No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Hydrolysis of Pyrethroids by Human and Rat Tissues: Examination of Intestinal, Liver and Serum Carboxylesterases
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 approximately 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 (approximately 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.
... While synthetic pyrethroids exhibit a low oral toxicity to mammals, they have been shown to produce neurotoxic effects, with toxicity being considered to be due to the parent pyrethroid (Gammon et al., 2012;Soderlund et al., 2002;Mikata et al., 2012). In both rat and human liver, pyrethroids are detoxified by metabolism by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes (Anand et al., 2006;Crow et al., 2007;Godin et al., 2006Godin et al., , 2007Hedges et al., 2019a,b;Kaneko, 2011;Mikata et al., 2012;Ross et al., 2006;Scollon et al., 2009;Soderlund et al., 2002). While CYP enzymes are localised in the microsomal fraction, CES enzymes are distributed in both the microsomal and cytosolic fractions (Boberg et al., 2017;Crow et al., 2007;Hedges et al., 2019a;Hines et al., 2016;Sato et al., 2012). ...
... In both rat and human liver, pyrethroids are detoxified by metabolism by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes (Anand et al., 2006;Crow et al., 2007;Godin et al., 2006Godin et al., , 2007Hedges et al., 2019a,b;Kaneko, 2011;Mikata et al., 2012;Ross et al., 2006;Scollon et al., 2009;Soderlund et al., 2002). While CYP enzymes are localised in the microsomal fraction, CES enzymes are distributed in both the microsomal and cytosolic fractions (Boberg et al., 2017;Crow et al., 2007;Hedges et al., 2019a;Hines et al., 2016;Sato et al., 2012). Pyrethroids can also be metabolised by CES enzymes present in rat plasma/serum (Anand et al., 2006;Crow et al., 2007;Godin et al., 2007). ...
... While CYP enzymes are localised in the microsomal fraction, CES enzymes are distributed in both the microsomal and cytosolic fractions (Boberg et al., 2017;Crow et al., 2007;Hedges et al., 2019a;Hines et al., 2016;Sato et al., 2012). Pyrethroids can also be metabolised by CES enzymes present in rat plasma/serum (Anand et al., 2006;Crow et al., 2007;Godin et al., 2007). ...
• The metabolism of bifenthrin (BIF), β-cyfluthrin (CYFL), λ-cyhalothrin (CYHA), cyphenothrin (CYPH) and esfenvalerate (ESF) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 90, 21 and 15 days and from adult humans. Pyrethroid metabolism was also studied with some human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes.
• All five pyrethroids were metabolised by adult (90 day old) rat hepatic microsomal CYP and CES enzymes and by cytosolic CES enzymes. The pyrethroids were also metabolised by human liver microsomes and cytosol. Some species differences were observed.
• Pyrethroid metabolism by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds.
• CYFL, CYHA, CYPH and ESF were metabolised by rat plasma CES enzymes, whereas none of the pyrethroids were metabolised by human plasma.
• This study demonstrates that the ability of male rats to metabolise these pyrethroids by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90 day old rats.
• All pyrethroids were metabolised by some of the human expressed CYP enzymes studied and apart from BIF were also metabolised by CES enzymes.
... Des constantes in vitro pour le métabolisme hépatique ont été utilisées (Godin et al., 2006). En revanche, aucun métabolisme sanguin n'a été supposé chez l'humain puisque le sérum humain ne contient pas les estérases qui métabolisent le composé parent (Crow et al., 2007;Godin et al., 2006). (Anadón et al., 1991;Tomalik-Scharte et al., 2005;Tornero-Velez et al., 2012;Willemin et al., 2016). ...
... Absorbed pyrethroids are eliminated from the body by metabolism. Metabolism in humans is rapid with a half-life in blood of few hours; it occurs in the liver and the intestines (Crow et al., 2007;Godin et al., 2007;. Metabolism was described by a first order process. ...
Les pyréthrinoïdes sont des insecticides ubiquitaires utilisés dans de nombreux domaines et par ce fait, les populations humaines sont susceptibles d’y être exposées par plusieurs médias environnementaux (air, sol, aliments, produits etc.) et plusieurs voies (inhalation, ingestion et contact cutané). Le composé parent est suspecté d’induire des perturbations neuronales et hormonales chez l’humain et certains composés sont plus toxiques que d’autres. Les concentrations urinaires de métabolites des pyréthrinoïdes sont souvent les seuls biomarqueurs d’une exposition humaine et ces métabolites étant parfois communs à plusieurs pyréthrinoïdes, les biomarqueurs sont le reflet d’une exposition globale et non à un pyréthrinoïde spécifique. Ces imprégnations doivent être mises en regard des doses d’expositions externes qui sont les valeurs de référence utilisées pour fixer des seuils réglementaires. Le travail consiste à développer une approche basée sur la modélisation PBPK des pyréthrinoides afin d’analyser les mesures de biomarqueurs dans les urines. Tout d’abord, un modèle PBPK global a été développé pour relier l'exposition externe de quatre pyréthrinoïdes (perméthrine, cyperméthrine, cyfluthrine et deltaméthrine) et de leurs isomères aux concentrations urinaires de métabolites (cis- et trans-DCCA, 3-PBA, F-PBA et DBCA). Le modèle permet de simuler les expositions cumulées agrégées par des voies multiples (inhalation, ingestion et contact cutané). Le modèle PBPK global a été évalué avec des données toxicocinétiques humaines et testé avec des expositions réalistes pour la population française. Le modèle a ensuite été utilisé pour estimer les expositions de la population générale française à partir de biomarqueurs d’exposition. L’étude ENNS réalisée par Santé Publique France a été utilisée. Nous avons mis en évidence des problèmes d’identifiabilité liés à la cyperméthrine et la perméthrine (i.e. partagent deux métabolites communs, mais pas de spécifique) et avons proposé une méthodologie basée sur les questionnaires de consommation alimentaire. Ce travail de thèse souligne la possibilité d’établir un modèle PBPK générique pour les pyréthrinoïdes et sur son applicabilité pour améliorer l'interprétation des biomarqueurs d'exposition aux pyréthrinoïdes.
... deltamethrin, DLM) containing a cyano moiety (Gammon et al., 2012;Soderlund et al., 2002;Soderlund, 2012). However, in mammals pyrethroids are rapidly and efficiently detoxified by metabolism by cytochrome P450 (CYP) and/or carboxylesterase (CES) enzymes Crow et al., 2007;Godin et al., 2006Godin et al., , 2007Hedges et al., 2019a,b;Kaneko, 2011;Ross et al., 2006;Scollon et al., 2009;Soderlund et al., 2002). ...
... Thus in this study no allowance was made for lymph and portal blood flow rates and possible metabolism of the two pyrethroids. In terms of metabolism, both DLM and CPM can be metabolised by hepatic CYP and CES enzymes and by serum/plasma CES enzymes Crow et al., 2007;Godin et al., 2006Godin et al., , 2007Hedges et al., 2019a;Ross et al., 2006;Scollon et al., 2009). Based on data obtained from studies on DLM metabolism in liver microsomes and plasma from adult male Sprague-Dawley rats , it was estimated that hepatic clearance accounted for over 90 % of total DLM metabolism . ...
The objective of this study was to obtain data on pathways of absorption of the synthetic pyrethroids deltamethrin (DLM) and cis-permethrin (CPM) following oral administration to rats. Adult male Sprague-Dawley rats with cannulated mesenteric lymph ducts and hepatic portal veins were given single doses of either 5 mg/kg DLM or 60 mg/kg CPM via the duodenum and lymph and portal blood samples collected for up to 300 min. The pyrethroid dosing vehicles (5 mL/kg body weight) were either corn oil or glycerol formal. Levels of DLM and CPM in lymph and portal blood samples were determined by high-performance liquid chromatography-mass spectrometry-mass spectrometry. Over the time period studied, levels of both DLM and CPM following administration in either corn oil or glycerol formal were greater in lymph than in portal blood. Lymphatic uptake of both DLM and CPM was enhanced following dosing in glycerol formal than in corn oil. The results of this study suggest that after oral administration to rats, these two pyrethroids are predominantly absorbed via the lymphatic system rather than via portal blood. The data obtained in this study thus support a recently developed physiologically-based pharmacokinetic (PBPK) model to evaluate age-related differences in pyrethroid pharmacokinetics in the rat, where it was assumed that absorption of pyrethroids was predominantly via lymphatic uptake.
... Nevertheless, it should be recognized that clearance of TRANS by plasma CaEs far exceeds the isomer's hepatic clearance in rats (Hedges et al., 2019). Human plasma lacks pyrethroid hydrolytic activity (Crow et al., 2007), so the marked disparity in isomer TK in rats in the present study may be less pronounced in humans. ...
... The apparent intrinsic clearance of each pyrethroid progressively increased with age in the 15-, 21-, and 90-day-old animals. trans-Permethrin is known to be hydrolyzed in rat liver by 2 CaEs (ie, hydrolases A and B) and in serum by a single isozyme (Crow et al., 2007). Hydrolases A and B are quite low from 1 to 2 weeks, "surge" between 2 and 4 weeks, and continue to increase until rats reach 8 weeks of age (Morgan et al., 1994). ...
Permethrin exposure of children and adults is widespread in many populations, but knowledge of its relative toxicokinetics (TK) and health risks in immature age groups is lacking. Studies were conducted in rats to determine the influence of immaturity and sex (on plasma and target organ dosimetry of each of the insecticide's two isomers, cis- and trans-permethrin (CIS and TRANS). Postnatal day (PND) 15, 21 and 90 (adult) Sprague-Dawley rats were orally administered a graduated series of doses of CIS and TRANS in corn oil. Serial sacrifices were conducted over 24 h to obtain plasma, brain, liver, skeletal muscle and fat profiles of CIS and TRANS. Levels of TRANS decreased relatively rapidly, despite administration of relatively high doses. Concentrations of each isomer in plasma, brain, and other tissues monitored were inversely proportional to the animals' age. The youngest pups exhibited 4-fold higher plasma and brain AUCs than did adults. Little difference was observed in the TK of CIS or TRANS between adult male and female rats, other than higher initial plasma and liver CIS levels in females. Elevated exposure of the immature brain appears to be instrumental in increased susceptibility to the acute neurotoxicity of high-dose permethrin (Cantalamessa, 1993), but it remains to be established whether age-dependent TK is relevant to long-term, low-level risks.
... Mammalian carboxylesterases (CESs) belong to the serine esterase superfamily, which are expressed ubiquitously in the endoplasmic reticulum and cytoplasm of tissues, including liver, intestine, lung and kidney [1]. The most primary physiological function of CESs identified to effectively catalyze the hydrolysis of various ester and amide-containing xenobiotics [2,3], for instance, many ester prodrugs (such as clopidogrel, irinotecan and oseltamivir) and environmental toxicants (such as pyrethroids) [4,5]. Multiple factors such as genetic polymorphism, age, drugs and disease status have been proved to alter CESs expression and function in tissues or individuals, and may ultimately cause a great impact on the therapeutic efficacy and toxicity of many drugs [6][7][8]. ...
... The CESs activity can be quantitatively determined by measuring the absorbance of phenol at 405 nm using a UV/VIS spectrophotometer. This method is simple, rapid, sensitive and could quantitatively determine the total CESs activity [5,33,34]. Nonetheless, the spectrophotometric analysis also has several severe shortcomings. At first, the overlapping wavelengths of absorption of substrates and products would result in interference during determination, so it is difficult to determine the relative activity of specific CESs in a complex biological sample or enzyme mixture by simple spectrophotometry. ...
... Further, hydrolytic metabolites of pyrethroids show greater estrogenic and anti-androgenic activity than the parent compounds (Tyler et al., 2000;McCarthy et al., 2006;Sun et al., 2007Sun et al., , 2014Tange et al., 2014). Carboxylesterase is known to hydrolyze pyrethroids Crow et al., 2007). Cis-/trans-permethrin is hydrolyzed to PBAlc, and further oxidized to PBAld and PBAcid by CYP and alcohol and aldehyde dehydrogenases (Choi et al., 2002;Nakamura et al., 2007). ...
... In this study, all the pyrethroid pesticides showed agonistic activity against PXR ( Fig. 2A), but the activity was decreased after incubation with rat liver microsomes (Fig. 2B). It is reported that type I pyrethroid is more easily hydrolyzed than type II by carboxylesterase (Crow et al., 2007), but we observed no systematic difference in the metabolic modification of PXR activity between type I and type II pyrethroids (Fig. 2B). The PXR activity of phenothrin (type I pyrethroid) was significantly decreased, whereas those of allethrin and bioresmethrin (type I pyrethroid) were slightly decreased by metabolism. ...
In this study, we used reporter gene assays in COS-1 cells to examine the activation of rat pregnane X receptor (PXR), rat constitutive androstane receptor (CAR) and rat peroxisome-proliferator activated receptor (PPAR)α by pyrethroid pesticides, and to understand the effects of metabolic modification on their activities. All eight pyrethroids tested in this study showed rat PXR agonistic activity; deltamethrin was the most potent, followed by cis-permethrin and cypermethrin. However, when the pyrethroids were incubated with rat liver microsomes, their rat PXR activities were decreased to various extents. Cis- and trans-permethrin showed weak rat CAR agonistic activity, while the other pyrethroids were inactive. However, fenvalerate showed dose-dependent inverse agonistic activity toward rat CAR, and this activity was reduced after metabolism. None of the pyrethroids showed rat PPARα agonistic activity, but a metabolite of cis-/trans-permethrin and phenothrin, 3-phenoxybenzoic acid, activated rat PPARα. Since PXR, CAR and PPARα regulate various xenobiotic/endobiotic-metabolizing enzymes, activation of these receptors by pyrethroids may result in endocrine disruption due to changes of hormone-metabolizing activities.
... Overviews of known substrates of HCE1 and HCE2 are given in the literature [2,4]. Also, natural substances and xenobiotiocs (e.g., insecticides) can bind as substrates or inhibitors, and are detoxified or affect drug metabolism [8,[16][17][18][19]. Among several other routinely administered drugs, irinotecan ({7-ethyl-10-[4-(1-piperidino)-1-piperidino]} carbonyloxycamptothecin) is a well-known substrate of HCE2 and RCE [2,6,12,20]. ...
