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Antithyroid activity of some naturally occurring isothiocyanates in vitro
Antithyroid Activity of Some Naturally Occurring
Isothiocyanates
In Vitro
By PAVEL LANGER ANDMONTE A. GREER Allyl-, methyl-, and butyl-isothiocyanate profoundly depressed the iodination of amino acids by rat thyroid lobes in an in vitro system. Allylisothiocyanate, being the most soluble of the three, was studied in concentrations up to 10-l M. At higher concentrations, allylisothiocyanate depressed thyroidal iodide concentration
as well as thyroidal biosynthesis. The effect of high concentrations was probably due to non-specific toxicity since it was not removed by leaching. Allylisothiocyanate, but not butylisothiocyanate, rapidly decomposed in aqueous solution. (Metabolism 17: No. 7, July, 596-605, 1968)
I
SOTHIOCYANATES (mustard oils) are present in the edible portions of many species of plants and in their seeds in the form of thioglycoside precursors. The isothiocyanates are formed from the aglycone liberated from the thioglycosides during enzymatic hydrolysis. Since the isothiocyanates are present in particularly high concentration in the cabbage family, members of which have been shown to have antithyroid activity in both animals and man, there has been recurring speculation that these compounds might be goitrogenie. However, there is no clear consensus as to whether these substances actually do possess antithyroid activity. Several authors, without presenting the exact conditions of their experiments, have reported that mustard oils have no effect upon the thyroid. lv2 Clements3 stated that benzyl isothiocyanate is goitrogenic in rats but gave no experimental details. Hercus and Purves4 speculated that the goitrogenic activity of Brassica seeds might be caused by thioglycosides. Wagner-Jauregg and Koch5 observed some goitrogenic activity when the thioglycoside, sinalbin, was administered simultaneously orally with the enzyme myrosinase. Bachelard and Trikoju@ reported that methyl-sulfonyl-propyl-isothiocyanate and n-propyl-isothiocyanate were weak inhibitors of radioiodine uptake in rats. In previous experiments we have observed thyroid hypertrophy when allylisothiocyanate was administered to rats in various doses for periods of 26 to is one of the most wide-spread isothiocyanates 60 days. 7,8 Allylisothiocyanate From the Division of Endocrinology, Department of Medicine, University of Oregon Medical School, Portland, Oregon. Supported by grants from the National Institutes of Health, U. S. Public Health Seruice. Receiued for publication July 27, 1967. PAVEL LANGER, M.D.: Institute of Endocrinology, Slovak Academy of Sciences, Rratishzva, Czechoslovakia. This work was carried out while DT. Lunger was a visiting scientist in th.e Division of Endocrinology, University of Oregon Medical School. MONTE A. GREER, M.D.: Professor of Medicine; Head, Division of Endocrinology, Department of Medicine, University of Oregon Medical School, Portland, Oregon. 596
ANTITHYROID
597
AmIVITY
in the plant kingdom; it therefore appeared worthwhile to investigate its antithyroid activity in more detail. Allylisothiocyanate deteriorates in aqueous solution and is also probably metabolized rapidly, thus we felt it would be most profitable to study its effect in an in vitro system where the material could be tested immediately after being put in solution and with minimal potential changes having taken place in the isothiocyanate molecule. Since the mustard oils are quite irritating, we also concluded it would be possible to study the effects of higher concentrations in vitro than in viva, MATERIALS
AND METHODS
Young, adult, male Holtzman rats were fed a low iodine diet (General Biochemicals, Inc.) containing approximately 30 pg. of iodine per Kg. for 7 days. The animals were killed with chloroform and the thyroid lobes were carefully removed, each lobe being placed in 2.5 ml. of Krebs-Ringer phosphate (KRP) solution containing the indicated test substance. Twenty-five PC. carrier-free 1311 in 0.5 ml. KRP were then added. The flasks were incubated for 15 minutes to 3 hours in a shaking water bath at 37” C. with air as the gas phase, The thyroid lobes were then removed, washed with I to 2 ml. of KRP and placed in tubes containing 0.5 ml. of 0.2 per cent methimazole in pH 8.5 phosphate buffer. Such rinsing removes approximately 2 per cent of the intrathyroidal iodide but none of the organicallybound 1slI.a The radioactivity in each lobe was measured in a well-type scintillation counter and the lobes were then homogenized. One-half ml of a 2 per cent solution of pancreatin (Viokase 4X) in the same phosphate buffer was added and the samples were hydrolyzed for 18 hours in a shaking water bath at 37’ C. under one to two drops of toluene. Aliquots of the hydrolysates containing 0.05 PC. were applied to strips of Whatman No. 3 chromatographic paper and developed in an ascending butanol-acetic acid-water (4:1:5) system. Chromatograms were scanned with an automatic scanner. Identity of the radioactive peaks was determined by comparing the localization of authentic stable compounds added to the hydrolysates on the paper. Iodinated ammo acids were detected with a diazotized sulfanilic acid spray reagent. The quantity of each radioiodinated substance was determined by planimetry of the scans. The “absolute” amount of radioiodinated material present was determined by multiplying the relative per cent of each compound expressed as the per cent of total thyroidal radioactivity times the radioiodine uptake. In each experiment two to four control lobes were incubated in KRP alone. The other lobes were incubated in varying concentrations of allylisothiocyanate (AITC), butylisothiocyanate (BITC) and methylisothiocyanate (MITC), ranging from 0.025 to 100 mM. In all experiments two lobes from separate animals were used for each concentration of test substance. The results from the duplicate lobes were averaged to ,give a mean value. AITC was obtained from Matheson, Coleman & Bell, BITC and MITC from Distillation Products Co., a division of Eastman Kodak. All specimens were stated to be of the highest obtainable purity (94-99 per cent). The isothiocyanates used are only slightly soluble in aqueous solution. The most soluble is AITC (1:500 at 20’ C). MITC is very slightly soluble and BITC is supposedly practically insoluble.la Therefore the investigations were studied in a wide concentration range from 0.025 to 100 mM only with AITC. MITC and BITC were used in a range from 0.1 to 0.5 mM, using aliquot volumes of solutions which were originally made in a larger quantity of KRP containing 1 per cent dimethylsulfoxide (DMSO). DMSO is a very effective solvent for many organic compounds and does not have any direct effect upon the thyroid itself in concentrations up to 5 per cent in vitro (Shimoda and Kendall, unpublished). As no data are available concerning the stability of isothiocyanates in an aqueous solution, a study was made of AITC and BITC under the conditions of our experiments. Five ~1. of these compounds were added to 25 ml. of KRP, either with or without 1 per cent DMSO, and incubated 10 hours at 37O C. in a shaking water bath. At various time intervals, l-ml. samples were taken, extracted three times with 3 ml. of ether, and 1 ml. of ammonia was
598
LANGEX
AND
GREER
0.250 1
0
0
0.200-o
0
BITC
0
O
\
Fig. L-Changes
in concentration of allyl- and butylisotbiocyanate during varying intervals of incubation in
KRP, as measured by absorption of thiourea derivatives.
