Download P096 Effectiveness of human cytochrome P450 1A1 expressed in

P096
Effectiveness of human cytochrome P450 1A1 expressed in eukaryotic and prokaryotic
systems in oxidation and DNA adduct formation by benzo[a]pyrene
Stiborová Marie, Moserová Michaela, Indra Radek, Frei Eva, Schmeister Heinz H., Arlt
Volker M.
Department of Biochemistry, Faculty of Science, Charles University in Prague, Albertov 6,
128 43 Prague 2; Division of Radiopharmaceutical Chemistry, German Cancer Research
Center (DKFZ), 69120 Heidelberg, Germany; Analytical and Enviromental Sciences
Division, MRC-HPA Centre for Enviroment and Health, King´s College London, London SE1
9NH, United Kingdom
E-mail: [email protected]
Sekce: Xenobiochemie a molekulární toxikologie
Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by
cytochrome P450 (CYP), predominantly by CYP1A1. Here, we investigated a role of human
CYP1A1 in BaP oxidation and formation of BaP-DNA adducts. Human CYP1A1 expressed
in the eukaryotic system (microsomes of insect cells transfected with human CYP1A1 and
NADPH:CYP reductase, POR - Supersomes™) and in the prokaryotic system of Escherichia
coli cells (E. coli membranes containing over-expressed human CYP1A1 and POR Bactosomes) were used as models. In the eukaryotic cells, CYP enzymes, including CYP1A1,
are components of a mixed-function oxidase (MFO, monooxygenase) system located in the
membrane of endoplasmic reticulum (microsomes). This enzymatic system also contains
other enzymes, the multidomain flavoprotein POR and cytochrome b5 accompanied by its
NADH:cytochrome b5 reductase (CBR). Because Supersomes™ are microsomes, they contain
beside the over-expressed human recombinant CYP1A1 with POR, also basic levels of CBR,
microsomal epoxide hydrolase (mEH) and cytochrome b5. In the case of the prokaryotic cells
of E. coli transfected with human CYP1A1 and POR, this CYP together with POR are
expressed in the cell membrane that does not contain any other enzymes of the MFO
monooxygenase system. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts
were generated by the CYP1A1-Supersomes™ system, both in the presence of NADPH and
in the presence of NADH. BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP3,6-dione, an unknown metabolite, and at least three BaP-DNA adducts were observed in the
system using CYP1A1 expressed with POR in E. coli plus NADPH, a cofactor of POR.
Because of the absence of mEH, no dihydrodiol metabolites of BaP were formed. When
CYP1A1 in E. coli membrane was incubated with a cofactor of CBR, NADH, no BaP
metabolites as well as BaP-DNA adducts were detectable. Only low levels of BaP-DNA
adducts were found in the presence of NADH when purified CBR and cytochrome b5 were
added into the E. coli enzymatic system. This finding suggests a role of the
NADH/cytochrome b5/CBR system as sole donor of electrons to CYP1A1 in its reaction cycle
substituting the system of POR with NADPH. The results found indicate some limitation in
the use of human CYP1A1 expressed in cells of the prokaryotic organism, E. coli. The
differences between enzyme (protein) compositions of the cell membrane of E. coli from
those of the microsomal membrane of Supersomes™, absenting several components of the
CYP monooxygenase system, seem to be responsible for the found results.
Supported by GACR (grant 15-02328S) and Charles University (grant UNCE 204025/2012).