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The extracellular domain of integrin αvβ3 contains a receptor for thyroid hormone and hormone analogs. The integrin is amply expressed by tumor cells and dividing blood vessel cells. The proangiogenic properties of thyroid hormone and the capacity of the hormone to promote cancer cell proliferation are functions regulated nongenomically by the hormone receptor on αvβ3. An L-thyroxine (T4) analog, tetraiodothyroacetic acid (tetrac), blocks binding of T4 and 3,5,3'-triiodo-L-thyronine (T3) by αvβ3 and inhibits angiogenic activity of thyroid hormone. Covalently bound to a 200 nm nanoparticle that limits its activity to the cell exterior, tetrac reformulated as Nanotetrac has additional effects mediated by αvβ3 beyond the inhibition of binding of T4 and T3 to the integrin. These actions of Nanotetrac include disruption of transcription of cell survival pathway genes, promotion of apoptosis by multiple mechanisms, and interruption of repair of double-strand deoxyribonucleic acid breaks caused by irradiation of cells. Among the genes whose expression is suppressed by Nanotetrac are EGFR, VEGFA, multiple cyclins, catenins, and multiple cytokines. Nanotetrac has been effective as a chemotherapeutic agent in preclinical studies of human cancer xenografts. The low concentrations of αvβ3 on the surface of quiescent nonmalignant cells have minimized toxicity of the agent in animal studies.

Hydroxytyrosol 1 is a natural polyphenolic antioxidant present in leafs and fruits of olive tree and in small amount in virgin olive oil. It presents high radical scavenging capacity so protecting cells against oxidative stress. Furthermore, it possesses antiatherogenic, anti-inflammatory and anti-platelet aggregation properties, reducing risk for CVD disease. However, 1 is highly hydrophilic and this has hampered its use as nutraceutical or antioxidant additive in oils or fats. Fatty acids hydroxytyrosyl esters has overcome these limitations. Continuing the research on new derivatives of this natural antioxidant, in order to test the effect on the antioxidant activity of the presence of a double hydroxytyrosyl moiety in a single molecule, we have developed the synthesis of a series of dihydroxytyrosyl esters of dicarboxilic acids. To obtain regioselectively the diesters 3 from 1 the catechol hydroxyls in 1 were previously protected as methyl orthoformate 2 and easily deprotected after esterification. Dihydroxytyrosyl esters 3 constitute a new class of antioxidants and were analyzed for their antioxidant activity. Both chemical (ABTS assay) and biological (DCF assay) experiments were carried out. In vitro studies evidenced in all the derivatives 3 a strong and lipophilicity-dependent enhancement of antioxidant capacity (TEAC) in comparison with that of the already known monohydroxytyrosyl esters of carboxylic fatty acids. In cell environment, MTT experiments demonstrated that these compounds do not inhibit cell proliferation, while either with L6 or HTP-1 cells, high antioxidant activities were measured, although not as strong as in in vitro measurements.

Administration of chemotherapy during pregnancy may represent a big risk factor for the developing brain, therefore we studied whether the transplacental transport of doxorubicin (DOX) may affect the development of neuroendocrine system. DOX (25 mg/kg; 3 times interaperitoneally/week) was given to pregnant rats during whole gestation period. The disturbances in neuroendocrine functions were investigated at gestation day (GD) 15 and 20 by following the maternal and fetal thyroid hormone levels, fetal nucleotides (ATP, ADP, AMP) levels and adenosine triphosphatase (Na(+), K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase) activities in two brain regions, cerebrum and cerebellum. In control group, the levels of maternal and fetal serum thyroxine (T4), triiodothyronine (T3), thyrotropin (TSH), and fetal serum growth hormone (GH) increased from days 15 to 20, whereas in the DOX group, a decrease in maternal and fetal T4, T3 and increase in TSH levels (hypothyroid status) were observed. Also, the levels of fetal GH decreased continuously from GD 15 to 20 with respect to control group. In cerebrum and cerebellum, the levels of fetal nucleotides and the activities of fetal ATPases in control group followed a synchronized course of development. The fetal hypothyroidism due to maternal administration of DOX decreased the levels of nucleotides, ATPases activities, and total adenylate, instead, the adenylate energy charge showed a trend to an increase in both brain regions at all ages tested. These alterations were dose- and age-dependent and this, in turn, may impair the nerve transmission. Finally, DOX may act as neuroendocrine disruptor causing hypothyroidism and fetal brain energetic dysfunction.

