ToxSci Advance Access originally published online on December 11, 2006
Toxicological Sciences 2007 96(1):154-161; doi:10.1093/toxsci/kfl183
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A Method to Determine Precise Benchmark Doses for Carbamate Anticholinesterases
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905
1 To whom correspondence should be addressed at Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Fax: (507) 284-9111: E-mail: lassiter.tommie{at}mayo.edu.
Received September 15, 2006; accepted December 5, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In determining benchmark doses for risk assessment and regulation of carbamate anticholinesterase pesticides like formetanate, oxamyl, and methomyl, one needs to quantitate low levels of cholinesterase inhibition. For improved accuracy while using fewer subjects, we developed an assay based on the recognized ability of carbamates to protect cholinesterase from irreversible inactivation. This assay measures enzyme that survives diisopropylfluorophosphate exposure in vitro and then reactivates by decarbamylation after small molecules are removed with size-exclusion centrifugation. The 99% silencing of unprotected cholinesterase yields a low background. Comparisons of recovered activity with initial activity (representing carbamate-free enzyme) use each sample as its own control. As a result, carbamate-protection assays can demonstrate a statistically significant 2–3% inhibition of brain cholinesterase in a single experimental group of modest size. When applied to brain samples from formetanate-treated rats, such an assay predicted a benchmark dose of 0.19 mg/kg for 10% inhibition (BMD10), with a lower 95% confidence limit of 0.15 mg/kg (BMDL10). Protection assays should enable precise determinations of benchmark doses for other carbamates, as well as accurate assessment of in vivo inhibition half-lives under low-dose scenarios.
Key Words: carbamate; benchmark dose; cholinesterase; rat; brain; reactivation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Carbamate insecticides like carbaryl, aldicarb, and methomyl have established uses as agricultural insecticides; but by preventing metabolism of acetylcholine at cholinergic synapses, they are also highly toxic to humans (Ecobichon, 2001
). Task forces of the International Life Sciences Institute and advisory panels of the U.S. Environmental Protection Agency (EPA) recently concluded that the N-methyl carbamates constitute a group of compounds with a common mechanism of toxicity: inhibition of acetylcholinesterase (AChE) by carbamylation of its active site (ILSI, 1999; USEPA, 2004). Under the U.S. Food Quality Protection Act of 1996, the Agency must assess the cumulative risk of exposure to these carbamates. Of particular concern is the issue of cumulative toxicity from multiple exposures to multiple agents within a single day.
A favored approach toward cumulative risk assessment involves relative potency factors. In order to combine effects from multiple compounds sharing a common mechanism, each exposure is normalized in terms of the given compound's potency relative to that of a reference compound. Normalized exposures are then simply summed. Useful points of departure for normalization are benchmark doses producing 10% inhibition of brain AChE (BMD10) and, particularly, their lower 95% confidence limits (BMDL10). These doses are thought to pose minimal risk while still producing measurable effects (USEPA, 2005). Comparisons of compounds in terms of BMD10 are inherently more accurate than those based on the No Observed Adverse Effect Level, or NOAEL, as discussed by Sand et al. (2006). Nonetheless, 10% inhibition is difficult to measure in a tissue that cannot be sampled serially to serve as its own control. As a result, in order to determine brain BMD10 values for a carbamate, one typically needs large numbers of animals, multiple doses, and extensive fitting of the data.
The reversible nature of carbamylation creates additional problems in assessment. Carbamylated AChE regenerates itself by cleaving the adducted group from the active site serine. Although this decarbamylation is 107- to 108-fold slower than the deacetylation associated with acetylcholine hydrolysis, regeneration will be more than half complete within an hour (Reiner, 1971; Reiner and Aldridge, 1967; Roufogalis and Thomas, 1969). Because of this rapid recovery, one must prepare samples quickly and with minimal dilution to avoid underestimating the inhibition in situ (Nostrandt et al., 1993).
