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K. Madhava Madyastha - One of the best experts on this subject based on the ideXlab platform.
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metabolic fate of s pulegone in rat
Xenobiotica, 1998Co-Authors: K. Madhava Madyastha, Nilesh W. GaikwadAbstract:1. S-(-)-pulegone was administered orally to rat (250 mg/kg) and the nature of the urinary metabolites was investigated. Eleven metabolites, namely S-(-)-Menthofuran, piperitone, piperitenone, p-cresol, 5-hydroxypulegone, 4-methylcyclohexenone, 3- methylcyclohexanone, isopulegone, pulegol, 7-hydroxypiperitone and benzoic acid, have been isolated from rat urine. It is assumed that Menthofuran, isopulegone and 4- methylcyclohexenone retain the stereochemistry of the parent compound, whereas in other metabolites the stereochemistry at the asymmetric centres is not known. 2. The relative amounts of various major metabolites present in the total urine extracts from the R-(+) and S-(-)-pulegone-treated rat were established by glc analyses. Urine samples of rats treated with R-(+)-pulegone contained higher levels of p-cresol and piperitenone than in similar experiment carried out with S-(-)-pulegone, whereas the levels of unmetabolized pulegone, piperitone and benzoic acid were considerably higher in the urine o...
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Effects of Menthofuran, a monoterpene furan on rat liver microsomal enzymes, in vivo.
Toxicology, 1994Co-Authors: K. Madhava Madyastha, C. Paul RajAbstract:Abstract Oral administration (250 mg/kg) of Menthofuran, a monoterpene furan, to rats once daily for 3 days caused hepatotoxicity as judged by a significant increase in serum glutamate pyruvate transaminase (SGPT) and decreases in glucose-6-phosphate and aminopyrine N -demethylase activities. Administration of Menthofuran also resulted in a decrease in the levels of liver microsomal cytochrome P-450, whereas cytochrome b 5 and NAD(P)H-cytochrome c reductase activities were not affected. These effects of methofuran were both dose- and time-dependent. retreatment of rats with phenobarbital (PB) prior to Menthofuran treatment potentiated hepatotoxicity suggesting that a PB-induced cytochrome P-450 catalyzed the formation of reactive metabolite(s) responsible for the hepatotoxicity.
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biotransformations of r pulegone and Menthofuran in vitro chemical basis for toxicity
Biochemical and Biophysical Research Communications, 1990Co-Authors: K. Madhava MadyasthaAbstract:Incubation of R-(+)-pulegone(I) with PB-induced rat liver microsomes in the presence of NADPH resulted in the formation of Menthofuran(II) and 2′-Z-[2′-keto-4′-methylcyclohexylidene] propanol (III, 9-hydroxy pulegone) as the major and minor metabolites, respectively. When isopulegone(IV) was used as the substrate, the major metabolite formed was shown to have identical GC-MS fragmentation pattern to that of synthetic 2-[2′-keto-4′-methylcyclohexyl]prop-2-en-1-ol (V) and the minor metabolite was shown to be Menthofuran (II). Transformation of Menthofuran (II) by microsomes in the presence of NADPH yielded a metabolite identified as 2-Z-(2′-keto-4′-methyl cyclohexylidene) propanal (VI, pulegone-8-aldehyde). Formation of this $\alpha$ , $\beta$ - unsaturated aldehyde was further confirmed by trapping it as cinnoline derivative by adding semicarbazide to the assay medium. The toxicity mediated by pulegone is discussed in the light of these observations.
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BIOTRANSFORMATIONS OF R-(+)-PULEGONE AND Menthofuran IN VITRO : CHEMICAL BASIS FOR TOXICITY
Biochemical and Biophysical Research Communications, 1990Co-Authors: K. Madhava MadyasthaAbstract:Incubation of R-(+)-pulegone(I) with PB-induced rat liver microsomes in the presence of NADPH resulted in the formation of Menthofuran(II) and 2′-Z-[2′-keto-4′-methylcyclohexylidene] propanol (III, 9-hydroxy pulegone) as the major and minor metabolites, respectively. When isopulegone(IV) was used as the substrate, the major metabolite formed was shown to have identical GC-MS fragmentation pattern to that of synthetic 2-[2′-keto-4′-methylcyclohexyl]prop-2-en-1-ol (V) and the minor metabolite was shown to be Menthofuran (II). Transformation of Menthofuran (II) by microsomes in the presence of NADPH yielded a metabolite identified as 2-Z-(2′-keto-4′-methyl cyclohexylidene) propanal (VI, pulegone-8-aldehyde). Formation of this $\alpha$ , $\beta$ - unsaturated aldehyde was further confirmed by trapping it as cinnoline derivative by adding semicarbazide to the assay medium. The toxicity mediated by pulegone is discussed in the light of these observations.
