Chem. Res. Toxicol. 1992,5, 726-733
726
Metabolism of Benzimidazoline-2-thionesby Rat Hepatic Microsomes and Hog Liver Flavin-Containing Monooxygenase Caroline J. Decker,+Daniel R. Doerge,*J and John. R. Cashmans Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709, National Center for Toxicological Research, Jefferson, Arkansas 72079, and Department of Pharmaceutical Chemistry, University of California, S a n Francisco, California 94143 Received May 1 I , 1992
The metabolism of benzimidazoline-2-thione(I) and the l-methyl(I1) and 1,3-dimethyl(III) derivatives was studied to elucidate the mechanisms of hepatic oxidation for this class of thionosulfur-containing xenobiotics. NADPH-dependent metabolism of I, 11, and I11 to the corresponding benzimidazoles Ia, IIa, and IIIa, respectively, was observed in dexamethasonepretreated rat hepatic microsomes. I11 was the only thiocarbamide converted to an amide metabolite (IIIb). The effects of heat and l-aminobenzotriazole pretreatment suggested that rat hepatic microsomal metabolism of I was catalyzed by the flavin-containing monoxygenase (FMO) only and that of I1 and I11by both FMO and cytochrome P450 isozymes (P450). Addition of 5.0 mM glutathione (GSH) blocked formation of all metabolites from I, 11, and 111. Highly purified hog liver FMO catalyzed formation of all metabolites observed in rat hepatic microsomal systems. Incubation of I11 with either rat liver microsomes or with highly purified hog liver FMO in the presence of [lsO1water led t o ca. 50% incorporation of [1803into IIIb. When [‘*O] molecular oxygen was used, ca. 8% incorporation of [lsO1 into IIIb was observed. Highly purified hog liver FMO also converted 1-111 to chemically reactive species that covalently bound to protein thiols. In the presence of hog liver FMO, the covalent binding pattern of radiolabeled 1-111 to bovine serum albumin was essentially identical to that observed for rat hepatic microsomes. The formation of IIIb and concomitant extrusion of the sulfur moiety of the molecule to species that covalently bind to microsomal proteins suggested a novel oxidation mechanism for I11 by both microsomal monooxygenases in which sulfur monoxide is a reactive species produced. While rat liver microsomal FMO alone catalyzed the metabolism of I, these studies suggested that P450 and FMO catalyzed formation of the same reactive intermediates from I1 and 111. Apparently, the reactivity of the S-oxygenated species is a major determinant in the ultimate formation of products and covalent binding t o proteins. The results of these studies are inconsistent with a general role for the formation of atomic sulfur as a reactive species and hydrodisulfide protein adducts during microsomal metabolism of thiocarbamides.
Introduction Organosulfur compounds including benzimidazoline2-thiones are substrates for oxidation by the hepatic microsomal monoxygenases, P4501 isozymes, and FMO (1-4). For some organosulfur compounds, oxidation results in the formation of reactive S-oxygenated intermediates that can initiate hepatotoxicity and other organspecific toxicities including inhibition of thyroid hormone synthesis (2, 3, 5 ) . However, incomplete information is available concerning the chemical and biochemical mechanisms of thiocarbamide S-oxygenation in hepatic microsomes. Thionosulfur compounds are metabolized by P450 to species that apparently fragment to form atomic sulfur (5-7). It is the covalent binding of this reactive species to proteins that is thought to initiate liver damage Corresponding author [tel (501) 543-7943, FAX (501) 543-71361. Burroughs Wellcome Co. t National Center for Toxicological Research. University of California. 1 Abbreviations: ABT, l-aminobenzotriazole; BSA, bovine serum albumin; DEX, dexamethasone; ETU, ethylenethiourea; FMO, flavincontaining monooxygenase; GSH, glutathione; GSSG, oxidized glutathione; IBSA, iodoacetamide-treated BSA; LC/MS, liquid chromatography/” spectrometry; LOD, limit of detection; LSC, liquid scintillation or methimazole; P450, counting; MMI, l-methylimidazoline-2-thione cytochrome P450 isozymes; TLC, thin-layer chromatography. +
and inactivate P450 (5). For example, MMI is an important drug used in anti-hyperthyroid therapy, and its use is sometimes limited by hepatotoxicity. It has been reported that MMI is metabolized by P450 and FMO to intermediates which bind covalently to microsomal proteins and inactivate cytochrome P450 enzymes (5-8). ETU is an impurity and metabolite from ethylenebis(dithi0carbamate) fungicides which produces thyroid and liver tumors in rodents (9). Hepatic microsomal FMO-catalyzed metabolism of [l4C1ETU results in covalent binding to microsomal proteins with the concomitant inactivation of P450 isozymes (2). Previous studies from this laboratory employed three pairs of [35S] and [“C]benzimidazoline-2-thionesto examine hepatic microsomal activation and covalent binding to microsomal proteins (3). The series of benzimidazoline-2-thiones (i.e., 1-111) was selected to systematically probe structure-reactivity differences in compounds that could be readily labeled with radioisotopes and detected by LC/UV. The present study was undertaken to identify the metabolites of these thiocarbamides in the presence of rat hepatic microsomalor highly purified hog liver FMO systems to understand the metabolism of these compounds in view of the P450-mediated oxidative 1992 American Chemical Society
Hepatic Metabolism of Thiocarbamides
desulfuration reactions proposed in earlier reports (5-7). In addition, possible differences between products from P450-catalyzed vs FMO-catalyzed reactions were investigated since P450 can effect one-electron oxidations while FMO catalyzes only two-electron oxygenations.
