Interactions of Rat Brain Acetylcholinesterase with the

63, 208 –213 (2001) Copyright © 2001 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Interactions of Rat Brain Acetylcholinesterase with the Detergent Triton X-100 and the Organophosphate Paraoxon Clint Rosenfeld, Ahmed Kousba, and Lester G. Sultatos 1 Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, New Jersey 07103 Received March 5, 2001; accepted June 15, 2001

The cholinesterases (acetylcholinesterase [E.C. 3.1.1.7]) and butyrylcholinesterase (E.C. 3.1.1.8) are thought to hydrolyze substrates such as acetylcholine and butyrylcholine by a PingPong Bi-Bi kinetic mechanism that can be viewed as an Ordered Uni-Bi reaction since water, the second substrate, is present in excess (Fig. 1, top and center panels; Aldridge and Reiner, 1972; Froede and Wilson, 1984, Wilson et al., 1950). 1

To whom correspondence should be addressed. Fax: (973) 972-4554. E-mail: [email protected].

The phosphorylation of the active center of these enzymes by organophosphates can be viewed kinetically, in the absence of aging, as analogous to the hydrolysis of substrate by cholinesterases. The major difference between normal substrate hydrolysis and enzyme inhibition by organophosphates is that reactivation of the acylated enzyme during catalysis of acetylcholinesterase occurs in a fraction of a second, while reactivation of phosphorylated enzyme occurs over a period of minutes to days (Sultatos, 1994). In the classic report by Main (1964), the Ordered Uni-Bi kinetic scheme for the interaction of organophosphates with acetylcholinesterase was used to derive a bimolecular rate constant, k i, that quantified the inhibitory power of an organophosphate towards acetylcholinesterase (Fig. 1, center and bottom panels). The derivation of this bimolecular rate constant included an affinity constant as well as a phosphorylation constant, and therefore recognized that the inhibitory power of an organophosphate could be a function of both binding affinity to the active site and the rate of phosphorylation (Main, 1964). This bimolecular rate constant continues to be used as the single best approach for comparing inhibitory powers of various organophosphates. Inhibition of the critical enzyme acetylcholinesterase with subsequent cholinergic crisis is the mechanism of acute toxicity of the organophosphorus insecticides (Mileson et al., 1998). Consequently, measurement of acetylcholinesterase activity is important for evaluating the mammalian toxicity of these commonly used insecticides. A variety of methodologies have been developed for quantifying acetylcholinesterase activity (as reviewed by Wilson et al., 1996), including detection of the thiol group formed by hydrolysis of acetylthiocholine (Ellman et al., 1961; Loof, 1992), measurement of the pH change upon hydrolysis of acetylcholine (Hestrin, 1949), and quantification of radiolabeled acetate following hydrolysis of radiolabeled acetylcholine (e.g., Johnson and Russell, 1975). Some of the advantages and disadvantages of these techniques have been discussed previously (Wilson et al., 1996). Mammalian cholinesterase activity has often been determined in toxicological studies in tissue homogenates in the presence of the nondenaturing detergent Triton X-100 at a

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Inhibition of the critical enzyme acetylcholinesterase (E.C. 3.1.1.7) with subsequent cholinergic crisis is the mechanism of acute toxicity of the organophosphorus insecticides (B. E. Mileson et al., 1998, Toxicol. Sci. 41, 8 –20). Consequently, measurement of acetylcholinesterase activity is important for evaluating the mammalian toxicity of this commonly used class of insecticides. While mammalian acetylcholinesterase activity has often been determined in tissue homogenates in the presence of the nondenaturing detergent Triton X-100 at a concentration of 1%, the potential actions of this detergent on the activity of this critical enzyme are not understood. In the current study, homogenization of rat brain in buffer containing 1% Triton X-100 slightly elevated the appV max for hydrolysis of acetylthiocholine, without affecting the appK m or the appK ss. However, the presence of both 1% Triton X-100 and paraoxon (at concentrations of 5 nM–100 nM) resulted in complex kinetic interactions with acetylcholinesterase, as evidenced by a curvilinear secondary plot for determination of the appk i. These results suggest that measurement of acetylcholinesterase activity in the presence of up to 1% Triton X-100, but in the absence of oxon, should pose no problems with regard to data interpretation, provided it is recognized that the detergent slightly elevates activity. However, measurement of acetylcholinesterase activity after enzyme was exposed simultaneously to Triton X-100 and oxon could be problematic. Caution is warranted when interpreting data where acetylcholinesterase activity was determined under such conditions since in the presence of 1% Triton X-100, the capacity of oxon to inhibit acetylcholinesterase might change as a function of oxon levels. Key Words: paraoxon; acetylcholinesterase; organophosphate; pesticide.

