Neopterin derivatives modulate toxicity of reactive species on Escherichia coli.

Neopterin and 7,8-dihydroneopterin are released by human monocytes/macrophages upon stimulation with interferon-gamma. In parallel, a panel of highly reactive species is produced by macrophages as part of their cytotoxic armature, which is directed against microbial and viral challenge and against malignant growth. Recently, neopterin and 7,8-dihydroneopterin were shown to modulate the action of reactive species in vitro. In this study we investigated the impact of neopterin and 7,8-dihydroneopterin on the toxicity of reactive species, namely chloramine-T, H2O2, hypochlorite, nitrite, and formaldehyde, respectively. We studied the growth inhibition of Escherichia Coli (E. coli) by these toxic agents and its modulation by neopterin and 7,8-dihydroneopterin. Bacterial growth was monitored by optical density of suspension cultures at 600 nm. Compared to control experiments, neopterin enhanced toxicity of all reactive species tested except formaldehyde, while 7,8-dihydroneopterin reduced activity of hypochlorite and chloramine-T. No significant impact of the pteridines could be established for H2O2-mediated and formaldehyde-mediated growth inhibition. The data support the concept that neopterin and 7,8-dihydroneopterin produced during immune response in humans could be important to modulate the action of reactive species released in parallel.

Inhibition of xanthine oxidase by pterins.

The effect of a panel of pterins on xanthine oxidase was investigated by measuring formation of urate from xanthine as well as formazan production from nitroblue tetrazolium. The pterin derivatives, depending on their chemical structure, decreased urate as well as formazan generation: 200 microM neopterin and biopterin suppressed urate formation (90% from baseline) and formazan production (80% from baseline) as well. Their reduced forms, 7,8-dihydroneopterin and 5,6,7,8-tetrahydrobiopterin, showed a lesser but still strongly diminishing influence (40% from baseline). Another oxidized pterin namely leukopterin showed only a weak inhibitory effect. Xanthopterin, a known substrate of xanthine oxidase, had a strong effect on urate formation (80% inhibition), but a lesser effect on formazan production (30% reduction). When iron-(III)-EDTA complex was added to the reaction mixture all the effects were more pronounced. Superoxide dismutase, which removes superoxide anion by dismutation into oxygen, decreased formazan production in addition to pterin derivatives and had a small but enhancing effect on urate formation. Also the reductant N-acetylcysteine had an additive effect to pterins to diminish formazan production in a dose-dependent way. The results of our study suggest that depending on their chemical structure pterins reduce superoxide anion generation by xanthine oxidase.

Neopterin derivatives modulate the nitration of tyrosine by peroxynitrite.

The impact of neopterin and 7,8-dihydroneopterin on peroxynitrite-induced nitration of l-tyrosine was studied. Neopterin derivatives and peroxynitrite are formed during immune response. Tyrosine nitration represents one major effect of nitric oxide-mediated cytotoxicity. Peroxynitrite formed in situ was co-incubated with tyrosine and neopterin or 7,8-dihydroneopterin or other pteridine derivatives, respectively. The nitration product, 3-nitro-l-tyrosine, was measured by HPLC via UV absorption at 360 nm. Neopterin (200 microM) increased the nitration rate between pH 4.0 and 5.5 up to +60%. 7,8-Dihydroneopterin inhibited tyrosine nitration over the whole pH range examined. In a series of various pteridine derivatives, neopterin and 7,8-dihydroneopterin achieved the strongest modulating effects on tyrosine nitration. Interactions of peroxynitrite with hydroxypropyl side chains of fully aromatic pterin derivatives may increase nitration, while partially hydrated pyrazino ring structures abate the reactivity of peroxynitrite. The results of this study suggest a potential impact of neopterin derivatives on peroxynitrite-mediated cytotoxicity.