174
Chem. Res. Toxicol. 1993,6, 174-179
Possible Role of Thioformamide as a Proximate Toxicant in the Nephrotoxicity of Thiabendazole and Related Thiazoles in Glutathione-Depleted Mice: Structure-Toxicity and Metabolic Studies T a m i o Mizutani,* K a o r u Yoshida, and Sadahiro Kawazoe Department of Food Science and Nutrition, Kyoto Prefectural University, Shimogamo, Kyoto 606, J a p a n Received November 17, 1992
In mice depleted of GSH by treatment with buthionine sulfoximine (BSO), thiabendazole (TBZ) causes renal injury characterized by an increase in serum ureanitrogen (SUN) concentration and by tubular necrosis. Previous studies have shown that TBZ requires metabolic activation before it produces nephrotoxicity and that the structure contributing to the toxicity of TBZ is the thiazole moiety of the molecule. TBZ and its thiazole analogues were examined for the ability to increase SUN concentration and serum alanine aminotransferase activity in GSHdepleted mice. Unsubstituted thiazole and thiazoles with 4- and/or 5-, and no 2-, substituents caused marked increases in SUN concentration, suggesting nephrotoxicity. Furthermore, the nephrotoxic potency of these thiazoles decreased with the increasing number and bulk of the 4- and/or 5-substituents. On the other hand, the target organ (the kidney or liver) and the toxic potency of 4-methylthiazoles were markedly altered with the type of substituents a t the 2-position. These observations and the known toxicity of thiono-sulfur compounds led us to the hypothesis that the nephrotoxic thiazoles, which lack 2-substituents, would undergo microsomal epoxidation of the C-4,5 double bond and, after being hydrolyzed, the resulting epoxide would then be decomposed to form thioformamide, a possibly toxic metabolite. Evidence for this hypothesis was provided by the results that thioformamide and tert-butylglyoxal as the accompanying fragment were identified as urinary metabolites in mice dosed with 4-tert-butylthiazole and that thioformamide caused a marked increase in SUN concentration when administered to mice in combination with BSO. Introduction
Thiabendazole [2-(4'-thiazolyl)benzimidazole, TBZ]' has an antifungal activity and has been widely used as an agricultural fungicide and a food preservative. TBZ is also used as an anthelmintic in the treatment of some parasite infections in domestic animals and humans. We have shown that treatment of normal male mice with TBZ causes no nephrotoxicity even at higher doses (up to 1200 mg/kg body wt) (I). In contrast, TBZ causes severe kidney injury in male mice depleted of GSH by pretreatment with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis (I). The injury is characterized by increases in kidney weight and serum urea nitrogen (SUN)concentration and by tubular necrosis. Moreover, we have shown that some thiazoles structurally simpler than TBZ, e.g., 4-methylthiazoleand 4-phenylthiazole,also produce nephrotoxicity in mice depleted of GSH and, hence, that the structure contributing to the nephrotoxicity of TBZ is the thiazole moiety of the molecule (2). Cytochrome P450dependent metabolism appears to be involved in the nephrotoxicityof TBZ and the related thiazoles, since the treatments with inhibitorsof cytochrome P450-dependent monooxygenases, such as piperonyl butoxide and methoxsalen, stronglyinhibit the nephrotoxicitycaused by these compounds (1,2). We designed the present study to elucidatethe structural features of thiazoles essential for nephrotoxic activity in I Abbreviations: BSO,buthionine sulfoximine; SALT, serum alanine aminotransferase; SUN,serum urea nitrogen; TBZ,thiabendazole.
