GRAHAM et al.
Effects of One-Year Administration of Ethylenethiourea upon the Thyroid of the Rat Stuart L. Graham,* Walter H. Hansen, Kent J. Davis,l and Carleene H. Perry
Male and female Charles River rats, approximately 5-weeks-old, were fed ethylenethiourea (ETU) for 2, 6, or 12 months a t levels of 0, 5, 25, 125, 250, or 500 ppm in the diet. Body weight, thyroid and other organ weights, thyroidal 1311 uptake, hematology, and histology were the criteria studied. Significant decreases in body weight and increases in thyroid weight were seen at the 125-, 250-, and 500-ppm levels. Uptake of I3lI
Dithiocarbamates have been used as fungicides against a variety of pathogenic plant fungi. Smith et al. (1953) reported thyroid changes in rats fed the ethylenebisdithiocarbamate fungicides throughout their life span. These compounds are unstable and one of the degradation products is ethylenethiourea (ETU). A possible degradation scheme for metallic derivatives of the ethylenebisdithiocarbamates has been suggested and is shown in Figure 1. Ludwig and Thorn (1958) speculated that ethylenebisdithiocarbamic acid readily forms ETU under highly alkaline conditions (pH 10.5). It is possible that the ETU obtained under these conditions is formed from ethylenethiuram monosulfide by the loss of a molecule of carbon disulfide. Until recently, the literature had only one reference to a toxicological study utilizing ETU. Seifter and Ehrich (1948) fed ETU to weanling rats a t 0.1% of the diet (1000 ppm) for 8 days and noted decreased growth, increased thyroid weight, and marked thyroid hyperplasia. Innes et al. (1969) revived the interest in ETU when they reported that this compound was tumorigenic for the liver; thyroids were not examined. Graham and Hansen (1972), in a short-term feeding study using male OsborneMendel rats, reported increases in thyroid weight, decreases in 1311 uptake, and, at high doses (500 and 750 ppm), thyroid hyperplasia. Ulland et al. (1972) reported a study in which rats were fed 350 or 175 ppm of ETU in their diets for 18 months, followed by a control diet for 6 months. They concluded that ETU has an action like that of a number of other thio compounds which cause thyroid carcinomas and indirectly affect the liver. This report describes the effects on the thyroid glands of male and female Charles River rats after 2, 6, and 12 months of ETU administration. MATERIALS AND METHODS
Five groups of 68 male and 68 female Charles River rats, approximately 5-weeks-old, were started on ETU diets. Control groups were fed the basic diet of Purina Ground Chow, while the test groups were maintained on the commercial diet containing added ETU (Lot No. 4876, K & K Laboratories, Plainview, N. Y.) a t levels of 5, 25, 125, 250, or 500 ppm. The rats were individually housed and food and water were provided ad libitum. Body weights and food consumption were recorded every 7 days. At the end of the 2-, 6-, and 12-month feeding periods, ten males and ten females a t each ETU level were given, intraperitoneally, 0.2 ml of physiologic saline prepared to contain approximately 5 pCi of 1311. The rats were fasted Bureau of Foods, Food and Drug Administration, DeDartment of Health. Education. and Welfare. Washington. D. C. 20204. Present address: Environmental Protection Agency, Washington, D. C. 20460. 0
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was significantly decreased in male rats after 12 months at 500 ppm but was increased in females. After 12 months, microscopic examination of the thyroids revealed the development of nodular hyperplasia a t dose levels of 125 ppm and higher; carcinomas were found a t dose levels of 250 and 500 ppm. The development of thyroid carcinomas indicates that, under the conditions of this study, ETU is a carcinogen.