ACETYLCHOLINESTERASE, TRITON X-100, AND PARAOXON

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MATERIALS AND METHODS Chemicals. Acetylthiocholine chloride (ATC), 5,5⬘-dithio-bis (2-nitrobenzoic acid; DTNB), and Triton X-100 (alkyl phenoxy polyethoxy ethanol) were purchased from Sigma Chemical Company (St. Louis, MO). Paraoxon (O,Odiethyl O-(p-nitrophenyl) phosphate) was purchased from Chem Services (West Chester, PA). Animals. Male Sprague-Dawley rats (300 –350 g; Tac:N(SD)fBR) were purchased from Taconic Farms (Germantown, NY). They were housed under standard laboratory conditions and given free access to water and food (Purina Rodent Chow 5001). All procedures involving animals were in accordance with protocols established in the NIH/NRC Guide and Use of Laboratory and were reviewed by the Institutional Animal Care and Use Committee at the New Jersey Medical School.

FIG. 1. Kinetic mechanism for the interaction of a substrate or an organophosphate (AB) with acetylcholinesterase (E). The top panel represents a Ping-Pong Bi-Bi kinetic mechanism. The 2 dots to the right of E represent reversible binding of AB to the active site vicinity, while the solid line to the right of E represents the covalent bond between S 200 at the active site and the acetyl or phosphoryl moiety (A) of AB. Reactivation of E occurs as a result of addition of water as a second substrate. The center panel represents a simplification of the Ping-Pong Bi-Bi mechanism to an Ordered Uni-Bi reaction since water is always present in excess. The lower panel represents the basis for the derivation of k i by Main (1964). K i is defined in terms of rate constants depicted in the center panel. In both panels, the first order rate constant k 3 represents reactivation of phosphorylated enzyme. The presence of the peripheral binding site (Taylor and Radic, 1994) is not included in these kinetic schemes. Similarly, the process of aging of phosphorylated enzyme is not included.

concentration of 1%. While the origins of this practice are not entirely clear, early studies showed that most cholinesterase and/or acetylcholinesterase activity from mammalian muscle and nerve tissue could be solubilized in 0.5% or 1% Triton X-100 (Bon et al., 1979; Hall, 1973; Vigny et al., 1979). Despite the long history of the use of 1% Triton X-100 in mammalian cholinesterase assays, little is known regarding the actions of this detergent on this important class of enzymes. Preliminary results from this laboratory have suggested that the effects of 1% Triton X-100 on the interaction of the organophosphate paraoxon and acetylcholinesterase are complex (Sultatos, 2000). Therefore the present study was undertaken to better understand the actions of 1% Triton X-100 on rat brain acetylcholinesterase and its interaction with the organophosphate paraoxon.

Incubations and assays. Following thawing, a 1 ml aliquot of homogenate was added to 5 ml of phosphate buffer with and without 1% Triton X-100. Acetylcholinesterase activity was measured by the Ellman assay (Ellman et al., 1961), modified for a 96-well microtiter plate reader (Mortensen et al., 1996). Assay volumes were reduced to 0.3 ml so that the assays could be performed in a 96-well plate reader (Nostrandt et al., 1993). Wells contained 0.25 ml sample (where sample is 400 ␮l diluted homogenate sample plus 4.6 ml buffer without Triton X-100), with ATC added to give a final concentration of 0.4 mM, and DTNB added to give a final concentration of 0.1 mM. Kinetic studies utilized ATC concentrations ranging from 0.1 mM to 100 mM. The absorbance at 412 nm was monitored over 30 – 40 min. The linear portion of the profile of increasing absorbance with time was fitted with the equation for a straight line, and the slope of this line was used as a measurement of uninhibited acetylcholinesterase activity. Incubations containing paraoxon included 200 ␮l of diluted rat brain homogenate (with and without 1% Triton X-100) in a total volume of 400 ␮l, with the indicated oxon concentrations (made up in phosphate buffer with and without 1% Triton X-100), as described by Kardos and Sultatos (2000). Incubations were terminated by dilution with 4.6 ml phosphate buffer without Triton X-100 since preliminary studies indicated that addition of 4.6 ml phosphate buffer with 1% Triton X-100 inhibited acetylcholinesterase activity (data not shown). Incubations were done at 23°C in a shaking water bath. Controls did not include organophosphate, or received the organophosphate at the end of the incubation, just before addition of buffer for the determination of activity. No differences in results were observed with or without the addition of 3 mM EDTA to the buffer to inhibit A-esterase (Sultatos, 1994; data not shown), so EDTA was not routinely included. Similarly, inclusion of 100 ␮M iso-OMPA to inhibit butyrylcholinesterase (Mortensen et al., 1998) had a negligible effect on the results (data not shown), and was therefore not routinely included in the incubations. Data were fit to the indicated equations by software Sigmaplot (SPSS Science Inc., Chicago, IL). Statistical analyses were performed with the software SigmaStat (SPSS Science Inc., Chicago, IL).

