A plant-based diet has many undeniable ecological and health benefits. As a food or food additive, soya is appealing because it is a complete protein that is cholesterol-free, lactose-free, high in fibre and rich in complex carbohydrates, antioxidants and unsaturated fats. Soya is also replete with phyto-oestrogens, which makes it a hormonally active food. For many, the consequences of this activity will be minimal, or even potentially beneficial, but for others the endocrine-disrupting properties of soya should not be discounted and health practitioners should be more broadly aware of this phenomenon and potential outcomes. The pros and cons of a phyto-oestrogen-rich diet on many aspects of human health, including breast and prostate cancer, reproductive maturation and function, cardiovascular health, bone health and menopausal symptoms have been reviewed previously by myself and others( Reference Patisaul and Jefferson 1 – Reference Messina 6 ). The present review specifically focuses on the endocrine-disrupting properties of soya isoflavones, particularly within the neuroendocrine system, and highlights our most recent findings along those lines.
Phyto-oestrogens are naturally occurring plant compounds that are structurally and/or functionally similar to mammalian oestrogens and their active metabolites( Reference Whitten, Kudo, Okubo and D'Mello 7 ). There are several phyto-oestrogen classes, but the most hormonally active are the phenolic compounds of which the isoflavones and coumestans are the most widely studied groups. Isoflavones are most abundant in soyabeans and other legumes but also found in berries, wine, grains, nuts and soya-fortified foods( Reference Kurzer and Xu 8 ). Although present as inactive glycoside conjugates (containing glucose or carbohydrate moieties) and unconjugated (aglycone) forms in food, only the latter are bioactive. Fermented soya, such as tempeh or miso, typically contains higher aglycone levels than other soya-based foods. Once consumed, isoflavones are rapidly metabolised and absorbed, entering systemic circulation predominantly as conjugates with limited bioavailability and bioactivity, leaving only a tiny fraction of the ‘free’ bioactive form in systemic circulation. Typically, metabolites are less bioactive than the parent compounds but equol, a metabolite of daidzein, is a notable exception( Reference Setchell, Brown and Lydeking-Olsen 9 ). At best, only 30–50 % of individuals are capable of bioconverting daidzein to its more oestrogenic metabolite equol with vegetarians and individuals of Asian origin being most likely( Reference Setchell, Brown and Desai 10 , Reference Lampe, Karr and Hutchins 11 ). Age and health status, particularly the use of antibiotics, can significantly impact the production and absorption of bioactive isoflavones, including equol.
Although they are structurally similar to anthropogenic endocrine-disrupting compounds (EDC) and behave similarly on numerous molecular and cellular targets, intake of soya phyto-oestrogens is broadly encouraged and regarded as healthy, while their synthetic counterparts are increasingly viewed with caution and met with repeated calls to ban or restrict their use (Fig. 1). This attitudinal discordance is almost entirely based on the source of the compounds (soya is ‘natural’, while synthetic chemicals are not) rather than the scientific evidence regarding their hormone-disrupting activities. While it is clear that for many people soya diets are a healthful option, particularly when meat and saturated fat intake is concomitantly reduced, a growing chorus of scepticism is cautioning that the health benefits popularly ascribed to soya may be overstated and minimally supported by robustly conducted, statistically sound scientific studies( Reference Balk, Chung and Chew 12 – Reference Sacks, Lichtenstein and Van Horn 14 ). It has also been recognised for nearly a century that phyto-oestrogens have endocrine-disrupting properties in vertebrates, including human subjects, and that exposure to these compounds may pose a risk to some groups, particularly infants and the unborn( Reference Rozman, Bhatia and Calafat 15 – Reference Barrett 19 ).
Fig. 1. Structures of some well-known anthropogenic and naturally occurring endocrine-disrupting compounds. BPA, bisphenol A; DDT, dichlorodiphenyltrichloroethane; DEPH, di(2-ethylhexyl)phthalate.
Endocrine-disrupting activities of phyto-oestrogens in vertebrates and human subjects
An EDC is defined by the Endocrine Society as a compound that interferes with any aspect of hormone action( Reference Diamanti-Kandarakis, Bourguignon and Giudice 20 ). The word ‘interferes’ is important because many things can have hormone action as part of maintaining homeostasis and interacting with the environment, such as the simple act of eating or standing in sunlight. An EDC is a compound that interferes with the way in which the pancreas responds to a meal, or disrupts the vitamin-D-producing capacity of sunbathed skin. In the case of isoflavones, the target of this ‘interference’ is primarily thyroid hormone and oestrogen. Although a formal definition has not yet been established, the term neuroendocrine disruption has been used to broadly describe chemical impacts on endocrine-related brain development and function( Reference Waye and Trudeau 21 , Reference Gore and Patisaul 22 ). In the case of phyto-oestrogens, the vast majority of research effort has concentrated on the reproductive neuroendocrine system, which includes the hypothalamic–pituitary–gonadal (HPG) axis. Importantly, neuroendocrine disruption is distinct from, and should not be conflated with, neurotoxicity, which characterises processes contributing to neuronal cell death and related downstream consequences (e.g. dopaminergic cell death and Parkinson's like symptoms) and peripheral neuropathies. Isoflavones are not neurotoxic.
That phyto-oestrogens are endocrine disrupting has been known since at least the 1940s when ewes grazing on clover rich pastures in Australia were observed to have abnormally high rates of infertility, abortion and reproductive abnormalities in their offspring( Reference Bennetts, Underwood and Shier 23 – Reference Adams 25 ). Consequently, management of phyto-oestrogen levels has been the subject of grazing/feeding practices within the agricultural community for decades, including, most recently, in aquaculture( Reference Green and Kelly 26 , Reference Gontier-Latonnelle, Cravedi and Laurentie 27 ). Phyto-oestrogens have proven to be potently endocrine disrupting for a wide range of vertebrates, including rodents( Reference Patisaul and Jefferson 1 , Reference Whitten, Naftolin and Hochberg 28 ), birds( Reference Leopold, Erwin and Oh 29 ), cheetahs( Reference Setchell, Gosselin and Welsh 30 ), multiple species of fish( Reference Sassi-Messai, Gibert and Bernard 31 , Reference Clotfelter and Rodriguez 32 ), and grazing mammals such as cattle, sheep and even the southern white rhinoceros( Reference Bennetts, Underwood and Shier 23 , Reference Adams 25 , Reference Tubbs, Hartig and Cardon 33 ).
