Kuhn U D, Splinter F K, Rost M, Müller D
Institute of Pharmacology und Toxicology, Friedrich Schiller University Jena, Germany.
Exp Toxicol Pathol. 1998 Sep;50(4-6):491-6. doi: 10.1016/S0940-2993(98)80039-0.
7-Ethoxycoumarin (EC) is widely used as a model substrate for monooxygenase function, its O-deethylation representing cytochrome P450 (P450) activity mainly of 1A but also of 2B isoforms. Reports on investigations of its own capacity to induce or suppress P450 activities, however, have not been found in biomedical literature. To avoid the influence of in vivo pharmacokinetics, studies can well be undertaken with liver slice incubation. Therefore in the present investigation precision-cut rat liver slices from male 43-63-day-old male HAN:Wistar outbred rats were incubated at 30 degrees C in carbogen saturated William's Medium E for 24 h. EC was added previously to final concentrations of 10, 25, 50, 75 or 100 microM. After incubation, homogenate was prepared from slices and used for model reactions (7-ethoxyresorufin O-deethylation [EROD] and 7-pentoxyresorufin O-depentylation [PROD]). EROD, indicating activities of 1A isoforms, was enhanced by incubation with EC at 25 and 50 microM to about doublefold but showed control or lower values at 75 and 100 microM. Incubation with beta-naphthoflavone in comparison led to variable increases (3-5-fold of controls). For PROD as an indicator of the phenobarbital inducible P450 isoforms 2B1 and 2B2 no enhancement was found, but a decrease by incubation with 75 and 100 microM EC. To further investigate the correlation between enzyme activity and gene expression after slice incubation, P450 1A1 mRNA content was measured by RT-PCR. Induced gene expression for 1A1 was seen with different EC concentrations to a variable extent, though not as strong as with BNF. Similar incubation with 4-methyl-7-ethoxycoumarin revealed an even stronger induction of EROD activity with maxima at about 10-32 microM, reaching BNF values. In contrast incubation with 7-benzyloxycoumarin had no evident inducing or suppressing effect, neither on EROD nor on PROD activity.
7-乙氧基香豆素（EC）被广泛用作单加氧酶功能的模型底物，其O-脱乙基反应代表细胞色素P450（P450）的活性，主要是1A亚型的活性，但也有2B亚型的活性。然而，在生物医学文献中尚未发现关于其自身诱导或抑制P450活性能力的研究报道。为避免体内药代动力学的影响，可以采用肝切片孵育进行研究。因此，在本研究中，将43 - 63日龄雄性HAN:Wistar远交系大鼠的精密切割肝切片在30℃下于含95%氧气和5%二氧化碳的饱和William's E培养基中孵育24小时。预先加入EC使其终浓度分别为10、25、50、75或100微摩尔/升。孵育后，从切片制备匀浆并用于模型反应（7-乙氧基试卤灵O-脱乙基反应[EROD]和7-戊氧基试卤灵O-脱戊基反应[PROD]）。EROD代表1A亚型的活性，与25和50微摩尔/升的EC孵育后增强至约两倍，但在75和100微摩尔/升时显示为对照值或更低。相比之下，与β-萘黄酮孵育导致不同程度的增加（为对照值的3 - 5倍）。对于作为苯巴比妥诱导的P450亚型2B1和2B2指标的PROD，未发现增强作用，但与75和100微摩尔/升的EC孵育后出现下降。为了进一步研究切片孵育后酶活性与基因表达之间的相关性，通过逆转录聚合酶链反应（RT-PCR）测量P450 1A1 mRNA含量。在不同EC浓度下均可见到1A1基因表达的诱导，但程度不同，尽管不如与β-萘黄酮孵育时强烈。与4-甲基-7-乙氧基香豆素进行类似孵育显示EROD活性诱导更强，最大值出现在约10 - 32微摩尔/升，达到β-萘黄酮的值。相比之下，与7-苄氧基香豆素孵育对EROD和PROD活性均无明显的诱导或抑制作用。
