Chem. Res. Toxicol. 1992,5, 791-796
79 1
Oxidative Conversion by Rat Liver Microsomes of 2-Naphthyl Isothiocyanate to %NaphthylIsocyanate, a Genotoxicant Mei-Sie Lee Department of Chemical Carcinogenesis, Michigan Cancer Foundation, 110 East Warren Avenue, Detroit, Michigan 48201 Received May 11,1992
The present study investigated the oxidative metabolism of 2-naphthyl isothiocyanate catalyzed by rat liver microsomes. Incubation of 2-naphthyl isothiocyanate, microsomes, and NADPH yielded either N,”-di-2-naphthylurea or, on inclusion of 2-aminofluorene in the incubations, N-2-naphthyl-N’-2-fluorenylurea.These ureas were formed by the production of 2-naphthyl isocyanate, which reacted with its hydrolysis product, 2-aminonaphthalene, to yield the symmetrical urea or, with 2-aminofluorene, to form the mixed urea. Formation of NJV-di2-naphthylthiourea was also observed, since 2-aminonaphthalene reacted with the substrate. Urea formation was dependent on microsomes, NADPH, and 02.Use of microsomes from rats previously treated with Aroclor increased urea formation 210-fold. The enzyme activity was inhibited by a-naphthoflavone, flavone, or CO and slightly inhibited by metyrapone, 7-ethoxycoumarin, or SKF-525A. It was not inhibited by methimazole or paraoxon. These data are consistent with a cytochrome P-450-dependent, oxidative desulfuration of the isothiocyanate to yield an isocyanate.
Introduction Organic isocyanates with a general formula of RN=C=O are extensively used in the manufacture of paints, pesticides, and urethanes ( I ) . Chemotherapeutic dialkylnitrogoureas (2)and alkylformamides that are used as industrial solvents (3)can be metabolized to isocyanates. Chemically, isocyanates are reactive electrophilic agents that are capable of modifying proteins ( 4 , 5 ) and nucleic acids (6-10); biologically, they can cause chromosome aberrations, sister-chromatid exchanges (11-14), mutations (10, 11, 15), and/or cancer (16, 17). A related class of compounds, the isothiocyanates (RN=C=S), possess a wide range of biological activities. For example, benzyl isothiocyanate has antibiotic activity against bacteria and fungi in vitro and has, consequently, been used as a medicine (reviewed in ref 18). Moreover, this and related isothiocyanates can be generated from a variety of vegetables, such as brussels sprouts, cabbage, cauliflower, broccoli, and turnips, by hydrolysis of their glucosinolates with myrosinase which may be present either in the vegetables or in the intestinal bacteria (1921). Several synthetic and dietary isothiocyanates can inhibit the metabolic activation of certain carcinogens in vitro (22-28) and tumorigenesis in experimental animals (22,29-35). Although relatively little attention has been focused on the possible adverse long-term effects exerted by isothiocyanates, a number of isothiocyanateshave been found to be mutagenic for Salmonella typhimurium TA100; among them, dietary allyl isothiocyanate is the most potent (36, 37). Furthermore, allyl isothiocyanate has been found to cause bladder tumors in F344 rats (38). Organic isothiocyanates are considerably less reactive than their isocyanate counterparts, and they have relatively little reactivity toward DNA bases.’ Although examples of oxidative removal of sulfur such as the conversions of 1 Unpublished
observations.
parathion to paraoxon (39) and a-naphthylthiourea to a-naphthylurea (40) through metabolism are known, the analogous metabolic conversion of an organic isothiocyanate to the more reactive isocyanate has not been demonstrated. The widespread exposure of humans to dietary isothiocyanates and the possibility that these agents might be converted enzymatically to more reactive isocyanates prompted the present study to determine whether such a pathway of metabolic activation might exist. The model isothiocyanate selected for this study was NITC2,since previous experimentshad clearly shown that the corresponding isocyanate reacted with the N4-amino group of cytosine with mutagenic consequences (10).
