ABSTRACT
Background: Deletions that encompass 2q31.1 have been proposed as a microdeletion syndrome with common clinical features, including intellectual disability/developmental delay, microcephaly, cleft palate, growth delay, and hand/foot anomalies. In addition, several genes within this region have been proposed as candidates for split hand-foot malformation 5 (SHFM5). Methods: To delineate the genotype-phenotype correlation between deletions of this region, we identified 14 individuals with deletions at 2q31.1 detected by microarray analysis for physical and developmental disabilities. Results: All subjects for whom detailed clinical records were available had neurological deficits of varying degree. Seven subjects with deletions encompassing the HOXD cluster had hand/foot anomalies of varying severity, including syndactyly, brachydactyly, and ectrodactyly. Of 7 subjects with deletions proximal to the HOXD cluster, 5 of which encompassed DLX1/DLX2, none had clinically significant hand/foot anomalies. In contrast to previous reports, the individuals in our study did not display a characteristic gestalt of dysmorphic facial features. Conclusion: The absence of hand/foot anomalies in any of the individuals with deletions of DLX1/DLX2 but not the HOXD cluster supports the hypothesis that haploinsufficiency of the HOXD cluster, rather than DLX1/DLX2, accounts for the skeletal abnormalities in subjects with 2q31.1 microdeletions.
ABSTRACT
1. The aim was to compare the metabolic activity of human CYP3A4 expressed in bacteria (E. coli), yeast (S. cerevisiae) and human lymphoblastoid cells (hBl), with the native CYP3A4 activity observed in a panel of human livers. 2. Three CYP3A4 substrates were selected for study: dextromethorphan (DEM), midazolam (MDZ) and diazepam (DZ). The substrate metabolism in each of the four systems was characterized by deriving the kinetic parameters K(m) or S(50), V(max) and intrinsic clearance (CL(int)) or maximum clearance (CL(max)) from the kinetic profiles; the latter differing by 100-fold across the three substrates. 3. The K(m) or S(50) for the formation of metabolites 3-methoxymorphinan (MEM), 1'-hydroxymidazolam (1'-OH MDZ) and 3-hydroxydiazepam (3HDZ) compared well in all systems. For CYP3A4-mediated metabolism of DEM, MDZ and DZ, the V(max) for hBl microsomes were generally 2-9-fold higher than the respective yeast and human liver microsomes and E. coli membrane preparations, resulting in greater CL(int) or CL(max). In the case of 3HDZ formation, non-linear kinetics were observed for E. coli, hBl microsomes and human liver microsomes, whereas the kinetics observed for S. cerevisiae were linear. 4. The use of native human liver microsomes for drug metabolic studies will always be preferable. However, owing to the limited availability of human tissues, we find it is reasonable to use any of the recombinant systems described herein, since all three recombinant systems gave good predictions of the native human liver enzyme activities.
Subject(s)
Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Escherichia coli/enzymology , Lymphocytes/enzymology , Saccharomyces cerevisiae/enzymology , Adult , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP3A , Dextromethorphan/pharmacology , Diazepam/pharmacology , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/pharmacology , Female , GABA Modulators/pharmacology , Humans , Kinetics , Liver/pathology , Male , Microsomes, Liver/metabolism , Midazolam/pharmacology , Middle Aged , Protein Binding , Recombinant Proteins/metabolism , Substrate Specificity , Temazepam/pharmacologyABSTRACT
Human and mouse liver microsomes and membranes isolated from Escherichia coli, which expressed cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6, were used to investigate CYP-mediated metabolism of five selected dietary flavonoids. In human and mouse liver microsomes kaempferol, apigenin and naringenin were hydroxylated at the 3'-position to yield their corresponding analogs quercetin, luteolin and eriodictyol, whereas hesperetin and tamarixetin were demethylated at the 4'-position to yield eriodictyol and quercetin, respectively. Microsomal flavonoid metabolism was potently inhibited by the CYP1A2 inhibitors, fluvoxamine and -naphthoflavone. Recombinant CYP1A2 was capable of metabolizing all five investigated flavonoids. CYP3A4 recombinant protein did not catalyze hesperetin demethylation, but showed similar metabolic profiles for the remaining compounds, as did human microsomes and recombinant CYP1A2, although the reaction rates in general were lower as compared to CYP1A2. CYP2C9 catalyzed the 4'-demethylation of tamarixetin, whereas CYP2D6 did not seem to play any role in the metabolism of the selected flavonoids. The major involvement in flavonoid metabolism of human CYP1A2, which mediates the formation of metabolites with different biochemical properties as compared to the parent compound and furthermore is known to be expressed very differently among individuals, raises the important question of whether individual differences in the CYP enzyme activity might affect the beneficial outcome of dietary flavonoids, rendering some individuals more or less refractory to the health-promoting potential of dietary flavonoids.