Mailander Paula C, Meza Jane L, Higginbotham Sheila, Chakravarti Dhrubajyoti
Eppley Institute for Research in Cancer and Allied Diseases, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA.
J Steroid Biochem Mol Biol. 2006 Nov;101(4-5):204-15. doi: 10.1016/j.jsbmb.2006.06.019. Epub 2006 Sep 18.
Evidence suggests that the genotoxic mechanism of estrogens (estrone/estradiol) in breast cancer involves their oxidation to 3,4-quinones and reaction with DNA to form depurinating N3Ade and N7Gua adducts. We examined whether estrogen genotoxicity is mutagenic in the mammary gland of the female ACI rat, a model for estrogen-dependent breast cancer. Mutagenesis was studied by PCR amplification of the H-ras1 gene (exons 1-2), cloning in pUC18, transforming Escherichia coli, and sequencing the inserts in plasmids from individual colonies. Mammary glands of both estrogen-responsive (ACI and DA) and resistant (Sprague-Dawley) rats contained pre-existing mutations at frequencies of (39.8-58.8)x10(-5), the majority (62.5-100%) of which were A.T to G.C transitions. Estradiol-3,4-quinone (200 nmol) treatment of ACI rats caused rapid (6h to 1 day) mutagenesis (frequency (83.3-156.1)x10(-5); A.T to G.C 70-73.3%). The estrogen-induced A.T to G.C mutations were detected as G.T heteroduplexes, as would be expected if N3Ade depurinations caused Gua misincorporations by erroneous repair. These heteroduplexes were identified by the T.G-DNA glycosylase (TDG) assay. TDG converts G.T heteroduplexes to G.abasic sites, rendering DNA templates refractory to PCR amplification. Consequently, A.T to G.C mutations present as G.T heteroduplexes in the DNA are eliminated from the spectra. TDG treatment of mammary DNA from estradiol-3,4-quinone-treated ACI rats brought A.T to G.C mutations down to pre-existing frequencies. Our results demonstrate that treatment with estradiol-3,4-quinone, an important metabolite of estrogens, produced A.T to G.C mutations in the DNA of the mammary gland of ACI rats.
有证据表明，雌激素（雌酮/雌二醇）在乳腺癌中的遗传毒性机制涉及它们氧化为3,4-醌并与DNA反应形成脱嘌呤的N3腺嘌呤和N7鸟嘌呤加合物。我们研究了雌激素遗传毒性在雌性ACI大鼠乳腺中是否具有致突变性，ACI大鼠是雌激素依赖性乳腺癌的模型。通过PCR扩增H-ras1基因（外显子1-2）、克隆到pUC18中、转化大肠杆菌以及对来自单个菌落的质粒中的插入片段进行测序来研究诱变作用。雌激素反应性（ACI和DA）和抗性（Sprague-Dawley）大鼠的乳腺中都存在预先存在的突变，频率为（39.8 - 58.8）×10⁻⁵，其中大多数（62.5 - 100%）是A.T到G.C的转换。用雌二醇-3,4-醌（200 nmol）处理ACI大鼠会导致快速（6小时至1天）诱变（频率为（83.3 - 156.1）×10⁻⁵；A.T到G.C为70 - 73.3%）。雌激素诱导的A.T到G.C突变被检测为G.T异源双链体，正如如果N3腺嘌呤脱嘌呤通过错误修复导致鸟嘌呤错配掺入所预期的那样。这些异源双链体通过T.G-DNA糖基化酶（TDG）测定法进行鉴定。TDG将G.T异源双链体转化为G.无碱基位点，使DNA模板难以进行PCR扩增。因此，DNA中以G.T异源双链体形式存在的A.T到G.C突变从谱图中消除。用TDG处理来自经雌二醇-3,4-醌处理的ACI大鼠的乳腺DNA，使A.T到G.C突变降至预先存在的频率。我们的结果表明，用雌激素的一种重要代谢产物雌二醇-3,4-醌处理会在ACI大鼠乳腺DNA中产生A.T到G.C突变。
