Reduction of amphetamine hydroxylamine and other aliphatic hydroxylamines by benzamidoxime reductase and human liver microsomes

For the reduction of N-hydroxylated derivatives of strongly basic functiona l groups, such as amidines, guanidines, and aminohydrazones, an oxygen-inse nsitive liver microsomal system, the benzamidoxime reductase, has been desc ribed. To reconstitute the complete activity of the benzamidoxime reductase , the system required cytochrome bs, NADH-cytochrome bs-reductase, and the benzamidoxime reductase, a cytochrome P450 enzyme, which has been purified to homogeneity from pig liver. It was not known if this enzyme system was a lso capable of reducing aliphatic hydroxylamines. The N-hydroxylation of al iphatic amines is a well-known metabolic process. It was of interest to stu dy the possibility of benzamidoxime reductase reducing N-hydroxylated metab olites of aliphatic amines back to the parent compound. Overall, N-hydroxyl ation and reduction would constitute a futile metabolic cycle. As examples of medicinally relevant compounds, the hydroxylamines of methamphetamine, a mphetamine, and N-methylamine as model compounds were investigated. Formati on of methamphetamine and amphetamine was analyzed by newly developed HPLC methods. All three hydroxylamines were easily reduced by benzamidoxime redu ctase to their parent amines with reduction rates of 220.6 nmol min(-1) (mg of protein)(-1) for methamphetamine, 5.25 nmol min(-1) (mg of protein)(-1) for amphetamine, and 153 nmol min(-1) (mg of protein)(-1) for N-methylhydr oxylamine. Administration of synthetic hydroxylamines of amphetamine and me thamphetamine to primary rat neuronal cultures produced frank cell toxicity . Compared with amphetamine or the oxime of amphetamine, the hydroxylamines were significantly more toxic to primary neuronal cells. The benzamidoxime reductase is therefore involved in the detoxication of these reactive hydr oxylamines.