Relative Toxicities of DNA Cross-Links and Monoadducts:  New Insights from Studies of Decarbamoyl Mitomycin C and Mitomycin C

Abstract

Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other than a selective defect in cross-link repair.

Strong Protein Adduct Trapping Accompanies Abolition of Acrolein-Mediated Hepatotoxicity by Hydralazine in Mice

“…A similar mechanism of toxic action involving a central role for cross-linking has been demonstrated for other xenobiotics with bifunctional reactivity (Palom et al, 2002). Evaluation of this possibility would require the development of new analytical tools to detect acrolein-linked macromolecules in biological systems, perhaps via similar approaches to those used to identify 4-hydroxynonenal-linked lysine residues in adducted proteins (Tsai et al, 1998;Xu et al, 2000).…”

γ-H2AX formation in response to interstrand crosslinks requires XPF in human cells

“…Unlike psoralens, mitomycin C forms both monoadducts and ICL in DNA at purine bases in the absence of UV irradiation, and ICL typically constitute a much lower percentage of the total adducts than can be achieved with psoralens and UVA [1,46]. As seen with HMT and UVA treatment, XP-F cells exhibited significantly lower γ-H2AX levels following mityomycin C treatment than GM637 cells (Fig.…”

Characterization of the Staphylococcus aureus Heat Shock, Cold Shock, Stringent, and SOS Responses and Their Effects on Log-Phase mRNA Turnover

“…Mitomycin C is an antimicrobial and anticancer agent that causes DNA intra-and interstrand cross-linking as well as monofunctional alkyl lesions and is a potent inducer of the bacterial SOS regulon (41). To define the optimal conditions for induction of the SOS regulon, log-phase UAMS-1 cells were treated with 0.5, 1.0, 2.5, or 5 g ml Ϫ1 mitomycin C for 30 min.…”