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Radioiodine-131 released from nuclear reactor accidents has dramatically increased the incidence of papillary thyroid cancer in exposed individuals. The deposition of ionizing radiation in cells results in double-strand DNA breaks (DSB) at fragile sites, and this early event can generate oncogenic rearrangements that eventually cause cancer. The aims of this study were to develop a method to show DNA DSBs induced by (131)I in thyroid cells; to test monovalent anions that are transported by the sodium/iodide symporter to determine whether they prevent (131)I-induced DSB; and to test other radioprotective agents for their effect on irradiated thyroid cells. Rat FRTL-5 thyroid cells were incubated with (131)I. DSBs were measured by nuclear immunofluorescence using antibodies to p53-binding protein 1 or γH2AX. Incubation with 1-10 μCi (131)I per milliliter for 90 min resulted in a dose-related increase of DSBs; the number of DSBs increased from a baseline of 4-15% before radiation to 65-90% after radiation. GH3 or CHO cells that do not transport iodide did not develop DSBs when incubated with (131)I. Incubation with 20-100 μm iodide or thiocyanate markedly attenuated DSBs. Perchlorate was about 6 times more potent than iodide or thiocyanate(.) The effects of the anions were much greater when each was added 30-120 min before the (131)I. Two natural organic compounds recently shown to provide radiation protection partially prevented DSBs caused by (131)I and had an additive effect with perchlorate. In conclusion, we developed a thyroid cell model to quantify the mitogenic effect of (131)I. (131)I causes DNA DSBs in FRTL-5 cells and had no effect on cells that do not transport iodide. Perchlorate, iodide, and thiocyanate protect against DSBs induced by (131)I.

In basal conditions, thyroid epithelial cells produce moderate amounts of reactive oxygen species (ROS) that are physiologically required for thyroid hormone synthesis. They are not necessarily toxic because they are continuously detoxified either in the process of hormone synthesis or by endogenous antioxidant systems. Using a rat model of goiter formation and iodine-induced involution, we found that compared with control thyroids, the oxidative stress, assessed by the detection of 4-hydroxynonenal, was strongly enhanced both in hyperplastic and involuting glands. The level of antioxidant defenses (glutathione peroxidases and peroxiredoxins) was also up-regulated in both groups, although somewhat less in the latter. Of note, increased oxidative stress came along with an inflammatory reaction, but only in involuting glands, suggesting that although antioxidant systems can adequately buffer a heavy load of ROS in goiter, it is not necessarily the case in involuting glands. The effects of 15-deoxy-Delta(12,14)-prostaglandin J2 (15dPGJ2), an endogenous ligand of peroxisome proliferated-activated receptor gamma (PPARgamma) with antiinflammatory properties, were then investigated in involuting glands. This drug strongly reduced both 4-hydroxynonenal staining and the inflammatory reaction, indicating that it can block iodine-induced cytotoxicity. When experiments were carried out with the PPARgamma antagonist, bisphenol A diglycidyl ether, 15dPGJ2-induced effects remained unchanged, suggesting that these effects were not mediated by PPARgamma. In conclusion, thyroid epithelial cells are well adapted to endogenously produced ROS in basal and goitrous conditions. In iodine-induced goiter involution, the increased oxidative stress is accompanied by inflammation that can be blocked by 15dPGJ2 through PPARgamma-independent protective effects.

