254
Chem. Res. Toxicol. 1989,2, 254-259
Isolation and Identification of 3-Hydroxy-3-methyloxindole, the Major Murine Metabolite of 3-Methylindole Gary L. Skiles, James D. Adams, Jr.,t and Garold S. Yost* Department of Pharmacology and Toxicology, 112 Skaggs Hall, University of Utah, Salt Lake City, U t a h 84112 Received January 4, 1989 3-Methylindole (3MI) is pneumotoxic to ruminants and rodents subsequent t o metabolic oxidative activation by cytochrome P-450 monooxygenases. Goats are much more susceptible than mice and rats to 3MI-mediated lung damage, and these differences in species susceptibility may be reflected by differences in the metabolic products of 3MI. Radioactive 3MI was administered ip t o Swiss-Webster mice, and the major nonpolar urinary metabolites were fractionated and separated by HPLC. Although 3-methyloxindole has been shown to be the major urinary metabolite of 3MI in goats, it was not detected in mouse urine. Instead, the major metabolite, 3-hydroxy-3-methyloxindole, was isolated and purified and its structure elucidated by lH and 13C NMR, mass spectrometry, and IR spectroscopy. This is the first identification of this highly oxidized indole from mammalian sources. The production of this metabolite may be indicative of the formation of an electrophilic methyleneoxindole intermediate, which could be responsible for pneumotoxicity in this species.
Scheme I. Known Metabolic Products in the Mammalian Metabolism of 3-Methylindole
Introduction The relationship between exposure to 3-methylindole (3MI)' and the development of acute pulmonary disease in ruminants has been firmly established. In cattle, acute bovine pulmonary edema and emphysema occur after intravenous or oral administration of 3M1, or after a switch of feeding conditions from a dry suummer pasture to lush green forage (1-3). Tryptophan from the forage is converted to 3MI by ruminal bacteria via the readily formed deamination product indole-3-acetic acid (IAA). The mechanism of formation has been postulated to occur by the decarboxylation of IAA by a Lactobacillus sp. which has been isolated from ruminal contents (4). The pulmonary damage elicited by 3MI can be shown to occur in several animal species including goats (5-7), horses (8,9), sheep (IO), and rodents (11-13). Ruminants are much more susceptible to 3MI-mediated pneumotoxicity than mice and rats, but rodents display the same organ (lung) and cell-specific (Clara) dose-dependent toxicity; the degree to which each species forms 3MI from tryptophan has not been established. Man is exposed to 3MI from cigarette smoke (14, 151, in various foodstuffs (16-18), and as a presumptive microorganism degradation product of tryptophan in the gastrointestinal tract. Pathological conditions, such as a malabsorption syndrome and Heinz body formation, have been correlated with unusually high urinary concentrations of 3MI metabolites such as 6-sulfatooxy-3-methylindole (19). The excretion of 3MI metabolites was reduced and the symptoms of toxicity ameliorated by treatment with oral antibiotics. There have been several reports of high 3MI metabolite levels in the urine of schizophrenic patients, but the etiology of these finding has not been conclusively determined. The majority of these patients were undergoing drug therapy, indicating a possible relationship with constipation as a result of reduced gastrointestinal motility due to phenobarbital or the antimuscarinic effects of phenothiazines. However, some un-
treated patients also had high 3MI urinary metabolite levels when compared to controls, indicating a possible relationship with the disease. Administration of oral tetracycline to both groups of patients eliminated the excretion of 3MI metabolites (20, 21). The metabolites of 3MI identified in humans and other mammals (Scheme I) are ring-hydroxylated sulfate conjugates (5), primarily the 6-sulfatooxy derivative, 3-methyl oxidation products such as indole-3-carbinol (6) and indole-3-carboxylic acid (7),and the pyrrole ring opened products 2-formamidoacetophenone (3) and 2-aminoacetophenone (4), as well as 3-methyloxindole (2) (22-25). The mechanism of pulmonary cellular damage by 3MI is believed to occur through the formation of an electrophilic intermediate produced by the oxidative metabolism by cytochrome P-450. This mechanism is supported by observations that in vivo and in vitro covalent binding of radioactive 3M1, as well as the in vivo toxicity of 3M1, was proportional to P-450 activity which was inhibited with
+Present address: School of Pharmacy, 1985 Zonal Ave., University of Southern California, Los Angeles, CA 90033.
