Blood Acylpeptide Hydrolase Activity Is a Sensitive Marker for Exposure to Some Organophosphate Toxicants
http://www.100md.com
《毒物学科学杂志》
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720–3112
ABSTRACT
Acylpeptide hydrolase (APH) unblocks N-acetyl peptides. It is a major serine hydrolase in rat blood, brain, and liver detected by derivatization with 3H-diisopropyl fluorophosphate (DFP) or a biotinylated fluorophosphonate. Although APH does not appear to be a primary target of acute poisoning by organophosphorus (OP) compounds, the inhibitor specificity of this secondary target is largely unknown. This study fills the gap and emphasizes blood APH as a potential marker of OP exposure. The most potent in vitro inhibitors for human erythrocyte and mouse brain APH are DFP (IC50 11–17 nM), chlorpyrifos oxon (IC50 21–71 nM), dichlorvos (IC50 230–560 nM), naled (IC50 370–870 nM), and their analogs with modified alkyl substituents. 3H-diisopropyl fluorophosphate is a potent inhibitor of mouse blood and brain APH in vivo (ED50 0.09–0.2 mg/kg and 0.02–0.03 mg/l for ip and vapor exposure, respectively). Mouse blood and brain APH and blood butyrylcholinesterase (BChE) are of similar sensitivity to DFP in vitro and in vivo (ip and vapor exposure), but APH inhibition is much more persistent in vivo (still >80% inhibition after 4 days). The inhibitory potency of OP pesticides in vivo in mice varies from APH selective (dichlorvos, naled, and trichlorfon), to APH and BChE selective (profenofos and tribufos), to ChE selective or nonselective (many commercial insecticides). Sarin administered ip at a lethal dose to guinea pigs inhibits blood acetylcholinesterase and BChE completely but erythrocyte APH only partially. Blood APH activity is therefore a sensitive marker for exposure to some but not all OP pesticides and chemical warfare agents.
Key Words: acylpeptide hydrolase; chlorpyrifos; dichlorvos; diisopropyl fluorophosphate; naled; trichlorfon (metrifonate).
Introduction
Acylpeptide hydrolase (APH, E.C. 3.4.19.1 [EC] ) unblocks N-acetyl peptides and conceivably acts on nascent forms during biosynthesis, as well as on bioactive peptides (Jones et al., 1994). This enzyme occurs in many tissues, including blood, brain, and liver. Human erythrocyte APH cleaves oxidized proteins (Fujino et al., 2000). Acylpeptide hydrolase and the proteasome act in coordination to clear cytotoxic denatured proteins from cells (Shimizu et al., 2004). The APHs isolated from various tissues and mammalian species are quite similar. Human erythrocyte and liver APH are highly homologous (96% identity for sequenced peptide fragments) (Fujino et al., 2000). Human liver APH is 92% identical to the corresponding proteins from pig and rat (Mitta et al., 1996). Nevertheless, although APHs from different sources are very similar, minor structural differences may result in varied sensitivity to inhibitors.
Acylpeptide hydrolase is a sensitive target for organophosphorus (OP) compounds. It is the major serine hydrolase in rat brain detected with 3H-diisopropyl fluorophosphate (DFP) (Richards et al., 1999, 2000). Erythrocyte APH is inhibited by DFP (Fujino et al., 2000), but its sensitivity to other OPs is unreported. Profiling of serine hydrolase activities in complex proteomes of numerous tissues and cell lines reveals prominent derivatization of APH by a biotinylated fluorophosphonate (Jessani et al., 2002; Kidd et al., 2001). Although it does not appear to be a target for OP acute poisoning (Duysen et al., 2001), the continued use of OP pesticides and concerns for chemical terrorism make understanding secondary targets such as APH critical.
This investigation considers APH as a sensitive enzyme and marker in blood for potential exposure to OP pesticides and chemical warfare agents. It also considers two other peptide hydrolases (dipeptidyl peptidase IV [DPP IV] and tissue plasminogen activator [t-PA]) known to be inhibited by DFP (Chmielewska et al., 1988; Kenny et al., 1976) but of unknown sensitivity to other OP toxicants.
Materials And Methods
Chemicals.
Caution: Some of the test compounds have high acute toxicity and others are delayed neurotoxicants in mice (Casida and Quistad, 2004; Wu and Casida, 1996). They were used under careful containment conditions. Mouse plasma, DFP, 5,5'-dithiobis(2-nitrobenzoic acid), CH3SO2-D-HHT-Gly-Arg-pNA·AcOH, Gly-Pro-p-nitroanilide, acetylthiocholine, and butyrylthiocholine were from Sigma (St. Louis, MO). N-Acetyl-L-alanyl-p-nitroanilide was from Bachem California (Torrance, CA). Chlorpyrifos oxon (CPO) was from Dow AgroSciences (Indianapolis, IN). Many of the pesticides, including dichlorvos and naled, were from Chem Service (West Chester, PA), and other pesticides and candidate inhibitors were from our previous investigations (Quistad et al., 2005). Compounds 13, 14, 16, 25, and 32 were synthesized by Karl J. Fisher of this laboratory. All candidate inhibitors were >95% purity.
Mouse and human samples.
Male Swiss-Webster mice (27–30 g) from Harlan Laboratories (Indianapolis, IN) were maintained under standard conditions with access to food and water ad libitum. The studies were carried out in accordance with the Guiding Principles in the Use of Animals in Toxicology as adopted by the Society of Toxicology in 1989. Some mice were treated ip with test compound in dimethyl sulfoxide (DMSO) (30 μl) or carrier solvent alone as a control and typically maintained for 4 h. Initial ip doses were chosen based on the highest tolerated level from previous investigations in this laboratory. Subsequent doses were reduced in a 100, 30, 10, 3, etc. mg/kg series for comparison of specific compounds at the same dose. Mice in studies of enzyme activity recovery were kept longer (4 h and 8 h, and 1, 2, 3, and 4 days for DFP and 4 days for naled, profenofos, and tribufos). Other mice were exposed for 10 min to DFP vapor by placing an individual in a liter jar with a loose lid (allowing air entry) and an inner strip of filter paper treated with DFP (0.03–1 mg) in acetone (25 μl). After the animal was sacrificed by cervical dislocation, mouse erythrocytes and serum were prepared from blood recovered by cardiac puncture and the brain was removed. Human blood with EDTA as the anticoagulant was used directly or after fractionation, i.e. erythrocytes, plasma and Ficoll-paque lymphocytes.
Guinea pig samples.
Male Hartley guinea pigs were treated with sarin subcutaneously at 70 μg/kg and sacrificed at 30 min (Hulet et al., 2002). Erythrocytes and plasma were frozen with dry ice and provided by John H. McDonough and Tsung-Ming Shih (US Army Medical Research Institute, Aberdeen Proving Grounds, MD) to the Berkeley laboratory for analysis.
Sample preparations.
Blood was centrifuged to separate serum and erythrocytes. Erythrocytes and whole blood were diluted with an equal volume of 100 mM Tris buffer (pH 7.4, 25°C) and frozen on dry ice to lyse cells, which were homogenized and diluted 1/20 for assay. Mouse brain was homogenized (20% w/v) in 50 mM Tris buffer (pH 8, 5°C) containing 0.2 mM EDTA. Homogenates were centrifuged at 700 x g for 10 min (pellet discarded) and the supernatant was assayed directly (APH or DPP IV) or after 1/20 dilution (acetylcholinesterase; AChE).
