Chem. Res. Toxicol. 1990, 3, 98-101
98
Mechanism of Thyroid Peroxidase Inhibition by Ethylenethiourea Daniel R. Doerge* and Richard S. Takazawa Department of Agricultural Biochemistry, University of Hawaii, Honolulu, Hawaii 96822 Received November 17, 1989
Ethylenethiourea (ETU) is a thyroid carcinogen present in foods formed by degradation and metabolism of ethylenebis[dithiocarbamate]fungicides. ETU inhibits thyroid peroxidase (TPX), the enzyme that catalyzes synthesis of thyroid hormones. Inhibition of TPX-catalyzed reactions by ETU occurs only in the presence of iodide ion with concomitant oxidative metabolism to imidazoline and bisulfite ion. Inhibition ceases upon consumption of E T U with no loss of enzymatic activity and negligible covalent binding of ETU to T P X . T P X inhibition by ETU is unlike that for derivatives of imidazoline-2-thione, which cause suicide inactivation via covalent binding to the prosthetic heme group. These results demonstrate a metabolic route for detoxication of E T U in the thyroid and suggest that low-level or intermittent exposure to ETU would have minimal effects on thyroid hormone production.
Introduction ETU' is a thyroid toxicant formed by decomposition and metabolism of the EBDC fungicides which are applied to approximately one-third of all fruits and vegetables in the US. (1). ETU has been designated a Class B oncogen (probable human carcinogen) by the U S . Environmental Protection Agency ( U S . EPA) because of its ability to cause thyroid cancer in laboratory animals and its structural similarity to other known thiocarbamide (thioureaderived) thyroid carcinogens (2). Thiocarbamides block thyroid function by inhibiting TPX, the enzyme that catalyzes the iodination and coupling of tyrosine residues in the synthesis of the thyroid hormones, T4and T, (3). TPX also catalyzes the oxidation of nonphysiological reducing substrates, e.g., guaiacol and ABTS. This report describes the mechanism by which TPX-catalyzed iodination reactions are inhibited by ETU, the concomitant detoxication of ETU, and the implications of this mechanism for the carcinogenic risks from dietary exposure to ETU. Experimental Sectlon All enzymes and reagents except those noted were obtained from commercial sources and used as received. T P X was solubilized from hog thyroid by trypsinization and deoxycholate (10% w/v) solubilization and purified t o homogeneity by Sephacryl S-300, Con-A-Sepharose, and DE-52 chromatography (4, 5). Purity was established by HPLC using a TSK 3000SW column, and the molecular weight of 92000 is consistent with the uncleaved form of detergent-solubilized T P X (4). T P X activity was determined spectrophotometrically (Hewlett Packard 8452 photodiode array) by using iodide ion, guaiacol ( 6 ) ,and ABTS (7)as substrates, and T P X concentration was determined from absorbance a t 412 nm (5). Hydrogen peroxide concentration was determined by iodometric titration (8). In some experiments, a hydrogen peroxide generating system consisting of glucose oxidase (10 pg/mL) plus glucose (1mg/mL) was employed. In a typical experiment T P X (4.4 nM) was incubated a t 22 "C in the presence Abbreviations: ABTS,2,2'-azinobis[benzothiazoline-6-sulfonic acid]; DIT, diiodotyrosine; EBDC, ethylenebis[dithiocarbamate]; ETU, ethylenethiourea; LPX, lactoperoxidase; MBI, benzimidazoline-2-thione;MIT, monoiodotyrosine; NOEL, no observed effect level; TPX, thyroid peroxidase; TSH, thyroid-stimulating hormone; T4,thyroxine; T,, triiodothyronine.