Evidence of endocrine disruption by soya in human subjects also dates back decades. Soya has been known to be goitrogenic for nearly a century( Reference McCarrison 34 , Reference Divi, Chang and Doerge 35 ) necessitating the addition of iodine to soya infant formula and other soya-rich foods. Both genistein and daidzein potently block thyroxine synthesis by serving as alternate substrates and blocking thyroid peroxidase catalysed tyrosine iodination. Soya also decreases absorption of synthetic thyroid hormone( Reference Messina and Redmond 36 ) potentially necessitating higher doses in hypothyroid patients. Thus, for these and other patients at risk for clinical or subclinical hypothyroid, compensatory iodine intake is advisable if soya is part of the regular diet. Additionally, although research regarding the relationship between soya intake and thyroid levels during pregnancy is extremely limited( Reference Li, Teng and Wang 37 ), because thyroid hormone is essential for normal brain development, pregnant women regularly consuming soya should be particularly mindful of this endocrine-disrupting property of soya.
Soya can also impact reproductive function in women. Suppression of circulating steroid hormone levels and attenuation of the preovulatory gonadotropin surge have been repeatedly observed and a 2009 meta-analysis concluded that isoflavone intake moderately increases cycle length and suppresses luteinising hormone and follicle-stimulating hormone levels( Reference Hooper, Ryder and Kurzer 38 ). A 2008 clinical case report described three women (aged 35–56 years) experiencing a suite of symptoms related to excessive soya intake (estimated to exceed 40 g/d), including abnormal uterine bleeding, endometrial pathology and dysmenorrhea, all of which resolved when soya intake was discontinued or reduced( Reference Chandrareddy, Muneyyirci-Delale and McFarlane 39 ). Importantly, as for all EDC, timing of exposure is important when considering the potential for long-term effects. The youngest of the three patients had been on a soya-rich diet since age 14 years and was experiencing secondary infertility, a condition that resolved and resulted in a pregnancy once she cut back on her soya consumption. Of even greater concern is what might happen in infant girls who consume high levels of soya, while their reproductive systems are still developing. Exposure earlier in life may have more lasting effects because disruption of the organisational actions of hormones may produce permanent structural and/or functional changes( Reference Gore, Chappell and Fenton 40 ).
The earliest evidence for developmental reproductive health effects came from two studies, conducted in the mid-1980s, which associated neonatal phyto-oestrogen exposure with thelarche before age 2 years in a population of Puerto Rican girls. A number of confounding factors, however, including the consumption of meat that had been fattened with potent oestrogens, including the notorious endocrine disruptor diethylstilbestrol, make the data problematic and difficult to interpret( Reference Freni-Titulaer, Cordero and Haddock 41 , Reference Schoental 42 ). A highly cited retrospective cohort study of 952 women found that young women reared on soya-based infant formula (248 women) as part of a controlled, University of Iowa feeding study, reported longer menstrual bleeding and menstrual discomfort than those who were fed a non-soya based formula (563 women)( Reference Strom, Schinnar and Ziegler 43 ). At the time the study was conducted, the women were too young to comprehensively examine pregnancy or fertility outcomes, but, now that nearly a decade has past, this area is ripe for reevaluation. Soya formula consumption has also been linked to a greater risk of developing uterine fibroids( Reference D'Aloisio, Baird and DeRoo 44 ). A prospective study reported oestrogenised vaginal epithelium in female soya formula-fed infants, an important finding confirming soya infant formula is oestrogenic in human subjects( Reference Bernbaum, Umbach and Ragan 45 ). Other studies, however, have found no link between soya infant formula and developmental reproductive parameters, including breast, ovarian or testes volume( Reference Bernbaum, Umbach and Ragan 45 , Reference Andres, Moore and Linam 46 ), and impacts of soya formula intake and on age at menarche are mixed( Reference D'Aloisio, DeRoo and Baird 47 , Reference Adgent, Daniels and Rogan 48 ). That soya is hormonally active is irrefutable. Whether or not soya intake, particularly during infancy, can have long-term health effects remains the subject of debate, but parents should be made aware of possible oestrogenic effects if they choose to feed their infants a soya-based formula.
Mechanisms of endocrine disruption by isoflavones
EDC can act via a myriad of mechanisms but the most fundamental include: (1) mimicking the effects of natural hormones by acting as a ligand at their binding sites; (2) antagonising the effect of these hormones by blocking their interaction with their physiological binding sites; (3) reacting directly and indirectly with the hormone in question; (4) altering the natural pattern of synthesis/degredation of hormones; or (5) altering cellular hormone receptor levels( Reference Gore, Chappell and Fenton 40 , Reference Frye, Bo and Calamandrei 49 , Reference Patisaul and Adewale 50 ). Isoflavones have been shown to interfere with oestrogen action via all of these. They also have other biological activities, which is not atypical as one of the hallmarks of EDC is that they simultaneously affect multiple hormonal systems, and act by multiple mechanisms. Genistein is thought to slow tumourigenesis, for example, via inhibition of protein tyrosine kinases and inhibition of DNA topoisomerases I and II, along with other chemoprotective mechanisms( Reference Patisaul and Jefferson 1 , Reference Messina 6 , Reference Mahmoud, Yang and Bosland 51 ). Phyto-oestrogens are also good antioxidants and anti-inflammatory agents.
