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

Jaime D'Agostino, Xiaoliang Zhuo, Mohammad Shadid, Daniel G. Morgan, Xiuling Zhang, W. Griffith Humphreys, Yue Zhong Shu, Garold S. Yost, Xinxin Ding

Research output: Contribution to journalArticle

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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 (Vmax = 1.5 nmol/min/nmol of P450; Km = 14 μM), MOI (Vmax = 1.9 nmol/min/nmol of P450; Km = 15 μM) and 3-glutathionyl-S-methylindole (Vmax = 0.7 nmol/min/nmol of P450; Km = 13 μM). 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 KI value of ∼10 μM and a kinact of 0.046 min-1. Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.

Original languageEnglish (US)
Pages (from-to)2018-2027
Number of pages10
JournalDrug Metabolism and Disposition
Volume37
Issue number10
DOIs
StatePublished - Oct 1 2009
Externally publishedYes

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Skatole
Aryl Hydrocarbon Hydroxylases
Pneumothorax
Substrate Specificity
Respiratory System
Cytochrome P-450 Enzyme System
Gene Expression
varespladib methyl
Glutathione
Ions
NADP
Suicide
Mass Spectrometry
Observation

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

Cite this

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'Agostino, Jaime; Zhuo, Xiaoliang; Shadid, Mohammad; Morgan, Daniel G.; Zhang, Xiuling; Humphreys, W. Griffith; Shu, Yue Zhong; Yost, Garold S.; Ding, Xinxin.

In: Drug Metabolism and Disposition, Vol. 37, No. 10, 01.10.2009, p. 2018-2027.

Research output: Contribution to journalArticle

D'Agostino, Jaime ; Zhuo, Xiaoliang ; Shadid, Mohammad ; Morgan, Daniel G. ; Zhang, Xiuling ; Humphreys, W. Griffith ; Shu, Yue Zhong ; Yost, Garold S. ; Ding, Xinxin. / 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: Drug Metabolism and Disposition. 2009 ; Vol. 37, No. 10. pp. 2018-2027.
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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 (Vmax = 1.5 nmol/min/nmol of P450; Km = 14 μM), MOI (Vmax = 1.9 nmol/min/nmol of P450; Km = 15 μM) and 3-glutathionyl-S-methylindole (Vmax = 0.7 nmol/min/nmol of P450; Km = 13 μM). 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 KI value of ∼10 μM and a kinact of 0.046 min-1. Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.",
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AU - Zhuo, Xiaoliang

AU - Shadid, Mohammad

AU - Morgan, Daniel G.

AU - Zhang, Xiuling

AU - Humphreys, W. Griffith

AU - Shu, Yue Zhong

AU - Yost, Garold S.

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N2 - 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 (Vmax = 1.5 nmol/min/nmol of P450; Km = 14 μM), MOI (Vmax = 1.9 nmol/min/nmol of P450; Km = 15 μM) and 3-glutathionyl-S-methylindole (Vmax = 0.7 nmol/min/nmol of P450; Km = 13 μM). 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 KI value of ∼10 μM and a kinact of 0.046 min-1. Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.

AB - 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 (Vmax = 1.5 nmol/min/nmol of P450; Km = 14 μM), MOI (Vmax = 1.9 nmol/min/nmol of P450; Km = 15 μM) and 3-glutathionyl-S-methylindole (Vmax = 0.7 nmol/min/nmol of P450; Km = 13 μM). 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 KI value of ∼10 μM and a kinact of 0.046 min-1. Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.

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