Materials and Methods Chemicals. GSH, DEX, NADPH, and catalase were purchased from Sigma Chemical Co. (St. Louis, MO). ABT was synthesized as previously described (2). Syntheses of unlabeled 1-111 and their metabolites were performed as previously described (3). The products were purified by recrystallization or preparative TLC on 1-mm silica Uniplates (Analtech, Newark, DE) using 5 7% methanol in chloroform as the developing solvent. All structural assignment structures were confirmed by electron impact particle beam LC/MS as described below. Rat Liver Microsomes and Hog Liver FMO Preparation. Male Sprague-Dawleyrats (220-25Og) were obtained from Bantin Kingman Labs (Gilroy, CA) and were pretreated with DEX (3). Microsomes were prepared as described previously (2). Hog liver FMO was purified chromatographically to homogeneity (195% ) as described in ref 10, and the protein content was determined by the BCA method ( 1 1 ) . The preparation contained no detectable P450 (heme absorbance or P450 peptide sequences, data not shown). FMO-dependent oxidation of MMI (2) was determined to be 1178 nmol/(mg of proteimmin). Covalent Binding. Incubations to assess the FMO-dependent covalent binding of thiocarbamides to BSA or IBSA (1.0 mL) contained 23 pg of FMO, 2.0 mM NADPH, 1.0 mM thiocarbamide (0.38-1.0 pCi/incubation), and 3.6 mg of BSA or IBSA in 50 mM phosphate buffer (pH 8.3). The specific activities for 1-111 (1-3 mCi/mmol) were determined by LC/UV and LSC, and purity was determined by TLC prior to use. Methods for the synthesis of [ W ] - and [35S]-I and -11 were reported previously (12). Synthesis of [W]-and [14C]-IIIwas performed using [Wlthiourea and [Wlmethyl iodide, respectively (Amersham Corp., Clearbrook, IL),with minor modifications from the methods described for I and I1 (3). Covalent binding studies were conducted for 30 min at 33 OC and were terminated by the addition of 3 volumes of 5% H2S04in methanol. IBSA was prepared and covalent binding determined as previously described (3). Metabolism Studies. Solutions containing 2.0 mg/mL DEXpretreated microsomal protein, 1.0 mM thiocarbamide, 1.5 mM diethylenetetraminepentaaceticacid, kl.0 mM NADPH, and f5.0 mM GSH in 0.1 M phosphate buffer (pH 7.4) in a total volume of 1.0 mL were incubated for 10 min at 37 "C. Formation of all metabolites was found to be linear to this time point. Hepatic microsomesfrom rats pretreated with DEX were chosen because of their greater propensity to effect S-oxygenation of thiocarbamides (2, 3). In some cases rat liver microsomes were preincubated with 3.0 mM ABT and 1.0 mM NADPH for 15 min and then centrifuged to recover the microsomes. This process led to 60-95 % loss of both erythromycin N-demethylase activity and microsomalheme content from DEX-pretreated microsomes (2). Microsomeswere heated at 37 "C for 1h prior to incubation with benzimidazoline-2-thionesto inactivate FMO preferentially. Under these conditions, FMO activity in untreated rat liver microsomes was decreased by 95% while the P450 activity of DEX-pretreated rat liver microsomes was decreased by 29% (2, 3). Incubations with I, 11,and I11 were terminated by the addition of 2.0 mL of ethyl acetate. Two subsequent ethyl acetate extractions (2.0 mL) were performed, the fractions were pooled and evaporated under nitrogen, and the residue was dissolved in 0.5 mL of methanol and filtered (0.45 pm) for LC analysis (10 pL). The extraction efficiency of known amounts of Ib, IIb, and IIIb from the microsomalincubations was determined to be 96 % , 9676, and 83%, respectively. The extraction efficiency for Ia and IIa was 99% and 101% , respectively. The concentration of IIIa and sulfate ion was determined using LC (10-pL aliquots) following centrifugation (1050oOg)of microsomal incubations.
