THE MECHANISM OF THE NITRIC OXIDE-MEDIATED ENHANCEMENT OF TERT-BUTYLHYDROPEROXIDE-INDUCED DNA SINGLE-STRAND BREAKAGE

1 Caffeine (Cf) enhances the DNA cleavage induced by tert-butylhydrope roxide (tB-OOH) in U937 cells via a mechanism involving Ca2+-dependent mitochondrial formation of DNA-damaging species (Guidarelli el al., 1 997b). Nitric oxide (NO) is not involved in this process since U937 ce lls do not express the constitutive nitric oxide synthase (cNOS). 2 Tr eatment with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP, 10 mu M), or S-nitrosoglutathiane (GSNO, 300 mu M), however, potentiated the DNA strand scission induced by 200 mu M tB-OOH. The DNA lesions ge nerated by tB-OOH alone, or combined with SNAP, were repaired with sup erimposable kinetics and were insensitive to anti-oxidants and peroxyn itrite scavengers but suppressed by iron chelators. 3 SNAP or GSNO did not cause mitochondrial Ca2+ accumulation but their enhancing effects on the tB-OOH-induced DNA strand scission were prevented by ruthenium red, an inhibitor of the calcium uniporter of mitochondria. Furthermo re, the enhancing effects of both SNAP and GSNO were identical to and not additive with those promoted by the Ca2+-mobilizing agents Cf or A TP. 4 The SNAP- or GSNO-mediated enhancement of the tB-OOH-induced DNA cleavage was abolished by the respiratory chain inhibitors rotenone a nd myxothiazol and was not apparent in respiration-deficient cells. 5 It is concluded that, in cells which do not express the enzyme cNOS, e xogenous NO enhances the accumulation of DNA single strand breaks indu ced by tB-OOH via a mechanism involving inhibition of complex III.