Pronounced Synergistic Promotion of N-Bis(2-hydroxypropyl)Nitrosamine-Initiated Thyroid Tumorigenesis in Rats Treated with Excess Soybean and Iodine-Deficient Diets

Akiyoshi Nishikawa*,1, Takako Ikeda*,{dagger}, Hwa-Young Son*, Kazushi Okazaki*, Takayoshi Imazawa*, Takashi Umemura*, Shuichi Kimura{dagger} and Masao Hirose*

* Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501, and {dagger} Showa Women's University, Tokyo 154-8533, Japan

1 To whom correspondence should be addressed at Division of Pathology, National Institute of Health Sciences, 1–18–1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. E-mail: nishikaw{at}nihs.go.jp.

Received March 21, 2005; accepted May 11, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have reported that excess soybean treatment and iodine deficiency synergistically interact, resulting in remarkable induction of thyroid hyperplasias in rats (Carcinogenesis [2000]. 21, 707–713,). In the present study, modifying effects of excess soybean and iodine-deficient diets were investigated in the post-initiation phase of N-bis(2-hydroxypropyl)nitrosamine [DHPN]-initiated thyroid tumorigenesis in rats. AIN-93G in which casein was replaced with gluten was used as a basal diet to avoid possible iodine contamination. In Experiment 1, F-344 rats of both sexes were sc injected with DHPN at a dose of 2800 mg/kg body weight and then fed a diet containing 0%, 0.8%, 4%, or 20% defatted soybean for 12 weeks, with proportional replacement of gluten by soybean flour. Although no thyroid proliferative lesions were found in any group, the absolute thyroid weights were significantly (p < 0.01) elevated with the 20% soybean treatment. In Experiment 2, after similar sc injection of DHPN, rats were fed a basal diet or a diet containing 20% soybean under iodine normal or deficient conditions for 12 weeks. Soybean feeding to both sexes under iodine deficient but not normal conditions dramatically enhanced the development of thyroid follicular adenomas (p < 0.01) and adenocarcinomas (p < 0.05), in good agreement with decrease in thyroxine and increase in thyroid-stimulating hormone. Thus co-exposure to excess soybean and iodine deficiency results in synergistic promotion of DHPN-initiated thyroid tumorigenesis in rats, of which mechanisms appear to primarily involve effects on serum hormone levels.

Key Words: soybean; iodine deficiency; thyroid tumor-promotion; synergism.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Development of thyroid goiter due to excessive soybean intake, especially in children and women, has been known from epidemiological studies for almost half a century (Hydovitz, 1960Go; Pinchera et al., 1965Go; Shepard et al., 1960Go). Addition of iodine supplements to commercial soy formulas in the 1960s eliminated the prevalence of hypothyroidism in soy-fed infants (Block et al., 1961Go), suggesting an interaction between the two agents, and experimentally, induction of goiter has been found in iodine-deficient rats maintained on a soybean diet (Kimura et al., 1976Go; McCarrison, 1933Go). In fact, iodine deficiency is a well-established 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 depression of serum thyroid hormone levels (Susan et al., 1996Go). The physiological changes underlying the goitrogenic activity of soybean protein in excess and the synergism with iodine deficiency have yet to be fully elucidated, although it has been suggested that dietary soybean may primarily stimulate serum TSH release from the rat pituitary (Ikeda et al., 2000Go).

