Isolation and Structure of a Covalent Cross-Link Adduct Between Mitomycin C and DNA

Abstract

A DNA cross-link adduct of the antitumor agent mitomycin C (MC) to DNA has been isolated and characterized; the results provide direct proof for bifunctional alkylation of DNA by MC. Exposure of MC to Micrococcus luteus DNA under reductive conditions and subsequent nuclease digestion yielded adducts formed between MC and deoxyguanosine residues. In addition to the two known monoadducts, a bisadduct was obtained. Reductive MC activation with Na2S2O4 (sodium dithionite) leads to exclusive bifunctional alkylation. The structure of the bisadduct was determined by spectroscopic methods that included proton magnetic resonance, differential Fourier transform infrared spectroscopy, and circular dichroism. Formation of the same bisadduct in vivo was demonstrated upon injection of rats with MC. Computer-generated models of the bisadduct that was incorporated into the center of the duplex B-DNA decamer d(CGTACGTACG)2 indicated that the bisadduct fit snugly into the minor groove with minimal distortion of DNA structure. A mechanistic analysis of the factors that govern monofunctional and bifunctional adduct formation is presented.

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

“…Typical substrates for repair by NER in other bacteria include intra- and interstrand cross-links, such as those generated by UV irradiation (37) and mitomycin C (38,39), respectively. Therefore, survival of the wild-type, uvrA mutant and complemented strains following exposure to these agents was assessed initially, and compared to that previously reported for a uvrB mutant (12).…”

Mechanism of differential sensitivity of human bladder cancer cells to mitomycin C and its analogue

“…MMC requires enzymatic bioactivation, and metabolic pathways for one-and two-electron reduction of MMC leading to the generation of cytotoxic species have been described (Iyer & Szybalski, 1963;Pan et al, 1984;Tomasz et al, 1987;Siegel et al, 1992). While many enzymes have been implicated in MMC bioactivation, NADPH cytochrome P450 reductase and DT diaphorase appear to be the key enzymes involved in its bioreductive activation (Keyes et al, 1984;Pan et al, 1984;Hoban et al, 1990;Gustafson & Pritsos, 1992;Siegel et al, 1992).…”

“…While many enzymes have been implicated in MMC bioactivation, NADPH cytochrome P450 reductase and DT diaphorase appear to be the key enzymes involved in its bioreductive activation (Keyes et al, 1984;Pan et al, 1984;Hoban et al, 1990;Gustafson & Pritsos, 1992;Siegel et al, 1992). Whereas the most abundant lesion produced by enzymatically activated MMC is the DNA monoadduct, MMC is believed to exert cytotoxic activity primarily through the formation of DNA-DNA cross-links (Long et al, 1984;Dorr et al, 1985;Tomasz et al, 1987). MMC-dependent oxyradicals have also been proposed to contribute to its cytotoxicity (Pan et al, 1984;Dusre et al, 1990).…”

Automatic scoring of sister chromatid exchanges by image analysis in a dose response experiment

“…To investigate the machine versus human dose-response, we measured with different observers the effect of Mitomycin C (MMC) on SCE frequency in human chromosomes in vitro. MMC was used because of its known mutagenic capacity, especially as an SCE inducer; it is a crosslinking agent, which acts as a bifunctional alkylating agent capable of inducing intrastrand and interstrand DNA crosslinks [Latt et aJ., 1975;Fujiwaraetal., 1977;Tomasz et al, 1987].…”