KO2 chemical transformation/mass spectrometry detection of covalent damage to the DNA of cultured human lymphocytes exposed to benzo[a]pyrene

Cultured human lymphocytes were exposed to benzo[a]pyrene (B[a]P), and diol epoxide-type DNA adducts arising from this chemical were detected by a method consisting of the following sequence of steps: (1) isolate the DNA; (2) subject the DNA to mild acid hydrolysis to release the polyaromatic moiety...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1993-07, Vol.65 (13), p.1723-1727
Main Authors: KARIMAN ALLAM, SAMY ABDEL-BAKY, GIESE, R. W
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Animals
Benzo(a)pyrene - analysis
Benzo(a)pyrene - toxicity
Biological and medical sciences
Cells, Cultured
DNA - analysis
DNA Adducts
DNA Damage
Dna, deoxyribonucleoproteins
Fundamental and applied biological sciences. Psychology
Humans
Hydrolysis
Lymphocytes - drug effects
Mass Spectrometry
Nucleic acids
Superoxides
Thymus Gland
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Summary:Cultured human lymphocytes were exposed to benzo[a]pyrene (B[a]P), and diol epoxide-type DNA adducts arising from this chemical were detected by a method consisting of the following sequence of steps: (1) isolate the DNA; (2) subject the DNA to mild acid hydrolysis to release the polyaromatic moiety as a tetrahydrotetrol; (3) add an internal standard; (4) oxidize the tetrahydrotetrol with potassium superoxide to pyrene-2,3-dicarboxylic acid; (5) derivatize the latter with pentafluorobenzyl bromide; (6) purify the diester product on a silica cartridge; and (7) detect this product by gas chromatography electron capture negative ion mass spectrometry. From the dose (1 microgram/mL) of B[a]P applied, five adducts in 10(7) normal nucleotides were found. Largely because steps 2-5 of the method take place sequentially in a single vial, the procedure is convenient and affords precise results. To demonstrate the potential of the method to detect KO2-susceptible polyaromatic hydrocarbon DNA adducts in general, including unknowns, it was also applied to picomole and femtomole amounts of a standard of chrysene-1,4-quinone using scanning and selected ion monitoring conditions, respectively, in the MS. Since standard products can be detected with selected ion monitoring at levels 10(4) below those encountered here (prior work), it should be possible in the future to extend the method to samples containing smaller amounts of such adducts.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac00061a015