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

Abstract

The metabolism of flucytosine (5FC) in two Aspergillus species (Aspergillus fumigatus and A. niger) was investigated by '9F nuclear magnetic resonance spectroscopy. In intact mycelia, 5FC was found to be deaminated to 5-fluorouracil and then transformed into fluoronucleotides; the catabolite a-fluoro-I8-alanine was also detected in A. fumigatus. Neither 5-fluoroorotic acid nor 5-fluoro-2'-deoxyuridine-5'-monophosphate was detected in perchloric acid extracts after any incubation with 5FC. 5FC, 5-fluorouracil, and the classical fluoronucleotides 5-fluorouridine-5'-mono-, di-, and triphosphates were identified in the acid-soluble pool. Two hydrolysis products of 5-fluorouracil incorporated into RNA, 5-fluorouridine-2'-monophosphate and 5-fluorouridine-3'-monophosphate, were found in the acid-insoluble pool. No significant differences in the metabolic transformation of 5FC were noted in the two species of Aspergillus. The main pathway of 5FC metabolism in the two species of Aspergillus studied is thus the biotransformation into ribofluoronucleotides and the subsequent incorporation of 5-fluorouridine-5'-triphosphate into RNA.Flucytosine (5FC) is an antimycotic agent used in the treatment of many fungal infections (11). Although the metabolic fate of 5FC has been extensively investigated in yeasts (15 and references cited therein), much less is known of it in filamentous fungi of the Aspergillus type (2,8,10, 13,14). 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. We therefore carried out an investigation of 5FC metabolism in two species of Aspergillus (Aspergillus fumigatus and A. niger) by 19F NMR. MATERIALS AND METHODSChemicals. SFO, 5FC, 5-fluorouracil (5FU), 5-fluorouridine, 5-fluoro-2'-deoxyuridine, OMP, UMP, dUMP, OMP decarboxylase (from bakers' yeast), and alkaline phosphatase (from calf intestine) were supplied by Sigma Chemical Co., St. Louis, Mo. 5-Fluorouridine-5'-mono-, di-, and triphosphates (FUMP, FUDP, and FUTP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) and 5-fluoro-2'-deoxyuridine-5'-diphosphate were purchased from Sierra Bioresearch, Tucson, Ariz. a-Fluoro-,-alanine was from Koch-Light Laboratories, Colnbrook, United Kingdom.Organisms, culture conditions, and PCA extract prepara- determined by the agar dilution method after 3 days of incubation at 37°C (9). The strains were maintained on Sabouraud agar mediu...

19 F Nuclear Magnetic Resonance Analysis of 5-Fluorouracil Metabolism in Four Differently Pigmented Strains of Nectria haematococca

“…This is an uncommon FU degradative pathway, since FUrd was not detected in culture media of Candida yeasts (3,4,18) or filamentous fungi (Aspergillus spp. [2]).…”

Noninvasive quantitation of cytosine deaminase transgene expression in human tumor xenografts with in vivo magnetic resonance spectroscopy

“…The quantitative nature of in vivo 19 F-MRS measurements has been confirmed with HPLC (10,11). Building on previous MRS studies of 5-FC metabolism in perfused yeast cultures (12)(13)(14)(15)(16), conversion of 5-FC to 5-FU by antibody-yeast CD conjugates has been observed with MRS (17).…”

Differences in metabolism of 5-fluorouracil and 5-fluorouridine and regulation by glucosamine in human colon cancer multicell tumor spheroids

“…No other signals were detected within the range of 44.27 ppm upfield and downfield relative to FUra, including the FdUMP resonance 0.23 ppm downfield from FUMP. 17 The FUrd resonance was centered at 3.83 ppm relative to FUra. 10 After experiments, cell-free perfusates gave rise only to 19 F resonances originating from FUra or FUrd, indicating that signals from metabolites originated entirely from MTS.…”