Zangar R C, Woodcroft K J, Kocarek T A, Novak R F
Institute of Chemical Toxicology, Wayne State University, Detroit, MI 48201, USA.
Drug Metab Dispos. 1995 Jul;23(7):681-7.
Investigation of the posttranscriptional mechanisms involved in the xenobiotic-mediated enhancement of cytochrome P450 2E1 (CYP2E1) expression has been limited by a lack of a functional primary hepatocyte cell culture system. We examined the effects of ciprofibrate (CIPRO) and pyridine (PYR) treatment on the expression of CYP2E1, P450 4A (CYP4A), and P450 2B (CYP2B) in primary rat hepatocytes cultured on Vitrogen or Matrigel substratum and in the presence of Chee's medium. Cells were cultured for 72 hr or longer before initiation of treatment. Northern blot analyses indicated that 24-hr CIPRO treatment enhanced the expression of CYP4A, and CYP2B mRNA in a concentration-dependent manner, with maximal induction of CYP2E1 mRNA (2- to 3-fold) and CYP4A1 mRNA (up to approximately 15-fold) monitored at 30-300 microM CIPRO. Maximal CYP2B mRNA levels (7- to 8-fold) were monitored at 300-1000 microM CIPRO. Treatment of hepatocytes for 24, 48, and 72 hr with 30 microM CIPRO showed progressive increases in CYP2B and CYP4A mRNA levels, with approximately 13- and 60-fold elevations in the respective mRNAs occurring at 72 hr posttreatment. In contrast, CYP2E1 mRNA levels were maximally elevated between 2- and 3-fold at both 24 and 48 hr and were returning to basal levels by 72 hr. Western blot analyses revealed that 24-hr PYR (25 mM) treatment of CIPRO-treated cells, in the absence of any further increase in CYP2E1 mRNA levels, increased CYP2E1 protein levels approximately 6- to 8-fold. PYR treatment also increased CYP2B mRNA and CYP2B1/2B2 protein levels approximately 16-fold relative to cells treated only with CIPRO.(ABSTRACT TRUNCATED AT 250 WORDS)
由于缺乏功能性原代肝细胞培养系统，对外源物质介导的细胞色素P450 2E1（CYP2E1）表达增强所涉及的转录后机制的研究受到了限制。我们研究了环丙贝特（CIPRO）和吡啶（PYR）处理对在Vitrogen或基质胶基质上培养且添加Chee培养基的原代大鼠肝细胞中CYP2E1、P450 4A（CYP4A）和P450 2B（CYP2B）表达的影响。在开始处理前，细胞培养72小时或更长时间。Northern印迹分析表明，24小时的CIPRO处理以浓度依赖的方式增强了CYP4A和CYP2B mRNA的表达，在30 - 300μM CIPRO时监测到CYP2E1 mRNA的最大诱导倍数为2至3倍，CYP4A1 mRNA的最大诱导倍数高达约15倍。在300 - 1000μM CIPRO时监测到CYP2B mRNA的最大水平为7至8倍。用30μM CIPRO处理肝细胞24、48和72小时，CYP2B和CYP4A mRNA水平呈逐渐升高，在处理后72小时，各自的mRNA分别升高约13倍和60倍。相比之下，CYP2E1 mRNA水平在24小时和48小时时最大升高2至3倍，到72小时时恢复到基础水平。Western印迹分析显示，在CIPRO处理的细胞中，24小时的PYR（25 mM）处理在CYP2E1 mRNA水平没有进一步增加的情况下，使CYP2E1蛋白水平增加了约6至8倍。相对于仅用CIPRO处理的细胞，PYR处理还使CYP2B mRNA和CYP2B1/2B2蛋白水平增加了约16倍。（摘要截短于250字）