Endocrine disrupting chemicals (EDCs), either plant constituents or contaminants deriving from industrial products, may interfere with the thyroid hormone (TH) axis. Here, we examined whether selected EDCs inhibit the key reactions of TH biosynthesis catalyzed by thyroid peroxidase (TPO). We used a novel in vitro assay based on human recombinant TPO (hrTPO) stably transfected into the human follicular thyroid carcinoma cell line FTC-238. F21388 (synthetic flavonoid), bisphenol A (building block for polycarbonates), and the UV filter benzophenone 2 (BP2) inhibited hrTPO. BP2 is contained in numerous cosmetics of daily use and may be in regular contact with human skin. Half-maximal inhibition in the guaiacol assay occurred at 450 nmol/liter BP2, a concentration 20- and 200-fold lower than those required in case of the TPO-inhibiting antithyroid drugs methimazole and propylthiouracil, respectively. BP2 at 300 nmol/liter combined with the TPO substrate H(2)O(2) (10 mumol/liter) inactivated hrTPO; this was, however, prevented by micromolar amounts of iodide. BP2 did not inhibit iodide uptake into FRTL-5 cells. In BP2-treated rats (333 and 1000 mg/kg body weight), serum total T(4) was significantly decreased and serum thyrotropin was significantly increased. TPO activities in the thyroids of treated animals were unchanged, a finding also described for methimazole and propylthiouracil. Thus, EDCs, most potently BP2, may disturb TH homeostasis by inhibiting or inactivating TPO, effects that are even more pronounced in the absence of iodide. This new challenge for endocrine regulation must be considered in the context of a still prevailing iodide deficiency in many parts of the world.

It is believed that a combination of environmental factors with mutagens induces carcinomas derived from thyroid follicular cells. In this study we tried to ascertain whether a single short-term exposure to external radiation is sufficient to induce thyroid carcinomas in rats under long-term high or low dietary iodine intake. Rats were tested over a period of 110 wk under high (approximately 10-fold of normal), normal, and low (approximately 0.1-fold of normal) daily iodine intake. Forty-day-old animals were subjected to single external radiation of 4 Gy or sham radiation. Thyroid function was tested weekly, and thyroid morphology was determined after 15, 35, 55, and 110 wk. Iodine deficiency, but not high iodine intake, led to a decrease in T(3) and T(4) plasma levels, but to an increase in TSH, which became significant after 9 and 11 wk of treatment, respectively. Both high and low iodine treatment significantly increased the proliferation rate and induced thyroid adenomas, but no malignancies after 55 and 110 wk. Radiation with 4 Gy resulted in a significant destruction of the follicular structure. Under high and low iodine intakes (50-80% of animals), but not under normal iodine supply, thyroid carcinomas were observed in irradiated rats. Thus, the increased proliferation rate induced under the experimental conditions described in this study is apparently not sufficient to cause thyroid carcinomas, but the presence of a mutagen-like radiation is required. This model may help to define genetic alterations long before histological changes are detectable.

Immunoreactive epidermal growth factor (EGF) has been detected in the thyroid, which raises questions concerning the source of thyroidal EGF and what affects its levels. We therefore have examined the effects of manipulating thyroid function on the immunoreactive EGF levels in plasma, thyroid, submaxillary gland (SMG), and kidney of adult male BALB/c mice, and we also analyzed the prepro-EGF messenger RNA in these tissues. Groups of six mice received daily injection of T4 (1 microgram, 5 micrograms, or 25 micrograms) or bovine TSH (1 mU) for up to 14 days. The plasma EGF concentration was 0.31 +/- 0.01 ng/ml in control animals, and T4 (5 micrograms) decreased the plasma levels by about one-half within 1 week. The thyroidal EGF was 0.50 +/- 0.14 ng/mg protein in control animals, and T4 (5 micrograms) increased the thyroidal EGF 8-fold within 1 week. There was a negative correlation between plasma and thyroidal EGF concentration (r = -0.93, P less than 0.01). Increasing doses of T4 also increased the SMG EGF content, while plasma levels fell (r = -0.88, P less than 0.01). The TSH treatment did not significantly alter the plasma or tissue EGF levels. Studies on mRNA prepared from these three tissues, using the reverse transcription and the polymerase chain reaction, indicated that the prepro-EGF mRNA was present in thyroid, SMG, and kidney. In conclusion, it appears that at least some thyroidal EGF is synthesized in the thyroid. Our observations that T4 increases the level of intrathyroidal EGF in a dose- and time-dependent fashion, while plasma levels fall, suggest the possibility that intrathyroidal EGF represents a shortloop feedback for the autocrine and/or paracrine regulation of thyroid function.