' Abbreviations: 3M1, 3-methylindole; HMOI,3-hydroxy-3-methyloxindole; GSH, glutathione; IAA, indole-3-acetic acid.
0893-228x189 12702-0254$01.50/0
d
/
o
NH
-dH -
d
1
4 R=H
I
NH o 2
/-OH
HO 5
i sulfate conjugates
0 1989 American Chemical Societv
"
0
%OH
dH 7
Chem.Res. Toxicol., Vol. 2,No.4,1989 255
Isolation of 3-Hydroxy-3-methyloxindole Scheme 11. Proposed Metabolic Pathway of 3-Methylindole in Mice To Form the Isolated Metabolite 3-Hydroxy-3-Methyloxindolea
tion of IAA by horseradish peroxidase. The electrophilic nature of this compound was demonstrated by facile, reversible addition of thiols including thiophenol and GSH (33).Horseradish peroxidase oxidation or photooxidation of IAA has been reported to produce “3-(hydroxymethy1)oxindole” (oxindole-3-carbinol), an isomer of HMOI, which was postulated to nonenzymatically dehydrate to 3-methyleneoxindole (33-35). 3-Methyleneoxindole (13) may be a compelling candidate for a metabolic intermediate that is reactive enough to result in covalent binding to proteins. The identification of 13, or a metabolite consistent with its formation, would provide important information about the proposed mechanisms of bioactivation.
Materials and Methods
11 -
P-;SO(OH*)
I 12 -
O Pathway A shows formation via a free-radical intermediate formed by one-electron oxidation by cytochrome P-450 with subsequent reaction with molecular oxygen and rearrangement by an oxygen rebound mechanism. Pathway B shows formation via cytochrome P-450 oxidation followed by hydroxylation without freeradical or peroxide intermediates.
SKF-525A, piperonyl butoxide, or the cytochrome P-450 suicide substrates 1-aminobenzotriazole and [ (cu-methylbenzyl)amino]benzotriazole (26-28).The in vivo covalent binding was inversely proportional to the concentrations of glutathione (GSH), which was depleted by pretreatment with diethyl maleate or increased by pretreatment with cysteine, a GSH precursor (29). Moreover, tissues that display the most pathology following 3MI administration were also maximally depleted of endogenous GSH levels (29). The exact chemical nature of the electrophilic intermediate has not been established. We have postulated (30) that 3MI is converted, via a two-electron oxidation process, to an electrophilic methylene imine (10, Scheme 11). This chemical species has two electrophilic centers, at the 2carbon and at the exocyclic methylene. In in vitro microsomal studies, a GSH adduct a t the 3-methyl carbon has been identified (30);however, the analogous conjugate at the 2-carbon has not been found. The in vivo and in vitro isolation of a free radical by spin trapping with phenyl-tert-butylnitrone indicates that the reaction probably occurs via two one-electron oxidative steps (31). Studies on the oxidation of IAA with horseradish peroxidase have demonstrated that a free radical (9) can be formed at the 3-carbon. Reaction of this intermediate with oxygen was proposed to result in the formation of the methylene imine intermediate (10) and superoxide radical (32). In other studies (33),3-methyleneoxindole (13, Scheme 11) has been identified as a product of the oxida-
Chemicals. 3-Methylindole was obtained from Sigma Chem(2.7 mCi/mmol) was ical Co., and [nethyl-14C]-3-methylindole custom synthesized by New England Nuclear. Isatin and CH3MgBr were obtained from Aldrich Chemical Co. 3-Methyloxindole and indole-3-carbinol were gifts from Dr. James R. Carlson, Washington State University. All other chemicals and reagents were of the highest purity available and obtained from local suppliers. Animal Dosing, Sample Collection, and Radiocarbon Quantitation. Male Swiss-Webster mice (Charles River) were housed in polyethylene/stainless steel cages for a minimum of a 24-h acclimation period during which rodent chow and tap water were provided ad libitum, and lighting was maintained on a 12-h light-dark cycle. Following a 12-h fast, the mice were dosed by ip injection with 400 mg/kg (the LD50 is 575 mg/kg) of 3MI or [14C]3MI(approximately 5 pCi per mouse) dissolved in corn oil. The mice were placed in Nalgene mouse metabolism cages, and the urine was collected for 24 h. The radiocarbon in the urine from the mice dosed with [14C]3MI was quantitated by liquid scintillation spectrometry on a Packard 3385 liquid scintillation spectrometer. Results were corrected for counting efficiency by the automatic external standard