Ontogenic expression of human carboxylesterase-2 and cytochrome P450 3A4 in liver and duodenum: Postnatal surge and organ-dependent regulation

Abstract

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1–198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner.

Introduction

Personalized medicine is an ultimate goal of health professionals and inter-individual variability presents the major challenge to achieve this goal (Patel et al., 2013, Preissner et al., 2013, Schleidgen et al., 2013, Stoll et al., 2013). While many factors are contributing to inter-individual variability, biotransformation is recognized as one of the major contributing factors (Preissner et al., 2013, Stoll et al., 2013). There are three types of biotransformation, commonly referred to as phase I (Lewis, 2003), phase II (Deenen et al., 2011) and phase III reactions (You, 2004). Phase I and II reactions are accomplished by drug-metabolizing enzymes. Phase III reactions, without chemical modifications, are accomplished by drug transporters. The human genome contains ∼150 biotransformation genes with known pharmacological and toxicological significance (Lewis, 2003, You, 2004, Deenen et al., 2011, Preissner et al., 2013, Stoll et al., 2013). Many biotransformation genes are expressed in a wide range of organs and tissues. However, the highest expression of many biotransformation genes occurs in the liver and the gastrointestinal (GI) tract (Xie et al., 2002, Zancanella et al., 2012). The expression of these genes, on the other hand, exhibits large inter-individual variability, up to 100-fold in some cases (Pearce et al., 2001). Genetic and environmental factors as well as disease status are known to regulate the expression of these genes (Song et al., 2005, Liu et al., 2008, Zanger et al., 2014).

We and other investigators have shown that the expression of biotransformation genes is developmentally regulated in rodents and humans (Morgan et al., 1994, Yang et al., 2009, Hines, 2013, Lu et al., 2013, Peng et al., 2013). Based on immunoblotting analysis (Morgan et al., 1994), one to two week old rats express no hydrolase A or B, two major rat carboxylesterases. Consistent with little expression of carboxylesterases, the intrinsic hydrolytic clearance of the pyrethroid deltamethrin in 10-day old rats is only ∼3% of adult rats (Anand et al., 2006). Even in 4-week old rats, the intrinsic clearance is less than half of that of adults (Anand et al., 2006). In addition, young animals are generally much more sensitive to pesticides such as organophosphates and pyrethroids (Sheets et al., 1994, Liu et al., 1999, Anand et al., 2006). Carboxylesterases are known to protect against these chemicals by hydrolysis in the case of pyrethroids or scavenging mechanism in the case of organophosphates. Human carboxylesterase-1 (CES1) and 2 (CES2) in the liver are developmentally regulated (Yang et al., 2009, Zhu et al., 2009, Shi et al., 2011).

We recently showed that the developmental regulation of human CES1 in the liver consists of a postnatal surge followed by an incremental increase throughout the entire adolescence (Yang et al., 2009, Zhu et al., 2009). Based on the level of CES1 mRNA, the postnatal surge of human CES1 is completed at the age of approximately two months (Zhu et al., 2009). The present study was undertaken to determine whether the ontogenic expression pattern of CES1 represents a common phenomenon among biotransformation genes in humans. This study focused on CES2 and cytochrome P450 3A4 (CYP3A4). In humans, CES1 and CES2 together represent as much as 90% of hydrolytic capacity toward drugs and other xenobiotics (Yan, 2012, Sanghani et al., 2009). CYP3A4 is a member of the cytochrome P450 mixed-function oxidase system (Klein and Zanger, 2013). This oxidase is involved in the metabolism of more than 50% drugs and other xenobiotics. CES2 and CYP3A4 in humans are functionally linked in terms of tissue distribution and coupled metabolism. For example, both CES2 and CYP3A4 are abundantly expressed in the liver and the GI tract (Xie et al., 2002, Fakhoury et al., 2005, Paine et al., 2006). Importantly, some drugs are metabolized by both CES2 and CYP3A4 and their relative activity has profound therapeutic consequences (Yang et al., 2007). The anticancer drug irinotecan, for example, is hydrolyzed to produce SN-38, a metabolite with potent anticancer activity. In contrast, irinotecan undergoes oxidation by CYP3A4 to produce two major oxidative metabolites: NPC 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecin and APC 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin. Both NPC and APC are less active than the parent compound (Fakhoury et al., 2005).

This study tested a large number of human liver and duodenal samples for the expression of CES2 and CYP3A4 by Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the liver, the levels of CES2 and CYP3A4 mRNA exhibited a postnatal surge by 2.7 and 29 fold, respectively. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. Nevertheless, CES2 and CYP3A4 protein increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum in humans. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2.