3 0.150- ! Z 4 2
T 0.100-
i
0.50-
l
III1 0
.\_
1
60 120
I
I
240
600
Minutes
added to the pooled ether extract. This was allowed to stand 24 hours and then evaporated to dryness. The residue was dissolved in 10 ml. of alcohol and the ultraviolet absorption of the solution measured over a range of 220 to 270 rnp. The thiourea derivatives formed by the isothiocyanateswith ammoniahave a maximal absorptionat 243 mp. Results can be most accurately expressed by subtracting the mean value of the extinctions at 230 and 2% rnk from the extinction at 243 mu to correct for non-specific absorption. The details of this method of determining isothiocyanates as thiourea derivatives are to be published &anger, in preparation). RESULTS
Stability of Isothiocyanates in KRP As shown in Fig. 1, AITC decomposes rather rapidly in aqueous solution. By two hours its concentration had already decreased to 50 per cent of the original value. On the other hand, the concentration of BITC remained unaltered during a lo-hour incubation. The pH of the KRP solutions did not change during the lo-hour incubation. This decomposition was not analyzed further. It may be related to the chemical structures of the compounds used. Because of the early rapid decomposition of AITC, the concentrations of this compound documented in these experiments should be considered as the initial concentration only. In those thyroid lobes analyzed at the end of 3 hours the antithyroid effect of a given concentration of AITC may actually be greater than indicated herein. Eflect on the Biosynthesis of Thyroid Hormones The changes produced in radioiodine uptake by increasing concentrations of the three isothiocyanates used are plotted in Fig. 2. The radioiodine uptake began to decrease at a concentration of approximately 0.1 mM with AITC and BITC. MITC was more potent. The 3hr thyroidal uptake of the radioiodine in the flask was reduced to less than 0.2 per cent at a concentration of 100 mM AITC.
ANTITHYROID
599
ACTIVITY
l
AITC
0 BITC A MITC
Concentration
-mM
Fig. S.-Changes produced by varving concentrations of three isothiocyanates tested on in vitro radioiodine uptake of rat thyroid lobes.
I
I-
mM
A
Fig. 3.-Changes produced in relative intrathyroidal composition of radioiodinated substances by increasing concentrations of allylisothiocyanate during 3-hour incubation of rat thyroid lobes with 1311.
