Dietary Iodine Deficiency As a Tumor Promoter and Carcinogen in Male F344/Ncr Rats1

[CANCER RESEARCH 46. 877-883, February 1986]

Dietary Iodine Deficiency as a Tumor Promoter and Carcinogen in Male F344/NCr Rats1

Masato Ohshima2 and Jerrold M. Ward3

Tumor Pathology and Pathogenesis Section, Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Frederick, Maryland 21701

ABSTRACT by 33 wk in that experiment (7), in several previous investigations by others it had been shown that more prolonged administration Groups of 6-wk-old male F344/NO rats received a single i.v. of IDD (6 to 20 mo) resulted in development of thyroid tumors in injection of either vehicle or A/-nitrosomethylurea (Cas: 684-93- 54 to 100% of female rats (11,14-16). 5) (MNU) at a dose of 41.2 mg/kg body weight. Two wk later, It is well known that the development of spontaneous pituitary groups of rats were placed on iodine-deficient, iodine-adequate, tumors is common in aging rats of various strains (17-20). or commercial (Wayne Lab Blox) diets, or one of these diets and Although pituitary tumors are readily induced in rodents within a without previous MNU injection. Animals were sacrificed at either relatively short period by prolonged estrogen administration (10, 52 or 77 wk, or when they became moribund. Carcinogen-treated 18, 21), induction of a high incidence of pituitary tumors by rats on the iodine-deficient diet for up to 52 wk had significantly chemical carcinogens has not been reported. We have now increased thyroid gland weights and increased incidences of investigated the morphology, incidence, and biological features both thyroid follicular cell carcinoma (90%) and diffuse pituitary of thyroid tumors and pituitary tumors in MNU-treated male F344 thyrotroph hyperplasia (90%) at 52 wk. The majority of the rats and the effects on these parameters of prolonged dietary follicular carcinomas were transplantable and invasive into the iodine deficiency over a period of up to 77 wk. mammary fat pad of weanling F344/NCr rats. No other tumors induced by MNU were affected by the iodine-deficient diets. Rats MATERIALS AND METHODS fed the iodine-deficient diet without MNU injection had a 40% incidence of thyroid follicular adenomas at 52 wk and 60% at 77 Animals. A total of 180 male F344/NO rats, 4 wk of age, were wk, and a 10% incidence of follicular carcinomas at 77 wk. Thus obtained from the Animal Genetics and Production Branch, Division of this experiment provided evidence that the iodine-deficient diet Cancer Treatment, National Cancer Institute, NCI-Frederick Cancer Re is a potent promoter of thyroid tumors initiated by MNU and search Facility, Frederick, MD. The rats were housed 4 per polycarbonate carcinogenic by itself. In addition, pituitary tumors were found in cage in filtered racks in a standard barrier facility and fed sterilizable 29 of the 58 rats treated with the carcinogen alone, compared WLB (Allied Mills, Inc., Chicago, IL) or a test diet as specified. During a 2-wk acclimation period, WLB and water were available ad libitum. to only 3 of the 20 rats in the control groups. The vast majority Chemicals and Test Diets. MNU (CAS:684-93-5; Ash Stevens, Inc., of these pituitary tumors contained prolactin that was demon Detroit, Ml) was dissolved at 40 ITIMin sodium citrate buffer at pH 5.5. strable by the avidin:biotin:peroxidase complex immunocyto- The IDD (Remington; Teklad, Inc., Madison, Wl) was used as the chemical technique. experimental diet. It was prepared commercially per kg as follows: whole ground corn (780 g); wheat gluten (180 g); Brewer's yeast (20 g); calcium

INTRODUCTION carbonate (10 g); and sodium chloride (10 g) (22). The IDD with 0.01 g of potassium iodate per kg of diet (IAD) and WLB were used as control Recently, there have been several reports concerning tumor diets. Vitamin K3 (as menadione; Teklad, Inc.) was added to the IDD and promotion or cocarcinogenesis in the thyroid gland (1-10). In the IAD at 5 mg/kg. majority of these studies, various chemical goitrogens were used Experimental Design. At 6 wk of age, rats were divided into 6 groups as promoters of thyroid tumors. Iodine-deficient diets have also of 30 rats each. In Experimental Groups 1 to 3, rats received a single i.v. injection of MNU at a dose of 0.4 mmol (41.2 mg)/kg body weight via been shown to have goitrogenic effects (11,12). There is also the tail vein. Controls (Groups 4 to 6) received i.v. injections of sodium some evidence that iodine deficiency or goiter may play a role in citrate buffer solution at a dose of 1.0 ml/kg body weight once at 6 wk the etiology of human thyroid cancer (13). In a previous report, of age. All rats were fed WLB for the first 2 wk following the injections. we demonstrated that dietary iodine deficiency is a potent pro Starting 2 wk after MNU injection, and continuously thereafter, rats were moter of thyroid tumors in the male F344 rat treated with an placed on IDD (Groups 1 and 4), or IAD (Groups 2 and 5), or WLB initiating dose of MNU4 (7). Although iodine deficiency alone, (Groups 3 and 6) for up to 77 wk. Rats were weighed weekly and were without MNU injection, did not induce thyroid tumors in male rats sacrificed when clinical signs (severe loss of body weight, dyspnea) indicated impending death or at Wk 52 or 77 of the experiment (Table