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 727 The recovery of known amounts of IIIa by this procedure was determined to be 96% LC analysis was performed usinga Perkin-Elmer 410 LC pump and a NovaPak C18 column (4-pm X 12-cmRadialPak cartridge, Waters Associates, Milford, MA) with a mobile phase consisting of 20% acetonitrile in water for Ib, 30% acetonitrile in water for IIb, and 40% acetonitrile in water for IIIb at a flow rate of 1.5 mL/min with UV detection at 284 nm. Iaand IIa were quantified using the same column with a mobile phase of 25% acetonitrile and 0.01 % triethylamine in water, a flow rate 1.5 mL/min, and UV detection a t 254 nm. Analysis of IIIa was performed using an OmniPac PCX-500 column (Dionex Corp., Sunnyvale, CA) with a mobile phase consisting of 50 mM ethylenediamine, 100 mM KCl, and 8% acetonitrile in water at a flow rate of 1.3 mL/ min with UV detection a t 270 nm. All thiocarbamide metabolites were quantified by comparison of peak heights with those from known amounts of authentic synthetic standards. Product identity of Ia, IIa, and IIIb was confirmed using online particle beam LC/MS using a VG Trio 2A mass spectrometer equipped with a LINC interface (VG BioTech, Altrincham, U.K.) using the same mobile phases and column as described above a t a flow rate of 0.5 mL/min. The temperature of the gas flow through the desolvation chamber was maintained at 30 "C, the source temperature was 200 "C, and typical operating pressures mbar a t the first-stage momentum were 3, 0.8, and 3 X separator pump, the second-stage momentum separator pump, and the ion source housing, respectively. Full-scan spectra were obtained using electron impact conditions (m/z 70-300 in 1 s ) . Hog liver FMO incubations (0.25mL) contained 39 pg of protein f2.0 mM NADPH, and h5.0 mM GSH in 50 mM phosphate buffer (pH 8.3). Incubations were carried out for 10 min at 33 "C and were terminated by extraction with ethyl acetate for Ia, IIa, and all amide products. For the analysis of IIIa, the reaction was stopped in a 50 "C water bath. The reaction mixture was then centrifuged through a 30-kDa cutoff membrane filter to remove protein prior to LC analysis of IIIa or sulfate ion. Sulfate ion concentrations were determined before and after hydrogen peroxide addition (0.1 M final concentration) to microsomalsupernatants or FMO incubations. Incubations were conducted as described above for 20 min in the presence of 10 mM mannitol and analyzed after passing samples through a octadecylsilyl solid-phase extraction cartridge (J. T. Baker Co., Phillipsburg, NJ). Sulfate ion was quantitated by ion chromatography using a Dionex AS4A SC anion-exchange column, a Dionex gradient LC, and suppressed ion conductivity detection. The mobile phase (2.0 mL/min) was 1.8 mM Na2C03 and 1.7 mM NaHC03, pH 10, and an anion membrane suppressor system (Dionex AMMS-1) was used. The regenerant solution was 32.4 mM H2SO4 applied at 2.0 mL/min. Peaks were quantified by comparing peak heights with those from injections of known amounts of sulfuric acid. Bisulfite ion analysis was performed using ion exclusion chromatography with amperometric detection as described above using an HPICE-AS4 column (Dionex Corp.) with 20 mM sulfuric acid as mobile phase at 0.9 mL,"in and a platinum working electrode (13). The LOD for bisulfite ion in aqueous standards was 25 pM. [lSO] Incorporation Studies. The incorporation of [l*O] from water and dioxygen was studied in incubations at 34-36 "C containing microsomes from DEX-pretreated rats or highly purified hog liver FMO as described above using 0.25-mL total reaction volume and a 30-min incubation. The substrate I11 was purified by preparative TLC immediately before incubations because of the slow conversion of I11 to IIIb observed in the solid state. Incubations with labeled water were performed at 50% atom excess ['SO] by diluting 98.9 % atom excess [180]water (Isotec Corp., Miamisburg, OH). Incubations with labeled dioxygen were performed using a Firestone valve (Aldrich Chemical Co., Milwaukee, WI) to alternatively evacuate and recharge the gas phase above a well-stirredsolution at 34-36 "C. Labeled dioxygen (98.4% atom excess,Isotec Corp.) was admitted into the reaction flask after five pump, nitrogen purging cycles. After a I
Decker et al.
