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

Abstract

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.

Figure 1

Hydrolysis of 2AG and PG-Gs by recombinant human CES 1 and 2. (A) Chemical scheme of 2AG hydrolysis. (B) LC-MS chromatograms of arachidonic acid (AA, m/z 303) obtained when 2AG was incubated with CES1 for 10-75 min. (C) Time course of AA formation when 2AG (250μM) was incubated in the presence (enzymatic) or absence (non-enzymatic) of CES1. (D) Substrate concentration vs. velocity (rate of 2AG turnover) curves for human recombinant CES1 and CES2. Curves are representative of at least 3 independent experiments. (E-G) Comparison of the hydrolytic activities of FAAH, CES1, and MAGL toward 2AG (10 μM), PGE₂-G (25 μM) or PGF_2α-G (25 μM). Recombinant human enzymes were incubated with (E) 2AG (10μM), (F) PGE₂-G (25μM), or (G) PGF_2α-G (25μM) for 15 min. Products were analyzed by LC-MS and quantified using internal standard (8-iso-PGF_2α-d₄ or AA-d₈). Data represent the mean ± SD of triplicate reactions. Different lower case letters indicate statistical differences between groups (p<0.05, one-way ANOVA and Tukey test).

Figure 2

THP1 monocytes hydrolyze the lipid mediators 2AG, PGE₂-G, or PGF_2α-G. (A) Intact THP1 cells were incubated in serum-free medium in the absence (control) and presence of exogenous 2AG (10μM) for 1 h. LC-MS chromatograms are shown. Solid black line (AA, m/z 303); dashed grey line (AA-d₈, m/z 310). (B) Inhibition of lipid glyceryl ester metabolism in THP1 monocyte lysates by S-3030. Control THP1 lysate hydrolysis activities for 2AG, PGF_2α-G, and PGE₂-G represent 2.5, 0.6, and 0.8 nmol product/min/mg protein, respectively, when using 25 μM substrate. (C) Serine hydrolase activity profile of THP1 monocyte lysate determined with FP-biotin (2μM, 1h, room temp). heat indicates lysates were boiled prior to addition of FP-biotin, native indicates lysates were not boiled. An endogenous biotin-containing protein is detected ~75kDa in both heat and native samples (band not shown) and is not dependent on FP-biotin treatment. (D) Activity profile of CES1 and 31-32kDa enzyme following incubation of THP1 lysate with JZL184 or 100μM CPO (positive control) for 30 min (37°C), followed by FP-biotin (2μM, 1h, room temp). (E) Inhibition of lipid glyceryl ester metabolism in THP1 monocyte lysates by JZL184. Bar graphs represent the mean ± SD of triplicate reactions. * p<0.05 for inhibitor-treated lysate versus vehicle-treated lysate, one-way ANOVA and Dunnett’s test.

Figure 3

Expression of 2AG hydrolytic enzymes in THP1 cells and sensitivity of CES1 and MAGL toward oxons. (A) THP1 macrophages express CES1 and MAGL, but do not express FAAH. PVDF membranes were probed using rabbit anti-CES1, rabbit anti-MAGL, or rabbit anti-FAAH antibodies. Antigen-antibody complexes were detected using goat anti-rabbit secondary antibody conjugated to HRP. Rabbit anti-βactin was used to verify equal protein loading (25μg per lane). +, rat liver microsomal protein used as a positive control for FAAH antibody. Normalized band intensities are shown below western blot. (B,C) Inhibition curves for CES1- and MAGL-mediated hydrolysis of 2AG by PO (open symbol) and CPO (closed symbol). Data represent the mean ± SD of triplicate reactions.

Figure 4

Treatment of intact THP1 macrophages with paraoxon inhibits the hydrolytic metabolism of 2AG. THP1 macrophages were pretreated with increasing amounts of PO (0.1-10 μM, 30 min), followed by addition of 2AG (10 μM, 60 min). Cells and culture medium were com-bined, spiked with internal standard (AA-d₈), and extracted with ethyl acetate (0.1% acetic acid) for LC-MS assay. Data represent the mean ± SD of triplicate plates. * p<0.05 for inhibitor-treated cells versus vehicle-treated cells, one-way ANOVA and Dunnett’s test. 2AG exists in aqueous solution as a mixture of two isomers, 2AG and 1(3)-AG (50).