RESULTS

Acetylthiocholine at concentrations of about 1 mM and higher resulted in progressively reduced acetylcholinesterase activity (Fig. 2). This observation has been made previously, and likely occurs as a result of allosteric modification of the


Tissue preparations. Rats were decapitated, and their brains were removed and placed on ice. They were weighed, and homogenized in 9 volumes of 100 mM sodium phosphate buffer (pH 7.4; phosphate buffer) with and without 1% Triton X-100, using a Polytron威 homogenizer (Brinkmann Instruments, Westbury, NY). The homogenates were stored in individual 1 ml aliquots at –70°. Preliminary studies detected no differences in fresh and frozen homogenates, provided homogenates were not kept frozen longer than 3 months (data not shown).

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sion of 1% Triton X-100 elevated slightly the appV max of acetylcholinesterase, without affecting the appK ss and appK m (Fig. 3). In the absence of Triton X-100, the determination of the app k i for paraoxon towards acetylcholinesterase in rat brain homogenate was uncomplicated, yielding primary and secondary plots, as well as app k i values comparable to those reported previously by other investigators (Figs. 4 and 5; Eto, 1974; Kardos and Sultatos, 2000; Wang and Murphy, 1982). Inclusion of 1% Triton X-100 within the homogenate and the incubations with paraoxon yielded apparently unremarkable primary plots (Fig. 6), but atypical, nonlinear secondary plots (Fig. 7). The nonlinear relationship between the inverse of the paraoxon concentration and the inverse of the slope of the acetylcholinesterase velocity curve precluded the calculation of an app k i for paraoxon in the presence of 1% Triton X-100.

FIG. 2. Hydrolysis of acetylthiocholine by rat brain homogenate as a function of substrate concentration and 1% Triton X-100. Each panel represents data from a single rat. The circles and triangles are hydrolysis rates in the absence and presence, respectively, of 1% Triton X-100. The solid lines represent curve fits to the Haldane equation v ⫽ V max/(1 ⫹ K m/[S] ⫹ [S]K ss), where K ss represents a substrate inhibition constant (Radic et al., 1993).

active site through binding of acetylthiocholine to the peripheral binding site (Radic et al., 1993). Hydrolysis of acetylthiocholine by acetylcholinesterase as a function of substrate concentration can be modeled by the Haldane equation (Fig. 2; Radic et al., 1992, 1993), where the concentration dependence is described in terms of the Michaelis-Menten constant, K m, and a substrate inhibition constant, K ss. In the current study, fitting of acetylcholinesterase activity (in the absence of detergent) as a function of acetylthiocholine concentration with the Haldane equation yielded values for appK m and appK ss in the same range as those reported previously for recombinant mouse wild-type acetylcholinesterase (Fig. 3; Radic et al., 1993). The presence of 1% Triton X-100 in the homogenate slightly increased acetylcholinesterase activity at nearly all concentrations of acetylthiocholine utilized (Fig. 2). Application of the Haldane equation to these data revealed that inclu-

While an early study reported that Triton X-100, at concentrations up to 8%, inhibited human whole blood cholinesterase activity (Stavinoha et al., 1969), subsequent studies failed to reproduce this effect (Bon et al., 1979; Hall, 1973; Vigny et al., 1979). However the current study revealed a biphasic effect of 1% Triton X-100 in rat brain homogenate hydrolysis of acetylthiocholine. The presence of 1% Triton X-100 during the preparation of the homogenate, in the incubations, and in the 4.6 mls of buffer utilized to terminate the incubations, inhibited hydrolysis of acetylthiocholine activity (data not shown), while the presence of the detergent in the homogenate and incubations, but not the buffer used to terminate the incubations, resulted in a slight elevation of the app V max (Fig. 3). Increased activity in the

FIG. 3. Effects of 1% Triton X-100 on kinetic parameters derived from hydrolysis rates presented in Figure 2. The bars represent the mean and standard deviation of an n ⫽ 3. Open bars show parameters determined in the absence of detergent (also indicated by the minus sign), while cross-hatched bars show parameters determined in the presence of detergent (also indicated by the plus sign). An asterisk indicates a significant difference (p ⬍ 0.05) from the corresponding kinetic parameter determined in the absence of detergent, by a paired t-test.


