Cisplatin intrastrand adducts sensitize DNA to base damage by hydrated electrons

Abstract

The oligonucleotide TTTTTGTGTTT with or without a cisplatin adduct was reacted with hydrated electrons generated by ionizing radiation. Hydroxyl radicals were quenched with ethylenediaminetetraacetic acid (EDTA), and the solutions were bubbled with wet nitrogen to eliminate oxygen, a scavenger of hydrated electrons. Prior to irradiation, the structure of the initial cisplatin adduct was identified by mass spectrometry as G-cisplatin-G. Radiation damage to DNA bases was quantified by high-performance liquid chromatography (HPLC), after enzymatic digestion of the TTTTTGTGTTT-cisplatin complex to deoxyribonucleosides. The masses of the platinum adducts following digestion and separation by HPLC were measured by mass spectrometry. Our results demonstrate that hydrated electrons induce damage to thymines as well as detachment of the cisplatin moiety from both guanines in the oligonucleotide. This detachment regenerates both unmodified guanine and damaged guanine, in equimolar amounts. At 1000 Gy, a net average of 2.5 thymines and 1 guanine are damaged for each platinum lost from the oligonucleotide. Given the extensive base damage that occurs for each cisplatin adduct lost, it is clear that, prior to undergoing detachment, these adducts must catalyze several cycles of reactions of hydrated electrons with DNA bases. It is likely that a single reaction leads to the loss of the cisplatin adduct and the damage observed on the guanine base; however, the damage to the thymine bases must require the continued presence of the cisplatin adduct, acting as a catalyst. To our knowledge, this is the first time that platinum-DNA adducts have been shown to have catalytic activity. We propose two pathways for the interaction of hydrated electrons with TTTTTGTGTTT-cisplatin: (1) the hydrated electron is initially captured by a thymine base and transferred by base to base electron hopping to the guanine site, where the cisplatin moiety detaches from the oligonucleotide via dissociative electron attachment, and (2) the hydrated electron interacts directly with the platinum-guanine adduct and induces detachment of the cisplatin moiety via dissociative electron attachment. Although the precise mechanism remains to be elucidated, our results provide important insights into the radiosensitization of DNA by cisplatin.

Figure 1

HPLC traces for deoxyribonucleosides from the oligonucleotides following reaction with hydrated electrons, generated by ionizing radiation. The oligonucleotides were irradiated with doses from 0 to 1000 Gy under conditions which quenched ^•OH radicals and favored hydrated electrons (i.e., reduced O₂) (see Materials and Methods). Afterward, the oligonucleotides were digested to deoxyribonucleosides (see Materials and Methods): (a) no irradiation with and without cisPt, (b) irradiation of the oligonucleotide without cisPt, and (c) with cisPt with different doses.

Figure 2

Radiation-induced loss of the dG-cisPt-dG adduct (red ●) and resulting formation of undamaged dG (black ■) and modified dG (green ▲) as revealed by digestion of the oligonucleotide to deoxyribonucleosides followed by HPLC analysis.

Figure 3

Percentage of modified (a) dG and (b) dT as a function of irradiation dose for ODN-GTG (black ■) and ODN-GTG-cisPt (red ●) following digestion to nucleosides.

Figure 4

Structure of dG-cisPt-dG following the digestion.

Figure 5

ESI-MS spectrum of dG-cisPt-dG, in negative mode.

Figure 6

dG-cisPt-dG: ESI-MS/MS spectrum of the peak at 760 in Figure 5 in negative mode.