Quantitative Measurement of Radiation-Induced Base Products in DNA Using Gas Chromatography-Mass Spectrometry

Abstract

Gas chromatography-mass spectrometry with selected-ion monitoring was used to study radiation-induced damage to DNA. Quantitative analysis of modified purine and pyrimidine bases resulting from exposure to ionizing radiation using this technique is dependent upon the selection of appropriate internal standards and calibration of the mass spectrometer for its response to known quantities of the internal standards and the products of interest. The compounds 6-azathymine and 8-azaadenine were found to be suitable internal standards for quantitative measurement of base damage in DNA. For the purpose of calibration of the mass spectrometer. relative molar response factors for intense characteristic ions were determined for the trimethylsilyl derivatives of 5-hydroxyuracil, thymine glycol, and 5,6-dihydrothymine using 6-azathymine, and for the trimethylsilyl derivatives of 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine using 8-azaadenine. Accurate measurement of the yield of radiation-induced modifications to the DNA bases is also dependent upon two chemical steps in which the purines and pyrimidines are released from the sugar-phosphate backbone and then derivatized to make them volatile for gas chromatography. The completeness of these reactions, in addition to assessing the stability of the modified DNA bases in acid and their trimethylsilylated derivatives over the time necessary to complete the experimental analysis was also examined. Application of this methodology to the measurement of radiation-induced base modification in heat-denatured, nitrous oxidesaturated aqueous solutions of DNA is presented.

The Chemistry of Free-Radical-Mediated DNA Damage

“…Most recently, hydrolysis of DNA, trimethylsilyl derivatization of the nucleobase products, and subsequent gas chromatography combined with mass spectrometry (GC-MS) using the single-ion monitoring (SIM) technique gave the most detailed analysis so far available. (91,92) They are compiled in Table V It can be seen from this table that a good number of products have indeed been detected and quantitatively determined. However, examination of the total yields show that under N 2 0, G( detected products) is only 0.8 x 10-7 mol J-1, while in the presence of oxygen (N 2 0/0 2 ) this yield is 1.4 x 10-7 mol J-1.…”

Damage to the bases in DNA induced by stimulated human neutrophils.

“…Authentic samples of isobarbituric acid (5-hydroxyuracil, Sigma), 4,6-diamino-5-formamidopyrimidine (Sigma), and 2-amino-6,8-dihydroxy-purine (8-hydroxyguanine; Chemical Dynamics Corp., South Plainfield, NJ), were available commercially. 8-hydroxyadenine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were synthesized as previously described (21). Thymine glycol was kindly provided by Dr. W. F. Blakely of the Armed Forces Radiobiology Research Institute, Bethesda, MD.…”

“…DNA base products were quantitatively measured using 6-azathymine and 8-azaadenine (both from Sigma Chemical Co.) as internal standards, and using the GC-MS/SIM technique as previously described (21). Aliquots of the internal standards were added to aliquots of DNA samples.…”

Ineffectiveness of a Fluorometric Method for Identifying Irradiated Food Based on Thymine Glycol

“…Other techniques that may have potential for detecting DNA base damage products in irradiated foods are presently being developed. These include an enzyme immunoassay technique (Jabir et al, 1989), gas chromatography-mass spectrometry with selected-ion monitoring (Fuciarelli et al, 1989(Fuciarelli et al, , 1990, and HPLC with electrochemical detection (HPLC-EC, Grootveld and Jain, 1989;Grootveld et al, 1990;Park et al, 1989;Berger et al, 1990). Although further development is required, these techniques appear to be capable of providing the sensitivity and specificity necessary for a practical test for identifying irradiated food.…”