Fungistatic Activity, Uptake and Incorporation of 5-Fluorocytosine in &lt;i&gt;Candida albicans, &lt;/i&gt;as Influenced by Pyrimidines and Purines. I. Reversal Experiments

Polak, Annemarie; Scholer, H. J.

doi:10.1159/000162642

Cited by 30 publications

(35 citation statements)

References 7 publications

(12 reference statements)

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“…This may seem surprising, as it has been reported that both transport through the fungus cell membrane and intracellular deamination take place rapidly (16); a large amount of radioactivity was found in the internal pool of C. albicans cells during the uptake phase, i.e., up to about 1 to 2 h after exposure to FC (14). We therefore studied the culture medium after 16 h of incubation with FC; a high percentage of residual FC (74%) was found compared with the percentage of 5FU excreted from the cells (26%).…”

Section: Resultsmentioning

confidence: 91%

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

Vialaneix¹,

Chouini²,

Malet‐Martino³

et al. 1986

Antimicrob Agents Chemother

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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;

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Section: Resultsmentioning

confidence: 91%

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

Vialaneix¹,

Chouini²,

Malet‐Martino³

et al. 1986

Antimicrob Agents Chemother

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“…Like YNB, RPMI is a synthetic and completely defined medium and is characterized by small lot-to-lot variation, resulting in high reproducibility of susceptibility tests, unlike AM3, which has considerable variation from lot to lot and source to source. By contrast the use of chemically complex undefined media such as SAB medium is not recommended (19,28) since undefined components that they may contain might interact with antifungal drugs (13,14,19,26). Furthermore, acidic media such as YNB and SAB, if they were used unbuffered, may inactivate amphotericin B, which is not stable at low pH (16,21).…”

Section: Discussionmentioning

confidence: 99%

Analysis of Growth Characteristics of Filamentous Fungi in Different Nutrient Media

et al. 2001

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A microbroth kinetic model based on turbidity measurements was developed in order to analyze the growth characteristics of three species of filamentous fungi (Rhizopus microsporus, Aspergillus fumigatus, and Scedosporium prolificans) characterized by different growth rates in five nutrient media (antibiotic medium 3, yeast nitrogen base medium, Sabouraud broth, RPMI 1640 alone, and RPMI 1640 with 2% glucose). In general, five distinct phases in the growth of filamentous fungi could be distinguished, namely, the lag phase, the first transition period, the log phase, the second transition period, and the stationary phase. The growth curves were smooth and were characterized by the presence of long transition periods. Among the different growth phases distinguished, the smallest variability in growth rates among the strains of each species was found during the log phase in all nutrient media. The different growth phases of filamentous fungi were barely distinguishable in RPMI 1640, in which the poorest growth was observed for all fungi even when the medium was supplemented with 2% glucose. R. microsporus and A. fumigatus grew better in Sabouraud and yeast nitrogen base medium than in RPMI 1640, with growth rates three to four times higher. None of the media provided optimal growth of S. prolificans. The germination of Rhizopus spores and Aspergillus and Scedosporium conidia commenced after 2 and 5 h of incubation, respectively. The elongation rates ranged from 39.6 to 26.7, 25.4 to 20.2, and 16.9 to 9.9 m/h for Rhizopus, Aspergillus, and Scedoporium hyphae, respectively. The germination of conidia and spores and the elongation rates of hyphae were enhanced in antibiotic medium 3 and delayed in yeast nitrogen base medium. In conclusion, the growth curves provide a useful tool to gain insight into the growth characteristics of filamentous fungi in different nutrient media and may help to optimize the methodology for antifungal susceptibility testing.

