Dramatic synergism between excess soybean intake and iodine deficiency on the development of rat thyroid hyperplasia
Takako Ikeda1,
Akiyoshi Nishikawa2,
Takayoshi Imazawa,
Shuichi Kimura1 and
Masao Hirose
Division of Pathology, National Institute of Health Sciences,1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 and
1 Showa Women's University, 1-7 Taishido, Setagaya-ku, Tokyo 154-8533, Japan
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Abstract
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The effects of defatted soybean and/or iodine-deficient diet feeding were investigated in female F344 rats. Rats were divided into four groups, each consisting of 10 animals, and fed basal AIN-93G diet in which the protein was exchanged for 20% gluten (Group 1), iodine-deficient gluten (Group 2), 20% defatted soybean (Group 3) and iodine-deficient defatted soybean (Group 4). At week 10, relative thyroid gland weights (mg/100 g body wt) were significantly (P < 0.01) higher in Groups 2 (15.5 ± 1.3) and 4 (81.7 ± 8.6) than in Group 1 (8.4 ± 2.0) and pituitary gland weights (mg/100 g body wt) were significantly (P < 0.01) higher in Groups 3 (9.1 ± 0.6) and 4 (9.7 ± 1.5) than in Group 1 (6.5 ± 1.5). Serum biochemical assays revealed thyroxine to be significantly (P < 0.05) lower in Groups 2 and 4 than in Group 1. On the other hand, serum thyroid-stimulating hormone (TSH) was significantly (P < 0.01) higher in Groups 3 and 4 than in Group 1. This was particularly striking for TSH (ng/ml) at week 10 in Group 4 (126 ± 11) as compared with Groups 1 (4.36 ± 0.30), 2 (4.84 ± 0.80) and 3 (5.78 ± 0.80). Histologically, marked diffuse follicular hyperplasia of the thyroid was evident in Group 4 rats. Proliferating cell nuclear antigen labeling indices (%) were significantly higher (P < 0.05) in Groups 2 (4.8 ± 2.5) and 4 (13.2 ± 1.1) than in Group 1 (0.4 ± 0.5). Ultrastructurally, severe disorganization and disarrangement of mitochondria were apparent in thyroid follicular cells of Group 4. In the anterior pituitary, dilated rough surfaced endoplasmic reticulum and increased secretory granules were remarkable in this group. Our results thus strongly suggest that dietary defatted soybean synergistically stimulates the growth of rat thyroid with iodine deficiency, partly through a pituitary-dependent pathway.
Abbreviations: H-E, hematoxylin and eosin; PCNA, proliferating cell nuclear antigen; r-ER, rough surfaced endoplasmic reticulum; SG, secretory granules; T3, triiodothyronine; T4, thyroxine; TSH, thyroid-stimulating hormone; T4-UDP-GT, T4-uridine disphosphate glucuronosyltransferase.
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Introduction
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Epidemiologically, a high intake of soy-based products is inversely associated with the risk of breast (1), prostate (2) and gastric cancers in humans (3). Dietary supplementation of soybean or soy-based products in fact inhibited chemically induced mammary tumorigenesis in female rats (4) and hepatocarcinogenesis in male mice (5), although in a few studies no effects on tumorigenesis were observed in other animal models (6). Soybean is a major dietary source of isoflavones (7), which exert a number of biological effects associated with cancer chemoprevention. These include anti-estrogenic activity (8), inhibition of protein tyrosine kinase (9) and topoisomerase activities (10) and inhibition of angiogenesis (11). Genistein, a major soy isoflavone, was found to inhibit the development of chemically induced mammary tumors in female rats when administered s.c. (12) or in the diet (11) and the development of colon tumors in male rats when supplemented in the diet (13).
On the other hand, the fact of thyroid enlargement due to excessive soybean intake, especially in women and children, has been known for half a century. It has also been reported that this can be reversed by a substantial increase in dietary iodine (14). Soybean is one of the main protein sources for vegetarian Buddhists in East Asia, particularly in Japan. Nevertheless, endemic goiter is rare, possibly because of sufficient consumption of iodine-rich seaweed (15). Several investigators have reported induction of goiter in iodine-deficient rats maintained on a soybean diet (16). Kimura et al. (17) also reported induction of thyroid carcinomas in rats fed an iodine-deficient diet containing 40% defatted soybean. Iodine deficiency is a well-known factor predisposing to thyroid hyperplasias and subsequently thyroid tumors, the mechanism being interpreted as stimulation of follicular cells by increased serum thyroid-stimulating hormone (TSH) following a reduction in thyroid hormone synthesis (18). However, the goitrogenic activity of soybean protein in excess and the synergism with iodine deficiency have not been fully elucidated. Therefore, in the present study the effects of soybean protein feeding alone and in combination with iodine deficiency were histopathologically, biochemically and ultrastructurally investigated in rats.
