James Margaret O, Lou Zhen, Rowland-Faux Laura, Celander Malin C
Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610-0485, USA.
Aquat Toxicol. 2005 May 15;72(4):361-71. doi: 10.1016/j.aquatox.2005.03.001.
Biotransformation in the intestine may influence the bioavailability and toxicity of ingested xenobiotics. The objective of this study was to examine the expression and catalytic properties of a constitutive cytochrome P450 (CYP) 3A-like protein along the intestine of channel catfish, Ictalurus punctatus. Fish were maintained on commercial chow or nutritionally complete semi-purified diets. Polyclonal antibodies generated against rainbow trout CYP3A proteins reacted strongly with catfish washed intestinal microsomes on Western blots showing a major protein band with MW of 59 kDa. In catfish maintained on a standard chow diet, the expression of this protein was higher in the proximal segment (0.101 +/- 0.031 units/mg protein, mean +/- S.D., n = 4) than in the distal part (0.032 +/- 0.023 units/mg protein). Microsomal testosterone 6beta-hydroxylation activity was monitored as the catalytic indicator of CYP3A, and was higher in proximal than distal catfish intestine (263 +/- 80.3 and 88.6 +/- 15.6 pmol/min/mg protein for proximal and distal, respectively, mean +/- S.D., n = 4). CYP3A protein levels and testosterone 6beta-hydroxylation activities were lower in microsomes from the proximal segment of intestine from catfish maintained on a semi-purified diet, compared with commercial chow, but again the proximal intestine had higher CYP3A and 6beta-hydroxylase activities than distal intestine. Testosterone 6beta-hydroxylase activities in all samples correlated with the CYP3A protein levels, r2 = 0.8. Testosterone 6beta-hydroxylation was inhibited by specific CYP3A inhibitors, ketoconazole (IC50 = 0.02 microM) and erythromycin (IC50 = 41 microM), as well as general CYP inhibitors, metyrapone (IC50 = 2.8 microM) and SKF-525A (IC50 = 25 microM). There was evidence for the involvement of CYP3A in the mono-oxygenation of benzo(a)pyrene and of (-)-benzo(a)pyrene-7,8-dihydrodiol in intestinal microsomes from catfish maintained on the semi-purified diet. Mono-oxygenation of both substrates was increased in a concentration-dependent manner by in vitro addition of alpha-naphthoflavone. Benzo(a)pyrene hydroxylase activities were higher in proximal than in distal intestine; 3.72 +/- 0.77 pmol/min/mg protein, mean +/- S.D., n = 5 and 1.45 +/- 0.42 in these respective segments. The results of this study strongly suggest that CYP3A is important in the first pass metabolism of dietary xenobiotics in untreated fish.
肠道中的生物转化可能会影响摄入的外源性物质的生物利用度和毒性。本研究的目的是检测斑点叉尾鮰肠道中一种组成型细胞色素P450（CYP）3A样蛋白的表达及催化特性。鱼分别投喂商业饲料或营养完全的半纯化饲料。针对虹鳟CYP3A蛋白产生的多克隆抗体在蛋白质免疫印迹中与鲶鱼洗涤后的肠道微粒体发生强烈反应，显示出一条主要蛋白带，分子量为59 kDa。在投喂标准商业饲料的鲶鱼中，该蛋白在近端肠段的表达（0.101±0.031单位/毫克蛋白，平均值±标准差，n = 4）高于远端肠段（0.032±0.023单位/毫克蛋白）。微粒体睾酮6β-羟基化活性作为CYP3A的催化指标进行监测，近端鲶鱼肠道中的活性高于远端（近端和远端分别为263±80.3和88.6±15.6 pmol/分钟/毫克蛋白，平均值±标准差，n = 4）。与商业饲料相比，投喂半纯化饲料的鲶鱼近端肠段微粒体中的CYP3A蛋白水平和睾酮6β-羟基化活性较低，但近端肠段的CYP3A和6β-羟化酶活性仍高于远端肠段。所有样品中的睾酮6β-羟化酶活性与CYP3A蛋白水平相关，r2 = 0.8。睾酮6β-羟基化受到特异性CYP3A抑制剂酮康唑（IC50 = 0.02 microM）和红霉素（IC50 = 41 microM）以及一般CYP抑制剂美替拉酮（IC50 = 2.8 microM）和SKF-525A（IC50 = 25 microM）的抑制。有证据表明，在投喂半纯化饲料的鲶鱼肠道微粒体中，CYP3A参与了苯并(a)芘和(-)-苯并(a)芘-7,8-二氢二醇的单加氧反应。体外添加α-萘黄酮后，两种底物的单加氧反应均呈浓度依赖性增加。苯并(a)芘羟化酶活性在近端肠段高于远端肠段；在这些相应肠段中分别为3.72±0.77 pmol/分钟/毫克蛋白，平均值±标准差，n = 5和1.45±0.42。本研究结果强烈表明，CYP3A在未处理鱼类饮食中外源性物质的首过代谢中起重要作用。