AITC
As shown in Fig. 3, concentrations of AITC greater than approximately 0.05 mM caused a decrease in the relative proportion of mono- and diiodotyrosine formed from 18iI during the S-hour incubation. Formation of diiodotyrosine was apparently completely inhibited at a concentration of 10 mM while formation of monoiodotyrosine was not completely prevented by a concentration up to 100 mM. Formation of iodothyronines was inhibited by even lower concentrations than was formation of diiodotyrosine. At concentrations >0.2
600
LANGE%
AND
GREER
mM AITC Fig. 4.-Data of Fig. 3 plotted to show radioiodinated substances formed by thyroid lobes as an “absolute” per cent of the 1811added to each incubation flask. mM AITC, an unidentiiied radioiodinated component which migrated at the front of the chromatograms began to appear. The quantity of this material formed was maximal at a concentration of 20 mM AITC, at which point it represented 20 per cent of the total radioiodine content of the thyroid. Although only the data from AITC are plotted, similar results were obtained with BITC and MITC in a range of 0.1 to OS mM. In Fig. 4 the radioactivity contained in each compound is plotted as a percent of the radioiodine added to the flask rather than as a percent of the total thyroidal radioiodine, as in Fig. 3. It can be seen that with the lower concentrations of AITC, as organic binding began to be inhibited, the concentration of iodide in the thyroid rose to a level approximately three-fold greater than that in the control lobes at a concentration of 0.2 mM AITC. Concentrations greater than 1 mM AITC caused a progressive decrease in the inorganic radioiodide content in the thyroid until at 100 mM AITC 1311- had reached a level approximately one-tenth that of the control lobes. To determine whether the disappearance of AITC from solution could be correlated with a corresponding decrease in the antithyroid activity of AITCcontaining media, flasks containing 1.5 x 10m3 and 3 x 10-s M AITC in KRP were incubated at 37” C for up to 22 hours before adding thyroid lobes and 1311. As seen in Table 1, the length of preincubation of the AITC solution was roughly inversely correlated with the degree of inhibition of thyroidal biosyn-
ANTITHYR0lL-J
Table L-Effect
of Preincubation on In Vitro Antithyroid Effect of AITC
Preincubation Time (hours ) AITC
0
0 0 0
1.5 X 10-Z M 3X 10-3 M 1.5 X 10-s M 3X 10-s M 1.5 X 10-Z M 3X10-3 M 1.5X 10-Z M 3X 10-3 M 0 1.5X 10-3 M 3X 10-3 M
1 1 2 2 3 3 22 22 22
601
ACEVEY
‘*II uptake (24
0
20.5 2.8 1.4 3.1 3.7 7.3 2.4 7.3 5.9 13.7 8.1 7.3
4.5 1.7 1.3 1.7 1.6 2.1 1.4 1.5 2.0 3.9 1.6 1.4
PercentIo_f Intrathyroi$;~I
9.2
54.0 60.4 51.6 57.5 33.9 57.3 44.4 44.6 9.0 49.6 54.1
38.5 26.4 15.0 27.4 22.5 36.4 22.1 30.0 30.3 42.3 25.4 21.6
DIT
-
32.9 9.9
3.1 10.6 10.7 18.3 8.9 13.1 14.8 32.2 12.3 9.8
Flasks preincubated for indicated times before adding individual thyroid lobes and 1311. Incubations after addition of 1311 were for 3 hours. AU results expressed as mean of duplicate values. 0 = Origin
I-
x Iodide
Table Z.-Effect Incubation Time (min. )
15 15 30 30 60 60 120 120 180 180
MIT = Monoiodotyrosine
DIT = Diiodotyrosine.
of AITC on In Vitro Thyroidal is11 Metabolism at Various Periods of Incubation
AITC
W Uptake (%)
0
0 + 0 + 0 + 0 + 0 +
1.37 0.56 3.87 1.64 6.58 2.07 8.78 2.66 12.94 2.43
3.0 0.4 3.1 0.3 3.1 0.4 3.3 0.4 3.4 1.0
Percent of Intrathyroidal Ia11 IMIT
28.4 86.3 21.7 89.6 9.1 85.2 5.8 82.4 6.1 67.6
41.7 5.4 48.7 5.2 53.9 7.7 51.4 9.0 47.0 16.6
DIT
16.9 2.0 20.8 1.4 27.8 3.1 32.2 3.4 32.0 9.5
One lobe from one animal incubated with, the other without, 3 x 10-3 M AITC in KRP for each indicated interval. Results are expressed as the mean of duplicate determinations. 0 = Origin I-- = Iodide
MIT = Monoiodotyrosine
DIT = Diiodotyrosine.
thesis produced, The most pronounced inhibition of both uptake and biosynthesis was seen with freshly added AITC. There was less difference apparent in solutions preincubated 2 to 22 hours. This is consistent with the more rapid disappearance of AITC from solution within the first 2 to 3 hours than at later times (Fig. 1). Thyroid inhibition could be demonstrated at the end of 15 minutes incubation of thyroid Iobes in a fresh AITC-KRP solution (TabIe 2). The decreased 1311 uptake as we11 as decreased organic binding of that *“lI which entered the thyroid produced by the concentration of AITC (3 x 1O-3 M) employed in this experiment indicates that iodide concentration by the thyroid lobes was decreased. If organic binding is completely blocked by drugs such as propylthiouracil or methimazole in the medium, no decrease in the
602
LANGER
Table 3.-Effect
of Percblorate and Allylisotbiocyanate 1311 from Blocked Thyroid Lobes
on Release of
Control
4.84
4 x lo-4 M ClO-,
0.30
1.5 X 10-2 M AITC lobes averaged
GREER
Final Ia11 content/lobe ( % of added l=I)
Group
Duplicate
AND
0.83
for each value.
Lobes incubated with 6 x added at end of first hour.
10-a M methimazole
and
1311
for 3 hours.
