Thiols Alter the Partitioning of Calicheamicin-Induced Deoxyribose 4‘-Oxidation Reactions in the Absence of DNA Radical Repair

Abstract

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).

The Chemical Toxicology of 2-Deoxyribose Oxidation in DNA

“…There are many approaches to quantifying or at least estimating total 2-deoxyribose oxidation events, including the plasmid nicking assay for in vitro studies and the comet assay for studies in cells. The former is a well-established technique that exploits the conversion of supercoiled plasmid DNA to nicked and linear forms following direct strand breaks and, after derivatization with agents such as putrescine, oxidized abasic sites – . Alternatively, oxidized purine and pyrimidine bases can be converted to strand breaks using formamidopyrimidine DNA glycosylase and endonuclease III, respectively .…”

DNA Binding and Cleavage Modes of Shishijimicin A

“…As already established for calicheamicin γ 1 I , these observations rule out the possibility of involvement of hydrogen peroxide and hydroxyl radicals in the shishijimicin A-mediated DNA-cleaving processes. However, in the presence of a commonly used scavenger of superoxide radical (O 2 • – ), dithiothreitol (DTT), the efficiency of DNA-cleavage by either shishijimicin A or calicheamicin γ 1 I was reduced, with single-strand cuts becoming dominant (Lane 9 in Figures a,b). This occurs, most likely, because DTT at the concentration employed (80 mM), apart from acting as superoxide radical scavenger, can also interfere with the fate of DNA radicals either by quenching them or leading to the formation of abasic sites, rather than strand cuts …”

Differential Oxidation of Deoxyribose in DNA by γ and α-Particle Radiation

“…superoxide, hydrogen peroxide, other oxidizing species) that interact with or influence the chemical reactivity of the initially formed carbon-centered radicals in the sugar. The partitioning of deoxyribose oxidation chemistry is known to be influenced by the biochemical environment of the initial carbon-centered radical in DNA damage produced by bleomycin and enediyne antibiotics (33)(34)(35).…”