In order to obviate some of the difficulties in determining BMD10 values for carbamate anticholinesterases in brain, we set about developing a new way to measure inhibition. The aim was to obtain radical increases in sensitivity and precision by eliminating background noise and by using each brain as its own control. We also sought to trap enzyme in the state in which it had been sampled, thereby preventing problems with regeneration during assay. In fact, we planned to take advantage of rapid regeneration by developing a "negative image" of the carbamylation state. Others have used related approaches to measure carbamate inhibition indirectly (French et al., 1977; Hunt and Hooper, 1993; Hunt et al., 1995). However past methods have been cumbersome, limited to red cells, or unsuited to detecting small effects.
Our approach was to determine, in rats exposed to carbamate, what fraction of the brain cholinesterase would escape subsequent in vitro inactivation by an irreversible organophosphate inhibitor. We needed to meet three criteria. One, carbamate-protected enzyme must be spared while unprotected enzyme is completely and permanently inactivated. Two, the protected enzyme must be rapidly and quantitatively separated both from residual carbamate and from free organophosphate. Three, separated enzyme must spontaneously reactivate before undergoing proteolysis or denaturation. As will be described, it proved possible to satisfy these criteria to a reasonable degree with a practical protocol.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
Three environmentally relevant agents from the "common mechanism group" of N-methyl carbamates were selected for this study based on ease of solubilization and other considerations. Oxamyl (under EPA consideration as a reference chemical for the group), methomyl, and formetanate were purchased from Chem Services (West Chester, PA). Tritiated acetylcholine iodide was from Perkin-Elmer (Boston, MA). Diisopropylfluorophosphate (DFP) was from Sigma-Aldrich (St Louis, MO).
Tissue collection.
Animal work was performed in conformity with the National Institutes of Health Guide to Care and Use of Animals under protocols approved by the Mayo Animal Care and Use Committee. The in vitro experiments used mouse brains (C57B6 x SJL/J background, both sexes) obtained under sodium pentobarbital anesthesia and stored at – 80°C (6–12 months). In vivo subjects were adult, male Long-Evans rats (300–400 g) dosed by gavage with formetanate (0.025–0.6 mg/kg in sterile 0.9% NaCl). Controls were gavaged with saline alone. After 70 min, the rats were euthanized with sodium pentobarbital followed by decapitation. Our standard protocol called for immediate processing of brains for assay. In some cases, however, brains were rapidly frozen and held up to a week at – 80°C. There is good evidence that AChE and carbamylated AChE are stable for months under such conditions (Hunter and Padilla, 1999). Fresh and frozen material gave similar results in our hands.
Cholinesterase activity.
Brain hemispheres truncated at the lower border of the medulla were disrupted with a Polytron homogenizer (30 s, maximum power) in 10 volumes of Tris HCl buffer, pH 7.4, containing 1% vol/vol Triton X 100. The resulting homogenates were used without centrifugation or filtration. For the preliminary in vitro experiments, cholinesterase activity was assayed by a microtiter version of the standard spectrophotometric assay (Ellman et al., 1961). A radiometric, minimal-dilution assay (Johnson and Russell, 1975; Nostrandt et al., 1993) was used in later experiments. For this assay, duplicate 50-µl brain samples were incubated at room temperature for 5 min (fresh homogenates) or 60 min (diluted effluents of spin columns—see below) with 50 µl of 3H-acetylcholine (100 nCi, 10 nmol). To avoid complex interactions, assays were run without selective inhibitors. We confirmed, however, that AChE accounted for > 98% of the activity in whole rat brain, as shown previously (Li et al., 2000).
Size-exclusion centrifugation.
Preloaded Micro "Bio-Spin" columns with Bio-Gel P-6 size-exclusion resin equilibrated in 10mM Tris buffer, pH 7.4 (Bio-Rad, Hercules, CA) were prepared just before use according to the supplier's instructions. Excess buffer was drained from the bottom of the tube for 2 min and any remaining excess buffer was removed by centrifugation at 1000 x g for 3 min. A 60-µl sample of brain homogenate was then loaded into the column and centrifuged at 1000 x g for 5 min. The effluent, deficient in small molecules, was saved for cholinesterase assay.
Statistics.
