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Oxidation of benzo(a)pyrene by cytochrome P450 1A1 expressed in prokaryotic and eukaryotic systems
Kroftová, Natálie ; Stiborová, Marie (advisor) ; Kubíčková, Božena (referee)
Benzo[a]pyrene (BaP) is a human carcinogen, which is metabolized by a variety of enzyms such as cytochrome P450 (CYP) and epoxide hydrolase. The aim of this work was to study BaP metabolism in vitro by the hepatic microsomal system of rats treated with CYP inducers and by human cytochrome P450 1A1 (CYP1A1) expressed in eukaryotic and prokaryotic systems. An eukaryotic expression system consisted of microsomes isolated from insect cells, whereas a prokaryotic expression system was formed by the membrane fragments of E. coli. In the case of recombinant human CYP1A1, we investigated the influence of cytochrome b5, NADPH:cytochrome P450 reductase (CPR) and epoxide hydrolase in BaP oxidation. Isolation and purification of rabbit hepatic CPR was another aim of this work. BaP metabolites were separated by HPLC. The results found in this work demostrate the fact that hepatic microsomal systems of rats treated with an inducer of CYP1A (Sudan I), an inducer of CYP2B (phenobarbital) and an inducer of CYP3A (PCN) exhibit higher efficiency of BaP oxidation than microsomes of control rats. BaP is oxidized by human CYP1A1 expressed in the eukaryotic system to six metabolites (BaP-9,10-dihydrodiol, BaP metabolite with unknown structure, BaP-7,8-dihydrodiol, BaP-1,6-dion, BaP-3,6-dion, BaP-3-ol), whereas by human...
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The comparison of the efficiency of cytochromes P450 expressed in prokaryotic and eukaryotic systems
Kroftová, Natálie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
v anglickém jazyce Cancer disease is a group of clinically diverse diseases characterized by uncontrolled proliferation of cancer cells incapable of differentiation. Tumor therapy consists of a combination of surgery, radiotherapy and chemotherapy utilizing cytostatic agents. The plant alkaloid ellipticine ranks among a group of cytostatic agents. Its mode of action is based on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts. Ellipticine is oxidized through cytochromes P450 catalysis into detoxication metabolites 9-hydroxyellipticine and 7-hydroxyellipticine, and into activation metabolites 12-hydroxyellipticine, 13-hydroxyellipticine and ellipticine N2 -oxide. The aim of practical part of this work was to compare the efficiency of human cytochromes P450 1A1/2 and 3A4 expressed in prokaryotic and eukaryotic systems in the process of ellipticine oxidation. Ellipticine metabolites were analysed using high performance liquid chromatography. It was found that cytochrome P450 1A1 expressed in the prokaryotic system catalyses predominantly the formation of 9-hydroxyellipticine and 7-hydroxy- ellipticine. The difference is minimal in the production of ellipticine metabolites catalysed by cytochromes P450 1A2 expressed in both cellular systems. The formation of...
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