Chem. Res. Toxicol. 1994, 7, 164-169
164
Inhibition of Peroxidase-CatalyzedReactions by Arylamines: Mechanism for the Anti-Thyroid Action of Sulfamethazine Daniel R. Doerge'pt and Caroline J. Decked Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, and Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709 Received August 23, 199P
Sulfonamide antibiotics, typified by sulfamethazine (SMZ), are widely used in veterinary practice. Sulfonamide residues in milk and meat products are of regulatory concern since SMZ is a thyroid carcinogen in rodents and sulfonamide-induced hypersensitivity reactions, including hypothyroidism, have been reported in humans. SMZ and other primary arylamines inhibited iodination reactions catalyzed by thyroid peroxidase (TPO) and the closely related lactoperoxidase (LPO). Inhibition of LPO-catalyzed triiodide ion formation by SMZ and other primary arylamines was complex as both apparent K, and V,, values were affected, but consistent with a rapid equilibrium binding mechanism. The apparent Ki for SMZ inhibition of TPO- and LPOcatalyzed iodide ion oxidation was approximately 0.42 and 0.11 mM, respectively. The corresponding Ki values for a series of para-substituted anilines correlated with the ease of one-electron N-oxidation as measured by ionization potentials determined from semiempirical molecular orbital calculations. The aniline derivatives containing electron-donatingsubstituents (e.g., p-CH,, p-OEt, p-C1) were converted by LPO to colored products characteristic of oneelectron oxidation. However, sulfonamides were not consumed in such reactions nor were any N-oxygenated derivatives formed in the absence of ascorbate (e.g., hydroxylamino, nitroso, nitro, azoxy). These observations suggest that the primary mechanism for sulfonamide-induced hypothyroidism is reversible inhibition of TPO-mediated thyroid hormone synthesis and not the formation and covalent binding of reactive N-oxygenated metabolites. These results are consistent with a hormonal mechanism for SMZ-induced thyroid carcinogenesis mediated by thyroid-stimulating hormone (TSH). This nongenotoxic mechanism predicts that a threshold dose exists for carcinogenic effects in the thyroid and suggests that minimal carcinogenic risks are associated with consumption of trace amounts of sulfonamide antibiotics in current food supplies.
Introduction Sulfonamide antibioticsare widely used prophylactically in veterinary practice to treat subacutebacterial infections in livestock and poultry throughout the lifetime of the animals (1). SMZl (see Figure 1)is the most widely used sulfonamide, and residues enter the human food chain through milk and meat products (2). This is of toxicological and regulatory concern because thyroid follicular adenomas result from chronic high-dose administration (1200-4800 ppm in feed) to rats and mice in 1-2-year bioassays (3). Sulfonamideshave also been associated with drug-induced allergic hypersensitivity reactions in humans and animals (4-6). In addition, the general role of antibiotic addition to animal feeds in producing resistance to antibiotics in human pathogens is unresolved (7). The function of the thyroid gland is synthesis of thyroid hormones, and TPO is the enzyme that catalyzes the
* Address correspondence to this author. Tel: (501) 543-7943; FAX: (501) 543-7136. t National Center for Toxicological Research. t Burroughe Wellcome Co. *Abstract published in Advance ACS Abstracts, February 1, 1994. Abbreviations: ABTS, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate);CPO, chloroperoxidase;DETPAC, diethylenetriaminepentaacetic acid;DIT, 3,5-diiodotyrosine;HOMO, highest occupied molecular orbital HRP, horseradish peroxidase; IC@, concentration yielding 50 5% inhibition; LPO, lactoperoxidase; MIT, 3-iodotryosine;SMZ ulfamethazine; SNM, sulfanilamide; thyroid hormones = thyroxine + &dothyronine; TPO, thyroid peroxidase.