method. High-Performance Liquid Chromatography. Quantitation and purification of the major nonpolar 3MI metabolite by HPLC was done on a Beckman System Gold liquid chromatograph. The metabolites of [‘*C]3MI that were partitioned into a n organic phase (described below) were quantitated, and preliminary chromatographic purification of the major nonpolar metabolite was done by reverse-phase HPLC on an Ultramex C-18, 5-pm, 250 X 4.6 mm column (Phenomenex, Rancho Palos Verdes, CA) with a convex gradient of 3-100% acetonitrile and water. For radiolabeled metabolites, the fractions were collected directly into scintillation vials containing scintillation cocktail and then quantitated by liquid scintillation spectrometry. Correlation of retention times detected by absorbance a t 254 nm and by radioactivity quantitation was done by plotting radioactive counts and overlaying the histogram onto the chromatogram that was produced from absorbance monitoring. Normal-phase HPLC purification of the nonpolar metabolite was done on a Ultramex CN, 5-pm, 250 X 4.6 mm column with an eluant of isocratic T H F and heptane (30:70). Metabolite Quantitation and Purification. Urine from four mice dosed with [14C]3MIwas pooled and partitioned into aqueous and organic fractions with a Waters Sep-Pak reverse-phase extraction cartridge. The cartridge was prepared by sequential washing with 0.05 M ammonium acetate (pH 6) and acetonitrile. After the cartridge was charged with a 1-mL aliquot of urine, it was rinsed with 1 mL of 0.05 M ammonium acetate to obtain the aqueous fraction and then eluted with 2 mL of acetonitrile to obtain the organic fraction. The organic fraction was analyzed by reverse-phase HPLC to determine the distribution of nonpolar metabolites. The urine from the mice dosed with the nonradiolabeled 3MI was extracted with three aliquots of ethyl acetate that were each equal to half of the volume of urine that was obtained. The organic fraction was evaporated under ah N2 stream a t room temperature and redissolved in acetonitrile/water (1:9), and the major nonpolar 3MI metabolite was purified by reverse-phase HPLC. The fraction containing the metabolite of
256 Chem. Res. Toxicol., Vol. 2, No. 4, 1989
Skiles et al. 1000
4
I
7
H
2a 4
6
7
10 0
U
0
1
I
I
I
I
4
8
12
16
20
MINUTES
Figure 1. HPLC chromatogram of the organic fraction of urine from mice treated with [l'CI-3-methylindole. The histogram represents the radioactivity in 0.5-min fractions of the eluant. interest was evaporated to dryness with N2.The sample was redissolved in THF/heptane (3:7) and then further purified by normal-phase HPLC. The fraction containing the purified metabolite w a ~evaporated to dryness, redissolved in chloroform, and evaporated to dryness again. The crystals obtained were then placed under high vacuum for 30 min to evaporate residual solvent from the purified extract. Urine from an untreated mouse was spiked with 8 mg of 3M1, which would be the amount of 3MI if the total dose of 400 mg/kg was excreted unchanged, and the urine was extracted as described above. Analysis by reverse-phase HPLC of the extracts from this experiment showed only 3MI without the formation of other compounds. Therefore, 3MI was not autoxidized during the workup process. Spectral Analysis. The metabolite isolated from the normal-phase HPLC purification was dissolved in chloroform-d (Aldrich Gold Label, 100 atom %) and analyzed by and 'H NMR on a Varian XL-500. Proton-decoupled and coupled carbon spectra were obtained to aid in chemical shift assignmentsof the 13Cspectra. A 'H COSY spectrum between 6.7 and 7.5 ppm was also obtained to confirm the chemical shift assignments of the aromatic protons. The mass spectrum was obtained on a VG7050E by direct-probe electron impact. The molecular weight was obtained by accelerating voltage ratio peak matching with the m/z 168.98882 fragment of perfluorokerosine. The IR spectrum was obtained on a Bio-Rad Digilab FT-IR with the sample dissolved in chloroform. The ORD spectrum was measured in acetonitrile and obtained on a Jafco J-20C instrument. Synthesis of 3-Hydroxy-3-methyloxindole. 3-Hydroxy-3methyloxindole was synthesizedaccording the procedure of Kohn and Ostersetzer (36)by adding CH3MgBrto isatin in anhydrous THF. The product was recrystallized from ether/CH2C12to produce a white crystalline solid with a mp of 160-161 O C [lit. (36)160 "C] in 65% purified yield.
