Pulse radiolysis of the DNA-binding bisbenzimidazole derivatives Hoechst 33258 and 33342 in aqueous solutions.
OBJECTIVE
The DNA-minor-groove-ligands bisbenzimidazoles Hoechst 33258 and 33342 have been reported to protect against radiation-induced DNA-strand breakage. In order to elucidate the mechanisms of protection by these DNA-binding compounds, pulse radiolysis studies on the reactions of the OH radical, the solvated electron and the H atom with Hoechst as well as OH-radical-induced nucleotide radical quenching by free Hoechst (model level) was investigated.


METHODS
The pulse radiolysis of Hoechst 33258 and 33342 was studied in N2O and N2O/O2-(4:1)-saturated aqueous solutions in the absence and presence of azide and bromide ions and nucleotides.


RESULTS
In a fully scavenged system (3 x 10(-2) mol x dm(-3) t-butanol, N2O/O2-saturated), a transient is formed which in the presence of phosphate buffer is no longer observed. This is assigned metastable quinonoid forms of Hoechst (lambdamax(Hoechst) = 340; lambdamax(transient) = 370 nm) which is generated in protonation/ deprotonation reactions by H+/OH- formed during the pulse. To prevent their formation 10(-3) mol x dm(-3) phosphate buffer was added in all other experiments. The transient spectra formed upon OH-radical attack (k=9 x 10(9) dm3 x mol(-1) x s(-1)) indicate that a major part of the primary OH-adduct radicals undergo rapid transformation (k approximately 5 x 10(5) x s(-1)), attributed to water elimination yielding an N-centered radical. This intermediate, also generated by N3. (k = 4 x 10(9) dm3 mol(-1) x s(-1)), subsequently complexes with a Hoechst molecule [k = 8 x 10(8) dm3 x mol(-1) x s(-1) epsilon(440 nm) = 1.4 x 10(4) dm3 mol(-1) x cm(-1)]. The N-centered radical does not react with O2 (k < 5 x 10(5) dm3 mol(-1) x s(-1)), but reacts readily with the superoxide radical (k= 1.0 x 10(9) dm3 x mol(-1) x s(-1)). Hoechst reacts with the peroxyl radicals derived from uridine (k approximately 5 x 10(6) dm3 x mol(-1) x s(-1)) or 5'-UMP (k approximately 1 x 10(7) dm3 mol(-1) x (s-1)), but not with the less oxidizing (e.g. methylperoxyl radical) yielding intermediates whose spectral properties are similar to those of the N-centered radical. However, they decay at a much lower rate (2k approximately 1 x 10(8) dm3 mol(-1) x s(-1)) than the N-centered radicals generated by N3. (2k= 1.1 x 10(9) dm3 x mol(-1) s(-1)), and it has been suggested that these peroxyl radicals form adducts rather than undergoing electron transfer. The H atom (k= 7 x 10(9) dm3 x mol(-1) x s(-1)) and the solvated electron (k= 2.3 x 10(10) dm3 x mol(-1) x s(-1)) yield, albeit noticeably different, H-adduct radicals which also strongly absorb in the 440 nm region. The reduction potential of Hoechst 33258 has been determined electrochemically at 0.84-0.90 V vs. NHE at pH 6.8.


CONCLUSIONS
Hoechst reacts fast only with strongly oxidizing radicals by electron transfer (e.g. with the adenine-and guanine-derived heteroatom-centered radicals), but also more slowly with nucleo-base-derived peroxyl radicals, here albeit via addition. This may have important implications with regard to its protection owing to DNA-radical quenching under oxic vs. anoxic conditions.
Adhikary
A
A
Max-Planck-Institut für Strahlenchemie, Mülheim an der Ruhr, Germany.
Bothe
E
E
Jain
V
V
Von Sonntag
C
C
D001562:Benzimidazoles / Q000737:chemistry*; D006690:Bisbenzimidazole / Q000737:chemistry*; D001966:Bromine / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005609:Free Radicals / Q000528:radiation effects; D006859:Hydrogen / Q000737:chemistry; D017665:Hydroxyl Radical; D008956:Models, Chemical; D009584:Nitrogen / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011675:Pulse Radiolysis; D011838:Radiation-Sensitizing Agents / Q000737:chemistry*; D013481:Superoxides / Q000737:chemistry
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D005609:Free Radicals; D011838:Radiation-Sensitizing Agents; D013481:Superoxides; D017665:Hydroxyl Radical; D006859:Hydrogen; D004247:DNA; C031000:benzimidazole; D006690:Bisbenzimidazole; D009584:Nitrogen; C017807:bisbenzimide ethoxide trihydrochloride; D010100:Oxygen; D001966:Bromine