- Email: [email protected]
The effect of 3-amino-1,2,4-triazole on hepatic and renal deiodination of l -thyroxine to 3,5,3′-triiodothyronine
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
60,
45-51
(1981)
The Effect of 3-Amino-1,2,4-triazole on Hepatic and Renal Deiodination of L-Thyroxine to 3,5,3’-Triiodothyroninel G. SCAMMELL
JONATHAN Department
of Physiology,
University
Received
October
of Florida,
J. FREGLY
AND MELVIN College
31, 1980; accepted
of Medicine,
March
Gainesville,
Florida
32610
21, 1981
The Effect of 3-Amino-1,2,4-triazole on Hepatic and Renal Deiodination of L-Thyroxine to 3,5,3’-Triiodothyronine. SCAMMELL, J. G., AND FREGLY, M. J. (1981). Toxicol. Appl. Pharmacol. 60, 45-51. An in vitro model has been employed to evaluate the effects of administration of aminotriazole (ATZ) both in vivo and in vitro on the rate of outer-ring deiodination of thyroxine (T4) to triiodothyronine (T3) by 2000g supematants of fresh hepatic and renal homogenates. Administration of ATZ (29 * 1 mg/kg body wtiday) in the diet for 2 weeks to male rats reduced hepatic and renal T3 generation to 7 and 15% of control, respectively. This was associated with a significant depression of serum T, and T, concentrations. Administration ofT, (5Opg/kg body wt/day) in combination with ATZ increased hepatic and renal T, generation to 135 and 182% of control levels, respectively. T, concentration in the serum of this group was significantly higher, while T, concentration was significantly lower, than in control rats. The addition of ATZ (lo-* or lo-* M) to the incubation medium in vitro did not affect significantly the T3 generation by control hepatic and renal homogenates. The results of this study suggest that the action of ATZ on peripheral deiodination of T, may be indirect and secondary to inhibition of the synthesis of thyroid hormones and resulting hypothyroidism.
Aminotriazole (3-amino-1,2,Ctriazole, ATZ) is a herbicide which has found widespread use in agriculture (Kroller, 1966; Sund, 1956). It is known to exert a number of physiological effects in mammals. ATZ is reported to protect animals against experimentally induced tumors, although it is also classified as a carcinogen (Feinstein et al., 1978). In addition, ATZ inhibits the enzymes Saminolevulinic acid dehydrase (Tschudy and Collins, 1957) and catalase (Margoliash and Novogrodsky ,1958), prostaglandin synthesis (Boeynaems et al., 1979), and the induction of hepatic cytochrome P-450 (Baron and Tephly, 1969). Of its many physiological effects, its antithyroid activity has received the most at-
tention. ATZ is goitrogenic, most probably as a result of inhibition of thyroid peroxidase (Taurog, 1970). However, investigations of the effect of ATZ on peripheral metabolism of thyroid hormones have yielded conflicting results. Administration of ATZ to rats in vivo significantly reduced deiodination of thyroxine (TJ by peripheral tissues (Lutherer, 1969). On the other hand, deiodination of T, by homogenates of liver in vitro was enhanced either by addition of ATZ in vitro or by administration of ATZ in rive prior to sacrifice (Galton and Ingbar, 1964). It is now possible to measure directly some of these deiodination reactions. As extrathyroidal 5’-monodeiodination of T, accounts for most of the daily production of triiodothyronine (T3) in the rat (Abrams and Larsen, 1973) and T, is considered to
’ Supported by Grant HL 14526-09from the National Heart, Lung, and Blood Institute. 45
0041-008X/81/100045-07$02.00/0 Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved
46
SCAMMELL
be the more potent of the thyroid hormones (Larsen and Frumess, 1977), the peripheral conversion of T4 to T3 has received considerable attention. An in vitro system, developed to study the enzymatic nature of this process, has demonstrated that a large proportion of peripheral 5’-monodeiodination occurs in liver and kidney (Chopra, 1977). Therefore, the purpose of this study was twofold. First, we wished to reassess the effect of administration of AT2 in vivo and in vitro, at doses similar to those used by the previous investigators (Lutherer, 1969: Galton and Ingbar, 1964), on the rate of monodeiodination of T, to T, by 2000g supernatants of hepatic and renal homogenates. Second, the ability of T, to reverse the changes resulting from administration of ATZ in I’~L’O was studied. METHODS Eighteen male Sprague-Dawley rats from a breeding colony maintained in our animal facility were used. The animals, weighing initially 210-250 g, were housed three per cage in a room maintained at 25 + 1°C and illuminated from 0600 to 1800 hr. All rats were allowed tap water and finely ground Purina Laboratory Chow ad libitum. The rats were divided randomly into three groups, each containing six rats. The first group served as a control, the second group received ATZ2 (0.5 g/kg food) mixed thoroughly into their food: while the third group received ATZ as above plus daily subcutaneous administration of 50 pg I-thyroxineVkg body wt/day. The thyroxine was prepared by dilution in several drops of 0.1 N NaOH and adjusted to a concentration of 50 pg I-thyroxine/ml by addition of physiological saline. The first two groups received a subcutaneous injection of identically prepared vehicle (1 ml/kg body wt/day). On the last 5 days of treatment body weight and food intake were measured to determine the amount of ATZ ingested daily. At the end of 2 weeks, 24 hr after the last injection, the rats were sacrificed by decapitation and the trunk blood collected. The serum was separated and stored at -20°C. The concentrations of T3 and T4 were later determined by a commercially available radioim* ICN Pharmaceuticals, Inc., Cleveland, Ohio. 3 Sigma Chemical Co., St. Louis, MO.
