Stoddart A M, Levine W G
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461.
Drug Metab Dispos. 1990 Jan-Feb;18(1):36-41.
This laboratory has investigated the azoreduction of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB), by hepatic microsomal cytochrome P-450 (P-450) and its specific induction by the hypolipidemic drug, clofibrate. To extend these studies further, a primary hepatocyte culture system was developed as a model. Hepatocytes isolated from male Sprague-Dawley rats were incubated in a basal medium containing fetal calf serum, insulin, and hydrocortisone for up to 96 hr with varying concentrations of clofibrate or nafenopin, a related hypolipidemic agent. Both DAB azoreductase and laurate hydroxylase activities decreased rapidly in control cultures. However, there was gradual marked induction of both activities in medium supplemented with clofibrate: hydrocortisone was required for induction. Nafenopin stabilized and induced DAB azoreductase and laurate hydroxylase activities, respectively. The responses of both activities were dose dependent. DAB azoreductase and laurate hydroxylase activities in control hepatocytes retained their ability to respond to clofibrate for up to 96 hr, although the response gradually diminished after 24 hr. In all cases, maximal induction of both enzyme activities was observed 72 hr after addition of drug. Phenobarbital and beta-naphthoflavone did not induce DAB azoreduction, although the normal induction of other P-450-catalyzed pathways, 7-ethoxycoumarin O-deethylation and ethlymorphine N-demethylation, were seen. Suppression of DAB azoreductase activity by inhibitors of P-450 activity confirmed the involvement of this enzyme in DAB azoreduction. The results demonstrate that a primary culture of rat hepatocytes is a useful model for studying the regulation of DAB azoreductase activity.
本实验室研究了肝微粒体细胞色素P-450（P-450）对肝癌致癌物N，N-二甲基-4-氨基偶氮苯（DAB）的偶氮还原作用，以及降血脂药物氯贝丁酯对其的特异性诱导作用。为进一步拓展这些研究，开发了一种原代肝细胞培养系统作为模型。从雄性Sprague-Dawley大鼠分离出的肝细胞，在含有胎牛血清、胰岛素和氢化可的松的基础培养基中，用不同浓度的氯贝丁酯或相关降血脂药物萘酚平孵育长达96小时。在对照培养物中，DAB偶氮还原酶和月桂酸羟化酶活性迅速下降。然而，在添加氯贝丁酯的培养基中，这两种活性逐渐显著诱导：诱导需要氢化可的松。萘酚平分别稳定并诱导DAB偶氮还原酶和月桂酸羟化酶活性。两种活性的反应均呈剂量依赖性。对照肝细胞中的DAB偶氮还原酶和月桂酸羟化酶活性在长达96小时内仍保持对氯贝丁酯的反应能力，尽管在24小时后反应逐渐减弱。在所有情况下，添加药物72小时后观察到两种酶活性的最大诱导。苯巴比妥和β-萘黄酮未诱导DAB偶氮还原，尽管观察到其他P-450催化途径（7-乙氧基香豆素O-脱乙基作用和乙基吗啡N-脱甲基作用）的正常诱导。P-450活性抑制剂对DAB偶氮还原酶活性的抑制证实了该酶参与DAB偶氮还原。结果表明，大鼠肝细胞原代培养是研究DAB偶氮还原酶活性调节的有用模型。