Previous studies have shown decreased T3 and increased T4 and rT3 serum levels in subjects treated with amiodarone. We studied the acute effect of amiodarone on the in vitro conversion of T4 to T3 in suspensions of isolated rat hepatocytes. Iodine and melperone, another class III antiarrhythmic drug, were included in the study as controls. Amiodarone (60 microM) totally blocked the formation of T3 from T4, whereas concentrations of 6 and 0.6 microM reduced T3 formation to 13.7 +/- 10.6% and 63.9 +/- 15.9% (+/- SD), respectively, compared with untreated controls. The drug solvent did not affect the conversion rate. Iodide (120 microM) had an inhibitory effect of approximately 10% compared with the controls, whereas melperone did not affect this in vitro conversion. Amiodarone (60 microM) caused a slight but significant reduction of the cellular uptake of [125I]T4 after 10 min of incubation, whereas the 60 min values were unaltered. Our study indicates that amiodarone inhibits the 5'-monodeiodination of T4 to T3 in a dose-related manner. It is suggested that the antiarrhythmic activity of the drug is related to its inhibitory effect on the conversion of T4 to T3.

The nonmetabolizable glucose analogs, [3H]2-deoxy-D-glucose and [3H]O-methyl-D-glucose, were used to determine whether iodide influences glucose transport in porcine cells in primary culture. Incubation with iodide (3 h) decreased basal glucose transport with a half-maximum at NaI 3 X 10(-5) M and maximum at 10(-4) M. Iodide (10(-6) M to 10(-4) M) also abolished the stimulatory effect of TSH (1 mU/ml) on glucose transport. The iodide effect on [3H]2-deoxy-D-glucose transport had the following characteristics: 1) it was abolished 24 h after incubation in iodide-free medium; 2) it was prevented by methimazole (3 mM), and correlated with newly formed organic iodine, 3) and it affected the maximum velocity (Vmax) of glucose transport, reducing it from 25.1 to 14.4 and 12.0 nmol/(min mg protein) at 10(-5) M and 10(-4) M NaI, without affecting the Michaelis-Menten constant (Km) (6mM). Iodide-treated cells had a reduced specific binding of [3H]cytochalasin B (38% and 47% with respect to control cells at 10(-5) M and 10(-4) M NaI). These data suggest that iodide treatment reduces the functional carriers mediating glucose transport in the thyroid.

We have explored the mechanisms whereby iodine inhibits thyroid growth using as models both the FRTL5 line of rat thyroid follicular cells that require TSH for growth and the M12 line of mutant cells that grow in the absence of TSH. Between 0.01-1.0 mM, NaI produced a dose-dependent inhibition of TSH stimulation of [3H]thymidine incorporation and replication in FRTL5 cells as well as spontaneous growth in M12 cells. Iodide also inhibited the cAMP-dependent growth of FRTL5 cells induced by forskolin and (Bu)2cAMP, as well as the cAMP-independent mitogenesis induced by insulin-like growth factor-I. The effect of iodide to inhibit both TSH- and insulin-like growth factor-I-stimulated growth in FRTL5 cells was abolished by concomitant culture with methimazole, and no iodide inhibition of growth was observed in L6 myoblasts and BRL 30E hepatocytes. Exposure of cells to iodide under conditions that resulted in inhibition of TSH-stimulated growth did not significantly alter the ability of TSH to increase the intracellular cAMP concentration, nor did iodide alter two responses to TSH in FRTL5 cells that depend upon an increase in cAMP concentration: down-regulation of TSH receptor and cytoskeletal reorganization. We conclude that iodide exerts its inhibitory action on the growth of thyroid cells at multiple loci related to both the cAMP-dependent and cAMP-independent pathways of mitogenic regulation.