Materials and Methods Caution: 2AN, 2AF, NITC, NIC, thiophosgene, and Aroclor 1254 are hazardous and should be handled carefully. Materials. 2AN, 2AF, thiophosgene, flavone, a-naphthoflaand metyraponewere from vone, 7-ethoxycoumarin,methimazole, Aldrich Chemical Co. (Milwaukee,WI). Aroclor 1254 was from the NCIRepository(Bethesda,MD). Trioctanoin was from Pfaltz and Bauer (Waterbury,CT). NADPH (type 111) was from Sigma Chemical Co. (St. Louis, MO). SKF-525Awas a gift from Dr. P. Hollenberg, Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI. Instrumentation and General Procedures for Chemical Analysis. TLC was performed using Whatman PE SIL G/UV silica gel polyester sheets with benzene/95% ethanol (201) as solvent. The chromatograms were viewed under UV light. UV spectrawere recorded using a Hewlett Packard 8452A diode array spectrophotometer. ‘H-NMR spectra were recorded with a GE Abbreviations: NITC, 2-naphthyl isothiocyanate; NIC, 2-naphthyl isocyanate;2AN, 2-aminonaphthalene;2AF, 2-aminofluorene;DNT, NJV’di-2-naphthyl thiourea; DNU, N,”-di-2-naphthylurea; NFT, N-2-naphthyl-N’-2-fluorenylthio- “U,N-2-naphthyl-N‘-2-fluorenylurea; FMO, flavin-containing monooxygenme; P-450,cytochrome P-450;TLC, thinlayer chromatography;HPLC ,high-performanceliquid chromatography; DMF, Nfl-dimethylformamide.
0893-228~/92/2705-0791$03.00/0 0 1992 American Chemical Society
792 Chem. Res. Toxicol., Vol. 5, No. 6,1992 NMR QE-300 instrument using DMSO-& solutions, and D 2 0 was added to observe the disappearance of NH protons; 6 values are ppm downfield from the signal of tetramethylsilane. Mass spectra were recorded using a Kratos MS80RFA mass spectrometer. Reverse-phase HPLC analyses of samples were carried out using a Perkin-Elmer Series 3B liquid chromatograph system interfaced with a Nelson Analytical Data Acquisition System for quantitation and with an LKB Model 2140 diode array detector for metabolite identification. Chemical Syntheses. DNU was prepared as described previously (41). NITC was synthesized by stirring 2AN (485 mg, 3.4 mmol) in 20 mL of CHC13 with thiophosgene (585 mg, 5.1 mmol) and 12 mL of 0.5 N NaOH a t room temperature for 0.5 h. The CHC13 layer was washed with water (3X). Evaporation of solvent gave 540 mg of product (86% yield) which was recrystallizedfrom hexanes: mp 57-59 "C [lit.mp 62-63 OC (42)l; TLCRf0.88; MS m/z (relative intensity) 185(M+,loo), 153 (8.6), 141 (3.9), 127 (39.1); high-resolution MS: calcd for M+, 185.02992: found, 185.0301. DNT was prepared by reaction of NITC (103 mg, 0.56 mmol) in 2 mL of dry acetone with 2AN (96 mg, 0.67 mmol) a t room temperature for 2 h. The product was filtered and washed thoroughly with acetone (155 mg, 84% yield): mp 200-202 "C [lit. mp 203 "C (43)l. NFT was prepared by reaction of NITC (60 mg, 0.32 mmol) with 2AF (60 mg, 0.33 mmol) in 4 mL of acetone a t 60 "C for 3 h. Removal of solvent gave 96 mg (70% yield) of product which was recrystallized from DMF/acetone/hexane to give an analytical sample: mp 186-187 274, 304 (e, 38 220, 35 220); "C; TLC Rf 0.8; UV (MeOH) ,A, lH-NMR 6 3.89 (s,2 H, Cg-protons of fluorene), 7.23-7.98 (m, 14 H, aromatic He), 9.97, 9.99 (e, 1 H each, NH groups); MS m/z (relative intensity) 223 (66.4), 185 (93.8), 181 (89.8), 165 (74.8), 143 (loo), 127 (42.5); high-resolution MS: calcd for M+ of 2-fluorenyl isothiocyanate, 223.04556; found, 223.0460; calcd for M+ of 2AN, 143.07349; found, 143.0731. Anal. Calcd for C24HlsNzS.0.5H20: C, 76.80; H, 5.07; N, 7.47; S, 8.53. Found: C, 76.94; H, 5.03; N, 7.32; S, 8.53. NFU was prepared by reaction of 2 AF (23 mg, 0.13 mmol) with freshly prepared NIC (10) (22 mg, 0.13 mmol) in 1mL of dry acetone a t room temperature for 2 h to give 36 mg (80% yield) of product which was triturated in hot MeOH/acetone to give an analytical sample: mp 302 "C dec; TLC Rf 0.59; UV (MeOH) A,, 244, 280 (sh), 298 nm (e, 40 670,44 090,49 200); NMR 6 3.88 (s,2 H, Cg-protonsof fluorene), 7.19-8.12 (m, 14 H, aromatic H's), 8.85, 