RESUMO
Tamoxifen is a known hepatocarcinogen in rats and is associated with an increased incidence of endometrial cancer in patients. One mechanism for these actions is via bioactivation, where reactive metabolites are generated that are capable of binding to DNA or protein. Several metabolites of tamoxifen have been identified that appear to predispose to adduct formation. These include alpha-hydroxytamoxifen, alpha,4-dihydroxytamoxifen, and alpha-hydroxy-N-desmethyltamoxifen. Previous studies have shown that cytochrome P450 (P450) enzymes play an important role in the biotransformation of tamoxifen. The aim of our work was to determine which P450 enzymes were capable of producing alpha-hydroxylated metabolites from tamoxifen. When tamoxifen (18 or 250 microM) was used as the substrate, P450 3A4, and to a lesser extent, P450 2D6, P450 2B6, P450 3A5, P450 2C9, and P450 2C19 all produced a metabolite with the same HPLC retention time as alpha-hydroxytamoxifen at either substrate concentration tested. This peak was well-separated from 4-hydroxy-N-desmethyltamoxifen, which eluted substantially later under the chromatographic conditions used. No alpha,4-dihydroxytamoxifen was detected in incubations with any of the forms with tamoxifen as substrate. However, when 4-hydroxytamoxifen (100 microM) was used as the substrate, P450 2B6, P450 3A4, P450 3A5, P450 1B1, P450 1A1, and P450 2D6 all produced detectable concentrations of alpha,4-dihydroxytamoxifen. These studies demonstrate that multiple human P450s, including forms found in the endometrium, may generate reactive metabolites in women undergoing tamoxifen therapy, which could subsequently play a role in the development of endometrial cancer.
Assuntos
Antineoplásicos Hormonais/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A , Humanos , Microssomos Hepáticos/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a,l]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450s (but not CYP1B1) (in T. ni cells), we found that CYP1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Carcinógenos/metabolismo , Citocromo P-450 CYP1A1/fisiologia , Sistema Enzimático do Citocromo P-450/fisiologia , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Salmonella typhimurium/metabolismo , Alelos , Biotransformação , Citocromo P-450 CYP1B1 , Humanos , Proteínas Recombinantes/farmacologiaRESUMO
1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg48 Ala119 Leu432, Arg48 Ala119 Val432 Gly48 Ala119 Leu432, Gly48 Ala119 Val432, Arg48 Ser119 Leu432, Arg48 Ser119 Val432, Gly48 Ser119 Leu432 and Gly48 Ser119 Va1432 (all with Asn453), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17beta-oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+.CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg48 variants tended to have higher activities for 17beta-oestradiol 4-hydroxylation than Gly48 variants, although there were no significant variations in 17beta-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17beta-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val432 forms than the corresponding Leu432 forms. 4. In contrast, Leu432 forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val432 forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17beta-oestradiol and benzo[a]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Benzo(a)pireno/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Estradiol/química , Estradiol/farmacologia , Oxigênio/metabolismo , Alelos , Animais , Catálise , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Humanos , Immunoblotting , Ferro/metabolismo , Cinética , Fígado/enzimologia , Microssomos Hepáticos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Plasmídeos/metabolismo , Polimorfismo Genético , Ligação Proteica , Coelhos , Ratos , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fatores de TempoRESUMO
We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC"lacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B(1) exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta-Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrome P450 enzyme involved in bioactivation of HCAs.
