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The radiation-induced "in-pulse" luminescence emission from solid DNA containing either metronidazole or a highly electron-affinic 5-nitrofuran in the range 3-2000 (w:w) base pairs per additive molecule has been investigated in vacuo at 293 K using electron pulses of energy below 260 keV. The luminescence intensity at 450 nm from DNA decreases with increasing content of the additive in the sample and approaches a limiting level at high concentrations of the additives. At these higher concentrations the limiting value represents about 50% of that observed from DNA alone. It is shown that the efficiency of the additives in reducing the luminescence intensity is dependent upon their redox potential E1(7); this dependence is consistent with these additives acting as electron acceptors. It is concluded that the ability of the electron acceptors to reduce the luminescence is related to the electron affinity of E1(7) of the acceptors and electron migration distances of at least 300 base pairs are proposed.

During the preparation of our recent paper (1) there was a question as to whether it is possible to fit the experimental data to a model or models where the migration distance of radiation-induced electrons in DNA molecules incorporated with misonidazole could be estimated. In this letter we describe five different models (Table I) which yield the electron migration distance in solid DNA. These models are based upon the experimentally obtained luminescence quenching curve by the trap misonidazole where the observed migration distance for 50% quenching has been estimated to be about 25 base pairs (1). In these models we have assumed that misonidazole, as an electron-affinic agent, is acting as an electron "trap." It is assumed that the misonidazole molecules are distributed randomly within the DNA. Electrons emitted from a site of ionization may travel along the DNA molecule, in either direction, for a distance a which varies in a stochastic fashion between electrons. If an electron encounters a misonidazole molecule within a distance a, it will be irreversibly trapped, irrespective of the depth of that trap. Simple probabilistic arguments show, using this model, that the proportion of elect ons which are not trapped and therefore can result in light emission is P, where

The efficacies of a series of potential prodrugs of RSU-1069 and its alkyl-aziridine analogues were assessed. These 1-(2-haloethylamino)-3-(2-nitro-1-imidazolyl)-2-propanol compounds were designed to cyclize in vivo to generate 2-nitro-imidazoles with aziridine (RSU-1069) or alkyl-substituted aziridine (RSU-1164, RB-7040, or RSU-1150) functions. Maximum tolerated single, intraperitoneal doses (MTD) were determined in C3H/He mice bearing subcutaneous KHT sarcomas, and a drug dose-response relationship for radiosensitization was established for each compound administered at the optimum time (45-60 min) before local irradiation of tumors with a 10-Gy dose of X-rays. The potentials of the compounds as bioreductive cytotoxins were studied by administering them immediately after irradiation. Tumor cell survival was measured 18-24 h after treatment in an in vitro soft agar clonogenic assay. Results of toxicity, radiosensitization, and bioreductive cytotoxicity assays for each of the prodrugs (RB-6171, RB-6172, RB-6173, RB-6174, and RB-6175) of the alkyl-substituted aziridines were entirely consistent with complete conversion to their respective target compounds. For example, RB-6171 (the prodrug form of RSU-1164) was only about four times less efficient than RSU-1069 as a radiosensitizer and bioreductive cytotoxin but had an MTD 7.5 times higher. In contrast, prodrugs of RSU-1069 (RB-6144 and RB-6145) were two- to threefold less toxic than their expected product. RB-6144 was a poor radiosensitizer and bioreductive agent compared with RSU-1069 and was similar to RB-6170, a nonalkylating nitroimidazole. This is consistent with the observation that there is limited conversion of RB-6144 to RSU-1069 in vitro. However, radiosensitization and bioreductive cytotoxicity produced by RB-6145 were only slightly less than the effects produced by RSU-1069; thus a therapeutic gain was achieved with RB-6145 in a murine tumor model.

Radiosensitization efficiencies for seven different 2-nitroimidazoles including Ro-07-0582 and its urinary metabolite, Ro-05-9963, and two 5-nitroimidazoles including metronidazole, have been determined in hypoxic Chinese Hamster cells, line V79-379A, X-irradiated in vitro. All the compounds were active hypoxic cell sensitizers with the enhancement ratios increasing with drug concentration. The 2-nitroimidazoles were all more efficient than the 5-nitroimidazoles. Overall, the efficiencies, defined as the concentration required to give a particular enhancement ratio, varied by a factor of about 200. Electron-affinities of the sensitizers were determined by pulse radiolysis as the one-electron reduction potentials and these correlate well with the sensitization efficiencies of the compounds. The correlation extends beyond the nitroimidazole series as is shown by data for p-nitroacetophenone, nifuroxime (a nitrofuran) and oxygen itself. The nitroimidazoles varied by a factor of 70 in their octanol/water part...