8.91 (s, 1 H each, NH groups); MS m/z (relative intensity) 207 (91.9), 181 (95.9), 169 (loo),143 (88.2). Anal. Calcd for C24HleN20: C, 82.29; H, 5.14; N, 8.00. Found: C, 82.37; H, 5.35; N, 7.92. Preparation of Microsomes. Male F344 rats (300-400 g) from Charles River (Wilmington, MA) were used in this study. For enzyme induction, rats were injected ip with Aroclor 1254 (500 mg/kg body weight/l.3 mL of trioctanoin) on day 1 and sacrificed on day 5; control animals were injected with trioctanoin only. Liver microsomes were prepared according to published procedure (44),except that the microsomes were washed with 10 mM sodium phosphate (pH 7.4) that contained 0.5 mM EDTA and then resuspended in the same buffer. The protein content was determined by the method of Bradford (45). Microsomes could be stored in liquid nitrogen for extended periods without apparent loss in activity. Enzyme Assays. Incubations were carried out in open glass tubes (16 X 100 mm) with 1 mL of solution [980 pL of 10 mM sodium phosphate (pH 7.4) that contained 0.5 mM EDTA and 20 wL of DMSO] a t 37 "C for 0-30 min. Incubation mixtures contained 0-1.0 mg of protein, 0.5 pmol of NITC, and 5 pmol of NADPH, with or without 0.2 pmol of 2AF. In order to determine whether the formation of ureas might be due to the oxidation of thioureas initially formed in the reaction mixtures, 0.2 wmol of NFT and 0.2 pmol of DNT were used as substrates under the same conditions. To explore the possibility of enzymatic hydrolysis of NITC to 2AN or NIC, 1.0 or 2.0 pmol of the esterase inhibitor paraoxon (46) was also used. In order to determine the nature of the enzyme responsible for this transformation, 0.51.0 @molof cytochrome P-450 inhibitors, Le., flavone, a-naph-
Lee A: Without 2AF
: With 2AF
Without Microsomes
5
4
1 gi
10 15 20 25 Retention Time (min)
30
Figure 1. Reverse-phase HPLC profiles of EtOAc extracts of incubation mixtures of NITC with NADPH and microsomes without 2AF (A) and with 2AF (B),and of NITC with NADPH alone (C). One milligram of Aroclor 1254-incuded rat liver microsomes was used in the incubation a t 37 "C for 30 min for (A) and (B). The EtOAc extracts were analyzed using HPLC solvent system I as described in Materials and Methods. thoflavone, 7-ethoxycoumarin, metyrapone or SKF-525A, or a FMO substrate, methimazole ( 4 3 , was included. Reaction was initiated by adding the substrate to the ice-cold mixture, and the tube was immediately placed in a shaking water bath. When gases such as argon, CO, CO/Oz mixtures, and Nz/Oz mixtures were used, the gas stream was passed over the mixtures with occasionalshaking starting 6 min prior to initiation of the reaction. Reactions were terminated by extraction with EtOAc (4 X 1 mL), and the residues produced by removal of the solvent with a Savant Speed Vac concentrator were redissolved in DMF and diluted withCH3CN for analysis by reverse-phase HPLC. HPLC effluents were monitored for absorption a t 254 nm, and a pBondapak Cle column (300 X 3.9 mm) was used with both solvent systems. System I was 30% C H ~ C N / H Z O to 63% CH3CN in 5 min linearly, a t 63 % CH3CN for 25 min, and then to 100% CH3CN linearly for 4 min; the flow rate was 0.8 mL/min. t~ values (min) are as follows: 2 AN, 12.68; 2AF, 14.63; DNT, 19.45; DNU, 21.02; NFT, 23.17; NFU, 25.80; NITC, 28.02. System11 ~ ~ 3 0 % CH30H/H20 to 70% CH30H in 5 min linearly, a t 70% CH30H for 27 min, and then to 100% CH30H in 5 min linearly; the flow rate was 1.2 mL/min. t~ values (min) are as follows: 2AN, 10.75; 2AF, 12.80; DNT, 17.15; DNU, 20.50; NFT, 22.45; NITC, 26.10; NFU, 29.15. HPLC analyses of each sample were carried out a t least twice using one solvent system. When inhibitors were included in the incubations, neither the inhibitors nor their metabolites interfered with the detection and quantitation of the desired products. The identifications of DNT, DNU, and NFU collected from HPLC were carried out by UV spectrophotometry and mass spectrometry.
