Doi A M, Holmes E, Kleinow K M
Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, South Stadium Drive, Baton Rouge, LA 70803, USA.
Aquat Toxicol. 2001 Nov 12;55(3-4):157-70. doi: 10.1016/s0166-445x(01)00180-1.
The p-glycoprotein (pgp)-mediated multixenobiotic resistance (MXR) mechanism of aquatic animals has been associated with protection against pollution. Recent studies in mammals suggest that intestinal pgp may modulate intestinal bioavailability of dietary xenobiotics. In order to further delineate this mechanism in the catfish, these studies: (1) examined the pgp-related distribution in the intestine and liver of catfish, (2) evaluated the MXR response following exposure to various dietary xenobiotics and a prototypic pgp inducer and (3) evaluated pgp functional activity in membrane vesicles, using prototypic substrates and inhibitors. For this purpose, catfish were exposed in vivo to the pgp inducer vincristine (VIN), and the xenobiotics beta-naphthoflavone (BNF), benzo[a]pyrene (BaP), and 3,4,3',4'-tetrachlorobiphenyl (TCB). Membrane vesicles, prepared from liver and intestine (proximal and distal sections) of control and exposed catfish, were subjected to SDS PAGE, Western Blot, and detection with the pgp C219 monoclonal antibody. Transport activity was evaluated in vitro using the pgp substrate [3H]vinblastine (VBL), and the pgp inhibitor verapamil (VP). Immunoblot studies demonstrated a pgp-related protein of approximately 170 kDa in the intestine and liver of catfish. This protein appears to be very susceptible to degradation, and was present in higher levels in the liver, in comparison to the intestine, where regional differences were not observed. Dietary exposure to the pgp substrate VIN, or the xenobiotics BNF, BaP, and TCB, did not appear to affect pgp-related reactivity. Transport studies with VBL indicate that the pgp-related protein of the catfish intestine displays classic pgp-mediated multidrug resistance (MDR) characteristics, such as energy-dependency, and sensitivity to VP. These studies suggest that the pgp-related protein in the catfish intestine and liver is not only immunochemically, but also functionally related to the mammalian MDR. Moreover, the results presented indicate that pgp-related reactivity and transport in intestinal vesicles of catfish may be influenced by factors including method sensitivity, sample collection, sample preparation, and immunoblot conditions.
水生动物的P-糖蛋白（pgp）介导的多异生素抗性（MXR）机制与抵御污染有关。近期在哺乳动物中的研究表明，肠道pgp可能会调节膳食异生素的肠道生物利用度。为了进一步阐明鲶鱼中的这一机制，这些研究：（1）检测了鲶鱼肠道和肝脏中与pgp相关的分布情况；（2）评估了暴露于各种膳食异生素和一种典型的pgp诱导剂后MXR的反应；（3）使用典型底物和抑制剂评估了膜囊泡中pgp的功能活性。为此，将鲶鱼在体内暴露于pgp诱导剂长春新碱（VIN）以及异生素β-萘黄酮（BNF）、苯并[a]芘（BaP）和3,4,3',4'-四氯联苯（TCB）。从对照鲶鱼和暴露鲶鱼的肝脏和肠道（近端和远端部分）制备的膜囊泡，进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS PAGE）、蛋白质免疫印迹法（Western Blot），并用pgp C219单克隆抗体进行检测。使用pgp底物[3H]长春碱（VBL）和pgp抑制剂维拉帕米（VP）在体外评估转运活性。免疫印迹研究表明，鲶鱼的肠道和肝脏中存在一种约170 kDa的与pgp相关的蛋白质。这种蛋白质似乎非常容易降解，与肠道相比，在肝脏中的含量更高，在肠道中未观察到区域差异。膳食暴露于pgp底物VIN或异生素BNF、BaP和TCB，似乎并未影响与pgp相关的反应性。用VBL进行的转运研究表明，鲶鱼肠道中与pgp相关的蛋白质表现出典型的pgp介导的多药耐药（MDR）特征，如能量依赖性和对VP的敏感性。这些研究表明，鲶鱼肠道和肝脏中与pgp相关的蛋白质不仅在免疫化学上，而且在功能上与哺乳动物的MDR相关。此外，所呈现的结果表明，鲶鱼肠道囊泡中与pgp相关的反应性和转运可能受到包括方法敏感性、样本采集、样本制备和免疫印迹条件等因素的影响。