H,N
0
im
98% pure by LC/UV and TLC. The identity and purity of SMZ-NHOH and SMZ-N02, prepared by known methods (16), and azoxySMZ, prepared by ferric chloride oxidation of SMZ-NHOH (Doerge and Pinto-Ferriera, unpublished), were established by TLC ( 5 % methanol in chloroform), LC with UV-spectral and amperometric detection (hydroxylamines), and FAB/MS. LC was performed using a GPM gradient pump (Dionex Co., Sunnyvale, CA) and UV (FOCUS variable wavelength, Spectra Physics, San Jose, CA) or pulsed electrochemical (Dionex) detection. UV-vis measurements were performed using a 8452A diode arrayspectrophotometer (Hewlett Packard, Palo Alto, CA). Stopped-flow measurements (dead time ca. 20 ms) were made using an SFA-11 Rapid Kinetics Accessory (Hi-Tech Scientific, Salisbury, U.K.). Peroxidase concentration was determined spectrophotometrically (15,17). Assays for oxidation of iodide ion, guaiacol, and ABTS were determined at pH 7.0 (100 mM phosphate) as previouslydescribed (I7), and initial rates were determined from the linear reaction phase ( 5 5 e). Oxidation rates were determined for a series of substrate concentrations with different fixed amounts of inhibitor present. Data were plotted and the linear regression lines computed by graphics software. The concentration of TPO was 7.6 nM in all assay systems. Peroxidasemediated tyrosine monoiodination was analyzed by LC/UV as previously described (1.9, and initial rates of MIT formation were determined during the linear phase of reaction (0.5 or 1min for 3 nM LPO or 4.6 nM TPO, respectively). LPO was used in the majority of studies reported here because it was available in the large amounts needed for all kinetic and oxidation studies. LPO-mediatedmetabolism of arylamineswas determined using 200 nM LPO, 200 p M hydrogen peroxide, and 100-500 p M arylamine in phosphate buffer (100 mM, pH 7.0). In some cases, ascorbate and/or DETPAC was added to final concentrations of 1 mM and 2 pM, respectively. Incubations were carried out at 22 "C for 15 min and were terminated by addition of 1400 units of bovine catalase. Analysis of 100-fiL aliquots by LC was performed immediately using a Radial Compression Module equipped with a NovaPak C18 cartridge (5 X 100 mm, 4-pm particles, Waters Associates, Milford, MA) using UV (260 nm) and amperometric (0.2 V vs Ag/AgCl) detectors in series. The
ss 00
2
4
6
0
SMZ (mM)
Figure 2. Inhibition of TPO-catalyzed MIT formation by SMZ. The initial rate of triiodide ion formation was determined spectrophotometrically (352 nm) for a fixed series of substrate concentrations and varying concentrations of SMZ. Inset: The double-reciprocal slopes were plotted vs SMZ concentration. mobile phases used were linear gradient programs comprised of acetonitrile and phosphate buffer (25 mM, pH 7.0) designed to separate N-oxidized metabolites (e.g., hydroxylamine, amine, nitro, nitroso, and azoxy). For sulfanilamide, the program was isocratic at 2% acetonitrile for 5 min, then an 18-min linear gradient to 25% acetonitrile. The retention times (min) for authentic standards were as follows: SNM-NHOH, 1.3; SNMNH2,1.9; SNM-N02,14.1;SNM-NO, 14.7. For SMZ,the program was isocratic at 10% acetonitrile for 1 min and then a 25-min linear gradient to 50% acetonitrile. The retention times (min) for authentic standards were as follows: SMZ-NHOH, 3.4; SMZNH2,5.7; SMZ-N02,11.3;azoxy-SMZ, 12.4,SMZ-NO,12.9. Under these conditions, the estimated detection limits using amperometry (S/N = 3) for SMZ-NHOH and sulfanilamide-NHOH were 0.3 and 0.1 pM, respectively. Oxidation of 4-chloroaniline (100fiM)was similarlyperformed using isocratic30% acetonitrile in 25 mM phosphate buffer (pH 7.0). The retention time for authentic (4-chlorophenyl)hydroxylaminewas 2.6 min, and the estimated detection limit was 0.05 pM. AM-1semiempirical molecular orbital calculations performed using a Tektronix CAChe computer-aided work station and vertical ionization potentials were calculated from the HOMO energies using Koopman's Theorem as previouslydescribed (18).
