Gamma-radiolysis study of the reductive activation of neocarzinostatin by the carboxyl radical.
The activation of the antitumor protein antibiotic neocarzinostatin (NCS) by the carboxyl radical CO-2, a one-electron donor obtained selectively from gamma-ray irradiation of nitrous oxide-saturated formate buffer, has been investigated in the presence and in the absence of DNA at pH 4.7 and pH 7.0. The reaction of NCS with CO-2 in the absence of DNA is followed by a marked red shift (420----441 nm) and a pronounced increase (X 8.8) of the fluorescence emission corresponding to the naphthalene moiety of the NCS chromophore. The light absorption spectrum shows in parallel a hypochromic change with considerable fine structure throughout the 250-400 nm wavelength range. When DNA is present, the fluorescence intensity at completion of the reaction is slightly reduced (by 5 to 15 per cent) and the maximum emission wavelength shifted to 436-438 nm. However, the bulk rate of reaction is not altered by DNA and is independent of the pH, of the temperature and of the concentration of NCS. The NCS concentration-independence of the reaction rate is consistent with a high intrinsic rate (k greater than 10(8)M-1 . s-1) for the reaction of CO-2 with the NCS chromophore. Complete reduction of the NCS chromophore involves a total of three electron-equivalents. The final product does not react with oxygen, shows no odd electron spin, and is unable to induce DNA strand scission. Its molecular state, however, is fundamentally different when gamma-ray irradiations are performed with DNA. This bears evidence of short-lived one electron or two-electrons reduced intermediates decaying via non-identical routes depending on the presence of the acceptor DNA. Actually, dose-related strand breaks appear in DNA exposed to the action of NCS and CO-2. Some NCS chromophore-DNA covalent adducts are also found. DNA strand breakage by CO-2-activated NCS is correlated with thymine release and is inhibited by a redox-stable intercalating agent. The DNA-nicking process thus bears resemblance to that reported by other authors using mercaptans to initiate reductive activation of the NCS chromophore. However, some spectral differences are observed between the CO-2-reacted and the thiol-treated chromophores. Moreover, thymine release and strand scission in DNA incubated with CO-2 and NCS proceed under anaerobic conditions. It is proposed that the strict oxygen requirement for DNA damage by NCS in the presence of mercaptans is due, at least partly, to competition between oxygen and thiols for reaction with the same primary deoxyribose radical resulting from DNA attack by the reductively activated NCS chromophore.
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000528:radiation effects*; D002245:Carbon Dioxide; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004229:Dithiothreitol / Q000494:pharmacology; D004355:Drug Stability; D005609:Free Radicals; D005720:Gamma Rays; D006863:Hydrogen-Ion Concentration; D007364:Intercalating Agents / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology; D011839:Radiation, Ionizing; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013941:Thymine / Q000378:metabolism; D009353:Zinostatin / Q000378:metabolism / Q000528:radiation effects*
D000903:Antibiotics, Antineoplastic; D005609:Free Radicals; D007364:Intercalating Agents; D002245:Carbon Dioxide; C041069:carboxyl radical; D004247:DNA; D009353:Zinostatin; D013941:Thymine; D010100:Oxygen; D004229:Dithiothreitol