RESUMO
1. Recent guidance from the US Food and Drug Administration (USFDA) has advocated testing of time-dependent inhibition of cytochrome P450 (CYP), which can be addressed by performing IC(50) shift as well as K(I)/k(inact) determinations. 2. Direct (IC(50), K(i)) and time-dependent inhibition (IC(50) shift, K(I)/k(inact)) assays were validated in human liver microsomes with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis for the following enzyme/substrate/inhibitor combinations: CYP1A2/phenacetin/alpha-naphthoflavone/furafylline, CYP2C8/amodiaquine/montelukast/gemfibrozil-1-O-beta-glucuronide, CYP2C9/diclofenac/sulfaphenazole/tienilic acid, CYP2C19/S-mephenytoin/S-benzylnirvanol/S-fluoxetine, CYP2D6/dextromethorphan/quinidine/paroxetine, and CYP3A4/midazolam/testosterone/ketoconazole/azamulin/verapamil/diltiazem. IC(50) shift assays were performed with two pre-incubation time points (10 and 30 min) to facilitate k(inact) assay design. 3. Data obtained show good agreement with literature values. For rapid acting inhibitors, such as azamulin/CYP3A4 and tienilic acid/CYP2C9, the IC(50) shifts were similar at both time points suggesting a short maximum pre-incubation time with closely spaced time points for the K(I)/k(inact) assay. Slow acting inhibitors (such as verapamil/CYP3A4 or S-fluoxetine/CYP2C19) showed an increase in IC(50) shift between 10 and 30 min suggesting a longer maximum pre-incubation time with wider spacing of time points for K(I)/k(inact). 4. The two-time point IC(50) shift experiment proved to be an excellent method for the selection of appropriate K(I)/k(inact) assay parameters and is suitable for the routine analysis of P450 inhibition by drug candidates.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Cromatografia Líquida , Inibidores Enzimáticos/toxicidade , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
This study demonstrates interplay among human hepatic cytochrome P450 (CYP) isoforms in transforming aromatic compounds from being prosubstrates of CYP3A4 into phenolic substrates. Incubation of methoxychlor with CYP2C19 yields the phenolic monodemethylated derivative (mono-OH-M). Additionally, CYP2C19 catalyzes the ortho-hydroxylation of mono-OH-M and of residual methoxychlor. CYP3A4 does not catalyze the O-demethylation or hydroxylation of methoxychlor, but does hydroxylate mono-OH-M (ortho to the phenolic hydroxyl) (Stresser DM and Kupfer D, Biochemistry 36: 2203-2210, 1997). A combination of reconstituted CYP2C19 and 3A4 in the same vessel elicits stimulation of the ortho-hydroxylation of mono-OH-M compared with 2C19 alone. It is unlikely that stimulation of hydroxylation was due to protein-protein interactions, generating more active P450(s), because progression of the stimulation was time-dependent. When reconstituted CYP3A4 was added to an ongoing incubation containing reconstituted 2C19, stimulation of catechol formation occurred. In another experiment, stimulatory activity was similar when 2C19 and 3A4 were reconstituted together in the same vesicles or separately. Cumulative evidence demonstrates that the stimulation of catechol formation resulted from CYP3A4-mediated ortho-hydroxylation of the phenolic metabolite(s) generated by CYP2C19. Similarly, estradiol 3-methyl ether is demethylated by CYP2C19 into estradiol, a CYP3A4 substrate for ortho-hydroxylation; there was significant stimulation of hydroxylation by combined 2C19 and 3A4. These findings demonstrate that pro-phenolic compounds (methoxychlor and estradiol 3-methyl ether) are prosubstrates of CYP3A4. Because catalysis may become evident only after prosubstrate conversion (by a different P450) into a substrate, caution is warranted when concluding a lack of catalytic involvement by a particular P450 isoform, based solely on data from the use of individual cDNA-expressed P450s.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Estradiol/análogos & derivados , Isoenzimas/metabolismo , Metoxicloro/metabolismo , Microssomos Hepáticos/metabolismo , Oxigenases de Função Mista/metabolismo , Esteroide 16-alfa-Hidroxilase , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/biossíntese , DNA Complementar/metabolismo , Estradiol/metabolismo , Humanos , Hidroxilação , Isoenzimas/biossíntese , Isoenzimas/genética , Microssomos Hepáticos/enzimologia , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Esteroide Hidroxilases/biossíntese , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
Rapid screening for cytochrome P450 inhibitors is part of the current paradigm for avoiding development of drugs likely to give clinical pharmacokinetic drug-drug interactions and associated toxicities. We have developed microtiter plate-based, direct, fluorometric assays for the activities of the principal human drug-metabolizing enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, as well as for CYP2A6, which is an important enzyme in environmental toxicology. These assays are rapid and compatible with existing high-throughput assay instrumentation. For CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, the potency of enzyme inhibition (IC50) is consistent regardless of the probe substrate or assay method employed. In contrast, CYP3A4 inhibition for an individual inhibitor shows significant differences in potency (>300-fold) depending on the probe substrate being used. We have investigated these differences through the use of several structurally distinct fluorescent substrates for CYP3A4 and several classical substrate probes (e.g., testosterone, nifedipine, and midazolam), with a panel of known, clinically significant, CYP3A4 inhibitors. The use of multiple probe substrates appears to be needed to characterize the inhibition potential of xenobiotics for CYP3A4.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/toxicidade , Fluorometria/métodos , Cumarínicos/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Concentração Inibidora 50 , Isoenzimas/análise , Isoenzimas/antagonistas & inibidores , Cinética , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/metabolismo , Xenobióticos/análise , Xenobióticos/metabolismo , Xenobióticos/toxicidadeRESUMO
