Binding of two hypoxia probes, [3H]misonidazole and AF-2 (2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide), was compared in Chinese hamster V79 spheroids incubated under different oxygen concentrations. Fluorescence-activated cell sorting based on Hoechst 33342 penetration was used to obtain populations of cells from different depths within the spheroid, and sorted cells were analyzed by cytofluorometry for AF-2 content and by liquid scintillation counting for [3H]misonidazole content. The patterns of AF-2 and misonidazole binding were very similar, with about 20-fold more localization of both drugs in anoxic compared to aerobic cells. Similar results were obtained when cells were sorted on the basis of AF-2 rather than Hoechst 33342 fluorescence. When mean cellular fluorescence of AF-2 was plotted versus cpm misonidazole/cell for different oxygen tensions, it appeared that oxygen was equally effective in inhibiting AF-2 and misonidazole binding. Internal cells of anoxic spheroids bound about twice as much AF-2 and misonidazole as external cells, apparently due to an increased rate of nitroreduction by chronically hypoxic cells. AF-2 was found to enhance the retention of misonidazole in oxic and hypoxic spheroids when both drugs were present.

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