsSinclair PR, Gorman N, Dalton T, Walton HS, Bement WJ, Sinclair JF, Smith AG, Nebert DW
JournalBiochem J
PubMed ID9461503
'In the present study we have investigated the putative requirement for the cytochrome P-450 isoform CYP1A2 in murine uroporphyria, by comparing Cyp1a2(-/-) knockout mice with Cyp1a2(+/+) wild-type mice. Uroporphyria was produced by injecting animals with iron-dextran and giving the porphyrin precursor 5-aminolaevulinic acid in the drinking water. Some animals also ... More
7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases.
'A series of 7-alkoxyquinolines was synthesized and tested as substrates with hepatic microsomes prepared from male Wistar rats. Microsomal O-dealkylation rates and kinetic constants were determined for the 7-alkoxyquinolines with microsomes from control, 3-methylcholanthrene (MC)-pretreated, and phenobarbitone (PB)-pretreated rats. Structure-activity relationship studies indicated that the 7-benzyloxyquinoline was the most rapidly ... More
Evaluation of alkoxyresorufins as fluorescent substrates for cytochrome P450 BM3 and site-directed mutants.
'In this study, the first fluorescent assay for bacterial cytochrome P450 BM3 (BM3) and mutants is described. BM3 mutants are potentially very versatile biocatalysts for the production of fine chemicals. A fluorescent assay would be very useful for the identification of nonnatural ligands in high-throughput inhibition assays. Because of the ... More
Molecular dimensions of the substrate binding site of cytochrome P-448.
AuthorsLewis DF, Ioannides C, Parke DV
JournalBiochem Pharmacol
PubMed ID3729975
'The molecular geometries of specific substrates, inhibitors and inducers of cytochrome P-448 activity were determined using computer-graphic techniques for use in defining the molecular dimensions of the substrate binding site of this enzyme. Specific substrates of cytochrome P-448 are essentially planar molecules characterised by a small depth and a large ... More
Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins).
AuthorsBurke MD, Mayer RT
JournalChem Biol Interact
PubMed ID6883573
'The metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (1-8C) was studied in hepatic microsomes of control, phenobarbitone-pretreated (PB) and 3-methylcholanthrene-pretreated (3MC) C57/BL10 mice. Phenoxazone and its ethers were hydroxylated and O-dealkylated respectively to a common metabolite, resorufin. The three categories of microsomes differed ... More
Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450.
'The individual members of a homologous series of phenoxazone ethers related to ethoxyresorufin were O-dealkylated, and the parent compound phenoxazone was ring-hydroxylated, each at different rates with hepatic microsomes of untreated rats. A structure-activity relationship (SAR) was plotted, relating the rate of O-dealkylation to the length and type of the ... More
Use of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers.
'Various fluorescent substrates have been used as specific indicators of induction or activity of different cytochrome P450 isozymes in both fish and mammalian species. In an attempt to identify additional definitive fluorescent substrates for use in fish, we examined a series of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as substrates in O-dealkylation ... More
Induction of liver monooxygenases by annatto and bixin in female rats.
AuthorsDe-Oliveira AC, Silva IB, Manhaes-Rocha DA, Paumgartten FJ
JournalBraz J Med Biol Res
PubMed ID12532234
'Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment (''colorau''). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment ... More
Multiple forms of cytochrome P-450 related to forms induced marginally by phenobarbital. Differences in structure and in the metabolism of alkoxyresorufins.
'The properties of five structurally related forms of cytochrome P-450 (PB1a, PB1b, PB2a, PB2b and PB2d) isolated from rats treated with phenobarbital have been compared with two forms isolated previously now termed ''PB1c'' and ''PB2c''. These enzymes were characterized by their marginal inducibility by phenobarbital and are clearly distinguishable from ... More
Inhibition of hepatic and cutaneous biotransformation of resorufin ethers following intraperitoneal administration of 1-aminobenzotriazole.
AuthorsOldfield NF, Mortillo M, Garland WA, Mico BA
JournalPharm Res
PubMed ID1409385
O-dealkylation of resorufin ethers as an indicator of hepatic cytochrome P-450 isoenzyme induction in the cotton rat (Sigmodon hispidus): a method for monitoring environmental contamination.
AuthorsElangbam CS, Qualls CW, Lochmiller RL
JournalBull Environ Contam Toxicol
PubMed ID1932860
Metabolism of alkoxyphenoxazones by channel catfish liver microsomes: effects of phenobarbital, Aroclor 1254 and 3-methylcholanthrene.
AuthorsAnkley GT, Reinert RE, Mayer RT, Burke MD, Agosin M
JournalBiochem Pharmacol
PubMed ID3109440
The effect of reciprocal active site mutations in human cytochromes P450 1A1 and 1A2 on alkoxyresorufin metabolism.
Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 --> Ala, were constructed, expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy- and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, ... More
Alkoxyresorufin O-dealkylases: association with the murine Ah locus.
AuthorsLum PY, Burke MD, Mayer RT, Ioannides C
JournalCancer Lett
PubMed ID3768851
The hepatic microsomal dealkylation of a series of alkoxyresorufins and the oxidation of phenoxazone to resorufin were investigated in C57BL/6 and DBA/2 mice of both sexes. In both strains of mice and in both sexes the dealkylation rate decreased with increasing length of the alkyl chain. With all alkoxyresorufins the ... More
Induced alkoxyresorufin-O-dealkylases in tilapias (Oreochromis niloticus) from Guandu river, Rio de Janeiro, Brazil.
