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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: paraoxon; acetylcholinesterase; organophosphate; pesticide.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 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.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals.
Male Sprague-Dawley rats (300350 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.
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® 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).
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 µl 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 3040 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 µl of diluted rat brain homogenate (with and without 1% Triton X-100) in a total volume of 400 µl, 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 µM 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 appki for the inhibition of acetylcholinesterase by paraoxon (as evidenced by nonlinear secondary plots, see Fig. 7
) suggests complex interactions between detergent, paraoxon, and enzyme. A changing slope in the secondary plots (Fig. 7
) might reflect an appki 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 concentrationconditions that preclude the determination of appki by the generally accepted approach first outlined by Main (1964; see also Figs. 47
).
In view of the results of the current report, the interpretation of acetylcholinesterase activities in the presence of 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).
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bon, S., Vigny, M., and Massoulie, J. (1979). Asymmetric and globular forms of acetylcholinesterase in mammals and birds. Proc. Natl. Acad. Sci. U.S.A. 76, 25462550.[Abstract]
Ellman, G. L., Courtney, D., Valentino A., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 8895.[ISI][Medline]
Eto, M. (1974). Organophosphorus Pesticides: Organic and Biological Chemistry. CRC Press, Cleveland.
Froede, H. C., and Wilson, I. B. (1984). Direct determination of acetyl-enzyme intermediate in the acetylcholinesterase-catalyzed hydrolysis of acetylcholine and acetylthiocholine. J. Biol. Chem. 259, 1101011013.
Hall, Z. W. (1973). Multiple forms of acetylcholinesterase and their distribution in endplate and non-endplate regions of rat diaphragm muscle. J. Neurobiol. 4, 343361.[ISI][Medline]
Hestrin, S. (1949). The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application. J. Biol. Chem. 189, 249261.[ISI]
Jaganathan, L., and Boopathy, R. (1998). Interaction of Triton X-100 with acyl pocket of butyrylcholinesterase: Effect on esterase activity and inhibitor sensitivity of the enzyme. Ind. J. Biochem. Biophys. 35, 142147.[ISI][Medline]
Johnson, C. D., and Russell, R. L. (1975). A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations. Anal. Biochem. 64, 229238.[ISI][Medline]
Kardos, S. A., and Sultatos, L. G. (2000). Interactions of the organophosphates paraoxon and methyl paraoxon with mouse brain acetylcholinesterase. Toxicol. Sci. 58, 118126.
Loof, I. (1992). Experience with the Ellman method: Proposal for a modification and an alternative method (PAP). In Proc. U.S. EPA Workshop on Cholinesterase Methodologies, Arlington, VA, December 45, 1991 (B.W. Wilson, B. Jaeger, and K. Baetcke, Eds.), pp. 119142. Office of U.S. EPA, Pesticide Programs, Washington, DC.
Main, A. R. (1964). Affinity and phosphorylation constants for the inhibition of esterases by organophosphates. Science 144, 992993.[ISI][Medline]
Mileson, B. E., Chambers, J. E., Chen, W. L., Dettburn, W., Ehrich, M., Eldefrawi, A. T., Gaylor, D. W., Hamernik, K., Hodgson, E., Karczmar, A. G., Padilla, S., Pope, C. N., Richardson, R. J., Saunders, D. R., Sheets, L. P., Sultatos, L. G., and Wallace, K. B. (1998). Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol. Sci. 41, 820.[Abstract]
Mortensen, S. R., Brimijoin, S., Hooper, M. J., and Padilla, S. (1998). Comparison of the in vitro sensitivity of rat acetylcholinesterase to chlorpyrifos-oxon: What do tissue IC50 values represent? Toxicol. Appl. Pharmacol. 148, 4649.[ISI][Medline]
Mortensen, S. R., Chanda, S. M., Hooper, M. J., and Padilla, S. (1996). Maturational differences in chlorpyrifos-oxonase activity may contribute to age-related sensitivity to chlorpyrifos. J. Biochem. Toxicol. 11, 279287.[Medline]
Nostrandt, A. C., Duncan, J. A., and Padilla, S. (1993). A modified spectrophotometric method appropriate for measuring cholinesterase activity in tissue from carbaryl-treated animals. Fundam. Appl. Toxicol. 21, 196203.[ISI][Medline]
Radic, Z., Gibney, G., Kawamoto, S., MacPhee-Quigley, K., Bongiorno, C., and Taylor, P. (1992).Expression of recombinant acetylcholinesterase in a baculovirus system: Kinetic properties of glutamate 199 mutants.Biochemistry 31, 97609767.[ISI][Medline]
Radic, Z., Pickering, N. A., Vellom, S. C., Camp, S., and Taylor, P. (1993). Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry 32, 1207412084.[ISI][Medline]
Stavinoha, W. B., Ryan, L. C., and Endecott, B. R. (1969). A study of the measurement of cholinesterase by constant-pH titration: The effect of hemolysins and a comparison with the electrometric technique. Toxicol. Appl. Pharmacol. 14, 469474.[ISI][Medline]
Sultatos, L. G. (1994). Mammalian toxicology of organophosphorus pesticides. J. Toxicol. Environ. Health 431, 271289.
Sultatos, L. G. (2000). Kinetic interactions of paraoxon with rat brain acetylcholin-esterase. Toxicologist 54, 338.
Taylor, P., and Radic, Z. (1994). The cholinesterases: From genes to proteins. Pharmacol. Toxicol. 34, 281320.
Vigny, M., Bon, S., Massoulie, J., and Gisiger, V. (1979). The subunit structure of mammalian acetylcholinesterase: Catalytic subunits, dissociating effect of proteolysis and disulphide reduction on the polymeric forms. J. Neurochem. 33, 559565.[ISI][Medline]
Wang, C., and Murphy, S. D. (1982). The role of non-critical binding proteins in the sensitivity of acetylcholinesterase from different species to diisopropyl flurophosphate (DFP) in vitro. Life Sci. 31, 139149.[ISI][Medline]
Wilson, I. B., Bergmann, F., and Nachmansohn, D. (1950). Acetylcholinesterase X. Mechanism of the catalysis of acylation reactions. J. Biol. Chem. 186, 781790.
Wilson, B. W., Padilla, S., Henderson, J. D., Brimijoin, S., Dass, P. D., Elliot, G., Jaeger, B., Lanz, D., Pearson, R., and Spies, R. (1996). Factors in standardizing automated cholinesterase assays. J. Toxicol. Environ. Health 48, 187195.[ISI][Medline]