Nucleic Acids Research

Thiol-activated neocarzinostatin chromophore abstracts tritium from the 5', but not from the 1' or 2' positions of deoxyribose in DNA and incorporates it into a stable, non-exchangeable form. The abstracted tritium remains covalently associated with the chromophore or its degradation product after treatment with acid or alkali, respectively. Drug activation and the consequent hydrogen abstraction reaction, presumably generating a carbon-centered radical at C-5', do not require molecular oxygen but have a dose-dependent relation with thiol. Under aerobic conditions, where base release and DNA strand breaks with nucleoside 5'-aldehyde at the 5'-ends are produced, hydrogen abstraction from C-5' parallels these parameters of DNA damage. It is possible to formulate a reaction scheme in which the carboncentered radical at C-5' is an intermediate in the fonnation of the various DNA damage products found under both aerobic and anaerobic conditions. INTRODUCTION The precise roles of thiol and 02, the two cofactors required (1) in DNA strand breakage by the nonprotein chromophore of the antitumor antibiotic neocarzinostatin (NCS) (2-5) remain incompletely understood. DNA strand breakage occurs only if all the reaction components are simultaneously present, indicative of the extreme lability of the intermediates involved. DNA damage is manifested mainly by the production of single strand breaks which bear a nucleoside 5'-aldehyde at their 5'-termini (6,7), alkali-labile breaks (7,8) and the release of free base (9,10). Labile DNA-NCS chromophore adducts with covalent linkage to the DNA sugar (11,12) appear as minor products and have properties consistent with their being precursors of the strand breaks in DNA. Oxidation at the C-5' of nucleosides in DNA is involved both in the production of the 5'-aldehyde (7) and the labile adducts (13). On the other hand, in the absence of 02 a stable DNA-NCS chromophore adduct is formed (12). Nitroaromatic radiation sensitizers such as misonidazole can substitute for 02 in DNA strand cleavage, but a strand gap is produced with phosphate at both ends (14). While there is ample evidence that thiol is involved in drug activation it © I RL Press Limited, Oxford, England. 1 637 Nucleic Acids Research is not known whether 02 is involved in the generation of an active species of the drug that attacks DNA, in addition to its role in "fixing" the nascent DNA lesion. It has been proposed that thiol addition to the chromophore (15) generates a reactive species, possibly a free radical, that abstracts a hydrogen from the 5'-carbon of deoxyribose in DNA to form a carbon-centered radical that in the presence of 02 undergoes subsequent reactions leading to 5'-aldehyde formation and a strand break (6,16). Support comes from the recent findings that 1) in an aerobic reaction in which DNA strand breaks are produced, thiolactivated NCS chromophore abstracts a 5'-hydrogen from 5'-[3HJthymidine-labeled poly(dA-dT). poly(dA-dT) and incorporates it in a non-exchangeable form (17), 2) dioxygen is the source of the oxygen incorporated into thymidine 5'-aldehyde at the DNA strand break (18), and 3) kinetic studies show that in the DNA-degrading reaction, reaction of the chromophore with thiol precedes the reaction with 02 (16). The experiments presented in this paper show that oxygen is not required in the activation of the drug for the hydrogen abstraction reaction and suggest a reaction scheme, involving the intermediacy of a carbon-centered radical at C-5', that is consistent with the various reaction products formed under both aerobic and anaerobic conditions. MATERIALS AND METHODS NCS (clinical form) kindly supplied by Dr. W.T. Bradner of the BristolMyers Company was dialyzed against distilled H20 and lyophilized after the addition of sodium citrate, pH 4.0 required to give a final level of 0.02 M prior to extraction of the chromophore with 100% methanol. The chromophore solution (0.3-0.4 mM) was stored in the dark at -70°. x DNA with radiolabels at various positions (5'E3H]; [5-methyl-3H]; 5methyl, 1',2'[3H]; [U-14C]) of thymidine was prepared as previously described (7). The specific activities of the DNAs, cpm per pg, are: [5-methyl-3H], 16540; 5'E3H], 33172; [U-14C], 990; 5-methyl, 1',2'r3H], 5434 of which 40% radioactivity is equally distributed in the 1' and 2' positions. Misonidazole was from Hoffmann-La Roche. Drug Treatment The standard reaction (0.3-0.75 ml) contained 10 nM sodium citrate, pH 4.0, 1 mM diethylenetriaminepentaacetic acid, 100 mM Tris-HCl, pH 9.0, 1 mM glutathione, and DNA and NCS chromophore at levels indicated in the legends. The anaerobic reactions were performed in micro Warburg vessels. The components of the reaction were split such that the chromophore was bound to DNA at the acidic pH and following removal of 02 from the system thiol and pH 9.0