DISCUSSION


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FIG. 4. Brain homogenate acetylcholinesterase activity following incubations with different paraoxon concentrations for a variety of time periods. Each panel represents data from a single rat. Each data point represents the velocity of thiocholine production from acetylthiocholine over 30 – 40 min, calculated as the change in optical density (OD) at 412 nm. The symbols indicate the following paraoxon concentrations: 5 nM, circles; 10 nM, squares; 50 nM, triangles; and 100 nM, diamonds. The straight lines represent the best fit from linear regression analyses. Slopes obtained from the regression analyses were used for the secondary plots in Figure 5.

presence of 1% Triton X-100 could result from a slight activation of acetylcholinesterase, since this same detergent has been reported to slightly activate butyrylcholinesterase by interacting with the hydrophobic region of the acyl pocket of the enzyme (Jaganathan and Boopathy, 1998). Similarly, solubilization of acetylcholinesterase from brain homogenate might also account for the detergent effect. Although the effect of 1% Triton X-100 on hydrolysis of acetylthiocholine by acetylcholinesterase was slight (Figs. 2 and 3), this detergent markedly affected the interaction of paraoxon and acetylcholinesterase (Fig. 7). The inability to determine an appk i for the inhibition of acetylcholinesterase by paraoxon (as evidenced by nonlinear secondary plots, see Fig. 7) suggests complex interactions between detergent, paraoxon,

FIG. 5. Secondary plot for k i determination for each rat utilized in Figure 4. Each symbol represents a slope value obtained from the corresponding curve in Figure 4. The appk is were obtained from the slopes of these secondary plots and were as follows: rat 1 (top panel) 0.0781 nM –1min –1; rat 2 (center panel) 0.0943 nM –1min –1; and rat 3 (bottom panel) 0.0528 nM –1min –1.


and enzyme. A changing slope in the secondary plots (Fig. 7) might reflect an appk i that changes as a function of the paraoxon concentration, in the presence of the detergent. However, the exact nature of these complex interactions cannot be determined from the data presented in the current report. It should be noted that a previous study from this laboratory (Kardos and Sultatos, 2000) suggested that paraoxon and methyl paraoxon interact reversibly with acetylcholinesterase at a site separate from the active site, thereby reducing the capacity of subsequent oxon molecules to phosphorylate the active site. The current study did not address the possible effect of detergent on this binding since this interaction can be quantified only at oxon concentrations that are similar to the enzyme concentration— conditions that preclude the determination of appk i by the generally accepted approach first outlined by Main (1964; see also Figs. 4 –7). In view of the results of the current report, the interpretation of acetylcholinesterase activities in the presence of

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1% Triton X-100 deserves consideration. Measurement of acetylcholinesterase activity in the presence of Triton X-100, but in the absence of oxon, should pose no problems with regard to data interpretation, provided it is recognized that the detergent slightly elevates activity. However, measurement of acetylcholinesterase activity after enzyme was exposed simultaneously to Triton X-100 and oxon could be problematic given the effects reported here. Caution is warranted when interpreting data where acetylcholinesterase activity was determined under such conditions since in the presence of 1% Triton X-100, the capacity of oxon to inhibit acetylcholinesterase might change as a function of oxon levels (Fig. 7).

REFERENCES

FIG. 6. Brain homogenate acetylcholinesterase activity in the presence of 1% Triton X-100 following incubation with different paraoxon concentrations for a variety of time periods. Each panel represents data from a single rat (the same rats utilized in Figure 4). Each data point represents the velocity of thiocholine production from acetylthiocholine over 30 – 40 min, calculated as the change in optical density (OD) at 412 nm. The symbols indicate the following paraoxon concentrations: 5 nM, circles; 10 nM, squares; 25 nM, hexagons; 50 nM, triangles; and 100 nM, diamonds. The straight lines represent the best fit from linear regression analyses. Slopes obtained from the regression analyses were used for the secondary plots in Figure 7.

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FIG. 7. The effects of 1% Triton X-100 on the secondary plot for k i determinations. Each symbol represents a slope value obtained from the corresponding curve in Figure 6. The paraoxon concentrations associated with each symbol were as follows: 5 nM, circles; 10 nM, squares; 25 nM, hexagons; 50 nM, triangles; and 100 nM, diamonds. Deviation from linearity suggests complex kinetic effects. The solid lines represent curve fits of the data to the equation 1/PO ⫽ A *e (B * 1/sl° pe). For the top panel (rat 1), A ⫽ 8.6569 ⫻ 10 – 4 and B ⫽ 0.1038, with a correlation coefficient of 0.99. For the center panel (rat 2), A ⫽ 0.0113 and B ⫽ 0.0308, with a correlation coefficient of 0.99. For the bottom panel (rat 3), A ⫽ 1.0630 ⫻ 10 –5 and B ⫽ 0.1581, with a correlation coefficient of 0.97.


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