Rats maintained on a low-iodine diet were hypophysectomized, and their diet was than enriched with iodide. Cyclic AMP (cAMP) concentrations achieved in their thyroids following in vitro TSH stimulation were significantly lower than those in the thyroids of control animals that did not receive dietary iodide enrichment. The addition of 0.1% methimazole (MMI) or 1% KC1O4 to the diet abolished this inhibitory effect of iodide. The administration of triiodothyronine in the died did not reproduce the inhibitory effect of iodide. The effect of iodide in vitro on the thyroid cAMP response to TSH was then investigated using paired thyroid lobes obtained from intact rats fed a low-iodine diet. During a 15-min incubation period, concentrations of iodide up to 10(-3)M, together with TSH (125 mU/ml), did not affect the thyroid cAMP response to TSH. In contrast, the preincubation of the lobes in 5 X 10(-5)M Nal for 2 h preceding a final 15-min incubation in medium containing TSH alone resulted in final cAMP concentrations significantly lower than those in paired lobes not exposed to iodide. Basal cAMP concentrations in thyroids not subjected to TSH stimulation were unaffected by preincubation in iodide. The inclusion of TSH during the preincubation period augmented the inhibitory effect of iodide on the final thyroid cAMP concentration achieved. The inclusion of MMI together with iodide during the preincubation period abolished the inhibitory effect of iodide on the final cAMP concentration achieved by TSH stimulation. Direct measurement of newly formed organic iodine in vitro demonstrated it to be inversely proportional to the final cAMP concentration achieved by TSH stimulation. The preincubation of thyroid lobes in iodide was without effect on the subsequent stimulation of cAMP by PGE1, or on the stimulation by F- of adenylate cyclase activity in the thyroid homogenate. The data support the concept of an as yet unknown organic form of iodine that limits thyroid adenylate cyclase responsiveness to TSH stimulation. This may, in part, explain the diverse, and generally inhibitory, actions of iodide on thyroid function.

Thyroid hormone biosynthesis depends upon the presence of adequate amounts of thyroidal iodine, and during fluctuations in dietary iodine intake, relatively constant thyroid hormone levels are maintained by various homeostatic mechanisms. These mechanisms include an enhancement of iodide pump efficiency and organification when iodine intake is limited, and significant decreases in iodide uptake and hormone synthesis when excess iodine intake occurs. The present study was designed to determine whether acclimation to different dietary iodine regimens is associated with changes in thyroid blood flow and to assess the time course of any such alterations in relation to pituitary-thyroid axis hormone levels. Male Sprague-Dawley rats were fed a diet containing low (LID), high (HID), or normal (CTR) iodine concentrations. Three, 7, 14, or 133 days after starting these dietary regimens, the animals were anesthetized with ketamine/pentobarbital, and thyroid blood flows were assessed using the reference sample version of the microsphere technique. At the same times and at weekly intervals throughout the 133 days of treatment, blood samples for the determination of TSH, T4, and T3 levels were obtained. Additionally, thyroidal immunoreactive vasoactive intestinal peptide (VIP) was measured at the end of the experiments. LID treatment increased thyroid blood flows to 240%, 350%, and 240% of levels in control rats at 7, 14, and 133 days of treatment, respectively. Thyroid weight was also elevated above levels in control animals at each of these times. A slight decrease in plasma T4 levels occurred over the 133 days of LID treatment; however, this dietary regimen did not alter circulating levels of T3 or TSH or thyroidal VIP concentration. HID treatment had opposite effects, in general, to those of LID. Thyroid blood flows were decreased by 34%, 56%, 46%, and 35% after 3, 7, 14, and 133 days of treatment with HID, respectively. Circulating levels of T4 were increased over the 133 days of HID treatment, whereas plasma levels of T3 and TSH and thyroid weights remained unchanged from those in control rats over this period of study. A small decrease in thyroidal VIP concentrations coincident with the decrease in thyroid blood flow was observed at the beginning of the HID treatment. Neither LID nor HID had any effect on blood pressure, cardiac output, or blood flow in other organs. These data demonstrate that acclimation to changes in dietary iodine intake in the rat include alterations in thyroid blood flow which are reciprocal to the iodine intake level and appear to be independent of circulating TSH levels.

