Free radicals and excited species in metabolic disorders and bioluminescence

Abstract

In the past three decades, remarkable advances were achieved in the comprehension of chemical mechanisms underlying the biochemistry and biology of chemiluminescence, bioluminescence and redox balance. The biological sources, molecular targets and fates of free radicaIs, peroxides, electronically excited molecules and other highly oxidizing species reactive in these processes were identified. In this context, our efforts centered on chemical and biochemical study of metabolites accumulated in certain metabolic disorders endowed with the ability to generate reactive oxygen species, particularly the superoxide anion radical, hydroxyl radical, and triplet ketones, when reacting with molecular oxygen both in the presence and absence of transition metal complexes. Arnong these metabolites, we have studied: (i) 6-hydroxydoparnine (A), a neurotoxin putatively important in schizophrenia and manic-depression; (ii) 5-arninolevulinic acid (B), a heme precursor hypothesized to behave as a pro-oxidant when accumulated in intermittent acute porphyria, hereditary tyrosinemia type I and lead poisoning; (iii) aminoacetone (C), an intermediate of threonine and glycine metabolism supposedly overproduced in threoninemia, cri-du-chat syndrome, and diabetes mellitus; (iv) succinylacetone (D), a tyrosine catabolite accumulated and excreted in hereditary tyrosinemia type I, where it acts as a strong inhibitor of 5-aminolevulinic acid dehydratase; and (v) acid 2-methylacetoacetic (E), derived from isoleucine and associated to ketoacidosis triggered by a metabolic defect of this amido acid. Reaction of metabolites A-C with molecular oxygen was found to yield oxygen radicals and hydrogen peroxide whereas D and E were shown to be chemi1uminescent due to the generation of triplet carbonyls. Interesting1y, metabolites (B-E) render a-dicarbonyls as final products (e.g., methylg1yoxal, 4,5-dioxovaIeric acid, biacetyl), a class of compounds predicted in the thirties by Szent-Gyorgyi to act as endogenous toxicants and, indeed, proved to be highly cytotoxic and genotoxic in the recent decades. Our plans encompass (i) to undertake a detailed mechanistic study of these reactions by oxyrnetry, EPR, photon-counting, and capilIary electrophoresis; (ii) to clarify the role of "free" iron/ferritin as cataIysis of these reactions; (iii) to examine the role of peroxynitrite, a toxicant but also a signaling species,and of cytochrome c, a peroxidase and apoptosis inducer, as initiators of the aerobic oxidation of compounds B-E; (iv) to study B-E-driven oxidative damage on macromolecules, supramolecular structures and organelles such as DNA, liposomes, mitochondria and synaptosomes; (v) to design and prepare triplet ketone quenchers to approach ultra-weak chemiluminescent biological systems; and (vi) to investigate the possible formation of fluorescent adducts of B-E reaction products with isolated model proteins and proteins of biological structures such as endothelium. In paralell, our work on beetle bioluminescence will continue, but emphasizing from now on its aspects related to the redox balance. The trehalose/trehalase system of larval Pyrearinus termitilluminans (Coleoptera: Elateridae) was chosen as the object of our attention because (i) trelaIose constitutes the principal carbohydrate in insects where acts as an antioxidant; (ii) strong evidence favor the notion that bioluminescence plays an auxiliary role in oxygen detoxification; and (iii) during the Winter, P. termitilluminans larvae experience a condition of water stress when membrane-associated threalose is expected to protect the cell integrity. We believe that the research proposal presented here can result in interesting and relevant information for the area of redox balance and provide foundation for an adequate scientific and technical formation of undergraduate and graduate students. (AU)