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Determination of O⁶-Butylguanine in DNA by Immunoaffinity Extraction/Gas Chromatography-Mass Spectrometry¹

Laboratorio di Farmacologia e Tossicologia Ambientali, Istituto di Ricerche Farmacologiche "Mario Negri," 20157 Milan, Italy [M. B., C. M., A. G., R. B., M. M., R. F., L. A.], and Dipartamente di Scienze Chimiche, Università di Camerino, 62032 Camerino (MC), Italy [P. G.]

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 µmol O⁶BuG/mol guanine, and in the second case O⁶BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 µmol O⁶BuG/mol guanine) than after N-nitrosodibutylamine (0.34 µmol 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.

¹ This work was supported by the Italian National Research Council, Special Project "Oncology," Contract 88.01115.44, and by NATO Grant 0675/88.

² To whom requests for reprints should be addressed, at Istituto di Richerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milan, Italy.

Received 3/12/90. Accepted 7/30/90.

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