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Flavonoids/metabolism , Animals , Biotransformation , Cytochrome P-450 Enzyme Inhibitors , Diet , Enzyme Inhibitors/pharmacology , Female , Food Analysis , Humans , In Vitro Techniques , Kinetics , Membranes/enzymology , Mice , Mice, Inbred C57BL , Microsomes, Liver/enzymology , Recombinant Proteins/metabolism , Species SpecificityABSTRACT
NADPH-P450 oxidoreductase (CPR) is essential for the activity of cytochrome P450 (P450). Previous studies demonstrated that CPR regulates the levels of various P450 isoforms in vitro. We investigated the mechanistic basis for this regulation. By transfection of Chinese hamster ovary DUKXB11 cells we obtained the cell line DUKX/2D6, which expressed human CYP2D6, a P450 isoform. Subsequently, DUKX/2D6 cells were transfected with human CPR cDNA to generate the cell line DUKX/2D6/CPR-3. Expression of recombinant CPR decreased the level of spectrally detectable CYP2D6 holoprotein in DUKX/2D6/CPR-3 cells by 70%, whereas the level of immunodetectable apoprotein remained unchanged. Addition of the radical scavenger DMSO increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 cells but not in DUKX/2D6 cells. A similar effect was noted when cells were grown in the presence of hemin. Importantly, combined treatment with DMSO and hemin increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 but not in DUKX/2D6 cells even further than either treatment alone. None of these treatments affected the level of immunodetectable CYP2D6. This demonstrates that expression of CPR increases production of damaging radicals but also that CPR may alter haem homoeostasis. In agreement with this, the activity of haem oxygenase, a rate-limiting enzyme in haem metabolism, was compared with that in DUKX/DHFR control cells (expressing dihydrofolate reductase), and was 3-fold higher in DUKX/2D6/CPR-3 but similar in DUKX/2D6 cells. Furthermore, treatment of cells with sodium arsenite increased levels of haem oxygenase concomitant with a marked decrease of spectrally detectable CYP2D6 and a rise in levels of ferritin, which sequesters free iron released from the destruction of haem. These data demonstrate that CPR regulates P450 activity by supplying electrons and also by altering P450 levels via radical-and haem oxygenase-mediated pathways.
Subject(s)
Cytochrome P-450 CYP2D6/metabolism , Heme Oxygenase (Decyclizing)/metabolism , NADPH-Ferrihemoprotein Reductase/metabolism , Animals , Base Sequence , CHO Cells , Cricetinae , DNA Primers/genetics , Dimethyl Sulfoxide/pharmacology , Hemin/pharmacology , Holoenzymes/metabolism , Homeostasis , Humans , In Vitro Techniques , NADPH-Ferrihemoprotein Reductase/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
Melatonin, the predominant product of the pineal gland, is involved in the maintenance of diurnal rhythms. Nocturnal blood concentrations of melatonin have been shown to be enhanced by fluvoxamine, but not by other serotonin reuptake inhibitors. Because fluvoxamine is an inhibitor of several cytochrome P450 (CYP) enzymes, the authors studied the biotransformation of melatonin and the effects of fluvoxamine on the metabolism of melatonin in vitro using human liver microsomes and recombinant human CYP isoenzymes. Melatonin was found to be almost exclusively metabolized by CYP1A2 to 6-hydroxymelatonin and N-acetylserotonin with a minimal contribution of CYP2C19. Both reactions were potently inhibited by fluvoxamine, with a Ki of 0.02 microM for the formation of 6-hydroxymelatonin and 0.05 microM for the formation of N-acetylserotonin. Other than fluvoxamine, fluoxetine, paroxetine, citalopram, imipramine, and desipramine were also tested at 2 and 20 microM. Among the other antidepressants, only paroxetine was able to affect the metabolism of melatonin at supratherapeutic concentrations of 20 microM, which did not reach by far the magnitude of the inhibitory potency of fluvoxamine. The authors concluded that fluvoxamine is a potent inhibitor of melatonin degradation. Because this inhibitory action is also found in vivo, fluvoxamine might be used as an enhancer of melatonin, which might offer new therapeutic possibilities of fluvoxamine.