Received 7/2/85; revised 9/9/85; accepted 10/23/85. 1). The costs of publication of this article were defrayed in part by the payment of Pathology. A complete necropsy was performed on each rat. The page charges. This article must therefore be hereby marked advertisement in mean body and thyroid gland weights as well as thyroid gland weight: accordance with 18 U.S.C. Section 1734 solely to indicate this fact. body weight ratio were determined at necropsy for each group. Data 'This work was supported in part by USPHS Contracts N01-CO-23910 from were evaluated by Duncan's multiple range test and Student's i test for the National Cancer Institute to Program Resources, Inc., and N01-CO-23912 to body and thyroid weights or by x2 analysis for comparison of tumor Information Management Services, Inc. 2 Present address: Nissei Hospital, Osaka, Japan. incidence (7). Thyroid, pituitary, and adrenal glands, lungs, eyes, thymus, 3 To whom requests for reprints should be addressed. 'The abbreviations used are: MNU, A/-nitrosomethylurea; ABC, avidin: and grossly visible lesions were examined microscopically. Tissues were biotin:peroxidase complex; FPPL, focal proliferative pituitary lesions; IAD, iodine- fixed in 10% buffered formalin, embedded in paraffin, sectioned, and adequate diet; IDD, iodine-deficient diet; TSH, thyroid-stimulating hormone; WLB, stained with hematoxylin:eosin. For the localization of TSH or prolactin, Wayne Lab Blox diet. formalin-fixed pituitary sections were stained by the ABC technique (7,

CANCER RESEARCH VOL. 46 FEBRUARY 1986 877

Table 1 Mean survival time, body weight, thyroid weight, and tumor incidence of male F344 rats given MNU and/or IDD wtSurvival wtAbsolute mental of tu of organs no. of period (mg/ mor-bear with tumor/ Group123456TreatmentMNU rats29283010(wk)52525252time37 (mg)541 wt)190100 g body ingrats29I.100)*'"28(100)Â°29rat2.0 *48+ 8aâ€¢ Â±45"318 Â±352*150 +122(11)c51 +0.6*3.5 +IDDMNU +786 Â±7"47 Â±1.6*3.0 +IADMNU Â±1392 +56343 Â±17030 Â±63(2)10 +8*52 (96.7)"5 Â±1.4*0.6 +WLBIDDIAOWLBEffective Â±989 Â±61365 Â±1179 Â±3(3)22 Â±0* Â±21" Â±6" Â±2*(10) Â±8 (50.0) Â±0.7 77 +0"52 394 +32"362 127 +43*49 34 + 12"(5)14 16(80.0)"1 1.2 +0.8*0.1 2010191119Experi7752775277Body 91+991+598 +0e73 Â±13"366 Â±6*41 Â±2"(10)11 (10.0)7 +0.30.4 Â±6"51 Â±72"393 Â±2"(5)6Â±1 (36.8)"1 +883 Â±526 Â±0.6"0.1 Â±2"76 Â±2693 Â±25474 +535 (10)7 (9.1)16(84.2)No. Â±0.31.3 Â±5"Initial88 Â±33Final293 Â±33Thyroid Â±3Relative Â±1 (5)No. + 0.8 * Mean Â±SD. " Statistically significant differences (P< 0.05) between this group and appropriate carcinogenor diet controls. c Numbers in parentheses, number of thyroid glands weighed. " Numbers in parentheses,percentageof rats bearingtumors. " The majority of these rats were sacrificed in good health at 52 or 77 wk.

