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
Monoclonal antibody-directed characterization of benzene, ethoxyresorufin and pentoxyresorufin metabolism in rat liver microsomes.
AuthorsNakajima T, Elovaara E, Park SS, Gelboin HV, Hietanen E, Vainio H
JournalBiochem Pharmacol
PubMed ID2403379
'The contribution of cytochrome P450IA, P450IIB, P450IICII and P450IIEI to the oxidative metabolism of benzene, 7-ethoxyresorufin and 7-pentoxyresorufin was investigated using monoclonal antibodies (MAb) in liver microsomes from fed, one-day fasted, phenobarbital (PB)-, 3-methylcholanthrene (MC)- and ethanol-treated rats. Overall catalytic activity varied with different pretreatments and thereby the contribution of ... 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
Regulation of CYP1A1 by indolo[3,2-b]carbazole in murine hepatoma cells.
AuthorsChen YH, Riby J, Srivastava P, Bartholomew J, Denison M, Bjeldanes L
JournalJ Biol Chem
PubMed ID7673247
'To determine the basis for unexpected differences in CYP1A1 inducing potencies and efficacies for the diet-derived indole derivative, indolo[3,2-b]carbazole (ICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we conducted a systematic analysis of events involved in the induced expression of CYP1A1 in murine hepatoma-derived cell lines (Hepa-1). In contrast to the effects of TCDD, ... 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
Intralobular localization of different cytochrome P-450 form dependent monooxygenase activities in the liver of normal and inducer-treated rats.
AuthorsSerasinghe P, Yamazaki H, Nishiguchi K, Serasinghe S, Nakanishi S
JournalInt J Biochem
PubMed ID1612185
'1. Isolated periportal (PP) and perivenous (PV) hepatocytes from normal and inducer-treated rat livers were used to examine the following: intralobular localization of cytochrome P-450IA, P-450IIB, P-450IIE and P-450IIIA dependent monooxygenase activities and effects of phenobarbital (PB), beta-naphthoflavone (BNF) and pregnenolone-16 alpha-carbonitrile (PCN) on the zonal induction of these monooxygenases. ... More
Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase.
AuthorsDutton DR, Reed GA, Parkinson A
JournalArch Biochem Biophys
PubMed ID2464338
'The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The ... More
Distribution of cytochrome P450 activities towards alkoxyresorufin derivatives in rat brain regions, subcellular fractions and isolated cerebral microvessels.
AuthorsPerrin R, Minn A, Ghersi-Egea JF, Grassiot MC, Siest G
JournalBiochem Pharmacol
PubMed ID2242042
'The regional and subcellular distributions of rat brain cytochrome P450 and cytochrome P450-dependent activities were examined. Cytochrome P450 was found to be mainly localized in mitochondria in all the six cerebral regions studied. The activities of the isoforms mostly implicated in drug metabolism, cytochromes P450 b and c, were measured ... More
Interactions among P450 enzymes when combined in reconstituted systems: formation of a 2B4-1A2 complex with a high affinity for NADPH-cytochrome P450 reductase.
AuthorsBackes WL, Batie CJ, Cawley GF
JournalBiochemistry
PubMed ID9737863
'The purpose of this study is to characterize the interactions among P450 1A2, P450 2B4, and P450 reductase in mixed reconstituted systems. Previously, our laboratory demonstrated that in the presence of certain substrates, 1A2 can influence the catalytic characteristics of 2B4 [Cawley et al. (1995) Biochemistry 34, 1244-1247]. The goal ... More
A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates.
AuthorsDonato MT, Gómez-Lechón MJ, Castell JV
JournalAnal Biochem
PubMed ID8238878
'This study deals with the development of a sensitive and simple microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes. The method is based on the metabolism by cells cultured in microwells of appropriate substrates at noncytotoxic concentrations (8 microM 7-ethoxyresorufin and 15 microM 7-pentoxyresorufin). ... 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
Human adult hepatocytes in primary monolayer culture. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism.
'The stabilities of several drug oxidation and conjugation pathways in human adult hepatocytes have been investigated during 72 hr culture. Cytochrome P-450-dependent mixed function oxidase was measured by the O-dealkylations of ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD), which are probes for different isozymes of cytochrome P-450 in the rat. ... More
Inhibition and inactivation of murine hepatic ethoxy- and pentoxyresorufin O-dealkylase by naturally occurring coumarins.
