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Formation of Cyclic 1,N²-Propanodeoxyguanosine and Thymidine Adducts in the Reaction of the Mutagen 2-Bromoacrolein with Calf Thymus DNA¹

University of Washington, Department of Medicinal Chemistry, Seattle, Washington 98195 [T. R. S., P. G. P., G. P. M., S. D. N.], and Center for Biopharmaceutical Sciences, Division of Toxicology, University of Leiden, P. O. Box 9503, 2300 RA Leiden, The Netherlands [J. H. N. M.]

The interaction of the mutagen 2-bromoacrolein (2BA) with DNA and thymidine was studied in vitro by reaction of [3-³H]2BA with thymidine, RNA, single-stranded DNA, and double-stranded DNA in phosphate buffer (pH 7.4). After purification of the nucleic acids, they were incubated at alkaline pH to convert any (hydroxybromo)propano(deoxy)-guanosine adducts to their dihydroxy analogues. After acid or enzymatic hydrolysis, the hydrolysates were analyzed by reversed-phase high-performance liquid chromatography. At a concentration of 1.6 mM, the fraction of 2BA that became covalently bound to DNA was 2.3% of the amount added. Only 3% of the radioactivity bound to DNA after extensive purification could be accounted for as cyclic 1,N²-(6,7-dihydroxy)-propanoguanine adducts. More 2BA became covalently bound to single-stranded DNA and RNA as compared with double-stranded DNA. However, high-performance liquid chromatographic analyses showed that formation of cyclic 1,N²-(6,7-dihydroxy)propanoguanine adducts was also a minor reaction with these macromolecules.

Because these data showed that other type(s) of reaction(s) are more important in the reaction of 2BA with nucleic acids, we have investigated the reaction of 2BA with other nucleosides. It was found that 2BA reacted well with thymidine in vitro, and the major product was identified by 500 MHz ¹H and 75.43 MHz ¹³C nuclear magnetic resonance and thermospray mass spectrometry as 3-(2''-bromo-3''-oxopropyl)thymidine. This adduct was unstable and decomposed upon storage. After enzymatic hydrolysis of [³H]2BA-modified double-stranded DNA and subsequent analysis of the hydrolysate by high-performance liquid chromatography, 22% of the covalently bound radioactivity to DNA coeluted with decomposition products of the 3-(bromooxypropyl)thymidine adduct. This indicates that reaction of 2BA with this nucleotide in DNA is a major reaction.

¹ Supported by NIH Grant ES-02728 (S. D. N.), a stipend from the Netherlands Organization for the Enhancement of Pure Science (ZWO), and a Fulbright grant (J. H. N. M.).

² To whom requests for reprints should be addressed.

Received 5/ 2/89. Revised 8/17/89. Accepted 8/21/89.