This review explores the relation between antioxidant defense system and reactive oxygen species (ROS) during the development and shows the effect of environmental pollutants on this process. In normal state, the decline in levels of free radicals is coupled with increased antioxidant and the reverse is true, but there is a critical balance between them during the development. Also, redox signaling induced by environmental pollutants (stressors) involves both alterations in antioxidant defenses and accumulation of ROS leading to oxidative stress which acts as a critical pathophysiological mechanism. This disturbance has deleterious effect on male/female reproductive functions, on the development of the blastocysts and on the health of the embryos, newborns (perinatal life) and adulthood. Also, this overview shows that sperm, egg, zygote or blastocyst derived during the abnormal production of ROS due to environmental pollutants may result into offspring with high risk of any type of diseases producing developmental delay, embryopathy, teratogenic changes and apoptosis. These early insults may then lead to an increased rate of miscarriage and congenital anomalies depending on free radicals signaling and cell-death pathways. Thus, maintaining the balance between antioxidants and ROS during pregnancy or lactation period may modulate normal fetal/neonates growth and development, and may play an important role in a healthy life for the newborns. However, this argument is still ambiguous because of the difficulties of to what degree oxidants could participate as signaling molecules controlling fundamental and developmentally relevant cellular processes such as proliferation, differentiation, and death.

P-glycoprotein (P-gp; multidrug resistance pump 1, MDR1; ABCB1) is a plasma membrane efflux pump that when activated in cancer cells exports chemotherapeutic agents. Transcription of the P-gp gene (MDR1) and activity of the P-gp protein are known to be affected by thyroid hormone. A cell surface receptor for thyroid hormone on integrin αvβ3 also binds tetraiodothyroacetic acid (tetrac), a derivative of L-thyroxine (T4) that blocks nongenomic actions of T4 and of 3,5,3′-triiodo-L-thyronine (T3) at αvβ3. Covalently bound to a nanoparticle, tetrac as nanotetrac acts at the integrin to increase intracellular residence time of chemotherapeutic agents such as doxorubicin and etoposide that are substrates of P-gp. This action chemosensitizes cancer cells. In this review, we examine possible molecular mechanisms for the inhibitory effect of nanotetrac on P-gp activity. Mechanisms for consideration include cancer cell acidification via action of tetrac/nanotetrac on the Na+/H+ exchanger (NHE1) and hormone analogue effects on calmodulin-dependent processes and on interactions of P-gp with epidermal growth factor (EGF) and osteopontin (OPN), apparently via αvβ3. Intracellular acidification and decreased H+ efflux induced by tetrac/nanotetrac via NHE1 is the most attractive explanation for the actions on P-gp and consequent increase in cancer cell retention of chemotherapeutic agent-ligands of MDR1 protein.

The sodium/proton (Na/H) exchanger, Na,K-ATPase, and Ca2+-ATPase are membrane ion pumps whose basal activities may be regulated by local nongenomic actions of thyroid hormone and hormone analogues via a hormone receptor on plasma membrane integrin αvβ3. System A amino acid transport and the activity of P-glycoprotein (P-gp; ABCB1), a multidrug efflux pump, are also modulated by thyroid hormone and αvβ3. Where signal transduction has been studied, the presence of the hormone at the receptor is transduced by mitogen-activated protein kinase (MAPK) isoforms (ERK1/2; p38) or phosphatidylinositol 3-kinase into local actions. The existence of the cell surface receptor offers opportunities to pharmacologically modify actions of these important transport functions with nanoparticulate formulations of T4 and T3 that do not enter the cell. Such formulations may reverse complex intracellular accumulations of H+, Na+, and Ca2+ that occur in clinical settings such as ischemia. In addition, nanoparticulate tetraiodothyroacetic acid (tetrac), a thyroid hormone analogue that inhibits binding of T4 and T3 to integrin αvβ3 as well as certain other functions of the integrin, may reverse P-gp-dependent resistance to anti-cancer drugs in tumor cells.

