The antiestrogen LY117018 is estrogenic in the fetal rat

LY117018 (LY) has high affinity for the adult rat uterine estrogen receptor, has little uterotrophic activity, and inhibits many estradiol (E2)-induced responses in the adult or immature uterus. In these studies, LY was injected into day 19 rat fetuses, with and without diethylstilbestrol (DES) or E2, to determine whether it could block the estrogen-induced teratogenesis. LY at 1, 25, or 50 micrograms/fetus failed to decrease the 15-70% incidences of oviduct malformation and cleft phallus induced by DES (2.5 micrograms/fetus) or E2 (50 micrograms/fetus). However, LY alone (1-50 micrograms/fetus) was more potent than E2 in eliciting these same urogenital malformations. LY also failed to compete in vitro for plasma protein-bound 3H-E2, and therefore, like DES, is more available than E2 for uptake into fetal tissues. Thus, in the fetus, unlike the adult, LY was an estrogen agonist, which indicates that the fetus has a very different sensitivity than the adult to estrogenic compounds.     

Additive growth-inhibitory effects of DL-alpha-difluoromethylornithine and antiestrogens on MCF-7 breast cancer cell line

We studied the growth inhibitory effects of DL-alpha-difluoromethylornithine, and antiestrogens (tamoxifen, 4-hydroxytamoxifen, trioxifene, keoxifene, and LY117018) as single agents and in combinations on the proliferation of a breast cancer cell line, MCF-7. At 0.1 mM difluoromethylornithine, the proliferation of MCF-7 cells was inhibited to 75 +/- 6% of the controls. Treatment of the cells with 0.1 microM 4-hydroxytamoxifen reduced cell growth to 72 +/- 4%. Combination of 0.1 mM difluoromethylornithine and 0.1 microM 4-hydroxytamoxifen reduced cell growth to 38 +/- 5%, indicating additive growth inhibitory effects. Similar additive effects were observed with all 5 antiestrogens in combination with difluoromethylornithine.     

Effects of oestrogen and the antioestrogens, tamoxifen and LY117018, on four oestrogen-regulated RNAs in the EFM-19 breast cancer cell line

The EFM-19 cell line is a new breast cancer cell line whose proliferation has been reported to be stimulated by oestrogens and inhibited by the antioestrogen tamoxifen. Oestrogen receptor mRNA levels are higher in EFM-19 cells than in other oestrogen-responsive cell lines. The levels of four oestrogen-inducible RNAs [pNR-1, pNR-2, pNR-25 and pNR-100] were measured in EFM-19 cells. Oestradiol treatment increased the levels of the four regulated RNAs between 3-fold (pNR-100) and greater than 100-fold (pNR-2). The induction was half maximal between 1.5 x 10(-11) and 1.5 x 10(-10) M oestradiol. The effects of two antioestrogens, tamoxifen and LY117018, were measured on the expression of the oestrogen-regulated RNAs. Tamoxifen was a partial oestrogen agonist for the induction of the pNR-1 and pNR-25 RNAs but had very little effect on the pNR-2 and pNR-100 RNA levels. The pNR-2 RNA levels were less induced by tamoxifen in EFM-19 cells than in MCF-7 cells. LY117018 did not increase the levels of any RNA. The oestrogen-induced levels of the four RNAs were reduced by both antioestrogens to the RNA levels present in cells treated with the antioestrogens alone. LY117018 was at least 100-fold more potent than tamoxifen as an oestrogen antagonist.     

Actions of oestrogens and antioestrogens on rat mammary gland development: relevance to breast cancer prevention

The proliferative actions of a series of antioestrogens on the development of the second thoracic mammary gland of ovariectomized immature Sprague-Dawley rats have been investigated. Evidence is presented that shows trans-tamoxifen, LY 117018 and LY 139481, like oestradiol-17 beta and cis-tamoxifen, promote full mammary gland ductal development and induce a high rate of cell proliferation in the undifferentiated epithelial cells of the terminal end buds, the main growth region for ductal growth. Conversely, ICI 164,384, a new antioestrogen, is without effect on ductal elongation. In vivo exposure of trans-tamoxifen and LY 117018 treated glands in medically castrated animals to the carcinogen DMBA, results in a high rate of mammary tumour development. Indeed, the actions of these so-called antioestrogens are equivalent to those observed in oestradiol-treated rats.     

