Oxidative DNA damage induced by equine estrogen metabolites: role of estrogen receptor alpha

Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases in 8-oxo-deoxyguanosine (8-oxo-dG), as measured by LC-MS-MS or by the Fpg comet assay, were only detected in the S30 cells, and the amount of this lesion could be enhanced by agents, which catalyze redox cycling (NADH) or deplete GSH (diethyl maleate). The role of the estrogen receptor in modulating DNA damage was further established in incubations with the ER antagonist tamoxifen, where decreases in 8-oxo-deoxyguanosine were observed. Another equine estrogen metabolite, 4,17 beta-hydroxyequilenin (4,17 beta-OHEN), was found to have the same cytotoxicity and a similar ability to induce reactive oxygen species (ROS), and caused the same oxidative DNA damage in S30 cells as compared to 4-OHEN. However, 4,17 beta-OHEN induced twice as much single strand DNA breaks in S30 cells compared to 4-OHEN. Also 4,17 beta-OHEN was more estrogenic than 4-OHEN as demonstrated by a higher binding affinity for ER alpha and an enhanced induction in activity of estrogen-dependent alkaline phosphatase in Ishikawa cells. These data suggest that the mechanism of DNA damage induced by equine estrogen metabolites could involve oxidative stress and that the estrogen receptor may play a role in this process.     

雌激素受体基因甲基化与脑出血的关系

目的:探讨伴或不伴颈动脉粥样硬化脑出血与雌激素受体α(ER-α)基因启动子区甲基化状态的关系,方法:入选124例脑出血(经颈动脉超声检查证实部分有颈动脉粥样硬化)的患者为实验组,47例健康体检者为对照组,采集上述入选病例和对照组肘静脉血;提取DNA,采用巢式甲基化特异性PCR(nMSP)检测ER-α基因启动子区甲基化状态.结果:脑出血组甲基化发生率显著高于对照组,差异具有统计学意义(X2=14.090,P=0.000).颈动脉有无粥样硬化的脑出血患者甲基化率差异无统计学意义.结论:脑出血患者ER-α基因启动子区甲基化可能与动脉粥样硬化有关,是促进脑出血发病的机制之一,颅内动脉粥样硬化与颈动脉硬化可能不平行.     

雌激素受体α基因多态性与子宫内膜异位症的相关性

目的探讨雌激素受体α（Estrogen receptorα ERα）基因多态性与子宫内膜异位症（Endometriosis EM）发病的相关性。方法选取107例经开腹或腹腔镜手术治疗后,病理确认为子宫内膜异位症的患者作为实验组,选取健康体检者80例作为正常对照组。提取DNA,采用聚合酶链式反应-限制性片段长度多态性（Polymerase chain reaction-restriction fragment length polymor-phism PCR-RFLP）的方法,检测ERα基因XbaⅠ和PvuⅡ位点多态性。结果 ERα基因多态性显示ERα-XbaⅠ基因多态性在子宫内膜异位症组与对照组间的分布则有显著性差异（P=0.039）,XX/Xx样本组51（47.66%）,对照组25（31.25%）,两者有显著性差异（P=0.024）。X等位基因患EM的风险是x等位基因的2.00倍（OR=2.00,95%CI：1.09-3.67）。而ERα-PvuⅡ基因多态性在EM与对照组间的分布差异无显著性,（P=0.285）。结论 ERα-XbaⅠ基因多态性在子宫内膜异位症组与对照组间的分布则有显著性差异（P=0.039）。提示ERα-XbaⅠ基因多态性与EM发病相关。     

