PHENYLBUTAZONE PEROXIDATIC METABOLISM AND CONJUGATION

Phenylbutazone, a nonsteroidal anti-inflammatory drug, elicits therape utic as well as toxic effects by unknown pathways. Phenylbutazone was shown to form a conjugate with the heterocyclic amine bladder carcinog en 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT). To understand further the reactivity of these compounds, this study was conducted to identif y the conjugate formed and determine the mechanism of conjugate format ion. Both prostaglandin H synthase and horseradish peroxidase catalyze d conjugate formation. This conjugate was identified by H-1-NMR to be zolyl]-4-butyl-1,2-diphenyl-3,5-pyrazolidinedione. Phenylbutazone-medi ated oxygen uptake was inhibited by ANFT (0.1 mM) and the spin traps 5 ,5-dimethyl-1-pyrroline-N-oxide (200 mM) and tert-nitrosobutane (4 mM) . By contrast, phenol (0.005 to 0.25 mM) and aminopyrine (0.4 mM) stim ulated oxygen uptake. None of these agents mediated oxygen uptake in t he absence of phenylbutazone. Conjugate formation was significantly in creased by phenol (0.005-0.25 mM) and aminopyrine (0.4 mM), as well as in the absence of oxygen. Conjugate formation was inhibited by 5,5-di methyl-1-pyrroline-N-oxide (200 mM), tert-nitrosobutane (4 mM), ascorb ic acid (2 mM), and 95% oxygen. Horseradish peroxidase initiated conju gate formation at much lower concentrations than it metabolized ANFT. The stoichiometric relationship between phenylbutazone and ANFT, with respect to conjugate formation, was complex. With the concentration of ANFT fixed at 0.05 mM, phenylbutazone exhibited saturation kinetics w ith a K(m) of 0.2 mM. In contrast, saturation kinetics were not observ ed with ANFT. K(m) values for ANFT varied with the concentration of ph enylbutazone used. A large excess of phenylbutazone was required relat ive to ANFT perhaps due to the reducing activity of ANFT. The results were interpreted as phenylbutazone being the peroxidatic substrate and the phenylbutazone carbon-centered radical formed by this process rea cting with ANFT to produce the conjugate. The results may have importa nt implications in the therapeutic and toxic effects of phenylbutazone , and in the chemoprevention of ANFT-induced bladder cancer.