ClO-,
and AITC
total 1311 accumulation by such blocked lobes compared to that of control lobes is seen until after 45SO-minute incubation (Greer and Stott, unpublished data). Such decreased uptake at early time intervals can, in our experience, be produced only by substances which inhibit iodide concentration. Efect
of AZTC on the Thyroid “Zodide Pump”
Thyroid lobes were incubated for one hour in 3 ml. KRP containing 200 pg. methimazole and 25 Z.LC. 1311.After one hour 200 pg. KClO, or 500 pg. AITC in 0.5 ml. KRP were added. Two lobes were removed just before addition of AITC or perchlorate and one hour after their addition. The results are presented in Table 3. Both perchlorate and AITC caused a discharge of 80 to 90 per cent of the iodide in the thyroid within a period of one hour. This dose of methimazole has previously been shown to prevent completely any detectable binding of radioiodine by thyroid lobes incubated in this fashion so that all loss of radioactivity can be safely assumed to be iodide. Chromatographic analysis of all thyroid lobes in this experiment also revealed that only 1311was present. Since the mustard oils are known to be very irritating, it was considered possible that the profound depression of 1311- concentration by the larger quantities of allylisothiocyanate might be a non-specific, irreversible phenomenon. Accordingly, thyroid lobes were preincubated for 30 minutes in KRP alone or in KRP containing allylisothiocyanate. Previous studies had shown that high concentrations of certain monovalent anions, such as perchlorate and thiocyanate, also inhibit both organic binding and iodide concentration by incubated thyroid lobes, but that such inhibition is largely reversed by leaching.ll We wished to compare the reversibility of the inhibition produced by such anions with that produced by AITC. To one-half the flasks, 1311 was then added and the accumulation of radioiodine over a 2-hour period was measured. The remaining lobes were rinsed with KRP and incubated in four fresh changes of KRP for 15 minutes each to leach out the inhibitor present. 1311 was then added for a e-hour incubation as above. The results are shown in Table 4. No change was produced in the uptake of the control lobes reincubated in the four changes of KRP. There was a slight increase in the radioiodine uptake and/or percent of intrathyroidal radioactivity organically bound (determined by chromatography of undigested ho-
ANTITHYROID
603
ACTIVITY
Table 4.-Effect -__
of Leaching on Z-hour 1311 Uptake of Thyroid Lobes Preincubated in AITC __ 131I
COIlCl3ltrdiOIl
of AITC 0
(Control)
Leached ___ .-
0
3 X 10-a M
0
94.3 93.4 93.9
+
20.8
94.4
0
11.4 17.0
73.7 79.8
+ 10-z M 3 X 10-S M 10-l M
% Ornanic binding
19.2 20.1 16.1
I1O-4 M
% Uptake
0
9.9
27.9
t
8.2
0
1.0
i
2.2 0.4 0.3
59.4 14.1 22.1 10.6 9.1
0
lDuplicate lobes averaged for each value.
~-.--
mogenatesg) at some concentrations of AITC, but recovery after leaching was much less marked than had been seen in the experiments with monovalent anions.ll DISCWSSION
Although somewhat equivocal results had previously been obtained from in vivo experiments, 7~~the present in vitro studies conclusively demonstrate that the three isothiocyanates studied inhibit thyroidal biosynthesis. The mustard oils investigated, particularly AITC, are widely distributed throughout the plant kingdom. l2 It is therefore possible that they may play a role of some significance in the production of goiter by ingestion of natural foodstuffs. A rapid decomposition of AITC in aqueous solution was noted. This may explain the relatively low activity of this compound in vivo observed in previous experiments. ?** A similar rapid decomposition of this material may occur in the animal organism. Any studies made in vivo (or in vitro) should therefore employ freshly prepared solutions. The antithyroid activity of the isothiocyanates studied is most probably due to inherent properties of these compounds rather than to those of degradation products. Although AITC disappeared fairly promptly from aqueous solution, its disappearance was not associated with conversion to a thiourea derivative since the method of detection herein employed involved a measurement of the AITC as a thiourea derivative. The disappearance of AITC from solution (Fig. 1) could be directly correlated with a loss of antithyroid activity of the solution (Table 1). Furthermore, the demonstrated inhibition of thyroidal biosynthesis within 15 minutes (Table 2) indicates that the mustard oil has a direct effect on thyroidal biosynthesis and does not exert its action through some secondary product of decomposition. We cannot be certain that some contaminant of the mustard oil solutions employed was not responsible for the observed inhibition of thyroidal biosynthesis. However, since definite thyroid inhibition was noted with concentrations
IANGER AND
604
GREER
effect of
REFERENCES 1. Marine, D., Baumann, E. J., Spence, A. W., and Cipra, A.: Further studies on etiology of goiter with particular reference to action of cyanides. Proc. Sot. Exp. Biol. Med. 29:772,1932. 2. Jensen, K. A., Conti, J., and Kjaer, A.: Isothiocyanates II. Volatile isothiocyanates in seeds and roots of various brassicae. Acta Chem. Stand. 7:1276, 1953. 3. Clements, F. W.: A goitrogenic factor in milk. Med. J. Aust. ii:645, 1957. 4. Hercus, C. E., and Purves, H. D.: Studies on endemic and experimental goitre. J. Hyg. 36:182, 1936.
5. Wagner-Jauregg, T., and Koch, J.: Z. Naturforsch. B 2:14, 1947. 6. Bachelard, H. S., and Trikojus, V. M.: Plant thioglycosides and the problem of endemic goitre in Australia. Nature 185:80, 1960. 7. Langer, P., and Stoic V.: Goitrogen activity of allylisothiocyanate-a widespread natural mustard oil. Endocrinology 76:151, 1965. 8. Langer, P.: Study of chemical mpresentatives of the goitrogenic activity of raw cabbage. Physio. Bohemoslov. 3:542, 1964. 9. Shimoda, S.: Comparison of the recov-
ANTITHYROID
ACTIVJTY
cry of inorganic iodide by paper electrophoresis or chromatography. Endocrinology 77:401, 1965. 10. Handbook of Chemistry and Physics, 44th Edition, Chemical Rubber Publishing Company, Cleveland, Ohio, 1962. 11. Greer, M. A., Stott, A. K., and Mtine, K. A.: Effect of thiocyanate, perchlorate and other anions on thyroidal iodine metabolism,
605 Endocrinology 79:237, 1966. 12. Kjaer, A.: In: Progress in the Chemistry of Natural Products, Vol. 18, Wien, Springer-Verlag, 1960, p. 123. 13. Ingbar, S. H.: The action of 1, 1, 3tricyano-2-amino-1-propene (U-9189) on the thyroid gland of the rat and its effects in human thyrotoxicosis. J. Clin. Endocr. 21: 128,196l.
