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2000 19: 511Hum Exp ToxicolH J Mason, C Sams, A J Stevenson and R Rawboneinhibition by organophosphorus pesticides

Rates of spontaneous reactivation and aging of acetyicholinesterase in human erythrocytes after  

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Human & Experimental Toxicology (2000) 19, 511-516c 2000 Arnold All rights reserved 0960-3271/00 $15.00

www.arnoldpublishers.com/journals

Rates of spontaneous reactivation and agingof acetyicholinesterase in human erythrocytesafter inhibition by organophosphoruspesticidesHJ Mason*", C Sams', AJ Stevenson' and R Rawbone2

'Health and Safety Laboratoiy, Broad Lane, Sheffield, S3 7HQ, UK; 2Health Directorate, Health and SafetyExecutive, Stanley Precinct, Bootle, L20 3QZ, UK

The in vitro rates of spontaneous reactivation and aging inhuman erythrocyte acetylcholinesterase were studied afterinhibition by a dimethoxy (RjR2) and diethoxy substituted(RjR2) organophosphate pesticide (OP) of general struc-ture R1R2P(O)X. These have been compared with data forhuman plasma cholinesterase previously reported using asimilar methodology.A significantly slower rate of aging for erythrocyteacetylcholinesterase was found compared to plasmacholinesterase, whether inhibited by dimethoxy ordiethoxy substituted OPs. For diethoxy OPs the rate ofspontaneous reactivation of the inhibited plasma enzymewas significantly slower than for the inhibited red cellenzyme. This acetylcholinesterase, and previously pub-lished plasma cholinesterase, data suggest that in practisea blood sample taken 30-40 h after significant acute OPexposure will still show inhibition in either plasma orerythrocyte cholinesterase when analysed, but that anyinhibited plasma enzyme is more likely to be in the agedform. In contrast a substantial proportion of the erythro-cyte acetylcholinesterase is found unaged and thereforesensitive to reactivation by oximes. Samples from an

occupational exposure where depressions in plasma orerythrocyte cholinesterase activity from baseline measure-ments were reactivated ex vivo using the oxime 2-PAMsupport this hypothesis. These data also confirm that theplasma enzyme is a more sensitive than erythrocyteacetylcholinesterase as an indicator of OP exposure andthus the potential value of ex vivo oxime reactivation oferythrocyte acetylcholinesterase in a blood sample toindicate subclinical OP exposure may be limited. However,this study is too small to draw conclusions on thesensitivity of ex vivo oxime reactivation of acetylcholines-terase as a novel biomarker of excessive OP absorption.Given that there is a better relationship between antic-holinergic symptoms and red cell acetylcholinesteraseinhibition, and that the slower resynthesis rate of any agedor inhibited red cell enzyme may be interpretatively usefulwhen venepuncture is delayed, it is suggested that red cellacetylcholinesterase activity does have a place in monitor-ing potential OP exposure. Human & ExperimentalToxicology (2000) 19, 511-516.

Keywords: cholinesterase; erythrocytes; organophosphates

Introduction

Organophosphorus compounds (OPs) form an impor-tant class of pesticides. Their mode of toxicity isthrough inhibition of acetylcholinesterase, a keyenzyme in the metabolism of the neurotransmitteracetylcholine within the cholinergic nervous system.Acute inhibition of acetylcholinesterase in the ner-

vous tissues and neuromuscular junctions of humansleads to a well-characterised set of symptoms.' Thesame enzyme, acetylcholinesterase (AChE), is alsofound in erythrocytes, whereas another enzyme

inhibited by OPs, called "pseudocholinesterase" or

"plasma cholinesterase" (pChE), is found in bloodplasma.