Enzyme assays.
Enzyme activity was determined colorimetrically with a microplate reader with 96-well plates (Versamax, Molecular Devices, Sunnyvale, CA). Samples were analyzed directly or after in vitro exposure to candidate inhibitors. Protein was determined by the Bradford (1976) method.
Analysis of data.
Results are reported as percent of control or as the concentration of compound inhibiting 50% of enzyme activity (IC50) as derived from two to three concentrations (above and below the IC50, each in triplicate) in the range of 15–85% enzyme inhibition. Results are reported as the mean ± SD. Bimolecular rate constants were calculated from plots of log % activity versus time and [inhibitor]/k versus [inhibitor] (Aldridge and Reiner, 1972).
APH assay.
A colorimetric procedure was used for APH assay (Jones et al., 1994) (Fig. 1). Homogenate (20 μl) was added to individual wells containing 100 mM Tris buffer (175 μl, pH 7.4, 25°C). Candidate inhibitors were added in DMSO (5 μl), and the mixture was incubated at 25°C for 15 min. N-Acetyl-L-alanyl-p-nitroanilide (1.5 mg/ml, 100 μl) was introduced, and absorbance from liberated p-nitroaniline was monitored (405 nm) at 37°C for 10 min. Activity was measured using 5 μl of serum/plasma or 360, 35, and 74–98 μg protein for brain, lymphocytes, and whole blood/erythrocytes, respectively. The vast majority of blood APH is in erythrocytes (>98 and >87% for human and mouse, respectively), and whole blood is suitable for assay because plasma lacks APH.
AChE assay.
The general method of Ellman et al. (1961) was used with acetylthiocholine as the substrate to measure the inhibition of AChE activity. Homogenate (20 μl) was added to individual wells containing 100 mM phosphate buffer (175 μl, pH 7.4, 25°C). For whole blood only, quinidine (20 μM final concentration) was preincubated at 25°C for 15 min to inhibit BChE (Wilson et al., 2002). Standard methods for monitoring human OP exposure involve assay of AChE in whole blood and BChE in plasma (Wilson et al., 2002). Because whole blood contains both AChE and BChE, the BChE component was minimized with a specific inhibitor (i.e., 20 μM quinidine). Candidate OP inhibitors were added in DMSO (5 μl), and the mixture was incubated at 25°C for 15 min. 5,5'-Dithiobis(2-nitrobenzoic acid) (1.76 mg/ml, 90 μl) and acetylthiocholine (2.6 mg/ml, 30 μl) were added with monitoring at 412 nm and 37°C for 10 min. Activity was measured using 18, 14, and 74–98 μg protein for brain, serum/plasma, and whole blood/erythrocytes, respectively.
BChE assay.
The conditions for AChE were used, except with butyrylthiocholine as the substrate (1.7 mg/ml, 30 μl). Activity was measured with 14 and 20 μg protein for human and mouse plasma/serum, respectively.
DPP IV assay.
The method monitors release of p-nitroaniline from a peptide substrate (Richards et al., 2000). Brain homogenate (20 μl) was added to individual wells containing 50 mM Tris buffer (pH 7.4, 25°C) with 1 mM dithiothreitol (185 μl). Candidate inhibitors were added in DMSO (5 μl), and the mixture was incubated at 25°C for 15 min. Gly-Pro-p-nitroanilide (0.82 mg/ml, 90 μl) was introduced, and absorbance was monitored at 405 nm (37°C, 10 min).
t-PA assay.
The procedure was from a product information bulletin of Sigma using t-PA chromogenic substrate (CH3SO2-D-HHT-Gly-Arg-pNA·AcOH). Human plasma (30 μl) was added to individual wells containing 50 mM Tris buffer (pH 8.4, 25°C), 30 mM imidazole, and 130 mM NaCl (215 μl). Candidate inhibitors were added in DMSO (5 μl), and the mixture was incubated at 25°C for 15 min. The chromogenic substrate (1.32 mg/ml, 60 μl) was added, and absorbance was monitored at 405 nm (25°C, 10 min).
Results
Similar In Vitro OP Inhibitor Specificity Profiles for APH of Human Erythrocytes and Mouse Brain
Five sets of compounds were examined with human erythrocyte and mouse brain APH to define the structure–activity relationships and optimize potency (Table 1). The two enzyme sources are generally of similar sensitivity (IC50 within 4-fold), except for two ethylphosphonates (3 and 5) and phosphonates 8 and 9 with long alkyl chains (C12 and C13), which are more potent on the erythrocyte enzyme. The degree of similarity for the erythrocyte and brain enzymes is evident from the IC50 correlation coefficient of 0.99 for the 25 compounds with values for both APH preparations.
The fluorophosphates and fluorophosphonates (1–10) are generally of very high potency with DFP (1) and dipentyl-DFP (2) the most active (IC50 0.009–0.017 μM). Lower potency is observed for O-alkyl alkylfluorophosphonates (3–9) and diphenyl phosphinofluoridate (10). The CPO homologs (11–16) are optimal at ethyl (12), but they maintain high potency from methyl through n-pentyl (IC50 0.02–0.86 μM), with isopropyl being less active (IC50 3.3 μM). The assays also included 18 other pesticides, oxons, and analogs (17–34). Except for dichlorvos-pentyl (17) (IC50 0.10–0.25 μM), they are generally less potent than DFP, CPO, and many analogs in the first two sets, yet IC50 values of 0.23–12 μM are evident with dichlorvos (18), naled (19), diazoxon (20), profenofos (21), 3-methylparaoxon (22), paraoxon (23), and acephate (24). Replacement of fluorine with 4-nitrophenoxy (25) as the leaving group in DFP reduces potency 900-fold. Similarly, replacement of fluorine with dichlorovinyloxy in the potent dipentyl analog of DFP (2) reduces potency by 9-fold. Trichlorfon (26) is less active (IC50 18 μM), and tribufos (27) and other pesticides/analogs (28–34) are considerably less active. The fourth set of compounds is the benzodioxaphosphorin oxides (35–39), all with a small range of IC50 values (0.18–1.6 μM). Finally, the sulfonyl fluorides (40, 41) have little or no activity.
Comparative Sensitivities In Vitro of APH, AChE, and BChE of Human and Mouse Blood to DFP and CPO
The inhibition of APH activity by DFP (3.3, 10, and 33 nM) is rapid. The bimolecular rate constants for human erythrocyte APH, plasma BChE, and erythrocyte AChE are 5 x 106, 4 x 106, and 9 x 103 (M–1min–1), respectively. The value for BChE agrees with previous literature (Main, 1964). Thus, DFP reacts at a similar rate with APH and BChE but >400-fold slower with AChE.
The in vitro structure–activity studies above with human erythrocytes indicate that APH is a potential marker for exposure to some OP pesticides, and the findings demonstrate the need for comparative sensitivity data for human and mouse blood in evaluating this model. Two of the most potent inhibitors of human erythrocyte APH (CPO and DFP) were therefore compared for inhibition in vitro of cholinesterases (ChEs) (Table 2). Blood from both humans and mice shows similar sensitivity for APH and AChE inhibition by CPO (IC50 32–80 nM), but APH is much more sensitive to DFP (IC50 9–11 nM), >500-fold greater compared to AChE. For inhibition of AChE by both CPO and DFP, the IC50 values differ by only 0.5–4.6-fold for erythrocytes and whole blood. With these overall similarities, whole blood is used for inhibition assay of both APH and AChE. Human and mouse plasma BChE differ little in sensitivity to DFP (IC50 11–26 nM), although human BChE is 70-fold more sensitive to CPO. Lymphocyte APH is a possible alternate preparation with which to monitor CPO inhibition (IC50 35 nM). These findings establish that APH in whole blood of mice is a convenient model to assess in vivo effects of DFP and some OP pesticides.