Species-specific pharmacology of antiestrogens: role of metabolism

The nonsteroidal antiestrogen tamoxifen exhibits a paradoxical species-specific pharmacology. The drug is a full estrogen in the mouse, a partial estrogen/antiestrogen in humans and the rat, and an antiestrogen in the chick oviduct. Inasmuch as tamoxifen has antiestrogenic effects in vitro, differential metabolism of tamoxifen to estrogens might occur in the species in which it has an estrogenic pharmacology. Tamoxifen or its metabolite 4-hydroxytamoxifen could lose the alkylaminoethane side chain to form the estrogenic compound metabolite E or bisphenol. Sensitive metabolic studies with [3H]tamoxifen in chicks, rats, and mice identified 4-hydroxytamoxifen as the major metabolite, but no potentially estrogenic metabolites were observed. Athymic mice with transplanted human breast tumors can be used to study the ability of tamoxifen to stimulate target tissue or tumor growth. Estradiol caused the growth of transplanted MCF-7 breast cancer cells into solid tumors and a uterotrophic response. However, tamoxifen does not support tumor growth when administered alone, although it stimulates uterine growth. Since a similar profile of metabolites is sequestered in human and mouse tissues, these studies strongly support the concept that the drug can selectively stimulate or inhibit events in the target tissues of different species without metabolic intervention.     

Antagonism of estrogen- and antiestrogen-induced uterine complement component C3 expression by ICI 164,384

The uterus of the immature rat synthesizes and secretes complement component C3 in response to estradiol treatment. This response occurs in the uterine epithelial cells and is also stimulated by several antiestrogens including tamoxifen and LY117018. The administration of a new antiestrogen ICI 164,384 blocked the estradiol as well as the antiestrogen-stimulated increases in uterine weight, epithelial cell height, C3 synthesis and C3 mRNA. ICI 164,384 demonstrated no agonist properties in terms of epithelial cell response as determined by C3 expression.     

Use of the immature rat uterotrophic assay for specific measurements of chorionic gonadotropins and follicle-stimulating hormones in vivo bioactivities

The uterine weight growth stimulation by equine Chorionic Gonadotropin (eCG/PMSG) was found to occur at much lower eCG concentrations than ovarian growth. Human Chorionic Gonadotropin (hCG) which has only LH activity, was found to be as active as eCG in the uterotrophic assay whereas equine Luteinizing Hormone (eLH) which has dual LH+FSH activities like eCG, exhibited a much lower potency. In contrast to hCG, porcine and ovine LH as well as pFSH and oFSH exhibited no uterotrophic activity indicating that only gonadotropins with both LH activity and long half-lives are active alone in this assay. The FSH preparations were nevertheless found to trigger a dose-dependent response, but only in the presence of a subactive dose of hCG. The uterotrophic activity of hCG was found to be suppressed in ovariectomized immature rats and to be diminished after injection of GnRH antagonist suggesting an indirect pathway implicating the hypothalamo-pituitary complex.The data in this report together with the analysis of literature suggest that choriogonadotropins exert their stimulatory role on uterine growth by an indirect mechanism involving an increase in ovarian FSH receptors and FSH release by the pituitary. At the lowest concentrations of hCG, the increase in ovarian FSH receptors without endogenous FSH release is thought to be responsible for the sensitivity of the uterotrophic assay to exogenous FSHs. In conclusion, the immature rat uterotrophic assay is a sensitive and convenient assay for eCG and hCG as well as for FSHs in the presence of a sub-active dose of hCG.     

