NAD(P)H-QUINONE OXIDOREDUCTASE(1) (DT DIAPHORASE) SPECIFICALLY PREVENTS THE FORMATION OF BENZO[A]PYRENE QUINONE-DNA ADDUCTS GENERATED BY CYTOCHROME P4501A1 AND P450 REDUCTASE
NAD(P)H-QUINONE OXIDOREDUCTASE(1) (DT DIAPHORASE) SPECIFICALLY PREVENTS THE FORMATION OF BENZO[A]PYRENE QUINONE-DNA ADDUCTS GENERATED BY CYTOCHROME P4501A1 AND P450 REDUCTASE
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DOI：
10.1073/pnas.91.18.8413
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发表时间：
1994-08-30
影响因子：
11.1
通讯作者：
JAISWAL, AK
中科院分区：
文献类型：
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作者：
JOSEPH, P;JAISWAL, AK
用质粒pMT 2-细胞色素P450 1A 1（CYP 1A 1）、pMT 2-细胞色素P450还原酶（P450还原酶）和pMT 2-NAD（P）H：醌氧化还原酶（1）（NQO（1）或DT心肌黄酶）单独或组合瞬时转染的猴肾COS 1细胞表达显著升高的相应酶水平。将转染的细胞均质化以破坏细胞膜而不影响细胞核，并与苯并[a]芘（BP）孵育以确定cDNA编码的酶在BP代谢活化和/或解毒中的作用。这些研究通过测量转染细胞形成DNA加合物的能力来进行，如通过P-32后标记和蛋白加合物检测所确定的。共转染COS 1细胞与编码CYP 1A 1和P450还原酶的cDNA导致八个不同的BP-DNA加合物。将编码NQO（1）的cDNA沿着与CYP 1A 1和P450还原酶一起转染，使DNA加合物的数目减少到6个。由于cDNA衍生的NQO（1）活性的存在，两种丢失的DNA加合物被特异性消除。随后用BP-1，6-醌、BP-3，6-醌和BP-6，12-醌进行的实验将这两种加合物鉴定为BP醌的加合物。在体外系统中，BP-3，6-醌与脱氧鸟苷（dG）产生两种加合物，但不与dA，dC和dT。此外，TLC板上BP-3，6-醌-dG加合物的位置对应于由cDNA衍生的NQO（1）阻止的位置，因此将这些加合物鉴定为dG的BP醌。此外，NQO（1）减少了转染COS 1细胞的CYP 1A 1和P450还原酶产生的蛋白质-BP加合物的量。这些结果表明，半醌可以直接与DNA结合，并证明NQO（1）活性可以特异性地减少由CYP 1A 1和P450还原酶产生的BP醌代谢物与DNA和蛋白质的结合。
Monkey kidney COS1 cells transiently transfected with plasmids pMT2-cytochrome P450 1A1 (CYP1A1), pMT2-cytochrome P450 reductase (P450 reductase), and pMT2-NAD(P)H:quinone oxidoreductase(1) (NQO(1) or DT diaphorase), individually or in combination, expressed significantly elevated levels of the respective enzyme(s). The transfected cells were homogenized to break cell membranes without affecting the nuclei and incubated with benzo[a]pyrene (BP) to determine the role of cDNA-encoded enzymes in metabolic activation and/or detoxification of BP. These studies were performed by measuring the capacity of the transfected cells to form DNA adducts as determined by P-32 postlabeling and protein adduct detection. Cotransfection of the COS1 cells with cDNAs encoding CYP1A1 and P450 reductase resulted in eight distinct BP-DNA adducts. Inclusion of cDNA encoding NQO(1) along with CYP1A1 and P450 reductase in transfection reduced the number of DNA adducts to six. The two lost DNA adducts were specifically eliminated due to the presence of cDNA-derived NQO(1) activity. Subsequent experiments with BP-1,6-quinone, BP-3,6-quinone, and BP-6,12-quinone identified these two adducts as those of BP quinones. In an in vitro system, BP-3,6-quinone produced two adducts with deoxyguanosine (dG) but not with dA, dC, and dT. Furthermore, the positions of BP-3,6-quinone-dG adducts on TLC plate correspond to those that are prevented by cDNA-derived NQO(1), thus identifying these adducts as BP quinones of dG. In addition, NQO(1) reduced the amount of protein-BP adducts generated by CYP1A1 and P450 reductase into transfected COS1 cells. These results show that semiquinones can directly bind to DNA and demonstrate that NQO(1) activity can specifically reduce the binding of quinone metabolites of BP generated by CYP1A1 and P450 reductase to DNA and protein.