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Blocking of in Vitro DNA Replication by Deoxycytidine Adducts of the Mutagen and Clastogen 2-Bromoacrolein¹

Division of Toxicology, Leiden/Amsterdam, Center for Drug Research [G. J. M. v. B., F. B. v. G., J. H. N. M.], Department of Radiation Genetics and Chemical Mutagenesis [H. V.], Sylvius Laboratories, P. O. Box 9503, 2300 RA Leiden The Netherlands; Department of Medicinal Chemistry, BG-20, University of Washington, Seattle, Washington 98195 [S. D. N.]; Drug Metabolism Research, Upjohn Laboratories, Kalamazoo, Michigan 49001 [P. G. P.]

Calf thymus single-stranded DNA was modified with 2-bromoacrolein (2BA), a genotoxic metabolite of tris(2,3-dibromopropyl)phosphate. This DNA was used as a template for in vitro DNA replication by T7-polymerase and Klenow fragment of Escherichia coli DNA polymerase I. Increasing levels of 2BA modification led to decreased DNA synthesis as measured by [methyl-³H]dTTP incorporation. M13 mp19 single-stranded DNA template modified with 2BA was used to determine the sites of termination of DNA replication by T7 polymerase and Klenow fragment of Escherichia coli DNA polymerase I. It was found that DNA replication stopped frequently before and occasionally opposite deoxycytidine nucleotides. These results indicated that an as yet unidentified deoxycytidine:2BA adduct may have been formed in the reaction of 2BA with M13 DNA. To investigate if such adducts were formed, we reacted 2BA with deoxycytidine in vitro at pH 4.4, and putative deoxycytidine:2BA adducts were isolated by high-performance liquid chromatography. They were characterized by ¹H and ¹³C nuclear magnetic resonance and with fast atom bombardment mass spectrometry as two diastereomeric 3-bromo-7-(ß-D-deoxyribofuranosyl)-3,4-dihydro-2-hydroxy-(2H,7H)[1,6-]pyrimidin-6-one adducts and a 3-bromo-7-(ß-deoxyribofuranosyl)-(4H,7H)-pyrimido[1,6-]pyrimidin6-one adduct. Only the latter adduct, however, was formed in the reaction of 2BA with calf thymus single-stranded DNA in vitro.

Tris(2,3-dibromopropyl)phosphate is clastogenic. Because clastogenicity may result from DNA adducts that block replication, the role of the presently identified deoxycytidine adducts of the reactive metabolite 2BA in the clastogenicity of tris(2,3-dibromopropyl)phosphate is discussed.

¹ This study was supported by NWO Grant 900-521-123 (J. H. N. M.), NIH Grant ES 02728 (S. D. N.), and NATO collaborative research Grant CRG 900275 (J. H. M. and S. D. N.).

² To whom requests for reprints should be addressed, at Division of Toxicology, Leiden/Amsterdam, Center for Drug Research, Sylvius Laboratories, P.O. Box 9503, 2300 RA Leiden, the Netherlands.

Received 2/15/93. Accepted 10/ 6/93.

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