of ETU (0-100 pM) and KI (5 mM) in 100 mM phosphate buffer, pH 7.0, containing NaCl ( 5 mM), 0.0025% Triton X-102, and 0.005% deoxycholate. Reaction was initiated by addition of hydrogen peroxide or glucose oxidase plus glucose. Tyrosine iodination was performed similarly in the presence of tyrosine (100 pM). ETU metabolism was determined and quantitated by HPLC (Hewlett Packard 1050 pump, 1040 diode array detector) using 10-gm Novapak silica (Waters Associates) with 12.5% acetonitrile/water as eluent a t 1.5 mL/min and UV detection a t 230 nm. Tyrosine iodination was determined by HPLC using 4-pm Novapak C18 (Waters Associates) with 33% methanol/10 mM phosphate, pH 3.0, as eluent a t 1.5 mL/min and detection a t 290 nm. Ions were determined and quantitated by ion chromatography using a Dionex GMP with an AS4A separator column and 3.5 mM sodium carbonate, pH 10, as eluent a t 2.0 mL/min and suppressed ion conductivity detection. Isotopically labeled ETU was synthesized by a published method (9) using l3CSZ (Cambridge Isotope Laboratories) or [14C]ethylenediamine(Amersham Corp.). [2J3C]ETU was 99% enriched and [4,5-14C]ETUhad a specific activity of 2.0 mCi/mmol (10). [14C]MBI was prepared with specific activity of 2.1 mCi/ mmol as previously described (11). NMR spectra were obtained by using a Nicolet NT300 spectrometer a t 76 mHz for 13C and 300 mHz for 'H. T P X (10 nM) was incubated a t 22 "C with [2-13C]ETU(200 pM) and KI (0 or 5 mM) and the reaction initiated with hydrogen peroxide (400 pM). After 30 s 500 units of bovine liver catalase catalase was added to quench the reaction and the solution was lyophilized and redissolved in 0.5 mL of DzO. The spectrum was obtained from >32 OOO acquisitions with broad-band proton decoupling and dioxane as internal reference ( 6 = 66.5 ppm). Covalent binding of ETU to T P X was determined by incubation of T P X (0.4 pM) and [14C]ETU (50 pM) with and without KI (5 mM), and the reaction was initiated by addition of hydrogen peroxide (200 pM) a t 22 "C. The reaction was quenched after 1min by addition of 500 units of catalase, the sample was applied to a Sephadex G-25 column (0.8 X 13.5 cm) equilibrated in the same buffer, T P X activity was assayed, and the amount of ETU was calculated from radioactivity present in 0.4-mL fractions. Determination of [14C]MBI binding to T P X was carried out similarly in the absence of KI.
Results ETU inhibits iodination reactions catalyzed by TPX. The assay systems used iodide ion and tyrosine as iodination acceptors with triiodide ion and MIT + DIT as the 0 1990 American Chemical Society
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 99
Thyroid Peroxidase Inhibition by Ethylenethiourea 2.0
0 PM ETU
//
1.6
Figure 1. Inhibition of TPX-catalyzed iodide ion oxidation by ETU. TPX was incubated with a hydrogen peroxide generating system and ETU at the concentration indicated, and triiodide ion formation was measured spectrophotometrically at 350 nm. TYR
a
a
E
*I
+ TPXII-IH2OZIETU
50
- - . . 2
I
7
3
4
TYR
5
6
+ TPXII.IHZ02
200
180
E
10
3
C A DIT
a
a
E 2
3
5
6
I
Figure 2. Inhibition of TPX-catalyzediodination of tyrosine by
ETU. TPX/hydrogen peroxide was incubated with KI and tyrosine f ETU, and iodination products were determined by HPLC. (A) Chromatogram of TPX-catalyzed tyrosine iodination products in the presence of ETU. (B)Chromatogram as in (A) without ETU. (C) Chromatogram of tyrosine, MIT, and DIT standards a t 100 pM each. respective products. ETU inhibition of iodide ion oxidation in the presence of a hydrogen peroxide generating system is characterized by a lag phase whose length is related to the amount of ETU present (Figure 1). Following the lag phase, iodination resumes at rates indistinguishable from the control irrespective of the original concentration of ETU. This is similar to the "reversible inhibition" of iodide ion oxidations reported for some antithyroid drugs (12). ETU also totally blocks TPX-catalyzed