Formation of a major DNA adduct of the mitomycin metabolite 2,7-diaminomitosene in EMT6 mouse mammary tumor cells treated with mitomycin C

Treatment of EMT6 mouse mammary tumor cells with [H-3]mitomycin C (MC) resu lts in the formation of six major DNA adducts, as described earlier using a n HPLC assay of H-3-labeled products of enzymatic hydrolysis of DNA isolate d from MC-treated cells. Four of these adducts were identified as monofunct ional and bifunctional guanine-N-2 adducts in the minor groove of DNA. In o rder to establish relationships between individual types of MC-DNA adducts and biological responses it is necessary to identify all of the adducts for med in cells. To this end we have now identified a predominant, previously unknown adduct formed in MC-treated EMT6 cells as a derivative not of MC, b ut of 2,7-diaminomitosene (2,7-DAM), the major bioreductive metabolite of M C. Rigorous proof demonstrates that it is a DNA major groove, guanine-N7 ad duct of 2,7-DAM, linked at C-IO Co DNA. The adduct is relatively stable at ambient temperature, but is readily depurinated upon heating. Its isolation from MC-treated cells indicates that MC is reductively metabolized to 2,7- DAM, which then undergoes further reductive activation to alkylate DNA, alo ng with the parent MC. Low MC:DNP. ratios were identified as a critical fac tor promoting 2,7-DAM adduct formation in an in vitro model calf thymus DNA /MC/reductase model system, as well as in MC-treated EMT6 cells. The 2,7-DA M-guanine-N7 DNA adduct appears to be relatively noncytotoxic, as indicated by the dramatically lower cytotoxicity of 2,7-DAM in comparison with MC in EMT6 cells. Like MC, 2,7-DAM exhibited slightly greater cytotoxicity to ce lls treated under hypoxic as compared to aerobic conditions. However, 2,7-D AM was markedly less cytotoxic than MC under bath aerobic and hypoxic condi tions. Thus: metabolic reduction of MC to 2,7-DAM represents a detoxificati on process. The differential effects of MC-DNA and 2,7-DAM-DNA. adducts sup port the concept that specific structural features of the DNA damage may pl ay a critical role in the cytotoxic response to a DNA-targeted chemotherape utic agent.