CYTOCHROME-P450 SPECIFICITIES OF ALKOXYRESORUFIN O-DEALKYLATION IN HUMAN AND RAT-LIVER

The O-dealkylations of ethoxyresorufin and pentoxyresorufin are widely used activity probes for measuring the cytochrome P450 forms, CYP1A1 and CYP2B1, respectively, and their induction by xenobiotics, but ther e is confusion in the literature about which P450 forms are detected i n human and rat liver microsomes by these and homologous alkoxyresoruf ins. High performance liquid chromatographic analysis confirmed that O -dealkylation to resorufin was the sole or predominant route of metabo lism for both short-chain and long-chain alkoxyresorufins and benzylox yresorufin by rat liver microsomes. The purified 3-methylcholanthrene (3MC)-induced rat P450 forms, CYP1A1 and CYP1A2, and a possible varian t form, CYP1A1, showed substrate selectivities for propoxyresorufin, methoxyresorufin and ethoxyresorufin, respectively. Purified phenobarb itone (PB)-induced CYP2B1 was selective for benzyloxyresorufin and pen toxyresorufin. Purified constitutive CYP2C6 was much less active than CYP2B1 or the CYP1A forms but showed distinctive selectivity for benzy loxy-, propoxy- and butoxyresorufin. CYP1A2 and CYP2C6 metabolised n-p ropoxy- and n-butoxyresorufin much more rapidly (8-23-fold) than iso-p ropoxy- and iso-butoxyresorufin, whereas CYP1A1 and CYP2B1 showed only small differences (2-5-fold) between the n- and iso-homologues and CY P1A1 and CYP2B2 did not discriminate between them. The results show t hat ratios between different alkoxyresorufin O-dealkylation (AROD) act ivities can be more useful than absolute values of single activities f or identifying P450 forms. Anti-P450 antibody and furafylline inhibiti on of rat liver microsomal AROD confirmed that ethoxyresorufin was a s elective probe for CYP1A1 in 3MC-induced and isosafrole (ISF)-induced microsomes and that pentoxy- and benzyloxyresorufins both selectively measured CYP2B1 in PB-induced and ISF-induced microsomes. Ethoxyresoru fin was not a selective probe for CYP1A in liver microsomes from untre ated or PB-induced rats, however, where it was metabolised mainly by C YP2C6 and CYP2B1, respectively. Pentoxyresorufin and benzyloryresorufi n were metabolised by several different P450 forms in non-induced rat liver microsomes but mainly by the CYP1A subfamily in 3MC-induced micr osomes and by CYP2B1 in PB- and ISF-induced microsomes. Although with purified rat P450s methoxyresorufin appeared not effectively to discri minate CYP1A2 from CYP1A1, CYP1A1 or CYP2C6, furafylline inhibition i ndicated that methoxyresorufin was a selective measure of CYP1A2 in un induced and 3MC-induced rat liver microsomes but not in ISF- or PB-ind uced microsomes. In human liver microsomes, antibody inhibition and fu rafylline inhibition showed that ethoxyresorufin and methoryresorufin were metabolised mainly by CYP1A2, whilst benzyloxyresorufin metabolis m was due mainly to the CYP3A subfamily but also involved CYP1A2 and C YP2A6. There was considerable interindividual variation in the roles o f different P450 forms in all three reactions in human liver.