EFFECTS OF GLUTATHIONE ON ALKYLATION AND CROSS-LINKING OF DNA BY MITOMYCIN-C - ISOLATION OF A TERNARY GLUTATHIONE-MITOMYCIN-DNA ADDUCT

Mitomycin C (MC), a clinically used antitumor antibiotic, is known to alkylate DNA monofunctionally, and to generate DNA interstrand cross-l inks by bifunctional alkylation. Both processes are dependent on the r eductive activation of MC. Glutathione (GSH) was shown here to cause t hree types of changes in the pattern of alkylation of DNA by MC: (i) G SH caused a decrease of both the overall covalent binding ratio of MC to Micrococcus luteus DNA and the extent of interstrand cross-linking of P-32-pBR322 DNA, as the concentration of GSH was increased in the r eaction media. Approximately 50% inhibition of cross-linking was obser ved at 20 mM GSH. It is likely that the inhibition is caused by the fo rmation of MC-GSH conjugates competing with DNA alkylation, since both processes are triggered by reductive activation of MC [Sharma, M., an d Tomasz, M. (1994) Chem. Rest Toxicol. (preceding paper in this issue )]. (ii) GSH causes a switch from monofuctional to bifunctional activa tion of MC by the prototype ''monofunctional'' MC-activating agents H- 2/PtO2 and NADPH:cytochrome c reductase/NADPH. This was seen by the pr edominance,of bisadducts (i.e., cross-linked adducts) instead of the u sual monoadducts in the enzymatic digests of MC-DNA complexes formed i n the presence of GSH, as analyzed by HPLC. This finding suggests that GSH participates in the bifunctional activation of MC in vivo. (iii) A ternary MC-GSH-DNA adduct (6) was formed in the presence of GSH both with M. luteus DNA and with a synthetic duplex oligonucleotide; in th is adduct the mitosene C1 is linked to N-2 of guanine and the mitosene C10 is linked to GSH via sulfur. Adduct 6 was also formed from the DN A-bound MC monoadduct 2a. The overall inhibitory effect of GSH on alky lation and cross-linking of DNA by MC may be significant in drug resis tance of tumor cells possessing elevated levels of GSH.