tert-Butoxyl radicals generate mainly 7,8-dihydro-8-oxoguanine in DNA.
Like hydroxyl radicals, alkoxyl radicals have been implicated in the generation of cellular oxidative DNA damage under physiological conditions; however, their genotoxic potential has not yet been established. We have analyzed the DNA damage induced by a photochemical source of tert-butoxyl radicals, the water soluble peroxy ester [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), using various repair endonucleases as probes. The irradiation (UV(360)) of BCBT in the presence of bacteriophage PM2 DNA was found to generate a DNA damage profile that consisted mostly of base modifications sensitive to the repair endonuclease Fpg protein. Approximately 90% of the modifications were identified as 7,8-dihydro-8-oxoguanine (8-oxoGua) residues by HPLC/ECD analysis. Oxidative pyrimidine modifications (sensitive to endonuclease III), sites of base loss (AP sites) and single-strand breaks were only minor modifications. Experiments with various scavengers and quenchers indicated that the DNA damage by BCBT+UV(360) was caused by tert-butoxyl radicals as the ultimate reactive species. The mutagenicity associated with the induced damage was analyzed in the gpt gene of plasmid pSV2gpt, which was exposed to BCBT+UV(360) and subsequently transfected into Escherichia 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 be characteristic for 8-oxoGua. In conclusion, alkoxyl radicals generated from BCBT+UV(360) induce 8-oxoGua in DNA with a higher selectivity than any other reactive oxygen species analyzed so far.
Mahler
H C
HC
Institute of Pharmacy, University of Mainz, Staudinger Weg 5, D-55099, Mainz, Germany.
Schulz
I
I
Adam
W
W
Grimm
G N
GN
Saha-Möller
C R
CR
Epe
B
B
D000438:Alcohols / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D019240:Corticoviridae / Q000235:genetics; D004249:DNA Damage; D004279:DNA, Viral / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010430:Pentosyltransferases; D010957:Plasmids / Q000235:genetics; D011506:Proteins; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D014466:Ultraviolet Rays
C115185:(4-(tert-butyldioxycarbonyl)benzyl)triethylammonium chloride; D000438:Alcohols; D001426:Bacterial Proteins; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D011506:Proteins; D000644:Quaternary Ammonium Compounds; C059688:alkoxyl radical; C024829:8-hydroxyguanine; D006147:Guanine; D010430:Pentosyltransferases; C071962:Gpt protein, E coli