GSH-depleted mice, to suggest chemical identity of the postulated active metabolite, and then to provide positive support for the suggestion. Experimental Procedures Chemicals. The compounds examined for toxicity are shown in Chart I. Each sample had a purity exceeding 99% as determined by HPLC analysis. Chemicals were purchased as follows: thiazole (la),4-methylthiazole (lb), TBZ (Id), and 2,4dimethylthiazole (lg) from Tokyo Kasei Kogyo Co. (Tokyo, Japan); thioacetamide (2b) from Wako Pure Chemical Industry Co. (Osaka, Japan). 5-Methylthiazole (le) and 4,bdimethylthiazole (10were synthesized according to the method of Kurkjy and Brown (3). 4-tert-Butylthiazole (IC) (4), 2-hydroxy-4methylthiazole(lh)(5),2-methoxy-4-methylthiazole (li)(6),2-@methoxyphenyl)-4-methylthiazole(lj) (7),thioformamide (2a) (8),p-methoxythiobenzamide (2c) (9),2-tert-butylquinoxaline (9) (10),andBSO (11)weresynthesizedaccording to thepublished procedure. Synthesis of 4-(pNitrophenyl)thiazole(8). A solution of thioformamide (2a)(1.0 g, 17 mmol) in EtOH (30mL) was added to a solution of p-nitrophenacyl bromide (2.9 g, 12 mmol) in EtOH (40 mL), and the mixture was heated to reflux for 1 h. EtOH was removed under reduced pressure, and the residue was partitioned between CHC& (100 mL) and HzO (100 mL). The CHC13 extract was dried over NazSOd and concentrated. The residue was chromatographed on silicagel with benzene as eluent. Recrystallization of the isolated product from EtOH gave 1.8 g (73%)of 8: mp 182-183 "C [lit. mp (12) 180 "C]; 'H NMR (60 MHz, CDC13) 6 8.94 (d, J = 1.8 Hz, 1 H, thiazolyl H-2), 8.34 (d, J = 9.0 Hz, 2 H, phenyl H), 8.08 (d, J = 9.0 Hz, 2 H, phenyl H), 7.76 (d, J = 1.8 Hz, 1 H, thiazolyl H-5). Anal. Calcd for
0S93-22S~/93/2706-0~74$04.00/0 0 1993 American Chemical Society
Thioformamide: A Toxic Metabolite of Thiazoles
Chart I. Structures of Compounds Examined for Toxicity
a:
H
b:
H
C:
H
d:
H
E:
CH3
t:
CH3
(I:
H
h:
H
I:
H
I:
H
HS2 N h R 2 a:
R=H
b:
R=W3
C:
R = CeH,-pOCH3
C ~ H ~ O ~ N C, Z S52.42; : H, 2.93; N, 13.58. Found: C, 52.14; H, 3.18; N, 13.54. Animals. Seven-week-oldmale ICR mice were obtained from Charles River Japan, Inc. (Kanagawa, Japan) and acclimatized to our laboratory conditions for 1week before being used. Mice were housed in aluminum boxes on a wood chip bedding (White Flake, Charles River Japan, Inc.) at a constant temperature (23 f 2 "C) and under a 12-h light/dark cycle. Except when stated otherwise,mice received food (Funabashi F-2, Funabashi Farms, Chiba, Japan) and water ad libitum. Assessment of Nephro- and Hepatotoxicity. Mice were treated ip with BSO (4 mmol/kg body wt) in water (20 mL/kg body wt) (8.00-9.00 h). One hour later, the treated animals received PO doses of thiazoles la-j in olive oil (10 mL/kg body wt) or sc doses of thioamides [thioformamide (2a) and thioacetamide (2b) in water (5 mL/kg body wt) and p-methoxythiobenzamide (2c) as a fine suspension in olive oil (5 mL/kg body wt)]. The animals were fasted for 16 h before dosing with test compounds and then for 2 h after administration. Blood was collected by cardiac puncture under pentobarbital anesthesia. The blood was allowed to clot at 37 "C for 1 h, and serum was prepared by centrifugation (9OOg). SUN concentrations and serum alanine aminotransferase (SALT) activities were measured with commercial kits, Urea-NB test Wako and GPT-UV test Wako (Wako Pure Chemical Industries), respectively. Study on the Urinary Metabolites of 4- tert-Butylthiazole (IC). Fifteen mice received IC (2 mmol/kg body wt) in olive oil (3 mL/kg bodywt) by PO intubation and were placed in metabolic cages. The urine was collected for 24 h. The urine sample was centrifuged to remove particulate material and passed through an octadecylsilicacartridge (Bond Elute LRC C18, Varian). The cartridge effluent (25 mL) was subjected to analysis for the presence of thioformamide (2a). The cartridge was eluted with MeOH-H20 (1:l)(5 mL), and the eluate was subjected to analysis for the presence of tert-butylglyoxal (512). (A) Identification of Thioformamide (2a). The effluent from the octadecylsilicacartridge was saturated with (NH4)2S04