Sidney D Nelson - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Rat Liver Proteins Adducted by Reactive Metabolites of Menthofuran
2016Co-Authors: Cyrus S. Khojasteh, Shimako Oishi, Dylan P. Hartley, Hirdesh Uppal, Kevin A. Ford, Sidney D NelsonAbstract:Pulegone is the major constituent of pennyroyal oil, a folkloric abortifacient that is associated with hepatotoxicity and, in severe cases, death. Cytochrome P450-mediated oxidation of pulegone generates Menthofuran, which is further oxidized to form electrophilic reactive intermediates, Menthofuran epoxide and the ring-opened γ-ketoenal, both of which can form adducts to hepatocellular proteins. Modification of hepatocellular proteins by the electrophilic reactive intermediates of Menthofuran has been implicated in hepatotoxicity caused by pennyroyal oil. Herein, we describe the identification of several proteins that are the likely targets of Menthofuran-derived reactive metabolites. These proteins were isolated from the livers of rats treated with a hepatotoxic dose of Menthofuran by two-dimensional gel electrophoresis (2D-gel) separation and detected by Western blot analysis using an antiserum developed to detect protein adducts resulting from Menthofuran bioactivation. The antibody-reacting proteins were excised from the 2D-gel and subjected to tryptic digestion for analysis of peptide fragments by LC-MS/MS. Although 10 spots were detected by Western blot analysis, only 4 were amenable to characterization by LC-MS/MS: serum albumin, mitochondrial aldehyde dehydrogenase (ALDH2), cytoplasmic malate dehydrogenase (MDH1), and mitochondrial ATP synthase subunit d. No direct adduct was detected, and, therefore, we complemented our analysis with enzyme activity determination. ALDH2 activity decreased by 88%, and ATP synthase complex V activity decreased by 34%, with no activity changes to MDH1. Although the relationship between these reactive metabolite adducted proteins and hepatotoxicity is not clear, these targeted enzymes are known to play critical roles in maintaining cellular homeostasis
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Characterization of rat liver proteins adducted by reactive metabolites of Menthofuran.
Chemical Research in Toxicology, 2012Co-Authors: S. Cyrus Khojasteh, Shimako Oishi, Dylan P. Hartley, Kevin A. Ford, Hirdesh Uppal, Sidney D NelsonAbstract:Pulegone is the major constituent of pennyroyal oil, a folkloric abortifacient that is associated with hepatotoxicity and, in severe cases, death. Cytochrome P450-mediated oxidation of pulegone generates Menthofuran, which is further oxidized to form electrophilic reactive intermediates, Menthofuran epoxide and the ring-opened γ-ketoenal, both of which can form adducts to hepatocellular proteins. Modification of hepatocellular proteins by the electrophilic reactive intermediates of Menthofuran has been implicated in hepatotoxicity caused by pennyroyal oil. Herein, we describe the identification of several proteins that are the likely targets of Menthofuran-derived reactive metabolites. These proteins were isolated from the livers of rats treated with a hepatotoxic dose of Menthofuran by two-dimensional gel electrophoresis (2D-gel) separation and detected by Western blot analysis using an antiserum developed to detect protein adducts resulting from Menthofuran bioactivation. The antibody-reacting proteins ...
