LOW INDUCIBILITY OF CYP1A ACTIVITY BY POLYCHLORINATED-BIPHENYLS (PCBS) IN FLOUNDER (PLATICHTHYS-FLESUS) - CHARACTERIZATION OF THE AH RECEPTOR AND THE ROLE OF CYP1A INHIBITION

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) fol lowing exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A i nducibility of flounder, involving molecular characterization of the A h receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners, Hepatic cytosolic AhR levels in flounder we re determined using hydroxylapatite, protamine sulfate adsorption anal ysis, or velocity sedimentation on sucrose gradients. AhR levels in fl ounder (similar to 2-7 fmol/mg protein) were much lower than observed generally in rodents (similar to 50-300 fmol/mg protein). Molecular ch aracterization of the flounder AhR was provided by first-strand cDNA s ynthesis and amplification of flounder hepatic poly(A)(+) RNA using RT -PCR. A 690-bp product was found, similar in size to a Fundulus AhR cD NA, The specificity of the 690-bp band was established by Southern blo tting and hybridization with a degenerate AhR oligonucleotide. The ded uced amino acid sequence of the flounder AhR fragment was 59-60% ident ical to mammalian AhR sequences. Although the AhR is present in flound er cytosol, we were unable to demonstrate detectable amounts of induci ble TCDD-AhR-DRE complex in gel-retardation assays. High induction lev els of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further charac terized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners o n rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-te traCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexa CB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K-m for ethoxyresorufin (0.095 mu M) in flounder is strik ingly close to K-i's found for the tested PCBs. This emphasizes the po ssible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is m ore efficient in metabolizing ethoxyresorufin than that of rat CYP1A. (C) 1998 Society of Toxicology.