728 Chem. Res. Toxicol., Vol. 5, No. 5, 1992
0-NADPH =+NADPH
"1
d A R T /I
0
Figure 1. Formation of Ia, IIa, IIIa, and IIIb by rat hepatic microsomes. Incubations were conducted as described in the Materials and Methods section using hepatic microsomes from DEX-pretreated rats. Ordinate values represent nmol of product formed in a 10-minincubationin 1-mLtotal volume. Data points are means f standard deviations of duplicatedeterminationsfrom two individual experiments (n = 4) with standard deviation error bars.
20-min period,the reaction was cooled on ice and then evacuated and recharged with nitrogen prior to solvent extraction. Concentrationand chromatography of the extracts from the metabolic incubations were performed as described above. Control experimentswith I11conducted in the absence of NADPH showed no formation of IIIb or any other product. Full-scan mass spectra were collected, and the areas under mass chromatograms for M+ and (M + 2)+ ions of IIIb (m/z 162 and 164, product respectively) were used to determine the ratio of formed. Percent incorporation was calculated from the following relationship: % incorporation = area(m/z 164)/[area(m/z 162) + area(m/z 164)]. Incorporationresults were normalized to the maximum incorporation possible on the basis of the 50% [l8O1water content or 98.4% [Wldioxygen content. Enzymatic formation of IIIb in [l6O]water and dioxygen gave less than 2% excess (M + 2)+ion, so this value wastaken asthe limit of detection for incorporation. The stability of [1'3O]-IIIb in exchange reactionswith [Wlwater and oxygenwas determined in complete enzymatic incubations (+NADPH). Complete enzymatic incubations containing I11 in which oxygen was replaced with dry nitrogen yielded negligible quantities of IIIb.
Results Rat Hepatic Microsomal Metabolism. Benzimidazole products (Ia, IIa, and IIIa) were formed in incubations of the thiocarbamides I, 11, and 111,respectively, with rat hepatic microsomes (Figure 1). Formation of Ia, IIa, and IIIa was completely blocked by addition of GSH. Heat treatment of microsomesunder conditions that inactivated FMO blocked Ia formation completely, but only partially attenuated IIa and IIIa formation. Pretreatment of microsomes with ABT and NADPH (to inactivate P450) attenuated IIa and IIIa production, but had no effect on formation of Ia. Formation of amide metabolites (Ib, IIb, and IIIb) was monitored in rat hepatic microsomes. Formation of IIIb was observed after a 10-min incubation, but neither Ib nor IIb was detected (LOD = 0.1 pM). Formation of IIIb was linearly dependent on time through at least 10 min
(data not shown). After 30 min, however, a small amount of Ib and IIb was observed from incubations with I and 11, respectively. In control experiments, incubation of microsomes and NADPH with low concentrations of Ia or IIa (25pM) produced Ib and IIb, respectively. Consistent with these data was the possibility that the Ib and IIb observed at later time points were produced via P450mediated oxidation of initially formed Ia and IIa, respectively. The observation of relatively high amounts of Ia and IIa at earlier time points and the complete inhibition of IIb formed from IIa by prior P450 inactivation with ABT (data not shown) supported this hypothesis. When microsomes were incubated with NADPH and IIIa, no formation of IIIb was observed. Heat inactivation or pretreatment of microsomes with ABT decreased IIIb formation, and addition of GSH totally blocked it (see Figure 1). FMO-Dependent Covalent Binding of Thiocarbamides to BSA. Previous studies demonstrated that rat hepatic microsomes converted 1-111 into species which bound covalently to BSA sulfhydryls (3). Radiolabeled thiocarbamides 1-111 were incubated with purified hog liver FMO in the presence of BSA, and covalent binding to protein was determined. Highly purified hog liver FMOdependent activation of 1-111 produced covalent binding patterns nearly identical to those observed with rat liver microsomes (see Figure 2). FMO Metabolism Studies. Metabolism of 1-111 by highly purified hog liver FMO produced a similar profile of metabolites to that observed in rat hepatic microsomal incubations (seeTable I). However, the amount of purified hog liver FMO-catalyzed benzimidazole formation (IIa > IIIa > Ia) was elevated relative to that observed in rat hepatic microsomes. Production of IIIbwas also catalyzed by highly purified hog liver FMO and occurred at levels comparable to those observed using rat hepatic microsomes. No formation of Ib or IIb was observed during incubations of I or I1 with highly purified hog liver FMO.