Inhibition of cytochromes P-450 (CYP) is a principal mechanism for metabolism-based drug interactions. In vitro methods for quantitatively measuring the extent of CYP inhibition are well-established. Classical methods use drug molecules as substrates and HPLC-based analysis. However, methodologies, which do not require HPLC separations for data acquisition generally offer higher throughputs and lower costs. Multiwell plate-based, direct, fluorometric assays for the activities of the five principal drug-metabolizing enzymes are available and parameters for the use of these substrates to measure CYP inhibition have been established. This methodology is quantitative, rapid, reproducible, and compatible with common high throughput screening instrumentation. This article describes approaches to establishing this methodology in a drug-discovery support program.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Omeprazol/análogos & derivados , 2-Piridinilmetilsulfinilbenzimidazóis , Animais , Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP3A , Fluorometria , Humanos , Lansoprazol , Oxigenases de Função Mista/antagonistas & inibidores , Omeprazol/farmacologiaRESUMO
Because little is known about the interactions between herbal products and standard medications, the effects of seven ginsenosides and two eleutherosides (active components of the ginseng root) on the catalytic activity of c-DNA expressed cytochrome P450 isoforms were studied in in vitro experiments. Increasing concentrations of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 and eleutherosides B and E were incubated with a panel of recombinant human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and their effects on the conversion of specific surrogate substrates measured fluorometrically in a 96-well plate format. For each test substance, the IC50 (the concentration required to inhibit the metabolism of the surrogate substrates by 50%) was estimated and this value compared with that obtained for positive control inhibitory drugs furafylline, sulfaphenazole, tryanylcypromine, quinidine, and ketoconizole. Of the components tested, three ginsenosides (Rd, Rc, and Rf) modified the activity of the recombinant enzymes. Ginsenoside Rd produced weak inhibitory activity against the surrogate substrates for CYP3A4 and CYP2D6 and even weaker inhibitory activity against the surrogate substrates for CYP2C19 and CYP2C9. The IC50 values of 58 and 74 uM for the two substrates for CYP3A4 are orders of magnitude higher than that for the potent inhibitor ketoconazole used as a positive control. Ginsenoside Rc produced an increase in the activity of CYP2C9 (70% at 200 uM) and ginsenoside Rf produced an increase in the activity of CYP3A4 (54% at 200 uM). The biological significance of this is unclear at this time. Enzyme "activation", the process by which direct addition of one compound to an enzyme enhances the rate of reaction of the substrate, has been observed in a number of cases with P450 enzymes; however, a matrix effect caused by the test compound fluorescing at the same wavelength as the metabolite of the marker substrate cannot be ruled out. In summary, these studies suggest that the ginsenosides and eleutherosides tested are not likely to inhibit the metabolism of coadministered medications in which the primary route of elimination is via cytochrome P450; the potential of ginsenosides to enhance the catalysis of certain substrates requires further investigation.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/biossíntese , Inibidores Enzimáticos/farmacologia , Ginsenosídeos , Panax/química , Extratos Vegetais/farmacologia , Plantas Medicinais , Saponinas/farmacologia , Catálise/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450 , Eleutherococcus , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
This study examined the significance of hepatic cytochrome P4501A (CYP1A) induction in the inhibition of aflatoxin B1 (AFB1)-DNA adduction by indole-3-carbinol (I3C) in rainbow trout. I3C, fed prior to [3H]AFB1 exposure, provided dose-dependent inhibition of hepatic AFB1-DNA binding, which appeared to vary inversely with hepatic CYP1A-mediated ethoxyresorufin O-deethylase (EROD) activity (r = -0.81, P = 0.051). However, 1000 ppm dietary 13C inhibited AFB1-DNA adduction without detectably inducing CYP1A protein or EROD activity. Dietary I3C was found to inhibit AFB1-DNA adduction by approximately 50%, whether [3H]AFB1 was injected ip 1, 2, 3, 5 or 7 days after the onset of I3C feeding, yet hepatic EROD activity was only transiently induced over this period and was not correlated with AFB1-DNA inhibition. Microsome-catalysed AFB1-DNA binding in vitro did correlate inversely with EROD activity in microsomes from control- and I3C-treated trout (r = -0.955, P = 0.01), but data obtained using microsomes from beta-naphthoflavone-treated trout suggest that this observation may not be indicative of a cause-and-effect relationship. I3C-mediated reduction in covalent binding was not due to I3C derivatives in the microsomal preparation or to reduced CYP protein levels, but may reflect a lower microsomal catalytic capacity for AFB1 epoxidation as a result of enzyme inactivation. In addition, the major I3C derivative found in liver, 3,3'-diindolylmethane, has been shown to be a non-competitive inhibitor of EROD, and of enzymes that catalyse AFB1 epoxidation. These findings indicate little, if any, role for CYP1A induction in the inhibition of AFB1 carcinogenicity in rainbow trout by levels of I3C likely to be encountered in cruciferous vegetables.
