Mechanistic studies of the cytochrome P450-dependent oxygenation and dehydrogenation of the pneumotoxin 3-methylindole

3-Methylindole is an extremely pneumotoxic compound tat requires P450-mediated dehydrogenation to an electrophilic intermediate, 3-methyleneindoline, to elicit its toxicity. The pulmonary cytochromes P450 2F1 (human) and 2F3 (goat) exclusively catalyze the dehydrogenation reaction, without formation of oxygenated metabolites. The mechanism that govern selectivity between cytochrome P450-mediated oxygenation and dehydrogenation of 3-methylinodole have not elucidated. The hypothesis for these studies was that differentiation between P450-medicated 2,3-oxygenation and 3-methyl dehydrogenation of 3-methylindole is controlled by unique active site differences exhibited by pulmonary P450 enzymes. The mechanisms of the formation of the P450-meditaed oxygenation-dependent metabolites 3-methyloxindole and 3-hydroxy-3-methyloxindole were determined. An epoxide, 2,3-epoxy-3-methylindoline was demonstrated as an intermediated in the formation of both metabolites. 3-Methyloxindole retained P450-introduced oxygen at position 2, formed by a mechanism involving and "NIH" shift. 3-Hydroxy-3-methyloxindole retained P-450-introduced oxygen at position 3, and probably originated from the epoxide, which also formed the electrophilic intermediated 3-hydrosxy-3-methylindolenine. The presence of this intermediate was confirmed through characterization of novel intra-molecular thioether conjugate with thioglycolic acid. These data are consistent with formation of the precursor epoxide, that reacts by multiple ring-opening mechanisms. Site directed mutagenesis of cytochrome P450 2F3 was performed to identify active site residues that direct of participate in the dehydrogenation of 3-methylindole. Additionally, a 3-dimensional homology model was constructed, based on the structure of rabbit P450 2C5, for 2F3. When mutations were made to SR 1, the mutant P450 enzyme was not produced. Enzymes with mutations to SRS 4, 5 and 6 were successfully expressed in bacteria and their functions were characterized. The SRS 4 mutant enzyme was catalytically identical to the wild type, with regard to 30methylindole dehydrogenation, but a 3-methylindole-oxygenase function was introduced to 2F3 when mutations were made in either SRS 5 or 6. Analysis of the homology model indicated that the sites of mutation were proximal to the active site. These studies have provided specific novel methods to distinguish oxygenation vs. dehydrogenation of 3-methylindole, and have identified critical SRS residues in 2F3 that help direct the unique dehydrogenation mechanism of the pulmonary enzyme.

MECHANlSTIC STUDIES OF THE CYTOCHROME P450-DEPENDENT OXYGENATION AND DEHYDROGENATION OF THE PNEUMOTOXIN 3-METHYLINDOLE by Konstantine William Skordos A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Pharmacology Department of Pharmacology and Toxicology The University of Utah December 2002 Copyright © Konstantine William Skordos 2002 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Konstantine W. Skordos This dissertation has been read by each member of the following supervisory committtee and by majority vote has been found to be satisfactory. Ch William K. Nichols Michael R. Franklin Darrell R. Davis THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Konstantine W. Skordos in its final form and have found that (1) its format, citations, and bibliographic style are consistent and acceptable: (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate Scho '} /? /J tJ~ f d()()~ ~~ ) arold S. Yost Date Approved for the Major Department Approved for the Graduate Council ABSTRACT 3-Methylindole is an extremely pneumotoxic compound that requires P450- mediated dehydrogenation to an electrophilic intermediate, 3-methyleneindolenine, to elicit its toxicity. The pulmonary cytochromes P450 2FI (human) and 2F3 (goat) exclusively catalyze the dehydrogenation reaction, without formation of oxygenated metabolites. The mechanisms that govern selectivity between cytochrome P450- mediated oxygenation and dehydrogenation of 3-methylindole have not been elucidated. The hypothesis for these studies was that differentiation between P450-mediated 2,3- oxygenation and 3-methyl dehydrogenation of 3 -methylindole is controlled by unique active site differences exhibited by pulmonary P450 enzymes. The mechanisms of the formation of the P450-mediated oxygenation-dependent metabolites 3-methyloxindole and 3-hydroxy-3-methyloxindole were determined. An epoxide, 2,3-epoxy-3- methylindoline was demonstrated as an intermediate in the formation of both metabolites. 3-Methyloxindole retained P450-introduced oxygen at position 2, formed by a mechanism involving an "NIIf' shift. 3-Hydroxy-3-methyloxindole retained P-450- introduced oxygen at position 3, and probably originated from the epoxide, which also formed the electrophilic intermediate 3-hydroxy-3-methylindolenine. The presence of this intermediate was confirmed through characterization of a novel intramolecular thioether conjugate with thioglycolic acid. These data are consistent with formation of the precursor epoxide, that reacts by multiple ring-opening mechanisms. Site directed mutagenesis of cytochrome P450 2F3 was performed to identify active site residues that direct or participate in the dehydrogenation of 3-methylindole. Additionally, a 3-dimensional homology model was constructed, based on the structure of rabbit P450 2C5, for 2F3. When mutations were made to SRS 1, the mutant P450 enzyme was not produced. Enzymes with mutations to SRS 4, 5, and 6 were successfully expressed in bacteria and their functions were characterized. The SRS 4 mutant enzyme was catalytically identical to the wild type, with regard to 3-methylindole dehydrogenation, but a 3-methylindole-oxygenase function was introduced to 2F3 when mutations were made in either SRS 5 or 6. Analysis of the homology model indicated that the sites of mutation were proximal to the active site. These studies have provided specific novel methods to distinguish oxygenation vs. dehydrogenation of 3- methyl indole, and have identified critical SRS residues in 2F3 that help direct the unique dehydrogenation mechanism of this pulmonary enzyme. v This manuscript is dedicated to my late father, Dr. William Konstantine Skordos, for his life-long adherence to the belief that you can do anything, if you put your mind to it. TABLE OF CONTENTS ABSTRACT ................................................................................................................... iv ACKNOWLEDGEMENTS ............................................................................................ ix Chapter 1. INRODUCTION .................................................................................................... 1 Cytochrome P450: Properties and Reactions ....................................................... 1 3-Methylindole: Toxicity and Bioactivation ...................................................... II General Importance ofP450-Catalyzed Dehydrogenation ................................. 19 The Cytochrome P450 2F Sub-family Enzymes ................................................ 22 Other P450 Isozyme Involvement in 3-Methylindole Metabolism ..................... 23 Research Objectives of this Study ..................................................................... 26 References ................. '" .................................................................................... 34 2. EVIDENCE SUPPORTING THE FORMATIONOF 2,3-EPOXY -3-METHYLINDOLENINE: A REACTIVE INTERMEDIATE OF THE PNEUMOTOXIN 3-METHYLINDOLE ........................................................................................... 39 Introduction ...................................................................................................... 40 Experimental Procedures .................................................................................. 42 Results ............................................................................................................. 44 Discussion ........................................................................................................ 46 References.... ........ . ....................... .... ...... ... ......... . . .. ... ............ ..... ...... . .............. 47 3. TIDOETHER ADDUCTS OF A NEW IMINE REACTIVE INTERMEDIATE OF THE PNEUMOTOXIN 3-METHYLINDOLE .................. 49 Introduction ...................................................................................................... 50 Experimental Procedures .................................................................................. 51 Results ............................................................................................................. 52 Discussion ........................................................................................................ 54 References ............................................................................................ , ........... 55 4. HETEROLOGOUS EXPRESSION OF THE PULMONARY CYTOCHROMES P450 2Fl AND 2F3 ................................................................ 56 Materials and Methods ..................................................................................... 59 Results ............................................................................................................. 88 Discussion ........................................................................................................ 97 References ...................................................................................................... 100 5. INTRODUCTION OF 3-METHYLINDOLE OXYGENASE ACTIVITY TO CYTOCHRO:MES P450 2F3 ..................................................... 104 Materials and Methods ................................................................................... 108 Results ........................................................................................................... 126 Discussion ...................................................................................................... 159 References ...................................................................................................... 167 6. CONCLUSIONS ................................................................................................ 171 References ...................................................................................................... 178 Vlll ACKNOWLEDGEMENTS I would like to thank the members of my thesis advisory committee for their time and valuable suggestions during this research project. In particular, I would like to thank Drs. William Nichols and Michael Franklin for their support and guidance, both at the undergraduate and graduate levels of my training. I am deeply grateful for the professional and personal support extended by my advisor, Dr. Garold Yost. I sincerely appreciate the unending optimism, encouragement and guidance he provided throughout my graduate training. I would like to thank Ms. Diane Lanza for her technical assistance throughout this research project. Her involvement is instrumental in the completion of any research endeavor in our laboratory. I would like to thank Dr. Eric Johnson for his assistance in the construction and interpretation of the three-dimensional model of cytochrome P450 2F3. I would also like to express my appreciation of the friendship and support provided by the members of my graduate school class, Mr. Brian Carr, Mr. Joseph Yeh and Dr. Matthew Barton. I am deeply grateful to my friends and family. Without the love, support and understanding provided my mother Susan, my sisters Angela and Amy and my wife LeAnne the completion of this research project would not have been possible. CHAPTER 1 INTRODUCTION Cytochrome P450: Properties and Reactions The cytochromes P450 are a superfamily of heme-thiolate enzymes that catalyze the oxidation of a wide variety of endogenous and xenobiotic compounds (Nelson et aI., 1996). The enzymes are in fact the major catalysts in the oxidation of xenobiotics, including therapeutic compounds (Guengerich, 2001a). Cytochromes P450 are present throughout the spectrum of biological organisms, from bacteria to humans (Nelson et aI., 1996). At present, there are 977 animal cytochrome P450 sequences, 60 of which are human (drnelson.utmem.edulCytochromeP450.html, accessed on June 22, 2002). This represents strong evolutionary pressure for the maintenance of the enzymes, and results in great diversity of enzyme function. In mammalian systems, the enzymes are membrane-bound, and typically are located in the endoplasmic reticulum (Dallner and Ernster, 1968) and the inner mitochondrial membrane (Black, 1992). The cytochromes P450 are expressed in the highest concentration in the liver (Meyer, 1996), but extrahepatic expression is also very important to the metabolism of drugs and biochemically-mediated toxicity (Krishna and Klotz, 1994). The enzymes catalyze a diverse array of oxidative reactions, which are involved in biosynthetic processes as well 2 as metabolism of xenobiotics. For the purposes of this introduction, only cytochrome P450 reactions that relate to xenobiotic metabolism will be described. These reactions are the most germane to a discussion of cytochrome P450's role in biochemical toxicity. The Cytochrome P450 Reaction The general reaction cycle for cytochrome P450-mediated oxidation is presented in Figure 1.1. The first step in the P450 reaction is binding of the substrate, which in some cases causes the heme spin state to change from a low to high state. This permits reduction of the heme iron from the resting fen-ic to the ferrous form, via the participation of cytochrome P450 reductase and NADPH. Next, molecular oxygen binds to the heme to give a ferrous dioxygen complex. A second electron is then transferred via cytochrome P450 reductase (through cytochrome bs in some cases) and NADPH, to give a peroxo-iron III complex. The distal oxygen is then protonated, resulting in the loss of water, and the formation of the active oxygen species. Debate still exists as the exact electronic identity of the active oxygen species. However, it is generally believed that the active oxidant is an oxo-iron IV, radical porphyrin cation, analogous to compound I of the related peroxidase enzymes. This conclusion has recently been substantiated by spectroscopic studies of model P450 active oxidant species (Harris et aI., 2001). In many cases, the next step is the introduction of the oxygen atom into the substrate, in the form of a hydroxyl group. Additional cytochrome P450-medated oxidation reactions include epoxidation, heteroatom oxygenation, dealkylation, dehydrogenation and others. The reactions most relevant to this discussion include hydroxylation, epoxidation and dehydrogenation. 