Detection and characterization of DNA adducts of 3-methylindole.

The pneumotoxin 3-methylindole is metabolized to the reactive intermediate 3-methyleneindolenine which has been shown to form adducts with glutathione and proteins. Reported here is the synthesis, detection, and characterization of nucleoside adducts of 3-methylindole. Adducted nucleoside standards were synthesized by the reaction of indole-3-carbinol with each of the four nucleosides under slightly acidic conditions, which catalyze the dehydration of indole-3-carbinol to 3-methyleneindolenine. Following solid phase extraction, the individual adducts were infused via an electrospray source into an ion trap mass spectrometer for molecular weight determination and characterization of the fragmentation patterns. The molecular ions and fragmentation of the dGuo, dAdo, and dCyd adducts were consistent with nucleophilic addition of the exocyclic primary amine of the nucleosides to the methylene carbon of 3-methyleneindolenine. The apparent chemical preference of this addition lead primarily to dAdo and dGuo adducts, with substantially less of the dCyd adduct formed. No adduct with dThd was detected. The adducts were purified by HPLC and subsequent NMR analysis of the dGuo and dCyd adducts confirmed the proposed structures. Mass spectral fragmentation of the three adducts produced primarily two ions which were the result of the loss of either the 3-methylindole moiety or the sugar. On a triple quadrupole electrospray mass spectrometer, the neutral loss of the sugar, [M + H - 116](+), was utilized for selected reaction monitoring of the calf thymus DNA adducts, formed by incubations of 3-methylindole with various microsomes (rat liver, goat lung, and human liver). All three adducts were detected from each of the microsomal incubations, following extraction and cleavage of the DNA to the nucleoside level. The dGuo adduct was the primary adduct formed, with smaller amounts of the dAdo and dCyd adducts. Rat hepatocytes incubated with 3-methylindole produced the same three adducts, in approximately the same proportions, while no adducts were detected in untreated hepatocytes. Microsomal incubations in the presence of ([3-(2)H(3)]-methyl)indole confirmed the formation and identification of the adducts as well as the fragmentation patterns. These results demonstrate that bioactivated 3-methylindole forms specific adducts with exogenous or intact cellular DNA, and indicates that 3-methylindole may be a potential mutagenic and/or carcinogenic chemical.

An ion exchange liquid chromatography/mass spectrometry method for the determination of reduced and oxidized glutathione and glutathione conjugates in hepatocytes.

A rugged LC-MS/MS method was developed to quantify reduced and oxidized glutathione (GSH and GSSG, respectively) in rat hepatocytes. In addition, GSH conjugates can be detected, characterized and measured in the same analysis. Samples were treated with acetonitrile and iodoacetic acid to precipitate proteins and trap free GSH, respectively. These highly polar analytes were separated by ion exchange chromatography using conditions that were developed to be amenable to electrospray ionization and provide baseline chromatographic resolution. A solvent gradient with a total run time of 13 min was used to elute the analytes, as well as any highly retained components in the samples that would otherwise accumulate on the HPLC column and degrade the chromatography. The analytes were detected using either selected ion monitoring (SIM) using an ion trap mass spectrometer or selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer. The ranges for quantification of GSH and GSSG using an ion trap were 0.651-488 microM and 0.817-327 microM, respectively. Using SRM with the triple quadrupole instrument, the ranges of quantification for GSH and GSSG were 0.163-163 microM and 0.0816-81.6 microM, respectively. The accuracy and precision for both methods were within 15%. The utility of the method was demonstrated by treating rat hepatocytes with model compounds menadione and precocene I. Menadione, which contains a quinone moiety that undergoes redox cycling and induces concentration- and time-dependent oxidative stress in hepatocytes, resulted in decreased GSH concentrations with concomitant increase in concentrations of GSSG, as well as a GSH-menadione conjugate. When hepatocytes were incubated with precocene I, a time-dependent decrease in GSH concentrations was observed with concomitant increase in a GSH-precocene conjugate. GSSG concentrations did not increase in the presence of precocene I, consistent with its lack of redox activity. This analytical method has general utility for simultaneously investigating the potential of test compounds to induce both oxidative stress from redox cycling in vitro and the formation of GSH conjugates.