Differences between treatment groups were assessed with Student t-test (p < .05 taken as statistically significant). Statistical confidence limits were obtained after regression analysis with StatView 4.5 (Abacus Concepts, Berkeley, CA). Curve fitting was accomplished with Sigma Plot 5.01 (Jandel Scientific, San Rafael, CA). Unless specifically indicated otherwise, all estimates of variability are reported below as SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Conditions for In Vitro Inhibition and Reactivation
The first task was finding how to separate cholinesterase rapidly from inhibitors. For this purpose, we examined size-exclusion centrifugation columns. Initially, high–molecular weight dextran blue mixed with 3H-acetylcholine (10 µCi) was loaded onto Bio-Spin columns in different volumes. With a loading volume of 60 µl, the centrifuged effluent contained little radiolabeled acetylcholine (< 5%) but retained most of the high–molecular weight marker. Using the same loading volume and homogenized brain as a carrier, over 95% of the labeled tracer was eliminated, but with independent brain samples from a total of 14 rats, the recovery of cholinesterase activity was 79 ± 2.2% (mean, SEM). This performance was judged adequate. We therefore investigated the stability of spin-separated cholinesterase under conditions that would allow extensive regeneration of carbamylated enzyme. Effluents from spin columns loaded with untreated brain homogenates were diluted 50-fold in sodium phosphate (0.1M, pH, 7.4) and incubated for long periods at room temperature. The average residual cholinesterase activity at 24 h proved to be 101 ± 3% of the immediate postcentrifugation value (n = 3). In light of these findings, we expected that one cycle of spin separation followed by dilution and overnight incubation would give us (1) 80% of the original enzyme, in a stable conformation, (2) over 1000-fold depletion of inhibitors from the assay mixture, and (3) near 100% recovery from carbamylation.
Next we addressed the means of irreversible phosphorylation. An agent with nearly instantaneous onset and rapid "aging" would be ideal, but we selected DFP for its comparative safety. Preliminary trials were conducted to determine the shortest exposure and the lowest concentration that would completely inhibit unprotected cholinesterase. With brain homogenates at room temperature, 99% inhibition required a 1-h exposure to 10µM DFP (Table 1). To confirm that this inhibition was permanent, treated samples were passed over spin columns and reassayed after 24 h. Spontaneous recovery was detectable but minimal under these conditions. The level of inhibition remained at 97.8% (actual recovery in 20 replicate samples: 1.2 ± 0.24%).
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In Vitro Test of Protection Assay
To determine if exposure to DFP followed by spin chromatography could be used in measuring carbamate protection, we incubated mouse brain homogenates for 1 h at room temperature with methomyl, oxamyl, or formetanate (0.25nM–5mM). One aliquot of each incubated sample was assayed immediately to determine residual cholinesterase activity (at 50-fold final dilution), while another aliquot was exposed to DFP (10µM, no further dilution). At the end of the hour, the DFP-exposed samples were centrifuged on the size-exclusion columns. Column effluents were vortex mixed, diluted an additional 50-fold, and left at room temperature for 20–24 h before assay of cholinesterase activity.
The enzyme assays showed that graded carbamate exposures gave graded protection of brain cholinesterase from DFP (Fig. 1). The low background activity in unprotected samples exposed to DFP allowed us to determine very small carbamate effects (Fig. 1). For example, exposure to 0.015µM oxamyl protected 2.4 ± 0.5% of the cholinesterase activity (p < .01). Maximal protection, relative to the activity in the untreated samples, was substantial for all three carbamates (84, 90, and 95% for oxamyl, methomyl, and formetanate, respectively). The response curves, however, were flattened at higher concentrations of carbamate. Because the time and dilution involved in spectrophotometric assay might have influenced outcomes, we repeated the formetanate experiments with a minimal-time, minimal-dilution radiometric assay (Johnson and Russell, 1975; Nostrandt et al., 1993). The resulting data better fit a standard occupancy model, even at carbamate concentrations well above the IC50 (Fig. 2).
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One issue remained. Comparisons with the raw cholinesterase activities showed that, as dosage increased, protection rose only about half as fast as inhibition. That is, the regression of protection versus inhibition had a slope near 0.5 (Fig. 3A). The disparity did not reflect a delayed phosphorylation by residual DFP in the diluted column effluents since internal standards added after centrifugation retained full activity. The likely cause, instead, was partial phosphorylation of carbamylated enzyme during the incubation with DFP, which exceeded the half-time for decarbamylation. A kinetic model developed to address this issue indicated that our conditions, in fact, allowed DFP to phosphorylate nearly half of the originally carbamylated enzyme.
