Spin-quenching by Oxygen of a Radiosensitizing Nitroxide Free Radical in Aqueous Solution

Stable organic nitroxide free radicals have recently become of considerable interest in radiobiology, since they have been found to increase the sensitivity of anoxic cells to radiation-induced reproductive death without affecting the aerobic sensitivity (Emmerson and Howard-Flanders 1964, 1965, Emmerson 1967 and Brustad 1968). Such characteristics make these compounds of potential interest in radiotherapy. Among the nitroxide free radicals studied to date, 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (often referred to as triacetoneamine-N-oxyl or TAN) appears to combine the most desirable qualities of relatively low toxicity with efficient radiosensitization. Emmerson and Howard-Flanders (1965) and Emmerson (1967) have suggested that TAN exerts its sensitizing effect by reacting with radiationinduced radicals in vital cellular constituents in the same way as does oxygen. Pulse radiolysis experiments have revealed that TAN interacts rapidly with OH-induced DNA transients (Willson and Emmerson 1970). We have recently investigated the nature of this radiation-induced interaction in dilute neutral aqueous solution. Our results show that TAN becomes covalently bonded to OH-induced DNA transients under anoxic conditions, whereas no binding occurs when irradiation is performed in the presence of oxygen. This lack of binding, as well as the absence of a radiosensitizing effect upon oxygenated cells, might most obviously be explained by competition between TAN and 02 for reaction with radiation-induced DNA transients. An alternative explanation, which so far has not been considered, could involve an interaction of oxygen with TAN to render the latter ineffective. However, it has been pointed out that the nitroxide free radicals do not react with oxygen, at least to form peroxides (Emmerson and Howard-Flanders 1964, Hoffmann and Henderson 1961). Nevertheless, since TAN is a free radical, its ability to react with radiation-induced DNA transients may conceivably be related to the unpaired electron localized on the NO group. We therefore decided to conduct an electron spin resonance (ESR) study to determine whether oxygen alters the free radical properties of TAN. TAN was synthesized from 2,2,6,6-tetramethyl-4-piperidone-HCl (Aldrich Chemical Company) according to published procedures (Briere, Lemarie and Rassat 1965), with some minor modifications. Our X-band ESR spectrometer with transmission H102 mode cavity has been described previously (Henriksen 1962). All E.S.R. measurements described here were carried out at 77°K. All