Peroxynitrite-mediated nitration of tyrosine and inactivation of the catalytic activity of cytochrome P450 2B1.
AuthorsRoberts ES, Lin H, Crowley JR, Vuletich JL, Osawa Y, Hollenberg PF
JournalChem Res Toxicol
PubMed ID9760281
'The addition of peroxynitrite to purified cytochrome P450 2B1 resulted in a concentration-dependent loss of the NADPH- and reductase-supported or tert-butylhydroperoxide-supported 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 with IC50 values of 39 and 210 microM, respectively. After incubation of P450 2B1 with 300 microM peroxynitrite, the heme moiety was not ... More
Inactivation of cytochrome P450s 2B1, 2B4, 2B6, and 2B11 by arylalkynes.
'The time-dependent loss of the 7-ethoxy-4-trifluoromethylcoumarin (EFC) O-deethylase activity of rat P450 2B1, rabbit P450 2B4, or dog P450 2B11 by 1-ethynylnaphthalene (1EN), 2-ethynylnaphthalene (2EN), 2-(1-propynyl)naphthalene (2PN), 1-ethynylanthracene (1EA), 2-ethynylanthracene, 2-ethynylphenanthrene, 3-ethynylphenanthrene, 9-ethynylphenanthrene (9EPh), 9-(1-propynyl)phenanthrene (9PPh), 4-ethynylpyrene (4EP), and 4-(1-propynyl)biphenyl (4PbP) was investigated. The rate constants for inactivation by the ... More
Development of a human lymphoblastoid cell line constitutively expressing human CYP1B1 cDNA: substrate specificity with model substrates and promutagens.
AuthorsCrespi CL, Penman BW, Steimel DT, Smith T, Yang CS, Sutter TR
JournalMutagenesis
PubMed ID9106248
'An AHH-1 TK+/- cell derivative was developed that stably expresses human cytochrome P4501B1 (CYP1B1) cDNA in an extrachromosomal vector which confers resistance to 1-histidinol and co-expresses NADPH cytochrome P450 oxidoreductase (OR). The CYP1B1-expressing cell line was designated h1B1/OR. Microsomes prepared from CYP1B1 cDNA expressing cells exhibit elevated levels of 7-ethoxy-resorufin ... More
Comparison of in vitro activities of biotransformation enzymes in pig, cattle, goat and sheep.
AuthorsSzotáková B, Baliharová V, Lamka J, Nozinová E, Wsól V, Velík J, Machala M, Neca J, Soucek P, Susová S, Skálová L
JournalRes Vet Sci
PubMed ID14659728
'In vitro activities of cytochromes P450 (7-alkyl/aryloxyresorufin dealkyl(aryl)ases, testosterone hydroxylase/oxidase, 6-chlorzoxazone hydroxylase, 7-methoxy-4-trifluoromethyl-coumarin demethylase, and lauric acid hydroxylases), reductases of carbonyl group (toward metyrapone, daunorubicin, glyceraldehyde, and 4-pyridine-carboxaldehyde) and conjugation enzymes (p-nitrophenol-UDP-glucuronosyl transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-tranferase) in young adults, males, non-castrated (N=6) farm animals were studied and compared. Presence of proteins ... More
Examination of purported probes of human CYP2B6.
AuthorsEkins S, VandenBranden M, Ring BJ, Wrighton SA
JournalPharmacogenetics
PubMed ID9241656
'7-Ethoxy-4-trifluoromethylcoumarin (7-EFC) was examined as a substrate for cytochrome P450 (P450) in microsomes from human livers and expressed in B-lymphoblastoid cells. The O-deethylation of 7-EFC to 7-hydroxy-4-trifluoromethylcoumarin (7-HFC) varied over a liver bank (n = 19) by a factor of 13 (40-507 pmol min-1 mg-1 protein). When compared with the ... More
Threonine-205 in the F helix of p450 2B1 contributes to androgen 16 beta-hydroxylation activity and mechanism-based inactivation.
