Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufinsin rat brain microsomes

Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was ca rried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B 2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for b oth the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufin s varied in different regions of the brain. Microsomes from the olfactory l obes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresor ufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities . The dealkylation was found to be inducer selective. While pretreatment wi th phenobarbital (PB; 80 mg/kg; i.p. x 5 days) resulted in significant indu ction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthre ne (MC; 30 mg/kg; i.p. x 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significan tly induced the activity of EROD (3 fold) while PB had no effect on it. Kin etic studies have shown that this increase in the activities following pret reatment with P450 inducers was associated with a significant increase in t he velocity of the reaction (V-max) of O-dealkylation. In vitro studies usi ng organic inhibitors and antibodies have further provided evidence that th e O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of a-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a conce ntration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the c ase of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selec tivity of P450 isoenzymes in rat brain microsomes.