The mechanism of deoxyribonucleic acid breakage induced by 4'-(9-acridinylamino)methanesulfon-m-anisidine and copper: role for cuprous ion and oxygen free radicals

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Abstract
4''-(9-Acridinylamino)methanesulfon-m-anisidide (mAMSA) which has antitumor activity interacts with Cu(II), as indicated by changes in the mAMSA absorption spectrum induced by Cu(II). The spectral changes are due to the oxidation of mAMSA by Cu(II), resulting in an oxidized mAMSA product and Cu(I). Two lines of evidence for the oxidation of mAMSA are as follows: the spectral changes induced by manganese oxide, an oxidizing agent, were similar to those induced by Cu(II), and the Cu(II)-induced spectral changes were reversed by a reducing agent, NADPH. TLC studies showed the oxidized mAMSA product to be N1-methylsulfonyl-N4-(9-acridinyl)-3-methoxy-2,5-cyclohexadiene-1,4-diimine (mAQDI). The involvement of Cu(I) in the reaction was demonstrated by the use of 2 Cu(I)-specific chelating agents, neocuproine and bathocuproine. Neocuproine or bathocuproine chelated the Cu(I) ions in the mixture, producing Cu(neocuproine)2+ complex or Cu(bathocuproine)2+ complex. The stoichiometry of mAMSA-Cu(II) interactions was determined by titrating the mAMSA-Cu(II) mixtures with bathocuproine. Job plots of the absorbance at 480 nm showed a clear end point at a Cu(II)/mAMSA ratio of 1.5/l, indicating that 1.5 equivalent ofCu(II) reacts with 1 equivalent of mAMSA to produce 1.5 equivalent of Cu(I). Cu(I) plays an important role in the mAMSA-Cu(II)-induced DNA breakage, since in the presence of neocuproine the DNA breakage is inhibited. Up to 200 .mu.M, Cu(I) by itself is virtually ineffective, in contrast to the mixture of mAMSA and Cu(II). mAMSA, aside from reducing Cu(II) to Cu(I), may play a role in mediating DNA breakage. The DNA breakage was reduced in partially anaerobic conditions, indicating the involvement of molecular O2. Catalase and 4,5-dihydroxy-1,2-benzenedisulfonate inhibited the DNA breakage completely, indicating that hydrogen peroxide and superoxide radicals mediate DNA breakage. 1,3-Diazabicyclo[2.2.2]octane partially inhibited the breakage, suggesting that singlet O2 is involved. DNA breakage was not inhibited by potassium iodide or mannitol, indicating that hydroxyl radicals are not involved. The available evidence supports a mechanism indicating the formation of a DNA.cntdot.mAMSA.cntdot.Cu(II) ternary complex and the subsequent oxidation-reduction of the complex to form DNA.cntdot.mAQDI.cntdot.Cu(I). The oxidation of the complexed Cu(I) may result in reduction of O2 to O2 free radicals which induce DNA breaks.