Steroid hormones such as the dominant physiologic estrogen, estradiol (E₂) have many effects on pituitary function, including regulation of most pituitary hormones and proliferation of several pituitary cell types 1 2 . The Fischer 344 (F344) rat has long been used as a model for investigating growth control of estrogen-responsive tissues (especially those prone to estrogen-induced tumors), by various estrogens, and related biological processes such as angiogenesis 3 4 . When female F344 rats are chronically treated with E₂ or the pharmaceutical estrogen diethylstilbestrol (DES), their pituitaries grow 10 to 20 times normal size and sometimes form a tumor by 10 weeks 5 6 .

Estrogens can increase the expression levels of basic fibroblast growth factor and pituitary tumor transforming gene products in F344 animals 1 2 , leading to prolactinoma development, vascularization, and increases in cell number, which have been identified as quantifiable genetic traits 5 7 .

The peptide hormone prolactin (PRL) is expressed in the pituitary of all mammals, and its major function in females is the stimulation of milk production by the mammary gland. Additional known functions include modulation of other aspects of reproduction, osmoregulation, growth, metabolism, and migratory and maternal behaviors 2 . Hyperprolactinomas release highly elevated plasma PRL levels leading to reproductive dysfunction in both males and females 8 9 . There is also a tight correlation between E₂ levels and growth hormone (GH) secretion by the pituitary. Serum GH responds to changes in E₂ levels during different life stages in women 10 11 and regulates body growth and composition, metabolism, bone density and pubertal development in both sexes 12 .

Phytoestrogens are plant-derived compounds that structurally and functionally mimic mammalian endogenous estrogens. These compounds have been considered candidate therapeutic or prevention agents for such diseases as reproductive system cancers, heart disease, menopausal symptoms, and osteoporosis - essentially mimicking the health benefits thought to be characteristic of endogenous estrogens, while counteracting the hazards 13 14 . Considering the numerous beneficial effects of estrogens, it is not surprising that phytoestrogens are considered possible complementary or alternative medicine treatments. However, some estrogens are associated with detrimental effects over life-long exposures. For instance, cumulative high exposures to endogenous, therapeutic, or environmental estrogens have been implicated in diseases such as breast cancer 2 15 16 17 . Recently, breast cancer incidence in a large human population was noted to be inversely correlated to the consumption of soy phytoestrogens in the diet 18 . Therefore, we need to carefully examine the beneficial vs. the detrimental biological effects of phytoestrogens in animal studies.

Isoflavones, including the components of soy bean-derived foods such as genistein and daidzein, are some of the most studied phytoestrogens. Part of the original reasoning behind proposing potential health benefits of phytoestrogens stemmed from the fact that those consuming "Asian diets" high in soy isoflavones seem to be less vulnerable to the diseases of both estrogen overexposure (cancers) and estrogen underexposure (osteoporosis, hot flashes, heart disease, depression, etc.). These benefits are thought to be diet-related rather than genetic, because when Asians move to Western countries and adopt their diets, their incidences of these diseases become similar to Westerners 19 20 . Coumestrol which is supplied by foods such as alfalfa sprouts, or is transmitted to the diet via red clover consumption by livestock, is also thought to have these beneficial effects. Resveratrol consumption, also speculated to be beneficial, could explain why populations that daily consume red wine (which contains high levels of resveratrol) benefit by having lower levels of diseases thought to be associated with estrogen deficits (eg. heart disease).

