Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 947203112
1 To whom correspondence should be addressed at Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 947203112. Fax (510) 642-6497. E-mail: ectl{at}nature.berkeley.edu.
Received April 1, 2005; accepted May 9, 2005
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ABSTRACT |
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Key Words: acylpeptide hydrolase; chlorpyrifos; dichlorvos; diisopropyl fluorophosphate; naled; trichlorfon (metrifonate).
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Introduction |
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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.
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Materials And Methods |
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Mouse and human samples.
Male Swiss-Webster mice (2730 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 (0.031 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 1585% 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 7498 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.
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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).
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Results |
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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 (M1min1), 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 structureactivity 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 3280 nM), but APH is much more sensitive to DFP (IC50 911 nM), >500-fold greater compared to AChE. For inhibition of AChE by both CPO and DFP, the IC50 values differ by only 0.54.6-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 1126 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.
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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 4053% 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).
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Discussion |
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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 04 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).
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ACKNOWLEDGMENTS |
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