Metabolism and Bioactivation of 3-Methylindole by Human Liver Microsomes

Abstract

Metabolism and bioactivation of 3-methylindole (3MI) were investigated in human liver microsomes. The metabolism of two deuterium-labeled analogues of 3MI permitted a relatively unambiguous identification of multiple metabolites and glutathione (GSH) adducts of reactive intermediates. A total of eight oxidized metabolites were detected, five of which were assigned as previously identified 3-methyloxindole, 3-hydroxy-3-methylindolenine, 3-hydroxy-3-methyloxindole, 5-hydroxy-3-methylindole, and 6-hydroxy-3-methylindole. Among the three new metabolites, one was either 4- or 7-OH-3-methylindole, and the other two were derived from additional oxidation on the phenyl ring of 3-methyloxindole. When GSH was added to the microsomal incubations, seven conjugates that had molecular ions corresponding to the incorporation of GSH and an atom of oxygen at m/z 453 (group I) were produced, and two additional conjugates had molecular ions at m/z 437 that corresponded to the incorporation of GSH with no additional oxygen (group II). Two conjugates in group I (m/z 453) were apparently derived by GSH addition to the 5,6-epoxide metabolite of 3-methyloxindole. These two GSH adducts were tentatively identified as 5-(glutathione-S-yl)-3-methyloxindole and 6-(glutathione-S-yl)-3-methyloxindole. The most abundant conjugate in group I was identified as 3-(glutathione-S-yl)-3-methyloxindole, which substantiated the presence of the putative 2,3-epoxy-3-methylindole intermediate. The remaining four adducts in group I were likely formed by conjugation of GSH at different positions of the phenyl ring, possibly via oxidation of 5-hydroxy-3-methylindole and 6-hydroxy-3-methylindole to two very interesting new electrophilic benzoquinone imine intermediates. For the group II conjugates (m/z 437), two isomers were identified as 2-(glutathione-S-yl)-3-methylindole and 3-(glutathione-S-yl-methyl)-indole. The former adduct was primarily derived from the 2,3-epoxide intermediate by thiol conjugation followed by dehydration. The latter adduct was consistent with our previously published work on the dehydrogenation of 3MI. In those studies, we showed that the reactive intermediate, 3-methylenenindolenine, was formed by hydrogen abstraction at the methyl group and was trapped with GSH. The putative dehydrogenation bioactivation mechanism is also substantiated by the finding that CYP2E1 selectively generated 2-(glutathione-S-yl)-3-methylindole but did not produce 3-(glutathione-S-yl-methyl)-indole. In summary, the results not only confirmed the formation of 2,3-epoxide-3-methylindole in human liver microsomes but also suggested that the phenolic metabolites of 3-methylindole were dehydrogenated to previously uncharacterized reactive intermediates.

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

“…In GSH-fortified metabolism reactions with 3MI or 3-d 2 -MI, we further demonstrated that the formation of GS-A3 and GS-A2 occurred primarily via 2,3-epoxidation. In a previous study, human liver microsomes were shown to be able to oxidize MOI to a number of metabolites, including GSH adducts in GSH-fortified reactions; this finding led the authors to speculate that further metabolism of MOI is also implicated in 3MI toxicity (Yan et al, 2007). The present studies have indicated that CYP2A13 can catalyze the hydroxylation of MOI; however, the main metabolite, Met1*, was not observed when 3MI was used as the substrate.…”

“…In fact, incubations of 3MI with goat lung microsomes and cytosol showed that the carbonyl oxygen of 3-hydroxy-3-methyloxindole was derived from water, not molecular oxygen, confirming catalysis of the HMI imine by aldehyde oxidase. We propose, on the basis of these data, that Met 4 is HMI, a reactive metabolite, which, although it is somewhat unstable (Diaz et al, 1999), has previously been detected in reactions of 3MI with human and pig liver microsomes (Diaz et al, 1999;Yan et al, 2007).…”

“…GS-A3 was not detected by high-resolution mass analysis. In an earlier report, incubations with human liver microsomes produced at least seven distinct GSH adducts detected at m/z 453 (Yan et al, 2007;Zhu et al, 2007); however, cleavage of the thioether bond in the MS 2 product ion spectrum is only consistent with 3-glutathionyl-S-3-methyloxindole. Therefore, we propose GS-A3 to be 3-glutathionyl-S-3-methyloxindole; its formation could be rationalized in terms of a nucleophilic attack of 2,3-epoxy-3MI by GSH at the C-3 position, followed by loss of water to form an imine and subsequent oxidation to a lactone (Scheme 1), as was suggested recently (Yan et al, 2007).…”

“…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.…”

“…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 metabolism of 3MI in human liver microsomes revealed additional metabolic pathways that can lead to formation of potentially toxic intermediates; one such pathway is oxidation of the benzene ring, resulting in benzoquinone intermediates (Yan et al, 2007). It is noteworthy that P450-catalyzed dehydrogenation of 3MI-containing compounds also has the potential to inactivate P450s (Kassahun et al…”