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 729
Hepatic Metabolism of Thiocarbamides 0 -NADPH m+NADPH
Figure 2. Highly purified hog liver FMO-mediated covalent binding of [14C]-I, [IC]-11, or [35S]-III to BSA or IBSA. Incubations were conducted a described in the Materials and Methods section, and the values represent the mean of two determinations. Table I. Formation of Thiocarbamide Metabolites by Highly Purified Hog Liver FMO. product formedb incubation condition Ia IIa IIIa IIIb 2.68 f 0.31 0.66 i 1.13 7.86i 0.62 -NADPH 1.79 i 0.93 21.0 f 2.9 151 i 7.9 113 i 21 24.5 f 9.9 +NADPH +NADPH+ GSH
2.14
2.20
0
5.48
Incubations were conducted as described in the Materials and Methods section. The product formed (rM product formed by 10min incubations in 1.0-mL total volume) represents the mean & standard deviation of three determinations, or one determination for the GSH data. b Product formation was determined by the LC method described in the Materials and Methods section.
Determination of Inorganic Sulfur Species. The formation of sulfate ion in incubations containing rat hepatic microsomes or highly purified hog liver FMO was measured after centrifugation (microsomes) or filtration (FMO) using ion chromatography. However,the presence of NADPH and other incubation components interfered with determination of bisulfite ion by colorimetric or ion exclusion LC/electrochemical detection methods. There fore, bisulfite ion was measured indirectly by determining sulfate ion before and after addition of hydrogen peroxide (100 mM final concentration) to reaction mixtures. Accordingly, the reaction was terminated by passing the solution through a Cl8 solid-phaseextraction cartridge to remove unreacted 1-111. Hydrogen peroxide wa8 then added to convert bisulfite to sulfate ion. This procedure quantitatively converted bisulfite to sulfate ion in control experiments. It is likely that this process also oxidized any intermediates present. Incubation of I11 with rat liver microsomes resulted in an NADPH-dependent increase in sulfate ion concentration concomitant to formation of IIIa and IIIb (see Table I1 for relative stoichiometries). Addition of hydrogen peroxide to these samples produced no increase in sulfate ion concentration. Incubation of I11 with hog liver FMO also produced an NADPH-dependent increase in sulfate ion concentration. No sulfite ion was detected in incubations of [36Sl-IIIand rat liver microsomes or highly purified hog liver FMO by monitoring radioactivity in ion exclusion chromatography fractions. Analysis of incubations containing I or I1 with rat liver microsomes or highly purified hog liver FMO showed the presence of negligible amounts of sulfate ion. However, when purified hog liver FMO incubationswere supplanted with hydrogen peroxide, sulfate ion was detected.
Table 11. Metabolite Stoichiometry for Rat Hepatic Microsomal and Highly Purified Hog Liver FMO Incubations with Benzimidazoline-2-thiones incubation relative product stoichiometv condition benzimidazole amideb bisulfite sulfate microsomes + I 1.0 N P ND ND FMO + I 1.0 ND 0.71 1.61 microsomes + I1 1.0 ND ND ND FMO + I1 1.0 ND 0.20 0.08 microsomes + I11 1.0 0.70 ND 0.78 FMO + I11 1.0 0.20 ND 0.62 a The stoichiometry of metabolites was normalized with respect to Ia, IIa, or IIIa from the values contained in Figure 1 or Table I. b Formation of Ib, IIb, or IIIb. Not detected.
