Cytochrome P4501B1-mediated estrogen metabolism results in estrogen-deoxyribonucleoside adduct formation
Cytochrome P4501B1-mediated estrogen metabolism results in estrogen-deoxyribonucleoside adduct formation
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DOI：
10.1158/0008-5472.can-06-2133
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发表时间：
2007-01-15
期刊：
影响因子：
11.2
通讯作者：
Parl, Fritz F.
中科院分区：
文献类型：
--
作者：
Belous, Alexandra R.;Hachey, David L.;Parl, Fritz F.
雌激素的氧化代谢与乳腺癌的发生有关；然而，对于雌激素引起DNA损伤从而引发乳腺癌的机制，人们知之甚少。为了确定母体激素17 β -雌二醇（E-2）的代谢如何导致DNA加合物的形成，我们使用重组，纯化的I相酶，细胞色素P450 1B1 (CYP1B1)，这是在乳腺组织中表达的，在2'-脱氧鸟苷或2'-脱氧腺苷存在下氧化E2。我们采用气相色谱和液相色谱串联质谱法测定了E-2、2-和4-儿茶酚雌激素（2- ohe2 - 4-OHE2）以及去嘌呤加合物4-OHE2-1(α, β)- n7 -鸟嘌呤（4-OHE2- n7 -gua）和4-OHE2-1(α, β)- n3 -腺嘌呤（4-OHE2- n3 -ade）。CYP1B1将E2氧化为儿茶酚4-OHE2和不稳定的醌4-羟基雌二醇-醌，产生4-OHE2- n7 - gua和4-OHE2- n3 - ade，并呈时间和浓度依赖性。由于反应性醌是作为cyp1b1介导的氧化反应的一部分产生的，因此加合物的形成遵循Michaelis-Menten动力学。在本实验条件下，4-OHE2-N7-Gua加合物（k -m, 4.6 +/- 0.7 μ mol/L; k(cat), 45 +/- 1.6/h）的产率是4-OHE2-N3-Ade加合物（k -m, 4.6 +/- 1.0 μ mol/L; k(cat), 30 +/- 1.5/h）的1.5倍。加合物的产量比4-OHE2的产量低两到三个数量级。该结果直接证明了cyp1b1介导的e -2诱导的加合物形成，为进一步研究雌激素致癌作用提供了实验依据。
The oxidative metabolism of estrogens has been implicated in the development of breast cancer; yet, relatively little is known about the mechanism by which estrogens cause DNA damage and thereby initiate mammary carcinogenesis. To determine how the metabolism of the parent hormone 17 beta-estradiol (E-2) leads to the formation of DNA adducts, we used the recombinant, purified phase I enzyme, cytochrome P450 1B1 (CYP1B1), which is expressed in breast tissue, to oxidize E2 in the presence of 2'-deoxyguanosine or 2'-deoxyadenosine. We used both gas and liquid chromatography with tandem mass spectrometry to measure E-2, the 2- and 4-catechol estrogens (2-OHE2 4-OHE2), and the depurinating adducts 4-OHE2-1(alpha,beta)-N7-guanine (4-OHE2-N7-Gua) and 4-OHE2-1(alpha,beta)-N3-adenine (4-OHE2-N3-Ade). CYP1B1 oxidized E2 to the catechol 4-OHE2 and the labile quinone 4-hydroxyestradiol-quinone to produce 4-OHE2-N7-Gua and 4-OHE2-N3-Ade in a time- and concentration-dependent manner. Because the reactive quinones were produced as part of the CYP1B1-mediated oxidation reaction, the adduct formation followed Michaelis-Menten kinetics. Under the conditions of the assay, the 4-OHE2-N7-Gua adduct (K-m, 4.6 +/- 0.7 mu mol/L; k(cat), 45 +/- 1.6/h) was produced 1.5 times more efficiently than the 4-OHE2-N3-Ade adduct (K-m, 4.6 +/- 1.0 mu mol/L; k(cat), 30 +/- 1.5/h). The production of adducts was two to three orders of magnitude lower than the 4-OHE2 production. The results present direct proof of CYP1B1-mediated, E-2-induced adduct formation and provide the experimental basis for future studies of estrogen carcinogenesis.