for 24 hr. Each animal was then given a barbiturate overdose and the thyroids, heart, liver, kidneys, spleen, brain, and testes, in the case of the males, were removed and weighed. The radioactivities of individually weighed thyroid pairs were determined with a y well counter. Reference standards were made a t the time of injection and all counts were compared with this standard as an average of three successive determinations of counts per minute (cpm). The uptake was determined after conventional corrections were made for radioactive decay. After 3 and 11 months, blood samples were collected from the tail vein of ten male and ten female rats a t each dietary level and hemoglobin, hematocrit, leukocyte counts, and leukocyte differential counts were determined. After the 6- and 12-month feeding periods, tissues were removed from all sacrificed animals and evaluated grossly, and the hematoxylin-eosin-stained (H & E ) paraffin sections of the formalin-fixed thyroids were then examined by light microscopy. All data were compiled and tested for significance by the two-tailed student t test. RESULTS AND DISCUSSION
Body Weights. Table I shows the effect of ETU on the body weights of rats fed the test diets for 2, 6, and 12 months. The body weights of males fed ETU for 2 months were significantly decreased from control values a t the 250- ( p < 0.01) and 500-ppm ( p < 0.001) levels; males fed ETU for 6 months had body weights which were decreased significantly at the 25- ( p < 0.001), 250- ( p < 0.01), and 500-ppm ( p < 0.001) levels. After 12 months, only the weights of male rats fed ETU at 500 ppm were significantly lower than the control value ( p < 0.001). The body weights of female rats fed ETU for 2 months were significantly decreased from control values a t the 25-, 125-, 250-, and 500-ppm levels (Table I), and after 6 and 12 months they were significantly decreased a t the 125-, 250-, and 500-ppm levels. Organ-to-Body Weight Ratios. 2 Months. The effects of ETU administration for 2 months on organ-to-body weight ratios of rats are shown in Table 11. Liver-to-body weight ratios of both males and females, as compared with control ratios, were increased significantly at the 125-, 250-, and 500-ppm levels. In males fed 500 ppm, the kidneys and testes ratios were significantly increased over control values. The greatest effect of ETU was seen in the thyroid gland; thyroid ratios of the male rats fed the 250and 500-ppm levels and females fed the 125-, 250-, and 500-ppm levels were significantly ( p < 0.001) elevated over control values. 6 Months. Table I11 gives the organ-to-body weight ratios of rats fed ETU for 6 months. In males, ratios for liver, kidneys, and testes were significantly elevated above those of the controls at the 125-, 250-, and 500-ppm levels,
ETU EFFECTS ON RAT THYROID Table I. Mean Body Weights (9) f S E of Rats Fed ETU in the Diet
H I
S
Dietary No. level, of ppm rats
2 months of diet
No. of rats
of rats
12months of diet
-1
CHI-N-C
I
No. 6 months of diet
U
II
%-4-*-S
'M
/
CH.-N-C-S
I H
0 5 25 125 250 500
68 68 68 68 67 68
419f5 421f4 411 f 4 421 f 5 403 f 46 327 f 4a
56 58 57 58 58 58
617f10 613f8 583 f 8a 601 f 9 579 f 8 6 509 f 7a
45 48 47 46 44 47
7 0 3 f 13 711f11 670 f 12 692 f 12 677 f 12 604 f 15a
47 48 46 48 46 46
4 6 6 f 11 4 6 4 f 11 442 f 10 429fllc 408 f g a 406 f 8a
-
68 68 68 68 68 68
270 267 259 254 245 231
f 3 f 3 f 36 f3a f3a f 3a
58 58 58 58 57 58
371 f 6 363f7 357 f 5 342f5a 331 f 5a 326 f 5a
/
+
MS
-
S /I
+
"i>=S
CS*+
S
CH,-N H
II
D I
Ethyiendhiunrm
EthylinethDYrW
MmOwIfIde
Figure 1. D e g r a d a t i o n s c h e m e for m e t a l l i c d e r i v a t i v e s of t h e ethylenebisdithiocarbamates
Significantly different from control value p < 0 001 il Slgnificantly different from controi value p < 0 01 Significantly different from control value p < 0 05 a
H
I1 S'
HI
io1 ZlNEB where M Z I ~ C ib) MANEB where M rmqaneso
Females
0 5 25 125 250 500
S
C%-N-C
I1 S
I
Males
I
'
as were thyroid-to-body weight ratios at the 250- and 500ppm levels. In females, statistically significant increases in liver-to-body weight ratios were seen at the 250- and 500-ppm levels and in thyroid ratios a t the 125-, 250-, and 500-ppm levels. 12 Months. The effects of ETU feeding for 12 months on organ-to-body weight ratios of rats are shown in Table IV. Thyroid-to-body weight ratios of both males and females fed the 125-, 250-, and 500-ppm levels were significantly increased as compared with control values. In females, liver and kidney ratios were elevated significantly over control values a t both the 250- and 500-ppm levels. Uptake of Radioactive Iodine. The uptakes of iodine (expressed as cpm/mg of tissue) 24 hr postinjection are
Table II. Mean Organ-to-Body Weight (g/kg) Ratios f S E of Charles River Rats Fed ETU for 2 Months (Ten Rats per Group) Dietary level, ppm
I-iver
Kidney
Spleen
Testes
Heart
Thyroida
Males
0 5
25 125 250 500
25.94 f 0.57 26.50 f 0.54 26.73 f 0.55 28.50 f 0 . 4 l C 29.58 f O.3lb 29.53 f 0.76c
6.48 f 0.14 6.62 f 0.13 6.54 f 0.13 6.72 f 0.20 6.66 f 0.16 7.00 f 0.136
1.73 f 0.09 1.52 f 0.07 1.67 f 0.09 1.53 f 0.11 1.51 f 0.09 1.62 f 0.17
2.94 f 0.10 3.13 f 0.16 2.85 f 0.09 2.83 f 0.1 1 2.92 f 0.10 2.89 f 0.13
8.22 f 0.25 7.60 f 0.15 7.47 f 0.236 7.47 f 0.26 7.79 f 0.22 10.78 f 0.55d
44.7 f 2 . 3 45.4 f 3.5 40.1 f 1.9 55.9 f 4.9 93.6 f 5.gd 102.9 f 8.3d
Females
0 5
25 125 250 500
25.40 f 0.49 24.93 f 0.56 25.50 f 0.37 27.60 f 0.07b 28.72 f 1 .27b 29.34 f 0.76d
6.48 f 0.14 6.37 f 0.13 6.88 f 0.15 6.73 f 0.22 6.92 f 0.32 6.91 f 0.43b
1.96 f 0.06 2.01 f 0.12 2.00 f 0.08 2.11 f 0.10 2.17 f 0.17 1.69 f 0.086
a Thyroid ratios are expressed as mg/kg. b Significantly different from controi, p different from control, p < 0.001.
