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Oxidation of ellipticine by human cytochromes P450 expressed in prokaryotic and eukaryotic systems
Vejvodová, Lucie ; Stiborová, Marie (advisor) ; Hýsková, Veronika (referee)
Ellipticine is an alkaloid with antitumor activity, whose mechanism of action is based on intercalation into DNA, inhibition of topoisomerase II and formation of covalent adducts with DNA, after its enzymatic activation by cytochromes P450 and/or peroxidases. Ellipticine is oxidized by cytochromes P450 to form up to five metabolites (7-hydroxy-, 9-hydroxy, 12- hydroxy-, 13-hydroxyellipticine and N2 -oxide ellipticine). 9-Hydroxy- and 7- hydroxyellipticine are considered to be detoxification metabolites, whereas 12-hydroxy-, 13- hydroxyellipticine and N2 -oxide of ellipticine are considered as activation metabolites, which are responsible for formation of covalent DNA adducts. The aim of this thesis was to examine the efficiency of human recombinant cytochromes P450 expressed in eukaryotic (SupersomesTM ) and two prokaryotic expression systems (Bactosomes) in oxidation of ellipticine. Cytochromes P450 expressed in prokaryotic systems differed in the amounts of "coexpressed" NADPH:CYP reductase. The resulting ellipticine metabolites were analyzed by HPLC. The results obtained in this thesis demonstrate that human cytochromes P450 2C9/2D6/2C19 expressed in prokaryotic or eukaryotic systems oxidize ellipticine to form up to four metabolites: 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and N2 -oxide...
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Oxidation of ellipticine by human cytochromes P450 expressed in prokaryotic and eukaryotic systems
Vejvodová, Lucie ; Stiborová, Marie (advisor) ; Hýsková, Veronika (referee)
Ellipticine is an alkaloid with antitumor activity, whose mechanism of action is based on intercalation into DNA, inhibition of topoisomerase II and formation of covalent adducts with DNA, after its enzymatic activation by cytochromes P450 and/or peroxidases. Ellipticine is oxidized by cytochromes P450 to form up to five metabolites (7-hydroxy-, 9-hydroxy, 12- hydroxy-, 13-hydroxyellipticine and N2 -oxide ellipticine). 9-Hydroxy- and 7- hydroxyellipticine are considered to be detoxification metabolites, whereas 12-hydroxy-, 13- hydroxyellipticine and N2 -oxide of ellipticine are considered as activation metabolites, which are responsible for formation of covalent DNA adducts. The aim of this thesis was to examine the efficiency of human recombinant cytochromes P450 expressed in eukaryotic (SupersomesTM ) and two prokaryotic expression systems (Bactosomes) in oxidation of ellipticine. Cytochromes P450 expressed in prokaryotic systems differed in the amounts of "coexpressed" NADPH:CYP reductase. The resulting ellipticine metabolites were analyzed by HPLC. The results obtained in this thesis demonstrate that human cytochromes P450 2C9/2D6/2C19 expressed in prokaryotic or eukaryotic systems oxidize ellipticine to form up to four metabolites: 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and N2 -oxide...
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