Isothiocyanates
In Vitro
By PAVEL LANGER ANDMONTE A. GREER Allyl-, methyl-, and butyl-isothiocyanate profoundly depressed the iodination of amino acids by rat thyroid lobes in an in vitro system. Allylisothiocyanate, being the most soluble of the three, was studied in concentrations up to 10-l M. At higher concentrations, allylisothiocyanate depressed thyroidal iodide concentration
as well as thyroidal biosynthesis. The effect of high concentrations was probably due to non-specific toxicity since it was not removed by leaching. Allylisothiocyanate, but not butylisothiocyanate, rapidly decomposed in aqueous solution. (Metabolism 17: No. 7, July, 596-605, 1968)
I
SOTHIOCYANATES (mustard oils) are present in the edible portions of many species of plants and in their seeds in the form of thioglycoside precursors. The isothiocyanates are formed from the aglycone liberated from the thioglycosides during enzymatic hydrolysis. Since the isothiocyanates are present in particularly high concentration in the cabbage family, members of which have been shown to have antithyroid activity in both animals and man, there has been recurring speculation that these compounds might be goitrogenie. However, there is no clear consensus as to whether these substances actually do possess antithyroid activity. Several authors, without presenting the exact conditions of their experiments, have reported that mustard oils have no effect upon the thyroid. lv2 Clements3 stated that benzyl isothiocyanate is goitrogenic in rats but gave no experimental details. Hercus and Purves4 speculated that the goitrogenic activity of Brassica seeds might be caused by thioglycosides. Wagner-Jauregg and Koch5 observed some goitrogenic activity when the thioglycoside, sinalbin, was administered simultaneously orally with the enzyme myrosinase. Bachelard and Trikoju@ reported that methyl-sulfonyl-propyl-isothiocyanate and n-propyl-isothiocyanate were weak inhibitors of radioiodine uptake in rats. In previous experiments we have observed thyroid hypertrophy when allylisothiocyanate was administered to rats in various doses for periods of 26 to is one of the most wide-spread isothiocyanates 60 days. 7,8 Allylisothiocyanate From the Division of Endocrinology, Department of Medicine, University of Oregon Medical School, Portland, Oregon. Supported by grants from the National Institutes of Health, U. S. Public Health Seruice. Receiued for publication July 27, 1967. PAVEL LANGER, M.D.: Institute of Endocrinology, Slovak Academy of Sciences, Rratishzva, Czechoslovakia. This work was carried out while DT. Lunger was a visiting scientist in th.e Division of Endocrinology, University of Oregon Medical School. MONTE A. GREER, M.D.: Professor of Medicine; Head, Division of Endocrinology, Department of Medicine, University of Oregon Medical School, Portland, Oregon. 596
ANTITHYROID
597
AmIVITY
in the plant kingdom; it therefore appeared worthwhile to investigate its antithyroid activity in more detail. Allylisothiocyanate deteriorates in aqueous solution and is also probably metabolized rapidly, thus we felt it would be most profitable to study its effect in an in vitro system where the material could be tested immediately after being put in solution and with minimal potential changes having taken place in the isothiocyanate molecule. Since the mustard oils are quite irritating, we also concluded it would be possible to study the effects of higher concentrations in vitro than in viva, MATERIALS
AND METHODS
Young, adult, male Holtzman rats were fed a low iodine diet (General Biochemicals, Inc.) containing approximately 30 pg. of iodine per Kg. for 7 days. The animals were killed with chloroform and the thyroid lobes were carefully removed, each lobe being placed in 2.5 ml. of Krebs-Ringer phosphate (KRP) solution containing the indicated test substance. Twenty-five PC. carrier-free 1311 in 0.5 ml. KRP were then added. The flasks were incubated for 15 minutes to 3 hours in a shaking water bath at 37” C. with air as the gas phase, The thyroid lobes were then removed, washed with I to 2 ml. of KRP and placed in tubes containing 0.5 ml. of 0.2 per cent methimazole in pH 8.5 phosphate buffer. Such rinsing removes approximately 2 per cent of the intrathyroidal iodide but none of the organicallybound 1slI.a The radioactivity in each lobe was measured in a well-type scintillation counter and the lobes were then homogenized. One-half ml of a 2 per cent solution of pancreatin (Viokase 4X) in the same phosphate buffer was added and the samples were hydrolyzed for 18 hours in a shaking water bath at 37’ C. under one to two drops of toluene. Aliquots of the hydrolysates containing 0.05 PC. were applied to strips of Whatman No. 3 chromatographic paper and developed in an ascending butanol-acetic acid-water (4:1:5) system. Chromatograms were scanned with an automatic scanner. Identity of the radioactive peaks was determined by comparing the localization of authentic stable compounds added to the hydrolysates on the paper. Iodinated ammo acids were detected with a diazotized sulfanilic acid spray reagent. The quantity of each radioiodinated substance was determined by planimetry of the scans. The “absolute” amount of radioiodinated material present was determined by multiplying the relative per cent of each compound expressed as the per cent of total thyroidal radioactivity times the radioiodine uptake. In each experiment two to four control lobes were incubated in KRP alone. The other lobes were incubated in varying concentrations of allylisothiocyanate (AITC), butylisothiocyanate (BITC) and methylisothiocyanate (MITC), ranging from 0.025 to 100 mM. In all experiments two lobes from separate animals were used for each concentration of test substance. The results from the duplicate lobes were averaged to ,give a mean value. AITC was obtained from Matheson, Coleman & Bell, BITC and MITC from Distillation Products Co., a division of Eastman Kodak. All specimens were stated to be of the highest obtainable purity (94-99 per cent). The isothiocyanates used are only slightly soluble in aqueous solution. The most soluble is AITC (1:500 at 20’ C). MITC is very slightly soluble and BITC is supposedly practically insoluble.la Therefore the investigations were studied in a wide concentration range from 0.025 to 100 mM only with AITC. MITC and BITC were used in a range from 0.1 to 0.5 mM, using aliquot volumes of solutions which were originally made in a larger quantity of KRP containing 1 per cent dimethylsulfoxide (DMSO). DMSO is a very effective solvent for many organic compounds and does not have any direct effect upon the thyroid itself in concentrations up to 5 per cent in vitro (Shimoda and Kendall, unpublished). As no data are available concerning the stability of isothiocyanates in an aqueous solution, a study was made of AITC and BITC under the conditions of our experiments. Five ~1. of these compounds were added to 25 ml. of KRP, either with or without 1 per cent DMSO, and incubated 10 hours at 37O C. in a shaking water bath. At various time intervals, l-ml. samples were taken, extracted three times with 3 ml. of ether, and 1 ml. of ammonia was
598
LANGEX
AND
GREER
0.250 1
0
0
0.200-o
0
BITC
0
O
\
Fig. L-Changes
in concentration of allyl- and butylisotbiocyanate during varying intervals of incubation in
KRP, as measured by absorption of thiourea derivatives.