Comparative Sensitivities In Vivo of APH, AChE and BChE in Mouse Blood and Brain DFP Administered ip and in the Vapor Phase
The potency of DFP was determined as an inhibitor of blood and brain APH and AChE and blood BChE in mice in vivo (Table 3). Blood and brain APH have similar sensitivity for vapor exposure, but brain APH is less inhibited after ip treatment at the lowest dose (0.1 mg/kg). Acylpeptide hydrolase and BChE are equally sensitive to DFP, both on ip treatment and after vapor exposure, i.e., 51–63% inhibition at 0.1 mg/kg ip and 0.03 mg/l; with these doses, erythrocyte AChE is 4- to 6-fold less inhibited and brain AChE is not affected. The effective dose for 50% inhibition (ED50) is 7–11-fold lower for blood APH and BChE than for AChE. Cholinergic symptoms occur with 62% inhibition of AChE in blood and brain (Table 3).
A separate study compared the enzymes relative to the persistence of DFP-induced inhibition. Treatment of mice ip at 0.3 mg/kg after 4 h inhibits 98% and 77% of the APH activity in blood and brain, respectively, whereas 29% and 82% of the AChE and BChE in blood are inhibited, respectively, and AChE in brain is unaffected (Table 3). Inhibited APH in blood recovers slowly (still >80% inhibited after 4 days; Fig. 2), whereas brain APH recovers about half its activity (t1/2) after 4 days (77 ± 14% and 34 ± 15% inhibition at 4 h and 4 days, respectively). Butyrylcholinesterase recovers much faster (t1/2 1–2 days). These mice with DFP at 0.3 mg/kg showed no overt toxic signs during the 4 days after treatment. Toxicity was also not observed at 1 mg/kg, a dose of DFP that caused 96% inhibition of APH in brain and blood but only 31% inhibition of AChE in brain.
The high sensitivity of blood APH relative to AChE and BChE observed with DFP in mice prompted a related study with sarin. Guinea pigs were chosen (instead of mice) because they are the most commonly used experimental models with sarin (Hulet et al., 2002) and a lethal dose would give the greatest opportunity for APH inhibition. In contrast to DFP in mice as noted above, administration of a lethal dose of sarin (70 μg/kg) to guinea pigs inhibits erythrocyte APH (41 ± 9%, n = 4) much less effectively than erythrocyte AChE and plasma BChE (97–98%).
OP Pesticides Administered ip
Seventeen OP pesticides were administered ip to mice to study the inhibition of APH, AChE, and BChE of blood and brain 4 h after treatment (Table 4). Blood APH is more sensitive than the ChEs for dichlorvos, naled, and trichlorfon. APH is particularly sensitive to dichlorvos and naled (ED50 3 mg/kg). Acylpeptide hydrolase and BChE are almost equally sensitive to profenofos and tribufos, whereas AChE is less inhibited. For 10 other OP insecticides, the ChEs are more sensitive than APH, but 40–53% inhibition of blood APH occurs for chlorpyrifos and diazinon at a toxic dose (30 mg/kg). Dimethoate and acephate inhibit APH, AChE, and BChE in blood and brain to similar degrees. In general, APH inhibition is more persistent than that of AChE or BChE, as evident from a comparison of blood and brain with naled, profenofos, and tribufos at 4 h and 4 days after ip treatment at 30 mg/kg (still 81% inhibition of blood APH after 4 days) (Table 4).
Comparative OP Sensitivities In Vitro of Mouse Brain DPP-IV and Human Plasma t-PA
Dipeptidyl peptidase IV and t-PA are serine hydrolases inhibited by DFP, as noted earlier and confirmed here, but of unknown sensitivity to other OP toxicants. Mouse brain DPP-IV and human plasma t-PA are considerably less OP sensitive (Table 5) than blood or brain APH (Table 1). With DPP-IV there are three O-alkyl alkylfluorophosphonates (3–5) with an IC50 of 0.9–1.9 μM and four others (2, 7, 9, and 10) in the range of 6.4–12 μM. The benzodioxaphosphorin oxides include two moderately potent inhibitors (IC50 28–73 μM), i.e., the ethylphosphonate (36) and S-pentyl phosphorothiolate (39). Tissue plasminogen activator from human plasma is of very low sensitivity, with only diphenyl phosphinofluoridate (10) giving an IC50 of <100 μM.
Discussion
Blood APH Activity as a Potential Marker for OP Exposure
Organophosphate pesticide exposure is usually measured by analysis of metabolites in urine or activity levels of BChE in plasma and AChE in blood (Eskenazi et al., 2004; Wilson, 2001). Urinary metabolites are diagnostic relative to exposure compound or class (e.g., dimethylphosphate). Inhibition of ChEs is more general and depends on individual OP potency, with BChE typically more sensitive as a marker but AChE more relevant to toxicity and often differing in the structure–activity relationships (Wilson, 2001). The present study with mice and DFP establishes that APH activity in blood is equally sensitive to plasma BChE, and that APH has the advantage of slower recovery, allowing detection of inhibition after longer times.
Seventeen OP pesticides were tested in mice (Table 4), including the top 10 by total pounds applied in California (California Environmental Protection Agency, 2005), using APH, AChE, and BChE as markers. With three insecticides (dichlorvos, naled, and trichlorfon) APH was the more sensitive marker in vivo in mice, and two others (profenofos and tribufos) APH and BChE had similar sensitivity. Ten OP pesticides were AChE and BChE selective. Dimethoate and acephate had similar potency on APH, AChE, and BChE. Thus, based on these studies with mice, APH may be a suitable marker for 4 of the 10 top insecticides used in California (dimethoate, acephate, naled, and tribufos), collectively applied at almost 1 million pounds annually. Current studies with guinea pigs indicate that APH is a less sensitive marker for sarin exposure than either AChE or BChE, but possible species differences in APH sensitivity are unknown.
Pharmacological Implications of APH Inhibition
Acylpeptide hydrolase plays a vital role in normal metabolism, evidenced by apoptosis occurring in human monoblastic U937 cells after APH inhibition by acetylleucine chloromethyl ketone (Yamaguchi et al., 1999). Reduced APH levels may be associated with cancer because, although normal cultured lung cells have APH, it is practically absent in small-cell lung carcinoma cell lines (Scaloni et al., 1992). No adverse toxicology was observed in the present study in mice 0–4 days after near chemical knockout of APH with DFP. The chemical warfare agent VX is toxic to mice lacking AChE, but APH is not the target (Duysen et al., 2001).