The primary mode of isoflavone endocrine disruption is interference with oestrogen. At almost the same instant that a second subform of the nuclear oestrogen receptor (ER) was discovered (termed ERβ) it was recognised that isoflavones bind and activate transcription via both forms (ERα and ERβ), but generally have a higher relative binding affinity for ERβ( Reference Kuiper, Lemmen and Carlsson 52 – Reference Kuiper, Carlsson and Grandien 55 ). Potency estimates vary by assay, but most isoflavones bind nuclear ER far more readily than their synthetic endocrine-disrupting counterparts including bisphenol a( Reference Kuiper, Lemmen and Carlsson 52 ). Exposure is also consistently higher, often orders of magnitude higher, making them one of the most significant EDC in the human landscape( Reference Patisaul and Jefferson 1 , Reference Rappaport and Smith 56 ). Once bound, isoflavones act as partial agonists, with activity varying across tissue types and local levels of endogenous oestrogen. ER subtype distribution varies across tissues and cell types, particularly in the brain, changes over the lifespan, and is sexually dimorphic( Reference Cao and Patisaul 57 – Reference Schwarz, Nugent and McCarthy 59 ). Because ERα and ERβ are differentially distributed throughout the body and the brain, including neuroendocrine pathways, which coordinate reproductive function, that isoflavones are more bioactive via ERβ is functionally significant( Reference Shughrue, Lane and Scrimo 60 – Reference Handa, Ogawa and Wang 64 ). ERα and ERβ regulate different aspects of reproduction, behaviour and neuroendocrine function across the lifespan, although their relative roles are more clearly elucidated in animal models than in human subjects, in some tissues than others and, in some cases, one sex than another( Reference Rissman 65 – Reference Jia, Dahlman-Wright and Gustafsson 68 ). For example, ERβ in the paraventricular nucleus of the hypothalamus (PVN), a region important for the coordination of reproductive, social and stress-related behaviours, suppresses anxiety-related behaviours and enhances production of the neuropeptide oxytocin (OT)( Reference Lund, Rovis and Chung 69 – Reference Acevedo-Rodriguez, Mani and Handa 71 ). ERβ is also expressed at higher levels than ERα in the basal forebrain, hippocampus, dorsal raphe and cerebral cortex in the adult( Reference Shughrue, Lane and Scrimo 60 , Reference Zhang, Cai and de Zhou 72 , Reference Fan, Xu and Warner 73 ), all brain regions critical to neuroendocrine function and mood-related behaviours. ERβ is particularly abundant in the prenatal brain and plays a key role in brain morphogenesis by affecting cortical layering and interneuron migration( Reference Fan, Xu and Warner 73 ).
Once bound to ER, phyto-oestrogens can initiate transcription classically through interactions with the oestrogen response element or by binding early immediate genes, such as Jun and Fos( Reference Kushner, Agard and Greene 74 ). Steroid hormones, particularly oestrogens, can also initiate rapid, non-genomic actions at the cell surface via a range of mechanisms, including the binding of specialised steroid membrane receptors or ion channel subunits( Reference Levin 75 – Reference Kow and Pfaff 78 ). The vast majority of rapid actions are thought to originate at oestrogen-binding sites at the extracellular surface of the cell membrane, meaning that a potential EDC does not have to enter the cell to be active. Binding then activates second messenger pathways leading to cellular responses such as increased intracellular calcium or cAMP levels, or promoting nitric oxide release resulting in the stimulation of signal transduction pathways important for neuronal signalling, differentiation and other cellular processes( Reference Belcher and Zsarnovszky 79 ). The best-known transmembrane ER, the G-protein-coupled oestrogen receptor, was cloned as the orphan receptor GPR30 two decades ago and is now known to be capable of binding a wide range of EDC, including genistein( Reference Thomas and Dong 80 ). The functional significance of this pathway, or its disruption, has yet to be fully described but G-protein-coupled oestrogen receptor plays an important role in rapid vascular oestrogen signalling along with ERα and ERβ( Reference Barton 81 ). Emerging data reveals that phyto-oestrogens have epigenetic activity and can alter activities of DNA and histone methyltransferases, NAD-dependent histone deacetylases and other modifiers of chromatin structure( Reference Shanle and Xu 82 – Reference Labinskyy, Csiszar and Veress 84 ).
Phyto-oestrogens have also been shown to interfere with the enzymes needed for steroid biosynthesis and/or degradation. Coumestrol, for example, attenuates the conversion of [3H]-estrone to [3H]-estradiol in vitro by inhibiting the enzyme 17β-hydroxysteroid oxidoreductase Type 1 in a dose-dependent fashion( Reference Franke and Custer 85 ). Genistein, though weaker, has a similar dose-dependent inhibitory effect. In rats, genistein can alter folliculogenesis, an outcome postulated to result, at least in part, from dysregulation of steroidogenic enzymes( Reference Patel, Zhou and Rattan 86 ). In porcine granulosa cells, genistein decreases the activity of cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase( Reference Piasecka-Srader, Kolomycka and Nynca 87 ). Genistein has also been characterised as a non-competitive inhibitor of 11β-hydroxysteroid dehydrogenase type 1, which produces bioactive glucocorticoids, such as cortisol, from inactive precursors( Reference Tagawa, Kubota and Kobayashi 88 ). Disruption of aromatase and 5α-reductase by a number of phyto-oestrogens has also been demonstrated in vitro but this potential activity in mammalian tissues remains controversial( Reference Jefferson, Patisaul and Williams 2 ). Disruption of biosynthetic/degradative enzymes could significantly alter local endogenous hormone levels but not manifest as a change in circulating hormone levels. This may be particularly important for brain and hormone-sensitive subregions such as the hypothalamus as growing evidence strongly suggests that neural cells have the capacity to synthesise steroid hormones de novo ( Reference Taves, Plumb and Sandkam 89 – Reference Fokidis, Adomat and Kharmate 91 ).
