Metabolic activation of aromatic amine mutagens by simultaneous expression of human cytochrome P450 1A2, NADPH-cytochrome P450 reductase, and N-acetyltransferase in Escherichia coli
Metabolic activation of aromatic amine mutagens by simultaneous expression of human cytochrome P450 1A2, NADPH-cytochrome P450 reductase, and N-acetyltransferase in Escherichia coli
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DOI：
10.1021/tx970171q
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发表时间：
1998-01-01
影响因子：
4.1
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Josephy, PD;Evans, DH;Guengerich, FP
我们描述了一种新的大肠杆菌菌株的构建，该菌株旨在生物活性芳香胺并以高灵敏度检测它们的致突变性。DJ4309菌株含有两个质粒，一个是pACYC184衍生的表达鼠伤寒沙门氏菌乙酰CoA的质粒：芳香胺N-乙酰转移酶(NAT)，另一个是pBR322衍生的表达人细胞色素P450 1A2和NADPH-细胞色素P450还原酶的质粒。这些酶的联合作用将芳香胺转化为活性的、诱变的N-乙酰氧基酯。该菌株还携带LacZ基因的突变副本(位于F‘因子上)，通过-(GPC)移码突变恢复为野生型基因。菌株DJ4309表达高水平的NAT和细胞色素P4501A2，对具有代表性的芳香胺的诱变非常敏感。在缺乏P450表达载体的亲本菌株中，DJ4309菌株的2-氨基蒽的诱变性高于大鼠肝匀浆9000g上清液(S9)。菌株DJ4309为检测诱变芳香胺和研究其在人P450 1A2中的代谢提供了一个有用的系统。
We describe the construction of a new strain of Escherichia coli designed to bioactivate aromatic amines and to detect their mutagenicity with high sensitivity. Strain DJ4309 bears two plasmids, a pACYC184-derived plasmid which expresses Salmonella typhimurium acetyl CoA:arylamine N-acetyltransferase (NAT) and a pBR322-derived plasmid which expresses human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase. The combined actions of these enzymes convert aromatic amines into reactive, mutagenic N-acetoxy esters. The strain also carries a mutated copy of the lacZ gene (on an F' factor) which reverts to the wildtype gene by a -(GpC) frameshift mutation. Strain DJ4309 expresses high levels of NAT and cytochrome P450 1A2 and is very sensitive to mutagenesis induced by representative aromatic amines. Mutagenicity of 2-aminoanthracene in strain DJ4309 is higher than can be obtained by rat liver homogenate 9000g supernatant(S9) activation in the parent strain lacking the P450 expression vector. Strain DJ4309 provides a useful system for detecting mutagenic aromatic amines and for studying their metabolism by human P450 1A2.