[WCEthylenethiourea: ] Distribution, Excretion, and Metabolism in Pregnant Rats JOSEPH A. RUDDICK, DAVID T. WILLIAMS, L. HIERLIHY AND K. S. KHERA Health Protection Branch, Bureau of Chemical Safety, Food Directorate, Tunney's Pasture, Ottawa, Ontario K I A OL2, Canada
ABSTRACT Following administration of single oral doses of [14C] ethylenethiourea (ETU) to pregnant rats maternal blood maintained peak radioactivity for 2 h, and the radioactivity was dispersed uniformly between the red blood cells and plasma. The level of radioactivity was distributed equally among several maternal tissues but was present in lower amounts in embryos. Twenty-four hours after treatment all tissues examined, except blood, were relatively clear of radioactivity and 72.8% of the total radioactivity given had been excreted in the urine. Elution patterns of metabolites from Sephadex separation suggested that ethylenethiourea was degraded very little. The teratological mechanism is discussed.
Ethylenethiourea (ETU) is one of the decomposition products and metabolites of the ethylenebis(dithi0carbamate) group of fungicides (Vonk, '75). It has been found to be teratogenic following multiple or single doses (Khera, '73; Ruddick and Khera, '75), carcinogenic (Ulland et al., '72), and goitrogenic (Graham and Hansen, '72). Metabolism of ETU in cows resulted in its conversion and breakdown to several products which appeared in the milk and urine. It was concluded that the carbon atoms from ETU also entered the general metabolic pool (Lyman, '72). In rats and guinea pigs 64.9 and 47.4% respectively, of the administered oral dose was excreted as ETU in the urine in 48 h (Newsome, '74). As noted above ETU is teratogenic but the teratological mechanism, as with other teratogens, remains to be determined. This report presents data regarding the distribution, excretion, and metabolism in rats of single oral teratogenic doses of [14C] ETU. MATERIALS AND METHODS
[4,5I4C]ETU was synthesized in our laboratories from ethylene [ 1,214CI diamine dihydrochloride (New England Nuclear, Boston, Massachusetts) as described by Allen et al. ('46). Chemical and label purity were determined by thin layer chromatography, melting point, and radiochromatography (Ruddick and Khera, '75); there were less TERATOLOGY, 1 3 : 35-40.
than 0.05% impurities. A single oral dose, containing 240 mg/kg ETU and 50 pCi/ kg [14C] ETU, was given for all experiments except for the blood-level and excretion studies for which radiolabel was reduced to 25 pCi/kg. All samples, except those from the Sephadex fractions, were prepared for radioactivity counting by being solubilized in a scintillation vial at room temperature with 1 ml of Soluene (Packard, Downers Grove, Illinois) and subsequent addition of 15 ml of Spectrafluor (Amersham/Searle, Arlington Heights, Illinois). Samples taken from the Sephadex fractions were dissolved in 10 ml of Aquasol (New England Nuclear, Boston, Massachusetts) and counted. Duplicate radioactivity counts of each sample were determined in a Beckman LS-230 scintillation counter. A n external standardization method was used for quench correction. Results are expressed as dpm/ mg of wet weight tissue or dpm/ml of blood and urine. Distribution and excretion. Wistar female rats (Woodland Farms, Guelph, Ontario, 175-200 g) were treated on day 1 1 , 12, or 15 of gestation (day 1 began the morning a sperm-positive smear was noted). Those dosed on day 11 or 12 were killed 6, 12, or 24 h after treatment. Samples of approximately 100 mg of maternal kidney and liver and of pooled embryos Received May 30, '75.Accepted Oct. 24,'75.