子宫内膜异位症与雌激素受体α基因多态性之间的相关性

目的探讨雌激素受体α（estrogen receptor,ERα）基因多态性与子宫内膜异位症（en-dometriosis,EMs）发生的关系,从而了解EMs的发病因素,为疾病的早期诊断及预防提供实验依据。方法选取214例经手术及病理检查确认为EMs的患者作为观察组,选取健康体检者160例作为正常对照组。采用分子生物学的方法分析ERα的Pvu、Xba酶切位点限制性片段长度多态性（re-striction fragment length polymorphism,RFLP）,观察ERα基因多态性在观察组与对照组中的基因型分布。采用PCR-RFLP的方法分析雌激素受体的多态性。RFLP用Pp（Pvu）和Xx（Xba）来表示。结果Xba多态性在观察组与对照组中的分布存在显著性差异（P=0.002）。观察组X等位基因频率为26.17%,对照组为18.75%,两组间频率比较有显著性差异（P=0.002）;x等位基因频率为73.83%,对照组为81.25%,两组间频率比较有显著性差异（P=0.002）。其中X与x的比值比（OR）值为2.25（95%可信区间1.56～3.23）。Pvu多态性在两组间分布比较,无显著性差异（P=0.081）,两组P与p等位基因频率分布比较,亦无显著性差异（P=0.069）。结论观察人群中X等位基因与EMs发病危险密切相关。     

No relationship exists between itai-itai disease and TA repeat polymorphisms of the estrogen receptor alpha gene

Itai-itai (ouch-ouch) disease is a syndrome accompanied by bone mineral disorders that may be related to oral cadmium exposure. Itai-itai predominantly affects postmenopausal women with a history of multiple childbirth. In a previous study we have examined the genotype distributions of PvuII and XbaI restriction fragment length polymorphisms of the estrogen receptor alpha (ER alpha) gene in patients with itai-itai disease and compared them with those of controls. However, no significant differences were shown between the genotype distributions of the patients and controls. In the present study, we determined the TA repeat polymorphisms of the patients and controls. The distributions of the patients were: HH 25.0%, HL 50.0%, and LL 25.0%; where HH includes two alleles with a high number of TA repeats (TA> or =16), HL includes one high number allele and one low number allele (TA< or =15), and LL includes two alleles with a low number of TA repeats. These patients' distributions were not significantly different from those of the controls. Although our sample number was limited, we concluded that a polymorphism variant of the ER alpha gene is not a predisposing factor for itai-itai disease.     

Expression of estrogen receptor alpha and progesterone receptor in normal human breast epithelium

BACKGROUND: Recently, the immunohistochemical detection of estrogen receptor alpha (ERalpha) expression in breast cancer has become a prerequisite for therapeutic decision-making, however, it remains unknown whether ERalpha or progesterone receptor (PgR) expression in histologically normal breast epithelium (NBE) adjacent to carcinoma can be a reliable internal positive control. PATIENTS AND METHODS: Tissues from a total of 220 breast cancer patients were investigated by immunohistochemistry of ERalpha and PgR expression in NBE adjacent to carcinoma, as well as in carcinoma. The expression pattern was divided into three groups: singular, one or two positive cells; scattered, scattered positive cells surrounded by negative cells; contiguous, ten or more positive cells in contact with each other. RESULTS: In NBE adjacent to carcinoma, the positivity of ERalpha and PgR was 99% (217 out of 220) and 89% (195 out of 220), respectively. The expression pattern of ERalpha and PgR was as follows: singular - 13 and 42 patients, scattered - 116 and 100 patients, and contiguous - 88 and 53 patients, respectively. The contiguous expression pattern of PgR was more frequently noted in premenopausal patients in contrast with ERalpha (p=0.0004). PgR expression was more frequently seen in premenopausal than postmenopausal patients (p=0.0034). PgR expression in carcinoma was more frequently seen in premenopausal than postmenopausal patients (p= 0.009). There was statistically significant correlation between PgR expression in carcinoma and NBE adjacent to carcinoma (p=0.0019). CONCLUSION: These findings suggest that more frequent PgR expression in NBE adjacent to carcinoma might be correlated with carcinogenesis in premenopausal breast cancer patients and that ERalpha expression, not PgR, in NBE adjacent to carcinoma could be a reliable internal positive control.     