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Metabolism and toxicity of Menthofuran in rat liver slices and in rats
Chemical Research in Toxicology, 2010Co-Authors: S. Cyrus Khojasteh, Shimako Oishi, Sidney D NelsonAbstract:Menthofuran is a monoterpene present in mint plants that is oxidized by mammalian cytochrome P450 (CYP) to hepatotoxic metabolites. Evidence has been presented that p-cresol and other unusual oxidative products are metabolites of Menthofuran in rats and that p-cresol may be responsible in part for the hepatotoxicity caused by Menthofuran [ Madyastha, K. M. and Raj, C. P. (1992) Drug Metab. Dispos. 20, 295 - 301]. In the present study, several oxidative metabolites of Menthofuran were characterized in rat and human liver microsomes and in rat liver slices exposed to cytotoxic concentrations of Menthofuran. Metabolites that were identified were monohydroxylation products of the furanyl and cyclohexyl groups, mintlactones and hydroxymintlactones, a reactive γ-ketoenal, and a glutathione conjugate. A similar spectrum of metabolites was found in urine 24 h after the administration of hepatotoxic doses of Menthofuran to rats. In no case was p-cresol (or any of the other reported unusual oxidative metabolites of Menthofuran) detected above background concentrations that were well below concentrations of p-cresol that cause cytotoxicity in rat liver slices. Thus, the major metabolites responsible for the hepatotoxic effects of Menthofuran appear to be a γ-ketoenal and/or epoxides formed by oxidation of the furan ring.
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metabolism of r pulegone and r Menthofuran by human liver cytochrome p 450s evidence for formation of a furan epoxide
Drug Metabolism and Disposition, 1999Co-Authors: Siamak C Khojastehbakht, Weiqiao Chen, Luke L Koenigs, Raimund M Peter, Sidney D NelsonAbstract:( R )-(+)-Pulegone, a monoterpene constituent of pennyroyal oil, is a hepatotoxin that has been used in folklore medicine as an abortifacient despite its potential lethal effects. Pulegone is metabolized by human liver cytochrome P-450s to Menthofuran, a proximate hepatotoxic metabolite of pulegone. Expressed human liver cytochrome (CYP) P-450s (1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4) were tested for their ability to catalyze the oxidations of pulegone and Menthofuran. Expressed CYP2E1, CYP1A2, and CYP2C19 oxidized pulegone to Menthofuran, with respective K m and V max values of 29 μM and 8.4 nmol/min/nmol P-450 for CYP2E1, 94 μM and 2.4 nmol/min/nmol P-450 for CYP1A2, and 31 μM and 1.5 nmol/min/nmol P-450 for CYP2C19. The human liver P-450s involved in the metabolism of Menthofuran are the same as pulegone except for the addition of CYP2A6. These P-450s were found to oxidize Menthofuran to a newly identified metabolite, 2-hydroxyMenthofuran, which is an intermediate in the formation of the known metabolites mintlactone and isomintlactone. Based on studies with 18 O 2 and H 2 18 O, 2-hydroxyMenthofuran arises predominantly from a dihydrodiol formed from a furan epoxide. CYP2E1, CYP1A2, and CYP2C19 oxidized Menthofuran with respective K m and V max values of 33 μM and 0.43 nmol/min/nmol P-450 for CYP2E1, 57 μM and 0.29 nmol/min/nmol P-450 for CYP1A2, and 62 μM and 0.26 nmol/min/nmol P-450 for CYP2C19.
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Metabolism of (R)-(+)-Pulegone and (R)-(+)-Menthofuran by Human Liver Cytochrome P-450s: Evidence for Formation of a Furan Epoxide
Drug Metabolism and Disposition, 1999Co-Authors: Siamak C. Khojasteh-bakht, Weiqiao Chen, Luke L Koenigs, Raimund M Peter, Sidney D NelsonAbstract:( R )-(+)-Pulegone, a monoterpene constituent of pennyroyal oil, is a hepatotoxin that has been used in folklore medicine as an abortifacient despite its potential lethal effects. Pulegone is metabolized by human liver cytochrome P-450s to Menthofuran, a proximate hepatotoxic metabolite of pulegone. Expressed human liver cytochrome (CYP) P-450s (1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4) were tested for their ability to catalyze the oxidations of pulegone and Menthofuran. Expressed CYP2E1, CYP1A2, and CYP2C19 oxidized pulegone to Menthofuran, with respective K m and V max values of 29 μM and 8.4 nmol/min/nmol P-450 for CYP2E1, 94 μM and 2.4 nmol/min/nmol P-450 for CYP1A2, and 31 μM and 1.5 nmol/min/nmol P-450 for CYP2C19. The human liver P-450s involved in the metabolism of Menthofuran are the same as pulegone except for the addition of CYP2A6. These P-450s were found to oxidize Menthofuran to a newly identified metabolite, 2-hydroxyMenthofuran, which is an intermediate in the formation of the known metabolites mintlactone and isomintlactone. Based on studies with 18 O 2 and H 2 18 O, 2-hydroxyMenthofuran arises predominantly from a dihydrodiol formed from a furan epoxide. CYP2E1, CYP1A2, and CYP2C19 oxidized Menthofuran with respective K m and V max values of 33 μM and 0.43 nmol/min/nmol P-450 for CYP2E1, 57 μM and 0.29 nmol/min/nmol P-450 for CYP1A2, and 62 μM and 0.26 nmol/min/nmol P-450 for CYP2C19.