3 Figure 1.1. The general reaction cycle for cytochrome P450 enzymes. Hydroxylation H erne resting state ROH \ Y 3+ Fe ~strate (RH) Fe3+ RH Fe3+ ROH nVia (NADPH and Cytochrome P450 i Dehydrogenation I C Reductase) -R=R'- ~ I' FeOH Fe2+ RH 3 + R· H20 I r02 4+ FeO RH ~ ;:'02RH H20 FeOOH ~ Fe2 +02- RH e- via (NADPH and Cytochrome P450 Reductase RH TT+J or Cytochrome b5) H ~ 5 Hydroxylation The general mechanism for cytochrome P450-mediated hydroxylation is presented in Figure 1.2. The reaction is believed to proceed by initial hydrogen atom abstraction from a carbon atom, leaving a carbon-centered radical. The active oxygen species then rebounds to the radical, leaving the hydroxylated form. Evidence for this reaction pathway exists from studies in which a high intrinsic deuterium isotope effect was observed when deuterium was substituted for hydrogen (Ortiz de Montellano, 1995). This is consistent with a reaction involving an initial step involving hydrogen atom abstraction. Additional evidence exists from studies of the stereochemistry of hydroxylation reactions. Stereochemical scrambling is observed for many hydroxylation reactions, indicating a non-concerted reaction (Ortiz de Montellano, 1995). Epoxidation Epoxidation reactions have been studied extensively due to their role in biochemical toxicity. Epoxide products can be unstable and form adducts with cellular nuc1eophiles such as proteins and DNA (Guengerich, 2001a). The precise chemical mechanism for epoxidation of aromatic compounds by cytochrome P450 enzymes has not been fully characterized. The epoxide in this case is probably a short-lived highly reactive intermediate. Strong evidence of its formation can come from characterization of hydroxylated glutathione conjugates (Guengerich, 2001a) and demonstration of a hydride or "NIH" shift (Guroff et a1., 1967) (Figure 1.2). However, an alternative P450- mediated oxygenation pathway has been proposed in which similar molecular migrations Figure 1.2. Examples ofP450-mediated hydroxylation and epoxidation. Panel A shows the hydroxylation of testosterone. Panel B shows the epoxidation of the fungal toxin aflatoxin. 6 7 A. OH OH + FeOH3 + o o Testosterone OH o OH 6P-Hydroxytestosterone B. o o o o P450 .. o Aflatoxin B I Epoxide ! DNA Ad~UCts Carcinogenesis 8 can occur (Darbyshire et aI., 1996). Therefore, demonstration of an "NIH" shift is not necessarily conclusive evidence of an epoxide' s existence. Dehydrogenation Cytochrome P450-mediated dehydrogenation reactions are much less common than oxygenation reactions. However, their importance to the study of drug metabolism and biochemically mediated toxicity is increasing. Mechanistic studies of cytochrome P450-mediated dehydrogenation have been performed for both fatty acid-like compounds (Rettie et aI., 1988; Guan et ai., 1998) and aromatic compounds (Skiles and Yost, 1996). Kinetic deuterium isotope data implicate a mechanism involving an initial hydrogen atom abstraction followed by a second electron abstraction from the substrate yielding the dehydrogenated product (Figure 1.3). Role of Cytochrome P450 Reactions The role of cytochrome P450 oxidation reactions is to increase the solubility of lipophilic compounds for either direct excretion from the body, or to provide a substrate for subsequent conjugative reactions. These reactions also facilitate excretion from the body. Therefore, in the general case, the role of cytochrome P450 is the detoxication of xenobiotic compounds, by facilitating excretion. However, P450-mediated oxidation reactions frequently lead to metabolic products that are more reactive than the parent compound, potentially to toxicity and carcinogenicity. The issue of P450-mediated toxicity is very important in the fields of toxicology and drug metabolism. The toxicity associated with the therapeutic compounds Figure 1.3. Examples ofP450-mediated hydroxylation with accompanying dehydrogenation reactions. Panel A shows the hydroxylation and dehydrogenation of 3-methylindole. Panel B shows the hydroxylation and dehydrogenation of valproic acid. 9 A. B. Pr C;VC02H H VPA P450 ... P450 ... ~OH VN~ ~gen Rebound Pr CH3VC02H H OH 4-0H-VPA Pr CH2VC02H H ~4,5_VPA --..-. Hepatotoxicity 10 11 valproic acid (Rettie et aI., 1987) and acetaminophen (Raucy et aI., 1989) are both directly attributable to cytochrome P450-dependent formation of highly reactive intermediates that form covalent adducts with cellular nuc1eophiles. The toxicity and carcinogenicity associated with certain environmental and industrial compounds is aIso attributable to P450 action. The toxicity of naphthalene (Yost et aI., 1989), ethylcarbamate (urethane) (Forkert and Lee, 1997), and aflatoxin (Guengerich et aI., 1996) results from cytochrome P450 bioactivation. 3-Methylindole: Toxicity and Bioactivation 3-Methylindole is an example of an environmental compound that exhibits profound pulmonary toxicity following bioactivation by cytochrome P450 enzymes (Bray and Carlson, 1979). 3-Methylindole is a degradation product of the amino acid tryptophan. Tryptophan is first converted to indole-3-acetic acid in the gut, and is subsequently converted to 3-methylindole by a Lactobacillus sp. (Figure 1.4) (Carlson et aI., 1972). The toxicity of 3-methylindole was first characterized in cattle, when grazing conditions were rapidly changed from sparse to lush pastures. A pulmonary disease was observed in the cattle, which resulted in pulmonary edema, respiratory failure and death (Hammond et aI., 1979). Further characterization revealed that the change in grazing conditions resulted in a perturbation in the ruminal microfloral environment. This permitted increased bacterial conversion of tryptophan to 3-methylindole, and its subsequent toxicity. It was discovered that the pulmonary toxicity could be prevented by treating the cattle with an antibiotic regimen, to eliminate the bacteria required for 3- methylindole formation (Hammond et aI., 1980). The pulmonary toxicity of 12 Figure 1.4. Formation of 3-methylindole from tryptophan. NH2 Tryptophan \ CH 'COOH COOH Indole-3-acetic acid 3-Methylindole 13 14 3-methylindole is observed in other species including sheep, rats and mice (Carlson et aI., 1972; Hammond et al., 1979; Huijzer et al., 1987). However, goats and cattle are the species most sensitive to 3-methylindole's pulmonary toxicity (Yost, 1989). An intravenous dose of30-40 mg/kg is usually lethal to goats. Under normal conditions, the toxicity of 3-methylindole is only observed in pulmonary tissue, despite the fact that it is formed in the gut. Therefore, it must undergo systemic circulation prior to reaching the lung. This indicates that the bioactivation process required for its toxicity probably occurs in the target organ (Foth, 1995). Humans are exposed to 3-methylindole though a variety of sources. Colonic fermentation of tryptophan to form 3-methylindole is believed to occur, and 3- methyl indole is detectable in human feces at a level of 16-1S0 J.lg/g (Karlin et aI., 1985; Dehnhard et aI., 1991). 3-Methylindole is a pyrolysis product present in cigarette smoke, 0.4-1.4 J.lg/cigarette (Wynder and Hoffman, 1967). Additionally, 3-methylindole is present in coffee and certain cheeses and seafood (Thornton-Manning, 1992). 3-Methylindole Bioactivation: Oxygenation and Dehydrogenation The cytochrome P4S0-mediated metabolism of 3-methylindole has been well characterized, and a metabolic pathway is presented in Figure 1.5. Metabolic studies have been performed in mice, goats and pigs (Skiles et aI., 1989; Smith et aI., 1993; Smith et al., 1996; Diaz et aI., 1999). The metabolism of 3-methylindole by cytochrome P4S0 enzymes occurs though two pathways, oxygenation and dehydrogenation (Yost, 1996). Cytochrome P4S0-mediated oxygenation of 3-methylindole produces the stable metabolites, 3-methyloxindole and 3-hydroxy-3-methyloxindole, that are conjugated with 15 Figure l.5. Metabolic pathway of 3-methylindole, showing putative reactive intermediates that may control the pneumotoxicity and/or carcinogenicity of this lung toxicant. P450 - 3-Methyl Oxidation Protein and DNA.. cQ~ Adducts- ~ h Pulmonary Toxicity .b- NH 3-Methy leneindolenine Dehydrogenation ... V~-J 3-Methy lindole P450 - 2,3 Oxygenation 1 H20 / Hydroxylation ~? ONl , ,... Protein and DNA Adducts? ~OH UN' Indole-3-caIbinol 2,3-Epoxy -3-methylindobne / V~-roJ= 3 -Methy loxidole ~ ~OH ~ Pro~nandDNA I .... c:9 h Adducts? .b- NH 3-Hydroxy -3-methy lindolenine 1 Aldehyde Oxidase o}:H ~ I 0 .b- NH 3-Hydroxy-3-methyloxindole -0'\ 17 glucuronic acid (Smith et ai., 1993; Smith et ai., 1996). The formation of these metabolites may involve one oxygenated intermediate in the form of the 2,3-epoxide, 2,3- epoxy-3-methylindoline. If produced, an epoxide of 3-methylindole may contribute to the toxicity observed for this compound. The toxicity of epoxide reactive intermediates has been introduced previously. Several mechanistic possibilities exist for the formation of each metabolite from a precursor epoxide. Epoxide ring opening to position 2 followed by tautomerization can form the metabolite 3-methyloxindole. This mechanism may be accompanied by the Nlli shift, previously described, of the 2-position hydrogen atom to position 3. Epoxide ring opening to position 3, can lead to the proposed reactive intermediate, 3-hydroxy-3-methylindolenine. This intermediate may participate in the toxicity of 3-methylindole. Additionally, this intermediate may be a substrate for the cytosolic enzyme aldehyde oxidase. Action of this enzyme would produce the stable metabolite 3-hydroxy-3-methyloxindole. This metabolite is in fact the major urinary metabolite formed from 3-methylindole in mice (Skiles et ai., 1989), and it is found in human urine as well (Albrecht et ai., 1989). Additional studies are required to elucidate the identity of the oxygenation-dependent precursor to these metabolites. At least some, and perhaps a majority of the toxicity of 3-methylindole IS attributable to P450-dependent dehydrogenation at the methyl position to form the intermediate 3 -methyleneindolenine (Skiles and Yost, 1996). The presence of this highly electrophilic intermediate was originally proposed and structurally characterized by analyzing its glutathione adduct, originally isolated from cattle, and later from microsomal incubations containing 3-methylindole (Nocerini et aI., 1985a; Nocerini et aI., 1985b; Yost et aI., 1986). Additional evidence of the metabolite's involvement in 18 pulmonary toxicity came from studies in which mice were treated with 3-methylindole that had been deuterated at the methyl position. Methyl-position deuteration of 3- methylindole attenuated the toxicity, demonstrating that oxidation at this position is requisite for toxicity (Huijzer et aI., 1987). Cellular protein binding of 3- methyleneindolenine has also been demonstrated in HepG2 cells that overexpress certain P450 isozymes (Thornton-Manning et aI., 1991; Thornton-Manning et aI., 1996). Antibodies to a mimetic of 3-methyleneindoleine-bound protein were generated and utilized for immunohistochemical analysis of tissues from animals that were exposed to 3-methylindole. Immunoreactive material was detectable in the pulmonary tissues of susceptible species, including humans (Kaster and Yost, 1997). Recently, DNA adducts of 3-methyleneindolenine have been isolated (Regal et aI., 2001). This potentially indicates that the compound has genotoxic properties as well. The mechanism of P450-mediated dehydrogenation of 3-methylindole was evaluated extensively using goat lung microsomes (Skiles and Yost, 1996). Direct evidence for a mechanism involving an initial hydrogen atom abstraction step that may be rate determining, was provided by intramolecular isotope effect data. Studies in which incubations were performed with 3-eHr methyl]-indole, demonstrated an intramolecular isotope effect (kH{kD) of 5.5 for the formation of3-methyleneindolenine. Dehydrogenation also leads to the stable, oxygenated metabolite indole-3- carbinol. Hydroxylation of the compound at the 3-methyl position yields the reasonably stable metabolite, indole-3-carbinol. At least two mechanisms for its formation exist, and they have been evaluated in detail (Skiles and Yost, 1996). The first involves an initial hydrogen atom abstraction from the methyl position followed by a very fast hydroxyl 19 rebound from the heme to produce the alcohoL The second mechanism again involves initial hydrogen atom abstraction from the methyl position. Rather than hydroxyl rebound from the heme, the dehydrogenated product is released and hydrates to form the alcohol. The contribution of each mechanism could be evaluated by performing incubations with 180 2 or H2 180 . Using this method, the source of oxygen atoms introduced into the product could be tracked. Cytochrome P450-introduced oxygen would originate from O2, while oxygen introduced by some other mechanism would originate from H20. Surprisingly, dehydrogenation followed by hydration accounted for 80% of indole-3-carbinol produced in metabolic incubations of goat lung microsomes. General Importance ofP450-Catalyzed Dehydrogenation As stated previously, dehydrogenation reactions are considered uncommon among cytochrome P450 enzymes (Guengerich, 2001b). However, as demonstrated by the example of 3-methylindole, they can participate in the generation of reactive electrophilic intermediates that directly participate in chemical toxicity. Another example is the dehydrogenation of valproic acid to form the L\ 4,5 -valproic acid intermediate. This intermediate was proposed to be an important mediator of the hepatotoxicity that is associated with this anticonvulsant agent (Rettie et ai., 1987). Additionally, characterization of the formation of this intermediate provided the first mechanistic study of the P450-catalyzed dehydrogenation process (Rettie et ai., 1988). When deuterium was substituted at position 4, a deuterium isotope effect of 5.6 was observed, both for the formation of the dehydrogenated product and the 4 position alcohol. This is consistent with a mechanism that is initiated by hydrogen atom 20 abstraction, followed by competition between hydroxyl rebound from the heme, and a second electron abstraction to form the dehydrogenated product. The study also demonstrated that the dehydrogenated product