MECHANISM OF TOXICITY OF FLUOROCYTOSINE AND FLUOROURACIL

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.