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SECTION OF BIOLOGICAL AND MEDICAL SCIENCES

IODINE METABOLISM AND BREAST CANCER” t

Bernard A. Eskin, M.D.

Department of Obstetrics and Gynecology The Medical College of Pennsylvania Philadelphia, Pa.

INTRODUCTION

Statistics favor an increased rate of breast cancer morbidity in regions of known endemic goiter. Demographic surveys of Japan and Iceland show low incidences of endemic nontoxic goiter and breast cancer, while Mexico and Thailand show high incidences of goiter and breast cancer.15 In addition, increased breast cancer in specific endemic-goiter regions in Poland, Switzerland, Australia, and the Soviet Union have been described in publications and through personal communications.~7~ 44

Iodine deficiency is not an uncommon condition. A summation by the World Health Organization shows that surprisingly large areas, even in the United States, have definite goitrous tendencies.26 When these areas are superimposed on a recent statistical map showing breast cancer mortality (by states) in the United States the similarity of high mortality regions to endemic-goiter areas is striking (FIGURE 1 ) . The only states where there is significant cancer without endemic goiter are Florida, South Dakota, and Oklahoma. However, the transient population found in Florida is derived from endemic-goiter states. Three states have endemic goiter but are low in breast cancer, and these are states with lowest population density and, hence, some problems in reliable statistical re- porting. The highest breast mortality rates in the United States are found in states bordering the Great Lakes, an area often called the “goiter belt.”

Endemic goiter has been reported in countries with presumably adequate dietary iodine and in the presence of iodine overload. Significant iodine deficiency might also be found in populations that do not exhibit any increase in goiter. In a study of an isolated island that was completely iodine-deficient, one region showed more significant goiter formation and abnormal iodine metabolic values than the other part.B Iodine deficiency, thus, appears to be a permissive condition for goiter development, although through adaptations this physical change may not express itself.

Thyroid hypofunction as a cause of mammary cancer is controversial; many references, divided equally between those who consider it one and those who do not, can be cited. In an age-related clinical population, less breast cancer occurred in a thyrotoxic group of women than in a myxedematous g r o ~ p . 3 ~

*This paper was presented at a meeting of the Section on March 9, 1970. tsupported, in part, by American Cancer Society grant T-410 and PHS Research

Grant CA 08615 from the National Cancer Institute. 911

912 Transactions New York Academy of Sciences

Clinical evidence exists that precancerous conditions, such as cystic nodular breasts, occurring in patients treated for clinical and subclinical hypothyroidism, show improvement after thyroid replacement therapy.6 Many workers feel that the techniques employed for evaluation of thyroid function in breast cancer studies are inadequate.38 A retrospective case-control study failed to clarify whether thyroid hypofunction had any specific relationship to breast cancer.36

These conflicting results may have arisen from a failure to consider thyroid kinetics influenced by local “trapping” of labeled hormone in hepatic and tumor tissues. Significant iodine uptakes are obtained in such extrathyroidal sites as the stomach, salivary glands, liver, tumor tissue, and ovaries.” In lactating breasts, organic iodination and iodine transport have been established, and evidence of these activities has been reported in nonlactating breasts.16

FIGURE 1. Breast cancer mortality (BCM) and endemic goiter (EG) in the United States.

Using a carefully controlled rat model, we are attempting to determine how iodine deficiency is related qualitatively and quantitatively to histological changes within breast tissues. Reproducible dysplasia and neoplasia are obtained in iodine deficiency, and a reversal of these breast lesions occurs with iodine replacement. Our results imply that iodine deficiency may be a factor in these thyroid-breast relationships.

The aim of this report is to present a series of basic and clinical experiments that: ( I ) define the effects of iodine lack on breast tissues and breast metabolism, (2) relate quantitative breast iodine changes to the occurrence of breast dysplasia or carcinoma, (3) localize the control of the iodine-deficiency- induced breast tissue changes, ( 4 ) describe histological and metabolic reversal of these lesions in the breast by replacement therapy, and ( 5 ) present results obtained in preliminary clinical research.

Eskin: Iodine Metabolism and Breast Cancer 913

DYSPLASIA AND NEOPLASM

Rats were subjected to iodine deficiency and hypothyroid states in order to determine breast tissue responses. These changes were studied with sex steroid stimulation and in the presence of a carcinogen.

Histological Changes in the Female Breast

During unrelated experiments, breasts of chronically iodine-deficient female rats treated with sex steroids were observed to be considerably larger than their normally fed, sex steroid-injected littermate controls.7 Since no report directly pertaining to this observation was found in the literature, we initiated a systematic investigation of the influence of iodine deficiency on the resting and sex hormone-treated breasts of female and male rats. The changes seen were compared with the breast tissue responses found in normally fed euthyroid and normally fed hypothyroid (propylthiouracil( PTU) -treated) rats.*

Method. One hundred and fifty-six Sprague-Dawley albino virgin rats initially weighing 180-228 g. were divided into four major categories: iodine- deficient, normally fed PTU-treated, iodine-deficient PTU-treated, and age- paired normally fed controls. Rats were made chronically iodine-deficient on the Remington diet, and deionized drinking water was given ad libitum for 12 weeks. On this regimen, rats grew and gained weight at the same rate as did their normally fed controls. Hypothyroidism was produced by the daily sub- cutaneous injection of 2 mg of PTU suspended in 0.5 cc of deionized water, with an equal quantity of gum acacia added as a dispersing agent over a similar period of time.

Each of the four major groups of rats was further separated into groups receiving sesame oil control, estradiol benzoate, or testosterone propionate. Estradiol benzoate (50 pg) and testosterone propionate (2.5 mg) were injected subcutaneously daily in 0.1 cc of sesame oil for a 20-day period. All animals received the same volume of water and sesame oil throughout.

At the end of the 20-day steroid period, the rats were injected with ten p c of carrier-free sodium iodide-1251 intraperitoneally 60 minutes before being killed with ether. The two inguinal and two inferior abdominal mammary glands of each rat were bilaterally dissected free and fixed in 10% formaldehyde solution. In addition, the thyroids were dissected, weighed, and fixed in formaldehyde, and venous blood was removed from the inferior vena cava. Counting was carried out in a well-type scintillation counter precalibrated for I25I.

Slides of the breast tissue and thyroid gland were prepared and stained with hematoxylin and eosin (H & E) . Resulting breast sections stained with hematoxylin and eosin were examined under high power ( x 100) by use of a reticle with concentric circles 0.5 mm to 12.0 mm. Mean acinus, duct, and lobule sizes were measured for at least six lobules on each slide for each rat. The average value for each group of experimental and control animals was finally expressed as a multiple of the result for the breast of the normal untreated female rat.

Morphological Results. Photomicrographs show the breast tissue of rats in the control (FIGURE 2) and treated (FIGURES 3-5) groups. The acinar, duct, and lobule areas of the breast tissue sections from the experimental animals were


. . c , , .! ,': J

FIGURE 2. Effect of hormone administration on breast tissue of a normally fed female rat: (A) no hormones; (B) estrogen; (C) testosterone. Stain: hematoxylin and eosin. x 15 (orig. mag. x 24).

measured. It can be seen that the changes described for the single-field photomicrographs parallel the objective results reproduced in TABLE 1. Estrogen and testosterone induce mild proliferation and dilatation of ducts and acini (FIGURE 2), testosterone having a slightly more pronounced effect. When these hormones are administered to iodine-deficient rats (FIGURE 3 ) , there is a distinct en- hancement of the estrogenic and androgenic change of both ducts and acinar elements, with marked cellular and cystic hyperplasia.