Erythrocyte AChE and plasma pChE are oftenmonitored in cases of accidental OP exposure and inworkers with high risk of exposure to OPs.1'2 OP-induced depressions in erythrocyte AChE are con-

sidered to reflect changes in nerve tissue more closelythan are changes in the plasma enzyme activity.3Inhibition of erythrocyte AchE activity is reasonablywell correlated with severity of symptoms in acute OPpoisoning, although the influence of toxicodynamicsin the distribution of OP between blood, storagedepots in fatty tissue and target nervous tissue isimportant in limiting the strength of this relationship.Large inhibitions in pChE have been noted in the

*Correspondence: HJ Mason, Health and Safety Laboratory, BroadLane, Sheffield, S3 7HQ, UKReceived 12 May 2000; revised 15 July 2000; accepted 24 July 2000

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absence of any significant symptoms of toxicity to thecholinergic nervous system.3 Thus pChE is consid-ered to be a more sensitive indicator of OP absorptionthan the red blood cell AChE.

Inhibition of both enzymes is caused by phosphor-ylation of the active site by an OP of general structure,R1R2P (O) X. In causing this phosphorylation the OPstructure is split and the alkyl phosphate moiety(R1R2P (0) -) is attached to a serine amino acidwithin the enzyme active site.4 Most OPs registeredas pesticides or medicines in the UK have dimethoxyor diethoxy groups as the alkyl groups (R1R2). Afterinhibition by an OP, pChE and erythrocyte AChE canundergo two fates. One leads to an "aged enzyme"where, after a molecular rearrangement of the alkylphosphate group attached to the serine residue, theenzyme is irreversibly inhibited and enzyme activitycan only return by synthesis of new enzyme. Alter-natively the inactivated enzyme can spontaneouslyreactivate back to the normal active enzyme. Thesetwo fates of inhibited, non- aged enzyme havedifferent rates of reaction, which are said to dependon the nature of the alkyl phosphate group of the OP.Spontaneous reactivation of inhibited enzyme can beaccelerated by the use of nucleophilic reagents, suchas oximes, and these reagents form the basis of clinicaltreatment of acute OP poisoning.5

The rates of spontaneous reactivation and aging forthe enzymes are important parameters which under-pin the value of these enzymes in the diagnosis of acutepoisoning or in the monitoring of OP exposure. Agingand spontaneous activation can continue both in vivoafter exposure has ceased and ex vivo in the time takenbetween venepuncture and laboratory analysis. Forthe erythrocyte enzyme, the replacement of any agedenzyme is dependent on the lifetime of the erythrocytein circulation (approx. 120 days) whereas the replace-ment rate of plasma ChE is considerably faster.6'7

Historically, the rates of aging and spontaneousreactivation have been studied in vitro for AChe fromvarious tissue and animal species.8- Studies inhuman material have not been prominent and themethods used to monitor the enzyme reaction rateswere imprecise compared to present analytical tech-niques. We have previously reported the in vitro ratesof aging and spontaneous reactivation in humanplasma ChE,`i which suggested that in the in vivosituation the majority of any inhibited plasma enzymewould be in the "aged" form, particularly where OPgets stored in lipophilic tissue and then leaches outover a period of time.We now report a series of in vitro experiments of the

rates of aging and spontaneous reactivation of AChEin native human erythrocytes. A similar experimentaltechnique has been used as that in the previous studyof pChE.1i The concordance of the results with data

from occupational exposure are discussed togetherwith the implications for the use of red blood cellAChE in monitoring OP exposure.

Methods

The basis of the experimental technique was thaterythrocyte AchE was inhibited by OP and then anyloosely bound OP removed by rapid, repeated wash-ing. The spontaneous return of AChE activity wasmonitored immediately after the final wash and attime points up to 72 h after the initial measurement.The increase in aged enzyme was investigated bydetermining the level of AChE inhibition at any timepoint that could not be reversed by addition ofpralidoxime chloride (2 -PAM).AChE activity for the in vitro experiments was

measured by a routinely used colorimetric assay on aCOBAS FARA automated analyser.7i2 The methodhas a long-term interbatch precision of approximately3.5%. For in vitro experiments, fresh whole humanblood, collected using EDTA anticoagulant, wascentrifuged and the plasma and white cells removed.The erythrocytes were resuspended in 0.9% sodiumchloride solution to give an approximate 50% suspen-sion and mixed thoroughly. A 1:50 dilution of thesewashed erythrocyte was measured for AChE activity.A diethoxy-OP (paraoxon-ethyl) and a dime-