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Kinetic Model
Concurrent decarbamylation and phosphorylation of cholinesterase is formally equivalent to first-order exponential absorption of drug from a depot, followed by first-order elimination from the central compartment. The underlying differential equations are readily solved if decarbamylation (like absorption) is treated as an essentially irreversible event. The following expression, adapted from Bowman and Rand (1980)
yields the fraction of carbamylated cholinesterase (F) that escapes phosphorylation during DFP exposure:
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The output of this equation does not depend on the concentration of carbamate or the starting level of inhibition. The only variable is time, t, and the only parameters are the rate constant for decarbamylation, kc, and the apparent rate constant for phosphorylation, kp. Since the latter depends on experimental conditions, we used the data in Table 1 to estimate an overall phosphorylation rate constant of 4.2 ± 0.4 h–1 for brain homogenate exposed to 10µM DFP. To derive an intrinsic, decarbamylation rate constant, samples exposed 1 h to 0.1µM formetanate were spin-separated, diluted 50-fold, and then assayed 5 to 60 min later. Nonlinear regression analysis of data from three independent experiments yielded a value of 1.1 ± 0.09 h–1. Given a 1-h exposure to DFP (t = 1), the fully parameterized rate equation predicts that 46% of carbamylated enzyme will escape phosphorylation. To calculate the initial level of carbamylation, one must therefore apply a correction factor of 1/0.46 = 2.2 to the activity recovered in the protection assay. With this correction, the dose-dependent increase in protection roughly mirrors the loss of activity in brain homogenates treated with formetanate (Fig. 3B).
Protection Assay for In Vivo Inhibition
The in vitro data indicated that a protection assay could also be used to characterize the inhibition of brain cholinesterase by carbamates delivered in vivo. To explore this possibility, we administered formetanate to rats by gavage in doses expected to span the BMD10. A 70-min delay between gavage and assay was selected for practical reasons. Trials showed near maximal inhibition at this point (Fig. 4). At harvest, each brain was homogenized and divided into two aliquots. Aliquot A was assayed immediately for total cholinesterase activity (5-min radiometric method), while aliquot B was exposed to DFP (10µM, for 1 h at room temperature) and then centrifuged over size-exclusion columns. Column effluents were diluted 50-fold as before, incubated 20–24 h at room temperature, and finally assayed for total cholinesterase activity (flow chart in Fig. 5).
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Since carbamates protect cholinesterase from DFP by occupying the enzyme's active site, we equated protection with carbamate-induced inhibition. Because carbamylated enzyme should regenerate fully overnight (20 half-lives), the original extent of carbamylation should be 2.2B/(0.8A + 2.2B). Simplifying this formula, we obtain the equation, C = 100 x B/(0.36A + B), where C is the original percentage of carbamylated enzyme, A is the cholinesterase activity measured in the fresh homogenate (from carbamate-free enzyme), and B is the activity recovered after spin separation and overnight incubation (from carbamate-protected enzyme). The factor of 0.8 corrects for the 20% loss of enzyme on the spin column, while the factor of 2.2 corrects for the predicted 54% phosphorylation of carbamylated enzyme. Applying this equation to the data from formetanate-treated rats resulted in a "protection curve" that rose in approximately linear fashion with dose (Fig. 6). At the highest dose, directly assayed cholinesterase activity was reduced 46% in formetanate-treated rats, as compared with untreated rats, a result almost identical to the value from the protection assay (42 ± 3%). At lower doses, the mean cholinesterase activities in formetanate-treated rats did not differ significantly from the means in untreated controls. By contrast, protection assays revealed significant inhibition at 0.1 mg/kg (2.4 ± 0.7%, p < .05) and 0.2 mg/kg (7.1 ± 1%). For a rigorous estimate of the formetanate BMD10, we fitted the whole range of data in a simple linear regression. The computations yielded a maximum likelihood estimate of 0.19 mg/kg, with 95% confidence limits of 0.15 and 0.23 mg/kg.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present results establish carbamate masking as a quantitative surrogate for carbamate-induced inhibition of cholinesterase. This method detects low levels of inhibition because it measures signal against a low background. It also offers improved precision because each sample provides its own control. The method can be generalized. A similar approach could characterize any toxicant that acts in a slowly reversible manner on any readily assayed target that also has a fast-acting, irreversible inhibitor. Carbamate masking lends itself to multiple variations. Other techniques could be used for molecular separation (e.g., ultrafiltration). One could also employ cholinesterase inhibitors that are faster and less reversible than DFP, like soman (French et al., 1977
) or phenyldichlorophosphate (Dawson and Poretski, 1977). Such agents require handling precautions and special facilities, rendering them unsuitable for many laboratories, but they might eliminate the need to correct for concurrent decarbamylation.