35
36
J. A. RUDDICK, D. T. WILLIAMS, L. HIERLIHY AND K. S. KHERA
and 0.02 ml of maternal blood and urine were taken from all rats and the amount of radioactivity in each sample determined. Females dosed on day 15 were killed 3 h later and in addition to the above tissues muscle and placenta samples were analyzed for radioactivity. Blood levels and excretion of radiolabel were studied on day 15 of gestation in 3 females isolated in metabolic cages. Duplicate 0.02 ml aliquots of blood were taken from the tail at 0.5, 1, 2, 4, 6, 12, 24, and 48 h after treatment. Total urine was collected for 0-12, 12-24,24-32, and 32-48 h after treatment with 0.02-ml aliquots processed for counting. Blood and embryo analysis. Whether radiolabeled ETU binds to red blood cells (RBC) was determined by taking 5 ml of heparinized blood from 2 females 2 h after oral dosage. One-milliliter aliquots of this blood were centrifuged at 1,500 g . The plasma was removed, combined with 0.5 ml of physiological saline, and 0.5 ml of 30% TCA shaken and left to stand for 30 min. Aliquots of 0.05 ml from the homogenate before and the supernatant after centrifuging for 10 min at 12,000 g were counted. The residual RBC were washed twice with physiological saline, the packed cells resuspended in 1 ml of 0.9% saline, and 0.02 ml analyzed for radioactivity. To other 1-ml aliquots of the collected maternal blood were added 1 ml of 0.9% saline followed by 0.5 ml of 40% trichloroacetic acid (TCA). The samples were shaken, left to stand for 30 min, shaken again, and 0.02 ml-aliquots removed for counting. The residual homogenates were centrifuged at 12,000 g for 10 min and 0.05 ml quantities from the supernatant removed for radioactivity counting. Binding of label to embryonic tissue was studied in pooled day-12 embryos (5 each at 6 and 12 h after treatment) and 5 day-15 embryos. The 3 groups of separately pooled embryos were sonicated in 1 ml of 0.9% NaCl and 0.5 ml of 15% TCA added. After standing for 30 min 0.05-ml samples of sonicate were counted. Following resuspension of the pellet bulk DNA, RNA, and protein supernatant were extracted (Ruddick and Runner, '74) and the amount of label in each fraction determined. Characterization of metabolites. Labeled metabolites were separated with a
Sephadex G-10 column (18 X 32 mm). The void volume (Vo) a s measured with dextran blue for the column was 10 ml, the flow rate was 0.5 ml/min, and the eluting solution was 0.9% NaC1. An LKB Ultrorack fraction collector (Fisher Scientific, Ottawa, Ontario) was used to collect 0.5-ml samples of which 0.1 ml from every second tube after the void volume was counted. Five milliliters of maternal urine or blood were precipitated with 1 ml of 10% TCA, centrifuged, and the supernatants flash evaporated to dryness at 30°C. The residues were redissolved in 2 ml of distilled HzO, charged to the column, and eluted. Seven day-12 or -15 embryos were sonicated in 4 ml of distilled HzO and were processed for column separation in the same manner as the maternal urine and blood. A 2-ml aliquot of the [14C] ETU dosing solution (40,000 cpmlml) was used to determine the elution volume of ETU. ETU eluted in fractions 85-95. To determine whether ETU or ethyleneurea was excreted in the urine, 30 pg of pooled eluted fractions 85-95 and 0.5 pl of 1 % ['W] ETU, ethyleneurea, and ETU were spotted on duplicate silica-gel thinlayer chromatography plates (Kodak). Each plate was developed 3 times in CHC13 saturated with water. One of the plates was exposed on an X-ray film (Kodak, RP Royal X - 0 Mat) for 20 days and developed. The other plate was developed in an iodine chamber. RESULTS
The level of radioactivity (mean f SD) in maternal kidney (25.3 & 4.5 dpmlmg) and liver (23.4 f 4.8 dpmlmg) and in embryos (12.3 f 5.3 dpmlmg) remained statistically unchanged at 6 and 12 h following administration of [l4C] ETU on day 11 of gestation (table 1). Twenty-four hours after treatment the radioactivity dropped to 3.7 f 1.2 and 3.9 f 1.2 dpm/ mg in maternal kidney and liver and to 1.8 f 0.5 dpmlmg in embryos. Maternal blood counts (dpmlml X 103) were 24.8 f 5.2, 20.6 f 2.5, and 2.2 t 0.7 for 6, 12, and 24 h respectively. Similar results were obtained following treatment on day 12 of gestation. Maternal liver and kidney at 6 and 12 h after treatment had a similar mean value, 17.5 3.6 dpmlmg, and decreased to 4.6 f
*
37
[14C] ETHYLENETHIOUREA I N PREGNANT RATS TABLE 1
Distribution of radioactivity in m a t e r n a l t i s s u e s a n d e mb r y o s . Va lu e s ( m e a n k S D ) we r e derived f r o m du p l i c a t e s a m p l e s of e a c h tissue t a k e n from 3 f e m a l e s killed at e a c h t i m e interval Day 12
D a y 11
6h
Kidney Liver Blood(X103)' Fetus 1
12 h
25.3 k 4.5 25.8 2 3.9 2 3 . 4 i 4 . 8 24.2t2.6 24.825.2 20.622.5 12.3k5.3 1 4 . 5 t 1 . 2
24 h
3.7 k 1.2 3.9k1.2 2.220.7 1.850.5
6h
12 h
19.1k4.4 1 6 . l c 4 . 1 18.8k4.2 15.3k2.9 16.0k3.9 12.2k2.6 12.1s2.9 6.5?1.1
Day 15 24 h
3 h
4.521.8 4.7s1.6 2.9k1.4 2,320.9
91.0212.7 89.9k 7.4 82.0-+ 8.3 73.025.6
dpm/ml, all other values are dpmlmg.