Subject(s)
Antidepressive Agents, Second-Generation/pharmacology , Cytochrome P-450 CYP1A2/metabolism , Fluvoxamine/pharmacology , Melatonin/analogs & derivatives , Melatonin/metabolism , Serotonin/metabolism , Cytochrome P-450 CYP1A2/drug effects , Enzyme Inhibitors/pharmacology , Humans , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Serotonin/analogs & derivatives , Theophylline/analogs & derivatives , Theophylline/pharmacologyABSTRACT
Some compounds used for phenotyping of cytochrome P450s are substrates of P-glycoprotein (pgp). It is likely that in these cases, the level of pgp modulates the metabolism of in vivo probes. To address this important issue, we have analyzed the effects of pgp on CYP3A4-mediated reactions in two newly established cell lines (3A4/HR/MDR(-) and 3A4/HR/MDR(+)), which express CYP3A4 in the absence and presence of pgp, respectively. In cultured cells, the presence of pgp increased the apparent K(m) for the 6beta-hydroxylase activity of CYP3A4 toward testosterone and cortisol by a factor of 1.7 and 4, respectively. These steroids are poor and good substrates of pgp, respectively, and cortisol 6beta-hydroxylase has been frequently used as an in vivo probe for CYP3A4. Interestingly, we also found that pgp modulated the inhibition of CYP3A4-mediated metabolism by several compounds in intact cells. Although quinidine inhibited testosterone 6beta-hydroxylase activity in membranes or in intact cells that expressed recombinant CYP3A4 in the absence of pgp, low concentrations of this compound increased CYP3A4 activity in intact cells that expressed pgp. These results imply that pharmacokinetic drug-drug interactions involving CYP3A4 can be influenced by pgp.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Antifungal Agents/pharmacology , Cardiotonic Agents/pharmacology , Cell Line , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Cytochrome P-450 CYP3A , Digoxin/pharmacology , Humans , Hydroxylation , Ketoconazole/pharmacology , NADPH-Ferrihemoprotein Reductase/metabolism , Phenotype , Steroid Hydroxylases/metabolism , TransfectionABSTRACT
A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent k(cat) of W676A is equivalent (90%) to the NADPH-dependent k(cat) of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent K(M)(NADPH) and K(M)(NADH) values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2'-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system.
