Perchlorate Ion Enhances Mouse Thyroid Responsiveness to Thyrotropin, Human Chorionic Gonadotropin and Long Acting Thyroid Stimulator BERNARD ROUSSET,* JACQUES ORGIAZZL AND REN& MORNEX Groupe de Physiopathologie France


Hopital de I'Antiquaille,

chlorate effect was obtained with a dose (12.5 ptg) which, when tested in different experimental conditions (MMI-blocked thyroid), discharged 80% of intrathyroidal radioiodide. Perchlorate exerted its augmenting effect by enhancing thryoid secretion: it increased plasma radioiodothyronines and radioiodide concentrations without decreasing the blood disappearance rates of iodide and iodothyronines. The potentiating effect of perchlorate probably takes place at a step prior to cyclic AMP action since it did not affect dbcAMP-stimulated secretion. The perchlorate effect may be indirect, through mobilization of minute amounts of intrathyroidal iodide. (Endocrinology 100: 1628, 1977)

ABSTRACT. Perchlorate treatment of mice increased by 1.5-2-fold the thyroid secretory response to TSH, hCG and LATS, in the McKenzie bioassay. Perchlorate alone did not increase basal plasma radioactivity. Perchlorate augmentation of the secretory response index was roughly proportional to the level of stimulation; it was similar for all three stimulators despite their different time courses of action which were unaltered by perchlorate; it was the same whether perchlorate administration preceded, coincided with or shortly followed injection of the stimulator, a finding in keeping with the slow clearance of this ion. The perchlorate effect was dose-related, although within a narrow range (6.25-12.5/xg/mouse). Near-maximal per-


OME anions including perchlorate, thiocyanate andfluoroborateare known to interfere with several aspects of the metabolism of iodide in the thyroid gland (1-8). They inhibit iodide uptake (1,2) and discharge free intrathyroidal iodide either trapped or released by deiodination of iodotyrosines (7,8). Moreover, at high concentration, they inhibit organification of iodide (6). In addition to these effects, we have found that, in mice prepared for the thyrotropin (TSH) bioassay by the method of McKenzie (9) perchlorate increases the stimulatory effect of TSH and other stimulators on thyroid secretion. The characteristics of this effect of perchlorate are reported in the present paper.

Received September 9, 1976. Supported in part by INSERM grant no. 74-1-132-04. Part of this work was presented at the 7th annual meeting of the European Thyroid Association, Helsinki, June 1976. Abbreviations: MMI: Methimazole; dbcAMP:b N6, 2'-0-dibutyryl cyclic adenosine-3',5'-monophosphate. * To whom reprint requests should be addressed.

69321 Lyon Cedex 1,

Materials and Methods The following modification of the McKenzie technique (9) was used for measurement of thyroid stimulation. Swiss albino mice (10-15 g) were fed a low iodine diet (Licence ANVAR 69-195-00, brevet Triantaphyllidis; iodide content: 2.5 /u.g/100 g) and given tap water ad libitum for 2 or 3 weeks. Starting just after ip injection of 5 fid of carrier free Na125I, 2 /Ltg of L-thyroxine (L-T 4 ) was administered sc daily for 3 days. Twenty-four hours after the last dose of T4, the test material was injected into a tail vein in 0.5 ml of saline. Control mice received saline. The animals were bled by orbital puncture just before and at various times after the injection depending on the nature of the test material. Sodium perchlorate, potassium thiocyanate or MMI was injected ip or iv in saline before, simultaneously with or after the stimulator. The radioactivity of 0.1 ml of blood was measured in a Packard Autogamma scintillation spectrometer. The ratio of the radioactivities of the post-injection and the preinjection blood samples multiplied by 100 was taken as the secretory response index (SRI). The significance of the difference between results was assessed by Student's t test.


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C104- AND STIMULATED THYROID SECRETION To assess the effect in mice of various doses of perchlorate on iodide uptake or discharge, experiments were carried out as described in Results. The thyroid glands were excised with the trachea under ether anesthesia and counted for radioactivity. Plasma 125I-labeled compounds were analyzed by paper chromatography. Blood samples of 0.1ml were collected in heparin (120IU/ml), pooled and centrifuged at 3000 rpm for 10 min. Plasma aliquots of 0.075 or 0.1 ml were chromatographed on 4 cm-wide strips of Whatman 3 MM filter paper in a butanol-acetic acid-water system for 18 h at room temperature according to Shimoda and Greer (10). The strips of paper were then dried and peaks of radioactivity were located using a chromatogram scanner (Packard). For quantitative measurement, areas corresponding to the peak of radioactivity were cut out and counted for radioactivity. The Rfs of iodide and iodothyronines were, respectively, 0.16 and 0.85. Bovine TSH (1 IU/13 mg) was kindly supplied by the Medical Research Council (Mill-Hill, Great Britain). LATS plasma was obtained from a patient with Graves' disease (patient D.) by plasmapheresis. hCG was purchased from ISH laboratories (Paris, France), sodium perchlorate from Fluka (Buchs, Switzerland), potassium thiocyanate from Merck (Darmstadt, Germany), dbcAMP and MMI from Sigma (St Louis, Missouri) and L-T4 from Calbiochem (San Diego, California). The low iodine diet was provided by Usines Alimentaires Rationnelles (Villemoisson,


