Development of a liquid chromatography electrospray ionization tandem mass spectrometry method for analysis of stable 4-hydroxyequilenin-DNA adducts in human breast cancer cells

Zhican Wang, Praneeth Edirisinghe, Johann Sohn, Zhihui Qin, Nicholas Geacintov, Gregory R J Thatcher, Judy L. Bolton

Research output: Contribution to journalArticle

Abstract

Estrogen - DNA adducts are potential biomarkers for assessing cancer risk and progression in estrogendependent cancer. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in hormone replacement therapy formulations, autoxidizes to a reactive o-quinone that subsequently causes DNA damage. The formation of stable stereoisomeric cyclic 4-OHEN-DNA adducts has been reported in vitro and in vivo, but their removal by DNA repair processes in cells has not been determined. Such studies have been hampered by low yields of cyclic adducts and poor reproducibility when treating cells in culture with 4-OHEN. These problems are attributed in part to the instability of 4-OHEN in aerobic, aqueous media. We show herein that low yields and reproducibility can be overcome by 4-OHEN diacetate as a novel, cell-permeable 4-OHEN precursor, in combination with a sensitive LC-MS/MS method developed for detecting adducts in human breast cancer cells. This method involves isolation of cellular DNA, DNA digestion to deoxynucleosides, followed by the addition of an isotope-labeled internal standard (4-OHEN-15N5-dG adduct) prior to analysis by LC-MS/MS. A concentration-dependent increase in adduct levels was observed in MCF-7 cells after exposure to 4-OHEN diacetate. The chemical stabilities of the adducts were also investigated to confirm that adducts were stable under assay conditions. In conclusion, this newly developed LC-MS/MS method allows detection and relative quantification of 4-OHEN-DNA adducts in human breast cancer cells, which could be adapted for adduct detection in human samples.

Original languageEnglish (US)
Pages (from-to)1129-1136
Number of pages8
JournalChemical Research in Toxicology
Volume22
Issue number6
DOIs
StatePublished - Jun 15 2009

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Electrospray ionization
DNA Adducts
Electrospray Ionization Mass Spectrometry
Liquid chromatography
Tandem Mass Spectrometry
Liquid Chromatography
Mass spectrometry
Cells
Breast Neoplasms
DNA
Estrogens
Hormone Replacement Therapy
Chemical stability
MCF-7 Cells
Tumor Biomarkers
Metabolites
Isotopes
DNA Repair
Horses
DNA Damage

ASJC Scopus subject areas

  • Toxicology

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Development of a liquid chromatography electrospray ionization tandem mass spectrometry method for analysis of stable 4-hydroxyequilenin-DNA adducts in human breast cancer cells. / Wang, Zhican; Edirisinghe, Praneeth; Sohn, Johann; Qin, Zhihui; Geacintov, Nicholas; Thatcher, Gregory R J; Bolton, Judy L.

In: Chemical Research in Toxicology, Vol. 22, No. 6, 15.06.2009, p. 1129-1136.

Research output: Contribution to journalArticle

Wang, Zhican ; Edirisinghe, Praneeth ; Sohn, Johann ; Qin, Zhihui ; Geacintov, Nicholas ; Thatcher, Gregory R J ; Bolton, Judy L. / Development of a liquid chromatography electrospray ionization tandem mass spectrometry method for analysis of stable 4-hydroxyequilenin-DNA adducts in human breast cancer cells. In: Chemical Research in Toxicology. 2009 ; Vol. 22, No. 6. pp. 1129-1136.
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AU - Qin, Zhihui

AU - Geacintov, Nicholas

AU - Thatcher, Gregory R J

AU - Bolton, Judy L.

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