Leo T. Burka - One of the best experts on this subject based on the ideXlab platform.
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14c labeled pulegone and metabolites binding to α2u globulin in kidneys of male f 344 rats
Journal of Toxicology and Environmental Health, 2007Co-Authors: Lingjen Chen Ferguson, Edward H Lebetkin, Horace D Parkinson, Susan J. Borghoff, Kenneth B Tomer, Leo T. BurkaAbstract:Pulegone is a major constituent of pennyroyal oil and a minor component of peppermint oil. Pulegone is biotransformed to Menthofuran and menthones (diastereomeric menthone and isomenthone) in pennyroyal and peppermint as well as in rodents. Pulegone and Menthofuran are hepatotoxic to rodents, and menthones are less toxic. The metabolism and disposition of pulegone and Menthofuran were previously studied in rodents, and higher concentrations of pulegone- and Menthofuran-derived radioactivity were observed in male than female rat kidney. One explanation is the association of pulegone and metabolites with a male rat-specific protein, α2u-globulin. To test this hypothesis, male and female rats were dosed orally with 14C-labeled pulegone (80 mg/kg, 120 μCi/kg) or Menthofuran (60 mg/kg, 120 μCi/kg) or menthones (80 mg/kg, 120 μCi/kg) in corn oil, and the kidney cytosol was prepared 24 h after dosing. An equilibrium dialysis experiment showed that in all three studies the radioactivity was associated with kidney...
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metabolism of r Menthofuran in fischer 344 rats identification of sulfonic acid metabolites
Drug Metabolism and Disposition, 2003Co-Authors: L.-j. Chen, Edward H Lebetkin, Leo T. BurkaAbstract:(R)-(+)-Menthofuran is a metabolite of (R)-(+)-pulegone, the chief constituent of pennyroyal oil. Menthofuran has been shown to account for a significant percentage of pulegone toxicity through further metabolism to a reactive intermediate, an enonal (2-Z-(2'-keto-4'-methylcyclohexylidene)propanal). Hydration of the enonal followed by a 1,4-dehydration and rearrangement gives rise to diastereomeric (-)-mintlactone and (+)-isomintlactone (mintlactones). We have conducted disposition studies on pulegone as part of the National Toxicology Program initiative in herbal medicines and dietary supplements, and have reported previously unknown urinary metabolites of pulegone. Comparative metabolism studies of 14C-labeled Menthofuran in Fischer-344 (F344) rats were carried out to determine urinary metabolites of pulegone that are derived from the Menthofuran pathway. Three sulfonic acid metabolites, namely, hexahydro-3,6-dimethyl-1-(2-sulfoethyl)-2H-indol-2-one, hexahydro-3,6-dimethyl-7a-sulfo-2(3H)-benzofuranone, and 2-sulfoMenthofuran, were identified in urine of treated rats. Formation of these metabolites may be derived from reactions of the enonal with taurine or glutathione (GSH) (or sulfite ion). Other identified urinary metabolites of Menthofuran could be attributed to further metabolism of mintlactones. Further hydroxylation of mintlactones could give 7a-hydroxymintlactone and 6,7a-dihydroxymintlactone. Glucuronidation or reduction of 7a-hydroxymintlactone could give rise to the major metabolites 7a-hydroxymintlactone glucuronide and 2-[2'-keto-4'-methylcyclohexyl]propionic acids. Glucuronidation or repeated hydroxylation/dehydration of 2-[2'-keto-4'-methylcyclohexyl]propionic acids could result in formation of hexahydro-3,6-dimethyl-7a-hydroxy-2(3H)-benzofuranone glucuronide and 2-(2'-hydroxy-4'-methylphenyl)propionic acid. 2-(Glutathion-S-yl)Menthofuran, a GSH conjugate of the enonal that has been partially characterized in bile of rats dosed with pulegone, is at most a minor biliary metabolite of Menthofuran in rats.
