Induction of P4502E1 by acetone in isolated rabbit hepatocytes. Role of increased protein and mRNA synthesis.
Induction of P4502E1 by acetone in isolated rabbit hepatocytes. Role of increased protein and mRNA synthesis.
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丙酮在离体兔肝细胞中诱导 P4502E1。
DOI：
10.1016/0006-2952(93)90049-3
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发表时间：
1993
影响因子：
5.8
通讯作者：
Raucy,JL
中科院分区：
文献类型：
--
作者：
Kraner,JC;Lasker,JM;Corcoran,GB;Ray,SD;Raucy,JL
外源物质(如乙醇、丙酮)诱导肝微粒体细胞色素P4502E1(2E1)表达的分子机制(S)尚有争议。所提出的机制包括由于2E1mRNA水平升高而增加酶合成的速率，增强先前存在的mRNAs的翻译，或稳定2E1蛋白。为了进一步评估这些事件中的哪一种，如果有的话，在2E1蛋白诱导的初始阶段，我们研究了丙酮处理对培养的兔肝细胞中2E1含量的影响，这是一种允许精确控制细胞里程的体外系统。取自雌性兔的肝细胞，接种于含血清的塑料培养皿中，培养至少48小时，存活率为90-100%。对培养至24小时的肝细胞微囊泡的免疫活性2E1含量和苯胺羟基酶活性的分析表明，2E1的表达与未培养细胞的微粒体相当，培养48小时后，2E1的水平仅下降了35%。此外，与未经处理的细胞相比，经17 mM丙酮处理24小时的细胞中分离的微粒体显示出苯胺羟基酶活性和2E1含量分别增加了53%和62%。为了解释这些增加的原因，通过首先将肝细胞暴露在添加35S标记的蛋氨酸和半胱氨酸([35S]Met/Cys)的培养液中，然后评估放射性标记对2E1蛋白的掺入，测定了未处理细胞或17 mM丙酮处理细胞中2E1蛋白的合成率。虽然未处理的细胞和丙酮处理的细胞在将[35S]Met/Cys掺入三氯乙酸可沉淀的微粒体蛋白方面没有发现差异，但免疫亲和纯化显示，丙酮将35S标记的氨基酸特异性地掺入2e1中，使其参入率增加到对照的200%。用转录抑制剂α-Amanitin处理肝细胞，显著抑制了丙酮介导的[35S]Met/Cys掺入2E1的增加。肝细胞核糖核酸分析显示，丙酮使2E1mRNA水平在6小时和24小时分别增加到对照水平的130%和160%，这种增加可以被α-Amanitin预先阻止。我们的结果表明，丙酮通过刺激2E1蛋白的去合成而增加培养的兔肝细胞的2E1蛋白水平。由于2E1合成的增加至少部分源于丙酮介导的肝细胞2E1mRNA含量的增加，并可被α-Amanitin抑制，因此，兔CYP2E1基因的转录激活显然参与了丙酮对2E1蛋白的诱导。
The molecular mechanism(s) underlying induction of the hepatic microsomal cytochrome P4502E1 (2E1) by xenobiotics (e.g. ethanol and acetone) is controversial. Proposed mechanisms include increased rates of enzyme synthesis due to elevated 2E1 mRNA levels, enhanced translation of preexisting mRNA, or stabilization of 2E1 protein. To further assess which, if any, of these events predominates during the initial stages of 2E1 protein induction, we investigated the effects of acetone treatment on 2E1 content in cultured rabbit hepatocytes, anin vitrosystem that allows for precise control of the cellular mileau. Hepatocytes harvested from female rabbits and plated on plastic dishes with serum-supplemented medium were 90–100% viable for at least 48 hr in culture. Analysis of irnmunoreactive 2E1 content and aniline hydroxylase activity in microsornes isolated from hepatocytes cultured for up to 24 hr revealed that 2E1 expression was equal to that of microsomes from unplated cells and by 48 hr of culture, 2E1 levels decreased by only 35%. Moreover, microsomes isolated from cells exposed to 17 mM acetone for 24 hr exhibited a 53 and 62% increase in aniline hydroxylase activity and 2E1 content, respectively, compared to untreated cells. To explain these increases, the rate of 2E1 protein synthesis was determined in untreated cells or in cells treated with 17 mM acetone by first exposing hepatocytes to medium supplemented with35S-labeled methionine and cysteine ([35S]Met/ Cys) and subsequently assessing radiolabel incorporation into 2E1 protein. While no difference was found between untreated and acetone-treated cells in the incorporation of [35S]Met/Cys into trichloroacetic acid-precipitable microsomal proteins, immunoaffinity purification of 2E1 revealed that incorporation of35S-labeled amino acids specifically into 2E1 was elevated by acetone to 200% of control values. Treatment of hepatocytes with the transcriptional inhibitor, α-amanitin, markedly inhibited this acetone-mediated increase in [35S]Met/Cys incorporation into 2E1. Analysis of hepatocyte RNA revealed that acetone increased 2E1 mRNA to 130 and 160% of control levels at 6 and 24 hr, respectively, and that these increases were prevented by pretreatment with α-amanitin. Our results indicate that acetone increases 2E1 protein levels in cultured rabbit hepatocytes by stimulating its rate ofde nouosynthesis. Since this increase in 2E1 synthesis stems, at least in part, from the acetone-mediated enhancement of hepatocyte 2E1 mRNA content and is inhibitable by α-amanitin, transcriptional activation of the rabbitCYP2E1gene is apparently involved in the induction of 2E1 protein by acetone.