Rats treated with PTU show mild glandular hyperplasia (FIGURE 4 ) . When

TABLE 1 AVERAGE SIZES OF ACINI, DUCTS, AND LOBULES I N EXPERIMENTAL GROUPS*

_ _ _ _ . ~ _ _ ~ __ .~

Experimental Conditions Size in Females Size in Males Diet PTU Steroid Acini Ducts Lobules Acini Ducts Lobules

Normal - None 1.0 1.0 1.0 1.0 1.0 1 .O Normal - Estrogen 3.0 3.0 4.0 1.5 6.0 2.0 Normal - Testosterone 4.0 2.0 5.0 1.5 1.5 5.3

Ideficient - None 1.0 0.5 1.0 1.0 0.5 2.5 Ideficient - Estrogen 2.0 3.5 6.0 3.0 11.0 3.7 [deficient - Testosterone 12.0 1.0 8.0 2.5 2.5 8.0

Normal + None 4.0 3.5 6.7 1.5 1.0 1.3 Normal + Estrogen 4.0 5.0 9.0 2.0 7.5 4.3 Normal + Testosterone 12.0 2.0 7.0 2.5 1.5 5.3

Ideficient + None 6.0 3.0 9.0 2.0 1.5 4.7

__ _______ ~

- __________________

1 deficient + Estrogen 8.0 6.0 11.0 1.0 5.5 2.7 [deficient + Testosterone 24.0 7.0 34.0 3.5 1.5 11.0 _ _ _ _ _ _ _ ~ ___ ____ ~- - _ _ _ _____

*Breast tissue was evaluated under high-power ( x 100) by use of a reticle with concentric measured circles. Results expressed as multiples of breast findings with normal diet as unity. There were seven females and six males per group.


FIGURE 3. Effect of hormone administration on breast tissue of a chronically iodine- deficient female rat: (A) no hormones; (B) estrogen; (C) testosterone. Stain: hematoxylin and eosin. x 15 (orig. mag. x 24).

PTU-treated normally fed rats receive estrogen, the breasts show marked ductal dilatation and papillary hyperplasia of the lining cells, while moderate cystic dilatation and secretion occur in the acini. The breasts of PTU-treated rats given testosterone show mild ductal growth with coalescent cystic acini filled with secretion. When PTU is administered to iodine-deficient rats (FIGURE 5) , ductal and acinar hyperplasia results. Marked ductal dilatation and papillary hyperplasia of the lining cells occur when estrogen is administered to PTU- treated iodine-deficient rats; their glands show striking dilatation and are filled

FIGURE 4. Effect of hormone administration on breast tissue of propylthiouracil-treated female rat: (A) no hormones; (B) estrogen; (C) testosterone. Stain: hematoxylin and eosin. x 16 (orig. mag. x 24).


FIGURE 5. Effect of hormone administration on breast tissue of chronically iodine- deficient, propylthiouracil-treated female rat: (A) no hormones; (B) estrogen; (C) testosterone. Stain: hematoxylin and eosin. x 16 (orig. mag. x 24).

with secretions. In the presence of testosterone, the PTU-treated iodine-deficient animals display such marked ductal and acinar proliferation and dilatation that macrocysts are formed.

Histological Changes in the Male Breast

Method. One hundred and twenty Sprague-Dawley albino male rats initially weighing 124-169 g. were divided and treated exactly as in the related female experiment.

Morphological Results. Photomicrographs of the breasts of male rats in the control (FIGURE 6) and treated groups (FIGURES 7-9) are reproduced. As was done in previous studies with the female breast tissue sections, acinar, duct, and lobule areas from the experimental animal$ were measured. As can be seen in TABLE 1 , the breast changes described for the single-field microphotographs parallel the objective results set down by reticle evaluation. The control group ( F I G U R E 6) showed the characteristic features of normal male rat breast. Normal male rats show an abundance of acinar elements in their breast lobules; however, these acini are composed of cells with prominent eosinophilic cyto- plasm which differ from the acinar cells of normal female rats. Under the stimulus of estrogen the ducts increase in size more than in the female rat breast under estrogen stimulation, although the lobular components are not as prominent. Testosterone treatment in the male has a slightly greater effect on acinar-cystic development than in the female.

When estrogen is given to the iodine-deficient male rat (FIGURE 7), breast tissues show a remarkable increase in duct size far beyond that anticipated and seen in the female. Testosterone causes a marked dilatation of acinar elements, with some ductule enlargement in the iodine-deficient rat breast. When PTU is given with estrogen (FIGURE 8 ) , a moderate increase in duct size occurs in the


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Eskin: Iodine Metabolism and Breast Cancer 92 1

breast. Testosterone in the presence of PTU causes an increase in glandular tissue greater than that seen in control animals.

When PTU is given to iodine-deficient male rats (FIGURE 9) , their breasts show a very marked dilatation of acini and ducts. When estrogen is given to iodine-deficient male rats treated with PTU, there is a marked increase in the number of ducts, although the duct size is smaller than seen in estrogen-treated rats on either an iodine-deficient diet or a normal diet plus PTU. However, when testosterone is given to the iodine-deficient PTU male rat, the breast tissues show an overwhelming glandular hyperplasia with dilatation of ducts and acini.

The variations seen between the female and male breast in groups treated with testosterone might be anticipated, since the male breast lacks “estrogen priming” considered necessary by some workers for testosterone-induced lobular hyperplasia.24 Some experiments in male and female castrate rats have shown that testosterone may act locally to produce tissue changes in the mammary gland.’ In summary, the breast tissue changes seen in female rats were of a similar general nature but did not completely parallel the results seen in male rats.

Gynecomastia and Male Breast Cancer

Thyroid abnormalities as a cause of gynecomastia remain controversial. Although cases of gynecomastia with hyperthyroidism and hypothyroidism have been reported, data are generally insufficient to exclude the possibility of fortui- tous associations. Most authors are satisfied that the reason for transient gynecomastia in hyperthyroid males treated with 1311 is weight gain in the recovery from the poor nutritional state induced by hyperthyr~idism.~O

The administration of estrogen to male rats induces some changes in the breast similar to gynecomastia, namely, growth of mammary ducts with an increase in cellularity and periductal stroma but without lobule formation. When estrogen is administered to chronic iodine-deficient male rats there is an exag- geration of the hormonal changes. Our results in the male rat suggest that iodine deficiency may be one etiological factor in gynecomastia.20

Comment. Stimulation of the lobules of the male and female rat breast by thyroidectomy and goitrogen treatment has been previously described.33.42 In addition, experiments have shown that administered estrogen or testosterone cause increased acinar development in rat breast tissue under the influence of thiouracil.4~ 29 Iodine deficiency, however, enhances the response of rat breast tissues to sex hormone injections; the breast changes seen in the iodine-deficient rat are clearly distinguishable from those induced by sex hormones in the hypothyroid and in the euthyroid rat. Estrogen and testosterone administration to iodine-deficient and/ or hypothyroid rats result in lesions that mimic human breast dysplasia.

