Abdolsamad Borazjani

Inactivation of lipid glyceryl ester metabolism in human THP1 monocytes/macrophages by activated organophosphorus insecticides: role of carboxylesterases 1 and 2.

Carboxylesterases (CES) have important roles in pesticide and drug metabolism and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad substrate specificity to hydrolyze endobiotics, such as cholesteryl esters and triacylglycerols. Here, we determined if two human CES isoforms, CES1 and CES2, hydrolyze the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide (AEA), and two prostaglandin glyceryl esters (PG-Gs), which are formed by COX-mediated oxygenation of 2AG. We show that recombinant CES1 and CES2 efficiently hydrolyze 2AG to arachidonic acid (AA) but not amide-containing AEA. Steady-state kinetic parameters for CES1- and CES2-mediated 2AG hydrolysis were, respectively, kcat, 59 and 43 min(-1); Km, 49 and 46 μM; and kcat/Km, 1.2 and 0.93 μM(-1) min(-1). kcat/Km values are comparable to published values for rat monoacylglycerol lipase (MAGL)-catalyzed 2AG hydrolysis. Furthermore, we show that CES1 and CES2 also efficiently hydrolyze PGE2-G and PGF2α-G. In addition, when cultured human THP1 macrophages were treated with exogenous 2AG or PG-G (10 μM, 1 h), significant quantities of AA or PGs were detected in the culture medium; however, the ability of macrophages to metabolize these compounds was inhibited (60-80%) following treatment with paraoxon, the toxic metabolite of the insecticide parathion. Incubation of THP1 cell lysates with small-molecule inhibitors targeting CES1 (thieno[3,2-e][1]benzothiophene-4,5-dione or JZL184) significantly reduced lipid glyceryl ester hydrolase activities (40-50% for 2AG and 80-95% for PG-Gs). Immunodepletion of CES1 also markedly reduced 2AG and PG-G hydrolase activities. These results suggested that CES1 is in part responsible for the hydrolysis of 2AG and PG-Gs in THP1 cells, although it did not rule out a role for other hydrolases, especially with regard to 2AG metabolism since a substantial portion of its hydrolysis was not inactivated by the inhibitors. An enzyme (Mr 31-32 kDa) of unknown function was detected by serine hydrolase activity profiling of THP1 cells and may be a candidate. Finally, the amounts of in situ generated 2AG and PG-Gs in macrophages were enhanced by treating the cells with bioactive metabolites of OP insecticides. Collectively, the results suggest that in addition to MAGL and fatty-acid amide hydrolase (FAAH), which have both been documented to terminate endocannabinoid signaling, CES may also have a role. Furthermore, since PG-Gs have been shown to possess biological activities in their own right, CES may represent an important enzyme class that regulates their in vivo levels.

Examination of the carboxylesterase phenotype in human liver

Carboxylesterases (CES) metabolize esters. Two CES isoforms are expressed in human liver (CES1 and CES2) and liver extracts are used in reaction phenotyping studies to discern interindividual metabolic variation. We tested the hypothesis that an individuals CES phenotype can be characterized by reporter substrates/probes that interrogate native CES1 and CES2 activities in liver and immunoblotting methods. We obtained 25 livers and found that CES1 is the main hydrolytic enzyme. Moreover, although CES1 protein levels were similar, we observed large interindividual variation in bioresmethrin hydrolysis rates (17-fold), a pyrethroid metabolized by CES1 but not CES2. Bioresmethrin hydrolysis rates did not correlate with CES1 protein levels. In contrast, procaine hydrolysis rates, a drug metabolized by CES2 but not CES1, were much less variant (3-fold). Using activity-based fluorophosphonate probes (FP-biotin), which covalently reacts with active serine hydrolases, CES1 protein was the most active enzyme in the livers. Finally, using bioorthogonal probes and click chemistry methodology, the half-life of CES 1 and 2 in cultured HepG2 cells was estimated at 96 h. The cause of the differential CES1 activities is unknown, but the underlying factors will be important to understand because several carboxylic acid ester drugs and environmental toxicants are metabolized by this enzyme.

Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon.

Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC(50) values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon>paraoxon>methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k(inact)/K(I)) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC(50) values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon>paraoxon>methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1 and CES2 respectively. Together, the results define the kinetics of inhibition of three important hydrolytic enzymes by activated metabolites of widely used agrochemicals.