France), 125I by CEA (Saclay, France) and [125I]T4 (30 fxCi/fjig) by the Radiochemical Centre (Amersham, England). Results Effect of perchlorate on the response to TSH, hCG, and LATS When perchlorate, 50 />ig/mouse, was injected simultaneously with TSH, or a known thyroid stimulating preparation of hCG (11), or LATS, it enhanced the response to each stimulator. However, injected alone in similarly prepared mice, perchlorate had no effect (Table 1). With the stimulators used in this experiment, the time between injection and maximum effect ranged from 2 h for TSH and 8 h for hCG to 20 h for LATS (12). It should be noted that the concomitant injection of perchlorate did not alter the characteristic time course of action of each stimulator. For TSH, the results of this study are depicted in Fig. 1. In another set of experiments, perchlorate was injected 15 min, 60 min or 4 h before or 15 min after TSH. As shown in Table 2, this did not modify the augmenting effect of perchlorate on TSH stimulation. The same was true for LATS when perchlorate was injected 3 h after t h e stimulator.

TAHLE 1. Effect of C1O4 on TSH, hCG and LATS-stimulated thyroid secretion in the McKenzie bioassay Secretory response index at various times after stimulator injection C1(V (50 (jig/mouse)



(6) (6)


96 ± 2 100 ± 5

77 ± 3 90 ± 14

TSH (0.5 mU/mouse)

(7) (7)


855 ± 66 1,417 ± 91*

570 ± 44 734 ± 57*

hCG (1600 IU/mouse)

(6) (6)


466 ± 70 981 ± 198*

LATS serum (125 /i.l/mouse)

(12) (12)

— +

454 ± 63 942 ± 88*

Groups Saline

20 h 87 ± 105 ±

4 8

— 371 ± 64 830 ± 102* 1,078 ± 156 1,509 ± 129*

Where indicated, C1O4 was injected with the stimulator at zero time. Numbers in parentheses indicate the number of mice per group. Values represent the mean ± SEM. * Significant difference from control: P < 0.05.

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jag/mouse was ineffective. Doses of 12.5 fig and above increased the SRI 1.5 to 2-fold. From 10 experiments, it was calculated that a dose of 50 /xg/mouse of perchlorate increased the response to 0.25 mU of TSH by 1.57 ± 0.09 times (mean ± SE; P < 0.01) and the response to LATS (60 /xl of plasma D.) by 1.73 ± 0.08 times (P < 0.01). In an attempt to link this effect of perchlorate on the stimulated thyroid secretion to other known effects of this ion, we tested the ability of similar doses of perchlorate to inhibit radioiodide uptake and to alter the discharge of intrathyroidal radioiodide. In the first experiment, groups of



500 TSH 0.25 mil/mouse






Endo i 1977 Vo! 100 , No 6




FIG. 1 Lack of alteration by C104~ of the time course of stimulation of thyroid secretion by TSH. C104~ was injected simultaneously with TSH. Values are the mean ± SEM of determinations in six mice.

Effect of graded doses of perchlorate Figure 2A illustrates the effect of varying doses of perchlorate on the SRI increase induced by a constant dose of TSH (0.20 mU per mouse). A dose of perchlorate of 6.2






perchlorate in doses ranging from 6.2 to to 200 fig, 15 min before injection of 125 I". The thyroids were excised 2 h after radioiodide administration and counted for radioactivity to determine the 125I~ uptake. As shown in Fig. 2B, the 2 h-125I" uptake was decreased by 50% by 12.5 fig of perchlorate and completely blocked by 200 fig. In the second experiment, T4-treated mice received MMI (2 mg/mouse, ip, and then

TABLE 2. Lack of alteration of the augmenting effect of C1O4~ by desynchronization of stimulator and C1O4~ injections Schedule of injections of stimulator and C1O4~


- 1 5 min





cio 4 -

cio 4 -

— —


Saline + TSH C1O4" + TSH


Secretory response index

+ 15 min


Exp 1

545 ± 5 5 864 ± 68* 694 ± 58 909 ± 61*


cio 4 -

695 ± 30 909 ± 34*

341 ± 41 687 ± 43*

654 ± 36 806 ± 55*

443 ± 60 639 ± 40* 385 ± 28 709 ± 50*


Exp 2


cio 4 -

424 ± 44 707 ± 58*

TSH (0.20 mU/mouse) or LATS plasma (125 /xl) was injected at zero time. C1O4 (200 /ig/mouse) or saline was injected iv before, simultaneously with or after the stimulator, as indicated. The secretory response index was determined 2 h after TSH injection or 20 h after LATS injection. Values are the mean ± SEM of determinations on 8 or 10 mice. * Significant difference from control: P < 0.05.