The development of antioxidants as useful drugs for the treatment of neurodegenerative diseases such as Alzheimer's is extremely challenging in medicinal chemistry. Coumarins have attracted great attention as possible therapeutic tools against oxygen radicals in human degenerative diseases. In order to establish the possible structure-antioxidant activity relationship, a series of twenty four 4-methylcoumarin derivatives were examined by employing reducing power measurements, and comparison with bond dissociation enthalpy and ionization potential calculations. Based on the reducing potency of 4-methylcoumarin derivatives with respect to trolox, these compounds were classified into five groups as “most active”, “more active”, “moderately active”, “less active” and “inactive” derivatives. The presence of hydroxyl groups is an essential requirement for the activity, and substitution of hydroxyl groups by methoxy groups leads to non-active derivatives. The results revealed that dihydroxyl groups in the ortho position show a better antioxidant activity with respect to dihydroxyl groups in the meta position. This is ascribed to the ability to construct more stable 4-methylcoumarin radical intermediates by rearrangement of intra-molecular hydrogen bonding. Our findings indicate that other important factors to enhance the antioxidant activity of coumarins are the number of hydroxyl groups, the presence of ester substitutions and a thiono functional group on the pyrone ring. However, bond dissociation enthalpy and ionization potential calculations alone are not sufficient to identify the best antioxidant structures. As a result, chemical and functional properties of molecules such as 4-methylcoumarins should be examined as a whole entity, considering all substitutions versus a single substitution to design functional compounds with good antioxidant activity.

Short-term effects of thyroid hormones on Na(+)-K(+)-ATPase activity of chick embryo hepatocytes during development: focus on signal transduction. Am J Physiol Cell Physiol 296: C4-C12, 2009. First published October 1, 2008; doi:10.1152/ajpcell.90604.2007.-Nongenomic effects of thyroid hormones on Na(+)-K(+)-ATPase activity were studied in chick embryo hepatocytes at two different developmental stages, 14 and 19 days of embryonal age, and the signal transduction pathways involved were characterized. Our data showed the following. 1) 3,5,3'- Triiodo-L-thyronine (T(3)) and 3,5-diiodo-L-thyronine (3,5-T(2)) rapidly induced a transient inhibitory effect on the Na(+)-K(+)-ATPase; the extent and duration depended on the developmental age of the cells. 2) 3,5-T(2) behaved as a true hormone and fully mimicked the effect of T(3). 3) Thyroxine had no effect at any of the developmental stages. 4) The inhibition of Na(+)-K(+)-ATPase was mediated by activation of protein kinase A, protein kinase C, and phosphoinositide 3-kinase, suggesting several modes of modulation of ATPase activity through phosphorylation at different sites. 5) The MAPK pathway did not seem to be involved in the early phase of hormone treatment. 6) The nonpermeant analog T(3)-agarose inhibited Na(+)-K(+)-ATPase activity in the same way as T(3), confirming that hormone signaling initiated at a receptor on the plasma membrane. From these results, it can be concluded that the cell response mechanisms change rapidly and drastically within the early phase of embryo growth. The differences found at the two stages probably reflect the different roles of thyroid hormones during development and differentiation.