AuthorsCai Y, Bennett D, Nair RV, Ceska O, Ashwood-Smith MJ, DiGiovanni J
JournalChem Res Toxicol
PubMed ID8117927
'The present study was designed to evaluate the effects of a series of natural coumarins on ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activities in vitro using hepatic tissues from SENCAR mice. Fifteen different coumarins were examined for potential modulating activities. Several naturally occurring coumarins, found in the human diet, ... More
Polycyclic aromatic hydrocarbons cause hepatic porphyria in iron-loaded C57BL/10 mice: comparison of uroporphyrinogen decarboxylase inhibition with induction of alkoxyphenoxazone dealkylations.
AuthorsFrancis JE, Smith AG
JournalBiochem Biophys Res Commun
PubMed ID3606611
'Multiple doses of beta-naphthoflavone to iron-loaded C57BL/10ScSn mice for 6 weeks caused inhibition of hepatic uroporphyrinogen decarboxylase and a porphyria indistinguishable from that previously only reported for polyhalogenated aromatic chemicals. beta-Naphthoflavone and other polycyclic aromatic hydrocarbon inducers of cytochrome P1-450-mediated ethoxyphenoxazone deethylation (ethoxyresorufin deethylase), benzo[a]pyrene, benz[a]anthracene, dibenz[ah]anthracene, 3-methylcholanthrene and alpha-naphthoflavone, ... 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
Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat.
'The O-dealkylation of pentoxyresorufin (7-pentoxyphenoxazone) by rat liver microsomes was examined. The reaction appeared highly specific for certain phenobarbital inducible forms of cytochrome P-450 and was increased 95- to 140-fold by animal pretreatment with phenobarbital (75 mg/kg/day, four ip injections) and approximately 50-fold by Aroclor 1254 (500 mg/kg, one ip ... 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
Suppression of levels of phenobarbital-inducible rat liver cytochrome P-450 by pituitary hormone.
AuthorsYamazoe Y, Shimada M, Murayama N, Kato R
JournalJ Biol Chem
PubMed ID3584119
'The effect of pituitary factor on the constitutive and inducible levels of hepatic phenobarbital (PB)-inducible major cytochrome P-450, P-450b and P-450e, in male and female rat livers was studied by immunoblot analyses. Although only trace amounts (approximately 4 pmol/mg protein) of P-450b and P-450e were detected in untreated adult rats, ... 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
Cytochrome P-450-dependent mixed-function oxidase and glutathione S-transferase activities in spontaneous obesity-diabetes.
AuthorsBarnett CR, Abbott RA, Bailey CJ, Flatt PR, Ioannides C
JournalBiochem Pharmacol
PubMed ID1575780
The effect of non-insulin-dependent diabetes on the hepatic microsomal cytochrome P450-dependent mixed-function oxidase system and on cytosolic glutathione S-transferase activity was determined using the spontaneously obese-diabetic (ob/ob) mouse model. The activities of the xenobiotic-metabolizing cytochrome P450 proteins were monitored by the use of chemical probes. Non-insulin-dependent diabetes did not influence ... More
Pre-translational induction of pentoxyresorufin O-depentylase by pyridine.
AuthorsPark KS, Sohn DH, Veech RL, Song BJ
JournalBiochem Biophys Res Commun
PubMed ID1610360
Pentoxyresorufin O-depentylase activity, mainly associated with phenobarbital-inducible cytochrome P450IIB1 (designated CYP2B1), was increased after a single treatment of pyridine (250 mg/kg, i.p.), and further increased by repeated treatments for 5 days. The catalytic activity and immunoreactive protein of CYP2B recognized by polyclonal antibodies were significantly induced by a relatively high ... 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
Fluorescence assay for per-cell estimation of cytochrome P-450-dependent monooxygenase activities in keratinocyte suspensions and cultures.
AuthorsReiners JJ, Cantu AR, Pavone A, Smith SC, Gardner CR, Laskin DL
JournalAnal Biochem
PubMed ID2221380
An assay was characterized that facilitated per-cell estimation of cytochrome P-450-dependent monooxygenase activities in whole-cell suspensions and cultures of murine epidermal keratinocytes (MEKs). 7-Ethoxycoumarin O-deethylase (7-ECD), 7-ethoxyresorufin O-deethylase (7-ERD), and 7-pentoxyresorufin O-deethylase (7-PRD) activities were monitored by fluorescent detection of their products. MEKs were made permeable by a freeze-thaw cycle, ... More
Induction by xenobiotics of phase I and phase II enzyme activities in the human keratinocyte cell line NCTC 2544.
