Generation of Acetyl Radicals by Diacetyl/Peroxynitrite
Fujiy, D.E.1; Massari, J.1; Ito, M.L.1; Vaz, S.M.1; Micke, G.A.2; Costa, A.C.O.2; Dutra, F.3; Bechara, E.J.H.1
1Departamento de Bioquímica, IQ-USP, SP; 2Departamento de Química Fundamental, IQ-USP, SP; 3Centro de Ciências Biológicas e da Saúde, UNICSUL, SP
Diacetyl as well as other a-dicarbonyl compounds, such as methylglyoxal and 4,5-dioxovaleric acid, are reportedly cytotoxic and genotoxic. Diacetyl is a food and cigarette contaminant that has also been associated with alcohol hepatotoxicity. In turn, peroxynitrite is a putative biological oxidant and carbonyl nucleophile which is formed by the diffusion-controlled reaction of the radical anion superoxide with nitric oxide (k2= 2 x 1010 M-1 s-1; pKa(ONOOH) = 6.8). We propose here that at nitrosative and carbonyl stress conditions peroxynitrite could add to a-dicarbonyls yielding reactive acyl radicals, capable to attach to proteins and DNA bases, triggering deleterious responses. Using diacetyl as an a-dicarbonyl model, we envisage a phosphate-catalyzed nucleophilic addition of peroxynitrite to diacetyl, followed by homolysis of the O-O bond of the diacetyl/peroxynitrite adduct yielding an oxyl radical. Subsequently, the oxyl radical would undergo b-cleavage of the C-CO bond producing acetate ion and acetyl radical.
Indeed, experiments monitored by UV/stopped-flow spectrophotometry showed that peroxynitrite reacts with diacetyl much faster (k2 = 1.4 x 105 M-1 s-1) than monocarbonyl substracts (k2 < 103 M-1 s-1), with a pH profile peaking at roughly 7.2. Polarographic experiments (oximetry) revealed consumption of O2 by the reaction at rates depending on the concentration of both peroxynitrite (mM range) and diacetyl (mM range). Formation of acetyl radical intermediate by the peroxynitrite/diacetyl system was attested to by EPR spin-trapping with MNP (triplet, aN = 8.3). Using HPLC/MS, stoichiometric amounts of acetate was found in the spent reaction mixture and, in the presence of deoxyguanosine, acetyl-deoxyguanosine was also detected.
These studies provide evidence that the peroxynitrite/diacetyl system yields acetyl radical intermediate and support our hypothesis that DNA radical acetylation may occur in vivo and be implicated in aging and metabolic errors where oxygen and nitrogen reactive species are possibly implicated.
Support: FAPESP, CNPq, PRONEX, Milênio Redoxoma.
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