In vitro reactions of aflatoxin B1-adducted DNA.

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Abstract
The chemical stability of aflatoxin B1 bound to calf thymus DNA was studied over a 48-h exposure to phosphate buffers at pH 6.8-8.0 (37.degree. C). Aliquots of the aflatoxin B1-modified DNA were acid-hydrolyzed and analyzed for the presence of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyflatoxin B1, 2,3-dihydro-2,3-dihydroxy-aflatoxin B1, and the tentatively identified, 2,3-dihydro-2-(N5-formyl)-2'',5'',6''-triamino-4''-oxo-N5-pyrimidyl-3-hydroxyflatoxin 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 1 residue/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 pH and the initial level of modification of the DNA. During the first 6 h 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 h of incubation at pH 7.0, the major reaction of the modified DNA was depurination of the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyflatoxin B1 adduct. At pH 8.0, the predominant reaction product formed in 48 h was the putative 2,3-dihydro-2-(N5-formyl-2'',5'',6''-triamino-4''-oxo-N5-pyrimidyl)-3-hydroxy-aflatoxin 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-hydroxyaflatoxin B1 and 2,3-dihydro-2-(8,9-dihydro-8-hydroxy-N7-guanyl)-3-hydroxyaflatoxin B1] were stable in DNA for at least 24 h at pH 6.8 and 7.4. Taken together with observed patterns of stability of aflatoxin B1 adducts in vivo, these observations strongly suggest the involvement of enzymatic repair processes in removal of the N7-guanyl adduct and emphasize the possible biological significance of the stable imidazole ring-opened adduct. [Aflatoxin B1 (AFB1) is toxic, hepatocarcinogenic and mutagenic in a wide range of organisms.].