Inhibition of poly(ADP-ribose) synthetase (PARS) and protection against peroxynitrite-induced cytotoxicity by zinc chelation

1 Peroxynitrite, a potent oxidant formed by the reaction of nitric oxide an d superoxide causes thymocyte necrosis, in part, via activation of the nucl ear enzyme poly(ADP-ribose) synthetase (PARS). The cytotoxic PARS pathway i nitiated by DNA strand breaks and excessive PARS activation has been shown to deplete cellular energy pools, leading to cell necrosis. Here we have in vestigated the effect of tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) a heavy metal chelator on peroxynitrite-induced cytotoxicity. 2 TPEN (10 mu M) abolished cell death induced by authentic peroxynitrite (2 5 mu M) and the peroxynitrite generating agent 3-morpholinosidnonimine (SIN -1, 250 mu M). Preincubation of TPEN with equimolar Zn2+ but not Ca2+ or Mg 2+ blocked the cytoprotective effect of the chelator. 3 TPEN (10 mu M) markedly reduced the peroxynitrite-induced decrease of mit ochondrial transmembrane potential, secondary superoxide production and mit ochondrial membrane damage, indicating that it acts proximal to mitochondri al alterations. 4 Although TPEN (1 - 300 mu M) did not scavenge peroxynitrite, it inhibited PARS activation in a dose-dependent manner. 5 The cytoprotective effect of TPEN is only partly mediated via PARS inhibi tion, as the chelator also protected PARS-deficient thymocytes from peroxyn itrite-induced death. 6 While being cytoprotective against peroxynitrite-induced necrotic death, TPEN (10 mu M), similar to other agents that inhibit PARS, enhanced apoptos is (at 5-6 h after exposure), as characterized by phosphatydilserine exposu re, caspase activation and DNA fragmentation. 7 In conclusion, the current data demonstrate that TPEN, most likely by zin c chelation, exerts protective effects against peroxynitrite-induced necros is. Its effects are, in part, mediated by inhibition of PARS.