Induction Time Course of Cytochromes P450 by Phenobarbital and 3-Methylcholanthrene Pretreatment in Liver Microsomes of Alligator mississippiensis Robin P. Ertl,* John J. Stegeman† and Gary W. Winston*‡ *DEPARTMENT OF BIOCHEMISTRY,LOUISIANA STATE UNIVERSITY,BATON ROUGE, LA 70803; AND †DEPARTMENT OF BIOLOGY,WOODS HOLE OCEANOGRAPHIC INSTITUTION, WOODS HOLE, MA 02543, U.S.A. ABSTRACT. Alligator mississippiensis has at least two classes of inducible hepatic microsomal cytochromes P450 (CYP): (1) those induced by 3-methylcholanthrene (3MC), and (2) those induced by phenobarbital (PB). The rates of induction by these xenobiotic compounds are significantly slower than those reported for mammals. Carbon monoxide binding, western blots, and enzymatic activity measurements indicated that at least 48 –72 hr are required to reach full induction. A methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone (resorufin) O-dealkylation (MROD, EROD, PROD, and BROD) profile was indicative of substrate selectivity typical of 3MC- and PB-induced P450s. MROD and BROD showed the greatest ability to discriminate between alligator hepatic microsomes induced by 3MC and PB, respectively. This is in contrast to mammals, in which EROD is a biomarker of polycyclic aromatic hydrocarbon exposure because of its ability to discriminate the induction of CYP 1A. In a similar manner, PROD is a highly preferred activity of CYP 2B in mammals; thus, it is used to indicate CYP 2B induction. The induction of P450 by PB is a general phenomenon in mammals and birds. To the best of our knowledge, this is the first report demonstrating PB induction of P450 activities typical of the mammalian CYP 2 family isoforms in alligator or any reptilian liver. The importance of this finding to the evolution of CYP 2 family regulation by PB is heightened by the fact that induction by this xenobiotic is not common to fish and other lower vertebrates (Ertl RP and Winston GW, Comp Biochem Physiol, in press). Although indicating the presence of CYP 1A- and CYP 2B-like isoforms in alligator, it remains to be established how closely related these alligator P450s are to mammalian isoforms. BIOCHEM PHARMACOL 55;9:1513–1521, 1998. © 1998 Elsevier Science Inc. KEY WORDS. alligator; cytochromes P450; induction time course; 3-methylcholanthrene; mixed function oxidase; phenobarbital The American alligator (Alligator mississipiensis) has be- come an important agricultural commodity for both its meat and its hide since its removal from the endangered species list. It is the most economically important cultured reptilian species in Louisiana, with 158,760 wild and farm-raised alligators harvested in 1995 [1]. In the same year, the United States, as a whole, harvested 208,332 alligators. Based on Louisiana commodity prices, the annual gross farm value is about $17,000,000 and $24,000,000 in Louisiana and the U.S., respectively. The effect and metabolism of drugs used in aquaculture of this organism are of particular importance in light of the mandate of the U.S. Food and Drug Administration calling for the investigation and development of minor animal use drugs and alteration of the guidelines to encourage appli- cation for drug approval in minor agricultural species [2– 8]. The absence of currently approved drugs stems from the comparatively low gross market value of these minor species, which precludes the amount of research previously required in the approval process. Diseases associated with the aquaculture of alligators and the effects of some classic major use drugs are starting to be investigated [9 –13]. The lack of approved, safe, and effective drugs for alligator aquaculture poses direct and indirect human health risks in light of the increasing use of these animals as agricultural commodities [5]. Understanding how this reptile metabolizes pollutants and other xenobiotics from farm runoff and chemical industry discharge into its environment [14, 15] is of concern, as exposure not only threatens this organism, but, as a food commodity it is also a potential source of human exposure. Reproductive failure in alligator populations in Florida has been linked to chlorinated hydrocarbon pesti- cide exposure [16 –19]. Such chlorinated hydrocarbons are P450 inducers and are estrogenic molecules that may ‡ Corresponding author: Dr. Gary W. Winston, Department of Toxicol- ogy, North Carolina State University, Box 7633, Raleigh, NC 27695- 7633. Tel. (919) 515-4377; FAX (919) 515-7169; E-mail: gary_ winston@ncsu.edu. Received 30 June 1997; accepted 25 November 1997. Biochemical Pharmacology, Vol. 55, pp. 1513–1521, 1998. ISSN 0006-2952/98/$19.00 + 0.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0006-2952(98)00003-3