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 175 and extracted with ether (4 X 20 mL). The organic extracts were dried over Na2SO4 and added to a solution of p-nitrophenacyl bromide (200 mg) in EtOH (25 mL). After removal of the ether by fractional distillation, the residual solution was heated for 2 h under reflux. The reaction mixture was evaporated to dryness by using Kudema-Danish concentrator and finally with a nitrogen stream. The residue was applied on a silica gel column (silica gel 60,70-230mesh, Merck; 14-mmi.d. x 14.5 cm). The column was washed with 100mL of benzene and then with 20 mL of hexaneether (8:2) and eluted with 50 mL of hexane-ether (82). The eluate (hereafter called derivatized fraction a) was analyzed by GC-MS for the presence of 4-@-nitrophenyl)thiazole (8) derivatized from 2a. (B)Identification of tert-Butylglyoxal(5c). To the eluate from the octadecylsilicacartridge was added o-phenylenediamine (100 mg) in 10 mL of acetic acid, and the mixture was heated at 80 "C for 1h. The mixture was diluted with water (20 mL) and extracted with CHzClz (3 X 20 mL). The organic phase was washed withO.1 N HCl(2 X 25mL) and with 5 7% aqueous NaHC03 (2 X 25 mL), dried over Na2S04, and evaporated. The residue (hereafter called derivatized fraction b) was analyzed by GCMS for the presence of 2-tert-butylquinoxaline (9) derivatized from 5c. GC-MS. Analyses of the derivatized metabolites were carried out by GC-MS using a Model 5890 Series I1 gas chromatograph (Hewlett-Packard, Palo Alto, CA) coupled to a Model JMSSX102A mass spectrometer (JEOL Ltd., Tokyo, Japan). A 0.2mm i.d. X 25-m fused silica capillary column (HP-5, HewlettPackard) was used for GC separations. The injector and the GC-MS interface temperatures were set at 210 and 230 "C, respectively. The samples were injected in the splitless mode with an initial column temperature of 80 "C for 1 min. The column temperature was programmed at a rate of 15 "C/min to 210 "C and then kept at 210 OC for 18 min. The carrier gas was helium with a flow rate of 0.7 mL/min. The mass spectrometer was operated in the electron impact ionization mode and scanned from 50 to 500 amu every 0.5 s with an ionization energy of 70 eV. Statistical Analysis. Data from the SUN concentrationassay were compared by a one-way analysis of variance combined with Dunnett's multiple-range test. Data from the SALT activity assay were analyzed by Kruskal-Wallis nonparametric analysis of variance followed by Mann-Whitney's U test. Differences were considered significant if p < 0.05.
Results Nephrotoxicity of Unsubstituted and 4- and/or 5-Substituted Thiazoles in Mice Depleted of GSH by BSO. Thiazole (la) and several 4- and/or 5-substituted thiazoles (lb-f) were evaluated for their nephrotoxicity. In a previous study, we have shown that the increases in relative kidney weight and SUN concentration in mice treated with TBZ in combination with BSO parallel the severity of tubular necrosis (I). In this study, therefore, changes in SUN concentration a t 24 h after the administration were used as an indication of nephrotoxicity. All the thiazoles (la-f) produced no sign of nephrotoxicity in the absence of BSO pretreatment (datanot shown). When examined in mice depleted of GSH by BSO, all the thiazoles produced marked and dose-dependent increases in SUN concentration. Among the compounds examined, however, there were marked differences in toxic potency based on the dose-effect curves for SUN concentration (Figure 1). The nephrotoxic potentials decreased with various substituents in the following order: unsubstituted (la) > 4-methyl (lb)> 5-methyl (le)> 4-tert-butyl (lc)and4-(2'benzimidazolyl) (Id) > 4,5-dimethyl (lf). Nephro- and Hepatotoxicity of 4-Methylthiazoles Having Different 2-Substituents in Mice Depleted
176 Chem. Res. Toxicol., Vol. 6, No. 2, 1993
Mizutani et al.