iodination of tyrosine (Figure 2). ETU, however, has no inhibitory effect on the rate or extent of TPX/ H20,-catalyzed oxidation of guaiacol or ABTS. HPLC analysis showed that TPX/hydrogen peroxide catalyzed the degradation of ETU during the lag phase in iodide ion oxidation. Only after all ETU was consumed did the formation of triiodide ion resume. Ion chromatography indicated that consumption of ETU proceeded with no decrease in the concentration of iodide ion, suggesting a catalytic role. Measured iodide ion concentrations were identical (within experimental error of 3.5%) for control incubations with 2 mM KI without hydrogen peroxide compared to incubations where 200 or 400 pM ETU (10% and 20% of the iodide ion concentration) was enzymatically oxidized. The products of TPX/hydrogen peroxide catalyzed metabolism were determined by using 2-13C-labeled ETU and NMR. The 13C NMR spectrum shows a single major product is formed during iodide ion dependent oxidation of ETU by TPX/hydrogen peroxide
PPM
TPX/hydrogen peroxide with KI and [13C]ETUas described under Experimental Section. (B) Incubation as in (A) of TPX/hydrogen peroxide with [ 13C]ETUwithout KI. Table I. Effect of ETU Preincubation on TPX Activity iodide ion oxidation rate) samplen
a
160
Figure 3. 13C NMR determination of iodide ion dependent metabolism of ETU catalyzed by TPX. (A) Incubation of
pM min-I
complete incubation system 8.69 f 0.16 (TPX/H202 + I- + ETU) without ETU 8.87 f 0.53 without iodide ion 8.43 f 0.28 without iodide ion and ETU 8.79 f 0.20 "TPX was incubated f ETU (100 pM) and KI (5 mM) as indicated. Reaction was initiated with hydrogen peroxide (100 pM), and after 100 s an aliquot was diluted 200-fold and the TPX activity determined as described under Experiment Section. *Mean f SD, n = 4.
(see Figure 3A). The resonance at 157 ppm was assigned to [2-13C]imidazoline on the basis of 'H and 13C NMR spectra of independently synthesized imidazoline (13,14). The minor resonances a t 165, 167, and 181 ppm are due to N-formylethylenediamine (the hydrolysis product of imidazoline), imidazolidinone, and unreacted ETU, respectively. Figure 3B shows that TPX/hydrogen peroxide catalyzes no detectable metabolism of ETU in the absence of iodide ion. The stoichiometry of ETU consumption during iodide ion dependent metabolism by T P X / hydrogen peroxide was determined by HPLC to be nearly 1:2 [0.44 f 0.02 mol of ETU/mol of hydrogen peroxide added (mean f SD, n = 5)]. The formation of bisulfite ion was confirmed by ion chromatography from ETU oxidation by T P X and LPX. The formation of bisulfite from LPX/hydrogen peroxide oxidation of ETU in ca. 40% yield was measured colorimetrically (15) although the detergents required in T P X incubations interfered with the color reaction. The final sulfur-containing product in the presence of excess hydrogen peroxide was sulfate ion in stoichiometric yield (0.97 f 0.08 mol of sulfate ion/mol of ETU, mean f SD, n = 3). The slightly low stoichiometry observed for ETU is probably from oxidative conversion of bisulfite ion to sulfate ion under the reaction/chromatography conditions. The possible oxidants are atmospheric oxygen, hydrogen peroxide, or the enzymatic iodinating species. Iodide ion dependent metabolism of ETU by T P X / hydrogen peroxide proceeds without loss of enzyme activity as determined following 200-fold dilution (Table I) or gel permeation chromatography (data not shown). Table I shows that incubation of TPX/ hydrogen peroxide with ETU and iodide ion results in no loss of enzyme activity under conditions where 1000 equiv of ETU is catalytically
Doerge and Takazawa
100 Chem. Res. Toxicol., Vol. 3, No. 2, 1990 Scheme I. Proposed Mechanism of ETU Metabolism by TPX
Imidazoline Sulfinic Acid
ETU
Imidazoline HSO,
Scheme 11. Proposed Mechanism of TPX Inhibition by ETU
0 Fe’
‘’
EO1 I - Tyr
TP X
1/2 equiv. ETU
1/2
1
3
5
7
9
11
13
15
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18
Fraction No.