AND FREGLY munoassay (RIA) kit.’ Livers and kidneys were dissected, washed in cold (4°C) 0.15 M phosphate buffer (pH 7.4), blotted, weighed, and homogenized in 3 vol of buffer for 30 set using a Tekmar SDT homogenizer. The homogenates were centrifuged at 20008 for 20 min at 4°C and the supernatant was used immediately. The term homogenate will henceforth refer to this supernatant preparation. The rate of T, to T, conversion in tissue homogenates was measured by a modification of a previously described method (Scammell ef al., 1980). Briefly, 200-~1 aliquots of homogenate were incubated at 37°C in duplicate with nonradioactive T, (6.4 pM) and dithiothreitoP (2.8 mM) in a l-ml incubation volume for 30 min. The reaction was stopped by addition of 2 ml of 95% ethanol to the incubation mixtures; the tubes were vortexed and immediately placed on ice. Samples were centrifuged and the supernatant was stored at -20°C until assayed for T, by RIA. T3 present in 0-min incubation tubes (containing T,, but not incubated) was subtracted from that measured in the test samples to derive the amount of T3 produced due to incubation of the homogenate and to account for T3 already present in the homogenates and commercial T,. Protein concentration was determined by the Biuret method (Bailey, 1967), using bovine albumin as a standard, and the results of the incubation were expressed as the mean amount of T, generated per milligram of protein per 30 min. To assess the effect of the addition of ATZ to the incubation mixture containing liver and kidney homogenates, 100 ~1 of buffer was replaced by an equal volume of buffer containing ATZ (lO-3 or 10-l M) and T, generation was determined as described above. Statistical analysis of the data was made by means of a one-way analysis of variance (Steel and Torrie, l%O). Comparison between individual groups was made by a t test using the pooled variance from the analysis of variance (Huntsberger, 1961). Significance was set at the 95% confidence interval.
RESULTS Control rats ingested 74 t 1 g of food/kg body wtlday, while the ATZ- and ATZ + T,-treated rats ingested 58 5 2 (SE) and 82 ‘-c 2 g/kg body wtiday, respectively. Therefore the amounts of ATZ ingested by the ATZ- and ATZ + T,-treated groups were 29 + 1 and 41 & 1 mg/kg body wtl day, respectively. Body weights of rats administered ATZ for 2 weeks were sig4 Diagnostic Products Co., Los Angeles, Calif.
AMINOTRIAZOLE
AND
THYROXINE TABLE
THE
EFFECTS
OF TREATMENT
WITH
EITHER
(ATZ + T,) ON BODY WEIGHT, THE WEIGHTS HEPATIC AND RENAL HOMOGENATES’
Body weight (!d Control ATZ-treated ATZ + T,-treated
311 + 6b 2&J + Y 291 i 4
1 (ATZ) AND KIDNEY,
AMINOTRIAZOLE
OF LIVER
4.7
METABOLISM
Liver weight (g/100 g body wt)
Kidney weight (g/100 g body wt)
3.% 2 0.04 3.67 c 0.09” 4.14 + 0.06
0.79 -t 0.02 0.67 + O.Old 0.86 2 0.01
PLUS THYROXINE CONCENTRATIONS OF
OR AMINOTRIAZOLE
AND ON PROTEIN
Liver homogenate, protein concentration (mg/ml)
Renal homogenate. protein concentration (mgiml) .26 i; I 27 fr I 2s t I -
71 t 3 63 f; ?’ 54 r I”
” There are six rats in each group. h Standard error of the mean. c Significantly different from control (p < 0.05). d Significantly different from control (p < 0.01).
nificantly less than control, while administration of T, in combination with ATZ resulted in body weights not different from control (Table 1). Hepatic and renal weights were significantly reduced by treatment with ATZ, while treatment with ATZ + T4 resulted in an increase in renal weight. The protein concentrations of hepatic homogenates of the ATZ- and ATZ + T,treated groups were significantly less than control, while those of the renal homogenates did not differ among the groups (Table 1). The rate of 5’-monodeiodination of T, by the 2000g supernatant of hepatic homogenates of the ATZ-treated group was 7% of control, while that of the ATZ + T,-treated group was 135% of control (Fig. 1A). In addition, treatment with ATZ resulted in a renal T:, generation which was 15% of control, while renal T:, generation of the ATZ + T,-treated rats was 182% of control (Fig. 1B). ATZ, added to the incubation mixture to achieve concentrations of lo+ or 10m4 M, did not affect significantly the T, generation of either control hepatic or renal homogenates (Table 2). Concentrations of both T, and T, in the serum of the ATZ-treated group were reduced significantly below control, while serum T4 concentration of the ATZ + T,treated group was significantly elevated above control (Table 3). However, serum
T, concentration of this group nificantly less than control.
was sag-
DISCUSSION The results of these studies suggest that dietary administration of ATZ for 2 weeks results in a considerable reduction of both renal and hepatic T, to T3 converting activities. Recent studies have indicated that outer-ring monodeiodination of T4 is enzymatic (Chopra, 1977), that it is stimulated by thiol-protective agents (Chopra, 1978). and that enzymatic activity is localized in plasma membranes and microsomes (Maciel rt al ., 1979). That the rates of renal and hepatic T, deiodination are similarly affected by administration of ATZ is not surTABLE
ro
2
THE EFFECT OF AMINOTRIAZOLE THE INCUBATION MEDIUM ON
RENAL
T, 5’-MONODEIODINASE
(ATZ) ADLJEI) HEPA~IC .\ND
ACTIVI-~Y -
T, S’-mono-
deiodinase activity” Hepatic Renal
ATZ (lo-’ M)
Control 100 + 13b (6) 100 t 15 (6)’
’ Results are expressed * Mean k SE. c Number of rats.
AT;< 110
90t 9 105 1?- 13
as percentage
2 MI --
908 106 2 12 of contra!.
SCAMMELL
CONTROL
ATZTREATED
AT2 + T,TREATED
CONTROL
ATZTREATED
AT2 + TTREATEb
FIG. 1. Effect of treatment with aminotriazole (ATZ) or aminotriazole plus thyroxine (AT2 + T,) on hepatic (A) and renal (B) TJ to Ts converting activity by 2OOOg supernatants of tissue homogenates. One and two asterisks indicate those values which were significantly different (p < 0.05 and p < 0.01, respectively) from control. One standard error is set off at each mean.
prising as the 5’-monodeiodinases of the kidney and liver of the rat are considered to be the same enzyme (Kaplan et al., 1979). In a previous study, administration of ATZ in vivo reduced total peripheral thyroid
AND
FREGLY
hormone deiodination, as measured by 1311 excretion after injection of 1311-labeled T4 (Lutherer, 1969). The present study indicates that the reduction in the activity of the outer-ring monodeiodinase, which deiodinates Tq, 3,3’,5’-triiodothyronine (rT3), and probably 3’,5’- and 3,3’-diiodothyronine as well (Chopra, 1981), is at least partially responsible for these previous findings. Whether the deiodination of other thyroid hormones is altered by administration of ATZ is not known. However, our findings are not in agreement with those of Galton and Ingbar (1964). These investigators showed that administration of ATZ in viva and in vitro increased in vitro T4 deiodination by hepatic but not renal homogenates of the mouse. In the present study the addition of ATZ to the incubation mixture had no effect on generation of T3 by either hepatic or renal homogenates. It is most likely that different assay conditions and different species can account for the disparities between the two studies. Incubations in the first study were carried out in 100% 02, while recent studies have shown that anaerobic conditions and reducing agents enhance enzyme activity (Chiraseveenuprapund et al., 1978). The conditions of incubation in the present study undoubtedly allowed greater enzyme activity to occur. The lack of an effect when TABLE
3
THEEFFECTSOFTREATMENTWITHAMINOTRIA~OLE OR AMINOTRIAZOLE PLUS THYROXINE (ATZ + T4) ON SERUM CONCENTRATIONS OF THYROXINE (T,) AND TRIIODOTHYRONINE(T~)
(ATZ)
Control ATZ-treated AT2 + T,-treated
No. of rats
Serum Ta concentration ba’dl)
Serum T3 concentration Wdl)
6 6 6
4.7 f 0.2” 0.9 + o.ob 6.1 -c 0.3b
170 * 3 81 -c 2b 1.54* 5”
a Standard error of the mean. b Significantly different from control (p < 0.01).
