1,10-PHENANTHROLINE STIMULATES INTERNUCLEOSOMAL DNA FRAGMENTATION IN ISOLATED RAT-LIVER NUCLEI BY PROMOTING THE REDOX ACTIVITY OF ENDOGENOUS COPPER IONS

Isolated rat-liver nuclei were incubated with a series of membrane-per meable metal-ion-complexing agents and examined for DNA damage. Of the reagents tested, only 1,10-phenanthroline (OF) and neocuproine (NC) w ere found to induce DNA fragmentation. Agarose-gel electrophoresis of the DNA fragments generated in the presence of OP revealed internucleo somal cleavage, which is widely considered to be a hallmark for the en zymic DNA digestion that occurs during apoptosis. Ascorbate, particula rly in the presence of hydrogen peroxide, increased the levels of frag mentation induced by OF. As. well as undergoing fragmentation, the DNA from nuclei was also found to contain 8-hydroxydeoxyguanosine, which indicates attack (oxidation) by the:hydroxyl radical. Complementary ex periments in vitro involving ESR determinations of hydroxyl radical fo rmation and measurements of DNA oxidation under biomimetic conditions demonstrated that Cu2+, but not Fe3+, forms a complex with either OP o r NC (but not the other complexing agents tested) that stimulates hydr oxyl radical formation and DNA damage in the presence of hydrogen pero xide and ascorbate, It is therefore proposed that OP in the nuclei inc ubations binds to Cu2+, which exists naturally in chromosomes, forming a complex that promotes hydroxyl-radical-dependent DNA fragmentation. These findings demonstrate the promotion of hydroxyl-radical-mediated DNA damage by endogenous Cu2+ and, perhaps more significantly, demons trate that the internucleosomal DNA 'laddering' that is often used as an indicator of apoptosis may also result from DNA fragmentation by no n-enzymic processes.