CALCIUM-DEPENDENT MITOCHONDRIAL FORMATION OF SPECIES MEDIATING DNA SINGLE-STRAND BREAKAGE IN U937 CELLS EXPOSED TU SUBLETHAL CONCENTRATIONSOF TERT-BUTYLHYDROPEROXIDE

Treatment of U937 cells with a sublethal albeit DNA-damaging concentra tion of tert-butylhydroperoxide (tB-OOH) enhanced mitochondrial Ca++ u ptake and ruthenium red (RR), a polycation that inhibits the calcium u niporter of mitochondria, significantly reduced the extent of DNA clea vage generated by the hydroperoxide. Release of Ca++ From the ryanodin e(Ry)/caffeine(Cf)-sensitive stores further increased mitochondrial Ca ++ uptake and elicited a parallel enhancement in DNA strand scission i nduced by tB-OOH that was prevented by both Ry and RR. DNA damage caus ed by tB-OOH alone or associated with either Cf or RR was prevented by iron chelators, insensitive to antioxidants and repaired with kinetic s superimposable with those observed after treatment with H2O2. Cf enh anced the DNA-damaging effects of tB-OOH in permeabilized cells as wel l, and similar effects were observed upon addition of CaCl2. Cf did no t further increase the formation of DNA lesions elicited by tB-OOH in the presence of CaCl2. The enhancing effects of Cf were prevented by R R and ryanodine, whereas those mediated by exogenous calcium were prev ented only by RR. DNA strand scission caused by tB-OOH alone or associ ated with Cf in the permeabilized cell system was severely inhibited b y ethylene glycol-bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic ac id. The mechanism(s) whereby Ca++ promotes the mitochondrial formation of species that will ultimately result in the formation of DNA lesion s was subsequently analyzed using intact as well as permeabilized cell s. Hydrogen peroxide was identified to be one of these species.