I00
80 W
In
0
a
m
0
60 LL
X
at
0
3:
z
2
n
4oc w
0
a
0
d
0 X W
20
TIME IN HOURS ( P O S T D O S I N G )
Fig. 1 A plot of the mean values of labeled activity in maternal blood and urine following an oral dose of [14C] ETU.
1.7 dpm/mg at 24 h. Radioactivity in maternal blood was 16.0 & 3.9 X lo3 and 12.2 2.6 x 103 dpmlml at 6 and 12 h, decreasing to 2.9 & 1.4 X 10 dpm/ml at 24 h. Counts in embryos were 12.1 f 2.9, 6.5 & 1.1, and 2.3 f 0.9 dpm/mg at 6, 12, and 24 h, respectively. Radioactivity in urine had a consistent mean value of 10.5 f 0.9 X 106 dpm/ml at all times examined and for treatment on day 11 or 12 of gestation. Counts in tissues (maternal liver, kidney, and muscle and placenta and embryo) taken 3 h after treatment on day 15 of gestation were statistically the same, among these samples, with a mean value of 81.4 & 7.9 dpm/mg (table 1). Maternal blood
*
had 82.0 & 8.3 X 103 dpm/ml and urine 14.1 +- 2.3 X lOSdpm/ml. For rats isolated in metabolic cages and treated on day 15 the radioactivity in maternal blood was 30.2 +- 4.9 X 103 dpm/ ml at 0.5 h and continued in this range until 2 h (28.8 & 0.4 X 10" dpm/ml) after treatment. At 4 h (24.6 f 2.1 X 103 dpmlml) the counts began to decrease and continued to do so until they were 1.2 & 0.2 X 10" dpm/ml at 48 h. The maternal blood half-life of radiolabel was calculated to be 10 h. Of the total radioactivity administered on day 15 of gestation the maternal urine contained 54.8% at 12 h, 72.8% at 24 h, and 84% at 48 h posttreatment (fig. 1).
38
J. A. RUDDICK, D. T. WILLIAMS, L. HIERLIHY AND K. S. KHERA
With or without the addition of TCA to maternal blood the label was distributed uniformly between RBC and plasma. The association between RBC and label was very frail since washing of centrifuged RBC removed the label. All of the label present in the plasma was TCA soluble. The radioisotope in the embryos was also TCA soluble. No label was detected in DNA, RNA, or protein fractions. Metabolites in day-12 embryos could not be characterized because too little label was present. However Sephadex separation of the TCA supernatant from day-15 embryos indicated a peak that corresponded to [ 14C] ETU. Profile characterization of labeled metabolites in maternal urine following all 3 days of treatment indicated the same pattern. There were 3 peaks; 2 overlapped, with peak maximums at fraction 42 and 52, while the third, peak maximum at fraction 90, corresponded to "4C] ETU. The ratio of counts between fractions 42, 52, and 90 was 4:1:600. Radiochromatography of TLC plates indicated predominantly ETU in eluted urine fraction 90 with a slight trace of ethyleneurea. Characterization of metabolites in the plasma revealed 2 peaks: an unknown at fraction 52, which was similar to one of the unknowns observed in urine, and a peak analogous to "4C] ETU. The ratio of counts between fractions 52 and 90 was 1:15. DISCUSSION
Reports on the biochemical behavior of thiourea and its derivative permitted a comparison with ETU's metabolism. Label from [ " T I ETU was absorbed r a p idly from the gastrointestinal tract. Radioactivity in maternal blood maintained peak activity from 0.5-2 h after treatment with uniform dispersion of the label; i.e., the label behaved as if it were a homogeneous solution. The eventual elimination of label from blood indicated that the label was not bound irreversibly to RBC. Phenylthiourea (Combs and Giri, '73) and thiourea (Williams and Kay, '45) have been observed to bind to RBC. None of the maternal organs examined, nor the embryos, selectively accumulated label. Three hours after treatment maternal liver, kidney, and muscle and placenta and embryo all had the same levels of
label and this similarity among tissues continued to 12 h after treatment. By 24 h all tissues except blood were relatively free of label. Shulman and Keating ('50) noted that rats injected ip with [3%] thiourea had label concentrations in tissue equally distributed at 6 h but differing somewhat at later times. The thyroid, which we did not examine, had the greatest affinity of all tissues for thiourea (Maloof and Soodak, '57; Shulman and Keating, '50) and ETU (Newsome, '74). There were no detectable binding of radiolabel in day-12 or -15 embryos. The radioactivity in embryos was apparently dispersed uniformly since no binding to either DNA, RNA, or protein was