Subject(s)
NADPH-Ferrihemoprotein Reductase/chemistry , NADPH-Ferrihemoprotein Reductase/metabolism , NAD/metabolism , Protein Engineering , Amino Acid Substitution , Binding Sites , Cytochrome c Group/metabolism , Enzyme Inhibitors/pharmacology , Escherichia coli , Ferricyanides/metabolism , Humans , Kinetics , Models, Biological , Mutation , NADP/metabolism , NADPH-Ferrihemoprotein Reductase/antagonists & inhibitors , NADPH-Ferrihemoprotein Reductase/genetics , Oxazines/metabolism , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrophotometry , Substrate SpecificityABSTRACT
The development of cancers of the breast, endometrium, ovaries and possibly prostate is modulated by steroid hormones. Many steroids and environmental carcinogens are subject to cytochrome P450 (P450)-mediated metabolism that generates reactive metabolites and modulates steroid potency, thereby influencing tumor initiation and promotion respectively. These pathways, which are modulated by polymorphisms in P450 genes, are therefore likely to play an important role in the etiology of hormone-related cancers. Several groups have evaluated genotypes of xenobiotic- and steroid-metabolizing P450 enzymes as risk factors for hormone-related cancers. Polymorphisms in P450s that are specifically involved in the metabolism of steroids appear to be single risk factors. The situation is less clear for xenobiotic-metabolizing P450s. For these genes, only combined genotypes of several P450s or combined genotypes of P450s together with other enzymes have been clearly correlated with disease frequency. Success in identifying the appropriate combination of candidate genes requires a thorough knowledge of the metabolic pathways and enzyme systems that control the initial stages of carcinogenesis, as will be illustrated in this review.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Gonadal Steroid Hormones/adverse effects , Neoplasms/genetics , Polymorphism, Genetic , Animals , Breast Neoplasms/genetics , Cytochrome P-450 Enzyme System/physiology , Genetic Predisposition to Disease , Gonadal Steroid Hormones/metabolism , Humans , Neoplasms/chemically induced , Neoplasms/enzymology , Neoplasms/etiology , Risk FactorsABSTRACT
Most drug metabolizing cytochrome P450s (P450) are predominantly expressed in the liver. In contrast, human CYP1B1 is an extrahepatic P450 which is overexpressed in many tumours and has been strongly implicated in the activation of carcinogens. Rare allelic variants of the CYP1B1 gene which encode an inactive protein have been identified. However, four polymorphisms which most likely do not abolish functionality have been described. In this report, we have characterized the functional consequences of these. A CYP1B1 cDNA, identical to a cDNA published previously, served as a template to introduce allelic changes either separately or in combination. The resulting effects on CYP1B1 activity were determined in membranes isolated from Escherichia coli which coexpressed CYP1B1 together with P450 reductase. None of the allelic changes affected the CYP1B1 expression level. The allelic changes Arg48 to Gly, Ala19 to Ser and Asn453 to Ser had little influence on the Vmax and the Km of the CYP1B1 mediated 2- and 4-hydroxylation of estradiol. In contrast, the Km of these metabolic pathways was increased at least three-fold by the allelic change Va432 to Leu or by simultaneously changing Val432 to Leu and Asn453 to Ser. However, these alterations had little effect on the kinetic parameters of other CYP1B1 mediated reactions such as the epoxidation of (-)-trans-(7R,8R)-benzo[a]pyrene 7,8-dihydrodiol as determined by (r-7,t-8,t-9,c-10)-benzo[a]pyrene tetraol formation, or such as the O-dealkylation of ethoxyresorufin and the 1'-hydroxylation of bufuralol. Molecular modelling suggests that amino acid residue 432 of CYP1B1 may be involved in the interaction between CYP1B1 and P450 reductase. Since 4-hydroxyestradiol has been implicated in hormonal carcinogenesis and CYP1B1 is expressed in target tissues, the data presented demonstrate that polymorphisms in CYP1B1 have the potential to affect disease susceptibility.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Carcinogens/metabolism , Cytochrome P-450 Enzyme System/genetics , Estradiol/analogs & derivatives , Estradiol/metabolism , Polymorphism, Genetic , Alleles , Base Sequence , Carcinogens/toxicity , Cytochrome P-450 CYP1B1 , DNA Primers , DNA, Complementary , Dihydroxydihydrobenzopyrenes/pharmacokinetics , Estradiol/toxicity , Estrogens, Catechol , Ethanolamines/pharmacokinetics , Humans , Hydroxylation , Kinetics , Mutagenesis, Site-Directed , Oxazines/pharmacokineticsABSTRACT
Vinca alkaloids are important chemotherapeutic agents, and their pharmacokinetic properties display significant interindividual variations, possibly due to CYP3A4-mediated metabolism. We have evaluated the relevance of this metabolism for the chemotherapeutic and the toxicological properties of these drugs. Analysis was performed using Chinese hamster ovary cell lines that expressed either CYP2D6 or CYP3A4. The latter cells metabolized vinblastine with a turnover number of 0.4 min(-1), resulting in a decreased cytotoxicity of this compound. Whereas vincristine and vinblastine at a concentration of 100 nM killed more than 90% of the parental cells, more than 50 and 35%, respectively, of cells that coexpressed CYP3A4 and cytochrome P450 (P450) reductase survived these treatments. No additional increase in cytotoxicity was noted above 100 nM. Similarly, preincubation of vinblastine with bacterial membranes that contained recombinant CYP3A4 and P450 reductase decreased the cytotoxicity of vinblastine for parental Chinese hamster ovary cells. We also demonstrate that the presence of vinblastine in a coculture of cells that expressed beta-galactosidase together with cells that expressed CYP3A4 strongly selected for the latter cells, resulting in an increased level of CYP3A4 in the surviving cell population. Similarly, treatment of the human colon adenocarcinoma cell line LS174T with vinblastine selected for a cell population with higher levels of endogenous CYP3A4 as revealed by immunohistochemistry without simultaneous increase of multidrug resistance protein 1 (MDR1). This is the first evidence that tumor P450s have the potential to contribute to the development of drug resistance during chemotherapy.
