Trent D. Lund

Neurobehavioral effects of dietary soy phytoestrogens

Phytoestrogens, plant-derived nonsteroidal estrogens found in high abundance in most soy food products, have been studied for their potential beneficial effects against hormone-dependent cancers and age-related diseases. However, little is known about the influence of phytoestrogens on the brain or behavior. This brief review describes mainly our own studies in rodents that have examined the influence of dietary soy isoflavones on certain aspects of brain structure, learning, memory and anxiety along with the brain androgen-metabolizing enzyme, aromatase. These studies used a commercially available diet rich in phytoestrogens (Phyto-rich) vs. a custom diet relatively free of phytoestrogens (Phyto-free). The phytoestrogen content of each diet was determined by high-performance liquid chromatography analysis, circulating plasma phytoestrogen levels were quantified by gas chromatography mass spectroscopy and concentrations of phytoestrogens in specific brain regions were measured by time-resolved fluoroimmunoassay (TR-FIA). Our studies showed that brain aromatase levels were not significantly altered by phytoestrogen diet treatments in perinatal, maternal or adult rats. However, volumes of the sexually dimorphic nucleus of the preoptic area (SDN-POA) were significantly affected by the Phyto-free diet treatment in male rats during adulthood, where SDN-POA volumes were smaller compared to Phyto-rich male values. Additionally, the Phyto-rich diet fed to adult male and female rats produced anxiolytic effects as assessed in the elevated plus maze vs. Phyto-free fed animals. Finally, when learning and memory parameters were examined in a radial arm maze testing visual-spatial memory (VSM), the diet treatments significantly changed the typical sexually dimorphic pattern of VSM. Specifically, adult Phyto-rich fed females outperformed Phyto-free fed females, while in males on the same diets, the opposite pattern of maze performance was observed. When female vs. male performance was compared, Phyto-rich females executed the VSM task in a manner similar to that of Phyto-free fed males, while Phyto-free fed females VSM was comparable to Phyto-rich males. These results indicate that consumption of dietary phytoestrogens resulting in very high plasma isoflavone levels (in many cases over a relatively short interval of consumption in adulthood) can significantly alter sexually dimorphic brain regions, anxiety, learning and memory. The findings of these studies identify the biological actions of phytoestrogens, specifically isoflavones and their metabolites, found in animal soy-containing diets on brain and behavior and implicate the importance of phytoestrogens given the recognized significance of estrogens in brain and neural disorders, such as Alzheimers disease, especially in women.

The Androgen 5α-Dihydrotestosterone and Its Metabolite 5α-Androstan-3β, 17β-Diol Inhibit the Hypothalamo–Pituitary–Adrenal Response to Stress by Acting through Estrogen Receptor β-Expressing Neurons in the Hypothalamus

Estrogen receptor β (ERβ) and androgen receptor (AR) are found in high levels within populations of neurons in the hypothalamus. To determine whether AR or ERβ plays a role in regulating hypothalamo–pituitary–adrenal (HPA) axis function by direct action on these neurons, we examined the effects of central implants of 17β-estradiol (E2), 5α-dihydrotestosterone (DHT), the DHT metabolite 5α-androstan-3β, 17β-diol (3β-diol), and several ER subtype-selective agonists on the corticosterone and adrenocorticotropin (ACTH) response to immobilization stress. In addition, activation of neurons in the paraventricular nucleus (PVN) was monitored by examining c-fos mRNA expression. Pellets containing these compounds were stereotaxically implanted near the PVN of gonadectomized male rats. Seven days later, animals were killed directly from their home cage (nonstressed) or were restrained for 30 min (stressed) before they were killed. Compared with controls, E2 and the ERα-selective agonists moxestrol and propyl-pyrazole-triol significantly increased the stress induced release of corticosterone and ACTH. In contrast, central administration of DHT, 3β-diol, and the ERβ-selective compound diarylpropionitrile significantly decreased the corticosterone and ACTH response to immobilization. Cotreatment with the ER antagonist tamoxifen completely blocked the effects of 3β-diol and partially blocked the effect of DHT, whereas the AR antagonist flutamide had no effect. Moreover, DHT, 3β-diol, and diarylpropionitrile treatment significantly decreased restraint-induced c-fos mRNA expression in the PVN. Together, these studies indicate that the inhibitory effects of DHT on HPA axis activity may be in part mediated via its conversion to 3β-diol and subsequent binding to ERβ.

