Thioether Adducts of a New Imine Reactive Intermediate of the Pneumotoxin 3-Methylindole

Skordos, Konstantine W.; Laycock, John D.; Yost, Garold S.

doi:10.1021/tx9801209

Cited by 29 publications

(34 citation statements)

References 8 publications

(12 reference statements)

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“…In one such experiment, 3-glutathionyl-S-3-methyloxindole, a thioether adduct at the C-3 position of the 2,3-epoxide, was identified in metabolic reactions of 3MI with GSH-fortified human liver microsome (Yan et al, 2007). Additional studies showed that 3-methyl-2-glutathionyl-Sindole was not derived primarily from MEI in human liver microso- mal reactions and that the corresponding NAC adduct was not primarily derived from MEI in goat lung microsomal reactions (Skordos et al, 1998a;Yan et al, 2007). The studies presented here have identified MOI and most likely HMI as products of CYP2A13-mediated 3MI metabolism, a result suggesting that CYP2A13 catalyzes the epoxidation of 3MI.…”

Section: Discussionmentioning

confidence: 94%

The Pneumotoxin 3-Methylindole Is a Substrate and a Mechanism-Based Inactivator of CYP2A13, a Human Cytochrome P450 Enzyme Preferentially Expressed in the Respiratory Tract

D’Agostino¹,

Zhuo²,

Shadid³

et al. 2009

Drug Metab Dispos

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ABSTRACT:3-Methylindole (3MI), a respiratory tract toxicant, can be metabolized by a number of cytochromes P450 (P450), primarily through either dehydrogenation or epoxidation of the indole. In the present study, we assessed the bioactivation of 3MI by recombinant CYP2A13, a human P450 predominantly expressed in the respiratory tract. Four metabolites were detected, and the two principal ones were identified as indole-3-carbinol (I-3-C) and 3-methyloxindole (MOI). Bioactivation of 3MI by CYP2A13 was verified by the observation of three glutathione (GSH) adducts designated as GS-A1 (glutathione adduct 1), GS-A2 (glutathione adduct 2), and GS-A3 (glutathione adduct 3) in a NADPH-and GSH-fortified reaction system. GS-A1 and GS-A2 gave the same molecular ion at m/z 437, an increase of 305 Da over 3MI. Their structures are assigned to be 3-glutathionyl-S-methylindole and 3-methyl-2-glutathionyl-S-indole, respectively, on the basis of the mass fragmentation data obtained by high-resolution mass spectrometry. Kinetic parameters were determined for the formation of I-3-C (V max ‫؍‬ 1.5 nmol/min/nmol of P450; K m ‫؍‬ 14 M), MOI (V max ‫؍‬ 1.9 nmol/min/nmol of P450; K m ‫؍‬ 15 M) and 3-glutathionyl-S-methylindole (V max ‫؍‬ 0.7 nmol/min/nmol of P450; K m ‫؍‬ 13 M). The structure of GS-A3, a minor adduct with a protonated molecular ion at m/z 453, is proposed to be 3-glutathionyl-S-3-methyloxindole. We also discovered that 3MI is a mechanism-based inactivator of CYP2A13, given that it produced a time-, cofactor-, and 3MI concentration-dependent loss of activity toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, with a relatively low K I value of ϳ10 M and a k inact of 0.046 min ؊1 . Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.3-Methylindole (3MI) is a potent pneumotoxicant and nasal toxicant in several animal species studied, including ruminants, rabbits, and rodents (Adams et al., 1988;Carlson and Yost, 1989;Yost, 1989;Gaskell, 1990). The pulmonary toxicity of 3MI has been attributed to bioactivation by cytochrome P450 (P450) enzymes, which catalyze the formation of reactive intermediates that can bind to cellular proteins (Yost, 1989) and DNA (Regal et al., 2001). Evidence for P450 involvement comes from studies on in vitro metabolism of 3MI using vaccinia-expressed P450s (Thornton-Manning et al., 1991Lanza and Yost, 2001), as well as studies in which P450 inhibitors were found to decrease covalent binding and toxicity associated with 3MI metabolism (Huijzer et al., 1989). Bioactivation of 3MI results in the formation of at least three potentially toxic species via two distinct pathways: 3-methyleneindolenine (MEI), via dehydrogenation, and both 2,3-epoxy-3MI and 3-hydroxy-3-methylindolenine (HMI), via epoxidation on the pyrrole moiety (Scheme 1). Extensive studies seemed to show that dehydrogenation of 3MI is the major route of metabolism that results in toxicity (Skiles and Yost, 1996; reviewed in Yost, 2001). A study of the me...