Structural essentials of xenoestrogen dialkyl phthalates to bind to the estrogen receptors

Xenoestrogen dialkyl phthalates, C(6)H(4)(COOC(n)H(m))(2), lack the phenolic hydroxyl group that is an essential structural component of the steroid A ring of 17 beta-estradiol. In order to examine whether dialkyl phthalates imitate the steroid structure, we have synthesized a series of 4-hydroxyl derivatives of dialkyl phthalates. The compounds were examined for their ability to displace [(3)H]17 beta-estradiol from the recombinant human estrogen receptor, which was expressed on Sf9 cells using the vaculovirus expression system. Dialkyl 4-hydroxyl phthalates were found to exhibit several-fold higher binding affinities compared to phthalates without the 4-hydroxyl group. From the analyses of receptor binding modes of dialkyl phthalates with and without the 4-hydroxyl group, it was deduced that the phthalic benzene ring mimics the steroid A ring. A biphasic binding curve observed for dicyclohexyl phthalate was also depicted by its 4-hydroxyl derivative, but it increased binding affinity only at the high affinity binding site. These data suggest that the phthalate benzene moiety recognizes the core of the estrogen receptor binding site and the hydrophobic interaction of the dialkyl moiety substantiates the binding characteristics of the phthalates. The present data indicate that even chemicals with slight structural analogy and weak receptor affinity can perturb the endocrine system when administered in high concentrations.     

Competitive binding of some alkyl p-hydroxybenzoates to human estrogen receptor alpha and beta

Alkyl p-hydroxybenzoates such as isobutyl p-hydroxybenzoate (PHBA-iBu), butyl p-hydroxybenzoate (PHBA-nBu), isopropyl p-hydroxybenzoate (PHBA-iPr), propyl p-hydroxybenzoate (PHBA-nPr), ethyl p-hydroxybenzoate (PHBA-Et), and methyl p-hydroxybenzoate (PHBA-Me) are widely used as preservatives, stabilizers and antiseptics for medical supplies, cosmetics, foodstuffs etc. We determined the binding affinity of alkyl p-hydroxybenzoates to human estrogen receptor alpha (ER alpha) and beta (ER beta) by non-RI receptor binding assays. PHBA-iBu had a high binding affinity for ER alpha (IC50: 6.0 x 10(-6) M, the relative binding affinity (RBA): 0.267) and ER beta (IC50: 5.0 x 10(-6) M, RBA: 0.340). These IC50 values and RBA were almost the same as those of bisphenol A. The ranking of the estrogenic potency of alkyl p-hydroxybenzoates for both ERs is different; that is, PHBA-iBu > PHBA-nBu[symbol: see text]PHBA-iPr[symbol: see text]PHBA-nPr > PHBA-Et > PHBA-Me. Alkyl p-hydroxybenzoates bound with equal relative affinity to both ER alpha and beta proteins. Alkyl p-hydroxybenzoate having a long alkyl side-chain showed a high affinity for ER alpha and beta. These findings suggest that p-hydroxybenzoates may be endocrine disruptors.     

Estrogen receptor ligands. II. Discovery of benzoxathiins as potent, selective estrogen receptor alpha modulators

The discovery and synthesis of dihydrobenzoxathiins as potent, ERalpha subtype selective ligands are described. The most active analogue, 4-D, was found to be 50-fold selective in a competitive binding assay and 100-fold selective in a transactivation assay in HEK-293 cells. The alpha selectivity was postulated to lie in the interaction of the sulfur atom of the benzoxathiin ring with the two discriminating residues in the binding pocket of the receptor isoforms.     

Effect of estrogen receptor agonists treatment in MPTP mice: evidence of neuroprotection by an ER alpha agonist