It is generally accepted that carcinoma occurs four times as often in dysplastic breasts as it does in normal breasts, although evidence for progression from benign dysplasia or malignant neoplasia is lacking.23 We feel that this experimental model may provide a means of studying these premalignant changes and determine what role the incorporation of iodine in tissues plays in breast dysplasia and neoplasia. While neither iodine insufficiency nor the


influence of sex hormones independently affects the breast sufficiently to account for these breast lesions, in combination a profound effect is seen.

Dietary Differences

Certain differences in pertinent metabolic responses have been found to occur when several iodine-deficient diets are given to rats." This experiment was done to ascertain whether our results were, in fact, due to the iodine-deficient state and not a dietary disturbance.1° Three low-iodine diets that have been shown to have none of the concomitant characteristics of hypothyroidism (growth rate, sexual activity, and other endocrine organ responses) were chosen.

Method. One hundred and sixty female CFN rats initially weighing 150 g were divided into four groups. Purina chow, used as the normal iodine (1.73 ppm of iodine) control diet, and three separate special diets, each containing less than 0.05 ppm-Remington diet (R), NF-30X low-iodine diet (NF) , and reconstituted chow without iodine (1DP)-were given for 16 weeks. Addition- ally, PTU was given as 4 mg/ 100 ml in the drinking water to one-half of each group for the same length of time.

Body weights were determined three times a week. At the end of 16 weeks of experimental treatment, the rats were killed with ether 60 minutes after an intraperitoneal injection of 10 ~c carrier-free sodium-12511. Dissection of thyroids and breasts was done as before, and blood was removed from the inferior vena cava. Thyroids and blood counts were made in a well-type scintillation counter. Protein-bound iodine (PBI) assays were done in pairs from each of the groups.

Results. As seen in TABLE 2, body growth, thyroid, and T / S ratio responses differed in the various iodine-deficient diets employed. PBI values are extremely low for R (0.1 @ l o 0 ml) but are low-normal for both NF-30X (2 .5 Pg/lOO ml) and IDP (2.0 Pg/lOO ml). With PTU, PBI is 0.2 Pg/lOO ml in R but decreases in N F (0.8 Pg/lOO ml) and IDP (0.5 @lo0 ml). The thyroid size increases with R and IDP but remains in the normal range with NF; all thyroids increase five to seven times with PTU.

Body growth is the same on all four diets for 16 weeks with the exception of the N F groups, which show a progressive decrease after 8 weeks. Growth is uniformly depressed in PTU-treated animals. Thyroid: serum ratios 1 hour after injection of 1251 are shown in TABLE 2. Endocrine glands show changes in growth and histology that also vary with the diet.

Comment. Although body growth, thyroid, and peripheral tissue responses differ with the various iodine-deficient diets employed, the degree of breast dysplasia seen was similar throughout the experiment by histological breast examination. This effect on the breast is influenced only by the lack of iodine. Further laboratory studies were done varying the length of time that iodine- deficient diets were employed; i.e., 3, 6, 12, 18, and 24 weeks, determining quantitatively what breast changes had occurred. While 12 weeks seemed maxi- mally effective, there were significant breast tissue changes beginning during the 6th week of deficiency. Changes in breast histology are evident after 4 days of estrogen therapy with duct enlargements, and by 6 days both estrogen and testosterone show significant tissue changes throughout the lobules. These find-

&kin: Iodine Metabolism and Breast Cancer 923

TABLE 2 METABOLIC RE-SPONSES TO VARIOUS IODINE-DEFICIENT DIETS

PBI Range Thyroid

No. of (& Body Weight* Weight* T/S Diet PTU Rats 100ml) (i3) (mg) Ratio*

Normal - 20 4.8-5.5 258.8 f 5.6 10.2 f 2.2 14.0 2 2.5

Normal + 20 1.6-1.8 200.0 f 14.1 74.0 rt 1.9 0.7 2 0.1 Remington - 20 0.0-0.1 221.0 & 11.2 64.4 -+ 1.0 144.2 -+ 30.4

~ ~

Remington + 20 0.1-0.1 194.2 -C 9.7 68.3 f 2.4 27.4 +- 6.2 NF-30X - 20 2.5-2.7 197.2 rt 3.9 59.0 f 0.8 132.8 f 52.5

NF30X + 20 0.7-1.0 181.2 f 13.1 73.0 f 3.3 34.8 -C 4.2 Deiodized

chow - 20 2.0-2.5 262.5 k 23.5 5 2 . 0 2 1.6 250 .02 34.8 Deiodized

chow + 20 0.4-0.5 225.4 f 25.8 79.0 f 3.8 25.4 2 6.2

*Values with standard deviation.

ings permit some latitude to our research, although controls are employed for each experiment.

Carcinogenic Influences

Time of Onset of Tumors. The development of mammary tumors in rats by orally feeding 7,12-dimethylbenz( a ) anthracene (DMBA) has been shown to be influenced by the hormonal state of the animal. Breast cancer can be produced in rats by gavage with a single dose of DMBA, and these tumors usually appear within 2 months. It has been shown that after treatment with DMBA fewer mammary tumors developed in thyroidectomized animals than in intact controls. This is considered attributable to the consequent smaller calorie intake, which influences the genesis of spontaneous and induced tum0rs.~5 Hypothyroidism chemically induced by PTU is capable of producing the same effect.21 Some workers suggest that modifications of response to carcinogens as a result of altered thyroid hormone levels were due principally to the growth-promoting stimulus of thyroid hormone or to the lack of it on established neoplastic cells.35 The rate of development of DMBA-induced breast cancer in rats made iodine-deficient and/or hypothyroid was used as another means of associating iodine metabolism with dysplasia and neop1asia.O

Method. Sprague-Dawley female rats obtained on the 40th day of age were randomly divided into six groups, (TABLE 3); 10 mg of DMBA, dissolved in 0.5 ml of sesame oil, was given once by gastric tube at 50 days of age to all the animals. None of the rats showed any marked ill-effects from the DMBA. Hypo- thyroidism was produced by giving 4 mg/ 100 ml of PTU in the drinking water, while Remington diet was given with double-deionized drinking water. TWO groups of animals (I1 and 111) were started on Remington diet or PTU 4 days before the gavage feeding of the DMBA, while groups IV, V, and VI were


TABLE 3

DMBA WITH IODINE DEFICIENCY AND/OR PTU MAMMARY GLAND TUMOR bCIDENCE IN RATS GIVEN

Aver- age

No. of Appearance of First Tumors

Palpable Tumors per Rat No. (days after DMBA) Day 60 of Mean f. After

Rats Range S.D. DMBA I Intact Controls 10 3 1 4 9 42.5 4.8 7.4

~~

Therapy 4 days before DMBA I1 Iodine-Deficient 6 24-33 26.6 5 4.2 7.8

111 PTU 6 24-35 29.6 2 4.2 7.2

Therapy 3 days after DMBA IV Iodine-Deficient 9 31-46 36.5 & 3.4 7.9 V PTU 10 3 1 4 7 37.3 2 2.4 8.1

VI Combination 9 3 3 4 6 37.1 2 2.3 5.4

started on their iodine-deficient PTU diet or combination 3 days after DMBA was given. All animals were killed 6 days after being given DMBA. The palpable nodules were carefully diagramed and removed at autopsy for histological study to determine the iype and extent of the lesions. Each of the palpable breast nodules was confirmed to be an epithelial neoplasm.