Equol Is a Novel Anti-Androgen that Inhibits Prostate Growth and Hormone Feedback

Abstract Equol (7-hydroxy-3[4′hydroxyphenyl]-chroman) is the major metabolite of the phytoestrogen daidzein, one of the main isoflavones found abundantly in soybeans and soy foods. Equol may be an important biologically active molecule based on recent studies demonstrating that equol can modulate reproductive function. In this study, we examined the effects of equol on prostate growth and LH secretion and determined some of the mechanisms by which it might act. Administration of equol to intact male rats for 4–7 days reduced ventral prostate and epididymal weight and increased circulating LH levels. Using binding assays, we determined that equol specifically binds 5α-dihydrotestosterone (DHT), but not testosterone, dehydroepiandrosterone, or estrogen with high affinity. Equol does not bind the prostatic androgen receptor, and has a modest affinity for recombinant estrogen receptor (ER) β, and no affinity for ERα. In castrated male rats treated with DHT, concomitant treatment with equol blocked DHTs trophic effects on the ventral prostate gland growth and inhibitory feedback effects on plasma LH levels without changes in circulating DHT. Therefore, equol can bind circulating DHT and sequester it from the androgen receptor, thus altering growth and physiological hormone responses that are regulated by androgens. These data suggest a novel model to explain equols biological properties. The significance of equols ability to specifically bind and sequester DHT from the androgen receptor have important ramifications in health and disease and may indicate a broad and important usage for equol in the treatment of androgen-mediated pathologies.

An alternate pathway for androgen regulation of brain function: Activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5α-androstane-3β,17β-diol

The complexity of gonadal steroid hormone actions is reflected in their broad and diverse effects on a host of integrated systems including reproductive physiology, sexual behavior, stress responses, immune function, cognition, and neural protection. Understanding the specific contributions of androgens and estrogens in neurons that mediate these important biological processes is central to the study of neuroendocrinology. Of particular interest in recent years has been the biological role of androgen metabolites. The goal of this review is to highlight recent data delineating the specific brain targets for the dihydrotestosterone metabolite, 5α-androstane, 3β, 17β-diol (3β-Diol). Studies using both in vitro and in vivo approaches provide compelling evidence that 3β-Diol is an important modulator of the stress response mediated by the hypothalmo-pituitary-adrenal axis. Further, the actions of 3β-Diol are mediated by estrogen receptors, and not androgen receptors, often through a canonical estrogen response element in the promoter of a given target gene. These novel findings compel us to re-evaluate the interpretation of past studies and the design of future experiments aimed at elucidating the specific effects of androgen receptor signaling pathways.

Visual spatial memory is enhanced in female rats (but inhibited in males) by dietary soy phytoestrogens

BackgroundIn learning and memory tasks, requiring visual spatial memory (VSM), males exhibit superior performance to females (a difference attributed to the hormonal influence of estrogen). This study examined the influence of phytoestrogens (estrogen-like plant compounds) on VSM, utilizing radial arm-maze methods to examine varying aspects of memory. Additionally, brain phytoestrogen, calbindin (CALB), and cyclooxygenase-2 (COX-2) levels were determined.ResultsFemale rats receiving lifelong exposure to a high-phytoestrogen containing diet (Phyto-600) acquired the maze faster than females fed a phytoestrogen-free diet (Phyto-free); in males the opposite diet effect was identified. In a separate experiment, at 80 days-of-age, animals fed the Phyto-600 diet lifelong either remained on the Phyto-600 or were changed to the Phyto-free diet until 120 days-of-age. Following the diet change Phyto-600 females outperformed females switched to the Phyto-free diet, while in males the opposite diet effect was identified.Furthermore, males fed the Phyto-600 diet had significantly higher phytoestrogen concentrations in a number of brain regions (frontal cortex, amygdala & cerebellum); in frontal cortex, expression of CALB (a neuroprotective calcium-binding protein) decreased while COX-2 (an inducible inflammatory factor prevalent in Alzheimers disease) increased.ConclusionsResults suggest that dietary phytoestrogens significantly sex-reversed the normal sexually dimorphic expression of VSM. Specifically, in tasks requiring the use of reference, but not working, memory, VSM was enhanced in females fed the Phyto-600 diet, whereas, in males VSM was inhibited by the same diet. These findings suggest that dietary soy derived phytoestrogens can influence learning and memory and alter the expression of proteins involved in neural protection and inflammation in rats.

Androgen Inhibits, While Oestrogen Enhances, Restraint-Induced Activation of Neuropeptide Neurones in the Paraventricular Nucleus of the Hypothalamus

The hormonal response to stress is enhanced by oestrogen but inhibited by androgens. To determine underlying changes in activity of neuropeptide neurones in the paraventricular nucleus of the hypothalamus (PVN), we examined the effect of oestrogen and androgen treatment on restraint‐induced c‐fos mRNA, corticotropin‐releasing hormone (CRH) heteronuclear RNA, and arginine vasopressin hnRNA expression in the PVN. Male rats were gonadectomized and injected with oestradiol benzoate (EB) or dihydrotestosterone propionate (DHTP; s.c., daily for 4 days). Rats were stressed by restraint for 10 min or 30 min before killing. Other rats were stressed for 30 min and then returned to their home cage for 20 min before killing. Corticosterone and adrenocorticotropic hormone responses to restraint stress were significantly greater in EB‐treated rats and lower in DHTP‐treated rats at the 30‐min timepoint compared to controls. c‐fos mRNA increases following stress were augmented by EB but inhibited by DHTP. CRH hnRNA expression increased significantly in the PVN in response to restraint stress, and this increase was augmented by EB treatment, but decreased by DHTP treatment. Vasopressin hnRNA expression was also increased in response to stress, and this increase was attenuated by DHTP. These findings indicate that gonadal hormones influence the reactivity of the hypothalamic‐pituitary adrenal axis to stress.

