tert-Butoxyl radicals generate mainly 7,8-dihydro-8-oxoguanine in DNA

Like hydroxyl radicals, alkoxyl radicals have been implicated in the genera tion of cellular oxidative DNA damage under physiological conditions; howev er, their genotoxic potential has not yet been established. We have analyze d the DNA damage induced by a photochemical source of tert-butoxyl radicals , the water soluble peroxy ester [4-(tert-butyldioxycarbonyl)-benzyl]trieth ylammonium chloride (BCBT), using various repair endonucleases as probes. T he irradiation (UV360) of BCBT in the presence of bacteriophage PM2 DNA was found to generate a DNA damage profile that consisted mostly of base modif ications sensitive to the repair endonuclease Fpg protein. Approximately 90 % of the modifications were identified as 7.8-dihydro-8-oxoguanine (8-oxoGu a) residues by HPLC/ECD analysis. Oxidative pyrimidine modifications (sensi tive to endonuclease III), sites of base loss (AP sites) and single-strand breaks were only minor modifications. Experiments with various scavengers a nd quenchers indicated that the DNA damage by BCBT + UV360 was caused by te rt-butoxyl radicals as the ultimate reactive species. The mutagenicity asso ciated with the induced damage was analyzed in the gpt gene of plasmid pSV2 gpt, which was exposed to BCBT + UV360 and subsequently transfected into Es cherichia coli. The results were in agreement with the specific generation of 8-oxoGua. Nearly all point mutations (20 out of 21) were found to be GC --> TA transversions known to he characteristic for 8-oxoGua. In conclusion , alkoxyl radicals generated from BCBT + UV360 induce 8-oxoGua in DNA with a higher selectivity than any other reactive oxygen species analyzed so far . (C) 2001 Elsevier Science B.V. All rights reserved.