Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

https://doi.org/10.1016/j.taap.2007.03.002Get rights and content

Abstract

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are ∼ 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts (∼ 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected. Together, these results demonstrate that extrahepatic esterolytic metabolism of specific pyrethroids may be significant. Moreover, hepatic cytosolic and microsomal hydrolytic metabolism should each be considered during the development of pharmacokinetic models that predict the disposition of pyrethroids and other esterified compounds.

Introduction

Carboxylesterases (CEs) are members of the α,β-serine hydrolase multigene family (Cygler et al., 1993). These enzymes catalyze the hydrolysis of esters, amides and thioesters and play an important role in endobiotic and xenobiotic metabolism (Satoh and Hosokawa, 1998). The two predominant CE isozymes in humans, hCE1 and hCE2, are expressed at high levels in the liver. hCE1 and hCE2 share 48% amino acid sequence homology and thus are placed in two separate CE classes, CES1 and CES2, respectively. The large quantities of CEs found in liver likely compensate for the fact that they are rather inefficient enzymes (Testa and Mayer, 2003). In addition to their abundant expression in liver, tissue-specific expression of human CE isozymes has been observed (Satoh et al., 2002). For example, hCE2 is expressed at relatively high levels in the small intestine, while hCE1 is not expressed in this tissue (Schwer et al., 1997). The selective expression of hCE2 in intestinal enterocytes may represent a defensive barrier that esterified xenobiotics need to breech before the organism is exposed following ingestion. Thus, xenobiotics that are metabolized by hCE2 in the small intestine may exhibit reduced bioavailability compared to compounds that are not metabolized by hCE2 (Imai, 2006). This may significantly affect the disposition of certain esterified xenobiotics following oral doses.

The physiological role(s) of CEs is an area of active research. Recent data strongly suggests a role for CEs in lipid metabolism utilizing their esterolytic activities (Dolinsky et al., 2004, Zhao et al., 2005). Many environmental toxicants, pharmaceuticals and illicit drugs are esterified compounds (reviewed in Potter and Wadkins, 2006). Examples include pyrethroid insecticides, phthalate esters, chemotherapeutic prodrugs (e.g., irinotecan) and narcotics such as cocaine and heroin. These compounds are cleared (or bioactivated in the case of irinotecan) from the body in part by the action of the CEs. Pyrethroid insecticides are esterified toxicants of particular importance because of their extensive use in agriculture (Hodgson and Levi, 1996) and public health practices (Takken, 2002). Pyrethroids will increasingly be used in agriculture as a result of the curtailed usage of organophosphate (OP) insecticides. As a result, human exposure to these compounds will likely increase in the future. The importance of esterases in the detoxification of pyrethroids has previously been demonstrated (Gaughan et al., 1980). For instance, when the OP compounds profenofos, sulprofos, O-ethyl O-(4-nitrophenyl) phenyl-phosphorothioate or S,S,S-tributyl phosphorothioate were administered to mice in vivo before dosing with the pyrethroid trans-permethrin, all four OP compounds strongly inhibited liver microsomal esterase activity and in turn increased the in vivo toxicity of the pyrethroid. This provides direct evidence for the importance of hydrolytic metabolism in pyrethroid detoxification.

