[CANCER RESEARCH 45, 2450-2455, June 1985] Evidencefrom Rat Hepatocytesof an UnrecognizedPathwayof 5-Fluorouracil Metabolismwiththe Formationof a GlucuronideDerivative1 Jean-Pierre Sommadossi,2 David S. Cross, David A. Gewirtz, I. David Goldman, Jean-Paul Cano, and Robert B. Diasio3 Department ot Pharmacology and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294 [J-P. S., R. B. D.J; Departments of Medicine and Pharmacology, Medical College of Virginia, Richmond, Virginia 23298 [D. S. C., D. A. G., I. D. G.J; and The Institut National de la Sante et de la Recherche Medicale U278, Laboratoire de Pharmacocinetique et Toxicocinetique, 73385 Marseille Cedex 5, France [J-P. S., J.-P. C.] ABSTRACT 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 metabo lites by measurement of radioactivity. As the extracellular con centration of FUra was increased above 30 UM, the intracellular levels of FUra increased, with detection of a new peak of radio activity 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 mw. Inhibition of FUra catabolism by 2 mw thymine resulted in a further increase in intracellular FUra (approaching the extracel lular 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 hepato cytes, although it was not bound to intracellular macromolecules. "G" was converted to FUra in the presence of ß-o-glucuronidase; this reaction was inhibited with the addition of saccharo-1,4-/3- lactone, a specific inhibitor of the 0-D-glucuronidase. This data, together with evidence from hepatocyte homogenates in which formation of "G" was shown to be dependent on the concentra tion 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. INTRODUCTION Since the synthesis of FUra4 25 years ago, most studies of fluoropyrimidine metabolism have concentrated on anabolism, focusing on the conversion of FUra or its nucleosides to ribosyl or deoxyribosyl nucleotides and the subsequent effects on DNA or RNA synthesis or function (2, 12-15, 19, 22, 23, 28). In 1This 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. 4 The abbreviations used are: FUra, 5-fluorouracil; FUH2, dihydrofluorouracil; PUPA, a-fluoro-0-ureidopropionic acid; FBAL, «-fluoro-0-alanine; "G," unknown metabolite of FUra thought to be a glucuronide derivative of FUra; HPLC, high- performance liquid chromatography; UDPGA, uridine 5'-diphosphoglucuronic acid. Received 10/22/84; revised 2/14/85; accepted 2/21/85. contrast, until recently, there have been few studies (3, 4, 21) that have examined FUra catabolism, despite the fact that, in humans, approximately 90% of administered FUra is metabolized via the pyrimidine catabolic pathway (2), with the major site of FUra catabolism believed to be in the liver (7). The development of a highly specific HPLC methodology that can rapidly resolve all of the known anabolites and catabolites of FUra has permitted a reexamination of fluoropyrimidine me tabolism (26). We have recently demonstrated the usefulness of the isolated rat hepatocyte model for studying fluoropyrimidine metabolism because of its excellent agreement with the meta bolic fate of FUra in humans (6). In a previously reported study (26), hepatocytes in suspension were exposed to 30 fiM FUra [a concentration corresponding to that found after systemic admin istration of FUra (2, 6)]. Intracellular and extracellular FUra and its metabolites were quantitated, demonstrating a transport- limited, rapid, and complete conversion of FUra to FUH2, with FUH2 being the major intracellular catabolite, with subsequent conversion to FUPA and then to FBAL, but with no evidence for FUra anabolism (26). In order to better understand the metabolism of FUra at the hepatic site with concentrations more representative of FUra levels achieved following various clinical regimens, including i.v. bolus injection (2), studies were undertaken with FUra concen trations higher than 30 /*M. In the present study, we demonstrate that, as extracellular FUra is increased, the catabolic enzyme dihydrouracil dehydrogenase (EC 1.3.1.2) becomes saturated, resulting in increased intracellular levels of FUra. However, this is not accompanied by evidence of increased anabolism. Most notable is the appearance of a new metabolite distinct from any of the known anabolites or catabolites of FUra that appears to be a glucuronide of the FUra base formed via a previously unrecognized metabolic pathway. MATERIALS AND METHODS Chemicals [6-3H]FUra (20 Ci/mmol) was obtained from Moravek Biochemicals, Inc. (City of Industry, CA) and purified by the HPLC technique described below. [Carboxyl-14C]inulin (2.5 Ci/g) was purchased from Amersham- Searie Corp. (Irvine, CA). FUra and authentic standards of FUH2, FUPA, and FBAL were kindly supplied by Hoffmann-La Roche Laboratories (Nutley, NJ and Basel, Switzerland). /3-o-Glucuronidase (from bovine liver, type B-3), saccharo-1,4-0-lactone, and thymine were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were reagent grade. CANCER RESEARCH VOL. 45 JUNE 1985 2450 Research. on December 27, 2015. © 1985 American Association for Cancer cancerres.aacrjournals.org Downloaded from