Subject(s)
Antineoplastic Agents, Phytogenic/metabolism , Cytochrome P-450 Enzyme System/physiology , Mixed Function Oxygenases/physiology , Vinblastine/metabolism , Vincristine/metabolism , Animals , CHO Cells , Cricetinae , Cytochrome P-450 CYP3A , Drug Resistance, Neoplasm , Humans , Inactivation, Metabolic , Vinblastine/toxicity , Vincristine/toxicityABSTRACT
Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Indans/pharmacology , Mixed Function Oxygenases/antagonists & inhibitors , Piperazines/pharmacology , Binding Sites , Catalysis , Cell Membrane/metabolism , Chromatography, High Pressure Liquid , Computer Simulation , Cyanogen Bromide/metabolism , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/isolation & purification , Cytochrome P-450 Enzyme System/metabolism , Dialysis , Escherichia coli/cytology , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/metabolism , HIV Protease Inhibitors/pharmacology , Humans , Hydroxylation , Indans/chemistry , Indans/metabolism , Microsomes, Liver/enzymology , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/isolation & purification , Mixed Function Oxygenases/metabolism , Models, Chemical , Models, Molecular , NADP/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Piperazines/chemistry , Piperazines/metabolism , Protein Binding , Protein Structure, Secondary , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/metabolismABSTRACT
Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wild-type CYP3A4. The A305F mutation abolished diazepam oxidase activity and reduced the S(50) and V(max) for erythromycin N-demethylase activity from 17 to 10 microM and from 3.2 to 1.2 pmol product/min/pmol P450, respectively. The V(max) for testosterone 6beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P450, whereas the S(50) increased from 33 to 125 microM. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S(50) increased from 9 to 42 microM. The K(i) for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 microM, from 0.052 to 0.73 microM, and from 0.043 to 2.2 microM by the A305F mutation when measured against erythromycin, nifedipine, and testosterone metabolism activities, respectively. Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 15-fold by the A305F mutation. In contrast, the A305S mutation increased testosterone 6beta-hydroxylase (V(max) = 2.9 pmol product/min/pmol P450) and erythromycin N-demethylase (V(max) = 5.1 pmol product/min/pmol P450) activities, but reduced nifedipine oxidase activity (V(max) = 4.6 pmol product/min/pmol P450). K(i) values for ketoconazole and other azole inhibitors were unchanged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the catalytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.