Acylpeptide hydrolase is a proposed target for cognitive-enhancing drugs such as metrifonate (Richards et al., 2000). Metrifonate-treated patients showed significant improvement, but it was withdrawn from development as an Alzheimer's disease drug because a reversible but clinically significant proximal weakness of limbs occurred in some individuals at high doses (Gauthier, 2001; Wynn and Cummings, 2004). Metrifonate is converted in vivo to dichlorvos as the active form (Fig. 3). Dichlorvos and naled (its dibrominated analog) are more potent than metrifonate as in vitro APH inhibitors, and they are also more effective in vivo in mice. Naled is proposed to undergo thiol-catalyzed conversion to dichlorvos as an activation mechanism for AChE inhibition (Eto, 1974) (Fig. 3). Glutathione (10 μM) enhances the potency of APH inhibition by naled, a finding consistent with the proposed formation of dichlorvos as the active product (data not shown).
Dipeptidyl peptidase IV and t-PA are candidate peptide hydrolase targets for OP inhibitors. Dipeptidyl peptidase IV belongs to a serine protease subfamily related to APH, and inhibitors of DPP IV are in clinical trials for treatment of type 2 diabetes (Rosenblum and Kozarich, 2003). Tissue plasminogen activator–based recombinant thrombolytic proteins represent a large market (>$100 million/year) (Roman, 2001), and OP inhibitors are therefore of interest and possible concern. However, although both DPP IV and t-PA react with certain OPs, no potent inhibitors were found in this investigation.
ACKNOWLEDGMENTS
This work was supported by grant R01 ES08762 from the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. R.K. was supported by a postdoctoral fellowship from the Swedish Research Council. We thank our Berkeley colleagues Nina T. Holland and Sarah C. Owen for providing human blood and lymphocytes and Daryl Wong for assistance with enzyme assays. Conflict of interest: none declared.
REFERENCES
Aldridge, W. N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates: Interactions of Esterases with Esters of Organophosphorus and Carbamic Acids. North-Holland Publishing, Amsterdam.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
California Environmental Protection Agency (2005). Department of Pesticide Regulation, 2003 Pesticide Use Data, Sacramento, CA.
Casida, J. E., and Quistad, G. B. (2004). Organophosphate toxicology: Safety aspects of nonacetylcholinesterase secondary targets. Chem. Res. Toxicol. 17, 1–16.
Chmielewska, J., Rnby, M., and Wiman, B. (1988). Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Biochem. J. 251, 327–332.
Duysen, E. G., Li, B., Xie, W., Schopfer, L. M., Anderson, R. S., Broomfield, C. A., and Lockridge, O. (2001). Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: Supersensitivity of acetylcholinesterase knockout mouse to VX lethality. J. Pharmacol. Exp. Ther. 299, 528–535.
Ellman, G. L., Courtney, K. D., Andres V., Jr., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95.
Eskenazi, B., Harley, K., Bradman, A., Weltzien, E., Jewell, N. P., Barr, D. B., Furlong, C. E., and Holland, N. T. (2004). Association of in utero organophosphate pesticide exposure and fetal growth and length of gestation in an agricultural population. Environ. Health Perspect. 112, 1116–1124.
Eto, M. (1974). Organophosphorus Pesticides: Organic and Biological Chemistry. CRC Press, Cleveland, OH.
Fujino, T., Watanabe, K., Beppu, M., Kikugawa, K., and Yasuda, H. (2000). Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase. Biochim. Biophys. Acta. 1478, 102–112.
Gauthier, S. (2001). Alzheimer's disease: Current and future therapeutic perspectives. Prog. Neuro-Psychopharmacol. Biol. Psychiat. 25, 73–89.
Hulet, S. W., McDonough, J. H., and Shih, T.-M. (2002). The dose-response effects of repeated subacute sarin exposure on guinea pigs. Pharmacol. Biochem. Behav. 72, 835–845.
Jessani, N., Liu, Y., Humphrey, M., and Cravatt, B. F. (2002). Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl. Acad. Sci. USA 99, 10335–10340.
Jones, W. M., Scaloni, A., and Manning, J. M., (1994). Acylaminoacyl-peptidase. Meth. Enzymol. 244, 227–231.
Kenny, A. J., Booth, A. G., George, S. G., Ingram, J., Kershaw, D., Wood, E. J., and Young, A. R. (1976). Dipeptidyl peptidase IV, a kidney brush-border serine peptidase. Biochem. J. 157, 169–182.
Kidd, D., Liu, Y., and Cravatt, B. F. (2001). Profiling serine hydrolase activities in complex proteomes. Biochemistry 40, 4005–4015.
Main, A. R. (1964). Affinity and phosphorylation constants for the inhibition of esterases by organophosphates. Science 144, 992–993.
Mitta, M., Ohnogi, H., Mizutani, S., Sakiyama, F., Kato, I., and Tsunasawa, S. (1996). The nucleotide sequence of human acylamino acid-releasing enzyme. DNA Res. 3, 31–35.
Quistad, G. B., Fisher, K. J., Owen, S. C., Klintenberg, R., and Casida, J. E. (2005). Platelet-activating factor acetylhydrolase: Selective inhibition by potent n-alkyl methylphosphonofluoridates. Toxicol. Appl. Pharmacol. 205, 149–156.
Richards, P. G., Johnson, M. K., and Ray, D. E. (2000). Identification of acylpeptide hydrolase as a sensitive site for reaction with organophosphorus compounds and a potential target for cognitive enhancing drugs. Mol. Pharmacol. 58, 577–583.
Richards, P., Johnson, M., Ray, D., and Walker, C. (1999). Novel protein targets for organophosphorus compounds. Chem.-Biol. Interact. 119–120, 503–511.
Roman, K. M. (2001). 2000 IMS health hospital pharmacy review: Leading dollar volume pharmaceuticals. Hospital Pharmacy 36, 708–716, 789.
Rosenblum, J. S., and Kozarich, J. W. (2003). Prolyl peptidases: A serine protease subfamily with high potential for drug discovery. Curr. Opin. Chem. Biol. 7, 496–504.
Scaloni, A., Jones, W., Pospischil, M., Sassa, S., Schneewind, O., Popowicz, A. M., Bossa, F., Graziano, S. L., and Manning J. M. (1992). Deficiency of acylpeptide hydrolase in small-cell lung carcinoma cell lines. J. Lab. Clin. Med. 120, 546–552.
Shimizu, K., Kiuchi, Y., Ando, K., Hayakawa, M., and Kikugawa, K. (2004). Coordination of oxidized protein hydrolase and the proteasome in clearance of cytotoxic denatured proteins. Biochem. Biophys. Res. Commun. 324, 140–146.
Wilson, B. W. (2001). Cholinesterases. In Handbook of Pesticide Toxicology (R. I. Krieger, Ed.), Vol. 2. Agents, pp. 967–985. Academic Press, San Diego.
Wilson, B. W., Henderson, J. D., Ramirez, A., and O'Malley, M. A. (2002). Standardization of clinical cholinesterase measurements. Intl. J. Toxicol. 21, 385–388.
Wu, S.-Y., and Casida, J. E. (1996). Subacute neurotoxicity induced in mice by potent organophosphorus neuropathy target esterase inhibitors. Toxicol. Appl. Pharmacol. 139, 195–202.
Wynn, Z. J., and Cummings, J. L. (2004). Cholinesterase inhibitor therapies and neuropsychiatric manifestations of Alzheimer's disease. Dement. Geriatr. Cogn. Disord. 17, 100–108.