Another mechanism by which phyto-oestrogens can perturb steroid bioavailability and transport is by altering sex hormone-binding globulin synthesis and availability. Isoflavones have long been known to appreciably stimulate sex hormone-binding globulin production, particularly in individuals who have levels on the low range of normal( Reference Pino, Valladares and Palma 92 ). Heightened sex hormone-binding globulin levels are thought to be one mechanism by which soya may lower breast cancer risk because bioavailable levels of circulating oestrogens are concomitantly reduced( Reference Low, Dunning and Dowsett 93 ). Similarly, suppression of circulating androgens, particularly dihydrotestosterone by equol, is hypothesised to be one way in which soya might protect against prostate cancer( Reference Mahmoud, Yang and Bosland 51 , Reference Tanaka, Fujimoto and Chihara 94 ). Notably, a subset of studies have found no impact of isoflavones on circulating sex hormone-binding globulin or steroid hormone levels in human subjects (e.g.( Reference Maskarinec, Williams and Inouye 95 )). One found suppressed luteal oestrogen levels following increased soya intake, but only in women of Asian descent( Reference Wu, Stanczyk and Hendrich 96 ), indicating ethnicity and/or the capacity to produce equol could be an underappreciated factor-mediating interindividual variability in responsiveness( Reference Atkinson, Newton and Stanczyk 97 , Reference Jackson, Greiwe and Schwen 98 ).
Endocrine-disrupting effects of soya isoflavones on the adult neuroendocrine system
Impacts on the mature reproductive axis in human subjects and other vertebrates have already been summarised and include altered serum hormone levels and suppression of ovulation. Elevated urine levels of genistein and daidzein have been associated with idopathic infertility and lower semen quality in Chinese men( Reference Xia, Chen and Zhu 99 ), and a slightly lower percentage of normal sperm in US men whose partners were attempting pregnancy( Reference Mumford, Kim and Chen 100 ), but supporting evidence in other populations or species for effects on spermatogenesis is limited. In contrast, the animal literature has explored a wider age range and a more diverse array of endocrine-disrupting effects.
Work in our laboratory focuses on neuroendocrine pathways underlying sexually dimorphic behaviours and, using a variety of animal models, we and others have shown that isoflavone intake interferes with oestrogen-mediated behaviours, including female sexual motivation. For example, consumption of a commercially prepared isoflavone supplement to adult female rats, at a dose that results in serum levels between those seen in Western and Asian (human) adults, attenuated lordosis (a reflexive posture indicating sexual receptivity) to the same degree as tamoxifen( Reference Patisaul, Luskin and Wilson 101 , Reference Patisaul, Dindo and Whitten 102 ). The supplement also suppressed proceptive behaviours even more profoundly than tamoxifen suggesting that soya isoflavones can suppress female sexual motivation and solicitation (Fig. 2). Administration of genistein alone did not recapitulate these effects( Reference Patisaul, Melby and Whitten 103 ). Whether or not libido is altered in human subjects appears to be completely unknown. Remarkably, a Pubmed search with the keywords ‘soya’ and ‘libido’ produced only nine published papers, not all of which were relevant. One was a case report describing a case of bilateral gynecomastia, erectile dysfunction and loss of libido in a 60-year-old man, which resolved when he discontinued drinking three quarts of soya milk daily( Reference Martinez and Lewi 104 ). Another reported a beneficial effect of soya protein dietary supplements on libido in post-menopausal women but there was an equally beneficial placebo effect suggesting that the soya effect was spurious( Reference Kotsopoulos, Dalais and Liang 105 ). Further inquiry revealed no studies, which have tackled this question in younger populations, or with a large-enough sample size to achieve reasonably robust statistical power. Given that soya appears to have a consistently suppressive effect on circulating steroid hormone levels it is not implausible that libido may also be suppressed but this appears to be, by and large, an unanswered question.
Fig. 2. (Colour online) In ovariectomised, hormone replaced female rats, sexual behaviour is suppressed by a soya isoflavone supplement. (a) Lordosis is a hallmark receptive posture in the rat and the frequency of lordosis in response to male mounting, which can be induced in ovariectomised females with progesterone (P) and estradiol benzoate (E), but not P alone. (b) In the presence of E and P tamoxifen (E + T+) or a soya supplement (E + S+) significantly decrease lordosis in female rats. (c) Similarly, proceptive behaviour, including hopping and darting, is also suppressed in hormonally replaced female rats on tamoxifen (E + T+) and, to an even greater degree, the soya isoflavone supplement (E + S+). *P ≤ 0·05; **P ≤ 0·01; means ± sem. Figure adapted from Patisaul et al.( Reference Patisaul, Luskin and Wilson 101 )
Mood and anxiety-related behaviours may also be impacted by adult soya intake( Reference Lephart, Setchell and Handa 106 ). In human subjects, nearly all studies along these lines have focused on post-menopausal women and evidence for improvement of mood is minimal and sporadic( Reference Thomas, Ismail and Taylor-Swanson 107 ). Results across animal studies are mixed and sex dependent with females generally showing decreased anxiety and males showing heightened( Reference Patisaul, Blum and Luskin 108 , Reference Patisaul 109 ). This pattern tends to abrogate or reverse expected sex differences in assessments of anxiety-related behaviours( Reference Patisaul, Sullivan and Radford 110 ) and appears to involve the neuropeptides OT and vasopressin (AVP). For example, male cynomolgus monkeys fed soya protein isolate containing 1·88 mg isoflavones/g protein over 18 months demonstrated higher frequencies of intense aggressive (67 % higher) and submissive (203 % higher) behaviours as well as a decreased proportion of time (68 % reduction) spent in physical contact with other monkeys( Reference Simon, Kaplan and Hu 111 ). Male rats maintained on a diet containing 150 µg/g genistein and daidzein displayed increased anxiety and elevated stress-induced plasma AVP and corticosterone levels( Reference Hartley, Edwards and Spiller 112 ). Increased hypothalamic AVP has also been reported in Sprague–Dawley rats fed a diet containing 1250 ppm genistein across the lifespan in a study run at a US Food and Drug Association research center( Reference Scallet, Wofford and Meredith 113 ). We found that the same isoflavone supplement found to suppress reproductive behavior and motivation in female rats (described earlier) abrogated the oestrogen-dependent up-regulation of OT receptors in the ventromedial nucleus of the hypothalamus and heightened ERβ expression levels in the PVN, an effect opposite to that of estradiol( Reference Patisaul, Dindo and Whitten 102 ).