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Materials and methods
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Animals and experimental design
Four-week-old specific pathogen-free F344/DuCrj female rats (Charles River Japan, Kanagawa, Japan) were kept in aluminum cages in an air-conditioned room (barrier system) at a temperature of 23 ± 2°C and a relative humidity of 60 ± 5% under a daily cycle of alternating 12 h periods of light and darkness. The animals were given ion exchanged water ad libitum. After a 1 week acclimatization period, rats were divided into four groups each consisting of 10 animals. The composition of the experimental diets is shown in Table I
. Casein was replaced with gluten or defatted soybean flour as alternative protein sources in the AIN-93G diet (Oriental Yeast Co., Tokyo, Japan) (19) in order to avoid possible contamination with iodine contained in casein sources. Group 1 received a modified AIN-93G diet in which the protein constituent was exchanged for 20% gluten, Group 2 iodine-deficient 20% gluten, Group 3 20% defatted soybean and Group 4 iodine-deficient 20% defatted soybean. At weeks 5 and 10, rats in Groups 14 were killed after being anesthetized with ether and blood was collected from the aorta (Figure 1
). At autopsy, major organs, including the thyroid and pituitary, were carefully examined macroscopically. After weighing, they were fixed in 10% phosphate-buffered formalin and sections stained with hematoxylin and eosin (H-E).
For the analysis of thyroid cell proliferation, sections were immunohistochemically stained (20) with anti-proliferating cell nuclear antigen (PCNA) antibody PC-10, obtained from Dakopatts (Glostrup, Denmark). The numbers of PCNA-positive nuclei (PCNA labeling indices) in 1000 cells from areas of proliferative follicular epithelial lesions were counted and expressed as percentages. For electron microscopical analysis, small portions of thyroid and pituitary tissues from two rats of each group were fixed in phosphate-buffered 2.5% glutaraldehyde for 2 h at 4°C, post-fixed in phosphate-buffered 1.0% osmium tetroxide for 1 h, dehydrated through a series of alcohols and embedded in Epon 812. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined under an electron microscope (JEM 1000CXS; JEOL, Tokyo). For hormone analysis, serum triiodothyronine (T3) and thyroxine (T4) were measured with a RIABEAD radio immunoassay kit (Dainabot, Tokyo) and serum TSH with a rat TSH kit (Amersham Life Science, USA).
Statistical analysis
Variance of data for lesion multiplicities, body weights and organ weights were estimated for homogeneity by the Bartlett's procedure. If the variance was homogeneous, the data were assessed by one way analysis of variance (ANOVA) techniques with the Student's t-test multiple comparison procedures. If not homogeneous, they were analyzed by the KruskalWallis test followed by the MannWhitney test.
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Results
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No animals died during the experimental period. Body weights were consistently higher in Groups 3 and 4 as compared with the Groups 1 and 2 values (Figure 2
) and those final body weights were significantly (P < 0.01) higher at weeks 5 and 10 (Table II
). As shown in Table II
, relative liver weights at weeks 5 and 10 were significantly (P < 0.05) higher in Groups 3 and 4 than in Group 1, except for the Group 4 value at week 10. Relative kidney weights at weeks 5 and 10 were significantly (P < 0.05) lower in Groups 3 and 4 than in Groups 1 and 2, except for the Group 3 value at week 10. Relative thyroid weights (mg/100 g body wt) at week 10 were significantly (P < 0.01) higher in Groups 2 (15.5 ± 1.3) and 4 (81.7 ± 8.6) than in Group 1 (8.4 ± 2.0), a remarkable increase being shown in Group 4 (Figure 3
). Relative pituitary weights (mg/100 g body wt) at week 10 were significantly (P < 0.05) higher in Groups 3 (9.1 ± 0.6) and 4 (9.7 ± 1.5) than in Group 1 (6.5 ± 1.5).
Table III
shows serum biochemical data. Serum T3 (ng/ml) levels at week 5 were significantly (P < 0.05) higher in Groups 2 (1.36 ± 0.18) and 4 (1.44 ± 0.22) than in Group 1 (1.10 ± 0.16), although this was no longer the case at week 10. Serum T4 (µg/dl) levels at weeks 5 and 10 were significantly (P < 0.05) lower in Groups 2 (1.56 ± 0.45 and 1.62 ± 0.73) and 4 (1.02 ± 0.04 and 1.20 ± 0.45) than in Group 1 (2.88 ± 0.63 and 3.60 ± 0.58). On the other hand, serum TSH (ng/ml) levels at weeks 5 and 10 were significantly (P < 0.01) higher in Groups 3 (5.00 ± 0.47 and 5.78 ± 0.80) and 4 (6.70 ± 0.80 and 126 ± 11) than in Group 1 (3.98 ± 0.41 and 4.36 ± 0.30). This was particularly striking for TSH at week 10 in Group 4 as compared with the other groups (Figure 3
).