Evidence supporting the formation of 2,3-epoxy-3-methylindoline: a reactive intermediate of the pneumotoxin 3-methylindole.

The existence of a cytochrome P450-dependent 2,3-epoxide of the potent pneumotoxin 3-methylindole was indirectly confirmed using stable isotope techniques and mass spectrometry. Determination of hydride shift and incorporation of labeled oxygen in 3-methyloxindole and 3-hydroxy-3-methyloxindole, metabolites that may be in part dependent on the presence of the epoxide, were utilized as indicators of the epoxide's existence. One mechanism for the formation of 3-methyloxindole involves cytochrome P450-mediated epoxidation followed by ring opening requiring a hydride shift from C-2 to C-3. Through incubations of goat lung microsomes with [2-2H]-3-methylindole, the retention of 2H in 3-methyloxindole was found to be 81%, indicating a majority of the oxindole was produced by the mechanism described above. 3-Hydroxy-3-methylindolenine is an imine reactive intermediate that could be produced by ring opening of the 2,3-epoxide. The imine may be oxidized to 3-hydroxy-3-methyloxindole by the cytosolic enzyme aldehyde oxidase. Activities of this putative detoxification enzyme were determined in both hepatic and pulmonary tissues from goats, rats, mice, and rabbits, but the activities could not be correlated to the relative susceptibilities of the four species to 3-methylindole toxicity. The 18O incorporation into either 3-methyloxindole or 3-hydroxy-3-methyloxindole from both 18O2 and H218O was determined. The 18O incorporation into 3-methyloxindole from 18O2 was 91%, strongly implicating a mechanism requiring cytochrome P450-mediated oxygenation. Incorporation of 18O into 3-hydroxy-3-methyloxindole indicated that the alcohol oxygen originated from molecular oxygen, also implicating an epoxide precursor. These studies demonstrate the existence of two new reactive intermediates of 3-methylindole and describe the mechanisms of their formation and fate.

Characterization of carbamazepine metabolism in a mouse model of carbamazepine teratogenicity.

The disposition of carbamazepine (CBZ) was investigated in the SWV mouse. A 14C-CBZ dose was administered to CBZ pretreated mice, and the distribution of radiolabeled material was determined. Twenty-four hours after the 14C-CBZ dose, 92.5% of the dose was accounted for in urine (56%), in the visera and carcass (22%), in feces (11%), and expired as 14CO2 (2%). CBZ metabolites present in hydrolyzed urine were also identified using a combination of spectroscopic techniques. CBZ, CBZ-10,11-epoxide (CBZE), 2- and 3-hydroxy-CBZ, methylsulfonyl-CBZ, and glucuronides of CBZ and CBZE accounted for 64% of total urinary radioactivity (0-24 hr) in CBZ pretreated mice. Minor metabolites of CBZ included novel cysteine and N-acetylcysteine conjugates of CBZ, as well as a methylsulfonyl conjugate of CBZE not previously reported. The urinary excretion of these thioether conjugates was increased in CBZ/phenobarbital pretreated mice and decreased in CBZ/stiripentol pretreated mice in comparison with CBZ-only treated mice. Preliminary studies of the effects of phenobarbital and stiripentol on the urinary abundance of these metabolites are consistent with the modulation of teratogenicity in the SWV mouse by the same pretreatments. These data suggest the formation of thioether metabolites of CBZ may be related to CBZ teratogenicity in the SWV mouse.

Effects of finasteride, a type 2 5-alpha reductase inhibitor, on fetal development in the rhesus monkey (Macaca mulatta).

