Cytochromes P450 in liver of the turtle Chrysemys picta picta and the induction and partial purification of CYP1A-like proteins.

Cytochromes P450 (CYP) in hepatic microsomes from the turtle Chrysemys picta picta and their response to inducers were examined. Freshly caught turtles had one protein (59 kDa) detected in western blot with monoclonal antibody 1-12-3 to scup CYP1A. That same band and a second band were detected with polyclonal anti-mouse Cyp1a1. Polyclonal anti-scup P450B (putative CYP2B) recognized three bands and anti-scup P450A (putative CYP3A), one band. TCB (3,3',4,4'-tetrachlorobiphenyl) at 5 mg kg-1 injected once induced EROD activity 3-fold. Repeated high-dose injections of TCB, 2,3,3',4,4'-pentachlorobiphenyl, Aroclor 1254 or beta-naphthoflavone induced CYP1A 20-fold and P450B-related proteins 2-3-fold. Rates of ethoxy- (EROD) methoxy- (MROD) and pentoxyresorufin O-dealkylases and benzo[a]pyrene (B[a]P) hydroxylase (AHH) were induced by these treatments, and were correlated with putative CYP1A content. Phenobarbital slightly elevated only MROD activity. Ethoxycoumarin (EC) O-deethylase rates were high, 1.6-2.2 nmol min-1 mg-1 in control and treated turtles, suggesting that EC is not a turtle CYP1A substrate. Highly induced EROD rates were 0.06 nmol min-1 mg-1, while AHH rates exceeded 4 nmol min-1 mg-1, suggesting that C. picta picta CYP1A may prefer PAH substrates. Induction of AHH was reflected in the formation of metabolites 3-OH-, 9-OH- and 7-OH-BP and BP-7,8-dihydrodiol (DHD). BP-4,5-DHD was not detected. Chromatographic procedures resolved the 59 kDa putative CYP1A from the second protein recognized by anti-Cyp1a1. The 59-kDa protein was also specifically and highly immunopurified by Mab 1-12-3. Thus, several CYP including two CYP1A-related proteins are expressed in turtle liver. Multiple CYP1A genes in reptiles may provide an insight into the origin of divergence in the CYP1A subfamily. Induction of a CYP1A may be a useful indicator of exposure to Ah receptor agonists in turtles.