and the alcohol do not interconvert. In the general case of dehydrogenation of fatty acid like substrates, the hydroxylation reaction predominates (Guengerich, 2001a). This was somewhat contrary to the observations of the dehydrogenation of 3-methylindole (Skiles and Yost, 1996). In this case the dehydrogenated product did hydrate to form the alcohol and in fact this was the major mechanism for its formation. However, at physiological pH, the alcohol does not dehydrate to form the dehydrogenated intermediate. This indicated that in the case of methyl position oxidation of 3-methylindole by cytochromes P450 present in goat lung microsomes, the dehydrogenation process predominates. Several other examples of cytochrome P450-mediated dehydrogenation reactions exist, often with deleterious consequences. Substituted alkoxy phenolic compounds, such as the dental anesthetic eugenol, are dehydrogenated to form very reactive quinone methide intermediates (Bolton et aI., 1995; Thompson et aI., 1995). The toxicity and ultimate carcinogenicity of ethylcarbamate is dehydrogenation-dependent (Forkert and Lee, 1997; Forkert et aI., 2001). Ethylcarbamate is dehydrogenated to form an olefin, vinylcarbamate. Vinylcarbamate is subsequently oxidized by cytochrome P450 to form an epoxide, which forms DNA adducts and is believed to be the ultimate carcinogen (Forkert et aI., 2001). In addition to their direct participation in the toxicity of certain compounds, dehydrogenation reactions may have effects on drug metabolism as well. As described in some detail, dehydrogenation reactions can form electrophilic intermediates that are more 21 reactive than the parent compound. The possibility exists that this reaction process leads to suicide inactivation of enzymes that catalyze it. Subsequent to formation, the dehydrogenated intermediate could interact with the enzyme, perhaps covalently. This may lead to the irreversible inhibition of the enzyme. This phenomenon has been demonstrated for the dehydrogenation of 3-methylindole and the human pulmonary P450 2Fl, which catalyzes the dehydrogenation reaction exclusively (Lanza et aI., 1999). The precise mechanism of inactivation has yet to be determined, but may act through a mechanism like the one described above. Conclusive evidence for a P450-inhibitory mechanism like this has been provided by characterization of the inhibition of cytochrome P450 1 A2 by the compound furafylline. (Kunze and Trager, 1993; Racha et ai., 1998) The mechanism of inhibition by this compound requires catalysis-dependent dehydrogenation to an imidazomethide intermediate. The intermediate then covalently modifies the enzyme, irreversibly inhibiting it. This mechanism may also playa role in the inhibition of cytochrome P450 2B6 by the anti-estrogenic compound tamoxifen (Sridar et aI., 2002). Dehydrogenation-dependent formation of a reactive quinone methide intermediate has been demonstrated for this compound (Zhang et aI., 2000), and could have the potential to covalently modify the enzyme (Notley et aI., 2002). The importance of cytochrome P450-mediated dehydrogenation reactions to the fields of toxicology and drug metabolism has been demonstrated. However, the mechanisms that control divergent processes of hydroxylation and second electron oxidation have not been elucidated. Thus, additional investigation of the nature of this interesting process is required. These studies could possibly provide information that can 22 assist in the prediction of enzyme/substrate combinations that are likely to proceed through this pathway. The Cytochrome P450 2F Subfamily Enzymes Human cytochrome P450 2F1 was cloned and initially characterized as a pulmonary selective enzyme in 1990 (Nhamburo et aI., 1990). Interest in the 2F1 enzyme in the context of pulmonary toxicity and 3-methylindole bioactivation came from studies ofHepG2 cells that express the 2F1 enzyme. Incubations of the cells with radio­labeled 3-methylindole exhibited a high degree of radioactive protein adduct, which was ultimately attributed to 3-methyleneindolenine formation (Thornton-Manning et aI., 1991; Thornton-Manning et aI., 1996). Subsequent studies of heterologously expressed 2F1 in a human lymphoblastoid cell line demonstrated that the enzyme exclusively dehydrogenated 3-methylindole to 3-methyleneindolenine and catalyzed the stereoselective epoxidation of naphthalene to the 1R,2S oxide (Lanza et aI., 1999). Both reactions are known to playa role in the pulmonary toxicity associated with these compounds. Additional substrates for the enzyme were ethoxycoumarin, propoxycoumarin, and pentoxyresorufin (Nhamburo et aI., 1990) and styrene (Nakajima eta!.,1994). As previously described, goats exhibit a high level of sensitivity to the pneumotoxicity associated with 3-methylindole. It was hypothesized that a 2F enzyme may be present in goats, and that it contributed to the bioactivation of 3-methylindole. A 2F transcript was subsequently identified from goat lung tissue. The gene was cloned and it exhibited 84% sequence identity to cytochrome P450 2F1 and was given the 23 designation cytochrome P450 2F3 (Wang et aI., 1998). Characterization of the enzyme demonstrated that it, like cytochrome P450 2F1, exclusively dehydrogenated 3- methylindole to form 3-methyleneindolenine and catalyzed the epoxidation of naphthalene to the 1R,2S-oxide (Wang et al., 1998). However, unlike 2F1, cytochrome P450 2F3 was not able to catalyze the deethylation of ethoxycoumarin. Other substrates were not found for cytochrome P450 2F3. Two additional members of the subfamily have been cloned. The mouse homologue, cytochrome P450 2F2, has been cloned and expressed in yeast (Ritter et aI., 1991) and insect cells (Shultz et al., 1999). This isozyme is expressed in the mouse respiratory system, but it is also present in the liver (Ritter et al., 1991). Cytochrome P450 2F2 has demonstrated the ability to bioactivate naphthalene (Shultz et al., 1999), 1- nitronaphthalene and 2-methylnaphthalene (Shultz et al., 2001). It is therefore believed to be important for the pulmonary toxicity associated with these compounds. A fourth member of the 2F family was recently cloned from the rat, and was designated cytochrome P450 2F4. The sequence for the enzyme is available though Genbank, but a formal publication of the enzyme's characterization has not been performed. A sequence alignment of the four members to the cytochrome P450 2F subfamily is presented in Figure 1.6. Other P450 Isozyme involvement in 3-Methylindole Metabolism Additional studies have been performed to test the involvement of major drug and xenobiotic metabolizing cytochrome P450 isozymes in the bioactivation of 3- methylindole (Lanza and Yost, 2001). A summary of the metabolites and intermediates Figure 1.6. Sequence alignment of the known members of the cytochrome P450 2F subfamily. 2FI-Human, 2F3-goat, 2F2-mouse and 2F4-rat. 24 1 50 25 2F1 MDSISTAILL LLLALVCLLL TLSSRDKGKL PPGPRPLSIL GNLLLLCSQD 2F3 MDSISTAILL LlLALICLLL TTSSKGKGRL PPGPRALPFL GNLLQLRSQD 2F2 MDGVSTAILL LLLAVISLSL TFSSRGKGQL PPGPKPLPIL GNLLQLRSQD 2F4 MDGVSTAILL LLLAVISLSL TFTSWGKGQL PPGPKPLPIL GNLLQLRSQD 51 100 2F1 MLTSLTKLSK EYGSMYTVHL GPRRVVVLSG YQAVKEALVD QGEEFSGRGD 2F3 MLTSLTKLSK EFGAVYTVYL GPRRVVVLSG YQAVKEALVD QAEEFSGRGD 2F2 LLTSLTKLSK EYGSVFTVYL GSRPVIVLSG YQTVKEALVD KGEEFSGRGA 2F4 LLTSLTKLSK DYGSVFTVYL GPRRVIVLSG YQTVKEALVD KGEEFSGRGS 101 150 2F1 YPAFFNFTKG NGIAFSSGDR WKVLRQFSIQ ILRNFGMGKR SIEERILEEG 2F3 YPAFFNFTKG NGIAFSNGDR WKALRKYSLQ ILRNFGMGKR TIEERILEEG 2F2 YPVFFNFTRG NGIAFSDGER WKILRRFSVQ ILRNFGMGKR SIEERILEEG 2F4 YPIFFNFTKG NGIAFSDGER WKILRRFSVQ ILRNFGMGKR SIEERILEEG 151 200 2F1 SFLLAELRKT EGEPFDPTFV LSRSVSNIIC SVLFGSRFDY DDERLLTIIR 2F3 HFLLEELRKT QGKPFDPTFV VSRSVSNIIC SVIFGSRFDY DDDRLLTIIH 2F2 SFLLEVLRKM EGKPFDPVFI LSRSVSNIIC SWFGSRFDY DDERLLTIIH 2F4 SFLLDVLRKT EGKPFDPVFI LSRSVSNIIC SVIFGSRFDY DDERLLTIIH 201 250 2F1 LINDNFQIMS SPWGELYDIF PSLLDWVPGP HQRIFQNFKC LRDLIAHSVH 2F3 LINENFQIMS SPWGEMYNIF PNLLDWVPGP HRRLFKNYGR MKNLIARSVR 2F2 FINDNFKIMS SPWGEMYNIF PSVLDWIPGP HKRLFRNFGG MKDLIARSVR 2F4 FINDNFQIMS SPWGEMYNIF PSLLDWVPGP HRRVFRNFGG MKDLIARSVR 251 300 2F1 DHQASLDPRS PRDFIQCFLT KMAEEKEDPL SHFHMDTLLM TTHNLLFGGT 2F3 EHQASLDPNS PRDFIDCFLT KMAQEKQDPL SHFFMDTLLM TTHNLLFGGT 2F2 EHQDSLDPNS PRDFIDCFLT KMAQEKQDPL SHFNMDTLLM TTHNLLFGGT 2F4 EHQDSLDPNS PRDFIDCFLT KMVQEKQDPL SHFNMDTLLM TTHNLLFGGT 301 350 2F1 KTVSTTLHHA FLALMKYPKV QARVQEEIDL WGRARLPAL KDRAAMPYTD 2F3 ETVGTTLRHA FRLLMKYPEV QVRVQEEIDR WGRERLPTV EDRAEMPYTD 2F2 ETVGTTLRHA FLILMKYPKV QARVQEEIDR WGRSRMPTL EDRTSMPYTD 2F4 ETVGTTLRHA FLILMKYPKV QARVQEEIDC WGRSRMPTL EDRASMPYTD 351 400 2F1 AVIHEVQRFA DIIPMNLPHR VTRDTAFRGF LIPKGTDVIT LLNTVHYDPS 2F3 AVIHEVQRFA DIIPMSLPHR VTRDTNFRGF TIPRGTDVIT LLNTVHYDPS 2F2 AVIHEVQRFA DVIPMNLPHR VTRDTPFRGF LIPKGTDVIT LLNTVHYDSD 2F4 AVIHEVQRFA DVIPMNLPHR VIRDTPFRGF LIPKGTDVIT LLNTVHYDSD 401 450 2F1 QFLTPQEFNP EHFLDANQSF KKSPAFMPFS AGRRLCLGES LARMELFLYL 2F3 QFLKPKEFNP EHFLDANMSF KKSPAFMPFS AGRRLCLGEA LARMELFLYL 2F2 QFKTPQEFNP EHFLDDNHSF KKSPAFMPFS AGRRLCLGEP LARMELFIYF 2F4 QFKTPQEFNP EHFLDANQSF KKSPAFMPFS AGRRLCLGEP LARMELFIYL 451 491 2F1 TAILQSFSLQ PLGAPEDIDL TPLSSGLGNL PRPFQLCLRP R 2F3 TAILQSFSLQ PLGAPEDIDL TPLSSGLGNV PRPYQLCVRA R 2F2 TSILQNFTLQ PLVDPEDIDL TPLSSGLGNL PRPFQLCMHI R 2F4 TSILQNFTLH PLVEPEDIDL TPLSSGLGNL PRPFQLCMRI R 26 formed by each isozyme is presented in Table 1.1. Interestingly, cytochrome P450 2El exclusively catalyzed the oxygenation of 3-methylindole, forming only 3- methyloxindole. Therefore, cytochrome P450 2E 1 may prove to be an important comparative tool in the design of studies to target the differential mechanisms of dehydrogenation and oxygenation. Research Objectives of this Study As presented in this introductory chapter, cytochrome P450-mediated bioactivation of 3-methylindole is required for the pulmonary toxicity that this compound exhibits. The P450 metabolism of 3-methylindole has been well characterized, but several mechanistic questions remain, that pertain to both oxygenation and dehydrogenation. The central hypothesis for these studies is that differentiation between P450-mediated 2,3-oxygenation and 3-methyl-dehydrogenation of 3-methylindole is controlled by unique active site differences exhibited by pulmonary P450 enzymes. This research project has two major components, both of which can provide a better understanding ofP450-meditated processes. Cytochrome P450-mediated oxygenation of 3-methylindole generates the stable metabolites 3-methyloxindole and 3-hydroxy-3-methyloxindole. A mechanistic investigation into the formation of these metabolites has not been performed and is required for a better understanding of the divergent pathways of oxygenation and dehydrogenation. The possibility exists that they originate from one precursor oxygenation-dependent intermediate. The formal hypothesis for this phase of the project is that an epoxide, 2,3-epoxy-3-methylindoline is formed, and is an intermediate in the Table 1. 1. The 3-methylindole metabolites formed by some of the major drug and xenobiotic metabolizing cytochrome P450 isozymes. Enzyme lAl lA2 lBl 2A6 2B6 2C19 2D6 2El 2Fl 2F3 (Goat) 3A4 3A5 4Bl (Rabbit) 3 -Methyleneindolenine ( dehydrogenation) 4 22 72 1.6 X Indole-3-Carbinol ( oxygenation) 42 100 85 X X X x 3-Methyloxindole ( oxygenation) 72 72 7 X X X X 98 X X X 27 All of the enzymes tested were the human isoforms, with the exception of2F3, which is goat and 4B 1 which is rabbit. The numbers indicate the V IK value, X indicates that the metabolite was formed but reaction kinetics were not determined. 28 formation of3-methyloxindole and 3-hydroxy-3-methyloxidole. Initial studies to test this hypothesis were performed by Skiles, who conducted elegant experiments to track the origin of the oxygen introduced into each metabolite (Skiles, 1992). Studies will be performed to examine the possibility that an "NIH" shift of the C-2 position hydrogen atom to position 3 occurs in the formation of 3-methyloxindole. This rearrangement mechanism is indicative of oxygenation reactions, including epoxidation. Additional studies will be performed in which P450-dependent reactive intermediates of 3- methyl indole will be trapped with various thiol nucleophiles. The oxygenation­dependent reactive intermediate 3-hydroxy-3-methylindolenine has been proposed, but a thorough investigation of its presence has not been performed. These studies will be performed with differentially deuterium-labeled 3-methylindole to differentiate between alternate pathways to the same electrophilic intermediate. Structurally identical adducts could arise via either dehydrogenation or oxygenation mechanisms (Figure 1.7). Based on the differential retention and loss of various deuterium labels, these studies can elucidate both the intermediate that formed the thiol-adducts, and also the mechanism required for the intermediate's formation. The next phase of this research project examines the dehydrogenation dependent metabolism of 3-methylindole. For the dehydrogenation of substrates, including 3- methylindole, the mechanism involving a net two-electron oxidation of the substrate involving an initial hydrogen atom abstraction has been characterized. However, the mechanisms that control the subsequent step in which a dehydrogenated vs. hydroxylated product is formed have not been characterized. These mechanisms may involve direct participation of active site residues. A residue or residues with a high potential for redox 29 Figure 1.7. Thiol trapping of electrophilc intermediates of 3-methylindole biotransforn1ation. A C-2 position adduct can form fron1 either P450-mediated dehydrogenation or oxygenation. Dehydrogenation / ~ v~i 3-~efhyleneindolenine ~ VN' 3-~ethylindole Oxygenation ~ ~OH I ~ G)h ~ NH 3-Hydroxy-3-methylindolenine R/ "- RSH R~ty ~ -H2~ ~SH ~~ UN' SR ~ SR VJ- ~OHSR Vm?