The peroxidation of liposomes by a haem peroxidase and hydrogen peroxide in the presence of indole-3-acetic acid and derivatives was investigated. It was found that these compounds can accelerate the lipid peroxidation up to 65 fold and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. The peroxyl radicals are formed by peroxidase-catalyzed oxidation of the enhancers to radical cations which undergo cleavage of the carbon-carbon bond on the side-chain to yield CO2 and carbon-centred radicals that rapidly add oxygen. In competition with decarboxylation, the radical cations deprotonate reversibly from the N1 position. Rates of decarboxylation, pka values and rate of reaction with the peroxidase compound I indicate consistent substituent effects which, however, can not be quantitatively related to the usual Hammett or Brown parameters. Assuming that the rate of decarboxylation of the radical cations taken is a measure of the electron density of the molecule (or radical), it is found that the efficiency of these compounds as enhancers of lipid peroxidation increases with increasing electron density, suggesting that, at least in the model system, the oxidation of the substrates is the limiting step in causing lipid peroxidation.

The rates of reaction of seven indole-3-acetic acid derivatives with horseradish peroxidase compound 1 at pH 5 were measured by stopped flow, and the reduction potentials and pKa of their radical cations were determined by pulse radiolysis. Reasonable correlation of these properties with Hammett substituent parameters was found, but not with Brown-Okamoto (theta +) parameters. The rates of reaction with compound I correlate well with the reduction potentials under the same conditions, with rates of reaction that increase by ca. 2.5 orders of magnitude with a 100 mV decrease in the reduction potential. This relationship is in agreement with that previously estimated for the reaction of compound I with phenols and anilines, suggesting that the rate of reaction depends solely on the reduction potential of the substrate radical, even for compounds of dissimilar structure.

Summary Numerous methods have been proposed for the detection of hypoxic cells using nitroimidazoles labelled with both radioactive and stable isotopes where the isotopic label becomes bound as a result of reductive metabolism of the nitro group. A new probe for hypoxia, 7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline, is described where an immunologically recognisable hapten (theophylline) is covalently linked to a 2-nitroimidazole. Bioreduction of the nitroimidazole leads to binding of bioreductive metabolites, and hence the theophylline side-chain, to intracellular molecules. Immunochemical procedures are then used to stain cells containing the bound theophylline using an FITC-conjugated anti-serum. Flow cytometric analysis of stained cells is facilitated by co-staining cellular DNA, which allows discrimination of single cells in the sample and rejection of cell clumps and debris. The alternative use of an immunoperoxidase-conjugated anti-serum has been used to demonstrate the localisation of hypoxic cells in frozen tumour sections.The existence of poorly oxygenated radioresistant cells in tumours is thought to be one of the factors contributing to local failure of radiotherapy and tumour regrowth (Gray et al., 1953;Churchill-Davidson et al., 1966;Bush et al., 1978;Mueller-Klieser et al., 1981;Dische, 1985;Dische et al., 1986;Overgaard et al., 1986). A simple, rapid clinical test for the presence of hypoxic cells in tumours could enable radiotherapy to be optimised for individual patients on the basis of the oxygen status of their tumours. Adjuncts to radiotherapy such as hypoxic cell sensitisers and hyperbaric or normobaric oxygen could then be administered to those patients most likely to benefit from them.Of the methods that have been proposed for determining the hypoxic fractions of tumours, several are based on the hypoxia-dependent bioreductive metabolism of a labelled 2-nitroimidazole which results in binding of labelled fragments of the original compound to cellular macromolecules. Various labels have been proposed, including 3H (Raleigh et al., 1985), 14C (Chapman et al., 1981;Franko & Chapman, 1982; Garrecht & Chapman, 1983), 75Br, 76Br, 7Br (Rasey et al., 1985 and '9F (Raleigh et al., 1986): 3H-misonidazole has been administered to small numbers of patients with treatment-resistant tumours (Urtasun et al., 1986). Identification of hypoxic cells using radiolabelling requires prolonged autoradiography to detect labelled regions of tumour sections. NMR detection of bioreductively bound metabolites of fluorinated nitroimidazoles in tissues has been demonstrated experimentally and such bound metabolites have also been detected by fluorescence immunohistochemistry (Raleigh et al., 1987).In this paper, we describe the use of the immunologically detectable hapten theophylline, covalently bound to a 2-nitroimidazole, as a method of identifying hypoxic cells. An isotopic label on the side-chain of misonidazole binds to cellular constituents as efficiently as a ring label (Raleigh et al., 1985) and an immun...

The effects of substituting 2-nitroimidazoles with groups carrying basic functions were studied. Prototropic, redox, lipophilicity and protein-binding properties were compared with the efficiency in radiosensitizing hypoxic Chinese hamster V79-379A cells in vitro and the cytotoxicities of the compounds after chronic aerobic exposure. Seventeen compounds were (2-nitro-1-imidazolyl)alkylamines in which the effects of changes in the terminating base and of alkyl chain length were investigated. About an order of magnitude increase in sensitization efficiency could be observed in some compounds without any increase in the aerobic cytotoxicity compared to simple, uncharged 2-nitroimidazoles such as misonidazole. The behaviour of five hydrazones was similar to that of uncharged analogues. The methiodide quaternary salts of two of the (2-nitro-1-imidazolyl)alkylamines showed that quaternization considerably reduced sensitization efficiency. (Nitro-1-imidazolyl)alkylamines appear worthy of further investigation as hypoxic cell radiosensitizers in vivo.