Accuracy
Our experiments predicted a BMD10 of 0.19 mg/kg for formetanate. The formetanate BMD10 estimated from studies performed by the National Health and Environmental Effects Research Laboratory (USEPA, 2005) was somewhat lower (0.1 mg/kg for brain cholinesterase). Our longer interval between dosing and sampling might explain that discrepancy. In any case, the present data yield a narrower range in confidence limits (1.5-fold from BMDL10 of 0.15 to the upper confidence bound on BMD10 of 0.23) than did the previous determination for formetanate (fivefold from BMDL10 of 0.045 to the upper confidence bound on BMD10 of 0.22).
Although carbamate masking yields data of improved precision, possibilities for systematic error deserve close attention. Of course, fresh homogenates must be assayed properly to minimize premature decarbamylation, which would overstate initial activity and reduce apparent protection. Decarbamylation is likely when assays are delayed, prolonged, or run at high dilution on strongly inhibited samples. This reaction probably explains the decreased slopes in the upper ranges of the dose-response curves in Figure 1. As Figure 2 indicates, however, premature decarbamylation can be minimized by rapid, minimal-dilution radiometric assays. Also, the protection assay, in contrast with conventional approaches, becomes less and less sensitive to such errors as one moves lower on the dose-response curve.
A minor, quantifiable problem in the assay was enzyme loss on the spin columns. Bio-Gel P-6 excludes proteins with molecular weight > 6000 while allowing > 99% of salts and small molecules to permeate. Hence, cholinesterase appears in the void volume while the peaks of DFP and carbamate are retarded. Unfortunately, the spin columns are too short to separate the peaks completely. To eliminate most of the inhibitor, one must truncate the sample and accept a partial loss of cholinesterase. Nonspecific binding to the P-6 resin may slightly increase this loss. The physics of separation, however, are identical with carbamate-inhibited, DFP-inhibited, and free cholinesterase molecules. In light of the column recovery data collected from 14 rat brains, we considered it reasonable to build a uniform 20% correction for recovery into all calculations.
Correction was also needed for spontaneous dephosphorylation, which increases the apparent level of carbamate protection. We detected approximately 1% spontaneous recovery after DFP treatment of a fresh, unprotected brain homogenate. This effect is unimportant at the mid and upper portions of the dose-response curve, but it distorts outcomes, in relative terms, when the dose of carbamate is small and most of the enzyme becomes phosphorylated. Using either contemporary or historical controls, one must subtract spontaneous reactivation in order to determine the true percentage of carbamate-protected cholinesterase. After applying formulas to calculate protection, we reduced measured recoveries by a total of 2% (1% for spontaneous recovery of phosphorylated enzyme and 1% for incomplete phosphorylation of unprotected enzyme).
A final source of potential error is unwanted phosphorylation. Internal standards applied to column eluates showed no inhibition, which means that lingering DFP did not reduce the apparent degree of carbamate protection. On the other hand, we found that the prolonged DFP exposure did phosphorylate some of the cholinesterase that was initially masked by carbamate but then decarbamylated and exposed its active site. Our kinetic model predicted that 46% of the originally carbamylated cholinesterase would remain protected in our assay. This estimate matched the actual regression slope of 0.48 for protection versus inhibition in vitro (Fig. 3). Overall, therefore, there is good reason to think that the protection assay provides an unbiased index of carbamylation for the purpose of estimating BMD10 values. Because the computations do involve correction factors, however, it is important to consider how the results would be affected by variability in the underlying parameters. That issue can be addressed by a sensitivity or uncertainty analysis.