noted. These results were similar to those observed for maternal blood. It is possible that the minute amounts of label in the whole embryo made detection in a specific fraction difficult. The rate of radiolabel elimination from embryo was similar to maternal liver and kidney. Pathological examination of embryos (unpublished data) indicated that the teratological inductions were accomplished within 18 h. In the present study, 24 h after treatment all maternal organs and embryos were almost free of label with 72.8% of the total label given being excreted in the urine. Newsome ('74) observed that after 24 h 61.2% of the total oral dose administered to rats was present as ETU in urine and 1% in feces. The implication is that ETU, unlike thiourea, is degraded very lettle. Desulfuration of thiourea to give rise to inorganic sulfate, thiosulfate, and protein-bound sulfur (Maloof and Spectar, '59; Maloof and Soodak, '57) as well as 3 5 s which concentrated in the fetal thyroid and trachea (Shepard, '63) has been reported. Profile characterization of label metabolites in our experiments suggested a conjugated product. The labeled metabolites did not a p pear to be formed via the metabolic pool since no general labeling was observed upon fractionation of embryonic DNA, RNA, and protein. TLC and radiochromatography of radioactive compounds in the urine indicated primarily ETU (99% ) with traces of ethyleneurea and 2 unknown metabolites. This is in contrast with cow which catabolize ETU to several compounds
[“C] ETHYLENETHIOUREA IN PREGNANT RATS
that are eliminated in the milk and urine as well as release carbon atoms of ETU into the general metabolic pool (Lyman, ’71). The teratological action of ETU appears to be initiated by ETU, per se, and not a degradation product. Other experiments in our laboratory (unpublished) have indicated that the imidazolidine ring with adjoining sulfur atom are essential for the teratogenicity of ETU. With no detectable binding of radiolabel to the embryonic DNA, RNA, or protein the suggestion is that ETU initiates the teratological event but other steps follow that produce the anomalies. Whether this process begins in the pregnant female or the embryo cannot be determine at this time. LITERATURE CITED Allen, C. F. H., Co. Edens and J. Von Allen 1946 Ethylenethiourea. Org. Synth., 26: 34-35. Combs, A. B., and S. N. Giri 1973 Trichloroacetic acid-induced binding of phenylthiourea to erythrocytes. J. Pharm. Sci., 62; 631-633. Graham, S. H., and W. H. Hansen 1972 Effects of short-term administration of ethylenethiourea upon thyroid function of the rat. Bull. Env. Contam. Toxic., 7: 19-25. Khera, K. S. 1973 Ethylenethiourea: teratogenicity study in rats and rabbits. Teratology, 7: 24 3-254. Lyman, W. R. 1971 Metabolic fate of Dithane
39
M-45. Coordination product of zinc ion and manganous ethylenebisdithiocarbamate. In: Int. Sym. Pesticide Terminal Residues. A. S. Tahori, ed. Butterworth, New York, pp. 243-256. Maloof, F., and M. Soodak 1957 The uptake and metabolism of S35 thiourea and thiouracil by the thyroid and other tissues. Endocrinology, 61 : 55 5-56 9. Maloof, F., and L. Spectar 1959 The desulfuration of thiourea by thyroid cytoplasmic particulate fractions. J. Biol. Chem., 234: 949-954. Newsome, W. H. 1974 The excretion of ethylenethiourea by rat and guinea pig. Bull. Env. Contam. Toxic., 11 ; 174-176. Ruddick, J. A,, and K. S. Khera 1975 Pattern of anomalies following single oral doses of ethylenethiourea to pregnant rats. Teratology, 12: 2 7 7-282. Ruddick, J. A., and M. N. Runner 1974 5-FU in chick embryos as a source of label for DNA and a depressant of protein synthesis. Teratology, 10: 39-46. Shepard, T. H. 1963 Metabolism of thioureau S35 by the fetal thyroid of the rat. Endocrinology, 72; 223-230. Schulman, Jr., J., and R. P. Keating 1950 Studies on the metabolism of thiourea. J. Biol. Chem., 183: 215-221. Ulland, B. M., J. H. Weisburger, E. K. Weisburger, J. M. Rice and R. Cypher 1972 Thyroid cancer in rats from ethylenethiourea intake. J. Nat. Cancer Inst., 49: 583-584. Williams, R. H., and G. A. Kay 1945 Absorg tion, distribution and excretion of thiourea. Am. J. Physiol., 143: 715-722. Vonk, J. W. 1975 Chemical Decomposition of Bisdithiocarbamate Fungicides and Their Metabolism by Plants and Microorganisms. Ph.D. Thesis. Elinkwijk BV, Utrecht, Holland.