thoxy-OP (azinphosmethyl-oxon) were used in thisexperimental work. Azinphos -methyl -oxon was usedrather than paraoxon -methyl as it is known to be amore potent inhibitor of AChE in intact erythrocyteand thus allows a smaller volume of solvent-based OPsolution to be used. Amounts of paraoxon-ethyl andazinphosmethyl - oxon were defined that produced90-95% inhibition of enzyme activity from initialexperimentation constructing the dose-inhibitionresponse curves for the two OPs.

Thus, solutions of 1.5 ,g azinphos-methyl-oxon(Promochem, UK) and 4.50 ,ug paraoxon-ethyl(Promochem) were blown to dryness under nitrogen.Nine milliliters of washed 50% suspension of ery-throcytes was added to each tube containing OP andincubated at room temperature (approximately 220C)for 30 min with gentle mixing. The erythrocytes werethen washed six times to remove unbound, excess OPby centrifuging and resuspending in 0.9% sodiumchloride solution, which was ice cold to minimisereactivation and ageing. After the final centrifugation,the erythrocytes were resuspended to 9 ml and mixedthoroughly. Subsequent incubations were carried outat 4, 22 and 370C. AChE activity was then measuredperiodically, as described above. 2-PAM-inducedreactivation at each time point was carried out bymaking 100 pl of the erythrocyte suspension 1 mM

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Actiwt

controh+2PAMconr

, hiMdA+2PAM

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Figure 1 Schematic diagram of in vitro experiments. 1 Representsthe activity measurements of the sample inhibited by OP withany residual OP removed. 2 Represents the activity measured inan aliquot of the OP inhibited sample reactivated with 2-PAM ata particular time-point. 3 Represents the activity measured inthe control sample which was subject to all manipulationsexcept not inhibited by OP. 4 Represents the activity in analiquot of the control sample reactivated with 2-PAM at aparticular time -point

with respect to 2-PAM and incubating at roomtemperature for 1 h before reanalysing for AChEactivity. This concentration of 2-PAM had beenshown to remove over 95% of inhibition in an unaged,but inhibited erythrocyte sample. An unpoisonedcontrol sample was also prepared and underwentparallel investigation.The rates of spontaneous reactivation and aging are

both proportional to the concentration of the phos-phorylated enzyme. The rates of aging can be definedby changes in the level of enzyme activity that cannotbe reactivated by oximes, such as 2-PAM. By combi-nation, substitution and solution of first order equa-tions describing the two reactions, it can be shown thatthe percentage of active enzyme (i.e., measuredactivity at a time point) is defined by the equation:

Y - 100 x (B/(B + A)(1- exp(-(B + A)t))

whereA = rate constant for aging, B = rate constant forspontaneous reactivation, t is time in hours and Y is

00; '=_ >_ _6aSc00E u

N

3a- c

X.0

0 10 20 30 40 50 60 70

Hours

Figure 2 Curve fit (Skrinjaric - Spoljar9) for return of red cell AChEactivity after inhibition by paraoxon (diethoxy-OP) and subse-quent removal of any residual OP. Data points shown forexperiments at 37°C

Reactivation and aging of acetylcholinesteraseHJ Mason et al

513

the measured percentage enzyme activity at any timepoint after enzyme poisoning by OP and removal ofresidual OP relative to the unpoisoned enzymeactivity. (i.e. Y= [ (activity of OP sample at time t) /(activity of control sample at time t) I x 100). Thederivation of this equation is described in detail inSkrinjaric - Spoljar.9 Figure 1 is a schematic diagram ofactivity measurements obtained in this project.PRISM V2. (Graphpad, USA) was used to fit themeasured activity data, adjusted by control data, tothe equation above.