In genetic male fetuses, dihydrotestosterone (DHT) plays an important role in normal prostatic and external genital differentiation. The enzyme steroid 5-alpha reductase (5 alpha R) catalyzes the conversion of testosterone (T) to DHT. The importance of 5 alpha R in sexual differentiation is evident from the study of human genetic males who congenitally lack this enzyme and consequently develop ambiguous genitalia. These individuals are specifically deficient in the type 2 isozyme, whereas the normal type 1 isozyme activity has been found. The purpose of this study was to determine 1) the suitability of the rhesus monkey for testing the safety of 5 alpha R inhibitors when administered during pregnancy and 2) the potential risk of administering a known type 2 5 alpha R inhibitor, finasteride, during the critical period of internal and external genital differentiation in rhesus monkeys. In vitro studies were also performed on selected rhesus monkey tissues to determine the distribution of the 5 alpha R isozymes. Gravid monkeys were treated once daily from gestational days (GD) 20 to 100. Sonographic monitoring was performed during the course of gestation to monitor viability, growth, and organ system development. Detailed fetal evaluations for developmental abnormalities were performed at term (GD 152 +/- 2). A group of 13 pregnant monkeys ("positive control") were given a high oral dose (2 mg/kg/day) of finasteride to demonstrate that inhibiting type 2 5 alpha R results in specific external genital abnormalities in male fetuses. Thirty-two pregnant monkeys were administered an intravenous (i.v.) formulation of finasteride at doses of 8, 80, or 800 ng/day. The highest i.v. dose selected was at least 60-750 times the semen levels of finasteride in man given orally 5 or 1 mg/day, respectively. Seventeen vehicle-control pregnant monkeys were also included. Administration of a high oral dose (2 mg/kg/day) of finasteride resulted in external genital abnormalities characterized by hypospadias, preputial adhesions to the glans, a small underdeveloped scrotum, a small penis, and a prominent midline raphe in male fetuses; however, no developmental abnormalities were seen in female fetuses. Similarly, no abnormalities were observed in either male or female fetuses of mothers given iv doses (8, 80, or 800 ng/day) of finasteride during pregnancy. The in utero sonographic findings in fetuses correlated with the gross findings at term. These studies have shown that external genital abnormalities can be produced in male monkey fetuses when exposed to a high oral dose (2 mg/kg/day) of finasteride, whereas no abnormalities were observed in fetuses exposed to the i.v. formulation of finasteride. Detailed in vitro studies demonstrated that the rhesus monkey also has two 5 alpha R isozymes (types 1 and 2) with a tissue distribution similar to that seen in man and, furthermore, that finasteride is a potent, mechanism-based inhibitor with selectivity for both human and rhesus type 2 5 alpha R. These studies have demonstrated that the monkey is a suitable model for assessing the safety of 5 alpha R inhibitors administered during pregnancy.

Teratogenicity of carbamazepine-10, 11-epoxide and oxcarbazepine in the SWV mouse.

The mechanism of carbamazepine (CBZ)-related teratogenicity was investigated in the SWV mouse by contrasting the effects of CBZ-10, 11-epoxide (CBZE) and oxcarbazepine (OXC) treatments. Dietary CBZE administration was initiated 2 weeks before mating and continued through day 18 of gestation. OXC was administered to pregnant dams by gavage on day 6 of gestation and continued through day 18 of gestation. Maternal plasma concentrations of CBZE ranged from 1.4 to 17.7 micrograms/ml and OXC ranged from 6.1 to 15.9 micrograms/ml. In comparison, clinical plasma concentrations of CBZE ranged from 1 to 2 micrograms/ml and OXC plasma concentrations were 1 microgram/ml or less. The incidence of malformation were 14%, 27% and 26% after daily CBZE doses of 300, 600 and 1000 mg/kg, respectively, compared with a 6% incidence in no-drug control mice, P < .05. The incidence of malformation was 8% after exposure at the highest tolerable dose of OXC (1100 mg/kg/day), compared with a 5% incidence in no-drug controls, P > .05. Phenobarbital cotreatment (45 mg/kg/day) with OXC (1100 mg/kg/day) did not lead to changes in the incidence of malformation when compared with OXC (1100 mg/kg/day) dosed alone. These data are consistent with a teratogenic CBZ metabolite, possibly CBZE, or with oxidation of CBZE or CBZ at positions on the aromatic ring leading to the formation of reactive intermediates such as arene oxides or quinones.

Mechanistic studies on the cytochrome P450-catalyzed dehydrogenation of 3-methylindole.

