Heather B. Patisaul

The pros and cons of phytoestrogens

Phytoestrogens are plant derived compounds found in a wide variety of foods, most notably soy. A litany of health benefits including a lowered risk of osteoporosis, heart disease, breast cancer, and menopausal symptoms, are frequently attributed to phytoestrogens but many are also considered endocrine disruptors, indicating that they have the potential to cause adverse health effects as well. Consequently, the question of whether or not phytoestrogens are beneficial or harmful to human health remains unresolved. The answer is likely complex and may depend on age, health status, and even the presence or absence of specific gut microflora. Clarity on this issue is needed because global consumption is rapidly increasing. Phytoestrogens are present in numerous dietary supplements and widely marketed as a natural alternative to estrogen replacement therapy. Soy infant formula now constitutes up to a third of the US market, and soy protein is now added to many processed foods. As weak estrogen agonists/antagonists with molecular and cellular properties similar to synthetic endocrine disruptors such as Bisphenol A (BPA), the phytoestrogens provide a useful model to comprehensively investigate the biological impact of endocrine disruptors in general. This review weighs the evidence for and against the purported health benefits and adverse effects of phytoestrogens.

Endocrine disrupters: a review of some sources, effects, and mechanisms of actions on behaviour and neuroendocrine systems.

Some environmental contaminants interact with hormones and may exert adverse consequences as a result of their actions as endocrine disrupting chemicals (EDCs). Exposure in people is typically a result of contamination of the food chain, inhalation of contaminated house dust or occupational exposure. EDCs include pesticides and herbicides (such as dichlorodiphenyl trichloroethane or its metabolites), methoxychlor, biocides, heat stabilisers and chemical catalysts (such as tributyltin), plastic contaminants (e.g. bisphenol A), pharmaceuticals (i.e. diethylstilbestrol; 17α‐ethinylestradiol) or dietary components (such as phytoestrogens). The goal of this review is to address the sources, effects and actions of EDCs, with an emphasis on topics discussed at the International Congress on Steroids and the Nervous System. EDCs may alter reproductively‐relevant or nonreproductive, sexually‐dimorphic behaviours. In addition, EDCs may have significant effects on neurodevelopmental processes, influencing the morphology of sexually‐dimorphic cerebral circuits. Exposure to EDCs is more dangerous if it occurs during specific ‘critical periods’ of life, such as intrauterine, perinatal, juvenile or puberty periods, when organisms are more sensitive to hormonal disruption, compared to other periods. However, exposure to EDCs in adulthood can also alter physiology. Several EDCs are xenoestrogens, which can alter serum lipid concentrations or metabolism enzymes that are necessary for converting cholesterol to steroid hormones. This can ultimately alter the production of oestradiol and/or other steroids. Finally, many EDCs may have actions via (or independent of) classic actions at cognate steroid receptors. EDCs may have effects through numerous other substrates, such as the aryl hydrocarbon receptor, the peroxisome proliferator‐activated receptor and the retinoid X receptor, signal transduction pathways, calcium influx and/or neurotransmitter receptors. Thus, EDCs, from varied sources, may have organisational effects during development and/or activational effects in adulthood that influence sexually‐dimorphic, reproductively‐relevant processes or other functions, by mimicking, antagonising or altering steroidal actions.

Regulation of estrogen receptor beta mRNA in the brain: opposite effects of 17β-estradiol and the phytoestrogen, coumestrol

Estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) are differentially distributed in the brain and likely mediate different estrogen-dependent processes. ERbeta is abundant in the bed nucleus of the stria terminalis, medial preoptic nucleus, paraventricular nucleus of the hypothalamus and the amygdala of the rat. In the paraventricular nucleus, which is devoid of ERalpha, ERbeta is colocalized with the neuropeptides, oxytocin and vasopressin, suggesting a potential functional role for ERbeta in the regulation of these peptides. We examined the regulation of ERbeta mRNA expression in the rat brain by 17beta-estradiol and the phytoestrogen, coumestrol. 17beta-Estradiol treatment decreased ERbeta mRNA in situ hybridization signal by 44.5% in the paraventricular nucleus of the hypothalamus (PVN), but had no effect in the bed nucleus of the stria terminalis (BnST) or the medial preoptic nucleus (MPA). In contrast, dietary exposure to coumestrol increased ERbeta mRNA signal by 47.5% in the PVN but had no effect in the BnST or the MPA. These data demonstrate that like ERalpha, ERbeta is down regulated by estrogen in a region specific manner in the rat brain. Furthermore, exposure to coumestrol may modulate ERbeta-dependent processes by acting as an anti-estrogen at ERbeta. This data contradicts results from cell transfection assays which suggest an estrogenic activity of coumestrol on ERbeta, indicating that the mode of action may be tissue specific, or that metabolism of dietary coumestrol may alter its effects. Because the highest concentrations of phytoestrogens are found in legumes, vegetables and grains, they are most prevalent in vegetarian and traditional Asian diets. Understanding the neuroendocrine effects of phytoestrogens is particularly important now that they are being marketed as a natural alternative to estrogen replacement therapy and sold in highly concentrated pills and powders.

Long-Term Effects of Environmental Endocrine Disruptors on Reproductive Physiology and Behavior

It is well established that, over the course of development, hormones shape the vertebrate brain such that sex specific physiology and behaviors emerge. Much of this occurs in discrete developmental windows that span gestation through the prenatal period, although it is now becoming clear that at least some of this process continues through puberty. Perturbation of this developmental progression can permanently alter the capacity for reproductive success. Wildlife studies have revealed that exposure to endocrine disrupting compounds (EDCs), either naturally occurring or man made, can profoundly alter reproductive physiology and ultimately impact entire populations. Laboratory studies in rodents and other species have elucidated some of the mechanisms by which this occurs and strongly indicate that humans are also vulnerable to disruption. Use of hormonally active compounds in human medicine has also unfortunately revealed that the developing fetus can be exposed to and affected by endocrine disruptors, and that it might take decades for adverse effects to manifest. Research within the field of environmental endocrine disruption has also contributed to the general understanding of how early life experiences can alter reproductive physiology and behavior through non-genomic, epigenetic mechanisms such as DNA methylation and histone acetylation. These types of effects have the potential to impact future generations if the germ line is affected. This review provides an overview of how exposure to EDCs, particularly those that interfere with estrogen action, impacts reproductive physiology and behaviors in vertebrates.

Neonatal Bisphenol-A Exposure Alters Rat Reproductive Development and Ovarian Morphology Without Impairing Activation of Gonadotropin-Releasing Hormone Neurons

Developmental exposure to endocrine-disrupting compounds is hypothesized to adversely affect female reproductive physiology by interfering with the organization of the hypothalamic-pituitary-gonadal axis. Here, we compared the effects of neonatal exposure to two environmentally relevant doses of the plastics component bisphenol-A (BPA; 50 μg/kg and 50 mg/kg) with the ESR1 (formerly known as ERalpha)-selective agonist 4,4′,4″-(4-propyl-[1H]pyrazole-1,3,5-triyl)trisphenol (PPT; 1 mg/kg) on the development of the female rat hypothalamus and ovary. An oil vehicle and estradiol benzoate (EB; 25 μg) were used as negative and positive controls. Exposure to EB, PPT, or the low dose of BPA advanced pubertal onset. A total of 67% of females exposed to the high BPA dose were acyclic by 15 wk after vaginal opening compared with 14% of those exposed to the low BPA dose, all of the EB- and PPT-treated females, and none of the control animals. Ovaries from the EB-treated females were undersized and showed no evidence of folliculogenesis, whereas ovaries from the PPT-treated females were characterized by large antral-like follicles, which did not appear to support ovulation. Severity of deficits within the BPA-treated groups increased with dose and included large antral-like follicles and lower numbers of corpora lutea. Sexual receptivity, examined after ovariectomy and hormone replacement, was normal in all groups except those neonatally exposed to EB. FOS induction in hypothalamic gonadotropic (GnRH) neurons after hormone priming was impaired in the EB- and PPT-treated groups but neither of the BPA-treated groups. Our data suggest that BPA disrupts ovarian development but not the ability of GnRH neurons to respond to steroid-positive feedback.

