Effect of Formation and Removal of Aflatoxin B1: DNA Adducts in IOTY2 Mouse Embryo Fibroblasts on Cell Viability

Confluent cultures of mouse embryo fibroblasts 10TY2 were shown to metabolize aflatoxin B1 (AFB1) to its ultimate form and to produce covalent AFBiiDNA adducts. After 16 hr at 0.2 fiM AFB1, the DNA adduct concentration was 7/imol adducts per mol DNA phosphate. The spectrum of specific AFBirDNA adducts in 10TY2 cells was qualitatively identical to that of other metabolically active mammalian tissues which had been studied previously. High-pressure liquid chromatography showed that acid hydrolysates of DNA extracted from 10T½ cells immediately after AFB1 treatment contained 66 to 80% 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1t 8 to 13% putative 2,3-di-hydro-2-(A/5-formyl-2,5#,6‘-triamino-4/-oxo-A/5-pyrimidyl)-3- hydroxyaflatoxin B1 (AFBi-triamino-Py), and other structurally unidentified minor components. Within 16 hr of posttreatment incubation of confluent 10T½ cultures, 50 to 60% of the AFB1 adducts disappeared from high-molecular-weight DNA. The extent and kinetics of adduct removal were similar for DNA in intact cells and DNA extracted from the cells immediately after AFB1 treatment and incubated under physiological conditions in vitro. Similar kinetics for AFB1-DNA in situ in the cell and free AFB1-DNA was also observed for the modification of the primary AFBi adducts to chemically more stable secondary lesions. The secondary lesions accumulated in the DNA and mostly gave rise to the putative AFB1-triamino-Py upon acid hydrolysis. It is concluded that the reactions of the AFB1 adducts in 10T1/2 cells are mostly spontaneous rather than enzyme catalyzed. A linear relationship was observed between the log of the colony-forming ability of 10T½ cells and the initial concentration of AFB1:DNA adducts. An initial adduct concentration of 1.9 jumol per ml DNA phosphate effects a decrease of the colony-forming ability to 37%. Holding the AFB1-treated 10½ cultures in confluency before replating at low cell densities for the determination of the colony-forming ability resulted in substantial but incomplete recovery of viability. The recovery of cell viability is attributed to the spontaneous removal of AFB1: DNA adducts, i.e., to “chemical repair.” The incompleteness of the recovery is attributed to the accumulation of a sizable fraction of persistent irrepairable AFB1 adducts, in particular of adducts which are released in the form of AFB1-triamino-Py upon acid hvdrolvsis.