3 0.150- ! Z 4 2
T 0.100-
i
0.50-
l
III1 0
.\_
1
60 120
I
I
240
600
Minutes
added to the pooled ether extract. This was allowed to stand 24 hours and then evaporated to dryness. The residue was dissolved in 10 ml. of alcohol and the ultraviolet absorption of the solution measured over a range of 220 to 270 rnp. The thiourea derivatives formed by the isothiocyanateswith ammoniahave a maximal absorptionat 243 mp. Results can be most accurately expressed by subtracting the mean value of the extinctions at 230 and 2% rnk from the extinction at 243 mu to correct for non-specific absorption. The details of this method of determining isothiocyanates as thiourea derivatives are to be published &anger, in preparation). RESULTS
Stability of Isothiocyanates in KRP As shown in Fig. 1, AITC decomposes rather rapidly in aqueous solution. By two hours its concentration had already decreased to 50 per cent of the original value. On the other hand, the concentration of BITC remained unaltered during a lo-hour incubation. The pH of the KRP solutions did not change during the lo-hour incubation. This decomposition was not analyzed further. It may be related to the chemical structures of the compounds used. Because of the early rapid decomposition of AITC, the concentrations of this compound documented in these experiments should be considered as the initial concentration only. In those thyroid lobes analyzed at the end of 3 hours the antithyroid effect of a given concentration of AITC may actually be greater than indicated herein. Eflect on the Biosynthesis of Thyroid Hormones The changes produced in radioiodine uptake by increasing concentrations of the three isothiocyanates used are plotted in Fig. 2. The radioiodine uptake began to decrease at a concentration of approximately 0.1 mM with AITC and BITC. MITC was more potent. The 3hr thyroidal uptake of the radioiodine in the flask was reduced to less than 0.2 per cent at a concentration of 100 mM AITC.
ANTITHYROID
599
ACTIVITY
l
AITC
0 BITC A MITC
Concentration
-mM
Fig. S.-Changes produced by varving concentrations of three isothiocyanates tested on in vitro radioiodine uptake of rat thyroid lobes.
I
I-
mM
A
Fig. 3.-Changes produced in relative intrathyroidal composition of radioiodinated substances by increasing concentrations of allylisothiocyanate during 3-hour incubation of rat thyroid lobes with 1311.
AITC
As shown in Fig. 3, concentrations of AITC greater than approximately 0.05 mM caused a decrease in the relative proportion of mono- and diiodotyrosine formed from 18iI during the S-hour incubation. Formation of diiodotyrosine was apparently completely inhibited at a concentration of 10 mM while formation of monoiodotyrosine was not completely prevented by a concentration up to 100 mM. Formation of iodothyronines was inhibited by even lower concentrations than was formation of diiodotyrosine. At concentrations >0.2
600
LANGE%
AND
GREER
mM AITC Fig. 4.-Data of Fig. 3 plotted to show radioiodinated substances formed by thyroid lobes as an “absolute” per cent of the 1811added to each incubation flask. mM AITC, an unidentiiied radioiodinated component which migrated at the front of the chromatograms began to appear. The quantity of this material formed was maximal at a concentration of 20 mM AITC, at which point it represented 20 per cent of the total radioiodine content of the thyroid. Although only the data from AITC are plotted, similar results were obtained with BITC and MITC in a range of 0.1 to OS mM. In Fig. 4 the radioactivity contained in each compound is plotted as a percent of the radioiodine added to the flask rather than as a percent of the total thyroidal radioiodine, as in Fig. 