Pro-drugs, which ideally release their active compound only at the site of action, i.e., in a cancer cell, are a promising approach towards an increased specificity and hence reduced side effects in chemotherapy. A popular form of pro-drugs is esters, which are activated upon their hydrolysis. Since carboxylesterases that catalyse such a hydrolysis reaction are also abundant in normal tissue, it is of great interest whether a putative pro-drug is a probable substrate of such an enzyme and hence bears the danger of being activated not just in the target environment, i.e., in cancer cells. In this work, we study the binding mode of carboxylesters of the drug molecule camptothecin, which is an inhibitor of topoisomerase I, of varying size to human carboxylesterase 2 (HCE2) by molecular docking and molecular dynamics simulations. A comparison to irinotecan, known to be a substrate of HCE2, shows that all three pro-drugs analysed in this work can bind to the HCE2 protein, but not in a pose that is well suited for subsequent hydrolysis. Our data suggest, moreover, that for the irinotecan substrate, a reactant-competent pose is stabilised once the initial proton transfer from the putative nucleophile Ser202 to the His431 of the catalytic triad has already occurred. Our simulation work also shows that it is important to go beyond the static models obtained from molecular docking and include the flexibility of enzyme–ligand complexes in solvents and at a finite temperature. Under such conditions, the pro-drugs studied in this work are unlikely to be hydrolysed by the HCE2 enzyme, indicating a low risk of undesired drug release in normal tissue.
... In general, pyrethroid toxicity is thought to be low in humans because of efficient metabolic detoxication. Pyrethroids are rapidly hydrolyzed by serum carboxylesterases (Crow et al., 2007); and human serum lacks carboxylesterase activity, suggesting that humans may have a reduced capacity to metabolize pyrethroids. However, a physiologically based pharmacokinetic study demonstrated that exposure to the pyrethroid deltamethrin was predicted to generate a 2-fold greater peak brain concentration in humans compared with rats (Godin et al., 2010). ...
Background: Deltamethrin (DM) is a potent product of type II pyrethriod insecticides causes developmental neurotoxicity that leads to lethality. The aim of the present work was to evaluate the possible postnatal influence of gestational exposure to DM on some behavioral aspects and the correspondent changes in whole brain catecholamines levels in both genders. Methods: Deltamethrin was administrated orally in a dose of 1mg/Kg/body weight from gestation day (GD) 14-20. After parturition, all dams and weaning rats were kept on standard diet and divided into male and female groups to be assessed for behavioural and neurochemical parameters at postnatal day (PND) 30. Results: The obtained results revealed that there was an obvious gender difference in different behavioral and neurochemical aspects among control groups. Prenatal exposure to DM decreased the learning ability in the T-maze. In addition, they induced antidepressant like effect in forced swimming test. In the open field test, DM induced increase in the emotionality state and decrease in the locomotor and exploration activities. The sociability and playing in social interaction test were also found to be affected by DM. Such behavioral effects were correlated with significant changes in monoamines levels and AChE activity in whole brain at PND 30. Conclusion: Overall, gender differences were found among normal subjects either in brain AchE activity and dopamine content as well as in some behavioral aspects. Deltamethrin affected both gender differently.
... Pyrethroids mainly get metabolized via hydrolytic pathway and Cytochrome P450 and further gets eliminated from the body [23,24]. Esterases (carboxylesterase 2/hCE2 in humans) and oxidation by Cytochrome P450s play an important role in metabolism and detoxification of pyrethroids [25][26][27][28][29]. Hydrolytic activity of these compounds deficits in human serum but takes place in rat serum, that may be because of the presence of a single carboxylesterase isozyme [30,31]. ...
Many scientists and researchers have published their work on the analysis of pesticides in biological as well as, non-biological matrices and their toxicokinetics using different separation techniques and detection procedures but no study gives a direct indication towards any “one” method or technique to carry out the procedure more effectively and efficiently. This review provides comprehensive information regarding variant bioanalytical techniques, their sensitivity, precision, and accuracy to determine the most efficient bioanalysis method for multiple pyrethroids. Since, these insecticides are extensively used in various sectors that lead to an extravagant exposure to humans, animals, and aquatic life, necessarily be showing some toxicities. These toxicities raise the need to monitor their toxicokinetics and assess the risks caused by cumulative as well as single pyrethroidal exposure. After carrying out the comparative study of the above mentioned parameters, it is evident that solid phase extraction procedure gives high percent recovery with minimum matrix interferences when analysed with LC-MS/MS for few pyrethroids residing in brain, whereas liquid-liquid extraction with GC-MS-NCI technique is reported to be most sensitive to quantitate a wide range of pyrethroids accumulating in blood and serum. This study can aid a reader or an analyst to understand different analytical procedures and to develop and optimize better determination techniques for pyrethroids to monitor their exposure and resulting effects.
... 18 The most important physiological function of CESs is to efficiently catalyze the hydrolysis of various lipid-and amide-containing xenobiotics, 19,20 including some ester prodrugs such as clopidogrel, irinotecan ciclopirox, and oseltamivir, as well as environmental toxicants such as pyrethroids. 21,22 Recent studies have shown that CES2 plays an important role in the development of colorectal, neuroblastoma, and pancreatic cancers. [13][14][15] However, the molecular function of CES2 in BRCA has not been fully elucidated. ...
Purpose
The expression and function of CES2 in breast cancer (BRCA) has not been fully elucidated. The purpose of this study was to investigate its clinical significance in BRCA.
Patients and Methods
Bioinformatics analysis tools and databases, including The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) databases, SURVIVAL packages, STRING database, Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Gene set variation analysis (GSVA), and Tumor Immunity Estimation Resource (TIMER), were utilized to measure the expression level and clarify the clinical significance of CES2 in BRCA. In addition, we verified the expression level of CES2 in BRCA at the cellular and tissue levels by Western blot, immunohistochemistry (IHC) and real-time fluorescence quantitative PCR assays. Furthermore, DDAB is the first reported near-infrared fluorescent probe that can be used to monitor CES2 in vivo. We applied the CES2-targeted fluorescent probe DDAB in BRCA for the first time and verified its physicochemical properties and labeling sorting ability by CCK-8, cytofluorimetric imaging, flow cytometry fluorescence detection, and isolated human tumor tissue imaging assays.
Results
The expression of CES2 was higher in normal tissues than that in BRCA tissues. Patients with lower CES2 expression in the BRCA T4 stage had a poorer prognosis. Finally, we applied the CES2-targeted fluorescent probe DDAB in BRCA for the first time, which was demonstrated to have good cellular imaging performance with low biological toxicity in BRCA cells and ex vivo human breast tumor tissue models.
Conclusion
CES2 can be considered a potential biomarker to predict the prognosis of breast cancer at stage T4 and might contribute to the development of immunological treatment strategies. Meanwhile, CES2 is able to distinguish between breast normal and tumor tissues, the CES2-targeting NIR fluorescent probe DDAB may have potential for surgical applications in BRCA.
... The activated activity of detoxification enzymes at higher temperature reduces the burden insecticides pose on organisms. Biotransformation of BC to CPF usually occurs through the hydrolysis of carboxylates/phosphates, resulting in the formation of metabolites with lower toxicity (Chanda et al., 2002;Crow et al., 2007). These metabolites are mainly related to the function of carboxylesterases. ...
In this study, the temperature-dependent chemical toxicity of three insecticides and the resulting thermoregulatory (TR) behavior of the lizard Eremias argus have been consolidated into the current risk assessment framework. According to acute dermal toxicity assays, an increase of ambient temperature from 15 °C to 35 °C decreased the acute dermal toxicity of beta-cyfluthrin (BC) but increased the toxicity of chlorpyrifos (CPF). The toxicity of avermectin (AVM) did not show significant temperature-dependent responses. Based on thermal preference trials, lizards changed their body temperature via TR behavior to adaptively reduce toxicity under sub-lethal doses, which can be understood as a “self-rescue” behavior attenuating lethal effects. However, the risk quotient indicated that the effectiveness of this “self-rescue” behavior is limited. Metabolomics analysis showed that six different metabolites (i.e., creatine, glutamate, succinate, N-acetylaspartate, acetylcholine, and lactate) contributed to TR behavior changes. Biochemical assays and insecticide residue results demonstrated that the temperature-dependent toxicity of BC, CPF, and AVM affected lizards in the three aspects of biotransformation, oxidative stress, and neurometabolic interference. This work clarifies the ecotoxicological impacts of representative insecticides on reptiles from toxicological understanding to risk relevance. This knowledge may improve ecological predictions of agrochemical applications in the context of global climate change.
... Increased activities of detoxification enzymes has been reported to account for insecticides resistance through metabolizing them before reaching their target site where toxicities are expressed or as a result of reduced target site sensitivity of DDT binding site sodium ion channel (Nwane et al., 2009) and carbamate binding site acetylcholinesterase (Weill et al., 2004). Esterases detoxify organophosphate, carbamates and synthetic pyretheroids through hydrolysis of ester bond and binding of insecticides to the active site of esterase (Crow et al., 2007). Insect cytochrome p450 monooxygenase metabolize exogenous compounds including insecticides and plant toxins leading to insecticides resistance (Wen et al., 2003) and higher tolerance to plant toxins (Li et al., 2002;Wen et al., 2003). ...
Evaluating levels of detoxifying enzymes is informative to governmental, nongovernmental organization and insecticides producing industries. In this work DDT and bendiocarb resistance status of Anopheles gambae breeds in Auyo (May-September, 2014) was studied. Larvae collected from residential sites (AR) and rice Irrigation sites (AI) of Auyo town in Auyo LGA Jigawa State Nigeria reared to adult was studied by WHO adult bioassay. Standard methods were adapted to determine the specific activities of insecticides detoxifying enzymes; glutathione s transferase (GST), esterase and monooxygenase in the insecticides resistant (r) and susceptible (s) vectors. In addition those tested with bendiocarb were analyzed for acetylcholinesterase activities. The finding of the study established high resistance status of the malarial vectors to DDT in both sites, low resistance status to bendiocarb in residential site and possible resistance to bendiocarb in irrigation site according to WHO interpretation. Significant (P<0.05) elevation in the activity of esterase and GST were seen
... AchE is key enzyme in detoxification of neurotoxic insecticides (their target is nervous system) and overproduction of AchE catalyzes the breakdown of acetylcholine and some other choline esters which acts as neurotransmitters (Byrne and Toscano, 2001). Nonspecific esterase (αesterase and βesterase) is another important enzyme, detoxify several pesticides by two main ways, hydrolysis of the ester bond and binding of the pesticide to the active site of esterase (Crow et al., 2007). GST plays critical role in detoxification of insecticides by conjugating reduced glutathione to the electrophilic centers of insecticides. ...
Spodoptera littoralis nucleopolyhedrosis virus (SlNPV) has been considered as a vital and safe alternative to chemical insecticides. It is effectively used in biological control of Spodoptera littoralis which is considered the most destructive and detrimental economic pest in Egypt. However, its slow speed of action limits its application among other bio-control agents. So, great attention was directed to solve this problem by using insecticide synergists. In this study the toxicity of both SlNPV and the insecticide synergist pipronyl butoxide (PBO) was investigated against 2 nd and 4 th instar S. littoralis larvae. Different concentrations of the tested viral isolate used (10 6 , 10 7 , 10 8 , 10 9 and 10 10 POB/ml) showed high mortality percentage ranging from 60 to 93% and 56 to 83% for the two tested instar larvae respectively 10 days post treatment. The three PBO concentrations used (0.1, 0.2 and 0.3%) revealed weak toxic effect as they produced 13, 20 and 26% mortality rate for the three tested concentrations respectively 4 days post treatment. To examine the biosafety of the three PBO concentrations (0.1, 0.2 and 0.3%), alone or in combination with SlNPV; in vivo animal studies, and in vitro cell line studies were applied. In vivo evaluations included histomorphometric and immunohistochemical alterations in Caspase-3, Proliferating cell nuclear antigen (PCNA), tumor necrosis factor-alpha (TNF-α) in hepatic and renal tissues, plus serum assessments of malondialdehyde (MDA) and total antioxidant capacity (TAC). The sulforhodamine B (SRB) cytotoxicity assay has been applied on OEC (Oral epithelial cell) and Vero (Green monkey kidney) cell lines. Results revealed that 0.1% PBO concentration was safe and didn't induce any pathogenicity, as a dose concentration-dependent decreased pathological changes by comparing 0.3%, 0.2% PBO treated rats. Furthermore, SlNPV was safe to rat tissues and cells and PBO didn't alter the safe nature of SlNPV on mammalian cells. According to the results of histopthological bio-safety, 0.1% of PBO was applied to 4 th instar larvae for estimation its effect on larval enzyme activity and the results proved significant differences in the metabolic tested enzymes between the control and treated larvae with inhibition in both of Actylcholin esterase (AchE), Glutathione S-transferase (GST), Alpha esterases (α-esterases), Beta esterases (B-esterases), and Glutamic oxaloacetic transaminase (GOT) and stimulation in Lactate dehydrogenase (LDH), Glutamic pyruvic transaminase (GPT) and Multi-function oxidases (MFO). Moreover, 0.1% of PBO has been mixed with viral LC 50 for 2 nd and 4 th instar larvae and the results showed highly synergistic effect of PBO as 100 and 96 % mortality rate respectively fourth day post treatment.
... Le profil toxicocinétique du 3-PBA, un des métabolites de la perméthrine a également été étudié chez le rat (Ueyama et al. 2010). En revanche, on dispose de données in vitro de métabolisme chez l'Homme et chez l'animal (Choi et al. 2002;Crow et al. 2007;Nakamura et al. 2007;Scollon et al. 2009;Takaku et al. 2011;Willemin et al. 2015). ...
Les études de biosurveillance ont montré une exposition plus élevée de la population française, y compris les femmes enceintes, aux pyréthrinoïdes par rapport à d'autres pays. Les pyréthrinoïdes sont des composés neurotoxiques qui agissent sur leurs organismes cibles en interférant sur la transmission du signal nerveux. Ces composés passent la barrière placentaire exposant le fœtus durant la période sensible du développement. Au sein de cette famille d'insecticides, la perméthrine (mélange des isomères cis et trans) est le composé le plus utilisé en milieu domestique. L'objectif de ce travail de thèse est de développer un modèle toxicocinétique basé sur la physiologie (PBPK) chez la rate gestante. Pour ce faire, nous avons tout d'abord mis au point une méthode de dosage LC-MS/MS des isomères de la perméthrine et de ses métabolites dans différentes matrices maternelles (foie, cerveau, sang, rein, graisse, glande mammaire, placenta, urine et fèces) et fœtales (foie, cerveau et sang). Cette méthode a ensuite été utilisée pour doser les composés d'intérêt dans le cadre d'une étude de toxicocinétique chez la rate gestante. Un modèle PBPK gestationnel chez le rat a été développé grâce à ces données de cinétique permettant ainsi d'estimer l'exposition maternelle et fœtale durant toute la période de la gestation. Ce modèle pourra par la suite être extrapolé à l'Homme, en vue de pouvoir estimer l'exposition maternelle et fœtale
... Compared to α-cypermethrin, permethrin levels demonstrated a more linear increase in serum concentrations in proportion to the dose administered (Fig. 1). However, serum and tissue concentrations of permethrin were notably lower relative to dose than those for αcypermethrin, consistent with a higher rate of the first-pass metabolism for cis-permethrin as compared to α-cypermethrin (Crow et al., 2007). In addition, previous reports have indicated a generally higher rate of metabolism in rats for the trans-versus cis-isomer of pyrethroids in general (Tornero-Velez et al., 2012). ...