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Metabolism of (R)-(+)-Menthofuran in Fischer-344 rats: identification of sulfonic acid metabolites.
Drug Metabolism and Disposition, 2003Co-Authors: L.-j. Chen, Edward H Lebetkin, Leo T. BurkaAbstract:(R)-(+)-Menthofuran is a metabolite of (R)-(+)-pulegone, the chief constituent of pennyroyal oil. Menthofuran has been shown to account for a significant percentage of pulegone toxicity through further metabolism to a reactive intermediate, an enonal (2-Z-(2'-keto-4'-methylcyclohexylidene)propanal). Hydration of the enonal followed by a 1,4-dehydration and rearrangement gives rise to diastereomeric (-)-mintlactone and (+)-isomintlactone (mintlactones). We have conducted disposition studies on pulegone as part of the National Toxicology Program initiative in herbal medicines and dietary supplements, and have reported previously unknown urinary metabolites of pulegone. Comparative metabolism studies of 14C-labeled Menthofuran in Fischer-344 (F344) rats were carried out to determine urinary metabolites of pulegone that are derived from the Menthofuran pathway. Three sulfonic acid metabolites, namely, hexahydro-3,6-dimethyl-1-(2-sulfoethyl)-2H-indol-2-one, hexahydro-3,6-dimethyl-7a-sulfo-2(3H)-benzofuranone, and 2-sulfoMenthofuran, were identified in urine of treated rats. Formation of these metabolites may be derived from reactions of the enonal with taurine or glutathione (GSH) (or sulfite ion). Other identified urinary metabolites of Menthofuran could be attributed to further metabolism of mintlactones. Further hydroxylation of mintlactones could give 7a-hydroxymintlactone and 6,7a-dihydroxymintlactone. Glucuronidation or reduction of 7a-hydroxymintlactone could give rise to the major metabolites 7a-hydroxymintlactone glucuronide and 2-[2'-keto-4'-methylcyclohexyl]propionic acids. Glucuronidation or repeated hydroxylation/dehydration of 2-[2'-keto-4'-methylcyclohexyl]propionic acids could result in formation of hexahydro-3,6-dimethyl-7a-hydroxy-2(3H)-benzofuranone glucuronide and 2-(2'-hydroxy-4'-methylphenyl)propionic acid. 2-(Glutathion-S-yl)Menthofuran, a GSH conjugate of the enonal that has been partially characterized in bile of rats dosed with pulegone, is at most a minor biliary metabolite of Menthofuran in rats.
Ari Tolonen - One of the best experts on this subject based on the ideXlab platform.
