Mechanisms of the reactions of some copper complexes in the presence of DNA with superoxide, hydrogen peroxide, and molecular oxygen.

TLDR: It is found that both copper(I1) and copper(1) complexes bind to DNA, and it is believed that the cuprous complex intercalates with DNA and that the subsequent oxidation by hydrogen peroxide causes the damage due to the formation of OH.

Abstract: The kinetics and the reaction mechanism of some copper complexes of l,IO-phenanthroline, 5-nitrc1, IO-phenanthroline, and 2,2’-bipyridine with 02-, Hz02, and O2 in the presence of calf thymus DNA have been investigated with use of the pulse radiolysis technique. We have found that both copper(I1) and copper(1) complexes bind to DNA. The ternary complexes react very slowly with 02relative to the free complexes, while the rates of the oxidation of free and bound cuprous complexes by H202 are almost the same. Therefore these ternary copper complexes turned out to be good catalysts of the reaction between 02and Hz02. A complex between the chelating agent 1,lO-phenanthroline (OP) and copper(I1) is able to induce the degradation of DNA in the presence of a reducing agent.’-’ N o primary sequence specificity is apparent in the scission reaction* which proceeds under a variety of experimental conditions. These include incubtation of DNA, OP, and copper(I1) ions with the following: (a) reducing agents such as thiol or ascorbate in the presence of molecular oxygen;’” (b) systems generating the superoxide radical in the presence of molecular o ~ y g e n ; ~ . ~ (c) NADH and hydrogen pero~ide;~.’ (d) reducing agents and hydrogen p e r o ~ i d e . ~ The degradation of DNA was inhibited by intercalating agents and any reagent which reduced the concentration of either the cuprous complex (e.g., neocupr~ine)*-~g~ or hydrogen peroxide (e.g., ~a ta lase) .~” The sensitivity of the reaction to other inhibitors depended on the pathway for the generation of the cuprous complex and hydrogen peroxide (e.g., superoxide dismutase (SOD) inhibited the reaction potentiated by NADH and hydrogen peroxide but had no effect where thiol and hydrogen peroxide were p r e ~ e n t . ~ ~ 5-NO2-OP and 5-CLOP were more effective than OP in cleaving DNA while 5-CH3-OP was less effective than OP under comparable conditions.’,’ The cuprous complex of 2,2’-bipyridine (bpy) was unable to degrade DNA at similar concentrations used for OP,3-5 although the coordination chemistry, the kinetics, and the mechanism of the oxidation of this complex by oxygen and hydrogen peroxide are similar to that of OP.93’0 Moreover, it is known that complexes of bpy as well as those of OP bind to The reaction mechanism for this process has not yet been determined. It is believed that the cuprous complex intercalates with DNA and that the subsequent oxidation by hydrogen peroxide causes the damage due to the formation of OH. at the binding site.4” The binding constants of the various copper complexes to DNA and the kinetics and mechanism of the oxidation of the ternary complexes by oxygen, hydrogen peroxide, and superoxide radicals have not yet been determined. The understanding of the kinetics and mechanism of these reactions may shed light on the mechDNA.” ( I ) DAurora, V.; Stern, A. M.; Sigman, D. S. BBRC 1977, 78, 170. (2) Sigman, D. S.; Graham, D. R.; DAurora, V.; Stern, A. M. J . Biol. (3) Doweny, K. M.; Que, B. G.; So, A. G. BBRC 1980, 93, 264. (4) Que, B. G.; Doweny, K. M.; So, A. G. Biochemistry 1980, 19, 5987. (5) Marshall, L. E.; Graham, D. R.; Reich, K. A.; Sigman, D. S . Bio(6) Gutteridge, J. M.; Halliwell, B. Biochem. Phormacol. 