Uncertainty Analysis
The central equation in our kinetic model contains two experimentally determined parameters. The parameter for recovery of enzyme on the spin column is "procedural," as actual recovery will vary slightly from run to run. The parameter defining fractional protection of carbamylated enzyme depends on intrinsic properties of DFP and human cholinesterase, but it may also be affected by experimental conditions like temperature. It is legitimate to ask how errors in these parameters would affect the model output. The parameters themselves have been estimated with care. Measured column recovery with 14 replicates was distributed in quasinormal fashion with a mean of 0.79, a median of 0.76, and a SD of 0.08. One therefore expects that most recoveries will fall within 25% of the measured mean value. Fractional protection is expected to vary less than column recovery. The key parameter in the protection equation is carbamate inhibition half-life (because the onset of DFP inhibition is relatively rapid). Having determined this parameter with a coefficient of variation of 12%, we conclude that most cases are likely to fall within 25% of the mean value of 0.46. It is straightforward to correct for errors of this magnitude.
A general form of the equation used to calculate the starting level of carbamylation, "C," is C = (B/R x F)/(A + (B/R x F)), where R is the column recovery parameter and F is the parameter defining the fraction of carbamylated enzyme actually protected by DFP. As carbamylation grows large (at BMD50 and above), errors in R and F have proportionately less effect on output. At BMD10 and below, however, a 25% error in either parameter alters output by nearly 25%. Nonetheless, it is important to note that the propagated error never becomes disproportionate but merely approaches one-to-one. In other words, the model exhibits stable behavior.
Detection Limits
Spontaneous recovery from DFP and the failure to inhibit 100% of unprotected enzyme are the two main factors limiting the sensitivity of this protection assay. We found their combined effect to be 2.2%, with a SD of 0.5% or less. If assays on six carbamate-treated rats were to show a similar 25% coefficient of variation, then a mean gross recovery of 3% would just meet the basic criterion for statistical significance by t-test (p < .05 vs. spontaneous recovery). Hence, the protection assay should detect inhibition as low as 1% (i.e., 3–2%) in a single group of rats. In contrast, simple comparisons of net activities in separate groups can easily fail to demonstrate a 10% inhibition. Thus, the apparent 10% group difference in raw brain cholinesterase activity of control rats and rats given the BMD10 dose of formetanate was far from significant, though the coefficients of variation were within our normal range (10–20%). To detect a 10% group difference would have required similar data from many more rats (more than 24 per group) or much tighter data from the same number of rats (coefficient of variation less than 10%).
The ability to quantitate low levels of inhibition by carbamate-protection assays opens up a range of new experimental possibilities. For example, it now becomes feasible to determine the biological half-life of cholinesterase inhibition in animals exposed to low doses of carbamates. Previous studies showed that in vivo recovery from moderately large doses of carbaryl can require 12 h in the rat (USEPA, 2005). Most probably, this slow course reflected the metabolic clearance of the pesticide rather than the intrinsic reactivation kinetics of cholinesterase. Because rat and human cholinesterase are likely to exhibit similar kinetics, the accepted rules for allometric scaling of pharmacokinetic data (Kirman et al., 2003; USEPA, 1992) should not be used to assess human health risks in low-exposure scenarios. Nonetheless, the capability of determining in vivo half-lives for inhibition in animals exposed to environmentally realistic levels of carbamates may prove useful.
Implications
A reduced limit of detection raises questions of physiological and toxicological relevance. A safe exposure is one that does not disturb homeostasis in cells and organisms. Cholinergic synapses have a wide margin of safety, and signs of cholinergic dysfunction are often absent until AChE activity declines by nearly half (Bignami et al., 1975; Gupta et al., 1986). Behavioral signs, typically the most sensitive index, generally do not appear until brain AChE is inhibited 20% or more (Sheets et al., 1997), though the pattern is sometimes more complex (Moser, 1999). Increased accuracy in measuring small levels of inhibition and their lower confidence limits (i.e., BMDL10) enables one to detect events that in themselves may be insufficient to perturb synaptic function but are precursors of toxicity or initial steps on the direct pathway to toxicity. A body of new information on low-dose effects might spur further thought about physiological criteria for safe levels of carbamate exposure and what truly constitutes an adverse effect of carbamates in the brain.
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