3. It can be seen that with the lower concentrations of AITC, as organic binding began to be inhibited, the concentration of iodide in the thyroid rose to a level approximately three-fold greater than that in the control lobes at a concentration of 0.2 mM AITC. Concentrations greater than 1 mM AITC caused a progressive decrease in the inorganic radioiodide content in the thyroid until at 100 mM AITC 1311- had reached a level approximately one-tenth that of the control lobes. To determine whether the disappearance of AITC from solution could be correlated with a corresponding decrease in the antithyroid activity of AITCcontaining media, flasks containing 1.5 x 10m3 and 3 x 10-s M AITC in KRP were incubated at 37” C for up to 22 hours before adding thyroid lobes and 1311. As seen in Table 1, the length of preincubation of the AITC solution was roughly inversely correlated with the degree of inhibition of thyroidal biosyn-
ANTITHYR0lL-J
Table L-Effect
of Preincubation on In Vitro Antithyroid Effect of AITC
Preincubation Time (hours ) AITC
0
0 0 0
1.5 X 10-Z M 3X 10-3 M 1.5 X 10-s M 3X 10-s M 1.5 X 10-Z M 3X10-3 M 1.5X 10-Z M 3X 10-3 M 0 1.5X 10-3 M 3X 10-3 M
1 1 2 2 3 3 22 22 22
601
ACEVEY
‘*II uptake (24
0
20.5 2.8 1.4 3.1 3.7 7.3 2.4 7.3 5.9 13.7 8.1 7.3
4.5 1.7 1.3 1.7 1.6 2.1 1.4 1.5 2.0 3.9 1.6 1.4
PercentIo_f Intrathyroi$;~I
9.2
54.0 60.4 51.6 57.5 33.9 57.3 44.4 44.6 9.0 49.6 54.1
38.5 26.4 15.0 27.4 22.5 36.4 22.1 30.0 30.3 42.3 25.4 21.6
DIT
-
32.9 9.9
3.1 10.6 10.7 18.3 8.9 13.1 14.8 32.2 12.3 9.8
Flasks preincubated for indicated times before adding individual thyroid lobes and 1311. Incubations after addition of 1311 were for 3 hours. AU results expressed as mean of duplicate values. 0 = Origin
I-
x Iodide
Table Z.-Effect Incubation Time (min. )
15 15 30 30 60 60 120 120 180 180
MIT = Monoiodotyrosine
DIT = Diiodotyrosine.
of AITC on In Vitro Thyroidal is11 Metabolism at Various Periods of Incubation
AITC
W Uptake (%)
0
0 + 0 + 0 + 0 + 0 +
1.37 0.56 3.87 1.64 6.58 2.07 8.78 2.66 12.94 2.43
3.0 0.4 3.1 0.3 3.1 0.4 3.3 0.4 3.4 1.0
Percent of Intrathyroidal Ia11 IMIT
28.4 86.3 21.7 89.6 9.1 85.2 5.8 82.4 6.1 67.6
41.7 5.4 48.7 5.2 53.9 7.7 51.4 9.0 47.0 16.6
DIT
16.9 2.0 20.8 1.4 27.8 3.1 32.2 3.4 32.0 9.5
One lobe from one animal incubated with, the other without, 3 x 10-3 M AITC in KRP for each indicated interval. Results are expressed as the mean of duplicate determinations. 0 = Origin I-- = Iodide
MIT = Monoiodotyrosine
DIT = Diiodotyrosine.
thesis produced, The most pronounced inhibition of both uptake and biosynthesis was seen with freshly added AITC. There was less difference apparent in solutions preincubated 2 to 22 hours. This is consistent with the more rapid disappearance of AITC from solution within the first 2 to 3 hours than at later times (Fig. 1). Thyroid inhibition could be demonstrated at the end of 15 minutes incubation of thyroid Iobes in a fresh AITC-KRP solution (TabIe 2). The decreased 1311 uptake as we11 as decreased organic binding of that *“lI which entered the thyroid produced by the concentration of AITC (3 x 1O-3 M) employed in this experiment indicates that iodide concentration by the thyroid lobes was decreased. If organic binding is completely blocked by drugs such as propylthiouracil or methimazole in the medium, no decrease in the
602
LANGER
Table 3.-Effect
of Percblorate and Allylisotbiocyanate 1311 from Blocked Thyroid Lobes
on Release of
Control
4.84
4 x lo-4 M ClO-,
0.30
1.5 X 10-2 M AITC lobes averaged
GREER
Final Ia11 content/lobe ( % of added l=I)
Group
Duplicate
AND
0.83
for each value.
Lobes incubated with 6 x added at end of first hour.
10-a M methimazole
and
1311
for 3 hours.