Paul J Davis,1,2 Hung-Yun Lin,2,3 Thangirala Sudha,2 Murat Yalcin,2,4 Heng-Yuan Tang,2 Aleck Hercbergs,5 John T Leith,6 Mary K Luidens,1 Osnat Ashur-Fabian,7,8 Sandra Incerpi,9 Shaker A Mousa2 1Department of Medicine, Albany Medical College, Albany, NY, USA; 2Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, NY, USA; 3PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan; 4Department of Physiology, Veterinary Medicine Faculty, Uludag University, Gorukle, Bursa, Turkey; 5Radiation Oncology, Cleveland Clinic, Cleveland, OH, USA; 6Rhode Island Nuclear Science Center, Narragansett, RI, USA; 7Translational Hemato-oncology Laboratory, Hematology Institute and Blood Bank, Meir Medical Center, Kfar-Saba, Israel; 8Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 9Department of Sciences, University of Roma Tre, Rome, Italy Abstract: The extracellular domain of integrin αvβ3 contains a receptor for thyroid hormone and hormone analogs. The integrin is amply expressed by tumor cells and dividing blood vessel cells. The proangiogenic properties of thyroid hormone and the capacity of the hormone to promote cancer cell proliferation are functions regulated nongenomically by the hormone receptor on αvβ3. An L-thyroxine (T4) analog, tetraiodothyroacetic acid (tetrac), blocks binding of T4 and 3,5,3'-triiodo-L-thyronine (T3) by αvβ3 and inhibits angiogenic activity of thyroid hormone. Covalently bound to a 200 nm nanoparticle that limits its activity to the cell exterior, tetrac reformulated as Nanotetrac has additional effects mediated by αvβ3 beyond the inhibition of binding of T4 and T3 to the integrin. These actions of Nanotetrac include disruption of transcription of cell survival pathway genes, promotion of apoptosis by multiple mechanisms, and interruption of repair of double-strand deoxyribonucleic acid breaks caused by irradiation of cells. Among the genes whose expression is suppressed by Nanotetrac are EGFR, VEGFA, multiple cyclins, catenins, and multiple cytokines. Nanotetrac has been effective as a chemotherapeutic agent in preclinical studies of human cancer xenografts. The low concentrations of αvβ3 on the surface of quiescent nonmalignant cells have minimized toxicity of the agent in animal studies. Keywords: integrin, thyroid hormone, thyroxine, antiangiogenesis, proapoptosis

Aim: Thyroid hormone through its membrane receptor, the integrin aVß3, has been reported to be able to modulate the activity of growth factors. We studied glucose uptake in L-6 myoblasts in order to determine whether thyroid hormone modulates the activity of IGF-1, perhaps through crosstalk between the integrin and IGF-1R.Methods: The carrier-mediated hexose uptake (cytochalasin B-inhibitable) was measured for 10 min at 37°C in Hepes-Buffered Saline by 10 µM 2-deoxy-[3H]-D-glucose, after 4 hours of serum depletion. Results: IGF-1 activated glucose uptake in L-6 cells by a PI 3-K-dependent, that is, wortmannin-sensitive, mechanism. Thyroid hormones, 3,5,3'-triiodo-L-thyronine (T3; 1 nM) and L-thyroxine (T4; 100 nM) both stimulated glucose uptake. In the presence of IGF-1, however, T3 and T4 inhibited the IGF-1 effect on glucose uptake by a pathway that was RGD-sensitive, suggesting the involvement of the cell surface receptor for thyroid hormone at the RGD (Arg-Gly-Asp) recognition site on integrin alphaVbeta3 in iodothyronine action on the effect of IGF-1. Echistatin, an RGD-sequence-containing inhibitor of integrins, blocked IGF-1 action on glucose uptake in L6 cells. Conclusions: There are two modes of crosstalk between the integrin receptor for thyroid hormone on integrin alphaVbeta3 and the IGF-1 receptor. One level of crosstalk allows T3 and T4 to modulate IGF-1 action and requires PI 3-K activation. Another mode of crosstalk between the integrin and IGF-1R is thyroid hormone-independent and echistatin-sensitive.

Oxidative stress may be broadly defined as an imbalance between oxidant production and the antioxidant capacity of the cell to prevent oxidative injury; it is a condition believed to be involved in a large number of human diseases including atherosclerosis, cancer and neurodegenerative neuroinflammatory diseases, and also in aging. The situation of oxidative stress results in the production of O2 metabolites known as Reactive Oxygen Species (ROS) due to their enhanced reactivity compared to molecular oxygen; the most common are the superoxide anion (O2-.), hydrogen peroxide (H2O2), and the highly reactive hydroxyl radical (OH.). In order to protect themselves from ROS damage cells possess several antioxidant enzymes, such as superoxide dismutase (SOD), catalase and glutathione peroxidase, but also low molecular weight antioxidants like vitamin C and vitamin E.Much evidence suggests that ROS are not only injurious by-products of cellular metabolism but also essential participants in cell signaling and regulation and it is now clear that small amounts of ROS act as messenger molecules in cell signal transduction pathways. The plasma membrane of eukaryotic cells in particular contains a variety of different ROS-producing oxidases and reductases, of which the best characterized are the superoxide-producing NADPH oxidases. It has been known for many years that these membrane redox activities can be changed rapidly by various hormones and growth factors, but the molecular mechanisms involved and the physiological importance of this phenomenon have only recently begun to be unveiled. We here summarize the state of the art on membrane-based ROS signaling in the pathways of several hormones, mainly insulin and thyroid hormones. The apparent paradox of ROS being essential biomolecules in the regulation of cellular functions, but also toxic by-products of metabolism, may be important for the pharmacological application of natural and synthetic antioxidants. We report results on the capability of coumarins, flavonoids and other polyphenols to modulate the ROS production by some hormones and the signal transduction pathways involved.