- w o 31 or radical-mediated reactions could participate in radical abstraction from the substrate to yield the dehydrogenated product (Figure 1.8). This has not been proposed for P450 reactions previously, and an investigation of this possibility is required. As stated previously, cytochromes P450 2Fl and 2F3 exclusively catalyze the dehydrogenation of 3-methylindole. This provides an excellent opportunity to examine the dehydrogenation process at the level of the enzyme itself. The formal hypothesis for the second phase of this project is that there are amino acid residues within the active site of cytochromes P450 2Fl and 2F3 that govern their specificity for 3-methylindole dehydrogenation. This hypothesis can be addressed and tested by completing several specific aims. First, a heterologous expression system that permits the production of sufficient amounts of enzyme for biochemical characterization is required. Both bacterial and insect cell expression systems will be investigated for their ability to produce active cytochrome P450 2Fl and 2F3. Next, through the analysis of prior mutagenesis studies and sequence comparisons with enzymes that do not catalyze the dehydrogenation reaction, such as cytochrome P450 2El, rational site and region directed mutants can be designed. Once expressed, these mutant enzymes can be tested for their catalytic properties with regard to both oxygenation and dehydrogenation of 3-methylindole. Additionally, using a cytochrome P450 enzyme for which a three-dimensional crystal structures exists, a three­dimensional homology model of the 2F enzymes can be constructed. The homology model can guide mutagenesis studies and aid in the interpretation of mutagenesis data. 32 Figure 1.8. A theoretical mechanism for cytochrome P450 2F-mediated dehydrogenation of 3-methylindole. The mechanism involves the participation of an active-site residue with the capacity to participate in radical-mediated reactions. A tyrosine residue is provided as an example, but tryptophan and other residues have been shown to participate in reactions of this type as well. A mechanism of P450 action in which an active-site residue/s participates in the catalysis has not previously been proposed. cAH r1C~2 7_' _ '\ h C;;,H HO;J Hydrogen atom abstracti on ..... I If H ~ -~ +4 \ ,.... 3-Methylindole docked in the active site of2Fl or 2F3 /Fe-..:... Ac~ive site tyrosine resIdue V~~ I H 0C~2 ~ \ ::::-... -H20 ..... ...... HO 3-Methyleneindolenine --F +1 / e-- Electron transfer to heme, product release and loss of water ~ / N O-J<c::,'H · H 0 CH2 ~ ~ ::::-... HO ~ ~ ~ ?H -/-Fe-+--1. Radi cal transfer to tyrosine resi due 0C~2 ~ ~ ::::-... 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Thornton-Manning J, Appleton ML, Gonzalez FJ and Yost GS (1996) Metabolism of3- methylindole by vaccinia-expressed P450 enzymes: correlation of 3- methyleneindolenine formation and protein-binding. J Pharmacol Exp Ther 276:21-29. Thornton-Manning JR, Ruangyuttikarn W, Gonzalez FJ and Yost GS (1991) Metabolic activation of the pneumotoxin, 3-methylindole, by vaccinia-expressed cytochrome P450s. Biochem Biophys Res Commun 181:100-107. Wang H, Lanza DL and Yost GS (1998) Cloning and expression of CYP2F3, a cytochrome P450 that bioactivates the selective pneumotoxins 3-methylindole and naphthalene. Arch Biochem Biophys 349:329-340. Wynder EL and Hoffman D (1967) Certain constituents of tobacco products. Academic Press, New York. Yost GS (1989) Mechanisms of 3-methylindole pneumotoxicity. Chem Res Toxicol 2:273-279. Yost GS (1996) Mechanisms of cytochrome P450-mediated formation of pneumotoxic electrophiles. Adv ExpMed Bioi 387:221-229. Yost GS, Buckpitt AR, Roth RA and McLemore TL (1989) Mechanisms of lung injury by systemically administered chemicals. Toxicol Appl Pharmacoll0l: 179-195. Yost GS, Nocerini MR, Carlson JR and Liberato DJ (1986) Structure of the adduct of glutathione and activated 3-methylindole. Adv Exp Med Bioi 197:373-380. Zhang F, Fan PW, Liu X, Shen L, van Breeman RB and Bolton JL (2000) Synthesis and reactivity of a potential carcinogenic metabolite of tamoxifen: 3,4- dihydroxytamoxifen-o-quinone. Chem Res ToxicoI13:53-62. CHAPTER 2 EVIDENCE SUPPORTING THE FORMATION OF 2,3-EPOXY- 3-METHYLINDOLENINE: A REACTIVE INTERlv1EDIATE OF THE PNEMOTOXIN 3-METHYLINDOLE Reproduced with permission from Chem. Res. Toxieo!. 1998, 11, 741-479. Copyright 1998 Am. Chern. Soc. Chern. Res. Toxicol. 1998,11, 741-749 Evidence Supporting the Formation of 2,3-Epoxy-3-methylindoline: A Reactive Intermediate of the Pneumotox:in 3-Methylindole Konstantine W. Skordos, Gary L. Skiles,t John D. Laycock,; Diane L. Lanza, and Garold S. Y ost* Department of Pharmacology and Toxicology, 112 Skaggs Hall, University of Utah, Salt Lake City, Utah 84112 Received November 21, 1997 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 of3-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 [2-2Hl-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 180 incorporation into either 3-methyloxindole or 3-hydroxy-3- methyloxindole from both 180 2 and H2 180 was determined. The 180 incorporation into 3-methyloxindole from 180 2 was 91%, strongly implicating a mechanism requiring cytochrome P450-mediated oxygenation. Incorporation of 180 into 3-hydroxy-3-methyloxindole indicated that the alcohol oxygen originated from molecular oxygen, also implicating an epoxide precursor. These studies demonstrate the existence oftwo new reactive intermediates of 3-methylindole and describe the mechanisms of their formation and fate. 40 741 Introduction 3-Methylindole, a degradation product of tryptophan, is a selective pulmonary toxicant that requires cyto­chrome P450 biotransformation to elicit its toxic effects (1). Most susceptible to 3-methylindole pneumotoxicity are goats and cattle, followed by mice, rats, and rabbits (2,3). Human susceptibility to 3-methylindole has not been thoroughly characterized to date, but the toxin is bioactivated by human microsomes and is a good sub­strate for human cytochrome P450 enzymes that are selectively expressed in lung tissue (4,5). Exposure to 3-methylindole occurs through ingestion of dietary tryp­tophan, followed by degradation to indole-3-acetic acid through decarboxylation and deamination. Indole-3- acetic acid is decarboxylated to 3-methylindole by a Lactobaccillus sp. bacterium in the gut (6). 3-Methylin­dole circulates systemically to the lung tissue where it is bioactivated to highly reactive intermediates (Scheme 1) that have the potential to aikylate proteins and other cellular macromolecules leading to toxicity (7). One well-established reactive intermediate of 3-meth­ylindole is 3-methyleneindolenine (8). This highly reac­tive methylene imine is produced by cytochrome P450- mediated dehydrogenation of3-methylindole at the methyl position (9). The mechanism appears to proceed by hydrogen atom abstraction followed by a one-electron oxidation step to produce the reactive intermediate which alkylates proteins (10) through a Michael-like addition reaction. ,. Correaponding author. Tel: [phone]. Fax: [phone]. E-mail: [e-mail]. t Present address: Biochemical and Investigative Toxicology, De­partment of Safety Assessment, WP45-319, Merck Research Labora­tories, West Point, PA 19486. * Present address: Performance Bioanalytical, 11545 Sorrento Val­ley Rd, Suite 315, San Diego, CA 92121. Another highly reactive cytochrome P450-dependent intermediate that could participate in the toxicity of 3-methylindole is the 2,3-epoxide. The 2,3-epoxide of indole may be an intermediate in the formation of 3-hydroxyindole conjugates or oxindole from indole in rats (II). The 2,3-epoxide of N-acyl-3-methylindole is formed by chemical oxidation with dimethyldioxirane and rearranges toN-acyl-3-methyloxindole (12). Thus, indi­rect evidence exists for the formation of indole 2,3- epoxides, but we are unaware of studies that conclusively demonstrate the formation of indole epoxides by P450· mediated oxidation. 2,3-Epoxy-3-methylindole could have at least three potential chemical fates: (1) direct alky­lation of cellular protein and/or nucleic acids, (2) ring opening, possibly accompanied by a "NIH shift" of the hydride at C-2, resulting in a stable oxindole, or (3) ring opening to produce an imine reactive intermediate, S0893-228x(97)OO208-7 CCC: $15.00 CO 1998 American Chemical Society Published on Web 0610211998 41 742 Chem. Res. Toxicol., Vol. 11, No.7, 1998 Skordos et ai. Scheme 1. Cytochrome P460-Mediated Bioactivation of S-Methylindolea Protein and Nucleic Acid AdduCIJI )-Melhylindole B lp4SO o:Y -P450 A c 3-Methyleneindolenine ~OH / V~ ~3-Epoxy-3-methylindoline 3-Hydroxy-3-metbylindolenine F! Aldehyde • Oxidase ~H I 0 NH Protein and Nucleic Acid Adducts 2-Hydroxy-)-methylindole )-Metbyloxindole 3-Hydroxy-3-methyloxindole apossible mechanisms include: dehydrogenation to 3-methyleneindolenine (pathway A); epoxidation (pathway B) followed by ring opening to C-2, resulting in the formation of 3-methyloxindole by both hydride shift (pathway E) and proton lOBS (pathway D) mechanisms; or epoxide ring opening to C-3leading to 3-hydroxy-3-methylindolenine (pathway C) and its oxidation by aldehyde oxidase (pathway F). The possibility of reactive intermediate conjugation with proteins and/or nucleic acids is indicated. 3-Methyleneindolenine is depicted in the protonated form based on considerations of the basicity of indolenine nitrogen atoms (22). Alphabetical discriminators for each possible pathway are kept consistent in subsequent schemes. 3-hydroxy-3-methylindolenine, which can alkylate cel­lular macromolecules or be detoxified by further oxidation to 3-hydroxy-3-methyloxindole. 3-Methyloxindole that is formed from 2,3-epoxy-3- methylindoline through the NIH shift. mechanism would retain the C-2 H-atom at C-3 (Scheme 2, B and E). However, another mechanism for the formation of3-meth­yloxindole from the epoxide involves loss of the H atom from C-2 and the formation of an enol that tautomerizes to the keto form, 3-methyloxindole (Schemes 1 and 2, 0). The mechanism of deuterium loss could also involve a "direct hydroxylation" mechanism wherein a formal epoxide is not formed (vide infra) during the oxidation of3-methylindole to 3-methyloxindole. Finally, a mech­anism for deuterium loss could occur through hydration of the dehydrogenated intermediate, 3-methyleneindo­lenine (Scheme 2, A). Therefore, characterization of the 3-methyloxindole formed from [2-2H)-3-methylindole can be used to support the production of the epoxide and to assess the extent of the hydride shift. mechanism versus other mechanisms in ring opening ofthe epoxide (Scheme 2). It is possible that P45O-mediated oxygenation of3-meth­ylindole by an addition/rearrangement mechanism oc­curs. This process would be similar to other aromatic hydroxylation reactions that have been proposed (13) to proceed through the addition of the triplet-like active oxygen species of reduced P450 to the ;rr system of the aromatic ring, followed by rearrangement to form the enol directly, without formal formation of the epoxide. In fact, initial addition to the indole ring may be electronically more favorable than addition to a benzene­like aromatic system that does not contain nitrogen, because of the higher electron density of the indole ring. However, distinguishing between these two mechanisms is quite difficult using the methods employed in the current report. Scheme 2. Potential Fates of the Deuterium. Label after Oxidation of [2·2H]-S.Methylindole by P450 Enzymes to Produce a-Methyloxindolea V~N?Dr 2-~-3-Methylilldole 2-1H.2.3-Epoxy-3-methylindoline E! NIH Shift ~DO VN.f 3.1H-3-Methyloxindole 3-Methyloxindole a Mechanisms include: epoxidation (pathway B) followed by ring opening involving a hydride shift (pathway E) and ring opening not involving a hydride shift. (pathway D) or dehydrogenation to 3-methyleneindolenine (pathway A) which is hydrated at C-2 and which subsequently aromatizes to the indole and undergoes enol! keto tautomerization to 3-methyloxindole. The source of oxygen incorporated into 3-methyloxin­dole and also 3-hydroxy-3-methyloxindole is another indicator ofthe proposed epoxide intermediate. 3-Meth­yloxindole that is produced via a cytochrome P450- dependent epoxide should incorporate 180 from molecular oxygen. Other mechanisms for the formation of3-meth- 42 3-Methylindole Epoxidation Chem. Res. Toxicol., Vol. 11, No.7, 1998 74S SchemeS. Mechanisms of 2,S·Epoxy..s.methylindoline Ring Opening" o1H 2,3-Epoxy-3-medlyJindoiillt y 'l ~,o ~ c:Jj:~ I NH H oJH ~ NH ojeH NH 1 I .H:p 1 o)-ONH H ~e ~NH ~I ~ H II 1 II 1 ~ 0:;0 ~ ~I H 3.Medlyloxindoie 3-Medlyloxindole l.MCI1hyloxindoie l·Hydroxy-3-medlylifldoienine 4Mechanisms include: proton loss and ring opening to C-2 (pathway D). followed by tautomerization to 3-methyloxindole; ring opening to C-2 (pathway E) involving a hydride shift from C-2 to C-3 forming 3-methyloxindole; hydration of the epoxide (pathway G) to the dihydrodiol that dehydrates and tautomerizes to 3-methyloxindole; and ring opening to C-3 (pathway C) forming 3-hydroxy-3- methylindolenine. yloxindole include incorporation of oxygen from water through the formation of a diol and subsequent dehydra­tion to S-methyloxindole (Scheme 3, G). Therefore, an assessment of the incorporation of 18() from either 180 2 or R2 180 should provide an indication of the presence of the epoxide, the extent of ring opening versus the hydration/dehydration mechanism, or the possibility that hydration of the methylene imine is a predominant mechanism. 3-Hydroxy-3-methylindolenine is another putative re­active intermediate of 3-methylindole which could be produced by epoxide ring opening (Scheme 1, C). 3-Ry­droxy- 3-methylindolenine may be oxidized to 3-hydroxy- 3-methyloxindole by the cytosolic enzyme aldehyde oxi­dase (EC 1.2.3.1) (14). Because 3-hydroxy-3-methyl­indolenine may be formed though the epoxide, the subsequent formation of 3-hydroxy-3-methyloxindole could be another indicator of the epoxide's existence. 3-Ry­droxy- 3-methyloxindole is in fact the major metabolite of 3-methylindole (15, 16). Therefore, aldehyde oxidase may be a major contributor in the detoxification of 3-methylindole. Differing activities of this cytosolic enzyme for the hydroxyindolenine could contribute sub­stantially to differences in species and organ susceptibil­ity to 3-methylindole. By supplementing goat lung microsomal incubations with cytosolic proteins that contain aldehyde oxidase from different species, produc­tion of 3-hydroxy-3-methyloxindole can be monitored, simultaneously evaluating the presence of the epoxide and the role of aldehyde oxidase in species-selective 3-methylindole detoxification. The present study investigated the possibility of the existence of an epoxide reactive intermediate of 3-meth­ylindole. Through the use of the stable isotopes 18() and 2H, the formation and metabolic fate of the epoxide, 2,3- epoxy-3-methylindoline, was monitored, indirectly con­firming its presence. ExperbnentruProcedures Chemicals. 3-Methylindole, N-acetyl-L-cysteine, indole·3- carbinol, nicotinamide adenine dinucleotide phosphate (NAD· PH). ammonium acetate. THF. D20, and formic acid were purchased from Sigma Chemical Co. (St. Louis. MO). 18()2 (50 atom %) was obtained from Cambridge Isotopes (Woburn, MAl. Acetanilide, p-hydroxyacetanilide (acetaminophen), and H218() (95 atom %) were purchased from Aldrich Chemical Co. (Mil­waukee, WI). 