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Evidence from Rat Hepatocytes of an Unrecognized Pathway of 5-Fluorouracil Metabolism with the Formation of a Glucuronide Derivative¹

Department of Pharmacology and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294 [J-P. S., R. B. D.]; Departments of Medicine and Pharmacology, Medical College of Virginia, Richmond, Virginia 23298 [D. S. C., D. A. G., I. D. G.]; and The Institut National de la Sante et de la Recherche Medicale U278, Laboratoire de Pharmacocinetique et Toxicocinetique, 13385 Marseille Cedex 5, France [J-P. S., J.-P. C.]

Isolated rat hepatocytes in suspension were exposed to [³H]-5-fluorouracil for intervals over 2 h, following which the cells were removed from the media and sonicated, and the cytoplasm was sampled. High-performance liquid chromatography was used to separate 5-fluorouracil (FUra) from its known anabolites and catabolites, with subsequent quantitation of these metabolites by measurement of radioactivity. As the extracellular concentration of FUra was increased above 30 µM, the intracellular levels of FUra increased, with detection of a new peak of radioactivity distinct from any of the known anabolites or catabolites. This new metabolite, "G," increased in concentration as the extracellular concentration of FUra was raised above 1 mM. Inhibition of FUra catabolism by 2 mM thymine resulted in a further increase in intracellular FUra (approaching the extracellular FUra concentration) and was accompanied by a further increase in the intracellular concentration of "G," demonstrating that "G" was not formed via the catabolic pathway. The increase in intracellular FUra and "G" was not accompanied by an increase in intracellular anabolites, suggesting that "G" was formed via a novel metabolic pathway. "G" was retained within the hepatocytes, although it was not bound to intracellular macromolecules. "G" was converted to FUra in the presence of ß-D-glucuronidase; this reaction was inhibited with the addition of saccharo-1,4-ß-lactone, a specific inhibitor of the ß-D-glucuronidase. This data, together with evidence from hepatocyte homogenates in which formation of "G" was shown to be dependent on the concentration of uridine-5'-diphosphoglucuronic acid, demonstrates that "G" is a glucuronide of FUra. The formation of "G" suggests that FUra is metabolized via a previously unrecognized metabolic pathway.

¹ This work was supported by NIH Grants CA 23412, CA 40530, CA 16906, and AM 18976.

² Supported in part by the National Cancer Institute, Scientist Exchange Program G 50111 (United States-France Cancer Program).

³ To whom requests for reprints should be addressed, at Division of Clinical Pharmacology, University of Alabama in Birmingham, Birmingham, AL 35294.

Received 10/22/84. Revised 2/14/85. Accepted 2/21/85.