Subject(s)
Alanine/metabolism , Amino Acid Substitution/genetics , Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Mixed Function Oxygenases/metabolism , Multienzyme Complexes/metabolism , Alanine/genetics , Binding Sites , Catalysis/drug effects , Clotrimazole/pharmacology , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/genetics , Diazepam/metabolism , Econazole/pharmacology , Erythromycin/metabolism , Humans , Imidazoles/chemistry , Imidazoles/metabolism , Imidazoles/pharmacology , Ketoconazole/metabolism , Ketoconazole/pharmacology , Kinetics , Miconazole/pharmacology , Mixed Function Oxygenases/antagonists & inhibitors , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Mutation/genetics , Nifedipine/metabolism , Oxidoreductases, N-Demethylating/antagonists & inhibitors , Oxidoreductases, N-Demethylating/chemistry , Oxidoreductases, N-Demethylating/genetics , Oxidoreductases, N-Demethylating/metabolism , Substrate Specificity , Testosterone/metabolismABSTRACT
The major drug-metabolizing human hepatic cytochrome P-450s (CYPs; CYP1A2, 2C9, 2C19, 2D6, and 3A4) coexpressed functionally in Escherichia coli with human NADPH-P-450 reductase have been validated as surrogates to their counterparts in human liver microsomes (HLM) using automated technology. The dealkylation of ethoxyresorufin, dextromethorphan, and erythromycin were all shown to be specific reactions for CYP1A2, CYP2D6, and CYP3A4 that allowed direct comparison with kinetic data for HLM. For CYP2C9 and CYP2C19, the kinetics for the discrete oxidations of naproxen and diazepam were compared to data obtained using established, commercial CYP preparations. Turnover numbers of CYPs expressed in E. coli toward these substrates were generally equal to or even greater than those of the major commercial suppliers [CYP1A2 (ethoxyresorufin), E. coli 0.6 +/- 0.2 min(-1) versus B lymphoblasts 0.4 +/- 0.1 min(-1); CYP2C9 (naproxen), 6.7 +/- 0.9 versus 4.9 min(-1); CYP2C19 (diazepam), 3.7 +/- 0.3 versus 0.2 +/- 0.1 min(-1); CYP2D6 (dextromethorphan), 4.7 +/- 0.1 versus 4.4 +/- 0.1 min(-1); CYP3A4 (erythromycin), 3 +/- 1.2 versus 1.6 min(-1)]. The apparent K(m) values for the specific reactions were also similar (K(m) ranges for expressed CYPs and HLM were: ethoxyresorufin 0.5-1.0 microM, dextromethorphan 1.3-5.9 microM, and erythromycin 18-57 microM), indicating little if any effect of N-terminal modification on the E. coli-expressed CYPs. The data generated for all the probe substrates by HLM and recombinant CYPs also agreed well with literature values. In summary, E. coli-expressed CYPs appear faithful surrogates for the native (HLM) enzyme, and these data suggest that such recombinant enzymes may be suitable for predictive human metabolism studies.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Escherichia coli/enzymology , Liver/enzymology , Pharmaceutical Preparations/metabolism , Cytochrome P-450 Enzyme System/chemistry , Humans , Isoenzymes/metabolism , Kinetics , Plasmids/metabolism , Predictive Value of Tests , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Cytochrome P450s (P450) form a superfamily of membrane-bound proteins that play a key role in the primary metabolism of both xenobiotics and endogenous compounds such as drugs and hormones, respectively. To be enzymically active, they require the presence of a second membrane-bound protein, NADPH P450 reductase, which transfers electrons from NADPH to the P450. Because of the diversity of P450 enzymes, much of the work on individual forms has been carried out on purified proteins, in vitro, which requires the use of complex reconstitution mixtures to allow the P450 to associate correctly with the NADPH P450 reductase. There is strong evidence from such reconstitution experiments that, when cytochrome b5 is included, the turnover of some substrates with certain P450s is increased. Here we demonstrate that allowing human P450 reductase, CYP3A4, and cytochrome b5 to associate in an in vivo-like system, by coexpressing all three proteins together in Escherichia coli for the first time, the turnover of both nifedipine and testosterone by CYP3A4 is increased in the presence of cytochrome b5. The turnover of testosterone was increased by 166% in whole cells and by 167% in preparations of bacterial membranes. The coexpression of cytochrome b5 also resulted in the stabilization of the P450 during substrate turnover in whole E. coli, with 109% of spectrally active CYP3A4 remaining in cells after 30 min in the presence of cytochrome b5 compared with 43% of the original P450 remaining in cells in the absence of cytochrome b5.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Cytochromes b5/metabolism , Mixed Function Oxygenases/metabolism , Cloning, Molecular , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Cytochromes b5/genetics , Enzyme Stability , Escherichia coli/genetics , Humans , Inactivation, Metabolic , Mixed Function Oxygenases/genetics , Nifedipine/metabolism , Recombinant Proteins/metabolism , Testosterone/metabolismABSTRACT
