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.

Mitomycin C, an antitumor antibiotic, is rapidly metabolized in the presence of rat liver microsomes. NADPH and anaerobic conditions are required for the process. The products isolated after reexposure to air are 2,7-diaminomitosene derivatives. Specifically, in the presence of inorganic phosphate, 1,2-cis- and -trans-2,7-diaminomitosene 1-phosphates, 1,2-cis- and -trans-2,7-diamino-1-hydroxymitosenes, and 2,7-diaminomitosene are formed. The last substance is a new mitomycin C derivative, and proof for its structure is presented. Mytomycin C has been previously postulated to be an alklating agent requiring reduction for activity (Iyer, V. N., & Szybalski, W. (1964) Science (Washington, D.C.) 145, 55]. The 1-phosphates above represent the first chemically characterized bioreductive alkylation products of the drug. 5'-Uridylic acid is alkylated analogously under these conditions, to give cis- and trans-2,7-diaminomitosene 1-(5'-uridylate), while the phosphodiester UpU and uridine itself are inert. Hydrogen gas/PtO2 gives the same results as microsomes/NADPH. The formation of the observed compounds indicates that enzymatic (or chemical) reduction of the quinone system of mitomycin C induces ring opening of the aziridine function, generating a reactive center at the C1 position as previously postulated by others (ibid.). The second alkylating center, also postulated, is not evident, however, under the conditions tested, indicating that the aziridine is the primary bioreductive alkylation function of mitomycin C. Identification of the products and mechanism of the microsomal anaerobic metabolism of mitomycin C are significant in view of the reported toxicity of the drug to anaerobic cancer cells.

The antitumor antibiotic mitomycin C is shown to form a covalent complex with calf thymus DNA under anaerobic conditions in the presence of either NADPH cytochrome c reductase/NADPH, xanthine oxidase/NADH, or the chemical reducing system H2/PtO2. Digestion of the complex with DNase I/snake venom diesterase/alkaline phosphatase yields a single mitomycin deoxyguanosine adduct as the major DNA alkylation product, identified as N2-(2",3,7"-diaminomitosen-l"a-yl) 2'-deoxyguanosine (Structure 2). Two minor adducts, 2-5% each of the total adduct pool, are isolated and identified as the 1"j8 stereoisomer of 2 (Structure 3), and 10"-decarbamoyl-2 (Structure 7). The same results were obtained with M13 DNA and poly(dG-dC) poly(dG-dC); however, in the latter case, a minor adduct apparently possessing two deoxyguanosine and one mitomycin unit is isolated. Digestion of the covalent mitomycin-calf thymus DNA complex with nuclease P1 yields four dinucleotide adducts, all of which consist of 2 linked at its 3' end to each of the four possible 5' nucleotides (A, T, G, and C). Upon treatment of each dinucleotide adduct with snake venom diesterase/alkaline phosphatase, 2 is released along with the corresponding free nucleoside. In apparent conflict with the present results, previous reports from another laboratory have indicated that modification of calf thymus DNA by mitomycin C under conditions identical to those described here result in the isolation of three mitomycin C mononucleotide adducts possessing linkages of the drug to N2 and 06 of guanine and N6 of adenine. Evidence is shown suggesting that the latter adducts are actually three of the above four dinucleotide derivatives of 2 obtained independently by us and, thus, all of them in fact possess an identical N2-mitosenylguanine adduct moiety. Model-building studies indicate an excellent fit of the guanine N2-linked drug molecule inside the minor groove of B-DNA with no appreciable distortion of the DNA structure.Mitomycin C (MC; Structure 1), a potent antibiotic and clinically used antitumor agent, is known to interact covalently with DNA in vivo and in vitro. This is manifested by the reversible melting behavior of MC-exposed DNA, attributed to formation of covalent crosslinks between the complementary strands (1) and by covalent association of the ultraviolet chromophore of MC to DNA (2). These processes require reduction of MC into a transiently activated form, which is thought to occur in cells and can be mimicked easily in vitro chemically or enzymatically (1). The cytotoxicity of MC is most likely a direct result of DNA alkylation, as indicated by the parallels in biological activity of MC with a number of known "DNA damaging agents": selective inhibition of DNA replication (1) and sister chromatid exchange (4), and cross-resistance or cross-hypersensitivity of bacterial (1, 5) and mammalian (6) cells to UV light and MC. The molecular nature of the covalent interactions between MC and DNA has remained elusive, mainly because of the difficulty of isolating low...

Synthetic oligodeoxyribonucleotides were reacted with mitomycin C (MC) under conditions which restricted MC to monofunctional alkylating activity. The yields of monofunctional alkylation of oligonucleotides with variable sequence were determined by enzymatic digestion of the reaction mixture to unreacted nucleosides and the product of alkylation, a MC-deoxyguanosine adduct (2), followed by quantitative analysis by HPLC. The relative yields of 2 reflected relative monoalkylation reactivities. They were compared in a series of oligonucleotides having the sequence 5'-NGN' in which the 5'-base was varied while the 3'-base was kept constant as T. Under Na2S2O4 activation conditions a striking enhancement of the yield was observed at the 5'-CG sequence: 36%, compared to 2% at 5'-AG and 4.1% at 5'-TG. The 5'-GG sequence also showed enhanced reactivity although to a lesser extent (14.7%). The enhancements were specific to the duplex state of the oligonucleotides. Using NADPH:cytochrome c reductase as the reducing agent gave similar results. MC activated by acidic pH also displayed 5'-CG alkylation specificity. 10-Decarbamoyl-MC activated by Na2S2O4 showed the same 5'-CG specificity as MC. Replacement of deoxyguanosine by deoxyinosine in the opposite strand at a 5'-CG site abolished the enhancement of alkylation. Such replacement at a 5'-GG site had a similar effect. It was found that the base 3' to the guanine had only a relatively modest modulating effect on the enhanced reactivity of the G at the 5'-CG sequence. This 3'-base effect appeared to be independent of the 5'-base of the 5'-NGN' triplet. The order of reactivity is 3'-(C greater than T greater than A).(ABSTRACT TRUNCATED AT 250 WORDS)