Pyrethroid insecticides are widely used throughout agriculture and household products. Recent studies suggest that prenatal exposure to these insecticides may adversely affect fetal development; however, little is known about the distribution of these chemicals in pregnant animals. The present study aimed to address this gap in knowledge by investigating the distribution of two commonly used pyrethroid insecticides, permethrin and α-cypermethrin, in maternal and fetal tissues of pregnant CD-1 mice. Dams were dosed from gestational days 6 to 16 via oral gavage with permethrin (1.5, 15, and 50 mg/kg), α-cypermethrin (0.3, 3, and 10 mg/kg), or corn oil vehicle. Pyrethroid levels were determined in gestational day 16 tissues collected 90 minutes after the final dose was administered. Across maternal tissues, levels of both pyrethroids were the highest in maternal ovaries, followed by liver and brain, respectively. In addition, levels of both pyrethroids in maternal tissues and placenta were significantly higher than those in the fetal body and amniotic fluid, suggesting that these compounds may exhibit low transfer across the mouse placenta. While additional toxicokinetic studies are needed to verify the time course of pyrethroids in the fetal compartment, these findings support investigation into indirect modes of action relevant to the effects of pyrethroids on mammalian fetal development.
... We recently identified THC, CBD, and cannabinol as potent in vitro inhibitors of carboxylesterase 1 (CES1), and THC and CBD were predicted to pose a risk of DDI when consumed concurrently with CES1 substrate medications (Qian et al., 2019a;Qian et al., 2019b). CES1 is an esterase predominantly expressed in the liver and is responsible for the majority of hepatic hydrolytic activity (Imai et al., 2006;Crow et al., 2007;Taketani et al., 2007;Basit et al., 2020). When examined by the absolute abundance in the liver, the expression of CES1 exceeds that of any major phase I or phase II drug-metabolizing enzymes (i.e., cytochrome P450s (P450s) and UDP-glucuronosyltransferases) suggesting its essential role in the disposition of both endogenous compounds and xenobiotics (He et al., 2019). ...
The use of cannabis products has increased substantially. Cannabis products have been perceived and investigated as potential treatments for attention-deficit/hyperactivity disorder (ADHD). Accordingly, co-administration of cannabis products and methylphenidate (MPH), a first-line medication for ADHD, is possible. Oral MPH undergoes extensive pre-systemic metabolism by carboxylesterase 1 (CES1), a hepatic enzyme which can be inhibited by two prominent cannabinoids, ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD). This prompts further investigation into the likelihood of clinical interactions between MPH and these two cannabinoids through CES1 inhibition. In the present study, inhibition parameters were obtained from a human liver S9 system and then incorporated into static and physiologically-based pharmacokinetic (PBPK) models for prediction of potential clinical significance. The inhibition of MPH hydrolysis by THC and CBD was reversible, with estimated unbound inhibition constants (Ki,u) of 0.031 and 0.091 µM, respectively. The static model predicted a mild increase in MPH exposure by concurrent THC (34%) and CBD (94%) from smoking a cannabis cigarette and ingestion of prescriptive CBD, respectively. PBPK models suggested no significant interactions between single doses of MPH and CBD (2.5 - 10 mg/kg) when administered simultaneously, while a mild interaction (AUC increased by up to 55% and Cmax by up to 45%) is likely if multiple doses of CBD (10 mg/kg twice daily) are administered. In conclusion, the pharmacokinetic disposition of MPH can be potentially influenced by THC and CBD under certain clinical scenarios. Whether the magnitude of predicted interactions translates into clinically relevant outcomes requires verification in an appropriately designed clinical study. Significance Statement This work demonstrated a potential mechanism of drug-drug interactions between methylphenidate (MPH) and two major cannabinoids (∆9-tetrahydrocannabinol [THC] and cannabidiol [CBD]) not previously reported. We predicted a mild interaction between MPH and THC when the cannabinoid exposure occurred via cannabis smoking. Mild interactions between MPH and CBD were predicted with multiple oral administrations of CBD.
... L-Allothreonine is a stereoisomer of threonine and an intermediate in plant glycine, serine, and threonine metabolism [121]. 3-Phenoxybenzyl alcohol is a mammalian metabolite of the insecticide permethrin produced by carboxylesterases [122,123]. Dihydroshikonofuran is a monoterpenoid product of the ubiquinone biosynthetic pathway that also produces shikonin, a plant pigment with anti-inflammatory, antibacterial, and wound-healing properties [124,125]. Phenethyl alcohol is a natural fragrance produced by rose, carnation, and other plants [126,127]. ...
Untargeted metabolomics has been increasingly used to evaluate metabolic alterations caused by diet, disease, or other factors in animals. The purpose of this exploratory study was to evaluate the impact of Cannabidiol (CBD) supplementation on the canine carboxyl and hydroxyl submetabolomes. Sixteen dogs (18.2 ± 3.4 kg BW) were utilized in a completely randomized design with treatments consisting of control and 4 mg CBD/kg BW/d. After 21 d of treatment, blood was collected approximately 2 h after morning treat consumption. Plasma collected from samples was analyzed using CIL/LC-MS-based untargeted metabolomics to analyze carboxyl- and hydroxyl-containing metabolites. Metabolites that differed (fold change (FC) ≥ 1.2 or ≤ 0.83 and FDR ≤ 0.05) between the two treatments were identified using a volcano plot. Biomarker analysis based on Receiver Operating Characteristic (ROC) curves was performed to identify biomarker candidates (area under ROC ≥ 0.90) of the effects of CBD supplementation. Volcano plot analysis revealed that 42 carboxyl-containing metabolites and 32 hydroxyl-containing metabolites were differentially altered (FC ≥ 1.2 or ≤ 0.83, FDR ≤ 0.05) by CBD; these metabolites were involved in the metabolism of lipids, amino acids, carbohydrates, and more. Biomarker analysis identified 23 carboxyl-containing metabolites and 15 hydroxyl-containing metabolites as candidate biomarkers of the effects of CBD (area under ROC ≥ 0.90; P<0.01). Results of this study indicate that 4 mg CBD/kg BW/d supplemented for 3-weeks altered the canine carboxyl and hydroxyl submetabolomes and may indicate potential mechanisms by which CBD exerts some of its effects. Future work is warranted to investigate these potential mechanisms.
... In this effect, it should be kept in mind that metabolism may be important in the gastrointestinal lumen associated with the microbial flora of the digestive system, which is due to the physicochemical properties. Pyrethroid group insecticides are also metabolized by this pathway (MIYAMOTO, 1976;SODERLUND and CASIDA, 1977;CROW et al., 2007;ANADÓN et al., 2009). C max and t max are among the parameters that can be indicative of absorption rate (SHEN, 2008;MERWE et al., 2012;GUPTA, 2016). ...
The toxicokinetics of single dose phenothrin were examined in rabbits. For this aim, a total of 14 New Zealand breed, 2 to 2.5 kg body weight, 6 month-old female rabbits were used. The animals were divided into two groups and each group had 7 animals. Phenothrin was administered intravenously to each animal in group 1, at a dose of 10 mg/kg b.w. and orally to each of the animals in group 2 at the same dose. Dimethyl sulfoxide was used as a solvent in application of phenothrin. Plasma phenothrin levels were measured by gas chromatography equipped with an ECD detector. Toxicokinetic evaluations were made according to the plasma phenothrin level-time curve. Phenothrin was found to be distributed according to the two-compartment open model. The values of elimination half-life (t1/2β), mean residence time (MRT) and area under the curve (AUC0→∞) after intravenous phenothrin administration were 2.57 ± 0.10 h, 2.79 ± 0.09 h and 6893.05 ± 261.26 ng/h/mL, respectively. On the other hand, the maximum plasma concentration (Cmax), time to reach Cmax (tmax), t1/2β, MRT and AUC0→∞ after oral administration were 185.71 ± 8.21 ng/mL, 1.21 ± 0.20 h, 4.24 ± 0.39 h, 6.65 ± 0.54 h and 1054.04 ± 65.90 ng/h/mL, respectively. The oral bioavailability of phenothrin was calculated as 15.29%. Mean residence time was short and oral bioavailability was low. This may be one of the reasons why phenothrin is included in safe pesticides.
... Only the K sI values were estimated as K sI is considered as a sensitive parameter according to previous PBPK models published for pyrethroids (58). For metabolic clearances, as the liver is the main site of metabolism (59), only metabolic rate constants for permethrin isomers in the liver were estimated. Blood and intestinal rate constant values were set to the optimized values of the adult model of Willemin et al. (38). ...
Biomonitoring studies have highlighted the exposure of pregnant women to pyrethroids based on the measurement of their metabolites in urine. Pyrethroids can cross the placental barrier and be distributed in the fetus as some pyrethroids were also measured in the meconium of newborns. Prenatal exposure to pyrethroids is suspected to alter the neurodevelopment of children, and animal studies have shown that early life exposure to permethrin, one of the most commonly used pyrethroid in household applications, can alter the brain development. This study aimed to characterize the fetal permethrin exposure throughout gestation in rats. We developed a pregnancy physiologically based pharmacokinetic (pPBPK) model that describes the maternal and fetal kinetics of the cis- and trans- isomers of permethrin during the whole gestation period. Pregnant Sprague–Dawley rats were exposed daily to permethrin (50 mg/kg) by oral route from the start of gestation to day 20. Permethrin isomers were quantified in the feces, kidney, mammary gland, fat, and placenta in dams and in both maternal and fetal blood, brain, and liver. Cis- and trans-permethrin were quantified in fetal blood and tissues, with higher concentrations for the cis-isomer. The pPBPK model was fitted to the toxicokinetic maternal and fetal data in a Bayesian framework. Several parameters were adjusted, such as hepatic clearances, partition coefficients, and intestinal absorption. Our work allowed to estimate the prenatal exposure to permethrin in rats, especially in the fetal brain, and to quantitatively estimate the placental transfer. These transfers could be extrapolated to humans and be incorporated in a human pPBPK model to estimate the fetal exposure to permethrin from biomonitoring data.
... Indeed, biochemical analyses carried out in the laboratory on several jassid samples showed that those coming from Ouangolodougou (North-East) produce high quantities of αesterases and β-esterases, compared to samples coming from Bouaké (South) [9,10] . This result means that jassid populations in the northeast would be better able to degrade some insecticides such as Carbamates, Pyrethroids and Organophosphorus, which are widely used by farmers [2,7,12] . Four groups of localities were defined from the multivariate analysis. ...
... DLM appears to be metabolized by similar enzymes in humans and rats (Rehman et al., 2014), but there are quantitative differences in the rate of metabolism across species and over the course of development. Humans have low serum CaE levels, whereas rodents have high levels (Crow et al., 2007;Li et al., 2005;Wang et al., 2018). The lower levels in humans result in slower DLM clearance. ...
Deltamethrin (DLM) is a Type II pyrethroid pesticide widely used in agriculture, homes, public spaces, and medicine. Epidemiological studies report that increased pyrethroid exposure during development is associated with neurobehavioral disorders. This raises concern about the safety of these chemicals for children. Few animal studies have explored the long-term effects of developmental exposure to DLM on the brain. Here we review the CNS effects of pyrethroids, with emphasis on DLM. Current data on behavioral and cognitive effects after developmental exposure are emphasized. Although, the acute mechanisms of action of DLM are known, how these translate to long-term effects is only beginning to be understood. But existing data clearly show there are lasting effects on locomotor activity, acoustic startle, learning and memory, apoptosis, and dopamine in mice and rats after early exposure. The most consistent neurochemical findings are reductions in the dopamine transporter and the dopamine D1 receptor. The data show that DLM is developmentally neurotoxic but more research on its mechanisms of long-term effects is needed.
... The studies on the properties of the purified carboxylesterases of plant seeds are scanty, on account of the difficulties involved in the purification. Esterase from seeds of Jatropha curcas has been purified to homogeneity and characterized as a carboxylesterase [23]. Similarly, two esterases have been purified from Mucuna pruriens and characterized as carboxylesterases [24]. ...
The present work is focus on physical and chemical properties of purified Carboxylesterase using the Seeds of Tamarindus Indica.The esterases are extracted from the germinating tamarind seeds using 50 mM phosphate buffer, pH 7 and purified. The Km with α-naphthyl acetate, β-naphthyl acetate and α-naphthyl butyrate as the substrates is 28.6 μM, 22.2 μM and 26.7 μM respectively. The Vmax for the same substrates is 7.1 x 10-3 µmole/min, 7.41 x 10-3 µmole/min and 8.00 x 10-3 µmole/min respectively. The enzymes optimally active at pH 7.0 and are stable between pH 5.0 to 8.0. The optimum temperature of esterase activity is 40˚C. The molecular weight of 27.5 kD as determined by SDS-PAGE, both in the presence and absence of β-mercaptothanol and is in close agreement with the molecular weight determined by gel-filtration on Sephadex G-100 (26.9 kD).
... Pesticide-degrading esterases have been investigated for their biochemical properties, such as optimal pH, temperature, inhibitors, molecular weight, and enzyme kinetic parameters Bhatt, 2019;Gangola et al., 2018;Bhatt et al., 2020c;Pandey et al., 2010;Crow et al., 2007;Stok et al., 2004;Wu et al., 2006;Zhai et al., Fig. 2. Detailed catalytic mechanisms of an esterase using the active site amino acid with the ester pesticide (cyhalothrin). Serine (Ser), Histidine (His), Glutamine (Glu), and Glycine (Gly) are participating in two-step catalysis reactions. ...
Ester-containing organophosphate, carbamate, and pyrethroid (OCP) pesticides are used worldwide to minimize the impact of pests and increase agricultural production. The toxicity of these chemicals to humans and other organisms has been widely reported. Chemically, these pesticides share an ester bond in their parent structures. A particular group of hydrolases, known as esterases, can catalyze the first step in ester-bond hydrolysis, and this initial regulatory metabolic reaction accelerates the degradation of OCP pesticides. Esterases can be naturally found in plants, animals, and microorganisms. Previous research on the esterase enzyme mechanisms revealed that the active sites of esterases contain serine residues that catalyze reactions via a nucleophilic attack on the substrates. In this review, we have compiled the previous research on esterases from different sources to determine and summarize the current knowledge of their properties, classifications, structures, mechanisms, and their applications in the removal of pesticides from the environment. This review will enhance the understanding of the scientific community when studying esterases and their applications for the degradation of broad-spectrum ester-containing pesticides.