Blood for these in vitro experimental studies wasobtained from volunteers within our laboratory whohad given informed consent as part of an EthicsCommittee approved project on OPs.

Occupational study

A number of workers in a factory preparing OP-containing products were undergoing regular biologi-cal monitoring and formal health surveillance by anoccupational physician.13 Due to an incident sevenworkers were considered likely to have had excessiveoccupational exposure to a dimethoxy-OP and gaveconsent to the occupational physician for furtherclinical/toxicological evaluation of any likely OPexposure. As part of this investigation the influenceof ex vivo 2-PAM reactivation was additionally madeto standard methodologies for measuring pChE andAChE were used;'2 by making an aliquot of eachsubject's plasma and washed erythrocyte 2 mM withrespect to 2-PAM and incubating for 2 h beforereanalysis for pChE and AChE activity.

Results

The data used to calculate each rate constant werecollected from three separate experiments using

+422C

- +37°C------ +4°C

0 10 20 30 40 50 60 70

Hours

Figure 3 Curve fit (Skrinjaric - Spoljar9) for return of red cell AChEactivity (spontaneous reactivation) after inhibition by azinphos -

methyl - oxon and removal of any residual OP. Data points shownfor experiments at 37°C

>_ 50-- ----- -+4C

= 7_+22°C' u o 40-

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A

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Table 1 First -order reaction rate constants for aging and spontaneous reactivation of human erythrocyte AChE after inhibition by OP Therate constants were derived from three separate experiments using reiterative curve fitting to the theoretical formula presented bySkrinjaric - Spoljar.9 Mean reaction rate and (95% confidence interval) shown.

Temperature (°C) Rate constant (1st-order) for aging (h-') Rate constant (lst-order) forspontaneous reactivation (h -

Diethoxy-OP (paraoxon)4 - 0.01033 ( 0.02146 to 0.000793) 0.002888 (0.001781-0.00399)22 0.001575 ( 0.006266 to 0.009417) 0.005379 (0.004174-0.006583)37 0.01498 (0.00731-0.0227) 0.01412 (0.01163-0.01662)

Dimethoxy-OP (azinphosmethyl-oxon)4 0.001706 (-0.01419 to 0.01761) 0.002944 (0.001626-0.004263)22 0.005775 ( 0.006841 to 0.01839) 0.006806 (0.004516-0.009095)37 0.02198 (0.01127-0.03269) 0.01888 (0.01494-0.02282)

between 39 and 52 time points. The calculated Although in general the confidence intervals for thepercentage aging at the initial time point for all half-lives of aging and spontaneous reactivation forexperiments was less than 5%. Figures 2 and 3 show red cell AChE were much wider than those for thethe fitted equations at three temperatures for the plasma enzyme, the mean half-lives of aging werediethoxy and dimethoxy-OP, respectively. All the significantly longer for the red cell enzyme. Overallmeasured data points at 37'C are shown to give a the repeatability of experiments for the red cellrepresentation of the curve fit. enzyme was not as good as for the plasma enzyme.

The rate constants for spontaneous reactivation For dimethoxy-OPs, where calculated data for theand aging are shown in Table 1. The correlation of plasma enzyme was available,"1 the half-life ofdetermination (R2) for the fitted data were all spontaneous reactivation of the red cell enzyme wasgreater than 0.69. Correlations were better for the significantly longer than for the plasma enzyme,higher temperature experiments; R2 being 0.92 and suggesting that the rate of spontaneous reactivation0.85 for diethoxy and dimethoxy-OPs, respectively, for plasma is faster. For diethoxy-OP and the plasmaat 37°C. At both 4 and 22°C the rate constants for enzyme," the Skrinjaric-Spoljar equation had failedaging were not statistically different from zero for to produce a result probably due to the very slowboth OPs. return of spontaneous enzyme activity. It is therefore