Table 111. Incorporation of [180] into IIIb* incubation conditions microsomes heated microsomes purified FMO IIIb control N2 control
% [I801 incorporationb '802 H2'80 49.8 56.9 52.5
* 5.6
2.5 not detectedc
7.2 & 2.7 8.9
6.3 1.8 not detected
0 I11 was incubated with rat hepatic microsomes or with highly purified hog liver FMO, and IIIb formation was determined as described in the Materials and Methods section. b Incorporation of ["%I] into IIIb was determined from the M+ and (M + 2)+ ions and the percent oxygen incorporation determined by normalizing to the [1801 content in water or molecular oxygen present in the incubation medium. Control incubations of I11 with rat liver microsomes in the presence of NADPH but with a nitrogen atmosphere produced negligible amounts of IIIb.
[1 8 0 ] Incorporation Studies. The incorporation of
[ W I from either dioxygen or water into IIIb was measured to identify the source of the amide oxygen in separate experiments. Approximately 50% of oxygen in IIIb was derived from [l801water and 8% from molecular oxygen. These levels of incorporation were essentially identical in (a) untreated microsomes, (b) heat-pretreated microsomes which selected for P450-catalyzed reactions, and (c) purified hog liver FMO (see Table 111). The concentration of IIIb produced was similar in all such incubations.
Discussion Monooxygenase-MediatedMetabolism of Benzimidazoline-2-thiones. Incubations of rat liver microsomes or highly purified hog liver FMO with I or I1 yielded a single product containing the benzimidazole nucleus (Ia or IIa). The rate of IIa formation catalyzed by purified hog liver FMO was approximately 6-fold higher than that for Ia (Table I). Rat liver microsomes also catalyzed formation of IIa at a faster rate than Ia (ca. 25% faster). The higher rate of oxidative metabolism for I1 to IIa may in part explain the lower levels of covalent binding of [W]I1 to microsomal proteins and BSA (see Figure 2 and ref 3). Incubations containing purified hog liver FMO with I and I1 produced bisulfite ion as the primary sulfurcontaining product. The small amounts of sulfate ion observed are consistent with the nonspecific oxidation of bisulfite to sulfate ion under the incubation/chromatography conditions. No sulfur-containing product from I and I1 was detected in rat liver microsomal incubations presumably because of the more complex nature of the biological matrix and the lower level of total products formed.
730 Chem. Res. Toricol., Vol. 5 , No. 5, 1992
Decker et al.
Scheme I. Proposed Mechanisms for Monooxygenase-Mediated Metabolism of I and I1 FMO NH
OH
NH
r-so
-I
OH
I-so2
1 -
NH
[I4C]AND [35Sl PROTEIN BINDING
Ia -
HS03
2 P450
ONy N
I
11 CH3 -
IIa cH3
FMO,
E O
N
OH
I
CH3
s=o
-
OH
I
II-SO? -
CH3
[35S]PROTEIN BINDING
N
Incubation of rat liver microsomes or highly purified hog liver FMO with I11 yielded 1,3-dimethylbenzimidazolium ion (IIIa) and the corresponding amide (IIIb). Sulfate ion was the sole sulfur-containing product identified. The relative stoichiometries for metabolite formation from 1-111 are shown in Table 11. Both enzyme systems yielded IIIb labeled to the same extent when [1803water or [180]molecular oxygen is used. Purified hog liver FMO effected covalent binding of radiolabeled 1-111 to BSA cysteine groups (Figure 2) similar to that previously observed in rat hepatic microsomal preparations (3). Therefore, both rat hepatic microsomal P450 and highly purified hog liver FMO effect the oxidative desulfuration of I11 to IIIa and IIIb with concomitant covalent binding to protein thiols. These findings give strong support to the hypothesis that both enzymes catalyze the formation of common reactive intermediates from 111. It is, therefore, the reactivity of the S-oxygenated intermediate, and not the differences in oxidative enzymatic mechanisms, that determines the nature of the reaction products (14-16). This is an important point since it is well appreciated that P450-catalyzed reactions are initiated by one-electron transfer from the oxoferryl heme as opposed to FMO which catalyzes a two-electron oxygen transfer from the hydroperoxyflavin (4, 14-16). The formation of metabolites from 1-111 catalyzed by rat liver microsomes and highly purified hog liver FMO was totally blocked by the addition of GSH to the reaction mixtures. This was accompanied by concomitant oxidation of GSH to GSSG (data not shown). Previous work showed that negligible NADPH-dependent oxidation of GSH occurs in the presence of microsomes (2, 3). This strongly suggests the involvement of diffusible reactive intermediates in all reaction pathways that are trapped by thiols. The probable reactive intermediate is the S-oxide (e.g., I-SO, 11-SO,11140)because previous studies demonstrated the facile reduction by thiols of thioamide S-oxides in vitro (17). Oxidative metabolism of ETU effected GSH oxidation in vitro (21,and MMI metabolism in vivo resulted in increased efflux of GSSG (18). These observations underscore the potential for GSH to detox-