... The carboxylesterase in humans can be divided into five subtypes, but the two main subtypes are human carboxylesterase 1A (hCES1A) and human carboxylesterase 2A (hCES2A). The amino acid sequences of hCES1A and hCES2A have 47% homology, and there are significant differences in substrate selectivity and tissue distribution [3][4][5]. hCES1A is mainly expressed in the liver and tends to hydrolyze substrates of small alcohol groups and large acyl groups such as enalapril, oseltamivir, imidazolidinium, clopidogrel [6][7][8]. However, hCES2A is mainly known as the enzyme expressed in the intestinal tract and involved in metabolism of large alcohol-based and small acyl substrates such as irinotecan, capecitabine, flutamide, procaine [9,10]. ...
Human Carboxylesterase 2A (hCES2A), one of the most important serine hydrolases, plays crucial roles in the hydrolysis and the metabolic activation of a wide range of esters and amides. Increasing evidence has indicated that potent inhibition on intestinal hCES2A may reduce the excessive accumulation of SN-38 (the hydrolytic metabolite of irinotecan with potent cytotoxicity) in the intestinal tract and thereby alleviate the intestinal toxicity triggered by irinotecan. In this study, more than sixty natural alkaloids have been collected and their inhibitory effects against hCES2A are assayed using a fluorescence-based biochemical assay. Following preliminary screening, seventeen alkaloids are found with strong to moderate hCES2A inhibition activity. Primary structure–activity relationships (SAR) analysis of natural isoquinoline alkaloids reveal that the benzo-1,3-dioxole group and the aromatic pyridine structure are beneficial for hCES2A inhibition. Further investigations demonstrate that a steroidal alkaloid reserpine exhibits strong hCES2A inhibition activity (IC50 = 0.94 μM) and high selectivity over other human serine hydrolases including hCES1A, dipeptidyl peptidase IV (DPP-IV), butyrylcholinesterase (BChE) and thrombin. Inhibition kinetic analyses demonstrated that reserpine acts as a non-competitive inhibitor against hCES2A-mediated FD hydrolysis. Molecular docking simulations demonstrated that the potent inhibition of hCES2A by reserpine could partially be attributed to its strong σ-π and S-π interactions between reserpine and hCES2A. Collectively, our findings suggest that reserpine is a potent and highly selective inhibitor of hCES2A, which can be served as a promising lead compound for the development of more efficacious and selective alkaloids-type hCES2A inhibitors for biomedical applications.
... With an identical external exposure, the internal (target tissue) concentration is lower in children than in adults resulting in lower extrapolated HEDs than observed rat PODs. Metabolism plays an important role in the detoxification and elimination of pyrethroids in both rats and humans, but species differences exist in the enzymes involved in pyrethroid metabolism (hepatic cytochrome P450 and carboxylesterases enzymes) as well as in the ontogeny of these enzymes (Boberg et al., 2017;Crow et al., 2007;Godin et al., 2006Godin et al., , 2007Hedges et al., 2019a,b;Hideo, 2012;Hines, 2007;Hines et al., 2016;Saghir et al., 2012;Scollon et al., 2009). At high doses, the species-specific immaturity of metabolizing enzymes in juvenile animals can result in the saturation of available metabolic capability, thus leading to higher pyrethroid concentrations in target tissues in juvenile rats compared with adults Kim et al., 2010). ...
The assessment of potentially sensitive populations is an important application of risk assessment. To address the concern for age-related sensitivity to pyrethroid insecticides, life-stage physiologically based pharmacokinetic (PBPK) modeling supported by in vitro to in vivo extrapolation (IVIVE) was conducted to predict age-dependent changes in target tissue exposure to eight pyrethroids. The purpose of this age-dependent dosimetry was to calculate a Data Derived Extrapolation Factor (DDEF) to address age-related pharmacokinetic differences for pyrethroids in humans. We developed a generic human PBPK model for pyrethroids based on our previously published rat model that was developed with in vivo rat data. The results demonstrated that the age-related differences in internal exposure to pyrethroids in the brain are largely determined by the differences in metabolic capacity and in physiology for pyrethroids between children and adults. The most important conclusion from our research is that, given an identical external exposure, the internal (target tissue) concentration is equal or lower in children than in adults in response to the same level of exposure to a pyrethroid. Our results show that, based on the use of the Life Stage PBPK models with eight pyrethroids, DDEF values are essentially close to 1, resulting in a DDEF for age-related pharmacokinetic differences of 1. For risk assessment purposes, this indicates that no additional adjustment factor is necessary to account for age-related pharmacokinetic differences for these pyrethroids.
... In contrast, the human model represented in this manuscript did not predict any substantial difference in brain C max in different age groups for any of the three case pyrethroids (Figure 7). Hepatic CYPs and CES are important metabolizing enzymes for detoxification and elimination of pyrethroids in both rats and humans Crow et al., 2007;Godin et al., 2006Godin et al., , 2007Scollon et al., 2009), but significant species differences exist in the relative contribution of various CYPs and CES enzymes (Godin et al., 2007;Scollon et al., 2009) as was confirmed by our in vitro expressed enzyme results. This species difference is likely one of the factors for the age-related sensitivity observed in rats but not in humans as the major enzyme for pyrethroid metabolism in human is CES, which shows a rapid maturation after birth. ...
To address concerns around age-related sensitivity to pyrethroids, a life-stage physiologically based pharmacokinetic (PBPK) model, supported by in vitro to in vivo extrapolation (IVIVE) was developed. The model was used to predict age-dependent changes in target tissue exposure of eight pyrethroids, deltamethrin (DLM), cis-permethrin (CPM), trans-permethrin (TPM), esfenvalerate, cyphenothrin, cyhalothrin, cyfluthrin and bifenthrin. A single model structure was used based on previous work in the rat. Intrinsic clearance (Clint) of each individual cytochrome P450 (CYP) or carboxylesterase (CES) enzyme that are active for a given pyrethroid were measured in vitro, then biologically scaled to obtain in vivo age-specific total hepatic CLint. These IVIVE results indicate that, except for bifenthrin, CES enzymes are largely responsible for human hepatic metabolism (>50% contribution). Given the high efficiency and rapid maturation of CESs, clearance of the pyrethroids is very efficient across ages, leading to a blood flow-limited metabolism. Together with age-specific physiological parameters, in particular liver blood flow, the efficient metabolic clearance of pyrethroids across ages results in comparable to or even lower internal exposure in the target tissue (i.e., brain) in children than that in adults in response to the same level of exposure to a given pyrethroid (Cmax ratio in brain between 1 and 25 year old = 0.69, 0.93 and 0.94 for DLM, bifenthrin and CPM, respectively). Our study demonstrated that a life-stage PBPK modeling approach, coupled with IVIVE, provides a robust framework for evaluating age-related differences in pharmacokinetics and internal target tissue exposure in humans for the pyrethroid class of chemicals.
... The detoxification of active ingredients by esterases is obtained by two main ways. On the one hand, the hydrolysis of the ester bond for carbamates and pyrethroids; and on the other hand, the binding of the pesticide to the active site of esterase for organophosphates (Crow et al., 2007;Holling -worth & Dong, 2008;Liu et al., 2011). ...
Cotton production is one of the main sources of income for the population and contributes to the socioeconomic development in the northern and central areas of Côte d'Ivoire. However, cotton crop is subject to large and diversified pest problems, including jassids. Heavy infestations of jassids are likely to induce the stopping of plant development or the shedding of reproductive organs. For the last decade, there has been an increase and persistence of leafhopper attacks throughout the crop cycle despite insecticide treatments. This study aims to determine the biochemical mechanism likely to be involved in insecticide susceptibility of jassids. A biochemical assay of enzyme activities was performed on adult populations of jassids collected from cotton fields in six different localities (Bouaké, Korhogo, Boundiali, Ferké, Ouangolo and Niakara) within the cotton growing area of Côte d'Ivoire. The method of Brogdon et al. (1997) was adopted with minor modifications. Alpha-esterase activity values ranged from 380.334 to 965.775 nmol α-naphtol/min/mg protein for Bouaké and Ouangolo respectively. Beta-esterase activity ranged from 398.640 nmol β-naphtol/min/mg protein for Bouaké to 1,528.885 nmol β-naphtol/min/mg protein for Ouangolo. Oxidase activity ranged from 0.016 nmol P450 EU/mg protein for Ferké to 0.038 nmol P450 EU/mg protein for Ouangolo. Glutathione-δ-transferases varied between 0.554 nmol GSH conj/min/mg protein for Ferké and 1.128 nmol GSH conj/min/mg protein for Niakara. ABSTRACT Cotton production is one of the main sources of income for the population and contributes to the socioeconomic development in the northern and central areas of Côte d'Ivoire. However, cotton crop is subject to large and diversified pest problems, including jassids. Heavy infestations of jassids are likely to induce the stopping of plant development or the shedding of reproductive organs. For the last decade, there has been an increase and persistence of leafhopper attacks throughout the crop cycle despite insecticide treatments. This study aims to determine the biochemical mechanism likely to be involved in insecticide susceptibility of jassids. A biochemical assay of enzyme activities was performed on adult populations of jassids collected from cotton fields in six different localities (Bouaké, Korhogo, Boundiali, Ferké, Ouangolo and Niakara) within the cotton growing area of Côte d'Ivoire. The method of Brogdon et al. (1997) was adopted with minor modifications. Alpha-esterase activity values ranged from 380.334 to 965.775 nmol α-naphtol/min/mg protein for Bouaké and Ouangolo respectively. Beta-esterase activity ranged from 398.640 nmol β-naphtol/min/mg protein for Bouaké to 1,528.885 nmol β-naphtol/min/mg protein for Ouangolo. Oxidase activity ranged from 0.016 nmol P450 EU/mg protein for Ferké to 0.038 nmol P450 EU/mg protein for Ouangolo. Glutathione-δ-transferases varied between 0.554 nmol GSH conj/min/mg protein for Ferké and 1.128 nmol GSH conj/min/mg protein for Niakara. The involvement of α-esterases and β-esterases in controlling the sensitivity level of jassids to active ingredients is thus highlighted. This observation suggests a rational selection of the active ingredients used to control these pests.
Human carboxylesterase 2 (hCES2A), one of the most important serine hydrolases distributed in the small intestine and colon, plays a crucial role in the hydrolysis of various prodrugs and esters. Accumulating evidence has demonstrated that the inhibition of hCES2A effectively alleviate the side effects induced by some hCES2A-substrate drugs, including delayed diarrhea caused by the anticancer drug irinotecan. Nonetheless, there is a scarcity of selective and effective inhibitors that are suitable for irinotecan-induced delayed diarrhea. Following screening of the in-house library, the lead compound 01 was identified with potent inhibition on hCES2A, which was further optimized to obtain LK-44 with potent inhibitory activity (IC50 = 5.02 ± 0.67 μM) and high selectivity on hCES2A. Molecular docking and molecular dynamics simulations indicated that LK-44 can formed stable hydrogen bonds with amino acids surrounding the active cavity of hCES2A. The results of inhibition kinetics studies unveiled that LK-44 inhibited hCES2A-mediated FD hydrolysis in a mixed inhibition manner, with a Ki value of 5.28 μM. Notably, LK-44 exhibited low toxicity towards HepG2 cells according to the MTT assay. Importantly, in vivo studies showed that LK-44 significantly reduced the side effects of irinotecan-induced diarrhea. These findings suggested that LK-44 is a potent inhibitor of hCES2A with high selectivity against hCES1A, which has potential as a lead compound for the development of more effective hCES2A inhibitors to mitigate irinotecan-induced delayed diarrhea.
Organophosphates (OPs), pyrethrins and fipronil, are acaricides commonly used in cattle, mainly as pour on formulations. Scant information is available on their potential interactions with hepatic xenobiotic metabolizing enzymes. This work aimed to evaluate in vitro the potential inhibitory effects of widely employed acaricides on catalytic activities mediated by hepatic cytochrome P450 (CYP) and flavin-monooxygenase (FMO) enzymes in cattle. Bovine (n = 4) liver microsomes were incubated in the absence (control assays) and in presence of different OPs (fenthion, chlorpyrifos, ethion, diazinon and dichlorvos), fipronil and cypermethrin at 0.1-100 μm. Five oxidative enzyme activities were assayed by spectrofluorimetric or HPLC methods: 7-ethoxyresorufin O-deethylase (for CYP1A1), methoxyresorufin O-demethylase (for CYP1A2), benzyloxyresorufin O-debenzylase (for CYP2B), testosterone 6-beta hydroxylase (for CYP3A) and benzydamine N-oxidase (for FMO). All acaricides, particularly phosphorothionate-containing OPs, inhibited to some extent more than one enzyme activity. The most frequent inhibitor was fenthion, which inhibited (p < .05) all enzyme activities tested (from 22% at 1 μm to 72% at 100 μm). However, low inhibitory potencies (IC50s higher than 7 μm) of all acaricides studied were observed against the catalytic activities assayed. Therefore, the risk of in vivo metabolic interactions due to inhibition of monooxygenases would be low under common husbandry conditions.
Worldwide, rates of metabolic diseases are rapidly increasing and environmental exposure to pesticides, pollutants and/or other chemicals may play a role. Reductions in Brown Adipose Tissue (BAT) thermogenesis, mediated in part by uncoupling protein 1 (Ucp1), are associated with metabolic diseases. In the current study, we investigated whether the pesticide deltamethrin (0.01-1 mg/kg bw/day) incorporated into a high-fat diet and fed to mice housed at either room temperature (21 °C) or thermoneutrality (29 °C) would suppress BAT activity and accelerate the development of metabolic disease. Importantly, thermoneutrality allows for more accurate modeling of human metabolic disease. We found that, 0.01mg/kg bw/day of deltamethrin induced weight loss, improved insulin sensitivity and increased energy expenditure, effects that were associated with increases in physical activity. In contrast, exposure to 0.1 and 1 mg/kg bw/day deltamethrin had no effect on any of the parameters examined. Deltamethrin treatment in mice did not alter molecular markers of BAT thermogenesis, despite observing suppression of UCP1 expression in cultured brown adipocytes. These data indicate that while deltamethrin inhibits UCP1 expression in vitro, 16wks exposure does not alter BAT thermogenesis markers nor exacerbates the development of obesity and insulin resistance in mice.
Benoxacor is a safener added to current-use herbicide formulations to protect the target crop from herbicidal toxicity. It is an emerging environmental contaminant that has been detected in surface waters, raising the possibility of human exposure via drinking water. Because it is not subject to the same regulations as active pesticide ingredients, its metabolism and toxicity in humans have not been studied. Here we investigate the enantioselective metabolism of benoxacor in human subcellular fractions. Pooled human liver microsomes (pHLM) and cytosol (pHLC) were incubated with racemic benoxacor for up to 30-minutes. Gas chromatographic analyses were used to measure the enantioselective depletion of benoxacor. pHLMs with and without nicotinamide adenine dinucleotide phosphate (NADPH, co-factor for cytochrome P450 enzymes [CYPs]) and pHLC with glutathione (GSH, co-factor for glutathione S-transferases [GSTs]) metabolized benoxacor. These results demonstrate that microsomal CYPs, microsomal carboxylesterase (CESs), and cytosolic GSTs metabolize benoxacor. Females were predicted to have a higher clearance of benoxacor by GSTs than males. Male and female pHLM incubations with NADPH showed enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). pHLM incubations without NADPH and pHLC incubations with GSH showed an enrichment of the second eluting enantiomer of benoxacor (E2-benoxacor). Our results indicate that human hepatic microsomal and cytosolic enzymes enantioselectively metabolize benoxacor, a fact that needs to be considered when investigating human exposures and toxicities of benoxacor.
