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

Abstract

Flucytosine (5-FC)-resistant strains were isolated from the haploid opportunistic pathogen Candida glabrata by UV-induced mutation and fluoropyrimidine selection. These strains were characterized biochemically, and the metabolism of fluorinated pyrimidines was studied by '9F nuclear magnetic resonance spectroscopy. No evidence was obtained from these studies for degradative metabolism of the fluorinated derivatives. In the parental susceptible strain of C. glabrata, 5-fluorouracil but not 5-FC was detected within the cells. 5-Fluorouracil was also present in the culture supernatant after incubation of the cells with 5-FC. The distribution of fluorinated derivatives within the 5-FC-resistant strains was consistent with their genotype. Two strains of C. glabrata which had only a partial loss of cytosine deaminase and UMP pyrophosphorylase activity had high levels of resistance to 5-FC. Both C. glabrata and Candida albicans were susceptible to 5-fluorouridine. This compound but not the anticancer drug 5-fluoro-2-deoxyuridine was shown to be transported into susceptible cells by a specific uridine permease.Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for identifying drug metabolites (33) and investigating drug-receptor interactions in cell-free solutions (13). It can also be used noninvasively to study metabolism in vivo in microorganisms, isolated tissues, intact animals, or humans (18). The practical application of NMR spectroscopy in these living systems, however, is limited by sensitivity considerations.The "9F nucleus has the second-highest NMR sensitivity of the stable nuclei (ca. 85% of that of 'H at constant field). It is the naturally occurring isotope of fluorine (100% natural abundance) and is not present in living systems to any significant degree. The (6) and, more recently, in the liver (22, 31), plasma, and urine (8) of patients undergoing chemotherapy. In addition, it has been used to demonstrate tumor trapping of 5-FU in tumorbearing humans and rabbits (32). NMR spectroscopy has also been successful in demonstrating the existence of two distinct pathways of 5-FU degradation in E. coli (14).'9F NMR spectroscopy has also been employed in a preliminary study of the uptake and metabolism of the clinical antifungal drug 5-flucytosine (5-FC) in the medically important yeast Candida albicans (10). 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. Similar studies have been reported with Aspergillus spp. in which it has been shown that the main pathway of 5-FC metabolism is via the pyrimidine salvage pathway to 5-fluorouridine triphosphate (5-FUTP). In Aspergillus fumig...

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