Assuntos
Aflatoxina B1/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Adutos de DNA/metabolismo , Indóis/farmacologia , Fígado/enzimologia , Oxirredutases/biossíntese , Aflatoxina B1/antagonistas & inibidores , Animais , Citocromo P-450 CYP1A1 , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Adutos de DNA/antagonistas & inibidores , Indução Enzimática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Oncorhynchus mykiss , Oxirredutases/antagonistas & inibidores , Oxirredutases/efeitos dos fármacos , Fatores de TempoAssuntos
Anticarcinógenos/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Regulação para Baixo , Indóis/metabolismo , Oxigenases/metabolismo , Compostos de Anilina/administração & dosagem , Compostos de Anilina/metabolismo , Animais , Anticarcinógenos/administração & dosagem , Interações Medicamentosas , Indução Enzimática , Indóis/administração & dosagem , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Nicotina/administração & dosagem , Nicotina/metabolismo , Ratos , Ratos Endogâmicos F344 , Tamoxifeno/administração & dosagem , Tamoxifeno/metabolismoRESUMO
CYP3A4 is the major human cytochrome P-450 in a superfamily of heme-thiolate proteins that catalyze the oxidation of numerous lipophilic compounds. In this investigation, we report that CYP3A4 requires a phenolic function for ortho hydroxylation of estradiol and mono-O-demethylated methoxychlor and that CYP3A4 aromatic hydroxylation in general may be dependent on the presence of a free phenolic group. Indeed, when methoxyls were present instead of phenolic hydroxyls, CYP3A4 essentially failed to catalyze ortho hydroxylation. By contrast, of eight additional cDNA-expressed P-450s (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, and 2E1) examined, only CYP1A2 and CYP2B6 could catalyze ortho hydroxylation of [o-3H]methoxychlor (7.2 and 14.6 pmol/90 min/pmol P-450, respectively), indicating that these isoforms do not require a phenolic hydroxyl for aromatic hydroxylation and that methoxyls do not sterically hinder catalysis by these CYPs. However, with [o-3H]mono-O-demethylated methoxychlor, containing a phenolic group, five isoforms (CYP1A2, 2B6, 2D6, 2E1, and 3A4) supported ortho hydroxylation. Of these, CYP3A4 exhibited by far the highest rate of hydroxylation at 87.8 pmol/90 min/pmol P-450. Further studies with [2-(3)H]estradiol 3-methyl ether and with [2-(3)H]estradiol revealed a similar and dramatic augmentation of CYP3A4-mediated C2 hydroxylase activity of approximately 75-fold by the presence of the phenolic group in the 3-position. The mechanism of augmentation by the phenolic hydroxyl does not appear to involve the acidic proton of estradiol, since CYP3A4-catalyzed estradiol 2-hydroxylation and testosterone 6-beta-hydroxylation were diminished to an equal extent when incubations were performed at increasing buffer pH values from 7 to 9. Both estradiol and its 3-methoxy derivative bound with similar affinity to cDNA-expressed, microsomal CYP3A4: spectral dissociation constants were 270 and 370 microM, respectively, and both compounds exhibited type I spectra. Thus, the disparities in aromatic hydroxylation rates between compounds containing phenolic hydroxyls and those with methoxyls cannot be explained by differences in their binding affinities. To explain the mode via which the phenolic hydroxyl facilitates ortho hydroxylation, a mechanism in which the phenolic moiety attacks the iron-oxo double bond of CYP3A4, resulting in oxygen transfer to the ortho position, is proposed. It is anticipated that these findings will assist in forecasting the CYP-mediated metabolic fate of phenolic compounds.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Estradiol/metabolismo , Metoxicloro/metabolismo , Oxigenases de Função Mista/metabolismo , Citocromo P-450 CYP3A , Humanos , Hidroxilação , MetilaçãoRESUMO
To study cytochrome P-450 (CYP) 2B6 contribution to methoxychlor metabolism within human liver microsomes and to initiate an investigation of CYP2B6 protein expression, we developed a polyclonal antibody targeted to a 20-residue peptide within that protein. The antibody was found to be highly sensitive and monospecific for CYP2B6 on immunoblots. Although many immunological studies have described the absence or low expression of CYP2B6 in human livers, in the present investigation, we have found this not to be the case. We immunoquantified CYP2B6 apoprotein expression in a panel of 28 livers and found concentrations ranging from 2 to 82 pmol/mg protein, with a mean value of 25 pmol/mg protein. Five livers ( approximately 18%) displayed relatively high levels of CYP2B6 (>40 pmol/mg protein). There were no sex-related differences, although the highest level was observed in a 1-week postpartum donor given several medications. A marked diminution in variability was found in individuals aged 56 or older (n = 12), but there were no age-related trends in mean CYP2B6 content. We suggest that CYP2B6 represents a significant portion of total CYP in human liver. The exquisite sensitivity of this antibody (fmol quantities are detected easily on immunoblots) may explain our detection of CYP2B6 in 100% of livers versus its detection in a limited number of livers by certain other investigators. The antibody also was found to immunoinhibit CYP2B6-catalyzed N-demethylation of (S)-mephenytoin in human liver microsomes by 68 to 79%. The utility of this antibody for determining human liver microsomal CYP2B6 contribution to the ortho-hydroxylation of methoxychlor was demonstrated.
