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Mechanisms Involved in the Regulation of CYP3A Ontogeny.

机译：CYP3A个体发育调控的机制。

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摘要

Interindividual drug metabolizing enzyme (DME) expression differences can impact susceptibility to adverse drug reactions. Of the DMEs, the cytochrome P4503A (CYP3A) subfamily is important because it is responsible for the metabolism of nearly 50% of commonly prescribed drugs that undergo oxidative transformation. During human liver development, the CYP3A subfamily exhibits age-specific expression patterns. CYP3A4 is expressed at low levels during the late second or third trimester in most individuals, followed by a substantial increase in expression after birth. Expression levels mature at one to two years of age. In contrast, CYP3A7 is present at the highest levels within the fetal liver and expression is non-detectable or exhibits low expression levels by one to two years of age. Because of the CYP3A4 and 3A7 age-specific transition during early development, the substantial interindividual expression variation during early life stages and the marked differences in substrate specificity between these two enzymes, neonates and young children are particularly susceptible to some xenobiotic-related adverse events.;Although DME ontogeny and interindividual expression differences are well recognized, translating this information into clinical relevance has been limited, in part, because the underlying mechanisms remain unknown, hampering our ability to predict metabolic capacity. Studies attempting to elucidate ontogeny mechanisms focusing on differential transcription factor regulation have been largely disappointing. More recent studies have assessed epigenetic mechanisms, such as chromatin structural dynamics or differential microRNA expression. Chromatin structural dynamics are important in development, and modified and variant histones affect these dynamics. Additionally, microRNAs exhibit age-specific expression patterns in human liver. Given past failures to implicate differential transcription factor expression as a possible regulatory mechanism, both of these epigenetic mechanisms are likely candidates for regulating CYP3A ontogeny and were the focus of the studies reported herein.;Chromatin structural dynamics were evaluated by measuring chromatin occupancy by modified and variant histones within the known major CYP3A4 and 3A7 regulatory regions. Chromatin immunoprecipitation with modified and variant histone specific antibodies was performed using pooled fetal (n=11) and adult (n=10) hepatic chromatin, followed by qPCR quantification.;Chromatin occupancy by modified histones representing bivalent chromatin, indicated by the occupancy by modified histones associated with both active and repressed transcription, was observed for CYP3A4 and most 3A7 regulatory regions in the adult and fetal liver. However, comparing the two life stages, the CYP3A4 regulatory regions had significantly greater occupancy by modified histones associated with repressed transcription in the fetal liver whereas some modified histones associated with active transcription exhibited greater occupancy in the adult. CYP3A7 regulatory regions also had significantly greater occupancy by modified histones associated with repressed transcription in the fetus.;Using microRNA target prediction algorithms, multiple microRNAs were identified that potentially targeted the CYP3A4 and 3A7 3'UTR. From previous work by others, several of these candidate microRNAs also exhibit a developmental expression pattern inverse to either CYP3A4 or 3A7, respectively. HEK293 cell co-transfections with reporter plasmid constructs containing the CYP3A4 or 3A7 3'UTR and candidate microRNAs were used to evaluate the ability of each miRNA to downregulate gene expression relative to a negative control. Many candidate microRNAs exhibited minor downregulation. Two microRNAs downregulated gene expression substantially but non-specifically.;Although these studies have provided some insight, the mechanisms regulating CYP3A4 and 3A7 ontogeny remain somewhat elusive. The occupancy by modified histones observed is consistent with chromatin structural dynamics contributing to CYP3A4 ontogeny, whereas the data is less conclusive regarding CYP3A7 ontogeny. However, interpretation of the latter data is confounded by the presence of substantial numbers of non-CYP3A expressing hematopoietic cells in fetal liver. The microRNAs investigated likely do not have a role in regulating either CYP3A4 or 3A7 ontogeny.
机译：个体间药物代谢酶（DME）的表达差异会影响药物不良反应的易感性。在DME中，细胞色素P4503A（CYP3A）亚家族非常重要，因为它负责将近50％的经过氧化转化的常用处方药物的代谢。在人类肝脏发育过程中，CYP3A亚家族表现出特定年龄的表达模式。 CYP3A4在大多数个体的中晚期或晚期都以低水平表达，随后在出生后表达显着增加。表达水平在一到两岁时成熟。相反，CYP3A7在胎儿肝脏中以最高水平存在，到一到两岁时其表达不可检测或表达水平低。由于CYP3A4和3A7在早期发育过程中出现了年龄特定的过渡，因此生命早期阶段个体间的表达差异很大，并且这两种酶，新生儿和幼儿之间底物特异性的显着差异特别容易受到某些与异种生物有关的不良事件的影响。 ；尽管DME个体发育和个体间表达差异得到了公认，但将这种信息转化为临床相关性受到了限制，部分原因是其潜在机制仍然未知，从而阻碍了我们预测代谢能力的能力。试图阐明专注于差异转录因子调控的个体发育机制的研究在很大程度上令人失望。最近的研究评估了表观遗传机制，例如染色质结构动力学或差异性microRNA表达。染色质的结构动力学在发育中很重要，修饰的和变异的组蛋白会影响这些动力学。另外，microRNA在人类肝脏中表现出特定年龄的表达模式。鉴于过去未能将差异转录因子表达作为可能的调控机制，这两种表观遗传机制均可能是调节CYP3A个体发育的候选者，并且是本文报道的研究重点。已知的主要CYP3A4和3A7调控区域内的变异组蛋白。使用合并的胎儿（n = 11）和成人（n = 10）肝染色质进行修饰的和变异的组蛋白特异性抗体进行染色质免疫沉淀，然后进行qPCR定量分析;修饰的组蛋白代表二价染色质的染色质占有率，通过修饰的占有率表示在成年人和胎儿肝脏中，CYP3A4和大多数3A7调节区均观察到与活性和受阻转录相关的组蛋白。但是，比较这两个生命阶段，CYP3A4调节区在胎儿肝脏中被抑制的转录相关的修饰组蛋白的占有率明显更高，而在某些情况下，与活性转录相关的修饰的组蛋白的占有率更高。 CYP3A7调控区通过修饰的组蛋白与胎儿转录受抑制相关的占有也显着增加。使用microRNA靶标预测算法，鉴定了多个可能靶向CYP3A4和3A7 3'UTR的microRNA。根据其他人的先前工作，这些候选微RNA中的几种还分别表现出与CYP3A4或3A7相反的发育表达模式。将HEK293细胞与包含CYP3A4或3A7 3'UTR的报告质粒构建体和候选微RNA共转染，以评估每种miRNA相对于阴性对照下调基因表达的能力。许多候选microRNA表现出轻微的下调。尽管有两项研究提供了一些见识，但调控CYP3A4和3A7个体发育的机制仍然不清楚，尽管有两个microRNA显着下调了基因表达，但非特异性。观察到的被修饰的组蛋白的占据与有助于CYP3A4个体发育的染色质结构动力学一致，而关于CYP3A7个体发育的数据尚无定论。然而，后一种数据的解释被胎儿肝脏中大量非CYP3A表达造血细胞的存在所困扰。所研究的microRNA可能在调节CYP3A4或3A7个体发育中不起作用。

著录项

  • 作者

    Giebel, Nicholas L.;

  • 作者单位

    The Medical College of Wisconsin.;

  • 授予单位 The Medical College of Wisconsin.;
  • 学科 Biology Molecular.;Biology Genetics.;Health Sciences Pharmacy.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 148 p.
  • 总页数 148
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 高分子化学（高聚物）;
  • 关键词

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