There are several different mechanisms underlying chemical promotion of thyroid tumor development in rodents. For example, sulfadimethoxine (SDM) inhibits thyroid hormone synthesis by inhibiting thyroid peroxidase (TPO) (Hill et al., 1989Go), and phenobarbital (PB) induces hepatic microsomal enzymes such as thyroxine (T4)-uridine disphosphate glucuronosyl transferase (T4-UDP-GT) responsible for T4 clearance in the liver (Hill et al., 1989Go; McClain et al., 1989Go). In both cases, subsequent increase in serum TSH stimulates thyroid follicular cell proliferation. We have reported that a combination of excess soybean and iodine deficiency synergistically results in remarkable induction of thyroid proliferative lesions in rats (Ikeda et al., 2000Go, 2001Go). In the present study, the modifying effects of multiple dose levels of soybean were first investigated in a two-stage thyroid carcinogenesis model in rats. Combined effects of soybean and iodine deficiency were then examined with reference to induction of thyroid tumors in rats.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals, diets, and chemicals.
The protocols for this study were approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences, Japan. A total of 160 5-week-old specific-pathogen-free F-344/DuCrj rats of both sexes purchased from Charles River Japan Inc. (Kanagawa, Japan) were maintained 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, they were used for Experiments 1 and 2. Casein was replaced with gluten as an alternative protein source in the basal diet AIN-93G (Oriental Yeast Co. Ltd., Tokyo, Japan) in order to avoid possible contamination with iodine contained in casein sources (Reeves et al., 1993Go). Defatted soybean containing 0.2% soy isoflavone was supplied from Oriental Yeast Co. Ltd. and N-bis(2-hydroxypropyl)nitrosamine [DHPN], a thyroid tumor initiator, was purchased from Nacalai Tesque Inc. (Kyoto, Japan).

Experimental designs.
In Experiment 1, rats were divided into four groups, each consisting of 10 males and 10 females. They were subcutaneously injected with DHPN at a dose of 2800 mg/kg body weight and then fed a diet containing 0.8%, 4%, or 20% defatted soybean or the basal diet alone for 12 weeks, the soybean flour proportionally replacing gluten. In Experiment 2, rats were also divided into four groups, each consisting of 10 males and 10 females. They were subcutaneously injected with DHPN at a dose of 2800 mg/kg body weight and then fed an iodine-normal or iodine-deficient diet supplemented with or without 20% soybean for 12 weeks.

Autopsy and measurements.
At week 12, all rats were killed under ether anesthesia after blood was collected from the aorta. At autopsy, the thyroid and pituitary glands were carefully examined macroscopically. After weighing, the thyroids were fixed in 10% phosphate-buffered formalin, and routinely processed for production of sections stained with hematoxylin and eosin (H-E). Thyroid proliferative lesions were histopathologically classified according to the criteria of Botts et al. (1991)Go. Serum triiodothyronine (T3) and T4 were measured with radioimmunoassay Riabead kits (Dainabot, Tokyo), and serum TSH was measured with a rat TSH kit (Amersham Life Science, Buckinghamshire, UK).

Statistical analysis.
Variance in data for body and organ weights, serum hormones, and tumor multiplicity were analyzed by Tukey's test for multiple comparisons. Tumor incidence data were evaluated by Fisher's exact probability test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of Soybean Alone Diets
No animals died during Experiment 1. As shown in Table 1, the final body weights were significantly (p < 0.01) increased in the 4% and 20% soybean-treated groups for males or females as compared to each basal diet group value, although food consumptions were comparable between groups (data not shown). The absolute and relative thyroid weights (thyroid to body weight ratios) were comparable between groups for males or females, except for the male 20% soybean-treated group, which had significantly (p < 0.01) increased absolute values as compared to the corresponding basal diet rats. The absolute and relative pituitary weights were comparable between groups for males and females, although the absolute weights showed a tendency for increase in the 20% soybean-treated males and 4% and 20% soybean-treated females. As shown in Table 2, the average serum T3, T4, and TSH levels were comparable between groups for males and females, with only a tendency for increase in TSH in the 4% and 20% males and the 20% females. Histopathologically, no thyroid neoplastic lesions were noted (data not shown).