ABSTRACT Following administration of single oral doses of [14C] ethylenethiourea (ETU) to pregnant rats maternal blood maintained peak radioactivity for 2 h, and the radioactivity was dispersed uniformly between the red blood cells and plasma. The level of radioactivity was distributed equally among several maternal tissues but was present in lower amounts in embryos. Twenty-four hours after treatment all tissues examined, except blood, were relatively clear of radioactivity and 72.8% of the total radioactivity given had been excreted in the urine. Elution patterns of metabolites from Sephadex separation suggested that ethylenethiourea was degraded very little. The teratological mechanism is discussed.
Ethylenethiourea (ETU) is one of the decomposition products and metabolites of the ethylenebis(dithi0carbamate) group of fungicides (Vonk, '75). It has been found to be teratogenic following multiple or single doses (Khera, '73; Ruddick and Khera, '75), carcinogenic (Ulland et al., '72), and goitrogenic (Graham and Hansen, '72). Metabolism of ETU in cows resulted in its conversion and breakdown to several products which appeared in the milk and urine. It was concluded that the carbon atoms from ETU also entered the general metabolic pool (Lyman, '72). In rats and guinea pigs 64.9 and 47.4% respectively, of the administered oral dose was excreted as ETU in the urine in 48 h (Newsome, '74). As noted above ETU is teratogenic but the teratological mechanism, as with other teratogens, remains to be determined. This report presents data regarding the distribution, excretion, and metabolism in rats of single oral teratogenic doses of [14C] ETU. MATERIALS AND METHODS
[4,5I4C]ETU was synthesized in our laboratories from ethylene [ 1,214CI diamine dihydrochloride (New England Nuclear, Boston, Massachusetts) as described by Allen et al. ('46). Chemical and label purity were determined by thin layer chromatography, melting point, and radiochromatography (Ruddick and Khera, '75); there were less TERATOLOGY, 1 3 : 35-40.
than 0.05% impurities. A single oral dose, containing 240 mg/kg ETU and 50 pCi/ kg [14C] ETU, was given for all experiments except for the blood-level and excretion studies for which radiolabel was reduced to 25 pCi/kg. All samples, except those from the Sephadex fractions, were prepared for radioactivity counting by being solubilized in a scintillation vial at room temperature with 1 ml of Soluene (Packard, Downers Grove, Illinois) and subsequent addition of 15 ml of Spectrafluor (Amersham/Searle, Arlington Heights, Illinois). Samples taken from the Sephadex fractions were dissolved in 10 ml of Aquasol (New England Nuclear, Boston, Massachusetts) and counted. Duplicate radioactivity counts of each sample were determined in a Beckman LS-230 scintillation counter. A n external standardization method was used for quench correction. Results are expressed as dpm/ mg of wet weight tissue or dpm/ml of blood and urine. Distribution and excretion. Wistar female rats (Woodland Farms, Guelph, Ontario, 175-200 g) were treated on day 1 1 , 12, or 15 of gestation (day 1 began the morning a sperm-positive smear was noted). Those dosed on day 11 or 12 were killed 6, 12, or 24 h after treatment. Samples of approximately 100 mg of maternal kidney and liver and of pooled embryos Received May 30, '75.Accepted Oct. 24,'75.
