Vol. 144, No. 1, 1987 April 14, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 432-437 DITHIOTHREITOL-INDUCED OXIDATIVE DAMAGE TO THYMINE AND DNA IN SOLUTION H. Gregg Claycamp Radiation Biology Program, University of Iowa, 14 Medical Laboratory, Iowa City, IA 52242 Received February 25, 1987 SUMMARY: Studies on dithiothreitol-induced oxidative damage to thymine and DNA in solution are reported. The major thymine products, cis- and trans- 5,6-dihydroxy-5,6-dihydrothymine (thymine glycols), are produced rapidly in 37°C neutral solutions of 10mM thymine and 10mM dithiothreitol. Iron-EDTA enhances while the iron chelator, di~thylenetriaminepentaacetic acid, inhibits the reaction. In experiments using ~H-TdR-labeled Escherichia coli DNA, DNA damage was measured as increased ethanol-soluble radioactivity after treatment of the DNA with 5mM dithiothreitol at 45°C. The findings are important with respect to current research interest in thiol radioprotection and thiol-plus-heat toxicity. © 1987AcademicPress, Inc. The occurrence of thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) in DNA in vitro or in vivo is generally considered to be evidence of oxidative damage to DNA (1,2). Oxidative DNA damage is implicated as an etiologic factor in a number of human diseases including cancer (3,4). The objective of the present communication is to demonstrate that significant yields of thymine glycol and DNA damage are generated from the oxidation of dithiothreitol in thymine or DNA solutions at physiological pH. More than a century has elapsed since thiol oxidation was noted by E. Baumann (5); yet, it was not until 1957 that active oxygen species (free radicals) were postulated as intermediaries in iron-catalyzed thiol oxidation reactions (6). Superoxide free radicals (02T) from thiol oxidation were measured by Misra (7), and Rowley and Halliwell (8) in 1981 verified the presence of -OH in thiol oxidation reactions. The fact that iron is a potent catalyst of thiol oxidation has been verified by a number of authors (5,6,8-10); the Fe-complex-oxygen recycling reactions are now often referred to as "iron-catalyzed Haber-Weiss reactions" (i0). Abbreviations: DTT , dithiothreitol; DHD = trans-4,5-hydroxy- 1,2-dithiane, DTPA i diethylenetriaminepentaacetic acid; KP , mono- and dibasic potassium phosphate buffer, i0 mM; pH 7. 0006-291X/87 $1.50 Copyright © 1987 by Academic Press, lnc. All rights of reproduction in any form reserved. 432