Figure 4. Binding of radiolabeled ETU and MBI to TPX. (A) TPX was incubated with KI, [“CIETU, and hydrogen peroxide and chromatographed on Sephadex G-25 as described under Experimental Section and the elution volume of the enzymatic activity determined. (B) Same reaction as (A) showing elution of radiolabeled ETU in the included (large molecules) and excluded (small molecules) fractions with (A)or without ( 7 )KI present in the incubation mixture. (C)Incubation and chromatography as in (B)with [‘%]MI31as described under Experimental Section. consumed. Table I also shows that incubation of TPX/ hydrogen peroxide with ETU in the absence of iodide ion results in no loss of activity. Iodide ion dependent metabolism of [14C]ETUproceeds without significant covalent binding to TPX. Figure 4A shows the separation of T P X from small molecules by gel permeation chromatography. At the excluded elution volume, only low levels of [I4C]ETU (50.1 mol/mol of TPX) elute bound to T P X during metabolic turnover conditions (Figure 4B). Similar low-level ETU binding to T P X is observed whether or not iodide ion is present, conditions where ETU metabolism is very different (see Figure 3). When TPX was inactivated with [14C]MBIand hydrogen peroxide in the absence of iodide ion, extensive radiolabel binding to T P X is observed (Figure 4C) as predicted for a suicide inhibitor (15).
Discussion Among the typical TPX substrates used (iodide ion, guaiacol, and ABTS) the inhibition by ETU is unique for iodide ion oxidation. Both iodide ion and ABTS are oxidized by the a-cation radical form of peroxidase compounds I (7, 16). Guaiacol is oxidized by both this form of T P X and the protein radical form of compound I analogous to cytochrome c peroxidase compound ES (16, 17). The fact that ETU inhibits iodination reactions and not ABTS or guaiacol oxidation suggests that ETU does not react with either form of TPX compound I but a different reactive enzyme intermediate, the enzymatic iodinating species (18).
rb
+
1 / 2 HSO;
The production of triiodide ion by TPXIhydrogen peroxide is totally blocked by ETU during an initial lag phase as seen in Figure 1. During the lag phase, the enzymatic iodinating intermediate is trapped by reaction with ETU, leading to its oxidative metabolism instead of iodination of the acceptor molecules which were measured (iodide ion, tyrosine). This appears to be the result of a rapid bimolecular reaction between the iodinating species and ETU since the inhibition was all or none during the lag phase at all concentrations tested. Following consumption of all ETU, triiodide ion formation resumes and the rate is unaffected. The oxidation of ETU by TPX/hydrogen peroxide catalyzed iodination produces imidazoline and bisulfite ion and proceeds with a stoichiometry of ca. 2 mol of hydrogen peroxide/mol of ETU. This suggests the formation of imidazoline-2-sulfinicacid as an unstable intermediate that undergoes hydrolytic cleavage (Scheme I). This is the only observed reaction since only minor amounts of other products are seen in the NMR spectrum (Figure 3A). The formation of this reactive intermediate does not affect the catalytic competence of TPX since no loss of enzyme activity is observed (Figure 1,Table I). In addition, negligible amounts of radiolabeled ETU become bound to T P X during the enzymatic oxidation reaction (Figure 4). Inhibition of TPX-catalyzed iodination results from the competition between acceptor (tyrosine in path 1,Scheme 11, or iodide ion) and ETU (path 2, Scheme 11) for the enzyme-generated iodinating intermediate (EOI). The reaction of ETU with EO1 results in catalytic destruction of the inhibitor. This alternate substrate inhibition by ETU is therefore transient, and TPX-catalyzed iodination reactions are blocked only when the amount of ETU exceeds twice that of hydrogen peroxide. The incubation of TPXIhydrogen peroxide with ETU in the absence of iodide ion also results in no loss of enzyme activity (Table I). This contrasts the action of therapeutic antithyroid drugs, e.g., l-methylimidazoline2-thione (methimazole), and model compounds based on benzimidazoline-2-thione,e.g., MBI. These thiocarbamides are suicide inhibitors of TPX and LPX in the absence of