Assuntos
Especificidade de Anticorpos , Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/imunologia , Imunoglobulina G/química , Oxirredutases N-Desmetilantes/imunologia , Peptídeos/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Citocromo P-450 CYP2B6 , Inibidores das Enzimas do Citocromo P-450 , Feminino , Humanos , Imunoglobulina G/farmacologia , Inseticidas/metabolismo , Masculino , Metoxicloro/metabolismo , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Peptídeos/farmacologia , CoelhosRESUMO
1,1,1-Trichloro-2,2-bis(4-methoxyphenyl)ethane (methoxychlor) is a widely used pesticide that is pro-estrogenic. We have elucidated the human cytochrome P450 enzymes responsible for conversion of methoxychlor into its major metabolite, the mono-O-demethylated derivative (mono-OH-M) that is estrogenic. Incubation of methoxychlor with microsomes from insect cells overexpressing either CYP1A2, CYP2C18, or CYP2C19 yielded mono-OH-M with turnover numbers of 14.9, 15.5, and 39.1 nmol/min/nmol of P450, respectively. CYP2B6 and CYP2C9 were much less active. Incubations with purified CYP2C19 and CYP2C18 resulted in formation of mono-OH-M, and also the bis-demethylated metabolite. Co-incubation of liver microsomes with methoxychlor and various P450 isoform-selective inhibitors suggested involvement of several P450s in mono-O-demethylation, including CYP1A2, CYP2A6, CYP2C9, and CYP2C19. A role for CYP2C19, CYP1A2, and CYP2A6 was also indicated by multivariate regression analysis of the mono-O-demethylase activity in a panel of human liver microsomes characterized for isoform-specific catalytic activities (R2 = 0.96). Based on the totality of the evidence, CYP2C19 appears to be the major catalyst of methoxychlor mono-O-demethylation. However, in individuals lacking functional CYP2C19 (e.g. the "poor metabolizer" phenotype), CYP1A2 may play the predominant role. CYP2A6, CYP2C9, and CYP2B6 probably contribute to a lesser extent. Although CYP2C18 is an efficient methoxychlor demethylase, its expression in liver is reportedly low or absent, suggesting a negligible role for this enzyme in methoxychlor metabolism. Lengthy incubations of liver microsomes with methoxychlor produced other secondary and tertiary metabolites. Efficient conversion of methoxychlor to estrogenic mono-OH-M by liver microsomes suggests that methoxychlor has the potential to be estrogenic in humans, as observed in several animal species.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Congêneres do Estradiol/metabolismo , Inseticidas/farmacocinética , Metoxicloro/farmacocinética , Animais , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C19 , Inibidores das Enzimas do Citocromo P-450 , Remoção de Radical Alquila , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Isoenzimas/metabolismo , Cinética , Masculino , Microssomos Hepáticos/metabolismo , Oxigenases de Função Mista/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Análise de RegressãoRESUMO
Aflatoxin B1 (AFB1) is a highly hepatotoxic and hepatocarcinogenic secondary metabolite of the grain mold Aspergillus flavus and related fungi. Indole-3-carbinol (I3C), found in cruciferous vegetables, can both inhibit and promote AFB1-induced carcinogenesis. We have examined the influence of dietary treatment with I3C and the well-known Ah receptor agonist beta-naphthoflavone (BNF) on the relative levels of different cytochrome P-450 (CYP) isoforms known to metabolize AFB1 in male Fischer 344 rats. After 7 days of feeding 0.2% I3C or 0.04% BNF, alone or in combination, the relative levels of hepatic CYP1A1, 1A2, 2B1/2, 2C11, and 3A were assessed by laser densitometry of Western blots. Both diets containing I3C markedly increased band densities of CYP1A1 (up to 24-fold), 1A2 (3.1-fold), and 3A1/2 (3.8-fold), and had lesser effects on the levels of 2B1/2 (1.8-fold) and no effect on CYP2C11. BNF also strongly increased band densities of CYP1A1 (12-fold) and 1A2 (2.7-fold), but had no effect on the levels of CYP2B1/2 or 3A1/2 band densities, and repressed those of CYP2C11 (2-fold). In addition, we examined the in vitro hepatic microsomal metabolism of AFB1 at 16, 124, and 512 microM substrate levels. Diets containing I3C elevated initial rates of AFM1 (a detoxication product) production 18.6- to 19.2-fold over control at 16 microM AFB1, which declined to 7.8- to 9.5-fold at 512 microM AFB1. The BNF-only diet gave similar, but less dramatic effects (5.9-fold at 16 microM AFB1, 3.5-fold at 512 microM AFB1).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aflatoxina B1/metabolismo , Aflatoxina B1/farmacocinética , Anticarcinógenos/farmacologia , Benzoflavonas/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Indóis/farmacologia , Aflatoxina B1/análogos & derivados , Aflatoxina M1/metabolismo , Aflatoxinas/metabolismo , Animais , Biotransformação , Western Blotting , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Inativação Metabólica , Isoenzimas/biossíntese , Isoenzimas/metabolismo , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos , Ratos Endogâmicos F344 , beta-NaftoflavonaRESUMO
