Abstract

Treatment of U937 cells with a sublethal albeit DNA-damaging concentration of tert-butylhydroperoxide (tB-OOH) enhanced mitochondrial Ca⁺⁺ uptake and ruthenium red (RR), a polycation that inhibits the calcium uniporter of mitochondria, significantly reduced the extent of DNA cleavage generated by the hydroperoxide. Release of Ca⁺⁺ from the ryanodine(Ry)/caffeine(Cf)-sensitive stores further increased mitochondrial Ca⁺⁺ uptake and elicited a parallel enhancement in DNA strand scission induced by tB-OOH that was prevented by both Ry and RR. DNA damage caused by tB-OOH alone or associated with either Cf or RR was prevented by iron chelators, insensitive to antioxidants and repaired with kinetics superimposable with those observed after treatment with H₂O₂. Cf enhanced the DNA-damaging effects of tB-OOH in permeabilized cells as well, and similar effects were observed upon addition of CaCl₂. Cf did not further increase the formation of DNA lesions elicited by tB-OOH in the presence of CaCl₂. The enhancing effects of Cf were prevented by RR and ryanodine, whereas those mediated by exogenous calcium were prevented only by RR. DNA strand scission caused by tB-OOH alone or associated with Cf in the permeabilized cell system was severely inhibited by ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid. The mechanism(s) whereby Ca⁺⁺promotes the mitochondrial formation of species that will ultimately result in the formation of DNA lesions was subsequently analyzed using intact as well as permeabilized cells. Hydrogen peroxide was identified to be one of these species.