Results Identification of Products from the Metabolism of
NITC. NITC incubated with NADPH and 1mg of liver microsomes from an Aroclor 1254-treated rat in the air at 37 "C for 30 min yielded EtOAc extracts that gave the
Oxidatiue Conversion of NITC to NIC
Chem. Res. Toricol., Vol. 5, No. 6, 1992 793
Table I. Metabolism of NITC with Rat Liver Microsomesa products (nmol/incubation)
incubation components
expt NITC microsomes NADPH 2AF DNT DNU NFU
I I1
+ + + + + +
++ + +
+ ++ + -
-
+
+ +
16.8 3.62 3.73 17.8 2.24 5.86
2.87 0.06 0.67 1.83 0.04 0.43
15.8 0.36 2.66
4 Microsomes (1mg) from an Aroclor-treated rat were used, and the incubations were for 30 min. 2AF was added to trap NIC as a mixed urea. Experimental details are provided in Materials and Methods. Values are the means of duplicate analyses of one incubation using reverse-phaseHPLC; repeat analyseswere generally within 15% of each other.
HPLC profile shown in Figure 1A. The retention times and UV spectra of peaks indicated as DNT and DNU were identical to those of synthetic standards. The materials collected from such HPLC runs were analyzed by mass spectrometry. The DNT sample gave no parent ion but fragmented in the same manner as an authentic sample to give 2AN and NITC (mlz 143 and 185). The small amounts of DNU present prevented the acquisition of a satisfactory mass spectrum. When 2AF was included in the incubation, EtOAc extracts of the incubation mixture gave the typical HPLC profile shown in Figure 1B. In addition to the two peaks identified as DNT and DNU as in Figure lA, the peak at 25.8 min was identified as NFU by comparing the retention time and UV spectrum with those of the authentic material. This material fragmented in the mass spectrometer in the same manner as an authentic sample to give 2-fluorenylisocyanate, 2AF, NIC, and 2AN (mlz 207, 181, 169, and 143) with no molecular ion present. Figure 1C shows the dependency on microsomes of this enzymatic reaction in the presence of 2AF. In the absence of microsomes, 2AF reacted with the substrate, but few other products were formed. NFT was produced from the reaction of 2AF and NITC in these incubations. In order to determine whether the disubstituted urea products, DNU and NFU, might be generated enzymatically from the initial products, DNT and NFT, these compounds were subjected to the same incubations in the presence or absence of 2AF. The results (data not shown) disclosed that the thioureas were stable under the incubation conditions and that no ureas were formed. These results suggest that reactive NIC was formed in the enzymatic incubation mixture, since the ureas were not produced by removal of sulfur from the thioureas. Possible metabolites from 2AN and 2AF were not identified; however, their presence did not interfere with the identification of products from NITC since independent experiments in the absence of NITC showed that their metabolites appeared below 15 min using the same HPLC conditions. Table I shows the product distributions of these two reactions. When either Aroclor 1254-induced microsomes or NADPH was omitted from the incubations, products diminished greatly. These results demonstrated the dependency of these reactions on both NADPH and microsomes. The formation of limited DNT in the absence of microsomes or NADPH may be due to the spontaneous hydrolysis of NITC to 2AN. Determinationof Optimal Enzymatic Conditions. Preliminary results showed that microsomes a t a concen-
Table 11. Effect of Aroclor Treatment on Metabolism of NITCa Droducta rnmol/
incubation source of microsomes comDonenta treated rata complete system -NADPH untreated complete system rata -NADPH
(Idg of proGin*miA)l DNT
NFU ~~-
0.97 k 0.35 (3)b 1.01 f 0.15 (3)bvc 0.12 A 0.10 (3)bJ' 0.00 (3)b 0.30, 0.13e 0.11,0.22e
0.17, 0.06ef
0.02,0.14e8
Incubations were carried out with NITC, 2AF, NADPH, and microsomes (0.3 or 0.4 mg) (complete system) or complete system without NADPH or without microsomes at 37 OC for 30 min. Experimental details are provided in Materials and Methods. b Values represent the means A standard deviations obtained withmicrosomes from 3 animals. The mean value of nmol/incubation from the 3 rata was 27 times as high as that of the complete system without microsomes. The mean value of nmol/incubation from the 3 rata was 40 % more than that of the complete system without microsomes. e Individual values from the microsomes of 2 rata are shown. f The mean value of nmol/incubation from the 2 rata was the same as that of the complete system without microsomes. 8 The mean value of nmol/incubation from the 2 rata was 7% more than that of the complete system without microsomes.