AMINOTRIAZOLE
AND
THYROXINE
ATZ was added in vitro suggests that the mechanism of action of this compound differs from that of propylthiouracil (PTU), which has been shown to inhibit peripheral 5’-monodeiodination at micromolar concentrations (Kaplan and Utiger, 1978a). It has been suggested that PTU inactivates the enzyme by formation of a disulfide bond (Leonard and Rosenberg, 1980). If this is the case, it is not surprising that ATZ does not affect 5’-monodeiodination by this mechanism since its molecular structure contains no sulfur. However, both PTU in an earlier study (Hershman and Van Middlesworth, 1962) and ATZ in the present study reduced peripheral deiodination of T, when they were administered to rats in ~~ivo. It is likely that PTU acts, at least partially, by direct inhibition of enzyme activity as discussed, but the mechanism of action of ATZ under these conditions is less certain. ATZ is thought to depress thyroid hormone synthesis by inhibition of the thyroid peroxidase enzyme (Taurog, 1970), and it reduces uptake of radioactive iodide by the thyroid gland at a dose similar to that used in this study (Alexander, 1959). Furthermore, a state of hypothyroidism reduces 5’-monodeiodinase activity in hepatic and renal homogenates (Kaplan and Utiger, 1978b). The major factor responsible for defective peripheral T, formation in hypothyroidism is a decrease in the activity of the 5’-deiodinase, although a reduction in the concentration of cofactors, glutathione and NADPH, may also contribute (Balsam et al., 1979). A marked hypothyroidism resulted from administration of ATZ in t*ivo in this study, as indicated by reduced body, hepatic, and renal weights, a reduced food intake, and reduced serum T, and T, concentrations. Therefore, it is possible that the effect of ATZ on hepatic and renal 5’-deiodination is indirect and secondary to inhibition of thyroid hormone synthesis. This suggestion is strengthened by the
METABOLISM
49
effect of administration of T, in addition to ATZ. Although this group of animals ingested over one-third more ATZ than the group receiving ATZ alone, most physiological parameters of thyroid status were returned to control or above control levels. While treatment with ATZ + T, resulted in hepatic and renal T, generation exceeding that of control, treatment of PTIJtreated rats with T, failed to return peripheral metabolism of T, to normal (Escobar de1 Rey and Morreale de Escobar, 1961). Although it is possible that ATZ exhibits a direct effect on hepatic and renal deiodination of T, by an unknown mechanism, the ability of T, to reverse the effects of administration of ATZ suggests that the enzymatic defect is due to hypothyroidism per se. However, the increased rate of ‘T:( generation by liver and kidneys under these conditions was not sufficient to return serum T, concentrations to normal, most likely due to continued suppression of T, production of thyroidal origin and loss of its contribution to circulating T3 concentration. The possibility that an increased rate of hepatic conjugation of thyroid hormones could account for this observation can be ruled out as this has been shown to be actually decreased under these conditions (Lutherer. 1969). Hence, other compounds which directly block only thyroid hormone synthesis would be expected to reduce peripheral metabolism of T, by a similar mechanism. In addition, the administration of ATZ resulted in a reduction in the protein concentration of 2OOOg supernatants of liver but not renal homogenates. It is uncertain whether ATZ is exhibiting either a direct effect on the liver or an indirect effect resulting from the induced hypothyroidism. However, recent evidence indicates that both intracellular and secretory protein production is impaired in livers of thyroidectomized rats (Peavy et al., 1981). In addition, part of this deficit persists even after replacement with T.,. The protein concen-
50
SCAMMELL
trations of the homogenates of the ATZ + T,-treated group also were significantly less than control, suggesting that at least part of this effect might be a result of hypothyroidism. Such an effect of hypothyrodism has not been demonstrated for the kidney. This is an area which deserves further study. Studies to determine the minimal dietary dose of ATZ which produces an effect on activity of the thyroid gland have been performed in this laboratory (Fregly, 1968). One-third of the measures of thyroid function were affected by a dose of 50 ppm ATZ in the food (one-tenth of that administered in this study). In future studies it would be worthwhile to establish a dose-response relationship between drug dose and concentrations of T3 and T, in the serum and T, deiodination. Indeed, some of the other effects of ATZ are dose related. Feinstein et al. (1978) have shown that dietary administration of ATZ produces hepatic cancer in mice. This effect has been observed characteristically when the intake of ATZ is at least 20-fold higher than that used in this study. However, nonmalignant adenomatous changes have been observed in the thyroid glands of rats, when the diet contained one-fifth the amount of ATZ used in this study (Jukes and Shaffer, 1960). This difference is especially apparent when one considers that the accumulation of *Clabeled ATZ is 4-fold greater in the liver than in the thyroid gland (Tjalve, 1975). It seems likely that the different sensitivities of the two tissues are related to different effects accompanying administration of the compound. It has been proposed that the carcinogenicity of ATZ is at least partially due to inhibition of the catalase enzyme and resultant accumulation of H,O, (Feinstein et al., 1978). On the other hand, the effect of ATZ on thyroid hyperplasia has been suggested to be a result of prolonged stimulation of the thyroid gland by thyrotropin, as a consequence of ATZ-dependent de-
AND FREGLY
pression of thyroid hormone synthesis (Jukes and Shaffer, 1960). ACKNOWLEDGMENTS We thank Mrs. Charlotte the figures.
