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Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems

ABSTRACT: Cytochrome P450 (CYP) 1A1 is the most important enzyme activating and detoxifying the human carcinogen benzo[a]pyrene (BaP). In the previous studies, we had shown that not only the canonic NADPH:CYP oxidoreductase (POR) can act as electron donor but also cytochrome b (5) and its reductase,...

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Autores principales: Stiborová, Marie, Indra, Radek, Moserová, Michaela, Bořek-Dohalská, Lucie, Hodek, Petr, Frei, Eva, Kopka, Klaus, Schmeiser, Heinz H., Arlt, Volker M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Vienna 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653725/
https://www.ncbi.nlm.nih.gov/pubmed/29104317
http://dx.doi.org/10.1007/s00706-017-2002-0
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author Stiborová, Marie
Indra, Radek
Moserová, Michaela
Bořek-Dohalská, Lucie
Hodek, Petr
Frei, Eva
Kopka, Klaus
Schmeiser, Heinz H.
Arlt, Volker M.
author_facet Stiborová, Marie
Indra, Radek
Moserová, Michaela
Bořek-Dohalská, Lucie
Hodek, Petr
Frei, Eva
Kopka, Klaus
Schmeiser, Heinz H.
Arlt, Volker M.
author_sort Stiborová, Marie
collection PubMed
description ABSTRACT: Cytochrome P450 (CYP) 1A1 is the most important enzyme activating and detoxifying the human carcinogen benzo[a]pyrene (BaP). In the previous studies, we had shown that not only the canonic NADPH:CYP oxidoreductase (POR) can act as electron donor but also cytochrome b (5) and its reductase, NADH:cytochrome b (5) reductase. Here, we studied the role of the expression system used on the metabolites generated and the levels of DNA adducts formed by activated BaP. We used an eukaryotic and a prokaryotic cellular system (Supersomes, microsomes isolated from insect cells, and Bactosomes, a membrane fraction of Escherichia coli, each transfected with cDNA of human CYP1A1 and POR). These were reconstituted with cytochrome b (5) with and without NADH:cytochrome b (5) reductase. We evaluated the effectiveness of each cofactor, NADPH and NADH, to mediate BaP metabolism. We found that both systems differ in catalysing the reactions activating and detoxifying BaP. Two BaP-derived DNA adducts were generated by the CYP1A1-Supersomes, both in the presence of NADPH and NADH, whereas NADPH but not NADH was able to support this reaction in the CYP1A1-Bactosomes. Seven BaP metabolites were found in Supersomes with NADPH or NADH, whereas NADPH but not NADH was able to generate five BaP metabolites in Bactosomes. Our study demonstrates different catalytic efficiencies of CYP1A1 expressed in prokaryotic and eukaryotic cells in BaP bioactivation indicating some limitations in the use of E. coli cells for such studies. GRAPHICAL ABSTRACT: [Image: see text]
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spelling pubmed-56537252017-11-01 Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems Stiborová, Marie Indra, Radek Moserová, Michaela Bořek-Dohalská, Lucie Hodek, Petr Frei, Eva Kopka, Klaus Schmeiser, Heinz H. Arlt, Volker M. Monatsh Chem Original Paper ABSTRACT: Cytochrome P450 (CYP) 1A1 is the most important enzyme activating and detoxifying the human carcinogen benzo[a]pyrene (BaP). In the previous studies, we had shown that not only the canonic NADPH:CYP oxidoreductase (POR) can act as electron donor but also cytochrome b (5) and its reductase, NADH:cytochrome b (5) reductase. Here, we studied the role of the expression system used on the metabolites generated and the levels of DNA adducts formed by activated BaP. We used an eukaryotic and a prokaryotic cellular system (Supersomes, microsomes isolated from insect cells, and Bactosomes, a membrane fraction of Escherichia coli, each transfected with cDNA of human CYP1A1 and POR). These were reconstituted with cytochrome b (5) with and without NADH:cytochrome b (5) reductase. We evaluated the effectiveness of each cofactor, NADPH and NADH, to mediate BaP metabolism. We found that both systems differ in catalysing the reactions activating and detoxifying BaP. Two BaP-derived DNA adducts were generated by the CYP1A1-Supersomes, both in the presence of NADPH and NADH, whereas NADPH but not NADH was able to support this reaction in the CYP1A1-Bactosomes. Seven BaP metabolites were found in Supersomes with NADPH or NADH, whereas NADPH but not NADH was able to generate five BaP metabolites in Bactosomes. Our study demonstrates different catalytic efficiencies of CYP1A1 expressed in prokaryotic and eukaryotic cells in BaP bioactivation indicating some limitations in the use of E. coli cells for such studies. GRAPHICAL ABSTRACT: [Image: see text] Springer Vienna 2017-07-10 2017 /pmc/articles/PMC5653725/ /pubmed/29104317 http://dx.doi.org/10.1007/s00706-017-2002-0 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Original Paper
Stiborová, Marie
Indra, Radek
Moserová, Michaela
Bořek-Dohalská, Lucie
Hodek, Petr
Frei, Eva
Kopka, Klaus
Schmeiser, Heinz H.
Arlt, Volker M.
Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems
title Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems
title_full Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems
title_fullStr Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems
title_full_unstemmed Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems
title_short Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems
title_sort comparison of human cytochrome p450 1a1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems
topic Original Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653725/
https://www.ncbi.nlm.nih.gov/pubmed/29104317
http://dx.doi.org/10.1007/s00706-017-2002-0
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