Acetylation of 2-aminofluorene derivatives by dog hepatic microsomes

Dog urinary bladder is a target organ of carcinogenic arylamines. However, dog hepatic and urothelial cytosols lack acetylation enzymes that are capable of activating N-hydroxy metabolites of arylamines, suggesting that other enzymes may be involved. In the present study, we found that dog liver microsomes were capable of N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), and that these activities were inhibited by paraoxon. The 0.25% Triton X-100 extractable fraction of microsomes was resolved on an ion-exchange column into three different proteins that retained these activities. Two of these proteins, designated as enzyme I and enzyme II, were further chromatographed on a Sephacryl S-300 column. As judged from the gel filtration profile, the mol. wt of enzyme I was approximately 180 kDa and that of enzyme II was greater than 700 kDa. SDS-PAGE analysis showed that the subunit weight of enzyme II was approximately 150 kDa. In addition to N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-OH-AAF, these three enzymes were capable of the deacetylation of 2-acetylaminofluorene, N-OH-AAF and 4-nitrophenyl acetate. The ability of these microsomal enzymes to activate N-hydroxylated aromatic amines and the presence of these enzymes in urothelial cells, reported previously, suggests that they may play an etiological role in the carcinogenicity of these agents in the dog.