PROTECTION AGAINST PEROXYNITRITE-INDUCED FIBROBLAST INJURY AND ARTHRITIS DEVELOPMENT BY INHIBITION OF POLY(ADP-RIBOSE) SYNTHASE

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and s uperoxide, induces DNA strand breakage, which activates the nuclear en zyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30), The cellular funct ion of PARS was determined in fibroblast lines from PARS knock; out an imals (PARS(-/-)) and corresponding wild type animals (PARS(+/+)), wit h the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyr one (INH2BP). We investigated the role of PARS in peroxynitrite-induce d fibroblast injury in vitro and also in the development of arthritis in vivo. Exposure of embryonic fibroblasts from the PARS(+/+) animals to peroxynitrite caused DNA single-stand breakage and PARS activation and caused an acute suppression of mitochondrial respiration, INH2BP p rotected the PARS(+/+) cells against the suppression of mitochondrial respiration in response to peroxynitrite (50-100 mu M). Similarly to P ARS inhibition with INH2BP, the PARS(-/-) cells were protected against peroxynitrite-induced injury, The protection against cellular injury by PARS(-/-) phenotype or INH2BP waned when cells were challenged with higher concentrations of the oxidant, Inhibition of PARS by INH2BP or by PARS(-/-) phenotype reduced inducible nitric-oxide synthase (iNOS; EC 1.14.13.39) mRNA levels and inhibited production of NO in immunost imulated cells, INH2BP had no peroxynitrite scavenging or hydroxyl rad ical scavenging effects, and it exerted no additional (nonspecific) ef fects in the PARS(-/-) cells, In collagen-induced arthritis, significa nt staining for nitrotyrosine, a marker of peroxynitrite formation, wa s found in the inflamed joints, Oral treat ment with INH2BP (0.5 g/kg, daily), starting at the onset of arthritis (day 25), delayed the deve lopment of the clinical signs at days 26-35 and improved histological status in the knee and paw, Our data demonstrate that deletion of PARS by genetic manipulation or pharmacological inhibition of PARS protect s against oxidant-induced cellular injury in vitro and exhibits anti-i nflammatory effects in vivo.