Results. The results are indicated in TABLE 3. A growth curve (FIGURE 10) for this time period shows that weight in rats with iodine deficiency alone is similar to that of the normal control. Hypothyroidism causes a decreased growth curve, while iodine deficiency with hypothyroidism shows no remarkable weight change after seven days.

GIM

3 0 0 .

ZbO -

140-

4b 60

FIGURE 10. Study of daily body weights (in grams) for major groups studied, extended for 100 days.

&kin: Iodine Metabolism and Breast Cancer 925

Comment. This experiment shows that both iodine deficiency and hypothyroidism have an influence on the rate of onset of palpable tumors in DMBA- induced breast carcinoma of rats only when it is given before treatment with DMBA. It would seem, then, that after the initial onset of palpable tumors, the average number of tumors seen in all groups rapidly equalizes, additional tumors appearing at a relatively regular rate except in those animals (VI) given both iodine deficiency and PTU. This latter group shows a marked decrease in body growth probably accounting for the reduction in the number of tumors. These results emphasize that factors relating to the thyroid modify the respon- siveness of the breast to carcinogenic influences. Again there is evidence that under conditions of reduced iodine availability the breasts respond remarkably differently to an extrinsic carcinogenic factor and, apparently, unfavorably.

Dose-Response Study. The interesting finding that the number of days prior to DMBA treatment seemed to have an influence on producing an earlier onset of breast cancer was studied. Doses of 2.5, 5.0, and 10.0 ing DMBA were given at one time to groups of Sprague-Dawley rats 4 or 10 days after or 3 days prior to the beginning of the iodine-deficiency diet. In all cases duplicate controls and PTU-controls were maintained. The results show that, a dosage of 2.5 mg gave the most apparent separation of data and that 3 days of therapy prior to DMBA showed the most significant effect.

Resistance to Estrogen Therapy

Sex steroid therapy in breast cancer is disappointing even in selected cases. The impetus for such clinical therapy has been the many published reports that show convincing evidence that estrogen and other steroids reduce or extinguish certain induced neoplasms in rats. This study considers how induced breast cancer might be effected by sex steroids in rats with iodine deficiency or thyroid hypofunction.

Method. Two hundred randomized Sprague-Dawley female rats were divided into four major groups as follows: (1) control-normal iodine diet (euthyroid) ; (2) iodine-deficient only (Remington diet with less than 0.05 ppm of iodine); (3) PTU-treated (hypothyroid); and (4) combination of iodine-deficient and PTU-treated.I2

From the experience of our prior studies, after 3 days of these treatments, DMBA, 10 mg in 0.2 ml sesame oil, was force-fed through a stomach tube to all rats. Daily palpations and diagraming of tumors were done and each tumor was carefully followed. Biopsy was done only if regression occurred. All rats developed mammary neoplasms that equalized, as previously seen, on the 60th day (TABLE 4) . The animals in each division were then randomly subdivided into three groups receiving, IM: (a) 0.1 ml sesame oil (S.O.) control; (b) 50 p g estradiol benzoate in 0.1 ml S.O.; or (c) 2.5 mg testosterone propionate in 0.1 ml S.O. (steroid control). After 20 days of these injections, autopsy was done and breast neoplastic incidence, size, and type of growth were carefully examined and compared with findings on palpation.

Histological Results. Certain basic neoplasms are common in DMBA induction. Two acute and one chronic histologic type of malignancy have been described in the literature. Since our time period was restricted to approximately


TABLE 4 ONSET OF NEOPLASMS

Median Day No. of Tumors per Rat of Onset Dav40 Dav60

Control 34 I deficient 25 Hypothyroid 30 Combination 23

1.8 4.9 1.9 4.8 1.6 4.4 1.9 4.7

80 days, only the acute types were seen. These have been previously described as clear-cell and papillary types of carcinoma. Our finding consisted of approximately 70: 30, respectively, of these types.

Tumor Statistics. During the steroid phase of this study, we noted from palpations that the expected decrease in tumor numbers and size occurred with estrogen in the nontreated controls. However, treatment with iodine deficiency alone and combined iodine deficiency and hypothyroidism resulted in a significant increase in tumor numbers (P value less than 0.001). Testoster- one was employed as a steroid control in this evaluation. At autopsy our palpation findings were confirmed, as seen in TABLE 5.

Comment. From these data, iodine deficiency with or without concomitant hypothyroidism seems to forestall the “therapeutic” effects of estrogen in DMBA-induced breast cancer. Abnormal iodine metabolism may be a prevent- able reason for the high percentage of failures by estrogen regimens in amelio- rating mammary gland carcinoma.

Summary. These studies have shown that iodine deficiency or hypothyroidism causes specific breast dysplasias in male and female rats. These tissue changes are augmented by estrogen or testosterone. Iodine deficiency particularly causes an earlier onset of induced breast cancer and prevents estrogen therapy from favorably affecting carcinogenesis. The breast tissue changes seen resemble human breast diseases.

PHYSIOLOGY

Physiological changes in breast tissue caused by iodine deficiency and hypothyroidism were studied. The techniques used were: ( 1 ) radioactive I* uptakes of breast tissue, (2) DNA and RNA determinations, and (3) histochemistries.

TABLE 5 TOTAL ADDITIONAL TUMORS AFTER STEROID THERAPY

No Steroid Estrogen Testosterone ~~

Control I-Deficien t Hypothyroid Combination

none none none

+ 1 3

none +8 none +13

+8 +13 +a +14


Radioactive Iodine Uptake of Breast Tissues

Significant iodine activity has been described in organs other than the thyroid gland. As previously stated, the gastric mucosa, salivary glands, ovary, and tumor tissues have been shown to have iodine activity. Breast tissue is a known site of iodine metabolism during lactation. Actually, soluble iodinating enzymes have been isolated in unpasteurized milk and iodinations of milk proteins in vivo have been described. Inorganic iodine has been reported to be concentrated by the lactating human breast.22

Surviving rat mammary tissues incorporate inorganic 1311 in moieties that are insoluble in trichloracetic acid, indicating that true organic iodination of mammary tissue does occur. It has also been shown that lactating mammary slices can transport inorganic iodine against a concentration gradient. No quantita- tions of the magnitude of both organic iodinations and iodine transport have been made. Organic iodination has been seen to occur in nonlactating rat breasts.lB Hence, in mammary tissue, as in the thyroid gland, an association of mechanisms may co-exist for concentrating iodide and for organic iodinations.

This series of laboratory experiments was initiated to determine whether there is change in breast tissue iodine in iodine inadequacy or hypothyroidism, particularly when influenced by sex hormones.

Method. Two hundred and thirty Sprague-Dawley females weighing approximately 200 g were used. In our studies, 10 pc of carrier-free sodium l25I was injected IP 60 minutes prior to killing of the rats with ether. Careful dis- sections of two abdominal and one inguinal breast were done bilaterally; one side was used for uptake and histology while the other side was immediately frozen for DNA-RNA evaluations. Venous blood was removed from the vena cava. In all cases the thyroid state of the animals was ascertained by PBI, thyroid/serum radioactive uptake ratios, and thyroid size.

The breasts were placed in 5 ml of formalin and counted in a well-type scintillation counter (Baird Atomic), precalibrated for l25I, and the final results expressed as count/minute/100 mg of tissue per 0.1 ml of blood. These counts were also repeated for tissue freed from fat in several studies. When the breast tissue was not defatted, inguinal fat pads were used for comparison with the breast tissue on a weight basis. Uptakes were interpreted on a relative basis between the groups in each experiment.

