Cytochrome P450 product complexes and glutathione consumption produced in isolated hepatocytes by norbenzphetamine and its N-oxidized congeners

Abstract

Incubation of liver microsomes from phenobarbital-treated rats with norbenzphetamine (I) and its two N-oxidized metabolites, N-hydroxynorbenzphetamine (II) and the corresponding nitrone (III), in the presence of NADPH and molecular oxygen, gave rise to the formation of cytochrome P450 product complexes characterized by maximal absorbance at 455 nm. The complex forming activity increased in the order I, II and III, with the nitrome (III) exhibiting an activity of about 60 per cent of that of N-hydroxyamphetamine (IV). The same relative order of complex forming activity was seen also when incubations were performed with hepatocytes isolated from phenobarbital-treated rats, but the complex formation was less rapid as well as less extensive. Liver tissues from untreated rats exhibited the same complex forming pattern, but with considerably lower activity. Incubations of hepatocytes with I, II and III caused a decrease in the cellular level of reduced glutathion (GSH) and II and III caused the most significant drops in the GSH level. The decrease in GSH was enhanced in hepatocytes from phenobarbital-treated rats. N-hydroxyamphetamine had no effect on the cellular GSH level and the amount of oxidized glutathione (GSSG) was unaffected by addition of I-IV. It is suggested that N-hydroxyamphetamine (IV), formed by hydrolysis of the nitrone (III), is the ultimate substrate for the reaction leading to complex formation. The results indicate that the nitrone (III) is a common intermediate in the reactions leading to the interaction with GSH and cytochrome P450 complex formation and by reacting with III, GSH decreases the concentration of cytochrome P450-binding norbenzphetamine metabolites in the hepatocyte.