Oxidative Conversion of 6-nitrocatecholamines to Nitrosating Products: a Possible Contributory Factor in Nitric Oxide and Catecholamine Neurotoxicity Associated 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 horseradish peroxidase (HRP)/H(2)O(2), lactoperoxidase (LPO)/H(2)O(2), Fe(2+)/H(2)O(2), Fe(2+)-EDTA/H(2)O(2) (Fenton reagent), HRP or Fe(2+)/EDTA in combination with D-glucose-glucose oxidase, or Fe(2+)/O(2), resulted in the smooth formation of yellowish-brown pigments positive to the Griess assay. In the case of 1, formation of the Griess positive pigment (GPP-1) promoted by HRP/H(2)O(2) proceeded through the intermediacy of two main dimeric species that could be isolated and identified as 3 and the isomer 4, featuring the 4-nitro-6,7-dihydroxyindole system linked to a unit of 1 through ether bonds. Spectroscopic (FAB-MS, (1)H NMR) and chemical analysis of GPP-1 indicated a mixture of oligomeric species related to 3 and 4 in which oxidative modification of the nitrocatechol 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 concentration- and pH-dependent nitrosation of 2,3-diaminonaphthalene, but very poor (up to 2%) nitration of 600 microM tyrosine. At pH 7.4, 1 exerted significant toxicity to PC12 cells, while GPP-1 proved virtually innocuous. By contrast, when assayed on Lactobacillus bulgaricus cells at pH 3.5, 1 was inactive whereas GGP-1 caused about 70% inhibition of cell growth. Overall, these results hint at novel pH-dependent mechanisms of nitrocatecholamine-induced cytotoxicity of possible relevance to ischemia- or inflammation-induced catecholaminergic neuron damage.