Edelstein for drawing
REFERENCES ABRAMS, G. M., AND LARSEN, P. R. (1973). Triiodothyronine and thyroxine in the serum and thyroid glands of iodine-deficient rats. J. Clin. Invest. 52, 2522-253 1. ALEXANDER, N. M. (1959). Antithyroid action of 3amino-1,2,4-triazole. J. Biol. Chem. 234, 148-150. BAILEY, J. L. (1%7). Techniques in Protein Chemistry. Elsevier, Amsterdam. BALSAM, A., SEXTON, F., AND INGBAR, S. H. (1979). On the mechanism of impaired in vitro generation of 3,5,3’-triiodothyronine from thyroxine in the livers of hypothyroid rats. Endocrinology 105, 11151121. BARON, J., AND TEPHLY, T. R. (1969). Effect of 3amino-1,2,4-triazole on the stimulation of hepatic microsomal heme synthesis and induction of hepatic microsomal oxidases produced by phenobarbital. Mol. Pharmacol. 5, 10-20. BOEYNAEMS, J. M., GALAND, N., AND DUMONT, J. E. (1979). In vitro inhibition of prostaglandin synthesis by antithyroid drugs. Biochem. Pharmacol.
28,3195-3198.
CHIRASEVEENUPRAPUND,
P.,
BUERGI,
U.,
GOSWANI,
A., AND ROSENBERG, 1. N. (1978). Conversion of l-thyroxine to triiodothyronine in rat kidney homogenate. Endocrinology 102, 612-622. CHOPRA, 1. J. (1977). A study of extrathyroidal conversion of thyroxine (T,) to 3,3’,5-triiodothyronine (T3) in vitro. Endocrinology 101, 453-463. CHOPRA, I. J. (1978). Sulfhydryl groups and the monodeiodination of thyroxine to triiodothyronine. Science
199, 904-906.
CHOPRA, I. J. (1981). Characteristics of outer ring (5’- or 3’-) monodeiodination of 3’,5’- or 3,3’diiodothyronine: Evidence suggesting one outer ring monodeiodinase for various iodothyronines. Endocrinology 108, 464-471. ESCOBAR DEL REY, F., AND MORREALE DE ESCOBAR, G. (l%l). The effect of propylthiouracil, methylthiouracil and thiouracil on the peripheral metabolism of l-thyroxine in thyroidectomized, l-thyroxine maintained rats. Endocrinolog)l 69, 456-465. FEINSTEIN,
R.
N.,
FRY,
R.
J. M.,
AND
STAFFELDT,
E. F. (1978). Carcinogenic and antitumor effects of
AMINOTRIAZOLE
AND THYROXINE
METABOLISM
51
aminotriazole on acatalasemic and normal catalase mice. J. Nat. Cancer Inst. 60, 1113-1116. FREGLY, M. J. (1968). Effect of aminotriazole on thyroid function in the rat. Toxicol. Appl. Phar-
LEONARD, J. L., AND ROSENBERG. I. N. (1980). Characterization of essential enzyme sulfhydryl groups of thyroxine 5’-deiodinase from rat kidney.
macol. GALTON,
LUTHERER, L. 0.
13, 271-286. V. A.. AND
of catalase inhibitors roxine. Endocrinology HERSHMAN,
J. M.,
H. (1964). Effect on the deiodination of thy-
INGBAR,
S.
14, 627-634. AND VAN MIDDLESWORTH,
L.
297.
KAPLAN, M. M., TATRO, J. B., BREITBART, R., AND LARSEN, P. R. (1979). Comparison of thyroxine and 3,3’,5’-triiodothyronine metabolism in rat kidney and liver homogenates. Metabolism 28, 1139- 1146. KAPLAN, M. M., AND UTIGER, R. D. (1978a). Iodothyronine metabolism in rat liver homogenates. J. fnvesr.
61, 459-471.
KAPLAN. M. M., AND UTIGER, R. D. (1978b). Iodothyronine metabolism in liver and kidney homogenates from hyperthyroid and hypothyroid rats. Endocrinology 103, 156-161. KROI-LER, E. (1966). Anwendung und Eigenscbaften des 3-amino-1,2,4-triazols im Hinblick auf seine Ruckstande in Lebensmitteln. Residue Ret’. 12, 162192.
LARSEN, P. R., AND FRUMESS, R. D. (1977). Comparison of the biological effects of thyroxine and triiodothyronine in the rat. Endocrinology 100, 980-988.
vascular Aspects Interrelationship
106, 444-451.
(1%9).
Metabolic and C’urdioThyroxine-Cutecholamine Rats. Doctoral Dissertation,
qf the in
University of Florida.
(1%2). Effect of antithyroid compounds on the deiodination of thyroxine in the rat. Endocrinology 71, 94- 100. HUNTSBERGER. D. V, (1961). Elements of Statistical fr@rnce. Allyn & Bacon, Boston. JUKES, T. H., AND SHAFFER, C. B. (l%O). Antithyroid effects of aminotriazole. Science 132, 296-
C/in.
Endocrinology
R. M. B., OZAWA, Y., AND CHOPRA, I. I. (1979). Subcellular localization of thyroxine and reverse triiodothyronine outer ring monodeiodinating activities Endocrinology 104, 365-371. MARGOLIASH, E., AND NOVOGRODSKY, A. (1958). .4 study of the inhibition of catalase by 3-amino1:2:4-triazole. Biochem. J. 68, 468-475. PEAVY, D. E., TAYLOR, J. M., AND JEFFERSON, L,. S. (1981). Protein synthesis in perfused rat liver following thyroidectomy and hormone treatment. Amer. J. Physio/. 240 (Endocrinol. Metab. 3), Et&E23. SCAMMELL, J. G., SHIVERICK, K. T., AND FREG~Y. M. J. (1980). In vitro hepatic deiodination of Ithyroxine to 3,5,3’-triiodothyronine in cold-acclimated rats. J. Appi. Physiol.: Respirat. Envrrtm.
MACIEL,
Exercise
Physiol.
49, 386-389.
STEEL, R. B. D., AND TORRIE, J. H. (l%O). Principles qf Procedures c!f Statistics. McGraw-Hill. New York. SUND, K. A. (1956). Residual activity of 3-amino1,2,4-triazole in soils. J. Agr. Food Chem. 4.57-60. TAUROG, A. (1970). Thyroid peroxidase and thyroxine biosynthesis. Rec. Progr. Hormone Re.v. 26, 189247.
TJALVE. H. (1975). The distribution of labelled aminotriazole in mice. Toxicology 3, 49-67. TSCHUDY, D. P., AND COLLINS, A. (1957). Effect of 3-amino-1,2,4-triazole on &aminolevulinic acid dehydrase activity. Science 126, 168.