Extranuclear or nongenomic effects of thyroid hormone are unaffected by the inhibitors of protein synthesis, and their time course cannot be explained by the interaction of the hormone molecule with nuclear receptors. Their site of action has been localized at the plasma membrane and identified very recently. In particular thyroid hormone has been reported to activate, by both genomic and non genomic mechanism, the Ca2+-ATPase, an ion pump that removes calcium from the cytosol and stores it in the sarcoplasmic reticulum. The decrease in intracellular Ca2+ generated during the systole leads to cardiac muscle relaxation. Given to all these important effects on the cardiovascular system, T3 can be also envisaged as a potent novel therapeutic agent as a inotropic drug. The Na/H exchanger, activated in L-6 myoblasts by the thyroid hormone through both genomic and nongenomic mechanism, is a ubiquitous plasma membrane integral protein exchanging extracellular Na+ with cytoplasmic H+ ions according to the concentration gradient; it does not require energy supply, but depends on the Na+/K+-ATPase. The transduction of the hormone signal in this nongenomic response requires tyrosine kinase-dependent phospholipase C activation and two different signaling pathways: 1) mobilization of intracellular calcium, through activation of IP3-receptors and without contributions from extracellular calcium or ryanodine receptors; 2) protein phosphorylation involving protein kinase C and mitogen-activated protein kinase (MAPK; ERK1/2), likely interfaces between genomic and nongenomic effects. The extranuclear actions should be taken into account when considering appropriate therapeutic intervention based on thyroid hormone both for their possible reinforcement of the nuclear responses and for their fast time onset, that might be of clinical relevance particularly for the cardiovascular system. Department of Biology, University of Rome “Roma Tre”, Viale Extranuclear or nongenomic effects of thyroid hormone are unaffected by the inhibitors of protein synthesis, and their time course cannot be explained by the interaction of the hormone molecule with nuclear receptors. Their site of action has been localized at the plasma membrane and identified very recently. In particular thyroid hormone has been reported to activate, by both genomic and non genomic mechanism, the Ca2+-ATPase, an ion pump that removes calcium from the cytosol and stores it in the sarcoplasmic reticulum. The decrease in intracellular Ca2+ generated during the systole leads to cardiac muscle relaxation. Given to all these important effects on the cardiovascular system, T3 can be also envisaged as a potent novel therapeutic agent as a inotropic drug. The Na/H exchanger, activated in L-6 myoblasts by the thyroid hormone through both genomic and nongenomic mechanism, is a ubiquitous plasma membrane integral protein exchanging extracellular Na+ with cytoplasmic H+ ions according to the concentration gradient; it does not require energy supply, but depends on the Na+/K+-ATPase. The transduction of the hormone signal in this nongenomic response requires tyrosine kinase-dependent phospholipase C activation and two different signaling pathways: 1) mobilization of intracellular calcium, through activation of IP3-receptors and without contributions from extracellular calcium or ryanodine receptors; 2) protein phosphorylation involving protein kinase C and mitogen-activated protein kinase (MAPK; ERK1/2)

