* Battelle, Pacific Northwest Division, Richland, Washington 99352, and University of Medicine and Dentistry New Jersey, Newark, New Jersey
Received March 2, 2004; accepted April 26, 2004
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ABSTRACT |
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Key Words: acetylcholinesterase; organophosphate insecticide; chloropyrifos oxon; paraoxon.
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INTRODUCTION |
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Concentration-dependent ki values have been attributed to the possible existence of a secondary binding site (Kardos and Sultatos, 2000), which may or may not be related to the peripheral binding site identified on the surface of the AChE molecule. Early studies of the hydrolysis of acetylcholine by AChE documented substrate inhibition by acetylcholine and suggested an allosteric mechanism (Changeux, 1966
). Taylor and Lappi (1975)
first identified a peripheral binding site for AChE, which likely results in substrate inhibition when occupied. Subsequent studies have established the existence of this peripheral binding site on the surface of the enzyme, about 20 Å from the entrance to the active site (Berman et al., 1980
). This peripheral binding site has been considered as an obligatory landing site for charged ligands, a part of the active site, an ionic strength sensor (Berman and Leonard, 1992
), and the site of excess substrate inhibition. Taylor and Radic (1994)
have suggested that ligand association with the peripheral binding site may prevent access of substrates to the active site by physical obstruction, charge repulsion with the association of a cationic ligand, or by an allosteric mechanism in which the active center conformation is altered. While several ligands in addition to acetylcholine have been shown to bind to the peripheral binding site, most OPs have been thought not to occupy this site since they have been reported to follow simple second-order kinetics (Friboulet et al., 1990
). However, Kardos and Sultatos (2000)
have suggested that PO and methyl PO likely bind to a site distinct from the active site and that occupation of the site makes subsequent phosphorylation of the active site more difficult. However, they did not determine if this putative secondary site is identical to or overlaps with the well-characterized peripheral binding site.
The aim of our study was to compare the dynamics of AChE inhibition following in vitro incubation with CPO or PO, over a wide range of concentrations, to help elucidate the presence of a peripheral binding site. The selection of CPO and PO was based on several factors including similarities in chemical structures, mechanism of actions, and common metabolic pathways as well as chemically identical enzyme inhibition complexes (Table 1).
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MATERIALS AND METHODS |
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Animals. Male Sprague-Dawley rats that were 3 months old (weighing 300350 g) were purchased from Charles River Lab Inc. (Raleigh, NC). Prior to use, animals were housed in solid-bottom cages with hardwood chips (Laboratory grade SANI chips, TEKLAD, Madison, WI) under standard laboratory conditions and given free access to water and food (PMI 5002, Certified Rodent Diet, Animal Specialities, Inc., Hubbard, OR). All procedures involving animals were in accordance with protocols established in the NIH/NRC Guide and Use of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee at Battelle, Pacific Northwest Division.
Tissue preparations. Rats were weighed, humanely sacrificed by CO2 asphyxiation, and individual rat brains were immediately removed, rinsed in ice-cold buffer, weighed, and homogenized in 9 volumes of 0.1 M phosphate buffer (pH 7.4) using a polytron homogenizer (Brinkman Instruments, Westbury, NY). The brain homogenates were stored as individual 1-ml aliquots at 80°C until the time of ChE activity determination. Preliminary studies detected no differences in AChE activity in both fresh and frozen homogenates, provided homogenates were not kept frozen longer than 3 months (data not shown).
Characterization of brain AChE activity profile and enzyme kinetics. The brain homogenates were thawed at room temperature and an aliquot was diluted in an additional 6 volumes of phosphate buffer. Next, 200 µl diluted homogenate were incubated with 200 µl phosphate buffer containing a range of CPO (1 x 10325 nM) or PO (5 x 104100 nM) concentrations in a shaker at room temperature for 024 h. The dilution of brain homogenate was used to place the absorbance between 0.1 and 1 OD/min. Reactions were terminated by the addition of 4.6 ml phosphate buffer to each brain sample. Brain AChE activity was determined by a modified Ellman method (Ellman et al., 1961) using a 96-well automated microplate spectrophotometer ELx808 equipped with a KC4 software package (Bio-Tek Instruments, Inc., Winooski, VT). Sample (250 µl/well) was transferred and 25 µl of DTNB and ATC were placed in each well for final concentrations of 0.1 and 0.4 mM of DTNB and ATC, respectively, and a final volume of 300 µl/well (Mortensen et al., 1996
; Nostrandt et al., 1993
). The control samples were incubated with phosphate buffer that did not include any oxon. The AChE activity described by the rate of ATC hydrolysis was monitored by following the absorbance profile at 405 nm over 3040 min. The slope of the linear regression of that profile was used to measure the remaining enzyme activity. The statistical analysis of the data was limited to a determination of a mean and standard deviation of three samples where appropriate by using the standard equations.