The mechanism of 3-methyleneindolenine (3MEI) formation from 3-methylindole (3MI) in goat lung microsomes was examined using stable isotope techniques. 3MEI is highly electrophilic, and its production is a principal factor in the systemic pneumotoxicity of 3MI. Noncompetitive intermolecular isotope effects of DV = 3.3 and D(V/K) = 1.1 obtained after deuterium substitution at the 3-methyl position indicated either that hydrogen abstraction from the methyl group was not the initial rate-limiting step or that this step was rate-limiting and was masked by a high forward commitment and low reverse commitment to catalysis. An intramolecular isotope effect of 5.5 demonstrated that hydrogen atom abstraction was probably the initial oxidative and rate-limiting step of 3MI bioactivation or that deprotonation of an aminium cation radical, produced by one-electron oxidation of the indole nitrogen, was rate-limiting. However, a mechanism which requires deprotonation of the aminium cation radical is probably precluded by an unusual requirement for specific base catalysis at a site in the cytochrome P450 enzyme other than the heme iron. The pattern of 18O incorporation into indole-3-carbinol from 18O2 and H(2)18O indicated that approximately 80% of the indole-3-carbinol was formed in goat lung microsomes by hydration of 3MEI. However, the inverse reaction, dehydration of indole-3-carbinol, did not significantly contribute to the formation of 3MEI. These results show that 3MEI was formed in a cytochrome P450-catalyzed dehydrogenation reaction in which the rate-limiting step was presumably hydrogen atom abstraction from the 3-methyl position. The ratio of the amounts of 3MEI to indole-3-carbinol formed (50:1) indicated that dehydrogenation of 3MI is an unusually facile process when compared to the dehydrogenation of other substrates catalyzed by cytochrome P450 enzymes.

Metabolism and bioactivation of 3-methylindole by Clara cells, alveolar macrophages, and subcellular fractions from rabbit lungs.

3-Methylindole (3MI), a fermentation product of tryptophan produced by intestinal and ruminal microflora, has been shown to cause pneumotoxicity in several species subsequent to cytochrome P450-mediated biotransformation. Among several species studied, rabbits are comparatively resistant to 3MI-induced pneumotoxicity, especially when compared to goats or mice. In this study, rabbit pulmonary cells and subcellular fractions were used to examine the metabolism and bioactivation of 3MI. A covalent-binding metabolite was produced in 3MI incubations by both Clara cells and macrophages. The addition of the cytochrome P450 inhibitor, 1-aminobenzotriazole, to these incubations inhibited the production of covalent-binding metabolite(s) by 94% in Clara cells and only 24% in macrophages. In incubations of Clara cells or macrophages with 3MI and N-acetylcysteine (NAC), a polar conjugate was detected and tentatively identified as an adduct of 3-hydroxy-3-methylindolenine (3H3MIN). Also identified were 3[(N-glutathione-S-yl)-methyl]-indole (3MI-GSH) and 3-methyloxindole (3MOI). In rabbit lung microsomal incubations with 3MI and glutathione (GSH), 3MI-GSH, 3MOI, indole-3-carbinol, and a GSH adduct of 3H3MIN were identified. The addition of cytosol to the microsomal incubations with GSH did not increase the rate of formation of the GSH adducts, indicating that cytosolic GSH-S-transferases are not essential in the formation of these metabolites. GSH significantly decreased the covalent binding of an electrophilic metabolite in microsomal incubations. These data suggest that GSH may be important in the mitigation of 3MI toxicity. Furthermore, the comparison of 3MI bioactivation to electrophilic intermediates in Clara cells and alveolar macrophages suggests that 3MI is metabolized by different oxidative pathways in the two different cell types, although the same metabolites were produced by the two cell types. This study shows that rabbit pulmonary enzymes are capable of bioactivating 3MI to reactive intermediates which become covalently bound to cellular macromolecules. This indicates that the relative resistance of rabbits to 3MI-induced pneumotoxicity is probably not due to differences in metabolic enzymes which convert 3MI to reactive intermediates.

Identification of goat and mouse urinary metabolites of the pneumotoxin, 3-methylindole.