Results Quantitation of Urinary Radiocarbon. Recovery of radiocarbon was virtually quantitative within the 24-h collection period. The urine was partitioned with a SepPak cartridge to separate 41.0% of the radioactivity into a polar fraction and 58.9% into a nonpolar fraction. Quantitation of the radioactive metabolites from the nonpolar fraction by HPLC and produced the majority of the "C as several peaks of relatively polar material, but 10.5% of the dose (17.8% of organic fraction) as a rela-
I
I
I
I
I
I
I
I
0
7
6
5
4
3
2
I
ppm
Figure 2. 500-MHz lH NMR in CDCls of the major nonpolar urinary metabolite of 3MI in mice. The bottom inset is the expanded spectrum of the aromatic region. Proton assignments are indicated by the structure of 3-hydroxy-3-methyloxindole in the top inset. tively nonpolar metabolite a t 13.6 min (Figure 1). The metabolite was partially purified by collecting the eluant from several injections of the urinary extract. The known caprine 3MI metabolites 3-methyloxindole (2) and indole-3-carbinol (6) (23) were available as standards and had different retention times than the metabolite a t 13.6 min; neither 2 nor 6 were detected in urine extracts from 3MI-treated mice. The major nonpolar metabolite was further purified by normal-phase HPLC to produce 20.3 mg of the crystalline compound from several injections of the partially purified metabolite. The metabolite eluted as a broad peak between 5.9 and 8.5 min. Spectral Characterization of Major Nonpolar Metabolite. The 500-MHz 'H NMR spectrum shown in Figure 2 displays a chemical shift and splitting pattern consistent with a structural assignment of 3-hydroxy-3methyloxindole (HMOI): 6 1.62 [s, 3 H, C(10)H3], 6.87 [dv J = 7.63 Hz, 1 H, C(7)H,], 7.09 [dd, J = 7.63, 7.63 Hz, 1 H, C(5)H,], 7.27 [dd, J = 7.63,7.63 Hz, 1H C(6)H1], 7.40 [d, J = 7.63 Hz, 1H, C(4)Hl]. The four aromatic protons split in the pattern characteristic of indolines. The assignments for the protons in the aromatic region were confirmed by a 'H COSY spectrum obtained from 6.7 to 7.5 ppm (not shown). The 13C NMR proton-decoupled spectrum (Figure 3) and the proton-coupled spectrum (not shown) showed nine carbon lines with chemical shifts and proton-coupled splitting consistent with the structural assignment of HMO1 6 24.990 [q, C(lO)], 73.871 [s, C(3)], 110.222 [d, C(7)], 123.401 [d, C(4)], 124.129 [d, C(5)], 129.826 [d, C(6)], 131.893 [ e , C(3a)], 139.900 [s, C(7a)], 179.885 [s, C(2)]. In addition, the carbon line at 6 179.885 (C-2) was split into a quartet in the proton-coupled spectrum due to long-range coupling to the C-10 methyl. The assignments for C-4 and C-5 are tentative because they have nearly identical chemical shifts. The directprobe electron impact mass spectrum (Figure 4) is also consistent with the structural assignment of HMO1 m/z (relative abundance) 163.1 (15.1), 148.0 (6.0), 146.1 (3.0), 145.1 (3.8), 135.1 (49.7), 120 (100.0), 92.0 (14.3), 77.0 (4.6). A molecular weight of 163.06294, obtained by peak matching, closely correlated with the theoretical HMOI
Chem.Res. Toxicol., Vol. 2, No.4, 1989 257
Isolation of 3-Hydroxy-3-methyloxindole
n
IO
180
160
140
120
100
80
60
40
20
ppm
Figure 3. Proton-decoupled 126-MHz 13CNMR in CDC13of the major nonpolar urinary metabolite of 3MI in mice. The inset shows the assignments for 3-hydroxy-3-methyloxindole. I20
40
4
I
20~~fl,,,L,,h;,
,,[Il,
,,,,,;
J,!il
1
,/
0 50
130
90
il,
,;,‘a
:f , I70
m/z
Figure 4. Direct-probe electron impact mass spectrum of the major nonpolar urinary metabolite of 3MI in mice. The molecular ion for 3-hydroxy-3-methyloxindoleis 163, and the inset shows the assignments for several fragments.