The metabolism and toxicity of current-use herbicide safeners remain understudied. We investigated the enantioselective metabolism of the safener benoxacor in Rhesus monkey subcellular fractions. Benoxacor was incubated with liver microsomes and cytosol from female and male monkeys (≤30 minutes). Benoxacor levels and enantiomeric fractions were determined with gas chromatography. Benoxacor was metabolized by microsomal cytochrome P450 enzymes (CYPs), cytosolic glutathione-S-transferases (GSTs), and microsomal and cytosolic carboxylesterase (CESs). CES-mediated microsomal metabolism followed the order males > females, whereas the CYP-mediated clearance followed the order females > males. CYP-mediated metabolism initially resulted in an enrichment of the second eluting benoxacor enantiomer (E2-benoxacor), whereas the first eluting benoxacor enantiomer (E1-benoxacor) was enriched after 10 or 30 minutes in female or male microsomal incubations. Benoxacor metabolism by GSTs was enantiospecific, with a total depletion of E1-benoxacor after approximately 20 minutes. Thus, the enantioselective metabolism of benoxacor by GSTs and CYPs may affect its toxicity.
Endoplasmic reticulum (ER) stress and neuroinflammation are involved in the pathogenesis of many neurodegenerative disorders. Previously, we reported that exposure to pyrethroid insecticide deltamethrin causes hippocampal ER stress, apoptosis, a reduction in neurogenesis, and learning deficits in adult male mice. Recently, we found that deltamethrin exposure also increases the markers of neuroinflammation in BV2 cells. Here, we investigated the potential mechanistic link between ER stress and neuroinflammation following exposure to deltamethrin. We found that repeated oral exposure to deltamethrin (3 mg/kg) for 30 days caused microglial activation and increased gene expressions and protein levels of TNF-α, IL-1β, IL-6, gp91phox, 4HNE, and iNOS in the hippocampus. These changes were preceded by the induction of ER stress, as the protein levels of CHOP, ATF-4, and GRP78 were significantly increased in the hippocampus. To determine whether induction of ER stress triggers the inflammatory response, we performed an additional experiment with mouse microglial cell (MMC) line. MMCs were treated with 0–5 µM deltamethrin for 24–48 h in the presence or absence of salubrinal, a pharmacological inhibitor of the ER stress factor eIF2α. We found that salubrinal (50 µM) prevented deltamethrin-induced ER stress, as indicated by decreased levels of CHOP and ATF-4, and attenuated the levels of GSH, 4-HNE, gp91phox, iNOS, ROS, TNF-α, IL-1β, and IL-6 in MMCs. Together, these results demonstrate that exposure to deltamethrin leads to ER stress-mediated neuroinflammation, which may subsequently contribute to neurodegeneration and cognitive impairment in mice.
The sensing of carboxylesterases (CESs) activity in living systems is of great importance for drug design and biomedical research. Herein, water-soluble AIE-active fluorescent organic nanoparticles (2B3OTs) containing hydrophilic lactose and hydrophobic BODIPY-conjugated tetraphenylethene moieties were rationally designed for ratiometrically fluorescent sensing of CESs with the meso-ester BODIPY as the recognition unit. 2B3OTs were cell membrane permeable and successfully applied to detect the real activities of endogenous CESs in living cells. More importantly, 2B3OTs could preferentially recognize hepatoma cells rather than normal liver cells, since the lactose is an efficient ligand for asialoglycoprotein receptors which are over expressed in tumor liver cells. It is first time that water-soluble AIE-active fluorescence organic nanoparticle were designed and utilized for real-time sensing of CESs activities in living cells.
In this work, a BODIPY-based multi-site red emission fluorescent probe MRS-BOD for the detection of carboxylesterase 1 (CES1) was designed and synthesized. MRS-BOD showed good biocompatibility and could be used to monitor the activity of CES1 in living cells. MRS-BOD emission is located in the red emission region, which has the advantages of less background interference and less damage to biological samples. MRS-BOD has multiple CES1 recognition sites, and the ultra-sensitive quantification of CES1 has been successfully achieved. The low detection limit of MRS-BOD was 2.5 ng/mL, and the fluorescence quantum yield was 0.49. This study can promote the development of fluorescent probes and help the identification of related diseases.
Deltamethrin (DEL) can be introduced into the food chain through bioaccumulation in Pacific oysters, and then potentially threaten human health. The objective of this study was to investigate the bioaccessibility of DEL in oysters with different cooking methods after simulated digestion. DEL content in different tissues of oysters going from high to low were gills, mantle, viscera, and adductor muscle. Bioaccessibility of DEL in oysters decreased after steaming (65%) or roasting (51%) treatments compared with raw oysters (82%), which indicated that roasting can be used as a recommended cooking method for oysters. In the simulated digestion process, the concentration of DEL in the digestive juice and the bioaccessibility of DEL were affected by the pH in the gastric phase. And the transport efficiency of DEL through the monolayer molecular membrane of NCM460 cells ranged from 35 to 45%. These results can help assess the potential harm to consumers of DEL in shellfish. Furthermore, it provides a reference for the impact of lipophilic toxins in seafood.
An aggregation-induced emission (AIE) fluorescent protein chromophore-based probe (CBZ-FP) for detection of human carboxylesterases (CESs) was designed and synthesized. CBZ-FP exhibited good cell permeability with a large stokes shift (116 nm) and can be applied to reveal the actual activities of CES1 in living cells associated with pesticides detoxification process. CBZ-FP can also serve as a fluorescence indicator of pesticide exposure in the way of hydrolyzing the carboxylic acid ester group in CBZ-FP. Therefore, CBZ-FP has high selectivity for CESs and can detect real-time activity of CES1 in biological samples. Molecular docking study was used to explore the binding of CESs and CBZ-FP. Finding that only one specific activity site of CESs can bind with probe. In view of the fact that, the biotransformation of drugs and poisons containing ester groups can carry out normally depending on CESs, Carboxylesterase probes are expected to contribute to the characterization of relevant disease.
Human and animal welfare primarily depends on the availability of food and surrounding environment. Over a century and half, the quest to identify agents that can enhance food production and protection from vector borne diseases resulted in the identification and use of a variety of pesticides, of which the pyrethroid based ones emerged as the best choice. Pesticides while improved the quality of life, on the other hand caused enormous health risks. Because of their percolation into drinking water and food chain and usage in domestic settings, humans unintentionally get exposed to the pesticides on a daily basis. The health hazards of almost all known pesticides at a variety of doses and exposure times are reported. This review provides a comprehensive summation on the historical, epidemiological, chemical and biological (physiological, biochemical and molecular) aspects of pyrethroid based insecticides. An overview of the available knowledge suggests that the synthetic pyrethroids vary in their chemical and toxic nature and pose health hazards that range from simple nausea to cancers. Despite large number of reports, studies that focused on identifying the health hazards using doses that are equivalent or relevant to human exposure are lacking. It is high time such studies are conducted to provide concrete evidence on the hazards of consuming pesticide contaminated food. Policy decisions to decrease the residual levels of pesticides in agricultural products and also to encourage organic farming is suggested.
Parabens are antimicrobial compounds used as preservatives in cosmetics, foods, and pharmaceuticals. Paraben exposure occurs through a variety of routes including dermal absorption, ingestion, and inhalation. Ester bond hydrolysis has been shown to be the predominant biotransformation for this chemical class. Here we evaluated a series of parabens of increasing alkyl chain length and branching in addition to the aryl side chain of phenyl paraben (PhP). We evaluated the parabens under full Michaelis-Menten parameters to obtain intrinsic clearance values and found different trends between human liver and skin, which correlate with the predominant esterase enzymes in those matrices, respectively. In liver, where carboxylesterase 1 (CES1) is the predominant esterase enzyme, the shorter chain parabens were more readily metabolized, while in skin, where carboxylesterase 2 (CES2) is the predominant esterase enzyme, the longer chain parabens were more readily metabolized. Alkyl chain branching reduced the hydrolysis rates relative to those for the straight chain compounds, while the addition of a phenyl group, as in PhP, showed an increase in hydrolysis, producing the highest observed hydrolysis rate for skin. These data summarize the structure-metabolism relationship for a series of parabens and contribute to the safety assessment of this class of compounds.
Herein, a lysosomal targeting [email protected] metal-organic framework (MOF) was fabricated using fluorescent protein chromophore-based probe (LysFP) for selectively detection of carboxylesterase 1 (CES1) in living cells. Unlike the regular small molecule fluorescent probes, [email protected] showed wide range pH tolerabiligy, high selectivity and sensitivity to CES1 in bio-samples, and was successfully applied to achieve the visual monitoring of CES1 activity in living cells. Low detection limit and high fluorescence quantum yield was calculated as 79 ng/mL and 0.76 for [email protected], respectively. Furthermore, [email protected] can also serve as a fluorescence indicator of organophosphates pesticide exposure in the way of hydrolyzing the carboxylic acid ester group in LysFP. This type of probe can inspire the development of fluorescent tools for further explore many pathological processes.
As the most important members of serine esterase hydrolase superfamily, mammalian carboxylesterases (CESs) are widely distributed in various tissues like liver, intestine, lung and kidney. Many physiological processes require the hydrolysis of CES with a variety of endogenous and exogenous ester drugs and environmental poisons. So, developing the methods that can detect CESs activity is of great significance. In last decades, huge breakthrough has been made in the development of specific detection technologies including chromatography, quantitative reverse transcription polymerase chain reaction (qRT‐PCR), proteomics and western blotting. However, these traditional methods for CESs detection have little effect on the spatiotemporal distribution and in situ activity of CES in living cells and in vivo. Compared with that, fluorogenic methods have been developed for real‐time monitoring CESs in biological environments. In this review, we summarized the recent reported CESs probes. The designing strategies as well as their applications are reviewed. We hope this review will inspire the development of novel specific CESs fluorescent probes.
• The kinetics of metabolism of deltamethrin (DLM) and cis- and trans-permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol.
• Apparent intrinsic clearance (CLint) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration. However, when apparent CLint values were corrected for nonspecific binding to allow calculation of unbound (i.e. corrected) CLint values, the unbound values did not vary greatly with microsomal protein concentration.
• Unbound CLint values for metabolism of 0.05-1 μM DLM in rat liver microsomes (CYP and CES enzymes) and cytosol (CES enzymes) were not significantly different from rates of DLM metabolism in isolated rat hepatocytes.
• This study demonstrates that the nonspecific binding of these highly lipophilic compounds needs to be taken into account in order to obtain accurate estimates of rates of in vitro metabolism of these pyrethroids. While DLM is rapidly metabolised in vitro, the hepatocyte membrane does not appear to represent a barrier to the absorption and hence subsequent hepatic metabolism of this pyrethroid.
In traditional Chinese medicine herbs, including Radix Salviae Miltiorrhizae (Danshen), Radix Puerariae Lobatae (Gegen), Radix Angelicae Sinensis (Danggui), and Rhizoma Chuanxiong (Chuanxiong) are widely used for the prevention and treatment of cardiovascular diseases and also often co-administered with western drugs as a part of integrative medicine practice. Carboxylesterase 1 (CES1) plays a pivotal role in the metabolisms of pro-drugs. Since (S)-2-(2-(6-dimethylamino)-benzothiazole)-4,5-dihydro-thiazole-4-carboxylate (NLMe) has recently been identified by us as a selective CES1 bioluminescent sensor, we developed a rapid method using this substrate for the direct measurement of CES1 activity in rats. This bioluminescence assay was applied to determine CES1 activity in rat tissues after a two-week oral administration of each of the four herbs noted above. The results demonstrated the presence of CES1 enzyme in rat blood and all tested tissues with much higher enzyme activity in blood, liver, kidney and heart than that in the small intestine, spleen, lung, pancreas, brain and stomach. In addition, the four herbs showed tissue-specific effects on rat CES1 expression. Based on the CES1 biodistribution and its changes after treatment in rats, the possibility that Danshen, Gegen and Danggui might alter CES1 activities in humans in blood and kidney should be considered. In summary, a selective and sensitive bioluminescence assay was developed to rapidly evaluate CES1 activity and the effects of orally administered TCMs in rats.
Lambda-cyhalothrin (LCT) is a broad-spectrum pesticide widely used in agriculture throughout the world. This pesticide is considered a potential contaminant of surface and underground water as well as food, posing a risk to ecosystems and humans. In this sense, we decided to evaluate the activity of enzymes belonging to the purinergic system, which is linked with regulation of extracellular nucleotides and nucleosides, such as adenosine triphosphate (ATP) and adenosine (Ado) molecules involved in the regulation of inflammatory response. However, there are no data concerning the effects of LCT exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. The aim of this study was to evaluate nucleotide hydrolysis by E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase), Ecto-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase), ecto-5′-nucleotidase and ecto-adenosine deaminase (E-ADA) in platelets and liver of adult rats on days 7, 30, 45 and 60 after daily gavage with 6.2 and 31.1 mg/kg bw of LCT. Gene expression patterns of NTPDases1–3 and 5′-nucleotidase were also determined in those tissues. In parallel, lambda-cyhalothrin metabolites [3-(2-chloro-3,3,3- trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic acid (CFMP), 4-hydroxyphenoxybenzoic acid (4-OH-3-PBA), and 3-phenoxybenzoic acid (3-PBA)] were measured in plasma. Results showed that exposure rats to LCT caused a significant increase in the assessed enzymes activities. Gene expression pattern of ectonucleotidases further revealed a significant increase in E-NTPDase1, E-NTPDase2, and E-NTPDase3 mRNA levels after LCT administration at all times. A dose-dependent increase in LCT metabolite levels was also observed but there no significant variations in levels from weeks to week, suggesting steady-steady equilibrium. Correlation analyses revealed that LCT metabolites in the liver and plasma were positively correlated with the adenine nucleotides hydrolyzing enzyme, E-ADA and E-NPP activities in platelets and liver of rats exposed to lambda-cyhalothin.
Our results show that LCT and its metabolites may affect purinergic enzymatic cascade and cause alterations in energy metabolism.
