The six
DNA adducts formed in EMT6 mouse mammary
tumor cells upon treatment with
mitomycin C (MC) fall into two groups: (1) four
guanine adducts of MC and (2) two
guanine adducts derived from
2,7-diaminomitosene (2,7-DAM), the major reductive metabolite of MC. The two groups of adducts were proposed to originate from two pathways arising from reductive activation of MC: (a) direct alkylation of
DNA and (b) formation of 2,7-DAM, which then alkylates
DNA. The aim of this study was to test the validity of this proposal and to evaluate the significance of alkylation of
DNA by 2,7-DAM. Treatment of the cells with 2,7-DAM itself yielded the same 2,7-DAM-guanine adducts as treatment with MC; however, 2,7-DAM was approximately 100-fold less cytotoxic than MC. The uptake and efflux of 2,7-DAM by EMT6 cells was comparable to that of MC, but 2,7-DAM alkylated
DNA with higher efficiency than MC. These results validate the two proposed pathways and show that formation of 2,7-DAM-DNA adducts in MC-treated cells represents a relatively non-toxic pathway of reductive metabolism of MC. A selective stimulatory effect of
dicumarol (
DIC) on 2,7-DAM-DNA adduct formation in EMT6 cells treated with MC was also investigated.
DIC had no effect on alkylation by MC in cell-free systems, nor did it have significant effects on adduct formation or cell survival for cells treated with 2,7-DAM. It is proposed that in the cell
DIC stimulates a
reductase enzyme located at subcellular sites where the activated MC species has no direct access to
DNA and therefore is diverted into the non-cytotoxic pathway, which leads to the formation of 2,7-DAM and its adducts.