Mechanisms Involved in the Regulation of CYP3A Ontogeny

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.