Table 2 Histological findings in the thyroid gland of male F344 rats given MNU and/or IDD lesionsGroup1 No. of rats with

mentalperiod(wk)52 fol-licular follic-ular C-cell thy ofrats29 hy-perplasia29(1hyper-plasia0 hyper-plasia0 carci car roid tu noma8 noma26 noma29(100)* cinoma1(3)mors29(1001* + IDD OO)'-* (28)* (90)" 23 (82)" 9 (32)* 23(82)* 2 MNU + IAD 52 28 0 2(7) 1(4) 0 0 23(82f 19(63)" 6 (20)" 20(67)* 20(67)* 3 MNU + WLB 52 30 0 3(10) 1(3) 1(3) 0 10(100)" 4 (40)* 4 (40)* 4(40)* 456TreatmentMNUIDDIAOWLBExperi52 10 2(20) 2(20) 0 0 0 20(100)"0000Focal 12(60)*0000Follicularcarci13(65)*0001(5)C-celladenoma0 7752775277Effectiveno.2010191119Diffuse 00000Focal 2(10)01(5)00Follicularade2(10)0001(5)Follicularadenomaor1(5)0002(7)C-cell2(10)01(5)01(5)Any14(70)*01(5)04(21)

Numbers in parentheses,percentage. " Statistically significant differences (P < 0.05) between this group and appropriate carcinogen or diet controls.

23) with the use of the Vectastain ABC kit (Vector Laboratories, Inc., incidences of which were significantly higher than in the respec Burtingame, CA) and using rabbit anti-rat TSH or prolactin antisera tive control groups (P < 0.05). The number of organs with tumor (National Hormone and Pituitary Program, Baltimore, MD) at a dilution of per rat in MNU treatment groups was also significantly higher 1:100 (TSH) or 1:200 (prolactin) with saline. than in control groups (P < 0.05). Transplantation. Portions (2 to 3 mm) of grossly visible thyroid lesions Thyroid Pathology. The histological findings in the thyroid from each of 9 rats in Group 1 (MNU + IDD) were transplanted into the inguinal mammary fat pads of weanling (4- to 5-wk-old) male or female gland in each group are summarized in Table 2. The histological F344/NCr rats (23). Transplant sites were palpated, rats were weighed features of thyroid lesions have been described in our previous weekly, and rats were maintained for up to 33 wk. Some of the trans publication (7). Most follicular adenomas in group 2 (MNU + IAD) planted tumors were used for a second transplantation passage. All at Wk 52 were well differentiated, and cystic, multi-locular, transplant recipients were necropsied, and tumors and lung were fixed papillary, and their epithelial linings were very thin (Fig. 1). The in 10% buffered formalin, embedded in paraffin, sectioned, and stained complex features of these lesions differentiated them from more with hematoxylinreosin. simple cystic hyperplastic lesions. Also carcinoma was occasion ally seen arising within these follicular cystadenomas (Fig. 2). RESULTS Mosf of the follicular adenomas and carcinomas induced by IDD only (Figs. 3 and 4) consisted of follicles of various sizes contain Survival Time, Body, and Thyroid Weights. Table 1 sum ing epithelial cells that were more cuboidal (Fig. 3) than those marizes the experimental survival times, the initial and final body found in tumors of Group 2 rats (MNU + IAD) (Fig. 1). At Wk 52, weights, thyroid weight, and the effective numbers of rats in the incidences of thyroid tumors (adenoma and carcinoma) were each treatment group. Only animals that survived for more than significantly increased in Group 1 (MNU + IDD) as compared 27 experimental wk were included in effective numbers. All rats with other groups (P < 0.05). The incidences of thyroid tumors that had received MNU (Groups 1 to 3) became moribund prior in Group 2 (MNU + IAD) and Group 3 (MNU + WLB) were to Wk 52. Survival time, body, and thyroid weights were related significantly higher than in Control Groups 5 and 6 (P < 0.05). to MNU and the iodine-deficient diet exposure (Table 1). Follicular adenoma was found in 4 (40%) rats of Group 4 (IDD) Incidence of Tumors. The incidences of tumor-bearing rats at Wk 52 and in 12 (60%) rats at Wk 77. Those incidences were and number of organs with tumors per rat are also shown in significantly higher than those in Control Groups 5 and 6 (P < Table 1. Most rats (96.7 to 100%) in MNU treatment groups 0.05). Two follicular carcinomas were found at 77 wk in IDD rats. (Groups 1 to 3) had tumors at up to 8 different organ sites, the MÃ©tastases were not found in any rat in any group. Diffuse

CANCER RESEARCH VOL. 46 FEBRUARY 1986 878

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Fig. 2. Follicularcarcinoma of thyroid gland in Group 2 (MNU + IAD) at Wk 52. Fig. 4. Follicular carcinoma of thyroid gland in Group 4 (IDD) at Wk 77. Note Carcinomaappeared in follicular cystadenoma. H & E, x 54. hyperplasticgoiter in adjacent thyroid. H & E, x 25.