35
36
J. A. RUDDICK, D. T. WILLIAMS, L. HIERLIHY AND K. S. KHERA
and 0.02 ml of maternal blood and urine were taken from all rats and the amount of radioactivity in each sample determined. Females dosed on day 15 were killed 3 h later and in addition to the above tissues muscle and placenta samples were analyzed for radioactivity. Blood levels and excretion of radiolabel were studied on day 15 of gestation in 3 females isolated in metabolic cages. Duplicate 0.02 ml aliquots of blood were taken from the tail at 0.5, 1, 2, 4, 6, 12, 24, and 48 h after treatment. Total urine was collected for 0-12, 12-24,24-32, and 32-48 h after treatment with 0.02-ml aliquots processed for counting. Blood and embryo analysis. Whether radiolabeled ETU binds to red blood cells (RBC) was determined by taking 5 ml of heparinized blood from 2 females 2 h after oral dosage. One-milliliter aliquots of this blood were centrifuged at 1,500 g . The plasma was removed, combined with 0.5 ml of physiological saline, and 0.5 ml of 30% TCA shaken and left to stand for 30 min. Aliquots of 0.05 ml from the homogenate before and the supernatant after centrifuging for 10 min at 12,000 g were counted. The residual RBC were washed twice with physiological saline, the packed cells resuspended in 1 ml of 0.9% saline, and 0.02 ml analyzed for radioactivity. To other 1-ml aliquots of the collected maternal blood were added 1 ml of 0.9% saline followed by 0.5 ml of 40% trichloroacetic acid (TCA). The samples were shaken, left to stand for 30 min, shaken again, and 0.02 ml-aliquots removed for counting. The residual homogenates were centrifuged at 12,000 g for 10 min and 0.05 ml quantities from the supernatant removed for radioactivity counting. Binding of label to embryonic tissue was studied in pooled day-12 embryos (5 each at 6 and 12 h after treatment) and 5 day-15 embryos. The 3 groups of separately pooled embryos were sonicated in 1 ml of 0.9% NaCl and 0.5 ml of 15% TCA added. After standing for 30 min 0.05-ml samples of sonicate were counted. Following resuspension of the pellet bulk DNA, RNA, and protein supernatant were extracted (Ruddick and Runner, '74) and the amount of label in each fraction determined. Characterization of metabolites. Labeled metabolites were separated with a
Sephadex G-10 column (18 X 32 mm). The void volume (Vo) a s measured with dextran blue for the column was 10 ml, the flow rate was 0.5 ml/min, and the eluting solution was 0.9% NaC1. An LKB Ultrorack fraction collector (Fisher Scientific, Ottawa, Ontario) was used to collect 0.5-ml samples of which 0.1 ml from every second tube after the void volume was counted. Five milliliters of maternal urine or blood were precipitated with 1 ml of 10% TCA, centrifuged, and the supernatants flash evaporated to dryness at 30°C. The residues were redissolved in 2 ml of distilled HzO, charged to the column, and eluted. Seven day-12 or -15 embryos were sonicated in 4 ml of distilled HzO and were processed for column separation in the same manner as the maternal urine and blood. A 2-ml aliquot of the [14C] ETU dosing solution (40,000 cpmlml) was used to determine the elution volume of ETU. ETU eluted in fractions 85-95. To determine whether ETU or ethyleneurea was excreted in the urine, 30 pg of pooled eluted fractions 85-95 and 0.5 pl of 1 % ['W] ETU, ethyleneurea, and ETU were spotted on duplicate silica-gel thinlayer chromatography plates (Kodak). Each plate was developed 3 times in CHC13 saturated with water. One of the plates was exposed on an X-ray film (Kodak, RP Royal X - 0 Mat) for 20 days and developed. The other plate was developed in an iodine chamber. RESULTS
The level of radioactivity (mean f SD) in maternal kidney (25.3 & 4.5 dpmlmg) and liver (23.4 f 4.8 dpmlmg) and in embryos (12.3 f 5.3 dpmlmg) remained statistically unchanged at 6 and 12 h following administration of [l4C] ETU on day 11 of gestation (table 1). Twenty-four hours after treatment the radioactivity dropped to 3.7 f 1.2 and 3.9 f 1.2 dpm/ mg in maternal kidney and liver and to 1.8 f 0.5 dpmlmg in embryos. Maternal blood counts (dpmlml X 103) were 24.8 f 5.2, 20.6 f 2.5, and 2.2 t 0.7 for 6, 12, and 24 h respectively. Similar results were obtained following treatment on day 12 of gestation. Maternal liver and kidney at 6 and 12 h after treatment had a similar mean value, 17.5 3.6 dpmlmg, and decreased to 4.6 f
*
37
[14C] ETHYLENETHIOUREA I N PREGNANT RATS TABLE 1
Distribution of radioactivity in m a t e r n a l t i s s u e s a n d e mb r y o s . Va lu e s ( m e a n k S D ) we r e derived f r o m du p l i c a t e s a m p l e s of e a c h tissue t a k e n from 3 f e m a l e s killed at e a c h t i m e interval Day 12
D a y 11
6h
Kidney Liver Blood(X103)' Fetus 1
12 h
25.3 k 4.5 25.8 2 3.9 2 3 . 4 i 4 . 8 24.2t2.6 24.825.2 20.622.5 12.3k5.3 1 4 . 5 t 1 . 2
24 h
3.7 k 1.2 3.9k1.2 2.220.7 1.850.5
6h
12 h
19.1k4.4 1 6 . l c 4 . 1 18.8k4.2 15.3k2.9 16.0k3.9 12.2k2.6 12.1s2.9 6.5?1.1
Day 15 24 h
3 h
4.521.8 4.7s1.6 2.9k1.4 2,320.9
91.0212.7 89.9k 7.4 82.0-+ 8.3 73.025.6
dpm/ml, all other values are dpmlmg.