Results SMZ inhibited LPO- and TPO-catalyzed iodination of free tyrosine as shown for TPO in Figure 2. T h e approximate IC60 value was 0.3 and 0.5 mM, respectively, and high concentrations of SMZ completely blocked MIT formation (see Figure 2) and DIT formation by both peroxidases (data not shown). It was determined that SMZ did not inactivate LPO or TPO, in t h e presence or absence of hydrogen peroxide, using preincubation assays t h a t measure suicide inhibition (17). It was further determined t h a t SMZ did not inhibit peroxidase-mediated triiodide ion formation by a n alternate substrate mechanism as previously described (15, 19). T h a t is, in t h e presence of a hydrogen peroxide generating system, no lag phase in iodination was observed at any concentration of SMZ. T h e effect of SMZ on t h e kinetics of peroxidasemediated iodination was determined using a spectrophotometric assay of triiodide ion formation (see Scheme 1). Figure 3 shows t h e family of double-reciprocal plots produced by TPO in the presence of increasing concentrations of SMZ. T h e intersection t o t h e right of t h e l / u axis is not typical b u t may be related t o the participation of t h e substrate, iodide ion, in two product-determining
166 Chem. Res. Toxicol., Vol. 7, No. 2, 1994
Doerge and Decker
Scheme 1. Ionization Reactions Catalyzed by LPO
-
LPO + HzOz
MIT
1
- 1r/’Tyrosine
LPO-CompoundI I-
LPO-01 I- 1, -+ I- 1; HOI
steps subsequent to oxidation (20). The inset to Figure 3 plots the double-reciprocal slopes vs SMZ concentration, and the replot is consistent with inhibition by rapid equilibrium binding where K , is increased and V, is decreased (21). The presence of high amounts of SMZ (18mM) caused complete inhibition of TPO-catalyzed iodide ion oxidation measured by triiodide ion (data not shown) and MIT formation (see Figure 2). The apparent Ki,0.42 mM, was approximated from the linear portion of the replot. Similar plots were obtained for SMZ-mediated inhibition of LPO-catalyzed triiodide ion formation; however, the slope replot was linear (Ki= 0.11 mM, datanot shown). The apparent Ki values determined from the slope replots roughly correspond with the ICSOSdetermined for MIT formation catalyzed by TPO and LPO (cf. Figure 2). Inhibition of LPO-mediated iodination was also measured with other primary arylamines using the same procedure, and linear slope replots from the double-reciprocal plots were obtained. A spectrophotometric MIT formation assay was used to confirm kinetic behavior and inhibition constants for those arylamines whose absorbance did not interfere in the assay (p-C1, p-CO2H). The apparent Ki values determined were similar to those determined from inhibition of triiodide ion formation (data not shown). SMZ also inhibited LPO-catalyzed oxidation of guaiacol and ABTS (data not shown). The mixed-type inhibition of guaiacol and ABTS oxidation by SMZ produced hyperbolic slope replots and apparent Ki values similar to those determined for triiodide ion oxidation (1.4 and 0.9 mM, respectively). It was observed that LPO and TPO catalyzed hydrogen peroxide-dependent formation of colored products from arylamines containing electron-donating para substituents (e.g., C1, CH3, OEt). This was accompanied by a decrease in arylamine concentration as determined by LC/UV. Incubation of SMZ or sulfanilamide with LPO and hydrogen peroxide (with and without iodide ion) gave no detectable changes in UV spectrum or losses in parent compound concentration by LC/UV. The possible formation of N-oxygenated products from arylamines was further investigated using LC with amperometric detection (0.2 V), a sensitive and selective technique for measuring arylhydroxylamines (22). No hydroxylamine products were observed for SMZ, sulfanilamide, or p-chloroaniline when incubated with LPO and hydrogen peroxide (at the levels of detection given in the Experimental Section). In addition, no other corresponding N-oxidation products were observed (see the Experimental Section). The addition of iodide ion to incubations had no effect on hydroxylamine formation. Recovery of added hydroxylamines to LPOIhydrogen peroxide followed by catalase was 93 %,93 % ,and 25 % for sulfanilamide, SMZ, and p-chloroaniline, respectively. In the presence of 1 mM ascorbate, LPO + hydrogen peroxide produced small but reproducible amounts of the hydroxylamine from 500 pM sulfanilamide (0.14 f 0.06
0
1
0.0
0.2
0.4
1/[1-1
0.6
0.8
1 .o
(mM)
Figure 3. Correlation of apparent mixed-type inhibition constants with vertical ionization potentials for p-substituted anilines. Apparent Ki values for inhibition of LPO-catalyzed triiodide ion formation were determined from the doublereciprocal slope vs [SMZ] replots. Vertical ionization potentials were determined from AM-1 semiempirical molecular orbital calculations.
