Noninvasive and quantitative 19F nuclear magnetic resonance study of flucytosine metabolism in Candida strains

Abstract

19F nuclear magnetic resonance was used for a noninvasive and quantitative study of flucytosine (FC; 5-fluorocytosine) metabolism in two strains of Candida albicans and one strain of Candida tropwcalis with various susceptibilities to FC. Three intracellular fluorinated metabolites were detected in the highly susceptible strain, F-nucleotides (Fnt), F-nucleosides, and 5-fluorouracil (5FU). Fnt were partially converted into 5FU when the spectra of the yeasts were recorded at 37°C without perfusion, but the intensities of the signals were not modified at 4 or 37°C if the cells were perfused. In the acid extract, the Fnt signal was resolved into three distinct peaks; none of them was attributable to 5-fluoro-2'-deoxyuridine-5'-monophosphate. The same signals were detected in the partially resistant strain, but only 5FU was observed in the highly resistant strain; the resistance of the latter strain therefore was primarily due to a defect in UMP pyrophosphorylase. At the end of the incubation period, only FC and released 5FU were present in the culture media. The concentration of the intracellular fluorinated metabolites was increased if the strain was susceptible to FC. The total amount of metabolized FC was very similar for the highly susceptible and the partially resistant strains, but the percentage of Fnt was much higher in the former (38%) than in the latter (8%); the mild resistance of the partially resistant strain therefore was attributed to the decreased activity of UMP pyrophosphorylase.Flucytosine (FC; 5-fluorocytosine) has gained an important place in the systemic treatment of deep-seated fungal infections in humans, in particular against candidiasis, cryptococcosis, and chromomycosis (16). Investigation of the intrafungal pathway of the drug is an important key to the understanding of the mechanisms of drug action and drug resistance. Previous studies of the FC mode of action have involved invasive techniques requiring extraction procedures and, most often, the use of labeled drugs (5,15,19,20).We used '9F nuclear magnetic resonance (NMR) to study the mode of action of FC. This noninvasive technique allows a direct analysis of heterogeneous biological samples without prior extraction and does not require a labeled drug, as the observed probe, the '9F nucleus, is an inherent part of the parent drug and its metabolites. Moreover, this technique permits simultaneous identification and quantification of all the fluorinated metabolites present in the analyzed sample (1). We have already applied this methodology to metabolism studies of antineoplastic fluoropyrimidines, 5-fluorouracil (5FU), and 5'-deoxy-5-fluorouridine in human biofluids and cultured tumor cells (12, 13;

19F nuclear magnetic resonance study of fluoropyrimidine metabolism in strains of Candida glabrata with specific defects in pyrimidine metabolism

“…These observations are similar to those determined with C. albicans (2,25). The issue of 5-FU efflux is particularly interesting because it could provide an alternative explanation for the mechanism of toxicity of 5-FC in humans.…”

“…A similar but more quantitative approach was adopted to study 5-FC metabolism in three C. albicans strains and one Candida tropicalis * Corresponding author. strain (25). Results from these studies complemented each other and were consistent with the assigned genotypes of the strains used; in addition, they established the basic conditions under which "9F NMR spectroscopy may be used to obtain significant information on 5-FC metabolism in viable cells of Candida spp.…”

“…Results of a recent study (7) suggested that toxicity of 5-FC could arise from conversion to 5-FU by human intestinal microflora. The results obtained in this and other studies (2,25) suggest that the pathogens themselves (C. glabrata and C. albicans) excrete large quantities of 5-FU into the surrounding medium and may contribute to any 5-FU-induced toxicity in the human host.…”

“…This latter compound can be converted to either 5-FUTP or to 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP), which, together or independently, determine the antifungal activity of 5-FC via production of aberrant RNA or inhibition of DNA synthesis, respectively (21,25) (Fig. 1).…”

Study of the metabolism of flucytosine in Aspergillus species by 19F nuclear magnetic resonance spectroscopy

“…Among the studies done, one in particular has held our attention as it proposed an unexpected pathway for 5FC metabolism, with 5-fluoroorotic acid (SFO) being the principal metabolite (14). We thought that fluorine-19 nuclear magnetic resonance ('9F NMR) spectroscopy, already used to study the metabolic transformation of 5FC in Candida yeasts (3,12), could lead to better knowledge of the metabolism of this antifungal agent and confirm or invalidate the results obtained by the authors cited above. Indeed, this technique allows the simultaneous detection of all fluorinated metabolites in a sample and does not require a labeled drug, as the observed probe, the 19F nucleus, is an inherent part of the parent drug and its metabolites.…”