Involvement of the PVN is consistent with both the oestrogen and thyroid-disrupting properties of soya, and the high potency of isoflavones at ERβ. The PVN, which contains little to no ERα but high levels of ERβ( Reference Hrabovszky, Kallo and Hajszan 114 ), is a primary site of OT and AVP production, peptide hormones important for social behaviour and the facilitation of sexual behaviour( Reference Ross and Young 115 ), as well as thyroid hormone releasing hormone and corticotropin-releasing hormone. Oestrogen-dependent stimulation of PVN OT and AVP production requires ERβ( Reference Patisaul, Scordalakes and Young 116 , Reference Nomura, McKenna and Korach 117 ). OT then binds to its receptor throughout the brain, including the ventromedial nucleus, a nucleus critical for mediating the lordosis response in females( Reference Pfaff 118 ). Isoflavone-related effects on these and other oestrogen dependent systems in the adult rodent brain have previously been reviewed( Reference Patisaul 109 , Reference Lephart, West and Weber 119 – Reference Patisaul and Polston 121 ) but a concerted focus on OT/AVP systems in human subjects remains lacking.
Evidence for developmental neuroendocrine disruption in animals and human subjects
Neuroendocrine disruption by soya isoflavones in mature neuroendocrine systems is by and large reversible with dietary modification and thus, with the exception of some hypersensitive groups such as hypothyroid and oncology patients, soya likely poses no long term health risk and may even confer modest benefits. Of greater concern is that phyto-oestrogens may interfere with the organisational role of oestrogen in the developing brain and reproductive system. Data from a diversity of animal models have repeatedly shown that manipulation of oestrogen during specific critical windows of development throughout gestation and early infancy leads to a myriad of adverse outcomes in the HPG axis including malformations in the ovary, uterus, mammary gland and prostate, early puberty, reduced fertility, disrupted brain organisation, and reproductive tract cancers( Reference Simerly 66 , Reference Newbold 122 – Reference Crain, Janssen and Edwards 126 ). The disruptor diethylstilbestrol story also starkly illustrates the broad spectrum of sex-specific consequences on neuroendocrine systems following fetal oestrogen exposure( Reference Reed and Fenton 127 , Reference Newbold 128 ). Although isoflavones and other EDC are far less potent than disruptor diethylstilbestrol, human exposure is ubiquitous and there is growing acceptance that EDC are contributing to adverse reproductive health trends in Western nations including median age at menarche, first breast development, and sexual precocity( Reference Gore, Chappell and Fenton 40 , Reference Aksglaede, Sorensen and Petersen 129 – Reference Mouritsen, Aksglaede and Sorensen 131 ). Advanced pubertal onset in girls adopted from developing countries by Western parents supports a role for environmental factors( Reference Aksglaede, Sorensen and Petersen 129 ). Emerging but controversial data suggest that EDC may also be shifting age at puberal onset in boys( Reference Zawatski and Lee 132 ). Among men, sperm counts in the USA and Europe appear to have declined by approximately half over the past 50 years( Reference Swan, Elkin and Fenster 133 , Reference Hauser 134 ) with upwards of 30 % in the subfertile range in places like Denmark where exposure to persistent environmental pollutants is particularly high( Reference Joensen, Jorgensen and Rajpert-De Meyts 135 ). A provocative but limited study associated increased incidence of hypospadias (malformation of the male external genitalia) with maternal vegetarianism( Reference North and Golding 136 ) but this effect has not been replicated. Synthetic EDC which interfere with androgen biosynthesis or activity are also associated with disorders of male genital development( Reference Skakkebaek, Rajpert-De Meyts and Main 137 , Reference Fisher 138 ) thus it is not implausible that equol may be endocrine disrupting in this regard. Increased prevalence of reproductive health disorders is likely not attributable to a single factor, not even a single environmental factor, but EDC are causal to some degree and isoflavones are hypothesised to play a contributing role( Reference Cederroth, Zimmermann and Nef 5 , Reference Gore, Chappell and Fenton 40 , Reference Massart, Parrino and Seppia 139 – Reference West, Anderson and McClure 141 ).
Disruption of reproductive tract development
The vast majority of studies exploring the impact of early life isoflavone exposure on HPG differentiation and function have used rodent models, with the compounds administered either prenatally to the pregnant dam or postnatally to the pups. This aspect of the literature has been extensively reviewed and will thus not be recapitulated in detail here but adverse outcomes in female rodents include disrupted timing of vaginal opening (pubertal onset), altered ovarian development, impaired oestrous cyclicity and ovulation, and disrupted HPG steroid feedback( Reference Patisaul and Jefferson 1 , Reference Jefferson, Patisaul and Williams 2 , Reference Cederroth, Zimmermann and Nef 5 , Reference Jefferson and Newbold 142 ). We have recently shown, for example, that female rats reared on a soya-rich diet across the lifespan (gestation through adulthood) have earlier pubertal onset (defined as the day of vaginal opening in the rat), and a greater number of corpera lutea post-puberty but took longer to establish regular oestrus cycles than their conspecifics on soya-free diet. Cycle regularity then degraded with time and soya-reared animals had a greater number of cystic follicles in early adulthood( Reference Patisaul, Mabrey and Adewale 143 ). Notably, not all pathology is readily obvious. Emerging evidence suggests that the oviductal and uterine environments in mice developmentally exposed to human-relevant gensitein levels are not suitable to maintain pregnancy, which manifests as the incapacity of the uterus to support implantation and embryonic development( Reference Jefferson, Padilla-Banks and Newbold 144 ). Moreover, embryo transfer experiments have shown that the uterus of genistein-treated mice is not capable of sustaining pregnancy even if the blastocysts arise from control mice( Reference Jefferson, Padilla-Banks and Goulding 145 ). These data are consistent with effects seen in sheep and other species suggesting that developmental isoflavone exposure induces permanent changes in the function of the female reproductive tract that may be subtle but can result in complete infertility, particularly as the animal ages.
