TOXICOLOGY AND APPLIED PHARMACOLOGY 14,108-113 (1969)

Thyroiditis in Rats Given Subcutaneous Injections of Trypan Blue

MELVIN D. REUBER

Laboratory of Biology, National Cancer Institute, Bethesda, Maryland 20014

Received July 3, 1968

Thyroiditis in Rats Given Subcutaneous Injections of Trypan Blue. REUBER, MELVIN D. (1969). Toxicol. Appl. Pharmacol. 14,108-l 13. Buffalo strain male and female rats were injected subcutaneously once a week with a 1% solution of trypan blue. The thyroid gland was enlarged in 6 of 12 female rats and 2 of 12 male rats after 5 injections. Histologically, the gland showed thyroiditis with lymphocytes, plasma cells, and hyper- plastic epithelial acinar cells.

Chronic thyroiditis has been observed in this laboratory in inbred Buffalo strain rats given 3-methylcholanthrene continuously in the diet (Glover and Reuber, 1968) or by a single subcutaneous injection (Reuber and Glover, 1968). This lesion also was present in Buffalo rats with cirrhosis of the liver induced by subcutaneous injections of carbon tetrachloride (Glover and Reuber, 1967) or a high fat, low protein, and choline- deficient diet (Reuber, 1968). The purpose of this paper is to describe the development of thyroiditis in the same strain of rat when given subcutaneous trypan blue.

METHODS

Inbred male and female Buffalo strain rats 12 weeks of age were used. Twelve rats of each sex were weighed and injected subcutaneously once a week with a 1 y0 solution of trypan blue in 0.9 % saline. The dosage was 1 ml per 200 g of body weight (50 mg/kg body weight). Twelve control animals of each sex were injected with the same amount pf saline once weekly. The diet was Rockland Laboratory pellets.

The experiment was terminated at the end of 5 weeks of injections. Complete necrop- sies were done, and tissues were fixed in 10% formalin. The thyroid, liver, kidney, spleen, heart, and testes were weighed. Sections of all tissues were stained with hema- toxylin and eosin. Sections of the thyroid gland were also stained with periodic acid- Schiff (PAS), Perls’ stain for hemosiderin, and Masson trichrome for connective tissue. Pituitaries were stained with PAS-orange G.

RESULTS

The gross and microscopic findings at autopsy are given in Table 1. Male animals with thyroiditis weighed 42 g less than the experimental animals without thyroid gland lesions and 45 g less than the control animals. The female animals with thyroiditis weighed 17 g less than the controls. Thyroiditis occurred in females (6 of 12) more often than in males (2 of 12). Each lobe of the largest gland observed in a male, meas-

108

TABL

E 1

Gro

up

Sex

Thyr

oid

glan

d

BOD

YAN

DTH

YRO

IDG

LAN

DW

EIG

HTS

Ave

rage

A

vera

ge

begi

nnin

g te

rmin

al

Num

ber

body

bo

dy

of

wei

ght

wei

ght

anim

als

(g)

69

;: 3 %

Thy

roid

gla

nd w

eigh

t (m

g)

F -_

_ __

_-

- R

ange

A

vera

ge

s 5 _-

___-

.- 4

Try

pan

blue

P

Thy

roid

itis

6 N

orm

al

6 19

2 19

7 44

-117

20

5 20

-30

79

2

27

Q

w

Con

trol

0

Nor

mal

12

19

0 21

4 22

-31

26

2 T

rypa

n bl

ue

8 T

hyro

iditi

s 2

278

254

41-1

60

100

5 N

orm

al

10

296

26-3

2 29

2 u

Con

trol

C

T N

orm

al

12

281

299

18-2

8 25

z t;

110 REUBER

ured 4 x 6 x 10 mm and weighed 80 mg. On gross examination the glands with thyroi- ditis did not appear to be adherent to adjacent organs. The tissue was firm and stained blue.

Histologically, the thyroiditis was classified as mild, moderate, or severe. There was a good correlation between the severity and the size of the gland. In 3 females the lesion was severe, in 2 moderate, and in 1, mild. One male had moderate, the other severe thyroiditis.

Mild thyroiditis. The gland was slightly enlarged. Lymphocytes with few plasma cells were seen focally in the interstitium. Acini were not unusual.

FIG. 1. Moderate thyroiditis. There are large numbers of lymphocytes and plasma cells in the inter- stitium. Acini in the center have hyperplastic cells. Parathyroid is seen at the top. Hematoxylin and eosin. x160.

Moderate thyroiditis. The left and right lobes each measured an average of 2 x 4 x 6 mm. Lymphocytes, plasma cells in various stages of maturation, and occasional macrophages containing hemosiderin involved the interstitium diffusely (Fig. 1). Epithelial acinar cells were hyperplastic in some of the acini. The cells were columnar with acidophilic cytoplasm, oval or round nuclei, and occasional mitoses.

Severe thyroiditis. Each lobe measured an average of 3 x 6 x 8 mm. The inflam- matory cells, predominantly plasma cells, were present throughout the interstitium. Sometimes clusters of cells resembled germinal centers. The cells of most of the acini were so hyperplastic that the lumens were often obscured (Figs. 2 and 3). Focally, lymphocytes and plasma cells invaded the basement membrane of the acini and ex- tended into adipose tissue adjacent to the gland. Fibrosis was not observed in the gland.

TRYPAN BLUE INDUCED THYROIDITIS III

FIG. 2. Severe thyroiditis. Hyperplasia of the epithelial cells is present in addition to the marked inflammation. A cluster of cells resembling a germinal center is present in the upper left. Hematoxylin and eosin. z 160.

FIG, 3. Severe thyroiditis. Cells lining acini are columnar with acidopholic cytoplasm and round or oval nuclei. Hematoxylin and eosin. x250.

