Nitroakridin 3582: a fluorescent nitroacridine stain for identifying hypoxic cells.

Abstract

The ability to determine the proportion of hypoxic cells within a tumour during treatment could lead to a more rational assessment of benefit when planning and executing radiotherapy. Until recently the only method available to determine the hypoxic fraction has been an indirect radiobiological assessment. This is applicable to experimental tumour systems but not to human patients. We have therefore sought a more direct and rapid quantitative method for detecting hypoxic cells which could eventually be applicable in the clinic. The method is based on the production of fluorescence in hypoxic cells which can be detected by microscopy or by using a flow cytometer. The rationale for the present work was based on the observations that:—(1) with certain nitroaromatic compounds the quantum efficiencies of fluorescence for the nitroreduction products (amines) are at least four orders of magnitude greater than that of the parent compound (McGlynn et al, 1969); (2) the amine is only produced under hypoxic conditions by cellular enzymes, because the initial reduction step to the nitro radical-anion, an obligate intermediate, is rapidly reversed in the presence of oxygen (Mason, 1982); (3) the reduction products formed from the bioreductive metabolism of misonidazole, metronidazole and nitrofurazone bind intracellularly in hypoxic cells. Radiolabelling of misonidazole or its analogues, followed by measurement of bound label using scintillation counting (Verghese et al, 1967), autoradiography (Chapman et al, 1981), or (potentially) external gamma scanning (Rasey etal, 1982) have been proposed as the basis for clinical detection and imaging of hypoxic cells in tumours.