A wide variety of pharmacological and toxicological properties of drugs are determined by cytochrome P450-mediated metabolism. Characterization of these pathways and of the P450 isoenzymes involved constitutes an essential part of drug development. Similarly, because P450s are catalyzing the toxication and detoxication of environmental pollutants, an understanding of these reactions facilitates risk assessment in environmental toxicology. Recently, a variety of recombinant expression systems has been employed to study the role of human P450s in these reactions. These include insect, bacterial, yeast, and mammalian models. As these were developed and characterized by different laboratories, evaluation of their merits and limitations is inherently difficult. To resolve this problem, we have established and characterized the latter three systems and present the key results here. In general, the catalytic properties of P450 isozymes in the various models were rather similar. However, taking technical considerations into account as well as the high level of functional expression of P450s achieved in bacteria make this system ideally suited for drug metabolism research, including the generation of milligram quantities of metabolites for structural determinations. For toxicological studies, however, expression of P450s in mammalian cells was most appropriate. This is exemplified here by studies into the role of human P450s in the activation and inactivation of chemotherapeutic drugs.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Pharmaceutical Preparations/metabolism , Amino Acid Sequence , Animals , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/metabolism , Drug-Related Side Effects and Adverse Reactions , Gene Expression , Humans , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
NADPH-cytochrome P-450 oxidoreductase (CPR) is essential for the catalytic activity of cytochrome P-450 (P-450). On a molar basis, the amount of P-450 exceeds that of CPR in human liver. In this study, we investigated whether drug-drug interactions can occur as a result of competition between P-450 isozymes for this ancillary protein. For this purpose, combinations of P-450 isozymes were coexpressed together with P-450 reductase in Escherichia coli. We show that testosterone inhibited the CYP2D6-mediated bufuralol 1'-hydroxylase activity in bacterial membranes containing both CYP2D6 and CYP3A4 but not in membranes containing CYP2D6 alone. Conversely, bufuralol inhibited the CYP3A4-mediated testosterone 6beta-hydroxylase activity in bacterial membranes containing both CYP3A4 and CYP2D6 but not in membranes containing only CYP3A4. In each case, inhibition was seen even at a P-450 to P-450 reductase ratio of 1.9:1, which is more favorable than the ratio of 4 reported for human liver. The physiological significance of this mechanism was demonstrated by the observation that testosterone inhibited several prototypical P-450 enzyme activities, such as bufuralol 1'-hydroxylase, coumarin 7-hydroxylase, and 7-ethoxyresorufin O-dealkylase, in human liver microsomes, but not if tested against a panel of bacterial membranes containing the human P-450 isozymes that mainly catalyze these reactions.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Ethanolamines/metabolism , NADPH-Ferrihemoprotein Reductase/metabolism , Testosterone/metabolism , Cytochrome P-450 CYP1A1/antagonists & inhibitors , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2A6 , Cytochrome P-450 CYP2D6/biosynthesis , Cytochrome P-450 CYP2D6/genetics , Cytochrome P-450 CYP2D6/metabolism , Cytochrome P-450 CYP2D6 Inhibitors , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/genetics , Drug Interactions , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Escherichia coli/genetics , Humans , In Vitro Techniques , Isoenzymes/antagonists & inhibitors , Isoenzymes/biosynthesis , Isoenzymes/genetics , Isoenzymes/metabolism , Membranes , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Mixed Function Oxygenases/antagonists & inhibitors , Mixed Function Oxygenases/biosynthesis , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , NADPH-Ferrihemoprotein Reductase/antagonists & inhibitors , NADPH-Ferrihemoprotein Reductase/biosynthesis , NADPH-Ferrihemoprotein Reductase/genetics , Plasmids , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Steroid Hydroxylases/antagonists & inhibitors , Steroid Hydroxylases/metabolism , Testosterone/pharmacologyABSTRACT