Yamaguchi, M., Kambayashi, D., Toda, J., Sano, T., Toyoshima, S., and Hojo, H. (1999). Acetylleucine chloromethyl ketone, an inhibitor of acylpeptide hydrolase, induces apoptosis of U937 cells. Biochem. Biophys. Res. Commun. 263, 139–142.(Gary B. Quistad, Rebecka )
ABSTRACT
Acylpeptide hydrolase (APH) unblocks N-acetyl peptides. It is a major serine hydrolase in rat blood, brain, and liver detected by derivatization with 3H-diisopropyl fluorophosphate (DFP) or a biotinylated fluorophosphonate. Although APH does not appear to be a primary target of acute poisoning by organophosphorus (OP) compounds, the inhibitor specificity of this secondary target is largely unknown. This study fills the gap and emphasizes blood APH as a potential marker of OP exposure. The most potent in vitro inhibitors for human erythrocyte and mouse brain APH are DFP (IC50 11–17 nM), chlorpyrifos oxon (IC50 21–71 nM), dichlorvos (IC50 230–560 nM), naled (IC50 370–870 nM), and their analogs with modified alkyl substituents. 3H-diisopropyl fluorophosphate is a potent inhibitor of mouse blood and brain APH in vivo (ED50 0.09–0.2 mg/kg and 0.02–0.03 mg/l for ip and vapor exposure, respectively). Mouse blood and brain APH and blood butyrylcholinesterase (BChE) are of similar sensitivity to DFP in vitro and in vivo (ip and vapor exposure), but APH inhibition is much more persistent in vivo (still >80% inhibition after 4 days). The inhibitory potency of OP pesticides in vivo in mice varies from APH selective (dichlorvos, naled, and trichlorfon), to APH and BChE selective (profenofos and tribufos), to ChE selective or nonselective (many commercial insecticides). Sarin administered ip at a lethal dose to guinea pigs inhibits blood acetylcholinesterase and BChE completely but erythrocyte APH only partially. Blood APH activity is therefore a sensitive marker for exposure to some but not all OP pesticides and chemical warfare agents.
Key Words: acylpeptide hydrolase; chlorpyrifos; dichlorvos; diisopropyl fluorophosphate; naled; trichlorfon (metrifonate).
Introduction
Acylpeptide hydrolase (APH, E.C. 3.4.19.1 [EC] ) unblocks N-acetyl peptides and conceivably acts on nascent forms during biosynthesis, as well as on bioactive peptides (Jones et al., 1994). This enzyme occurs in many tissues, including blood, brain, and liver. Human erythrocyte APH cleaves oxidized proteins (Fujino et al., 2000). Acylpeptide hydrolase and the proteasome act in coordination to clear cytotoxic denatured proteins from cells (Shimizu et al., 2004). The APHs isolated from various tissues and mammalian species are quite similar. Human erythrocyte and liver APH are highly homologous (96% identity for sequenced peptide fragments) (Fujino et al., 2000). Human liver APH is 92% identical to the corresponding proteins from pig and rat (Mitta et al., 1996). Nevertheless, although APHs from different sources are very similar, minor structural differences may result in varied sensitivity to inhibitors.
Acylpeptide hydrolase is a sensitive target for organophosphorus (OP) compounds. It is the major serine hydrolase in rat brain detected with 3H-diisopropyl fluorophosphate (DFP) (Richards et al., 1999, 2000). Erythrocyte APH is inhibited by DFP (Fujino et al., 2000), but its sensitivity to other OPs is unreported. Profiling of serine hydrolase activities in complex proteomes of numerous tissues and cell lines reveals prominent derivatization of APH by a biotinylated fluorophosphonate (Jessani et al., 2002; Kidd et al., 2001). Although it does not appear to be a target for OP acute poisoning (Duysen et al., 2001), the continued use of OP pesticides and concerns for chemical terrorism make understanding secondary targets such as APH critical.
This investigation considers APH as a sensitive enzyme and marker in blood for potential exposure to OP pesticides and chemical warfare agents. It also considers two other peptide hydrolases (dipeptidyl peptidase IV [DPP IV] and tissue plasminogen activator [t-PA]) known to be inhibited by DFP (Chmielewska et al., 1988; Kenny et al., 1976) but of unknown sensitivity to other OP toxicants.
Materials And Methods
Chemicals.
Caution: Some of the test compounds have high acute toxicity and others are delayed neurotoxicants in mice (Casida and Quistad, 2004; Wu and Casida, 1996). They were used under careful containment conditions. Mouse plasma, DFP, 5,5'-dithiobis(2-nitrobenzoic acid), CH3SO2-D-HHT-Gly-Arg-pNA·AcOH, Gly-Pro-p-nitroanilide, acetylthiocholine, and butyrylthiocholine were from Sigma (St. Louis, MO). N-Acetyl-L-alanyl-p-nitroanilide was from Bachem California (Torrance, CA). Chlorpyrifos oxon (CPO) was from Dow AgroSciences (Indianapolis, IN). Many of the pesticides, including dichlorvos and naled, were from Chem Service (West Chester, PA), and other pesticides and candidate inhibitors were from our previous investigations (Quistad et al., 2005). Compounds 13, 14, 16, 25, and 32 were synthesized by Karl J. Fisher of this laboratory. All candidate inhibitors were >95% purity.
Mouse and human samples.
Male Swiss-Webster mice (27–30 g) from Harlan Laboratories (Indianapolis, IN) were maintained under standard conditions with access to food and water ad libitum. The studies were carried out in accordance with the Guiding Principles in the Use of Animals in Toxicology as adopted by the Society of Toxicology in 1989. Some mice were treated ip with test compound in dimethyl sulfoxide (DMSO) (30 μl) or carrier solvent alone as a control and typically maintained for 4 h. Initial ip doses were chosen based on the highest tolerated level from previous investigations in this laboratory. Subsequent doses were reduced in a 100, 30, 10, 3, etc. mg/kg series for comparison of specific compounds at the same dose. Mice in studies of enzyme activity recovery were kept longer (4 h and 8 h, and 1, 2, 3, and 4 days for DFP and 4 days for naled, profenofos, and tribufos). Other mice were exposed for 10 min to DFP vapor by placing an individual in a liter jar with a loose lid (allowing air entry) and an inner strip of filter paper treated with DFP (0.03–1 mg) in acetone (25 μl). After the animal was sacrificed by cervical dislocation, mouse erythrocytes and serum were prepared from blood recovered by cardiac puncture and the brain was removed. Human blood with EDTA as the anticoagulant was used directly or after fractionation, i.e. erythrocytes, plasma and Ficoll-paque lymphocytes.
Guinea pig samples.
Male Hartley guinea pigs were treated with sarin subcutaneously at 70 μg/kg and sacrificed at 30 min (Hulet et al., 2002). Erythrocytes and plasma were frozen with dry ice and provided by John H. McDonough and Tsung-Ming Shih (US Army Medical Research Institute, Aberdeen Proving Grounds, MD) to the Berkeley laboratory for analysis.
Sample preparations.
Blood was centrifuged to separate serum and erythrocytes. Erythrocytes and whole blood were diluted with an equal volume of 100 mM Tris buffer (pH 7.4, 25°C) and frozen on dry ice to lyse cells, which were homogenized and diluted 1/20 for assay. Mouse brain was homogenized (20% w/v) in 50 mM Tris buffer (pH 8, 5°C) containing 0.2 mM EDTA. Homogenates were centrifuged at 700 x g for 10 min (pellet discarded) and the supernatant was assayed directly (APH or DPP IV) or after 1/20 dilution (acetylcholinesterase; AChE).
Enzyme assays.
