CYTOCHROMES P450 IN LIVER OF THE TURTLE CHRYSEMYS-PICTA PICTA AND THEINDUCTION 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 cau ght turtles had one protein (59 kDa) detected in western blot with mon oclonal antibody 1-12-3 to scup CYP1A. That same band and a second ban d were detected with polyclonal anti-mouse Cyp1a1. Polyclonal anti-scu p P450B (putative CYP2B) recognized three bands and anti-scup P450A (p utative 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 be ta-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 substra te. 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-dihydrod iol (DHD). Bp-4,5-DHD was not: detected. Chromatographic procedures re solved the 59 kDa putative CYP1A from the second protein recognized by anti-Cyp1a1. The 59-kDa protein was also specifically and highly immu nopurified by Mab 1-12-3. Thus, several CYP including two CYP1A-relate d proteins are expressed in turtle liver. Multiple CYP1A genes in rept iles 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. (C) 1998 Elsevier Science B.V. Al l rights reserved.