The 50% inhibitory concentration values (IC50) for CPO and PO were determined under identical experimental conditions with respect to homogenate dilution, homogenate/buffer incubation ratio and time, and substrate concentrations. Diluted brain homogenate (200 µl) was incubated with equal amounts of buffer containing a range of oxon concentrations in a shaker at room temperature for 5 min; the reactions were terminated and AChE activity was determined as described above. Control samples incubated with buffer but containing no oxon were also included. The IC50 values were calculated using the pharmacokinetic software WinNonlin version 1.1 (Pharsite Corp., Cary, NC). The first-order (h1) reactivation rate constant (kr) was determined by transforming the percentage of AChE activity into the percentage of AChE inhibition where the slope of the linear regression of the natural log (ln) of the terminal portion of the curve equals the kr (Levine and Murphy, 1977).
Pharmacodynamic model development. Pharmacodynamic models describing the in vitro interaction of the oxons with AChE were developed in SIMuSOLV® (Trademark of the Dow Chemical Co., Midland, MI), as described previously (Kardos and Sultatos, 2000). The first model, referred to as the active binding site model, used the inhibitory rate constant (ki) based on the equation derived by Main (1964)
; the second model, referred to as the peripheral binding site model, included equations describing a secondary oxon binding site on the AChE molecule (Fig. 1). The differential equations that describe the active binding site model were as follows (Kardos and Sultatos, 2000
):
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To solve the active binding site model equations (Equations 1 3), the initial AChE active site concentration and kr values had to be determined experimentally. Titration of brain AChE enzyme with known oxon concentrations (5 x 1040.01 nM) was used to estimate the total concentration of active sites while kr was determined as described above (Kardos and Sultatos, 2000
; Kousba et al., 2003
; Levine and Murphy, 1977
). The initial AChE active site estimation was calculated based on the enzyme inhibition obtained for a given oxon concentration. The model optimization was used to estimate the final rat brain AChE active site concentration and the bimolecular inhibition rate constant (ki; nM1 h1) of CPO and PO toward rat brain AChE as a function of varying oxon concentrations (5 x 104100 nM). The ki was also estimated using the method of Main (1964)
at higher CPO and PO concentrations (1100 nM), selected to give a maximum inhibition ranging from 1090% over a 5- to 30-min incubation period. The log percentage of activity was plotted against time and the slopes of each log plot were calculated using linear regression. These slopes were used for the final ki calculation.
To solve the peripheral binding site model equations (Equations 4 8), the initial values for the model parameters (ki, ki2, kr, and total enzyme concentration) were based on the active binding site model optimization. Estimates of k1 and k2 and the final parameter values for ki, ki2, kr, and total enzyme concentration were determined by optimization of the peripheral binding site model against several experimental data sets. The peripheral binding site model was only applied to fit the whole data sets for AChE inhibition by PO due to the robust amount of data that were generated. Also, comparison of the ki estimated values for the more limited data of AChE inhibition by CPO at different concentrations was consistent with the same relationship observed following incubation with PO.
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RESULTS |
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The final parameter estimates were used in the peripheral binding site model and the model was optimized simultaneously against AChE inhibition at several PO concentrations. The peripheral binding site model projected slight changes in the initial estimate of AChE active site concentration (0.0014 vs. 0.0015 nM determined by the active binding site model) and ki (1450 vs. 420 nM1h1 determined by the active binding site model), which may be attributed to the fact that the active binding site model did not account for the oxon molecules bound to the peripheral binding site. Figure 9 shows the peripheral binding site model simulations against AChE inhibition at 1 x 103, 2, and 10 nM PO. Figure 9 indicates that the inclusion of a peripheral binding site for the AChE molecule resulted in an adequate description of the enzyme inhibition and that the existence of such a site exerted a significant role in modulating the dynamic behavior of AChE.