Species differences exist in the expression of CEs. These differences can impact the extrapolation of animal studies to humans. Rats and mice, the two most widely used animal models in pharmacokinetic studies, express high levels of CEs in their plasma. In contrast, human plasma has no detectable CE enzyme (Li et al., 2005). This difference may significantly affect the disposition of esterified xenobiotics and thus prevent the accurate prediction of the pharmacokinetic behavior of such compounds in humans. Another difference is the large number and complex distribution of CE isozymes present in the rodent liver, which contrasts to the less complex situation in human liver, where hCE1 and hCE2 are the two major isoforms expressed. The metabolism of pyrethroids by rodent and human hepatic microsomes, which possess high concentrations of CEs, has been investigated in depth (Soderlund and Casida, 1977, Choi et al., 2002, Anand et al., 2006, Godin et al., 2006, Ross et al., 2006). However, other tissues in humans and rats, such as adipose tissue, contain lipases that may contribute to pyrethroid metabolism but have not been adequately studied. In addition, pancreatic lipases (also called carboxyl ester lipases, EC 3.1.1.3), secreted into the lumen of the small intestine to facilitate the hydrolysis of triacylglycerols and cholesteryl esters (Hui and Howles, 2002), may also contribute to pyrethroid metabolism following oral exposures. The enzymatic activities of these digestive lipases are stimulated by bile salts, which are present in the intestinal lumen at high concentrations.

The first objective of this study was to investigate further the expression and hydrolytic activity of CEs present in the soluble hepatic fraction (i.e., cytosol) and in various other human and rat tissues that are likely important in the disposition and clearance of pyrethroid insecticides. We have focused on trans-permethrin and bioresmethrin because they are prototypical type 1 pyrethroids and widely used in agriculture. We have made comparisons to some type 2 pyrethroids when appropriate; however, a more in depth study of deltamethrin and esfenvalerate will be forthcoming (Godin et al., personal communication). The tissues investigated include human intestinal microsomes, rat intestinal microsomes and cytosol, human and rat hepatic microsomes and cytosol, and human and rat serum. An additional objective of this study was to determine the substrate specificity and kinetic properties of purified rat serum carboxylesterase. The final objective was to investigate the hypothesis that lipases, which are expressed at high levels in adipose tissue, can hydrolyze pyrethroids since these compounds are hydrophobic and likely to partition into adipose reservoirs. We also examined a model pancreatic lipase, which is secreted into the lumen of the gastrointestinal tract, because it will likely encounter pyrethroid esters following oral exposures.

Section snippets

Chemicals

The pyrethroids shown in Fig. 1 were obtained from Chem Service (West Chester, PA). Type 1 pyrethroids included 1RS trans-permethrin (98% pure, 93% trans and 5% cis) , 1RS cis-permethrin (purity 99%) and 1R trans-resmethrin (bioresmethrin, 99% pure, 97% trans and 2% cis); type 2 pyrethroids, which contain a cyano functionality on the α carbon, included alpha-cypermethrin (99% pure, mixture of isomers), lambda-cyhalothrin (99% pure, mixture of isomers) and deltamethrin (99% pure). 1RS/1R and cis/

Pyrethroid hydrolysis catalyzed by human and rat intestinal microsomes

A pool of human intestinal microsomes (n = 1) was found to effectively hydrolyze trans-permethrin (specific activity of 1.88 ± 0.55 nmol/min/mg protein); however, bioresmethrin and deltamethrin were not metabolized to any appreciable extent (see Figs. 2A and B). trans-Permethrin was hydrolyzed nearly 20-fold faster than deltamethrin. These results are consistent with the substrate specificity of the hCE2 enzyme, which is the major CE isoform found in human small intestine. Pure hCE2 was previously

Discussion

Biotransformation of pyrethroid insecticides by CEs accounts for a significant fraction of the clearance of these compounds from the body (Godin et al., 2006, Nishi et al., 2006, Ross et al., 2006). Further studies that examine the functional activities of intestinal, liver and serum CEs are important to understand better the systemic availability and disposition of pyrethroid insecticides in exposed mammals. Moreover, physiologically based pharmacokinetic (PBPK) models of pyrethroids may

Acknowledgments

The project described was supported by Grant Number P20RR017661 (J.A.C. and M.K.R.) from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. P.M Potter was supported by NIH CA76202, CA79763, CA108775, CA98468, a Cancer Center Core Grant P30 CA-21765 and the American Lebanese Syrian Associated Charities. We gratefully

References (36)

Cited by (174)

View all citing articles on Scopus
View full text