AuthorsGelardi A, Morini F, Dusatti F, Penco S, Ferro M
JournalToxicol In Vitro
PubMed ID11698172
This study analyses the expression and induction of several drug-metabolising enzyme activities involved in either phase I or phase II biotransformations in NCTC 2544 human keratinocytes. The phase I activities 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depenthylase (PROD) were easily detectable in basal conditions. During incubations lasting up ... More
Phenobarbital induction of CYP1A1 gene expression in a primary culture of rainbow trout hepatocytes.
In mammals, phenobarbital (PB) is an in vivo inducer of the cytochrome P4502B (CYP2B) family, whereas in teleosts PB induction of cytochrome P450 is unclear. We show that teleost cytochrome P4502K1 (CYP2K1) protein levels and 7-pentoxyresorufin-O-deethylase activity were not induced by exposure of primary cultures of rainbow trout hepatocytes to ... More
Probing the active site of cytochrome P450 2B1: metabolism of 7-alkoxycoumarins by the wild type and five site-directed mutants.
AuthorsKobayashi Y, Fang X, Szklarz GD, Halpert JR
JournalBiochemistry
PubMed ID9578551
A series of 7-alkoxycoumarins (chain length of 1-7 carbon atoms) was utilized as active site probes of purified Escherichia coli-expressed cytochrome P450 2B1 wild type and five site-directed mutants (I114V, F206L, V363A, V363L, and G478S). The production of 7-hydroxycoumarin, the O-dealkylation product, by the wild-type enzyme exhibited a rank order ... More
Dealkylation of 7-methoxycoumarin as assay for measuring constitutive and phenobarbital-inducible cytochrome P450s.
Six alkyl ethers of 7-hydroxycoumarin, ranging from methoxy- to hexoxycoumarin, were studied for their NADPH-dependent metabolism by liver microsomes of male rats treated with phenobarbital (PB) or 3-methyl-cholanthrene (MC). The six alkyl ethers were metabolized by both types of microsomes, forming 7-hydroxycoumarin as the major product. Among the test compounds, ... More
Induction of alkoxyresorufin O-dealkylases and UDP-glucuronosyl transferase by phenobarbital and 3-methylcholanthrene in primary cultures of porcine ciliary epithelial cells.
AuthorsSakamoto S, Shichi H
JournalBiochem Pharmacol
PubMed ID1900006
Our previous studies have shown that drug-metabolizing activities in the eye are highest in the ciliary body, a tissue responsible for aqueous humor production. In this work, we have separated nonpigmented epithelial (NPE) cells and pigmented epithelial (PE) cells from porcine ciliary body and determined basal and induced activities of ... More
Assessment of DE-71, a commercial polybrominated diphenyl ether (PBDE) mixture, in the EDSP male and female pubertal protocols.
AuthorsStoker TE, Laws SC, Crofton KM, Hedge JM, Ferrell JM, Cooper RL
JournalToxicol Sci
PubMed ID14999130
DE-71, a commercial mixture, was used to test the sensitivity of the female and male pubertal protocol to detect thyroid active chemicals. These protocols are being evaluated for the U.S. EPA's Endocrine Disruptor Screening Program as part of a Tier I Screening Battery. To examine the ability of these protocols ... 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
Effects of polychlorinated biphenyls, kanechlor-500, on serum thyroid hormone levels in rats and mice.
AuthorsKato Y, Haraguchi K, Yamazaki T, Ito Y, Miyajima S, Nemoto K, Koga N, Kimura R, Degawa M
JournalToxicol Sci
PubMed ID12660360
Effects of a commercial polychlorinated biphenyls mixture, Kanechlor-500 (KC500), on the levels of serum thyroid hormones such as total thyroxine (T4) and triiodothyronine (T3) were examined comparatively in male Wistar rats and ddy mice. Serum T4 levels were significantly decreased in both rats and mice 4 days after a single ... More
Nitric oxide is a mediator of the decrease in cytochrome P450-dependent metabolism caused by immunostimulants.
AuthorsKhatsenko OG, Gross SS, Rifkind AB, Vane JR
JournalProc Natl Acad Sci U S A
PubMed ID7504296
Bacterial lipopolysaccharide (LPS) and a diverse array of other immunostimulants and cytokines suppress the metabolism of endogenous and exogenous substances by reducing activity of the hepatic cytochrome P450 mixed-function oxidase system. Although this effect of immunostimulants was first described almost 40 yr ago, the mechanism is obscure. Immunostimulants are now ... More
Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.
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 ... 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