Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1.

Aoyama, Toru; Yamano, Shigeru; Guzelian, Philip S.; Gelboin, Harry V.; Gonzalez, Frank J.

doi:10.1073/pnas.87.12.4790

Cited by 201 publications

(88 citation statements)

References 19 publications

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“…A fruitful alternative consists of the expression of cloned cDNA, coding for individual forms of P450 enzymes, in an adequate heterologous system. Several human liver P450 from the subfamilies involved in xenobiotic oxidation have already been expressed in different systems, including recombinant simian virus 40 in COS cells and references therein; Romkes et al, 1991;Veronese et al, 1991), recombinant vaccinia viruses in human hepatoma Hep G2 cells (Aoyama et al, 1990a and1990b, and references therein) and recombinant plasmids in the yeast Succharomyces cerevisiae (Yasumori et al, 1989;Brian et al, 1989;Brian et al, 1990;Renaud et al, 1990;Eugster et al, 1990;Ching et al, 1991; Truan, G., Cullin, C., Reisdorf, P., Urban, P. and Pompon, D., unpublished results). P450 NF25 (CYP3A4) is important in pharmacology and toxicology, not only because it is probably the major form of human liver (Guengerich and Turvy, 1991) but also because it is involved in the metabolism of numerous widely used drugs such as nifedipine (Guengerich et al, 1986a), erythromycin and troleandomycin (Renaud et al, 1990 and references therein), quinidine (Guengerich et al, 1986b), cyclosporin A (Kronbach et al, 1988;Aoyama et al, 1989;Combalbert et al, 1989), 1 7a-ethynylestradiol (Guengerich, 1988), midazolam (Kronbach et al, 1989), lidocaine (Bargetzi et al, 1989;Imaoka et al, 1990), and diltiazem (Pichard et al, 1990).…”

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confidence: 99%

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Peyronneau

Renaud

Truan

et al. 1992

European Journal of Biochemistry

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Human liver P450 NF25 (CYP3A4) had been previously expressed in Succhuromyces cerevisiue using the inducible GALIO-CYCl promoter and the phosphoglycerate kinase gene terminator [Renaud, J. p., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194,. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72,463 -4721 increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25 -iron-metabolite complexes. %fold, g-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6@-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b, always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25 -iron-metabolite complex. A P450 Fe(I1)-(RNO) complex was obtained upon oxidation of Nhydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase. These results show that specific activities of yeast-expressed P450 NF25 may be artificially low, owing to limiting amounts of the associated microsomal redox proteins and emphasize the importance of controlling the amounts of the different components of the monooxygenase complex in order to optimize these catalytic activities, especially when the expression system is to be used for demonstrating metabolic capacities towards new substrates.

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confidence: 99%

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Peyronneau

Renaud

Truan

et al. 1992

European Journal of Biochemistry

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“…These cells contain ample intracellular membrane and NADPH-P450 oxidoreductase to support P450 catalytic activity. Infected cells were then analyzed for their capacities to activate the procarcinogen aflatoxin B1 (Aoyama et al 1990). Out of twelve P450s analyzed, CYP1A2, CYP2A6, CYP2B6, CYP3A3 and CYP3A4 all converted aflatoxin B1 to metabolites capable of binding to DNA and producing histidine revertants in the Ames test.…”

Section: Results and Discussion Cdna-directed Expression Of Human Cytmentioning

confidence: 99%

“…P450 expression was analyzed using Western immunoblotting and standard enzyme assays. Aflatoxin B 1 activation by vaccinia-expressed human P450s was determined using in situ DNA binding and the Ames test (Aoyama et al 1990). In the latter case, the common rat 89 preparations, used To quantify P450 mRNA, RNase protection vas performed as shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Human Cytochrome P450 Catalytic Activities and Expression.

Gonzalez

Crespi²,

Czerwinski

et al. 1992

Tohoku J. Exp. Med.