2s
'"1
01 0.01
I ! "25
,It,
1 1
176
a
1-
I,,
,
,
,
,
?e
,
-4 149 25
T m (MJ I
I
0.1
1
i
5
?e6
Dose (mmol/kg)
Figure 1. SUN concentration 24 h after administration of different doses of thiazole (la), 4-methylthiazole (lb), 4-tertbutylthiazole (IC), TBZ (Id), 5-methylthiazole (le), or 4,5dimethylthiazole (If) in mice depleted of GSH by BSO. Mice were treated with BSO (4 mmolikg body wt, ip) and received thiazoles (PO) 1 h later. Values, except that for If (1.5mmolikg body wt), represent means f SE from 2-6 mice; treatment with If (1.5 mmol/kg body wt) killed two out of three mice. The dotted area shows the 95% confidence interval for the SUN control values. Table I. SUN Concentration and SALT Activity 24 h after Administration of 2-Substituted 4-Methylthiazoles in Mice Depleted of GSH by Treatment with BSOn dose, SALT, SUN, Karmen mmollkg mg body wt % unitslmL compound 21.6 f 2.3 27.5 f 5.5 control 181 f 53.8* 339 f 96.gb 0.3 4-methylthiazole (lb) 34.0 k 14.2 1690 f 447b 1.5 2,4-dimethylthiazole(lg) 1.5 2-hydroxy-4-methylthiazole (1h) 1.0 2-methoxy-4-methylthiazole (li) 2-(p-methoxyphenyl)-4-methyl- 1.5 thiazole (lj)
J
8
23.2 f 3.5 24.4 f 6.0 53.3 f 16.4 5860 f 658b 16.5 f 1.4 7700 f 3860b
I
Mice were treated withBSO (4mmol/kgbodywt, ip) andreceived thiazoles (PO) 1 h later. Values are means h SE from 4 or 5 mice. * Significantly different from control group ( p < 0.05).
of GSH by BSO. A previous study has shown that 4-methylthiazole (lb) in combination with BSO produces injury in the liver, as evidenced by an increase in SALT activity, as well as in the kidney (2). Therefore, the effects of various 2-substituted 4-methylthiazoles on SUN concentration and SALT activity were compared to those of lb, the reference compound (Table I). lb caused a severe increase (8.4-fold) in SUN concentration and a moderate increase (12-fold) in SALT activity at a dose of 0.3 mmol/ kg body wt. 2,4-Dimethylthiazole (lg) did not produce any change in SUN concentration even at a dose (1.5 mmol/ kg body wt) 5 times higher than that for lb but caused a marked increase (61-fold) in SALT activity. Similarly, 2-methoxy-4-methylthiazole(li) (1.0 mmollkg body wt) and 2-(p-methoxyphenyl)-Cmethylthiazole (lj) (1.5 mmoll kg bodywt) had no effect on SUN concentration but caused marked increases (210-and 280-fold,respectively)in SALT activity. In contrast with these thiazoles, 2-hydroxy-4methylthiazole (lh) (up to 1.5 mmol/kg body wt) was totally ineffective in inducing nephro- or hepatotoxicity as judged by SUN concentration or SALT activity, respectively. Identification of Thioformamide (2a) as a Urinary Metabolite of 4-tert-Butylthiazole (IC). The presence of 2a in the urine of mice dosed with IC was determined by condensing 2a with p-nitrophenacyl bromide, forming 4-@-nitrophenyl)thiazole(8) (Scheme I). This procedure is based on Hantzsch's method for the synthesis of thiazoles
2a
8
(13). The rates of the reaction of phenacyl bromides with thioamides are increased by substituting an electronwithdrawing group in the para position of phenacyl bromide (14). We, therefore, used p-nitrophenacyl bromide as a trapping reagent for 2a. Although no quantitative study was made, pilot experiments with synthetic 2a indicated that the derivatization procedure with p-nitrophenacyl bromide is effective for the isolation and identification of this unstable and low molecular weight metabolite. Panel A in Figure 2 shows the reconstructed ion chromatograms for the urinary fraction (derivatized fraction a) derivatized withp-nitrophenacyl bromide. The total ion chromatogram and single ion traces at mlz 206 and 176 equally showed a peak (M - 1)with a retention time identical with that of the authentic standard, 4-(pnitropheny1)thiazole (8). The mass spectrum of peak M - 1 (Figure 2B) was very similar to that of the authentic sample (Figure 2 0 , with a molecular ion at mlz 206, an M - NO peak a t m/z 176, an M - NO2 peak at m/z 160, an M - NO - CO peak at mlz 148, and an M - NO2 - HCN
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 177
Thioformamide: A Toxic Metabolite of Thiazoles
.. M-2
Table 11. SUN Concentration and SALT Activity 24 h after Administration of Thioamides in Mice Depleted of GSH by Treatment with BSOB dose, SALT, mmol/kg SUN, Karmen compound bodywt mg % units/mL control 21.9 f 0.9 28.6 f 3.7 thioformamide (2a) 2.0 110 f 29.5b 120 f 44.gb thioacetamide (2b) 0.5 35.6 f 6.6 2610 f 580b p-methoxythiobenzamide (2c) 0.5 36.7 f 5.0 5060 f 307b Mice were treated withBSO (4mmoVkgbodywt, ip) andreceived thioamides (sc) 1 h later. Values are means i SE from 5-10 mice. * Significantly different from control group QJ < 0.05). ~
6
I
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8
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188 110 120
,;:
138
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!,
110
158 1 6 8
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, ;a0
, 198 200
,
,
2 1 0 220
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Figure 3. Panel A GC-MS reconstructed ion chromatogram of derivatized urinary extract (derivatized fraction b) from mice dosed with 4-tert-butylthiazole (IC). Panels B and C: Electron impact mass spectra of (B)derivatized metabolite M - 2 and (C) 2-tert-butylquinoxaline (9) derivative of synthetic tert-butylglyoxal (5c).
