Sudan I (1-phenylazo-2-hydroxynaphthol) is a suspected human carcinogen causing tumors in the livers and urinary bladders of rats, mice, and rabbits. Here, we investigated for the first time the influence of Sudan I exposure on the expression of several biotransformation enzymes in the livers, kidneys, and lungs of rats concomitantly at the mRNA and protein levels and assayed their enzymatic activities. We also studied its effect on the formation of Sudan I-derived DNA adducts in vitro. Sudan I increased the total amounts of cytochrome P450 (P450) in all organs tested. Western blots using antibodies raised against various P450s, NADPH:P450 reductase, and NAD(P)H:quinone oxidoreductase 1 (NQO1) showed that the expression of P450 1A1 and NQO1 was induced in the liver, kidney, and lung of rats treated with Sudan I. The higher protein levels correlated with increased enzyme activities of P450 1A1/2 and NQO1. Furthermore, 9.9-, 5.9-, and 2.8-fold increases in the formation of Sudan I oxidative metabolites catalyzed by microsomes isolated from the liver, kidney, and lung, respectively, of rats treated with Sudan I were found. The relative amounts of P450 1A and NQO1 mRNA, measured by real-time polymerase chain reaction (RT-PCR) analysis, demonstrated that Sudan I induced the expression of P450 1A1 and NQO1 mRNA in the liver, kidney, and lung, and of P450 1A2 mRNA in kidney and lung. Finally, microsomes isolated from livers, kidneys, and lungs of Sudan I exposed rats more effectively catalyzed the formation of Sudan I-DNA adducts than microsomes from organs of control rats. This was attributable to the higher P450 1A1 expression. Because P450 1A1 is playing a major role in the bioactivation of Sudan I in rat and human systems, its induction by Sudan I may have a profound effect on cancer risk by this azo dye. In addition, the induction of P450 1A1/2 and NQO1 enzymes can influence individual human susceptibility to other environmental carcinogens and have an effect on cancer risk.
- MeSH
- adukty DNA účinky léků metabolismus MeSH
- barvicí látky metabolismus MeSH
- cytochrom P-450 CYP1A1 genetika metabolismus MeSH
- cytosol účinky léků enzymologie MeSH
- játra účinky léků enzymologie MeSH
- karcinogeny metabolismus MeSH
- krysa rodu rattus MeSH
- ledviny účinky léků enzymologie MeSH
- messenger RNA genetika MeSH
- mikrozomy účinky léků enzymologie MeSH
- NAD(P)H dehydrogenasa (chinon) genetika metabolismus MeSH
- naftoly metabolismus MeSH
- plíce účinky léků enzymologie MeSH
- potkani Wistar MeSH
- upregulace účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytochrome b(5) (b(5)) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b(5) on CYP-catalyzed reactions, but also that of the apo-cytochrome b(5) (apo-b(5)). Therefore, the apo-b(5) protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b(5) was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5 alpha cells. The gene sequence was verified by DNA sequencing. The sequence coding b(5) was cleaved from pUC19 by NdeI and XhoI restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b(5) was induced with isopropyl beta-D-1-thiogalactopyranoside (IPTG). The b(5) protein, produced predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE-Sepharose. Using such procedures, the homogenous preparation of apo-b(5) protein was obtained. Oxidized and reduced forms of the apo-b(5) reconstituted with heme exhibit the same absorbance spectra as native b(5). The prepared recombinant apo-b(5) reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b(5) is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b(5).
- MeSH
- apoenzymy genetika metabolismus MeSH
- chromatografie iontoměničová MeSH
- cytochrom P-450 CYP3A metabolismus MeSH
- cytochromy b5 genetika metabolismus MeSH
- Escherichia coli genetika MeSH
- hem metabolismus MeSH
- klonování DNA MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- naftoly metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Ellipticine is an antineoplastic agent whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II, and formation of covalent DNA adducts mediated by cytochromes P450 (P450s) and peroxidases. Here, this drug was found to induce CYP1A1 and/or 1A2 enzymes and their enzymatic activities in livers, lungs, and kidneys of rats treated (i.p.) with ellipticine. The induction is transient. In the absence of repeated administration of ellipticine, the levels and activities of the induced CYP1A decreased almost to the basal level 2 weeks after treatment. The ellipticine-mediated CYP1A induction increases the DNA adduct formation by the compound. When microsomal fractions from livers, kidneys, and lungs of rats treated with ellipticine were incubated with ellipticine, DNA adduct formation, measured by (32)P-postlabeling analysis, was up to 3.8-fold higher in incubations with microsomes from pretreated rats than with controls. The observed stimulation of DNA adduct formation by ellipticine was attributed to induction of CYP1A1 and/or 1A2-mediated increase in ellipticine oxidative activation to 13-hydroxy- and 12-hydroxyellipticine, the metabolites generating two major DNA adducts in human and rat livers. In addition to these metabolites, increased formation of the excretion products 9-hydroxy- and 7-hydroxyellipticine was also observed in microsomes of rats treated with ellipticine. Taken together, these results demonstrate for the first time that by inducing CYP1A1/2, ellipticine increases its own metabolism, leading both to an activation of this drug to reactive species-forming DNA adducts and to detoxication metabolites, thereby modulating to some extent its pharmacological and/or genotoxic potential.
- MeSH
- adukty DNA MeSH
- cytochrom P-450 CYP1A1 biosyntéza genetika MeSH
- cytochrom P-450 CYP1A2 biosyntéza genetika MeSH
- cytochromy MeSH
- elipticiny farmakologie MeSH
- enzymová indukce účinky léků MeSH
- játra metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- ledviny metabolismus účinky léků MeSH
- messenger RNA metabolismus MeSH
- mikrozomy enzymologie MeSH
- plíce metabolismus účinky léků MeSH
- potkani Wistar MeSH
- protinádorové látky farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
Ellipticine induces formation of two DNA adducts in leukemia HL-60 and CCRF-CEM cells, identical with deoxyguanosine adducts generated by ellipticine metabolites 13-hydroxyellipticine and 12-hydroxyellipticine in vitro and in vivo. The ellipticine cytotoxicity to HL-60 (IC(50)=0.64microM) and CCRF-CEM cells (IC(50)=4.7microM) correlates with levels of DNA adducts. The different expressions of enzymes activating ellipticine in cells explain this finding. While cytochrome P450 1A1 and cyclooxygenase-1 are expressed in both cells, HL-60 cells express also high levels of another activator, myeloperoxidase. The results suggest the adduct formation as a new mode of antitumor action of ellipticine for leukemia.
- MeSH
- benzopyren * chemie toxicita MeSH
- biotransformace MeSH
- cytochrom P-450 CYP1A1 * farmakokinetika terapeutické užití MeSH
- hepatocyty enzymologie účinky léků MeSH
- jaterní mikrozomy MeSH
- játra metabolismus MeSH
- karcinogeny MeSH
- krysa rodu rattus MeSH
- myši knockoutované MeSH
- NADPH-cytochrom c-reduktasa * MeSH
- polycyklické aromatické uhlovodíky chemie škodlivé účinky toxicita MeSH
- systém (enzymů) cytochromů P-450 farmakokinetika terapeutické užití MeSH
- umlčování genů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