pM, 0.03% conversion) and p-chloroaniline (0.1 f 0.02 pM, 0.03 % ). However, no corresponding hydroxylamine formation from SMZ was observed under any condition. It was further observed that TPO- and LPO-catalyzed oxidation of iodide ion, guaiacol, or ABTS was completely blocked by ascorbate under these conditions. The addition of DETPAC, an octadentate ferric ion chelator, to sulfanilamide/LPO incubations containing ascorbate caused ca. 50 % reduction in hydroxylamine formation (duplicate experiments). DETPAC had no effect on LPO-catalyzed iodide ion oxidation and did not reduce the amount of (6chlorophenyl)hydroxylamine formed in the presence of ascorbate.
Discussion The kinetics observed for SMZ inhibition of LPO- and TPO-catalyzed oxidation reactions were consistent with rapid equilibrium binding of enzyme and inhibitor to form reversible complexes that change both apparent K, and V,, values (21). The inhibition of LPO by SMZ is consistent with the binding of inhibitor to both free and iodide ion-bound LPO because the slope replot is linear and the reaction velocity can be driven to zero at high inhibitor concentration. This suggests that the enzymeSMZ-iodide ion complex is not catalytically competent (21). In contrast, SMZ inhibition of LPO-catalyzed guaiacol and ABTS oxidation produced hyperbolic slope replots. This kinetic mechanism is consistent with an enzyme-inhibitor complex that can bind and effect oxidation of substrate (21). The concave-upward slope replot shown in Figure 3 for SMZ inhibition of TPOcatalyzed iodide ion oxidation could be consistent with inhibitor binding to two sites. This would result in greater inhibition at high concentrations of SMZ when both inhibitor binding sites are occupied. Further data are required to confirm this possibility. Inhibition constants estimated for inhibition of LPO- and TPO-catalyzed triiodide ion formation by SMZ are of the same order (0.11 vs 0.42 mM, respectively). This is consistent with the many qualitative similarities between these enzymes (23). These values are similar to those determined with LPO for guaiacol and ABTS (1.4 and 0.9 mM, respectively). There is spectroscopic evidence in the literature for the formation of complexes between native LPO and aromatic
Chem. Res. Toxicol., Vol. 7, No. 2, 1994 167
Sulfamethazine Inhibition of Peroxidases
compounds (e.g., phenols, arylamines) and iodide ion (24) as well as kinetic results consistent with the binding of tyrosine to LPO (20). Similar interactions between SMZ and either LPO, compound I, or LPO-01 are possible, and such interactions could affect the enzyme kinetics as observed here. However, it should be noted that the Kd values determined spectroscopically with natiue LPO are ca. 100-fold larger than the apparent Kis determined kinetically for SMZ. This emphasizes the potential for differences in binding properties between native and oxidized peroxidase species. The kinetic results described here are not those predicted from the classical peroxidase ping-pong cycle described for HRP (25). Due to the irreversible step in this mechanism, substrate K , is not relevant as the rate for one-electron oxidation of reducing cosubstrates (e.g., guaiacol and ABTS) is determined by the second-order rate for reaction between peroxidase compound I1 and substrate. For iodide ion oxidation, a two-electron oxidation is catalyzed by peroxidase compound I. In the case of LPO and TPO, an enzyme-bound hypoiodite species has been implicated in catalysis of iodination reactions (20,26). In peroxidase-catalyzed iodide ion oxidation (see Scheme l),other possible chemical iodinating species (I2 and HOI but not 13-)have been identified (20). Sun and Dunford showed that, at high [I-] > 0.3 mM), the ratedetermining step is protonation of LPO-01 to give HOI which reacts with I- to give 12 then I3-. At the [I-] used in the present study (1-10 mM), this rate-determining step is certainly operative. Under these conditions, compounds that bind to LPO, compound I, or LPO-01 could alter the apparent binding constant for iodide ion or the maximal rate of triiodide ion formation or both. This interpretation is consistent with the findings of Libby et al., who noted that saturation kinetics and noncompetitive inhibition can result when substrate reacts exclusively with an enzyme-generated