tration of 0.5 mg per 1-mL incubation reached the saturation point. When the concentration was decreased to 0.4 mg and below, a roughly linear response was observed for the formation of NFU when NITC was metabolized in the presence of 2AF. Thus, it was reasonable to carry out other experiments using0.3 or 0.4 mg of microsomal protein per incubation. When the time course from 0 to 30 min was carried out using 0.4 mg of microsomes, formation of NFU increased with time up to 30 min. Thus, for the ease of quantitation of products by HPLC analysis, a 30-min incubation time was used for later experiments. Inducibility of the Enzyme(s) Responsible for the Conversion Reaction. In order to determine whether the enzyme(@ responsible for this reaction is (are) inducible, incubations were carried out with induced or uninduced liver microsomes. Data presented in Table I1 clearly indicate that the transformation of NITC to NIC is mediated by enzyme(s) inducible with Aroclor 1254 and the Aroclor induction increased the formation of DNT and NFU by at least -3- and 10-fold, respectively. Effects of Argon and Paraoxon on the Formation of DNT and NFU from the Metabolism of NITC in the Presence of 2AF. Argon was used to exclude oxygen from the incubation mixture to determine if formation of both products is oxygen-dependent. DNT is produced from the reaction of NITC with 2AN which could conceivably be derived through either reduction or hydrolysis of NITC. Since NIC may also be a result of the hydrolytic reaction of NITC, paraoxon (46),an inhibitor of hydrolytic enzymes, was used. Results of these experiments are shown in Table 111. Argon reduced the formation of NFU by49 5% and that of DNT by 25 '3%. Thus, these two products were derived at least in part from an oxidative metabolism. Paraoxon did not inhibit the production of these compounds, thereby supporting the conclusion that these products did not come from a hydrolytic process. However, how paraoxon increased the production of DNT is not clear. Effects of Oxidative Enzyme Inhibitors on the Formation of NFU from Metabolism of NITC in the Presence of 2AF. In order to determine whether either cytochrome P-450 or FMO was involved in these conversions, typical P-450 inhibitors, such as a-naphthoflavone,
794 Chem. Res. Toxicol., Vol. 5,No.6,1992
Lee
Table 111. Effects of Argon and Paraoxon on the Formation of DNT and NFU from NITC Metabolisma inhibitors none argon paraoxon
concentration (mM)
1 2
Droduct ( % of control) DNT NFU 100 100 64,85 50,52 155 f 15 102 f 9 99,173 96,94
"Incubations were carried out with the complete system as described in Table I1 for 30 min using microsomes from Aroclortreated rata. Experimental details are provided in Materials and Methods. Values are the mean f standard deviation of three independent incubations or the individual values from two incubations.
