GENERATION OF HYDROXYL RADICALS BY NUCLEOHISTONE-BOUND METAL-ADRIAMYCIN COMPLEXES

A recently developed method has been utilized to demonstrate the gener ation of hydroxyl radicals (HO.) in the immediate proximity of DNA by copper(II)/iron(III)-adriamycin in the presence of ascorbate and hydro gen peroxide. SECCA, a succinylated derivative of coumarin, generates the fluorescent 7-hydroxy-SECCA following reaction with HO.. SECCA was coupled to polylysine or to histone H1 and then complexed to DNA. Whe n HO. was generated in the proximity of DNA by polylysine-coupled iodi ne-125, which emits short range Auger electrons, 7-hydroxy-SECCA was p roduced. DMSO was only moderately efficient in reducing the fluorescen ce induction, demonstrating the ''local'' generation of HO. in this sy stem. Copper(II)/iron(III)-adriamycin in the presence of ascorbate and hydrogen peroxide generated the fluorescent 7-hydroxy-SECCA both when SECCA was free in solution and when SECCA was DNA-conjugated. With SE CCA free in solution, the fluorescence induction was almost eliminated in the presence of HO. scavengers (ethanol, tert-butanol or DMSO) and the relative efficiency of the scavengers in reducing the fluorescenc e followed their rate constant with HO.. Furthermore, SECCA incubated with a singlet oxygen-generating compound demonstrated no fluorescence induction. When SECCA was positioned in close proximity to DNA as a S ECCA-histone-H1-DNA complex, the relative efficiency of the scavengers in reducing the fluorescence still. followed their rate constant with HO.; overall however the scavengers were much less effective in reduc ing the fluorescence, due presumably to the formation of HO. radical i n the immediate vicinity of DNA. These data suggest that copper(II)/ir on(III)-adriamycin produces HO. in the presence of ascorbate and hydro gen peroxide whether unbound or bound to DNA and suggest that in the l atter case scavengers would not prevent HO. from attacking chromatin. In addition, the ability of DMSO to trap HO. was shown to decrease as the conformation of the H1-DNA complex becomes more compact indicating the strong dependence of the trapping ability on chromatin conformati on.