The long-term objective of this research proposals is to improve the clinical effectiveness of the fluoropyrimidine drugs through and increased understanding of the biochemical and pharmacologic factors that determine toxicity. There are 4 major specific aims: I) Examination of fluoropyrimidine metabolism in the liver with assessment of the quantitative importance of catabolism as well as clarification of a potentially novel pathway of fluorouracil (FUra) metabolism (? conjugation-glucuronide). The catabolic enzymes will be purified from rat and human liver followed by kinetic assessment. Attempts will be made to confirm the identity of the unknown FUra metabolite formed in rat liver. Is this or other novel metabolites formed in human liver? Fluoropyrimidine metabolism will be re-examined using reversed-phase ion pair HPLC at the cellular (hepatocyte and nonhepatocyte), in vivo (with selective catheterization of portal, hepatic, and biliary vessels), and inclinical studies; II) Examination of toxicity of dihydrofluorouracil (FUH2). Human tumor (breast + colon) and human bone marrow will be examined to confirm our preliminary observations of selective toxicity of FUH2. The cellular metabolic fate of radiolabeled FUH2 to be prepared enzymatically will be examined in susceptible cells. An attempt will be made to determine the mechanism of activation of FUH2; III) Examination of the effect of FUra incorporation into DNA on DNA structure and the potential correlation with DNA-directed component of FUra toxicity. An assessment will be made of the effect on parental as well as newly synthesized DNA, with an assessment of whether chain termination of further DNA elongation occurs, whether initiation of new chain synthesis is inhibited, whether FUra is incorporated into double as well as single stranded DNA, and whether fragmentation of DNA correlates with timing and duration of toxicity. The correlation of DNA structural damage with inhibition of thymidylate synthetase activity and DNA-directed toxicity will be carried out with regard to recovery as well as toxicity; and IV) The biochemical basis of fluorocytosine (FC) toxicity in mammalian cells will be examined. Bone marrow cells exposed to toxic concentrations of FC will be examined to determine if FUra or FUra metabolites are formed. An attempt will be made to confirm whether FC itself is incorporated as FC into RNA and possibly DNA. The possible modulation of FC-induced bone marrow toxicity by allopurinol will be examined in a proposed clinical protocol.
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