Low inducibility of CYP1A activity by polychlorinated biphenyls (PCBs) in flounder (Platichthys flesus): characterization of the Ah receptor and the role of CYP1A inhibition.
Low inducibility of CYP1A activity by polychlorinated biphenyls (PCBs) in flounder (Platichthys flesus): characterization of the Ah receptor and the role of CYP1A inhibition.
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DOI：
10.1006/toxs.1998.2455
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发表时间：
1998-06
期刊：
影响因子：
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通讯作者：
H. Besselink;M. Denison;M. E. Hahn;S. Karchner;A. Vethaak;J. Koeman;A. Brouwer
中科院分区：
文献类型：
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作者：
H. Besselink;M. Denison;M. E. Hahn;S. Karchner;A. Vethaak;J. Koeman;A. Brouwer
几项研究报告了欧洲比目鱼（Platichthys flesus）暴露于多氯联苯（PCB）混合物后，肝脏细胞色素P4501 A（CYP 1A）活性的低诱导。在这里，我们报告的机制研究，了解这种低CYP 1A诱导的比目鱼，涉及的Ah受体（AhR）途径的分子特征，以及抑制CYP 1A催化活性的PCB同系物。肝细胞质AhR水平在比目鱼测定使用羟基磷灰石，硫酸鱼精蛋白吸附分析，或速度沉降蔗糖梯度。比目鱼中的AhR水平（约2-7 fmol/mg蛋白质）远低于啮齿动物中通常观察到的水平（约50-300 fmol/mg蛋白质）。通过第一链cDNA合成和RT-PCR扩增比目鱼肝脏Poly（A）+ RNA，提供了比目鱼AhR的分子特征。一个690 bp的产物被发现，类似于一个底AhR cDNA的大小。通过Southern印迹和与简并AhR寡核苷酸的杂交建立了690-bp条带的特异性。推测的比目鱼AhR片段的氨基酸序列与哺乳动物AhR序列的同源性为59-60%。虽然AhR是存在于比目鱼胞质溶胶中，我们无法证明可检测量的诱导TCDD-AhR-DRE复合物在凝胶阻滞试验。先前已发现比目鱼暴露于2，3，7，8-四氯二苯并-对-二恶英（TCDD）后，CYP 1A蛋白和相关EROD活性的诱导水平升高。相比之下，多氯联苯混合物对CYP 1A催化活性的诱导仍然出乎意料地低。因此，我们进一步表征了PCB同系物对比目鱼CYP 1A活性的抑制潜力，并将其与PCB同系物对大鼠CYP 1A活性的抑制作用进行了比较。体外分析表明，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和商业PCB混合物Clophen A50是比目鱼和大鼠肝微粒体CYP 1A催化活性的有效竞争性抑制剂。比目鱼中乙氧基试卤灵（0.095 μ M）的K（m）与检测的PCB的Ki值惊人地接近。这强调了可能参与多氯联苯同源物在抑制EROD活动在PHAH暴露的鱼。最后，我们的数据表明，比目鱼CYP 1A是更有效地代谢乙氧基试卤灵比大鼠CYP 1A。
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.