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TABLE 1 Body, Thyroid, and Pituitary Weights in Rats Receiving 0.8%, 4%, or 20% Soybean (SB) Diet Following N-bis(2-hydroxypropyl)Nitrosamine (DHPN) (Experiment 1)

 

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TABLE 2 Data for Serum Ts, T4 and TSH levels in Rats Receiving 0.8%, 4%, or 20% Soybean (SB) Diet Following DHPN (Exp. 1)

 
Effects of Soybean Diet and Iodine-Deficient Diet on Organ Weights
A few animals died of pituitary adenomas or from other spontaneous causes during Experiment 2. As shown in Table 3, the final body weights in both sexes were significantly (p < 0.05 or 0.01) increased (or showed a tendency for increase) in the soybean-treated groups regardless of iodine deficiency as compared to each basal diet group value, although food consumption was comparable between groups (data not shown). The absolute thyroid weights for males were significantly (p < 0.05) higher, and those for females showed a tendency toward increase in the groups receiving the iodine-deficient diet with or without 20% soybean flour, as compared to the respective basal diet group values. Iodine deficiency also increased the relative thyroid weights 4.2-fold (p < 0.01) in males and 2.7-fold (p < 0.05) in females. The two exposures in combination further increased the relative thyroid weights 3.6 fold in males and 4.1 fold (p < 0.01) in females, as compared to the iodine deficiency alone. The absolute pituitary weights were also significantly (p < 0.01) increased in males by the combined treatment, with a similar tendency in females. The relative pituitary weights were comparable between groups for both sexes.


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TABLE 3 Body, Thyroid, and Pituitary Weights in Rats Receiving Iodine-Deficient (ID) Diet and/or 20% Soybean (SB) Diet Following DHPN (Experiment 2)

 
Effects of Soybean Diet and Iodine-Deficient Diet on Hormone Levels
As shown in Table 4, serum T4 levels were significantly (p < 0.01) decreased by the iodine-deficient diet in both sexes, and they were further reduced by the combination of the iodine-deficient diet with soybean treatment. In contrast, serum TSH levels showed a tendency for increase with the iodine-deficient diet in both sexes, and further elevated (p < 0.01) by the combination. The diet of soybean alone did not affect the serum T4 or TSH levels in either sex, consistent with Experiment 1. Serum T3 levels were slightly but significantly (p < 0.05) increased by the iodine-deficient diet in males, but they were decreased (p < 0.01) by the combination with soybean. Serum T3 levels in females were comparable in all groups.


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TABLE 4 Data for Serum T3, T4 and TSH in Rats Receiving Iodine-Deficient (ID) Diet and/or 20% Soybean (SB) Diet Following DHPN (Exp. 2)

 
Effects of Soybean Diet and Iodine-Deficient Diet on Thyroid Tumorigenesis
As shown in Table 5, the histopathologically assessed incidences of thyroid follicular adenocarcinomas were significantly (p < 0.05) increased in the combined exposure groups of both sexes. The incidences of thyroid follicular adenomas were also significantly (p < 0.01) increased in the iodine-deficient males, with or without soybean, and in the female combined-treatment group, as compared to the respective basal diet alone group values. The multiplicities of thyroid adenomas were also synergistically (p < 0.01) increased in the combined treatment groups of both sexes.


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TABLE 5 Incidence and Multiplicity Data for Thyroid Tumors in Rats Receiving Iodine-Deficient (ID) Diet and/or 20% Soybean (SB) Diet Following DHPN (Experiment 2)

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously we reported a high intake (20%) of soybean to dramatically and synergistically increase the development of thyroid follicular hyperplasia in rats in combination with iodine deficiency (Ikeda et al., 2000Go). In the present study, after initiation with DHPN, soybean feeding at a dose level of 20% to both sexes under iodine-deficient conditions dramatically enhanced the development of thyroid follicular tumors as compared to DHPN alone, although soybean feeding at 0.8–20% did not clearly modulate DHPN-initiated thyroid tumorigenesis under normal iodine conditions. These findings were in good alignment with data for serum hormone levels, such as decreased T4 and increased TSH. Modulation of sex hormones would not be involved in the synergistic promotion, because no apparent sex differences were found in the effects of excess soybean diets and/or iodine-deficient diets on thyroid-related hormone levels and thyroid tumorigenesis.