1982. 31, 2801. (7) Reich, K. A,; Marshall, L. E.; Graham, D. R.; Sigman, D. S. J . Am. (8) Pope, L. M.; Reich, K. A,; Graham, D. R.; Sigman, D. S . J . Biol. (9) Goldstein, S.; Czapski, G. J . Am. Chem. Sot . 1983, 105, 7276. (IO) Goldstein, S . ; Czapski, G. Inorg. Chem. 1985, 24, 1087. ( 1 I ) Howe-Grant, M.; Lippard, S . J. Biochemisfry 1979, 18, 5762. Chem. 1979, 254, 12269. chemistry 1981, 20, 244. Chem. SOC. 1981, 103, 3582. Chem. 1982, 257, 12121. anism of DNA cleavage initiated by the various copper complexes. Experimental Section Materials. All chemicals employed were of analytical grade and were used as received: calf thymus DNA, type I, 2,2’-bipyridine, and sodium formate (Sigma Chemical Co.), l,lO-phenanthroline, 5-nitrophenanthroline (Fluka), H202 (Merck), SOD (Diagnostic Data Int.), cupric sulfate, monosodium and disodium phosphate (Mallinckrodt). All solutions were prepared in distilled water which was further purified by a Millipore reagent grade water system. A stock solution of DNA was prepared as 1 mg/mL containing 1 mM sodium phosphate buffer at pH 7. The concentration of DNA per nucleic acid phosphate was determined spectrophotometrically at 260 nm with z = 6875 M-’ cm-‘.I2 The cuprous complexes were generated by using the pulse radiolysis technique in oxygenated solutions containing 0.02 M sodium formate and 1 mM sodium phosphate buffer at pH 7. Under these conditions all the radicals formed by irradiation reduce the cupric c~mplexes . ’~’~ Kinetic studies were followed at 435 nm, where the various cuprous complexes a b s ~ r b . ~ . ’ ~ The concentration of H202 was determined with ferrous ~ u l f a t e . ’ ~ Apparatm. UV-visible absorption spectra were recorded with a Bauch and Lomb Model Spectronic 2000 spectrophotometer. The pulse radiolysis setup consisted of a Varian 7715 linear accelerator. The pulse duration ranged from 0.1 to 1.5 p s with a 200 mA current of 5 MeV electrons. The total concentration of the various cuprous complexes produced per pulse (1-15 pM) was evaluated with the use of a (0P),Cu2+ dosimeter. The yield of (OP)2Cu+ in oxygenated formate solution was assumed to be G = 6.05 and c = 6770 M-’ cm-’ at 435 Irradiation was carried out in a 2 cm long optical spectrosil cell with use of three light passes. A 150-W xenon lamp was used as the analytical light source and appropriate light filters were used to avoid photochemistry and to eliminate any scattered light. The detection system included a grating monochromator and an IP28 photomultiplier. The signal was transferred to a Nova 1200 minicomputer via either a Biomation 8100 or an analog-to-digital converter. The analysis of the data was carried out with the Nova 1200 minicomputer. Results and Discussion A. ”be Reduction of Copper(I1) by 0, in the Presence of DNA. In the irradiation of aqueous solutions containing formate ions and oxygen, the superoxide radical is p r ~ d u c e d . ’ ~ . ’ ~ As the pK of H02 is 4.8,I4J6 the reducing radical is mainly 02at pH 7. When the cupric complexes of OP, 5-N02-OP, or bpy (CuL?’) are present in excess relative to [02-],, reaction 1 takes place: ki CUL22+ + 0 2 CUL2+ + 0 2 (1) (12) Felsenfeld, G.; Hirschman, S. Z . J . Mol. Biol. 1965, 13, 407. (13) Matheson, M. S.; Dorfman, L. M. “Pulse Radiolysis”; MIT Press: (14) Bielski, B. H. J. Photochem. Photobiol. 1978, 28, 645. (15) Holm, N. W.; Berry, R. J. “Manual on Radiation Chemistry“; Marcel (16) Behar, D.; Czapski, G.; Rabani, J . ; Dorfman, L. M.; Schwartz, H. Cambridge, MA, 1969. Dekker Inc.: New York, 1970; pp 313-317. A. J. Phys. Chem. 1970, 74, 3209. 0002-7863/86/ 1508-2244$01.50/0