Effect of Methylthiouracil Administration on Nucleic Acids in Rabbit Thyroid

EFFECT OF METHYLTHIOURACIL ADMINISTRATION ON NUCLEIC ACIDS IN RABBIT THYROID NOBUO SUZUKI* Department of Biochemistry,School of Medicine,University of Nagoya Since the experimental reports by Richter and Clisby (1) and Kennedy (2), it has been demonstrated by many authors that the administration of thiourea and its derivatives produce thyroid enlargement involving hypertrophy and hyperplasia of the epithelial cell and colloid loss in various kinds of animals. Although such substances as antithyroid agents functionally give rise to a total hypothyrosis of the animal (3, 4), the follicular cell morphologically does not show the picture of the resting state, but shows on the contrary a hyperactive state. Thiourea and its derivatives have been shown to exert antithyroidal action through their reducing power and preferential reactivity with iodine, which inhibits the oxidative conversion of iodide to iodine-the first step of hormone synthesis (5, 6) and the formation of diiodotyrosine and also the oxidative coupling of two molecules of diiodotyrosine to one molecule of thyroxine-the second and third step of hormone synthesis (7, 8, etc.). It is generally observed that during the course of thiouracil treatment patients are often led into granulocytopenia and in rare cases even fatal agranulocytosis. This toxic effect was confirmed by animal experiments (9, 10), and Tinacci (11) demonstrated that thiouracil blocks the process of cell division of bone-marrow. On the other hand, according to Du Chaliot (12) a grain of wheat in the presence of methylthiouracil does not germinate or worse yet even sprout, and De Ritis and Scalfi (13) observed partial or complete inhibition of the growth of Staphy- lococci in the culture-medium containing thiourea in proportion to the dose. Further Wolff and Karlin (14) demonstrated that thiouracil inhibited partially the growth of Lactobacillus casei in Roberts and Smell's medium and that this action was antagonized by the addition of uracil which is one of the structural units of ribonucleic acid-pyrimidine base. This competitive antagonism is considered to be analogous to that which exists between sulfanilamide and p- aminobnnzoic acid (15). From the above mentioned experimental reports it seems probable that thiouracil in general may interfere with the metabolism of nucleic acids in the cells of organisms, although at the present time it is not known whether nucleic acids in the thyroid gland play any role in the synthesisof the hormone, especially at the stage of formation of thyreoglobulin which is synthesized from diiodotyrosine and thyroxine through peptide-linkage and is stored as such in the Received for publication July 29, 1953. *鈴木信雄 279 280 N. SUZUKI colloid of the follicles. I find it interesting to study and note the effect of methylthiouracil upon nucleic acids in the thyroid, since it is believed that ribonucleic acid is closely concerned with the synthesis of proteins in the cell (16, 17). PROCEDURE 30 mg. of 4-methyl-2-thiouracil per 1 kg. of body weight was injected daily by the subcutaneous route into 18 male rabbits weighing 2,100-2,700 g. that were kept outdoors in winter. They (3 rabbits) and a control were killed by a blow on the head and the subsequent bleedings at 1, 2, 7, 15, 30 and 60 days respectively; the thyroid glands were dissected and freed of connective tissue, weighed on a Hartman-Braun torsion balance, and then a piece of each gland was examined histologically (stained with hematoxylin and eosin) and each remaining tissue was appropriated for the chemical estimation of both desoxy- ribonucleic and ribonucleic acid phosphorus (DNAP and RNAP) by Schmidt and Thannhauser's method (18). RESULTS On histological examination, just as many authors have descrived, that soon after the injection of the drug, colloid in the follicle decreased in amount, epithelial cells rapidly grew taller in height, the nucleous became so large and prominent and the cytoplasm inclined to stain intensely basophilic. These alterations attained maximal intensity at 15 days-colloid loss was virtually complete, the epithelial cell was almost exclusively of the columnar type and basophilic changes of cytoplasm were most marked throughout the whole period. By this time the hyperplasia became so intense that in many areas cord-like masses of cells proliferated among the follicles and papillary growths gave rise to new follicles. After 30 days however these intensely active histological findings retrograded a little; and the basophilic tincture of cytoplasm faded very much and at 30 days colloids once lost began to reappear in most follicles (see figure 1-4). Nucleic acids in whole thyroid (absolute amount). Following daily injections of methylthiouracil, both DNAP and RNAP increased with a gain in thyroid