Differences in 4-hydroxyestradiol levels in leukocytes are related to CYP1A1(∗)2C, CYP1B1(∗)3 and COMT Val158Met allelic variants.

Exposure to estrogen and its metabolites, including catechol estrogens (CEs) and catechol estrogen quinones (CE-Qs) is closely related to breast cancer. Polymorphisms of the genes involved in the catechol estrogens metabolism pathway (CEMP) have been shown to affect the production of CEs and CE-Qs. In this study, we measured the induction of CYP1A1, CYP1B1, COMT, and GSTP1 by 17ß-estradiol (17ß-E2) in leukocytes with CYP1A1(∗)2C, CYP1B1(∗)3, COMT Val158Met and GSTP1 Ile105Val polymorphisms by semi quantitative RT-PCR and compared the values to those of leukocytes with wild type alleles; we also compared the differences in formation of 4- hydroxyestradiol (4-OHE2) and DNA-adducts. The data show that in the leukocytes with mutant alleles treatment with 17ß-E2 up-regulates CYP1A1 and CYP1B1 and down-regulates COMT mRNA levels, resulting in major increments in 4-OHE2 levels compared to leukocytes with wild-type alleles. Therefore, we propose induction levels of gene expression and intracellular 4-OHE2 concentrations associated with allelic variants in response to exposure of 17ß-E2 as a noninvasive biomarker that can help determine the risk of developing non-hereditary breast cancer in women.

Polymorphisms of catechol estrogens metabolism pathway genes and breast cancer risk in Mexican women.

Breast cancer is associated to estrogen exposure. Allelic variants involved in estrogen metabolism might change the risk of developing this neoplasia. We examined the potential association of breast cancer risk in Mexican women with the polymorphisms CYP1A1 rs1048943, CYP1B1 rs1056836, COMT rs4680, GSTP1 rs1695, GSTT1 null and GSTM1 null which are involved in estrogen metabolism pathway. This study included 150 cases and 150 controls. A significant association was observed between, CYP1A1 rs1048943 (OR = 1.95, C.I. 1.13-3.36) and GSTP1 rs1695 (OR = 2.39, C.I. 1.24-4.24) polymorphisms with the risk of breast cancer. This risk was increased when the women were stratified according to their menopausal status. The results show that breast cancer risk significantly increases in women with 3-6 risk polymorphisms (OR = 3.75, C.I. 1.44-9.74).

Catechol estrogens as biomarkers for mammary gland cancer.

The origin of human tumors has been attributed to the exposure to several environmental chemicals and implicated in the increase of incidence in breast cancer. Progression of breast cancer follows a complex multistep process that seems to depend on various exogenous and endogenous factors. The aim of this study was to examine the effects of the organo-phosphorous pesticide malathion in the presence of estrogen on neoplastic transformation of rat mammary glands. Virgin female rats were sacrificed after 30, 124 and 240 days of 5-day injections twice a day. There were four groups: i) control, ii) malathion (22 mg/100 g body weight, BW), iii) 17ß-estradiol (30 µg/100 g BW) and iv) combination of both. Progressive alterations in ducts were observed by the effect of malathion in comparison to control after 240 days. Ducts markedly increased in size and number of cells per square millimeter and tumors similar to ductal carcinoma were originated. The increase in number of proliferative ducts per square millimeter was significantly (P<0.05) higher in malathion-treated animals compared to the other groups. Progressive alterations in lobules with estrogen treatment were found after 240 days. Lobules became markedly abnormal, referred to as secretory lobules, increased in number and size and the tumors originated were similar to lobular carcinoma. The increase in number of secretory lobules was significantly (P<0.05) higher in estrogen- treated animals compared to the other groups. Treatment with the combination of malathion and estrogen gave rise to tumors constituted of both proliferative ducts and secretory lobules as well as formation of estrogen metabolites such as 2 and 4 catechol estrogens in the blood of the animals after 240 days. We concluded that morphological changes and alterations in the blood of the animals can be used as biomarkers for mammary gland cancer.

Contribution of alpha2-adrenoceptors to the mitogenic effect of catecholestrogen in human breast cancer MCF-7 cells.