3-Methyloxindole was a generous gift from Dr. James Carlson. Washington State University. 3-Hydroxy-3- methyloxindole and 12-2HI-3-methylindole were synthesized as described below. Synthesis. 3-Hydroxy-3-methyloxindole was prepared by adding CHsMgBr to indole-2.3-dione in anhydrous THF accord­ing to a published method (17). The product was recrystallized from ether/CH2Ch and produced white crystals with a melting point of 160-161 °C, in 65% purified yield. [2-2Hl-3-Methylindole was prepared by a modification of a published method (18). 3-Methylindole (15.2 mmol) was added to a flask which was evacuated and flushed with argon three times. THF (50 mL) was added to give a homogeneous mixture which was cooled to -70°C. n-Butyllithium (6.1 mL of a 2.5 M hexane solution) was added, and a colorless precipitate formed. The solution was warmed to 25°C over 10 min, and C02 was bubbled into the solution with vigorous stirring for 3 min to produce a colorless homogeneous solution. The solution was allowed to stand at 25 "C for 5 min at which point the solvent was removed under reduced pressure below 25 "C to give a colorless salt (lithium 3-methylindole-1-carboxylate) which was dried under a vacuum. The flask was again evacuated and flushed with argon three times. THF was added to give a homogeneous pale-yellow solution which was cooled to -70°C. and tert-butyllithium (10 mLofa 1.7 M pentane solution) was slowly added to give a pale­orange solution oflithium 2-lithio-3-methylindole-1-carboxylate. 744 Chern. Res. Toxicol., Vol. 11, No.7, 1998 This solution was warmed to -20 ·C and kept at this temper· ature for 1 h. The solution was cooled to -70 ·C, and D20 (0.2 mL) was added to form 12·2H1-3-methylindole-l-carboxylate. The solution was kept at -70 ·C for 3 h and then slowly quenched with aqueous ammonium sulfate. The solution was warmed to 25 DC, and 2 N sulfuric acid was added to bring the solution to pH 4. The solution was extracted twice with diethyl ether, washed with water, dried over anhydrous MgS04• and filtered, and the solvent was evaporated under reduced pressure to give crude 12-2HI-3-methylindole-l-carboxylic acid as a pale-yellow solid. The solid was then heated, yielding the decarboxylated product 12-2Hl-3-methylindole. The product was purified by column chromatography on silica gel using hexane as an eluent. The purified yield of 12·2HI-3-methylindole (mp 96-97 ·C) was 90%, and deuterium incorporation was 83%, measured in triplicate by LCIMS of the molecular ion cluster at 148/149 mlz. Microsomal Preparation. Goat lungs were obtained from three male goats. The lungs were perfused in situ with ice­cold 0.05 M phosphate buffer containing 1 mM EDT A and 1.15% KCl and then frozen at .. 70 ·C. Microsomes were prepared by standard centrifugal isolation procedures (4). Mouse livers were obtained from four male Swiss-Webster mice that were sacri­ficed by cervical dislocation. The livers were removed. and microsomes were prepared by standard centrifugal isolation procedures. For experiments requiring cytosolic protein, the cytosolic fraction from the microsomal preparation was used in each case. In all cases the cytosolic fraction was the supernatant from the first 105000g centrifugation step. This procedure was performed for the goat and mouse lung and liver tissues from animals described above. Cytosolic fractions were also obtained in an analogous manner from the lung and liver tissues from three male Sprague-Dawley rats and five male New Zealand white rabbits. Incubations To Determine Extent of Hydride Shift Mechanism in the Formation of 3·Methyloxindole. Incu· bations contained 4 mM NADPH, goat lung microsomes con­taining 1.1 nmol of cytochrome P4501mL, and either 0.5 mM 3-methylindole or 0.5 mM [2-2H1-3-methylindole. Incubations were performed in 0.01 M ammonium acetate, pH 7.4 in a final volume of 1 mL. Incubations were also performed in the presence of 4 mM N-actetyl-L-cysteine. The reaction was performed at 37°C in a shaking water bath for 30 min and stopped by immersion in an ice bath and by the addition of an equivalent volume of ice-cold acetonitrile to both stop the reaction and precipitate protein. The precipitated protein was separated from the reaction mixture by centrifugation at 3100g in a Beckman GPR tabletop centrifuge. The supernatant was removed and concentrated to 200 ilL in a Savant SpeedVac (model SVCI00) for analysis by LCIMS. 1110 Incorporation into 3-Methylo:dnd.ole and a.Hy­droxy- 3-methylo:dndole from l~ and HII8(), Incubations contained 1 mM 3-methylindole, goat lung microsomes contain­ing 1.1 nmol of cytochrome P4501mL, 1 mM NADPH, and 0.05 M phosphate buffer in a final volume of 3 mL. Some incubations also contained 8 mM N-acetylcysteine. The incubations were performed in an air-tight four-flask glass manifold apparatus that was constructed so that it could be evacuated, be flushed with ultrapure N2. and have 18()2 (50 atom %) introduced from a lecture bottle. 3-Methylindole was incubated with goat lung microsomes in two of the flasks, and acetanilide was incubated with mouse liver microsomes in the other two. Hydroxylation of acetanilide to p-hydroxyacetanilide was utilized as a positive control for incorporation from molecular oxygen. 18() Incorpora­tion into p-hydroxyacetanilide was normalized to 100%. Incu­bations contained mouse liver microsomes because goat lung microsomes do not hydroxylate acetanilide to a large enough extent for reliable quantification. After adding all incubation components except substrate into the flasks, the system was cooled on ice and then evacuated and flushed with N2 at least 10 times. The 180:z was introduced, substrate was added, and the system was heated to 37°C for the 30·min incubation. The reactions were stopped by immersing the flasks into a dry ice! 43 Skord08 et at. acetone bath, and the flasks were removed from the manifold. Three milliliters of ethyl acetate was added to the frozen incubation solutions, and as they thawed, they were mixed to denature protein and prevent further metabolism. The ethyl acetate layer was removed, and the aqueous solutions were extracted twice more with ethyl acetate. The organic extracts were pooled, evaporated to dryness under an N 2 stream at ambient temperature, and then derivatized in MSTFAIaceto­nitrile for analysis by GeIMS. For incorporation from H211l(), incubations were performed in 0.05 M phosphate buffer that was prepared using water enriched with H218(). The net isotopic enrichment in the incubation mixture was 44.7 atom %, and the final volume of the incubations was 0.5 mL. Incubations also contained goat lung microsomes (1.1 nmoVmL cytochrome P450) and 1 mM NADPH. For assessment of incorporation into 3-hydroxy-3- methyloxindole, mouse liver microsomes and cytosol were utilized because goat lung enzymes did not produce a significant amount of this metabolite. 3-Methylindole was added, and a 50-ilL aliquot was removed from each incubation mixture and placed on ice. The incubation was then performed at 37°C for 30 min and stopped by the addition of 500 ilL of ice-cold ethyl acetate. The organic layers were removed, and the samples were extracted twice more with ethyl acetate. The extracts were pooled, evaporated to dryness, and derivatized with MSTFA for analysis by GCIMS. Five microliters of benzoyl chloride was added to the 50-ilL aliquots that were removed just prior to incubation. The incorporation of 180 into benzoic acid, in the conversion from benzoyl chloride in water, was normalized to 100%. The aliquots were then extracted three times with ethyl acetate. The extracts were pooled, evaporated to dryness, and derivatized with MSTF A for GCIMS analysis. Aldehyde O:ddaae Activity. Oxidation of N-methylnico­tinamide, the model substrate for aldehyde oxidase, to N-meth­yl- 2-pyridone-5-carboxamide (2-pyridone) and N-methyl-4- pyridone-3-carboxamide (4-pyridone) was assayed in lung and liver tissues of goat, mouse, and rabbit (19). The activity of aldehyde oxidase in rat tissues was not measured with this substrate. Product formation was monitored by UV analysis (300 nm) using a Gilford spectrophotometer. Potassium phos­phate buffer <0.1 M, pH 7.8) (700 mL), and 0.005% EDTA were added to the cuvettes. Bovine serum albumin (50 ilL of a 25 mglmL solution) and catalase (50 ilL of a 0.25 mglmL solution) were added. The N-methylnicotinamide concentration was 0.5 M. An appropriate volume of water was added such that the cuvette volume was 1 mL at the time of the assay. The cuvette was then placed in a 25°C water bath for 5 min; once the temperature of the solution was equilibrated, the cytosolic fractions were added. The cuvette was then placed in the spectrophotometer, and the absorbance was monitored at 300 nm. The change in absorbance was converted to ,umol of product formed using an extinction coefficient of 4.23 x 103 mM-1 cm -1. 3-Hydroxy-3-methyloxindole was quantified in incubations that generated 3-hydroxy-3-methylindolenine as a substrate for aldehyde oxidase. Incubations contained 0.5 mM 3-methylin­dole, 4 mM NADPH, goat lung microsomes containing 5 nmol of cytochrome P4501mL, and cytosolic protein, 1 mg/mL, from either liver or lung fractions from goat, mouse, rat, or rabbit. The higher P450 content was utilized because it was necessary to maximize the amount of 3-hydroxy-3-methylindolenine that was produced from 3-methylindole. Incubations alae contained 20 ilL of a 1 mM solution of 3-phenyloxindole for use as an internal standard (retention time of 15.2 min). Peak area ratios of 3-hydroxy-3-methyloxindole to 3-phenyloxindole were com­pared to a standard curve generated with synthetic 3-hydroxy- 3-methyloxindole for quantification of 3-hydroxy-3-methyloxin­dole formation. Incubations were performed at 37 DC in a shaking water bath for 15 min and were stopped by immersion in an ice bath and by the addition of an equal volume of ice­cold acetonitrile. The precipitated protein was removed by centrifugation, and the supernatant was concentrated for analysis by HPLC. 44 3-Methylindole Epoxidation Chem. Res. Toxieol., Vol. 11, No.7, 1998 745 Table 1. 18() Incorporation into 3-MethyloIindole, 3-Hydroxy-S-methyloIindole, and APAP from 1802 metabolite NAC 18()o 180 1 1802 1802 incorporation (%) APAP correction (%) APAP 0.52 0.01 0.49 ± 0.01 N/A 98 APAP + 0.50 0.01 0.50 ± 0.01 N/A 100 3MOI 0.64 = 0.01 0.36 ± 0.01 N/A 73 74 3MOI + 0.55 = 0.01 0.45:1: 0.01 N/A 90 91 APAP' 0.52 = 0.01 0.47 ± 0.01 N/A 95 3H3MOIa 0.49:1: 0.01 0.41 :::: 0.01 0.02 ± 0.001 82,4.20 87, 4.40 a These experiments were performed using mouse liver microsomes and cytosol. b Percentage of 3-hydroxy-3-methyloxindole that incorporated two atoms of oxygen from molecular oxygen. N/A, not applicable; APAP, p-hydroxyacetanilide; 3MOI, 3-methyloxindole; 3H3MOI, 3-hydroxy-3-methyloxindole. Incubations were performed in an air-tight system that was evacuated prior to 1802 introduction. Incorporation of18() into p-hydroxyacetanilide (APAP) was normalized to 100%, and metabolite incorporation is corrected for this value. Liquid Chromatography for Analysis of S-Hydroxy-3. methyloxindole. A Beckman HPLC with dual 114M pumps and a 421A pump controller was utilized. Metabolites were separated using a reversed·phase Ultramex C·18, 5-,um, 250- x 4.6-mm column (Phenomonex, Torrance, CAl by a gradient that began at 90% aqueous (0.01 M ammonium acetate, pH 6.0), 10% organic (acetonitrile). The gradient proceeded with linear increases to 35% organic after 5 min, 50% organic at 10 min, 55% organic at 15 min, 95% organic at 19 min, isocratic for 2 min, and back to 10% organic at 25 min. The column was reequilibrated for 7 min between injections. 3-Hydroxy-3. methyloxindole was detected using a Hewlett-Packard 1040A diode array detector and a Hewlett-Packard 9000 series, Pascal­based workstation. The compound's retention time was 7.5 min, and it was positively identified by its characteristic pattern of absorbance as compared to a synthetic standard. GCIMS of ISO-Labeled S-Methyloxindole and p-Hy­droxyacetanilide. The ratios of metabolically produced iso· topomers were determined by mass spectrometry with a Finni­gan MAT 4500 GCIMS. The GC was fitted with a 30-m DB-5 microbore column with O.25-mm film thickness (J&W Scientific). The carrier gas was hydrogen at a flow rate of ca. 30 m 5-1. The GC oven temperature profile started at 100 °C for 2.0 min and was ramped at 10 °C min-1 for 13.0 min to 230 °C and then to 300 °C at 20 °C min-I before returning to 100 °C. The injector, ionizer, and transfer line temperatures were 270,120, and 280 °C, respectively. Ionization of the derivatized ISO-labeled 3- methyloxindole (retention time 11.3 min) and p-hydroxyacet· anilide (retention time 8.6 min) was accomplished by methane chemical ionization. The mass spectrometer was operated in the positive ion mode, and each sample was analyzed in triplicate. Direct Probe Electron Impact MS of3-[18())Hyclroxy-3- methyloxindole. Electron impact mass spectra were recorded on a VG Micromass 7050E double focusing high-resolution mass spectrometer with VG Data System 2000. The mass spectrom­eter was operated in the direct probe electron impact mode, and ionization was achieved with a 70-e V electron beam. LCJMS Analysis of S-Methyloxindole Formed from [2-IH]-S·Methylindole. The incubation mixtures from the NIH shift-tracking experiments were analyzed by LCIMS. The preconcentrated mixtures were injected using a Leap Technolo­gies CTC·A2ooS autosampler into a Waters 626 liquid chro­matograph. Metabolites were separated using a Phenomenex Ultramex 5 CIS IP (250-mm x 2.1-mm, 5-,um) column by the same instrument-controlled gradient described above for liquid chromatography. In this case, however. the organic phase was acetonitrile, containing 0.1% (vlv) formic acid, and the aqueous phase was H20, containing 0.1% (v/v) formic acid. Mass spectrometry was performed using a Finnigan model TSQ-7000 triple-quadrupole mass spectrometer with an atmospheric pres­sure ionization interface to the liquid chromatograph. A capil· lary temperature of 175 °C and a vaporizer temperature of 500 °C were used. A corona discharge voltage of 2.84 kV at 4.9 rnA was used with the sheath gas pressure set at 70 psi N2. Results Nm Shift-Tracking Experiments_ The molecular ion for synthetic 3-methyloxindole had a mJz of 148 and a retention time of 12.5 min. After incubation of goat lung microsomes with [2-2Hl-3-methylindole, metaboli­cally formed 3-methyloxindole with a mJz of 149 was produced by an