There is a surprising paucity of data on the impact of developmental isoflavone exposure on male neuroendocrine physiology (reviewed in( Reference Cederroth, Auger and Zimmermann 146 )). There is some sporadic evidence in animal models that developmental isoflavone exposure affects testicular function, but many studies find no effects, which makes it challenging to draw definitive conclusions( Reference Cederroth, Auger and Zimmermann 4 ). A transformative pair of high-impact studies, which greatly contributed to health advisories in Europe, was conducted in marmosets. Twins were fed either soya or milk formula. Males on the soya diet had lower serum testosterone concentrations and higher numbers of Leydig cells then their milk-fed twins. As adults, the soya fed marmosets had larger testes and lower serum testosterone levels, demonstrating that the impacts were persistent( Reference Tan, Walker and Morris 147 , Reference Sharpe, Martin and Morris 148 ), but fertility was not compromised. Two rat studies conducted using classical toxicological testing parameters and long-term multi-generational oral exposure protocols spanning gestation through adulthood linked genistein with abnormalities in spermatogenesis( Reference Eustache, Mondon and Canivenc-Lavier 149 , Reference Delclos, Bucci and Lomax 150 ). One also found genistein-related alterations in sperm motility and a reduction in litter size accompanied by evidence of post-implantation embryo loss when the adult rats underwent fertility testing( Reference Eustache, Mondon and Canivenc-Lavier 149 ). Chronically exposed males have also been shown to develop mammary gland hypertrophy at doses at or above 11 mg/kg, and mammary gland hyperplasia at doses at or above 29 mg/kg (ductal/alveolar hyperplasia was observed in females as well)( Reference Delclos and Newbold 151 ). This effect was confirmed in a subsequent study by a different research group even though exposure was restricted to the perinatal period, suggesting that the sensitive period of exposure is pre-pubertal( Reference Boberg, Mandrup and Jacobsen 152 ). The male mammary gland may be one of the most sensitive targets for endocrine disruption but is rarely examined in EDC studies, leading some to advocate for its inclusion in chemical test guideline studies and risk assessment( Reference Rudel, Fenton and Ackerman 153 , Reference Fenton 154 ).
Disruption of brain sexual differentiation and neuroendocrine organisation
Work in our laboratory focuses on sexually dimorphic, oestrogen-sensitive hypothalamic systems and we have repeatedly shown that the sex-specific ontogeny of these systems is vulnerable to synthetic and naturally occurring EDC including soya isoflavones( Reference Patisaul and Jefferson 1 , Reference Patisaul and Polston 121 , Reference Rebuli and Patisaul 155 ). In rodents, hormone mediated morphological and functional organisation within the neuroendocrine system occur during a series of well-defined critical periods spanning gestation through puberty( Reference Simerly 66 , Reference Gorski 124 , Reference Cooke, Hegstrom and Villeneuve 156 ). Although most sex differences are established during prenatal and neonatal development, in the rat new cells (neurons and glia) are added to sexually dimorphic nuclei during adolescence in response to steroid hormone treatments( Reference Ahmed, Zehr and Schulz 157 , Reference Sisk 158 ), demonstrating the long-term sensitivity of sexually dimorphic brain regions to steroid hormone-mediated signalling. Interference with the hormone-sensitive organisation of neuroendocrine pathways could result in irreversible developmental defects and disruption of sex-typical behaviours, emphasising that development is likely the most susceptible periods for EDC exposure over the lifespan. Although it does not readily transfer lactationally, genistein efficiently crosses the rat placenta and the bioactive aglycone form of genistein is present in the fetal brain at levels comparable to circulating levels in the dam( Reference Doerge, Churchwell and Chang 159 , Reference Doerge, Twaddle and Churchwell 160 ). Moreover, the transfer of genistein to the brain from systemic circulation appears to be more efficient in prenatal animals than adults( Reference Chang, Churchwell and Delclos 161 ) demonstrating that it and other isoflavone phyto-oestrogens are capable of directly interfering with the organisation of neuroendocrine signalling pathways in the developing brain.
The sexually dimorphic brain region most frequently used as a biomarker of endocrine disruption in rats is the sexually dimorphic nucleus of the preoptic area (SDN-POA). The volume of the SDN-POA is enhanced by estradiol aromatised from perinatal, testicular androgen( Reference Gorski 124 ), is five to six times larger in males than females( Reference Gorski, Gordon and Shryne 162 ), and is thought to play a role in male reproductive behaviours and mate choice. Although both ERα and ERβ are expressed in the SDN-POA across the lifespan, ERα appears to play a dominant role in masculinising SDN-POA morphometrics( Reference Perez, Chen and Mufson 61 , Reference Shughrue, Lane and Merchenthaler 163 , Reference Patchev, Gotz and Rohde 164 ), a process which has now been elucidated in detail and is largely complete by the second week of life( Reference Wright, Schwarz and Dean 67 , Reference Sakuma 165 ). In rats, numerous studies have queried the extent to which soya isoflavones alter SDN-POA volume in both sexes and, while not always in complete accordance, the data are generally consistent with oestrogenic effects. For example, when administered prenatally through adulthood, genistein increases SDN-POA volume in males but not females( Reference Scallet, Divine and Newbold 166 ). No enhancement, however, was observed in males exposed from birth through weaning( Reference Lewis, Brooks and Milburn 167 ) or in males exposed on only the first few days of life( Reference Patisaul, Fortino and Polston 168 ) suggesting that exposure must be ongoing to maintain the enlargement. Masculinising effects on female SDN-POA volume have only been observed following high-dose exposure( Reference Faber and Hughes 169 ) and some studies have not found genistein to be endocrine disrupting in the female rat SDN-POA, even at doses high enough to be uterotrophic( Reference Lewis, Brooks and Milburn 167 , Reference Masutomi, Shibutani and Takagi 170 ).