60.0 f 2.9 66.8 f 3.5 66.2 f 3.3 78.4 f 3.3d 105.5 f 5.3d 171.3 f 15.5d
3.10 f 0.09 2.86 f 0.08 3.02 f 0.03 3.14 f 0.06 3.12 f 0.13 2.86 f 0.06
< 0.05. C Significantly
different from control, p
< 0.01.
Significantly
Table 1 11 . Mean Organ-to-Body Weight (g/kg) Ratios f S E of Charles River Rats Fed ETU for 6 Months (Ten Rats per Group) Dietary level, ppm
Liver
Kidney
Spleen
Heart
Testes
Thyroida
Males
0 5 25 125 250 500
23.30 f 0.37 23.61 f 0.63 24.82 f 0.13 29.89 f 0.966 29.35 f 0.766 30.98 f 1.15b
5.66 f 0.10 5.81 f 0.23 5.62 f 0.18 6.52 f 0.23c 6.12 f 0.18d 6.22 f 0.21d
1.35 f 0.08 1.32 f 0.07 1.31 f 0.06 1.43 f 0.08 1.44 f 0.07 1.31 f 0.09
2.64 iz 0.06 2.65 f 0.11 2.61 f 0.09 2.66 f 0.10 2.75 f 0.06 2.76 f 0.08
6.28 f 0.17 6.27 f 0.33 6.02 f 0.31 6.83 f 0.32d 6.88 f 0.16d 7.72 f 0.246
51.5 f 3.3 45.1 f 2.4 44.3 f 2.3 61.9 f 4.1 79.9 f 6.1L 139.5 f 14.9O
Females
0 5 25 125 250 500
31 .OO i 0.68 32.14 f 1.21 28.68 f 0.8W 31.74 f 1.41 33.75 f 1.09d 35.58 f 0.64b
6.07 f 0.20 6.15 f 0.19 5.90 f 0.18 6.11 f 0.11 6.40 f 0.18 6.40 f 0.27
a Thyroid ratios are expressed as mg/kg. different from control, R < 0.05.
1.83 f 0.23 1.65 f 0.07 1.59 f 0.07 1.50 f 0.07 1.60 f 0.11 1.57 f 0.07
Significantly different from control, p
59.1 f 3.1 65.9 f 4.4 59.8 f 3.8 72.0 f 4.6d 9 3 . 5 f 4.1° 174.6 f 21 .56
2.81 f 0.09 3.04 f 0.09 2.90 f 0.12 3.00 f 0.14 3.11 f 0.12 3.01 f 0.12
< 0.001,
significantly different from controi. p
< 0.01.
Significantly
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GRAHAM e t al Table IV. Mean Organ Weight-to-Body Weight (g/kg) Ratios f SE of Charles River Rats Fed ETU for 12 Months (Ten Rats per Group) ~~
Dietary level. o m ~
~~
~~
Liver
Kidney
Ween
Heart
Testes
Thy ro Ida
~
Males 0
5 25 125 250 500
26.96 f 1.22 25.38 f 0.69 24.89 f 1.09 31.13 f 1.246 27.88 f 1.36 28.32 f 1.21
5.52 5.24 5.57 5.77 5.73 5.40
1.55 f 0.07 1.57 f 0.07 1.65 f 0.05 1.50 f 0.10 1.60 f 0.14 1.22 f 0.09c
f 0.27 f 0.15
f 0.21 f 0.22 f 0.31 f 0.20
2.42 2.56 2.44 2.47 2.42 2.32
f 0.09
2.66 2.65 2.57 2.60 3.08 2.77
f 0.13 f 0.13 f 0.09 f 0.1 1 f 0.156 f 0.07
44.3 f 2.0 43.9 f 3 . 3 48.8 f 2.9 52.9 f 3.5b 87.7 f 13.2C 779.0 f 231 .46
5.56 f 0.20 5.99 f 0.21 5.80 f 0.28 5.76 f 0.29 5.76 f 0.18 6.23 f 0.26
f 0.08
f 0.07 f 0.10 f 0.16 f 0.1 1
Females 0 5 25 125 25od 500
5.29 5.32 5.52 5.53 6.21 6.24
25.73 f 1.55 22.68 f 0.76b 24.07 f 0.82 26.97 f 1.21 29.78 f 1.42* 30.64 f 1.676
1.60 f 0.09 1.85 f 0.19 1.37 f 0.06 1.68 f 0.1 1 1.50 f 0.08 1.61 f 0.10
f 0.22 f 0.27
f 0.26 f 0.25
f 0.306 f 0.2a6
Thyroid ratios are expressed as mg/kg. b Significantly different from control, p e Significantly different from control, p < 0.001, a
%I2
< 0.05. C Significantly
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