The half-lives for aging erythrocyte AChE inhib- likely that for the red cell enzyme the rate ofited by dimethoxy and diethoxy OPs at 37°C are spontaneous reactivation is considerably faster thancalculated from Table 1 as 31.5 (95% CI= 21.2-61.5) for plasma ChE after diethoxy-OP inhibition.and 46.3 (95% CI=30.5-94.8) h. These contrastwith much shorter half-lives for the aging of plasmaChe at 37°C calculated as 5.5 and 12.6 h in our Data from occupational exposureprevious paper." Table 2 compares the half-lives ofaging and reactivation for red blood cell AChE and Seven workers gave depressions in plasma ChE that,plasma ChE defined from this project and the earlier using our current laboratory criteria,'4 would bestudy that used similar methodologies and calcula- considered significant (Table 3). Only 1 of 7 subjectstion techniques. showed a statistically significant increase in plasma

Table 2 Mean half-lives of aging and spontaneous reactivation for red cell cholinesterase and plasma pChE

Half-life aging (h) Half-life reactivation (h) Half-life aging (h) Half-life reactivation (h)

RBC acetylcholinesterase Plasma cholinesterase

Diethoxy- OP22°C 440 (70-inf) 129 (105-166) 39 (36-41) NA very long?37°C 46 (31-94) 49 (41-60) 13 (12-14) NAverylong?

Dimethoxy- OP22°C 120 (38-inf) 102 (76-153) 22 (18-25) 63 (54-75)37°C 32 (21-61) 37 (30-46) 6 (5-8) 22 (18-26)

The latter data is from Mason."1 Data have been derived using similar experimental methodologies.

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Table 3 Comparison of plasma ChE activity (Chet) and red blood cell AChE (AChEt), and again after incubation for at least 2 h of the samesample made 2 mM with respect to 2-PAM (Che2- PAM, AChe2-PAM)

Subject or % pChE % pChE depression Che2 PAM-Chet Iul Red cell AChE % Red cell AchE % AChE2- PAM-AChEt IuIsample depression from baseline depression from depression baseline

from baseline after 2- PAM baseline after 2PAM

1 37 38 - 30 9 6 4702 15 12 10 9 5 10063 45 39 110 8 3 8134 39 39 0 8 4 5885 59 56 80 13** 4 15236 26 9* 600* 14** 3 21217 75 74 20 27** 9 2600H20 (2) - - 20;50 - - - 80; 55In vitro poisoned 91 7 2690 93 5 17,220sample

Samples were from seven workers monitored for suspected occupational exposure to organophosphorus pesticides. Two water samples andnormal plasma sample, poisoned in vitro immediately before 2-PAM addition, were also analysed. *Represents pChE showing a significantex vivo reactivation by 2-PAM. **Represents samples showing significant depressions in red cell AChE activity.

ChE activity following ex vivo 2-PAM addition. This isin contrast to a control plasma poisoned in vitroimmediately before 2 -PAM reactivation where 2-PAM removed 84% of the inhibition. The remainingsix subjects showed no effect of 2-PAM reactivationon pChE.

Of the seven subjects with depression in plasmaChE, only three subjects (Table 3) showed asignificant depression in red cell AChE using ourlaboratory criteria.14 However, all seven erythrocytesamples showed some increase in enzyme activitycaused by 2-PAM addition. The three samples thatshowed significant depressions in red cell acetylcho-linsterase activity also showed substantial reactiva-tion in enzyme activity by 2-PAM addition.

Discussion

The in vitro data from intact erythrocyte suggests thatthe rate of spontaneous reactivation and aging areslower than the existing limited published data.Skrinjaric - Spoljar,9 using different experimentaltechniques, found rates of spontaneous reactivationand aging for dimethoxy OPs of 0.816 and 0.178 h-1 .These rates are considerably faster than our data fordimethoxy OPs of 0.0188 h- 1 for spontaneous rates ofreactivation and for aging of 0.02198 h- 1 for AChE inintact erythrocyte. Their technique employed a highdilution of inhibited enzyme rather than our extensivewashing of inhibited erythrocyte to remove any freeOP. We have used a similar experimental technique tothat used in our previous study on plasma ChE.1" Itwould be surprising if the rate of spontaneousreactivation is as fast as noted by Skrinjaric - Spoljaras this would suggest that after a single exposureerythrocyte AChe would very rapidly spontaneouslyreactivate to normal activity. In our experience of

investigating subclinical OP incidental exposures,any inhibition of erythrocyte AChE is usually lessthan that found in pChE, but it is often still apparenteven when the blood sample is taken 1-2 days afterexposure.