Assuntos
Acetiltransferases/metabolismo , Carcinógenos/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Compostos Heterocíclicos/toxicidade , Mutagênicos/toxicidade , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Salmonella typhimurium/enzimologia , Acetiltransferases/genética , Aminas/toxicidade , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Ativação Enzimática , Fluorenos , Humanos , Isoenzimas , Testes de Mutagenicidade/métodos , NADPH-Ferri-Hemoproteína Redutase/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Sensibilidade e EspecificidadeAssuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Acetaminofen/farmacocinética , Analgésicos não Narcóticos/farmacocinética , Animais , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Glutationa S-Transferase pi , Glutationa Transferase/genética , Humanos , Inativação Metabólica , Isoenzimas/genética , Camundongos , Camundongos KnockoutRESUMO
The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Biotecnologia/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Animais , Biotecnologia/tendências , Citocromo P-450 CYP2A6 , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/metabolismo , Humanos , Índigo Carmim , Indóis/metabolismo , Oxigenases de Função Mista/genética , Modelos Químicos , Plantas Geneticamente Modificadas , Proteínas Recombinantes/metabolismoRESUMO
Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the body in substantial amounts. The compound is known to be oxidized to indoxyl and excreted in urine as indoxyl (3-hydroxyindole) sulfate. Further oxidation and dimerization of indoxyl leads to the formation of indigoid pigments. We report the definitive identification of the pigments indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, (1)H NMR, and mass spectrometry. P450 2A6 was most active in the formation of these two pigments, followed by P450s 2C19 and 2E1. Additional products of indole metabolism were characterized by HPLC/UV and mass spectrometry. Indoxyl (3-hydroxyindole) was observed as a transient product of P450 2A6-mediated metabolism; isatin, 6-hydroxyindole, and dioxindole accumulated at low levels. Oxindole was the predominant product formed by P450s 2A6, 2E1, and 2C19 and was not transformed further. A stable end product was assigned the structure 6H-oxazolo[3,2-a:4, 5-b']diindole by UV, (1)H NMR, and mass spectrometry, and we conclude that P450s can catalyze the oxidative coupling of indoles to form this dimeric conjugate. On the basis of these results, we propose that the P450/NADPH-P450 reductase system can catalyze oxidation of indole to a variety of products.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Indóis/metabolismo , Animais , Citocromo P-450 CYP2A6 , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Índigo Carmim , Indóis/química , Isatina/química , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredução , Pigmentos Biológicos/química , Ratos , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Alquilação , Animais , Cromatografia Líquida , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas Fúngicas , Humanos , NADH NADPH Oxirredutases/metabolismo , Coelhos , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta-estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala-Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4-hydroxylase activity among four different variant forms of CYP1B1. Thus, inter-individual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Neoplasias da Mama/genética , Carcinoma de Células Escamosas/genética , Carcinoma/genética , Sistema Enzimático do Citocromo P-450/genética , Neoplasias Pulmonares/genética , Polimorfismo Genético/genética , Adenocarcinoma/enzimologia , Adenocarcinoma/epidemiologia , Adenocarcinoma/genética , Neoplasias da Mama/enzimologia , Neoplasias da Mama/epidemiologia , Carcinoma/enzimologia , Carcinoma/epidemiologia , Carcinoma de Células Grandes/enzimologia , Carcinoma de Células Grandes/epidemiologia , Carcinoma de Células Grandes/genética , Carcinoma de Células Pequenas/enzimologia , Carcinoma de Células Pequenas/epidemiologia , Carcinoma de Células Pequenas/genética , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/epidemiologia , Catálise , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/metabolismo , Estradiol/metabolismo , Feminino , Frequência do Gene , Variação Genética , Genótipo , Humanos , Incidência , Japão/epidemiologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/epidemiologia , Masculino , Polimorfismo Conformacional de Fita Simples , Valores de Referência , Medição de Risco , Esteroide Hidroxilases/metabolismoRESUMO
Four polymorphic human cytochrome P450 (CYP) 1B1 allelic variants, namely Arg48,Ala119,Leu432,Asn453, Arg48,Ser119,Leu432,Asn453, Arg48, Ala119,Val432,Asn-453 and Arg48,Ser119,Val432,Asn453, were expressed in Escherichia coli together with human NADPH-P450 reductase and the recombinant proteins (in bacterial membranes) were used to assess whether CYP1B1 polymorphisms affect catalytic activities towards a variety of P450 substrates, including diverse procarcinogens and steroid hormones. Activities for activation of 19 procarcinogens to DNA-damaging products by these four CYP1B1 variants in a Salmonella typhimurium NM2009 umu response system were found to be essentially similar, except that a Arg48, Ser119,Leu432,Asn453 variant was slightly more active (1.2- to 1.5-fold) than the other three CYP1B1 enzymes in catalyzing