mass of 163.06332 for a difference of 0.38 ppm. The IR spectrum was also consistent with the structural assignment: 1624,1726,2575,3204,3562 cm-‘. The ORD of the compound showed a negative band at 237 nm with a molar rotation of [MI = -6450”. The spectral characteristics and chromatographic behavior of the purified HMOI were identical with those of synthetic HMOI, except that the ORD of the synthetic material was not measured.
Discussion The highly oxidized, yet relatively nonpolar and nonionic metabolite HMOI (12) was readily isolated from the urine of mice dosed with 3MI. The spectra obtained clearly show a structural assignment consistent with HMOI. An alternative structure for the metabolite, 3-(hydroxymethy1)oxindole (oxindole-3-carbinol), is ruled out by (1) integration of three C-10 methyl protons on the ‘H NMR spectrum; (2) splitting of the C-10 resonance into a quartet in the proton-coupled 13C NMR spectrum; (3) the downfield shift of C-3, but not (2-10, in the proton-decoupled 13CNMR spectrum; and (4) loss of either an OH or a CH3 but not a CHzOH fragment in the mass spectrum. The identification of HMOI as a metabolic product of 3MI in mice is the first report of this metabolite in a mammalian species. There have been several reports of
IAA degradation to “3-(hydroxymethyl)oxindole” by photooxidation (s), horseradish peroxidase oxidation (30, and bacteria (33).One description of the treatment of IAA with fungal extracts (37)has presented evidence for the formation of HMOI. The structure of the oxindole product from all of the IAA decomposition studies may be HMOI since the structural assignments were based solely on UV and IR spectroscopy and the UV and IR spectra of “3(hydroxymethy1)oxindole” from these reports and HMOI from our studies appear to be identical. These studies with IAA in nonmammalian enzymatic and nonenzymatic systems may indicate a mechanism for 3MI oxidation a t the 2- and 3-positions via formation of free radicals and a peroxide. The occurrence of HMOI as a metabolic product of 3MI in a mammalian system suggests the possibility of a common mechanism of formation of HMOI between 3MI oxidation and the oxidation of IAA, although there is no direct evidence for this mechanism. The mammalian pathway may include the formation of a nitrogen-centered free radical (8) by one-electron cytochrome P-450 oxidation (Scheme 11, pathway A). Rearrangement to the carboncentered free radical (9) can be expected to occur since it is highly favored due to resonance stabilization at the benzylic position. Subsequent reaction of the carboncentered free radical with molecular oxygen, probably after release from the active site of the enzyme, could form a hydroperoxide (1 1) that could rearrange by an oxygen rebound mechanism via the peroxidase activity of cytochrome P-450 (38)to form HMOI (12, Scheme 11, pathway A). Such a rebound mechanism has been proposed to occur during the isomerization of butylated hydroxytoluene hydroperoxide (39). The production of HMOI from a hydroperoxide may also occur nonenzymatically although evidence suggests that such autoxidations of 3-hydroperoxide 3-substituted indoles normally result in pyrrole ring opening via dioxetane or oxazine intermediates (40). Autoxidation of a hydroperoxide may be responsible for the formation of the known 3MI metabolites 2-formamidoacetophenone (3) and 2-aminoacetophenone (4). Since the isolated HMOI produced a negative ORD band, it is probably a single enantiomer and its formation must be enzyme-mediated. Therefore, the hydroperoxide intermediate (11) must also be formed stereoselectively, which argues against the dissociation of the free-radical intermediate 9 from the active site of the enzyme. In addition, the ORD spectrum of HMOI argues against the autoxidation of the hydroperoxide as a viable mechanism of formation of HMOI. An alternative pathway that does not include the formation of free-radical or electrophilic intemediates is also possible (Scheme 11, pathway B). Cytochrome P-450 catalyzed oxidation of 3MI to form 3-methyloxindole (2), followed by hydroxylation a t the 3-carbon methine, could also result in the formation of HMOI. Significant evidence exists, however, that cytochrome P-450 mediated oxidation of 3MI in mammalian systems proceeds through the formation of free radicals. The identification of free radicals from in vitro and in vivo studies of 3MI metabolism (31, 32) and the ease of one-electron oxidation of indoles (41) are supportive of pathway A. The degradation of IAA has been postulated to include the dehydration of 3-(hydroxymethy1)oxindole to form 3-methyleneoxindole (13), a reaction that was believed to occur nonenzymatically (35). 3-Methyleneoxindole has been shown to be an inhibitor of enzymatic activity via sulfhydryl group binding (33). Such a dehydration may also be possible with 3-hydroxy-3-methyloxindole to pro-
258 Chem. Res. Toxicol., Vol. 2, No. 4 , 1989
duce an electrophile. The previously postulated toxic intermediate, the methylene imine (lo), may also be a byproduct of pathway B. A second one-electron oxidation of the free radical by P-450 could be expected to result in the formation of the methylene imine. These electrophiles could be responsible for in vivo covalent binding to macromolecules leading to toxicity, as well as serve as substrates for GSH conjugation. In summary, these results have demonstrated the formation of a unique metabolic product of 3MI in mice. The formation of this product probably proceeds through free-radical intermediates which could alternatively form an electrophilic methylene imine derivative of 3MI. The HMOI metabolite may also undergo dehydration to form the electrophilic 3-methyleneoxindole. The mechanism of formation of HMOI is not fully understood, but a cytochrome P-450 catalyzed reaction involving oxygen rebound of a peroxide is consistent with previously reported 3MI metabolic intermediates and the chemistry of indole oxidation.
Acknowledgment. We are grateful to Dr. Sidney Williamson for the synthesis of HMOI and to Dr. James R. Carlson, Washington State University, and Dr. Carl F. Albrecht, University of Stellenbosch, for helpful suggestions concerning this research. This work was supported by Grant HL13645 from the United States Public Health Service, National Institutes of Health. G.S.Y. is a United States Public Health Service Research Career Development Awardee (HL02119). Registry No. 1, 83-34-1; 12, 3040-34-4.
