ADDUCTS OF MITOMYCIN-C AND DNA IN EMT6 MOUSE MAMMARY-TUMOR CELLS - EFFECTS OF HYPOXIA AND DICUMAROL ON ADDUCT PATTERNS

6-CH3-H-3-Mitomycin C (MC) was used to identify MC-DNA adducts formed in EMT6 mouse mammary tumor cells. DNA was isolated from cells treated with H-3-MC. The DNA was enzymatically digested, and the digest was a nalyzed for H-3-labeled adducts by high performance liquid chromatogra phy. All four major adducts previously isolated and characterized in c ell-free systems were detected: two different monoadducts and two bisa dducts forming DNA-interstrand and DNA-intrastrand cross-links, respec tively. No MC-DNA adducts other than the DNA interstrand cross-link ha d been shown previously to be formed in living cells. A MC-deoxyguanos ine adduct of unknown structure was also detected in DNA from EMT6 cel ls; this adduct was also formed with purified EMT6 DNA. High performan ce liquid chromatography analysis was further applied to study the rel ationship between DNA adducts and cytotoxicity. The number of adducts increased with the concentration of MC in both aerobic and hypoxic cel ls. At a constant drug level, more adducts were observed in cells trea ted under hypoxic conditions than in cells treated aerobically; at 2 m uM MC, 4.8 x 10(-7) and 3.1 x 10(-7) adducts/nucleotide were observed under hypoxic and aerobic conditions, respectively. The increased addu ct frequency under hypoxia correlates with the known increased cytotox icity of MC to EMT6 cells under hypoxic conditions. In addition, a hig her ratio of cross-linked adducts to monoadducts was observed in hypox ic cells. The high performance liquid chromatography techniques were a lso used to examine the effects of dicumarol (DIC) on adduct patterns in cells treated simultaneously with H-3-MC. The MC-DNA adduct frequen cies in DIC-treated cells were increased 1.5-fold under hypoxia and de creased 1.6-fold under aerobic conditions from those observed without DIC. This finding correlates with the known DIC-induced increase and d ecrease in the cytotoxicity of MC in hypoxic and aerobic EMT6 cells, r espectively. The monoadduct resulting from monofunctionally activated MC was suppressed by DIC under both hypoxic and aerobic conditions. In addition, DIC induced the selective formation of an unknown DNA-assoc iated radiolabeled substance in hypoxic cells; this is hypothesized to be a cytotoxic DNA lesion produced by a DIC-stimulated oxido-reductas e. The methodology developed to measure MC adduct patterns may be usef ul as an indicator of distinct enzymatic activation processes for this drug.