Identification of Palmitoyl Protein Thioesterase 1 in Human THP1 Monocytes and Macrophages and Characterization of Unique Biochemical Activities for This Enzyme

The profiles of serine hydrolases in human and mouse macrophages are similar yet different. For instance, human macrophages express high levels of carboxylesterase 1 (CES1), whereas mouse macrophages have minimal amounts of the orthologous murine CES1. On the other hand, macrophages from both species exhibit limited expression of the canonical 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, MAGL. Our previous study showed CES1 was partly responsible for the hydrolysis of 2-AG (50%) and prostaglandin glyceryl esters (PG-Gs) (80-95%) in human THP1 monocytes and macrophages. However, MAGL and other endocannabinoid hydrolases, FAAH, ABHD6, and ABHD12, did not have a role because of limited expression or no expression. Thus, another enzyme was hypothesized to be responsible for the remaining 2-AG hydrolysis activity following chemical inhibition and immunodepletion of CES1 (previous study) or CES1 gene knockdown (this study). Here we identified two candidate serine hydrolases in THP1 cell lysates by activity-based protein profiling (ABPP)-MUDPIT and Western blotting: cathepsin G and palmitoyl protein thioesterase 1 (PPT1). Both proteins exhibited electrophoretic properties similar to those of a serine hydrolase in THP1 cells detected by gel-based ABPP at 31-32 kDa; however, only PPT1 exhibited lipolytic activity and hydrolyzed 2-AG in vitro. Interestingly, PPT1 was strongly expressed in THP1 cells but was significantly less reactive than cathepsin G toward the activity-based probe, fluorophosphonate-biotin. KIAA1363, another serine hydrolase, was also identified in THP1 cells but did not have significant lipolytic activity. On the basis of chemoproteomic profiling, immunodepletion studies, and chemical inhibitor profiles, we estimated that PPT1 contributed 32-40% of 2-AG hydrolysis activity in the THP1 cell line. In addition, pure recombinant PPT1 catalyzed the hydrolysis of 2-AG, PGE2-G, and PGF2α-G, although the catalytic efficiency of hydrolysis of 2-AG by PPT1 was ~10-fold lower than that of CES1. PPT1 was also insensitive to several chemical inhibitors that potently inhibit CES1, such as organophosphate poisons and JZL184. This is the first report to document the expression of PPT1 in a human monocyte and macrophage cell line and to show PPT1 can hydrolyze the natural substrates 2-AG and PG-Gs. These findings suggest that PPT1 may participate in endocannabinoid metabolism within specific cellular contexts and highlights the functional redundancy often exhibited by enzymes involved in lipid metabolism.

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids.

Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC50=33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC(50)=8.1 microM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (K(iapp)=10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (K(iapp)=1.7 microM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo.

Covalent Inhibition of Recombinant Human Carboxylesterase 1 and 2 and Monoacylglycerol Lipase by the Carbamates JZL184 and URB597

Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metabolism of several pharmaceuticals. Recently, carbamate compounds were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate constants for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimolecular rate constants (k(inact)/K(i)) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 10(3) M(-1) s(-1) and 4.5 (±1.3) × 10(3) M(-1) s(-1), respectively]. However, k(inact)/K(i) for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 10(2) M(-1) s(-1) and 3.9 (±1.0) × 10(3) M(-1) s(-1), respectively]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also determined kinetic constants for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metabolism.

Effect of Developmental Chlorpyrifos Exposure on Endocannabinoid Metabolizing Enzymes in the Brain of Juvenile Rats

The endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamide (AEA or anandamide) play vital roles during nervous system development including regulating axonal guidance and synaptogenesis. The enzymatic degradation of 2-AG and AEA is highly susceptible to inhibition by organophosphate compounds in vitro. Furthermore, acute in vivo exposure of adult animals to the agricultural insecticide chlorpyrifos (CPS) caused moderate inhibition of both 2-AG and AEA hydrolysis. However, the effects of repeated exposure to lower levels of CPS, especially during development, on endocannabinoid metabolism in the brain is not known. To examine this, rat pups were orally exposed daily from postnatal days 10-16 to either 1.0, 2.5, or 5.0 mg/kg CPS. Body weight gain was reduced by 5.0 mg/kg on all days of treatment whereas 2.5 mg/kg reduced the weight gain only on the last two days of treatment. At 4-h postexposure on day 16, forebrain cholinesterase (ChE) activity and hydrolysis of 2-AG and AEA were inhibited in a dose-related manner, and the extent of inhibition from highest to lowest level was AEA hydrolysis > ChE activity > 2-AG hydrolysis. The extent of inhibition of AEA hydrolysis was approximately twice than that of ChE activity with AEA hydrolysis being virtually eliminated by 2.5 and 5.0 mg/kg and 1.0 mg/kg causing 40% inhibition. The sensitivity of AEA hydrolysis, compared with canonical targets such as ChE activity, suggests a potential alternative developmental target for CPS. Inhibition of AEA hydrolysis could result in accumulation of endocannabinoids, which could alter normal endocannabinoid transmission during brain maturation.