To investigate the potential role of the COOH-terminal peptides in retaining a family of soluble carboxylesterases in the lumen of the endoplasmic reticulum, the pI 6.1 esterase cDNA has been cloned into the pKCR3 vector for transient expression in COS cells. The plasmid-encoded product appeared to be identical to the authentic enzyme: it was active on alpha-naphthyl acetate and behaved as a homotrimer of noncovalently bound 60-kDa subunits which contain a single, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide chain. This enzyme was retained in the transgenic COS cells. In contrast, a mutated form ending in HVER-COOH was secreted, indicating that the natural terminus HVEL-COOH contains topogenic information, with the ultimate Leu residue as an essential part. Variants of pI 6.1 esterase ending in HIEL-COOH, or HTEL-COOH were retained in cells to the same extent as the wild-type protein. Therefore, the sequences HIEL and HTEL present at the COOH termini of several liver esterases (rabbit forms 1 and 2, human esterase, mouse egasyn, and rat pI 6.4 esterase) most likely have a function in their localization in the endoplasmic reticulum. Finally, an HDEL-COOH variant of pI 6.1 esterase was also normally retained, demonstrating that this signal can be correctly decoded by the sorting machinery of mammalian cells. Cell retention signals of the type HXEL-COOH appear to be common in higher eukaryotes and tolerate considerable variation at the antepenultimate X residue.
Pesticides are high-volume, widely used, environmental chemicals and there is continuous debate concerning their possible role in many chronic human health effects. Because of their known structures, known rates of application, and the presence of a large occupationally exposed population, they are not only important in their own right but are ideal models for the effects of environmental chemicals on the population in general. For reasons that are not always clear, this potential has not been realized. These exposed populations represent an underused asset in the study of the human health effects of environmental contaminants. Chronic effects thought to involve pesticides include carcinogenesis, neurotoxicity, and reproductive and development effects. In this paper we attempt to summarize this concern and, relying to a large extent on studies in our own laboratory, to indicate the importance and present status of studies of the mammalian metabolism of pesticides and indicate the need for further use of this model. Aspects considered include the role of pesticides as substrates for xenobiotic-metabolizing enzymes such as cytochrome P450 and the flavin-containing monooxygenase and their role as inducers or inhibitors of metabolic enzymes. The interaction of pesticides with complex multienzyme pathways, the role of biological characteristics, particularly gender, in pesticide metabolism, and the special role of pesticides at portals of entry and in target tissues are also considered.
One challenge for veterinarians, animal facilities and research scientists is the making of physiological estimates appropriate to a variety of species for which data are often either completely lacking or are incomplete. Our intent in compiling the data in this paper is to provide the best possible database of normal physiological and anatomical values primarily (though not exclusively) for four common mammalian model species: mouse, rat, dog and man. In order to make those data as accessible and applicable as possible, we have presented the results of this study in the form of body-size dependent allometric equations in which some variable (Y) is expressed as a dependent function of body mass (M) in the power-law equation, Y = aM(b). By compiling these data, it is apparent that the resultant equations are quantitatively grouped (with similar slope or 'b' values). These emergent patterns provide insights into body-size dependent 'principles of design' that seem to dictate several aspects of design and function across species among all mammals. In general, the weights of most individual organs scale as a constant fraction of body mass (i.e. the body mass exponent, b approximately equal to 1.0). Biological rates (e.g. heart rate, respiratory rate) scale as b approximately equal to -1/4. Finally, volume-rates (the product of volume and rate) such as cardiac output, ventilation and oxygen uptake vary as b approximately equal to 3/4.
Carboxyl ester lipase (C33347), previously named cholesterol esterase or bile salt-stimulated (or dependent) lipase, is a lipolytic enzyme capable of hydrolyzing cholesteryl esters, tri-, di-, and mono-acylglycerols, phospholipids, lysophospholipids, and ceramide. The active site catalytic triad of serine-histidine-aspartate is centrally located within the enzyme structure and is partially covered by a surface loop. The carboxyl terminus of the protein regulates enzymatic activity by forming hydrogen bonds with the surface loop to partially shield the active site. Bile salt binding to the loop domain frees the active site for accessibility by water-insoluble substrates. CEL is synthesized primarily in the pancreas and lactating mammary gland, but the enzyme is also expressed in liver, macrophages, and in the vessel wall. In the gastrointestinal tract, CEL serves as a compensatory protein to other lipolytic enzymes for complete digestion and absorption of lipid nutrients. Importantly, CEL also participates in chylomicron assembly and secretion, in a mechanism mediated through its ceramide hydrolytic activity. Cell culture studies suggest a role for CEL in lipoprotein metabolism and oxidized LDL-induced atherosclerosis.
Thus, this enzyme, which has a wide substrate reactivity and diffuse anatomic distribution, may have multiple functions in lipid and lipoprotein metabolism, and atherosclerosis.
Recent scientific advances have revealed the identity of several enzymes involved in the synthesis, storage and catabolism of intracellular neutral lipid storage droplets. An enzyme that hydrolyzes stored triacylglycerol (TG), triacylglycerol hydrolase (TGH), was purified from porcine, human and murine liver microsomes. In rodents, TGH is highly expressed in liver as well as heart, kidney, small intestine and adipose tissues, while in humans TGH is mainly expressed in the liver, adipose and small intestine. TGH localizes to the endoplasmic reticulum and lipid droplets. The TGH genes are located within a cluster of carboxylesterase genes on human and mouse chromosomes 16 and 8, respectively. TGH hydrolyzes stored TG, and in the liver, the lipolytic products are made available for VLDL-TG synthesis. Inhibition of TGH activity also inhibits TG and apolipoprotein B secretion by primary hepatocytes. A role for TGH in basal TG lipolysis in adipocytes has been proposed. TGH expression and activity is both developmentally and hormonally regulated. A model for the function of TGH is presented and discussed with respect to tissue specific functions.
Hydrolysis of intracellular cholesteryl esters (CEs) represents the first step in the removal of cholesterol from lipid-laden foam cells associated with atherosclerotic lesions. Neutral cholesteryl ester hydrolase (CEH) catalyzes this reaction, and we recently cloned the cDNA for the human macrophage CEH and demonstrated increased mobilization of intracellular CE droplets by CEH overexpression. The present study was undertaken to test the hypothesis that for CE hydrolysis, CEH must become associated with the surface of the cytoplasmic lipid droplets. Our data show the redistribution of CEH from cytosol to lipid droplets upon lipid loading of human THP-1 macrophages. Depletion of triacylglycerol (TG) by incubation with the acyl-CoA synthetase inhibitor Triacsin D had no effect on CEH association with the lipid droplets, suggesting that CEH associates with mixed (CE + TG) as well as TG-depleted CE droplets. However, CEH had 2.5-fold higher activity when mixed droplets were used as substrate in an in vitro assay, consistent with the reported higher cholesterol efflux from cells containing mixed isotropic droplets. Perilipin as well as adipophilin, two lipid droplet-associated proteins, were also present on the lipid droplets in THP-1 macrophages.
In conclusion, CEH associates with its intracellular substrate (lipid droplets) and hydrolyzes CE more efficiently from mixed droplets.
The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.
Hydrolase activity from human liver and small intestine microsomes was compared with that of recombinant human carboxylesterases, hCE-1 and hCE-2. Although both hCE-1 and hCE-2 are present in human liver, the dominant component was found to be hCE-1, whereas the hydrolase activity of the human small intestine was found to be predominantly hCE-2. hCE-2 has a limited ability to hydrolyze large acyl compound substrates. Interestingly, propranolol derivatives, good substrates for hCE-2, were easily hydrolyzed by substitution of the methyl group on the 2-position of the acyl moiety, but were barely hydrolyzed when the methyl group was substituted on the 3-position. These findings suggest that hCE-2 does not easily form acylated intermediates because of conformational interference in its active site. In contrast, hCE-1 could hydrolyze a variety of substrates. The hydrolytic activity of hCE-2 increased with increasing alcohol chain length in benzoic acid derivative substrates, whereas hCE-1 preferentially catalyzed the hydrolysis of substrates with short alcohol chains. Kinetic data showed that the determining factor for the rate of hydrolysis of p-aminobenzoic acid esters was V(max) for hCE-1 and K(m) for hCE-2. Furthermore, the addition of hydrophobic alcohols to the reaction mixture with p-aminobenzoic acid propyl ester induced high and low levels of transesterification by hCE-1 and hCE-2, respectively. When considering the substrate specificities of hCE-1, it is necessary to consider the transesterification ability of hCE-1, in addition to the binding structure of the substrate in the active site of the enzyme.
Pyrethroids are neurotoxic pesticides whose pharmacokinetic behavior plays a role in their potency. This study examined the elimination of esfenvalerate and deltamethrin from rat and human liver microsomes. A parent depletion approach in the presence and absence of NADPH was used to assess species differences in biotransformation pathways, rates of elimination, and intrinsic hepatic clearance. Esfenvalerate was eliminated primarily via NADPH-dependent oxidative metabolism in both rat and human liver microsomes. The intrinsic hepatic clearance (CL(INT)) of esfenvalerate was estimated to be 3-fold greater in rodents than in humans on a per kilogram body weight basis. Deltamethrin was also eliminated primarily via NADPH-dependent oxidative metabolism in rat liver microsomes; however, in human liver microsomes, deltamethrin was eliminated almost entirely via NADPH-independent hydrolytic metabolism. The CL(INT) for deltamethrin was estimated to be 2-fold more rapid in humans than in rats on a per kilogram body weight basis. Metabolism by purified rat and human carboxylesterases (CEs) were used to further examine the species differences in hydrolysis of deltamethrin and esfenvalerate. Results of CE metabolism revealed that human carboxylesterase 1 (hCE-1) was markedly more active toward deltamethrin than the class 1 rat CEs hydrolase A and B and the class 2 human CE (hCE-2); however, hydrolase A metabolized esfenvalerate 2-fold faster than hCE-1, whereas hydrolase B and hCE-1 hydrolyzed esfenvalerate at equal rates. These studies demonstrate a significant species difference in the in vitro pathways of biotransformation of deltamethrin in rat and human liver microsomes, which is due in part to differences in the intrinsic activities of rat and human carboxylestersases.
Studies on the metabolism rates of 44 pyrethroids and 24 model compounds in mouse liver microsomal systems serve to divide the substrates into three groups based on their ease of hydrolysis and oxidation. Primary alcohol esters of trans-substituted cyclopropanecarboxylic acids are most rapidly metabolized with hydrolysis generally serving as the major component of the total metabolism rate. Although hydrolyzed slowly or not at detectable rates, the primary alcohol cis-substituted cyclopropanecarboxylates, tetramethylcyclopropanecarboxylates, and p-chlorophenyl-α-isopropylacetates are rapidly oxidized. The highly insecticidal α-cyano-3-phenoxybenzyl esters are least susceptible to metabolic attack due to both reduced esterase rates attributable to α substitution in the alcohol moiety and reduced oxidase rates for which no adequate explanation is currently available. Other structural modifications in the acid and alcohol moieties are less important in determining the metabolism rates. The substrate specificities of the microsomal esterases and oxidases are compared with in vivo pyrethroid structure-biodegradability relationships.
Permethrin is a pyrethroid insecticide widely used in agriculture and public health. It has been suggested that permethrin may interact with other chemicals used during military deployments and, as a result, be a potential cause of Gulf War Related Illness. To determine the causal relationship between permethrin and human health effects, the basic enzymatic pathway of permethrin metabolism in humans should be understood. In the present study we report that trans-permethrin is metabolized in human liver fractions, producing phenoxybenzyl alcohol (PBOH) and phenoxybenzoic acid (PBCOOH). We identified human alcohol (ADH) and aldehyde dehydrogenases (ALDH) as the enzymes involved in the oxidation of phenoxybenzyl alcohol, the permethrin hydrolysis product, to phenoxybenzoic acid by way of phenoxybenzaldehyde (PBCHO). Cis-permethrin was not significantly metabolized in human liver fractions. Cytochrome P450 isoforms were not involved either in the hydrolysis of trans-permethrin or in the oxidation of PBOH to PBCOOH. Purified ADH isozymes oxidized PBOH to PBCHO and PBOH was a preferred substrate to ethyl alcohol. Purified ALDH was responsible for PBCHO oxidation to PBCOOH with similar substrate affinity to a previously known substrate, benzyl alcohol. Based on these observations, it appears that PBOH is oxidized to PBCHO by ADH and subsequently to PBCOOH by ALDH, although PBCHO does not accumulate during microsomal incubation. In order to analyze permethrin and its metabolites, previous HPLC-UV methods had to be re-validated and modified. The resulting refined HPLC-UV method is described in detail.
The organophosphorus pesticides profenofos, sulprofos, O-ethyl O-(4-nitrophenyl) phenylphosphonothioate (EPN), and S,S,S,-tributyl phosphorotrithioate (DEF) administered intraperitoneally to mice at 0.5 to 5 mg/kg strongly inhibit the liver microsomal esterase(s) hydrolyzing trans-permethrin. Profenofos, EPN, and DEF at 25 mg/kg increase the intraperitoneal toxicity of fenvalerate > 25-fold and of malathion > 100-fold. Topically applied profenofos, sulprofos, and DEF significantly synergize the toxicity of cis-cypermethrin to cabbage looper larvae and house fly adults but these phosphorus compounds are much less effective in synergizing the toxicity of trans-permethrin. The magnitude of synergism appears to depend on the species, organophosphorus compound, and pyrethroid involved. Profenofos, sulprofos, and EPN do not significantly alter the persistence of trans-permethrin on bean foliage.
Separate esterase activities of rat and mouse liver microsomes hydrolyzing malathion, trans-permethrin, and cis-permethrin were differentiated on the basis of their sensitivities to inhibition by paraoxon and α-naphthyl N-propylcarbamate (NPC). In rat liver microsomes, the malathionhydrolyzing activity was more sensitive to both inhibitors and showed a different time course of NPC inhibition than the activities hydrolyzing the permethrin isomers. Paraoxon completely inhibited trans-permethrin hydrolysis, but only partially inhibited that of cis-permethrin. The paraoxonsensitive trans- and cis-permethrin-hydrolyzing activities were not differentially inhibited, but separate inhibition curves were obtained for the inhibition of trans- and cis-permethrin hydrolysis by NPC. The mouse liver esterase activity hydrolyzing trans-permethrin showed a similar paraoxon sensitivity to that of rat liver, but that the paraoxon-sensitive portion of the cis-permethrinhydrolyzing activity was 5.5-fold less sensitive to paraoxon than the corresponding rat liver activity and was clearly differentiated from the mouse liver trans-permethrin-hydrolyzing activity. The mouse liver malathion-hydrolyzing activity was 100-fold less sensitive to paraoxon and 14-fold less sensitive to NPC than the corresponding rat liver activity. Rat and mouse liver esterase activities hydrolyzed trans- and cis-permethrin at similar rates under standard assay conditions, but mouse liver esterases were 10-fold less active in hydrolyzing malathion. The higher specific activity of rat liver malathion-hydrolyzing esterases resulted from the greater apparent affinity and maximum velocity for malathion hydrolysis. These results demonstrate that the hydrolysis of malathion, trans-permethrin, and cis-permethrin by rat and mouse liver microsomal preparations involves several esterases with differing substrate specificities and inhibitor sensitivities.
