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 superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS(-/-)) and corresponding wild-type animals (PARS(+/+)), with the aid of lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH₂BP). We investigated the role of PARS in peroxynitrite-induced 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 protected the PARS(+/+) cells against the suppression of mitochondrial respiration in response to peroxynitrite (50-100 μM). Similarly to PARS 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 immunostimulated cells. INH₂BP had no peroxynitrite scavenging or hydroxyl radical scavenging effects, and it exerted no additional (nonspecific) effects in the PARS(-/-) cells. In collagen-induced arthritis, significant staining for nitrotyrosine, a marker of peroxynitrite formation, was found in the inflamed joints. Oral treatment with INH₂BP (0.5 g/kg, daily), starting at the onset of arthritis (day 25), delayed the development 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 protects against oxidant-induced cellular injury in vitro and exhibits anti-inflammatory effects in vivo.