There are two basic mechanisms whereby chemicals induce thyroid gland tumors in rodents. One, classified as genotoxic, involves direct carcinogenic effects at the DNA level in follicular cells. The other, termed non-genotoxic, involves the so-called negative feedback system of the thyroid–pituitary–hypothalamus axis, in which decreased circulating T3 or T4 levels due to disruption of thyroid function or accelerated turnover lead to increased TSH and its prolonged stimulation of follicular epithelium (Capen, 1997Go; Hill et al., 1989Go; McClain, 1992Go). A number of non-genotoxic factors may contribute to decreases in circulating thyroid hormone levels, through inhibition of iodine transport into the thyroid, iodine oxidation or organification defects resulting from inhibition of TPO, inhibition of thyroid hormone secretion, or increased hormone inactivation by hepatic microsomal enzymes such as T4-UDP-GT (Capen, 1997Go; Hill et al., 1989Go). Thus, it has been concluded that thyroid tumor promotion is consistently mediated by chronic increase of TSH secretion from the pituitary (Ikeda et al., 2001Go).

Iodine-deficient diets produce enlargement of the thyroid, an increase in monoiodotyrosine/diiodotyrosine, and a progressive increase in the T3/T4 molar ratio (Fukuda et al., 1975Go; Martin et al., 1985Go). Sustained decrease in intracellular iodine concentration (Wolff, 1969Go) resulting from dietary iodine deficiency inhibits thyroglobulin hydrolysis (Bagchi et al., 1985Go), tyrosine iodination by thyroid peroxidase (Nunez and Pommier, 1982Go), or accumulation of cAMP in the thyroid (Van Sande et al., 1975Go). In our previous study (Ikeda et al., 2000Go), iodine deficiency alone reduced serum T4 levels and inversely increased TSH levels, whereas excess soybean increased both serum T4 and TSH, suggesting primary stimulation of TSH release from the pituitary of rats under certain conditions. In the present study using DHPN as an initiator, however, 20% soybean feeding only showed a tendency to increase serum TSH levels, with no effects on T4. One explanation might be that the pretreatment with DHPN affected hormone metabolism (Onodera et al., 1994Go). In our previous study, either SDM or PB treatment reduced both serum T3 and T4 levels and increased TSH levels, but co-treatment with soybean rather attenuated changes in these parameters, suggesting that excess soybean may induce thyroid proliferation by acting on the pituitary and/or hypothalamus (Ikeda et al., 2001Go). Our previous and present findings regarding specific interactions with iodine deficiency are also suggestive of different modes of action of the various thyroid tumor-promoters (Ikeda et al., 2001Go).

It has been reported that soy isoflavones like genistein inactivate rat as well as human thyroid peroxidase in vitro, but measures of thyroid function in vivo such as serum levels of T3, T4, and TSH; thyroid weights; and thyroid histopathology may all be normal (Doerge and Chang, 2002Go; Doerge and Sheehan, 2002Go). This is in line with our previous data demonstrating that both genistein and soy isoflavone lack modulating effects on thyroid tumorigenesis in rats initiated with DHPN (Son et al., 2000aGo, 2000bGo, 2001Go). A recent epidemiological study conducted in the San Francisco Bay Area suggested that consumption of traditional and non-traditional soy-based foods and alfalfa sprouts may actually be associated with a reduced risk of thyroid cancer (Horn-Ross et al., 2002Go). It has also been suggested that in post-menopausal women soy protein may have only minor effects on thyroid hormone values that are unlikely to be clinically important (Persky et al., 2002Go). Therefore, additional factors such as iodine deficiency could be necessary for soy to cause overt thyroid effects (Doerge and Chang, 2002Go; Doerge and Sheehan, 2002Go). Although safety assessment of soy products may not be required, the possibility that widely consumed soy products may cause harm in the human population via goitrogenic activity might be a safety concern, especially in iodine-deficient areas.

