Induction and Purification of Cytochrome-P450 1A1 from 3-Methylcholanthrene-Treated Tilapia, Oreochromis niloticus × Oreochromis aureus

Pretreatments of freshwater fish tilapia, Oreochromis niloticus × O. aureus, with 3-methylcholanthrene (3-MC) and polychlorinated biphenyls (PCBs) increased liver microsomal cytochromes P450 (P450) and b5 contents, benzo[a]pyrene hydroxylation, and 7-ethoxyresorufin and 7-ethoxycoumarin O-dealkylations. The pretreatments also increased gill microsomal benzo[a]pyrene and 7-ethoxyresorufin oxidations. Immunoblot analysis of liver and gill microsomes revealed that 3-MC and PCBs induced a protein recognized by the mouse monoclonal antibody (MAb) 1-12-3 against scup P450 1A1. Northern analysis of liver and gill RNA showed that 3-MC and PCBs increased the intensity of 2.9-kb- and 1.5-kb-sized mRNA bands hybridizable to a trout P450 1A1 cDNA probe. Pretreatment with phenobarbital was without effects on the monooxygenase activity or protein or mRNA levels in liver and gill. A 3-MC-inducible P450 hemoprotein (Mr = 59,000) and a NADPH-cytochrome P450 reductase flavoprotein (Mr = 74,000) were purified from liver microsomes. The tilapia P450 hemoprotein showed an absorption maximum at 447 nm in CO-difference spectrum and a strong immunoreactivity with MAb 1-12-3. A reconstituted tilapia monooxygenase system consisting of P450 and NADPH-cytochrome P450 reductase was effective in the catalysis of 7-ethoxyresorufin, benzo[a]pyrene, and 7-ethoxycoumarin oxidations, but not in N-nitrosodimethylamine demethylation. These results show that 3-MC and PCBs can induce P450 1A1 in tilapia liver and gill and the tilapia P450 is highly similar to other teleost P450 1A1 with respect to spectral, immunochemical, and catalytic properties.