Repair and Expression of Aflatoxin B1-Induced DNA Damage
The chemical properties, cellular repair and biological expression of aflatoxin B1 (AFB1)-DNA adducts were investigated in mouse embryo fibroblasts 10T1/2, human epithelioid lung cells A549 and human skin fibroblasts (NF). These studies were complicated by the inherent chemical instability of AFB1-DNA adducts. In order to distinguish enzymatic reactions from spontaneous decomposition it was necessary to compare routinely the reactions occurring intracellularly to those of free AFB1-DNA in vitro and of AFB1-DNA in situ in excision repair deficient Xeroderma pigmentosum (XPA) fibroblasts. Following treatment of very actively metabolizing 10T1/2 cells with the procarcinogen AFB1 or of NF and A549 with microsome activated AFB1, approximately 90% of the adducts corresponded to 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-AFB1 (AFB1-N 7 -Gua). AFB1-DNA adducts were introduced preferentially in nucleosomal-linker relative to — core DNA both after cellular metabolic activation of AFB1 and exogenous activation by microsomes. For the determination of the nucleosomal distribution, the primary adducts, AFB1-N7-Gua, were transformed into the chemically more stable secondary products 2,3-dihydro-2(N5-formyl–2′,5′,6′-triamino-4′-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-triamino-Py) by a short exposure of the AFB1-treated cells to pH 9.5 medium.
Adduct removal was studied as a function of post-treatment incubation. The following conclusions were reached using the reactions of free AFB1-DNA in vitro and AFB1-DNA in XPA fibroblasts as reference: 1) AFB1-N7-Gua is removed spontaneously and enzymatically in 10T1/2, NF and A549 cells. 2) AFB1-triamino-Py is formed in a spontaneous secondary reaction from AFB1-N7-Gua and accumulates in the DNA. It represents the major persistent lesion at prolonged post-treatment incubation.
The major target for the cytotoxic effect of AFB1 in 10T1/2 cells was identified as DNA. In particular, confluent holding experiments showed that removal of AFB1-adducts lead to partial recovery of viability. The incompleteness of recovery is probably due to the accumulation of irrepairable AFB1-triamino-Py adducts in the DNA. The continuous decrease in the concentration of total AFB1 adducts during confluent holding was not reflected in a corresponding, continuous decrease in malignant transformation of 10T1/2 cells.The number of transformed foci per dish increased during the first 16 h of confluent holding and later decreased below the initial value by 40 h. These results exemplify the complex relationship between the efficiency of transformation, the changes in adduct concentration and the recovery of cell viability as a function of confluent holding.
KeywordsHigh Pressure Liquid Chromatography Colony Form Ability Mouse Embryo Fibroblast Cellular Repair Cellular Metabolic Activation
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