Brain androgen and progesterone metabolizing enzymes: biosynthesis, distribution and function.

This review summarizes the biosynthesis, cell type-distribution and function of brain aromatase cytochrome P450 (P450aro) and 5alpha-reductase enzymes. This overview covers the impact of the steroid products of the P450aro and 5alpha-reductase enzymes in establishing sexually dimorphic brain structures, specifically the sexually dimorphic nucleus of the preoptic area (SDN) and the anteroventral periventricular nucleus (AVPV). Additionally, since metabolites of the P450aro and 5alpha-reductase enzymes are known to regulate the calcium-binding protein, calbindin (CALB), CALB is reviewed in relationship to its potential role in determining sexually dimorphic brain structures. Finally, recent reports indicate that phytoestrogens inhibit P450aro and 5alpha-reductase activities in peripheral tissue sites, therefore, the effects of phytoestrogens on brain P450aro and 5alpha-reductase are briefly considered and the impact of consuming a high vs. a low phytoestrogen diet on visual spatial memory in male and female rats is presented.

Dietary soy phytoestrogens produce anxiolytic effects in the elevated plus-maze.

Naturally occurring estrogen-like molecules in plants (phytoestrogens), present via soy, in animal diets, exert many of the biological responses evoked by physiological estrogens. This study characterized the effects of dietary phytoestrogens on the expression of body weight, consummatory behavior, and anxiety (as expressed in the elevated plus-maze). Phytoestrogens produced anxiolytic effects in both male and female Long-Evans rats. Additionally, phytoestrogens decreased body weight but increased consumption of food and/or water.

Dihydrotestosterone may inhibit hypothalamo–pituitary–adrenal activity by acting through estrogen receptor in the male mouse

The corticosterone (CORT) response to environmental perturbation has been shown to be enhanced by estrogen but inhibited by the androgen dihydrotestosterone (DHT). However, the mechanism of androgens action has not been identified. This study examined the effects of estradiol benzoate (EB), the non-aromatizable androgen DHT, and the DHT metabolite 5alpha-androstan-3beta, 17beta-diol (3beta-diol) on the corticosterone response to stress. Adult male CBB6/F1 mice were gonadectomized and injected subcutaneously (once a day for 4 days) with the above compounds (controls received oil vehicle injections). Animals (within treatments) were randomly assigned to stress or non-stress conditions. The non-stress animals were taken directly from their home cages and killed. Animals were stressed by a 30 min restraint prior to being killed. Hormone levels were determined in plasma via radioimmunoassay. In agreement with previous studies, the CORT response to immobilization was enhanced by EB and inhibited by DHT. Surprisingly, 3beta-diol inhibited the CORT response similar to the effect of DHT. In a second study, concomitant injections of the androgen receptor antagonist flutamide only partially blocked DHTs, but had no effect on 3beta-diols, inhibitory action. In contrast, injections with the estrogen receptor antagonist tamoxifen completely blocked the effects of 3beta-diol and partially blocked DHTs effect. Taken together these studies suggest that DHTs inhibitory effects may be, at least in part, via the estrogen receptor, through its conversion to 3beta-diol. These studies also suggest that the DHT metabolites may be functionally relevant when considering hormonal responses to stress.

Altered sexually dimorphic nucleus of the preoptic area (SDN-POA) volume in adult Long-Evans rats by dietary soy phytoestrogens.

Naturally occurring estrogen-like molecules in plants (phytoestrogens), present via soy, in animal diets can alter morphology and physiology in rodents. Phytoestrogens have the ability to bind estrogen receptors and exert many of the biological responses evoked by physiological estrogens. This study characterized the effects of dietary phytoestrogens on the expression of body and prostate weight, circulating testosterone and estradiol levels, puberty onset, vaginal cyclicity, and volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in Long-Evans rats. Using different experimental protocols, animals were fed either a phytoestrogen-rich (Phyto-600) or a phytoestrogen-free (Phyto-free) diet. Animals fed the Phyto-600 diet displayed significantly decreased body weights (in males and females), prostate weights and delayed puberty in females compared to that of animals fed the Phyto-free diet. Circulating testosterone or estradiol levels in males or estrous cyclicity were not altered by the diets. The volume of the SDN-POA was significantly altered by a change in diet at 80 days of age where one-half of the males or females fed the Phyto-600 diet (from birth) were switched to the Phyto-free diet until 120 days of age. Males initially fed a Phyto-600 diet but changed to a Phyto-free diet had significantly smaller SDN-POA volumes compared to males fed the Phyto-600 diet (long-term). These data suggest that consumption of phytoestrogens via a soy diet, significantly: (1) decreases body and prostate weight, (2) delays puberty onset, and (3) alters SDN-POA volumes during adulthood.