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Cytochromes P450 are a large group of membraneassociated heme protein monooxygenases, most of which are responsible for metabolizing foreign compounds. Chemical carcinogens, which are ingested or absorbed into the body as inert forms, are metabolically activated by P450s to electrophilic metabolites capable of binding to and mutating DNA. Different P450 forms are responsible for activation of the various classes of chemical carcinogens including the arylamines, polycyclic aromatic hydrocarbons, nitrosamines and afiatoxins. Thus, the cellular constituency and levels of P450s could determine the fate of a particular carcinogen and the risk of humans to exposure. To study the catalytic activities of human P450s, human P450 cDNAs were cloned and expressed into active enzymes using cultured cells. By both transient and stable cDNA expression systems, several human P450s were found to be capable of metabolically-activating the human hepatocarcinogen afiatoxin B1. These cDNA expression systems can also be used to determine whether an unknown chemical will be activated by a human P450 and thus be toxic or mutagenic in humans. To assess the extent of interindividual variation in P450 expression, probes developed from P450 cDNAs are being used to quantify levels of P450 mRNAs in various human tissues. Studies using RNase protection revealed that the closely related CYP2B6 and CYP2B7 mRNAs could be independently quantified in liver and lung, respectively. This procedure can be used to examine expression of different P450 genes in banks of human tissue specimens.

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“…P450 enzymes have been shown to play major roles in activating these carcinogens, based on the analysis of formation of chemically reactive metabolites, DNA adduct and damage, chromosomal abbreviation, and bacterial mutagenicity and genotoxicity assays such as Ames and umu test systems (2)(3)(4)(5)(6)(7)(8). Our previous studies using umu genotoxicity assay with human P450 enzymes in conjunction with the results obtained from Ames mutagenicity assay and other detection systems reported so far (6,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) have suggested that human CYP1A1, 1A2, 1B1, 2A6, 2A13, 2E1, and 3A4 are major enzymes involved in the activation of various environmental carcinogens including PAHs and tobacco-related nitrosamines (Table 1). In this review, we first describe in vivo studies on the roles of CYP1 and 2A enzymes in the formation of tumors caused by various chemical carcinogens; these are reported using (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31).…”

Section: T Shimadamentioning

confidence: 99%

Inhibition of Carcinogen-Activating Cytochrome P450 Enzymes by Xenobiotic Chemicals in Relation to Antimutagenicity and Anticarcinogenicity

Shimada

2017

ToxicolRes

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A variety of xenobiotic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), aryl-and heterocyclic amines and tobacco related nitrosamines, are ubiquitous environmental carcinogens and are required to be activated to chemically reactive metabolites by xenobiotic-metabolizing enzymes, including cytochrome P450 (P450 or CYP), in order to initiate cell transformation. Of various human P450 enzymes determined to date, CYP1A1, 1A2, 1B1, 2A13, 2A6, 2E1, and 3A4 are reported to play critical roles in the bioactivation of these carcinogenic chemicals. In vivo studies have shown that disruption of Cyp1b1 and Cyp2a5 genes in mice resulted in suppression of tumor formation caused by 7,12-dimethylbenz[a]anthracene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, respectively. In addition, specific inhibitors for CYP1 and 2A enzymes are able to suppress tumor formation caused by several carcinogens in experimental animals in vivo, when these inhibitors are applied before or just after the administration of carcinogens. In this review, we describe recent progress, including our own studies done during past decade, on the nature of inhibitors of human CYP1 and CYP2A enzymes that have been shown to activate carcinogenic PAHs and tobacco-related nitrosamines, respectively, in humans. The inhibitors considered here include a variety of carcinogenic and/or non-carcinogenic PAHs and acethylenic PAHs, many flavonoid derivatives, derivatives of naphthalene, phenanthrene, biphenyl, and pyrene and chemopreventive organoselenium compounds, such as benzyl selenocyanate and benzyl selenocyanate; o-XSC, 1,2-, 1,3-, and 1,4-phenylenebis(methylene)selenocyanate.

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Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1.

Cited by 201 publications

References 19 publications

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Analysis of Human Cytochrome P450 Catalytic Activities and Expression.

Inhibition of Carcinogen-Activating Cytochrome P450 Enzymes by Xenobiotic Chemicals in Relation to Antimutagenicity and Anticarcinogenicity

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Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1.

Cited by 201 publications

References 19 publications

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b5

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b5

Analysis of Human Cytochrome P450 Catalytic Activities and Expression.

Inhibition of Carcinogen-Activating Cytochrome P450 Enzymes by Xenobiotic Chemicals in Relation to Antimutagenicity and Anticarcinogenicity

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Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