NIH shift of the deuterium from position 2 to position 3, a process which is consistent with epoxide ring opening. Incubations were performed in the pres­ence and absence of the thiol N-acetylcysteine in order to trap the component of 3-methyloxindole that may form via hydration of3-methyleneindolenine, a mechanism not requiring hydride migration, as its thioether adduct. The deuterium retention in 3-methyloxindole was 71 ± 1.7% in the absence of N-acetylcysteine and 81 ± 2.5% in its presence. Percentages are the mean ± standard devia­tion ofthree incubations. An N-acetylcysteine adduct of the epoxide or the ring-opened 3-hydroxy-3-methylindo­lenine product was observed, but no products that appeared to be dihydrodiols were observed by LCIMS analysis. Analysis of the thiol adducts of these products is currently under investigation. Ill() Incorporation into 3-Methyloxindole and a-By­droxy- 3-methyloxindole. The ratios of the isotopomers and amounts of 180 incorporated into p-hydroxyacetanil­ide and 3-methyloxindole when 3-methylindole was incubated with and without N-acetylcysteine in an atmosphere that was 50 atom % 180 2 are shown in Table 1. These results demonstrated that incorporation of oxygen in 3-methyloxindole was 74% and 91% from molecular oxygen in the absence and presence of N­acetylcysteine, respectively. Also shown are results of 180 incorporation into 3-hydroxy-3-methyloxindole incu­bated as described above with the exception that mouse liver microsomes and cytosol, as opposed to goat lung microsomes alone, were used. Molecular oxygen incor­poration into 3-hydroxy-3-methyloxindole was 87% for one atom and 4.4% for two atoms of oxygen. The incorporation results were corrected for the theoretical amount of Ill() which should be incorporated into p­hydroxyacetanilide (100% incorporation) and mUltiplied by 2 to correct for the fact that only 50% of the O2 was labeled with 180. These corrections provided a measure­ment of the total amount of i8() in 3-hydroxy-3-methy­loxindole that originated from molecular oxygen. The incorporation of 18() from H2 180 is shown in Table 2. Incorporation results are shown for 3-methyloxindole and 3-hydroxy-3-methyloxindole. For these incubations the positive control for incorporation was benzoic acid which is rapidly formed from benzoyl chloride in water; it is assumed that 180 incorporation is 100%. Again, because goat lung microsomes do not produce 3-hydroxy- 746 Chem. Res. Toxicol., Vol. 11, No.7, 1998 Table 2. 1110 Incorporaiion into a-Meihyloxindole, a-Hydrosy.s-meihyloxindole, and BA from H2180 BA correction metabolite 1800 18()1 18()2 (%) BA 0.55 ± 0.03 0.44 ± 0.10 0 3MOI 0.94 ± 0.04 0.03 ± 0.03 N/A 6.44 BAa 0.60 ± 0.02 0.37 ± 0.01 0 3H3MOIa 0.57 ± 0.03 0.40 ± 0.02 0.02 ± 0.03 107 a These experiments were performed with goat lung micro80mes and cytosol. N/A, not applicable; BA, benzoic acid produced from benzoyl chloride; 3MOI, 3-methyloxindole; 3H3MOI, 3-hydroxy- 3-methyloxindole. Incubations were performed in buffer that had been prepared with H218Q to produce a final isotopic enrichment of 44.7%. Incorporation ofl8() into benzoic acid (BA) was normal­ized to 100%, and metabolite incorporation is corrected for this value. 14a/uo 100 170/122- ..c..: 80 ~ 60 .,.. ... ., 40 II) a: ~'. ~:r= 1l$/l37- K\I - 163/165 92 120 163 135 149 180 ",/z Figure 1. Electron impact, direct probe, high-resolution mass spectrum of 3-[180Ihydroxy-3-methyloxindole, formed by cyto­chrome P450 oxidation in the presence of 18Q2 (50 atom %). 3-methyloxindole to an appreciable extent, mouse liver enzymes were used in this case. Only 6.44% of the oxygen in 3-methyloxindole was shown to be derived from water. Essentially all of one atom (107%) of oxygen in 3-hydroxy·3-methyloxindole came from water. The re­sults were corrected for the amount of 180 incorporated into benzoic acid from benzoyl chloride (100% theoretical incorporation) and also multiplied by 2.24 to correct for the fact that the enrichment of H218() in the incubation mixture was 44.7 atom %. Electron Impact Mass Spectrum. of 180-Labeled 3-Bydroxy-S-Methyloxindole. The direct probe elec­tron impact mass spectrum of 3-hydroxy-3-methyloxin­dole that was formed by mouse liver microsomes under a 50 atom % 18() atmosphere is shown in Figure 1. The major ions in the spectrum have the following mlz, assignments, and relative intensities, respectively: mlz 163/165/167, M+ (58, 52, 6.0); 1481150. [M - CH31+ (15, 14); 1351137, [M - CO]+ (53, 47); 120/122, [M - CHa- COJ+ (lOO, 91). Aldebyde Oxidase Experiments. Products of N­methylnicotinamide oxidation by aldehyde oxidase present in the cytosolic fractions of goat, mouse, and rabbit lung and liver tissues are shown in Figure 2. The enzyme present in goat pulmonary and hepatic tissues oxidized the model substrate to a much lesser extent than that in the rabbit tissues. The enzyme from mouse lung cytosol did not oxidize the substrate to any measurable level, but the mouse liver cytosol had an activity that was lower than that of the goat and higher than that of the rabbit. 45 Skordos et ai. 16 I.Goat-~ 1 i.Mouse _CI1t 4 iORabbit : .e c ~12 E a. c 810 III E 0 IL. II 8 c 0 :s! f6 "c III II g 4 "·c ~ C'o! 2 0 Lung Liver Figure 2. Aldehyde oxidase-catalyzed oxidation of N-meth­ylnicotinamide to 2-pyridone and 4-pyridone. Error bars repre­sent standard deviations of two or more replicates from at least three animals. Lung Uver Figure 3. Aldehyde oxidase-catalyzed oxidation of 3-hydroxy- 3-methylindolenine to 3-hydroxy-3-methyloxindole. Error bars represent standard deviations of two or more replicates from at least three animals. For both liver and lung tissues, the rabbit exhibited the greatest activity. As expected (14), the activities in liver cytosols were much higher than in the lung tissues for all species. Aldehyde oxidase-catalyzed conversion of 3-hydroxy- 3-methylindolenine to 3-hydroxy-3-methyloxindole was monitored by HPLC. The results are reported in Figure 3. The amount of substrate for the aldehyde oxidase reaction, 3-hydroxy-3-methylindolenine, was normalized in that all incubations contained the same concentration of goat lung microsomal cytochrome P450. Therefore, the production of 3-hydroxy-3-methylindolenine from cyto- 3-Methylindole Epoxidation chrome P450-mediated metabolism of 3-methylindole should have been relatively constant. The source of aldehyde oxidase was the cytosolic protein fractions from both liver and lung from goats, mice, rats, and rabbits. 3-Hydroxy-3-methyloxindole was not produced in incuba­tions that contained microsomes alone or cytosolic protein alone (data not shown). Surprisingly, both liver and lung from the goat, the most susceptible species to 3-meth­ylindole pneumotoxicity, exhibited the highest aldehyde oxidase activities for this substrate. The activity in mouse lung was extremely low, while the hepatic activity from this species was nearly as high as that of the goat. The rat exhibited the lowest activities for both lung and liver. The rabbit exhibited an activity nearly as high as the goat for lung, but the activity was lower than both goat and mouse activity for the liver. Expectedly, incubations containing liver cytosol had much higher aldehyde oxidase activities than lung cytosol. Discussion Cytochrome P450-mediated epoxidation reactions play a critical role in the biotransformation and subsequent toxicity of many xenobiotics. Perhaps one of the most notable is aflatoxin B1• This fungal metabolite is biotrans­formed by cytochrome P450 CYP3A forms to its 8,9- epoxide (20) in human lung tissues. The carcinogenicity of 1,3-butadiene is also linked to mono- and diepoxides that are produced by cytochrome P450 enzymes (21). Therefore, given that cytochrome P450 enzymes have the potential to oxygenate 3-methylindole to its 2,3-epoxide, it is reasonable to hypothesize that this 2,3-epoxide could play some role in the pneumotoxicity of this compound. Data in this work provide evidence for the existence and the chemical and metabolic fate of this epoxide. It is possible that oxidation of 3-methylindole to 3-methyloxindole or 3-hydroxy-3-methylindolenine by cytochrome P450 enzymes occurs without the interven­tion of a formal epoxide or that the epoxide is formed by ring closure of a tetrahedral intermediate, formed by addition (13) of a triplet-like FeO species to the 2- or 3-position of the indole moiety. As stated previously, the techniques employed in the current investigations do not permit us to conclude unequivocally that the 2,3-epoxide is released from the active site of the enzyme(s). Studies have shown (12), however, that 2,3-epoxides of 2- and 3-substituted N-acylindoles can be synthesized chemi­cally, and some of these epoxides are stable overnight at o DC. When these indole 2,3-epoxides are warmed to room temperature, they form substituted oxindoles. Thus, these studies demonstrate that 2,a-epoxy-a-meth­ylindoline may be a reasonably stable intermediate, and its formation and release by P450 enzyme{s) are not unreasonable mechanisms ofP450-mediated oxygenation of a-methylindole. a-Methyloxindole is a stable metabolite of3-methylin­dole that theoretically can be formed from the 2,a-epoxide (Scheme a, D, E, G). As described above, one mechanism for a-methyloxindole formation is through the epoxide that undergoes an NIH shift. Ring opening of the epoxide probably occurs by another mechanism as well, which does not require a hydride shift for formation of the oxindole; this mechanism invokes hydrogen loss from position 2 and ring opening to the enol with subsequent tautomerization to the keto form. Another mechanism for 3-methyloxindole formation likely involves the hydra- Chem. Res. Toxicol., Vol. 11, No.7, 1998 747 tion of 3-methyleneindolenine at position 2 and a sub­sequent tautomerization. The studies presented here show that all of the possible mechanisms for a-methyl­oxindole formation occur, at least to a small extent. 46 180 Incorporation from molecular oxygen into 3-meth­yloxindole demonstrated that 74% of the metabolite contained the label. When this experiment was per­formed in the presence of N-acetylcysteine, 91% of the metabolite contained the label. This confirms that multiple mechanisms for this metabolite's formation are likely occurring (Scheme 2, A). When N-acetylcysteine is present in the incubation, one or more reactive intermediates, e.g., a-methyleneindolenine, that lead to the oxindole by incorporation of oxygen from water are being trapped as the N-acetylcysteine thioether conjugate before the metabolite forms. Therefore, in the presence of N-acetylcysteine, 91% of the oxindole is probably produced from the epoxide. This leaves 9% whose mechanism of formation is unexplained but could be produced by hydration of the epoxide followed by a dehydration step in which the 180 label is lost (Scheme 3, 0). A worldwide shortage of 180 at the time these experiments were performed necessitated the use of extremely small incubation volumes and did not permit experiments in both the absence and presence of N­acetylcysteine for studies of 180 incorporation from H2 180. As shown in Table 2, the incorporation of 180 into 3-methyloxindole from water was roughly 6%, a value much smaller than the expected 26% based on incorpora­tion from air in the absence of N-acetylcysteine. An extremely small amount of a-methyloxindole was pro­duced in these incubations, probably due to the small incubation volumes. Therefore, reliable quantification of this metabolite was extremely difficult, and poor analyti­cal precision led to a wide range in the incorporation results. Consequently, 20% of the oxygen incorporated into 3-methyloxindole in the absence of N-acetylcysteine was unidentified. a-Methyloxindole's retention of 2H, when [2-2Hl-a­methylindole was used as the substrate in the incubation, also indicates the presence of the epoxide and that at least one mechanism for ring opening involves an NIH shift. In the absence of N-acetylcysteine the 2H retention by 3-methyloxindole was 71%, and in its presence the retention went up to 81%, an increase of 10%. This is somewhat inconsistent with the ISO incorporation data, which displayed an increase in lSO incorporation of 17% in the presence of N-acetylcysteine. In both cases N­acetylcysteine is presumably depleting the fraction of a-methyloxindole that forms by hydration of another reactive intermediate (3-methyleneindolenine). 