Beneficial effects of 17 beta-estradiol (17 beta-E(2)) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced striatal dopamine (DA) depletion are well documented but the mechanisms implicated are poorly understood. The present experiments investigated the effect of estrogen receptor (ER) agonists treatment in MPTP mice as compared to 17 beta-E(2). The agonists specific for each subtype were 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT) (ER alpha agonist), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and Delta 3-diol (5-androsten-3 beta, 17 beta-diol, also known as 5-androstenediol, androstenediol or hermaphrodiol) (ER beta agonists). Biogenic amines were assayed by HPLC with electrochemical detection. 8 mg/kg of MPTP was administered to give a moderate depletion of striatal DA and its metabolite dihydroxyphenylacetic acid (DOPAC). Protection against MPTP-induced striatal DA and DOPAC depletion was obtained with PPT and 17 beta-E(2) but not with DPN or Delta 3-diol. The striatal dopamine transporter (DAT) was assayed by autoradiography with [(125)I]RTI-121-specific binding. A positive and significant correlation was observed between striatal DA concentrations and [(125)I]RTI-121-specific binding, suggesting that estrogenic treatment that prevented the MPTP-induced DA depletion also prevented loss of DAT. The effect of PPT suggests the implication of an ER alpha in the estrogenic neuroprotection against MPTP. Pointing out which ER is implicated in neuroprotection becomes helpful in designing more specific estrogenic drugs for protection of the aging brain.     

Differential response of estrogen receptors alpha and beta to SP500263, a novel potent selective estrogen receptor modulator

We determined the differential response of a novel SERM, SP500263, on estrogen receptor (ER) alpha and the more recently cloned ER-beta. Because of the high homology of amino acid residues in the ligand-binding domain of ER-alpha and ER-beta, we were not surprised to find that SP500263 binds to both ERs equally well. In contrast, SP500263 acts as a strong estrogen agonist in a strictly ER-alpha-specific manner in U2OS osteosarcoma cell lines blocking the production of interleukin (IL) 6 and granulocyte macrophage colony-stimulating factor. SP500263 also blocked IL-6 production in primary bone cells. The mechanism of this inhibition is different from the classic estrogen stimulation involving an estrogen response element (ERE). SP500263 does not activate gene expression through an ERE. In contrast to the results observed in U2OS cells, SP500263 acts as a strong estrogen antagonist in an MCF-7 breast cancer proliferation assay. Therefore, SP500263 is a member of a series of next-generation SERMs with functional selectivity toward ER-alpha and a mixed agonist/antagonist profile in a bone cell assay versus a breast cancer assay. The panel of assays described herein allow for the development of receptor-specific ligands that may be further developed into novel pharmaceuticals with an improved profile for the treatments of osteoporosis and breast cancer.     

Genistein impairs early testosterone production in fetal mouse testis via estrogen receptor alpha

The widespread consumption of soy-based products raises the issue of the reproductive toxicity of phytoestrogens. Indeed, it is well known that genistein, an isoflavone found in soybeans and soy products, mimics the actions of estrogens and that the fetal testis is responsive to estrogens. Therefore we investigated whether genistein could have deleterious effects on fetal testis. Using organ cultures of fetal testes from wild type and ERα or ERβ knock-out mice we show that genistein inhibits testosterone secretion by fetal Leydig cells during early fetal development (E12.5), within the “masculinization programming window”. This effect occurs through an ERα-dependent mechanism and starting at 10 nM genistein, a concentration which is compatible with human consumption. No effect of genistein on the number of gonocytes was detected at any of the studied developmental stages. These results suggest that fetal exposure to phytoestrogens can affect the development and function of the male reproductive system.     

Interaction between estrogen receptor alpha and insulin/IGF signaling in breast cancer

Estrogens and insulin/Insulin like growth factor 1 (IGF-I) have potent positive effects on the proliferation of mammary epithelial cells and estrogen-dependent breast cancer cells. A cooperative crosstalk between estrogens and insulin/IGF-I signaling pathways exists and it plays a critical role in breast carcinogenesis, tumor cell proliferation, differentiation and survival through the modulation of multiple biological events. The biological effects of estrogens are mainly mediated by the activation of estrogen receptor (ERalpha) whose activity is deeply influenced by the insulin/IGF-I signaling pathway. On the other hand, estrogens enhance insulin signaling by increasing the expression and/or the functional activity of some proteins involved in the insulin/IGF-I pathway. This review will focus on the critical node of the IGF-I network involved in the crosstalk with ERalpha and implicated in breast cancer development and progression.