112 REUBER

There were cysts with flattened epithelial lining cells and sloughed cells in the lumens in the thyroid glands not showing inflammatory cells. They were observed in the glands of 4 males and 1 female given trypan blue.

In the animals with thyroiditis the basophils in the pituitary contained large hyaline droplets within the cytoplasm. In the adrenal the fasciculata was narrowed with lipid depletion of the cells and the reticularis widened in all animals given trypan blue. Animals injected with trypan blue also had plasma cells in lymph nodes, as well as extramedullary hematopoiesis in the spleen. There were small islands of reticuloendo- thelial cells in the portal areas in the liver. The cells of the distal convoluted tubules of the kidney were swollen focally.

Control animals did not have lesions of the thyroid gland or other organs.

DISCUSSION

The thyroiditis in animals given trypan blue has the same gross and histologic characteristics as that observed in rats given oral or subcutaneous methylcholanthrene (Glover and Reuber, 1968; Reuber and Glover, 1968) and in rats with cirrhosis of the liver induced by carbon tetrachloride or diet (Glover and Reuber, 1967; Reuber, 1968). It also is similar to that produced in rats with homologous or heterologous thyroid extract or thyroglobulin (Jones and Roitt, 1961). The lesion has been observed in the rabbit, guinea pig, and dog immunized in the same way (Terplan et al., 1960).

Trypan blue is known to influence the function of the thyroid gland. The chemical decreases the weight of the thyroid gland, the serum protein-bound iodine, the uptake of radioactive iodine (Yamada, 1960), and the basal metabolic rate in rats (Christie, 1966). Exogenous thyroid-stimulating hormone (TSH) restored hormone secretion by the thyroid gland (Yamada, 1960). Trypan blue also suppresses the thyroxine binding capacity of plasma (Crispell et al., 1957).

Size of the thyroid gland in the results obtained by Yamada (1960) and those ob- served in this laboratory might be related to a number of variables. There were dif- ferences in the sex and strain of rat, in the route of administration of trypan blue, and in the duration of the experiments.

Trypan blue alters the relative percentage of normal serum proteins (Christie, 1964) and induces the formation of an abnormal serum protein in the rat (Yamada, 1959; Paoletti Ed al., 1962).

It is not known whether the effect of trypan blue on the thyroid gland is direct or by some other means. It has been suggested that the chemical may compete with thy- roxine for serum protein binding sites. The thyroxine would then inhibit the secretion of TSH and result in decreased functioning of the thyroid gland (Shimoda et al., 1962; Yamada, 1960). However, Christie (1964) felt that trypan blue may have increased the TSH and inhibited the release of thyroglobulin from the thyroid gland.

Thyroiditis occurs more frequently in women than in men (Roitt and Donioch, 1960). It is of interest that the thyroiditis in animals given trypan blue in this experi- ment occurred more often in females than in males. This difference in the sexes was also observed with the thyroiditis induced in rats by other chemicals (Glover and Reuber, 1967, 1968 ; Reuber and Glover, 1968).

TRYPAN BLUE INDUCED THYROIDITIS 113

REFERENCES

CHRISTIE, G. A. (1964). The teratogenic effect of trypan blue and its effect on the thyrohypo- physeal axis in the rat. J. Anat. 98, 377-384.

CHRISTIE, G. A. (1966). Influence of thyroid function on the teratogenic activity of trypan blue in the rat. J. Anat. 100,361-368.

CRISPELL, K. R., COLEMAN, J., and HYER, H. (1957). Factors affecting the binding capacity of human erythrocytes for I131 labeled l-thyroxine and I-triiodothyronine. J. Clin. Endocrinol. Metab. 17, 1305-1312.

GLOVER, E. L., and REUBER, M. D. (1967). Chronic thyroiditis in Buffalo rats with carbon tetrachloride-induced cirrhosis. Endocrinology 80, 361-364.

GLOVER, E. L., and REUBER, M. D. (1968). Chronic thyroiditis in Buffalo strain rats ingesting 3-methylcholanthrene. Arch. Pathol. in press.

JONES, H. E. H., and Rorrr, I. M. (1961). Experimental auto-immune thyroiditis in the rat. Brit. J. Exptl. Pathol. 42, 546-557.

PAOLETTI, C., RIou, G., and TRUHAUT, R. (1962). Electrophoretic pattern of plasma protein in rats treated with trypan blue and ethionine. Nature 193, 784-785.

REUBER, M. D. (1968). Thyroiditis in Buffalo strain rats with dietary-induced cirrhosis. Brit. J. Exptl. Pathol. in press.

REUBER, M. D., and CLOVER, E. L. (1968). Thyroiditis in rats injected subcutaneously with 3-methylcholanthrene. Proc. Sot. Exptl. Biol. Med. in press.

ROITT, I. M., and DONIACH, D. (1960). Thyroid auto-immunity. Brit. Med. Bull. 16, 152-158. SHIMODA, S., TOMIZAWA, T., YAMADA, T., and SCHICHIJO, K. (1962). Further studies on the

inhibitory effect of diazo dyes on thyroid function in the rat. Endocrinology 71, 414-421. TERPLAN, K. L., WITEBSKY, E., ROSE, N. R., PAINE, J. R., and EGAN, R. W. (1960). Experi-

mental thyroiditis in rabbits, guinea pigs, and dogs, following immunization with thyroid extracts of their own and of heterologous species. Am. J. Pathol. 36,213-239.

YAMADA, T. (1959). Abnormal serum protein observed in trypan blue-treated rats. Proc. Sot. Exptl. Biol. Med. 101, 566-568.

YAMADA, T. (1960). The effect of trypan blue on thyroid function in the rat. Endocrinology 67,204-2 I 1.