Subchronic exposure to phytoestrogens alone and in combination with diethylstilbestrol - pituitary tumor induction in Fischer 344 rats

Abstract

Background: Subchronic administration of the potent pharmaceutical estrogen diethylstilbestrol (DES) to female Fischer 344 (F344) rats induces growth of large, hemorrhagic pituitaries that progress to tumors. Phytoestrogens (dietary plant estrogens) are hypothesized to be potential tumor inhibitors in tissues prone to estrogen-induced cancers, and have been suggested as "safer" estrogen replacements. However, it is unknown if they might themselves establish or exacerbate the growth of estrogen-responsive cancers, such as in pituitary.

Methods: We implanted rats with silastic capsules containing 5 mg of four different phytoestrogens - either coumestrol, daidzein, genistein, or trans-resveratrol, in the presence or absence of DES. We examined pituitary and other organ weights, blood levels of prolactin (PRL) and growth hormone (GH), body weights, and pituitary tissue histology.

Results: Blood level measurements of the administered phytoestrogens confirmed successful exposure of the animals to high levels of these compounds. By themselves, no phytoestrogen increased pituitary weights or serum PRL levels after 10 weeks of treatment. DES, genistein, and resveratrol increased GH levels during this time. Phytoestrogens neither changed any wet organ weight (uterus, ovary, cervix, liver, and kidney) after 10 weeks of treatment, nor reversed the adverse effects of DES on pituitaries, GH and PRL levels, or body weight gain after 8 weeks of co-treatment. However, they did reverse the DES-induced weight increase on the ovary and cervix. Morphometric examination of pituitaries revealed that treatment with DES, either alone or in combination with phytoestrogens, caused gross structural changes that included decreases in tissue cell density, increases in vascularity, and multiple hemorrhagic areas. DES, especially in combination with phytoestrogens, caused the development of larger and more heterogeneous nuclear sizes in pituitary.

Conclusions: High levels of phytoestrogens by themselves did not cause pituitary precancerous growth or change weights of other estrogen-sensitive organs, though when combined with DES, they counteracted the growth effects of DES on reproductive organs. In the pituitary, phytoestrogens did not reverse the effects of DES, but they did increase the sizes and size heterogeneity of nuclei. Therefore, phytoestrogens may oppose some but not all estrogen-responsive tissue abnormalities caused by DES overstimulation, and appear to exacerbate DES-induced nuclear changes.

Figure 1

Pituitary weight and functions, and body weight in animals treated with DES or phytoestrogens. A. wet pituitary weights; B. serum PRL levels; C. serum GH levels; and D. body weights of animals treated with DES (diethylstibesterol), Cou (coumesterol), Dai (daidzein), Gen (genistein), or Resv (trans-resveratrol) for 10 weeks. * = p < 0.05 compared to control animals treated with cholesterol (Chol) only. n = 10 animals for each group.

Figure 2

DES- or phytoestrogen-induced wet organ weights. The wet weight of organs from animals treated with DES, Cou, Dai, Gen, and Resv for 10 weeks including A. uterus; B. ovary; C. cervix; D. kidney; and E. liver. The abbreviations are as defined in the legend of Figure 1. * = p < 0.05 compared to control (Chol) animals. n = 10 animals for each group.

Figure 3

Pituitary weight and functions, and body weight in animals treated with DES + phytoestrogens. A. The wet weights of pituitaries; B. serum PRL levels; C. serum GH levels; and D. body weight, from animals treated without (open bar) or with DES (dark bars) in combination with Chol (control), Cou, Dai, Gen, and Resv for 8 weeks. The abbreviations are as defined in the legend of Figure 1. * = p < 0.05 compared to control (Chol) animals without DES treatment. n = 10 animals for each group. There were no statistically significant differences between animals treated with DES alone, compared to DES + phytoestrogens.

Figure 4

Wet organ weights as affected by DES treatment in combination with phytoestrogens. The weight of reproductive organs including: A. uterus; B. ovary; C. cervix; and non-reproductive organs: D. kidney; and E. liver, from animals treated without (open bar) or with DES (dark bars) in combination with Chol (control), Cou, Dai, Gen, and Resv for 8 weeks. The abbreviations are as defined in the legend of Figure 1. * = p < 0.05 compared to control (Chol) animals without DES treatment. # = p < 0.05 vs. DES alone. n = 10 animals for each group.

Figure 5

Representative stained pituitary tissues from animals treated with DES, daidzein, or the combination: The hematoxylin and eosin staining of pituitary tissues were recorded at 400× magnification from animals treated with A. cholesterol; B. DES; C. daidzein; or D. daidzein + DES. Note the large vascular and hemorrhagic areas (black arrows in B and D) resulting from DES treatments, regardless of whether daidzein (or other phytoestrogens) were administered in combination. Also note the larger nuclear size in animals treated with daidzein + DES (blue arrowheads in D). Combination treatments with DES plus other phytoestrogens gave similar results.

Figure 6

Nuclear morphometric changes in rat anterior pituitaries induced by phytoestrogens, DES, or combination treatments. Average nuclear areas were estimated as described in methods (n = 4-8 animals for each treatment group), * = p < 0.05 vs. control (Chol), # = p < 0.05 vs. DES alone.

Figure 7

Nuclear size histogram of pituitary cells: Individual nuclei (n = 1200-3000 for each group) measurements for anterior pituitaries were plotted as the 100-bin histogram, reflecting their variability and distribution by size. The Y axis shows the number of nuclei counted and X-axis shows the ranked nuclear size. See table 2 for the percentages of cells with large nuclear sizes (> 800).