An additional area of focus for our studies is the anterior ventral periventricular nucleus (AVPV), which, like the SDN-POA, is sexually differentiated by endogenous gonadal hormones during a series of pre- and perinatal critical periods but is larger in females than males( Reference Davis, Shryne and Gorski 171 , Reference Cooke, Hegstrom and Villeneuve 172 ). The presence and density of the two ER subtypes varies across species but both are present in the rat( Reference Cao and Patisaul 58 ). The AVPV is essential for coordinating the preovulatory gonadatropin surge and plays a central role in female sexual behaviour( Reference Gorski, Mennin and Kubo 173 – Reference Semaan and Kauffman 175 ). In human subjects, the neural machinery controlling gonadotropin pulsatility is functional by the end of the first trimester( Reference Grumbach 176 ), while in rodents this system does not fully sexually differentiate until the first few days of the neonatal period( Reference Davis, Shryne and Gorski 171 ). In male rodents, testicular androgen is aromatised to oestrogen in the brain, and it is this locally derived oestrogen, working primarily through ERα-dependent pathways, that is primarily responsible for defeminising/masculinising the AVPV( Reference Wright, Schwarz and Dean 67 , Reference McCarthy, Wright and Schwarz 177 , Reference Schwarz and McCarthy 178 ). At birth exogenous oestrogen administration can defeminise the female AVPV and surrounding structures thereby eliminating lordosis and the capacity for steroid-positive feedback. By extension, if endogenous oestrogen is blocked in males, either by castration, by aromatase inhibition, or antagonism of hypothalamic ER, the AVPV and surrounding structures fails to defeminise and the capacity to elicit lordosis and a gonadal surge remains. Therefore, interference with oestrogen at birth, in either sex, can result in the improper differentiation and function of the HPG axis across the lifespan.
We have shown that subcutaneous administration of 10 mg/kg genistein, a dose that is approximately equivalent to the total amount of isoflavones ingested by infants fed soya formula, over the first 4 d of life, advances vaginal opening and compromises the ability to maintain a regular oestrous cycle in female rats( Reference Bateman and Patisaul 179 ). This outcome was accompanied by an impaired ability to stimulate gonadotropin releasing hormone activity (as measured by the co-immunoreactivity of gonadotropin releasing hormone and Fos) following ovariectomy and hormone priming. We have further shown that neonatal exposure to 10 mg/kg genistein significantly decreases the density of kisspeptin immunoreactive fibres in the AVPV of female rats( Reference Patisaul, Todd and Mickens 180 , Reference Bateman and Patisaul 181 ). Exciting work over the past decade has identified kisspeptin neurons as the primary gatekeepers of gonadotropin releasing hormone release in many species, including human subjects( Reference Oakley, Clifton and Steiner 182 , Reference Piet, de Croft and Liu 183 ). Therefore, our findings suggest that disrupted organisation of kisspeptin signalling pathways may be a novel mechanism by which isoflavones and other EDC may induce a suite of HPG-related abnormalities, including advanced pubertal onset, irregular oestrous cycles and premature anovulation( Reference Patisaul 184 ).
How much is too much: human isoflavone intake, metabolism and excretion
Ultimately risk of harm comes down to two primary factors: dose and timing of exposure. Undoubtedly, development is the most sensitive period for the endocrine-disrupting consequences of soya isoflavone exposure, thus it is not surprising that concerns have been expressed regarding the safety of soya-based infant formula. Initially developed as an alternative to bovine milk formulas for babies with milk allergy, use of soya infant formula in the USA is a popular choice and constitutes an estimated 25 % of the formula market( Reference Badger, Ronis and Hakkak 185 – Reference Barrett 187 ). The safety of soya formula has been rigorously discussed from several perspectives, and a litany of review articles and position papers have been published on the subject( Reference Bhatia and Greer 188 – Reference Chen and Rogan 195 ). Societies including the American Academy of Pediatrics and the European Society for Pediatric Gastroenterology Hepatology and Nutrition Committee on Nutrition have issued guidelines recommending against the exclusive use of soya formula except in the rare cases of true milk allergy or lactose intolerance. The US National Toxicology Programme completed its most recent safety assessment of soya infant formula in 2010 (monograph available at http://ntp.niehs.nih.gov/pubhealth/hat/noms/formula/index.html) and concluded there is ‘minimal concern for adverse developmental effects.’ For comparison, this is the same level of concern initially expressed for bisphenol a until the Food and Drug Association elevated that advisory to ‘some concern’ in January, 2010 based on new data (and then subsequently lowered it again). Notably, the National Toxicology Programme could not issue a conclusive recommendation regarding potential long-term reproductive effects of soya infant formula largely because of limited and poor-quality human data. An apparent lack of adverse effects is one reason why so many consumers, clinicians and public health agencies consider regular use of soya formula to be safe, even beneficial. However, the absence of evidence is not evidence of safety so this problematic data gap regarding the long-term impacts of soya formula use remains in serious need of attention.
When considering the potential safety of soya formula, it is frequently argued that Asian populations have been consuming soya for centuries, with no obvious consequences. This argument fails to recognise, however, that exposure patterns differ in key ways between Asians consuming a traditional soya-rich diet and Caucasians eating a typical Western diet( Reference Badger, Ronis and Hakkak 185 ). This timing of exposure is critical. In a traditional Asian diet, soya consumption is moderate across the entire lifespan, but because isoflavones do not effectively transfer via lactation, exposure in breastfeeding infants is extremely low. By contrast, Western babies on soya infant formula have their highest exposures in the first year of life then exposure rapidly plummets. In that regard the two populations are not comparable because their exposure patterns during a critical window of development are so dramatically different. Other diet and lifestyle differences may also be confounding when evaluating the potential health benefits and risks of soya. For example, Asian populations on traditional diets eat less processed foods, considerably higher levels of seafood and lower levels of animal fat than Western populations.