AuthorsLin HL, Zhang H, Waskell L, Hollenberg PF
JournalJ Pharmacol Exp Ther
PubMed ID12721329
'Four mutants of Thr-205 in cytochrome p450 2B1 were constructed and expressed in Escherichia coli. The Ser-, Ala-, and Val-mutants displayed stable reduced CO difference spectra and were able to metabolize 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, androstenedione, and benzphetamine. The Arg-mutant displayed an unstable reduced CO difference spectrum at 450 nm, was concomitantly ... More
Metabolism of N-nitrosobenzylmethylamine by human cytochrome P-450 enzymes.
AuthorsMorse MA, Lu J, Stoner GD, Murphy SE, Peterson LA
JournalJ Toxicol Environ Health A
PubMed ID10616189
'N-Nitrosobenzylmethylamine (NBzMA) is a potent esophageal carcinogen in rodents, and has been found as a dietary contaminant in certain areas of China where esophageal cancer is endemic. To determine which cytochrome P-450 enzymes in humans are primarily responsible for NBzMA metabolism, microsomes from lymphoblastoid cell lines expressing a panel of ... More
The mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine.
AuthorsJushchyshyn MI, Kent UM, Hollenberg PF
JournalDrug Metab Dispos
PubMed ID12485952
'Phencyclidine (PCP) was analyzed for its ability to inactivate human cytochrome p450 (p450) 2B6. PCP inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of p450 2B6 in a concentration-, time-, and NADPH-dependent manner and exhibited pseudo-first order kinetics. The K(I) was 10 microM, k(inact) was 0.01 min(-1), which corresponds to a t(1/2) of ... More
Mechanistic studies with N-benzyl-1-aminobenzotriazole-inactivated CYP2B1: differential effects on the metabolism of 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, and benzphetamine.
'Mechanistic studies with N-benzyl-1-aminobenzotriazole (BBT)-inactivated cytochrome P450 2B1 were conducted to determine which step(s) in the reaction cycle had been compromised. Stopped-flow studies, formation of the oxy-ferro intermediate, and analysis of products suggested that the reductive process was slower with the BBT-modified enzyme. The reduced rate of reduction alone could ... More
Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation.
AuthorsXie HJ, Yasar U, Lundgren S, Griskevicius L, Terelius Y, Hassan M, Rane A
JournalPharmacogenomics J
PubMed ID12629583
'The role of polymorphic CYP2B6 in cyclophosphamide (CPA) bioactivation was investigated in human liver microsomes. A total of 67 human liver specimens were first genotyped with respect to the CYP2B6*5 and CYP2B6*6 variant alleles. CYP2B6 apoprotein levels in 55 liver microsomal preparations were assessed by immunoblotting. 4-Hydroxy-CPA and hydroxy-bupropion were ... More
Mechanism-based inactivation of cytochrome P450 2B1 by 8-methoxypsoralen and several other furanocoumarins.
AuthorsKoenigs LL, Trager WF
JournalBiochemistry
PubMed ID9748325
'Of several furanocoumarins [5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 5-hydroxypsoralen (5-OH-P), 8-hydroxypsoralen (8-OH-P), 4'',5''-dihydro-8-MOP (DH-8-MOP), and psoralen (P)] tested as mechanism-based inactivators (MBIs) of purified reconstituted cytochrome P450 (P450) 2B1, 8-MOP was found to be the most potent (KI, kinact, and partition ratio of 2.9 microM, 0.34 min-1, and 1.3, respectively). The ... More
Further characterization of the expression in liver and catalytic activity of CYP2B6.
AuthorsEkins S, Vandenbranden M, Ring BJ, Gillespie JS, Yang TJ, Gelboin HV, Wrighton SA
JournalJ Pharmacol Exp Ther
PubMed ID9732386
'Previous studies in this laboratory have determined the lack of specificity of several antibody and substrate probes of CYP2B6. The goals of the current study were to examine the expression of CYP2B6 in a bank of human liver microsome (HLM) samples using a new specific monoclonal antibody (MAb 49-10-20) and ... More
Interaction of buprenorphine and its metabolite norbuprenorphine with cytochromes p450 in vitro.