Figure 5

Treatment of intact THP1 macrophages with paraoxon inhibits the hydrolytic metabolism of PGE₂-G and PGF_2α-G. THP1 macrophages were pretreated with increasing amounts of PO (0.1-10 μM, 30 min), followed by addition of PGE₂-G (10 μM, 60 min) (A) or PGF_2α-G (10 μM, 60 min) (B). Cells and culture medium were combined, spiked with internal standard (8-iso-PGF_2α-d₄), and extracted with ethyl acetate (0.1% acetic acid) for LC-MS assay. Data represent the mean ± SD of triplicate plates. * p<0.05 for inhibitor-treated cells versus vehicle-treated cells, one-way ANOVA and Dunnett’s test.

Figure 6

Immunoprecipitation of CES1 from THP1 monocyte lysates significantly reduces the hydrolysis of lipid glyceryl esters. CES1 was immunoprecipitated from cell lysates using rabbit anti-CES1 IgG antibodies. As a negative control, lysates were incubated with preimmune IgG antibodies. (A) Following centrifugation of antibody-lysate incubates, aliquots of the supernatants were run on SDS-PAGE to detect CES1 and β-actin by western blot (lanes 1 and 2) or MAGL (lanes 3 and 4). β-Actin was probed to verify equal gel loading of lysate proteins. (B) Supernatants were also treated with FP-biotin to examine the serine hydrolase activity profile. THP1 indicates lysates prepared in 50 mM Tris-HCl (pH 7.4) buffer, THP1* indicates lysates were solubilized in Tris-HCl (pH 7.4) buffer containing 0.5% Brij. (C,D) Supernatants were incubated with fixed amounts of PG-G (25 μM) for 30 min and ethyl acetate extracts analyzed by LC-MS to determine the amounts of hydrolysis product formed (PGE₂ and PGF_2α). (E) Alternatively, supernatants were incubated with 2AG (10 μM) for 30 min. Data are from at least two independent IP experiments, each reaction with substrate was performed in duplicate or triplicate. * p<0.05 for CES1-depleted lysate versus control lysate, Student’s t-test.

Figure 7

Murine and human macrophage cell lines produce PG-Gs. (A,B) Murine J774 macrophages were primed with LPS (1 μg/ml) for 5 h, followed by addition of 2AG (10 μM) (A) or ionomycin (5 μM) (B). After 30 min, the culture medium was removed and extracted for LC-MS/MS analysis (chromatograms for PGF_2α-G are shown). (C) Human THP1 macrophages were primed with LPS (1 μg/ml) for 5 h, followed by addition of 2AG. No PG-Gs were detected. (D) If macrophages were pretreated with CPO (1 μM) prior to adding 2AG, then PG-Gs were detected. (E) If the non-specific COX inhibitor indomethacin (3 μM) was added along with CPO prior to 2AG addition, then no PG-Gs were produced. (F) A chromatogram of the authentic standard of PGF_2α-G is shown. Chromatograms shown are representative of three independent experiments. As seen with 2AG, PG-Gs exist in aqueous solution as a mixture of two isomers, 2-PG-G and 1(3)-PG-G (see Figure 10).

Figure 8

PO significantly elevates the amounts of PG-Gs formed in situ by THP1 macrophages subsequently exposed to 2AG. THP1 macrophages were treated with lipopolysaccharide (LPS, 1 μg/ml) for 5 h. Culture medium was removed and cells pre-treated with paraoxon (PO, 1 μM) or vehicle (ethanol) in serum-free medium for 30 min. After 30 min, 2AG (10 μM) was directly added to culture medium and cells incubated for an additional 30 min. Culture medium was re-moved and extracted for LC-MS/MS analysis of PGF_2α-G (A,C) and PGE₂-G (B,D). Data represent the mean ± SD of triplicate plates in a single experiment, and are representative of three independent experiments. * p<0.05 for PO-treated cells versus vehicle-treated cells, Student’s t-test.

Figure 9

CPO-pretreated THP1 macrophages have elevated ionomycin-stimulated PG-G levels. (A) LPS priming (1 μg/ml, 5 h) of THP1 macrophages induced COX-2, as determined by western blot. (B) Prereatment of macrophages with 1 μM CPO followed by ionomycin stimulation caused PG-Gs and 2AG levels to be elevated. (C) Prereatment of macrophages with 1 μM PO followed by ionomycin stimulation did not elevate PG-G and 2AG levels. Data represent the mean ± SD of triplicate plates in a single experiment, and are representative of three independent experiments. * p<0.05 for CPO-treated cells versus vehicle-treated cells, Student’s t-test.

Figure 10

Cross talk between endocannabinoids and PG-Gs in THP1 macrophages. Abbreviations: cPLA₂, cytosolic phospholipase A₂; PLC, phospholipase C; PLD, phospholipase D; DAGL, diacylglycerol lipase; COX, cyclooxygenase; PGES, prostaglandin E₂ synthase.