Thyroid Peroxidase Inhibition by Ethylenethiourea
iodide ion and rapidly inactivate these peroxidases in the presence of hydrogen peroxide by covalent binding to the prosthetic heme group (15). Incubation of TPX/hydrogen peroxide with MBI results in enzyme inactivation and covalent binding to T P X as shown in Figure 4. These findings provide a biochemical basis for the existence of a NOEL reported for the thyroid toxicity of ETU during chronic high-level dosing of rodents (19). The effects of low-level or intermittent exposure to ETU would be very different since, as shown here, ETU is detoxified by TPX, leaving the capacity for thyroid hormone production undiminished. The existence of a NOEL has important implications for the carcinogenic risks associated with dietary exposure to ETU. In the absence of evidence for a NOEL, regulators must assume the worst case scenario where one molecule of a genotoxic carcinogen is sufficient to initiate cancer by modification of target DNA (the one-hit model) (20). The current theory of thyroid follicular cell carcinogenesis from antithyroid chemicals centers on perturbation of the hormonal regulatory system (3). Thyroid inhibitors reduce the output of T, and T,, causing compensatory release of excess TSH from the anterior pituitary. This chronic growth stimulus to the thyroid is throught to induce a loss in cellular differentiation that can eventually lead to cancer. However, the toxicological effects on the thyroid are predicted to be very different for ETU as opposed to suicide inhibitors like methimazole (15)and the herbicide amitrole (21) although all three act by inhibiting synthesis of thyroid hormones. Since suicide inhibitors inactivate T P X by covalent binding, de novo protein synthesis is required to restore lost enzyme activity. However, ETU is oxidatively metabolized to noninhibitory products by TPX, leaving the capacity for thyroid hormone synthesis undiminished. Therefore, the disruption of thyroid/pituitary homeostasis by ETU is of much shorter duration. A recent National Academy of Sciences assessment of carcinogenic risks from human foods examined the EBDC fungicides (1). This assessment did not use a NOEL for ETU, and as a result the carcinogenic risks estimated for EBDC contamination of foods were among the highest for all pesticides examined. EBDC’s are currently under the Special Review process by the U S . EPA to determine whether food uses should be banned because of unacceptable cancer risks to consumers (2). EBDC’s are generally acknowledged to be highly effective agents for the control of fungal pests on a wide variety of crops especially in warm humid areas (1). The results of this study show that the thyroid has the capacity to detoxify ETU and suggest minimal adverse affects on the thyroid from exposure to the trace levels of ETU currently present in foods. Acknowledgment. The support of NIEHS Grant ES04622 and USDA Grant 88WRPIAP-17 and assistance of the University of Hawaii, Manoa, NMR facility are gratefully acknowledged. This is Journal Series No. 3396 from the Hawaii Institute of Tropical Agriculture and Human Resources.
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 101 Registry No. ETU, 96-45-7; TPX, 9031-28-1; I-, 20461-54-5.
