Peroxidase-mediated dealkylation of tamoxifen, detected by electrospray ionization-mass spectrometry, and activation to form DNA adducts

Nilesh W. Gaikwad, William J. Bodell

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Tamoxifen (TAM) is extensively used for the treatment and prevention of breast cancer. Associated with TAM treatment is a two- to eightfold increase in risk of endometrial cancer. To understand the mechanisms associated with this increased risk several pathways for TAM metabolism and DNA adduct formation have been studied. The purpose of this study was to investigate the role of peroxidase enzymes in the metabolism of TAM and its activation to form DNA adducts. Using advanced tandem mass spectrometry we have investigated the peroxidase-mediated metabolism of TAM. Incubation of TAM with horseradish peroxidase (HRP) and H 2O 2 produced multiple metabolites. Electrospray ionization-MS/MS analysis of the metabolites demonstrated a peak at 301.3 m/z with daughter ions at 183.0, 166.9, 128.9, and 120.9 m/z, which identified the metabolite as metabolite E (ME). The levels of ME were significantly inhibited by the addition of ascorbic acid to the incubation mixture. Co-incubation of either TAM or ME and DNA with HRP and H 2O 2 produced three DNA adducts with a RAL of 1.97 ± 0.01 × 10 - 7 and 8.45 ± 2.7 × 10 - 7. Oxidation of ME with MnO 2 produced metabolite E quinone methide (MEQM). Furthermore, incubation of either TAM or ME with HRP and H 2O 2 resulted in formation of MEQM. Reaction of calf thymus DNA with MEQM produced three DNA adducts with a RAL of 9.8 ± 1.0 × 10 - 7. Rechromatography analyses indicated that DNA adducts 1, 2, and 3 formed in the HRP activation of either TAM or ME were the same as those formed by the chemical reaction of DNA with MEQM. The results of these studies demonstrate that peroxidase enzymes can both metabolize TAM to form the primary metabolite ME and activate ME to a quinone methide intermediate, which reacts with DNA to form adducts. It is possible that peroxidase enzymes or peroxidase-like activity in endometrium could contribute to the formation of DNA damage and genotoxic effects in endometrium after TAM administration.

Original languageEnglish (US)
Pages (from-to)340-347
Number of pages8
JournalFree Radical Biology and Medicine
Volume52
Issue number2
DOIs
StatePublished - Jan 15 2012

Fingerprint

Dealkylation
Electrospray ionization
DNA Adducts
Electrospray Ionization Mass Spectrometry
Tamoxifen
Metabolites
Peroxidase
Mass spectrometry
Chemical activation
Horseradish Peroxidase
Endometrium
DNA
Enzymes
Metabolism
Endometrial Neoplasms
Tandem Mass Spectrometry
Ascorbic Acid
DNA Damage
quinone methide
Ions

Keywords

  • DNA adducts
  • ESI-MS/MS
  • Free radicals
  • HRP
  • Metabolite E
  • Peroxidase
  • Quinone methide
  • Tamoxifen
  • UPLC

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

Cite this

Peroxidase-mediated dealkylation of tamoxifen, detected by electrospray ionization-mass spectrometry, and activation to form DNA adducts. / Gaikwad, Nilesh W.; Bodell, William J.

In: Free Radical Biology and Medicine, Vol. 52, No. 2, 15.01.2012, p. 340-347.

Research output: Contribution to journalArticle

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abstract = "Tamoxifen (TAM) is extensively used for the treatment and prevention of breast cancer. Associated with TAM treatment is a two- to eightfold increase in risk of endometrial cancer. To understand the mechanisms associated with this increased risk several pathways for TAM metabolism and DNA adduct formation have been studied. The purpose of this study was to investigate the role of peroxidase enzymes in the metabolism of TAM and its activation to form DNA adducts. Using advanced tandem mass spectrometry we have investigated the peroxidase-mediated metabolism of TAM. Incubation of TAM with horseradish peroxidase (HRP) and H 2O 2 produced multiple metabolites. Electrospray ionization-MS/MS analysis of the metabolites demonstrated a peak at 301.3 m/z with daughter ions at 183.0, 166.9, 128.9, and 120.9 m/z, which identified the metabolite as metabolite E (ME). The levels of ME were significantly inhibited by the addition of ascorbic acid to the incubation mixture. Co-incubation of either TAM or ME and DNA with HRP and H 2O 2 produced three DNA adducts with a RAL of 1.97 ± 0.01 × 10 - 7 and 8.45 ± 2.7 × 10 - 7. Oxidation of ME with MnO 2 produced metabolite E quinone methide (MEQM). Furthermore, incubation of either TAM or ME with HRP and H 2O 2 resulted in formation of MEQM. Reaction of calf thymus DNA with MEQM produced three DNA adducts with a RAL of 9.8 ± 1.0 × 10 - 7. Rechromatography analyses indicated that DNA adducts 1, 2, and 3 formed in the HRP activation of either TAM or ME were the same as those formed by the chemical reaction of DNA with MEQM. The results of these studies demonstrate that peroxidase enzymes can both metabolize TAM to form the primary metabolite ME and activate ME to a quinone methide intermediate, which reacts with DNA to form adducts. It is possible that peroxidase enzymes or peroxidase-like activity in endometrium could contribute to the formation of DNA damage and genotoxic effects in endometrium after TAM administration.",
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N2 - Tamoxifen (TAM) is extensively used for the treatment and prevention of breast cancer. Associated with TAM treatment is a two- to eightfold increase in risk of endometrial cancer. To understand the mechanisms associated with this increased risk several pathways for TAM metabolism and DNA adduct formation have been studied. The purpose of this study was to investigate the role of peroxidase enzymes in the metabolism of TAM and its activation to form DNA adducts. Using advanced tandem mass spectrometry we have investigated the peroxidase-mediated metabolism of TAM. Incubation of TAM with horseradish peroxidase (HRP) and H 2O 2 produced multiple metabolites. Electrospray ionization-MS/MS analysis of the metabolites demonstrated a peak at 301.3 m/z with daughter ions at 183.0, 166.9, 128.9, and 120.9 m/z, which identified the metabolite as metabolite E (ME). The levels of ME were significantly inhibited by the addition of ascorbic acid to the incubation mixture. Co-incubation of either TAM or ME and DNA with HRP and H 2O 2 produced three DNA adducts with a RAL of 1.97 ± 0.01 × 10 - 7 and 8.45 ± 2.7 × 10 - 7. Oxidation of ME with MnO 2 produced metabolite E quinone methide (MEQM). Furthermore, incubation of either TAM or ME with HRP and H 2O 2 resulted in formation of MEQM. Reaction of calf thymus DNA with MEQM produced three DNA adducts with a RAL of 9.8 ± 1.0 × 10 - 7. Rechromatography analyses indicated that DNA adducts 1, 2, and 3 formed in the HRP activation of either TAM or ME were the same as those formed by the chemical reaction of DNA with MEQM. The results of these studies demonstrate that peroxidase enzymes can both metabolize TAM to form the primary metabolite ME and activate ME to a quinone methide intermediate, which reacts with DNA to form adducts. It is possible that peroxidase enzymes or peroxidase-like activity in endometrium could contribute to the formation of DNA damage and genotoxic effects in endometrium after TAM administration.

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