Human carboxylesterases HCE1 and HCE2: Ontogenic expression, inter-individual variability and differential hydrolysis of oseltamivir, aspirin, deltamethrin and permethrin☆
Introduction
Carboxylesterases constitute a class of enzymes that hydrolyze chemicals containing such functional groups as a carboxylic acid ester, amide and thioester [1]. These enzymes are known to play important roles in drug metabolism and insecticide detoxication. Two major human carboxylesterases (HCE1 and HCE2) are abundantly expressed in the liver, whereas HCE2 is predominately expressed in the gastrointestinal tract [1], [2]. In addition to the difference in tissue distribution, these two enzymes differ markedly in the hydrolysis of certain drugs. For example, HCE1, but not HCE2, rapidly hydrolyzes the anti-influenza viral agent oseltamivir [3], [4]. In contrast, HCE2, but not HCE1, rapidly hydrolyzes the anticancer agent irinotecan [5]. In addition to hydrolyzing numerous compounds, carboxylesterases catalyze transesterification. In the presence of ethyl alcohol, HCE1 effectively converts the anti-platelet agent clopidogrel (a methyl ester) into ethyl clopidogrel [4].
The expression of carboxylesterase is altered by xenobiotics and pathological conditions. In human primary hepatocytes, therapeutic agents such as dexamethasone and phenobarbital cause a slight or moderate induction of HCE1 and HCE2 [6]. Dexamethasone and phenobarbital also alter the expression of rat carboxylesterases [7]. However, the pattern of the alteration is different. Phenobarbital moderately induces rat carboxylesterases (hydrolase A and hydrolase B), whereas dexamethasone profoundly suppresses the expression of these enzymes [7]. Suppression also occurs in human primary hepatocytes treated with the pro-inflammatory cytokine interleukin-6 (IL-6), and the suppression is achieved by transcriptional repression [8]. More importantly, the IL-6 mediated suppression strongly alters cellular responsiveness to therapeutic agents such as clopidogrel, irinotecan, and oseltamivir [8]. Hydrolysis of clopidogrel represents inactivation. In contrast, hydrolysis of irinotecan and oseltamivir leads to the formation of therapeutically active metabolites thus represents activation [5], [9].
The expression of carboxylesterases is regulated in a developmental manner, and it seems likely that these enzymes are developmentally regulated in humans as well. One to two week old rats express no hydrolase A or B based on immunoblotting analysis [7]. Consistent with the low level expression of carboxylesterases, the intrinsic clearance of the pyrethroid deltamethrin through hydrolysis in 10-day-old rats is only ∼3% of adult rats [10]. Even in 4-week-old rats, the intrinsic clearance is less than half of that of adult rats [10]. In addition, young animals are generally much more sensitive to pesticides such as organophosphates and pyrethroids [11], [12], [13]. Carboxylesterases are known to protect against these chemicals by hydrolysis in the case of pyrethroids or scavenging mechanism in the case of organophoaphates. The developmental regulation of human carboxylesterases remains to be established. A previous report by Pope et. al. [14] observed that infants differ from adults in the expression and hydrolytic activity of carboxylesterases, but the difference was statistically insignificant [14]. The Pope’s study, however, used a small number of samples and had only five samples for each group.
In the current study, we analyzed a total of 104 individual liver samples for the expression patterns of HCE1 and HCE2. These samples were grouped according to age: adults (>18 years old), children (0–10) and fetuses. Multiple experimental approaches were used including RT-qPCR, Western analysis and enzymatic assays. The fetuses expressed lower carboxylesterases than the children, and the children expressed lower carboxylesterases than the adults. Overall, the expression of both HCE1 and HCE2 showed a large inter-individual variability with the largest variability in the fetal group.
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Chemicals and supplies
Acetaminophen, aspirin, naproxen, and salicylic acid were purchased from Sigma (St. Louis, MO). Clopidogrel and clopidogrel carboxylate were from ChemPacific (Baltimore, MD). Oseltamivir and oseltamivir carboxylate were from Toronto Research Chemicals (Canada). Deltamethrin and permethrin were purchased from ChemService (West Chester, PA). Deltamethrin had a purity of 99%, and permethrin was from a batch that contained a mixture of cis- and trans-isomers at a ratio of 46% and 52%, respectively.
Ontogenic expression of human carboxylesterases HCE1 and HCE2 by RT-qPCR
In this study, we first evaluated the expression patterns of human carboxylesterases HCE1 and HCE2 in various age groups. A total of 104 RNA samples were analyzed including 48 samples from fetuses (82–224 gestation days), 34 from children (0 days–10 years of age) and 22 from adults (≥18). The levels of HCE1 and HCE2 mRNA were determined with RT-qPCR, and the results are summarized in Fig. 1 and Table 2. Overall, the adult group had the highest levels of HCE1 and HCE2 mRNA, and the fetal group
Discussion
Carboxylesterases constitute a class of hydrolytic enzymes that play important roles in the metabolism of therapeutic agents and detoxication of insecticides [1]. In this study, we analyzed a large number of individual liver samples for the expression patterns of HCE1 and HCE2, two human carboxylesterases predominately expressed in the liver. Overall, the adult group expressed significantly higher HCE1 and HCE2 than the child group or the fetal group. The age-related expression was confirmed on
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This work was supported by NIH grants R01ES07965 and R01GM61988 (BY) and R01CA053596 (YJW).