AuthorsZhang W, Ramamoorthy Y, Tyndale RF, Sellers EM
JournalDrug Metab Dispos
PubMed ID12756210
'Buprenorphine is a thebaine derivative used in the treatment of heroin and other opiate addictions. In this study, the selective probe reactions for each of the major hepatic cytochromes P450 (P450s) were used to evaluate the effect of buprenorphine and its main metabolite norbuprenorphine on the activity of these P450s. ... More
A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man. Direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin.
AuthorsButers JT, Schiller CD, Chou RC
JournalBiochem Pharmacol
PubMed ID8240414
'The O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC) by liver microsomes has been assessed as a method for monitoring the activity of cytochrome P450. The principle advantage of this substrate is the formation of a fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC) which can be assayed directly in the reaction medium. For rat microsomes the deethylated ... More
Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes.
AuthorsDonato MT, Jiménez N, Castell JV, Gómez-Lechón MJ
JournalDrug Metab Dispos
PubMed ID15205384
'In this study we describe a battery of fluorescence assays for rapid measurement in intact cells of the activity of nine cytochromes P450 (P450s) involved in drug metabolism. The assays are based on the direct incubation of monolayers of cells expressing individual P450 enzymes with a fluorogenic substrate followed by ... More
Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450.
AuthorsBusby WF, Ackermann JM, Crespi CL
JournalDrug Metab Dispos
PubMed ID9929510
'The effects of methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied in vitro on nine individual, cDNAexpressed cytochrome P-450 activities (phenacetin O-deethylase for CYP1A1 and CYP1A2, coumarin 7-hydroxylase for CYP2A6, testosterone 6beta-hydroxylase for CYP3A4, 7-ethoxy-4-trifluoromethylcoumarin deethylase for CYP2B6, paclitaxel 6alpha-hydroxylase for CYP2C8, diclofenac 4''-hydroxylase for CYP2C9, S-mephenytoin 4-hydroxylase for ... More
Mechanism-based inactivation of cytochrome P450 2B1 by N-benzyl-1-aminobenzotriazole.
AuthorsKent UM, Bend JR, Chamberlin BA, Gage DA, Hollenberg PF
JournalChem Res Toxicol
PubMed ID9168259
'The kinetics of inactivation of cytochrome P450 2B1, the major phenobarbital inducible rat hepatic P450, by N-benzyl-1-aminobenzotriazole (BBT) were characterized. Purified, reconstituted P450 2B1 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylase activity was inhibited by BBT in a mechanism-based manner. The loss of O-deethylase activity followed pseudo-first-order kinetics and was NADPH and BBT dependent. ... More
High-performance liquid chromatographic method for measurement of cytochrome P450-mediated metabolism of 7-ethoxy-4-trifluoromethylcoumarin.
AuthorsMorse MA, Lu J
JournalJ Chromatogr B Biomed Sci Appl
PubMed ID9653975
An HPLC method for analysis of deethylation of 7-ethoxy-4-trifluoromethylcoumarin (ETFMC), a substrate of various enzymes of the cytochrome P450 superfamily, was developed. ETFMC was incubated at 37 degrees C with human hepatic microsomes or microsomes prepared from a lymphoblastoid cell line that expresses human CYP2B6. Under these conditions, the highly ... More
Effects of cytochrome P450 inhibitors on the biotransformation of fluorogenic substrates by adult male rat liver microsomes and cDNA-expressed rat cytochrome P450 isoforms.
AuthorsMakaji E, Trambitas CS, Shen P, Holloway AC, Crankshaw DJ,
JournalToxicol Sci
PubMed ID19858067
We have evaluated the use of a panel of six fluorogenic cytochrome P450 (CYP) substrates as a potential tool for rapid screening for global changes in CYP activity in rats under different physiological conditions. The biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 7-benzyloxy-4-(trifluoromethyl)-coumarin, 7-benzyloxyquinoline, 3-cyano-7-ethoxycoumarin, 7-methoxy-4-(trifluoromethyl)-coumarin, and 7-ethoxy-4-trifluoromethyl-coumarin by microsomes from adult male ... More
A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin analog.