freely dissociable species (27). In this case, trimethylphenol oxidation was effected by C12 generated by chloroperoxidase-catalyzed C1- oxidation in competition with peroxidation of catechol. Mixed-type inhibition of HRP-catalyzed iodide ion and guaiacol oxidation by thioanisole has also been reported (28). Thioanisole, at concentrations where negligible S-oxidation occurs, affected both K , and V,, that were determined from the linear transformation of MichaelisMenten steady-state rate data. Furthermore, the k&Km determined for guaiacol by this method was similar to k2, the second-order rate constant determined for its reaction with HRP compound 11. This suggests concordance of kinetic mechanisms (rapid equilibrium vs ping-pong with an irreversible step) under these conditions. Therefore, the use of Michaelis-Menten kinetics to analyze peroxidase-catalyzed reactions (including inhibition and activation), which is common in the literature, may reflect subtle undescribed changes in the kinetic behavior similar to those described by Sun and Dunford for LPO-mediated iodide ion oxidation (see above and ref 20). Similar mixed-type inhibition was also observed for LPO-mediated iodide ion oxidation by a series of parasubstituted primary arylamines. As shown in Figure 4, the log of apparent Ki correlated with the AM-1-calculated one-electron oxidation potentials. The enhancement of inhibitory potency by electron-donating substituents and the high degree of correlation (r = 0.90) suggest that binding results in partial electron transfer from the
n
%
-9
-1
.o
0.0
1 .o
2.0
I
3.0
log K i ( P M )
Figure 4. Correlation of apparent mixed-type inhibition constants with vertical ionization potentials for p-substituted
anilines. Apparent Ki values for inhibition of LPO-catalyzed triiodide ion formation were determined from the doublereciprocal slopevis [SMZ] replots. Vertical ionization potentials were determined from AM-1 semiempirical molecular orbital calculations. arylamine nitrogen to an electron-deficientenzyme species, Le., the oxoferryl heme of LPO compound I. A similar correlation was reported for the interaction of sulfurcontaining substrates and suicide inhibitors with LPO using both AM-1-calculated (18) and experimentally derived voltammetric peak potentials (29). Further evidence was obtained for this inhibition mechanism. Arylamines containing electron-donating para substituents were substrates for LPO and TPO in the absence of iodide ion. For these arylamines, the electron-transfer reaction is complete. The uncharacterized colored products observed were consistent with the single-electron oxidation chemistry previously reported for interaction of arylamines with peroxidases and other oxidants (30). However, arylamines containing electronwithdrawing groups (e.g., SMZ) were not peroxidase substrates. Moreover, the corresponding arylhydroxylamines were not observed in incubations of SMZ, sulfanilamide, or p-chloroaniline containing high levels of LPO (200 nM) and arylamine (500 pM) . These arylamines were chosen to include a substrate for one-electron oxidation @-chloroaniline) and the two nonsubstrate sulfonamides. These findings preclude a mechanism in which oxygen rebound occurs from the LPO-oxoferryl heme to an initially formed N-cation radical (see Scheme 2). This is different from the N-oxygenation catalyzed by related hemoproteins CPO (31)and cytochrome P450 (32) but similar to the previously described one-electron oxidation of arylamines by peroxidases (30). Given the many similarities in oxidation mechanism between LPO and TPO (23), especially reaction with many reducing cosubstrates, we conclude that arylhydroxylamines are not primary reaction products from one-electron oxidation of arylamines effected by these peroxidases. Previous workers described TPO-dependent formation of N-hydroxysulfamethoxazole in vitro and linked its formation to allergic hypersensitivity reactions in the thyroid, including hypothyroidism (6).These experiments were conducted in the presence of ascorbate (1mM) to stabilize the putative hydroxylamine product. However, ascorbate is a well-known substrate for peroxidases (33) and reacts readily with radical species in solution (34). The present study determined that ascorbate completely blocked all LPO- and TPO-catalyzed reactions investigated under analogous conditions (e.g., iodide ion, guaiacol, and