CANCER RESEARCH VOL. 46 FEBRUARY 1986 879

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Table 3 Histological findings in the pituitary gland of male F344 rats given MNU and/or IDD lesionsGroup No. of rats with

tumorsAdenoma4(14) mental tive period(wk)52 no. of Treatment1 rats29 (focal)0 MNU + IDD (90)6' Â° 6(21)c 22 (79)c 2 MNU + IAD 52 28 0 4(14) 15(54f 1(4) 13 (43)c 16(53)c 3 MNU + WLB 52 30 0 0 4(13) 1(3) 0 4IDD5 52 10 0 0 3(30) 1(10) 2(20) 0 5(50) 77 20 0 1(5) 8(40) 0 5(25) 0 13(65) IAD 52 10 1(10) 0 0 0 1(10) 0 1(10) 77 19 2(11) 3(16) 6(32) 1(5) 3(16) 0 13(68) 6 WLBExperi 52 11 0 0 0 1(9) 1(9) 0 1(9) 77Effec 19HyperplasiaBasophilDiffuse260Focal01(4) 2(11)Chromophobe5(26)Cyst1(3) 4(21)Pituitary8(42)Carcinoma00AnyFPPL"4(14)15(79) " Focal proliferative pituitary lesions include focal hyperplasia, adenoma, and carcinoma. 6 Numbers in parentheses, percentage. c Statistically significant differences (P < 0.05) between this group and appropriate carcinogen or diet controls. follicular hyperplasia was observed in all rats on IDD (Groups 1 in the recipient was seen, but no lung mÃ©tastaseswere found in and 4). rats receiving the first transplant passage. One carcinoma of the Transplantation of Thyroid Tumors. All primary thyroid tu second passage grew more rapidly and was accompanied by mors that were transplanted from rats receiving MNU and IDD lung metastasis 16 wk after transplantation (Figs. 5 and 6). were follicular carcinomas. Transplants were successful in 15 of Pituitary Pathology. The histological findings in the pituitary 18 rats. After 15 ~ 33 wk, 13 transplanted lesions (66.7%) grew gland in each group are summarized in Table 3. The incidence as follicular carcinomas, all but one with invasion (Fig. 5). The of diffuse basophil cell (thyrotroph) hyperplasia was significantly average tumor latency was 112 days (range, 29 to 231 days). greater in Group 1 (MNU + IDD) than in any other groups (P < Transplanted tumors attained diameters of 3 cm in some cases. 0.05). The incidence of pituitary tumors (adenoma and carci The histological appearance of the transplanted tumors was noma) in rats of Groups 2 and 3, receiving MNU, combined was similar to that of the primary tumors. Invasion of adjacent tissue significantly higher than in rats of Groups 5 and 6 combined that

