Measurement of the incorporation and repair of exogenous 5-hydroxymethyl-2′-deoxyuridine in human cells in culture using gas chromatography-negative chemical ionization-mass spectrometry

Daniel K. Rogstad, Agus Darwanto, Jason L. Herring, Katherine Noyes Rogstad, Artur Burdzy, Scott R. Hadley, Jonathan W. Neidigh, Lawrence Sowers

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage. More recently, the corresponding deoxynucleoside, 5-hydroxymethyl-2′-deoxyuridine (HmdU), has proven to be valuable for the introduction of controlled amounts of a single type of damage lesion into the DNA of replicating cells, which is subsequently repaired by the base excision repair pathway. Complicating the study of HmU formation and repair, however, is the known chemical reactivity of the hydroxymethyl group of HmU under conditions used to hydrolyze DNA. In the work reported here, this chemical property has been exploited by creating conditions that convert HmU to the corresponding methoxymethyluracil (MmU) derivative that can be further derivatized to the 3,5-bis-(trifluoromethyl)benzyl analogue. This derivatized compound can be detected by gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) with good sensitivity. Using isotopically enriched exogenous HmdU and human osteosarcoma cells (U2OS) in culture, we demonstrate that this method allows for the measurement of HmU in DNA formed from the incorporation of exogenous HmdU. We further demonstrate that the addition of isotopically enriched uridine to the culture medium allows for the simultaneous measurement of DNA replication and repair kinetics. This sensitive and facile method should prove valuable for studies on DNA oxidation damage and repair in living cells.

Original languageEnglish (US)
Pages (from-to)1787-1796
Number of pages10
JournalChemical Research in Toxicology
Volume20
Issue number12
DOIs
StatePublished - Dec 2007
Externally publishedYes

Fingerprint

Cell culture
Gas chromatography
Gas Chromatography
Ionization
Mass spectrometry
Mass Spectrometry
Repair
Cell Culture Techniques
Cells
DNA Repair
Deoxyuridine
DNA
DNA Damage
Thymine
Uridine
Oxidation
Osteosarcoma
DNA Replication
Culture Media
Chemical reactivity

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

Measurement of the incorporation and repair of exogenous 5-hydroxymethyl-2′-deoxyuridine in human cells in culture using gas chromatography-negative chemical ionization-mass spectrometry. / Rogstad, Daniel K.; Darwanto, Agus; Herring, Jason L.; Rogstad, Katherine Noyes; Burdzy, Artur; Hadley, Scott R.; Neidigh, Jonathan W.; Sowers, Lawrence.

In: Chemical Research in Toxicology, Vol. 20, No. 12, 12.2007, p. 1787-1796.

Research output: Contribution to journalArticle

Rogstad, Daniel K. ; Darwanto, Agus ; Herring, Jason L. ; Rogstad, Katherine Noyes ; Burdzy, Artur ; Hadley, Scott R. ; Neidigh, Jonathan W. ; Sowers, Lawrence. / Measurement of the incorporation and repair of exogenous 5-hydroxymethyl-2′-deoxyuridine in human cells in culture using gas chromatography-negative chemical ionization-mass spectrometry. In: Chemical Research in Toxicology. 2007 ; Vol. 20, No. 12. pp. 1787-1796.
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abstract = "The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage. More recently, the corresponding deoxynucleoside, 5-hydroxymethyl-2′-deoxyuridine (HmdU), has proven to be valuable for the introduction of controlled amounts of a single type of damage lesion into the DNA of replicating cells, which is subsequently repaired by the base excision repair pathway. Complicating the study of HmU formation and repair, however, is the known chemical reactivity of the hydroxymethyl group of HmU under conditions used to hydrolyze DNA. In the work reported here, this chemical property has been exploited by creating conditions that convert HmU to the corresponding methoxymethyluracil (MmU) derivative that can be further derivatized to the 3,5-bis-(trifluoromethyl)benzyl analogue. This derivatized compound can be detected by gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) with good sensitivity. Using isotopically enriched exogenous HmdU and human osteosarcoma cells (U2OS) in culture, we demonstrate that this method allows for the measurement of HmU in DNA formed from the incorporation of exogenous HmdU. We further demonstrate that the addition of isotopically enriched uridine to the culture medium allows for the simultaneous measurement of DNA replication and repair kinetics. This sensitive and facile method should prove valuable for studies on DNA oxidation damage and repair in living cells.",
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AU - Herring, Jason L.

AU - Rogstad, Katherine Noyes

AU - Burdzy, Artur

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