In conclusion, our results clearly indicate that co-treatment with excess soybean and a diet deficient in iodine synergistically promotes DHPN-induced thyroid tumorigenesis in rats, with influence on hormone levels as a likely mechanism.


    ACKNOWLEDGMENTS
 
This work was supported in part by a grant (H11-Seikatsu-018) for Research on Environmental Health from the Ministry of Health, Labour and Welfare of Japan. Conflict of interest: none declared.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bagchi, N., Shivers, B., and Brown, T. R. (1985). Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine. Acta Endocrinol. 108, 511–517.[ISI][Medline]

Block, R. J. R., Mandal, H., Howard, H. W., Bauer, C. D., and Anderson, D. (1961). The curative action of iodine on soybean goiter and the changes in the distribution of iodo-amino acids in the serum and thyroid gland digests. Arch. Biochem. Biophys. 93, 15–24.[CrossRef][ISI]

Botts, S., Jokinen, M. P., Isaack, K. R., Meuten, D. J., and Tanaka, N. (1991). Proliferative lesions of the thyroid and parathyroid glands. E-3. Guide for Toxicologic Pathology, pp. 1–12, STP/ARP/AFIP, Washington DC.

Capen, C. C. (1997). Mechanistic data and risk assessment of selected toxic end points of the thyroid gland. Toxicol. Pathol. 25, 39–48.[ISI][Medline]

Doerge, D. R., and Chang, H. C. (2002). Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 777, 269–279.[ISI][Medline]

Doerge, D. R., and Sheehan, D. M. (2002). Goitrogenic and estrogenic activity of soy isoflavones. Environ. Health Perspect. 110(Suppl 3), 349–353.

Fukuda, H., Yasuda, M., Greer, M. A., Kutas, M., and Greer, S. E. (1975). Changes in plasma thyroxine, triiodothyronine, and TSH during adaptation to iodine deficiency in the rat. Endocrinology 97, 307–314.[Abstract]

Hill, R. N., Erdreich, L. S., Paynter, O. E., Roberts, P. A., Rosenthal, S. L., and Wilkinson, C. F. (1989). Thyroid follicular cell carcinogenesis: a review. Fundam. Appl. Toxicol., 12, 629–697.[CrossRef][ISI][Medline]

Horn-Ross, P. L., Hoggatt, K. J., and Lee, M. M. (2002). Phytoestrogens and thyroid cancer risk: The San Francisco Bay Area thyroid cancer study. Cancer Epidemiol. Biomarkers Prev. 11, 43–49.[Abstract/Free Full Text]

Hydovitz, J. D. (1960) Occurrence of goiter in an infant on a soy diet. N. Engl. J. Med. 262, 351–353.[ISI][Medline]

Ikeda, T., Nishikawa, A., Imazawa, T., Kimura, S., and Hirose, M. (2000). Dramatic synergism between excess soybean intake and iodine deficiency on the development of rat thyroid hyperplasia. Carcinogenesis 21, 707–713.[Abstract/Free Full Text]

Ikeda, T., Nishikawa, A., Son, H.-Y., Nakamura, H., Miyauchi, M., Imazawa, T., Kimura, S., and Hirose, M. (2001). Synergistic effects of high-dose soybean intake with iodine deficiency, but not sulfadimethoxine or phenobarbital, on rat thyroid proliferation. Jpn. J. Cancer Res. 92, 390–395.[ISI][Medline]

Kimura, S., Suwa, J., Ito, B., and Sato, H. (1976). Development of malignant goiter by defatted soybean with iodine-free diet in rats. Gann 67, 763–765.[ISI][Medline]

Martin, D. W., Jr., Mayes, P. A., Rodwell, V. W., and Granner, D. K. (1985). "20th Harper's Review of Biochemistry," Lange Medical Publications, Los Altos, CA.