Additionally, breast radioactive iodine uptake evaluation was carried out in some experiments by carefully homogenizing a known weight of breast tissue in Krebs solution and hydrolyzing the defatted homogenate in 5 ml of I-N NaOH. Bray’s solution was added to this cocktail, and these breasts were counted in a Packard Tri-Carb Liquid Scintillation Spectrometer (No. 33 10) set for 1251. These are compared as net counts/minute/lOO mg of breast tissue. Fatty tissue loss was compensated.

Results. The data from the uptake studies are shown in TABLE 6 (well counter) and TABLE 7 (Tri-Carb counter). Two different counting and prepara- tion techniques were used. The responses were similar regardless of the method.

(1 ) Iodine deficiency shows an uptake that is slightly greater or similar to normal; PTU increases one to two times normal. The combination is usually high but not remarkably greater than PTU.

From these tables, it can be seen that:


TABLE 6 BREAST 1261 UPTAKE: CTS/MIN/100 MG BREAST TISSUE BY WELL COUNTER

Diet Control Estrogen Testosterone IV:148 (71 rats)

Normal 210 (63)* 930 (32) 980 (30) ID 241 (32) 1,697 (106) 405 (16) PTU 343 (65) 400 (70) 1,249 (40) ID-PTU 330 (85) 1,190 (45) 3,228 (100)

V:92 (60 rats) Normal 546 (18) 1,030 (256) 620 (15) ID 586 (32) 2,920 (220) 1,240 (190) PTU 726 (62) 1,000 (304) 834 (105) ID-PTU 500 (77) 1,770 (390) 3,850 (390)

VI:38 (59 rats) Normal 170 (10) 1,080 (210) 2,240 (340) ID 100 (10) 1,440 (130) 2,220 (310) PTU 200 (10) 1,780 (240) 2,790 (131) ID-PTU 442 (15) 1,780 (190) 3,240 (720)

*S.D.

(2) Estrogen causes a two-to-five times increase over normals and five-to-ten times increase over iodine deficiency alone (P value less than 0.001). Estrogen causes a mild effect with PTU usually less than two times. The combination causes a two-to-four times increase over controls when estrogen is given. (3) Testosterone causes two-to-four times increase, although only minimally with iodine deficiency. Both PTU and the combination show a remarkable increase (P value less than 0.001).

Iodine deficiency and hypothyroidism change breast iodine uptakes. These differences in iodine counts above the normal control are consistent with the qualitative tissue findings of hyperplasia, dysplasia, and neoplasia seen in the breast.

Since female breast tissues are normally sensitive to estrogen stimu!ation, the fact that iodine-deficient breast tissue is dec t ed more by estrogen and quite remarkably less by testosterone is consistent with clinical expectations. PTU-induced hypothyroidism shows an opposite effect. Studies with thyroidectomy show changes similar to PTU treatment.

Thus, the increasing iodine uptakes seen with dysplasias parallel the in-

TABLE 7 BREAST 1261 UPTARE: CTS/MIN/100 M O BREAST TISSUE BY 'TRI-CARB COUNTER

Diet Control Estrogen VI:60 (48 rats)

Normal 270 (23)* 1330 (262) ID 330 (70) 2050 (604) PTU 1140 (96) 2120 (299) ID-mu 770 (102j 4980 ( i47 i j

*S.D.


creasing cellularity in the breast and the diminishing iodine values. These uptakes differentiate between iodine deficiency and hypothyroidism as well. The intracellular iodine effects will require further clarification.

R N A and D N A Determinations

Specific breast tissue differences in desoxyribonucleic acid (DNA) values from paired strain controls in rats with various altered thyroid conditions have been seen. These workers studied the experimental changes in rat mammary glands using DNA an an index of tissue g r o ~ t h . 3 ~ The techniques employed were used in our project with only slight modifications. This gave us a series of basic values to use, although we always employed our own weight-paired, age-paired, and strain controls.

RNA synthesis has been previously utilized for diagnosis of precancerous and cancerous breast diseases. DNA activity is greatest in least differentiated and most rapidly growing tumors.39 We utilized these nucleic acid determinations in order to provide further measurable evidence of the morphological tissue changes caused by iodine deficiency in the breast. In addition, these data should reflect the cellular metabolic responses seen in breast tissues that have become dysplastic or neoplastic because of iodine inadequacy.

Method. Frozen breast tissue was rendered dry and fat-free (DFFT). DNA assay was then performed according to the technique of Webb and Levyq1 and the RNA assay method employed was that of C e r i ~ t t i . ~ The results were calculated as p g DNA/mg DFFT or p g RNA/mg DFFT. DNA/RNA ratios were obtained in order to evaluate total nucleic acid activity.

Results. The DNA results obtained with each of the basic experimental groups are shown in TABLE 8. In this control series, DNA increased with both ID (P less than 0.01 ) and PTU (P less than 0.001 ) .

It was felt that greater differences might be obtained if the work were repeated using a single stimulatory dose of DMBA. This series was primed with 10 mg of DMBA by gavage three days after the start of an iodine-deficient diet and/or PTU therapy. The rats were killed after 16 weeks of treatment. These data were analyzed by log transformations in order to equalize variances within the groups.

The results from the experiment (TABLE 9) show that an elevation of DNA occurred with ID (P less than 0.001) and PTU ( P less than 0.001) from the normal. An algebraic increase occurs when both ID and PTU are given. In RNA it was seen that an increase occurs with ID greater than PTU ( P less

TABLE 8 DNA VALUES IN BREAST TISSUE

pg DNA/mg Diet DFFT

Normal 15.8 Iodine-deficient (ID) 21.6 Normal + PTU 38.4 ID + F'TU 39.5


TABLE 9 DNA/RNA VALUES IN BREAST TISSUE WITH DMBA

- pg DNA/mg pg RNA/mg DNA/RNA

Diet DFFT DFFT Ratio Normal 50.0 47.8 1.04

Normal + PTU 363.9 58.5 6.24 ID + PTU 755.1 215.7 3.50 Coefficient of variation 21 % 12% 19% (analysis of variance)

Iodinedeficient (ID) 158.5 155.6 1.02

than 0.01). There is a greater increase with both I D and PTU, but this is not as large as with DNA. The DNA/RNA ratio remains low for I D but elevates when PTU is alone or with ID. An analysis of variance is used statistically, as indicated on the Table.

Quantitative measurement of DNA and RNA per cell would be more ac- curate in light of existing hyperplasia. Nevertheless, the results seen are sufficiently significant to permit speculation on the differences between the DNA values in these conditions. There is a buildup of DNA and RNA with iodine deficiency that seems remarkably different from the findings of DNA increases without the concomitant RNA formation seen with hypothyroidism. There- fore, it appears that changes in iodine conditions are reflected in cellular components in a manner that seems to duplicate the abnormal tissue changes that are seen.

Histochemistry

Procedures were carried out primarily to characterize the dysplasias and neoplasias seen and to determine whether an analysis of the secretions could be made. Standard staining procedures consisting of nile blue sulphate, schiff reagent, and alkaline phosphatase were performed.'* No attempts at quanti- tation were made.

The qualitative histochemical studies described show that the secretions within the cystic areas of the mammary glands contain glycogen, glycoprotein, triglycerides, free fatty acids, and lipoproteins. In addition, an increase in alkaline phosphatase staining was seen in glandular areas under the influence of iodine deficiency over control and PTU sections.

