Analysis of benzo[a]pyrene metabolites formed by rat hepatic microsomes using high pressure liquid chromatography: optimization of the method

A simple and sensitive method was developed to separate the carcinogenic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), and six of its oxidation metabolites generated by rat hepatic microsomes enriched with cytochrome P450 (CYP) 1A1, by high pressure liquid chromatography (HPLC). The HPLC method, using an acetonitrile/water gradient as mobile phase and UV detection, provided appropriate separation and detection of both mono- and di-hydroxylated metabolites of BaP as well as BaP diones formed by rat hepatic microsomes and the parental BaP. In this enzymatic system, 3-hydroxy BaP, 9-hydroxy BaP, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-9,10-dihydrodiol and BaP-dione were generated. Among them the mono-hydroxylated BaP metabolite, 3-hydroxy BaP followed by di-hydroxylated BaP products, BaP-7,8-dihydrodiol and BaP-9,10-dihydrodiol, predominated, while BaP-dione was a minor metabolite. This HPLC method will be useful for further defining the roles of the CYP1A1 enzyme with both in vitro and in vivo models in understanding its real role in activation and detoxification of BaP

The study of activation and detoxication metabolism of the anticancer drug ellipticine by the cytochrome P450 system in vitro and in vivo…

Gen. Physiol. Biophys. (2006), 25, 245-261 245 Oxidation Pattern of the Anticancer Drug Ellipticine by Hepatic Microsomes - Similarity Between Human and Rat Systems M. Stiborová1 , L. Bořek-Dohalská1 , D. Aimová1 , V. Kotrbová1 , K. Kukačková1 , K. Janouchová1 , M. Rupertová1 , H. Ryšlavá1 , J. Hudeček1 and E. Frei2 1 Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic 2 Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany Abstract. Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellip- ticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12- hydroxy-, 13-hydroxyellipticine and ellipticine N2 -oxide. However, only rat micro- somes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N2 -oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in..

The study of activation and detoxication metabolism of the anticancer drug ellipticine by the cytochrome P450 system in vitro and in vivo…

Gen. Physiol. Biophys. (2006), 25, 245-261 245 Oxidation Pattern of the Anticancer Drug Ellipticine by Hepatic Microsomes - Similarity Between Human and Rat Systems M. Stiborová1 , L. Bořek-Dohalská1 , D. Aimová1 , V. Kotrbová1 , K. Kukačková1 , K. Janouchová1 , M. Rupertová1 , H. Ryšlavá1 , J. Hudeček1 and E. Frei2 1 Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic 2 Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany Abstract. Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellip- ticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12- hydroxy-, 13-hydroxyellipticine and ellipticine N2 -oxide. However, only rat micro- somes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N2 -oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in...RNDr. Věra Kotrbová Cíl disertačnípráce cÍLDIsERTačnÍpnÁcn |. Cílem předkládané disertačnípráce bylo rozšířenísoučasných znalostí l " oblasti metabolismu protinádorového léčivaellipticinu jako zásadníhoI I faktoru determinujícího mechanismus působenía cíloýúčinektohoto léčiva. V rámci disertačnípráce byly řešeny následující problematiky: . Metabolismus ellipticinu cytochromy P450 jaterních mikrosomů modelových organismů . Metabolismus ellipticinu purifikovanými cytochomy P450 . Studium indukčníhopotenciálu ellipticinu vůčicytochromům P450 podrodiny lA . Vliv cytochromu b5 na oxidaci ellipticinu katalyzovanou cýochromy P450 lAl atA2 . objasnění mechanismu působenícytochromu b5 na cytochromy P450 lAl a lA2 . Příprava a charakterizace majoritního aduktu ellipticinu s DNA tvořeného jeho metabolitem, l3-hydroxyellipticinem, s deoxyguanosinemDepartment of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

The anticancer drug ellipticine activated with cytochrome P450 mediates DNA damage determining its pharmacological efficiencies:Studies with rats, Hepatic Cytochrome P450 Reductase Null (HRN™) mice and pure enzymes

Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models