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) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels 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 characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, 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 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.
Besselink
H T
HT
Department of Food Technology and Nutritional Sciences, Agricultural University, Wageningen, The Netherlands.
Denison
M S
MS
Hahn
M E
ME
Karchner
S I
SI
Vethaak
A D
AD
Koeman
J H
JH
Brouwer
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D017931:DNA Primers; D004790:Enzyme Induction; D005432:Flounder; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D011078:Polychlorinated Biphenyls / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018336:Receptors, Aryl Hydrocarbon; D011078:Polychlorinated Biphenyls; D019363:Cytochrome P-450 CYP1A1