Table IV. Effects of Inhibitors of Oxidative Enzymes on the Formation of NFU from Metabolism of N I T 0 concns NFU inhibitors (mM) or (% of inhibitors or gases gas ratios control) or gases none 0 100 metyrapone a-naphtho0.5 37 f 10 flavone 1.0 27,27 methimazole flavone 0.5 41 f 3 1.0 30 f 5 Nz/Oz 7-ethoxy0.5 7 1 f 5 CO/Oz coumarin 1.0 6 1 f 8 CO/Oz SKF-525A 0.5 74f 2 N 2 / 0 ~ 1.0 6 3 f 6 CO/Oz
concns (mM) NFU or gas ( % of ratios control) 0.5 84,78 1.0 69,65 0.5 107,94 1.0 99, 97 80:20 100b 8020 6 4 k 7 1oO:O 1 0 f 8 9010 l00C 9010 4 7 f 2
"Incubations were carried out with the complete system as described in Table I1 using Aroclor-induced rat liver microsomes for 30min. Experimental details are provided in Materials and Methods. Values are the mean f standard deviation of at least three independent incubations or the individual values of two incubations. As control for the experiments with CO/Oz = 8020 and 100:O. As control for the experiments with CO/Oz = 9010.
7-ethoxycoumarin, metyrapone, SKF-525A, and flavone, were incorporated into the incubations. A substrate of FMO, methimazole, which would be expected to decrease the metabolism of NITC by this enzyme (47), was also used. Reactions were also carried out in CO/Oz atmospheres using Nz/O2 mixtures as controls. The results are shown in Table IV. The inhibitory effects on the formation of NFU follow the order of a-naphthoflavone > flavone > 7-ethoxycoumarin, SKF-525A > metyrapone; methimazole had no effect. CO inhibited the conversion, and the inhibition increased as the concentration of CO increased. These results suggest that the transformation is mediated by a P-450 enzyme and not by a FMO enzyme.
Discussion The results reported here demonstrate the enzymatic oxidative desulfuration of NITC to NIC (see Scheme I for the reaction pathways). The decrease in reaction under low oxygen tension produced by both argon and CO atomspheres showed that this was primarily an oxidative process. Since the putative product NIC is a very reactive entity, it readily hydrolyzes to 2AN. Subsequently, 2AN can react with the substrate to produce DNT, or with NIC to give a small amount of DNU. When 2AF was included in the incubation to provide a higher and more constant level of amine, NFU was also produced to directly confirm the generation of NIC. Furthermore, DNU and NFU must have come from the formation of NIC intermediate, since they did not come from DNT and NFT through the
Scheme I 0
j2AF
s
w~~-t-~~w NFT
2AN
oxidative desulfuration pathway in this study. This is consistent with the finding that diphenylthiourea cannot be converted to diphenylurea enzymatically although oxidative desulfuration occurred with phenylthiourea (48). It is unlikelythat enzymatic hydrolysiscould convert NITC to NIC or 2AN, since paraoxon did not inhibit the formation of products. This reaction is microsome-, NADPH-, and oxygen-dependent; clearly, a reductive process is unlikely to be involved. Results from the inhibition experiments indicated that a P-450 enzyme rather than FMO is involved and the P-450 is inducible by Aroclor 1254. The transformation described here is a hitherto chemically uncharacterized enzymatic reaction. Although ElHawari and Plaa (49) had shown previously that the NADPH-dependent protein binding of 1-[3H]naphthyl isothiocyanate (labeled in the 4-position of the ring) or l-[l4CInaphthyl isothiocyanate (labeled in the isothiocyanate moiety) required the oxidative conversion of the compound to a metabolite in rat liver microsomes by a P-450 system, they did not succeed in identifying the reactive binding species, presumably l-naphthyl isocyanate by analogy to the present observations. This oxidative desulfuration reaction seems to be common, at least, for aryl isothiocyanates, since phenyl isothiocyanate and NITC inhibited the NADPH-mediated protein binding of l-naphthyl isothiocyanate (49). Without activation (Le., no NADPH), a low level of binding of l-naphthyl isothiocyanate was also observed, and this is consistent with the fact that isothiocyanates themselves are reactive toward protein, especially with sulfhydryl groups (50). Increased binding of an enzymatically generated isocyanate is expected, since the product is much more reactive than l-naphthyl isothiocyanate toward the nucleophilic groups in protein, e.g., phenolic, sulfhydryl, and amino groups. They also found that the enzyme(s) responsible for this activation was(were) inducible with phenobarbital or 3-methylcholanthrene (49). Metabolism of l-naphthylthiourea by rat liver and lung microsomes has been reported by Lee et al. using [carbon~l-~WI - and [%]-labeled compounds (40). They found that this thiourea was converted to l-naphthylurea and atomic sulfur at least in part by a P-450 system; half of the atomic sulfur bound to the microsomes by reacting with cysteine side chains of the proteins to form a hydrodisulfide. The loss of P-450 and monooxygenase activity seen on incubation of liver microsomes with l-naphthylthiourea is likely the result of binding of the atomic sulfur top-450, Hunter and Neal (51)have studied the inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thiono sulfur-containing compounds. From their results and those of previous studies with parathion and CS2 (reviewed in ref 51) they also postulated that the inhibitory effects of these com-