The different activities and the bioavailability of phytoestrogens vary depending on factors such as the route of administration, dosage, individual metabolism, co-ingestion of other substances, and phytoestrogen levels present in intake foods 21 22 . For example, in Japanese men and women consuming a traditional diet, the plasma isoflavone concentrations can be as high as 0.2 to 1 μM. In Europe and North America, plasma concentrations for isoflavones are between 0.005 and 0.4 μM 22 23 24 . The blood concentrations for coumesterol can be from 0.01 (reported as an average from food intake, Malaysia 25 ) to 0.5 μM (resulting from taking dietary supplements 24 ). Resveratrol, a compound which has very low bioavailability and is rapidly metabolized in humans 26 27 , has serum concentrations (for the compound and its metabolites) as high as 2 μM 28 . In the present studies, these four phytoestrogens were provided via sustained slow release directly to the circulation, bypassing gut microflora and hepatic first pass metabolism, which can have a major impact on the biological potency of phytoestrogens. We examined estrogenic effects on size and architecture of multiple organs (both reproductive and not), and on body weights, in female F344 rats. In particular we focused on the size, structure, cellular composition, and function of the pituitaries, as this tissue is used to monitor carcinogenic estrogenic effects in this animal model. Our studies investigate whether phytoestrogens mimic, inhibit, or exacerbate the known effects of DES.

Cholesterol, flavone, DES, trans-resveratrol, coumesterol, daidzein, and genistein were obtained from Fluka (Milwaukee, WI) or Sigma Chemical Co. (St. Louis, MO). The high purity grade solvents and silica gel were purchased from Fisher Scientific (Pittsburgh, PA).

These studies examined organ size and functional responses to several phytoestrogens, in comparison to the actions of a well-studied pharmaceutical estrogen-induced model for carcinogenicity. The stimulatory effects of DES treatment on pituitary function were robust (as expected for this well established model) in these young (21 day old) female F344 rats treated with estrogens for ~2 months. Large impacts on pituitary functions and reproductive maturation was expected in animals whose human life-span equivalency approximated the beginning of puberty. DES treatment increased the size, as well as caused structural changes, in the pituitary. However, phytoestrogen treatments, even at these high concentrations, neither caused the same effects, nor attenuated the effects of DES. DES treatment also increased serum PRL and GH levels, and two phytoestrogens, genistein and resveratrol, also caused significant increases in serum GH levels. Genistein was previously reported to increase GH in ewes and rats 35 36 . These kinds of effects may be related to estrogenic effects on stature in both boys and girls 37 . Although daidzein is very similar in chemical structure to genistein, it did not increase the serum GH levels. This may be due to the lower serum daidzein levels at 10 weeks compared to genistein, or to the broader signaling capabilities of genistein (for instance to also inhibit tyrosine kinases 36 ). These effects are clearly separable from the effects on pituitary weight in our study, as no phytoestrogens were able to change the pituitary weights. Known effects of GH and PRL do not explain the phytoestrogen-induced reversal of DES-induced growth on the cervix and ovary that we observed. However, as we have shown previously in pituitary tumor cell lines, some of the abilities of phytoestrogens to change tissue growth patterns could be related to their capacity to differentially activate caspases and several mitogen-activated protein kinases 38 39 via nongenomic signaling mechanisms.

The average nuclear size in pituitary cells (which reflects their functional state) is known to be changed due to perturbations in hormonal status 40 41 42 . Although the average nuclear size from pituitaries of DES-treated or phytoestrogen-treated animals showed no difference compared to cholesterol-treated control animals, the observed changes in nuclear size heterogeneity with DES (and especially with DES in combination with all phytoestrogens), suggested that the functions of pituitary cells had been altered. High serum PRL and GH levels as well as pituitary size increases in animals treated with DES suggest that nuclear heterogeneity can be a marker for peptide hormone production and/or growth in the pituitary, though elevated GH levels caused by genistein did not correlate with such nuclear changes. It is interesting that phytoestrogens increased nuclear area only when their actions were superimposed on DES effects, and that the corresponding serum PRL and GH levels did not reflect any additional changes due to these combinations. Increases in nuclear size measured by tissue morphometry have been previously correlated to high risk pre-invasive breast lesions 43 , and pituitary tumors are known to have large pleiomorphic nuclei 44 . Estrogens have also been shown recently to alter nuclear morphology related to MAPK activation and changes in cell division machinery 45 . Though the nuclear size changes we observed are not directly connected to a known health risk, they could signal some tissue growth instabilities brought on by excessive and diverse estrogenic exposure which would require more exposure time to manifest their effects. However, the levels of exposure that we examined were very high and were intended to demonstrate actions in an experimental situation where DES was known to cause pituitary tumorigenesis over a short time period.

