Thiols alter the partitioning of calicheamicin-induced deoxyribose 4'-oxidation reactions in the absence of DNA radical repair.

Cellular thiols have been proposed to play a protective role in oxidative DNA damage by quenching radical species in solution and by repairing deoxyribose and nucleobase radicals. There is also evidence for participation of thiols in reactions after formation of the DNA radical. Previous studies with neocarzinostatin, a thiol-dependent DNA-cleaving enediyne, revealed that the structure and charge of the activating thiol influence the partitioning of deoxyribose 4'-oxidation reactions between a 3'-phosphoglycolate residue and the alternative 4'-keto-1'-aldehyde abasic site [Kappen, L. S., et al. (1991) Biochemistry 30, 2034-2042; Dedon, P. C., et al. (1992) Biochemistry 31, 1917-1927]. However, interpretation of these results is confounded by the formation of a neocarzinostatin-thiol conjugate that could alter the position of the activated drug in the minor groove and quench drug radicals. Using the DNA-cleaving enediynes calicheamicin gamma(1)(I) and Ø, which are identical except for their trigger moieties, we now present a more definitive study of the role of thiol structure in the partitioning of the deoxyribose 4'-oxidation reaction. In the absence of thiols, calicheamicin Ø, which can undergo hydrolytic or reductive activation, generated 4'-oxidation products consisting of 26% 3'-phosphoglycolate residues, 33% 3'-phosphate-ended fragments, and 41% abasic sites (determined as the 3'-phosphopyridazine derivative). Using a series of thiols of varying size and charge, we found that, at concentrations that do not quench drug or DNA radicals, the negatively charged thiols glutathione and thioglycolate did not significantly affect the baseline proportions of the 4'-oxidation products. However, neutral thiols (O-ethylglutathione, methyl thioglycolate, 2-mercaptoethanol, and dithiothreitol) and, to a greater extent, the positively charged aminoethanethiol inhibited the production of 3'-phosphoglycolate residues with a proportional increase in the number of abasic sites. The effect of the thiols on the quantities of single- and double-stranded DNA lesions produced by calicheamicin gamma(1)(I) was also investigated since 3'-phosphoglycolate residues produced by calicheamicin exist only in double-stranded DNA lesions, and the thiol effects could have resulted from quenching of drug or DNA radicals. These studies revealed that, at thiol concentrations found to alter deoxyribose 4'-oxidation reactions, there was no apparent quenching of drug radicals or repair of DNA radicals. Thus, the effects of the thiols on the deoxyribose 4'-oxidation chemistry are due to reactions with a key intermediate in the phosphoglycolate- and abasic site-generating pathways. These results also suggest that cellular glutathione plays a relatively minor role in the chemistry of deoxyribose 4'-oxidation, which has implications for other oxidative reactions occurring in the minor groove of DNA (e.g., deoxyribose 5'- and 1'-oxidation).