An in vitro radiometric assay selective for inducible CYP2B activity is described. The assay is based on the quantification of 3H2O release that occurs during o-ring hydroxylation of [o-3H]methoxychlor by liver microsomes in the presence of NADPH. 3H2O is isolated by removing > 99.9% of the parent compound and organic metabolites by facile charcoal extraction and filtration. There was no evidence for an NIH shift during ring hydroxylation, and there was little or no isotope effect. Selectivity for CYP2B was demonstrated using liver microsomes prepared from rats and mice treated with inducers of different CYP isoforms. Ring hydroxylation of [o-3H]methoxychlor was elevated 11.4-fold over control values in liver microsomes from male rats treated with phenobarbital. With mice, phenobarbital treatment elevated liver microsomal ring hydroxylation 7.1-fold. Clofibrate, 3-methylcholanthrene, or beta-naphthoflavone treatment of male rats or pyridine treatment of female rats did not elevate liver microsomal ring-hydroxylase activity, indicating that CYP4A, 1A, and 2E1 do not support this reaction. In female rats, dexamethasone and pregnenolone-16 alpha-carbonitrile treatment elevated ring hydroxylation up to 5.5- and 3.2-fold, respectively, an activity that may be attributed to CYP2B induction in those animals. Incubation of liver microsomes from phenobarbital-treated males with monospecific anti-CYP2B monoclonal antibodies (Mab) inhibited ring-hydroxylase activity up to 86%, demonstrating predominantly CYP2B-mediated catalysis. An 86% inhibition by these Mabs was also observed using liver microsomes from male mice treated with phenobarbital, indicating the assay is not limited to rats. The CYP2B mechanism-based inhibitor orphenadrine caused a 76% decline in activity, providing further evidence for CYP2B involvement. Unlike other CYP2B-selective assays, this method may be readily adapted to in vivo studies, by measuring urinary excretion of 3H2O as an indication of total body CYP2B activity.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/análise , Metoxicloro/química , Microssomos Hepáticos/enzimologia , Radiometria/métodos , Esteroide Hidroxilases/análise , Animais , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática/efeitos dos fármacos , Hidroxilação , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Sondas Moleculares , Estrutura Molecular , Ratos , Esteroide Hidroxilases/biossíntese , Esteroide Hidroxilases/metabolismo , Especificidade por Substrato , TrítioRESUMO
The organochlorine pesticide methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane] (MXC) has been classified as a proestrogen in mammals and fish, requiring demethylation prior to eliciting estrogenic activity or binding to the estrogen receptor. While microsomal demethylation occurs readily in the liver of fish, little is known about the enzyme(s) responsible or the effect of cytochrome P450 (CYP) inducers, other than those of CYP1A and CYP2K, on biotransformation. Consequently, male channel catfish were pretreated with MXC or beta-naphthoflavone (BNF), alone and in combination, to determine their effects on CYP protein expression, MXC biotransformation by hepatic microsomes, microsomal protein binding, and MXC estrogenic activity as determined by serum vitellogenin and 17beta-estradiol. Liver microsomes of both treated and untreated mature male catfish catalyzed formation of monodemethylated MXC, bisdemethylated MXC, as well as ring-hydroxylated metabolites. Pretreatment with BNF did not affect MXC metabolite profiles, overall rates of MXC biotransformation, or microsomal proteins recognized by anti-trout CYP2K1, but had the expected effect of inducing CYP1A and associated ethoxyresorufin O-deethylase activity. By contrast, pretreatment with MXC, alone or in combination with BNF, significantly reduced rates of MXC biotransformation and binding to liver microsomal protein. MXC/BNF cotreatment followed by MXC significantly induced serum vitellogenin, whereas MXC treatment alone led to a nonsignificant increase in vitellogenin and a significant increase in serum 17beta-estradiol. Thus, estrogenic activity elicited by cotreatment with MXC and BNF can occur despite diminished capacity of liver microsomes to catalyze formation of estrogenic demethylated metabolites or metabolites that bind microsomal protein. Possible mechanisms of MXC-induced attenuation of CYP-dependent metabolism are discussed.
