Cellular metabolism of fluorescent nitroheterocycles

Since nitroheterocycles are preferentially metabolized and bound in hypoxic cells, we have examined more than 2 dozen nitroheterocycles as potential fluorescent probes for hypoxia. Using flow cytometry, several patterns of cellular fluorescence (CF) have been observed; for most nitroheterocycles, CF was several fold higher for anoxic than for aerobic cells (which was not predicted based on comparison of the fluorescence spectra of parent drug and reduced products). CF gradually increased when cells were exposed to 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) or to 4-nitroquinoline-1-oxide (4-NQO), and cells remained fluorescent when the drug was washed off. In contrast, cells exposed to trans-5-amino-3-[5-nitro-2-furyl)vinyl-1,2,4-oxadiazole (NFVO) lost fluorescence with a half-time of 60 minutes. Cells exposed to nitrofurazone (NF-7) reached maximum fluorescence within 30 minutes and then lost fluorescence, even in the presence of the drug. Finally, cells exposed to 3-nitropyrene (NP-3) were initially more fluorescent when incubated under aerobic conditions than anoxic conditions; however, after 2 hours in the presence of NP-3, anoxic cells continued to increase in fluorescence while aerobic cells lost fluorescence. Differences in the patterns of cellular accumulation of fluorescent nitroheterocycles were accompanied by differences in the toxicity and metabolism of these drugs. Therefore, chemical studies alone do not allow us to predict the potential of a compound as a hypoxic probe; studies at the cellular level are also essential.