weight and an early increase in RNAP was disproportionally marked (see table 1). Nucleic acids per 100 g. of fresh thyroid (relative amount). With daily administration of the drug, RNAP increased rapidly, reaching a maximal level on the 15th day (55% increase over the control level), and thereafter it decreased gradually but never below the normal level. On the contrary DNAP decreased gradually from the first, attaining a minimal level on the 30th day (31% decrease below the control level) and on the 60th day it increased a little (see Table I). DISCUSSION AND CONCLUSION As stated above it is now believed that high concentrations of nucleic acids in cells represent active protein synthesis, in the sense of either the production NUCLEIC ACIDS IN THYROID 281 FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 1. Normal rabbit thyroid. •~120 FIG. 2. Rabbit thyroid after 7 days' treatment with methylthiouracil. •~120 FIG. 3. Rabbit thyroid after 15 days' treatment with methylthiouracil. •~120 FIG, 4. Rabbit thyroid after 60 days' treatment with methylthiouracil. •~120 282 N. SUZUKI TABLE 1. Thyroid Weight and Nucleic Acids in the Thyroid of Rabbits after Methylthiouracil Administration of gene proteins in the chromosomes (DNA), or the production of major cyto- plasmic proteins for cellular growth or for secretion (RNA). However in such a case where drug administration results in the inhibition of hormone synthesis being followed by cellular hyperplasia and hypertrophy of its gland, the simple interpretation of the variations in the amounts of nucleic acids in the thyroid seems to be unjustified. It should also be pointed out that according to a chemical method an enlargement of cell-size, even if unaccompanied by an alteration in nucleic acids per cell, would result in a decrease in these per unit weight of tissue. Therefore a reduction in DN AP content per 100 g. of thyroid as in this experiment, does not necessarily mean a true reduction per cell, but an increase in the amount of RNAP per 100 g. of tissue reflects also the increase per cell. kelf.Abek and kei.abek (19) stated that in albino rats treated with methylthiouracil in daily doses of 50 mg., the quantities of DNA and RNA (by des- oxyribose and ribose estimation) per 1 g. of thyroid were reduced respectively by 7.2% and 11% of normal value after 3 days and by 41.6% and 49.2% after 10 days, on the other hand the volumes of the follicular cells and nuclei were increased about 3 times, and 2.7 times the normal value after 3 and 10 days respectively. From their experiments, however, it cannot be deduced that methylthiouracil administration brought about a true decrease in the amount of nucleic acids (both RNA and DNA) per cell. Using the Unna-Pappenheim mixture in conjunction with the ribonuclease test, Petronelli (20) found histochemically that methylthiouracil administration to guinea pigs in daily doses of 250 mg. for 15 days resulted in an increase in RNA content in the cytoplasm of the cubic epithelial cell in the smaller follicles and a decrease in RNA content in that of the epithelial cell in the large follicles of the thyroid gland. It should however be taken into consideration that by the histochemical method one can find only the relative distribution of RNA in the cell, since an enlargement of cell-size even without any change in RNA content per cell would cause a decrease in the density of RNA in the cell and apparantly a lighter colouring than in a normal one ensues. In conclusion, a moderate amount of methylthiouracil does not bring about a decrease in nucleic acids of the thyroid but causes a remarkable increase in RNAP per 100 g. of the tissue which is well in accord with the morphological NUCLEIC ACIDS IN THYROID 283 activity of the epithelial cell (an increase in cell height, large nucleolus and intense basophilic staining). A decrease in DNAP per 100 g. of the tissue does not necessarily represent a true decrease per cell. An increase in DNAP and RNAP in the whole thyroid seems to reflect respectively hyperplasia and hypertrophy of epithelial cells of the gland. ACKNOWLEDGEMENT The author is very grateful to Prof. Dr. K. Hotta for his kind advice and encouragement and to Mr. Aoki for the preparation of histological samples. RE FERENCES 1. RICHTER, C. P. AND CLISBY,K. H. Proc. Soc. ext. Biol. and Med. 48: 684, 1941. 2. KENNEDY,T. H. Nature 150: 233, 1942. 3. AsTWooD, E. B., SULLIVAN,J., BISSEL,A. AND TYSLOWITZ,R. Endocrinol. 32: 210, 1943. 4. MACKENZIE,C. G. AND MACKENZIE,J. B. Endocrinol. 32: 185, 1943. 5. CAMPBELL,D., LANDGREBE,F. W. AND MORGAN,T. N. Lancet 246: 630, 1944. 6. MILLER, W. H., ROBLIN, R. 0. AND ASTWOOD,E. B. J. Amer. chem. Soc. 67: 2201, 1945. 7. KESTON, A. S., GOLDSMITH,E. D., GORDON,A. S. AND CHARIPPER, H. A. J. biol. Chem. 152: 241, 1944. 8. FRANKLIN, A.

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