Catecholestrogens are estrogen metabolites formed by hydroxylation of 17beta-estradiol and estrone at either the C-2 or C-4 position, rivaling the parent estrogens in concentration. The objective of the present work was to assess if their catechol group could make them induce proliferation of human breast cancer cells via alpha(2)-adrenoceptors. In competition studies in human breast cancer MCF-7 cells, high concentrations of 2-hydroxy-estradiol (2-OH-E(2)), 2-hydroxy-estrone (2-OH-E(1)) and 4-hydroxy-estrone (4-OH-E(1)) competed for [(3)H]-rauwolscine binding, whereas 4-hydroxy-estradiol (4-OH-E(2)) did not. The contribution of alpha(2)-adrenoceptors and estrogen receptors (ERs) in proliferation enhancement was analyzed with specific antagonists. The specific alpha(2)-adrenergic antagonist yohimbine partially reversed the effect of catecholestrogens except 4-OH-E(2). The selective ER downregulator ICI-182780 or fulvestrant partially or totally reversed the effect of all hydroxylated catecholestrogens. When analyzing the effect of the combination of both antagonists in MCF-7, the contribution of the alpha(2)-adrenoceptors and ERs for 2-OH-E(2), 2-OH-E(1) and 4-OH-E(1) was mixed, whereas for 4-OH-E(2), the only receptor implied was an ER. In MDA-MB-231 cells (ER-alpha negative) the proliferation stimulation by these three catecholestrogens and reversal by the adrenergic antagonist was also observed. It can be concluded that alpha(2)-adrenoceptors contribute at least in part to the mitogenic effect of 2-OH-E(2), 2-OH-E(1) and 4-OH-E(1).

Catechol-o-methyltransferase and methoxyestradiols participate in the intraoviductal nongenomic pathway through which estradiol accelerates egg transport in cycling rats.

Estradiol (E(2)) accelerates oviductal egg transport through intraoviductal nongenomic pathways in cyclic rats and through genomic pathways in pregnant rats. This shift in pathways, which we have provisionally designated as intracellular path shifting (IPS), is caused by mating-associated signals and represents a novel and hitherto unrecognized phenomenon. The mechanism underlying IPS is currently under investigation. Using microarray analysis, we identified several genes the expression levels of which changed in the rat oviduct within 6 hours of mating. Among these genes, the mRNA level for the enzyme catechol-O-methyltransferase (COMT), which produces methoxyestradiols from hydroxyestradiols, decreased 6-fold, as confirmed by real-time PCR. O-methylation of 2-hydroxyestradiol was up to 4-fold higher in oviductal protein extracts from cyclic rats than from pregnant rats and was blocked by OR486, which is a selective inhibitor of COMT. The levels in the rat oviduct of mRNA and protein for cytochrome P450 isoforms 1A1 and 1B1, which form hydroxyestradiols, were detected by RT-PCR and Western blotting. We explored whether methoxyestradiols participate in the pathways involved in E(2)-accelerated egg transport. Intrabursal application of OR486 prevented E(2) from accelerating egg transport in cyclic rats but not in pregnant rats, whereas 2-methoxyestradiol (2ME) and 4-methoxyestradiol mimicked the effect of E(2) on egg transport in cyclic rats but not in pregnant rats. The effect of 2ME on egg transport was blocked by intrabursal administration of the protein kinase inhibitor H-89 or the antiestrogen ICI 182780, but not by actinomycin D or OR486. We conclude that in the absence of mating, COMT-mediated formation of methoxyestradiols in the oviduct is essential for the nongenomic pathway through which E(2) accelerates egg transport in the rat oviduct. Yet unidentified mating-associated signals, which act directly on oviductal cells, shut down the E(2) nongenomic signaling pathway upstream and downstream of methoxyestradiols. These findings highlight a physiological role for methoxyestradiols in the female genital tract, thereby confirming the occurrence of and providing a partial explanation for the mechanism underlying IPS.