Assuntos
Estrogênios/sangue , Metoxicloro/farmacologia , Metoxicloro/farmacocinética , beta-Naftoflavona/farmacologia , Animais , Biotransformação/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática/efeitos dos fármacos , Estradiol/sangue , Ictaluridae , Injeções Intraperitoneais , Masculino , Metoxicloro/administração & dosagem , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Vitelogeninas/sangue , beta-Naftoflavona/administração & dosagemRESUMO
Our laboratory has previously shown that dietary administration of indole-3-carbinol (I3C) to male Fischer 344 rats has the very unusual property of inducing hepatic levels of a number of cytochrome P450s (CYPs), especially CYP1A1, while markedly inhibiting the levels of flavin-containing monooxygenase (FMO) 1 protein and its catalytic activity. We hypothesized that rats fed I3C or 3,3'-diindolylmethane (DIM), one of its major acid condensation products formed in vivo, should exhibit a marked shift in the metabolic profiles of drugs or xenobiotics that are substrates for both monooxygenase systems. Male rats were fed AIN-76A powdered diets containing 0, 1000, or 2500 ppm I3C or DIM for 4 weeks. Dietary I3C and DIM reduced FMO1 protein levels (8% reduction with I3C and 84% with DIM at 1000 ppm, and 90% reduction with I3C and 97% with DIM at 2500 ppm) in hepatic microsomes. The ratio of FMO (N-oxygenation)- to CYP (N-demethylation)-mediated metabolism of N,N-dimethylaniline decreased in liver microsomes from I3C- or DIM-fed rats from near unity to 0.02 at the highest dietary doses. FMO-mediated N-oxygenation (nicotine N-1'-oxide) was decreased, whereas CYP-mediated (nornicotine and nicotine delta (1,5)-iminium ion) metabolism of nicotine was unchanged in liver microsomes from rats fed I3C or DIM. Similarly, the ratio of FMO to CYP metabolites of tamoxifen decreased due to a reduction in N-oxygenation. This study demonstrates alteration of FMO- and CYP-mediated drug metabolism in vitro by dietary I3C or DIM and suggests the potential for altered toxicity of tamoxifen and nicotine in vivo.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Indóis/metabolismo , Oxigenases/metabolismo , Compostos de Anilina/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Dieta , Regulação para Baixo , Interações Medicamentosas , Indução Enzimática , Masculino , Microssomos Hepáticos/metabolismo , Nicotina/metabolismo , Ratos , Ratos Endogâmicos F344 , Tamoxifeno/metabolismoRESUMO
This study describes the disposition and excretion of indole-3-carbinol (I3C), a natural dietary tumor modulator and candidate chemopreventive agent, in male Fisher 344 rats after continuous dietary or a single oral administration. Steady-state urinary and fecal excretion were attained 40 and 112 hr, respectively, after commencing continuous exposure. These two routes accounted for approximately 75% of the administered dose, of which 77% appeared in feces. After 7 days of 2,000 ppm dietary I3C, a mean of 1,154 microM I3C eq was found in liver, of which 17% was present as extractable, unbound I3C derivatives. Total equivalents in liver decreased to 643 and 411 microM 24 and 48 hr later, respectively, for animals returned to control diet. Mean levels of I3C eq in lung decreased from 436 to 219 microM, and blood levels decreased from 320 to 208 microM over the same 48-hr period. After administration of 1 mmol/kg radioinert I3C (a comparable daily dose as in the feeding study) for 6 days, animals were given 1 mmol/kg [3H]I3C. Mean liver levels were 257, 283, and 541 microM I3C eq at 1.5, 3, and 6 hr after dosing, and these levels represented 0.97%, 1.34%, and 2.45% of the total I3C dose administered, respectively. Concentrations of I3C eq changed little in blood, kidney, tongue, or lung over this time period. HPLC analysis of ethyl acetate extracts of liver from rats given an oral dose revealed 24 distinct [3H]I3C-derived peaks. Two of the predominant peaks were identified as 3,3'-diindolylmethane (I33', a linear dimer of I3C) and [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LT, a linear trimer). A novel I3C metabolite was identified as 1-(3-hydroxymethyl)-indolyl-3-indolylmethane (HI-IM). Hepatic levels of these metabolites and three major, but unidentified, products were between 1.0 and 13.1 microM; highest levels were observed at 6 hr or, for HI-IM, at 1.5 hr postdosing. Parent I3C was not detected in liver extracts, whereas the potent Ah receptor agonist 3,2-b-indolocarbazole (ICZ) was estimated at 1.6 nM. These data suggest that neither I33', LT, or ICZ alone reach sufficient hepatic concentration to account for cytochrome P450IA induction by dietary I3C, or provide effective inhibition of microsomal bioactivation of the hepatocarcinogen aflatoxin B1; however, the total hepatic mixture of I3C derivatives may be sufficient to provide both modulatory responses in the rat.