References (1) Hammond, A. C., Bradley, B. J., Yokoyama, M. T., Carlson, J. R., and Dickinson, E. 0. (1979) 3-Methylindole and naturally occurring acute bovine pulmonary edema and emphysema. Am. J . Vet. Res. 40, 1398-1401. (2) Carlson J. R., and Breeze, R. G. (1983) Cause and prevention of acute pulmonary edema and emphysema in cattle. In Handbook of Natural Toxins. Plant and Fungal Toxins (Keeler, R. F., and Tu, A. T., Eds.) Vol. 1, pp 85-115, Markel Dekker, New York. (3) Carlson, J. R., Nocerini, M. R., and Breeze, R. G. (1984) The role of metabolism in 3-methylindole-induced acute lung injury. In Progress in Tryptophan and Serotonin Research (Schlossberger, H. G., Kochen, W., Linzen, B., and Steinhart, J., Eds.) pp 483-489, de Gruyter and Co., Berlin. (4) Yokoyama, M. T., and Carlson, J. R. (1974) Dissimulation of tryptophan and related indolic compounds by ruminal microorganisms in vitro. Appl. Microbiol. 27, 1540-1548. (5) Huang, T. W., Carlson, J. R., Bray, T. M., and Bradley, B. J. (1977) 3-Methylindole-induced pulmonary injury in goats. Am. J . Pathol. 87, 647-666. (6) Bradley, B. J., and Carlson, J. R. (1980) Ultrastructural pulmonary changes induced by intravenous administered 3-methylindole in goats. Am. J . Pathol. 99, 551-560. (7) Mesina, J. E., Bisgard, G. E., and Robinson, G. M. (1984) Pulmonary function changes in goats given 3-methylindole orally. Am. J . Vet. Res. 45, 1526-1531. (8) Turk, M. A., Breeze, R, G., and Gallina, A. M. (1983) Pathologic changes in 3-methylindole-induced equine bronchiolitis. Am. J . Pathol. 110, 209-218. (9) Turk, M. A., and Thomas, D. E. (1986) Effects of phenobarbital treatment on 3-methylindole toxicosis in ponies. Am. J . Vet. Res. 47,901-905. (10) Bradley, B. J., Carlson, J. R., and Dickinson, E. 0. (1978) 3-Methylindole-induced pulmonary edema and emphysema in sheep. Am. J. Vet. Res. 39, 1355-1358. (11) Turk, M. A. M., Flory, W., and Henk, W. G. (1984) Dose response in 3-methylindole-induced bronchiolar epithelial necrosis in mice. Res. Commun. Chem. Pathol. Pharmacol. 46, 351-362. (12) Kiorpes, A. L., Keith, I. M., and Dubielzig, R. R. (1988) Pulmonary changes in rats following administration of 3-methylindole in cremophore EL. Histol. Histopathol 3, 125-132. (13) Adams, J. D., Laegreid, W. W., Huijzer, J. C., Hayman, C., and Yost, G. S. (1988) Pathology and glutathione status in 3-methyl-
Skiles et al. indole-treated rodents. Res. Commun. Chem. Pathol. Pharmacol. 60, 323-335. (14) Hoffman, D., and Rathkamp, G. (1970) Quantitative determination of 1-alkylindoles in cigarette smoke. Anal. Chem. 42, 366-370. (15) Wynder, E. L., and Hoffman, D. (1967) VIII: Certain constituents of tobacco products. In Tobacco and Tobacco Smoke, Studies in Experimental Carcinogenesis, pp 377-379, Academic Press, New York. (16) Silwar, R., Kamperschroer, H., and tressl, R. (1987) Gas chromatographic-mass spectrometric study of roasted coffee aromaquantitative determination of steam volatile aroma constituents. Chem. Mikrobiol., Technol. Lebensm. 10, 176-187. (17) Schulz H. (1986) Determination of indole and skatole in sea food by high-performance liquid chromatography (HPLC). 2. Lebensm.-Unters. Forsch. 183, 331-334. (18) Kowalewska, J., Zelazowska, H., Babuchowski, A., Hammond, E. G., Glatz, B. A., and Ross, F. (1985) Isolation of aroma-bearing material from Lactobacillus helveticus culture and cheese. J. Dairy Sci. 68, 2165-2171. (19) Horning, J. R., and Dalgliesh, D. E. (1959) The association of skatole-forming bacteria in the intestinal tract with the malabsorption syndrome and certain anaemias. Biochem. J. 70, 13P14P. (20) Leyton, G. B. (1958) Indolic compounds in the urine of schizophrenics. Br. Med. J . 