ify thiocarbamide metabolites as well as the potential for high doses of thiocarbamides to effect GSH depletion in organs with significant monooxygenase activity. Monooxygenase-Mediated Metabolism of I. The prominent effect of heat inactivation and lack of effect of ABT pretreatment on the rat hepatic microsomal metabolism of I to Ia suggests that FMO-mediated Soxygenation initiates product formation. In previously published experiments using rat liver microsomes, similar ABT and heat pretreatment effects were observed on the covalent binding of radiolabeled I (3). In addition, equal binding of P4C1and [35S] portions of I was observed and equal amounts of bound [l4C1and 135S]were released by thiophilic reagents. It waa concluded that the binding of I to protein thiols resulted from the reaction of an FMOproduced sulfenic acid metabolite (see Scheme I). The metabolism and binding of I observed with either rat liver microsomes or purified hog liver FMO are consistent with the initial formation of a sulfenic acid (see Scheme I). Sulfenic acid formation has been proposed for rat liver microsomal metabolism of ETU (2) and the metabolism of deacetylated spironolactone by rat liver microsomes and purified hog liver FMO (15). No amide desulfuration binding product (Ib) was observed and no excess [36S] occurred. We conclude that although the sulfenic acid I-SO is sufficiently relative to covalently modify protein thiol groups, it is sufficiently stable to be oxidized again by FMO to I-SO2, which then decomposes to Ia and sulfite ion. Monooxygenase-Mediated Metabolism of 11. The effects of heat and ABT pretreatment on rat hepatic microsomal metabolism of I1 are consistent with a role for both P450 and FMO. This conclusion was also reached for the covalent binding of radiolabeled I1 to microsomal proteins (3). Highly purified hog liver FMO also catalyzes the formation IIa (Table I) and effects similar levels of covalent binding to BSA as detected in rat liver microsomes (see Figure 2 and ref 3). These results are also consistent with the proposed formation of sulfenic acids in hepatic microsomes (2, 3, 15).
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 731
Hepatic Metabolism of Thiocarbamides
Scheme 11. Proposed Mechanisms for Monooxygenase-Mediated Metabolism of I11 CH3
, I11 -
III-so -
CH3
CH3 PROTEIN BINDING
J CH3 N+
o n N
CH3 V -
HS04
vr H+I
CH3
Aslight excess in the covalent binding of [35Sl-vs [l4C1I1to microsomal proteins was previouslyreported (3).This observation could be consistent with the formation and reactions of singlet atomic sulfur, as previously proposed for the metabolism of a phosphorothionate (parathion), carbon disulfide, and the thiocarbamides, MMI and naphthylthiourea (5). The microsomal metabolism of parathion, carbon disulfide, and naphthylthiourea was reported to yield the corresponding oxygen-containing desulfurated products: paraoxon, carbonyl sulfide, and naphthylurea (5). For these substrates, a mechanism of singlet atomic sulfur extrusion from an oxathiirane-like intermediate was proposed. However, MMI did not give the analogous oxygen-containing product but, instead, N-methylimidazole as the desulfuration product (7). In this case, a mechanism of singlet atomic sulfur formation via disproportionation of sulfur monoxide was proposed (5). However, the formation of hydrodisulfide adducts (RSSH) between atomic sulfur and protein thiols was proposed for all the above compounds. In agreement with previous reports for the microsomal oxidation of MMI (3, the metabolism of I1 produced none of the amide desulfuration product, IIb (LOD0.1 pM), in rat liver microsomes or highly purified hog liver FMO incubations. This finding is also inconsistent with an oxathiirane mechanism. The slight excess [35Slbinding of I1 previously observed (3)was too low to demonstrate release by cyanide ion (data not shown). This suggested that single atomic sulfur addition to thiols to form hydrodisulfide adducts was minimal for 11. Indeed, most of the covalent binding of [35Sl-IIwas consistent with the reactions of the sulfenic acid metabolite (11430;see Scheme I). The lack of measurable hydrodisulfide adduct formation observed here differs from previous results with MMI (7)where it was concluded that ca. 50% of the [35Sl binding was due to atomic sulfur. A possible source of this difference may be the content of FMO present in the microsomal samples examined. In the present study, significant effort was employed to preserve high levels of FMO. The fact that purified FMO and microsomes produce similar metabolites and covalent binding in the presence of I1 is also inconsistent with an oxathiirane mechanism.
