Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

Gary W. Winston


Alligator mississippiensis contains all of the components of the hepatic microsomal mixed function oxidase system. In general, basal and induced levels of P450 specific content, immunochemically detected abundance and examined activities are less than in mammals. In alligator at least two classes of P450s are inducible by xenobiotics: those induced by 3-methylcholanthrene (3MC) and those induced by phenobarbital (PB). PB and 3MC induce multiple P450 isoforms as detected by western blot and enzymatic analyses. The induced level of alkoxyphenoxazone O-dealkylation (AROD) was 10- to 100-fold lower in alligator than in rat, with the exception of phenylbenzyloxyphenoxazone O-dealkylation. In contrast to mammals methoxyphenoxazone and benzyloxyphenoxazone exhibited the greatest ability of AROD substrates to discriminate between 3MC- and PB-induced isoforms. Carbon monoxide binding, western blots, and enzymatic activity indicates that the PB and 3MC time-courses take at least 48-72 h to reach full induction, longer than found in mammals. In contrast to rat, aroclor and tetrachlorobiphenyl yielded faint induction in western blots and along with clofibrate exhibited only minor alterations in AROD activity. Clofibrate also failed to induce lauric acid hydroxylation, a CYP 4A activity. Classical inhibitors of P450 inhibited AROD activity of the expected P450 isoforms. While antibodies used across phylogenetic classes lost specificity they only crossreacted within a gene family. Western blot, enzymatic and N-terminal sequence analysis indicates that the purified PB-induced P450 is a CYP 2 family isoform. Whether alligator isoforms are transcriptionally regulated remains unanswered as northern blots were inconclusive. The contention that alligator CYP 1A isoforms are transcriptionally regulated is supported by the presence of Ah receptors. The markedly different responses to single and multiple xenobiotics, lower or absent activities, altered discrimination factors, loss of antibody specificity and slower rates of induction challenges the view of how reptiles respond to xenobiotics and the interpretation of P450 assays as biomarkers of environmental pollution. While indicating the presence of alligator P450s in several families, the number of isoforms present, the breadth of substrates and whether these P450s are alligator homologues/orthologues of mammalian isoforms remain to be established.