Chem. Res. Toxicol., Vol. 5, No. 6, 1992 795
Oxidative Conversion of NITC to NIC pounds may be the result of the mixed-function oxidasecatalyzed release and covalent protein binding of atomic sulfur as was likewise suggested by De Matteis (52)on the loss of P-450 in the incubation of phenylthiourea and 1-naphthyl isothiocyanate with phenobarbitone-treated rat liver microsomes in the presence of NADPH. The present results, together with the previous observations by others, suggest that one may hypothesize that isothiocyanates can be metabolically activated to isocyanates and sulfur atoms by a P-450 system(s), and the protein binding of isothiocyanates and their metabolites can cause losses of P-450 activities. The inhibitory effect of isothiocyanates on nitrosamine carcinogenesis is thought to be the result of the inhibition of the metabolic activation of the procarcinogens to DNAmodifying species by P-450 systems although the exact mechanism is not fully understood (25-28).The hypothesis presented here may explain the effect of phenethyl isothiocyanate on microsomal N-nitrosodimethylamine metabolism, since the inhibition of nitrosodimethylamine demethylase activity by this compound was greatly enhanced when NADPH was preincubated with the isothiocyanate and microsomes (28). The present study suggests that NITC can be converted oxidatively to NIC, which has been shown to be mutagenic (IO), and to its hydrolysis product, 2AN, which is a carcinogenic aromatic amine (53). Thus, this type of conversion has the potential of causing genetic effects. Isothiocyanates can be generated from a variety of vegetables (19-21). These dietary isothiocyanates (RN=C=S) encompass diverse structural features that include mutagenic compounds such as R = allyl, n-butyl, ethyl, and benzyl (36),as well as methyl isothiocyanate, which is a potential precursor of genotoxic methyl isocyanate (11,13-16),if metabolic activation of this type were to take place. At the present time, it is not known whether the metabolic conversion of dietary isothiocyanates to reactive isocyanates occurs. Even if this activation were to be demonstrated, the instability of most isocyanates in aqueous media would limit their half-lives in biological systems and, consequently, the distance through which they could exert adverse biological effects. However, recent studies revealed that isocyanatescan reversiblyform glutathione conjugates (reviewed in ref 54). It is speculated that, in biological systems, these conjugates can be transported and, under suitable conditions,the isocyanates can be released enzymatically or nonenzymatically to modify macromolecules and elicit toxic effects (54). Thus, conjugation of chemically reactive compounds with glutathione may not always result in detoxification of these substances in a classical sense. To the contrary, these conjugations may represent an intriguing mechanism for the transport of certain short-lived electrophiles, such as isocyanates, in vivo. The difficulty of preserving and transporting the reactive isocyanates in biologicalsystems once they are formed from the oxidative desulfuration of isothiocyanates may be circumvented by this mechanism. Alternatively, isothiocyanates may be subject to metabolic activation pathways in a wide range of potential target tissues because their relatively greater stabilites permit their distribution. Acknowledgment. I thank Dr. C. M. King for helpful discussions and Mrs. E. Thomas-Weber for her assistance in the preparation of the manuscript. I also thank Dr. M.
B. Ksebati and Ms. M. B. Kempff from the Central Instrumentation Facility of the Comprehensive Cancer Center of Metropolitan Detroit for measuring the NMR and mass spectra, respectively. This work from the A. Alfred Taubman Facility was supported by NIH Grant CA-37885and an institutional grant from the United Way of Detroit. References (1) Reisch, M. S. (1991) Higher paint sales brighten profits outlook. Chem. Eng. News, October 14, 29-58. (2) Baril, B. B., Baril, E. F., Laszlo, J., and Wheeler, G. P. (1975)
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Registry No. NITC, 1636-33-5;NIC, 2243-54-1;P-450,903551-2.