vn 02
I CH3
[35S]PROTEIN BINDING
IIIb -
N
CH3
IIIa CH3 -
It has been reported that carbon disulfide and parathion are converted by P450 catalysis to atomic sulfur via an oxathiirane (phosphoxathiirane) intermediate (5). However, carbon disulfide and parathion are not substrates for purified FMO (4). Oxidative desulfuration mediated by FMO is only known for phosphonate compounds (e.g., fonofos), and for these compounds, the putative site of oxygenation is phosphorus and not sulfur (19). Furthermore, the S-oxygenation of aromatic thioureas and MMI to sulfinic acids is catalyzed by hog liver FMO ( 1 , 4 ) ,and we find evidence for sulfinic acid formation (14302 and II-SO2) from I and I1 as discussed below. These observations strongly suggest that oxidative desulfuration of I1 does not proceed via an oxathiirane intermediate. Monooxygenase-Mediated Metabolism of 111. Metabolism of I11in rat liver microsomes is catalyzed by both P450 (Figure 1)and FMO and results in ca. 5-fold excess [35Slover [14C] covalent binding to rat liver microsomal proteins (3). Moreover, I11was unique in the present study since metabolism by rat liver microsomes and highly purified hog liver FMO produced the corresponding amide desulfuration product, IIIb. An oxathiirane mechanism predicts excess [35S]binding and the formation of the amide desulfuration product (cf. IV, Scheme 11). However, identical metabolic products, including IIIb (see Table I), and covalent binding of -111to BSA thiols are observed in incubations with rat liver microsomes (3) and highly purified hog liver FMO (Figure 2). This is evidence against a P450-specific desulfuration mechanism as discussed above. Incubation of I11 with rat liver microsomes or highly purified hog liver FMO yielded IIIb in which 8% of the oxygen was derived from dioxygen but >50 % was derived from water. The low level of incorporation from molecular oxygen into IIIb also suggests that the involvement of an oxathiirane mechanism for I11 is minimal. This is in contrast to the P450-mediated oxygenation of parathion by rat liver microsomes, which yielded essentially stoichiometric incorporation of ['801 from molecular oxygen into paraoxon (5). That result gave strong support for the formation of atomic sulfur via the phosphoxathiirane mechanism (5). However, microsomal oxidation of carbon disulfide produced carbonyl sulfide in which all the oxygen
732 Chem. Res. Toricol., Vol. 5,No.5, 1992
atoms were derived from water (20). The incorporation of ["303from water into carbonyl sulfide was inconsistent with an oxathiirane mechanism because carbonyl sulfide did not exchange with solvent. Unfortunately, the mechanism of this reaction has not been further elaborated. The low level of [la01 incorporation into IIIb from labeled dioxygen relative to that from labeled water could also be consistent with rapid exchange of water into a reactive intermediate (e.g., 111-SO)or a hydrolytic reaction since IIIb itself is stable to the reaction/isolation conditions employed. The hydrolytic reactivity of intermediates like 111-SOis unknown, but [la01 thiobenzamide-S-oxide was stable to [ l a 0 3 exchange under similar conditions (21). Hydrolysis of the sulfonyl betaine V by addition of water to C-2 can be eliminated as a possible reaction because this reaction would yield sulfite ion. Hydrolysis of 111-SO may be possible, but product analysis and literature precedent do not support this possibility. The lack of quantitative ['80] incorporation into IIIb (water + dioxygen = 60%, see Table 111) could be the result of [1801 isotope effects on the hydrolysis or exchange of an intermediate like 111-SO. We conclude that singlet atomic sulfur binding alone cannot adequately account for the covalent binding of P5S]-111. Evidence for multiple desulfuration reactions is that the amount of incorporation of dioxygen into IIIb (8% X 20 nmol = 1.6 nmol) is lower than the amount of excess [35S]binding (ca. 10 nmol, ref 3). The binding of atomic sulfur to protein thiols yields hydrodisulfide adducts as previously inferred by measuring the cyanide ion-mediated release of bound [35S]as thiocyanate ion (5). In our previous study (3),cyanide ion was ineffective in releasing bound radiolabel from microsomal proteins following incubations with [35Sl-III (