Studies were conducted to define more clearly the site in the thyroid adenylate cyclase complex at which iodine exerts its inhibitory effect on activation of this enzyme by TSH. Iodine- and TSH-induced desensitization were additive. Dissociation was observed between the rates of recovery from TSH- and iodine-induced desensitization. Cycloheximide (10(-4) M) prevented recovery from the inhibitory effect of iodine on thyroid adenylate cyclase activation. Preincubation of freshly isolated dog thyroid follicles in 10(-4) M iodide decreased the subsequent cAMP response to cholera toxin (0.5 micrograms/ml) stimulation. This effect of iodide was prevented by 3 mM methimazole. Thyroid adenylate cyclase regulatory protein (Ns) activity was assessed by the ability of detergent extracts of thyroid plasma membranes to reconstitute adenylate cyclase responsiveness to isoproterenol in N-deficient S49 cyc- plasma membranes. Thyroid Ns activities were similar in control and iodide-pretreated thyroid cells. The inhibitory effect of iodine on TSH activation of thyroid cAMP generation was additive to that of inhibition via the alpha 2- adrenergic pathway and also additive to inhibition by 2',5'-dideoxyadenosine (an adenosine P-site agonist). Preincubation of freshly dispersed dog thyroid cells in 10(-4) M NaI reduced the cAMP response to stimulation by 100 microM forskolin. These data provide evidence that in iodine-induced TSH desensitization in the thyroid; 1) TSH receptor function is normal, 2) the regulatory protein (Ns) in the adenylate cyclase stimulatory pathway is functionally unaltered, 3) iodine does not exert its effect via the regulatory protein (Ni) in the pathway that inhibits adenylate cyclase activation, 4) iodine does not act via the adenosine P-site inhibitory pathway, 5) the action of iodine is at or near the adenylate cyclase catalytic unit, and 6) new protein synthesis is necessary for recovery from iodine desensitization.

Delayed morphological changes induced in mouse hyperplastic thyroid by refeeding iodine were analyzed by light and electron microscopy, stereology, and autoradiography. Thyroid hyperplasia was induced by a low iodine diet supplemented with 0.25% propylthiouracil for 10 days. Involution was obtained by discontinuing the propylthiouracil and returning either to a moderate iodine diet [(MID) 1 microgram I/day] or to an iodine-rich diet [(HID) 10 micrograms I/day] for 40 days. In other experiments, three cycles of hyperplasia (8 days) and subsequent involution (8 days) with MID or HID were brought about. Control animals were fed MID or HID. All animals were killed when 12-14 weeks old after injection of 10-50 microCi 125I. Double labeling, with repeated injections of [3H]thymidine from day 0 to day 7 of involution followed by 125I injection 4 h before killing, was also performed. When involutions were performed with MID, most morphological variables returned to control values. However, when involution was brought about with HID, the glandular weight, the number of follicles, and the relative volume of follicular lumina remained larger than in controls. Moreover, the 125I-labeling pattern of the follicles was altered. The proportions of unlabeled, and unevenly or partly labeled, follicles, which were fewer than 5% in control groups, represented 25-35% of all follicles after involution with HID, whereas they were unchanged with MID. In unlabeled follicles the epithelium was flattened, with a reduced number of microvilli. Partly labeled follicles were of two types. In some follicles a persistent ring reaction was observed, suggesting an abnormally slow mixing of thyroglobulin. In others, the 125I labeling was restricted to areas adjacent to the apex of a reduced number of cells, suggesting that some cells were iodinating thyroglobulin, whereas others were not. There was no relationship between the follicular 125I labeling and the frequency of [3H]thymidine-labeled cells. These results indicate that refeeding iodine excess after hyperplasia leads to the formation of a colloid goiter with new follicles, and to an increased heterogeneity of iodine metabolism among follicles and among cells.

In order to obtain further information about the stimulatory action of excess iodide on thyroid hormone secretion in thyroxine (T4)-treated rats, experiments were performed in hypophysectomized rats, or rats treated with graded doses of T4 or triiodothyronine (T3).T3 as well as T4 played a permissive role in the production of the iodide effect in normal animals, but T3 was more effective than T4. Excess iodide stimulated thyroid hormone secretion in hypophysectomized animals, this finding being compatible with the hypothesis that, by inhibiting TSH secretion, T3 and T4 produced a condition in which excess iodide stimulated thyroid hormone secretion in intact rats. However, T4 played an additional role in thyroid hormone secretion by acting directly on the thyroid. In hypophysectomized animals, small doses of T4 stimulated thyroid hormone secretion, and this action was additive to that of excess iodide, whereas large doses of T4 were inhibitory and reduced the effectiveness of excess iodide. The stimulatory action on thyroid hormone secretion was specific for iodide and was not shared by other anions. The action of excess iodide was blocked by methimazole. We suggest that excess iodide stimulates thyroid hormone secretion by increasing intrathyroidal concentrations of cyclic AMP in the absence of TSH, and that this increase in cyclic AMP concentration is blocked by methimazole.

