Wortelboer H M, de Kruif C A, van Iersel A A, Falke H E, Noordhoek J, Blaauboer B J
UTOX, Research Institute of Toxicology (RITOX), University of Utrecht, The Netherlands.
Biochem Pharmacol. 1990 Dec 1;40(11):2525-34. doi: 10.1016/0006-2952(90)90095-3.
Changes in the isoenzyme pattern of cytochrome P450 during culture were investigated in primary cultures of rat hepatocytes, measuring specific enzyme activities in microsomes prepared from cultured cells as well as in intact monolayers. Assays of 7-ethoxyresorufin O-deethylation (EROD), 7-pentoxyresorufin O-depentylation (PROD), aniline 4-hydroxylation (AH) and the specific regioselective hydroxylation of testosterone were used as representatives of the activities of seven isoenzymes of cytochrome P450. The isoenzyme profile expressed as catalytic activities was qualitatively and quantitatively similar in microsomes obtained from freshly isolated hepatocytes in comparison with microsomes obtained from whole livers of untreated rats. There was a relatively high activity in EROD, AH and the oxidation of testosterone at the 7 alpha, 2 alpha, 6 beta, 16 alpha and 17 sites (androstenedione). During culture, these microsomal enzyme activities declined at a similar rate to ca. 50% of the activities of microsomes prepared from freshly isolated hepatocytes after 24 hr and to 15% after 96 hr. The overall decline of cytochrome P450-dependent activities during culture was not accompanied with gross changes in catalytic profile. Determining the same drug-metabolizing activities directly in intact hepatocyte monolayers revealed a much higher metabolic rate for all measured P450-dependent activities. The profile of the catalytic activities was essentially the same as measured in microsomes prepared from cultured hepatocytes. The relatively low activity towards the 7 alpha site of testosterone measured in intact hepatocytes, however, remained constant during culture. Determination of enzyme activities directly in intact hepatocytes is a convenient way of studying changes in monooxygenase activities of different P450 isoenzymes in vitro.
在大鼠肝细胞原代培养物中研究了细胞色素P450同工酶模式在培养过程中的变化，通过测量从培养细胞制备的微粒体以及完整单层细胞中的特定酶活性来进行研究。7-乙氧基异吩恶唑酮O-脱乙基酶（EROD）、7-戊氧基异吩恶唑酮O-脱戊基酶（PROD）、苯胺4-羟化酶（AH）以及睾酮的特定区域选择性羟化反应的测定被用作细胞色素P450七种同工酶活性的代表。与从未经处理大鼠的全肝获得的微粒体相比，以催化活性表示的同工酶谱在从新鲜分离的肝细胞获得的微粒体中在定性和定量上相似。EROD、AH以及睾酮在7α、2α、6β、16α和17位点（雄烯二酮）的氧化反应具有相对较高的活性。在培养过程中，这些微粒体酶活性以相似的速率下降，24小时后降至从新鲜分离的肝细胞制备的微粒体活性的约50%，96小时后降至15%。培养过程中细胞色素P450依赖性活性的总体下降并未伴随着催化谱的显著变化。直接在完整的肝细胞单层中测定相同的药物代谢活性显示，所有测量的P450依赖性活性的代谢率要高得多。催化活性谱与从培养的肝细胞制备的微粒体中测量的基本相同。然而，在完整肝细胞中测得的对睾酮7α位点的相对较低活性在培养过程中保持恒定。直接在完整肝细胞中测定酶活性是体外研究不同P450同工酶单加氧酶活性变化的一种便捷方法。
