Penclomedine (PEN) is a synthetic
pyridine derivative that has been selected for clinical development based on its activity against human and mouse
breast tumors implanted in mice. Its mechanism of action was unclear, and we were interested in determining its mechanism of cytotoxicity in vitro and in vivo. We found
chromosome breaks, gaps, and exchanges in P388
ascites cells from BD2F1 mice treated with 200 mg/kg PEN. Maximal observed damage occurred 24 hr after
drug administration. Alkaline elution indicated only limited
DNA strand breaks and interstrand cross-linking. In vitro, PEN (75 micrograms/mL) inhibited
RNA and
DNA syntheses almost completely. In addition, incubation of [14C]PEN with rat liver S-9 fraction in the presence of
calf thymus DNA resulted in the stable transfer of radioactivity to
DNA. Addition of
butylated hydroxytoluene, a
free radical scavenger, to the incubation mixture inhibited the binding of
drug to
DNA, implicating
free radicals as the ultimate reactive species. These data suggest that PEN can be metabolized to
free radical,
DNA-reactive products, and that its cytotoxicity is due to chromosomal damage produced by monofunctional alkylation. As an alternate mechanism, the ability of PEN to inhibit cellular
dihydroorotate dehydrogenase was explored. Although PEN is an inhibitor of this
enzyme in cells in vivo, in vitro, and in isolated cell sonicates, HPLC analyses of
ribonucleotide triphosphate pools in P388 cells showed that all triphosphates had increased, especially
UTP. Addition of
uridine to the cell culture failed to prevent PEN-mediated cytotoxicity, suggesting that inhibition of de novo
pyrimidine biosynthesis was not likely to be an important mechanism of action of this
drug. These data suggest that PEN is activated in cells to a
free radical that binds
DNA.