Biology and mode of action of pure antioestrogens

The properties of a series of 7 alpha-alkyl analogues of oestradiol are described. Studies of chemical structure and activity in the immature rat uterotrophic/antiuterotrophic assay revealed that molecules containing a terminal functional group (acid, alcohol, amine, amide) linked to the steroid by a decamethylene bridge possess both oestradiol agonist and antagonist activity. However, certain amides, exemplified by the compound ICI 164,384 [N-n-butyl-11-(3,17 beta-dihydroxyoestra-1,3,5(10)-trien-7 alpha-yl)-N-methylundecanamide], were devoid of oestrogenic activity but possessed potent antioestrogenic activity. Comparison of receptor binding and biological potency of steroid 7 alpha- and 7 beta-isomers showed that activity is confined largely to the 7 alpha-isomer. Comparison of the effects of tamoxifen and ICI 164,384 on progesterone receptor (PR) concentration in the rat uterus showed that, unlike tamoxifen, ICI 164,384 did not induce PR and blocked induction of PR by oestradiol. Chronic treatment of mature female rats with ICI 164,384 led to an ovariectomy-like regression of the uterus without affecting LH secretion or the rate of growth. ICI 164,384 was also an effective antitumour agent in rats bearing carcinogen-induced mammary tumours.     

Developmental toxicity and uterotrophic studies with di-2-ethylhexyl terephthalate

BACKGROUND: These studies were conducted to evaluate the potential adverse effects of di-2-ethylhexyl terephthalate (DEHT) exposure on in utero development in mice and rats. In addition, a uterotrophic assay for estrogenic activity was conducted in sexually immature rats. METHODS: In the developmental toxicity studies, diet containing DEHT was fed to four groups of mated female Crl:CD(SD)IGS BR rats (25/group) from gestation day (GD) 0-20 or Crl:CD1(ICR) mice (25/group) from GD 0-18. Concentrations within the feed were 0, 0.3, 0.6, and 1.0% for the rats and 0, 0.1, 0.3, and 0.7% for the mice. Laparohysterectomies were carried out on the last day of exposure and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. The fetuses were weighed, sexed, and examined for external, visceral and skeletal malformations, and developmental variations. The dose rate from dietary DEHT exposure was 0, 226, 458, and 747 mg/kg/day in the rats and 197, 592, and 1382 mg/kg/day in the mice for the control, low, mid, and high-exposure groups, respectively. RESULTS: DEHT exposure did not affect clinical observations. A slight reduction in body weight gain was noted in the high-dose level rat group; the remaining groups were unaffected. At necropsy, increased liver weights were noted in the high-dose rat group and the mid- and high-dose mouse groups. Mean numbers of implantation sites and viable fetuses, mean fetal weights, and mean litter proportions of preimplantation loss, early resorptions, late resorptions, and fetal sex ratios were unaffected by DEHT exposures. No test article-related malformations or variations were observed at any concentration level in the rat and mouse developmental toxicity studies. In the uterotrophic assay for estrogenic activity, sexually immature female rats received oral gavage doses 20, 200, or 2000 mg DEHT/kg bw/day from postnatal day (PND) 19-21. A slight reduction in rate of body weight gain was noted on the first day of dosing in the high dose group, but no other indications of toxicity were evident. DEHT exposure did not affect wet or blotted uterine weight parameters in any of these dose groups. The NOEL for developmental toxicity in rats was 747 mg/kg/day and 1382 mg/kg/day in mice. The NOEL for estrogenic activity was 2000 mg/kg/day. The NOEL for maternal toxicity was 458 mg/kg/day in rats and 197 mg/kg/day in mice. CONCLUSIONS: The lack of adverse developmental effects with DEHT exposure are in contrast to the adverse developmental effects noted after di-2-ethylhexyl phthalate (DEHP) exposure. The difference between the effects noted with the ortho-constituent (DEHP) and the lack of effects reported with the para-constituent (DEHT) is due most likely to differences in metabolism and the formation of the stable monoester, mono-2-ethylhexyl phthalate (MEHP) from the DEHP moiety.     