activation of (+)- and (-)-benzo[a]pyrene-7, 8-diols, 7,12-dimethylbenz[a]anthracene-3,4-diol, benzo[g]chrysene-11,12-diol, benzo[b]fluoranthene-9,10-diol, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminofluorene. Kinetic analysis of 17beta-estradiol hydroxylation showed that V(max) values for 4-hydroxylation ranged between 0.9 and 1.5 nmol/min/nmol P450 for 4-hydroxylation and 0.3 and 0.6 nmol/min/nmol P450 for 2-hydroxylation in these CYP1B1 variants, with K(m) values ranging from 1 to 9 microM. Interestingly, the ratio of product formation of 4-hydroxyestradiol to 2-hydroxyestradiol was higher for the Val432 variants of CYP1B1 variants than the Leu432 variants of the enzyme. The same trend was noted in the ratio of estrone 4-hydroxylation to estrone 2-hydroxylation catalyzed by CYP1B1 variants. Mutation in the CYP1B1 genes also affected the K(m) and V(max) values in the 6beta-hydroxylation of testosterone and 6beta- and 16alpha-hydroxylation of progesterone. These results indicate that the polymorphisms in the human CYP1B1 gene cause some alterations in catalytic function towards procarcinogens and steroid hormones and thus may make some contribution to susceptibilities of individuals towards mammary and lung cancers in humans.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Carcinógenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Alelos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/enzimologia , Estradiol/metabolismo , Estrona/metabolismo , Humanos , Hidroxilação , Progesterona/metabolismo , Esteroide 16-alfa-Hidroxilase , Testosterona/metabolismoRESUMO
Recombinant human enzymes expressed in membranes obtained from Escherichia coli transformed with cytochrome P450 (P450) and NADPH-P450 reductase cDNAs were used to identify the human P450 enzymes that are most active in catalyzing the oxidative transformation of benzo[a]pyrene in vitro. Activation of benzo[a]pyrene to genotoxic products that cause induction of umu gene expression in Salmonella typhimurium NM2009 by P450 1A1 and P450 1B1 enzymes was found to be enhanced by inclusion of purified epoxide hydrolase (isolated from rat or human livers) with the reaction mixture. High-performance liquid chromatographic analysis showed that P450 1B1 catalyzed benzo[a]pyrene to trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene at level of approximately 3 nmol min(-)(1) nmol of P450(-)(1) only when epoxide hydrolase was present and P450 1A1 (with the hydrolase) was able to catalyze benzo[a]pyrene at one-tenth of the activity catalyzed by P450 1B1. Kinetic analysis showed that ratio of V(max) to K(m) for the formation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in this assay system was 3.2-fold higher in CYP1B1 than in CYP1A1. Other human P450s (including P450s 1A2, 2E1, and 3A4) were found to have very low or undetectable activities toward the formation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene. A reconstituted system containing purified P450 1B1, rabbit liver NADPH-P450 reductase, and human liver epoxide hydrolase was found to catalyze benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at a rate of 0.86 nmol min(-)(1) nmol of P450(-)(1); the activities were found to be largely dependent on the presence of sodium cholate in the system. These results suggest that P450 1B1 is a principal enzyme in catalyzing the oxidation of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and that the catalytic functions of P450 1B1 may determine the susceptibilities of individuals to benzo[a]pyrene carcinogenesis.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Benzo(a)pireno/metabolismo , Carcinógenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Di-Hidroxi-Di-Hidrobenzopirenos/metabolismo , Epóxido Hidrolases/metabolismo , Fígado/enzimologia , Animais , Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1B1 , Humanos , Testes de Mutagenicidade , Coelhos , Ratos , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/metabolismoRESUMO
Human cytochrome P450 (P450) 1B1 was expressed in Escherichia coli at a level of 200 nmol/liter culture using a pCW vector by removal of codons 2-4 and modification of the nucleotide sequence of the resulting N-terminal seven codons; a similar level of expression was found with a bicistronic construct that also expressed human NADPH-P450 reductase. P450 1B1 was purified (from the monocistronic system) to electrophoretic homogeneity and a specific content of 9.2 nmol P450/mg protein using DEAE, CM, and hydroxylapatite chromatography. The absolute spectra showed a considerable fraction of high-spin iron and little cytochrome P420. The catalytic activity of the purified enzyme was considerably enhanced in the presence of cholate. Both reconstituted P450 1B1 and the bacterial membranes prepared from the bicistronic vector system had similar7-ethoxyresorufin O-deethylation activities; as expected, 17beta-estradiol was hydroxylated primarily at the 4-position. The ability of human P450 1B1 to activate several heterocyclic amines and polycyclic hydrocarbon dihydrodiols was confirmed with reconstituted P450 1B1 and the P450 1B1 membranes in which NADPH-P450 reductase was coexpressed.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Carcinógenos/metabolismo , Catálise , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Escherichia coli/enzimologia , Vetores Genéticos/síntese química , Humanos , Proteínas Recombinantes/isolamento & purificação , EspectrofotometriaRESUMO
Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation. We report the development of bacterial expression systems for P450 3A7. Maximal yields (up to 50 nmol P450/liter culture) were obtained with a construct in which the 5'-terminus of the 3A7 cDNA was modified to include the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and to incorporate several downstream amino acid substitutions derived from the P450 3A5 sequence. This sequence also appeared optimal for expression of P450 3A4 and 3A5. Recombinant P450 3A7 was partially purified using ion-exchange and hydroxylapatite chromatography and reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, and lipids. Activity comparable to that of P450 3A4 was demonstrated toward a number of procarcinogens. An alternative approach was used to further characterize recombinant 3A7 due to low yields of recombinant protein in the expression and poor recovery in the purification. P450 3A7 was subcloned into a bicistronic vector containing human NADPH-cytochrome P450 reductase and expressed in bacteria. Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation).
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/biossíntese , NADH NADPH Oxirredutases/biossíntese , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Apoproteínas/biossíntese , Apoproteínas/genética , Sequência de Bases , Biotransformação , Carcinógenos/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Genes , Vetores Genéticos , Humanos , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Mutagênese , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH-Ferri-Hemoproteína Redutase , Especificidade por SubstratoRESUMO
Human cytochrome P450 (P450) 1B1 (CYP1B1) has recently been shown to be an important enzyme in the activation of diverse procarcinogens such as arylarenes, nitroarenes, and arylamines to reactive metabolites that cause DNA damage in the cells. However, it is not known whether this P450 enzyme also plays roles in the oxidation of certain drugs or model substrates commonly used in P450 assays. We examined the substrate oxidation activities of recombinant human CYP1B1 in yeast microsomes and compared these activities with those catalyzed by reconstituted systems containing recombinant CYP1A1 and CYP1A2 which were isolated from membranes of Escherichia coli in which respective cDNAs have been expressed. Catalytic activities towards some of the model substrates of other human P450 enzymes including CYP2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 were also determined and compared. CYP1B1 catalyzed benzo[a]pyrene 3-hydroxylation at rates lower than those of CYP1A1 but higher than those of CYP1A2. The activity towards 7-ethoxyresorufin O-deethylation catalyzed by CYP1B1 was about one-tenth of that of CYP1A1, but the Km values were lower for CYP1B1 than those for CYP1A1 and CYP1A2. CYP1B1 was also able to catalyze the oxidation of theophylline and caffeine, two prototypic substrates for CYP1A2. CYP1B1 did not oxidize other typical P450 substrates such as coumarin, tolbutamide, S-mephenytoin, chlorzoxazone, nifedipine, and testosterone, while low rates of oxidation of bufuralol and 7-ethoxycoumarin were found for CYP1B1. These results indicate that CYP1B1 has catalytic activities overlapping CYP1A1 and CYP1A2 with respect to the oxidation of drugs and model P450 substrates, although the relative catalytic roles in these three P450 enzymes differ depending upon the substrates examined. A distinct marker activity of CYP1B1 has not been identified.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Xenobióticos/metabolismo , Anticorpos/imunologia , Catálise , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/imunologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/imunologia , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/imunologia , Escherichia coli/genética , Humanos , Cinética , Microssomos Hepáticos/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
Bacterial assays were used to examine the activation of 14 known procarcinogens by cytochrome P450 (P450) enzymes. Human P450s 1A1, 1A2 and 3A4 were expressed in Escherichia coli with slight modification of their N-terminal sequences. Genotoxicity was measured by the induction of the SOS response in Salmonella typhimurium NM2009 (TA1535/pSK1002/pNM12), which contains a umuC regulatory sequence attached to the lacZ reporter gene. Conditions for analysis were examined using E. coli membranes and purified enzymes. Membrane fractions, fortified with NADPH-P450 reductase, were found to be useful preparations for measuring activation of the procarcinogens. Conditions of linearity were established for these assays and the systems were applied to several particular problems related to bioactivation of procarcinogens by P450s. The patterns of activation of the 14 individual chemicals were consistent with the literature developed using human liver microsomes, purified liver P450s and other approaches. The P450s expressed in bacterial membranes could be inhibited by antibodies. 7,8-Benzoflavone inhibited P450s 1A1 and 1A2 and stimulated P450 3A4 in the membranes. The contributions of P450s 1A1 and 1A2 were distinguished with some of the arylamines and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Recombinant P450 3A4 was found to be more active than P450 1A2 in the activation of aflatoxin B1 at all substrate concentrations examined.