AND
APPLIED
PHARMACOLOGY
60,
45-51
(1981)
The Effect of 3-Amino-1,2,4-triazole on Hepatic and Renal Deiodination of L-Thyroxine to 3,5,3’-Triiodothyroninel G. SCAMMELL
JONATHAN Department
of Physiology,
University
Received
October
of Florida,
J. FREGLY
AND MELVIN College
31, 1980; accepted
of Medicine,
March
Gainesville,
Florida
32610
21, 1981
The Effect of 3-Amino-1,2,4-triazole on Hepatic and Renal Deiodination of L-Thyroxine to 3,5,3’-Triiodothyronine. SCAMMELL, J. G., AND FREGLY, M. J. (1981). Toxicol. Appl. Pharmacol. 60, 45-51. An in vitro model has been employed to evaluate the effects of administration of aminotriazole (ATZ) both in vivo and in vitro on the rate of outer-ring deiodination of thyroxine (T4) to triiodothyronine (T3) by 2000g supematants of fresh hepatic and renal homogenates. Administration of ATZ (29 * 1 mg/kg body wtiday) in the diet for 2 weeks to male rats reduced hepatic and renal T3 generation to 7 and 15% of control, respectively. This was associated with a significant depression of serum T, and T, concentrations. Administration ofT, (5Opg/kg body wt/day) in combination with ATZ increased hepatic and renal T, generation to 135 and 182% of control levels, respectively. T, concentration in the serum of this group was significantly higher, while T, concentration was significantly lower, than in control rats. The addition of ATZ (lo-* or lo-* M) to the incubation medium in vitro did not affect significantly the T3 generation by control hepatic and renal homogenates. The results of this study suggest that the action of ATZ on peripheral deiodination of T, may be indirect and secondary to inhibition of the synthesis of thyroid hormones and resulting hypothyroidism.
Aminotriazole (3-amino-1,2,Ctriazole, ATZ) is a herbicide which has found widespread use in agriculture (Kroller, 1966; Sund, 1956). It is known to exert a number of physiological effects in mammals. ATZ is reported to protect animals against experimentally induced tumors, although it is also classified as a carcinogen (Feinstein et al., 1978). In addition, ATZ inhibits the enzymes Saminolevulinic acid dehydrase (Tschudy and Collins, 1957) and catalase (Margoliash and Novogrodsky ,1958), prostaglandin synthesis (Boeynaems et al., 1979), and the induction of hepatic cytochrome P-450 (Baron and Tephly, 1969). Of its many physiological effects, its antithyroid activity has received the most at-
tention. ATZ is goitrogenic, most probably as a result of inhibition of thyroid peroxidase (Taurog, 1970). However, investigations of the effect of ATZ on peripheral metabolism of thyroid hormones have yielded conflicting results. Administration of ATZ to rats in vivo significantly reduced deiodination of thyroxine (TJ by peripheral tissues (Lutherer, 1969). On the other hand, deiodination of T, by homogenates of liver in vitro was enhanced either by addition of ATZ in vitro or by administration of ATZ in rive prior to sacrifice (Galton and Ingbar, 1964). It is now possible to measure directly some of these deiodination reactions. As extrathyroidal 5’-monodeiodination of T, accounts for most of the daily production of triiodothyronine (T3) in the rat (Abrams and Larsen, 1973) and T, is considered to
’ Supported by Grant HL 14526-09from the National Heart, Lung, and Blood Institute. 45
0041-008X/81/100045-07$02.00/0 Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved
46
SCAMMELL
be the more potent of the thyroid hormones (Larsen and Frumess, 1977), the peripheral conversion of T4 to T3 has received considerable attention. An in vitro system, developed to study the enzymatic nature of this process, has demonstrated that a large proportion of peripheral 5’-monodeiodination occurs in liver and kidney (Chopra, 1977). Therefore, the purpose of this study was twofold. First, we wished to reassess the effect of administration of AT2 in vivo and in vitro, at doses similar to those used by the previous investigators (Lutherer, 1969: Galton and Ingbar, 1964), on the rate of monodeiodination of T, to T, by 2000g supernatants of hepatic and renal homogenates. Second, the ability of T, to reverse the changes resulting from administration of ATZ in I’~L’O was studied. METHODS Eighteen male Sprague-Dawley rats from a breeding colony maintained in our animal facility were used. The animals, weighing initially 210-250 g, were housed three per cage in a room maintained at 25 + 1°C and illuminated from 0600 to 1800 hr. All rats were allowed tap water and finely ground Purina Laboratory Chow ad libitum. The rats were divided randomly into three groups, each containing six rats. The first group served as a control, the second group received ATZ2 (0.5 g/kg food) mixed thoroughly into their food: while the third group received ATZ as above plus daily subcutaneous administration of 50 pg I-thyroxineVkg body wt/day. The thyroxine was prepared by dilution in several drops of 0.1 N NaOH and adjusted to a concentration of 50 pg I-thyroxine/ml by addition of physiological saline. The first two groups received a subcutaneous injection of identically prepared vehicle (1 ml/kg body wt/day). On the last 5 days of treatment body weight and food intake were measured to determine the amount of ATZ ingested daily. At the end of 2 weeks, 24 hr after the last injection, the rats were sacrificed by decapitation and the trunk blood collected. The serum was separated and stored at -20°C. The concentrations of T3 and T4 were later determined by a commercially available radioim* ICN Pharmaceuticals, Inc., Cleveland, Ohio. 3 Sigma Chemical Co., St. Louis, MO.
AND FREGLY munoassay (RIA) kit.’ Livers and kidneys were dissected, washed in cold (4°C) 0.15 M phosphate buffer (pH 7.4), blotted, weighed, and homogenized in 3 vol of buffer for 30 set using a Tekmar SDT homogenizer. The homogenates were centrifuged at 20008 for 20 min at 4°C and the supernatant was used immediately. The term homogenate will henceforth refer to this supernatant preparation. The rate of T, to T, conversion in tissue homogenates was measured by a modification of a previously described method (Scammell ef al., 1980). Briefly, 200-~1 aliquots of homogenate were incubated at 37°C in duplicate with nonradioactive T, (6.4 pM) and dithiothreitoP (2.8 mM) in a l-ml incubation volume for 30 min. The reaction was stopped by addition of 2 ml of 95% ethanol to the incubation mixtures; the tubes were vortexed and immediately placed on ice. Samples were centrifuged and the supernatant was stored at -20°C until assayed for T, by RIA. T3 present in 0-min incubation tubes (containing T,, but not incubated) was subtracted from that measured in the test samples to derive the amount of T3 produced due to incubation of the homogenate and to account for T3 already present in the homogenates and commercial T,. Protein concentration was determined by the Biuret method (Bailey, 1967), using bovine albumin as a standard, and the results of the incubation were expressed as the mean amount of T, generated per milligram of protein per 30 min. To assess the effect of the addition of ATZ to the incubation mixture containing liver and kidney homogenates, 100 ~1 of buffer was replaced by an equal volume of buffer containing ATZ (lO-3 or 10-l M) and T, generation was determined as described above. Statistical analysis of the data was made by means of a one-way analysis of variance (Steel and Torrie, l%O). Comparison between individual groups was made by a t test using the pooled variance from the analysis of variance (Huntsberger, 1961). Significance was set at the 95% confidence interval.