CONTROL OF BREAST IODINE METABOLISM

The mammary gland iodine activity that is seen may be subject to either thyroid-gland, central (pituitary), or intrinsic-breast-cellular regulation. In order to localize this control we have begun to study rat breast tissues in: thyroidectomized animals, hypopnysectomized rats with and without TSH replacement, and hypophysectomized-thyroidectomized rats with and without TSH therapy.

Induced Hypothyroidism and Thyroidectomy

We have always done parallel experiments with iodine deficiency and thyroid hypofunction, usually by treating the rat with PTU. It was necessary to separate

Eskin: Iodine Metabolism and Breast Cancer 93 1

TABLE 10 LABORATORY RESULTS IN ALTERED IODINE STATES

Normal PBI (pg/% ) < 0.2 1 .O-I .5 4.0-5.5

~- I Deficiency Hypothyroid (PTU)

Thyroid/serum 224.4 0.2 18.3

T,-I (column) 0.3 0.9 3.2 *I ratio

14-18 1

Weights (mg) 70-80 89-120

these conditions to delineate clearly the effect of iodine metabolism. Laboratory tests done for both these metabolic abnormalities were PBI, T, by column, thyroid/serum I3lI or lnaI uptake ratio, and thyroid weight. Obvious differences in these parameters in these altered iodine and thyroid states are seen. The average changes seen are noted in TABLE lo.

The results obtained with PTU-induced hypothyroidism have been described consistently along with our studies throughout this presentation. In order to determine what effects could be expected when there was no thyroid present, thyroidectomized rats were studied with and without iodine deficiency under the influences of sex hormones. This experiment also provides evidence that the results seen with PTU in our research were not pharmacologic effects of the drug but hypothyroidism. This additional hypothyroid condition further demon- strates that breast changes seen were not mediated through any direct thyroid action.

Method. Thirty-six Sprague-Dawley rats were thyroidectomized at 150 to 175 g. Postoperative care was given for 10 days, following which the animals were randomly subdivided into those receiving normal diet and those receiving an iodine-deficient diet. Further division of the groups were made into those receiving injections of sesame oil control, estrogen, or testosterone IM for 20 days prior to autopsy. Weights were measured biweekly. Diets were maintained for a total of 16 weeks. The rats were killed with ether, and breasts and other routine organs were dissected. Evaluation of the tracheal region for possible thyroid remnants was routinely done.

Results. The body weights were generally depressed below those of our normal age-weight groups. The combined thyroidectomized-iodine-deficient groups were generally lighter than thyroidectomy alone, The PBI results were uniformly low, less than 3.5 &% with thyroidectomy and less than 0.5 pg/% in the combined groups.

Histologically, the thyroidectomized rat shows glandular breast development similar to those described with PTU (FIGURE 11). Estrogen and testosterone increase both duct size and lobular elements. With both thyroidectomy and iodine deficiency (FIGURE 12) there is a modest glandular increase with very large cystic changes. Estrogen and testosterone produce breast changes similar to those seen in PTU-treated animals.

Comment. These results show that the absence of thyroid directly influences breast tissue changes. Hypothyroid conditions, therefore, seem to have the same results at the breast level whether induced with PTU or by surgical extirpa-


0 U

s E 8


tion of the thyroid gland. Iodine deficiency has a unique effect on breast tissue, and, whether in the presence of the thyroid or not, it also produces specific breast tissue changes.

Hypophysectomy Studies

Some published studies suggest that certain trophic hormones from the pituitary act as carcinogens on the breast. Since many pituitary hormones have a direct or indirect effect on the mammary gland, they have been considered to produce or enhance breast tumor formation. The most obvious are prolactin, which initiates lactation, and growth hormone, a factor in growth throughout the body, both of which seem to be related to one another chemically. Sub- stantial evidence is available concerning the presence of a marnmotrophic hormone (Furth) that responds at the breast level to produce neoplastic changes. Other hormones suggested are the adrenocorticotrophic and the gonadotrophic.

Thyrotropin (TSH) has played an unknown role until this time.37 TSH increases in PTU treatment, iodine deficiency and thyroidectomy. Thus, the influence of TSH on the mammary gland iodine metabolism may be critical. For this reason we have begun to study the role of TSH.

Method. Eighty hypophysectomized female rats were divided equally into four major groups and maintained on normal diet, fed a Remington iodine- deficient diet, treated with PTU, or given both regimens for 12 weeks. Forty intact age-weight Sprague-Dawley female controls were similarly divided and maintained. The hypophysectomized rat groups were further subdivided randomly, and half were given TSH replacement for 4 days prior to autopsy. The TSH used was NIH-obtained (S, ovine), given in doses of 0.75 units per animal every 12 hours for 5 days prior to autopsy. The breasts were stimulated with 50 Mg of estrogen daily for 7 days prior to autopsy.

At the time of killing by ether, the breasts and thyroids were dissected. In addition, tissues from the area about the sella turcica were removed for determination of possible residual pituitary tissue. Blood was removed from the vena cava.

Results. The histological sections were read and rated by several observers for both studies. The intact controls showed anticipated changes in ducts, as seen in previous experiments (FIGURES 2-5). However, hypophysectomized rats uniformly evidenced decreased duct hyperplasia in all of the groups (FIGURE 13).

When TSH was given to the hypophysectomized experimental rats, the breast tissue showed proliferative changes comparable with the intact control groups (FIGURE 14). This experiment was repeated with forty additional rats. Similar results were obtained.

Comment. TSH therefore seems to play a role in the induction of mammary gland dysplasia in altered thyroid states and iodine deficiency, possibly through direct control of iodine metabolism at the breast.11 Surviving slice studies al- ready cited have shown some iodine metabolic transformation and protein bind- ing even in non-lactating breasts. Central stimulation by TSH may promote breast tissue activities through organic iodide synthesis.


Hypophysectomy and Thyroidectomy Combined

Since experiments on hypophysectomized animals stimulated with estrogen showed that the breast changes were obliterated, but returned when TSH was given, it seemed relevant to determine what effect both hypophysectomy and thyroidectomy would have on the breast with and without TSH replacement.

A preliminary study was done using only 12 hypophysectomized-thyroidectomized female Sprague-Dawley rats. All the animals were placed on Reming- ton diet as previously described. During the last five days, one-half of the group was given 0.75 units of TSH ( S , ovine, NIH) every 12 hours subcutaneously. In addition, 50 p g of estradiol benzoate was given in oil daily for the last 7 days to all of the animals.

The animals were killed with ether and the breasts dissected as in previous experiments. The breast tissues were sectioned and stained routinely:

Results. Histological sections of breasts on iodine deficiency with estrogen alone showed minimal ductular response. When TSH was given as described in the protocol above there was an obvious increase in ductular enlargement and cellular hyperplasia. These results are preliminary and require repetition.

Summary. It would seem from our results that TSH has a significant effect on the pathogenesis of breast clysplasia as seen in iodine deficiency. This response occurs with or without the presence of the thyroid gland. In light of previous evidence that breast tissue changes are associated with iodine activity, these results suggest that TSH may control specific iodination processes in brsast tissue.

