Phytoestrogen research has grown rapidly in recent years due to its potential health benefits in preventing age-related and hormone-dependent cancers [26–37]. In contrast very little research has been conducted to identify phytoestrogen effects on cognitive behavior. Because phytoestrogens are present in virtually all natural-ingredient rodent diets that use soy as a source of protein [36, 38], and since these compounds are endocrine-active [27, 41–49], it is important to determine whether the amounts present in rodent diets are sufficient to affect sexually dimorphic spatial ability.
The present study addressed these issues, in part, by investigating the influence of dietary phytoestrogens (present in a normal rodent diet), on visual spatial ability. In tasks requiring the use of spatial skills, a sexually dimorphic difference has been consistently demonstrated in which males reliably outperform females [6–12]. This sex difference is most likely due to the presence of testosterone or more likely its metabolite estradiol, in brain [13–20]. Because phytoestrogens have the ability to bind estrogen receptors and alter many of the biological responses that are evoked by physiological estrogens [25, 39–46], we determined phytoestrogens affects on VSM in adult rats. Measurements of accuracy in acquisition (determined as trials required for shaping to a predetermined criterion) of the radial maze were diet dependent in that males (in experiment 1) fed the phytoestrogen-free diet and females (in experiment 2) fed the phytoestrogen-rich diet acquired the maze in fewer days than females or males fed the alternate diet. It should be noted that the dietary effects on body weight are the same for both male and female rats and therefore, do not account for the sexually dimorphic maze performance . There was, however, no significant dietary effects observed in the 8-arm task performance in either males (experiment 1) or females (experiment 2). Measures of accuracy on the baited/unbaited four-arm task (experiment 3) demonstrated that a diet change in young adult animals (a change from Phyto-600 to Phyto-free) had a positive influence on the accuracy in males, but a negative influence on the accuracy in females. In a companion paper, the impact of hormonal manipulation and dietary phytoestrogens' influence on VSM is examined and validates several aspects of the findings from the present study.
The discrepancies in maze performance were expressed in two ways: (1) Phyto-600 males (exposed lifelong to the diet) committed more reference errors than males switched to the Phyto-free diet and (2) lifelong Phyto-600 fed males and females had increased mobility within the maze which seemed to be advantageous to females but disruptive to males. It is intriguing to speculate that, the increase in reference errors, but not working or working/reference errors, suggests that disruption of maze performance is occurring within the frontal cortex (but not the hippocampus) of lifelong Phyto-600 fed males. The drop in performance is directly correlated to phytoestrogen levels in brain and the effects of phytoestrogens on brain CALB and COX-2 levels, in that phytoestrogens were identified as having a negative relationship with CALB (-0.942) and a positive relationship with COX-2 (0.974). In all other studies conducted previously the determination of phytoestrogens in the diet, circulating in plasma and especially in brain have not been performed together. The exception to this line of investigation has been our laboratory, where, in general, we obtained similar values previously to those quantified in the present study [39, 42], when overlapping diet exposure and brain regions were examined in Long-Evans and Sprague-Dawley rats [40, 51].