References (1) National Research Council (1987) in Regulating Pesticides in
Food. The Delaney Paradox.. _DD- 208-214, National Academy Press, Washington,bC. (2) US. EPA (1987) EBDC Fungicides; Initiation of Special Review. Fed. Regist. 52, 27172-27177. (3) Hill, R N., Erdreich, L. S., Paynter, 0. E., Roberts, P. A,, Rosenthal, S. L., and Wilkinson, c. F. (1989) Thyroid Follicular Cell Carcinogenesis. Fundam. Appl. Toxicol. 629,629-697. (4) Lukat, G. S., Jabro, M. N., Rogers, K. R., and Goff, H. M. (1988) Electron Paramagnetic Resonance of Thyroid Peroxidase. Biochim. Biophys. Acta 954,265-270. (5) Ohtaki, S., Nakagawa, H., Nakamura, M., and Yamasaki, Y. (1982) Reactions of Purified Hog Thyroid Peroxidase with H20z, Tyrosine and Methylmercaptoimidazole (Goitrogen) in Comparison with Bovine Lactoperoxidase. J . Biol. Chem. 257,761-766. (6) Morrison, M. (1970) Iodination of Tyrosine: Isolation of Lactoperoxidase. Methods Enzymol. 17A,653-657. (7) Shindler, J. S., Childs, R. E., and Bardsley, W. G. (1976) Peroxidase from Human Cervical Mucus. Eur. J . Biochem. 65, 325-331. (8) Kolthoff, I. M., Sandell, E. B., Meehan, E. J., and Bruckenstein, S. (1969) in Quantitatioe Chemical Analysis. - DD 842-860, Macmillan, Londbn. (9) Allen, C. F. H., Edens, C. O., and Van Allan, J. (1955) EthylenDD_394-395. ethiourea. In Organic Syntheses. Collect. Vol. 111. . Wiley, New York: (10) Doerge, D. R., Cooray, N. M., Yee, A. B. K., and Niemczura, W. P. (1990) Synthesis of Isotopically-Labelled Ethylenethiourea. J . Labelled Compds. Radiopharm. (in press). (11) Doerge, D. R. (1988) Synthesis of I4C- and 35S-Labelled 2Mercaptobenzimidazoles. Ibid. 25,985-990. (12) Engler, H., Taurog, A., Luthy, C., and Dorris, M. (1983) Reversible and Irreversible Inhibition of Thyroid Peroxidase-Catalyzed Iodination by Thioureylene Drugs. Endocrinology 112, 86-95. (13) Grundmann, C., and Kreutzberger, A. (1955) The Ring Cleavage of s-Triazines by Primary Amines. A New Method for the Synthesis of Heterocycles. J . Am. Chem. SOC. 77, 6559-6564. (14) Marshall, W. D. (1979) Oxidative Degradation of Ethylenethiourea (ETU) and ETU Progenitors by Hydrogen Peroxide and Hypochlorite. J . Agric. Food Chem. 27,295-299. (15) Doerge, D. R. (1988) Mechanism-Based Inhibition of Lactoperoxidase by Thiocarbamide Goitrogens. Identification of Turnover and Inactivation Pathways. Biochemistry 27, 3697-3700. (16) Nakamura, M., Yamazaki, I., Kotani, T., and Ohtaki, S. (1985) Thyroid Peroxidase Selects the Mechanism of either 1- or 2Electron Oxidation of Phenols, Depending on Their Substituents. J. Biol. Chem. 260,13546-13552. (17) Virion, A., Courtin, F., Deme, D., Michot, J. L., Kaniewski, J., and Pommier, J. (1985) Spectral Characteristics and Catalytic Properties of Thyroid Peroxidase-H202Compounds in the Iodination and Coupling Reactions. Arch. Biochem. Biophys. 242, 41-47. (18) Magnusson, R. P., Taurog, A., and Dorris, M. L. (1984) Mechanisms of Thyroid Peroxidase- and Lactoperoxidase-catalyzed Reactions Involving Iodide. J . Biol. Chem. 259, 13783-13790. (19) O’Neil, W. M., and Marshall, W. D. (1984) Goitrogenic Effects of Ethylenethiourea on Rat Thyroid. Pestic. Biochem. Physiol. 21, 92-101. (20) Grice, H. C. (1984) in Current Issues in Toxicology, pp 49-65, Springer Verlag, New York. (21) Doerge, D. R., and Niemczura, W. P. (1989) Suicide Inactivation of Lactoperoxidase by 3-Amino-l,2,4-triazole. Chem. Res. Toxicol. 2, 100-103. I