RESULTS Control rats ingested 74 t 1 g of food/kg body wtlday, while the ATZ- and ATZ + T,-treated rats ingested 58 5 2 (SE) and 82 ‘-c 2 g/kg body wtiday, respectively. Therefore the amounts of ATZ ingested by the ATZ- and ATZ + T,-treated groups were 29 + 1 and 41 & 1 mg/kg body wtl day, respectively. Body weights of rats administered ATZ for 2 weeks were sig4 Diagnostic Products Co., Los Angeles, Calif.
AMINOTRIAZOLE
AND
THYROXINE TABLE
THE
EFFECTS
OF TREATMENT
WITH
EITHER
(ATZ + T,) ON BODY WEIGHT, THE WEIGHTS HEPATIC AND RENAL HOMOGENATES’
Body weight (!d Control ATZ-treated ATZ + T,-treated
311 + 6b 2&J + Y 291 i 4
1 (ATZ) AND KIDNEY,
AMINOTRIAZOLE
OF LIVER
4.7
METABOLISM
Liver weight (g/100 g body wt)
Kidney weight (g/100 g body wt)
3.% 2 0.04 3.67 c 0.09” 4.14 + 0.06
0.79 -t 0.02 0.67 + O.Old 0.86 2 0.01
PLUS THYROXINE CONCENTRATIONS OF
OR AMINOTRIAZOLE
AND ON PROTEIN
Liver homogenate, protein concentration (mg/ml)
Renal homogenate. protein concentration (mgiml) .26 i; I 27 fr I 2s t I -
71 t 3 63 f; ?’ 54 r I”
” There are six rats in each group. h Standard error of the mean. c Significantly different from control (p < 0.05). d Significantly different from control (p < 0.01).
nificantly less than control, while administration of T, in combination with ATZ resulted in body weights not different from control (Table 1). Hepatic and renal weights were significantly reduced by treatment with ATZ, while treatment with ATZ + T4 resulted in an increase in renal weight. The protein concentrations of hepatic homogenates of the ATZ- and ATZ + T,treated groups were significantly less than control, while those of the renal homogenates did not differ among the groups (Table 1). The rate of 5’-monodeiodination of T, by the 2000g supernatant of hepatic homogenates of the ATZ-treated group was 7% of control, while that of the ATZ + T,-treated group was 135% of control (Fig. 1A). In addition, treatment with ATZ resulted in a renal T:, generation which was 15% of control, while renal T:, generation of the ATZ + T,-treated rats was 182% of control (Fig. 1B). ATZ, added to the incubation mixture to achieve concentrations of lo+ or 10m4 M, did not affect significantly the T, generation of either control hepatic or renal homogenates (Table 2). Concentrations of both T, and T, in the serum of the ATZ-treated group were reduced significantly below control, while serum T4 concentration of the ATZ + T,treated group was significantly elevated above control (Table 3). However, serum
T, concentration of this group nificantly less than control.
was sag-
DISCUSSION The results of these studies suggest that dietary administration of ATZ for 2 weeks results in a considerable reduction of both renal and hepatic T, to T3 converting activities. Recent studies have indicated that outer-ring monodeiodination of T4 is enzymatic (Chopra, 1977), that it is stimulated by thiol-protective agents (Chopra, 1978). and that enzymatic activity is localized in plasma membranes and microsomes (Maciel rt al ., 1979). That the rates of renal and hepatic T, deiodination are similarly affected by administration of ATZ is not surTABLE
ro
2
THE EFFECT OF AMINOTRIAZOLE THE INCUBATION MEDIUM ON
RENAL
T, 5’-MONODEIODINASE
(ATZ) ADLJEI) HEPA~IC .\ND
ACTIVI-~Y -
T, S’-mono-
deiodinase activity” Hepatic Renal
ATZ (lo-’ M)
Control 100 + 13b (6) 100 t 15 (6)’
’ Results are expressed * Mean k SE. c Number of rats.
AT;< 110
90t 9 105 1?- 13
as percentage
2 MI --
908 106 2 12 of contra!.
SCAMMELL
CONTROL
ATZTREATED
AT2 + T,TREATED
CONTROL
ATZTREATED
AT2 + TTREATEb
FIG. 1. Effect of treatment with aminotriazole (ATZ) or aminotriazole plus thyroxine (AT2 + T,) on hepatic (A) and renal (B) TJ to Ts converting activity by 2OOOg supernatants of tissue homogenates. One and two asterisks indicate those values which were significantly different (p < 0.05 and p < 0.01, respectively) from control. One standard error is set off at each mean.
prising as the 5’-monodeiodinases of the kidney and liver of the rat are considered to be the same enzyme (Kaplan et al., 1979). In a previous study, administration of ATZ in vivo reduced total peripheral thyroid
AND
FREGLY
hormone deiodination, as measured by 1311 excretion after injection of 1311-labeled T4 (Lutherer, 1969). The present study indicates that the reduction in the activity of the outer-ring monodeiodinase, which deiodinates Tq, 3,3’,5’-triiodothyronine (rT3), and probably 3’,5’- and 3,3’-diiodothyronine as well (Chopra, 1981), is at least partially responsible for these previous findings. Whether the deiodination of other thyroid hormones is altered by administration of ATZ is not known. However, our findings are not in agreement with those of Galton and Ingbar (1964). These investigators showed that administration of ATZ in viva and in vitro increased in vitro T4 deiodination by hepatic but not renal homogenates of the mouse. In the present study the addition of ATZ to the incubation mixture had no effect on generation of T3 by either hepatic or renal homogenates. It is most likely that different assay conditions and different species can account for the disparities between the two studies. Incubations in the first study were carried out in 100% 02, while recent studies have shown that anaerobic conditions and reducing agents enhance enzyme activity (Chiraseveenuprapund et al., 1978). The conditions of incubation in the present study undoubtedly allowed greater enzyme activity to occur. The lack of an effect when TABLE
3
THEEFFECTSOFTREATMENTWITHAMINOTRIA~OLE OR AMINOTRIAZOLE PLUS THYROXINE (ATZ + T4) ON SERUM CONCENTRATIONS OF THYROXINE (T,) AND TRIIODOTHYRONINE(T~)
(ATZ)
Control ATZ-treated AT2 + T,-treated
No. of rats
Serum Ta concentration ba’dl)
Serum T3 concentration Wdl)
6 6 6
4.7 f 0.2” 0.9 + o.ob 6.1 -c 0.3b
170 * 3 81 -c 2b 1.54* 5”
a Standard error of the mean. b Significantly different from control (p < 0.01).