IODINE REPLACEMENT THERAPY

Since iodine deficiency causes specific breast changes, it is necessary that evidence be presented to show that normal tissue findings would be obtained in rats given iodine replacement. Two conditions were considered: (A) replacement of iodine concurrently with the dietary regimens and (B) iodine therapy after the breast changes have occurred.

Concurrent Dietary Replacement of Iodine

Method. Three separate diets: Remington diet (R), NF-30X diet (NF) , and reconstituted chow without iodine (IDP), each containing less than 0.05 ppm of iodine, were given for 16 weeks to three groups of female CFN rats initially weighing 150 g. Purina chow was used as the “normal iodine” (1.73 ppm) control diet. During the last three weeks, estrogen, testosterone, or sesame oil was given IM to equally divided groups on each dietary regimen. Iodine in 1.73 ppm was added to one-half of the rats on the control diet and in 3.46 ppm to one-half of the rats on each of the iodine-deficient diets.

Results. The degree of breast dysplasia seen was similar in all dietary regimens as previously described. However, breast tissues in the various iodine-deficiency groups when given iodine dietary replacement were similar to the normal diet controls.


FIGURE 13. Effect of estrogen administration on breast tissue of hypophysectomized female rat: (A) normal-fed; ( B ) iodinedeficient; (C) propylthiouracil-treated, normal-fed; (D) iodine-deficient, propylthiouracil-treated. Stain: hematoxylin and eosin. x 18 (orig. mag. x 24).


FIGURE 14. Effect of estrogen administration on breast tissue of hypophysectomized female rat treated with TSH: (A) normal-fed; (B) iodine-deficient; (C) propylthiouracil-treated, normal-fed; (D) iodinedeficient, propylthiouracil-treated. Stain: hematoxylin and eosin x 18 (orig. mag. x 24).


Comment. Thus, iodine deprivation, using several iodine-deficient diets, sensitizes the rat breast to sex-hormone stimulation. The addition of iodine to these diets effectively prevents these breast-dysplastic changes.

Therapeutic Iodine Replacement

Since deficiency diets with concomitant iodine treatment showed normal breast histology, this series of experiments was devised to determine specifically whether iodine replacement therapy could reverse breast abnormalities produced by iodine deficiency once they were producd.13

Many workers have shown that giving iodized salt to goiter patients in some areas with endemic disease did not completely treat the metabolic problems that arise in these p o p ~ l a t i o n s . ~ ~ ~ 3 1 ~ ~ ~ Iodized oil injections have been given in some areas of endemic goiter, with reduction in the goiter size and correction of elevated TSH values.2 Limited improvement is believed by some workers to occur because of failure to increase the efficiency of the intrathyroidal iodine utilization in the deficiency state, a factor that may play a role in the development of endemic goiter initially.2A Therapeutic failures in some regions have been attributed to presence of goitrogens in the food or water and genetic potentiaLl7 In the presence of breast disease, rat studies by several workers have shown that thyroxine replacement is ineffectual in metastatic breast carcinoma, although the thyroid may atrophy and lose its functi0n.~3

Method. Fifty-six Sprague-Dawley female rats were divided into eight groups of seven each. All groups were given the low-iodine test diet (purina chow without iodine) for 6 weeks. During the last 20 days of this period, all animals received 50 pg per day estradiol benzoate in 0.1 ml sesame oil IM. The control groups were killed at 6 weeks and 8 weeks.

The remaining groups received iodine replacement for 3, 7, or 14 days either by the addition of 3.46 ppm of iodine to the test diet or by daily intraperitoneal injections of 0.2 ml per rat of 20 mg of sodium iodide (Lilly) in distilled water. Animals were weighed at least twice per week.

At the end of the indicated time periods, each group was injected with lZ5I to measure iodine uptake. They were killed by ether 60 minutes later, and the breasts and thyroids were dissected and weighed. Blood was withdrawn from the inferior vena cava. Blood, thyroids, and breasts were assayed for lz5I uptake in a well-type scintillation counter. The breasts were additionally counted in a Tri-Carb scintillation counter. Histologic sections of the breast tissues and thyroids were studied.

Histological Results. FIGURES 15-1 7 show by microphotographs the histology seen using this experimental design. The sections chosen were considered the mean representation by examination of all the slides in that particular group. FIGURE I5 shows the control results: (a ) breast histology as normally seen

in the presence of estrogen therapy, (b) sections seen when 20 days of estrogen is given to rats made iodine-deficient by diet for 6 weeks, and (c) sections seen when 20 da;s of estrogen is given to rats made iodine-deficient for 8 weeks; (b) and (c) show similar areas of dysplasia with evidence of neoplastic changes. FIGURE 16 shows the breast tissue changes that occurred when iodine wa.

injected daily for 3, 7, and 14 days. Estrogen was injected for 20 days prior to autopsy. The animals had been made iodine-deficient for 6 weeks prior to the


.^

Eskin: Iodine Metabolism and Breast Cancer

lu 0

94 1

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P B c .- a2 7)


initiation of their iodide therapy. A gradual improvement in the appearance of the breast sections occurs as compared with those seen in the control estrogen slides. The change seems to occur more by decreased alveoli formation than duct-size reduction. The difference at 14 days is more evident, particularly when compared with the control estrogen effect.

FIGURE 17 shows microphotographs of breasts after the addition of iodine to the diet for 3, 7, and 14 days. Estrogen had been given for 20 days prior to autopsy. Again the decrease in alveolar change was most apparent at 14 days of therapy.

From these sections, short-term replacement showed histologic evidence of a progressive return towards the control tissue.

Thyroid Weights and Uptake Studies. TABLE 11 shows the thyroid weights obtained in both 6- and &week control iodine-deficient-estrogen therapy and after the various short-term iodine replacements. From these data, thyroid weight decreased the longer iodide treatment was continued.

TABLE 11 THYROID W ~ r c m s AND UPTAKE STUDIES

Thyroid Weights Thyroid/Serum (me) *I Ratio

Control Iodinedeficient

6-week 32.2 k 3.16* 255 k 28 8-week 31.5 ? 0.97 376 f 54

Injection iodine 3-day 29.1 f 4.60 7day 28.6 2 1.26

14-day 20.1 k 1.87

~~ ~

245 k 53 321 k 26 312 2 41

Dietary iodine 3day 7-day

23.2 +- 3.48 25.7 ? 1.90

330 2 54 197 ? 12

14day 21.6 k 0.87 369 2 18

*S.D. values

It appears from the FIGURE that there is a significant decrease with both iodide injection and dietary iodide at 14 days (P value less than 0.001), although varying degrees of significance occur on shorter-term therapy. This shows a marked decrease in the need for the compensatory thyroid hyperplasia required with iodine deficiency.

Thyroid uptakes uniformly decreased with both dietary and injectable-iodide treatment. The serum uptakes similarly decreased; therefore, the thyroid-serum iodine uptake ratios continued to be relatively high.

Breast Uptakes. TABLE 12 shows the breast 1251 uptakes as gauged by both the Tri-Carb and the well-type scintillation counting methods. At three days, Tri-Carb uptakes were elevated markedly above control values. Thereafter the uptakes decreased relatively to the 3-day level, being lowest after 14 days of therapy.


