Kranendonk M, Carreira F, Theisen P, Laires A, Fisher C W, Rueff J, Estabrook R W, Vermeulen N P
Leiden/Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Vrije Universiteit van Amsterdam, De Boelelaan 1083, 1081 HV, Amsterdam, Netherlands.
Mutat Res. 1999 Apr 26;441(1):73-83. doi: 10.1016/s1383-5718(99)00032-7.
We report here on the genetic engineering of four new Escherichia coli tester bacteria, coexpressing human CYP1A1, CYP2A6, CYP3A4 or CYP3A5 with human NADPH cytochrome P450 reductase (RED) by a biplasmid coexpression system, recently developed to express human CYP1A2 in the tester strain MTC. The four new strains were compared for CYP- and RED-expression levels and CYP activities with the formerly developed CYP1A2 expressing strain. CYP1A2 and CYP2A6 were expressed at the highest, CYP1A1 at the lowest and CYP3A4 and CYP3A5 at intermediate expression levels. Membranes of all five tester bacteria demonstrated similar RED-expression levels, except for the two CYP3A-containing bacteria which demonstrated slightly increased RED-levels. CYP-activities were determined as ethoxyresorufin deethylase (CYP1A1 and CYP1A2), coumarin 7-hydroxylase (CYP2A6) and erythromycin N-demethylase (CYP3A4 and CYP3A5) activities. Reaction rates were comparable with those obtained previously for these CYP-enzymes, except for CYP3A5 which demonstrated a lower activity. Benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene demonstrated mutagenicity in the CYP1A1 expressing strain with mutagenic activities, respectively, approximately 10-fold and 100-fold higher as compared with those obtained with the use of rat liver S9 fraction. Aflatoxin B1 demonstrated a significant mutagenicity with all CYP expressing strains, albeit lower as compared to those obtained with the use of rat liver S9. CYP1A2 was approximately 3-fold more effective in generating a mutagenic response of AFB1 as compared to CYP3A4. CYP3A5 and CYP3A4 demonstrated comparable capacities in AFB1 bioactivation which was equal as found for CYP1A1. It is concluded that these four new strains contain stable CYP- and RED-expression, significant CYP-activities and demonstrated significant bioactivation activities with several diagnostic carcinogens.
我们在此报告通过双质粒共表达系统对四种新型大肠杆菌测试菌进行基因工程改造的情况，该系统最近用于在测试菌株MTC中表达人CYP1A2，可使人CYP1A1、CYP2A6、CYP3A4或CYP3A5与人NADPH细胞色素P450还原酶（RED）共表达。将这四种新菌株的CYP和RED表达水平以及CYP活性与先前开发的表达CYP1A2的菌株进行了比较。CYP1A2和CYP2A6表达水平最高，CYP1A1最低，CYP3A4和CYP3A5处于中等表达水平。除了两种含CYP3A的细菌RED水平略有升高外，所有五种测试菌的膜均显示出相似的RED表达水平。CYP活性通过乙氧异吩唑酮脱乙基酶（CYP1A1和CYP1A2）、香豆素7-羟化酶（CYP2A6）和红霉素N-脱甲基酶（CYP3A4和CYP3A5）活性来测定。反应速率与先前这些CYP酶获得的反应速率相当，除了CYP3A5活性较低。苯并[a]芘和7,12-二甲基苯并[a]蒽在表达CYP1A1的菌株中表现出致突变性，其诱变活性分别比使用大鼠肝脏S9组分时高约10倍和100倍。黄曲霉毒素B1在所有表达CYP的菌株中均表现出显著的致突变性，尽管比使用大鼠肝脏S9时低。与CYP3A4相比，CYP1A2在产生AFB1诱变反应方面的效率约高3倍。CYP3A5和CYP3A4在AFB1生物活化方面表现出相当的能力，与CYP1A1相同。结论是，这四种新菌株具有稳定的CYP和RED表达、显著的CYP活性，并在几种诊断性致癌物中表现出显著的生物活化活性。