So how much is too much? There is no ‘typical’ level of isoflavone intake as consumption patterns vary widely across populations, and geographic regions. For Asians, vegetarians and other groups in which soya is foundational to the diet, isoflavone consumption can be as high as 100 mg/d (intake range is about 0·3–1·5 mg/kg body weight)( Reference Messina 6 , Reference Setchell, Zimmer-Nechemias and Cai 192 , Reference Franke, Custer and Tanaka 196 – Reference Mortensen, Kulling and Schwartz 198 ). Western diet intake estimates range from 1 to 3 mg/d( Reference Mortensen, Kulling and Schwartz 198 – Reference Horn-Ross, Barnes and Lee 201 ). For their weight, infants exclusively fed soya-based formula have the highest mean daily consumption of total isoflavones, ranging from 6 to 9 mg/kg body weight per d in 4-month-old infants, an amount that is up to seven times higher than Asians consuming a traditional soya-based diet.
The isoflavone content of an array of foods and food products is now available via online databases (reviewed in:( Reference Schwarz, Sontag and Plumb 202 )) including one maintained by the USDA( 203 ). Food isoflavone content varies widely, even in the same foods, because of local and/or seasonal differences in growing conditions so its difficulty to accurately estimate intake( Reference Mortensen, Kulling and Schwartz 198 ). Additionally, soya is found in upwards of 60 % of processed foods and ground meats( 204 ). Textured soya protein (50–70 % soya protein) is used as a meat substitute or filler for hotdogs, hamburgers, sausages and other meat products( Reference Thomas and Lutz 205 , Reference Senti 206 ), while soya protein isolate (90 % soya protein) is frequently used to enrich energy bars and sports drinks (particularly those advertising high protein levels), cereals, granola bars, infant formula, imitation dairy products, ice cream and cheese. Soya isoflavones and other phyto-oestrogens are also widely available as dietary supplements( Reference Setchell, Brown and Desai 207 , Reference Piotrowska, Jakobkiewicz-Banecka and Wegrzyn 208 ), typically containing concentrations far higher than those found in food( Reference Cao, Calafat and Doerge 209 ).
Not surprisingly, blood isoflavone levels also vary widely, and can be orders of magnitude different between individuals based on dietary preferences and individual differences in phyto-oestrogen absorption and metabolism( Reference Hutchins, Slavin and Lampe 210 – Reference Rolwand, Wiseman and Sanders 212 ). Blood genistein levels are generally in the range of 25 ng/ml for Asian women, slightly less for vegetarian women, and under 2 µg/ml for US women( Reference Verkasalo, Appleby and Allen 213 ). Isoflavones can pass from mother to fetus through the placenta, and have been found in human umbilical cord blood and amniotic fluid at levels comparable with concentrations seen in maternal plasma, demonstrating that fetal exposure can be significant( Reference Adlercreutz, Yamada and Wahala 214 ). Infants on soya formula can have plasma levels exceeding 1000 ng/ml( Reference Cao, Calafat and Doerge 209 ) which is 13 000–22 000 times higher than their own endogenous oestrogen levels, 50–100 times higher than oestradiol levels in pregnant women, and 3000 times higher than oestradiol levels at ovulation( Reference Badger, Ronis and Hakkak 185 , Reference Winter, Hughes and Reyes 215 , Reference Setchell, Zimmer-Nechemias and Cai 216 ). In contrast, infants fed cow's milk formula or human breast milk have plasma isoflavone levels of 9·4 and 4·7 ng/ml, respectively( Reference Setchell, Zimmer-Nechemias and Cai 192 , Reference Franke, Custer and Tanaka 196 , Reference Setchell, Zimmer-Nechemias and Cai 216 ). Notably, levels in infants and vegetarians easily far surpass, sometimes by several orders of magnitude, internal levels other endocrine disruptors of concern, including bisphenol a and phthalates( Reference Crain, Janssen and Edwards 126 ).
Conclusions and recommendations
Soya isoflavones are clearly endocrine disrupting, but although they are similar to their synthetic brethren in terms of their cellular and molecular mechanisms of action on neuroendocrine structure and function, and the scope of adverse outcomes they can inflict, society embraces these compounds at the same time it rejects, often with vigour, exposure to their synthetic brethren. Thus, phyto-oestrogens both challenge our attitudes regarding EDC and highlight how profoundly the direction and interpretation of research and available data can be influenced by source. While some beneficial effects might be conferred by including moderate levels of dietary soya, particularly in adults eating a diet high in saturated fat and animal protein, the potentially adverse effects of these compounds for some groups are likely underappreciated. An abundance of animal data unequivocally demonstrates that soya isoflavone exposure, at doses and plasma concentrations attainable in human subjects, including soya-reared infants, can permanently alter the structure and function of neuroendocrine pathways in both sexes. Infants fed soya formula have the highest exposure to any non-pharmacological source of oestrogen-like compounds, and yet greater anxiety surrounds compounds like bisphenol a and the phthalates which have far lower potency on neuroendocrine targets and to which exposure is far lower. Although relatively few adverse effects have been reported, that is somewhat a consequence of lack of data rather than lack of measurable effects. Although unsatisfying, a parsimonious approach to soya intake is to follow the classic adage and consume in moderation. Development of dietary guidelines should consider the endocrine-disrupting properties of soya and other hormonally active foods, particularly for vulnerable groups such as pregnant women and hypothyroid individuals.
Financial Support
This effort was supported by NIEHS R21ES021233 to H. P. B. and pilot funds from the NCSU Center for Human Health and the Environment.
Conflicts of Interest
None.
Authorship
The author had sole responsibility for all aspects of preparation of this paper.
