Liver microsomal cytochromes P450-dependent alkoxyphenoxazone O-dealkylation in vitro by alligator and rat: activities, inhibition, substrate preference, and discrimination factors.

Six substituted alkoxyphenoxazones (resorufins) and four inhibitors of P450-dependent mixed-function oxygenases (MFO) were used to probe the breadth and extent of P450 metabolism induced by pretreatment with five xenobiotic chemicals in liver microsomes of the American alligator, Alligator mississippiensis. Phenobarbital (PB), 3-methylcholanthrene (3MC), and PB-3MC co-pretreatment elicited major induction of alligator MFO activity measured by alkoxyresorufin O-dealkylation (AROD). The induced levels of activities observed with appropriate substrate, 7-ethoxy, 7-methoxy, 2-phenylbenzyloxy, 7-pentoxy, or 7-benzyloxyresorufin (EROD, MROD, PBROD, PROD and BROD, respectively), were 10 to 100 times lower in alligator as compared to rat. The exception was a higher level of isopropoxyresorufin O-dealkylation (IPROD) in alligator. The induction regimes used in alligator and rat revealed marked differences in substrate preference, discrimination factors (DF) for various inducible P450 isoforms. EROD, a classic indicator of CYP1A activity in rat, had a low DF in alligator. MROD was the best discriminator in alligator of CYP1A-type induction. In contrast to rats, pretreatment of alligators with Aroclor 1254, 2,2',4,4' tetrachlorobiphenyl, and clofibrate caused minor alterations in AROD relative to untreated controls. The inhibitors, alpha-napthaflavone, 1-ethynylpyrene, SKF 525A, and 9-ethynylphenanthrene, inhibited AROD activity of the expected P450 isoform. For example, 10 microM alpha-napthaflavone inhibited liver microsomal EROD catalyzed by 3MC-inducible isoforms from alligator by 90% and from rat by 97%. Similarly, 10 microM SKF 525A inhibited PROD catalyzed by PB-inducible isoforms by 63% and 79% in alligator and rat liver microsomes, respectively. To the best of our knowledge, the present studies are the first to show PB induction of P450 activities typical of the mammalian CYP2 family and their inhibition with classical inhibitors in alligator liver. While our data indicate metabolism of P450 substrates with preferences to certain isoforms, it remains to be established which isoforms exert catalytic function in alligator and whether these are homologues or orthologues of mammalian isoforms.