CANCER RESEARCH VOL. 46 FEBRUARY 1986 880

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Fig. 8. Portion of pituitary adenoma (prolactinoma) immunoreactive for prolactin. Fig. 7. Focal prolactin cell hyperplasia in pituitary gland of rat given MNU. ABC Note solid pattern and some large cells. ABC immunocytochemistry, hematoxylin. immunocytochemistry, hematoxylin. x 330. x 330. had received no MNU (P < 0.05). The incidences of FPPL, decreased survival of rats in this group. The incidence of inter including focal basophil or chromophobe hyperplasia, adenoma, stitial cell tumor in Group 6 (WLB) was significantly higher than and carcinoma were significantly greater than in Groups 5 or 6 in Groups 4 and 5 (IDD or IAD; P < 0.05). (P < 0.05). Most basophil hyperplasia was associated with cytomegalic and vacuolar change, and these cells were immu- DISCUSSION noreactive for TSH (7). FPPL including focal hyperplasia (Fig. 7) and adenoma (Fig. 8) were almost always immunoreactive for The results of the present study confirm our previous findings prolactin, but not for TSH, and a few FPPL were negative for (7) that dietary iodine deficiency has a pronounced promoting both TSH and prolactin. Some adenomas with large bizarre cells effect on thyroid carcinogenesis in rats. Until recently, the carcin- were also immunoreactive for prolactin (Fig. 8). ogenicity of iodine deficiency for the thyroid had been primarily Pathology of Other Tissues. Proliferative lesions in various reported in female rats (11, 14-16). The incidence of thyroid organs of rats in each group are summarized in Table 4. A variety tumors (especially follicular adenoma) in these reports ranged of other tumors were found in MNU treatment groups (Groups 1 from 52 ~ 100%. In several of these reports, however, it is to 3) and control groups at Wk 77. There were no tumors found unclear if C-cell (parafollicular cell) tumors were induced by the except for those of the pituitary and thyroid glands in control dietary deficiency or if spontaneous tumors could be distin rats at Wk 52. The incidences of any proliferative lung lesions guished from induced tumors, since these papers were published (focal alveolar type II cell hyperplasia, adenoma, and adenocar- prior to the current classification systems for thyroid tumors (19, cinoma) in rats of MNU treatment groups were 59 ~ 96% and 23). In our experiments, it was clearly demonstrated that dietary significantly higher than those in control groups (P < 0.05). The iodine deficiency for 52 wk, in and of itself, led to goiter followed incidences of skin tumors in MNU treatment groups were 52 ~ by thyroid follicular neoplasms, but not C-cell neoplasms, in male 82% and significantly higher than those in control groups (P < F344 rats. Of those rats consuming the IDD diet, 70% developed 0.05). Oral tumors (papilloma and adenocarcinoma of tongue thyroid tumors after 77 wk (12 of 20 rats had follicular adenomas, and mucous membrane) were found in 19 of 58 rats (33%) in and 2 rats had follicular carcinomas). Our results are in agree Groups 2 and 3 (MNU + IAD or WLB) and were significantly ment with those of previous reports, but more clearly define the more common than in Groups 5 and 6 (IAD or WLB; P < 0.05). sequence of events. In our study, the majority of thyroid tumors The incidences of proliferative lung lesions and oral tumors in induced by the IDD were benign and were different morphologi Group 1 (MNU + IDD) were significantly lower than in Groups 2 cally than those in MNU-treated rats. Although the incidence of and 3 (MNU + IAD or WLB; P < 0.05), in part, because of follicular carcinoma induced by the IDD only in our present study

CANCER RESEARCH VOL. 46 FEBRUARY 1986 881

Table4 Incidence of proliferative lesions at various body sites of male F344 rats treated with MNU or IDD at 52 or 77 wk groups3SkinLungOral No. of rats with lesions in the following

(MNU+ (MNU + (MNU + (IDD, (IAD, (WLB, IDD, 29 IAD, 28 WLB, 30 20 rats 19 rats 19 rats typeEpidermaland lesion ratstotal)12(41)*'c2(7)03(10)12(41)c1(3)2(7)1(3)01(3)00003(10)02(7)001(3)00002(7)001(3)002ratstotal)19(68)Â°5(18)03(11)24ratstotal)12(40)c2(7)1(3)6(20)22total)00001(5)001(5)000001(5)0001(5)00000001(5)001(5)05total)1(5)000001(5)00000000000001(5)001(5)0000006total)1(5)0006(32)1(5)00009 cystPapillomaSebaceousinclusion

adenomaSquamous carcinomaFocalcell hyperpla-siaAdenomaAdenocarcinomaPapiltomaAdenocarcinomaOdontomaInterstitialalveolartype II cell (86)c6(21)2(7)8(29)c02(7)03(11)1(4)3(11)01(4)001(4)0001(4)0002(7)01(4)2(7)3(73)c2(7)2(7)10(33)c1(3)1(3)06(20)003(10)2(7)6(20)0001(3)1(3)00002(7)01(3)1(3)4

cavityTestisKidneyZymbal's

tumorMesenchymalcell (47f0000000002(11)000000000 tumorRenal celladenomaSquamous glandStomachIntestineLiverPancreasThymusSpleenParathyroidAdrenalPreputialpapillomaSquamouscell carcinomaPapillomaAdenocarcinomaHepatccellularcell

adenomaHemangtomaThymomaLarge

leukemiaHemangiomaHemangiosarcomaAdenomaCorticalgranular lymphocyte

adenomaPheochromocytomaAdenomaAstrocytomaSchwannomaOsteosarcoma1 glandBrainSpinal cordBoneSite

a Groups 1 to 3 are at Wk 52. Groups 4 to 6 are at Wk 77. No tumors developed in Groups 4 to 6 at Wk 52 except for thyroid and pituitary tumors. " Numbersin parentheses,percentage. c Statistically significant differences (P < 0.05) between this group and appropriate carcinogenor diet controls.