As reported, phytoestrogens were 47 times higher in the frontal cortex of Phyto-600 vs. Phyto-free fed males where there is an abundance of ER-β receptors , while phytoestrogen levels were similar in these animals in the hippocampal region. Furthermore males fed the Phyto-600 diet displayed a significant decrease in frontal cortical CALB levels in comparison to males receiving the Phyto-free diet. As CALB plays an important role in regulating intraneuronal cellular calcium, which protects against neurodegenerative disease and defends against apoptosis [48–52], a decrease in frontal cortex CALB may result in cognitive impairment due to cell death or abnormal calcium homeostasis [48–52]. Furthermore, frontal cortical COX-2 was shown to significantly increase in males fed the Phyto-600 diet. Because the expression of COX-2 is associated with key pathophysiologic event(s) in Alzheimer's disease [53–55], the significant increase in COX-2 expression in the frontal cortex may be relevant to the cognitive decline seen in the Phyto-600 males. In this regard, it is known that the hormonal action of estrogens may be mediated by the differential expression of estrogen receptors alpha vs betta in brain structures that activate or inhibit cell death mechanisms . Although, this parameter was not directly investigated in this study, the differing influence that dietary phytoestrogens had on CALB (significant decrease) and COX-2 (significant increase) in the male frontal cortex, suggests that phytoestrogens may activate programmed cell death. In support of this notion, since the Phyto-600 fed males displayed a 47-fold greater phytoestrogen levels in the frontal cortex (vs. Phyto-free males) and the greater affinity of phytoestrogens is for ER-β > ER-α, it has been shown that estrogen-related neuronal apoptosis is determined by ER-β and the Fas/Fas ligand system . In fact isoflavones have been shown to cause apoptosis in rat primary cortical neurons in vitro via a calcium dependant mechanism . Furthermore, the action of phytoestrogens may be tissue site specific. As our results show phytoestrogens are more prevalent and have greater affinity for certain brain structures and areas, therefore, phytoestrogens may act both as agonist and antagonist in a site-specific manner or in other words as natural SERM-type molecules [35, 38–42].
Taken together these findings suggest that soy dietary phytoestrogens present in the animal diets or the lack thereof, for a relatively short interval even in young adult animals, can significantly influence sexually dimorphic cognitive behavior. Dietary phytoestrogens sex reversed VSM, as expressed in the radial-arm maze by enhancing spatial memory in females but inhibited this ability in males, this finding corresponds to research regarding increased dementia observed in aged men consuming high tofu levels . However, while it is important to establish the influence of dietary phytoestrogens on brain function and behavior in animal models, the true significance in humans remains to be determined as it relates to the present findings.
Finally, as established by the above findings, of particular importance which deserves emphasis is that the Phyto-600 diet, used in this study, is a typical rat chow formulation that is similar to other rodent diet products that are used in many laboratories, whereas, phytoestrogen-free (Phyto-free) diets are rarely used. As previously reported, Thigpen et al.  and Brown and Setchell  determined the source and concentration of phytoestrogens in rodent diets. The results of this study showed that phytoestrogen concentrations vary widely among diets (between 200 μg/g and 600 μg/g of phytoestrogens). Additionally, one of our laboratories has quantified phytoestrogen plasma levels in a variety of physiologic conditions validating the importance of soy-derived phytoestrogens via rodent chow diets . Therefore not all diets have the same phytoestrogen levels, however, all diets, which have soy as their main protein source, contain phytoestrogens. It is very unusual to consider the influence of diet in hormone sensitive research investigations. This may be a critical error due to the complicating and/or confounding role phytoestrogens play. For example, we have previously shown that soy dietary phytoestrogens significantly alter body weight, food and water intake levels, puberty onset in females and prostate weights . Also, we have preliminary (unpublished) data demonstrating the effects of soy-derived phytoestrogens on several endocrine and metabolic parameters. However, brain aromatase and circulating plasma estradiol levels in male rats does not appear to be influenced by dietary phytoestrogens [22, 41, 51]. As the results from this research imply, phytoestrogens have considerable effects on hormonally sensitive parameters and their influence may be, in part, responsible for many reported sexual dimorphisms such as, the establishment and plasticity of sexually dimorphic brain structures like the sexually dimorphic nucleus of the preoptic area (SDN-POA) and the anteroventral periventricular nucleus (AVPV) [40, 41].
Therefore, dietary soy derived phytoestrogens can influence brain, cognition and memory in a manner that was previously unknown, where soy phytoestrogens enhance memory in intact females but inhibit memory in intact males. Further research is warranted in order to examine this important and growing research field of endocrine disruptors to determine the true significance of phytoestrogens influence that appears to be hormonal and gender dependent.