Yields of radiation-induced base products in DNA: effects of DNA conformation and gassing conditions.

Gas chromatography-mass spectrometry with selected-ion monitoring was used to measure the yields of radiation-induced base products in aqueous solutions of native or heat-denatured DNA irradiated in the dose range 20-100 Gy. These DNA solutions were saturated with nitrous oxide, nitrogen, air or 20% oxygen in nitrous oxide during irradiation. The products measured were as follows: 5,6-dihydrothymine; 5-hydroxy-5,6-dihydrothymine; 5,6-dihydrothymine (thymine glycol); 5-hydroxy-5,6-dihydrocytosine; 5,6-dihydroxy-5,6- dihydrocytosine (cytosine glycol); 4,6-diamino-5-formamidopyrimidine; 7,8-dihydro-8-oxoadenine (8-hydroxyadenine); 2,6-diamino-4-hydroxy-5- formamidopyrimidine; and 7,8-dihydro-8-oxoguanine (8-hydroxyguanine). In oxygenated solutions, 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine and 5-hydroxy-5,6-dihydrocytosine were not formed. The yields of all products, other than 5,6-dihydrothymine, were greater in irradiated DNA samples from N2O-saturated solutions than from N2-saturated solutions. In N2-saturated solutions the yield of 8-hydroxyadenine was low and 8-hydroxyguanine was undetectable. Yields of pyrimidine products in heat-denatured DNA were greater than those in native DNA using all types of gases. However, the effects of DNA conformation on the yields of purine products were dependent on the type of gas used to saturate the irradiated DNA solutions. Yields of formamidopyrimidines were generally lower in solutions of DNA irradiated in the native than in the heat-denatured conformation. In air-saturated solutions of DNA, yields of 8-hydroxypurines were not influenced greatly by DNA conformation. In DNA solutions saturated with N2O/O2, 8-hydroxypurine formation was more favourable in the heat-denatured conformation than in the native conformation. On the other hand, in deoxygenated solutions, formation of 8-hydroxypurines was favoured in the native conformation. Data indicate that DNA conformation and the type of gas used to saturate the irradiated solutions have a profound influence on yields of base products in DNA.

Hydrogen peroxide-induced base damage in deoxyribonucleic acid.

Aqueous solutions of calf thymus deoxyribonucleic acid (DNA) were exposed to hydrogen peroxide in the presence of air. Base products formed in DNA were identified and quantitated following acid hydrolysis and trimethylsilylation using gas chromatography-mass spectrometry. The yields of these products were dependent upon the hydrogen peroxide concentration, and increased in the following order: 8-hydroxyadenine, cytosine glycol, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine, thymine glycol, and 4,6-diamino-5-formamidopyrimidine. Previous studies have shown that these compounds are typically formed in DNA in aqueous solution by hydroxyl radicals generated by ionizing radiation. Hydrogen peroxide is thought to participate in a Fenton-like reaction with transition metals, which are readily bound to DNA in trace quantities, resulting in the production of hydroxyl radicals close to the DNA. This proposed mechanism was examined by exposing DNA to hydrogen peroxide either in the presence of a hydroxyl radical scavenger or following pretreatment of DNA with metal-ion chelators. The results indicate that trace quantities of transition metal ions can react readily with hydrogen peroxide to produce radical species. The production of radical species was monitored by determining the altered bases that resulted from the reaction between radicals and DNA. The yields of the base products were reduced by 40 to 60% with 10 mmol dm-3 of dimethyl sulfoxide. A 100-fold increase in the concentration of dimethyl sulfoxide did not result in a further reduction in hydrogen peroxide-induced base damage. DNA which was freed from bound metal ions by pretreatment with metal ion chelators followed by exhaustive dialysis was found to be an ineffective substrate for hydrogen peroxide. The yields of base products measured in this DNA were at background levels. These results support the role of metal ions bound to DNA in the site-specific formation of highly reactive radical species, most likely hydroxyl radicals, in hydrogen peroxide-induced damage to the bases in DNA.

Quantitative measurement of radiation-induced base products in DNA using gas chromatography-mass spectrometry.

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.