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DISCUSSION |
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Prior to Kardos and Sultatos (2000), ki determination studies were based on the method of Main (1964)
, assuming that the in vitro interaction between cholinesterase and OP approximated first-order conditions with respect to uninhibited enzyme concentration. Main (1964)
used organophosphate concentrations much higher than the tissue homogenate cholinesterase (ChE) concentration, yielding a single ki estimate of enzyme inhibition. The approach we used was a pharmacodynamic model describing the in vitro inhibition kinetics of AChE by CPO and PO (Kardos and Sultatos, 2000
; Kousba et al., 2003
). The pharmacodynamic model was coded to estimate the ki under both first- and second-order conditions through model optimizations against experimental data. This method was proposed as a more robust technique for ki estimation (Kardos and Sultatos, 2000
; Kousba et al., 2003
) and the results indicated a substantial difference in the ki values compared with previous studies (Amitai et al., 1998
; Carr and Chambers, 1996
; Rosenfeld et al., 2001
). Although AChE inhibition, using high oxon concentrations, was markedly different, spontaneous reactivation of the inhibited enzyme was similar (Fig. 3). This is not surprising since the enzyme-inhibited form of AChE is similar in both situations and spontaneous reactivation mainly occurs due to hydrolysis of the phosphorylated bond. The kr value of 0.091 ± 0.023 h1 determined for AChE inhibited by PO is similar to the 0.070 h1 reported by Kardos and Sultatos (2000)
for mouse brain AChE, but slightly higher than 0.024 h1 reported by Levine and Murphy (1977)
in rats. The kr values of 0.0840.087 h1 determined for AChE inhibited by CPO are higher than those reported by Carr and Chambers (1996)
, who estimated similar kr values (0.0140 and 0.0148 h1) for AChE inhibited by CPO and PO at 37°C. Pope et al. (1991)
reported that, among different OP insecticides, cholinesterase recovery in vivo is probably related to differences in absorption, biotransformation, and, most importantly, spontaneous enzyme reactivation. The ki values describing AChE inhibition following incubation with 1 pM oxon were >1000 and 10,000 times higher than those values determined using the nM range concentrations for CPO and PO, respectively (compare Figs. 2 and 7). However, the ki values determined for both CPO and PO at higher oxon concentrations (1100 nM; Figs. 5, 7, and 8) were approximately similar to previously reported values. These results suggest that the pharmacodynamic modeling is a sound approach for ki estimation over a broad range of oxon concentrations.
As shown in Figure 2, the active binding site model optimization reasonably described AChE inhibition at a 1 x 103 nM oxon concentration; however, using those ki values resulted in an overestimation of AChE inhibition at higher oxon concentrations. The estimated ki values that described AChE inhibition at 1 x 103 nM oxon concentrations were more than three orders of magnitude greater that the estimated ki at nM concentrations. In essence, the high oxon concentrations resulted in a lower ki value, which reflects a lower capacity to phosphorylate the serine OH group of the active site of AChE. At lower oxon concentrations, the phosphorylation of AChE was more efficient (compare Figs. 2 and 7). Likewise, Kardos and Sultatos (2000) estimated a 10-fold higher ki value for brain AChE incubation with 0.1 nM PO compared with the ki value following 100 nM.
As described by Kardos and Sultatos (2000), several explanations for the oxon concentrationdependent ki values have been considered, including the existence of multiple forms of AChE and excess substrate inhibition of AChE. Whereas several isoforms of AChE have been identified, all are products of one gene; their catalytic cores are identical, exhibiting similar susceptibility to inhibition, and only differ in their mechanism of anchoring (Friboulet et al., 1990
; Sussman et al., 1991
). This implies that isoforms of AChE are unlikely to differ in their response to inhibition by different organophosphate concentrations by more than four orders of magnitude, as shown in Figures 1 and 5. A more plausible explanation, consistent with the results obtained in this study, is based on AChE being inhibited by its own substrate, acetylcholine, as a result of allosteric modification or blockage of the active site. Allosteric modification or blockage are due to acetylcholine binding to a peripheral site and preventing access of other substrate molecules to the active site (Taylor and Radic, 1994
). The occupation of the proposed secondary binding site may prevent the substrates from reaching the active site of the AChE molecule, resulting in slower rates of phosphorylation of AChE at high oxon concentrations (Fig. 1).