Doerge and Decker
168 Chem. Res. Toxicol., Val. 7, No. 2, 1994
Scheme 2. Proposed Mechanism for Inhibition of Peroxidase-Catalyzed &actions by Primary Arylamines ? ... :H N-& H Compound1
Fc" TPO or LPO
N-Oxidizcd Polymeric Rod~cts (Electron-Donating Substituents)
= Protoporphyrin IX
ABTS oxidation). In addition, LPO-dependent hydroxylamine formation was not observed for SMZ in the presence or absence of ascorbate. Furthermore, the ascorbate-dependent formation of N-hydroxysulfanilamide was attenuated by addition of DETPAC, a ferric ion chelator. The reduction of chelated ferric ion to ferrous ion by ascorbate and the requirement of these species in molecular oxygen- and hydrogen peroxide-dependent lipid peroxidation have been reported (35). These observations suggest that adventitious N-oxidation, effected by trace amounts of transition metal ions present in aerobic solution, and not an LPO-mediated reaction, was involved in the ascorbate-dependent reactions that do produce hydroxylamines. The uncertainties in determining enzymatic N-oxygenation products from arylamines in the presence of ascorbate have been discussed previously (31, 36). Scheme 2 shows a mechanism that is consistent with the data presented here for LPO and TPO. Arylamines block the iodination and phenolic coupling reactions required for thyroid hormone synthesis by kinetics consistent with a form of noncompetitive inhibition. Since SMZ inhibits triiodide ion formation and guaiacol/ABTS oxidation by mixed-type mechanisms, it presumably interacts with an enzymatic species distinct from LPO-01 and compound 11, respectively. This also suggests LPO compound I as the site for interaction with SMZ (see above). These results demonstrate reversible inhibition of TPOcatalyzed iodination by SMZ. These results are consistent with the data of Takayama et al.that suggested competitive inhibition of TPO-catalyzed guaiacol oxidation by sulfamonomethoxine in rat and monkey thyroid microsomes (37). The relatively high Ki value observed with purified hog TPO predicts minimimal inhibition at low concentrations of SMZ. However, there is considerable interspecies variation with respect to TPO inhibition by sulfonamides. Rat microsomal TPO appears to be much more sensitive to inhibition by sulfonamides than solubilized hog (this work) or monkey microsomal TPO (37). Corresponding data regarding sulfonamide inhibition of human TPO are currently not available. A mechanism of reversible TPO inhibition by SMZ is sufficient to explain the blockade of thyroid hormone synthesis and elevated thyroid-stimulating hormone observed in rodents during chronic high-dose feeding studies (38). This is consistent with a subsequent proliferative response in the thyroid due to the presence of excess thyroid-stimulating hormone, and this response alone is
sufficient to produce thyroid tumors ( 1 0 , l l ) . Therefore, increased secretion of thyroid-stimulating hormone and subsequent proliferative changes in the thyroid should occur only after prolonged exposure to high doses. These are the conditions that prevailed in chronic high-dose bioassays in which SMZ produced thyroid tumors in rodents (e.g., 1000 ppm in feed) ( 3 ) . In contrast, typical exposure of humans to SMZ in meat and milk products is at low levels (ca. 5 ppb) and intermittent in nature (39). In addition, since N-hydroxy-SMZ or related metabolites could not be detected in this in vitro model study, it is unlikely that significant formation and binding of genotoxic SMZ metabolites occur in the thyroid. The inability of LPO to produce potentially genotoxic N-oxygenated metabolites from SMZ is also consistent with a thyroidstimulating hormone-mediated (secondary)mechanism for initiation of thyroid carcinogenesis. A nongenotoxic mechanism implies, but does not prove, that a threshold dose exists below which no carcinogenic effect is observed (40). These results of this study suggest that minimal risks of thyroid carcinogenesis are associated with dietary exposure to the small amounts of SMZ present in food.
Acknowledgment. We thank Dr.M. E. Brewster for calculating arslamine vertical ionization potentials and R. S. Takkawa for purifying hog TPO.
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