AuthorsDeLuca JG, Dysart GR, Rasnick D, Bradley MO
JournalBiochem Pharmacol
PubMed ID3259881
The microsomal O-deethylation of a novel coumarin analog, 7-ethoxy-4-trifluoromethylcoumarin (EFC), to a fluorescent product was characterized. Results indicate that this analog provides a rapid, convenient and highly sensitive means to assay cytochrome P-450-mediated metabolism. Like microsomal 7-ethoxycoumarin (7-EC) O-deethylation, EFC O-deethylation responded to both phenobarbital was greater than that seen ... More
Development of a human lymphoblastoid cell line constitutively expressing human CYP1A1 cDNA: substrate specificity with model substrates and promutagens.
AHH-1 TK+/- cell derivatives were developed that stably express human CYP1A1 cDNA, and an AHH-1 TK+/- derivative expressing higher levels of CYP1A2 cDNA in extrachromosomal vectors which confer resistance to 1-histidinol. The CYP1A1-expressing cell lines, designated h1A1 and h1A1v2, differ by containing one and two CYP1A1 cDNA expression units per ... More
Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes.
AuthorsGhosal A, Hapangama N, Yuan Y, Lu X, Horne D, Patrick JE, Zbaida S
JournalBiopharm Drug Dispos
PubMed ID14689466
Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results ... More
Role of the alanine at position 363 of cytochrome P450 2B2 in influencing the NADPH- and hydroperoxide-supported activities.
Escherichia coli was used to express the two closely related cytochromes P450 2B1 and 2B2 and two mutants of 2B2 in which residues Gly-303 and Ala-363 were replaced by Ser and Val, respectively. The expressed proteins were partially purified and assayed for benzphetamine and n-octylamine (NOA) binding and 7-ethoxy-4-trifluoromethylcoumarin O-deethylation ... More
Mechanistic studies of 9-ethynylphenanthrene-inactivated cytochrome P450 2B1.
The mechanism of inactivation of the major phenobarbital-inducible cytochrome P450 of rat liver, P450 2B1, by 9-ethynylphenanthrene (9EPh) has been investigated. Matrix-assisted laser desorption ionization-mass spectrometry analysis of the cyanogen bromide-generated peptides from 9EPh-inactivated P450 2B1 confirmed the addition of a phenanthrylacetyl group to the peptide corresponding to residues 290 ... More
Metabolic inactivation of cytochrome P4502B1 by phencyclidine: immunochemical and radiochemical analyses of the protective effects of glutathione.
AuthorsSharma U, Roberts ES, Kent UM, Owens SM, Hollenberg PF
JournalDrug Metab Dispos
PubMed ID9029055
Phencyclidine (PCP) inactivates the 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity of P4502B1 in a reconstituted system containing NADPH-cytochrome P450 (P450) reductase (reductase) and L-alpha-phosphatidylcholine, dilauroyl in a time-, concentration-, and NADPH-dependent manner. Catalytic activity of the enzyme could not be restored upon reconstitution with fresh reductase, indicating that the effect was on the ... More
Structure-function analysis of human cytochrome P-450 2B6 using a novel substrate, site-directed mutagenesis, and molecular modeling.
AuthorsDomanski TL, Schultz KM, Roussel F, Stevens JC, Halpert JR
JournalJ Pharmacol Exp Ther
PubMed ID10454488
The structural basis for functional differences between human cytochrome P-450 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. Two ... More
Significance of glycine 478 in the metabolism of N-benzyl-1-aminobenzotriazole to reactive intermediates by cytochrome P450 2B1.