Rat brain AChE activity following incubation with a high oxon concentration showed more in vitro inhibition by CPO than PO, as evidenced by the lower IC50 and higher ki values for CPO compared with those of PO. The estimated in vitro IC50 for CPO was 10 times lower than for PO (8.98 vs. 81.66 nM, respectively), which is consistent with previously reported relationships (Atteberry et al., 1997
; Mortensen et al., 1998
). The ki values determined for CPO and PO inhibition of AChE in this study were also consistent with Amitai et al. (1998)
, who estimated a CPO ki of 911 times greater than for PO. The basis of this inhibitory potency difference is not fully understood since the enzyme inhibition complex for the two substrates are chemically identical (Table 1). Carr and Chambers (1996)
suggested that the potency difference may be attributed to the differences in the interaction of the leaving group with the active site, whereby the association of the leaving group moiety with the anionic site may produce an environment that affects the rate of phosphorylation of the active site by inducing conformational changes in the tertiary structure of the enzyme. In contrast to the observed in vitro potency for AChE inhibition, parathion was appreciably (>30 times) more acutely toxic than chlorpyrifos (LD50 values of 413 and 82155 mg/kg, respectively; Gaines, 1969
). This in vivo potency difference is primarily a reflection of the higher capacity for metabolic detoxification of chlorpyrifos compared to parathion (Atteberry et al., 1997
; Chambers et al., 1990
; Chanda et al., 1998
).
Of particular interest in our study was the observation that, at low oxon concentrations, comparable ki values for CPO and PO were obtained versus a 10- to 15-fold difference following incubation at a nM oxon concentration range (Figs. 2 and 7). The lowest oxon concentration (1 pM) used in this study was two orders of magnitude lower than what Kardos and Sultatos (2000) used and has facilitated the comparison of the inhibitory potency of CPO and PO at disparate (high vs. low) oxon concentrations with respect to the potential secondary binding site occupation by the oxons. Following incubation with high oxon concentrations, all peripheral binding sites were assumed to be fully occupied; at low concentrations, the peripheral binding sites were assumed to be minimally occupied, as evidenced by partial AChE inhibition. A possible explanation for this phenomenon is that the difference in AChE phosphorylation could be attributed to a difference in the occupancy of the peripheral binding site (excess substrate inhibition site). At low oxon concentration, both oxons have similar accessibility to the enzyme active site yielding similar ki. Since CPO and PO share a common diethyl phosphate metabolite that is responsible for binding and inactivation of AChE, it is reasonable to assume that they would exhibit similar inhibition kinetics for AChE. Therefore, the similarity in inhibitory capacity at low oxon concentration is biologically plausible based on the stochiometric interaction of OP with cholinesterase, suggesting that other chemically related phosphorothioate insecticides would behave similarly following in vitro interaction with AChE. Nevertheless, additional in vitro studies are needed for validation.
The results of this study suggest the presence of a peripheral binding site that may play an important role in determining the biological interaction of AChE with CPO and PO and potentially other OP insecticides. This may be of particular relevance in understanding the dynamics associated with exposures at low, environmentally relevant levels. Our data clearly characterized the different ki values for the inhibition of rat brain AChE by PO and CPO under various in vitro kinetic scenarios and indicated that the estimated ki values are significantly affected by the oxon concentrations, which may be explained by the presence of a proposed secondary binding site on AChE. Still, more studies are needed to further assess the cholinesterase response to other insecticides in different species. Also, it should be noted that changing the phosphorylation capacity of the oxon molecules following in vitro incubation of AChE with different oxon concentrations has not been confirmed with the in vivo exposure studies; therefore, the possibility of an in vivo concentration effect needs more investigation.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Battelle, Pacific Northwest Division, 902 Battelle Boulevard, P.O. Box 999, Richland, WA 99352. Fax: (509) 376-9064. E-mail: charles.timchalk{at}pnl.gov.
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