The mode of action of cytotoxic and antitumor 1-nitroacridines. II. In vivo enzyme-mediated covalent binding of a 1-nitroacridine derivative, ledakrin or nitracrine, with DNA and other macromolecules of mammalian or bacterial cells

Abstract

Earlier evidence, in Part I of this paper, has shown that cytotoxic and antitumor 1-nitroacridines did not primarily exert their potent inhibitory effects on cultured mammalian cells by physicochemical binding with DNA, although it undoubtedly occurred (Chem.-Biol. Interact., 43 (1983) 131). As a result it was investigated (i) whether 9- 14C- or 1′- 14C-labeled derivatives of their representative, 1-nitro-9-/3′-dimethylamino- n-propylamino/acridine (Ledakrin or Nitracrine), were capable of covalent binding with nucleic acids and other suitable macromolecules in target cells in vivo and/or (ii) whether activation of the agent in the cell was a necessary prerequisite for such binding. Using the criteria of resistance to exhaustive extractions with trichloroacetic acid and/or organic solvents, [ 14C]Ledakrin was found to bind covalently, with relatively little discrimination, with: (i) intracellular macromolecules, including DNA, of cultured tumor HeLa cells (370–2500 DNA base pairs/one Ledakrin molecule; (ii) experimental animal tumor Ehrlich ascites (Eat) cells in vivo (650–5880 DNA base pairs/one Ledakrin molecule); (iii) bacterial Bacillus subtilis SB 1058 cells (7000–33 000 Ledakrin links/one cell genome); (iv) NADPH-fortified rat liver homogenates in vitro (25.6 nmol/mg microsomal protein under air). These results far exceed the common levels reported for alkylating agents or chemical carcinogens. Unlike [ ethyl- 14C]quinacrine, compared in vitro, covalent macromolecules binding with Ledakrin in vitro, and most probably in vivo, can be equated to NADPH-dependent activation(s) by oxidoreductase systems and the presence of DNA alone was not satisfactory in itself to attain Ledakrin binding. Fractionation of the enzymatic digest of 14C-associated DNA, isolated from Eat cells exposed in vivo to [9- 14C]Ledakrin, by Sephadex LH-20 column chromatography followed by mass spectrometry analyses of modified nucleosides, indicated that both mono- and dinucleosidical Ledakrin metabolites were the products of an in vivo reaction. This implied that the lethal reaction of the drug could be its cross-linking of the target macromolecules and/or its monofunctional attack on vitally important cellular components.