The cytotoxic effect of '5-fluorouracil (5-FU) on yeast cells is thought to be mainly via a misincorporation of fluoropyrimidines into both RNA and DNA, not only DNA damage via inhibition of thymidylate synthase (TYMS) by FdUMP. However, some studies on Saccharomyces cerevisiae show a drastic decrease in ATP concentration under oxidative stress, together with a decrease of concentration of other tri- and diphosphates. This raises a question if hydrolysis of 5-fluoro-2-deoxyuridine diphosphate (FdUDP) under oxidative stress could not lead to the presence of FdUMP and the activation of so-called "thymine-less death" route. We attempted to answer this question with in silico modeling of 5-FU metabolic pathways, based on new experimental results, where the stages of intracellular metabolism of 5-FU in Saccharomyces cerevisiae were tracked by a combination of 19F and 31P NMR spectroscopic study. We have identified 5-fluorouracil, its nucleosides and nucleotides, and subsequent di- and/or triphosphates. Additionally, another wide 19F signal, assigned to fluorinated unstructured short RNA, has been also identified in the spectra. The concentration of individual metabolites was found to vary substantially within hours, however, the initial steady-state was preserved only for an hour, until the ATP concentration dropped by a half, which was monitored independently via 31P NMR spectra. After then, the catabolic process leading from triphosphates through monophosphates and nucleosides back to 5-FU was observed. These results imply careful design and interpretation of studies in 5-FU metabolism in yeast.

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