(10%) is still higher than that of spontaneous follicular carcinoma the vast majority showed invasiveness into adjacent connective in aging male F344 rats (0.72%) (19), the prolonged administra tissue. These results confirmed that these thyroid tumors in tion of the IDD after a single injection of MNU induced thyroid duced by MNU and the IDD were malignant, even in the absence follicular carcinomas after a significantly shorter latency period of pulmonary mÃ©tastases. and at higher incidence and multiplicity when compared with rats Spontaneous pituitary tumors are rare in rats under 18 mo of receiving only the IDD. In addition, most thyroid tumors induced age and become more common in older rats (18, 19). In the by MNU in rats on the IAD were benign. Therefore, it is suggested present study, although the incidence of pituitary tumors in that iodine deficiency may act primarily as a potent promoter control rats was higher than that seen in other studies (19), the rather than as an initiator of carcinogenesis or as a weak com incidence of pituitary tumors in MNU-treated rats was signifi plete carcinogen. From the findings of our previous study (7) and cantly higher than in our controls. Therefore, these results sug the present investigation, the quantitation and timed sequence gest for the first time that MNU is carcinogenic for the pituitary of these events can readily be seen. If rats are killed early in the gland, as well as the thyroid gland and many other tissues. In experiment (20 to 33 wk), IDD does not induce tumors. In rats rats given MNU alone (Groups 2 and 3), 29 of 38 rats (76%) with killed between 52 and 77 wk, IDD caused thyroid tumors. Like FPPL had thyroid tumors, and 65% (28 of 43 rats) with thyroid other nongenotoxic agents (24), the IDD is, by itself, both a tumors had FPPL. On the other hand, among control rats potent tumor promoter and a weak carcinogen. We suggest that (Groups 5 and 6), only 19% (5 of 27 rats) had FPPL associated the potent hyperplastic stimulus of IDD on the thyroid could play with thyroid tumors, and 83% (5 of 6 rats) of rats with thyroid a significant role in tumor promotion. Its role, however, in tumor tumors had FPPL. Surprisingly, however, the vast majority of promotion and carcinogenesis is undefined in both rats and MNU-treated rats developed tumors which contained prolactin humans (13, 25-27), although a working hypothesis is that a and not TSH, even in rats that received IDD. Previously, basophil thyroid-stimulating hormone may be the mediator of carcinogen adenomas of the pituitary gland producing TSH were induced in esis and tumor promotion (28). rats by radioiodine (29). Our studies provide evidence that MNU Successful transplantation of a primary tumor does not nec is also carcinogenic for the rat pituitary gland. essarily indicate that the primary tumor is malignant, although malignant tumors can be differentiated from benign tumors by their invasiveness in transplant recipients (23). In the present ACKNOWLEDGMENTS study, 83% of the thyroid tumors induced by MNU and the IDD We thank Peter Lynch, Rosemary Riggs, Dan Logsden, Areitha Smith, Paul that were tested for transplantability were transplantable, and Donovan, Fred Argilan, Cindy Harris, Teddy Thompson, Kim Forsman, Kathy

CANCER RESEARCH VOL. 46 FEBRUARY 1986 882

Breeze, and Larry Ostby for excellent technical assistance. Dr. Michael Waalkes 13. McTiernan, A. M., Weiss, N. S., and Daling, J. R. Incidence of thyroid cancer and Joyce Vincent provided important editorial assistance. in women in relation to previous exposure to radiation therapy and history of thyroid disease. J. Nati. Cancer Inst., 73: 575-581, 1984. 14. Isler, H.. Leblond, C. P., and Axelrod, A. A. Influence of age and of iodine REFERENCES intake on the production of thyroid tumors in the rat. J. Nati. Cancer Inst., 27: 1065-1081,1958. 1. Hiasa, Y., Ohshima, M., Kitahori, Y., Yuasa, T., Fujita, T., and Iwata, C. 15. Isler, H. Effect of iodine on thyroid tumors induced in the rat by a low-iodine Promoting effects of 3-amino-1,2,4-trizole on the development of thyroid diet. J. Nati. Cancer Inst., 23: 675-693,1959. tumors in rats treated with W-bis(2-hydroxypropyl)nitrosamine. Carcinogenesis 16. Kimura, S., Suwa, J., Ito, M., and Sato, H. 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CANCER RESEARCH VOL. 46 FEBRUARY 1986

883

Masato Ohshima and Jerrold M. Ward

Cancer Res 1986;46:877-883.

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