Histopathologically, the most prominent lesions were observed in the thyroids of Group 4, in which diffuse follicular hyperplasia and a lack of colloid were evident (Figure 4D
). In the thyroids of Group 2, mild to moderate irregularity of hypertrophic follicular cells and decreased colloid were observed (Figure 4B
). However, in the thyroids of Group 3, the change was limited to mild irregularity of thyroid follicles (Figure 4C
). The livers and pituitaries of all groups did not demonstrate apparent histological changes. Table IV
summarizes data for cell proliferation. The PCNA labeling indices (%) in Groups 2 (4.8 ± 2.5) and 4 (13.2 ± 1.1) at week 10 were significantly higher (P < 0.05) than the Group 1 value (0.4 ± 0.5).

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Fig. 4. (A) Histology of the thyroid of a rat in Group 1. Colloid-rich uniform follicles are lined by a layer of flat epithelial cells. H-E. x360. (B) Histology of the thyroid of a rat in Group 2. Mild to moderate irregularity and hypertrophy of follicular cells and decrease in colloid are evident. H-E. x360. (C) Histology of the thyroid of a rat in Group 3. Note mild irregularity of follicles. H-E. x360. (D) Histology of the thyroid of a rat in Group 4. Marked diffuse follicular hyperplesia is apparent almost without colloid. H-E. x360.
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Electron microscopical findings are summarized in Table V
. The most prominent lesions were again observed in the thyroids of Group 4, characterized by dilated rough surfaced endoplasmic reticulum (r-ER), increased mitochondria with disintegrated and elongated cristae and an almost complete disappearance of colloid (Figure 5D
). In the thyroids of Group 2, dilated r-ER, increased mitochondria with disintegrated cristae and cytoplasmic blebbing were evident (Figure 5B
). However, in Group 3 (Figure 5C
), the appearance was as in Group 1 (Figure 5A
), except for occasional dilated r-ER. Prominent lesions observed in the pituitaries of Group 4 were dilated r-ER of mammosomatotrophs and increased prolactin secretory granules (SG) (Figure 6D
). In the pituitaries of Group 3, dilated r-ER of mammosomatotrophs were also evident (Figure 6C
). However, in the pituitaries of Group 2 (Figure 6B
), there were no ultrastructural changes as compared with Group 1 (Figure 6A
).


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Fig. 5. (A) Electron micrograph of normal thyroid follicular cells in Group 1. x3400. (B) Follicular cells with blebbing, cytoplasmic processes extending into the follicular lumen, in Group 2. Hypertrophic follicular cells contain dilated r-ER and colloid droplets. x5600. (C) Papillary projections of hyperplastic follicular cells extending into the follicular lumen lined by tall columnar cells in Group 3. x3400. (D) Thyroid follicular cells showing expanded dilated r-ER and vacuolated mitochondria with disrupted cristae. x8500.
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Fig. 6. (A) Ultrastructural morphology of the pituitary in a Group 1 rat showing a normal appearance. x4250. (B) Ultrastructural morphology of the pituitary of a Group 2 rat showing a normal looking appearance. x3400. (C) Ultrastructural morphology of the pituitary of Group 3, showing extended cisternae of r-ER and increased SG. x5100. (D) Ultrastructural morphology of the pituitary of Group 4, showing dilated cisternae of r-ER and SG moderately diminished in number. x2550.
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Discussion
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In the present study, soybean feeding and iodine deficiency synergistically induced thyroid follicular cell hyperplasia in rats. Iodine deficiency alone reduced serum T4 levels and resulted in a tendency for increased serum TSH levels consistently at weeks 5 and 10. In contrast, soybean diet by itself significantly increased serum TSH levels although it rather showed a tendency for increased serum T4. Regarding the influence on endocrine organs, the severity of weight changes and morphological effects in the thyroid was greater in the iodine deficiency alone group than in the soybean alone group. However, ultrastructural changes in the pituitary were only evident in the soybean-treated animals, even at week 10. In addition to marked induction of thyroid follicular hyperplasias, the combined soybean and iodine deficiency treatment caused a dramatic increase in serum TSH levels, thyroid weight and thyroid and pituitary ultrastructural changes as compared with the individual treatments alone.