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“…For most strains, there was a positive correlation between the degree of 5-fluorocytosine susceptibility and the inhibition of biosynthesis of both RNA and DNA, incorporation of 5-fluorouracil into RNA, inhibition of ribosomal protein synthesis, and levels of 5-fluorodeoxyuridine monophosphate. However, in some strains with a similar degree of 5-fluorocytosine resistance, either reduced incorporation of 5-fluorouracil or reduced 5-fluorodeoxyuridine monophosphate levels occurred, suggesting that these two mechanisms are not necessarily linked to each other and that both may be responsible for 5-fluorocytosine activity.The mechanism of action of the antifungal drug 5-fluorocytosine (5-FC) has been investigated in yeasts (7,8,12), aspergilli (12), and dematiaceous fungi (17). It has been suggested that the activity of 5-FC after uptake by the fungi and intracellular deamination is a consequence of intrafungal formation of two metabolites, 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate (FURTP) (11,15).…”

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confidence: 99%

“…The mechanism of action of the antifungal drug 5-fluorocytosine (5-FC) has been investigated in yeasts (7,8,12), aspergilli (12), and dematiaceous fungi (17). It has been suggested that the activity of 5-FC after uptake by the fungi and intracellular deamination is a consequence of intrafungal formation of two metabolites, 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate (FURTP) (11,15).…”

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confidence: 99%

Mechanisms of action of 5-fluorocytosine

Waldorf¹,

Polak²

1983

Antimicrob Agents Chemother

124

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5-Fluorouracil and 5-fluorodeoxyuridine monophosphate levels were estimated in 75 isolates of Candida albicans to determine whether 5-fluorocytosine susceptibility could be ideally correlated with the intrafungal formation of both 5-fluorodeoxyuridine monophosphate and 5-fluorouridine triphosphate or a reciprocal formation of the two metabolites to prove the mechanism of 5-fluorocytosine activity. Using the results of four in vitro susceptibility tests, we separated isolates of C. albicans into susceptibility groups. For most strains, there was a positive correlation between the degree of 5-fluorocytosine susceptibility and the inhibition of biosynthesis of both RNA and DNA, incorporation of 5-fluorouracil into RNA, inhibition of ribosomal protein synthesis, and levels of 5-fluorodeoxyuridine monophosphate. However, in some strains with a similar degree of 5-fluorocytosine resistance, either reduced incorporation of 5-fluorouracil or reduced 5-fluorodeoxyuridine monophosphate levels occurred, suggesting that these two mechanisms are not necessarily linked to each other and that both may be responsible for 5-fluorocytosine activity.The mechanism of action of the antifungal drug 5-fluorocytosine (5-FC) has been investigated in yeasts (7,8,12), aspergilli (12), and dematiaceous fungi (17). It has been suggested that the activity of 5-FC after uptake by the fungi and intracellular deamination is a consequence of intrafungal formation of two metabolites, 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate (FURTP) (11,15).FdUMP is a potent inhibitor of thymidylate synthetase (5), causing an impairment of DNA synthesis in bacteria and tumor cells. Furthermore, in one 5-FC-susceptible strain of Candida albicans, in the presence of 5-FC, FdUMP was formed, followed by a decrease in the thymidylate synthetase activity (2). Moreover, the addition of 5-FC to both hyphal and yeast phases of C. albicans cultures resulted in an immediate inhibition in the increase of DNA (12).FURTP is incorporated into fungal RNA in place of uridylic acid (11, 15), which alters amino acylation of tRNA in vitro (4) and is believed to cause unbalanced internal pool and disturbed synthesis of proteins and carbohydrates (6, 13, 14). There is a statistical correlation between incorporation of the fluorinated metabolite into RNA and the antifungal activity of 5-FC, although there are considerable individual variations in incorporation in mutants with similar 5-FC susceptibility (1, 7, 10). Whether these two suggested mechanisms of 5-FC activity (FdUMP and FURTP) are linked or independent of each other is still unknown. The formation of FdUMP and decreased thymidylate activity may explain the antifungal 5-FC activity, the incorporation of FURTP in RNA being only incidental, or the converse. The purpose of this study was to determine on which of these mechanisms the activity of 5-FC is based.

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Fungistatic Activity, Uptake and Incorporation of 5-Fluorocytosine in <i>Candida albicans, </i>as Influenced by Pyrimidines and Purines. I. Reversal Experiments

Cited by 30 publications

References 7 publications

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

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

Analysis of Growth Characteristics of Filamentous Fungi in Different Nutrient Media

Mechanisms of action of 5-fluorocytosine

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