Scheme 11. Derivatization of tert-Butylglyoxal (5c) with &Phenylenediamine
5c
9
peak at m/z 133. These results thus unambiguously demonstrate the occurrence of thioformamide (2a) in the urine as a metabolite of 4-tert-butylthiazole (IC). Identification of tert-Butylglyoxal (5c) as a Urinary Metabolite of 4-tert-Butylthiazole (IC). The production of 5c as a urinary metabolite of lc was examined by GC-MS after derivatization to its quinoxaline derivative, 2-tert-butylquinoxaline (9) (Scheme 11). Panel A in Figure 3 shows the reconstructed ion chromatograms for the urinary fraction (derivatized fraction b) derivatized with o-phenylenediamine. The total ion chromatogram and selected ion chromatogram at mlz 186 both showed a single peak (M - 2) with a retention time identical with that of authentic 9. The mass spectra of peak M - 2 (Figure 3B) and the authentic sample (Figure 3C) were in good agreement with respect to typical peaks, a molecular ion at mlz 186, an M - CH3 peak at mlz 171, an M - CH3 HCN peak at m/z 144, and an M - C(CH3)3peak at mlz 129. Thus, the formation of tert-butylglyoxal (5c) as a
urinary metabolite of 4-tert-butylthiazole (IC) was confirmed. Nephro- and Hepatotoxicity of Thioamides i n Mice Depleted of GSH by BSO. Thioformamide (2a), thioacetamide (2b),and p-methoxythiobenzamide (2c) were examined for their ability to increase SUN concentration and SALT activity in mice depleted of GSH by treatment with BSO. Table I1 shows the effects of the thioamides at their minimal effective doses. Treatment with 2a produced a marked increase in SUN concentration at a dose of 2.0 mmol/kg body wt. In contrast, SALT activity was only slightly (4.2-fold),but significantly, increased by the same dose of 2a. On the other hand, 2b at a dose as low as 0.5 mmol/kg body w t caused a marked increase (91-fold) in SALT activity but resulted in no change in SUN concentration. Similarly, 2c (0.5mmol/kg body wt) produced a marked increase (180-fold) in SALT activity but had no effect on SUN concentration. In the absence of BSO pretreatment, 2a failed to produce any change in SUN concentration or SALT activity, whereas 2b and 2c produced increases in SALT activity, although the increases were smaller than those observed after treatment in combination with BSO (data not shown).
Discussion In order to gain insight into the chemical nature of the postulated active metabolite of TBZand other nephrotoxic thiazoles, we studied structure-toxicity relations for a number of thiazole derivatives in BSO-pretreated mice. Previously, we have reported in detail on the tissue GSH depletion produced by the BSO pretreatment regimen employed in the present study (I). Briefly, the pretreatment with BSO reduces renal and hepatic GSH concentrations to 33 % and 37 % ,respectively, of the control after 2 h. In the first series of experiments, unsubstituted thiazole (la) and thiazoles (lb-f) with 4- and/or 5-, and no 2-, substituents were equally effective in inducing nephrotoxicity in GSH-depleted mice (Figure 1). Furthermore, the nephrotoxic potency of the thiazoles appears to decrease with the increasing number and bulk of the 4- and/or 5-substituents (Figure 1). Therefore, this sequence of relative toxicity might be the result of the steric substituent effect on the relative rates of obligatory metabolic activation steps. The second series of experiments demonstrates that the type of substituents at the 2-position is critically important in determining both the target organ (the kidney or liver) and the toxic potency of 4-methylthiazoles (Table I). It is, therefore, most likely that a structure including the C-2 moiety of thiazole ring is closely related to the formation of metabolite(s) responsible for the toxicity.
