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ToxSci Advance Access originally published online on August 25, 2004
Toxicological Sciences 2004 82(2):497-503; doi:10.1093/toxsci/kfh262
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Toxicological Sciences vol. 82 no. 2 © Society of Toxicology 2004; all rights reserved.

DNA Cross-Linking by Dehydromonocrotaline Lacks Apparent Base Sequence Preference

W. Kurt Rieben, Jr. and Roger A. Coulombe, Jr.1

Graduate Toxicology Program, and Department of Veterinary Sciences, Animal Science 213, Utah State University, Logan, Utah 84322-4620

Received June 8, 2004; accepted August 23, 2004

Pyrrolizidine alkaloids (PAs) are ubiquitous plant toxins, many of which, upon oxidation by hepatic mixed-function oxidases, become reactive bifunctional pyrrolic electrophiles that form DNA-DNA and DNA-protein cross-links. The anti-mitotic, toxic, and carcinogenic action of PAs is thought to be caused, at least in part, by these cross-links. We wished to determine whether the activated PA pyrrole dehydromonocrotaline (DHMO) exhibits base sequence preferences when cross-linked to a set of model duplex poly A-T 14-mer oligonucleotides with varying internal and/or end 5'-d(CG), 5'-d(GC), 5'-d(TA), 5'-d(CGCG), or 5'-d(GCGC) sequences. DHMO-DNA cross-links were assessed by electrophoretic mobility shift assay (EMSA) of 32P endlabeled oligonucleotides and by HPLC analysis of cross-linked DNAs enzymatically digested to their constituent deoxynucleosides. The degree of DNA cross-links depended upon the concentration of the pyrrole, but not on the base sequence of the oligonucleotide target. Likewise, HPLC chromatograms of cross-linked and digested DNAs showed no discernible sequence preference for any nucleotide. Added glutathione, tyrosine, cysteine, and aspartic acid, but not phenylalanine, threonine, serine, lysine, or methionine competed with DNA as alternate nucleophiles for cross-linking by DHMO. From these data it appears that DHMO exhibits no strong base preference when forming cross-links with DNA, and that some cellular nucleophiles can inhibit DNA cross-link formation.

Key Words: dehydromonocrotaline; DNA cross-linking; sequence specificity.


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