An aryl hydrocarbon receptor agonist amplifies the mitogenic actions of estradiol in granulosa cells: evidence of involvement of the cognate receptors.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that, besides mediating toxic responses, may have a central role in ovarian physiology. Studying the actions of AHR ligands on granulosa cells function, we have found that beta-naphthoflavone amplifies the comitogenic actions of FSH and 17beta-estradiol in a dose-dependent manner. This amplification was even greater in cells that overexpress the AHR and was reversed by cotreatment with the AHR antagonist alpha-naphthoflavone, suggesting that this effect is mediated by the AHR. The estrogen receptor is likewise implicated in this phenomenon, because a pure antiestrogen abolished the described synergism. However, the more traditional inhibitory AHR-estrogen receptor interaction was observed on the estrogen response element-driven transcriptional activity. On the other hand, alpha-naphthoflavone inhibited dose-dependently the mitogenic actions of FSH and 17beta-estradiol. Beta-naphthoflavone induced the expression of Cyp1a1 and Cyp1b1 transcripts, two well-characterized AHR-inducible genes that code for hydroxylases that metabolize estradiol to catecholestrogens. Nevertheless, the positive effect of beta-naphthoflavone on proliferation was not caused by increased metabolism of estradiol to catecholestrogens, because these compounds inhibited the hormonally stimulated DNA synthesis. This latter inhibition exerted by catecholestrogens suggests that these hydroxylases would play a regulatory point in granulosa cell proliferation. Our study indicates that AHR ligands modulate the proliferation of rat granulosa cells, and demonstrates for the first time that an agonist of this receptor is able to amplify the comitogenic action of classical hormones through a mechanism that might implicate a positive cross-talk between the AHR and the estrogen receptor pathways.

Exposure of bovine oocytes to the endogenous metabolite 2-methoxyestradiol during in vitro maturation inhibits early embryonic development.

Catecholestrogens are endogenous metabolites that have been shown to modulate granulosa, theca, and luteal cell function in some species. The present study was aimed at determining the possible role of these steroids on oocyte maturation. Cumulus-enclosed bovine oocytes were matured for 24 h, fertilized, and then cultured for 8 days. Whereas estradiol was without effect, addition of catecholestrogens (2-hydroxyestradiol, 4-hydroxyestradiol, and 2-methoxyestradiol [2-MOE2]) to the maturation medium did not affect the cleavage rate but was associated with a decrease in blastocyst production on Day 8. Although 2-MOE2 was also able to inhibit blastocyst formation when added during embryo culture, the effects were less pronounced than those seen when the steroid was added only during maturation. In agreement with the known ability of 2-MOE2 to bind tubulin at the colchicine site, marked alterations were observed in the spindle assembly of oocytes exposed to 2-MOE2 during maturation, which lead to gross chromosomal aberrations after fertilization and consequent developmental arrest at the morula stage. Moreover, that the blastocyst rate was not affected when meiosis was blocked with roscovitine during 2-MOE2 exposure is consistent with the idea that altered nuclear maturation is the cause of the low developmental competence. Because 2-MOE2 could be increased in follicular fluid in response to aryl hydrocarbon-receptor ligands, such as some environmental contaminants, our results show that abnormally high intraovarian levels of catecholestrogens could have a deleterious effect on oocyte maturation and early embryonic development arising from the alterations in the meiotic spindle.

Effect of 2-hydroxyoestradiol on insulin secretion in normal rat pancreatic islets.

The possible action of 2-hydroxyoestradiol (2-OHE2) on glucose-induced insulin secretion was evaluated in pancreatic islets isolated from normal rats by collagenase digestion and incubated in KRB buffer. Insulin output in response to either 3.3 or 16.6 mM glucose was measured by radioimmunoassay in the absence or presence of different concentrations of 2-OHE2, norepinephrine (NE), or oestradiol. Islets were also incubated with 2-OHE2, NE, or oestradiol plus a fixed concentration (1 microM) of the alpha 2-adrenergic-receptor blocking agent yohimbine. The results showed that 2-OHE2, oestradiol and NE within a range of 0.1 to 20 microM inhibited glucose-induced insulin secretion in a dose-dependent manner: Ki (microM): 0.04 +/- 0.0001, 0.04 +/- 0.0002, and 0.01 +/- 9.1 E-6 respectively. This suppression was significantly reversed by yohimbine. Contrary to NE and 2-OHE2, oestradiol at lower concentrations (increasing within a range of 0.001 to 0.05 microM) in incubation medium in the same experimental conditions had a significant stimulatory effect on insulin secretion. Thus, it would appear that catecholoestrogens suppress islet insulin release via alpha 2-adrenergic receptors, which suggests that oestrogens may exert a dual modulatory effect on insulin secretion by enhancing release via direct interaction with the cytosolic-oestrogen receptor and inhibiting release after their local hydroxylation and the interaction of their new catechol moiety with alpha 2-adrenergic receptors. Our results suggest that these compounds may play a complementary role to CAs as negative modulators, and they also provide a broader scope for understanding the effect of oestrogens and/or their metabolites in the control of endocrine functions other than those related to reproduction.