Our use of this animal model for carcinogenesis demonstrated the expected estrogenic overstimulation by DES on the reproductive organs of female F344 rats. Others have reported that estradiol or DES treatment increases uterine wet weight, epithelial thickness, loose density stroma, and development of more uterine glands 46 . They also regulate cervical epithelial cell proliferation 47 and increases ovarian wet weight 48 , including in F344 rats. However, phytoestrogens by themselves had none of these effects at the organ weight level, though they were able to suppress the weight gain effects of DES in ovaries and cervices after 8 weeks of co-treatment. So where reproductive organs show estrogenic effects, phytoestrogens may be an effective foil.

Only DES caused lower body weights in our study, which could not be reversed by phytoestrogens. Sex hormones are known to regulate rat body weights 49 ; possible mechanisms include decreased metabolism and decrease lipoprotein lipase activity in adipose tissues 50 . Other studies have suggested that estrogens, especially at high concentrations, can cause nausea, indirectly contributing to poor eating and weight loss, which could contribute to the prevalence of anorexia in adolescent girls 51 . None of the high dose phytoestrogens in our study caused weight loss effects similar to DES, at least not for such subchronic exposures.

There is a concern that phytoestrogens present in animal feeds may affect experimental outcomes and also cause phytoestrogen exposures to humans consuming livestock fed on such diets 19 21 . Blood phytoestrogen content in our control animals was not detectable, and if present, negligible compared to the high concentrations delivered by our implants. The fact that the levels of these compounds were undetectable in the cholesterol-fed control animals using our sensitive extraction and assay methods, correlates with the amounts attributable to animal feed previously studied 52 .

The increase in serum levels of coumesterol, daidzein, and genistein from 4 to 10 weeks suggests bioaccumulation, as has been suggested by previous studies 53 . Though factors such as species, age, developmental status, gender, diet, dose, route of administration, and metabolism all influence the ultimate phytoestrogen exposure, limiting the effectiveness of comparisons between studies in both rodents and humans, the levels of serum phytoestrogens that we report are comparable to high human levels. It appears that even such high doses of phytoestrogens did not cause effects in the organs or hormonal systems that we examined, except to alter some microscopic tissue architecture when in combination with a carcinogenic estrogen (DES). However, with some exceptions on reproductive organs, neither did they counteract the effects of this known potent estrogenic carcinogen.

Our studies demonstrated that phytoestrogens by themselves, even in very high subchronic doses (that could match, however, accumulations in human blood via dietary intake 22 23 24 25 28 ), did not cause the growth of pituitaries to sizes associated with tumor development, nor did they cause the growth of other reproductive, nonreproductive, or xenoestrogen-processing metabolic organs (liver or kidney). Though the phytoestrogens were unable to attenuate the effects of DES treatment on PRL and GH levels, pituitary weight, and body weight, they were able to reverse the DES-induced reproductive organ weight gains. So, it is these effects which may contribute to any protection such estrogens may give from endogenous or environmental estrogen overexposure. Concerns that have been raised about safety of these compounds as dietary supplements are not supported by many of our results, though the carcinogenic estrogen + phytoestrogen effects that we showed on nuclear size and heterogeneity should be studied further.

The authors declare that they have no competing interests.

DN and BSK analyzed serum phytoestrogens level in these studies, MK performed the morphometric analysis of pituitaries, and YJJ carried out the rest of studies. YJJ and CSW both participated in the design of the study and statistical analyses. All authors read and approved the final manuscript.