Immunochemical analysis of liver microsomal cytochromes P450 of the American alligator, Alligator mississippiensis

Ten antibodies raised against various mammalian and fish cytochromes P450 (CYP) enzymes were used to probe the effects of xenobiotic pretreatment on liver microsomes of the American alligator, Alligator mississippiensis. Pretreatment with phenobarbital (PB), 3-methyIcholanthrene (3MC), and PB plus 3MC elicited significant induction of multiple CYP enzymes in alligator, as detected by antibodies to CYP1A, CYP2B, CYP2C, CYP2E, CYP2K, and CYP3A. In contrast to the rat, 3MC treatment induced alligator liver microsomes that were immunoreactive with antibodies to CYP2 family enzymes. Induction of CYP enzymes was not as apparent with the Aroclor 1254 (ARO), and 2,2',4,4' tetrachlorobiphenyl (TCB) pretreatment used; fewer CYP enzymes primarily detected with antibodies against CYP2C or CYP2E were observed. Clofibrate (CLO; 80 mg/kg Days 1-4), markedly induced CYP4A in rat but this induction was not apparent in alligator. A purified PE-induced alligator liver microsomal CYP enzyme cross-reacted with several antibodies raised against CYP2 family enzymes but did not cross-react with antibodies raised against other CYP families. This indicates the PB-inducible CYP in alligator shares some epitope homology with several CYP2-family enzymes from other animals. These experiments demonstrate the usefulness and limitations of using antibodies across phylogenetic classes. While indicating the presence of CYP enzymes that have epitope homology with CYP1A, CYP2, CYP3 and CYP4 enzymes in alligator, it remains to be established whether these CYP forms are alligator orthologues of mammalian enzymes. In all cases, the relative abundance of alligator liver microsomal CYP as determined by immunoblot analysis appeared lower than found in rat. The presence and induction of CYP indicated by immunochemical analysis, corroborated previously reported enzymatic studies of the same microsomal preparations (Ertl et al., 1998a). Thus, increases in CYP protein by the various inducers employed were paralled by the increases in CYP enzyme-specific or selective activities, e.g., induction of CYP1A protein corresponded with induction of EROD. (C) 1999 Academic Press.