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Section: Discussionmentioning

confidence: 94%

The Pneumotoxin 3-Methylindole Is a Substrate and a Mechanism-Based Inactivator of CYP2A13, a Human Cytochrome P450 Enzyme Preferentially Expressed in the Respiratory Tract

D’Agostino¹,

Zhuo²,

Shadid³

et al. 2009

Drug Metab Dispos

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“…Even with Ser which produced adducts without S9, adducts levels were higher with S9, indicating that metabolic activation also plays an important role in Ser-induced adducts. In the case of 3MI, cytochrome P450-mediated epoxidation of the imidazole ring or hydroxylation/dehydrogenation of the methyl group has been shown to produce reactive intermediates, which can form adducts with glutathione, N-acetylcysteine, proteins, and DNA [Skiles and Yost, 1996;Kaster and Yost, 1997;Skordos et al, 1998;Regal et al, 2001]. These routes of metabolic activation may also be involved in the formation of electrophiles from Mel, Ser, and Tryp.…”

Section: Discussionmentioning

confidence: 99%

Genotoxicity of naturally occurring indole compounds: correlation between covalent DNA binding and other genotoxicity tests

Reddy

Storer

Laws

et al. 2002

Environ and Mol Mutagen

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3-Methylindole (3MI), melatonin (Mel), serotonin (Ser), and tryptamine (Tryp) were evaluated in vitro for their potential to induce DNA adducts, DNA strand breaks, chromosomal aberrations (Abs), inhibition of DNA synthesis, and mutations. All compounds produced DNA adducts in calf thymus DNA in the presence of rat liver S9. In cultured rat hepatocytes, all produced DNA adducts but none induced DNA strand breaks. In Chinese hamster ovary cells, 3MI and Mel produced DNA adducts, Abs, and inhibition of DNA synthesis with and without S9, except that Mel without S9 did not form adducts. Ser formed DNA adducts, was an equivocal Abs inducer, and suppressed DNA synthesis. Tryp induced neither adducts nor Abs, but did suppress DNA synthesis with S9. Ser and Tryp were less cytotoxic than 3MI and Mel. Mel, Ser, and Tryp failed to induce mutations in Salmonella and E. coli strains with or without S9. 3MI and Mel produced DNA adducts but not mutations in Salmonella TA100 with S9. 3MI and its metabolite indole 3-carbinol also did not induce mutations in a shuttle vector system in human cells. The lack of correlation between DNA adducts and other genotoxicity endpoints for these indole compounds may be due to the higher sensitivity of the (32)P-postlabeling adduct assay or it may indicate that the indole-DNA adducts per se are not mutagenic and are not able to induce strand breaks or alkali-labile lesions. The indole-induced Abs may result from cytotoxicity and suppression of DNA synthesis with minimal if any contribution from DNA adducts.

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“…NAC has a simpler structure compared with GSH, making the analysis of MS/MS spectra of the trapped adducts less complicated. In addition, NAC has been used successfully for trapping reactive imine intermediates (Skordos et al, 1998a). NAC adducts of PCP were formed from incubations with 2B6 and observed at R t 18.9 and 19.13 min with an m/z value of 437 (data not shown).…”

Section: Shebley Et Almentioning

confidence: 99%

SELECTIVE PATHWAYS FOR THE METABOLISM OF PHENCYCLIDINE BY CYTOCHROME P450 2B ENZYMES: IDENTIFICATION OF ELECTROPHILIC METABOLITES, GLUTATHIONE, AND N-ACETYL CYSTEINE ADDUCTS

Shebley¹,

Jushchyshyn

Hollenberg³

2005

Drug Metab Dispos

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ABSTRACT:The metabolism of phencyclidine (PCP) has been studied previously in cytochrome P450 (P450)-containing microsomal systems. However, the reactive intermediate(s) that covalently binds to the P450 and leads to inactivation or leaves the active site to modify other proteins has not been identified. In this study two electrophilic intermediates of PCP were identified by mass spectrometry and by trapping with reduced glutathione (GSH) or N-acetyl cysteine (NAC). The tentative structures of these electrophilic intermediates were determined using mass spectrometry. P450s 2B1 and 2B4 formed a metabolite that exhibited an m/z of 240 corresponding to the mass of the 2,3-dihydropyridinium species of PCP or its conjugate base, the 1,2-dihydropyridine. Chemical reduction of the incubation mixture using NaBH

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Thioether Adducts of a New Imine Reactive Intermediate of the Pneumotoxin 3-Methylindole

Cited by 29 publications

References 8 publications

The Pneumotoxin 3-Methylindole Is a Substrate and a Mechanism-Based Inactivator of CYP2A13, a Human Cytochrome P450 Enzyme Preferentially Expressed in the Respiratory Tract

The Pneumotoxin 3-Methylindole Is a Substrate and a Mechanism-Based Inactivator of CYP2A13, a Human Cytochrome P450 Enzyme Preferentially Expressed in the Respiratory Tract

Genotoxicity of naturally occurring indole compounds: correlation between covalent DNA binding and other genotoxicity tests

SELECTIVE PATHWAYS FOR THE METABOLISM OF PHENCYCLIDINE BY CYTOCHROME P450 2B ENZYMES: IDENTIFICATION OF ELECTROPHILIC METABOLITES, GLUTATHIONE, AND N-ACETYL CYSTEINE ADDUCTS

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