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tandem mass spectrometric analysis of s and n linked glutathione conjugates of pulegone and Menthofuran and identification of p450 enzymes mediating their formation
Rapid Communications in Mass Spectrometry, 2016Co-Authors: Toni Lassila, Sampo Mattila, Miia Turpeinen, Olavi Pelkonen, Ari TolonenAbstract:Menthofuran is a hepatotoxin and a major metabolite of pulegone, a monoterpene found in the essential oils of many mint species. It is bioactivated by cytochrome P450 (CYP) enzymes to reactive metabolites, which may further react with glutathione to form S-linked and N-linked conjugates. The tandem mass spectrometric (MS/MS) fragmentation pathways of rarely observed N-linked conjugates, and the differences to fragmentation of S-linked conjugates, have not been reported in the literature previously, although this information is essential to enable comprehensive MS/MS-based screening methods covering the both types of conjugates.(R)-(+)-Pulegone, (S)-(-)-pulegone, and Menthofuran were incubated with a human liver S9 fraction with glutathione (GSH) as the trapping agent. Conjugates were searched with ultra-performance liquid chromatography (UPLC)/orbitrap MS and their MS/MS spectra were measured both in the negative and positive ionization polarities. Menthofuran was also incubated with recombinant human CYP enzymes and GSH to elucidate the CYPs responsible for the formation of the reactive metabolites.Four GSH conjugates of Menthofuran were detected and identified as S- and N-linked conjugates based on MS/MS spectra. N-linked conjugates lacked the characteristic fragments of S-linked conjugates and commonly produced fragments that retained parts of glutamic acid. CYP1A2, 2B6 and 3A4 were observed to produce more GSH conjugates than other CYP isoforms.Furans can form reactive aldehydes that react in Schiff-base fashion with the free glutamyl-amine of GSH to form N-linked conjugates that have distinct MS/MS spectra from S-linked adducts. This should be taken into account when setting up LC/MS/MS-based detection of glutathione conjugates to screen for reactive metabolites, at least for compounds with a furan moiety. Neutral loss scanning of 178.0412 Da and 290.0573 Da in the positive ionization mode, or neutral loss scanning of 256.0695 Da and 290.0573 Da and precursor ion scanning of m/z 143.0462 in the negative ionization mode, is recommended. Copyright © 2016 John Wiley & Sons, Ltd.
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Tandem mass spectrometric analysis of S- and N-linked glutathione conjugates of pulegone and Menthofuran and identification of P450 enzymes mediating their formation.
Rapid Communications in Mass Spectrometry, 2016Co-Authors: Toni Lassila, Sampo Mattila, Miia Turpeinen, Olavi Pelkonen, Ari TolonenAbstract:RATIONALE Menthofuran is a hepatotoxin and a major metabolite of pulegone, a monoterpene found in the essential oils of many mint species. It is bioactivated by cytochrome P450 (CYP) enzymes to reactive metabolites, which may further react with glutathione to form S-linked and N-linked conjugates. The tandem mass spectrometric (MS/MS) fragmentation pathways of rarely observed N-linked conjugates, and the differences to fragmentation of S-linked conjugates, have not been reported in the literature previously, although this information is essential to enable comprehensive MS/MS-based screening methods covering the both types of conjugates. METHODS (R)-(+)-Pulegone, (S)-(-)-pulegone, and Menthofuran were incubated with a human liver S9 fraction with glutathione (GSH) as the trapping agent. Conjugates were searched with ultra-performance liquid chromatography (UPLC)/orbitrap MS and their MS/MS spectra were measured both in the negative and positive ionization polarities. Menthofuran was also incubated with recombinant human CYP enzymes and GSH to elucidate the CYPs responsible for the formation of the reactive metabolites. RESULTS Four GSH conjugates of Menthofuran were detected and identified as S- and N-linked conjugates based on MS/MS spectra. N-linked conjugates lacked the characteristic fragments of S-linked conjugates and commonly produced fragments that retained parts of glutamic acid. CYP1A2, 2B6 and 3A4 were observed to produce more GSH conjugates than other CYP isoforms. CONLUSIONS Furans can form reactive aldehydes that react in Schiff-base fashion with the free glutamyl-amine of GSH to form N-linked conjugates that have distinct MS/MS spectra from S-linked adducts. This should be taken into account when setting up LC/MS/MS-based detection of glutathione conjugates to screen for reactive metabolites, at least for compounds with a furan moiety. Neutral loss scanning of 178.0412 Da and 290.0573 Da in the positive ionization mode, or neutral loss scanning of 256.0695 Da and 290.0573 Da and precursor ion scanning of m/z 143.0462 in the negative ionization mode, is recommended. Copyright © 2016 John Wiley & Sons, Ltd.
Miia Turpeinen - One of the best experts on this subject based on the ideXlab platform.
