Development of a Sensitive Method for the Determination of 5,6-Dihydrothymidine Formed by Gamma Irradiation of DNA below 0.1 kGy

Abstract

A sensitive method was developed to determine 5,6-dihydrothymidine (DHdThd) produced by relatively low-dose (<0.1 kGy) gamma irradiation of DNA. Previously, we had examined a method to determine the irradiation history of foods measured by DHdThd levels, which is a radiolytic product formed in the DNA of foods. The method included: (1) DNA extraction from food, (2) enzymatic digestion of DNA into nucleosides, and (3) measurement of DHdThd using liquid chromatography-tandem mass spectrometry. This method could detect relatively high-dose (≥0.5 kGy) irradiation history with regard to the sterilization of both animal- and plant-based food items. However, detection of relatively low-dose (<0.5 kGy) irradiation in foods for quarantine or sprouting inhibition would require more sensitivity, since lower the dose, lesser the DHdThd produced. To enable such detection, in this study, we aimed to introduce the purification and concentration of large amounts of degraded DNA in aqueous solutions, using solid-phase extraction columns, into the existing protocol, and succeeded in increasing the sensitivity of DHdThd by approximately 30-fold. Using this method, DHdThd could be detected, in a dose-dependent manner, in 60–150 Gy gamma-irradiated aqueous solution of thymidine and salmon sperm DNA. The method could be applicable for the detection of gamma irradiation of foods at doses of several tens of Gy.