Identification of radiation-induced thymine derivatives in DNA

A methodology for the separation of radiation-induced thymine derivatives in DNA using high pressure liquid chromatography is presented. DNA was subjected to enzymatic hydrolysis yielding 2'-deoxyribonucleosides and the hydrolysate cochromatographed with marker compounds. Confirmation of the presence of derivatives was accomplished by chromatography on Sephadex LH-20 and microderivatization. The method separates free bases from nucleosides allowing for identification of spontaneously released bases or those released through the action of repair enzymes. The results indicate that most of the thymine derivatives formed in irradiated cellular DNA were the same as those found in DNA irradiated in solution. However, the major cellular derivative was not present in the latter. This derivative was identified as 5-hydroxymethyl-2'-deoxyuridine (HMdU). HMdU has previously been shown to be cytotoxic to cells in culture and caused diarrhea and bone marrow failure when administered to mice. Thus, the presence of this radiation-induced thymine derivative in cellular DNA correlates with the known effects of ionizing radiation on cells and animals.