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tandem mass spectrometric analysis of s and n linked glutathione conjugates of pulegone and Menthofuran and identification of p450 enzymes mediating their formation
Rapid Communications in Mass Spectrometry, 2016Co-Authors: Toni Lassila, Sampo Mattila, Miia Turpeinen, Olavi Pelkonen, Ari TolonenAbstract:Menthofuran is a hepatotoxin and a major metabolite of pulegone, a monoterpene found in the essential oils of many mint species. It is bioactivated by cytochrome P450 (CYP) enzymes to reactive metabolites, which may further react with glutathione to form S-linked and N-linked conjugates. The tandem mass spectrometric (MS/MS) fragmentation pathways of rarely observed N-linked conjugates, and the differences to fragmentation of S-linked conjugates, have not been reported in the literature previously, although this information is essential to enable comprehensive MS/MS-based screening methods covering the both types of conjugates.(R)-(+)-Pulegone, (S)-(-)-pulegone, and Menthofuran were incubated with a human liver S9 fraction with glutathione (GSH) as the trapping agent. Conjugates were searched with ultra-performance liquid chromatography (UPLC)/orbitrap MS and their MS/MS spectra were measured both in the negative and positive ionization polarities. Menthofuran was also incubated with recombinant human CYP enzymes and GSH to elucidate the CYPs responsible for the formation of the reactive metabolites.Four GSH conjugates of Menthofuran were detected and identified as S- and N-linked conjugates based on MS/MS spectra. N-linked conjugates lacked the characteristic fragments of S-linked conjugates and commonly produced fragments that retained parts of glutamic acid. CYP1A2, 2B6 and 3A4 were observed to produce more GSH conjugates than other CYP isoforms.Furans can form reactive aldehydes that react in Schiff-base fashion with the free glutamyl-amine of GSH to form N-linked conjugates that have distinct MS/MS spectra from S-linked adducts. This should be taken into account when setting up LC/MS/MS-based detection of glutathione conjugates to screen for reactive metabolites, at least for compounds with a furan moiety. Neutral loss scanning of 178.0412 Da and 290.0573 Da in the positive ionization mode, or neutral loss scanning of 256.0695 Da and 290.0573 Da and precursor ion scanning of m/z 143.0462 in the negative ionization mode, is recommended. Copyright © 2016 John Wiley & Sons, Ltd.
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Tandem mass spectrometric analysis of S- and N-linked glutathione conjugates of pulegone and Menthofuran and identification of P450 enzymes mediating their formation.
Rapid Communications in Mass Spectrometry, 2016Co-Authors: Toni Lassila, Sampo Mattila, Miia Turpeinen, Olavi Pelkonen, Ari TolonenAbstract:RATIONALE Menthofuran is a hepatotoxin and a major metabolite of pulegone, a monoterpene found in the essential oils of many mint species. It is bioactivated by cytochrome P450 (CYP) enzymes to reactive metabolites, which may further react with glutathione to form S-linked and N-linked conjugates. The tandem mass spectrometric (MS/MS) fragmentation pathways of rarely observed N-linked conjugates, and the differences to fragmentation of S-linked conjugates, have not been reported in the literature previously, although this information is essential to enable comprehensive MS/MS-based screening methods covering the both types of conjugates. METHODS (R)-(+)-Pulegone, (S)-(-)-pulegone, and Menthofuran were incubated with a human liver S9 fraction with glutathione (GSH) as the trapping agent. Conjugates were searched with ultra-performance liquid chromatography (UPLC)/orbitrap MS and their MS/MS spectra were measured both in the negative and positive ionization polarities. Menthofuran was also incubated with recombinant human CYP enzymes and GSH to elucidate the CYPs responsible for the formation of the reactive metabolites. RESULTS Four GSH conjugates of Menthofuran were detected and identified as S- and N-linked conjugates based on MS/MS spectra. N-linked conjugates lacked the characteristic fragments of S-linked conjugates and commonly produced fragments that retained parts of glutamic acid. CYP1A2, 2B6 and 3A4 were observed to produce more GSH conjugates than other CYP isoforms. CONLUSIONS Furans can form reactive aldehydes that react in Schiff-base fashion with the free glutamyl-amine of GSH to form N-linked conjugates that have distinct MS/MS spectra from S-linked adducts. This should be taken into account when setting up LC/MS/MS-based detection of glutathione conjugates to screen for reactive metabolites, at least for compounds with a furan moiety. Neutral loss scanning of 178.0412 Da and 290.0573 Da in the positive ionization mode, or neutral loss scanning of 256.0695 Da and 290.0573 Da and precursor ion scanning of m/z 143.0462 in the negative ionization mode, is recommended. Copyright © 2016 John Wiley & Sons, Ltd.