Duarte Maria Paula, Palma Bernardo Brito, Gilep Andrei A, Laires António, Oliveira José Santos, Usanov Sergey A, Rueff José, Kranendonk Michel
Department of Genetics, Faculty of Medical Sciences, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal.
Mutagenesis. 2005 Mar;20(2):93-100. doi: 10.1093/mutage/gei012. Epub 2005 Feb 22.
Cytochrome b(5) (b(5)) is increasingly recognized to be of importance for specific cytochrome P450 (CYP) activities. We developed human b(5)/CYP-competent mutagenicity tester bacteria to study the role of b(5) in the bioactivation activity of human CYP. These new tester bacteria were derived from the previously engineered human CYP-competent Escherichia coli K12 tester strain MTC, containing a bi-plasmid system for the co-expression of a specific CYP form (CYP1A2, 2A6 or 2E1) with human b(5), and human NADPH cytochrome P450 reductase (RED), resulting in the strain BTC-b(5)-1A2, BTC-b(5)-2A6 and BTC-b(5)-2E1, respectively. The relative content of b(5) with CYP and RED in these three BTC-b(5)-CYP strains demonstrated physiologically relevant co-expression levels and typical CYP-specific activities could be determined with their specific chemical probes. These strains were applied in mutagenicity assays along with their corresponding b(5)-void strains to determine the effect of b(5) on the CYP1A2-, CYP2A6- and CYP2E1-mediated bioactivation of several promutagens. For CYP1A2, of the 5 compounds tested [2-aminoanthracene (2AA), 1-aminopyrene, 6-aminochrysene, 2-amino-3-methylimidazo(4,5-f)quinoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)], only the mutagenicity of 2AA was slightly increased ( approximately 1.5-fold) in the presence of b(5). The CYP2E1- and CYP2A6-dependent mutagenicity of N-nitrosodiethylamine increased approximately 3- and 23-fold, respectively when the bacteria contained b(5). The CYP2A6-mediated mutagenicity of NNK increased approximately 9-fold when co-expressed with b(5). The stimulatory effect of b(5) on the bioactivation of N-nitrosodi-n-propylamine was most striking. The mutagenicity of this procarcinogen was completely dependent on the co-expression of b(5) with CYP2A6 or CYP2E1. This demonstrates the prominent role of b(5) in the bioactivation of this carcinogen.
细胞色素b(5)（b(5)）对于特定的细胞色素P450（CYP）活性的重要性日益受到认可。我们构建了具有人b(5)/CYP活性的致突变性测试细菌，以研究b(5)在人CYP生物活化活性中的作用。这些新型测试细菌源自先前构建的具有人CYP活性的大肠杆菌K12测试菌株MTC，其含有双质粒系统，用于共表达特定的CYP形式（CYP1A2、2A6或2E1）与人b(5)以及人NADPH细胞色素P450还原酶（RED），分别产生了菌株BTC-b(5)-1A2、BTC-b(5)-2A6和BTC-b(5)-2E1。这三种BTC-b(5)-CYP菌株中b(5)与CYP和RED的相对含量显示出与生理相关的共表达水平，并且可以用其特定的化学探针测定典型的CYP特异性活性。这些菌株及其相应的无b(5)菌株被应用于致突变性试验，以确定b(5)对几种前诱变剂的CYP1A2-、CYP2A6-和CYP2E1介导的生物活化的影响。对于CYP1A2，在测试的5种化合物[2-氨基蒽（2AA）、1-氨基芘、6-氨基 Chrysene、2-氨基-3-甲基咪唑并(4,5-f)喹啉和4-(甲基亚硝基氨基)-1-(3-吡啶基)-1-丁酮（NNK）]中，仅2AA的致突变性在b(5)存在时略有增加（约1.5倍）。当细菌含有b(5)时，N-亚硝基二乙胺的CYP2E1-和CYP2A6-依赖性致突变性分别增加了约3倍和23倍。与b(5)共表达时，NNK的CYP2A6介导的致突变性增加了约9倍。b(5)对N-亚硝基二正丙胺生物活化的刺激作用最为显著。这种前致癌物的致突变性完全依赖于b(5)与CYP2A6或CYP2E1的共表达。这证明了b(5)在这种致癌物生物活化中的突出作用。
