Oxidative conversion of 6-nitrocatecholamines to nitrosating products: A possible contributory factor in nitric oxide and catecholamine neurotoxicityassociated with oxidative stress and acidosis

Oxidation of 6-nitrodopamine (1) and 6-nitronorepinephrine (2), as well as of the model compounds 4-nitrocatechol and 4-methyl-5-nitrocatechol, with h orseradish peroxidase (HRP)/H2O2, lactoperoxidase (LPO)/H2O2, Fe2+/H2O2, Fe 2+-EDTA/H2O2 (Fenton reagent), HRP or Fe2+/EDTA in combination with D-gluco se-glucose oxidase, or Fe2+/O-2, resulted in the smooth formation of yellow ish-brown pigments positive to the Griess assay. In the case of 1, formatio n of the Griess positive pigment (GPP-1) promoted by HRP/H2O2 proceeded thr ough the intermediacy of two main dimeric species that could be isolated an d identified as 3 and the isomer 4, featuring the 4-nitro-6,7-dihydroxyindo le system linked to a unit of I through ether bonds. Spectroscopic (FAB-MS, H-1 NMR) and chemical analysis of GPP-1 indicated a mixture of oligomeric species related to 3 and 4 in which oxidative modification of the nitrocate chol moiety of 1 led to the generation of reactive nitro groups supposedly linked to sp(3) hybridized carbons. In the pH range 3-6, GPP-1 induced conc entration- and pH-dependent nitrosation of 2,3-diaminonaphthalene, but very poor (up to 2%) nitration of 600 muM tyrosine. At pH 7.4, 1 exerted signif icant toxicity to PC12 cells, while GPP-1 proved virtually innocuous. By co ntrast, when assayed on Lactobacillus bulgaricus cells at pH 3.5, 1 was ina ctive whereas GGP-1 caused about 70% inhibition of cell growth. Overall, th ese results hint at novel pH-dependent mechanisms of nitrocatecholamine-ind uced cytotoxicity of possible relevance to ischemia- or inflammation-induce d catecholaminergic neuron damage.