1f3-meth­yleneinodolenine is the precursor of the oxindole, lSO from molecular oxygen would not be incorporated and 2H would not be retained. Therefore, it might be expected that changes in 180 incorporation and 2H retention, in the presence of N-acetylcysteine, would be of the same magnitude. However, one might not necessarily expect the absolute percentage changes to be the same due to the possibility that multiple mechanisms of epoxide ring opening may be occurring that lead to the oxindole, perhaps not involving an NIH shift. Ring opening leaving oxygen at position 2, can lead to an enol that tautomerizes to the oxindole, resulting in the loss of the label to water. (Scheme a, 0). Another possibility involves cytochrome P450-mediated direct hydroxylation of 3-methylindole at 748 Chem. Res. Toxicol., Vol. 11, No.7, 1998 C-2 through an addition-rearrangement pathway (13) to form the enol which also tautomerizes to the keto form (3-methyloxindole). In both cases, the oxygen incorpo· rated into the oxindole does, in fact, arise from molecular oxygen, but 2H at position 2 would be lost. 3-Hydroxy-3-methylindolenine is another reactive in­termediate of 3-methylindole that likely results from epoxide ring opening leaving the oxygen at position 3. This transient electrophilic intermediate appears to be oxidized to the stable metabolite, 3-hydroxy-3-methylox­indole, by the cytosolic enzyme aldehyde oxidase. Both the 180 incorporation characteristics and the putative detoxification pathway were investigated in this work. The incorporation of molecular oxygen into 3-hydroxy- 3-methyloxindole was higher than that of 3-methyloxin­dole, 87% versus 74%. The ratios of the 1800, 1801, and 180 2 isotopomers indicate that, except for a minor amount of incorporation of two oxygen atoms that originated from molecular oxygen, the vast majority of the compound was formed by incorporation of only one atom from molecular oxygen. There are several possible 'scenarios for oxygen incorporation into the metabolite, but the one that best fits the data is one in which one site incorporates oxygen from molecular oxygen and the other incorporates oxygen from water. In this case the expected ratios of the isotopomers 1800, 180 1, and I8()2 in 3-hydroxy-3-methyl­oxindole, from either molecular oxygen or water, would be 1:1:0. This scenario best fits the data as presented in Tables 1 and 2. The very small amount of the 180 2 isoto po mer was probably produced by an uncharacterized mechanism that involves incorporation of two oxygen atoms from molecular oxygen. The mass spectral data (Figure 1) also support a mechanism for 3-hydroxy-3-methyloxindole formation that involves incorporation of only one oxygen from molecular oxygen and that the site of incorporation is the alcohol oxygen. This also supports a mechanism involv­ing the epoxide that ring-opens to position 3. The only fragmentation mechanism resulting in the M - 28 ions at mlz 135/137 is loss of CO; thus, the retention of approximately an equal isotopomeric ratio (18() atom % of H20 was 50%) in the mlz 135/137 fragment cluster indicates that the site of incorporation must be the alcohol at C 3 (Figure 1). Aldehyde oxidase appears to catalyze the oxidation of 3-hydroxy-3-methylindolenine to 3-hydroxy-3-methylox­indole; therefore, it is reasonable to hypothesize that the enzyme plays an important role in the detoxification of this 3-methylindole reactive intermediate. Presumably, animal species with higher pulmonary aldehyde oxidase activities for this substrate, e.g., rabbits (14), would be less susceptible to 3-methylindole-induced pneumotox­icity. When the model substrate (N-methylnicotinamide) for this enzyme was utilized, the pattern of enzyme activity for the species and tissue types appeared to confirm this hypothesis; i.e., rabbits had higher enzyme activities in both lung and liver than mice or goats. However, in incubations with 3-methylindole, goats, the most susceptible species, exhibited the highest aldehyde oxidase activities for this substrate in both liver and lung cytosol. Thus, N-methylnicotinamide does not appear to be a good surrogate substrate for 3-hydroxy-3-methylin­dolenine; the isozyme of aldehyde oxidase that catalyzes the oxidation of 3-hydroxy-3-methylindolenine may be different from the isozyme that catalyzes the oxidation of N-methylnicotinamide. If 3-hydroxy-3-methylindole- 47 Skordos et al. nine plays a role in the toxicity of 3-methylindole, then aldehyde oxidase is probably involved in detoxification processes and may be protective of hepatic cells. How­ever, the relative order of species sensitivities to the pneumotoxic effects of 3-methylindole does not correlate to a relative lack of aldehyde oxidase activity in pulmo­nary tissues of sensitive species. Presented here is an indirect confirmation that a cytochrome P450-dependent 2,3-epoxide of 3-methylin­dole exists and can be characterized by monitoring its decomposition products. Additional work is required to elucidate the epoxide's role and the role of 3-hydroxy-3- methylindolenine in 3-methylindole-mediated pneumo­toxicity. An analysis of the protein and/or nucleic acid alkylation sites for each of the proposed reactive inter­mediates is essential to an understanding of the cascade of events that likely lead to the observed toxicity. Work describing the nature ofthiol conjugates ofthese reactive intermediates is underway. Acknowledgment. This work was supported by United States Public Health Service Grant HL13645 from the National Heart, Lung and Blood Institute and by a United States Public Health Service Research Career Development Award (Grant HL02119) to G.S.Y. The authors gratefully acknowledge the technical assistance of Sandra J. Lehman and Dr. Pradipta Jeshti. We are grateful to Drs. Douglas E. Rollins and Roger L. Foltz of the Center for Human Toxicology for providing mass spectrometry instrumentation. References (1) 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. (2) Carlson, J. R., and Yost, G. S. (1989) 3-Methylindole-induced acute lung injury resulting from ruminal fermentation of tryptophan. In Toxicants of Plo.nt Origin (Cheeke, P.R., Ed.) Vol. 3, pp 107- 123, CRC Press, Boca Raton, FL. (3) Adams, J. D., Laegreid, W. W., Huijzer, J. C., Hayman, C., and Yost, G. S. (1988) Pathology and glutathione status in 3-meth­ylindole- treated rodents. Res. Commun. Chern. Po.thol. PhanTl4COl. 60, 323-335. (4) Ruangyuttikarn, W., Appleton, M. L., and Yost, G. S. (1991) Metabolism of 3-methylindoJe in human tissues. Drug Metab. Dispos. 19, 977-984. (5) Thornton-Manning, J. R., Appleton, M. L., Gonzalez, F. J., and Yost, G. S. (1996) Metabolism of 3-methylindole by vaccinia­expressed P450 enzymes: correlation of 3-methyleneindolenine formation and protein-binding. J. Pharmacal. Exp. Titer. 276, 21- 29. (6) Yokoyama, M. T., and Carlson, J. R. (1974) Dissimilation of tryptophan and related indolic compounds by ruminal microor­ganisms. AppL Microbial. 27, 1540-1548. (7) Nocerini. M. R., Carlson, J. R., and Yost, G. S. (1985) Glutathione adduct formation with microsomally activated metabolites of the pulmonary alkylating and cytotoxic agent, 3-methyliodole. Toxi· col. Appl. Pharmacol. 81,75-84. (8) 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 metbide is the electrophilic intermediate. DrUB Metab. Disp08. 13, 690-694. (9) Skiles, G. L., and Yost, G. S. (1996) Mechanistic studies on the cytochrome P450-catalyzed dehydrogenation of 3-methylindole. Chern. Res. Toxicol. 9, 291-297. (10) Ruangyuttikarn. W., Skiles, G. L., and Yost, G. S. (1992) Ident.ification of a cysteinyJ adduct of oxidized 3-methylindole from goat lung and human liver microsomal proteins. Chern. Res. Toxicol. 6, 713-719. (11) King, L. J., Parke, D. V., and Williams, R. T. (1966) The metabolism of 12-14Clindole in the rat. Biochem. J. 9B, 266-277. (12) Zhang, X .• and Foote, C. S. (1993) Dimethyldioxirane oxidation of indole derivatives. Formation of novel indole-2.3-epoxides and 3-Methylindole Epoxidation a versatile synthetic route to indolinones and indolines. J. Am. Chem. Soc. lIS, 8867-8868. (13) Darbyshire, J. F., Iyer, K. R., Grogan, J., Korzekwa, K. R., and Trager, W. F. (1996) Selectively deuterated warfarin. Substrate probe for the mechanism of aromatic hydroxylation catalyzed by cytochrome P450. Drug Metab. Dispos. 24, 1038-1045. (14) Beedham, C. (987) Molybdenum hydroxylases: biological dis­tribution and substrate-inhibitor specificity. In Progress in Me­dicinal Chemistry (Ellis, G. P., West, G. B., Eds.) pp 85-127, Elsevier Science, Amsterdam. (15) Smith, D. J., Skiles, G. L., Appleton, M. L., Carlson, J. R., and Yost, G. S. (1993) Metabolic fate of 3-methylindole in goats and mice. Xenobiotica 23, 1025-1044. (16) Skiles, G. L., Adams, J. D., Jr., and Yost, G. S. (1989) Isolation and identification of 3-hydroxy-3-methyloxindole, the major mu­rine metabolite of 3-methylindole. Chem. Res. Toxicol. 2, 254- 259. (17) Kohn, M., and Ostersetzer, A. (1911) Einige neue abkommLing desdioxindoLs. Monatsh. Chem. 32, 905-916. 48 Chem. Res. Toxicol., Vol. 11, No.7, 1998 749 (1B) Katritzky, A. R., Akutagawa, K., and Jones, R. A. (1988) Carbon dioxide: a reagent for the protection of nucleophilic centres and the simultaneous activation to electrophilic attack Part XII. One-pot conversion of 3-methylindole into 2-formyl-3-methyindole. Synth. Commun. 18, 1151-115B. (19) Felsted, R. L., and Chaykin, S. (1971) Rabbit liver Nl-methylni­cotinamide oxidase. In Methods of Enzymology (McCormick, D. B., and Wright, L. D., Eds.) pp 216-222, Academic Press, New York. (20) Kelly, J. D., Eaton, D. L., Guengerich, F. P., and Coulombe, R. A, Jr. (1997) Aflatoxin Bl activation in human lung. TO%icol. Appl. Pharmacal. 144, 88-95. (21) Himmelstein, M. W., Acquavella, J. F., Recio, L .• Medinsky, M. A .• and Bond, J. A (1997) TOxicology and epidemiology of 1,3- butadiene. Crit. Rev. TO%icol. 27, 1-108. (22) Albright, J. D., and Snyder, H. R. (1959) Reaction of optically active indole mannich bases. J. Am. Chem. Soc. 81, 2239-2245. TX9702087 CHAPTER 3 TIllOETHER ADDUCTS OF A NEW IMINE REACTIVE INTERMEDIATE OF THE PNEUMOTOXIN 3-METHYLINDOLE Reproduced with permission from Chem. Res. Toxieol. 1998, ii, 1326-1331. Copyright 1998 Am. Chern. Soc. 50 1326 Chem. Res. Toxicol. 1998, II, 1326-1331 Thioether Adducts of a New Imine Reactive Intermediate of the Pneumotoxin 3-Methylindole Konstantine W. Skordos, John D. Laycock,t and Garold S. Yost* Department of Pharmacology and Toxicology, 112 Skaggs Hall, University of Utah, Salt Lake City, Utah 84112·5820 Received May 26, 1998 Cytochrome P450 enzymes can potentially oxygenate 3-methylindole to form 2,3-epoxy-3- methylindoline which could rearrange to the stable metabolite 3-methyloxindole or open to form 3-hydroxy-3-methylindolenine, a putative electrophilic imine. The purpose of the current work was to determine if the imine was formed, and to characterize it via its adducts with thiol nucleophiles. Thiols were added to incubations of goat lung microsomes with 3-meth­ylindole and deuterated analogues of 3-methylindole to trap the imine intermediate as its thioether conjugates. The N-acetylcysteine conjugate of 3-hydroxy-3-methylindolenine was detectable by LeIMB, but a molecular ion was not observed because the adduct rapidly dehydrated to form the 2-substituted indole. However, the imine was B-alkylated, and the intermediate carbinol was intramolecularly trapped using thioglycolic acid as a trapping agent that induced cyclocondensation to a lactone. The retention of one atom of deuterium from [2-2Hl-3-methylindole and three from 3.[2H3-methyllindole substantiated the mechanism in which the lactone adduct was produced by sulfur addition to either 3-hydroxy-3-methylindo­lenine or the epoxide. Tandem mass spectrometry of the lactone adduct produced a daughter ion spectrum consistent with this adduct. These studies demonstrated the existence of a new reactive intermediate of3-methylindole, 3-hydroxy-3-methylindolenine, which may playa role in the pneumotoxicity of this chemical. Introduction 3-Methylindole is a rumina! degradation product of the essential amino acid tryptophan, and is selectively toxic to pulmonary tissues following biotransformation by cytochrome P450 enzymes (1). Goats, cattle, and other ruminants are by far the most susceptible to 3-methylin­dole's toxicity, but mice, rats, and rabbits are also susceptible to much lesser extent (2, 3). 3-Methylindole's toxicity is most apparent in cattle when grazing condi· tions are changed rapidly from sparse to lush pastures. The cattle develop acute pulmonary edema that has been attributed to the production and subsequent bioactivation of3·methylindole (4). 3-Methylindole circulates systemi­cally to the pulmonary tissues where it is bioactivated by cytochrome P450 enzymes to several reactive inter­mediates. These electrophilic intermediates have the potential to alkyl ate cellular macromolecules including proteins and nucleic acids, initiating the cascade of events that leads to toxicity (5). 3-Methylindole toxicity has not been fully evaluated in humans, but the toxin is a substrate for human cytochrome P450 enzymes, some of which are selectively expressed in human lung tissues (6, 7). The toxicity of 3-methylindole has predominantly been attributed to the action of 3-methyleneindolenine, a cytochrome P450-dependent reactive intermediate that is produced by dehydrogenation at the methyl group * Corresponding author. Telephone: [phone]. Fax: 801585· 3945. Email: gyOlltiideans.pharm.utah.edu. t Present address: Performance Bioanalytical, 11545 Sorrento Val· ley Rd, Suite 315, San Diego, CA 92121. (Scheme 1) (8). The mechanism for its formation pro­ceeds by hydrogen atom abstraction from the methyl position followed by a one-electron oxidation that pro­duces the soft electrophile that can alkylate proteins at nucleophilic sites by a Michael-like addition (Scheme 1) (9). The imines are depicted in their protonated form based on the basicity of indolenine nitrogen atoms (10). Cytochrome P450 has recently been shown to oxygen· ate 3-methylindole to its 2,3-epoxide (Scheme 1) (11). The fate of the epoxide may include alkylation reactions, rearrangement to form the stable oxindole, or rearrange­ment to form 3-hydroxy-3-methylindolenine. 3-Hydroxy- 3-methylindolenine is another electrophilic iminium in­termediate that may participate in alkylation reactions, or undergo further oxidation by the cytosolic enzyme aldehyde oxidase to 3-hydroxy-3-methyloxindole (Scheme 1) (11). 3-Hydroxy-3-methyloxindole is in fact the pre­dominant urinary metabolite of 3-methylindole in all species (12, 13). Given the prevalence of the metabolite 3-hydroxy-3-methyloxindole, 3-hydroxy-3-methylindole­nine may be a prominent reactive intermediate of 3-me­thylindole. A complete investigation and confirmation of the existence of 3-hydroxy-3-methylindolenine as a reactive intermediate of 3-methylindole have not been accom­plished to date. The work presented here utilizes reac­tive thiols to trap the intermediate as a thioether conjugate. Using goat lung microsomes to provide a P450-containing system, experiments were performed using both N-acetylcysteine and thioglycolic acid to trap the reactive intermediates of 3-methylindole. 10.1021/tx9801209 CCC: $15.00 01998 American Chemical Society Published on Web 10/07/1998 Reactive Intermediates of 3-Methylindole Chern. Res. Toxicol., Vol. 11, No. 11, 1998 1327 Scheme 1. 3-Methylindole Reactive Intermediates and Their Thiol COrQugateso Dehydrogenation ~ Med!yletIC imine ,cd'" l . ~, 6 A NH 'n,f] Oxygenation l:! cd=o 3-Methyloxindole 4 Dehydrogenation and oxygenation pathways of cytochrome P450-mediated oxidation are shown. 3MI, 3-methylindole; methylene imine, 3-methyleneindolenine; 2,3-epoxide, 2,3-epoxy-3-methylindoline; hydroxyindolenine, 3-hydroxy-3-methylindolenine; RSH, N-acetylcysteine. Scheme 2. Intramolecular Trapping of 3-Hydroxy-3-methylindolenine with Thioglycolic Acid- ~OH .U, il HydroxyindoiCllline TGA 4 Hydroxyindolenine, 3-hydroxy-3-methylindolenine; TGA, thio­glycolic acid. Liquid chromatography in conjunction with mass spectrometry was used to detect and perform structural characterization of the various thiol adducts of 3-meth­ylindole reactive intermediates. N-Acetylcysteine was used in an attempt to trap the intermediates directly. Thioether adducts would be expected both at the meth­ylene carbon and at C-2 from 3-methyleneindolenine and at C-2 from 3-hydroxy-3-methylindolenine. Similar to N-acetylcysteine, thioglycolic acid could be used to trap the reactive intermediates directly, but in the case of 3-hydroxy-3-methylindolenine the thiol adduct has the potential to undergo cyc1ocondensation, trapping the alcohol oxygen as a thiolactone (Scheme 2). Deuterated analogues of 3-methylindole were used to evaluate the structures of the electrophiles and their mechanisms of formation through P450-mediated dehydrogenation and oxygenation. Experimental Procedures Chemieais. 