The activity of fish monooxygenases has been extensively used as a monitoring tool to detect contamination of water bodies by cytochrome P450-inducing agents. In this study we evaluated the activities of ethoxy- (EROD), methoxy- (MROD) and pentoxy- (PROD) resorufin-O-dealkylases in the liver of Nile tilapias (Oreochromis niloticus) collected at the ... More
Induction and decline of hepatic cytochromes P4501A1 and 1A2 in rats exposed to hyperoxia are not paralleled by changes in glutathione S-transferase-alpha.
AuthorsMoorthy B, Nguyen UT, Gupta S, Stewart KD, Welty SE, Smith CV
JournalToxicol Lett
PubMed ID9020404
We investigated the effects of hyperoxia on the activities of hepatic ethoxyresorufin O-deethylase (EROD) (CYP1A1), methoxyresorufin O-demethylase (MROD) (CYP1A2), and glutathione transferase-alpha (GST-alpha), and the status of protein thiols (PSH) in male Sprague-Dawley rats. Twenty-four h of hyperoxia more than doubled EROD and MROD activities, which were increased 7.6- and ... More
Characterization of house fly microsomal mixed function oxidases: inhibition by juvenile hormone i and piperonyl butoxide.
AuthorsFisher CW, Mayer RT
JournalToxicology
PubMed ID6814016
The microsomal mixed function oxidase system of the house fly (Musca domestica [L.]) was characterized with respect to N-demethylation of p-chloromethylaniline, O-demethylation of methoxyresorufin, epoxidation of aldrin and the formation of a metabolite-cytochrome P-450 complex during oxidation of piperonyl butoxide (PB). The inhibition of these reactions by juvenile hormone I ... More
Usefulness of statistic experimental designs in enzymology: example with recombinant hCYP3A4 and 1A2.
AuthorsBournique B, Petry M, Gousset G
JournalAnal Biochem
PubMed ID10585740
First, the effects of 10 incubation factors were screened altogether on nifedipine dehydrogenase (NIF) and methoxyresorufin O-deethylase (MROD) activities catalyzed by recombinant human CYP3A4 and 1A2, respectively. Using a statistic experimental design, only 36 assays were needed to be exhaustive. Eight factors influenced CYP3A4-mediated NIF activity: buffer type, pH, temperature, ... More
Functional differentiation of hepatocyte-like spheroid structures from putative liver progenitor cells in three-dimensional peptide scaffolds.
AuthorsSemino CE, Merok JR, Crane GG, Panagiotakos G, Zhang S
JournalDifferentiation
PubMed ID12823227
We investigated the ability of a new type of biological material, the self-assembling peptide scaffold, to foster tissue-like function by a putative adult rat hepatocyte progenitor cell line, Lig-8. In conventional adherent petri-dish cultures, Lig-8 cells divide exponentially, express markers for definitive endoderm HNF3 beta and hepatocyte lineage, including CK8 ... More
Expression of house fly CYP6A1 and NADPH-cytochrome P450 reductase in Escherichia coli and reconstitution of an insecticide-metabolizing P450 system.
AuthorsAndersen JF, Utermohlen JG, Feyereisen R
JournalBiochemistry
PubMed ID8117673
The house fly (Musca domestica) cytochrome P450 gene CYP6A1 was expressed in Escherichia coli. The native protein was produced at a level of 0.25-0.34 mumol/L (15-20 mg/L) of culture with approximately 50% of the P450 being associated with the membrane fraction. The CYP6A1 protein was characterized spectrally and purified by ... More
A comparative study of constitutive and induced alkoxyresorufin O-dealkylation and individual cytochrome P450 forms in cynomolgus monkey (Macaca fascicularis), human, mouse, rat and hamster liver microsomes.
AuthorsWeaver RJ, Thompson S, Smith G, Dickins M, Elcombe CR, Mayer RT, Burke MD
JournalBiochem Pharmacol
PubMed ID8135852
The expression of constitutive and inducible cytochrome P450 forms was measured in cynomolgus monkey liver and compared with man, rat, mouse and hamster. Four alkoxyresorufin O-dealkylation (AROD) activities widely used as indicators of P450 induction were measured: methoxyresorufin O-demethylation (MROD), ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-dealkylation (PROD) and benzyloxyresorufin O-dealkylation (BROD). ... More
Characterization of substrate binding to cytochrome P450 1A1 using molecular modeling and kinetic analyses: case of residue 382.
Key residue Val-382 in P450 1A1 has been predicted to interact with the alkoxy chain of resorufin derivatives. Therefore, we undertook a detailed analysis of substrate mobility in the active site of the P450 1A1 homology model and assessed the effect of mutations at position 382. Dynamic trajectories of 7-methoxy-, ... More
Liver microsomal cytochrome P-450-dependent O-dealkylation reaction in various animals.
AuthorsMatsubara T, Otsubo S, Yoshihara E
JournalJpn J Pharmacol
PubMed ID6645114
Liver microsomal O-dealkylation activity was determined using O-methyl, O-ethyl and O-propyl derivatives of p-nitrophenol, 7-hydroxycoumarin (umbelliferon) and 7-hydroxyphenoxazone (resorufin) as substrates. Microsomal O-dealkylation activities of p-nitrophenol and 7-hydroxycoumarin O-alkyl derivatives were of similar levels, but the activities of 7-hydroxyphenoxazone O-alkyl derivatives were very low compared with those of other substrates. ... More
Induction of cytochrome P450, generation of oxidative stress and in vitro cell-transforming and DNA-damaging activities by glucoraphanin, the bioprecursor of the chemopreventive agent sulforaphane found in broccoli.
The reduced cancer risk that appears to be linked to a diet rich in fruits and vegetables has fueled the belief that regular intake of isolated phytochemicals could potentially prevent cancer. In recent years, the glucosinolate metabolites derived from cruciferous vegetables, such as the isothiocyanate sulforaphane in broccoli, have gained ... More