Enzyme activity was determined colorimetrically with a microplate reader with 96-well plates (Versamax, Molecular Devices, Sunnyvale, CA). Samples were analyzed directly or after in vitro exposure to candidate inhibitors. Protein was determined by the Bradford (1976) method.
Analysis of data.
Results are reported as percent of control or as the concentration of compound inhibiting 50% of enzyme activity (IC50) as derived from two to three concentrations (above and below the IC50, each in triplicate) in the range of 15–85% enzyme inhibition. Results are reported as the mean ± SD. Bimolecular rate constants were calculated from plots of log % activity versus time and [inhibitor]/k versus [inhibitor] (Aldridge and Reiner, 1972).
APH assay.
A colorimetric procedure was used for APH assay (Jones et al., 1994) (Fig. 1). Homogenate (20 μl) was added to individual wells containing 100 mM Tris buffer (175 μl, pH 7.4, 25°C). Candidate inhibitors were added in DMSO (5 μl), and the mixture was incubated at 25°C for 15 min. N-Acetyl-L-alanyl-p-nitroanilide (1.5 mg/ml, 100 μl) was introduced, and absorbance from liberated p-nitroaniline was monitored (405 nm) at 37°C for 10 min. Activity was measured using 5 μl of serum/plasma or 360, 35, and 74–98 μg protein for brain, lymphocytes, and whole blood/erythrocytes, respectively. The vast majority of blood APH is in erythrocytes (>98 and >87% for human and mouse, respectively), and whole blood is suitable for assay because plasma lacks APH.
AChE assay.
The general method of Ellman et al. (1961) was used with acetylthiocholine as the substrate to measure the inhibition of AChE activity. Homogenate (20 μl) was added to individual wells containing 100 mM phosphate buffer (175 μl, pH 7.4, 25°C). For whole blood only, quinidine (20 μM final concentration) was preincubated at 25°C for 15 min to inhibit BChE (Wilson et al., 2002). Standard methods for monitoring human OP exposure involve assay of AChE in whole blood and BChE in plasma (Wilson et al., 2002). Because whole blood contains both AChE and BChE, the BChE component was minimized with a specific inhibitor (i.e., 20 μM quinidine). Candidate OP inhibitors were added in DMSO (5 μl), and the mixture was incubated at 25°C for 15 min. 5,5'-Dithiobis(2-nitrobenzoic acid) (1.76 mg/ml, 90 μl) and acetylthiocholine (2.6 mg/ml, 30 μl) were added with monitoring at 412 nm and 37°C for 10 min. Activity was measured using 18, 14, and 74–98 μg protein for brain, serum/plasma, and whole blood/erythrocytes, respectively.
BChE assay.
The conditions for AChE were used, except with butyrylthiocholine as the substrate (1.7 mg/ml, 30 μl). Activity was measured with 14 and 20 μg protein for human and mouse plasma/serum, respectively.
DPP IV assay.
The method monitors release of p-nitroaniline from a peptide substrate (Richards et al., 2000). Brain homogenate (20 μl) was added to individual wells containing 50 mM Tris buffer (pH 7.4, 25°C) with 1 mM dithiothreitol (185 μl). Candidate inhibitors were added in DMSO (5 μl), and the mixture was incubated at 25°C for 15 min. Gly-Pro-p-nitroanilide (0.82 mg/ml, 90 μl) was introduced, and absorbance was monitored at 405 nm (37°C, 10 min).
t-PA assay.
The procedure was from a product information bulletin of Sigma using t-PA chromogenic substrate (CH3SO2-D-HHT-Gly-Arg-pNA·AcOH). Human plasma (30 μl) was added to individual wells containing 50 mM Tris buffer (pH 8.4, 25°C), 30 mM imidazole, and 130 mM NaCl (215 μl). Candidate inhibitors were added in DMSO (5 μl), and the mixture was incubated at 25°C for 15 min. The chromogenic substrate (1.32 mg/ml, 60 μl) was added, and absorbance was monitored at 405 nm (25°C, 10 min).
Results
Similar In Vitro OP Inhibitor Specificity Profiles for APH of Human Erythrocytes and Mouse Brain
Five sets of compounds were examined with human erythrocyte and mouse brain APH to define the structure–activity relationships and optimize potency (Table 1). The two enzyme sources are generally of similar sensitivity (IC50 within 4-fold), except for two ethylphosphonates (3 and 5) and phosphonates 8 and 9 with long alkyl chains (C12 and C13), which are more potent on the erythrocyte enzyme. The degree of similarity for the erythrocyte and brain enzymes is evident from the IC50 correlation coefficient of 0.99 for the 25 compounds with values for both APH preparations.
The fluorophosphates and fluorophosphonates (1–10) are generally of very high potency with DFP (1) and dipentyl-DFP (2) the most active (IC50 0.009–0.017 μM). Lower potency is observed for O-alkyl alkylfluorophosphonates (3–9) and diphenyl phosphinofluoridate (10). The CPO homologs (11–16) are optimal at ethyl (12), but they maintain high potency from methyl through n-pentyl (IC50 0.02–0.86 μM), with isopropyl being less active (IC50 3.3 μM). The assays also included 18 other pesticides, oxons, and analogs (17–34). Except for dichlorvos-pentyl (17) (IC50 0.10–0.25 μM), they are generally less potent than DFP, CPO, and many analogs in the first two sets, yet IC50 values of 0.23–12 μM are evident with dichlorvos (18), naled (19), diazoxon (20), profenofos (21), 3-methylparaoxon (22), paraoxon (23), and acephate (24). Replacement of fluorine with 4-nitrophenoxy (25) as the leaving group in DFP reduces potency 900-fold. Similarly, replacement of fluorine with dichlorovinyloxy in the potent dipentyl analog of DFP (2) reduces potency by 9-fold. Trichlorfon (26) is less active (IC50 18 μM), and tribufos (27) and other pesticides/analogs (28–34) are considerably less active. The fourth set of compounds is the benzodioxaphosphorin oxides (35–39), all with a small range of IC50 values (0.18–1.6 μM). Finally, the sulfonyl fluorides (40, 41) have little or no activity.
Comparative Sensitivities In Vitro of APH, AChE, and BChE of Human and Mouse Blood to DFP and CPO
The inhibition of APH activity by DFP (3.3, 10, and 33 nM) is rapid. The bimolecular rate constants for human erythrocyte APH, plasma BChE, and erythrocyte AChE are 5 x 106, 4 x 106, and 9 x 103 (M–1min–1), respectively. The value for BChE agrees with previous literature (Main, 1964). Thus, DFP reacts at a similar rate with APH and BChE but >400-fold slower with AChE.
The in vitro structure–activity studies above with human erythrocytes indicate that APH is a potential marker for exposure to some OP pesticides, and the findings demonstrate the need for comparative sensitivity data for human and mouse blood in evaluating this model. Two of the most potent inhibitors of human erythrocyte APH (CPO and DFP) were therefore compared for inhibition in vitro of cholinesterases (ChEs) (Table 2). Blood from both humans and mice shows similar sensitivity for APH and AChE inhibition by CPO (IC50 32–80 nM), but APH is much more sensitive to DFP (IC50 9–11 nM), >500-fold greater compared to AChE. For inhibition of AChE by both CPO and DFP, the IC50 values differ by only 0.5–4.6-fold for erythrocytes and whole blood. With these overall similarities, whole blood is used for inhibition assay of both APH and AChE. Human and mouse plasma BChE differ little in sensitivity to DFP (IC50 11–26 nM), although human BChE is 70-fold more sensitive to CPO. Lymphocyte APH is a possible alternate preparation with which to monitor CPO inhibition (IC50 35 nM). These findings establish that APH in whole blood of mice is a convenient model to assess in vivo effects of DFP and some OP pesticides.
