Reduction of Nilutamide by NO Synthases: Implications for the Adverse Effects of This Nitroaromatic Antiandrogen Drug

Nitric oxide synthases (NOSs) are flavohemeproteins that catalyze the oxidation of l-arginine to l-citrulline with formation of the widespread signal molecule NO. Beside their fundamental role in NO biosynthesis, these enzymes are also involved in the formation of reactive oxygen species and in the interactions with some xenobiotic compounds. Nilutamide is a nonsteroidal antiandrogen that behaves as a competitive antagonist of the androgen receptors and is proposed in the treatment of metastatic prostatic carcinoma. However, therapeutic effects of nilutamide are overshadowed by the occurrence of several adverse reactions mediated by toxic mechanism(s), which remain(s) poorly investigated. Here, we studied the interaction of NOSs with nilutamide. Our results show that the purified recombinant neuronal NOS reduced the nitroaromatic nilutamide to the corresponding hydroxylamine. The reduction of nilutamide catalyzed by neuronal NOS proceeded with intermediate formation of a nitro anion free radical easily observed by EPR, was insensitive to the addition of the usual heme ligands and l-arginine analogues, but strongly inhibited by O(2) and a flavin/NADPH binding inhibitor. Involvement of the reductase domain of nNOS in the reduction of nilutamide was confirmed by (i) the ability of the isolated reductase domain of nNOS to catalyze the reaction and (ii) the stimulating effect of Ca(2+)/calmodulin on the accumulation of hydroxylamine and nitro anion radical. In a similar manner, the recombinant inducible and endothelial NOS isoforms also displayed nitroreductase activity, albeit with lower yields. The selective reduction of nilutamide to its hydroxylamino derivative by the NOSs could explain some of the toxic effects of this drug.