Assuntos
Anticarcinógenos/farmacologia , Anticarcinógenos/farmacocinética , Indóis/farmacologia , Indóis/farmacocinética , Administração Oral , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , Esvaziamento Gástrico/fisiologia , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Ratos , Ratos Endogâmicos F344 , Distribuição TecidualRESUMO
Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several mechanisms of inhibition may exist. We reported previously that 3,3'-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent ethoxyresorufin O-deethylase with Ki values in the low micromolar range. We now report a similar potent inhibition by 3,3'-diindolylmethane of rat and human CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential activity assays. 3,3'-Diindolylmethane also inhibited in vitro CYP-mediated metabolism of the ubiquitous food contaminant and potent hepatocarcinogen, aflatoxin B1. There was no inhibition of cytochrome c reductase. In addition, we found 3,3'-diindolylmethane to be a substrate for rat hepatic microsomal monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These observations indicate that 3,3'-diindolylmethane can inhibit the catalytic activities of a range of CYP isoforms from lower and higher vertebrates in vitro. This broadly based inhibition of CYP-mediated activation of procarcinogens may be an indole-3-carbinol anticarcinogenic mechanism applicable to all species, including humans.
Assuntos
Anticarcinógenos/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Aflatoxina B1/antagonistas & inibidores , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Citocromo P-450 CYP1A1 , Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2B1 , Humanos , Indóis/metabolismo , Masculino , Microssomos Hepáticos/enzimologia , Oxirredutases/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344RESUMO
Aflatoxin B1 (AFB1), a metabolite of the grain mold Aspergillus flavus, is a potent hepatocarcinogen and widespread contaminant of human food supplies. AFB1-induced tumors or preneoplastic lesions in experimental animals can be inhibited by cotreatment with several compounds, including indole-3-carbinol (I3C), a component of cruciferous vegetables, and the well-known Ah receptor agonist beta-naphthoflavone (BNF). This study examines the influence of these two agents on the AFB1-glutathione detoxication pathway and AFB1-DNA adduction in rat liver. After 7 days of feeding approximately equally inhibitory doses of I3C (0.2%) or BNF (0.04%) alone or in combination, male Fischer 344 rats were administered [3H]AFB1 (0.5 mg/kg, 480 microCi/kg) intraperitoneally and killed 2 hr later. All three experimental diets inhibited in vivo AFB1-DNA adduction (BNF, 46%; I3C, 68%; combined, 51%). Based on Western blots using antibodies specific for the glutathione S-transferase (GST), subunit Yc2 (subunit 10) appeared to be substantially elevated by the diets containing I3C (I3C diet, 4.0-fold increase in band density; combined diet, 2.8-fold). The BNF diet appeared to elevate Yc2 to a lesser extent (2.2-fold increase in band density).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aflatoxina B1/análogos & derivados , Aflatoxina B1/metabolismo , Anticarcinógenos/farmacologia , Adutos de DNA , DNA/metabolismo , Glutationa Transferase/biossíntese , Glutationa Transferase/efeitos dos fármacos , Indóis/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Animais , Benzoflavonas/farmacologia , Western Blotting , Citosol/enzimologia , Indução Enzimática , Glutationa Transferase/metabolismo , Isomerismo , Fígado/metabolismo , Substâncias Macromoleculares , Masculino , Ratos , Ratos Endogâmicos F344 , beta-NaftoflavonaRESUMO
The microsomal flavin-containing monooxygenases (FMO) represent a family of xenobiotic-metabolizing enzymes with distinct tissue- and species-specific patterns of expression. Expression for two FMO isoforms (FMO1 and FMO2) in rabbit was characterized by determining mRNA levels, protein levels and catalytic activity in male and female liver, lung, kidney, esophagus, intestine, nasal mucosa (maxilloturbinates and ethmoturbinates) and gonadal tissue. Northern blot hybridization analyses performed with cDNA probes for each isoform showed marked differences in mRNA expression between tissues: FMO1 expression was highest in liver and intestine, followed by ethmoturbinates, maxilloturbinates and low but detectable levels in female kidney; FMO2 expression was highest in lung, followed by maxilloturbinates, ethmoturbinates, esophagus and kidney. More sex-related differences were observed for FMO2, with higher levels of mRNA in female esophagus, nasal mucosa and kidney. Western blot analyses showed similar patterns of expression at the protein level. Microsomal catalytic activities determined by [14C]-DMA N-oxide formation also indicated tissue- and sex-related differences in substrate metabolism by FMO. Analysis of tissue-specific FMO catalytic activity was also performed using thiocarbamides as isoform-specific probes. Microsomes from those tissues containing FMO2, but not FMO1, failed to catalyze oxidation of the larger (van der Waals surface area greater than 178 A) FMO1-specific thiocarbamides. The results of this study demonstrate that tissue-specific control mechanisms play a more dominant role in the overall constitutive regulation of FMO than other potential factors, such as hormonal influences. Elucidation of the mechanisms controlling FMO tissue-specific expression will lead to a better understanding of target organ specificity for xenobiotic detoxication or bioactivation.