Rat serum carboxylesterase [carboxylic ester hydrolase, EC 3.1.1.1] was purified by ammonium sulfate precipitation, chromatography on DEAE-cellulose, QAE Sephadex A-50 and brushite, and gel filtration on Sephadex G-200. This purified enzyme was shown to be a single protein band on slab gel electrophoresis and its final specific activity was 49.5 units/mg protein. This enzyme was very sensitive to diisopropylfluorophosphate (DFP) but not to p-chloromercuribenzoate (PCMB), eserine, o-iodosobenzoate, NaF or ethylenediamine tetraacetic acid (EDTA). The pH profile of the reactions catalysed by this enzyme showed broad optimum between pH 6.0 and 8.8. The activity of purified enzyme was not affected by Ca2+, Mg2+, Mn2+, Cu2+, Zn2+, Co2+, and Cd2+ at 1 mM concentration. The molecular weight measured by gel filtration was approximately 84,000 and the isoelectric point was 4.4. The enzymatic properties were not changed by neuraminidase treatment with regard to heat stability, pH optimum, sensitivity to metal ions and inhibitors, and Km values for p-nitrophenylesters of different acyl C-chain length.
1. Synthetic pyrethroids, based on the naturally-occurring insecticidal components of pyrethrum extract, emerged in the 1970s as the fourth major chemical class of synthetic insecticides. They are widely used today in the control of agriculture and household pests and disease vectors. 2. Early efforts in the design of synthetic analogues focused on the need to identify novel structural moieties that preserved or enhanced intrinsic insecticidal activity while eliminating known sites of metabolic and photolytic attack in the natural compounds. Subsequent efforts focused on achieving high levels of insecticidal activity while minimizing costs of synthesis and retaining desirable levels of selective toxicity. 3. The synthetic compounds obtained in these efforts constitute a group of insecticides having unprecedented biological activity against target species with low acute toxicity to mammals. 4. The evolutionary development of the pyrethroids illustrates how knowledge of metabolic fate can contribute to the design of novel insecticides with improved insecticidal activity and selective toxicity.
The tissue distribution of cocaine methyl esterase and ethanol-dependent ethyl transferase activities was determined in the rat and compared to the tissue distribution of three distinct non-specific hydrolases. Rates of formation of benzoylecgonine from cocaine and cocaethylene from ethanol and cocaine were measured in serum and tissue homogenate-supernatants of the brain, heart, kidney, liver, lung and spleen. The tissue distribution of three nonspecific esterases, A, B and C, was defined by nondenaturing gel electrophoresis and measuring the hydrolysis of 4-methylumbelliferyl acetate in the gels. Immunoreactive protein was localized by using Western blot analysis with polyclonal rabbit antihuman liver cocaine methyl esterase antibody after denaturing and nondenaturing gel electrophoresis. The rat liver, lung, kidney and heart exhibited cocaine methyl esterase and ethyl transferase activities and immunoreactive protein. The brain had cocaine methyl esterase activity but no ethyl transferase activity; neither activity was found in serum or spleen. The dominant immunoreactive bands in the liver, lung, kidney and heart comigrated with the 59 kD band of purified human liver cocaine methyl esterase. The rat liver, lung and kidney exhibited a band of nonspecific esterase activity that migrated with purified human liver cocaine methyl esterase and rat hydrolase A. These observations suggest that rat hydrolase A is similar to human cocaine methyl esterase. The lack of straight forward correlation between cocaine methyl esterase activity and immunoreactive protein and nonspecific esterase activity suggests that more than one enzyme catalyzes the hydrolysis of cocaine to benzoylecgonine in the rat.
Multiple forms of carboxylesterase have been identified in rat liver, and five carboxylesterases (designated hydrolases A, B, C, S, and egasyn) have been cloned. Hydrolases A, B, C, and egasyn all have a C-terminal consensus sequence (HXEL) for retaining proteins in the endoplasmic reticulum, and these carboxylesterases are found in rat liver microsomes. In contrast, hydrolase S lacks this C-terminal consensus sequence and is presumed to be secreted. In order to test this hypothesis, a polyclonal antibody was raised against recombinant hydrolase S from cDNA-directed expression in Escherichia coli. In addition to hydrolases A, B, and C (57-59 kDa), this antibody recognized a 67-kDa protein in rat liver microsomes and a 71-kDa protein in rat serum. The 71-kDa protein detected in rat serum was also detected in the extracellular medium from primary cultures of rat hepatocytes. Non-denaturing gel electrophoresis with staining for esterase activity showed that a serum carboxylesterase comigrated with the 71-kDa protein. Immunoprecipitation of the 71-kDa enzyme from rat serum decreased esterase activity toward 1-naphthylacetate and para-nitrophenylacetate. The 71-kDa protein immunoprecipitated from rat serum had an N-terminal amino acid sequence identical to that predicted from the cDNA encoding hydrolase S, providing further evidence that hydrolase S is synthesized in and secreted by the liver. The levels of the 67-kDa protein in rat liver microsomes and the levels of the 71-kDa protein in rat serum were co-regulated. Deglycosylation of microsomes and serum converted the 67- and 71-kDa proteins to a 58-kDa peptide, which matches the molecular mass calculated from the cDNA for hydrolase S. These results suggest that the 67-kDa protein in liver microsomes is a precursor form of hydrolase S that undergoes further glycosylation before being secreted into serum. In rats, liver appears to be the only source of hydrolase S because no mRNA encoding hydrolase S could be detected in several extrahepatic tissues. Serum carboxylesterases have been found to play an important role in lipid metabolism and detoxication of organophosphates, therefore, the secretion of hydrolase S and the modulation of its expression by xenobiotics may have physiological as well as toxicological significance.
The enzymatic hydrolysis of para-nitrophenylacetate by rat liver microsomes is predominantly catalyzed by two esterases: one with high affinity (Km approximately 25 microM) and one with low affinity (Km approximately 400 microM) for the substrate. Two kinetically distinct esterases were similarly detected in liver microsomes from mouse, hamster, guinea pig, rabbit, cat, cynomolgus monkey, and human, but only the high-affinity enzyme was detectable in dog liver microsomes. The tissue distribution of these kinetically distinct esterases was examined in rats. High-affinity (Km 20-35 microM esterase activity toward para-nitrophenylacetate was detected in testis, lung, prostate, and pancreas. The activity in testicular microsomes was comparable to that in liver microsomes. Low-affinity (Km 200-700 microM) esterase activity was detected in kidney, small intestine, lung, spleen, heart, and brain. The activity in kidney microsomes was comparable to that in liver microsomes. The high-affinity esterase in testicular and liver microsomes was highly sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF), whereas the low-affinity esterase in kidney and liver microsomes was relatively resistant. These results suggested that rat liver microsomes contain two esterases with high activity toward para-nitrophenylacetate, a PMSF-sensitive esterase with high substrate affinity, and a PMSF-insensitive esterase with low substrate affinity. In support of the hypothesis, we have purified and characterized two esterases, designated hydrolases A and B, which appear be the only abundant enzymes in rat liver microsome that rapidly hydrolyze para-nitrophenylacetate. Hydrolase A hydrolyzed para-nitrophenylacetate with high affinity (Km approximately 25 microM), and was inhibited by extremely low concentrations of PMSF (IC50 approximately 100 nM). In contrast, hydrolase B hydrolyzed para-nitrophenylacetate with low affinity (Km approximately 400 microM) and was inhibited only by relatively high concentrations of PMSF (IC50 approximately 100 microM Paraoxon, the active metabolite of parathion, and cresylbenzodioxaphosphorin oxide, the active metabolite tri-ortho-tolylphosphate, completely inhibited the hydrolysis of pra-nitrophenylacetate by rat liver microsomes and by hydrolases A and B, whereas the sulfhydryl agent, para-chloromercurobenzoate, was not inhibition. These results suggest that hydrolases A and B are both serine esterases. The N-terminal amino acid sequence of hydrolases A and B were similar but distinct (23 the first 30 amino acid residues were identical), indicating that these two esterases are isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)
Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures.
The purified enzyme hydrolyzed cholesteryl oleate, cholesteryl linoleate, and triolein at similar rates over a broad range of concentrations. Hydrolytic activity was relatively low with p-nitrophenyl acetate, but much higher with PNP-esters of the more lipophilic C4-C18 fatty acids, in sharp contrast to microsomal esterases which hydrolyze PNP-acetate more efficiently. Zn2+, Cu2+, Cd2+, Hg2+, and phenylmethylsulfonyl fluoride inhibited, whereas N-ethyl maleimide and iodoacetamide stimulated activity of the pure enzyme. Limited trypsin digestion selectively inhibited cholesteryl esterase activity with retention of activity toward PNP-octanoate, suggesting involvement of a trypsin-labile loop in the lipophilic substrate binding pocket.
A full-length cDNA coding for a putative intestinal carboxylesterase (iCE) was isolated from a human small intestine cDNA library. The cDNA has an open reading frame of 559 amino acids with up to 65% homology to other carboxylesterases of different mammalian species. The deduced amino-acid sequence contains many structural features, that are highly conserved among all carboxylesterase isoenzymes, like the serine esterase active site, an ER-retention signal and one Asn-Xxx-Thr site for N-linked carbohydrate addition. Northern blot analysis revealed that the corresponding mRNA is 3.4-3.6 kb in size and is preferentially expressed in human intestine with a weak signal also in liver. Analysis of cells from the gastrointestinal tract unveiled site-specific, transcriptional regulation of iCE, with higher expression in small intestine and lower expression in colon and rectum. The high expression in small intestine is attributable to a higher expression in jejunum compared to duodenum and ileum.
Multiple carboxylesterases (EC 3.1.1.1) play an important role in the hydrolytic biotransformation of a vast number of structurally diverse drugs. These enzymes are major determinants of the pharmacokinetic behavior of most therapeutic agents containing ester or amide bonds. Carboxylesterase activity can be influenced by interactions of a variety of compounds either directly or at the level of enzyme regulation. Since a significant number of drugs are metabolized by carboxylesterase, altering the activity of this enzyme class has important clinical implications. Drug elimination decreases and the incidence of drug-drug interactions increases when two or more drugs compete for hydrolysis by the same carboxylesterase isozyme. Exposure to environmental pollutants or to lipophilic drugs can result in induction of carboxylesterase activity. Therefore, the use of drugs known to increase the microsomal expression of a particular carboxylesterase, and thus to increase associated drug hydrolysis capacity in humans, requires caution. Mammalian carboxylesterases represent a multigene family, the products of which are localized in the endoplasmic reticulum of many tissues. A comparison of the nucleotide and amino acid sequence of the mammalian carboxylesterases shows that all forms expressed in the rat can be assigned to one of three gene subfamilies with structural identities of more than 70% within each subfamily. Considerable confusion exists in the scientific community in regards to a systematic nomenclature and classification of mammalian carboxylesterase. Until recently, adequate sequence information has not been available such that valid links among the mammalian carboxylesterase gene family or evolutionary relationships could be established. However, sufficient basic data are now available to support such a novel classification system.
Expression of a rabbit liver carboxylesterase has been achieved in several different model systems including Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Spodoptera frugiperda, and COS7 cells. Although, recombinant protein was observed in E. coli sonicates, little or no enzymatic activity was detected. Similarly, no activity was observed following expression in S. cerevisiae. In contrast, active protein was produced in P. pastoris, from S. frugiperda following baculoviral infection and in COS7 cells following transient transfection of plasmid DNA. For the preparation of small amounts of protein for kinetic and biochemical studies, enzyme expressed in P. pastoris has proved sufficient. However, to produce large amounts of carboxylesterase for structural studies, baculoviral-mediated expression of a secreted form of the protein in S. frugiperda was the most efficient. Using this system, we have generated and purified milligram quantities of essentially pure protein. These results demonstrate that the choice of in vitro system for the generation of large amounts of active carboxylesterase, and probably most endoplasmic reticulum processed proteins, is crucial for high level expression and subsequent purification.
This article reports on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 2001 Experimental Biology meeting. Current developments in molecular-based studies into the structure and function of cholinesterases, carboxylesterases, and paraoxonases are described. This article covers mechanisms of regulation of gene expression of the various esterases by developmental factors and xenobiotics, as well as the interplay between physiological and chemical regulation of enzyme activity.
Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.
The use of insecticide-treated bednets (ITNs) has been widely adopted as an important method for malaria control. Few data exist on effects of ITNs on mosquito biology and ecology, other than the development of insecticide resistance against the insecticides used. There is no hard evidence that the insecticide resistance recorded is the result of insecticidal use on bednets or from agricultural use. Resistance against pyrethroids, the preferred class of insecticides for ITN use, has been recorded from countries in Asia, Africa and South America. Resistance is expressed as reduced excito-repellency and mortality of mosquitoes exposed to insecticide-treated materials. In the absence of resistance, however, most studies on ITN effects report a reduced survival of adult mosquitoes as well as mass killing. Other effects are highly variable, and shifts in time of biting, feeding site and blood hosts have occasionally been reported, but not in proportion to the scale of ITN use. In general, a reduced sporozoite rate is recorded in ITN programmes. Because many of the anticipated behavioural effects caused by insecticidal use will be avoided by the use of untreated nets, studies on the efficacy of untreated nets are required. Examples are presented in which untreated nets provided a reasonable degree of protection against malaria.