I00
80 W
In
0
a
m
0
60 LL
X
at
0
3:
z
2
n
4oc w
0
a
0
d
0 X W
20
TIME IN HOURS ( P O S T D O S I N G )
Fig. 1 A plot of the mean values of labeled activity in maternal blood and urine following an oral dose of [14C] ETU.
1.7 dpm/mg at 24 h. Radioactivity in maternal blood was 16.0 & 3.9 X lo3 and 12.2 2.6 x 103 dpmlml at 6 and 12 h, decreasing to 2.9 & 1.4 X 10 dpm/ml at 24 h. Counts in embryos were 12.1 f 2.9, 6.5 & 1.1, and 2.3 f 0.9 dpm/mg at 6, 12, and 24 h, respectively. Radioactivity in urine had a consistent mean value of 10.5 f 0.9 X 106 dpm/ml at all times examined and for treatment on day 11 or 12 of gestation. Counts in tissues (maternal liver, kidney, and muscle and placenta and embryo) taken 3 h after treatment on day 15 of gestation were statistically the same, among these samples, with a mean value of 81.4 & 7.9 dpm/mg (table 1). Maternal blood
*
had 82.0 & 8.3 X 103 dpm/ml and urine 14.1 +- 2.3 X lOSdpm/ml. For rats isolated in metabolic cages and treated on day 15 the radioactivity in maternal blood was 30.2 +- 4.9 X 103 dpm/ ml at 0.5 h and continued in this range until 2 h (28.8 & 0.4 X 10" dpm/ml) after treatment. At 4 h (24.6 f 2.1 X 103 dpmlml) the counts began to decrease and continued to do so until they were 1.2 & 0.2 X 10" dpm/ml at 48 h. The maternal blood half-life of radiolabel was calculated to be 10 h. Of the total radioactivity administered on day 15 of gestation the maternal urine contained 54.8% at 12 h, 72.8% at 24 h, and 84% at 48 h posttreatment (fig. 1).
38
J. A. RUDDICK, D. T. WILLIAMS, L. HIERLIHY AND K. S. KHERA
With or without the addition of TCA to maternal blood the label was distributed uniformly between RBC and plasma. The association between RBC and label was very frail since washing of centrifuged RBC removed the label. All of the label present in the plasma was TCA soluble. The radioisotope in the embryos was also TCA soluble. No label was detected in DNA, RNA, or protein fractions. Metabolites in day-12 embryos could not be characterized because too little label was present. However Sephadex separation of the TCA supernatant from day-15 embryos indicated a peak that corresponded to [ 14C] ETU. Profile characterization of labeled metabolites in maternal urine following all 3 days of treatment indicated the same pattern. There were 3 peaks; 2 overlapped, with peak maximums at fraction 42 and 52, while the third, peak maximum at fraction 90, corresponded to "4C] ETU. The ratio of counts between fractions 42, 52, and 90 was 4:1:600. Radiochromatography of TLC plates indicated predominantly ETU in eluted urine fraction 90 with a slight trace of ethyleneurea. Characterization of metabolites in the plasma revealed 2 peaks: an unknown at fraction 52, which was similar to one of the unknowns observed in urine, and a peak analogous to "4C] ETU. The ratio of counts between fractions 52 and 90 was 1:15. DISCUSSION
Reports on the biochemical behavior of thiourea and its derivative permitted a comparison with ETU's metabolism. Label from [ " T I ETU was absorbed r a p idly from the gastrointestinal tract. Radioactivity in maternal blood maintained peak activity from 0.5-2 h after treatment with uniform dispersion of the label; i.e., the label behaved as if it were a homogeneous solution. The eventual elimination of label from blood indicated that the label was not bound irreversibly to RBC. Phenylthiourea (Combs and Giri, '73) and thiourea (Williams and Kay, '45) have been observed to bind to RBC. None of the maternal organs examined, nor the embryos, selectively accumulated label. Three hours after treatment maternal liver, kidney, and muscle and placenta and embryo all had the same levels of
label and this similarity among tissues continued to 12 h after treatment. By 24 h all tissues except blood were relatively free of label. Shulman and Keating ('50) noted that rats injected ip with [3%] thiourea had label concentrations in tissue equally distributed at 6 h but differing somewhat at later times. The thyroid, which we did not examine, had the greatest affinity of all tissues for thiourea (Maloof and Soodak, '57; Shulman and Keating, '50) and ETU (Newsome, '74). There were no detectable binding of radiolabel in day-12 or -15 embryos. The radioactivity in embryos was apparently dispersed uniformly since no binding to either DNA, RNA, or protein was noted. These results were