Sex and gonadal activity modify the effect of 2-hydroxyestradiol on hypothalamic GABA uptake in the rat.

The aim of this study was to determine the changes induced by sex and sexual steroids on the effect of the catecholestrogen 2-hydroxyestradiol (2OHE2) upon hypothalamic GABA uptake. For this purpose we have measured [3H]-GABA uptake by crude synaptosomal fractions obtained from normal female and male rats and from ovariectomized and virilized female rats in the presence or absence of increasing concentrations (0.1 to 100 microM) of 20HE2. The results presented in this paper demonstrate that the effect of the catecholestrogen varied according to sex: it potentiated the specific [3H]-GABA uptake in female rats, whereas it clearly inhibited the uptake in male and virilized rats. The enhancing effect of the catecholestrogen was not affected by ovariectomy, but a higher specific GABA uptake was observed in the ovariectomized animals. The present study provides the first evidence that the effect of 2OHE2 on hypothalamic GABA uptake depends on sex, thus suggesting the existence of a sexual dimorphism. Further studies in this field are required to elucidate the physiological significance and the underlying mechanism of the mentioned effect.

Effect of catecholestrogens on [3H]-GABA uptake by hypothalamic crude synaptosomes.

Catecholestrogens (CE), 2-hydroxyestradiol, 2-hydroxyestrone and primary estrogens, estradiol and estrone were tested in their ability to compete for the high affinity uptake of [3H]-GABA into crude synaptosomal fractions. Aliquots of the crude synaptosomal fraction obtained from normal rats were incubated for 10 min at 37 degrees C with [3H]-GABA in the presence, or absence, of estrogens and catecholestrogens. Neither estradiol nor estrone modified the specific [3H]-GABA uptake into crude synaptosomal fractions. On the contrary, CE significantly affected the specific [3H]-GABA uptake in a dose-dependent manner: low concentrations of CE enhanced the uptake; this effect disappeared with high concentrations of the compounds. The stimulatory effect of CE on [3H]-GABA uptake was blocked when samples were coincubated with nipecotic acid, thus suggesting that this effect is specific rather than the result of non-specific interactions of CE with the hypothalamic membrane.

Influences of estradiol and of catechol and non-catechol estrogens on the output of prostaglandins in uteri from spayed rats.

The effects of 17-beta estradiol and of some catechol and non-catechol-estrogens on the synthesis and output of prostaglandins (PGs) E and F by uteri from ovariectomized rats, were explored. Uteri from castrated animals released twice as much PGE than PGF. When uterine tissue was obtained from spayed rats injected prior to sacrifice with a low dose of 17-beta estradiol (0.5 + 1.0 microgram, on two consecutive days), the output of PGE diminished significantly. With a higher dose of the hormone (0.5 + 50.0 micrograms) the depressive influence on the synthesis and release of PGE was even more marked, whereas the output of PGF rose significantly. Low or high doses of estrone or of estriol failed to affect the release of either one of the PGs determined. On the other hand, 2-0H-estradiol at a low dose had no action but at a higher one inhibited the release of PGE without influencing PGF. Neither low nor high doses of 2-0H estriol or of 2-0H estrone affected the synthesis and release of uterine PGs. It was also observed that all the compounds tested evoked a significant uterotrophic action. It appears plausible that some catechol metabolites of 17-beta estradiol, but not other catechol-estrogens, could be involved in the mechanism of action of 17-beta estradiol modulating the production of PGs by the rat uterus.