Optimal time and dosage of phenyl N-tert-butyl nitrone (PBN) for the inhibition of nitric oxide synthase induction in mice

We have previously reported that phenyl N-tert-butyl nitrone (PEN) inhibits the induction of inducible nitric oxide synthase (iNOS) and, thus, prevents the overproduction of nitric oxide (NO), resulting in the reduction of endotoxin-mediated death in mice. In this study, to examine the effect of PEN in detail, we investigated the dose- and administration-timing dependence of PEN on endotoxin-induced NO generation in mice. NO generation was monitored in the mouse liver after administration of lipopolysaccharide (LPS) by the in vivo NO-spin trapping method using the iron complex of N-methyl-D-glucamine dithiocarbamate (MGD) as a spin trap, followed by ex vivo EPR measurement of the liver tissue. PEN was effective in reducing liver NO generation monitored 6 h after endotoxin injection when it was administered shortly before or after LPS injection. The maximum inhibition of liver NO was obtained when PEN was administered 30 min before LPS injection. ID50 for the inhibition was estimated to be approximately 200 mg/kg when the LPS dose of 50 mg/kg was used. Expression of mRNA for iNOS in the liver as estimated by reverse transcription polymerase chain reaction was decreased when PEN was given 30 min before LPS injection, indicating that the reduction of expression of iNOS protein by PEN, which has been shown previously, is at least in part caused by a decrease in mRNA expression. Copyright (C) 1996 Elsevier Science Inc.