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C104- AND STIMULATED THYROID SECRETION 0.6 mg/ml in drinking water) and 15 h later, an ip injection of 125I~. Perchlorate was administered iv 2 h after the radioiodide injection and the thyroids were collected 1 h later. A dose of 12.5 fig of perchlorate discharged 80% of intrathyroidal 125J- (Fig, 2C). The curves representing the effect of graded doses of perchlorate on the discharge of radioiodide and on the TSH-stimulated secretory response index were very similar: 12.5 fig of perchlorate induced a nearly maximal effect on both parameters. Effect of perchlorate on plasma radioiodothyronine and radioiodide concentrations


tio was not significantly altered by perchlorate. It ranged between 4.2 and 5.6 2 h after TSH injection but between 11.0 and 14.2 20 h after LATS injection, an observation we have previously reported (12). Since there was a possibility that perchlorate could increase plasma radioiodocompounds by altering their plasma disappearance rate, we studied the blood clearance of radioiodide and radiothyroxine in mice treated with perchlorate. T 4 -treated mice received MMI ip (2 mg and 0.5 mg 15 h and 1 h prior to 125I~ or [125I]T4 injection, respectively). Perchlorate (200 fig/ mouse) was injected ip 1 h before the iv

As shown in Table 3, both the plasma concentrations of radioiodide and radioiodothyronines were higher in mice injected with TSH or LATS and perchlorate than in mice injected with the stimulator alone. Increases in the concentrations of each plasma radiodinated compound were proportional to that of SRI. Accordingly, the plasma radioiodothyronine-radioiodide ra-

FIG. 2. Comparison of the action of graded doses of CIO.," on the TSH-stimulated SRI (A) and on thyroidal radioiodide metabolism: uptake (B) and discharge (C) in mice prepared for the McKenzie bioassay. (A) Mice were injected with 0.20 mU TSH and C104~ at the indicated doses. The C104~induced augmentation of the secretory response to TSH is expressed as per cent of the response to TSH alone. (B) and (C) Different experimental conditions were employed. (B) Mice were injected iv with various doses of C104- and 15 min later with 5/xCi of 125 r, ip; 2 h later, the thyroids were excised and counted for radioactivity; radioiodide thyroidal uptake was expressed as per cent of the thyroid radioactivity of control mice not receiving C104": (C) Mice were first treated with MMI, then 15 h later injected with 1 2 5 r and 2 h later with graded doses of C104"; thyroid radioactivity was measured 1 h later; at each dose of C104~, iodide discharge was expressed as Thyroid


I in control - Thyroid 125I in C1O4"-treated mice Thyroid 125I in control mice Control mice received saline. Each point represents the mean of determinations on 6 animals.


Oj 12.525 6.2 NaCIO* (po/mouse)

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Endo • 1977 Vol 100. No 6



TABLE 3. Effect of perchlorate on plasma radioiodothyronine and radioiodide concentrations Plasma radiocompounds Secretory response index

Blood radioactivity cpm/0.1 ml

706 ± 97 (12)

1,561 ± 215

TSH 0.25 mU/mouse + ClOr 12.5 /ig/mouse

1,157 ± 139 (12)*

TSH 0.25 mU/mouse + C1O4" 50 jug/mouse TSH 0.25 mU/mouse + C1O4" 200 /ig/mouse



TSH 0.25 mU/mouse


[125I]Iodothyronines (B) cpm/0.1 ml


274 ± 69

1,536 ± 19

5.6 ± 1.6

2,560 ± 308*

532 ±


2,830 ± 137*

5.3 ± 0.2

1,065 ± 60 (12)*

2,358 ± 132*

552 ± 90*

3,026 ± 176*

5.5 ± 0.6

1,164 ± 99 (12)*

2,576 ± 219*

715 ± 48*

3,027 ±221*

4.2 ± 0.4

l-Iodide (A) cpm/0.1 ml

714 ± 86 (12)

2,500 ± 302

259 ± 14

3,676 ±

1,127 ± 115 (18)*

3,947 ± 403*

441 ± 42*

4,792 ± 182*

11.0 * 1.1

LATS 250 /il/mouse

1,253 ± 86 (12)

4,376 ± 302

470 ± 112

6,242 ± 17

13.3 ± 3.3

LATS 250 /xl/mouse + C1O4" 50 /ig/mouse

2,037 ± 147 (18)*

7,133 ± 514*

721, ± 64*

10,137 i 500*

14.0 ± 1.7

LATS 125 fil/mouse


B m

LATS 125 /il/mouse + CIO.," 50 ng/mouse


14.2 ± 1.0

Mice prepared for the McKenzie bioassay were injected with C1O

Perchlorate ion enhances mouse thyroid responsiveness to thyrotropin, human chorionic gonadotropin and long acting thyroid stimulator.

Perchlorate Ion Enhances Mouse Thyroid Responsiveness to Thyrotropin, Human Chorionic Gonadotropin and Long Acting Thyroid Stimulator BERNARD ROUSSET,...
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