Gonzalez Raymond J, Tarloff Joan B
Division of Pharmacology and Toxicology, Department of Pharmaceutical Sciences, University of the Sciences in Philadelphia, 600 South 43rd Street, Philadelphia, PA 19104, USA.
Toxicol In Vitro. 2004 Dec;18(6):887-94. doi: 10.1016/j.tiv.2004.05.001.
LLC-PK1 cells are frequently used in toxicology research, but little information is available concerning the capacity of these cells to metabolize xenobiotics. We examined the expression and activities of cytochromes P450 (P450) 1A1/1A2 (CYP 1A1/1A2), 2E1 (CYP 2E1), flavin monooxygenase (FMO), 5-lipoxygenase (5-LO) and prostaglandin H synthase (PHS)-associated cyclooxygenase-1 (COX-1). We prepared S9 fractions from LLC-PK1 cells, rat liver, and rat kidney, and measured enzyme activities using ethoxyresorufin O-deethylation (EROD) for CYP 1A1/1A2 and ethoxycoumarin O-deethylation (ECOD) for CYP 2E1, benzydamine N-oxidation (BNO) for FMO, leukotriene B(4) (LTB(4)) formation for 5-LO, and thromboxane B(2) (TXB(2)) formation for COX-1 activities. To assure that product formation was due to enzymatic activity, we used the following inhibitors: 1-aminobenzotriazole (ABT) for P450, methimazole for FMO, caffeic acid for 5-LO and acetylsalicylic acid (ASA) for COX-1. We also performed Western blot analysis to confirm our observations. All five enzyme activities were demonstrable in rat liver at much greater levels than in rat kidney S9 fractions. Activities in LLC-PK1 cells were significantly lower than activities in rat liver S9 fraction and generally less than activities in rat kidney S9 fraction. Enzyme inhibitors decreased product formation in all three tissues and Western blot analysis supported our observations of low enzyme activity in LLC-PK1 cells. These results indicate that LLC-PK1 cells have very low content of relevant drug-metabolizing enzyme activities.
LLC-PK1细胞常用于毒理学研究，但关于这些细胞代谢外源性物质的能力，目前所知甚少。我们研究了细胞色素P450（P450）1A1/1A2（CYP 1A1/1A2）、2E1（CYP 2E1）、黄素单加氧酶（FMO）、5-脂氧合酶（5-LO）和前列腺素H合酶（PHS）相关的环氧化酶-1（COX-1）的表达和活性。我们从LLC-PK1细胞、大鼠肝脏和大鼠肾脏中制备了S9组分，并使用乙氧芴香豆素O-脱乙基化（EROD）法测定CYP 1A1/1A2的酶活性，乙氧香豆素O-脱乙基化（ECOD）法测定CYP 2E1的酶活性，苄达明N-氧化（BNO）法测定FMO的酶活性，白三烯B4（LTB4）生成法测定5-LO的酶活性，血栓素B2（TXB2）生成法测定COX-1的活性。为确保产物形成是由于酶活性，我们使用了以下抑制剂：1-氨基苯并三唑（ABT）抑制P450，甲巯咪唑抑制FMO，咖啡酸抑制5-LO，乙酰水杨酸（ASA）抑制COX-1。我们还进行了蛋白质印迹分析以证实我们的观察结果。所有五种酶活性在大鼠肝脏中的水平均远高于大鼠肾脏S9组分中的水平。LLC-PK1细胞中的活性明显低于大鼠肝脏S9组分中的活性，且通常低于大鼠肾脏S9组分中的活性。酶抑制剂降低了所有三种组织中的产物形成，蛋白质印迹分析支持了我们关于LLC-PK1细胞中酶活性较低的观察结果。这些结果表明，LLC-PK1细胞中相关药物代谢酶活性的含量非常低。