3-Methylindole, N-acetylcysteine, thioglycolic acid, nicotinamide adenine dinucleotide phosphate (NADPH), ammonium acetate, and formic acid were purchased from Sigma Chemical Co. (St. Louis, MO). 3-Hydroxy-3-methyloxindole and [2.2HI-3-methylindole were prepared by a method described by Skordos et a1. (11). 3-MINAC and 3·f2Ha-methylJindole were synthesized by published methods (14) and (15), respectively. Microsomal Preparation. Goat lungs were obtained from mature male goats. The lungs were perfused in situ with ice­cold 0.05 M phosphate bufTercontaining 1 mM EDTAand 1.15% KCI and then frozen at -70 ·C. Microsomes were prepared by standard centrifugal isolation procedures (16). IncubatiolUl for Thiol Trapping of a.Methylindole Re­active Intermediates. Incubations contained, 4 mM NADPH, goat lung microsomes containing 1.1 nmol of cytochrome P4501 mL, and either 0.5 mM 3-methylindole, 0.5 mM !2-2HJ-3- methylindole, or 0.5 mM 3-[2Ha-methyllindole. Incubations were performed in 0.01 M ammonium acetate, pH 7.4, in a final volume of 1 mL. Incubations were also performed in the presence of 4 mM N-acetylcysteine or 2 mM thioglycolic acid as thiol trapping agents. The reactions were performed at 37 ·C in a shaking water bath for 30 min and stopped by immersion in an ice bath and by the addition of an equivalent volume of ice-cold acetonitrile to both stop the reaction and precipitate protein. The precipitated protein was separated from the reaction mixture by centrifugation at 3100g in a Beckman GPR tabletop centrifuge. The supernatant was removed and con­centrated to 2oo,uL in a Savant SpeedVac SVC100 for analysis by LCIMS. Liquid Cbromatography/Mass Spectrometry (LC'JMS). The supernatant fractions from the thiol trapping experiments were analyzed by the LCIMS technique described by Skordos et al. (11). The mixtures were injected using a Leap Technolo­gies CTC-A200S auto-sampler into a Waters 626 liquid chro­matograph. Metabolites were separated using a Phenomenex Ultramex 5,ut C18 IP (250 mm x 2.1 mm, 5,um) and a gradient of 10-35% organic after 5 min, 50% organic at 10 min, 55% organic at 15 min, 95% organic at 19 min, isocratic for 2 min, and back to 10% organic at 25 min. The column was reequili­brated for 7 min between injections. The organic phase was acetonitrile, containing 0.1% (v/v) formic acid, and the aqueous phase contained 0.1% (v/v) formic acid. Mass spectrometry was performed using a Finnigan model TSQ-7000 triple stage quadrupole mass spectrometer with an atmospheric pressure ionization interface to the liquid chromatograph. A capillary temperature of 175 ·C and a vaporizer temperature of 500 ·C were used. A corona discharge voltage of 2.84 kV at 4.9,uA was used with the sheath gas pressure set at 70 psi N2. Samples were also analyzed byelectrospray ionization with the capillary at a temperature of224.7·C and a voltage of 4.5 kV at 13.6 pA. Negative ions were monitored in this technique. Tandem Mass Spectrometry of the Thioglyeolic Acid ConJugate. Liquid chromatography was performed as de- 51 52 1328 Chem. Res. ToxicoZ., Vol. 11, No. 11, 1998 Skordos et oZ. Substrate RSH mJz Intensity 3MI 291 Leoa 8.4xl02 e d"b OO..-{) 0 A- o gO e 0,. e,,= r j (y(SR n c0-SR 3MI + 291 VNH. .& Nil r 0. A 7.4x1OS 2d3MI + 291 j A OO-SR .& NN r 7.lxlOS 2d3MI + 292 j o:Nfil : I\. r ba 6 1.2xlO5 ] o,c.SK 3Mld3 + 293 C0. 11 ~. .r Nil fJ. ~~H 1.6x I ()4 3Mld3 + 294 j b, of" J 7.6x1Os 111:00 11:40 1l:10 15:80 16:40 11:10 10:00 Figure 1. Ion chromatograms from LCIMS analysis of3-methylindole incubations with goat lung microsomes using N-acetylcysteine as a trapping agent. Negative ions generated by electrospray ionization were detected. Chromatograms are representative of three independent replicates. Abbreviations: 3MI, 3-methylindole; 2d3MI, 12-2Hj-3-methylindole; 3MIdS, 3-12Ha-methyllindole; RSH, N-acetylcysteine; *, first isotope peak of mJz 291. scribed above, and ionization was accomplished by atmospheric pressure chemical ionization. Tandem mass spectrometry of the thioglycolic acid adduct was perfonned using the first quadru­pole to select parent ions, mlz 222 in this case, for collision­induced dissociation. The radio frequency quadrupole was used as a collision cell using Ar as the collision gas. Positive daughter ions were monitored by the third quadrupole. Results Trapping with N.Acetylcysteine. Incubations per­formed with goat lung microsomes, 3-methylindole, and N-acetylcysteine were analyzed by LCIMS, detecting negative ions, [M-H]-, which were generated byelec­trospray ionization. Three N-acetylcysteine ad ducts of 3-methylindole reactive intermediates were expected. One anticipated adduct was at the methylene position of3-methyleneindolenine, another was an adduct at C-2 of 3-methyleneindolenine, and the third was the adduct at C-2 of 3-hydroxy-3-methylindolenine. A pure standard of the first was available for analysis and was found to have a retention time of approximately 13 min and a m/z of 291 under these conditions. The second would have the same m/z as the first but should not have the same retention time. The third would have a m/z of 309. Only two N-acetylcysteine-dependent peaks were ob­served (Figure 1). One had a retention time ofI3:11 min and a m/z of 291, and the other had a retention time of 14:56 min and a mlz of 291. The early eluting adduct appeared to be produced by thiol addition to the meth­ylene position of3-methyleneindolenine; the other adduct was probably formed by thiol addition to C~2 of 3-meth­yleneindolenine, or by thiol addition to C-2 of3-hydroxy- 3-methylindolenine that subsequently dehydrated as a result of chemical manipulation or the ionization tech­nique. Thus, this peak had a mlz of 291, 18 mass units fewer than the expected mlz of 309. Incubations were then performed with [2-2H]-3-meth­ylindole. Again, two N-acetylcysteine-dependent peaks were observed (Figure 1). In this case, however, the adduct eluting at 13:11 min had a mlz of 292, indicating that it had retained the 2H atom at the C-2 position. This is consistent with the assignment of this peak to the methyl position adduct of 3-methyleneindolenine because this reactive intermediate and subsequently formed adducts were produced by dehydrogenation and adduct formation at the methyl position (Scheme 3). The 2H atom at the C-2 position was lost in the adduct eluting at 15:00 min; its mlz remained at 291. This was also consistent with the assignment of this peak as the thiol adduct of3-methyleneindolenine at C-2 or the dehydrated adduct of3-hydroxy-3-methylindolenine at C-2. In either case, the 2H atom at position 2 would be lost because the C-2 adduct of 3-methyleneindolenine would lose the 2H atom during tautomerization to the indole. The adduct of 3-hydroxy-3-methylindolenine would also lose the 2H as it dehydrates to re-form the indole (Scheme 3). The assignments of the peaks formed as thiol adducts of N-acetylcysteine were further confIrmed from the results of incubations with 3- [2H3-methyl]indole. The adduct eluting at 13:20 min had a mlz of 293, indicating that it had lost one of the methyl 2H atoms (Figure 1). Again, this is consistent with the assignment of this peak as the methylene adduct of 3-methyleneindolenine. The reactive intermediate was presumably generated by a cytochrome P450-catalyzed dehydrogenation at the meth­yl position, and would therefore have lost one of the methyl2H atoms (Scheme 3). These incubations permit­ted positive identification of the peak at 15:04 min. The peak was in fact composed of both the C-2 adduct of 3-methyleneindolenine and, to a much greater extent, the adduct of 3-hydroxy-3-methylinolenine that had lost water. The peak had components with mlz of 293 and 294, with the m/z at 294 having a nearly 50-fold greater intensity than the one at 293. These adducts were chemically identical, but were probably formed from completely different cytochrome P450-dependent reactive Reactive Intermediates of 3-Methylindok Scheme S. Expected Man Shifts of N-AcetylcysteiDe Adduct. Produced from IncubatioDs with [2-'H)-S-Methylindole <Panel A) or S-PZHs-methyl]iDdole (Panel B)a OO-D / 2d3MI ~ o:}-D cr;. I HAC 00:: 1 NAC -UOD ~ SR UN~ IM-H)" • m.'& 191 1 RSH -UOD 0 J Vpui-SR IM-Itr •• U93 (M-Ur ... U94 a 2d3MI, 12-2H1-3-methylindole; 3MId3, 3-12Hs-methyllindole; RBH. N-acetylcysteine. intermediates. The use ofLCIMS and the trideuterated substrate permitted the resolution of these adducts. The adduct at mJz 293 was likely formed by N-acetylcysteine addition to C-2 of the dehydrogenation product, 3-meth­yleneindolenine, indicated by the retention of only two of the three methyl deuterium atoms. The adduct at mJz 294 retained all three methyl deuterium atoms and was probably formed by thiol addition to a reactive intermedi­ate other than 3-methyleneindolenine. probably 3-hy­droxy- 3-methylindolenine, which had subsequently lost a molecule of H20 after N-acetylcysteine addition. An N-acetylcysteine-dependent peak was also observed at a retention time of 19:11 and appeared to have a mJz of 291. This was shown to be a fragment ion of an N-acetylcysteine adduct of a reactive 3-methylindole dimer with a mlz of 420. Trapping with Thioglycolic Acid. Incubations of goat lung microsomes with 3-methylindole and thiogly­colic acid were also analyzed by LCIMS; however, in this case, positive ions, [M + H]+, generated by atmospheric pressure chemical ionization, were detected. Three thioglycolic acid adducts of a-methylindole reactive in­termediates were expected. Again, one should be a methyl adduct of 3-methyleneindolenine and another 53 Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1329 should form by thiol addition to C-2 of both a-methyl­eneindolenine and 3-hydroxy-3-methylindolenine. The last adduct should be the thiolactone that forms by cyclocondensation following addition to the 2 position of 3-hydroxy-3-methylindolenine. The first has an expected mJz of 222 , the second at 240, and the last at 222 (Scheme 2). Three thioglycolic acid-dependent peaks were observed. A pair of peaks eluted very close to one another, sepa­rated by less than 0.5 min, and was centered at a retention time of 12:43 min (Figure 2). Both peaks had a mlz of 222, but the earlier of the two was considerably less intense than the latter. The third eluted at a retention time of 16:51 min and also had a mlz of 222. An adduct was not observed at mlz 240. Since the adducts eluting at 12:43 min had very similar chromato­graphic properties, it is likely that they are the diaster­eomers of the putative cyclic thioglycolic acid adduct. The adduct that eluted at 16:51 min could possibly have been either a C-2 adduct, formed by mechanisms discussed above, or an adduct at the methyl position. Incubations were then performed with [2-2Hl-a-meth­ylindole. The pair of adducts eluting at 12:50 min retained the C-2 deuterium atom, and had a mlz of 223 (Figure 2). This is consistent with the assignment of these peaks as the diastereomers of the putative cyclic adduct, because the deuterium atom at C-2 should be retained in this product. However, the adduct at 17:00 min lost the C-2 deuterium atom and had a mlz of 222. Therefore. the peak could not be assigned to the methyl adduct of 3-methyleneindolenine because this adduct would retain the C-2 deuterium. It must therefore be either a C-2 adduct of 3-methyleneindolenine or a dehy­drated adduct of 3-hydroxy-3-methylindolenine. The pair of adducts eluting at 12:5a min had a mlz of 225 when mcubations were performed with 3-[2H3- methyllindole, indicating that they had retained all three methyl deuterium atoms and could not have formed from the dehydrogenation product (Figure 2). This confirms the assignment of these peaks as diastereomers of the cyclic thioglycolic acid adduct, and therefore could have only originated from addition to 3-hydroxy-3-methyl­indolenine. The adduct eluting at 17:02 min also had a mlz of 225 which indicated that it had retained all three methyl deuterium atoms, and therefore did not originate from addition of the thiol to C-2 of 3-methyleneindole­nine. It too must have originated from addition to position 2 of 3-hydroxy-3-methylindolenine followed by dehydration re-forming the indole, as seen with the N-acetylcysteine adduct. Unexpectedly, an adduct of3-methyleneindolenine was not observed for any of the trapping experiments with thioglycolic acid. Tandem Mass Spectrometry of the Putative Cy­clic Adduct. Positive daughter ions of the thioglycolic acid adduct eluting at 12:43 min with a mJz of 222 were detected after collision-induced dissociation. The MSIMS spectrum is shown in Figure a. The fragment ion that supports the proposed structural assignment is at mJz 147.8. The generation of a fragment of a thioglycolic acid adduct with mJz 147.8 is consistent with the proposed structure. Other prominent ions were observed at mJz 130.8, 117.7, and 105.7. 1330 Chern. Res. Toxicol., Vol. 11, No. 11, 1998 Substrate TGA nUl i 3MI ,k".t 222 0 3Ml + uo~ 222 cQ-s oA 2d3MI + 222j 0 2d3MI + 223j o~ cls~ ANtill, 3MId3 + 3Mld3 + It t. Intensity • _de. M ,r 2.1x104 ~ c:}·')'1 2.8xlO' Acc i-'i(~13.8xlO' 54 Skordos et al. 10:N 11:" 13:20 15:00 .6: .. 0 11:20 2O:N Figure 2. Ion chromatograms from LCIMS analysis of 3-methylindole incubations with goat lung microsomes using thioglycolic acid as a trapping agent. Positive ions generated by atmospheric pressure chemical ionization were detected. Chromatograms are representative of three independent replicates. Abbreviations: 3MI, 3-methylindole; 2d3Ml, 12-2Hl-3-methylindole; 3MId3. 3-[2Ha­methyllindole; TGA. thioglycolic acid. 100 In 20 00i ;;;~\-<; I ',s h NH IM+HI-• • 1" 211.9 1110 150 m/z 100 Figure 3. MSIMS spectrum of the cyclic thioglycolic adduct. Daughter ions of mlz 222 are shown. Discussion The contribution of cytochrome P45O-mediated oxy­genation in the metabolism of 3-methylindole was re­cently confirmed with the characterization of 2,3-epoxy- 3-methylindoline (11). The metabolic fate of the epoxide intermediate includes chemical rearrangement to a stable metabolite, 3-methyloxindole. An additional rearrange­ment mechanism could produce 3-hydroxy-3-methyl­indolenine, a reactive imine that could playa role in the pneumotoxicity of 3-methylindole. The formation of 3-hydroxy-3-methylindolenine is not necessarily depend­ent on a precursor epoxide and in fact may proceed by an additi