AMINOTRIAZOLE
AND
THYROXINE
ATZ was added in vitro suggests that the mechanism of action of this compound differs from that of propylthiouracil (PTU), which has been shown to inhibit peripheral 5’-monodeiodination at micromolar concentrations (Kaplan and Utiger, 1978a). It has been suggested that PTU inactivates the enzyme by formation of a disulfide bond (Leonard and Rosenberg, 1980). If this is the case, it is not surprising that ATZ does not affect 5’-monodeiodination by this mechanism since its molecular structure contains no sulfur. However, both PTU in an earlier study (Hershman and Van Middlesworth, 1962) and ATZ in the present study reduced peripheral deiodination of T, when they were administered to rats in ~~ivo. It is likely that PTU acts, at least partially, by direct inhibition of enzyme activity as discussed, but the mechanism of action of ATZ under these conditions is less certain. ATZ is thought to depress thyroid hormone synthesis by inhibition of the thyroid peroxidase enzyme (Taurog, 1970), and it reduces uptake of radioactive iodide by the thyroid gland at a dose similar to that used in this study (Alexander, 1959). Furthermore, a state of hypothyroidism reduces 5’-monodeiodinase activity in hepatic and renal homogenates (Kaplan and Utiger, 1978b). The major factor responsible for defective peripheral T, formation in hypothyroidism is a decrease in the activity of the 5’-deiodinase, although a reduction in the concentration of cofactors, glutathione and NADPH, may also contribute (Balsam et al., 1979). A marked hypothyroidism resulted from administration of ATZ in t*ivo in this study, as indicated by reduced body, hepatic, and renal weights, a reduced food intake, and reduced serum T, and T, concentrations. Therefore, it is possible that the effect of ATZ on hepatic and renal 5’-deiodination is indirect and secondary to inhibition of thyroid hormone synthesis. This suggestion is strengthened by the
METABOLISM
49
effect of administration of T, in addition to ATZ. Although this group of animals ingested over one-third more ATZ than the group receiving ATZ alone, most physiological parameters of thyroid status were returned to control or above control levels. While treatment with ATZ + T, resulted in hepatic and renal T, generation exceeding that of control, treatment of PTIJtreated rats with T, failed to return peripheral metabolism of T, to normal (Escobar de1 Rey and Morreale de Escobar, 1961). Although it is possible that ATZ exhibits a direct effect on hepatic and renal deiodination of T, by an unknown mechanism, the ability of T, to reverse the effects of administration of ATZ suggests that the enzymatic defect is due to hypothyroidism per se. However, the increased rate of ‘T:( generation by liver and kidneys under these conditions was not sufficient to return serum T, concentrations to normal, most likely due to continued suppression of T, production of thyroidal origin and loss of its contribution to circulating T3 concentration. The possibility that an increased rate of hepatic conjugation of thyroid hormones could account for this observation can be ruled out as this has been shown to be actually decreased under these conditions (Lutherer. 1969). Hence, other compounds which directly block only thyroid hormone synthesis would be expected to reduce peripheral metabolism of T, by a similar mechanism. In addition, the administration of ATZ resulted in a reduction in the protein concentration of 2OOOg supernatants of liver but not renal homogenates. It is uncertain whether ATZ is exhibiting either a direct effect on the liver or an indirect effect resulting from the induced hypothyroidism. However, recent evidence indicates that both intracellular and secretory protein production is impaired in livers of thyroidectomized rats (Peavy et al., 1981). In addition, part of this deficit persists even after replacement with T.,. The protein concen-
50
SCAMMELL
trations of the homogenates of the ATZ + T,-treated group also were significantly less than control, suggesting that at least part of this effect might be a result of hypothyroidism. Such an effect of hypothyrodism has not been demonstrated for the kidney. This is an area which deserves further study. Studies to determine the minimal dietary dose of ATZ which produces an effect on activity of the thyroid gland have been performed in this laboratory (Fregly, 1968). One-third of the measures of thyroid function were affected by a dose of 50 ppm ATZ in the food (one-tenth of that administered in this study). In future studies it would be worthwhile to establish a dose-response relationship between drug dose and concentrations of T3 and T, in the serum and T, deiodination. Indeed, some of the other effects of ATZ are dose related. Feinstein et al. (1978) have shown that dietary administration of ATZ produces hepatic cancer in mice. This effect has been observed characteristically when the intake of ATZ is at least 20-fold higher than that used in this study. However, nonmalignant adenomatous changes have been observed in the thyroid glands of rats, when the diet contained one-fifth the amount of ATZ used in this study (Jukes and Shaffer, 1960). This difference is especially apparent when one considers that the accumulation of *Clabeled ATZ is 4-fold greater in the liver than in the thyroid gland (Tjalve, 1975). It seems likely that the different sensitivities of the two tissues are related to different effects accompanying administration of the compound. It has been proposed that the carcinogenicity of ATZ is at least partially due to inhibition of the catalase enzyme and resultant accumulation of H,O, (Feinstein et al., 1978). On the other hand, the effect of ATZ on thyroid hyperplasia has been suggested to be a result of prolonged stimulation of the thyroid gland by thyrotropin, as a consequence of ATZ-dependent de-
AND FREGLY
pression of thyroid hormone synthesis (Jukes and Shaffer, 1960). ACKNOWLEDGMENTS We thank Mrs. Charlotte the figures.