ClO-,
and AITC
total 1311 accumulation by such blocked lobes compared to that of control lobes is seen until after 45SO-minute incubation (Greer and Stott, unpublished data). Such decreased uptake at early time intervals can, in our experience, be produced only by substances which inhibit iodide concentration. Efect
of AZTC on the Thyroid “Zodide Pump”
Thyroid lobes were incubated for one hour in 3 ml. KRP containing 200 pg. methimazole and 25 Z.LC. 1311.After one hour 200 pg. KClO, or 500 pg. AITC in 0.5 ml. KRP were added. Two lobes were removed just before addition of AITC or perchlorate and one hour after their addition. The results are presented in Table 3. Both perchlorate and AITC caused a discharge of 80 to 90 per cent of the iodide in the thyroid within a period of one hour. This dose of methimazole has previously been shown to prevent completely any detectable binding of radioiodine by thyroid lobes incubated in this fashion so that all loss of radioactivity can be safely assumed to be iodide. Chromatographic analysis of all thyroid lobes in this experiment also revealed that only 1311was present. Since the mustard oils are known to be very irritating, it was considered possible that the profound depression of 1311- concentration by the larger quantities of allylisothiocyanate might be a non-specific, irreversible phenomenon. Accordingly, thyroid lobes were preincubated for 30 minutes in KRP alone or in KRP containing allylisothiocyanate. Previous studies had shown that high concentrations of certain monovalent anions, such as perchlorate and thiocyanate, also inhibit both organic binding and iodide concentration by incubated thyroid lobes, but that such inhibition is largely reversed by leaching.ll We wished to compare the reversibility of the inhibition produced by such anions with that produced by AITC. To one-half the flasks, 1311 was then added and the accumulation of radioiodine over a 2-hour period was measured. The remaining lobes were rinsed with KRP and incubated in four fresh changes of KRP for 15 minutes each to leach out the inhibitor present. 1311 was then added for a e-hour incubation as above. The results are shown in Table 4. No change was produced in the uptake of the control lobes reincubated in the four changes of KRP. There was a slight increase in the radioiodine uptake and/or percent of intrathyroidal radioactivity organically bound (determined by chromatography of undigested ho-
ANTITHYROID
603
ACTIVITY
Table 4.-Effect -__
of Leaching on Z-hour 1311 Uptake of Thyroid Lobes Preincubated in AITC __ 131I
COIlCl3ltrdiOIl
of AITC 0
(Control)
Leached ___ .-
0
3 X 10-a M
0
94.3 93.4 93.9
+
20.8
94.4
0
11.4 17.0
73.7 79.8
+ 10-z M 3 X 10-S M 10-l M
% Ornanic binding
19.2 20.1 16.1
I1O-4 M
% Uptake
0
9.9
27.9
t
8.2
0
1.0
i
2.2 0.4 0.3
59.4 14.1 22.1 10.6 9.1
0
lDuplicate lobes averaged for each value.
~-.--
mogenatesg) at some concentrations of AITC, but recovery after leaching was much less marked than had been seen in the experiments with monovalent anions.ll DISCWSSION
Although somewhat equivocal results had previously been obtained from in vivo experiments, 7~~the present in vitro studies conclusively demonstrate that the three isothiocyanates studied inhibit thyroidal biosynthesis. The mustard oils investigated, particularly AITC, are widely distributed throughout the plant kingdom. l2 It is therefore possible that they may play a role of some significance in the production of goiter by ingestion of natural foodstuffs. A rapid decomposition of AITC in aqueous solution was noted. This may explain the relatively low activity of this compound in vivo observed in previous experiments. ?** A similar rapid decomposition of this material may occur in the animal organism. Any studies made in vivo (or in vitro) should therefore employ freshly prepared solutions. The antithyroid activity of the isothiocyanates studied is most probably due to inherent properties of these compounds rather than to those of degradation products. Although AITC disappeared fairly promptly from aqueous solution, its disappearance was not associated with conversion to a thiourea derivative since the method of detection herein employed involved a measurement of the AITC as a thiourea derivative. The disappearance of AITC from solution (Fig. 1) could be directly correlated with a loss of antithyroid activity of the solution (Table 1). Furthermore, the demonstrated inhibition of thyroidal biosynthesis within 15 minutes (Table 2) indicates that the mustard oil has a direct effect on thyroidal biosynthesis and does not exert its action through some secondary product of decomposition. We cannot be certain that some contaminant of the mustard oil solutions employed was not responsible for the observed inhibition of thyroidal biosynthesis. However, since definite thyroid inhibition was noted with concentrations
IANGER AND
604
GREER
effect of
REFERENCES 1. Marine, D., Baumann, E. J., Spence, A. W., and Cipra, A.: Further studies on etiology of goiter with particular reference to action of cyanides. Proc. Sot. Exp. Biol. Med. 29:772,1932. 2. Jensen, K. A., Conti, J., and Kjaer, A.: Isothiocyanates II. Volatile isothiocyanates in seeds and roots of various brassicae. Acta Chem. Stand. 7:1276, 1953. 3. Clements, F. W.: A goitrogenic factor in milk. Med. J. Aust. ii:645, 1957. 4. Hercus, C. E., and Purves, H. D.: Studies on endemic and experimental goitre. J. Hyg. 36:182, 1936.
5. Wagner-Jauregg, T., and Koch, J.: Z. Naturforsch. B 2:14, 1947. 6. Bachelard, H. S., and Trikojus, V. M.: Plant thioglycosides and the problem of endemic goitre in Australia. Nature 185:80, 1960. 7. Langer, P., and Stoic V.: Goitrogen activity of allylisothiocyanate-a widespread natural mustard oil. Endocrinology 76:151, 1965. 8. Langer, P.: Study of chemical mpresentatives of the goitrogenic activity of raw cabbage. Physio. Bohemoslov. 3:542, 1964. 9. Shimoda, S.: Comparison of the recov-
ANTITHYROID
ACTIVJTY
cry of inorganic iodide by paper electrophoresis or chromatography. Endocrinology 77:401, 1965. 10. Handbook of Chemistry and Physics, 44th Edition, Chemical Rubber Publishing Company, Cleveland, Ohio, 1962. 11. Greer, M. A., Stott, A. K., and Mtine, K. A.: Effect of thiocyanate, perchlorate and other anions on thyroidal iodine metabolism,
605 Endocrinology 79:237, 1966. 12. Kjaer, A.: In: Progress in the Chemistry of Natural Products, Vol. 18, Wien, Springer-Verlag, 1960, p. 123. 13. Ingbar, S. H.: The action of 1, 1, 3tricyano-2-amino-1-propene (U-9189) on the thyroid gland of the rat and its effects in human thyrotoxicosis. J. Clin. Endocr. 21: 128,196l.