The naturally occurring C-17 fatty acid esters of estradiol are long-acting estrogens

C-17 fatty acid esters of estradiol are naturally occurring biosynthetic metabolites of estradiol. A representative component of this family of esters, estradiol-17-stearate, was studied in order to determine the estrogenic properties of these unusual hydrophobic steroids. Following the classical estrogen bioassay, a solution of this ester in oil was injected subcutaneously into immature rats once a day for 3 days. There was little effect on the uterus on the first day after the third injection. However, on subsequent days a large stimulation of uterine growth occurred. The course of this estrogenic effect was exactly opposite to that obtained with estradiol. In order to eliminate the possibility that this effect on the time course of estrogenic stimulation was caused by increased solubility of the hydrophobic esters in the carrier oil, the steroids were administered to adult ovariectomized animals in aqueous medium via a single intravenous injection. The uterotrophic response to estradiol was maximal at 12 h and was completely dissipated in 48-60 h. Estradiol-17-stearate produced a uterotrophic effect of twice the duration of estradiol. In the immature rat, aqueous intravenous injections of estradiol-17-stearate produced a greater uterotrophic effect than estradiol and this effect was still maximal 96 h later. In addition, this single injection of estradiol-17-stearate advanced the time of vaginal opening, a marker for puberty in the female rat. The mechanism of the prolonged estrogenic stimulation was investigated by studying the steroidal content of the uterus after injecting [3H]estradiol and [3H]estradiol-17 -stearate i.v. into immature rats. At 1 and 4 h there was significantly more radioactivity in the uteri of the [3H]estradiol treated animals. At later times (8 h and onwards) the total radioactivity in the uterus did not differ appreciably between the two groups. However at these later times, the amount of [3H]estradiol was far greater in the uteri of animals receiving [3H]estradiol-17-stearate. Consequently, the prolonged estrogenic effects of the endogenous C-17 fatty acid esters of estradiol are caused by the increased duration of the estrogenic signal. It is hypothesized that one of the roles of the fatty acid is to protect the steroid nucleus from metabolism and thereby prolong the life of the parent C18 steroid. Thus, the results of these experiments are consistent with the family of endogenous alkyl esters of estradiol having a physiological role as long-acting estrogens.     

Antiestrogenic and antitumor properties of the new triphenylethylene derivative toremifene in the rat

The effects of toremifene, a new triphenylethylene derivative, on the uterus and DMBA-induced mammary tumors in rats were compared to tamoxifen. The ability of toremifene to compete with [3H]estradiol for cytoplasmic estrogen receptor from rat uterus was similar to tamoxifen, the IC50 being 26 and 23 microM respectively. In immature intact rats the two compounds, administered orally for three consecutive days, had similar intrinsic partial estrogenic efficacy, at 50 mg/kg, about 40% of that of estradiol benzoate (EB). However, at doses less than or equal to 10 mg/kg, the estrogenic effect of toremifene was seen at doses about 40 times higher than that of tamoxifen. The two compounds, administered together with a standard dose of EB, expressed the same maximal antiestrogenic efficacy (about 65% inhibition) at 50 mg/kg. However, the minimal effective antiestrogenic dose of toremifene was about 10 times that of tamoxifen and the ratio between antiestrogenic/estrogenic properties was favourable to toremifene. The duration of the antiestrogenic (antiuterotrophic) effect of a single oral dose (10 mg/kg) of the two compounds proved similar: at least 4 days in intact rats and 3 days in ovariectomized rats. In DMBA-induced tumor bearing rats toremifene was administered p.o., 6 times/week for 4 weeks at 0.08, 0.4, 2, 10 and 50 mg/kg. It was effective at the doses of 2, 10 and 50 mg/kg, inducing 39, 35 and 46% tumor regressions. The activity of toremifene at the minimal effective dose of 2 mg/kg was then compared with that of tamoxifen given at the same dose level. The compounds had comparable activity (47 vs 44% tumor regressions).     