The rates of aging and reactivation are temperaturedependent. But the rates of spontaneous reactivationand aging are such that, in practise, sending orkeeping blood samples overnight at room temperatureor lower for immediate laboratory analysis will notradically alter the level of aging or inhibition found inthe sample. Keeping samples at 4°C between vene-puncture and analysis even for a period of severaldays will largely eliminate the influence of sponta-neous reactivation or aging of erythrocyte AChE orplasma ChE.

The limited data on the 2-PAM ex vivo reactivationof blood samples from workers exposed to OPsconfirms that the plasma enzyme is more likely tobe inhibited to a significant degree than erythrocyteAChE. Unfortunately, the wide interindividual varia-tion in plasma ChE means that a significant depres-sion in a single activity measurement can only bedefined in relation to an unexposed value in the sameindividual. The relatively fast rate of aging found inthe in vitro experiments means that in practise anyinhibited pChE is likely to be aged and nonreacti-vable. This was confirmed in the seven workersoverexposed to a dimethoxy OP. The results for redcell cholinesterase, although this is a less sensitiveindex of OP exposure, would suggest that the ex vivo2-PAM reactivation of erythrocyte AChE in a bloodsample could be used to confirm significant acute OPexposure for a subject. However, further work isneeded to define the changes in AChE activity,caused by ex vivo oxime reactivation, that could bedefined as greater than expected from normalanalytical variation.

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2 Larsen 0, Hanel H. Effects of exposure to organopho-sphorus compounds on cholinesterase in workersremoving poisonous depots. Scand J Environ Health1982; 8: 222-228.

3 World Health Organization. Organophosphorus insecti-cides: a general introduction. WHO, Geneva, 1986.

4 Gallo M, Lawryk N. Organic phosphorus pesticides. In:Hayes W, Laws E (Eds.), Handbook of PesticideToxicology. Academic Press, San Diego, 1991, pp.

917-1124.5 Johnson M, Vale J. Clinical management of acuteorganophosphate poisoning: an overview. In: BallantyneB, Marrs T (Eds.), Organophosphates and Carbamates:Clinical and Experimental Toxicology. Butterworth andHeinmann, Oxford, 1992, pp. 528-535.

6 Whittaker M. Cholinesterase. Karger, Basel, 1986.7 Mason HJ. The recovery of plasma cholinesterase anderythrocyte acetylcholinesterase in workers after over-exposure to dichlorvos. Occup Med (London) 50(5): 343-347.

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9 Skrinjaric- Spoljar M, Simeon V, Reiner E. Spontaneousreactivation and aging of dimethylphosphorylated acet-ylcholinesterase and cholinesterase. Biochim BiophysActa 1972; 315: 363-369.

10 Witter R, TB G. Rate of formation in-vivo of theunreactivable form of brain cholinesterase in chickensgiven DDVP or malathion. Biochem Pharmacol 1963; 12:1421-1427.

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12 Lewis P, Lowing R, Gompertz D. Automated discretekinetic method for erythrocyte acetylcholinesterase andplasma cholinesterase. Clin Chem 1981; 27: 926-929.

13 Health and Safety Executive. Biological monitoring ofworkers exposed to organo - phosphorus pesticidesMS17. Revised 2000.

14 Mason HJ, Lewis P. Intra-individual variation in plasmaand erythrocyte cholinesterase activities and the mon-itoring of uptake of organophosphorus pesticides. J SocOccup Med 1989; 39: 121-124.

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