In vitro reactions of aflatoxin B₁-adducted DNA

The chemical stability of aflatoxin B₁ bound to calf thymus DNA was studied over a 48-hour exposure to phosphate buffers at pH 6.8-8.0 (37°C). During this time, aliquots of the aflatoxin B₁-modified DNA were acid-hydrolyzed and analyzed for the presence of r184w1.w2 =2,3-dihydroxy-2-(n⁵-formyl-2',5',6'-triamino-4'-oxo-n⁵-p yrimidyl-3-hydroxyaflatoxin r185w1 =2,3-dihydro-2-(8,9-dihydro-8-hydroxy-n⁷-guanyl)-3-hydroxy aflatoxin l atoxin B₁ and 2,3-dihydro-2-(8.9-dihydro-8-hydroxy-N⁷-guanyl)-3-hydroxyaflatoxin B₁. Initial experiments determined the stability of 2,3-dihydro-2(N⁷-guanyl)-3-hydroxyaflatoxin B₁ in DNA at levels of modification of one residue per 60 and 1500 nucleotides. The acid-hydrolysis products obtained from these modified nucleic acids were qualitatively similar, but their proportional concentrations were different. These quantitative differences were dependent upon both pH and the initial level of modification of the DNA. During the first 6 hr of incubation, under all conditions examined, the formation of 2,3-dihydro-2,3-dihydroxyaflatoxin B₁ was responsible for the initial decrease of the 2,3-dihydro-2-(N⁷-guanyl)-3-hydroxyaflatoxin B₁ adduct in DNA. After 48 hr of incubation at pH 7.0 the major reaction of the modified DNA was depurination of the 2,3-dihydro-2-(N⁷-guanyl)-3-hydroxyaflatoxin B₁ adduct. However, at pH 8.0, the predominant reaction product formed in 48hr was the putative 2,3-dihydro-2-(N⁵-formyl-2',5',6'-triamino-4'-oxo-N⁵-pyrimidyl)-3-hydroxy-aaflatoxin B₁. The putative DNA-bound products resulting from the elimination of the positive charge in the imidazole ring of the aflatoxin-N⁷-guanine adduct [2,3-dihydro-2-(N⁵-formyl-2',5',6'-triamino-4'-oxo-N⁵-pyrimidyl)-3-hydroxy-aflatoxin r206w1 =2,3-dihydro-2-(8,9-dihydro-8-hydroxy-N⁷-guanyl)-3-hydroxy aflatoxin B₁] B₁ and 2,3-dihydro-2-(8.9-dihydro-8-hydroxy-N⁷-guanyl)-3-hydroxyaflatoxinB₁] were found to be stable in DNA for at least 24 hr at both pH 6.8 and 7.4. Taken together with observed patterns of stability of aflatoxin B₁ adduct in vivo, these observations strongly suggest the involvement of enzymatic repair processes in removal of the N⁷-guanyl adduct and also emphasize the possible biological significance of the stable imidazole ring-opened adduct.