Evaluation of the ‘side door’ in carboxylesterase-mediated catalysis and inhibition

Abstract Structures of mammalian carboxylesterases (CEs) reveal the presence of a ‘side door’ that is proposed to act as an alternative pore for the trafficking of substrates and products. p-Nitrobenzyl esterase (pnb CE) from Bacillus subtilis exhibits close structural homology and a similar side-door domain as mammalian CEs. We investigated the role of a specific ‘gate’ residue at the side door (i.e., Leu 362) during pnb CE-catalyzed hydrolysis of model esters, pesticides, and lipids. Recombinant pnb CE proteins containing mutations at position 362 demonstrated markedly lower k cat and k cat/K m values. The mutation with the most significant impact on catalysis was the L362R mutant (k cat/K m was 22-fold lower). Moreover, the ability of the L362R mutant to be inhibited by organophosphates (OP) was also lower. Investigation into the altered catalytic proficiency using pH-activity studies indicated that the catalytic triad of the mutant enzyme was preserved. Furthermore, viscosity variation and carbamate inhibition experiments indicated that rates of substrate association and acylation/deacylation were lower. Finally, recombinant CEs were found to possess lipolytic activity toward cholesteryl oleate and 2-arachidonylglycerol. In summary, the L362R mutant CE markedly slowed the rate of ester hydrolysis and was less sensitive to OP inhibition. The apparent causes of the diminished catalysis are discussed.

Enzymatic activity of human carboxylesterases.

The carboxylesterases (CEs) are hydrolytic enzymes that metabolize xenobiotics that contain ester, thioester, or amide bonds. CEs are ubiquitously expressed but are found in highest concentration in membrane‐enriched fractions of the liver. This unit describes assays used to measure the enzymatic activity and tissue distribution of human CEs. Both spectrophotometric and HPLC‐based assays are described in detail. Several of these methods are rapid and easy to perform. These methods will be useful to investigators who are interested in studying the metabolism and disposition of esterified drugs and environmental chemicals. Curr. Protoc. Toxicol. 33:4.24.1‐4.24.14.

Lipopolysaccharide suppresses carboxylesterase 2g activity and 2-arachidonoylglycerol hydrolysis: A possible mechanism to regulate inflammation.

Inflammation is an important part of the innate immune response and is involved in the healing of many disease processes; however, chronic inflammation is a harmful component of many diseases. The regulatory mechanisms of inflammation are incompletely understood. One possible regulatory mechanism is the endocannabinoid system. Endocannabinoids such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA) are generally anti-inflammatory via engagement of the cannabinoid receptor 2 (CB2) on innate cells; therefore, preventing the degradation of endocannabinoids by specific serine hydrolases such as fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), and carboxylesterases (CES) might decrease inflammation. We hypothesized that the activities of these catabolic enzymes would decrease with a subsequent increase in 2-AG and AEA in a model of inflammation. Mice were injected with lipopolysaccharide (LPS) for 6 or 24h, and inflammation was confirmed by an increase in interleukin-6 (il6) and il17 gene expression. Activity-based protein profiling (ABPP) of serine hydrolases showed no significant difference in various serine hydrolase activities in brain or liver, whereas a modest decrease in Ces activity in spleen after LPS administration was noted. 2-AG hydrolase activity in the spleen was also decreased at 6h post LPS, which was corroborated by LPS treatment of splenocytes ex vivo. ABPP-MudPIT proteomic analysis suggested that the decreased 2-AG hydrolysis in spleen was due to a reduction in Ces2g activity. These studies suggest that the endocannabinoid system could be activated via suppression of a 2-AG catabolic enzyme in response to inflammatory stimuli as one mechanism to limit inflammation.