178 Chem. Res. Toxicol., Vol. 6, No. 2, 1993
Mizutani et al.
Scheme 111. Proposed Pathway of Metabolic Activation of Nephrotoxic Thiazoles in Mice r R.
L
la-f
a:
H
H
b:
CH3
H
C:
C(CH&
H
d:
2-benzimidazolyi
H
e:
H
CH3
1 :
CHg
CH3
3a-t
It has been shown that chlormethiazol [4-methyl-5(chloroethyl)thiazolel is metabolized to ita 2-hydroxylated metabolites in rats both in vivo and in vitro (15, 16). However, participation of such a metabolic pathway in the nephrotoxicity of thiazoles presently examined seems unlikely because 2-hydroxy-4-methylthiazole (1h) was nontoxic even at a higher dose (Table I). Moreover, the effectiveness of various 4- and/or 5-substituted thiazoles (lb-f) in producing renal injury (Figure 1) excludes the possibility that direct hydroxylation at the 4- or &position of thiazole ring is involved in the metabolic activation processes. These considerations suggest that a ring cleavage reaction of thiazoles to yield a toxic species including the C-2 unit is involved in the toxicity caused by the nephrotoxic thiazoles. In addition, the well-known toxic properties of various thiono-sulfur compounds such as thioamides and thioureas (17) lead to the idea that some thiono-sulfur-containing compound might be a metabolite responsible for the nephrotoxicity of thiazoles examined in this study. A possible mechanism for the postulated ring cleavage is shown in Scheme 111. Initially, the thiazoles (la-f) may be oxidized by cytochrome P450 enzymes to form an epoxide (3). The enzymatic or nonenzymatic hydrolysis of the epoxide (3) would produce an unstable hemiketallike metabolite (41, which is expected to undergo spontaneous ring cleavage to form an a-dicarbonyl compound (5) and thioformamide (2a). Previous studies on the hepatotoxicity of thioamides, namely, thioacetamide and thiobenzamide, have shown that their oxidative biotransformation, mediated mainly by the flavin-containing monooxygenase, primarily to the S-oxides and then possibly to the S-dioxides is a necessary step in inducing the toxicity (18-20). Therefore, 2a, if formed, would be further biotransformed to its S-oxidized metabolites (6 and 7) before it produces nephrotoxicity. To provide experimental evidence for the postulated metabolic pathways, we chose 4-tert-butylthiazole (IC)as a representative of the nephrotoxic thiazoles and studied its metabolism. When IC was administered to mice, a ring cleavage product, thioformamide (2a), and the accompanying fragment, tert-butylglyoxal (5c), were identified as urinary metabolites after being trapped as 4-@-nitropheny1)thiazole(8) and 2-tert-butylquinoxaline (91, respectively (Figures 2 and 3). Together with these results, the observation that 2a is nephrotoxic to mice depleted of GSH by BSO (Table 11) strengthens our
I
2a
hypothesis (Scheme 111)that this product is a proximate metabolite responsible for the nephrotoxicity of thiazoles. Unexpectedly, 2a, a putative proximate nephrotoxicant generated from the nephrotoxic thiazoles (la-f), was found to be less toxic than most of the parent thiazoles (Figure 1 and Table 11). However, this discrepancy may be ascribed to possible differences between 2a and thiazoles la-f in the extent of excretion and distribution to the target organ because 2a has much lower lipophilicity than the parent thiazoles. Moreover, other lines of evidence are consistent with the proposed mechanism for the nephrotoxicity. First, we have observed that cytochrome P450-dependent monooxygenase inhibitors such as methoxsalen and piperonyl butoxide prevent the nephrotoxicity of TBZ ( 1 ) and 4-methylthiazole (2);these observations suggest that at least one oxidative biotransformation step is required prior to the formation of any toxic thiazole metabolite(s), and, therefore, agree with suggesting the involvement of epoxidation in the toxification processes of the thiazoles. Second, the steric substituent effect observed in the nephrotoxic potency of 4- and/or 5-substituted thiazoles (Figure 1)can be explained on the basis of the steric effect of the substituents on the epoxidation of thiazoles. Increasing