Determination of O6-butylguanine in DNA by immunoaffinity extraction/gas chromatography-mass spectrometry
- Istituto di Ricerche Farmacologiche, Mario Negri, Milan (Italy)
A sensitive, specific, and rapid method for quantitating the minor adduct O6-butylguanine (O6BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against O6BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific O6-alkylguanine. After addition of O6BuG and its deuterium labeled analogue (O6BuG-D7), used as internal standard, hydrolyzed DNA was applied on the immunoaffinity column and washed with water, and the immunoadsorbed butylated guanines were eluted with acetone/water cetome/water (95/5) before gas chromatographic derivatization. O6BuG and O6BuG-D7 were analyzed and quantitated by high resolution gas chromatography-negative ion chemical ionization-mass spectrometry as their pentafluorobenzyl-trimethylsilyl derivatives. Immunoaffinity column capacity and O6BuG recovery from this column were 1.53 nmol O6BuG/column and 62 +/- 5%, respectively. The method was applied to evaluate O6BuG levels in DNA butylated in vitro with 10 mM N-nitroso-Nr-butylurea or isolated from rats given an i.p. dose of 185 mg/kg N-nitroso-N-butylurea or N-nitrosodibutylamine. In the first case the level of modifications present in calf thymus DNA was 104 mumol O6BuG/mol guanine, and in the second case O6BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 mumol O6BuG/mol guanine) than after N-nitrosodibutylamine (0.34 mumol O6BuG/mol guanine) treatment. These results indicate that O6BuG formed in vivo can be isolated and quantitated by this method, which may also be useful for studying DNA damage and repair mechanisms.
- OSTI ID:
- 6140955
- Journal Information:
- Cancer Research; (USA), Vol. 50:21; ISSN 0008-5472
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
DNA ADDUCTS
BIOSYNTHESIS
GUANINE
ENZYME IMMUNOASSAY
NITROSO COMPOUNDS
METABOLISM
ANTIBODIES
DEUTERIUM
DNA
GAS CHROMATOGRAPHY
LIVER
MASS SPECTROSCOPY
TRACER TECHNIQUES
ADDUCTS
AMINES
AROMATICS
AZAARENES
BIOASSAY
BODY
CHROMATOGRAPHY
DIGESTIVE SYSTEM
GLANDS
HETEROCYCLIC COMPOUNDS
HYDROGEN ISOTOPES
HYDROXY COMPOUNDS
IMMUNOASSAY
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PURINES
SEPARATION PROCESSES
SPECTROSCOPY
STABLE ISOTOPES
SYNTHESIS
560300* - Chemicals Metabolism & Toxicology
550201 - Biochemistry- Tracer Techniques