similar to those observed for maternal blood. It is possible that the minute amounts of label in the whole embryo made detection in a specific fraction difficult. The rate of radiolabel elimination from embryo was similar to maternal liver and kidney. Pathological examination of embryos (unpublished data) indicated that the teratological inductions were accomplished within 18 h. In the present study, 24 h after treatment all maternal organs and embryos were almost free of label with 72.8% of the total label given being excreted in the urine. Newsome ('74) observed that after 24 h 61.2% of the total oral dose administered to rats was present as ETU in urine and 1% in feces. The implication is that ETU, unlike thiourea, is degraded very lettle. Desulfuration of thiourea to give rise to inorganic sulfate, thiosulfate, and protein-bound sulfur (Maloof and Spectar, '59; Maloof and Soodak, '57) as well as 3 5 s which concentrated in the fetal thyroid and trachea (Shepard, '63) has been reported. Profile characterization of label metabolites in our experiments suggested a conjugated product. The labeled metabolites did not a p pear to be formed via the metabolic pool since no general labeling was observed upon fractionation of embryonic DNA, RNA, and protein. TLC and radiochromatography of radioactive compounds in the urine indicated primarily ETU (99% ) with traces of ethyleneurea and 2 unknown metabolites. This is in contrast with cow which catabolize ETU to several compounds
[“C] ETHYLENETHIOUREA IN PREGNANT RATS
that are eliminated in the milk and urine as well as release carbon atoms of ETU into the general metabolic pool (Lyman, ’71). The teratological action of ETU appears to be initiated by ETU, per se, and not a degradation product. Other experiments in our laboratory (unpublished) have indicated that the imidazolidine ring with adjoining sulfur atom are essential for the teratogenicity of ETU. With no detectable binding of radiolabel to the embryonic DNA, RNA, or protein the suggestion is that ETU initiates the teratological event but other steps follow that produce the anomalies. Whether this process begins in the pregnant female or the embryo cannot be determine at this time. LITERATURE CITED Allen, C. F. H., Co. Edens and J. Von Allen 1946 Ethylenethiourea. Org. Synth., 26: 34-35. Combs, A. B., and S. N. Giri 1973 Trichloroacetic acid-induced binding of phenylthiourea to erythrocytes. J. Pharm. Sci., 62; 631-633. Graham, S. H., and W. H. Hansen 1972 Effects of short-term administration of ethylenethiourea upon thyroid function of the rat. Bull. Env. Contam. Toxic., 7: 19-25. Khera, K. S. 1973 Ethylenethiourea: teratogenicity study in rats and rabbits. Teratology, 7: 24 3-254. Lyman, W. R. 1971 Metabolic fate of Dithane
39
M-45. Coordination product of zinc ion and manganous ethylenebisdithiocarbamate. In: Int. Sym. Pesticide Terminal Residues. A. S. Tahori, ed. Butterworth, New York, pp. 243-256. Maloof, F., and M. Soodak 1957 The uptake and metabolism of S35 thiourea and thiouracil by the thyroid and other tissues. Endocrinology, 61 : 55 5-56 9. Maloof, F., and L. Spectar 1959 The desulfuration of thiourea by thyroid cytoplasmic particulate fractions. J. Biol. Chem., 234: 949-954. Newsome, W. H. 1974 The excretion of ethylenethiourea by rat and guinea pig. Bull. Env. Contam. Toxic., 11 ; 174-176. Ruddick, J. A,, and K. S. Khera 1975 Pattern of anomalies following single oral doses of ethylenethiourea to pregnant rats. Teratology, 12: 2 7 7-282. Ruddick, J. A., and M. N. Runner 1974 5-FU in chick embryos as a source of label for DNA and a depressant of protein synthesis. Teratology, 10: 39-46. Shepard, T. H. 1963 Metabolism of thioureau S35 by the fetal thyroid of the rat. Endocrinology, 72; 223-230. Schulman, Jr., J., and R. P. Keating 1950 Studies on the metabolism of thiourea. J. Biol. Chem., 183: 215-221. Ulland, B. M., J. H. Weisburger, E. K. Weisburger, J. M. Rice and R. Cypher 1972 Thyroid cancer in rats from ethylenethiourea intake. J. Nat. Cancer Inst., 49: 583-584. Williams, R. H., and G. A. Kay 1945 Absorg tion, distribution and excretion of thiourea. Am. J. Physiol., 143: 715-722. Vonk, J. W. 1975 Chemical Decomposition of Bisdithiocarbamate Fungicides and Their Metabolism by Plants and Microorganisms. Ph.D. Thesis. Elinkwijk BV, Utrecht, Holland.
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