Edelstein for drawing
REFERENCES ABRAMS, G. M., AND LARSEN, P. R. (1973). Triiodothyronine and thyroxine in the serum and thyroid glands of iodine-deficient rats. J. Clin. Invest. 52, 2522-253 1. ALEXANDER, N. M. (1959). Antithyroid action of 3amino-1,2,4-triazole. J. Biol. Chem. 234, 148-150. BAILEY, J. L. (1%7). Techniques in Protein Chemistry. Elsevier, Amsterdam. BALSAM, A., SEXTON, F., AND INGBAR, S. H. (1979). On the mechanism of impaired in vitro generation of 3,5,3’-triiodothyronine from thyroxine in the livers of hypothyroid rats. Endocrinology 105, 11151121. BARON, J., AND TEPHLY, T. R. (1969). Effect of 3amino-1,2,4-triazole on the stimulation of hepatic microsomal heme synthesis and induction of hepatic microsomal oxidases produced by phenobarbital. Mol. Pharmacol. 5, 10-20. BOEYNAEMS, J. M., GALAND, N., AND DUMONT, J. E. (1979). In vitro inhibition of prostaglandin synthesis by antithyroid drugs. Biochem. Pharmacol.
28,3195-3198.
CHIRASEVEENUPRAPUND,
P.,
BUERGI,
U.,
GOSWANI,
A., AND ROSENBERG, 1. N. (1978). Conversion of l-thyroxine to triiodothyronine in rat kidney homogenate. Endocrinology 102, 612-622. CHOPRA, 1. J. (1977). A study of extrathyroidal conversion of thyroxine (T,) to 3,3’,5-triiodothyronine (T3) in vitro. Endocrinology 101, 453-463. CHOPRA, I. J. (1978). Sulfhydryl groups and the monodeiodination of thyroxine to triiodothyronine. Science
199, 904-906.
CHOPRA, I. J. (1981). Characteristics of outer ring (5’- or 3’-) monodeiodination of 3’,5’- or 3,3’diiodothyronine: Evidence suggesting one outer ring monodeiodinase for various iodothyronines. Endocrinology 108, 464-471. ESCOBAR DEL REY, F., AND MORREALE DE ESCOBAR, G. (l%l). The effect of propylthiouracil, methylthiouracil and thiouracil on the peripheral metabolism of l-thyroxine in thyroidectomized, l-thyroxine maintained rats. Endocrinolog)l 69, 456-465. FEINSTEIN,
R.
N.,
FRY,
R.
J. M.,
AND
STAFFELDT,
E. F. (1978). Carcinogenic and antitumor effects of
AMINOTRIAZOLE
AND THYROXINE
METABOLISM
51
aminotriazole on acatalasemic and normal catalase mice. J. Nat. Cancer Inst. 60, 1113-1116. FREGLY, M. J. (1968). Effect of aminotriazole on thyroid function in the rat. Toxicol. Appl. Phar-
LEONARD, J. L., AND ROSENBERG. I. N. (1980). Characterization of essential enzyme sulfhydryl groups of thyroxine 5’-deiodinase from rat kidney.
macol. GALTON,
LUTHERER, L. 0.
13, 271-286. V. A.. AND
of catalase inhibitors roxine. Endocrinology HERSHMAN,
J. M.,
H. (1964). Effect on the deiodination of thy-
INGBAR,
S.
14, 627-634. AND VAN MIDDLESWORTH,
L.
297.
KAPLAN, M. M., TATRO, J. B., BREITBART, R., AND LARSEN, P. R. (1979). Comparison of thyroxine and 3,3’,5’-triiodothyronine metabolism in rat kidney and liver homogenates. Metabolism 28, 1139- 1146. KAPLAN, M. M., AND UTIGER, R. D. (1978a). Iodothyronine metabolism in rat liver homogenates. J. fnvesr.
61, 459-471.
KAPLAN. M. M., AND UTIGER, R. D. (1978b). Iodothyronine metabolism in liver and kidney homogenates from hyperthyroid and hypothyroid rats. Endocrinology 103, 156-161. KROI-LER, E. (1966). Anwendung und Eigenscbaften des 3-amino-1,2,4-triazols im Hinblick auf seine Ruckstande in Lebensmitteln. Residue Ret’. 12, 162192.
LARSEN, P. R., AND FRUMESS, R. D. (1977). Comparison of the biological effects of thyroxine and triiodothyronine in the rat. Endocrinology 100, 980-988.
vascular Aspects Interrelationship
106, 444-451.
(1%9).
Metabolic and C’urdioThyroxine-Cutecholamine Rats. Doctoral Dissertation,
qf the in
University of Florida.
(1%2). Effect of antithyroid compounds on the deiodination of thyroxine in the rat. Endocrinology 71, 94- 100. HUNTSBERGER. D. V, (1961). Elements of Statistical fr@rnce. Allyn & Bacon, Boston. JUKES, T. H., AND SHAFFER, C. B. (l%O). Antithyroid effects of aminotriazole. Science 132, 296-
C/in.
Endocrinology
R. M. B., OZAWA, Y., AND CHOPRA, I. I. (1979). Subcellular localization of thyroxine and reverse triiodothyronine outer ring monodeiodinating activities Endocrinology 104, 365-371. MARGOLIASH, E., AND NOVOGRODSKY, A. (1958). .4 study of the inhibition of catalase by 3-amino1:2:4-triazole. Biochem. J. 68, 468-475. PEAVY, D. E., TAYLOR, J. M., AND JEFFERSON, L,. S. (1981). Protein synthesis in perfused rat liver following thyroidectomy and hormone treatment. Amer. J. Physio/. 240 (Endocrinol. Metab. 3), Et&E23. SCAMMELL, J. G., SHIVERICK, K. T., AND FREG~Y. M. J. (1980). In vitro hepatic deiodination of Ithyroxine to 3,5,3’-triiodothyronine in cold-acclimated rats. J. Appi. Physiol.: Respirat. Envrrtm.
MACIEL,
Exercise
Physiol.
49, 386-389.
STEEL, R. B. D., AND TORRIE, J. H. (l%O). Principles qf Procedures c!f Statistics. McGraw-Hill. New York. SUND, K. A. (1956). Residual activity of 3-amino1,2,4-triazole in soils. J. Agr. Food Chem. 4.57-60. TAUROG, A. (1970). Thyroid peroxidase and thyroxine biosynthesis. Rec. Progr. Hormone Re.v. 26, 189247.
TJALVE. H. (1975). The distribution of labelled aminotriazole in mice. Toxicology 3, 49-67. TSCHUDY, D. P., AND COLLINS, A. (1957). Effect of 3-amino-1,2,4-triazole on &aminolevulinic acid dehydrase activity. Science 126, 168.