Accumulation and Endocrine Disrupting Effects of the Flame Retardant Mixture Firemaster® 550 in Rats: An Exploratory Assessment

Firemaster® 550 (FM 550), a fire‐retardant mixture used in foam‐based products, was recently identified as a common contaminant in household dust. The chemical structures of its principle components suggest they have endocrine disrupting activity, but nothing is known about their physiological effects at environmentally relevant exposure levels. The goal of this exploratory study was to evaluate accumulation, metabolism and endocrine disrupting effects of FM 550 in rats exposed to 100 or 1000 µg/day across gestation and lactation. FM 550 components accumulated in tissues of exposed dams and offspring and induced phenotypic hallmarks associated with metabolic syndrome in the offspring. Effects included increased serum thyroxine levels and reduced hepatic carboxylesterease activity in dams, and advanced female puberty, weight gain, male cardiac hypertrophy, and altered exploratory behaviors in offspring. Results of this study are the first to implicate FM 550 as an endocrine disruptor and an obesogen at environmentally relevant levels. #x000A9; 2012 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:124‐136, 2013; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21439

Neonatal exposure to endocrine active compounds or an ERβ agonist increases adult anxiety and aggression in gonadally intact male rats

Endocrine active compounds (EACs) have been shown to influence a number of reproductive endpoints but less is known about how they might affect other hormone dependent behaviors including anxiety and aggression. Recent evidence suggests that these effects may be mediated through the beta form of the estrogen receptor (ERbeta). Using male Long Evans rats, we sought to determine how neonatal exposure to EACs affects anxiety and aggression in adulthood. Anxiety was assessed using the elevated plus maze and aggression was assessed 8 weeks later using the resident intruder test. To gain insight into which ER subtype (ERalpha vs ERbeta) might be mediating these effects we used agonists specific for ERalpha (1,3,5-tris(4-Hydroxyphenyl)-4-propyl-1H-pyrazole (PPT)) or ERbeta (Diarylpropionitrile (DPN)) as additional treatment groups. For these experiments the synthetic EAC bisphenol-A (BPA) and the phytoestrogen metabolite equol (EQ) were used. Male neonates were injected with either 0.05 ml sesame oil (control), 50 microg estradiol benzoate (EB), 1 mg/kg DPN, 1 mg/kg PPT, 50 microg/kg BPA, or 10 mg/kg EQ daily for 4 days beginning on the day of birth (PND 0). Compared to the oil treated controls, significantly fewer open arm entries were made by the males neonatally treated with DPN, EQ, or BPA. The DPN and EQ treated males were also more aggressive compared to the controls. These findings suggest that neonatal exposure to EACs with agonistic activity on ERbeta may influence affective behavior in adulthood, including anxiety and aggression.

Disrupted female reproductive physiology following neonatal exposure to phytoestrogens or estrogen specific ligands is associated with decreased GnRH activation and kisspeptin fiber density in the hypothalamus.

It is well established that estrogen administration during neonatal development can advance pubertal onset and prevent the maintenance of regular estrous cycles in female rats. This treatment paradigm also eliminates the preovulatory rise of gonadotropin releasing hormone (GnRH). It remains unclear, however, through which of the two primary forms of the estrogen receptor (ERalpha or ERbeta) this effect is mediated. It is also unclear whether endocrine disrupting compounds (EDCs) can produce similar effects. Here we compared the effect of neonatal exposure to estradiol benzoate (EB), the ERalpha specific agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), the ERbeta specific agonist diarylpropionitrile (DPN) and the naturally occurring EDCs genistein (GEN) and equol (EQ) on pubertal onset, estrous cyclicity, GnRH activation, and kisspeptin content in the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei. Vaginal opening was significantly advanced by EB and GEN. By 10 weeks post-puberty, irregular estrous cycles were observed in all groups except the control group. GnRH activation, as measured by the percentage of immunopositive GnRH neurons that were also immunopositive for Fos, was significantly lower in all treatment groups except the DPN group compared to the control group. GnRH activation was absent in the PPT group. These data suggest that neonatal exposure to EDCs can suppress GnRH activity in adulthood, and that ERalpha plays a pivotal role in this process. Kisspeptins (KISS) have recently been characterized to be potent stimulators of GnRH secretion. Therefore we quantified the density of KISS immunolabeled fibers in the AVPV and ARC. In the AVPV, KISS fiber density was significantly lower in the EB and GEN groups compared to the control group but only in the EB and PPT groups in the ARC. The data suggest that decreased stimulation of GnRH neurons by KISS could be a mechanism by which EDCs can impair female reproductive function.