Assuntos
Oxigenases/metabolismo , Animais , Sequência de Bases , Feminino , Expressão Gênica , Masculino , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/química , Oxigenases/genética , RNA Mensageiro/genética , Coelhos , Distribuição TecidualRESUMO
Inhibition of cytochrome P450 catalytic activity is a principal mechanism for pharmacokinetic drug-drug interactions. Rapid, in vitro testing for cytochrome P450 inhibition potential is part of the current paradigm for identifying drug candidates likely to give such interactions. We have explored the extent that qualitative and quantitative inhibition parameters are dependent on the cytochrome P450 (CYP) 3A4 probe substrate. Inhibition potential (e.g., IC(50) values from 8-point inhibition curves) or activation potential for most compounds varied dramatically depending on the fluorometric probe substrates for CYP3A4 [benzyloxyresorufin (BzRes), 7-benzyloxy-4-trifluoromethylcoumarin (BFC), 7-benzyloxyquinoline (BQ), and dibenzylfluorescein (DBF)]. For 21 compounds that were primarily inhibitors, the range of IC(50) values for the four substrates varied from 2.1- to 195-fold with an average of 29-fold. While the rank order of sensitivity among the fluorometric substrates varied among the individual inhibitors, on average, BFC dealkylation was the most sensitive to inhibition, while BQ dealkylation was least sensitive. Partial inhibition was observed with BzRes and BQ but not for BFC and DBF. BzRes was more prone to activation, whereas dramatic changes in IC(50) values were observed when the BQ concentration was below the S(50). Three different correlation analyses indicated that IC(50) values with BFC, BQ, and DBF correlated well with each other, whereas the response with BzRes correlated more weakly with the other substrates. One of these correlation analyses was extended to the percent inhibition of 10 microM inhibitor with the standard CYP3A4 probe substrates testosterone, midazolam, and nifedipine. In this analysis the responses with BQ, BFC and DBF correlated well with testosterone and midazolam but more poorly with nifedipine. In the aggregate, BFC and DBF appear more suitable as an initial screen for CYP3A4 inhibition. However, the substrate-dependent effects reported here and by others indicate that all CYP3A4 inhibition data should be interpreted with caution.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Corantes Fluorescentes/farmacocinética , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/metabolismo , Algoritmos , Bloqueadores dos Canais de Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP3A , Moduladores GABAérgicos/metabolismo , Humanos , Técnicas In Vitro , Cinética , Espectrometria de Massas , Midazolam/metabolismo , Nifedipino/metabolismo , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Testosterona/metabolismoRESUMO
Recently, a novel nonfluorescent probe 3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4-methylcoumarin (AMMC), which produces a fluorescent metabolite AMHC (3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-hydroxy-4-methylcoumarin) was used with microsomes containing recombinant enzymes (rCYP) to monitor CYP2D6 inhibition in a microtiter plate assay. This article describes the studies that were performed in human liver microsomes (HLM) to establish the selectivity of AMMC toward CYP2D6. Metabolism studies in HLM showed that AMMC was converted to one metabolite identified by mass spectrometry as AMHC. Kinetic studies indicated an apparent K(m) of 3 microM with a V(max) of 20 pmol/min. mg of protein for the O-demethylation reaction. The O-demethylation of AMMC in HLM was inhibited significantly in the presence of a CYP2D6 inhibitory antibody. Using a panel of various HLM preparations (n = 12), a good correlation (r(2) = 0.95) was obtained between AMMC O-demethylation and bufuralol metabolism, a known CYP2D6 substrate, but not with probes for the other major xenobiotic metabolizing CYPs. Finally, only rCYP2D6 showed detectable metabolism in experiments conducted with rCYPs using AMMC at a concentration of 1.5 microM (near K(m)). However, at a concentration of 25 microM AMMC, rCYP1A also contributed significantly to the formation of AMHC. Knowing the experimental conditions under which AMMC was selective for CYP2D6, a microtiter assay was developed to study the inhibition of various compounds in HLM using the fluorescence of AMHC as an indication of CYP2D6 activity. The inhibition potential of various chemicals was found to be comparable to those determined using the standard CYP2D6 probe, bufuralol, which requires high-performance liquid chromatography separation for the analysis of its CYP2D6-mediated 1'-hydoxylated metabolite.