2, 1136-1139. (21) Nakaro, A., and Ball, M. (1960) The appearance of a skatole derivative in the urine of schizophrenics. J. Neru. Ment. Dis. 130, 317-419. (22) Acheson, R. M., and Hands, A. R. (1961) 6-Sulphatoxyskatole in human urine. Biochim. Biophys. Acta 51, 579-581. (23) Hammond, A. C., Carlson, J. R., and Willett, J. D. (1979) The metabolism and disposition of 3-methylindole in goats. Life Sci. 25, 1301-1306. (24) Frydman, R. B., Tomaro, M. L., and Frydman, B. (1972) Pyrrolooxygenases: isolation, properties, and products formed. Biochim. Biophys. Acta 284, 63-79. (25) Frydman, B., Frydman, R. B., and Tomaro, M. L. (1973) Pyrrolooxygenases: a new type of oxidases. Mol. Cell. Biochem. 2, 121-136. (26) Huijzer, J. C., Adams, J. D., Jr., Jaw, J.-Y., and Yost, G. S. (1989) Inhibition of 3-methylindole bioactivation by the cytochrome P-450 suicide substrates 1-aminobenzotriazole and amethylbenzylaminobenzotriazole. Drug Metab. Dispos. 17,37-42. (27) Bray, T. M., and Carlson, J. R. (1979) Role of mixed-function oxidase in 3-methylindole-induced acute pulmonary edema in goats. Am. J . Vet. Res. 40, 1268-1272. (28) Hanafy, M. S. M., and Bogan, J. A. (1980) The covalent binding of 3-methylindole metabolites to bovine tissue. Life Sci. 27, 1225-1231. (29) Nocerini, M. R., Carlson, J. R., and Breeze, R. G. (1983) Effect of glutathione status on covalent binding and pneumotoxicity of 3-methylindole in goats. Life Sci. 32, 449-458. (30) Nocerini, M. R., Yost, G. S., Carlson, J. R., Liberato, D. J., and Breeze, R. G. (1985) Structure of the glutathione adduct of activated 3-methylindole indicates that an imine methide is the electrophilic intermediate. Drug Metab. Dispos. 13, 690-694. (31) Bray, T. M., and Kubow, S. (1985) Involvement of free radicals in the mechanism of 3-methylindole-induced pulmonary toxicity: an example of metabolic activation in chemically induced lung disease. Enuiron. Health Perspect. 64, 61-67. (32) Mottley, C., and Mason, R. P. (1986) An electron spin resonance study of free radical intermediates in the oxidation of indole acetic acid by horseradish peroxidase. J. Biol. Chem. 261,16860-16864. (33) Still, C. C., Fukuyama, T. T., and Moyed, H. S. (1965) Inhibitory oxidation products of indole-3-acetic acid. J. Biol. Chem. 240, 2612-2618. (34) Hinman, R. L., Bauman, C., and Lang, J. (1961) The conversion of indole-3-acetic acid to 3-methyleneoxindole in the presence of peroxidase. Biochem. Biophys. Res. Commun. 5, 250-254. (35) Fukuyama, T. T., and Moyed, H. S. (1964) Inhibition of cell growth by photooxidation products of indole-3-acetic acid. J. Biol. Chem. 239, 2392-2397. (36) Kohn, M., and Ostersetzer, A. (1911) Einige neue Abkommling des Dioxindols. Monatsh. Chem. 32, 905-916. (37) Pilet, P.-E. (1962) L'hydroxy-3 methyl-3 oxo-2 indole, un des produits possibles du catabolisme de l'acide 8-indolylacetique. Bull. SOC.Chim. Biol. 44, 875-886. (38) Fish, K. M., Avaria, G. E., and Groves, J. T. (1988) Rearrangement of alkyl hydroperoxides mediated by cytochrome P-
Isolation of 3-Hydroxy-3-methyloxindole 450 evidence for the oxygen rebound mechanism. In Microsomes and Drug Oxidations (Minor, J. O., Birkett, D. J., Drew, R., May, B. K., and McManus, M. E., Eds.) pp 176-183, Taylor and Francis, Philadelphia, PA. (39) Thompson, J. A.,and Wand, M. D. (1985)Interaction of cytochrome P-450 with a hydroperoxide derived from butylated hy-
Chem. Res. Toxicol., Vol. 2, No. 4, 1989 259 droxytoluene. J. Biol. Chem. 260, 10637-10644. (40) Matsuura, T. (1977)Bio-mimetic oxygenation. Tetrahedron, 33,2869-2905. (41) Merenyi, G.,Lind, J., and Shen, X. (1988)Electron transfer from indoles, phenol, and sulfite @Os2-)to chlorine dioxide (C102). J. Phys. Chem. 92,134-137.
