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

IF 1.7 4区化学 Q3 CHEMISTRY, MULTIDISCIPLINARY

Monatshefte Fur Chemie Pub Date : 2017-01-01 Epub Date: 2017-07-10 DOI:10.1007/s00706-017-2002-0

Marie Stiborová, Radek Indra, Michaela Moserová, Lucie Bořek-Dohalská, Petr Hodek, Eva Frei, Klaus Kopka, Heinz H Schmeiser, Volker M Arlt

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引用次数: 0

Abstract

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 b₅ and its reductase, NADH:cytochrome b₅ 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 b₅ with and without NADH:cytochrome b₅ 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.

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原核和真核表达系统中人细胞色素 P450 1A1 催化苯并[a]芘氧化作用的比较。

摘要：细胞色素 P450（CYP）1A1 是活化和解毒人类致癌物苯并[a]芘（BaP）的最重要酶。在以前的研究中，我们已经证明不仅能子 NADPH:CYP 氧化还原酶（POR）能作为电子供体，细胞色素 b5 及其还原酶 NADH:cytochrome b5 还原酶也能作为电子供体。在这里，我们研究了所使用的表达系统对活化 BaP 产生的代谢物和 DNA 加合物水平的作用。我们使用了一个真核细胞系统和一个原核细胞系统（Supersomes，从昆虫细胞中分离出来的微粒体；Bactosomes，大肠杆菌的膜部分，分别转染了人类 CYP1A1 和 POR 的 cDNA）。我们用细胞色素 b5 与 NADH:cytochrome b5 还原酶或不与 NADH:cytochrome b5 还原酶进行了重组。我们评估了 NADPH 和 NADH 这两种辅助因子介导 BaP 代谢的有效性。我们发现，这两种系统在催化活化和解毒 BaP 的反应方面存在差异。在有 NADPH 和 NADH 存在的情况下，CYP1A1-超微体产生了两种源于 BaP 的 DNA 加合物，而在 CYP1A1-菌体中，NADPH 而非 NADH 能够支持这一反应。在有 NADPH 或 NADH 的超微体中发现了七种 BaP 代谢物，而在菌体中 NADPH 而非 NADH 能够生成五种 BaP 代谢物。我们的研究表明，原核细胞和真核细胞中表达的 CYP1A1 在 BaP 生物活化过程中具有不同的催化效率，这表明使用大肠杆菌细胞进行此类研究存在一定的局限性：

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来源期刊

Monatshefte Fur Chemie 化学-化学综合

CiteScore

3.70

自引率

5.60%

发文量

116

审稿时长

3.3 months

期刊介绍： "Monatshefte für Chemie/Chemical Monthly" was originally conceived as an Austrian journal of chemistry. It has evolved into an international journal covering all branches of chemistry. Featuring the most recent advances in research in analytical chemistry, biochemistry, inorganic, medicinal, organic, physical, structural, and theoretical chemistry, Chemical Monthly publishes refereed original papers and a section entitled "Short Communications". Reviews, symposia in print, and issues devoted to special fields will also be considered.