Proliferation assays for estrogenicity testing with high predictive value for the in vivo uterotrophic effect

Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. In the present study four different human cell lines derived from three different female estrogen-sensitive tissues, i.e. breast (MCF-7/BOS and T47D), endometrial (ECC-1) and ovarian (BG-1) cells, were characterised by investigating their relative ERα and ERβ amounts, as the ERα/ERβ ratio is a dominant factor determining their estrogen-dependent proliferative responses. All four cell lines clearly expressed the ERα type and a very low but detectable amount of ERβ on both the mRNA and protein level, with the T47D cell line expressing the highest level of the ERβ type. Subsequently, a set of reference compounds representing different modes of estrogen action and estrogenic potency were used to investigate the proliferative response in the four cell lines, to determine which cell line most accurately predicts the effect observed in vivo. All four cell lines revealed a reasonable to good correlation with the in vivo uterotrophic effect, with the correlation being highest for the MCF-7/BOS cell line (R2=0.85). The main differences between the in vivo uterotrophic assay and the in vitro proliferation assays were observed for tamoxifen and testosterone. The proliferative response of the MCF-7/BOS cells to testosterone was partially caused by its conversion to estradiol by aromatase or via androstenedione to estrone. It is concluded that of the four cell lines tested, the best assay to include in an integrated testing strategy for replacement of the in vivo uterotrophic assay is the human MCF-7/BOS breast cancer cell line.     

Toxoplasma gondii: decreased resistance to infection in mice due to estrogen

Exposure to pharmacological concentrations of potent estrogenic compounds, including 17 beta-estradiol, diethylstilbestrol, and alpha-dienestrol, increased the susceptibility of mice to Toxoplasma gondii as measured by brain cyst formation. Compounds with weak estrogenic activity or other hormonal activity, including 5 alpha-dihydrotestosterone, progesterone, and zearalanol, did not alter host resistance to infection. The ability of estrogens to alter susceptibility was inhibited by the estrogen antagonist, tamoxifen. The restoration of ovariectomized mice with normal physiological concentrations of estrogen had no effect on subsequent infection with T. gondii. These results indicate that pharmacological, but not physiological, levels of estrogen selectively alter host resistance to T. gondii, possibly through hormonal events.     

Comparison of the estrogenic potencies of standardized soy extracts by immature rat uterotrophic bioassay.

Soy phytoestrogens, isoflavones, are a primary class of plant-based estrogen alternatives being sold over the counter nowadays. Genistein, daidzein and glycitein are the major isoflavones found in soybeans, as aglycones and glycosides. Each isoflavone shows distinctive estrogenic activity and pharmacokinetics. Soy dry extracts, employed as pharmaceutical raw material for manufacturing isoflavone supplements, are standardized to contain 40% of total isoflavones, but the amount of each isoflavone is highly diverse. The influence of these compositional differences on the estrogenic potency of soy extracts was evaluated by uterotrophic bioassay. Five commercial samples of standardized soy dry extract, homogeneously suspended in arachis oil, were administered per os in serial doses (125-4150 mg/kg bw/day) to immature female rats for 3 days. Soy extract samples with considerable diversity in isoflavone composition revealed different estrogenic potencies. Our results indicate a need of standardization of the individual isoflavone content in soy extracts.     

Estrogenic activities of nitrophenols in diesel exhaust particles

We recently isolated 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP) from diesel exhaust particles (DEP) and identified them as vasodilators. Because these compounds are alkylphenolic derivatives that might mimic hormones, we evaluated their estrogenic activity by using recombinant yeast screens, myometrial contractility assays, and in vivo uterotrophic assays. Recombinant yeast screen assays showed that both PNMC and PNMPP possess estrogenic activity. Furthermore, ovariectomized 25-day-old immature female rats injected with PNMC and PNMPP subcutaneously for 2 days showed significant increases in uterine weight among those receiving 100 mg/kg PNMC and 0.1 and 1.0 mg/kg PNMPP. To clarify further the estrogenic activity of PNMC and PNMPP, rat uterine horns were monitored in organ bath chambers for myometrial contractility in response to oxytocin (OT). Significant differences occurred in the initial and maximum contractilities to OT at 0.25 and 25 mIU/ml in uterine horns obtained from animals treated with 100 mg/kg PNMC and in the maximum contractilities to OT at 0.025, 0.25, and 25 mIU/ml in those from rats treated with 0.1 mg/kg PNMPP. These results clearly demonstrated that PNMC and PNMPP in DEP have estrogenic activity both in vitro and in vivo and might therefore be considered as endocrine-disrupting chemicals.     

