Ellipticine is a pro-drug, whose activation is dependent on its oxidation by cytochromes P450 (CYP) and peroxidases. Cytochrome b(5) alters the ratio of ellipticine metabolites formed by isolated reconstituted CYP1A1 and 1A2, favoring formation of 12-hydroxy- and 13-hydroxyellipticine metabolites implicated in ellipticine-DNA adduct formation, at the expense of 9-hydroxy- and 7-hydroxyellipticine that are detoxication products. Cytochrome b(5) enhances the production of 12-hydroxy and 13-hydroxyellipticine. The change in metabolite ratio results in an increased formation of covalent ellipticine-DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. This finding explains previous apparent discrepancies found with isolated enzymes and in vivo, where CYP1A enzymatic activation correlated with ellipticine-DNA-adduct levels while isolated CYP1A1 or 1A2 in reconstituted systems were much less effective than CYP3A4. The effect of cytochrome b(5) might be even more pronounced in vivo, since, as we show here, ellipticine increases levels of cytochrome b(5) in rat liver. Our results demonstrate that both the native 3D structure of cytochrome b(5) and the presence of the heme as an electron transfer agent in this protein enable a shift in ellipticine metabolites formed by CYP1A1/2.
- MeSH
- adukty DNA metabolismus MeSH
- antitumorózní látky fytogenní chemie metabolismus farmakologie MeSH
- cytochrom P-450 CYP1A1 metabolismus MeSH
- cytochromy metabolismus MeSH
- elipticiny chemie metabolismus farmakologie MeSH
- hemoproteiny metabolismus MeSH
- injekce intraperitoneální MeSH
- jaterní mikrozomy účinky léků enzymologie metabolismus MeSH
- krysa rodu rattus MeSH
- metabolická inaktivace MeSH
- oxidace-redukce MeSH
- potkani Wistar MeSH
- transportní proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming covalent DNA adducts. The target of this study was to investigate a role of CYP and peroxidase enzymes in ellipticine oxidative activation in rats, a suitable model mimicking the fate of ellipticine in humans, in details. The contribution of pulmonary and renal CYP- and peroxidase enzymes to ellipticine metabolic activation is investigated and compared with that found in the liver. METHODS: Ellipticine oxidation and DNA adduct formation in vitro were investigated using microsomes isolated from liver, lung and kidney of rats, either control (untreated) or treated i.p. with a single dose of 40 mg of ellipticine per kg of body weight. HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites. Inhibitors of CYPs and cyclooxygenase (prostaglandin H synthase, COX) were used to characterize the enzymes participating in ellipticine oxidative activation in rat liver, lung and kidney. Ellipticine-derived DNA adducts were detected by 32P-postlabeling. RESULTS: Using α-naphthoflavone, furafylline and ketoconazole, inhibitors of CYP1A, 1A2 and 3A, respectively, we found that the CYP1A and 3A enzymes play a major role in ellipticine activation to species forming DNA adducts in liver microsomes. Because of lower expression of these enzymes in lungs and kidneys, even after their induction by ellipticine, they play a minor role in ellipticine activation in these extrahepatic tissues. Arachidonic acid, a cofactor of COX, increased ellipticine activation in the microsomes of extrahepatic tissues. In addition, indomethacin, an inhibitor of COX, efficiently inhibited formation of ellipticine-derived DNA adduct in these microsomes. Based on these results, we attribute the higher activation of ellipticine in lung and kidney microsomes to COX than to CYP enzymes. CONCLUSION: The results demonstrate that whereas CYP enzymes of 1A and 3A subfamilies are the major enzymes activating ellipticine in rat livers, peroxidase COX plays a significant role in this process in lungs and kidneys.
- MeSH
- adukty DNA účinky léků MeSH
- antitumorózní látky metabolismus MeSH
- biotransformace účinky léků MeSH
- elipticiny metabolismus MeSH
- játra metabolismus MeSH
- krysa rodu rattus MeSH
- ledviny metabolismus MeSH
- mikrozomy účinky léků metabolismus MeSH
- modely u zvířat MeSH
- oxidace-redukce účinky léků MeSH
- peroxidasy fyziologie MeSH
- plíce metabolismus MeSH
- potkani Wistar MeSH
- systém (enzymů) cytochromů P-450 fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of activity of several cytochromes P450 (CYP). To elucidate the mechanism of such modulations it is necessary to evaluate not only the effect of native cyt b5, but also that of apo-cyt b5. To prepare apo-cyt b5, heme transfer from native cyt b5 to a protein with higher affinity toward the heme, the horse heart apo-myoglobin, was utilized. METHODS: Butanone extraction was employed to prepare apo-myoglobin. Apo-cyt b5 was separated from myoglobin by chromatography on DEAE-Sepharose. Mass spectrometry was utilized to characterize proteins eluted from DEAE- Sepharose. RESULTS: The prepared apo-myoglobin was incubated with the cyt b5 at pH 4.2 that is the optimal pH for heme transfer from cyt b5 into apo-myoglobin. The apo-cyt b5 protein was separated from myoglobin present in the reaction mixture by chromatography on a column of DEAE-Sepharose. Using such a procedure, 16% yield of apo-cyt b5 that did not contain any heme in its molecule was obtained from the native rabbit cyt b5. Oxidized and reduced forms of the apo-b5 reconstituted with heme exhibit the same absorbance spectra as native cyt b5. The prepared apo-cyt b5 reconstituted with heme can receive electrons from NADPH:CYP reductase. CONCLUSION: A biologically active apo-cyt b5 was prepared using transfer of heme from cyt b5 to horse heart apo-myoglobin by the procedure described here.
- MeSH
- absorpce MeSH
- apoproteiny chemie genetika MeSH
- butanony MeSH
- chromatografie MeSH
- cytochromy b5 chemie genetika metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- elektrony MeSH
- hem chemie MeSH
- hmotnostní spektrometrie MeSH
- koncentrace vodíkových iontů MeSH
- koně MeSH
- králíci MeSH
- molekulární sekvence - údaje MeSH
- myoglobin chemie genetika MeSH
- myokard chemie MeSH
- NADPH-cytochrom c-reduktasa chemie MeSH
- oxidace-redukce MeSH
- sekvence aminokyselin MeSH
- spektrální analýza MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- financování organizované MeSH
- Publikační typ
- abstrakty 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
- antitumorózní látky farmakologie 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
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- MeSH
- antitumorózní látky fytogenní MeSH
- antitumorózní látky MeSH
- cytochrom P-450 CYP1A1 * MeSH
- cytochrom P-450 CYP1A2 * MeSH
- cytochromreduktasy MeSH
- elipticiny * farmakokinetika MeSH
- jaterní mikrozomy * MeSH
- klinické laboratorní techniky metody využití MeSH
- králíci MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- NADPH-cytochrom c-reduktasa * MeSH
- rozpřahující látky MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