Assuntos
Carcinógenos/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Testes de Mutagenicidade/métodos , Sequência de Aminoácidos , Benzoflavonas/farmacologia , Biotransformação , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Microssomos Hepáticos/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/farmacologiaRESUMO
Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens. The purification of the protein from human liver is difficult, and we report the development of a system for relatively high-level expression in Escherichia coli. A cDNA was prepared from liver cDNA by polymerase chain reaction methods and several variants with modified 5'-termini were constructed. Analysis of seven of these indicated that the highest levels of expression were found when the first 21 codons of the native sequence were deleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT). Levels of 40-nmol membrane-bound P450 2E1 (liter culture)-1 were routinely recovered. The recombinant P450 2E1 was purified to electrophoretic homogeneity from the bacterial membranes in two ion-exchange steps in > 80% yield. Ferric P450 2E1 was isolated in a mixed spin state. The enzyme was active in chlorzoxazone 6-hydroxylation; the addition of human liver cytochrome b5 lowered the Km for the substrate and increased Vmax. N-Terminal amino acid sequence analysis yielded the expected first 21 residues. The expression system should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Sequência de Bases , Catálise , Clorzoxazona/metabolismo , Clonagem Molecular , Citocromo P-450 CYP2E1 , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Escherichia coli/genética , Humanos , Hidroxilação , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Oxirredutases N-Desmetilantes/isolamento & purificação , RNA Mensageiro/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência , EspectrofotometriaRESUMO
A full-length human cytochrome P450 (P450) 3A4 cDNA clone and four derivatives in which the N-terminus was modified were inserted into a pCW vector and used to transform Escherichia coli DH5 alpha cells. Little expression was seen with the native sequence; the highest level of expression (range of 40-110 membrane-bound nmol P450 liter-1) was achieved with a construct (NF14) in which residues 3-12 were deleted. In all of the constructs P450 was found primarily in the membranes. The modified P450 3A4 (construct NF14) showed typical P450 hemoprotein spectra. The protein was purified to electrophoretic homogeneity in a five-step procedure [nominally 23 nmol P450 (mg protein)-1]. For most purposes it was found to be more practical to purify the modified P450 3A4 to approximately 70% homogeneity [nominally 15 nmol P450 (mg protein)-1] in a simple two-step process. The modified P450 3A4 (NF14) or P450 3A4 purified from human liver could be mixed with rabbit liver NADPH-P450 reductase to achieve catalytic activities nearly as high as those found in human liver microsomes (on a nmol P450 basis), but the optimal reconstitution conditions included not only a mixture of phosphatidylserine, L-alpha-dilauroyl- and L-alpha-dioleoyl-sn-glycero-3-phosphocholines, cholate, and cytochrome b5 suggested by others but also glutathione during the preincubation. Several other thiols were found not to substitute in this role. Good catalytic activity was seen for nifedipine oxidation, testosterone 6 beta-hydroxylation, and the 8,9-epoxidation and 3 alpha-hydroxylation of aflatoxin B1, reactions previously ascribed to the enzyme. These procedures provide a relatively convenient and reliable means of producing, purifying, and reconstituting a catalytically active and useful derivative of P450 3A4, a human P450 enzyme that has many roles in the oxidation of drugs and other xenobiotic chemicals.