Radioisotope Research Center, Department of Neurology and Medical Genetics Research Center, Nara Medical University School of Medicine, Kashihara, Nara 634-8521, Japan.
Nucleic Acids Res. 2010 Jul;38(12):e133. doi: 10.1093/nar/gkq233. Epub 2010 Apr 20.
Estrogen-DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN-DNA adducts. Although the formation of 4-OHEN-DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN-DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN-dA adducts and of 4-OHEN-dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose-response between known amounts of 4-OHEN-DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10(8) bases in 1 microg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN-DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN-DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN-DNA adducts in mammalian cells.
雌激素-DNA 加合物是评估与雌激素相关癌症风险和发展的潜在生物标志物。 4- 羟基马烯雌酮 (4-OHEN) 和 4- 羟基马烯醌 (4-OHEQ) 是常见激素替代疗法 (HRT) 制剂中马雌激素的代谢物，能够产生大量的 4-OHEN-DNA 加合物。虽然已经报道了 4-OHEN-DNA 加合物的形成，但尚未在哺乳动物细胞中对其进行定量检测。为了定量检测这些 DNA 加合物，我们生成了一种针对 4-OHEN-DNA 加合物的新型单克隆抗体 (4OHEN-1)。该抗体识别的主要表位是 DNA 中 4-OHEN-dA 加合物和 4-OHEN-dC 加合物的一种立体异构体。用 4OHEN-1 进行的免疫测定显示，已知量的 4-OHEN-DNA 加合物与该抗体结合之间存在线性剂量反应，在 1μg DNA 样品中检测到的下限约为 5 个加合物/10(8)个碱基。在人乳腺癌细胞中，定量免疫测定显示 4-OHEN 产生的 4-OHEN-DNA 加合物比 4-OHEQ 多五倍。此外，在 HRT 的小鼠模型中，口服 Premarin 以时间依赖性方式增加了各种组织（包括子宫和卵巢）中 4-OHEN-DNA 加合物的水平。因此，我们成功建立了一种用于定量检测哺乳动物细胞中 4-OHEN-DNA 加合物的新型免疫测定法。
