Peroxynitrite-induced DNA damage in the supF gene: correlation with the mutational spectrum

Tissue inflammation and chronic infection lead to the overproduction of nit ric oxide and superoxide. These two species rapidly combine to yield peroxy nitrite (ONOO-), a powerful oxidizing and nitrating agent that is thought b e involved in both cell death and an increased cancer risk observed for inf lamed tissues. ONOO- has been shown to induce single-strand breaks and base damage in DNA and is mutagenic in the supF gene, inducing primarily G to T transversions clustered at the 5' end of the gene. The mutagenicity of ONO O- is believed to result from chemical modifications at guanine nucleobases leading to miscoding DNA lesions. In the present work, we applied a combin ation of molecular and analytical techniques in an attempt to identify biol ogically important DNA modifications induced by ONOO-. pUC19 plasmid treate d with ONOO- contained single-strand breaks resulting from direct sugar dam age at the DNA backbone, as well as abasic sites and nucleobase modificatio ns repaired by Fpg glycosylase. The presence of carbon dioxide in the react ion mixture shifted the ONOO- reactivity towards reactions at nucleobases, while suppressing the oxidation of deoxyribose. To further study the chemis try of the ONOO- interactions with DNA, synthetic oligonucleotides represen ting the mutation-prone region of the supF gene were treated with ONOO-, an d the products were analyzed by liquid chromatography-negative ion electros pray ionization mass spectrometry (LC-ESI- MS) and tandem mass spectrometry . 8-Nitroguanine (8-nitro-G) was formed in ONOO--treated oligonucleotides i n a dose-dependent manner with a maximum at a ratio of [ONOO-]: [DNA] = 10 and a decline at higher ONOO- concentrations, suggesting further reactions of 8-nitro-G with ONOO-. 8-Nitro-G was spontaneously released from oligonuc leotides (t(1/2) = 1 h at 37 degrees C) and, when present in DNA, was not r ecognized by Fpg glycosylase. To obtain more detailed information on ONOO-- induced DNA damage, a restriction fragment from the pSP189 plasmid containi ng the supF gene (135 base pairs) was [P-32]-end-labeled and treated with O NOO-. PAGE analysis of the products revealed sequence-specific lesions at g uanine nucleobases, including the sites of mutational "hotspots." These les ions were repaired by Fpg glycosylase and cleaved by hot piperidine treatme nt, but they were resistant to depurination at 90 degrees C. Since 8-nitro- G is subject to spontaneous depurination, and 8-oxo-guanine is not efficien tly cleaved by piperidine, these results suggest that alternative DNA lesio n(s) contribute to ONOO- mutagenicity. Further investigation of the identit ies of DNA modifications responsible for the adverse biological effects of ONOO- is underway in our laboratory. (C) 2000 Elsevier Science B.V. All rig hts reserved.