The drinking water disinfection byproduct bromodichloromethane (CHBrCl(2)) was previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study to investigate the DNA covalent binding potential of reactive intermediates generated by GSTT1-1-mediated metabolism of CHBrCl(2). First, rodent hepatic cytosol incubations containing [(14)C]CHBrCl(2), supplemented glutathione (GSH), and calf thymus DNA resulted in approximately 3-fold (rat liver cytosol) and 7-fold (mouse liver cytosol) greater amounts of total radioactivity (RAD) associated with the purified DNA as compared to a control (absence of rodent cytosol) following liquid scintillation counting (LSC) of isolated DNA. The relative increase in DNA labeling is consistent with the conjugation activity of these rodent cytosols toward CHBrCl(2). Second, exposure of GSTT1-1-expressing S. typhimurium to [(14)C]CHBrCl(2) resulted in a concentration-dependent increase of bacterial DNA-associated total radioactivity. Characterization of DNA-associated radioactivity could not be assigned to a specific deoxynucleoside adduct(s) following enzymatic hydrolysis of DNA and subsequent HPLC analysis. A possible explanation for this observation was formation of a 'transient' adduct that was unstable in the DNA isolation and hydrolysis procedures employed. To circumvent problems of adduct instability, reactions of [(14)C]CHBrCl(2) with GSH catalyzed by recombinant rat GSTT1-1 were performed in the presence of calf thymus DNA or, alternatively, the model nucleophile deoxyguanosine. Hydroxyapatite chromatography of [(14)C]-labeled DNA or HPLC chromatography of [(14)C]-labeled deoxyguanosine derivatives demonstrated the covalent binding of [(14)C]CHBrCl(2)-derived metabolites to DNA and deoxyguanosine in low yield (approximately 0.02% of [(14)C]CHBrCl(2) biotransformed by GSTT1-1 resulted in DNA adducts). Cytochrome P450 (CYP)- and GST-catalyzed biotransformation of CHBrCl(2) in rat tissues (kidney and large intestine) that develop tumors following chronic CHBrCl(2) exposure were compared with rat liver (a nontarget tissue). Rat liver had a significant capacity to detoxify CHBrCl(2) (to carbon dioxide) compared with kidney and large intestine as a result of CYP-catalyzed oxidation, liver was approximately 16-fold more efficient than kidney and large intestine when intrinsic clearance values (V(max)/K(m)) were compared. In contrast, the efficiency of GST-mediated GSH conjugation of CHBrCl(2) in kidney and large intestine was only slightly lower than liver (approximately 2- to 4-fold lower), thus, the relative amounts of reactive intermediates that are produced with the capacity to covalently modify DNA may be enhanced in these extrahepatic tissues. The significance of these findings is that conjugation of CHBrCl(2) with GSH can result in the covalent modification of DNA and that cancer target tissues in rats have a much reduced detoxification capacity, but only a modest decrease in bioactivation capacity, as compared to the liver (a nontarget tissue in rats).
Permethrin, a pyrethroid insecticide, is one of several deployment-related chemicals that have been suggested as causative agents for Gulf War related illnesses. Hydrolysis of trans-permethrin (tPMT) is a major route of detoxication and a potential locus for interactions with chemicals with similar use patterns. This study examined the potential inhibitory effects of chlorpyrifos, carbaryl, pyridostigmine bromide and the insect repellent N,N-diethyl-m-toluamide (DEET) on tPMT hydrolysis in human liver fractions. Although chlorpyrifos was not inhibitory, its toxic metabolite, chlorpyrifos oxon, strongly and irreversibly inhibited tPMT hydrolysis at low concentrations (cytosolic and microsomal Ki values of 3 and 16 nM, respectively). Carbaryl, a known anticholinesterase agent, showed non-competitive inhibition kinetics, with Ki values two orders of magnitude higher than those for chlorpyrifos oxon. Although DEET was much less effective than either chlorpyrifos oxon or carbaryl, equimolar concentrations inhibited up to 45% of tPMT hydrolysis. Pyridostigmine bromide showed no inhibitory effects. This study suggests that interaction potential between organophosphorus and pyrethroid insecticides should be considered in safety assessments when both insecticides are deployed simultaneously.
Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2) > 0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.
Lipases and esterases constitute a large category of enzymes. They are ubiquitous in nature, found in bacteria, fungi, and animals. The family members address a wide variety of structurally diverse substrates. Appropriately, a large number of assays have been developed to analyze their activity in vitro. Here, we present an overview of these enzymes, along with protocols for common assays performed in solution. An emphasis is placed on assays for enzymes that can hydrolyze triacylglycerols. (c) 2005 Elsevier Inc. All rights reserved.
To examine the antitumor activity and the pharmacokinetics of CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) in a plasma esterase-deficient scid mouse model, bearing human tumor xenografts.
Plasma carboxylesterase (CE)-deficient mice were bred with scid animals to develop a strain that would allow growth of human tumor xenografts. Following xenotransplantation, the effect of the plasma esterase on antitumor activity following CPT-11 administration was assessed. In addition, detailed pharmacokinetic studies examining plasma and biliary disposition of CPT-11 and its metabolites were performed.
In mice lacking plasma carboxylesterase, the mean SN-38 systemic exposures were approximately fourfold less than that observed in control animals. Consistent with the pharmacokinetic data, four to fivefold more CPT-11 was required to induce regressions in human Rh30 xenografts grown in esterase-deficient scid mice, as opposed to those grown in scid animals. Additionally, the route of elimination of CPT-11, SN-38, and SN-38 glucuronide (SN-38G) was principally in the bile.
The pharmacokinetic profile for CPT-11 and its metabolites in the esterase-deficient mice more closely reflects that seen in humans. Hence, these mice may represent a more accurate model for antitumor studies with this drug and other agents metabolized by CEs.
Deltamethrin, a widely used type II pyrethroid insecticide, is a relatively potent neurotoxicant. While the toxicity has been extensively examined, toxicokinetic studies of deltamethrin and most other pyrethroids are very limited. The aims of this study were to identify, characterize, and assess the relative contributions of esterases and cytochrome P450s (CYP450s) responsible for deltamethrin metabolism by measuring deltamethrin disappearance following incubation of various concentrations (2 to 400 microM) in plasma (esterases) and liver microsomes (esterases and CYP450s) prepared from adult male rats. While the carboxylesterase metabolism in plasma and liver was characterized using an inhibitor, tetra isopropyl pyrophosphoramide (isoOMPA), CYP450 metabolism was characterized using the cofactor, NADPH. Michaelis-Menten rate constants were calculated using linear and nonlinear regression as applicable. The metabolic efficiency of these pathways was estimated by calculating intrinsic clearance (Vmax/Km). In plasma, isoOMPA completely inhibited deltamethrin biotransformation at concentrations (2 and 20 microM of deltamethrin) that are 2- to 10-fold higher than previously reported peak blood levels in deltamethrin-poisoned rats. For carboxylesterase-mediated deltamethrin metabolism in plasma, Vmax=325.3+/-53.4 nmol/h/ml and Km=165.4+/-41.9 microM. Calcium chelation by EGTA did not inhibit deltamethrin metabolism in plasma or liver microsomes, indicating that A-esterases do not metabolize deltamethrin. In liver microsomes, esterase-mediated deltamethrin metabolism was completely inhibited by isoOMPA, confirming the role of carboxylesterases. The rate constants for liver carboxylesterases were Vmax=1981.8+/-132.3 nmol/h/g liver and Km=172.5+/-22.5 microM. Liver microsomal CYP450-mediated biotransformation of deltamethrin was a higher capacity (Vmax=2611.3+/-134.1 nmol/h/g liver) and higher affinity (Km=74.9+/-5.9 microM) process than carboxylesterase (plasma or liver) detoxification. Genetically engineered individual rat CYP450s (Supersomes) were used to identify specific CYP450 isozyme(s) involved in the deltamethrin metabolism. CYP1A2, CYP1A1, and CYP2C11 in decreasing order of importance quantitatively, metabolized deltamethrin. Intrinsic clearance by liver CYP450s (35.5) was more efficient than that by liver (12.0) or plasma carboxylesterases (2.4).
The goal of this work was to identify the esterases in human plasma and to clarify common misconceptions. The method for identifying esterases was nondenaturing gradient gel electrophoresis stained for esterase activity. We report that human plasma contains four esterases: butyrylcholinesterase (EC 3.1.1.8), paraoxonase (EC 3.1.8.1), acetylcholinesterase (EC 3.1.1.7), and albumin. Butyrylcholinesterase (BChE), paraoxonase (PON1), and albumin are in high enough concentrations to contribute significantly to ester hydrolysis. However, only trace amounts of acetylcholinesterase (AChE) are present. Monomeric AChE is seen in wild-type as well as in silent BChE plasma. Albumin has esterase activity with alpha- and beta-naphthylacetate as well as with p-nitrophenyl acetate. Misconception #1 is that human plasma contains carboxylesterase. We demonstrate that human plasma contains no carboxylesterase (EC 3.1.1.1), in contrast to mouse, rat, rabbit, horse, cat, and tiger that have high amounts of plasma carboxylesterase. Misconception #2 is that lab animals have BChE but no AChE in their plasma. We demonstrate that mice, unlike humans, have substantial amounts of soluble AChE as well as BChE in their plasma. Plasma from AChE and BChE knockout mice allowed identification of AChE and BChE bands without the use of inhibitors. Human BChE is irreversibly inhibited by diisopropylfluorophosphate, echothiophate, and paraoxon, but mouse BChE spontaneously reactivates. Since human plasma contains no carboxylesterase, only BChE, PON1, and albumin esterases need to be considered when evaluating hydrolysis of an ester drug in human plasma.
Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of alpha-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against alpha-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity.
Carboxylesterases hydrolyze a large array of endogenous and exogenous ester-containing compounds, including pyrethroid insecticides. Herein, we report the specific activities and kinetic parameters of human carboxylesterase (hCE)-1 and hCE-2 using authentic pyrethroids and pyrethroid-like, fluorescent surrogates. Both hCE-1 and hCE-2 hydrolyzed type I and II pyrethroids with strong stereoselectivity. For example, the trans-isomers of permethrin and cypermethrin were hydrolyzed much faster than corresponding cis-counterparts by both enzymes. Kinetic values of hCE-1 and hCE-2 were determined using cypermethrin and 11 stereoisomers of the pyrethroid-like, fluorescent surrogates. K(m) values for the authentic pyrethroids and fluorescent surrogates were in general lower than those for other ester-containing substrates of hCEs. The pyrethroid-like, fluorescent surrogates were hydrolyzed at rates similar to the authentic pyrethroids by both enzymes, suggesting the potential of these compounds as tools for high throughput screening of esterases that hydrolyze pyrethroids.
Pyrethroid chemicals are attractive alternatives to the organophosphates (OPs) because of their selective toxicity against pests rather than mammals. The carboxylesterases (CEs) are hepatic enzymes that metabolize ester-containing xenobiotics such as pyrethroids. The primary aim of this study was to gain insight into the catalytic properties of the CE enzymes in humans that metabolize pyrethroids, while a secondary aim was to investigate pyrethroid metabolism using CEs from other mammalian species. Pure human CEs (hCE-1 and hCE-2), a rabbit CE (rCE), and two rat CEs (Hydrolases A and B) were used to study the hydrolytic metabolism of the following pyrethroids: 1Rtrans-resmethrin (bioresmethrin), 1RStrans-permethrin, and 1RScis-permethrin. hCE-1 and hCE-2 hydrolyzed trans-permethrin 8- and 28-fold more efficiently than cis-permethrin (when k(cat)/K(m) values were compared), respectively. In contrast, hydrolysis of bioresmethrin was catalyzed efficiently by hCE-1, but not by hCE-2. The kinetic parameters for the pure rat and rabbit CEs were qualitatively similar to the human CEs when hydrolysis rates of the investigated pyrethroids were evaluated. Further, a comparison of pyrethroid hydrolysis by hepatic microsomes from rats, mice, and humans indicated that the rates for each compound were similar between species, which further supports the use of rodent models for pyrethroid metabolism studies. An eight-fold range in hydrolytic rates for 11 individual human liver samples toward trans-permethrin was also found, although this variability was not related to the levels of hCE-1 protein in each sample. We also determined that the CE inhibitor 2-chloro-3,4-dimethoxybenzil blocked hCE-2-catalyzed trans-permethrin hydrolysis 36 times more potently than hCE-1. Thus, this inhibitor will be useful in future studies that examine CE-mediated metabolism of pyrethroids. While there are likely other esterases in human liver that hydrolyze pyrethroids, the results of this study clearly demonstrate that hCE-1 and hCE-2 are human pyrethroid-hydrolyzing CEs.
Carboxylesterases (CE) are ubiquitous enzymes responsible for the detoxification of xenobiotics. Many therapeutically useful drugs are metabolized by these proteins which impacts upon the efficiency of drug treatment. In some instances, CEs convert inactive prodrugs to active metabolites, a process that is essential for biological activity. Such compounds include the anticancer agents CPT-11 (3) and capecitabine (4), the antibiotics Ceftin (9) and Vantin, as well as the illicit street drug heroin (6). However, more commonly, CEs hydrolyze many esterified drugs to inactive products that are then excreted. Agents such as flestolol (11), meperidine (5), lidocaine (8) and cocaine (7), are all hydrolyzed and inactivated by these enzymes. Therefore the efficacy of esterified drugs will be dependent upon the distribution and catalytic activity of different CEs. In this review, we examine the structural aspects of CEs and their roles in drug detoxification and propose that modulation of CE activity may allow for improvements in, and potentiation of, drug efficacy.
Human carboxylesterase 1 (hCE-1, CES1A1, HU1) and carboxylesterase 2 (hCE-2, hiCE, HU3) are a serine esterase involved in both drug metabolism and activation. Although both hCE-1 and hCE-2 are present in several organs, the hydrolase activity of liver and small intestine is predominantly attributed to hCE-1 and hCE-2, respectively. The substrate specificity of hCE-1 and hCE-2 is significantly different. hCE-1 mainly hydrolyzes a substrate with a small alcohol group and large acyl group, but its wide active pocket sometimes allows it to act on structurally distinct compounds of either large or small alcohol moiety. In contrast, hCE-2 recognizes a substrate with a large alcohol group and small acyl group, and its substrate specificity may be restricted by a capability of acyl-hCE-2 conjugate formation due to the presence of conformational interference in the active pocket. Furthermore, hCE-1 shows high transesterification activity, especially with hydrophobic alcohol, but negligible for hCE-2. Transesterification may be a reason for the substrate specificity of hCE-1 that hardly hydrolyzes a substrate with hydrophobic alcohol group, because transesterification can progress at the same time when a compound is hydrolyzed by hCE-1.
The mammalian carboxylesterases: from molecules to functions
- T Satoh
- M Hosokawa
Satoh T, Hosokawa M. The mammalian carboxylesterases: from molecules to functions. Annu Rev
Pharmacol Toxicol 1998;38:257–288. [PubMed: 9597156]
Techniques to measure lipase and esterase activity in vitro
- D Gilham
- R Lehner
Gilham D, Lehner R. Techniques to measure lipase and esterase activity in vitro. Methods 2005;36:139–
147. [PubMed: 15893936]
Hydrolysis in Drug and Prodrug Metabolism: Chemistry, Biochemistry, and Enzymology
- B Testa
- Jm Mayer
Testa, B.; Mayer, JM. Hydrolysis in Drug and Prodrug Metabolism: Chemistry, Biochemistry, and
Enzymology. Verlag Chimica Acta; Zurich, Switzerland: 2003.
Identification and characterization of novel benzil (diphenylethane-1,2-dione)analogues as inhibitors of mammalian carboxylesterases
- Rm Wadkins
- Jl Hyatt
- X Wei
- Kj Yoon
- M Wierdl
- Cc Edwards
Wadkins RM, Hyatt JL, Wei X, Yoon KJ, Wierdl M, Edwards CC, et al. Identification and
characterization of novel benzil (diphenylethane-1,2-dione)analogues as inhibitors of mammalian
carboxylesterases. J Med Chem 2005;48:2906–2915. [PubMed: 15828829]