substitution in a carbon-carbon double bond is likely to reduce the rate of epoxidation catalyzed by cytochrome P450 enzymes (21). Third, thioacetamide (2b) and p-methoxythiobenzamide (2c) caused severe hepatotoxicity but not nephrotoxicity (Table 11);moreover, 2b (17)and 2c (19)have been reported as hepatotoxicanta in rata. Therefore, if we assume that 2,4-dimethylthiazole (lg) and 2-@-methoxyphenyl)-4-methylthiazole(lj) also undergo ring cleavage to form thioamides 2b and 2c, respectively, by the same mechanism as that proposed for the nephrotoxic thiazoles, the observed hepatotoxicity and the lack of nephrotoxicity of lg and l j (Table I) can reasonably be understood. Some 2-acetylaminothiazoles, e.g., 2-(acetylamino)-4methylthiazole, have been reported to produce acetylthiourea as a minor metabolite in rata (22). This finding indicates the possibility that the cleavage of a thiazole ring in the manner described here for the nephro- and hepatotoxic thiazoles takes place in the metabolism of a wider rage of thiazole compounds. Interestingly, in the present study all the thiazoles (laf) without 2-substituents and their putative metabolite, thioformamide (2a), equally caused toxicity mainly in the
Thioformamide: A Toxic Metabolite of Thiazoles
kidney, while some thiazoles having 2-substituenta, namely, 2,4-dimethylthiazole (lg), 2-@-methoxyphenyl)-4methylthiazole (1j),and 2-methoxy-4-methylthiazole (li), caused toxicity exclusively in the liver. Thioacetamide (2b) and p-methoxythiobenzamide (2c), possible toxic metabolites of l g and lj, respectively, also caused toxicity only in the liver. Previously, we have presented circumstantial evidence that a toxic metabolite responsible for the nephrotoxicity of thiazoles such as TBZ and 4-methylthiazole is generated directly in the kidney but not in the liver (2). However, it is unlikely that the observed differences in target organ selectivity among the compounds arise from differences in organ distribution of the parent compounds because no clear differences in lipophilic and hydrophilic properties exist between the nephrotoxic and hepatotoxiccompounds. Thus, a more possible reason for the different target organ selectivity may reside in a difference in activity between both organs to generate ultimate toxic species from these compounds. It is well-known that GSH is involved in the detoxification of many foreign compounds and that their toxic effects often are enhanced in animals with depressed tissue GSH levels. In general, the enhancement of the toxicity has been considered to result from the inability of the target cells to detoxify the reactive metabolites responsible for cell damage (23). In the present study, thioformamide (2a),as well as ita parent thiazoles, required GSH depletion in producing nephrotoxicity when administered to mice (Table 11). It is therefore likely that GSH plays a protective role toward the nephrotoxic thiazoles by inactivating reactive metabolite(@ generated by further metabolism of thioformamide; this metabolite can attack the cellular constituents to result in the nephrotoxicity in GSHdepleted mice. As mentioned above, the thioamide S-dioxides have been postulated as ultimate toxic metabolites in the hepatotoxicity caused by thioacetamide and thiobenzamide (18-20). In the present case, however, the suggestion on the ultimate toxic species formed via thioformamide (2a) (Scheme 111)is only a tentative one; the exact nature of the ultimate nephrotoxicant and the chemical mechanism of its interaction with GSH remain to be elucidated. Acknowledgment. We thank Takako Iguchi, Tom0 Ikeda, Sachiko Kinefuchi, and Michiko Ishihara for their excellent technical assistance. References Mizutani, T., Ito, K., Nomura, H., and Nakanishi, K. (1990) Nephrotoxicity of thiabendazole in mice depleted of glutathione by treatment with DL-buthioninesulphoximine. Food Chem. Toricol. 28,169-177. Mizutani, T., Yoshida, K., and Ito, K. (1992) Nephrotoxicity of thiazoles structurally related to thiabendazole in mice depleted of
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