In vivo test systems for the quantitative and qualitative analysis of the biological activity of phytoestrogens

Many compounds of plant origin with the ability to bind to the estrogen receptor have been identified in the last decades. One of the most extensively used in vivo assays to characterise the estrogenic potency of these phytoestrogens and mechanisms of their action is the rodent uterotrophic assay. Various protocols exist for this test system, using immature, hypophysectomized, or ovariectomized rats and mice and oral or subcutaneous administration of the test compound. However, just monitoring the ability of a compound to stimulate uterine growth is not sufficient to characterize its estrogenicity. Over the last decades, an increasing number of estrogen sensitive tissues has been identified. Moreover, a variety of different molecular mechanisms have been discovered for the action of estrogens, including non-genomic actions. Therefore, an in vivo test design for estrogenicity should include an analysis of several estrogen sensitive parameters in different estrogen sensitive tissues. To distinguish between agonistic and antagonistic properties of a substance, combinations of the test compound with estrogens and antiestrogens should be analyzed. A reasonable supplement to this enhanced uterotrophic assay are selected estrogen sensitive tumor models, which can be used to test for potential chemopreventive properties of phytoestrogens.     

Antagonism of estrogen action with a new benzothiophene derived antiestrogen

A new benzothiophene derived antiestrogen, LY139481, inhibited the uterotropic action of estradiol in a dose related fashion, and at 1 mg per day suppressed more than 90 percent of estradiol's activity in immature rats. LY139481 induced minimal uterotropic activity, and that activity declined in relation to dose. The relative binding affinity of LY139481 for rat uterine cytosol estrogen receptors was greater than that of estradiol in competitive assays and increased in relation to temperature (2.9 +/- 0.5 x estradiol at 30 degrees C). LY139481 caused estradiol-induced uterine hypertrophy to regress in a manner similar to that which resulted from withdrawal of estradiol treatment. Three successive daily injections of LY139481 slightly increased uterine weight, and blocked additional uterotropic action in response to estradiol and LY139481 administration on subsequent days. Furthermore, ten daily injections of estradiol alone did not increase uterine weight in animals pretreated with LY139481 for three days. In contrast, LY139481 did not prevent the partial uterotropic action of tamoxifen administration.     

Target specificity of selective estrogen receptor modulators within human endometrial cancer cells

Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that function as antagonists in some tissues, but have either partial or full agonist activity in others. SERMs often display variable partial agonist activity in uterine tissues and this activity can be displayed in uterine cell lines such as the human Ishikawa endometrial adenocarcinoma cell line. In this study, we compared the effects of several ER ligands including some SERMs on alkaline phosphatase (AP) activity and the expression of an ER target gene, the progesterone receptor (PR), in Ishikawa cells. As expected, estradiol (E2) was a potent and efficacious activator of both AP activity and PR mRNA expression. 4-Hydroxytamoxifen (4OHT) stimulated AP activity to a level 47% of that of E2 (100nM), while CP 336156 (lasofoxifene) increased AP activity 18%. A benzothiophene, such as LY 117018, a raloxifene analog, stimulated AP even less with values approximately 11% of E2-stimulated levels. A pure antiestrogen, ICI 182,780 did not stimulate AP activity. Interestingly, when we examined the ability of these compounds to increase the expression of the ER target gene, PR, a different rank order of efficacy was detected. After E2, CP 336156 was the most efficacious in increasing PR mRNA with a maximal stimulation of 20% of E2 levels, while 4OHT stimulated only 17%. LY 117018 increased PR mRNA expression 8% while ICI 182,780 did not increase PR mRNA expression at all. These data illustrate the target specificity that a SERM is able to display within a single cell type independent of "tissue specificity" and differential levels of expression of various cofactors. While 4OHT is 160% more active than CP 336156 in terms of inducing AP activity in the Ishikawa cells, CP 336156 has equivalent activity as 4OHT when one examines the ability of these SERMs to induce PR mRNA expression. Since the stimulation of Ishikawa cells by ER ligands is often used to assess the potential in vivo uterotrophic activity, these data indicate that examination of several endpoints in these cells may be necessary in order to fully characterize the activity of SERMs.