McCarrison, R. (1933). The goitrogenic action of soybean and groundnut. Ind. J. Med. Res. 21, 179–181.

McClain, R. M., Levin, A. A., Posch, R. C., and Downing, J. M. (1989). The effect of phenobarbital on the metabolism and excretion of thyroxine in rats. Toxicol. Appl. Phamacol. 99, 216–228.[CrossRef][ISI][Medline]

McClain, R. M. (1992). Thyroid gland neoplasia: Genotoxic mechanisms. Toxicol. Lett. 64/65, 397–408.[CrossRef]

Nunez, J., and Pommier, J. (1982). Formation of thyroid hormones. Vitam. Horm. 39, 175–229.[ISI][Medline]

Onodera, H., Mitsumori, K., Takahashi, M., Shimo. T., Yasuhara, K., Kitaura, K., Takahashi, M., and Hayashi, Y. (1994). Thyroid proliferative lesions induced by anti-thyroid drugs in rats are not always accompanied by sustained increases in serum TSH. J. Toxicol. Sci. 19, 227–234.[Medline]

Persky, V. W., Turyk, M. E., Wang, L., Freels, S., Chatterton, R., Jr., Barnes, S., Erdman, J., Jr., Sepkovic, D. W., Bradlow, H. L., and Potter, S. (2002). Effect of soy protein on endogenous hormones in postmenopausal women. Am. J. Clin. Nutr. 75, 145–153.[Abstract/Free Full Text]

Pinchera, A., MacGillivray, M. H., Crawford, J. D., and Freeman, A. G. (1965) Thyroid refractoriness in an athyreotic cretin fed soybean formula. N. Engl. J. Med. 265, 83–87.

Reeves, P. G., Nielsen, F. G., and Fahey, G. C. (1993). AIN-93 purified diets for laboratory rodents: Final report of the American Institute of Nutrition Ad Hoc Writing Committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951.[ISI][Medline]

Shepard, T. H., Pyne, G. E., Kirschvink, J. F., and McLean, C. M. (1960). Soybean goiter. N. Engl. J. Med. 262, 1099–1103.[ISI]

Son, H. Y., Nishikawa, A., Ikeda, T., Furukawa, F., and Hirose, M. (2000b). Lack of modification by environmental estrogenic compounds of thyroid carcinogenesis in ovariectomized rats pretreated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Jpn. J. Cancer Res. 91, 966–972.[ISI][Medline]

Son, H. Y., Nishikawa, A., Ikeda, T., Nakamura, H., Miyauchi, M., Imazawa, T., Furukawa, F., and Hirose, M. (2000a). Lack of modifying effects of environmental estrogenic compounds on the development of thyroid proliferative lesions in male rats pretreated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Jpn. J. Cancer Res. 91, 899–905.[ISI][Medline]

Son, H. Y., Nishikawa, A., Ikeda, T., Imazawa, T., Kimura, S., and Hirose, M. (2001). Lack of effect of soy isoflavone on thyroid hyperplasia in rats receiving an iodine-deficient diet. Jpn. J. Cancer Res. 92, 103–108.[ISI][Medline]

Susan, M. P., Joy, P., and Berber, J. (1996). Soy protein concentrate and isolated soy protein similarly lower blood serum cholesterol but differently affect thyroid hormones in hamsters. J. Nutr. 126, 2007–2011.[ISI][Medline]

Van Sande, J., Grenier, G., Willems, C., and Dumont, J. E. (1975). Inhibition by iodide of the activation of the thyroid cyclic 3',5'-AMP system. Endocrinology 96, 781–786.[Abstract]

Wolff, J. (1969). Iodide goiter and the pharmacologic effects of excess iodide. Am. J. Med. 47, 101–124.[CrossRef][ISI][Medline]





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