Comparative Sensitivities In Vivo of APH, AChE and BChE in Mouse Blood and Brain DFP Administered ip and in the Vapor Phase
The potency of DFP was determined as an inhibitor of blood and brain APH and AChE and blood BChE in mice in vivo (Table 3). Blood and brain APH have similar sensitivity for vapor exposure, but brain APH is less inhibited after ip treatment at the lowest dose (0.1 mg/kg). Acylpeptide hydrolase and BChE are equally sensitive to DFP, both on ip treatment and after vapor exposure, i.e., 51–63% inhibition at 0.1 mg/kg ip and 0.03 mg/l; with these doses, erythrocyte AChE is 4- to 6-fold less inhibited and brain AChE is not affected. The effective dose for 50% inhibition (ED50) is 7–11-fold lower for blood APH and BChE than for AChE. Cholinergic symptoms occur with 62% inhibition of AChE in blood and brain (Table 3).
A separate study compared the enzymes relative to the persistence of DFP-induced inhibition. Treatment of mice ip at 0.3 mg/kg after 4 h inhibits 98% and 77% of the APH activity in blood and brain, respectively, whereas 29% and 82% of the AChE and BChE in blood are inhibited, respectively, and AChE in brain is unaffected (Table 3). Inhibited APH in blood recovers slowly (still >80% inhibited after 4 days; Fig. 2), whereas brain APH recovers about half its activity (t1/2) after 4 days (77 ± 14% and 34 ± 15% inhibition at 4 h and 4 days, respectively). Butyrylcholinesterase recovers much faster (t1/2 1–2 days). These mice with DFP at 0.3 mg/kg showed no overt toxic signs during the 4 days after treatment. Toxicity was also not observed at 1 mg/kg, a dose of DFP that caused 96% inhibition of APH in brain and blood but only 31% inhibition of AChE in brain.
The high sensitivity of blood APH relative to AChE and BChE observed with DFP in mice prompted a related study with sarin. Guinea pigs were chosen (instead of mice) because they are the most commonly used experimental models with sarin (Hulet et al., 2002) and a lethal dose would give the greatest opportunity for APH inhibition. In contrast to DFP in mice as noted above, administration of a lethal dose of sarin (70 μg/kg) to guinea pigs inhibits erythrocyte APH (41 ± 9%, n = 4) much less effectively than erythrocyte AChE and plasma BChE (97–98%).
OP Pesticides Administered ip
Seventeen OP pesticides were administered ip to mice to study the inhibition of APH, AChE, and BChE of blood and brain 4 h after treatment (Table 4). Blood APH is more sensitive than the ChEs for dichlorvos, naled, and trichlorfon. APH is particularly sensitive to dichlorvos and naled (ED50 3 mg/kg). Acylpeptide hydrolase and BChE are almost equally sensitive to profenofos and tribufos, whereas AChE is less inhibited. For 10 other OP insecticides, the ChEs are more sensitive than APH, but 40–53% inhibition of blood APH occurs for chlorpyrifos and diazinon at a toxic dose (30 mg/kg). Dimethoate and acephate inhibit APH, AChE, and BChE in blood and brain to similar degrees. In general, APH inhibition is more persistent than that of AChE or BChE, as evident from a comparison of blood and brain with naled, profenofos, and tribufos at 4 h and 4 days after ip treatment at 30 mg/kg (still 81% inhibition of blood APH after 4 days) (Table 4).
Comparative OP Sensitivities In Vitro of Mouse Brain DPP-IV and Human Plasma t-PA
Dipeptidyl peptidase IV and t-PA are serine hydrolases inhibited by DFP, as noted earlier and confirmed here, but of unknown sensitivity to other OP toxicants. Mouse brain DPP-IV and human plasma t-PA are considerably less OP sensitive (Table 5) than blood or brain APH (Table 1). With DPP-IV there are three O-alkyl alkylfluorophosphonates (3–5) with an IC50 of 0.9–1.9 μM and four others (2, 7, 9, and 10) in the range of 6.4–12 μM. The benzodioxaphosphorin oxides include two moderately potent inhibitors (IC50 28–73 μM), i.e., the ethylphosphonate (36) and S-pentyl phosphorothiolate (39). Tissue plasminogen activator from human plasma is of very low sensitivity, with only diphenyl phosphinofluoridate (10) giving an IC50 of <100 μM.
Discussion
Blood APH Activity as a Potential Marker for OP Exposure
Organophosphate pesticide exposure is usually measured by analysis of metabolites in urine or activity levels of BChE in plasma and AChE in blood (Eskenazi et al., 2004; Wilson, 2001). Urinary metabolites are diagnostic relative to exposure compound or class (e.g., dimethylphosphate). Inhibition of ChEs is more general and depends on individual OP potency, with BChE typically more sensitive as a marker but AChE more relevant to toxicity and often differing in the structure–activity relationships (Wilson, 2001). The present study with mice and DFP establishes that APH activity in blood is equally sensitive to plasma BChE, and that APH has the advantage of slower recovery, allowing detection of inhibition after longer times.
Seventeen OP pesticides were tested in mice (Table 4), including the top 10 by total pounds applied in California (California Environmental Protection Agency, 2005), using APH, AChE, and BChE as markers. With three insecticides (dichlorvos, naled, and trichlorfon) APH was the more sensitive marker in vivo in mice, and two others (profenofos and tribufos) APH and BChE had similar sensitivity. Ten OP pesticides were AChE and BChE selective. Dimethoate and acephate had similar potency on APH, AChE, and BChE. Thus, based on these studies with mice, APH may be a suitable marker for 4 of the 10 top insecticides used in California (dimethoate, acephate, naled, and tribufos), collectively applied at almost 1 million pounds annually. Current studies with guinea pigs indicate that APH is a less sensitive marker for sarin exposure than either AChE or BChE, but possible species differences in APH sensitivity are unknown.
Pharmacological Implications of APH Inhibition
Acylpeptide hydrolase plays a vital role in normal metabolism, evidenced by apoptosis occurring in human monoblastic U937 cells after APH inhibition by acetylleucine chloromethyl ketone (Yamaguchi et al., 1999). Reduced APH levels may be associated with cancer because, although normal cultured lung cells have APH, it is practically absent in small-cell lung carcinoma cell lines (Scaloni et al., 1992). No adverse toxicology was observed in the present study in mice 0–4 days after near chemical knockout of APH with DFP. The chemical warfare agent VX is toxic to mice lacking AChE, but APH is not the target (Duysen et al., 2001).
Acylpeptide hydrolase is a proposed target for cognitive-enhancing drugs such as metrifonate (Richards et al., 2000). Metrifonate-treated patients showed significant improvement, but it was withdrawn from development as an Alzheimer's disease drug because a reversible but clinically significant proximal weakness of limbs occurred in some individuals at high doses (Gauthier, 2001; Wynn and Cummings, 2004). Metrifonate is converted in vivo to dichlorvos as the active form (Fig. 3). Dichlorvos and naled (its dibrominated analog) are more potent than metrifonate as in vitro APH inhibitors, and they are also more effective in vivo in mice. Naled is proposed to undergo thiol-catalyzed conversion to dichlorvos as an activation mechanism for AChE inhibition (Eto, 1974) (Fig. 3). Glutathione (10 μM) enhances the potency of APH inhibition by naled, a finding consistent with the proposed formation of dichlorvos as the active product (data not shown).