Generally, TSH from the pituitary gland is considered to play an important role in enhancement of thyroid tumor development. Its production and release are controlled by thyroid hormone levels in the blood, via negative feedback of the pituitarythyroid axis (21,22). Many factors may contribute to a decrease in circulating thyroid hormone levels. For example, inhibition of iodide transport into the thyroid, iodide oxidation (22), iodine organification or coupling catalyzed by thyroid peroxidase (22) or hormone inactivation by hepatic microsomal enzymes such as T4-uridine diphosphate glucuronosyltransferase (T4-UDP-GT) can all cause reduced thyroid hormone levels (23). A decrease in circulating thyroid hormone levels caused by chronic iodine deficiency or severe thyroid dysfunction leads to increased release of TSH from the pituitary. Consequently, continuous stimulation of the thyroid occurs. In rodents, especially in rats, long-term administration of a low iodine diet can cause follicular hyperplasia (22). In the present experiment, PCNA labeling indices in the follicular epithelium and thyroid weight were significantly increased by an iodine-deficient diet alone.
Goitrogenic effects of a soybean diet in animals were reported in 1933 (16). They were initially explained by excessive loss of T4 in the feces (24). As in animals, an increase in the uptake of 131I by the thyroid was demonstrated in infants on a soy diet (25). The addition of iodine supplementation to commercial soy formulas in the 1960s has eliminated the prevalence of hypothyroidism in soy-fed infants (26). However, the underlying mechanism for the development of goiter with a soybean diet is not fully understood. In the present experiment, the soybean diet by itself significantly increased serum TSH levels but the tendency for an increase in serum T4 does not suggest the same negative feedback mechanism as with iodine deficiency. It is known that large amounts of phytoestrogenic isoflavones such as genistein and daidzein are contained in soybeans and estradiol-3-benzoate leads to an increase in the sensitivity of the TSH response to TSH-releasing hormone (27). Subcutaneous treatment with ethinylestradiol enhances N-methyl-N-nitrosourea-initiated thyroid carcinogenesis in castrated F344 rats fed an iodine-deficient diet (28). Thus it has been suggested that ethinylestradiol acts directly on thyroid tissue via estrogen receptors and partly through hypersecretion of TSH in the pituitary gland (28). However, we recently found that a high isoflavone diet does not induce goiter under iodine deficiency conditions (unpublished data). The fact of increased SG and dilated r-ER without a decrease in T4 level in the soybean-fed groups indicates that an unknown compound in soybean may directly stimulate TSH release from the anterior pituitary or through the hypothalamic pathway. It is known that isoflavones inhibit thyroid peroxidase, which catalyzes a reaction essential for thyroid hormone synthesis (29). In addition, it cannot be ruled out that soybean feeding could stimulate hepatic microsomal enzymes such as T4-UDP-GT, which inactivate thyroid hormones (23), as suggested by the increases in liver weight. Thus, a soybean diet may induce thyroid proliferation by several different mechanisms in the rat, with possible contributions of the anterior pituitary and/or hypothalamic pathways.
In order to avoid possible contamination by iodine contained in casein, as a protein source casein in the diet was replaced by gluten (30). Body weight differences between soybean-fed and gluten-fed rats could be caused by their nutritional efficacy (31). Although effects of gluten on the the hypothalamuspituitary axis in the peculiar disease coeliac sprue are known (32), there are no reports regarding an influence of gluten on the pituitarythyroid axis under normal conditions except for one reporting an effect on serum hormones of lysine deficiency due to gluten feeding (33). In addition, the average pituitary weight (6.5 mg%) of rats fed gluten was comparable with the background data (6.46.7 mg%) for 5- to 20-week-old female F344/DuCrj rats supplied to some institutes from Charles River Japan (unpublished data). Taken together, our pathological observations strongly support the idea that gluten does not damage tissues, including the pituitary, nor contain factors inhibiting the pituitary axis. The histological and ultrastructural features of the pituitary were intact in rats fed gluten and ultrastructural changes of the pituitary were only noted in the soybean-fed animals.
It is still unknown why combined soybean excess and iodine deficiency cause a dramatic increase in serum TSH levels, in thyroid weight and histopathological changes at week 10. It is speculated that dietary defatted soybean may primarily stimulate serum TSH levels by stimulating release from the pituitary of rats under iodine deficiency conditions, while thyroid gland growth is synergistically enhanced by further stimulation of TSH production due to a decrease in T4. Further studies are required to test this hypothesis.
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Acknowledgments
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This work was supported in part by a Grant-in-Aid (H11-Seikatsu-018) for Research on Environmental Health from the Ministry of Health and Welfare of Japan.
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Notes
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2 To whom correspondence should be addressed. Email: nishikaw{at}nihs.go.jp 
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Received June 24, 1999;
revised October 19, 1999;
accepted November 25, 1999.