1. Urine from goats dosed i.v. with 3-methylindole (3MI; 15 mg/kg) or [methyl-14C] 3MI (15 mg/kg, 0.5 microCi/kg) contained at least 11 metabolites of 3MI. 2. Goat metabolized 3MI to sulfate conjugates of 4- or 7-hydroxy-3-methyloxindole, 5- or 6-hydroxy-3-methyloxindole, and 3,5- or 6-dihydroxy-3-methyloxindole; glucuronic acid conjugates of indole-3-carboxylic acid and 4- or 7-hydroxy-3-methyloxindole; and unconjugated 3-hydroxy-3-methyloxindole. Diastereoisomeric glucuronic acid conjugates of 3-hydroxy-3-methyloxindole were also identified in goat urine. 3. Urine from mice dosed i.p. with 3MI (400 mg/kg) or [ring-UL-14C] 3MI (400 mg/kg, 125 microCi/kg) contained at least six metabolites of 3MI. 4. Mice metabolized 3MI to glucuronic acid conjugates of 3,5- or 6-dihydroxy-3-methyloxindole, 5- or 6-hydroxy-3-methyloxindole, and indole-3-carboxylic acid; and unconjugated indole-3-carboxylic acid. Unconjugated 3-hydroxy-3-methyloxindole was identified in mouse urine in a previous report. 5. Both goats and mice metabolized 3MI to a mercapturate, 3-[(N-acetyl-L-cystine-S-yl)methyl]indole, which has been previously identified and was confirmed in this study. 6. 3-Methyloxindole was not identified in the urine of either goats or mice. 7. The major pathways of 3MI biotransformation in goats and mice is the formation of mono- and dihydroxy-3-methyloxindoles and their subsequent conjugation with glucuronic acid or sulfate. 8. There are no apparent qualitative differences in the biotransformation of 3MI between goats and mice that can account for their different sensitivities to 3MI-induced lung injury.

Identification of a cysteinyl adduct of oxidized 3-methylindole from goat lung and human liver microsomal proteins.

3-Methylindole is a selective pneumotoxin that is oxidized by cytochrome P-450 enzymes to a reactive intermediate. 3-Methyleneidolenine, a methylene imine electrophile, is the postulated reactive intermediate, and it binds to proteins, a reaction that probably initiates the pneumotoxicity of 3-methylindole. Thioether adducts of this electrophile are formed with glutathione in vitro, but the identity of the adducted electrophile with amino acid residues of microsomal proteins had not previously been determined. 3-Methylindole was incubated with NADPH and goat lung or human liver microsomal proteins, and the proteins were hydrolyzed. 3-(Cystein-S-ylmethyl)indole was isolated and identified as the major amino acid adduct of 3-methyleneindolenine, demonstrating that cysteine thiols preferentially attack the exocyclic methylene position and result in a covalently (thioether) attached 3-methylindole residue to these pulmonary and hepatic proteins. These results demonstrate that the putative methylene imine intermediate is indeed the active electrophile that binds to proteins and presumably initiates the toxic events.

Isolation of a mercapturate adduct produced subsequent to glutathione conjugation of bioactivated 3-methylindole.

Bioactivation of the pneumotoxin 3-methylindole (3MI) to a methylene imine intermediate has been demonstrated previously by trapping the electrophile with glutathione in goat lung microsomal incubations. To determine whether the same bioactivation process occurs in whole animals, 3MI was administered to goats, mice, and rats, and the urinary metabolites from these three species were analyzed by HPLC for the presence of the mercapturate that would be expected as the processed and excreted form of the 3MI-glutathione adduct. The mercapturate, 3-[(N-acetylcysteine-S-yl)-methyl]indole (3MI-NAC), was identified in the urine from all three species and was isolated from rat urine for structural identification by uv, NMR, and mass spectrometry. Synthetic 3MI-NAC had uv, NMR, and chromatographic characteristics identical to the isolated metabolite. The presence of this mercapturate in the urine of treated animals unequivocally demonstrates that 3MI is bioactivated to the methylene imine in vivo and that the glutathione adduct is also formed, presumably to detoxify the methylene imine.

Isolation and identification of 3-hydroxy-3-methyloxindole, the major murine metabolite of 3-methylindole.

3-Methylindole (3MI) is pneumotoxic to ruminants and rodents subsequent to 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 to 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 1H 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.