AuthorsKent UM, Hanna IH, Szklarz GD, Vaz AD, Halpert JR, Bend JR, Hollenberg PF
JournalBiochemistry
PubMed ID9305960
The effect of mutating Gly 478 to Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of the wild-type enzyme was completely inactivated by incubating with 1 microM BBT. The G478A mutant, however, was not inactivated by incubating with up to 10 ... More
P450 active site architecture and reversibility: inactivation of cytochromes P450 2B4 and 2B4 T302A by tert-butyl acetylenes.
AuthorsBlobaum AL, Harris DL, Hollenberg PF
JournalBiochemistry
PubMed ID15751959
The inactivations of P450 2B4 and the T302A mutant of 2B4 by tert-butyl acetylene (tBA) and the inactivation of 2B4 T302A by tert-butyl 1-methyl-2-propynyl ether (tBMP) have been investigated. tBA and tBMP inactivated both enzymes in a mechanism-based manner with the losses in enzymatic activity corresponding closely to losses in ... More
Inactivation of cytochrome P4502B1 by the monoamine oxidase inhibitors R-(-)-deprenyl and clorgyline.
AuthorsSharma U, Roberts ES, Hollenberg PF
JournalDrug Metab Dispos
PubMed ID8781784
The monoamine oxidase inhibitors R-(-)-deprenyl (deprenyl) and clorgyline inactivated the 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity of purified cytochrome P4502B1 (P4502B1) in a reconstituted system containing P4502B1, NADPH-cytochrome P450 oxidoreductase, and L-alpha-phosphatidylcholine dilauroyl as the lipid. The inactivation was time- and concentration-dependent. The inactivation required NADPH, demonstrated saturation kinetics, and no lag time ... More
Metabolism of N,N',N"-triethylenethiophosphoramide by CYP2B1 and CYP2B6 results in the inactivation of both isoforms by two distinct mechanisms.
AuthorsHarleton E, Webster M, Bumpus NN, Kent UM, Rae JM, Hollenberg PF
JournalJ Pharmacol Exp Ther
PubMed ID15121764
The anticancer drug N,N,"N"-triethylenethiophosphoramide (tTEPA) inactivated CYP2B6 and CYP2B1 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner indicative of mechanism-based inactivation. The KI value for the inactivation of CYP2B1 was 38 microM, the kinact was 0.3 min(-1), and the t1/2 value was 2.5 min. Spectral carbon monoxide ... More
A role for threonine 302 in the mechanism-based inactivation of P450 2B4 by 2-ethynylnaphthalene.
2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of P450 2B4 that covalently modifies an amino acid in the peptide Glu273-Met314 with a 2-naphthylacetyl group [Roberts et al. (1994) Biochemistry 33, 3766-3771]. Truncated 2B4 lacking amino acids 2-27, 2B4 (Delta2-27), was expressed in Escherichia coli, purified, and found to catalyze the oxidation ... More
Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by 2-phenyl-2-(1-piperidinyl)propane.
AuthorsChun J, Kent UM, Moss RM, Sayre LM, Hollenberg PF
JournalDrug Metab Dispos
PubMed ID10901699
2-Phenyl-2-(1-piperidinyl)propane (PPP), an analog of phencyclidine, was tested for its ability to inactivate cytochrome P450s (P450s) 2B1 and 2B6. PPP inactivated the 7-(benzyloxy)resorufin O-dealkylation activity of liver microsomes obtained from phenobarbital-induced rats with a K(I) of 11 microM. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified rat liver P450 2B1 and expressed ... More
Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation.
AuthorsCode EL, Crespi CL, Penman BW, Gonzalez FJ, Chang TK, Waxman DJ
JournalDrug Metab Dispos
PubMed ID9280407
The level of expression and interindividual variation in human hepatic microsomal cytochrome P450 (CYP) 2B6 was characterized using a polyclonal antibody (WB-2B6) raised against rat CYP2B1. Immunoblot analysis using cDNA-expressed human CYPs revealed strong cross-reactivity of this antibody with CYP2B6 (limit of detection < 0.05 pmol) and only minor cross-reactivities ... More