Five nitro-substituted imidazoles and three nitro-substituted pyrazoles have been investigated as radiosensitizers for hypoxic mammalian cells cultured and irradiated in vitro. All the compounds sensitize, but the pyrazoles are less effective than the imidazoles. There is no sensitization of cells irradiated in the presence of oxygen. One of the two 2-nitroimidazoles, Ro-07-0582, is much more effective than any of the other compounds and shows a large degree of sensitization in the submillimolar range. At 4 mM concn, the enhancement ratio is about 2.5 which is near the value for the oxygen enhancement ratio for these cells. The cytotoxicity of this sensitizer is low and the sensitizing properties are not diminished by increasing the protein concentration of the medium. The maximum enhancement ratio for this sensitizer is independent of the position of the cells in the mitotic cycle.

ADAMS, G. E., BISBY, R. H., CUNDALL, R. B., REDPATH, J. L., AND WILLSON, R. L. Selective Free Radical Reactions with Proteins and Enzymes: The Inactivation of Ribonuclease. Radiat. Res. 49, 290-299 (1972). The effect of free radicals derived from thiocyanate, bromide, and carbonate ions on the activity of ribonuclease have been studied in neutral and alkaline solutions. Thiocyanate ions and, to a lesser extent, carbonate ions, protect ribonuclease against radiation-induced inactivation, although the presence of bromide ions has little effect. The absolute rate constants for the reactions of these radical anions with ribonuclease have been measured as a function of pH and salt concentration, and the transient absorption spectra of the reaction products have been determined. The results suggest that damage to a histidine residue, or residues, leads directly to loss of activity and support previous conclusions that exposed tyrosine residues in the enzyme are not essential for activity.

ADAMS, G. E., BAVERSTOCK, K. F., CUNDALL, R. B., AND REDPATH, J. L. Radiation Effects on a-Chymotrypsin in Aqueous Solution: Pulse Radiolysis and Inactivation Studies, Radiat. Res. 54, 375-387, (1973). Pulse radiolysis and steady-state inactivation methods have been used to study the separate roles of H, OH, and e.q- in the radiation-induced inactivation of aqueous a-chymotrypsin solutions. The effects of nitrous oxide and t-butanol used as selective free radical scavengers allow the inactivating efficiencies per unit G-value of OH, H, and eaq- radicals to be estimated; these are 0.137, 0.13, and 0.055, respectively, in neutral solution. The transient spectra observed show that both OH radicals and hydrogen atoms react mainly with tryptophan residues. Only about 15% of the hydrated electrons react with the cystine disulfide linkages; rupture of some of these linkages may lead to inactivation. It is suggested that the protective action of oxygen is due partly to the reactions of 02 with enzyme radicals formed initially by reaction with OH.

time scale of the oxygen effect in irradiated Serratia marcescens. The bacteria were held on a Millipore filter inside a chamber which was usually flushed with humidified nitrogen and were exposed to an explosion of oxygen released into the chamber through a fastacting solenoid-operated valve. The bacteria were irradiated with a single 2-psec pulse of 1.8 MeV electrons from a linear accelerator. The arrival of oxygen at the bacteria was timed to occur at a preset interval before or after the electron pulse. The time resolution was 100 ,sec. After fixed doses of 10, 19, and 28 krads per pulse, the bacterial survival was measured as a function of the oxygen contact time. It was found that, when oxygen contact occurred at any instant before or up to the time of the electron pulse, full sensitization was obtained. However, bacteria exposed to oxygen at times after the electron pulse showed decreasing sensitization as the time interval increased, the survival approaching the anoxic control value at times greater than 2 msec. The data show that, in Serratia marcescens irradiated under anoxic conditions, oxygen-dependent damage decays approximately exponentially with a half-life of about 500 ,usec.