Oxytocin, but not oxytocin receptor, is regulated by oestrogen receptor β in the female mouse hypothalamus

In the female rat, oestrogen receptor (ER) β is colocalized with both oxytocin‐ and vasopressin‐producing neurones in the paraventricular nucleus of the hypothalamus (PVN). In this study, we demonstrate that the same pattern of colocalization between ERβ and oxytocin exists in the female mouse. Because this nucleus contains only a negligible quantity of ERα, it is likely that the oestrogen‐dependent regulation of oxytocin and vasopressin synthesis in the PVN is mediated by ERβ. Thus, we compared the effect of ovarian hormones on oxytocin and vasopressin mRNA expression in the PVN of wild‐type (WT) and ERβ knockout (βERKO) mice. We also compared the effects of ovarian hormones on oxytocin receptor (OTR) expression in the medial amygdala (MeA) and ventromedial nucleus of the hypothalamus (VMN) in female WT and βERKO mice. Ovariectomized mice underwent long‐term treatment with oestradiol or oil. Progesterone was given concurrently on the final 7 days of treatment, and all mice were killed 48 h after the final progesterone injection. In the PVN, hormone treatment increased oxytocin mRNA expression in WT but not βERKO females. These results suggest that ERβ is necessary for the regulation of the expression of oxytocin in the PVN. Hormone treatment had no effect on vasopressin mRNA expression in the PVN, but significantly increased OTR binding in both the VMN and the MeA in both genotypes. Collectively, our data show region and peptide specific regulation by ERα and ERβ in the mouse hypothalamus.

Impact of neonatal exposure to the ERα agonist PPT, bisphenol-A or phytoestrogens on hypothalamic kisspeptin fiber density in male and female rats

Neonatal exposure to endocrine disrupting compounds (EDCs) can impair reproductive physiology, but the specific mechanisms by which this occurs remain largely unknown. Growing evidence suggests that kisspeptin (KISS) neurons play a significant role in the regulation of pubertal onset and ovulation, therefore disruption of KISS signaling could be a mechanism by which EDCs impair reproductive maturation and function. We have previously demonstrated that neonatal exposure to phytoestrogens decreases KISS fiber density in the anterior hypothalamus of female rats, an effect which was associated with early persistent estrus and the impaired activation gonadotropin releasing hormone (GnRH) neurons. The goals of the present study were to (1) determine if an ERalpha selective agonist (PPT) or bisphenol-A (BPA) could produce similar effects on hypothalamic KISS content in female rats and (2) to determine if male KISS fiber density was also vulnerable to disruption by EDCs. We first examined the effects of neonatal exposure to PPT, a low (50 microg/kg bw) BPA dose, and a high (50 mg/kg bw) BPA dose on KISS immunoreactivity (-ir) in the anterior ventral periventricular (AVPV) and arcuate (ARC) nuclei of adult female rats, using estradiol benzoate (EB) and a sesame oil vehicle as controls. AVPV KISS-ir, following ovariectomy (OVX) and hormone priming, was significantly lower in the EB and PPT groups but not the BPA groups. ARC KISS-ir levels were significantly diminished in the EB and high dose BPA groups, and there was a nonsignificant trend for lower KISS-ir in the PPT group. We next examined effects of neonatal exposure to a low (50 microg/kg bw) dose of BPA and the phytoestrogens genistein (GEN) and equol (EQ) on KISS-ir in the AVPV and ARC of adult male rats, using OVX females as an additional control group. None of the compounds affected KISS-ir in the male hypothalamus. Our results suggest that the organization of hypothalamic KISS fibers may be vulnerable to disruption by EDC exposure and that females might be more sensitive than males.