Dipeptidyl peptidase IV and t-PA are candidate peptide hydrolase targets for OP inhibitors. Dipeptidyl peptidase IV belongs to a serine protease subfamily related to APH, and inhibitors of DPP IV are in clinical trials for treatment of type 2 diabetes (Rosenblum and Kozarich, 2003). Tissue plasminogen activator–based recombinant thrombolytic proteins represent a large market (>$100 million/year) (Roman, 2001), and OP inhibitors are therefore of interest and possible concern. However, although both DPP IV and t-PA react with certain OPs, no potent inhibitors were found in this investigation.
ACKNOWLEDGMENTS
This work was supported by grant R01 ES08762 from the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. R.K. was supported by a postdoctoral fellowship from the Swedish Research Council. We thank our Berkeley colleagues Nina T. Holland and Sarah C. Owen for providing human blood and lymphocytes and Daryl Wong for assistance with enzyme assays. Conflict of interest: none declared.
REFERENCES
Aldridge, W. N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates: Interactions of Esterases with Esters of Organophosphorus and Carbamic Acids. North-Holland Publishing, Amsterdam.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
California Environmental Protection Agency (2005). Department of Pesticide Regulation, 2003 Pesticide Use Data, Sacramento, CA.
Casida, J. E., and Quistad, G. B. (2004). Organophosphate toxicology: Safety aspects of nonacetylcholinesterase secondary targets. Chem. Res. Toxicol. 17, 1–16.
Chmielewska, J., Rnby, M., and Wiman, B. (1988). Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Biochem. J. 251, 327–332.
Duysen, E. G., Li, B., Xie, W., Schopfer, L. M., Anderson, R. S., Broomfield, C. A., and Lockridge, O. (2001). Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: Supersensitivity of acetylcholinesterase knockout mouse to VX lethality. J. Pharmacol. Exp. Ther. 299, 528–535.
Ellman, G. L., Courtney, K. D., Andres V., Jr., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95.
Eskenazi, B., Harley, K., Bradman, A., Weltzien, E., Jewell, N. P., Barr, D. B., Furlong, C. E., and Holland, N. T. (2004). Association of in utero organophosphate pesticide exposure and fetal growth and length of gestation in an agricultural population. Environ. Health Perspect. 112, 1116–1124.
Eto, M. (1974). Organophosphorus Pesticides: Organic and Biological Chemistry. CRC Press, Cleveland, OH.
Fujino, T., Watanabe, K., Beppu, M., Kikugawa, K., and Yasuda, H. (2000). Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase. Biochim. Biophys. Acta. 1478, 102–112.
Gauthier, S. (2001). Alzheimer's disease: Current and future therapeutic perspectives. Prog. Neuro-Psychopharmacol. Biol. Psychiat. 25, 73–89.
Hulet, S. W., McDonough, J. H., and Shih, T.-M. (2002). The dose-response effects of repeated subacute sarin exposure on guinea pigs. Pharmacol. Biochem. Behav. 72, 835–845.
Jessani, N., Liu, Y., Humphrey, M., and Cravatt, B. F. (2002). Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl. Acad. Sci. USA 99, 10335–10340.
Jones, W. M., Scaloni, A., and Manning, J. M., (1994). Acylaminoacyl-peptidase. Meth. Enzymol. 244, 227–231.
Kenny, A. J., Booth, A. G., George, S. G., Ingram, J., Kershaw, D., Wood, E. J., and Young, A. R. (1976). Dipeptidyl peptidase IV, a kidney brush-border serine peptidase. Biochem. J. 157, 169–182.
Kidd, D., Liu, Y., and Cravatt, B. F. (2001). Profiling serine hydrolase activities in complex proteomes. Biochemistry 40, 4005–4015.
Main, A. R. (1964). Affinity and phosphorylation constants for the inhibition of esterases by organophosphates. Science 144, 992–993.
Mitta, M., Ohnogi, H., Mizutani, S., Sakiyama, F., Kato, I., and Tsunasawa, S. (1996). The nucleotide sequence of human acylamino acid-releasing enzyme. DNA Res. 3, 31–35.
Quistad, G. B., Fisher, K. J., Owen, S. C., Klintenberg, R., and Casida, J. E. (2005). Platelet-activating factor acetylhydrolase: Selective inhibition by potent n-alkyl methylphosphonofluoridates. Toxicol. Appl. Pharmacol. 205, 149–156.
Richards, P. G., Johnson, M. K., and Ray, D. E. (2000). Identification of acylpeptide hydrolase as a sensitive site for reaction with organophosphorus compounds and a potential target for cognitive enhancing drugs. Mol. Pharmacol. 58, 577–583.
Richards, P., Johnson, M., Ray, D., and Walker, C. (1999). Novel protein targets for organophosphorus compounds. Chem.-Biol. Interact. 119–120, 503–511.
Roman, K. M. (2001). 2000 IMS health hospital pharmacy review: Leading dollar volume pharmaceuticals. Hospital Pharmacy 36, 708–716, 789.
Rosenblum, J. S., and Kozarich, J. W. (2003). Prolyl peptidases: A serine protease subfamily with high potential for drug discovery. Curr. Opin. Chem. Biol. 7, 496–504.
Scaloni, A., Jones, W., Pospischil, M., Sassa, S., Schneewind, O., Popowicz, A. M., Bossa, F., Graziano, S. L., and Manning J. M. (1992). Deficiency of acylpeptide hydrolase in small-cell lung carcinoma cell lines. J. Lab. Clin. Med. 120, 546–552.
Shimizu, K., Kiuchi, Y., Ando, K., Hayakawa, M., and Kikugawa, K. (2004). Coordination of oxidized protein hydrolase and the proteasome in clearance of cytotoxic denatured proteins. Biochem. Biophys. Res. Commun. 324, 140–146.
Wilson, B. W. (2001). Cholinesterases. In Handbook of Pesticide Toxicology (R. I. Krieger, Ed.), Vol. 2. Agents, pp. 967–985. Academic Press, San Diego.
Wilson, B. W., Henderson, J. D., Ramirez, A., and O'Malley, M. A. (2002). Standardization of clinical cholinesterase measurements. Intl. J. Toxicol. 21, 385–388.
Wu, S.-Y., and Casida, J. E. (1996). Subacute neurotoxicity induced in mice by potent organophosphorus neuropathy target esterase inhibitors. Toxicol. Appl. Pharmacol. 139, 195–202.
Wynn, Z. J., and Cummings, J. L. (2004). Cholinesterase inhibitor therapies and neuropsychiatric manifestations of Alzheimer's disease. Dement. Geriatr. Cogn. Disord. 17, 100–108.
Yamaguchi, M., Kambayashi, D., Toda, J., Sano, T., Toyoshima, S., and Hojo, H. (1999). Acetylleucine chloromethyl ketone, an inhibitor of acylpeptide hydrolase, induces apoptosis of U937 cells. Biochem. Biophys. Res. Commun. 263, 139–142.(Gary B. Quistad, Rebecka )