Microsomal activation of benzo[a]pyrene to mutagens by Alligator mississippiensis in vitro: induction by 3-methylcholanthrene

Abstract

The metabolism of benzo[a]pyrene (BaP) to mutagens by liver microsomes of the American alligator, Alligator mississippiensis, was studied as a function of induction by pretreatment with 3-methylcholanthrene (3MC). 3MC-pretreatment greatly enhanced the microsomal-mediated oxidation of BaP to mutagens in the umu mutagenicity assay. Analysis of the BaP metabolite profile by reversed-phase HPLC showed that this increase in mutagenicity of BaP corresponded to an increase in the formation of BaP-diols, tetrols and diol epoxide as compared with untreated alligators. SDS-polyacrylamide gel electrophoresis of the microsomal fractions indicated the presence of additional protein bands within the 50kDa molecular weight region in microsomes of 3MC-pretreated alligators. Western blot analysis revealed the presence of a protein band of about 50 kDa that cross-reacted with a polyclonal antibody to rabbit P450 LM4 and a monoclonal antibody to scup P450E. The former recognizes both CYPIA1 and CYPIA2 in liver microsomes from 3MC-treated rats and the latter is believed to be orthologous to these isoforms; thus, our data suggest the 3MC-inducible liver microsomal P450 is the alligator orthologue of one of these isoforms. The results also suggest that 3MC induces P450 in A. mississippiensis with structural and functional similarity to forms present in mammals.