Fluorescent probes for cellular hypoxia: lack of transfer of fluorescence between cells in vitro

Fluorescent nitroheterocycles may be useful as probes for cellular hypoxia. Reductive metabolism of AF-2 (cis 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide) and NFVO (trans-5-amino-3((5-nitro-2-furyl)vinyl)1,2,4-oxadiazole) results in intracellular accumulation of fluorescent molecules; the mean cellular fluorescence has previously been shown to be related to the cellular oxygen content during incubation in vitro. However, factors in addition to oxygen and nitroreductive activity may also affect cellular accumulation of these drugs. The stability of cellular fluorescence and possible diffusion of metabolites were examined by flow cytometric analysis of mouse and hamster fibroblasts exposed to NFVO and AF-2. Incubation of cells with 14C-AF-2 allowed calibration of the flow cytometer for AF-2 fluorescence; 5 X 10(8) molecules/cell resulted in double the spontaneous cellular fluorescence. Cellular fluorescence was stable for days after exposure to AF-2, and no evidence of transfer between exposed and unexposed cells was observed. For concentrations resulting in less than 5 X 10(9) AF-2 adducts/cell, all of the metabolites could be accounted for intracellularly. Therefore, it is unlikely that transfer of reduced nitroheterocycles occurs between cells.