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 CIHR Author ManuscriptCIHR Author Manuscript CIHR Author Manuscript 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 TTTTTGTGTTTcisplatin: (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.

Radiosensitization of DNA by Cisplatin Adducts Results from an Increase in the Rate Constant for the Reaction with Hydrated Electrons and Formation of PtI

“…Nevertheless, when the irradiated oligonucleotide was digested to deoxyribonucleosides followed by HPLC analysis, the detachment of cisPt by e − aq was observed. These results are consistent with the previous observation by HPLC and gel sequencing analysis of radiolysis products from longer 11-mer oligonucleotides; 20,22 as well the observation of the strong enhancement of single and double strand break formation due to e − aq in γ-irradiated plasmid DNA-cisplatin complexes. 19 Taken together, these results strongly suggest that, in cells, the radiosensitization of DNA by cisplatin adducts probably results, at least in part, from the increase in the rate constant of the reaction of e − aq with DNA and formation Pt I .…”

“…19 Moreover, capture of e − aq by the base also creates base damage, such as 5,6-dihydrothymine, which is much more important in DNA-cisPt complexes than in DNA without cisPt. 22 …”

Transient Anions in Radiobiology and Radiotherapy: From Gaseous Biomolecules to Condensed Organic and Biomolecular Solids

“…The results demonstrated that hydrated electrons induce damage to thymines as well as detachment of the cisplatin moiety from both guanines in the oligonucleotide. The amount of free cisplatin (i.e., the cleavage of two Pt-G bonds) was found to be much larger than that of the products resulting from the cleavage of a single bond [181,182]. The errors represent the deviation of three identical measurements. )…”

“…This type of radiosensitization was investigated in more detail by irradiating with a γ source the oligonucleotide TTTTTGTTGTTT with or without cisplatin bound to the guanines [181]. Using scavengers and by eliminating oxygen, the oligonucleotide was shown to react with hydrated electrons.…”

Cisplatin Radiosensitization of DNA Irradiated with 2–20 eV Electrons: Role of Transient Anions

“…Such a resonance in solution would have much lower energy owing to polarization of the molecular orbitals of electrons and vibrational and phonon modes of surrounding water molecules (i.e., polaron formation); binding of cisplatin to DNA could also lower the resonance energy. Thus, according to the results of Behmand et al, 44,45 the formation of TNI at the site of cisplatin could considerably increase the formation of radicals owing to detachment of the Pt drug and damage to the bases. Assuming that the core-excited resonances at 4.6, 9.6, and 13.6 eV have a behavior similar to the lowest energy TNI in cisplatin-DNA complexes, these higher energy anion states could also strongly amplify the formation of radicals from DNA, in the presence of cisplatin.…”