INDUCTION OF RAT HEPATIC CYTOCHROME-P450 2B SUBFAMILY BY AZIDOPHENOBARBITAL, AS A POSSIBLE PHOTOAFFINITY PROBE FOR THE PUTATIVE PHENOBARBITAL RECEPTOR - COMPARATIVE-STUDY WITH MODIFIED PHENOBARBITALS WITH DIFFERENT FUNCTIONAL-GROUPS

To study the specific target to which phenobarbital (PB) binds, result ing in the induction of cytochrome P450, we prepared two azido-PBs (AZ PBs) as photoaffinity ligands. The azido substituent was introduced at the para-or meta-position of the PB aromatic ring. In this study, we estimated the utility of these compounds by examining their inducing a ctivities in vivo in rats. Induction was assessed by immunoblotting wi th anti-CYP2B1/2 antibody and measuring testosterone-metabolizing acti vity, using hepatic microsomes, Administration of p-AZPB to rats incre ased hepatic CYP2B1/2 protein and testosterone 16 beta-hydroxylase act ivity, although the effects were less than those of unmodified PB, m-A ZPB showed no effect in the induction of CYP2B1/2, To assess the speci ficity of the effects of substituents, we compared the inducing activi ties of p/m-nitro-PBs, p/m-amino-PBs, and p/m-hydroxy-PBs with those o f AZPBs. The results showed that p-nitro-PB, m-amino-PB, and p-hydroxy -PB were also potent inducers for CYP2B1/2, with lower activity than t hat of unmodified PB, whereas the other three isomers had no effect. T hese results suggest that 1) the absence of any substituents on the ar omatic ring of PB is needed for maximal inducing activity and 2) subst itution at: the meta-position of the PB aromatic ring tends to reduce effectiveness as an inducer more than does substitution at the para-po sition. Because p-amino-PB and p-acetylamino-PB, the minor and major m etabolites of p-AZPB, respectively, were without effect in the inducti on of CYP2B1/2, the effect of p-AZPB was considered to be due to the u nchanged compound itself. The present study demonstrates that, based o n the weak but positive ability to induce CYP2B1/2, 9-AZPB may be a us eful tool for identifying the putative PB receptor.