Studies were carried out to characterize the cyclic nucleotide phosphodiesterase from rat calvaria. 25-40% of the total enzyme activity was membrane-bound. pH, magnesium, and temperature requirements conformed closely to those established for phosphodiesterase from other tissues. Kinetic evidence was found for dual enzyme activities with different substrate affinities for both the particulate and soluble enzyme. Apparent Kms for the soluble enzyme (3.5 times 10-6 and 2.5 times 10-5M) approximated those for the particulate enzyme (5.7 times 10-6 and 2.5 times 10-5M). L-Thyroxine, 10-5M, inhibited competitively the low- and high-Km enzymes from both the particulate and soluble fractions (Ki equals 1.7 times 10-5M). T4 was more potent an inhibitor than T3 with all enzyme fractions, but this relationship could be altered by adding protein to the incubation mixtures. Tests of diverse thyroid hormone analogues showed that 1) T4 and its derivatives were more potent than T3 and its analogues; 2) acetic and propionic acid derivatives were more potent than the thyronines; 3) "reverse T3," an antagonist of some T3 actions, also inhibited phosphodiesterase. These effects were not attributable to chelation, and were not duplicated by iodide or by other physiologically inactive thyroid hormone analogues.

Involution of thyroid hyperplasia was induced in mice by discontinuing a goitrogenic treatment (low iodine diet plus 0.25% propylthiouracil for 10 days) and returning either to a moderate iodine diet (MID; 1 microgram I/day) alone or associated with T3 administration (1 microgram/day) or to a high iodine diet (HID; 10 micrograms I/day) alone or associated with T3 treatment. Thyroid involution was studied by morphological, stereological, and biochemical methods after 2, 4, 6, and 8 days of involution. Age-paired, HID-fed animals were used as controls. When the involution was induced by MID, the glands resumed a normal morphological aspect. The synthesis and secretion of T3 were highly stimulated on day 2, but decreased thereafter. Plasma T4 levels reached a plateau at 50% of the control value from days 2-8. The administration of T3 together with MID accelerated the involution of hyperplasia and colloid accumulation in the follicular lumina. The synthesis and secretion of T3 and T4 remained lower than those in controls. When the involution was induced by HID, the thyroid weight remained higher than that in controls or in any involuting groups. The number of follicles and epithelial cells as well as the glandular thyroglobulin content were twice the control values. A Wolff-Chaikoff effect was evident on day 4, and hypothyroidism persisted. When HID was supplemented with T3 treatment, glandular weight and morphology were normal, but the Wolff-Chaikoff effect occurred earlier. In conclusion, the iodine dose given after a goitrogenic treatment must be carefully controlled; a high but physiological dose can have deleterious effects, whereas a small dose is beneficial. T3 prevents the deleterious effects of HID, but the thyroid enters a resting state.

From the earliest detectable development of fetal pituitary-thyroid function (day 18-19 of gestation) through the first postnatal day, there was a higher degree of stimulation of the pituitary-thyroid axis in the fetuses of rats fed a low iodine diet (LID) than in those of rats fed a high iodine diet (HID). Significant differences between the two groups were consistently observed in relative thyroid size, plasma TSH, 4-h thyroid radioiodine uptake, and the labeled iodoamino acid composition of thyroid digests. Plasma T4 concentration was lower in both LID and HID fetuses and pups than in the HID mothers. Plasma T3 was not detectable (less than 20 ng/dl) in the fetuses of either group, nor was labeled T3 in the thyroid digests. Body weight, plasma T4, and pituitary TSH content were usually lower in the LID than the HID animals of comparable age; however, these differences were not consistently statistically significant (P less than 0.05). We conclude that iodine deficiency causes a marked stimulation of TSH secretion and, consequently, of thyroid growth and metabolism from the earliest development of fetal pituitary-thyroid function.