Data from: Metabolites of n-Butylparaben and iso-Butylparaben exhibit estrogenic properties in MCF-7 and T47D human breast cancer cell lines

Two oxidized metabolites of n-butylparaben (BuP) and iso-butylparaben (IsoBuP) discovered in human urine samples exhibit structural similarity to endogenous estrogens. We hypothesized that these metabolites bind to the human estrogen receptor (ER) and promote estrogen signaling. We tested this using models of ER-mediated cellular proliferation. The estrogenic properties of 3-hydroxy n-butyl 4-hydroxybenzoate (3OH) and 2-hydroxy iso-butyl 4-hydroxybenzoate (2OH) were determined using the ER-positive, estrogen-dependent human breast cancer cell lines MCF-7 and T47D. The 3OH metabolite induced cellular proliferation with EC50 of 8.2 μM in MCF-7 cells. The EC50 for 3OH in T47D cells could not be reached. The 2OH metabolite induced proliferation with EC50 of 2.2 μM and 43.0 μM in MCF-7 and T47D cells, respectively. The EC50 for the parental IsoBuP and BuP was 0.30 μM and 1.2 μM in MCF-7 cells, respectively. The expression of a pro-proliferative, estrogen-inducible gene (GREB1) was induced by these compounds and blocked by co-administration of an ER antagonist (ICI 182,780), confirming the ER-dependence of these effects. The metabolites promoted significant ER-dependent transcriptional activity of an ERE-luciferase reporter construct at 10 and 20 μM for 2OH and 10 for 3OH μM. Computational docking studies showed that the paraben compounds exhibited the potential for favorable ligand binding domain interactions with human ERα in a manner similar to known x-ray crystal structures of 17β-estradiol in complex with ERα. We conclude that the hydroxylated metabolites of n-butyl- and iso-butylparaben are weak estrogens and should be considered as additional components of potential endocrine disrupting effects upon paraben exposure.

本研究在人体尿液样本中检出的尼泊金丁酯（n-butylparaben, BuP）与尼泊金异丁酯（iso-butylparaben, IsoBuP）的两种氧化代谢物，其结构与内源性雌激素具有高度相似性。我们提出假说：此类代谢物可结合人雌激素受体（estrogen receptor, ER）并激活雌激素信号转导通路。为验证该假说，我们采用ER介导的细胞增殖模型开展实验。我们借助雌激素受体阳性、雌激素依赖型人乳腺癌细胞系MCF-7与T47D，对3-羟基-4-羟基苯甲酸丁酯（3-hydroxy n-butyl 4-hydroxybenzoate, 3OH）与2-羟基-4-羟基苯甲酸异丁酯（2-hydroxy iso-butyl 4-hydroxybenzoate, 2OH）的雌激素活性进行了测定。在MCF-7细胞中，3OH代谢物可诱导细胞增殖，其半数有效浓度（EC50）为8.2 μM；而在T47D细胞中，未检测到3OH的EC50值。2OH代谢物在MCF-7与T47D细胞中的EC50分别为2.2 μM与43.0 μM。母体化合物IsoBuP与BuP在MCF-7细胞中的EC50分别为0.30 μM与1.2 μM。上述化合物均可诱导促增殖雌激素诱导基因（GREB1）的表达，且该效应可通过共施予雌激素受体拮抗剂（ICI 182,780）予以阻断，证实了上述效应的ER依赖性。在10 μM与20 μM浓度下，2OH与3OH可分别显著诱导雌激素反应元件-荧光素酶报告基因构建体的ER依赖性转录活性（3OH测试浓度为10 μM）。分子对接研究显示，此类对羟基苯甲酸酯类化合物可与人雌激素受体α（ERα）的配体结合域产生良好的相互作用，其结合模式与已解析的17β-雌二醇-ERα复合物X射线晶体结构高度相似。综上，尼泊金丁酯与尼泊金异丁酯的羟基化代谢物属于弱雌激素类物质，在对羟基苯甲酸酯暴露引发的潜在内分泌干扰效应中，此类代谢物应被视为额外的贡献组分。