TABLE 12 BREAST 1261 UPTAKES: COUNTS/100 M G BREAST TXSSUE/MIN

Tri-Carb X SD - Well -

X SD Control Iodine-deficient

6-week 945.0 243.5 74.0 7.7 8-week 991.0 172.5 - -

Injection iodine 3day 1,612.0 284.5 63.0 3.0 7day 1,115.0 171.5 37.0 3.7

14day 1,518.5 197.5 24.0 3.0 Dietary iodine

3-day 7day

14-dav

1,830.0 1,363.0 1.183.5

175.5 346.0 185.5

53.0 5.1 43.0 2.2 17.0 1.3

Well-counter results showed a gradual diminution of iodine uptake as replacement therapy was given.

Comment. Simultaneous iodiue replacement to rats on an iodine-deficient diet prevents breast dysplasia. However, once dysplasia or neoplasia due to iodine deficiency is established, chronic therapy by iodlde injection or dietary iodide may be required. The therapeutic course is characterized by an initial latent period (3-7 days), then a gradual histologic improvement. Specifically, the ductular hyperplasia is reduced.

This tissue improvement seems to correspond to such metabolic changes as decreased thyroid size, decreased breast 1251 uptake, and decreased thyroid and serum uptakes.

From these data we conclude that breast dysplasia and neoplasia caused by iodide deficiency improves gradually on short-term iodide replacement therapy after an apparently latent period. Injectable iodine thus far appears more effective than diet. This seems pertinent, since published clinical studies show that iodine in oil injections decrease TSH in patients with goiter in endemic regions. We have also begun evaluating therapy with triiodothyronine (T,) and L-thyroxine (T4) singly and together.

CLINICAL EVALUATION

In the United States, breast dysplasia occurs in about 25 per cent of the adult female population and breast cancer produces the highest rate of cancer mortality among American women. References dealing with iodine metabolism and clinical breast disease have been described. We have begun specific pro- grams aimed at clarifying the relationship of iodine metabolism to human breast dysplasia and neoplasia. These clinical experiments will parallel our laboratory research.

Breast Dysplasia Studies

Patients complaining of breast discomfort and showing any pertinent thyroid signs or symptoms were objectively evaluated with mammography and thermog-


raphy. In those women with iodine deficiency or hypothyroid disease, adequate therapy was given, usually utilizing iodine replacement or L-thyroxine medica- tion. After a reasonable therapeutic period, both mammography and thermography and thyroid laboratory tests were repeated. Changes in mammographs and thermograms were read by radiologists at a single institution without prejudice to the case. No specific controls were employed, and the patient was compared only with her own prior laboratory values.

Results. Within this preliminary framework, it was seen that in 10 selected cases (TABLE 13) breast dysplasia described as adenosis, fibroadenoma, or cystic disease of the breast on mammography and thermography decreased or disappeared after adequate replacement therapy had been given. Two patients considered as iodine-deficient were relieved of prolonged breast tenderness after adequate iodine intake.

Five patients described only minimal difficulty until started on estrogen therapy. Thereafter breast tenderness persisted even after the steroid was dis- continued. Thyroid therapy improved their mammograms.

Comment. Iodine inadequacy as a cause has been suggested by us in progressive breast disease and induced carcinoma. Sex steroids cause a profound effect in the mammary glands. Therefore, this is important, since estrogen is increas- ingly used for contraception and postmenopausal medical maintenance. Since no evaluation of iodine status is usually made before this therapy is given, sensitive women could be exposed to a greater risk of dysplasia or neoplasia.

The use of iodine replacement and T4 therapy in these cases seemed to be effective and at least temporarily improved the breast condition. There is need for further basic information on these therapeutic regimens and longer periods of follow-up on these patients.

Iodine-Uptake Studies in Breast Tissues

Patients with thyroid abnormalities admitted for biopsy of breast lesions sus- pected as carcinomatous are being studied by breast-iodine uptakes in a manner similar to those described in our laboratory work. Other thyroid and iodine parameters are likewise carefully determined on all these subjects. In a few cases thus far studied the results show that where there is a history of thyroid disease the breast counts show a higher iodine uptake with disease tissue than in histologically normal tissue from the same patient.

SUMMARY AND CONCLUSIONS

By means of basic investigation in rats, a relationship has been established between iodine deficiency and breast diseases, particularly in the presence of sex steroids. These lesions differ histologically and metabolically from those seen in hypothyroid states. Evidence of iodine in the breast is shown, which quantitatively varies according to the breast changes seen and the generalized iodine status of the rat. These findings agree with published evidence of breast iodine concentration and organic iodinations and add the possibility of pituitary TSH control of this iodination.

Breast dysplasia and neoplasia are influenced by the available iodine. Carcino- genesis occurs earlier and estrogen therapy is unsuccessful in iodine deficiency.


TABLE 13 CLINICAL FINDINGS IN PATIENTS WITH BREAST AND THYROID ABNORMALITIES

Mammogram/ Pt. age Thermogram T1 Thyroid Thyroid PARA Diagnosis PBI Col. Uptake Rx sx W027 Cysticdisease 5/68 2.7 - 0 Thyrox 1964 1001 (nocomplaints) 4/69 7.0 - 0 T, 0.25 for colloid

goiter EC33 Cysticdisease 7/67 3.2 3.0 0 Many 6015 Neg 6/68 7.2 6.4 0 T10.3 None

Nen 6/69 7.4 - 0 T, 0.3 None - F B 38 Cysticdisease 7/65 2.4 - 0 Few 5005 Adenosis 8/67 4.2 - 0 T, 0.3 Few

Neg 2/69 7.2 - N T10.3 None RF 22 Adenosis,

fibroadenoma 8/69 5.6 3.5 0 Few 0000 Adenosis,

reduced 1/70 6.2 4.5 0 ThyUSP None MG 41 Fibrocystic

3003 Nen 2/70 - 6.8 N T.0.25 None disease 9/68 4.0 3.0 + Few -

MH 23 Adrenosis, severe 5/69 - 2.9 0 Many

0000 Normal 2/70 - 5.6 0 T1 0.25 None EM 30 Adenosis;

2002 Mild adenosis cystic disease 1/67 2.9 2.6 + ID Goiter

only 9/67 4.4 3.8 0 T10.3 Goiter None 5/68 7.0 - 0 T10.3 Improved

PM 44 Cysticdisease 4/69 3.5 3.2 4- Many 2002 Singularcyst 1/70 6.2 5.8 N T.0.3 None

ID 0000 Neg 6/68 4.5 - 0 T40.3 None

Iatrogenic -24 Cysticdisease 11/67 3.0 - + JR28 Cysticdisease 4/66 2.8 - 0 Few 3003 5/67 6.4 - 0 ThyUSP Few

Addition of iodine to iodine-deficient diets prevents breast dysplasia; once breast lesions are established, only chronic iodine replacement manages the dysplasia that is formed. Therefore, iodine metabolism in the breast may be a factor in the changes responsible for breast tumorogenesis. The demographic and statistical evidence relating iodine deficiency and breast cancer is compelling. Clinical statistical studies have further directed our attention to this area. Specific preliminary clinical studies using mammography and thermography show that there can be improvement with adequate thyroid or iodine therapy. Estrogen acts adversely under these conditions, which should cause further caution in its generalized use.

The work presented points to a new factor in breast dysplasia and cancer that seems to be amenable to prevention and therapy.


ACKNOWLEDGMENTS

Estrogen (estradiol benzoate) and testosterone (testosterone propionate) were the sex steroids used throughout this report and were generously supplied by the Schering Corporation and propylthiouracil by Lederle Laboratories, Inc.

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