A general scheme for the purification of baculovirus-expressed cytochrome P450s (P450s) from the crude insect cell pastes has been designed which renders the P450s suitable for analysis by high-performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESI-MS). An HPLC/ESI-MS procedure has been developed to analyze small amounts of intact purified P450 (P450s cam-HT, 1A1, 1A2, 2A6, 2B1, 2C9, 2C9 C175R, 3A4, 3A4-HT) and rat NADPH cytochrome P450 reductase (P450 reductase). The experimentally determined and predicted (based on the amino acid sequences) molecular masses (MMs) of the various proteins had identical rank orders. For each individual protein, the difference between the experimentally determined (+/-SD, based on experiments performed on at least 3 different days) and predicted MMs ranged from 0.002 to 0.035%. Each experimentally determined MM had a standard deviation of less than 0.09% (based on the charge state distribution). Application of this HPLC/ESI-MS technique made the detection of the covalent modification to P450 2C9 following mechanism-based inactivation by tienilic acid possible. In the absence of glutathione, three P450 2C9 species were detected that produced ESI mass spectra corresponding to native P450 2C9 and both a monoadduct and a diadduct of tienilic acid to P450 2C9. In the presence of glutathione, only native P450 2C9 and the monoadduct were detected. Based on the observed mass shifts for the P450 2C9/tienilic acid adducts, a mechanism for the inactivation of P450 2C9 by tienilic acid is proposed.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/chemistry , Steroid 16-alpha-Hydroxylase , Steroid Hydroxylases/chemistry , Ticrynafen/chemistry , Animals , Baculoviridae/genetics , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/isolation & purification , Electron Spin Resonance Spectroscopy , Electrons , Enzyme Activation , Genetic Vectors , Humans , Rats , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Steroid Hydroxylases/antagonists & inhibitors , Steroid Hydroxylases/genetics , Steroid Hydroxylases/isolation & purificationABSTRACT
Some xenobiotics induce membrane-bound drug metabolizing enzymes (Xme) and a profound proliferation of the endoplasmic reticulum (ER) in vivo. However these effects are much weaker in vitro, possibly due to absence of certain transcription factors. We tested the possibility that ER proliferation can affect the level of ER-resident enzymes even in the absence of transcriptional activation. For this purpose we analysed the effects of compactin, which has been shown to induce ER proliferation in vitro, on recombinant Xme, which were expressed from a constitutive viral promoter. High levels of recombinant UDP-glucuronosyltransferase UGT1A6 were achieved by amplification of the UGT1A6 cDNA using the dihydrofolate reductase cDNA as selectable marker in DHFR- CHO cells. Treatment of the resulting cell lines with lipoprotein-deficient serum in the absence and presence of compactin for 5 days resulted in a 1.3- and 2.3-fold, respectively, increase of the UGT enzyme activity towards 4-methylumbelliferone, paralleled by an induction of immunoreactive UGT1A6 protein. Similarly, treatment with this 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor increased the endogenous P450 reductase activity 2.6-fold, concomitant with an increase of immunodetectable protein. As expected compactin induced the level of 3-hydroxy-3-methylglutaryl-CoA reductase. Increased levels of this protein have been associated with a proliferation of the ER. Compactin treatment of a separate cell line that expressed recombinant human P450 reductase increased this enzyme activity fivefold. Pulse-chase experiments revealed that the induction of the recombinant Xme by compactin was most likely due to decreased protein degradation. Our results show that enzyme systems unrelated to those involved in cholesterol biosynthesis are affected by compounds known to affect membrane biogenesis. Since this effect extends to heterologously expressed enzymes, it also provides an efficient means by which to increase the levels of recombinant ER proteins.
Subject(s)
Endoplasmic Reticulum/drug effects , Enzyme Inhibitors/pharmacology , Glucuronosyltransferase/metabolism , Hydroxymethylglutaryl CoA Reductases/metabolism , Lovastatin/analogs & derivatives , Recombinant Proteins/metabolism , Animals , Blood Proteins/pharmacology , CHO Cells , Cell Line , Cricetinae , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/metabolism , Enzyme Induction/drug effects , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Immunohistochemistry , Lovastatin/pharmacology , NADH, NADPH Oxidoreductases/metabolism , NADPH-Ferrihemoprotein Reductase , RNA, Messenger/metabolism , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolism , TransfectionABSTRACT
Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)-configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.