Extranuclear effects of thyroid hormones L-T3 and L-T4 are now widely recognized, but in the last few years evidence for a similar activity of a new thyroid hormone, 3,5-diiodo-L-thyronine (3,5-T2), has been reported. The 3,5-T2 is a deiodination metabolite of L-T3 that is able to mimic some important rapid nongenomical effects of L-T3. High levels of 3,5-T2 have been found in connection with some types of brain tumors and other nonthyroidal illnesses, such as liver cirrhosis, and it appears likely that 3,5-T2 may have a specific physiological role. Previous results from our laboratory demonstrated that 3,5-T2 in the physiological range (around 0.1 nM) mimics L-T3 activation of the Na/H exchanger, causing an increase in the intracellular pH of L-6 myoblasts (Incerpi et al., Endocrinology 140:693-689, 1999) and chick embryo hepatocytes (Incerpi et al., Endocrinology, 143:1660-1668, 2002). We here show that 3,5-T2 increases the fluorescence of diclorofluorescein in L-6 myoblasts, probably due to the production of reactive oxygen species (ROS). The increase is partially prevented by extracellular superoxide dismutase (SOD) or by treatment of cells with an inhibitor of the plasma membrane NADPH oxidase, that is believed to be an important source of superoxide formation both inside and outside cells. Experiments with Electron Paramagnetic Resonance spectroscopy, using Cu,Zn SOD as a probe, show that 3,5-T2 over a wide concentration range induces superoxide formation, whereas other analogs of thyroid hormones do not give the same effect. The results also indicate that superoxide is produced on the outside of the cell membrane, probably due to activation of the plasma membrane NADPH oxidase. Experiments are in progress in order to establish whether ROS production induced by 3,5-T2 represents a physiological signal, possibly as part of a response to a pathological condition.

Nongenomic effects are nowadays described for most hormones of the nuclear receptor superfamily, such as thyroid hormones (T3, T4). These effects are mainly membrane –located, such as calcium mobilization, glucose uptake, intracellular pH, activation of PKC, and the MAPK pathway; they show a time-course of seconds to minutes. They may be mediated by specific receptors on the plasma membrane of different cells. In these experiments it is important to use agonist hormones conjugated to non-diffusible anchor molecules, such as Agarose. The hormone binds to the membrane and cannot enter the cell: this is an easy way to study membrane effects. T3-Agarose was prepared as reported at the website: www.amershambiosciences.com. T3-Agarose is a membrane-impermeant derivative based on the use of activated CH Sepharose 4BTM, a pre-activated medium for covalent immobilization of either proteins or other ligands containing primary amino groups. The group to be coupled is the amino group (-NH2) and the matrix Agarose (4%), the coupling conditions are pH 5-10, 4-25 °C, 1-4 h. The contamination by free T3 is very low, about 1 mol free T3/10E5 mol T3-Agarose as assessed by RIA. T3-Agarose (1 nM) was able to elicit nongenomic effects of thyroid hormone, in fact it increased intracellular pH and gave rise to an increase in intracellular calcium concentration similar to that obtained with the same concentrations of T3 (1 nM) in L-6 myoblasts. Nuclear responses were not affected by T3-Agarose. The results appear promising and the same technique will be used in the near future also for T4 and analogs that mimic thyroid hormone effects.

Low physiological concentrations of 17beta-estradiol increased the intracellular pH of rat aortic smooth muscle cells by a rapid nongenomic mechanism. This effect was due to stimulation of the Na+/H+ exchanger activity, measured using the intracellular pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The 17beta-estradiol gave rise to a bell-shaped dose response, with a maximum at 10(-12)M and no significant effect at 10(-9)M. The specificity of the effect was verified by the use of the Na+/H+ exchanger inhibitor 5-(ethyl-N-isopropyl) amiloride and the lack of effect of the isomer 17alpha-estradiol. Inhibitors of the nuclear estrogen receptors, tamoxifen and ICI 182,780, completely prevented activation of the exchanger by 17beta-estradiol. The effect of low estrogen concentrations on the intracellular pH was mimicked by both norepinephrine and phenylephrine, suggesting a connection between the increase of intracellular pH and the muscle contraction process. The transduction mechanism for this nongenomic effect of estrogens did not involve modulation of the cAMP content, whereas inositol 1,4,5-trisphosphate, protein kinase C and MAPK pathways appear to play a role, as indicated by both pharmacological approaches and immunoblot experiments on protein kinase C translocation and ERK phosphorylation. These results for the first time provide evidence for a nongenomic effect of low physiological concentrations of 17beta-estradiol on intracellular pH that, together with other factors, may contribute to the development of hypertension and atherosclerosis in men and postmenopausal women and increase the risk of cardiovascular disease. Paradoxically, the lack of stimulation at high physiological estradiol levels could explain the protective effects found in premenopausal women.